D-negative Blood Research Articles

Genotyped RhD+ red cells for D-positive patients with sickle cell disease with conventional RHD and unexpected anti-D

AbstractAnti-D can occur in D-positive patients who inherit RHD genetic variants encoding partial D antigen expression, but unexpected anti-D is also found in the plasma of patients with sickle cell disease who have conventional RHD gene(s) and are transfused with units from Black donors. These anti-D are likely stimulated by variant Rh expressed on donor cells; however, patients with anti-D, regardless of cause, are transfused for a lifetime with D-negative (Rh-negative) blood. This results in significant increased use of Rh-negative units, especially for those requiring chronic transfusion, which can strain Rh-negative blood inventories. We tested whether D-positive patients who made anti-D and had conventional RhD by RHD genotyping could safely be returned to D-positive transfusions without anti-D reappearance or compromised red blood cell survival using RHD genotype-matched units from Black donors. Five patients receiving chronic red cell exchange received an increasing number of D-positive units per procedure with a total of 72 D-positive RHD genotyped units transfused, with no anti-D restimulation. Unexpected anti-C and anti-E were identified during the study associated with donors with variant RHCE alleles. RH genotyping of D-positive units for transfusion may improve use and allocation of valuable Black donor units and reduce demand for Rh-negative blood. This trial was registered at www.clinicaltrials.gov as NCT04156906.

Rare occurrence of RHD null alleles with Del expression among serologically D-negative blood donors

Background/Aim: An investigation into the diversity of serologically D-negative blood donors in the Republic of Srpska entity of Bosnia and Herzegovina forms the basis of this research. The primary purpose of the study was the examination of RHD variants over a period of five years. Methods: A comprehensive depiction of the RHD distribution in D-negative blood donors is achieved through a combination of serological observations and DNA testing (PCR-SSP with fluorometric signal detection), involving 74,149 blood donors. The adsorption/elution method was used to confirm the Del phenotype. Results: A small fraction (0.31 %) of the serologically D-negative blood donors was found to contain eight different RHD alleles. The Del phenotype of the RHD*01N.03 and RHD*01EL.44 alleles was highlighted, challenging the common perception that these alleles are associated exclusively with a D-negative expression. Conclusion: The importance of molecular methods in analysing and understanding Del variants, which typically elude conventional serological assays , is underscored by the findings. A group of donors seemingly having the RHD*01 allele but who lacked D antigen expression was encountered, hinting at the potential presence of still unidentified, possibly geographically restricted, RHD variants or alterations in other genes responsible for the expression of Rh proteins in the erythrocyte membrane.

There is an urgent need to adopt a pull-flow logic for the supply of RBCs to meet patients' needs: A single center study.

Despite major demographic changes, several decisions and initiatives, among which Patient Blood Management, have led to a significant reduction in the transfusion of packed red blood cells (RBCs) in Belgium, as it has been observed in many countries. Unfortunately, not all blood groups were proportionately impacted and shortage in O D-negative RBCs is regularly or chronically observed. The goal of this study was to examine how to optimize the use and the supply of O D-negative blood in our academic hospital. All blood transfusions performed at Cliniques universitaires Saint-Luc between January 1, 2019 and December 31, 2021 were reviewed. The blood group of the patients was compared with the blood group of the RBCs actually supplied and transfused. 49.823 RBCs transfusions were analyzed. The patients' needs didn't reflect those of a Caucasian population, with an increase of O (47.9%) and B (10.3%) for the ABO blood group, and a quite high proportion of R0r (8.6%) for the Rh blood group. Only two thirds of O D-negative RBCs were transfused to O rr or R0r patients. The application of PBM and the ethnic imbalance between blood donor and patient populations are two important risk factors for chronic shortages of O D-negative blood. To adapt blood component resources, it is essential to have a complete picture of the real needs of patients according to their blood group profile. Blood donor centers must adapt to the evolving needs of hospitals in order to plan future supplies in a "pull-flow" approach.

Generation of Rh D-negative blood using CRISPR/Cas9.

Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD-/- cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD-/- derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.

Prevalence of Rhesus negative blood group in surgical patients in a tertiary hospital in Rivers State, Nigeria

Background: The Rhesus-D negative blood group is not as common in Africans when compared to Caucasians thus the need to optimize surgical patients prior to surgical procedures. Blood group “O” Rh-D negative blood is a universal donor hence its importance in emergency situations where there is no time to type and cross match blood.This is because surgical patients may need blood transfusion pre-operatively, intra-operatively or post-operatively. Aim: To determine the prevalence of rhesus negative blood group in surgical patients in the Rivers State University Teaching Hospital (RSUTH). Method: This was a one-year retrospective study of surgical patients (Surgery and Obstetrics/Gynaecology departments) of the RSUTH. The patients comprised of all the consecutive cases of the surgeries in these departments for the period under review. Ethical clearance was obtained from ethical committee of the Rivers State Hospital Management Board. Structured profoma was used to extract information from patients’ case notes and analyzed using SPSS version 25. Result: A total of 370 patients were attended to pre-operatively. There were 146 (39.5%) males and 224 (60.5%) females. The mean age was 31 years. The age range was 22 years to 56 years. One hundred and ninety four (52.4%) were obstetrics and gynaecological surgeries while 176 (47.6 %) were non-gynaecological surgeries. The commonest indication for surgery was caesarean representing 126 (34.1%) of the subject.The distribution of Rh-D positive blood and Rh-D negative blood were 337 (91.7%) and 33 (8.9%) respectively. The prevalence of Rh-D negative blood group was 33 (8.9%) with blood group O Rh-D negative as the commonest 15 (4.1%). Conclusion: Our showed the prevalence of Rh-D negative blood group in surgical patients at the Rivers State Teaching Hospital as 8.9%. Rhesus–D “O” negative blood group was the most common representing 4.1% of Rhesus D-negative blood group. This makes it important in emergency surgical practice as universal donor when there may be to time for typing and matching prior blood transfusion.

Serologically D-negative blood donors in Thailand: molecular variants and diagnostic strategy.

Discriminating individuals with "Asian type DEL" from those who are "true D-negative" (D-) among serologically D- donors/patients in Asia would be very valuable, as clinical outcomes are different in these groups. Here we investigated the molecular basis of D-negativity in Thai blood donors, designing a specific strategy for this purpose. After routine testing, a total of 1,270 serologically D- blood donors originating from Central, Northeastern and South Thailand underwent analysis of the RHD gene by (i) quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF); (ii) direct sequencing of exon 9 to identify the c.1227G>A variant defining the Asian type DEL allele; and (iii) direct sequencing of the other exons. The most common observation was whole deletion of the gene (i.e. RHD*01N.01; allele frequency: 86.81%), followed by the Asian type DEL allele (RHD*01EL.01; 7.60%) and a D-negative hybrid allele (RHD*01N.03; 3.46%). Four novel alleles, including one with a 13.1 kilobase-deletion, were identified and characterized. All but one RHD*01EL.01 allele carriers (183/184) were C-positive (C+), suggesting that this latter subset may be screened specifically when investigating the c.1227G>A variant, which can be identified with 100% accuracy by a specific Tm-shift genotyping assay. On the basis of our extensive molecular findings, we have designed a dedicated diagnostic strategy based on Rh C antigen typing followed by a genotyping test. Implementation of this method in all or selected groups of serologically D- donors/patients will contribute to improve the management of transfusion and pregnancy in Thailand.

Secondary alloanti-D immunization post transfusion of “Asia type” DEL red blood cells

Background“Asia type” DEL red blood cells (RBCs) express a very weak D antigen and cannot be detected by routine RhD typing. Thus, it is routinely typed as D-negative (D–) blood group and transfused to D– recipients. Here we described a case of secondary alloanti-D immunization that was associated with transfusion of DEL RBCs to D– recipients and was initially considered as primary alloanti-D immunization. Case presentationA 44-year-old D– woman (G2P2) with adenomyosis and anemia underwent transabdominal hysterectomy. She received four units of D– RBCs before operation. Before transfusion, the alloantibody screening test was negative. Four days after the first transfusion, she needed another RBC transfusion. Unexpectedly, the routine pre-transfusion alloantibody screening test became positive and anti-D (titer, 128-fold) was identified, indicating an alloanti-D immunization. The anti-D developed four days after the first transfusion was unexplained, so alloantibody identification was performed on the sample collected before the first transfusion, and weak anti-D combined with anti-E, which was not detectable during the previous routine pre-transfusion alloantibody screening test with non-enzyme-treated screening cells, was identified using bromelain-treated panel cells. The remaining blood samples of first transfusion in bag tails from two donors were collected for RHD genotyping analysis. One donor was later identified as “Asia type” DEL having RHD* 1227 A/01 N.01 genotype. ConclusionCaution should be applied when we conclude that transfusion of “Asia type” DEL RBCs to true D– recipients could induce primary alloanti-D immunization, especially if the short time interval between transfusion and detection of anti-D is observed.

Implementation of Low Titer Whole Blood for French overseas operations: O positive or negative products in massive hemorrhage?

Massive hemorrhage is the leading preventable cause of death during military operations. During these operations, the delay between initial treatment and arrival at a surgical facility is considerably longer than in Metropolitan settings. This increased prehospital period requires the availability of blood products during the prehospital stage and justifies the availability of Low Titer Whole Blood (LTOWB) for surgical facilities. This product is a fully authorized labile blood product processed by the French Military Blood Institute according to French regulations. Its shelf life is 21 days when stored between 2 and 6°C. It provides the three products necessary for the transfusion management of war injuries in a single product and in physiological proportions. The low anti-A and anti-B titers (<1/64) make LTOWB compatible with any recipient. However, the RhD antigen remains an issue due to its potential harm in cases of transfusion to a D-negative childbearing-age woman due to the potential risk of fetal-maternal incompatibility. This risk must be balanced with the availability of D-negative blood products. Considering the epidemiology of war injuries and LTOWB use guidelines, in addition to the current knowledge on anti-RhD fetal-maternal alloimmunization, the harm-benefit assessment favors the use of RhD-positive LTOWB during overseas operations. Follow-up of childbearing recipients and setup of countermeasures to prevent alloimmunization in those cases remain key points of transfusion safety.

Lack of alloimmunization to the D antigen in D‐negative orthotopic liver transplant recipients receiving D‐positive red blood cells perioperatively

D-negative patients undergoing orthotopic liver transplantation (OLT) might require a large number of red blood cell (RBC) units, which can impact the inventory of D-negative blood. The blood bank might need to supply these patients with D-positive RBCs because of inventory constraints. This study evaluates the prevalence of anti-D formation in D-negative OLT patients who received D-positive RBCs perioperatively, as this will assist in successful patient blood management. This was a retrospective study performed at a single academic medical centre. Electronic medical records for all 1052 consecutive patients who underwent OLT from January 2007 through December 2017 were reviewed. D-negative patients who were transfused perioperatively with D-positive RBCs and had antibody screening at least 30 days after transfusion were included. Of a total of 155 D-negative patients, 23 (14.8%) received D-positive RBCs perioperatively. Seventeen patients were included in the study. The median age was 54 years (range 36-67 years); 13 (76.5%) were male. The median number of D-positive RBC units transfused perioperatively was 7 (range 1-66 units). There was no evidence of D alloimmunization in any patient after a median serologic follow-up of 49.5months (range 31 days to 127.7months). The average number of antibody screening post OLT was 7.29. Our study showed that transfusion of D-positive RBCs in D-negative OLT recipients is a safe and acceptable practice in the setting of immunosuppression. This practice allows the conservation of D-negative RBC inventory.

Emergency Clinicians interpretation and application of Anti-D guidelines

Objective: The objective of this study was to audit the use of anti-D immunoglobulin (anti-D) against the current Australian guidelines in one large inner-city referral hospital over three years and critique the practice identified. Background: Pregnant patients who have a D-negative (RhD negative) blood type are at risk of D alloimmunisation if a potentially sensitising event occurs during pregnancy or birth. The administration of anti-D Ig can prevent complications related to alloimmunisation. Potentially sensitising events commonly present to the emergency department requiring the administration of anti-D Ig in line with current guidelines. Study Design and Methods: This is a retrospective cohort study of all patients who received anti-D Ig in a large inner-city emergency department (ED) over three years (July 2014 – June 2017). Indications for administration were scrutinised against current guidelines by experienced clinicians. Results: A total of 228 patients received anti-D Ig, with the majority being less than twelve weeks in gestation (169, 74.1%). Anti-D Ig was administered without support from the guidelines in 81 (35.5%) patients, with a lack of documented sensitising event in 77 (95%) of these cases. Discussion and Conclusion: There were inconsistencies amongst clinicians who prescribe anti-D Ig in the ED, and a lack of the application of current guidelines. This may stem from a lack of empirical evidence about the need for anti-D Ig in the most common group presenting to EDs, those under twelve weeks in gestation. Current guidelines also fail to take into consideration future need, which could be incorporated in future, ED specific anti-D Ig guidelines. Implications for research, policy, and practice: This audit identified overuse of anti-D Ig in the ED. This may stem from the absence of evidence for its use in pregnant patients under 12 weeks in gestation. To reduce unsupported use, further data on alloimmunisation rates following potentially sensitising events in early pregnancy would be helpful. Additional guidelines specific to patients under 12 weeks in gestation, and presenting to the emergency department may reduce some unsupported usage. What is already known about the topic? Pregnant patients who have an D-negative blood type are at risk of D alloimmunisation when a sensitising event occurs. It is common for patients to present to the emergency department with complications in early pregnancy Previous work has shown that there is variation in anti-D Ig administration in the emergency department. What this paper adds: There is significant use of anti-D Ig in the emergency department that is outside of current guidelines. The current guidelines may not serve the needs of the majority of presentations in the emergency department. Further data on alloimmunisation rates following potentially sensitising events in patients less then twelve weeks of gestation would be useful.

Fetomaternal hemorrhage assessment in Rh-negative patients undergoing dilation and evacuation between 20 and 24 weeks’ gestational age: A retrospective cohort study

ObjectiveTo estimate the rate of requiring more than one 300-mcg Rh D immune globulin dose for fetomaternal hemorrhage (FMH) at the time of second-trimester dilation and evacuation (D + E). Study designWe performed a retrospective cohort analysis of patients at greater than 20 weeks’ gestation who underwent D + E, had Rh D-negative blood type, and received FMH quantification testing. ResultsOf 25 eligible patients, 24 had negative quantification of FMH; one had positive quantification that did not meet the clinical threshold for additional dosing. ConclusionsThe absolute risk of requiring additional Rh D immune globulin after D+E for pregnancies greater than 20 weeks’ gestation was 0%.

The Experience of RHD Genotyping in D-negative Blood Donors

The Experience of <i>RHD</i> Genotyping in D-negative Blood Donors

Prevalence of Rhesus D negativity among reproductive age women in Southern Ethiopia: a cross-sectional study

BackgroundThe Rhesus (Rh) blood group system is the next most clinically significant blood group system following the ABO blood group. Rh D-negative women are at risk of alloimmunization following exposure to Rh D-positive blood. The exposure of Rh D-negative women to Rh D-positive fetal blood may cause hemolytic disease of the fetus or new-born due to Rh incompatibility. Knowing Rh blood phenotype has paramount importance to prevent the risk of sensitization and bad obstetric outcome in Rh D-negative women. Despite the aforementioned fact, the distribution of Rh D-negative phenotype of women was not explored in Arba Minch Zuria district, southern Ethiopia. This study was aimed to assess the prevalence of Rh D-negative blood phenotype among reproductive-age women in Arba Minch Zuria district, southern Ethiopia.Methods and materialsA community-based cross-sectional study was conducted among reproductive-age women in Arba Minch Zuria district, Southern Ethiopia from March to April 2019. Socio-demographic data were collected using an interviewer-administered semi-structured questionnaire and blood phenotype determination was done by laboratory technicians using the slide method principle aseptically and Statistical Package for Social Science (SPSS) version 21 was used for analysis.ResultThe data were collected from 417 study participants with a 98.8% response rate. This study revealed that 2.1%, 1.9%, 1.2%, and 1% of study participants with blood group O, A, B, and AB were Rh D negative, respectively. In this study, the overall prevalence of Rh D negative phenotype was found 6.2% among reproductive-age women in Arba Minch Zuria district, Southern Ethiopia.ConclusionsThis study showed a high prevalence of Rh D negative factor among reproductive-age women in Arba Minch Zuria district. Therefore, counseling of reproductive age women on the importance of Rh D factor status determination would be worthy to avoid the potential risk of sensitization among Rh D negative women in order to prevent hemolytic disease of the fetus and new-born.

Establishment of iPSC (KRIBBi001-A) from CD34+ group O D-negative bone marrow blood.

Human induced pluripotent stem cells with indefinite propagation in vitro provide a potential donor source of cells including erythroid cells for human therapy. Since group O D-negative (RhD-) blood cells are considered as universal donors for transfusion, it is compelling to derive iPSC line from group O/RhD- sample as a new cellular source to generate universal RBCs. The resulting iPSC line derived from group O/RhD- somatic source showed typical features of pluripotent stem cells and could provide an unprecedented cellular tool to develop universal therapeutics for blood transfusion.

D variants in the population of D-negative blood donors in the north-eastern region of Croatia.

The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

Transfusion of D positive red cells to selected D-negative patients

BackgroundTo transfuse packed red blood cells isogroup ABO D is a usual transfusion practice. However, when there is not enough D-negative blood available, we can transfuse positive red blood cells to negative patients. Immunocompetent D-negative individuals may develop serologically detectable anti-D antibodies within 3 months after exposure to D-positive red blood cells. Material and methodOver the last 18 years, we have experienced situations of D-negative blood cell scarcity. In these situations, we have applied a clinical assistance protocol, selecting patients with lower risk of alloimmunization and chronic transfusion requirements. We have retrospectively evaluated this policy for the use of D-positive red blood cells in D-negative patients, focussing on alloimmunization and mortality. ResultsApplying the protocol, 3% of D-negative patients were transfused with D-positive units, with an alloimmunization rate of 12.3%. The rate of alloimmunization was higher in the younger age group and in those transfused with more units. No haemolytic reactions were reported. Mortality in the alloimmunized group was lower. ConclusionThe use of D-positive red blood cells in selected D-negative patients does not induce adverse reactions, is a safe practice and allows saving of a product that is sometimes limited.

Blood Components Management: An Analysis on the Blood Transfusion Medicine in Zabol, Sistan and Baluchistan Province of Iran

Introduction: Blood components are commonly used during surgical operations; however, limited sources are globally available in this regard. The present study aimed to assess blood product usage and wastage in Amir-Almomenin hospital, Zabol, Iran. Methods: A total of3883 ordered blood components were retrospectively analyzed in AmirAlmomenin hospital, Zabol, Iran (January) 2017-(July)2018. The data were analyzed using the SPSS software, version 18. Results: The results demonstrated that the most frequent ordered blood products included packed red blood cells (PRBCs, 2097 units, 54%), followed by fresh frozen plasma (823 units, 21.2%), platelet concentrates (757 units, 19.5%), and cryoprecipitate (206 units, 5.2%), respectively. Intensive care unit department had the highest records of orders (34.2%) and the ratio of crossmatched blood to transfused blood (C/T) was 1.73. In addition, based on the results, the total amount of component wastage was 2.03% with the highest and lowest percentage for PRBCs (59.6%) and cryoprecipitate (4.35%), respectively. The highest rate of wastage was related to the delivery ward (8.23%). There was no return from pediatrics, dialysis, pediatric critical care unit, critical care unit, and gastroenterology wards. Further, a significant difference was observed between the returned rates of D-positive and D-negative blood components with higher rates belonging to D-negative products (P=0.001). Conclusion: In general, due to the 2.03% wastage rate, there is an indispensable need regarding implementing sufficient supervision and assigning vigilant policies on the hospital-based transfusion policies in order to optimize the blood product management.

Strategies to identify candidates for D variant genotyping.

RhD variants have altered D epitopes and/or decreased antigen copies per red cell. Individuals carrying these variants may test antigen negative, weakly positive, or positive by serology, and may or may not be at risk of alloimmunisation after exposure. There have been recommendations to perform RHD genotyping of patients, pregnant women and females of childbearing potential with serological weak D phenotype, to guide prophylactic use of Rh immune globulin (RhIG), and better conserve D-negative blood products. The purpose of this study was to evaluate the performance of a set of empirical criteria to identify such patients. A two-method strategy of gel testing (GT) and tube testing (TT) was used for Rh typing of patients with no historical blood type in the present institution. A monoclonal-polyclonal blend anti-D was used for Rh typing by TT at immediate spin. Three empirical criteria were used to identify candidates for genotyping: C1: discrepancy between the two test methods and a GT reaction strength >2+ stronger than TT; C2: weak serological reaction, defined as reaction strength ≤2+ regardless of testing method if both GT and TT were performed or reaction strength ≤2+ if only GT was performed, or reaction strength ≤1+ if only TT was performed; C3: presence of anti-D in D-positive patients with no history of RhIG use in the preceding 3 months and in whom alloanti-D is suspected. Overall, 50 patients, ranging from newly born to 93 years old, were identified. Genomic testing confirmed D variants in 49/50 cases with a positive predictive value of 98%. This two-method strategy is a powerful screening tool for identifying candidates for RHD genotyping. This strategy meets the current requirements of two blood type determinations/two specimens in pre-transfusion testing while simultaneously identifying candidates for RHD genotyping with a minimal increase in work load and cost.

Prevalence of Rhesus D-negative blood type and the challenges of Rhesus D immunoprophylaxis among obstetric population in Ogbomoso, Southwestern Nigeria

Background: Rhesus incompatibility could pose a major problem in pregnancy, and it could be the cause of obstetric failure in a handful of women. Implementation of programs for antenatal and postnatal (Rhesus D) RhD immune globulin prophylaxis has led to a significant reduction in the frequency of maternal RhD alloimmunization and associated fetal and neonatal complications. Aims: To determine the prevalence of RhD negative among the pregnant population attending the antenatal clinic of a young tertiary health institution in Ogbomoso, a semi-urban town in southwestern Nigeria, and also the challenges faced by this sub-population of pregnant women. Materials and Methods: A retrospective review of the antenatal and labour records of obstetric patient attending Ladoke Akintola University of Technology Teaching Hospital (LTH), Ogbomoso, Nigeria. Variables were expressed as percentages. Result: Of the 596 booked patient who had their blood group systems determined, 563 women (94.5%) were RhD-positive, and 33(5.5%) women were RhD-negative. Almost 50% of the Rh-negative pregnant women were primipara. Twenty-three (69.7%) of the study population had a previous delivery or abortion, but only 9 (39.1%) of these had the Rhesus anti-D immunoglobulin following the delivery or the abortion. One of the RhD-negative women had been sensitized to the RhD positive antigen from the previous delivery at the time of booking the index pregnancy. Of the study population that delivered in our facility, only 11 (33.3%) received the anti-D immunoglobulin after the delivery, and financial inability to purchase the anti-D immunoglobulin was a major reason for refusing the immunoprophylaxis. Conclusion: Although the prevalence of RhD negative still remains low, and the rate of Rh-immunoprophylaxis remains quite low in our obstetric population, the risk of haemolytic disease of the newborn with its attendant perinatal morbidity and mortality is real in our community. The anti-D immunoglobulin should be made available and affordable.

Dried blood spots of pooled samples for RHD gene screening in blood donors of mixed ancestry.

In this study, we present a strategy for RHD gene screening based on real-time polymerase chain reaction (PCR) using dried blood spots of pooled samples. Molecular analysis of blood donors may be used to detect RHD variants among the presumed D-negative individuals. RHD genotyping using pooled samples is a strategy to test a large number of samples at a more reasonable cost. RHD gene detection based on real-time PCR using dried blood spots of pooled samples was standardised and used to evaluate 1550 Brazilian blood donors phenotyped as RhD-negative. Positive results were re-evaluated by retesting single samples using real-time PCR and conventional multiplex PCR to amplify five RHD-specific exons. PCR-sequence-specific primers was used to amplify RHDψ allele. We devised a strategy for RHD gene screening using dried blood spots of five pooled samples. Among 1550 serologically D-negative blood donors, 58 (3.74%) had the RHD gene. The non-functional RHDψ allele was detected in 47 samples (3.02%). The present method is a promising strategy to detect the RHD gene among presumed RhD-negative blood donors, particularly for populations with African ancestry.