Fixation on Histology

When the Coronavirus hit this past Spring, histologists were understandably curious whether additional precautions needed to be taken to protect themselves from contracting the deadly virus while handling positive specimens. Articles on the topic, including Tony Henwood’s timely publication, Coronavirus disinfection in histopathology, discuss the effectiveness of routine histology laboratory disinfection processes and point to the importance of universal precautions. In histology, we can never be sure what a specimen may test positive for, so universal precautions are implemented to protect ourselves from the possibility of exposure.

One of the few things that formalin does not inactive however is CJD, Creutzfeldt-Jakob Disease, making exposure to this fatal neurodegenerative disorder one of the top histology fears. CJD is a prion disease. Prion is a relatively new term, coined in 1982 by Stanley Prusiner to describe the polypeptide proteins in our bodies that are devoid of genetic material. Normal healthy humans have prions in their body, and these are referred to as normal cellular Prion Protein (PrPc). These normal prions however can mutate into a misfolded shape. Once they are misshapen, they have the ability to spread their misfolded shape to normal proteins of the same type by contact, as opposed to by genetic reproduction, causing rapid damage. It is these bad prions that are responsible for animal ailments such as Mad Cow Disease and Chronic Wasting Disease in deer, as well as the human equivalent, CJD.

Despite prions not being categorized until the 1980’s, the disease CJD was initially discovered in the 1920’s as German neurologists began to notice cases of the neurodegenerative disease. Upon examination under the microscope, they found that the brain tissue had numerous holes, giving it a sponge like appearance. This appearance was similar to results found in cases of Scrapie in sheep and the term transmissible spongiform encephalopathy (TSE) was given to describe the broad category of disease that Mad Cow, Scrapie and CJD fall into.

So, what do you do if your lab gets a suspected or confirmed CJD case? First, it is worth noting that according to the CDC, no cases of equipment related CJD have been reported since 1976 when modern sterilization procedures were implemented, and no cases have been reported related to surfaces like walls or countertops, though you should still cover the workspace with plastic backed absorbent pads. There have also been no reported cases of histotechs getting CJD as a result of working with infected tissue. There is negligible risk for cutaneous exposure to intact skin or even mucous membranes other than eyes. Where there is greater potential risk is if it comes in contact with an open cut on your skin, or if you are cut with the needle or scalpel that is working with the tissue. The most serious risk comes from entry into the eye.

This is where PPE is extremely important. Most of the exposure risk described above can be reduced by wearing safety glasses and mask or face shield, protective gear like a disposable fluid resistant gown, as well as two pair nitrile gloves with the addition of Kevlar gloves if you’re working with sharps. Change your outer gloves between tasks. Keep the area restricted to essential personnel. Trust us, no one is going to come near you if you put up a sign that says “Caution, processing CJD!”.

When it comes to your equipment, use disposable instruments where possible. Even disposable materials will need to be disinfected before disposing of them, so it is essential to know what is effective at decontamination. The chemical disinfectants you’re used to, including alcohol, formalin, hydrogen peroxide, ammonia, hydrochloric acid, are NOT effective. Neither are gaseous disinfectants like ethylene oxide or formaldehyde. Boiling, dry heat, or UV light are also not going to do the trick. So, what does work? Kill it with fire! Incineration is the preferred method for disposable instruments, materials and waste. For things that you can’t dispose of, autoclaving is recommended. Autoclaving will require longer times, and higher temperature and pressure. The chemicals used for cleaning are going to be sodium hypochlorite (bleach) and sodium hydroxide. For the bleach solution you will want to use 2% solution bleach, which means 2 parts of 5.35% bleach (most commercial household bleaches), and 3 parts water. Whether you use bleach or sodium hydroxide depends on what you’re decontaminating. Bleach is corrosive to stainless steel but does not corrode aluminum, while sodium hydroxide is corrosive to aluminum and corrosive to some stainless steel but less so than bleach.

The other chemical you will see used in conjunction with CJD is formic acid. As we mentioned, your traditional fixatives, such as formalin or glutaraldehyde do not inactive CJD, so CJD tissue fixed in traditional fixatives should be treated with the same caution as you would give un-fixed fresh tissue. We still need the fixation however to preserve the tissue. After NBF fixation, you must treat the tissue in cassettes with formic acid for 60 mins with continuous, gentle agitation, in a hood or in front of a back-draft ventilation. After the acid, the cassettes are transferred to fresh 10% NBF for 48 hours. Treatment with formic acid greater than or equal to 96% formic acid reduces the infectivity to negligible levels, but it doesn’t destroy all prions. This percentage is important, as a lot of the formic acids that are sold are between 85-90% formic acid and are not as effective.

If you plan on processing a CJD case, inform everyone; all sections of the lab, the pathologist, the safety coordinator. Check with your institution’s waste management team and infectious waste hauler as well to make sure you follow their protocols for disposal of the hazardous material, and to make sure they don’t have any restrictions that would prevent you from processing this tissue. You can find additional information on full safety precautions for CJD on the CDC and WHO websites.

The alternative to in house processing is sending it out. In 1997, the National Prion Disease Pathology Surveillance Center was established at Case Western Reserve University in Ohio. If your lab has a suspected CJD case, Case Western is a fantastic resource. They accept shipments of samples for diagnosis. Information about this service can be found on their website.