Abo blood group system

Abo blood group system

1. ABO BLOOD GROUP SYSTEM P SUNIL KUMAR Dept.of Haematology St.John’s Medical College Hospital Bangalore 1 SUNIL KUMAR.P

2. HISTORY ………??? SUNIL KUMAR.P 2

3. HISTORY ABO blood group system was discovered by Karl Landsteiner in 1900-1901. It is the most important blood group system for transfusion practice. A fourth blood group AB was discovered by Landsteiner’s associates,Von Decastello and Sturli in 1902. The four groups are determined by the presence or absence of blood group antigens on the RBCs and accordingly an individual’s group is A,B,AB or O In 1911, Von Dungern and Hirszfeld showed that group A could be divided into 2 main sub-groups A 1 and A 2 3 SUNIL KUMAR.P

4. Landsteiner’s Law…….. SUNIL KUMAR.P 4

5. Landsteiner concluded from his observation that…….. 1. Individuals who lack the antigens in of ABO system, have the corresponding Ab in their serum. 2. Ab’s of ABO system are usually “naturally occuring “ IgM type. 3.These are capable of causing intravascular haemolysis in patients, if transfused with incompatible blood. 4.Thus , an error in ABO grouping o donor or recipient can be fatal. 5 SUNIL KUMAR.P

6. Naturally occuring ………. There is a enough evidence to suggest that the Anti-A and Anti-B are stimulated by substances that are ubiquitous in nature. Eg; bacteria, pollen or other substances present in nature. 6 SUNIL KUMAR.P

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9. THE ABO ANTIGENS AND CORRESPONDING ANTIBODIES Antigen on RBC Antibody in plasma/serum Blood group A Anti-B A B Anti-A B AB None AB None Anti-A and Anti-B O 9 SUNIL KUMAR.P

10. GENETICS OF ABO SYSTEM SUNIL KUMAR.P 10

11. GENETICS In order to understand the ABO blood group system one must have a basic knowledge of genetic inheritance. The ABO system follows the mendelian laws of inheritance . 11 SUNIL KUMAR.P

12. GENETICS The locus of ABO gene is on chromosome 9 occupied by one of the three major allelic genes i.e. A,B or O , thus there is gene responsible for the specificity of our ABO blood groups. Other gene which plays an important role in the determination of ABO blood groups is H gene with locus on chromosome 19. Each individual inherits two ABO genes , one from each parent and these genes determine the ABO antigen present on their red cells The A and B genes are dominant while O is recessive thus is not directly detected. Absence of A and B antigens on the cells indicates O blood group. The serological typing reveals the phenotype and and the family studies reveal the genotype 12 SUNIL KUMAR.P

13. The ABO antigens are not fully developed at birth and hence the cells react weakly against the antisera till the age of 6-18 month. By 2-4 years of age A and B antigens expression is fully developed and remain fairly consistent throughout life. 13 SUNIL KUMAR.P

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15. Phenotype and genotype in ABO blood group system Phenotype Genotype A AA,AO B BB,BO O OO AB AB 15 SUNIL KUMAR.P

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17. BIOCHEMISTRY OF ABO SYSTEM.. SUNIL KUMAR.P 17

18. BIOCHEMISTRY The A and B antigens are basically glycoproteins formed from a basic precursor by addition of terminal sugar. The basic precursor substance is converted to H substance by enzyme L-fucosyl transferase (a product of H gene) by addition of a terminal sugar L-fucose. H substance is converted to A and B antigen by N-acetyl galactosaminyl and D-galactosyl transferase by addition of terminal sugars N-acetyl galactosamine and D-galactose resp. Thus it can be seen that genes do not synthesize antigens directly. The products of A,B and H genes are transferase enzymes. The O gene is an amorph (no gene product) and hence group O cells contains only H substance. Some H substance remains unconverted ,thus , all A and/or B cells normally contain some H substance along with A and B antigen. The amount of H substance in order of decreasing quantity in the red cells is. O>A 2 >B>A 2 B>A 1 >A 1 B cells 18 SUNIL KUMAR.P

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21. BOMBAY BLOOD GROUP In 1952, Bhende , Bhatia and Deshpande discovered a new blood group called Bombay group (Oh). This results from the homozygous hh condition, h being amorph, has no effect on precursor substance and hence is not converted to H substance. The A and B gene specified transferase enzymes cannot act directly on the precursor substance. As a result red cells of Bombay group (Oh) have no A, B and H antigens but the plasma has Anti-A and Anti-B. The cells react with all other cells except those of the same group (Oh). Bombay group is easily detected by the following tests: Negative reaction with anti H lectin, anti A and anti B 21 SUNIL KUMAR.P

22. ABH ANTIGENS A,B and H antigens are present not only on the red cells but widely distributed throughout the tissues except the CNS. A,B and H antigenicity is determined by specific sugars linked to the terminal portion of oligosaccharides. These are present on glycoproteins or glycolipids. In the red cell membrane, both glycoproteins or glycolipids with ABH activity are present. In the plasma only glycolipid in soluble form are found. 22 SUNIL KUMAR.P

23. Subgroups of A and AB , B A and AB have been divided into subgroups A 1 A 2, A 1 B and A 2 B depending upon the reaction with anti-A 1 Lectin or human anti-A 1 serum. Weak subgroups of A include A 3 ,A x ,A m and A intermediate. Weak subgroups of B- B 3 ,B x ,B m but they are rare. 23 SUNIL KUMAR.P

24. Antibodies of ABO system In the ABO system, once the antigen is absent from the cells, the corresponding antibody is present in the serum. These antibodies begin to appear during the first few 3-4 months of life, probably from exposure to ABH antigen-like substances in the environment. Because of this, these antibodies are called “naturally occuring”. Anti-A or Anti-B antibodies are naturally occuring and are mostly IgM. However some IgG antibodies are also present. IgG anti-A and anti-B are found more commonly in group O individuals than A or B individuals. O group individuals may have high titer of anti-A and anti-B as they are both IgM and IgG and often referred to as high titer O group individuals. 24 SUNIL KUMAR.P

25. ABO TYPING TECHNIQUES DIFFERENT METHODS SUNIL KUMAR.P 25

26. ABO TYPING TECHNIQUES The Method / techniques are : 1.Slide Method 2.Tube Method or Technique 3.DiaMed ID Microtyping System (Gel System) 4.Microplate Technique 5.Glass Microbeads Method 26 SUNIL KUMAR.P

27. SLIDE TESTING This maybe used for emergency ABO typing tests or for preliminary typing tests particularly in outdoor camps where a centrifuge is not available. REAGENTS REQUIRED : Monoclonal anti-A , anti-B sera. 27 SUNIL KUMAR.P

28. METHOD : The slide test maybe performed on clean microscopic slide Put one drop of anti-A and anti-B sera separately on the labeled slide Add 1 drop of whole blood of test sample to each drop of typing serum. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the slide. Rock/rotate the slide and leave the test for 2 mins at room temp. Then rock again and look for agglutination. Record the results. SUNIL KUMAR.P 28

29. Disadvantages It is not recommended as a routine tests because it is not reliable for weakly reactive antigens on cells and for serum typing tests with low titer anti-A/anti-B. It is less sensitive than the tube test. Drying of the reaction mixture can cause aggregation of cells that may be misinterpreted as agglutination. 29 SUNIL KUMAR.P

30. INTERPRETATION OF ABO BLOOD GROUPS 30 SUNIL KUMAR.P

31. TUBE TESTING It is of two types : Sedimentation method Spin tube method 1.SEDIMENTATION METHOD CELL GROUPING OR FORWARD GROUPING 31 SUNIL KUMAR.P

32. PRINCIPLE: Cell grouping is done to detect the antigen present on the red cells using commercially available antisera. SPECIMEN : Blood collected with or without anticoagulant may be used. EDTA anticoagulated blood is preferred. Random sample can be used. REAGENTS AND EQUIPMENTS: Refrigerator to store blood samples and reagents at 2-4 degree Centrifuge Testtubes , microscope Anti-A and Anti-B sera, 0.9% saline SUNIL KUMAR.P 32

33. METHOD Prepare an appropriate 2-5% cell suspension of test sample in normal saline. Set up 3 rows of test tubes and label them . Add two drops of anti-A in the tube labeled A, Add two drops of anti-B in the tube labeled B Add two drops of anti-AB in tube labeled AB. Add 1 drop of 2-5% of test sample in each tube. Mix the contents of the tube gently and centrifuge for 1min at 1000rpm. Gently resuspend the cell buttons and examine for agglutination. Read, interpret and record the results. 33 SUNIL KUMAR.P

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35. SERUM GROUPING (Reverse grouping) PRINCIPLE : It is done to detect the presence of ABO blood group antibody in the serum using pooled A,B and O cells. SPECIMEN REQUIRED: Serum from clotted blood less than 48 hr old. TYPE OF SPECIMEN: Randon non – fasting sample 35 SUNIL KUMAR.P

36. REAGENTS AND EQUIPMENTS: Centrifuge Microscope Testtubes Group A,B and O pooled cells 0.9% saline SUNIL KUMAR.P 36

37. PREPARATION OF POOLED CELLS (A,B and O) Take 3 clean testtubes and label as ‘A’ cell, ‘B’ cell and ‘O’ cell To the tube labeled as ‘A’ cell, add 3-4 drops of 3-4 different individual’s samples of ‘A’ Rh positive red cells. To the tube labeled as ‘B’ cell, add 3-4 drops of 3-4 different individual’s samples of ‘B’ Rh positive red cells. To the tube labeled as ‘AB’ ,add 3-4 drops of 3-4 different individual’s samples of ‘AB’ Rh positive red cells. To all the tubes add isotonic saline (3/4 th of the tube ), mix well and centrifuge for 5 mins at 3000-4000 rpm. Discard the clear supernatant from top with the help of a pasteur pipette, resuspend and repeat step 5, 3 times. After the third wash a suspension of A cell, B cell and O cell is made. 37 SUNIL KUMAR.P

38. PREPARATION OF 5% RED CELL SUSPENSION: To 20 ml normal saline add 1 ml of sedimented RBCs. Pooled cells are prepared freshly everyday SUNIL KUMAR.P 38

39. METHOD : Label 3 tubes as A, B and O. Add 2 drops of serum to each tube. Add 1 drop of 5% suspension of ‘A’ reagent cells to the tube labeled as A. Add 1 drop of 5% suspension of ‘B’ reagent cells to the tube labeled as B. Add 1 drop of 5% suspension of ‘O’ reagent cells to the tube labeled as O. Mix the contents of the tubes gently and centrifuge for 1 min at 1000 rpm. Gently resuspend the cell button and examine for agglutination(positive). Read, interpret and record the results. Compare the test results with those obtained in the forward grouping. 39 SUNIL KUMAR.P

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41. Reactions of test red cells with Reaction of serum with pooled cells Interpretation (Blood group of test cells) Anti-A Anti-B Anti-AB A B O + + + - - - AB + - + - + - A - + + + - - B - - - + + - O - - - + + + Oh 41 SUNIL KUMAR.P

42. GRADING OF AGGLUTINATION REACTION ++++ : Single clump of agglutination with no free cells +++ : 3 or 4 individual clumps with few free cells ++ : Many fairly large clumps with many free cells + : Fine granular appearance visually, but definite small clumps (10-15 cells) per low power field. W : 2-3 cells sticking together per low power field, uneven distribution. O : All cells are free +H haemolysis (partial or total) must be interpreted as positive. 42 SUNIL KUMAR.P

43. ADVANTAGES OF TUBE METHOD It allows longer incubation of antigen and antibody mixture without drying. Tubes can be centrifuged which enhances antigen and antibody reaction Weaker antigens and antibodies can be detected Less reagents are required Results can be read comfortably as there is no drying. SPIN TUBE METHOD All steps are similar to sedimentation technique except in step 4 of the sedimentation technique, the serum and cell mixture is centrifuged after 5-10 mins incubation at room temp and see the results. It is very useful in urgent cases. 43 SUNIL KUMAR.P

44. ANTI-A1 LECTIN TEST: *RBCs possessing the A Ag can be subdivided into A 1 and A 2 . Group A Ags which are agglutinated with Anti-A 1 lectin are of the subgroup A 1 , those which are not agglutinated fall into subgroups weaker than A 1 , the majority being classified as A 2 . *Group AB RBCs can be similarly classified as A,B and A 2 B. SAMPLE: EDTA anticoagulated blood METHODS: 1.SLIDE METHOD: *Prepare a 10% suspension of the RBCs to be tested in isotonic saline. *On a glass slide at room temp, place 1 drop of Anti-A 1 lectin *Add 2 drops of the red cell suspension, mix well. *Gently tilt the slide back and forth and observe for agglutination. *Tests that show agglutination within 2 mins are confirmed as A 2 /A 2 B. 44 SUNIL KUMAR.P

45. 2. TUBE METHOD Prepare a 5% suspension of the RBCs in isotonic saline. Label 2 test tubes as test and control. To both test tubes add 1 drop of Anti-A 1 lectin. To the tube labelled ‘test’, add 1 drop of 5% suspension of the patient. To the tube labelled ‘control’, add 1 drop of 5% of A 1 cells. Mix well, centrifuge at 1000rpm for 1 min. Agglutination should be seen in the control tube If no agglutination in the tube labelled as ‘test’, it is confirmed that the patient is A 2 . If there is agglutination in the tube labelled ‘test’, it is confirmed that the patient is A 1 . 45 SUNIL KUMAR.P

46. ANTI-H LECTIN TEST *RBCs possessing the H antigen are agglutinated with anti-H lectin. Those which are not agglutinated by anti-H lectin are classified as Bombay phenotype (Oh). *The general pattern of strength of agglutination of red cells with anti-H lectin is as follows: O>A 2 >A 2 B> B> A 1 >A 1 B METHODS: 1.SLIDE METHOD *Use whole blood or prepare a suspension of red cells of an approx equal concentration to whole blood in their own serum or plasma. *On a glass slide at room temp, place 1 drop of anti-H lectin. *Add 1 drop of whole blood, mix well, gently tilt the slide back and forth and observe for agglutination. *Test that show no agglutination within 2 mins are considered negative. 46 SUNIL KUMAR.P

47. 2. TUBE METHOD: i) Prepare a 5% suspension of the RBCs in isotonic saline. ii) To the testtube add 1 drop of anti-H lectin. iii) Add 1 drop of the cell suspension , mix well and centrifuge at 1000 rpm for 1 min. iv) Gently dislodge the cell button and observe for agglutination. GROUPING OF CORD OR INFANT BLOOD Special precautions should be taken while testing cord or new-born infant blood since ABO antigens are not fully developed and allo -agglutinins are usually absent. Cord RBCs should be washed 3 times to avoid error due to Wharton’s jelly. Serum grouping is not recommended in new born upto 3 months. 47 SUNIL KUMAR.P

48. GEL TECHNOLOGY Gel technology can be used for any immunohematological test that has hemagglutination as its endpoint such as 1. ABO and Rh typing 2. Typing for other blood group system 3. Antibody sreening and identification 4. Compatibilty testing including crossmatching PRINCIPLE: The basic principle is that instead of testtube , the serum and cell reaction takes place in a microtube consisting of a reaction chamber that narrows to become a column Red cells or mixture of cells & serum (as appropriate) are added into the gel The cells are always added prior to the serum so that the serum does not come into contact with the gel This eliminates the need for washing as in conventional techniques Incubation takes place followed by centrifugation under strictly controlled conditions 48 SUNIL KUMAR.P

49. METHODS: 1.FORWARD GROUPING ABO AND Rh DETERMINATION i) A 5% suspension of the test red cell is prepared in LISS ii) 50µl of this suspension is added to each of the microtubes (1-4 having A nti-A, Anti-B, Anti-AB and Anti-D). iii)After incubation at room temp for 10 min s the card is centrifuged. iv) Read results 2.REVERSE GROUPING ABO SERUM GROUPING i)For reverse grouping 50µl of known 5% cell suspension of A1 and B cells are added to the respective microtubes (5 & 6 resp ) ii) 25µl of each patients serum is added to microtubes 5 & 6 iii) Incubate at room temp for 10 mins iv) The card is then centrifuged and the results are read 49 SUNIL KUMAR.P

50. ADVANTAGES The gel technique is simple, reliable, rapid to use, reproducible and sensitive Greater uniformity between repeat test Less specimen volume are needed to perform a large number number of test The cards have a shelf life of 1 year and easy storage at room temp (18-25̊ ̊c) DISADVANTAGES Special centrifuge to accommodate the microtube cards Special incubators to incubate the microtube cards Expensive 50 SUNIL KUMAR.P

51. MICROPLATE TECHNIQUE Microplate technique has been successfully applied in blood bank laboratory All routine test of blood grouping, antibody screening and cross-matching can be done in microplate without loss of sensitivity of antibodies or any problem in blood grouping PRINCIPLE: M icroplate is a matrix of 96 short testubes. Principle of agglutination by test-tube method is also applicable here. Micro plate maybe rigid or flexible either with U-shaped or V-shaped wells. U-bottom plates are more widely used because results can be read by observing the characteristics of resuspended RBCs or the streaming pattern of RBCs when the plate is placed at an angle. 51 SUNIL KUMAR.P

52. METHODS 1.SALINE TECHNIQUE 2.ENZYME TECHNIQUE 3.INDIRECT ANTIGLOBULIN TEST (IAT) 4.DIRECT ANTIGLOBULIN TEST (DAT) ADVANTAGES It is cost effective for testing large number of blood samples Enhanced sensitivity of reaction Saving in reagents and equipments Less laboratory space required Less time for technologists Results can be observed visually or can be evaluated with automatic photometric readers which eliminates human errors 52 SUNIL KUMAR.P

53. GLASS MICROBEADS TECHNOLOGY This system is for irregular antibody screening The test is performed in a microcolumn prefilled with glass microbeads in suspension of antihuman globulin serum, any diagnostic reagent or neutral isotonic solution. The sensitized cells are trapped by the microbead suspension during column centrifugation. The detection of sensitized cell is based on the seiving effect of glass microbeads. Cassettes of different programs are available like ABO-Rh(D) typing with ABO reverse grouping AHG polyspecific (anti -IgG + anti-C3d) AHG-IgG monospecific Cross-matching ABO-Rh(D) tying in newborn 53 SUNIL KUMAR.P

54. ADVANTAGES Minimum incubation time of 10 mins for antibody screening or cross-matching More objective, consistent and reproducible interpretation of results DISADVANTAGES Special centrifuge to accommodate glass beads cassettes Special incubators to incubate the glass bead cassettes Expensive 54 SUNIL KUMAR.P

55. PROBLEMS IN ABO GROUPING Problems arise when there is discrepancy between results of cell and serum grouping and are related to one or more of the following EQUIPMENTS AND GLASSWARES An inaccurate centrifuge may result in over or under-centrifuge to give false-positive or negative results Dirty glasswares may give false positive result Thermostatic equipment may not have proper temperature REAGENTS AND SPECIMEN Substandard or deteriorated grouping sera due to improper storage Contaminated or haemolysed reagents 55 SUNIL KUMAR.P

56. TECHNIQUES Any of the following can lead to incorrect result i)Failure to add proper reagent, cell or serum ii)Incorrect cell: serum ratio iii)High ambient temperature in the lab iv)Incorrect reading or interpretation of test results v)Failure to confirm negative results under the microscope may cause false negative result vi)Failure to interpret haemolysis as a positive reaction causes false negative results 56 SUNIL KUMAR.P

57. RED CELL MEDIATED DISCREPANCIES 1.Weakening or loss of antigens due to age, diseases such as leukemias and other malignancies and inherited weak expression of A and B antigens which lead to false negative results 2.Acquired B antigen like activity is associated with gram negative septicemia, intestinal obstruction and carcinoma of colon or rectum. Red cells may give false positive reaction with anti-B serum 3.Polyagglutinable cells 4.Antibody coated on red cells as in HDN,AIHA, incompatible blood transfusions may mask the antigen on the red cell surface give false negative results 5.Presence of 2 separate cell population as in chimera e.g. a patient recently transfused with non-specific blood group (massive or exchange transfusion), bone marrow transplant or fraternal twins which lead to mixed reaction 6.Substances in plasma or serum-excess blood group substances, Wharton’s jelly 7.Reagents maybe weak or deteriorated 57 SUNIL KUMAR.P

58. SERUM MEDIATED DISCREPANCIES Unexpected antibodies in serum like anti-A 1 in A 2 or A 2 B individuals, anti –I, anti-H or anti-P1 cause problems in serum grouping Auto-agglutinins Rouleaux formation Absence or loss of antibodies in serum Haemolysis SOLVING PROBLEMS/DISCREPANCIES Once a discrepancy is detected in ABO cells and serum grouping, repeat the test before additional investigations are carried out Quality assurance of reagents, correct technique, careful observation and interpretation of results resolve many problems 58 SUNIL KUMAR.P

59. THANK YOU… 59 SUNIL KUMAR.P