Véronique Deneys

Age-related changes in human blood lymphocyte subpopulations

Flow cytometric analysis of major lymphocyte populations and their subsets reveals age-related changes in the cellular human immune system. Immunophenotypic markers were evaluated in 110 normal pediatric subjects, divided into groups of newborn infants, infants aged 2 days to 11 months, and children aged 1 to 6 years and 7 to 17 years; results were then compared with those obtained from 101 normal adults aged 18 to 70 years. Comparisons among age groups from newborn infants through adults reveal progressive declines in the absolute numbers of leukocytes, total lymphocytes, and T, B, and natural killer (NK) cells. The percentages of T cells within the total lymphocyte population increase with age, in both CD4+ and CD8+ subsets. Percentages of B and NK cells are higher in newborn infants than in adults. The expression of the activation markers interleukin-2R and HLA-DR on T cells increases with age, as does the NK-associated expression of CD57 on CD8 cells. The proportions of B lymphocytes that coexpress CD5 or CDw78 decrease with age, whereas expression of Leu-8 and CD23 increases. The proportion of CD4 cells bearing the CD45RA and Leu-8 markers is consistently lower in adults than in children. These data may serve as a reference range for studies of pediatric subjects.

Developmental and maturational changes in human blood lymphocyte subpopulations

Recent application of flow cytometric immunophenotyping to childhood disease states has highlighted the need for reliable lymphocyte data ranges in normal infants and children. In the management of pediatric human immunodeficiency virus (HIV) disease and in the diagnosis of DiGeorge syndrome, T-cell enumeration plays a vital role and extrapolation from adult normal range data has been misleading. Here, the findings of a multicenter study of 110 normal pediatric subjects divided into cohorts of newborns, infants (ages 2 days to 11 months) and children (1 to 6 years and 7 to 17 years) are presented. These age divisions were chosen to reflect maturational events in the immune system, such as antigenic challenges and vaccination. Pediatric results were compared to those of 101 normal adults evaluated at the same centers using the same methods.

Association of Localized Intravascular Coagulopathy With Venous Malformations

OBJECTIVEnTo determine which venous malformations (VMs) are at risk for coagulopathy. Venous malformations are slow-flow vascular malformations present at birth, and localized intravascular coagulopathy (LIC) causes pain and thrombosis within a lesion and severe bleeding during surgical procedures.nnnDESIGNnProspective convenience sample accrued from 2 multidisciplinary sites in Brussels, Belgium, and Caen, France.nnnPARTICIPANTSnThe study population comprised 140 patients with clinical data and coagulation parameters. Magnetic resonance imaging was performed for 110 patients.nnnMAIN OUTCOME MEASUREnMeasurement of D-dimer levels.nnnRESULTSnOf the 140 participants, 59 (42%) showed high D-dimer levels, 36 (61%) of whom had levels higher than 1.0 microg/mL. Six of the participants had low fibrinogen levels. In univariate analysis, large surface, presence of palpable phleboliths, and truncal localization were associated with high D-dimer levels. In the multivariate analysis, only large surface area and presence of phleboliths remained independently associated with high D-dimer levels. Severe LIC, characterized by concomitant low fibrinogen level, was associated with extensive venous malformations of the extremities.nnnCONCLUSIONSnLocalized intravascular coagulopathy is statistically significantly associated with large and/or deep venous malformations that affect any location, which can have a palpable phlebolith. These patients are at risk of local pain due to thrombosis. Lesions with elevated D-dimer levels associated with low fibrinogen levels (severe LIC) commonly affect an extremity and have a high risk of hemorrhage. Low-molecular-weight heparin can be used both to treat the pain caused by LIC and to prevent decompensation of severe LIC to disseminated intravascular coagulopathy.

Bedside transfusion errors : a prospective survey by the Belgium SAnGUIS group

The true incidence of bedside transfusion errors, i.e. those happening when blood products have left the blood bank, is underestimated because published figures rely on reporting of clinically relevant events or on indirect methods. The SAnGUIS project assessing blood practice in a prospective and randomized fashion for 6 elective surgical procedures gave the opportunity to trace all transfused units and to identify steps at risk during blood delivery in surgery. We considered transfusion of a wrong unit as a major error and poor execution or documentation as a recording error.

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). Matutess 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.

Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.

BACKGROUND AND OBJECTIVESnFreezing 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.nnnMATERIAL AND METHODSnBlood samples from 15 CLL patients were analyzed freshly and after freezing at -196 degrees C, without separation, and thawing. Results were compared by Students paired t-test.nnnRESULTSnThe 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 Matutess score was modified in 6 cases out of 15 (40%).nnnCONCLUSIONnCryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression.

Pretransplant helper T-lymphocyte determination in bone marrow donors: acute graft-versus-host disease prediction and relation with long-term survival

Helper T‐lymphocyte precursor (HTLp) frequency from 19 allogeneic bone marrow donors was tested to detect weak antigenic differences with the recipient, and then compared to the outcome. HTLp frequency was estimated in limiting dilution cultures, and HLA‐DR and CDu200380 expression by stimulating cells was measured by flow cytometry. 12/19 patients experienced acute graft‐versus‐host disease (aGVHD) grade II–IV. A good correlation was found between high pretransplant HTLp frequency and grade II–IV aGVHD (median: 1/55848 PBMNC for II–IV GVHD versus 1/184346 for 0–I GVHD; Pu2003=u20030.008). Sensitivity was 82%, specificity 63%, negative predictive value 71% and positive predictive value 75%. Long‐term survivors also had a lower HTLp median frequency (1/143354) when compared with patients who died as a result of the transplant procedure (1/22100, Pu2003<u20030.001). No correlation was found between HTLp frequency and HLA‐DR or CD80 expression by patients cells. We conclude that HTLp frequency estimation can predict, although poorly, acute GVHD risk and long‐term survival.

Prophylactic platelet transfusions.

R. N. I. Pietersz, H. W. Reesink, S. Panzer, M. P. Gilbertson, M. E. Borosak, E. M. Wood, G. C. Leitner, W. Rabitsch, C. Ay, M. Lambermont, V. Deneys, D. Sondag, V. Compernolle, D. Legrand, A. François, R. Tardivel, F. Garban, R. B. Sawant, P. Rebulla, M. Handa, H. Ohto, J.-L. H. Kerkhoffs, A. Brand, E. Zhiburt, J. Cid, G. Escolar, M. Lozano, L. Puig, F. Knutson, H. Hallböök, N. Lubenow, L. Estcourt, S. Stanworth, M. F. Murphy, L. Williams, D.L. Mraz, R.L. Ross & E. Snyder

Belgian consensus recommendations for flow cytometric immunophenotyping

This paper summarises the guidelines and recommendations that were generated during a number of discussion forums attended by the majority of Belgian cytometry laboratory professionals. These forums focused on the rational and optimal use of flow cytometric evaluations in the clinical laboratory setting. The aim was to improve the coherence of the testing panels and the quality of the results and -as such-the clinical diagnostic information. It was also the aim to provide the Belgian prescribing physician and interested laymen with an updated overview of the flow cytometric possibilities. Emphasis is placed on immunophenotyping of haematological malignancies, hematopoietic progenitor cell counting and follow-up of the viral infection caused by the human immunodeficiency virus.

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.