RESUMEN
OBJECTIVES: Follicular lymphoma (FL) is the most common adult non-Hodgkin's lymphoma. Diagnosis is based on morphology and can be confirmed by cytogenetic, flow cytometry (FCM) or molecular studies. Despite all these complementary approaches, diagnosis sometimes remains difficult. The purpose of the present work was to characterise the expression of new specific follicular cells markers which allows us to target specifically the abnormal FL cell population in FCM. METHODS: A total of 153 samples from healthy subjects and from patients with chronic B-cell lymphoproliferative disorders were analysed by FCM in the same conditions for purpose of comparison. RESULTS: We showed that CD44 is weakly expressed in FL cells compared with peripheral blood mononuclear cell from normal blood donors and others cells from B lymphoproliferative diseases. We nevertheless observed bone marrow samples where some immature B-cell population express CD44 with lower fluorescence intensity. Therefore, we developed a double antibody combination, using CD44 and CD38, which allowed us to separate the normal immature cells from the pathological population using FCM. CONCLUSION: This new phenotypic approach offers an accurate (sensitivity and specificity of 93% and 96%, respectively), fast and low sample consuming method for the diagnosis of FL.
Asunto(s)
ADP-Ribosil Ciclasa/análisis , Anticuerpos/inmunología , Antígenos CD/análisis , Linfocitos B/inmunología , Citometría de Flujo/métodos , Receptores de Hialuranos/análisis , Linfoma Folicular/diagnóstico , ADP-Ribosil Ciclasa/inmunología , ADP-Ribosil Ciclasa 1 , Antígenos CD/inmunología , Médula Ósea/inmunología , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Receptores de Hialuranos/inmunología , Inmunofenotipificación , Ganglios Linfáticos/inmunología , Trastornos Linfoproliferativos/inmunología , Sensibilidad y EspecificidadRESUMEN
Integration of morphological and immunophenotypic data is critical in achieving diagnosis accuracy and minimising interobserver interpretative discrepancies. The aim of this work was to compare the immunophenotype and the morphology of chronic lymphocytic leukaemia and mantle cell lymphoma, to help in the differential diagnosis of CD5 positive monoclonal B cells. Frozen/thawed samples from 91 patients were analysed retrospectively. Fresh samples from 17 mixed/atypical CLL and 13 MCL were tested to corroborate the results. Markers were analysed as percentage (%) of positive B lymphocyte subpopulation, and in terms of median fluorescence intensity (MFI). Matutes's CLL score clearly allowed distinguishing between classical CLL on the one hand, and atypical CLL and MCL on the other hand. The percentage of CD54-positive cells and the median fluorescence intensity of CD20 and CD54 were the only parameters which were significantly higher in MCL than in atypical CLL (P < 0.05), allowing an immunological distinction between these two entities. Nevertheless, due to a quenching problem when using CD20 and CD54 together, and because CD18 showed a statistically different expression between classical and atypical CLL, the combination of CD18/CD54 has been preferred and showed a different pattern in the three entities. Immunophenotyping could be helpful in the differential diagnosis of CD5-positive B cell chronic lymphoproliferative disorders with atypical features that do not fit exactly into any of the morphologic proposed groups.
Asunto(s)
Antígenos CD20/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Leucemia Linfoide/inmunología , Linfoma de Células del Manto/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Antígenos CD5/análisis , Diagnóstico Diferencial , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Leucemia Linfoide/diagnóstico , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Cord blood (CB) transplantations are associated with low graft-versus-host disease (GVHD). The pathophysiology of GVHD involves interaction and activation of different cell types, as lymphocytes and monocytes, and results in a cascade of cytokine production. After antigen or mitogen stimulation, CB monocytes release lower levels of cytokines than adult blood (AB) monocytes. In this study, the detection of intracellular IL-1 beta and TNF-alpha produced by monocytes was evaluated in response to tuberculin PPD to investigate whether the reduced capacity of CB monocytes to secrete cytokines could be related to an impaired functional activity and to a particular phenotypic profile. Results showed that the percentage of CD64(+)monocytes producing intracellular IL-1 beta and TNF-alpha was significantly lower in CB and that the phenotypic profile of CB monocytes producing these cytokine (CD64(+)CD14(+)) was different to that of AB monocytes (CD64(+)CD14(+), CD64(+)CD33(+) and CD64(+) CD45RO(+)). These results suggest that the lower capacity of CB monocyte populations to produce IL-1 beta and TNF-alpha might be due to a functional immaturity of CB monocytes at the cellular level as reflected by the different phenotypic profile of CB monocytes.
Asunto(s)
Citocinas/metabolismo , Sangre Fetal/citología , Monocitos/metabolismo , Citocinas/genética , Sangre Fetal/química , Sangre Fetal/metabolismo , Citometría de Flujo , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Subgrupos Linfocitarios , Monocitos/química , Monocitos/efectos de los fármacos , Fenotipo , Tuberculina/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Freezing is a practical approach for cell preservation for retrospective studies. The aim of this work was to check the cryopreservation impact on B cell chronic lymphocytic leukaemia phenotype. MATERIAL AND METHODS: Blood samples from 15 CLL patients were analyzed freshly and after freezing at -196 degrees C, without separation, and thawing. Results were compared by Student's paired t-test. RESULTS: The phenotype of fresh CLL cells was as follows: CD19+, CD5+, faint CD20, CD23+/-, weak CD22 and sIg, CD37+, HLA-DR+, FMC7-. After cryopreservation, the percentage of CD5 and CD23 positive cells decreased, whereas HLA-DR positive cells increased moderately. The CLL Matutes's score was modified in 6 cases out of 15 (40%). CONCLUSION: Cryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression.
Asunto(s)
Conservación de la Sangre , Criopreservación , Leucemia de Células B/sangre , Leucemia de Células B/inmunología , Adulto , Antígenos CD/sangre , Linfocitos B/inmunología , Antígenos CD5/sangre , Estudios de Evaluación como Asunto , Citometría de Flujo , Antígenos HLA-DR/sangre , Humanos , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Inmunofenotipificación , Receptores de IgE/sangreRESUMEN
Because 2-chloro-2'-deoxyadenosine (CdA) is active in B-chronic lymphocytic leukemia (B-CLL), and may interfere with DNA repair, we investigated the potentiating effect of CdA on the cytotoxicity induced in vitro in B-CLL lymphocytes by cyclophosphamide (CP) derivatives, which induce DNA damage by DNA cross-linking. Exposure to CdA at clinically achievable concentrations for 2 h, followed by mafosfamide (MAF) or 4-hydroxycyclophosphamide (4HC) for 22 h, resulted in synergistic cytotoxicity in the majority of B-CLL samples tested. Synergy between CdA and MAF was observed in cell samples of sensitive/untreated patients, as well as in cells of resistant/pretreated patients, particularly at the highest concentrations of MAF. In the cells treated with CdA and MAF, we observed loss in ATP and hallmarks of apoptosis, as evidenced by cellular morphology and high molecular weight DNA fragmentation. The synergy could be explained neither by an influence of MAF on the phosphorylation of CdA, nor by an increase in the incorporation of CdA into DNA in the presence of MAF. The in vitro synergy between CdA and CP derivatives provides a rationale for the use of this association in B-CLL patients.