組織適合性検査としてのヒト末梢血白血球混合培養

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Attempts were made to elaborate a technique of the mixed leukocyte culture (MLC) and to determine whether this technique can be used as a quantitative in vitro histocompatibility test on human beings. Responding cells were suspended in TC 199 containing 20% autologous serum at the concentration of 1 x 10^6 cells/1.3 ml. Stimulating cells prepared by treatment with mitomycin C were suspended in the same manner. MLC were set up by mixing each 0.65 ml of responding cells from 2 persons (two way) or each 0.65 ml of responding cells from one person and stimulating cells from the other (one way). After 168 hours of incubation, 1.3 μc of tritiated thymidine was added to each culture. Cultures were harvested in 2 hours and radioactivity of cultured cells was measured by liquid scintillation counter after discarding unincorporated tritiated thymidine. Following results were obtained. 1) The highest radioactivity of MLC reaction is obtained on the 7th day after the initiation of MLC. 2) The leukocytes should be subjected to the initiation of culture within 23 hours after collection of peripheral blood. 3) Homologous serum and erythrocytes present in MLC as admixture have no stimulating effect on cultured leukocyte, and do not exert any appreciable effect on the result of MLC. 4) Abnormally low cpm values are obtained in the cases of polymorphonuclear leukocytosis. After purification of lymphocytes using tetron fiber column, cpm values are normalized. 5) The combinations in which no stimulation is obtained in mutual one way and two way reactions are certified to be MLC identical. To confirm the reliability of MLC as a histocompatibility test, MLC was performed in various pairs (monozygotic twins, dizygotic twins, siblings, parent-child and unrelated subjects).

組織適合性検査としてのヒト末梢血白血球混合培養

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