or clotted whole blood may be used. The blood sample

Reactions of Common Erythrocyte Antibodies

Antibody Saline Albumin AHG Enzyme In Vitro Optimal C0

For Medium Medium Test Test Hemolysis 4 24 37

H, l M S F S S M S F

A, B, A, B M F F M S M S F

Lua M S F F N M S M

Lub S S M F N R S M

M, N M S S F N M M F

P1 M S S S F M S F

PP1Pk M M M M M S M M

Lea

, Leb M S S M S M S F

S, s S S M S N F S M

K, k, Jsa

 Jsb F S M F N F S M

C, D, E, c, e, S S M M N F S M

Fya

, Fyb F F M F N N F M

should be tested as soon as possible after collection and

should not be stored.

For Indirect Antiglobulin Test

Serum not more than 48 hours old should be used for

testing purpose.

Additional Material Required

For Direct Antiglobulin Test

Test tubes (12 × 75 mm), Pasteur pipettes, centrifuge,

isotonic saline, Coomb’s control cells, optical aid.

For Indirect Antiglobulin Test

Test tubes (12 × 75 mm), Pasteur pipettes, centrifuge,

incubator (37°C), isotonic saline, Erybank bovine serum

albumin, reagent red blood cells for antibody detection

and antibody identification, Coomb’s control cells, optical

aid.

Procedure

Bring reagent to room temperature before testing.

Direct Antiglobulin Test

1. Prepare a 5% suspension of red blood cells to be tested

in isotonic saline.

2. Pipette one drop of the cell suspension into a test tube.

3. Fill the tube with fresh isotonic saline and centrifuge

for 30 seconds at 3400 rpm (1000 g).

4. Decant and repeat this washing at least thrice.

5. Add two drops of Erybank anti-human IgG monospecific Coomb’s reagent and mix well.

6. Centrifuge for one minute at 1000 rpm (125 g) or for

20 seconds at 3400 rpm (1000 g).

7. Very gently, resuspend the cell button observing for

agglutination macroscopically.

8. To all negative results, add one drop of Coomb’s control

cells and observe for agglutination.

Indirect Antiglobulin Test for Antibody Identification

1. Prepare 5% suspension of reagent red blood cells to

be tested in isotonic saline.

2. Pipette two drops of serum to be tested in an

appropriately labeled test tube.

3. Pipette one drop of 5% reagent red blood cell

suspension and mix well.

4. If required, add two drops of Erybank bovine serum

albumin reagent and mix well and incubate at 37°C for

15 minutes.

5. If enhancement medium is not being used, incubate

the tube at 37°C for 30 minutes.

6. After incubation, wash the cells thoroughly with

isotonic saline for minimum three times. Decant

completely after last wash.

7. Add two drops of Erybank anti-human IgG

monospecific Coomb’s reagent into the test tube

containing the sedimented cells and mix well.

8. Centrifuge for one minute at 1000 rpm (125 g) or 20

seconds at 3400 rpm (1000 g).

9. Very gently, resuspend the cell button and observe for

agglutination macroscopically.

Interpretation of Results

Direct Antiglobulin Test

Agglutination of red blood cells is a positive result and

indicates presence of human IgG on the red blood cells.

No agglutination is a negative test result and indicates

absence of human IgG on red blood cells.

Indirect Antiglobulin Test

Agglutination of red blood cells is a positive result and

indicates presence of antibody against the antigen in the

serum under test.

No agglutination of red blood cells is a negative result

and indicates absence of antibody against the antigen in

the serum under test.

Remarks

1. To all negative test results, after the antiglobulin test,

one drop of Coomb’s control cells should be added. If

Coomb’s control cells do not agglutinate then the test

must be repeated.

2. Red blood cells showing a positive direct antiglobulin

test cannot be used for the indirect antiglobulin test.

3. It is recommended that anti-IgG activity of anti-human

IgG monospecific Coomb’s reagent be tested from

time to time preferably on a daily basis using Coomb’s

control cells as a positive control.

4. All glassware used in the test should be scrupulously

clean, dry and free from contamination with human

serum.

5. Contaminated bovine serum albumin, saline or

glassware may inactivate anti-human IgG monospecific Coomb’s reagent.

6. Use of various drugs and certain diseases (such as

megaloblastic anemia) are known to be associated

with a positive direct antiglobulin test.

7. Cord cells obtained from a newborn exhibiting

hemolytic disease of the newborn, especially due to

ABO incompatibility may give false negative results.

8. Erybank anti-human IgG monospecific Coomb’s

reagent is free from anti-T activity.

9. As undercentrifugation or overcentrifugation could

lead to erroneous results, it is recommended that each

laboratory calibrate its own equipment and determine

the time required for achieving the desired results.

356 Concise Book of Medical Laboratory Technology: Methods and Interpretations ANTIHUMAN GLOBULIN REAGENT FOR DIRECT

AND INDIRECT ANTIGLOBULIN TESTS

(Eryclone® from Tulip)

Summary

Generally antibodies involved in transfusion reactions are

of two types, namely, the complete and the incomplete,

whereas the complete antibodies agglutinate red cells in

saline medium, the incomplete type of antibody sensitizes

red cells without agglutination. Usually, IgM class of

antibodies and IgG1 and IgG3 type of IgG antibodies fix

complement. Cell lysis, in vivo is mediated through the

complement system and the complement component C3b

is further acted upon to produce C3d.

In the direct antiglobulin tests, anti-human globulin

reagent is used to detect antibodies adsorbed to the

red blood cells in vivo. In the indirect antiglobulin tests,

Anti-human globulin reagent is used to detect antibodies

adsorbed to the red blood cells in vitro. Anti-human

globulin reagent is useful for compatibility testing,

antibody detection, antibody identification, umbilical

cord red blood testing and detection of the Du variant of

the human red blood cell antigen D(Rho).

Reagents

Eryclone anti-human globulin is a balanced ready-touse blend of highly purified immunoglobulins. It contains

Anti-human IgG antibodies and antibodies reactive with

human complement components C3b and C3d. These

anti-complement antibodies are IgM class monoclonals

and they impart the necessary sensitivity to the reagent.

Each batch of reagent undergoes rigorous quality control

at various stages of manufacture for its specificity, avidity

and titer.

Reagent Storage and Stability

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

b. 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 the antigen and antibody involved.

Eryclone anti-human globulin reagent would react with

red cells sensitized with gammaglobulins or components

of human complement involved and cause agglutination

of the 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. Eryclone reagent are not from human sources, hence,

contamination due to HBsAg and HIV is practically

excluded.

Sample Collection and Storage

No special preparation of the 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, titrated

or dotted whole blood may be used. The blood sample

should be tested as soon as possible after collection and

should not be stored.

For Indirect Antiglobulin Test

Serum, not more than 48 hours old, should be used. Donor

units may be tested up to the end of their dating.