Red Cell Genotyping Research Articles

Lost in the Ds: Unexplained Loss of Rh(D) Antigen Expression By Serological Testing In Two Patients

Abstract Introduction/Objective While the loss of ABOH antigens has been well documented, reduced-to-absent expression of the RHD antigen is comparatively rare. Here, we describe two cases with D antigen loss presenting associated clinical settings, smear results, and red blood cell (RBC) genotyping findings to elucidate this unusual phenomenon. Methods/Case Report Cases (n=2) were retrospectively reviewed based on discovery during routine serological testing in our Blood Bank. RhD blood typing was performed via gel methods [Grifols, Los Angeles, CA]. Clinical information and laboratory data were extracted via the electronic medical record. Case 1 is a 79-year-old female with metastatic breast cancer. She initially typed as D positive (4+) in at least two separate analyses, but her D front type progressively became weaker until turning entirely negative after several weeks. Spherocytes and stomatocytes were present on her peripheral blood smear. This patient received 10 D+ red blood cell (RBC) units without anti-D formation. RBC genotyping revealed traditional “dce” by standard polymerase chain reaction (PCR). Case 2 is a 61-year-old male with a history of pre-B acute lymphocytic leukemia post-stem cell transplant, myelodysplastic syndrome, and progressive loss of D antigen expression (4+ to 0). Standard RBC genotyping revealed “Dce” by PCR. The peripheral blood smear was significant for RBC anisopoikilocytosis with increased stomatocytes, spherocytes, and ovalocytes. Results (if a Case Study enter NA) NA Conclusion The disappearance of the D antigen is rare, and our experience suggests an association between malignancies (both hematopoietic and non-hematopoietic) and D antigenic loss. Beyond these disease associations, however, loss of RhD is not otherwise readily explained in either case. Further analysis to test for other RHAG and RHCE mutational variants is an ongoing area of investigation in these cases, as more sensitive testing may reconcile the traditional PCR results with our initial detection of the D antigen.

Red Cell Genotyping Helps in Managing a Multi-Transfused Patient with Serologically Inconclusive Results

Red Cell Genotyping Helps in Managing a Multi-Transfused Patient with Serologically Inconclusive Results

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

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

Breakpoint regions of an RHD-CE(4-9)-D allele and a rare JK allele in a Pacific Islander individual.

Among 710 RHD alleles, 3 alleles have been shown to express CcEe antigens and, among 67 hybrid alleles of the RHD gene, 2 alleles have evolved to include RHCE exons 4-9. No breakpoint region had been described for such RHD-CE(4-9)-D hybrid alleles. In the Kidd blood group system, the JK*02N.01 null allele is found with high prevalence in the Polynesian population. We investigated a self-identified Pacific Islander with discrepant serologic and molecular results for his C and Jkb antigens. Another 8 samples with genotype-phenotype discrepancies in the Kidd blood group system were assessed. A combination of published molecular methods and commercial kits were applied to analyze the RHD, RHCE, and SLC14A1 gene sequences, as were hemagglutination tests to determine the serologic phenotypes. Nucleotide sequencing of the RHD gene in the index case, including relevant intron stretches, and cDNA identified an RHD-CE(4-9)-D hybrid allele. Nucleotide sequencing of his RHCE gene confirmed the presence of 2 RHCE*ce alleles despite expressing the C antigen. Sequencing of his SLC14A1 gene documented the JK*02N.01 null allele. In the other 8 samples, 5 previously known SLC14A1 nucleotide substitutions were identified. The JK*02N.17 allele was determined to be Jkb-positive. We determined the 2 breakpoint regions of his RHD-CE(4-9)-D hybrid allele, which was likely distinct from the 2 previously published hybrid alleles due to the differences in the linked RHCE allele. His RHD variant was shown to express the C antigen. An SLC14A1 substitution was underlying his unexpected Jkb-negative phenotype. In a quality improvement project, we resolved 8 samples with similarly discrepant results between Jk serology and red cell genotyping.

DNA Reference Reagents for Genotyping RH Variants

Patients who carry RH blood group variants may develop Rh alloantibodies requiring matched red cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at CBER/U.S. FDA. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles including commercial platforms and laboratory developed testing such as Sanger sequencing, next generation sequencing, and third generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation.

Cases of RhD variants RhD*DAU2/DAU6 and RhD*weak D type 4.1 in pregnant women in Saudi Arabia.

The D antigen is one of the most immunogenic and clinically significant antigens of the Rh blood group system due to its various genotypes that encode for more than 450 different variants. Accurate RhD typing and D variant identification is crucial specially in prenatal screening during pregnancy. Women with RhD -ve phenotype are eligible to Rh immune globulin (RhIG) prophylaxis for the prevention of anti-D alloimmunization and hemolytic disease of the fetus and newborn (HDFN). However, there are some women who possess RhD variant alleles, who are mistakenly grouped as RhD positive and considered not eligible for RhIG prophylaxis, putting them at risk of anti-D alloimmunization and consequently leading to HDFN during subsequent pregnancies. Here, we describe two cases of RhD variants DAU2/DAU6 and Weak D type 4.1 in obstetric patients who were grouped as RhD +ve with negative antibody screening during routine serologic testing. Weak/Partial D molecular analysis using genomic DNA Red Cell Genotyping (RCG) revealed that both patients had RhD variants, one of which DAU2/DAU6 allele associated with anti-D alloimmunization. According to routine testing neither patients received RhIG or transfusion. In this case report we document to our knowledge the first reported cases of RhD variants among pregnant women in Saudi Arabia.

Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies

The serologic evaluation of autoimmune hemolytic anemia (AIHA) confirms the clinical diagnosis, helps distinguish the type of AIHA, and identifies whether any underlying alloantibodies are present that might complicate the selection of the safest blood for any needed transfusion. The spectrum of testing is generally dependent on the amount and class (immunoglobulin G or M) of autoantibody as well as the resources and methodologies where testing is performed. The approach may range from routine pretransfusion testing, including the direct antiglobulin test, to advanced techniques such as adsorptions, elution, and red cell genotyping. When transfusion is needed, the selection of the optimal unit of red blood cells is based on urgency and whether time allows for the completion of sophisticated serologic and molecular testing methods. From the start of when AIHA is suspected until the completion of testing, communication among the clinical team and medical laboratory scientists in the transfusion service and immunohematology reference laboratory is critical as testing can take several hours and the need for transfusion may be urgent. The frequent exchange of information including the patient's transfusion history and clinical status, the progress of testing, and any available results is invaluable for timely diagnosis, ongoing management of the patient, and the safety of transfusion if required before testing is complete.

New Areas of Consensus for the Management of Sickle Cell Disease (SCD) in Pregnancy: An International Delphi Panel

Introduction and aim Sickle cell disease (SCD) is an autosomal recessive disorder characterized by a single nucleotide substitution in the beta-globin gene, resulting in the production of hemoglobin (Hb) S, and is the most common hematologic genetic disorder worldwide. As a result of Hb polymerization, oxidative stress and chronic hemolysis, complications occur during pregnancy for women with SCD, impacting both maternal and fetal health. We aimed to identify areas of consensus for the management of pregnancy-related complications in SCD, with a focus on prophylactic transfusion therapy (PTT). Methods A Delphi panel consisting of 13 experts from 4 countries replied to 2 rounds of questions (1 panelist dropped out between the 1st and 2nd survey). The 1st survey (October-November 2021) consisted of open-ended questions, while the 2nd one (April-May 2022, used to identify the degree of consensus or changes in opinion among panelists) was comprised of multiple-choice or closed questions based on the answers from the 1st survey. The thresholds were: <9/12 panelists (<75%) for no consensus, 9-10/12 panelists (75-90%) for consensus, and ≥11/12 panelists (>90%) for strong consensus. The Delphi panel covered topics where no global standardization or little evidence exists to guide management and prevention of maternal complications during pregnancy in SCD. These included antenatal care, hydroxyurea (HU) therapy, transfusion, prevention and treatment of complications, delivery and follow-up, bottlenecks and knowledge gaps. Results The panelists achieved strong consensus in the following areas of management: antenatal care (specifically, that pregnant women with SCD should be followed at least every 4 weeks by a multi-disciplinary team of specialists who would decide on the required tests and treatments), HU (discontinuation ≥3 months before conception; experts would not recommend discontinuation of pregnancy in case of exposure to HU during pregnancy), delivery and follow-up (that a multidisciplinary team should decide on labor induction or elective cesarean section). For the following areas of management, no consensus was achieved: continuation of HU therapy (could be considered during pregnancy/lactation if the risks of stopping HU therapy outweigh the benefits and if the patient understands the risks and agrees with continuing therapy), Hb level targeted prior to delivery, or preferred transfusion modality (simple transfusions versus automated red cell exchange [aRCX]) for a pregnant woman with SCD who was not on PTT during pregnancy but needed to be transfused prior to delivery. The input collected for transfusion (including details on when to recommend PTT) is summarized in the Table. The experts felt that target Hb and hematocrit should be set by clinical experience and would be impacted by the time interval between transfusions and the clinical indication for transfusion. There was no consensus on the most suitable transfusion modality. The experts identified a strong interest to prioritize the use of routine PTT in pregnant women with SCD as an area of research. Conclusion The experts did not achieve uniform consensus to support the standardized use of PTT in pregnant women with SCD. They achieved consensus that PTT should be routinely used in pregnant women with ≥2 acute vaso-occlusive pain episodes in a 4-week period regardless of genotype, with a preference for peripheral venous over central venous access, and the use of aRCX over simple transfusion (target HbS <30%) for the prevention of transfusional iron overload. The experts also achieved a strong consensus that prophylactic red cell exchanges should be used to reduce frequency of acute pain episodes in women taking high-dose opioids daily, and recommended consideration of on-demand transfusions for acute fetal complications. Consensus was not achieved on frequency of prophylactic transfusions, timing of PTT initiation during pregnancy in relation to HU discontinuation, target hematocrit levels, methods for the prevention of alloimmunization (extended red cell phenotyping versus genotyping) and the use of PTT for secondary prevention of fetal complications and pregnancy loss. In general, the areas of non-consensus seemed to correspond to areas with the widest knowledge gaps and lack of evidence, highlighting the need for further research in the use of PTT in pregnant women with SCD. Funding: Terumo Blood and Cell Technologies Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Throughput Next-Generation Sequencing of the Kidd Blood Group: Unexpected Antigen Expression Properties of Four Alleles and Detection of Novel Variants

Background: The blood supply for patients with foreign ethnic backgrounds can be challenging, as they often have blood group and HPA patterns that differ from the variants prevalent in the German population. In addition, hemoglobinopathies requiring regular blood transfusion may be more common in such populations. High-throughput genotyping tests can facilitate the identification of the most compatible blood products, thereby reducing the risk of transfusion reactions. The present study reports the results of a molecular study for the Kidd (JK) blood group. Allele frequencies and antigen prevalence data are presented for >8,000 individuals of various origins. Material and Methods: More than 8,000 blood donors were genotyped for 22 blood group systems and 5 HPA genes using an amplicon-based next-generation sequencing (NGS) approach. As part of the test system, we focused on the JK system in more detail. Double-ARMS PCR analysis was performed for the haplotype phasing of the JK1/JK2 and two more common synonymous polymorphisms. We performed transcript analysis to detect potential alternative splice products. For a subset of samples, a comparison between serotype and red cell genotype was conducted. Allele frequencies were determined for geographically different panels of individuals. Results: We successfully genotyped the JK blood group for 99.6% of the samples. Haplotype phasing revealed 96 different alleles. For several alleles that carry one of the synonymous SNVs c.588A>G and c.810G>A, we could not confirm the reported JK phenotypes. We found a higher frequency of JK:1 alleles for all populations except Iraqis. JK*01W.01 alleles were more common in the Asian groups and sub-Saharan Africans. A variant of the allele JK*02N.01 was present exclusively in Southeast Asians. Conclusion: Genotyping for JK antigens with a targeted NGS assay can easily be performed in routine. The interpretation that c.588A>G leads to a weak phenotype and c.810G>A to a null phenotype is questionable. IDs as well as the descriptions of alleles carrying these SNVs should be revised in the ISBT JK table.

Haemoglobin S testing using HEA BeadChip™ technology: Lifeblood comparison with clinical diagnosis.

Red cell antigen genotyping is commonly performed on patients requiring chronic transfusion support, such as sickle cell disease and thalassaemia. The Immucor HEA BeadChip™ test, in addition to assessing red cell antigen expression, can also detect the haemoglobin S (HbS) mutation. Our aim was to compare HbS results using HEA BeadChip™ performed at the Australian Red Cross Lifeblood with conventional haemoglobin studies. Patients with thalassaemia and sickle cell trait (SCT) or disease (SCD) referred for red cell genotyping between 2017 and 2019 were assessed. The HbS result obtained from HEA BeadChip™ was compared with that obtained from high-performance liquid chromatography (HPLC) performed by the referring pathology provider. One-hundred and nineteen cases had comparable HPLC and HEA BeadChip™ results. On HEA BeadChip™ testing, 40 cases showed a negative HbS result, 31 cases showed HbS+ and 47 cases showed HbS++. There was one case with 'low signal' result. Of the negative HbS cases, there was none with SCT. The HbS+ group comprised a mixture of SCT and SCD due to compound heterozygosity for HbS and β-thalassaemia mutations. The HbS++ group comprised predominantly SCD due to homozygosity for HbS. HEA BeadChip™ is an accurate screening test for the detection of HbS. There were no false positives or false negatives. The identification of donors with the HbS mutation through the targeted genotyping programme would enable early intervention, improved donor management and reduced wastage.

Transfusion support for a woman with RHD*09.01.02 and the novel RHD*01W.161 allele in trans.

According to recent work group recommendations, individuals with the serologic weak D phenotypes should be RHD genotyped and individuals with molecular weak D types 1, 2, 3, 4.0, or 4.1 should be treated as D+. We report an African American woman with a long-standing history of metrorrhagia, who presented for infertility evaluation. Blood grouping showed AB with a possible subgroup of A, based on mixed-field agglutination, and a serologic weak D phenotype. Results from routine red cell genotyping for the RHD gene was incongruent with the serologic RhCE phenotype. For the surgical procedure, the patient was hence scheduled to receive group AB, D- RBC transfusions. Subsequent molecular analysis identified the ABO*A2.01 and ABO*B.01 alleles for the ABO genotype and the novel RHD allele [NG_007494.1(RHD):c.611T>A] along with an RHD*09.01.02 allele for the RHD genotype. Using a panel of monoclonal anti-D reagents, we showed the novel RHD(I204K) allele to represent a serologic weak D phenotype, despite occurring as a compound heterozygote, designated RHD*weak D type 161 (RHD*01W.161). Individuals with a weak D type 4.2 allele are prone to anti-D immunization, while the immunization potential of novel RHD alleles is difficult to predict. For now, patients should be treated as D- in transfusion and pregnancy management, when they harbor a novel RHD allele along with any weak D allele other than weak D types 1, 2, 3, 4.0, or 4.1. This study exemplifies strategies for how and when a laboratory should proceed from routine genotyping to nucleotide sequencing before any decisions on transfusion practice is made.

High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

Background: High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. Methods: Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. Conclusions: We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

Molecular blood group screening in Omani blood donors.

Blood group genotyping has been used in different populations. This study aims at evaluating the genotypes of common blood group antigens in the Omani blood donors and to assess the concordance rate with obtained phenotypes. Blood samples from 180 Omani donors were evaluated. Samples were typed by serological methods for the five blood group systems MNS, RH (RHD/RHCE), KEL, FY and JK. Samples were genotyped using RBC-FluoGene vERYfy eXtend kit (inno-train©). Predicted phenotypic variants for 70 red blood cell antigens among the MNS, RH (RHD/RHCE), KEL, FY, JK, DO, LU, YT, DI, VEL, CO and KN blood group systems were assessed. Simultaneous phenotype and genotype results were available in 130 subjects. Concordance rate was >95% in all blood group systems with exception of Fy(b+) (87%). Homozygous GATA-1 mutation leading to erythroid silencing FY*02N.01 (resulting in the Fy(b-)ES phenotype) was detected in 81/112 (72%) of genotyped samples. In addition, discrepant Fyb phenotype/genotype result was obtained in 14/112 samples; 13 of which has a heterozygous GATA-1 mutation and one sample with a wild GATA genotype. D and partial e c.733C>G variants expressing the V+VS+ phenotype were found in 22/121 (18.2%) and 14/120 (11.7%) of the samples, respectively. Di(a-b+), Js(a-b+), Yt(a+b-) and Kn(a+b-) genotype frequencies were 99.4%, 95.8%, 91.9% and 97.7%, respectively. In conclusion, we report a high frequency of FY*02N.01 allele due to homozygous c.-67T>C GATA-1 single-nucleotide variation. This is the first study reporting the detailed distribution of common and rare red cell genotypes in Omani blood donors.

Molecular red cell genotyping of rare blood donors in South Africa to enhance rare donor-patient blood matching.

BackgroundMolecular red cell genotyping is devoid of serology limitations such as the scarcity of rare antisera and the possibility of inconclusive results due to biological interferences. Blood incompatibility can result in immune transfusion reactions such as haemolytic transfusion reactions or haemolytic disease of the foetus and newborn.ObjectiveThe study aimed to use molecular red cell genotyping to identify rare blood group donors among South African blood donors.MethodsRed cell genotyping data were extracted retrospectively from the BIDS XT genotyping software in the Immunohaematology Reference Laboratory from January 2015 to August 2016. The ID CORE XT genotyping assay was used to identify the single nucleotide polymorphisms of 10 blood groups system alleles in 150 donors. Associations between the resultant genotypes and predicted phenotypes, ABO group, RhD type, race group and gender were studied.ResultsSignificant red cell genetic variability was noted among the numerous South African donor genotypes identified in this study. Genotyping further confirmed the presence of at least one of the 16 rare genotypes in 50 donors. Group O Black donors were associated with two rare blood types, while several other rare blood types were found only in White donors, supporting an association between ABO/Rh subtype, race group and rare blood types.ConclusionTargeted screening of donors for antigen-negative rare blood units for patients should be done to reduce the risk of haemolytic transfusion reactions and haemolytic disease of the foetus and newborn.

Optimal blood issuing by comprehensive matching

Mass-scale red blood cell genotyping of donors and transfusion recipients increases the availability of extended antigen matched red blood cell units. Therefore, a new mathematical framework is developed that can be applied for general blood groups (i.e., beyond the ABO, RhD blood groups). It determines which red blood cell units should be issued from inventory, such that requests from hospitals can be satisfied with antigen compatible red blood cell units, shortages for future requests are avoided, and outdating is prevented. The optimization model consists of two steps: a binary vector representation for general blood groups and the formulation of the inventory allocation problem as a minimum cost flow problem. The potential practical performance of the optimization model is evaluated by iterative simulations, based on historical data on supply and demand of red blood cell units in the Netherlands. When including the fourteen clinically most relevant antigens, more than 90% of all requests can be satisfied with antigen identical red blood cell units. Shortages and outdating can be kept restricted and could even be reduced to virtually zero, when only the ABO, RhD blood groups are considered.

1110 Predictive value of elevated MCA doppler in patients with alloimmunization

To evaluate pregnancy outcomes after abnormal middle cerebral artery peak systolic value (MCA PSV) measurement managed with percutaneous umbilical blood sampling (PUBS) or delivery as compared to repeat MCA PSV within 7 days. We performed a descriptive study including all patients in a single tertiary care center between 2006 and 2016 with a MCA PSV > 1.5 multiples of the median (MoM) in the setting of maternal red cell alloimmunization. The index visit was the first exam with MCA PSV > 1.5 MoM. We excluded cases with fetal hydrops prior to the index visit or abnormal MCA PSV at > 35 weeks. Patients undergoing immediate intervention (PUBS or delivery) (group 1) were compared to patients with repeat MCA PSV performed within 7 days (group 2). All scans were interpreted by board-certified Maternal-Fetal Medicine physicians. Fifty patients were identified. 19 underwent intervention based on the first abnormal MCA PSV (group 1) and 31 underwent repeat MCA PSV (group 2). The gestational ages (GA) or MCA PSV at index exam did not vary between the groups (table 1). Fetal red blood cell genotypes were infrequently known in this population. MCA PSV values at index exam were not different regardless of anemia severity (none, mild, moderate /severe). In group 1, 14/19 (74%) had anemia, with 8/19 (42%) having moderate/severe anemia (Figure 1). In group 2, 11/31 patients underwent PUBS for a persistently abnormal MCA PSV, all of whom had anemia which was moderate/severe in 7/11 (64%). Of the remaining 20 patients (65%) in group 2, 19/20 (95%) never underwent PUBS or developed moderate/severe anemia. One fetus had an abnormal MCA PSV 13 days after the index and was transfused for severe anemia. Of patients who underwent repeat MCA PSV testing rather than immediate intervention, 65% had normal repeat MCA PSVs and safely avoided PUBS. Persistently elevated MCA PSVs were more predictive of anemia than single elevated values preceding intervention. Other factors contributing to the decision to immediately pursue intervention should be further evaluated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

USING DROPLET DIGITAL PCR TO SCREEN FOR RARE BLOOD DONORS: PROOF OF PRINCIPLE

Abstract Background Digital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). Methods Nucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. Results DI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. Conclusion The proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Using droplet digital PCR to screen for rare blood donors: Proof of principle

BackgroundDigital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). MethodsNucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. ResultsDI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. ConclusionThe proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

ACKR1 Alleles at 5.6 kb in a Well-Characterized Renewable US Food and Drug Administration (FDA) Reference Panel for Standardization of Blood Group Genotyping

The glycoprotein encoded by the ACKR1 gene expresses the Duffy blood group antigens and is a receptor for malaria parasites. We recently described 18 long-range ACKR1 alleles in an autochthonous population of a malaria endemic region. Extending this work, we sequenced the gene in a 53-sample repository established by the US Food and Drug Administration (FDA) as reference reagents for blood group genotyping. The FDA samples have been characterized for 19 genes; however, long-range haplotype information for these genes, including ACKR1, was lacking. We used a hybrid approach, novel for this type of gene, to characterize ACKR1 by combining two next-generation sequencing technologies, the short-read massively parallel sequencing and the long-read nanopore sequencing. The expedient integration of data from both next-generation sequencing systems were necessary and sufficient to allow determination of all 25 long-range ACKR1 alleles found in the 53 samples accurately. All 25 alleles identified in our current FDA cohort were novel and, unexpectedly, none had been observed among the 18 alleles in our previous study. The alleles will be useful for validation, calibration, and proficiency testing of red cell genotyping. The lack of any overlap between the ACKR1 alleles in the two studies documents differences in mutation rate and recombination frequency among populations. The exact haplotype and their interethnic or interpopulation dissimilarities can influence disease susceptibility and therapy.

Identification of ACKR1 variants associated with altered Duffy phenotype expression in blood donors from southern Brazil

The atypical chemokine receptor 1 gene (ACKR1) is responsible for the clinically significant Duffy blood group. The main antigens of this system, Fya and Fyb, can be related to a null or weak expression of the DARC protein. In the present work, we aimed to identify ACKR1 gene variants in blood donors from southern Brazil based on discrepancies between their serological and molecular typing results. Then, we analyzed the association of these variants with the expression of the Duffy phenotype. The Fy antigen types were determined via hemagglutination and real-time PCR (c.125 G > A, c.265C > T and c.−67T > C SNPs) tests in a sample composed of 382 regular repetitive voluntary blood donors to the Blood Bank of Hospital de Clínicas de Porto Alegre. An inconclusive correlation between phenotype-genotype analyses was found in 11 (2.88 %) donors, and the entire ACKR1 gene was sequenced in these samples. Our investigation found 11 genetic variants, four of which (c.−541C > T, c.21 + 150C > T, c.22−58A > G, and c.298 G > A SNPs) seem to have putative functional effects on the structure and expression of DARC undertaken for in silico analysis (SIFT, PolyPhen-2 and RegulomeDB). Molecular events can result in apparent discrepancies between red cell genotypes and phenotypes. Our findings provided insight into the molecular background of FY antigens to improve technical approaches for red cell genotyping.