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Safety with Special Stains

Maureen Doran, B.A., M.S., HTL(ASCP)

Histology laboratories perform a wide range of special stains that include multiple chemicals many of which have unique hazards. This workshop will cover the appropriate handling of dyes and reagents used in special stains. The discussion will consist of reviewing staining protocols to determine the hazards of stain mixtures. We will examine options for minimizing exposure without compromising quality by alternate stain protocols, engineering controls, and work practices and personnel protective equipment.

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Learning Objectives

  1. Recognize chemical hazards in histology staining protocols.
  2. Evaluate standard operating procedures for handling staining reagents.
  3. Select appropriate engineering controls, work practices & PPE.

Webinar Transcription

Maureen Doran B.A., M.S., HTL (ASCP):

We want to, in this presentation, recognize the chemical hazards in histology staining protocols. We're all aware that we use hazardous chemicals and the lab. However, do we consider the potential hazards of these chemicals and the mixtures that we use in our special stains? We also would like to evaluate or maybe reevaluate standard operational procedures for handling some of our staining reagents. And lastly, we want to make sure that we can select the appropriate engineering controls, work practices, and personal protective equipment to handle our stains safely.

Are we using a one size fits all safety protocol? And is the best that we can do? In our talk today, we're going to focus on several special stains that are used in histology. We want to look at some of the ones that are commonly used. We are going to talk about a few of the carbohydrates and connective tissue stains, along with Oil Red O that is used for fats and lipids. We will also cover stains for tissue pigments, microorganisms, like the Gram stain and AFB, and also for our fungi, which we usually use our GMS.

Exposure assessment

When we talk about the hazards of our staining protocols, we want to assess our exposure. Our exposure assessment, or analysis, involves looking at each chemical and considering the following questions. So this is the who, what, when, and where scenario. Who are the employees exposed? Is it everyone in our lab? Or are there tasks where only certain employees are exposed? So what are they exposed to? What are the chemical hazards? Are they characterized as flammables, corrosives or maybe carcinogenic? And how does the physical form of death chemical that we are using, maybe liquid, solid, or gas, affect the exposure? And when does this exposure occur? Is it continuous? Or is it intermittent? Or only perform any special stains at a certain time during the day? Where does the exposure occur? Mixtures may affect where it occurs. Maybe we’re using this in the main lab or maybe by a draft wall or a fume hood.

So when we evaluate our exposures, we want to look at the regulatory limits that define exposures in the workplace. There is agencies have reviewed chemical hazards and have set limits for exposures to those chemicals in the workplace. These occupational exposure limits are identified as threshold limit values. Two commonly used values are the time weighted average, abbreviated as TWA, and the short term exposure limit, STEL. The TWA is the average exposure to a contaminant to which workers are exposed. STEL is a time weighted average that occurs when we’re going over short exposure of our short period of time. Usually, this is 15 minutes. Remember that all of these exposures are going to exclude those individuals that are hypersensitive or maybe those that have preexisting conditions.

Alcian Blue

This is a stain that is used to demonstrate acid mucopolysaccharides and mucins. And you can see that we want to see them stained as this turquoise blue, as seen in this slide. This stain protocol has minimal hazards. Alcian Blue is an anthraquinone dye that contains copper. And it is difficult to dissolve. The dye powder is combustible, though, and it is an eye or respiratory tract irritant. The dye content of the powder is variable and it is unstable at room temperature is or higher. Storing this dye at 4° improves the stability of both the powder dye and also the working solution.

The working solution is corrosive due to the fact that we are adding acid to lower the pH. And so this actually brings our TWA to 10 parts per million. The next stain that we are going to look at is our aldehyde fuchsin. This is a Gomori’s recipe and this actually stains several structures, including elastic fibers and the beta cells and pancreas. We usually like to see this stain as a very dark fuchsia color. Aldehyde fuchsin contains 0.5% basic fuchsin and this is dissolved in 70% ethanol and we add 1% hydrochloric acid, making this solution both flammable and corrosive. Paraldehyde is a flammable solvent that is used to ripen the stain. It is also a severe skin and eye irritant. It is a sedative and is listed as a controlled drug agent and the United States. Basic fuchsin is used in several histology stains and it is characterized as a possible carcinogenic to humans. It only has a TWA of 5 mg. There is evidence that inhalation or ingestion of basic fuchsin can cause a blood disorder that impairs our hemoglobin. And we're going to talk a lot about basic fuchsin, since it is in several of our staining recipes.

Periodic Acid Schiff

We're going to move on to our periodic acid Schiff reaction for carbohydrate that demonstrates neutral mucopolysaccharides. We like to see them as a bright pink, if you see the staining here. Periodic acid is a corrosive and it’s a strong oxidizer. So this is our pretreatment. It’s extremely destructive to mucous membranes, eyes, and skin. Standard PPE includes goggles and a face shield. And that is required if there is any potential for exposure to those mucous membranes are eyes. Examples of potential exposure in our lab may be filling or pouring off the periodic acid from the stainer or weighing out the solid.

Chemical pneumonitis and pulmonary edema have been reported with chronic exposure to periodic acid. So periodic acid functions as the oxidizer of the carbohydrates, changing them to aldehydes. And this is where our Schiff’s reagent detects them. Schiff’s reagent is made up in the same percent of basic fuchsin as Gomori’s aldehyde fuchsin and it adds also hydrochloric acid. Except here, we are also adding 1% potassium metabisulfite. And this is a highly irritating material. It’s also an experimental tumorigen and all sulfites, which are derivatives of sulfuric acid, react with water or acids to produce a toxic and corrosive material. This indicates that we need to do this in a ventilated system. Remember, some individuals are dangerously sensitive to sulfites. Symptoms include facial flushing, bronchoconstriction, and shock. Basic fuchsin is incompatible with strong oxidizing agents, so it should not be combined with periodic acid for storage or discard purposes.

Carbol fuchsin

Here, we're going to talk about one of the most common staining procedures for the demonstration of acid fast bacillus. And this is our Ziehl-Neelsen procedure. Carbol fuchsin is the staining agent in the stain. It contains 5% phenol, 10% ethanol, and 1% basic fuchsin. Basic fuchsin is sometimes referred to as magenta for basic red nine. In 1850, magenta was one of the first synthetic dyes to be manufactured. According to IARC, which is the International Agency for Research on Carcinogens, occupational exposure to magenta during production is carcinogenic to humans, making it a category group 1. IARC reviewed these exposures in 2010 and found a 23 fold increase in the urinary bladder tumors and people engaged in the manufacture of magenta or basic fuchsin.

In Italy in 1985, bladder cancer was associated with employment in these industries, with potential magenta exposure. For all other exposures to basic fuchsin, IARC places it in a group 2B category as possibly carcinogenic to humans. So in this stain, we're going to mix our basic fuchsin with phenol. So let’s take a look at that phenol component of carbo fuchsin. Phenol has good warning properties but it has a distinctive odor has detected well below the permissible exposure limit of 5 ppm. Phenol is rapidly absorbed by the skin and dilutions of 1 to 2% can cause burns. Absorption of phenolic liquid can be very rapid and can cause death within 30 minutes to several hours by dermal exposure of as little as 64 cm. This statement is sometimes included on many of our SDSs. It’s pretty specific.

So get these details come from an actual case? Well, yes they did. A young man accidentally broke a bottle of dilute phenol in his pocket. The contents ran down his thigh. He attempted to clean the phenol off his pants with ethanol and he did not remove the pants. The ethanol actually helped the phenol absorption. He actually showed signs of systemic poisoning within five minutes. He died within hours. Lesser exposures to phenol can also cause damage to the liver, kidneys, pancreas, and spleen. So not only are we using concentrate on a dilute form of phenol that is similar to what this young man was using, we are actually heating the phenol. So by heating it, we are actually increasing the absorption. So we want to remember in this stain, we are using a possible carcinogen. We’re also taking it and we're increasing the temperature. And so we're increasing our exposure to this.

Phenol has higher risks in handling it at room temperature. So if heating the solutions, maybe we want to consider using secondary containment. For sure, we want to make sure that we have protective glove material and all our skin is protected. In this example here, you can see that the tech is using secondary containment when she is heating the AFB in the microwave. However, there are still areas of her skin exposed.

So proper glove protection is an important safety precaution when doing many of our special stains but especially with our AFB staining. There are a number of things to consider when selecting glove protection. Permeation rate, this is the diffusion or spread of chemicals through the glove material. Sometimes, it’s difficult to interpret our permeation rate when working with mixtures of chemicals because on the charts and the information we have, usually the chemicals are listed separately, not as mixtures. Sometimes the mixture can be more aggressive towards glove material than anyone mixture alone. Breakthrough time is the time required before appearance of the chemical on the inside of the glove. And remember, this may not be noticeable to you.

Gauge refers to the glove’s thickness. Usually, the thicker the glove, the better the barrier. And working with phenol, it’s recommended that we use 8 mm thickness nitrile gloves. Our normal gloves are 4 mm thick, so double gloving is an option here. Remember, if exposure occurs, remove contaminated gloves and or clothing immediately. Glycerol and vegetable oil are recommended for removal of dermal contamination of phenol because water and enhances the absorption. After cleaning with glycerol or oil, wash the contaminated area for 15 minutes with soap and water. In many of our labs, where there’s a lot of crossover of our duties, and some of us are still performing some cytology in the lab and we do the Ziehl-Neelsen sometimes on slides that we're looking for tuberculosis.

Just this year, a tragic accident happened in March at the Zimbabwe University. A young man who was a bright and upcoming student, Emmanuel Tsuro, died from severe burns when he was performing the Ziehl-Neelsen stain. A fire broke out and ignited and he had run down the hall. Somebody had actually tackled him and put out the fire. Unfortunately, he did die of burns a couple of days later. He had done this procedure many, many times. And they're not quite sure what caused the fire to occur. But let’s just remember that just because we perform something many, many times does not mean that that hazard still does not exist.

Gram staining

This is used to demonstrate both positive and negative bacteria. In microbiology, this is a quick and simple procedure. However, in histology, it is more difficult because we need to decolorize the tissue in order to visualize the stained bacteria. Most Gram staining recipes, and there’s many of them, use crystal violet, which is a toxic irritant as a powder but safely manageable in our 1% aqueous solution that we use in this stain. Most of the hazards involved in our Gram stain for us occur with our decolorization steps. The first decolorization solution in our Gram stain here is galecal [phonetic] solution, which contains 2% formalin and acetic acid mixture. So again, we have something that is toxic and then it’s also a corrosive. Any concentration of formalin over 1% falls under our formaldehyde standards and our guidelines for usage.

The picric acid acetone mixture, which is also used in this step, poses some unique hazards in our histology labs. Acetone is a flammable liquid and it has a high evaporation rate. Picric acid, on the other hand, is a nitro dye that is explosive when dry. So caution must be taken to assure that in this mixture, the acetone does not evaporate, leaving the dried by potentially explosive picric acid in the container. Many labs up have now substituted a safer tetrazine acetic acid solution to replace the picric acid and acetone.

Gomori’s iron

Gomori’s iron is a stain that we use quite often to detect iron. It’s a histochemical reaction that uses hydrogen, uses hydrochloric acid and potassium ferrocyanide to demonstrate ferric irons and ferritin. The acid lowers the pH below 2.0 and splits off the protein, allowing the potassium ferrocyanide to combine with the ferric iron to form the Prussian blue reaction we see here in the picture. However, lowering the pH of potassium ferrocyanide below 2.0 potentially produce dangerous hydrogen cyanide fumes. So we are doing this every day when we make up this solution because making up fresh. We add the acid to the potassium ferrocyanide right before we perform our Gomori’s iron. So our Gomori’s iron reaction should only be done in a vented hood. Using secondary containment when heating or transferring the reagent is recommended to prevent any inhalation exposure.

Grocott’s methenamine silver

Grocott’s methenamine silver is used to demonstrate fungus and tissue. Chromic acid is used to oxidize the fungal wall polysaccharides to aldehydes, which are demonstrated by reduction of an alkaline hexamine silver complex. In this stain, actually, our silver is fairly dilute. It’s only 0.25%. And we add this to a methionine solution off about 0.1%. Remember, if more than one gram of silver is accumulated in the body, it can cause a condition argyria to develop, which is a permanent cosmetic condition of the skin or maybe internal organs in which they turn a blue gray in color.

In our GMS, we use this methenamine in our silver solution and this is a flammable solid. Actually, it sublimes, meaning it converts directly from solid to gas. And that’s one of the reasons why, when you’re storing methenamine, and it’s not in secondary containment, you can always kind of smell that amine coming off of it. It is listed as an experimental tumorigen and there has been some human mutagenic data cited. To this methenamine silver solution, we're also going to add sodium borate. And remember, borates are poisons. They're often used in ant poisons and they do affect the central nervous system.

Chromic acid is our oxidizer in this stain. And it is one of our most powerful oxidizers. It is also a human carcinogen via inhalation. It is also a severe eye, skin, and mucous membrane irritant. Penetration of the skin can cause painless ulcers. They referred to them as chrome holes and they can have delayed healing. Chromic acid ignites on contact with alcohols. Chromic acid also can cause adverse effects to the skin, respiratory tract, and kidneys. It has only a PEL of 0.05 ppm. And there’s been a lot of information documented on exposure to chromic acid because of chrome plating and industry. But it wasn’t until 1990 that they realized even a small burn from chromic acid can cause systemic effects. So in 1990, a man sustained a severe burn measuring only 8 x 6 cm, with a 1% solution of chromic acid. In this area, he actually had to have the skin excised and have skin grafts. 10 days later after the incident, he actually was hospitalized and he exhibited systemic poisoning with acute renal failure.

Verhoeff-van Gieson stain

Verhoeff-van Gieson stain uses an iron hematoxylin to stain elastic fibers black. This working Verhoeff solution it’s made fresh by adding ferric chloride, a corrosive, to Weigert's iodine, which has a 5% alcoholic hematoxylin. So hematoxylin is one of our natural dyes and is used extensively in histology labs. It may be harmful via inhalation from handling the dried powder and actually might want to have some dermal protection when we are using this. It is considered an equivocal tumorigen, indicating marginal increase in neoplasms, which are chemically related. And there is some cumulative data cited. The iodine portion of our Weigert’s is a poison by ingestion and by inhalation. But this is not usually an issue due to the low volatility of iodine at room temperature. However, once opened, it is difficult to reseal. Secondary containment or sealing the container with a wax is suggested.

The counter stain that we use in Verhoeff’s is van Geison’s. In this stain, van Geison’s is a 2% of acid fuchsin and it’s dissolved in a saturated picric acid. So this is actually fairly concentrated. Our Bouin’s fixative contains one third less picric acid. The picric acid is that strong oxidizer that, again, is explosive when dry. The more concentrated the solution, the more likely it is to dry out. If storing van Geison’s solution, check periodically to assure that the solution has not evaporated. And make sure when we’re handling the container before resealing, we wipe off the threads of the lids and make sure that there is no outside contamination on the container. Because picric acid is such a strong oxidizer, do not discard with other reagents. Keep it separate.

Steiner stain

This is used to demonstrate spirochetes. In this stain, uranyl nitrate as traditionally used to sensitize the bacteria, making them able to precipitate silver from the silver nitrate solution. Uranyl nitrate is corrosive to the eyes, skin, and mucous membranes. It’s also a poison by ingestion and inhalation. Moderately soluble, it can be absorbed through the skin. Chemical toxicity causes irreversible kidney damage. It is also difficult to dispose of, at least in the US, because it is categorized as a radioactive by our Environmental Protection Agency. Many labs have successfully substituted zinc formalin as a sensitizer to replace uranyl nitrate. And yes, zinc formalin is still hazardous because it contains formaldehyde. However, most of us have formaldehyde already in the lab, so we have standard operational procedures in place for that chemical.

Masson’s trichrome

This is one of the popular trichromes used to differentiate muscle and collagen. Bouin’s fixative, which we’ve talked about before, which contains picric acid, formalin, and acetic acid, is used as a pretreatment in this stain. Many people that are using Masson’s trichrome will heat the Bouin’s as the pretreatment. So extreme caution should be used if we are heating our Bouin’s. Increased temperatures will increase volatilization of the formalin and acetic acid, thus increasing the potential exposure and also the potential for drying out of the picric acid. This stain also contains Biebrich scarlet, which we also mix with acid fuchsin. The Biebrich scarlet belongs to a chemical dye group called the benzidine-based dyes or nitro dyes. Dyes in this group are human carcinogens and pose increased risks of chronic toxicity. All the other components of Masson’s trichrome can be handled, usually, with our routine laboratory safety measures.

Standard operational procedure template for carcinogens

This differs from our other standard operational procedures because we have additional requirements, as we need to post warning signs. We have to limit access. We need to have designated work areas, and we have to keep usage records. And these are things that we do commonly with our formaldehyde. Along with these additional things, we have to comply with the global harmonization system on the labeling of our chemicals. This rule came in effect in December 2013 and we had until June 2016 to make sure that we were in total compliance.

Here is an example of a GSH label. This is for our formalin. So as you can see here, we are using our placards and we have three here, so we have the one that is actually the acute toxicity. That’s our skull and cross bones. Our carcinogen, so that’s our little man with a heart here, and our corrosive. Also, at a minimum on a formalin label, we must have the words danger formaldehyde, cancer hazard, and causes skin, eye, and respiratory irritation. This label would be on anything that has formalin in it that is greater than 1%. So those mixtures of special stains, which do contain formalin, must also have this label.

We also must post warning signs at all entrances and access ways in regulated areas where everyone levels exceed the PEL or the STEL. And this is just an example of the posting. In regards to examples of designated areas or sometimes referred to these as restricted areas, so the example here, an automated tissue processor, would be considered a designated area because it contains formalin in the retort. And it has a potential for exceeding the permissible exposure limit when the retort is open.

So in regard to our formaldehyde monitoring, we want to remember from our standard that we need to do initial baseline monitoring. And this is done anytime there is a potential for exposure of greater than 0.1%. So remember, you have to do this if there is a potential. Just because you're using your work practices, your engineering controls, and you're below that limit, you have to assume that if you have formaldehyde and you don’t have those engineering controls that they are still is a potential. You need to identify all workers exposed but all the monitoring can be done if you have one designated employee, as long as that employee is representative of the employees that are exposed. So that means you can pick one person. You don’t have to monitor everyone. But the person has to do the duties that all of the people would have done so that it’s representative of the exposure.

And when do we have to repeat this exposure? We need to repeat it if there’s a change in location or work practices that affect the exposure or if an employee complains of signs or symptoms of exposure. Also, with our formaldehyde standard, we have to have a training program. And so all employees possibly exposed to 0.1% formalin have to be trained. We have to do this at initial assignment or again, if there’s a change in exposure. It has to be accessible and at no cost to our employees. And it has to include an explanation of the contents and the safety data sheets. We have to describe the surveillance system, if that’s needed, and what’s on our medical disease questionnaire. We also have to include all the jobs with potential exposure, the standard operational procedures and controls, and what our emergency procedures are.

Congo red

Briefly, before we mentioned the Biebrich scarlet was a benzidine-based dye. So Congo red is also in this group. Actually, we use Congo red to demonstrate amyloid deposits that appeared as those pinkish plaques under light microscopy that are not actually positive until we view them under polarized light as apple green. Congo red actually is used more in the textile industry than in histology. It’s used as a cotton dye. So what are our concerns about these dyes?

Benzidine has been listed as a known carcinogen since 1970. It was one of the first chemicals for which an association of occupational exposure, an increased incidence of urinary bladder cancer in humans, was reported. As far back as in 1973, the United States halted benzidine production and restricted imports. And the late 70s, the IARC evaluated exposure to benzidine-based dyes in the textile industry. They used biological exposure indices to assess the exposure. These measurements indicate the concentration of the chemical marker. In this case, it was benzidine in the blood or urine. And this indicated exposure.

It was found that workers handling benzidine-based dyes with their protective equipment had increased levels of benzidine in their urine up to two weeks after exposure if and in their blood up to four months. In 1978, NIOSH concluded that all benzidine-based dyes should be considered as potential human carcinogens and the production, use, storage, packaging and distribution should be discontinued. The diagram you see here at the bottom corner is of the benzidine dye molecule. So researchers at this time theorize that somehow, this azo link that is between the two benzidine molecules was somehow released to free benzidine in the body.

So here, I’ve listed some of the most important agencies that define carcinogens. It is these agencies that dictate our regulatory guidelines and standards. There is not total agreement on all the listings between groups. But 90% of all the chemicals are listed in each one of these agencies. So when we are talking about human carcinogens, we’re talking about in the first group, the IARC, these are the group 1, which are known to be carcinogenic, and also group 2A and 2B. In the European Union, it’s also the ones that are in group 1A and 1B. And then, we have the National Toxicology Program, which lists them as known to be carcinogenic or reasonably anticipated to be carcinogenic. And then, the last one is the American Conference of Governmental Industrial Hygienists and they also have top two categories are confirmed human carcinogen and a suspected human carcinogen.

We're going to take a little bit deeper look into these benzidine-based dyes. We’ve been told they are carcinogenic. So let’s think about what that actually means. Cancer risks are difficult to evaluate because other factors, besides exposure, play a role in that risk. Genetics is an uncontrollable factor, where some of our individual habits, like diet, exercise, maybe smoking, those are all controllable habits. Occupational exposure we may feel is a little of both. One factor affecting routes of occupational exposure to benzidine-based dyes depends on the dye form. Is in a powder or a solution? Dye powders have increased inhalation exposure and solutions will have more of a dermal route of exposure.

When we look at these, we also want to consider, in our exposure, what’s the intensity? Is the concentration high? Do we add two mixtures which are solvents that may be are more absorbed through the skin or maybe increase the volatility? And again, temperatures, we increase the temperature it may increase our exposure. And if we have a high pH or a low pH, that may also increase the exposure. What’s our duration of exposure? Are we using this all day every day or are we just using it for brief periods? And again, our routes of exposure, so the form is going to affect whether it’s inhalation, ingestion, absorption, or injection. Our azo dye activity, we have found that that bond is broken. The intestinal bacteria, skin absorption through the bacteria on our skin and it actually will have systemic effects. And the target organ is usually the urinary bladder.

We want to remember that benzidine is one of those things that had a skin notation listed with this exposure. This warns us that is insignificant dermal absorption and a potential for systemic toxicity from that dermal absorption. So appropriate while protection and good work practices, such as you see here as washing your hands after you remove gloves, is essential. In 1983, NOISH published a publication called The Health Hazards from Exposure to Benzidine Dyes. They noted in this paper the relatively large particle size of the dye powders causes inhalation of the dyes to be deposited largely in the upper respiratory tract. And then, they are swallowed and ingested. The intestinal bacteria are capable of reducing the azo bond, releasing free benzidine into the body. The most effective control of dye powders were feasible is at the source of contamination by enclosure of the operation or a local exhaust system.

Here you see a balance and closure that is used to contain carcinogenic powders during the weighing and dispensing process. They are using things here such as benzidine-based dyes and also para formaldehyde, which is a powdered form of formalin. A very effective method also for reducing inhalation exposures of powders is eliminating the dye in the powder form and actually purchasing a liquid solution. This is an example of dye drift. This actually occurred in a lab that is not far from me. Here you see there is a balance enclosure over the scale. They had just recently gotten this enclosure. Before, they were waiting out there chemicals right here on the countertop and they, of course would clean them off after they were used.

Here you see there’s an old cryostat that had not been functioning. They had put it to the side because they needed to have it serviced. And so it sat here for a couple of months before they had gotten the balance enclosure. When it came time for them to take the machine out to be serviced, they suggested, let’s just wipe it down, make sure it’s clean. And here you can see the tech went over and wiped off the top of the cryostat. And here were many, many different colors of dyes. So these dyes had drifted without this containment from the balance area out all the way over to the machine over here on the wall. And they had no idea how long it had taken for these dyes to accumulate over here but it had to be less than two months. And so that makes you wonder how many of these dyes had been aerosoled that they didn’t know of and how many of these dyes were actually benzidine-based dyes.

If we're not able to have some kind of containment system and we’re weighing out dry powders, our last line of defense is actually our respirators. And remember, this is not the best scenario because when we’re using our respirators, number one, we have to have a respirator program. It includes a medical evaluation for the person that has to be wearing a respirator. It also includes fit testing. And when we’re doing this, only the wearer is protected in this area. So it has to be isolated. And also, we have to have very stringent controls for decontamination and that is done with the respirator on before we take it off.

Oil Red O

It is used to stain fats and lipids. It is usually used on frozen sections and this, too, is a benzidine-based dye. So our stock solution all of our Oil Red O it’s made up in isopropanol. Isopropanol is in the same family as ethanol and it is flammable. But it only has an exposure limit of 400 ppm, whereas our ethanol has an exposure limit of 1000 ppm. The Oil Red O dye is also a respiratory sensitizer and has been documented to cause asthma. We wanted to think about, when we talked about our exposure to these benzidine-based dyes and we talked about it at the industrial facility and you would think yes, I will agree that they, even if they have protective equipment, they were probably handling large volumes of these benzidine-based dyes.

And that is true. But in 1986, the American Journal of Medicine published an article on cancer risks among artists. The author stated continued exposure to small amounts of many different chemicals is not only relevant to chemist but also artists who use it dyes and pigments. It has been found that for this occupational group, bladder can start mortalities increased twofold over the general population. So let’s think about this. I’m willing to say that on my daily routine in my histology lab, I may not be exposed to the levels benzidine-based dyes as they were in the manufacturing industry. But I’m pretty sure that my level of exposure may be the same or more than artists that are using these dyes and pigments in their work.

So it’s unfortunate that NIOSH, in this study they did in the late 80s, included artists. They actually sent out 453 letters to places where they thought they tracked where the artist had bought these dyes, warning them of the cancer risks of these benzidine-based dyes. Unfortunately, they missed our population, which probably exceeded 10,000 at that time. We’ll go back to our Oil Red O. Here we have a solvent that is flammable and also would make it more absorption to our skin, again, we want to make sure we’re using good protection for our glove so that we protect our dermal exposure. Again, since it is a regulated carcinogen, we need to use it in a designated area. So we have a little sign saying that we're using carcinogens in this area. It can be a small spot in our fume hood. It could also be the entire lab.

Also, when we're using carcinogens, we want to make sure that they go by the guidelines and that we have the caution labels on where we're storing them. Sometimes we may have to put them in secondary containment and separate them from some of the other dyes and powders that we are using. When we're thinking about our safety, we want to remember that when we look at our safety, who is the key in this? It is us. It’s employee participation. Sure, we need our procedures and protocols and our chemical safety training and our chemical inventory. But if we are not going to adopt these procedures and take them seriously, our puzzle is not going to fit.

Question & Answers

First, are reagents with more than one safety symbol, for example, corrosive, explosive, and toxic, should we give them more importance in the storage cabinet? Also, some reagents have noncompatible symbols, those that shouldn’t be stored together. How should we proceed with those?

This is a complicated issue but when you're looking at reagents for storage, you're not going to look at the toxic part because that refers to you when you're using it. So you want to look at it, basically, from the storage part. So this comes out a lot, especially when we look at acid alcohol. We have this alcohol mixture with a 1% solution of acid in it. Actually, it’s the flammable because it’s mostly flammable. It’s usually 70 to 100% flammable with only 1% acetic acid in it. So actually, your acid alcohol should be stored with your flammables, not with your corrosives. Kind of look at it as a storage point and then, when you take it out, you're looking more asset toxicity portion of it.

Are we required to monitor for formaldehyde on all shifts?

That depends. If there is a different exposure to the formaldehyde on those different shifts, yes, you are. So if routinely, you leave your processing until the end or even if the person is on the morning shift before you come in has to change out the processor and that’s a different level of exposure, then yes, you will have to monitor on all those levels.

Can you repeat the name of the publication exposure to benzidine dyes?

Oh, yes, I can. Actually, there’s many of them and there’s one that’s put out called the Benzidine Dye Initiative and I think that was actually put out by the International Agency on Research for Carcinogens, IARC. The other one is a NIOSH publication from 1986. And all of these, you just have to Google them and they will pop up. Some of them are actually listed on the CDC and in the US. And OSHA also has several publications on benzidine-based dyes.

Is it acceptable to discard all reagents from special stains into one container from the automatic stainer?

It is not. So when you think about it, when we’ve talked about all of these reagents, number one, you don’t want to discard your oxidizers with anything else. And if you have picric acid, you want to separate that one exclusively because it has an explosive hazard. So if you only have one container for discard from your special stains, you are running the risk of having incompatible chemicals react with each other that could cause a potential danger. And from a financial point of view, you want to separate out those things that are going to be more expensive to discard. So you want to separate out your silver and your chromium and things like that because if you just put a small amount of that into a large container, you're going to pay for 55 gallons at the higher rate rather than taking it and putting it into a small quart container and only paying for a quart for that more expensive stuff to be disposed of.

Do I have to do exposure assessment for benzidine-based dyes if we use them in our lab?

Yes. So since anything that is considered a regulated carcinogen has to have exposure assessment in those different standard operational procedures. And so that would include your formal LAN annual benzidine-based dyes and also the basic fuchsin.

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About the presenter

Maureen Doran, B.A., M.S., HTL(ASCP)

Maureen Doran is a histologist/co-owner of Saffron Scientific Histology Services (SSHS). Prior to working at SSHS, Maureen worked at SIUC School of Medicine as the Director of the Histology Center for 32 years. She has presented numerous continuing education workshops, lectures and teleconferences for NSH, ASCP and the University of Texas Health Science Center in San Antonio.
Maureen is currently President of the Illinois Society for Histotechnologists. Maureen is a member of NSH Health & Safety committee and served as committee chair from 1996-2016. She received the SIU award for Civil Service employee of the year and SIUC Outstanding Civil Service Teaching Support Award. She received the Histotechnologist Of The Year award from the Illinois Society for Histotechnologists and was the recipient of the J.B. McCormick award by NSH.

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