Check the patient’s history for the presence of such disorders

7. False positive Coombs test is seen in blood with high

reticulocyte count

Check the history of the patient for high reticulocyte count

Blood Banking (Immunohematology) 379

Problem: False Negative Results

Possible causes Solutions

1. Insufficient washing of the sample may lead to neutralization

of anti-human globulin by the globulin fraction of the serum

Washing must be carried out thoroughly and for the number of times

as mentioned in the pack insert to ensure complete removal of free

IgG from the sample

2. Serum residues remaining in the poorly washed glassware

can cause neutralization of anti-human globulin

Only clean and dry glassware must be used for testing

3. Anti-human globulin reagent not working To all negative test results, after the antiglobulin phase Coombs

control cells should be added. If the Coombs control cells do not

agglutinate then the test should be repeated using fresh anti-human

globulin

4. Contaminated bovine serum albumin may inactivate

antihuman globulin

Ensure that the bovine serum albumin, saline and glassware are free

from contamination

5. Anti-human globulin may be neutralized with human globulin Check the Anti-IgG of the human globulin reagent using Coomb’s

control cells

6. Interrupted or delayed testing. Too much time has elapsed

between washing the erythrocytes and adding anti-human

globulin reagent, so that antibodies have eluted. The Coombs

serum has in fact reacted with the antibody but agglutination

is not visible, since the antibodies are no longer bound to the

erythrocytes

The washing should be undertaken as quickly as possible to minimize

the elution of antibody from the cells

Addition of the anti-human globulin followed by centrifugation and

reading of results should be in immediate succession

7. If plasma is used in the indirect antiglobulin test, the

complement dependant antibodies may not be detected due

to the absence of calcium

Do not use plasma. Serum not more than 48 hours old should be used

for the indirect antiglobulin test

8. Improper centrifugation Under centrifugation leads to false negative results. Resuspension

of centrifuged cells vigorously breaks up weak agglutination leading

to false negative results. Centrifugation should be carried out at the

appropriate speed for an amount of time as given in package insert

9. If the red cells are few the reaction is difficult to read Ensure that 5% suspension of red cells is used in the tests as per the

instructions given in the package insert

10. Omission of anti-human globulin in the test by mistake Ensure that all the reagents are added properly as per the instructions

given in the pack insert. Eryclone AHG offers the advantage of being

color coded so that it helps in identification

11. The incubation temperature was not the optimal one for

the antibodies. It was either too high or too low, so that the

antibody coating of the erythrocytes did not occur

Incubate at the optimal temperatures as per the instructions given in

the pack insert

12. Only one drop of anti-human globulin reagent may have been

used for the Du

 test. If one drop is used then residual anti-D

from previous incubation or excess wash solution may

neutralize/dilute the reagent affecting its reactivity and giving

rise to false negative reactions

Ensure that two drop of anti-human globulin reagent are used for the

Du

 test

13. Anti-human globulin reagent has deteriorated Confirm the results using Coomb’s control cells

14. Cord cells sensitized heavily with anti-D yield a false positive

result in direct antiglobulin test

Check the cells for sensitization before testing

380 Concise Book of Medical Laboratory Technology: Methods and Interpretations Anti-A1 Lectin

Problem: False positive results

Possible causes Solutions

1. Bacterial contamination (some

bacteria 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. Particles of dust, debris, chemicals

or detergents on the slide or

in the tube giving non-specific

agglutination

Clean and dry glassware should be used while carrying out the test

3. 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 one minute and not beyond as

drying may be interpreted as positive result

4. Excessive centrifugation in case of

tube test

Ensure that centrifugation is carried out for either one-minute at 1000 rpm 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 alibrate its equipment at regular intervals

Problem: Hemolysis of red blood cells

Possible causes Solutions

1. Use of wet slides and tubes Wet glassware can cause hemolysis of RBC’s. Ensure that only dry glassware is used for

testing

Problem: False negative results

Possible causes Solutions

1. Whole blood red cells used directly

for the slide test

10% red cell suspension should be used for the slide test as mentioned in the pack insert.

2. Storage of antiseras at higher/lower

temperatures than specified

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

Antibody activity decreases at lower temperatures

Do not freeze the reagents

3. Blood sample stored for too long Check the period of time for which the blood sample has been stored

Anticoagulated blood using various anticoagulants should be tested within the mentioned

time period (when stored at 2–8°C)

• EDTA or heparin—2 days

• Sodium oxalate or sodium citrate—14 days