ISSN 1866-8836
Клеточная терапия и трансплантация

DT-03. Determination of weak D antigen in blood donors using molecular genetic typing

Svetlana V. Gavrovskaya, Natalia V. Mineeva, Elena A. Sysoeva

Russian Research Institute of Hematology and Transfusiology, St. Petersburg, Russia


Contact: Svetlana V. Gavrovskaya, phone: +7 (921) 747-50-52, e-mail: mousemoon@mail.ru

doi 10.18620/ctt-1866-8836-2023-12-3-1-176

Summary

Determination of antigen D is an important component of testing donors and recipients for the prevention of post-transfusion complications (PTO) of hemolytic type. The antigen D expression depends on the number of antigenic determinants (epitopes) on erythrocytes. In healthy individuals with Rh-positive blood affiliation, all D antigen epitopes normally expressed. A decrease in the number of epitopes D leads to weak agglutination or its absence when testing by serological methods. Due to the high polymorphism of the RHD gene, there are variants of the D antigen which are difficult to diagnose by serological methods, for example, weak D expression (D weak). Donors with D weak types 1, 2, 3 are Rh-positive, and the blood components harvested from them cannot be used for transfusions to Rh-negative recipients. If the donor has a D weak, the use of only serological methods can lead to a false conclusion on the Rh-negative affiliation. The use of molecular genetic research methods in immunohematology makes it possible to identify variants of antigen D. Our goal was to determine Rh-affiliation of donors using genotyping in the difficult-to-diagnose cases.

Materials and methods

The study material consisted of 20 blood samples from blood donors (13 males and 7 females, median age 37) at the Russian Research Institute of Hematology and Transfusiology, which showed ambiguous results when determining Rh affiliation. Serologically, the RBC D antigen was determined by gel technology in DiaClon ABO/D+ Reverse Grouping ID cards containing anti-D IgM (BioRad, USA), and by indirect antiglobulin test (NAGT) with ID-DiaClon anti-D IgG reagent (BioRad, USA). The results of agglutination test were evaluated from 1+ to 4+ points. Genetic studies were carried out by real-time polymerase chain reaction using reagents RBC-Gene D weak/variant (Inno-Train, Germany) which allows identification of weak D types 1; 1.1; 2; 3; 4; 4.0; 4.1; 4.2 (DAR); 5; 11; 14; 15 17. Genomic DNA was isolated from whole blood using the DNA-Sorb-B reagents (AmpliSens, Russia).

Results

In the ID cards containing anti-D IgM, agglutination was absent in 5 cases, and the results were doubtful (+/-) in 4 samples. Weak agglutination (1+ to 2+) was observed in 11 samples. With NAGT testing, RBC agglutination was not observed in 5 samples, in the rest of blood samples it varied from 1+ to 3+. As a result of molecular genetic typing, the presence of D weak antigens was revealed in 12 persons. The specificity of D weak was represented by following types: 1; 1.1; 2; 3. RHD*D weak type 1 was detected in 2 donors, RHD*D weak type 1.1. was found in three cases. RHD*D weak type 2 was detected in 2 donors. RHD*D weak type 3 was more common (5 cases, 25%). Other types of RHD*D weak antigen, i.e., 4, 4.0, 4.1, 4.2 (DAR), 5, 11, 14, 15, 17 were not detected. The RHD gene was not found in 3 cases, Rh-negative affiliation was established; the common D antigen was detected in 5 cases.

Conclusion

Five donors were verified to have Rh-negative affiliation by serological tests. However, only 3 of them lacked the RHD gene, allelic variants of the RHD*D weak were detected in 2 cases. Molecular genetic typing made it possible to reliably establish the Rh affiliation of donors.

Keywords

Rhesus antigen detection, D group antigen, D weak.



Supplement 12-3
09/30/2023

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doi 10.18620/ctt-1866-8836-2023-12-3-1-176

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