[Preparation of partially fixed erythrocytes and its application for immunohematologic examination]

A suspension of washed human erythrocytes (2%) in PBS was mixed with an equal volume of 1 mM glutaraldehyde (GA) and allowed to stand at laboratory temperatures, followed by washing with normal saline. The agglutinability of the erythrocytes toward anti-A, anti-B, anti-M and anti-N reagents remained unchanged after GA treatment shorter than 20 minutes, and the agglutinability toward anti-C, anti-c, anti-D, anti-E, anti-e, anti-Lea, anti-Leb and anti-P1 did not decrease after treatment for 10 minutes. GA treatment for longer periods of time than the above caused a decrease of the reactivities. The agglutinability toward an anti-H (Ulex europaeus) and other lectins increased after 10 to 30 minutes of GA treatment and decreased after 40 minutes or more of exposure to GA. These results indicate that the immunologic agglutinability of erythrocytes were practically unchanged after a 10 minutes treatment with 1 mM GA (a mild fixation procedure hereafter called "partial fixation"). The properties of the partially fixed erythrocytes were closely similar to those of untreated erythrocytes as regards cell volume, membrane fragility in hypotonic solutions (measured by a modification of the Ribiere method), and membrane fragility under continuous shaking. The resistance of the partially fixed erythrocytes heating up to 50 degrees C was superior to that of the untreated erythrocytes. From these results, the deformability of the partially fixed erythrocytes was concluded to be similar to that of untreated erythrocytes. After storage for 6 months at 4 degrees C in Alsever solution containing adenine and inosine, the shapes of the untreated erythrocytes changed to "spherocytes" or "echinocytes", whereas the partially fixed cells retained the original discocyte shape. Blood grouping laboratory tests were performed with the partially fixed erythrocytes as indicators. Both anti-A and anti-B agglutinins in normal human sera could be detected without difficulty. Presence of irregular agglutinins, such as anti-H and anti-N, in healthy donors' sera could also be detected, as with the freshly obtained erythrocytes. The detection limits of an incomplete anti-D agglutinin were equal to those in the tests with untreated erythrocytes. The partially fixed erythrocyte (stored at 4 degrees C for 2.5 to 3 months) were used as indicators of ABO-blood grouping from blood stains, saliva stains and hairs by the agglutinin-inhibition test, absorption-elution test or mixed agglutination test. The results obtained were practically equal to those of the tests with freshly obtained erythrocytes, indicating the availabilities of the partially fixed erythrocytes.