Principle: | Clinical Significance: N.A. | Specimen: N.A. | Materials: | Reagents: | Standardization: N.A. | Procedure: | Results Derivation: | Expected Result(s) and/or | Quality Control: | Reference:

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Centrifuge Calibration, Serologic

I. Principle:

Calibrating the centrifuge evaluates the behavior of cells in solution of different viscosity. The addition of antihuman globulin (AHG) to rbcs may require centrifugation conditions different from those for immediate agglutination because AHG is added to a dry rbc button. This procedure is performed on every serologic centrifuge on receipt, once per year as routine function verification and whenever a significant adjustment or repair to the instrument or a change in technique has occurred.

II. Clinical Significance: N.A.

III. Specimen: N.A.

IV. Materials:

  1. 12 x 75 mm test tubes (CardinalHealth #T1290-3)
  2. Blood bank droppers (CardinalHealth #P5201-3)
  3. Serologic centrifuge
  4. Cellwasher
  5. Serologic pipette (1 ml and 5 ml)
  6. Volumetric flask (250 ml)

V. Reagents:

  1. Anti Human Serum, IgG
  2. 0.85% Saline (CardinalHealth #B3158-6)
  3. Plasma from a group A individual
    Dilute the plasma with saline to yield a 1+ reaction in immediate spin phase.

    Note: The dilution will vary depending upon the strength of the individual’s natural antibody.
  4. Group A reagent red blood cells (Affirmagen-Ortho)
  5. Group B reagent red blood cells (Affirmagen-Ortho)
  6. Rh(D) positive red blood cells (Selectogen-Ortho)
  7. Rh(D) negative red blood cells (Affirmagen-Ortho)
  8. Dilute anti D (yielding 1+ reaction in antiglobulin phase). The dilute anti D used for quality control may be used. Refer to the “Quality Control of Blood Bank Reagents;Testing & Recording” procedure.

VI. Standardization: N.A.

VII. Procedure:

The centrifuge calibration is performed annually. The calibration procedure must also be performed upon installation of any new serologic centrifuge.

  1. Immediate agglutination for saline-active antibodies:
    The plasma from a group A individual is diluted to give a 1+ immediate spin reaction. The diluted plasma is then mixed with group B (Positive reaction) and group A (Negative reaction) red blood cells and centrifuged at high speed for 10, 15, 20, 30 and 40 seconds.
    1. Label ten 12 x 75 mm test tubes as follows: 10+, 10-, 15+, 15-, 20+,
      20-, 30+, 30-, 40+ and 40-.
    2. Add 2 drops of the diluted plasma from a group A individual to each of the tubes using a blood bank dropper.
    3. Add 1 drop of group A reagent red blood cells to each of the tubes labeled - (Neg) and 1 drop of group B reagent red blood cells to the tube labeled 10+.
    4. Centrifuge the tubes labeled 10+ and 10- on high speed for 10 seconds.
    5. Remove the tubes from the centrifuge, observe the tubes and record the answers on the worksheet to the following question:
      1. Is the supernatant fluid clear?
      2. Is there clear delineation of the cell button?
      3. Are the cells easily re-suspended?
      4. What is the strength of agglutination?
      5. Is the negative tube negative?
    6. Repeat the test with each pair of tubes, adding the B reagent red blood cells to the tube marked +, just prior to centrifuging. Centrifuge each pair on high speed for the time in seconds specified on each tube. (i.e.: 15+ and 15- for 15 seconds; 20+ and 20- for 20 seconds; etc…). Record the answers to the 5 questions from step E for each pair of tubes immediately after removing them from the centrifuge.
    7. Record the "Optimum Centrifugation Time" for the saline-active antibodies. This will be the least centrifugation time that meets the criteria stated in E.a - e. (i.e.: If the 20 seconds spin is the least centrifugation time in which all the criteria in step E are met, then 20 seconds is the optimum centrifugation time.)
  2. Washing and antiglobulin testing:
    Reagent anti D is diluted with saline to yield a 1+ reaction at the antiglobulin phase of testing. The dilute anti D is mixed with Rh(D) positive reagent red blood cells and incubated at 37° C. for 15 minutes. Rh(D) negative cells (Ortho Affirmagens A or B cells) are incubated with the same dilute anti D as a negative control. The tubes are then washed, centrifuging during the wash cycle on high speed for 30, 45, 60, 90 and 120 seconds. Anti human serum is then added after the wash cycle is completed. The tubes are centrifuged on high speed for 10, 15, 20, 30 and 40 seconds.

    Note: The centrifugation time for the Sorvall Cellwasher I and II are pre-set and are not adjustable. The Serologic centrifuges (Serofuge, Immufuge, etc...) will be the only centrifuges in which the actual wash procedure will be tested at the various centrifuge times.
    1. Label ten 12 x 75 mm test tubes as follows: (30)10+, (30)10-; (45)15+, (45)15-; (60)20+, (60)20-; (90)30+, (90)30-; (120)40+ and (120)40-.
    2. Add 2 drops of dilute anti D to each tube.
    3. Add 1 drop of Rh(D) positive reagent (Selectogen Ortho) red blood cells to each of the tubes labeled +.
    4. Add 1 drop of Rh(D) negative reagent (Affirmagen Ortho) red blood cells to the tubes labeled -.
    5. Incubate all 10 tubes at 37° C. for 15 to 30 minutes.
      Note: If calibrating an automatic cell washer, place all tubes into the cell washer to wash three times. (It is not necessary to label tubes with wash times for this automatic device). Skip to L.
    6. Fill the tubes labeled (30)10+ and (30)10- with saline and centrifuge on high speed for 30 seconds.
    7. After this first wash, remove the tubes from the centrifuge, observe the tubes and record the answers to the following questions:
      1. Do the red cells form a clearly delineated cell button?
      2. Are there cells trailing up the side of the tube?
      3. After decanting the saline, is the cell button easily re-suspended in the residual fluid?
    8. Wash the 4 remaining pair of tubes once centrifuging each pair on high speed for the times specified on each tube, (i.e.: (45)15+ and (45)15- for 45 seconds; (60)20+ and (60)20- for 60 seconds; etc…). Record the answers to the 3 questions from step G for each pair of tubes immediately after removing them from the centrifuge.
    9. Record the "Optimum Centrifugation Time" for the washing procedure. This will be the least centrifugation time that meets the criteria stated in 2.G.a - c. (i.e.: If the 60 seconds spin is the least centrifugation time in which all the criteria in step G are met, then 60 seconds is the optimum centrifugation time.)
    10. Repeat the washing process on all pairs two more times, using the time determined in step H to be optimum or use an automatic cell washer for the final two washings.
    11. Decant the supernatant saline thoroughly after the last wash and blot the rim of each tube dry with a lint free material. (i.e.: gauze, paper towel)
      Note: step K is not necessary if using an automatic cell washer.
    12. Add anti human serum to each of the tubes and centrifuge each pair on high speed for the time specified (i.e.: (30)10+ and (30)10- for 10 seconds; (45)15+ and (45)15- for 15 seconds; etc…).
    13. Remove the pair of tubes from the centrifuge, observe the tubes and record the answer to the 5 criteria questions specified in section 1.E..
    14. Record the "Optimum Centrifugation Time" for the antiglobulin test. This will be the least centrifugation time that meets the criteria stated in 1.E.a - e. (i.e.: If the 20 seconds spin is the least centrifugation time in which all the criteria in step 1.E. are met, then 20 seconds is the optimum centrifugation time.)
    15. Add coombs control cells to each tube yielding a negative reaction after antiglobulin testing.
    16. Mix the contents of the tubes by gentle shaking and centrifuge on high speed for the time determined to be optimum for the antiglobulin phase of testing.
    17. Record yes or no if the coombs control cells reacted on the worksheet for each tube.
    18. If the tubes do not yield a positive reaction at this point the antiglobulin phase of testing must be repeated.
  3. Post the following information on each centrifuge: centrifuge name, date calibrated and optimum centrifugation times for the saline-active immediate spin, antiglobulin phase spin and the wash procedure spin (if applicable).

VIII. Results Derivation:

  1. Immediate agglutination saline-active antibodies, and antiglobulin reacting antibodies:
    The optimum time for immediate spin is the least time required for centrifugation to fulfill these 5 criteria:
    1. Clear supernatant fluid
    2. Clearly delineated cell button
    3. Cell button is easily re-suspended
    4. Strength of agglutination is 1+
    5. Negative tube is negative
  2. Washing procedure:
    The optimum time for washing is the least time required for centrifugation to fulfill these 3 criteria:
    1. Clearly delineated cell button
    2. No Cells trail up the side of the tube
    3. Cell button is easily re-suspended after decanting

IX. Expected Result(s) and/or Critical Values: N.A.

X. Quality Control:

  1. A positive and negative reaction is tested at each phase.
  2. Coombs control cells are added to all negative antiglobulin phase reactions to verify reactivity of the anti human serum.

XI. Reference:

  1. Committee on Technical Manual, AABB Technical Manual, American Association of Blood Banks, Washington D.C., 11th Edition; pp 752-753; 1993.

 

  1. 2-17-86 M. English
  2. 2-27-91 S. Wallace (Revised: format; VII, VIII)
  3. 11-12-96 M. Burger (Removed High Protein antibody calibration; Revised worksheet)
  4. December 2000 M. Burger (Revised: IV.)

 

Instrument Specialist:

Technical Director:


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