(P-IG-45) The RHAG*01N.26 Variant does not Lead to Rh-null but to Rh-mod Phenotype: The Importance of Absorption and Elusion Assays

Background/Case Studies: Rh deficiency phenotypes are rare and expressed by an absent (Rh-null) or decreased (Rh-mod) expression of Rh antigens. Regulatory Rh-null phenotype is produced by homozygote or compound heterozygote mutations in the RHAG gene with normal RHD and RHCE. Rh-mod phenotype shares this exact mechanism, so it can only be distinguished from Rh-null by specific antibodies (Anti-Rh17 and Anti-Rh-29) or absorption and elution techniques. A female blood donor who seemingly lacked Rh antigens was founded during the routine screening. This study aimed to analyze this donor's molecular background and red blood cell antigen expression.

Study

Design/Methods: Red blood cell serotyping and antibody screening/identification were performed, and the expression levels of Rh antigens were determined by absorption and elusion tests. A complete blood count and smear, reticulocyte count, osmotic fragility test, and fetal hemoglobin detection were also conducted. The genomic DNA was extracted from total blood, and Whole Exome Sequencing was performed to analyze the RHD, RHCE, and RHAG genes.

Results/Findings: ABO and Rh typing showed an O Rh negative result, with the Rh phenotype showing no expression of any Rh antigen. The extended RBC phenotyping showed K-, k+, Kp(a-b+) Jk(a-b+), M+N+S+s-, Fy(a+b+), Le(a+), P1+. Irregular antibody screening was negative. The absorption and elution test assays demonstrated an extremely weak expression of D (2+), c (+/-), and e (+/-, 1+) antigens, but not E antigen. Additional anti-E absorption and elution tests with a DccEE sample (positive control) showed a decreased agglutination (2+), suggesting a decreased sensitivity of the antiserum (MS260). The individual had the following RH genotype: RHD*01.01/RHD*01El.32 (RHD*D/RHD*DEL32), RHCE*01/RHCE*03 (RHCE*ce/RHCE*cE), and the Rh-null variant, RHAG*01.N26 (c.544A>G; p.Gly182Ser) was homozygote. Thus, the individual had an Rh-mod phenotype. In addition, increased osmotic fragility, reticulocytosis (corrected reticulocytes 3.0%), and stomatocytosis (1+), with no anemia or fetal hemoglobin, were observed.

Conclusions: Although the variant RHAG*01.N26 has been previously described as conducing to Rh-null phenotype, the absorption and elution tests in this blood donor showed an extremely weak expression of D, c, and e antigens. Thus, this variant did not conduce to Rh-null but to Rh-mod phenotype, with increased osmotic fragility, reticulocytosis, and stomatocytosis but no anemia or fetal hemoglobin. The negative results of the anti-E absorption and elusion test were due to low sensitivity, as previously reported in a family with Rh-mod phenotype. However, the absorption and elution tests are fundamental in analyzing the genotype/phenotype correlation in variants within the Rh system.

Importance of research: The RHAG*01.N26 variant was recently described as causing the Rh-null phenotype. However, genetic and serological analysis of a female blood donor demonstrated homozygosity for this variant, along with extremely weak expression of Rh antigens determined by absorption and elution assays. Therefore, this variant is not the cause of Rh-null but of an Rh-mod phenotype.