Antiglobulin Phase

1. Only the albumin tubes (A) are tested in the antiglobulin phase.

2. Wash the mixture of red blood cells and serum thoroughly with isotonic saline for a minimum of three times.

Decant completely after the last wash

3. Place two drops of Eryclone anti-human globulin

reagent into the test tube containing the sedimented

cells and mixwell.

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

seconds at 3400 rpm (1000 g).

5. Very gently, resuspend the cells and observe for

agglutination macroscopically.

Antibody Titration Test

1. a. Prepare a 5% suspension of red blood cells with

specific antigen reacting with antibody to be

titrated, in Erybank bovine serum albumin

reagent.

b. Also, prepare a 5% suspension of patient red cells

in Erybank bovine serum albumin reagent.

2. Label ten test tubes (1 to 10) and make progressive

dilutions of the patients serum as indicated below:

a. Pipette 0.1 mL of AB neutral serum into each test

tube except the first tube.

b. Pipette 0.1 mL of the patient serum into first two

tubes only.

c. After mixing the contents of the second tube

thoroughly, transfer 0.1 mL of the mixture to

the third tube. Continue the serial dilution by

transfer up to tube No. ten. Discard 0.1 mL of the

mixture from the last tube.

3. To tubes No. one through to nine, add one drop of

albumin suspended selected red blood cells, (as

prepared in point No. 1 (a) above) and mix well.

4. To tube No. ten, add one drop of patient’s red cells

suspended in albumin (as prepared in point No.1 (b)

above) and mix well.

5. lncubate all the tubes at 37°C for a minimum of 15

minutes.

Blood Banking (Immunohematology) 327

6. Centrifuge all the tubes for one minute at 1000 rpm or

20 seconds at 3400 rpm (1000 g).

7. Very gently, resuspend the cell buttons and observe

for agglutination macroscopically.

8. Antiglobulin test should be performed on all tubes

which do not show a very strong agglutination.

Interpretation of Results

Compatibility Test

In all phases of the compatibility test, if no agglutination or

hemolysis is observed, then the patient and the donor may

be considered compatible. If hemolysis or agglutination

at any point till the completion of the antiglobulin

phase is observed, the patient and donor are considered

incompatible.

Antibody Titration Test

The end point of the titration is the reciprocal of the

dilution in the last tube showing agglutination.

Remarks

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

the complement-dependent antibodies may not be

detected due to the absence of calcium.

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

phase, one drop of Coomb’s control cells should be

added. If the Coomb’s control cells do not agglutinate,

then the compatibility test must be repeated.

3. Red blood cells showing a positive direct antiglobulin

test should not be used for the indirect antiglobulin

test.

4. Bovine serum albumin will not bring about agglutination of red cells by all IgG blood grouping typing

antibodies.

5. As undercentrifugation or overcentrifugation can lead

to erroneous results, it is recommended that each

laboratory calibrates its own equipment and determine

the time required for achieving the desired results.

6. After usage, the reagent should be immediately

recapped and replaced at 2–8°C storage.

CONCENTRATED ISO-OSMOTIC

PHOSPHATE BUFFERED SALINE FOR

SEROLOGICAL APPLICATIONS

(Osmosol from Tulip)

Summary

The pH of reaction medium is an important factor in

antigen-antibody interaction. It has been observed that

irrigation/infusion saline solution of low pH, isotonic saline

autoclaved and stored in plastic containers could severely

compromise the sensitivity and specificity of antiglobulin

test when used as wash solutions/resuspension medium

in immunohematological procedures. Hence, careful

consideration should be given to the source, pH and storage

container of isotonic saline solutions intended for use in

immunohematological procedures. Usage of buffered

isotonic phosphate saline such as Osmosol maintains

the pH at 7.0–7.2 thereby improving the sensitivity and

specificity of tests employed in immunohematological

procedures.

Reagent

Osmosol is a concentrated 20X buffered isotonic

phosphate saline useful for preparing iso-osmotic saline

preparation, especially for immunohematological use.

Inclusion of sodium azide in the final formulation prevents

contamination during use.

Reagent Storage and Stability

Store the reagent at room temperature. Once opened,

store at 2–8°C.

The shelf-life of the concentrated Osmosol reagent is as

per the expiry date mentioned on the reagent vial label.

Upon dilution, the isotonic buffered saline solution so

obtained is stable for at least a month provided it is not

contaminated during use.

Principle

Osmosol with osmolarity similar to blood serum or

plasma, incorporating phosphate buffer maintains red

blood cell membrane integrity and optimum pH of the

reaction medium for antigen-antibody reaction during

immunohematological tests thereby improving the

sensitivity and specificity of the test.

Note

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

2. The reagent contains 0.2% sodium azide as preservative. Avoid contact with skin and mucosa. On

disposal flush with large quantities of water.

3. Extreme turbidity may indicate contamination. Such

reagent must be discarded.

Additional Material Required

Distilled water for blood bank use, pH paper capable for

reading pH at 6.5–7.5 or pH meter, sterile and scrupulously

clean glasswares for preparing the isotonic buffered saline

solution.

328 Concise Book of Medical Laboratory Technology: Methods and Interpretations Method of Preparation

1. Invert the contents of Osmosol vial into a scrupulously

clean, sterile glass jar/bottle. Make the volume to

500 mL with distilled water. Gently mix the contents.

Alternatively, if lesser quantity of isotonic buffered

saline is required then dilute 1 part of concentrated

Osmosol with 19 parts of distilled water. The glasswares

used for preparing the isotonic buffered saline should

be sterile and scrupulously clean.

2. Check the pH of isotonic buffered saline. The pH of the

isotonic buffered saline should be in the range 6.9–7.2.

3. The isotonic buffered saline so obtained is readyto-use for washing and preparing red blood cell

suspension, dilution medium for antibodies in

serological applications.