Diagnosis of acquired aplastic anemia

Abstract

Since the introduction of the concept of aplastic anemia (AA) by Paul Ehrlich in 1888 and despite the current better understanding of the underlying mechanisms involved in this disease, a clear delimitation among BM failure syndromes is still a matter of debate. The diagnosis of AA can be difficult basically due to the overlapping morphological characteristics with other BM failure disorders. This paper reviews critical data relevant to the diagnosis of acquired AA and recommends work out steps and main considerations to determine severity and characterization of the disease. The diagnostic challenge in the differentiation between AA and hypoplastic myelodysplastic syndromes is also addressed. The definition of the response criteria to treatment belongs to the diagnostic tasks and it is included in this review as well as an overview of novel tools for the diagnosis of AA.

Main

The diagnosis of aplastic anemia (AA) is defined by the coexistence of pancytopenia with persistent and unexplained reduced marrow hematopoietic cellularity, with no major dysplastic signs and fat cell replacement Radiotherapy and chemotherapy-induced aplasia do not belong to AA. For the diagnosis of AA, there are no specific markers and the diagnosis is reached by exclusion of other reasonable entities. The diagnosis of AA can be difficult basically due to the overlapping morphological characteristics with other BM failure disorders.1, 2, 3 To reach the final diagnosis repeated marrow investigations are sometimes needed. During the work out process, a number of diseases should be considered in the differential diagnosis, namely: hypoplastic myelodysplastic syndromes (MDS), hypoplastic AML and ALL, large granular lymphocytosis, paroxysmal nocturnal hemoglobinuria (PNH) and congenital marrow failure syndromes, such as Fanconi anemia and dyskeratosis congenita1 (Table 1). There are three diagnostic steps to define AA: (I) confirm the suspicion of AA and exclude other BM failure diseases; (II) define the severity of the disease and (III) characterize the AA.

Table 1 Aplastic anemia, main differential diagnosis entities

Confirm the suspicion of AA and exclude other BM failure diseases

For the diagnosis of AA, the presence of pancytopenia and the proof of an empty BM without major dysplastic changes are mandatory. All evaluations needed for the diagnosis of AA are listed in Table 2. AA is clinically characterized by no enlarged lymph nodes, no splenomegaly and no hepatomegaly. Pancytopenia is the main manifestation in the peripheral blood. Anemia is accompanied by reticulocytopenia, and macrocytosis is a common feature. The RBCs should not present relevant anisocytosis and poikilocytosis. Lymphocyte count is usually preserved. In early stages isolated cytopenia, particularly thrombocytopenia can be seen. Monocytopenia can be present and imposes the differential diagnosis of hairy cell leukemia. Careful examination of the blood film is needed to exclude dysplastic neutrophils, the presence of erythroblasts, immature myeloid cells, abnormal platelets or other abnormal cells, such as hairy cells. Fetal Hb can be increased in AA,4 in children pre-transfusional increase in fetal Hb imposes the differential diagnosis with myeloproliferative/MDSs like juvenile myelomonocytic leukemia or other subtype of MDS.5, 6

Table 2 Suggested diagnostic workup for AA

Aspiration and BM biopsy provide the diagnostic clues. Although aspiration allows a better discrimination of cellular morphology, particularly by the assessment of dysplasia, trephine is crucial to assess overall cellularity, topography of hematopoietic cells and to exclude abnormal infiltrates by examining at least 4–5 undistorted fields under × 100 magnifications. Dry tap is unusual and suggests diagnoses other than AA. BM cellularity in AA, despite its definition is more often reduced rather than completely absent (Figure 1). Hypocellularity in AA has been set at <30% hematopoietic cells. However, this definition has been established mainly for children and young adults. In healthy elderly patients, BM cellularity is physiologically decreased.7 Therefore, cutoff <30% may not be applicable to elderly patients. It is not rare to find aplastic subcortical areas in a biopsy of an otherwise healthy patient. Increase in stromal cells such as plasma cells, lymphocytes sometimes forming follicles and mast cells are typical but confounding findings. Stromal cells hence have to be excluded in the global evaluation of marrow cellularity. Presence of ‘hot spots’ (Figure 1) with dominating erythropoiesis and a certain degree of dyserythropoiesis is a typical feature in AA. Immunostaining has become mandatory for topographic identification of various cell population, including increased blast cells, and might eventually identify the unusual association with lymphoma.8 Flow cytometry of BM may contribute to identifying abnormal populations.

Figure 1
figure1

BM histology: aplastic BM histology (a), and hypocellular bone marrow with ‘hot spots’ (b) from a patient with severe AA. (Picture courtesy of Professor Stephan Dirnhofer, Institute of Pathology, University Hospital of Basel, Basel, Switzerland.)

Cytogenetic investigations should be systematically performed in AA patients. Cytogenetic abnormalities can be present in up to 12% of otherwise typical AA patients.9 Abnormal cytogenetic clones often are small, may arise during the course of the disease,10, 11 or may be transient and disappear spontaneously/after immunosuppression. An abnormal cytogenetic clone does not necessarily imply the diagnosis of MDS or AML. In marrow failure syndromes, cytogenetic evaluation may be difficult because of the lack of metaphases. In such a situation, FISH analysis seeking for specific chromosomes aberrations is recommended. Most frequent anomalies include trisomy 8, trisomy 6, 5q−, anomalies of chromosome 7 and 13.

The distinction between AA and hypoplastic MDS may be a difficult diagnostic task. Both marrow failure syndromes show markedly hypocellular BM with increased fat cells.12, 13 The absence of dysplasia particularly of the megakaryopoiesis and the absence of blast cells, as well as the lack of increased numbers of CD34 and CD117-positive cells by immunohistochemistry is the most conspicuous element supporting the diagnosis of AA. Nevertheless, in many cases changes in the karyotype, for instance, the presence of a monosomy 7, are the only criterion in favor of a hypoplastic MDS. Main characteristics of both entities are summarized in Table 3. In children, one of the most prominent features for identifying refractory cytopenia is the presence of erythropoiesis with uni- or multifocal clustering of predominantly immature forms with increased mitoses, occasionally with atypia. The granulopoiesis is typically sparsely dispersed or lacking and megakaryopoiesis usually decreased with micromegakaryocytes or other dysplastic changes.14 Despite similar clinical presentations, distinct cytokine profiles were observed between AA and hypocellular MDS. Characteristic pattern of cytokines such as TPO and chemokine (C–C motif) ligand 3 might in future be useful tools to better discriminate both entities in clinical practice.15

Table 3 Main diagnostic characteristics of aplastic anemia and hypoplastic myelodysplastic syndromes

For the diagnosis of AA, a positive family history including other members affected with anemia, macrocytosis, cytopenia or malignancies should invoke an inherited BM failure syndrome. A normal clinical examination does not definitively rule out a ‘cryptic’ dyskeratosis congenita16, 17 or a non-classical Fanconi anemia.18 The presence of unusual clinical features should alert the possibility of a congenital form of AA. Peripheral blood lymphocytes should be tested for spontaneous and diepoxybutane or mitomycin C-induced chromosomal breakage to identify or exclude Fanconi anemia. This should be performed in all marrow failure patients who are candidates to hematopoietic SCT. Screening should also include sibling donors of Fanconi anemia patients. Telomere shortening is a consistent and typical finding of dyskeratosis congenita. Telomere length measurement of leukocytes from peripheral blood can be considered in any marrow failure syndrome, but it does not belong yet to standard screening.

Define the severity of the disease

Once the diagnosis of AA has been established, the severity of the disease has to be defined. The severity is based exclusively on values of the peripheral blood. Accordingly, from Table 4, three groups of AA are defined7, 19, 20 severe AA, very severe AA and non-severe AA.

Table 4 Definition of disease severity based on peripheral values and BM findings

Characterize the AA

AA and PNH

There is a close correlation between AA and PNH.21, 22 Patients with typical PNH can develop AA in the course of their disease and patients with AA often present a PNH clone.23 Even the presence of a very small PNH clone is a strong argument for a marrow failure syndrome. Flow cytometry is the Gold Standard method for screening and diagnosis of PNH.24 This is currently best achieved by analysis of glycophosphatidylinositol (GPI)-linked Ags using MoAbs and fluorescent aerolysin.25, 26 About 40–50% of patients with acquired AA have a detectable PNH clone. Most clones are small and patients do not have symptoms related to PNH. In some patients, the PNH clone can increase after immunosuppressive treatment. In such case, the patient may present the typical symptoms and complications of the disease.23 PNH clone size measurements should be performed at presentation and followed-up on serial monitoring every 6–12 months.

AA and HLA-DR2/HLA-DRB1*15

HLA-DR typing might be useful for predicting a response to immunosuppression in AA patients. HLA-DR227 and particularly HLA-DRB1*15 (DRB1*1501 and DRB1*1502)28 have been associated with some characteristics and to affect the outcome of AA. Patients possessing HLA-DR15 tend to be older and actually >50% of the patients with HLA-DRB1*1502 are older than 40 years of age. In Japanese patients, DRB1*1501 seems to be associated with the presence of a small population of PNH-type cells and a good response to the immunosuppressive therapy (IST).29 In a recent study on 37 Korean patients with severe AA, responders to immunosuppressive treatment had a significantly higher HLA-DR15 and lower DR4 frequency compared with non-responders.30 The response rates in the best (DR15(+)/DR4(−)), intermediate, and poor response groups (DR15(−)/DR4(+)) were 88.9%, 38.5% and 0%, respectively (P=0.00001). At the allelic level, DRB1*1501 and closely linked DQB1*0602 were associated with a good response and DRB1*0405 and closely linked DQB1*0401 with a poor response to IST.

Hepatitis-associated AA

Seronegative hepatitis is documented in 5–10% of patients with acquired AA.31, 32, 33, 34, 35 It typically occurs in young, healthy males with severe but self-limited liver inflammation. A common inciting infectious cause could be involved both in the liver disease and in the BM failure.31 Indeed, in hepatitis-associated AA, similar skewed T-cell repertoires have been detected in the liver and in the peripheral blood lymphocytes.36 Patients with post-hepatitis AA do not respond differently to immunosuppressive treatment compared with patients with idiopathic acquired AA.

AA associated with other autoimmune disorders

Associations of AA with other autoimmune disease (AID) have been shown in single-case reports.37, 38, 39 In a single-center report, 5.3% of the patients had an AID before the diagnosis of AA and 4.5% of them developed an AID after diagnosis and treatment for AA.40 AID can appear at any time before or after the AA. The frequency of a concomitant AID seems higher in older AA patients as >25% of AA patients diagnosed after 50 years of age presented a concomitant AID. In a large multicenter study of the Severe Aplastic Anemia Working Party (SAAWP) of the European Group for Blood and Marrow Transplantation (EBMT), 50 of 1251 AA patients had an AID.41 Whether or not the immunosuppression applied to treat the AA has an influence on the outcome of the AID still remains a controversial topic. In consideration of the frequency of a concomitant AID in AA patients, it is unlikely that both diseases appear together just by chance.

Response criteria of AA

The definition of the response criteria to treatment belongs to the diagnostic tasks. The response criteria of AA depend in part on the severity of the disease before treatment. A complete response (CR) is defined in any case as a normalization of the blood values, which are neutrophil count 1.5 × 109/L, platelet count 150 × 109/L and Hb 120 g/L. A partial response (PR) includes, transfusion independency in patients who needed transfusions before treatment, with either improvement of the severity degree of the AA, or neutrophil counts increasing 0.5 × 109/L and platelet count 200 × 109/L, in case the values were lower before treatment. Non-response is considered the persistence of transfusion dependency or of values lower than those mentioned above. The definition of PR in AA is not fully satisfactory because it includes patients with very good and stable PR and other patients who have barely obtained values above the minimal criteria.

Future directions in the diagnosis of acquired AA

Since the introduction of the concept of AA by Paul Ehrlich in 1888 and despite the current better understanding of the underlying mechanisms involved in this disease a clear delineation among BM failures is still a matter of debate. The diagnostic challenge for the forthcoming years is to identify AA patients at risk of failing IST, and therefore being candidates for early hematopoietic SCT, and to improve diagnostic standardization of inherited BM failures. During the past years, a number of studies focused on parameters to predict response after IST but were not able to define refractoriness. Baseline absolute reticulocyte and lymphocyte counts were defined as simple predictors of response following IST.42 In children, lower WBC count (<2.0 × 109/L) was the most significant predictive marker of better response.43 In patients receiving G-CSF, the lack of a neutrophil response (<0.5 × 109/L) by day 30 was associated with significantly lower response rate and survival.44 Patients refractory to two consecutive courses of anti-thymocyte globulin (ATG) have a very low chance to respond to a third course of anti-thymocyte globulin, and may be suitable candidates for novel therapeutic options.45

A recent publication showed that telomere shortening in AA patients was associated with both numerical and structural chromosome abnormalities. Patients with shorter telomeres were at higher risk of malignant transformation. Shorter average telomere lengths inversely correlated with monosomy 7 at diagnosis (Figure 2).46 This result might have clinical implications because affected individuals may benefit from therapies that eventually eliminate the shortest (and dysfunctional) telomeres. A measurement of telomere length arises indeed as an interesting prognostic tool in acquired AA.

Figure 2
figure2

Telomere length in AA patients. Probability of evolution to MDS and/or acute leukemia in patients with acquired AA and treated with immunosuppression, according to pre-treatment age-adjusted telomere length. Patients were divided into quartiles based on age-adjusted telomere length (first quartile, shorter telomeres; and fourth quartile, longer telomeres). Patients in the shorter quartile (first) had a higher chance to evolve to myelodysplasia and/or leukemia (24.5%) at 5 years in comparison with patients in the second (10.7%), third (4.8%) and fourth (9.7%) quartiles (log-rank, P¼0.06). Modified from Calado et al.46

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Acknowledgements

ERG spa, SAAR Depositi Oleari Portuali, Rimorchiatori Riuniti and Cambiaso and Risso are acknowledged for their support to the activity of the Hematology Unit of G Gaslini Children’s Hospital, Genova, Italy.

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Rovó, A., Tichelli, A. & Dufour, C. Diagnosis of acquired aplastic anemia. Bone Marrow Transplant 48, 162–167 (2013). https://doi.org/10.1038/bmt.2012.230

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