Tracking the follicular lymphoma cells in flow cytometry: characterisation of a new useful antibody combination.

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.

Atypical lymphocytic leukemia and mantle cell lymphoma immunologically very close: flow cytometric distinction by the use of CD20 and CD54 expression.

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.

Reference values for peripheral blood B-lymphocyte subpopulations: a basis for multiparametric immunophenotyping of abnormal lymphocytes.

BACKGROUND AND OBJECTIVES: Immunophenotyping has become a useful tool for the differential diagnosis of chronic B-cell lymphoproliferative disorders. The aim of this work was to determine reference values of normal B-cell subpopulations. MATERIAL AND METHODS: Blood samples from 38 healthy volunteers were analyzed by multidimensional flow cytometry, using a panel of directly conjugated antibodies. Results were expressed as percent of positive B cells and as median fluorescence intensity, an indirect assessment of the expression level. RESULTS: CD20, CD22, CD24, CD40, CD79a, CD79b, FMC7, CD11a, CD18, CD44 were positive in the whole B cell population, whereas CD10, CD86, CD103, CD154 and FasL were almost absent from the B-lymphocyte population. 75% were IgD positive. The kappa/lambda ratio was 1.5. CD5, CD23, CD25, CD38, CD43, CD54, CD62L, CD80 and CD95 were positive in different B-cell subpopulations. The utility of all these markers in the differential diagnosis of chronic B-cell lymphoproliferative disorders is discussed. CONCLUSION: In order to interpret a pathological immunophenotype, it is necessary to refer to quantitative and qualitative values of normal B-cell subpopulations.

Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.

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.

The immune profile of multiple sclerosis: T-lymphocyte effects predominate over all other factors in cyclophosphamide-treated patients.

It is widely believed that multiple sclerosis is a T-cell mediated autoimmune disease associated with abnormalities in immunoregulation. This large, prospective study evaluated the lymphocyte immunophenotypic profile of 246 MS patients, divided clinically into a remitting/relapsing group (n = 176) and a progressive group (n = 70), and compared their results to those of 117 healthy controls. All patients were found to have significantly elevated percentage and absolute numbers of IL2R+CD3+ cells as well as depressed percentages of CD45RA+CD4+ cells. However, when the factor of treatment with cyclophosphamide (CY) versus no treatment or treatment with other agents was used to group patients, dramatic declines in both percentages and absolute numbers of CD45RA+CD4+ cells were discovered. These declines were associated specifically with CY and and could be explained by this factor independent of the clinical state of the patient. The effects were seen in patients undergoing current treatment or in those exposed to CY in the near or remote past. These findings highlight the confounding effect of specific treatments on the immune profile of MS patients groups and suggest that there may be important implications for cellular function and clinical outcome in these and other patient groups.

Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates.

Reduction of leucocytes in blood components, achieved by filtration, may reduce the risk of HLA alloimmunization, virus transmission, and febrile reactions. Nevertheless, white blood cell (WBC) concentrations obtained in this way can be too low to be accurately counted with automated and manual techniques. We describe here a rapid, reliable, and very sensitive method for counting low leucocyte numbers using flow cytometry. WBC nuclei are labelled by propidium iodide. A known amount of fluorescein- and phycoerythrin-conjugated beads (Standard Brite, Coulter) is added as an indicator of the examined volume. The time required for analyzing one tube is approximately 5 min. This method was first validated by comparing WBC counts (120-2,570/microliters) assessed on a haemocytometer (Bürker) and by flow cytometry. The second step was to determine the sensitivity of the method: we diluted normal platelet concentrates with phosphate-buffered saline. The comparison of expected with observed values showed a very good correlation up to a concentration of 1 WBC/microliter. This technique is an accurate method that can be applied in blood bank quality controls and in clinical studies.