Every pathology department and associated histology laboratory must have a chemical monitoring program.  It may be part of a larger Chemical Hygiene Plan.  Laboratory employees must be kept safe by providing information on dangers, explaining the ways in which employees can protect themselves, and providing annual training to reinforce this information.  This is not just a good plan – in most instances, this plan is backed by laws and regulations.  This current blog will continue the ideas of the previous one, where we discussed engineering controls such as grossing hoods used in laboratories to provide safe work stations.

OSHA – The Formaldehyde Standard

OSHA uses different types of limits for airborne exposures, which reflect Permissible Exposure Limits (PEL). 

Time Weighted Average (TWA) is the airborne concentration averaged over eight consecutive hours.  The TWA for formaldehyde is 0.75 ppm.  No employee may be exposed to more than 0.75 ppm of formaldehyde over eight hours.

Short Term Exposure Limit (STEL) is the airborne concentration averaged over the worst 15 minutes.  The STEL for formaldehyde is 2 ppm.

OSHA has also set an Action Level for formaldehyde of 0.5 ppm averaged over 8 hours.  If this limit is reached or exceeded, the employee must be notified and steps taken immediately to adjust procedures that result in a decrease of the airborne concentration.

Employee monitoring is required to document the exposures existing in the laboratory.  Initial monitoring of each exposed employee is required, unless monitoring has been done previously for a particular job description.  Monitoring must be repeated if there is a change in work procedures or control systems since initial monitoring.  If an employee displays any exposure symptoms (i.e. respiratory, dermal, etc.) the employee must be monitored immediately.

If either the TWA or STEL is exceeded, the employer can:

• Install engineering controls, such as increased ventilation and/or use specially designed filters for the laboratory air.
• Change work practice controls, such as procedural changes.
• Provide and require use of respirators.

Periodic employee monitoring depends on the initial monitoring results.

• If exposure levels are at or above the Action Level of 0.5 ppm, immediate monitoring must be done for each employee or job description and continued until the issue is resolved.
• If the STEL is exceeded, immediate monitoring must occur and continue until the issue is resolved.

Periodic monitoring may be discontinued if two successive samples taken at least seven days apart are below the Action Level and STEL.

Recommendation:  Even if your laboratory monitoring results are satisfactory, and you are not required to perform any additional monitoring, it is recommended that you continue annual chemical monitoring for laboratory employees.  This benefits both the employer and the laboratory employees by demonstrating the commitment to a safe work environment. If your laboratory is larger than eight employees, you can select two employees from each job classification (i.e. accessioning, grossing, lab aide, histotechnician) to be monitored.  The results are valid for every employee in each job classification, as the tasks are the same for each.  For laboratories that have less than eight employees, it is recommended that every employee participate in the chemical monitoring process.

Required:  Safety goggles, gloves and an impervious gown, as well as access to an eyewash station and safety shower are required when handling formalin, regardless of the exposure limit.

Remember: 

  

The above label must be present on EVERY container and specimen bottle of formaldehyde. It is there for a reason.  Formaldehyde is a POISON and CARCINOGEN.  You must handle it with proper personal protective equipment (PPE) using a ventilated work station…always…without exception.

Histology laboratory personnel who change out tissue processors, and who perform the surgical grossing of specimens, are especially prone to formaldehyde liquid and vapor exposure.  Personnel must wear the PPE described above while handling formaldehyde.  Additionally, when pouring containers of formaldehyde (i.e. from on board tissue processors, into waste containers), employees should be performing this function in a fume hood to prevent exposure.  Alternatively, employees may be fit-tested for a respirator to wear during the handling of formaldehyde.  Goggles, rather than safety glasses, are the eye protection of choice, since they protect against splashes and vapors.

REFERENCES:

1. Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill  Livingstone, NY.  Fourth edition. 1996
2. Theory and Practice of Histotechnology.  DC Sheehan, BB Hrapchak.  CV Mosby Company, St. Louis. First edition. 1980.
3. Dermatopathology Laboratory Techniques.  CM Chapman, I Dimenstein.  2016.  Amazon.com
4. Dapson and Dapson.  Hazardous Materials in the Histopathology Laboratory.  Anatech Ltd.

Ok. I know the previous Chemistry blog may have traumatized you a bit. But hang in there – we’re almost done.

The dyes used in histology are chemical dyes that react in accordance with the basic rules of chemistry.  Whether you are performing a routine hematoxylin and eosin (H&E) stain, or a special stain for fungi, the staining methods are based on underlying chemical principles.

The most basic chemical principle is that of pH, which is a figure expressing the acidity or alkalinity of a solution on a logarithmic scale on which 7 is neutral, lower values are more acidic, and higher values more alkaline (basic).  The “p” stands for potential and the “H” stands for hydrogen.  pH is the negative log of hydrogen ion concentration in a water-based solution. Figure 1 shows the pH scale, with some examples of acidic and basic solutions.  Acidic solutions have extra “H+ “molecules, and basic solutions have extra “OH- “molecules.  Remember from the last blog? Rule #1 in chemistry is: opposites attract.

Basic and Acidic Dyes

The most widely used histological stains differentiate between the acidic and basic components of cells and tissues.  Basic dyes have a net positive charge. They bind to components of cells and tissues that are negatively charged.  Examples include: hematoxylin, phosphate groups of nucleic acids (DNA and RNA), sulfate groups of some polysaccharides (glycosaminoglycans) and some proteins (mucus)

Tissue components that stain with basic dyes are referred to as basophilic.

Acidic dyes have a net negative charge. They bind to components of cells and tissues that are positively charged.  Examples include: eosin and ionized amino groups in proteins (side chains of lysine and arginine).

Tissue components that stain with acid dyes are referred to as acidophilic.

The first use of a dye is credited to Antonie van Leeuwenhoek (1673) who worked with “saffron”, a natural dye extracted from saffron crocus to stain histological structures, but genuine work on histological dye staining started not before the second half of the 19th century when C Weigher, J Gerlach, P Erlich and H Gierke systematically studied dyes for histology. At that time coloring materials were still of natural origin from which carmine or cochineal was the most used dye.

The human eye is able to perceive wavelengths of light between 400 and 700 nm. Dyes appear colored because they absorb a particular wavelength in the visible region, and the eye senses the reflected light as the complementary color.

According to their sources, coloring agents are discerned as natural or as synthetic dyes. So-called general stains will dye the tissue uniformly (indifferently) while selective stains have affinity for special cell or tissue components. A new chapter in the history of staining began when WH Perkin discovered the first aniline dye (from extracts of coal tar) in 1856 which would revolutionize the dying industry.

Dyes can be roughly divided into acidic dyes and basic dyes. Basic dyes stain acidic components, and acidic dyes stain mainly basic components.  Histological staining is usually done by staining cut sections inasmuch as a dye in solution is offered to bind to defined tissue structures. Progressive and regressive techniques can be differentiated including direct and indirect procedures.

One can stain (operate) with mixtures of dyes simultaneously or successively in order to discriminate different tissue textures by the respective dyes used. So, double, triple and multiple stainings can be achieved.  Many dyes have only poor affinity to tissues, but this can be overcome by the use of metal salts. Those enhancing compounds are then called “mordants”.  A uniform theory of histological dye staining does not exist. This is because the mechanisms of dye binding to the different tissue components are quite heterogenous.

Hematoxylin & Eosin (H&E)

Hematoxylin and eosin, H&E or HE stain is the most commonly used technique in histology. This stain works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features.

Hematoxylin is a positively charged, blue dye complex. It stains basophilic structures, such as nuclei.  Eosin is a negatively charged, pink dye. It stains acidophilic (also known as eosinophilic) structures.  Some structures do not stain well with H&E. Hydrophobic structures tend to remain clear (such as those rich in fats).

The hallmark of an excellent H&E stain is the presence of all nuclei having been stained with precise differentiation, showing chromatin material, and nucleoli.  In addition, the eosin stain should result in three shades of pink: (1) bright pink-red blood cells, (2) medium pink staining of compact connective tissue and (3) light pink staining of loose connective tissue.  Figure 2 shows such and H&E stain.

If you have been able to follow the chemical principles in this blog, and the previous one, you should now have a basic understanding of the chemical principles underlying the fixation, processing and the H&E stain.  Additional personal experience with regard to resolving chemistry issues in your laboratory will add to your general knowledge of chemical principles.

 

References

1. Theory and Practice of Histological Techniques. Chapter 10.  JD Bancroft, A Stevens ed.  Churchill Livingstone, NY.  Fourth edition. 1996
2. Theory and Practice of Histotechnology.  Chapter 9.   DC Sheehan, BB Hrapchak.  CV Mosby Company, St. Louis. First edition. 1980.
3. CM Chapman. Histology Study Group. Presented at Region I meeting,     hosted by MaSH.  2014.

In order to be a competent histologist that has the ability to troubleshoot processing and staining issues in the histology laboratory, you must have a basic understanding of how atoms and molecules bind together and react.

Atoms are the smallest building blocks of molecules.  Atoms are composed of a nucleus, which contains protons and neutron, that are surrounded by electrons.  Protons have a positive charge, electrons have a negative charge, and neutrons are neutral – they have zero charge.  One of the basic rules in chemistry is: opposites attract.  This rule is the basis for atoms staying together: the negative electrons are attracted to the positive protons located inside the nucleus.

Back when I was in school (when the world was considered to be flat), atomic structure was likened to the planets orbiting the son: the nucleus was the sun, and the electrons orbited around it.  Today, the facts support the idea of an “electron cloud” around each nucleus.  Atoms may “share” their electron cloud with the same, or other different atoms, which binds them into molecules.

Figure 1 and 2 show how two separate hydrogen atoms bind together to make a hydrogen molecule.  Hydrogen atoms are very stable when sharing their one electron, and this principle extends to all atoms: they “like” their first binding level of to be filled with two electrons.  A carbon atom has a second, outer level that wants to be filled with eight electrons.  It has four, so is always “looking” for four other atoms to share electrons with (Figure 3).

Histology involves using formaldehyde to chemically “fix” dynamic, living tissue into a static “snapshot” of cellular activity. The cells in your body are currently metabolizing energy sources, and performing chemical reactions to ensure that all of your bodily functions continue, and you stay alive. When tissue is removed from the body (i.e. surgery or biopsy), the cells no longer receive oxygen from the blood, and the cells begin to die, and autolyze (i.e. break down). The fixation process “fixes” the tissue, and stops the autolysis process, thereby preserving the cellular structure and tissue architecture, for subsequent processing into a paraffin block.

At the molecular level, formaldehyde is a simple molecule, consisting of one carbon atom joined to two hydrogen atoms with a single bond, and one oxygen atom with a double bond (Figure 4) . Carbon is stable when it forms a total of four bonds. A double bond contains a lot of energy – similar to compressing a spring. The bond wants to “spring apart” to release the energy. It does this by “springing apart’ the double bond, to provide two single bonds, which immediately bind two other molecules. This is what is meant by the term “cross linking” fixation, as relates to formaldehyde. The formaldehyde molecule cross links molecules within the protein structure of the cells (Figure 5).

Why do we need to know this?

Because living tissues are made up primarily of carbon, hydrogen and oxygen, and these are the molecules of biochemistry. Histologists need to know the chemistry of fixation, processing and staining.

Once the tissue is fixed in formalin, the proteins within are cross linked and stabilized. The tissue is in a solution of 4% formaldehyde in 96% water – similar to the natural water content of the human body. In routine histology, the final goal is to embed the tissue in a paraffin wax block. Water and wax do not mix. In order to be able to infiltrate the tissue with wax, and embed it in a paraffin block, the water has to be removed; the tissue must be dehydrated.

Dehydration is usually accomplished by using a graded series of alcohols to remove the water, and replace with 100% alcohol. Alcohol and wax do not mix. Therefore, histologists can use an “intermediate substance”, to bridge the gap between alcohol and wax. For most laboratories, this substance is xylene – although there are now substitutes that can be used.

The molecular structure of xylene is shown in Figure 6. You can see that it is a “hybrid” molecule. The center is a “ring’ of carbon atoms, with alternating single and double bonds. The exterior is made up of single bonds to hydrogen. This unique structure allows xylene to mix with both alcohol and paraffin. This brings us to the second rule of chemistry: “like dissolves like”. The middle ring of xylene is described as “organic”, which is like the organic ring structure of paraffin. The exterior is a straight chain “inorganic” structure, which is like the structure of alcohol.

The principles above form the basis of understanding the chemical basis of fixation and processing of tissues in histology. Once you understand them, you can troubleshoot fixation and processing issues that will occur in your laboratory.

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REFERENCES:

1. Theory and Practice of Histological Techniques. Chapter 10. JD Bancroft, A Stevens ed. Churchill Livingstone, NY. Fourth edition. 1996
2. Theory and Practice of Histotechnology. Chapter 9. DC Sheehan, BB Hrapchak. CV Mosby Company, St. Louis. First edition. 1980.
3. CM Chapman. Histology Study Group. Presented at Region I meeting, hosted by MaSH. 2014.