blood grouping.pptx

blood grouping and its techniques

1. BLOOD GROUPING METHODOLOGY

2. Learning Objectives : Understanding inheritance , synthesis of various antigens and antibodies and their clinical significance in ABO and Rh blood group systems . U nderstanding practical aspects of ABO and Rh blood grouping . Discrepancies and resolutions related to ABO blood grouping.

3. Human Blood group systems : A blood group also called a blood type . Classification of blood group is based on presence or absence of inherited antigenic substances on the surface of red blood cells (RBCs). T hese antigens maybe proteins , carbohydrate , glycoproteins , or glycolipids depending on the blood group system . Thirty three major blood group systems were recognised by the international Society of blood transfusion ( ISBT ) in October 2012 . In addition to the ABO a ntigens and Rhesu s antigens , many other antigens are expressed on the red blood cell surface membrane .

4. Blood group systems :

5. ABO Blood Groups : The ABO blood group system is most well known and clinically important blood group system in human blood transfusion . ABO blood types are also present in some other animals for example rodent s and apes such as a chimpanzee s, bonobos and Gorillas. Determination of AB O blood groups depend upon the immunological reaction between antigen and antibody . Relative frequency of different blood types ( WORLD ) O- 47% ,A- 41% ,B -09% , AB – 3%

6. History: Austrian immunologist Karl Landsteiner discovered the ABO blood group system in 1901 . L andsteiner was awarded in the 1930 Nobel Prize in Physiology or medicine for his work .

7. Landsteiner’s law : If an ‘ antigen ’ is present on patient ’ s red blood cells , the corresponding antibody will NOT be present in the patient ’ s plasma , under ‘ normal condition ’.

8. Inheritance and genetic of ABO blood groups : The ABO gene is autosomal ( the gene is not on either sex chromosomes ). The ABO gene locus is located on chromosome 9 . A and B blood group s are dominant over O blood group . A and B group genes are co-dominant . E ach person has two copies of genes coding for their ABO blood group (one maternal and one paternal in origin ).

9. A B O and H antigen genetics : The pr esence or absence of the H, A, B antigen s is controlled by the H and ABO genes . LOCATION : The presence or absence o f the ABH antigens on the red blood cell membrane is controlled by the H gene. T he presence o r absence of ABH antigens in secretion is indirectly controlled by the Se gene .

10. ABO Antigen Genetics: H gene- H and h alleles (h is an amorph ) Se gene- Se and se alleles (se is an amorph ) ABO genes- A , B and O alleles.

11. Formation of the H antigen :

12. Formation of the A & B antigen :

13. Genetics : The H antigen is found on the RBC when you have the Hh or HH genotype , but NOT f rom the hh genotypes . The A antigen is found on the RBC when you have the Hh , HH and A/A, A/O , or A/B genotypes . The B antigen is found on the RBC When you have the Hh , HH and B/B, B/O or A/B genotypes .

14. ABO Antigens in Secretions: Secretions include body fluids like plasma, saliva, synovial fluid, ect . Blood Group Substance are soluble antigens (A,B and H) that can be found in all body secretions ( except CSF ). This is controlled by the H and Se genes .

15. Secretors Non secretors If the Se allele is inherited as SeSe or Sese , the person is called a “ secretor ”. - 80 % of the population are secretors . Individuals who inherit the sese gene are called “ non secretors “ -The se allele is an amorph ( nothing expressed ) 2. Secretors express soluble forms of the H antigen in secretions that can then be converted into A or B antigens. 2. sese individuals do not convert antigen precursors to H antigen and has neither soluble H antigen nor soluble A or B antigen in body fluids.

16. Bombay blood group: First reported by Bhende in Bombay in 1952 . A bsence of H , A and B antigen s . No agglutination with anti-A and anti- B or anti-H antisera. P resence of all 3 antibodies i.e. Anti-H , an ti - A and anti-B in the serum . No A, B or H substances present in saliva . In compatible with any ABO blood groups , compatible with Bombay phenotype only .

17. Development of antigens: All the ABH antigens develop as early as day 37 of fetal life but do not increase very much in a strength during gestation period . Red cell of newborn carry 25 to 50 % of number of antigenic sites found on adult RBC . Although cord red cells can be ABO grouped, the reactions may be a bit weaker than expected . A or B antigen expression fully developed at 2-4 years of age and remain constant throughout life .

18. Development of antibodies : Not present in newborn , appear in the first years of life (4- 6 month usually ), reach adult level at 5-10 years of age , decreases in elderly . Na turally occurring as they do not need any antigenic stimulus .

19. The ABO blood group system O n presence or absence of antigen A and antigen B, blood is divided into four groups A,B, AB,O group .

20. ABO Subgroups: ABO subgroups differ in the amount of antigen present on the red blood cell membrane . - subgroups have less antigen . S ubgroups are the result of less effective enzymes . T hey are not as efficient in converting H antigen to A or B antigens. Subgroups of A are more common than subgroups of B .

21. Subgroups of A & B Antigen : The 2 principle subgroups of A are: A1 & A2 - Both react strongly with the reagent anti –A. - To distinguish A1 from A2 red cells, the lectin Dolichos biflorus is used (anti-A1) - 80% of group A or AB individuals are subgroup A1 and A1B . - 20% are A2 and A2B . B subgroups occur less than A subgroups. -Example : B3,Bx,Bm and Bel .

22. A2 Phenomenon : Why is the A2 phenomenon important ? - 8% of A2 and 25% of A2B individuals may produce an anti-A1 antibodies in the serum . -This may cause discrepancies when a cross match is done (incompatible). - H owever , these anti - A1 antibodies are cold reacting and therefore may not cause problems routinely .

23. ABO antibodies: Ig M is the predominant antibody in Group A and Group B individuals . -Anti-A -Anti-B IgG ( with some Ig M ) is the predominant antibody in Group O individuals . -Anti-A,B (with some anti-A and anti-B) Newborns may passively acquire maternal antibodies ( IgG crosses placenta) -Reverse grouping (with serum) should not be performed on newborns or cord blood.

24. Rh Blood group system : The Rh blood group is the 2 nd most important blood group system . R h blood group system consists of 40 defined blood-g roup antigens , among them there are six common types of R h antigen s. Ea ch of which is called as Rh factor . Th ese types are designated C, D, E, c, d, and e .

25. Rh antigen cont … The “ type D” antigen is widely prevalent in the population and considerably more antigenic than the other R h antigens. A nyone who has this type of antigen is said to be Rh positive , whereas a person who does not have type D antigen is said to be Rh negative . The antigen was discovered by Karl landsteiner and Alexander Wiener in 1940 in rhesus monkeys hence the name ‘ Rh factor ‘.

26. ABO Typing techniques: Slide test Tube technique Microplate Gel card system

27. Clinical Application of B lood G rouping : Safe Blood transfusion . Preventing h emolytic disease of newborn ( Rh incompatibility in newborns). To solve the legal disputes related to paternity disputes . M edicolegal use . S usceptibility of various diseases . Group O - duodenal carcinoma Group A - Carcinoma of stomach, pancreas & salivary glands. R outine health checkup .

28. Principle of blood grouping : Blood grouping is done on the basis of agglutination . Agglutination means the collection of separate particle like RBC in to clumps or masses . Agglutination occurs if an antigen is mixed with its corresponding antibody which is called isoagglutinin i .e. occurs when A antigen is mixed with anti -a or when B antigen is mixed with anti-b .

29. Practical aspects of ABO grouping : Routine ABO grouping must include both serum and cell testing as each test serves as a check on the other . Tubes , slide should be dry and labeled properly . S erum / antisera should always be added BEFORE addin g cells . R esults should be recorded immediately after the observation . H aemolysis i s interpreted as positive result .

30. Blood sample for blood grouping : BLOOD SAMPLE : Clearly lab e led blood samples in sterile tubes ( Plane or EDTA ). T est should be performed on fresh sample for best results . In case the test cannot be performed immediately , sample can be stored at 4 ° C and should be tested within 48 hours . N o signs of hemolysis should be there .

31. Red cell suspension s for blood grouping: 2-5 % : Test tube method 0.8-1% : Gel technology 1% : Microplate

32. Slide Test ABO ANTISERA Blood sample

33. SLIDE TEST :- method A cl ean and dry glass slide is divided into two sections with a glass marking pencil . Th e sections are labeled a nti -A & anti-B to identify the antisera . P lace 1 drop of anti-A serum and 1 drop of anti-B serum in the centre of the corresponding section of the slide . A ntiserum must be taken first to ensure that no rea gent are missed.

34. Samples added to the slide- 3.Add 1 drop of blood sample to be tested to each drop of antiserum . 4. Mix antiserum and blood by using a separate stick or a separate corner of a slide for each section over an area of about 1 inch of diameter .

35. Observe for agglutination- Sample 1- A positive Sample 2- B positive 5.By tilting the slide backwards and forwards , examine for agglutination after two minutes .

36. Slide test: cont …. Result : -P ositive (+): L ittle clumps of RBC are seen floating in a clear liquid . -Ne gative (-) : Re d cell s are floating homogeneously in a uniform suspension. Interpretation: Forward (cell) grouping Reverse (serum) grouping Interpretation Anti-A serum Anti-B serum A-1 cells B cells Blood group + - - + A - + + - B - - + + O + + - - AB “+” Agglutination of red cells “-” No Agglutination of red cells

37. Slide grouping: Advantage Quick and needs only simple equipments . Preliminary typing tests. Uses during camps and in case of emergency . Disadvantages Less sensitivity. Drying of reaction giving to false positive results.

38. TEST TUBE METHOD :Recommended method (Gold standard) More reliable than slide test. Allows longer incubation of antigen and antibody mixture without drying . T ubes can be centrifuged to enhance reaction . C an detect weaker antigen-antibody reactions . TEST TUBE METHOD

39. 2 Steps in ABO grouping 2.1 CELL GROUPING (forward Grouping) Tests the p atients RBC’s with known anti-A and anti-B to determine the ‘ Antigen ’ expressed. 2.2 SERUM GROUPING (Reverse grouping) Tests the patient’s serum with known A and B cells to determine the presence of “ antibody ”.

40. CELL GROUPING (FORWARD GROUPING) Prepare 2-5 % suspension of test sample in normal saline . S et 3 tubes , labelled as A , B, D . Ad d 2 drops of antisera (anti A, an ti B and anti D ) in three different tubes. Add 1 drop of 2 - 5% cell suspension ( ratio 2 : 1 ) Mi x contents well and centrifuge at 1000- 1500 rpm for 1 minute . Ob serve for h emolysis . Gently disperce cell button and check for agglutination . C onfirm negative result under the microscope .

41. SERUM GROUPING (REVERSE GROUPING) Prepare 2- 5% suspension of pooled cell A, B, O. L abel 3 tubes A cell , B cells and O cells . P lace 2 drops of serum in each tube . Add 1 drop of cell suspension ( A cell to A tube, B cells to B tube, and 1drop of O cells to O tube) C entrifuge tube at 150 0 rpm for 1 minute . G ently disperse for agglutination . Ne gative results check by microscope .

42. Microplate method : It is sensitive and ideal for testing large number of blood samples . Microplate is a poly styrene plate consisting of 96 micr o wells of either U - or V - shape . The principal is same as for agglutination in test tubes . M ore sensitive to detect weaker antigen - antibody reactions . Mi croplate can be incubated and centrifuged . Re sults can be photographed for a rchival storage . Th ere is significant savings in time and in the cost of disposable s and reagent s. M icroplates can be adapted for automation .

43. Gel or Column agglutination Technology : One gel card is basically a set of six microtubes . Each microtube has an upper reaction chamber and the section containing dextran acrylamide gel ( which functions both as a reaction medium and a size filter ) and anti serum . A fter addition of red cell to the top of the tube , haemagglutinates ( if formed ) are trapped at the top of the tube ( positive test ). N on - agglutinated cells pass through the gel and form a button at the bottom of the tube ( negative test ). Th e method is standardized and has defined e nd point leading to better interpretation of the resu lts . The technology is suitable for automation .

44. R h blood group system :

45. Rh (D) Antigen : Rh refers to the presence or absence of the D antigen on the red blood cell . Frequency in Indian population 92-95% Rh positive. Unlike the ABO system , individuals who lack the D antigen DO NOT naturally produce anti - D . P roduction of antibody to D require exposure to the antigen . Th e D antigen is very immunogenic , i . e. individuals exposed to it w ill very likely make an antibody to it . F or this reason all individuals are type for D , if negative must receive R h (D) negative blood .

46. Rh antibodies : All Rh antibodies are immune in nature , developed after immunizing event . React at 37 °C and require anti globin to demonstrate the reaction. G enerally do not react at room temperature in saline . Mo st are IgG in nature therefore can cross the placenta. A ll are important in HDN and delayed HTR .

47. Rh typing: Normal typing for R h antigens only includes typing of Rh( D ). Th e result of this typing determine the Rh status of the cells (Rh- positive or Rh- negative ). It i s recommended to use two monoclonal anti-D se ra from two different manufacturers labeled as D1 and D2 , e specially to confirm all Rh negatives .

48. Monoclonal anti -D: Three types IgM anti-D monoclonal reagent . B lend of I g M and IgG monoclonal antibodies reagent . M onoclonal I g G anti -D. I g M antibodies are highly specific and saline reacting equally at both , room temperature and at 37 °C , but unr eliable for detection of weak D . B lended antibodies are no w routinely used and can be used for detecting w eak D.

49. Tube technique for Rh typing : The method of tube / slide technique for Rh typing is similar to ABO blood grouping . For all RhD negative test on blood Donor , Du test recommended .

50. Weak D: Inheritance of D genes which result in lowered densities of D antigen s on RBC membrane, gene codes for less D.

51. Method for weak D testing : Add 1 drop of 2- 5% suspension of D negative red cells to a test tube and add 2 drops of anti D ( b lend of Ig G + IgM ). I ncubat e at 37 °C for 30 minutes . W ashed three times with normal saline . M ake dry red full button and add poly specific AHG r eagent . Lo ok for agglutination . RESULTS : If there is agglutination Du positive . if there is no agglutination Du negative .

52. Significance of weak D: W eak D is much less antigenic in comparison to D, however , S u ch red cells may be destroyed if transfused to a patient already having anti-D. Hence weak D donor units are labeled as R h positive . T he weak D positive recipients are classified as R h negative and safety transf used with Rh negative blood .

53. ABO Grouping D iscrepancies :

54. ABO Grouping D iscrepancies : Can be due to:- Technical discrepancies Clinical discrepancies

55. Technical discrepancy : Clerical errors . Missing identification of blood specimen . Mixing of blood samples . C ontaminated reagents or not following manufacturer’s instructions . C ontamination or dirty glasswares .

56. Clinical discrepancies: Group 1 discrepancy Overall most common discrepancy . Mainly seen in Reverse grouping due to weak / missing antibodies . Commonly associated conditions are - Newborns - E derly patients. Resolution For newborns , only forward grouping is done till 4 months of age . T hese discrepancies can be solved b y enhancing the serum grouping reaction . T his can be achieved by - incubating the cell s serum mixture at low temperature (4°C for 15 - 30 minutes ) OR by prolonging incubation at room temperature ( 1/2 hr-1hr at 22°C)

57. Group 2 Discrepancy Due to missing or weak antigens Least common discrepancy overall The cause are: - Subgroups of A or B. Resolution Subgroup of A and B can be solved by -R epeating blood grouping by using w ashed cells -Us e of anti AB antisera and anti A1 Lectin

58. Group 3 discrepancy This is du e to proteins or plasma abnormalities . The causes are : - Elevated levels plasma globulins as seen in Multiple Myeloma, Waldenstrom’s Macroglobulinemia and Hodgkin’s Lymphoma . -E levated level of fibrinogen . -Us e of Plasma expanders such as Dextran. - Wharton’s jelly i n cord blood samples. Resolution The main problem is due to Rouleaux formation , which is resolved by washing the cell with normal saline 6 to 8 times and confirming it with microscopic examination . If the serum /reverse grouping is affected performs salin e replacement technique .

59. Group 4 discrepancy Polyagglutination : this is due to exposure of h idden erythrocyte antigens (T antigen in bacterial or viral infections). P atient with cold auto anti bodies . A2 or A2B i ndividual with A1 antibodies. N aturally occurring or irregular antibodies reactive at room temperature . Resolution C old auto agglutination : - Warm saline wash (at 37 °C – 40 ° C ) of auto agglutinated cells - Pre warming of sera and reagent c ells at ( 37 °C).

60. Thankyou