PMC10078764_01
Detection of an aberrant myeloid blast population in a hypocellular, hemodiluted bone marrow specimen. bone marrow (BM) smears (MGG, 40x magnification) in Case 1 were hemodiluted and extremely low-cellular with 4% blasts (a). Immunohistochemical staining of the BM aspirate demonstrates an increase in CD34+ blast cells (b). Flow cytometric analysis shows an expanded myeloid progenitor population (CD45dim/CD34+/CD117+) with aberrant loss of CD13 and HLA-DR and lack of MPO (c-f). Nucleated cells are depicted in pale gray, myeloid progenitor cells are highlighted in dark gray [Color figure can be viewed at wileyonlinelibrary.com]
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BM smears were hemodiluted and of poor cellularity (Figure 1a), but displayed dysplastic changes in the granulocytic lineage, mild dyserythropoiesis without any ring sideroblasts, and 4% of blasts. Flow cytometric analysis following ELN recommendations (Porwit et al., ; Porwit et al., ; van der Velden et al., ) demonstrated an expanded myeloid progenitor population (14% of total BM cells) with several abnormalities, including lack of CD13, HLA-DR and myeloperoxidase (MPO) expression (Figure 1c-f), a decreased fraction of B cell precursors within the CD34+ compartment, aberrant ratio of CD45 expression of lymphocytes vs. that of the myeloid progenitors (mean fluorescence intensity) and decreased sideward light scatter (SSC) of granulocytic cells (as ratio to lymphocyte SSC). The embedded BM aspirate clot showed 30% cellularity, the presence of micromegakaryocytes (dysmegakaryopoiesis), a diffuse increase of CD34+ blasts (>10%, Figure 1b) together with a strong nuclear p53 staining in <5% of BM cells by immunohistochemistry.
PMC10078764_02
Immunophenotyping demonstrates aberrancies in hypocellular bone marrow. Immunophenotypic analysis of (a) myeloid progenitor cells (gated on CD34+/CD45dim +/ HLA-DR+/CD13+), (b) granulocytic cells (gated on dim CD45/SSC), and (c) erythroid cells (gated on negative to weak CD45/SSC/CD36+). The myeloid precursor cells (black) showed heterogeneous CD117 expression, ranging from negative to positive, and partly aberrant expression of CD11b (black square); back-gating of CD11b+ cells showed that these cells were heterogeneous CD117+. The granulocytic cells (black) showed slightly abnormal maturation (reference images of normal granulocytes are shown in dark gray) and lacked the most mature (CD10+) cells. The erythroid cells (black) showed increased heterogeneity of CD71 expression
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T cells and 1% NK cells), but no abnormalities were detected. The percentage of myeloid progenitors (CD34+/CD117+/CD13+) was 0.2% and these cells partly showed abnormal expression of CD11b and reduced expression of CD117 (Figure 2a). Analysis of the granulocytic lineage (18% of cells) showed slightly aberrant maturation with reduced frequency of the most mature (CD10+) cells (Figure 2b). The more mature erythroid cells showed increased heterogeneity for CD71 expression (Figure 2c).
PMC10078764_03
Aberrant antigen expression on the CD34+ myeloid progenitor population. (a) Increased CD45 expression on myeloid progenitors (in blue; reference line of normal progenitors in gray); increased fraction of erythroid cells (in dark red); and hypogranular granulocytes (in pink); lymphocytes are shown in green. (b) Reduced CD71 expression as well as an increased CD71 coefficient of variation in the erythroid lineage (in dark red). Residual normal erythroid population is highlighted in bright green. (c) Histograms showing aberrant expressions of several markers on myeloid progenitors (in blue; gray reference lines represent normal myeloid progenitors, reference lymphocyte expression in green) [Color figure can be viewed at wileyonlinelibrary.com]
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Whilst modestly increased by FCM and no increase by cytomorphology the myeloid progenitors (CD34+/CD117+/HLADR+/CD33+) showed multiple phenotypic abnormalities including aberrant expression of CD7, CD11b and CD123. (Figure 3).
PMC10078764_04
Immunophenotypic aberrancies in a case of MDS/CMML. (a) Myeloid progenitors (0.17% of nucleated cells, in blue) with an abnormal homogenous (decreased CV [arrow]) and increased expression of CD117; expression of CD33 was decreased (not shown). (b) Low frequency of CD7 expression (12%) on myeloid progenitors (square), although not representing a tight cluster. Normally, CD13 versus CD7 expression on CD34+ myeloid progenitors show an L-shaped pattern, part of the CD7+CD13- cells may be non-myeloid cells, that is, precursor T cells (CD34+), double positive cells are absent. The latter are regarded as aberrant when present but only as a tight cluster. Here, CD13+CD7+ cells are distributed too randomly to represent an aberrant cluster, hence, CD7 expression was deemed negative. (c) Granulocytes (in pink) dimly express CD14, monocytes are displayed as reference in orange. (d,e) Aberrancies in monocytes (orange): Low-to-absent, heterogeneous CD13 expression in (d). (dashed line represents normal reference); CD56 expression in (e). (square). CD56 may be upregulated upon activation. (f) Monocyte partitioning in peripheral blood by CD14 and CD16; classical monocytes constituted 93.7% of the population (CD14+, CD16-, orange); slan+ non-classical monocytes (detected by M-DC-8 antibody) represented 0.98% (in green). No aberrancies were detected in the erythroid lineage (not shown) [Color figure can be viewed at wileyonlinelibrary.com]
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Flow cytometric analysis of BM cells revealed 0.17% of myeloid progenitor cells with an aberrant immunophenotype, aberrancies in maturing granulocytes and monocytes (Figure 4). Blood monocyte partitioning by FCM supports this conclusion (Figure 4f): classical monocytes were close to but not >94%; slan+ monocytes were characteristically below 1.7%, thus in keeping with CMML in an inflammatory background (Selimoglu-Buet et al., ; Tarfi et al., ).