ABO Discrepancies

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ABO Discrepancies other problems

• Reneé Wilkins, PhD, MLS(ASCP)CM
• CLS 325/435
• School of Health Related Professions
• University of Mississippi Medical Center

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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

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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

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Why?

• Reaction strengths could be weaker than expected
• Some reactions may be missing in the reverse or
  forward typings
• Extra reactions may occur

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(No Transcript)
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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

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Discrepancy ?

• If a real discrepancy is encountered, the results
  must be recorded
• However, the interpretation is delayed until the
  discrepancy is RESOLVED

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Errors
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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

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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

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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

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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

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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
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ABO discrepancies
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ABO Discrepancies

• Problems with RBCs
• Weak-reacting/Missing antigens
• Extra antigens
• Mixed field reactions
• Problems with SERUM
• Weak-reacting/Missing antibodies
• Extra antibodies

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May cause all reactions
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Forward Grouping Problems
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Red Cell Problems

• Affect the forward grouping results
• Missing or weak antigens
• Extra antigens
• Mixed field reactions

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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)

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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

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Forward GroupingExtra Antigens

• Acquired B
• B(A) phenotype
• Rouleaux
• Polyagglutination
• Whartons Jelly

EXAMPLE
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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)

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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
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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)

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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

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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

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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)

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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.

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Reverse Grouping Problems
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Reverse Grouping

• Affect the reverse grouping results
• Missing or weak antibodies
• Extra antibodies

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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

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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

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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

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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

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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

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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)

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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
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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

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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)

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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??

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Resolving ABO Discrepancies

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

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Lets practice !
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Example 1
Problem Causes Resolution
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Example 2
Problem Causes Resolution
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Example 3
Problem Causes Resolution
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Example 4
Problem Causes Resolution
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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)

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Example 5
Problem Causes Resolution
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Example 6
Problem Causes Resolution
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Example 7
Problem Causes Resolution
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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.

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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.