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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. [ABSTRACT FROM AUTHOR]

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.

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.