Recovery of functional human lymphocytes from Leukotrap filters.
We report here a method by which functional human PBL may be recovered from a blood product preparation unit (Leukotrap) which is normally employed by blood banks to prepare a leukocyte-poor blood product and which is commonly discarded after use. Blood filters which were back-flushed with cold, phosphate-buffered saline (PBS) and the resulting buffy coats collected and separated on Histopaque gradients yielded an average of 2.67 x 10(8) (+/- 1.17 x 10(8] cells per filter processed (n = 38), with viability greater than 95%. The percentages of cell types recovered before or after Histopaque separation closely approximated those reported for freshly drawn blood when examined using differential staining and morphological criteria. Fluorescent antibody analysis revealed a normal distribution of Leu-4 (T), Leu-12 (B), Leu-3a (T helper), Leu-2a (T suppressor) and Leu-M3 (macrophage) positive cell subsets. Recovered lymphocytes were fully responsive to phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed (PWM) mitogens. Random donor lymphocytes which were co-cultured in a two-way mixed lymphocyte response (MLR) were fully capable of proliferative responses compared to the same populations cultured alone. The results from our study indicate that Leukotrap filters provide a readily available, easily processed and convenient source of functional human PBL for use in the in vitro study of the human immune system.
Longley
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RE
Immunology Program, Harbor Branch Oceanographic Institute, Fort Pierce, FL 34946.
Stewart
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D
D002469:Cell Separation / Q000379:methods*; D005374:Filtration / Q000295:instrumentation; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes