1. Preleukemic states and acute myeloblastic leukemia Check the clinical history of the patient
2. Acquired loss of antigens occur in healthy elderly
1. Transfusion of blood of a different group. (e.g. group O
to group A or more significantly group A to group O)
It is extremely important to verify the cause of mixed field agglutination. An
investigation of the patient’s history may help to verify the cause of mixed
2. Chimera (this is a condition where an individual
possesses more than one population of red cells)
This may be caused by exchange of blood cells during early fetal life of
nonidentical twins or may be artificially induced by compatible but not same
blood group. Check the history of the patient
3. Blood group antigens altered by bacterial enzymes.
Some group A individuals with intestinal obstruction,
carcinoma of the colon or rectum and other disorders
of the lower intestine acquire a B-like antigen
Check the history of the patient for the presence of any of these conditions
and perform reverse grouping in case of acquired B to confirm the blood
Problem: Delayed agglutination
1. Subgroups of A, other than A1 such as A2, A3, Ax, etc.
have weakly expressed A antigenic sites on the red
cells, hence these red cells give weak reaction with
anti-A reagent as compared to A1 cells
Check these cells with Anti-A1 lectin, these cells should not react with anti-A1
Graded reaction helps the clinician to differentiate between strong (A1) and
other weaker subgroups of A group
2. Reagents used immediately after removing from the
The reagent vial must be brought to room temperature prior to starting the
3. The antigen and antibody are not present in optimal
The sample volume and the reagent volume dispensed should be as per the
instructions given in the protocol
376 Concise Book of Medical Laboratory Technology: Methods and Interpretations Rh Typing
Problem: False positive results
1. Bacterial contamination (some bacteria will
agglutinate all red cell samples thereby giving
Check the reagents for turbidity. Extreme turbidity may indicate bacterial
contamination. Such contaminated reagents should not be used for testing
2. The antisera may be contaminated with
another antibody. There may have been
accidental mixing of antisera; this could be
Check the color of the cap on the antisera bottle. Also check the antisera with the blood
of a person whose blood group is known
3. Particles of dust, debris, chemicals or
detergents on the slide or in the tube giving
Clean and dry glassware should be used while carrying out the test
4. Peripheral drying or fibrin strands were
mistaken for agglutination in case of slide test
The test should not be carried out directly under the fan. The results should be read
within 2 minutes. The vial should be capped immediately after use
seconds at 3400 rpm. Centrifugation should be adequate to produce a cell button with
a clear supernatant but without packing the cells so tightly that they are difficult to
dislodge. Each laboratory must calibrate its equipment at regular intervals
6. Rouleaux formation in case of tube test may
be mistaken for agglutination. Rouleaux
formation is said to occur when the red
cells appear like a stack of coins. Rouleaux
formation may be caused by the following
Rouleaux formation can be distinguished from agglutination by adding 2 drops of
saline to the reaction mixture; if the clumping of cells dissolves then it indicates
Check the history of the patient for the conditions like multiple myeloma.
The suspension of the cells prepared should be as per the instructions in the package
Problem: False negative results
1. Storage of antiseras at higher/lower
Reagents should be stored at 2–8°C when non in use. Thermal damage due to faulty
storage may result in a loss of reactivity. Check the turbidity of the reagents; extreme
turbidity may be due to thermal damage. Do not freeze the reagents
2. Blood sample stored for too long than
Check the period of time for which the blood sample has been stored. Anticoagulated
blood using various anticoagulants should be tested within the below mentioned time
• Sodium oxalate or sodium citrate—14 days
3. Use of hemolyzed samples Check the samples before use. Do not sue hemolyzed samples.
4. Prozoning (zone of antibody excess) or
postzoning (zone of antigen excess)
The antigen and the antibody should be present in optimal concentrations of the
agglutination to be seen properly. Check the sample volume and the reagent volume
used. Both the sample and reagent volume should be equal in slide test and a 5%
suspension of cells should be used in tube test. Ensure that there are no air bubbles
while dispensing samples and reagents
Extreme turbidity may be due to microbial contamination
6. Blocking effect when the cells are coated, e.g.
in hemolytic disease of the newborn (HDN),
acquired immune hemolytic anemia (IHA)
Check the history of the patient of the presence of disorders in which the cells may be
Blood Banking (Immunohematology) 377
should be read again after 5 minutes, so that weak antigens are not overlooked
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