Aplastic Anemia

Paul Ehrlich introduced the concept of aplastic anemia in 1888 when he reported the case of a pregnant woman who died of bone marrow failure. However, it was not until 1904 that Anatole Chauffard named this disorder aplastic anemia.

The theoretical basis for marrow failure includes primary defects in or damage to the stem cell or the marrow microenvironment.[8, 9, 10, 11, 12]  This condition is characterized by both qualitative loss in functions as well as quantitative loss in stem cell numbers. The distinction between acquired and inherited disease may present a clinical challenge, but more than 80% of cases are acquired. External insults (eg, infections, radiation, drugs) may disrupt stem cell homeostasis in marrow environment, leading to altered growth. Clinical and laboratory observations suggest that acquired aplastic anemia is an autoimmune disease.

On morphologic evaluation, the hematopoietic elements in the bone marrow are less than 25%, and they are largely replaced with fat cells. Flow cytometry shows that the CD34 cell population, which contains the stem cells and the early committed progenitors, is substantially reduced.[10] Fas-mediated apoptosis of CD34+ progenitor cells causes stem cell depletion.[13, 14]  

Data from in vitro colony-culture assays suggest profound functional loss of the hematopoietic progenitors, so much so that they are unresponsive even to high levels of hematopoietic growth factors.

Previously, it had been hypothesized that aplastic anemia may be due to a defect at various levels, such as an intrinsic defect of hematopoietic cells; external injury to hematopoietic cells; and defective stroma, which is critical for normal proliferation and functioning of hematopoietic cells. Theoretically, all of these mechanisms could be responsible for aplastic anemia. This theory was the basis of many in vitro stem cell culture experiments using a crossover design in which stem cells from patients with aplastic anemia were cultured with normal stroma and vice versa. The conclusions from these studies led to the understanding that stem cell defect is the central mechanism in the majority of patients with aplastic anemia.[15, 16]

In patients with severe aplastic anemia, stromal cells have normal function, including growth factor production. Adequate stromal function is implicit in the success of hematopoietic cell transplantation (HCT) in aplastic anemia, because the stromal elements are almost entirely (frequently) of host origin.

The role of an immune dysfunction was suggested in 1970, when autologous recovery was documented in a patient with aplastic anemia who failed to engraft after HCT. Mathe proposed that the immunosuppressive regimen used for conditioning promoted the return of normal marrow function. Since then, numerous studies have shown that, in approximately 70% of patients with acquired aplastic anemia, immunosuppressive therapy improves marrow function.[11, 17, 18, 19, 20]

Immunity is genetically regulated (by immune response genes), and it is also influenced by environment (eg, nutrition, aging, previous exposure).[21, 22] Although the inciting antigens that breach immune tolerance with subsequent autoimmunity are unknown, human leukocyte antigen (HLA)-DR2 is overrepresented among European and United States patients with aplastic anemia, and its presence is predictive of a better response to cyclosporine.

Suppression of hematopoiesis is likely mediated by an expanded population of CD8+ HLA-DR+, cytotoxic T lymphocytes (CTLs) that are frequently detectable in the blood and bone marrow of patients with aplastic anemia. These cells produce inhibitory cytokines, such as gamma-interferon and tumor necrosis factor, which can suppress progenitor cell growth. Polymorphisms in these cytokine genes that are associated with an increased immune response are more prevalent in patients with aplastic anemia. These cytokines suppress hematopoiesis by affecting the mitotic cycle and cell killing by inducing Fas-mediated apoptosis.

In addition, such cytokines induce nitric oxide synthase and nitric oxide production by marrow cells, which contributes to immune-mediated cytotoxicity and the elimination of hematopoietic cells. Hirano et al reported that CD8+ cytotoxic T cells raised against kinectin-derived peptides suppress colony-forming units (CFUs) in an HLA class I–restricted fashion, findings that suggest kinectin may be a candidate autoantigen in the pathophysiology of aplastic anemia.[23]

Constitutive expression of Tbet, a transcriptional regulator that is critical to type 1 T helper cell (Th1) polarization, occurs in a majority of aplastic anemia patients.[17] Perforin is a cytolytic protein expressed mainly in activated cytotoxic lymphocytes and natural-killer cells. Mutations in the perforin gene are responsible for some cases of familial hemophagocytosis[24] ; mutations in SAP, a gene encoding for a small modulator protein that inhibits undefined-interferon production, underlie X-linked lymphoproliferation, a fatal illness associated with an aberrant immune response to herpesviruses and aplastic anemia. Perforin and SAP protein levels are markedly diminished in some cases of acquired aplastic anemia.

The transcription factors FOXP3 and NFAT1 have key roles in regulatory T-cell (Treg) development and function, and Tregs play a role in autoimmunity. Tregs are decreased at presentation in almost all patients with aplastic anemia; FOXP3 protein and messenger RNA levels also are significantly lower in patients with this condition, and NFAT1 protein levels are decreased or absent.[25]

Variations in telomere length in peripheral blood cells, especially neutrophils, have been reported in severe aplastic anemia, but the clinical significance of this finding is uncertain. A faster reduction in telomere length in aplastic anemia leads to decreased expression of cell cycle checkpoint genes such as CDK2/6 and MYC, and a high number of mutations is observed in the telomere reverse transcriptase (TERT) gene.[26, 27]

However, although telomere length was unrelated to response, it has been associated with the risk of relapse, clonal evolution, and overall survival in patients with severe aplastic anemia.[28] Studies in murine models also suggest a role of thrombopoietin (TPO) and associated signalling pathway during normal hematopoiesis. Raised levels of TPO have been observed in aplastic anemia, most likely due to compensatory response to diminished stem cells function.[29, 30] Moreover, a decrease in TPO levels is seen after immunosuppressive therapy.[31]

Congenital or inherited causes

Congenital or inherited causes of aplastic anemia are responsible for at least 25% of cases in children and for perhaps up to 10% of adults.[32] Patients may have dysmorphic features or physical stigmata, but marrow failure may be the initial presenting feature. Several loci have been identified that are associated not only with increased susceptibility to aplastic anemia but also with other physical findings.

Fanconi anemia

Fanconi anemia is characterized by the following:

• Multiple congenital anomalies (60-75%): Short stature, abnormal skin pigmentation, malformations of the thumbs with or without dysplastic or absent radii, as well as microphthalmos and malformations of the heart, kidneys, intestines, and ears

• Bone marrow failure: Thrombocytopenia, leukopenia, or aplastic anemia; most patients with Fanconi anemia have bone marrow failure by adulthood

• Cancer: Hematologic malignancies are common with Fanconi anemia and myelodysplasia, with acute myeloid leukemia (AML) being the most common; solid tumors such as those in squamous cell head and neck cancer, female genital tumors, and liver tumors are also seen; Fanconi anemia is associated with increased chromosomal breakage and abnormal sister chromatid exchange in the presence of agents such as diepoxybutane or mitomycin C

• Predominantly autosomal recessive inheritance pattern: Fifteen genes are now known to be causative for Fanconi anemia; only 1 of these— FANCB (X-linked recessive)—is not inherited in an autosomal recessive manner

• Association between bone marrow progression and a complex pattern of recurrent chromosomal abnormalities: Some chromosomal abnormalities are commonly found in non–Fanconi anemia myelodysplastic syndrome (MDS) and secondary AML (eg, -7/7q or RUNX1 abnormalities), whereas others are specific for Fanconi anemia (eg, 1q+ and 3q+)

Dyskeratosis congenita

Dyskeratosis congenita is characterized by the diagnostic physical triad of dysplastic nails, lacy reticular pigmentation of the upper torso, and oral leukoplakia. However, patients may have dyskeratosis congenita without the triad. The following are also features of this condition:

• Some signs of premature aging, such as early graying of the hair
• Progressive bone marrow failure at any age, which can cause any combination of cytopenias, including aplastic anemia
• Malignancy: Common; frequently MDS or AML; solid tumors such as head and neck cancer or genital cancers can also be seen
• Pulmonary fibrosis
• Autosomal dominant, autosomal recessive, and X-linked inheritance patterns; 6 genes are known to cause this disorder

Familial aplastic anemia

This is an isolated aplastic anemia. Mutations have been found in the TERC and TERT genes and are thought to confer a susceptibility to aplastic anemia. These genes encode proteins that are part of the telomerase apparatus that restores repeated regions in the telomere.[33]

Cartilage-hair hypoplasia

Cartilage-hair hypoplasia, which is caused by mutations in the RMRP gene, is inherited in an autosomal recessive manner. This condition is characterized by the following:

• Short stature with short and bowed limbs
• Sparse, lightly pigmented hair
• Variably severe immune deficiency
• Anemia during childhood
• Hematopoietic malignancies, as well as malignancies of the skin, eyes, and liver
• Gastrointestinal malformations and malabsorption

Pearson syndrome

Pearson syndrome causes sideroblastic anemia and exocrine pancreatic dysfunction. This condition results from mitochondrial DNA deletions.

Thrombocytopenia-absent radius syndrome

Thrombocytopenia-absent radius (TAR) syndrome is characterized by deletions located at chromosome 1q21.1 (which are typically about 200kb in size). Patients have bilateral absence of the radii with presence of the thumbs, as well as thrombocytopenia. Other congenital anomalies can also occur (eg, cardiac disease, skeletal anomalies, urogenital anomalies).

Shwachman-Diamond syndrome

Shwachman-Diamond syndrome is caused by mutations in the SBDS gene and is inherited in an autosomal recessive manner. This disease is characterized by dysfunction of the exocrine pancreas with malabsorption and growth failure, as well as cytopenias of single or multiple lineage. Patients with Shwachman-Diamond syndrome also have an increased risk of MDS and AML.[34]

Dubowitz syndrome

Dubowitz syndrome is characterized by intrauterine growth retardation, extremely short stature, and wizened facial appearance. Patients also have microcephaly and mild developmental delay. Dubowitz syndrome is also associated with eczema, immune deficiency, and aplastic anemia. Malignancy is more common with this disorder, particularly lymphoma and neuroblastoma. No single gene has been implicated as responsible for this syndrome.[35]

Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is characterized by a normochromic macrocytic anemia that can be isolated, or can be associated with growth retardation or congenital malformation in the upper limbs, heart, and genitourinary systems. In a small minority of patients, DBA can progress to aplastic anemia. Nine genes have been found to be causative for DBA, and they are inherited in an autosomal dominant manner.[36] Approximately 50% of cases are inherited from a parent, and about 50% result from de novo mutations.

Acquired causes

Acquired causes of aplastic anemia (80%) include the following:

• Idiopathic factors
• Infections, with pathogens such as hepatitis viruses, ^[37] Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), parvovirus, and mycobacteria
• Exposure to ionizing radiation
• Exposure to toxic chemicals, such as benzene or pesticides ^[38]
• Transfusional graft versus host disease (GVHD)
• Orthotopic liver transplantation for fulminant hepatitis
• Pregnancy
• Eosinophilic fasciitis
• Anorexia
• Severe nutritional deficiencies (vitamin B12, folate)
• Paroxysmal nocturnal hemoglobinuria (PNH)
• MDS
• Acute lymphoblastic leukemia (ALL)(rarely)

Drugs and elements (eg, chloramphenicol, gold) may cause aplasia of the marrow. The immune mechanism does not account for the marrow failure in idiosyncratic drug reactions. In such cases, direct toxicity may occur, perhaps due to genetically determined differences in metabolic detoxification pathways. For example, the null phenotype of certain glutathione transferases is overrepresented among patients with aplastic anemia.

PNH is caused by an acquired genetic defect affecting the PIGA gene and limited to the stem cell compartment. Mutations in the PIGA gene render cells of hematopoietic origin sensitive to increased complement lysis. Approximately one third of patients with aplastic anemia have evidence of PNH at presentation, as detected by means of flow cytometry.[39] Furthermore, patients whose disease responds after immunosuppressive therapy may recover with clonal hematopoiesis and PNH.

Very rarely, aplastic anemia has been reported following vaccination. Case reports describe aplastic anemia—de novo or relapse—following vaccination against hepatitis B, influenza, varicella, and COVID-19.[40, 41, 42]

United States statistics

No accurate prospective data are available regarding the incidence of aplastic anemia in the United States. Findings from several retrospective studies usually overlap those from Europe and suggest that the incidence is 0.6-6.1 cases per million population; this rate is largely based on data from reviews of death registries.

International statistics

The annual incidence of aplastic anemia in Europe, as detailed in large, formal epidemiologic studies, is 2 cases per million population.[43] Aplastic anemia is thought to be more common in Asia than in the West. The incidence was accurately determined to be 4 cases per million population in Bangkok,[44] but based on prospective studies, it may actually be closer to 6 cases per million population in the rural areas of Thailand. This increased incidence may be related to environmental factors, such as increased exposure to toxic chemicals, rather than to genetic factors, because this increase is not observed in people of Asian ancestry who are living in the United States.

Race-, sex-, and age-related demographics

Although no racial predisposition for aplastic anemia is reported in the United States, the prevalence is increased in the Far East. The male-to-female ratio for acquired aplastic anemia is approximately 1:1, although a male preponderance has been reported in studies from Asia.[45]

Although aplastic anemia occurs in all age groups, a small peak in the incidence is observed in childhood because of the inclusion of inherited marrow-failure syndromes. A second peak is observed in people aged 20-25 years.

The outcome of patients with aplastic anemia has substantially improved because of improved supportive care. The natural history of aplastic anemia suggests that a small number of patients may spontaneously recover with supportive care[46] ; however, observation and/or supportive care alone is rarely indicated. Specific therapies, including hematopoietic cell transplantation (HCT) and immunosuppressive regiimens, can be curative, particularly in cases with an identical HLA family donor, where overall survival probability is as high as 90%.[47] ​ 

The estimated 10-year survival rate for the typical patient receiving immunosuppression is 68%, compared with 73% for HCT.[48] However, there is a significantly improved outcome for HCT over time, for matched sibling and alternative donors, and with younger age.[48] In cases of immunosuppression, relapse and late clonal disease are risks.

In a single-institution analysis of 183 patients who received immunosuppressive treatments for severe aplastic anemia, the telomere length of peripheral blood leukocytes was unrelated to treatment response. In a multivariate analysis, however, telomere length inversely correlated with risk of relapse, clonal evolution, and overall survival.[28] In a study of 330 patients with aplastic anemia (235 acquired, 85 Fanconi anemia, and 10 Diamond-Blackfan anemia) who underwent HCT from unrelated donors found that longer telomere length in donor leukocytes was associated with increased 5-year survival, but patient leukocyte telomere length was not associated with survival.[49]

In a Japanese study of 64 children with aplastic anemia, multivariate analysis showed that the following were significant variables for poor response to immunosuppressive therapy[50] :

• Telomere length shorter than -1.0 standard deviations (hazard ratio [HR]: 22.0; 95% confidence interval [CI]: 4.19-115; P< 0.001)
• Platelet count at diagnosis less than 25 × 10 ⁹/L (HR: 13.9; 95%CI: 2.00-96.1; P = 0.008),
• Interval from diagnosis to immunosuppressive therapy longer than 25 days (HR: 4.81; 95%CI: 1.15-20.1; P = 0.031)

Mortality/morbidity

Infection and bleeding are major causes of morbidity and mortality from aplastic anemia. Patients who undergo HCT have additional issues related to acute and chronic toxicity from the conditioning regimen and graft versus host disease (GVHD), as well as a potential for graft failure.[22, 51, 52, 53, 54, 55]

In approximately 25-30% of patients with aplastic anemia, the condition does not respond to immunosuppression. In cases with a treatment response, relapse and late-onset clonal disease, such as paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndrome (MDS), and leukemia, are risks—regardless of the treatment response or degree of response.[18, 56, 57, 58, 59]

Kulasekararaj and colleagues reported that the presence of somatic mutations (including ASXL1, DNMT3A, and BCOR) in patients with aplastic anemia for more than 6 months was associated with 40% risk of transformation to MDS. Nearly a fifth of patients with aplastic anemia have mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS.[60]

In a Japanese study of 427 patients (16-72 years old) with aplastic anemia who underwent unrelated-donor bone marrow transplantation, outcome was significantly inferior in patients whose donors were 40 years of age or older than in those with younger donors. In the older donor group, overall survival was significantly inferior (adjusted hazard ratio, 1.64), the incidence of fatal infection was significantly higher (13.7% vs. 7.5%), and primary engraftment failure and acute GVHD occurred significantly more often (9.7% vs. 5.0% and 27.1% vs. 19.7%, respectively).[61]

An Australian population-based cohort study of adults receiving allogeneic HCT reported an elevated secondary cancer risk in several patient groups, including those transplanted for severe aplastic anemia. Overall, in patients alive 2 years after transplantation (n=1463), the cumulative incidence of late mortality was 22.2% at 10 years and the risk of death relative to the matched general population was 13.8.[62]

A study of 663 patients with severe aplastic anemia, 95 of whom developed clonal evolution, found that pre-treatment age > 48 years and absolute neutrophil count > 0.87 × 109/L were strong predictors of high-risk evolution (defined as presence of an overt myeloid neoplasm, or any chromosome 7 abnormality or complex karyotype). Splicing factors and RUNX1 somatic variants were detected exclusively at high-risk evolution; RUNX1, splicing factors, and ASXL1 somatic mutations detected at 6 months after immunosuppressive therapy predicted high-risk evolution.[63]

Five-year overall survival (OS) was 37% in patients with high-risk clonal evolution, versus 94% in those with low-risk. OS was improved in high-risk patients who underwent HCT.[63]

The clinical presentation of patients with aplastic anemia includes symptoms related to the decrease in bone marrow production of hematopoietic cells. The onset is insidious, and the initial symptom is frequently related to anemia or bleeding, although fever or infections may be noted at presentation. Specific manifestations include the following:

• Anemia: May manifest as pallor, headache, palpitations, dyspnea, fatigue, or foot swelling
• Thrombocytopenia: May result in mucosal and gingival bleeding or petechial rashes
• Neutropenia: May manifest as overt infections, recurrent infections, or mouth and pharyngeal ulcerations

Most cases of aplastic anemia are idiopathic,[5] and the search for an etiologic agent is often unproductive. Nevertheless, obtain an appropriately detailed work history, with emphasis on solvent exposure, as well as a family, environmental, and infectious disease history.

In the absence of obvious phenotypic features, the presentation of a patient with an inherited marrow-failure syndrome is subtle, and a thorough family history may first suggest the condition, as well as potentially identify rarer inherited marrow-failure syndromes.

With regard to environmental agents, the time course of aplastic anemia and exposure to the offending agent varies greatly, and only rarely is an environmental etiology identified.

Physical examination may show signs of anemia (eg, pallor, tachycardia) and of thrombocytopenia (eg, petechiae, purpura, ecchymoses). Overt signs of infection are usually not apparent at diagnosis. A subset of patients with aplastic anemia present with jaundice and evidence of hepatitis.[1, 2] Findings of adenopathy or organomegaly should suggest an alternative diagnosis (eg, hepatosplenomegaly and supraclavicular adenopathy are observed more frequently in cases of leukemia and lymphoma than in cases of aplastic anemia).

In any case of suspected aplastic anemia, look for physical stigmata of inherited marrow-failure syndromes, such as the following:

• Abnormal skin pigmentation
• Short stature
• Renal, cardiac, and gastrointestinal (GI) abnormalities
• Microcephaly
• Microphthalmos
• Hypogonadism
• Skeletal anomalies

The oral pharynx, hands, and nail beds should be carefully examined for clues of inherited bone marrow-failure syndromes. Oral leukoplakia in dyskeratosis congenita is shown in the image below.

Oral leukoplakia in dyskeratosis congenita.

Aplastic anemia is diagnosed with blood and bone marrow studies. This condition is defined by the finding of a hypoplastic bone marrow that has fatty replacement and that may have relatively increased nonhematopoietic elements, such as mast cells. Careful examination is necessary to exclude metastatic tumor foci on biopsy, as occasionally metastatic tumor deposits may cause pancytopenia. Diagnosis also involves ruling out other causes of pancytopenia.

Carefully evaluate dysplasia to rule out myelodysplastic syndrome (MDS), although some degree of dysplasia may be present in aplastic anemia. Because abnormal cytogenetic clones can occur in up to 12% of patients with aplastic anemia, the presence of some clones in otherwise typical cases of aplastic anemia does not necessarily signify a diagnosis of MDS or acute myeloid leukemia (AML). However, the presence of the monosomy 7 clone indicates a high risk of transformation to MDS or AML.[5]

Varying degrees of progressive cytopenia are observed in all three hematopoietic lineages. Reduction in platelet and leukocyte counts may precede the fall in hemoglobin. Peripheral smear is unremarkable except for the presence of mild macrocytosis. The degree of cytopenia is useful in assessing the severity of aplastic anemia. A decreased reticulocyte count is a characteristic finding.

A peripheral blood smear may be helpful in distinguishing aplasia from infiltrative disease causes. Teardrop cells, poikilocytes, and leukoerythroblastic changes suggest an infiltrative process.

Peripheral blood tests in patients with suspected aplastic anemia may include the following:

• Hemoglobin electrophoresis and blood-group testing
• Biochemical profile
• Serology
• Fluorescence-activated cell sorter (FACS) profiling
• Fluorescent-labeled inactive toxin aerolysin (FLAER) testing
• Chromosomal breakage analysis 
• Histocompatibility testing
• Emerging diagnostic tests

Hemoglobin electrophoresis and blood-group testing may show elevated levels of fetal hemoglobin (HbF) and red cell I antigen, suggesting stress erythropoiesis. Pretransfusion assessment of HbF levels may help in identifying the cause of aplasia in many cases of inherited bone marrow failure syndromes. A positive Coombs test may point to autoimmune hemolytic anemia.

Although a biochemical profile has limited value in evaluation of the etiology and differential diagnosis of aplastic anemia, an analysis of kidney function, as well as measurement of transaminase, bilirubin, and lactate dehydrogenase (LDH) levels, can indicate relevant kidney or liver diseases. Abnormal liver function test (LFT) results may indicate antecedent/ongoing hepatitis as well as features suggestive of active hemolysis.

Serologic testing for hepatitis and other viral entities, such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV), may be useful. An autoimmune disease evaluation for evidence of collagen-vascular disease may be performed.

Aplastic anemia often occurs together with paroxysmal nocturnal hemoglobinuria (PNH).[64] Screening for PNH involves detection of deficiency of  glycosylphosphatidyl-inositol (GPI) anchored proteins, such as CD14, CD16 and CD24, as well as fluorescent aerolysin (FLAER) for white blood cells, and CD55 and CD59 for red cell analysis, on flowcytometry.

FLAER is a highly sensitive flow cytometry test for PNH that uses whole blood and binds specifically to glycophosphatidylinositol (GPI) anchor proteins in peripheral blood granulocytes.[65, 66] In PNH, mutation of the PIGA anchor protein gene results in a lack of the GPI anchors. Thus, the lack of FLAER binding to granulocytes is sufficient for the diagnosis of PNH.[65, 66] The disadvantage of the test is in measuring binding in the absence of adequate granulocytes—such as in severe aplastic anemia, when the number of circulating granulocytes is extremely low.

Increased chromosomal breakage in the presence of DNA cross-linking agents such as diepoxybutane or mitomycin C is observed in cases with inherited bone marrow failure syndromes (IBMFS). Analysis is performed on metaphase of peripheral blood lymphocytes. Results are reported as aberrations per cell and the number of cells with breaks or radial forms. If the results of a breakage study are normal but there is a high index of suspicion for IBMFS, the test should be performed on skin fibroblasts.

This test is required even in the absence of phenotypic features of Fanconi anemia, because up to 50% of those patients may not have any clinical stigmata. Characteristic findings on chest x-ray and imaging of hands/forearms may help identify various IBMFS.

Newer tests, such as next-generation sequencing (NGS) may help differentiate aplastic anemia from MDS and also identify a subset of patients with a susceptibility for future progression to MDS/AML.[67]

Histocompatibility testing should be conducted early to identify potential related donors, especially those for young patients. Because the extent of previous transfusion has been shown to significantly affect the outcomes of patients undergoing hematopoietic cell transplantation (HCT) for aplastic anemia, the rapidity with which these data are obtained is crucial.

Hosokawa et al identified three microRNAs that are dysregulated (>1.5-fold change) in acquired aplastic anemia, compared with healthy controls, and could be used in diagnosis: miR-150-5p, miR-146b-5p, and miR-1. Plasma levels of miR-150-5p and miR-146b-5p are elevated in aplastic anemia, whereas levels of miR-1 are decreased. Because miR-150-5p expression decreased significantly after successful immunosuppressive therapy, but did not change in non-responders, these authors propose that miR-150-5p could be used for disease monitoring.[68]

In a study of pediatric patients with very severe aplastic anemia, Fang et al found that the percentage of CD20+ B-cells in peripheral blood was higher than that in healthy children (26.26% ± 10.53% versus 15.2 ± 3.7%; P < 0.01), whereas the percentage of regulatory T cells (Tregs) was lower than that in healthy children (2.63% ± 0.94% versus 4.83% ± 1.42%; P < 0.001). After treatment, the percentage of CD20+ B cells decreased, and the percentage of Tregs significantly increased. The authors suggest CD20+ B cells and Tregs as potential markers for evaluating therapeutic efficacy and prognosis in these cases.[69]

Bone marrow biopsy is performed in addition to aspiration to assess cellularity qualitatively and quantitatively. Aspiration samples alone may appear hypocellular because of technical reasons (eg, dilution with peripheral blood), or they may appear hypercellular because of areas of focal residual hematopoiesis. By comparison, core biopsy better reveals cellularity. A trephine biopsy of at least 2 cm is essential to assess overall cellularity and morphology of residual hemopoietic cells and to exclude abnormal infiltrates.

In aplastic anemia, hypocellular marrow particles are observed. The specimen is considered hypocellular if it is less than 30% cellular in individuals younger than 60 years or less than 20% cellular in those older than 60 years (see the following image). Some dyserythropoiesis with megaloblastosis may be observed in aplastic anemia. Focal hyperplasia of erythroid cells may sometimes be observed, in the background of generalized hypocellularity. Small aggregates of lymphoid cells may also be observed in few conditions (autoimmune diseases).

Aplastic anemia. Low-power view of hematoxylin-eosin–stained bone marrow showing hypocellularity, with increased adipose tissue and decreased hematopoietic cells in the marrow space.

Bone marrow culture may be useful in diagnosing mycobacterial and viral infections. However, the yield is generally low. Currently, alternative studies include polymerase chain reaction (PCR) assay, but the value of this technique is unclear in this setting. Leukemia and metastatic cancers may also be diagnosed with bone marrow examination.

Myelodysplastic syndrome

Aplastic anemia must be differentiated from myelodysplastic syndrome (MDS). The bone marrow in patients with aplastic anemia may have hyperplastic pockets, which can sometimes be confused with MDS; moreover, hypoplasia of bone marrow may be present in cases of hypoplastic  MDS.[70] However, in aplastic anemia, CD34 evaluation always reveals a low count; further, ringed sideroblasts, myeloblasts, and dysplastic megakaryocytes are never seen in aplastic anemia but are often seen in MDS.

Characteristic bone marrow abnormalities that are often found in MDS include the following:

• Dyserythropoietic red blood cells (RBCs)
• Neutrophils with hypogranulation, hypolobulation, or apoptotic nuclei reaching to the edges of the cytoplasm
• Dysplastic megakaryocytes (easily highlighted on immunohistochemistry by CD41 and CD61)
• Increased or decreased cellularity
• Increase in blasts

Myelodysplastic features are usually observed in hematopoietic precursors and progeny. Islands of immature cells or abnormal localization of immature progenitors (ALIP) indicate MDS. Patients with MDS may have megakaryocytic abnormalities (micromegakaryocytes, megakaryocytes with dyskaryorrhexis), greater than 15%/5% ring sideroblasts (observed only on iron stains), and granulocytic abnormalities (pseudo–Pelger-Huët cells, hypogranulation, excess of blasts). On occasion, marrow fibrosis may be observed. Monocytes are similarly hypogranular, and their nuclei may contain nucleoli.

Chromosomal rearrangements are considered diagnostic of MDS, with trisomies of 8 and 21 and deletions of 5, 7, and 20 being the most common. However, the conventional karyotype technique reveals abnormalities in only about 50% of patients with MDS. Additionally, fluorescence in situ hybridization (FISH) may be used to visualize chromosomal abnormalities in interphase cells. Note that in hypoplastic marrows, obtaining sufficient sample for karyotyping is often difficult.

Although aplastic anemia is characterized by hypocellularity of bone marrow, a small minority of patients have pockets of hypercellularity, and a bone marrow biopsy may give misleading results if the sample is taken from one of those hypercellular areas. Overall bone marrow cellularity may be assessed by evaluation of magnetic resonance imaging (MRI) scans of the marrow areas of the axial skeleton.

Matcuk et al reported that calculations of bone marrow cellularity based on T1 signal intensity measurements from MRI show statistically significant correlation with determinations of cellularity from bone marrow biopsy. Cellularity increased from T11 to S1 and decreased with patient age.[71]

Staging of aplastic anemia is based on the criteria of the International Aplastic Anemia Study Group (IAASG), also known as modified Camitta criteria.

Severe aplastic anemia (SAA) is defined as marrow cellularity < 25% (or 25–50% with < 30% residual hematopoietic cells), plus at least two of the following peripheral blood findings:

• Neutrophils less than 0.5 × 10 ⁹
• Platelets less than 20 × 10 ⁹/L
• Reticulocytes less than 20 × 10 ⁹/L

Very severe aplastic anemia (VSAA) is defined as as marrow cellularity < 25% (or 25–50% with < 30% residual hematopoietic cells), plus at least two of the following peripheral blood findings:

• Neutrophils less than 0.2 × 10 ⁹/L
• Platelets less than 20 × 10 ⁹/L
• Reticulocytes less than 20 × 10 ⁹/L

Non-severe aplastic anemia (NSAA) is defined as aplastic anemia not fulfilling the criteria for SAA or VSAA.

Therapy for aplastic anemia may consist of supportive care only, immunosuppressive therapy, or hematopoietic cell transplantation (HCT). Severe and very severe aplastic anemia (SAA and VSAA, respectively; see Workup/Staging) have a mortality rate of greater than 70% with supportive care alone[72] and are therefore a hematologic emergency. Treatment should be instituted promptly for SAA or VSAA, and clinicians must stress the need for patient compliance with therapy.

Inpatient care for patients with aplastic anemia may be needed during periods of infection and for specific therapies, such as antithymocyte globulin (ATG) or HCT. In addition, iron chelation may be required in chronically transfused patients who develop elevated serum ferritin levels above 1000 µg/L.[5]  Phlebotomy can be done to decrease the iron overload post transplantation in aplastic anemia patients.[5]

The British Committee for Standards in Haematology recommends treating infection or uncontrolled bleeding before administering immunosuppressive therapy, including in patients scheduled for HCT.[5] In the presence of severe infection, however, it may be necessary to proceed directly to HCT to provide the patient with the best chance for early neutrophil recovery.[5]  

Both the British Committee for Standards in Haematology and the Pediatric Haemato-Oncology Italian Association recommend HCT from a matched sibling donor for severe aplastic anemia. If a matched donor is not available, options include immunosuppressive therapy or unrelated donor HCT.[6, 7]

Approximately one third of patients with aplastic anemia do not respond to immunosuppression. The thrombopoietin receptor agonist eltrombopag is approved for use in patients with severe aplastic anemia who fail to respond adequately to immunosuppressive therapy. Independent of response or degree of response, risks include relapse and late-onset clonal disease, such as paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndrome (MDS), or leukemia.[18, 56, 57, 58, 59]

Pregnant women with aplastic anemia have a 33% risk of relapse.[5] Provide supportive care in these patients, maintain the platelet count above 20 × 109/L, if possible, and consider administering cyclosporine.[5]

Note that monotherapy with hematopoietic growth factors (eg, recombinant human erythropoietin [rHuEPO], granulocyte colony-stimulating factor [G-CSF]) is not recommended for newly diagnosed patients.[5]

Frequent outpatient follow-up for patients with aplastic anemia is needed to monitor blood counts and any adverse effects of various drugs. Transfusions of packed red blood cells (RBCs) and platelets are administered on an outpatient basis.

Transfer considerations

Patients with aplastic anemia should be treated by physicians who are experts in the care of immunocompromised patients and in consultation with a hematologist and/or an HCT physician.

Patients with aplastic anemia require transfusion support until the diagnosis is established and specific therapy can be instituted. The British Committee for Standards in Haematology recommends prophylactic transfusions in patients whose platelet counts fall below 10 × 109/L (or < 20 × 109/L if bleeding or febrile).[5] However, it is important that transfusions be guided by the patient’s clinical status and not by numbers alone. Avoiding transfusions from family members is important because of possible sensitization against non-HLA (human leukocyte antigen) tissue antigens of potential donors.

For patients in whom hematopoietic cell transplantation (HCT) may be attempted, transfusions should be used judiciously because minimally transfused subjects have achieved superior therapeutic outcomes.

If using blood-bank support, attempt to minimize the risk of cytomegalovirus (CMV) infection. The blood products should, if possible, undergo leukocyte reduction to prevent alloimmunization and CMV transmission and should be irradiated to prevent transfusion-associated graft versus host disease (GVHD) in HCT candidates.

The British Committee for Standards in Haematology also recommends irradiated blood products for all patients receiving antithymocyte globulin (ATG) therapy. In patients with life-threatening neutropenic sepsis, the committee suggests consideration of irradiated granulocyte transfusions.[5]

Development of a transfusion plan in consultation with a physician who is experienced in the management of aplastic anemia is essential.

Infections are a major cause of mortality in patients with aplastic anemia.[73, 74] Risk factors include prolonged neutropenia and the indwelling catheters used for specific therapy. Fungal infections, especially those due to Aspergillus species, pose a major risk. Patients should maintain hygiene to reduce infection risk.

The British Committee for Standards in Haematology recommends prophylactic antibiotic and antifungal agents for patients whose neutrophil counts are below 0.2 ×109/L.[5] Patients on immunosuppressive therapy should receive prophylactic antiviral therapy.[5] Empirical antibiotic therapy should be broad based, with gram-negative and staphylococcal coverage based on local microbial sensitivities. Especially consider including antipseudomonal coverage at the start of treatment for patients with febrile neutropenia; also consider early introduction of antifungal agents for individuals with persistent fever.

However, the strategy of empiric antibiotic use has also resulted in the development of resistant organisms and thus is not favored by some clinicians.[75]

Cytokine support with granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) may be considered in refractory infections, although the benefit of this therapy should be weighed against its cost and efficacy.[10, 76, 77, 78] Discontinue cytokine support after 1 week if the neutrophil count does not rise.[5]

Central venous catheter placement is required prior to hematopoietic cell transplantation (HCT). Note that the following recommendations do not apply to patients with Fanconi anemia and other types of inherited marrow failure;[5] these patients require special consideration.

HCT using an HLA-matched sibling donor

Human leukocyte antigen (HLA)-matched sibling-donor HCT is the treatment of choice for a young patient with severe or very severe aplastic anemia (SAA or VSAA, respectively), being generally accepted for patients younger than 50 years.[5] Persons undergoing this procedure do not require irradiation-based conditioning regimens.[5]

A study of 692 German patients with SAA who received transplants from HLA-matched siblings found that in patients younger than 20 years, rates of chronic graft versus host disease (GVHD) and overall mortality were higher after transplantation of peripheral blood progenitor cell (PBPC) grafts than after bone marrow transplants.[79] A multinational study of patients with SAA who received HCT from an HLA-matched sibling donor concluded that although bone marrow should definitely be the preferred graft source for these patients, PBPCs may be an acceptable alternative in countries with limited resources where patients present later in their disease course and risks of graft failure and infective complications are high.[80]

Although evidence suggests that stem cells from bone marrow afford better outcomes compared with PBPCs, an additional consideration is the perspective of the donor, who must be informed of the difference between the methods of harvesting. Bone marrow harvesting is usually performed with the donor under general anesthesia, while with PBPC harvesting the donor is awake and connected via large-bore intravenous catheters to an apheresis machine, which separates out the stem cells (for descriptions of the two methods, see Bone Marrow Donor Procedure).

Along with the risks associated with anesthesia, bone marrow donors typically experience moderate pain for several days following the procedure. PBPC donors usually experience bone pain, which may be severe, from the filgrastim-induced bone marrow stimulation used to mobilize stem cells in advance of the procedure.

One of the major problems of HCT in aplastic anemia is the high rate of rejection (10%; range, 5-50%). The rejection rate correlates positively with the number of transfusions the patient received and the duration of his or her disease prior to transplantation.

Past attempts to reduce rejection rates included use of a higher stem cell dose, as well as the addition of total body irradiation to cyclophosphamide conditioning. Although that was associated with a reduced incidence of graft rejection, the benefit was negated by high transplant-related mortality (TRM) due to an increase in GVHD.

Currently, antithymocyte globulin (ATG) with cyclophosphamide is a commonly used conditioning regimen for transplantations in aplastic anemia. The addition of ATG to cyclophosphamide for conditioning has resulted in infrequent graft rejections, as well as improved overall survival.[20, 76, 77, 81]

Fludarabine- and cyclophosphamide-based reduced-intensity conditioning (RIC) regimens with or without ATG reduced rejection and improved outcome in Indian patients undergoing allogeneic HCT for SAA.[82] When compared with 26 patients previously transplanted using cyclophosphamide/antilymphocyte globulin, RIC regimens led to faster neutrophil engraftment (12 vs 16 days, respectively), with significantly lower rejection rates (2.9% vs 30.7%, respectively), and superior event-free (82.8% vs 38.4%, respectively) and overall (82.8% vs 46.1%, respectively) survival rates.[82]

Standard conditioning regimens use a cyclophosphamide dose of 200 mg/kg, which is known to be associated with significant organ toxicity. In a multicenter phase I-II study of adult patients with SAA receiving bone marrow grafts from unrelated donors, Anderlini et al found that a regimen of cyclophosphamide in doses of 50 or 100 mg/kg, combined with total body irradiation 2 Gy, fludarabine, and ATG, provided effective conditioning and few early deaths.[83]

The occurrence of GVHD as a complication of HCT is positively correlated with increasing age of the patient. Grafts depleted of T cells reduce the risk of GVHD but increase the risk of graft failure. The addition of cyclosporine along with methotrexate is the standard GVHD approach for matched siblings.[81] However, very few comparative data exist, and no data have reported an alternative approach to be superior to this regimen.

Although horse ATG is better than rabbit ATG as an immunosuppressive therapy, a multinational study of ATG used as part of the conditioning regimen, which included 546 HLA-matched sibling HCT transplants for severe aplastic anemia, found that 3-year overall survival rates were not significantly different with rabbit versus horse ATG. However, the day-100 incidences of both acute and chronic GVHD were higher with horse than with rabbit ATG (acute GHVD, 17% versus 6%, respectively, P < 0.001; chronic GVHD, 20% versus 9%, P < 0.001). The authors concluded that these results support the use of rabbit ATG for bone marrow conditioning.[84]

Early referral to a transplantation center at diagnosis is recommended for all young patients, even if they lack a suitable related donor, because transplant planning needs to be done even if patients are receiving immunosuppressive therapy, given the significant failure rate of that approach.[21]

Fertility

Patients receiving high-dose cyclophosphamide conditioning in allogeneic HCT from an HLA-identical sibling donor for aplastic anemia have relatively well-preserved fertility.[5] Provide these patients with appropriate contraceptive advice to prevent unintended pregnancies.

Longer-term data are limited for patients with fludarabine-based regimens. Therefore, discussions related to potential fertility preservation, including cryopreservation of sperm and oocytes/embryos, is recommended.[5] Cyclosporine is safe for use in pregnancy.

HCT using an unrelated donor

Unrelated-donor HCT is currently justified only if the donor is a full match and only if immunosuppressive therapy fails (failure of ≥1 course of immunosuppressive therapy) or treatment as part of a clinical trial fails.[5]

High-resolution allelic matching, however, has improved outcomes in unrelated-donor HCT, especially in younger patients. Maury et al found that results for young patients who are fully HLA matched at the allelic level with their donor are comparable to those observed after stem cell transplantation from a related donor.[85]

GVHD

In the first cohorts transplanted, HCT using an unrelated donor was associated with very high mortality due to high rates of graft failure, infection, and GVHD. This poor outcome resulted primarily from the use of less stringent HLA matching in addition to the fact that these first patients had long-term disease, a history of infection, iron overload, transfusion resistance, and other related factors. However, more recent reports suggest a better outcome after unrelated transplants, an improvement that is due mainly to high-resolution HLA testing, optimization of the conditioning regimen, better supportive care, and better management of GVHD.

A retrospective study of comparative data from Japan indicated similar overall survival in children and young adults with aplastic anemia who received transplants from either a sibling or an unrelated donor, although rates of acute and chronic GVHD were significantly higher in the group receiving unrelated transplants.[86]

Due to the rates of GVHD in unrelated donor transplantation, this procedure is not preferred over immunosuppressive therapy.[52]

In unrelated donor HCT, partial T-cell depletion may decrease the risk of severe GVHD while still maintaining sufficient donor T lymphocytes to ensure engraftment.[52]

Graft failure

In a study that evaluated data for unrelated matched HCT versus mismatched transplants, the probability of graft failure at 100 days after using a 1-antigen mismatched, related donor was 21%, whereas the probability was 25% for a greater-than-1-antigen mismatched, related donor; 15% for a matched, unrelated donor; and 18% for a mismatched, unrelated donor.[21]

Conditioning regimens

In a multinational study that included 287 patients with severe aplastic anemia who received ATG as part of their conditioning regimen for unrelated-donor HCT, rabbit ATG resulted in a lower incidence of acute (but not chronic) GVHD and better survival (3-year overall survival, 83% for rabbit ATG versus 75% for horse ATG; P=0.02). The authors concluded that these data support the use of rabbit ATG in this setting.[84] Typical regimens in this study population were horse ATG, cyclophosphamide, and low-dose total-body irradiation (TBI); and rabbit ATG, cyclophosphamide, fludarabine, and TBI.

It should be noted, however, that early results from a cyclophosphamide de-escalation study in a fludarabine-based conditioning regimen for unrelated donor HCT demonstrated life-threatening adverse events (excessive organ toxicity) at predefined cyclophosphamide dose levels.[87] The investigators reported an association between such toxicity and cyclophosphamide 150 mg/kg plus TBI at 2 Gy, fludarabine at 120 mg/m2, and ATG.[87]

Conditioning regimens without TBI have also been studied. These include fludarabine, ATG, and cyclophosphamide[53] and fludarabine, low-dose cyclophosphamide, and alemtuzumab.[88] Hamad and colleagues reported on HCT using a conditioning regimen with intermediate-dose alemtuzumab (50 to 60 mg) and high-dose cyclophosphamide or fludarabine in 41 adult patients with aplastic anemia, and reported excellent survival with a favorable impact on GVHD and long-term health outcomes, but frequent viral complications. At 3 years, survival was 96% in patients younger than 40 years of age and 67% in those 40 years and older.[89]

Although the optimal regimen for unrelated-donor HCT remains unclear, the British Committee for Standards in Haematology indicates that a non–irradiation-based fludarabine regimen appears to be favored for younger patients.[5]

According to a study by Samarasinghe et al, a conditioning regimen with fludarabine, cyclophosphamide, and alemtuzumab with matched unrelated-donor (MUD) HCT appears to be very well-suited in children with severe aplastic anemia and has excellent outcomes.[90] The investigators suggested that MUD HCT may be a reasonable alternative when immunosuppressive therapy fails.[90] In a study of fludarabine, low-dose cyclophosphamide, and alemtuzumab as a conditioning regimen for HCT (21 transplants from unrelated donors, with 3 of those from HLA 9/10 mismatch donors and 6 from matched sibling donors), Sheth et al reported similar outcomes in patients aged 50 years or older and those younger than 50 years.[88]

Haploidentical HCT

Nonmyeloablative peripheral blood stem cell transplantation from HLA-haploidentical family donors has been proposed as rescue therapy in patients with refractory SAA or VSAA.[91] A systematic review and meta-analysis concluded that haploidentical HCT is a promising approach in idiopathic aplastic anemia, with high engraftment rates and reduced complication rates. In 15 studies that included 577 patients, successful engraftment occurred in 97.3% of patients (95% confidence interval [CI], 95.9-98.7). Grades II-IV acute and chronic GVHD were reported in 26.6% and 25.0% of patients, respectively. The pooled incidence of transplant-related mortality was 6.7% per year (95% CI, 4.0-9.4%).[92]

Rates of successful engraftment were higher with reduced-intensity conditioning compared with nonmyeloablative conditioning (97.7% vs 91.7%, P = 0.03) and rates of acute GVHD were lower (29.5% vs 18.7%, P = 0.008). No differences in the incidence rates of chronic GVHD or mortality were found. Posttransplantation regimens containing cyclophosphamide were associated with reduced rates of acute GVHD, cytomegalovirus (CMV) viremia, and CMV disease in initially viremic patients, compared with methotrexate-containing regimens and other regimens. The authors of this study recommend prospective trials to identify the preferred conditioning regimen, GVHD prophylaxis, and graft source for haploidentical HCT for aplastic anemia.[92]

Early studies of unrelated umbilical cord blood transplantation (UCBT) reported high graft failure rates. However, subsequent studies of UCBT using conditioning regimens that included low-dose total-body irradiation (TBI) yielded improvements in engraftment and overall survival.[93]

A nationwide prospective phase II study from France concluded that UCBT is a valuable option for young adults with refractory severe aplastic anemia (SAA) and no available matched unrelated donors. The conditioning regimen comprised fludarabine, cyclophosphamide, antithymocyte globulin (ATG), and 2-Gy TBI. Engraftment occurred in 23 of the 26 patients in the study, and all 23 were alive at 1 year, for an overall survival rate of 88.5%. The cumulative incidences of grade II-IV acute and chronic graft-versus-host disease were 45.8% and 36%, respectively.[94]

Du et al reported successful use of an immunoablative conditioning regimen for UCBT that does not include TBI and does not include ATG, as previous studies have found reduced engraftment and survival rates with conditioning regimens containing ATG. In their 15 patients, the first 10 received fludarabine and cyclophosphamide, to which busulfan was added in the subsequent 5 patients. Sustained engraftment was observed in 13 patients and after median follow-up of 33.8 months, 12 patients remained alive.[93]

Immunosuppressive therapy using antithymocyte globulin (ATG) and cyclosporine is used as first-line therapy[95] for patients with severe or very severe aplastic anemia (SAA or VSAA, respectively) who are older than 50 years (35-50 years in presence of comorbidities) and as second-line therapy in younger patients with SAA or VSAA if a human leukocyte antigen (HLA)–matched sibling donor is not available. Immunosuppressive therapy is also recommended in patients with nonsevere aplastic anemia who are transfusion dependent.[5] Immunosuppressive therapy is associated with an overall response rate of 60-80% and a 5-year survival rate of 75% in most reports, but event-free survival rates are in the range of 35-50%.

Up to 50% of patients with aplastic anemia demonstrate small paroxysmal nocturnal hemoglobinuria (PNH) clones in the absence of evidence of hemolysis.[5] Some, but not all, studies have found evidence that the presence of a PNH clone predicts a better response to immunosuppressive therapy. Patients with a significant PNH clone who are receiving immunosuppressive therapy, especially ATG, should be actively monitored for signs of hemolysis. In patients with a history of PNH-associated thrombosis, use of ATG is not recommended.

Adding granulocyte colony-stimulating factor (G-CSF) to ATG and cyclosporine does not provide a survival advantage, but the lack of a neutrophil response by day 30 in patients receiving G-CSF has been associated with significantly lower rate of response to immunosuppressive therapy, and  this early identification of probable nonresponders may stimulate an urgent transplant approach.[96] A study from the European Group of Blood and Marrow Transplantation found that adding G-CSF to ATG plus cyclosporine had no meaningful long-term effect. On median follow-up of 11.7 years, there was no significant difference in overall survival, event-free survival, or incidence of clonal disorders.[97]

Central venous catheter placement may be required before the administration of immunosuppressive therapy, and patients with these catheters should be treated as inpatients. Patients may require blood product support during ATG therapy, as well as close monitoring for allergic or anaphylactic signs and symptoms and for prophylaxis and treatment of fevers.[5]

ATG sources

ATG can be derived from horses or rabbits. Scheinberg et al reported a large difference in the rate of hematologic response at 6 months in favor of horse ATG (68%), as compared with rabbit ATG (37%), in 120 patients with aplastic anemia.[98] Overall survival at 3 years also differed, with a survival rate of 96% in the horse-ATG group, compared with 76% in the rabbit-ATG group when data were censored at the time of stem-cell transplantation, and 94% versus 70% in the respective groups when stem-cell–transplantation events were not censored.[98]

A meta-analysis by Hayakawa et al of 13 studies comparing horse ATG with rabbit ATG for immunosuppressive therapy in severe aplastic anemia concluded that horse ATG results in a higher response rate (P=0.015); further, a sensitivity analysis showed higher early mortality with rabbit ATG.[99] Hence, these authors consider horse ATG the preferred choice in this setting.

However, other retrospective studies have failed to show significant differences between horse ATG and rabbit ATG.[96] In a study of 955 patients with aplastic anemia who received rabbit ATG and cyclosporine as first line treatment (492 patients treated from 2001 to 2008 and 463 treated from 2009 to 2012, responses rose from 37% at 90 days to 52% at 180 days and 65% at 365 days. Mortality within 90 days was 5.7% in the 2001-2008 arm and 2.4% in the 2009-2012 arm. Response rates at 6 months were highest in patients treated within 30 days after diagnosis, and in patients younger than 21 years.[100]

Cyclosporine

In a study of immunosuppressive therapy with cyclosporine alone, response rates were 45% overall, 16% for VSSA, 47% for SAA, and 85% for moderate aplastic anemia.[101] The only predictor of response to cyclosporine was an absolute neutrophil count (ANC) of less than 200/mm3. A prospective study from India concluded that for resource-poor patients, cyclosporine monotherapy, in a dosage of 5 mg/kg/day, is a relatively safe treatment option for aplastic anemia.[102]

Response and complications

The treatment response in aplastic anemia, unlike in other autoimmune diseases, is slow. At least 4-12 weeks is usually needed to observe early improvement, and the patient may continue to improve slowly thereafter. Late responses may also occur. About 50% of patients respond by 3 months after ATG administration, and about 75% respond by 6 months. Full hematologic recovery is rare, but most patients improve to a functional hematologic recovery and become transfusion independent.

Failure to respond, relapse, and clonal evolution are major complications of immunosuppressive therapy for aplastic anemia.[96] If the patient has not responded to a first course of ATG, then a second course may be given, using either the same preparation or another one. Approximately 30-60% of patients may respond to a second course of ATG (60% responses in relapsed and 35% in refractory patients).[103, 104, 105, 106, 107, 108, 109]

To reduce the risk of relapse, continue cyclosporine for a minimum of 12 months after achieving maximal hematologic response, with a very slow tapering by 25 mg every 3 months thereafter.[5] Approximately one third of patients have a relapse, most often at the time of cyclosporine taper. About one third of responders are cyclosporine dependent. Of patients without a response or who relapse, 40-50% respond to a second course of immunosuppressive therapy.

The risk of some form of clonal disease other than paroxysmal nocturnal hemoglobinuria (PNH) is 15-30% and may be due to the inability of these therapies to completely correct bone marrow function, a missed diagnosis of myelodysplastic syndrome (MDS), or the fact that the stem cells under proliferative stress may be more prone than other cells to mutation. MDS that evolves from aplastic anemia treated with immunosuppressive therapy responds similarly to hematopoietic stem cell transplantation as does de novo MDS,[110] so patients who have received immunosuppressive therapy for severe aplastic anemia should have regular assessment of marrow cells, possibly at yearly intervals.[96]

High-dose cyclophosphamide

At present, the use of high-dose cyclophosphamide should be limited to clinical trials.[96] Preliminary data suggested that high-dose cyclophosphamide may result in durable remissions in some patients with aplastic anemia. However, some of these patients develop PNH and cytogenetic abnormalities on follow-up. A prospective, randomized study comparing high-dose cyclophosphamide plus cyclosporine with ATG plus cyclosporine in 31 patients with previously untreated severe aplastic anemia was terminated early because of very high mortality and fungal infections in the cyclophosphamide arm.[111]

A long-term follow-up study of high-dose cyclophosphamide treatment showed better results in treatment-naive patients than in those with refractory disease. At 10 years, in 44 treatment-naive SAA patients and 23 patients with refractory SAA, overall actuarial survival was 88% versus 62%, the response rate was 71% versus 48%, and the actuarial event-free survival was 58% versus 27%, respectively. 

Eltrobombopag (Promacta) is an oral thrombopoietin receptor agonist that was approved in 2014 for patients with severe aplastic anemia that failed to respond adequately to at least one prior immunosuppressive therapy (IST) regimen. Eltrombopag probably acts in aplastic anemia by stimulating the small number of residual stem cells in these patients' bone marrow. Approval of eltrombopag for this indication was supported by a phase II study in which 41% of patients experienced a hematologic response in at least one lineage (ie, platelets, red blood cells, neutrophils) after 12 weeks of treatment.[112, 113]  In the extension phase of the study, three patients achieved a multi-lineage response. Four of those patients subsequently tapered off treatment and maintained the response (median followup 8.1 months, range 7.2-10.6 months).[112, 113]

Eltrombopag has also shown benefit as first-line therapy. Townsley et al combined standard IST with eltrombopag in previously untreated patients with severe aplastic anemia, using three different regimens. The best response was noted in patients receiving eltrombopag from day 1 to 6 months, along with horse ATG on days 1 to 4 and daily cyclosporine from day 1 to 6 months. In this cohort, at 6 months, complete responses were seen in 58% of patients and hematologic responses in 94%. This compared with 10% and 66% rates, respectively, observed in a historical cohort treated with IST alone. In addition, the improved blood counts were accompanied by increased marrow cellularity and hematopoietic progenitor numbers. No additional increase in risk of clonal evolution (15%) was noted with the addition of eltrombopag to ATG and cyclosporine.[114]

Geng et al reported on upfront use of eltrombopag in two pediatric patients with non-severe aplastic anemia. Both patients achieved hematologic response with eltrombopag monotherapy.[115]

In  2018, the FDA expanded approval for eltrombopag to include use as first-line therapy, in combination with standard IST, for adult and pediatric patients 2 years and older with severe aplastic anemia.[116]

Romiplostim

Romiplostim is a peptibody (Fc antibody fusion protein) with thrombopoietin receptor (also known as c-MPL) agonist activity that stimulates endogenous thrombopoietin production, promoting the proliferation and differentiation of megakaryocytes in the bone marrow. As an alternative to eltrombopag in frontline treatment for severe aplastic anemia, romiplostim is still under evaluation in ongoing trials (NCT03957694). In an open-label randomized phase 2 trial in refractory aplastic anemia patients previously treated with IST, romiplostim at dose of 10 mcg/kg/week showed responses in around 70% patients at 9 weeks[117] . Present studies are focusing on effectiveness of high-dose romiplostim (20 mcg/kg/week) in eltrombopag-refractory aplastic anemia.[118]

The diet for the patient with aplastic anemia who has neutropenia or who is receiving immunosuppressive therapy should be tailored carefully to exclude raw meats, dairy products, or fruits and vegetables that are likely to be colonized by bacteria, fungus, or molds. Furthermore, a salt-limited diet is recommended during therapy with steroids or cyclosporine.

The patient should avoid any activity that increases the risk of trauma during periods of thrombocytopenia. Menstruating women are also advised to be on hormonal treatment to avoid menstrual cycles that are likely to be heavy due to thrombocytopenia.

Inform patients of the increased risk of community-acquired infections during periods of neutropenia and lymphopenia. Patients should maintain hygiene to reduce the risks of infection.

The goals of pharmacotherapy in cases of aplastic anemia are to reduce morbidity and prevent complications. Chelating agents are used to reduce iron overload in patients undergoing repeated transfusions. Immunosuppressive agents are used in conditioning regimens for hematopoietic stem cell transplantation; immunosuppression is also used for non-transplant therapy, most often with the combination of antithymocyte globulin (ATG) and cyclosporine.

Hematopoietic support with eltrombopag, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) may be considered in refractory infections, although the cost and efficacy of this therapy should be weighed.[10, 76, 77, 78, 112, 113]

Class Summary

Immunosuppressive agents are used in the conditioning regimen for patients undergoing hematopoietic stem cell transplantation for aplastic anemia, and as therapy in patients with severe disease who are not eligible for transplantation or lack a suitable donor, as well as in patients with nonsevere disease who are transfusion dependent.

Cyclosporine (Sandimmune, Neoral)

Cyclosporine is a cyclic polypeptide that suppresses some humoral immunity and, to a greater extent, cell-mediated immune reactions (eg, delayed hypersensitivity, allograft rejection, experimental allergic encephalomyelitis, graft versus host disease) for a variety of organs.

For children and adults, base the initial dosing on the ideal body weight and subsequently adjust for levels. Frequent monitoring of drug levels is needed. To convert to the oral dose, use an intravenous (IV)-to-oral correction factor of 1:4. The dosage and duration of therapy may vary with different protocols. When used without hematopoietic growth factor in children, ATG and cyclosporine-based immunosuppressive therapy has been shown to lead to an excellent response and survival rate with low incidence of clonal evolution.

Methylprednisolone (Medrol, Solu-Medrol)

Steroids ameliorate the delayed effects of anaphylactoid reactions and may limit biphasic anaphylaxis. In severe serum sickness (mediated by immune complexes), parenteral steroids may reduce the inflammatory effects. Hence, methylprednisolone is used with antithymocyte globulin (ATG) to decrease the adverse effects (eg, allergic reactions, serum sickness). Also, this agent has an additional immunosuppressive effect. High doses or long duration may be needed if serum sickness occurs with ATG. The doses and duration may vary with different protocols.

Alemtuzumab (Campath, MabCampath)

Alemtuzumab is a recombinant monoclonal antibody against CD52 (lymphocyte antigen). This agent promotes antibody-dependent lysis.

Lymphocyte immune globulin, equine (Atgam)

Lymphocyte immune globulin inhibits the cell-mediated immune response by altering T-cell function or by eliminating antigen-reactive cells. There are few prospective, randomized data to suggest a single schedule that is superior, but experience suggests that a 4- to 5-day infusion is associated with less toxicity than older 7- to 10-day schedules.

Cyclophosphamide (Cytoxan)

Cyclophosphamide is chemically related to nitrogen mustards. As an alkylating agent, the mechanism of action of the active metabolites may involve cross-linking of DNA, which may interfere with the growth of normal and neoplastic cells. Monitor carefully; used only on an investigational basis.

Antithymocyte globulin, rabbit (Thymoglobulin)

Antithymocyte globulin (ATG) may modify T-cell function. The dose and duration of therapy vary with the investigational protocols.

Class Summary

Eltrombopag has gained FDA approval for severe aplastic anemia. It is indicated for first-line treatment, in combination with standard immunosuppressive therapy, and as second-line treatment in patients who fail to respond adequately to at least 1 prior immunosuppressive therapy.

Several preliminary studies have demonstrated that the addition of cytokines (eg, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) may hasten the neutrophil recovery and that these agents may improve response rate and survival, although long-term use may increase the risk of clonal evolution.

Eltrombopag (Promacta)

Eltrombopag is a thrombopoietin (TPO)-receptor agonist that interacts with human TPO receptor transmembrane domain of human TPO-receptor. It initiates signaling cascades that induce proliferation and differentiation of megakaryocytes from bone marrow progenitor cells. It is indicated for severe aplastic anemia in patients who fail to respond adequately to at least 1 prior immunosuppressive therapy.

Sargramostim (Leukine)

A recombinant human GM-CSF, sargramostim can stimulate production of neutrophils and activate mature granulocytes and macrophages. The dose and frequency of administration vary with the investigational protocol.

Filgrastim (Neupogen)

Filgrastim is a G-CSF that activates and stimulates the production, maturation, migration, and cytotoxicity of neutrophils.

Class Summary

Antimetabolites are antineoplastic agent that inhibit cell growth and proliferation.

Fludarabine (Fludara)

Fludarabine contains fludarabine phosphate, a fluorinated nucleotide analogue of the antiviral agent vidarabine, 9-b-D-arabinofuranosyladenine (ara-A) that enters the cell and is phosphorylated to form the active metabolite 2-fluoro-ara-adenosine triphosphate, which inhibits deoxyribonucleic acid (DNA) synthesis. Specifically, this agent inhibits DNA polymerase, DNA primase, DNA ligase, and ribonucleotide reductase, as well as ribonucleic acid (RNA) function, RNA processing, and mRNA translation. Fludarabine also activates apoptosis.

Class Summary

Chelating agents eliminate iron overload from transfusions.

Deferoxamine (Desferal)

Deferoxamine chelates iron by forming a stable complex that prevents the iron from entering into further chemical reactions; it also chelates iron readily from ferritin and hemosiderin but not readily from transferrin. Desferoxamine does not combine with the iron from cytochromes and hemoglobin. The chelate is readily soluble and is renally excreted.

Deferasirox (Exjade)

Deferasirox chelates trivalent iron. This agent is used to treat chronic iron overload due to blood transfusions. Monitor patients' renal and hepatic function.