A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.

BACKGROUND: Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V-FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells. METHODS: Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus-containing cells are selected using CD45-phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy-coupled dye (ECD) labeling. Apoptosis is detected using Annexin V-FITC with 7-amino-Actinomycin-D (7-AAD) to distinguish early apoptotic from late apoptotic lymphocytes. RESULTS: We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%. CONCLUSIONS: We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies.

Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

Oligoclonal peripheral T-cell lymphocytosis as a result of aberrant T-cell development in a cortical thymoma.

A 42-year-old man presented with a locally invasive cortical thymoma. Before chemotherapy was commenced 36 months after presentation, an unusual peripheral lymphocytosis of 19 x 10(9)/l had slowly developed over time. After the first course of chemotherapy, the lymphocytosis showed a sharp decline to normal absolute cell numbers and subsequently remained at normal levels. Currently, the patient is in stable partial remission and doing well. Immunophenotypic analysis showed a mature T-cell phenotype with 78% TcR-a beta and 16% TcR-gamma delta in the absence of an immature component. Pretreatment Southern blot analysis of peripheral blood mononuclear cells showed an oligoclonal pattern with 13-20 rearranged fragments of different intensity for the TcR beta-gene. TcR gamma also showed a pattern compatible with an oligoclonal proliferation. After treatment, after normalization of absolute blood counts, the distribution of T-cell subsets still showed a slightly aberrant pattern. Immunophenotypic analysis of a blood sample taken 6 months later, also at normal absolute cell counts, showed an increase of thymocytes as well as of mature T cells with a polyclonal pattern on Southern blot analysis. These findings may be interpreted as the result of aberrant positive and negative selection and development of thymocytes in the microenvironment of neoplastic thymic epithelial cells and clonal selection through continuous peripheral stimulation. Moreover, this case stresses the importance of integrated interpretation of clinical, morphological, immunophenotypical, and molecular data to gain insight in unusual clinical problems.