I don’t have formalin, what is the best substitute to transport a biopsy specimen……………………………………………

This article is part of the series “With love, Oral Pathologist”, they are love letters to oral surgeons, dentists, and clinicians who seek our services and who need a diagnosis to manage their patients.

One of the emergency calls I receive frequently from surgeons is, “I don’t have formalin, what’s the second best solution to use to transport the specimen to the laboratory?”. Before answering this question, let’s take a step back and understand “Tissue fixation” and “Tissue processing”.

(PS: If you are on the go and want an answer straight away for the above question, scroll down to the end of the article for the summary.)

Why tissue fixation and processing?

Many surgeons and clinicians are wonderful cooks and I am sure you have your encounters in the kitchen. Let me ask you a question, do you find tomato easier to cut evenly or a cucumber?

Yes, definitely a cucumber can be sliced more evenly with ease than a tomato. From the counters of the kitchen to a histopathology laboratory room, same rationale can be applied. The tissue embedded within a paraffin wax block or frozen block is easier to cut into thin 4 micron section using a microtome. The sectioned thin tissue section is laid on a glass slide and it is colored using stains like hematoxylin and eosin to be visualized under a microscope. In summary, tissue fixation and processing is a process of converting soft tissue in its original form from ‘a tomato like’ consistency to ‘a cucumber like’ consistency which enables the tissue to be cut into very thin sections.

Once a tissue bit is excised from the oral cavity, the tissue bit is cut off from its vascular channels. Thus, the tissue bit does not receive oxygen and nutrients, leading to autolysis and putrefaction secondary to the micro-organism from the external environment. Tissue fixation (using formalin or other fixatives) arrest any such changes within the tissue and makes it structurally as natural as possible. Chemically it also sterilizes the tissue by killing off the putrefactive micro-organisms. Autolysis is prevented and structure of the tissue is maintained by cross-linking or coagulation of the structural and other proteins.

This also makes the tissue more permeable to the subsequent reagents which are used to tissue processing and staining. To summarize, main property of a fixative is to disinfect/sterilize the tissue, kill all the microbes and fix the tissue in its original form.

Tissue processing: A journey of love and hate between tissue and water molecules

Tissue processing is a journey of the tissue from being hydrophilic to being hydrophobic so that paraffin wax, which is required to stabilize the tissue, can easily flow into minute spaces within the tissue. Molten wax is comparable to oil that is not submersible in water. After a biopsy, tissue is filled up with interstitial fluid and blood. Here the fluid and the blood is comparable to water and the paraffin wax is comparable to oil. The tissue is not submersible or compatible with paraffin wax, just like water and oil. Tissue processing is the process of making tissue more permeable to wax by making it hydrophobic.

The process involves passing the tissue bit through dehydrating solutions from 10% diluted alcohol to 100% alcohol gradually, at the end of which tissue loses residual water. The dehydrated tissue is now moved to Xylene for ‘clearing’ and later into molten paraffin wax containers where wax flows into lucent corners of the tissue. Finally, the tissue is embedded into the paraffin wax block which dries up a solid block of tissue. This block is mounted on microtome, a machine that slices the tissue into thin sections. The thin sections are moved onto to water bath from where they are picked up on glass slides to the staining booth. Here, the tissue sections are converted to be hydrophilic where it absorbs stains according to the pH of the tissue. Before it is mounted permanently on to slide and covered with a cover slip, it is made hydrophobic again! So, the process of tissue processing and staining is a journey of love (hydrophilic) and hate (hydrophobic) of tissue with water molecules.

The slide thus prepared is then placed under the microscope and the pathologist makes observations and provides a diagnosis. Refer the schematic diagram for visual understanding.

10% Formalin: The humble fixative

Commercially available formaldehyde is 37% in strength/concentration. When 10 ml of this solution is diluted with 90 ml of distilled water, you get 10% Formalin but technically it is 3.7% Formalin. However, the popular ‘10% Formalin’ name has stayed in usage even though it is a misnomer. It is similar to many misnomers in pathology that are too popular not to use it. You can order Formaldehyde or ready made 10% Formalin online or laboratory chemical retail and wholesale shops. The 10% formalin is available in chemist shop as well. Store the chemicals in a cool, dry place and make sure it is labelled and the content of the same is understood by the other staff in the department or the dental clinic.

You can carry 10% formalin in your surgery kit in airtight labeled bottle. Never leave your formalin in car especially on a hot day as fumes maybe released and there is risk of inhalation and lung complication. Remove the surgery kit or your bag from the car every time you park your car. Keep checking the formalin levels in the bottle and replenish it accordingly.

Where to procure 10% Formalin at times of emergency…when you don’t have it in your surgery kit

1. A popular chemist in your area will have stock of 10% formalin. Before planning a biopsy, a call or visit to the place will help.

2. You can visit a well-known laboratory in your place where 10% formalin will be available. You can borrow it once, and try your luck after that.

Nth hour, biopsy tissue is out and there is no formalin! Just remember, “SALINE IS THE ENEMY OF TISSUE PROCESSING FOR HISTOLOGIC SECTIONS”

Once the biopsy is out, wash it thoroughly under running tap water or a bath with saline to remove the blood around it. Now look around the clinic for there is always one alternative solution that is available in the clinic which is used for disinfection and can also coagulate proteins. Yes, you are correct! Surgical spirit containing the Methylated Spirit (95%) disinfectant solution can perform both these functions. Alcohol is considered to be a fixative but not as good as the 10% Formalin. As a post graduate, I had conducted experiments on animal tissue for fixation and processing artefacts and it was found that storing tissue in surgical spirit for less than 30 minutes and followed by regular 10% Formalin fixation does not change tissue architecture. On the other hand, it is observed that on storing tissue in saline for more than 15 minutes, the epithelium separates from the deeper connective tissue rendering the biopsy tissue useless for reporting. So if you are able to transfer the tissue to 10% formalin or send the tissue to the nearest laboratory or to an oral pathologist within 30 minutes, surgical spirit can be used as a temporary transport material.

Special conditions where formalin fixation does not help

1. Immunofluorescence: In cases like Pemphigus, pemphigoid or any vesiculobullous lesion where direct immunofluorescence is planned, avoid formalin. Here, plan for a biopsy and use “Michele’s Solution’ for transportation. For more details contact the author personally.

2. Molecular Studies: It is easier to extract RNA and DNA from unfixed tissues. Here, freezing the tissue helps. For more details contact the author personally.

Summary of the article: Give me the answer straight away!

The humble surgical spirit in the clinic can be used, surgical spirit not only disinfects the tissue by killing the microorganism and prevent putrefaction, it also fixes the tissue by the action of protein coagulation and arrests autolysis. This can be used as a transport medium for only 30 minutes and post that it needs to be transferred to 10% Formalin promptly.

Saline is the enemy of tissue processing, never ever use it as fixative or transportation media for histopathological examination.

Avoid formalin when you are sending the tissue for immunofluorescence examination in case of Autoimmune diseases like Pemphigus, Pemphigoid etc. Michel’s transport medium or freeze dried biopsy are used as formalin makes the tissue autofluorescence.

For DNA and molecular studies again use a freezing method as it is easier to extract from an unfixed tissue.