Prevalence and Distribution of ABO and RhD Antigens along with Its Subgroups & Rare Types in Greater Gwalior Region

Among a total of 29 blood group systems and over 600 different blood group antigens discovered so far, ABO and Rhesus are the most important blood group systems. The most significance rhesus antigen is Rh-D because of its immunogenicity. The sound knowledge of ABO and Rh-D antigens and its distribution in population is essential for the effective management of blood transfusion services, in population genetic studies, in resolving medico-legal issues and more importantly in compatibility test in blood transfusion practice. Study is aimed to provide data on ABO and Rh-D distribution in Gwalior region (Madhya Pradesh) and its comparison with related studies in India and abroad. Total 90,000 samples in 4 years from January 2004 to December 2007 were grouped for ABO, Rh-D and their subgroups at Blood Bank of Gajra Raja Medical College, along with Emergency Blood Bank, Gwalior, Madhya Pradesh, India. Blood group distribution among these samples showed phenotypes A, B, AB and O as 22.7%, 37.5%, 9% and 30.8% respectively. A2 and A2B were 8.0% and 8.6% respectively. The Rh-D negative phenotype was found 8.9% and D variants/weaker D was 0.036% among the sample tested. In cord blood samples, the incidence of A2 and A2B were 18.6% and 20.2% respectively.


Introduction
Among a total of 29 blood group systems and over 600 different blood group antigens discovered so far, ABO and Rhesus are the most important blood group systems [1].Landsteiner and his colleagues classified ABO group system into four blood groups (A, B, AB and O) depending on presence or absence of A and B antigens on red cells [2,3].
Group A individuals were further subdivided into A 1 , A 2 and other rare types like A 3 , A intermediate (int.),A x , Am, A bantu, A end, Ay, A finland (fin), A el, Ah (Hpartially deficient, non-secretor), and A weak.Blood group of A individual divided into two subgroup A 1 and A 2 on the basis of strength of the antigen in which former being stronger [4].Group A red cells which react with both anti-A and Anti-A 1 are classified as A 1 .A 1 constituted approximately 80% of entire A blood group popu-lation and group A cell which react with anti-A and not agglutinate with anti-A 1 are classified as A 2 , making up of remaining 20% [5].Subgroup of B are very rare and occur less frequently than subgroup of A and they are B 3 , B x , B m , Bh (H-partially deficient, non-secretor).
The Rh antigen is present in almost 85% of human whites [6].The original Rh antigen was referred to as D and related ones are C and E; the antithetically related antigens are called c and e [6].
Other blood group systems are Lewis, P, I, MNSs, Kell, Duffy, Kidd and many more.Bombay (oh) phenotype was discovered in 1952 [7].Para-Bombay subject shows weaker reaction with anti H reagent [8].
Despite the long list of several other blood groups discovered so far, for the safety of blood/blood products transfusion till date [9], the knowledge and distribution of ABO and Rh-D blood group are essential for effective management of blood bank inventory [10] as well as useful in population genetic studies, researching population migration patterns and resolving certain medicolegal issues [9].
A limited study investigated the ABO and Rh-D blood group frequency in Madhya Pradesh (M.P), especially in and around Gwalior region of India.This study was aimed to provide data on ABO and Rh-D blood group distribution with their subtypes and weaker variants in Gwalior and surrounding region and compare their frequency in other parts of India and abroad.

Material and Method
Total 90,000 blood samples were grouped for complete ABO and Rh-D grouping along with subgroup panel over 4 years (Jan. 2004to Dec. 2007).Blood samples were received from blood donor, blood recipient, antenatal OPD, neonates, cord blood and people who came for routine medical checkup at blood bank, Gajra Raja medical college & JAH groups of hospital and emergency blood bank, Gwalior (M.P) India.All entries were double checked by each author.
ABO and Rh-D grouping were carried out by standard tile and tube technique with appropriate positive and negative control.The tile technique was done by using one drop of 5% RBC suspension mixed with 2 drops of appropriate antiserum, rocked gently and looked for agglutination.In tube method, it was done by taking one drop of 5% cell suspension mixed with one drop of antiserum, incubate for 10 minutes at appropriate temperature and centrifuged for one minute at 1500 revolution per minute (rpm) and examined macroscopically and also under microscope for confirmation of agglutination.
Reverse grouping was done by using pooled A and B cells [11].For complete ABO grouping (forward and reverse) anti A, anti A 1 , anti B, anti AB, anti H antiserum and A, B, O pooled cells were used.
For Rh typing anti-D (R o & R 1 ) antiserum were used.For confirmation of blood grouping & Rh-D typing supportive tests like Indirect Coombs Test (ICT), saliva grouping and absorption elution test, were also performed when necessary.
Data on frequency of ABO and Rh-D blood groups were reported in simple percentages.
Amongst 33,748 cases of blood group B, weak B variants were found in 4 (0.011%) and 1 case (0.0029%) of pseudo B was also reported.Out of 8101 cases of blood group AB; 7405 (91.4%) were A 1 B and 696 (8.6%) were A 2 B while 3 cases (0.04%) off A 3 B was also reported (Table 1).Incidence of irregular antibody, anti-A 1 was 10% in A 2 and 21% in A 2 B cases, detected by agglutination method.
Out of 90,000 samples, 8980 samples were collected from cord blood/neonates and showed 2103 cases (23.4%) of A blood group, 3322 cases (36.9%) of B, 2754 cases  A x -1 (0.047%), A 3 B-1 (0.37%) were also reported (Table 2).Bombay Group (Oh) and its variants were not reported in the present study.
In 90,000 blood samples, incidence of Rh-D negative was 8.9% (8010) and Rh-D positive was 91.1% (81,990) (Figure 2).D U variant/weaker D were observed in 0.036% (34).Apparently, there was difference in distribution of ABO and Rh-D phenotypes among the samples obtained from different sources.
The most uncommon blood group in our study was AB and it is also observed in other parts of India and abroad.The incidence of A 2 and A 2 B was 8% and 8.6% in our study which is more than studies conducted in India.In south India it is A 2 -3.01%and A 2 B-1.43% [15] and in Andhra Pradesh it is less than 4% [21].Significance of weaker variant like A 2 , A 2 B and other subgroups of A and AB is that they may have anti-A 1 , which gives an incorrect ABO blood typing in reverse grouping and mistyped AB as B and A as O. Substantial qualitative differences between A 1 and A 2 antigen is also exist.A 2 and A 2 B individuals can not recognize A 1 antigens as being part of there own red cells make up and are immunologically stimulated to produce specific Anti-A 1 antibody that does not cross react with A 2 red cells.A 1 and A2 are interchangeable as far as transfusion purposes are concerned.Both types will react with anti-A serum, so a mistake in typing will usually not be made.There is a very small percentage of A 2 and large group of A 2 B people who produce antiA 1 , which gives an incorrect blood typing result for ABO type, it become critical as failure to detect a weak subgroup of A may result in a patient or donor being mistyped as group AB or B. They may also be mistyped as group O but this is not a serious problem.
Surprisingly incidence of A 2 and A 2 B in neonatal/cord blood sample was 18.6% and 20.2% respectively and it is much more than reported in adults.Most of the group A infants appears to be A 2 at birth, because ABO antigens are not fully developed on the red cells at this time.However no difficulty is usually encountered in grouping cord red cells because most of the reagent contains potent anti-A and anti-AB.Most of the cord blood of A 2 cells will eventually group as A 1 after few months [22].We have also reported weaker variants of B like weak B and pseudo B.
Subgroups of A and B differentiated by using the following reactions: 1) Cell's agglutination with antiserum like, anti A, anti A 1 , anti B, anti AB, and anti H sera; 2) Presence of A, B & H substance in saliva; 3) Adsorption elution studies; 4) Presence or absence of antibodies in the serum.Incidence of Rh-D negative in present study is 8.9% which is more than observed in other part of India like South India-5.42% [15], Lodha tribe in Midnapore district of West Bengal 0.5% ( 16), Assam >2% [23], Northern India i.e.U.P and Punjab-8.7%[17].It is higher in European population (15%) and in Nigeria it is 6% [24].
The discrepancy between our finding and that reported from other area may be attributed to ethnic difference among the population of Gwalior and surrounding region.

Conclusion
We concluded that among the various ABO blood groups, group B is the commonest; followed by O and A while AB is the least common type.The prevalence of Rh-D negative is higher than the other studies.The blood groups A 2 and A 2 B are more frequent in cord blood/neonates than in adults.

Figure 2 .
Figure 2. Distribution of various Rh phenotypes among the samples studied (90,000).