Large scale preparation of leukocyte concentrates and further purification of lymphocytes and granulocytes with the IBM 2991 cell processor.

A procedure is described for the large-scale production of pure suspensions of human lymphocytes and granulocytes. Platelet-poor buffy-coats, obtained by centrifugation of six units of ABO-compatible ACD blood, were pooled and centrifuged again, resulting in a crude leukocyte concentrate containing approximately 60 per cent of the original amount of leukocytes. Further fractionation of such crude leukocyte concentrates was achieved by scaling up Bøyum's Ficoll-Isopaque density gradient technique with the aid of an IBM 2991 blood cell processor. In this way 2.9 +/- 0.3 X 10(9) mononuclear leukocytes, consisting of 90 +/- 0.7 per cent lymphocytes and 5.3 +/- 0.3 X 10(9) polymorphonuclear leukocytes of 92 +/- 0.7 per cent purity could be isolated in one run. The overall yield was 36 per cent of the original amount of leukocytes.

A comparative study of the production of antibodies against human blood cells in animals of various species.

In view of the importance of the species of animals used for the preparation of specific heteroantisera against human blood cells, comparative immunization experiments were carried out in goats, rabbit, chickens, guinea pigs, and rats, using lysates of leukocytes, granulocytes, lymphocytes, monocytes, and thrombocytes. The antisera obtained were tested by the indirect immunofluorescence technique. The strongest antisera were obtained in goats, but these antisera required extensive absorptions to make them specific for the immunizing cell, whereas rabbit antisera needed only few absorptions. Antisera from both of these animals could be rendered specific for lymphocytes, granulocytes and platelets. In the guinea pig only immunization with monocytes and granulocytes resulted in useful antisera, which were all specific for granulocytes after appropriate absorptions. Antisera obtained from rats could be rendered specific for granulocytes and lymphocytes by absorption. Antibodies against platelets could not be detected in any of the rat antisera. Chickens, however, produced strong anti-platelet antibodies. A specific anti-granulocyte antiserum could also be obtained in these animals. Specific anti-monocyte antisera could not be prepared from any of the animal sera. Immunizations with leukocyte lysate resulted in granulocyte-specific antisera in most animals but not in chickens. All antisera produced in chickens were specific for platelets. It can be concluded from these results that animals of different species may react very differenly to the antigens of some human blood cells.

Preparation and specificity testing of a rabbit anti-human thymocyte serum (with 1 colour plate).

The preparation of a specific anti-T cell serum, applicable in the indirect immunofluorescence technique on cell suspensions, smears of peripheral blood and bone marrow and on tissue sections is described. Rabbits were immunized with thymocytes; after removal of antibodies against species-specific antigens and antigens common to leucocytes by absorptions with red cells and granulocytes, specificity for thymocytes was obtained by repeated absorptions with CLL cells, used as B cell equivalents. Subsequently, the IgG fraction of the absorbed antiserum was isolated. This antibody preparation was tested with various types of blood cells as well as with cell suspensions depleted or enriched in T cells. For the study of tissue sections it had to be absorbed with liver powder. When studying lymphatic tissue it was found to stain only thymus-dependent areas in the specimens tested.

Specific antisera against human blood cells applicable in the indirect immunofluorescence technique.

The preparation of antisera against various cells of the human peripheral blood applicable in the indirect immunofluorescence technique (IIFT) is described. Such antisera will be a great interest for the study of cell-specific membrane antigens, for example during haematopoiesis. Purified erythrocytes, lymphocytes, neutrophils, monocytes, and thrombocytes from healthy donors were injected into rabbits. The antisera thus produced were not spontaneously specific. Only by extensive absorption of the crude antisera with purified cells from healthy donors was it possible to obtain antisera that were specific for erythrocytes, lymphocytes, and thrombocytes. By injection of small doses of leukocyte lysate and by absorption of the resulting antiserum with mononclear cells a specific antineutrophil serum was produced. So far it has not been possible to prepare a specific anti-monocyte antiserum. The specific antisera were applicable in the IIFT on paraformaldehyde-fixed cells in suspension and on cells on slides.