ISOLATION OF HUMAN PERIPHERAL-BLOOD MONOCYTES - COMPARATIVE METHODOLOGICAL STUDY

Monocytes were isloted from 8.0 ml of peripheral human blood by 4 different procedures: (I) Dextran sedimentation followed by bovine serum albumin buoyant density centrifugation (BSA-BDC) of supernatant cells. (II) Metrizoate/polysucrose buoyant density centrifugation (M/P-BDC). (III) M/P-BDC followed by formation of rosettes by interface cells with sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (AET) and a second M/P-BDC. (IV) M/P-BDC with subsequent incubation of interface cells on plastic petri dishes and removal of adherent cells by a rubber policeman. The degree of purification of monocytes was 28, 21, 65 and 24% and the monocyte yield amounted to 51, 97, 58 and 9% after procedures I, II, III and IV, respectively. Cell viability was 99-100% after procedures I II annd III and 70% after procedure IV. Contamination with granulocytes was less than 2% for all procedures, while procedure III resulted in heavy contamination of isolated cells with SRBC. Monocytes isolated by procedures III and IV showed reduced ingestion of latex compared with cells from procedures I and II. Procedure II was by far the most rapid and simple technique of the 4 and resulted in the best preservation of the esterase-staining properties of monocytes. There was a marked day-to-day and person-to-person variation of peripheral and monocyte counts. The numbers of monocytes in blood smears were found to be highest when cells were identified by staining for non-specific esterase. Morphological identification after staining with Wright's stain yielded on the average only 85% of the number found using the enzyme stain. These two methods for identification of monocytes showed excellent agreement when cells were counted after cytocentrifugation. © 1979.