Quality Control and Standard Operating Procedures

Abstract

Recently, systematic reviews have found quantitative evidence that low study quality may have introduced a bias into preclinical stroke research. Monitoring, auditing, and standard operating procedures (SOPs) are already key elements of quality control in randomized clinical trials and will hopefully be widely adopted by preclinical stroke research in the near future. Increasingly, funding bodies and review boards overseeing animal experiments are taking a proactive stance and demand auditable quality control measures in preclinical research. Every good quality control system is based on its SOPs. This chapter introduces the concept of quality control by using SOPs and provides practical advice on how to write them for experimental stroke research. Write down what you do; do what is written down!

Appendix to SOP

• 1. Entry qualification experiment for mouse MCAO surgeons

• 2. Randomized selection of animals from cage and concealment of treatment allocation

  • 2a. Pharmacological study

  • 2b. Genetically manipulated animals

• 3. Temperature control

• 4. Outcome assessment

• 5. Physiological parameters

• 6. Quasi-sterile surgery

1. Entry qualification experiment for mouse MCAO surgeons

New surgeons need to demonstrate in a series of experiments that they perform the MCAO operation within 15 min. Reproducibility is verified by induction of a certain infarct volume within a standard deviation of 40 %. Mortality must not exceed 10 % within 24 h.

2. Randomized selection of animals from cage and concealment of treatment allocation

2a. Pharmacological study:

Animals in cage are marked with bar/dot code at the beginning of the procedure. Computer program (random number generator) selects an animal and assigns it to the concealed treatment arm (“A,” “B,” etc.).

Stock solution or pharmaceutical ready for application is prepared by assistant and randomly assigned code (“A,” “B,” etc.).

2b. Genetically manipulated animals:

Animals in both cages (e.g., knockout/wild type) are marked with bar/dot code at the beginning of the procedure. Computer program (random number generator) selects an animal and assigns it to the concealed experimental arm (“A,” “B,” etc.), blinded intervention whenever possible.

3. Temperature control

The body temperature of mice during surgery is maintained at 36.5 °C ± 0.5 °C using a temperature-controlled heating plate. Maintain a body temperature of 36.5 +/- 0.5 °C also after reperfusion (for 2 h) using a heated recovery box set at thermoneutral temperature (30–31 °C).

4. Outcome assessment

Infarct volume should be evaluated blinded. Functional outcome (more than Bederson Score!) should be assessed as well. Mortality and exclusion of animals have to be reported, including specific causes for exclusion.

For exclusion, follow the inclusion exclusion criteria SOP.

Main criteria:

no stroke, indicated by absence of functional deficit like circling behavior during occlusion time, insufficient Doppler flow reduction or missing functional deficit or missing infarct in MRI at 24 h after MCAo

Problems during induction of MCAo (excessive bleeding, prolonged operation time ≥15 min, thread placement).

CAVE:

Especially in genetically manipulated animals, be aware of vascular alterations, which might directly affect stroke outcome.

5. Physiological parameters

MABP, HR, blood gases, and CBF should be measured in selected animals.

6. Quasi-sterile surgery

Prior to surgery, the surgeon has to scrub his hands. It is advisable to wear clean gown and non-sterile gloves at all times the animal is being handled. The surgeon has to wear a clean gown, cap, and mask during surgery. Surgical gloves ought to be worn. If gloves cannot be used, a surgical hand scrub from tips to elbows must precede every operation.

The necessary components of aseptic techniques in rodents include also sterile instruments and separate surgical and animal prep areas. The use of glass bead sterilizer for re-sterilization of instruments during repetitive procedures is recommended.

• All instruments used must be sterilized prior to each group of surgeries.

• Instruments must be kept on sterile nonporous drapes during use.

• Separate instruments should be used for skin and tissue handling.

• Instruments must be cleaned of blood and debris by brushing or wiping with sterile water or saline and sterile gauze sponges between surgeries (best cleaned with distilled water).

• If contamination has occurred, instruments must be placed in 70 % ethanol or a glass bead sterilizer for the appropriate period of time for the method used to be effective (or the instrument pack replaced by a new sterile instrument pack) between animals.

• If 70 % ethanol is used, instruments must be rinsed with sterile water or saline before being used on the next animal.

• Surgical gloves and blades should be changed after contamination.

• Following surgery, all instruments must be thoroughly cleaned and rinsed.

7. Postoperative care:

Please see maximizing animal welfare in experimental rodent stroke SOP.