Bone Marrow as a Source of Cells for Paroxysmal Nocturnal Hemoglobinuria Detection.

OBJECTIVES: To determine fluorescently labeled aerolysin (FLAER) binding and glycophosphatidylinositol-anchored protein expression in bone marrow (BM) cells of healthy volunteers and patients with paroxysmal nocturnal hemoglobinuria (PNH) detected in peripheral blood (PB); compare PNH clone size in BM and PB; and detect PNH in BM by commonly used antibodies. METHODS: Flow cytometry analysis of FLAER binding to leukocytes and expression of CD55/CD59 in erythrocytes. Analysis of CD16 in neutrophils and CD14 in monocytes in BM. RESULTS: FLAER binds to all normal BM leukocytes, and binding increases with cell maturation. In PNH, lymphocytic clones are consistently smaller than clones of other BM cells. PNH clones are detectable in mature BM leukocytes with high specificity and sensitivity using common antibodies. CONCLUSIONS: PNH clone sizes measured in mature BM leukocytes and in PB are comparable, making BM suitable for PNH assessment. We further demonstrate that commonly used reagents (not FLAER or CD55/CD59) can reliably identify abnormalities of BM neutrophils and monocytes consistent with PNH cells.

Increased immature hematopoietic progenitor cells CD34+/CD38dim in myelodysplasia.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal disorders affecting hematopoietic progenitor cells (HPC). Despite the relevance of clonal CD34+ cells in developing MDS, only few studies analyze the phenotype of this cell population. The aim of this study was to evaluate phenotypic changes on HPC in MDS that could reflect abnormalities in the differentiation process of stem cells. METHODS: We analyzed the expression of CD38 and HLA-DR on CD34+ cells by flow cytometry in 36 patients with MDS, as well as in healthy donors (n = 12) and patients with other hematological disorders: non-Hodgkin lymphomas and multiple myeloma, both in complete remission (CR) (n = 32); acute lymphoblastic leukemia in CR (n = 17); de novo acute myeloblastic leukemia (AML) at diagnosis (n = 22) and in CR (n = 37); and AML secondary to MDS at diagnosis (n = 19). Cases with available karyotype were grouped according to the International Prognostic Scoring System (IPSS). RESULTS: Compared to normal BM, the fraction of immature HPC, characterized as CD34+bright, intermediate FSC/SSC, and CD38dim, was significantly increased in high risk MDS and secondary AML, but not in low risk MDS, (P < or = 0.001, P = 0.03, and P = 0.7). De novo AML showed decreased immature HPC. High numbers of immature HPC correlated with higher IPSS risk groups (P = 0.05) and showed significant impact on disease progression (P = 0.03). CONCLUSION: Our study confirms that evaluation of CD38 expression pattern on HPC is an easy and reproducible test that allows evaluating the immature subset of progenitor cells. Increased immature HPC in high risk MDS and secondary AML may reflect blocked differentiation of CD34+ cells in these diseases.

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting.

BACKGROUND: Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. METHODS: A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. RESULTS/CONCLUSION: Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 10(6) and 5 × 10(6) bone marrow cells to be measured, respectively.

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells.

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor kappaB (NF-kappaB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-kappaB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-kappaB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.

Is assessment of surface CD38 expression worthwhile as a prognostic factor in chronic lymphocytic leukemia patients?

We studied the clinical impact of CD38 expression in 226 chronic lymphocytic leukemia patients (CLL) at disease presentation and during follow up to determine its prognostic significance, progression free survival (PFS) and overall survival (OS), and to verify whether this parameter changed over time. Various patients' characteristics were studied including gender, Rai and Binet stages, immunoglobulin light chain expression, lymphocyte doubling time and CD38 expression. After a median follow up of 53 months (range 6-282), 62% CD38 positive(+) patients required therapy. PFS and OS at 84 months were significantly lower for CD38(+) patients: 20 and 71% respectively, compared to CD38 negative(-): 70 and 96%. At multivariate analysis CD38(+) showed to be the best factor for predicting progression: HR 3.3, 95%CI 2.10-5.14, p = 0.000. Its expression did not change in 98% re-evaluated patients. We confirm that CD38(+) is a stable parameter for the identification of CLL patients with a more aggressive disease course.

Fine needle aspiration for flow cytometry immunophenotyping of non Hodgkin lymphoma

The aim was to evaluate the usefulness of lymph node biopsies obtained by fine needle aspiration (FNA) for immunophenotyping of non Hodgkin lymphoma (NHL). Seventeen superficial and deep lymph node samples were fractioned for conventional cytological examination and immunophenotyping studies. Out of ten NHL, nine were readily detected by flow cytometry (FC), while failure on the remaining case was due to selective loss of large cell population, which is liable to occur with this procedure. A single case, which proved negative for all markers employed, was finally diagnosed by immunohistochemistry as germ cell tumor. The other six cases, presenting lymphoid population without phenotypic abnormalities, were diagnosed by cytology and/or histology as Hodgkin disease or hyperpiasic disorders. To conclude, FC immunophenotyping seems to improve the efficacy of FNA in NHL diagnosis, whereas for Hodgkin disease and hyperplasic disorders, classic morphological criteria are more useful for differential diagnosis. Although FNA for FC immunophenotyping cannot replace histopathological examination for NHL diagnosis, it proves to be a useful tool for staging and follow up, making surgical procedures for sample collection unnecesary.

Primer estudio multicéntrico realizado por el Grupo Rioplatense de Citometría de Flujo: control de calidad en las mediciones de subpoblaciones de linfocitos T / First multicentric study by Grupo Rioplatense de Citometría de Flujo : quality control of lymphocyte subsets

El control de calidad se efectuó sobre los valores obtenidos, relativos y absolutos, de linfocitos T y de sus subpoblaciones CD4+ y CD8+ en muestras de sangre de pacientes infectados con el virus de la inmunodeficiencia humana (HIV). El estudio incluyó dieciocho centros: diez utilizaron citómetros de flujo de Becton Dickinson, tres de Coulter y 5 de Ortho que representan a 17 laboratorios de Argentina y a uno de Uruguay. Los siguientes programas se utilizaron para analizar los datos : SimulSET, Paint a Gate (Becton Dickinson), Profile II, XL System (Coulter), ImmunoCount Trio y Combo Cytoron (Ortho). Se obtuvieron muestras de sangre periférica en horas de la mañana (8 a 10 hs) de 10 voluntarios normales (por serología y hemograma) y de 10 pacientes HIV positivos con valores previos de CD4 que variaron entre 200-350 células por microlito y fueron procesadas dentro de las 12 horas. Cada centro obtuvo los valores relativos con el procedimiento técnico habitual y el de los valores absolutos utilizando el hemograma propio. Además, en un contador hematológico Cell-Dyn 3500 se obtuvo para cada muestra el hemograma correspondiente considerado de referencia. Los valores absolutos medios, obtenidos en cada centro con el hemograma propio, para los linfocitos T y los de sus subpoblaciones fueron significativamente diferentes. No hubo diferencias significativas para los valores porcentuales entre los diferentes centros ni para los valores absolutos obtenidos con el hemograma de referencia. Concluimos que las diferencias en los valores absolutos de los linfocitos T y sus subpoblaciones dependen del recuento hematológico empleado.