The Sheep‐Cell Rosette Test on Human Peripheral Blood Lymphocytes: An Analysis of Some Variable Factors in the Technique

Abstract

Summary. Results obtained in the sheep cell‐rosette test for identification of human T lymphocytes are strongly dependant on the origin of the sheep erythrocytes. Other factors which influence rosette formation are the duration of incubation of the lymphocyte erythrocyte mixture and the concentration of serum used. The most reproducible results, giving a range of 55–75% RFC among the peripheral blood lymphocytes of healthy adults, are obtained by leaving the cell mixture overnight at room temperature in the presence of 10–25% serum.The spontaneous sheepcell rosette test appears to be a useful means of identifying human T lymphocytes (Jondal et al, 1972; Papamichail et al, 1972; Wortis et al, 1973; Yata et al, 1973); there are, however, striking discrepancies in the percentage of rosette‐forming cells (RFC) reported among the peripheral blood lymphocytes of healthy donors for the figures quoted range from under 5% (Bach et al, 1969) to almost 80% (Bentwich et al, 1973). There are many points of variation between the techniques employed (Bach et al, 1969; Coombs et al. 1970; Brain et al, 1970; Lay et al, 1971; Jondal et al, 1972; Papamichail et al, 1972; Wybran et al, 1973; Yata et al, 1973; Bentwich et al, 1973) but the importance of such technical variations is largely unknown (Urbaniak et al, 1973). In the present study, several of the points of variance between published techniques have been analysed.

Lymphocyte transformation induced by autologous cells. IV. Human T-lymphocyte proliferation induced by autologous or allogeneic non-T lymphocytes.

“…Preparation of Lymphocyte Suspensions. 50 ml of venous blood from healthy volunteers were drawn into a plastic syringe containing 10 ) and collected by centrifugation at 150 g for 10 rain at 20°C. The cells were then resuspended in RPMI 1640 (Microbiological Associates) with 100 U penicillin/ml, 100 ~g streptomycin/ml, L-glutamine 2 mM (Microbiological Associates), and 20% FBS at a concentration of 2 million lymphocytes per ml.…”

Immunological studies of Aging. III. Cytokinetic basis for the impaired response of lymphocytes from aged humans to plant lectins.

“…Statistical analysis of data comparing the function of lymphocytes from old or young persons made use of the paired Student's t test or the sign test (15). staining for peroxidase granules (16), and T cells were assayed by SRBC rosetting (9). Based upon assays of 10 paired lymphocyte preparations from old and young subjects, monocytes made up 15-20% and T lymphocytes made up 45-50% of the mononuclear cell preparation.…”

Malignant histiocytosis occurring in patients with acute lymphocytic leukemia

“…17. 20 The results were as follows: 43% marked as T (thymic) cells, 12.5% as B (bursa-equivalent) cells, and the remainder as null cells. The leukemia failed to respond to therapy and the patient died in February 1977.…”