4. Referring to Table 11.9, add 0.2 mL each of 2%

suspension of sensitized cells with anti-D (IgG)

dilutions in their respective tubes (vertical rows).

5. Mix well all the tubes.

6. Centrifuge the tubes at 3000 rpm for 20 seconds.

7. Gently dislodge the cell button and observe for

agglutination.

8. Chart the results as given in Table 11.8.

i. Add 0.2 mL neat AHG reagent.

ii. Similarly add dilutions of AHG in respective

tubes.

iii. Referring to Table 11.9, add 0.2 mL each of 2%

suspension of complement-coated cells to all

the tubes containing AHG reagent with dilutions.

iv Mix well all the tubes.

v. Centrifuge the tubes at 3000 rpm for 20 seconds.

vi. Gently dislodge the cell button and observe for

agglutination.

Preparation of Coomb’s Control Cells

Reagents and materials required:

1. Anti-D (IgG)

2. Freshly collected O Rho(D) positive cells

3. Normal saline

4. Test tubes 12 × 100 mm

5. Pipettes 1 mL and 5 mL

6. Table centrifuge, timer

7. Water bath or laboratory incubator.

Procedure

Since commercially available anti-D (IgG) reagent have

albumin titer of 256–512, diluting anti-D (IgG) reagent 1:40

to 1:50 in normal saline is enough to achieve sensitization

with O Rho(D) positive cells:

1. Take equal volume of 1:40 to 1:50 diluted anti-D (IgG)

in a 12 × 75 mm test tube and 3% cell suspension of O

Rho(D) positive cells.

2. Mix well and incubate at 37°C for 30 minutes. Periodic

mixing during 30 minutes interval ensures thorough

sensitization.

3. Remove the supernatant and resuspend the cell button

in 5 mL of normal saline.

4. Centrifuge at 3000 rpm for 2–3 minutes.

5. Repeat the washing (steps 3 and 4) at least four times.

6. Resuspend the cell button in normal saline to obtain

a 3% suspension of Coomb’s control cells.

ANTI-HUMAN IgG MONOSPECIFIC

COOMB’S REAGENT FOR DIRECT AND

INDIRECT ANTIGLOBULIN TEST

(Courtesy: Tulip’s Erybank Range)

Summary

Generally, antibodies involved in transfusion reactions

are of two types namely, the complete and incomplete,

whereas the complete antibodies agglutinate red cells

TABLE 11.8: Chequerboard titration

Dilution of AHG 2% cell suspension of complement coated Cells

N

1:2

1:4

1:8

1:16

1:32

Dilution of AHG 3% Rho (D) positive senstitized cells

N 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512

N

1:2

1:4

1:8

1:16

1:32

TABLE 11.9: Complement potency titration

354 Concise Book of Medical Laboratory Technology: Methods and Interpretations in saline medium, the incomplete type of antibodies

sensitizes red cells without agglutination.

In the direct antiglobulin tests, Anti-human globulin

reagent is used to detect antibodies adsorbed to the red

blood cells in vivo. After direct antiglobulin testing with

a polyspecific reagent reveals globulins, anti-human IgG

monospecific Coombs reagent is used to characterize the

coating proteins.

In the indirect antiglobulin tests, anti-human

globulin reagent is used to detect antibodies adsorbed

to red blood cells in vitro. Anti-human IgG monospecific

Coombs reagent is used in indirect antiglobulin testing

to distinguish patterns of reactivity in a single serum

containing complement-binding and non complementbinding antibodies.

Anti-human IgG monospecific Coombs reagent is

useful for antibody detection, antibody identification and

umbilical cord red blood testing.

Reagent

Erybank anti-human IgG monospecific Coomb’s reagent

is a ready-to-use reagent containing antibodies reactive

with human gamma globulins. Each batch of reagent

undergoes rigorous quality control at various stages of

manufacture for its specificity, avidity and titer.

Reagent Storage and Stability

1. Store the reagent at 2–8°C. Do not freeze.

2. The shelf life of the reagent is as per the expiry date

mentioned on the reagent vial label.

Principle

Normal human red blood cells, in presence of antibody

directed towards the antigen they possess, may fail to

agglutinate and become sensitized. This may be due to

the particular nature of antigen and antibody involved.

Erybank Anti-human IgG monospecific Coomb’s reagent

would react with red cells sensitized with gamma globulins

and cause agglutination of red blood cells.

Note

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. The reagent contains sodium azide 0.1% as preservative.

Avoid contact with skin and mucosa. On disposal flush

with large quantities of water.

3. Extreme turbidity may indicate microbial contamination or denaturation of protein due to thermal

damage. Such reagent should be discarded.

4. Erybank anti-human IgG monospecific Coomb’s

reagent is not from human source, hence, contamination

due to HBsAg and HIV is practically excluded.

Sample Collection and Storage

No special preparation of patient is required prior to

sample collection by approved techniques. Do not use

hemolyzed samples.

For Direct Antiglobulin Test

Blood drawn into EDTA is preferred but oxalated, citrated