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 :
|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|
|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|
|White women, y|
|Black men, y|
|Black women, y|
|White men, y|
|White women, y|
|Black men, y|
|Black women, y|
|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|
|White women, y|
|Black men, y|
|Black women, y|
|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||café-au-lait spots, skeletal
anomalies (thumb and radius),
short stature, microcephaly
|DEB or MMC
|dyskeratosis congenita (DC)||nail dystrophy, macular
or reticular hypopigmentation, mucosal leukoplakia
|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
dysostosis, short stature,
||congenital anomalies (thumb),
short stature, cardiac
(ventricular or atrial septal defects)
|genetic||increased HbF; increased RBC, increased eADA|
> 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 laboratoryref1,
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 studyref.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 associationref.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 functionref.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 developmentref.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 agoref.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 seriesref1, 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 survivalref14 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 1980sref15 and the recently completed Thai NHLBI Aplastic Anemia Study in Bangkok and a northeast rural regionref.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 patientsref.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
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 patientsref.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 failedref.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 Youngref20). The effector cells were identified by immunophenotyping as activated cytotoxic T cells expressing Th1 cytokines, especially -interferon. CD8 cells containing intracellular interferon may now be measured directly in the circulationref,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 clonotyperef.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 deathref.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 milieuref.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 deathref.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 experimentsref.26 In a minor histocompatibility antigen-discordant model, marrow destruction resulted from activity of an expanded H60 antigen-specific T-cell cloneref.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 cyclosporineref1, 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 -308ref1, ref2,30,31 homozygosity for a variable number of dinucleotide repeats in the gene encoding -interferonref,32 and polymorphisms in the interleukin 6 generef.33 Constitutive expression of T-bet, a transcriptional regulator that is critical to Th1 polarization, occurs in a majority of aplastic anemia patientsref.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 marrowref.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 cellsref,36 for which there is some preliminary experimental supportref1, 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-ICsref,39
or cobblestoneforming cellsref,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 elementsref1,
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 -interferonref.44
One peculiar feature of white blood cells in aplastic anemia is short telomeresref1,
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
and later TERT mutationsref49
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
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 controlsref.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 telomeresref51;
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 damageref.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
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.
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 1990sref67 (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 cyclosporineref1,
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 ATGref.70 Comparable figures for hematologic response rates have come from Europeref69 and Japanref.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 Germanref,75 Japaneseref,71 and Chineseref76 trials, compared with about 50% survival for adults older than 60 years in the collective European experienceref.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 commonref.69 Reinstitution of cyclosporine usually reverses declining blood counts, but when required, a second round of horseref78 or rabbitref79 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, ref270,80; 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 therapyref.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
and much larger, randomized studies of G-CSFref1,
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.
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 armref,85 and both relapse and cytogenetic evolution were observedref.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 Baltimoreref.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 ATGref.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 improvedref87;
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% reportedref.83
A third course of immunosuppression may benefit only patients who showed
some response to a previous treatmentref.88
Response to retreatment correlates to a better survival compared with refractory
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 requiredref.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 combinationref,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 toxicityref.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 receptorref,91 and this activity may provide a mechanism of action for their effects on marrow function.
Hematopoietic stem-cell transplantation
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 therapyref.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 alemtuzumabref110 may reduce the frequency and severity of acute GVHD, a predictor of chronic GVHD.
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 adultsref.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 GVHDref.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.
|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|
|"clinical" deficiency||"subclinical" deficiency|
|clinical signs and symptoms||Present (by definition) but:
|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
||not identifiable in at least half of cases
|course||progressive in almost all cases
||unknown, but appears to be slow (many years)
|management||full diagnostic evaluation is mandatory but its nature
and extent are debated.
Therapeutic intervention is mandatory
|diagnostic evaluation is mandatory
therapeutic intervention is probably advisable
|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.|
|Imerslund-Gräsbeck Syndrome (MGA1)||cblE||cblG||methylenetetrahydrofolate reductase (MTHFR) (severe deficiency)|
|gene product(s)||cubilin amnionless||methionine synthase reductase||methionine synthase||methylenetetrahydrofolate reductase|
|serum folate||normal||normal||normal||normal or low|
|homocysteine elevation||not a major feature||yes||yes||yes|
|common polymorphisms (postulated to be associated with common diseases)||66A=>G
|infant (6-11 months)||
|child (1-2 years)||
|female (14-30 years)||
|female, 60-65 years||
|male, 12 and older||
|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.|
|maximum dose (mg iron)||500–1000||125|
|test dose required||yes||no|
|administration time||2–4 hours||10 min|
|anaphylactic reactions||uncommon (0.61%)||very rare (0.04%)|
|delayed reactions||common (2.5%)||uncommon (0.4%)|
||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|
|type of porphyria||neurovisceral||clinical manifestationref|
|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%)|
|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||-|
|coproporphyrinogen III oxidase||HCP||ALA, PBG, COPRO||COPRO (PROTO)||COPRO||-|
|protoporphyrinogen oxidase||VP||ALA, PBG, COPRO||PROTO (COPRO)||PROTO (COPRO)||-|
||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)|
|Cooperative Study for Sickle Cell Disease (CSSCD)ref||58||266||21.8%||16.8-26.8|
|Cumulative numbers from CSSCD,
French Cohort and London Cohort
|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
|role of transcranial Doppler||Associated with an abnormal
|Not necessarily associated with an abnormal TCD
|treatment||Blood transfusion therapy to keep hemoglobin S < 30%ref||No established treatment
Focus of silent cerebral infarct transfusion (SIT) Trial
|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.|