BLOOD GROUP TYPING: ABO AND RH (D) BLOOD GROUPS Contents 0 Introduction 1 ABO Blood Group System 2 RhBlood Group System 3 Phenotyping of ABO and Rh Blood Groups Materials Required Preparation of 3% RBC Phenotyping of ABO and Rh Blood Groups Detection of Weak D Phenotype 13.4.1 Materials Required 1342 Procedure 14 References Learning Objectives fier reading this unit, you will be able to > — Elucidate about the importance of ABO and Rh blood groups in blood transfusions, and > Acquire knowledge of phenotyping of ABO and Rh blood group system involving forward and reverse phenotyping 1.0 INTRODUCTION Determination of blood group is called blood group typing. Blood groups are red blood cell (RBC) antigen containing blood systems. Blood groups are determined using RBCS. As per the International society of Blood ‘Transfusion (www isbtweb.org) report dated 30" June, 2021, 43 blood groups have been identified in humans. Information of blood groups isused for various applications such as, as a tool for genetic relationship between populations and migration, and to investigate the population characteristics and diversity in populations. Investigations of human variation aid in tracing origin, medically significant variants and to eliminate racial prejudices. Blood group typing is based on the principle of agglutination (clumping) of RBC containing antigen (for example AorB or ABor Rh(D)) when react with antibody (for example: anti-A or anti-B or anti-AB, anti Rh (D)) present in the plasma, broadly indicating interaction of antigen and antibody. Clumping of RBC indicates the presence of respective blood group. Each individual carries only one type of antigen and antibody for a particular blood group Antigen is any substance that induces an immune response. In case of blood groups, antigens are polysaccharide or proteins in nature which are encoded by Contributed by Dr. SAA Latheef. Department of Genetics and Biotechnology, Osmania University Hyderabad conser algenes located mostly on autosomes or sex chromosomes (X or Y). Antibody is a protein produced by plasma cell, a differentiated B lymphocyte in response to immune response induced by antigen. Blood group antibodies (natural occurring substances) found on immunoglobulin M molecules in plasma are formed in the first year of human life when exposed to food, bacteria and virus. In laboratories, for teaching purpose, blood groups are determined using voluntary donor RBCs and commercial antibodies. In blood banks, before blood transfusion, blood group typing is done for both the donors and recipients/patients and also using RBC of donor with serum of recipient, a procedure known as cross matching. Depending on the need of type of blood component (RBC, plasma and platelets) transfusions of particular blood component are carried out. With respect to blood transfusions in humans, 9 blood groups (ABO, Rh , MNS, Kidd, Kell, P, Lewis, Duffy and Lutheran) are important for the purpose of blood component transfusions. For the present unit, determination of ABO and Rh blood groups are described 1.1. ABO BLOOD GROUP SYSTEM In 1901, Karl Landsteiner discovered A, B, O of ABO blood group system while the AB blood group was discovered by de Castello and Steini in 1902. For the discovery of A, B and O blood groups of ABO system, Karl Landsteiner was awarded Nobel Prize in Physiology or Medicine in the year 1930. The gene for ABO system is located on the chromosome 9. This system has four blood groups (A, B, AB and ©) which are determined based on the presence and absence of antigens and antibodies. Antigens and antibodies are present in the blood (the former present on the red cells and the latter in blood plasma). The antigens are designated as A and B and the antibodies as anti-A and anti-B. Group A person carries antigen A and antibody anti-B. Group B person carries. antigen B and antibody anti-A. Group O individuals possess both the antibodies and lack any antigen while group AB individuals carry both the antigens but lack any antibody. Individuals with O blood group can donate any blood component (RBC, plasma, and platelets) to individuals carrying all blood groups. Carriers of A blood group can donate any blood component to either A or AB; carriers of B blood group can donate blood components to the carriers of B and AB and individuals of AB blood group can donate blood components to the carriers of AB blood group. In other words, we can understand this as carriers of A blood group can receive any blood component from carriers of A as well as O blood groups. Carriers of B blood group can receive the blood components from B as well as O blood groups. Carriers of © blood group can receive blood components only from O blood group, while carriers of AB blood group can receive blood components from all blood groups, A, B, AB and O. On the basis of the above explanations, we can conclude that while blood group O (-ve) is a universal donor, blood group AB (+ve) is a universal recipient. conser alThree genes namely A, B, and O controls the system. A and B allele are co- dominant and both A and B are dominant to O which is recessive. Inheritance of ABO follows Mendelian pattern. In World population the frequency of blood group O (63%) is dominant followed by A(21%)and B (16%). Indian populations are characterized by predominance of ‘B’ blood group (Venkatramana, 2012). Among Europeans, the Blood group ‘A’ is dominant, ‘B’ in Asia and ‘O” in South America, Siberia and some areas of Switzerland (Farhud and Yeganeh,2013). In India, first investigations on blood groups were done on soldiers by Hirschfield in 1919. ABO blood group is a good example of multiple alleles which was discovered by Felix Bernstein in 1924. ABO blood group was the first studied polymorphism in humans and it was the data of this marker that was used for the construction of evolutionary tree. Blood groups were also studied for susceptibility to diseases among its carriers. For example, it was observed that carriers of ‘O” blood group were found to have higher risk of developing gastric and duodenal ulcer, cholera and other types of diarrhoea than carriers of other blood groups. Among carriers of A and B blood groups, more B than A blood groups carriers were found to be susceptible to cholera. Carriers of A and AB were shown to be at risk of getting infected with small pox than people carrying other blood groups. Carriers of A blood group were shown to be susceptible to gastric cancer (Sarkar, 2012; Dean, 2015). In forensic labs using blood groups, paternity disputes are resolved and suspected criminal identity is confirmed. 1.2. RH BLOOD GROUP SYSTEM Rh blood group was discovered by Karl Landsteiner and Weiner in 1940.This blood group got its name because of discovery of Rh factor. Discoverers of this blood group injected RBC of Rhesus monkey into the Rabbit and after a series of injections, plasma of immunized Rabbits caused clumping of RBC of not only Rhesus monkey and but also of humans. Since then, Rh factor name was retained for Rh (D) antigen. This blood group also came into limelight due to its association with Erythroblastosis fetalis, a haemolytic disease of the new born. This occurs due to the production of IgG antibodies, in plasma of Rh- (negative) mother against D antigen of foetus, which enter through placenta during labour into the foetus and causes haemolysis, leads to anaemia and death of the foetus, mostly in second pregnancy. Rh blood group is a complex blood group in humans and contains 56 antigens (www.isbtweb.org). Among them the notable are D, C, E, c, and e (Dean, 2015). These antigens are encoded by RHD and RCHE genes harboured on chromosome 1. RHD gene encodes RHD antigen whereas RHCE gene encodes CE antigens in varying combinations such as Cece, CE or cECx (RH9), Cw (RH8) and VS (RH20). Carriers of Rh (D) antigen are called Rh+ (positive) and those lacking it are labelled as Rh-(negative). Rh+ is predominant in Asians and Rh- in Caucasians. C and E antigens are predominant in Asians; c and e antigens in Blacks and Caucasians (Dean, 2015). Rh antigens are only expressed on RBC only. conser al1.39 PHENOTYPING OF ABO AND RH BLOOD GROUPS Phenotyping or determination of blood groups is carried out on porcelain tile/ glass slide/ microcentrifuge tube /microplate. Phenotyping of blood groups is done using commercially available antisera. 1.3.1 Materials Required 1) 3% Red Blood Cells (weight/volume) (W/V) 2) 0.9% (W/V) saline 3) Labtop centrifuge 4) Polystyrene/glass tubes (SmL) 5) Micro/mini centrifuge 6) Microcentrifuge tubes (ImL) 7 8) Micropipettes (100ul and ImL) 9) Micropipette tips (100p1 and ImL) 10) Commercial antibodies (antisera-A, B, H and D) 11) Microcentrifuge tube stand 12) Double distilled water 13) Glass beaker 14) Glass bottle Microcentrifuge tubes (2mL) 1.3.2 Preparation of 3% RBC 1) A SmL venous blood is drawn from human volunteer and the drawn blood is slowly released through the walls of SmI. falcon tube or glass tube containing anticoagulant (sodium citrate (160mg) or Ethylene diamine tetra acetic acid (10mg) or heparin (751U/mL) and mixed well by rolling the tube gently on palm or bench for 5 to 6 minutes. a Centrifuge the falcon/glass tube at 1000 revolutions per minute (rpm) in labtop centrifuge. 3) The supernatant formed in falcon/glass tube is collected into the 2mL microcentrifuge tubes. 4) 100 mL of 0.9% saline (W/V) is prepared by adding 900mg of sodium Chloride (NaCl) into a glass beaker containing 100mL of double distilled water and store in glass bottle at room temperature. conser alTable 1.1: Interpretation of ABO System Results ‘ABO Blood group ‘Anti-A Anti-B ‘Anti-H Result ‘Agglutination present | Agglutination | Agglutination _ | A blood group absent, absent, ‘Agglutination absent |Agglutination | Agglutination |B blood group resent absent, ‘Agglutination present | Agglutination | Agglutination _ | AB blood group present absent. ‘Agglutination absent | Agglutination | Agglutination _ | O blood group absent present Rh blood group ‘Anti-D ‘Agglutination present | Rh positive Ai 1.3.4 Detection of Weak D Phenotype Ifa particular individual is identified as Rh-(negative) there is a possibility of presence of weak D (Du)+ (positive) antigen in that individual. To confirm if weak D (Du)+ antigen is present, indirect agglutination test is performed 1.3.4.1 Materials Required 1) Anti human globulin 2) 0.9% saline 3) Minicentrifuge/Microcentrifuge 4) Micropipette (ImL) 5) Micropipette tips (ImL) 6) Water bath 7) Microcentrifuge tube stand 8) Microscope 1.3.4.2 Procedure 1) Incubate the microcentrifuge tube (ImL) determined as Rh-(negative) as per procedure described in 1.3.3 at 37°C in water bath for 30minutes. 2) Centrifuge the microcentrifuge tube for 1 minute at 1000 rpm in minicentrifuge. 3) If agglutination of RBC is seen, consider it as Rh+ (positive). If no agglutination, proceeds to further steps. 4) Add 8001 of 0.9% saline into Rh- containing microcentrifuge tubes using ImL micropipette and centrifuge the microcentrifuge tube at 1000rpm in conser alminicentrifuge and remove the supernatant (liquid). Repeat this step three times. 5) Add 200 pl of anti human globulin to the microcentrifuge tube with ImL pipette. 6) Gently shake the microcentrifuge tube with tapping the finger and examine for the presence of agglutination of RBC under the light or microscope. 7) If agglutination of RBC is seen in microcentrifuge tube, the presence of weak D (Du)+ is confirmed and in the absence of it, it is determined as Rh- (negative), Fig. 1.9: Water bath (Source: hutps://en wikipedia org/wiki/Laboratory_water_bath#/media/File Shaking_water_bath_2015 JPG) wy road 7 (Source: https://www quirumed comv/ie/professional-binocular-microscope-achromatic html) cance si bt14 REFERENCES Dean, L. (2005). Blood groups and red cell antigens. National Center for Biotechnology Information (US). Chapter 5, The ABO blood group. Available from: https://www.ncbi.nlm.nih.gov/books/NBK2267/ Farhud, D.D. Zarif Yeganeh, M. (2013), A brief history of human blood groups. Iran J Public Health. 42(1):1-6. Guidance manual on ABO and Rh blood grouping (2013). National Institute of Biologicals, Ministry of Health and Family Welfare, Government of India. Available at _hitp://nib.gov.in/guidance_document/Guidance_manucal_QC_ ABO_Rh_blood_grouping_26_03_2013.pdf. International society of Blood Transfusion. Available at(www.isbtweb.org) Latheef, S.A.A. (2021). Determination of Al, A2, B, O, M, N and Rh Blood Groups. Unit 2 (Practical Manual), In Biological Diversity of Human Populations (BANC 107), IGNOU, Available athttp://egyankosh.ac.in/ bitstream/123456789/73698/ | /Unit-2.pdf Sarkar, R.N (2012). Applications of genetic polymorphism. Unit 3. In Practising Anthropology (MANI-003), IGNOU. pp 28-39. Venkatramana P. (2012). Human genetics and society. Unit2. In Practising Anthropology (MANI-003), IGNOU. pp 19-27. conser al5) To the falcon/glass tube from which supernatant is removed, add 3.5mL of 0.9% saline with ImL micropipette. 6) Centrifuge the falcon/glass tube at 2500 rpm for 3 minutes in labtop centrifuge. Repeat this step several times till the supernatant in falcon/glass tube appear as transparent. 7) For preparing 2mL volume of 3% RBC, 601 of RBC suspension is added to the 2mL microcentrifuge tube containing 19401 of 0.9% saline using either ImL or 100u micropipette. 8) Mix the contents in 2mL microcentrifuge tube with 1 mL micropipette several times by aspirating and dispensing into the microcentrifuge tube. A Fig. 1.1: Labtop Centrifuge (Source: bitps://ww.biomallin/productVlabtop-centrifuge-Ire 2St-lre-25t) Fig. 1.2: Micro or mini centrifuge (Source: https //www pipette com/Microcentrfuges) Fig. 1.3: Polystyrene 5 mL tubes (Source: https /wwrw.intscientific.com/product/pbt081/) conser alFig. 1.4: Glass tubes (SmL) (Source: bps: /isww universalmediealine com/ 1 3mm-X- smm-Smi-test-tubes htm!) Fig. 1.5: Variable volume micropipette (Source: hitps://ien labbox. com/product/var ble-volume. nicropipette-easy-40-elite/) Fig. 1.6: Micropipette tips in boxes (Source: https://indolabutama,com/catalogue/pipette-tips/) cance si blFig. 1.7: Microcentrifuge tubes (Source: Starlab.com) 1.3.3 Phenotyping of ABO and Rh Blood Groups 1) Take 4 microcentrifuge tubes (ImL) and label them as A, B, H and D. 2) Add 100p! of antisera- A, B, H and D into each of 4 microcentrifuge tubes. 3) Add 100 pl of 3% RBC suspension to each of 4 microcentrifuge tubes 4) Centrifuge the microcentrifuge tubes for 1 minute at 1000 rpm in minicentrifuge 5) Formation of agglutination of RBC in microcentrifuge tubes indicates the presence of particular blood group. Results are interpreted as shown in Table 1.1. If there is an agglutination in microcentrifuge labelled A, it is A blood group; if there is an agglutination in the microcentrifuge tube labelled B, it is B blood group; if agglutination found in both A and B labelled microcentrifuge tubes then it is considered as AB blood group and if there is agglutination in the tube labelled H, it is O group. If agglutination found in microcentrifuge labelled D, indicated presence of Rh+ (positive) blood group. Big clump (aggluti Small clumps small clump Fig. 1.8: Intensity of Aggluti ion (Source: NIB, Guidance manual, 2013) conser al