Use and Functional Verification of Automatic

and Micro-PipettesModule 7:

Quality Assurance of Analytical Phase

Objectives

Upon completion of this lesson the student will be able to:

• Discuss the appropriate usage of:– Automatic/micropipette– Repipettor

• Explain the need to verify pipette calibration periodically

Objectives

• Describe methods used to verify pipette calibration

• List the statistical calculations needed to determine pipette precision and accuracy

• Calculate statistical data to verify pipette accuracy and precision

Reasons for Pipetting

Pipetting is a part of all types of laboratory testing.

• Immunology-Serology• Microbiology• Body Fluid testing• Hematology• Blood Transfusion Medicine• Biochemistry/Clinical Chemistry

Specific Reasons for Pipetting

• Dilutions of patient samples when the concentration of a substance is too high to be accurately measured

• Preparation of stock standards to yield various target concentrations for working standards

• Reconstitution of controls used to assess instrument performance and validity of test results.

• Dilution or preparation of reagents used in the testing method.

Uses of Micropipettes

• Glass pipettes are not highly accurate at dispensing < 1 milliliter (1 mL)

• Automatic pipettes are both accurate and precise for small volumes

• These may be adjustable digital pipettes

Role of Pipettes• Accurate pipetting is a critical skill required

of all clinical laboratory scientists• Good pipetting skills are required to

prepare accurate and precise dilutions of:– Reagents – Patient specimens – For quantitative measurements in the clinical

laboratory

Good laboratory practice requires accurate and precise pipetting. It is part of Quality Assurance.

Micropipettes: Small Volumes Dispensed

A micro-volume pipette is used when pipetting < 1.0 mL

Advantages and Disadvantages

• Micropipettes Don’t– Need a suction bulb for aspiration– Need as much hand-eye coordination

• Micropipettes Do– Seem easier for most people to operate

• Disadvantages– Require proper training for use– Require periodic validation of calibration– Sometimes require cleaning and lubrication

Types of Micropipettes

• Types of micro-pipettes– Air Displacement– Positive Displacement– Repipettor

Multi-Channel and Adjustable Micropipettes

Mechanical Micropipettes

• There are 2 basic types of mechanical or manual micropipettes– Piston (air displacement)

• The piston style may have 2 stops, in which the lowest position is to ensure that air blows out the last drop

• Note: a one-stop piston micropipette is easiest to use but prone to having too much of the sample retained behind in the pipette

– Syringe (positive displacement)

Quality Assurance Micropipette Procedures

• Always follow the procedural instructions provided by the manufacturer for the proper use of a micropipette

Quality Assurance of Micropipette Use

• Select the appropriate pipette for the volume required

• Select the appropriate pipette tip • Ensure that there are enough tips for the

procedure• Ensure that the pipettor, tips, and

specimen are at the same temperature

Volume Adjustment Knob:

Digital Volume Indicator:

Pipettors – 3 Volumes:

Operating the Micropipette

Step 1: Set the Volume

Operating the MicropipetteStep 1: Read the Volume

How to Read the Volume Indicator             

(a): P-20 Model 6.86 l = 0.00686 or 6.86 x 10-3 ml  

(b): P-200 Model 132.4 l = 0.1324 or 1.324 x 10-1 ml

(c): P-1000 Model 262 l= 0.262 or 2.62 x 10-1 ml

 

Setting Adjustable Volume

• Course and fine setting of the pipette line. Because the course and fine settings are separate, it is not necessary to "wind" the pipette as would be done on a screw-type adjustment

Example of tip sizes:

Attaching the disposable tip:

Operating the MicropipetteStep 2: Attach the Disposable Tip

Operating the Micropipette Steps 3-6

• Step 3: Depress the Plunger to the First Stop

• Step 4: Immerse Tip in Sample

• Step 5: Draw up the sample

• Step 6: Pause before withdrawing tip– Wait a few seconds

Mechanical Micropipette Procedures

• Particularly important is removing excess fluid from the outside of the pipette tip– For syringe pipettes

• The filled syringe should be dipped in distilled water to remove excess fluid clinging to the outside

– For piston pipettes• The outside of the pipette should be carefully wiped

with a disposable tissue, taking care not to touch the bottom of the pipette tip

Proper Droplet Removal WRONG Droplet Removal

 

Operating the MicropipetteStep 7: Wiping the Tip

(a) Start Dispensing 

(b) 1st Stop = Dispense

(c) 2nd Stop = Expel

Operating the MicropipetteStep 8: Dispense the

Sample

Press ejector button to discard tip.

Operating the Micropipette Steps 9-11

• Step 9: Withdraw the Pipette

• Step 10: Release the Plunger

• Step 11: Discard the Tip

Pipette: Use and Care

• Discard contaminated tips in appropriate container after completion of task

• Handle carefully• Store in an appropriate rack or stand• DO NOT RE-USE

– Pipette tips – Graduated plastic bulb transfer pipettes

Routine Maintenance

• Check at the beginning of each workday for dust and dirt on the outside surfaces. Pay particular attention to the tip cone

• Clean with 70% isopropyl alcohol• In addition to routine calibration,

pipettes should be cleaned and lubricated as recommended by the manufacturer

Routine Maintenance

• Inspect the pipette for scratches or other damage to the nose cone

• Check for leaks by aspirating the maximum listed volume of distilled water

• If the pipette is dropped or you suspect any type of damage, do calibration check

Mechanical Pipette Maintenance

• Maintain complete records of pipette calibration function check– Include serial and other identifying numbers

• Annual check of calibration with gravimetric or spectrophotometric method

Mechanical Pipette Maintenance

• The accuracy and precision should be checked the first time of use and periodically thereafter

• Accuracy and precision should be checked at least once every year– If either fails, it is important to follow the

manufacturer’s instructions

See job aid for complete maintenance procedures

Pipette: Troubleshooting

Problem Potential Cause Action

Leakage • Tip(s) incorrectly attached

• Foreign articles between the tip and cone

• O-ring damaged• Scratches or hairline fractures on the shaft

• Attach firmly

• Clean tip cones

• Change the O-ring• Contact distributor; use replacement pipette

Application 1: Troubleshooting

• A technologist observed that when using the pipette method for analysis, the volume of sample dispensed into each tube seemed to look different.

Question:• What is the next step the technologist

should take?Answer:• Check the precision and accuracy of the

pipette

Pipette: Troubleshooting

Problem Potential Cause ActionInaccurate Dispensing

• Incorrect operation• Tip incorrectly attached• Pipette calibrated at room temperature, used with cold liquid

• Tip immersed too deep• Tip at an angle while aspirating

• Follow manufacturer’s instructions carefully

• Firmly attach tip• Calibrate and use at same temperature

Immerse only 2 – 3 mm• Hold vertically

How to Organize Pipette Calibration Check

• If a site has an analytic balance on-site, do calibration at the site

• If no analytic balance is available on-site– Send pipette for calibration to a nearby site

with a balance

• An optional procedure using a spectrophotometer may be used

Precision Pipettes Require

Performance Checks• Performed periodically (at least once per

year)• Required supplies:

– Pipette– Pipette tips– Analytical balance (micrograms)– Weigh boats– Distilled or deionsed water

Testing Pipetting Accuracy

• The accuracy of pipetting should be evaluated periodically (i.e. every 6-12 months)

• Retain the evaluation records for each pipette for quality management purposes

• Select a volume normally used in the procedure

• Using the pipetting technique, pipette replicates of distilled water and record the weights

Testing Pipette Accuracy• The following procedure can be used to test

how accurately the pipette measures volume (water is used because the weight of water is 1g/mL):– Using the correct pipetting technique, pipette 10

replicates of 50 μl of distilled water and record the weight to the nearest 0.0000 g

– Calculate the mean weight – The mean should fall within 0.0490 and 0.0510– A correction factor for the room temperature may

be needed to convert weight to volume.

Checking Pipette Precision

• Set the pipette to 50 µL• Using pipetting technique, pipette 10

replicates of distilled water and record the weights

• A correction factor for the room temperature may be needed to convert weight to volume.

• Calculate the mean, standard deviation and coefficient of variation (CV)– The CV for replicates should be < 0.3%– Standard deviation (s) may be compared with

published requirement

Gravimetric Calibration Requirements

Pipette Volume tested

(µl)

Gravimetric Measurement

Accuracy(mg)

Standard Deviation

Precision (mg)

≥ 0,2-10 ≤ 0,001 ≤ 0,002

≥ 10-100 ≤ 0,01 ≤ 0,02

≥ 100-1000 ≤ 0,1 ≤ 0,2

≥ 1000 ≤ 0,1 ≤ 0,2

Pipetting

0.0000 g0.0495 g0.1001 g0.1499 g

Gravimetric Measurements

Calculation of First Weight From Empty Weighing Boat

Calculations of Second Weight From Combined Aliquots 1 and 2

Calculations of Weights 3-10 From Combined Aliquots

Gravimetric Data of 50 uL Micropipette

Weight in g.

Convert to mL and adjust for temperature

0.0495 Mean in mL0.0506 Mean (g) ?

0.0498

0.0501 Mean g x Factor =

0.0506 Mean (mL) ?

0.0492 s ?

0.0498 %CV ?

0.05070.05020.0491

Temperature Conversion for Water Density

Multiple correction factor by mass in g to determine corrected volume in mL.T (°C) Corr. Fact. (mL/g) T (°C) Corr. Fact. (mL/g) 17 1.0023 23 1.0035 18 1.0025 24 1.0038 19 1.0027 25 1.0040 20 1.0029 26 1.0043 21 1.0031 27 1.0046 22 1.0033 28 1.0048

Calculations for Precision and Accuracy

N

xx

• Arithmetic mean

• Standard deviation

• Coefficient of variation

1

22

N

Nx

xSD

100% xSDCV

Application 2: Practice with Precision and Accuracy Calculation

• In order to be precise for patient testing, the 50 uL pipette was required to have a % coefficient of variation of < 2% and accuracy was required to have < 1% error rate.

• A quick precision and accuracy study was performed with the following results:

Volumes obtained from gravimetric testing of distilled water corrected for tempearuature

0.048 mL0.045 mL0.051 mL0.049 mL0.053 mL

Application 2: Precision and Accuracy Calculation

• The mean, s and %CV should be determined.

• The % accuracy or mean volume error is determined using the following formula:

(Expected volume -Mean of Measured volume) / Expected volume X 100% Remember that 1 g = 1 mL or I mg = 1 uL

In order to be precise for patient testing, the 50 uL pipette was required to have a % CV < 2% and accuracy was required to have a <1% error rate.

Volumes obtained from gravimetric testing of distilled water

0.048 mL0.045 mL0.051 mL0.049 mL0.053 mL

Application 2: Precision and Accuracy Calculation

Questions:1. What is the mean? 2. What is the standard

deviation? 3. What is the coefficient of

variation (%C.V.)? 4. What is the accuracy error

of this pipette?5. Is this pipette acceptable

to use? Why or why not?Work in groups to determine

these results.

• The pipette was required to have a % CV < 2% and accuracy was required to have a <1% error rate.

Volumes obtained from gravimetric testing of

distilled water0.048 mL0.045 mL0.051 mL0.049 mL0.053 mL

Application 2 Debrief: Precision CalculationQuestions Answers

1. What is the mean in uL?

2. What is the coefficient of variation (%C.V.)?

3. What is the standard deviation in uL?

4. What is the accuracy error of this pipette?

5. Why or why not is this pipette acceptable to use?

0.0492 mL or 49.2 uL

6.1%

0.003 mL or 3 uL

[(0.05 – 0.0492)/0.05] x 100 = 1.6%

Not acceptable since accuracy error > 1% and precision > 2%

Summary

• Discuss the advantage of using volumetric equipment when preparing standard solutions, diluting controls and patient samples

• Discuss the appropriate usage of automatic/ micropipettes for quality

• Discuss the need to verify pipette calibration (precision and accuracy) periodically

• Discuss the main procedure used to verify pipette calibration