1. ABO Discrepancies & other problems
Prepared By
Ahmad Shihada Silmi Msc, FIBMS
Medical Technology Dept
Islamic University of Gaza

2. Importance
It is important for students to recognize discrepant results and how to (basically) resolve them
Remember, the ABO system is the most important blood group system in relation to transfusions
Misinterpreting ABO discrepancies could be life threatening to patients

3. Discrepancies
A discrepancy occurs when the red cell testing does NOT match the serum testing results
In other words, the forward does NOT match the reverse

4. Why? Reaction strengths could be weaker than expected
Some reactions may be missing in the reverse or forward typing
Extra reactions may occur

5. Patient
Anti-A
Anti-B
A1 Cells
B Cells 1 4+ 1+ 2 3 4 3+

6. What do you do? Identify the problem
Most of the time, the problem is technical
Mislabeled tube
Failure to add reagent
Either repeat test on same sample, request a new sample, or wash cells
Other times, there is a real discrepancy due to problems with the patient’s red cells or serum

7. Discrepancy ?
If a real discrepancy is encountered, the results must be recorded
However, the interpretation is delayed until the discrepancy is RESOLVED

8. Errors

9. Technical Errors Clerical errors Reagent or equipment problems
Mislabeled tubes
Patient misidentification
Inaccurate interpretations recorded
Transcription error
Computer entry error
Reagent or equipment problems
Using expired reagents
Using an uncalibrated centrifuge
Contaminated or hemolyzed reagents
Incorrect storage temperatures
Procedural errors
Reagents not added
Manufacturer’s directions not followed
RBC suspensions incorrect concentration
Cell buttons not resuspended before grading agglutination
Clerical errors can result in fatalities. Please develop a set order when performing tests. This will eliminate mix-ups with tubes and prevent serious errors in reporting results.

10. Clotting deficiencies
Serum that does not clot may be due to:
Low platelet counts
Anticoagulant therapy (Heparin, Aspirin, etc)
Factor deficiencies
Serum that does not clot completely before testing is prone to developing fibrin clots that may mimic agglutination
Thrombin can be added to serum to activate clot formation
OR, tubes containing EDTA can be used
Protamine sulfate can also be added to neutralize heparin in the serum.

11. Contaminated samples or reagents
Sample contamination
Microbial growth in tube
Reagent contamination
Bacterial growth causes cloudy or discolored appearance…do not use if you see this!
Reagents contaminated with other reagents (don’t touch side of tube when dispensing)
Saline should be changed regularly

12. Equipment problems
Routine maintenance should be performed on a regular basis (daily, weekly, etc)
Keep instruments like centrifuges, thermometers, and timers calibrated
Uncalibrated serofuges can cause false results

13. Hemolysis Detected in serum after centrifugation (red)
Important if not documented
Can result from:
Complement binding
Anti-A, anti-B, anti-H, and anti-Lea
Bacterial contamination
Red supernatant

14. ABO discrepancies

15. ABO Discrepancies Problems with RBCs Problems with serum
Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions
Problems with serum
Weak-reacting/Missing antibodies
Extra antibodies

16. May cause all + reactions

17. Forward Grouping Problems

18. Red Cell Problems Affect the forward grouping results
Missing or weak antigens
Extra antigens
Mixed field reactions

19. Forward Grouping: Missing or Weak antigens
ABO Subgroups
Disease (leukemia, Hodgkin’s disease)
Anti-A
Anti-B
A1 Cells
B Cells 4+
Group O
Group A
Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

20. Subgroups of A (or B)
Subgroups of A account for a small portion of the A population (B subgroups rarer)
These subgroups have less antigen sites on the surface of the red blood cell
As a result, they show weakened (or missing) reactions when tested with commercial antisera
Resolution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups

21. Forward Grouping: Extra Antigens
Acquired B
B(A) phenotype
Rouleaux
Polyagglutination
Wharton’s Jelly
Anti-A
Anti-B
A1 Cells
B Cells 4+ 1+
EXAMPLE

22. Acquired B Phenomenon
Cause: there are two causes of acquired B phenomenon:
In vivo, patients with bacterial infections and often cancer of the colon or rectum may develop a false B-like antigen.
The mechanism: The bacterial produce a deacetylase (enzyme) which chemically alters the terminal sugar of A antigens (N-acetyl-D-galactosamine) into D-galactosamine.
Because the terminal sugar of the B antigen is galactose, anti-B antisera will cross react with the B-like D-galactosamine antigen. Because of this, in vivo, only group A people can develop an acquired B-like antigen. The condition is transient and disappears when the infection is cured.

23. Acquired B
Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….
Acquired B Phenotype
Group A individual
Galactosamine results from the deacetylating reaction, resembling D-galactose (found in Group B individuals). This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still technically it is “extra”). Patients should receive Group A units. Acquired B usually goes away when the condition resolves.
N-acetyl galactosamine
Galactosamine now resembles D-galactose (found in Group B)
Bacterial enzyme removes acetyl group

24. Another mechanism
In vitro, blood specimens can get an acquired B-like antigen if they are bacterially contaminated. This is because the membranes of some bacteria (e.g., E. coli and P. vulgaris ) have determinants which are chemically similar to the B antigen.
In this case, anti-B antisera is actually reacting with the bacterial antigens which have attached to the red cells.
In vitro, both group O and group A cells can acquire the B-like antigen. Note: most examples of acquired B phenomenon detected in the blood bank happen in vivo to group A people only.

25. ES4 Anti-B antisera
The use of monoclonal ABO typing antisera (specifically an anti-B clone designated "ES4") initially caused an increase in acquired B phenomenon.
because
The ES4 monoclonals can detect even a small number of galactosamine molecules on red cells. However, the reactions are particularly sensitive to pH and can be reduced (not eliminated totally) if the pH is lowered, something that the manufacturers have done.

26. Typical reaction pattern
The reactions with anti-B are weaker than expected (e.g., 1+ or 2+). The patient's autocontrol is negative even though anti-B is present (patient is group A).
The patient's own anti-B will not recognize and agglutinate the B-like antigen, but everyone else's anti-B (including the typing sera) will.

27. Resolution of acquired B
Check the past records in case the patient is a known group A.
Check the diagnosis for bacterial infection (with or without Cancer of the colon or rectum).
Test the red cells with anti-B reagent acidified to pH 6.0
If using human polyclonal reagents, redo the ABO group using monoclonal anti-A and anti-B typing sera, which may resolve the problem.
If using monoclonal reagents, redo the ABO group using human polyclonal anti-A and anti-B typing sera.
Do autologous control it should give negative result.
Try secretor status studies (usually not necessary). If the patient is group A and a secretor, he will secrete A and H antigens only.

28. Implication in blood transfusion
Group A people (especially children with a small blood volume) who have acquired B phenomenon should receive group A washed red cells (or group O washed red cells).
The red cells should be washed to remove all traces of donor anti-B which can react with the patient's B-like antigens.

29. Acquired B Phenotype Limited mainly to Group A1 individuals with:
Lower GI tract disease
Cancer of colon/rectum
Intestinal obstruction
Gram negative septicemia (i.e. E. coli)

30. Resolving Acquired B Check patient diagnosis: Infection?
Some manufacturers produce anti-B reagent that does not react with acquired B
Test patients serum with their own RBCs
The patients own anti-B will not react with the acquired B antigen on their red cell (autologous testing)

31. B(A) phenotype Similar to acquired B
Patient is Group B with an apparent extra A antigen
The B gene transfers small amounts of the A sugar to the H antigen
Sometimes certain anti-A reagents will detect these trace amount of A antigen
Resolution: test with another anti-A reagent from another manufacturer

32. Other reasons for “extra” antigens
Polyagglutination – agglutination of RBCs with human antisera no matter what blood type
Due to bacterial infections
Expression of hidden T antigens react with antisera
Rouleaux – extra serum proteins
Wharton’s Jelly – gelatinous substance derived from connective tissue that is found in cord blood and may cause false agglutination (Remember: only forward typing is performed on cord blood)
Wash red cells or request new sample from heel, etc

33. Forward Grouping: Mixed Field Agglutination
Results from two different cell populations
Agglutinates are seen with a background of unagglutinated cells
All groups transfused with Group O cells
Bone marrow/stem cell recipients
A3 phenotype
Anti-A
Anti-B
A1 Cells
B Cells 2+ 4+

34. Mixed Field Agglutination

35. Reverse Grouping Problems

36. Reverse Grouping Affect the reverse grouping results
Missing or weak antibodies
Extra antibodies

37. Reverse Grouping: Missing or Weak antibodies
Newborns
Do not form antibodies until later
Elderly
Weakened antibody activity
Hypogammaglobulinemia
Little or no antibody production (i.e. immunocompromised)
Often shows NO agglutination on reverse groupings

38. Missing or Weak antibodies
Example
Anti-A
Anti-B
A1 cells
B cells
Tentative group #1 +4 - A #2
B or AB #3 O
#1: Patient is a newborn: Anti-A and anti-B are not present at birth and develop about 3-6 months of age. (Usually the reverse group is not done when grouping newborns.)
#2: Patient is very elderly: Anti-A and anti-B levels decrease in old age because levels of immunoglobulins decrease. Because the levels may only be decreased and not totally missing, further investigation can be done. (Note: It would be unusual for an elderly person to totally lack ABO antibodies in the absence of an immune disorder.)
#3: Patient has a- or hypogammaglobulinemia: Anti-A and anti-B will be weak or missing in patients with a gammaglobulinemia or hypogammaglobulinemia.

39. Resolution Check the age of the patient
Repeat the ABO group at 4°C [anti-A and anti-B react best at 4°C].
QC required: because all persons have a harmless auto-anti-I reactive at 4°C, include an autocontrol. (Auto-anti-I may agglutinate the A1 cells, the B cells, and the patient's own cells at 4°C.)
Check the diagnosis.
If undiagnosed, have gammaglobulin levels tested

40. Resolving Weak or Missing antibodies
Determine patients age, diagnosis
Incubate serum testing for 15 minutes (RT) to enhance antibody reactions
If negative, place serum testing at 4°C for 5 minutes with autologous control (a.k.a. Autocontrol, AC)
This is called a “mini-cold” panel and should enhance the reactivity of the antibodies

41. Reverse Grouping: Extra Antibodies
Cold antibodies (allo- or auto-)
Cold antibodies may include anti-I, H, M, N, P, Lewis
Rouleaux
Anti-A1 in an A2 or A2B individual

42. Cold antibodies
Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing
Alloantibodies are made against foreign red cells
Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination with red cells at room temperature and below. The autocontrol will be positive.
Resolution: warming tube to 37° and washing red cells can disperse agglutination; breaking the IgM bonds.

43. Rouleaux Can cause both extra antigens and extra antibodies
“stack of coins” appearance
May falsely appear as agglutination due to the increase of serum proteins (globulins)
Stronger at IS and weak reaction at 37°C and no agglutination at AHG phase
Associated with:
Multiple meloma
Waldenstrom’s macroglobulinemia (WM)
Hydroxyethyl starch (HES), dextran, etc
Agglutination at AHG should not occur because cells have been washed three times

45. Resolving Rouleaux Remove proteins!
If the forward grouping is affected, wash cells to remove protein and repeat test
If the reverse grouping is affected, perform saline replacement technique (more common)
Cells (reagent) and serum (patient) centrifuged to allow antigen and antibody to react (if present)
Serum is removed and replaced by an equal volume of saline (saline disperses cells)*
Tube is mixed, centrifuged, and reexamined for agglutination (macro and micro)
*some procedures suggest only 2 drops of saline.

46. Extra Antibodies Example Anti-A Anti-B A1 cells B cells
Tentative group #1 +4 - +1 A #2 +2 AB #3 B
Anti-A1 in A2 or A2B people: examples #1 and #2 illustrate the presence of anti-A1. The autocontrol (not shown) would be negative.
Irregular IgM Alloantibodies: All three examples could represent the presence of irregular IgM alloantibodies such as anti-M, -N, -Lea, -Leb, or -P1. The A1 cells (or B cells) may be agglutinating because they are positive for the corresponding antigen. The autocontrol (not shown) would be negative.

47. Example Anti-A Anti-B A1 cells B cells Tentative group #1 +4 - +1 A #2+2 AB #3 B
Rouleaux: providing both cells in the reverse grouping show agglutination (examples #1 and #3), the discrepancy could be due to Rouleaux. The autocontrol (not shown) would be positive.
Causes: Rouleaux is a type of false agglutination caused by an increase in serum globulins. This can occur in diseases such as multiple myeloma or macroglobulinemia or can be caused by infusion of macromolecular substances such as dextran or polyvinyl pyrollidone (PVP), which are used as blood volume expanders.
Autoanti-I: Many people have a harmless autoanti-I that is IgM and reacts best at 4°C. The harmless autoanti-I of most people will not react above 10°-15°C, but some people have an autoanti-I that can react at RT and cause unexpected agglutination in both cells of the reverse serum group (examples #1 and #3).

48. Anti-A1
Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody
A2 (or A2B) individuals have less antigen sites than A1 individuals
The antibody is a naturally occurring IgM
Reacts with A1 Cells, but not A2 Cells
+ A1 cells
AGGLUTINATION
Anti-A1 from patient
+ A2 cells
NO AGGLUTINATION

49. Resolving anti-A1 discrepancy
2 steps:
Typing patient RBCs with Anti-A1 lectin
Repeat reverse grouping with A2 Cells instead of A1 Cells
Both results should yield NO agglutination
Anti-A
Anti-B
A1 Cells
B Cells 4+ 2+

50. Resolution of discrepancies caused by anti-A1
First step:
We must determine if the person is group A1 or group A2. (If group A1, the discrepancy with the A1 cells is NOT due to anti-A1).
To do this, we antigen type the person's red cells with the anti-A1 lectin which is Dolichos biflorus . If the red cells agglutinate, the person is group A1. If the red cells do not agglutinate, the person is not group A1, and probably is group A2 assuming the red cells reacted strongly (3+ or 4+ with anti-A).

51. Resolution of discrepancies caused by anti-A1
Second step:
If the person appears to be group A2, we must prove that the extra antibody is anti-A1, and not some other IgM irregular antibody.
To do this we test the person's serum against a panel of 3 A1 cells and 3 A2 cells. If anti-A1 is present, only the A1 cells should agglutinate.

52. Resolution of discrepancies caused by anti-A1
NOTE:
3 A1 and 3 A2 cells are required in order to ensure that the antibody reacting is anti-A1 and not some other antibody.
With 3 cells of each group we can achieve a statistical probability of 95% that the right antibody has been identified.
For example, if only one A1 cell and one A2 cell were tested, by chance, another antibody like anti-M or anti-P1 could react with the A1 cells (if they were M+ or P1+), but not the A2 cells (if they were M- or P1-).

53. Others…
The Bombay phenotype (extremely RARE) results when hh is inherited
These individuals do not have any antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-H
Basically, NO forward reaction and POSITIVE reverse
Resolution: test with anti-H lectin (Bombay’s don’t have H and will not react)

54. Finding the problem… Forward type tests for the antigen (red cell)
Reverse type tests for the antibody (serum)
Identify what the patient types as in both Forward & Reverse Groupings
Is there a weaker than usual reaction?
Is it a missing, weak, or extra reaction??

55. Resolving ABO Discrepancies
Get the patient’s history:
age
Recent transplant
Recent transfusion
Patient medications
The list goes on….

56. Let’s practice !

57. Example 1 Anti-A Anti-B A1 Cells B Cells 3+ 1+ Problem: Causes:1+
Problem: Reverse grouping, weakened patient antibody
Causes: Age related or weakened immune system
Resolution: Incubate at Room Temperature minutes and respin. Check patient history.
Problem:
Causes:
Resolution:

58. Example 2 Anti-A Anti-B A1 Cells B Cells 3+ 1+ 4+ Problem: Causes:4+
Problem: 1+ reaction with anti-B. Appears to have additional antigens.
Causes: Acquired B antigen
Resolution: Patient history – bowel obstruction, carcinoma of colon/rectum. (E. coli)
Problem:
Causes:
Resolution:

59. Example 3 Anti-A Anti-B A1 Cells B Cells 2+ 0+ 1+ 4+ Problem: Causes:
Problem: Weak forward with anti-A and 1+ reaction with A1 cells
Causes: 1) Subgroup of A (A2 with anti-A1)
2) unexpected cold reacting antibody to antigen on reagent A1 cells
Resolution: 1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells
2) an unexpected cold antibody would be detected in the antibody screen
Problem:
Causes:
Resolution:

60. Example 4 Anti-A Anti-B A1 Cells B Cells 3+ Problem: Causes:3+
Problem: missing antigen in forward grouping. Patient appears as group A in reverse grouping
Causes: A subgroup
Resolution: extend incubation time because this may enhance the reaction. Test with a polyclonal or monoclonal blend of anti-A,B (may contain subgroup antigens)…..
Problem:
Causes:
Resolution:

61. Example 4 Anti-A,B Patient RBC 1+ Probably a subgroup of A (Ax)
if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)

62. Example 5 Anti-A Anti-B A1 Cells B Cells 2+mf 3+ Problem: Causes:
2+mf 3+
Problem: strength of anti-B is weaker than expected; reverse indicates a group B individual
Causes: Group B individual transfused with group O cells
Resolution: recent transfusion? Bone marrow/stem cell transplant? Find what ABO type the patient was prior to transfusion
Problem:
Causes:
Resolution:

63. Example 6 Anti-A Anti-B A1 Cells B Cells 4+ 1+ Problem: Causes:1+
Problem: Forward shows AB individual, Reverse shows weaker “extra” reaction with B cells (looks like a group A)
Causes: Possible cold allo- or autoantibody (patient may have an antibody to another blood group system; A1 and B cells may have the antigens to these antibodies) (allo: P, M, N, Lewis) (auto: I or IH)
Resolution: screen for antibodies using Screening Cells and an autocontrol
Problem:
Causes:
Resolution:

64. Example 7 Anti-A Anti-B A1 Cells B Cells Problem: Causes: Resolution:
Problem: Reverse grouping, missing patient antibody (probably group O with no antibodies)
Causes: Age related or weakened immune system
Resolution: Incubate at Room Temperature minutes and respin. Check patient history.
Problem:
Causes:
Resolution:

65. Screening Cells (I and II)
Example 8
Screening Cells (I and II)
Autocontrol (AC)
Conclusion
Patient Serum 1
Pos
Neg
Cold alloantibody
Patient Serum 2
Cold autoantibody
if alloantibody – antibody ID techniques
if autoantibody – special procedures (minicold panel, prewarming techniques

66. References
Rudmann, S. V. (2005). Textbook of Blood Banking and Transfusion Medicine (2nd Ed.). Philadelphia, PA: Elsevier Saunders.
Blaney, K. D. and Howard, P. R. (2000). Basic & Applied Concepts of Immunohematology. St. Louis, MO: Mosby, Inc.