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Background: D antigen variants may be grouped into partial D, weak D, and DEL types. Cumulative phenotype frequencies of these D variants may approach 1% in certain European regions. Unambiguous and quick identification of D variants is of immediate clinical relevance, with implications for transfusion strategy., Study Design and Methods: A total of 628 samples with ambiguous serologic results from different immunohematology laboratories throughout the Flanders region, Belgium, were genotyped using a commercially available weak D typing approach. After exclusion of detectable weak D types, molecular RHD exon scanning was performed for the remaining samples, and RHD sequencing was performed in two particular cases., Results: Of all samples investigated, 424 (67.5%) were positive for weak D Type 1, 2, or 3, and 22 cases (3.5%) typed weak D Type 4.0/4.1/4.3, 4.2, 5, 11, 15, or 17. Another 49 (7.8%) samples were partial D variants, with a major proportion being category DVI types (n = 27). One RHD(S103P) sample was identified as high-grade partial D, with DIII-like phenotype and anti-D and anti-C immunization. Additionally, a novel DVI Type 3 (A399T) variant was found. Of the remaining 133 samples mainly tested because of ambiguous serologic D typing results due to recent transfusion, 32 (5.1%) were negative for RHD, and 101 (16.1%) were indistinguishable from wild-type RHD and not investigated further., Conclusion: Despite the enormous diversity of RHD alleles, first-line weak D genotyping was remarkably informative, allowing for rapid classification of most samples with conspicuous RhD phenotype in Flanders. The clinical implications are discussed., (© 2014 AABB.)

Background: Among more than 300 mutated alleles identified so far within the RHD gene, almost 40 are assumed to alter cellular splicing and therefore may have a direct effect on Rh phenotype both at the quantitative and at the qualitative levels. Functional data are, however, mostly unavailable to assess the direct involvement of splicing defect in the underlying physiology., Study Design and Methods: We generated plasmid constructs to carry out an exhaustive investigation of 38 RHD variants located within or in the vicinity of exon-intron junctions by a minigene splicing assay, further characterized the transcript structures by sequencing, and identified cryptic sites activated by the genetic defect. Bioinformatics predictions were carried out in parallel and compared with the functional data., Results: For the first time we demonstrate that a product including the full-length Exon 9 is transcribed in the presence of the c.1227G>A substitution frequently carried by Asians with DEL phenotype and confirmed that splicing is altered in the RHD*weak D Type 2 allele, a rare variant most commonly found in Caucasians., Conclusion: Overall we 1) show significant correlation between functional analyses, bioinformatics predictions, and phenotypes, when available, especially for variants in close proximity of the consensus splice sites; 2) classify the variations as splicing or nonsplicing variants; and 3) provide functional data to further improve bioinformatics splicing tools. Conversely assessment of seven silent exonic variants was mainly inconclusive., (© 2015 AABB.)

It is standard practice for pregnant RhD-negative women who have not already formed anti-D to receive antepartum Rh immunoprophylaxis and, if they deliver an RhD-positive neonate, to receive postpartum Rh immunoprophylaxis. An estimated 0.6% to 1.0% of white women have red blood cells that express a serologic weak D phenotype. Of these women, approximately 80% will have a weak D type 1, 2, or 3 that could be managed safely as RhD-positive. Surveys of laboratory practice reveal a lack of standards for interpreting the RhD type for women with a serologic weak D and for determining their need for Rh immunoprophylaxis. RhD genotyping is recommended to determine the molecular basis of serologic weak D phenotypes in pregnant women as a basis for determining their candidacy for Rh immunoprophylaxis.

Objectives: To analyse anti-D alloimmunisation in pregnant women with D-elute (DEL) phenotype in China, for developing a predictive model to evaluate whether a person with the DEL phenotype can receive RhD-positive blood., Background: Alloanti-D acquired by pregnancy or transfusion is one of the major causes of both haemolytic disease among newborns and haemolytic transfusion reactions. To date, there is little data available about the antigenic properties and immunogenicity of extremely weak D variants known as DEL., Methods: RHD genotyping and D epitope mapping were performed using gene sequencing and comprehensive immunohaematological methods, respectively. DEL pregnant women carrying an RhD-positive fetus were tested for the presence of alloanti-D., Results: A total of 130 of 142 (91·5%) pregnant women with a DEL phenotype were confirmed to carry the RHD (K409K) allele. Among 12 DEL women who appeared to have RHD-CE-D hybrid alleles, there were 1 RHD-CE (4-7)-D, 7 RHD-CE(4-9)-D, and 4 RHD-CE (2-5)-D alleles. Alloanti-D antibodies were detected in 6 of 142 DEL women, and all the six women had the partial DEL phenotype., Conclusion: The data indicate that partial DEL women appear at risk of alloimmunization to the D antigen. RhD immune globulin prophylaxis is necessary for partial DEL women. Partial DEL patients should receive only RhD-negative RBCs, whereas DEL patients with complete expression of antigen can safely receive RhD-positive RBCs., (© 2015 British Blood Transfusion Society.)

Background: Recent retrospective studies indicate that D- recipients of D+ apheresis platelets (PLTs) are not alloimmunized to D. Our hospital policy is to offer RhIG to D- women of childbearing age who received D+ apheresis PLTs but not to other D- recipients of D+ apheresis PLTs. We instituted prospective surveillance of the D- recipients who were not given RhIG., Study Design and Methods: All apheresis PLT recipients were prospectively entered into a database that recorded the patient's age, sex, diagnosis, D status, apheresis PLT transfusions, and antibody screen results from before and after PLT transfusions. Data are reported for PLTs transfused between October 16, 2012, and April 16, 2014, and antibody screens obtained through June 16, 2014. The analysis excludes neonates; women not more than 50 years of age; and patients who also received D+ red blood cells, received only D- PLTs, received RhIG, were previously alloimmunized to D, and did not have a follow-up antibody screen after the first D-incompatible apheresis PLT transfusion., Results: A total of 158 of 1107 apheresis PLT recipients were D-. Seventy-nine were eligible for analysis based on the exclusion criteria listed above. None became alloimmunized to D during the observation interval. In 45 (57%) cases the last follow-up antibody screen was obtained at least 4 weeks after the first D-incompatible apheresis PLT transfusion., Conclusion: Prospective surveillance confirms prior retrospective observations that D- patients do not appear to risk D alloimmunization after receiving D+ apheresis PLTs., (© 2014 AABB.)

Compulsory exposure to genetically foreign maternal tissue imprints in offspring sustained tolerance to noninherited maternal antigens (NIMA). Immunological tolerance to NIMA was first described by Dr. Ray D. Owen for women genetically negative for erythrocyte rhesus (Rh) antigen with reduced sensitization from developmental Rh exposure by their mothers. Extending this analysis to HLA haplotypes has uncovered the exciting potential for therapeutically exploiting NIMA-specific tolerance naturally engrained in mammalian reproduction for improved clinical outcomes after allogeneic transplantation. Herein, we summarize emerging scientific concepts stemming from tolerance to NIMA that includes postnatal maintenance of microchimeric maternal origin cells in offspring, expanded accumulation of immune suppressive regulatory T cells with NIMA-specificity, along with teleological benefits and immunological consequences of NIMA-specific tolerance conserved across mammalian species.

Background: In Pakistan routine blood group typing of thalassemia patients identifies ABO and Rh(D) antigens only. Therefore, other antigen incompatibilities between blood donor and blood recipient may cause alloimmunisation., Objective: The aim of this study was to estimate the frequency of alloimmunisation and to evaluate the risk factors associated with its development in beta (β)-thalassemia patients receiving regular blood transfusions., Materials and Methods: In total 162 β thalassemia patients were included in this study. An extended red cell antigen panel was performed to detect antibodies. Patients received red cell concentrates, which were matched for ABO and Rh(D) antigens. Clinical and laboratory data were collected and analysed to estimate the frequency of alloantibodies and the factors influencing immunisation in patients on regular blood transfusion., Results: The median age of patients was 6·7 (range: 0·5-25) years. A total of 14 (8·6%) patients developed alloantibodies against red cell antigens. The most frequently occurring alloantibodies was anti-E (2·5%), anti-K (1·8%), anti-e (1·2%) and anti-D (0·6%). Five (3·1%) patients developed more than one red blood cell (RBC) alloantibody. Age at first transfusion in alloimmunised patients was 1·22 ± 0·87 years. The frequency of blood transfusion in alloimmunised patients was 23 ± 8·81 days and in those without alloimmunisation was 31·8 ± 16 days (p = 0·02). Logistic regression analysis showed no independent risk factor associated with alloimmunisation., Conclusion: The frequency of transfusion was increased in patients who developed alloantibodies. Typing patients and donors to match for Rh and Kell antigens would prevent more than 90% of RBC alloantibodies and reduce the frequency of transfusion in thalassemia patients., (© 2015 British Blood Transfusion Society.)

Evaluation of fetomaternal hemorrhage (FMH) in the immediate postpartum period is critical for the timely administration of Rh immunoglobulin (RhIG) prophylaxis to minimize the risk of alloimmunization in D-negative mothers of D-positive newborns. We report a series of two clinically-unsuspected cases of massive FMHs identified at our university medical center. Retrospective records of two cases of massive FMH were investigated using the electronic medical record. After positive fetal bleed screens, flow cytometric analysis for hemoglobin F was performed to quantify the volume of the hemorrhages in both cases. Flow cytometric enumeration with anti-D was also performed in one case. The two patients had 209.5 and 75 mL of fetal blood in circulation, resulting in 8 and 4 doses of RhIG administered, respectively. For the former patient, flow cytometric analysis with anti-D ruled out hereditary persistence of fetal hemoglobin and supported the fetal origin of the red cells. Due to the clinically-silent nature of both hemorrhages, further evaluation of the newborns' blood was not performed. These cases highlight the importance of rapidly obtaining accurate measurements of fetal blood loss via flow cytometric analysis in cases of FMH, particularly in clinically-unsuspected cases, to ensure timely administration of adequate immunoprophylaxis to D-negative mothers., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: AABB Standard 5.27 requires transfusion services to have a process for urgent release of blood before completion of compatibility testing. Our institution endorses a policy for the emergency release of group O, D+ red blood cells (RBC; O+ RBC) to males and females at least 50 years of age. Our emergency department (ED) stocks 4 O- RBC units. To determine if O+ RBCs can replace ED O- RBCs, we performed a retrospective review., Study Design and Methods: Patients admitted to the ED between January 2001 and August 2011 and transfused emergency-release O- RBCs were identified. Data were collected on sex, age, length of stay, clinical status, ABO/Rh, RBC transfusions, and RBC antibody screen results., Results: A total of 498 ED O- RBC units were transfused to 268 patients (168 male, 100 female). A total of 322 units were transfused to males and 114 to females at least 50 years of age. Thirty-nine (14%) were D- with 18 receiving O+ RBCs. A total of 109 had follow-up antibody screens; one D- patient developed alloanti-D., Conclusions: The findings support the placement of O+ RBCs in the ED. The majority of ED O- RBCs (88%) went to patients who qualified for O+ RBCs; a minority (1.5%) of patients were D- females less than 50 years of age. The rate of alloimmunization was low., (© 2014 AABB.)

A 5-year-old male with sickle cell disease presented with pain, dark urine, and fatigue 10 days after a red blood cell (RBC) transfusion. Laboratory evaluation demonstrated severe anemia, blood type O+, and anti-D in the serum. Anti-D in a D+ patient led to RH genotyping, which revealed homozygosity for RHD*DAU4 that encodes partial D antigen. Anti-D in this patient whose RBCs exclusively express partial D caused a delayed hemolytic transfusion reaction after exposure to D+ RBCs. The finding of anti-D in a D+patient should be investigated by molecular methods to help distinguish an alloantibody from an autoantibody.

Clinicians sometimes are confronted with the challenge of transfusing haemorrhaging Rhesus (Rh) D negative patients with Rh D positive blood to save their lives. There are concerns about alloimmunization and future haemolytic disease of the newborn in women of the reproductive age. Another fear is transfusion reaction if they receive another Rh D positive blood in future. We present a 32-year-old Rh D negative woman, who had postpartum haemorrhage in her first pregnancy and was transfused with Rh D positive blood because of unavailability of Rh D negative blood. She did not receive anti D immunoglobin but subsequently had a normal term pregnancy of an Rh positive fetus without any detectable anti D antibodies throughout the pregnancy. In life threatening situations from obstetric haemorrhage, transfusion of Rh D negative women with Rh D positive blood should be considered as the last resort.

Purpose: The pharmacologic properties of Rhesus (Rh) immune globulin (RhIG) and clinical data on its effectiveness in preventing Rh-antigen alloimmunization in pregnant women are reviewed., Summary: RhIG is a human plasma derivative that targets red blood cells (RBCs) positive for Rh(O) antigen (also called D antigen). In the United States and other countries, the widespread use of RhIG has markedly reduced the occurrence of hemolytic disease of the fetus and newborn (HDFN), a devastating condition caused by D-antigen sensitization of a pregnant woman via exposure to fetal RBCs (usually during detachment of the placenta in labor) that results in a maternal immune response leading to severe hemolysis in the fetus. Routine administration of RhIG at 26-30 weeks' gestation and again within 72 hours of delivery has been shown to be highly effective in preventing maternal Rh alloimmunization, with very low rates of D-antigen sensitization (in the range of 0-2.2%) reported in multiple studies of at-risk women. The four RhIG products currently available in the United States have common clinical indications but differ in certain attributes. Pharmacists can play an important role in guiding other clinicians on the rationale for the use of RhIG, important differences between products, and appropriate timing of RhIG therapy., Conclusion: Routine administration of RhIG to women at risk for Rh alloimmunization is clinically effective and has made HDFN a rare clinical event. The available RhIG products are not the same and should be carefully reviewed to ensure that they are administered safely., (Copyright © 2015 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

The reported frequency of D alloimmunization in D- recipients after transfusion of D+ platelets varies. This study was designed to determine the frequency of D alloimmunization, previously reported to be an average of 5 ± 2%. A primary anti-D immune response was defined as the detection of anti-D ≥ 28 d following the first D+ platelet transfusion. Data were collected on 485 D- recipients of D+ platelets in 11 centres between 2010 and 2012. Their median age was 60 (range 2-100) years. Diagnoses included: haematological (203/485, 42%), oncological (64/485, 13%) and other diseases (218/485, 45%). Only 7/485 (1·44%; 95% CI 0·58-2·97%) recipients had a primary anti-D response after a median serological follow-up of 77 d (range: 28-2111). There were no statistically significant differences between the primary anti-D formers and the other patients, in terms of gender, age, receipt of immunosuppressive therapy, proportion of patients with haematological/oncological diseases, transfusion of whole blood-derived or apheresis platelets or both, and total number of transfused platelet products. This is the largest study with the longest follow-up of D alloimmunization following D+ platelet transfusion. The low frequency of D alloimmunization should be considered when deciding whether to administer Rh Immune Globulin to D- males and D- females without childbearing potential after transfusion of D+ platelets., (© 2014 John Wiley & Sons Ltd.)

Hemoglobinopathies are significant health problems in Iraq, including its Northern Kurdistan region. One of the essential components of management of these disorders is regular lifelong blood transfusions. The latter is associated with several complications including red cell alloimmunization. No study has looked at the frequency of alloimmunization and its associations in the country. To address the latter issue, 401 multi transfused patients [311 with β-thalassemia (β-thal) syndrome and 90 with sickle cell disease], registered at a large thalassemia care center in Iraqi Kurdistan had their records reviewed, and their sera tested for atypical antibodies using screening and extended red cell panels. Red cell alloimmunization was detected in 18 patients (4.5%) with a total of 20 alloantibodies, while no autoantibodies were detected. The most frequent alloantibody was anti-E, followed by anti-D, anti-K, anti-C(w), anti-C, anti-c and anti-Le(a). Ethnicity was an important predictor of alloimmunization, while age at start of transfusion (>2 vs. ≤2 years) (p = 0.005), Rhesus D (RhD) negative status (p = 0.0017) and history of previous transfusion reactions (p = 0.007) showed a statistically significant higher rate of alloimmunization. However, patients' age, gender, number of units transfused, underlying diagnosis and splenectomy were not significantly associated with alloimmunization. Based on our observations, measures to reduce alloimmunization rates may include extended matching for Rhesus and Kell antigens and early initiation of blood transfusions.

Background: The frequency and molecular basis of the D variants have been reported in the Caucasian and African populations, but relatively little information was known in the Chinese population. Here, a study was investigated in Chinese persons with weak or discrepant D serologic typing., Study Design and Methods: D variant was typed with a serologic method. The full coding regions of RHD of these variants were amplified with polymerase chain reaction and then directly sequenced. RHD zygosity test was performed using the hybrid Rhesus box technique and a multiplex ligation-dependent probe amplification (MLPA) assay was also used to analyze the variant alleles and RHD gene copy number., Results: Twelve distinct RHD mutation alleles were found in 32 D variant individuals, with eight weak D and four partial D alleles. Weak D Type 15 and DVI Type 3 were the major weak D and partial D alleles in Zhejiang Han persons. Three novel weak D alleles (RHD weak D 95A, 779G, and 670G) and one new partial D allele (RHD130-132 del TCT) were identified. The results of RHD zygosity in three individuals disagreed between the RHD zygosity test and the MLPA assay. The most known variant alleles can be detected, but four novel alleles were missed using the RH-MLPA assay., Conclusion: The molecular basis and zygosity of D variants in Zhejiang Han persons were analyzed, and four novel RHD alleles were identified. These data extend the information of D variants and may help to improve the transfusion strategy of the D variants., (© 2014 AABB.)

Background: Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced., Materials and Methods: RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined., Results: We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC., Discussion: In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered.

Background: This study aimed to assess fetomaternal hemorrhage (FMH) among RhD negative pregnant mothers using two techniques, Kleihauer-Betke (KBT) and Flow cytometry (FCM). To determine if patient-specific doses of prophylactic anti-D warrant further investigation in Ethiopia and wider Africa., Methods: Hospital- based cross-sectional study was conducted among 75 RhD negative pregnant mothers using convenient sampling technique., Result: FMH has been detected in 52% and 60% by KBT and FCM techniques, respectively. The volume of FMH quantified in the majority of the cases (92.5% and 87%) was <10 mL fetal blood while >30 mL in 1.3% (1/75) and 2.7% (2/75) as calculated by KBT and FCM, respectively. The FMH calculated by the two methods have good correlation; r = 0.828 (p = 0.000) for categorized and r = 0.897 (p = 0.000) for continuous values and the agreement between the FCM and KBT was moderate with kappa (κ) value of 0.53 (p = 0.000)., Conclusion: Most of FMH calculated (<10 mL) could have been neutralized by lower doses which might have lower costs than administering 300 μg dose which is currently in practice in our country for affording mothers. Besides, it also showed that the volume of FMH was >30 mL in 1.3% and 2.7% of the cases as calculated by KBT and FCM, respectively, which need more than 300 μg dose RhIG for neutralization. Further investigation into the cost- effectiveness and scalability of patient- specific dosing of prophylactic anti-D appears warranted.

Rhesus (Rh) antigens are not expressed on platelets but residual red cells carry the risk of anti-D iso-immunization in transfusion recipients of platelet concentrates (PC). The main theoretical risk associated with this reaction relates to female subjects due to potential obstetrical situations of maternal-foetal Rh incompatibility. Isogroup PC transfusion in this system is therefore advised. However, logistical constraints impose frequent Rh-incompatible transfusions that require the recommendation of anti-Rh immunoglobulin in a girl of childbearing age in this situation. This recommendation, already restricted to a group of patients deserves to be questioned over a decade after being issued. Data from published reports are difficult to interpret because of the heterogeneity of the few series (CP type, immune status, timing of biological tests) but the current techniques for preparing products and most common use of CP apheresis limited the risk of immunization. Moreover, platelet transfusions are particularly relevant to immunocompromised populations which, to what extent (heavy chemotherapy and/or hematopoietic stem cells recipients) seems to be protected from this risk. It is noteworthy that the clinical consequences that may be expected from such immunization are not reported. Although some authors emphasize significant isoimmunization rates (maximum 19%), the heterogeneous conditions and the lack of evidence of clinical consequence suggest evaluating the recommendations or revising them towards more targeted indications of seroprophylaxis., (Copyright © 2014. Published by Elsevier SAS.)

Background: The RhD antigen is very immunogenic and is a significant cause of hemolytic disease of the newborn (HDN). The RHD-CE(8-9)-D hybrid allele is commonly associated with a D- phenotype. Here, we report a case of high-titer maternal anti-D and late onset of HDN in a newborn carrying a RHD-CE(9)-D variant supposedly encoding the same partial D antigen as the RHD-CE(8-9)-D allele, but with significant expression of D antigen., Study Design and Methods: To elucidate the blood group antigen background of the case, we carried out serologic, flow cytometric, and genetics studies of the newborn and his father., Conclusion: Individuals carrying the RHD-CE(9)-D allele do express D antigen, but do so at significantly lower levels than those carrying the more common D+ phenotypes (e.g., DCe/dce). It may mitigate and delay otherwise severe HDN in pregnancies complicated by high-titer anti-D., (© 2014 AABB.)

Background: The D- phenotype is mainly caused by the complete deletion of the RHD gene in Caucasians. However, a plethora of allelic variants have been described among D- individuals from different ethnic groups., Study Design and Methods: A cohort of 1314 routine serologically D- samples from white Argentineans was studied by molecular methods., Results: Among the 1314 D- samples, 2.1% showed RHD-specific amplifications. One hybrid Rhesus box was detected in all D-/RHD+ samples, suggesting a hemizygous status. The RHDΨ was found in 0.7% of rr samples while DEL and null variants were detected in 16.7% of the D- samples expressing C and/or E antigens. The variants associated with the C antigen were seven RHD-CE-D(s) , two RHD(1-2)-CE(2-9)-D(10), two previously unreported RHD(329T>C)-CE(3-9)-D null alleles, one RHD(M295I), and one new RHCE(1-2)-RHD(3361del11 -10) null allele whereas those associated with the E antigen were five RHD(46T>C) and one novel RHD(581insG) null allele responsible for a premature stop codon., Conclusions: The prevalence of D-/RHD+ samples is higher than that observed in Europeans. More than 50% of the RHD alleles found were represented by RHDψ and RHD-CE-D(s) showing the African contribution to the genetic pool of the admixed population analyzed. Interestingly, three new alleles were found, two of them being hybrid structures between previously described RHD variants recombined with RHCE sequences. The knowledge of the RHD allele repertoire in our population allowed the implementation of reliable typing and transfusion strategies for a better management of patients and pregnant women., (© 2014 AABB.)

Background & Objectives: The development of alloantibodies can significantly complicate transfusion therapy and results in difficulties in cross-matching of blood. Most literature on alloimmunization is limited to multitransfused individuals, with very few studies on the general hospital patients. This study was aimed at assessing the frequency and type of unexpected red cell antibodies in the general patient population at a multispecialty tertiary care centre in New Delhi, India., Methods: The results of 49,077 antibody screening tests carried out on patients, from January 2009 to December 2012 were analyzed. The clinical and transfusion records were reviewed. The data were compiled and statistically analysed., Results: A total of 49,077 (29,917; 60.96% males and 19,160; 39.04% females) patient samples were screened for the presence of unexpected antibodies. Antibody screening was positive in 403 patients (0.82%). In the serum samples of 164 patients only autoantibodies were identified, 27 revealed autoantibodies with one or more underlying alloantibodies, while 212 patients had only alloantibody/ies in their serum. The overall alloimmunization rate was 0.49 per cent. Antibodies against the Rh system were the most frequent (64.1%), the most common alloantibody identified being anti E (37.2%), followed by anti D (19.2%)., Interpretation & Conclusions: Since clinically significant antibodies are frequently detected in our patient population, antibody screening and if required, identification is the need of the hour. Since antibodies against the common Rh and Kell blood group antigens are the most frequent, provision of Rh and Kell matched red cells may be of protective value.

Data on blood group phenotypes are important for blood transfusion programs, for disease association and population genetics studies. This study aimed at reporting the phenotypic and allelic distribution of ABO and Rhesus (Rh) groups in various ethnolinguistic groups in the Cameroonians. We obtained ABO and Rhesus blood groups and self-identified ethnicity from 14,546 Cameroonian students. Ethnicity was classified in seven major ethnolinguistic groups: Afro-Asiatic, Nilo-Saharan, Niger-Kordofanian/West Atlantic, Niger-Kordofanian/Adamawa-Ubangui, Niger-Kordofanian/Benue-Congo/Bantu/Grassfield, Niger-Kordofanian/Benue-Congo/Bantu/Mbam and Niger-Kordofanian/Benue-Congo/Bantu/Equatorial. ABO allelic frequencies were determined using the Bernstein method. Differences in phenotypic distribution of blood groups were assessed using the chi-square test; a P value <0.05 being considered as statistically significant. The frequencies of the antigens of blood groups O, A, B and AB were 48.62%, 25.07%, 21.86% and 4.45%, respectively. Rhesus-positive was 96.32%. The allelic frequencies of O, A and B genes were 0.6978, 0.1605 and 0.1416, respectively. Phenotypic frequencies of the blood groups in the general study population and in the different ethnolinguistic groups were in agreement with Hardy-Weinberg equilibrium expectations (P > 0.05). The frequencies of O, A, and B blood phenotypes were significantly lower, respectively, in the Nilo-Saharan group (P = 0.009), the Niger-Kordofanian/Benue-Congo/Bantu groups (P = 0.021) and the Niger-Kordofanian/West-Atlantic group. AB blood group was most frequent in the Niger-Kordofanian/Adamawa-Ubangui group (P = 0.024). Our study provides the first data on ethnic distribution of ABO and Rhesus blood groups in the Cameroonian population and suggests that its general profile is similar to those of several sub-Saharan African populations. We found some significant differences in phenotypic distribution amongst major ethnolinguistic groups. These data may be important for blood donor recruitment policy and blood transfusion service in Cameroon., (© 2014 John Wiley & Sons Ltd.)

Purpose of the Study: Use of matched red blood cell (RBC) concentrates is imperative in patients with RBC allo-antibodies (Abs) and when platelet (PLT) specific allo-Abs are present additional difficulties occur for PLT transfusions. In order to evaluate the prevalence of the PLT and RBC allo-Abs association, a study on patients with PLT specific allo-Acs was performed. This association is not a rare event., Patients and Methods: In the database of a PLT immunohaematology laboratory, patients with PLT specific allo-Abs were selected and the presence and specificity of RBC allo-Abs was evaluated., Results: Six hundred and eighty seven patients (673 females, 14 males) with PLT specific allo-Abs were found. Six hundred and seventy-five patients (98.3%) had PLT specific allo-Abs with only one specificity. Anti-HPA-5b was the most frequent (539 cases). Twenty-nine (4.2%) patients had also RBC allo-Abs, including 27 females (93.1%) and two males. Seventy (58.6%) had RBC allo-Abs with only one specificity, 10 several and two unknown. Among the first, RBC allo-Abs directed against Rhesus blood group antigens were predominant (11 cases [64.7%]). Among the 29 patients with associated PLT and RBC allo-Abs, 15 (51.7%) were 50 or more years old and 14 (48.3%) under 50., Conclusion: In PLT specific alloimmunized patients, detection of RBC alloimmunization is not a rare event. When RBC and PLT transfusions are required, the supply of matched RBC and PLT concentrates is more difficult., (Copyright © 2014. Published by Elsevier SAS.)

Context: Thalassemia is a major health problem in Egypt. Red blood cell alloimmunization is an important complication in transfusion-dependent patients., Objectives: To determine alloimmunization prevalence in Egyptian patients with β-thalassemia and to evaluate risk factors that could influence alloimmunization, with the hope of minimizing transfusion-associated risks in those patients., Design: Records of 272 patients with β-thalassemia who are receiving regular blood transfusions, matched for ABO-Rh(D), were analyzed. Alloantibody identification was performed by DiaMed-ID microtyping system. Autoantibodies were detected by direct Coombs test., Results: Alloimmunization incidence was 22.8% with 123 alloantibodies detected in 62 patients. The most common alloantibody was Rh-related (37.4%; 46 of 123), comprising anti-E (14.6%; 18 of 123), anti-D (8.9%; 11 of 123), anti-C (8.9%; 11 of 123), and anti-c (4.9%; 6 of 123), followed by anti-Kell (26%; 32 of 123), → anti-MNS (9.8%; 12 of 123), → anti-Kidd (8.9%; 11 of 123) → anti-Duffy (8.1%; 10 of 123), → anti-Le (5.7%; 7 of 123), → anti-Lu (2.4%; 3 of 123), and → anti-P1 (1.6%; 2 of 123). Anti-D antibodies developed in 34.5% of all Rh-negative patients. Eighty percent of all anti-D antibodies developed in patients older than 18 years. Males had the highest alloimmunization incidence. Alloimmunization incidence increased with the number of units transfused (P = .01). Patients who received unfiltered blood had a higher alloimmunization rate than did those who always received leukoreduced blood (P < .001). Splenectomized patients had a higher alloimmunization rate (32%; 40 of 125) than did those who did not have a splenectomy (16.3%; 24 of 147; P = .003). Autoantibodies occurred in 1.5% (4 of 272) of all patients., Conclusion: Transfusion of leukoreduced and phenotypically matched cells for selective antigens may help reduce expenses and risks of alloimmunization in patients with thalassemia.

Objective: To estimate the potential economic benefit of reduced indirect antiglobulin screening for Rh(D)-negative pregnant women., Methods: A chart review of all Rh(D)-negative mothers delivering at the University of Washington from 2002 to 2012 was conducted to determine the rate of gestational seroconversion to anti-D antibodies before 28 weeks of gestation. A decision tree was constructed to estimate the economic effects of eliminating the indirect antiglobulin screen at 28 weeks of gestation and instead immunizing all Rh(D)-negative, anti-D antibody-negative women with anti-D immune globulin at that time. A theoretical cohort of 100,000 women was modeled. Probabilities and costs were derived from published literature, chart review, and expert opinion. Univariate sensitivity analyses followed by a Monte Carlo analysis examined assumptions and uncertainties in our model across entire distributions., Results: The seroconversion rate of development of anti-D antibodies before 28 weeks of gestation in the cohort analyzed was 0.099% (2/2,029 women). From a societal perspective, the expected cost savings from implementing the reduced indirect antiglobulin screening strategy, per 100,000 women, ranged from $6 to $7.7 million. The overall cost savings for implementing this strategy in the United States for 1 year ranged from $34.7 to $35.6 million. This strategy remained cost-beneficial when varying our parameters (eg, anti-D immune globulin, antibody test cost) to their logical extremes. The Monte Carlo analysis verified the cost savings of our strategy., Conclusion: The updated seroconversion rate and our model suggest that eliminating the 28-week antibody screen would be cost-beneficial from a societal perspective while posing minimal potential harm to the recipients.

D variants, collectively called DEL, express trace amounts of D antigen which is considered to be serologically detectable only by adsorption-elution techniques. We detected six cases of DEL phenotype by indirect antiglobulin test, in Dalmatia County of Croatia by routine serological testing of D antigen of new blood donors. RH genotyping found that all six donors carry allele RHD(M295I), RH genotype CcDdee. D antigen densities of D variants were very low, between 26 and 44 D antigens per red blood cell. The frequency of D variants detected by IAT allele RHD(M295I) was 1:272 in D negative donors. Obviously, DEL phenotype is more common in some parts of European population than initially thought. In conclusion, our routine serological testing of D antigen can detect extremely weak D antigen, even RBCs with DEL phenotype and antigen density as low as 26 D antigens per RBC., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions., (© 2013 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.

Objective: To determine transfusion compatibility of maternal RBCs for her neonate up to 4 weeks of age, irrespective of maternal-neonatal ABO mismatch., Methods: This was a prospective observational study involving eligible mothers with their neonates delivered in participating site from 1 July 2012 till 31 December 2012. Mother's blood was collected before child birth. Neonatal blood sample was collected from placental end, shortly after birth. Blood Groups of mother-baby pair were individually tested for ABO and Rh-D groups. Pairs with negative Rh-D antigen/s or with same ABO blood groups were excluded. Thus, 28 pairs with both maternal and neonatal samples positive for Rh-D antigen and with different maternal neonatal ABO blood groups were included in the study. Blood samples were collected at birth and at 4 weeks. Cross matching was done at birth and at 4 weeks for each pair with standard blood bank protocols., Results: All 28 pairs showed positive compatibility with standard blood bank cross-matching protocols at birth and at 4 weeks., Conclusions: Maternal blood irrespective of ABO compatibility might be a viable and potentially acceptable option for her new born baby in neonatal period. This may be especially important in developing world with limited blood bank resources.

Aim: To estimate the current incidence of maternal sensitisation to Rh(D) and examine reasons for prophylaxis failures., Method: Retrospective chart review of new sensitisations to Rh(D) detected in antenatal records, between 2005 and 2012 in Christchurch, New Zealand and systematic examination of circumstances likely to have caused prophylaxis failures., Results: Fifty-four new sensitisations in an at-risk population of about 4624 in 8 years means an incidence of roughly 1.1%. In 86.6% of 45 sensitisations where information was available, there was a recognised sensitising event including previous deliveries while in 13.3% there were none. Of those with recognised sensitising events, 46.1% had anti-D prophylaxis per local guidelines, in 12.8%, prophylaxis was given though it did not conform, entirely, to guideline. No prophylaxis at all was given to 41% despite a sensitising event being recognised., Conclusion: The incidence of maternal sensitisation to Rh(D) in Christchurch, New Zealand, is as expected given our prophylaxis regimen. Half the sensitisations were associated with complete or partial failure to follow local guidelines. Better adherence to this may reduce incidence of sensitisation. It is also thrice as high as might be expected with a routine antenatal anti-D prophylaxis (RAADP) program. An economic analysis of RAADP in New Zealand will be useful.

In an immunohematology reference laboratory, samples received for antibody identification react in many different ways requiring a variety of approaches. Sometimes, the clues from initial testing can lead to faulty assumptions and misdirection. Fortunately, a well-supplied reference laboratory will have access to a variety of techniques and reagents that, when used together, can reveal the true identity of the antibodies involved. We present a case of a patient sample with an apparent group AB, D+ blood type showing strong reactivity with all cells tested in the forward and reverse ABO, in the D testing as well as in a three-cell antibody screen. The initial assumption was that the plasma contained a cold autoantibody. Subsequent testing, including the use of gel column technology, ficin-treated cells, and antisera for phenotyping, showed the apparent cold autoantibody to bea red herring. Additional tube testing at immediate spin, 37°C,and indirect antiglobulin test (IAT) revealed the presence of four alloantibodies: anti-M and anti-E reacting at immediate spin, 37°C, and IAT plus anti-Fy(a) and anti-Jk(b) reacting at lAT.

To conserve D- red blood cells (RBCs), our facility developed a policy for transfusion of D+ units to D- patients, particularly in trauma situations. To our knowledge, this is the first study looking at D-mismatched RBC transfusion in trauma patients. We developed guidelines for the transfusion of D-mismatched RBCs. patients were followed by anti body screening and direct antiglobulin testing. Twenty-six patients were identified, and 57.7 percent of the cases were the result of trauma. Follow-up ranged from 7 to 455 days. The trauma cohort had a follow-up of 7 to 102 days. Overall, patients were transfused with 1 to 36 units of D-mismatched RBCs. Three patients produced alloanti-D, resulting in a 20 percent rate of antibody formation rate of 11.5 percent compared with 21.4 to 30.4 percent in previous studies. Approximately 58 percent of our study population was composed of trauma patients. Within that cohort, the formation of anti-D was 20 percent, similar to the rate seen in previous studies looking at primarily non-trauma populations.

Background: There is an ongoing controversy regarding provision of usually matched blood (i.e. matched for ABO-D antigens) or phenotypically matched blood (also matched for Rh and Kell antigens) for multiply transfused thalassaemics, especially in developing countries. A pilot study conducted at our centre revealed an alloimmunisation rate of 3.79% with Rh and Kell alloantibodies accounting for 90% of all antibodies. The present cross-sectional study was conducted to assess the impact of a policy of partial better matching (for Rh cDE and Kell antigens) of blood on alloimmunisation in thalassaemics., Material and Methods: In this cross-sectional study three groups of patients were considered. Group 1 comprised 211 thalassaemics who received usually matched (UM) blood until April 2009. Their rates of alloimmunisation have already been published in a prior study. Group 2 consisted of 46 thalassaemics who were enrolled after April 2009 and have received partially better matched (PBM) blood (matched for ABO, Rh cDE and Kell antigens) since the initiation of transfusion therapy. Group 3 (UM→PBM) comprised the patients from group 1 who, from April 2009, were given partial better matched blood. Antibody screening (using a 3-cell panel) and antibody identification (11-cell panel) were carried out to detect the presence of alloantibodies., Results: None of the thalassaemic patients in group 2 (PBM) developed alloantibodies. Eight thalassaemics in group 3 (UM→PBM) developed new alloantibodies (after April 2009)., Discussion: According to the results of the present study, providing at least partially better matched blood appears to improve the efficacy of transfusion for chronically transfused thalassaemics. Large-scale, comprehensive, multicentre studies need to be carried out to formulate realistic, evidence-based, economically feasible transfusion policies for thalassaemic children based on the red blood cell antigen profile of the population.

Objective: To determine the antenatal course of severe red cell alloimmunisation in pregnancies requiring intrauterine fetal transfusion., Study Design: A retrospective cohort study over 16 years in a single national quaternary fetal medicine centre. From 1996 to 2011, 242 red cell intrauterine transfusions (IUT) were performed in 102 alloimmunised pregnancies. Antibody type was categorized into Rh(D) and non-Rh(D) (including Rh(c), Kell and Rh(E)). Women with Rh(D) antibodies were further stratified into those with and without additional red cell antibodies. Data were compared using the Mann-Whitney U and Fisher's exact tests. Two-tailed P values at the 5% level were considered significant., Results: Comparing Rh(D) and non-Rh(D) pregnancies, there were no differences in either gestational age or fetal haemoglobin at first IUT, number of transfusions required, gestation at delivery, caesarean delivery rates or perinatal losses. In women sensitized to Rh(D), the presence of additional antibodies did not influence the degree of fetal anaemia or the first transfusion-delivery interval, although rates of fetal hydrops were higher in the presence of multiple antibodies. The "procedure-related" loss rate was 1.7% per procedure in our institution., Conclusion: Antibody status does not appear to influence clinical outcomes following fetal transfusion for alloimmunisation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: As cord blood (CB) is being used frequently as a source for heamtopoetic stem cell transplantation defining risk factors for transplantation outcome is an important issue., Procedure: The data of all single unit CB transplantation preformed in Israel from 1992 to 2011 were collected. The risk factors for myeloid engraftment, event free survival (EFS) and overall survival (OS) were studied in 87 children., Results: There were 49 children with hematological malignancies and 38 with non-malignant diseases. Cumulative rate of neutrophil recovery was 78.3%, while median time to myeloid recovery was 26 days. The incidence of platelet engraftment at 150 days was 53%, and the median time to platelet recovery was 36 days. ABO blood group matching between CB unit and recipient was associated with superior myeloid engraftment. Acute graft versus host disease of grades II-IV occurred in 33% of the patients. Chronic graft versus host disease occurred in 16% of patients. Probabilities of EFS and OS at 1 year were 45% and 57%, respectively. Factors associated with inferior OS were Rh major mismatch versus matched Rh and transplantation from unrelated donor versus related donor., Conclusions: These results indicate that matching of ABO blood groups is an important factor that affects engraftment, and also that Rh matching seem to have an impact on OS, which was not previously described in the setting of CB transplantation., (© 2013 Wiley Periodicals, Inc.)

Background: The D typing strategies in several European countries protect carriers of D category VI (DVI) from anti-D immunization but not carriers of other partial D. Besides DVI, one of the clinically most important partial D is D category IV (DIV). A detailed description and direct comparison of the different DIV types was missing., Study Design and Methods: RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels., Results: DIV comprises several variants of the D antigen with distinct serology, molecular structures, evolutionary origins, and ethnic prevalences. The DIV phenotype is determined by 350H shared by all, but not limited to, DIV variants which are further divided into DIVa and DIVb. The DIVa phenotype is expressed by DIV Type 1.0 harboring 350H and the dispersed amino acids 62F, 137V, and 152T. The DIVb phenotype is expressed by DIV Type 3 to Type 5 representing RHD-CE-D hybrids. Four of the six postulated DIV variants were encountered among 23 DIV samples analyzed. Of 12 DIV carriers with anti-D, 10 were female and seven likely immunized by pregnancy. Two DIV-related alleles are newly described: DWN, which differs from DIV Type 4 by 350D and epitope pattern. DNT carries 152T, known to cause a large D antigen density., Conclusion: DIV alleles arose from at least two independent evolutionary events. DIV Type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV Type 2 to Type 5 with DIVb phenotype arose from more recent gene conversions. Anti-D immunization, especially dreaded in pregnancies, will be avoided not only in carriers of DVI but also in carriers of other D variants like DIV, if our proposed D typing strategy is adopted., (© 2013 American Association of Blood Banks.)

Rhesus (Rh) hemolytic disease of the newborn represents a broad spectrum of symptoms in the fetus and newborn, ranging from mild to severe hemolytic anemia and hydrops fetalis. Cholestasis is a common problem in infants with immune hydrops fetalis (IHF). The aim of this study was to evaluate the incidence and course of cholestasis in infants with IHF due to Rh alloimmunization. Infants with IHF during the 10-year follow-up were retrospectively included in the study. Demographics, laboratory parameters, and prenatal and postnatal interventions were recorded. The incidence of cholestasis and certain risk factors were investigated. A total of 30 infants with IHF with a mean gestational age 33 ± 2.6 weeks were included. Of these, 15 infants (50%) survived to discharge. The incidence of cholestasis was 60% (18/30). Cholestasis was diagnosed within a median 3 (0-7) days. All cholestatic infants who survived recovered within three months. In conclusion, cholestasis in IHF is frequent, transient and has an early onset.

Background: Molecular variant RHD allele analysis is best complemented by detailed characterization of the associated D phenotype., Study Design and Methods: Variant D types were characterized using molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen quantification., Results: We discovered three novel weak D types termed weak D Types 45.1, 75, and 76 with RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches; antigen densities of approximately 1.990, 900, and 240 D sites per red blood cell were found, respectively. Adsorption-elution technique-supported D epitope mapping of these three weak D types demonstrated the expression of all tested D epitopes. Initial molecular typing of the three investigated samples by RHD gene exon scanning polymerase chain reaction using sequence-specific priming yielded a negative reaction for A1193 located in RHD Exon 9 and could be explained by specific mutations for weak D Types 45.1 (C818T, G1195A), 75 (G1194C), and 76 (A1215C)., Conclusion: All novel weak D types expressed all tested D epitopes. It is of interest that for weak D Types 45.1, 75, and 76, similar alleles with a maximal divergence of one amino acid only, that is, weak D Types 45, 41, and 68, respectively, have been reported so far., (© 2013 American Association of Blood Banks.)

Background: Rh discrepancies are a problem during routine testing because of partial and weak D phenotypes. Some blood units with weak and partial D expression may escape detection by serology. Limitations of serology can be overcome by molecular typing. The objective of study was to compare currently used serologic methods with molecular analysis to determine the potential application of molecular methods to improve D typing strategies and to estimate the frequency of weak D types among the Arab population., Study Design and Methods: Fifty blood donor and patient samples with discrepant results of D phenotyping were subjected to routine serology to define the D phenotype including monoclonal anti-D immunoglobulin M and indirect antiglobulin test. Commercially available panels of monoclonal anti-D were used for identification of partial D and weak D phenotypes. Genomic DNA was evaluated using allele-specific amplification polymerase chain reaction with sequence-specific primers to define weak D type., Results: Molecular typing confirmed most of the serology results; three samples that were not clear-cut serologically were identified by molecular typing, two samples as weak D Type 4.2 (DAR), and one sample as weak D Type 4.0. Another two samples identified by serologic panel as weak D were unresolved by molecular typing. A sample with partial D Type II by serology revealed a Weak D Type 4.0 by molecular typing. Results interestingly showed the high frequency of weak D Type 4.2 (DAR) in Egypt., Conclusion: RHD molecular typing can solve discrepancies during routine testing due to partial and weak D phenotypes for better transfusion outcome., (© 2013 American Association of Blood Banks.)

Background: Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D- by standard serology hence permitting incompatible transfusion to D- recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D- Polish donor population and to characterize its molecular background., Study Design and Methods: Plasma pools collected from 31,200 consecutive Polish donors typed as D- were tested by real-time polymerase chain reaction (PCR) for the presence of RHD-specific markers located in Intron 4 and Exons 7 and 10. RHD+ individuals were characterized by PCR or cDNA sequencing and serology., Results: Plasma cross-pool strategy revealed 63 RHD+ donors harboring RHD*01N.03 (n = 17), RHD*15 (n = 12), RHD*11 (n = 7), RHD*DEL8 (n = 3), RHD*01W.2 (n = 3), RHD-CE(10) (n = 3), RHD*01W.3, RHD*01W.9, RHD*01N.05, RHD*01N.07, RHD*01N.23, and RHD(IVS1-29G>C) and two novel alleles, RHD*(767C>G) (n = 3) and RHD*(1029C>A). Among 47 cases available for serology, 27 were shown to express the D antigen, Conclusion: 1) Plasma cross-pool strategy is a reliable and cost-effective tool for RHD screening. 2) Only 0.2% of D- Polish donors carry some fragments of the RHD gene; all of them were C or E+. 3) Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D- recipient., (© 2013 American Association of Blood Banks.)

Background: The partial D variant DAR1 (weak D Type 4.2) is caused by three single-point mutations, 602C>G, 667T>G, and 1025T>C. Here we report a molecular study on different D variants belonging to the DAR category (DAR1, DAR1.2, DAR1.3, and DAR2) and their serologic data., Study Design and Methods: A total of 42 samples belonging to the DAR category were screened for the presence of the silent mutations 744C>T and 957G>A. The samples were phenotyped for RhD and RhCE, characterized for RhD epitope expression, and sequenced for RHD exons. Flow cytometry was performed to determine RhD antigen density., Results: The silent mutation 744C>T was found in all six samples previously typed as RHD*DAR2 (602C>G, 667T>G, 957G>A, 1025T>C). In addition to the three nucleotide changes originally reported for the RHD*DAR1 allele, the silent mutations 744C>T and 957G>A were found in 14 of 16 samples previously typed as RHD*DAR1. In the remaining two samples one additional silent mutation, 744C>T, was found. Serologically the DAR1.2 and DAR1.3 samples analyzed in this study showed no distinct difference in their anti-D reaction pattern compared to each other. The anti-D reaction pattern of DARA/DAR2 showed some distinct differences compared to those of DAR1.2 and DAR1.3., Conclusion: RHD*DARA and RHD*DAR2 are the same allele. Furthermore, the alleles RHD*DAR1.2 and RHD*DAR1.3 both exist; however, the silent mutation 957G>A (V319) showed no influence on the RhD phenotype., (© 2013 American Association of Blood Banks.)

Background: Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening., Methods: Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104)., Results: The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10-39, n = 1317)., Conclusion: The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.