This document discusses blood group systems, specifically ABO and Rh blood groups. It provides details on: - The antigens found on red blood cell membranes that determine blood type - Landsteiner's discovery of the ABO blood group system in 1900 and the four main blood types (A, B, AB, and O) - The antigens and antibodies present in each blood type - Rh blood group system including the Rho(D) antigen and typing only for Rho(D) to determine Rh status - Techniques for blood typing including tube, slide, microplate, and newer gel/cassette methods - Interpreting and resolving discrepancies in blood typing results

1. Blood Group
Systems
(ABO &
Rh)
Dr. Topon Narzary, MD (Path)
Assistant Professor, Pathology,
DMC & H, Diphu

2. Human Blood Groups
• Red cell membranes have antigens (glycoproteins)
on their external surfaces
• These antigens are
- unique to the individual
- recognized as foreign if transfused into another
individual
- promote agglutination of red cells if combine with
antibody
- more than 30 such antigen systems discovered
• Presence or absence of these antigens is used to
classify blood groups
• Major blood groups – ABO & Rh
• Minor blood groups – Kell, Kidd, Duffy etc.

3. ABO Blood Groups
• The most well known and medically important blood
types are in the ABO group.
• They were discovered in 1900 by Karl Landsteiner.
• The ABO blood group consists of
- Two antigens (A & B) on the surface of the RBCs
- Two antibodies in the plasma (anti-A & anti-B)
• An individual with ABO blood may have various types
of antigens and spontaneously preformed antibodies.

4. Landsteiner's law : ‘The plasma contains natural
antibodies to A or B, if these antigens are absent
from the red cells of that person.’

5. Type A
 A person with Type A blood has A antigens on the surface of their
red blood cells.
 A-Type individual does not make antibodies against A antigens.
 A-Type individual makes antibodies against B antigens.
A
A
A
A

6. Type B
 A person with Type B blood has B antigens on the surface of
their red blood cells.
 If Type A blood is introduced into the bloodstream of a B
individual, the transfused red blood cells will be destroyed by
complement-mediated lysis.
B
B
B

7. Type O
 A person with Type O blood has no antigens on the surface of their
red blood cells.
 Type O individuals are known as Universal donors because Type O
blood is not subject to antibody destruction.
 Type O individuals can receive only Type O blood because they have
antibodies to A and B antigens.

8. Type AB
 Type AB individuals are Universal recipients
because their blood cells have both A and B
antigens.
 Type AB individuals can donate to only Type
AB individuals.
A
A
A B
B

9. Blood has pre-formed antibodies opposite Blood Type

10. Practical aspects of ABO grouping
• Routine ABO grouping must include both Cell & Serum
Testing as each test serves as a check on the other.
• Test should be done at room temperature or lower;
testing at 37oC weakens the reactions.
• Tubes, slides should be dry and labeled properly.
• Serum should always be added before adding cells.
• Results should be recorded immediately after
observation.
• Hemolysis is interpreted as positive result.

11. ABO Typing Techniques
Blood sample:
• Clearly labeled blood samples in sterile tubes (Plain & EDTA).
• Can be stored at 4oC & should be tested with in 48 hours.
• No signs of hemolysis should be there.
• If serum is not completely separated, centrifuge the tube at
1000-3000 rpm for 3 mins.
• Take patient’s cell in pre labeled tube, wash the cells thrice
with normal saline and make 2-5% suspension.
Techniques:
• Slide test
• Tube technique
• Diamed ID microtyping system
• Micro plate technique

12. Slide Testing
(NOT RECOMMENDED AS A ROUTINE)
- less sensitive
- not reliable for weakly reactive antigens and antibodies
- drying of reaction mixture can cause aggregation -
false positive
- used for emergency ABO Typing (in outdoor camps
where Centrifuge is not available)

13. Method:
• Put 1 drop of anti-A and 1 drop of anti- B separately on clean
pre labeled slide/ tile
• Add 1 drop of 40-50% suspension of test red cells or whole
blood to each drop of typing antisera
• Mix the cells and reagent using a clean stick, spread each
mixture evenly on the slide over an area of about 15 mm
diameter
• Leave the test for 2 mins. at room temp(20-24oC)
• Rotate the slide again & look for agglutination
• Record the results

14. Anti D
Anti B
Anti A

18. Anti-A
Anti-B
Anti-D

19. Anti-B
Anti-A
Anti-D

20. Tube Technique
Recommended method
• Allows longer incubation of antigen and antibody
mixture without drying
• Tubes can be centrifuged to enhance reaction
• Can detect weaker antigen / antibody
• Results can be read comfortably as there is no
drying
Cell grouping (Forward grouping)
Tests the patient’s red cells with known Anti-A & Anti-B to
determine the antigen expressed
Serum grouping (Reverse grouping)
Test the patient’s serum with known A & B cells to determine the
presence of antibody

21. Method
• Set up three rows of clean test tubes and label them
• Add 2 vol of anti- A & anti-B in the tubes labeled as A & B, respectively
• Add one vol of 2-5% suspension of test red cells in each tube
• Mix the contents of each tube by gentle shaking and leave at room temp
(20-24oC) for 5 mins.
• Centrifuge the tubes at 1000 rpm for 1 min
• Observe the supernatant fluid for agglutination against a well lighted
background
• Gently disperse the cell button and check for agglutination against a well
lighted background
• Record results immediately
Forward grouping
Patient’s RBC
Anti-A Anti-B Clump
A B

22. • Tests the patient’s serum/ plasma with known A
& B red cells to determine antibody produced
• Acts as a confirmation of the forward group
Method
• Label three clean test tubes as A cell, B cell &
O cell
• Add 2 vol of test serum in each tube
• Add one vol of 2-5% suspension of reagent red
cells in respective tubes
• Mix the contents of each tube by gentle
shaking and leave at room temp (20-24oC) for 5
mins.
• Centrifuge & record the results similarly as for
Cell grouping
Reverse grouping
Patient’s Serum
A cells B cells
Clump ?

23. Reaction of Cells
tested with
Reaction of Serum
tested against ABO
Group
% North
Indian
Pop.
% US
White
Pop.
Anti-A Anti-B A Cells B Cells
1 0 0 + + O 32 45
2 + 0 0 + A 22 40
3 0 + + 0 B 38 11
4 + + 0 0 AB 8 4
Interpretation of Forward & Reverse grouping

24. Grading of agglutination reaction
+4 Single clump of agglutination with no free cells
+3 Three or four individual clumps with few free cells
+2 Many fairly large clumps with few free cells
+1 Fine granular appearance visually, but definite small
clumps per low power field
+w 2 or 3 cells sticking together per low power field
0 All cells are free
+H Hemolysis (partial or total) must be interpreted as positive

25. 4+ 1+
3+ 2+ 0
Grading strength of agglutination

26. Forward grouping
/ Cell grouping
Rh grouping Reverse grouping
/ Sera grouping

27. Use of 5%
Cell
suspension
for Tube
grouping

28. Preparation of
5%
known
A cell, B cell
suspension
for
Serum grouping

29. Showing 4+ agglutination in Anti A

30. No agglutination in Anti B

31. Showing 4+ agglutination in Anti D1 & Anti D2

32. Showing 4+ agglutination in B Cells

33. Microplate method
• Is ideal for testing large number of blood
samples.
• There is significant saving in time, in the
cost of disposables and reagents.
• Microplates are intended to be disposable
however they can be reused after cleaning
them properly making sure that all foreign
proteins are removed.

34. Microplates

35. Adding of
sample in
the micro
plate

36. Reaction in the microplate after 1 hour incubation at
room temperature

37. Gel Technology
• Gels held in microtube
contained in a plastic
card.
• Each mictotubes contain
about 35ul Sephadex gel
prepared in a buffer
solution such as LISS or
Saline.
• 6 of these microtubes are
embedded in a plastic card.
Gel card

38. Gel Technology
 Instead of a test tube the
serum and cell reaction takes
place in a microtube.
 Gel matrix acts as a sieve
which separates the red cells
from the suspension media
during the centrifugation
phase.

39. Negative reaction results in
the formation of a pellet of
cells at the bottom of the
microtube whereas Positive
reaction shows
(agglutination) trapped on
top.

40. Cassettes
Barcode with all
Information’s about
the cassette
Expiry Date
Lot Number
Anti Sera containing in the
column

41. 4 +
0.5 +
3 +
2 +
1 +
Neg
Neg
Automatic Reading and Interpretation

42. Result Reading

43.  Glass beads function as a filter to trap agglutinated red
cells
 Non agglutinated red cells pass freely through the
column
Positive Reaction
Negative Reaction
Column Agglutination Technology (CAT)

44. Column Agglutination Technology

45. Problems in ABO Grouping
Discrepancy between Cell and Serum grouping may be
due to following reasons
 CLERICAL ERRORS
 IDENTIFICATION ERRORS
 IMPROPER TECHNIQUES
 RED CELL PROBLEMS
 Newborns – weak antigen expression
 SERUM PROBLEMS
 Multiple myeloma – rouleaux formation

46. Technical problems
 Glassware, Reagents, Equipment
 Dirty glassware, contaminated or outdated reagent,
temperature not proper
 Cell concentrations
 Too high or too low concentration
 Centrifugation
 Carelessness -
 Patient identification
 Sample identification
 Reading and recording results

47. Patient: X Age: 30 yrs Sex: Male
1 Routine grouping
Anti
-A
Anti
-B
Anti-
D
Infe
r
Ac Bc Oc Infer
2+ 0 4+ A+ 1+ 4+ 0 O
Possibilities
 Subgroup of A
 No transfusion history - Passively acquired ABO antibody r/o
2 Resolution (Lectins, A2 cells)
Anti-
A
Anti-B Anti-
A1
Infer A1c A2c Bc Oc Infer
2+ 0 0 A2 1+ 0 4+ O Anti-A1
3 Confirmation of weak subgroups
 Adsorption using A1 cells
 Eluate tested with 3 known A1, B and O cells
 Saliva inhibition studies

48.  Rule out Clerical error
 Rule out Technical error
 History – age, diagnosis, pregnancy, drug &
transfusion
 Repeat test using
 Washed RBC
 Antisera from different Lot no.
 Antisera from different manufacturer
Resolution of ABO Blood Group
discrepancies

49. Additional Tests:
 A2, O or cord cells if required may be used
 Anti AB antisera
 Lectins (anti A1, anti H)
 Increasing serum : cell ratio
 Increasing incubation time
 Decreasing incubation temperature
 Including auto control
 Saliva secretor status
 Adsorption elution test
Resolution of ABO Group discrepancies
….contd.

50. √
×
×
×
AB
√
√
×
×
B
√
×
√
×
A
√
√
√
√
O
AB
B
A
O
Groups
Recipient
Donor
ABO Blood Donor & Recipient Compatibility
[ Table below shows who can donate blood to whom, and
who can receive blood from whom ]
×
×
×

51. Rh Blood Group System
 Rho ( D )
 rh’ ( C )
 hr’ ( c )
 rh’’ ( E )
 hr’’ ( e )
Antigens

52. Rho (D) antigen
 A very potent antigen (50% may form antibody
to exposure)
 95% positive - Rh positive
 05% negative - Rh negative
 No allele found

53. Rh Antibodies
 Most antibodies react at 37o C and require a
Coomb’s procedure to demonstrate the reaction
 Some react at saline and room temperature
 Most are IgG
 None fix Complement
 All are important in HDN and HTR

54. Rh Typing
 Normal typing for Rh antigens only includes
typing for Rho (D).
 The result of this typing determines the Rh
status of the cells (Rh - positive or Rh -
negative).
 Some Rh typing sera is diluted in high protein
solutions and may require a negative control.

55. Thank You
Dr. Topon Narzary, MD (Path)
Assistant Professor, Pathology,
DMC & H, Diphu