Aplastic Anemia Workup

Updated: Dec 05, 2018
  • Author: Sameer Bakhshi, MD; Chief Editor: Emmanuel C Besa, MD  more...
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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. Carefully consider dysplasia to rule out myelodysplastic syndrome (MDS), although some degree of dysplasia may be present in aplastic anemia.


Complete Blood Cell Count and Peripheral Smears

A paucity of platelets, red blood cells (RBCs), granulocytes, monocytes, and reticulocytes is found in patients with aplastic anemia. Mild macrocytosis is occasionally observed. The degree of cytopenia is useful in assessing the severity of aplastic anemia.

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


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

  • Diepoxybutane incubation

  • Histocompatibility testing

Hemoglobin electrophoresis and blood-group testing may show elevated levels of fetal hemoglobin and red cell I antigen, suggesting stress erythropoiesis. These findings are observed in aplastic anemia and in other marrow-failure states and are often proportional to the macrocytosis. A positive Coombs test may point to autoimmune hemolytic anemia.

Although a biochemical profile has limited value in examination 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 renal or hepatic diseases. Liver function test (LFT) results can indicate 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). [47] Although the Ham test, or the sucrose hemolysis test, was frequently performed in the past to diagnose PNH, it has been replaced by FACS profiling of phosphatidylinositol glycan class A (PIGA) anchor proteins, such as CD55 and CD59. This study is more accurate than the Ham test for excluding PNH.

FLAER is also a highly sensitive flow cytometry test for PNH that uses whole blood and binds specifically to glycophosphatidylinositol (GPI) anchor proteins in peripheral blood granulocytes. [48, 49] In PNH, mutation of the PIGA anchor proteins results in a lack of the GPI anchors. Thus, the lack of FLAER binding to granulocytes is sufficient for the diagnosis of PNH. [48, 49] 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.

Diepoxybutane incubation is performed to assess chromosomal breakage in Fanconi anemia and is available only in reference laboratories. This test is required even in the absence of phenotypic features of Fanconi anemia, because up to 50% of patients may not have any clinical stigmata.

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. [50]

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 (P < 0.01), whereas the percentage of regulatory T cells (Tregs) was lower than that in healthy children (P < 0.001). After treatment, the percentage of CD20+ B cells was decreased, and the percentage of Tregs was significantly increased. The authors suggest CD20+ B cells and Tregs as potential markers for evaluating therapeutic efficacy and prognosis in these cases. [51]


Bone Marrow Aspiration and Biopsy

Bone marrow biopsy is performed in addition to aspiration to assess cellularity qualitatively and quantitatively. In aplastic anemia, the specimens are hypocellular. 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. The specimen is considered hypocellular if it is less than 30% cellular in individuals younger than 60 years or if it is less than 20% cellular in those older than 60 years (see the following image). Some dyserythropoiesis with megaloblastosis may be observed in aplastic anemia.

Low power, H and E showing a hypocellular bone mar Low power, H and E showing a hypocellular bone marrow 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 some cases of MDS. [52] 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
  • Increased or decreased cellularity

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 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.


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. [53]



Staging of aplastic anemia is based on the criteria of the International Aplastic Anemia Study Group (IAASG).

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 × 109/L

  • Platelets less than 20 × 109/L

  • Reticulocytes less than 20 × 109/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 × 109/L

  • Platelets less than 20 × 109/L

  • Reticulocytes less than 20 × 109/L