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.

Intracellular cytokine profile of cord and adult blood monocytes.

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.

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.

Expression of HLA-DR, CAM and co-stimulatory molecules on cord blood monocytes.

Umbilical cord blood (CB) transplantations are associated with a lower risk of severe graft-versus-host disease (GVHD) compared to BMT. GVHD is an immune reaction that involves interaction between cell surface molecules resulting in cell activation and release of many cytokines. Monocytes are known to be an important source of cell adhesion (CAM) and co-stimulatory molecules which play a crucial role in the efficient activation of T and B cells. We analyzed the phenotype of CB monocytes in the presence or absence of an inflammatory signal (rIFN-gamma) and compared them to adult blood (AB); the expression of HLA-DR and 17 different markers (CD11a, CD11b, CD11c, CD18, CD29, CD40, CD44, CD49a, CD49d, CD49e, CD49f, CD54, CD58, CD62L, CD80, CD86 and CD102) was measured by flow cytometry. Statistical analysis showed that, compared to AB, CB monocytes did not express CD11b, CD11c, CD49d and after stimulation with rIFNgamma, they lost the expression of CD58 and CD102, whereas CD80 and CD86 expression was induced. The analysis of fluorescence intensity (MFI) revealed that CB monocytes expressed some CAM (CD29, CD54, CD102) with a lower intensity than AB monocytes except CD44. In conclusion, absence and reduced expression of some markers argue for a different phenotypic profile of CB monocytes compared to AB monocytes, which might partly contribute to their impaired immune response and to the low incidence of GVHD observed after CB transplantations. However, CB monocytes expressed CD80 and CD86 co-stimulatory molecules, but this expression did not prove a normal co-stimulatory function.

[Hematopoietic recovery as a function of the number of autografted CD34+ cells: a retrospective study of 66 patients with malignant hematologic diseases]. / Reprise Hématopoïétique en fonction du nombre de cellules CD34+ autogreffées: étude rétrospective chez 66 patients atteints d'hémopathies malignes.

Engraftment in relation to infused CD34+ cell number was retrospectively analysed in 66 patients with hematological diseases: non-Hodgkin's lymphoma (n = 33), multiple myeloma (n = 21), acute myelogenous leukemia (n = 7), Hodgkin's disease (n = 4) and myelodysplastic syndrome (n = 1). Progenitor cells were mobilized with rhG-CSF, alone or in association with chemotherapy. The cells were harvested by leukapheresis until at least 2 x 10(6) CD34+/kg body weight were obtained. A total of 194 leukaphereses were performed (median = 3 per patient, range 1-9). A median of 3.40 x 10(8) nucleated cells/kg (range 0.31-27.59) and a median of 7.15 x 10(6) CD34+ cell/kg (range 1.31-115.70) were transplanted. Regardless of transfusional support or patient diagnosis, engraftment was rapid in patients who had received > or = 5 x 10(6) CD34+ cell/kg. In this case, absolute neutrophil blood count > or = 0.5 x 10(9)/l was obtained on day 12 post graft (range 7-19) and platelet count > or = 20 x 10(9)/l was also reached after the same median time interval (range 8-121). From the present results, a minimal threshold of 5 x 10(6) CD34+ cell/kg appears to be suitable for providing rapid and complete hematopoieitc reconstitution in patients exposed to high doses of chemotherapy with or without total body irradiation. Furthermore, administration of rhG-CSF during post-graft period significantly decreased the neutrophil time recovery (P = 0.002) but not that of platelets (P > 0.05).

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.

Influence of body mass index, sex and age on serum alanine aminotransferase (ALT) level in healthy blood donors.

BACKGROUND AND AIM: Serum alanine aminotransferase (ALT) level is the most common screening test as part of a routine evaluation of liver damage. In order to determine the factors influencing this liver function test in normal subjects, the relationship between ALT level and gender, age and body mass index (BMI) was studied in a large population of healthy blood donors. METHODS: This population included 9,420 volunteer blood donors (4,488 men and 4,932 women aged from 18 to 70 years) selected on the basis of negative answers to a detailed medical questionnaire including past medical history, drug and alcohol consumption, on the absence of clinical signs of liver disease, on the negativity of serological testing for hepatitis B and C virus and HIV. RESULTS: In the overall population, the mean serum ALT value was 21.8 I.U./L and the mean BMI was 24.4 kg/m2. There was a positive significant correlation between serum ALT level and BMI (Pearson r = 0.54; p < 0.001) and between ALT and age (Pearson r = 0.25; p < 0.001). A major sex-difference in ALT value was observed, the mean ALT value being higher in men than in women (26.8 +/- 13.6 vs. 17.2 +/- 8.1 I.U./L, p < 0.0001). In both sexes, ALT level was significantly correlated with BMI (Pearson r = 0.45 in men and r = 0.37 in women; p < 0.001). In women a consistent rise in BMI and ALT value with increasing age was observed whereas in men BMI and ALT level only increased with age up to the fifth decade. IN CONCLUSION: There was a significant positive correlation between ALT and BMI regardless the gender in a population of healthy volunteer blood donors. Moreover, at the same age and the same BMI, ALT was significantly lower in women than in men suggesting that the normal range for ALT value should be adjusted for gender. So gender and BMI have to be considered in the interpretation of ALT values.

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 CD 80 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; P = 0.008). 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, P < 0.001). No correlation was found between HTLp frequency and HLA-DR or CD80 expression by patient's cells. We conclude that HTLp frequency estimation can predict, although poorly, acute GVHD risk and long-term survival.

Comparison of seroprevalences of human herpesvirus-6 and -7 in healthy blood donors from nine countries.

BACKGROUND AND OBJECTIVES: The purpose of the study was to register antibody prevalences of HHV-7 in various locations of the world in comparison to the closely related HHV-6. MATERIALS AND METHODS: Sera of healthy blood donors from nine countries in five continents were titered by indirect immunofluorescent assays using HHV-6 infected HSB2 and HHV-7 infected SupT1 cells. RESULTS: Antibody prevalence for HHV-7 is high (75-98%) in practically all countries except for Northern Japan (44%), with no simple correlation to elevated HHV-6 antibody titers. There were regions of low, intermediate and high mean antibody titers against HHV-7 such as 78.5-91.3 for Belgium, Israel, Japan, USA and Australia, 175.4-182.6 for Mexico and Cologne/Germany, and 389.2 for South Africa for which geographic characteristics may be responsible. CONCLUSION: HHV-7, similar to HHV-6, is a widespread human herpesvirus with elevated antibody titers in the healthy human population essentially everywhere. The data warrant further studies to evaluate its possible pathologic potential, preferentially in persons with defective immune responses.

Improved IL-2 detection for determination of helper T lymphocyte precursor frequency in limiting dilution assay.

In the context of allogeneic bone marrow transplantation, an accurate estimate of the risk of developing graft-versus-host disease (GVHD) is of major interest. The pre-transplant frequency of donor's helper T-lymphocyte precursors (HTLp) directed against host's antigens may be helpful in predicting this risk. This technique relies on an indirect measurement of interleukin-2 (IL-2) secreted by the HTLp, as assessed by the proliferation of an IL-2 dependent cell line. Many authors use the murine CTLL-2 cell line in this assay, but these cells do not respond to the presence of minute amounts of IL-2 in the culture medium, and thus do not discriminate between the absence or the presence of very low levels of IL-2. We therefore decided to compare CTLL-2 with another IL-2 dependent cell line, the murine A9.12 cell line. A comparison was made using serial dilutions of recombinant human IL-2, limiting dilutions of baby hamster kidney (BHK) cells transfected with human IL-2 gene and in the context of clinical tests performed for the detection of pre-transplant HTLp. Both the sensitivity and reliability of the tests were better using A9.12. We conclude that the A9.12 cell line might be a more suitable tool for pre-transplant HTLp determinations before allogeneic bone marrow transplantation or whenever low IL-2 levels are to be measured.

Circadian and seasonal variations of hematopoiesis in cord blood.

Cord blood hematopoietic progenitors undergo circadian and seasonal variations. The lowest values are obtained between 4:00 and 12:00, as well as between May and August. This represents the first observation of such rhythms before birth.

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells.

Ex vivo expanded bone marrow CD34+DR- cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1alpha (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR- cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61 + megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR- cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR- expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR- cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1alpha had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR- cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.

A seroepidemiological study on toxoplasmosis.

Using the microparticle capture enzyme-immunoassay (MEIA) based on IMx technology (Abbott), we determined the current prevalence of toxoplasmosis in 784 pregnant women followed up during 1990, and in 1,839 randomly selected blood donors. They all came from the Brabant Wallon area and the South-East of Brussels. Specimens yielding low IgG immunity (6-15 units) [corrected], were further tested with a sensitive direct agglutination assay (Toxo-Screen DA, bioMerieux). Overall, the prevalence was 67% among blood donors and 50% in pregnant women. In blood donors, the prevalence in women was not statistically different from the prevalence in men: X2 = 2.95 NS. In the two populations, a progressive age-related increasing prevalence of up to 60% for pregnant women and 77% for blood donors was observed. In females, the prevalence was higher among female blood donors than among pregnant women: 63% versus 50%, X2 = 16, P < 0.001. However, when the prevalences were compared within three age subgroups of women (< or = 33 yrs, 34 to 41 yrs, > or = 42 yrs), there were no statistically significant differences between pregnant women and blood donors. Thus, the overall observed difference was due to an age effect. Therefore, the distribution of IgG titers was established in each of the six age and sex subgroups. The 25th, 50th and 75th percentiles of those distributions ranged between 14 IU and 20 IU, 24 IU and 35 IU, and 40 IU and 64 IU, respectively. The annual seroconversion rate was 0.8% in pregnant women, against 0.2% amongst non-immune blood donors over 3 months. In conclusion, our findings confirm the general prevalence of 50% of toxoplasmosis and an annual seroconversion rate of 0.8% in these two populations.

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.

[Immunologic tolerance of fetal allograft]. / Tolérance immunologique de l'allogreffe foetale.

In mammals, reproduction involves two potentially incompatible mechanisms: viviparity and development of a competent immune system. Thus the maternal and foetal organisms must respond by developing immunologic tolerance. The phenomenon does not involve total immunosuppression, but includes several highly precise processes initiated at conception. It is known that cell- and humour-mediated processes occur but their relative importance remains to be elucidated. Cytokines, especially those mediating T-helper2 cell response appear to play a predominant role in inducing immunologic tolerance to the foetal allograft. A better understanding of these mechanisms could have major implications in the diagnosis and treatment of repeated miscarriage and unexplained infertility.

HLA class II genotyping: two assay systems compared.

In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.

Long-term culture of autologous transplanted bone marrow for acute myeloid leukaemia: evidence for an in vitro haemopoietic defect and lack of correlation with the speed of engraftment.

Haematological recovery after autologous bone marrow transplantation (BMT) for acute myeloid leukaemia (AML) is often delayed and available laboratory assays cannot accurately predict speed of engraftment. By using the long-term bone marrow culture (LTBMC) method, we attempted to define the haemopoietic defect underlying this slow engraftment, and assessed the usefulness of LTBMC in predicting engraftment. Cryopreserved bone marrow (BM) harvested from three different groups (AML autografts, n = 18; autografts for non-leukaemic diseases, n = 23; normal donors. n = 10) were cultured and their growth was compared and correlated with the speed of engraftment. In the AML autografts, non-adherent and adherent progenitor production was significantly reduced compared with normal BM during the whole culture period (P < 0.01). None of the LTBMC parameters was found to correlate with engraftment after autologous BMT. The non-leukaemic autografts showed progenitor production intermediate between normal and AML autografts. Their progenitor content at the end of the culture period (reflecting the stem cell pool) was not statistically different from normal BM. In this group, most of the progenitor cell contents, during LTBMC correlated with neutrophil (rs = -0.618 to -0.879, P < 0.01) and platelet (rs = -0.479 to -0.707, P < 0.02) recovery. The conclusion drawn from these results is that AML autografts are defective in their ability to sustain in vitro haemopoiesis, but this in vivo defect does not correlate with the slow haemopoietic recovery after autologous BMT. In contrast, LTBMC of autografts for non-leukaemic diseases, whose defect affects the stem cell pool to a lesser extent than BM in AML correlates with the speed of engraftment.

Transplantation of umbilical cord blood in a refractory lymphoma.

A successful cord blood transplantation combined with hematopoietic growth factor was performed in a boy presenting with refractory mediastinal T-cell lymphoma. Cord blood cells were collected from an HLA-identical sibling at the time of delivery. A transient and corticosensitive acute grade II graft versus host disease was observed. One year after transplantation, the child is still in remission with complete engraftment. This is the first report of cord blood transplantation in a patient with refractory lymphoma.

Anti-hepatitis C virus positivity (HCV) in Belgian volunteer blood donors: source of contamination and HCV-testing among donors and related recipients.

We investigated a group of Belgian HCV-100 Elisa positive volunteer blood donors for potential sources of contamination and the presence of liver biochemical abnormalities. In addition, results of serological testing and liver biochemistry of their related blood products recipients were also analysed. In blood donors, anti-HCV-100 repeat reactive rate was 0.77% with a 34% rate of abnormal liver function tests. A potential source of parenteral exposure was found in all donors with RIBA-confirmed HCV-100 positivity. Among recipients, anti-HCV-100 RIBA-2 positive blood product donations were associated with RIBA-2 seroconversion, a history of transfusion in donors being of high predictive value of infectivity.