4. Reagent red cells may be stored as whole blood with
CPD or ACD but more usually they are stored as
washed cells in a preservative solution. A modified
Alsever’s solution, with added inosine and with
antibiotics is commonly used permitting satisfactory
storage for at least 35 days at 4°C.
¾ Clotted whole blood should be tested within 14 days
¾ Anticoagulated blood using various anticoagulants
should be tested within specific time limits as follows:
• Sodium citrate or sodium oxalate: 14 days
¾ Prior to testing, check whether pipettes and other
instruments used are well calibrated and in proper
¾ The blood grouping reagents contain 0.1% sodium
azide as preservative. Avoid contact of the reagent
with skin and mucosa. On disposal flush with large
¾ Adhere to appropriate storage conditions of the reagent
as mentioned in the respective package inserts.
Common Causes of False Negative and
False Positive Results in ABO Testing
¾ Reagent or test serum not added to a tube
¾ Hemolysis not identified as a positive reaction
¾ Inappropriate ratio of serum (or reagent) to red cells
¾ Tests not centrifuged sufficiently
¾ Tests incubated at temperatures above 20–24°C
¾ Incorrect interpretation of test results.
¾ Use of contaminated reagents, red cells or saline
¾ Incorrect interpretation of results.
Interpretation of Agglutination Reactions
To avoid false readings and to standardize recording of
results, tests should be read with the aid of the microscope
TABLE 11.10: Interpretation of agglutination reactions
++++ One complete mass of agglutination, readily visible on the
slide before microscopic examination
+++ Large separate masses of agglutination, readily visible on
the slide before microscopic examination
++ Smaller agglutinates, still readily visible on the slide
before examination under the microscope
+ A granular appearance, just visible on the slide before
examination under the microscope. The microscope
reveals big clumps of agglutinates of more than 20 cells
(+) Smaller clumps (12–20) cells only detectable by use of
GW Good weak—clumps of 8–12 cells only detectable
W Weak—Weak reactions with uniform distribution of small
? Sticky—uneven distribution of cells, 2 or 3 sticking
together, more noticeable when the cells are “rolling” on
0 or - All cells free and evenly distributed. The 0 sign is
preferred since—can easily be altered to +
MF/NF Mixed filed or negative field—agglutinates in a field of free
cells, e.g. the type of reaction observed with mixed bloods
374 Concise Book of Medical Laboratory Technology: Methods and Interpretations ABO Grouping
Problem: False positive results
1. Bacterial contamination (some bacteria such as
Staphylococcus aureus will agglutinate all red cell
samples thereby giving false positive results)
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 due to the interchanging of
caps. For example, if either anti-A, anti-B or anti-A, B is
contaminated with anti-D as anti-D reagent is colorless
and contamination is not visually observed in such a
case the anti A or anti B or anti A, B reagent will give
false positive results with all Rh positive blood groups
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. If anti-A, anti-B
or anti-A, B vial is contaminated with anti D, then the anti-A, anti-B or
anti-A, B should be checked with Rh negative blood sample, if it gives no
agglutination thereby confirming anti-D contamination or else it
could be some other contamination
3. Particles of dust, debris, chemicals or detergents
on the slide or in the tube giving non-specific
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 vials should be
capped immediately after use. The results of the test should be read at
2 minutes not beyond as drying may be interpreted as positive result. Except
in case of anti-A1 lectin, the agglutination should be observed at one minute
or 20 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 interval its
Problem: False negative results
1. Storage of antisera at higher/lower temperatures than
Reagents should be stored at 2–8°C when not in use. Thermal damage due
to faulty storage may result in a loss of reactivity. Check the turbidity of the
reagents; extreme turbidity may also be due to thermal damage. Antibody
activity decreases at lower temperatures. Do not freeze the reagents
Anticoagulated blood using various anticoagulants should be tested within
the below mentioned time period (when stored at 2–8°C)
• Sodium oxalate or sodium citrate—14 days
3. Use of hemolyzed samples Check the samples before use. Do not use hemolyzed samples
4. Prozoning (zone of antibody excess) or postzoning
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
5. Outdated or contaminated reagent Check the expiry date of the reagent. Also check the reagent for
contamination. Extreme turbidity may be due to microbial contamination
Blood Banking (Immunohematology) 375
6. Blocking effect when the cells are coated, e.g. in
hemolytic disease of the newborn (HDN), acquired
Check the history of the patient for the presence of disorders in which case
the cells may be coated in vivo. This can be established by doing a DAT on
7. Weak antigens In the tube test, every tube with negative reaction should be centrifuged and
the results should be read again after 5 minutes, so that weak antigens are
8. Under centrifugation Centrifugation should be carried out for 1 minute at 1000 rpm or for 20
seconds at 3400 rpm as per recommended procedures to give enough time
9. Vigorous shaking for resuspension of cells after
Resuspend the cells slowly and gently after centrifugation. Each laboratory
must calibrate its equipment at regular intervals
10. In case of A1 lectin, A antigen is not fully expressed on
the red cells of newborns below one year of age
Check the age of the patient. If below 1 year repeat typing after child reaches
Problem: Hemolysis or red blood cells
1. Use of wet slides and tubes Wet glassware can cause hemolysis of RBC’s. Ensure that only dry
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