Antibody Detection

Agglutination or hemolysis indicates that the antibody

has reacted with the corresponding red blood cell antigen.

No agglutination or hemolysis indicates the absence of

corresponding antibody.

Remarks

1. As undercentrifugation or over-centrifugation 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.

2. Erroneous results may also occur due to improper red

blood cell concentration, improper incubation time or

temperature while performing the test.

3. The ionic strength of the test system is dependent

on the amount of serum used. Alteration of the ionic

strength of Tuliss procedure by addition of excess

human serum will increase the ionic strength and

decrease the sensitivity of the test system.

4. The performance of Tuliss reagent should be periodically evaluated with a known LISS enhanced antibody

and the corresponding antigen for positive result

and red cell lacking the corresponding antigen for

negative result.

5. To all negative test results after the antiglobulin test

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

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

then the test must be repeated.

6. Low ionic strength media have been used to enhance

many antigen-antibody reactions. However, not all

antibodies are reactive in a LISS test system. Some

weakly reactive IgM antibodies of ABO system may not

be detected in the system employing low ionic strength

media.

STABILIZED, ACTIVATED PAPAIN ENZYME

SOLUTION FOR SEROLOGICAL APPLICATIONS

(Liquipap from Tulip)

Summary

Enzyme treatment enhances the reactivity of red blood

cells with certain antibodies of Rh, Kidd, Lewis and

P systems. Certain clinically significant antibodies of Rh

and Kidd systems can be detected only with enzyme treated

cells. Traditionally, papain needs to be prepared fresh for

use and long-term storage at 20°C is recommended. This

leads to frequent reagent preparation, lot to lot variation

of and strict quality control to assess adequate and correct

performance. Stabilized papain solution overcomes this

limitation. Thus, an activated, stabilized papain enzyme

solution is useful in detecting clinically significant

antibodies for specific serological studies. Proteolytic

activity of papain destroys blood group antigens notably

M, N, S, Fya

 and Fyb

, a property which may be useful for the

identification and separation of mixed antibodies.

Reagent

Liquipap is a stabilized ready-to-use papain reagent useful

for serological applications such as antibody screening,

antibody detection and cross-match techniques.

Reagent Storage

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

The sialic acid molecules present in the red cell membrane

impart a net negative charge to the surface of the red cell.

Due to the negative charge a repulsive force exists between

two red blood cells, which is termed as the ‘zeta potential’.

Proteolytic enzymes, such as papain, reduce the red blood

cell surface charge by cleaving the sialic acid molecules

from the polysaccharide chains on the red blood cell

membrane. Also, the enzyme treatment causes spicule

formation on the red cell thereby exposing the red blood

cell antigens on the surface. This dual action of reduction

in the ‘zeta potential’ and exposure of the red blood cell

antigens on the surface enhances the agglutination

reaction.

Note

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

2. The reagent contains 0.1% sodium azide as a preservative. Avoid contamination with skin and mucosa. On

disposal flush with large quantities of water.

3. Do not freeze or expose the reagent to elevated

temperatures. After usage, immediately replace the

reagent vial back to 2–8°C.

4. Marked turbidity may indicate reagent deterioration

or contamination, such reagent should not be used.

Do not use the reagent beyond expiry date.

362 Concise Book of Medical Laboratory Technology: Methods and Interpretations Sample Collection and Preparation

No special preparation is required prior to sample

collection by approved techniques. Serum samples may

be stored at 2–8°C up to 3 hours if not tested immediately.

Do not use hemolyzed samples.

Red blood cells used for detecting antibodies should

preferably be fresh.

Additional Material Required

¾ Test tubes (10 × 75 mm), Pasteur pipettes, laboratory

centrifuge, incubator (37°C), isotonic saline/isotonic

buffered saline (Available from Tulip: Osmosol), donor

red blood cells and recipient serum for cross-match,

reagent red blood cells for antibody detection, optical

aid.

Procedure

Bring all the reagent to room temperature (25–30°C) before

testing.