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ABO AND Rh TYPING IS PERFORMED BY TWO METHOD:- 1.SLIDE METHOD:- Aim: – ABO grouping and Rh typing using the slide method. Objective: – ABO grouping and Rh typing using the slide method, which is a standard procedure for determining an individual’s ABO and Rh blood types. Principle: – ABO grouping and Rh typing performed by antisera based on the principle of agglutination. Normal human red cell processing antigen will clump in the presence of corresponding antibody (Anti A, Anti B, Anti D). Requirement:- *whole blood sample (EDTA) *Disposable gloves *Clean glass slide *ABO grouping and Rh typing reagents *Toothpicks or wooden sticks *Droppers of pipettes *Absorbent paper towels *Timer or stopwatch *Blood typing record sheet. Preparation:- Ensure that you are working in a well-lit and clean laboratory environment Label each glass slide with the patient’s identification or sample number Put on disposable gloves to maintain aseptic conditions ABO blood grouping procedure:-*Take three clean and dry glass slides *Place one drop of patient’s blood on each slide *Add a drop of Anti A reagent on first slide anti B on second slide, Anti D on third slide. *Mix the blood and serum gently using separate wooden sticks. Observation and record the reaction: – *if blood agglutinates (clumps)with anti A serum then the patient blood group is A. *If blood agglutinates with anti B serum, then the patient blood group is B. *If blood agglutinates with anti A and anti B both serums, then the blood group is AB. *If blood do not agglutinate with both anti A and anti B serum, then the blood group is O. Antisera A Antisera B Blood group + – A – + B + + AB – – O Rh blood grouping procedure: – Take clean and dry glass slide Place a fresh drop of the patient blood on slide Add a drop of anti Rh (anti D) serum. Mix gently with wooden stick. Observation and record the reactions: – if the blood agglutinates with anti Rh serum, then the patient blood Rh positive e.g. (A+, B+, AB+, O+) If blood is not agglutinate with anti Rh serum, then the patient blood Rh negative e.g. (A-, B-, AB-, O-) Antisera D Blood group + Rh positive – Rh negative   Clinical significance: – ABO grouping and Rh typing is important in: – *.Blood transfusion *.Used for the personal identification and paternity exclusion (before DNA testing) *.Before a person donates blood. *.Before an organ and tissue transplant *.Before surgery *.To show whether to people could be blood relatives *.To check the identity of a person suspected of committing a crime. Note: – The ABO and Rh typing determination is used for the transfusion safety, pregnancy management, transplantation, forensic identification, and understanding disease susceptibility. TEST TUBE METHOD:- AIM: – ABO grouping and Rh typing by test tube method. Principle: –In the ABO grouping and Rh typing using the antisera is based on the principle of agglutination. Normal human red cell processing antigen will clump in the presence of corresponding antibody. Requirements: – blood group tube Pasteur pipettes. Centrifuge. Reagent. Normal saline. Blood sample. Procedure: – Prepare 5% red cell suspension for ABO grouping and Rh typing. Mix 5 drops(0.05ml) of sediment red cell with 2 ml of normal saline. Centrifuge at 1500 rpm for 1–2-minute discard the supernatant part wash 3 time with normal saline. Add 4ml of normal saline to sedimented red blood cells. Take 3 test tube label them as A, B, D. Place 1 drop of anti A into ‘A’ tube, one drop of anti B into the ‘B’ tube. one drop of anti D in ‘D’ tube. Add one drop of RBC suspension to each tube. Gently shake each tube to mix the contents. And then centrifuge tube at 1500 rpm for 1 minute. The RBC will form a button or pellet at the bottom of each test tube. Gently resuspend the RBC button and examine agglutination macro and microscopically. Observation: – Antisera A Antisera B Blood group + – A – + B + + AB – – O Antisera D Blood group + Rh positive – Rh negative

Human blood group system:- Rh are major, the most important of all the blood group system. All people (with some exception) of ABO system can be divided in 4 major groups in this system they are A, B, AB and O group. This determines by the reaction of two different reagent, known Anti A, and Anti B. For example: – Forword grouping or cell grouping by using known Anti A and Anti B results obtained are as follow. Red cell sample  Reagent  Reaction Blood group  Red cell  Anti A  Agglutination  A groupThere are nearly 300 blood group systems so for discovered. The ABO and Red cell  Anti B  No agglutination  Red cell  Anti A  No agglutination  B group  Red cell  Anti B  Agglutination  Red cell  Anti A  Agglutination  AB group  Red cell Anti B  Agglutination  Red cell  Anti A  No agglutination  O group  Red cell  Anti B  No agglutination  ABO BLOOD GROUP SYSTEM:- Karl Landsteiner, an Austrian scientist discovered the ABO blood group system in the year 1900.in his experiments, he mixed different blood types and noted that the plasma from certain blood type produced agglutinates. Which were caused by the absence of molecules on red blood cells and resulting in antibodies to defeat that molecules. He made a note of the agglutination and divided the blood type in 4 different groups. For the discovery of ABO blood group, he was awarded the Nobel price. ABO and Rh blood group: – During blood transfusion, two group system are examine the ABO and Rh (Rhesus)system. ABO BLOOD GROUP: – The ABO system is based on the presence or absence of two antigens (A and B) on the surface of red blood cells (RBCs). The presence of A,B, or O antigen on red cell is determined by the inheritance of the allelic genes A, B, and O on chromosome 9, which are inherited in pairs as Mendelian dominants.  it classified into 4 types  *Group A → RBCs have A antigen, plasma has anti-B antibodies *Group B → RBCs have B antigen, plasma has anti-A antibodies *Group AB → RBCs have both A and B antigens, plasma has no antibodies (universal recipient) *Group O → RBCs have no antigens; plasma has both anti-A and anti-B antibody The ABO group system is important in blood transfusion during blood donation as mismatching of blood group can lead to clumping of RBCs with various disorders.  It is important for blood cells matching during transfusing i.e. donor -recipient compatibility is necessary.  ABO antibody: – ABO antibodies are usually IgM, which are cold reacting. These antibodies don’t cross the placenta and can bind complements. The majority of anti A from group B person or individual and anti B from group A person or individual contain IgM antibody with minor amount of IgG or IgA present. Antisera used in ABO test procedure: – In ABO test procedure antigens present on an individual cells are determining method is called forward typing or direct typing and consist of testing the unknown cells with known antisera and then observing for agglutination. Anti A antiserum: – The serum is obtained from “B” type individuals, since there is a “natural” occurrence of anti A agglutinin in their serum. Anti B antiserum: –  The serum is obtained from “A” type individuals where there is a natural occurrence of anti B agglutinin in the serum. Anti A, B typing serum: – This anti serum is obtained from “O” Type individuals. Anti A1: – This serum is obtained from human sources or plant lectins. This serum determines A1 and A1 B person from other A and AB types. Anti H: – this serum is obtained from lectin (plant source) this antiserum determines Bombay (oh)types (who lack H substance 2. Rh (Rhesus) system: –  The Rh system is the second most important blood group system after the ABO system in transfusion medicine. About two-third of the papulation contain the third antigen on the surface of their red blood cells known as Rh factor or Rh antigen. It was first discovered in 1940 by Landsteiner and Wiener in his experiment using the blood of Rhesus monkeys NOTE: –  The Rh system is a complex blood group system, mainly determined by the D antigen, playing a vital role in transfusion safety and in preventing haemolytic disease of the newborn The Rh system has more than 49 antigens, but the most significant is the D antigen. If a person has the D antigen on their red blood cell, then the person is Rh positive (Rh+). If D antigen absent on their red blood cell, then the person is Rh negative (Rh-) Some other important Rh antigens are C, c, E, and e. The Rh system is inherited genetically. It is controlled by two closely linked genes on chromosome 1.      RHD gene     –        produces the D antigen.       RHCE gene –     produces C/c and E/e antigen. Haemolytic disease (break down of RBC called haemolytic disease) of the newborn (HDN):-occurs when an Rh- mother carries an Rh+ foetus. the mother’s immune system may produce anti D antibodies that destroyed fatal red blood cells. Blood Transfusion Reactions:If Rh⁻ patients receive Rh⁺ blood, they may form antibodies (anti-D), leading to haemolysis in future transfusions. That’s why Rh typing and cross-matching are essential before blood transfusion. Rh⁻ mothers are given anti-D immunoglobulin after delivery or miscarriage to prevent sensitization. Note: – Bombay blood group is discovered by Y.M Bende in 1950.

Blood banking index:- 1. Blood Bank Management & Documentation of Packaging:- Reception, Indexing & Recording Decontamination of blood bags, workbench, andal and immune antibodies) Distribution of ABO antigens (A, B, and H Antigens) on red cells and antibodies in the serum. Rh blood group system Sources of errors in ABO grouping  instruments Sterilization of transfusion sets (physical & Chemical) 2. Discovery of Human Blood Group:- Theory of inheritance and nomenclature of ABO and Rh blood group system Subgroup of ABO system and Bombay group. 3. Technique for Determination of Various Blood Groups and Rh factors:- Determination of various blood groups (NaturABO hemolytic disease 4. Cross Matching and Complications of Blood Transfusion Cross math Immunological complications, non-immunological complications. History of blood bank:- The history of Blood Bank began in 1616 when William Harvey  Discovered that blood circulation through the body. In 1665, a transfusion of blood from lamp saved a young patient’s life. This led to- animal to human transfusion becoming common. The first blood bank was established in a Leningrad hospital in 1932. IN 1937, Bernard Fantus was the director of the therapeutics at cook county hospital in Chicago. Established the first hospital blood bank in the United States. Fantus created a hospital laboratory that preserved refrigerated and stored donor blood.  He also coined the term “blood bank”. The first blood bank in India was established in Kolkata in March 1942. The red cross managed the blood bank at all India institute of Hygiene and public health. The first successful transfusion of human blood to a patient was performed in 1818 by British obstetrician James Blundell.   Blundell’s transfusion was to treat postpartum hemorrhage.  In 1901, three main blood groups were discovered in 1902, the blood group is discovered in 1907. Cross matching was first used in 1914; the first non-directed transfusion was performed. In 1917, the first blood depot was established -: Universal safety rules for blood bank technicians: –  Working in blood bank is a noble profession that’s required almost care and precautions. Blood bank technicians play a crucial role in ensuring the safety of both donors and recipients. To maintain a lifesaving legacy, it is essential for these technicians to follow universal safety rules. Personal protective equipment (PPE): – Always wearing appropriate PPE including Gloves Lab Coat / Gown / Apron Face Protection Head Cover / Cap Shoe Covers / Closed Shoes Additional PPE (when needed) Safety goggles When handling blood and blood products. 1.Hand hygiene: – Hand hygiene is one of the most critical infection control practices in blood bank, since staff handle human blood, component, and samples that may carry infectious agents like HIV, HCV. Wash your hands frequently and thoroughly with soap and water, apply 3-5 ml of sanitizer, rub for 20-30 second until dry should contain 60-95% alcohol. 2. Sharp object safety: – Prefer safety engineered needles and syringes with retractable or shielded tips. Do not recap needles after use. Avoid passing sharps directly from hand to hand (use trays). Dispose of used needles, scalpels, and glass pieces immediately after use in puncture-proof, leak-proof, clearly labelled sharps containers. Keep the blood collection area clean, uncluttered, and well-lit. Handle glass blood bottles, vacutainers, and pipettes carefully to prevent breakage.   Regular staff training on safe handling, disposal, and emergency procedures.   Posters and reminders in the blood bank to reinforce best practices.   3.Infection control:- Follow standard precaution to prevent the spread of infectious disease treat all blood product are potentially infectious. 4. Labelling: – Ensure proper labelling of all blood products and specimens confirm the information and levels match the requisition forms. 5..Blood typing and cross match: -Double check patients’identification and blood compatibility before transfusion to prevent error. Blood typing is the process of determining a person’s blood group based on the presence or absence of specific antigens on red blood cells (RBCs). Crossmatching is done before a blood transfusion to ensure compatibility between donor and recipient blood. It prevents haemolytic transfusion reactions. 6. Blood donation: – blood donation is the voluntary process of giving blood, which is then used for transfusions or to make blood components (like plasma, platelets, or red blood cells) for patients in need. If involved in the collection process, ensure that all blood donation equipment sterile and used according to established procedures 7. Equipment maintenance: – Regularly inspect and maintained equipment like refrigerator, freezer, and centrifuge to ensure the integrity of blood products 8.Storage:- Follow strict instructions of guidelines for storing blood products ensure proper temperature control and monitor for any signs of spoilage or contamination. 9.Emergency procedures: – Stay calm and follow the laboratory’s Standard Operating Procedures (SOPs) Protect yourself first using PPE (gloves, mask, apron) Alert staff and supervisor immediately Document the incident properly 10.Disposal: – Dispose of biohazardous waste including contaminants materials and sharps, accordance with rule -regulation and guidelines. 11.Training and education: – Stay current with blood banking procedures and safety practices throughout the regular training and continuing education. 12.Quality control:- Participate in quality control and assurance programs to maintain the highest standards of safety and accuracy. 13.Documantaion: – Maintain accurate records of all blood product handling testing and transfusions. Document any incidents or deviations from standards procedures 14. confidentially: – Maintain strict confidently of patient information and blood donor records. 15.Communication: – Clearly and accurately communicate with healthcare providers and other staff involved in the blood transfusion process. 16.Safety data sheets (SDS): – Familiarize yourself with safety data sheets for all chemicals and reagents used in the blood bank follow safety instructions and guidelines. 17. zero tolerance for risky behaviour: – Report any unsafe practices or deviations from safety protocols to your supervisor immediately 18.contnuous monitoring: – Regularly monitor and assess safety practices to identify and address potential risks and improvement. Donor selection:- Pre-donation counselling by trained staff should be made available maintaining privacy and confidentiality pre donation information should be included. Modes of transmission leading to risk behaviour and self-exclusion for patients’ safety. Alternative testing site Test carried out on donated blood.