A method for the recognition and separation of human blood monocytes on density gradients.

The density distribution of human mononuclear blood leukocytes was studied in order to define the optimal conditions for the separation of monocytes and lymphocytes by isopycnic centrifugation. Under standardized conditions, two populations of cells with partially overlapping, normally distributed densities were consistently found. The cells with the lowest density were recognized as monocytes, using phagocytosis and size distribution analysis as criteria. Since the density of monocytes continuously increased during the centrifugation, optimal separation of monocytes and lymphocytes could only be achieved by limiting the time of centrifugation to 10 min at 2200 g and 4 degrees C. The separation on discontinuous density gradients decreased when the load exceeded 8 X 10(6) mononuclear cells per sq cm. Analysis of the composition of the two cell populations obtained after separation on a three-layer discontinuous gradient revealed that the contamination of the monocytes with lymphocytes was due to the partial overlapping density distributions of both cell types. A small and a large scale method for isolation of monocytes from blood on discontinuous density gradients are presented. Under the described conditions, a preparation of functionally intact monocytes can be obtained which is comparable, both in yield and purity, to those obtained by methods based on surface adherence without the drawbacks of the latter methods.

Size distribution, electronic recognition, and counting of human blood monocytes.

During a study on the separation of human blood monocytes from lymphocytes, a method was developed to recognize and count monocytes by electronic means. Lightscattering (Cytograf, Bio/Physics), and changes in electrical resistance (Channelyzer, Coulter) were used to size mononuclear leukocytes directly in cell suspensions. Both methods revealed a size distribution profile in which two populations of mononuclear leukocytes could be distinguished. The largest cells were virtually eliminated after phagocytosis of iron particles. We confirmed that these cells were monocytes by three different criteria: the intracellular lysozyme activity, the number of phagocytes, and the percentage of cells with kidney-shaped nuclei. The highly significant correlations we found showed that monocytes could be recognized and counted by electronic sizing. For this method, purified mononuclear leukocyte preparations had to be used, since the presence of erythrocytes, platelets, and polymorphonuclear cells interfered. Statistical analysis revealed that electronic sizing permitted discrimination of differences in monocyte content of 4.5%, with a probability of 95%. It was calculated that this sensitivity of electronic monocyte counting was about three times higher than the sensitivity of microscopic methods. Since 100,000 cells can be sized within a few seconds, not only the efficiency of the preparation but also minor changes in the size of monocytes and lymphocytes introduced during the isolation can be followed.

Correlation of PPD and BCG-induced leukocyte migration inhibition, delayed cutaneous hypersensitivity, lymphocyte transformation in vitro and humoral antibodies to PPD in man.

In a study of healthy human individuals a complete lack of correlation between the results of the agarose leukocyte migration inhibition (LMI) test, using purified protein derivative (of tuberculin) (PPD) and Bacillus Calmette Guérin (BCG) as antigens, and delayed cutaneous hypersensitivity and lymphocyte transformation in vitro to PPD was found. There was a reasonable correlation between PPD- and BCG-induced LMI. Antibodies to PPD proved to have no influence on PPD-induced LMI. Purified polymorphonuclear leukocytes, whether derived from donors sensitive to PPD in the agarose LMI test or from nonsensitive donors, did not show migration inhibition to PPD. It was concluded that polymorphonuclear leukocytes and lymphocytes need to be present simultaneously for migration inhibition of peripheral blood leukocytes by PPD. Furthermore, because a consistent relation with conventional parameters of cell-mediated immunity was lacking, it is doubtful whether the agarose LMI test can be considered as an alternative parameter of this kind of immunity.