| 1. |
- Clausen, Frederik Banch, et al.
(författare)
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External quality assessment of noninvasive fetal RHD genotyping
- 2020
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Ingår i: Vox Sanguinis. - : Wiley. - 0042-9007 .- 1423-0410. ; 115:5, s. 466-471
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Tidskriftsartikel (refereegranskat)abstract
- Background and objectives: Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories. Materials and methods: Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly. Results: Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory. Conclusion: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting.
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| 2. |
- Clausen, Frederik Banch, et al.
(författare)
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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
- 2022
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Ingår i: Vox Sanguinis. - : Wiley. - 0042-9007 .- 1423-0410. ; 117:2, s. 157-165
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 3. |
- Gassner, Christoph, et al.
(författare)
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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
- 2022
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Ingår i: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 117:11, s. 1332-1344
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Tidskriftsartikel (refereegranskat)abstract
- 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/).
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| 4. |
- Hellberg, Åsa, et al.
(författare)
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A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype
- 2023
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Ingår i: Vox Sanguinis. - : John Wiley & Sons. - 0042-9007 .- 1423-0410. ; 118:8, s. 690-694
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Tidskriftsartikel (refereegranskat)abstract
- Background and ObjectivesThe extremely rare Rhnull phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rhnull cells have altered expression of glycophorin B and LW glycoprotein.Materials and MethodsFour unrelated Rhnull individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele-specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in-house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation.ResultsAnti-Rh29 was identified in all four individuals. Extended typing with anti-S and anti-s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti-s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3′-end of intron 6, c.946 −2a>g in all samples. RHD/RHCE showed no alterations.ConclusionA novel Nordic Rhnull allele was identified. In addition, it was shown that s+ Rhnull red blood cells are not only U− but also have qualitative changes in their s antigen expression.
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| 5. |
- Hult, Annika K, et al.
(författare)
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A new missense variant in exon 7 of the ABO gene, c.662G>A, in a family with B w phenotype.
- 2022
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Ingår i: Transfusion. - : Wiley. - 1537-2995 .- 0041-1132. ; 62:10, s. 55-58
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Tidskriftsartikel (refereegranskat)abstract
- 1 BACKGROUNDWeak expression of ABO antigens is encountered in the clinical laboratory occasionally, and subgroups of A are more commonly observed in Europeans than subgroups of B. To date, weakly expressing B variant phenotypes have been associated with 38 different alleles according to ISBT (https://www.isbtweb.org/resource/001aboalleles.html). This number is an underrepresentation since there have been several reports of aberrant B expression due to variant alleles since the last update of the ISBT allele table. The current study was initiated by an unusual blood group typing result in a 55-year-old male patient of Czech origin and previously reported as an abstract.12 BRIEF METHODSBlood grouping was performed according to standard blood banking practice, initially using an automatic analyzer (Galileo, Immucor) followed by confirmation with manual gel (BioRad; DG-Gel) and tube agglutination techniques. Initial genotyping analysis was done using a PCR-SSP kit (Innotrain), microarray (BloodChip Reference, Progenika) and subsequently verified by expanded PCR-ASP and PCR-RFLP as described previously.2, 3 ABO exons 1–7 and splice sites were amplified and analyzed, together with the product(s) of PCR-ASP for exons 6–7, by Sanger sequencing.4 A single nucleotide variation (SNV) was detected, and the localization of the affected amino acid is visualized in a 3D-model of ABO glycosyltransferase by Cn3D (v.4.3.1, www.ncbi.nih.gov) and a detailed view obtained by AlphaFold.5, 6 Flow cytometry testing with monoclonal ABO reagents was performed as described previously.73 RESULTSThe proband's red blood cells (RBCs) initially typed as group O but the plasma typing gave negative or weak reactions with test RBCs of group B, depending on the method used, Table 1. An ABO*B.01/O.01.01 genotype was revealed, normally consistent with group B. Screening for selected A and B subgroup allele markers was negative.2 After informed consent, samples from family members were drawn and further investigation was performed.In samples from the proband, his sister and niece, sequence analysis revealed heterozygosity for a SNV in ABO exon 7, c.662G>A (no rs number available) in an otherwise normal ABO*B.01 allele. Significantly weakened B antigen expression was observed in all three individuals. An overview of serological testing and genetic results is shown in Table 1.SNV c.662G>A encodes an amino acid change, p.Gly221Asp. The glycine residue is completely evolutionarily conserved among the members of the GT6 family of glycosyltransferases8 and centrally located in the enzyme, seven amino acids away from the DVD motif (pp. 211–213) that coordinates the Mn2+ ion and the UDP part of the UDP-galactose donor substrate (Figure 1A). However, it is not directly interfering with the catalytic site. Instead, the change of the small neutral glycine to the bulkier and charged aspartic acid is predicted to abolish selected hydrogen bonds and is therefore hypothesized to destabilize the protein conformation (Figure 1B).5, 6
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| 6. |
- Jakobsen, Marianne A., et al.
(författare)
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A novel ABO allele with a 21-bp duplication identified in two unrelated European individuals with weak A expression
- 2020
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Ingår i: Transfusion Medicine. - : Wiley. - 0958-7578 .- 1365-3148. ; 30:6, s. 508-512
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: To carry out genetic and serological analyses of a Swiss blood donor and a Danish patient carrying an aberrant ABO phenotype with weak A expression. Background: ABO is the most clinically important blood group system but also one of the most complex. The system antigens are determined by carbohydrate structures generated by A and B glycosyltransferases encoded by the ABO gene. Genetic variants of ABO may encode a glycosyltransferase with reduced activity, leading to weak expression of A antigen. Methods: Samples from two individuals were examined using genetic testing and extended immunohaematological evaluation, including standard serological methods, flow cytometry and analysis of plasma glycosyltransferase activity. Results: Both individuals were serologically determined to be AweakB. Genetic testing revealed that both were heterozygous for a novel ABO*A1.01-like allele with an in-frame duplication of 21 nucleotides in exon 7 (c.543_563dup), leading to the insertion of seven amino acids (QDVSMRR). Flow cytometric testing of native red blood cells (RBCs) showed very weak A antigen expression. This was in accordance with the enzyme activity test. Conclusion: In summary, we describe a novel A allele with a duplication of 21 nucleotides in exon 7 that significantly decreases the enzyme activity and leads to very weak expression of A antigen. (200 words).
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| 7. |
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| 8. |
- Ricci Hagman, Jennifer, et al.
(författare)
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Truncated glycosyltransferase coding regions in novel ABO alleles give rise to weak A or B blood group expression and discrepant typing results
- 2023
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Ingår i: Transfusion. - 1537-2995. ; 63:10, s. 1951-1961
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundCorrect ABO blood-group matching between donor and patient is crucial for safe transfusions. We investigated the underlying reason causing inconclusive ABO serology in samples referred to our laboratory.Study Design and MethodsFlow cytometric analysis, ABO genotyping, and sequencing were used to characterize ABO-discrepant blood samples (n = 13). ABO gene variants were inserted in a GFP-containing bicistronic vector to assess A/B expression following overexpression in HeLa cells.ResultsSeven novel alleles with nonsense mutations predicted to truncate the encoded ABO glycosyltransferases were identified. While these variants could represent O alleles, serology showed signs of ABO glycosyltransferase activity. ABO*A1.01-related alleles displayed remarkably characteristic percentages of A-positive cells for samples with the same variant: c.42C>A (p.Cys14*; 10%), c.102C>A (p.Tyr34*; 31%–32%, n = 2), c.106dup (p.Val36Glyfs*21; 16%–17%, n = 3) or c.181_182ins (p.Leu61Argfs*21; 12%–13%, n = 2). Transfection studies confirmed significantly decreased A expression compared to wild type. The remaining variants were found on ABO*B.01 background: c.1_5dup (pGly3Trpfs*20), c.15dup (p.Arg6Alafs*51) or c.496del (p.Thr166Profs*26). Although the absence of plasma anti-B was noted overall, B antigen expression was barely detected on erythrocytes. Overexpression confirmed decreased B in two variants compared to wildtype while c.1_5dup only showed a non-significant downward trend.ConclusionSamples displaying aberrant ABO serology revealed seven principally interesting alleles. Despite the presence of truncating mutations, normally resulting in null alleles, low levels of ABO antigens were detectable where alterations affected ABO exons 1–4 but not exon 7. This is compatible with the previously proposed concept that alternative start codons in early exons can be used to initiate the translation of functional ABO glycosyltransferase.
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| 9. |
- Stenfelt, Linn, et al.
(författare)
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Glycoproteomic and Phenotypic Elucidation of B4GALNT2 Expression Variants in the SID Histo-Blood Group System
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067 .- 1661-6596. ; 23:7
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Tidskriftsartikel (refereegranskat)abstract
- The Sd(a) histo-blood group antigen (GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a-) individuals (2-4% of Europeans) may produce anti-Sd(a), which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a-), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a-) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SIDnull candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sd(a) synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a-) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg beta 4GalNAc-T2 variant causes Sd(a)-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.
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| 10. |
- Stenfelt, Linn, et al.
(författare)
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The P1PK blood group system : revisited and resolved
- 2020
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Ingår i: Immunohematology. - 0894-203X. ; 36:3, s. 99-103
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Tidskriftsartikel (refereegranskat)abstract
- CONCLUSIONS: This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25-33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.
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