ABO DISCREPANCIES & OTHER PROBLEMS
Nawsherwan SadiqHematopathologyCollege of MedicineHawler Medical university2012-2013
IMPORTANCE
It is important to recognize discrepant results and how to resolve them
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
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 contaminationRed
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
A/B Subgroup
Disease (cancer)
Acquired B
B(A) Phenotype
O Transfusion
Bone Marrow Transplant
YoungElderly
Immunocompromised
Cold Autoantibody
Anti-A1
Rouleaux
Cold Alloantibody
RouleauxMay cause all + reactions
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
Anti-A Anti-B A1 Cells B Cells
0 0 0 4+
ABO Subgroups Disease (leukemia, Hodgkin’s
disease)
• Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)
Group O Group A
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
FORWARD GROUPING:EXTRA ANTIGENS
Anti-A Anti-B A1 Cells
B Cells
4+ 1+ 0 4+
Acquired B B(A) phenotype Rouleaux Polyagglutination
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)
ACQUIRED B
Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….
Group A individual
N-acetyl galactosamine
Acquired B
Phenotype
Bacterial enzyme removes acetyl group
Galactosamine now resembles
D-galactose (found in Group B)
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
Anti-A Anti-B A1 Cells B Cells
0 2+ mf 4+ 0
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)
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 CellsAnti-A1 from
patient
+ A1 cells
+ A2 cells
AGGLUTINATION
NO AGGLUTINATION
RESOLVING ANTI-A1 DISCREPANCY
Anti-A Anti-B A1 Cells
B Cells
4+ 0 2+ 4+
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
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 Cells (I and II)
Autocontrol (AC)
Conclusion
Patient Serum 1
Pos Neg Cold alloantibody
Patient Serum 2
Pos Pos Cold 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.
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