Phenotypic and Genotypic Association of the Duffy Blood Group Antigens (Fy^a, Fy^b) in Healthy Blood Donors

By Sadia Ali¹, Fahim Akhtar², Rehan Ahmad Khan Lodhi¹, Nargis Sabir²

Affiliations

1. Department of Haematology, Armed Forces Institute of Transfusion, Rawalpindi, Pakistan
2. Department of Microbiology, Armed Forces Institute of Transfusion, Rawalpindi, Pakistan

doi: 10.29271/jcpsp.2025.08.953

ABSTRACT
Objective: To determine the Duffy blood group antigens (Fy^a, Fy^b) phenotypic and genotypic distribution among blood donors reporting in a tertiary care transfusion centre in the northern region of Pakistan.
Study Design: A cross-sectional study.
Place and Duration of the Study: Department of Immunohaematology and Molecular Biology, Armed Forces Institute of Transfusion, Rawalpindi, Pakistan, from August 2023 to March 2024.
Methodology: The total number of blood donors recruited was 287, using the WHO sample size calculator. ABO, Rhesus (Rh), and Duffy blood group phenotyping were performed using commercially available antisera. Genotyping of the Duffy blood group was carried out using polymerase chain reaction specific sequence of primers (PCR-SSP). Statistical Package for the Social Sciences (SPSS) version 22 was used to analyse the data. For association analysis, the Chi-square test was used.
Results: The most common phenotype observed was (a+ b-), found in 53.3% of all Duffy phenotypes, followed by a+ b+ (31.7%), a- b+ (13.2%), and a- b- (1.7%). The frequency of Duffy genotypes showed a non-significant correlation with Duffy phenotypes.
Conclusion: In the catchment area of the studied centre in Northern Pakistan, the phenotypic frequency of (a+ b-) is the highest of all Duffy phenotypes i.e., (53.3%), followed by a+ b+, a- b+, and a- b-, respectively. Serological and PCR-SSP methods were found to be in complete agreement. PCR could serve as an alternative approach for identifying the Duffy blood group, especially in individuals with low antigen expression.

Key Words: Duffy blood group, Alloimmunisation, Genotype, Phenotype, Transfusion.

INTRODUCTION

The blood group systems are characterised by the presence or absence of specific antigens on the red blood cell surface, which exhibit genetic variation in their structure and biological functions. According to the ISBT, there are currently 346 distinct antigens distributed among 36 diverse blood group systems. ABO and Rhesus (Rh) are the most important blood group system  in  the  context  of  clinical  transfusion.^1,2

The Duffy blood group system, designated as ISBT number 008/symbol (FY), was first reported in 1950 when anti-Fy^a was detected in a 43-year patient with haemophilia, who developed jaundice following a transfusion of three packed red blood cell units. This patient was the first documented individual to develop anti-Fy^a antibodies. Subsequently, in the following year, anti-Fy^b antibodies  were  identified  in  a  multiparous  woman.^3,4

Two decades later, the rest of the Duffy antigens (FY3, FY4, FY5, and FY6) were discovered. Among these antigens, only FY3 seems to possess clinical significance. Duffy blood group antigens are encoded by the atypical chemokine receptor 1 (ACKR1) gene, which is situated on the long arm of chromosome 1 (1q22-1q23).^5,6 The Duffy glycoprotein contains two terminals as follows: the N-terminal, which is a short acidic region rich in aspartic acid and glutamic acid residues and responsible for the binding of Duffy glycoprotein to the Duffy receptor on the red blood cell surface,⁷ and the H terminal, which is longer and more hydrophobic that contains a number of N-linked glycosylation sites and augments the expression of H antigen on the red cell surface .

The glycosylated gene product binds multiple chemokines. Duffy phenotypes exhibit differences among variable ethnic groups. The presence of the Duffy null phenotype Fy (a- b-) is most prevalent among indivisuals of Black ethnicity and rare among the Caucasian and Asian populations.⁸ Plasmodium vivax invades the red cells through the Duffy antigen, and individuals with the Duffy null phenotype Fy (a- b-) are resistant to this malarial parasite. There are three alleles Fy^a, Fy^b (code for respective antigens), and Fy, (a silent allele in this blood group system).^9,10

The aim of this study was to determine the phenotypic and genotypic distribution of the Duffy blood group in blood donors reporting at a referral transfusion centre. In Pakistan, this blood group has not been well studied. Therefore, this study will be helpful in determining genotypic and phenotypic distribution of the aforementioned blood group and predicting the potential risk of developing haemolytic transfusion reactions associated with the Duffy blood group. This database will enable early stratification  of  the  individuals  into  different  risk  groups.

METHODOLOGY

The study was carried out at the Department of Immunohaematology and Molecular Biology, Armed Forces Institute of Transfusion, Rawalpindi, Pakistan, from August 2023 to March 2024. It is a cross-sectional study, conducted following IRB (Institutional Review Board) approval. Using the WHO calculator, the sample size was calculated to be 287, based on a prevalence P1 of 17.8% and prevalence P2 of 4.2%. The sampling technique was non-probability convenience sampling. All healthy donors were included in the study irrespective of their age and gender. All donors deferred after the physical examination, and donors of platelet-apheresis were excluded. SPSS version 22 was used for statistical analysis. Frequency and percentage were used to express qualitative variables while mean and standard deviation were used for quantitative variables. The Chi-square test was used to check the association of phenotype with genotype. A p-value of ≤0.05 was considered as significant.

The qualitative and quantitative data were collected by observation, interpretation, recording of demographic variables, and laboratory testing. A 2 ml blood sample was collected from the antecubital vein of each donor for phenotyping and transferred to an EDTA tube. Commercially available antisera for Fy^a and Fy^b (LORNE UK) were used with the tube method to determine the Duffy blood group. The indirect anti-human globulin (IAHG) method was utilised for this purpose. For genotyping, a 2-3 ml venous sample was collected in an EDTA tube for PCR. DNA was extracted using the Chelex method. The extracted DNA was amplified using the PCR–SSP (sequence-specific primer) technique, with an AB7 2700 thermal cycler. Human growth hormone was used as an internal control. Positive and negative controls were run with each batch. The amplified product was transferred to a polyacrylamide gel for electrophoresis. After staining and drying  of  the  bands,  the  results  were  interpreted.

Table I: Association between genotype and phenotype (n = 287).
 

Phenotypes	Genotypes				p-values
	(a+ b-) n = 154	(a+ b+) n = 87	(a- b+) n = 39	(a- b-) n = 7
a+ b-	151 (98.1%)	0 (0.0%)	0 (0.0%)	2 (28.6%)	<0.001
a+ b+	3 (1.9%)	87 (100.0%)	1 (2.6%)	0 (0.0%)
a- b+	0 (0.0%)	0 (0.0%)	38 (97.4%)	0 (0.0%)
a- b-	0 (0.0%)	0 (0.0%)	0 (0.0%)	5 (71.4%)
The Chi-square test was used.

Table II: The association of genotypic with demographic and clinical characteristics in study participants (n = 287).

Parameters	Genotypes				p-values
	(a+ b-) n = 154	(a+ b+) n = 87	(a- b+) n = 39	(a- b-) n = 7
Gender
Male	148 (96.1%)	86 (98.9%)	39 (100.0%)	6 (85.7%)	0.107
Female	6 (3.9%)	1 (1.1%)	0 (0.0%)	1 (14.3%)
Age
≤28 Years	81 (52.6%)	39 (44.8%)	19 (48.7%)	3 (42.9%)	0.686
>28 Years	73 (47.4%)	48 (55.2%)	20 (51.3%)	4 (57.1%)
Ethinicity	-	-	-	-	-
Punjabi	106 (68.8%)	60 (69.0%)	29 (74.4%)	5 (71.4%)	0.869
Pashtun	25 (16.2%)	15 (17.2%)	5 (12.8%)	2 (28.6%)	-
Kashmiri	17 (11.0%)	9 (10.3%)	2 (5.1%)	0 (0.0%)	-
Balouchi	1 (0.6%)	1 (1.1%)	2 (5.1%)	0 (0.0%)	-
Chittrali	3 (1.9%)	1 (1.1%)	0 (0.0%)	0 (0.0%)	-
Gilgitti	2 (1.3%)	1 (1.1%)	1 (1.1%)	0 (0.0%)	-
Blood group	-
A	43 (27.9%)	16 (18.4%)	7 (17.9%)	2 (28.6%)	0.200
B	61 (39.6%)	28 (32.2%)	10 (25.6%)	3 (42.9%)	-
O	40 (26.0%)	34 (39.1%)	17 (43.6%)	2 (28.6%)	-
AB	10 (6.5%)	9 (10.3%)	5 (12.8%)	0 (0.0%)	-
Rh blood group	-
Positive	144 (93.5%)	76 (87.4%)	34 (87.2%)	7 (100.0%)	0.266
Negative	10 (6.5%)	11 (12.6%)	5 (12.8%)	0 (0.0%)	-
The Chi-square test was used.

Table III: The association of phenotype with demographic and clinical characteristics in study participants (n = 287).

Parameters	Phenotypes				p-values
	(a+ b-) n = 153	(a+ b+) n = 91	(a- b+) n = 38	(a- b-) n = 5
Gender
Male	147 (96.1%)	90 (98.9%)	38 (100.0%)	4 (80.0%)	0.041
Female	6 (3.9%)	1 (1.1%)	0 (0.0%)	1 (20.0%)
Age in years
≤28 years	80 (52.6%)	40 (44.0%)	19 (50.0%)	3 (60.0%)	0.611
>28 years	73 (47.4%)	51 (56.0%)	19 (50.0%)	2 (40.0%)
Ethnicity
Punjabi	107 (69.9%)	62 (68.1%)	28 (73.7%)	3 (60.0%)	0.672
Pashtun	24 (15.7%)	16 (17.6%)	5 (13.2%)	2 (40.0%)
Kashmiri	17 (11.1%)	9 (9.9%)	2 (5.3%)	0 (0.0%)
Balouchi	0 (0.0%)	2 (2.2%)	2 (2.2%)	0 (0.0%)
Chittrali	3 (2.0%)	1 (1.1%)	0 (0.0%)	0 (0.0%)
Gilgitti	2 (1.3%)	1 (1.1%)	1 (2.6%)	0 (0.0%)
Blood group
A	44 (28.8%)	16 (17.6%)	7 (18.4%)	1 (20.0%)	0.130
B	61 (39.9%)	29 (31.9%)	10 (26.3%)	2 (40.0%)
O	38 (24.8%)	37 (40.7%)	16 (42.1%)	2 (40.0%)
AB	10 (6.5%)	9 (9.9%)	5 (13.2%)	0 (0.0%)
Rh blood group
Positive	143 (93.5%)	80 (87.9%)	33 (86.8%)	5 (100.0%)	0.325
Negative	10 (6.5%)	11 (12.1%)	5 (13.2%)	0 (0.0%)
The Chi-square test was used.

RESULTS

A total of 287 blood donors participated in this study. The mean age was 29.48 ± 7.36 years, ranging from 18 to 50 years. Out of the total donors, 279 (97.2%) were males and 8 (2.8%) were females. The majority of participants 200 (69.7%) were Punjabi, 47 (16.4%) were Pashtun, 28 (9.8%) were Kashmiri, 4 (1.4%) Balouchi, 4 (1.4%) Chitrali, and 4 (1.4%) Gilgitti. Regarding blood group, 102 participants (35.5%) had B blood group, followed by O (n = 93; 32.4%), A (n = 68; 23.7%), and AB (n = 24; 8.4%). Furthermore, out of the total donors, 261 (90.9%) donors were Rh D positive, and 26 (9.1%) were Rh D negative. Phenotyping of the participants revealed 153 (53.3%) with an a+ b- profile, 91 (31.7%) a+ b+, 38 (13.2%) a- b+, and 5 (1.7%) a- b- profiles. In constrast, the molecular study showed 154 (53.7%) a+ b-, 87 (30.3%) a+ b+, 39 (13.6%) a- b+, and 7 (2.4%) a- b- genotype. Out of 287 donors, the majority showed concordance between genotype and phenotype, as shown in Table I. However, six discordant samples were observed, including two a+ b-, three a+ b+, and one a+ b+ on phenotyping, which were a- b-, a+ b-, and a- b+ on genotyping, respectively. The association between the frequency of genotype and phenotype showed p <0.001, which is a statistically significant as shown in Table I.

No association was observed between genotype and demographic and clinical characteristics of study participants as shown in Table II.

However, gender showed a significant association with phenotype (p = 0.041), while age (p = 0.611), ethnicity (p = 0.672), blood group (p = 0.130), and Rh blood group (p = 0.325) showed no significant association with phenotype, as shown in Table III.

DISCUSSION

The frequency of Duffy phenotypes varies among different populations and ethnic groups. Incompatibilities are responsible for alloimmunisation, leading to haemolytic transfusion reactions and haemolytic disease of the foetus and newborn, with varying degrees of severity. Antigen exposure may occur through transfusions or pregnancies.^11,12

In Pakistan, the scarcity of data regarding phenotypic and genotypic prevalence of Duffy blood group antigens serves as a primary motivator for designing this study. The Armed Forces Institute of Transfusion is located in the northern region of the country, where it caters to the needs of diverse population comprising multiple ethnic backgrounds. This study will be helpful in forming a database for blood banks and transfusion centres for various ethnic groups within the population.^13-15 It can further assist in the development of cell panels and in providing antigen-negative blood products to the alloimmunised individuals.

In this study, 287 healthy blood donors were investigated for the phenotypic and genotypic distribution of the Duffy blood group. This research was conducted in Northern Pakistan due to the limited availability of data in this region.

In a study conducted by Langhi Junior, 250 individuals were taken for investigation of malaria, out of which 83.6% showed a history of malaria.

The Fy a+ b+ blood type was detected in 38.8% and a- b- in 2.8% individuals. On genotyping, FY*A / FY*B was seen in 52%, FY*A / FY*A in 18%, and 4.4% of the cases had homozygous mutation of c.1-67>TC. Those with a+ b- and FYA*/ FYB* had 11.8% of homozygous c.1-67T>C mutation.^16,17 Notably, one individual had Fy (a+ b-), FY*A/FY*B, and homozygous c.1-67T>C while another had Fy (a+ b-), FY*A/ FY*A, and heterozygous c.1-67T>C. The Pearson’s chi-square was used for statistical analysis, considering p-value <0.05 as significant, which came out to be <0.001.¹⁸

Despite the belief that Duffy-negative expression on red blood cells protects Africans from P. vivax malaria,¹⁹ Albsheer et al. conducted a study in Sudan in 2019 and found that the parasite infects Duffy-negative individuals as well. Out of 992 cases, 190 were P. vivax positive. Duffy Fy (a- b+), being the most prevalent phenotype, represented 67.9% of infections, while Duffy-negative a- b- accounted for 17.9%. Infection rates were significantly higher in a- b+ and a+ b- compared to Duffy-negative individuals, emphasising the prevalence of P. vivax in Duffy-negatives with low parasitemia. The significant p value was taken as <0.01.²⁰

In Brazil, researchers investigated the relationship between Duffy blood group proteins and susceptibility to P. vivax in a malaria-endemic area. Among 244 individuals, those with the FYA allele showed a higher frequency in vivax malaria cases. The most common genotype was FYAFYB, and the null genotype was absent in infected individuals but present in non- infected ones. A discordance of 11.5% between genotype and phenotype was observed when the Chi-square test was applied to calculate the p-value.^21,22

A limitation to this study is the lack of genotyping of other antigens within the same blood group system. Moreover, the absence of an advance molecular research on Duffy polymorphism, which could provide insights into human evolutionary adaptation to malaria, particularly in malaria-endemic countries.

CONCLUSION

In the studied regional centre of Northern Pakistan, the phenotypic frequency of (a+ b-) is the highest among all Duffy phenotypes (53.3%), followed by other phenotypes. The serological and the PCR-SSP methods showed perfect conformity in this study. PCR might serve as an alternative method for identifying the Duffy blood group, especially in cases with low antigen expression. Given the risk of haemolytic transfusion reactions and HDFN with anti-Duffy and anti-bodies, it is crucial to provide antigen-negative blood to prevent complications. Further research could explore the genetic factors influencing Duffy phenotypes distribution in this region. Expanding the use of advanced molecular techniques could enhance our understanding, ultimately reforming transfusion practices and improving patient outcomes.

ETHICAL  APPROVAL:
Ethical approval was obtained from the Ethics Review Committee of the Armed Forces Institute of Transfusion, Rawalpindi, Pakistan (Certificate No: AFIT-ERC-24-50).
 

PATIENTS’  CONSENT:
Informed consent was obtained from the participants prior to their enrolment in the study.

COMPETING  INTEREST:
The authors declared no conflict of interest.

AUTHORS’  CONTRIBUTION:
SA: Conception, design, data collection, acquisition, analysis, and writing of the study.
FA, RAKL: Drafting and critical revision of the work.
NS: Data collection and statistical analysis.
All authors approved the final version of the manuscript to be published.

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