Human neutrophil antigen‐1, -3, -4, and -5 allele and genotype frequencies in the Croatian blood donor population and their clinical significance

Abstract

Objectives

Human neutrophil antigens (HNAs) and antibodies play an important role in allo- and autoimmunity associated with immune neutropenia and transfusion reactions. The aim of this study was to determine the HNA-1, -3, -4 and -5 allele and genotype frequencies in the Croatian blood donor population to assess the role of HNA-1, -3, -4, and -5 alleles in the development of neonatal alloimmune neutropenia and antibody-mediated transfusion-related acute lung injury.

Material and methods

A total of 371 blood samples from unselected healthy blood donors were analyzed. Samples from all 371 donors were genotyped for HNA-1, samples from 160 donors were genotyped for HNA-3, and samples from 142 donors were genotyped for HNA-4 and HNA-5 using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method.

Results

The frequencies of the FCGR3B*01, FCGR3B*02 and FCGR3B*03 HNA-1 alleles were 0.393, 0.607 and 0.022, and of the SLC44A2*01 and SLC44A2*02 HNA-3 alleles 0.781 and 0.219, respectively. The frequencies of the ITGAM*01 and ITGAM*02 HNA-4 alleles were 0.796 and 0.204, and of the ITGAL*01 and ITGAL*02 HNA-5 alleles 0.718 and 0.282, respectively.

Conclusion

These are the first results on the HNA allele and genotype frequencies in the Croatian blood donor population. We observed no deviations from previous reports on Caucasian populations. Determination of the HNA antigen frequencies in the population is important to estimate the risk of alloimmunization to HNA, especially the risk of fetal-maternal incompatibility and alloantibody production by transfusion of the HNA incompatible blood components.

Introduction

Human neutrophil antigens (HNAs) and antibodies play an important role in allo- and autoimmunity. They are associated with a variety of clinical conditions, including neonatal alloimmune neutropenia (NAIN), autoimmune neutropenia, drug-induced immune neutropenia, immune neutropenia after bone marrow transplantation, and antibody-mediated transfusion-related acute lung injury (TRALI) [1].

The first granulocyte-specific antigen was described in 1966 by Lalezari & Bernard in a case of neonatal immune neutropenia [2]. Over years, a number of granulocyte antigens have been described and characterized at the biochemical and molecular level.

The HNA nomenclature is based on the glycoprotein location of various antigens and the nomenclature of alleles corresponds to the Guidelines of the International Workshop on Human Gene Mapping [3]. According to this nomenclature, currently there are five official groups/systems: HNA-1, -2, -3, -4 and -5, consisting of fourteen alleles located on the corresponding glycoproteins [4], [5]. The HNA-1, -2, -3, -4 and -5 antigens are located on CD16, CD177, choline transporter-like protein-2 (CTL2), CD11b and CD11a glycoproteins. They are encoded by the Fc Gamma Receptor IIIb (FCGR3B), CD177, Solute Carrier Family 44 Member 2 (SLC44A2), integrin alpha M (ITGAM) and integrin alpha L (ITGAL), respectively. The HNA-1 system consists of five FCGR3B alleles and one FCGR3B*null allele that indicates FCGR3B deficiency and occurs among individuals with a homozygous chromosomal deletion involving the FCGR3B gene deficiency [6].

Determination of neutrophil antigens by serologic phenotyping techniques (immunofluorescence test, IF; monoclonal antibody immobilization of neutrophil antigen enzyme immunoassay, MAINA-EIA) is time-consuming and dependent on the availability of specific polyclonal antisera of alloimmunized patients. The methods based on DNA technology sequence-based typing (SBT), polymerase chain reaction (PCR) sequence-specific oligonucleotide probes (SSOP), PCR sequence-specific primers (SSP), and PCR restriction fragment length polymorphism (RFLP) have enabled rapid and accurate determination of HNA genotypes and alleles. Since the 1990s, the allele specific PCR (PCR-SSP) is the most widely used molecular method for determination of HNA genotypes [7].

The frequencies of HNA alleles have been widely studied worldwide, and studies have revealed that they vary among different populations. In the Caucasian population, FCGR3B*01 is less frequent than FCGR3B*02 (0.32–0.42 vs 0.56–0.67) [8], [9], [10], [11], [12], whereas in the Asian population, FCGR3B*01 is more frequent than FCGR3B*02 (0.61–0.68 vs 0.32–0.39) [13], [14], [15], [16]. The FCGR3B*03 allele is found in the Caucasian population with a frequency of 0.01–0.04 and appears to be absent in Asians. The FCGR3B*null phenotype is rare in the Caucasian population (<0.01) [8], [9], [10], [11], and nearly absent in the Asian population [13], [15], [16], but is common in the sub-Saharan African populations, where the FCGR3B*03 antigen is also prevalent [8], [17], [18], [19]. Unlike the ITGAM*02 frequency in the Caucasian population of 0.09–0.015 [8], [9], [10], [11], [12], it seems to be very low in the Asian population, i.e., 0–0.005 [13], [14], [15], [16].

Evaluation of the HNA genotype and allele frequencies can give useful information for estimating the rate of HNA alloimmunization and help in the diagnosis of HNA alloimmunization which can lead to neutropenia or transfusion reactions.

There are no previous studies on the HNA frequencies in the Croatian population. Our study aimed to determine the frequencies of genotypes and alleles encoding HNA-1, -3, -4 and -5 in the Croatian blood donor population and compare them to the previously published findings in other populations. Another aim was to assess the role of HNA-1, -3, -4 and -5 allele frequencies in the development of NAIN and antibody-mediated TRALI in our population.