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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 5  |  Issue : 2  |  Page : 192-196

Serological evaluation and differentiation of subgroups of “A” and “AB” in healthy blood donor population in Eastern India


Department of Transfusion Medicine, Apollo Gleneagles Hospitals, Kolkata, West Bengal, India

Date of Submission21-Apr-2020
Date of Decision16-Jun-2020
Date of Acceptance14-Sep-2020
Date of Web Publication13-Nov-2020

Correspondence Address:
Sudipta Sekhar Das
Department of Transfusion Medicine, Apollo Gleneagles Hospitals, Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/GJTM.GJTM_34_20

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  Abstract 


Background and Objectives: Weak subgroups of A antigen in A or AB blood group can be differentiated by various immunohematological investigations. These weak phenotypes mainly result from weaker expression of an alternate weak allele present at the ABO loci. Weak subgroups of A antigen either reacts weakly with anti-A antisera such as A3, Ax, and Aendor do not react at all such as Am, Ay, and Ael. In this study, we characterized and differentiated the broad and weak subgroups of A in ABO blood group system through various immunohematological investigations. Methods: The prospective study included 67,954 healthy blood donors. Initial blood grouping and antibody screening of all donor samples were performed using an automated solid-phase assay. Samples showing blood group discrepancy or weaker agglutination were subjected to battery of serological investigation including saliva hemagglutination inhibition test and adsorption-elution procedures. Results: Among 16,034 A group donors, A1were 13,276 (82.8%), A22750 (17.15%), and weak subgroups were 8 (0.05%). Among AB group, A1B donors were 5621 (84.93%), A2B 996 (15.05%), and weak subgroups of AB were 1 (0.015%). The unexpected naturally occurring anti-A1antibodies (1.9%) were of IgM type optimally reactive at 4°C with titer ranging from 1 to 8. Red cells' agglutination with anti-A and anti-AB varied from Wk + to 2+ with or without mixed field agglutination in A3, Ax, and Aendphenotypes. While saliva study revealed specific substances, adsorption-elution demonstrated “A” antigen specificity in different strengths in Am, Ay, and AmB phenotypes. Conclusion: The subgroups of A and AB are attributed to a reduced number of A antigen sites on the red cells. We conclude that differentiating weak subgroups of “A” by serological assays is possible to a great extend with technical expertise; however, molecular genotyping is essential because it confirms the result.

Keywords: Adsorption-elution, anti-A1 lectin, saliva test, subgroups, weak subgroups


How to cite this article:
Das SS, Biswas RN, Safi M, Zaman RU. Serological evaluation and differentiation of subgroups of “A” and “AB” in healthy blood donor population in Eastern India. Glob J Transfus Med 2020;5:192-6

How to cite this URL:
Das SS, Biswas RN, Safi M, Zaman RU. Serological evaluation and differentiation of subgroups of “A” and “AB” in healthy blood donor population in Eastern India. Glob J Transfus Med [serial online] 2020 [cited 2020 Nov 27];5:192-6. Available from: https://www.gjtmonline.com/text.asp?2020/5/2/192/300619




  Introduction Top


Karl Landsteiner discovered the ABO blood group system in 1901.[1] It is the most important blood group system for clinical transfusion medicine.[2] In ABO blood grouping, the individual's red blood cells (RBCs) express A, B, AB, or no antigen, and the serum, respectively, contains the naturally occurring antibody anti-B, anti-A, no antibody, or anti-A and anti-B both. These naturally occurring antibodies in serum cause major barrier against ABO incompatible blood transfusion and organ transplantation. ABO locus present on the long arm of chromosome 9 encodes blood group-specific transferases, which transfer N-acetylgalactosamine and/or D-galactose sugar terminally to the H antigen for the formation of A antigen or B antigen, respectively.[3] The two major subgroups of A and AB, namely, A1 or A1B and A2 or A2B are differentiated on the basis of reactivity of A1 cells with both anti-A and anti-A1 where A2 cells do not react with anti-A1 lectins (Dolichos biflorus). Weak subgroups of A antigen in A or AB blood group can be differentiated by various immunohematological investigations as discussed by previous authors.[4],[5] These weak phenotypes mainly result from the weaker expression of an alternate weak allele present at the ABO loci. Weak subgroups of A antigen either react weakly with anti-A antisera such as A3, Ax, and Aend or do not react at all such as Am, Ay, and Ael.[4],[5],[6],[7],[8]

Objectives

Here, we studied the frequency of subgroups of A and AB in the healthy blood donor population of Eastern India and serologically characterized and differentiated the weak subgroups based on various immunohematological investigations.


  Materials and Methods Top


The prospective study conducted from January 2013 to December 2019 in a tertiary care hospital blood bank in Eastern India included 67,954 healthy blood donors.

Ethics

Ethical clearance was duly taken from the institute ethical committee vide letter no 2020/05/025328A, dated 18.5.2020.

All donors were selected for blood donations after proper testing and screening as mandated in the Drugs and Cosmetics Act, India.[9] As per departmental standard operating procedure (SOP), blood samples both in EDTA and plain vials were collected for mandatory testing to determine suitability of the blood units. Donors with a history of diseases, twin sibling, and transplantation were excluded from the study.

Initial blood grouping and antibody screening of all donor samples were performed using automated solid-phase assay (Immucor Galileo, USA). Any sample showing blood group discrepancy or weaker agglutination was subjected to further investigation as per departmental SOP using conventional tube technique. All A group and AB group samples were subjected to testing using anti-A1 lectin to differentiate A1 from A2 and other weaker variants. Samples showing weaker (≤2+), mixed field, or no agglutination with anti-A were further tested with monoclonal anti-AB and anti-H lectin.[4],[5],[7] Blood samples that failed to agglutinate with both anti-A and anti-AB even after incubation were subjected to adsorption-elution procedures.[4],[5],[7] Unexpected naturally occurring antibodies in serum such as anti-A1 when detected were further characterized for antibody titer, thermal amplitude, and immunoglobulin type based on dithiothreitol treatment of serum. Hemagglutination inhibition test for secretor status of saliva was done to detect ABH substance in A and AB group donors revealing weak subgroups of A.[4],[7]

Statistical analysis

Statistical analysis was done using the SPSS statistical package software (IBM SPSS, 2015, Armonk, New York, USA). Data were initially captured in MS Office Excel worksheet and then imported to the SPSS datasheet. Results were calculated as the number of cases (N) or expressed as a percentage (%). The range of values was calculated wherever applicable.


  Results Top


In 67,954 healthy donors under study, A, B, O, and AB blood group was found in 16,034 (23.6%), 23,181 (34.1%), 22121 (32.5%), and 6618 (9.7%) donors, respectively. Total Rh-positive donors were 64,929 (95.5%). Among A group donors, A1 were 13,276 (82.8%), A22750 (17.15%), and weak subgroups were 8 (0.05%). Among AB group, A1B donors were 5621 (84.93%), A2B 996 (15.05%), and weak subgroups of AB were 1 (0.015%). [Figure 1] depicts the detailed investigation workflow of A and AB subgroups. Investigations comprising testing RBC with anti-A1, anti-H, and anti-AB followed by saliva and adsorption-elution studies formed the primary basis of subgroup characterization. All unexpected naturally occurring anti-A1 antibodies were of IgM type and they were observed in 52 (1.9%) and 193 (19.4%) donors of A2 and A2B, respectively [Table 1]. The maximum titer of anti-A1 was found to be 8 reacting optimally at 4°C. [Table 2] describes the serological evaluation of weak subgroups of A and AB phenotypes. Where red cells' agglutination with anti-A and anti-AB varied from Wk+ to 2+ with or without mixed field agglutination in A3, Ax, and Aend phenotypes, saliva study revealed specific substances. Adsorption-elution demonstrated “A” antigen specificity in different strengths in Am, Ay, and AmB phenotypes.
Figure 1: Flowchart depicting detailed investigation of A and AB subgroups

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Table 1: Serological evaluation of A2 and A2B subgroups

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Table 2: Serological evaluation of weak subgroups of “A” and “AB” phenotypes

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  Discussion Top


The subgroups of A and AB are attributed to a reduced number of A antigen sites on the red cells. Where A1 is a potent gene that creates over 8 lakhs A1 antigen sites on the adult A1 red cells, A2 gene produces only 240,000–290,000 A2 antigen sites on adult A2 red cells.[5],[10] These quantitative differences have also been attributed to differences in concentration of N-acetylgalactosaminyltransferase in A1 and A2 individuals.[10] Approximately 80% of individuals of group A or AB are A1 or A1B and remaining 20% constitute A2 or A2B along with their weak subgroups.[4],[5] In the present study, A2 and A2B were observed in 17.15% and 15.05% of individuals, respectively. A prevalence of 1.85% A2 and 10.5% A2B was observed by Shastry and Bhatin their blood donor population.[11] Similarly, the prevalence of A2 and A2B as studied by Sharma et al. and Bangera et al. was found to be 8% and 8.6%, 1.3% and 12.7%, respectively.[12],[13] Similarly, workers in the past described varied figures of A2 and A2B prevalence in their population.[14],[15],[16]

Unexpected naturally occurring anti-A1 antibodies are seen in 1%–8% of A1 and 22%–35% of A2B individuals.[4],[5] Authors in the past described that anti-A1 antibody could really be antibody against Ac and Ad determinants, which A2 individuals lack. This anti-A1 reacts with A1 cells, which contains the Ac and Ad determinants and fails agglutination with A2 cells that actually lack Ac and Ad.[5] Anti-A1 was observed in 1.9% and 19.4% in A2 and A2B donors, respectively, in the present study. Where Giriyan et al. found anti-A1 in 0.4% of A2 and 25% of A2B blood donors, Shastry and Bhat demonstrated naturally occurring anti-A1 in 1.8% of A2 and 3.75% of A2B individuals.[11],[15] Unexpected anti-A1 in A2, A2B, and weak subgroups of A, which may cause a discrepancy in ABO testing and incompatibility in cross matches reacts better or only at a temperature well below 37°C.[4] We in the present study observed anti-A1 in 246 blood donors, which were IgM types and optimally reactive at colder temperatures (4°C > 22°C) with a maximum titer of 8. We observed anti-A1 in a young blood donor who was serologically characterized as Ax phenotype. Previous workers found that, although individuals with weak subgroups of “A” may produce anti-A1, the prevalence is comparatively higher in Ax phenotype.[17],[18]

Authors in the past commented that subgroups are mainly of academic interest and group “A” phenotype demonstrating weaker serologic reactivity than A2 is designated weak subgroups. A battery of test that included reactivity of red cells with anti-A, anti-AB, and anti-H followed by secretor study and adsorption-elution tests can be used to subdivide “A” group individuals into weak phenotypes such as A3, Ax, Aend, Am, Ay, and Ael.[5] Evidence for quantitative differences was first demonstrated by a gradual decrease in the standard agglutinability of “weak A” RBC with human anti-A (B) sera, from A3 red cells (63% ± 10%) to Ax(33% ± 10%), Aend(10% ± 5%) then Am, Ay, and Ael(0%) and second by direct measurement of A antigen site densities, the mean values being, respectively, 35,000 A sites/RBC in A3, 4800 in Ax, 3500 in Aend, and 700 A antigen sites/RBC in Am and Ael.[19] While Das et al. demonstrated “Ay” phenotype in a healthy female by serological means, however, authors commented that serologically “Ay” is almost similar to “Am” and even absorption-elution test fails to differentiate the two phenotypes.[20] Additional special procedures such as serum glycosyltransferase enzyme studies, “A” substance quantification, and molecular genetic analysis can be performed for differentiation of weak subgroups more accurately.[5],[21],[22] Huang et al. in a large study consisting of 1.45 million individuals identified ten novel weak ABO subgroup alleles in the Chinese population. They observed that GTA mutant p.L339P may lead to a weak A phenotype by changing the local conformation of GTA and reducing its stability.[21]

Thakral et al. observed 0.02% incidence of weak subgroups in their A, B, and AB blood donor population.[18] We found nine weak subgroups of “A” (0.01%) in our study [Table 2], which is in accordance with result observed by Shastry and Bhat.[11] Weak subgroup A3 has been estimated to be 1 in 90,000 in the Canadian blood donor population and 1 in 16,667 in the French donor population.[23],[24] In the current study, the frequency of A3, Am, and Ay phenotypes was estimated to be 1 in 33,977. Where frequency of Ax phenotype was observed in 1 in 40,000–1 in 77,000 populations in various French studies, the frequency of Ax, Aend, and AmB in the present study was found to be 1 in 67,954 each.[24] Unlike the other weak subgroups of A, “Ay” has a recessive mode of inheritance and can be observed in siblings. Therefore, family (pedigree) study is an important step in the evaluation of “Ay” phenotype. It is hypothesized that the presence of a recessive regulatory gene “yy” in double dose results in suppression of the A gene, leading to the development of Ay phenotype.[25] Frequency of ABO phenotypes, their subgroups including the weak ones, varies geographically according to the ethnic population and the population under study. Furthermore, variation in frequency in different studies from the same geographical location depends on the testing methodology and test sensitivity. Nair et al. performed molecular genotyping in a 54-year-old patient and confirmed the ABO status as Aw06/O13, which was serologically similar to Ax.[22] Due to the lack of molecular genotyping facility, the characterization of subgroups including the weak ones in the present study has been performed serologically following standardized protocols discussed elsewhere.[4],[5] However, more multicentric large studies are needed in the country or in similar ethnic populations to accurately comment on the frequency and prevalence of various broad and weak subgroups of ABO.


  Conclusion Top


We conclude that differentiating weak subgroups of “A” by serological assays is possible to a great extend with technical expertise; however, molecular genotyping is essential because it confirms the result. Mistyping weak subgroups of A as “O” group leads to (a) reporting and documenting wrong ABO blood group in a donor, (b) packed red cell units wrongly labeled as “O” and erroneously transfused to a “O” group patient may lead to hemolytic transfusion reaction, and (c) patient of weak A subgroup mistyped as “O” and transfused “O” whole blood may experience adverse transfuse reaction. In addition, unexpected naturally occurring anti-A1 may cause ABO group discrepancy, interfere with compatibility testing, and if not detected may cause transfusion reaction if clinically significant. Moreover, plasma containing unexpected naturally occurring anti-A1 should be managed as per blood bank policy and SOP. Differentiating subgroup of A also enables transfusion and transplant facilities to plan organ transplantations appropriately. Therefore, blood centers in the developing countries including India should establish simple procedures to detect and differentiate weak subgroups and develop procedures to ensure safe blood transfusion and transplantation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Landsteiner K. Uber agglutinationserscheinungen normalen menschlichen blutes. Wien Klin Wschr 1901;14:1132-4.  Back to cited text no. 1
    
2.
Storry JR, Olsson ML. The ABO blood group system revisited: A review and update. Immunohematology 2009;25:48-59.  Back to cited text no. 2
    
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Seltsam A, Hallensleben M, Kollmann A, Burkhart J, Blasczyk R. Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles. Transfusion 2003;43:428-39.  Back to cited text no. 3
    
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Fung MK, editor. American Association of Blood Banks Technical Manual. 18th ed. Bethesda, Maryland, USA: ABO, H and Lewis Blood Groups and Structurally Related Antigens; 2014. p. 291-315.  Back to cited text no. 4
    
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Harmening DM. ABO Blood Group System. Modern Blood Banking and Transfusion Practices. 6th ed. Philadelphia, USA: F. A. Davis Company; 2012. p. 119-148.  Back to cited text no. 5
    
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Cartron JP, Reyes F, Gourdin MF, Garretta M, Salmon CH. Antigen site distribution among weak A' red cell populations. A study of A3, Ax and Aend variants. Immunology 1977;32:233-44.  Back to cited text no. 6
    
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Bercher ME. Red cell typing. In: American Association of Blood Banks Edition. Technical Manual. 15th ed. Bethesda, Maryland: AABB; 2005. p. 731-50.  Back to cited text no. 7
    
8.
Lopez M, Benali J, Bony V, Salmon C. Activity of IgG and IgM ABO antibodies against some weak A (A3, Ax, Aend) and weak B (B3, Bx) red cells. Vox Sang 1979;37:281-5.  Back to cited text no. 8
    
9.
Malik V. Drugs and Cosmetics Act, 1940. 13th ed. Lucknow, India: Eastern Book Company; 2001.  Back to cited text no. 9
    
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Schachter H, Michaels MA, Tilley CA, Crookston MC, Crookston JH. Qualitative differences in the N-acetyl-D-galactosaminyltransferases produced by human A1 and A2 genes. Proc Natl Acad Sci U S A 1973;70:220-4.  Back to cited text no. 10
    
11.
Shastry S, Bhat S. Imbalance in A2 and A2B phenotype frequency of ABO group in South India. Blood Transfus 2010;8:267-70.  Back to cited text no. 11
    
12.
Sharma DC, Rai S, Iyenger S, Jain B, Sao S. Prevalence and distribution of ABO and Rh-D antigens along with its subgroups and rare types in greater Gwalior region. Open J Blood Dis 2013;3:69-73.  Back to cited text no. 12
    
13.
Bangera IS, Fernandes H, Swethadri G, Naik K. Prevalence of A2 sub group in A and AB blood groups and the transfusion implications. Asian J Transfus Sci 2007;1:103.  Back to cited text no. 13
    
14.
Mahapatra S, Mishra D, Sahoo D, Sahoo BB. Study of prevalence of A2, A2B along with major ABO blood groups to minimize the transfusion reactions. Int J Sci Res 2016;5:189-90.  Back to cited text no. 14
    
15.
Giriyan SS, Agrawal A, Bajpai R, Nirala NK. A1 and A2 sub-types of blood group 'A': A reflection of their prevalence in North Karnataka region. J Clin Diagn Res 2017;11:EC40-2.  Back to cited text no. 15
    
16.
Hussain R, Fareed M, Shah A, Afzal M. Prevalence and gene frequencies of A1A2BO and Rh (D) blood group alleles among some Muslim populations of North India. Egypt J Med Hum Genet 2013;14:69-76.  Back to cited text no. 16
    
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Cartron JP, Gerbal A, Hughes-Jones NC, Salmon C. 'Weak A' phenotypes. Relationship between red cell agglutinability and antigen site density. Immunology 1974;27:723-7.  Back to cited text no. 17
    
18.
Thakral B, Saluja K, Bajpai M, Sharma RR, Marwaha N. Importance of weak ABO subgroups. Lab Med 2005;36:32-4.  Back to cited text no. 18
    
19.
Cartron JP. Quantitative and thermodynamic study of weak A erythrocyte phenotypes. Rev Fr Transfus Immunohematol 1976;19:35-54.  Back to cited text no. 19
    
20.
Das SS, Zaman RU, Safi M, Sen S, Sardar TP, Ghosh S. Investigating weak A subgroups in a healthy lady: The blood bank limitations. Asian J Transfus Sci 2014;8:62-3.  Back to cited text no. 20
[PUBMED]  [Full text]  
21.
Huang H, Jin S, Liu X, Wang Z, Lu Q, Fan L, et al. Molecular genetic analysis of weak ABO subgroups in the Chinese population reveals ten novel ABO subgroup alleles. Blood Transfus 2019;17:217-22.  Back to cited text no. 21
    
22.
Nair R, Gogri H, Kulkarni S, Gupta D. Detection of a rare subgroup of A phenotype while resolving ABO discrepancy. Asian J Transfus Sci 2019;13:129-31.  Back to cited text no. 22
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23.
Garretta M, Muller A, Gener J, Matte C, Moullec J. Reliability in automatic determination of the ABO group by the groupamatic system. Vox Sang 1974;27:141-55.  Back to cited text no. 23
    
24.
Reed TE. The frequency and nature of blood group A3. Transfusion 1964;4:457-60.  Back to cited text no. 24
    
25.
Meng QB. Genetic analysis of an individual of Ay serologic phenotype. Nan Fang Yi Ke Da Xue Xue Bao 2009;29:1615-6.  Back to cited text no. 25
    


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