In the previous Blog “Dermatopathology 101”, a clear outline of material was provided which ensured the skin’s histology presentation as an organ in the final microscope slide. We discussed basic skin histology, and important aspects of embedding and microtomy.

In this Blog “Dermatopathology 102”, we will continue the discussion of the handling of skin specimens for dermatopathology to ensure the preparation of optimum microscope slides.

Tissue Processing

As mentioned previously, skin specimens are actually composed of three tissue types: epidermis, dermis and adipose.  Additionally, most skin specimens are small.  Given these factors, it is important to avoid over-processing skin specimens.  Over-processing may lead to tissues being hard and “scratchy” during microtomy.  The schedule detailed below for Routine Processing is optimal for all skin biopsies and excisions, as long as the pieces are no more than 3 mm thick.  Additionally, a Rapid Processing schedule is provided for skin specimens / biopsies and/or small tissues measuring less than 3 mm.  This schedule is ideal for “rush” biopsy specimens.

Routine Tissue Processing Schedule for Skin Specimens

Notes

• The above schedule is for a closed system, pressure vacuum tissue processor.
• Most skin specimens are small and submitted in formaldehyde. This procedure assumes the specimens are already fixed.
• All stations use pressure / vacuum.
• Paraffin stations only are heated.

Rapid Tissue Processing Schedule for Skin Specimens

Notes

• The above schedule is for a closed system, pressure vacuum tissue processor.
• Suitable skin specimens are small biopsies submitted in formaldehyde. This procedure assumes the specimens are already completely fixed and measure no greater than 3 cm in the greatest dimension with the remaining dimensions smaller.
• This procedure is not suitable for excisional specimens.
• All stations use pressure / vacuum.
• Paraffin stations only are heated.
• Microwave assisted processing of small biopsies may also be successful. The use of microwaves, combined with the omission of xylene, results in processing times of one to two hours.
• Specimens for demonstration of urate crystals (i.e. suspected gout) should be fixed in Carnoy’s fixative, or 100% alcohol, and processed from that point to prevent exposure to any water which may dissolve the crystals.

The principles of proper embedding for microtomy

There can be four main options of approaching the skin block by the microtomy blade, or four variants of skin specimens’ microtomy, as shown in Figure 1.

Dermatopathology is a subject heading in pathology all unto itself.  The intrinsic nature of dermatopathology specimens received in a laboratory necessitates a clear understanding of the material due to importance of the skin’s histology presentation as an organ. The goal of the histologist in the preparation of dermatopathology slides is to ensure that the entire area of skin which may contain pathology is represented in the final microscope slide.

Basic Skin Histology

Skin anatomy and histology must be understood by histologists, and is displayed in Figure 1. Two major reasons for this are:

• The pathologist must be able to see the dermal-epidermal junction. The vast majority of skin pathology takes place in this area.
• Skin is an organ system, composed of three major tissue areas: epidermis, dermis and sub-cutaneous (adipose). This has ramifications for processing and microtomy.

Dermis / Epidermis

Skin may be thought of as being comprised of two layers: an outer epidermis and lower dermis.  Adipose tissue may or may not be present below the dermis.

Notice in Figure 1 that the two layers interlock by folds in the epidermis (rete ridges) and dermis projections (dermal papillae).  The epidermis sits on the basement membrane, whereon rests the basal layer of the epidermis.

The morphology (anatomy and histology) of the skin varies depending on the anatomical site. For example, hair follicles extend through the dermis into subcutis (subcutaneous fat).  The skin of the face has numerous pilosebaceous elements, and the skin of the trunk has thick reticular dermis. The palms and soles have a thick compact cornified layer where usual basket-weave pattern is not seen. Muscle fibers are more prominent in the dermis of genitalia and areola. These anatomical particularities determine specifics in laboratory techniques. For example, longer fixation times and softening techniques may be necessary for specimens with a prevalence of keratotic structures.

From a technical point, the dermal / epidermal junction (DEJ) requires distinct presentation and should be clearly present in the final micro slide.  Additionally,

all skin layers on a micro slide ought to be equal in morphology presentation.

Embedding

It is always a good idea to standardize procedures in the histology laboratory whenever possible.  This provides the histologist with continuity in the work stream and results in increased efficiency.  Embedding should be standardized in that the epidermis should be facing away from the histologist, and located at a slight angle.  Additionally, the cassette back should be placed onto the mold with the number panel to the histologists left (Figures 2 and 3).

Figure 3 – The cassette back is placed onto
the mold with the number panel to the left.

Microtomy

When loading a block into the microtome for cutting, the block should be placed into the chuck with the number panel to the right.  This will insure that the knife cuts from dermis to epidermis; that is from the softest tissue to the hardest.  This will prevent tearing and “rolling up” of the epidermis.  Sections should be cut 4 to 5 microns thick, and a very slow stroke should be used to prevent the epidermis tearing away from the dermis.  Two millimeter punch specimens should be surface cut and examined unstained under the light microscope to ensure proper orientation.

Blocks of tissue that contain excess keratin and/or are very hard may be soaked in a solution of simple detergent.  This is done, of course, after the block has been faced off to expose the tissue.  After picking up the sections on a microscope slide and allowing them to drain, they should be placed into a conventional oven for 30-45 minutes at 60 C.  After cooling, they can be stained.

The above article is based on excerpts from CM Chapman and IB Dimenstein.  Dermatopathology Laboratory Techniques.  Copyright 2015.  In press.  Please contact the author at [email protected] to reserve a first print run copy of this valuable reference.

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. Luna L.   Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology.  3^rd ed., McGraw-Hill, New York, 1968, page 10.
4. Chapman CM. Dermatopathology: A Guide for the Histologist.  Copyright 2003.
5. Chapman CM and Dimenstein IB.  Dermatopathology Laboratory Techniques.  Copyright 2015.  In press.

Neuropathology is a histology specialty unto itself. Most hospitals and healthcare facilities will refer patients to a neuropathology center. Specimens can be procured by neurosurgery (i.e. tumor samples, brain biopsies, peripheral nerve biopsies, skeletal muscle biopsy) or post mortem (i.e. whole brain or spinal cord).

While your laboratory may not receive neuropathology specimens, there may be times when you might be consulted regarding fixation and processing of such specimens. Brain and spinal cord biopsies are tiny in nature, and the specimens are usually “sticky”. Parafilm or a piece of polythene, folded over to prevent drying, may be used to receive and transport fresh specimens. These may subsequently be frozen for rapid frozen section diagnosis.

Alternatively, 10% neutral buffered formalin may be used as a fixative for routine processing of larger tissue samples (i.e. post mortem specimens) into paraffin blocks. Subsequent processing protocols employ long processing times, to facilitate complete dehydration and infiltration of paraffin into the myelin.

It is important to note that brain, spinal and nerve tissue may contain transmissible spongiform encephalopathies (TSE) or prion diseases, which are neurodegenerative diseases that can affect humans and a variety of domestic and wild animal species. Prions are the causative agent of Creutzfeldt-Jakob disease in humans. The resulting holes in the brain tissue are remarkable (Figure 1 and 2). These infective prion agents are not inactivated by 10% formalin, so they remain potentially infectious, even after routine processing into paraffin. Formalin-fixed and paraffin-embedded tissues, especially of the brain, remain infectious. Treatment of formalin-fixed tissues from suspected cases for 30 min in 96% formic acid or phenol before histopathologic processing is considered to inactivate the prions, but such treatment may severely distort the microscopic neuropathology (Ref 4).
The safest method for ensuring that there is no risk of residual infectivity on contaminated instruments and other materials is to discard and destroy them by incineration.

The following is taken from two references: (1) National Geographic, “The Invisible War on the Brain.” February 2015, and (2) the “Neurologic Rehabilitation Institute at Brookhaven Hospital’ website.

“Military Brain Injury Study (Brookhaven Institute) Taking a play from the playbook of the National Football League, the US Military has created a “Brain Bank” repository program for the study of Traumatic Brain Injury (TBI) in veterans called the Center for Neuroscience and Regenerative Medicine Brain Tissue Repository for Traumatic Brain Injury. The donations are entirely voluntary and the study is not allowed to approach family members of the deceased to request donations to the study.

Since the launching of the study launched in June of 2013, the program has received numerous requests for information along with brain donations. The lab is attempting to collect several hundred brains from soldiers with and without a history of TBI. The goal of the research is to study the sections of the brain that are most susceptible to damage, as well as possible symptoms related to those damaged areas.

Per a recent article in Forbes magazine, Dr. Daniel Perl, the director of the repository cautioned “against the impulse to lump soldiers and football players together before the repository research is completed. Football players may bash their heads on a regular basis, but they aren’t exposed to explosions. “It’s important for us to not just say, ‘Okay he’s got tau, he’s like a football player.’”

Discovery of the similarities and differences between combat trauma injuries and those found in the NFL studies could prove to be very interesting.

 

References:

1. National Geographic. “The Invisible War on the Brain.” February 2015.
2. Neurologic Rehabilitation Institute at Brookhaven Hospital
3. http://www.traumaticbraininjury.net/military-brain-injury-study/
4. www.cdc.org

Whether you work in a hospital laboratory or a private reference laboratory, you probably receive specimens of prostate gland. In a hospital setting, the specimen may be received from a transurethral resection of the prostate, or a surgical resection of the entire prostate gland. In both cases, large pieces of prostate tissue are submitted to the pathology laboratory. In a private reference laboratory, usually needle core biopsies of the prostate are received (see Figure 1). These specimens usually measure approximately 1 mm in diameter by 1 cm long (Figure 2). Multiple needle core biopsies are usually procured from different areas within the prostate.

The prostate gland is comprised of many glands, with associated ducts, surrounded by connective tissue (Figure 3). The pathologist must assess the glandular areas and determine if there are any cancer cells present. Sometimes the diagnosis can be made with a hematoxylin and eosin (H&E) stained slide (Figure 4). Other times, the H&E slide may not be conclusive (Figure 5). In this case, immunohistochemical (IHC) stains must be performed.

A multi- antibody IHC stain can be used on prostate sections to assist in demonstrating if cancer cells are present. A high molecular weight cytokeratin cocktail of clones CK5 and CK14 are used to stain basal epithelia in the prostate gland. The P504S protein is known to be expressed by prostate adenocarcinoma cells, but not in benign prostate cells. It may also be produced by high grade prostatic intraepithelial neoplasia (PIN). Finally, the p63 antibody is added to the cocktail to detect normal cells, and is not produced by malignant tumor cells. The results of the staining patterns are used to confirm or rule out prostate cancer and prostatic intraepithelial neoplasia (Figure 6).

Initial receipt and handling of prostate needle cores must be done with care. The cores are small, fragile and easily damaged. Many times they are received fragmented. The exact number of cores and fragments must be documented during the surgical grossing procedure. These specimens must be wrapped, or contained in some way to prevent escape from the tissue cassette.

Processing may be done using routine tissue processing through alcohols, xylene and paraffin. A short biopsy protocol should be used to prevent over-dehydration of the tissue. Alternatively, a microwave assisted processor may be used. The omission of xylene during this processing protocol ensures that needle cores remain soft for optimal cutting.

The same care taken in surgical grossing must be utilized during the embedding procedure. Tissues must be unwrapped and handled gently to prevent fragmentation of the cores. Also, the cores must be embedded completely flat, to ensure accurate representation of the tissue in the final microscope slide.

Great care must be taken by histologists when cutting prostate needle cores. Usually, multiple, shallow levels are taken and picked up on 5 separate slides. Slides 1, 3 and 5 are stained with H&E. Slides 2 and 4 are held, unstained, for use in the IHC staining with the PIN 4 cocktail. Preservation of tissue is of the utmost importance.

The resulting stained slides are able to be used to obtain the maximum amount of information regarding any pathology present in these tiny specimens. Pathologists are able to assess and grade the prostate cancer, if present. This information is relayed to the clinician to help determine the treatment and prognosis of the patient.

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. www.mayoclinic.org