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ABO Discrepancies

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Title: ABO Discrepancies


1
ABO Discrepancies other problems
  • ReneĆ© Wilkins, PhD, MLS(ASCP)CM
  • CLS 325/435
  • School of Health Related Professions
  • University of Mississippi Medical Center

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 typings
  • Extra reactions may occur

5
(No Transcript)
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 patients 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
  • 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
  • Manufacturers directions not followed
  • RBC suspensions incorrect concentration
  • Cell buttons not resuspended before grading
    agglutination

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

11
Contaminated samples or reagents
  • Sample contamination
  • Microbial growth in tube
  • Reagent contamination
  • Bacterial growth causes cloudy or discolored
    appearancedo not use if you see this!
  • Reagents contaminated with other reagents (dont
    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
  • 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 GroupingMissing or Weak antigens
  • ABO Subgroups
  • Disease (leukemia, Hodgkins disease)

Group O
Group A
  • Since the forward and reverse dont 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 GroupingExtra Antigens
  • Acquired B
  • B(A) phenotype
  • Rouleaux
  • Polyagglutination
  • Whartons Jelly

EXAMPLE
22
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)

23
Acquired B
  • Bacteria (E. coli) have a deacetylating enzyme
    that effects the A sugar.

Acquired B Phenotype
Group A individual
N-acetyl galactosamine
Galactosamine now resembles D-galactose (found
in Group B)
Bacterial enzyme removes acetyl group
24
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)

25
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

26
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
  • Whartons 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

27
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)

28
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 patients 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.

29
Reverse Grouping Problems
30
Reverse Grouping
  • Affect the reverse grouping results
  • Missing or weak antibodies
  • Extra antibodies

31
Reverse GroupingMissing 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

32
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 4C 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

33
Reverse GroupingExtra 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

34
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

35
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 37C and no
    agglutination at AHG phase
  • Associated with
  • Multiple meloma
  • Waldenstroms macroglobulinemia (WM)
  • Hydroxyethyl starch (HES), dextran, etc

36
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)

37
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
38
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

39
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 (Bombays
    dont have H and will not react)

40
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??

41
Resolving ABO Discrepancies
  • Get the patients history
  • age
  • Recent transplant
  • Recent transfusion
  • Patient medications
  • The list goes on.

42
Lets practice !
43
Example 1
Problem Causes Resolution
44
Example 2
Problem Causes Resolution
45
Example 3
Problem Causes Resolution
46
Example 4
Problem Causes Resolution
47
Example 4
  • 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)

48
Example 5
Problem Causes Resolution
49
Example 6
Problem Causes Resolution
50
Example 7
Problem Causes Resolution
51
Example 6
  • 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.

52
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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