Original Article

Genotyping of Human Platelet Antigen-1 to -5 and -15 by Polymerase Chain Reaction with Sequence-specific Primers (PCR-SSP) and Real-time PCR in Azeri Blood Donors


Human platelet antigens (HPAs) are glycoproteins on the platelet surface that a single nucleotide mutation in the coding region gene could lead to the variation of different HPA polymorphisms. These antigens have shown variation among different races and may trigger immune responses during blood transfusion and pregnancy. Genotyping of HPAs is useful for managing these reactions and establishing a platelet registry to decrease platelet transfusion reactions. This study aimed to compare allelic and genotype frequencies of human platelet antigens in the Azeri ethnicity by TaqMan Real-time and polymerase chain reaction with sequence-specific primers (PCR-SSP) methods.
DNA was extracted from the whole blood of 100 Azeri blood donors in the Ardabil Blood Transfusion Center. Genotyping of HPA-1 to -5 and -15 was performed by TaqMan Real-time PCR, and PCR-SSP and consistency of results were evaluated.
The results of PCR-SSP and TaqMan Real-time PCR showed complete consistency. The allele frequencies were 91.5% and 8.5% for HPA-1a and -1b; 88% and 12% for HPA-2a and -2b; 58% and 42 % for HPA-3a and -3b; 100% for HPA-4a; 91% and 9% for HPA-5a and -5b; 56.5% and 43.5% for HPA-15a and -15b alleles.
Not incompatibility was detected in HPAs genotyping by PCR-SSP and TaqMan Real-time PCR so that real-time PCR can be used as a robust and quick method for HPA genotyping. We found differences between Azeri blood donors and previously reported HPAs alleles’ frequency in other ethnicities in the country. This fact highlights the need for a platelet registry to recruit platelet donors from different ethnicities and increase the number of donors by using faster methods.

1. Ghoshal K, Bhattacharyya M. Overview of platelet physiology: its hemostatic and nonhemostatic role in disease pathogenesis. ScientificWorldJournal. 2014;2014:781857.
2. Curtis BR. Genotyping for human platelet alloantigen polymorphisms: applications in the diagnosis of alloimmune platelet disorders. Semin ThrombHemost. 2008;34(6):539-48.
3. Mangerona CM, Garcia FB, Moraes-Souza H. Frequency of human platelet antigens (HPA)-1, -2, -5 and -15 in Brazilian blood donors and establishment of a panel of HPA-typed donors. Transfus Med. 2015;25(3):189-94.
4. Porcelijn L, Huiskes E, de Haas M. Progress and development of platelet antibody detection. TransfusApher Sci. 2019:102705.
5. Kopko PM, Warner P, Kresie L, Pancoska C. Methods for the selection of platelet products for alloimmune‐refractory patients. Transfusion. 2015;55(2):235-44.
6. Bhatti F, Uddin M, Ahmed A, Bugert P. Human platelet antigen polymorphisms (HPA‐1,‐2,‐3,‐4,‐5 and‐15) in major ethnic groups of Pakistan. Transfus Med. 2010;20(2):78-87.
7. Castro Vd, Origa AF, Annichino‐Bizzacchi JM, Soares M, Menezes R, Gonçalves MdS, et al. Frequencies of platelet‐specific alloantigen systems 1–5 in three distinct ethnic groups in Brazil. Eur J Immunogenet. 1999;26(5):355-60.
8. Xu X, Liu Y, Ying Y, Tao S, Hong X, Zhu F, et al. Human platelet antigen allele frequencies and new mutations on platelet glycoprotein genes in the Chinese Han population. Transfus Med. 2011;21(5):330-7.
9. Mojaat N, Halle L, Proulle V, Hmida S, Ben Hamed L, Boukef K, et al. Gene frequencies of human platelet antigens in the Tunisian population. Tissue Antigens. 1999;54(2):201-4.
10. Stroncek DF, Rebulla P. Platelet transfusions. Lancet. 2007;370(9585):427-38.
11. Pai S-C, Burnouf T, Chen J-W, Lin L-I. Human platelet antigen alleles in 998 Taiwanese blood donors determined by sequence-specific primer polymerase chain reaction. Biomed Res Int. 2013;2013:973789.
12. Londero D, Miani M, Rinaldi C, Totis V, Angelis V. Extensive human platelet specific antigens typing of blood donors of different geographical origin to manage platelet transfusion in alloimmunized patients: Experience from a transfusion center in Northeastern Italy. Int J Blood TransfusImmunohematol. 2018;8:4-11.
13. Wang J, Xia W, Deng J, Xu X, Shao Y, Ding H, et al. Analysis of platelet‐reactive alloantibodies and evaluation of cross‐match‐compatible platelets for the management of patients with transfusion refractoriness. Transfus Med. 2018;28(1):40-6.
14. Feng M, Liu D, Shen W, Wang J, Guo Z, Zhang X, et al. Establishment of an HPA‐1‐to‐16‐typed platelet donor registry in China. Transfus Med. 2006;16(5):369-74.
15. Vorholt SM, Hamker N, Sparka H, Enczmann J, Zeiler T, Reimer T, et al. High-Throughput Screening of Blood Donors for Twelve Human Platelet Antigen Systems Using Next-Generation Sequencing Reveals Detection of Rare Polymorphisms and Two Novel Protein-Changing Variants. Transfus Med Hemother. 2020;47(1):33-44.
16. Nozarimirakolaei M, Zadsar M, Samiee S, Shaiegan M. Iranian Turkmen blood donors determining the HPA-1 gene frequency in 1397. Sci J Iran Blood Transfus Organ.17(1):12-9.
17. Meyer O, Hildebrandt M, Schulz B, Blasczyk R, Salama A. Simultaneous genotyping of human platelet antigens (HPA) 1 through 6 using new sequence‐specific primers for HPA‐5. Transfusion. 1999;39(11-12):1256-8.
18. Cavanagh G, Dunn A, Chapman C, Metcalfe P. HPA genotyping by PCR sequence‐specific priming (PCR–SSP): a streamlined method for rapid routine investigations. Transfus Med. 1997;7(1):41-5.
19. Schuh AC, Watkins NA, Nguyen Q, Harmer NJ, Lin M, Prosper JY, et al. A tyrosine703serine polymorphism of CD109 defines the Gov platelet alloantigens. Blood. 2002;99(5):1692-8.
20. McBride SE. Real-time PCR assays for high-throughput human platelet antigen typing. Methods Mol Biol. 2009;496(5):39-49.
21. Jones D, Bunce M, Fuggle S, Young NT, Marshall S. Human platelet alloantigens (HPAs): PCR‐SSP genotyping of a UK population for 15 HPA alleles. Eur J Immunogenet. 2003;30(6):415-9.
22. Steffensen R, Kaczan E, Varming K, Jersild C. Frequency of platelet‐specific alloantigens in a Danish population. Tissue Antigens. 1996;48(2):93-6.
23. Klüter H, Fehlau K, Panzer S. Rapid typing for human platelet antigen systems‐1,‐2,‐3 and‐5 by PCR amplification with sequence‐specific primers. Vox Sang. 1996;71(2):121-5.
24. Merzoni J, Fagundes I, Lunardi L, Lindenau JR, Gil B, Jobim M, et al. Human platelet antigen genotyping of platelet donors in southern Brazil. Int J Immunogenet. 2015;42(5):329-35.
25. Hauck‐Dlimi B, Hammon K, Eckstein R, Ott S, Zimmermann R, Dengler T, et al. Human platelet antigen genotypes in Turkish and Caucasian blood donors in Germany. Tissue Antigens. 2012;80(3):214-8.
26. De La Vega Elena C, Nogues N, Fernandez Montoya A, Chialina S, Blanzaco P, Theiller E, et al. Human platelet‐specific antigens frequencies in the Argentinean population. Transfus Med. 2008;18(2):83-90.
27. Drzewek K, Brojer E, Zupańska B. The frequency of human platelet antigen (HPA) genotypes in the Polish population. Transfus Med. 1998;8(4):339-42.
28. Brouk H, Halle L, Bertrand G, Neche F, Ouelaa H, Kaplan C. Human platelet antigen allele frequencies in different Algerian populations. Tissue Antigens. 2010;75(6):673-8.
29. Chiba AK, Bordin JO, Kuwano ST, Figueiredo MS, Carvalho KI, Vieira-Filho JP, et al. Platelet alloantigen frequencies in Amazon Indians and Brazilian blood donors. Transfus Med. 2000;10(3):207-12.
30. Nie YM, Zhou HJ, Fu YS, Wang CX, Ma JP. The allele frequencies of HPA 1‐16 determined by PCR‐SSP in Chinese Cantonese donors. Transfus Med. 2010;20(6):376-82.
31. Lyou JY, Chen YJ, Hu HY, Lin JS, Tzeng CH. PCR with sequence‐specific primer‐based simultaneous genotyping of human platelet antigen‐1 to‐13w. Transfusion. 2002;42(8):1089-95.
32. An QX, Li CY, Xu LJ, Zhang XQ, Bai YJ, Shao ZJ, et al. High‐throughput simultaneous genotyping of human platelet antigen‐1 to‐16 by using suspension array. Transfusion. 2013;53(11):2722-8.
33. Ruan L, Pei B, Li Q. Multicolor real‐time polymerase chain reaction genotyping of six human platelet antigens using displacing probes. Transfusion. 2007;47(9):1637-42.
34. Seo D, Park SS, Kim D, Furihata K, Ueno I, Han K-S. Gene frequencies of eight human platelet-specific antigens in Koreans. Transfus Med. 1998;8(2):129-32.
35. Silvestre APA, Zacarias JMV, Guelsin GAS, Visentainer JEL, Sell AM. Genetic polymorphisms of human platelet antigens in Euro-African and Japanese descendants from Parana, Southern Brazil. Platelets. 2017;28(6):607-10.
36. Ferrer G, Muniz‐Diaz E, Aluja M, Arilla M, Martinez C, Nogues R, et al. Analysis of human platelet antigen systems in a Moroccan Berber population. Transfus Med. 2002;12(1):49-54.
37. Al‐Subaie AM, Al‐Absi IK, Al‐Ola K, Saidi S, Fawaz NA, Almawi WY. Gene frequencies of human platelet alloantigens in Bahraini Arabs. Am J Hematol. 2007;82(3):242-4.
38. Halle L, Bigot A, Mulen‐Imandy G, M'Bayo K, Jaeger G, Anani L, et al. HPA polymorphism in sub‐Saharan African populations: Beninese, Cameroonians, Congolese, and Pygmies. Tissue Antigens. 2005;65(3):295-8.
39. Madani T, Samiei S, Ataei Z, Kavari M, Babaei Gr, Mostakhdemin M, et al. Platelet Antigens Frequency In Blood Donors: Comparison Of Molecular Detection With Elisa Method ( For Hpa-1a ). Sci J Iran Blood Transfus Organ. 2007;4)15):3.
40. Ficko T, Galvani V, Rupreht R, Dovc T, Rožman P. Real‐time PCR genotyping of human platelet alloantigens HPA‐1, HPA‐2, HPA‐3 and HPA‐5 is superior to the standard PCR‐SSP method. Transfus Med. 2004;14(6):425-32.
41. Meyer O, Abou‐Chaker K, Heymann G, Bombard S, Kiesewetter H, Salama A. Human platelet antigen genotyping by using sequence‐specific primers and the particle gel agglutination assay. Vox Sang. 2005;88(4):271-4.
42. Hurd C, Cavanagh G, Schuh A, Ouwehand W, Metcalfe P. Genotyping for platelet‐specific antigens: techniques for the detection of single nucleotide polymorphisms. Vox Sang. 2002;83(1):1-12.
43. Simsek S, Faber NM, Bleeker PM, Vlekke AB, Huiskes E, Goldschmeding R, et al. Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis. Blood. 1993;81(3):835-40.
44. Holensteiner A, Walchshofer S, Adler A, Kittl E-M, Mayr WR, Panzer S. Human platelet antigen gene frequencies in the Austrian population. Haemostasis. 1995;25(3):133-6.
45. Kekomäki S, Partanen J, Kekomäki R. Platelet alloantigens HPA‐1,‐2,‐3,‐5 and‐6b in Finns. Transfus Med. 1995;5(3):193-8.
46. Tanaka S, Taniue A, Nagao N, Ohnoki S, Shibata H, Okubo Y, et al. Simultaneous DNA typing of human platelet antigens 2, 3 and 4 by an allele‐specific PCR method. Vox Sang. 1995;68(4):225-30.
47. Tanaka S, Ohnoki S, Shibata H, Okubo Y, Yamaguchi H, Shibata Y. Gene frequencies of human platelet antigens on glycoprotein IIIa in Japanese. Transfusion. 1996;36(9):813-7.
48. Roz˘ man P, Drabbels J, Schipper R, Doxiadis I, Stein S, Claas F. Genotyping for human platelet‐specific antigens HPA‐1,‐2,‐3,‐4 and‐5 in the Slovenian population reveals a slightly increased frequency of HPA‐1b and HPA‐2b as compared to other European populations. Eur J Immunogenet. 1999;26(4):265-9.
49. Cardone J, Chiba A, Boturão‐Neto E, Vieira‐Filho J, Bordin J. Gene frequencies of the HPA‐15 (Gov) platelet alloantigen system in Brazilians. Transfus Med. 2004;14(6):433-7.
50. Eyada TK, Amin DG, Samih I, Khedr SM. Human platelet antigen 1, 2 and 5 gene polymorphisms in Egyptians and their potential association with susceptibility to immune thrombocytopenic purpura in Egyptian patients. Hematology. 2018;23(2):111-6.
51. Ahmadzadeh Shad G, Zadsar M, Shayegan M, Samiee S, Zare A. HPA-1 gene polymorphism in Iranian couples with history of recurrent abortion. Sci J Iran Blood Transfus Organ. 2018;15(4):293-300.
52. Shaiegan M, Ghasemi A, Madani T, Samiee S, Bayat B, Mohammadi S, et al. Gene frequencies of human platelet antigen-1 to -5 and -15 in the Iranian population. ISBT Science Series. 2020;15(2):273-80.
53. Ghasemi A, Zadsar M, Shaiegan M, Samiei S, Namvar A, Rasouli M, et al. Human platelet antigens polymorphisms; association to the development of liver fibrosis in patients with chronic hepatitis C. J Med Virol. 2020;92(1):45-52.
54. Mostakhdemin Hosseini M, Samiee S, Shaiegan M, Mohammadi S, Jalaiekhoo H, Tabatabiepanah P, et al. Evaluation of platelet antigens and antibodies in patients with platelet refractoriness. Sci J Iran Blood Transfus Organ. 2019;16(2):91-102.
55. Madani T. Survey of Human Platelet Antigens In Iranian blood donors, MSC thesis in hematology and blood banking, Supervisor Shaeigan M: High institute of education and research in transfusion medicine, Iran, Tehran. Unpublished data; 2007.
56. Safizadeh F. The Dynamics of Ethnic Identity in Iranian Azerbaijan. Journal of Pedagogy, Pluralism, and Practice. 2013;5(1):55.
IssueVol 20 No 3 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijaai.v20i3.6333
Blood donors Human platelet antigens Real-time polymerase chain reaction

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Azizi SG, Samiee S, Shaiegan M, Zadsar M. Genotyping of Human Platelet Antigen-1 to -5 and -15 by Polymerase Chain Reaction with Sequence-specific Primers (PCR-SSP) and Real-time PCR in Azeri Blood Donors. Iran J Allergy Asthma Immunol. 2021;20(3):350-363.