Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation

Abstract

Multiparameter immunophenotypic analysis of neoplastic cells has proven to be of great help for the investigation of minimal residual disease in acute leukemias; however, its utility has not been systematically explored in B cell chronic lymphoproliferative disorders. The aim of the present study was to investigate the incidence of phenotypic aberrations in a series of 467 consecutive leukemic B cell chronic lymphoproliferative disorders through the comparison of the phenotypic characteristics of tumor vs normal peripheral blood (n = 10) and bone marrow (n = 10) B cells, in order to explore the applicability of this strategy for minimal residual disease monitoring. An additional goal of our study was to evaluate the sensitivity of multiparameter flow cytometry for the detection of minimal residual disease in leukemic B cell chronic lymphoproliferative disorders through dilutional experiments (n = 19). From the patients analyzed 382 corresponded to B cell chronic lymphocytic leukemia/small lymphocytic lymphoma (353 typical and 29 atypical); five to prolymphocytic leukemia; 13 to hairy cell leukemias; 12 to lymphoplasmacytic lymphomas; 14 to splenic marginal zone lymphomas; 22 were follicular lymphomas; and 19 mantle cell lymphomas. The following triple stainings were systematically applied to both normal and leukemic samples: FMC7/CD5/CD19, CD22/CD23/CD19, CD103/CD25/CD19, CD10/CD11c/CD19 and sIg/sIg /CD19. Overall, 98% of the leukemic B cell chronic lymphoproliferative disorders cases displayed aberrant phenotypes at diagnosis with no significant differences being found between cases analyzed in peripheral blood vs bone marrow samples. The most common types of aberrant criteria detected included asynchronous antigen expression (92%) and antigen over-expression (54%); abnormally light scatter characteristics were found in 17% of the cases. Most of the cases studied (90%) displayed four or more phenotypic aberrations. Once patients were divided according to the different diagnostic subgroups, the overall incidence of aberrant phenotypes ranged from 79 to 80% among atypical B cell chronic lymphocytic leukemia/small lymphocytic lymphoma and prolymphocytic leukemia to 97% of follicular lymphoma and 100% of typical B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, hairy cell leukemia, lymphoplasmacytic lymphomas, splenic marginal zone lymphomas and mantle cell lymphomas. Based on the aberrant phenotypes detected unique four-color stainings could be built for the specific identification of aberrant phenotypes. These include CD22/CD23/CD19/CD5 and sIg /sIg /CD19/CD5 for lymphocytic leukemia/small lymphocytic lymphoma and prolymphocytic leukemia, CD103/CD25 or CD22/CD19/CD11c for hairy cell leukemia, FMC7/CD22/CD19/CD103 and sIg /sIg /CD22/CD19 for splenic marginal zone lymphomas, CD22/CD23/CD19/CD10 for follicular lymphomas and CD10/CD22/CD19/CD5 for mantle cell lymphomas. Serial dilutional experiments showed that the sensitivity level of immunophenotyping ranges between 10 −4 and 10 −5 . In summary, the p...

Quantitative analysis of the expression of glycosylphosphatidylinositol‐anchored proteins during the maturation of different hematopoietic cell compartments of normal bone marrow

“…Such discrepancies could be due to the use of less sensitive fluorochromes, MAb reagents, and instruments. As expected (14), the expression of CD16 was detectable on metamyelocytes, reaching the highest levels on bands/mature neutrophils, while expression of CD87 was only detectable at this latter stage of neutrophil maturation, as expected (6).…”

“…For all stainings described above a well-established stain-and-then-lyse protocol (13,14) was used, except for those combinations of MAb which included the CD55, CD58, and CD59 reagents; for these latter stainings a previously described lyse-wash-and-then-stain protocol was employed (13) in order to lyse non-nucleated red cells prior to the staining procedure, and then avoid their bind to these MAb. Expression of CD55, CD58, and CD59 on red cells from PB was evaluated using a nonlyse-non-wash protocol, previously described in detail (13).…”

Marginal zone B‐cell lymphoma: A retrospective immunophenotypic analysis

“…Most cases were additionally positive for CD22 and FMC7 whereas CD10, CD23, and CD38 were positive in only one-third of cases. CD11c was positive in 40-50% of cases (18,19) and was overexpressed as compared to normal B-cells in 30% of these cases (20). Unlike previous publications (19,20), we did not see expression of CD103 in SMZL.…”

“…CD11c was positive in 40-50% of cases (18,19) and was overexpressed as compared to normal B-cells in 30% of these cases (20). Unlike previous publications (19,20), we did not see expression of CD103 in SMZL. The explanation for this discrepancy is uncertain, but it may be secondary to relatively small number of cases studied.…”

Non‐CLL‐like monoclonal B‐cell lymphocytosis in the general population: Prevalence and phenotypic/genetic characteristics

“…A total amount of around 4 mL/case of EDTA-anticoagulated PB were immunophenotyped using a previously described direct immunofluorescence stain-and-then-lyse technique (14,15), with the following multicolor antibody stainings: (1) CD20-pacific blue (PB)/CD45-pacific orange (PO)/CD8-fluorescein isothiocyanate (FITC)þanti-surface immunoglobulin (sIg)k-FITC/CD56-phycoerythrin (PE) þ anti-sIgj-PE/CD4-peridinin chlorophyll proteincyanin 5.5 (PerCPCy5.5)/CD19-PE-cyanin 7 (PE-Cy7)/ CD3-allophycocyanin (APC)/CD38-AlexaFluor 700 (AF700); (2) CD20-PB/CD45-PO/Cybcl2-FITC/CD23-PE/CD19-PerCPCy5.5/ CD10/-PE-Cy7/CD5-APC/CD38-AF700; and (3) CD20-PB/anti-sIgk-FITC/anti-sIgj-PE/CD19-PerCPCy5.5/ CD10-PE-Cy7/CD5-APC. In those cases in which a clonal…”