COMPOSITION OF BONE MARROW SUBPOPULATIONS ANALYZED BY FLOW CYTOMETRY REVEALS DIFFERENCES BETWEEN PEDIATRIC APLASTIC ANEMIA AND REFRACTORY CYTOPENIA

Background:

Aplastic anemia (AA) is a disease with immunological pathogenesis. Refractory cytopenia of childhood (RCC), the most common subtype of MDS in children, is regarded as a clonal disease. Typically RCC is characterized by hypoplastic bone marrow (BM). Immunological mechanisms play a role in at least part of RCC patients. Evaluation of composition of BM populations by flow cytometry (FC) might be challenging since detailed knowledge about physiological composition in childhood is limited.

Aims:

Our aim is to characterize BM by FC in AA and RCC with the focus on T cells and to define reference composition of subpopulations in physiological bone marrow.

Methods:

We measured BM samples by FC in 34 AA patients (median age 10.3, 1.1-18y) and 26 RCC patients (median age 11, 4.2-18y) diagnosed in 2005-2013. We analyzed granulocytes, lymphocytes, precursors CD34^pos, CD117^pos, B cells including evaluation of immature CD10^pos cells, T cells (CD3^pos4^pos, CD3^pos8^pos) including activation status by HLA DR expression. We also analyzed follow-up BM samples to evaluate changes following immunosuppression (IST).

We established 2 reference categories: BM samples from patients examined within staging of solid tumor and a healthy donor (group A, 28 patients, median age 2.6, 0-17y) and patients at least 2 years after stem cell transplantation (group B, in total 22 patients, median age 11, 2.6-23y).

For the aim of detailed characterization of T cells we introduced new polychromatic panel including markers describing T cell subsets (CD3, CD4, CD8), activation (CD57, HLA DR), cytotoxic function (Granzyme B, Perforin) and distribution from naive to memory stage (CD28, CD27, CD45RA).  We measured >300 samples including 6 AA and 5 RCC paired samples before and after IST to evaluate differences in T cell composition.

Mann-Whitney test was used for statistical analyses (p<0.05 for all bellow mentioned results).

Results:

Comparing both reference categories, we found higher percentage of lymphocytes and precursors CD34^pos and CD117^pos in patients in group A.  We observed higher percentage of lymphocytes, B cells and precursors in control patients younger than 1 year. Proportion of CD8^pos T cells out of all T cells and their activation was higher in patients older than 5 years.

Patients with AA have significantly higher number of lymphocytes compared to RCC and all reference samples. RCC patients have also higher percentage of lymphocytes compared to all reference samples. When RCC was compared to group A only the difference was not significant.

Total B cells were significantly decreased in RCC compared to AA and reference group, however in both AA and RCC proportion of immature CD10^pos B cells was significantly decreased. Following IST CD10^pos B cells tended to increase in the majority of treated patients. Precursors CD34^pos and CD117^pos were decreased in both AA and RCC, but in AA patients they were significantly lower compared to RCC.

T cells were increased in both AA and RCC patients, but significantly higher activation of cytotoxic T cells we observed only in AA.

By T cell-oriented panel we found decrease of activated CD8^pos T cells together with increase of naive CD8^pos27^pos28^pos45RA^pos cells in AA patients with good response to IST.

Summary / Conclusion:

We found reproducible differences in composition of BM populations analyzed by FC in AA and RCC patients. Decreased percentage of precursors and increased number of lymphocytes was present in both RCC and AA patients. It is important for analyses of BM composition to define control samples, which match also age characteristics.

Supported by GAUK23010, MHCZ for conceptual development 00064203 University Hospital Motol, GAČR P301/10/1877, UNCE 204012

Abstrakta v časopise Haematologica 2013, Suppl1

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