Comparison of ABO Blood Group, Rh Factor, and Genotype Frequency Among University Students in Liberia and Pakistan: A Cross‐Sectional Study

ABSTRACT

Background

Understanding the ABO and RhD blood group systems is important for safe blood transfusions and preventing hemolytic transfusion reactions caused by incompatible blood types. This study compared the phenotypes and calculated genotypes of ABO and RhD blood group systems among students from Cuttington University (CU), Liberia, and the University of Narowal (UON), Pakistan.

Methods

We conducted a cross‐sectional study with 1017 participants (517 from CU and 500 from UON). Capillary blood samples were collected and analyzed using standard antigen‐antibody agglutination test kits. Phenotype frequencies were determined, while genotype and allele frequencies were estimated under the Hardy–Weinberg equilibrium.

Results

In Liberia, blood group O (54%) was the most prevalent, followed by B (22.4%), A (15.8%), and AB (7.4%) blood groups. Blood group B (37.2%) predominated in Pakistan, followed by O (27.6%), A (27.2%), and AB (8.0%) blood groups. RhD‐positive individuals were the most reported in both populations. Similarly, the homozygous ii genotype was the most frequent, followed by IᴮIᴮ and IᴬIᴬ, whereas the heterozygous genotypes followed the order Iᴮi > Iᴬi > IᴬIᴮ. The statistical analysis of the Liberia and Pakistan populations yielded (χ² = 21.06, p < 0.001; χ² = 4.44, p = 0.217, respectively).

Conclusion

The observed differences in phenotype distribution, despite similar numerical genotypic patterns, highlight the underlying genetic variability within the population. These findings provide valuable insights for possible transfusion medicine, population genetics, and health planning across regions.

1. Introduction

In 1901, Landsteiner discovered the ABO blood group system in humans, and in 1939, he and Wiener discovered the Rhesus (Rh) factor in red blood cells. Of the more than 48 human blood group systems, the ABO and Rh systems are the most clinically significant, as transfusion reactions are mostly due to a mismatch of blood types [1]. Thereby, understanding these blood group systems is important for safe blood transfusions practice and prevention of hemolytic transfusion reactions [2]. On the other hand, it leads to a better blood bank management system, which might contribute to modern health care services [3].

The ABO blood group system categorizes the human blood into four main blood types: A, B, AB, and O, based on the presence or absence of antigens A and B on the surface of red blood cells [4]. This blood group system is controlled by three primary alleles (A, B, and O) of a single gene [5]. The O allele is recessive to the A and B alleles because it encodes a non‐functional enzyme. On the other hand, the A and B alleles are co‐dominant with each other, leading to the AB phenotype. Despite the excessiveness of the O allele, it is the most prevalent globally, while blood group AB is the least.

The genotypes corresponding to the phenotypes are I^AI^A (A), I^Ai (A), I^BI^B (B), I^Bi (B), I^AI^B (AB), and ii (O) [6].

The ABO blood group frequencies vary across populations owing to migration patterns and genetic makeup in different regions of the world [7]. Similarly, the Rh factor, named after its discovery in Rhesus monkeys, indicates whether a blood is Rh‐positive (indicating the presence of the D antigen) or Rh‐negative (indicating the absence of the D antigen). RhD and RhCE are the two related proteins of the Rh blood group system, expressed as D and CE antigens, respectively [8]. RhD incompatibility is an important factor that requires careful clinical attention during pregnancy [9]. Combining ABO phenotypes with RhD status results in eight common blood group types: A^{+/ −}, B^{+/ −}, AB^{+/ −}, and O^{+/ −} [6, 10, 11].

This study determined the frequency distributions of the ABO and Rh blood group systems among university students and provides data for phenotype‐genotype frequencies for cross‐continental comparisons.

2. Materials and Methods

The ABO and RhD blood group systems were analyzed in the biology laboratories of Cuttington University (Liberia) and the University of Narowal (Pakistan). Commercial antisera (anti‐A, anti‐B, and anti‐D) were used for the analysis. Blood group phenotypes (A, B, AB, and O) were determined using anti‐A and anti‐B antibodies. The Rh factor status (positive or negative) was determined using an anti‐D reagent, adhering to standard slide agglutination procedures.

Quality control testing was performed to ensure reagent reliability using samples from individuals with previously confirmed ABO and RhD blood group results obtained from the hospital. The reagents and testing procedures were considered valid after producing results identical to those of the reference records (Figure 1). All laboratory procedures were performed by licensed laboratory personnel under standard operating conditions at the laboratories.

Figure 1.

Phenotypes of positive ABO blood groups diagnosed for validation. These agglutination patterns were observed for ABO and RhD blood typing using standard anti‐A, anti‐B, and anti‐D reagents with a focus on positive blood.

2.1. Study Design and Participants

This cross‐sectional study was conducted among selected undergraduate students at two universities in Liberia and Pakistan, during the study period. Participants were recruited using a convenience sampling approach from classrooms and common campus areas.

Eligible participants were aged ≥ 18 years, provided written informed consent, and had no self‐reported history of hematological disorders or blood transfusions within the preceding 3 months. A total of 1017 students were enrolled for data collection, with 517 from CU, and 500 from the UON.

2.2. Blood Collection and ABO/RhD Typing

Capillary blood samples were collected under aseptic conditions using sterile lancets. The fingertip (third or fourth finger of the non‐dominant hand) was disinfected with 70% alcohol before the puncture.

ABO and RhD blood typing were performed using standard antigen–antibody slide agglutination techniques. A drop of blood was placed on a labeled slide (A, B, and D), followed by the addition of antiserum. The combined blood and antiserum were gently mixed using separate applicator sticks and observed for agglutination within 30–60 s.

2.3. Genotype and Statistical Analysis

The allele frequencies of A, B, and O were denoted as p, q, and r, respectively, for statistical analysis. The letter “p” was used to represent “D”, while “q” represented “d” for the determination of the RhD blood group system allele frequencies. Under Hardy–Weinberg equilibrium (HWE), the allele frequencies satisfy the equation (p + q + r)² = p² + q² + r² + 2pq + 2pr + 2qr, which sums up to one (1) [9].

After determining the population size (n), the phenotype frequencies were calculated. Genotype frequencies were obtained from phenotypic distributions under established allele–genotype relationships, in which each phenotype represented one or more underlying genotypes. That is, “A” = p² + 2pr (AA/AO, or I^A I^A/I^A i), “B” = q² + 2qr (BB/BO or I^B I^B/I^B i), “AB” = 2pq (AB, or I^A I^B), “O” = r² (OO, or ii) [6] (Table 1). The HWE was tested under the EM (Expectation–Maximization) algorithm to determine individual blood type frequency. The observed and expected phenotypic counts were analyzed through the IBM SPSS package version 27.0. The goodness‐of‐fit chi‐square test guided the agreement between the observed and expected frequencies [9, 12].

Table 1.

Blood types and their corresponding genotypes and allele frequencies.

Blood type	Genotype	Phenotype frequency formula
A	IAIA and IAi	P2+2pr
B	IBIB and IBi	q2+2qr
AB	IAIB	2pq
O	ii	r2

3. Results

The table above presents the results of the current study along with data from earlier publications. Double (Pakistan data) or single (Liberia data) underlines represent studies with the same order of occurrence in line with our study.

4. Discussion

This study analyzed the ABO and RhD blood groups distribution, where blood groups O & B predominated in Liberia and Pakistan, respectively (Table 2), with AB being the least frequent in both populations. These findings aligned with previously reported regional studies, which show blood types (O & B) are generally more prevalent in the African populations [13] and the South Asian populations, respectively [14] (Table 4).

Table 2.

ABO phenotype frequencies and HWE analysis.

Country	Phenotype	Observed count	Observed frequency	Expected count	Expected frequency
Liberia (N = 517)	A	82	0.16	98.43	0.19
	B	116	0.22	132.17	0.27
	AB	38	0.07	20.23	0.04
	O	281	0.54	266.16	0.54
	χ 2 (df = 3) 21.06		p < 0.001	—	—
Pakistan (N = 500)	A	136	0.27	125.93	0.25
	B	186	0.37	176.45	0.35
	AB	40	0.08	51.70	0.10
	O	138	0.28	146.15	0.28
	χ 2 (df = 3) 4.444		p = 0.217	—	—

Table 4.

Comparative analysis of the frequency of ABO phenotype and rhesus antigens in this study and those done by others.

Country	ABO blood group				Rhesus factor		Order	Reference
	A	B	AB	O	RhD+	RhD−
Liberia	15.80	22.40	7.40	54.40	98.00	2.00	O > B > A > AB	This study
Pakistan	27.20	37.20	8.00	27.60	88.00	12.00	B > O > A > AB	This study
Northern India	21.73	39.84	9.33	29.10	95.71	4.29	B > O > A > AB	Anifowoshe et al., [15]
Mauritania	28.28	18.56	4.05	49.10	94.23	5.77	O > A > B > AB
Morocco	32.86	15.80	4.53	46.80	91.00	9.00	O > A > B > AB
Cameroun	25.07	21.86	4.45	48.62	96.32	3.68	O > A > B > AB
Madagascar	22.61	29.66	6.13	41.60	98.90	1.10	O > B > A > AB
Guinea	22.54	23.86	4.72	48.88	95.94	4.06	O > B > A > AB
Ethiopia	28.11	23.35	5.44	43.08	92.06	7.94	O > A > B > AB
Iran	28.48	24.71	6.60	40.21	92.38	7.62	O > A > B > AB
Bangladesh	27.00	34.00	10.00	28.00	99.00	1.00	B > O > A > AB
Colombia	51.18	8.66	3.14	37.00	91.33	8.66	A > O > B > AB
Nigeria	22.77	20.64	3.66	52.93	94.90	5.10	O > A > B > AB
Winneba	23.50	17.50	3.00	56	92.20	7.80	O > A > B > AB	Koomson et al., [16]
Nigeria	26.70	17.90	3.60	51.8	95.20	4.80	O > A > B > AB	Oladeinde et al., [13]
Libya	26.27	21.31	18.74	33.68	68.11	31.89	O > A > B > AB	Regeai, Grenat [9]
India	23.88	37.38	9.97	29.27	94.90	5.01	B > O > A > AB	Sinha [14]
Ethiopia	32.50	23.80	6.30	37.5	96.60	3.40	O > A > B > AB	Tsega [17]

The occurrence of different blood types in each population may reflect underlying genetic variations shaped by population structure, historical migration patterns, and demographic factors, although the study did not investigate these parameters in detail.

HWE analysis revealed that the observed genotype frequencies in Pakistan did not significantly deviate from the expected values (χ ² = 4.44, df = 3, p = 0.217), indicating relative genetic stability within the sampled population. In contrast, the Liberian population showed a significant deviation from HWE (χ ² = 21.06, df = 3, p < 0.001), which may be attributed to sampling effects, population substructure, or other factors, such as non‐random mating.

Additionally, the chi‐square goodness‐of‐fit analysis showed that certain blood types differed significantly between the two countries (Table 2). The EM‐estimated allele and genotype frequencies for both populations, however, had the same order of occurrence. Specific patterns were observed for the alleles, homozygous and heterozygous genotypic frequencies (Table 3). Given the use of convenience sampling, these findings should be interpreted with caution.

Table 3.

EM‐estimated allele and genotype frequencies.

Country	Allele frequencies			Genotype frequencies
	P	q	r	p 2	q 2	r 2	2pq	2pr	2qr
Liberia	0.12	0.16	0.74	0.01	0.03	0.54	0.04	0.18	0.24
Pakistan	0.20	0.26	0.53	0.04	0.07	0.28	0.10	0.21	0.28

Studying these data is important as the prevalence of specific blood groups in each population may have implications for blood transfusion services, particularly in planning donor recruitment and managing blood bank services. The consistently low frequency of AB and RhD− blood types further underscores the need for targeted donor strategies for rare blood types.

While these results are scientifically obtained, we acknowledge that this study has several limitations. For example, the use of convenience sampling and restriction to undergraduate students may limit the generalizability of the findings to a broader population of students. Additionally, the study relied on descriptive analysis without advanced genetic or statistical modeling, which restricted the depth of analysis that could be made regarding population genetics.

5. Conclusion

The ABO and RhD blood groups studied in both populations revealed that blood types O and B predominated in Liberia and Pakistan, respectively. Blood group AB remained consistently the least common, whereas the RhD‐positive phenotype was the most prevalent in both settings. Despite the variations in ABO phenotype frequencies, the allele and genotype (heterozygous and homozygous) frequencies followed the same distribution pattern across the two populations.

These findings provide useful baseline data on the ABO and RhD blood group systems and highlight the need for large‐scale population‐based studies to further observe these patterns to determine the underlying genetic and epidemiological factors involved.

6. Future Direction

Since blood transfusion requires a blood type compatibility test, knowing one's own blood type can reduce the time spent labeling someone as incompatible for blood donation during screening. On the other hand, the genotypes of parents are also crucial in determining the blood type of their offspring. Other researchers must include calculations of genotypic frequencies in a study of this nature to validate the order of occurrence across populations. While the phenotypic appearance of blood types might differ, the genotypes may occur in the same order. Further studies are required to validate this observation.

Author Contributions

Augustine S. Samorlu: Conceptualization, investigation, methodology, validation, visualization, writing – review and editing, software, data curation, supervision, project administration, formal analysis, resources. Muhammad Sajid Saleem: Investigation, funding acquisition, writing – original draft, validation, visualization, writing – review and editing, data curation, resources.

Ethics Statement

The Ethical Research Committee of the University of Narowal was consulted and granted approval (reference number UON/Z/25/32) on January 7, 2025. Prior to the start of blood collection, informed consent was acquired from each willing participant. Being enrolled as a student at Cuttington University and the University of Narowal was a requirement for inclusion. Informed consent was obtained from all participants.

Conflicts of Interest

The authors declare no conflicts of interest.

Transparency Statement

The lead author, Augustine S. Samorlu, affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.