A new way of the Coombs test using flow cytometry-based assay to assess erythrocytes-bound IgG antibodies in the human and rabbit model

Abstract

The Coombs test is important in hematology for detecting erythrocyte-bound IgG antibodies or in serm through agglutination methods, but its sensitivity and specificity are limited. Flow cytometry provides a more precise and sensitive alternative for quantitatively assessing RBC-bound IgG antibodies. This assessment is crucial for evaluating the risk of hemolytic reactions and ensuring safe transfusions. This study aimed to explore a new method for the detection of RBC-bound IgG antibodies in rabbits following the injection of human red blood cells. Rabbits serum treated with 2-mercaptoethanol (2-ME) were serially diluted at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, and 1:2048. These diluted samples were then reacted with O-type red blood cells (RBCs). Serum samples from healthy individuals were used as the control group. The tubes were kept in a water bath at 37°C for 30 min incubation. After incubation, the samples were analyzed using a flow cytometry-based assay. Additionally, the traditional Coombs tube method was used and the strength of IgG antibody and agglutination was graded. The results were analyzed using a flow cytometry-based assay, and the agglutination strength was determined using the Coombs traditional tube method for RBC-bound IgG antibodies. A significant difference was found between the rabbits serum and normal control groups (p < 0.001). IgG titers increased significantly after 1 month of immunization in rabbits compared to the titers observed after 1 week. The serum Anti-D stability test showed a coefficient of variation (CV) of 7.74%, indicating good stability of the test results. In this study, we concluded that the flow cytometry-based assay for detecting RBC-bound IgG antibodies was accurate, sensitive, and had positional value in clinical laboratories and research centers.

Keywords: agglutination; antihuman globulin; flow cytometry; immunization; serum.

Figure 1.

Control (a) and original sample (b) serum agglutination results analyzed on flow cytometry.

Figure 2.

Antibody titers of rabbit sera against RBC-O after injection of 1-week and 1-month period.

Figure 3.

RBCs agglutination titer: (a) 1 week: the peak was higher indicating lower agglutination titers, (b) end of month: the peak was lower which indicated stronger agglutination titers.

Figure 4.

Grading of agglutination titers based on Coombs tube test method.