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

Aims: As a consequence of global migration flows the risk of HPA alloimmunization may be increased for recipients of platelets from ethnically diverse donors. In this study, the frequencies of HPA in donors of different geographical origin were determined in a Northeastern Italy Transfusion Department. Methods: One hundred and ninety-nine apheresis platelet donors, including Europeans (n = 154) and North Africans (n = 29), were enrolled. DNA was extracted with a commercial kit (Bee-Robotic). Extensive HPA genotyping was performed with BloodChip ID HPA (ProgenikaGrifols). Chi-squared test for Hardy-Weinberg equilibrium was used to compare frequencies. Results: The allele a was clearly predominant for HPA 1-11 in both European and North African donors. The allele b was absent for HPA 6-7-810-11 in Europeans and for HPA 4-6-7-8-9-10-11 in North Africans. For HPA-15, allele b was more frequent than allele a in North Africans. One case of HPA-4ab and one case of HPA-9abw were Donatella Londero1, Mauro Miani1, Cristina Rinaldi1, Vivianna Totis1, Vincenzo de Angelis1 Affiliations: 1Immunohematology Laboratory, Department of Transfusion Medicine, Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy. Corresponding Author: Dr. Donatella Londero, Immunohematology Laboratory, Department of Transfusion Medicine, Azienda Sanitaria Universitaria Integrata di Udine, Via Pozzuolo 330, 33100 Udine, Italy; Email: donatella.londero@ asuiud.sanita.fvg.it Received: 24 October 2017 Accepted: 21 December 2017 Published: 22 January 2018 detected in Europeans. All HPA genotypes were consistent with Hardy-Weinberg equilibrium. Gene frequencies between both ethnic groups were similar excepting HPA-2 (p = 0.0342). Conclusion: In our population, there should not be clinically relevant increased risk of posttransfusion purpura or platelet transfusion refractoriness secondary to antibodies to HPAs for recipients of platelet concentrates from blood donors of European and North African ethnicity.


INTRODUCTION
Human platelet specific antigens (HPAs) are immunogenic structures located on the membrane of platelets. Transfusion, pregnancy or transplantation can expose individuals to allogeneic HPAs and elicit an immune response. Alloantibodies against HPA are mainly involved in fetal and neonatal alloimmune thrombocytopenia (FNAIT), platelet transfusion refractoriness and, rarely, in post-transfusion purpura [1][2][3][4].
In recent years, a rise in platelet transfusion has been observed. Factors that may explain this trend include an increase in the general population, an ageing population, an increase in the incidence and prevalence of hematological malignancies, and changes in the management of hematology-oncology patients [18]. These latter factors account for the majority of platelet transfusion refractoriness [19], which is altogether prevalent among 15-25% of hematology-oncology patients. Refractoriness has been linked to inferior clinical outcomes, including bleeding and mortality, as well as higher health care costs [19].
To date, our blood donor population in Friuli-Venezia Giulia (FVG) region (Northeastern Italy) consists of Europeans, North Africans, and to a smaller extent, Sub-Saharans and Asians, as a consequence of global migration flows. This population ethnicity profile can be found in many other places of Europe. Differences in the ethnicity of blood donors and transfusion recipients are a key factor in immunization of some multitransfused patients to blood group antigens. Therefore, prospective matching requires the recruitment of donors from the same ethnic group as the patient population. Regarding HPA compatibility, extensive HPA typing of blood donors and the knowledge of HPA frequencies in different human populations are important tools to guide decision-making concerning platelet transfusion.
The aim of this study was to compare the frequency distribution of HPA alleles among ethnically diverse apheresis platelet donors to determine whether an increased HPA alloimmunization risk exists for recipients of platelets from blood donors of other geographical origin, regardless HLA mismatch, and to determine the need to have previously mass-scale genotyped suitable donors available. Currently, no similar study has been yet carried out in Italian blood donors.

Study design
One hundred ninety-nine volunteer platelet apheresis donors belonging to Transfusion Departments of Friuli-Venezia Giulia were enrolled in the study between May 2013 and December 2013. Since the study was performed in terms of prevention of alloimmunization management, no refractory patients but only donors were considered for enrolment. The sole inclusion criterion was ≥ 2 previous platelet apheresis donations in the last two years. Donors were classified by the investigator according to their European, African (North or Sub-Saharan) and Asian geographical origin.
All blood samples were collected after informed consent was given at the act of blood donation. The research was approved by the Hospital Ethics Committee. All procedures were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Procedures
Peripheral venous blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes and genomic DNA was extracted by using a commercial kit (Bee-Robotic DNA extraction kit, Caernarfon, UK), according to the protocol recommended by the manufacturer. The concentration and quality of the DNA obtained were analyzed using a spectrophotometer (Nanodrop, ThermoFisher Scientific, Wilmington DE, USA).
Human platelet specific antigens molecular typing was performed using BloodChip ® ID HPA (Progenika Biopharma, a Grifols Company, Derio, Spain) a genotyping assay that uses Luminex xMAP ® technology (Luminex Corporation, Austin, TX, USA) which identifies polymorphisms of 12 Human Platelet Systems (HPA 1-11, 15); the BLOODchip lD Software (BIDS) interprets the quantified signals and produces a file with the genotype results for each of the SNPs included, it also converts the genotypes into predicted phenotypes for the antigens tested. All results were compared with published data [5].
Luminex xMAP technology was validated by comparing results from 96 donor DNA samples analyzed by single specific primer-polymerase chain reaction (SSP-PCR) and Luminex xMAP.

Statistics
Genotype frequencies were estimated by direct counting. A chi-squared test for Hardy-Weinberg equilibrium was calculated to compare the HPA genotype frequencies between our different populations and literature data (IPD-HPA Sequence Database). A p-value less than 0.05 was considered to be statistically significant.  Table 1 illustrates the human platelet specific antigens allele frequencies among the 199 blood platelet apheresis donors belonging to different donor groups, estimated by direct counting. The average age was of 42 years (range 19-67 years). Since Sub-Saharan African and Asian donor samples were not numerous, only European (N = 154) and North African samples (N = 29) were included in statistical analysis. The results of comparison between the observed and expected genotype frequencies are presented in Table 2 for European donors and in Table  3 for North African donors. All tested allele frequencies followed the Hardy-Weinberg equilibrium.

International
Allele a was clearly predominant for HPA 1-11 in both studied groups and allele b was absent for HPA 6-7-8-10-11 in Europeans and for HPA 4-6-7-8-9-10-11 in North Africans. With regard to HPA-15, allele b was more frequent than allele a in North Africans. One case of HPA-4ab and one case of HPA-9abw were detected in European donors. The presence of the HPA-9abw genotype was confirmed by sequencing ( Figure 1).
Regarding comparisons with known populations, our European donors were compared with Italian donors (except for HPA-15 where we used Austrian population) and North African donors were compared with Moroccan people (as 76% of our donors came from Morocco). No significant difference (p-value ≥ 0.05) was found.
In the comparison using the chi-square test for homogeneity, there was no significant difference between our two studied ethnic groups (p-value ≥ 0.05) except for HPA-2 (p-value = 0.0342) ( Table 4).
During Luminex xMAP technology validation, no sample required repetition because of a failure to reach all internal quality control requirements. Results were concordant in all cases.

DISCUSSION
Methods to manage immunological refractoriness include the use of HLA-and/or HPA-matched platelets and platelet cross-matching [17,20], to increase the platelet count. For HPA involving antibodies, genotyping of both donors and recipients is an important tool in the identification of compatible platelets [21,22]. Therefore, as the start of a recruitment policy, in our study we analyzed the frequency of HPA expression in the FVG Transfusion Medicine Departments blood donors from different geographical origin (European, African and Asian).
The first result from our study is the validation of the Luminex xMAP, an innovative methodology for HPA genotyping which allows the simultaneous typing of 12 antigen systems instead of the six usually evaluated by SSP-PCR; results from DNA sequencing carried out by PCR were always consistent with those from the Luminex xMAP. This result allowed us to introduce a reliable and affordable genotyping analysis in immunohematology laboratory, thus facilitating the creation of a database for HPA genotyped donors.
When considering the results on HPA genotypes in donors, our preliminary results failed to detect a significant difference in the relative frequency of HPA antigen expression in European and North-African blood donors donating at FVG Blood Transfusion Departments, except for HPA-2 system (Europeans HPA-2a 0.945 HPA-2b 0.055; North-Africans HPA-2a 0.845 HPA-2b 0.155; p-value = 0.0342), with North-Africans exhibiting somewhat higher frequency of HPA-2b, as recently demonstrated also for Egyptian [23] and Algerian populations [24]. We also observed a predominance of allele a in platelet antigens from HPA-1 to HPA-11 and the absence of allele b for HPA-6,-7,-8,-10,-11 in both ethnicities, as also previously reported by Conti et al. [25]. Moreover, in donors originating from North Africa we noticed the absence of allele b for HPA-4,-9.

Author Contributions
Donatella Londero -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published Mauro Miani -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published Cristina Rinaldi -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published Vivianna Totis -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published Vincenzo de Angelis -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published

Guarantor of Submission
The corresponding author is the guarantor of submission.