Aplastic Anemia Workup

Updated: Jan 29, 2021
  • Author: Sameer Bakhshi, MD; Chief Editor: Emmanuel C Besa, MD  more...
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Workup

Approach Considerations

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]

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Complete Blood Cell Count and Peripheral Smears

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.

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Peripheral Blood Testing

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). [58] 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. [59, 60] 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. [59, 60] 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. [61]

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. [62]

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. [63]

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Bone Marrow Aspiration and Biopsy

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-eos 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. [64] 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.

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Magnetic Resonance Imaging

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. [65]

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Staging

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.

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