Standard Laboratory Techniques in Immunology and Serology

Standard Laboratory Techniques in Immunology and Serology

A. Pipetting

Definition of Terms

• Adjustment: Altering the pipette for accuracy in dispensed volume.
• Air Displacement Pipettes: Designed for common use with aqueous solutions.
• Aspirate: Draw up a sample into the pipette.
• Blow-out: Empty the pipette tip completely.
• Calibration check: Verifying the difference between dispensed and selected volumes.
• Dispense: Delivering the sample.
• Positive Displacement Pipettes: Used for high viscosity and volatile liquids.

Automatic Pipettes

• Most commonly used in clinical laboratories, particularly in immunology and serology.
• Advantages: Higher accuracy and less cleaning required.

Types of Pipettes

• Air Displacement: Utilizes a piston mechanism; disposable tips required; not in contact with liquids. Affected by temperature and atmospheric changes.
• Positive Displacement: Moves piston within the pipette tip; requires rinsing/blotting between samples due to carryover risks.
• Dispensing Pipette: Draws from a reservoir to dispense repeatedly; can be motorized, handheld, or attached to a dilutor.

Techniques, Care, and Maintenance

1. Inspect pipettes for dust at the beginning of the day, clean with 70% ethanol if necessary.
2. Set volume within the pipette's specified range.
3. Hold pipette with the finger rest on your index finger for optimal control.
4. Ensure the pipette, tip, and liquid are at the same temperature for accuracy.
5. Use only recommended high-quality contamination-free polypropylene tips.
6. Tips are single-use; do not attempt to clean for reuse.
7. Pre-rinse tips 1-3 times to improve accuracy, especially with positive displacement.
8. Avoid horizontal pipetting to prevent contamination.
9. Use gloves and tip ejectors to minimize finger contamination.
10. Store pipettes upright in stands.
11. Regularly check calibration; annually or every three months for frequent use.
12. Follow manufacturer instructions for recalibration.

Pipetting Techniques for Improved Accuracy

• Dispensing: Improve accuracy up to 1% by honing dispensing skills; touch the vessel wall while dispensing.
• Immersion Depth: Correct immersion depth enhances accuracy by up to 5%. Avoid over-immersion or too shallow positioning.
• Rhythm and Speed: Maintaining consistent rhythm increases accuracy by up to 5%; avoid fast or jerky movements.
• Immersion Angle: Keep close to vertical for accuracy improvements up to 2.5%.
• Pre-rinsing: Neutralizes capillary effects, improving accuracy by up to 0.2%.

Instrument/Environment Considerations

• Setting the Micrometer: Adjusting volume should involve specific dialing techniques to avoid backlash.
• Volume vs. Range: Aspiration/dispensation within 35%-100% of a pipette’s nominal volume improves accuracy.
• Hand Warming: Rest pipette on a stand to avoid hand warmth affecting results.
• Temperature: Maintain a consistent temperature (ideally 21.5°C) for best results; equilibrate samples with the room temperature.
• Ergonomics: Implement good posture and switch hands to reduce fatigue and increase productivity.

B. Preventing Cross Contamination

1. Pipette-to-Sample: Use sterilized tips and change after each use to avoid contamination.
2. Sample-to-Pipette: Keep pipette vertical and use filter tips to prevent liquid from entering the pipette body.
3. Sample-to-Sample (Carryover): Change tips after each sample to prevent mixing.

Common Sources of Error

• Evaporation of volatile liquids; ensure a saturated air environment.
• High-density liquids: adjust technique accordingly.
• Viscous liquids may adhere to tips; use techniques to mitigate this.
• Hydrophilic tips may require pre-wetting.

C. Centrifugation

• Definition: A process that uses centrifugal force to separate solids from liquids.
• Equipment: Uses a centrifuge with a rotor, carriers, and enclosed metal covering.

Types of Centrifuge

1. Microcentrifuges: For small volumes; used in hematology and molecular biology.
2. Benchtop Centrifuges: Suitable for low-speed separations of bodily fluids.
3. Ultracentrifuges: Spin at high speeds; used in blood banks.
4. Industrial Centrifuges: Handle large volumes continuously.
5. Low-speed Centrifuges: General purpose use.
6. Speed Centrifuges: Collect larger cellular organelles and precipitated proteins.
7. Refrigerated Centrifuges: Temperature controlled, beneficial for molecular biology.
8. Continuous Flow Centrifuges: Allow large volume centrifugation without affecting sedimentation rate.

Centrifugal Force

• Expressed as relative centrifugal force (RCF) or gravities (g), influenced by:
  • Mass
  • Speed (rpm)
  • Radius
• Formula: RCF = 1.118 imes 10^{-5} imes r imes (rpm)^2
  • Where ( r ) is the radius in centimeters from the centrifuge axis to the bottom of the test tube shield.