Anti-D Alloimmunization in Index Pregnancy after Appropriate Rho(D) Immune Globulin Injection in Two Obese Rh-Negative Patients.

Background The rhesus factor D (RhD)-negative patients who give birth to an RhD-positive newborn or who are otherwise exposed to RhD-positive red blood cells are at risk of developing anti-D antibodies. These antibodies may cause hemolytic disease of the fetus and newborn (HDFN). During pregnancy, prevention of alloimmunization is completed with a Rho(D) immune globulin (RhIg). Cases We report two cases, where obese patients developed alloimmunization, with high neonatal titers, after appropriate RhIG prophylaxis during the index pregnancy. Conclusion Our cases demonstrate cases of anti D-alloimmunization in an index pregnancy, with high neonatal titers. Both patients are obese, with BMI > 35 mg/m 2 . Key Points RhIG can be administered via intramuscular or intravenous formulations. Overall, it appears that both formulations are equally effective. The optimal administration, especially with obese women, is not clearly established.Our cases demonstrate that obesity is a risk factor for failure of RhIG, and could lead to an increase in HDFN.

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.

International collaborative study to assess new stocks of candidate reference preparations to control the level of anti-D in IVIG

The level of anti-D antibodies in human immunoglobulin products for intravenous administration (IVIG) is controlled by the direct haemagglutination method prescribed by the European Pharmacopoeia (Ph. Eur.) that requires 2 control reference reagents. The World Health Organization (WHO) positive control International Reference Reagent (IRR; 02/228) with a nominal titre of 8 defines the highest acceptable titre, while the negative control preparation (02/226) has a nominal titre of <2. Working reference preparations (04/132 and 04/140) were subsequently established as Biological Reference Preparations (BRPs) for the Ph. Eur., and for distribution by the United States Food and Drug Administration (US FDA) and the National Institute for Biological Standards and Control (NIBSC). Due to diminishing stocks of these working reference preparations across the 3 institutions, a joint international study was organised to establish harmonised replacement batches. Sixteen laboratories contributed data to the study to evaluate positive and negative candidate replacement batches (13/148 and 12/300, respectively) against the WHO positive and negative control IRRs and the current working reference preparations (BRPs). The results show that the candidate reference preparations (13/148 and 12/300) are indistinguishable from the corresponding IRRs and current BRPs. The candidate preparations 13/148 and 12/300 were adopted by the Ph. Eur. Commission as Immunoglobulin (anti-D antibodies test) BRP batch 2 and Immunoglobulin (anti-D antibodies test negative control) BRP batch 2 with nominal haemagglutination titres of 8 and <2, respectively. The same materials were also adopted as NIBSC and US FDA reference preparations, thus ensuring full harmonisation.

Why do RhD negative pregnant women still become anti-D immunized despite prophylaxis with anti-D immunoglobulin?

Maternal allo-anti-D in RhD negative pregnant women may cause mild to severe hemolytic disease of the fetus and newborn. Although several other antibodies may also destroy red blood cells of the fetus and newborn, preventive measures with anti-D immunoglobulin are only available for D antigen. Targeted antenatal care together with postpartum prophylaxis with anti-D immunoglobulin has significantly reduced the D-alloimmunization risk. Potentially sensitizing events like trauma to the pregnant abdomen, vaginal bleeding, and amniocentesis may lead to fetomaternal hemorrhage and necessitate additional doses. Despite comprehensive programs with these targeted measures, allo-anti-D is still the most common reason for severe hemolytic disease of the fetus and newborn. Where do we fail then? Here, in this review, I would therefore like to discuss the reasons for D-alloimmunizations hoping that the greater focus will pave the way for further reduction in the number of pregnancy-related allo-anti-Ds.

Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms.

The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 µg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

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.

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.

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.

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

'Old is gold' does conventional test tube method still reign supreme? An immuno-haematological survey of anti-D detection and titration in ante-natal cases among major hospitals across India.

INTRODUCTION: Anti-D detection and titration plays a major role in RhD negative antenatal cases both, for monitoring maternal as well as fetal status as well as initiation of early therapeutic interventions, such as intra-uterine transfusions (IUT) to improve maternal as well as fetal morbidity and mortality and reduce the adverse effects of haemolytic disease of fetus and newborn (HDFN). We conducted a survey focusing on the policies and procedures of anti-D detection and titration among major tertiary care centres across India. METHODOLOGY: The survey was drafted by a working group of transfusion medicine and immunohematology specialists from six different centres in India. Data were obtained via the use of an online questionnaire. RESULTS: Results were categorised into four categories, Hospital information, immuno-haematological testing methodology, clinical significance of anti-D testing and the role of transfusion medicine specialists. The survey highlighted the modalities as well as the methodologies of anti-D detection and titration in antenatal women across different major tertiary care centres in India. CONCLUSION: This survey provided a unique snapshot of the prevalent methodologies being employed by major tertiary care centres across the country for detection and titration of anti-D levels as well as the important role it plays in the therapy of affected antenatal women to minimise adverse effects on the fetus.

Antenatal RHD screening to guide antenatal anti-D immunoprophylaxis in non-immunized D- pregnant women.

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.