ABO Discrepancies & other

problems
cls.umc.edu/COURSES/CLS325/Week5/Discrepancies.ppt

Reneé Wilkins, PhD, MLS(ASCP)CM

CLS 325/435
School of Health Related Professions
University of Mississippi Medical Center
 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
 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
 Why?
• Reaction strengths could be weaker than
expected
• Some reactions may be missing in the reverse
or forward typings
• Extra reactions may occur
Patient Anti-A Anti-B A1 Cells B Cells

1 4+ 1+ 0 4+

2 0 4+ 1+ 0

3 4+ 4+ 1+ 0

4 0 3+ 0 0
 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
 Discrepancy ?
• If a real discrepancy is encountered, the
results must be recorded

• However, the interpretation is delayed until

the discrepancy is RESOLVED
Errors
 Technical Errors
• Clerical errors
– 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
 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
 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
 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
 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
ABO discrepancies
 ABO Discrepancies
Problems with RBCs
Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions
Problems with SERUM
Weak-reacting/Missing antibodies
Extra antibodies
 Grouping

Forward Reverse

Missing/Weak Extra Mixed Field Missing/Weak Extra

Young Cold
A/B Subgroup Acquired B O Transfusion Elderly
Immunocompromised Autoantibody

Disease Bone Marrow Cold

B(A) Phenotype
(cancer) Transplant Alloantibody

Rouleaux Rouleaux
May cause all + reactions

Anti-A1
Forward Grouping Problems
 Red Cell Problems
• Affect the forward grouping results
– Missing or weak antigens
– Extra antigens
– Mixed field reactions
 Forward Grouping:
Missing or Weak antigens
• ABO Subgroups
• Disease (leukemia, Hodgkin’s disease)

Anti-A Anti-B A1 Cells B Cells

0 0 0 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)
 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 may type as group O
• 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
 Forward Grouping:
Extra Antigens
• Acquired B
• B(A) phenotype
• Rouleaux
• Polyagglutination
• Wharton’s Jelly
Anti-A Anti-B A1 B
Cells Cells
4+ 1+ 0 4+

EXAMPLE
 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)
Problems with The Forward Grouping:

Extra ABO antigens

Acquired ‘B’ Antigen
a) 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.
1) Secondary to bowel obstruction or carcinoma of
the bowel
 Acquired B
• Bacteria (E. coli) have a deacetylating enzyme
that effects the A sugar….

Group A Acquired
individual B
Phenotype

N-acetyl galactosamine Galactosamine

now resembles
D-galactose (found
Bacterial enzyme in Group B)
removes acetyl group
 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)
 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
 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
 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 (sometimes B3)

Anti-A Anti-B A1 Cells B Cells

0 2+ mf 4+ 0
 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.
Reverse Grouping Problems
 Reverse Grouping
• Affect the reverse grouping results
– Missing or weak antibodies
– Extra antibodies
 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
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
 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
 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
 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
 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)

 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
 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 B
Cells Cells
4+ 0 2+ 4+
 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)
 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??
 Resolving ABO Discrepancies
• Get the patient’s history:
– age
– Recent transplant
– Recent transfusion
– Patient medications
– The list goes on….
Let’s practice !
 Example 1
Anti-A Anti-B A1 Cells B Cells

3+ 0 0 1+

Problem:
Causes:
Resolution:
 Example 2
Anti-A Anti-B A1 Cells B Cells

3+ 1+ 0 4+

Problem:
Causes:
Resolution:
 Example 3
Anti-A Anti-B A1 Cells B Cells

2+ 0+ 1+ 4+

Problem:
Causes:
Resolution:
 Example 4
Anti-A Anti-B A1 Cells B Cells

0 0 0 3+

Problem:
Causes:
Resolution:
 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)
 Example 5
Anti-A Anti-B A1 Cells B Cells

0 2+mf 3+ 0

Problem:
Causes:
Resolution:
 Example 6
Anti-A Anti-B A1 Cells B Cells

4+ 4+ 0 1+

Problem:
Causes:
Resolution:
 Example 7
Anti-A Anti-B A1 Cells B Cells

0 0 0 0

Problem:
Causes:
Resolution:
 Example 6

Screening Autocontrol Conclusion

Cells (AC)
(I and II)
Patient Pos Neg Cold
Serum 1 alloantibody
Patient Pos Pos Cold
Serum 2 autoantibody

• if alloantibody – antibody ID techniques

• if autoantibody – special procedures (minicold panel, prewarming
techniques); no prior transfusions. If they have had a recent transfusion, then
it could be an alloantibody.
 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. (2009). Basic & Applied Concepts of
Immunohematology. St. Louis, MO: Mosby, Inc.