anti-D Research Articles

Anti-D prophylaxis should protect all newborns from haemolytic disease, regardless of their country of residence.

Anti-D prophylaxis should protect all newborns from haemolytic disease, regardless of their country of residence.

Evaluating RhD Assessment By Automated Methodology: A Potential ‘Blind Spot’ For RhD Variant Identification

Abstract Background Automated blood typing platforms are ideal for blood donor centers, given their broad recognition of RhD antigen variants, resulting in products from such donors to be labeled as RhD positive. However, this ‘blind spot’ for variants may be problematic for hospital-based transfusion services, since RhD variants may require provision of RhD- RBC units and Rh immune globulin. The extent to which automated methods accurately characterize variant D antigen expression is not known. As such, the objective of our study was to assess D typing results, as well as RHD genotyping, among two automated testing platforms at our facility. Methods The study site is a single tertiary care center with analysis done from 12/2020-1/2024. At our facility, all females <60 years of age with RhD antigen testing results of ≤2+ agglutination (0-4+ scale) via automated methods [solid phase testing 12/20-4/22 (Neo, Immucor, Norcross, GA) and gel testing 5/22-1/24 (Erytra, Grifols, Emeryville, CA)] are assessed by a tube RhD antigen test (Grifols reagents). If tube testing confirms a ≤2+ result (0-4+ scale), molecular variant D analysis is performed (Versiti, Milwaukee, WI). Patients typed as Rh negative with anti-D antibodies, or those with inconsistent D typing, are also subjected to genetic D testing. The Wilcoxon matched pairs signed rank test was employed for comparative data (P< 0.05 was deemed significant). Results Amongst tested patients, 73 demonstrated ≤2+ agglutination via an automated typing method, leading to variant D genetic analysis. Of these, 71% (n=52) harbored variant D antigens. Within this subgroup, 29 (56%) subjects exhibited variants associated with potential allo-anti-D development (n=5 partial D variants; n=24 weak D variants). Notably, 4 patients with partial D and 1 with weak D demonstrated allo-anti-D antibodies. Of 23 patients with variant D who underwent automated gel testing, 20 concurrent tube testing results were available. Gel testing revealed significantly stronger agglutination compared to tube testing (p < 0.001). In contrast, for paired solid phase and tube testing (n=11), there was no statistically significant difference in agglutination strength (p = 0.28). Conclusion Automated testing, particularly gel-based platforms, exhibits reduced sensitivity to RhD antigen variant identification when compared to tube testing, placing subsets of patients potentially at risk for forming allo-anti-D. Implementing protocols for verification of D status could enhance the sensitivity of variant D detection for facilities that largely rely on automated methods for antigen typing.

Perioperative selection of blood and components for substitution: Immunohaematological assessment of immunization risk and methods for selection of optimally compatible blood

The aim of this study is to assess the risk for immune and hematologic adverse events in perioperative transfusion of blood components. Our second goal is to propose methods for the selection of an optimally compatible donor in order to limit the negative impact of complications following blood component transfusion on the hospital outcome of patients admitted to clinics of general, gastrointestinal, thoracic, and vascular surgery. The largest group of patients included in this study were patients with gastrointestinal disorders (614 patients; 31%) and patients with diseases of the organs of the blood and lymphatic system (589 patients; 30%). This was followed by patients with diseases of the skin and subcutaneous tissue (368 patients; 18%) and patients with benign and malignant neoplasms (365 patients; 18%). The remaining 65 patients (3%) were diagnosed with diseases of the endocrine system, trauma patients, and patients with genitourinary disorders. The methods used for optimal selection of compatible blood components in this study are enzyme tests, Coombs tests, and tests in agglutinating medium. The study shows that clinically significant antibodies, which could provoke a post-transfusion hemolytic reaction, were detected in 4.3% of the screened patients. In the majority of patients, the specificity of antierythrocyte antibodies cannot be established. For those patients where the specificity of anti-erythrocyte antibodies could be established, the type of antibody and the antigen system to which it belonged were as follows (in descending order): 1. Anti-erythrocyte antibodies belonging to the Rh system: 37% (n = 17) of all screened patients (1.6% of all patients), including anti-E type (5 patients), anti-D (8 patients), anti-C-1, anti-C (2 patients), and anti-Cw (1 patient). 2. In 3 (6% or anti-erythrocyte antibodies belonging to other erythrocyte antigen systems—Kidd-1 patients, Lewis-2 patients—6% of the screened patients (0.3% of all patients). 3. In 57% (n = 26) of those screened (2.4% of all patients), alloantibodies without any known specificity were identified. The largest number of identified antibodies were directed against the D antigen belonging to the Rh system. Most of the anti-D alloantibodies found in Rhesus D (-) negative patients with no history of prior transfusion of D (+) positive red blood cell (RBC) concentrate possibly have resulted from prior Rh isoimmunization during pregnancy, provided that anti-D antibodies persist for a long time after birth—in some of the studied patients, 15–20 or more years. A greater number of alloimmunizations were found in women—28 (61%), compared to men—18 (39%), due to previous sensitization with different blood group antigens during pregnancy. This study shows that timely diagnosis is essential for the selection of appropriate RBC concentrate, for the avoidance of adverse reactions, and for the improvement of hematological parameters after transfusion of blood components. Graphical abstract:

Antenatal anti-D prophylaxis and D antigen negativity in pregnant women of the UAE: a cross-sectional analysis from the Mutaba’ah Study

ObjectiveThis study aimed to determine the prevalence of the negative D antigen phenotype, adherence to routine antenatal anti-D immunoglobulin prophylaxis (RAADP) administration and D antigen sensitisation among pregnant women in...

Clinical Characteristics and Outcomes of Intrauterine Blood Transfusion (IUT) for Infectious Etiologies.

Congenital viral infection may result in fetal anemia and thrombocytopenia. While intrauterine blood transfusions (IUTs) are more commonly performed for Rh alloimmunization, reports using IUT for infection have varying success. Our primary objective was to characterize the outcomes of patients undergoing IUT for infectious etiologies at our center compared with Rh disease. This was a case series of patients undergoing IUT from 2012-2023. Infectious etiologies were identified by maternal serologies and confirmed by amniotic fluid polymerase chain reactions (PCR). Clinical outcomes were obtained from electronic medical records. During the study period, 70 patients underwent IUT, 34% (24/70) for Rh alloimmunization and 17% (12/70) for infection. Those with infectious etiologies were more likely to be diagnosed at earlier gestational ages (22 vs. 25weeks, p=0.04), with hydrops (75 vs. 33%, p=0.03), and thrombocytopenia (27±33×103 vs. 163±112×103, p<0.01). Perinatal death was significantly greater in cases of CMV (4/5, 80%) compared to parvovirus (1/7, 14%) or Rh alloimmunization (5/24, 21%) (p=0.02). Anemias and thrombocytopenias related to fetal infection may be indications for IUT. Compared with Rh alloimmunization, IUT in fetal infections was performed significantly earlier, and hydrops were more common at the time of IUT. In the case of CMV, greater rates of IUFD (80%) were observed. Patients should be counseled on the various outcomes by indication.

Incidence and risk factors of neutropenia in neonates with hemolytic disease of the newborn

Background Hemolytic disease of the newborn (HDN) is a less recognized cause of neonatal neutropenia. Therefore, this study aims to estimate the incidence of neutropenia and identify associated factors in infants with HDN at a tertiary care center. Methods This retrospective cohort study included infants with HDN who presented at a tertiary care center in Saudi Arabia between March 2008 and September 2023. Neutropenia was defined as an absolute neutrophil count of less than 1.5 μL. Results Among 339 neonates with HDN, 50.1% were male, and 49.9% were female. Rh isoimmunization was the most common antibody type, observed in 58.7% of cases. The severity of HDN was classified as mild in 62.6% of neonates, moderate in 33.3%, and severe in 4.1%. Neutropenia was more prevalent in moderate-to-severe HDN cases (P = 0.047). The incidence rate of neutropenia was 4.1 per 1,000 person-days, with 7.4% of neonates (25/339) being neutropenic at birth. Among these, 17 out of 25 neonates showed resolution within 2 days. Multivariate analysis identified male gender (P = 0.022), low gestational age (P = 0.008), low birth weight (P = 0.039), and the need for exchange transfusion (P = 0.036) as significant risk factors for neutropenia. Conclusion Neutropenia in infants with HDN, irrespective of antibody type, is generally a benign, self-limiting condition. This condition predominantly affects male neonates with moderate-to-severe HDN and prematurity and can be managed conservatively.

Single-exon fetal RHD genotyping: a 31-month follow up in the obstetric population of Western Sweden.

The Rh blood group system is highly complex, polymorphic, and immunogenic. The presence of RHD gene variants in RhD negative pregnant women is a challenge in fetal RHD genotyping as it may influence the antenatal management of anti-D prophylaxis. The aim of this study was to determine the efficiency of a non-invasive single-exon approach in the obstetric population of Western Sweden in a 31-month follow up. The frequency and type of maternal RHD variants were explored and the relation to the ethnicity was elucidated. Discrepant results between fetal RHD genotyping and serological blood group typing of newborns were investigated and clarified. RHD exon 4 was analysed with quantitative real-time PCR technique in a total of 6,948 blood samples from RhD negative women in early pregnancy. All cases with suspected maternal RHD gene and discrepant results observed in newborn samples, were further investigated using both serological and molecular technologies. A total of 43 samples (0.6%) had inconclusive fetal genotyping result due the presence of a maternal RHD gene. These findings were in most cases (>66%) observed in pregnant women of non-European ancestry. Additionally, two novel RHD alleles were found. Seven discrepant results between fetal RHD genotype and serological RhD type of the newborns, were shown to be related to D antigen variants in newborns. Assay sensitivity was 99.95%, specificity 100%, and accuracy 99.97%. The single-exon approach for fetal RHD screening early in pregnancy is an appropriate choice in the population of Western Sweden, with a very low frequency of inconclusive results caused by the presence of maternal RHD gene variants. Due to the high sensitivity, specificity, and accuracy of the test, serological typing of neonates born to RhD negative women has no longer been performed at our laboratory since June 2023.

Management and Treatment Outcomes of Hemolytic Disease of the Fetus and Newborn (HDFN)-A Retrospective Cohort Study.

Background: Hemolytic disease of the fetus and newborn (HDFN) is caused by maternal antibodies attacking fetal blood cell antigens. Despite routine antenatal anti-D prophylaxis, intrauterine transfusions (IUTs) are still needed in some HDFN cases. Methods: We conducted a retrospective cohort study on newborns with HDFN born in the 1st Department of Obstetrics and Gynecology of the Medical University of Warsaw. We analyzed 274 neonates with HDFN, identifying 46 who required IUT due to fetal anemia and 228 who did not. The laboratory results, management, and outcomes were compared between these groups. Results: Comparative analysis showed that newborns treated with IUT were more likely to have significant anemia, hyperbilirubinemia, and iron overload, indicated by a high ferritin concentration. These neonates more often required top-up transfusions, phototherapy, intravenous immunoglobulin infusions, and exchange transfusions. The length of stay was longer for newborns who received IUT. Conclusions: HDFN requiring IUT is associated with a greater number of complications in the neonatal period and more often requires additional treatment compared to HDFN not requiring IUT.

Rh disease in Mexico: evaluating regional and institutional differences in treatment availability and disease management.

Rh disease occurs following maternal alloimmunization, which can develop due to RhD blood group antigen incompatibility between a mother and her fetus. Despite developing robust clinical protocols for effective immunoprophylaxis over the last 50+ years, a significant global burden of Rh disease still exists, particularly in low/middle-income countries such as Mexico. This study examined disparities in the allocation of maternal and child health resources, as well as clinical knowledge regarding Rh disease, to gain insight into why Rh disease remains prevalent in Mexico. To this end, an 11-question survey was sent to members of the Federación Mexicana de Colegios de Obstetricia y Ginecología (FEMECOG) to evaluate their knowledge of the availability and implementation of anti-RhD immunoglobulin prophylaxis in their practices and institutions, and about managing Rh disease by monitoring fetal anemia risk and providing intrauterine treatment when necessary. Responses were separated by region, and chi-square two-by-two contingency tests were performed to evaluate regional and institutional differences. Significant variations in prevention and treatment were found within the Mexican healthcare system, particularly, with regard to providing anti-RhD immunoglobulin to prevent alloimmunization, which is critically important for preventing Rh disease. Specifically, Regions 5, 6, and 7 were most lacking in this regard. This study highlights differences in the Mexican healthcare system in preventing and treating Rh disease. Closing the gap in the availability of anti-RhD immunoglobulin should take priority in future efforts aimed at providing equitable care, because this will lead to the more preferable outcome of preventing Rh disease, rather than forcing patients to seek out more complex measures for treating Rh disease after it develops. These data can be used to create strategies to understand and eliminate these healthcare disparities.

Disease severity in subsequent pregnancies with RhD immunization: A nationwide cohort.

To evaluate the severity of haemolytic disease of the foetus and newborn (HDFN) in subsequent pregnancies with RhD immunization and to identify predictive factors for severe disease. Nationwide prospective cohort study, including all pregnant women with RhD antibodies. All women with at least two pregnancies with RhD antibodies and RhD-positive foetuses were selected. The main outcome measure was the severity of HDFN in the first and subsequent pregnancy at risk. A subgroup analysis was performed for the group of women where RhD antibodies developed after giving birth to an RhD-positive child and thus after receiving anti-D at least twice (group A) or during the first pregnancy at risk for immunization (group B). Sixty-two RhD immunized women with a total of 150 RhD-positive children were included. The severity of HDFN increased for the whole group significantly in the subsequent pregnancy (p < 0.001), although it remained equal or even decreased in 44% of women. When antibodies were already detected at first trimester screening in the first immunized pregnancy, after giving birth to an RhD-positive child (group A), severe HDFN in the next pregnancy was uncommon (22%). Especially when no therapy or only non-intensive phototherapy was indicated during the first immunized pregnancy (6%) or if the antibody-dependent cell-mediated cytotoxicity result remained <10%. Contrarily, women with a negative first trimester screening and RhD antibodies detected later during the first pregnancy of an RhD-positive child (group B), often before they had ever received anti-D prophylaxis, were most prone for severe disease in a subsequent pregnancy (48%). RhD-mediated HDFN in a subsequent pregnancy is generally more severe than in the first pregnancy at risk and can be estimated using moment of antibody detection and severity in the first immunized pregnancy. Women developing antibodies in their first pregnancy of an RhD-positive child are at highest risk of severe disease in the next pregnancy.

Feto-maternal outcome of Rh-Negative pregnancy presenting in a tertiary healthcare centre

Background: The term "Rhesus incompatibility" describes the mismatched Rh types of the mother and the fetus. It is linked to the onset of hemolytic disease of the newborn (HDN) ranging from haemolytic anemia to hydrops fetalis and also related to maternal Rh sensitization. The disease incidence is currently declining globally, having dropped from 1.3%–1.7% in the 1980s to 0.17% in the 1990s1. Erythroblastosis foetalis is one of the most terrible consequences of an ABO incompatible or Rh incompatible pregnancy. About 10% of all Rh-negative pregnancies result in Rh incompatibility in the Rh-negative mother carrying a Rh-positive fetus. Methods: This observational study was carried out among 60 Rh negative pregnant women attending our antenatal clinic and delivered in our institution from October 2022 to March 2024.The Rh negative women were followed up with a series of investigations such as Indirect Coombs Test (ICT), MCA-PSV and with regular antenatal care (ANC). After birth neonates were followed up with blood grouping and Rh typing, direct coombs test (DCT), duration of phototherapy, duration of NICU stay , need for immunoglobulin and exchange transfusion were recorded to evaluate the maternal and neonatal outcomes.The data was collected and tabulated in Microsoft excel sheet and the percentages were calculated. Results: This study included a total of 60 Rh negative mothers ,where 48 (80%) of them were aged less than 30 years and 12 of them (20%) were more than 30 years of age. Antenatally, 51 (85%) women were ICT negative and received RAADP, 9(15%) women were ICT positive and anti D titres were less than 1:16 followed up with MCA-PSV which was normal/less than 1.5MoM. The total preterm births were 6(10%).The total admissions to NICU were 12(20%) in our study. All the 9 babies born to ICT positive mothers turned out to be Direct Coombs test (DCT) positive and were managed with double surface phototherapy. For 2(3.3%) babies, human immunoglobulin was given. Only 1(1.6%) baby required exchange transfusion. Conclusion: We conclude that, severe hyperbilirubinemia and hydrops foetalis, which were observed previously are drastically reduced with recent advances and the use of Routine antenatal Anti D prophylaxis (RAADP). Neonatal morbidity and mortality reduced drastically with newer advances like immunoglobulin reducing the need for exchange transfusion and better NICU care. ICT positivity of 15% despite of postnatal immunisation suggest that there is chances of silent fetomaternal haemorrhage during antenatal period, health care professionals should be more knowledgeble with prenatal screening, the value of blood grouping and Rh typing, anti-D immunisation following sensitising events like MTP, abortion, ectopic pregnancy, ECV and regular implementation of RAADP in clinical practice.

Good Perinatal Outcome of Rhesus Incompatibility in Multigravida without Anti-D Injection Therapy: A Rare Case Report

Good Perinatal Outcome of Rhesus Incompatibility in Multigravida without Anti-D Injection Therapy: A Rare Case Report

RhD-Alloimmunization in Adult and Pediatric Trauma Patients

The actual risk of providing RhD-positive units to RhD-negative recipients remains debatable. There is no standard of care in the United States (US) to guide transfusion decisions regarding RhD type for patients with an unknown blood type, except for women of childbearing age and neonates. The risk of alloantibody formation by an RhD-negative patient exposed to RhD-positive blood is reported to be from 3% to 70%. Due to such wide variations, this review was undertaken to determine the prevalence of anti-D alloimmunization in trauma patients who are RhD-negative and were transfused RhD-positive blood products. This study used the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) approach to answer the question, “In trauma patients who were transfused blood, what is the prevalence of alloimmunization to the D-antigen?” The review included all published articles through April 3, 2022 in databases. Articles published after the search period found by the authors were added to the manuscript if they addressed the primary question and there was unanimous consensus. There were 1683 full-text articles that met the search criteria, with 19 studies meeting eligibility criteria. In addition, 57 references were added after the search period had closed. The incidence of anti-D alloimmunization in adult trauma patients receiving whole blood varied from 7.8% to 42.7%. In contrast, incidence varied in patients receiving red blood cells (RBCs), from 0 to 94%, depending on number of categories analyzed. Anti-D alloimmunization with platelet transfusions varied from 0% to 19%. The alloimmunization rate increased with age and was detected only in children older than 5 years. Recent guidelines recommend the administration of Rh immune globulin (RhIG) to all traumatically injured patients who are both RhD-negative and pregnant. However, there is no specific guidance focused on the RhD-negative patient, pregnant or nonpregnant, and who have received RhD-positive red blood cells (RBC) and platelets. While numerous studies have attempted to evaluate the frequency of RhD alloimmunization rate in trauma settings, emerging data suggests that many factors affect this phenomenon. Additionally, the role of RhIG administration in cases of RhD-incompatible transfusions within the trauma setting adds complexity. As our trajectory propels us towards precision medicine and tailored transfusion practices, gaining a big data approach becomes indispensable.

Genetic diversity of 1,845 rhesus macaques improves genetic variation interpretation and identifies disease models

Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which may lead to the development of rhesus disease models. Through phenotyping of macaques carrying a pathogenic OPA1:p.A8S variant, we identify a genetic model of autosomal dominant optic atrophy. Finally, we present a public website housing variant and genotype data from over two thousand rhesus macaques.

Assessment of Pregnant Women's Knowledge Regarding Rhesus Incompatibility

Assessment of Pregnant Women's Knowledge Regarding Rhesus Incompatibility

40 years of researching the Del phenotype results in a change of transfusion practice.

Anti-D cannot agglutinate red cells of any Del phenotype in routine serology. Many individuals with East Asian ancestry who type D-negative in serology harbor a Del phenotype. Almost all such individuals carry one distinct DEL variant, dubbed Asian-type DEL, known as RHD*01EL.01, RHD*DEL1, RHD:c.1227G>A, formerly known as RHD(K409K). Clinical evidence strongly suggests that Asian-type DEL individuals can safely be transfused with RhD-positive blood and do not need anti-D prophylaxis in pregnancy.

Does high body mass index (>25 kg/m2) or weight (>80 kg) reduce the effectiveness of anti-D prophylaxis in Rh(D)-negative pregnant women? A systematic review and meta-analysis.

Haemolytic disease of the foetus and newborn (HDFN) occurs when maternal antibodies, often triggered by foetal antigens, destroy foetal and neonatal red blood cells. Factors like antibody strength, quantity and gestational age influence HDFN severity. Routine antenatal anti-D prophylaxis (RAADP) has significantly reduced HDFN cases. However, the effect of overweight/obesity (body mass index [BMI] > 25/30 kg/m2) on anti-D prophylaxis efficacy remains unclear. This systematic review will examine the impact of BMI on anti D prophylaxis effectiveness in Rh(D) negative pregnant women. We conducted a systematic review and meta-analysis following Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) protocols. We searched databases from 1996 to 2023, focusing on studies exploring the link between high BMI/weight and anti-D serum levels in Rh(D)-negative pregnant women with Rh(D)-positive foetuses. Ten eligible studies were included, three suitable for meta-analysis. Study quality was assessed using the Strengthening the Reporting Observation Studies in Epidemiology (STROBE) checklist. Statistical analyses included Pearson correlation coefficients and risk differences. Our meta-analysis revealed a significant negative correlation (r = -0.59, 95% confidence interval [CI]: -0.83 to -0.35, p = 0.007) between high BMI/weight and serial anti-D levels in in Rh(D)-negative pregnant women with Rh(D)-positive foetuses. High BMI/weight had lower odds of serial anti-D level exceeding 30 ng/mL (arcsine risk difference [ARD] = 0.376, 95% CI: 0.143-0.610, p = 0.002). Heterogeneity among studies was low (I2 = 0). While our analysis suggests a potential linkage between high BMI/weight and reduced efficacy of anti-D prophylaxis, caution is warranted due to study limitations. Variability in study design and confounding factors necessitate careful interpretation. Further research is needed to confirm these findings and refine clinical recommendations.

Knowledge, attitude and prevention practice of Rhesus incompatibility among women of childbearing age in Rumueme Community of Port Harcourt

Knowledge, attitude and prevention practice of Rhesus incompatibility among women of childbearing age in Rumueme Community of Port Harcourt

When and why is red blood cell genotyping applicable in transfusion medicine: a systematic review of the literature.

This review aims to provide a better understanding of when and why red blood cell (RBC) genotyping is applicable in transfusion medicine. Articles published within the last 8 years in peer-reviewed journals were reviewed in a systematic manner. RBC genotyping has many applications in transfusion medicine including predicting a patient's antigen profile when serologic methods cannot be used, such as in a recently transfused patient, in the presence of autoantibody, or when serologic reagents are not available. RBC genotyping is used in prenatal care to determine zygosity and guide the administration of Rh immune globulin in pregnant women to prevent hemolytic disease of the fetus and newborn. In donor testing, RBC genotyping is used for resolving ABO/D discrepancies for better donor retention or for identifying donors negative for high-prevalence antigens to increase blood availability and compatibility for patients requiring rare blood. RBC genotyping is helpful to immunohematology reference laboratory staff performing complex antibody workups and is recommended for determining the antigen profiles of patients and prospective donors for accurate matching for C, E, and K in multiply transfused patients. Such testing is also used to determine patients or donors with variant alleles in the Rh blood group system. Information from this testing aides in complex antibody identification as well as sourcing rare allele-matched RBC units. While RBC genotyping is useful in transfusion medicine, there are limitations to its implementation in transfusion services, including test availability, turn-around time, and cost.

Serologic profiling of D variants in donor routine: unveiling the impact on false-negative results and alloimmunization.

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.