Cell-Free DNA Analysis for the Determination of Fetal Red Blood Cell Antigen Genotype in Individuals With Alloimmunized Pregnancies.

OBJECTIVE: To evaluate the accuracy of next-generation sequencing-based quantitative cell-free DNA analysis for fetal antigen genotyping in individuals with alloimmunized pregnancies undergoing clinical testing in practices across the United States as early as 10 weeks of gestation, with the objective of identifying individuals with pregnancies at risk for hemolytic disease of the fetus and newborn and guiding management. METHODS: This prospective cohort study included patients with alloimmunized pregnancies undergoing clinical fetal antigen cell-free DNA analysis between 10 0/7 and 37 0/7 weeks of gestation at 120 clinical sites. Both the pregnant person with the alloimmunized pregnancy and the neonates resulting from the pregnancies were included. The laboratory issued the cell-free DNA results prospectively as a part of clinical care. After delivery, neonatal buccal swabs collected between 0 and 270 days of life were sent to an outside independent laboratory for antigen genotyping. The outside laboratory was blinded to the fetal cell-free DNA results, and the results were compared. Concordance was reported for the fetal antigen cell-free DNA analysis for antigens to which the pregnant person was alloimmunized and for all antigens for which the pregnant person was genotype negative. RESULTS: A total of 156 pregnant people who received clinically ordered cell-free DNA fetal antigen testing provided neonatal buccal swabs for genotyping after delivery. Overall, 15.4% of participants were Hispanic, 9.0% were non-Hispanic Black, 65.4% were non-Hispanic White, 4.5% were Asian, 1.3% were more than one race or ethnicity, and 4.5% were unknown. The median gestational age at the time of testing was 16.4 weeks with a median fetal fraction of 11.1%. Concordance between cell-free DNA analysis results and neonatal genotype was determined for 465 antigen calls for the following antigens: K1 (n=143), E (124), C (60), Fy a (50), c (47), and D(RhD) (41). These 465 calls included 145 in which the fetus was antigen positive and 320 in which the fetus was antigen negative. We observed complete concordance between prenatal fetal antigen cell-free DNA analysis results and neonatal genotypes for the 465 calls, resulting in 100% sensitivity, specificity, and accuracy. CONCLUSION: In a diverse multicenter cohort, cell-free DNA analysis was highly sensitive and specific for determining fetal antigen genotype as early as 10 weeks of gestation in individuals with alloimmunized pregnancies. Taken together with previously published evidence, this study supports the implementation of cell-free DNA testing to manage individuals with alloimmunized pregnancies in the United States.

DNA Reference Reagents for Genotyping RH Variants.

Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood 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 the Center for Biologics Evaluation and Research/US Food and Drug Administration. 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.

Decreasing parvovirus B19 and hepatitis A nucleic acid test positivity rates in Canadian plasma donors following the initiation of COVID-19 restriction in March 2020.

BACKGROUND AND OBJECTIVES: In Canada, plasma sent for fractionation is tested for both parvovirus B19 (B19V) and hepatitis A virus (HAV). This study compared positivity rates of B19 and HAV nucleic acid tests (NATs) in Canadian plasma samples for the pre-COVID-19 restriction era (2015 to end of February 2020 [Q1] 2020) and the post-COVID-19 restriction era. MATERIALS AND METHODS: Pooled EDTA plasma specimens were tested within 24 months of blood draw using the Procleix Panther System (Grifols Diagnostic Solutions Inc, San Diego, CA, USA) for B19V and HAV detection. Reactive pools were resolved by individual specimen testing. RESULTS: Between 1 January 2015, and 31 March 2022, 3,928,619 specimens from Canadian plasma donors were tested for B19V. For the same period, 3,922,954 specimens were tested for HAV. To account for a lag in specimen testing for up to 24 months, the data were divided into: (1) a pre-pandemic period (1 January 2015-31 March 2020; B19V tested n = 2,412,701, B19V NAT-positive n = 240 [0.01%], HAV tested n = 2,407,036, HAV NAT-positive n = 26 [0.001%]); (2) a two-year mixed-impact period (1 April 2020-31 March 2022; B19V tested n = 968,250, B19V NAT-positive n = 14 [0.001%], HAV tested n = 968,250, HAV NAT-positive n = 2 [0.0002%]); and (3) a pandemic-impact period (1 April 2022-31 March, 2023; B19V tested n = 597,668, B19V NAT-positive n = 3 [0.0005%], HAV tested n = 597,668, HAV NAT-positive n = 1 [0.0002%]). CONCLUSION: The percentage of B19V- and HAV-positive donations was significantly reduced from the pre-pandemic period to the pandemic-impact period.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

A case of Tn polyagglutination discovered by an ABO blood group discrepancy.

BACKGROUND: Tn syndrome is an acquired form of polyagglutination arising from somatic mutations of hematopoietic stem cells. Tn red blood cells (RBCs) are agglutinable by naturally occurring anti-Tn antibodies in most adult sera. Current ABO typing reagents are monoclonal and do not detect polyagglutination on forward typing. However, herein we describe a case of Tn activation that was suspected due to cross-reactivity with a monoclonal anti-A reagent. STUDY DESIGN AND METHODS: A 63-year-old man with myeloproliferative neoplasm, who historically typed as group O, demonstrated unexpected mixed field reactivity with anti-A reagent using a gel-based method. However, manual tube testing was consistent with the patient's historical group O type. RESULTS: Lectin testing demonstrated reactivity with Salvia sclarea and Glycine soja, but not Arachis hypogea. The patient's RBCs produced positive crossmatches with healthy donor sera, but reactivity was eliminated by ficin pretreatment of the RBCs. Ficin treatment also resolved typing discrepancies on gel-based typing. No reactivity was noted using cord blood sera, and N antigen expression was diminished upon phenotyping. Tn activation was confirmed by detection of a novel 4-nucleotide deletion (c.395-398del) in exon 3 of C1GALT1C1 resulting in a truncated glycosyltransferase. CONCLUSION: This case of acquired Tn polyagglutination due to a novel mutation was first suspected from an ABO phenotyping discrepancy. It highlights the cross-reactivity of anti-A reagent with Tn antigen when tested on a common gel-based method. Furthermore, the case demonstrates that review of patient history and test information can clarify discrepancies and guide resolution.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Molecular blood group screening in Omani blood donors.

BACKGROUND AND OBJECTIVES: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.

Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34+ CD41+ CD235ab+ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers-CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.

Red cell genotyping of rare blood donors: donation behaviour and data visualization.

BACKGROUND: The continual identification of rare blood among donors is critical to support national programs like the American Rare Donor Program (ARDP). Some blood centres require consent from donors to be registered with a national registry. This situation provides an opportunity to determine whether a donor's willingness to register is associated with a change in donation behaviour. METHODS: Rare donors were identified by molecular typing. The average number of donations per year was compared for each donor prior to and after receiving a consent letter. Donors were categorized as either accepting or declining the request. Non-parametric t tests compared the statistical significance within and between categories. Rare types were overlaid with consensus data to look for trends using data visualization techniques. RESULTS: A total of 270 molecularly typed rare donors received letters over 4 years. Half of the donors (132, 49%) agreed to participate in the ARDP. Overall, donation frequency increased after the letter when enrolled. Both Caucasian and non-Caucasian donors increased their donations after enrolling providing an additional 159 red blood cell units over 3 years. Declining participation did not change donation frequency. Data visualization showed that enrolled donors were more affluent, high school and college educated, and lived in their home for longer periods of time. CONCLUSION: A donor's willingness to enrol in the ARDP was associated with a post-response increase in donation frequency. New interventions to reach non-Caucasian donors may be a prerequisite to increase donation frequency and a willingness to be a rare blood donor.

SCAR: The high-prevalence antigen 013.008 in the Scianna blood group system.

BACKGROUND: The Scianna (SC) blood group system comprises seven antigens. They reside on the erythroblast membrane-associated glycoprotein (ERMAP). The ERMAP and RHCE genes are juxtaposed to each other on chromosome 1. We report a novel SC antigen. STUDY DESIGN AND METHODS: Blood samples came from a patient and his two sisters in Saudi Arabia. To investigate the antibody specificity we used the column agglutination technique and soluble recombinant ERMAP protein. The significance of anti-SCAR was evaluated by the transfusion history and a monocyte monolayer assay. We determined the genomic sequence of ERMAP and RHCE genes. RESULTS: The patient's serum showed an antibody of titer 8 against a high-prevalence antigen. The soluble recombinant ERMAP protein inhibited the antibody. The propositus genotyped homozygous for an ERMAP:c.424C>G variant, for which his sisters were heterozygous. The c.424C>G variant occurred in the SC*01 allele in one haplotype with the RHCE*03 (RHCE*cE) allele. No signs of hemolysis occurred following an incompatible blood transfusion. The monocyte monolayer assay was negative. CONCLUSIONS: We characterized a high-prevalence antigen, with the proposed name "SCAR," which is the eighth antigen of the Scianna blood group system (proposed designation 013.008). Individuals homozygous for ERMAP:p.(Gln142Glu) protein variant can produce anti-SCAR. Although we did not observe any sign of hemolysis at this time, the anti-SCAR prompted a change of the treatment regimen. A review of the known reports indicated that all SC alloantibodies of sufficient titer should be considered capable of causing hemolysis.

Two Prevalent ∼100-kb GYPB Deletions Causative of the GPB-Deficient Blood Group MNS Phenotype S-s-U- in Black Africans.

The U antigen (MNS5) is one of 49 antigens belonging to the MNS blood group system (ISBT002) carried on glycophorins A (GPA) and B (GPB). U is present on the red blood cells in almost all Europeans and Asians but absent in approximately 1.0% of Black Africans. U negativity coincides with negativity for S (MNS3) and s (MNS4) on GPB, thus be called S-s-U-, and is thought to arise from homozygous deletion of GYPB. Little is known about the molecular background of these deletions. Bioinformatic analysis of the 1000 Genomes Project data revealed several candidate regions with apparent deletions in GYPB. Highly specific Gap-PCRs, only resulting in positive amplification from DNAs with deletions present, allowed for the exact genetic localization of 3 different breakpoints; 110.24- and 103.26-kb deletions were proven to be the most frequent in Black Americans and Africans. Among 157 CEPH DNAs, deletions in 6 out of 8 African ethnicities were present. Allele frequencies of the deletions within African ethnicities varied greatly and reached a cumulative 23.3% among the Mbuti Pygmy people from the Congo. Similar observations were made for U+var alleles, known to cause strongly reduced GPB expression. The 110- and 103-kb deletional GYPB haplotypes were found to represent the most prevalent hereditary factors causative of the MNS blood group phenotype S-s-U-. Respective GYPB deletions are now accessible by molecular detection of homo- and hemizygous transmission.

Three non-classical mechanisms for anemic disease of the fetus and newborn, based on maternal anti-Kell, anti-Ge3, anti-M, and anti-Jra cases.

Maternal alloantibody-mediated hemolytic disease of the fetus and newborn (HDFN) ranges from no or mild symptoms to severe hydrops and intrauterine fetal demise. Hemolytic anti-D-mediated HDFN proceeds via a long-known mechanism, to which three other pathways to fetal/neonatal anemia may be added: (0) Fetal erythrocyte destruction can proceed by extravascular phagocytosis. (1) An apoptotic pathway has been described for anti-Kell, and anti-Ge3. (2) Erythropoietic suppression may arise from altered or deformed erythroblast architecture in anti-M-mediated disease. (3) Clonal escape from erythropoietic suppression is hypothesized to arise from maternal anti-Jra immune pressure, albeit this requires further elucidation. Alloantibody-mediated anemic disease of the fetus and newborn (ADFN) is a designation we favor for cases when hemolysis or hyperbilirubinemia are not the dominant features, such as those provoked by anti-Kell, anti-Ge3, anti-M, and anti-Jra.

Predictive modeling of complex ABO glycan phenotypes by lectin microarrays.

Serological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.

Complement activating ABO anti-A IgM/IgG act synergistically to cause erythrophagocytosis: implications among minor ABO incompatible transfusions.

BACKGROUND: Typically minor ABO incompatible platelet products are transfused without any incident, yet serious hemolytic transfusion reactions occur. To mitigate these events, ABO 'low titer' products are used for minor ABO incompatible transfusions. We sought to understand the role of IgM/IgG and complement activation by anti-A on extravascular hemolysis. METHODS: Samples evaluated included (i) Group O plasma from a blood donor whose apheresis platelet product resulted in an extravascular transfusion reaction, (ii) Group O plasma from 12 healthy donors with matching titers that activated complement (N = 6) or not (N = 6), and (iii) Group O sera from 10 patients with anti-A hemolysin activity. A flow cytometric monocyte erythrophagocytosis assay was developed using monocytes isolated by immunomagnetic CD14-positive selection from ACD whole blood of healthy donors. Monocytes were frozen at - 80 °C in 10% dimethyl sulfoxide/FBS and then thawed/reconstituted on the day of use. Monocytes were co-incubated with anti-A-sensitized fluorescently-labeled Group A1 + RBCs with and without fresh Group A serum as a source of complement C3, and erythrophagocytosis was analyzed by flow cytometry. The dependency of IgM/IgG anti-A and complement C3 activation for RBC erythrophagocytosis was studied. Anti-A IgG subclass specificities were examined for specific samples. RESULTS: The plasma and sera had variable direct agglutinating (IgM) and indirect (IgG) titers. None of 12 selected samples showed monocyte-dependent erythrophagocytosis with or without complement activation. The donor sample causing a hemolytic transfusion reaction and 2 of the 10 patient sera with hemolysin activity showed significant erythrophagocytosis (> 10%) only when complement C3 was activated. The single donor plasma and two sera demonstrating significant erythrophagocytosis had high IgM (≥ 128) and IgG titers (> 1024). The donor plasma anti-A was IgG1, while the patient sera were an IgG3 and an IgG1 plus IgG2. CONCLUSION: High anti-A IgM/IgG titers act synergistically to cause significant monocyte erythrophagocytosis by activating complement C3, thus engaging both Fcγ- and CR1-receptors.