Calibrating Serologic Centrifuges
Each centrifuge should be calibrated upon receipt & after adjustment or repair . Calibrating the centrifuge evaluates the behavior of cells in solutions of different viscosity , it doesn’t test the reactivity of different antibodies .
A- For immediate agglutination :
1- use serum containing an antibody that produces +1 agglutination macroscopically .
2- select one sample of red cells positive for the appropriate antigen & one negative sample . prepare a fresh suspension of red cells in the concentration routinely used in the laboratory ( 2 – 5% ) .
a- For saline – active antibodies : serum from group A person ( anti –B) diluted with 6% albumin to give +1 macroscopic agglutination ( 3 ml of 22% bovine albumin + 1 ml normal saline = 6% bovine albumin .
Positive control : group B red cells in 2-5% saline suspension .
Negative control : group A red cells in 2- 5% saline suspension .
b- For high protein antibodies : 1 part anti –D diluted with 25 –30 parts of 22% or 30% albumin to give +1 macroscopic agglutination .
· positive control : D – positive red cells in 2-5 %saline suspension .
· negative control : D - negative red cells in 2-5% saline suspension .
3- For each set of tests saline & albumin prepare five 10x75 mm tubes for positive reactions & a duplicate for negative reactions . add the serum & test cells to each tube just before centrifugation .
4- In pairs ,one positive & one negative , centrifuge the tubes for different times ( 10 ,20 ,30 seconds ) observe each tube for agglutination & record observations .
5- The optimum time of centrifugation is the least time required to fulfill these criteria :
a- Agglutination in the positive tubes is as strong as determined in preparing reagents .
b- There is no agglutination or ambiguity in the negative tubes .
c- The red cell button is clearly delineated & the periphery is sharply defined , not fuzzy .
d- The supernatant fluid is clear .
e- The red cell button is easily resuspended .
For Washing & Antiglobulin Testing :
The addition of antihuman globulin (AHG) serum to cells may require centrifugation conditions different from those for immediate agglutination because AHG is added to a dry cell button . The only fluid in the tube is the AHG serum itself .
Centrifugation conditions appropriate for both washing & AHG reactions can be determined in one procedure . Note that this procedure doesn’t monitor the completeness of washing , use of globulin –coated cells to control negative AHG reactions provides this check . The procedure described below addresses only the mechanics of centrifugation .
Materials :
1- Antiglobulin serum , unmodified .
2- Positive control : 2-5% saline suspension of D- positive cells incubated for 15 min. at 37c with anti D diluted to give +1 microscopic agglutination after addition of antiglobulin serum .
3- Negative control: 2-5% suspension of the same D- positive cells incubated for 15 min. at 37c with 6% albumin .
4- Saline , large volumes .
Method :
1- prepare 5 pairs of tubes with positive & negative controls .
2- Fill tubes with saline & centrifuge pairs for different times 30,45,60,90,120 seconds , the red cells should form a clearly delineated button with no cells trailing up the side of the tube .
After the saline has been decanted , the cell button should be easily resuspended in the residual fluid . The least time that accomplishes these goals is the optimum time for washing .
3- Repeat washing process on all pairs two more times using time determined to be optimum .
4- Decant supernatant saline thoroughly & blot rims dry .
5- Add AHG to each of the pairs & centrifuge for different times e.g 10,15 ,20,30 ,& 45 seconds .
6- Select optimum time according to criteria described in step 5 of previous procedure .
بقلم
عادل عبد الحليم السلايمه
أخصائي طب مخبري
مسؤول مختبر جودة المياه / بلدية الخليل
السبت 29 نوفمبر - 8:30
