5. Mix well and centrifuge for one minute at 1000 rpm

(125 g) or 20 seconds at 3400 rpm (1000 g).

Blood Banking (Immunohematology) 325

6. Gently resuspend the cell button, observing for agglutination macroscopically.

Interpretation of Results

Slide and Tube Tests

Agglutination is a positive test result and indicates the

presence of H antigen. No agglutination is a negative test

result and indicates the absence of H antigen and the red

cells being of Bombay phenotype (Oh).

Tube Test (Secretor Status)

Agglutination of the red cells indicates that the anti-H has

not been neutralized and the patient is a non-secretor. No

agglutination of the red cells indicates the anti-H has been

neutralized and the patient is a secretor.

Remarks

1. Do not interpret peripheral drying or fibrin strands as

agglutination.

2. It is recommended that known negative and positive

cells must be included as controls with each test series.

3. As undercentrifugation or overcentrifugation could

lead to erroneous results, it is recommended that each

laboratory calibrate its own equipment and the time

required for achieving the desired results.

Other Requirements

1. Normal saline solution.

2. Bovine or human albumin (22% or 30%).

3. Reagent red blood cells: Suspensions of red cells in

which presence or absence of significant blood group

antigens has been determined may be collected

periodically from suitable donors or be purchased

commercially wherever available.

4. Enzymes: Bromelin, ficin, papain, trypsin.

PHYSIOLOGICAL SALINE SOLUTION FOR

SEROLOGICAL APPLICATIONS

(Sodium Chloride 0.9% w/v)

(Courtesy: Tulip’s Erybank Range)

Summary

In blood group serology, for detection of either antigens

or antibodies, physiological saline (0.85% to 0.9% w/v) is

being extensively used. However, it is important that the

physiological saline used should be compatible with red

blood cell membrane integrity.

Reagent

Erybank natrium chloride 0.9% w/v solution is standardized for its serological applications. Reagent contains 0.1%

sodium azide as preservative.

Principle

Red blood cell lysis or shrinkage is observed in case of

diluents used which contain very low salt concentration

or very high salt concentration respectively. So optimal

salt concentration is very essential to maintain the

red cell membrane integrity. Sodium chloride with

0.9% w/v concentration is observed as the optimal salt

concentration. It is routinely used as diluent for serological

applications.

Note

1. Store the reagent at RT, it also can be stored at 4–8°C.

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

3. Avoid contact with skin and mucosa. On disposal, flush

with large quantities of water.

Uses

1. The reagent is used for the suspension of red blood

cells.

2. The reagent is used as a diluent for the antibodies.

3. Washing of red blood cells.

4. Making dilutions of reagent or samples for testing.

BOVINE SERUM ALBUMIN 22% SOLUTION FOR

SEROLOGICAL APPLICATIONS

(Courtesy: Tulip’s Erybank Range)

Summary

Bovine serum albumin is mainly used to enhance the

reactivity of blood grouping and typing antibodies in

direct agglutination tests. bovine albumin also enhances

the reactivity and sensitivity of indirect antiglobulin test

which is used for compatibility testing, antibody screening, identification and titration.

Reagent

Erybank bovine serum albumin is manufactured from

selected raw bovine serum, its protein concentration

and pH adjusted to 22% and 7.1 (± 0.1) respectively. Its

conductivity is controlled specifically for serological

applications.

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

Agglutination of antibody coated red cells depends

upon the class and type of antibody involved and

the characteristics of the reaction medium such as

326 Concise Book of Medical Laboratory Technology: Methods and Interpretations ionic strength and pH. Incomplete antibodies of IgG

class, especially those with Rh specificity, agglutinate

red cells if the zeta potential between the red cells is

adjusted by addition of colloids and salts such as bovine

serum albumin (BSA). Addition of BSA enhances such

immunological reactions and increases test sensitivity.

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.

Sample Collection and Preparation

No special preparation of the patient is required prior

to sample collection by approved techniques. Samples

should be stored at 2-8°C if not tested immediately. Do not

use hemolyzed samples. Donor units can be tested up to

the end of their dating. For the indirect antiglobulin test,

serum from fresh clotted whole blood should be used.

Additional Material Required for Compatibility

Testing

Test tubes (10 × 75 mm), 0.2 mL serological pipettes,

Pasteur pipettes, human red blood cells with specific

antigen reacting with the antibody to be titrated, centrifuge,

incubator, isotonic saline, anti-humanglobulin reagent

such as Eryclone anti-human globulin reagent, Coomb’s

control cells (Refer Eryclone anti-human globulin pack

insert), AB neutral human serum.

Broad-spectrum Compatibility Test

Major Crossmatch Procedure

Initial Phase

1. Label two test tubes as A (for albumin) and B (for

saline), depending upon the number of donors to be

cross matched, as many pairs of such labeled tubes

would be required.

2. Prepare a 5% suspension of the red cells to be tested

in isotonic saline.

3. Pipette two drops of recipient serum in both the

labeled test tubes.

4. Pipette one drop of donor red cells in both the labeled

test tubes and mix well.

5. Only to the albumin tube (A), add two drops of

Erybankbovine serum albumin reagent and mix well.

6. Centrifuge both the tubes for one minute at 1000 rpm

(125 g) or for 20 seconds at 3400 rpm (1000 g).

7. First observe for hemolysis. Resuspend the cell button

and observe for agglutination macroscopically.

8. Proceed to incubation phase.

Incubation Phase

1. Incubate the saline tube at room temperature and the

albumin tube at 37°C for 15 minutes.

2. First observe for hemolysis. Resuspend the cell button

and observe for agglutination macroscopically.

3. Proceed to the antiglobulin phase.