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Background: Immunohematology skill education is an important part of the transfusion medicine professional training. We tried to solve the difficulty of obtaining suitable and sufficient positive samples in the immunohematology education., Methods: Different identification panels and panel cells were created by RhD-positive red blood cells (RBCs) and RhD-negative RBCs, according to the underlying antibodies. Diluted anti-D reagent was used as simulated plasma for identification., Results: The antibody identification of single antibody with dose-effect and two antibodies present at the same time were successfully simulated., Conclusions: It is a practical and cheap method for antibody identification training to use RhD blood group, especially when positive samples are short.

Objective To analyze the serological characteristics and clinical significance of IgG anti-D and anti-C combined antibodies and anti-E and anti-c combined antibodies in patients negative for RhDC and RhEc antigens. Methods The clinical data and laboratory results of 12 cases with two types of irregular antibodies were recorded and analyzed, including age, sex, history of blood transfusion/pregnancy, ABO and RhD blood group identification, Rh antigen typing, irregular antibody screening, antibody-specific identification, absorption-elution tests, antibody titer determination and cross-matching tests. Results Among the 12 patients, the mean age was 51.4±16.9 years. Nine patients had a history of blood transfusion; eight patients had a history of pregnancy; five patients had both. Serological tests showed positive antibody screening and incompatible cross-matching. The results of antibody-specific identification and absorption-elution tests showed the presence of both IgG anti-D and anti-C antibodies in three patients, with anti-D titers at 16-32, and anti-C titers at 8-16. Nine patients had both IgG anti-E and anti-c antibodies, with the titers of anti-E and anti-c antibodies at 8-16. From the patients with combined anti-D and anti-C antibodies, suspended red blood cells of ABO identical type, RhD negative and other Rh antigens as ccee were selected. From patients with combined anti-E and anti-c antibodies, suspended red blood cells of ABO identical type, RhD positive and other Rh antigens as CCee were selected. Cross-matching blood test results showed no agglutination or hemolysis in saline, polycoagulant and anti-human globulin media. Conclusion Blood transfusion and/or pregnancy are the primary causes of irregular antibodies in two Rh systems, leading to positive antibody screening and cross-match incompatibility. Routine compatibility transfusion of Rh antigens, based on ABO homotypic blood transfusion, is of great value and significance for the safety of clinical blood transfusion and the prevention of hemolytic disease of the newborn.

Maternal alloantibodies directed against fetal red blood cell (RBC) antigens may cause potentially life-threatening haemolytic disease of the fetus and newborn (HDFN). Dutch transfusion guidelines therefore prescribe preventive cEK matching for all (pre-)fertile females. To quantify the impact of cEK matching, we compared overall and antigen-specific cumulative RBC alloimmunisation incidences in females and males aged <45 years. Among a multicentre cohort comprised of patients who received their first and subsequent RBC unit between 2005 and 2019, first-formed RBC alloantibodies were detected in 47 of 2998 (1·6%) females and 49 of 2507 (2·0%) males. Comparing females and males, overall alloimmunisation incidences were comparable (3·1% [95% confidence interval (CI) 2·1-4·4] versus 3·5% (95% CI 2·4-4·9, P = 0·853) after 10 units transfused). However, cEK alloimmunisation incidences were significantly lower among females (0·6% (95% CI 0·3-1.5) versus 2·2% (95% CI 1·5-3·4, P = 0·001) after 10 units transfused). Yet, despite cEK-matching guidelines being in effect, 6·5%, 3·6% and 0·2% of all RBC units remained mismatched for c, E or K antigens respectively. Most of these mismatches were almost always due to emergency settings. Even though cEK alloimmunisation was not prevented completely, implementation of cEK matching resulted in an alloantigen-exposure risk reduction of up to 98%., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Platelets express ABO antigens and are collected in plasma, which contains ABO antibodies as would be consistent with the donor ABO group. Platelet ABO antigens that are incompatible with recipient ABO antibodies may have accelerated clearance from circulation and result in lower count increments. ABO antibodies that are passively transferred from donor plasma may result in hemolysis of recipient red blood cells. Although platelets do not express Rh antigens, they contain small numbers of intact red blood cells or fragments, which can lead to alloimmunization in the recipient. Alloimmunization to the RhD antigen may occur when platelets obtained from RhD-positive donors are transfused to RhD-negative recipients. All of these compatibility considerations must be balanced against the available supply, which may be limited due to the 5- to 7-day shelf life of platelets. This articles describes considerations for platelet ABO and RhD selection for platelet transfusions, including the impact of major ABO incompatibility on count increments, the risks of hemolysis associated with minor ABO incompatibility, and the risk of RhD alloimmunization when RhD-negative patients receive platelets obtained from RhD-positive donors., Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests., (© 2020 by The American Society of Hematology.)

Although the electronic cross-matching of blood has been widely applied in some developed countries and regions, concern over the risk of undetected red blood cell (RBC) antibodies has limited its application in mainland China.This study was performed to explore the missed detection rate of RBC antibodies in a Chinese population from 2011 to 2016. If the results of 2 consecutive tests of ABO/RhD blood group identification were consistent and antibody screening results were negative, electronic cross-matching of the blood was performed. In addition, traditional serological cross-matching of blood (polybrene method) and a parallel experiment for electronic cross-matching of blood were performed to analyze the missed detection of unexpected RBC antibodies in blood donors and recipients.Using the polybrene method, 40,228 blood samples were tested by parallel traditional serological cross-matching of blood; among these samples, blood compatibility was found in 40,222 cases, primary incompatibility (incompatibility of the donor's erythrocytes with the recipient's serum) was found in 6 cases, and no secondary incompatibility was found. Identification of antibody specificity was performed using panel cells, and all unexpected RBC antibodies were confirmed as anti-Mur alloantibodies in the MNS system.Further improvements in the erythrocyte antigenic spectrum, especially the Mur antigen in Asian populations, are expected to ensure the safety of implementing electronic cross-matching in China.

RhD negative pregnant women who carry an RhD positive fetus are at risk of immunization against the D antigen, which may result in hemolytic disease of the fetus and the newborn. Predicting the fetal RhD status by noninvasive antenatal screening for the fetal RhD gene (RHD) can guide targeted use of antenatal anti-D prophylaxis.Cell-free fetal DNA is extracted from maternal plasma from RhD negative pregnant women at a gestational age of 25 weeks. A real-time PCR-based detection of two RHD exons enables reliable prediction of the fetal RhD status to determine the administration of antenatal prophylaxis, as well as postnatal prophylaxis.

Background: Resolving ambiguous results of D antigen typing is crucial for appropriate and rational administration of anti-D immunoprophylaxis and transfusion practice in obstetric population. The aim of the study was to establish selection criteria of anti-D reagents for our population., Methods: A total of 12 689 samples from primiparous women in Split-Dalmatia County, Croatia, were typed for RhD antigen during the period of 5 years. Ambiguous results were submitted to additional serologic investigation and genotyping. RHD genotyping was performed by commercial genotyping kits (Ready Gene weak D ® and Ready gene CDE, Inno-Train, Kronberg, Germany). Relative frequencies and accompanying 95% confidence intervals were used to estimate the prevalence of variants., Results: The prevalence of D variants was 0.42% (95% CI 0.31; 0.53). The most common partial D variant was D Va (RHD*05.05), with the prevalence of 0.08% (95% CI 0.03; 0.13). All weak D variants were weak D types 1, 2 and 3 (RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3). Weak D samples were distinguishable from partial D in routine typing due to the difference in reactivity of partial D samples with clones D7B8 and RUM-1. Cell line RUM-1 gives weak or negative reactions with partial DVa category., Conclusion: The most common partial D variant in our population is DVa. It is recommended to use cell lines which do not strongly agglutinate DVa variant in routine RhD typing. The appropriate choice of reagents will enable the serology methods to recognize the cases in which RHD genotyping is required., (© 2017 Wiley Periodicals, Inc.)

ABO subtypes and RhD variants are the most studied blood groups in China. Some of the polymorphisms in these two blood groups have direct clinical relevance. Molecular diagnosis of blood group polymorphisms is underway in China. In addition, research groups have developed methods such as screening for blood group mimetic peptides using phage display technology. New reagents, akin to antibodies directed against RhD and ABO, are being investigated using aptamer-based techniques. Progress is also being made in the development of synthetic exoglycosidases for conversion of group A and/or B antigens to group O. Development of methoxy-polyethylene-glycol modified red cells has been successful in vitro but has not reached clinical application. In this paper, we summarize red cell immunohematology research that has been conducted in China., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically depends on results of serologic D typing. Interpretation and follow-up of weak D serology is variable. Recent recommendations promote genotyping for RHD status determination in those with weak D serology. Canadian Blood Services performs comprehensive serologic prenatal testing in four provinces. Genotyping is used to determine D typing in patients with weak D., Study Design and Methods: A serologic algorithm identified which patients require genotyping for RHD determination. Genotyping was performed on one of two commercially available platforms., Results: Only 0.4% of D- patients met criteria for genotyping. Sixty-one percent were weak D Type 1, 2, or 3. Thirty percent had a partial or weak D other than Type 1, 2, or 3. Eleven had variants which remained unresolved. Seventeen were D+ and four were D-., Conclusions: Genotyping of patients with weak D serology led to an identified genotype in most patients. RhIG administration was avoided in 66% who were weak D Type 1, 2, or 3 or were D+. The use of a serologic algorithm to select patients for RHD genotyping identifies a majority of patients with weak D types not at risk for alloimmunization. This approach limits the number of genotyping investigations and the cost of providing classification for weak D types., (© 2016 AABB.)

Background: The knowledge of D variants in patients and donors is important because anti-D alloimmunization can occur in some but not all individuals who express a variant RHD allele. Serologic distinction of RhD discrepancies is not always straightforward, which makes molecular analysis highly desirable., Methods: A group of 223 subjects, 129 patients, and 94 blood donors was identified and analyzed on the basis of a D typing discrepancy. The D antigen expression was evaluated by tube and gel hemagglutination with four anti-D reagents. PCR-single specific primer (SSP), multiplex PCR, RHD BeadChip (Immucor), or sequencing were used for molecular analysis., Results: In total, 168/223 (75%) weak D and 55/223 (25%) partial D variants were identified. Hemagglutination results varied in methods and anti-D reagents used in this process. There was no standard serologic reactivity identified, which could predict what type of D variant would be identified. Among weak D samples, types 1-3 were the most common, while DAR and DVI were most prevalent among partial D samples., Conclusion: Our results show that discrepancies found in the serologic typing should be investigated by molecular methods in order to determine the D variant involved and also to distinguish between weak D and partial D. The knowledge of the distribution of weak D types and partial D among populations is important for D- patients and pregnant women management., (© 2016 Wiley Periodicals, Inc.)

We present a novel methodology for automated ABO Rh-D blood typing using simple morphological image processing algorithms to be used in conjunction with a fabric strip based rapid diagnostic test. Images of the fabric strip post testing are acquired using low cost mobile phones and the proposed algorithm proceeds to automatically identify the blood type by processing the images using steps comprising of noise reduction, range filtering and empirically derived heuristics. The ultimate goal is to provide a simple mobile phone application to enable automated, rapid and accessible blood type detection at the point-of-care.

The accurate and reliable typing of blood groups is essential prior to blood transfusion. While current blood typing methods are well established, results are subjective and heavily reliant on analysis by trained personnel. Techniques for quantifying blood group antibody-antigen interactions are also very limited. Many biosensing systems rely on surface plasmon resonance (SPR) detection to quantify biomolecular interactions. While SPR has been widely used for characterizing antibody-antigen interactions, measuring antibody interactions with whole cells is significantly less common. Previous studies utilized SPR for blood group antigen detection, however, showed poor regeneration causing loss of functionality after a single use. In this study, a fully regenerable, multi-functional platform for quantitative blood group typing via SPR detection is achieved by immobilizing anti-human IgG antibody to the sensor surface, which binds to the Fc region of human IgG antibodies. The surface becomes an interchangeable platform capable of quantifying the blood group interactions between red blood cells (RBCs) and IgG antibodies. As with indirect antiglobulin tests (IAT), which use IgG antibodies for detection, IgG antibodies are initially incubated with RBCs. This facilitates binding to the immobilized monolayer and allows for quantitative blood group detection. Using the D-antigen as an example, a clear distinction between positive (>500 RU) and negative (<100 RU) RBCs is achieved using anti-D IgG. Complete regeneration of the anti-human IgG surface is also successful, showing negligible degradation of the surface after more than 100 regenerations. This novel approach is validated with human-sourced whole blood samples to demonstrate an interesting alternative for quantitative blood grouping using SPR analysis., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

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: 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.

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.)

Flow cytometry was previously applied for analysis of Rh(D) antigen density, therefore it was suggested that the flow cytometry may be used for routine detection of weak D positive phenotypes. This study was purposed to evaluate its practicability. Six weak D positive and 7 DEL individuals were detected by using saline, IAT and absorption/elution test from 2010 to 2011 years. By RHD genotyping, zygosity analysis and sequencing, 3 cases of weak D type 15, 3 cases of partial D type DVI-III and 7 cases of DEL carrying RHD1227A alleles were identified. Taking 2 normal Rh(D)-positive and 2 D-negative samples as controls, all the samples were tested by using flow cytometry, and the median fluorescence intensities were observed as well. The results indicated that all weak D type 15 and partial D type DVI samples were detected to be positive by flow cytometry, as compared with 2 Rh(D)-negative samples (P < 0.05). Seven 7 DEL samples were tested to be negative (P > 0.05), although one of 7 DEL was tested as "±" in IAT and strong positive in absorption/elution. The RHD zygosity analysis showed this DEL individual as RHD(+)/RHD(+) homozygote. It is concluded that the sensitivity of detecting D antigen by flow cytometry is similar to that of IAT, but lower than absorption/elution test. As for detecting weak D or partial D antigens, IAT is easier than flow cytometry; as for identifying DEL, the flow cytometry is not sensitive enough.

Background: Titration is a semiquantitative method used to estimate red blood cell (RBC) alloantibody reactivity. The conventional tube test (CTT) technique is the traditional method for performing titration studies. The gel microcolumn assay (GMA) is also a sensitive method to detect RBC alloantibodies. The aim of this study was to compare a GMA with the CTT technique in the performance of Rh and K alloantibody titration., Study Design and Methods: Patient serum samples that contained an RBC alloantibody with a singular specificity were identified by routine blood bank workflow. Parallel titration studies were performed on these samples by both the CTT method and a GMA (ID-Micro Typing System anti-IgG gel card, Micro Typing Systems, Inc., an Ortho-Clinical Diagnostics Company)., Results: Forty-eight samples were included, including 11 anti-D, five anti-c, 13 anti-E, one anti-C, three anti-e, and 15 anti-K. Overall, the two methods generated identical results in 21 of 48 samples. For 42 samples (87.5%) the two methods generated results that were within one serial dilution, and for the remaining six samples, results were within two dilutions., Conclusion: GMA systems may perform comparably to the CTT in titrating alloantibodies to Rh and Kell antigens., (© 2012 American Association of Blood Banks.)

Because of the variability of D expression, one method may be inadequate to correctly classify donors with variant RHD alleles. We evaluated the use of a solid -phase automated platform (ImmucorGamma Galileo) to confirm D- test results obtained on first-time donors on the Beckman Coulter PK7300 automated microplate test system. Samples with discordant results were analyzed by serologic tube methods, RHD genotyping using the BLOODchip platform (Progenika) and, if necessary, sequencing. We estimated the number of cases of alloimmunization in women younger than 50 years likelyto be prevented by the addition of Galileo testing. From May 2011 to May 2012, 910,220 donor samples were tested; 15,441 were first-time donors with concordant D- results. Five donors tested D- on the PK7300 and weak D+ on the Galileo; one was found to be a false positive on further testing. On manual testing, the other four donors had positive indirect antiglobulin test results with one to three of the antisera used and were C+. On BLOODchip testing, two donors were classified as D+, and two were assigned a "no call". D variants included weak D type 67, weak D type 9, and two novel variants. Approximately 10 percent of D- units are transfused to women younger that 50 years. Assuming an alloimmunization rate of 30 percent, use of the Galileo would prevent approximately one alloimmunization every 5 to 6 years in this patient group. We conclude that the yield of preventing alloimmunization in this population by adding a second automated seologic testing platform is very low.

Background: Whether anti-D produced by individuals with a weak D phenotype are allo- or autoantibodies remains a matter of debate even though blood transfusion practice is impacted. The aim of our study was to determine the serologic features of anti-D in individuals expressing the most frequent weak D type in Caucasians that are weak D Type 1 or weak D Type 2, to assess whether anti-D were allo- or autoantibodies., Study Design and Methods: Serologic D typing and molecular analysis enabled the including of 121 weak D Type 1 individuals and 99 weak D Type 2 individuals in our study. Serologic features of anti-D included autologous controls, direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum on autologous red blood cells (RBCs)., Results: Serologic D typing showed a variable reactivity of RBCs expressing weak D Type 1 or weak D Type 2 (4+ to 0). Anti-D was identified in six weak D Type 1 and six weak D Type 2 individuals, respectively. The serologic data were in favor of autoantibodies., Conclusion: A complete anti-D investigation in individuals with a D variant (weak D or partial D identified by molecular analysis) should be systematically performed before any valid conclusion on the nature of the antibody. Transfusing weak D Type 1 or weak D Type 2 patients with D+ RBC units should be recommended. Weak D Type 1 or weak D Type 2 pregnant women do not need anti-D immunoprophylaxis., (© 2011 American Association of Blood Banks.)

Red blood cell transfusion therapy is a key component in the treatment of patients with sickle cell disease (SCD). There is no universal standard of care for the appropriate selection of RBC products for patients with SCD. A number of programs extend antigen matching to E and C in the Rh system, and to K, and some attempt to transfuse blood from African-American donors. Although these varied approaches reduce the rate of alloimmunization, patients continue to develop Rh antibodies. Molecular DNA-based analyses of patients alloimmunized to the Rh system, despite serologic Rh antigen matching, invariably reveal altered RH alleles. The prevalence of altered RH alleles in patients with SCD suggests an important emerging role for molecular methods in expanding matching of patients and donors in the Rh system for this patient population., (Copyright © 2011. Published by Elsevier Ltd.)

Rh discrepancies are a problem during routine testing because of partial D or weak D phenotypes. Panels of monoclonal antibodies (MoAb) are being developed to identify D variants such as partial D and weak D when there are anomalous D typing results; however, molecular characterization offers a more specific classification of weak and partial D. The weak D and partial D phenotypes are caused by many different RHD alleles encoding aberrant D proteins, resulting in distinct serologic phenotypes and the possibility of anti-D immunization. We evaluated currently used serologic methods and reagents to detect and identify D variants and correlated the results with molecular analyses. A total of 306 blood samples from Brazilian blood donors and patients with discrepant results in routine D typing were analyzed. In total, 166 (54.2%) weak D, 136 (44.4%) partial D, 3 (1%) DEL, and 1 (0.3%) DHAR variants were identified. Among weak D samples, 76 weak D type 1 (45.8%), 75 weak D type 2 (45.2%), 13 weak D type 3 (7.8%), and 2 weak D type 5 (1.2%) alleles were found. Among the partial D samples, 49 type 4.0 weak partial D (36%), 9 DAR (6.6%), 24 DFR (17.6%), 6 DBT (4.4%), 1 DHMi (0.73%), 26 DVI (19%), 14 DVa (10.3%), 5 DIVb (3.7%), and 2 DVII (1.5%) were observed. Two samples identified as DEL by adsorption-elution were characterized by molecular analyses as RHD(IVS5–38DEL4) and one sample was characterized as RHD(K409K). One sample was characterized as DHAR, a CE variant positive with some monoclonal anti-D. Our results showed that the use of different methods and anti-D reagents in the serologic routine analysis revealed D variants that can be further investigated. Molecular methods can help to differentiate between partial D and weak D and to characterize the weak D types, providing additional information of value in the determination of D phenotypes. This distinction is important for optimized management of D– RBC units and for the prevention of anti-D–related hemolytic disease of the fetus and newborn.

Objectives: To evaluate the efficiency of blood tests (blood group, direct antiglobulin test) to assess severe hyperbilirubinemia in full-term newborns, delivered from mothers with rhesus negative or O group and to determine clinical and biological factors that may improve the prediction characteristics of this blood test., Patients and Methods: We included all the full-term newborns, delivered from mothers with rhesus negative or O group, in a tertiary maternity ward, in 2005, from January6th to December31st., Results: One thousand and ninety-two children were included. Newborns of A, B or AB group delivered from a mother 0 were at increased risk of presenting severe hyperbilirubinemia (OR=2.35 [1.22-4.52]). The negative predictive value was 96%. Yet, the determination of the Coombs test does not increase NPV., Conclusion: Systematic performance of blood test for newborns delivered from mother with O group does increase the ability to predict severe hyperbilirubinemia in a newborn infant. Direct antiglobulin test systematic performance remains questionable., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

HDN attributed to the rare Rh variants has become more and more significant caused by anti-D, but the compatible blood is usually very difficult to obtain when exchange transfusion is required. We treated a 10-hour neonate of O, D + C + c - E - e+ blood group with severe HDN due to anti-Rh17 with least incompatible blood typed O, D + C - c + E + e-. The neonatal hemolysis was relieved obviously and bilirubin was reduced gradually after exchange transfusion. The infant was discharged in good health 13 days after birth with 135.0 g/L, 28.0 micromol/L and 10.7 micromol/L of Hb, total bilirubin and direct bilirubin, respectively. No sequelae were observed in a three-year follow-up. The result suggesting that the least incompatible blood is an alternative choice for exchange transfusion in severe HDN due to anti-Rh17 in case that Rh17 antigen-negative blood is unavailable.

Monkeys are frequently used in experimental transplantation research because of their physical traits and availability. As ABO incompatibility may result in humoral injury, it is important to identify the ABO blood typing of monkeys before transplantation. However, monkeys lack expression of ABH antigens on red blood cells, which makes accurate determination of the blood type difficult. The gel agglutination assay has been widely used as a routine blood grouping test clinically for more than 10 years. In this study, we evaluated the efficacy and the interference factors of using the gel system (including the direct gel system and the reverse gel system) for ABO typing in rhesus monkeys (n = 38) and cynomolgus monkeys (n = 26). Immunohistochemistry assay was used to obtain the accurate blood type data of monkeys. The results revealed that the direct gel system was ineffective in blood typing of monkeys, whereas the reverse gel system assay, which is based on preabsorbed serum, provided reproducible results that were confirmed by histologic analysis. We conclude that the reverse gel system assay with use of preabsorbed serum is a simple and reliable method for ABO typing of monkeys.

Background: QWALYS 2 is a fully automated system for ABO/D grouping, Rh phenotyping, K typing, and antibody screening (ABS). Its new erythrocyte-magnetized technology (EMT) is based on the use of magnetic nanoparticles and avoids centrifugation and washing steps., Study Design and Methods: Overall 499 blood samples were tested with our routine blood bank methods for ABO/D grouping, 313 samples for Rh phenotyping and K typing (microtiter plates; Olympus PK 7200), and 478 samples for ABS (gel centrifugation technique, DiaMed). All samples were tested in parallel with the EMT., Results: In 496 of 499 samples (99.4%), a complete concordance between the observed (QWALYS 2) and the expected results for ABO/D grouping was found. One sample with a weak A in an AB blood group and 2 samples with a weak D were not detected by the QWALYS system. Rh phenotyping and K tests revealed a 100% concordance. In the two ABS techniques, 427 samples were negative in both and 15 samples showed the same antibody specificity in both. Three immunoglobulin M antibodies were as expected negative in EMT and positive by DiaMed. In 32 cases (6.7%), false-positive reactions were observed by EMT due to 22 unspecific reactions (4.6%) and 10 lipemic or fibrinic plasmas (2.1%). One autoantibody was found by EMT only., Conclusion: The EMT is reliably suited to ABO/D grouping, Rh phenotyping, and K testing and is suitable to detect immunoglobulin G red blood cell alloantibodies as well. The rate of false-positive reactions in ABS due to lipemic and fibrinic samples needs to be reduced.

Background: Erythrocyte-magnetized technology (EMT) is a new fully automated method for ABO-RH-K phenotyping and antibody detection. The magnetization of red cells avoids centrifugation and washing phases. This report describes the results of an evaluation of this new technology on its specific automated system., Study Design and Methods: ABO-RH-K phenotyping was compared between EMT and a semiautomated routine method (liquid microplate for ABO-D and microcolumn system for RH-K) on 311 patients' samples. The overall performance of the new method was further assessed in daily routine on a total of 11,022 samples during 3 months in two different laboratories. Antibody detection was evaluated on 624 consecutive patients' samples and on 118 frozen samples containing specific antibodies in comparison with commercial microcolumn systems., Results: Eight of 311 ABO-RH-K tests (2.6%) were not interpreted by EMT. Seven of them were weak antigen or reverse grouping reactions showing a negative result with the routine method. On a 3-month follow-up, 216 of 11,022 tests (1.96%) were not interpreted by the system, 75 percent of them being due to weak or mixed-field reactions. EMT was better in detecting ABO-D mixed-field reaction than routine microplate method. Detection of clinically significant antibodies was similar between EMT and microcolumn. In contrast, EMT detected a markedly lower rate of presumed nonsignificant antibodies. The system presents an overall high reliability., Conclusion: EMT is tailored to meet the needs of the transfusion service and represents an important advance in the field of immunohematology.

Background: To date more than 100 variant D types have been reported and the frequencies vary among populations. Blood donor typing should reveal all donors expressing D antigens, while patient typing should prevent the development of anti-D in patients with a D- or variant D blood type. Serotyping is the standard method to assign transfusion strategies, whereas molecular classification offers a more specific grouping of weak and partial D., Study Design and Methods: Blood donor and patient samples with discrepant results of D phenotyping were collected to investigate the frequency of weak D subtypes in Denmark and to evaluate currently used serologic methods., Results: Nine different weak D types were identified among the 101 samples. Weak D Types 1, 2, and 3 constituted 80 percent of the analyzed samples and 10 percent of the samples identified as weak D from serology were actually partial D., Conclusion: The distribution of weak D types in Denmark was found to resemble the distribution in Northern Germany in respect to the three most prevalent weak D types. Correctly defining all samples that show weak reactions in D typing as weak D or partial D is not possible with serotyping alone; genotyping offers the only exact categorization. Serology is superior for routine blood typing, however.

Background: Weak D Types 1, 2, and 3 recipients cannot be immunized when exposed to D antigen. Molecular biology is very efficient to type weak D variants but rarely implemented in daily practice. The serologic typing practice of weak D in a Caucasian patient population was analyzed and a transfusion strategy is proposed., Study Design and Methods: Samples typed either ddCcee or ddccEe in routine laboratories were tested with the indirect antiglobulin test (D(u) test). D(u)-positive samples were screened for weak D alleles Types 1, 2, and 3 and further tested with immunoglobulin M (IgM) anti-D reagents, used in a fully automated device., Results: A total of 468 of 55,162 samples were found to be ddCcee or ddccEe. Ninety-three expressed weak D after the D(u) test leading to D+ assignment for transfusion. Seventy-three percent of D(u)-positive samples were weak D alleles Type 1, 2, or 3. Almost all weak D Types 1, 2, and 3 were positive with IgM reagents in gel matrix with an automated device. Other variants that could be potentially associated with anti-D alloimmunization, however, were also positive., Conclusion: Serology is very sensitive to detect weak D Types 1, 2, and 3, but there is no cutoff to distinguish variants of clinical significance. When molecular analysis is not available, it is proposed that a D+ status for blood recipients found to be weak D with a sensitive method be assigned, except for women of childbearing age or younger, because of the remaining possibility to be partial D or other rare weak D who can be immunized.

RH is the most complex of all 29 blood group systems. New discoveries relating to the RHD gene, and an appreciation of its variant phenotypes such as weak D and partial D, have challenged the way that D status is assigned to both blood donors and blood product recipients. This concise review introduces the current concepts of weak D and partial D and how the identification of these variants has influenced the testing methods for the D antigen. We demonstrate how molecular tests of the RHD gene can and should be used in resolving serological discrepancies, in particular in pregnant women.

It is well known that certain combinations of alloantibodies are frequently found together. Patients with sickle cell disease (SCD) are mostly ofAfrican ancestry,and they may make anti-hrB. A transfusion of hrB- blood is often achieved by using e- (R2R2) RBCs; it is generally believed that hrB- patients readily make anti-E or a"broad-spectrum" anti-Rh34 (-HrB). We describe two multiply transfused D+ patients with SCD and a history of anti-hrB who subsequently produced anti- D. This raises the question whether anti-hrB together with anti-D is a more common antibody combination than anti-hrB with anti-E or anti-Rh34.

The role of enzyme-treated cells (ETCs) in red blood cell (RBC) antibody screening has been the subject of controversy, and its place in the clinical routine remains to be determined. In this work, plasma samples containing anti-RH1 (anti-D; N = 10), anti-RH2 (anti-C; N = 10), or anti-RH3 (anti-E; N = 10) antibodies were studied. The samples were diluted in nonbuffered or buffered normal saline, as well as in a pool of AB plasma samples. Titers and scores were determined by means of the gel test, using the indirect antiglobulin test (IAT) as well as ETCs, with R(0)r, r'r, or r''r test cells. Our results showed that compared to the IAT, ETCs allowed a clearer detection of anti-RH2 and anti-RH3, but not of anti-RH1 antibodies. Based on our study, it is not clear whether the ETC phase of the gel test should be maintained for RBC antibody screening., (2007 Wiley-Liss, Inc.)

Background: Pegylation of red blood cells (RBCs) has been the primary focus of research on the immunocamouflage of cell. The aim of this study was to demonstrate pegylation homogeneity, its shielding effect on D antigens, and its storage stability. In addition, methoxypolyethylene glycol (mPEG)-modified RBCs (mPEG-RBCs) were tested serologically against a panel of serum samples that was difficult to match to find a solution to the difficulty in matching., Study Design and Methods: In this study, fluorescein-PEG and a confocal laser scanning microscope were used to monitor PEG attachment on RBC population and observe reaction homogeneity, the stability of mPEG combined with RBCs in vitro was evaluated by the RBC ghost agglutination test, the pegylation sites on membrane were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with two dye methods, and the effect of pegylation on D antigen was detected by immunoblotting techniques. Compatibility tests were carried out between 66 cases of serum with difficulty in blood matching and mPEG-camouflaged RBCs by use of four blood matching methods including direct agglutination, indirect antiglobulin test (IAT), microtyping gel cards (MTS), and the manual polybrene technique (MPT)., Results: The results indicated the homogeneity of pegylation, the absence of RhD protein in mPEG-modified D+ RBCs by Western blotting, and attachment of PEG to RBCs after 30 days of storage, while RBCs still remained antigenically silent. All pegylation RBCs showed a negative reaction with ABO-matched patients' serum samples by direct agglutination, IAT, and MTS, which indicated that pegylation RBCs and patients' serum samples were compatible. MPT was not suitable for detecting blood matching of mPEG-RBCs, because modification changed the RBCs' biophysical properties., Conclusion: In conclusion, mPEG-RBCs have acceptable in vitro properties and provide a useful solution to problems with clinical blood matching, although such masking leaves much to be desired.

The RH blood group is the most polymorphic and immunogenic blood group system. The RH locus is composed of 2 highly homologous genes: the RHD gene encoding the D polypeptide; and the RHCE gene, encoding C or c together with either E or e polypeptides. Numerous variants exist for both RHD and RHCE genes. Among them we were interested in the serological and molecular definition of numerous pre-published RHCE variants encountered in different populations. The identification of these variants is crucial to ensure the transfusion safety for patients expressing these variants. Here we propose a procedure to identify some of them and discuss the adequate transfusion strategy. This procedure has enable us to identify new variants to be presented at the symposium.

Background and Objectives: The aim of this study was to evaluate a lyophilized monoclonal immunoglobulin M (IgM) anti-D preparation for use as an International Standard to specify a recommended minimum acceptable potency of anti-D blood-grouping reagents., Materials and Methods: The candidate International Standard (99/836) for specifying the minimum potency of anti-D blood-grouping reagents was evaluated against a wide range of commercial anti-D blood-grouping reagents in an international collaborative study involving 20 laboratories in 13 countries. Laboratories titrated reconstituted 99/836, in parallel with as many commercial anti-D blood-grouping reagents as were available to them, in tube tests according to specified haemagglutination methodology for low-protein (e.g. monoclonal IgM) and high-protein (e.g. polyclonal) reagents. The ratios of the mean end-point titres of the reagents to that of 99/836 within each laboratory were calculated., Results: The ratios of the mean titres of the low-protein reagents to the mean titre of 99/836 within a laboratory fell between 0.25 and 2 for 43 of the 45 low-protein anti-D reagents tested (i.e. the potencies of the low-protein reagents compared with 99/836 were between a 1:4 dilution of 99/836 to twice as potent as 99/836). The ratios of the mean titres of the high-protein reagents to the mean titre of 99/836 within a laboratory fell within 0.125 and 1 for eight out of the 10 high protein reagents tested., Conclusions: By international consensus, a 1:3 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of low-protein anti-D blood-grouping reagents. A 1:8 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of high-protein anti-D blood-grouping reagents. On the basis of the results presented here, 99/836 was established by the World Health Organization as the 1st International Standard for specifying the minimum potency of anti-D blood-grouping reagents, in tube tests.

Background: Recent molecular studies of the RHD gene have revealed that D(el) individuals retain a grossly intact RHD gene or have a portion of RHD in their genomes. No D(el) phenotype has yet been shown to induce a primary or secondary alloanti-D immunization, however., Case Report: A 67-year-old D- Japanese woman with a history of allosensitization from transfusion of D+ red blood cells (RBCs) was negative for anti-D at admission. After she received RBCs from 19 apparently D- donors, she developed anti-D with an 8-fold titer. The titer of anti-D increased further to 128-fold after transfusions of cross-match-compatible D- negative RBCs from 40 donors over the next 2 years. Two of 59 donors were found to be RHD gene-positive and antigen D- with a D(el) phenotype, that is, RHD(K409K)., Conclusion: This is the first case in which RBCs having the D(el) phenotype induced a secondary alloanti-D immunization. A D- donor with the RHD(K409K) allele was associated with the development of anti-D. Adverse episodes or evidence of hemolysis was not observed after the transfusion of RHD(K409K) RBCs. Further clinical evidence is needed to reveal whether the D(el) phenotype has a clinically relevant potential for anti-D immunization.

Background: Weak D expression is caused by a large number of RHD alleles. Increasingly recommendations for D+ or D- transfusions are based on polymerase chain reaction (PCR) identification of certain RHD alleles. Possible sources of error are rare D variants that are inadvertently carrying known polymorphisms of frequent weak D types., Study Design and Methods: Weak D donors were checked by direct column agglutination. In donors with unusually weak expression of D, the molecular weak D type was determined by weak D PCR and nucleotide sequencing. The serologic profile of a weak D type 1 variant was determined by agglutination serology and flow cytometry., Results: Several donors in whom direct agglutination barely revealed any D expression were shown to carry the new RHD(L18V,V270G) allele dubbed weak D type 1.1. Initially, such donors had been mistyped as weak D type 1 by PCR. In a systematic study, weak D type 1.1 was shown to be present in 7 of 23 donors with very weak D expression who all lived in a restricted area of Northern Germany. Although weak D type 1.1 was typed D- or barely D+ by direct agglutination, it was easily detected by antiglobulin technique and was shown to carry about 600 antigens D per red blood cell., Conclusion: The observation of weak D type 1.1 with its distinct phenotype pinpointed to two general problems of current RHD genotyping strategies: Mistyping of alleles with additional mutations and striking geographic variation of the allele distributions.

Background: Uncommonly, antibodies that appear to exhibit antigenic specificity on red blood cell (RBC) panels fail to maintain specificity following alloadsorption (i.e., they mimic antigenic specificity). Understanding both the pitfalls and the proper pathways to establish the diagnosis and to interpret the clinical significance of these mimicking antibodies is important for patient management., Case Report: A 68-year-old woman was admitted with dyspnea, anemia, bilateral pulmonary emboli, and metastatic ovarian cancer. Blood bank evaluation identified anti-E reactivity in the patient's plasma sample and a positive direct antiglobulin test (DAT)., Results: The DAT was positive for immunoglobulin G and negative for C3b. An eluate of the RBCs showed E-antigen specificity on a RBC antibody panel. Repeat serologic testing with RBC antibody panels with adsorbed patient plasma showed removal of apparent anti-E reactivity with either E-antigen-positive or E-antigen-negative RBC stroma., Conclusion: A mimicking autoantibody with apparent E-antigen specificity was identified in the plasma sample of a woman with newly diagnosed ovarian cancer. Despite their relative low frequency, mimicking antibodies, whether auto- or alloantibodies, may interfere with the timely issuance of compatible blood products and may confuse laboratory and clinical staff. Determining the clinical significance of the antibody, by taking into account the RBC phenotype of the patient and the antigen prevalence in the general population, guides the extent of workup required to best utilize resources while assuring patient safety.

Objective: Hemolytic disease of the fetus and of the newborn can be caused by incompatibility of maternal and fetal erythrocytes for Rh (D) or other blood type antigens. Routine antibody screening is advocated in all pregnant women, irrespective of whether they are Rh (D)-positive or Rh (D)-negative, to look for clinically significant alloantibodies other than Rh (D) that might cause hemolytic disease of the newborn. The purpose of this study was to assess the incidence of blood type antibodies other than Rh (D) in pregnant women attending for prenatal care in a typical urban population., Methods: A retrospective analysis was undertaken of the charts of all pregnant women followed throughout their entire pregnancy at our Women's Health Center from 1 January 1999 to 30 April 2002., Results: There were 1265 pregnant women included in the study: 465 had blood type A (36.7%), 269 type B (21.3%), 424 type O (33.5%) and 107 type AB (8.5%). A total of 1156 were Rh (D)-positive (91.4%) and 109 were Rh (D)-negative (8.6%). Of the Rh (D)-positive women, 522 (41.3%) underwent routine antibody screening in the first trimester. Only one woman (0.2%) had a positive antibody screen. Of the 109 Rh (D)-negative women, one (0.9%) had a positive third trimester screen with a negative first trimester screen., Conclusion: Routine antibody screening of Rh (D)-positive women is probably not warranted from a clinical cost-benefit perspective.