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Dilutions; Sample And Control

The following standardized protocol is to be used when diluting patient specimen and control material unless the written procedure for the analyte being measured calls for some specific variation.

Macro dilutions; sample vol > 0.5 ml

Micro dilutions ; sample vol. ≤ 0.5 ml

I. Micro Dilutions Using Rainin™ Pipettes:

Note: Proper execution of the following instructions requires complete familiarization with the “Two-Stop Micropipettor Pipetting Techniques procedure.

  1. Always use the same pipette to deliver the sample and the diluent.
  2. Pipette the diluent first, use the reverse technique.
  3. Change the pipette tip and then, using the forward-rinse technique, deliver the sample aliquot into the diluent rinsing 5 times.
  4. Cap the dilution vial and invert gently 5 times to mix.

    Example: A 1:5 dilution of serum using a 20 µl Rainin pipette:
    1. Deliver four, 20 µl aliquots of the diluent into the dilution vial using the reverse technique.
    2. Change the pipette tip.
    3. Using the forward-rinse technique, add one 20 µl aliquot of the serum into the diluent and rinse 5 times. Remember, aspiration of the sample must be slow and even!
    4. Cap the dilution vial and invert gently 5 times to mix.
    5. The result from the analyzer must then be multiplied by a factor of 5. (Refer to individual testing procedures for specifics regarding documentation of the initial result, reported result and dilution correction calculations.)

II. Macro Dilutions Requiring Sample Volumes ≥ 0.5 Ml:

  1. Use only Ostwald Folin Class A volumetric T.D. pipettes (etched ring-blow out) for the sample aliquot.
    Note: Ostwald Folin pipettes are similar to volumetric pipettes but have their bulb closer to the delivery tip, thereby reducing the surface area in contact with the liquid. These pipettes are used for accurate measurement of viscous fluids such as blood or serum. In contrast to a volumetric pipette, an Ostwald Folin pipette has an etched ring near the mouthpiece, indicating that it is a blowout pipette. The liquid is blown out of the pipette only after the blood or serum has drained freely to the last drop in the delivery tip. Controlled, slow drainage is required with all viscous solutions so that no residual film is left on the walls of the pipette. The top of the meniscus must be read when the pipette is filled with opaque fluids such as blood.2.
  2. If the diluent volume is ≤ 3 ml, use a volumetric Class A T.D. pipette for the diluent.
    Note: A volumetric transfer pipette is calibrated to deliver accurately a fixed volume of a dilute aqueous solution such as a standard, a filtrate, or a nonviscous sample. These pipettes are allowed to drain by gravity.2.
  3. If the diluent volume is > 3 ml but ≤ 5 ml, a 5 ml serologic pipette should be used for the diluent. If the diluent volume is > 5 ml but ≤ 10 ml, a 10 ml serologic pipette should be used for the diluent.
    Note: The serologic pipette has an etched ring near the mouthpiece of the pipette, signifying that it is a blowout pipette, and has graduation marks down to the tip. Serologic pipettes are principally used for the transfer of reagents and are not generally considered accurate enough to pipette samples and standards.2.
  4. The sample aliquot is delivered into the dilution vial first. The Ostwald Folin volumetric pipette (etched ringed-blow out) is held vertically with its’ tip resting against the wall of the dilution vial and the sample aliquot is allowed to drain slowly and completely into the vial. Sample remaining in the tip of the pipette following the drain step is then blown into the vial using a pipette bulb.
  5. Deliver the appropriate diluent aliquot into the dilution vial. If a volumetric pipette is used, diluent remaining in the pipette tip following the drain step is discarded. If a serologic pipette is used and the total volume of the pipette is required, diluent remaining in the tip of the pipette following the drain step must be blown into the dilution vial with a pipette bulb.
  6. Cap the dilution vial and invert gently 5 times to mix.
    Example: A 1:10 serum dilution with a serum volume of 0.5 ml:
    1. Deliver the 0.5 ml sample aliquot first using a 0.5 ml, Ostwald Folin volumetric Class A pipette (etched ring). The pipette must be held straight up and down with its’ tip resting at an oblique angle against the side of the dilution vial. Blow out the residual sample into the dilution vial using a pipette bulb.
    2. With a 5.0 ml serologic pipette, add 4.5 ml of the diluent. Discard the diluent remaining in the pipette.
      Note: Serologic pipettes are always filled to their calibrated capacity and allowed to drain down to the desired aliquot.
    3. Cap the dilution vial and invert gently 5 times to mix.
    4. The result from the analyzer must then be multiplied by a factor of 10. (See individual testing procedures for specifics regarding documentation of the initial result, reported result and dilution correction calculations.)

III. References:

    1. S. Raymond: Dubuque Pathology Associates.
    2. Textbook of Clinical Chemistry, Edited by Norbert W. Tietz. W.B. Saunders Company. 1986.

 

June 1985 S. Raymond

July 1993 S. Hosch

May 1994 L. McGovern (Revised II., Added III.)

 

Comprehensive Review:

Pathologist:

Technical Director:

 

Interim Review:
May 2010 (no changes)
May 2011 J. Heiar (no changes)

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