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.
 Understanding 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).
 These 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 antigens and Rhesus 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 rodents
and apes such as a chimpanzees, bonobos and Gorillas.
 Determination of ABO 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.
 Landsteiner 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 groups are dominant over O blood group.
 A and B group genes are co-dominant.
 Each person has two copies of genes coding for their ABO blood group (one
maternal and one paternal in origin).

9. ABO and H antigen genetics:
 The presence or absence of the H, A, B antigens is controlled by the H and ABO
genes.
LOCATION:
 The presence or absence of the ABH antigens on the red blood cell membrane is
controlled by the H gene.
 The presence or 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 from 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
1. If the Se allele is inherited as SeSe or
Sese, the person is called a “secretor”.
-80% of the population are secretors.
1. 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.
 Absence of H, A and B antigens. No agglutination with anti-A and anti-B or anti-H
antisera.
 Presence of all 3 antibodies i.e. Anti-H, anti-A and anti-B in the serum.
 No A, B or H substances present in saliva.
 Incompatible 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.
 Naturally occurring as they do not need any antigenic stimulus.

19. The ABO blood group system
• On 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.
 Subgroups are the result of less effective enzymes. They 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).
- However, 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 2nd most important blood group system.
 Rh blood group system consists of 40 defined blood-group antigens,
among them there are six common types of Rh antigens.
 Each of which is called as Rh factor .
These 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 Rh antigens.
 Anyone 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 Blood Grouping:
 Safe Blood transfusion.
 Preventing hemolytic disease of newborn (Rh incompatibility in newborns).
 To solve the legal disputes related to paternity disputes .
 Medicolegal use .
 Susceptibility of various diseases .
Group O - duodenal carcinoma
Group A - Carcinoma of stomach, pancreas & salivary glands.
 Routine 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 into 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.
 Serum / antisera should always be added BEFORE adding cells.
 Results should be recorded immediately after the observation.
 Haemolysis is interpreted as positive result.

30. Blood sample for blood grouping:
BLOOD SAMPLE:
 Clearly labeled blood samples in sterile tubes ( Plane or EDTA).
 Test 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.
 No signs of hemolysis should be there.

31. Red cell suspensions 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
1.A clean and dry glass slide is divided into two
sections with a glass marking pencil. The
sections are labeled anti-A & anti-B to identify
the antisera.
2. Place 1 drop of anti-A serum and 1 drop of
anti-B serum in the centre of the corresponding
section of the slide. Antiserum must be taken
first to ensure that no reagent 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 :
-Positive(+): Little clumps of RBC are seen floating in a clear liquid.
-Negative(-) : Red cells 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.
 Tubes can be centrifuged to enhance
reaction.
 Can detect weaker antigen-antibody
reactions.
TEST TUBE METHOD

39. 2 Steps in ABO grouping
2.1 CELL GROUPING (forward
Grouping)
Tests the patients 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.
 Set 3 tubes, labelled as A, B, D .
 Add 2 drops of antisera (anti A, anti B and anti D ) in three different tubes.
 Add 1 drop of 2-5% cell suspension ( ratio 2 : 1 )
 Mix contents well and centrifuge at 1000-1500 rpm for 1 minute.
 Observe for hemolysis.
 Gently disperce cell button and check for agglutination.
 Confirm negative result under the microscope.

42. SERUM GROUPING (REVERSE GROUPING)
 Prepare 2-5% suspension of pooled cell A, B, O.
 Label 3 tubes A cell, B cells and O cells .
 Place 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)
 Centrifuge tube at 1500 rpm for 1 minute.
 Gently disperse for agglutination.
 Negative results check by microscope.

44. Microplate method:
 It is sensitive and ideal for testing large number of blood samples.
 Microplate is a polystyrene plate consisting of 96 micro wells of
either U- or V- shape.
 The principal is same as for agglutination in test tubes.
 More sensitive to detect weaker antigen-antibody reactions.
 Microplate can be incubated and centrifuged.
 Results can be photographed for archival storage.
 There is significant savings in time and in the cost of disposables
and reagents.
 Microplates can be adapted for automation.

45. 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.
 After addition of red cell to the top of the tube, haemagglutinates ( if formed )
are trapped at the top of the tube ( positive test ).
 Non-agglutinated cells pass through the gel and form a button at the bottom of
the tube (negative test).
 The method is standardized and has defined end point leading to better
interpretation of the results.
 The technology is suitable for automation.

46. Rh blood group system:

47. 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.
 Production of antibody to D require exposure to the antigen.
 The D antigen is very immunogenic, i.e. individuals exposed to it will very likely make an
antibody to it .
 For this reason all individuals are type for D, if negative must receive Rh (D) negative blood.

48. 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.
 Generally do not react at room temperature in saline.
 Most are IgG in nature therefore can cross the placenta.
 All are important in HDN and delayed HTR.

49. Rh typing:
 Normal typing for Rh antigens only includes typing of Rh(D).
 The result of this typing determine the Rh status of the cells (Rh-positive
or Rh-negative).
 It is recommended to use two monoclonal anti-D sera from two different
manufacturers labeled as D1 and D2 , especially to confirm all Rh
negatives.

50. Monoclonal anti-D:
Three types
1. IgM anti-D monoclonal reagent.
2. Blend of IgM and IgG monoclonal antibodies reagent.
3. Monoclonal IgG anti-D.
 IgM antibodies are highly specific and saline reacting equally at both, room
temperature and at 37°C ,but unreliable for detection of weak D.
 Blended antibodies are now routinely used and can be used for detecting
weak D.

51. 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.

52. Weak D:
 Inheritance of D genes which result in lowered densities of D
antigens on RBC membrane, gene codes for less D.

53. 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 ( blend of IgG + IgM ).
 Incubate at 37°C for 30 minutes.
 Washed three times with normal saline.
 Make dry red full button and add polyspecific AHG reagent.
 Look for agglutination.
RESULTS :
 If there is agglutination Du positive.
 if there is no agglutination Du negative.

54. Significance of weak D:
 Weak D is much less antigenic in comparison to D, however, Such red cells
may be destroyed if transfused to a patient already having anti-D. Hence weak D
donor units are labeled as Rh positive.
 The weak D positive recipients are classified as Rh negative and safety
transfused with Rh negative blood.

55. ABO Grouping Discrepancies:

56. ABO Grouping Discrepancies:
 Can be due to:-
1. Technical discrepancies
2. Clinical discrepancies

57. Technical discrepancy:
 Clerical errors.
 Missing identification of blood specimen.
 Mixing of blood samples.
 Contaminated reagents or not following manufacturer’s instructions.
 Contamination or dirty glasswares.

58. Clinical discrepancies:
Group 1 discrepancy
 Overall most common discrepancy.
 Mainly seen in Reverse grouping due
to weak/missing antibodies.
 Commonly associated conditions are
- Newborns
- Ederly patients.
Resolution
 For newborns, only forward grouping is done till 4
months of age.
 These discrepancies can be solved by enhancing the
serum grouping reaction.
 This can be achieved by
-incubating the cells 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)

59. 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
-Repeating blood grouping by using
washed cells
-Use of anti AB antisera and anti A1
Lectin

60. Group 3 discrepancy
 This is due to proteins or plasma abnormalities.
 The causes are:
-Elevated levels plasma globulins as seen in
Multiple Myeloma,
Waldenstrom’s Macroglobulinemia and
Hodgkin’s Lymphoma .
-Elevated level of fibrinogen.
-Use of Plasma expanders such as Dextran.
-Wharton’s jelly in 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 saline replacement
technique.

61. Group 4 discrepancy
 Polyagglutination: this is due to
exposure of hidden erythrocyte
antigens (T antigen in bacterial or viral
infections).
 Patient with cold auto antibodies.
 A2 or A2B individual with A1
antibodies.
 Naturally occurring or irregular
antibodies reactive at room temperature .
Resolution
 Cold auto agglutination:
-Warm saline wash(at 37°C – 40° C) of
auto agglutinated cells
-Pre warming of sera and reagent cells
at (37°C).

62. Thankyou