Antibody identification | scientistlaboratory

Antibody Identification (Panoscreen)

OBJECTIVES:
1) To screen antibody in patient’s serum.
2) To identify any unexpected antibodies in the patient’s serum.
3) This is needed for transfusion purposes and is an important component of compatibility testing

PROCEDURE:
A) Room Temperature (Immediate Spin) Phase
1. Label ten test tubes as I, II, III until X.
2. Add 2 drops of patient serum in each tube and 1 drop of screeing cell according to the label of tube. Example, panoscreen I in tube label I.
3. Mix and centrifuge for 15 seconds at 3400 rpm.
4. Gently resuspend the cell button and read macroscopically.
5. Record result. Then, proceed with the next phase.

B) 37ºC Phase
1. Add 2 drops of enhancement solution such as LISS (Low Ionic Salt Solution) to each of test tubes.
2. Incubate for 15 minutes at 37ºC.
3. Centrifuge for 15 seconds at 3400 rpm.
4. Gently resuspend the cell button and read macroscopically. Record result.

C) Antiglobulin Phase (AHG)
1. Wash all of the test tubes from the 37ºC phase 3 times with normal saline.
2. After the last wash, decant all saline and add 2 drops of AHG reagent.
3. Mix and centrifuge for 15 seconds at 3400 rpm.
4. Gently resuspend the cell button and examine for any agglutination. Record result.
5. If any of the tubes demonstrate no agglutination, add 1 drop of Coombs control cell to the tube.
6. Centrifuge the tubes for 15 seconds at 3400 rpm. Observe and grade for agglutination. Agglutination must occur. The test is invalid if the control cells do not agglutinate and the test must be repeated.
7. Record result.

RESULT INTERPRETATION:

To determine antibody specificity, use the following protocol:

Look at each negative cell and cross off all antigens that are present (positive) on that cell. Use X for homozygous cells, and / for heterozygous cells.
Eliminate antigens along the top of the cell panel by crossing off all that have been crossed off at least three times in the antigen matrix, ideally with at least one of them homozygous for the antigen. Do not eliminate antigens based on only one or two heterozygous cells crossed off, especially if you are getting different strength reactions on different cells.
Exceptions to the above policy include:

May rule out Kell based on 3 heterozygous cells - no need for a homozygous cross-off
May rule out low frequency antigens (Cw, V, VS, Kpa, Jsa, Lua) based on only one cross-off, whether homozygous or heterozygous
May also rule out low-frequency antigens if there are no cells positive for them on the panel
If anti-D is present, you may rule out anti-C or anti-E based on three heterozygous cells (r'r for C and r"r for E)

4. From the antigens not crossed off, look for a pattern of agglutination matching the pattern you got in the test. This should identify the antibody specificity.
5. At this point there still may be one or more low-frequency antigens not crossed off. If the cell that has the low-frequency antigen also has the antigen that corresponds to the antibody you believe you have identified, you may now cross off this low frequency antigen, because the positive reaction on this cell is most likely due to the antibody you have identified.
6. Often you are unable to rule out the possibility of a second or third antibody because the corresponding antigens are all present on the same cells. For example, you have identified Anti-Jka but can't eliminate Anti-Kell because all Kell positive cells are also Jka positive. Check other cell panels and find 3 other cells that are Kell positive and Jka negative. A negative result when testing these cells with the patient's serum eliminates that antibody; a positive result confirms the second antibody.
7. When the antibody(ies) have been identified, be sure there are at least 3 cells that are possess that antigen and give a positive reaction, and at least 3 cells that are lack that antigen and give a negative reaction. Always check the results of the screening cells. You may have to test more cells from other panels.
8. As a final confirmation of the antibody specificity, if the autocontrol is negative or only weakly positive, type the patient's cells for the antigen. The result should be negative, unless the patient has been recently transfused. The result will then be a mixed field.
9. If you get different reactions at different phases of testing; if you get different strengths of reactions on different cells, or if your results don't form a pattern corresponding to one of the antigens on the matrix, consider multiple antibodies. See (6) above.
10. If you get a pattern of reactivity not matching any of the antigens on the matrix, the antibody may be showing dosage and reacting only with homozygous cells. This is most often seen in the MNS and Kidd systems, but can be seen in other systems. Eliminate only the non-reactive homozygous cells; the pattern of reaction may then match an antigen present only on the remaining homozygous cells. See (2) above.

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