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PubMed1988

Automated contrast fluorometry. I. Lymphocytotoxicity testing.

Automated contrast fluorometry has been carried out in lymphocytotoxicity testing using a computer-controlled microscope and direct comparison of the data with controls. The method uses heparinized lymphocytes and permits simultaneous evaluation of both fluorescein and ethidium bromide fluorescence by calculating the quotient between the green/red measurements. After comparison of the quotient with that of control samples, the raw data are automatically transformed into cytotoxicity scores. Fast and reliable measurements with minimum background fluorescence can be obtained for up to 24 hours after performance of the assay by controlling complement reactivity and the exposure time of the cells to the fluorescein. The evaluation of 15,204 cytotoxic reactions showed excellent correlation between the automated and visual results of 125 tissue typings using 120 different antibodies and of 17 HLA antibody titers using case-by-case analysis of variance. The values for the automated cytotoxicity readings were slightly higher and showed more intermediate scores than those of the visual readings suggesting a high sensitivity and objectivity of the automated system. In conclusion, this is a methods paper comparing parallel lymphocytotoxicity testing using automated and manual methods. The automated method was found to be more sensitive and faster than the manual method.

Automation↗
PubMed1976

A simple method for the cryopreservation of lymphocytes. Retention of specific immune effector functions by frozen-stored cells.

A simple, quick and inexpensive method for cryostorage of lymphocytes is discribed. When injected into appropriate normal recipients, frozen-stored rat and sheep lymphocytes caused GVH and NLT reactions respectively. Sheep lymphocytes remained viable after several months storage and functioned satisfactorily as 51Cr-labelled target cells in assays for cytotoxic antibodies. Lymphocytes, including immunoblasts, drained from sheep efferent lymphatics during immune responses to injected murine P815 cells or allogeneic lymphocytes survived freezing; when thawed the cells retained specific immune cytotoxic effector functions including the ability to secrete complement-dependent and leucocyte-dependent antibodies

Animals↗
PubMed1975

Recognition by pregnancy serums of non-HL-A alloantigens selectively expressed on B lymphocytes.

A group of alloantibodies are found in pregnancy sera which react with antigens present on B lymphocytes and monocytes but are not detectable on the vast majority of unstimulated T cells. This specificity distinguishes them from HL-A antibodies which react with both cell types. They were readily recognized through indirect fluorescent antibody analysis by employing the combination of B-cell lymphoid lines and normal peripheral blood T cells. Different sera gave a variety of patterns of reactivity with a panel of 11 lymphoid lines. Similar differential patterns were also observed with normal B cells from different individuals particularly after concentrating the B cells. The antibodies were also cytotoxic to B cells and this procedure gave parallel results to the fluorescence method. The pattern of reactions obtained indicated a very heterogeneous system similar to that for HL-A. Special study of certain of the sera provided evidence that the lymphocyte-defined determinants of the mixed lymphocyte reaction system were involved. For convenience the term HL-B has been employed for these antigens.

Antibody Specificity↗