Dermatopathology 102

Dermatopathology 102

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

STATION #

REAGENT

TIME (min)

TEMP

1 10% formaldehyde 30 ambient
2 50% alcohol 30
3 80% alcohol 30
4 95% alcohol 30
5 95% alcohol 30
6 100% alcohol 42
7 100% alcohol 42
8 100% alcohol 42
9 xylene 45
10 xylene 45
11 paraffin 30 60 C
12 paraffin 30 60 C
13 paraffin 30 60 C

 

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

 

STATION #

REAGENT

TIME (min)

TEMP

1 10% formaldehyde 5 ambient
2 50% alcohol 5
3 80% alcohol 5
4 95% alcohol 5
5 95% alcohol 10
6 100% alcohol 15
7 100% alcohol 15
8 100% alcohol 20
9 xylene 20
10 xylene 20
11 paraffin 20 60 C
12 paraffin 20 60 C
13 paraffin 20 60 C

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.

Blog-300x149

Dermatology 101

Dermatology 101

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-2-300x190

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.

Dermatology 101

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

Skin Cancer

In order to successfully prepare slides of skin specimens, the histologist must understand basic skin histology.  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 specimens are composed of three major tissue areas: epidermis, dermis and sub-cutaneous (adipose). This has ramifications for processing and cutting.

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.

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 (Figure 1).

Cells of the Epidermis

The basal cells that sit on the basement membrane divide and move up toward the surface of the epidermis as they differentiate into stratified squamous epithelium in a controlled fashion, replacing the epidermis as it is worn away, damaged, etc.  If they grow out of control, they become a basal cell carcinoma (BCC).  Similarly, if any of the squamous cells begin to grow out of control, a squamous cell carcinoma (SCC) may result.  Usually, these cancerous cells remain in place, and are removed surgically to cure the patient.  Very rarely, these cancers may metastasize and spread throughout the patient’s body.

Within the epidermis are many specialized cells, including melanocytes that contain the pigment melanin.  Each melanocyte is associated with several basal keratinocytes via dendritic processes.  The melanocyte transfers melanin into these cells.  The melanin provides protection from ultraviolet (UV) light to the underlying cells.  The more UV exposure, the more melanin is produced, and results in a darkening of the skin, or “tan”.  Excessive exposure to the sun, and UV light, can damage the DNA of all epidermal cells, causing them to grow and divide out of control.  This can result in a basal cell carcinoma, squamous cell carcinoma or malignant melanoma, depending upon the original cell type affected.  While BCC and SCC rarely metastasize (i.e. spread) in a patient, malignant melanoma has a very high rate of metastasis.  Thus, it is important to diagnose these cancers early and cure them by surgical removal.  As a histologist, we will see that you play a critical role in helping to cure patients of skin cancer.

Malignant melanoma is, historically, a treatment resistant cancer.  However, recently, there has been a breakthrough in treatment with the use of targeted therapies and immunotherapies.  Currently, metastatic melanoma can be treated with potentially curative treatment.

Melanoma is an aggressive and treatment resistant cancer, accounting for 75% of all skin cancer deaths.  The cancer arises from normal melanocytes, which produce melanin in the skin, meninges and mucosal epithelia.  Two main types of pigment are produced by melanocytes: melanin (brown/black) and pheomelanin (red) (Figure 2).  Melanoma risk factors include family history, immunosuppression, and ultraviolet light exposure.

A substance called “vemurafenib” was the first targeted therapy to show promise as a treatment for melanoma.  The treatment shows rapid stabilization of the disease in patients with the proper BRAF gene mutations, with limited survival (i.e. 5 – 7 months).

Immune response is important in patients with melanoma.  In fact, it has been noticed for years that patients undergoing immune therapy for a disease, may develop melanoma as a secondary complication.  Experiments using melanoma vaccines and treatments with nonspecific immune stimulants have proved to be not highly beneficial.  However, work with T cell treatments and blocking monoclonal antibodies have shown more promise.

There are certainly more successful treatments on the horizon as researchers work toward a therapy for patients with malignant melanoma.

References

  1. Lo and Fisher. The melanoma revolution: From UV carcinogenesis to a new era in therapeutics. Science. Vol 346, Issue 6212, pp. 945-949.  21 November 2014.
  1. Chapman CM. Dermatopathology: A Guide for the Histologist. Copyright 2003.

Bone Specimens

Bone specimens received in the laboratory can generate comments that range from:

“This block is impossible to cut!”… to …“This block cuts like butter!”

How can one tissue behave like Dr. Jekyll and the other like Mr. Hyde?  As with many things in histology, the answer is in the details.  Similar to dermatopathology, the histologist must understand the histology of bone tissue in order to produce optimal microscope slides.

Everyone knows that bone is basically hard and brittle.  This characteristic is due to the cell biology of bone growth and development.  Bone collagen is laid down in bands that are parallel to one another.  These bands, or lamellae, become mineralized with a polysaccharide containing calcium and phosphate which provides strength necessary in cortical bone to provide support to the skeleton.

Trabecular bone is mineralized in a similar fashion, however, many spaces remain where bone marrow is located.  The bone marrow stem cells divide and grow into the specialized blood cell components (Figure 1).

Bone is a dynamic, living tissue.  New bone is made by osteoblasts located on the surface of newly formed bone.  The most recent material is not mineralized and is referred to as the osteoid seam.  This material is mineralized later to form mature bone.  Osteoclasts are also located on the bone surface.  These are large, multinucleated cells responsible for “eating up” mature bone to release calcium into the blood stream.  If the balance between these two bone cell types is disturbed, disease may result.  Osteoporosis is a disease where the osteoclast activity outpaces the osteoblast activity; weak, porotic bones prone to breakage, can result (Figure 2).

The million dollar question in the histology laboratory is: How can we prepare bone specimens to be cut from a paraffin block on a rotary microtome, and keep the sections on the slide for optimal staining?

As with all specimens, the answer begins with proper fixation.  The “20 to 1” rule applies in that the bone specimen should be placed into unbuffered formalin fixative, in a volume that is 20 times that of the size of the specimen.  Then, since bone is very dense, the fixation time should be measured in days – not hours; with an average of 3-5 days of fixation for larger specimens.

Once the specimen is fixed, it must be decalcified.  Methods must be used that will remove the calcium from the bone tissue, to render it soft and amenable to subsequent processing and cutting.  There are methods for processing mineralized bone into plastic, however these methods are not within the scope of this discussion.

Before we discuss what can be used as decalcifying agents, we must understand that all act in the same way.  That is, the solutions are used to leach out and remove calcium ions from the tissue.  To that end, specimens should be placed into a decalcifying solution that is 100 times the volume of the specimen, thereby provided plenty of solution.  Since the solution will now become saturated with calcium during the process, it should be changed every 24 hours.

Large pieces of bone should be cut with a scalpel into smaller pieces as they become softer, yielding pieces of tissue not exceeding 2 mm in thickness.  Finally, the solution should be agitated using a rotary shaker, or stirring mechanism as shown in Figure 3.

So then, what decalcifying solution should be used?  Below are a few that are in use today:

Rapid decalcifer:  These solutions usually contain hydrochloric and/or nitric acid, and are used on bone marrow biopsies.  The advantage of rapid decalcifying over some hours is offset by decreased histology quality, and possibility of antigenicity damage.

Bouin’s solution:  The picric acid contained in Bouin’s solution can decalcify bone in small bone marrow biopsies, as described above.  The disadvantage is that picric acid is a dangerous chemical to use and dispose of.

10% formic acid in 10% unbuffered formaldehyde:  This method yields the best histology.  The disadvantage of this solution, is the time it takes to decalcify is measured in days.

Chelating agents:  These are mostly used in research applications, as histology and antigenicity are maximally preserved.  However, the time frame is measured in weeks.

The endpoint of decalcification can be determined subjectively, via manual manipulation of the tissue to determine softness.  Conversely, the chemical method detailed in the procedure “Determining the Endpoint of Decalcification, found later in this blog, can be used (Luna, 1968).  After decalcification is complete, the tissue should be rinsed thoroughly in running tap water.  Processing should be done using a procedure for large, dense tissues.

During microtomy, if there are times that the decalcification seems to be incomplete, the histologist may soak the block face in decalcification solution for 30 minutes to an hour.  Then, after rinsing, the first few sections should cut easier and be floated out on the water bath for section pick up.  Finally, microscope slides coated with 0.5% gelatin should be used to pick up sections.  This will help the sections to adhere to the microscope slide during staining.  Some eosin background staining on the slide may result, but it does not affect the final H and E stain.

By following the above guidelines, all of your bone specimens should now;

“Cut like butter!”

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. Chapman CM: Histology Hardball: Solutions for Hard Tissue.  Advance for Medical Laboratory Professionals: Vol. 24 No2, February 20, 2012.
  1. Luna L.   Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology.  3rd ed., McGraw-Hill, New York, 1968, page 10.
Neuropathology

Neuropathology

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.

fig1fig2fig3blastinbrain
 

 

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
Prostate Histology

Prostate Histology

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.

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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