Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Flow cytometric detection of aneuploid CD38(++) plasmacells and CD19(+) B-lymphocytes in bone marrow, peripheral blood and PBSC harvest in multiple myeloma patients.

DNA aneuploidy has been used as a genetic marker of malignancy in multiple myeloma (MM). CD38 and CD138 expression and absence of CD22 and CD19 may define plasmacells (PC). Several authors support evidences of circulating plasmacells, and their role in relapse after autologous stem cell transplantation has been hypothesised. The existence of B-lymphocytes belonging to the myeloma clone is still controversial. If CD19 or CD22 positive B-lymphocytes are part of the myeloma clone, there should be evidence of myeloma-specific genetic markers in this population. Using DNA content measurement in combination with CD19 or CD38 detection in a multiparametric flow cytometry analysis, we studied bone marrow and peripheral blood of 10 aneuploid MM patients. In the bone marrows of all these 10 aneuploid patients (100%), we detected CD38(++) aneuploid plasmacells ( 27 +/- 17%, mean +/- S.D.) and a small number of CD19(+) aneuploid lymphocytes ( 0.11 +/- 0.074%). In 100% of these patients, we also detected CD38(++) aneuploid circulating plasmacells ( 0.6 +/- 0.9 %) and a small number of CD19(+) aneuploid lymphocytes (0.03 +/- 0.04%). In this study, we detected aneuploid CD19(+) lymphocytes and CD38(++) plasmacells in bone marrow and peripheral blood of all MM patients. A crucial role for the detection of aneuploid CD19(+) cells was played by the acquisition of a sufficient number of CD19(+) lymphocytes by using a "live gate" acquisition and "continuous gating" analysis. With the techniques used in this study, it was possible to detect aneuploid B lymphoid cells among normal diploid B cells. The significance of this finding is controversial and opened to different interpretations.