Background/Case Studies: We utilize an algorithm to identify weak/variable D antigen expression in females < age 55 years in order to give recommendations for RhIG administration and transfusion. Samples reacting 1+ to 3+ in QuidelOrtho Vision ID-MT Gel are repeated using tube testing. If tube Anti-D testing results ≤ 2+, the sample is referred for RHD genotyping. We report the serologic and molecular investigation of two patients identified via this algorithm.
Study
Design/Methods: Blood samples (S1 and S2) were analyzed by QuidelOrtho Vision ID-MTS Gel and standard tube methods. Sample RBCs were tested with different Anti-D reagents; inconsistent results prompted referral for RHD genotyping. Antibody screen/identification was performed by QuidelOrtho Vision MTS Gel anti-IgG. DNA was isolated from peripheral blood (QIAGEN). Genotyping included: RHD Beadchip (Werfen); targeted RH NGS (Illumina); Sanger sequencing to confirm variants detected by NGS.
Results/Findings: Table 1 summarizes findings.S1, 35-y-old pregnant G 3 P 2002 White female; no known transfusions. Prenatal testing reactivity with MTS Gel Anti-D Card: 3-4+. Tube testing reactivity with Anti-D reagents: 1-2+ at immediate spin (IS) and 4+ after 10 min room temperature incubation (RT) with Ortho BioClone; 2-3+ at IS and 3+ after 10 min RT with Immucor Series 4; 1+ at IS and 3+ at indirect antiglobulin test (IAT) with BioRad Seraclone. Patient’s antibody screen was negative by MTS Gel anti-IgG. RHD BeadChip did not identify any variants. RH NGS identified only one RHD gene with variant, c.953G >C (p.Arg318Pro) in exon 7 of RHD that was confirmed by Sanger sequencing. S2, 16-y-old Hispanic, Latino, or Spanish Origin female trauma patient; no known pregnancies or transfusions. MTS Gel Anti-D Card reacted 2+. Tube testing reactivity with Anti-D reagents: negative to microscopic at IS and 2+ at IAT with Ortho Bioclone; negative at IS and 2+ at IAT with Immucor Gamma-clone; microscopic at IS and 3+ at IAT with Bio-Rad blend. Patient’s antibody screen was negative by MTS Gel anti-IgG. No known pregnancies or transfusions. RHD BeadChip did not identify any variants. RH NGS detected only one RHD gene, with an undescribed variant in exon 4: c.535T >G (p.Phe179Val).
Conclusions: An algorithm to identify weak/variable D antigen expression led to the detection of two new single-variant alleles, RHD*953C and RHD*535G. The variants are predicted to alter amino acids that reside in the intracellular (p.318Pro by c.953C) or transmembrane regions (p.179Val by c.535G) of the red cell membrane. Given that these were new variants with uncertain alloimmunization risk, both patients were considered D-negative for clinical purposes. S1 received RhIG at 30 weeks and cord blood testing showed the neonate to be strongly D+ with a negative direct antiglobulin test (DAT) by MTS Gel anti-IgG.
