53 GammaZyme-B Bromelin Solution™

(Example of one stage enzyme technique)

Principle

Proteolytic enzymes modify red cells. The effect is partly due to the removal of sialic acid from the membrane, which reduces the net surface charge causing a decrease in repulsion between antigen and antibody. A one-stage test involves incubation of the plasma to be tested, the enzyme, and the red cells to be treated. In a two-stage test, washed red cells are combined with the chosen enzyme; the enzyme is then removed by washing, the “new” treated cells are incubated with plasma. The one stage approach has certain disadvantages; which include enzyme inhibition due to the presence of enzyme inhibitors in plasma and due to the ability of proteolytic enzymes to cleave immunoglobulins. A two-stage technique is accordingly considered to be generally more sensitive than a one stage procedure, although somewhat less convenient to carry out. If a one-stage method is used, bromelin is the enzyme most often chosen, as it appears to be less susceptible to the inhibitory effects of human plasma.

The use of bromelin in a one-stage test procedure enhances the reactivity of some blood group antigens (ex. Rh, P, Lewis and Kidd), thereby improving accessibility of the corresponding antibodies. At the same time, some blood group antigens are inactivated by bromelin treatment (ex. M, N, Fya, Fyb, Xga), which diminishes the reactivity of the cells with the corresponding antibodies. These features can be applied to facilitate the detection of weak examples of certain antibodies, and to aid in the recognition of antibody mixtures by inhibiting the reactivity of certain specificities.

Specimen

Serum or plasma to be tested.

Reagents

1. Reagent red cells
2. Gammazyme-B Stable Bromelin Solution

Procedure

1. Add 2 drops of plasma to an appropriately labeled tube.
2. Add 1 drop of a 3% to 4% saline suspension of reagent red cells (panel cells). Washed Donor or patient red cells. Commercial reagent red cells can be used directly from the vial.
3. Add one drop (or 2 if enzymes are expired in student labs) of GammaZyme-B and mix well.
4. Incubate at 37°C for 10 minutes.
5. Centrifuge for 20 seconds, gently re-suspend the cells and observe for agglutination and hemolysis.
6. Grade and record results immediately while holding tubes in hand.
7. Wash the cells 4 times with saline.
8. Add 2 drops of AHG to the dry cell button.
9. Mix thoroughly but gently and centrifuge for 20 seconds.
10. Gently re-suspend the cell button and examine macroscopically.
11. Grade and record results immediately while holding tubes in hand.
12. Confirm validity of negative tests by adding CCs. Centrifuge, re-suspend, and read for agglutination; record results.

Interpretation

1. The presence of agglutination/hemolysis constitutes a positive test.
2. Microscopic examination is not recommended for routine use and is particularly inappropriate with enzyme-enhanced tests because false-positive reactions will often be detected.

Notes

1. Proper controls (auto) are essential in the performance of all laboratory procedures. In particular when testing serums by an enzyme test procedure, an autologous control using the patient’s own red cells is recommended as an aid in recognizing auto-antibodies, most of which are enhanced in their reactivity when an enzyme test procedure is applied. The auto control will also show if the cells have been “over treated with enzymes. If the auto is positive in enzymes but not with regular screening the enzyme panel results are not valid.
2. A dilute antibody should be run as a control as well with the procedure. It should be an antibody that shows enhanced reactivity in an enzyme test system. It should not be an antibody diluted in albumin, as it must be representative of the enzyme inhibitors present in fresh human serum that may diminish the effectiveness of the test.
3. An enzyme test procedure must not be used as the sole method of detecting unexpected antibodies as some blood group antigens are destroyed by enzymes (sometimes the destruction or inactivation of these antigens may be only partial)