1. Practical Blood Bank
Lab 2
ABO Discrepancies

2. ABO Discrepancy
Definition: When the results of the forward grouping (patient cells) do not correspond to the results of the reverse grouping (patient serum) or abnormal reactivity is present (i.e. Mixed Field):
Strength of reaction
Weak or missing
Additional reactions
Abnormal reactions

3. HINT
ABO forward and reverse reactions are typically very strong: 3+ to 4+. Weaker reactions should immediately send up red flags indicating that something is wrong.
Since production of ABO antigens is genetically controlled they are less vulnerable to problems than does the production of ABO antibodies.
Therefore we see more problems in which grouping: Forward or Reverse?

4. Patient Anti-A Anti-B A1-Cells B-Cells A 4+ 1+ B C D 3+B C D 3+
Patient A: Additional reaction with anti-B and patients cells.
Patient B: Weak reaction with patients serum and A1-cells.
Patient C: Additional reaction with patients serum and A1-cells.
Patient D: Missing reactions with patients serum A1-cells

5. Forward Grouping Problems

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

7. Mixed Field reactions Chimera: Two cell populations
Causes: Recent transfusion (O cells to an A patient), Bone marrow transplant
(Testing using Serum or plasma , suspended Patient RBCs)
Can cause non specific aggregation of RBC’s
Increased serum proteins: Multiple Myeloma patient
Contamination in cord blood sample: Wharton’s jelly
Infusion of macromolecular solutions: Dextran, etc.

8. Mixed Field Agglutination (Post transfusion)
~ (ABO Testing) Can be seen in A, B and AB individuals who have received O units. The antisera reacts with the patient’s RBCs, but not with the transfused O cells.
~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.

9. Weakened Antigen Expression
Potent cold reacting autoagglutinins
Antibody coats patient RBC and agglutinate spontaneously in the diluent
Subgroups of A or B
Some leukemia’s, Hodgkin's disease
Excess soluble A and B blood group substances
Carcinoma of the stomach and/or carcinoma of the pancreas

10. Missing or Weak antigens
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)

11. 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

12. Extra ABO antigens Acquired ‘B’ Antigen
Microbial deacetylating enzymes such as E. coli cleave off the N-Acetyl of the Group A N-acetyl-D-galactosamine immunodominant sugar. The remaining D-galactosamine becomes similar enough to the Group B D-galactose immunodominant sugar that it does react with reagent anti-B.
Secondary to bowel obstruction or carcinoma of the bowel

13. Polyagglutinable state
Exposure of ‘crypt’ or buried antigens (T, Tk, etc.) by inheritance or bacterial enzymes – RBC’s agglutinate with most sera.
Exposure of T, Tn and Tk (etc.) antigens. Antibodies to these antigens are present in virtually all human antisera. If using human source anti-A and anti-B these cells will agglutinate.

14. 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

15. 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)

16. Acquired B
Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….
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.

17. 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)

18. 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

19. 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

20. Reverse Grouping Problems

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

22. Unexpectedly Weakened Antibodies
Immunodeficient due to therapy or disease
Immunosuppressive drugs
Certain leukemia’s (CLL) or lymphoma’s (malignant lymphomas) have hypogammaglobulinemia (Little or no antibody production)
Age related
Very young: <6 months of age (Newborns)
Very old: >65 years of age (Weakened Abs Activity)
Dilutional Effect
Plasma Exchange, Transfusion, etc. dilutes out patient antibodies
Hypogammaglobulinemia: Often shows NO agglutination on reverse groupings

23. 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

24. 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

25. Extra Antibodies :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 with 2-ME will also disperse cells

26. 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

27. 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 (UMMC)

28. 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

29. 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+

30. 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)

31. Popular LAB CAUSES Of ABO Discrepancies
Poorly labeled specimen OR test tubes
Patient RBC suspension too heavy or light
Wrong specimen put in Patient’s labeled test tubes
Oh? Is hemolysis really a Pos. Rx’n?
Wrong results recorded on Pt. Form
Didn’t follow manufacturer’s instructions
Poor centrifugation: over or under!

32. Popular LAB CAUSES Of ABO Discrepancies
Didn’t add:
Patient Serum
Reagents
Correct Reagent
Reaction Reading:
Shaking tubes while looking elsewhere
Shaking tubes too hard
Shaking tubes too gently or not completely re- suspending cell button

33. When an ABO Discrepancy is encountered:
Results must be recorded, but interpretation of the ABO group must be delayed until the discrepancy is resolved…by you!
Begin follow up by getting an accurate patient history – age, medications, diagnosis, etc.
Repeat testing to rule out tech errors such as mislabeling, adding reagents, wrong patient sample, etc.

34. Resolving ABO Discrepancies
Repeat testing on the same sample…
Repeat testing using saline suspended and/or washed patient red blood cell’s. Saline Replacement.
From the beginning: re-label tubes, re-drop patient and reagent drops, etc.
Many labs make the patients red blood cell suspension with the patient’s serum/plasma. If the patient has increased plasma proteins it can cause non-specific red cell aggregation.

35. Weak or missing reactions?
Mislabeled or contaminated specimen:
Incubate test system at room temperature for minutes! Get patient history.
Redraw Patient!!
ALL of the above: any labeling error may account for the problem and needs to be redrawn.
Drawn above an IV?

36. Resolving ABO Discrepancies
Call the floor!!!
Get patient history.
Recent transplant: two cell populations
dilutional effect
Patient medication
etc.

37. Test patient cells with anti-A1 (Dolichos biflorus), anti-A,B or anti-H (Ulex europaeus)
Test patient serum with A1 or A2 cells
For suspected subgroups of A
Ditto!

38. Review Antibody Screening tests
Allo antibody or cold reactive allo or auto Ab
Incubate tests and controls for minutes room temperature
Can react with reagent A1 and B cells
Should strengthen weakened ABO antibody reactivity! WHY?

39. Anti-A
Anti-B
A1-Cells
B-Cells 3+ 1+
Problem: Reverse grouping - weakened patient antibody
Causes: Age related (>65, infant), immunosuppressed or immunocompromised,
Resolution: Incubate Room Temperature minutes and respin. Check Patient history.

40. Anti-A
Anti-B
A1-Cells
B-Cells 3+ 1+ 4+
Problem: 1+ Reaction with Anti-B. Appears to have additional antigens.
Causes: Acquired ‘B’ antigen.
Resolution: Patient history – bowel obstruction, carcinoma of the bowel. (E. coli deacetylation of the Group A antigen.)

41. Problem: Weak forward anti-A and 1+ reaction with A1 Cells.
Anti-B
A1-Cells
B-Cells 2+ 1+ 4+
Problem: Weak forward anti-A and 1+ reaction with A1 Cells.
Causes:
Subgroup of A – A2 with anti-A1.
Unexpected cold reacting antibody to antigen on reagent A1 cells.
Resolution:
Test patient cells with anti-A1 lectin and with patient serum test A2 cells
Antibody screen should demonstrate unexpected cold reacting antibody.

42. Let’s practice !

43. EXAMPLES of ABO Discrepancies and Possible Resolution
Forward:
Reverse:
Screening
Anti-A
Anti-B
A1 Cells
B Cells
Cells
Autocontrol:
Possible Causes
Possible Resolutions 1
Group O newborn; elderly patient; low immunoglobulin levels
Incubate tests at 4°C, check age of patient 2 4+ 2+
Rouleaux; cold autoantibody
Wash RBCs and repeat testing; test for cold antibodies 3 1+
Probable A2 subgroup with anti-A1
Test with anti-A1 and anti-H lectins and A2 cells 4 3+
Probable A2B subgroup with anti-A1 5
Probable Oh (Bombay)
Test with anti-H lectin; may sent to reference lab for confirmation 6
Probable acquired B phenotype
Investigate patient history; test with anti-B lectin if available 7
Probable alloantibody
Perform antibody identification (antibody panel) 8
Probable group B with cold autoantibody
Test for cold antibodies and identify if appropriate
Adapted from Table 3-11: Flynn, J. C. (1998). Essentials of Immunohematology. Philadelphia: W.B. Saunders Company.

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

45. 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:

46. 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:

47. 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:

48. Example 4
Anti-A,B
Patient RBC 1+
Problem:
Causes:
Resolution:

49. Example 5 Anti-A Anti-B A1 Cells B Cells 2+mf 3+ Problem: Causes:
2+mf 3+
Problem:
Causes:
Resolution:
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

50. 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 (we’ll talk later about Ab screens)
Problem:
Causes:
Resolution:

51. 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: