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Variant D antigens can cause variable serologic results when typing with Anti-D reagents. There is limited information regarding the ability of Anti-D reagents to differentiate between D variants defined by RHD genotyping. This study was performed to determine if a panel of 20 U.S. Food and Drug Administration-licensed Anti-D reagents can identify molecularly defined D variants. Red blood cells from 119 donors carrying variant RHD alleles were tested at immediate spin (IS) and/or by the indirect antiglobuin test (IAT) using conventional test tube and/or column agglutination technology. Reaction strength at IS and IAT was reviewed to determine whether a pattern of reactivity could be correlated with a specific D variant. Agglutination results from each sample with each Anti-D reagent were combined to assess overall reactivity. The sample set consisted of 21 D variants, based on prior RHD genotyping. Of these variants, nine categories had three or more samples used for analysis ( N = 102); 25 RHD*01W.1, 15 RHD*01W.2, 14 RHD*01W.3, 17 RHD*09.01, 14 RHD*09.03, 4 RHD*01W.4, 23 RHD*07, 4 RHD*10.05, and 6 reference allele RHD*01. As expected, IS showed more negative or weak reactions, and IAT produced more positive reactions with 3+/4+ agglutination strength. RHD*01W.3 samples showed strongest reactivity at IS and IAT. Greatest variation in reactivity was observed with RHD*01W.2, showing weakest overall reactivity at IS. All weak D types had at least one sample that yielded a negative result and one sample with 4+ agglutination at IS. Although there were general patterns of reactivity for each variant tested, no one pattern defined all samples carrying the same RHD allele. This study demonstrated that even with 20 different Anti-D reagents, serologic testing alone is insufficient to define weak or partial D types, characterize the risk for alloanti-D, or determine candidacy for Rh immune globulin. The results illustrate how multiple Anti-D reagents can be used to identify samples that should be reflexed to molecular testing., (© 2024 Crystal Theiler et al., published by Sciendo.)

The high number of D variants can lead to the unnecessary use of Rh immune globulin, overuse of D- RBC units, and anti-D allommunization. D variant prevalence varies among ethnic groups, and knowledge of the main variants present in a specific population, their behavior in serologic tests, and their impact on clinical practice is crucial to define the best serologic tests for routine use. The present study aimed to explore the serologic profile of D variants and to determine which variants are most associated with false-negative D typing results and alloimmunization. Donor samples were selected in two study periods. During the first period, D typing was performed on a semi-automated instrument in microplates, and weak D tests were conducted in tube or gel tests. In the second period, D typing was carried out using an automated instrument with microplates, and weak D tests were performed in solid phase. Samples from patients typed as D+ with anti-D were also selected. All samples were characterized by molecular testing. A total of 37 RHD variants were identified. Discrepancies and atypical reactivity without anti-D formation were observed in 83.4 percent of the samples, discrepant D typing results between donations were seen in 12.3 percent, and D+ patients with anti-D comprised 4.3 percent. DAR1.2 was the most prevalent variant. Weak D type 38 was responsible for 75 percent of discrepant samples, followed by weak D type 11, predominantly detected by solid phase. Among the D variants related to alloimmunization, DIVa was the most prevalent, which was not recognized by serologic testing; the same was true for DIIIc. The results highlight the importance of selecting tests for donor screening capable of detecting weak D types 38 and 11, especially in populations where these variants are more prevalent. In pre-transfusion testing, it is crucial that D typing reagents demonstrate weak reactivity with DAR variants; having a serologic strategy to recognize DIVa and DIIIc is also valuable., (© 2024 Carine P. Arnoni et al., published by Sciendo.)

In pregnancy, D- pregnant women may be at risk of becoming immunized against D when carrying a D+ fetus, which may eventually lead to hemolytic disease of the fetus and newborn. Administrating antenatal and postnatal anti-D immunoglobulin prophylaxis decreases the risk of immunization substantially. Noninvasive fetal RHD genotyping, based on testing cell-free DNA extracted from maternal plasma, offers a reliable tool to predict the fetal RhD phenotype during pregnancy. Used as a screening program, antenatal RHD screening can guide the administration of antenatal prophylaxis in non-immunized D- pregnant women so that unnecessary prophylaxis is avoided in those women who carry a D- fetus. In Europe, antenatal RHD screening programs have been running since 2009, demonstrating high test accuracies and program feasibility. In this review, an overview is provided of current state-of-the-art antenatal RHD screening, which includes discussions on the rationale for its implementation, methodology, detection strategies, and test performance. The performance of antenatal RHD screening in a routine setting is characterized by high accuracy, with a high diagnostic sensitivity of ≥99.9 percent. The result of using antenatal RHD screening is that 97-99 percent of the women who carry a D- fetus avoid unnecessary prophylaxis. As such, this activity contributes to avoiding unnecessary treatment and saves valuable anti-D immunoglobulin, which has a shortage worldwide. The main challenges for a reliable noninvasive fetal RHD genotyping assay are low cell-free DNA levels, the genetics of the Rh blood group system, and choosing an appropriate detection strategy for an admixed population. In many parts of the world, however, the main challenge is to improve the basic care for D- pregnant women., (© 2024 Frederik B. Clausen, published by Sciendo.)

Despite the existence of long-standing, well-organized programs for Rh immune globulin (RhIG) prophylaxis, immune anti-D continues to be detected in the D– perinatal population. Between 2006 and 2008, 91 prenatal patients, found to have a previously unidentified anti-D, were followed up with a survey to their treating physician and with additional serologic testing where possible. The physician survey requested pregnancy and RhIG history information, including recent or distant potential alloimmunizing events, and the physicians were asked their opinion on the likely cause for the anti-D. Based on survey responses, updated RhIG information, and results of follow-up serology, anti-D was determined to be attributable to previously unreported RhIG in 44 of 91 (48.3%) cases and to active immunization (immune anti-D) in 36 of 91 cases (39.6%). A probable cause for alloimmunization was reported in 14 of 52 (26.9%) returned surveys. Anti-D alloimmunization continues to occur in our prenatal population despite a comprehensive approach to RhIG therapy. Observations from this prospective patient management strategy include the need for improved application of guidelines for RhIG administration and improved quality of information provided to laboratories assessing RhIG eligibility. A laboratory process for prospective follow-up when unexpected anti-D is detected in pregnancy is recommended.

A blood requisition for double-volume exchange transfusion was received for a 2-day-old male child born to a 29-year-old multiparous female (P2002) referred to our institute having neonatal jaundice with encephalopathy; no maternal sample was received. the neonatal blood sample was typed as group A, D-, and the direct antiglobulin test (DAT) was strongly positive (4+) using the gel method. Mono-specific DAT showed the presence of IgG antibodies on neonatal red blood cells (RBCs). Acid elution and gentle heat elution (at 56°C) confirmed the presence of anti-D on neonatal RBCs. The baby received two exchange transfusions with group O, D-, packed RBCs compatible with his own serum. Later, on day 3, the neonate's mother was typed as group AB, D-, and her serum revealed the presence of alloanti-D, -C, and -S reactive in the anti-human globulin phase. The anti-D titer was 1024. this report highlights the "blocking" phenomenon caused by maternal anti-D in a case of hemolytic disease of fetus and newborn with a positive DAT.