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- Alattar, AG, et al.
(author)
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Gene editing of CD34+ progenitor cells from single blood donor waste bags to create cultured early erythroid cells for study of blood group knock-outs
- 2020
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In: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 115:Suppl. s1, s. 363-363
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Conference paper (peer-reviewed)abstract
- Background: Many blood group antigens are carried by red cell surface proteins, thefunctions of which are not yet fully characterized or in some cases completelyunknown. By investigating red cells with naturally-occurring blood group variants,much has been learnt about the underlying molecules. However, interindividualvariation affecting other molecules than the one(s) of interest may confound orotherwise hamper such studies. As an alternative, a reductionistic approach whereonly a single factor differs between test and control cells significantly facilitatesinterpretation of functional studies. Using various siRNA, shRNA and various gene-editing tools blood group expression can be manipulated and useful modelsdeveloped. Applying the latter on primary hematopoietic stem and progenitor cells(HSPCs) can be challenging.Aims: We evaluated a protocol for gene editing of CD44 using a CRISPR/Cas9hybrid system on HSPCs isolated from blood donation leukocyte waste bags fromsingle donors to develop a model for study of blood group molecular function inerythropoiesis.Methods: Peripheral blood mononuclear cells (PBMCs) were from anonymizedleucocyte waste bags obtained after whole blood unit processing in the Reveosautomated blood component system. Cells were harvested following Lymphoprepgradient separation and CD34+HSPCs enriched and collected using magnetic beads.CD34+cells were cultured in 2-phase culture medium to generate erythroid cellsfrom HSPCs (Vidovic, Vox Sang 2017). For CRISPR/Cas9 gene editing, a short guideRNA (sgRNA) targeting CD44 was designed and cloned into the lentiCRISPR v2vector (Addgene plasmid #52961). A non-targeting sgRNA cloned into the vectorwas used as control. Lentivirus particles were produced in the human 293T cell lineas described previously (Galeev, Methods Mol Biol 2017). Equal number of CD34+cells were transduced 24 hours after collection using RetroNectin following theRetroNectin-Bound Virus (RBV) Infection Method according to manufacturer’sprotocol. Cells were transduced at a multiplicity of infection (MOI) of 10 with atarget transduction efficiency of 20–30%. Cells were cultured at 37°C, 5% CO2 for72 hours in phase I culture medium and then electroporated with Cas9 mRNA usingthe ECM 830 Electroporation System as described previously for cord blood-derivedCD34+cells (Backstrom, Exp Hematol 2019). GFP+CD44-edited cell frequencieswere monitored by flow cytometry at day 7 of the HSPC expansion phase and atday 14 of the erythroid expansion-differentiation phase using antibodies againsterythroid-specific cell surface markers GPA and Band3 in addition to CD49d toassess the erythroid development stage.Results: We tested the above protocol and observed that the frequencies of editedcells lacking CD44 expression within the GFP+population at day 7 of culture were10–25% while the edited frequencies were increased at day 14 of culture to 60–80%within the GFP+cells. Whilst this stage corresponds to erythroblasts, earlier or laterstages can be tested.Summary/Conclusions: The previously established hybrid system for CRISPR/Cas9gene editing in cord blood-derived CD34+HSPCs, which combines lentiviraldelivery of the sgRNA with transient delivery of Cas9 mRNA by electroporation, isalso applicable to primary human adult HSPCs from Reveos-processed single-donorwhole blood donation, resulting in a traceable high-yield gene-editing system tostudy blood group function and erythroid development
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| 5. |
- Clausen, Frederik Banch, et al.
(author)
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External quality assessment of noninvasive fetal RHD genotyping
- 2020
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In: Vox Sanguinis. - : Wiley. - 0042-9007 .- 1423-0410. ; 115:5, s. 466-471
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Journal article (peer-reviewed)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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| 6. |
- Clausen, Frederik Banch, et al.
(author)
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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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In: Vox Sanguinis. - : Wiley. - 0042-9007 .- 1423-0410. ; 117:2, s. 157-165
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Research review (peer-reviewed)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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| 7. |
- Gassner, Christoph, et al.
(author)
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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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In: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 117:11, s. 1332-1344
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Journal article (peer-reviewed)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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| 8. |
- Hellberg, Åsa, et al.
(author)
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A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype
- 2023
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In: Vox Sanguinis. - : John Wiley & Sons. - 0042-9007 .- 1423-0410. ; 118:8, s. 690-694
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Journal article (peer-reviewed)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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| 9. |
- Hult, Andreas, Med Dr, 1981-, et al.
(author)
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In vitro phagocytosis of liquid-stored red blood cells requires serum and can be inhibited with fucoidan and dextran sulphate
- 2020
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In: Vox Sanguinis. - : John Wiley & Sons. - 0042-9007 .- 1423-0410. ; 115:8, s. 647-654
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Journal article (peer-reviewed)abstract
- BACKGROUND AND OBJECTIVES: Red-blood-cells (RBCs) undergo structural and metabolic changes with prolonged storage, which ultimately may decrease their survival after transfusion. Although the storage-induced damage to RBCs has been rather well described biochemically, little is known about the mechanisms underlying the recognition and rapid clearance of the damaged cells by macrophages.MATERIALS AND METHODS: We, here, used a murine model for cold (+4°C) RBC storage and transfusion. Phagocytosis of human or murine RBCs, liquid stored for 6-8 weeks or 10-14 days, respectively, was investigated in murine peritoneal macrophages.RESULTS: The effects of storage on murine RBCs resembled that described for stored human RBCs with regard to decreased adenosine triphosphate (ATP) levels, accumulation of microparticles (MPs) during storage, and RBC recovery kinetics after transfusion. Under serum-free conditions, phagocytosis of stored human or murine RBCs in vitro was reduced by 70-75%, as compared with that in the presence of heat-inactivated fetal calf serum (FCS). Human serum promoted phagocytosis of stored human RBCs similar to that seen with FCS. By adding fucoidan or dextran sulphate (blockers of scavenger receptors class A (SR-A)), phagocytosis of human or murine RBCs was reduced by more than 90%. Phagocytosis of stored human RBCs was also sensitive to inhibition by the phosphatidylinositol 3 kinase-inhibitor LY294002, the ERK1/2-inhibitor PD98059, or the p38 MAPK-inhibitor SB203580.CONCLUSION: RBCs damaged during liquid storage may be recognized by macrophage SR-A and serum-dependent mechanisms. This species-independent recognition mechanism may help to further understand the rapid clearance of stored RBCs shortly after transfusion.
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| 10. |
- Jongruamklang, Philaiphon, et al.
(author)
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Characterization of GYP*Mur and novel GYP*Bun-like hybrids in Thai blood donors reveals a qualitatively altered s antigen
- 2020
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In: Vox Sanguinis. - : Wiley. - 0042-9007 .- 1423-0410. ; 115:5, s. 472-477
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Journal article (peer-reviewed)abstract
- Background and objectives: The Mi(a+) GP(B-A-B) hybrid phenotypes occur with a prevalence of 2%–23% across Southeast Asia. While the s antigen is alleged to be altered, no evidence for specific variants is known. Screening using a monoclonal IgM anti-s mistyped six S‒s+ RBC units as S‒s‒. Further, alloanti-s was identified in an S+s+ patient. Our objective was to investigate the s antigen further. Materials and methods: DNA from 63 Thai blood donor samples PCR-positive for a GYP(B-A-B) hybrid was sequenced with primers spanning GYPB exons 3–4. Flow cytometry was used for semiquantitative analysis of s expression and correlated with the glycophorin genotype. Results: DNA sequencing showed that GYP*Mur was carried by 56/63 samples (88·9%) of which 5/56 lacked normal GYPB: three of these were GYP*Mur homozygotes, one was a compound heterozygote carrying GYP*Mur and a GYP*Bun-like allele (designated GYP*Thai), and the fifth sample carried GYP*Mur and another GYP*Bun-like allele. Seven samples (7/63) were GYP*Thai heterozygotes. IgM monoclonal anti-s (P3BER) did not react with the s antigen carried by GP.Mur or GP.Bun, whereas two IgG anti-s showed enhanced reactivity. Conclusions: We confirmed that GYP*Mur is the most frequent variant in Thai blood donors and also identified GYP*Thai with a frequency of 1·1%. We showed that s antigen on Mi(a+) GP(B-A-B) hybrids is qualitatively altered and should be considered when selecting reagents for phenotyping where such hybrids are prevalent, endemically and in blood centres worldwide.
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