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Calibrating Serologic Centrifuge

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  • Calibrating Serologic Centrifuge

    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

  • #2
    الله يبارك في مجهوداتك

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    • #3
      اخوي Anti virus شكرا جزيلا لك

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      • #4
        شكرا لك على موضوعك الجميل

        الى اللقاء

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        • #5
          بارك الله فيك وفي جهودك.....

          طريقة فريده وجيده, رغم انه هناك طرق اسهل ....حسبABB
          اما البريطانين لهم طرق معقده ...

          المهم الوصول إلى نتيجه واحده ....


          لكن سوالي ...كل كم من الزمن تتطبقون calibration

          كل اسبوع او اكثر....؟؟

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