Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group.

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174-179, 2000.

Quantitation of CD38 expression using QuantiBRITE beads.

The QuantiBRITE bead method was compared with the CD4 biological calibration method for quantitation of CD38 expression on CD8+ T-lymphocytes of Multicenter AIDS Cohort Study participants. Results were expressed as CD38 antibodies bound per cell (ABCs) and were the same with the two methods provided two conditions were met. These were the use of repurified (> 95% of the monoclonal antibodies [mAbs] have 1 phycoerythrin [PE] molecule per mAb) CD38-PE for both methods and use of repurified CD4-PE to calculate the relative fluorescence intensity multiplier for the CD4 biological calibration method. Our results indicate that the prognostic significance of CD38 values obtained using the QuantiBRITE method can be interpreted using previously published reports (Liu et al.: J Acquir Immune Defic Syndr Hum Retrovirol 16:83-92, 1997 and 18:332-340, 1998). Sample preparation using NH4Cl and FACS lysing solution gave similar results for CD38 relative fluorescence intensity. Dilution into either phosphate-buffered saline with 2% fetal calf serum and 0.1% sodium azide or fixation in 1% paraformaldehyde for 1 or 24 h also gave similar results. In experiments using Raji cells, which express high levels of CD38, the valence of binding of the intact Leu 17 antibody was approximately 68% bivalent and approximately 32% monovalent. This emphasizes the complexity of determining antigen density from ABCs. We conclude that repurified PE conjugates of CD38, which can be consistently made, together with QuantiBRITE PE beads, provide a convenient and reliable method for quantitation of CD38 expression as ABCs.

The mdm-2 gene is induced in response to UV light in a p53-dependent manner.

Irradiation of mammalian cells with UV light results in a dose-dependent accumulation of the p53 tumor-suppressor gene product that is evident within 2 hr. UV treatment causes a dramatic increase in p53-specific transcriptional transactivation activity and an increase in expression of the p53-responsive gene mdm-2. UV-stimulated mdm-2 expression is not directly correlated with the level of p53 protein in a cell because mdm-2 induction is delayed at high UV doses even though p53 levels rise almost immediately. Cells lacking p53 protein do not respond to UV by increasing their expression of mdm-2. The delayed induction of mdm-2 at high UV doses suggests that, in addition to p53 protein levels, other factors contribute to the regulation of mdm-2 expression following UV treatment. The time of induction of mdm-2 in cells treated with UV light correlates with recovery of normal rates of DNA synthesis, presumably after DNA repair. These data indicate a possible role for mdm-2 in cell cycle progression.

Cytotoxic T lymphocyte lines reactive against murine plasmacytoma antigens: dissociation of cytotoxicity and Lyt-2 expression.

The relationship between cell surface antigen expression and function in cytotoxic T cells directed against a non-H-2-restricted plasmacytoma antigen was examined using Lyt-2- variants of a continuous T cell line. This cytotoxic cell line originally had expressed Lyt-2 on all the cells as detected by flow microfluorometry, but after several months of culture, Lyt-2- variants spontaneously developed. Using cell sorting and limiting dilution cloning, Lyt-2- and Lyt-2+ cell lines were obtained, cultured and analyzed. These analyses indicated that such cytotoxic cells had similar activity regardless of Lyt-2 phenotype. This observation supports the concept that the Lyt-2 molecule is not obligatory for recognition and killing by cytotoxic T cells and emphasizes the need to clarify the precise relationship between cell surface phenotype and T cell function.