Mar Bellido

Flow cytometry using the monoclonal antibody CD10-Pe/Cy5 is a useful tool to identify follicular lymphoma cells

Abstract: Follicular lymphoma (FL) is a specific entity defined by characteristic histology, phenotype and molecular rearrangements. Classically, reactivity for CD19, CD10, and strong positivity for the surface light chain immunoglobulin (SIg) are considered to be phenotypic signs typically expressed in FL. In practice, this pattern is difficult to identify since most neoplastic cells analysed by flow cytometry (FC) show weak intensity for CD19‐Pe/Cy5 and for SIg and negativity for CD10‐FITC. We used triple antigen combinations including two monoclonal antibodies (MoAbs) against CD10 (CD10‐FITC and CD10‐Pe/Cy5) and a long‐distance polymerase chain reaction (PCR) approach to establish the phenotypic pattern of neoplastic cells carrying t(14;18)(q32;q21). Neoplastic cells showed the following immunophenotype: stronger reactivity against CD20 than against CD19, positivity for CD22 and SIg and negativity for CD5, CD11c and CD10‐FITC. Characteristically, CD10‐Pe/Cy5 was expressed in all the samples with positive bcl‐2/JH rearrangements. In FL, there was a high correlation between histologic diagnosis and reactivity against CD10‐Pe/Cy5 (96% cases). In diffuse large cell lymphomas (DLCL), CD10‐Pe/Cy5 identified positive cases with t(14;18)(q32;q21) chromosomal translocation, whereas Burkitt lymphomas showed all cases reactivity against CD10‐Pe/Cy5. In conclusion, CD10‐Pe/Cy5 is a useful antibody for identifying neoplastic cells carrying t(14;18)(q32;q21) in FL and DLCL. In combination with other MoAbs, anti‐CD10 (HI10a, Cy‐Chrome) can be used to identify a characteristic phenotypic profile of FL against other lymphoproliferative disorders.

Reticulocyte recovery is faster in allogeneic and autologous peripheral blood stem cell transplantation than in bone marrow transplantation

To the Editor: Over recent years automated methods have allowed a more accurate quantitative measurement of the reticulocytes (RET) and, more importantly, a new parameter has emerged in the study of the biology of RET (1). Specifically, the automated flow cytometry RET counters subdivide the circulating RET into 3 maturational stages. The more immature fractions have a higher RNA content (1, 2), and their rise in the early stages of hematopoietic recovery following intensive chemotherapy or bone marrow transplantation (BMT) has been shown to be the earliest objective sign of marrow recovery (3-6). Since granulocytes and platelets recover earlier in peripheral blood stem cell transplant (PBSCT) than in BMT, we performed a prospective study to determine whether or not absolute and immature RET recover earlier following PBSCT than BMT (both allogeneic and autologous) and whether this rise preceds granulocytic recovery, as seen in BMT. Twenty-three consecutive patients undergoing autologous transplantation (13 BMT and 10 PBSCT) who successfully engrafted were prospectively studied (AUTO group). The allogeneic (ALLO) group included 19 consecutive patients who received an allogeneic graft (9 PBSCT and 10 BMT) from an HLA-identical sibling and who successfully engrafted. All autologous BMT recipients received granulocyte-colony-stimulating factor (G-CSF, 5 pg/kg sbc per day) from d+l and until stable neutrophil engraftment, but none of the other groups received any growth factor. Prophylaxis for graft-versus-host disease included cyclosporine A and a short course of methotrexate in all allogeneic grafts, without T-cell depletion of the graft in any case. The Sysmex R-2000 (TOA Company, Kobe, Japan) was used to quantify the absolute RET count and its maturation fractions. The high and medium fluorescence ratio RET fractions (HFR and MFR, respectively) have a high RNA content and include immature RET which are expressed as the percentage of total RET. Thus, the percentage of HFR and MFR is referred to as the immature RET fraction (IRF). Peripheral blood counts, absolute RET counts and IRF were closely monitored following the transplant. The following criteria were used to define the day of recovery of neutrophils, RET and I R F the first of at least 3 consecutive days with an absolute neutrophil count (ANC) >O.5x1O9/1 and the first of at least 3 consecutive da s with an absolute RET count of at least 0 . 0 2 ~ lo& (absolute RET recovery) and an IRF >lo% (expressed as the percentage of total circulating RET). The recovery kinetics of the IRF and the neutrophil recovery were compared in each group (ALLO and AUTO) between the PBSCT and BMT recipients. Statistical analyses were performed using Students t-test for paired and non-paired data for the comparison of the recovery of the ANC and IRF between groups. The results were expressed as the mean of the day of recovery and the 95% CI, using a p value <0.05 as the level of significance. Table 1 summarizes the results obtained in all the groups. In the AUTO group patients who received BMT recovered IRF on d 13.5 (95% CI, 11.2-20.2), absolute RET on d 26.9 (95% CI, 19.734.1) and the ANC on d 22.6 (95% CI, 16.6-30.4). Patients who received PBSCT recovered IRF on d 10.1 (95% CI, 8.7-12.7), absolute RET on d 15.8 (95% CI, 12.6-19) and the ANC on d 14.7 (95% CI, 11.7-18.2). There were no statistically significant differences in the recovery of IRF between autologous BMT and PBSCT, although a trend for a faster recovery in the PBSCT was observed (see Table 1). The absolute RET recovery was significantly faster in PBSCT than in BMT recipients. More importantly, the IRF recovered before the ANC in both subgroups; thus, an IRF >lo% was reached 9.1 d (95% CI, 4.6-13.5) before an ANC >O.5x1O9/1 in the PBSCT subgroup, while they recovered 4.6d (95% CI, 1.6-7.7) earlier in the BMT subgroup. Finally, as expected, the ANC

Clonal Heterogeneity Assessed by Flow Cytometry in B-Cell Lymphomas Arising From Germinal Centers

Patients with mature follicular B-cell lymphomas develop aggressive non-Hodgkin lymphomas (NHLs) during disease progression. It is controversial whether most diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) emerge as de novo lymphomas or from an original follicular lymphoma. To distinguish clonally related populations in aggressive NHL, we studied the immunophenotypic features of 18 consecutive samples from 16 patients. Three flow cytometric patterns were distinguished: (1) a homogeneous neoplastic population of large B cells with phenotypic features of follicular center cells; (2) 2 atypical populations of B cells, small monoclonal B cells, and large B cells with loss of some surface antigens; and (3) 2 clonal populations of small and large B cells sharing the same light-chain isotype. The 3 flow cytometric patterns were observed, respectively, in de novo DLBCL and BL, transformation into BL, and transformation into DLBCL. Flow cytometric data can provide valuable information about the natural history of NHL.