Pipette Calibration - Guidelines

SMILE

JohnsHopkinsUniversity

Baltimore, MDUSA

Pipette Calibration - Guidelines

Audit:

Questions pertaining to pipette calibration can be found in:

• EquipmentSection

CAP Accreditation Checklist:

Questions pertaining to the calibration of micro pipettes can be found in:

• Chemistry and Toxicology Checklist section:
• Hematology and Coagulation Checklist section:
• Limited Service Laboratory Checklist section:
• Microbiology Checklist section:

Background Information:

Micropipettes are precision laboratory instruments. They require routine maintenance and calibration to ensure accuracy and precision. This is determined using gravimetric methodology by using an analytical balance and weighing the amount of water delivered by the pipette. For the purpose of calibration, milligrams and milliliters are considered to be equivalent. Calculation of percent duplication and percent delivery is used to determine the precision and accuracy of the pipette. Tolerance limits for pipettes are usually established by referring to the manufacturer’s guidelines although some general guidelines are available if manufacturer’s ranges cannot be obtained.

Resources

1. Clinical and Laboratory Standards Institute (CLSI) HIS-A2. A quality system model for healthcare; approved guideline – second edition.
2. JohnsHopkinsHospital Clinical Laboratory Procedure Manual (1995). Eppendorf Pipette Calibration
3. Operating Instructions for Eppendorf Pipette. (ND). Brinkman Instruments Inc. N.Y.
4. Westgard, J.O., PhD. (2002). Basic QC Practices: 2nd Edition. Westgard QC Inc. Madison, WI.

Pipette Calibration - Policy

Pipette Calibration - Process

Process forPipette Calibration

• Staff Technologist
• Department Supervisors

• QA Technologist
• Lead Technologist

• QA Technologist
• Department Supervisors

Pipette Calibration - SOP

Associated Forms:

I acknowledge that I have read, understand and agree to follow this SOP.

Purpose:

Micropipettes are precision laboratory instruments. They require routine maintenance and calibration to ensure accuracy and precision. This procedure establishes standards for pipette calibration to ensure they perform with acceptable accuracy and precision.

Frequency:

• Before pipettes are newly placed into service
• Periodically;suggested interval is quarterly or according to manufacturer guidelines
• After cleaning
• Anytime a pipette becomes dismantled and repaired
• As a means of troubleshooting assay problems

Supplies:

• Micropipettes for calibration
• Analytical balance with maintenance completed
• Pipette tips for appropriate pipettevolumes
• Weigh boats
• Worksheet for Micropipette Calibration
• Reagent grade, room temperaturewater in a clean glass beaker
• Thermometer
• Calculator
• Peanut Oil(Optional)

Procedure:

1. Pipettes 5-100 ul
2. Record ambient room temperature to determine water density to be used in calculations.
3. Place a plastic weigh boat and pipette tip on the balance and tare the balance.
4. Place the tip onto the pipette requiring calibration. Pipette room temperature reagent grade water into the tip using good pipetting technique.
5. Turn the pipette in a horizontal position and remove the tip taking care not to lose any of the water.
6. Weigh the tip and water on the same plastic weigh boat and record the result.
7. Repeat steps again to obtain a second result.
8. Pipettes 100 – 1000 ul
9. Record ambient room temperature to determine water density to be used in calculations.
10. Place a plastic weigh boat on the balance and tare the balance.
11. Pipette room temperature reagent grade water into the tip using good pipetting technique.
12. Dispense the reagent grade water onto the weigh boat.
13. Allow the balance to equilibrate and record the reading.
14. Repeat steps again to obtain a second result.

Procedural Notes:

• Avoid handling pipette tips with your fingers. Use a lint free wipe or forceps whenever possible.
• Two readings are required. Two fresh pipette tips must be used to confirm accuracy.
• Peanut Oil can be used to prevent evaporation of water. Simply place a small amount of the oil in the weigh boat in procedural step 2 (A or B).

• One weigh boat may be used for multiple readings with a tare performed between each weight measurement. Multiple pipette calibrations will require the boat to be emptied and re-zeroed. Reminder: The balance will not allow the tare of a negative reading. The balance must be re-zeroed.

Calculations:

Density of Water:at 20ºC = 0.998

at 25ºC = 0.997

at 30ºC = 0.996

Precision:

To check duplicates:weight 1 – weight 2 = ∆ weight

weight 1 + weight 2 = weight

2

∆ weight = % difference

weight

Sample Calculation: 0.3006 - 0.3010 = -0.0004

0.3006 + 0.3010 = 0.3008

2

-.0004 X 100 = -0.13%

0.3008

Accuracy:

weight x 100 = % Delivery

(Pipette volume (mL)) x density of water

Sample Calculation: .3008 x 100 = 100.6%

.300 x .997

(density of water at 25ºC)

Expected Results:

Acceptable accuracy and precision ranges for pipettes should be based on manufacturers guidelines stated in the pipette package insert. Accuracy and precision ranges of plus or minus 4% for pipettes 5-50 ul and plus or minus 2% for pipettes 51-1000 ul are a good guideline

Interpretation of Results:

1. Pipettes not meeting the established acceptance criteria for accuracy or precision should receive routine maintenance before being calibrated again.
2. If a pipette cannot meet the acceptance criteria it should be removed from service until it can be serviced by the manufacturer or an approved vendor.
3. Pipettes that do meet the established acceptance ranges should be labeled with a sticker that records the actual accuracy of the pipette, the date of calibration and the initials.

References:

1. Clinical Laboratory Standards Institute (CLSI). Clinical Laboratory Technical Procedure Manuals; Fourth Edition. CLSI Document GP2-A4 (ISBN 1-56238-458-9). Clinical and Laboratory Standards Institute, Wayne, PA
2. Clinical and Laboratory Standards Institute (CLSI) HIS-A2. A quality system model for healthcare; approved guideline – second edition.
3. JohnsHopkinsHospital Clinical Laboratory Procedure Manual (1995). Eppendorf Pipette Calibration
4. Operating Instructions for Eppendorf Pipette. (ND). Brinkman Instruments Inc. N.Y.
5. Westgard, J.O., PhD. (2002). Basic QC Practices: 2nd Edition. Westgard QC Inc. Madison, WI.

Associated SOP:

Analytical Balance Operation and Maintenance SOP and Appendices

Appendices:

Appendix A: Pipette Calibration worksheet

Appendix B: (Suggested): Appropriate pipette package inserts

Pipette Calibration Version#: 1.0Page 1 of 8