CELLCentral - Health & Safety

Procedures for the Cryosectioning of Fresh (unfixed) Human Tissues

Introduction

The cryostat with its sharp microtome blade presents a cutting hazard to the user. Since the cryostat is also an expensive, precision piece of equipment, for reasons of both safety and good work practice, all users of the cryostat are given an induction on its use prior to working independently.

In addition to this, the cryosectioning of fresh human tissue, which may contain biological hazards, requires the following appropriate safety procedures. The following procedures for the cryosectioning of fresh human tissue in the School of Anatomy and Human Biology are based on previous experience, consultation with the UWA Health and Safety Office, an inspection of current cryosectioning procedures at the PathCentre and an inspection of our procedures by the UWA Biological Safety Officer.

Procedures

Immunization. Hepatitis B Immunization is recommended for all staff and students who handle human blood and body tissue
Storage. Samples are stored at -80 degrees , in capped tubes and appropriately labeled according to the regulations for storage of Fresh Human Tissue
Signage. A sign stating Care Human Tissue Sectioning in Progress, Do Not Touch is placed on the cryostat while this work is in progress. In addition a sign informing users that human tissue is cut on this cryostat is permanently displayed.
Booking. The cryostat is booked for the whole day once this procedure has commenced to allow sufficient time for sectioning and cleaning / disinfecting on completion. Therefore other users do not use the cryostat until the next day.
Transporting samples. Small samples are transported into the Histology Laboratory, kept cold on dry ice or frozen ice brick, contained in a closed container such as a small esky. Flask containing liquid nitrogen can also be used.
Cuts or abrasions. If the user has any cuts or abrasions these must be covered with a non-porous, waterproof dressing.
Gloves. Disposable, water-impermeable gloves must be worn.
Lab coat. A disposable lab coat must be worn
Face shield. A face shield must be worn to prevent blood or body fluids/substances entering the body through mucosal contact (eg splashes of body substances to the mouth, nose, eyes or non-intact skin).
Cryostat number 2 The newer Leica CM3050S cryostat is to be used for human tissue cutting so this work is confined to one machine.
Cutting Procedure At every opportunity close the glass sliding window as this not only helps keep the temperature and humidity low for better sectioning, but is also safer as the specimen and blade are shielded from the user.
Plastic covers and aluminum foil are used inside the cryostat chamber to help prevent small tissue pieces lodging where they are difficult to remove.
Specimen chucks are isolated solely for human sectioning and stored separately from the regularly used chucks.
Sectioning equipment only the equipment necessary for this work is placed inside the cryostat chamber ie brush, forceps etc. This collection of instruments is set aside exclusively for human tissue sectioning.
Cryostat sections must be labeled Human on each microscope slide and transported in a labeled slide box or slide tray.
Cryostat Day Book is filled out indicating date and the user cryosectioning fresh human tissue.

Waste Disposal / Cleaning / Disinfecting

Blades & microscope slide disposal On completion the microtome blade (together with any other blades or sharps) are carefully removed and disposed of in a small yellow Medical Sharps Waste container. Any unwanted microscope slides with tissue sections are also disposed of in this container which is then closed and placed in the large yellow biological waste bin for incineration.
Waste disposal All sectioning waste and the aluminium foil is removed from the cryostat and placed in the Biological Contaminated Waste For Autoclaving waste bag and labeled Human Waste which is then autoclaved as soon as possible and placed in the yellow biological waste bin for incineration. Any disposables such as gloves, tissues, paper towels, disposable gown or fluid shields etc are also disposed of in this way.
Cryostat chamber cleaning / disinfecting The cryostat chamber, microtome, sample holder and any equipment are thoroughly cleaned with 70% ethanol. Waste ethanol is disposed of down the flushing sink. Once the chamber has dried, the microtome, sample holder and chamber equipment are thoroughly sprayed with Cryofect and left to soak for at least 15 minutes before wiping off with tissues which are disposed of as for waste disposal above. Cryofect is a Leica product specifically manufactured for the disinfecting of cryostats. Cryofect contains 72% ethanol and 0.16% chlorhexidindigluconat (see reference)
Equipment cleaning The brushes, forceps, thermal block, chucks etc. all need to be soaked for at least 20 minutes in 70% ethanol before being thoroughly cleaned with water and detergent. All autoclavable items are also autoclaved.
Hands Washed As per normal procedures hands are washed before leaving the lab.

In Case of an accident

In the case of a cut injury or splash exposure promptly flush the cut under running water or in the case of a splash rinse the eyes, mouth and nose (if affected) using the eyewash tap. Pat-dry and in the case of a cut, apply a sterile dressing. Seek immediate medical advice. An accident report form must also be completed for the School.

References

UWA Safety & Health site on Biological Hazards
http://www.safety.uwa.edu.au/policies/biological_hazards

National Code of Practice for the Control of Work-related Exposure to Hepatitis and HIV (Blood-borne) Viruses [NOHSC:2010(2003)] 2nd Ed Dec 2003
http://www.nohsc.gov.au/ohslegalobligations/nationalstandards/aidshepb/default.htm

UWA Immunisation Policy
http://www.safety.uwa.edu.au/policies/immunisation

Cryofect
http://www.hbu.de/products/Cryofect.htm