1.
ABO DISCREPENCIES
Dr. BIPIN B PALLATH
06/09/2016

2.
FORWARD BLOOD GROUPING
• Can be done by Slide method, tile method, or
tube method
• Slide or tile method is usually used in case of
emergencies or in camps
• The most accepted method for routine
grouping is tube method

3.
• Steps of tube method
1. Get the sample, see for any blood lysis
2. Separate Cell and Serum
3. Wash the Cell 3 times with normal saline and
clear the supernatant away.
4. Make 5% cell suspension by taking 19 drops
of normal saline and 1 drop of red cell.

4.
5. Take 4 test tubes and mark as Anti A, Anti B ,
Anti AB. And Control
6 Add one drop of corresponding antisera to
the test tubes and Normal saline to control
7.Add one drop of 5% Red cell suspension to
the above test tubes

5.
8. Mix it gently and wait for 10 min, and
centrifuge at 1000 rpm for 1 min
9. Then look for agglutination. Free cell
suspension indicates negative results.
10. Negative cell results should be conformed
with microscope.

6.
SERUM GROUPING
Reverse Grouping
• A, B, O cell reagents are prepared by pooling
three sample of each group
• Wash each with normal saline for 3 times
and make 5% solution of each by adding 19
drops of saline and 1 drop of red cell

7.
• Procedure
1. Take 3 test tubes and mark A, B, O
2. Place 2 drops of test serum to each test
tubes
3. Put one drop of A, B, O cells into
corresponding test tubes
4. Mix the test tubes and keep it in room
temperature for 10min

8.
5. Centrifuge at 1000 rpm for 1 min
Examine the tubes for haemolysis or
agglutination which indicates positive results

9.
Grading of results
• One solid aggregate - +4
• Several large aggregate of cells - +3
• Medium sized aggregates of cells - +2
• Small aggregates in reddish back ground - +1
• Microscopic aggregates only - +W
• No aggregate - 0

10.
DISCREPANCY
• 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

11.
MAY BE BECAUSE
• Reaction strengths could be weaker than
expected
• Some reactions may be missing in the
reverse or forward typing
• Extra reactions may occur

12.
THINGS TO 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
• Some times, there is a real discrepancy due to
problems with the patient’s red cells or serum

13.
ERRORS

14.
Technical Errors
• Clerical errors
–Mislabeled tubes
–Patient misidentification
–Inaccurate interpretations recorded
–Transcription error
–Computer entry error

15.
• Reagent or equipment problems
–Using expired reagents
–Using an uncalibrated centrifuge
–Contaminated or hemolysed
reagents
–Incorrect storage temperatures

16.
• Procedural errors
–Reagents not added
–Manufacturer’s directions not
followed
–RBC suspensions incorrect
concentration
–Cell buttons not resuspended
before grading agglutination

17.
CLOTTING DEFICIENCIES
• Serum that does not clot may be
due to:
–Low platelet counts
–Anticoagulant therapy (Heparin,
Aspirin, etc)
–Factor deficiencies

18.
• 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
• Tubes containing EDTA can be used

19.
Contaminated samples or reagents
• Sample contamination
– Microbial growth in tube
• Reagent contamination
– Bacterial growth causes cloudy or discolored
appearance, so do not use if you see this.
– Reagents contaminated with other reagents (don’t
touch side of tube when dispensing)

20.
Equipment problems
• Routine maintenance should be
performed on a regular basis (daily,
weekly, etc)
• Keep instruments like centrifuges,
thermometers, and timers calibrated
–Uncalibrated centrifuges can cause
false results

21.
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

22.
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