Monitoring the SARS-CoV-2 Pandemic: Prevalence of Antibodies in a Large, Repetitive Cross-Sectional Study of Blood Donors in Germany-Results from the SeBluCo Study 2020-2022.

SARS-CoV-2 serosurveillance is important to adapt infection control measures and estimate the degree of underreporting. Blood donor samples can be used as a proxy for the healthy adult population. In a repeated cross-sectional study from April 2020 to April 2021, September 2021, and April/May 2022, 13 blood establishments collected 134,510 anonymised specimens from blood donors in 28 study regions across Germany. These were tested for antibodies against the SARS-CoV-2 spike protein and nucleocapsid, including neutralising capacity. Seroprevalence was adjusted for test performance and sampling and weighted for demographic differences between the sample and the general population. Seroprevalence estimates were compared to notified COVID-19 cases. The overall adjusted SARS-CoV-2 seroprevalence remained below 2% until December 2020 and increased to 18.1% in April 2021, 89.4% in September 2021, and to 100% in April/May 2022. Neutralising capacity was found in 74% of all positive specimens until April 2021 and in 98% in April/May 2022. Our serosurveillance allowed for repeated estimations of underreporting from the early stage of the pandemic onwards. Underreporting ranged between factors 5.1 and 1.1 in the first two waves of the pandemic and remained well below 2 afterwards, indicating an adequate test strategy and notification system in Germany.

New ultraviolet C light-based method for pathogen inactivation of red blood cell units.

BACKGROUND: Pathogen inactivation (PI) technologies for platelet concentrates and plasma are steadily becoming more established, but new PI treatment options for red blood cells (RBCs), the most commonly used blood component, still need to be developed. We present a novel approach to inactivating pathogens in RBC units employing ultraviolet C (UVC) light. METHODS: Whole blood-derived leukoreduced RBCs suspended in PAGGS-C, a third generation additive solution, served as test samples, and RBCs in PAGGS-C or SAG-M as controls. Vigorous agitation and hematocrit reduction by diluting the RBCs with additional additive solution during illumination ensured that UVC light penetrated and inactivated the nine bacteria and eight virus species tested. Bacterial and viral infectivity assays and in vitro analyses were performed to evaluate the system's PI capacity and to measure the RBC quality, metabolic, functional, and blood group serological parameters of UVC-treated versus untreated RBCs during 36-day storage. RESULTS: UVC treatment of RBCs in the PAGGS-C additive solution did not alter RBC antigen expression, but significantly influenced some in vitro parameters. Compared to controls, hemolysis was higher in UVC-treated RBC units, but was still below 0.8% at 36 days of storage. Extracellular potassium increased early after PI treatment and reached ≤70 mmol/L by the end of storage. UVC-treated RBC units had higher glucose and 2,3-diphosphoglycerate levels than controls. CONCLUSION: As UVC irradiation efficiently reduces the infectivity of relevant bacteria and viruses while maintaining the quality of RBCs, the proposed method offers a new approach for PI of RBC concentrates.

Two novel antithetical KN blood group antigens may contribute to more than a quarter of all KN antisera in Europe.

BACKGROUND: All antigens described in the KN blood group system are located in the long homologous repeat D (LHR-D) of complement receptor 1 (CR1). While there have been reports that some sera react only with the long homologous repeat C (LHR-C), the antigens in LHR-C are unknown. STUDY DESIGN AND METHODS: Recombinant LHR-C and LHR-D were used to identify antibodies directed against LHR-C of CR1, into which a point mutation was introduced to characterize the underlying blood group antigens. In addition, database studies to define haplotypes of CR1 were performed. RESULTS: Several antisera were identified that were specific against CR1 p.1208His and against CR1 p.1208Arg, located in LHR-C. Fifteen KN haplotypes were found in the Ensembl genome browser. It was shown that due to a linkage disequilibrium anti-CR1 p.1208His may be mistaken for anti-KCAM. CONCLUSION: A novel antithetical KN blood group antigen pair was found at position p.1208 of CR1, for which the names DACY and YCAD are proposed. Antibodies against these two novel antigens seem to contribute to more than a quarter of all KN sera in Europe.

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting.

BACKGROUND: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. METHODS: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. RESULTS: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. CONCLUSIONS: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

Molecular typing for blood group antigens within 40 min by direct polymerase chain reaction from plasma or serum.

Determining blood group antigens by serological methods may be unreliable in certain situations, such as in patients after chronic or massive transfusion. Red cell genotyping offers a complementary approach, but current methods may take much longer than conventional serological typing, limiting their utility in urgent situations. To narrow this gap, we devised a rapid method using direct polymerase chain reaction (PCR) amplification while avoiding the DNA extraction step. DNA was amplified by PCR directly from plasma or serum of blood donors followed by a melting curve analysis in a capillary rapid-cycle PCR assay. We evaluated the single nucleotide polymorphisms underlying the clinically relevant Fya , Fyb , Jka and Jkb antigens, with our analysis being completed within 40 min of receiving a plasma or serum sample. The positive predictive value was 100% and the negative predictive value at least 84%. Direct PCR with melting point analysis allowed faster red cell genotyping to predict blood group antigens than any previous molecular method. Our assay may be used as a screening tool with subsequent confirmatory testing, within the limitations of the false-negative rate. With fast turnaround times, the rapid-cycle PCR assay may eventually be developed and applied to red cell genotyping in the hospital setting.

The Rhesus Site.

The Rhesus Site is a resource for information of the 'Rhesus' blood group. It is intended for specialists and non-specialists. The website details research in the field relevant for transfusion medicine, immunohematology, and molecular research. Link areas guide to important publications and to methodological resources for Rhesus. Many data originally presented at The Rhesus Site have been formally published later. The 'RhesusBase' section represents the largest database for RHD alleles; the 'RhesusSurveillance' section details the results of the largest prospective observational study on anti-D immunization events in D-positive patients. Visitors to the website are encouraged to explore the intricacies of the most complex blood group gene locus.

RHD PCR of D-Negative Blood Donors.

SUMMARY: RHD PCR of blood donors may be used to reveal weak D, partial D, DEL and chimeric D+/D- donors among presumed D-negative blood donors. Units donated by such donors pose a definite yet low risk for anti-D immunization of transfusion recipients. The frequency of DEL donors among D-negative donors is 1:350 to 1:2,000 in Europe and up to 1:5 in Asian countries. Different strategies for RHD PCR of blood donors have been used. Probably, the most cost-efficient implementation is replacement of sensitive D antigen testing with the indirect antiglobulin test by RHD PCR in pools which might even reduce total testing cost.

D category IV: a group of clinically relevant and phylogenetically diverse partial D.

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

Evaluation of single-nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups.

BACKGROUND: Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. STUDY DESIGN AND METHODS: DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma. RESULTS: The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. CONCLUSION: Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples.

Molecular testing in transfusion medicine.

IMPORTANCE OF THE FIELD: Molecular blood group prediction, including mass scale donor genotyping, is becoming a routine part of transfusion medicine. AREAS COVERED IN THIS REVIEW: This review covers transfusion-related applications of the molecular prediction of antigens of red blood cells. Forensic and prenatal applications are not covered. The review is mainly based on the personal experience of the author in the field. A PubMed literature search without temporal limit was performed using the search item 'molecular testing transfusion' and complemented by 'related articles' searches of key publications. WHAT THE READER WILL GAIN: The readers will understand the general principles and specific problems arising from the use of molecular testing for blood group antigen prediction, get an overview of methods used at present, and learn which methods are suitable for which specific applications. TAKE HOME MESSAGE: At present, molecular methods complement serologic antigen determination and are an invaluable tool for specific diagnostic problems. Mass-scale molecular antigen prediction of blood donors is beginning to improve blood supply for specific patient groups.