Identifi cation of ABO, RH and KEL blood group antigens with serology and genotyping methods

Introduction. Blood transfusion is a strong practice in traumatology, internal medicine, haematology, obstetrics and transplantation, which demands safety of haemotransfusion with estimating the red blood cell group antigens in donor and recipient blood. Routine immunotyping techniques usually provide for an antigen identification to weak subgroups, albeit with certain inherent limitations of serology tests that can be overcome in a genotyping approach.

Aim — performance assessment of serology and genotyping methods in the ABO, RH and KEL blood group identification.

Materials and methods. A total of 55,489 donor and 1,898 patient blood samples have been analysed. Ambiguous cases of chimerism, panagglutination and inconsistent results were tackled with genotyping. Serology tests were performed with gel cards. Whole blood DNA extraction was performed with Qiagen chemistry. Allele-specific PCR was used for the erythrocyte ABO, RH and KEL antigen genotyping with BAG Diagnostics commercial kits and a 2% agarose gel product detection. Sanger sequencing was used to complement genotyping.

Results. A combined use of serology tests and genotyping allowed a successful erythrocyte antigen-based blood group and Rh-status assignment in 26 donors and patients with ambiguous blood typing.

Conclusion. Genotyping coupled with serologic methods can be advised in a hampered blood group identification.

1. Javadzadeh Javadzadeh S.H., Hayati A. Blood group discrepancies at a regional blood center. Int J Hematol Oncol Stem Cell Res. 2020; 14(1): 38–44.

2. Kaur G., Kaur P., Basu S., Kaur R. Blood group discrepancies at a tertiary care centre — analysis and resolution. Int J Lab Hematol. 2014; 36(4): 481–7. DOI: 10.1111/ijlh.12176.

3. Garratty G. Do we need to be more concerned about weak D antigens? Transfusion. 2005; 45(10): 1547–51. DOI: 10.1111/j.1537-2995.2005.00625.x.

4. Golovkina L.L., Stremoukhova A.G., Pushkina T.D., et al. Molecular techniques for erythrocyte antigen blood typing. Spravochnik zavedujushego KDL. 2018; 9: 46–56 (In Russian).

5. Goebel M., Halm-Heinrich I., Parkner A., et al. Novel ABO gene variant leads to discrepant results in forward/reverse and molecular blood grouping. Transfus Med Hemother. 2013; 40(6): 454–8. DOI: 10.1159/000356378.

6. https://www.ncbi.nlm.nih.gov/snp/rs573234689#frequency_tab

7. https://www.ncbi.nlm.nih.gov/snp/rs782058388#frequency_tab

8. Yip S.P. Sequence variation at the human ABO locus. Ann Hum Genet. 2002; 66(1): 1–27. DOI: 10.1017/S0003480001008995.

9. Olsson M.L., Chester M.A. Heterogeneity of the blood group Ax allele: Genetic recombination of common alleles can result in the Ax phenotype. Transfus Med. 1998; 8(3): 231–8. DOI: 10.1046/j.1365-3148.1998.00161.x.

10. Ogasawara K., Yabe R., Uchikawa M., et al. Molecular genetic analysis of variant phenotypes of the ABO blood group system. Blood. 1996; 88(7): 2732–7.

11. Seltsam A., Das Gupta C., Bade-Doeding C., Blasczyk R. A weak blood group A phenotype caused by a translation-initiator mutation in the ABO gene. Transfusion. 2006; 46(3): 434–40. DOI: 10.1111/j.1537-2995.2006.00740.x.

12. Storry J.R., Olsson M.L. Genetic basis of blood group diversity. Br J Haematol. 2004; 126(6): 759–71. DOI: 10.1111/j.1365-2141.2004.05065.x.

13. Donskov S.I., Morokov V.A. Human blood groups: an immunoserologic guide. Moscow: Binom. 2011. 1016 p. (In Russian)

14. Golovkina L.L., Kalandarov R.S., Stremoukhova A.G., et al. Distinguishing A1 and A2 subgroups of ABO system: biological background and serologic strategy. Russian journal of hematology and transfusiology. 2019; 64(4): 504–515. DOI: 10.35754/0234-5730-2019-64-4-504-515 (In Russian).

15. Zhiburt E.B. Textbook. Transfusiology. Saint-Petersburg: Piter; 2002: 736 p. (In Russian).

16. Seltsam A., Hallensleben M., Kollmann A., Blasczyk R. The nature of diversity and diversification at the ABO locus. Blood. 2003; 102(8): 3035–42. DOI: 10.1182/blood-2003-03-0955.

17. Khorshidfar M., Chegini A., Pourfathollah A.A., et al. Establishing blood group genotyping to resolve ABO discrepancies in Iran. Indian J Hematol Blood Transfus. 2019; 35: 538–43. DOI: 10.1007/s12288-018-1044-8.

18. Hosseini-Maaf B., Hellberg A., Chester A.M., et al. Structural basis for change in activity of naturally occurring mutants of six new identified B-subgroup alleles in human ABO. Transfusion. 2007; 47(5): 864–75. DOI: 10.1111/j.1537-2995.2007.01203.x.

19. Mineeva N.V. Fundamentals of immunohaematology. Human blood groups. 2nd edition. Saint-Petersburg. 2007. 188 p. (In Russian).

20. Flegel W.A. Molecular genetics and clinical applications for RH. Transfus Apher Sci. 2011; 44(1): 81–91. DOI: 10.1016/j.transci.2010.12.013.

21. Flegel W. The genetics of the Rhesus Blood Group system. Blood transfusion. 2007; 5: 50–7. DOI: 10.2450/2007.0011-07.

22. Golovkina L.L., Stremoukhova A.G., Pushkina T.D., et al. Molecular and serologic traits of weak antigen D types in Rhesus system. Terapevticheskiy arkhiv. 2016; 88(7): 78–83. DOI: 10.17116/terarkh201688778-83f (In Russian).

23. Shao C.P., Maas J.H., Su Y.Q., et al. Molecular background of Rh D-positive, D-negative, Del and weak D phenotypes in Chinese. Vox Sang. 2002; 83(2): 156–61. DOI: 10.1046/j.1423-0410.2002.00192.x.

24. Wagner F.F., Gassner C., Müller T.H., et al. Molecular basis of weak D phenotypes. Blood. 1999; 93(1): 385–93.

25. Wagner F.F., Frohmajer A., Flegel W.A. RHD positive haplotypes in D negative Europeans. BMC Genet. 2001; 2: 10. DOI: 10.1186/1471-2156-2-10.

26. Sandler S.G., Flegel W.A., Westhoff C.M., et al.; College of American Pathologists Transfusion Medicine Resource Committee Work Group. It’s time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion. 2015; 55(3): 680–9. DOI: 10.1111/trf.12941.

27. Levitt J., ed. Standards for blood banks and transfusion service. 29th ed. American Association of Blood Banks; Bethesda, MD: 2014. http://www.aabb.org/sa/standards/Pages/standards-resources.aspx.

28. Flegel W.A. How I manage donors and patients with a weak D phenotype. Curr Opin Hematol. 2006; 13: 476–83. DOI: 10.1097/01.moh.0000245694.70135.c3.

29. Pham B.N., Roussel M., Peyrard T., et al. Anti-D investigations in individuals expressing weak D Type 1 or weak D Type 2: allo- or autoantibodies? Transfusion. 2011; 51(12): 2679–85. DOI: 10.1111/j.1537-2995.2011.03207.x.

30. Decree of the Government of the Russian Federation No. 797 of 22 June, 2019, “On approval of the rules for procurement, storage, transportation and clinical use of donor blood and its components and on expiration of certain acts of the Government of the Russian Federation”. (In Russian).

31. Wagner F.F. RHD PCR of D-negative blood donors. Transfus Med Hemother. 2013; 40(3): 172–81. DOI: 10.1159/000351604.

32. Golovkina L.L., Stremoukhova A.G., Pushkina T.D., et al. First observation of Rhnull Rh-phenotype in Russia. Spravochnik zavedujushego KDL. 2015; 10: 14– 200 (In Russian).

33. Flatt J.F., Musa R.H., Ayob Y., et al. Study of the D phenotype reveals erythrocyte membrane alterations in the absence of RHCE. Br J Haematol. 2012; 158: 262–73. DOI: 10.1111/j.1365-2141.2012.09149.x.

34. Pour M.S.S., Soleimany S., Ghasemimehr N., et al. A case report of a rare Rh phenotype: D–. Indian J Hematol Blood Transfus. 2019; 35: 402–4. DOI: 10.1007/s12288-019-01089-7.

35. Denomme G.A. Prospects for the provision of genotyped blood for transfusion. Br J Haematol. 2013; 163: 3–9. DOI: 10.1111/bjh.12476.

36. Flegel W.A., Wagner F.F. DEL. Blood Transfus. 2020; 18(3): 159–62. DOI: 10.2450/2020.0296-19.

37. Standards for molecular testing for red cell, platelet, and neutrophil antigens. 5th ed. American Association of Blood Banks; Bethesda, MD: 2020. http://www.aabb.org/sa/standards/Pages/standards-resources.aspx.

38. Denomme G.A., Flegel W.A. Applying molecular immunohematology discoveries to standards of practice in blood banks: Now is the time. Transfusion. 2008; 48(11): 2461–75. DOI: 10.1111/j.1537-2995.2008.01855.x.

39. van der Schoot C.E., Veldhuisen B., de Haas M. Will genotyping replace serology in future routine blood grouping? – Opinion 5. Transfus Med Hemother. 2009; 36(3): 234–5. DOI: 10.1159/000214840.

40. Reid M.E., Denomme G.A. DNA-based methods in the immunohematology reference laboratory. Transfus Apher Sci. 2011; 44(1): 65–72. DOI: 10.1016/j.transci.2010.12.011