Enumeration of T and B lymphocytes in whole peripheral blood: absence of a null cell population.

Alkaline phosphatase-labelled F(ab')2 polyvalent anti-human immunoglobulin stained a mean of 12% (s.d. 4.2) of lymphocytes in the whole peripheral blood of 15 normal individuals. However, when the sensitivity of detection of the bound anti-immunoglobulin reagent was enhanced by adding complexes of alkaline phosphatase with F(ab')2 anti-alkaline phosphatase, a mean of 22.1% (s.d. 7.8) of lymphocytes were positive. The mean number of T lymphocytes demonstrated in the same blood samples using a monoclonal anti-T lymphocyte antibody (OKT3) was 78% (s.d. 4.1) and was not increased by immunoenzyme enhancement. In five individuals the blood was washed at 37 degrees C to remove passively adsorbed IgG and was then studied using the enhanced method together with monoclonal anti-kappa and anti-lambda antibodies. The mean +/- s.d. number of kappa-positive lymphocytes was 15.5 +/- 4.6% and lambda-positive was 7.9 +/- 1.1%. The sum of these was the same as the number of cells stained either with anti-kappa and anti-lambda together or with the conventional polyvalent anti-immunoglobulin, confirming that the enhancement procedure was detecting integral membrane immunoglobulin and not passively adsorbed IgG. Application to the same blood sample of both the anti-T cell antibody and the enhancement procedure with polyvalent anti-immunoglobulin stained 99-100% of lymphocytes. The present observations confirm that there are two populations among the B-lymphocytes, the B-major cells with readily demonstrable surface immunoglobulin and the B-minor cells on which surface immunoglobulin is demonstrable only by very sensitive techniques (Haegert & Coombs, 1979). The B-major and B-minor cells together account for all the non-T lymphocytes and there are virtually no so-called 'null' cells in normal peripheral blood. These findings have significant implications for the use of surface membrane immunoglobulin as a marker in the typing of normal and abnormal lymphocyte populations.