Abnormal intracellular level of Bax in CD3+ cells from untreated B-cell chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disease caused by impaired apoptosis regulation that leads to an abnormal survival and an accumulation of B-lymphocytes. Anti-apoptotic Bcl-2 and proapoptotic Bax proteins are involved in the highly regulated mechanism of cell death. Bax and Bcl-2 intracellular levels were analyzed both in CD19+ and CD3+ cells from 28 B-CLL de novo patients and compared with cells from healthy donors. Our results were expressed as a ratio (Bax/Bcl-2) obtained by dividing Bax mean fluorescence intensity (MFI) and Bcl-2 MFI; obviously, a lower ratio is associated with an anti-apoptotic status, while a higher index correlates to apoptosis activation. In CD19+ B-CLL cells, the Bax/Bcl-2 ratio was lower than in the CD19+ normal counterpart (1.3 versus 3.51; P<.05), mainly due to a Bcl-2 over expression (17.65 versus 9.02; P<.001). In CD3+ cells from B-CLL patients, the Bax/Bcl-2 ratio was lower than in normal CD3+ cells (7.89 versus 8.96; P<.005), most importantly as a result of Bax suppression (77.22 versus 96.63; P<.001). These study data show an apoptosis inhibition not only in CD19+ cells, but also in CD3+ cells, suggesting a pivotal role of T-cells in B-CLL pathogenesis.

Determination of antibodies to extractable nuclear antigens by commercial kits: a multicenter study.

Several enzyme immunoassays for serum antibodies to extractable nuclear antigen have recently become available. The aim of this study was to evaluate the results obtained with: (1) the same kit under different conditions; (2) different enzyme immunoassays; (3) Western blot and enzyme immunoassays. Twenty-five sera from patients with autoimmune disorders were tested in five different laboratories by one Western blot and four enzyme immunoassay commercial kits. The different methods produced comparable qualitative results. However, semiquantitative evaluation, based on a cut-off value (index), yielded different results due both to laboratory conditions and to the kits employed. Standardization of commercial products and methods should be improved so that the results of different laboratories can be compared and large-scale and follow-up studies conducted. Western blot analysis could also be useful to analyze complex reactivities, although greater experience is necessary to interpret these results correctly.

Evaluation of minimal residual disease in high risk childhood acute lymphoblastic leukemia using an immunological approach during complete remission. AIEOP Cooperative Group for Immunology of Acute Leukemias.

BACKGROUND: Sensitive methods for detecting residual disease may complement conventional morphology while monitoring the response to treatment in leukemia patients. METHODS: We studied minimal residual disease (MRD) by selecting via a colony assay a peripheral blood (PB) cell population enriched in putative malignant cells and detecting occult leukemic cells by double immunologic analysis performed on colonyforming cells (CFC). Using this combined technique we assayed the PB of high risk children with acute lymphoblastic leukemia (ALL) in order to demonstrate possible differences in "the residual tumor cell burden" among leukemia patients and to correlate these with a 3-year clinical follow-up. RESULTS: In all 22 patients positive results were obtained for up to 18 months following induction chemotherapy at times of apparent hematologic remission. Common ALL (cALL) patients exhibited a mean of 9.6% cAlla+ cells (range: 2% to 30%), whereas cALL antigen (cALLA) and cALLA/Tdt or CD1a/Tdt combination were never found in the colonies derived from healthy individuals. Six out of 22 cALL patients expressed a mean of 16.5% cALLA+/Tdt+ CFC (range: 2% to 35%). Five T-ALL children presented a mean of 24% CD1a/Tdt+ cells (range: 8% to 44%). Extensive follow-up indicates a correlation between the percentage of CFC Tdt+/lymphoid marker+ cells (more than 10%) and subsequent clinical relapse. In one patient relapse occurred after 16 months with a dramatic increase in the number of leukemic cells. In contrast, the decline in malignant cells, observed in two cases, predicted a favourable course. Five patients tested before autologous bone marrow transplantation (ABMT) presented high number of positive cells and relapsed at various times. CONCLUSIONS: We conclude that this approach to the study of MRD could be valuable in monitoring the efficacy of chemotherapy, as well in evaluating the quality of purged marrow.