30 Blood Groups: ABO, Rh and MN systems

Dr. SAA Latheef and Dr. P. Venkatramana

Contents:

 

1. Introduction

 

2. Importance of screening blood groups

 

3. ABO blood group

 

3.1. Genetics of ABO blood group

 

4. Rh blood group

 

4.1. Genetics of Rh blood group

 

5. MNS blood group

 

5.1. Genetics of MNS blood group

 

6. Phenotyping of ABO, Rh and MNS blood groups 6.1. ABO Phenotyping

 

6.1.2 Detection of ABH secretion

 

6.2. Rh Phenotyping

 

6.3. MNS Phenotyping

 

7. Calculation of ABO, Rh and MNS allele frequency

 

7.1. ABO allele frequency and genotype calculations

 

7.2. Rh alleles (D and d) frequency calculation

 

7.3. MN allele frequency calculation

 

8. Summary

 

 

Learning Outcomes:

 

By reading this module you will be able to:

  •  discuss why physical anthropologists study blood groups and the importance of screening blood groups;
  •  appreciate ABO, Rh and MN blood groups and genetical aspects;
  •  know how to phenotype and interpret ABO, Rh and MN blood groups; and
  •  learn how to calculate allele frequencies of ABO, Rh and MN

 

  1. Introduction

 

Up to 19th century, physical anthropologists were occupied with descriptive, old or classical studies. In 20th century, physical anthropologists entered into analytical phase with a focus on reorientation in methodology and comprehension (Reddy, 2015). Currently, physical anthropologists are engaged in both descriptive and analytical studies besides embracing modern technologies for studying human population variations. Studying of human population variations help us in understanding our origins, medically relevant variations and also serve as antidote against racial prejudices (Bodmer, 2015). Physical anthropologists use various traits like skin colour, hair colour, colour blindness, taste sensitivity to phenylthiocarbamide and blood groups such as ABO, MN, Rh etc. to study variations among populations. As of now, there are 35 blood groups in human beings. It was observed that 44 genes and 1568 alleles encode for antigens in the 35 blood groups (McBean et al., 2014). ABO was the first blood group system which was discovered by Karl Landsteiner in 1900 and also the first polymorphic trait studied. Gene frequency data of ABO blood groups formed the basis for constructing first human evolutionary tree by Edwards and Cavalli-Sfroza (Seagurel et al., 2012; Bodmer, 2015). Blood group investigations on Indian soldiers was first carried out by Hirschfield in 1919 followed by Bais and Verhoef in 1927 (Majumdar and Roy, 1982) on Indian people. Blood groups are inherited characters of red cell surface that are detected by specific alloantibodies (Daniels, 2009).

  1. Importance of screening for blood groups

Transfusion means administration of blood components. Blood is transfused as components such as packed RBC, plasma, platelets and immunoglobins. First successful human to human transfusion was performed by Blundel in 1829 (Blundell, 1829). Blood components are transfused to prevent haemorrhages and to improve oxygen delivery to tissues (Sharma et al., 2011). Packed RBC is transfused in conditions such as road accident victims, anaemia (Hemoglobin < 9 g/dl), sickle cell anaemia, acute blood loss (>1500mL or 30% of blood volume); gamma irradiated red cells to immunocompromised patients, lymphoma patients, stem-cell/marrow transplants and unborn children undergoing intrauterine transfusion; and leuko (white blood cell) reduced red cell to reduce the risk of cytomegalovirus, Epstein-barr virus , human T-lymphotropic virus infections and febrile reactions. Platelets are administered to the patients of thrombocytopenia or platelet function defects or in preoperative setting when quantitative or qualitative defects of platelets are suspected as the cause of bleeding. Fresh frozen plasma is transfused in patients with multiple coagulation factor deficiencies, disseminated intravascular coagulation, congenital factor deficiencies and in thrombotic thrombocytopenic purpura (TTP) and haemolytic uremic syndrome along with plasmapheresis. In conditions of primary immune deficiency, B-cell chronic lymphocytic leukemia, idiopathic thrombocytopenia purpura, pediatric human immunodeficiency virus infection, Kawasaki syndrome and Guillain-Barre syndrome, transfusion of immunoglobulin is recommended (Sharma et al., 2011; Arya et al.,2011).

 

In India, 8 million units of blood or its components are transfused annually (Makroo et al., 2013). Incompatibility of recipient serum antibodies and donor blood group antigen can cause hemolytic transfusion reactions and haemolytic disease of newborn. Hemolytic transfusion reactions are due to immunogenic and non-immunogenic. The non-immunogenic causes are bacterial overgrowth, improper storing, infusion with incompatible medications, and infusion of blood through lines containing hypotonic solutions or small-bore intravenous tubes. The immunogenic cause are of two types i.e. acute or delayed (Sharma et al., 2011). In acute haemolytic transfusions, destruction of RBC(red blood cells) takes place within 24 hours. Acute type of reactions are classified as mild (urticaria, rash and itching), moderate (flusinghing, rigors, fever, restlessness, tachycardia, anxiety itching, palpitations, mild dyspnoea and headache) and life threatening(rigors, fever, restlessness, hypotension, tachycardia, haemoglobinuria, unexplained bleeding, anxiety, chest pain, pain near infusion site, respiratory distress loin/back pain, headache and breathlessness). Delayed type of haemolytic transfusion reactions occur after 24 hours to up to one year and due to reactivation of pre-existing antibodies against antigens on the transfused red cells. The delayed type of reactions includes alloimmunisation, multiple organ dysfunction syndrome, graft versus host disease, post transfusion purpura and iron overload (Sahu et al., 2014)

The destruction of RBC is also known as hemolysis which may be intra or extracellular. Intravascualr hemolysis is the occurrence of destruction of RBC and release its contents in the circulation due damage to the endothelium, complement fixation, activation on the cell surface and infectious agents. The extravascular hemolysis, is the removal or destruction of RBC with membrane alterations by the macrophages of the liver and spleen (Dhaliwal et al.,2004).

 

To ensure foetus survival till it matures, antibodies from mother are transferred to fetal circulation. Hemolytic disease of newborn (HDN) is caused when Rh negative mother become pregnant for Rh positive children second time. Hemolysis takes place when mother produce antibodies against D antigen (Rh blood group) antigen of foetus which are of IgG type, cross placenta and reach foetus. HDN is also caused by ABO incompatibility when mother with O blood group become pregnant for the foetus of A,B or AB. Mother with O blood group has anti-A and anti-B antibodies, produce antibodies of IgG type against fetal RBC antigen, cross placenta and cause hemolysis of fetal RBC. It was observed that fetal RBC express less of ABO antigens compared to adults and expression of ABO antigen by other fetal tissue, there is a less chance of binding maternal antiobodies to the target fetal RBC antigens. Occurrence of HDN due to the production of antibodies against antigens of the blood groups such as Kell, Kidd, Duffy, MNS and s were also observed (Dean, 2005).

It was observed that prevalence of alloantibodies ranges from 0-3% in randomly tested patients. Alloantibodies (antibodies developed against antigens lacked by the patient) are encountered in compatibility testing against antigens related to Rh, Kell, Kidd, Duffy and MNs blood groups. Alloantibodies are found in patients of thalassaemia, sickle cell anaemia, dialysis and cancer who

receive multiple transfusions. Antibodies are screened in transfusion centres using cells prepared from foreign donors in this kind of scenario antibodies against minor antigens go undetected. Presence of alloantibodies can also be detected in foreign patients ( who are coming for various treatments due to cost effective health care) transfused with blood components of Indian donors. There is grave need for availability of data on the prevalence of various blood group antigens in Indian populations to avoid the occurrence of alloantibodies (Makroo et al., 2013). Individuals with Bombay blood group don’t have A,B and H antigens on their RBC and have anti-A,B and H can be haemolytic and react with all blood ABO antigens and they have to be transfused with Bombay blood group or its components only (Shahshahani et al., 2013). Apart from importance of blood groups in blood transfusion, matching of recipients and donor blood group antigens is essential in organ transplantation. ABO blood group compatibility is the one of the criteria along with HLA, in organ transplantations of kidney, liver and heart (Rydberg, 2001).

Association of ABO blood groups and diseases were observed: Blood group A1 and pancreatic cancer (Wolpin et al., 2010), blood group A and hepatitis B virus infection, HIV infection and pancreatic cancer( Mohammadali and Pourfathollah, 2014; Wang et al., 2012), non-O blood group and deep vein thrombosis, arterial thrombosis, myocardial infarction, coronary artery disease, renal cancer in women, non-melanoma skin cancer, lung cancer, higher levels of Willebrand factor and coagulation factor VIII (Franchini and Lippi, 2015; Amjadi et al., 2015; Liumbruno et al., 2013); blood group A and helicobacter pylori infection(Wang et al., 2012); blood group A and AB and nasopharyngeal cancer (Sheng et al., 2013); blood group O and malaria, helicobacter pylori, cholera, peptic ulcer, hepatitis C and norovirus infection(Fry et al., 2007; Boren et al., 1993;Swerdlow et al., 1994; Aird et al., 1954; Aljooani et al., 2012; Huston et al., 2002); blood group B and malaria and type 2 diabetes (Panda et al., 2011; Qureshi and Bhatti, 2003); AB blood group and HIV-2 infection and influenza A and B (Abdulazeez et al., 2008; Aho et al., 1980; Naikhin et al., 1989). Investigations on association of blood groups and longevity showed conflicting reports, some studies showed increased survival O blood group individuals, whereas, other studies showed statistically significant increased survival of b blood group individuals (Mangoli et al., 2015).  Blood group screening is also used for studying genetic relationships between populations by physical anthropologists, to resolve paternity disputes and to exclude crime suspects by police and forensic personnel (Yamamoto, 2004).

 

  1. ABO blood group: ABO blood group system was discovered by Karl Landsteiner in 1901 and was awarded Nobel Prize for his seminal contribution in 1930. ABO blood groups are divided into four groups i.e A, B, AB and O based on the presence and absence of antigen on RBC. ABO antigens are oligosaccharides expressed on RBC as glycoproteins and glycolipids (Yamamoto et al., 2014). ABO antigens function physiologically as enzymes (3-α-D-GalNAc/Gal-transferases (A/B)) (Cartron and Colin, 2001).Blood group A has antigen A and antibody anti-B, B blood group has antigen B and antibody anti-A, AB blood has antigens A and B and no antibodies, whereas O blood group has no antigens but contains antibodies anti-A and anti-B. High frequency of O and B blood are observed in southern and northern parts of India(Suresh et al.,2015; group Antibodies of ABO are called natural antibodies (immunoglobulins produced by B lymphocytes of B1 type without stimulation(Schwartz-Albiez et al.,2009)) and issoagglutinins (structurally related antibodies which cause clumping of red cells). These antibodies are produced in the gut after contact with bacteria and virus carrying A-like and B like antigens (Ségurel et al., 2012). The natural antibodies belong to the class of IgM,IgG3 and IgA immunoglobulins (Panda and Ding,2015). ABO antibodies are detected by three months and reach adult levels by 5-10 years(Daniels,2002). Immunoglobulins are five types such as IgG,IgM, IgA, IgD and IgE and the main function of these immunoglobulins is to combine with antigen and mediate biological effects(Poole, 2001). Induced immuglobulins belong to the class of IgG and are produced in response to fetal RBC in pregnany and as alloantibodies in recipient’s blood in response to antigen which he/she lacks.

On RBC of humans, H antigen is present, which is a precursor for the formation of ABO antigens. The levels of H antigen present in carriers of O blood group, whereas least levels were found in AB carrying RBC. H antigen is formed by the binding of Fucosylation of terminal galactosyl residue of its precursor and this is catalyze by α1,2 fucosyltransferase. (Daniels, 2009; Das et al., 2011). H antigen exists as homo or heterozygous (HH or Hh) form. Individual who lack this are known to have homozygous null recessive allele (hh). These carrier of this type are known as having Bombay blood group (oh group) named after the city in which it was first discovered by Bhende and co-workers in 1952. Bombay blood group has no antigens but has anti-A, anti-B and anti-H antibodies (IgM an IgG type). Transfusion of ABO blood group in these individuals causes haemolytic transfusion reaction and they have to be transfused with Bombay blood or its components only. Another type of blood group is known as para Bombay blood group. The characteristic features of this blood group are weak expression of A,B, H antigens on red cells, weak reaction to antisera to A,B,H, presence of H antigen in serum and secretions. Presence of it detected at 4°C, by adsorption and elution or by anti-H lectin. Frequency of both Bomaby and ParaBombay blood groups in India is 1 in 10,000 people (Chen et al., 2004; Jonnavithula et al., 2013 and Chacko et al., 2011). International society of blood transfusion based in Amsterdam, Netherlands, revise terminology on blood groups and propose guidelines for safe blood transfusion.

 

ABO blood group system has six genotypes: A (AA,AO),B (BB,BO), AB and OO (Zhang et al., 2015). Bombay blood group genotype is hh (Das et al., 2011) whereas paraBombay blood group is (H), Se/Se or Se/se, or se/se(Yashovardhan et al., 2012; Chacko et al.,2011; Jonnavithula et al., 2013). Variations in ABO antigens were observed and were termed as subtypes.Antigen Subtypes in various blood group antigens as follows: A(A1,A2, A3,Ax,Afinn,Aend, Abantu, Am, Aw, Ay), B(B1,B2,B4, B3,BX,Bv,Bel,Bw), and O(O1, OIV ,O2, O3,O4, O5,O6 ., AB(A1B,A2B) and variants of ABO blood group system include cis-AB and B(A)(Blood group antigen gene mutation database). A1,A2 react with anti-A but the reaction is stronger for A1. A1 react with anti-A1 found in serum of A1B and A2B carriers. A1 transferase has greater activity than A2 transferase and has different pH optimum.A2 has single nucleotide deletion in codon before stop codon and this leads to the loss of 21 amino acid residues at C terminus which is responsible for low activity of A2 transferase (Daniels, 2009). In one Indian study, high frequency of A1, O1 and O1V was observed (Ray et al., 2014). Variation in antigen polymorphism is due to point mutations, deletion and recombination of genes(Kaneko et al., 1997).Anti1 lectin purified from seeds of Dolicos biflorus agglutinate RBC carrying A1 or A1B antigens(Hussain et al., 2013).

 

3.1. Genetics of ABO blood group: A and B in A/O and B/O are dominant, A and B in AB are codominant, O in A/O and B/O are autosomal recessive (Suresh et al., 2015). ABO gene (alpha 1-3-N-acetylgalactosaminyltransferase and alpha 1-3-galactosyltransferase) is located on q arm of chromosome 9 at position 34.2. It has seven exons and 6 introns. Exons span over 18kb of genomic DNA. The size of exons ranges from 28 to 688 bp, whereas, size of introns ranges from 554 to 12982. Of the exons such as exon 6 and 7 cover most of the coding sequence(1062bp).

 

The ABO has three alleles A,B and O. A and B allele encode alpha 1–3–N–Acetylgalactosaminyltransferase (A-transferase) and alpha 1–3–Galactosyltransferase (designated B-transferase). A transferase catalyze the transfer of N-acetylgalactose amine from the donor substrate uridine diphosphate N-acetyl-D-galactosamine to the fucosylated galactosyl resiue of H antigen and form A antigen. B transferase catalyze the transfer of UDP-galactose to the fucosylated galactose of H antigen and form B antigen (Daniels, 2009; Yamamoto et al., 2014). O allele do not encode transferase for the formation of antigen due to deletion of guanine at position 258 in the coding region near N-terminus of the protein, resulting frame shift generating a translational stop signal at codon 117 and this allele encodes a truncated protein lacking catalytic activity (Blumenfield,2003; Daniels, 2009) and this occurs on exon 6. A and B tranferases differ by four amino acid substitutions (Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala) (Yamamoto et al., 2014). Two alterations(Leu266Met, and Gly268Ala) determine the A or B specificity of enzyme. An erythroid specific regulatoray element(+5.8-kb site) is found in the first intron at positions +5653 to +6154 and DNA changes in the regulatory element specify number of haplotypes each linked to specific ABO allele. The activity of regulatory element depends on the binding of GATA-1 or 2 and RUNX1 transcription factors. Between nucleotides -149 and -2 relative to the translations start site, two ABO promoters are located(Blood group antigen gene mutation database). Fucosyl transferase 1(H gene) and 2(Secretor) genes(two variant transcripts of the geneFUT1,FUT2) are located on q arm of chromosome 19 at position 13.33, encodes proteins involved in the formation of H antigen and soluble H antigen which are required for the formation of A,B antigens and it soluble forms(Daniels, 2009; Genetics home reference). These two forms vary in substrate specificity (Yamamoto et al., 2014).Non-sectors have nonsense mutation in FUT2 converting the codon for Trp143 to a translation stop codon. Mutations in FUT1 is responsible for Bombay blood group. Secretors of Bombay phenotypes have traces of H,A or B on their RBC(Daniels,2009). A transferase sequence is referred as reference sequence and compared with B transferases or blood group subtypes (Ray et al., 2014). Coding sequence of O(O1) is identical to A sequence(Daniels,2009).OIV undergo single nucleotide deletion and differ by O1 by nine nucleotide changes in the coding sequence.O2 allele encode Gly268Arg substitution. Though it has Leu266 in the gene product like A transferase but only the Arg268 residue blocks the access to N-acetylgalactoseamine (Daniels, 2009)but may have minimal A transferase activity. Missense mutation of 491T>A, and nonsense mutations of 695 G>T, 826C>T and 948C>G are responsible for the H blood group deficiencies in Bombay and para-Bombay individuals (Kaneko et al., 1997;Chen et al., 2004).

  1. Rh Blood group: Rh is a largest and complex blood group system in humans with 51 antigens and 493 alleles (Daniels, 2009; Flegel, 2011; Blood group antigen gene mutation database). This blood group system was discovered by Landsteiner and Weiner in 1940. In blood transfusion, after ABO, Rh blood group is of importance. In fact, Rh blood group came into spotlight because of its association with haemolytic disease of newborn when a woman had transfusion reaction when she was administered with the blood of her husband after she had a delivered a stillbirth child. Landsteiner and Weiner during their haematological experiments had found that serum collected from rabbit and guinea pig immunized with rhesus monkey (Macaca mulatta) caused clumping of RBC of 85% of humans (Avent and Reid, 2001). The original terms given by the discoverers such Rh factor (antigen) and anti-Rh (serum) were retained and used now also.

4.1. Genetics of Rh blood group

 

Antigens of Rh blood group are proteins and are encoded by genes RHD and RHCE located closely on the p arm of the chromosome 1 at position 36.1. The 3’ ends of both genes are oriented in opposite direction and are separated by 30kb region harboured by SMP1 gene. Homologous sequence of 10 kb are located known as Rhesus boxes are located at the 5’ and 3’ ends of RHD and in these regions, only in Rh negative persons deletion of RHD gene occur by unequal homologous recombination(Blood group antigen gene mutation database(BGAGMD). Both RHD and RHCE genes have 10 exons, spread over 75kb DNA sequence, similar in 97% of sequence and considered to be evolved by gene duplications. RHD encode D antigen, whereas RHCE encode CE antigens in different combinations (ce, Ce, cE or CE) and also Cw (RH8),Cx(RH9) and VS(RH20) .RhD and RhCE proteins vary in 32-35 amino acids sequence. Each protein has 12 transmembrane protein segments with internal termini and six loops extending outside RBC membrane (Connie, 2007; Daniels, 2009; Flegel, 2011). In a hospital study on 3073 blood donors conducted in India has shown the incidence of D, C, c and e in the following percentages: 93.6%, 87%, 58% and 20% (Makroo et al., 2013). D antigen is considered as more immunogenic and its absence is considered as Rh negative. In multricentric study carried out in India has shown 94.61% of subjects were Rh positive and rest were Rh negative (Agrawal et al., 2014).

 

Rh associated glycoprotein(RHAG) present in RBC are essential for targeting of RhCE and RhD proteins to the membrane and mutations in RHAG cause loss of Rh antigens. RHAG analogues such as RhBG and RhCG are found in liver, kidney, brain and skin (Westhoff, 2007).RHAG gene is located on p arm of chromosome 6 at position 12.3, has 10 exons and share 30% of sequence of RHD/RHCE genes. Antigen of RHAG is called Rh50glycoprotein, whereas, antigen of RHD/RHCE are collectively termed Rh30. Rh30 polypeptide is palmitolyated (attachment of palmitic acid to cystein residues on membrane) and Rh50 undergo N-glycosylation at site (Asn 37)(BGAGMD). Components of Rh and RhAG protein complex in RBC includes band 3, glycophorin A and B, ICAM-4, CD47 and integrin associated antigen. Rh proteins act as structural components of glucose transporter1, dimers of band 3, glycophorin C, the Duffy, Kell glycoproteins and XK components of cytoskeleton. Rh Proposed roles of RhD and RhCE proteins includes membrane integrity, transport of CO2 , whereas RhAG in ammonia transport, gas exchange across the plasma membrane and in mutated form in hereditary stomatocytosis (Flegel, 2011).

 

For description of Rh blood group, three classifications described by Fisher and Race (1962), Wiener and Wexler(1963) and International society of blood transfusion are in usage, which are based on the assumptions of the aforementioned researchers on the inheritance of antigens. Fisher and Race  thought that Rh system is composed of closely linked genes or alleles, D, C or c and E or e at first, second and third locus and proposed DCE system. Winer proposed that antigens of Rh were the products of single gene and labelled major five antigens as Rh0, rh’,rh”,hr’ and hr”and this system became obsolete, later revised terminology was putforwarded such as R1, R2,R0,Rz for Rh positive and r’,r”and ry for Rh negative. R1, R2,R0,z stand for Ce, cE,0 and CE, whereas r’,r”and y stand for Ce,cE an CE. According ISBT terminology, D=Rh1, C=Rh2, E=Rh3, c=Rh4 and e=Rh5. Presence of antigen (D) as Rh: 1 and absence of antigen as (D-)

Rh:-1. RH genes (RHD,RHCE, RHAG and its analogues RHBG and RHCG) are indicated in capital letters with and without italics; antigens as D, C,c, E and e; alleles as(*) RHCE*ce, RHce*Ce and proteins as RhD, RhCE, RhAG, RhBG and RhcG (Westhoff, 2007).

 

In RHD gene more than 200 alleles were reported which are due to single nucleotide polymorphism or hybridization of RHD/RCHE. Phylogenetically RHD alleles are categorized into four clusters namely Eurasian D cluster and African clusters: DIVa, DAU and weak D type 4 based on the allele that differ from consensus RHD allele(Flegel,2011). Immunotyping studies have shown that antigen D contain at least 30 epitopes(Daniels, 2009). Serologically, D antigen are classified into six categories from DII-DVII.Based on the phenotypic correlation with molecular variations, RHD alleles have been categorized into partial D, weak D type, DEL and non-functional alleles(Flegel, 2011). In partial D type some D epitopes are missing, carriers of partial D antigen produce anti-D upon exposure to normal D antigen R. Some amino acid substitutions on extracellular loop affect linear D epitopes or 3D confirmation of loop. Partial D type are identified using monoclonal antibodies directed to domains or loops of the RBC. RBC type as D positive. Of the six categories, DII-VII, DII and DVII are the result of extracellular amino acid substitutions, whereas, DIII-DVI are the products of RHD-CE-D hybrid alleles.Partial D are included in D categories because they share exofacial amino acid substitutions in different spatial arrangements. Carriers of partial have very low or no risk of anti-D immunisation(Daniels,2009;Westhoff, 2007; Flegel, 2011). In India, prevalence of weak and partial were found to be 0.2% and 0.03%(Jain et al., 2014)

 

In carriers of weak D, epitopes are expressed weakly and RBC reacts with anti-D after an extended testing period in indirect antiglobulin test. Weak expression of epitopes is due to amino acid substitutions in transmembrane or intracellular segments and cause problems in integration of protein to the RBC membrane. Antigen expression is reduced in carriers of weak D. As of now, 80 weak D types including subtypes are found. Except in some weak D types (1-3, 4/4.115,4.2 (DAR),7) the risk of anti-D immunization is weak. Common weak D types don’t produce allo-antiD(Daniels,2009; Westhoff, 2007;Flegel, 2011).

 

In Del type D, epitopes are expressed very weakly and found only when anti-D is adsorbed and eluted from RBC. This is due to RHD (K406K) allele containing C1225 nucleotide substitution in exon 9 which caluse missplicing of RNA that result in minor form of transcript for translation. In 30% of East Asian Rh negative residents carry this mutations and hence referred as “Asian type DEL”. RBC with DEL type D produces 200 or less copies of D antigen. Other genetic changes observed in DEL type D is amino acid change M295I. When DEL positive donors wrongly labelled as D negative it can cause anti-D alloimmunization. Genotype an phenotype discrepancies are observed in carriers of DEL alleles. Due importance is given when fetal blood group gentotyping depends on the ethnicity of parents (Westhoff, 2007;Flegel, 2011).

 

D negative haplotype is due to deletion of RHD gene. Some carriers of D negative haplotype contain nonfunctional RHD allele such RHD pseudo gene and RHD-CD-E allele Cdes(Flegel, 2011). Majority of Rh negatives were observed in Caucasians (15%-17%) followed by Black Africans (5%) and Asians (3%)(Westhoff, 2007). In Africa, three haplotypes are responsible for D negative phenotypes. They are RHD deletion with normal RCHE, 37bp duplication of last 19 nucleotides of intron 3 and 18 nucloetides of exon 4 of RHD gene and hybrid gene RHD–CE–Ds(Daniels, 2009).

Rhnull phenotype is due to inheritance of non-functional RHCE and RHD alleles. D an CE antigens are not expressed. Alterations in RHAG can also cause the loss of Rhd and RhCE proteins(Flegel,2011). Frequency of this phenotype is 1 in million. Cardinal features of carriers of Rhnull phenotypes are stomatocytosis, spherocytosis, increased osmotic fragility, altered phospholipid asymmetry, altered cell volume, defective cation flexes, elevated Na+/K+ ATPase activity, shortened survival of RBC in vivo and mild compensated hemolytic anemia. On the basis of pattern of inheritance, Rhnull is of two types i.e. amorph and regulator. Amorph is due to molecular changes in RCHE in combination with RHD and have no RHD and inactive RCHE, whereas regulator type is caused by molecular defects in RHAG(Avent and Reid,2000) and have inactive RHAG gene. Rhnull when become pregnant and transfused form anti-Rh29. Serum of the carriers of Rhnull agglutinate RBC of all except from Rhnull (Westhoff).

 

C and c antigens are distinguished by four amino acids and the change from serine to proline occurs at position 103. Change from proline to alanine at position 226 differentiate E and e antigens (Westhoff, 2007). C and c and E and e antigens are antithetical antigens (if one antigen present other antigen is absent). E is less frequent than e antigen in populations. RHCE encode Cw and Cx both differ by single amino acid change and RBC typed as C+. Haplotype (C)ces encode altered C and e antigen and RBC type as C+, e+. Carriers of this phenotype produce anti-C, anti-e or anti-Ce and these multiple antibody specificities are called anti-hrB. Homo or heterozygous (C)ces with altered Rh haplotype on other chromosome is termed as hrB-. Altered RHce genes are observed in Blacks and mixed group populations. Altered alleles type as e positive and amino acid changes involve tryptophan in exon 1 and 5. Homozygous persons for these alleles show no high incidence of Rh antigens such as hrB and hrS.RBC with ceS alleles have hrB, whereas RBC with ceMO, ceEK alleles have hrS- . Variant RHce are associated with RHD-CE-D(D-negative hybrid encoding altered C antigen or linked to variant RHD such as DIII, DAU and DAR. Carriers of these alleles produce alloanti –C and anti- D, though their RBC are positive for these Rh antigens (Westhoff, 2007). Expression of e antigen is affected by amino acid change such as presence of valine than leucine at position 245, cystein than tryptophan at position 16 and deletion of arginine at position 229. Amino acid substitution of valine than leucine at position 245 causes the simultaneous presence of both VS and V antigens. Replacement of cystein in place of glycine at position 336 prevent the expression of V antigen in presence of VS. Replacement of RCHE encoding region of E/e antigen by RHD equivalent and loss of E/e was observed in in DCw- and DC- phenotypes (Avent and Reid, 2000). When RCHE may not or may encode E,e, C or c antigens they are called deleted or partially deleted phenotypes mainly observed in Caucasian children born out of consanguineous marriages. G antigen is encoded by exon 2 of RHD and RCHE genes, RBC type as D+ and C+ and also positive for G antigen. Anti-G appear as anti-D and anti-C and these can distinguished by adsorption and elution experiments. Compound antigens such as Ce(rhi), cE(Rh27), ce(f) and CE(Rh22) are encoded by haplotypes such as DCe(R1) and Ce(r’), DcE (R2) and cE(r’’), Dce(R0) and ce(r),DCE(Rz) and CE(ry)(Westhoff online source).

 

Apart from its Rh blood group role in haemolytic disease of newborn, in patients of myeloid leukemia, myeloid metaplasia, polycythemia or myelofibrosis have two types of Rh types were observed. Loss of Rh antigens was observed in patients with myeloid leukemia, change from D positive to negative was also found (Cherif-Zahar et al., 1998; Avent and Reid, 2000).

 

  1. MNS blood group: After ABO, MNS system is oldest blood group system discovered. After Rh blood group system, it is a complex system containing 46 antigens. MNS was first blood group for which molecular basis both at the protein and gene level was attempted. It is the blood group system in which most of the variants are immunogenic (BGMUT, Reid, 2009; Lomas-Francis, 2011). MNS blood group system is one of the nine blood group systems which are clinically significant because of their role in haemolytic transfusion reactions and haemolytic disease of newborn (Lamba et al., 2013). In 1927, Landsteiner and Levine discovered MN antigen by immunizing rabbit with human RBC and later discovered anti-M and anti-N.MNS antigens are developed at birth. MN blood group was named after second and fifth letter of the word ‘immune’. S antigen was discovered in 1947 in the serum of patient from Sydney city and antithetical antigen ‘s’ discovered in 1950. Another antigen ‘U’ was discovered 1953 and named U due to its universal distribution. Other antigens were named after the person in whom antigen was discovered (Reid, 2009).Low prevalent antigen which were under miltenberger series or system were found to be expressed by hybrid alleles of glycophorin involved in MNS system and merged with MNS system. A convention was started to name glycophorin as GP and genes as GYP (Lomass-Francis, 2011). In one Indian study, prevalence of M, N, S and S antigens were found to be 88.7%, 65.4%, 54.8% and 88.7% (Makroo et al., 2013). Another study from Gujarat on blood donors observed the prevalence of various MNS antigens in the following proportions: M+N+(39.13%),M+N-(37.39%), M-N+(23.48%). S+s+( 24.35%) and donors, S+s-( 8.69%), and S-s+( 66.96%)(Kahar and Patel, 2014).

MN antigens are found on glycophorin A, whereas Ss antigens are born by glycophorin B. One million copies of glycophorin A are available on RBC. Glycophorin A is a type 1 glycoprotein composed of 131 amino acids (aa). It is associated with anion exchange protein1 of the AE1. It has amino terminal extracellular domain (72 aa), single transmembrane domain and cytoplasmic domain (36 aa) and densely glycosylated with one N glycon and 15 O glycans. In RBC, the availability of glycophorin B copies are in the range of 170,000-250,000. Glycophorin B is composed of 72 aa. It has extracellular domain (44 aa) and 11 O-glycon sites. Glycophorin B is a component of AE1/band 3-ankyrin macrocomplex. Modified O glycan structures are found in both glycophorins such enzyme disialyted, congenital glycosylation deficient and substitutions by N-acetylglucosamine and N-acetylgalactoseamine. O glycan residues constitute 60% of sialic acid on RBC and responsible for negative charge of RBC. Both M and N antigens differ at two amino acids positions of 1 and 5. Ss antigen than MN antigens on intact RBC are resistant to trypsin enzyme cleavage (Cooling, 2015). GPA and GPB provide a glycan coat to the RBC, act as receptor for complement, cytokines, bacteria, viruses and may regulate the integrity of the RBC membrane(BGMUT).

 

5.1. Genetics of MNS blood group: Glychophorin A(GYPA), B(GYPB) and E(GYPE) genes occur as a cluster(A-B-E) spanning 330kb on long arm(q) of chromosome 4. Glycophorin A is encoded by exon by exonA2-A7, whereas, mature glycophorin B is encoded by exon B2 and B4-6. GYPE gene participate in gene arrangements forming variant alleles. GYPE has six exons (3 and 4 pseudo exons).Because of homologous nature and closely related gene recombinations and gene conversions can form new antigens. Difference between S and s is due to single amino acid substitutions (Met29Thr). Cells lacking glycophorin A and glycophorin B have reduced sialic acid levels and En(a-) and S-s-U- phenotypes. The former phenotype is due to recombination between exons A2 and B2 and deletion of exons A3 to A7. The later phenotype result from recombination and deletion of exons B2 and B4/B5. Recombination between GYPA and GYPE and resultant loss of expression of GYPA and GYPB form Mk phenotype(M-N- S-s-U-)(Cooling, 2015). Vr, Mta, Ria, Nya, Or, ERIK, Osa,ENEP ⁄ HAG, ENAV ⁄ MARS, ENEV and MNTD variants in GYPA and S ⁄ s, MV, sD and Mit variants in GYPB result due to single nucleotide substitutions. M/N in GYPA and ‘N’ in GYPB variants are attributed to two or more nucleotide substitutions. Variants such as Sta, Dantu, Hil, TSEN, MINY and SAT are caused by unequal crossing over. He, Mia, Mc, Vw⁄ Hut ⁄ ENEH, Mur, Mg, Me, Sta, Hil, Hop, Nob, DANE, MINY, MUT and ENDA variants results due to gene conversions during DNA reapir (Lomas-Francis, 2011 and Reid, 2009). Various variant phenotypes are observed due to hybrid genes. GP.Hil and GP.JL are encoded by GYP*(A-B) hybrid gene. Phenotypes GP.VW, GP.Hut, GP.Nob, GP.Joh and GP.Dane are encoded by GYP*(A-B-A) hybrid gene, whereas GP.Mur, GP.Hop, GP.Bun and GP.HF are encoded by a GYP*(A-B-A)hybrid gene (Lomas-Francis, 2011).

Glycophorin variants can be detected by treatment with enzymes(GPA sensitive to trypsin but resistant to α-chymotrypsin, whereas, GPB resistance to trypsin and sensitive to α-chymotrypsin), immunotyping studies and immunobloting using monoclonal antibodies (to know the expressed portions of GPA/GPB), testing with lectins(to detect altered glycosylation), SDS polyacrylamide gel electrophoresis( to check altered mobility), PCR based restriction fragment length polymorphism and sequence specific primer assay and cloning and sequencing of GYP exons (to detect variants at molecular(DNA) level) and inhibition studies with synthetic peptides and analysis of trypsin digested products of GPA and,GPB (to find antigenic determinants revealed by specific antibodies). Antibodies to low prevalence MNs antigens such as anti-Mia, -Vw, -Hil, -Hut and -Mur have  been implicated in  haemolytic disease of the foetus and newborn and delayed type of haemolytic transfusion reactions. For expression of high prevalence of MNS antigens(Wrb) requires 75-99 amino acids of GPA but individuals carrying phenotypes such as GP.Mur, GP.Bun, GP.HF, GP.Hop, GP.Hil and GP.JL lack these amino acids and make alloantibodies which cause haemolytic transfusion reactions and finding a suitable donor for blood transfusion may be tiresome(Lomas-Francis, 2011; Reid, 2009).

 

Phenotyping of ABO, Rh and MNS blood groups: In manual methods blood grouping is done using glass slide/porcelain tile or test tube or microplate. Each method has merits and demerits. Glass slide or porcelain tile are used in emergency blood grouping or in general population testing. This method is not ideal for detecting weak antigens and serum grouping using low titre anti-A and anti-B. Demerits of this method include low sensitivity, aggregation of cells due to drying of reaction mixture and difficulty in interpreting weak reactions. Various types of microplates are available such V, U or flat bottom shaped and U type plates are preferred for blood grouping because ease in reading results. Each plate contain 96 wells and accommodate the upto 300 volume of reaction mixture. Advantages of microplate is cost effective, small volume can be used, easy to handle, large number of samples can be tested and automation can be achieved to reduce errors. Merits of test tube (plastic or glass) are avoid drying up of contents, more sensitive than slide, facilitate detection of weak antigens and antibodies and hygienic(NIB manual for ABO and Rh blood grouping).

 

6.1. ABO phenotyping: Forward typing is done to detect the presence of antigen on RBC (A,B and AB using anti-A,B and AB) whereas reverse typing( detecting antibodies in serum using control cells of A,B,AB and O) is done to detect antibodies in serum. One millilitre of Anticoagulated (EDTA, sodium citrate and heparin) blood is collected and stored at room temperature till processing is done. If long time is required for serotyping can be stored at 2-8°C for one day. Immediate serotyping is preferred. Anticoagulated blood sample is centrifuged at 5000 rpm for 5 minutes at room temperature. Plasma (supernatant) is separated using tip of micropipette. RBC present in the suspension is washed with the double the volume of normal saline(0.9% saline) until clear supernatant appears. Here test tube method is given for blood group typing. For forward typing 3% (v/v) of red cell suspension(for 100 µl total volume=97 µl of normal saline and 3 µl of RBC suspension) prepared using normal saline. One positive control can be used mixing 100 µl of serum and 50 µl of RBC suspension of same sample(NIB manual). Antisera are used when at room temperature.

 

Forward grouping

 

Reagents: Anti-A, Anti-B, Anti-A,B, Anti-H

 

Procedure: Take 5ml test tubes and label them as Anti-A, Anti-B, Anti-AB and anti-H. Add 100 µl each of Anti-A, Anti-B, Anti-AB and anti-H. To this add 100 µl of 3% RBC cell suspension and centrifuge at 1000rpm for 2 minutes. If clumping is observed in any test tube it can be interpreted as per table 1.

 

Reverse grouping:

Reagents: A, B and O type RBC

 

Procedure: Add 100 µl of serum in three test tubes labelled as A, B and O. Add 50 µl of 3% RBC suspension. Mix the contents by gently shaking and then centrifuging at 1000rpm for 2 minutes. To define the strength of reaction, scoring is given. If clumping is observed in any test tube it can be interpreted as per table 1.

 

Scoring for agglutination (Figure 1) NIB manual):

 

4+:  1 big clump

 

3+: 2 or 3 clumps

 

2+: many small clumps with clear supernatant

 

1+: many small clumps with turbid supernatant

 

W: granular suspension Zero

 

or -: smooth suspension

 

H: partial or complete hemolytis(positive reaction)

 

6.1.2. Detection of ABH secretors (Venkatramana,2012): ABH secretors are individual who secrete ABH in body fluids such as saliva, swet, tears, semen and breast milk. Carriers of A,B,AB and O blood group secretor secrete A an H, B and H, A,B and H and H antigens in body fluids. Presence of antigens can be detected by absorption-inhibition test. Of all the body fluid saliva can be obtained easily and in good volume.

 

Absorption-inhibition test:

 

Principle: ABH antigens in saliva bind to the titrated antisera at initial state of the test and prevent from agglutinating RBC added at the final step carrying the same antigen. Reagents: anti-A, anti-B and anti-H, 5%RBC suspension.

 

Preparation of titrated antisera: Owing to excess antibodies in conventional antisera cause agglutination irrespective of secretor status of the subject. Anitsera is is diluted upto 1:64 using normal saline. To each dilution, a drop of 5% of RBC suspension is added and incubated for 10-15 minutes. The titrated antisera is determined one dilution before the dilution at which least activity was observed. Procedure: The individual whose secretor status is going to be tested is asked to wash the mouth with water and instructed to keep a small cotton swab under the tongue for 2 minutes. Then cotton swab is squeezed to collect the saliva. The collected saliva in test tube is boiled on water container kept on heater for 30 minutes to inactivate salivary enzymes. After cooling, the saliva is centrifuged for 10 minutes at 3000 rpm and supernatant is collected and kept at 4°C until used. One drop of supernatant is added to one drop of titrated antisera on porcelain tile and incubated for 1 hour at room temperature. To prevent dehydration, the porcelain tile is kep on petridish. To this mixture, one drop of 5% RBC suspension is added and incubated for 30 minutes. Absence of agglutination is suggested as ABO secretor and presence of agglutination indicate ABO non-secretor.

 

6.2. RhD phenotyping

 

Reagents: 3% RBC suspension, Anti-D (IgM + IgG) Blend of D1 and D2 from two manufactures, anti-E, anti-e, anti-C, anti-c, anti human globulin.

 

Procedure: Take six 5mL test tubes and label it as D1,D2, C, c, E and e. Using a micropipette dispense 100 µl of D1,D2 and 50 µl of anti-C, c, E and e into the test tube. Add 50 µl of 3% RBC into all these test tubes. Mix the contents gently and centrifuge at 1000rpm. In negative results observe under microscope, indirect agglutination test can be performed to detect weak D antigen(NIB manual).

 

Indirect agglutination test:

 

Reagents: antihuman globulin

 

Procedure: The test tube which showed negative result is incubated in incubator or water bath at 37°C for 30 minutes. Centrifuge the contents at 1000 rpm(revolutions per minute) for 1 minute. If agglutination is observed considered it as D positive. If negative result is observed, the RBC cell suspension is washed three types with normal saline by centrifuging the contents at 1000 rpm and remove supernatant. Add 200 µl of antihuman globulin and centrifuge the contents at 1000rpm for 1 min. If agglutination is found the sample is typed as Rh positive otherwise terme as Rh negative(NIB manual).Direct agglutination test known as comb’s test is used to determine wheter haemolytic anemia is caused by antibodies attached to the RBC. Interpretation of Rh blood group is shown in table 2 following NIB manual which is reproduced with minor modification.

 

 

6.3. MNS phenotyping (Kruzel and Immucor Gamma, USA):

 

Reagents: 3% RBC suspension, anti-M, anti-N, anti-S, anti-s

 

Procedure: Take four 5ml test tubes and label it as M, N, S and s. Dispense 100 µl of 3% RBC suspension using micropipette into the tubes. To this add 100 µl of anti-M, anti-N, anti-S, anti-s. Mix the contents by gently shaking and incubate at room temperature for 15 minutes. Centrifuge the contents at room temperature at 1000 rpm for 1 minute. Examine for agglutination. If agglutination is present the tube may be termed as positive for particular antigen otherwise considered as negative.

  1. Calculation of ABO, Rh and MN allele frequency:

 

7.1. ABO allele frequency and genotype calculations

Hypothetically let us assume that prevalence of A, B and O blood group is 20% (0.2), 30% (0.3) and 40%(0.4) in particular population. To calculate allele frequency in this case the Hardy-Weinberg equation is (p + q + r)2 =1 or p2+ q2+r2+2pq+ 2pr+2qr=1

Here A,B and O alleles are p,q and r respectively.

IA= 0.2 ,IB=0.3 and Io=0.4

P2 is allele frequency of IA,IA(A blood group)=(0.2)2 =0.04 q2is allele frequency of IB,IB(B blood group)=(0.3)2=0.09 r2 is allele frequency of IO,IO(O blood group)=(0.4)2=0.16

2pq is allele frequency of IA,IB(AB blood group)=2*(0.2)*(0.3)=0.12 2pr is allele frequency of IA,IO(A blood group)=2*(0.2)*(0.4)=0.16 2qr is allele frequency of IB,IO(B blood group)=2*(0.3)*(0.4)=0.24 A allele frequency=0.04 + 0.16=0.2 B allele frequency=0.09 + 0.24=0.33

O allele frequency=0.16

AB allele frequency=0.12

7.2. Rh alleles (D and d) frequency calculation

Assume that in a population of 50, there were 35(0.35) Rh positive (D) and 15(0.15) Rh negative (d).

Allele frequency is calculated using Hardy-Weinberg equation (p + q)=1

Dominant allele (D) =p, recessive allele(d)= q

q=√q2=0.15=0.38, p=1-q=1-0.38=0.62

7.3. MN allele frequency calculation

Assume that in a population of 100, there were 60(0.6) M and 40(0.4) were typed N. Allele frequency is calculate using Hardy-Weinberg equation (p + q)=1 Dominant allele(D) =p, recessive allele(d)= q

q=√q2=0.4=0.632, p=1-q=1-0.632=0.368

 

Summary

 

In 20th century, physical anthropologists entered into analytical phase with a focus on reorientation in methodology and comprehension.

Currently, physical anthropologists are engaged in both descriptive and analytical studies besides embracing modern technologies for studying human population variations.

Studying of human population variations help us in understanding our origins, medically relevant variations and also serve as antidote against racial prejudices.

Physical anthropologists use various traits like skin colour, hair colour, colour blindness, taste sensitivity to phenylthiocarbamide and blood groups such as ABO, MN, Rh etc. to study variations among populations.

Blood groups are inherited characters of red cell surface that are detected by specific alloantibodies.

As of now, there are 35 blood groups in human beings. It was observed that 44 genes and 1568 alleles encode for antigens in the 35 blood groups.

Blood is transfused as components such as packed RBC, plasma, platelets and immunoglobins. Blood components are transfused to prevent haemorrhages and to improve oxygen delivery to tissues.

Incompatibility of recipient serum antibodies and donor blood group antigen can cause hemolytic transfusion reactions and haemolytic disease of newborn.

Association of ABO blood groups and diseases were observed: Blood group A1 and pancreatic cancer; blood group A and hepatitis B virus infection, HIV infection and pancreatic cancer; non-O blood group and deep vein thrombosis, arterial thrombosis, myocardial infarction, coronary artery disease, renal cancer in women, non-melanoma skin cancer, lung cancer, higher levels of Willebrand factor and coagulation factor VIII; blood group A and helicobacter pylori infection; blood group A and AB and nasopharyngeal cancer; blood group O and malaria, helicobacter pylori, cholera, peptic ulcer, hepatitis C and norovirus infection; blood group B and malaria and type 2 diabetes; AB blood group and HIV-2 infection and influenza A and B.

 

Blood group screening is also used for studying genetic relationships between populations by physical anthropologists, to resolve paternity disputes and to exclude crime suspects by police and forensic personnel.