Ann Lab Med.  2024 Sep;44(5):418-425. 10.3343/alm.2023.0325.

Evaluating the TaqMan Jr^a -Genotyping Method for Rapidly Predicting the Presence of Anti-Jr^a Antibodies

• Koo YK
• Kwon SS
• Suh EJ
• Kim NH
• Kim HK
• Cho YK
• Choi SJ
• Kim S
• Lee KA

• KMID: 2559148
• DOI: http://doi.org/10.3343/alm.2023.0325

Abstract

Background
The Jr a antigen is a high-prevalence red blood cell (RBC) antigen. Reports on cases of fatal hemolytic disease of the fetus and newborn and acute hemolytic transfusion reactions suggest that antibodies against Jr a (anti-Jr^a ) have potential clinical significance. Identifying anti-Jr^a is challenging owing to a lack of commercially available antisera. We developed an alternative approach to rapidly predict the presence of anti-Jr^a using the TaqMan single-nucleotide polymorphism (SNP)-genotyping method.
Methods
Residual peripheral blood samples from 10 patients suspected of having the anti-Jr ^a were collected. Two samples with confirmed Jr(a–) RBCs and anti-Jr^a were used to validate the TaqMan genotyping assay by comparing the genotyping results with direct sequencing. The accuracy of the assay in predicting the presence of anti-Jr^a was verified through crossmatching with in-house Jr(a–) O+ RBCs.
Results
The TaqMan-genotyping method was validated with two Jr(a–) RBC- and anti-Jr^a -confirmed samples that showed concordant Jr a genotyping and direct sequencing results. Jr^a genotyping for the remaining samples and crossmatching the serum samples with inhouse Jr(a–) O+ RBCs showed consistent results.
Conclusions
We validated a rapid, simple, accurate, and cost-effective method for predicting the presence of anti-Jr^a using a TaqMan-based SNP-genotyping assay. Implementing this method in routine practice in clinical laboratories will assist in solving difficult problems regarding alloantibodies to high-prevalence RBC antigens and ultimately aid in providing safe and timely transfusions and proper patient care.

Keyword

Antibody identification; Anti-Jr^a; Genotyping; High-prevalence antigen; Jr(a–); Single-nucleotide polymorphism; TaqMan assay

Figure

• 

  Fig. 1 Genotyping results for S1 obtained with a TaqMan SNP assay and by direct sequencing. (A) Amplification plot of the TaqMan SNP-genotyping assay for rs72552713 (ABCG2*01N.01; c.376C>T, p.Gln126*). The increase in the fluorescent intensity of the FAM signal and not the VIC signal indicates homozygosity for c.376C>T. (B) Sequencing chromatogram obtained by directly sequencing ABCG2 exon 4. The blue arrow indicates the position showing homozygosity for c.376C>T. Abbreviations: S1, sample 1; SNP, single-nucleotide polymorphism.

• 

  Fig. 2 TaqMan SNP-genotyping assay results visualized on an allelic discrimination plot for the ABCG2 reference allele (allele 1, VIC, ABCG2*01, c.376C) versus the ABCG2 null allele (allele 2, FAM, ABCG2*01N.01, c.376C>T). (A) The samples plotted in the upper left corner had high FAM fluorescence but low VIC fluorescence, indicating homozygosity for ABCG2*01N.01. The blue dots correspond to S1–S8. (B) The samples plotted in the lower right corner had increased VIC fluorescence but not increased FAM fluorescence, indicating homozygosity for ABCG2*01. The red dots correspond to S9 and S10. Abbreviation: SNP, single-nucleotide polymorphism.

• 

  Fig. 3 Crossmatching results between two serum samples and the two in-house Jr(a–) O+ RBCs. (A) Representative crossmatching results for S6 showing no agglutination. (B) Representative crossmatching results for S9, showing agglutination. Abbreviations: RBCs, red blood cells; S6, sample 6.

Reference

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