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[Preparation of partially fixed erythrocytes and its application for immunohematologic examination]
1Department of Forensic Medicine, Juntendo University School of Medicine, Tokyo, Japan.
Insights
Partially fixing human erythrocytes with glutaraldehyde preserves their agglutinability and shape, making them stable for blood grouping tests. These fixed red blood cells maintain integrity during storage and various testing methods.
Area of Science:
- Hematology
- Immunoserology
- Biochemistry
Context:
- Human erythrocytes (red blood cells) are crucial for blood group serology.
- Standard erythrocyte preparations can degrade over time, impacting test reliability.
- Mild chemical fixation may offer a solution for preserving erythrocyte integrity.
Purpose:
- To investigate the effects of mild glutaraldehyde fixation on human erythrocyte properties.
- To assess the stability and agglutinability of partially fixed erythrocytes for blood grouping.
- To evaluate the utility of partially fixed erythrocytes in various blood grouping laboratory tests.
Summary:
- Treatment of erythrocytes with 1 mM glutaraldehyde for 10 minutes (partial fixation) minimally altered agglutinability with most blood group antibodies.
- Partially fixed erythrocytes maintained cell volume, hypotonic fragility, and deformability, with enhanced heat resistance compared to untreated cells.
- Partially fixed erythrocytes retained their discocyte shape after 6 months of storage, unlike untreated cells which became spherocytes or echinocytes.
Impact:
- Partially fixed erythrocytes serve as stable, reliable indicators for ABO and Rh blood grouping, including detection of irregular antibodies.
- These fixed cells demonstrate efficacy in forensic serology for blood, saliva, and hair analysis using various testing methods.
- The findings support the use of partially fixed erythrocytes as a viable alternative to fresh cells, enhancing laboratory efficiency and diagnostic accuracy.
Abstract:
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.