Bone Marrow Immunophenotyping By Flow Cytometry in Refractory Cytopenia of Childhood

Refractory cytopenia of childhood (RCC) is the most common type of childhood myelodysplastic syndrome (MDS). Because the majority of patients with RCC have a normal karyotype and a hypocellular bone marrow, differentiating RCC from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia ((v)SAA) can be challenging. The histopathological differentiation between RCC and (v)SAA is mainly based on the presence of patchy erythropoiesis with defective maturation and/or the presence of micromegakaryocytes in RCC, and the absence of erythropoiesis and megakaryopoiesis in (v)SAA. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable additional diagnostic tool in differentiating MDS from non-clonal cytopenias in adults, but in childhood MDS, only a limited number of flow cytometric immunophenotyping studies have been reported.

Here, we performed the first comprehensive flow cytometric analysis of myeloid and lymphoid progenitor cells, maturing granulocytes, monocytes and erythrocytes in bone marrow aspirates obtained from a large prospective cohort of 81 RCC patients, collected by the European Working Group of MDS in Childhood (EWOG-MDS). RCC was diagnosed according to WHO criteria and confirmed by central review of bone marrow morphology and histology. Bone marrow aspirates obtained from healthy adult individuals (n=9) and pediatric patients with (v)SAA (n=17) or advanced MDS (n=7) were used as controls. We employed a 7-tube, 6-color flow cytometry protocol, and data analysis was performed largely in line with European LeukemiaNet recommendations for flow cytometry in MDS.

RCC patients had a strongly reduced myeloid compartment compared to healthy controls, but not as severe as (v)SAA patients. In the majority of RCC patients, immature myeloid and/or lymphoid cells were reduced in numbers, but still detectable, while in the vast majority of (v)SAA patients, myeloid blast cells and CD34⁺B-cell precursors were absent: both cell types were absent in 27 of 81 RCC patients (33%) and in 15 of 17 (v)SAA patients (88%). Furthermore, the number of flow cytometric abnormalities was significantly higher in RCC patients than in healthy controls and in pediatric patients with (v)SAA, but lower than in advanced MDS. The most frequently occurring flow cytometric abnormalities in RCC were the heterogeneous expression of CD71 and CD36 on immature erythroid cells. Two or more flow cytometric abnormalities were detected in 49 of 81 RCC patients (60%), and in 2 of 17 (v)SAA patients (12%). If a diagnosis of RCC was considered if myeloid blast cells and/or CD34+ B-cell precursors were present, or if two or more immunophenotypic abnormalities were detected, 61 of 81 RCC patients (84%) could be correctly classified, using histopathology as gold standard, whereas the specificity of this combination, using (v)SAA as a control group, was 76% (13 of 17 (v)SAA patients). No significant associations were detected between the presence of flow cytometric abnormalities (defined as two or more abnormalities) in RCC patients and age or sex, the presence of HLA-DR15, bone marrow cellularity, transfusion dependency at diagnosis, the presence of a PNH clone, or skewing of the T-cell receptor Vβ chain. Of interest, although only 5 patients with monosomy 7 were included in the present study, all of them displayed at least 2 flow cytometric abnormalities. RCC with monosomy 7 confers a high risk of progression to AML, but histopathologically, no differences can be detected between RCC cases with or without monosomy 7.

In conclusion, our results indicate that, although flow cytometric abnormalities in RCC patients are present at a relatively low frequency, flow cytometric immunophenotyping might be a relevant addition to histopathology and cytogenetic analysis in the diagnostic work-up of RCC.