Biomedical subjects
Publications and source records attributed to G E Marti.
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In order to quantitate a previously noted decrease in CD20 fluorescence intensity (FI) on B-CLL lymphocytes, binding capacities [BC x 10(3) +/- 1SD = number of antibodies bound per cell] were calculated. The mean (N = 5) BC x 10(3) +/- 1SD of CD20 reagents for normal B-PBL and B-CLL lymphocytes confirmed this observation. B-PBL and B-CLL were 56 +/- 11 and 61 +/- 14, and 19 +/- 15 and 18 +/- 16, respectively, for Leu 16 and B1. Although adequate compensation standards for the determination of CD5 and CD20 coexpression are not available, qualitatively, the density of CD5 on both normal B-PBL and B-CLL is less compared to the expression of CD5 by normal T cells. CD5 expression on B-CLL seems to be linked to the lower levels of CD20, whereas CD5 expression may appear to be absent on CLL lymphocytes expressing normal levels of CD20. Levels of CD20 in B-CLL suggest involvement of one or two genes (alleles) whose decreased expression may be linked to CD5 expression.
We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WB) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lower values. Autofluorescence values were comparable by all methods except Immuno-lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45-stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q-Prep uniformly gave the highest purity of CD45-positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45-stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.
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A flow cytometric analysis of five B cell chronic lymphocytic leukemic (B-CLL) cell lines was undertaken using 129 unknown reagents from the blind panel (BP) and 72 reagents from the known CD panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B cell section (Vienna, 1989). The five cell lines examined were: SeD (PNAS 75, 5706, 1978), B-CLL-LCL (BLOOD 71, 9, 1988), JVM-HH and JVM-2(INT J CAN 38, 531, 1986), and WR#1 (TH and BD). The reagents were #1-129 (blinded panel) and reagents 1-44 and 53-84 (CD panel with CD23 reagents missing). Positivity was defined as greater than 30% of the cells having a three fold increase or more in mean channel fluorescence. Fourty-three reagents of the blinded panel were negative by these criteria while all remaining reagents were positive on all five lines. SeD showed the lowest reactivity; B-CLL-LCL and JVM-2 showed the most reactivity; JVM-HH and WR#1 were intermediate. The known CD panel confirmed the reactivity of the blinded panel. An average immunophenotype was constructed and compared to published normal EBV lymphoblastoid cell lines and several differences were noted. There was an absence or significant decrease in the expression of CD19, CD21, CD22 and CD37 while there was an increased expression of CD38, CD54, CD74 and CD76. The heterogeneity observed between the B-CLL lines may in part be due to polymorphisms but is more likely to represent the underlying heterogeneity seen in common and familial B-CLL.2+öff
Site-selective cyclic AMP (cAMP) analogues have been shown to inhibit growth and induce differentiation in several human leukemia cell lines. However, detailed studies of the effects exerted by cAMP analogues on cell cycle kinetics have been lacking. We have examined the effects of 8-Cl-cAMP and N6-benzyl-cAMP on the cell cycle kinetics of the HL-60 human promyelocytic leukemia cell line. A cell cycle study was performed by univariate DNA analysis after 24-72 h of treatment with noncytotoxic concentrations of 8-Cl-cAMP and N6-benzyl-cAMP capable of inducing 50-60% growth inhibition in these cells. HL-60 cells treated with 5 microM 8-Cl-cAMP showed no significant change in the cell distribution in the cycle as compared to the untreated control cells, whereas the treatment with 10 microM N6-benzyl-cAMP transiently increased the percentage of cells in the G0/G1 phase after 48 h, followed by a partial recovery at 72 h. Combined treatment with low doses of 8-Cl-cAMP and N6-benzyl-cAMP, each of which alone produced 20% growth inhibition, exerted a growth inhibitory effect of 65% and delayed increase of the G0/G1 phase by 72 h. To better understand the cell cycle effects induced by 8-Cl-cAMP, flow cytometric analysis of bromodeoxyuridine incorporation was also performed. 8-Cl-cAMP treatment exhibited a slowing down of the cell cycle; thus, the delayed appearance of the G0/G1 cell accumulation after combined treatment could be due to this effect of 8-Cl-cAMP on the HL-60 cell cycle. At a toxic dose, 8-Cl-cAMP brought about a G2M block, whereas N6-benzyl-cAMP brought about an increase of the G0/G1 compartment. G2M block produced by toxic doses of 8-Cl-cAMP was not related to its adenosine metabolite since 8-Cl-adenosine did not produce any specific block in the cell cycle. Our results show, for the first time, that these site-selective cAMP analogues could affect cell cycle kinetics at different points. These data may provide the basis for combination treatments involving cAMP analogues and other agents in the treatment of human leukemia.
The expression of the interleukin-2 (IL-2) receptor was studied in neoplastic cells derived from acute leukemias, T-cell lymphoblastic lymphomas, peripheral T-cell lymphomas, chronic lymphocytic leukemias, well-differentiated lymphocytic lymphomas, and established cell lines by both flow cytometric analysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) after affinity crosslinking of radiolabeled IL-2. Cells from most acute leukemias (19 of 22), irrespective of their subtype (T, common or nonlymphoid leukemias), as well as T-cell lymphoblastic lymphomas and peripheral T-cell lymphomas expressed only the p70-75 beta subunit of the IL-2 receptor. Cells from the more mature B-cell neoplasms, chronic lymphocytic leukemia, and well-differentiated lymphocytic lymphoma, expressed predominantly alpha beta IL-2 receptors (11 of 14). In contrast to these results, most cell lines established from hematopoietic malignancies do not express either chain of the IL-2 receptor. Further studies are necessary to determine the exact function of the IL-2R p70-75 beta subunit in immature hematopoietic cells, but its wide distribution throughout the hematopoietic system suggests that IL-2 may play a role in the early stages of hematopoiesis.
A previously described patient with X-linked agammaglobulinemia and growth hormone deficiency developed an echovirus-associated meningoencephalitis and dermatomyositis-like syndrome while being treated with intramuscular gamma globulin and human growth hormone. Initiation of high-dose intravenous gamma globulin resulted in resolution of the clinical symptoms and the patient has remained asymptomatic over the past 55 months. Lymphocyte phenotype analysis at the time of presentation with echovirus infection revealed an increase in CD2+, CD16+, HNK-1+ lymphocytes, a decrease in CD4+ T cells as well as absence of B cells. This elevation in the LGL/NK phenotype resolved with clinical improvement. In addition, there was evidence of lymphocyte activation following the development of echovirus infection (increase in HLA-DR expression and elevated serum IL-2 receptor levels) which resolved with clinical improvement. A muscle biopsy obtained during the period of the dermatomyositis-like syndrome demonstrated a CD8+ lymphocytic infiltrate very similar to the observations in classical dermatomyositis. Taken together, these findings suggest that growth hormone therapy in this patient failed to alter the humoral immunodeficiency. In addition, serum IL-2 receptor levels and lymphocyte phenotyping may be useful adjuncts for monitoring echovirus disease in immunodeficient patients.
A case of lymphoma in a middle-aged woman with Gaucher's disease, a mu-kappa monoclonal gammopathy, peripheral lymphocytosis, and massive splenomegaly is described. There was no evidence of Gaucher's disease in the splenectomy specimen. Instead, the splenic architecture was effaced by a small lymphocytic lymphoma (SLL), with a few foci of large cell lymphoma (LCL). Immunoperoxidase studies showed that both lymphomatous components had a mu-kappa immunophenotype. Flow cytometric analysis of peripheral blood and spleen lymphocyte surface markers confirmed monoclonality. The intensity of surface immunoglobulin expression and the results of other cell marker studies in the SLL component were consistent with a stage of differentiation beyond that typical of chronic lymphocytic leukemia (CLL). The presence of abundant cytoplasmic immunoglobulin in the SLL and the association with monoclonal gammopathy were more consistent with Waldenström's macroglobulinemia or plasmacytoid lymphocytic lymphoma. The immunohistochemical and flow cytometric findings suggest that the SLL component evolved clonally or underwent Richter's transformation to LCL. Although Gaucher's disease has been associated with a variety of B-cell disorders, the subsequent transformation to a high-grade lymphoma has not been previously reported.
A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.
Sera from 23 individuals with Gaucher disease (GD) were analyzed for hypergammaglobulinemia and oligoclonal and monoclonal gammopathies. Serum IgG level was elevated in 15/23 (65%) patients, and a diffuse hypergammaglobulinemia was present in 10/23 (43%) patients. An oligoclonal gammopathy was noted in six patients, and a monoclonal gammopathy in two. Lymphocyte subset analysis was also carried out in eight individuals with GD. Four of five individuals showed increased surface Ig-positive lymphocytes, while 7/7 were positive for either increased CD19- and/or CD20-positive lymphocytes. An eighth patient was found to have a B-cell leukemia. Statistical analysis of kappa and lambda histograms were suggestive of a monoclonal excess. However, restriction enzyme analysis of four individuals with GD and increased B cells failed to show any evidence of Ig gene rearrangements. Serum Ig abnormalities and perhaps B-cell lymphocytosis appear to be common in the GD patient population and are not associated with circulating monoclonal lymphocytes.
Peripheral blood mononuclear cells (PBMC) from normal individuals were examined using 16 pairs of FITC and phycoerythrin (PE) directly conjugated monoclonal antibodies. Each pair of reagents was used to evaluate a conventional lymphocyte gate as well as open (non) gate of monocyte depleted PBMC. Parallel studies using the same panel of monoclonal antibodies were carried out on selected, nonmonocyte depleted samples. The major findings of this analysis were that 1,000-1,200 lymphocytes in a 10,000 cell analysis are found outside the lymphocyte gate and of these approximately 2/5 are CD16 positive LGL/NK cells, 2/5 are CD3 positive T cells, and 1/5 are CD19/CD20 positive B cells. Thus, it appears that 10-15% of the lymphoid cells fall outside of the conventional lymphocyte gate, and in certain settings monocyte depletion may be useful to perform more complete evaluation of the total lymphoid cell population obtained after ficoll-hypaque separation.
A flow cytometric analysis of human platelet surface antigens was carried out using a panel of monoclonal antibody reagents. The reagents used were specific either for the GPIb or the GP IIb/IIIa complex, surface immunoglobulin, or von Willebrand factor (vWf). Indirect surface immunophenotypes were determined using an EPICS V flow cytometer and the monoclonal antibodies 6D1, 10E5, Plt-1, UR1663, anti-IgG, and anti-vWf. Platelets were obtained from normal individuals or patients with either Bernard-Soulier syndrome (BSS) or Glanzmann's thrombasthenia (GT). Normal platelets were positive for 10E5, 6D1, Plt-1, and UR1663 and showed negligible activity for anti-IgG and anti-vWf. Platelets from individuals with BSS showed a marked reduction in 6D1, while the platelets of a patient with GT showed a marked reduction in binding of 10E5, Plt-1, and UR1663. Differences between histograms for normal platelets and for platelets from individuals with BSS or GT were evaluated using the Kolmogorov-Smirnov test. Compared to normal platelets, the BSS and GT platelets contain at least 35-fold less of the GPIb and GP IIb/IIIa complex respectively. Flow cytometry is a useful and precise method for the study of normal and abnormal surface platelet phenotypes.
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The results of lymphocyte immunophenotyping in a variety of autoimmune disorders confirm major T cell immunoregulatory defects. The defects associated with autoreactive T cells appear to exist at the level/interface of the CD4 inducer of suppression and the CD8 effector cell. Although activated CD4 cells are occasionally found, subpopulations of activated CD8 cells are seen more commonly. A similar observation has been made in a subpopulation of patients with common variable hypogammaglobulinemia. In conjunction with antigen-specific T cell clones, we anticipate that flow cytometry will continue to aid in the further dissection of these HLA-restricted, anti-idiotype-controlled and pharmacological-mediated reactions. The known immunological distinction between AML and ALL are such that blast immunophenotyping will confirm and complement the clinical and morphological diagnosis in the vast majority of patients. With regards to chronic lymphocytosis in general and CLL in particular, flow cytometry offers an unusual opportunity to characterize lineage, monoclonality, stage of differentiation, presence or absence of activation antigens, aneuploidy and oncogene expression. Flow cytometry will continue to contribute to our understanding of the etiology and pathogenesis of CLL.
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In an effort to develop an assay which would rapidly detect and analyze circulating immune complexes, we have adapted the Raji cell radioimmunoassay (RIA) to a flow cytometric (FCM) analysis. The advantages of immune complex analysis by FCM are many. Foremost is the effectiveness and efficiency of the FCM method relative to the radioimmunoassay (RIA) method. The data demonstrated that FCM detection is three times more sensitive than RIA detection. Only populations of viable Raji cells bearing immune complexes are analyzed because parameters of the FCM analysis permitted the elimination (gating out) of dead cells. The determinations are rapid and the data are immediately available for several additional analyses. Because of the availability of many fluorescent monoclonal antibodies to complement components, viral antigens, light chains and immunoglobulin isotypes, it is possible to detect many components that might be present in the Raji cell bound complexes. Finally, the Raji cells can be characterized in different stages of their cell cycle to generate information about the state of the cells and the density of the receptors involved in binding the complexes.