AB0 Direct Determination Determination AB0 direct
highlights the presence of A and B antigens on red blood cells by means of a typing agglutination reaction. It 's the basic method and simple, it can also be performed in emergency on a few drops of capillary blood obtained by puncture of the earlobe or digital .(....)
Reagents:
· Antiserum A Anti B and commercial
[1] (keep refrigerated 2-6 ° C)
Standard:
· blood typing: anticoagulated (citrate or in a tube with EDTA is the preferred material) or fresh capillary (before clotting) or whole blood clot
[2] have 2 alternative methods:
· On white plate or slide:
> lay on the plate a drop of blood diluted + 1-2 drops of antiserum
> stir with a bar of plastic or glass cleaner
> tilting the plate to wait 30-60 seconds (or slide)
verify your agglutination.
• In test tube:
> put in a test tube one drop of cell suspension at 3-5% in saline
[3] + 1-2 drops of antiserum
> centrifuge at low rpm (1000 xg approximately) for 30 seconds
> resuspend the bottom with light taps and rotation of the tube - tip and roll-to highlight the agglutination.
Interpretation:
§ §
presence of the agglutination = not = no agglutination antigen
Example: • Anti
A + = blood agglutination
• Anti B + blood = no agglutination
· Interpretation: A group
antisera on the market today are usually made of monoclonal antibodies, highly active, specific and stable even in optimal storage conditions. The reaction agglutination waiting with these commercial antisera is obvious to the naked eye and does not require the use of microscopic examination.
rarely possible false positives
[4] , that is of real or apparent agglutination reactions that do not match the presence of the antigen, in this case A or B. The technique in test tubes with diluted red blood cells reduces false positives and questionable results.
E 'should perform regular quality control of the antisera with RBC notes (A, B, and 0) and most importantly it is essential to preserve the antisera in a working refrigerator and regularly check and verify their validity.
From a purely cultural point of view, we recall the existence rare subgroups of A and B that can give false-positive or questionable direct evidence.
[1] You can also use the antiserum anti-A, B, which agglutinate red blood cells both A and B. This serves as confirmation to the reactions observed with the 'anti-A el' anti-B. Today with the monoclonal antisera, more sensitive and specific than polyclonal human use in the past, the use of the antiserum anti-A, B is no longer indispensable. [2] E 'can also be used for the determination of coagulated blood group. After clot retraction test cells trapped the clot can be released and resuspended in serum breaking the branches of fibrin with a Pasteur pipette. [3] To prepare a cell suspension at 3-5% mix a drop of whole blood in 15 drops of saline solution or a drop of packed cells in 30 of saline solution. [4] False positive direct determination: this may be due to stacking of red blood cells (to be distinguished from agglutination), or you can see true agglutination caused by auto-antibodies in the blood plasma by typing, antibodies that affect temperature environment (called "cold"). In both cases, these factors present in blood plasma to be determined and are seen especially in the case of the technique on plate. In test-tube technique the plasma is very dilute, so the possibility of these false positives is also reduced in the test tube, the result is more clearly interpretable. Therefore, whenever possible, the test-tube technique is preferable.
In any case to exclude false-positive indirect evidence, particularly if the blood agglutinates with all the antisera available (in case of group AB + beware!), You should perform a self-control.
