Docosahexaenoic acid-induced alteration of Thy-1 and CD8 expression on murine splenocytes.

Here we test whether the incorporation of docosahexaenoic acid (DHA, 22:6), an (n-3) fatty acid, into lymphocyte membranes affects the expression of the surface proteins Thy-1.2 and CD8. DHA was incorporated into splenocytes by three methods: feeding mice diets containing menhaden (fish) oil, fusing splenocytes with DHA-containing phosphatidylcholine vesicles, and culturing splenocytes with DHA. Thy-1.2 and CD8 expression were measured by flow cytometry and complement-mediated lysis using a panel of monoclonal antibodies. As (n-3) fatty acid incorporation into the lymphocytes increased, the expression of one Thy-1.2 epitope and one CD8 epitope decreased; the expression of two CD8 epitopes increased. Although diet-induced changes in surface protein expression may result from selective migration of cell populations or the diet's effect on protein biosynthesis, fusion with lipid vesicles demonstrated that DHA-containing phospholipids can mediate a direct and immediate effect. The decrease in Thy-1.2 expression was sustained for more than a week after removal of (n-3) fatty acids from the diet, most likely due to retention of membrane-bound (n-3) fatty acids. Because Thy-1.2 and CD8 participate in T cell activation, modulation of their expression by DHA suggests that DHA, when serving as a membrane structural element, may alter immune function.

Quantitation of isolated pancreatic islets using imaging technology.

An automated image analysis routine was developed for the quantitative assessment of isolated pancreatic islets. This approach offers the advantages of standardization, increased precision of EIN determinations, batch analysis without user interaction, and the ability to customize the analysis for specialized requirements. Finally, images may be archived for later review or transmitted electronically for off-site analysis.

Issues for quality assurance in clinical flow cytometry.

An increasing number of clinical laboratories are using flow cytometry to analyze cells stained with fluorescent antibodies or dyes. Many articles discuss the analytical performance of immunofluorescent assays and DNA staining procedures. However, quality assurance transcends the performance of analytical methods and includes preanalytic, analytic, and postanalytic phases of the test procedure. This review focuses on preanalytic and analytic factors that affect the results of flow cytometric analysis of cells stained with fluorescent antibody or dyes specific for nucleic acid. The article reviews the controls used to assess instrument performance followed by a discussion of biological variables, and reagent, methodological, and instrumental factors that may affect the interpretation of these results.

Three immunoassay methods evaluated for quantifying prealbumin (transthyretin) in serum.

We developed an immunoturbidimetric assay for prealbumin on the Cobas Bio centrifugal analyzer and compared results from this assay with those from a rate nephelometric assay (Beckman Instruments Inc.) and a radial immunodiffusion kit (Behring Diagnostics). All three assays were evaluated for precision, linearity, and correlation to each other for analysis of sera from pediatric patients. All assays gave similar results for patients' samples. Values were higher by the radial immunodiffusion assay than by the other two methods, which gave similar results for the same specimens. We conclude that the immunoturbidimetric and rate nephelometric assay for prealbumin are acceptable alternatives for quantifying prealbumin in serum and also have a faster turnaround time than radial immunodiffusion.

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears.

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).

A micromodification of the CH50 test for the classical pathway of complement.

A micromodification of the CH50 assay of Kabat and Mayer is described in which the total reaction volume has been reduced five-fold. This assay utilizes sheep red blood cells pooled from multiple animals rather than from a single animal to avoid screening procedures of red blood cells from individual sheep. Analysis of natural hemolysin activity in normal serum revealed a correlation with elevated CH50 titers. However, this correlation does not appear to effect the clinical interpretation of the results.

Soluble factors in tolerance and contact sensitivity to 2,4-dinitrofluorobenzene in mice. IV. Characterization of migration inhibition factor-producing lymphocytes and genetic requirements for activation.

The production of migration inhibition factor (MIF) in vitro by lymph node cells from mice with contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) was investigated. MIF activity of cell-free culture supernatants was measured using a micro, indirect "hanging-drop" assay system. We found that DNFB-sensitized lymph node cells are stimulated to produce MIF by co-culture with DNP-labeled spleen cells or splenic adherent cells. The stimulation was quantitatively antigen-specific, as co-culture with TNP-spleen cells or TNP-splenic adherent cells induced only low levels of MIF activity. Pretreating the immune lymph node cells with different antisera plus complement, before addition of DNP-spleen cells, showed that MIF production is dependent on Ia- T cells. Additional experiments showed that in order for the T cells to be stimulated, homology at the I-A subregion of the major histocompatibility complex between the T cells and DNP-spleen cells is required. Collectively, these results correlate with our previous finding that transfer of contact sensitivity is mediated by Ia- T cells and indicate that both tests, i.e., transfer in vivo and MIF production in vitro, are measuring effector functions of the same T cell subset.