Table of contents :

An examination of the blood smear (or film) may be requested by physicians or initiated by laboratory staff. With the development of sophisticated automated blood-cell analyzers, the proportion of blood-count samples that require a blood smear has steadily diminished and in many clinical settings is now < 10-15%. Nevertheless, the blood smear remains a crucial diagnostic aid. The proportion of requests for a complete blood count that generate a blood smear is determined by local policies and sometimes by financial and regulatory as well as medical considerations. For maximal information to be derived from a blood smear, the examination should be performed by an experienced and skilled person, either a laboratory scientist or a medically qualified hematologist or pathologist. In Europe, only laboratory-trained staff members generally "read" a blood smear, whereas in the USA, physicians have often done this. Increasingly, regulatory controls limit the role of physicians who are not laboratory-certified. Nevertheless, it is important for physicians to know what pathologists or laboratory hematologists are looking for and should be looking for in a smear. In comparison with the procedure for an automated blood count, the examination of a blood smear is a labor-intensive and therefore relatively expensive investigation and must be used judiciously. A physician-initiated request for a blood smear is usually a response to perceived clinical features or to an abnormality shown in a previous CBC. A laboratory-initiated request for a blood smear is usually the result of an abnormality in the CBC or a response to "flags" produced by an automated instrument. Less often, it is a response to clinical details given with the request for a CBC when the physician has not specifically requested examination of a smear. For example, a laboratory might have a policy of always examining a blood smear if the clinical details indicate lymphadenopathy or splenomegaly. The International Society for Laboratory Hematology (ISLH) has published consensus criteria for the laboratory-initiated review of blood smears on the basis of the results of the automated blood count. The indications for smear review differ according to the age and sex of the patient, whether the request is an initial or a subsequent one, and whether there has been a clinically significant change from a previous validated result (referred to as a failed delta check). All laboratories should have a protocol for the examination of a laboratory-initiated blood smear, which can reasonably be based on the criteria of the International Society for Laboratory Hematology. Regulatory groups should permit the examination of a blood smear when such protocols indicate that it is necessary. There are numerous valid reasons for a clinician to request a blood smear, and these differ somewhat from the reasons why laboratory workers initiate a blood-smear examination. Sometimes it is possible for a definitive diagnosis to be made from a blood smear. Clinical indications for examination of a blood smear :

• anemia, unexplained jaundice, or both : in patients with anemia, physician-initiated examinations of blood smears are usually performed in response to clinical features or to a previously abnormal CBC. The presence of unexplained jaundice, particularly if unconjugated hyperbilirubinemia is also present, is an additional reason for a blood-smear examination. Laboratory-initiated examinations of blood smears for patients with anemia are usually the result of a laboratory policy according to which a blood smear is ordered whenever the hemoglobin concentration is unexpectedly low. This policy should be encouraged, since the consideration of the blood smear and the red-cell indices is a logical first step in the investigation of any unexplained anemia^ref. Initiating a smear as a reflex test also means that a further blood sample does not have to be taken for this purpose. Modern automated instruments impart valuable information about the nature of anemia. They provide not only a red-cell count, MCV, MCH, and the MCHC but also newer variables that give information that previously could be derived only from a blood smear. These variables usually include the RDW, which correlates on a blood smear with anisocytosis, and they may also include the hemoglobin-distribution width and the percentages of hypochromic and hyperchromic cells, which correlate with anisochromasia, hypochromia, and hyperchromia. A variety of histograms and scatterplots give a visual representation of red-cell characteristics. It may be possible to detect increased numbers of hyperchromic cells (spherocytes or irregularly contracted cells), small hyperchromic cells (microspherocytes), hypochromic microcytic cells, large normochromic cells (normally hemoglobinized macrocytes), and hypochromic macrocytes (either reticulocytes or dysplastic red cells). Despite this wealth of information, there are still morphologic abnormalities that are critical in the differential diagnosis of anemia and that can be determined only from a blood smear. Particularly important is the detection of variations in cell shape and of red-cell inclusions, such as Howell–Jolly bodies, Pappenheimer bodies, and basophilic stippling or punctate basophilia.
• hemolytic anemia : red-cell shape is of considerable diagnostic importance. Some types of hemolytic anemia yield such a distinctive blood smear that the smear is often sufficient for diagnosis. This is true of hereditary elliptocytosis (which is only infrequently associated with anemia; numerous elliptocytes and smaller numbers of ovalocytes), hereditary pyropoikilocytosis (striking poikilocytosis, with elliptocytes, ovalocytes, and fragments), and Southeast Asian ovalocytosis (moderate poikilocytosis, with the poikilocytes including several macro-ovalocytes). The presence of spherocytes is not diagnostically specific, since this may result from hereditary spherocytosis (numerous spherocytes (hyperchromatic cells with a regular outline)), or immune hemolytic anemia. Nevertheless, consideration of the clinical features, together with the results of a direct antiglobulin test, in patients with spherocytes will generally indicate the correct diagnosis. Microangiopathic hemolytic anemia resulting from cyclosporine therapy shows numerous red-cell fragments. Microspherocytes may be present in low numbers in patients with a spherocytic hemolytic anemia but are also characteristic of burns and of microangiopathic hemolytic anemia. The detection of a microangiopathic hemolytic anemia is of considerable clinical significance, since this type of anemia may indicate pregnancy-associated hypertension, disseminated cancer, chronic disseminated intravascular coagulation, the hemolytic–uremic syndrome, or thrombotic thrombocytopenic purpura; the latter 2 conditions both require urgent diagnosis so that appropriate management can be initiated. In microangiopathic hemolytic anemia, examination of the blood smear is also important to validate the platelet count, since red-cell fragments and platelets may be of similar size. Most automated instruments cannot make this distinction. A minority of automated instruments that measure both the size and the refractive index of small particles in the blood sample can make this distinction and can be used to exclude red-cell fragmentation; however, although the fragment "flag" on such instruments is sensitive, it is not specific. Hence, a blood smear is still advised for validation^ref. Blood-smear features similar to those seen in microangiopathic hemolytic anemia are also a feature of mechanical hemolytic anemia, such as that associated with a leaking prosthetic valve, and provide important evidence of this cause of hemolytic anemia. A blood smear is particularly important in the diagnosis of acute hemolysis induced by oxidant damage. The characteristic feature is the presence of keratocytes, or "bite" cells, "blister" cells, and irregularly contracted cells; the latter must be distinguished from spherocytes because of the quite different diagnostic significance. These irregularly contracted cells share with spherocytes the lack of central pallor but differ in that they have an irregular outline. Oxidant-induced hemolysis is most often seen in glucose-6-phosphate dehydrogenase (G6PD) deficiency but can also occur with other defects in the pentose shunt or in glutathione synthesis and when oxidant exposure overwhelms normal protective mechanisms. Oxidant damage may be exogenous, as in exposure to oxidant chemicals or drugs (most often dapsone), or endogenous, as in Wilson's disease^ref. G6PD deficiency affects millions of persons worldwide. A blood smear is important for the diagnosis of this condition, for 2 reasons. First, it is available far more rapidly than are the results of a G6PD assay and, when considered together with the patient's ethnic origin and clinical history, permits a provisional diagnosis. Second, a blood smear can suggest the diagnosis of G6PD deficiency even if a G6PD assay is normal. Normal G6PD activity may be found after acute hemolysis in G6PD-deficient persons in 2 situations. First, men of African or African-American ancestry who are hemizygous for the A– alloenzyme (which is present at normal levels in reticulocytes) can have normal G6PD levels because the reticulocyte count is high after hemolysis. Second, female carriers — for example, those who are hemizygous for the common Mediterranean variant of G6PD — can have a normal assay result after an acute hemolytic episode because the abnormal cells have lysed preferentially, leaving mainly the cells expressing the normal allele in the circulation. In both of these circumstances, the observation of a typical blood smear in an appropriate clinical setting is an indication to repeat the assay once the acute hemolytic episode is over. Other features may aid in the differential diagnosis of hemolytic anemia. For example, the presence of red-cell agglutinates usually indicates the presence of a cold agglutinin, and erythrophagocytosis is often a feature of paroxysmal cold hemoglobinuria
• macrocytic anemia :
• the blood smear is of great importance in the differential diagnosis of macrocytic anemias. For patients in whom there is a deficiency of vitamin B₁₂ or folic acid, the blood smear shows not only anisocytosis and macrocytes but also oval macrocytes and hypersegmented neutrophils. When the anemia is more severe, there may be marked poikilocytosis, with teardrop poikilocytes and red-cell fragments. Although these deficiency states are now usually recognized on the basis of assays of vitamin B₁₂ and folic acid, the blood smear remains important for two reasons. First, it permits a speedy provisional diagnosis, and initiation of appropriate treatment in severely anemic patients while assay results are pending. Second, occasionally there are patients with a clinically significant vitamin B₁₂ deficiency despite a normal assay result. This discrepancy occurs because much of the vitamin B₁₂ that is measured in the assay is bound to haptocorrin, whereas the functional vitamin B₁₂, which is bound to transcobalamin, contributes much less to the assay of total B₁₂. Similarly, acute folic acid deficiency sometimes develops in patients even though the total red-cell folate level remains normal. The observation of a blood smear that is typical of megaloblastic anemia despite normal assays is an indication that further investigation and a trial of treatment are needed. Liver disease and excess ethanol consumption are common causes of macrocytosis, with the blood smear usually showing round rather than oval macrocytes and lacking hypersegmented neutrophils; target cells and stomatocytes may also be present.
• in elderly patients, the myelodysplastic syndromes are an important cause of macrocytosis. Blood-smear features that may point to the diagnosis include anisocytosis, poikilocytosis, macrocytes, stomatocytes, hypogranular or hypolobulated neutrophils, blast cells, giant or hypogranular platelets, Pappenheimer bodies, and the presence of a minor population of hypochromic microcytic cells, leading to a dimorphic smear
• macrocytic anemia resulting from congenital dyserythropoietic anemia also yields a characteristic blood smear, with striking poikilocytosis. When macrocytosis is the result of hemolysis or recent blood loss, the blood smear shows polychromasia, which results from an increased reticulocyte count.
• microcytic anemia : the blood smear is generally less important. Red-cell indices and serum ferritin levels, sometimes supplemented by markers of inflammation, that are interpreted in the context of clinical features, permit the diagnosis of the majority of cases. However, it is important to note that the presence of Pappenheimer bodies and red-cell dimorphism in the sideroblastic anemias and of prominent basophilic stippling in cases of lead poisoning and in some types of thalassemia is diagnostically significant.
• hemoglobinopathy (sickle cell disease-dactylitis or sudden splenic enlargement and pallor in a young child or, in an older child or adult limb, abdominal or chest pain) and thalassemia: a blood smear is useful in the diagnosis and differential diagnosis of sickle cell disease, particularly if there is an urgent need for diagnosis and if the results of hemoglobin electrophoresis or high-performance liquid chromatography are not instantly available. Patients with sickle cell anemia (in which there is homozygosity for hemoglobin S) have anemia, but those with compound heterozygosity for hemoglobin S and hemoglobin C may have a normal hemoglobin level, and the condition thus may be confused with sickle cell trait if a blood smear is not examined. Consideration of the blood-smear features, of the hemoglobin level, and of the results of a sickle cell solubility test usually permits an accurate diagnosis^{ref1, ref2}. The blood smear of a compound heterozygote usually shows target cells, irregularly contracted cells, and boat-shaped cells but few classic sickle cells; typical hemoglobin SC poikilocytes (formed only when hemoglobin S and hemoglobin C are both present) are often seen. Sometimes the blood smear of a compound heterozygote shows only target cells and irregularly contracted cells and cannot be distinguished from the smear in hemoglobin C homozygosity; a positive sickle cell solubility test permits these conditions to be distinguished in an emergency situation (e.g., preoperatively). A blood smear is also important in the diagnosis of an unstable hemoglobin, with irregularly contracted cells and macrocytosis being characteristic of this condition; sometimes there is coexisting thrombocytopenia.
• thrombocytopenia(e.g. petechiae, abnormal bruising) or neutropenia(e.g. unexplained or severe infection) : falsely low platelet counts may be the result of small clots, platelet clumping, platelet satellitism, or abnormally large platelets. Fibrin strands indicate that thrombocytopenia is likely to be factitious. Underlying causes that may be revealed by the blood smear include the May–Hegglin anomaly,microangiopathic thrombopathies, and leukemias and lymphomas.
• thrombocytosis: high platelet counts should be confirmed microscopically with a blood smear; falsely high counts may be the result of other particles (red-cell fragments, fragments of leukemic cells, or fungi (e.g. Candida glabrata)) being counted as platelets^{ref1, ref2, ref3, ref4}. Also look for evidence of a myeloproliferative disorder, such as giant platelets, or an increase in the basophil count; the latter is not reliably detected by automated counters. A sudden, unexpected improvement in the platelet count also should be confirmed by blood-smear examination, since such an improvement may be factitious^ref
• lymphoma or other lymphoproliferative disorder (LPD)-lymphadenopathy, splenomegaly, enlarged thymus or bone marrow failure : blood smears must always be examined when there is unexplained leukocytosis, lymphocytosis, or monocytosisor when the flagging system of an automated instrument suggests the presence of blast cells. Depending on the instrument and the practice of the local laboratory, a flag for atypical or variant lymphocytes may also be an indication for examination of a blood smear, since this flag is sometimes indicative of the presence of blast cells. Low rather than high counts likewise are an indication for a smear, since they may be indicative of aplastic anemia, acute leukemia, hairy-cell leukemia, or infiltration of nonhematopoietic malignant cells into the bone marrow. The role of the blood smear in the diagnosis of leukemia and lymphoma is to suggest a likely diagnosis or range of diagnoses, to indicate which additional tests should be performed, and to provide a morphologic context without which immunophenotyping and other sophisticated investigations cannot be interpreted. For 2 conditions, Burkitt's lymphoma(basophilic vacuolated lymphoma cells) and acute promyelocytic leukemia(bilobed leukemic promyelocytes), a blood smear is of particular importance because it facilitates rapid diagnosis and specific treatment.
• myeloproliferative disorder (MPD)-splenomegaly, plethora, itching, weight loss
• suspicion of disseminated intravascular coagulation (DIC)
• acute or recent onset renal failureor unexplained renal enlargement, particularly in a child
• on retinal examination, hemorrhages, exudates, signs of hyperviscosity
• bacterialor parasiticdisease that can be diagnosed from a blood smear
• disseminated non-hematopoietic cancer-weight loss, malaise, bone pain
• general ill health, often with malaise and fever, suggesting infectious mononucleosisor other viral infection or inflammatory or malignant disease
• probable factitious results : members of the laboratory staff should always initiate a blood smear if an automated instrument produces a highly improbable result. Such results may be factitious, resulting from the accidental freezing or heating of the blood, from hyperlipidemia, or from the presence of cold agglutinins, a cryoglobulin, bacteria, or fungi. Factitious results also may stem from unusual characteristics of the blood cells or the plasma, such as a pseudo-neutropenia caused by a myeloperoxidase deficiency that occurs when the automated instrument employs a peroxidase reaction for the identification of neutrophils, eosinophils, and monocytes. Falsely low counts also may result from neutrophil or platelet clumping or from platelet satellitism.

RBC inclusions
• Howell-Jolly bodies : smooth, round purple staining remnants of nuclear chromatin due to incomplete nucleus expulsion

Aetiology :
• hemolytic anemia
• after splenectomy
• megaloblastic anemia (multiple)
They can be removed in spleen by pitting and culling

• chromatin dust : small red granules, smaller than Howell's bodies, sometimes seen at the periphery of stained erythrocytes
• Cabot's ring bodies : red-purple staining threadlike filaments in the shape of a ring or figure 8 in stained red blood cell, often in association with basophilic stippling. They may also appear as granules in a linear array rather than as complete rings. Cabot rings are thought to be microtubules from a mitotic spindle or remnants of the nuclear membrane.

Aetiology :
• severe anemia including megaloblastic anemia
• leukemia
• lead poisoning.
They are distinguished from the ring forms of Plasmodium by their larger size and by the absence of a red chromatin mass

• Heinz-Ehrlich bodies or granules : coccoid inclusion bodies resulting from oxidative injury to and precipitation of hemoglobin

Aetiology :
• Hb H
• Hb Köln
• enzyme deficiencies (G6PD)
• a-thalassemia
• lead poisoning
Refractile in fresh blood smears, they are not visible when stained with Romanowsky dyes but may be stained supravitally, e.g. with methylene blue.

• Pappenheimer bodies : very fine dark violet staining (basophilic), separated or connected granules present in the cytoplasm of the erythrocyte, particularly often near the periphery of the red cell. They must be confirmed with an iron stain. Probably these are the equivalent to nonhaeme iron granules of siderocytes.


Aetiology :
• sideroblastic anemia
• megaloblastic anemia
• ethanol
• following splenectomy
• some hemoglobinopathies
• sickle cell anemia (SCA)
• siderosomes : ferritin and hemosiderin granules
• hectoid bodies : sickle cell anemia (SCA)
• protozoal inclusions : Giemsa is the stain of choice for blood parasites
• babesial inclusions (Babesia spp.) : both thick and thin blood smears should be prepared. At least 200 to 300 oil immersion fields should be examined before the smears are considered negative (use the 100X oil immersion objective). The parasites appear as pleomorphic, ringlike structures (ring forms). They can resemble the early forms of malarial parasites, particularly Plasmodium falciparum(but falciparum rings are rarely extracellular, babesia parasites tend to be much more pleomorphic and may be 5-6 rings per cell vs. commonly 2 in falciparum cases). The classic arrangement of the 4 rings (Maltese cross) is not always seen. One negative set of blood smears does not rule out a Babesia infection


• leishmanial inclusions from Leishmania spp.


• plasmodial inclusions
• Maurer spots from Plasmodium falciparum


• Schüffner's dots or granules from Plasmodium vivax (arrow). Malaria male microgametes (arrowhead) are produced from the gametocytes by exflagellation, which takes place in the mosquito immediately after the blood meal. Exflagellation is induced by a temperature decrease and by an increase in pH, which occur on passage from the human bloodstream to the mosquito digestive tract. Microgametes are rarely seen in human blood and seem to be produced in vitro when laboratory conditions favor exflagellation during the preparation of blood smears; familiarity with their appearance may prevent diagnostic confusion with organisms such as trypanosomes and spirochetes^ref


• Ziemann granules : from Plasmodium malariae
• granules from Plasmodium knowlesi



• abnormally dried RBCs


• xerocyte : an erythrocyte that is dehydrated and has decreased cations due to abnormal permeability of the membrane that allows leakage of potassium ions and water out of the cell

Aetiology : hereditary xerocytosis
• poikilocyte : an abnormally shaped erythrocyte, such as a
• crenation / crenulation : the notched appearance of an erythrocyte caused by its shrinkage after suspension in a hypertonic solution, creating a burr cell or erythrocyte / crenated erythrocyte, crenocyte, and echinocyte : a spiculed erythrocyte that has multiple small projections evenly spaced over the cell circumference

Aetiology :
• uremia
• gastric carcinoma
• bleeding peptic ulcer

• acanthocytes (spur cells) : red cells with finger-like (thorny) projections due to an increased ratio of free cholesterol to phospholipid in the erythrocyte membranes. These acanthocytes undergo rapid splenic destruction and consequently have a shortened survival. Recent studies have indicated that alcoholic iron overload may be associated with spur cell anaemia rather than hereditary haemochromatosis. Patients usually need frequent blood transfusions and the prognosis is extremely poor. Liver transplantation, which improves hepatic function and resolves spur cell anaemia, has been the most effective treatment.


Aetiology :
• abetalipoproteinemia
• spur cell anaemia (acanthocytosis), a rare acquired haemolytic anaemia observed mainly in the end stages of alcoholic liver cirrhosis^ref
• after splenectomy
• McLeod syndrome
• drepanocyte (sickle cell)


Aetiology :
• sickle cell anemia (SCA)
• elliptocytes are red blood cells that are oval or cigar shaped


Aetiology :
• various anemias
• found in large amounts in hereditary elliptocytosis.
• codocyte / target cell or erythrocyte / Mexican hat cell or erythrocyte / leptocyte (?) are abnormally thin erythrocytes that when stained show a dark central color spot in the area of pallor and a peripheral ring of hemoglobin, separated by a pale unstained ring containing less hemoglobin, resembling a target


Aetiology :
• congenital and acquired anemias
• certain hemoglobinopathies
• liver disease, especially obstructive jaundice
• other disorders
• post-splenectomystate
• many hemolytic anemias, especially sickle cell anemia (SCA), HbC disease, and thalassemia
• dacryocyte / teardrop cell : an abnormal erythrocyte shaped like a teardrop


Aetiology :
• myelofibrosis with myeloid metaplasia and other myeloproliferative disorders
• pernicious anemia
• thalassemia
• some hemolytic anemias
• helmet cell


• schistocytes / keratocytes (bite cells) are red blood cell fragments that result from membrane damage encountered during passage through vessels


Aetiology :
• hemolytic anemias caused by
• microangiopathic hemolytic anemia (TTP and HUS)
• physical agents
• homozygous thalassemia
• severe burns
• uremia
• disseminated intravascular coagulation (DIC)
• spherocytes are almost spherical in shape. They are not biconcave like a normal red blood cell and do not have the central area of pallor which a normal red cell shows.


• microspherocytes
• hereditary spherocytosis
• burns
• microangiopathic hemolytic anemia

Aetiology :
• hemolytic anemia
• Rh deficiency syndrome
• ABO hemolytic disease of the newborn (HDN)
• hereditary spherocytosis
In some hereditary red cell enzyme deficiencies, the spherocytes may have many fine needle-like projections on the surface of the cell
• stomatocytes : an abnormal erythrocyte in which a slit-like, rectangular or mouthlike area replaces the normal circle of pallor, usually due to intracellular edema. These cells have lost the indentation on one side


Aetiology : stomatocytosis / hydrocytosis
• liver disease
• Rh deficiency syndrome
• b-sitosterolemia
• electrolyte imbalance
• cardiovascular disease
• hereditary stomatocytosis
• poikiloblast : an abnormally shaped erythroblast.
• asynclitism / dyserythropoiesis : defective development of erythrocytes, such as ...
• poikilocytosis / poikilocythemia : presence of poikilocytes in the blood
• pyropoikilocytosis : presence in the blood of numerous bizarre-shaped heat-sensitive poikilocytes.

Aetiology : hereditary pyropoikilocytosis
• size :
• macrocyte : MCV > 100 fL (Ø = 9-12 µm)

Aetiology :
• megaloblastic anemia (MCV > 120 fL). When associated with vitamin B₁₂or folic acid deficiency, the macrocytes may appear slightly oval in shape
• azathioprine^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}
• aplastic anemia (also normochromic, anisopoikilocytosis with dacryocytosis, leukoerythroblastosis)
• erythroblastopenia (also normochromic)
• 5q syndrome
• refractory anaemia with ringed sideroblasts (hypochromic microcytes usually constitute only a minor population of cells but their presence is a clue to the correct diagnosis)
• liver disease/chronic ethanol abuse

• normocyte : 81 < MCV < 99 fL

Aetiology :
• normal
• anemia of chronic disease (ACD) (also normochromic)
• myelodysplastic syndromes (MDS) (also normochromic, leukocytosis with relative neutropenia, and thrombocytopenia)
• liver disease/uremia
• hypometabolic anemia (hypothyroidism, hypoadrenalism, protein deficiency)
• acute bleeding
• hemolysis
• microcytes : MCV < 80 fL (Ø < 6 mm)
• microcythemia / microcytosis : a condition in which the erythrocytes are smaller than normal
• micronormoblast : an abnormal red cell precursor in which there has been defective hemoglobin synthesis, characterized by a narrow rim of cytoplasm and an overdeveloped pyknotic nucleus
Aetiology :
• anemia of chronic disease (ACD) (also hypochromic)
• impaired Hb synthesis (also hypochromic, increase in [soluble CD71 / TfR]_blood and decrease in [reticulocytes]_blood)
• sideropenic anemia (also anisopoikilocytosis); > 30% of cases of iron-deficiency anemia will be misdiagnosed if physicians rely on MCV and MCHC only
• sideroblastic anemia
• thalassemias
• hemoglobin Lepore trait
• hemoglobin E trait or homozygous
• hemoglobin H disease
• combination of above (double heterozygotes)
• anisocytosis / anisopoikilocytosis : presence in the blood of erythrocytes with excessive variation in size (RDW > )
• myelofibrosis with myeloid metaplasia
• aplastic anemia
• essential pernicious anemia
• sideropenic anemia
• thalassemia (also hypochromia and target cells)

• hemoglobin concentration :
• hypochromia : MCHC < 32 g / dL
• leptocyte (hypochromic microcytic red cell)


• achromocyte / demilune body / achromic erythrocyte : a crescent-shaped red cell artifact that stains more faintly than intact red cells
• ghost or shadow cell / erythroclast : a degenerating or fragmented erythrocyte with no hemoglobin
Aetiology :
• anemia of chronic disease (ACD) (also microcytic)
• impaired Hb synthesis (also microcytic, increase in [soluble CD71 / TfR]_blood and reticulocytopenia)
• sideropenic anemia (also anisopoikilocytosis); > 30% of cases of iron-deficiency anemia will be misdiagnosed if physicians rely on MCV and MCHC only
• PNH
• sideroblastic anemia
• thalassemias
• normochromia : MCHC = 33-36 g / dL

Aetiology :
• normal
• aplastic anemia (also macrocytic, anisopoikilocytosis with dacryocytosis, leukoerythroblastosis)
• erythroblastopenia (also macrocytic)
• myelodysplastic syndromes (MDS)(also normocytic, leukocytosiswith relative neutropenia, and thrombocytopenia)
• hyperchromia / polychromasia : MCHC> 36 g / dL


• anisochromasia : hemoglobin-distribution width >
• rouleaux formation occurs when RBCs form stacks or rolls

Aetiology :
• artifact (such as a result of not preparing the blood smear soon enough after placing the blood on the slide)
• hyperfibrinogenemia
• plasma cell dyscrasias (PCD)

• erythroblastosis / erythroblastemia : the presence in the peripheral blood of abnormally large numbers of erythroblasts (nucleated red blood cells (NRBCs))

Aetiology :
• previous studies have reported an association between acute and chronic hypoxia and presence of fetal heart rate accelerations. In vitro placental NRBC counts correlate with umbilical cord NRBCs and suggest that antenatal evaluation of fetal NRBCs could be achieved by placental FNAB^ref.
• reticulocytosis : an increase in the number of reticulocytes in the peripheral blood.
• reticulocyte response : increase in the formation of reticulocytes in response to a bone marrow stimulus, such as that provided by administration of a hematinic.
• reticulocytopenia : a decrease in the number of reticulocytes in the blood.
• basophilia : the reaction of immature erythrocytes to basic dyes so that they become stippled
• basophilia punctata / punctate basophilia / basophilic stippling : basophilia in which erythrocytes are blue and stippled with round, dark-blue granules on smears stained with supra vital stains such as brilliant cresyl blue, representing precipitated ribosomes and mitochondria. many course blue-purple staining granules within the red blood cell


Aetiology :
• lead
• poisoning with certain other heavy metals
• exposure to some drugs
• severe burns
• ethanol
• anemias with impaired hemoglobin synthesis
• megaloblastic anemia
• refractory anemia
• severe bacterial infection, septicemia
Laboratory examinations : stippled cell (an erythrocyte containing granules of varying size and shape, taking a basic or bluish stain with Wright's stain, as in punctate basophilia)
• diffuse basophilia : basophilia in which erythrocytes are blue-gray and not stippled.
• anemias (HGB < 13 g/dL or HCT < 39% in males; HGB < 12 g/dL or HCT < 35% in females)

Lower limits of normal of hemoglobin concentration of the blood of adult men and women as assessed by various sources :

Source	Men, g/dL	Women, g/dL	Percent normal below cutoff	Effect of race
WHO (Blanc B, Finch CA, Hallberg L, et al. Nutritional anaemias. Report of a WHO Scientific Group. WHO Tech Rep Ser. 1968;405: 1-40)	13	12	Not provided	Not provided
Jandl (Jandl JH. Blood. Boston, MA: Little, Brown and Company; 1996)	14.2	12.2	2.5	Discussed
Williams (Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U. Williams Hematology. New York, NY: McGraw-Hill; 2001)	14.0	12.3	2.5	Not provided
Wintrobe (Lee GR, Foerster J, Lukens J, Paraskevas F, Greer JP, Rodgers GM. Wintrobe's Clinical Hematology. Baltimore, MD: Williams and Wilkins; 1998)	13.2	11.6	Not provided	Not provided
Rapaport (Rapaport SI. Introduction to Hematology. Philadelphia, PA: JB Lippincott Company; 1987)	14	12	Not provided	Not provided
Goyette (Goyette RE. Hematology. A Comprehensive Guide to the Diagnosis and Treatment of Blood Disorders. Los Angeles, CA: Practice Management Information Corporation (PMIC); 1997)	13.2	11.7	5	Blacks' hemoglobin 0.5 g/dL lower
Tietz (Tietz NW. Clinical Guide to Laboratory Tests. Philadelphia, PA: WB Saunders Co; 1995)	13.2	11.7	Not provided	Not provided
Hoffman et al (Hoffman R, Benz EJ Jr, Shattil SJ, Furie B. Hematology: Basic Principles and Practice. New York, NY: Churchill-Livingstone; 2004)	13.5	12.0	2.5	Not provided

Mean hemoglobin (g/dL) by sex and age in white and black subjects :

	Scripps-Kaiser(collected in the San Diego area between 1998 and 2002)ref1, ref2			NHANES III(the third US National Health and Nutrition Examination Survey database)
	No.	Mean Hgb	SD	No.	Mean Hgb	SD
White men, y
20-29	250	15.43	0.77	332	15.51	0.96
30-39	894	15.24	0.85	389	15.29	0.95
40-49	2219	15.20	0.89	379	15.20	0.99
50-59	3346	15.10	0.93	356	15.17	1.01
60-69	3073	15.02	0.95	364	14.94	1.04
70-79	1976	14.75	1.07	315	14.70	1.15
80+	466	14.36	1.20	255	14.29	1.31
White women, y
20-29	240	13.41	0.80	322	13.57	0.88
30-39	809	13.59	0.86	402	13.66	0.86
40-49	1917	13.58	0.86	321	13.58	0.94
50-59	2829	13.62	0.86	312	13.76	0.94
60-69	3006	13.63	0.88	320	13.66	1.04
70-79	2051	13.66	0.94	421	13.68	0.97
80+	427	13.54	0.95	342	13.44	1.21
Black men, y
20-29	27	14.83	0.76	403	14.84	0.96
30-39	87	14.76	1.02	394	14.61	1.18
40-49	163	14.58	1.06	290	14.50	1.07
50-59	157	14.46	1.08	166	14.25	1.27
60-69	103	14.27	0.98	137	14.19	1.02
70+	32	14.44	1.06	98	13.75	1.15
Black women, y
20-29	17	13.26	0.75	321	12.78	0.92
30-39	68	12.97	0.93	339	12.77	1.11
40-49	120	12.88	0.91	244	12.87	1.05
50-59	135	12.96	0.89	146	13.07	0.95
60-69	90	13.03	0.91	161	12.89	1.01
70+	30	12.93	0.88	135	12.65	1.11

Proposed lower limits of normal for hemoglobin concentration of the blood for white and black adults :

Group	Hemoglobin, g/dL
White men, y
20-59	13.7
60+	13.2
White women, y
20-49	12.2
50+	12.2
Black men, y
20-59	12.9
60+	12.7
Black women, y
20-49	11.5
50+	11.5

Based on Scripps-Kaiser data for the 5th percentiles given in Table 2. NHANES data are considered to be confirmatory.
The ethnic origin of the individuals designated as white in these 2 databases is probably very similar. White Americans are a genetic mixture of persons from various parts of Europe and the Middle East. Data regarding ancestral origin are available in the Scripps Kaiser database but not in NHANES-III, so that direct comparison is not possible. Among black Americans there are varying degrees of admixture with non-African ancestry. The NHANES database attempts to reach a nationwide balance. It is likely that more of the black population of San Diego originates from the Northeast and the Midwest; therefore, San Diego may have greater white admixture than the NHANES sample. However, this has not been documented by the study of genetic markers in either data set. The NHANES-III database has recently been "mined" for hematologic values. The data in these compilations are valuable, but unfortunately the hemoglobin values in one report are derived from the entire population, not corrected for subjects with disease states that may be associated with anemia (Hollowell JG, Van Assendelft OW, Gunter EW, Lewis BG, Najjar M, Pfeiffer C. Hematological and iron-related analytes: reference data for persons aged 1 year and over: United States, 1988-94. Vital Health Stat. 2005;11: 1-156). The other report uses exclusion criteria that are so draconian that less than 40% of the subjects remain in  the data set^ref. Moreover, data derived from white subjects are not provided. In these and many other previous analyses of hematologic data the subjects have been divided into age in deciles. This type of analysis is shown in Table 2. Because there are virtually no differences in hemoglobin values of men in the age range of 20 to 59 or in women aged 20 through 49 in our data or in the analysis of the NHANES-III hemoglobin values,12 we have used these age ranges representing younger adults, and the ages beyond these ranges to represent older adults. In any analysis, elimination of subjects from consideration is constrained by the data that are available. Both the Scripps-Kaiser and the NHANES databases included transferrin saturation and serum ferritin levels, and we were therefore able to exclude subjects with a transferrin saturation of less than 16% or a serum ferritin level less than 10 µg/L. This serves to eliminate most subjects with iron deficiency anemia and also many of those with the anemia of chronic inflammation, in which the transferrin saturation is also usually low. We were also able to determine whether excessive alcohol intake, diabetes mellitus, renal failure, or inflammatory markers (C-reactive protein in NHANES, erythrocyte sedimentation rate in Scripps-Kaiser, elevated serum ferritin, or leukocytosis) affected the hemoglobin concentrations in these populations. None of these had an effect in the younger subjects, but in older subjects a serum creatinine of more than 106 µM (1.4 mg/dL) and elevated inflammatory markers (ESR > 30 in women and ESR > 20 in men or CRP > 1 mg/dL) were associated with significantly lower mean hemoglobin values. Older subjects with elevated creatinine and ESR or CRP were thus excluded from our calculations. Pregnant women were also excluded. Overall, the percentage of subjects excluded from the analysis ranged from 10% to 30%, except for black subjects in NHANES, in which the exclusion rate was approximately 50% for older men and 40% for women. The mean and 1.65 standard deviation below the mean of the hemoglobin values of the white subjects are shown in Figure 2; this provides the hemoglobin concentration below which only 5% of normal subjects in the population will be found, given the Gaussian distribution of hemoglobin values. Table 3 presents the lower limit of normal for white and black men and women. These data are presented to indicate the 2.5th and the 5th percentile as the actual percentile in the data set and as the predicted value based on the assumption that the hemoglobin values have a Gaussian distribution, as assumed in Figure 2. This assumption is clearly valid, as shown by the excellent correspondence between the predicted cutoff and the actual cutoff based on the number of cases found. The results obtained from the 2 databases are in very good agreement, but there are some minor differences that reach statistical significance. In particular, the mean hemoglobin of older men is slightly higher in the Scripps-Kaiser data than in the NHANES data. The latter difference in Table 3 is clearly an artifact resulting from the consolidation of all subjects older than 60 years into one group. As shown in Table 2, there are a higher proportion of men > 80 years in the NHANES survey than in the Scripps-Kaiser data, and there is a slight but well-documented decrease in the mean hemoglobin concentration of each decile among men (Hollowell JG, Van Assendelft OW, Gunter EW, Lewis BG, Najjar M, Pfeiffer C. Hematological and iron-related analytes: reference data for persons aged 1 year and over: United States, 1988-94. Vital Health Stat. 2005;11: 1-156). Some minor discrepancies could also be due to differences in ethnic origin within populations studied. This would obviously be true with the population designated "black," because it is well known that the genes of individuals classified as African American could be anywhere between 10% and 90% of European origin, with most of the remaining genome being of African origin. As pointed out earlier, differences in the degree of the European admixture could well account for the small differences in hemoglobin concentrations documented in the 2 data sets. This difference in genetic composition could also extend to the population designated "white": we have previously shown that there is a difference of about 2.8 g/L (0.28 g/dL) in the average hemoglobin concentration of a white subject with northern versus southern European ancestry^ref. This differential between northern and southern Europeans is maintained when the exclusion criteria used here are applied (data not shown). How is it that the WHO standard, so widely used in epidemiologic studies, is so seriously flawed? In fairness to the Expert Committee, a group of distinguished hematologists and nutritionists, it is clear that it was not their intent to set up a hallowed standard for all to follow. The values defining anemia are given in a small table and are presented without decimal points, clearly as approximations to be used within the context of the nutritional studies that were the principal topic of the meeting. The few references that are given to document this choice of numbers are, in general, surveys of the relatively small number of subjects, without documentation of methodology, and without efforts being made to remove patients with anemia in calculating the values. To further put these studies into the context of the times (Beutler E. Hemoglobin level and red blood cell count findings in normal women. JAMA. 1958; 167: 1551), the dilution of blood, sometimes obtained by finger puncture^ref, was carried out with pipettes that were often inaccurate. Cyanmethemoglobin standards were not widely available, and calibration required oxygenation of a blood sample to serve as a standard with measurement of the amount of oxygen released by acid in a van Slyke apparatus. Needless to say, this procedure was not always carried out, and, as a consequence, results were often unreliable. In view of these difficulties, perhaps we should marvel not only at the fact that the WHO standard is still used but also that it was not further from the mark. What, then, are reasonable benchmarks for anemia for a clinician to use today, based on today's improved laboratory practice and the data that we have been able to marshal from the Scripps-Kaiser and the NHANES study? The suggested lower limits of normal are summarized in Table 4. Based mostly on the larger Scripps-Kaiser database, but confirmed by the NHANES data, it would seem that a hemoglobin concentration below 137 g/L (13.7 g/dL) in a white man aged between 20 and 60 years would have only an approximately 5% chance of being a normal value. For older men, this hemoglobin value would be 132 g/L (13.2 g/dL). The corresponding value for women of all ages would be 122 g/L (12.2 g/dL). As shown in Table 1 and in a number of previous studies^{ref1, ref2},17,18 the lower limit of normal of hemoglobin concentrations of African Americans are appreciably lower. Although some of this difference is a result of the high frequency of -thalassemia in this population, other, as-yet-unidentified genes are involved, and because the gene frequency for -thalassemia is extraordinarily high in the black population, and the diagnosis is not readily made, we judge it best not to censor such cases in determining the normal range for the black population
The mean and -1.65 standard deviations of the hemoglobin values of the white subjects in the Scripps-Kaiser and NHANES-III databases after applying the criteria described in the text. The bottom of the error bars represents the hemoglobin value below which only 5% of normal values would be predicted to be present :

Lower limits of normal for hemoglobin concentration of the blood in g/dL of younger (age 20-59 for men; 20-49 for women) and older white and black adults :

	Scripps-Kaiser					NHANES-III
	No.	2.5% actual	2.5% normal distribution	5% actual	5% normal distribution	No.	2.5% actual	2.5% normal distribution	5% actual	5% normal distribution
White men, y
20-59	6709	13.4	13.4	13.7	13.7	1456	13.4	13.4	13.8	13.7
60+	5515	12.8	12.8	13.2	13.2	934	12.2	12.4	12.8	12.7
White women, y
20-49	2966	11.9	11.9	12.2	12.2	1045	12.0	11.9	12.2	12.1
50+	8313	11.9	11.9	12.2	12.2	1395	11.5	11.6	12.0	11.9
Black men, y
20-59	434	12.6	12.5	12.9	12.9	1253	12.3	12.4	12.8	12.8
60+	135	—	12.4	—	12.7	235	11.4	11.9	11.8	12.2
Black women, y
20-49	205	11.2	11.2	11.5	11.5	904	10.9	10.8	11.3	11.1
50+	255	11.2	11.2	11.5	11.5	442	11.0	10.9	11.3	11.2

Classification :
• according to MCV :
• < 80 fL (Ø < 6 mm) : microcytic anemia
• iron deficiency anemia
• PNH
• thalassemia
• myelodysplastic syndromes (MDS)
• hemoglobin Lepore trait
• hemoglobin E trait
• hemoglobin H disease
• combination of above (double heterozygotes)
• homozygous hemoglobin E
• sideroblastic anemia
• anemia of chronic disease
• 81 < x < 99 fL : normocytic anemia
• acute or chronic inflammation
• hypometabolic anemia (hypothyroidism, hypoadrenalism, sustained starvation)
• liver disease/uremia
• protein deficiency
• acute bleeding
• hemolysis
• > 100 fL (Ø > 9 mm)^ref : macrocytic anemia
• 5q syndrome
• megaloblastic anemia : MCV may exceed 150 fl. The RBCs vary considerably in size and shape. The macrocytes tend to be oval. Serum vitamin B12 determination remains the best test for unmasking vitamin B12 deficiency. It should be ordered in conjunction with serum and red cell folate determinations. When vitamin B₁₂ deficiency has been established, a Schilling test or plasma uptake test is indicated to pinpoint the cause.
• reticulocytosis : MCV < 110 fl; high reticulocyte count
• hemolytic anemia other than PNH (increase in [LDH₁ and LDH₂]_serum, [soluble CD71 / TfR]_plasma and [reticulocytes]_blood)
• uremia
• liver disease: abnormal liver function test, mild and uniform macrocytosis. The RBCs are round
• copper deficiency(associated with neutropenia, normal platelet count, and elevated serum ferritin and EPO levels)^ref
• Fanconi anemia (FA) or syndrome
• Shwachman-Diamond syndrome (SDS)
• dyskeratosis congenita (DC)
• according to MCHC :
• < 32 g / dL : hypochromic anemia
• 33 < x < 36 g / dL : normochromic anemia
• no hyperchromic anemia exists !
Compensation mechanisms : metabolic acidosis and hypercapnia => increase in 2,3-BPG
Symptoms & signs : severity directly relates to
• anemization rate and is mainly due to mechanisms that tend to compensate hypoxia :
• peripheral ischaemia (hemometakinesis in favour of vital organs) => pallor, tachycardia => hyperdynamic circulation=> functional heart murmurs when in clinostasis (disappearing in orthostasis, when venous blood return decreases), dizziness, diaphoresis, tachypnea, fatigue, amaurosis.
• pale palmar crests : HGB< 8 g/dl
• pale lip mucosae : HGB < 8-10 g/dl (conjunctival mucosae are normal)
• hyperdynamic ventricular output, tachycardia, systolic ejection murmur over pulmonary area, systodiastolic venous murmurs over neck disappearing after mild pression upstream of jugular vein (differential diagnosis with carotid artery stenosis > 75%)
• increased [EPO]_blood (except in chronic renal failure !) => aspecific stimulation on hematopoiesis, causing thrombocytosisand leukocytosis(except in hypoproliferative anemias !)
• jaundice
• splenomegaly
• lymphadenomegaly
• petechias
• concurrent cardiovascular diseases (IHD, CVD, lower limb obliterating arteriopathy)
Laboratory examinations :
• measure ABP in orthostatism and clinostatism
• reticulocyte index
Aetiology :
• hypoproliferative anemias : decrease in [soluble CD71 / TfR]_blood and [reticulocytes]_blood
• impaired medullary space
• myelophthisis / myelophthisic anemia / secondary myelofibrosis (normocytic and normochromic)
• impaired DNA synthesis
• impaired HSCs
• aplastic anemia (AA) / pure red cell aplasia (PRCA) : severe normochromic, normocytic anemia, reticulocytosis, and erythroblastopenia in bone marrow that produces the other cellular elements in a normal way (for general physicians) (normochromic and macrocytic, anisopoikilocytosis with dacryocytosis, leukoerythroblastosis). As the genetic basis and molecular pathogenesis of many of the inherited marrow failure syndromes are identified, it has become clear that some of these disorders may present as bone marrow failure or myelodysplasia (MDS) as well as epithelial malignancies well into adulthood. Distinguishing inherited aplastic anemia from acquired is not simply an esoteric exercise. The particular diagnosis will have an impact on proper management strategies and therapeutic approaches.
• congenital hypoplastic anemia (65%) : many inherited marrow failure syndromes may result in pancytopenia

disease		signs  (nonhematopoietic)	screening  tests	biomarkers
disorders frequently associated with multilineage bone marrow failure. Both are associated with a high risk of MDS/AML, and, when adults with these disorders are diagnosed, they commonly present with either hematopoietic (MDS/AML) or epithelial neoplasms. Because the initial diagnosis of both of these disorders has been made in individuals in their 5th and 6th decades of life, they must be considered even in adults with any one of the following: subtle but characteristic physical anomalies, hematologic cytopenias, unexplained macrocytosis, MDS/AML, or squamous cell cancer even in the absence of severe pancytopenia or a positive family history	Fanconi anemia (FA) or syndrome Estren-Dameshek variant	café-au-lait spots, skeletal  anomalies (thumb and radius),  short stature, microcephaly	DEB or MMC  sensitivity  (chromosomal breakage)	increased HbF;  macrocytosis
	dyskeratosis congenita (DC)	nail dystrophy, macular  or reticular hypopigmentation, mucosal leukoplakia	genetic	increased HbF; macrocytosis
inherited diseases associated with failure of single hematopoietic lineages : only occasionally evolve to hypoplastic bone marrows and pancytopenia. For this reason only rare patients may be ascertained in the later pancytopenic stages. Clonal evolution to MDS and AML has been described in both DBA and SDS. They rarely present in adulthood and most often begin with a failure of a single hematopoietic lineage.	neutropenia (e.g., Shwachman-Diamond syndrome (SDS))	exocrine pancreatic insufficiency, metaphyseal  dysostosis, short stature,  hepatic dysfunction	genetic  serum trypsinogen  & isoamylase	increased HbF; macrocytosis
	anemia (Diamond-Blackfan anemia (DBA))	congenital anomalies (thumb), short stature, cardiac  (ventricular or atrial septal defects)	genetic	increased HbF; increased RBC, increased eADA

• physiologic anemia is a common and normal finding in newborn infants
• anemia of prematurity : in preterm infants requiring transfusions of RBCs for, transfusion with 20 mL/kg RBCs produces a significantly greater increase in hemoglobin and hematocrit levels than does a transfusion with 10 mL/kg, without any detrimental effects on pulmonary function.
• Zinsser-Engman-Cole syndrome / congenital dyskeratosis
• Chediak-Higashi syndrome (CHS)
• Kostmann syndrome(severe congenital neutropenia), in which pancytopenia occurs only very rarely
• congenital amegakaryocytic thrombocytopenia
• congenital dyserythropoietic anemia
• Dubowitz and Seckel syndromes
• Pearson syndrome
• cartilage-hair hypoplasia or reticular dysgenesis
For more information regarding these entities: Alter BP. Inherited bone marrow failure syndromes. In: Nathan DG, Orkin SH, Ginsburg D, Look AT, eds. Hematology of Infancy and Childhood. Philadelphia: W.B. Saunders Co.; 2003:280–365
• acquired AA (35%) : pancytopenia and a hypocellular bone marrow

Epidemiology : preferentially affects young adults < 30 years and individuals aged > 60 years^ref.
Aetiology :
• idiopathic (50%)
• acute form that is self-limited :
• drugs :
• chloramphenicol
• quinacrine
• tolbutamide
• chlorpropamide
• phenylbutazone
• Au salts
• acetazolamide
• acetylsalicylic acid
• chlordiazepoxide
• chlorpheniramine
• chlorothiazide
• chlorpromazine
• diphenylidantoine / phenytoin / phenantoine
• mepazine
• penicillin
• pyrimethamine
• KClO₄
• sulfomethoxypyridazine
• sulfonamides
• ticlopidine
• xanthine oxidase inhibitors
• physical causes :
• X-rays
• g-rays
• viruses :
• B₁₉ virus
• alemtuzumab-induced PRCA^ref
• rituximab-associated PRCA^{ref1, ref2, ref3}
• HHV-4 / EBV(granular lymphocyte proliferative disorders (GLPD))
• HAV
• HBV
• HCV
• dengue fever virus
• HIV-1
• bacteria :
• Mycobacterium tuberculosis
• Brucella melitensis
• chronic forms secondary to immune disorders / autoimmune-mediated apoptosis / non-infectious PRCA (NI-PRCA) (immune-mediate pure red cell aplasia linked to HLA-DR2). If HSC are mutated in PIGA, it may lead to paroxismal nocturnal hemoglobinuria (PNH).
• thymoma (5-10% of PRCA cases) (thymectomy cures 50% of cases)
• hepatitis-associated aplastic anemia (HAA) (2-5%^ref) : the development of hematopoietic failure with bone marrow hypocellularity within 6 months of an episode of hepatitis. Following orthotopic liver transplantation for non-A, non-B, non-C hepatitis in young patients: 23% to 8% of non-A, non-B, non-C hepatitis patients receiving transplants developed aplastic anemia, compared with < 1% of all liver transplant patients^{ref1, ref2}. Most often affecting young males, the hepatitis generally follows a benign course, but the onset of aplastic anemia 2 to 3 months later can be explosive and is usually fatal if untreated^ref. The presumed infectious cause of the hepatitis is unknown, but most cases are seronegative for known hepatitis viruses, including HAV, HBV, HCV, and HGV^{ref1, ref2, ref3, ref4}. The prevalence of HGV RNA was significantly higher in transfused patients than in untransfused patients with idiopathic aplastic anemia or HA-aplastic anemia, suggesting that transfusions were a major source of HGV RNA in the serum of these patients. However, the increased prevalence of HGV RNA in untransfused patients with aplastic anemia, whether associated with hepatitis or not, compared with normal blood donors^{ref1, ref2, ref3}. 10 cases of HAA seen at the NIH were reported that had evidence of lymphocyte activation; 70% responded to immunosuppression with ATG and cyclosporine for patients who do not have an HLA-matched related donor available for allogeneic HSCT^ref. Apart from case reports, the presence of lymphocyte activation^{ref1, ref2}, and the clinical response to either immunosuppression or bone marrow transplantation^ref, little is known of the immunopathogenesis of this syndrome : there is antigen-driven T-cell expansion in HAA and achievement of a normal T-cell repertoire during recovery from HAA^ref
• epoetin-a-induced antibody (Ab)-mediated PRCA due to the production of neutralizing EPO antibodies was very rare prior to 1998^ref, but a large increase in the number of global cases was observed from 1999 to 2002 in European patients treated with s.c. epoetin for the anaemia associated with chronic kidney disease (CKD). Onset after 7-14 months of EPO, responsive to discontinuation. Incidence = 19 cases per 100,000 treated patients. The number of global cases of this immunological form of PRCA has declined precipitously since 2003 following increased awareness of this disorder and changes in the handling and administration of the formulation of epoetin that was associated with the majority of cases. Current recommendations state that patients should stop treatment immediately following a diagnosis of Ab-mediated PRCA and not resume treatment with the same or another ESA. The feasibility of re-treatment following remission from PRCA or during ongoing immunosuppressive therapy is currently under investigation^ref. The PRCA upsurge coincided with removal of human serum albumin from epoetin-a in 1998 and its replacement with glycine and polysorbate 80, which can induce the release of organic compounds with adjuvant properties from the rubber stoppers of prefilled syringes^ref (Sharma B, Bader F, Templeman T, Lisi P, Ryan M, Heavner GA. Technical investigations into the cause of the increased incidence of antibody-mediated pure red cell aplasia associated with Eprex. Eur J Hosp Pharm 2004;5:86-91). Although the immunogenic potential of this particular product may have been enhanced by the way the product was stored, handled and administered, it should be noted that the subcutaneous route of administration does not confer immunogenicity per se. The possible role of micelle (polysorbate 80 + epoetin-a) formation in the PRCA upsurge with Eprex is currently being investigated^ref.
Pathogenesis : immune-mediated destruction of blood-forming cells. This is manifested in vitro by coculture inhibition of hematopoietic colony formation by patient’s lymphocytes. In vivo, one can detect circulating activated CTLs and increased production of cytokines typical of the T_h1 response, especially IFN-g, TNF-a, and IL-2^ref. Acting locally in the BM, activated T cells likely induce Fas-mediated cell death of hematopoietic progenitor and stem cells, ultimately resulting in a marked decrease in the number of these cells and severe pancytopenia. In support of this hypothesis, we have shown that many patients with sAA have increased expression of CD8 lymphocyte IFN-g demonstrated by both flow cytometry^ref and microarray analysis^ref. Examination of TcRVß subfamilies has shown expansion of specific Vß subfamilies in many patients with AA suggestive of an antigen-driven immune response; spectratyping shows skewing indicative of oligoclonal expansion within these Vß subfamilies^{ref1, ref2}. In some patients, genetic abnormalities may place them at risk for AA. Recently, mutations have been found in the telomerase TERC gene and the reverse transcriptase itself (TERT gene) in a group of AA patients with short telomeres and markedly reduced telomerase activity^ref. Patients’ relatives carrying these mutations also have mild peripheral blood count abnormalities and a normal or reduced stem cell compartment. This disorder may result in progressive telomere shortening leading to early progenitor senescence. Telomerase complex gene mutations may represent a genetic risk factor for bone marrow failure. The fact that some family members with the mutation are asymptomatic and a number of patients respond to immunotherapy, suggests that at least in some cases an additional acquired factor (e.g., immune attack) is required to cause bone marrow failure in some of these patients.


> 25 years have passed since our first, highly speculative review of aplastic anemia; in that fortunately obscure publication, pathophysiology was addressed tentatively and immunosuppressive therapies hardly at all. The article did reflect both the dismal prospects for patients with the severe form of marrow failure and the formidable practical difficulties of experimentation in a rare disorder in which the cells of interest had disappeared. Aplastic anemia was considered heterogenous in origin and virtually impossible to study systematically. At the bedside, the clinical emphasis was the identification of a putative causal factor— exposure to benzene or a culpable pharmaceutical—to allow classification in an otherwise doomed patient. As progenitor assays were developed, diverse factors could be held theoretically responsible for failure to form colonies in tissue culture, ranging from quantitative and qualitative defects in stem cells and blocks in differentiation to a lack of stroma support or inadequate cytokine production, or the effects of a chemical poison.

In the intervening decades, our understanding of aplastic anemia has cohered around a unified immune mechanism of hematopoietic-cell destruction, which was inferred from but also has informed effective immunosuppressive therapies for the disease (Figure 1). Technical advances in cell biology, flow cytometry, molecular biology, and immunology have provided methods to measure numbers and function of very limited numbers of cells. As a result, we have a more unified and rational view of aplastic anemia's pathophysiology; the disease is understood in its relation to other related marrow failure syndromes; and in many important respects an unusual blood syndrome can model more common autoimmune diseases of other organ systems (Figure 2). Particularly satisfying is that aplastic anemia is now amenable to cure or amelioration in most patients, based both on high-quality clinical trials and mechanistic insights from the experimental laboratory^{ref1, ref2, ref3, ref4, ref5, ref6}.1-6
 

    Etiologies
Clinical associations : since Ehrlich's description of the first case of aplastic anemia in a pregnant woman,126 precipitating factors have been sought from the individual patient's history. An enormous literature, dating from the beginning of the 20th century, described chemical- and drug-induced disease, stimulated by observations of the effects of benzene on blood counts, of dipyrone's association with agranulocytosis, and a seeming epidemic of aplastic anemia after the introduction of chloramphenicol. These associations are worth reassessment in the context of the immune hypothesis of marrow failure. Pregnancy appears a real association, as deduced more from the documented improvement of blood counts with its termination than from formal epidemiologic study^ref.7 The unusual syndrome of eosinophilic fasciitis also is strongly linked to aplastic anemia. Five to 10% of cases of aplastic anemia follow an episode of seronegative hepatitis, in which immune activation is inferred from the pattern of T-cell activation, cytokine production, and HLA association^ref.8 Despite intensive efforts, including sophisticated molecular and immunologic approaches and animal inoculations, an infectious agent has not been identified. Benzene, or more correctly its metabolites, is a marrow toxin in animals and humans, but in the West benzene exposure as an etiology of aplastic anemia is now rare. Ancient case reports and series leave doubt as to whether marrow failure in benzene workers was not often myelodysplasia rather than aplastic anemia. Additionally, benzene also has effects on immune function^ref.9
Pathophysiology of acquired aplastic anemia. The figure stresses the crucial and related roles of the hematopoietic stem-cell compartment as a target for the immune response. An inciting event, such as a virus or medical drug, provokes an aberrant immune response, triggering oligoclonal expansion of cytotoxic T cells that destroy hematopoietic stem cells (left panel, Onset). Bone marrow transplantation or immunosuppressive therapy leads to complete response (CR) or partial response (PR) by eradicating or suppressing pathogenic T-cell clones (middle panel, Recovery). Relapse occurs with recurrence of the immune response, and the immunologically stressed and depleted stem-cell compartment also allows selection of abnormal hematopoietic clones that manifest as paroxysmal nocturnal hemoglobinuria, myelodysplasia (MDS), and occasionally acute myelogenous leukemia (AML) (right panel, Late Disease).

Of greatest practical import is the relationship of medical drug use to aplastic anemia—unpredictable marrow failure in this setting is devastating to the patient and physician and has serious legal ramifications for pharmaceutical drug development^ref.10 The study of idiosyncratic drug reactions, by definition extremely rare, is difficult. That genetic differences in drug metabolism, especially in detoxification of reactive intermediate compounds, underlie susceptibility is best supported by one study of a single individual exposed to carbamazepine, published more than 20 years ago^ref.11 Overrepresentation of deletions in the drug-metabolizing glutathione-S-transferase genes (GSTM1, GSTT1, which would increase concentrations of toxic drug intermediates) has been observed in some series^{ref1, ref2}.12,13 Astonishingly, no satisfactory mechanism has been developed for the most notorious pharmaceutical, chloramphenicol, or for other heavily inculpated agents such as penicillamine or gold. Many drugs on "black lists" also more commonly cause mild marrow suppression, and possibly regular but only modest destruction of marrow cells is a prerequisite for a much more infrequent immune response to an exposed neoantigen. A parallel mechanism is supported by the clinical observation of little difference between patients with idiopathic aplastic anemia and those with an assumed drug etiology, in demographics, response to therapy, or survival^ref14 In contrast, very few chemotherapeutic agents, despite being designed as cell poisons and administered in milligram or gram quantities, directly result in irreversible marrow destruction without obvious effects on other organs. Claims of permanent aplastic anemia after idiosyncratic exposure to minuscule quantities of chloramphenicol, for example (as in ophthalmic solutions), more likely reflect observation and reporting biases than a mechanism of extreme sensitivity to a hidden metabolite.

Venn diagram of the clinical and pathophysiologic relationships among the bone marrow failure syndromes, leukemia, and autoimmune diseases. Overlapping circles indicate difficulties in diagnostic discrimination and shared underlying mechanisms.

Epidemiology : 2 more reliable approaches to identifying etiology are epidemiology and laboratory identification of antigens. Unfortunately, neither has yielded conclusive results. Two large, controlled, population-based studies have been conducted, the International Aplastic Anemia and Agranulocytosis Study in Europe and Israel in the 1980s^ref15 and the recently completed Thai NHLBI Aplastic Anemia Study in Bangkok and a northeast rural region^ref.16 The incidence of aplastic anemia in the West is 2/million and about 2- to 3-fold higher in Asia. Benzene and pesticides, while significantly associated, accounted for only a small number of cases in both studies, and medical drugs have a negligible role in Asia. In rural Thailand, exposure to nonbottled water, as well as to certain animals (ducks and geese), to animal fertilizer, and also to pesticides, suggested an infectious etiology.

Autoantigens : a few putative antigens have been teased from screening antibodies in patients' sera against a peptide library (by expression of genes in fetal liver or leukemic-cell lines). Kinectin, a widely expressed protein, bound to antibodies from about 40% of aplastic patients^ref.17 Another antigen that bound to antibodies, in a smaller minority of marrow failure patients, was diazepam-binding related protein-1, an enzyme essential in the oxidation of unsaturated fatty acids and broadly distributed in tissue^refs.18 The relevance of these autoantibodies to a cellular pathophysiology of aplastic anemia is unclear. For kinectin, reactive cytotoxic T cells could be generated in vitro and inhibited human hematopoietic colony formation, but antikinectin T cells were not found in patients^ref.17 For diazepam-binding related protein-1, a putative T-cell epitope derived from this protein could stimulate cytotoxic T cells obtained from one patient, and T-cell precursors with peptide-binding activity were present in 2 cases.
Pathophysiology : in most cases, aplastic anemia is an immune-mediated disease. Cellular and molecular pathways have been mapped in some detail for both effector (T lymphocyte) and target (hematopoietic stem and progenitor) cells (Figure 3). Exposure to specific environmental precipitants, diverse host genetic risk factors, and individual differences in the characteristics of the immune response likely account for the disease's infrequency, variations in its clinical behavior, and patterns of responsiveness to treatment.
Immune-mediated T-cell destruction of marrow : an immune mechanism was inferred decades ago from the recovery of hematopoiesis in patients who failed to engraft after stem-cell transplantation, when renewal of autologous blood-cell production was credited to the conditioning regimen. Also suggestive was that the majority of syngeneic transplantations in which bone marrow was infused without conditioning failed^ref.19 The responsiveness of aplastic anemia to immunosuppressive therapies remains the best evidence of an underlying immune pathophysiology: the majority of patients show hematologic improvement after only transient T-cell depletion by antithymocyte globulins (ATGs); relapse also usually responds to ATG; and dependence of adequate blood counts on administration of very low doses of cyclosporine is not infrequent. As immunosuppression has intensified, from early attempts with corticosteroids to aggressive strategies such as high-dose cyclophosphamide, and the proportion of responders has risen, the willingness to ascribe an immunologic mechanism also increased. Indeed, little distinguishes responders to immunologic therapy from refractory patients (other than age, as children show higher rates of recovery and survival). A nonimmune pathophysiology has been inferred from a failure to respond to immunosuppression, but refractoriness to therapy is also consistent with very severe stem-cell depletion, a "spent" immune response, or immunologic mechanisms not susceptible to current therapies.

In early laboratory experiments, removal of lymphocytes from aplastic bone marrows improved colony numbers in tissue culture, and their addition to normal marrow inhibited hematopoiesis in vitro (reviewed in Young^ref20). The effector cells were identified by immunophenotyping as activated cytotoxic T cells expressing T_h1 cytokines, especially -interferon. CD8 cells containing intracellular interferon may now be measured directly in the circulation^ref,21 and oligoclonal expansion of CD8⁺ CD28^- cells, defined by (1) flow cytometric analysis for T-cell receptor (TCR) V subfamilies; (2) spectratyping to detect skewing of CDR3 length; and (3) sequencing of the CDR3 region to establish a molecular clonotype^ref.22 In general, patients at presentation demonstrate oligoclonal expansions of a few V subfamilies, which diminish or disappear with successful therapy; original clones re-emerge with relapse, sometimes accompanied by new clones, consistent with spreading of the immune response. Very occasionally, a large clone persists in remission, perhaps evidence of T-cell tolerance.

Immune destruction of hematopoiesis. Antigens are presented to T lymphocytes by antigenpresenting cells (APCs), which trigger T cells to activate and proliferate. T-bet, a transcription factor, binds to the interferon- (INF-) promoter region and induces gene expression. SAP binds to Fyn and modulates SLAM activity on IFN- expression, diminishing gene transcription. Patients with aplastic anemia show constitutive T-bet expression and low SAP levels. IFN- and TNF- up-regulate other T cells' cellular receptors and also the Fas receptor. Increased production of interleukin-2 leads to polyclonal expansion of T cells. Activation of Fas receptor by the Fas ligand leads to apoptosis of target cells. Some effects of IFN- are mediated through IRF-1, which inhibits the transcription of cellular genes and entry into the cell cycle. IFN- is a potent inducer of many cellular genes, including inducible nitric oxide synthase (NOS), and production of the toxic gas nitric oxide (NO) may further diffuse toxic effects. These events ultimately lead to reduced cell cycling and cell death by apoptosis.
The impact of T-cell attack on marrow can be modeled in vitro and in vivo. -Interferon (and tumor necrosis factor-) in increasing doses reduce numbers of human hematopoietic progenitors assayed in vitro; the cytokines efficiently induce apoptosis in CD34 target cells, at least partially through the Fas-dependent pathway of cell death^ref.23 In long-term culture of human bone marrow, in which stromal cells were engineered to constitutively express -interferon, the output of long-term culture-initiating cells (LTCI-ICs) was markedly diminished, despite low concentrations of the cytokine in the media, consistent with local amplification of toxicity in the marrow milieu^ref.24 Immune-mediated marrow failure has been modeled in the mouse: infusion of parental lymph node cells into F₁ hybrid donors caused pancytopenia, profound marrow aplasia, and death^ref.25 Not only a murine version of ATG and cyclosporine but also monoclonal antibodies to -interferon and tumor necrosis factor abrogated hematologic disease, rescuing animals. A powerful "innocent bystander" effect, in which activated cytotoxic T cells kill genetically identical targets, was present in secondary transplantation experiments^ref.26 In a minor histocompatibility antigen-discordant model, marrow destruction resulted from activity of an expanded H60 antigen-specific T-cell clone^ref.27
Why T cells are activated in aplastic anemia is unclear. HLA-DR2 is overrepresented among patients, suggesting a role for antigen recognition, and its presence is predictive of a better response to cyclosporine^{ref1, ref2}.28,29 Polymorphisms in cytokine genes, associated with an increased immune response, also are more prevalent: a nucleotide polymorphism in the tumor necrosis factor- (TNF2) promoter at -308^{ref1, ref2},30,31 homozygosity for a variable number of dinucleotide repeats in the gene encoding -interferon^ref,32 and polymorphisms in the interleukin 6 gene^ref.33 Constitutive expression of T-bet, a transcriptional regulator that is critical to Th1 polarization, occurs in a majority of aplastic anemia patients^ref.34 Mutations in PRF1, the gene for perforin, are responsible for some cases of familial hemophagocytosis; mutations in SAP, a gene encoding for a small modulator protein that inhibits -interferon production, underlie X-linked lymphoproliferation, a fatal illness associated with an aberrant immune response to herpesviruses and aplastic anemia. Perforin is overexpressed in aplastic marrow^ref.35 We have detected heterozygous mutations in PRF1 in 5 adults with severe aplasia and hemophagocytosis of the marrow, and SAP protein levels are markedly diminished in a majority of acquired aplastic anemia cases (E. Solomou, unpublished data, June 2006). These alterations in nucleotide sequence and in gene regulation suggest a genetic basis for aberrant T-cell activation in bone marrow failure. Genome-wide transcriptional analysis of T cells from aplastic anemia patients has implicated components of innate immunity in aplastic anemia, including TLRs and NK cells^ref,36 for which there is some preliminary experimental support^{ref1, ref2}.37,38

Hematopoiesis : immune attack leads to marrow failure. "Anhematopoiesis" was inferred from the empty appearance of the marrow at autopsy by the earliest observers of the disease. The pallor of the modern biopsy core or empty spicules of an aspirate, few or no CD34 cells on flow cytometry, and minimal numbers of colonies derived from committed progenitors in semisolid media all reflect the severe reduction in hematopoietic cells that defines the disease. Stem-cell "surrogate"—really correlative—assays, LTC-ICs^ref,39 or cobblestoneforming cells^ref,40 which measure a primitive infrequent and quiescent multipotential progenitor cell, also show marked deficiency, and from the product of the low percentage of marrow cellularity and the scant numbers of LTC-ICs per mononuclear cell, suggest that only a small percentage of residual early hematopoietic cells remains in severely affected patients at presentation. Qualititative features of these few cells, as measured, for example, by poor colony formation per CD34 cell or inadequate response to hematopoietic growth factors, are harder to interpret, although recent genetic studies have suggested explanatory mechanisms (see next paragraph). The reduced number and function of the marrow is secondary to cell destruction, and apoptosis is prevalent among the few remaining elements^{ref1, ref2}.41,42 Microarray of the scant CD34 cells from marrow failure patients revealed a transcriptome in which genes involved in apoptosis, cell death, and immune regulation were up-regulated43; this transcriptional signature was reproduced in normal CD34 cells exposed to -interferon^ref.44 One peculiar feature of white blood cells in aplastic anemia is short telomeres^{ref1, ref2}.45,46 Telomere shortening was initially most easily blamed on stem-cell exhaustion. However, the discovery, first by linkage analysis in large pedigrees, that the X-linked form of dyskeratosis congenita was due to mutations in DKC1 and subsequently purposeful identification of mutations in TERC in some autosomal dominant patients with this constitutional marrow failure syndrome indicated a genetic basis for telomere deficiency. Central to the repair machinery is an RNA template, encoded by TERC, on which telomerase, a reverse transcriptase encoded by TERT, elongates the nucleotide repeat structure; other proteins, including the DKC1 gene product dyskerin, are associated with the telomere repair complex. Systematic surveys of DNA disclosed first TERC^{ref1, ref2}47,48 and later TERT mutations^ref49 in some patients with apparently acquired aplastic anemia, including older adults. Family members who share the mutation, despite normal or near-normal blood counts, have hypocellular marrows, reduced CD34-cell counts and poor hematopoietic colony formation, increased hematopoietic growth factor levels, and of course short telomeres; however, their clinical presentation is much later than in typical dyskeratosis congenita, and they lack typical physical anomalies^{ref1, ref2}.47,49 Chromosomes are also protected by several proteins that bind directly to telomeres, and polymorphisms in their genes (TERF1, TERF2) are also more or less prevalent in aplastic anemia compared with healthy controls^ref.50 A few of our patients also have heterozygous mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. Almost all children with this form of constitutional aplastic anemia are compound heterozygotes for mutations in SBDS, and their white cells have extremely short telomeres^ref51; however, the SBDS gene product has not been directly linked to the telomere repair complex or to telomere binding. A parsimonious inference from all these data is that inherited mutations in genes that repair or protect telomeres are genetic risk factors in acquired aplastic anemia, probably because they confer a quantitatively reduced hematopoietic stem-cell compartment that may also be qualitatively inadequate to sustain immune-mediated damage^ref.52 Telomeres are short in one third to one half of aplastic anemia patients,45,46 but mutations have been identified in only < 10% of cases. The most interesting explanation is involvement of other genes, including genes for other members of the large repair complex, telomere binding proteins, still obscure components of the alternative repair system, and some DNA helicases. Alternatively, telomere shortening may be secondary to stem-cell replication.
Clonal evolution : clinically, aplastic anemia may coexist or appear to evolve to other hematologic diseases that are characterized by proliferation of distinctive cell clones, as in paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplasia (MDS; Figure 2). The mechanisms linking immune-mediated and premalignant pathophysiologies are not elucidated in marrow failure or in parallel circumstances (chronic hepatitis and hepatocellular carcinoma, ulcerative colitis and colon cancer, and many others). The presence of tiny clones at the time of diagnosis of aplastic anemia, detected using extremely sensitive assays—phenotypic (flow cytometry for PNH) or cytogenetic (fluorescent in situ hybridization for MDS)—also creates the problems of disease classification and patient diagnosis.
 

• PNH. > 50% of patients at presentation with pancytopenia have expanded populations of PNH cells, easily detected by flow cytometry due to the absence of glycosylphosphatidylinositol-linked membrane proteins, the result of somatic PIG-A gene mutations^ref.53 Most clones are small and do not lead to clinical manifestations of hemolysis or thrombosis, but classic PNH can be dominated by marrow failure (the "aplastic anemia/PNH syndrome"), and all PNH patients show evidence of underlying hematopoietic deficiency. The global absence of a large number of cell-surface proteins in PNH has been hypothesized to allow "escape" and survival of a pre-existing mutant clone. Association of an expanded PNH clone with HLA-DR229 and with autoantibodies^ref,18 and as a predictor of responsiveness to immunosuppressive therapies^ref,54 suggests that the escape is from immune attack. However, there is little concrete experimental evidence of reproducible differences in either differential immune responsiveness or susceptibility of PNH clones compared with phenotypically normal target-cell populations^{ref1, ref2}.55,56 In contrast to cells of normal phenotype, the marrow PNH clone retains its proliferative capacity in tissue culture and does not overexpress Fas^ref57; comparison by microarray shows that residual cells of normal phenotype in the PNH bone marrow up-regulate the same apoptosis and cell-death genes as do CD34 cells in aplastic marrow, while the PIG-A- clone appears transcriptionally similar to CD34 cells from healthy donors^ref.58 Despite a few provocative studies^{ref1, ref2, ref3},37,55,59 no satisfying mechanism to explain clonal escape has convincing empiric support (see Young53 for review).
• MDS. Stereotypical patterns of aneuploidy develop in a minority of patients over time: monosomy 7 or trisomy 8 is most characteristic.60 Trisomy 8 is MDS with many immune abnormalities that resemble aplastic anemia. Patients respond to immunosuppressive therapies, and oligoclonal T-cell expansions are usually present^ref.61 T-cell oligoclones appear to recognize the aneuploid cells, and specifically WT1 antigen that they express at high levels^ref,62 but the target cells are not killed due to up-regulation of antiapoptosis genes, including c-myc, survivin, and CDK1^ref.63 For trisomy 8, abnormal cells targeted by the immune system appear to be selected for their capacity to survive cytotoxic lymphocyte attack. Monosomy 7 is also a frequent cytogenetic abnormality in aplastic anemia but has a poorer prognosis, with patients usually succumbing to refractory cytopenias or evolving to acute leukemia^ref.64 Emergence of monosomy 7 has been linked to exogenous use of G-CSF in aplastic anemia^ref,65 as occurs in treated severe congenital neutropenia. Laboratory studies of marrow from aplastic anemia and myelodysplasia patients suggest that monosomy 7 clones expand in an abnormal cytokine milieu: high G-CSF concentrations lead to selection of cells that bear a short isoform of the G-CSF receptor that signals proliferation but not differentiation^ref.66


Treatment
Immunosuppression : antithymocyte globulin (ATG) and cyclosporine (combined or intensive immunosuppression). For aplastic anemia that is severe, as defined by peripheral-blood counts, definitive therapies are immunosuppression or stem-cell transplantation; immunosuppressive therapies are most widely used because of lack of histocompatible sibling donors, patient age, and the immediate cost of transplantation. Even in responding patients, blood counts often remain below normal but adequate to avoid transfusion and to prevent infection. Most specialists use an ATG-based regimen in combination with cyclosporine, based on the outcomes of relatively large studies performed in the 1990s^ref67 (Table 1). The larger experience is with ATG produced in horses, although a rabbit ATG, recently approved for use in the United States, is more potent by weight and in the treatment of graft rejection after solid organ transplantations (a current NIH trial is directly comparing these 2 ATGs as first therapy in severe aplastic anemia). ATG is cytolytic: lymphocyte numbers consistently decline during the first few days of infusion and then return to pretreatment levels in a week or 2. ATGs are produced by immunizing animals against human thymocytes, not lymphocytes, and the mix of antibody specificities plus direct experimentation suggests that ATG may be immunomodulatory as well as lymphocytotoxic, perhaps producing a state of tolerance by preferential depletion of activated T cells. The toxicity of ATG is allergic, related to administration of a heterologous protein, and there is little added infection risk beyond the neutropenia intrinsic to the disease. ATG doses and regimens are empiric and traditional. By administration of a larger dose over fewer days, immune complex formation and consequent serum sickness are minimized, as patients usually do not produce their own antibodies to the foreign protein until a week or 10 days after exposure.
Intensive immunosuppression (ATG plus cyclosporine) for severe aplastic anemia

Cyclosporine's selective effect on T-cell function is due to direct inhibition on the expression of nuclear regulatory proteins, resulting in reduced T-cell proliferation and activation. While severe aplastic anemia can respond to cyclosporine alone, it is less effective than either ATG alone or ATG plus cyclosporine^{ref1, ref2}.73,74 As with ATG, doses and length of treatment have not been formally established. Cyclosporine has many side effects, but most are manageable by dose reduction; permanent kidney damage is unusual with monitoring (to maintain blood levels at nadir of about 200 ng/mL). Maintenance of blood counts may be achieved with very low doses of cyclosporine, such that drug levels in blood are undetectable and toxicity is minimal, even with years of treatment.
Outcomes of combined immunosuppressive therapy. Reported hematologic response rates vary, at least in part due to lack of consensus on parameters (transfusion independence, absolute or relative improvement in blood counts) and defined landmarks. In our experience, improvement of blood counts so that the criteria for severity are no longer met highly correlates with termination of transfusions, freedom from neutropenic infection, and better survival. By this standard, about 60% of patients are responders at 3 or 6 months after initiation of horse ATG^ref.70 Comparable figures for hematologic response rates have come from Europe^ref69 and Japan^ref.71 Responders have much better survival prospects than do nonresponders. Long-term prognosis is predicted by the robustness of the early blood count response (defined as either platelets or reticulocytes > 50 x 10⁹/L [50 000/µL] 3 months after treatment): about 50% of patients who are treated with horse ATG have a robust response and almost all of them will survive long term. Outcomes of immunosuppressive therapy are related to patient age: 5-year survival of more than 90% of children has been reported in recent German^ref,75 Japanese^ref,71 and Chinese^ref76 trials, compared with about 50% survival for adults older than 60 years in the collective European experience^ref.77

Relapse, defined conservatively as a requirement for additional immunosuppression and not necessarily recurrent pancytopenia, is not uncommon, occurring in 30% to 40% of responding patients. Relapse defined by renewed need for transfusion was estimated at 12% of European patients at 3 years, but prolonged cyclosporine dependency among all patients was common^ref.69 Reinstitution of cyclosporine usually reverses declining blood counts, but when required, a second round of horse^ref78 or rabbit^ref79 ATG is usually effective. In our experience, relapse does not confer a poor prognosis, but it is obviously inconvenient and may not always be remediable. Molecular analysis of the T-cell response in aplastic anemia, discussed in "Pathophysiology," suggests that the major reason for relapse is incomplete eradication of pathogenic clones by ATG.

More serious than relapse is evolution of aplastic anemia to another clonal hematologic disease, PNH, myelodysplasia, and leukemia. Small PNH clones present at diagnosis usually remain stable over time but may expand sufficiently to produce symptomatic hemolysis. For myelodysplasia and leukemia, the cumulative long-term rate of clonal evolution is about 15%^{ref1, ref2}70,80; evolution is not inevitable in aplastic anemia, and some cytogenetic abnormalities may be transient or, as with trisomy 8, responsive to immunosuppressive treatments. As discussed in "Clonal evolution," emergence of monosomy 7 may be favored in severely neutropenic patients who require chronic G-CSF therapy^ref.66

Improving on ATG and cyclosporine. Growth factors. Historically, intensification of immunosuppression has increased response rates. However, attempts to improve on ATG plus cyclosporine have been frustratingly disappointing. Megadoses of methylprednisolone only added toxicities. Small pilots of GM-CSF^ref81 and much larger, randomized studies of G-CSF^{ref1, ref2}71,82 as routine additions to ATG and cyclosporine have been negative to date; improved neutrophil counts did not translate into a higher rate of recovery or even less infection. A very large ongoing European study of G-CSF should definitively answer efficacy and safety concerns.
Other immunosuppressive drugs. As the addition of cyclosporine clearly improved outcomes compared with the use of ATG alone, other immunosuppressive drugs might be predicted, based on their mode of action, animal studies, and experience in other human diseases and with organ transplantation, to be effective.

• Mycophenolate mofetil is a tolerizing agent, as it selectively depletes activated cells by inhibition of inosine monophosphate dehydrogenase, a critical enzyme of the purine salvage pathway, therefore blocking activated lymphocyte proliferation. Nonetheless, its addition to ATG and cyclosporine in an NIH trial of 104 patients did not change hematologic response (about 62%), relapse (37%), or evolution rates; at best, there was a modest sparing of cyclosporine usage^ref.72 The 4-year survival rate for all treated patients was 80%—almost certainly due to better supportive care rather than any new drug effect.
• Sirolimus, which blocks the serine-threonine kinase known as mammalian target of rapamycin is synergistic with cyclosporine in tissue culture and in clinical transplantation. Again, when tested in a randomized protocol in combination with ATG and cyclosporine in aplastic anemia, results were not superior to these agents only (P.S., unpublished data, January 2006).


Cyclophosphamide. As with ATG, recovery of blood counts can occur after a failed bone marrow transplantation preceded by conditioning with cyclophosphamide. High-dose cyclophosphamide was used intermittently by investigators at Johns Hopkins University (Baltimore, MD) in the 1980s during periods in which ATG apparently was not available to them; in their most recent update, of 38 previously untreated patients, the response rate was 74% and survival estimated at 86%^{ref1, ref2}.83,84 In contrast, an NIH randomized study was halted early due to the development of fungal infections and a much higher death rate in the cyclophosphamide arm^ref,85 and both relapse and cytogenetic evolution were observed^ref.86 The major toxicity of high-dose cyclophosphamide, prolonged neutropenia with concomitant susceptibility to infection, is now addressed by the Baltimore investigators by routine antimicrobial prophylaxis and prolonged G-CSF administration. Cyclophosphamide therapy does not eradicate PNH clones, and relapses now have been observed in Baltimore^ref.84 In the absence of another randomized trial, comparison of data from a small, single-center pilot with historical and more general results is problematic; it is especially difficult to exclude biased patient selection, both explicit (such as exclusion of those unlikely to respond or with a generally poor prognosis or older patients) and implicit (inability to treat uninsured individuals or foreign citizens).

Management of refractory aplastic anemia. There is no established algorithm for the management of patients who have failed to respond to ATG^ref.67 Transplantation from an alternative donor is offered by many centers to children who have failed a single course of immunosuppression and to adults after 2 rounds of ATG therapy. Response rates to second ATG have ranged from 22% to 64%^ref.78 In an Italian study of rabbit ATG as second therapy, 23 (77%) of 30 improved^ref87; in the NIH experience, the proportion responding was closer to 30%^ref.79 Cyclophosphamide also has been administered in this setting, with a response rate of about 50% reported^ref.83 A third course of immunosuppression may benefit only patients who showed some response to a previous treatment^ref.88 Response to retreatment correlates to a better survival compared with refractory patients^ref.79 The improved survival of patients who are refractory to immunosuppression, due to better supportive care, complicates the decision to undertake high-risk transplantation. We have tested alemtuzumab, a humanized monoclonal antibody specific for CD52, an antigen present on all lymphocytes; alemtuzumab induces profound immunosuppression by lymphocytotoxicity and has been effective in lymphoproliferative diseases, GVHD, and autoimmune disorders. To date, 4 of 8 patients who were refractory to treatment with horse ATG have responded to alemtuzumab, and toxicity has been modest (P.S., unpublished data, January 2006); we are now testing alemtuzumab in a randomized comparison with both horse and rabbit ATG in severe aplastic anemia at presentation.
Treatment of moderate pancytopenia. Clinically, the course of moderate aplastic anemia is variable: some patients progress to severe disease, others remain stable and may not require intervention; regular transfusions may not be required^ref.89 Very few clinical trials have specifically addressed moderate disease. Immunosuppression can reverse moderate pancytopenia and alleviate transfusion requirements; ATG and cyclosporine are more effective in combination^ref,73 but in practice are often used sequentially. Daclizumab, a humanized monoclonal antibody to the IL-2R, improved blood counts and relieved transfusion requirements in 6 of 16 evaluable patients; the outpatient regimen had little toxicity^ref.90 When there is residual hematopoietic function, androgens may be effective (although male hormones have failed most rigorous trials in severe aplastic anemia). Some moderate aplastic anemia likely results from telomere gene mutations and stem-cell exhaustion. In vitro, androgens increase telomerase activity in human lymphocytes and CD34 cells, acting through the estradiol receptor^ref,91 and this activity may provide a mechanism of action for their effects on marrow function.
Hematopoietic stem-cell transplantation

• Allogeneic HLA-matched sibling donor transplantation. Hematopoietic and immune system cells are replaced by stem-cell transplantation; conditioning with cyclophosphamide is not myeloablative but is sufficiently immunosuppressive to prevent and to eliminate residual host marrow by a graft-versus-marrow effect. Allogeneic transplant from a matched sibling donor cures the great majority of patients: the most recent cohort reported to the IBMTR showed 77% 5-year survival^ref,107 and in children, and patients undergoing transplantation who are minimally transfused, survival of 80% to 90% may be routinely achieved. Graft rejection, a historic problem in the application of transplantation to aplastic anemia (most dramatically manifest in rejection of unprepared syngeneic stem cells), is now not frequent in patients who undergo transplantation early and with a modest transfusion burden, likely a benefit of less immunogenic blood products (leukocyte-depleted erythrocytes, for example) from fewer donors (platelets collected by cytopheresis). Conditioning regimens that do not include irradiation now regularly achieve engraftment and avoid many of irradiation's long-term complications, especially late cancers. In a recent series of 81 patients who were prepared by cyclophosphamide plus ATG, sustained engraftment was achieved by 96%, and 3 of the 4 patients who initially rejected the transplant successfully underwent a retransplantation; 88% of the patients survived long term^ref.104 The combination of cyclophosphamide plus fludarabine, with or without ATG, has achieved high rates of graft acceptance and survival even in heavily transfused patients who received a transplant of mobilized peripheral-blood stem cells, months after proving refractory to immunosuppressive drugs^{ref1, ref2}.106,108

Allogeneic sibling transplantation for severe aplastic anemia

GvHD remains a serious problem for older patients, even with routine cyclosporine prophylaxis. In the IBMTR, rates of severe GVHD doubled in adults compared with children (15-20% for recipients  20 years of age to 40-45% for > 20 years of age)^ref.107 In Seattle, chronic graft-versus-host disease developed in 41% of patients who had survived more than 2 years after transplantation, tripling the risk of death and often requiring years of immunosuppressive therapy^ref.109 Even with resolution, chronic GVHD remains a risk factor for late complications such as growth and endocrine system effects, pulmonary disease, cataracts, neurologic dysfunction, and secondary malignancy. Addition of ATG105 and more recently its substitution by alemtuzumab^ref110 may reduce the frequency and severity of acute GVHD, a predictor of chronic GVHD.

• Matched unrelated donor transplant. A matched sibling donor is available in only 20% to 30% of cases. As the outcome in aplastic patients who have failed a single round of ATG has been poor, alternative sources of hematopoietic stem cells have been sought, usually from now very large donor registries. Outcomes of 318 alternative donor transplants performed from 1988 to 1998 recently have been summarized for the European registry92: for matched unrelated donors, the rejection rate was 15% and for grades II to IV GVHD, 48%, and 5-year survival was estimated at 39%. From diverse registry data (collected through EBMT, IBMTR, and the National Marrow Donor Program)^{ref1, ref2, ref3},92,107,121 the mortality rate is about twice that observed in matched sibling transplants; even with predominantly younger patients as recipients, age is probably the most powerful influence on survival, but also important are the closeness of the class I HLA match and the length of time from diagnosis. A retrospective analysis from the Japan Marrow Donor Program suggested that patients with the most favorable characteristics and conditioned with a minimal dose of radiation might anticipate survival comparable with matched sibling transplants^ref.113


Alternative donor stem-cell transplantation for severe aplastic anemia : prospective trials have enrolled fewer patients but have better results (perhaps due to superior protocols, but both careful patient selection and publication bias are likely important). In contrast to allogeneic sibling transplants, transplants from unrelated donors still require irradiation to ensure engraftment, due both to source of the donor cells and the transfusion status of the recipient. In a recent multicenter study, 62 patients with severe aplastic anemia who were refractory to immunosuppressive therapy underwent matched unrelated stem-cell transplantation following conditioning with cyclophosphamide, ATG, and TBI; graft failure occurred in 2%, acute grades II to IV GVHD was observed in 70%, chronic GVHD was observed in 52%, and overall survival was 61%. 25 patients who lacked an HLA-identical donor received an HLA-nonidentical stem-cell graft: 88% showed sustained engraftment, and overall survival was 44%^ref.120 The interval from diagnosis to transplantation in this study did not impact survival.
A European protocol substituted irradiation with fludarabine for unrelated and mismatched family donors: 73% were estimated to survive 2 years; while GVHD rates were relatively low, perhaps due to absence of radiation damage, graft rejection occurred in about one third of the older children and younger adults^ref.117 Children's Hospital of Milwaukee pioneered a rigorous conditioning regimen of cytosine arabinoside, cyclophosphamide, and total body irradiation, which produced long-term survival of about 50% with very little GVHD^ref.122 Other single-institution protocols have used a diversity of strategies to improve graft acceptance and reduce GVHD: T-cell depletion, CD34-cell purification, alemtuzumab, chemotherapy and monoclonal antibodies in combination; while almost exclusively enrolling small numbers of children and still preliminary, survival and morbidity may rival results of conventional sibling transplants. In current practice, unrelated transplant is offered for children who have failed a single course of immunosuppression and to adults who are refractory to multiple courses of ATG and alternative therapies such as androgens. Studies with longer follow-up of larger numbers of patients are crucial to establish the optimal conditioning regimen and to define which patients will benefit and especially how early unrelated transplantation should be performed.

• Other stem-cell sources. HLA mismatching is better tolerated for umbilical cord transplantations, making them in theory widely applicable. Published data for this procedure in marrow failure is limited, and almost all recipients have been small children, due to the small numbers of stem cells contained in a single cord-blood sample. In a report from the National Marrow Donor Program, engraftment occurred in less than half of 19 recipients, and almost all the patients died from transplant-related causes or survived due to autologous reconstitution or a second transplantation^ref.123 Surprisingly, 5 of 6 Chinese adult aplastic anemia patients successfully engrafted, and 4 survived^ref.116 Advocates of this approach are using pooled donations to increase stem-cell numbers. Family members are almost always available to the patient as a stem-cell source, but survival after haploidentical transplantation has been poor. More recently, engraftment was achieved in 3 children using the St Jude protocol, but one ultimately rejected and mixed chimerism in the others required further immunosuppression and donor lymphocyte infusions^ref.124
The treatment of severe aplastic anemia, whether by allogeneic HSCT or immunosuppression, has improved dramatically over the last 25 years, and long-term survival of > 75% of patients can be anticipated with either therapy^ref.125 For transplantation, the immediate challenge is the extension of stem-cell replacement to all patients, regardless of age, with a histocompatible sibling, and to others who lack a family donor using alternative stem-cell sources. The ability to achieve engraftment under these difficult circumstances may require conditioning regimens in which complications, particularly second malignancies, may not be apparent for many years. More optimistically, donor selection based on high-resolution histocompatibility typing may improve outcomes. The success of umbilical cord-blood transplantations, with their low risk of GVHD, may be enhanced by larger pools and histocompatibility matching. For immunosuppression, many new drugs and biologics have yet to be tested in aplastic anemia. Again, the costs of intensification need to be balanced against the benefits of higher hematologic response rates and lower rates of relapse and evolution. Repeated courses of immunosuppression offer the possibility of blood-count restitution to 75% to > 90% of patients, based on the range of published hematologic recovery rates with initial horse ATG followed by rabbit ATG, alemtuzumab, or cyclophosphamide. If residual stem-cell numbers are limiting, ex vivo expansion of hematopoiesis may be possible, as for example using Hox box proteins. Quantitative and practical measurements of oligoclonal T-cell activity and of hematopoietic stem-cell number and function would allow laboratory testing to guide treatment decisions. Ultimately, definition of genetic risk factors, affecting hematopoietic-cell function and the immune response, will clarify how agents in the environment initiate and perpetuate the marrow destruction of aplastic anemia.
Differential diagnosis : a family history of aplasia when present is strongly suggestive of a constitutive bone marrow failure syndrome, but may be negative in recessively inherited disorders (Alter BP. Inherited bone marrow failure syndromes: introduction. In: Young NS, Alter BP, eds. Aplastic Anemia, Acquired and Inherited. Philadelphia: W.B. Saunders; 1994:271–274). Determining whether a patient has AA or hypoplastic MDS may be difficult and sometimes arbitrary in the absence of cytogenetic abnormalities. This distinction may not be critical as substantial overlap exists between MDS and AA; younger patients with hypoplastic MDS of recent onset may be as likely to respond to immunosuppression as patients with AA^ref. Patients with splenomegaly, an unusual finding in AA, should always undergo peripheral blood phenotyping to exclude hairy cell leukemia, which can sometimes mimic the symptoms of AA (i.e., pancytopenia or hypocellular bone marrow).
Laboratory examinations : for treatment purposes, AA is categorically divided into :
• moderate AA (mAA) : patients with AA who do not fulfill the criteria for severe disease—those with ANC < 1200/µL, a reticulocyte count < 60,000/µL and, platelet count < 80,000/µL.
• severe AA (sAA) requires that the individual fulfill
• at least 1 out of 2 major criteria :
• severe bone marrow hypocellularity
• lymphoplasmacytic bone marrow infiltrate with hematopoietic residue < 30%
• at least 2 out of 3 minor criteria^ref:
• platelet count < 20,000 cells/µL
• reticulocyte count < 60,000 cells/µL (or corrected count < 1%) or transfusion-dependence
• ANC < 500 cells/µL
• very severe AA : ANC < 100 cells/µL
Prognosis : 6-month survival < 20%
Treatment : the urgency with which therapy is instituted is dictated by the current ANC and the duration of severe neutropenia, which most closely correlates with survival. Although patients with anemia and thrombocytopenia may be supported by red cell and platelet transfusions, very severe neutropenia (ANC < 200/µL) is associated with a high mortality rate and the risk of life-threatening infection. When patients present with higher ANC, there is more time to explore treatment options. Standard treatment for AA entails immunosuppressionor allogeneic HSCT. Various immunosuppressive drugs, including antithymocyte globulin (ATG), cyclosporine (CSA), and high doses of cyclophosphamide or corticosteroids, have produced hematologic improvement in the majority of patients with life-threatening cytopenias^ref. Responses usually correspond to independence from the need for transfusion and increased neutrophil counts to levels adequate to prevent infection. Factors such as the patient age, the availability of a matched sibling donor, and risk factors such as active infections or heavy transfusion burden should be considered in determining whether a patient is best treated by transplant or immunosuppression. Any of these factors might lead the patient and his or her physician to choose immunosuppression rather than HSCT. Younger patients generally tolerate transplantation better and have fewer long-term problems with GVHD. In this population the risks of cGVHD and TRM must be weighed against the high probability of relapse and the risk of developing clonal disease if they are treated with immunosuppression. Older patients and patients with comorbid conditions who may tolerate transplantation less well are generally offered a course of immunosuppression, while younger patients with a matched sibling are advised to pursue transplantation. One restrospective analysis supports this management. Although there were no differences in survival between BMT and immunosuppressive therapy (63% vs 61%) in the overall patient population, significant differences were apparent in favor of BMT for patients younger than 20 (64% vs 38%). Patients older than 20 years with neutrophil counts 0.2–0.5 x 10⁹/L appeared to benefit from immunosuppression in comparison to BMT. In general, patients with severely depressed neutrophil counts tended to do better with BMT, because of the shorter duration of time required for resolution of neutropenia (it should be kept in mind that patients receiving immunosuppressive therapy may respond with improvement of neutropenia as late as 6 months after initiation of therapy). For the middle-aged patient with a matched sibling donor, the recommendation regarding therapy should be made after considering the general health of the patient, the severity of disease, and the desires of the patient. Non-neutropenic patients may be supported for long periods of time without substantial morbidity except for the problem of iron overload. A treatment algorithm may be used as a guide in caring for patients with sAA.

• immunosuppressive therapy : most immunosuppressive regimens employ either horse ATG or rabbit antilymphocyte globulin (ALG). The addition of CsAto the treatment regimen results in better response rates^ref. A National Institutes of Health (NIH) study^ref as well as a European study, combining ATG with CSA resulted in response rates close to 70%. The NIH regimen consists of horse ATG (40 mg/kg per day for 4 days) and 12 mg/kg (adults) and 15 mg/kg in children per day (in divided dose) of CSA for 6 months. Corticosteroids are added (1 mg/kg of prednisone) during the first 2 weeks to ameliorate the serum sickness associated with ATG administration. Addition of G-CSFdoes not improve the survival rate. Response generally occurs within 6 months and improvement is usually observed between 1 and 2 months. Transfusion independence occurs at 2–3 months, but very late responses are possible. Response rates do not appear to be influenced by the presumed etiology of the disease (e.g., post-hepatitis, toxic exposure, paroxysmal nocturnal hemoglobinuria (PNH)/AA syndrome). Administration of ATG may be associated with anaphylactic reactions that are not always predicted by a positive skin test. Both ATG and ALG result in rapid reduction in the number of circulating lymphocytes to levels < 10% of the initial value. Although lymphopenia only persists for several days, the numbers of activated lymphocytes remain decreased for an extended period. Transient depression in platelet and neutrophil counts also can occur. Responses to immunotherapy frequently do not result in completely normal counts but confer transfusion independence or ameliorate the patient’s risk for severe infection due to neutropenia. Some patients who respond to immunotherapy may continue to be dependent on CSA and may develop worsening cytopenias upon discontinuation of the drug. In these patients, the CSA dose should be tapered to the lowest dose possible that maintains acceptable counts. CSA causes renal toxicity and hypertension. Irreversible renal damage may occur as a result of interstitial fibrosis and tubular atrophy, both of which appear to be related to the duration of therapy and the size of the doses received. The efficacy of daclizumabis being tested in patients who require continued immunosuppression. Development of tests predictive of response to immunotherapy would prove useful in helping manage patients unlikely to respond to other therapies. Some studies have proposed predictive tests based on hematopoietic colony formation with and without added T cells, in vitro response of progenitor cells to ALG, IFN-g mRNA measurements in marrow cells, and measurement of early progenitor cell numbers post-ATG treatment. None of these tests adequately distinguishes the 30% of nonresponders. Based on the assumption that most responding patients would show evidence of immune activation prior to treatment, we examined CD8 IFN-g levels in a cohort of sAA patients undergoing therapy with ATG/CSA. Levels of CD8 intracellular IFN-g were able to distinguish most responders from nonresponders^ref. 96% of patients with a population of CD8 cells expressing IFN-g responded to therapy while 32% of patients not expressing IFN-g responded. The results need to be validated by further study. Other clinical features discriminating responders from nonresponders have been studied. Patients with very severe AA (ANC < 100 cells/µL) receiving immunosuppression have inferior survivals to those with severe AA^ref. Patients failing to respond to G-CSFalso have an inferior response rate^ref. It has been suggested that this subgroup of patients be considered first for HSCT if a matched sibling donor is available. Relapse among responders to immunosuppressive therapy is common. In one European study, the relapse rate was 35% at 14 years^ref, but about half of these patients responded to a second course of immunotherapy. In the NIH study many patients required continued immunosuppresion^ref. In many cases patients responded after simply increasing the dose of CsA or reinstituting CsA therapy. Because of the high response to a second course of immunosuppression, relapse does not appear to influence survival. An uncontrolled study suggested that high-dose cyclophosphamide produces a higher rate of complete response, free of the late complications such as relapse and clonal evolution^ref. In a randomized controlled trial comparing ATG to high-dose cyclosphosphamide, the 2 drugs did not show statistically different response rates. Cyclosphosphamidetreatment also did not eliminate relapse or clonal evoluation^ref; this treatment also resulted in severe prolonged neutropenia and life-threatening fungal infections in some patients, necessitating termination of the trial^ref. Patients with moderate AA have successfully received less aggressive treatment such as androgens and growth factors, but these treatments do not clearly address the immune pathology and may only result in improvement in cytopenias without affecting progenitor cell destruction. Patients may respond to less aggressive immunosuppression. A small study using daclizumab, demonstrated a response rate of around 50% in previously untreated patients^ref. This antibody against the IL-2R has the advantage of being directed specifically against activated lymphocytes and can be administered as an outpatient. The dose is 1 mg/kg given every 2 weeks for 5 treatments. No significant side effects were seen in patients with moderate AA and responses were durable. Patients not responding to daclizumab may respond to more intensive immunosuppression with ATG.
• treatment of refractory severe AA : patients not responding to immunosuppressive therapy who have a matched sibling donor and no significant medical problems may be offered HSCT. Unfortunately, patients with significant transfusion history may have increased transplant morbidity. Generally, refractory patients without a matched related sibling donor and those who do not opt for transplantation should receive another course of ATG. Some investigators have attempted to increase the degree of immunosuppression, but these efforts have led to increased toxicity without significant improvements in responses. A trial using daclizumabfor refractory AA as well as CSA-dependent AA is currently underway at the NIH. Male anabolic hormones may be used as the third-line therapy with response rates as high as 50–75% in some studies. Some patients do respond to a combination of hematopoietic growth factors, especially when these are chronically administered over prolonged periods of time.
Clonal evolution : about 10% of patients with AA treated with immunosuppression develop clonal disease in the 7-year period following treatment^{ref1, ref2}. Bone marrow cytogenetic abnormalities can be demonstrated years following successful treatment and may, in fact, be part of the natural history of AA. Androgen-treated patients also have been reported to develop clonal abnormalities. Of 122 patients treated with ATG/CSA at NIH, 13 developed cytogenetic abnormalities (monosomy 7 in 9 patients, 7p deletion in 1, and trisomy 8 in 2). Monosomy 7 generally occurred in those with a minimal clinical response or in those who relapsed with severe pancytopenia. 7 of these patients died, 4 of bone marrow failure and 3 of leukemic progression. Patients with trisomy 8 generally are responsive to immunosuppression but may become dependent on CSA to maintain good counts. In a small study of patients with trisomy 8 in whom FISH was performed before and 6 months following immunosuppressive therapy, all 30 patients tested demonstrated increases in the proportion of cells with trisomy 8, though patients with other cytogenetic abnormalities had no change in the proportion of their cytopenias. Years of clinical stability were noted in these patients, some of whom have normal bone marrow histology. In the NIH study, only age was associated with risk for clonal evolution—each decade of life increasing the likelihood of evolution by 40%. Evolution to monosomy 7 correlated with the platelet count at 3 months (3.5-fold increased risk for patients with platelets < 50 x 10³/µL). Although selected retrospective analyses have correlated development of monosomy 7 with sustained administration of G-CSF^ref, other studies have failed to demonstrate such association. In the NIH study patients not receiving G-CSF developed monosomy 7. Confounding factors include the high endogenous levels of G-CSF in marrow failure patients as well as the likelihood that the most refractory patients are treated with G-CSF in a non-prospective trial (refractory patients are more likely to develop monosomy 7 even when not treated with G-CSF). Data suggest that many of these patients may have had a monosomy 7 clone at disease presentation, not detected by cytogenetics but demonstrable by FISH^ref. In vitro data suggest that the existing monosomy 7 clone may preferentially expand when exposed to pharmacologic doses of G-CSF, while normal bone marrow cells are unaffected. The reasons for the increased frequency of clonal abnormalities in bone marrow failure are unclear. In a number of these patients, karyotypic abnormalities may be distinguished by FISH at disease presentation. No abnormality in major checkpoint genes has been detected in these patients that might predispose to aneuploidy. Cells with karyotypic abnormalities or the PIG-A mutation may have growth or survival advantages over normal cells in a bone marrow subject to immune attack. With regard to PNH, previous evidence implies a growth advantage for PNH clones relative to "normal" marrow from the same individual; this relative growth advantage may be related to decreased susceptibility to immune attack, as GPI⁺ (normal) CD34 cells express apoptotic markers while those of the PNH clone do not. With regard to MDS, trisomy 8 cells have both a proliferative and survival advantage over normal cells from the same subject, relating to altered gene expression and upregulation of anti-apoptotic proteins. Should monosomy 7 cells be resistant to apoptosis, this may confer a survival advantage. In patients with trisomy 8, T cells with selective cytotoxicity for the abnormal clone were demonstrated. Although the full implications of this finding are unclear, one may envision a scenario where changes in chromosome 8 number are associated with altered antigen expression, leading to immune recognition and later resulting in destruction of normal innocent bystander progenitor cells and bone marrow failure. Certainly much more work needs to be done in order to understand the problem of clonality.
• treatment of PNH
• matched-related allogeneic HSCT: there has been a steady improvement in the results of BMT in this disorder related to improved conditioning regimens and drugs to control GVHD. 2 recent studies using CSA/methotrexate for GVHD prophylaxis estimate 5-year disease-free survivals of 88%^ref and 94%^ref. Chronic GVHD has been the major factor responsible for the decreased survival rate of adults when compared to children. In one study, 41% of adult patients who survived > 2 years had chronic GVHD. Their mortality was 3 times higher than for patients without this complication. Graft rejection constitutes a greater problem in patients with AA than in those receiving a transplant for other disorders. The increase in the rate of rejection may be related to the immune pathophysiology of AA, as well as to the less intensive conditioning regimens used in this patient population. Most transplant centers reserve total body irradiation for the unrelated BMT, as it has not been shown to confer a survival advantage over cytotoxic therapy alone for the matched related transplant recipient. While intensification of the conditioning regimen results in a lower graft rejection rate, in general it does not affect long-term survival. BMT is associated with several late complications, including solid tumors and MDS. Although most of these late complications are related to total body irradiation (TBI)(e.g., in a French study, solid tumors have developed in the radiation field of 5 of 147 patients), the risk of solid malignancy remains increased in BMT patients who were not treated with TBI. This risk appears to be equivalent in patients treated with immunosuppressive therapy and those treated with BMT (without TBI).
• matched-unrelated allogeneic HSCT: although allogeneic BMT is an option for young patients with a matched sibling donor, only 30% of patients have such a donor. For those young patients not responding to immunosuppression, matched-unrelated allogeneic transplantation may be considered an option. In a large European study, the survival for sibling-matched donors was was 86% compared to 43% for matched unrelated donors^ref. For patients with a single mismatch it is 25%, and for 2- or 3-locus mismatches it is 11%. In a recent study from Seattle, addition of TBI to the conditioning regimen increased the survival to 50% in patients receiving a partially matched donor graft^ref. Similarly, improved results have been obtained at Children’s Hospital in Milwaukee where pediatric patients receiving a TCD graft were conditioned with a preparative regimen including cytosine arabinoside, cyclophosphamide and TBI (a survival rate of 54% at 3 years was achieved)^ref. In general, results of unrelated BMT for adults have been disappointing due to higher rates of graft rejection and GVHD. In patients receiving matched unrelated donor transplants, factors such as age, degree of match, and type of conditioning regimen have a greater impact on survival than in patients receiving a matched sibling donor graft. TCD of the donor graft in combination with a rigorous conditioning regimen including TBI increases the survival and decreases the prevalence of chronic GVHD. However, many adults have difficulty tolerating the conditioning regimen that includes TBI. There is a higher frequency of malignant disease in these patients when compared to those not treated with TBI. Conditioning regimens :
• cyclophosphamideand antithymocyte globulin^ref
• reduced-intensity conditioning for sAA :
• fludarabine (120 mg/m²), cyclophosphamide (1,200 mg/m²) and antithymocyte globulin (7.5 mg/kg)^ref
• fludarabine (total dose 180 mg/m²), cyclophosphamide (total dose 120 mg/kg), and antithymocyte globulin (total dose 40 mg/kg) : 13 patients. All except 1 patient received multiple transfusions and had failed prior immunosuppressive treatment. 12 out of 13 patients achieved sustained engraftment. 1 patient was not evaluable for engraftment because of early death on day +10. None of the patients developed graft failure. Mucositis of mild-to-moderate severity was the only observed regimen-related toxicity. The cumulative incidence of acute GvHD grade II-IV and III-IV was 8.3% and 0%, respectively. With a median follow-up period of 45 months, the 5-year OS probability was 84%. 8 out of 11 surviving patients have been followed for > 1 year and only 1 developed limited chronic GvHD. All patients enjoy a normal life style, with a Karnofsky score of 100%, and all except 3, followed for 3, 5 and 6 months respectively, are free of any immunosuppressive medication^ref
• G-CSF and GM-CSF
• AR agonists : oxymetolone 3-5 mg/kg/day or fluoxymesterone 1 mg/kg/day
Prognosis : prognostic factors :
• PLT < 20,000 / mL
• reticulocyte index < 1%
• ANC < 500 / mL
• hematopoietic cells in bone marrow < 30%
If < 2 above mentioned conditions => 10% self-limiting.
If > 2 above mentioned conditions 6-mo OS = 20% => HSCT.
Evolution : acute myeloblastic leukemia (AML), responsive to treatment with bolus methylprednisolone, prednisolone, and androgens, G-CSF, cyclosporine, and methenolone^ref
• pure erythroid anemia / erythroblastopenia : abnormal deficiency of the erythroblasts (macrocytic and normochromic)
• Diamond-Blackfan anemia (DBA)
• acquired erythroblastopenia :
• transient erythroblastopenia of childhood (TEC) : temporary aplasia of erythropoietic tissue in young children, usually with anemia

Aetiology
• familial TEC is associated with the chromosome 19q13.2 region but not caused by mutations in coding sequences of the ribosomal protein S19 (RPS19) gene, which causes Diamond-Blackfan anemia (DBA)^ref
• viral illness within the previous 2 months : anyway HHV-6, B₁₉ virus^ref, HHV-4 / EBV, and HHV-5 / CMV are not common causative agents of TEC^ref.
Prognosis : the condition almost always resolves spontaneously without recurring.
• acute acquired erythroblastopenia (benign)
• Owren type
• Gasser type
• chronic acquired erythroblastopenia (thymoma-associated)
• congenital dyserythropoietic anemias (CDAs)

	heredity	locus	PB	BM	siderosis	acidified serum test	extra hematological symptoms
type I	autosomal recessive	CDAN1 (15q15.1-15.3)	macrocytosis (75%), aniso poikilocytosis	binucleated erythroblasts, megaloblasts, internuclear chromatin bridges, mitotic shapes, intracytoplasmatic bridges, large nuclear pores due to partial loss of nuclear envelope or invaginations	+	-	short stature, syndactyly, hypoplasia or agenesis of one or more distal phalanges
type II / hereditary erythroblast multinuclearity with positive acidified serum test (HEMPAS)	autosomal recessive	CDAN2 (20q11.2)	aniso poikilocytosis, basophilic stippling	binucleated erythroblasts (10-40%), rare multinucleated (up to 4 nuclei) erythroblasts, multipolar mitoses, karyorexis	-	+	mediastinal masses due to extramedullary erythropoiesis, bone hyperplasia of skull (erythropoietic bone marrow expansion)
type III	autosomal dominant / sporadic	CDAN3 (15q22)	anisocytosis (macrocytosis), basophilic stippling	giant multinucleated erythroblasts, karyorexis, nuclear vesicles	+	-	sometimes mongoloid facies
type IV	autosomal recessive / dominant sporadic ?		severe anemia at birth or during childhood	normoblastic erythroid hyperplasia, irregular or karyorectic nuclei, no protein precipitates in erythroblasts			transfusion-dependent
type V	variable		no anemia, normal MCV	severe dyserythropoiesis, sometimes with moderate megaloblastosis			indirect hyperbilirubinemia
type VI	autosomal recesive/dominant sporadic ?		no anemia, severe megalocytosis (MCV 119-125 fl)	erythroid hyperplasia with megaloblastosis unresponsive to B12 and folates			-
type VII	autosomal recesive/dominant sporadic ?		severe anemia at birth	normoblastic erythroid hyperplasia. Irregular nuclear shape, intraerythrocytary inclusions, exclusion of b-thalassemia trait in parents			transfusion-dependent
Majeed syndrome	autosomal recessive	LPIN2 (18p)	hypochromic and microcytic				chronic recurrent multifocal osteomyelitis (CRMO), neutrophilic dermatosis or Sweet syndrome

Therapy : RBC transfusions; splenectomy when associated to hemolysis.
• anemia of chronic disease (ACD) (normocytic & normochromic or microcytic & hypochromic)

Aetiology : chronic ...
• inflammatory diseases(rheumatoid arthritis) : hepcidin production is increased up to 100-fold and this may account for the sequestration of iron in macrophages
• infectious diseases(subacute bacterial endocarditis, tuberculosis)
• neoplasms(expecially carcinomas and lymphomas)
Pathogenesis :
• hepcidin and cytokines downregulate ferroportin expression => iron retention in monocytes => withdrawal of iron from the sites of erythropoiesis and the circulation (hypoferremia and hyperferritinemia)^ref
• bone marrow progenitor inhibition
• low serum levels and poor response to EPO
• pro-inflammatory cytokines :
• IL-1band TNF-a induce hypoferremia by modulating macrophage iron metabolism via induction of ferritin biosynthesis
• the T_h1 cytokine IFN-gblocks erythropoiesis directly although its role in iron homeostasis is controversial
• anti-inflammatory cytokines (IL-4and IL-13may contribute to the diversion of iron traffic by increasing iron uptake and storage in activated macrophages)
ACD may also hold some benefit by reducing iron availability to invading pathogens, thus limiting their growth, and by stimulating cell mediated immune function. The latter can be referred to the correction of an inhibitory effect of iron towards IFN-g induced immune effector pathways in macrophages. In ACD, the imbalances of iron homeostasis lead to reduced serum iron concentration and transferrin saturation, whereas ferritin levels, reflecting body iron stores, are often elevated. In addition, Tf concentrations are usually normal, and serum TfR levels are slightly increased.
Laboratory examinations :
• hyposideremia (< 75 mg/dL or < 13 mmol/L for males; < 65 mg/dL or < 11 mmol/L for males)
• ferritinemia = 30-200 ng/ml
• MCV > 70 fl^ref
• normal transferrin saturation
Therapy :
• IL-10
• blood transfusions for rapid correction of haemoglobin levels
• rHuEPO
• iron status in patients with CRF has varied widely over the past half century. Transfusional iron overload was invariable until the introduction of hemodialysis when iron deficiency emerged due to dialyzer blood loss. Vigorous parenteral iron therapy led to reports of significant iron overload but with the introduction of rHuEPO therapy, iron deficiency again became widespread. Vigorous parenteral iron therapy has again raised concern about iatrogenic iron overload (Fishbane S. Intravenous iron therapy: reweighing risk and reward. Semin Dialysis. 1999;12:5–8). The term functional iron deficiency has arisen mainly in the nephrology literature in reference to the IDE induced by rHuEPO therapy in dialysis patients with residual iron stores^ref. The diagnosis is usually based on one or more screening measurements. Aggressive parenteral iron therapy is commonly advised for its treatment on the assumption that functional iron deficiency is the major cause of resistance to rHuEPO therapy. While this is undoubtedly true in many patients, the laboratory features are also typical of the anemia of chronic disease and there is sparse information about the role of inflammation in so-called functional iron deficiency. The sTfR is not an optimal guide to the need for additional parenteral iron because of the enhancing effect of rHuEPO on erythropoiesis and consequently on the sTfR. More data is needed on the extent to which inflammation contributes to functional iron deficiency and whether a rise in sTfR concentration in patients on stable doses of rHuEPO can be used as a guide to parenteral iron therapy. In a dialysis patient without laboratory evidence of inflammation, the amount of iron that should be given during the first 2–3 weeks of initiating rHuEPO therapy can be calculated from the anticipated increase in circulating hemoglobin (roughly 4 mg iron/kg body weight for each 10 g/L hemoglobin rise) minus iron stores based on 8 mg available iron per µg/L serum ferritin or 3–4 mg/µg/L serum ferritin if the CRP is elevated. After achieving a hematological response, parenteral iron should be continued to maintain a serum ferritin > 100 µg/L if the CRP is normal and > 200–300 µg/L if elevated. There is evidence that serum ferritin values above 500–800 µg/L increase the risk of infections in hemodialysis patients due to promotion of pathogen growth and impairment of immune function^ref and this risk could be even greater in pancytopenic patients given rHuEPO therapy who are already at increased risk of infection
• chronic renal failureanemia (normochromic and normocytic, echinocytes, schistocytes, acanthocytes)

Pathogenesis :
• decreased peripheral half-life due to accumulation of toxic catabolytes
• EPO-resistance
• decreased EPOincretion
• polymenorrhea
• hemorrhagic gastritis
• excessively frequent blood sampling
• megaloblastic anemias (macrocytosis with MCV > 120 fL; Howell-Jolly bodies; Cabot's rings) (uneffective erythropoiesis due to deficiency of TTP, which impairs DNA synthesis but not RNA synthesis => protein accumulation)
• essential pernicious anemia / Addison-Biermer disease (anisopoikilocytosis).

Epidemiology : the peak incidence is in the sixth and seventh decades^{ref1, ref2, ref3}.
Aetiology : vitamin B₁₂deficiency
Symptoms & signs (after 3-4 years of malabsorption) :
• pallor, decrease in hemopoiesis : thrombocytopenia (=> purpura), neutropenia and elderly granulocytes in Arneth scheme
• mild jaundice
• malaise
• Hunter's glossitis / atrophic glossitis
• defects in myelin synthesis => posterolateral sclerosis / subacute combined degeneration of the spinal cord
Laboratory examinations^ref :
• [vitamin B₁₂]_plasma
• Schilling test (for gastrointestinal absorption of vitamin B₁₂) : a measured amount of both ⁵⁷Co- and ⁵⁸Co-vitamin B₁₂ is given orally, followed by a parenteral (i.m.) flushing dose of the nonradioactive vitamin to saturate TCs and other binding enzymes, and the percentage of radioactivity is determined in the urine excreted over a 24-hour period : if total radioactivity is < 7% than administered, low absorption in terminal ileum occurred. The test is usually performed 3 times: first with added intrinsic factor, then without it, and then after antibiotic therapy. The results are used in the diagnosis of pernicious anemia and other disorders of vitamin B₁₂ metabolism.
• deoxyuridine suppression test
• [methylmalonic acid (MMA)]_urine
• peripheral blood smear
• hypersegmented neutrophils (Figure A, arrow)
• macroovalocytes (Figure A, arrowheads)
• bone marrow aspirate : "blue bone marrow" due to presence of highly basophilic blasts
• megaloblastic erythroid precursors (Figure B, arrowheads)
• giant bands

subclinical deficiency^ref make it worthy of clinicians’ attention: (a) Its frequency is at least 10-fold that of clinically expressed cobalamin deficiency, and it affects millions of people. (b) Occasional patients, although asymptomatic, nevertheless have subtle electrophysiologic findings that suggest that neurologic changes sometimes lurk under the asymptomatic veneer^{ref1, ref2}. An unknown but probably small proportion of cases may eventually progress to overt clinical manifestations; the proportion is probably highest in that minority who have underlying classical malabsorptive disease, such as pernicious anemia (lack of intrinsic factor).
Comparison of clinical and subclinical cobalamin deficiency.

	"clinical" deficiency	"subclinical" deficiency
clinical signs and symptoms	Present (by definition) but:  - not every manifestation (e.g., hematologic, neurologic) appears in every patient - the manifestations may be mild.	absent (by definition) but- some patients may have electrophysiologic neurologic changes.
cobalamin levels	low in 97% of cases (< 200 ng/L; < 148 pmol/L) and often very low (< 100 ng/L; < 74 pmol/L).	usually low, but can be low-normal (250–350 ng/L; 185–258 pmol/L).
metabolic abnormalities	present in 99% of cases. often severe (serum methylmalonic acid (MMA) >1000 nmol/L or >1.0 µmol/L; plasma total homocysteine (tHcy) > 50 µmol/L)  All metabolic tests usually abnormal (MMA in 98% and tHcy in 96% of cases).	at least 1 abnormality present, by definition. Usually mild (MMA 300–800 nmol/L or 0.3–0.8 µmol/L; tHcy 15–25 µmol/L).  Some metabolic tests may be normal.
causes of the deficiency	identifiable in almost all cases  - malabsorption of free cobalamin in > 90% of cases (abnormal Schilling test) - dietary causes are rare (except infants of mothers with deficiency). - preclinical stage of free cobalamin malabsorption (e.g., pernicious anemia) in a few cases.	not identifiable in at least half of cases  - food-bound cobalamin malabsorption in 30%–40% of cases (normal Schilling test) - dietary causes may occur, but frequency is unknown and probably low
course	progressive in almost all cases  - usually takes several years to appear, but progresses more rapidly once symptoms appear.	unknown, but appears to be slow (many years)  - some patients remain asymptomatic >10 years after biochemical abnormality appears - progression may be more rapid in those few cases with free cobalamin malabsorption.
management	full diagnostic evaluation is mandatory but its nature and extent are debated.  Therapeutic intervention is mandatory  - dose and route depend on underlying cause (parenteral or large daily oral doses needed when malabsorption of free cobalamin present).	diagnostic evaluation is mandatory  therapeutic intervention is probably advisable  - dose unclear, may depend on underlying cause (larger oral doses may be needed in some).
frequency of entity	uncommon (even in the elderly, the highest at-risk group).  < 10% of all low cobalamin levels are associated with clinical signs of deficiency.	found in 10%–20% of the elderly; also present in younger persons but proportion is much lower.  ~70% of low cobalamin levels and ~30% of low-normal levels are thought to represent subclinical deficiency.
Cobalamin values are shown in ng/L values, which are used by most clinical laboratories, and pmol/L, which are used in many publications (conversion factor: ng cobalamin x 0.738 = pmol). Methylmalonic acid values are given in nmol/L and µmol/L values, either of which are used by different clinical laboratories and publications. Reference intervals vary widely among laboratories and methods for all tests. The values shown here are based on the following, fairly common cutpoints for abnormality: serum cobalamin < 200 ng/L; serum MMA > 280 nmol/L; plasma tHcy > 15 µmol/L in men, and > 13 in women.

The sometimes unstated and often undifferentiated focus on subclinical deficiency in many large studies of cobalamin status in healthy populations has helped blur the distinctions between subclinical and clinical cobalamin deficiency and foster the impression that conclusions derived from the former automatically extend to the latter. Without minimizing the potential importance of subclinical deficiency, its medical impact remains poorly understood. However, because it is encountered so frequently nowadays, physicians faced with the potentially cobalamin-deficient patient may benefit from distinguishing between the 2 deficiency states, which are part of a continuum but often differ in kind as much as extent and pose diverse diagnostic, clinical, and management characteristics, problems, and goals. Although it is daunting and probably unnecessary to actively seek out new asymptomatic cases by screening, it is axiomatic that any patient identified during a medical encounter must always be investigated. An increasingly common clinical challenge is how to respond to a patient without the typical manifestations of cobalamin deficiency or one in whom an inexplicably abnormal cobalamin-related test result is found. Possible approaches to the array of patient types and deficiency states will follow a brief, practical review of the available diagnostic armamentarium.
Tests of cobalamin status :
• serum cobalamin : long familiarity, wide availability, and low cost have contributed to the favored role of cobalamin as the diagnostic mainstay for assessing cobalamin status. Despite limitations of specificity and considerable controversy about sensitivity, which is actually 97% for clinical cobalamin deficiency^ref, no universally accepted replacement has emerged. Recommendations have emerged for broadening the cobalamin level criteria for deficiency. This issue has important ramifications and will be discussed later. Common causes, other than cobalamin deficiency, for abnormal cobalamin, methylmalonic acid, and homocysteine levels :
• cobalamin
• idiopathic
• pregnancy (the test abnormality particular to the entity can be severe sometimes; cobalamin level can be < 100 ng/L in some entities, whereas MMA can be > 1000 nmol/L in others, and homocysteine can be > 50 µmol/L in still others)
• mild transcobalamin I deficiency
• miscellaneous disease associations (e.g., HIV infection, multiple myeloma)
• folate deficiency
• miscellaneous drugs (e.g., anticonvulsants, oral contraceptives)
• severe transcobalamin I deficiency (the test abnormality particular to the entity can be severe sometimes; cobalamin level can be < 100 ng/L in some entities, whereas MMA can be > 1000 nmol/L in others, and homocysteine can be > 50 µmol/L in still others.)
• [abnormal results may be obtained because of the use of a possibly improper reference range by the laboratory or because of assay error. The reference interval controversy about cobalamin is discussed in some detail in the text, but issues exist for the other assays also (this category is listed last in each column because the frequency of the problem is difficult to compare with other causes.)]
• methylmalonic acid :
• renal insufficiency
• volume contraction, which has often been given as the explanation for some elevated MMA results that are otherwise hard to explain (e.g., in occasional patients with folate deficiency). Although volume contraction is a very plausible mechanism, evidence to support the explanation has rarely been provided
• infancy
• ? mild MMA-related enzyme defects
• ? bacterial contamination of the gut
• severe enzyme defects (e.g., mutase deficiency) (the test abnormality particular to the entity can be severe sometimes; cobalamin level can be < 100 ng/L in some entities, whereas MMA can be > 1000 nmol/L in others, and homocysteine can be > 50 µmol/L in still others)
• [abnormal results may be obtained because of the use of a possibly improper reference range by the laboratory or because of assay error. The reference interval controversy about cobalamin is discussed in some detail in the text, but issues exist for the other assays also. (This category is listed last in each column because the frequency of the problem is difficult to compare with other causes.)]
• homocysteine :
• incorrect sample or processing
• renal insufficiency/ increased creatinine
• folate responsiveness (homocysteine levels improve very often after folate supplementation even when evidence for folate deficiency is not found. The very common improvement may have many explanations (e.g., pharmacologic effect of folate in mild genetic conditions; subclinical folate deficiency))
• age, sex, life style factors (some of these may be surrogates for other influences)
• enzyme polymorphisms (e.g., MTHFR)
• alcohol abuse(hard liquor) (the test abnormality particular to the entity can be severe sometimes; cobalamin level can be < 100 ng/L in some entities, whereas MMA can be > 1000 nmol/L in others, and homocysteine can be > 50 µmol/L in still others)
• folate deficiency(the test abnormality particular to the entity can be severe sometimes; cobalamin level can be < 100 ng/L in some entities, whereas MMA can be > 1000 nmol/L in others, and homocysteine can be > 50 µmol/L in still others)
• vitamin B₆ deficiency
• hypothyroidism
• various drugs (e.g., isoniazid)
• various disease associations (e.g., renal transplant, leukemia)
• inborn errors of homocysteine metabolism (the test abnormality particular to the entity can be severe sometimes; cobalamin level can be < 100 ng/L in some entities, whereas MMA can be > 1000 nmol/L in others, and homocysteine can be > 50 µmol/L in still others.)
• [abnormal results may be obtained because of the use of a possibly improper reference range by the laboratory or because of assay error. The reference interval controversy about cobalamin is discussed in some detail in the text, but issues exist for the other assays also. (This category is listed last in each column because the frequency of the problem is difficult to compare with other causes.)]
Paradoxically, modern technology and economics in clinical chemistry have inhibited assessment of the new generation of cobalamin assays. Whereas dozens of independent studies had established and scrutinized the older microbiologic and radioisotopic assays, the literature is virtually silent on the commercially protected, immunologically based chemiluminescence assays that have displaced the older methods. As a result, we have limited understanding of how well cobalamin assays perform in clinical laboratories today. An additional potential source of confusion is the literature’s expression of cobalamin values in pmol/L, whereas all clinical laboratories use ng/L (ng x 0.738 = pmol, so that 200 ng/L = 148 pmol/L).
• plasma total homocysteine (tHcy) : elevation of tHcy is a very sensitive indicator of clinical cobalamin deficiency^{ref1, ref2}. Levels rise early in the course, preceding symptoms, progress as deficiency worsens, and do not recover until cobalamin therapy is given. However, it has the major flaw of poor specificity. Cobalamin deficiency accounts for only a minority of all high tHcy levels, common causes for which can be confused with it (e.g., folate deficiency), may be occult (e.g., alcohol abuse)^{ref1, ref2}, or reflect personal and life style characteristics^ref. The reference interval is not universally agreed,8 and creatinine levels must always be taken into account when interpreting mildly elevated tHcy. Assay methods are generally reliable, and cost has come down greatly. However, inappropriate sample collection and processing are major technical causes of slightly elevated levels (Rasmussen K, Moller J. Methodologies of testing. In: Carmel R, Jacobsen DW, eds. Homocysteine in health and disease. Cambridge, UK: Cambridge University Press; 2001:199–211). Anticoagulated plasma, not serum, must be used and centrifugation must be done within an hour (serum levels rise as much as 10% per hour’s delay). These requirements are not always met, a failure that often escapes the attention of the ordering physician.
• methylmalonic acid (MMA) : elevation of MMA is at least as sensitive a marker for clinical cobalamin deficiency as tHcy^ref (Carmel R. Cobalamin deficiency. In: Carmel R, Jacobsen DW, eds. Homocysteine in health and disease. Cambridge, UK: Cambridge University Press; 2001:289–305) and is more specific^ref, which favors the diagnostic use of MMA. Nevertheless, an automatic assumption that an isolated elevation of MMA indicates cobalamin deficiency invokes circular reasoning and must be resisted. There are other, often poorly understood, causes for MMA elevation than cobalamin deficiency; impaired renal status is an important influence^{ref1, ref2}. Serum MMA reference intervals, like those for tHcy, continue to be controversial (Rasmussen K, Moller J, Lyngbak M, Pedersen AMH, Dybkjaer L. Age- and gender-specific reference intervals for total homocysteine and methylmalonic acid in plasma before and after vitamin supplementation. Clin Chem. 1996;630–636) and have shrunk sharply in recent years, necessitating caution when interpreting older data in publications and patients’ charts. The major limitations in MMA use are practical: assay complexity, high cost, limited availability, and often slow turnaround time. MMA can also be sensitively assayed in urine, but this approach is not used widely.
• holo-transcobalamin II : most cobalamin in serum is carried on transcobalamin (TC) I, also called haptocorrin, which does not deliver its cobalamin to tissues. Therefore, measuring the smaller cobalamin fraction attached to TC II (i.e., holo-TC II) is theoretically attractive because it represents the cobalamin available to cells. However, initial claims that low holo-TC II is the earliest marker of cobalamin deficiency and a "surrogate Schilling test" were poorly documented. Specificity of low levels may also be limited^ref, and other influences on holo-TC II are not well understood yet^ref. The older crude methods have been replaced by immunoassays, one of which is available commercially^ref but is not approved for clinical use in the US. It is too early to determine whether holo-TC II provides any practical advantage over the measurement of serum cobalamin^ref
Response to cobalamin therapy : therapeutic response can and should serve as the court of last resort, especially when the diagnosis remains in doubt. Conditions that ensure reliable assessment are the following: (a) monotherapy; (b) appropriate dose and route (e.g., small oral doses of cobalamin have no diagnostic value if malabsorption exists); (c) criteria for response must be appropriate as well as objective: originally normal clinical status cannot improve (exception: mean corpuscular volume (MCV) within normal range can decrease slightly after therapy sometimes), and metabolic changes must not be the sole criterion of improvement if clinical problems were present; and (d) the approximately 20% rate of reported improvement of mild metabolite abnormalities after placebo administration^{ref1, ref2, ref3} illustrates that the phenomenon of regression to the mean, wherein abnormal results tend to drift toward normal upon retesting, spontaneous fluctuations, and the possibility of confounding events, must be borne in mind when assessing test result changes.
Tests of causes of cobalamin deficiency : identifying what caused the cobalamin deficiency, although a process distinct from diagnosing the deficiency itself, serves many important purposes. However, the ability to diagnose cobalamin malabsorption has become impaired. The availability of Schilling tests has decreased sharply in recent years, and assay of intrinsic factor in gastric juice is rarely done. These gaps leave assays for antibody to intrinsic factor and gastrin or pentagastrin I levels as major diagnostic substitutes, but these blood tests identify only somewhat more than half of cases of pernicious anemia^ref (Carmel R. Cobalamin deficiency. In: Carmel R, Jacobsen DW, eds. Homocysteine in health and disease. Cambridge, UK: Cambridge University Press; 2001:289–305; Chanarin I. The megaloblastic anaemias. 2nd ed. Oxford, UK: Blackwell Scientific; 1979). The diagnosis of ileal malabsorption cannot be made without Schilling tests. The milder but more common defect of food-cobalamin malabsorption also is not tested clinically today, and neither blood tests nor gastric studies provide reliable substitutes^{ref1, ref2}. Approximately 15% of low cobalamin levels, especially those unaccompanied by evidence of cobalamin deficiency, appear to be associated with mild or severe TC I deficiency^ref. Diagnosis requires TC I immunoassay. Suggestions that substandard elevation of serum cobalamin levels after oral cobalamin therapy helps diagnose either cobalamin malabsorption or TC I deficiency remain unproven.
The diagnostic approach to the patient : no diagnostic gold standard exists today. All tests have disadvantages, including the purest candidates, clinical abnormalities or elevated MMA levels that are corrected by cobalamin therapy. Serum cobalamin is the most commonly used screening test. Despite its shortcomings, it has proved quite sensitive in clinically deficient patients, but it is less reliable in subclinical deficiency. No single diagnostic algorithm fits everyone either, in part because requirements and goals vary with different patients. It may be more realistic to discuss options and adapting strategies to clinical contexts. 2 not at all mutually exclusive strategies may be considered initially, especially when the clinical picture is unclear. One strategy is to require abnormal results in a minimum of 2 tests in order to prove the diagnosis of cobalamin deficiency, especially in any patient in whom the diagnosis of cobalamin deficiency is doubtful after clinical assessment. However, the 2 tests must not share any common causes of falsely abnormal results. For example, tHcy and MMA levels make an unsatisfactory pair, because both rise in the presence of renal insufficiency; cobalamin and holo-TC II are not a good pair because both reflect facets of circulating cobalamin. An acceptable combination might be cobalamin and MMA levels. If the clinical dilemma is serious, the physician need not be limited to only 2 tests. Moreover, 2 tests do not suffice whenever an inborn error of metabolism is suspected. MMA, tHcy, and cobalamin are needed to screen such patients. A second strategy or modification of the first strategy is to tailor testing to the nature of the clinical problem being faced and its diagnostic requirements. Several representative patient encounters illustrate such nuances and how they can influence the diagnostic approach:
• the patient with typical clinical signs and/or symptoms, whether mild or severe (e.g., megaloblastic anemia; myeloneuropathy typical for cobalamin deficiency). The main diagnostic goal here is to confirm the strong clinical impression that cobalamin deficiency is indeed responsible. For this purpose, 1 test usually suffices. The chief risk of relying on 1 test is that attempting additional tests, should that become necessary, often becomes unreliable after therapy is given. The most practical candidate may be cobalamin assay, which is cheap and readily available. Moreover, falsely normal levels occur in only 2.9% of patients who have clinically obvious deficiency^ref. Cobalamin levels can be falsely low in many circumstances but, unless due to folate deficiency (Chanarin I. The megaloblastic anaemias. 2nd ed. Oxford, UK: Blackwell Scientific; 1979) those are unlikely to coincide with megaloblastic anemia; falsely low cobalamin levels might coincide on rare occasions with unrelated neurologic dysfunction, such as in multiple sclerosis^ref or HIV infection^ref. Diagnostic attention must be directed to the underlying cause as soon as the diagnosis of cobalamin deficiency is made. Malabsorption of free cobalamin explains > 90% of cases of clinical deficiency^ref (Chanarin I. The megaloblastic anaemias. 2nd ed. Oxford, UK: Blackwell Scientific; 1979). Addressing the causal disorder, whether malabsorptive or other, will also serve to confirm or throw doubt on the diagnosis of deficiency made in these patients.
• the patient with clinical findings for which cobalamin deficiency is an unlikely explanation but must be ruled out (e.g., anemia or neurologic problems of uncertain nature). The main diagnostic goal here is to ensure that if cobalamin deficiency exists, it is not missed. This can be a particularly common dilemma in patients with neuropsychiatric problems, in whom low cobalamin levels often may not represent deficiency^ref. Full metabolic testing, including MMA and tHcy, is often advisable because atypical clinical findings provide no anchor. If deficiency is indeed responsible for the symptoms, the metabolic findings are often as striking as in category 1, in which metabolic abnormalities are rarely very mild^{ref1, ref2}. For reasons discussed in the preceding paragraph, the cause of deficiency must be sought next.
• the asymptomatic patient with a condition known to cause cobalamin deficiency (e.g., ileal disease, gastric surgery, veganism). The main diagnostic goal is to determine whether cobalamin deficiency has developed yet in an asymptomatic patient known to be at risk. MMA assay is the test of choice because metabolic changes often precede low cobalamin levels in the evolution of the expected deficiency^{ref1, ref2, ref3}; tHcy is acceptable as a cheaper alternative for this purpose. Metabolic abnormalities are typically mild to moderate in the asymptomatic state.
• the asymptomatic patient accidentally found to have a low cobalamin level or other biochemical abnormality (e.g., a low cobalamin level found while screening because the patient is elderly; an elevated tHcy found during cardiovascular risk evaluation). The main diagnostic goal is to determine whether cobalamin deficiency exists; establishing whether it poses a continuing or progressive risk to the patient is an important subsequent goal, tends to depend on what caused the deficiency, and helps determine how long therapy will be needed. MMA is the best choice to diagnose deficiency because it is more specific than tHcy and also appears to be abnormal more frequently than tHcy^ref. An elevated MMA that is not due to renal failure requires further diagnostic work, combining clinical reassessment, additional testing, evaluation for pernicious anemia or other causes of deficiency, and a monitored therapeutic trial. A normal MMA level tends to rule out cobalamin deficiency as the cause of the original finding.
Other diagnostic issues :
• cobalamin level criteria for deficiency : the traditional cutpoint of 200 ng/L was derived from many studies in which unequivocally cobalamin-deficient patients, defined by clinical and hematologic criteria, were compared with healthy controls (Chanarin I. The megaloblastic anaemias. 2nd ed. Oxford, UK: Blackwell Scientific; 1979). Studies of clinically affected, unequivocally cobalamin-deficient patients confirm that cobalamin levels are almost always low^ref (Chanarin I. The megaloblastic anaemias. 2nd ed. Oxford, UK: Blackwell Scientific; 1979). Normal levels were reported in only 2.9% of such cases,4 although a 5.2% rate within a subset of the latter cases has been more widely cited. Widespread metabolic testing subsequently found mild MMA and/or tHcy abnormalities in a sizeable minority of seemingly healthy volunteers who had low-normal cobalamin levels (up to 300 or 350 ng/L)^{ref1, ref2, ref3, ref4, ref5}. The assumption that the mild, isolated metabolic abnormalities (most patients had no clinical evidence for cobalamin deficiency) represented subclinical deficiency was fortified by the frequent demonstration that the metabolic abnormalities disappeared after cobalamin therapy. The recommendation was made to raise the cobalamin cutpoint to 300 or 350 ng/L in order to capture those otherwise unrecognized cases of presumptive deficiency^ref. That important study of Framingham residents found that 32% of those with cobalamin levels up to 350 ng/L had abnormal tHcy and/or MMA levels (14% had elevated tHcy alone and 28% elevated MMA alone). Other studies and surveys have adopted this cutpoint, as have some clinical laboratories and clinicians. The validity of the strategy can be questioned, however, especially in the clinical setting^ref. There is a great difference between being aware that cobalamin-deficient patients sometimes have low-normal rather than low cobalamin levels and the wholesale clinical redefinition, with all its complications, of an established test’s criteria in order to capture additional cases along with many "noncases." The objections include the following: (a) The assumption that an isolated metabolite abnormality that often has alternative explanations constitutes adequate proof of deficiency can be invalid, especially when an independent gold standard is lacking. (b) Specificity of low cobalamin is a greater problem than sensitivity in patients with clinical deficiency (at 97%^ref, the sensitivity is identical to that of MMA at 98% and tHcy at 96%)^ref; any advantage brought by raising the cutpoint to include low-normal levels is counterbalanced by greatly worsened cobalamin level specificity. (c) The gain in new cases will be almost entirely in subclinical cobalamin deficiency, which vastly predominates among persons with low-normal cobalamin levels (200 to 300 or 350 ng/L); relatively few additional cases of clinical deficiency will be found. (d) Metabolic abnormality suggestive of subclinical deficiency is also found in about 15% of all elderly persons with unquestionably normal cobalamin levels (> 350 ng/L)^{ref1, ref2}, and the absolute number of such persons dwarfs those with low and low-normal levels combined; therefore, manipulating the cobalamin cutpoint will still not ensure detection of all or perhaps even most cases of subclinical deficiency. (e) The most serious argument against raising the cobalamin level cutpoint to 350 ng/L may be that whereas 58%–78% of persons with cobalamin levels < 200 ng/L have metabolic evidence for deficiency^{ref1, ref2}, the odds are reversed in persons with low-normal cobalamin levels, only 32–35% of whom have such metabolic evidence^{ref1, ref2}. Most persons with low-normal levels, therefore, are not just asymptomatic but 65–68% of them have normal cobalamin status by all metabolic criteria. (f) The resulting 65–68% probability of mislabeling normal patients as deficient by moving the cobalamin cutpoint to 350 ng/L in order to identify the 32–35% minority that has usually mild, subclinical deficiency has potentially great implications. The scope can be made explicit by estimating the absolute numbers. One third of elderly Americans, or 15 million, have low-normal cobalamin levels (as do 15% of younger adults, an even larger absolute number)^ref. All 15 million of these elderly people (and even larger numbers of nonelderly adults) would be labeled as cobalamin-deficient as a result, but 65%–68% or about 10 million of them (and even more nonelderly adults) will have entirely normal cobalamin status. (g) Many considerations must enter into setting screening strategies^ref. The misidentification of those 10 million nondeficient elderly persons as deficient is justifiable only if the other 5 million (32%–35%) thereby clearly benefit clinically. However, it is undetermined if and how those 5 million persons, most of whom by far have subclinical, not clinical, deficiency, will benefit^{ref1, ref2}. This is obviously an important clinical issue, and its resolution requires more information and careful analysis than is currently available in order to arrive at a reasoned consensus.
• use of tests to monitor response : monitoring response to therapy is important but often neglected. Clinical monitoring suffices in most patients with clinical deficiency, keeping in mind, however, that neurologic deficits are sometimes irreversible. Biochemical monitoring is usually reserved for patients who are asymptomatic or have equivocal clinical changes or other circumstances. Because serum cobalamin levels rise instantly regardless of cobalamin’s effectiveness, they have no monitoring value. MMA or the cheaper tHcy assay, however, do. Their levels begin to decline within a few days of effective therapy^{ref1, ref2}, which also creates a brief posttherapy window for reassessment, if needed. Responding to cobalamin but not folate therapy, the metabolite levels become normal within a week or 2.
• identification of the cause of cobalamin deficiency : it has been argued that most cobalamin deficiency results from pernicious anemia (although this argument is now known to apply only to clinical^ref, not subclinical^{ref1, ref2}, deficiency) and that other causes often do not alter the therapeutic approach. A more compelling argument is that testing for malabsorption with the Schilling test is onerous, subject to error, and, in any case, increasingly unavailable, that the logistics and cost of testing the many patients now being identified as cobalamin deficient are prohibitive, and that traditional gastroenterological tests are not helpful in most cases of subclinical deficiency^ref. Nevertheless, differentiating between pernicious anemia, ileal disease, bacterial overgrowth, or inborn errors of metabolism in the cobalamin-deficient patient provides important clinical information. Direct therapy of some diseases is possible (e.g., tropical sprue, bacterial overgrowth); endoscopic screening for gastric malignancy is recommended in pernicious anemia but not other disorders^ref38; and the diagnoses can modify not only prognosis and management but the cobalamin therapy itself. Finally, identifying the cause often affirms the diagnosis of cobalamin deficiency, and failure to find a cause warrants reexamination of the diagnosis of deficiency. The presenting clinical picture can help guide the diagnostic approaches to the underlying cause. Anyone with clinical cobalamin deficiency has a > 90% likelihood of having gastrointestinal disease with malabsorption of free cobalamin^ref (Chanarin I. The megaloblastic anaemias. 2nd ed. Oxford, UK: Blackwell Scientific; 1979). Because pernicious anemia is the most common of those diseases^ref, any patient with clinical cobalamin deficiency can be tested immediately and cheaply for intrinsic factor antibody (parietal cell antibody is diagnostically unreliable)^ref and serum gastrin. A positive intrinsic factor antibody (50%–70% sensitivity and near 100% specificity, as long as the sample is not obtained within a few days of cobalamin injection) can be taken as very suggestive of pernicious anemia, whereas lack of serum gastrin or pentagastrin I abnormality raises doubt about the diagnosis^ref. The Schilling test can be sought in those with normal blood test results and will also help identify other gastrointestinal causes of deficiency. The major uncertainties concern the approach to the much greater numbers of patients who have presumptive subclinical cobalamin deficiency^ref. Because latent pernicious anemia may explain a small fraction of such cases and is potentially serious^ref, the blood tests mentioned earlier are still recommended. However, very few other patients with subclinical deficiency will have malabsorption of free cobalamin, making the Schilling test advisable in only selected cases of subclinical deficiency rather than as a routine tool. Approximately 30–40% of such patients will have food-cobalamin malabsorption^ref, testing for which is clinically unavailable; no endoscopic or blood tests are reliable^{ref1, ref2}. This condition is generally stationary for many years but is occasionally a precursor of pernicious anemia^ref; pernicious anemia probably always passes through a stage of food-cobalamin malabsorption but not all patients reach the end-stage of pernicious anemia. Other than in strict vegetarians, dietary cobalamin insufficiency is rare. However, even long-standing veganism usually causes only subclinical cobalamin deficiency^{ref1, ref2}, except in the severely symptomatic infants born to and breast-fed by vegetarian or otherwise cobalamin depleted mothers whose own deficiency is most often subclinical^ref. TC I deficiency may account for 15% of unexplained low cobalamin levels, especially those with no metabolic evidence of cobalamin deficiency at all^ref. The causes in the remaining patients with low cobalamin levels (i.e., about half of all cases), whether accompanied by metabolic abnormalities or not, have not been identified.
The impact of diagnosis on management : clinical cobalamin deficiency is a medical disease, and any patient identified in a medical encounter must be approached and managed from that perspective. A distinction may be therapeutically helpful, too, between patients who have clinical cobalamin deficiency, usually a result of severe malabsorption affecting the intrinsic factor–mediated process, and those with subclinical deficiency, who usually have either only partial malabsorption (e.g., food-cobalamin malabsorption) or normal absorption. Different therapeutic doses, routes, and durations are needed for the various subgroups and further individualized for patients. Broad nutrition-based solutions for subclinical cobalamin deficiency are being proposed (e.g., routine cobalamin supplementation, food fortification), but many of the schemes may leave all or most persons with malabsorption unprotected. For this and other reasons, disorder-based considerations are needed even in formulating population-wide policies.
Therapy : vitamin B₁₂ or directly folic acid
• folic acid deficiency anemia

Aetiology :
• nutritional deficit (no raw vegetables in diet)
• chelating compounds in goat milk
• increased consumption
• thalassemia
• pregnancy
• drugs : cotrimoxazole
• alcoholism
• hemodialysis
• CHF
Laboratory examinations :
• bone marrow aspirate shows basophilic ground glass-like cells.
• [folates]_plasma
Therapy : 5-15 mg/day folic acid p.o.
The folate-cobalamin connection :
• vascular disease and homocysteine : homocysteine is normally present in the plasma at low concentrations (5–15 µmol/L). There is now abundant evidence that a sustained elevation in plasma homocysteine (hyperhomocysteinemia) is an established independent risk factor for vascular disease, including thrombosis^ref. It is not clear, however, whether this association is causal or merely serves as a surrogate marker of underlying conditions that cause both vascular disease and elevation of plasma homocysteine level^{ref1, ref2} (Green R. Homocysteine and Occlusive Vascular Disease: Culprit or Bystander? Prev Cardiol. 1998;1:31–33). Even if causally associated, whether hyperhomocysteinemia leads to or follows vascular disease has not been established definitively. Metabolism of homocysteine occurs along 2 major enzymatic pathways, either remethylation or transsulfuration, which require folate and vitamin B₁₂ or vitamin B₆ respectively. Disturbances of homocysteine metabolism leading to hyperhomocysteinemia can result from inherited or nutritional disorders^{ref1, ref2} as well as from various metabolic diseases, such as chronic renal disease and hypothyroidism^ref. Even within the normal range for creatinine there is a correlation with homocysteine and the variation in homocysteine encountered in hypothyroidism is explained, at least in part, by the reduced creatinine clearance associated with hypothyroidism. Among the various causes of hyperhomocysteinemia only those caused by vitamin deficiencies are amenable to simple and relatively safe correction. Consequently, interventional approaches using vitamin supplementation provide a promising avenue through which to explore the possible amelioration of vascular disease risk or recurrence, through lowering homocysteine levels. Yet, the recent successful outcome of this approach8 still does not provide clear evidence for a direct role of homocysteine in vascular disease, thrombosis, and premature vascular disease. Reduction of cardiovascular disease risk with concomitant decrease in homocysteine levels does not connote a direct cause-and-effect relationship between elevated homocysteine and vascular damage.
• causes and effects of hyperhomocysteinemia : severe hyperhomocysteinemia associated with homocystinuria due to inborn errors of metabolism is unquestionably associated with precocious atherosclerosis and both arterial and venous thrombosis^{ref1, ref2}. In these patients, the main cause of mortality and morbidity is thromboembolism, followed by cerebrovascular accident, peripheral arterial thrombosis, and myocardial infarction^{ref1, ref2}. Severe degrees of hyperhomocysteinemia (> 50 µmol/L) are most often the result of homozygosity for inborn errors of metabolism in children and in young adults. Among older adults, however, acquired disorders including chronic renal disease and vitamin B₁₂ and other nutrient deficiencies are more likely causes of severe hyperhomocysteinemia. Mild (15–20 µmol/L) or moderate (25–50 µmol/L) degrees of hyperhomocysteinemia are generally the result of acquired disorders. The most frequent causes are deficiencies of 1 or more of the 3 vitamins that are required as cofactors or substrates for homocysteine metabolism. Serum homocysteine levels show a significant inverse correlation with serum B₁₂, folate, and B₆ and even individuals with low-normal B₁₂, folate, or B₆ concentrations may have elevated levels of homocysteine^{ref1, ref2, ref3}. Certain populations also show a high frequency of heterozygosity for mutant forms of key enzymes in the homocysteine metabolic pathways that may be associated with hyperhomocysteinemia^{ref1, ref2}. Of several genetic mutations and disorders associated with hyperhomocysteinemia, those affecting methylenetetrahydrofolate reductase (MTHFR) have attracted the most attention. MTHFR is required for the generation of methyltetrahydrofolate, the required cosubstrate for remethylation of homocysteine to methionine. Mutations in MTHFR can therefore result in varying degrees of hyperhomocysteinemia^{ref1, ref2}. There are a variety of uncommon mutations in the MTHFR gene resulting in deficient enzyme activity and disorders of varying clinical severity. These should be distinguished from the far more prevalent but clinically less severe polymorphisms of MTHFR. The most common polymorphism in MTHFR is a 677CT substitution that is associated with reduced enzyme activity^ref. Homozygotes for this mutation frequently also have low plasma and red cell folate^ref. In some studies, an increased risk of vascular disease has been reported in individuals homozygous, as well as heterozygous, for the 677T allele. Whereas many studies have been negative for such an association, from a meta-analysis it was concluded that the combined odds ratio for coronary heart disease associated with homozygosity for the 677T allele was significantly increased in the setting of low folate status^ref. In a recent case-control multicenter study on patients with vascular disease, although the frequency of the TT genotype in cases was not different from the controls, adjustment for traditional risk factors resulted in a significant odds ratio for risk of vascular disease in the TT group^ref. Because this risk was attenuated by adjustment for plasma folate and homocysteine, the authors concluded that the modest risk increase was attributable mainly to increased homocysteine and low plasma folate in individuals with this genotype. Furthermore, heterozygosity for the C677T mutation has been reported to carry an increased risk for thrombosis when it coexists with hetererozygosity for the factor V Leiden mutation, a genetic defect that results in activated protein C resistance and a hypercoagulable state^ref. Moderate or mild hyperhomocysteinemia, caused either by heterozygosity or homozygosity for genetic defects^ref or by nutritional deficiency of the vitamins involved in homocysteine metabolism, is an independent risk factor for stroke, coronary, or peripheral artery disease^{ref1, ref2, ref3, ref4, ref5, ref6}. For each 5 µmol/L increment in total plasma homocysteine (approximately 1 standard deviation of the mean for the normal population), there is a corresponding increase of about 40% in the relative risk of developing coronary artery disease^ref. This increase is comparable to the risk associated with the same proportional increment in cholesterol and equates homocysteine and cholesterol as risk factors for ischemic heart disease. The relative risk/odds ratio for cerebrovascular and peripheral arterial disease associated with elevated homocysteine appears even greater than it is for coronary artery disease. The European Concerted Action Project on Hyperhomocysteinemia found that subjects in the top one fifth of the population for plasma homocysteine had a relative risk for vascular disease of 2.2 compared with the lower four fifths, and plasma homocysteine was shown to confer an independent risk of vascular disease similar to that of smoking or hyperlipidemia^ref.

The mechanism by which homocysteine might cause thrombosis and vascular damage is not known. The pathological changes attributed to homocysteine that are seen in association with severe hyperhomocysteinemia are indicative of a profound disruption of normal endothelial cell function or blood coagulation, but even in this setting, the mechanism whereby homocysteine produces these effects remains obscure. Homocysteine in various concentrations has been shown to affect platelets, monocytes, endothelial cells, vascular smooth muscle cells, cytokines, and adhesion molecules. Homocysteine has also been shown to inhibit anticoagulant, as well as to promote procoagulant pathways, in vitro^{ref1, ref2, ref3} (Green R. Homocysteine and coagulation factors: basic interactions and clinical studies. In: Homocysteine and Vascular Disease, K Robinson, ed. Kluwer Academic Publishers, Dordrecht, The Netherlands. 2000:349–370)
Identification, Investigation, and Management of Hyperhomocysteinemia : the identification of hyperhomocysteinemia usually takes place through simple measurement of plasma levels of the compound. Even when basal homocysteine levels are within the normal range, derangements in homocysteine metabolism may be present and may be uncovered by determining peak plasma homocysteine levels following administration of an oral methionine load^{ref1, ref2}. In general, impairment of the remethylation pathway is more likely to result in increased fasting levels of homocysteine, whereas transsulfuration pathway defects are more often associated with abnormal responses to methionine loading. There is no general agreement on whether plasma homocysteine should be measured as part of any assessment of a patient with a precocious or unexplained vascular occlusive event. It is not clear how often an elevated level of homocysteine is the only identifiable risk factor associated with the occurrence of a precocious vascular event. Finding hyperhomocysteinemia merits further investigation of the possible cause through measurement of relevant B-vitamin, thyroid, and renal functional status. This serves both to identify the possible presence of a previously unrecognized underlying disorder and to institute appropriate treatment to correct the hyperhomocysteinemia as well as its cause, where possible. The systematic workup of patients found to have hyperhomocysteinemia is important because of the need to uncover its proximal cause (vitamin deficiency or metabolic disorder) as well as the underlying disease process. Lowering of homocysteine through administration of appropriate vitamin supplements is generally safe, efficacious and inexpensive. Although the results of some long-term interventional studies have not yet appeared, there is mounting evidence that vitamin supplements may ameliorate cardiovascular disease complications among at-risk patient groups^{ref1, ref2} as will be discussed below. There is clear evidence that homocysteine levels can be lowered by the administration of supplemental folate, with or without pyridoxine^{ref1, ref2}. However, the need for supplementation of B vitamin intake in the population at large remains controversial. The conclusion reached in a meta-analysis of randomized trials of homocysteine-lowering B vitamin supplements was that 0.5- to 5-mg folic acid supplements lower homocysteine levels on average by 25%. The addition of vitamin B₁₂ (0.5 mg) produces a further reduction in homocysteine of approximately 7%, whereas vitamin B₆ (16.5 mg) had no significant effect^ref. It is not yet established whether lowering of homocysteine per se by increased intake of these vitamins results in a diminution of cardiovascular risk. Recently published data strongly support a protective role for high folate and vitamin B6 intake against ischemic heart disease^ref. In studies on at-risk groups, the administration of a folate, B₆, and B₁₂ supplement reduced the restenosis rate following coronary artery bypass surgery^ref, as well as the progression of carotid atherosclerosis in hyperhomocysteinemic renal transplant recipients^ref. The prospect of modifying risk of cardiovascular disease in the general population by simple nutritional intervention is appealing and, if substantiated, could constitute a substantial advance in the control of a major cause of morbidity and mortality worldwide.
Is homocysteine merely a marker for preexisting underlying cardiovascular disease? It remains possible that homocysteine does not, itself, directly cause vascular damage. Rather, it may be merely a biochemical marker of some related process, perhaps even an acute phase reactant, indicative of underlying inflammation in the process of atherogenesis. Conclusions drawn from an extensive meta-analysis of 43 studies published through 1998, were that although most cross-sectional and case-control studies found higher mean homocysteine levels or a greater frequency of high homocysteine levels in persons with cardiovascular disease, the results of most prospective studies indicated a smaller or no association. This supports the possibility that homocysteine may be a marker of atherogenesis or an indicator of factors more directly linked to cardiovascular disease risk^ref. The associations as well as the known or presumed cause and effect relationships between homocysteine, deficiencies of B vitamins, metabolic disorders and vascular disease are illustrated in Figure 2. Possible interrelationship between hyperhomocysteinemia and vascular disease.
Is homocysteine merely a marker for folate deficiency? Evidence for an association between folate deficiency and cardiovascular disease
Folate deficiency is associated with raised homocysteine, and raised homocysteine is associated with increased risk of cardiovascular disease. In a few studies, folate nutritional status (either intake or blood levels) is correlated directly with cardiovascular disease risk. In the Nutrition Canada Survey involving over 5000 men and women a statistically significant association between serum folate levels and risk of fatal coronary heart disease was found. In the same study there was a 69% increase in relative risk of fatal coronary heart disease among the population third with the lowest serum folate compared with the third that had the highest serum folate^ref.
A prospective cohort study involving over 80,000 women as part of the Nurses’ Health Study noted a significant inverse correlation between the dietary intake of folate (and vitamin B₆) and mortality and morbidity from cardiovascular disease over a 14-year follow-up period. There was a 31% reduction in relative risk of coronary artery disease among women in the highest compared with the lowest quintile of folate intake. The authors of this study also calculated that each 100 µg/day increase in folate intake was associated with a 5.8% lower risk of coronary heart disease^ref. This figure is in close agreement with that derived by Boushey and coworkers^ref, who estimated that increasing folate intake by 100 µg/day would reduce homocysteine levels by 6% and the risk of coronary heart disease by 5%. Folate consumption was shown to correlate inversely with both homocysteine level and carotid artery thickening. It is not yet known whether lowering homocysteine by increased intake of folate results in a diminution of cardiovascular risk, even though evidence is accumulating that this might be the case. This evidence includes the studies, cited above, that found lower blood folate levels are predictive for coronary artery disease. There is, therefore, a distinct possibility that cardiovascular disease incidence might be reduced in the general population by improvement of folate nutrition. Already there is clear evidence that folate status in the general population has been markedly improved in the postfortification era^{ref1, ref2}. From the prospective Nurses’ Health Study it was predicted that an increase in folate intake would have a favorable impact on coronary heart disease rates with a maximum benefit achieved at a folate intake of at least 400 µg/day^ref. Ward and associates^ref administered 3 graded doses of folic acid (100 µg, 200 µg and 400 µg daily) to groups of subjects separated into tertiles according to plasma homocysteine levels. The group with the highest homocysteine showed a progressive fall in homocysteine at each escalated dose of folic acid. A large-scale longitudinal intervention trial will be required to directly address the question of whether improvement in folate nutrition through increased intake will lower cardiovascular disease risk in the general population. Such a study would need to be conducted in a country where folate fortification of the food supply has not been undertaken. Folate fortification of the food supply was introduced in the US in 1998 as a means to reduce the prevalence of neural tube defect pregnancies. The nutritional modification of the population-at-large could potentially have a beneficial effect on the prevalence of ischemic vascular disease. Despite the reasonable anticipation that homocysteine levels will fall in the population as a whole, the overall effect of this change in nutritional status on the prevalence of cardiovascular disease-related deaths and disease will need to be evaluated. Even if a change in cardiovascular death rates occurs, it will be difficult to attribute this to a cause-and-effect relationship with either homocysteine levels or folate status^ref. Also, considerable genetic heterogeneity exists with respect to homocysteine metabolism, in particular because of MTHFR polymorphisms. Folate requirements may, therefore, vary considerably between individuals and different levels of intake may be required to control hyperhomocysteinemia and modify its potential effects throughout the population^{ref1, ref2}
The contribution of vitamin B₁₂ deficiency to hyperhomocysteinemia : vitamin B₁₂ deficiency is common and B₁₂ deficiency is a significant public health problem, particularly among the elderly. In the US there are 37 million people over age 65 and conservative estimates indicate that 2–3% of this population has or will develop pernicious anemia caused by failure of gastric intrinsic factor production and consequent B₁₂ malabsorption^ref (Chanarin I. The Megaloblastic Anemias, 2nd edition. Oxford, UK: Blackwell Scientific Publications; 1979). If undetected, this can result in progressive B₁₂ deficiency with its consequent effects on the blood and nervous system. Additionally, there is evidence that the prevalence among the elderly population of borderline and suboptimal B₁₂ status leading ultimately, in some cases, to frank B₁₂ deficiency may be as high as 20–30% among the elderly. The most common cause is believed to be food B₁₂ malabsorption caused by chronic gastritis, gastric atrophy, and the use of proton pump inhibitors^ref. Recently, a high prevalence of B₁₂ deficiency has also been observed in both children and young adults in diverse locations, such as Guatemala, India, and Israel^{ref1, ref2, ref3}. The causes of B₁₂ deficiency in these populations are unclear, but may be related to a combination of low intake and unrecognized malabsorption. The classic pathophysiological manifestations of B₁₂ deficiency include megaloblastic anemia and neurological complications that run a gamut from peripheral neuropathy to depression, cognitive disturbances, and dementia^{ref1, ref2}. More recent evidence suggests that B₁₂ deficiency may contribute to the risk of vascular disease (through its association with elevated levels of homocysteine in the blood). Since the introduction of folic acid fortification of the US diet, there has been a dramatic reduction in the prevalence rates of folate deficiency as well as hyperhomocysteinemia. This has resulted in a substantial change in the proportional risk of raised levels of homocysteine attributable to either folate or B₁₂ deficiency. B₁₂ deficiency now ranks well above folate deficiency as the primary modifiable cause of hyperhomocysteinemia^{ref1, ref2}. To further complicate this interrelationship between folate and vitamin B₁₂, it is possible that the risks of B₁₂ deficiency may be accentuated by folic acid fortification. The importance of recognizing B₁₂ deficiency and conditions that cause B₁₂ malabsorption is therefore magnified by the addition of folic acid to the US food supply as well as the widespread use of nutritional supplements containing folic acid. A recent national advisory committee on B vitamin intake has cautioned on the need for increased vigilance to detect B₁₂ deficiency and its causes, particularly among the elderly in the wake of the folic acid fortification initiative (Food and Nutrition Board—Institute of Medicine. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington, DC: National Academy Press; 1998). This is based on the observations that folic acid can reverse megaloblastic anemia caused by B₁₂ deficiency while neurological degeneration may progress unabated and that B12 deficiency in the elderly is often not suspected until anemia develops. It is therefore unknown whether the supplemented level of folic acid in the food supply will increase the incidence of neurological damage due to undiagnosed B₁₂ deficiency. Several authoritative sources have cautioned that there is no known safe dose of supplemental folic acid in patients with untreated B₁₂ deficiency (Chanarin I. The Megaloblastic Anemias, 2nd edition. Oxford, UK: Blackwell Scientific Publications; 1979; Food and Nutrition Board—Institute of Medicine. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington, DC: National Academy Press; 1998). Because of the precipitous introduction of folic acid fortification in the USA, there had been no opportunity to properly evaluate this risk. A recently published study attempts to address this issue by comparing the prevalence of anemia among patients with low B₁₂ concentrations for 9 years spanning the period before and after the folic acid fortification era. Since the proportion without anemia did not increase significantly over this time, the authors concluded that food fortification has not masked the hematological manifestations of vitamin B₁₂ deficiency^ref. Theoretically, it is also possible that the masking of B₁₂ deficiency by increased levels of folate intake may obscure the presence and delay the correction of the B₁₂ deficiency, allowing the persistence and progression of hyperhomocysteinemia among the elderly, a population already at risk for vascular disease. Assuming an agonistic role for hyperhomocysteinemia in vascular damage, then the institution of folic acid fortification necessitates increased consideration of B₁₂ status and vigilance for B₁₂ deficiency, particularly in older adults. Elucidation of this question will need to come from properly conducted clinical trials in countries that have not mandated folic acid fortification.
Genetic diseases and polymorphisms : the inborn errors of folate and cobalamin absorption and metabolism, reviewed comprehensively elsewhere (Rosenblatt D, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Scriver CR, Beaudet AL, Sly WS, et al. The metabolic & molecular bases of inherited disease. 8th ed. New York: McGraw-Hill; 2001:3897–3933) usually come to clinical attention because of hematological or neurological manifestations. The most common cause of low serum cobalamin and anemia in an infant is nutritional, usually seen in the breast-fed infant of either a vegetarian mother, or of a mother with sub-clinical pernicious anemia^ref. Because this disorder is acquired, it will not be discussed here. Recent advances in genetics and genomics have resulted both in the discovery of mutations in genes involved in rare inborn errors of metabolism and in the discovery of polymorphisms in these genes. These polymorphisms have been promoted as role players in the interaction between genes and environment that underlie many common diseases. The present section will first give an overview of the known inborn errors of folate and cobalamin absorption and metabolism. It will then focus on four rare disorders, one involving cobalamin absorption (Imerslund-Gräsbeck syndrome) and three involving folate and cobalamin metabolism: deficiencies of methionine synthase (cblG), methionine synthase reductase (cblE), and MTHFR. It will also examine the 677 CT polymorphism in the MTHFR gene, as a model for the study of the relationships between polymorphisms and common disease. These disorders illustrate how advances in genetics have increased our understanding of the biology underlying disease states and the challenge of integrating this information into clinical practice. Selected features of inherited diseases discussed :

	Imerslund-Gräsbeck Syndrome (MGA1)	cblE	cblG	methylenetetrahydrofolate reductase (MTHFR) (severe deficiency)
OMIM No.	261100	236270	250940	236250
gene(s)	CUBM AMN	MTRR	MTR	MTHFR
chromosome(s)	10p12.1 14q32	5p15.2-p15.3	1q43	1p36.3
gene product(s)	cubilin amnionless	methionine synthase reductase	methionine synthase	methylenetetrahydrofolate reductase
serum cobalamin	low	normal	normal	normal
serum folate	normal	normal	normal	normal or low
homocysteine elevation	not a major feature	yes	yes	yes
megaloblastic anemia	yes	yes	yes	no
developmental delay	no	yes	yes	yes
proteinuria	yes	no	no	no
common polymorphisms (postulated to be associated with common diseases)		66A=>G  1298A=>C	2756A=>G	677C=>T

Patients with inherited disorders of cobalamin transport are not able to deliver cobalamin to cells. These rare diseases are important because they are more easily treated than some of the disorders of intracellular metabolism, and they must be considered in the differential diagnosis of anemia, neurological problems, and even infection. The defect may be at the level of gastric intrinsic factor synthesis, intestinal transport (Imerslund-Gräsbeck syndrome) or at the level of a circulating cobalamin binding protein (TC I or TC II). Except for those with TC II deficiency, patients will have a low serum cobalamin level, and except for those with TC I deficiency, the Schilling test will be abnormal. Elevated levels of homocysteine and methylmalonic acid may be found in these transport disorders, but they tend not to be as high as in the disorders of intracellular metabolism. It should be noted that TC I deficiency is not associated with clinical cobalamin deficiency, or with elevated levels of these metabolites. It is extremely important to consider the diagnosis of TC II deficiency in the anemic infant because the disease is relatively easily treatable. Since serum cobalamin levels are often normal (reflecting cobalamin bound to TC I), direct measurement of total TC II levels must be requested.
The critical laboratory findings among patients with inborn errors of intracellular cobalamin metabolism are hyperhomocysteinemia and methylmalonic acidemia, either alone or in combination. It is reasonable to test for these metabolites in infants, children or adults with developmental delay or regression, anemia, thrombosis, metabolic acidosis, and a wide variety of neurological and psychiatric manifestations. Usually the serum cobalamin level is within the reference range. Hyperhomocysteinemia results from the abnormal function of the enzyme methionine synthase (cblG) or as a result of the inability to produce its cofactor, methylcobalamin (MeCbl) (cblE). Methylmalonic acidemia occurs because of the abnormal function of methylmalonyl coenzyme A (CoA) mutase (mut) or an inability to produce its cofactor adenosylcobalamin (AdoCbl) (cblA, cblB, cblH). Those patients who cannot produce either MeCbl or AdoCbl have both hyperhomocysteinemia and methylmalonic acidemia (cblC, cblD, cblF). Only those patients with impaired methionine synthase or MeCbl synthesis will have megaloblastic anemia (cblC, cblD, cblE, cblF, cblG). Whereas most patients present in infancy or childhood, some can present in adolescence or adulthood^{ref1, ref2, ref3}

Cobalamin metabolism in mammalian cells. The pathways by which vitamin B₁₂ (cobalamin) is taken up by cells and converted to its active coenzyme derivatives are shown. The steps affected by inborn errors of cobalamin metabolism (cblA-cblH, mut), as well as MTHFR deficiency are shown. The methionine synthase reaction (cblG) is shown twice in order to separate folate and homocysteine metabolism.
These inborn errors of intracellular metabolism have been classified by complementation analysis in cultured human fibroblasts into complementation groups: mut and cblA-cblH^{ref1, ref2}. In this test, the patient’s fibroblasts and a fibroblast line having a known defect are fused by exposure to polyethylene glycol. In general, if the defects in the two cell lines affect different loci, there will be a correction toward normal cobalamin metabolism in fused cultures as compared to nonfused cultures. Correction is not seen if the defects in the two cell lines affect the same genetic locus (Watkins D, Rosenblatt DS. Cobalamin and inborn errors of cobalamin absorption and metabolism. The Endocrinologist. 2001;11:98–104). The genes for some of these complementation groups are known: mut (MUT)^ref, cblA (MMAA)^ref, cblB (MMAB)^ref, cblE (MTRR)^ref, and cblG (MTR)^{ref1, ref2, ref3}. In contrast to the inborn errors of cobalamin transport and metabolism, there are only three validated inherited disorders affecting folate. Hereditary folate malabsorption presents early in life with severe anemia and developmental delay. Serum folate levels are low. Treatment with high levels of reduced folate is required not only to correct the anemia, but also to get folate across the blood brain barrier. Glutamate formiminotransferase deficiency has a variable phenotype. Although the original patients had megaloblastic anemia and developmental delay, some later patients had only large amounts of formiminoglutamate excretion with little else. The first mutations in an affected patient have been described in the FTCD gene on chromosome 21^ref. MTHFR deficiency is the most common inborn error of folate metabolism and will be discussed below in more detail.
Imerslund-Gräsbeck Syndrome (MGA1) : patients with this syndrome usually present with megaloblastic anemia between 1 and 5 years of age, although some patients have been much older (Watkins D, Rosenblatt DS. Cobalamin and inborn errors of cobalamin absorption and metabolism. The Endocrinologist. 2001;11:98–104). Many patients have proteinuria of the tubular type, as well as decreased levels of serum cobalamin in the presence of normal levels of gastric intrinsic factor. The abnormal pattern in the Schilling test result is characteristic of the inability of the enterocyte to absorb the intrinsic factor-cobalamin complex. > 250 patients have been published. Whereas the original patients were described from Finland and Norway, numbers of patients have been described from the Middle East. There have been interesting developments in our understanding of the molecular basis of this disorder, which has locus heterogeneity. The first of these was the finding that in all affected Finnish families studied, the disease is caused by mutations in the cubilin (CUBN) gene on chromosome 10p12.1 that encodes for a receptor for the intrinsic factor-cobalamin complex^ref. The investigators who discovered this gene also found that the frequency of the disease appeared to be decreasing in recent generations, and postulated that some environmental alteration such as diet may affect expression of the disease. Although the reason for the proteinuria is not known, cubilin is highly expressed in the kidney. Another observation in this disease is that mutations in CUBN were not found in Norwegian patients showing the same phenotype. Through linkage studies, a candidate gene was located on the long arm of chromosome 14; mutations were found in the AMN gene in the Norwegian patients^ref. This gene emerged as a candidate because of an expression pattern similar to that of CUBN, with high levels of expression in both small intestine and kidney. What was of particular interest is that inactivation of the mouse AMN gene results in an embryonic lethal condition in which the embryos have no amnion, whereas mutations in humans result only in the mild MGA1 phenotype. Thus this rare inherited disorder gives evidence for genetic/environment interaction, genetic heterogeneity, and "moonlighting proteins" (proteins that have multiple roles). The function of such proteins can vary with cell type, cellular localization, oligomeric state, differential binding sites, or the cellular concentration of a ligand, substrate, cofactor, or product^ref. In the case of AMN, it is postulated that the 5' end of the gene product is needed for cobalamin absorption, whereas the 3' end is needed for embryonic development^ref. Methionine Synthase [cblG, MTR gene product] Deficiency and Methionine Synthase Reductase [cblE, MTRR gene product] Deficiency
In the cell, cobalamin is converted to AdoCbl in the mitochondrion and to MeCbl in the cytoplasm. Synthesis of MeCbl occurs following binding of cobalamin to the target enzyme, methionine synthase (Watkins D, Rosenblatt DS. Cobalamin and inborn errors of cobalamin absorption and metabolism. The Endocrinologist. 2001;11:98–104). Patients in the cblG and cblE complementation groups have hyperhomocysteinemia and homocystinuria but low levels of methionine and no methylmalonic aciduria. Clinically, it is not possible to differentiate these diseases and classification is usually done by means of somatic cell complementation analysis. Megaloblastic anemia is common, but often neurological and even psychiatric symptoms are more prominent features. These include developmental delay, cerebral atrophy, alterations in muscle tone, ataxia, seizures and EEG abnormalities, nystagmus, and blindness. Although presentation is typically in the first years of life, some patients have only presented as adults, including a 21-year-old woman who had an initial diagnosis of multiple sclerosis^ref. It is therefore important for the adult as well as the pediatric hematologist to be aware of these diseases when faced with a patient with megaloblastic anemia and neurological findings. Treatment of cblG and cblE is with systemic hydroxycobalamin, and usually results in a rapid hematological response, although the neurological changes may not be reversible. The genes responsible for both cblG and cblE have been cloned. The MTR gene, which encodes methionine synthase, is localized on chromosome 1q4313–15 and causal mutations were identified in cblG patients, including a common P1173 mutation^ref. The MTRR gene on chromosome 5p15.2–15.3 encodes a member of the flavodoxin NADP⁺ reductase family and causal mutations have been found in cblE patients^{ref1, ref2}. Before the description of the cblE patients, the existence of this function was not known in mammalian cells.
Methylenetetrahydrofolate reductase deficiency : MTHFR catalyzes the reduction of methylenetetrahydrofolate, the single carbon donor for thymidine synthesis, to methyltetrahydrofolate, a methyl donor for the remethylation of homocysteine to methionine^ref (Rosenblatt D, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Scriver CR, Beaudet AL, Sly WS, et al. The metabolic & molecular bases of inherited disease. 8th ed. New York: McGraw-Hill; 2001:3897–3933). Because the reaction catalyzed by MTHFR is not reversible, folates can return to the pool of functional reduced folates, which are needed for both purine and pyrimidine metabolism, only through the cobalamin-dependent methionine synthase reaction. Severe MTHFR deficiency is the most common inborn error of folate metabolism with more than 85 published cases^ref (Rosenblatt D, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Scriver CR, Beaudet AL, Sly WS, et al. The metabolic & molecular bases of inherited disease. 8th ed. New York: McGraw-Hill; 2001:3897–3933). The major laboratory findings are hyperhomocysteinemia and hypomethioninemia. Because there is no deficiency of methylenetetrahydrofolate, the substrate for MTHFR and thymidylate synthase, patients with severe MTHFR deficiency do not have megaloblastic anemia. This helps differentiate them from patients with cblE and cblG. Clinical presentation can occur at any age from the neonatal period to adulthood, although most cases have been described in infants. The predominant findings are neurological, with apnea, developmental delay and seizures being common, and are probably related to the low levels of methionine in the brain. Another common feature is thrombosis, probably mediated through the elevated levels of homocysteine. Some adults have been completely without symptoms and only identified in a family after diagnosis of the proband. The gene on chromosome 1p36.3 has been cloned^ref and to date 33 mutations have been found in 32 patients^ref. Mutations causing severe deficiency are different from the polymorphisms described below. Treatment is very difficult in severe MTHFR deficiency, with betaine being the most useful therapy.
MTHFR polymorphisms : in early studies of patients with severe MTHFR deficiency, we showed in confluent cultured fibroblasts, that the residual specific enzyme activity in some patients was thermolabile^ref. In subsequent studies in the general population, Kang et al reported individuals with a thermolabile MTHFR and postulated an increased incidence of thermolabile MTHFR among patients with heart disease^ref. After the MTHFR gene was cloned^ref, the cause of the thermolabile enzyme in the general population^ref, as well as in the severely affected patients with a thermolabile enzyme^ref, was shown to be a common polymorphism, 677CT, that results in the change of an alanine to a valine. This polymorphism in the catalytic domain of MTHFR results in a 50% to 60% decrease in the specific activity of MTHFR when it is present in the homozygous state. The gene frequency for 677CT varies among ethnic groups with the T allele having a frequency of around 30% in Europeans and Japanese but only a frequency of around 11% in African Americans^ref. A second polymorphism, 1298AC, changes a glutamate to an alanine in the C-terminal regulatory domain of MTHFR and is associated with an approximately 35% decrease in MTHFR activity, but not with thermolability^{ref1, ref2}. The C allele frequency is about 18% among Asian populations, and about 30% in Western Europe^ref. It is now known that some of the effects of the 677CT polymorphism on folate and homocysteine levels can be altered by folate intake^ref, and that studies on the effect of this polymorphism can be confounded by the fortification of cereal grains with folic acid^ref. After the discovery that thermolability of MTHFR in the general population was associated with the 677CT alteration, it was relatively easy to perform large studies of this common polymorphism using small amounts of DNA. The types of diseases that have been studied, and for which a correlation with the polymorphism has been attempted, include neural tube defects, Alzheimer’s disease, colon cancer, leukemia, cardiovascular disease, diabetes mellitus, Down syndrome, and complications of pregnancy^{ref1, ref2, ref3, ref4, ref5, ref6}. There is good evidence that hyperhomocysteinemia is an independent risk factor for vascular disease^ref, but there is less evidence that lowering homocysteine levels reduces the risk. The results of prospective trials of vitamin therapy should clarify this issue in the next few years. Early studies showing an effect of 677CT on heart disease were difficult to interpret because the polymorphism was not always associated with elevated homocysteine levels. Recent studies have clarified that the polymorphism is only associated with elevated homocysteine levels in patients with low folate intake^{ref1, ref2}. It is thus possible that the effect of the polymorphism depends on the nutritional status of the individual^ref. There are reports that 677CT may be protective for colon cancer^ref and leukemia^{ref1, ref2}. Early studies suggest that the T allele may be protective for lymphocytic leukemia, but not for myeloid leukemia^ref. Studies are beginning to look at the 1298AC polymorphism in MTHFR^ref as well as at polymorphisms in a number of other genes involved in folate and cobalamin metabolism such as TC II, 776CG, MTR, 2756AG, and MTRR, 66AG^{ref1, ref2, ref3}. This list is only a fraction of the number of polymorphisms affecting genes in this pathway. It is difficult to predict the usefulness of the increasing number of these polymorphisms in clinical practice. Because each may interact with others, it will require large population studies to sort out both the genetic and environmental interactions. It seems reasonable to have data on these polymorphisms in the clinical record as part of the investigation of, for example, recurrent neural tube defects or of hyperhomocysteinemia. At this point, the presence or absence of a particular polymorphism in a gene of the folate or cobalamin pathway is not a complete explanation of an individual phenotype for one of the common multifactorial diseases such as cancer or heart disease.
• impaired Hb synthesis : increase in [soluble CD71 / TfR]_blood (which relates to [TfR]), decrease in [reticulocytes]_blood, microcytic and hypochromic
• iron (Fe²⁺) deficiency anemia (IDA) / sideropenic anemia (uneffective erythropoiesis) (anisopoikilocytosis).

See iron metabolism.
Aetiology :
• inadequate dietary iron intake
• starvation
• changes in patterns of food consumption in the USA from 1980 to 1992 show a marked decrease in consumption of food that has fair to moderate iron content (fish, seafood, pork, beef, lamb and other meats) and a marked increase in consumption of iron-poor food (potato chips, snacks, nuts, rice, pasta, oatmeal, cereals)
• vegetarianism
Daily dietary iron intake in the USA, mean values for selected groups :

age and sex	estimated Fe intake/day [mg]	RDA [mg]	% of RDA
infant (6-11 months)	11.9	6	200
child (1-2 years)	8.4	10	84
female (14-30 years)	10.5	15	70
female, pregnant	14	30	47
female, lactating	14	15	93
female, 60-65 years	10.2	10	102
male, 12 and older	> 12	12	> 100

Iron content of infant food :
• liquid food [mg/l]
• breast milk : 1.1
• evaporated milk : 1.8
• evaporated milk, diluted 13:19 : 0.7
• whole cows' milk : 0.7
• Similac, "low iron" : 1.3
• Similac, "with iron" : 11.5
• Enfamil, "low iron" : 0.45
• Enfamil, "with iron" : 11.5
• semisolid food [mg/serving]
• various cereals : 6.75
• vegetables purees : 0.0-0.60
• meat and vegetables mixes purees : 0.30-1.20
• decreased absorption :
• post-gastrectomysyndrome
• atrophic gastritis
• celiac disease
• chelating agents
• congenital atransferrinemia
• infections / infestations
• Ancylostoma duodenale
• Necator americanus
• Schistosoma spp.
• Trichuris trichiura
• Ascaris lumbricoides
• Giardia lamblia
• divalent metals transporter 1 (DMT-1) / NRAMP2 / DCT-1 mutations (poorly responsive to oral iron treatment, with liver iron overload associated paradoxically with normal to moderately elevated serum ferritin levels)^ref
• GTG deletion in exon 5, leading to the V114 in-frame deletion in transmembrane domain 2
• G=>T substitution in exon 8 leading to the G212V replacement in transmembrane domain 5
• increased loss : chronic hemorrhage
• respiratory tract :
• lung carcinoma
• epistaxis
• idiopathic pulmonary hemosiderosis
• infections
• teleangiectases
• alimentary tract :
• esophageal varices
• stomach :
• angiodysplasia
• antral vascular ectasia
• gastric carcinoma
• gastritis
• hemangioma
• hiatal hernia
• hypergastrinemia
• leiomyoma (Menetrier disease)
• mucosal hypertrophy
• gastric ulcer
• gastric varices
• biliary tract :
• aberrant pancreas
• biliary carcinoma
• cholelithiasis
• intrahepatic bleeding
• ruptured aneurysm
• trauma
• small bowel :
• aberrant pancreas
• intestinal angiodysplasia
• small bowel carcinoma
• helminthiasis
• hemangioma
• intussusception
• leiomyoma
• Meckel diverticulum
• polyp
• regional enteritis
• telangiectasia
• ulcer
• vascular occlusion
• volvolus
• colon :
• amebiasis
• angiodysplasia
• carcinoma
• diverticulum
• hemangioma
• polyp
• teleangiectasia
• ulcerative colitis
• rectum :
• angiodysplasia
• rectal carcinoma
• hemorrhoids
• ulceration
• genitourinary tract :
• kidney : hematuria
• renal carcinoma
• Goodpasture disease
• inflammatory disease
• uterus
• menstruations (RDA : 1.5 mg / die)
• uterine leiomyoma
• uterine carcinoma
• hemoglobinuria
• march hemoglobinuria (macrocytosis, echinocytes)
• paroxysmal cold hemoglobinuria
• paroxysmal nocturnal hemoglobinuria
• valvular hemolysis
• phlebotomy
• blood donation
• nosocomial
• self-induced (l'asthenia de Ferjol)
• therapeutic (e.g. in polycythemia vera)
• pregnancy (RDA : 2.4 mg / die = 700 mg lost/pregnancy)
Symptoms & signs : asymptomatic => malaise, gastrointestinal affections, irritability (Fe is cofactor for MAOs), glossitis, esophagitis and dysphagy (Plummer-Vinson syndrome), koilonychia, angular cheilitis, chlorosis, accumulation of protoporphyrin IX => fluorescence.
Laboratory examinations : test characteristics based on optimal cutoff values determined by receiver operator curve (ROC) analysis

	sensitivity (%)	specificity (%)	PPV (%)	NPV (%)	ROC area (± 95% confidence interval was calculated by using the Wilcoxon statistic according to Hanley and McNeil)
CHr (< 28.0 pg)	60.7	76.0	58.6	77.6	0.642 ± 0.15
CHr*	73.9	73.3	58.6	84.6	0.735 ± 0.14
Ferritin (< 50 mg/L)	42.3	93.6	78.6	74.6	0.660 ± 0.14
Ferritin*	52.4	92.9	78.6	79.6	0.690 ± 0.15
Tf sat (< 13%)	62.5	73.8	57.7	77.5	0.660 ± 0.15
Tf sat*	65.0	70.3	54.2	78.8	0.637 ± 0.16
MCV (< 81 fL)	25.9	94.0	70.0	70.1	0.505 ± 0.15
MCV*	31.8	93.3	70.0	73.7	0.570 ± 0.15
*Values determined after excluding patients with MCV > 100 fL.

• screening measurements that detect iron deficient erythropoiesis (IDE) :
• hemoglobin
• [hyposideremia (< 75 mg/dL or < 13 mmol/L for males; < 65 mg/dL or < 11 mmol/L for males)]
• (apo)transferrin saturation has the advantages of low cost and wide availability. However, marked diurnal variation and the numerous clinical disorders that affect the transferrin saturation limit its clinical utility. A normal or elevated value is as useful for excluding iron deficiency anemia as a low value is for diagnosing it. 50% deficiency in Fe storages cause saturation lowering at 15%, but symptoms appear only when Fe²⁺ storages are reduced to 30% (as exceptions are so common it is not reliable for differential diagnosis with anemia of chronic disease)
• TIBC > 410 mg_Fe / dL_serum or > 73.4 µmol / L_serum => [apotransferrin]_plasma> 360 mg / dL_serum.
• mean corpuscular volume (MCV) is one of the last parameters to change with the onset of IDE. > 30% of cases of IDA will be misdiagnosed if physicians rely on MCV (expecially when < 70 fl^ref) and MCHC only
• percentage hypochromic erythrocytes measured by selected hematology analyzers is earlier than MCV, but it is anyway a relatively late indicator of IDE^ref
• reticulocyte hemoglobin content (CHr) (the product of the hemoglobin concentration and the cell volume) is a more stable parameter than reticulocyte hemoglobin concentration and declines within a few days of the onset of IDE because of the short 1–2 day lifespan of circulating reticulocytes^{ref1, ref2, ref3}. Falsely normal values of the CHr can be observed with an elevated MCV or thalassemia^ref but the main limitation to its wider use is that it can be measured by only one model of analyzer (Bayer Advia). The low specificity of the percent hypochromic erythrocytes and the CHr is reflected in a recent study reporting that 62% of predominantly anemic patients had abnormal values on admission to hospital^ref However, when patients with MCV > 100 fL are excluded, receiver operator curve analysis of CHr, ferritin, transferrin saturation, and MCV demonstrates that CHr has the highest overall sensitivity and specificity of these peripheral blood tests for predicting the absence of bone marrow iron stores^ref. The CHr is an early indicator of response to iron therapy, increasing within 2 to 4 days of the initiation of intravenous iron therapy^ref. Thus, in patients receiving iron, the CHr may normalize before bone marrow iron stores return, resulting in false-negative test values. Thalassemia is a recognized cause of low CHr^ref
• zinc protoporphyrin (ZPP), a product of abnormal heme synthesis, is a simple and precise measure of IDE but still not widely used for clinical purposes^ref. One important advantage is the ability to measure the ratio of ZPP/heme directly on a drop of whole blood using a dedicated portable instrument called a hematofluorimeter. The ZPP is ideally suited for field surveys of iron status or pediatric and obstetrical clinics where uncomplicated iron deficiency is relatively common. The ZPP is redundant in laboratories equipped for percentage hypochromic red cell measurements because the two tests provide similar information. ZPP increases with lead toxicity and falsely elevated values and can occur in patients with elevated serum bilirubin or on regular hemodialysis, although the interference can be eliminated with prior washing of the red cells^ref.
• increased erythrocyte zinc protoporphyrin (does not differentiate among chronic lead poisoning and anemia of chronic disease)
• definitive measurements that evaluate tissue iron status :
• storage iron :
• serum (apo)ferritin < 30 µg/L (or ng/ml) (10-20 ng/ml in iron deficiency without anemia) is a universally available and well-standardized measurement that has been the single most important laboratory measure of iron status during the past quarter century. Phlebotomy studies in normal subjects have demonstrated that 1 µg/L serum ferritin corresponds to 8–10 mg or 120 µg storage iron/kg body weight^ref, although a log transformation gives a more accurate estimate^ref. Numerous studies have demonstrated its superiority over other iron-related measurements in identifying iron deficiency anemia. In 55 studies culled from 1179 relevant citations, receiver-operator characteristic curves in 2579 subjects gave a mean area for the serum ferritin of 0.95 ± 0.1 (95% confidence limits) as compared with 0.77 for the ZPP, 0.76 for the MCV, and 0.74 for the transferrin saturation^ref. The well-known limitation of the serum ferritin is the elevation in values independent of iron status that occur with acute or chronic inflammation, malignancy, liver disease, and alcoholism. In Gaucher disease the serum ferritin concentration is commonly in the range of thousands mg/l. When one of these conditions coexists with iron deficiency, as they often do, the serum ferritin concentration is commonly in the normal range; interpretation of results of this assay then become difficult. In patients with rheumatoid arthritis who are anemic, concomitant iron deficiency may be suspected when the serum ferritin concentration is < 60 mg/l. Moderate increase in serum ferritin concentration occur in patients with hepatitis and ESRD. Oral or parenteral iron administration also increases serum ferritin concentration. This appears to be particularly a problem in infants given oral iron. In adults with iron deficiency who were given oral iron in a dose of 60 mg of elemental iron thrice daily, the serum ferritin concentration remained < 10 mg/ for 2-3 weeks. However, for adults who have taken oral iron medication for > 3 weeks, the serum ferritin assay would be of no value in confirming a diagnosis of iron deficiency. Parenteral admnistration of iron dextran results in a rise in serum ferritin concentration to normal or supranormal values within 24 hours, and this effect persists for > 1 month. It has been proposed that measurement of the %saturation of serum ferritin may be a better indicator of iron deficiency or overload than the serum ferritin alone. As yet, there has been limited experience with this test.
• absence of stainable hemosiderin in the bone marrow aspirate is the gold standard : [Fe²⁺] => [Fe³⁺]_{bone marrow} with Perls staining : many hematologists still rely on the assessment of stainable iron on aspirated marrow smears or biopsy for the definitive diagnosis of iron deficiency anemia. Although still widely regarded as the gold standard for the diagnosis of iron deficiency, the reliability of the marrow iron stain is often suboptimal when used for routine clinical purposes. In a recent study, 108 consecutive bone marrow specimens from unselected hematology patients reported to have absent iron were reviewed^ref. 33% of the reports were incorrect due either to an inadequate specimen or detectable iron stores, and < 50% of the patients with absent marrow iron had clinical evidence that supported the diagnosis of iron deficiency anemia. In another recent review of iron stains, high intra-observer variability in pathological diagnosis led the authors to conclude that the bone marrow is not a perfect gold standard^ref. It should also be noted that the bone marrow is no longer reliable in diagnosing iron deficiency anemia after parenteral iron therapy. While marrow iron staining continues to play a critical role in validating newer laboratory measurements of iron status when performed and reviewed under standardized conditions in prospective studies by experienced investigators, bone marrow examinations should seldom be performed solely to diagnose iron deficiency because of the expense, discomfort, and technical pitfalls with this approach.
• tissue iron :
• serum soluble CD71 / TfR (sTfR): TfRs are membrane glycoproteins that serve as the gateway for circulating transferrin iron to the interior of all body cells. In addition to erythroid precursors that contain 80% of the total body receptor mass, rapidly dividing cells and the placenta contain a high density of receptors. The synthesis of ferritin and transferrin receptors is precisely regulated by a common mechanism involving the iron response protein and a nucleotide sequence termed the iron response element. The sTfR, first discovered in 1986 by Kohgo and coworkers^ref, is a truncated monome of the cellular receptor lacking the first 100 amino acids and composed of the extracellular domain, which circulates in the form of a complex of transferrin and its receptor. The erythroblasts rather than reticulocytes are the main source of serum sTfR. Serum sTfR levels average 5.0+/-1.0 mg/l in normal subjects but the various commercial assays give disparate values because of the lack of an international standard^ref. The most important determinant of sTfR levels appears to be marrow erythropoietic activity (total mass of erythroid precursors) which can cause variations up to 8 times below and up to 20 times above average normal values^ref. Soluble TfR levels are decreased in situations characterized by diminished erythropoietic activity, and are increased when erythropoiesis is stimulated by hemolysis or ineffective erythropoiesis. Measurements of sTfR are very helpful to investigate the pathophysiology of anemia, quantitatively evaluating the absolute rate of erythropoiesis and the adequacy of marrow proliferative capacity for any given degree of anemia, and to monitor the erythropoietic response to various forms of therapy, in particular allowing to predict response early when changes in hemoglobin are not yet apparent. The only determinant of the sTfR other than the erythroid precursor mass is tissue iron deficiency, which increases the sTfR in proportion to the severity of iron deficit^ref, but remain normal in the anemia of inflammation, and thus may be of considerable help in the differential diagnosis of microcytic anemia. This is particularly useful to identify concomitant iron deficiency in a patient with inflammation because ferritin values are then generally normal. Elevated sTfR levels are also the characteristic feature of functional iron deficiency, a situation defined by tissue iron deficiency despite adequate iron stores. The sTfR/ferritin ratio can thus describe iron availability over a wide range of iron stores. With the exception of chronic lymphocytic leukemia (CLL) and high-grade non-Hodgkin's lymphoma and possibly hepatocellular carcinoma, sTfR levels are not increased in patients with malignancies. Results may be confounded by poorly understood variations in patients with malignancies, in whom the sTfR concentration is reduced, and in patients with rheumatoid arthritis or thalassemia trait, in whom, in the absence of iron deficiency, it is increased. Thus, to be clinically useful, separate reference ("normal") ranges need to be defined for sTfR in these common conditions.
• additional tests :
• therapeutic trial iron is a reasonable approach in otherwise healthy populations, such as teenage girls and pregnant women, at high risk of deficiency. However, in most clinical settings, it is preferable to make a definitive diagnosis at the outset
• reduced ⁵⁷Co absorption : the urinary excretion of ⁵⁷Co, following an oral dose, is greater in iron-deficient subjects than in normal subjects. This appears to be a sensitive index of increased iron absorption, as occurs in iron-deficiency, in iron-loading states (e.g. hemochromatosis), or folllowing recent blood loss. It is not specific for iron deficiency
• iron tolerance tests : the patient receives an oral dose or inorganic iron compound (ferrous sulphate 325 mg or 1 capsule), and after 3 hours the change in the serum iron concentration is measured. In iron deficiency, there is an increased rate of absorption of the test dose, and this is often reflected in a more rapid increase and a higher plateau than in normal subjects. However, this test has quite limited practical application.
• FOBT
• high serum HbA_1c despite normal glycemia (7.4 vs. 5.9%). In patients with IDA, HbA_1c decreased significantly after iron treatment from a mean of 7.4% to 6.2%. Iron deficiency must be corrected before any diagnostic or therapeutic decision is made based on HbA_1c^{ref1, ref2}
• reduced erythrocyte ferritin concentration (increased in thalassemia and sideroblastic anemia). These changes appear to parallel those of serum ferritin concentration. Although it has been suggested that basic red cell ferritin is not influenced by inflammation, and could therefore detect iron deficiency when the serum ferritin concentration was normal in the elderly, comparative studies show that erythrocyte ferritin and serum ferritin concentration are both elevated in chronic disease. While erythrocyte ferritin determinations appear to have no more value than those of serum ferritin, the combination of both was more effective in the diagnosis of iron deficiency
Isolated iron deficiency : isolated or uncomplicated iron deficiency in the absence of other diseases that influence measurements of iron status is seen most often with rapid growth or during gestation, and in patients with excessive uterine or gastrointestinal blood loss. The key laboratory measurement for its identification is the serum ferritin. A low hemoglobin concentration in a patient with a serum ferritin < 30 µg/L is diagnostic of iron deficiency anemia. One drawback of relying solely on a low hemoglobin and serum ferritin is that milder iron deficiency without anemia goes undetected. Individuals with baseline hemoglobin values in the upper normal range must lose 20–30% of their body iron before iron deficiency can be detected by anemia. A method for measuring body iron quantitatively using the log(sTfR/serum ferritin) ratio that permits detection of mild tissue iron deficiency has been described recently^ref. The method estimates in mg/kg the surplus of storage iron in replete individuals or the tissue deficit in those with iron deficiency. Serial measurements in an individual remain constant over several months allowing those at risk of recurrent iron deficiency to be monitored prospectively. The relevance of iron deficiency without anemia that can be assessed with this approach has been unclear largely because of the difficulty in identifying it. Greater improvement in exercise performance was observed in iron-depleted nonanemic females given iron supplements as compared with unsupplemented controls despite no effect on hemoglobin values^ref. Mild iron deficiency without anemia could explain chronic fatigue in some individuals^ref (Beutler E, Larsh SE, Gurney JM. Iron therapy in chronically fatigued, nonanemic women: a double-blind study. Ann Intern Med. 1960;52:378–394). It is also preferable to detect declining tissue iron levels in individuals with recurrent iron deficiency before overt anemia develops.
Iron deficiency and chronic disease : the diagnosis of iron deficiency would be simple if it were not for the many clinical disorders that influence the internal iron cycle. The anemia of chronic disease is a common hematological disorder that is easier to recognize than it is to define. Because it alters screening tests for iron status in the same manner as true iron deficiency, the distinction between the anemia of chronic disease (ACD) and IDA requires tissue-related iron measurements. To avoid the need for bone marrow examinations in patients in whom IDA is suspected, reliance is often placed on the serum ferritin concentration despite the well-known elevation with acute or chronic inflammation. The optimal cut-off values of the serum ferritin to distinguish iron deficiency anemia from the anemia of chronic disease was examined in a landmark study in 259 anemic patients over 65 years of age^ref. In 36% of patients with iron deficiency anemia based on bone marrow examination, the serum ferritin was the only test of several that added useful diagnostic information. Only 2 of 49 patients with serum ferritin < 18 µg/L did not have IDA and only 8 of 116 with a serum ferritin > 100 µg/L had iron deficiency anemia. Between 18 and 100 µg/L, 40% had iron deficiency anemia although only 1 such patient had a serum ferritin > 45 µg/L. The authors later proposed serum ferritin values < 40 and < 70 µg/L to diagnose iron deficiency anemia in anemic patients without and with inflammation respectively^ref. A serum ferritin < 50 µg/L has been proposed as the best cutoff to identify iron deficiency anemia in patients with liver disease^ref. Because the sTfR concentration remains normal in patients with the ACD^ref, it is an invaluable addition to the serum ferritin measurement. The sTfR cannot only distinguish IDA from the ACD but it can also identify IDA when it occurs in patients with the ACD. In 129 consecutive anemic patients receiving a bone marrow for stainable iron, IDA was identified in 48, the ACD in 64 and both disorders in 17^ref. The sTfR was normal in all patients with the ACD, elevated in 41 of 48 patients with IDA and in 13 of 17 patients with both the ACD and iron deficiency. The separation between the 3 groups was further improved using the sTfR/log(serum ferritin) ratio; no patients with IDA overlapped those with ACD and all but 1 patient with both the ACD and IDA had higher values than patients with the ACD only. A recent study has indicated that even better discrimination can be obtained with the log(sTfR/serum ferritin)^ref as described above for quantifying body iron. Use of the receptor/ferritin ratio can eliminate the need for bone marrow examination to detect iron deficiency in patients with chronic inflammatory joint or bowel disease who are usually reluctant to undergo this unpleasant procedure.
Prevention : iron fortification of white flour, white bread, rolls, and buns in the USA is made with metallic ("reduced) iron (20 mg/lb = 44.1 ppm = 0.00441% w/v), which has poor absorbability, while no amount of ferrous sulfate (which has fair absorbability) is added
Therapy : iron replacement therapy
• oral iron therapy (preferable) :
• dietary therapy : the patient should be encouraged to eat a diversified diet supplying all nutritional requirements
• 1 325 mg tablet = 105 mg iron taken daily without food is as effective as 2-3 tablets with meals, and the disparity is even greater in patients with atrophic gastritis, chronic suppression of gastric acid secretion, and gastric stapling or bypass surgery. The major obstacle to successful oral therapy is the nausea and epigastric discomfort that occurs 30–60 minutes after taking iron, symptoms that are dose-related but often subside after 2–3 days with continued treatment. Reducing the dose or taking a tablet at bedtime is usually helpful in reducing side effects. Diarrhea or constipation are not dose-related and should be managed symptomatically. Commercial iron preparations promoted on the basis of fewer side effects are invariably less well absorbed. A brief follow-up clinic visit 2–3 weeks after initiating oral therapy can be helpful in tailoring an oral iron regimen in patients having troublesome side effects. A cycle should last 6 months (1 cycle per month for 10 days a month if polymenorrhea)
• parenteral iron therapy : the main indications for parenteral iron are uncontrolled blood loss (polymenorrhea), intolerance to oral iron (< 100 mg/day, flu-like syndrome at high doses), anaphylaxis, intestinal malabsorption (gastroresection), and poor adherence to an oral regimen. It causes an increase in reticulocyte count after 3-4 days (peak at day 10). Iron malabsorption can be detected by observing an increase in serum iron < 100 µg% over baseline in a fasting patient 1 or 2 hours after taking 60 mg iron as ferrous sulfate. Until recently, iron dextran has been the only parenteral iron preparation available in the USA. It is a low molecular weight dextran complexed with ferric oxide and supplied as a dark brown solution containing 50 mg iron per mL. Although intramuscular administration is still recommended by current manufacturers, intravenous (IV) administration is preferred by most physicians because of the ability to administer larger doses. In hemodialysis patients in whom multiple treatments can be given conveniently, 6–10 injections of 100 mg iron can be administered on consecutive dialysis days^ref. In other patients, a major advantage of iron dextran is the ability to administer relatively large doses of 500–2000 mg iron on a single occasion^{ref1, ref2}. After diluting the dose in 500 mL normal saline, and premedicating with diphenylhydramine with or without steroids, the first 20–30 mL is given slowly over 5 minutes as a test dose. If no allergic reaction occurs within the first 15–30 minutes, the remainder of the dose is given over the next 3–4 hours. The major drawback of iron dextran is a severe anaphylactic reaction that occurs within a few minutes of initiating the infusion and is sometimes fatal. At least 30 deaths have been attributed to iron dextran use in the US since 1976^ref and because anaphylaxis can occur in those who have not reacted to iron dextran in the past, a test dose is always required. Another drawback of iron dextran is a less serious delayed reaction occurring 24–48 hours after the infusion. Characterized by myalgia, arthralgia, headache, and malaise, delayed reactions occured in over 10% of patients given total dose infusions^ref. These symptoms usually respond promptly to nonsteroidal anti-inflammatory drugs but the reaction can be severe and prolonged in patients with inflammatory joint disease.
• sodium ferric gluconate (SFG) : this form of parenteral iron was approved in the US in 1999 under the Food and Drug Administration’s priority drug review, although it has been used in Europe for many years. SFG in sucrose is a stable macromolecular complex supplied as a deep red solution in 5 mL ampoules containing 62.5 mg elemental iron. The current product label advises administration of 5–10 mL as a direct IV push at a rate of 12.5 mg per min for 10 minutes for a recommended dose of 125 mg iron. No test dose is required. Unlike iron dextran, which can require several weeks for complete processing by macrophages, roughly 80% of SFG is delivered to transferrin within 24 hours. One report suggested that saturation of circulating transferrin can be exceeded following the infusion of SFG^ref but this was later explained by the release iron from the ferric gluconate during the serum iron determination^ref. The appeal of SFG administration is avoidance of the risk of the serious anaphylactic reactions associated with iron dextran. In a review of data from the World Health Organization and pharmaceutical manufacturers, no deaths were reported with 25 million infusions of SFG as compared with 31 deaths from about half the number of administratons of iron dextran^ref. Adverse reactions with SFG in a multicenter, randomized, crossover, double-blind, placebo-controlled prospective study in 25^ref hemodialysis patients was recently reported^ref. Adverse events were similar between SFG and placebo groups, 12.3% versus 9.8%. Life-threatening events (immediate reactions requiring resuscitation measures) and drug intolerance rates (any event that precluded further SFG infusions) were compared to historical adverse events in 3768 patient exposures to iron dextran. A life-threatening event was observed in a single patient given SFG as compared with 23 patients given iron dextran while drug intolerance occurred in 11 patients receiving SFG as compared with 64 patients given iron dextran. These results are a compelling reason for using SFG rather iron dextran in hemodialysis patients and in most patients requiring parenteral iron. The feasibility of large single doses with iron dextran provides a useful alternative in noncompliant patients or those who are seriously inconvenienced by multiple iron infusions. Comparison of iron dextran and sodium ferric gluconate (SFG) :

	iron dextran	SFG
maximum dose (mg iron)	500–1000	125
test dose required	yes	no
administration time	2–4 hours	10 min
utilization time	weeks	days
anaphylactic reactions	uncommon (0.61%)	very rare (0.04%)
delayed reactions	common (2.5%)	uncommon (0.4%)

• iron sucrose (saccharate) was approved for use in the US in late 2000, providing a third option for intravenous iron therapy. Iron sucrose has also been used for several decades outside of the US although recently published experience is more limited than with SFG. Iron sucrose is supplied as an aqueous brown solution in 5 mL vials containing 100 mg of elemental iron. No test dose is required. It is recommended that 5 ml iron sucrose (100 mg iron) either be infused directly or first diluted in 100 mL normal saline and infused over 15 min to reduce the risk of hypotensive episodes. The safety profiles of iron sucrose and SFG are similar although most studies have evaluated efficacy rather than adverse reactions.
Causes of unresponsiveness to therapy :
• wrong diagnosis
• poor compliance
• malabsorption
• medullary suppression
• losses > supplementation
• iron-refractory iron deficienc anemia (IRIDA) : mutations in matriptase 2 (Tmprss6)
• impaired heme synthesis = sideroblastic anemias (uneffective erythropoiesis)

Aetiology :
• hereditary sideroblastic anemias :
• d-ALA synthetase 2 deficiency (mitochondrial heredity)
• porphyria : any of a group of disturbances of porphyrin metabolism.

Classification :
• the porphyrias are usually classified as either hepatic or erythropoietic based on the principal site of expression of the enzymatic defect.
• erythropoietic porphyria : porphyria in which excessive formation of porphyrin or its precursors occurs in bone marrow erythroblasts. It occurs in both humans and cattle.
• congenital erythropoietic porphyria (CEP) / congenital photosensitive porphyria / Günther disease / erythropoietic uroporphyria (most common form)

Aetiology : an autosomal recessive deficiency of uroporphyrinogen III synthetase in which increased synthesis of uroporphyrinogen I relative to uroporphyrinogen III occurs in bone marrow erythroblasts
Symptoms & signs : cutaneous photosensitivity, leading to mutilating skin lesions. Erythrodontiaand hypertrichosis are invariably present; eye involvement include proptosis, tense eyelid, nonreactive pupil, cataract, and a lack of light perception in his left eye^ref
Laboratory examinations : hemolytic anemia and splenomegaly, and by greatly increased urinary excretion of uroporphyrin I and coproporphyrin I
Therapy : ascorbic acid and beta carotene, prompt treatment of all skin infections
Prognosis : without treatment, the worst forms can be grotesque, ultimately exacting the kind of hideous disfigurement one might expect of the undead. The victims' ears and nose get eaten away. Their lips and gums erode to reveal red, fanglike teeth. Their skin acquires a patchwork of scars, dense pigmentation and deathly pale hues, reflecting underlying anemia. Because anemia can be treated with blood transfusions, some historians speculate that in the dark ages people with porphyria might have tried drinking blood as a folk remedy, underlying truth in vampire stories. Whatever the truth of this claim, those with congenital erythropoietic porphyria would certainly have learned not to venture outside during the day. They might have learned to avoid garlic, too, for some chemicals in garlic are thought to exacerbate the symptoms of the disease porphyria, turning a mild attack into an agonizing reaction.
• erythrohepatic or erythropoietic porphyria (EPP) / hepatoerythropoietic porphyria (HEP) / protoporphyria (less common).  It is classified as an erythropoietic or erythrohepatic form of porphyria depending on whether the latter form is used in a given system

Aetiology : autosomal dominant disorder seen in both humans and cattle; there is partial deficiency of ferrochelatase
Symptoms & signs : photosensitive skin changes vary widely, ranging from a burning or pruritic sensation to erythema, plaquelike edema, and wheals.
Laboratory examinations : increased levels of protoporphyrin in the erythrocytes, plasma, liver, and feces
• erythropoietic coproporphyria : an extremely rare erythropoietic porphyria characterized by mild skin photosensitivity and elevated erythrocyte coproporphyrin III levels
• hepatic porphyria : porphyria in which the excess formation of porphyrin or its precursors is found in the liver; it includes :
• acute, inducible
• ALA dehydratase deficiency (ADP)
• Swedish or acute intermittent porphyria (AIP) / pyrroloporphyria

Aetiology : autosomal dominant defect in hydroxymethylbilane synthase / porphobilinogen deaminase^D
Symptoms & signs : recurrent attacks of abdominal pain, gastrointestinal dysfunction, psychic and emotional (porphyrismus) and neurologic disturbances
Laboratory examinations : excessive amounts of d-aminolevulinic acid and porphobilinogen in the urine
• hereditary coproporphyria (HCP)
• harderoporphyria : a severe variant of hereditary coproporphyria differing in having earlier onset of attacks, excretion of harderoporphyrin in the feces, and virtual absence of coproporphyrinogen oxidase activity
Aetiology : autosomal dominant trait, half-normal activity of coproporphyrinogen oxidase (CPO), a mitochondrial enzyme catalyzing the antepenultimate step in heme biosynthesis; deletion of residues (392-418) encoded by exon six disrupts dimerization. Conversely, harderoporphyria-causing K404E mutation precludes a type I b-turn from retaining the substrate for the second decarboxylation cycle^ref.
Symptoms & signs : recurrent attacks of gastroenterologic and neurologic dysfunction similar to those of acute intermittent porphyria, and sometimes by cutaneous photosensitivity
Laboratory examinations : coproporphyrin III is excreted constantly in the feces and intermittently, with d-aminolevulinic acid and porphobilinogen, in the urine
• porphyria variegata / South African genetic, mixed or variegate porphyria (VP) / protocoproporphyria / porphyria cutanea tarda hereditaria

Aetiology : autosomal dominant deficiency of protoporphyrinogen oxidase
Symptoms & signs : various combinations of chronic cutaneous photosensitivity with lesions and extreme skin fragility, attacks of abdominal pain, neuropathy, and gastrointestinal dysfunction
Laboratory examinations : fecal protoporphyrin and coproporphyrin are elevated continually and urinary coproporphyrin, d-aminolevulinic acid, and porphobilinogen are elevated during acute attacks
• chronic
• porphyria cutanea tarda (PCT) symptomatica / cutaneous hepatic porphyria : the most common form of porphyria

Aetiology : is debated, but 2 types, which may not be allelic, are generally recognized:
• an autosomal dominant (or familial) form in which enzyme activity is reduced to half normal in liver, erythrocytes, and fibroblasts
• a sporadic (but probably also familial) form in which the reduction is confined to the liver
Both types are believed to be heterozygous and clinical expression occurs in adulthood, precipitated by disease or environmental factors; a more severe homozygous form begins in childhood and is called hepatoerythropoietic porphyria (HEP) : a severe homozygous form of PCT believed to result from an autosomal dominant defect in uroporphyrinogen decarboxylase activity; it is clinically identical to porphyria cutanea tarda but onset is in early childhood and enzyme activity in liver, erythrocytes, and fibroblasts is virtually absent.
Symptoms & signs : cutaneous photosensitivity that causes scarring dermal-epidermal vesicles, hyperpigmentation, facial hypertrichosis, and sometimes sclerodermatous thickenings and alopecia; it is frequently associated with ethanol abuse, liver disease, or hepatic secondary hemochromatosis
Laboratory examinations : urinary levels of uroporphyrin and coproporphyrin are increased and activity of uroporphyrinogen decarboxylase is decreased
• erythrohepatic porphyria : porphyria in which abnormal overproduction of porphyrins and precursors occurs in both liver and bone marrow; when this category is used in classifying porphyrias, it usually includes only hepatoerythropoietic protoporphyria.
• classification according to major clinical manifestations
• abdominal pain (neurovisceral) :
• ADP
• AIP
• HCP
• VP
• cutaneous (photosensitivity, damage) :
• CEP
• EPP
• HCP
• HEP
• PCT
• VP
Each of the porphyrias is due to the deficiency of a specific enzyme involved in heme synthesis. The first and normally rate-controlling step is condensation of glycine and succinyl CoA to form 5-aminolevulinate (ALA), catalyzed by the mitochondrial enzyme ALA synthase. There are 2 forms of ALA synthase, the ubiquitous housekeeping form 1 and the erythroid-specific form 2. These 2 forms are products of separate genes and are under quite different regulation. ALA synthase-1 can be upregulated markedly by transcriptional and post-transcriptional mechanisms, and an "uncontrolled" upregulation of this enzyme in the liver is believed to be the biochemical sine qua non of acute porphyric attacks. Upregulation of ALA synthase-1 occurs due to 3 main known causes:
• lipophilic drugs and chemicals, that interact with nuclear receptors and that, in turn, increase activation of drug-response elements (RES) in the upstream enhancer of ALA synthase-1^ref
• hypoglycemia or deficiency of other gluconeogenic compounds that suppress gene expression of ALA synthase-1 (the so-called "glucose effect"^ref).
• deficiency of heme, which not only represses transcription of the ALA synthase-1 gene^ref, but also blocks translocation of the enzyme into mitochondria and decreases the stability of its mRNA^{ref1, ref2}
• downregulation of ALA synthase-1 by avoidance or removal of inducing drugs and chemicals by nutritional means (high carbohydrate intakes) and by administration of exogenous heme remains the cornerstone of management of the acute porphyrias.
There is no porphyria associated with a defect in ALA synthase-1, but mutations of the X-linked ALA synthase-2 (the erythroid form) are causative for X-linked sideroblastic anemia. Indeed, one would expect the opposite, namely inadequate porphyrin synthesis, if this enzyme were markedly deficient in activity. It is highly unlikely that such defects would be compatible with life. The last 3 enzymes of the pathway, namely, coproporphyrinogen III oxidase, protoporphyrinogen oxidase, and ferrochelatase, are also located within mitochondria, whereas the 2nd–5th enzymes are found in the cytoplasm or soluble fraction of the cell.

disease (abbreviation)	inheritance	deficient enzymes (synonyms; sequence in pathway)	subcellular locations	enzyme activity % of normal	number of known mutations according to Human Gene Mutation Database as of 14 Oct 2004	gene locus	OMIM
AIP	autosomal dominant	PBG deaminase, formerly known as uroporphyrinogen I synthase (HMB synthase; third)	cytosolic	~ 50	227	11q23.3	176000
HCP	autosomal dominant	coproporphyrinogen oxidase (sixth)	mitochondrial	~ 50	36	3q12	+121300
VP	autosomal dominant	protoporphyrinogen oxidase (seventh)	mitochondrial	~ 50	120	1q22	#176200
ADP	autosomal recessive	ALA dehydratase (porphobilinogen syntase; second)	cytosolic	~ 5	7	9q34	+125270

Acute intermittent porphyria is the most prevalent and 5-aminolevulinic acid-dehydratase deficient porphyria is the least prevalent of these diseases in the USA
Pathogenesis of acute attacks : the enzyme deficiencies are partial (usually about 50% of normal in 3 of the acute porphyrias and usually < 5% of normal in the fourth [ADP]), and the residual enzymatic activity is usually sufficient to maintain adequate hepatic heme synthesis. Activity of ALA dehydratase normally greatly exceeds that of the other enzymes of the heme biosynthetic pathway in the liver. Therefore, a much more severe deficiency in the activity of this enzyme (< 5% of normal) is usually needed to cause manifestations of ALA dehydratase deficiency porphyria. Such enzymatic defects are envisioned to predispose affected persons to the deleterious influences of factors that may trigger acute attacks, including drugs, such as barbiturates, hydantoins, rifampin, progestins, endogenous steroid hormones (especially progesterone), prolonged severe fasting or dieting, alcohol, and other intercurrent illnesses or stress. All of these can either increase the demand for hepatic heme or otherwise reduce the regulatory pool for heme and thus induce or upregulate synthesis of ALA synthase. Because hepatic ALA synthase-1 is normally rate-controlling, when production of heme pathway intermediates increases, the inherited partial enzymatic deficiency more distal in the pathway becomes rate limiting, and proximal intermediates accumulate. For reasons that are complex and not fully understood, intermediates distal to the inherited enzyme deficiency or their products may also accumulate.

The regulatory heme pool acts to stimulate (+) or down-regulate (–) the indicated steps. Those steps indicated as not within the nucleus or mitochondrion take place in the cytosol. The dashed line indicates a still controversial regulatory effect of heme to decrease transcription of the gene of ALA synthase 1. Although the precise pathogenic mechanisms underlying the neurologic damage in the acute porphyrias remain imperfectly understood, it seems likely that symptoms result primarily from accumulation of the porphyrin precursors, rather than deficiency of heme in nerve or muscle tissue^{ref1, ref2}. Symptomatic acute porphyria rarely becomes clinically manifest prior to puberty (highlighting the importance of endogenous steroid hormones, especially progesterone and less so estrogens), and the disease is more often clinically manifested in women, often with cyclical attacks linked to menstrual cycles. Very rare instances of homozygous acute porphyria with severe neurologic manifestations beginning in childhood have been described^{ref1, ref2, ref3}. Perhaps the most persuasive evidence that overproduction of hepatic ALA and/or porphobilinogen (PBG) are central is the clinical observation of their unique association with diseases that present with neurovisceral attacks. Further evidence was the rapid and dramatic improvement in chronic debilitating neurovisceral attacks that occurred following liver transplantation in a young woman with AIP^ref. A patient with a diagnosis of variegate porphyria, who underwent liver transplantation for alcoholic cirrhosis, similarly experienced biochemical improvement after liver transplantation^ref. In contrast, liver transplantation was not beneficial clinically or biochemically in a child with severe ALA dehydratase deficiency, suggesting that ALA overproduction did not arise chiefly from the patient’s native liver^ref
Symptoms & signs : the major clinical features consist of attacks of neurovisceral symptoms and cutaneous manifestations. In some forms, liver damage may also occur and an increased risk of hepatocellular carcinoma (HCC). This risk is increased in porphyria cutanea tarda, hepatoerythropoietic porphyria, and in all of the acute hepatic porphyrias, as well as in erythropoietic protoporphyria. The most common presenting symptom of acute porphyria is abdominal pain. This is usually colicky in nature and in the lower abdomen. It lasts hours to days. The most common sign in acute porphyric attacks is tachycardia. Among neurologic manifestations, evidence of autonomic neuropathywith a sympathomimetic state characterized by tachycardia and systemic arterial hypertension is most common. Severe constipationis also very common. Peripheral neuropathy, which is predominantly a motor neuropathy, initially affecting mainly the proximal rather than distal muscles, is next in order of frequency. Sensory loss over the trunk is also common, although it is often overlooked. Neuropsychiatric manifestations of anxiety, depression, insomnia, disorientation, hallucinations, and paranoia occur somewhat less commonly as do cranial nerve defects, seizures, or coma, and cerebellar, optic nerve, basal ganglion or pyramidal tract involvement. Another common manifestation is hyponatremia, which is usually due to vomiting and replacement of fluid lost with hypotonic solutions such as intravenous glucose without electrolytes, and to excessive secretion of ADH. Although it is often said that this represents SIADH, when plasma and blood volumes of patients with acute porphyria were measured, they were found to be decreased^ref. In the setting of plasma volume depletion, secretion of ADH can not truly be said to be inappropriate. Sudden death, probably due to cardiac arrhythmia, may occur during acute attacks^ref. Death can also be a consequence of progressive paralysis, including paralysis of respiratory muscles and bulbar paralysis with complicating disorders such as pneumonias and aspiration.

type of porphyria	neurovisceral	clinical manifestationref
		cutaneous	liver damage
ADP	yes	no	no
AIP	yes	no	no
HCP	yes	yes (bullae, fragility)	no
VP	yes	yes (bullae, fragility)	no
PCT	no	yes (bullae, fragility)	yes
HEP	+/–	yes (bullae, fragility)	yes
CEP	no	yes (bullae, fragility)	occasional
EPP	EPP with end-stage liver disease, especially just after liver transplantation, may rarely be associated with neurovisceral manifestations	yes (urticaria, erythema)	yes (10%)

Common symptoms & signs^ref :
• gastrointestinal :
• abdominal pain (85–95%) : usually unremitting (for hours or longer) and poorly localized but can be cramping. Neurologic in origin and rarely accompanied by peritoneal signs, fever, or leukocytosis.
• vomiting (43–88%) : nausea and vomiting often accompany abdominal pain.
• constipation (48–84%) : may be accompanied by bladder paresis.
• diarrhea (5–12%)
• neurologic :
• pain in extremities, back, chest, neck, or head (50–70%) : pain may begin in the chest or back and move to the abdomen. Extremity pain indicates involvement of sensory nerves, with objective sensory loss reported in 10%–40% of cases
• paresis (42–68%) : may occur early or late during a severe attack. Muscle weakness usually begins proximally rather than distally and more often in the upper than lower extremities.
• respiratory paralysis (9–20%) : preceded by progressive peripheral motor neuropathy and paresis.
• mental symptoms (40–58%) : may range from minor behavioral changes to agitation, confusion, hallucinations, and depression.
• convulsions (10–20%) : a central neurologic manifestation of porphyria or due to hyponatremia, which often results from syndrome of inappropriate antidiuretic hormone secretion or sodium depletion.
• cardiovascular :
• tachycardia (64–85%) : may warrant treatment ot control rate, if symptomatic
• systemic arterial hypertension (36–55%) : may require treatment during acute attacks, and sometimes becomes chronic.
Laboratory examinations : acute porphyria should be considered early on when patients present to the emergency room or other urgent care settings with compatible symptoms and signs. Too often, acute porphyria is considered only after expensive, time-consuming, unproductive searches for other causes of abdominal complaints have been carried out, sometimes including ill-advised and unnecessary surgery. Careful personal history may reveal recurrent episodes of colicky, usually lower, abdominal pain lasting for hours to days with associated obstipation. ER records of tachycardia and systemic arterial hypertension provide helpful clues. Attacks that occur cyclically in menstruating women, with onset during the bruited phase of the menstrual cycle, are suggestive, although more likely due to endometriosis than acute porphyrias. Severe stress, starvation, "crash" dieting, alcohol excess, or intercurrent infections may trigger acute porphyric attacks. A positive family history of porphyria, if established at a reputable center, is important, although usually not elicited. For several of the diseases, many patients are biochemically silent ("latent") carriers of the enzymatic defects for most of their lives.

		urine	stool	plasma	RBC
ALA dehydratase	ADP	ALA	-	ALA	ZnPROTO
PBG deaminase	AIP	ALA, PBG	-	ALA, PBG	-
uroporphyrinogen III sythase (cosynthase)	CEP	URO, COPRO	COPRO	URO	URO, COPRO
uroporphyrinogen III decarboxylase	PCT	URO	ISOCOPRO	URO	-
	HEP				ZnPROTO
coproporphyrinogen III oxidase	HCP	ALA, PBG, COPRO	COPRO (PROTO)	COPRO	-
protoporphyrinogen oxidase	VP	ALA, PBG, COPRO	PROTO (COPRO)	PROTO (COPRO)	-
ferrochelatase	(E)PP	-	PROTO	PROTO	PROTO

The key to establishing or excluding the diagnosis is rapid and simple testing for increased porphobilinogen (PBG) in the urine. This should be done in a single freshly passed urine to which no preservatives are added. Classically, the qualitative test for PBG in the urine has been the Watson-Schwartz test, although the Hoesch test is also useful and may be less prone to misinterpretation^ref. The commercially available Trace^® PBG kit (Trace American/Trace Diagnostics, Louisville, Colorado) is useful and includes a color chart for semi-quantitative estimation of levels of urinary PBG. These levels are generally markedly increased (20–200 mg or 220–880 µmol/day with a typical reference range of 0–4 mg or 0–18 µmol/day). In all cases of acute porphyric syndromes except for the very rare porphyria due to ALA dehydratase deficiency or in patients with symptoms due to lead poisoning or hereditary tyrosinemia type I, markedly elevated urinary porphobilinogen is expected and readily detectable. If the porphobilinogen level is increased, second line testing including erythrocytic PBG deaminase levels, urinary, fecal, and plasma porphyrin levels will establish the precise disorder of porphyrin metabolism. The most useful tests are the erythrocytic PBG deaminase level for AIP, the urine and fecal porphyrin levels for HCP, and the plasma porphyrin level and fluorescence pattern for VP. Worthy of emphasis is that about 10% of patients with AIP have normal erythrocytic PBG deaminase levels. There is a single gene encoding PBG deaminase, but erythroid cells utilize different promoters and transcriptional sites than hepatocytes and other cells. As a result, the erythrocyte enzyme lacks the amino terminal residues encoded by exon 1. Therefore, in the 10% or so of patients with AIP who have mutations in exon 1, erythrocytic PBG deaminase activities are entirely normal. Another limitation is that there is a large range of variation in activity of the enzyme, and there is an overlap between normals and carriers of the deficiency. Laboratory findings that differentiate AIP, HCP, and VP (the findings listed are considered diagnostic for AIP when porphobilinogen level is increased and for HCP and VP even when porphobilinogen levels may have returned to normal) :

disease	erythrocyte porphobilinogen deaminase levels	urine porphyrin levels	fecal porphyrin levels	plasma porphyrin levels
AIP	decreased by ~ 50% (in ~ 90% of cases)	increased, mostly uroporphyrin	normal or slightly increased	normal or slightly increased
HCP	normal	increased, mostly coproporphyrin	increased, mostly coproporphyrin (mostly coproporphyrin III)	usually normal
VP	normal	increased, mostly coproporphyrin	increased, mostly coproporphyrin (mostly coproporphyrin III) and protoporphyrin	increased, characteristic fluorescence peak (a simple test, which consists of fluorescence scanning of diluted plasma at neutral pH, readily differentiates variegate porphyria from other porphyrias that cause elevated plasma porphyrin levels and cutaneous photosensitivity. A plasma porphyrin level determination is the most sensitive porphyrin measurement for detecting variegate porphyria, including asymptomatic cases)

Diagnosis of ALA dehydratase deficiency, in which PBG is normal or only slightly increased, requires measurement of urinary ALA, which can be done on the same urine sample. Associated findings include elevations in urinary coproporphyrin and erythrocyte Zn protoporphyrin and markedly deficient erythrocyte ALA dehydratase activity. Other causes of this enzyme deficiency must be excluded.
After the above first- and second-line biochemical studies have established the presence and type of acute porphyria, DNA studies can identify the disease-causing mutation or mutations in the defective gene. Demonstration of a mutation in each ALA dehydratase allele is important for confirming the diagnosis of porphyria due to ALA dehydratase deficiency. If a mutation has been found in a proband, rapid and accurate testing of asymptomatic-at-risk family members can be carried out. DNA-based studies are rather widely available in European counties at central, government supported laboratories. Mutational analysis for patients and family members with acute porphyria in the USA is currently available through the Department of Human Genetics, Mt. Sinai School of Medicine, New York, NY (contact Dr. Kenneth Astrin).
Therapy :
• removal of inciting factors, such as one of the many drugs, alcohol, toxins or chemicals that can trigger or exacerbate acute porphyrias, is essential
• nutritional supplementation, including > 300 g of glucose given daily, is important because glucose suppresses activity of ALA synthase-1^ref. Due to nausea, vomiting, disordered bowel motility, etc., such supplemental glucose usually must be administered intravenously^ref
• because of the danger of severe hyponatremia, sodium should be administered and levels of serum sodium should be monitored frequently
• frequent checks of neurological status, especia and hypomagnesemially looking for evidence of the development of impairment of swallowing, coughing, and respiration. Prompt transfer to an intensive care unit for close monitoring and provision of respiratory support and other intensive nursing care may be life saving. Monitor for hypoventilation (rising arterial PpCO₂) or hypomagnesemia and treat vigorously if found
• tachycardia and/or systemic arterial hypertension can be treated cautiously with b-adrenergic blocking agents such as propranolol (40–240 mg/day) or nadolol (60-120 mg/day). Caution must be exercised when using b blockers because hypotension and serious bradycardia have been reported even after low doses^ref
• analgesia in the form of parenteral administration of morphine (3–12 mg/dose) or meperidine (50–200 mg/dose) is recommended, because these agents have been found to be safe and effective. Such analgesics may depress respiratory drive, such that monitoring of respiratory adequacy (e.g., arterial blood gases) is important.
• addition of a phenothiazine (e.g., chlorpromazine, 10–15 mg/dose) enhances the analgesic and sedative effects of narcotics and helps to treat the agitation and anxiety that often accompany acute porphyric attacks, and are also effective for nausea and vomiting
• if seizures supervene, the treatments of choice are benzodiazepines, gabapentin, and vigabatrin
• i.v. hematin 3–4 mg/kg/day for 3–5 days given as quickly as it can be obtained, is the treatment of choice since the early 1970s for any patient who is ill enough to require hospital admission for AIP. In the USA, hemin in the form of Panhematin^® (Ovation Pharmaceuticals, Deerfield, IL) is the only preparation available since 1983. Although the product labeling recommends an initial trial of i.v. glucose, heme should be given without delay. Lyophilized hematin, reconstituted with sterile water, rapidly degrades and it is hypothesized that the degradation products lead to morbidities such as thrombophlebitis, thrombocytopenia, and transient anticoagulation. Reconstitution with human serum albumin produces a well-tolerated hematin preparation and improves its stability significantly^ref. The clearance of i.v. hematin infusions are shown to fit a two-compartment model consisting of a rapid initial rate followed by a slower and prolonged second phase. This model is supported by the evidence demonstrating that hematin is first bound by hemopexin and, upon saturation, second by albumin. The highest i.v. human hematin dose reported in the literature was 12.2 mg/kg (1000 mg) and resulted in acute gastrointestinal pain, paresthesia, and acute tubercular necrosis. The patient's renal function returned to normal over the following 15 hours^ref. Heme arginate is available commercially from Leiras Oy of Turku, Finland and Orphan Europe (UK) Ltd under the tradename Normosang^®, which is approved in many other countries, is stable in concentrated solution and somewhat less irritating to veins, although it, too, is commonly given with human serum albumin.
Prevention and follow-up : all patients with acute porphyria should carry Medic Alert bracelets and wallet cards, as well as a list of safe and unsafe drugs, so that these are readily available to emergency response personnel in the event of accident or incapacity. For women with frequent cyclical attacks, use of gonadotropin-releasing hormone (GNRH) analogsis frequently highly effective. Other women have benefited from the use of low-dose estrogen and progesterone such as in low-dose birth control pills or estrogen patches. Estrogen patches can also be used to help prevent menopausal symptoms in women receiving GNRH analogs. Although pregnancy produces increased levels of progesterone, most women with acute porphyria tolerate pregnancy remarkably well. Some patients, most of them women with cyclical attacks, seem to require and benefit from regular prophylactic infusions of hemin. These may be given only once a month, shortly before the usual onset of symptoms, or they may be administered weekly or biweekly, especially if the attacks are frequent and mostly unrelated to the cycle. The frequency and doses used are empiric and dependent chiefly upon the symptoms of the patient. Because of the increased risk of development of hepatocellular carcinoma (HCC) in the hepatic porphyrias, it is becoming more common to do surveillance screening for HCC in affected persons. This is usually done with semiannual serum alphafetoprotein and liver ultrasound (perhaps, alternating with 4-phase, helical, dynamic CT scan). The cost-effectiveness of such surveillance is not established, but the practice has gained in popularity nonetheless.
Laboratory examinations : marked increase in formation and excretion of porphyrins or their precursors
• acquired sideroblastic anemias :
• myelodysplastic syndromes (MDS)
• lead
• zinc
• drugs (isoniazid, ...)
• pyridoxine (vitamin B₆) deficiency(normal activity of erythrocyte aspartate transaminase indicates adequate pyridoxine status
• long-term ethanol ingestion
Laboratory examinations :
• ferritinemia = 30-200 ng/ml
• Perls' staining shows sideroblast : a normoblast that contains granules of iron in the form of ferritin in its cytoplasm and stains with Prussian blue.
• ringed sideroblasts : an abnormal sideroblast containing many Fe³⁺ granules in its mitochondria in a ring shape around the periphery of the nucleus


• myelodysplastic syndromes (MDS)(uneffective erythropoiesis) (normocytic and normochromic)
• thalassemias
• peripheral hemolysis / erythrocytolysis / erythrolysis / hematolysis : disruption of the integrity of the erythrocyte membrane causing release of hemoglobin; it may be caused by bacterial hemolysins, by antibodies that cause complement-dependent lysis, by placing erythrocytes in a hypotonic solution, or by defects in the cell membrane => acute or chronic hemolytic anemias / Hayem-Widal syndrome: increase in [LDH₁ and LDH₂]_serum, [soluble CD71 / TfR]_plasma and [reticulocytes]_blood => macrocytosis(except for microcytosis in PNH). Decrease in Hb glycosylation due to decreased time for reactions. Anemia occurs only if RBC t_1/2 < 15 days (showable with ⁵¹Cr release test).

Aetiology :
• intrinsic aetiology
• Rh deficiency syndrome^ref : chronic hemolytic anemia in persons who have either absent (Rh_null) or markedly reduced (Rh_mod) Rh antigen expression. There is mild to moderate hemolytic anemia; it is marked by spherocytosis, stomatocytosis, and increased osmotic fragility. Mutations in the Rh^ref and RhAG genes have been associated with this syndrome.
• hemoglobinopathies / hemoglobin disease : any of the heredity conditions caused by the presence of abnormal hemoglobins in the blood
• fast hemoglobins : those with greater mobility on electrophoresis (in an alkaline buffer) than normal hemoglobin A, such as hemoglobin K, J, or N.
• slow hemoglobins : those less mobile on electrophoresis (in an alkaline buffer) than normal hemoglobin A, such as :
• hemoglobin S : the most common abnormal hemoglobin, having valine substituted for glutamic acid at position 6 of the b chain. The delineation of the abnormality in molecular structure was a milestone in biochemical genetics, paving the way for other investigations demonstrating how substitutions of a single amino acid could produce significant clinical effects. The asymptomatic heterozygous state is called sickle cell trait, and the homozygous state results in sickle cell disease (SCD) : any of the diseases associated with the presence of hemoglobin S and sickle cells
• drepanocytosis / sickle cell anemia (SCA) : HbS (point mutation in b-globin : 6 Glu=>Val), when in T status and under hypoxic conditions, may polymerize forming a tactoid that (ir)reversibly damages cell membrane => microthrombui and extravascular hemolysis. It may be coupled to a- or b-thalassemia, HbC, HbD or HbE.
• sickle cell–hemoglobin C disease / hemoglobin SC disease : a genetically determined anemia in which the erythrocytes contain both hemoglobin S and hemoglobin C; symptoms are similar to but less severe than those of sickle cell anemia and may include abdominal and skeletal pain, splenomegaly, splenic infarction, and infarctions or deformities of bone
• sickle cell–hemoglobin D disease / hemoglobin SD disease : a genetically determined anemia in which the erythrocytes contain both hemoglobin S and hemoglobin D, with symptoms like those of mild sickle cell anemia
• sickle cell–thalassemia disease / microdrepanocytosis / microdrepanocytic disease / hemoglobin S–thalassemia / sickle cell–thalassemia / thalassemia–sickle cell disease : any of several hereditary anemias involving simultaneous heterozygosity for hemoglobin S and a thalassemia gene; symptoms resemble those of sickle cell anemia
Pathogenesis : erythrostasis (the stoppage of erythrocytes in the capillaries) => although in SCD two thirds of hemolysis occurs extravascularly, the remaining one third of red cells hemolyze intravascularly, potentially releasing as much as 10 g hemoglobin per day into blood plasma^ref. KCl cotransport (KCC) activity contributes to pathological dehydration in sickle (SS) RBC. KCC activation by urea was measured in SS and normal (AA) RBC as Cl-dependent Rb influx. KCC-mediated volume reduction was assessed by measuring reticulocyte cellular hemoglobin concentration (CHC) cytometrically. Urea activated KCC fluxes in fresh RBC to levels seen in swollen cells, although SS RBC required lower urea concentrations than normal (AA) RBC. Little additional KCC stimulation by urea occurred in swollen AA or SS RBC. The pH dependence of KCC in "euvolemic" SS RBC treated with urea was similar to that in swollen cells. Urea triggered volume reduction in SS and AA reticulocytes, establishing a higher CHC. Volume reduction was Cl-dependent, and limited by the KCC inhibitor, di-indenyl-oxyalkanoic acid. Final CHC depended on urea concentration, but not on initial CHC. Under all activation conditions, volume reduction was exaggerated in SS reticulocytes and produced higher CHC than in AA reticulocytes. The sulfhydryl reducing agent, dithiothreitol, normalized the sensitivity of KCC activation to urea in SS RBC, and mitigated the urea-stimulated volume decrease in SS reticulocytes, suggesting that the dysfunctional activity of KCC in SS RBC was due in part to reversible sulfhydryl oxidation^ref.
Symptoms & signs :
• heterozygous : asymptomatic, spontaneous hematuria
• homozygous : chronic hemolysis since postnatal week 8-10 (when protective HbF drops) + ...
• bilirubinic calculosis
• acute chest syndrome (ACS) : often due to a bacterial infectionsor to infarction of lung tissue; characteristics include severe chest pain, dyspnea, tachypnea, fever, excessive leukocytosis, pulmonary edema, and sometimes petechiaeon the chest or conjunctivae as well as fat emboli.
• sickle cell crisis : a broad term used to describe several different acute conditions occurring with SCD, including
• aplastic crisis : the most common type of sickle cell crisis, a transient condition marked by sudden disappearance of erythroblasts from the bone marrow; it develops under various circumstances, including certain hemolytic states and infections.
• hemolytic crisis : a rare type of sickle cell crisis in which there is acute red cell destruction leading to jaundice
• vaso-occlusive crisis (VOC): a type of sickle cell crisis in which there is severe pain due to infarctions, which may be in the bones, joints, lungs, liver, spleen, kidney, eye, or CNS. This robust hemolytic rate increases even further during VOC. Elegant biochemical studies performed 35 years ago have demonstrated significant increases in serum LDH and plasma hemoglobin levels, the gold standard marker of intravascular hemolysis, during VOC^{ref1, ref2}. This hyper-hemolysis recently has been confirmed by labeled RBC studies that reveal further decreases in red blood cell survival during VOC in patients with sickle cell disease^ref. A state of nitric oxide (NO) resistance develops due to consumption of NO by plasma cell-free hemoglobin^{ref1, ref2}. Arginase depletes plasma arginine, the substrate for NO production by NO synthase, further compromising NO bioavailability^{ref1, ref2, ref3}. NO also appears to be consumed by reactive oxygen species generated by the high levels of xanthine oxidase activity and NADPH oxidase activity that accompany sickle cell disease^{ref1, ref2, ref3, ref4}. This state of reduced NO bioavailability is associated with impaired blood flow physiology in patients and mice with sickle cell disease, and also in healthy dogs with induced intravascular hemolytic anemia^{ref1, ref2, ref3, ref4,ref5, ref6, ref7}
• silent cerebral infarcts (SCI) are the most common form of neurologic injury in children with SCA and are increasingly recognized as a major cause of school problems, lower intelligence quotient (IQ) and other neurocognitive deficits; thus, SCI represent a current and evolving concern for students with SCA, their family, teachers and practicing hematologists. SCI are defined as an abnormal MRI^ref of the brain with increased signal intensity in multiple T2wi and no history or physical findings of a focal neurologic deficit lasting more than 24 hours^ref. SCI occur in 22% of children with SCA by 14 years of age who have not been identified as having a clinically evident stroke (overt stroke)^ref; these children are characterized by an increase in risk for further neurologic progression, including overt strokes^ref, new or progressive MRI lesions^ref, poor academic attainment^ref, or lower IQ when compared to patients with SCA with normal MRI examinations^ref or siblings without SCA^ref. Despite evidence that SCI are prominent in SCA, associated with significant morbidity and a significant risk factor for progressive neurologic disease, the optimal evaluation and treatment remain vague.

Prevalence and risk factors for silent cerebral infarcts : in children with SCA, SCI are prominent in infants and toddlers younger than 4 years of age, and cumulative prevalence continues to increase until at least 14 years of age. In an unselected group of 39 children with SCI less than 4 years of age, Wang et al demonstrated an incidence of 11% SCI.6 In the Cooperative Study for Sickle Cell Disease (CSSCD), an infant cohort of 266 was identified and followed for approximately 14 years. Each patient had at least one study-mandated brain MRI at age 6 years or older, and patients with overt strokes were excluded in the determination of the prevalence of SCI. Among the group of children with SCA, 22% had SCI. This prevalence is consistent with that reported in similar studies in Europe^ref. Table 1 describes the prevalence of SCI in 3 different cohorts of children with SCA. Children with hemoglobin SC disease and S Beta + thalassemia are also associated with an increased incidence of SCI, 6% (7 of 120) and 15% (3 of 20), respectively. Prevalence of silent cerebral infarct at institutions that performed surveillance magnetic resonance imaging (MRI) examinations.

	number of silent cerebral infarcts	total number  of patients	prevalence	confidence  interval
Cooperative Study for Sickle Cell Disease (CSSCD)ref	58	266	21.8%	16.8-26.8
French Cohortref	23	155	15%	9.4–20.6
London Cohortref	16	64	25%	14.4–35.6
Cumulative numbers from CSSCD,  French Cohort and London Cohort	97	485	20%	16.4–23.6

In addition to a high incidence in SCA, SCI infarcts result in progressive neurologic morbidity, either new or progressive cerebral injury, compared to children without SCI. Using complete MRI histories from 266 children with SCA, participants with SCI had an increased incidence of new stroke (1.03/100 patient years) and new or more extensive SCI (7.07/100 patient years) relative to stroke incidence among all children without SCI (0.54/100 patient years).
Clinically useful risk factors that will allow clinicians to predict what group of children will have a SCI have not been established. The CSSCD, study included 42 patients with SCI and patients without SCI. Low pain event rate, history of seizure, leukocyte count > 11.8 x 10⁹/L, and the SEN globin gene haplotype were associated with an increased incidence of SCI7; however, the ability of these factors to predict a SCI is unreliable. Preliminary data suggest that genetic modifiers may influence the risk of SCI, but replication studies along with evidence for a mechanism for the association are needed^ref. Other than the presence of an elevated transcranial Doppler (TCD) measurement, the greatest risk factor for overt stroke is the presence of an SCI. Again in the CSSCD, 5 (8.1%) of 62 patients with SCI had strokes for an incidence of 1.45 per 100 patient-years compared with 1 (0.5%) of 186 patients without prior SCI, for an incidence of 0.11 per 100 patient-years (P = .006)^ref. In an earlier study by CSSCD investigators, transient ischemic attack (TIA) was identified as a risk factor for strokes; however, this analysis did not account for the presence of SCI. When the same data set was later evaluated for the presence of SCI, it was determined that all patients with strokes and a prior TIA also had SCI, and the only patient who had a stroke without prior SCI had systemic lupus erythematosus^ref. Taken together, these data reinforce the importance of SCI as a significant risk factor for an overt stroke.
Neurological imaging of silent cerebral infarcts :
• radiologic definition of SCI : SCI is diagnosed on the basis of an abnormal MRI of the brain and a normal neurological examination, without a history or physical findings associated with an overt stroke. An early definition of SCI was established in the CSSCD1 and by Glauser et al^ref. The only significant difference between the 2 definitions is the requirement of an evaluation by a pediatric neurologist in the study by Glauser et al as opposed to a clinical evaluation by a pediatric hematologist in the study by the CSSCD. The CSSCD investigators defined SCI as an area of abnormally increased signal intensity on the intermediate and T2-weighted pulse sequences. The area of abnormal signal had to have an appearance consistent with infarction in addition to the abnormal signal. This definition of SCI was refined in practice to include a focal 3-mm or larger area of abnormally increased signal intensity on the T2-weighted image in more than one view.
• MRI mimics of silent cerebral infarcts : while the definition of the infarct-like lesion has been established, there are MRI signal abnormalities that mimic SCI but have distinct etiologies (ADEM, sequelae of periventricular leukomalacia, dilated perivascular spaces, and delayed myelination in the terminal zones).
• other MRI observations in sickle cell disease : the relationship between SCI and other patterns of brain injury that can be seen in children with SCA is not well defined. In part, this is because SCI can only be identified with surveillance MRI evaluations, which have only recently become routine in some clinical centers. A common finding is cerebral atrophy. This is a non-specific finding that serves as a marker for disease severity in the brain. Another common finding is Moya-Moya, a description that comes from the Japanese for "puff of smoke" because of the angiographic appearance of secondary extensive collateral formation.
• transcranial Doppler measurement as a predictor of silent cerebral infarct : a National Heart, Lung, and Blood Institute (NHLBI) sponsored Stroke Prevention Trial in Sickle Cell Anemia (STOP) demonstrated that blood transfusion therapy in patients with increased TCD velocities of the middle cerebral artery or terminal portion of the internal carotid (> 200 cm/sec) could prevent overt strokes^ref. Children were randomly assigned to an intervention regimen with transfusion therapy to maintain the Hgb S percentage below 30% or to standard care (observation)^ref. Of note, 7 of 10 patients in the observation arm who developed overt strokes had SCI at the start of the study, suggesting that the presence of both an elevated TCD velocity (> 200 cm/sec) and SCI may increase the risk of an overt stroke. Due to the design and size of the study, the investigators could not determine whether the presence of SCI alone or in combination with an elevated TCD measurement increased the risk of overt stroke. A post hoc analysis suggested that both the presence of SCI and elevated TCD measurement were important risk factors; however, the numbers were too small to determine whether the risk of overt stroke was different in patients with and without SCI. No prospective study has been designed to assess the utility of TCD measurement for identifying SCI. In the best study to date addressing the association between TCD measurement and SCI, Wang et al^ref compared the results of patients that participated in both the CSSCD natural history study and the STOP trial. A total of 78 children with SCD (mean age 11 years) were common to the natural history study and the STOP trial. Patients who experienced an overt stroke were excluded from the analysis. MRI findings were classified as normal or SCI. Results of TCD measurements were classified as normal, conditional or abnormal (> 200 cm/sec), based on the time-averaged maximum mean flow velocity in the proximal middle cerebral and distal internal carotid artery. Among 17 patients with SCI, only 1 had conditional and 4 had abnormal TCD velocities. In this small sample, no statistical association was found between TCD measurements and MRI findings. The study did not address whether the combination of a conditional TCD measurement (high normal > 170 cm/sec, < 200 cm/sec) and an SCI was a risk factor for future neurologic injury. Current National Institute of Health (NIH) recommendations are to identify children who are at greatest risk of developing overt strokes, not SCI^ref
• cognitive sequelae of SCIs : children with SCI have significant cognitive deficits that are intermediate in magnitude compared to children with overt strokes and those with normal MRI. Children with SCI have IQs in the low 80s, whereas children with overt strokes or normal MRI have IQs in the 70s and 90s, respectively^ref. Schatz et al demonstrated that 80% of children with SCA and SCI were found to have clinically significant impairments in at least one neurocognitive domain, when compared to 30% of the children with SCA and a normal MRI of the brain^ref. Other studies have shown that the majority of SCI related to SCA affect frontal brain regions^{ref1, ref2}. In addition, frontal lobe infarcts, whether overt or silent in children with SCA, have been associated with impairments in attention^ref (Craft S, Schatz J, Glauser T, Lee B, DeBaun M. The effects of bifrontal stroke during childhood on visual attention: Evidence from children with sickle cell disease. Developmental Neuropsychology. 1994;10:285–297) and various aspects of executive ability^ref, such as working memory^ref (Brandling-Bennett E, White DA, Armstrong M, Christ SE, DeBaun MR. Patterns of verbal long-term and working memory performance reveal deficits in strategic processing in children with frontal infarcts related to sickle cell disease. Develop Neuropsychol. 2003;24:1–12) and strategic long-term memory processing (Brandling-Bennett E, White DA, Armstrong M, Christ SE, DeBaun MR. Patterns of verbal long-term and working memory performance reveal deficits in strategic processing in children with frontal infarcts related to sickle cell disease. Develop Neuropsychol. 2003;24:1–12). These studies provide guidance for future research examining specific neurocognitive impairments in children with SCI. Given the high prevalence of SCI coupled with significant cognitive impairment, cognitive remediation of brain injury is becoming an important area of research. In the first study assessing whether cognitive remediation is even feasible in children with SCD, 6 children were identified with cerebral infarcts in the frontal lobes (either overt or silent strokes) and placed into a structured 6-week cognitive remediation program^ref. All of the children in the study were provided with general academic tutoring. Three of the children were provided with additional training using specific strategies to enhance working memory and long-term memory (silent rehearsal to facilitate working memory and semantic organization to facilitate long-term memory). In comparing memory performance pre- and post-intervention, the investigators found that the scores of children who received specific strategy training improved considerably, whereas the cognitive scores of children who received only academic tutoring remained relatively unchanged. Although the sample size in this study was quite small, these early findings optimistically suggest that, in addition to identifying cognitive impairments in SCI, interventional strategies may assist children in overcoming, circumventing, or compensating for their impairments.
• treatment for silent cerebral infarcts : blood transfusion therapy has been the mainstay of intervention for primary and secondary stroke prevention in children with SCD. Blood transfusion therapy to increase total hemoglobin while keeping the Hgb S percentage < 30% has several physiologic benefits. Increasing the hemoglobin concentration with hemoglobin A improves the blood’s oxygen carrying capacity, lowers the fraction of cells that contain hemoglobin S by dilution, and reduces the erythropoietic drive that produces sickle hemoglobin. These physiologic factors combine to be an effective strategy for reducing the onset of stroke in patients who have an elevated TCD12 and in preventing second stroke^ref. The relative risk reduction for the benefit of blood transfusion therapy for primary or secondary stroke prevention is approximately 85%, justifying the burdensome task of at least monthly blood transfusion therapy and deferoxamine administration 5 of 7 nights or more. No therapy has been established for the treatment of SCI; however, preliminary evidence to support the possible benefits of blood transfusion therapy for SCI was recently published. As part of a post hoc analysis from the STOP trial, Pegelow et al identified individuals with SCA with both abnormal TCD measurement and SCI^ref. Among this group of 47 patients, 2 groups were compared: those that were randomly allocated to receive (n = 18) or not receive (n = 29) blood transfusion therapy. None of the 18 patients receiving transfusion therapy who had a baseline lesion seen on MRI (SCI) developed new SCI lesions or overt strokes. In contrast, among the 29 individuals who had baseline SCI lesions who were not treated, 52% (15 of 29) of this group subsequently developed new or worse SCI lesions (n = 6) or suffered strokes (n = 9) over a 36-month period^ref. Thus, in patients with both an elevated TCD velocity measurement and baseline SCI, blood transfusion therapy showed a 100% relative risk reduction for the frequency of new overt strokes or subsequent SCI. This study strongly supports the use of blood transfusion therapy in children with both SCI and an elevated TCD measurement. However, there are no data to support the use of blood transfusion therapy in children with SCI and TCD measurements with time-averaged maximum mean flow velocity measurements < 200 cm/sec. Only a clinical trial can determine whether children with SCA and normal TCD measurements will benefit from blood transfusion therapy. Available evidence would suggest that blood transfusion therapy may prevent further neurologic injury in patients with SCI. However, we do not know if these patients will benefit from this treatment. SCI lesions are typically smaller than those present in overt stroke and may represent microvascular pathology, as opposed to overt strokes, which are generally associated with larger ischemic cerebral areas and stenosis or occlusion of the internal carotid and medium size cerebral vessels. Further, we do not know if the benefit of blood transfusion therapy will outweigh the risks and inconveniences of monthly transfusion. Given this background and the state of equipoise among pediatric hematologists as to whether blood transfusion therapy should be implemented, the Silent Cerebral Infarct Transfusion (SIT) Trial has been funded by the National Institutes of Health (NIH) and the National Institute of Neurologic Disease and Stroke. The primary hypothesis of this trial is that prophylactic blood transfusion therapy in children with SCI will result in at least an 86% reduction in the proportion of patients with clinically evident strokes or new or progressive SCI. Approximately 1800 children with SCA will be screened with an MRI of the brain to identify 200 children with SCI and TCD measurement less than 200 cm/sec. Among this group, 100 participants will be randomly allocated to either blood transfusion or observation for a total of 36 months. Secondary outcome measures will include cognitive assessment and a formal risk/benefit analysis. After the completion of the SIT trial, we should know whether blood transfusion therapy is effective and, if so, whether the benefits outweigh the adverse consequences.
• other treatments : no treatment for SCI has been established, although several should be considered in practice. Probably the most often recommended therapy for SCI is hydroxyurea; however, significant caution must be exercised in its use, because no formal trial has been initiated to demonstrate efficacy. Further, such a trial may not be feasible, given the cost, total number of patients that would be required for identifying individuals with SCI, and the eventual size of the trial (presumably more than the 1800 participants in the SIT trial). Thus, only anecdotal experience and indirect evidence for support will justify using hydroxyurea for treatment of SCI. Another therapeutic option is a stem cell transplant from a matched related donor. If successful, this strategy is curative; however, only few patients have HLA–matched full siblings, making this option less practical for most children with SCA. Future studies using alternative stem cell choices in unrelated donors may be a viable option for the majority of children with SCA.
In summary, SCI in SCA represents a prevalent and morbid condition, with high risk for progression with either new SCI or subsequent strokes, and is associated with lower IQ and other neurocognitive impairments. Optimal treatments for SCI have not been established. Past and ongoing clinical trials have contributed to our understanding of SCI, and promise to continue to do so. The role of transfusions for SCI is currently being explored in a large NIH-NINDS–funded clinical trial that should establish risk factors for SCI, the indications and risk/benefit ratio of blood therapy for this disorder. Summary of clinical features contrasting and comparing overt strokes and silent cerebral infarcts :

	overt strokes	silent cerebral infarcts
frequency prior to 14th birthday	9%ref	22%ref
average age of onset	7.7 yearsref	Prior to 6 years of ageref
Wechsler Intelligent Scale  for Children-revised, Full Scale  Intelligence Quotient	70.8ref	82.8ref
role of transcranial Doppler	Associated with an abnormal  TCD velocityref	Not necessarily associated with an abnormal TCD  velocityref
treatment	Blood transfusion therapy to keep hemoglobin S < 30%ref	No established treatment  Focus of silent cerebral infarct transfusion (SIT) Trial

The 9% and 22% risk of infarct and SCI are not mutually exclusive. The incidence of SCIs excludes patients who had an overt stroke; however, patients with an overt stroke may have had SCI.
• pulmonary arterial hypertension : doppler echocardiography screening reveals a tricuspid regurgitant jet velocity (TRV) of 2.5 m/s or greater in 33% of adults with sickle cell disease^ref. Pulmonary hypertension in these patients is associated with a 10-fold increased risk for early mortality. Elevated pulmonary artery pressures in patients with SCD have been associated with low hemoglobin concentration, high levels of serum LDH, elevated systolic systemic blood pressure, history of priapism, renal insufficiency, and markers of iron overload^ref
• malleolar ulcers (50%)
• hepatomegaly
• splenic infarctions => spleen fibrosis and hypotrophy (ecception to the rule hemolysis = splenomegalia !) => asplenism => infections
• hypostenuria, renal papillary necrosis and spontaneous hematuria
• priapism due to venous engorgement
Laboratory examinations :
• Itano-Pauling sickling test
• sickle cell / drepanocyte / meniscocyte (an erythrocyte shaped like a sickle or crescent owing to the presence of HbS)
Therapy :
• EPO
• red cell exchange
• administration of blood transfusionsevery 3 to 4 weeks to children with sickle cell anemia who are at high risk of stroke (abnormalities on transcranial Doppler ultrasonographic examination) reduces their rate of first-time stroke by 90%. Regular transfusions cannot be discontinued, according to results of the Optimizing Primary Stroke Prevention in Children with Sickle Cell Anemia (STOP II) trial. Discontinuation of transfusion for the prevention of stroke in children with sickle cell disease results in a high rate of reversion to abnormal blood-flow velocities on Doppler studies and stroke^ref.
• in order to prevent hypoxic crises during surgical operations, blood has to be diluted reducing HbS at 60%. Exposure to environmental or passive tobacco smokeincreased the risk of sickle cell crisis by 90%.
• gene replacement or gene reactivation therapies for SCD typically target the mutant b^S-globin subunits of hemoglobin-S (a₂b₂^S) for substitution by nonpathological b-like globins. The adverse properties of hemoglobin-S can be reversed by exchanging its normal b-globin subunits for z-globin, an endogenous, developmentally silenced, non-b-like globin^ref
• promoterless small-hairpin RNA (shRNA) encoded within the intron of a recombinant g-globin gene. Expression of both g-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage–specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34⁺ cells from patients with sickle cell anemia led to erythroid-specific expression of the g-globin transgene and concomitant reduction of endogenous S transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny^ref
• HbS polymerization : deoxygenation causes HbS (a₂ß₂^S) to polymerize—the proximate cause of sickle cell disease and a necessary but insufficient precursor of the disease phenotype. HbS and its polymer induce a panoply of cellular and tissue injuries. Neither the fetal hemoglobin (HbF) tetramer (a₂g₂) nor the a₂ß^Sg hybrid tetramer is incorporated into the HbS polymer, providing the rationale for treatments aimed at increasing HbF concentration. Several classes of drug, when titrated optimally, can increase levels of HbF in most patients with SCA. Only one is approved for treating SCA.
• hydrodyurea, the sole US Food and Drug Administration (FDA)–approved drug for treating sickle cell anemia, should be used in all adults where indications for this treatment are present^ref. Unfortunately, for complex reasons, only a fraction of patients who might benefit from treatment receive it. Hydroxyurea increases HbF in sickle cell anemia because its cytotoxicity causes erythroid regeneration and perhaps because its metabolism leads to NO–related increases in soluble guanylate cyclase (sGC) with an increase of cGMP that augments g-globin gene expression^ref. A multicenter trial of hydroxyurea in adults with sickle cell anemia, where the drug was given at sub-toxic doses, showed that hydroxyurea reduced the incidence of pain and acute chest syndrome by nearly half, with little risk seen during more than 9 years of observation. Cumulative mortality was reduced nearly 40%, and a favorable result was related to the ability of the drug to increase HbF and reduce painful episodes and acute chest syndrome^ref. No relationship between decrements in neutrophil counts and mortality was found. In infants, children and adolescents with sickle cell anemia, the HbF response to hydroxyurea is more robust than in adults. In a study of more than 100 children who received maximal drug doses, HbF increased to almost 20% and the treatment effects were sustained for 7 years without clinically important toxicity^{ref1, ref2}. HbF levels achieved during treatment were associated with baseline HbF level, hemoglobin level, reticulocyte count, and leukocyte count and with compliance to treatment. According to some experts, pushing the drug dose to near toxic levels is not necessary for a clinically beneficial result. Some have proposed using hydroxuyrea for secondary prevention of stroke in children^ref. Hydroxyurea may also conserve resting energy expenditure by curbing the hypermetabolic state observed in children with sickle cell disease^ref.  Hydroxyurea is potentially mutagenic and carcinogenic^ref. Cancer and leukemia have been reported in hydroxyurea-treated sickle cell disease patients, but whether the incidence is higher than in the general population is not known^ref. The relative risk of leukemia in hydroxyurea-treated SCA is much less than the observed risk in myeloproliferative disorders. To put this into perspective, the risk of death from the complications of adult sickle cell disease appears to be at least 10-times greater than the possible incidence of leukemia in hydroxyurea-treated sickle cell anemia patients. Questions remain about the clinical benefits of hydroxyurea in HbSC disease. The pathophysiology of HbSC differs from that of sickle cell anemia in some aspects so one might expect that the response to hydroxyurea treatment may also differ. While cell density was reduced, mean corpuscular volume increased and hemolysis apparently reduced, HbF increases were inconsistent^{ref1, ref2}. No randomized clinical trials have studied the effectiveness of hydroxyurea in HbSC disease so that a decision to treat patients with this genotype is a matter of clinical judgment.
• decitabine: a less-toxic analog of 5-azacytidine, 5-aza-2'-deoxycytidine (decitabine), may affect HbF levels by causing hypomethylation of the g-globin genes. In 8 symptomatic sickle cell anemia patients who failed to respond to hydroxyurea, decitibine treatment (0.2 mg/kg subcutaneously 1–3 times per week for 2 6-week cycles) led to an increase in HbF from 6.5% to 20.4%, with an increase in hemoglobin concentration from 7.6 to 9.6 g/dL and a fall in reticulocytes from 231 to 163 x 10⁹/L^ref. Clinical trials of this agent are planned in patients with sickle cell anemia and ß thalassemia.
• short chain fatty acids : by acting as inhibitors of histone deacetylase (HDAC)and causing histone hyperacetylation and changes in chromatin structure, short-chain fatty acids, their derivatives, and other compounds with HDAC activity can enhance -globin gene expression in erythroid cells of patients with sickle cell anemia and ß thalassemia. Very low concentrations of one HDAC inhibitor, an analog of trichostatin A, induced g-globin gene expression in an erythroleukemia cell line transfected with a reporter construct and in erythroid colonies of normal adults^ref. In the most advanced clinical trials of HDAC inhibitors, still only Phase II studies, arginine butyrate given by infusion once or twice a month was associated with a mean increase in HbF from 7% to 21% in 11 of 15 patients with sickle cell anemia. In some individuals, this level was maintained for 1–2 years^ref. No HDAC inhibitor of any class other than butyrate and phenylbutyrate has yet been used clinically.
• sickle erythrocyte damage and dehydration : a distinguishing feature of sickle cell disease is the presence of dense, dehydrated erythrocytes and abnormal reticulocytes. Since polymerization of HbS is uniquely dependent on the cellular concentration of HbS, the increased cellular hemoglobin concentration of dehydrated sickle erythrocytes markedly enhances the tendency for HbS polymerization and cell sickling. 4 pathways have been implicated in the dehydration of sickle erythrocytes, and modulation of these pathways, especially the Gardos channel and K⁺-Cl^– co-transport, is a potential means of treatment.
• inhibition of K⁺-Cl^– co-transport: oral magnesium supplementation inhibits erythrocyte K⁺-Cl^– co-transport in vivo. Following studies that showed a beneficial effect on the erythrocyte membrane of transgenic sickle mice, a 6-month clinical trial of oral Mg pidolate improved erythrocyte hydration and was associated with a reduction in the number of painful days^ref. Studies to evaluate the effects of long-term magnesium supplementation in adult and pediatric patients with sickle cell anemia are ongoing, and additional studies are planned in HbSC disease patients where activated K⁺-Cl^– co-transport and cell dehydration are likely to play a major pathophysiological role.
• inhibition of KCNN4 / putative erythrocyte intermediate conductance calcium-activated potassium Gardos channel: clotrimazole is an inhibitor of the human red cell Gardos channel but its use was associated with dysuria and reversible hepatocellular toxicity. ICA 17043, a clotrimazole derivative lacking the toxic imidazole residue, was a 10-fold more potent blocker of the Gardos channel than the native drug. In a recently completed Phase II trial of this agent in patients with sickle cell disease, cell density and hemolysis were decreased while hemoglobin concentration was increased. To see whether clinical benefit accrues from these erythrocyte and hematological changes will require a Phase III clinical trial
• inhibition of other channels : movement of K⁺ via the Gardos channel requires the parallel movement of Cl^– anions to maintain electroneutrality. High-affinity blockers of Cl^– conductance can reversibly block human erythrocyte chloride conductance in vitro without directly affecting the Gardos channel or the K⁺-Cl^– co-transport. In sickle mice treated with a Cl^– conductance inhibitor, packed cell volume (PCV) increased and mean corpuscular hemoglobin concentration (MCHC) decreased with an increase in cell K⁺. A selective loss of the most dense erythrocytes, with a shift from sickled to well-hydrated discoid erythrocytes, was seen. Clinical trials of this class of agent have not been done
• anti-adherence therapy : cellular damage enables adhesive interactions between sickle cells, endothelial cells and leukocytes. As endothelium is perturbed, vasoconstriction may be favored as nitric oxide (NO) production is impaired. Several agents directed at the endothelial receptors for sickle erythrocytes or leukocytes may interrupt cell-cell interactions. Anti-adherence therapy for sickle cell disease targets the abnormal interactions among erythrocytes, endothelial cells, leukocytes and platelets that are part of the pathophysiology of the disease process. Potential anti-adherence agents have been studied in acute painful events, where, through poorly understood mechanisms, they restore microvascular circulation and improve tissue ischemia. In Phase II studies of a non-ionic surfactant copolymer, poloxamer 188, this agent reduced the duration and increased the resolution of acute painful episodes, an effect especially notable in children less than 15 years old and in patients receiving hydroxyurea^ref. Whether poloxamer 188 exerts its effects by modifying interactions of sickle cells or other blood cells to endothelium is not known. Its clinical effectiveness as a single agent to treat or prevent vasoocclusive complications is, at best, modest, and currently no additional trials of this agent are ongoing.  Endothelium-dependent vasodilation is disturbed in sickle cell disease^ref. Superoxide generation induces chronic inflammation that may be inhibited by apolipoprotein (apo) A-1 mimetics. L-4F, an apoA-1 mimetic, inhibited superoxide production and improved vasodilation in sickle mice^ref. While a mechanism of action is not yet totally clear, this agent may preserve endothelial function and endothelial nitric oxide synthase (eNOS) activity. This type of drug may have widespread application in vascular diseases including sickle cell anemia^ref. Most agents that might disrupt the adhesive interactions and inflammation hypothesized to presage sickle vasoocclusion have not been studied clinically. Novel methods of modulating cellular interactions in sickle cell disease :
• sulfasalazine : NF-kB inhibitor; decreases endothelial activation^{ref1, ref2}
• a_Vß₃ antibodies block vWF and thrombospondin mediated adherence^ref
• anionic polysaccharides Inhibit thrombospondin-mediated adhesion to the endothelium^ref
• oxygenated perflubron-based fluorocarbon emulsion increase O₂ delivery and unsickle deformed cells^ref
• IgG inhibit WBC adherence and WBC-RBC interaction^ref
• heparin inhibits P-selectin–RBC interaction^ref
• statins inhibit tissue factor expression; induce NOS^ref
• nitric oxide: inflammation, reperfusion injury, oxidant radical production : neutrophils mediate inflammation and tissue damage. Neutrophil numbers are increased and they may be abnormally activated and adherent. An "oxidant" environment may also be present in the sickle erythrocyte and endothelium. Novel ways of countering oxidant-induced injury have been proposed. A potent vasodilator, NO is an important regulator of vascular tone and, because of its interaction with hemoglobin, blood vessels and blood cells, has been hypothesized to have several advantageous effects in sickle cell disease. Generated from L-arginine by NO synthases, NO activates sGC, to produce the second messenger, cGMP. NO inhibits the adhesive interactions among platelets, leukocytes and sickle erythrocytes and decreases VCAM-1 expression in endothelial cells. NO metabolites were decreased in severe sickle cell vasoocclusive crises. Plasma L-arginine and serum L-arginine levels were normal in children in the steady-state and fell during vasoocclusive crisis while NO levels were normal at presentation but decreased during hospitalization. 3 young patients with acute chest syndrome showed an increase in PaO₂ and a decrease in pulmonary artery pressure after inhaling NO. However, in each case, standard treatment for this complication was also given making the results difficult to interpret. While a biologically sound rationale exists for using NO in the acute chest syndrome and pulmonary hypertension of sickle cell disease, a controlled trial of this treatment has not been reported. It should be remembered that this approach could be deleterious as NO can be metabolized to damaging oxidants like nitrite (NO₂^–) and peroxynitrite (ONOO^–). In a clinical trial, inhaled NO was associated with a small reduction in pain score and opioid use in children with acute painful episodes^ref
Future goals of drug treatment : our growing knowledge of the human genome and its variation among populations and individuals raises the possibility of being able to know very early—even antenatally—the genetic susceptibility to developing one or another of the many subphenotypes of sickle cell disease and intervening to forestall anticipated complications. Understanding the genetic predispositions to a phenotype many also help the design of innovative treatments. Pharmacogenomics may further help tailoring a treatment to a patient. Preliminary work has associated several genetic polymorphisms in a diverse set of genes to selected complications of sickle cell disease :
• pharmacogenomics
• genotype patients to detect "high-risk" alleles for disease complications
• KL, VCAM1, HLA, LDLR, TNF, IL4R, ADRB2 and CVA
• NOS3 and ACS
• BMP6, ANXA2, EDN1, TGFBR and AVN
• PPH1, BMPR2, ACVRLK1, SLC6A4, PPARG and PH
• CYP2D, OPRM1 and Opioid Response
• develop interactive networks of "high-risk" alleles
• develop novel therapeutics based on "global" high-risk alleles and "complication-specific" alleles
• genotype-based early identification and treatment of likely complications
Prognosis : despite its straightforward Mendelian inheritance, is a highly phenotypically variable disease. Some individuals with SS have frequent vaso-occlusive complications and die young, whereas others seem little affected by the disease and have a normal lifespan. Therefore, risk factors and prediction models are needed to best counsel patients and families and to formulate optimal therapeutic plans. This section briefly reviews classic studies of disease modifiers and focuses on investigations in the past 5 years that have elucidated new risk factors or defined early risk prediction models.
• past studies : the CSSCD identified or confirmed many of the currently known risk factors, however incomplete, for common complications of SS. For example, low Hgb F concentration and leukocytosis are associated with increased risk of early death, acute chest syndrome (ACS), and painful episodes. Patients with a low steady-state Hgb concentration, compared to those with higher Hgb concentration, are more likely to suffer early death and stroke but are less likely to have ACS and pain. Both avascular necrosis (AVN) and sickle retinopathy are associated with a higher steady-state Hgb concentration. Other known disease modifiers are the bglobin cluster haplotype and a thalassemia. These established risk factors are reviewed elsewhere^ref. Predictors of adverse outcomes identified in the the Cooperative Study of Sickle Cell Disease (CSSCD) :
• reduced Hgb concentration => death, stroke, leg ulcers
• increased Hgb concentration => pain, acute chest syndrome (ACS), avascular necrosis (AVN)
• reduced Hgb F concentration => death, ACS, pain, leg ulcers
• increased steady-state WBC => death, ACS
• a-thalassemia present => AVN
• a-thalassemia absent => stroke
• increased pain rate => adult death, AVN
• acute anemia => death, stroke
• recent studies :
• risk factors for stroke : stroke prediction for individuals with SS was recently transformed when Adams et al demonstrated the utility of transcranial Doppler (TCD) ultrasonography in primary stroke prevention^ref. TCD is used to measure flow velocities in the intracranial carotid and cerebral arteries. Stenosis of these vessels, which may predispose to stroke, produces locally increased flow velocities that are detectable by TCD. Thus, children at risk of stroke can be started on a regimen of chronic red blood cell transfusions to prevent stroke. Although clearly predictive, the utility and universal acceptance of TCD screening is limited by the inconvenience and morbidity of chronic transfusions. Also, despite abnormally increased TCD velocities, some children do not develop a stroke. Approximately 10 children will have to be chronically transfused to prevent 1 stroke in 1 child each year. Thus, other predictors of stroke are still needed. Although a prior stroke is one of the strongest risk factors for stroke in SS, the prognostic significance of clinically "silent" strokes is now becoming clear. Children with SS in the CSSCD who had silent strokes identified at 6 years of age had a 14-fold increased incidence of subsequent new overt stroke (1.45 vs 0.11 per 100 patient-years, P = 0.006) during a mean follow-up of 5.2 years^ref. A multivariate model showed that silent stroke was the strongest independent risk factor for overt stroke (hazard ratio = 7.2, P = 0.027)^ref. MRI of the brain to identify silent strokes might, therefore, prove to be another useful screening measure to identify young children with SS who have an increased risk of overt stroke. The familial risk of stroke in SS was recently confirmed^ref, but the molecular genetic basis of stroke risk has been investigated only recently. Styles et al found an association between specific HLA alleles and either increased or decreased risk of stroke^ref, and similar findings were reported in an analysis of CSSCD participants^ref. Polymorphisms of genes associated with adhesion, thrombophilia, inflammation, and regulation of blood pressure have also been associated with risk of stroke^ref. Of interest, a polymorphism in VCAM-1 (G1238C) protected against stroke in patients with SS (odds ratio 0.35, 95% confidence interval [CI], 0.15– 0.83; P < 0.04)^ref. The hypothesis that nocturnal hypoxemia could predict central nervous system (CNS) events, including stroke, transient ischemic attacks, and seizure, in individuals with sickle cell disease has been tested^ref. Mean overnight oxygen saturation was found to be independently associated with the time to a CNS event (hazard ratio 0.82 for every 1% increase in saturation; P = 0.003). Screening for and appropriate management of nocturnal hypoxemia might, therefore, be useful to predict stroke and possibly to prevent it. Wierenga et al reported an increased risk of cerebrovascular accidents among individuals with SS who had aplastic crises due to parvovirus B₁₉ virus^ref. From this retrospective analysis, the crude risk of cerebrovascular episodes was 58-fold greater in the 5-week interval after parvovirus infection. Many of the episodes were coincident with the acute anemia, suggesting a mechanism, but several individuals had seizures and one had cortical blindness in the 2–5 weeks following infection. The authors suggest that the development of a parvovirus vaccine for humans might decrease the risk of stroke. Furthermore, an increased awareness of potential neurologic complications of parvovirus infection is needed.
• risk factors for acute chest syndrome : ACS is a descriptive name for a variety of acute pulmonary illnesses in individuals with sickle cell disease (SCD). ACS often occurs as a complication of another SCD-related illness, such as a painful crisis, and may not be apparent at the time of admission to the hospital. The prediction of impending ACS would permit preventive therapeutic interventions, such as red blood cell transfusions, to decrease morbidity and mortality. Secretory phospholipase A₂ (sPLA₂) is an enzyme that cleaves fatty acids, including arachidonic acid, from triglycerides, and this enzyme may be a biomarker for impending ACS. The release of free fatty acids and the pro-inflammatory arachidonic acid in the lungs likely contributes to the pathogenesis of ACS, especially in cases of pulmonary fat embolism. Styles et al showed that the serum concentration of sPLA₂ increases before ACS is clinically apparent, peaks at the clinical onset of ACS, and declines during its resolution^ref. These data are promising, but the published evidence for a predictive role of sPLA2 for ACS is limited to an analysis of about 20 patients, so further prospective study of this predictor is needed.
• risk factors for severe disease or early death : Miller et al tested many putative risk factors in the CSSCD infant cohort^ref. To this end, they defined four adverse outcomes: death, stroke, recurrent pain episodes (greater than 2 per year on average over the course of follow-up) and recurrent ACS (greater than 1 per year over the course of follow-up). Infants with SS or sickle-ß₀-thalassemia (Sß₀) who had a Hgb concentration < 7 g/dL in steady-state during the second year of life, those with an increased WBC count, and those who had dactylitis before 1 year of age were at significantly increased risk of an adverse outcome. This model is notable because it uses easily identifiable predictors in early life, but its clinical utility is limited because only 3% of the cohort had a combination of factors that predicted a 2-fold or greater risk of an adverse outcome. Also, it is unclear if this model is still valid, because the second most common adverse outcome, childhood death from SS, is becoming uncommon. For example, most of the deaths (56%) in this study were from infection, which is now a rare event because of universal newborn screening, prophylactic penicillin, and the conjugated pneumococcal vaccine^ref. Considering that leukocytosis predisposes to more severe disease in SS, Okpala et al hypothesized that the biological basis could be that the adherence of leukocytes to vascular endothelium, mediated by adhesion molecules, facilitates vaso-occlusion^ref. Compared to those without, patients who had complications of SCD showed high expression of Mß₂ integrin by neutrophils and high expression of L-selectin by lymphocytes and neutrophils (P < 0.03). The ß₂ integrin, CD18, was highly expressed by neutrophils in patients with sickle nephropathy (P = 0.018), and L-selectin was highly expressed by lymphocytes in those with stroke (P = 0.03). Monocyte L-selectin increased in sickle cell crisis relative to steady state (P = 0.04). They also found that hydroxyurea therapy decreased the expression of certain adhesion molecules coincident with clinical improvement and before significant induction of Hgb F. Whether polymorphisms in adhesion molecules or differences in their expression can be used to predict a severe clinical course remains to be determined. Gladwin et al studied the prevalence and effect on survival of pulmonary hypertension among individuals with SCD^ref. Using echocardiographic measurement of tricuspid regurgitant jet velocity, the investigators found that 32% of their subjects had pulmonary hypertension. A tricuspid regurgitant jet velocity of  2.5 m/s, as compared with a velocity of < 2.5 m/s, was strongly associated with an increased risk of death (rate ratio, 10.1; 95% CI, 2.2–47.0; P < 0.001). Thus, echocardiographic screening might be warranted for all adults with SCD to identify a high-risk group that might benefit from intervention. Interestingly, hydroxyurea therapy was not associated with a decreased tricuspid regurgitant jet velocity, and there was no significant association between the Hgb F or the WBC count, independent risk factors for death in SCD, and tricuspid regurgitant jet velocity.
The explanations for the phenotypic heterogeneity of SS remain incomplete, but the answers will likely be found among the multitude of genes that are co-inherited with the sickle (ß_S) mutation. That is, to understand a monogenic disease like SS, one must consider the molecular genetic context of the causative mutation in each individual^ref. Indeed, to understand phenotypes, the paradigm of a single gene disorder does not hold. Toward this end, genomic and proteomic profiling methods are promising, but they are still in their infancy. Until newer, comprehensive methods are available, we must continue to rely upon simple tools that include individual clinical features, laboratory values, and a small number of genetic polymorphisms.
Prolonged survival in homozygous SCD is more common than previously thought. A Jamaican study^ref in 1968 described 60 patients who were 30 years of age or older, and the Cooperative Study of Sickle Cell Disease in the USA^ref estimated a median survival of 42 to 48 years. The Sickle Cell Clinic at the University of the West Indies has treated 102 patients (64.7% women) who survived beyond their 60th birthday; of these patients, 58 are now dead, 4 have emigrated, and 40 are still alive. The diagnosis of homozygous SCD was based on criteria that excluded sickle cell–a⁰-thalassemia or sickle cell–HPFH. None of the patients received hydroxyurea, and only two patients with renal impairment received regular transfusions. The ages of the patients ranged from 60.2 to 85.6 years, with a marginal excess of women (67.5%, P=0.01). Sickle cell–a⁺-thalassemia was homozygous in 12% of patients and heterozygous in 39%, with a normal alpha-gene number in 46%; the trend failed to reach statistical significance (2=2.4, P=0.12). The Benin b-globin haplotype was homozygous in 76% of the patients, and none had the Bantu, Senegal, or Asian haplotype. Fetal hemoglobin levels were on average 4.9% higher among these patients than values extrapolated from the Jamaican Cohort Study of Sickle Cell Disease (P<0.001) and exceeded 10% in 16 women (24%) and in 5 men (14%), which suggests that higher fetal hemoglobin levels probably conferred protection in childhood. Family studies showed that 13 of 30 siblings of patients with homozygous sickle cell disease (43%) were over the age of 60 years, but their median survival (56.9 years) did not significantly exceed the median survival of 44.6 years in the Sickle Cell Clinic, as calculated by a standard statistical adjustment (P=0.07), or 56.3 years, as calculated by an adjustment for death rate (P=0.34)^ref. Hemoglobin values declined in both men and women, but more steeply in men (mean decrease, 2.6 g/dl) than in women (2.3 g/dl), and fell by 0.02 g/dl for every 10-unit increase in the creatinine level (P=0.001), or by 0.6 g/dl for every 1-unit increase in the natural-log–transformed creatinine level. Renal impairment increased with age^ref and affected 34 of 40 patients (85%), on the basis of a sensitive definition (Thompson J, Reid M, Hambleton IR, Serjeant GR. Albuminuria and renal function in homozygous sickle cell disease: observations from a cohort study. Arch Intern Med (in press)) An incidental presentation occurred in 16 patients (40%). 7 patients reported having had no bone-pain crises (18%); bone pain decreased with age in 30 of the remaining 33 patients; 58% of the patients had a history of leg ulcers. Pregnancy had a benign outcome, with 78 of 96 pregnancies (81%) resulting in live births. In these elderly survivors, high fetal hemoglobin levels and possible familial clustering were consistent with genetic factors. Some features, such as an incidental presentation and successful pregnancy outcomes, suggested an intrinsically mild course, whereas other features, such as severe bone pain, were ameliorated with age. In this group of patients, -thalassemia did not promote survival, and the group was too homogeneous to show any effect of the b-globin haplotype. The major clinical problems emerging with age were renal impairment and decreased levels of hemoglobin^ref.
Web resources :
• The Red Lane Forum
• hemoglobin D : any of several abnormal hemoglobins, all characterized by electrophoretic mobility equal to that of hemoglobin S on paper or cellulose acetate but unequal on acid agar gel
• heterozygous state is clinically silent
• homozygous state manifests as hemoglobin D disease, characterized by mild hemolytic anemia with numerous target cells in the peripheral blood.
• hemoglobin D-Los Angeles or Punjab (the most common one) : glycine substituted for glutamic acid at position 121 of the b chain
• hemoglobin D-Iran
• > 80 diseases due to unstable tertiary or quaternary Hb structure, usually owing to substitution or deletion of at least one amino acid (many have increased oxygen affinity and some have Heinz bodies; affected individuals often have Heinz body anemia or some other type of extravascular hemolytic anemia) and many other asymptomatic alleles ! E.g. :
• hemoglobin C : a common abnormal hemoglobin in which lysine replaces glutamic acid at position 6 of the b chains; it was one of the earliest hemoglobins to have its molecular abnormality defined. The asymptomatic heterozygous state is called hemoglobin C trait, and the homozygous state manifests as hemoglobin C disease, characterized by splenomegaly, mild to moderate hemolytic anemia, recurrent jaundice, and increased numbers of target cells and reticulocytes in the peripheral blood.
• hemoglobin C–thalassemia disease : a hereditary disorder involving simultaneous heterozygosity for HbC and thalassemia, manifested by mild hemolytic anemia and persistent splenomegaly
• hemoglobin E : an abnormal hemoglobin with lysine substituted for glutamic acid at position 26 of the b chain, seen most often in Southeast Asia, especially Thailand
• homozygous state is termed hemoglobin E disease : many patients are asymptomatic, but others have mild hemolytic anemia, usually without splenomegaly, and increased numbers of normochromic target cells in the peripheral blood. Without further testing, benign homozygous E disease can be misidentified as hemoglobin E-ß-thalassemia.
• heterozygous state is clinically silent
• hemoglobin E–thalassemia disease : a hereditary condition involving simultaneous heterozygosity for HbE and thalassemia. Worldwide, HbE-ß-thalassemia may be the most important hemoglobinopathy because of the high gene frequencies for both HbE and ß-thalassemia^ref. Since the classic description of HbE-ß-thalassemia by Chernoff, it has been noted to be an important hemoglobin disorder in the Indian subcontinent and Southeast Asia. It has recently replaced ß-thalassemia as the most common disorder in many regions, including coastal North America^{ref1, ref2, ref3}. HbE is synthesized at a mildly reduced rate and in its homozygous state is similar to ß-thalassemia trait. The homozygous form is very common, with thousands of cases already detected in North America through newborn screening programs. The compound heterozygote state of HbE-ß-thalassemia results in a variable, often severe anemia, with the phenotype ranging from a complete lack of symptoms to transfusion-dependence^{ref1, ref2, ref3, ref4}
• 50% of the patients are phenotypically similar to patients with thalassemia major who require regular transfusion therapy
• 50% resembles thalassemia intermedia, including patients that when grow older
• become transfusion-dependent
• remain asymptomatic
The marked variability and clinical course in Hb E-ß-thalassemia is largely unexplained. The variability is probably due, in part, to whether the patient is heterozygous for ß⁺-thalassemia or ß₀-thalassemia mutations. Co-inheritance of a-thalassemia mutations occurs in 15% of patients and may also modulate severity. HbF level is the strongest predictor of morbidity^{ref1, ref2, ref3, ref4}. Although the basis of increased HbF is usually unknown, inheritance of a ß-thalassemia chromosome with the XmnI ^+/+ genotype of the G-gamma globin gene is an example of an associated mutation that is responsible for increased HbF and a milder clinical course^{ref1, ref2, ref3}.
Pathogenesis : ineffective erythropoiesis, apoptosis, and oxidative damage are central components of the disease and its shortened red cell survival^{ref1, ref2, ref3}. The instability of HbE and the expression of a hemoglobin stabilizing protein do not appear to be major factors in the pathophysiology^ref. Non-transfusional iron overload can result in widespread organ injury in the absence of markedly elevated ferritin levels^ref (Pakbaz Z FR, Fung E, Harmatz P, Vichinsky E. Serum Ferritin Underestimates the Liver Iron Concentration. Pediatric Blood and Cancer. 2004;42:497)
Symptoms & signs : mild hemolytic anemia and persistent splenomegaly; a chronic hypercoagulable state may result in pulmonary thrombosis and other thromboembolic events. Splenectomy and increased exposure of erythrocyte phosphatidylserine appear to increase the prothrombotic risk^{ref1, ref2}. Oxidative damage with deficiency of antioxidants may also be important in the cellular pathophysiology of this disease^ref. The availability of prenatal diagnosis, chronic transfusion, bone marrow transplants and chemotherapy adds to the importance of characterizing the clinical course of HbE-ß-thalassemia. In earlier reports by Chernoff and Wasi, patients were classified as having severe thalassemia if they manifested with massive abdominal enlargement, growth failure and marked skeletal deformities. There was a uniformly increased risk of life-threatening infections (Pythium insidiosum). Patients suffered from recurrent anemic events due to hypersplenism, aplastic crises and megaloblastic anemia^ref. As most patients could not be transfused, they developed large tumor-like masses secondary to massive erythroid hyperplasia. When these masses impinged on the spinal cord, they caused paraplegiaand other complications. As noted above, severe iron overload may develop despite the absence of regular transfusion therapy. Early reports on the hematologic findings of E-ß-thalassemia stress the severity of the illness and report a mean hemoglobin of 6.3 g/dL. However, the thalassemia-related severity of HbE-ß-thalassemia in these reported patients may be overestimated because of the additional effects of acquired causes of anemia, such as infection and poor nutrition, which were endemic during that period in the studied region. In recent studies of HbE-ß₀-thalassemia, the hemoglobin concentrations ranged from 3 to 13 g/dL with an average of 7.7 g/dL. Even with screening for genetic modifiers, early predictors of severity have yet to be defined. The time of onset and the severity of anemia may be useful in predicting the long-term clinical phenotype^{ref1, ref2, ref3}. Severe anemia that begins at 6–12 months suggests a thalassemia major phenotype. A stable clinical condition at 5 years of age is generally sustained into at least early adulthood
Therapy : in the past, splenectomy was routinely performed in an attempt to increase hemoglobin levels. Splenectomy is often less successful than expected. Increasing evidence of the risk of thromboembolism in splenectomized patients has led to reconsideration of the role of splenectomy. The bone marrow expansion and increased metabolic rate that are found in HbE-ß₀-thalassemia result in growth failure, delay in secondary sex development and osteoporosis. In older patients, severe bone pain may lead to the initiation of transfusion therapy. Iron overload in nontransfused patients is common, secondary to increased gastrointestinal absorption of iron^ref. End organ failure secondary to iron overload may not be suspected because the serum ferritin level is disproportionately low. The cause of the lack of correlation between serum ferritin levels and liver iron stores is unknown. Vitamin C deficiency may play a role. Quantitative liver iron measurements are necessary to identify iron overload even in nontransfused patients with Hb E-ß₀-thalassemia. Cardiac disease and pulmonary disease are the most common causes of death in Hb E-ß-thalassemia. Increased cardiac output, severe anemia and, in some cases, thromboembolism result in cardiac failure and pulmonary hypertension. Transfusion therapy appears to reverse this process in some patients^ref. Patients with HbE-ß-thalassemia may be excellent candidates for agents directed at elevating Hb F production. A small increase in the steady state hemoglobin concentration might be of major clinical benefit. Pilot data suggest that some patients sustain long-term benefit from hydroxyurea, short chain fatty acids such as butyrate, or EPO^ref. However, individual responses are inconsistent.
• hemoglobin J (Capetown) : an abnormal hemoglobin; it has increased oxygen affinity and is associated with erythrocytosis
• hemoglobin K
• hemoglobin M : any of several abnormal hemoglobins having amino acid substitutions in the a or b chains and all associated with methemoglobinemia. In the most common type due to substitution of both distal and proximal (58) His residues with Tyr in a-globin > 1% Hb has Fe³⁺
• hemoglobin N
• hemoglobin Aichi : HbA₁ His50Arg^{ref1, ref2}
• hemoglobin Albany-Georgia : HbA₁ Lys11Asp^ref (The survey of abnormal hemoglobin in Kobe district. Jpn. J. Hum. Genet. 28: 127-128, 1983)
• hemoglobin Anantharaj : HbA₁ Lys11Asp^ref
• hemoglobin Ann Arbor : HbA₁ Leu80Arg^{ref1, ref2}
• hemoglobin Arja : HbA₁ Asp47Asn^ref
• hemoglobin Atago : HbA₁ Asp85Tyr^{ref1, ref2} (An amino acid substitution in Hb Atago, an abnormal human hemoglobin. J. Jpn. Biochem. Soc. 42: 341-349, 1970)
• hemoglobin Attleboro : HbA₁ Ser138Pro^ref
• See McDonald et al. (1990).
• hemoglobin Aztec : HbA₁ Met76Thr^ref
• See Shelton et al. (1985).
• hemoglobin Bari : HbA₁ His45Gln^ref
• See Marinucci et al. (1980).
• hemoglobin Beijing : HbA₁ Lys16Asn^ref
• See Liang et al. (1982).
• hemoglobin Bibba : HbA₁ Leu136Pro^ref
• See Kleihauer et al. (1968). (This is actually an allelic variant of the HBA2 gene; see 141850.0030.)
• hemoglobin Bourmedes : HbA₁ Pro37Arg^ref
• See Dahmane-Arbane et al. (1987).
• hemoglobin Broussais : HbA₁ Lys90Asn^ref
• hemoglobin J (Broussais)
• hemoglobin Tagawa I
See de Traverse et al. (1966), Yanase et al. (1968), Vella et al. (1970), and Fleming et al. (1978).
• hemoglobin Catonsville : HbA₁ Pro37Glu/Thr38^ref
• See Virshup et al. (1988). Moo-Penn et al. (1989) identified insertion of a glutamic acid residue between proline-37 and threonine-38 in an unstable hemoglobin variant. The PCR-amplified fragment of the variant gene showed insertion of a GAA codon. In the normal alpha-globin gene cluster, GAG is the codon for glutamic acid. Moo-Penn et al. (1989) suggested that this mutation may have resulted from nonhomologous nonallelic gene conversion.
• hemoglobin Chad : HbA₁ Glu23Lys^ref
• See Boyer et al. (1968).
• hemoglobin Chapel Hill : HbA₁ Asp74Gly^ref
• See Orringer et al. (1976).
• hemoglobin Chesapeake : HbA₁ Arg92Leu^ref
• See Clegg et al. (1966) and Harano et al. (1983). Polycythemia is the only clinical feature. This was the first polycythemia-producing variant to be described (Charache et al., 1966).
• hemoglobin Chiapas : HbA₁ Pro114Arg^ref
• See Jones et al. (1968).
• hemoglobin Chicago : HbA₁ Leu136Met^ref
• See Bowman et al. (1986).
• hemoglobin Chongqing : HbA₁ Leu2Arg^ref
• See Zeng et al. (1984).
• hemoglobin Contaldo : HbA₁ His103Arg^ref Unstable hemoglobin due to disruption of hydrogen bond between alpha 103 (his) and beta 108 (asn) (Sciarratta et al., 1984).
• hemoglobin Cordele : HbA₁ Asp47Ala^ref
• See Nakatsuji et al. (1984).
• hemoglobin Dagestan : HbA₁ Lys60Glu^ref
• See Spivak et al. (1981) and Lacombe et al. (1987).
• hemoglobin Dallas : HbA₁ Asn97Lys^ref
• See Dysert et al. (1982).
• hemoglobin Daneshgah-Tehran : HbA₁ His72Arg^ref
• See Rahbar et al. (1973) and de Weinstein et al. (1985).
• hemoglobin Denmark Hill : HbA₁ Pro95Ala^ref
• See Wiltshire et al. (1972).
• hemoglobin Duan : HbA₁ Asp75Ala^ref
• See Liang et al. (1981, 1988).
• hemoglobin Dunn : HbA₁ Asp6Asn^ref
• See Jue et al. (1979) and Baklouti et al. (1988).
• hemoglobin Etobicoke : HbA₁ Ser84Arg^ref
• See Crookston et al. (1969) and Headlee et al. (1983).
• hemoglobin Evanston : HbA₁ Trp14Arg^ref. Honig et al. (1982) first described Hb Evanston in 2 black families. See also Moo-Penn et al. (1983). Harteveld et al. (2004) found this rare variant alone and in the presence of common alpha-thalassemia deletions in 3 independent Asian cases.
• hemoglobin Ferndown : HbA₁ Asp6Val^ref
• See Lee-Potter et al. (1981).
• hemoglobin Fontainebleau : HbA₁ Ala21Pro^ref. Wajcman et al. (1989) found this substitution in an Italian family. The substitution produced no change in the stability or oxygen binding properties of the hemoglobin molecule. The electrophoretic properties were, furthermore, identical to those of Hb A, with the exception of isoelectric focusing in which the variant migrated like Hb A1c. Hb J(Nyanza), another substitution at position alpha-21, likewise causes no hematologic disorder.
• hemoglobin Fort de France : HbA₁ His45Arg^ref
• See Braconnier et al. (1977). Cash et al. (1989) confirmed that this is a mutant of the HBA1 gene.
• hemoglobin G (Audhali) : HbA₁ Gly23Val^ref
• See Marengo-Rowe et al. (1968).
• hemoglobin G (Fort Worth) : HbA₁ Glu27Gly^ref. This variant was described in 2 black families. Unusually low (5%) concentration was found in heterozygotes, perhaps because of decreased ability of the abnormal alpha chain to form dimers with beta chains. See Schneider et al. (1971) and Carstairs et al. (1985).
• hemoglobin G (Georgia) : HbA₁ Pro95Leu^ref
• See Huisman et al. (1970)
• hemoglobin G (Norfolk) : HbA₁ Asp85Asn^ref
• See Cohen-Solal et al. (1975) and Lorkin et al. (1975).
• hemoglobin G (Pest) : HbA₁ Asp74Asn^ref Hb G (Pest) and Hb J (Buda) (141850.0008), both alpha-chain mutants, occurred together in a Hungarian male with erythrocytosis. The occurrence of some normal Hb A in this man showed the existence of at least 2 alpha loci. See Brimhall et al. (1970, 1974) and Hollan et al. (1972). Using polymerase chain reaction (PCR) to amplify selectively alpha-1 and alpha-2-globin cDNAs, Mamalaki et al. (1990) then hybridized the cDNAs to synthetic oligonucleotides specific for either the normal or the mutated sequence. Using this approach, the alpha-globin structural mutants J-Buda and G-Pest were found to be encoded by the alpha-2 and the alpha-1-globin genes, respectively. The substitution in G-Pest was a change from GAC to AAC at codon 74.
• hemoglobin G (Taichung) : HbA₁ Asp74His^ref
• hemoglobin Q (HEMOGLOBIN Q (THAILAND)
• HEMOGLOBIN MAHIDOL
• HEMOGLOBIN ASABARA
• HEMOGLOBIN KURASHIKI
• See Vella et al. (1958), Gammack et al. (1961), Lie-Injo et al. (1966, 1979); Blackwell and Liu (1970), Pootrakul and Dixon (1970), Lorkin et al. (1970), Iuchi et al. (1978), and Higgs et al. (1980). Zeng et al. (1992) demonstrated that the mutation is due to a GAC-to-CAC change in codon 74 of the HBA1 gene. They developed a simple and accurate method for diagnosis of the Hb Q (Thailand) variant based on restriction enzyme analysis.
• hemoglobin G (Waimanalo) : HbA₁ Asp64Asn^ref
• hemoglobin Aida. See Blackwell et al. (1973) and Bunn et al. (1978). Schiliro et al. (1991) found this variant in a Filipino mother and child living in Sicily. They showed no hematologic abnormalities.
• hemoglobin Garden State : HbA₁ Ala82Asp^ref
• See Winter et al. (1978).
• hemoglobin Grady : HbA₁ 3 aa ins, 118THR-GLU-PHE119^ref
• hemoglobin Dakar : at the time it was first studied by Huisman et al. (1974), hemoglobin Grady was unique in having an insertion of threonine-glutamic acid-phenylalanine between amino acids 118 and 119 of the alpha chain. Several hemoglobins with deletions were then known (Leiden, Lyon, Freiburg, Niteroi, Tochigi, St. Antoine, Tours and Gun Hill). Scott et al. (1981) found no evidence of an extra (fifth) alpha gene. They argued, therefore, that if, as supposed, Hb Grady arose by unequal crossing over, the event occurred between alleles rather than between the separate alpha-1 and alpha-2 loci. The glu-phe-thr insertion is a repeat of normal residues 116, 117 and 118. See Cleek et al. (1983). Substitution of glutamine for histidine at alpha 112 was thought to be the change in hemoglobin Dakar; however, on restudy the hemoglobin was found to be identical to Hb Grady (Garel et al., 1976).
• hemoglobin Guangzhou : HbA₁ Asp64Gly^ref
• hemoglobin Hangzhou
• See Jen and Liu (1987), Zhou et al. (1987), and Li et al. (1990)
• hemoglobin Guizhou : HbA₁ Pro77Arg^ref
• hemoglobin Utsonomiya
• See Hattori et al. (1985).
• hemoglobin Handa : HbA₁ Lys90Met^ref
• hemoglobin Munakata
• See Harano et al. (1982) and Sugihara et al. (1983).
• hemoglobin Handsworth : HbA₁ Gly18Arg^ref
• See Griffiths et al. (1977), Chih-chuan et al. (1981), and Al-Awamy et al. (1985)
• hemoglobin Harbin : HbA₁ Lys16Met^ref
• See Zeng et al. (1984).
• hemoglobin Hekinan : HbA₁ Glu27Asp^ref
• See Harano et al. (1988). Using dot-blot analysis of amplified DNA with (32)p-labeled probes, Zhao et al. (1990) located the mutation in codon 27 of the minor alpha-1 globin gene and showed that the change involved a GAG (glutamic acid)-to-GAT (aspartic acid) mutation. Their patients were 3 Chinese women from Macau. In Thailand, Ngiwsara et al. (2004) described 2 unrelated cases of compound heterozygosity for Hb Hekinan and alpha-thalassemia.
• hemoglobin Hirosaki : HbA₁ Phe43Leu^ref
• See Ohba et al. (1975, 1978).
• hemoglobin Hobart : HbA₁ His20Arg^ref
• See Fleming et al. (1987).
• hemoglobin Hopkins 2 : HbA₁ His112Asp^ref
• Fast hemoglobin. See Smith and Torbert (1958), Itano and Robinson (1960), Bradley et al. (1961), Ostertag et al. (1972), Clegg and Charache (1978).
• hemoglobin I : HbA₁ Lys16Glu^ref
• hemoglobin I (Burlington)
• hemoglobin I (Philadelphia)
• hemoglobin I (Skamania)
• hemoglobin I (Texas) : fast hemoglobin. Substitution of aspartic acid for lysine at alpha 16 was first reported by Murayama (1962). However, Crick pointed out that this substitution could not be accomplished by change in one base. Restudy by Beale and Lehmann (1965) and by Schneider et al. (1966) showed substitution of glutamic acid for lysine. Hemoglobin I was thought to show sickling but this has been shown to be due to faulty technique (Schneider et al., 1967). See Rucknagel et al. (1955), Schwartz et al. (1957), Itano and Robinson (1959, 1960), Ranney et al. (1962), O'Brien et al. (1964), Thompson et al. (1965), Schneider et al. (1966), Bowman and Barnett (1967), Baur (1968), Labossiere and Vella (1971), Fleming et al. (1978), and Liebhaber et al. (1984). The hemoglobin I mutation is curious in that the mutation is present in HBA2 (141850.0011) as well as in HBA1
• hemoglobin Iwata : HbA₁ His87Arg^ref
• See Shibata et al. (1980) and Liu et al. (1983).
• hemoglobin J (Abidjan) : HbA₁ Gly51Asp^ref
• See Cabannes et al. (1972).
• hemoglobin J (Anatolia) : HbA₁ Lys61Thr^ref
• See Giordano et al. (1990).
• hemoglobin J (Birmingham) : HbA₁ Ala120Glu^ref
• hemoglobin J (Meerut)
• See Kamuzora and Lehmann (1974) and Blackwell et al. (1974).
• hemoglobin J (Camaguey) : HbA₁ Arg141Gly^ref
• See Martinez et al. (1978). Romero et al. (1995) found this hemoglobin variant in 3 Spanish families. The original description by Martinez et al. (1978) was in a Cuban family of Spanish ancestry.
• hemoglobin J (Cape Town) : HbA₁ Arg92Gln^ref
• See Botha et al. (1966), Harano et al. (1983), and Lambridis et al. (1986).
• hemoglobin  J( Cubujuqui) : HbA₁ Arg141Ser^ref
• See Saenz et al. (1977) and Moo-Penn et al. (1981).
• hemoglobin J (Habana) : HbA₁ Ala71Glu^ref
• See Colombo et al. (1974) and Ohba et al. (1983).
• hemoglobin J (Kurosh) : HbA₁ Ala19Asp^ref
• See Rahbar et al. (1976).
• hemoglobin J (Medellin) : HbA₁ Gly22Asp^ref
• See Gottlieb et al. (1964).
• hemoglobin J (Nyanza) : HbA₁ Ala21Asp^ref
• See Kendall et al. (1973).
• hemoglobin J (Paris 1) : HbA₁ Ala12Asp^ref
• hemoglobin J (Aljezur) : see Rosa et al. (1966), Trincao et al. (1968), and Marinucci et al. (1979).
• hemoglobin J (Rajappen) : HbA₁ Lys90Thr^ref
• See Hyde et al. (1971).
• hemoglobin J (Rovigo) : HbA₁ Ala53Asp^ref
• See Alberti et al. (1974) and Moo-Penn et al. (1978)
• hemoglobin J (Singa) : HbA₁ Asn78Asp^ref
• See Wong et al. (1984).
• hemoglobin J (Singapore) : HbA₁ Asn78Asp and Ala79Gly^ref. Since no simple frameshift mechanism could be imagined, the possibility of 2 separate mutations was favored by Blackwell et al. (1972), who suggested that 2 separate hemoglobins, appropriately called Hb J (Singa) and Hb J (Pore), will be discovered eventually. Double mutation on the same chromosome would seem more likely than crossing-over in a compound heterozygote since the 2 codons involved are contiguous.
• hemoglobin J (Tashikuergan) : HbA₁ Ala19Glu^ref
• See Houjun et al. (1984). Li et al. (1990) found this variant in populations in the Silk Road region of China.
• .0077 HEMOGLOBIN J (TONGARIKI) [HBA1, ALA115ASP]
• See Gajdusek et al. (1967) and Beaven et al. (1972). A homozygous individual had only anomalous hemoglobin suggesting the existence of only one alpha locus in Melanesians (Abramson et al., 1970).
• .0078 HEMOGLOBIN J (TORONTO) [HBA1, ALA5ASP]
• See Crookston et al. (1965).
• .0079 HEMOGLOBIN JACKSON [HBA1, LYS127ASN]
• See Moo-Penn et al. (1976).
• .0080 HEMOGLOBIN KARACHI [HBA1, ALA5PRO]
• See Ahmad et al. (1986).
• .0081 HEMOGLOBIN KARIYA [HBA1, LYS40GLU]
• See Harano et al. (1983) and Imai et al. (1989).
• .0082 HEMOGLOBIN KAWACHI [HBA1, PRO44ARG]
• See Harano et al. (1982).
• .0083 HEMOGLOBIN KOELLIKER [HBA1, ARG141DEL]
• HEMOGLOBIN F (KOELLIKER)
• Not a genetic change. The C-terminal amino acid, 141, of the alpha chain (arginine) is missing, probably from the action of a carboxypeptidase present in normal plasma. This unusual fast hemoglobin is observed in persons with hemolysis. The change can occur in fetal hemoglobin also (Kohne et al., 1977). See Marti et al. (1967) and Schiliro et al. (1982).
• .0084 HEMOGLOBIN KOKURA [HBA1, ASP47GLY]
• HEMOGLOBIN BEILINSON
• HEMOGLOBIN MICHIGAN-I
• HEMOGLOBIN MICHIGAN-II
• HEMOGLOBIN L (GASLINI)
• HEMOGLOBIN TAGAWA II
• HEMOGLOBIN UMI
• HEMOGLOBIN MUGINO
• HEMOGLOBIN YUKUHASHI-2
• See Yamaoka et al. (1960), Ooya et al. (1961), Sumida (1975), and Ohba et al. (1982). The change is in TP IV (DeVries et al., 1963).
• .0086 HEMOGLOBIN L (PERSIAN GULF) [HBA1, GLY57ARG]
• See Rahbar et al. (1969).
• .0087 HEMOGLOBIN LEGNANO [HBA1, ARG141LEU]
• See Mavilio et al. (1978).
• .0088 HEMOGLOBIN LE LAMENTIN [HBA1, HIS20GLN]
• See Sellaye et al. (1982), Harano et al. (1983), and Malcorra-Azpiazu et al. (1988).
• .0089 HEMOGLOBIN LILLE [HBA1, ASP74ALA]
• See Djoumessi et al. (1981) and Lu et al. (1984).
• .0090 HEMOGLOBIN LOIRE [HBA1, ALA88SER]
• This variant was discovered in a 10-year-old Algerian boy born in Loire. The child had erythrocytosis and microcytosis, the latter being due to iron deficiency (Baklouti et al., 1988).
• .0091 HEMOGLOBIN LUXEMBOURG [HBA1, TYR24HIS]
• Groff et al. (1989) found this substitution in association with mild hemolytic anemia and increased indirect bilirubinemia in a family originating from The Netherlands.
• .0092 HEMOGLOBIN M (BOSTON) [HBA1, HIS58TYR]
• HEMOGLOBIN GOTHENBURG
• HEMOGLOBIN M (GOTHENBURG)
• HEMOGLOBIN M (OSAKA)
• HEMOGLOBIN M (KISKUNHALAS)
• The aberrant hemoglobins associated with methemoglobinemia are referred to as hemoglobin M. Most of the hemoglobin M variants have substitutions of histidine at alpha 58, alpha 87, beta 63, or beta 92. These 4 amino acids are critical to the binding of the heme group. The exception is hemoglobin M (Milwaukee-1). See Gerald et al. (1957), Hansen et al. (1960), Gerald and Efron (1961), Betke (1962), Hayashi et al. (1964), Shimizu et al. (1965), Suzuki et al. (1965), Hollan et al. (1967), and Pulsinelli et al. (1973).
• .0093 HEMOGLOBIN M (IWATE) [HBA1, HIS87TYR]
• HEMOGLOBIN M (KANKAKEE)
• HEMOGLOBIN M (OLDENBURG)
• HEMOGLOBIN M (SENDAI)
• Hb Iwate was the first variant hemoglobin found in Japan (Shibata et al., 1960). Familial cyanosis had been recognized for about 200 years in the prefecture of Iwate in Honshu, where about 70 affected persons were identified in the 1950s. It was called 'kuchikuro,' or 'blackmouth.' In each form of methemoglobinemia, the heme iron is stabilized in the ferric form. Patients with the Hb M alpha forms are cyanotic at birth; those with the Hb M beta forms are usually not cyanotic until they are 3 months of age. Horst et al. (1987) showed that the Iwate mutation involves the alpha-1 globin gene. Specifically, they demonstrated a CAC-to-TAC mutation in codon 87 of that gene. They showed that the Iwate mutation can be identified directly on RsaI digestion. See Meyering et al. (1960), Shibata et al. (1961), Gerald and Efron (1961), Miyaji et al. (1962), Heller (1962), Heller et al. (1962), Tonz et al. (1962), Shibata (1964), Tamura (1964), Shimizu et al. (1965), Pik and Tonz (1966), Maggio et al. (1981), and Mayne et al. (1986).
• Ameri et al. (1999) likewise determined that the molecular defect in 2 patients with Hb M (Kankakee) was his87 to tyr in the HBA1 gene. The proportion of Hb M (Kankakee) observed was higher than that predicted for an alpha-1-globin variant. They presented evidence suggesting that the greater-than-expected proportion of Hb M (Kankakee) results from preferential association of the electronegative beta-globin chains with the alpha-(M)-globin chains that are more electropositive than normal alpha-globin chains.
• .0094 MOVED TO 141850.0047
• .0095 HEMOGLOBIN MATSUE-OKI [HBA1, ASP75ASN]
• See Ohba et al. (1977) and Yi-Tao et al. (1982).
• .0096 HEMOGLOBIN MEMPHIS [HBA1, GLU23GLN]
• Substitution of glutamine for glutamic acid at alpha 23. A hemoglobin S homozygote who also carries this abnormal hemoglobin has a mild form of sickle cell anemia. See Kraus et al. (1965, 1967) and Cooper et al. (1973).
• .0097 HEMOGLOBIN MEXICO [HBA1, GLN54GLU]
• HEMOGLOBIN J
• HEMOGLOBIN J (MEXICO)
• HEMOGLOBIN J (PARIS 2)
• HEMOGLOBIN UPPSALA
• Fast hemoglobin. See Jones et al. (1963, 1968), Beckman et al. (1966), Labie and Rosa (1966), Quattrin and Ventruto (1967), Fessas et al. (1969), and Trabuchet et al. (1982).
• .0098 HEMOGLOBIN MILLEDGEVILLE [HBA1, PRO44LEU]
• See Honig et al. (1980).
• .0099 HEMOGLOBIN MIYANO [HBA1, THR41SER]
• See Ohba et al. (1989).
• .0100 HEMOGLOBIN MIZUSHI [HBA1, ASP75GLY]
• No hematologic abnormality. See Iuchi et al. (1980).
• .0101 HEMOGLOBIN MOABIT [HBA1, LEU86ARG]
• See Knuth et al. (1979).
• .0104 HEMOGLOBIN NECKER ENFANTS-MALADES [HBA1, HIS20TYR]
• This variant was detected by chromatography in the course of screening diabetics for Hb A1c (Wajcman et al., 1980).
• .0105 HEMOGLOBIN NIGERIA [HBA1, SER81CYS]
• See Honig et al. (1978).
• .0106 HEMOGLOBIN NOKO [HBA1, MET76LYS]
• See Shibata et al. (1981).
• .0107 HEMOGLOBIN NORFOLK [HBA1, GLY57ASP]
• HEMOGLOBIN J (NORFOLK)
• HEMOGLOBIN KAGOSHIMA
• HEMOGLOBIN NISHIK
• Fast hemoglobin. See Ager et al. (1958), Baglioni (1962), Huntsman et al. (1963), Hanada et al. (1964), Imamura (1966), and Lehmann and Carrell (1969).
• .0108 HEMOGLOBIN NOUAKCHOTT [HBA1, PRO114LEU]
• See Wajcman et al. (1989).
• .0109 HEMOGLOBIN NUNOBIKI [HBA1, ARG141CYS]
• This hemoglobin showed an extremely high oxygen affinity. The patient, who had 'marginal erythrocytosis,' was shown to have 13.1% Hb Nunobiki (Shimasaki, 1985).
• .0110 HEMOGLOBIN O (INDONESIA) [HBA1, GLU116LYS]
• HEMOGLOBIN O (BUGINESE-X)
• HEMOGLOBIN BUGINESE-X
• HEMOGLOBIN O (OLIVIERE)
• HEMOGLOBIN OLIVIERE
• See Lie-Injo and Sadono (1958), Baglioni and Lehmann (1962), and Sansone et al. (1970).
• Daud et al. (2001) investigated the occurrence of hemoglobin O (Indonesia) in related ethnic populations of the Indonesian archipelago. Nineteen individuals heterozygous for this variant were identified in 4 ethnic populations. The level of Hb O (Indonesia) in 17 of the individuals was 11.6 +/- 1.0%, significantly lower than the expected 17 to 22%, indicating the instability of Hb O (Indonesia).
• .0111 HEMOGLOBIN O (PADOVA) [HBA1, GLU30LYS]
• See Vettore et al. (1974), Kilinc et al. (1985), and Martin et al. (1990). Schnedl et al. (1997) showed that the silent hemoglobin O Padova mutation causes an additional peak on high performance liquid chromatography (HPLC) and falsely low HbA(1c) values (glycated hemoglobin) when measured by HPLC. HPLC is the gold standard for evaluation of glycated hemoglobin in diabetes mellitus.
• .0112 HEMOGLOBIN OGI [HBA1, LEU34ARG]
• HEMOGLOBIN QUEENS
• See Sugihara et al. (1982), Moo-Penn et al. (1982), and Yongsuwan et al. (1987). This has been shown to be a mutation of the HBA1 gene (Cash et al., 1989).
• .0113 HEMOGLOBIN OLEANDER [HBA1, GLU116GLN]
• See Schneider et al. (1980).
• .0114 HEMOGLOBIN OTTAWA [HBA1, GLY15ARG]
• HEMOGLOBIN SIAM
• See Vella et al. (1974) and Pootrakul et al. (1974).
• Yodsowan et al. (2000) studied this variant in a 21-year-old Thai female and her mother. Turbpaiboon et al. (2002) reported a fourth case of Hb Siam in a healthy Thai female and concluded that there is no alpha-thalassemic effect of the variant.
• .0115 HEMOGLOBIN OWARI [HBA1, VAL121MET]
• This is a neutral-to-neutral change; it was detected in the course of mass screening by isoelectric focusing (Harano et al., 1986).
• .0116 HEMOGLOBIN PERSPOLIS [HBA1, ASP64TYR]
• See Rahbar et al. (1976).
• .0117 HEMOGLOBIN PETAH TIKVA [HBA1, ALA110ASP]
• See Honig et al. (1981).
• .0118 HEMOGLOBIN PONTOISE [HBA1, ALA63ASP]
• HEMOGLOBIN J (PONTOISE)
• See Thillet et al. (1977) and Gonzalez-Redondo et al. (1987).
• .0119 HEMOGLOBIN PORT PHILLIP [HBA1, LEU91PRO]
• See Brennan et al. (1977).
• .0120 MOVED TO 141850.0055
• .0121 HEMOGLOBIN Q (INDIA) [HBA1, ASP64HIS]
• See Sukumaran et al. (1972) and Schmidt et al. (1976).
• .0122 HEMOGLOBIN Q (IRAN) [HBA1, ASP75HIS]
• See Lorkin et al. (1970), Lie-Injo et al. (1979), and Higgs et al. (1980).
• .0123 MOVED TO 141850.0052
• .0124 HEMOGLOBIN REIMS [HBA1, GLU23GLY]
• See Bardakdjian-Michau et al. (1989).
• .0125 HEMOGLOBIN RUSS [HBA1, GLY51ARG]
• See Huisman and Sydenstricker (1962) and Reynolds and Huisman (1966). This has been shown to be a mutation of the HBA1 gene (Cash et al., 1989).
• .0126 HEMOGLOBIN SASSARI [HBA1, ASP126HIS]
• Masala et al. (1987) first described this variant as an electrophoretically slow-moving hemoglobin in 2 brothers affected by erythrocytosis with slight microcytosis. In a large screening program involving 20,000 people in the city of Sassari and its surrounding area in Sardinia, Masala (1992) found the variant in 3 other apparently unrelated subjects. A male of German origin was identified by Bardakdjian-Michau et al. (1990) as a carrier of the same mutation. Sanna et al. (1994) demonstrated that the adult variant has increased oxygen affinity, a dramatic reduction of homotropic interactions, and a significant decrease of the effect of 2,3-diphosphoglycerate (35% lower than that observed for Hb A). The fetal variant also showed increased oxygen affinity compared with normal Hb F and an almost abolished heme-heme interaction.
• Paglietti et al. (1998) demonstrated that Hb Sassari results from a GAC (asp)-to-CAC (his) mutation in the HBA1 gene.
• .0127 HEMOGLOBIN SAVARIA [HBA1, SER49ARG]
• See Szelenyi et al. (1980), Juricic et al. (1985), Ojwang et al. (1985), and Suarez et al. (1985).
• .0128 HEMOGLOBIN SAWARA [HBA1, ASP6ALA]
• No pathologic effects were observed (Sumida et al., 1973; Sumida, 1975).
• .0130 HEMOGLOBIN SETIF [HBA1, ASP94TYR]
• See Wajcman et al. (1972), Nozari et al. (1977), Al-Awamy et al. (1985), and Abdo (1989). Schiliro et al. (1991) found this hemoglobin variant in Sicily.
• Dincol et al. (2003) stated that Hb Setif was first described in an Algerian family (Wajcman et al., 1972) and subsequently in Iranian, African, Saudi Arabian, and Maltese populations. They identified the variant in a Turkish family. Heterozygotes were asymptomatic.
• .0131 HEMOGLOBIN SHAARE ZEDEK [HBA1, LYS56GLU]
• See Abramov et al. (1980).
• .0132 HEMOGLOBIN SHENYANG [HBA1, ALA26GLU]
• See Zeng et al. (1982) and Yi et al. (1989).
• .0133 HEMOGLOBIN SHIMONOSEKI [HBA1, GLN54ARG]
• HEMOGLOBIN HIKOSHIMA
• See Yamaoka et al. (1960) and Hanada and Rucknagel (1964).
• .0134 HEMOGLOBIN SHUANGFENG [HBA1, GLU27LYS]
• See Liang et al. (1981).
• .0135 HEMOGLOBIN SINGAPORE [HBA1, ARG141PRO]
• See Clegg et al. (1969).
• .0137 HEMOGLOBIN ST. CLAUDE [HBA1, LYS127THR]
• See Vella et al. (1974).
• .0138 HEMOGLOBIN ST. LUKE'S [HBA1, PRO95ARG]
• See Bannister et al. (1972).
• Felice (2003) cited evidence that Hb St. Luke's is a mutation of the HBA1 gene.
• .0139 HEMOGLOBIN STANLEYVILLE-II [HBA1, ASN78LYS]
• See Van Ros et al. (1968), North et al. (1980), and Rhoda et al. (1983). Costa et al. (1991) described a family with 1 homozygote and 3 heterozygotes for Hb Stanleyville II. The pattern of restriction fragments demonstrated an associated 3.7-kb alpha-globin gene deletion.
• .0140 HEMOGLOBIN STRUMICA [HBA1, HIS112ARG]
• HEMOGLOBIN SERBIA
• See Niazi et al. (1975) and Beksedic et al. (1975).
• .0143 HEMOGLOBIN SUNSHINE SETH [HBA1, ASP94HIS]
• See Schroeder et al. (1979).
• .0144 HEMOGLOBIN SURESNES [HBA1, ARG141HIS]
• See Poyart et al. (1976) and Saenz et al. (1978).
• .0145 HEMOGLOBIN SWAN RIVER [HBA1, ASP6GLY]
• See Moo-Penn et al. (1987). Harano et al. (1996) observed this variant in a Japanese man with mild polycythemia.
• .0147 HEMOGLOBIN THAILAND [HBA1, LYS56THR]
• See Pootrakul et al. (1977).
• .0148 HEMOGLOBIN TITUSVILLE [HBA1, ASP94ASN]
• See Schneider et al. (1975).
• .0149 HEMOGLOBIN TOKONAME [HBA1, LYS139THR]
• See Harano et al. (1983).
• .0150 HEMOGLOBIN TORINO [HBA1, PHE43VAL]
• See Beretta et al. (1968) and Prato et al. (1970).
• .0151 HEMOGLOBIN TOTTORI [HBA1, GLY59VAL]
• See Nakatsuji et al. (1981).
• .0152 HEMOGLOBIN TOYAMA [HBA1, LEU136ARG]
• HEINZ BODY HEMOLYTIC ANEMIA
• This hemoglobin variant is associated with congenital Heinz body anemia (Ohba et al., 1987).
• .0153 HEMOGLOBIN TWIN PEAKS [HBA1, LEU113HIS]
• See Guis et al. (1985). This has been shown to be a mutation of the HBA1 gene (Cash et al., 1989).
• .0154 HEMOGLOBIN UBE-2 [HBA1, ASN68ASP]
• See Miyaji et al. (1967). In Turkey, Bilginer et al. (1984) found the first instance of Hb Ube-2 outside Japan. It occurred in other members of the family.
• Cotton et al. (2000) found this rare variant during universal neonatal screening. The patients had normal hematologic parameters. The variant was found in twins and an older sister and in the father; both parents were of Belgian ancestry.
• Shin et al. (2002) described the disorder in a Taiwanese subject.
• .0155 HEMOGLOBIN UBE-4 [HBA1, GLU116ALA]
• See Ohba et al. (1978).
• .0156 HEMOGLOBIN WESTMEAD [HBA1, HIS122GLN]
• This variant was found in a Chinese woman (Fleming et al., 1980). See Liang et al. (1988).
• .0157 HEMOGLOBIN WINNIPEG [HBA1, ASP75TYR]
• See Vella et al. (1973) and Nakatsuji et al. (1983). This has been shown to be a mutation of the HBA1 gene (Cash et al., 1989).
• .0158 HEMOGLOBIN WOODVILLE [HBA1, ASP6TYR]
• Since alpha-6 asp is involved in salt linkage with alpha-127 lys of the same chain, the increased oxygen affinity of hemoglobin variants at this position probably reflects loss of this salt bridge in the deoxy state. Similar changes have been observed for Hb St. Claude which also cannot form the salt bridge because of substitution of threonine for lysine at alpha-127. See Como et al. (1986).
• .0159 HEMOGLOBIN WUMING [HBA1, LYS11GLN]
• HEMOGLOBIN J (WENCHANG-WUMING)
• See Zeng et al. (1981). Qualtieri et al. (1995) found this fast-migrating hemoglobin variant in a pregnant woman living in Italy.
• .0160 HEMOGLOBIN ZAMBIA [HBA1, LYS60ASN]
• See Barclay et al. (1969).
• .0161 HEMOGLOBIN BELLIARD [HBA1, LYS56ASN]
• See Wajcman et al. (1990).
• .0162 HEMOGLOBIN TONOSHO [HBA1, ALA110THR]
• In the course of measuring hemoglobin A1c by automated cation exchange high performance liquid chromatography, Ohba et al. (1990) detected a new alpha-chain variant: substitution of alanine by threonine at position 110. The abnormal alpha chain comprised about 14% of the total alpha chain.
• .0163 HEMOGLOBIN FUKUTOMI [HBA1, ASP126VAL]
• This hemoglobin, which has a high affinity for oxygen, was detected in a Japanese male during a screening survey. The proband was a 53-year-old man with liver cirrhosis and hemorrhagic gastritis (Hidaka et al., 1990).
• .0164 HEMOGLOBIN PORT HURON [HBA1, LYS56ARG]
• Zwerdling et al. (1991) investigated the structural abnormality of a putative Hb E detected in an African-American family with no apparent Asian ancestry. The tryptic peptide map formed by high performance liquid chromatography showed that the electrophoretic variant was indeed the beta glu26-to-lys mutation of Hb E. In addition, however, the tryptic map showed an abnormal alpha peptide. The second mutation was a substitution of arginine for lysine at residue 56 of the alpha chain. The variant was clinically silent.
• .0166 HEMOGLOBIN PAVIE [HBA1, VAL135GLU]
• See Wajcman et al. (1990).
• .0167 HEMOGLOBIN QUESTEMBERT [HBA1, SER131PRO]
• See Wajcman et al. (1990, 1993).
• .0168 HEMOGLOBIN THIONVILLE [HBA1, NH2 EXTENSION, VAL1GLU]
• See Vasseur et al. (1990). Substitution of glutamic acid for valine as the first residue in the mature protein is accompanied by retention of the initiator methionine residue. This may be the only known hemoglobin variant with an NH2-extension in the alpha-globin chain. Hb Marseille (141900.0171), Hb Doha (141900.0069), and Hb South Florida (141900.0266) are examples of hemoglobin variants with an NH2-extension due to retention of the initiator methionine in the beta-globin chain. Each is due to mutation in the first or second residue of the mature protein. Vasseur et al. (1992) found that elongation of the NH2-terminus of the alpha-chain, due to inhibition of cleavage of the initiator methionine which is then acetylated, modifies the 3-dimensional structure of hemoglobin at a region that is known to have an important role in the allosteric regulation of oxygen binding. Hb Thionville has a lowered affinity for oxygen. In contrast, response to 2,3-diphosphoglycerate is normal.
• .0169 HEMOGLOBIN KANAGAWA [HBA1, LYS40MET]
• In the course of a high performance liquid chromatography survey of Hb A1c, Miyashita et al. (1992) detected a new hemoglobin in a 70-year-old Japanese male with cerebral infarction and erythremia. Further studies revealed a lys40-to-met mutation. The variant showed increased oxygen affinity, decreased heme-heme interaction, and a lowered 2,3-diphosphoglycerate effect.
• (Erythemia, a now almost obsolete synonym for polycythemia and erythrocytosis, means increased red blood cell mass.)
• .0170 HEMOGLOBIN TURRIFF [HBA1, LYS99GLU]
• In a diabetic woman of Scottish ancestry, Langdown et al. (1992) detected a new hemoglobin variant in the course of determining Hb A1c by high performance liquid chromatography. The abnormal hemoglobin chromatographed with the Hb A1c fraction. Family studies showed that a lys99-to-glu mutation, which was not associated with any hematologic disturbance, had occurred de novo. An AAG-to-GAG mutation was presumed and was not assigned to either the alpha-2- or alpha-1-globin chain.
• The Hb A(1c) level in the patient of Langdown et al. (1992) was found to be very high. In a Japanese individual, Harano et al. (2003) likewise found an unexpectedly high Hb A(1c) level as measured by an automatic Hb A(1c) analyzer and found by DNA sequencing a change in the first nucleotide of codon 99 (AAG-GAG) of the Hb A1 gene.
• .0171 HEMOGLOBIN ZAIRE [HBA1, 15-BP TANDEM REPEAT]
• Hemoglobin Zaire was found in a 36-year-old patient from Zaire during a systematic hemoglobin study. Wajcman et al. (1992) demonstrated that the abnormality was the insertion of 5 amino acids--his, leu, pro, ala, glu--between glu116 and phe117 of the alpha-globin chain. This sequence represented a tandem repeat of the 5 amino acid residues from 112 through 116, located at the end of the GH corner of the molecule. Hemoglobin Grady (141800.0045) involves the insertion of 3 amino acids as repeats of residues 116, 117 and 118. Unequal crossing over between alleles rather than between the separate alpha-1 and alpha-2 loci was thought to be the mechanism in that case and possibly in the case of Hb Zaire as well.
• .0172 HEMOGLOBIN LUTON [HBA1, HIS89LEU]
• In a newborn infant and the father, a 35-year-old Pakistani man, Williamson et al. (1992) described a new hemoglobin with high oxygen affinity. The high affinity hemoglobin mutation was identified by HPLC peptide mapping and amino acid sequencing; leucine was substituted for histidine at amino acid position 89. The mutation occurred at the end of the F helix (FG1), a part of the hemoglobin structure critical in determining oxygen affinity since it is directly linked to the heme iron through the proximal histidine residue F8. This was the first example of a mutation at this position of the alpha chain of hemoglobin, although there were 2 high affinity mutants that involved the structurally equivalent amino acid (beta94 asp) of the beta chain: Hb Barcelona (beta94 his; 141900.0016) and Hb Bunbury (beta94 asn; 141900.0035). The new hemoglobin was called Hb Luton for the name of the hospital where the proband was originally treated. The proband was a neonate in whom 2 abnormal hemoglobin bands were found, the 2 bands being the mutant forms of fetal and adult hemoglobins containing the anomalous alpha globin. The father had microcytosis as well as mild polycythemia and was shown to have an accompanying alpha-thalassemia trait due to deletion of a single alpha-globin gene.
• .0173 HEMOGLOBIN OZIERI [HBA1, ALA71VAL]
• During a screening for hemoglobinopathies in Sardinia, Ferranti et al. (1993) found a new 'silent' hemoglobin variant in 5 apparently unrelated newborn babies. The variant was detected by means of isoelectric focusing (IEF), and further study revealed a valine for alanine substitution at position 71 of the alpha-globin chain. The substitution indicated that a C-to-T transition had occurred in the GCG codon for alanine which contains one of the 35 unmethylated CpG dinucleotides of the HBA1 gene. This observation brought to 13 the number of variants due to mutation in the CpGs of the HBA1 gene and raised the possibility that unmethylated CpGs, like methylated ones, may be hotspots for mutations.
• .0174 HEMOGLOBIN ADANA [HBA1, GLY59ASP]
• In 3 Turkish children with severe thalassemia, Curuk et al. (1992) found a GGC-to-GAC mutation in codon 59 of the HBA1 gene resulting in a replacement of glycine by aspartic acid. The combination of an alpha-thal-1 deletion with the unstable Hb Adana resulted in a severe type of Hb H disease.
• .0175 HEMOGLOBIN AL-AIN ABU DHABI [HBA1, GLY18ASP]
• During a routine program of hemoglobin screening performed in the United Arab Emirates, Abbes et al. (1992) found an electrophoretically fast-moving variant in a 9-month-old girl and in several members of her family. Amino acid sequencing demonstrated that the new variant had a gly18-to-asp substitution. Its functional properties were normal.
• .0176 HEMOGLOBIN POITIERS [HBA1, HIS45ASP]
• Hb Poitiers was discovered by Bardakdjian et al. (1994) in a 9-year-old French Caucasian boy who suffered from chronic anemia. The molecular defect consists of a missense mutation at codon 45 of the HBA1 gene, changing histidine to aspartate. Hb Poitiers displays a 2-fold increased oxygen affinity, a slightly decreased heme-heme interaction, and a slightly faster autooxidation rate. In adult hemoglobin (Hb A), the histidine residue at position 45 of the alpha-globin gene is the only polar contact between the heme group and globin. This position, however, seems to allow for moderate variation without dramatic consequences on the function of hemoglobin. His45 is replaced by glutamine in Hb Bari (141800.0009) and by arginine in Hb Fort de France (141800.0034).
• .0177 MOVED TO 141850.0062
• .0178 HEMOGLOBIN CAEN [HBA1, VAL132GLY]
• Wajcman et al. (1993) discovered the Hb Caen variant in a 25-year-old French Caucasian woman suffering from a mild chronic hemolytic anemia. Trypsin degradation of the isolated hemoglobin alpha chain followed by high performance liquid chromatography indicated that the valine residue at position 132 was replaced by glycine.
• .0179 HEMOGLOBIN YUDA [HBA1, ALA130ASP]
• Hb Yuda was discovered in a 65-year-old Japanese female with noninsulin-dependent diabetes mellitus (Fujisawa et al., 1992). Gas phase Edman degradation indicated that the abnormal hemoglobin alpha chain has a substitution of aspartic acid for alanine at residue 130. Hb Yuda has a very low oxygen affinity and slightly decreased cooperative subunit interaction.
• .0180 HEMOGLOBIN CAPA [HBA1, ASP94GLY]
• Hb Capa was discovered in a 28-year-old female in Turkey who was being treated for chronic iron deficiency anemia. The hemoglobin showed abnormal electrophoretic mobility and was mildly unstable in a heat denaturation test. The molecular change was a GAC-to-GGC transition in codon 94, resulting in substitution of glycine for aspartic acid. Three other substitutions of asp-94 are known: Hb Setif (141800.0130), Hb Titusville (141800.0148), and Hb Sunshine Seth (141800.0143). All 4 variants exhibit mild instability.
• .0181 HEMOGLOBIN MONTEFIORE [HBA1, ASP126TYR]
• Wajcman et al. (1992) demonstrated an asp126-to-tyr change in the HBA1 gene in an individual of Puerto Rican descent. At physiologic pH (7.4), the oxygen binding of the patient's red blood cells revealed a 40% reduction. Hb Montefiore appears to have lower cooperativity than other characterized alpha-126 mutants: aspartic acid is replaced by asparagine in Hb Tarrant (141800.0146), by histidine in Hb Sassari (141800.0126), and by valine in Hb Fukutomi (141800.0163).
• .0182 HEMOGLOBIN ROUEN [HBA1, TYR140HIS]
• HEMOGLOBIN ETHIOPIA
• A tyr140-to-his mutation in the HBA1 gene was discovered and characterized in a French patient with polycythemia vera by Wajcman et al. (1992) and in a newborn baby of Ethiopian descent by Webber et al. (1992). This mutation provides an example of an alteration of the C-terminus of the alpha chain, a region involved in the mechanisms of allosteric regulation. Hb Rouen has increased oxygen affinity and decreased cooperativity. A complementary tyr145-to-his mutation (Hb Bethesda; 141900.0022) in the hemoglobin beta chain has more dramatic effects, suggesting that the alpha and beta chains play unequal roles in the overall function of hemoglobin.
• .0183 HEMOGLOBIN MELUSINE [HBA1, PRO114SER]
• Hb Melusine was found in an Algerian patient during a systematic screening for hemoglobinopathies in Luxembourg. Using isoelectric focusing and reverse phase high performance liquid chromatography (RP-HPLC), Wajcman et al. (1993) determined that the molecular mutation at amino acid position 114 of the HBA1 gene changed the residue from proline to serine.
• .0184 HEMOGLOBIN TAYBE [HBA1, THR38DEL OR THR39DEL]
• Girodon et al. (1992) reported the characterization of Hb Taybe, a hemoglobin variant discovered in a young Arabic woman suffering since birth from a severe and highly regenerative hemolytic anemia. DNA amplification and sequencing of the HBA1 gene indicated a 3-bp deletion (encoding threonine) at amino acid position 38 or 39. This variant increases the hydrophobicity of the amino acid chain, and it is quite unstable.
• .0185 HEMOGLOBIN CEMENELUM [HBA1, ARG92TRP]
• Wajcman et al. (1994) described a missense mutation involving the same codon as that involved in Hb Chesapeake (141800.0018), the first high oxygen affinity hemoglobin variant to be described in association with polycythemia (Charache et al., 1966). Hb Chesapeake has an arg92-to-leu substitution; Hb Cemenelum has an arg92-to-trp substitution. Hb J (Cape Town) (141800.0063) has a substitution (arg92-to-gln) in the same codon. Hb Cemenelum was discovered in a French diabetic patient with no hematologic abnormalities. The purified abnormal hemoglobin, like Hb J (Cape Town), displayed only a 1.5- to 2-fold increased oxygen affinity. The findings demonstrate that the degree to which the functional properties are altered by changes in key residues at the alpha-beta interface depends upon the specific residue occupying this position.
• .0186 HEMOGLOBIN RAMONA [HBA1, TYR24CYS]
• Hb Ramona was accidentally detected by isoelectrofocusing in a pregnant woman of part Spanish descent; its mobility was slightly faster than that of Hb A. A TAT-to-TGT change was found at codon 24, corresponding to a replacement of tyrosine by cysteine.
• .0187 HEMOGLOBIN TATRAS [HBA1, LYS7ASN]
• In a 72-year-old woman born in Czechoslovakia, Wajcman et al. (1994) found a lys7-to-asn mutation when investigating the basis for an abnormal level of Hb A1c. No abnormal hematologic features were observed.
• .0188 HEMOGLOBIN LISBON [HBA1, GLU23ASP]
• In a 31-year-old man of Portuguese origin who had suffered from diabetes mellitus since the age of 15 years, Wajcman et al. (1994) found an abnormal hemoglobin during measurement of Hb A1c by an isoelectrofocusing study. There were no abnormal hematologic features.
• .0189 HEMOGLOBIN ROANNE [HBA1, ASP94GLU]
• Kister et al. (1995) described a new hemoglobin variant in a 73-year-old woman from Roanne in central France. She suffered from mild chronic hemolytic anemia. An asp94-to-glu substitution was found in the alpha-1 chain. Aspartate-94 is involved in several contacts, both in the deoxy- and oxy-structures of the hemoglobin.
• .0190 HEMOGLOBIN MALHACEN [HBA1, ALA123SER]
• Kazanetz et al. (1995) observed this variant hemoglobin in an adult male in Granada, Spain, who was evaluated because of severe iron deficiency anemia. Sequencing of the HBA1 gene showed 2 nucleotide changes. One was a simple polymorphism, as both GCG and GCT code for alanine (at codon 120). The second mutation was a GCC-to-TCC change at codon 123 resulting in replacement of alanine by serine. The replacement caused slight differences in the IEF and reversed-phase HPLC experiments, but the stability of the hemoglobin was normal. Family studies were not performed; thus, whether the 2 mutations were in coupling or repulsion was not known.
• .0191 HEMOGLOBIN TUNIS-BIZERTE [HBA1, LEU129PRO]
• In 3 members of a Tunisian family, Darbellay et al. (1995) identified a leu129-to-pro substitution in the HBA1 gene by sequencing the entirety of the HBA2 and HBA1 genes. In the heterozygous state, the variant was manifested by microcytosis, whereas the homozygous state showed moderate anemia with marked microcytosis.
• .0192 MOVED TO 141850.0068
• .0193 HEMOGLOBIN BOIS GUILLAUME [HBA1, ALA65VAL]
• By tiny abnormalities observed during isoelectrofocusing, Wajcman et al. (1995) identified this electrophoretically silent variant in 3 members of a Caucasian-French family. This hemoglobin was the first alpha-chain variant that involved position 64. In the beta chain, the corresponding position, E14, is also occupied by an alanine residue; in Hb Seattle (141900.0256), it is replaced by aspartic acid (ala70-to-asp).
• .0194 HEMOGLOBIN MANTES-LA-JOLIE [HBA1, ALA79THR]
• Wajcman et al. (1995) found this variant hemoglobin during a systematic study of the iron status in a 6-month-old baby and his mother who originated from Chad in North Central Africa.
• .0195 HEMOGLOBIN MOSELLA [HBA1, ALA111THR]
• Wajcman et al. (1995) found this variant in a 35-year-old pregnant woman of Caucasian origin who lived in Luxembourg. The abnormal Hb was also found in one of her daughters.
• .0196 HEMOGLOBIN FUCHU-I [HBA1, HIS72TYR]
• At the Fuchu Municipal Medical Center in Tokyo, Harano et al. (1995) identified 2 Hb variants in the course of assaying glycated hemoglobin, Hb A(1c), of the peripheral blood by cation exchange HPLC. Structural analyses demonstrated that 1 patient had a his72-to-tyr substitution and the other an asn97-to-his substitution (141800.0197) of the alpha-globin chain. These were named Hb Fuchu-I and Hb Fuchu-II, respectively. Both were healthy adults.
• .0197 HEMOGLOBIN FUCHU-II [HBA1, ASN97HIS]
• See 141800.0196.
• .0198 HEMOGLOBIN GOUDA [HBA1, HIS72GLN ]
• In a 54-year-old Dutch woman under treatment for diabetes mellitus, Giordano et al. (1996) incidentally found a silent alpha-chain variant on testing for glycated hemoglobin. A CAC-to-CAA transversion was predicted to result in substitution of glutamine for histidine at residue 72 in the HBA1 gene.
• .0199 HEMOGLOBIN J (BISKRA) [HBA1, 24-BP DEL]
• Wajcman et al. (1998) described Hb J-Biskra, a variant hemoglobin consisting of deletion of 24 nucleotides from the HBA1 gene and 8 amino acid residues from the alpha-globin chain: residues 50-57, 51-58, or 52-59. This variant was mildly unstable in vitro only, and there was no hematologic or biochemical evidence of hemolysis in affected family members. Wajcman et al. (1998) stated that this was the largest deletion reported to that time in a hemoglobin molecule that is expressed at an almost normal level in the red blood cell.
• .0200 HEMOGLOBIN GODAVARI [HBA1, PRO95THR]
• Hb Godavari is the fourth example of a substitution involving neutral residues at position 95 of the alpha-1 chain. In all of these variants, the electrophoretic pattern suggested that the structural modification unmasks a charged residue in the alpha-1/beta-2 contact area. The other examples are Hb Denmark Hill, pro95 to ala (141800.0027); Hb G (Georgia), and pro95 to leu (141800.0038). Hb Godavari shared the same electrophoretic properties as these variants, but displayed minimal alterations of the oxygen-binding properties. Wajcman et al. (1998) identified Hb Godavari in 2 families of different ethnic origin. The first case, found in the Netherlands, involved an Indian patient. The second case was identified a few months later in an African family from Mali, living in France.
• .0201 HEMOGLOBIN OITA [HBA1, HIS45PRO ]
• Hamaguchi et al. (1998) reported a neutral (silent) hemoglobin variant, designated Hb Oita, in which a change from CAC to CCC caused a his45-to-pro substitution. In Hb Bari (141800.0009), his45 is replaced by gln. In Hb Fort de France (141800.0034), his45 is replaced by arg. In Hb Portiers (141800.0176), his45 is replaced by asp.
• .0202 HEMOGLOBIN AGHIA SOPHIA [HBA1, VAL62DEL]
• In a Greek child with Hb H disease, Traeger-Synodinos et al. (1999) found deletion of codon 62 of the alpha-1 gene, leading to alpha-plus-thalassemia. Codon 62 encodes a valine residue at the E11 alpha helix, which is located in the interior of the heme pocket. Substitutions of this valine with other amino acid residues in the alpha as well as beta polypeptide chains lead, in the heterozygous carrier, either to Hb M disease or to congenital nonspherocytic hemolytic anemia. Traeger-Synodinos et al. (1999) assumed that deletion of val at position 62 disrupted the conformation of the alpha chain to such an extent that the mutated subunit was rapidly removed by proteolysis. The final result was an alpha-thalassemia phenotype rather than an unstable hemoglobin syndrome. This conclusion was supported by the apparent absence of an abnormal alpha chain in the peripheral blood of the patient. Hb Evans (141850.0006) is a val62-to-met mutation of the HBA2 gene and was found in a patient with mild hemolytic anemia. Four amino acid substitutions at position 67(E11)val of the beta chain lead to instability of the Hb tetramer and an anemia of variable degrees in the heterozygotes. One of these substitutions, val67 to glu (141900.0163), results in the stable Hb M-Milwaukee-I.
• .0203 HEMOGLOBIN CHAROLLES [HBA1, HIS103TYR ]
• Lacan et al. (1999) detected Hb Charolles in a 46-year-old patient who presented with microcytosis and hypochromia. It was easily detected by isoelectrofocusing and high performance liquid chromatography. It accounted for 11% of the total hemoglobin. The amino acid change resulted from a CAC-to-TAC change in codon 103.
• .0204 HEMOGLOBIN ROUBAIX [HBA1, VAL55LEU ]
• In a French family from the north of France, Prehu et al. (1999) found a new HBA1 variant in 5 members. The variant was initially detected during measurement of glycated hemoglobin in a woman originating from Roubaix. Codon 55 in exon 2 was found to have a heterozygous change from GTT (val) to CTT (leu). This was a neutral variant.
• .0205 HEMOGLOBIN DOUALA [HBA1, SER3PHE]
• In a woman from Cameroon, Prehu et al. (2001) identified a new hemoglobin variant, designated Hb Douala, with a C-to-T transition (TCT-TTT) in the HBA1 gene, resulting in a ser3-to-phe (S3F) amino acid substitution. The patient was also heterozygous for Hb S (141900.0243) and for a 3.7-kb deletional alpha-thalassemia.
• .0206 THALASSEMIA, ALPHA-PLUS [HBA1, 21-BP INS-DUP ]
• In a patient of Iranian descent with the hematologic profile of alpha-plus-thalassemia characterized by mild microcytosis, Waye et al. (2001) found a 21-bp insertion/duplication that gave rise to a predicted alpha-globin chain containing a duplication of amino acid residues 93-99.
• .0207 THALASSEMIA, ALPHA-PLUS [HBA1, 33-BP DEL ]
• In a patient of Greek descent with the hematologic profile of alpha-plus-thalassemia characterized by mild microcytosis, Waye et al. (2001) found a 33-bp deletion in the HBA1 gene resulting in a predicted alpha-globin chain missing amino acid residues 64-74.
• .0208 HEMOGLOBIN DELFZICHT [HBA1, ASN9LYS]
• Harteveld et al. (2002) reported a 69-year-old Dutch woman monitored for diabetes mellitus in whom Hb A(L1c) analysis revealed a clinically silent hemoglobin variant, asn9 to lys (N9K), due to an AAC-to-AAG transversion in heterozygous state. The mutation was identical to that found at the same position in the HBA2 gene that leads to a variant named Hb Park Ridge (141850.0048).
• .0209 HEMOGLOBIN SARATOGA SPRINGS [HBA1, LYS40ASN ]
• In a 34-year-old Caucasian male of Swedish ancestry who lived in Saratoga Springs, New York, Hoyer et al. (2003) identified a hemoglobin variant with abnormal oxygen affinity, designated Hb Saratoga Springs. There was no family history of erythrocytosis. The patient had no smoking history. A change of codon 40 of the HBA1 gene from AAG to AAC resulted in a lys40-to-asn (K40N) change. Lys40 is replaced by glu in Hb Kariya (141800.0081), and by met in Hb Kanagawa (141800.0169). Both of these hemoglobins had been shown to have increased oxygen affinity, but neither was associated with erythrocytosis.
• .0210 HEMOGLOBIN DIE [HBA1, VAL93ALA ]
• In a 7-year-old girl living near the town of Die in southeast France, Lacan et al. (2004) identified a val93-to-ala (V93A) mutation in the HBA1 gene. The family was of French Caucasian origin.
• .0211 HEMOGLOBIN BEZIERS [HBA1, LYS99ASN ]
• In a 72-year-old woman of French Caucasian origin living in the city of Beziers in the south of France, Lacan et al. (2004) identified a lys99-to-asn (K99N) mutation in the HBA1 gene. The variant was found during the determination of Hb A(1c) by high performance liquid chromatography (HPLC) in this diabetic patient. Hematologic data were normal, without hepatomegaly or splenomegaly.
• .0212 HEMOGLOBIN BUFFALO [HBA1, HIS89GLN ]
• In a 32-year-old Somali male living in the Netherlands who was being monitored for diabetes mellitus, Harteveld et al. (2004) identified Hb S (141900.0243) in heterozygous state and a heterozygous C-to-G transversion in the HBA1 gene, resulting in a his89-to-gln (H89Q) substitution. The H89Q mutation had previously been described in a Yemenite woman and 2 apparently unrelated Somali males (Hoyer et al., 2002), and had been designated Hb Buffalo. No hematologic abnormality had been associated with the allelic variant in this or other cases. In addition to Hb Buffalo, 4 amino acid substitutions had been reported at codon 89: Hb Luton (his89 to leu; 141800.0172), Hb Villeurbanne (his89 to tyr; 141800.0213), Hb Tokyo (his89 to pro; 141800.0214), and Hb Tamano (his89 to arg; 141800.0215).
• .0213 HEMOGLOBIN VILLEURBANNE [HBA1, HIS89TYR ]
• Deon et al. (1997) identified a his89-to-tyr (H89Y) mutation in the HBA1 gene as the defect in Hb Villeurbanne.
• .0214 HEMOGLOBIN TOKYO [HBA1, HIS89PRO ]
• Harteveld et al. (2004) stated that Hb Tokyo carries a his89-to-pro (H89P) mutation in the HBA1 gene.
• .0215 HEMOGLOBIN TAMANO [HBA1, HIS89ARG ]
• Harteveld et al. (2004) stated that Hb Tamano carries a his89-to-arg (H89R) mutation in the HBA1 gene.
• .0216 HEMOGLOBIN RICCARTON [HBA1, GLY51SER ]
• In a 4-year-old Caucasian boy investigated for fatigue and microcytosis, Brennan et al. (2005) found a GGC-to-AGC transition at codon 51 in the HBA1 gene, resulting in a gly51-to-ser substitution (G51S). The mutation was thought not to be the cause of the microcytosis as it was detected also in the boy's father who had normal red cell indices.
• .0217 HEMOGLOBIN OEGSTGEEST [HBA1, CYS104SER ]
• In an 8-year-old black female of Surinamese origin with a mild alpha-thalassemia phenotype, Harteveld et al. (2005) identified homozygosity for a TGC-to-AGC transversion in the HBA1 gene, resulting in a cys104-to-ser substitution. Cysteine-104 is involved in alpha/beta globin contact and had been described as a critical amino acid of the HBA2 chain when substituted by a tyrosine (cys104 to tyr) in Hb Sallanches (141850.0031).
• .9999 HEMOGLOBIN ALPHA VARIANTS, MOLECULAR DEFECT UNKNOWN
• HEMOGLOBIN J (INDIA). See Raper (1957).
• HEMOGLOBIN J (MALAYA). See Lehmann (1962).
• HEMOGLOBIN K (CALCUTTA). Fast hemoglobin. See Lehmann (1962).
• HEMOGLOBIN K (MADRAS). See Ager and Lehmann (1957).
• HEMOGLOBIN KARAMOJO. See Allbrook et al. (1965).
• HEMOGLOBIN L (BOMBAY). See Sukumaran and Pik (1965).
• HEMOGLOBIN M (RESERVE). Reduced oxygen affinity and decreased reversible oxygen-binding capacity (Overly et al., 1967).
• HEMOGLOBIN N, ALPHA TYPE. An alpha chain anomaly was deduced from molecular hybridization experiments with canine hemoglobin (Silvestroni et al., 1963). Other hemoglobin N variants have a beta change.
• HEMOGLOBIN NICOSIA. See Fessas et al. (1965).

 
• hemoglobin Bristol: Hb < 7 g/dl +Heinz body
• hemoglobin Chesapeake : an abnormal hemoglobin in which leucine is substituted for arginine in the a chain, resulting in high oxygen affinity so that the individual has erythrocytosis.
• hemoglobin Ferrara
• hemoglobin Gun Hill : an unstable hemoglobin with a segmental deletion of amino acids in the b chain that causes inability to bind heme and mild hemolytic anemia.
• hemoglobin Hammersmith : Hb < 7 g/dl +Heinz body
• hemoglobin Hiroshima : an abnormal hemoglobin; it has increased oxygen affinity and is associated with erythrocytosis.
• hemoglobin Indianapolis (defect in b-globin) => a₂b^Ind₂ and a₂b^Indb tetramers; Hb < 7 g/dl + Heinz body
• hemoglobin Kansas : an abnormal hemoglobin with threonine substituted for asparagine at position 102 of the b chain, resulting in decreased oxygen affinity and cyanosis
• hemoglobin Kempsey : an abnormal hemoglobin; it has increased oxygen affinity and is associated with erythrocytosis.
• hemoglobin Knossos
• hemoglobin Köln : an unstable hemoglobin that has methionine substituted for valine at position 95 of the b chain, usually resulting in Heinz body anemia.
• hemoglobin Olmsted
• hemoglobin Olympia is an electrophoretically silent mutant form of hemoglobin which leads to erythrocytosis
• hemoglobin Rainier : an abnormal hemoglobin in which histidine replaces tyrosine at position 145 in the b chain; it has increased oxygen affinity and is associated with erythrocytosis.^ref
• hemoglobin Seattle : an abnormal hemoglobin in which glutamic acid is substituted for alanine at position 76 of the b chain; it has decreased oxygen affinity.
• hemoglobin Yakima : an abnormal hemoglobin in which histidine is substituted for aspartic acid at position 99 of the b chain; it has increased oxygen affinity and is associated with erythrocytosis.
• hemoglobin Ypsilanti : an abnormal hemoglobin; it has increased oxygen affinity and is associated with erythrocytosis.
• crossover hemoglobin : an abnormal hemoglobin that has a globin chain formed from parts of 2 chains that have undergone crossover, such as
• hemoglobin Lepore : any of several abnormal crossover hemoglobins having 2 normal a chains and 2 globin chains that have portions of a d chain at the N terminus and portions of a b chain at the C terminus
• homozygous individuals have about 90% hemoglobin F, 10% hemoglobin Lepore, and no Hemoglobin A or A₂, which results in thalassemia major
• heterozygotes have varying amounts of hemoglobin Lepore and hemoglobin A and may have mild anemia
• hemoglobin anti-Lepore : an abnormal crossover hemoglobin similar to hemoglobin Lepore but whose non-a chains have fusion in the opposite configuration from those of hemoglobin Lepore (b chain portions at the N terminus and d chain portions at the C terminus); most individuals with this hemoglobin have predominantly normal hemoglobin and do not suffer from anemia or thalassemia.
• hemoglobin Kenya : an abnormal type of crossover hemoglobin in which the non-a chain has a g chain portion at the N terminus and a b chain portion at the C terminus, resulting in ß-thalassemia.
Laboratory examinations :
• PO₂ (according to Hb-O₂ affinity)
• isopropanol precipitation test
• heat stability
Therapy : folic acid 1 mg/day to compensate accelerated erythropoiesis
• structural protein defects (inherited as autosomal dominant traits) : erythrocyte membrane gene defects in disorders of the erythrocyte membrane :

gene	disorder1	comment
ankyrin 1	HS	most common cause of typical dominant HS.
band 3	HS, SAO, nonimmune hydrops fetalis (NIHF)	"pincered" spherocytes seen on smear presplenectomy. SAO due to 9 AA deletion.
a spectrin, erythrocytic	HS, HE, HPP, NIHF	location of mutation in spectrin determines clinical phenotype. a spectrin mutations are most common cause of typical HE.
ß spectrin	HS, HE, HPP, NIHF	"acanthocytic" spherocytes seen on smear presplenectomy. Location of mutation in spectrin determines clinical phenotype.
protein 4.2	HS	common in recessively inherited, HS in Japan.
protein 4.1	HE	an uncommon cause of HE.
glycophorin C	HE	concomitant protein 4.1 deficiency is basis of HE in glycophorin C defects.

• hereditary spherocytosis (HS) : presence of spherocytes in the blood (normocytosis due to balance with microspherocytes !). The HS syndromes are a group of inherited disorders characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear^ref.

Epidemiology : HS is found worldwide. It is the most common inherited anemia in individuals of northern European descent, affecting approximately 1 in 1000–2500 individuals depending on the diagnostic criteria.
Aetiology : membrane loss is due to defects in one of several membrane proteins, including ankyrin 1, erythrocyte membrane portein band 3 (band 3Tambau : point mutation 2102 T>C, changing methionine at position 663 to lysine^ref), a spectrin, erythrocytic, ß spectrin, and erythrocyte membrane protein band 4.2. Erythrocyte membranes from HS patients demonstrate qualitative and/or quantitative abnormalities of these proteins, most commonly combined spectrin and ankyrin deficiency, followed by band 3 deficiency, isolated spectrin deficiency, and protein 4.2 deficiency. The genes responsible for HS include ankyrin, ß spectrin, band 3 protein,  spectrin, and protein 4.2^ref. In approximately 70% of HS patients, inheritance is autosomal dominant. In this group of "typical" HS patients, ankyrin mutations are the most common cause of HS, followed by mutations in band 3 and ß spectrin. Over the past few years, numerous point mutations, defects in mRNA processing, and gene deletions have been described in these genes. Except for a few rare exceptions, HS mutations are private, i.e., each individual kindred has a unique mutation. In the remaining patients, inheritance is nondominant. Cases with autosomal recessive inheritance are due to defects in either -spectrin or protein 4.2. In other non-dominant cases, genetic studies have revealed that an unexpectedly large number of nondominant cases are due to de novo mutations in the HS genes^ref. A few cases of "double dominant" HS due to defects in band 3 or spectrin that result in fetal death or severe hemolytic anemia presenting in the neonatal period have been reported.
Pathogenesis : the primary defect in hereditary spherocytosis is a deficiency of membrane surface area. Decreased surface area may be produced by 2 different mechanisms:
• defects of spectrin, ankyrin, or protein 4.2 lead to reduced density of the membrane skeleton, destabilizing the overlying lipid bilayer and releasing band 3-containing microvesicles.
• defects of band 3 lead to band 3 deficiency and loss of its lipid-stabilizing effect. This results in the loss of band 3-free microvesicles.
Both pathways result in membrane loss, decreased surface area, and formation of spherocytes with decreased deformability^ref. These deformed erythrocytes become trapped in the hostile environment of the spleen where splenic conditioning inflicts further membrane damage, amplifying the cycle of membrane injury. The splenic environment is hostile to erythrocytes. Low pH, low levels of glucose and ATP, and high local concentrations of toxic free radicals produced by adjacent phagocytes all contribute to membrane damage..

Symptoms & signs : vary widely. "Typical" HS is marked by evidence of extravascular hemolysis with anemia, reticulocytosis, splenomegaly, Minkowski-Chauffard hemolytic jaundice, and cholelithiasis, evidence of spherocytosis with spherocytes on peripheral smear and increased erythrocyte osmotic fragility, and a positive family history. The degree of hemolysis varies widely, from asymptomatic patients who are diagnosed incidentally to severe, transfusion-dependent patients. Most HS patients have incompletely compensated hemolysis and mild to moderate anemia. The anemia is often asymptomatic except for pallor and/or fatigue. Jaundice is seen at some time in about 50% of patients, usually in association with viral infections. Splenomegaly is detectable in most older children and adults. About 25% of HS patients have compensated hemolysis and little or no anemia. Most of these patients are asymptomatic except during times of increased erythroid stress, compensating for their hemolysis with increased erythropoiesis. Finally, a small group of HS patients have poorly compensated hemolysis and severe anemia. These patients may develop complications of severe uncompensated anemia including growth retardation, delayed sexual maturation, and extramedullary "tumors," skin (malleolar) ulcers, or thalassemic facies. Many of these patients are transfusion dependent. Complications of cholelithiasis include biliary obstruction, cholecystitis and cholangitis^ref. As in other inherited hemolytic anemias, recent studies have shown that co-inheritance of Gilbert syndrome increases the risk of neonatal jaundice and cholelithiasis in HS patients^ref
• hemolytic crises are characterized by anemia, jaundice, increased splenomegaly, and reticulocytosis. Common in children, they are typically associated with viral illness and rarely require intervention
• deglobulinization crisis are characterized clinically by the acute onset of fever, abdominal pain, and vomiting, associated with reticulocytopenia, leukopenia, thrombocytopenia, and erythroblastopenia.
• aplastic crises occur after virally induced bone marrow suppression, most commonly parvovirus B₁₉. In patients with severe HS, aplastic crisis may lead to severe anemia with serious complications including congestive heart failure or even death. Aplastic crisis may also be the event that brings an HS individual to medical attention, particularly the asymptomatic one with normally compensated hemolysis
• megaloblastic crisis occurs due to deficiency of folic acid in patients with increased folate demands, e.g., the pregnant patient, growing children, or the elderly, or after an aplastic crisis
Initial assessment of a patient with suspected HS should include a family history and questions about history of anemia, jaundice, gallstones and splenectomy. Physical examination should seek signs such as scleral icterus, jaundice, and splenomegaly. After diagnosing a patient with HS, family members should be examined for the presence of HS.
Laboratory examinations :
• cytomorphology : typical HS patients have obvious spherocytes lacking central pallor on peripheral blood smear. Less commonly, only a few spherocytes are present or, in severe cases, there are numerous small, dense spherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis. Specific morphologic findings are associated with certain membrane protein defects such as pincered erythrocytes (band 3), spherocytic acanthocytes (ß spectrin), or spherostomatocytes (protein 4.2)
• mild to moderate hemolytic anemia (reticulocytosis, increased bilirubin, increased LDH, increased urinary and fecal urobilinogen, and decreased haptoglobin)
• MCHC is increased (between 35% and 38%) due to relative cellular dehydration in approximately 50% of patients, but all HS patients have some xerocytes
• incubated osmotic fragility (OF) test is considered the gold standard in diagnosing HS in a patient with Coombs-negative spherocytic hemolytic anemia, particularly one of Northern European descent or with a positive family history of undiagnosed anemia. After incubation at 37°C for 24 hours, HS red cells lose membrane surface area more readily than normal cells because their membranes are leaky and unstable. This exposes the membrane defect upon OF testing. When the spleen is present, a subpopulation of fragile erythrocytes that have been conditioned by the spleen form the "tail" of the OF curve that disappears after splenectomy. OF testing suffers from poor sensitivity as ~20% of mild cases of HS are missed after incubation


• other analyses such as the autohemolysis test, the hypertonic cryohemolysis test, and the acidified glycerol test suffer from lack of specificity and are not widely used
• flow cytometric analysis of eosin-5-maleimide binding to erythrocytes, a reflector of relative amounts of Rh-related integral membrane proteins and band 3, has recently been explored as a screening test for HS diagnosis^ref
• specialized testing, such as membrane protein quantitation, ektacytometry, and genetic analyses, are available for studying difficult cases or when additional information is desired
Therapy :
• patients with mild HS and compensated hemolysis can be followed carefully and referred for splenectomy if clinically indicated. The treatment of patients with mild to moderate HS and gallstones is also debatable, particularly since new treatments for cholelithiasis including laparoscopic cholecystectomy, endoscopic sphincterotomy, and extracorporal choletripsy, lower the risk of this complication.
• splenectomy cures or markedly improves the anemia in most HS patients. Even patients with severe HS exhibit significant clinical improvement. Postsplenectomy, erythrocyte life-span normalizes, transfusion requirements abate, and the incidence of cholelithiasis is reduced. Reticulocyte counts fall to normal or almost normal levels, spherocytosis and altered osmotic fragility persist, but the "tail" of the osmotic fragility curve, created by conditioning of a subpopulation of spherocytes by the spleen, disappears^ref. Laparoscopic splenectomy is now the surgical method of choice, resulting in less postoperative discomfort, quicker recovery time, shorter hospitalization, and decreased costs^{ref1, ref2}. Even huge spleens can be removed laparoscopically. Recently, partial splenectomy via laparotomy has been advocated for infants and young children with severe, usually transfusion-dependent, anemia^{ref1, ref2}. The goal is to palliate the hemolysis and anemia while maintaining residual splenic immune function. Long-term data for this procedure are lacking, but rapid regrowth of the spleen may limit its effectiveness in some cases^ref. Operative complications of splenectomy include local infection, bleeding, and pancreatitis. Overwhelming postsplenectomy infection (OPSI), typically from encapsulated organisms, is an uncommon but significant late complication of splenectomy especially in the first few years of life. The introduction of pneumococcal vaccines and the promotion of early antibiotic therapy for febrile children who have had a splenectomy have led to decreases in the incidence of OPSI. Previously, splenectomy was considered "routine" in HS patients. However, the risk of OPSI, the emergence of penicillin-resistant pneumococci, and growing recognition of increased risk of cardiovascular disease, particularly thrombosis and pulmonary arterial hypertension, have led to reevaluation of the role of splenectomy in HS^{ref1, ref2}. Recent HS management guidelines acknowledge these important considerations when entertaining splenectomy and recommend detailed discussion between health care providers, patient, and family^ref. One approach is to splenectomize all patients with severe spherocytosis and all patients who suffer from significant signs or symptoms of anemia including growth failure, skeletal changes, leg ulcers, and extramedullary hematopoietic tumors. Other candidates for splenectomy are older HS patients who suffer vascular compromise of vital organs. Whether patients with moderate HS and compensated, asymptomatic anemia should have a splenectomy remains controversial
• xerocytosis : presence of xerocytes in the blood.

Aetiology :
• hereditary xerocytosis : a hereditary type of hemolytic anemia characterized by xerocytes, sometimes containing phosphatidylcholine.
• hereditary stomatocytosis : a hereditary type of hemolytic anemia characterized by stomatocytes

Aetiology :
• stomatocytosis I
• stomatocytosis II
• pseudohyperkalemia Cardiff / cryohydrocytosis / cold-sensitive stomatocytosis : a leaky cell disorder in which the erythrocytes show very marked swelling and lysis when stored at 0 or 4°C; the curve is shouldered
• pseudohyperkalemia Chiswick or Falkirk : the curve is shouldered
• pseudohyperkalemia Edinburgh : the curve has a shallow slope
• dehydrated hereditary stomatocytosis (DHS)
• DHS, pseudohyperkalemia and perinatal edema
• stomatin-deficient cryohydrocytosis with mental retardation, seizures, cataracts, and massive hepatosplenomegaly
Laboratory examinations :
• cytomorphology : erythrocytes with a mouth-shaped (stoma) area of central pallor on peripheral blood smear^ref. Stomatocytosis is associated with abnormalities in red cell cation permeability that lead to changes in red cell volume, which may be either increased (hydrocytosis) or decreased (xerocytosis), or, in some cases, near normal
Pathogenesis : poorly understood and the molecular basis (or bases) of this group of disorders is unknown
• over-hydrated HS / hydrocytosis syndromes are characterized by significant stomatocytosis, severe hemolysis, macrocytosis (110–150 fL), elevated erythrocyte sodium concentration, reduced potassium concentration, and increased total sodium plus potassium content^ref. The excess cations elevate cell water, producing large, osmotically fragile cells with a low MCHC (24%–30%). The clinical severity of overhydrated HSt (OHSt) is variable; some patients experience hemolysis and anemia while others are asymptomatic. Stomatin, an integral membrane protein, is decreased or absent from the erythrocyte membranes of affected patients, due to maturational loss in the bone marrow and in the circulation^ref. Stomatin gene mutations have not been found in unrelated stomatocytosis patients deficient in this protein
• dehydrated stomatocytosis syndromes / xerocytosis are characterized by contracted and spiculated red cells, variable numbers of stomatocytes, and target cells on peripheral blood smear^{ref1, ref2}. Most patients have nearly normal erythrocyte morphology, with only a few target cells and an occasional echinocyte or stomatocyte. The MCHC and MCV (95–115 fL) are increased and the osmotic fragility is decreased. Erythrocyte potassium concentration and total monovalent cation content are decreased. The gene for xerocytosis has been mapped to 16q23–q24^ref. A clinical syndrome of xerocytosis, perinatal ascites, and pseudohyperkalemia has been described^ref. Genetic studies of patients with this constellation of disorders and with isolated pseudohyperkalemia also shows linkage to this region^ref.
Hydrocytosis and xerocytosis represent the extremes of a spectrum of red cell permeability defects. Patients with features of both conditions have been reported with variability in the severity of permeability defects, stomatin deficiency, hemolysis, anemia, and numbers of stomatocytes^{ref1, ref2}. This suggests that hereditary stomatocytosis is a complex collection of syndromes caused by various molecular defects
Symptoms & signs :
Therapy : the majority of patients are able to maintain an adequate hemoglobin level, so that splenectomy is not required; an unusual and important characteristic of the stomatocytosis syndromes is a marked predisposition to venous thrombosis after splenectomy^ref. Stomatocytosis patients, both over-hydrated and dehydrated, have developed hypercoagulability after splenectomy, leading to catastrophic thrombotic episodes or chronic pulmonary hypertension. In some cases, this has been attributed to increased erythrocyte-endothelial cell adhesion due to erythrocyte phosphatidylserine exposure^{ref1, ref2}.
Differential diagnosis :
• Rh deficiency syndrome
• b-sitosterolemia
• hereditary pyropoikilocytosis (HPP) : an autosomal recessive blood disorder in which there are many different types of heat-sensitive poikilocytes (pyropoikilocytosis) and erythrocyte fragments, with severe hemolytic anemia, reminiscent of that seen in patients after a thermal burn. It resembles severe hereditary elliptocytosis, but there are few if any elliptocytes. There is a strong association between HE and HPP

Epidemiology : up to a third of family members of HPP patients have HE.
Aetiology : spectrin a^D
• hereditary elliptocytosis / Dresbach's syndrome => extravascular hemolysis
• Southeast Asian ovalocytosis (SAO) is an unusual, dominantly inherited HE variant found in Malaysia, Papua New Guinea, the Philippines, and other parts of Southeast Asia and is now also seen in Europe and North America as a result of immigration. Rounded elliptocytes, or ovalocytes, and characteristic stomatocytes with longitudinal slits are found on peripheral blood smear. Most SAO patients are asymptomatic, although a few experience mild hemolysis. Compared to other membrane defects, SAO red cells are rigid and hyperstable, rather than unstable. The cause of SAO is a 27 bp genomic deletion leading to in-frame deletion of 9 amino acids in band 3. SAO is most prevalent in areas endemic for malaria and various studies have demonstrated that the condition confers some protection against malaria, particularly cerebral malaria.

Aetiology : mutations in erythrocyte membrane portein band 3D27^ref
Epidemiology : common in individuals of African and Mediterranean descent, presumably because elliptocytes confer some resistance to malaria. Worldwide, the incidence of HE is estimated at 1:2,000–4,000, approaching 1:100 in parts of Africa. The actual incidence is unknown, as most patients are asymptomatic. Up to a third of family members of HPP patients have HE. Many HPP patients experience severe hemolytic anemia in childhood that evolves into typical HE later in life
Aetiology : Similar to HS, study of erythrocyte membrane proteins in these disorders has identified qualitative and/or quantitative abnormalities of various erythrocyte membrane proteins^{ref1, ref2, ref3}. These include spectrin a and ß spectrin, protein 4.1 and glycophorin C. The majority of defects occur in spectrin, the principal structural protein of the erythrocyte membrane skeleton. Spectrin is composed of heterodimers of the related but nonidentical proteins a and ß spectrin that self-associate into tetramers and higher-order oligomers. Spectrin integrity is critical for erythrocyte membrane stability and erythrocyte shape and function. Structural and functional defects of protein 4.1 appear to disrupt spectrin-actin contacts in the membrane skeleton. Glycophorin variants are also deficient in protein 4.1. The pathogenesis of the formation of elliptocytes in these syndromes is unknown. HE is inherited in an autosomal dominant pattern with rare cases of de novo mutations^{ref1, ref2}. Abnormalities of either a or ß spectrin associated with the majority of cases of HE/ HPP are due to mutations in the spectrin heterodimer self-association site^{ref1, ref2}. Most of these mutations are missense mutations at, or very near, highly conserved residues of spectrin. In contrast to HS, the HE/HPP syndromes, while also heterogeneous, have been associated with distinct spectrin mutations in persons of similar genetic backgrounds, suggesting a "founder effect" for these mutations. There is great clinical phenotypic heterogeneity in individuals with the same spectrin mutation, even in individuals from the same kindred. Some of this variability is attributable to modifier alleles, such as LELY (Low Expression Lyon).
Pathogenesis : the principal defect in HE/HPP erythrocytes is mechanical weakness or fragility of the erythrocyte membrane skeleton^ref
Symptoms & signs : ranging from asymptomatic carriers to patients with severe, life-threatening anemia. Most patients with "typical" HE are asymptomatic and are diagnosed incidentally during testing for unrelated conditions. It is this subset of HE patients with decreased red cell lifespan who experience hemolysis, anemia, splenomegaly and intermittent jaundice. Many of these patients have parents with typical HE and thus are homozygotes or compound heterozygotes for defects inherited from each of the parents. Interestingly, symptomatology may vary between members of the same family; indeed, it may vary in the same individual at different times.
Laboratory examinations : the hallmark of HE is the presence of cigar-shaped elliptocytes on peripheral blood smear. These normochromic, normocytic elliptocytes may number from few to 100%; the degree of hemolysis does not correlate with the number of elliptocytes present. Spherocytes, stomatocytes and fragmented cells may also be seen. The osmotic fragility is abnormal in severe HE and HPP. Other laboratory findings in HE are similar to those found in other hemolytic anemias and are nonspecific markers of increased erythrocyte production and destruction. In difficult cases or cases desiring a molecular diagnosis, specialized testing such as erythrocyte membrane protein quantitation and genetic testing are available. Erythrocyte life span is normal in most patients, decreased in only ~ 10% of patients.
Differential diagnosis : elliptocytes may be seen in association with several disorders including megaloblastic anemias, hypochromic microcytic anemias, myleodysplastic syndromes and myelofibrosis.
Therapy : rarely required^ref. In a few cases, occasional red blood cell transfusions are required. Similar to HS, in severe cases of HE and HPP splenectomy is essentially curative. The indications for splenectomy in HS are viewed as applicable for HE and HPP. Complications in severe cases, e.g., cholelithiasis, hemolytic, aplastic, or megaloblastic crises, are similar to those in HS.
• McLeod syndrome

Aetiology : in some individuals having the McLeod phenotype of blood
Symptoms & signs : mild hemolytic anemia with acanthocytes, elevated serum CPK, and sometimes muscle wasting and neurological defects. A few cases have manifested as X-linked types of chronic granulomatous disease
• paroxismal nocturnal hemoglobinuria (PNH)^{ref1, ref2}

Aetiology : may result from one of > 100 different point mutations in the PIGA gene on Xp, coding for a phosphatidylinositol glycan type A, required for transfer of glucosamine from UDP-N-acetyl glucosamine onto phosphatidyl inositol in GPI anchor synthesis^{ref1, ref2, ref3}. As eventual autoimmunity leads to apoptosis through GPI-linked proteins, this evenience positively selects the somatic (not germinal !) mutant that doesn't express PIGA. As among GPI-dependent proteins are also CD55 / DAF(which controls the early part of the complement cascade by regulating the activity of the C3 and C5 convertases) and CD59 / MIRL (inhibits terminal complement by preventing the incorporation of C9 onto C5b-8 and therefore preventing the formation of the membrane attack complex (MAC)). While inherited isolated CD55 deficiency (Inab- phenotype) does not cause hemolysis, in 1990 an individual with inherited isolated deficiency of CD59 was described with many features similar to classic PNH, such as intravascular hemolysis with hemoglobinuria and thrombosis of the cerebral veins^ref. Thus CD59 deficiency is the abnormality that is responsible for the hemolysis and thrombosis classic of PNH. Although this abnormal cell population is often found to be monoclonal, it is not unusual that 1 or even several PIG-A mutant clones coexist in the same patient. Therefore it has been suggested that the PIG-A gene may be hypermutable in PNH. In 5 patients with PNH the intrinsic rate of somatic mutations (µ) ranged from 1.24 to 11.2 x 10^-7, against a normal range of 2.4 - 29.6 x 10^-7 mutations per cell division : genetic instability of the PIG-A gene is not a factor in the pathogenesis of PNH^ref
• a novel disease characterized by a propensity to venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In 2 unrelated kindreds, a point mutation (c => g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif. This mutation substantially reduces transcription of PIGM and blocks mannosylation of GPI, leading to partial but severe deficiency of GPI. These findings indicate that biosynthesis of GPI is essential to maintain homeostasis of blood coagulation and neurological function^ref
Pathogenesis : GPI-deficient cells with pig-a mutations occur very frequently at low levels in normal individuals but do not expand in competition with the normal hematopoietic cells. Dacie first proposed that in order to develop PNH 2 things were required: first, the occurrence of a GPI-deficient clone arising in a multipotent hematopoietic stem cell; second, a second event that encourages the expansion of the PNH clone over the residual normal hematopoiesis—a relative growth advantage for the PNH cells^ref. The clue to this second event is the close relationship between aplastic anemia and PNH. It appears that normal hematopoiesis is suppressed by the immune system, presumably either directly or indirectly through one or more GPI-linked antigens, and therefore this attack spares the GPI-deficient PNH clone. Thus, in an environment where there is intense pressure for hematopoiesis (aplastic anemia), the PNH clone is driven to produce mature hematopoietic cells and expands to fill the void left by the aplastic process. The exact mechanism for the relative growth advantage of PNH cells remains unclear but is the subject of considerable scientific activity. It is likely that the symptoms of PNH during a paroxysm, such as esophageal spasm and abdominal pain, are caused by smooth muscle dysfunction due to disturbances in the metabolism of nitric oxide (NO). These symptoms are identical to those observed when free hemoglobin was given to normal individuals during the early attempts to produce artificial blood and can be induced by the inhibition of NO synthetasein normal individuals. Haptoglobin efficiently removes free hemoglobin and is necessary because the function of hemoglobin depends upon its correct compartmentalization in red cells. Free hemoglobin, as is seen in hemolytic PNH, is an extremely efficient scavenger of NO with 10⁶ times greater affinity of the heme moiety for NO than that for oxygen. Thus intravascular hemolysis absorbs NO, disturbs smooth muscle function and causes the symptoms seen during a paroxysm. GPI-deficient platelets are more easily activated by complement than normal platelets. Thus PNH platelets, which comprise the vast majority of platelets in almost all hemolytic patients, similar to the proportion of PNH neutrophils, undergo microvesiculation at far lower concentrations of activated complement leading to greater prothrombinase activity and to thrombus formation^ref. Alternative mechanisms have been suggested, such as deficiency of urokinase plasminogen activator receptor from PNH neutrophils or directly due to intravascular hemolysis, but the platelet abnormalities appear to be the most important factor^{ref1, ref2}.
• classic PNH : patients with classic PNH have clinical evidence of intravascular hemolysis (reticulocytosis, abnormally high concentration of serum lactate dehydrogenase [LDH] and indirect bilirubin, and abnormally low concentration of serum haptoglobin) but have no evidence of another defined bone marrow abnormality. A cellular marrow with erythroid hyperplasia and normal or near-normal morphology, but without nonrandom karyotypic abnormalities, is consistent with classic PNH
• PNH in the setting of another specified bone marrow disorder (eg, PNH/aplastic anemia or PNH/refractory anemia-MDS) : the patients in this subcategory have clinical and laboratory evidence of hemolysis but also have concomitantly, or have had a history of, a defined underlying marrow abnormality. Bone marrow analysis and cytogenetics are used to determine if PNH arose in association with aplastic anemia, myelodysplastic syndrome (MDS), or other myelopathy (eg, myelofibrosis). Standard criteria are used for diagnosis of the bone marrow abnormality (eg, aplastic anemia, MDS, or myelofibrosis). Finding nonrandom karyotypic abnormalities that are associated with a specific bone marrow abnormality may contribute diagnostically (eg, abnormalities of chromosomes 5q, 7, and 20q are associated with MDS).
• PNH-subclinical (PNH-sc) in the setting of another specified bone marrow disorder (eg, PNH-sc/aplastic anemia) : patients with PNH-sc have no clinical or laboratory evidence of hemolysis. Small populations of GPI-AP–deficient hematopoietic cells (peripheral blood erythrocytes, granulocytes, or both) are detected by very sensitive flow cytometric analysis. PNH-sc is observed in association with bone marrow failure syndromes, particularly aplastic anemia and refractory anemia-MDS
Symptoms & signs :
• chronic complement-mediated intravascular hemolysis of GPI-proteins^- RBCs and platelets (=> thrombocytopenia) that is punctuated by episodes, or paroxysms, during which there is a marked increase in the intensity of hemolysis with macroscopic hemoglobinuria, expecially during night, when plasma pH lowers.
• venous thrombosis, which has a predilection for the intra-abdominal and cerebral veins. In 2 historical series of patients, approximately 50% experienced venous thrombosis at some time during their disease and a third of patients died as a result of thrombosis^{ref1, ref2}. The risk of thrombosis is greater in patients from Europe and the USA than in patients from the Far East^ref. A possible explanation is that aplastic anemia is more prevalent in the Far East and therefore patients are more likely to have PNH/AA rather than hemolytic disease. Alternatively patients from different ethnic groups may have different additional inherited prothrombotic traits—however, no correlation between the inherited thrombophilia and thrombosis in PNH has been demonstrated^ref. Risk appears to correlate with the size of the neutrophil PNH clone
• intravascular hemolysis => rise in free plasma hemoglobin => absorption of nitric oxide (NO) => impaired smooth muscle function =>
• abdominal pain
• dysphagia
• erectile failure
• severe lethargy
• 50% suffer from pulmonary arterial hypertension (ASH 2005)
• complications : Pythium insidiosum
Laboratory examinations :
• minimal essential criteria
• evidence of a population of peripheral blood cells (erythrocytes, granulocytes, or preferably both) deficient in GPI-APs. Flow cytometric analysis of peripheral blood erythrocytes, granulocytes, or both using primary antibodies against GPI-APs or the FLAER assay reveals a population of hematopoietic cells that is partially or completely deficient in all GPI-APs. Aerolysin is a protein from Aeromonas hydrophila that binds to the GPI structure^ref. When used for flow cytometric analysis, leukocytes that express GPI-APs bind the fluorescently labeled aerolysin (FLAER). PNH leukocytes do not bind FLAER because they do not express GPI-APs. Therefore, PNH leukocytes are identified in the flow cytometric histogram as a population of cells with absent or dim fluorescence. FLAER cannot be used for analysis of erythrocytes because, for unknown reasons, the reagent does not bind well to human red cells under the conditions required for the assay. The gold standard diagnostic test for PNH is the analysis of GPI-linked molecules on the surface of hematopoietic cells by flow cytometry^ref. An unusual feature of flow cytometry for PNH diagnosis is that a positive test is defined by an absence of antigens rather than the aberrant expression of antigens as seen in most other flow cytometric applications. However in almost all PNH patients there is at least a small residuum of normal cells, which acts as an internal control to demonstrate that the staining and gating strategy are correct. The minimum requirement to diagnose PNH is the demonstration that a proportion of red cells are deficient in at least two different GPI-linked molecules. The proportion of GPI-linked deficient granulocytes is not affected by either hemolysis or transfusions and therefore gives the best estimate of the actual proportion of PNH hematopoiesis. In addition to establishing the diagnosis of PNH, flow cytometry also provides information regarding the severity of deficiency of the antigens from PNH red cells. PNH Type III red cells are completely deficient in GPI-linked molecules and are 15 to 25 times more sensitive to activated complement (compared to normal) whereas PNH Type II red cells have a partial deficiency (PNH Type II cells) and are only 3 to 5 times as sensitive. The size of the PNH clone and type of PNH cells are important determinants of the clinical presentation. For example, patients with hemolytic anemia and macroscopic hemoglobinuria will almost always have over 10% PNH Type III red cells and a majority, often over 95%, of PNH neutrophils. The risk of thrombosis is directly related to the proportion of PNH neutrophils, and this information has been used to stratify patients when deciding who should receive warfarin as primary prophylaxis against venous thrombosis.
• CD14 on monocytes
• CD16 on neutrophils
• CD55 and CD59 on RBCs, neutrophils, monocytes, and lymphocytes
• complete blood count, reticulocyte count, serum concentration of lactate dehydrogenase (LDH), bilirubin (fractionated), and haptoglobin
• bone marrow aspirate, biopsy, and cytogenetics
• recommended supporting studies :
• medical history with emphasis on symptoms of hemoglobinuria, thromboembolic disease, dysphagia, odynophagia, abdominal pain, and male impotence
• determination by flow cytometry of the proportion of PNH I, PNH II, and PNH III erythrocytes (hemolysis depends on both the proportion and type of abnormal erythrocytes)
• PNH type I cells : normal or nearly normal sensitivity to complement
• PNH type II cells (partial deficiency) : require about 25% as much complement as normal cells for an equal amount of lysis
• PNH type III cells (complete deficiency) have about 7% the normal sensitivity to complement
• determination by flow cytometry of the proportion of GPI-AP-deficient granulocytes (determining the contribution of PIGA mutant hematopoiesis may influence therapeutic decisions such as prophylactic anticoagulation)
• serum iron studies (iron concentration, total iron binding capacity, and ferritin concentration) showing iron deficiency anemia (microcytic hypochromic)
• serum concentration of creatinine and blood urea nitrogen (BUN)
• serum concentration of erythropoietin
• urine hemosiderin showing intravascular hemolysis
• HLA type (the association between HLA type and subclinical PNH may have prognostic and therapeutic implications)
• Ham's acidified serum test : PNH red cells have an increased sensitivity to complement
• reduced leukocyte alkaline phosphatase (LAP) (differential diagnosis with CML) and leukopenia
• sucrose hemolysis test
• association with aplastic anemia
• reticulocytosis
• reduced erythrocyte acetylcholinesterase
Therapy : most patients with PNH/AA have only a small GPI-negative clone, which is clinically insignificant. For those with significant clones successful immunosuppressive therapy may improve hematopoiesis so that the bone marrow is able to at least partially compensate for ongoing hemolysis.
• conventional therapeutic strategies :
• in patients with purely hemolytic PNH, the bone marrow is frequently able to compensate fully for the chronic hemolysis. These patients generally only require folate and sometimes iron supplements
• when hemolysis becomes debilitating, steroids have been anecdotally associated with some success, but their chronic usage should be avoided.
• supportive care with transfusionsas required
• the only curative strategy is allogeneic hematopoietic stem cell transplantation but this carries a considerable risk of mortality from the reported series^ref and, in view of the fact that a proportion of patients will eventually experience a spontaneous remission of PNH and with the advent of potentially effective novel therapies, this should only be considered in selected cases, such as those with a syngeneic donor or with associated bone marrow failure. In these patients the indications for transplantation are similar to those for aplastic anemia. The occurrence of venous thrombosis affecting a major vessel is a life-threatening occurrence and the advent of reduced-intensity conditioning allogeneic stem cell transplantation, which carries a lower risk than conventional bone marrow transplantation (BMT), has led to this approach being used in some patients with life-threatening thromboses.
• patients with cytopenias due to associated aplastic anemia will often respond to immunosuppressive therapy with antilymphocyte globulinand/or cyclosporine
• prevention and treatment of thrombosis : the first thrombosis in a patient with PNH heralds a significant deterioration in the patient’s health and a worsening prognosis. Reports of tissue plasminogen activator (tPA)for intra-abdominal venous thrombosis in PNH show that the thrombus can be cleared effectively in a proportion of patients^ref. The standard of care after established venous thrombosis in PNH is life-long full anticoagulation. In view of the high risk of thrombosis and the fact that patients have decreased quality of life following the first thrombosis, selected patients could be considered for warfarin as primary prophylaxis prior to the thrombosis. Hall et al recently reported a retrospective analysis comparing 39 patients at high risk of venous thrombosis treated with warfarin as primary prophylaxis to 56 patients with similar sized clones who were not treated with warfarin^ref. The incidence of thrombosis at 10 years in the group of patients not on warfarin was 36.5%, which was statistically significantly higher than the warfarin group in whom no patients had a thrombosis. However, 2 patients in the warfarin group had major hemorrhages, with 1 dying as a direct result of an intracranial bleed. Despite this being a retrospective analysis and the fact that the patients were not randomized the results suggest that primary prophylaxis may have a role. The data are compelling, particularly in view of the fact that because the patients in the warfarin group were selected for anticoagulation they would be expected to have at least as high a risk of thrombosis as the non-warfarin group. PNH patients with neutrophil clone sizes of over 50%, platelet counts above 100 x 10⁹/L and no other contraindication for warfarin therapy should be considered for primary prophylaxis. There are no studies of antiplatelet drugs, such as aspirin or clopidogrel, in PNH.
• inhibition of the complement cascade : the 3 complement pathways all converge to cleave C5 into C5a, a potent anaphylatoxin, and C5b. C5b is the initial molecule of terminal complement and binds C6, then C7, and then C8. C5b-8 forms the scaffold for C9 molecules, which bind to each other to form the MAC. The MAC forms a pore in the cell membrane, which results in cell lysis. CD59 prevents the incorporation of C9 onto C5b-8. A remarkable feature of the complement cascade is that the MAC appears to be largely redundant in normal adults. Individuals with inherited deficiency of any of the complement molecules prior to C5 are vulnerable to both pyogenic organisms and to autoimmune disorders. In contrast, deficiency of any of the molecules after C5 remarkably has little phenotype. The only apparent sequela is an increased risk of infection by encapsulated organisms, namely Haemophilus influenzae, Neisseria meningitidis and Neisseria gonorrhoea. In the case of N. meningitidis, although the deficient individuals have an increased risk of suffering infection they also have a significantly lower mortality from infection when compared to complement-replete individuals.
• C5 is a good therapeutic target, because blockade here would not only prevent the creation of MAC but would also prevent the release of the potent anaphylatoxin C5a. Eculizumab is a humanized chimeric antibody against C5 with a completely nonfunctional Fc domain. Eculizumab has a high affinity for C5 and thus when bound remains so until the complex is removed from the circulation. Recently a pilot study of eculizumab in 11 patients with transfusion-dependent PNH was reported^ref. The antibody was given at a dose of 600 mg every week for 4 weeks and then at a dose of 900 mg every other week. The drug is given by intravenous infusion over 30 minutes and was extremely well tolerated. The parameters of intravascular hemolysis, namely LDH and AST, immediately fell to normal or near normal levels. The mean LDH prior to eculizumab was 3111 ± 598 IU/L (normal range 150–480) and this fell by the first week to 594 ± 32 IU/L. There was a significant improvement in the quality of life when comparing before the start of eculizumab and at week 12. There was also a significant reduction in mean transfusion requirement for the whole group from 2.1 units per month to 0.6 units per month. The benefit was most evident in patients with normal or near normal platelet counts and therefore no evidence of co-existent clinically significant marrow failure. There was also a complete cessation of hemoglobinuria immediately after commencing eculizumab. The proportion of PNH red cells in patients not on eculizumab is usually significantly lower than the proportion of PNH neutrophils because of the effect of transfusions and also the selective hemolysis of PNH red cells compared to their normal counterparts. The reduction or cessation of transfusions and control of hemolysis in patients on eculizumab leads to an increase in the proportion of PNH red cells toward the level of the PNH neutrophils. The median proportion of PNH type III red cells increased from 36.7% ± 5.9% prior to eculizumab to 59.2% ± 8.2% by week 12 of therapy. This raises the concern that stopping eculizumab for any reason might render the patient susceptible to a massive hemolytic attack and profound anemia. Two of the patients on eculizumab have had a breakthrough from complement blockade immediately prior to the next dose of eculizumab with a return of hemoglobinuria. Although both suffered hemolysis, the severity of the attack was not too extreme and in both the complement was reblocked immediately after the next dose of eculizumab and remained so on a slightly more frequent dosing regimen. Although eculizumab appears to almost completely stop the intravascular hemolysis, the patients’ hemoglobin does not return to normal (it usually plateaus between 10 and 12 g/dL), the bilirubin and reticulocytes remain elevated and the haptoglobin initially becomes detectable but reverts to undetectable within a few weeks of the start of therapy in almost all patients. Thus a continuing low level of well-compensated extravascular hemolysis persists, suggesting that the hemolysis in PNH is not only due to terminal complement activity but that there is also a component of extravascular hemolysis due to complement activity prior to C5. Eculizumab appears to be a highly promising therapy for PNH and is now the subject of a Phase III randomized clinical trial. The complement cascade and eculizumab : the complement cascade culminating in the production of the membrane attack complex that results in the lysis of the cell. The cleavage of C5 is the pivotal point of the pathway. Inherited deficiencies prior to C5 result in recurrent pyogenic infections and autoimmune disorders, whereas deficiencies after C5 have remarkably little effect except for an increased risk of infection by encapsulated organisms. The site of blockage of eculizumab is demonstrated. In the double-blind, randomized, placebo-controlled, multicenter, phase 3 TRIUMPH trial involving 87 patients, intravascular hemolysis was reduced during 6 months of treatment and stabilization of hemoglobin levels in the absence of transfusions was achieved in 49%. Even among patients receiving eculizumab in whom transfusion independence was not reached, the number of units of packed red cells transfused was reduced by 44%, as compared with patients in the placebo group. Residual low-level hemolysis in some patients despite terminal-complement blockade may be caused by an inherent decrease in the survival of PNH type III erythrocytes^ref or may be due to the fact that these cells are opsonized with C3b, which mediates extravascular clearance through the reticuloendothelial system^ref.


• replacement of complement regulatory proteins on PNH cells : an alternative therapeutic strategy in PNH could be to replace CD59, the deficient complement regulatory protein. In the first instance a gene therapeutic^ref approach might appear attractive. However, introducing the pig-a gene into the PNH hematopoietic stem cell is far from trivial and would simply render the "corrected" cell as a target for the aplastic process that was the reason for the proliferation of the PNH clone. An alternative strategy would be to simply replace CD59 on the PNH red cell surface. It is impractical to extract GPI-linked CD59 from cell membranes as it is extremely tightly bound and does not re-attach well to cells in the presence of albumin. CD59 is functional when bound to the cell by alternative mechanisms, such as a transmembrane linkage, but again this approach is impractical^ref. Sah et al^ref22 have recently reported the use of an alternative artificial glycolipid anchor (Prodaptin) to anchor CD59 into the cell. Prodaptin-CD59 has been shown to effectively coat PNH cells in vitro and murine cells in vivo and to restore the cell’s resistance to complement. This approach is attractive because the complement system is not blocked and the increased risk of Neisseria meningitidis, which is seen in complement deficient individuals, is no longer an issue.
In view of the infrequency of PNH it is extremely difficult to perform large or randomized trials of therapeutic interventions. In addition, the advent of complement inhibitors, such as eculizumab, or the routine use of prophylactic anticoagulation promises to alter the natural history of PNH. In fact, the approval process of eculizumab as a therapeutic agent will be on the basis of relatively small and short clinical trials. Thus few patients are likely to have received more than 3 years of eculizumab
Prognosis : spontaneous remission occurs in approximately 15% of hemolytic patients^ref
Web resources : Global Registry for PNH
Laboratory examinations : a flow cytometric method has been described for quantitating the fluorescence intensity of intact red cells after incubation with the dye eosin-5'-maleimide (EMA), which binds specifically to the anion transport protein (band-3) at lysine-430^ref
• enzymopathies (inherited as recessive traits) ...
• ... in Warburg-Dickens-Horecker or pentose phosphate pathway
• favism / glucose-6-phosphate dehydrogenase (G6PD) deficiency

Epidemiology : prevalence : 1:10,000,one of the most prevalent disease-causing mutations worldwide (Beutler E. Glucose-6-phosphate dehydrogenase deficiency and other red cell enzyme abnormalities. In: Beutler E, Lichtman MA, Coller BS, et al, eds. Williams Hematology, 6th ed. 2001:527–547; Prchal JT and Gregg XT. Red cell enzymopathies. In: Hoffman R, Benz E, eds. Hematology: Basic Principles and Practice, 4th ed. 2005:653–659)
Aetiology : G6PD enzymatic activity^D (> 300 different point mutations : 68 Val=>Met; 126 Asn=>Asp; 188 Ser=>Phe; ...)
Most of the G6PD isoenzymes with decreased activity are associated with only moderate health risks without a significant effect on longevity. G6PD is a housekeeping enzyme essential for basic cellular functions, including protecting red cell proteins from oxidative damage. Oxidant damage of hemoglobin leads to the precipitation of hemoglobin, which may be morphologically recognized as Heinz bodies. The enzymatic activity of G6PD generates NADPH that is utilized for glutathione reduction. Reduced glutathione restores hemoglobin to the soluble form. Thus, maintaining a high ratio of reduced-to-oxidized glutathione represents the major defense against oxidative damage of hemoglobin. Reticulocytes have five times higher G6PD enzyme activity than the oldest erythrocyte subpopulation. G6PD is encoded on the X chromosome and thus is subject to dosage compensation by X chromosome inactivation, a phenomenon that was discovered by studies of carrier females for G6PD deficiency. Further investigations of this phenomenon have played a pivotal role in our understanding of the hierarchy of hematopoiesis, clonality of malignant neoplasms, and the mechanism of X chromosome inactivation.
Symptoms & signs : G6PD B is the wild-type allele. > 300 G6PD variants have been defined; most are sporadic but some occur at a high frequency. G6PD variants can be divided into 3 categories based on the type of hemolysis they cause
• acute intermittent hemolytic anemia (most); some of these variants are endemic. Mutations associated with acute intermittent hemolysis or no hemolysis are scattered throughout the gene. Of the G6PD deficient variants that occur at a high frequency, the best known are African G6PD A– and the Mediterranean variants (referred to as G6PD Mediterranean); several variants are also present in Southeast Asia. All of these variants cause acute intermittent hemolysis. G6PD A+ has a gene frequency of 11%, compared to that of 20% for G6PD A– among African Americans. The G6PD A– mutation (202G=> A) arose on a G6PD A+ chromosome (376A=>G). There is no clinical or laboratory evidence of hemolysis unless the individual is exposed to oxidants (drugs), infections or fava beans. Predictably, exposure to the oxidants that cause hemolysis in the acute hemolytic G6PD variants further exacerbates hemolysis in patients with chronic hemolytic G6PD variants. The most common offending agents are :
• fava bean ingestion (hence the name)
• infections
• drugs
• acetylsalicylic acid (ASA) ?
• acetanilide
• nalidixic acid
• chloramphenicol
• doxorubicin
• phenacetin
• isosorbide dinitrate
• isobutyl nitrite
• toluidine blue
• nitrates and nitrites
• methylene blue
• naphthalene
• trinitrotoluene
• nitrofurantoin (Furadantin)
• phenazopyridine (Pyridium)
• phenylhydrazine
• primaquine
• sulfacetamide
• sulfamethoxazole(Gantanol)
• sulfanilamide
• sulfapyridine
In spite of continuation of the drug or persistence of infection, hemolysis is short lasting, presumably due to the elimination of a subpopulation of red cells with very low G6PD activity. The younger red cells and reticulocytes, which have higher G6PD activity, are typically not hemolyzed in mild variants such as G6PD A–.
• chronic hemolytic anemia (very rare) : the severity. Mutations associated with chronic hemolysis tend to cluster in the vicinity of the NADP-binding domain of the G6PD gene of hemolysis is highly variable, ranging from mild to transfusion dependent
• no obvious risk of hemolysis. G6PD A+, a high frequency African-specific polymorphic allele, has almost normal enzyme activity and is not associated with hemolysis
In individuals with a G6PD variant, the generation of NADPH and reduction of glutathione is variably impaired depending on the type of the G6PD mutation
Grading :
• class I : 0 < enzymatic activity < 5 %
• class II : 5 < enzymatic activity < 10 %
• class III : 10 < enzymatic activity < 60 %
Evolutionary benefit of G6PD deficiency : the geographic distribution of populations with a high gene frequency of deficient variants overlaps closely with the prevalence of malaria, suggesting that G6PD deficiency might be protective against malaria. However, the mechanism of protection is unknown. Early studies of females heterozygous for G6PD deficiency showed higher levels of malaria parasites in normal compared to G6PD-deficient red cells. Although malaria invasion of the cells was similar, the growth of the parasites in the G6PD-deficient cells was inhibited^{ref1, ref2}. Different results were found, however, in studies of the Mediterranean variant, where no differences in malaria invasion and growth of G6PD-deficient and non-deficient cells were found^ref. Similarly, conflicting results have been reported about whether hemizygous G6PD-deficient males have protection against malaria^{ref1, ref2, ref3}. The most likely mechanism of malarial protection may be increased phagocytosis of G6PD-deficient erythrocytes containing the early ring-stage parasites. In the ring-stage parasite-infected cells, the level of reduced glutathione was lower in the G6PD-deficient cells compared with normal red cells, leading to membrane damage of deficient cells containing parasites that may be preferentially targeted for destruction^ref
Laboratory examinations :
• the enzymatic activity of G6PD can be assessed by a rapid fluorescent screening test or by quantitative spectrophotometric analysis (Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods, 2nd ed. New York: Grune and Stratton; 1975). However, false-negative results are possible in milder forms of G6PD deficiency, especially if enzymatic analysis is performed shortly after resolution of acute hemolytic episodes when reticulocytes, which have much higher enzymatic activity, predominate and if a screening test rather than a quantitative spectrophotometric analysis of the enzyme activity is used. Females heterozygous for G6PD deficiency are particularly difficult to diagnose by enzymatic assays, but now that the nucleotide substitutions of many G6PD-deficient isoenzymes have been established, molecular diagnostic methods can be used for the diagnosis of females who are heterozygous for common variants
• if oxidative stress is mild => extravascular hemolysis; if it is severe => also intravascular hemolysis.
• cytomorphology : during the hemolytic episodes, either normal red cell morphology or nonspecific abnormalities (anisocytosis, polychromasia) may be observed. The morphological sequelae of the oxidative denaturation of hemoglobin, i.e., Heinz bodies, may be seen either directly on microscopic evaluation of the blood film or after the red cells are preincubated with oxidants such as phenylhydrazine. Bite cells are often cited as pathognomonic for G6PD deficiency; however, this has not been substantiated by any rigorous study known to these authors and the authors of this chapter have never seen "bite cells" during either acute hemolytic crisis or in the rare G6PD variants that are associated with chronic hemolytic states. In contrast we have seen rare "bite cells" in other hemolytic anemias of known and unknown cause wherein we carefully excluded G6PD deficiency. Thus, unless specific association data are provided we consider these cells as non-specific morphological abnormality that is falsely misinterpreted as diagnostic of G6PD deficiency
Therapy : drugs that are known to precipitate hemolysis in G6PD-deficient subjects should be avoided. In subjects with G6PD A– deficiency, hemolysis is typically short-lasting in spite of continuous use of the offending agent. This is not always the case in the more severe Mediterranean variant of G6PD deficiency, and the precipitating agent should always be withdrawn. When anemia is severe and symptomatic, blood transfusion may be necessary. Folate supplementation should be provided in those patients with chronic hemolysis.
• glutathione synthetase^D
• glutathione reductase^D
• ... in anaerobic Embden-Meyerhof-Parnas pathway / glycolysis :
• pyruvate kinase, liver and RBC deficiency : the molecular biology of pyruvate kinase (PK) is complex. 4 different PK isoenzymes are generated by the use of alternative promoters of 2 distinct genes (PK LR and PK M) that have variable expression in different tissues. The R isoform is unique to erythrocytes and gradually replaces the M2 isoform found in early erythroid and myeloid progenitors. PK converts phosphoenol pyruvate to lactate, generating ATP. The activity of PK is increased by an interaction with fructose diphosphate (FDP) that changes the conformational structure of PK.

Epidemiology : PK deficiency is the most common erythrocyte enzyme defect causing chronic congenital hemolytic anemia
Pathogenesis : the mechanism of hemolysis is not clear. Although it has been postulated that the defect in ATP generation contributes to the hemolytic process, this explanation is insufficient because ATP deficiency is difficult to demonstrate in many patients, and other disorders with more severe ATP deficiency are not associated with significant hemolysis. > 100 mutations of PK have been reported. These mutations are scattered throughout the coding regions of the genes^ref, and there appears to be no correlation with the location of the mutation and the severity of the hemolytic anemia^ref. Patients with hemolytic anemia who undergo splenectomy, with a resultant decrease in the hemolysis and improvement of anemia, have a higher number of reticulocytes than they did before the splenectomy. This perplexing observation indicates that our knowledge of the regulation of erythropoiesis and reticulocyte kinetics remains incomplete; however, it has been suggested that reticulocytes, having some mitochondria, have the capacity to make mitochondrial ATP, but as soon as this capacity is lost they are destroyed. The metabolic disturbances in PK deficiency affect both the survival of red cells and the maturation of erythroid progenitors (so far studied only in splenic progenitors). This results in their premature cell death, i.e., apoptosis, as demonstrated in a splenectomized PK deficient patient^ref. The same group made a similar observation in a PK deficient mouse^ref. It remains to be established whether the apoptosis of erythroid progenitors in PK deficiency extends to marrow erythroblasts, if this observation accounts for the previously unexplained post-splenectomy reticulocytosis, and if PK activity has any role in the apoptotic pathway in general^ref.
Malaria protection : PK deficiency is associated with protection against malariain mice^ref. Evidence for a similar effect in humans remains to be proven; however, there is no indication of a positive selection pressure in the geographic distribution of PK deficiency.
Symptoms & signs : the severity of hemolysis in PK-deficient patients is highly variable, ranging from life-threatening transfusion-requiring hemolytic anemia present at birth to a mild fully compensated hemolytic process without anemia. Affected individuals are either homozygous for the same mutation or compound heterozygotes for 2 different PK defects. PK deficiency is distributed worldwide, but it has been reported that the gene is more common among people of northern European extraction and perhaps Chinese and certain other ethnic and racial groups. A high frequency of this disorder has been well-documented among Pennsylvania Amish. Patients with severe hemolysis may be chronically jaundiced and may develop the clinical complications of chronic hemolytic states, including gallstones, transient aplastic anemia crises (often due to parvovirus infection), folate deficiency, and, infrequently, skin ulcers. Splenomegaly is frequently seen but not invariable
Laboratory examinations : there are no specific clinical findings or morphological abnormalities in PK deficiency and no routinely available laboratory measurements aid in diagnosis. A screening test utilizing crude hemolysate with one concentration substrate has been employed for detection of pyruvate deficiency, but it may miss those exceedingly rare PK variants and those more common PK characterized by increased KM variants with abnormal FDP interaction. Specialized laboratories can perform quantitative PK enzyme analysis with various concentrations of substrate with and without FDP. Due to the large number of the mutations and their low prevalence, it is difficult to replace these tests with molecular diagnostic methods.
Therapy : the beneficial effect of splenectomy on hemolysis is well documented; typically, the degree of hemolysis and anemia is ameliorated and in severe cases the transfusion requirement is generally abolished. Unless the patient is transfusion dependent, it is advisable to delay splenectomy until after the age of 3 years when the risk of pneumococcal, Hemophilus influenzae, and meningococcal infections declines.
• hexokinase 1^D
• triosephosphate isomerase 1^D
• pyrimidine 5' nucleotidase (cN-III) deficiency : red cell pyrimidine 5' nucleotidase is a member of a family containing at least 7 genes encoding 5' nucleotidases^ref. Its proper designation is cytosolic 5:Nucleosidase II (cN-III). Other members of this family are responsible for activation of antiviral compounds (cN-II), and ecto-5' nucleosidase (eN), also known as CD73, which plays a role in T-cell activation and cell adhesion. Because of the familiarity of the term pyrimidine 5' nucleotidase to hematologists, cN-III will be referred to here as pyrimidine 5' nucleotidase. Inherited deficiency of pyrimidine 5' nucleotidase is the third most common enzymatic deficiency resulting in hemolysis. Pyrimidine 5' nucleotidase participates in RNA degradation in reticulocytes. The accumulation of pyrimidines in the red cells is presumed to be toxic and a cause of hemolysis. However, recent evidence suggests that deficiency of pyrimidine 5' nucleotidase is at least in part compensated in vivo by other nucleosidases or perhaps other nucleotide metabolic pathways^ref. Pyrimidine 5' nucleotidase deficiency is inherited in an autosomal recessive fashion and is the only congenital hemolytic anemia due to a red cell enzyme deficiency that has a specific, consistent morphological abnormality—basophilic stippling. Lead is a powerful inhibitor of pyrimidine 5' nucleotidase and determination of lead levels should be included whenever the constellation of hemolytic anemia, pyrimidine 5' nucleotidase deficiency, and basophilic stippling is found. Lead-induced acquired pyrimidine 5' nucleotidase deficiency is treatable, unlike the congenital deficiency for which no therapy is available. Several clinical reports of the co-inheritance of hemoglobin E and pyrimidine 5' nucleotidase deficiency leading to severe hemolytic anemia suggest that this enzyme is particularly susceptible to oxidative damage resulting from the instability of hemoglobin E^ref.
• cytochrome b5R deficiency : the most common cause of congenital methemoglobinemia is due to deficiency of the b5R enzyme. These individuals have a decreased ability to reduce the methemoglobin that is formed continuously at physiologic rates. b5R is a constitutively expressed enzyme, a product of a single gene that produces multiple transcripts. There are 2 types of deficiency. Because the b5R enzyme is encoded by a single gene, the suggested explanation for the 2 types of b5R deficiency is that in type I the abnormal gene product is produced at a normal rate but is unstable; as a result, only mature red cells, which cannot synthesize proteins, are affected. By contrast, when mutations cause underproduction of the enzyme or an enzyme with decreased enzymatic activity, the b5R deficiency is generalized to all cell types (type II).
• inherited in an autosomal recessive pattern, type I b5R deficiency is found worldwide, but it is endemic in some populations such as the Athabascan Indians, Navajo Indians, and Yakutsk natives of Siberia. In other ethnic and racial groups, the defect occurs sporadically. Homozygotes or compound heterozygotes have methemoglobin concentrations of 10-35% and appear cyanotic but are usually asymptomatic even with levels up to 40%. Life expectancy is not shortened, and pregnancies occur normally. Significant compensatory elevation of hemoglobin concentration (polycythemia) is sometimes observed. The b5R activity of the erythrocytes of heterozygotes is approximately 50% of normal. Although this activity level is sufficient to maintain normal methemoglobin levels during normal conditions, oxidant stress can overwhelm the erythrocyte’s capacity to reduce methemoglobin and produce acute symptomatic methemoglobinemia
• 10-15% of cases of enzymopenic congenital methemoglobinemia are type II, which is caused by a general deficiency of b5R in all cell types. Type II b5R deficiency is found sporadically worldwide. The main symptoms are cyanosis, mental retardation, and severe developmental delay. Life expectancy is significantly shortened due to the neurological complications, the pathophysiological basis of which remains unexplained. The cyanosis can be effectively treated with methylene blue or ascorbic acid, as in type I b5R deficiency; however, treatment is not indicated except for cosmetic reasons because it has no effect on the neurologic abnormalities. Amniotic cells contain easily measurable b5R activity; thus, prenatal diagnosis of homozygous b5R deficiency is feasible. In summary, congenital methemoglobinemias, with the exception of type II congenital methemoglobinemias, are asymptomatic and only of cosmetic concern to some. In contrast, comparable level of methemoglobin in patients with acquired methemoglobinemia is symptomatic due to severe acute tissue hypoxia and may be life-threatening.
• hereditary high red cell membrane phosphatidylcholine hemolytic anemia (HPCHA) is a rare disease among hemolytic anemias. A dominantly transmitted chronic anemia characterized by an increase of erythrocyte membrane phosphatidylcholine (PC) and cholesterol with decrease of phosphatidylethanolamine (PE). On peripheral blood smears stomatocytes and target cells are evident. The MCV may be increased, but the life span of red cells is decreased. The osmotic fragility is normal or increased, but splenectomy is not effective. These two points are different from other hereditary hemolytic anemias. As in other hemolytic anemias, gallstone and biliary tract disease more frequently occur than in the general population. An imbalance in membrane phospholipid content is attributed to a defect in the enzyme required for the transfer of membrane fatty acid from PC to PE. As a result, cation flux glycolysis and membrane cation pump activity are increased. These abnormality is probably for hemolysis^ref. It appears likely that hereditary xerocytosis and HPCHA both represent a spectrum of disorders resulting from a variety of defects that produce the same general pattern of abnormalities in cation content and membrane phospholipid composition^ref
Non-enzymatic functions of red cell enzymes : there is a growing recognition that glycolytic enzymes have diverse, non-enzymatic functions including mobilization of cell movement, control of apoptosis, and interaction and modulation of regulation of oncogenes (comprehensively reviewed elsewhere)^ref. Some of these discoveries have stemmed from the seminal work of Otto Warburg on respiration and energy metabolism of cancer cells^{ref1, ref2} and their different regulation by hypoxia, which is now partially explained by their constitutive up-regulation of the master regulator of hypoxic responses; i.e., hypoxia inducible factor (HIF)^ref. One of the newly discovered functions of glycolytic enzymes is their role in tumor metastasis and cell motility. Glucose-6-phosphate isomerase acts as an autocrine motility factor (AMF) in cell migration. This activity has been studied in melanoma and it appears to be important in cancer metastasis^{ref1, ref2}. Other glycolytic enzymes have a role in the regulation of apoptosis. Mitochondrial hexokinase appears to inhibit apoptosis via interaction with the pro-apoptotic protein BAD^{ref1, ref2}, while glyceraldehyde phosphate dehydrogenase (GAPD) appears to have a pro-apoptotic function^{ref1, ref2}. In addition, some glycolytic enzymes, including hexokinase, GAPD, enolase, and lactic dehydrogenase, have important roles in the regulation of the transcription of an array of genes by multiple mechanisms^ref. It remains to be determined whether the non-erythroid clinical phenotypes of mutations of some red cell enzyme genes can be explained by their newly discovered non-glycolytic roles. However, the recent discovery of apoptotic erythroid progenitors in the spleen of a pyruvate kinase-deficient individual^ref suggests that other gene mutations previously considered to be hemolysis-specific may have other functions that contribute to the disease phenotype.
• extrinsic aetiology
• physical aetiology => poikilocytes / burr cells
• microangiopathic hemolytic anemias
• cavernous hemangiomatosis or Kasabach-Merritt syndrome
• disseminated intravascular coagulopathy (DIC)
• thrombotic thrombocytopenic purpura (TTP)
• hemolytic uremic syndrome (HUS)
• valvulopathies
• running (RBCs are deformated during hyperextension of plantary arc)
• burns
• chemical aetiology
• arsine (SA) (AsH₃)
• methan chloride CH₃Cl
• trinitrotoluene
• spider venoms
• snake venom(venom hemolysis)
• hypophosphatemia
• infectious aetiology : hemolysin / erythrocytolysin / erythrolysin
• immunitary aetiology : direct or indirect Coombs agglutinations (indirect one tells us if a transfusion will be successful) :
• IgG (hot incomplete) => extravascular hemolysis occured
• IgM (cold complete) => intravascular hemolysis occured
• type II hypersensitivity to
• methyldopaor penicillin
• isoAbs
• transfusion reactions(usually AB0 Ags)
• erythroblastosis fetalis or neonatorum / congenital or hemolytic anemia of newborn / hemolytic disease of the newborn (HDN)
• autoAbs (autoimmune hemolytic anemia (AIHA))
• heterophile hemolysin : a hemolysin that has affinity for erythrocytes of animal species in addition to the one for which it is specific.
• immune hemolysin : a hemolysin produced by deliberate immunization of an animal with blood or blood cells foreign to it, such as the rabbit anti-sheep red blood cell serum (hemolysin) that is used in complement fixation tests.
• immune hemolysis : the lysis by complement of erythrocytes sensitized as a consequence of interaction with specific antibody to the erythrocytes.
• passive hemolysis : the lysis of erythrocytes on which antigen has been adsorbed in the presence of complement and antiserum to that antigen.
Pathogenesis : single hit theory : the theory that hemolysis results from a single complement-induced lesion of the erythrocyte surface, rather than that lesions at several sites are necessary.
Symptoms & signs depend on ...
• site of hemolysis :
• extravascular hemolysis (i.e. increased erythrocatheresis in spleen and liver; e.g. immune hemolytic anemia) => splenomegaly, increase in unconjugated bilirubin, mildly elevated LDH₁ and LDH₂

‡
• intravascular hemolysis (e.g. thrombotic thrombocytopenic purpura, paroxysmal nocturnal hemoglobinuria and hypophosphatemia) => increase in [hemoglobin]_plasma(gold standard : > 1 mg/dL ; when > 50 mg/dL => pink serum), hemoglobinuria (on the contrary of hematuria, urine looks brilliant), sideruria (Fe accumulates also into flaking cells), acholuria, pleiochromic stools, substantially elevated LDH₁ and LDH₂ and AST^ref
• rate of onset :
• acute : malaise, dyspnea, tachycardia, nausea, abdominal pain (similar to acute pancreatitis)
• chronic :
• jaundice
• bones deformation due to bone marrow hyperplasiaformation of gall stones: increased excretion of bile pigments leads to formation of gall stones. The patient may get biliary colic and develop cholecystitis. Obstructive jaundice may be superadded to hemolytic jaundice thereby creating diagnostic dilemma. A young patient with gall stones should always be investigated for hemolytic anemia.
• hemolytic facies
• hypersplenism and hyposplenism or asplenia: certain hemolytic anemias like thalassemia have progressive enlargement of spleen and this manifests as increased transfusion requirements and require splenectomy to reduce transfusion requirement. As against this, in conditions like sickle cell anemia, there is hyposplenia or asplenia i.e. reduced splenic function. This is due to repeated splenic infarcts leading to splenic atrophy. These patients are prone to infections (like splenectomised patients) with capsulated organisms like pneumococci.
• [elevated HbF => reduced peripheral oxygen cession =>] hyperkinetic syndrome with opening of arterovenous anastomoses => chronic venous failure => nonhealing malleolar ulcers : this is commonly seen in sickle cell disease, thalassemia major and hereditary spherocytosis. Maintaining normal hemoglobin with transfusions along with local therapy promotes healing in majority of cases.
• iron overload and iron deficiency : iron derived from hemolysed red cells is retained in the body and re-utilized for hemoglobin synthesis. Anemia and increased erythropoietic activity increase iron absorption. Over the years this iron accumulates and gets deposited in organs like liver, pancreas, heart, testis etc. leading to their dysfunction. Thus patients may have cirrhosis, diabetes, cardiac failure and sterility. The common practice of giving iron to any anemic patient without any investigations could thus be hazardous and should be avoided at all costs. In patients suffering from thalassemia this problem of iron overload is more marked because of multiple transfusions and requires chelation therapy to reduce the body iron stores. As against this patients with chronic hemoglobinuria (e.g. those with severe valvular heart disease and mechanical heart valves) have iron deficiency because with every gram of hemoglobin lost in urine the patient loses 3.4 mg of iron. These patients require iron supplements.
• transfusion transmitted diseases
Laboratory examinations :
• indirect (unconjugated) hyperbilirubinemia : a raised bilirubin is usually present in hemolytic anemia but absence of jaundice does not rule out hemolytic anemia, as it depends on the hepatic reserve which enables to excrete the increased bilirubin load. Differential diagnosis with Gilbert syndrome and Crigler-Najar syndromes
• hemoglobinemia and hemoglobinuria : in cases with intravascular hemolysis, plasma hemoglobin levels rise and when it exceeds renal threshold, there is hemoglobinuria. The patient complains of passing "cola coloured urine." Spectroscopic examination of urine will show bands of oxyhemoglobin
• hemosiderinuria : iron resulting from breakdown of hemoglobin in the renal tubular cells is passed in urine as hemosiderin. This can be demonstrated by centrifuging urine and staining the sediment with Prussian blue which demonstrates bluish intracellular as well as extracellular granules. Hemosiderinuria is seen in chronic intravascular hemolysis.
• examination of blood smear : macrocytosis, polychromasia, punctuates basophilia and nucleated red cells are usually seen in hemolytic anemia and indicate increased erythropoietic activity. Presence of spherocytes points towards hereditary spherocytosis or autoimmune hemolysis. Presence of marked anisopoikilocytosis, hypochromia and target cells should prompt the clinician to investigate for thalassemia syndromes. Malarial parasites should always be looked for in all patients with suspected intravascular hemolysis. Fragmented red cells and schistocytes suggest microangiopathic hemolytic anemia, cardiac hemolytic anemia or chronic intravascular hemolysis. Irreversibly sickled cells may be seen in sickle cell disease.
• reticulocytosis : the reticulocyte count is elevated in majority of cases of hemolytic anemia and this can be demonstrated by supravital staining of the red cells.
• skeletal radiological abnormalities : in congenital chronic hemolytic anemias, marked marrow hypertrophy leads to bony changes which are evident radiologically. Lateral skull x rays show "hiar on end" appearance common in thalassemia. In tubular bones of the extremities, marrow hyperplasia causes thinning of the cortex, decreased density of medulla and coarse trabecular pattern. These changes can be best demonstrated in the metacarpal bones and the ribs.
• reduced haptoglobin
• increased LDH₁ and LDH₂ (substantially in intravascular, mild in extravascular)
• increased RDW (anisocytosis) and MCV (macrocytosis) in chronic hemolytic anemias
• reduced HbA_1c^{ref1, ref2, ref3}. HbA_1c decreased in inverse proportion to the increase in erythrocyte creatine because of a shortened mean age of erythrocytes. The abnormally decreased HbA_1c value could be assessed with erythrocyte creatine^ref.
• increased sTfR^ref
Therapy : splenectomy
• hemorrhages
Laboratory examinations :
• Türk's cell
• target cell
• polycythemias / erythrocythemia / hypercythemia / hypererythrocythemia (sometimes wrongly named polyglobulia) : an increase in the total red cell mass of the blood => polycytosis
• absolute polycythemia : an increase in RBC mass (> 36 ml/kg in the male or > 32 mL/kg in the female, measured with ⁵¹Cr assay) caused by a sustained overactivity of the erythroid component of the bone marrow
• primary polycythemia
• polycythemia vera
Laboratory examinations : decreased [EPO]_plasma
• secondary polycythemia / erythrocytosis / polyglobulia : any absolute increase in the total RBC other than polycythemia vera, occurring as a physiologic response to tissue hypoxia. Increase in blood viscosity is dangerous when Hct > 60 %.
• compensatory and appropriate secondary polycythemia, adjusting for systemic chronic hypoxia
• hemoglobinopathies with increased oxygen affinity :
• hemoglobin Chesapeake
• hemoglobin Yakima
• hemoglobin J (Capetown)
• hemoglobin Kempsey
• hemoglobin Rainier
• hemoglobin Ypsilanti
• hemoglobin Hiroshima
• hemoglobin Olympia is an electrophoretically silent mutant form of hemoglobin which leads to erythrocytosis
• thalassemia minor leads to 'microcytic polycythemia' . More often thalassemia minor presents as refractory hypochromic anemia
The heterozygotes show polycythemia; hence the phenotype is dominant
• inappropriate secondary polycythemia
• nephropathies (renal cysts, hydronephrosis, renal arterial stenosis (RAS), focal glomerulonephritis, renal transplantation)
• paraneoplastic syndrome due to ectopic EPOincretion
• hypernephroma
• ectopic incretion :
• hepatocellular carcinoma (HCC)
• cerebellar hemangioblastoma (usually found in the posterior fossa of the cranial cavity, retina, retroperitoneum^ref, and spinal cord)
• meningioma
• ovarian carcinoma
• pheochromocytoma
• uterine fibromyoma
• Bartter's syndrome
• Cushing syndrome
• factitious polycytemia (EPOor AR agonists used as doping)
• autosomal recessive bening erythrocytosis / Chuvash polycythemia is an autosomal recessive disorder that is endemic to the mid-Volga River region in Chuvashian population of the capital of the Chuvash Republic, Shupashkar (in Russian, Chebokshary)

Aetiology : homozygosity with respect to a C-->T missense mutation in VHL on chromosome 3p25, causing an arginine-to-tryptophan change at amino-acid residue 200 (Arg²⁰⁰Trp) => high erythropoietin levels^ref
Pathogenesis : the protein VHL modulates the ubiquitination and subsequent destruction of HIF-1a. The Arg²⁰⁰Trp substitution impairs the interaction of VHL with HIF-1a, reducing the rate of degradation of HIF-1a and resulting in increased expression of downstream target genes including EPO, SLC2A1 / GLUT1, transferrin, transferrin receptor and VEGF^ref
• autosomal dominant benign erythrocytosis / familial polycythemia / pure primitive erythrocytosis

Aetiology : truncation of the EPOR, which eliminates an important, phosphatase-binding negative signaling region^ref
• defect in the mechanisms for maintaining intra-erythrocytic pH
• one form of dominant erythrocytosis may be due to a mutation in the regulation of 2-3 diphosphoglycerate (Familial erythrocytosis. Birth Defects Orig. Art. Ser. 8(3): 39-45, 1972)
• increased hematocrit (60–75%) was observed after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGFR ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of EPO >40-fold through a HIF-1–independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte–endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo^ref
Laboratory examinations : increased [EPO]_plasma
Therapy : ACEIfor renal transplantation
Laboratory examinations :
• increased HGB and HCT
• [EPO]_plasma
• HbO₂
• anamnesis of tobacco smoking
• tumour screening : chest X-rays and posterior cranial fossa X-rays
• arterial blood gases
Therapy : erythrocytapheresis, phlebotomy
• relative polycythemia / spurious polycythemia / pseudopolycythemia : a decrease in plasma volume without the change in RBC mass of absolute polycythemia, so that the erythrocytes become more concentrated (elevated hematocrit). It occurs in both :
• acute relative polycythemia : a transient condition due to marked loss of body fluid, lowered fluid intake, or a combination
• chronic relative, hypertonic, or spurious polycythemia / benign or stress erythrocytosis, polycythemia, or polycytosis / pseudopolycythemia / pseudopolyglobulia / polycythemia hypertonica / Gaisböck's disease : a chronic type of relative polycythemia, seen most often in middle-aged, mildly obese males who are active, anxiety-prone, and hypertensive, occurring without the characteristic symptoms associated with polycythemia vera, i.e., without leukocytosis, splenomegaly, and thrombocytosis
Symptoms & signs : hypertonia polycythaemica
Laboratory examinations : HGB > 18 mg/dL and HCT > 52 in males; HGB > 16 mg/dL and HCT > 42 in females; HCT > in males and > 55 in females are highly suggestive