Life's Blood

by M. Schroeder and M. Jensen

ABO DISCREPANCIES

DEFINITION:

Any deviation from the expected pattern of antigen on the cell and the opposite antibody in the serum .

What are their ABO types ? ? ? ? ? ? ?

ABO Discrepancies MUST BE RESOLVED

• In RECIPIENTS the discrepancies must be resolved before any blood component is transfused. If not resolved before blood is needed, transfuse Group O (O NEGATIVE if there is a discrepancy in the Rh type also).
• In DONORS the discrepancies must be resolved before any blood is labeled with a blood t;ype..

GENERAL RULES TO RESOLVE:

1. Always re-test first.
    
2. Check for clerical/technical errors
    
3. Weakest reaction is usually the one in doubt.
    
4. Check results of the screening cells.
    
5. Check the patient’s age.
    
6. Check the diagnosis
    
7. Check the transfusion history.

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KINDS OF DISCREPANCIES:

CLERICAL ERRORS (TRANSCRIPTION ERRORS)

Clerical errors are the most common. 

• If you do not record the results as you read each tube, you run the risk of recording incorrect results.  Always check which tube you are reading and record the results immediately. 
• Make sure you are recording the results on the right worksheet.  One way to prevent this error is to minimize the times you are working with more than one patient or donor at a time. 
• Recording results in the wrong spot on worksheet could occur when you put some of the serum results in the cell typing area or vice versa.  Be sure you have techniques that will prevent you from performing this error. (This is why our labeling procedure uses capital A and B for the forward type and a₁C and bC for the reverse typing)

TECHNICAL ERRORS

There are a number of technical errors that may also occur:

• Sample mix-up such as wrong serum tube with wrong clot or 3% suspension.
• Failure to add serum or reagent can lead to technical errors where no reaction is occurring where one is expected.  Remember for both ABO and Rh always add your reagent antisera and serum before adding cells.
• Addition of wrong reagent such as screening cells, which are O, instead of A₁ and B cells can lead to significant technical errors.
• Contaminated reagents could result in either false negative or false positive results depending on whether the reagent added neutralized or added to the reactivity of the original reagent.
• Under centrifugation could lead to a negative reaction since the cells are not encouraged adequately to bind with the antibody.  Over centrifugation can lead to you reading the reaction as positive while there is still a button on the bottom of the tube or your shaking to dislodge the button broke up the agglutination reaction.
• Warming the test could result in a false negative reaction since ABO antibodies are IgMs that react better in the cold.
• Too many cells in your cell suspension can lead to decreased or negative reactions since there are too many cells for the number of antibodies present in the reagents.  Remember we want to be in the zone of equivalence for our reactions.
• Failure to detect weak results can occur if you are not watching the reactions while you are shaking them out or if you shake too hard. 
• Failure to detect hemolysis can be a definite problem.  Remember a positive reaction can be hemolysis as well as agglutination since the antigen-antibody reaction can bind complement.  When complement is bound it can lead to hemolysis that is also an indication of a positive reaction.
• Dirty glassware can cause the cells to artificially clump.

PROBLEMS WITH SERUM TESTING

So where is the problem if it is actually a true discrepancy between the ABO cell type and the ABO serum test?  Serum testing is more common than problems with cell typing.  This is either manifest as an extra antibody present or an expected antibody missing

PROBLEMS WITH RED CELL TESTING

There are a number of problems that can occur with the red cell testing including:

• Mixed-field agglutination
• Weak or missing antigens
• Unexpected antigen
• Polyagglutinable cells

The steps to follow to resolve this discrepancy is to:

1. Check birth date since newborns and the elderly are more likely to demonstrate this discrepancy.  Newborn antibodies are not present until at least 6 months.   DON'T ATTEMPT TO SERUM-CONFIRM NEWBORNS.  As individuals ages they  may also lose their ability to maintain their antibody levels.  Therefore, the very elderly have decreased antibody levels.
2. Check diagnosis since patient conditions such as:  Immune deficiencies,  Chemotherapy,  Radiation Therapy,  and  Bone marrow transplantation may explain the missing antibodies.

Resolution of Missing Antibodies:

1. Add two more drops of serum just in case you forgot to add them the first time and centrifuge.  If negative then incubate in cold (4-18^oC) 15-30 MINUTES
    
2. Include autocontrol to rule out interference from natural anti-I when incubating at (4-18^oC).
    
3. At 4^oC Anti-A and Anti-B enhanced since they are saline, cold-acting antibodies as seen in this example for an O individual.

4. Compare this with a 4^oC Auto-Anti-I enhanced would have a positive autocontrol as seen in the example below

5. Group A or Group B can serve as its own negative control.
    
6. 4^oC Anti-B enhanced is shown below:

7. 4^oC Anti-I enhanced on the other hand would have a positive autocontrol.

8. If anti-I enhanced along with anti-A or anti-B, can re-set up and incubate at 18^oC.  As seen in this example of 18^oC: Anti-B enhanced, anti-I nonreactive

Presence  Of Unexpected Anti-A

The presence of Anti-A₁ should be suspected when the antibody is reactive against the A cells but not the screening cells at immediate spin as seen in the example below.

Naturally anti-A₁ occurs in subgroups of A or are passively-transfused from Group O platelets and other blood products.

How to Resolve the Issue of Unexpected Anti-A:

1. Check recent transfusion history for group O products, (especially platelets) that would explain the presence of this antibody.
    
2. Test patient cells with lectin-A₁. Subgroups will be negative with this reagent but A₁cells will be positive.

   Lectin + A₁ CELL = 4+

   Lectin + A subgroups CELLS = 0
    

3. Test patient serum with three A₁ cells and three A₂ cells and if it is an anti-A₁  the following reactions will occur:

   Anti-A₁: SERUM + A₁ CELLS = +

   SERUM + A₂ CELLS = 0
   
   Anti-A₁ will react only with the A₁ cells but not with the A₂ cells
    

4.  In the case of passive Anti-A from Group O Platelets the reactions would be the following:

   SERUM + A₁ CELLS = +

   SERUM + A₂ CELLS = +
   
   In this case if the antibody is strong enough you may need to transfuse group O blood .

UNEXPECTED A OR B ANTIBODY WHEN THE IMMEDIATE SPIN ANTIBODY SCREENING IS POSITIVE

You may have a positive reaction with the reagent A₁ or B cell that is due to a room-temperature antibody reacting with an antigen other than A or B on the cells

How to Resolve the Issue of Unexpected Anti-A that is probably another antibody due to the results of the Antibody Screening:

1. Identify the antibody by performing an identification panel at room temperature.
2. Pre-warm away (use caution) the effect of this antibody by doing the reverse typing with prewarmed serum and reagent cells.
3. Type reagent A₁ or B cell for the corresponding antigen once the antibody is identified.

For example, if the patient had an anti-N that was showing up at room temperature according to the antibody identification process, you would then type for N on the reagent cells used for the reverse typing.  If anti-N is causing your problem, then the cells should have N antigen present.

ROULEAUX FORMATION GIVING UNEXPECTED AGGLUTINATION IN ALL SERUM TESTS

Rouleaux can give unexpected agglutination in all serum tests

Rouleaux may also give false positive cell typing if strong enough and cells are insufficiently washed.  This phenomenon is due to alteration in serum protein concentration such as:

• Multiple myeloma
• Macroglobulinemia
• Liver disease (decreased albumin)
• Also seen with volume expanders

Characteristics of rouleaux is that it:

• Looks like agglutination macroscopically
• Microscopically it appears as "stacks of coins"

How would you resolve rouleaux problems?

Do saline replacement technique:

1. Re-centrifuge the test tube.
2. Draw off serum without disturbing cell button
3. Add two drops of saline
4. Resuspend
5. Rouleax disperses in saline; TRUE AGGLUTINATION REMAINS

RESOLVING PROBLEMS WITH CELL TYPING

Mixed-field agglutination

• Mixed cell populations resulting from massive transfusion of another blood group such as an B individual receiving "O" red blood cell donor units since the transfusion center did not have enough B donor units.
• Bone marrow transplant patients may have both some of their original type of cells and the type of the bone marrow transplant.
• Weak subgroups of A₃ traditionally give a mixed field reaction.
• Rarely the condition called chimerism due to intrauterine exchange of erythrocyte precursors between twins or 2 fertilized eggs fuse into one individual.

You should try to find cause of mixed field agglutination before setting up blood to transfuse so be sure to check the patient's transfusion records and clinical history.  If it appears to be a weak subgroup performed the tests discussed under Unexpected Anti-A

Weak or missing antigen

Weak, or missing, antigen may be due to v ery weak subgroup of A or B, loss of transferase in acute leukemia, massive transfusion of GROUP O, or bone marrow transplant 

How would you resolve a weak, or missing, antigen?

• Obtain recent transfusion history and any clinical history of bone marrow transplant
• Read forward grouping microscopically
• Use anti-A,B and incubate at 4-22^oC at least 15 minutes
• Use monoclonal antisera that is known to react with antigens like A_x and B_x
• Perform specialized tests if the above steps do not resolve the problem:

Specialized tests would include absorption/elution techniques and saliva studies.

Acquired B antigen

Acquired B antigens are seen in problems with the colon or infections with Gram-negative rods

Bacterial enzymes modify the "A" antigen to a "B" antigen and the patient forward types as an AB but reverses as an A. 

How would you resolve a possible acquired B antigen?

• Set-up an autocontrol.  The patient's own anti-B will not agglutinate their own AB cells.
• Check clinical history to evidence of colon problems or Gram-negative rods.
• Check monoclonal anti-B product inserts since some will not react with B acquired antisera
• Acidify some reagents anti-B to pH 6 and re-test.  Modified (acquired) B antigens will not react in the acidified antiserum, normal B antigens will still react

Polyagglutinable cells

Most monoclonal anti-A and anti-B will show problems with polyagglutinable cells if it is a problem with the cell membrane that leads to the agglutination.  The most likely causes of  due to Wharton's Jelly, found in cord blood, and strong positive direct antiglobulin test due to a cold agglutinin.  In the case of the strong positive DAT, it would appear to be an AB in the forward type and an O on reverse.

1. WHARTON'S JELLY

• Coats newborn cord cells and the child's type may appear AB.  You do not do a reverse on newborn blood since they have not made any anti-A or anit-B yet.
• If the baby types as an AB  recheck by washing cells several times and re-testing since you need to make sure you have removed the Wharton's Jelly and the baby is truly an AB.  Better yet ALWAYS WASH CORD BLOOD AT LEAST 4 TO 5 X'S BEFORE DETERMINING THE TYPE OF THE BABY.

2. Strong positive DAT

• May be seen in cold auto-immune hemolytic anemia
• If due to cold agglutinin, wash several times in warm saline and re-test
• Cells washed 3X at 37^oC would probably look like this:

PROBLEMS WITH BOTH CELLS AND SERUM

Strong cold auto-agglutinins

A strong cold auto-agglutinin is most often due to strong auto-anti-I. 

1. To resolve cell typing difficulties:

• Wash cells 3-4X with warm (37^oC) saline
• Re-test warm-washed cells

2. To resolve serum typing difficulties:

• Perform serum testing at 37^oC (Use caution that weak isoagglutinins (anti-A and anti-B) are not missed using this technique)
• Autoabsorb cold agglutinins onto patient cells at 4^oC.

OBJECTIVES - ABO DISCREPANCIES

1. Describe how an ABO discrepancy would be recognized.
2. Explain what must be done if an ABO discrepancy cannot be resolved before the patient requires a transfusion.
3. Explain what must be done when a discrepancy arises in a blood donor.
4. List at least 6 technical or clerical errors that may cause ABO discrepancies.
5. Describe the reactions, list the clinical situations in which they may occur, and explain how to resolve each of the following causes of ABO discrepancies:
   *Decreased immunoglobulin levels
   *Weak subgroups of A with anti-A₁
   *Passively transfused anti-A₁
   *Unexpected alloantibody reacting at room temperature
   *Loss of A or B antigen
   *Acquired B antigen
   *Rouleaux
   *Cold agglutinins
    
6. List at least three causes for mixed-field agglutination.

Performance objectives:

1. Recognize when an ABO discrepancy exists.
2. Given any sample of blood showing an ABO discrepancy, correctly identify and perform the necessary procedures to resolve the discrepancy.

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