Abstract
ABO (ABH) antigen expression on red cell antigens and secretion of ABH substances in body fluids are governed by different genes. The presence or absence of the Secretor gene is the basis of secretor status positivity and negativity, respectively. Determination of the secretor status has an essential role in the medical field. It was a prospective observational study to determine the association of secretor and nonsecretor status with Lewis phenotyping among voluntary blood donors. It was conducted from Jan 2020 to December 2021 at the Department of Transfusion Medicine in a tertiary care hospital in South India. Among 318 donors, secretors were 229 (72%), and nonsecretors were 89 (28%). The Lewis phenotype was consistent with secretor status, i.e., All Le (a-b +) were secretors accounting for 213 (66.9%), and All Le(a + b-) were nonsecretors accounting for 80(25.1%)donors. In the Le(a-b-) phenotype, 16(5.0%) were secretors with Se positivity but defective Lewis expression, and 9(2.8%) were nonsecretors with sese. Detection of Lewis phenotype indirectly reflects the blood group secretor status. Hence, in case of discrepancies in ABO blood groups, Bombay and Para Bombay groups, testing for Le(b) confirmation will be useful.
Keywords: – Lewis blood group, Le a, Le b, Secretor
Introduction
The landmark discovery of ABO blood groups by Sir Karl Landsteiner in 1900 heralded a sea change in blood transfusion practices [1]. For the discovery of the ABO blood group system, he was awarded a Nobel prize in 1930 [2]. The term “blood group” refers to the entire blood group system containing red blood cell antigens specifically controlled by a series of genes that can be linked closely to the same chromosome or allelic. The peculiarity of the ABO blood group is the antigens are expressed on red cells. These antigens (ABH substances) are also secreted in body secretions in persons called secretors. The natural antibodies anti A and Anti B are detected in plasma. The expression of ABO antigens on the red cells and ABH substances secretion in body fluids are dependent on different genes. The presence or absence of the secretor gene is the basis for secretor status, with a co-expression of Le (b) also confirming the secretor [2]. ABO (ABH) antigen expression on red cell antigens and secretion of ABH substances in body fluids are governed by different genes. The presence or absence of the Secretor gene is the basis of secretor status positivity and negativity, respectively; it also determines the co-expression of Lewis b antigen {Le (b)}. Secretors being Le (b) positive. Hence in case of discrepancies of ABO blood groups, confirmation of Le(b), Bombay, and Para Bombay groups, testing for secretor status becomes an important testing method to resolve the same.
The importance of these well-defined antigens is increasingly being sought in disease, forensic pathology, and population genetics. In this regard, baseline data on the variable expression of blood group antigens concerning secretor status in the Pondicherry (South India) population is unavailable. This study was done for the first time in Pondicherry, and only a few studies were done in India. This study will provide a proportion of secretor and nonsecretor status in the voluntary blood donors population attending the blood Centre and resolve any ABO blood group discrepancies. The present study aims to identify secretor and nonsecretor status association with Lewis phenotyping among voluntary blood donors.
Material and Methods
Study Design
The present study is a prospective observational study conducted at the Department of Transfusion Medicine in a tertiary care hospital of South India from January 2020 to December 2021. Ethical approval was obtained from the institute ethics committee ie JIP/IEC/2019/519.
Study Participants
Inclusion criteria were all Voluntary blood donors attending our blood center for donating whole blood, while exclusion criteria were the donors who tested seropositive to Transfusion Transmissible Infections.
Sample Size Calculation
Sample size was calculated using Open Epi Software Version 3.0. With the expected prevalence of secretors as 75% based on previous study, [3] the sample size is estimated at 289 with a 95% confidence level and 5% absolute precision. Considering 10% loss due to wastage, discards, etc., the sample size was recalculated to be Three hundred and eighteen.
Sampling Technique
Consent was taken from voluntary blood donors. Every Monday (1stworking day of the week) -one A, Two B, and two O blood group donors have been taken along with one A or AB donor (alternate week) -A, B, O, and AB in the ratio of 2:4:4:1 expecting the prevalence of ABO blood groups in south India to be—blood group O (39%), B (33%), A (21%), AB (7%). Five ml saliva sample was collected from the donors.
Selection of Blood Grouping Reagent Dilution [4]
To 1 drop of each reagent antisera (anti A, anti B, anti H), one drop of 2% to 5% saline suspension of corresponding pooled cells was added. The tubes were centrifuged at 1000 rpm for 1 min and examined macroscopically for agglutination. The reaction was graded accordingly. The highest dilution giving 2 + reactions has been selected for the hemagglutination inhibitor test.
Inhibition Test for Secretor Status
Four test tubes have been labelled as saline control, test sample, secretor, and nonsecretor. One drop of appropriately diluted blood grouping reagent has been added to each tube. One drop of saline has been added to the saline control tube. One drop of the saliva of the known secretor, known nonsecretor, and test saliva has been added to the respective tubes. The contents were mixed well and incubated at room temperature for 8–10 min. One drop of 2%—5% pooled A group of red cells was added to each tube and incubated for 30 min at room temperature. The tubes have been centrifuged at 1000 rpm for 1 min and examined macroscopically for agglutination. (Table 1, Figs. 1 and 2).
Table 1.
Interpretation of results after inhibition test for secretor status
| Testing with specific Antisera | ||||
|---|---|---|---|---|
| Unknown Saliva | Se saliva (A Substance present) | Non-Se Saliva (A Substance Not present) | Saline (Dilution Control) | Interpretation |
| 2+ | 0 | 2+ | 2+ | Non-secretor |
| 0 | 0 | 2+ | 2+ | Secretor |
Fig. 1.
Determination of secretor status by hemagglutination inhibition method
Fig. 2.
Agglutination in Test sample (TS) tube indicating Nonsecretor. [Se-Secretor, NSe-Non-secretor, TS-Test Sample, SC—Saline Control]
Gel card method: Lewis(a) Lewis(b) gel cards have been taken, and donor ID details were labeled. Five percent Red cell suspension has been prepared and incubated at room temperature for 10 min. 10 ul of red cell suspension was added to gel cards. Gel cards were suspended to centrifuge for 10 min, and the results were recorded. (Fig. 3).
Fig. 3.
Lewis phenotyping gel card showing Le (a-b +) in ID 17030, ID 17031 indicating secretor and Le (a + b-) in ID 17037 indicating nonsecretor
Statistical Analysis
The distribution of categorical data such as blood group, Rh factor, gender, and Lewis phenotyping was expressed as frequency and percentage. The distribution of secretor nonsecretor status has been expressed as frequency and percentage. Chi square test was used for association between secretor status with Lewis phenotype.
Results
During the study period, a total of 27,014 donors donated to our blood center among them, 18,909(70.0%) were voluntary, and 8,105(30.0%) were replacement blood donors. Males were 25,663(94.9%) while female were 1,350 (5.1%). As per the sample size calculated, the study population comprised 318 donors, and all were voluntary blood donors. All the 318 donors were Transfusion-transmitted Infection non-reactive; hence no sample was discarded. The majority ABO and Rh blood group comprising O + were 126 (39.6%), lowest with AB + 19 (5.9%), and in Rh negative O negative- 2(0.6%), B negative-1 (0.3%) in total 318 study participants. Saliva testing showed that 229(72.0%) were secretors and 89 (28.0%) were nonsecretors among 318 blood donors (Table 2). Lewis phenotyping distribution among the study population with Le (a-b +) phenotype were 213 (66.9%) secretors, Le(a + b-) are 80 (25.1%) were nonsecretors and Le(a-b-) a mixed population of secretors and nonsecretors were 25(7.8%). (Table 3 and Table 4).
Table 2.
Distribution of secretor status prevalence among study participants
| Blood Donors (n = 318) | ||
|---|---|---|
| Secretor Status | No | % |
| Secretors | 229 | 72% |
| Nonsecretors | 89 | 28% |
Table 3.
Lewis Phenotype distribution among the study population
| Lewis Phenotyping | Blood Donors (no = 318) | |
|---|---|---|
| Le(a-b +) | 213 | 66.9% |
| Le(a + b-) | 80 | 25.1% |
| Le(a-b-) | 25 | 7.8% |
Table 4.
Lewis Phenotype distribution with secretor status among the study participants
| Blood Donors (318) | ||||||
|---|---|---|---|---|---|---|
| Lewis Phenotyping |
Le(a-b +) (213) |
Le(a + b-) (80) |
Le(a-b-) (25) |
|||
| Secretors | 213 | 66.9% | NIL | 16 | 5.0% | |
| Non-Secretors | NIL | 80 | 25.1% | 9 | 2.8 | |
Lewis phenotyping distribution among the study population was studied [Table 5]. The association between Lewis Phenotype distribution with age, gender, and blood group in the study population was statistically not significant (p < 0.05).
Table 5.
Association of Lewis phenotyping with Age, Gender, and blood group
| Lewis Phenotyping, [n = 318] | |||
|---|---|---|---|
| AGE(YEARS) |
Le(a-b +) (213) (P = 0.19) |
Le(a + b-) (80) (P = 0.72) |
Le(a-b-) (25) (P = 0.74) |
| 18–25 (114) | 72(63.1%) | 35(30.7%) | 7(6.1%) |
| > 25–30 (77) | 46(59.7%) | 20(25.9%) | 11(14.2%) |
| > 30–35 (63) | 42(66.6%) | 15(23.8%) | 6(9.5%) |
| > 35–40 (42) | 33(78.5%) | 8(19.0%) | 1(2.3%) |
| > 40–45 (16) | 15(93.7%) | 1(6.3%) | 0 |
| > 45–50 (3) | 2(66.6%) | 1(33.3%) | 0 |
| > 50 (3) | 3(100%) | 0 | 0 |
| Gender | |||
| Males (306) | 202(66.0%) | 79(26%) | 25(8%) |
| Females (12) | 11(91.6%) | 1(8.4%) | 0 |
| Blood Group | |||
| O (n = 128) | 75(58.5%) | 37(28.9) | 16(12.5%) |
| B (n = 108) | 77(71.2%) | 25(23.1%) | 6(5.5%) |
| A (n = 63) | 42(66.6%) | 18(28.5%) | 3(4.7%) |
| AB (n = 19) | 19(100%) | 0 | 0 |
Discussion
Lewis blood group which is considered as minor blood group and Le antibodies are usually clinically not significant. Presence or absence of Le a, Le b phenotype can help to know secretor status. In present study we found Le a + b- phenotype has concordance with nonsecretor while Le a-b + has concordance with secretors. Secretor status is indicated by the presence of (SeSe) and the absence of (se) gene, which secretes ABH antigen soluble substance. Usually, H antigens are present on red blood cells and secretions involving two different fucosyl transferase enzymes encoded by chromosome 19, FUT-1 and FUT -2. Therefore, these genes are also referred to as the H gene and the Se gene. H antigen is formed in RBCs when H (FUT 1) gene-encoded with fucosyl transferase attaches fucose via Alpha 1, 2 linkages to terminal galactose of type 2 precursor chains. H antigens in secretions are formed when Se gene-encoded fucosyltransferase attaches fucose via Alpha 1,2 linkage to the galactose of type 1 precursor chains in secretory tissues [4]. In the absence of a functional Lewis gene (le le), neither Le a nor Le b is synthesized, leading to Le (a– b–) or null phenotype. Type 1 chain ABH antigens may still be synthesized and secreted in individuals who have inherited at least one functional Se allele (one functional FUT2 allele). So, Le (a-b-) can be both secretor and non-secretor. In a study conducted on voluntary blood donors in India, the frequency of secretors was 75%, and non-secretors was 25%, respectively, similar to our findings. Further, 65% of the participants were Le (a-b +) positive, 20% were Le (a + b-), and the remaining 15% were Le (a-b-), which correlated with the ABH antigen secretor status [3].
In the present study, we assessed the frequency of Lewis blood group association with age, gender, and ABO blood group. We found no statistical significance. In most of the studies, above association were hardly assessed [2, 3]. Lewis antigens are synthesized in intestinal epithelial cells and circulate in plasma either in free or bound to lipoproteins or passively attached to the Red blood cell (RBC) membrane. Le (a) and Le (b) are synthesized stepwise by two separate fucosyl transferases in which Fucose moieties are added to the type 1 glycoprotein chain. These enzymes are coded by Le (FUT3), which adds Fucose to N-acetyl glucosamine Se (FUT2). Also, it adds fucose to the Gal (galactose) moiety. The enzyme encoded by Le (FUT3) is responsible for the synthesis of Le (a), resulting in the Le (a + b-) phenotyping [5]. Enzymes encoded by Se(FUT2) synthesize Le(a) to Le(b) by the addition of another Fucose molecule, resulting in a Le(a-b +) phenotype. Individuals who have mutations in Le (FUT3) do not make Le(a), and because of no Se (FUT2) enzyme activity, they cannot synthesize Le(b), resulting in the formation of Le (a-b-) phenotype [3]. In the present study, we got Le(b) 72%, Le(a) 22%, and 6% for Le (a-b-) and zero for Le (a + b +). These findings are nearly similar to other studies from Asia [3, 6]. In a study from Bangladesh on blood donors, they reported 60% of secretors and 40% of nonsecretors, 19% were Le (a + b-), 53% were Le(a-b +), 26% were Le (a-b-) and 2% were Le (a + b +) [5]. However, our sample size was much larger in comparison to those studies.
The Lewis blood type is divided into two groups. Lewis positive (Le (a + b-) or Le (a-b +) phenotypes) and Lewis negative (Le (a-b-) phenotypes. The Lewis blood group determinants are structurally related to determinants of the ABO and H/h blood group systems, and the outcome of Lewis typing can be used to determine ABH secretor status among Lewis-positive individuals because FUT1 provides the glycans required for glycosyltransferase conversion into the Lewis antigen in addition to ABH. The phenotype will be Le (a-b +) in the presence of FUT2 alleles that express type 1 H determinants but Le(a + b-) in the absence of the FUT2 gene.
ABH secretors are always Le (a-b +) in Lewis-positive persons when they convert all of their Lewis (a) antigens into Lewis (b) antigens. On either hand, ABH non-secretors are invariably Le (a + b-) among Lewis positives because they lack the FUT2-dependent glycosyltransferase necessary for all of this. Lewis type cannot be used to detect ABH secretor status since a small percentage of the population (from 1 and 8%, based on race) will be Lewis negative. The same has been demonstrated by our results. Five percent of our study population were Le b- (Le a-b-) still they were secretors (Table 4). Although it is commonly assumed that ABH secretor status is all-or-nothing, this is not always true. There will often be some active A or B blood group component in the saliva of certain ABH non-secretors (known as partial or weak secretors). However, the quantity and quality of these chemicals are substantially decreased, predisposing them to functional issues similar to other nonsecretors [7, 8]. In present study we did not face partial secretors. Significant amounts of blood-type antigens (ABO and Lewis) released into saliva have various benefits. The typical mucosa of secretors in the stomach mucosa of healthy people has a consistent distribution of blood-type antigens in the pits. Non-secretory mucosa displays little staining for these blood-type antigens. However, it does show considerable amounts of the I(Ma) antigen. As will be shown when immunity is explored, this predisposition to express the I(Ma) antigen will influence antibody capacities [9].
Most of the previous studies shown that majority of the study participants were Le(a-b +), the secretors, similar to our research. Akhter et al. reported two cases of Lewis (a-b-) in their study while our study had 25 cases. Frequency of Lewis Phenotyping in A, B, AB, and O blood group individual in our study shows ABO blood group distribution with the frequency of Lewis Phenotyping among the blood donors with secretors L(a-b +) are B > O > A > AB. In non-secretors, the L(a + b-) frequency is O > B > A. In our study, there are no blood grouping discrepancies in the study participants. We found no rare blood groups like Bombay or Para Bombay blood groups during the study period.
Lewis antigens are not intrinsic red cell antigens but adsorbed from plasma. Le (a-b-) creates a curious association with secretor status, which is unique. Two hundred thirteen donors are Le(a-b +) who are pure secretors, 80 are Le (a + b-) and are nonsecretors, but 25 donors are negative for both Le(a) and Le(b). Of them, 16 are secretors, which shows that they have either defective Lewis antigens or antigens that are not adsorbed on red cells. The rest of the 9 are mainly secretor negative. Le (a- b-) phenotyping distributions among the study population are all males, of which 16(64%) were secretors, and 9(36%) were nonsecretors. No female in the study population had the Le (a- b-) phenotype. The chi-square test was applied to find an association between secretor status and le(a-b-) phenotyping, but it was not statistically significant(P-value = 0.353). The strength of the present study was that we had large sample size, also the haemagglutination inhibition method used by us was simple to perform and can be done in any laboratory center. The limitation was none of the bombay or parabombay blood group were in the test samples.
Conclusion
The present study has shown Le a + b- has cent percent concordance with nonsecretor while Le a-b + has concordance with secretor status. This will help in the easy identification of the secretors, which is further helpful in blood group discrepancies, confirming the Bombay blood group, forensic pathology, and population genetics.
Funding
This work was supported by an intramural research fund from the Jawaharlal Institute Postgraduate Medical Education & Research (JIPMER), Pondicherry, India.
Declarations
Conflicts of Interest
There are no conflicts of interest.
Footnotes
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