In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.
The objective of this study was to investigate the sensitivity, specificity and reproducibility of some frequently used apoptosis assays. The degree of apoptosis was tested in two T-lymphoblastoid cell lines, HSB and Jurkat, in which apoptosis was induced by ionizing radiation. HSB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h after irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respectively. Four frequently used flow cytometric techniques were evaluated: (i) Annexin V/Propidium Iodide assay, detecting the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, simultaneously with preservation of the membrane integrity; (ii) Terminal deoxynucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labelling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA-flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) and (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibody against 7A6 antigen, a protein, which becomes exposed upon the mitochondrial membrane during apoptosis. As a general standard for identifying that apoptosis had occurred, the cells were assessed for the presence of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology. Results were as follows: Fluorescein Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytometry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture conditions in suspension. The expression of 7A6 antigen on the mitochondrial membrane appeared to be not specific for apoptotic cell death.
To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.
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