07/06/2023

TESTING OF
ABO & Rh
Blod Groups:
Discrepancies

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TESTING OF ABO BLOOD GROUP SYSTEM

O A AB B

DISCREPANCIES IN THE ABO SYSTEM

❑ ABO discrepancies occur when unexpected reactions are
obtained in the forward and/or reverse grouping.
❑ This can be due to problems in patients serum, RBC or both.
❑ The unexpected reaction(s) may be due to an extra positive
reaction or a weak or missing reaction in the forward and
reverse grouping.
❑ All ABO discrepancies must be resolved prior to reporting a
patient or donor ABO group.

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TECHNICAL ERRORS IN ABO PHENOTYPING

❑ These errors are defined as a failure to apply appropriate
technical judgment to adjust technique as needed to
produce the desired outcome from the performance of a
technical process. (www.labce.com)

Never forget to check the label!

Reagent should be added FIRST!

Do a quality control
testing!

TECHNICAL ERRORS IN ABO PHENOTYPING

It is important to make sure any and

all technical factors that may have
given rise to the ABO discrepancy are
reviewed and corrected.

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A B AB O
GENERAL RULE IN RESOLVING
ABO DISCREPANCY
1. Weaker reactions is always an indication of discrepancy!
2. If initial testing was performed using RBCs suspended in serum or plasma,
repeat testing of the same sample using a saline suspension of RBCs can
usually resolve the ABO discrepancy.
3. Rule out any possibilities of technical errors.
4. It is also essential to acquire information regarding the patient’s age, diagnosis,
transfusion history, medications, and history of pregnancy.
5. When a discrepancy is encountered, results must be recorded but interpretation
of the ABO type must be delayed until it is resolved.
6. If the specimen is from the donor unit, the unit must be quarantined!
7. If the discrepancy is identified on the recipient, it might be necessary to
transfuse group O compatible red cells.

ABO DISCREPANCIES: GROUP I

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ABO DISCREPANCIES: GROUP I

❑ Group I discrepancies are associated with unexpected reactions
in the reverse grouping due to weakly reacting or missing antibodies.
❑ One of the reasons for the missing or weak isoagglutinins is that the
patient has depressed antibody production or cannot produce the ABO
antibodies. Common populations with Group I Discrepancy:
1. Age (Newborn or Elderly)
2. Patients with Leukemia (CLL or Lymphoma)
3. Patients under immunosuppressive therapy (hypogammaglobulinemia)
4. Patients with congenital or acquired agammaglobulinemia or ID
5. Patients with bone marrow or hematopoietic progenitor stem cell
(HPC) transplants.
6. Patients whose existing ABO antibodies may have been diluted by
plasma transfusion or exchange transfusion.
7. ABO subgroups

ADVANCED CONCEPT: GROUP I DISCREPANCY

❑ Chimerism is defined as the presence of two cell populations in a single individual.

❑ Detecting a separate cell population may be easy or difficult, depending on what
percentage of cells of the minor population are present. Reaction is always mixed field.
❑ True chimerism occurs only in twins and is rarely found.
❑ If the patient or donor has no history of a twin, then the chimera may be due to dispermy
(two sperm fertilizing one egg) and indicates mosaicism.
❑ Artificial chimera are due to: (a) Blood transfusions, (b) Transplanted bone marrow or
hematopoietic progenitor stem cells (HPC) of a different ABO type, (c) Exchange
transfusions, (d) Fetal-maternal bleeding

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RESOLUTION OF GROUP I DISCREPANCY

1. Obtain the patient clinical information and history.
2. Enhance the reaction of weak & missing Ab through incubation.
a. Incubate he patient serum with A1 & B cells at RT for 15-30 mins.
b. If no reaction, incubate at 4OC for 15-30 mins.
c. Auto-control and O check cells should be performed. (detect presence of other
isoagglutinins such anti-I).
✓ Mixed-field agglutination may look like small to large agglutinates
with unagglutinated cells and can also appear as a “halo” or “puff of
smoke” of unagglutinated RBCs as the RBC button is dislodged from
the test tube bottom.
✓ Causes: receiving non-ABO-type specific RBCs, ABO subgroups
(A3), and bone marrow or hematopoietic stem cell transplantation.

A B AB O
ABO DISCREPANCIES: GROUP II

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ABO DISCREPANCIES: GROUP II

❑ Group II discrepancies are associated with unexpected reactions in
the forward grouping due to weakly reacting or missing antigens.
❑ Causes of discrepancies in this group:
1. Subgroups of A or B may be present
2. Leukemias may yield weakened A or B antigens and Hodgkin’s disease which
mimic the depression of antigens found in leukemia.
3. “acquired B” phenomenon (associated with disease of the digestive tract)
4. carcinoma in the stomach & pancreas: excess amounts of blood group
specific soluble (BGSS) substances present in the plasma
5. Antibodies to low-prevalence antigens in reagent anti-A or anti-B
can also cause weakly reactive or missing reactions
in RBC grouping.

ABO DISCREPANCIES: GROUP II

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RESOLUTION OF GROUP II DISCREPANCY

1. Obtain the patient clinical information and history.
2. Enhance the reaction of weak & missing Ag through incubation.
a. Incubate the patient RBC with antisera at RT for 15-30 mins.
b. If no reaction, incubate at 4OC for 15-30 mins.
c. Auto-control (-ve) and O check cells (-ve) should be performed.
3. Lower the pH of the ES4 (anti-B antisera)
4. RBCs can be pre-treated with enzymes or acetic anhydride.
5. Secretor studies can be performed to confirm O A AB B
presence of acquired antigens.
6. Washing the patient cells free of the BGSS
substances with saline.
7. Repeat forward typing and use anti-sera with a
different lot number.

ABO DISCREPANCIES: GROUP III

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ABO DISCREPANCIES: GROUP III

❑ Group III discrepancies between forward and reverse groupings are caused by
protein or plasma abnormalities and result in rouleaux formation or
pseudoagglutination.
❑ Discrepancy is attributable to the following:
1. Elevated levels of globulin: multiple myeloma, Waldenström’s
macroglobulinemia, other plasma cell dyscrasias, and certain moderately
advanced cases of Hodgkin’s lymphomas
2. Elevated levels of fibrinogen/plasma proteins
3. Plasma expanders, such as dextran and polyvinylpyrrolidone
4. Wharton’s jelly in cord blood samples
5. Rouleaux formation

RESOLUTION OF GROUP III DISCREPANCY

1. Obtain the patient clinical information and history.

2. Perform Saline Replacement Technique
3. Wash cord cells six to eight times with saline should
alleviate spontaneous rouleaux due to Wharton’s jelly.

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ABO DISCREPANCIES: GROUP IV

ABO DISCREPANCIES: GROUP IV

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ABO DISCREPANCIES: GROUP IV

❑Group IV discrepancies between forward and reverse
groupings are due to miscellaneous problems due to:
1. Cold reactive autoantibodies
2. Circulating RBCs of more than one ABO group due to RBC
transfusion or marrow/stem cell transplant
3. Unexpected ABO isoagglutinins (Anti-A1, A1B, & Anti-H)
4. Unexpected non-ABO alloantibodies
5. Antibodies against acriflavine
6. Inheriting three AB genes (cis AB gene)

RESOLUTION OF GROUP IV DISCREPANCY

1. The patient’s RBCs could be incubated at 37°C for a short period, then
washed with saline at 37°C three times and retyped.
2. Treat the patient’s RBCs with 0.01 M dithiothreitol (DTT) to disperse IgM-related
agglutination.
3. The reagent RBCs and serum can be warmed to 37°C for 10 to 15 minutes,
mixed, tested, and read at 37°C.
4. If the reverse typing is still negative, a cold autoabsorption (patient cells with
patient serum) could be performed to remove the cold autoantibody from the
serum. Serum
5. Serum grouping can be repeated using at least three examples of A1, A2, B cells,
O cells; and an autologous control.
6. The patient’s RBCs can be tested with Dolichos biflorus to confirm
the presence of the ABO subgroup.

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A B AB O
ABO DISCREPANCIES: GROUP II

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TECHNICAL OF Rh BLOOD GROUP SYSTEM

❑ High Protein Reagents & Controls
✓ Some Rh reagents used in slide, rapid tube or microplate test contains high
concentration of protein (20-24%) and other macromolecular additives. These are
prepared from pooled human sera.
✓ Prone to false-positive result, use according to manufacturers’ direction

❑ Low-Protein Reagents and Controls

✓ Most of Rh routine Rh reagents are low-protein monoclonal IgM antibodies.
✓ Prone to false-positive result but not as common as high-protein reagents.

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CLASSROOM ACTIVITY!!!

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