Common laboratory techniques

Aim: Standard Laboratory Techniques for Blood Collection, Sample Processing, Anesthetic Administration, and Humane Euthanasia in Laboratory Animals

References:

1. CPCSEA Guidelines for Laboratory Animal Facility, Ministry of Environment, Forest and Climate Change, Govt. of India
2. Institutional Animal Ethics Committee (IAEC) SOPs
3. AVMA Guidelines for the Euthanasia of Animals

Objective:

To provide a standardized, ethically guided protocol for key laboratory techniques in animal studies, covering blood withdrawal, sample processing, anesthesia, and euthanasia, with emphasis on animal welfare, procedural safety, and regulatory adherence.

Materials Required:

• Sterile syringes, needles (various gauges)
• Capillary tubes
• Blood collection tubes (with and without anticoagulants)
• Centrifuge
• Anesthetic agents (e.g., isoflurane, ketamine, xylazine)
• Euthanasia agents (e.g., sodium pentobarbital, carbon dioxide)
• Personal protective equipment (PPE: gloves, masks, lab coats)
• Animal restraining tools
• Disinfectants and antiseptics
• Logbooks or digital records for documentation

Procedures:

1. Blood Withdrawal:

Preparation: Before withdrawing blood, it is essential to sterilize all instruments and ensure the working area is clean to maintain aseptic conditions. The use of personal protective equipment (PPE) is necessary to prevent contamination and ensure the safety of personnel. Additionally, the animal should be calmed to minimize stress, which can be achieved through gentle handling or sedation if required.

Techniques and Sites:

Rodents:

Tail vein: Heat tail to dilate vessels; disinfect; use a fine needle (25–27G).

Retro-orbital sinus: Perform under anesthesia using a capillary tube; insert medially with care.

Cardiac puncture: Performed under deep anesthesia; insert needle between ribs into the heart.

Rabbits:

Marginal ear vein: Clean area; insert 21–23G needle and withdraw blood slowly.

Central auricular artery: Requires anesthesia due to sensitivity; avoid hemorrhage.

Post-Collection Care:

1. Apply light pressure to the puncture site.
2. Observe the animal for signs of pain, distress, or bleeding.
3. Record procedure details, volume of blood withdrawn, and any complications.

2. Serum and Plasma Separation:

Sample Collection: Use red-top tubes for serum (no anticoagulant) and purple/green-top tubes for plasma (contain EDTA or heparin). Allow serum tubes to clot at room temperature for 30 minutes prior to centrifugation.

Centrifugation: Balance tubes and centrifuge at 1500–2000 × g for 10–15 minutes. Carefully extract supernatant (serum/plasma) with a pipette without disturbing the cell layer.

Storage: Store at −20°C or −80°C for long-term preservation. Clearly label each tube with sample ID, date, and type.

3. Anesthetics:

Preparation and Dosing: Select an appropriate anesthetic based on species, procedure, and individual animal health. Dose calculation must be precise, based on body weight and route of administration.

Commonly Used Anesthetics:

Isoflurane (inhalational): Use induction chamber with 2–5% isoflurane in oxygen. Maintenance at 1–3% during procedure.

Ketamine-Xylazine (injectable): Typical dosage: ketamine 80–100 mg/kg + xylazine 5–10 mg/kg, administered IP or IM. Monitor reflexes (toe pinch, corneal reflex) to assess depth.

Monitoring & Recovery:

• Monitor respiration, heart rate, and temperature continuously.
• Post-procedure, place animals in a warm, quiet space until fully conscious and mobile.

4. Euthanasia:

Preparation: Select a humane method based on species, animal condition, and institutional guidelines. Gather and check all required materials beforehand.

Common Methods:

Sodium Pentobarbital (Injectable):

Dose: 50–100 mg/kg IV; ensure complete loss of consciousness and absence of vital signs.

CO₂ Inhalation: Gradual filling of the euthanasia chamber (20–30% volume/min); observe cessation of respiration for ≥2 minutes.

Cervical Dislocation: Used for small rodents under deep anesthesia; dislocate cervical vertebrae swiftly.

Confirmation and Disposal:

1. Confirm death by absence of respiration, heartbeat, and reflexes.
2. If needed, apply secondary method (e.g., decapitation or exsanguination).
3. Dispose of remains per institutional biosafety protocols.
4. Document all relevant details in the animal’s health record.

Safety and Ethical Considerations:

1. Personnel must be trained and certified in handling and experimental techniques.
2. Always adhere to IAEC and CPCSEA-approved protocols.
3. Apply the principles of the 3Rs—Replacement, Reduction, Refinement—to promote ethical animal use.
4. Maintain detailed records for traceability and accountability.