Antibody sensitization in vivo gives a positive direct antiglobulin test as an indicator of the presence of antibody molecules on the red blood cells (RBCs) surface. It has diagnostic importance in clinical conditions such as hemolytic disease of the fetus and newborn, autoimmune hemolytic anemia, or hemolytic transfusion reaction. The cell-bound antibody molecules could be eluted to test the serological specificity of the antibody involved in the disease process.
There are several approaches to elute antibodies from the sensitized RBCs. Selection of methods depends on the nature of the antigen involved. Carbohydrate-based antigens can easily give up antibodies by simply applying heat at 56°C. However, the antigens with lipid moiety are denatured leaving the antibody molecule free. Nathalang et al. found the ether elution technique superior to eluting Rh antibodies, while the acid elution technique superior to eluting Miltenberger (Mi (a)) antibodies. On the other hand, Panzer et al. observed ether method as inferior to other methods such as xylene and chloroform.
We found ether as the most efficient reagent to prepare eluate from the IgG antibody-sensitized RBCs. However, this method is not as popular with others for a fear of explosion while handling the ether. More so, it gives dense hemoglobin content to the eluate, making it difficult to read the results, particularly if one uses the devices such as gel/column agglutination technology. To obviate this problem, we have adopted a modified approach in testing as outlined here.
Ether eluate was prepared from the sensitized RBCs mainly as laid down in the original article with slight modification. In brief, the sensitized RBCs were washed using chilled normal saline. The packed RBCs were mixed with an equal volume of phosphate buffer saline, pH 6 in a glass tube, and further added a half volume of ether. The tube was plugged and the content was vigorously shaken for a couple of minutes, followed by carefully removing the plug to prevent an outflow of the effervescing content. The tube was centrifuged at 3000 rpm for 3 min. The top layer of ether was discarded. The eluate was aspirated by piercing the long nasal pipette through the layer of RBC ghost, poured in a broad-mouth container, and left at 37°C for 30 min to get rid of traces of ether. Removal of ether's smell was taken as an indicator that the eluate was free from the ether. The content was centrifuged hard and the supernatant was saved in a clean glass tube and used for antibody testing.
As an inherent problem, the eluate so obtained had good amount of hemoglobin pigment. As the method is used to elute the cell-bound IgG antibody to be tested by Indirect antiglobulin test (IAT), the dense hemoglobin content in eluate does not bother if the test is carried out by tube method that involves extensive washing of the sensitized RBCs in the test. However, the eluate, if tested by a more sensitive methods like gel column technology that does not involve the washing steps of the sensitized RBCs, may pose difficulty in viewing the results. The RBC agglutinates as a sign of the positive results would not be well appreciated in the thick red background of hemoglobin being present in the test material. To obviate this problem, we adopted a slightly different approach. Test was primarily incubated in the tube, and the sensitized RBCs were washed using normal saline and the washed sensitized RBCs were superimposed onto the gel column and centrifuge appropriately. The agglutination pattern on three test samples is clearly appreciated by the modified approach with no background hemoglobin tinge [Figure 1].
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