Comparison of cultures of human lymphocytes obtained following NH₄Cl induced red blood cell lysis and Ficoll-Hypaque density gradient centrifugation

Lymphocyte thymidine uptake in leucocytes prepared by NH₄Cl induced RBC lysis was compared with that of lymphocytes obtained following Ficoll-Hypaque centrifugation using polynomial regression dose response curves which adjusted for inherent subject variability. Eleven male subjects were studied. Heparinized blood was either layered on Ficoll-Hypaque or treated with 0.87% NH₄Cl to induce RBC lysis. Phytohemagglutinin-P (PHA) dose response curves were determined for each subject using triplicate 5 day cultures. For each subject, at each PHA concentration ranging from 12.5 to 167 μg/ml, triplicate mean cpm of Ficoll-Hypaque treated lymphocytes and NH₄Cl treated lymphocytes were compared by the 2 tailed Student's T test. Responses of cultures of NH₄Cl treated lymphocytes were significantly different (<0.05) from the responses of cultures of Ficoll-Hypaque treated lymphocytes for at least 2 PHA doses in 8 of the 11 subjects. The quadratic dose response curves were also significantly different (p<0.05) for 9 of the 11 subjects. A repeated measured quadratic regression was used to determine the best fit dose response curves for each of the experimental conditions. For Ficoll-Hypaque treated lymphocytes, mean cpm=44,977 ± 1,400.4 (PHA concentration)-5.90 (PHA concentration ²⁺ subject effect; s.e.=22,256 for NH₄Cl treated lymphocytes, mean cpm=58,039 +1,002.8 (PHA concentration)-5.32 (PHA concentration)² + subject effect; s.e.=37,966. The p values from the F tests for significance of these quadratic regressions are <0.0001 and >0.28, respecively. The s.e. of the quadratic regressions are different, F=2.91, d.f.=30,30; p<0.001. The lack of significance of the NH₄Cl treated lymphocytes' dose response quadratic regression and its larger s.e. indicates that the NH₄Cl treated lymphocyte culture dose response curve is less predictable than that of Ficoll-Hypaque treated lymphocytes.