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. 2025 Jan-Dec:39:3946320241305270.
doi: 10.1177/03946320241305270.

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

Anwar Ullah  1 , Xuewei Ding  1 , Xia Qi  1 , Hui Liu  1
Affiliations

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

Anwar Ullah et al. Int J Immunopathol Pharmacol. 2025 Jan-Dec.

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.

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Conflict of interest statement

Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Control (a) and original sample (b) serum agglutination results analyzed on flow cytometry.
Figure 2.
Figure 2.
Antibody titers of rabbit sera against RBC-O after injection of 1-week and 1-month period.
Figure 3.
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.
Figure 4.
Grading of agglutination titers based on Coombs tube test method.
See this image and copyright information in PMC

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