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• Too heavy red cell concentration may mask weak

agglutination. Too light suspension may be difficult

to read

• Improper/insufficient serum-cell ratio

¾ Complement

• Rare antibodies, notably Anti-Jka

 and Anti-Jkb may

only be detected when polyspecific Anti-human

globulin reagent is used and active complement is

present

¾ Saline

• Low pH of saline solution can decrease sensitivity of

Anti-human globulin test. Optimal wash solution for

most antibodies is pH 7.0–7.2. It has been observed

that commercially available infusion saline/

saline stored in plastic containers can seriously

compromise the sensitivity of anti-human globulin

test. Saline stored in plastic containers and further

autoclaved leads to leaching of certain chemicals

which shifts the pH to the acidic side and impacts the

sensitivity of anti-human globulin test. Preferably,

use phosphate buffered saline as wash solution or

suspending medium

• Some antibodies may require saline to be at specific

temperature to retain antibody on red blood cell. Use

37°C or 4°C saline.

False Positive Results

¾ Particles or contaminants

• Dust or dirt in glassware may cause clumping of cells.

Fibrin or precipitates in test serum may similarly

produce cell clumps that mimic agglutination

¾ Improper procedure

• Overcentrifugation may pack cells so tightly that they

do not easily disperse and appear positive.

344 Concise Book of Medical Laboratory Technology: Methods and Interpretations • Centrifugation of test with polyethylene glycol or

positively charged polymers prior to washing may

create clumps that do not disperse

¾ Cells with positive DAT result

• Cells that are positive by DAT will also be positive

in any indirect antiglobulin test. In such cases,

antibodies should be eluted from the sensitized cells

¾ Complement

• Complement components, primarily C4, may

bind to cells from clots or from CPDA-1 donor

segments during storage at 4°C and occasionally at

higher temperature. For DATs, use red blood cells

anticoagulated with EDTA, ACD or CPD

• Samples collected in scratched glass tubes can lead

to spurious activation of complement

• Complement may attach to cells in specimens

collected from infusion lines used to administer

dextrose containing solutions. Strongest reactions

are seen when large bore needles are used or when

sample volume is less than 0.5 mL.

Coomb’s Control Cells/Complement Coated Cells

Coomb’s control cells should be used routinely in direct

and indirect anti-human globulin test. Coomb’s control

reagent is anti-D IgG sensitized, washed and made up to a

5% suspension. Coomb’s control cells are used for:

¾ Procedural validation of tests employing Coomb’s

reagent. Coomb’s control cells are added after

performing anti-human globulin test. To a negative

result after addition of Coomb’s control cells,

agglutination indicates that AHG was indeed added and

that it has not been neutralized.

¾ Functional validation of Coomb’s reagent. The

performance of Coomb’s reagent can be validated as a

quality control measure on routine basis.

Similarly, complement-coated cells can also be

prepared in vitro. Thus, complement-coated cells can also

be used for functional validation of Coomb’s reagent.

Now with commercially available red blood cell

stabilizing solution, the Coomb’s control cells and

complement-coated cells can be prepared in situ and

stored in cell stabilizing solution for long-term storage and

use.

Indirect Anti-human Globulin Test for the Detection

of Red Blood Cell Antibodies

Saline Phase Indirect Anti-human Globulin Test

Specimen

Serum or plasma may be used. Preferably, freshly collected

serum should be used.

Reagents

1. Normal saline

2. Polyspecific AHG or monospecific anti-human IgG

reagent

3. Coomb’s control cells

4. Donor cells/reagent red blood cells.

Procedure

1. To properly labeled test tubes add two drops of serum.

2. Add one drop of reagent red blood cells or donor red

blood cells as a 2 to 5% saline suspension to each tube

and mix well.

3. Centrifuge for 15 to 20 seconds at approximately 900

to 1000 g. Observe for hemolysis and/or agglutination.

Grade and record the results.

4. Incubate at 37°C for 30 to 60 minutes.

5. Centrifuge for 15 to 20 seconds at approximately 900 to

1000 g and observe for hemolysis and/or agglutination.

Grade and record the results.

6. Wash the red blood cells three or four times with saline

and completely decant after the final wash.

7. Add AHG reagent to the cell button according to the

manufacturer’s instructions. Mix well.

8. Centrifuge for 15 to 20 seconds at approximately 900

to 1000 g and observe for reaction. Grade and record

the results.

9. Confirm the validity of negative tests by adding

Coomb’s control cells.

Albumin Phase Indirect Anti-human Globulin Test

Specimen

Serum or plasma may be used. Preferably, freshly collected

serum should be used.

Reagents

1. Normal saline

2. Bovine albumin (22 or 30%)

3. Polyspecific AHG or monospecific anti-human IgG

reagent