Report on the first Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia.

On October 16, 1996, the first Latin American Consensus Conference for the Immunophenotyping of Leukemia took place in Puebla, Mexico, with representatives from 10 countries of the region and two external consultants. This document summarizes the major conclusions for which scientific consensus was achieved. The purpose of disseminating these guidelines to the international community is based on the potential interest for other countries with similar social conditions and economical restrictions.

[1st Latin American consensus conference on immunologic typing of leukemias]. / Primera conferencia latinoamericana de consenso en la tipificación inmunológica de leucemias.

On October 16, 1996, the first Latinamerican Consensus Conference for the Immunophenotyping of Leukemia took place in the City of Puebla, Mexico, with representatives from ten countries of the region, and two external consultants. This document summarizes the major conclusions where scientific consensus was achieved.

Flow cytometric analysis of lymphomas and acute leukemias.

In summary, flow cytometry is highly applicable to the detection and classification of leukemias and lymphomas due to the ease with which single-cell suspensions may be made. Composite immunophenotypic analysis is essential for classifying leukemias once the disease is detected by traditional means. In contrast, in the detection of lymphoproliferative diseases, the composite immunophenotype is itself diagnostic of the disease process. Characterization of size, as measured by light scatter and by DNA ploidy and cell cycle analysis, contributes to the further subdivision of lymphoproliferative disorders. Specifically, small, monoclonal cells that are diploid with a synthetic fraction of 5% are characteristic of low-grade lymphomas. Admixtures of large and small cells wherein the large cells are monoclonal and the small cells are either monoclonal or heterogeneous may be seen in intermediate lymphomas. In this category, DNA ploidy is variable and the total synthetic fraction is usually between 5% and 15%. High-grade lymphomas, with the exception of the immunoblastic category, are usually of intermediate size, are diploid or near-diploid, and exhibit synthetic fractions greater than 15%. Interestingly, few reactive T cells are seen. Ongoing efforts to standardize procedures will eventually result in more widespread applicability together with improved understanding of the attributes and limitations of this technology. The most important consideration, however, is that the technology is useless in the absence of a working knowledge of the biology of the diseases to be characterized. Conversely, the complexity of flow cytometry is sufficient to warrant rigorous training of laboratory professionals in this field.

Reduced expression of CD20 antigen as a characteristic marker for chronic lymphocytic leukemia.

The surface antigens expressed by the cells of chronic lymphocytic leukemia (CLL) are well known. Most CLL are monoclonal B-cell lymphoproliferative disorders characterized by the coexpression of B-cell antigens and CD5, an antigen present predominantly on T cells. Very little attention, however, has been paid to the quantitative characteristics of the expression of B-cell antigens in CLL. In this study, we used flow cytometry to analyze the expression of CD20, a well-known B-cell-associated antigen, in lymphocytes from 42 cases of CLL and its tissue counterpart, small lymphocytic lymphoma (SLL), and compared the results with results obtained from the analysis of 21 follicular lymphomas, 20 hyperplastic reactive nodes, and 26 samples of normal peripheral blood. The intensity of CD20 expression in the CLL/SLL cells was significantly lower than that of B cells in the other categories. This antigen expression abnormality does not appear to be a universal phenomenon in CLL/SLL, since CD19, another pan-B antigen, was expressed in CLL/SLL at levels higher than those in follicular lymphomas and comparable to those in reactive lymph nodes. These results indicate that the low CD20 expression can be used as a marker for CLL/SLL. The few cases exhibiting intense CD20 expression may represent a biologically different disease. CLL/SLL cells faintly expressing CD20 also show concomitant low CD5 expression in a manner not observed in normal CD5-expressing B cells.

Biclonal composite lymphoma. A multiparameter flow cytometric analysis.

A case of composite, biclonal lymphoma detected and characterized by multiparameter flow cytometric analysis is presented. Analysis of cell surface immunophenotype and cell size, as assessed by forward light scatter, revealed that two populations of cells were present. The small cells were monoclonal kappa-positive cells admixed with reactive T and B cells. The large cells reacted solely with anti-lambda antibodies. Dual-color and dual-parameter (surface vs DNA) analysis further showed that the small, kappa-positive cells coexpressed CD5 and were diploid, with an estimated synthetic (S) fraction of 2.2%. The predicted histologic pattern was malignant lymphoma, small lymphocytic. In contrast, the large lambda-positive cells were both hyperdiploid and tetraploid with an estimated S fraction of 18%. On the basis of this multiparametric analysis, the predicted histologic pattern for the latter component was malignant lymphoma, diffuse large-cell type. Subsequent histologic examination confirmed the predicted pattern in both cases.

High-grade B-cell lymphoma presenting as polyserosal disease. Diagnosis by flow cytometry.

Two patients presenting with anasarca were found to have aggressive B-cell lymphoma. No bulky disease was detected. The diagnosis was rapidly established by the flow cytometric analysis of cell surface immunophenotype and cell cycle fractions of pleural or peritoneal cells. Such presentation of lymphoma is unusual and previously undescribed, and it may have a significant negative prognostic impact. The authors' observations indicate that lymphoma be included in the differential diagnosis of anasarca and that flow cytometry can be useful for a fast confirmation of the diagnosis.

Immunophenotypic characterization of an unusual T-cell lymphoma presenting as anterior uveitis. A clinicopathologic case report.

A 54-year-old woman presented with a unilateral, anterior uveitis that progressed to hypopyon over 4 months despite treatment with steroids. One hundred percent of the cells collected from aspirates of the anterior chamber of the affected eye were morphologically large granular lymphocytes. The aspirated cells were demonstrated by flow cytometry to be a uniform population of T lymphocytes with a diploid genome and an S fraction of 2.3%. On further investigation, the patient was found to have an extensive abdominal malignant lymphoma with the same immunophenotype but different morphologic features than the anterior chamber lymphoid infiltrate. In contrast to the cells in the anterior chamber, the abdominal tumor was highly aggressive as indicated by the cellular morphologic features and the S fraction of 43%. DNA hybridization studies of the abdominal lymphoma demonstrated a T beta 2 T-cell receptor gene rearrangement. The use of these modern diagnostic methods should facilitate the diagnosis of intraocular lymphomas and may have important therapeutic and prognostic implications in the future.

3-deaza-adenosine inhibition of stimulus-response coupling in human polymorphonuclear leukocytes.

In an effort to define better the functional role of S-adenosyl-methionine-mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3-deaza-adenosine (3-DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl-leucyl-phenylalanine (FMLP)-induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3'-dipropylthiocarbocyanine (di-S-C3-(5)) as an optical probe of membrane potential we observed that 3-DZA at concentrations that inhibit FMLP-induced O2- production does not significantly alter FMLP-induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3-DZA on FMLP-induced PMN pinocytosis and to a lesser degree on FMLP-induced degranulation. However, pretreatment of PMNs with 3-DZA did not alter FMLP-induced changes in Quin-2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor-induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor-induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor-induced methylation reactions. Furthermore, 3-DZA did not alter phorbol myristate acetate induced O2- production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2- production.

Reduced oxidative function in gingival crevicular neutrophils in periodontal disease.

Measurable amounts of viable and functional polymorphonuclear neutrophils (PMNs) are recovered from pooled washings of the gingival crevice of healthy individuals. In the present study, we have assessed the ability of the PMNs removed from single healthy or diseased pocket sites to mount an oxidative burst when challenged with phorbol myristate acetate (PMA) and compared these activities with each other and with those obtained with autologous peripheral-blood PMNs. The oxidative burst after PMA stimulation was evaluated by using methods developed for the flow cytometer. The results showed that the PMNs collected from untreated disease sites were minimally responsive to PMA when compared with peripheral-blood PMNs collected at the same time from the same individual. Thus, whereas the peripheral-blood PMNs exhibited significantly lower resting oxidative product formation and a 500% increase when stimulated with PMA, all gingival-crevicular PMNs exhibited significantly higher resting formation of oxidized products but only a 150% increase after PMA stimulation. PMNs obtained from a consistently healthy site had significantly higher resting production of oxidized products and were able to mount the greatest absolute increase in oxidized products after PMA stimulation when compared with PMNs collected from diseases sites. Mechanical debridement of these diseased sites, which both reduced the bacterial numbers and restored clinical health, resulted in the recovery of gingival-crevicular PMNs that exhibited an oxidative burst more typical of that observed in PMNs obtained from healthy gingival sites and from the peripheral blood. This suggested that the PMNs collected from the diseased sites either had been exhausted by the large numbers of bacteria present in these sites or had been specifically inhibited by these bacteria.

Quantitative assessment of neutrophil function by flow cytometry.

The use of flow cytometry (FCM) to quantitatively assess neutrophil function is reviewed. The methodology is capable of measuring a number of parameters involved in the oxidative pathways that form the basis of the activated neutrophil's contribution to the host defense mechanism. These events are summarized and some findings, such as in patients with chronic granulomatous disease, are discussed. FCM study of neutrophil function requires smaller numbers of cells than do traditional methods, which makes it particularly useful in the assessment of small fluid samples or in the evaluation of multiple parameters, and has the advantage that cell purification procedures are not essential.

Oxidative product formation in irradiated neutrophils. A flow cytometric analysis.

The effect of irradiation on neutrophil oxidative function was evaluated using a flow cytometric assay of intracellular hydrogen peroxide (H2O2) production. This assay quantitates the H2O2-dependent conversion of the nonfluorescent compound, 2'-7'-dichlorofluorescein (DCFH), into fluorescent 2'-7'-dichlorofluorescein (DCF) on a single-cell basis. Intracellular H2O2 production in response to stimulation with phorbol myristate acetate was not affected by neutrophil irradiation at doses up to 2500 rad. In addition, irradiation of intracellular DCFH and aqueous 2'-7'-dichlorofluorescein diacetate (DCFH-DA) resulted in DCF production, which suggested that oxidative molecules produced by aqueous radiolysis were detected by this assay. This study indicates that radiation doses of 1500 to 2500 rad, which are sufficient to prevent induction of graft-versus-host disease by transfused blood components, are not deleterious to neutrophil oxidative metabolism.

Measurement of antineutrophil antibodies by flow cytometry: simultaneous detection of antibodies against monocytes and lymphocytes.

We describe a flow cytometric technique which detects the presence of antineutrophil antibodies (NABs) in human serum. The technique provides a qualitative as well as a semiquantitative screen and provides an excellent method for monitoring the presence of antineutrophil antibodies in patients with suspected autoimmune neutropenia. Using light-scatter gating, individual populations consisting of neutrophils, monocytes, and lymphocytes can be examined simultaneously for the presence of antibody. The methodology utilizes an indirect immunofluorescence technique with FITC-labeled goat antihuman F(ab')2 antibody and fixed leukocyte suspensions. Furthermore, by utilizing class-specific FITC-labeled second antibody, significant information can be ascertained regarding the class, cell specificity, and quantity of detected antibody. Formalin fixation of neutrophils prevented pinocytosis of the fluorochrome, significantly reducing background fluorescence. Twenty-five normal subjects provided baseline antibody levels for each class. Of 92 patients with suspected autoimmune neutropenia, 27 had class IgG alone and seven were positive for both IgG and IgM class NABs. During treatment, IgG levels varied. IgM NABs alone were detected in four patients. Fifty-four patients had undetectable antibody. Antibodies were detected against monocytes in several of the IgG-positive patients. Two sera contained both IgA and IgG NABs. One serum contained IgA and IgG antibodies against monocytes. No IgD antibodies were detected in any sera tested. Some sera tested contained antibodies against lymphocytes--however, only in those sera which also contained antibodies to other cell types.

Site-dependent phenotypic heterogeneity in peripheral T-cell lymphoma.

Peripheral T-cell lymphomas constitute a heterogeneous population of postthymic T-cell malignancies. Characteristically, they present a varied phenotypic expression, which can be helpful in establishing the diagnosis. A case of a peripheral T-cell lymphoma in a 76-year-old man is described. The malignant cells in the skin and bone marrow were of the T4 (helper/inducer) phenotype, yet they did not express pan-T-cell antigens, such as T11, or functional E rosettes. In a biopsy specimen from a lymph node, however, the malignant cells had a helper/inducer phenotype and also expressed the pan-T-cell antigens T11 and Leu-5. Additionally, the malignant cells from the lymph node formed E rosettes. This study demonstrates the phenotypic heterogeneity of malignant T cells, which appears to be site-dependent.