Grocott Hexamine-Silver Special Stain For Fungus – Method and Tips

The Grocott Hexamine-Silver special stain is the method of choice for a large majority of histopathology laboratories for the demonstration of all fungi. The formalin-fixed sections are exposed to chromic acid which reacts with fungal cell wall polysaccharide components to form chromic acid-aldehydes. These then reduced by a hexamine-silver solution at an alkaline pH. This causes them to be selectively blackened.  It should be noted that this method is not specific for fungi but rarely fails to demonstrate any fungi within the test tissue.

 

Below is the author’s method of choice.

Solutions

5% aqueous chromic acid (chromium trioxide)

1% aqueous sodium metabisulphite

Stock hexamine-silver solution = 100ml 3% aqueous hexamine + 5% aqueous silver nitrate.

Working hexamine-silver solution = 2ml 5% aqueous sodium tetraborate (borax) + 25ml distilled water. Mix, then add 25ml stock hexamine-silver solution.

0.1% aqueous gold chloride

5% sodium thiosulphate

0.2% light green in 0.2% acetic acid

Method

1. Take sections to water.

2. Treat sections with chromic acid for 1 hour.

3. Wash thoroughly in tap water.

4. Treat with sodium metabisulphite solution for 1 minute.

5. Wash well in tap water.

6. Wash well in several changes of distilled water.

7. Treat sections with working hexamine solution (preheated in coplin jar at 56 degrees Celsius) at 56 degrees Celsius for 10-20 minutes. Check control sections to see if fungi are a dark brown colour, if not return to solution checking regularly at 3 minute intervals until correct colour achieved.

8. Wash in several changes of distilled water.

9. Treat sections with 0.1% aqueous gold chloride for 3 minutes.

10. Wash well in distilled water.

11. Treat sections with 5% sodium thiosulphate for 5 minutes.

12. Wash well with tap water.

13. Counterstain with 0.2% light green in 0.2% acetic acid for 1 minute.

14. Wash well in tap water.

15. Dehydrate, clear and mount.

Tips

- As with all other silver stains wash everything that you are going to use thoroughly with distilled water.

- Store the stock hexamine-silver solution at 4 degrees Celsius away from sunlight. It will keep for 1-2 months. If a white precipitate forms give it a good shake and it should redissolve.

- Do not extend the time in chromic acid too long as this can over oxidize the carbohydrates to carboxylic acid and therefore not take up the silver stain.

- Do not reduce the time in chromic acid as this will lead to under oxidation and therefore no take up of the silver stain.

- The chromic acid can be reused but its efficiency will decrease after each use.

- If the control sections are failing to stain even after extending the staining time in the heated hexamine-silver solution you have more than likely forgotten to add the borax. If so, you can just add it and continue with the stain.

- If you have a large amount of silver precipitate over your sections it is probably due to using low-grade silver.

- If you forget the sodium thiosulphate step you will not realise until after retrieving the slide from storage further in the future, as the remaining silver not removed will react with sunlight turning black.

- Try to keep your counterstain fairly light as dark counterstaining can mask fungal elements.

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Perls’ Technique For The Demonstration of Haemosiderin – Method and Tips

Iron is absorbed in the duodenum by cells called enterocytes. It is then stored or combined with a transport protein molecule. This iron-protein complex is then taken to the bone marrow where the iron is incorporated into the substance known as haemoglobin which is involved in oxygen transportation.

Iron can be stored in the bone marrow and spleen in its ferric state (Fe³⁺) as haemosiderin when combined with protein. When haemoglobin is broken down by tissues this results in the formation of haemosiderin.

When there is excess iron in the body haemosiderin can be found deposited in organs that are involved with iron storage such as the spleen, bone marrow and liver. This condition is known as haemosiderosis. A condition called haemochromatosis exists where the body indiscriminately absorbs iron resulting in the deposition of copious amounts of haemosiderin in many tissues. Haemosiderin founds in histology sections is usually derived from the breakdown of damaged erythrocytes and can also be found absorbed by macrophages (siderophages).

The method used by the wide majority of histology laboratories for the demonstration of haemosiderin is the Perls’ technique. This method works by the hydrochloric acid (HCL) splitting off the bound protein which then allows the potassium ferrocyanide to bind with the Fe³⁺ and form ferric ferrocyanide (Prussian blue).

Below is the author’s favoured method.

SOLUTIONS

2% hydrochloric acid (HCL)

2% aqueous potassium ferrocyanide

1% aqueous neutral red

METHOD

1. Take sections to water.

2. Mix equals parts of HCL and potassium ferrocyanide and filter onto sections. Leave for 15 minutes.

3. Wash for 5 minutes in running tap water.

4. Counterstain with neutral red for 1 minute.

5. Dehydrate, clear and mount.

TIPS

- Always include a positive control.

- Stronger staining results can be found by carrying out step 2 at higher temperatures (e.g. 37-56°C). This can result in false positive results. This author has found room temperature to suffice.

- The washing step (step 3) should not be decreased below 5 minutes as thorough washing is required to prevent a heavy dye precipitate resulting from the neutral red counterstain.

- The author has found neutral red to be the best counterstain. Do not use safranin as this can stain the Prussian blue granules a dark purple colour.

- Always mount in a DPX-type mountant as other mounting media results in fading of the stain.

- A common artefact is the presence of blue granules on and around the section. This can be due to expired HCL or potassium ferrocyanide. It can also be due to iron contaminants in the tap water. 

This can be fixed by replacing all steps from the cutting of the sections to the mounting of the stained slide that involve tap water with distilled water.

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Modified Ziehl–Neelsen Stain For Leprosy Bacilli – Method and Tips

Leprosy bacilli in comparison with tubercle bacilli are much less acid- and alcohol-fast. The leprosy bacilli’s lipid envelope is also much more affected by the fat solvents traditionally used to dewax sections (i.e. Xylene). Due to these factors a modification on the standard Ziehl-Neelsen technique is used for the demonstration of leprosy bacilli. 

 

Below is the author’s preferred technique. 

SOLUTIONS  

Dewaxing solution – equal parts of liquid paraffin and rectified turpentine 

Carbol Fuchsin – as per standard Ziehl-Neelsen technique

 

Methylene blue counterstain – as per standard Ziehl-Neelsen technique

 

10% sulphuric acid 

METHOD 

1. Dewax in ‘dewaxing solution’ described above for 30 minutes. 

2. Blot dry and wash in running water for approximately 10 minutes. 

3. Stain with filtered Carbol Fuchsin for 30 minutes at room temperature. 

4. Wash well in tap water. 

5. Differentiate in 10% sulphuric acid until section is pale pink. 

6. Wash well in tap water.

7. Counterstain with Methylene Blue for 15 seconds. 

8. Wash well in tap water. 

9. Blot dry, clear and mount. 

TIPS 

- This author always puts sections on ‘sticky’ slides to prevent any floating off. 

- There are many variations on the ‘softer dewaxing solution’ for the modified Ziehl-Neelsen technique for leprosy bacilli including:

- Two parts xylene to one part vegetable oil / clove oil / groundnut oil / olive oil / cottonseed oil. 

- Residual oil on the section after washing prevents shrinkage of the section. 

- Place slides directly from heater into dewaxing solution as this helps quicken the dewaxing.

- Some methods use a weaker acid-alcohol solution for differentiation, but this author prefers 10% sulphuric acid as it is quicker. 

- Don’t be alarmed when the section is placed into the sulphuric acid as it will turn a black colour. It will return to a pink colour when placed back in water.

- Ensure the counterstain colour isn’t too intense as this can mask some leprosy bacilli and even turn them a purple colour. 

- This is author lets the sections dry after washing after counterstaining and then directly mounts them.

I welcome any other tips and comments.

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Lack of UV-A Protection In Daily Moisturising Creams

Came across another interesting article in the May edition of ‘The Archives of Dermatology’ 2011. The article is entitled ‘Lack of UV-A Protection In Daily Moisturising Creams’ on page 618. 

 

Ultraviolet radiation (UV) contains UVA, UVB and UVC subtypes. The major source of UV exposure for humans is sunlight. The earths ozone layer blocks approximately 98% of all UV radiation and the 2% which reaches the earths surface 99% is of the UVA subtype. UVB can cause direct DNA damage whereas UVA causes indirect damage of DNA via the formation of free radicals. Therefore it is important any sunscreen solution contains both UVA and UVB filters. 

This article reported the estimated long-range UVA1 protection of 29 creams.

Major points of note from the article include

- Most daily facial creams contain ingredients known as UV filters claiming broad spectrum UV protection.

- Sun protection factor (SPF) doesn’t reflect UV-A1 protection.

- UVA penetrates window glass whereas UVB is blocked, therefore women working indoors need to protect themselves from UVA exposure.

- Of the 29 creams 6 didn’t contain any UVA1 filters.

I recommend reading the full article as it is an interesting read. Below is a link to the article.

Lack of UV-A Protection in Daily Moisturizing Creams
Steven Q. Wang; Jacqueline M. Goulart; Henry W. Lim
Arch Dermatol. 2011;147(5):618-620

 

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Verhoeff Van Gieson Elastin Special Stain – Method and Tips

Elastin is a connective tissue protein which allows the tissues of the body to return to their original shape after distortion or stretching. Elastin fibres can be of varying size and diameter and are particularly well seen histologically in sites such as the lung, heart, blood vessels and the dermis.

 

Histological demonstration of elastin fibres (or lack of them) are important in diagnostic pathology for conditions such as arteriosclerosis, temporal arteritis and elastosis. Fine elastic fibres are not so easily seen on standard haemtoxylin and eosin (H+E) staining therefore special stains which demonstrate elastin clearly are vital.

There are many elastin special stain techniques such as Weigert-Type, Orcein, Aldehyde-Fuchsin and Verhoeff’s. The most common is Verhoeff’s technique of staining elastin due to its quick method and strong elastin colour result. Below is the author’s favoured method for demonstrating elastin which is a version of the Verhoeff’s.

SOLUTIONS

Verhoeff’s solution – (5ml 5% alcoholic haematoxylin) + (2ml 10% aqueous ferric cholride) + (2ml Lugol’s iodine) MAKE IMMEDIATELY PRIOR TO USE.

Note – Lugol’s iodine = 2g potassium iodine dissolved in ~4ml of distilled water, then dissolve 1g iodine, then make up to 100ml.

2 % aqueous ferric chloride

Van Gieson counterstain = (100ml saturated aqueous picric acid) + (1% aqueous acid fuchsin), boil for 3 mins then filter.

METHOD

1. Take sections to water.

2. Stain with Verhoeff’s solution for 15-20 mins.

3. Wash well in tap water.

4. Differentiate in 2% aqueous ferric chloride until only elastin fibres remain darkly stained.

5. Wash in tap water for 5 mins.

6. Counterstain with Van Gieson for 3 mins.

7. Dehydrate, clear and mount.

TIPS

- aim for slight under-differentiation as the Van Gieson stain will continue the differentiation though more slowly.

- dehydrate quickly as alcohol can leach some of the Van Gieson stain from the section. You can accelerate dehydration by blotting the section with filter paper.

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Persistent Melanocytic Nevi: A Review and Analysis of 205 cases

Great article in the June 2011 issue of the ‘Journal of Cutaneous Pathology’ regarding persistent melanocytic naevi. 

Good reading for those interested in the subject.

 

Things of note are

- female predominance (reason unclear)

- back is the most common site followed by abdomen then chest

- mean time between original biopsy then biopsy of persistent naevus was 9.7 months

- dysplastic naevi were most likely to recur

- persistent melanocytic naevi were more likely to be initially removed via shave biopsy

Link to the article below

http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0560.2011.01692.x/abstract

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Melanoma Research – Sex differences in survival of cutaneous melanoma are age dependent

Came across an interesting article in the latest issue of the Melanoma Research journal regarding differences in survival rates based on sex. It has been previously observed that women have a better survival rate for melanoma than men. This has also been observed in other cancers such as lung adenocarcinoma and colon cancer.

 

The study reveals that the slight survival benefit women with melanoma experience, disappears after the age of 60. This is mirrored, but also conflicts with other studies referenced within the article.

Proposed reasons for this female survival benefit include women being more prudent in the personal examination of the skin, women having a greater percentage of lower limbs melanomas which are associated with a better prognosis and immune gender differences.

Below is a link to the article abstract

http://journals.lww.com/melanomaresearch/Abstract/2011/06000/Sex_differences_in_survival_of_cutaneous_melanoma.11.aspx

 I recommend getting the whole article if it is possible.

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Periodic-Acid Schiff Diastase (PASD) Special Stain – Method and Tips

In my previous post I covered the Periodic Acid-Schiff reaction (PAS) special stain which is by far the most common stain performed in a routine histology laboratory. A variation on this technique call the Periodic Acid-Schiff Reaction with diastase digestion (PASD) is another commonly performed special stain which I will be covering in this post.

 

The variation on the PAS technique involves simply exposing the section to the diastase enzyme amylase prior to continuing with the standard PAS method. The term ‘diastase’ refers to any enzyme that catalyses the breakdown of starch into maltose the dextrose. The diastase enzyme acts by cleaving the a-glucosidic 1-4 linkages of starch or glycogen (aka animal starch) leading to the formation of maltose and dextrose (maltose and dextrose are water-soluble sugars). So when sections are pre-exposed to diastase before commencing the PAS technique the glycogen within the tissue is broken down into maltose and dextrose which are dissolved and washed away when the section is rinsed sufficiently in tap water. 

The diagnostic purpose of performing the PASD technique include 

-         the removal of glycogen to make it easier to identify mucins stained by the PAS technique

-         analysis of glycogen deposits within the liver 

-         highlighting a thickened basement membrane for example in lupus

Solutions

Diastase solution – 1 part human saliva to 9 parts distilled water. 

1% aqueous periodic acid – from PAS method.

Schiffs Reagent – from PAS method.

Method

1. Take sections to water.

2. Expose sections to diastase solution for 30 minutes at room temperature.

3. Wash sections thoroughly in tap water.

3. Continue with PAS method from step 2.

Tips

- Always run a PAS and a PASD control with every batch of PASD stains to ensure your diastase solution is working. This author has found a glycogen rich liver control to be most sufficient. This author also runs a PAS and PASD for all PASD requests.

- Commercial amylase is available instead of using a saliva solution. Commerical amylase sometimes requires different incubation temperatures and conditions so check this before using. This author has found that a saliva solution is easiest due to its ease of preparation, availability and plus it is free.

- Put all sections onto to ‘sticky’ slides ie. Superfrost plus slides or their equivalent as the saliva solution causing some lifting of the section from the slide. This is reportedly more prevalent in sections exposed to the commercial amylase solution.

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Periodic-Acid Schiff (PAS) Special Stain – Method and Tips

The Periodic-Acid Schiff (PAS) technique (and its numerous variations) is by far the most commonly performed special stain within the histopathology laboratory, therefore knowledge of its method is a vital arrow in any medical scientist’s quiver of knowledge.

 

The PAS technique is most commonly used to highlight molecules with a high percentage carbohydrate content such as mucins, glycogen, fungi and the basement membrane in skin. 

The PAS method works by exposing the tissue to periodic acid. This acts an oxidizing agent which oxidizes vicinal (neighbouring) glycol groups or amino/alkylamino derivatives. This oxidation creates dialdehydes.These dialdehydes when exposed to Schiff’s reagent create an insoluble magenta compound which is similar to the basic fuchsin dye within the Schiff’s reagent.

SOLUTIONS

Schiffs reagent 

1% aqueous periodic acid

METHOD

1. Take sections to water.

2. Expose sections to periodic acid solution for 10-15 mins.

3. Rinse well in tap water.

4. Expose sections to Schiff’s reagent for 10-15 mins.

5. Wash in running tap water for 5-10 mins.

6. Counterstain with a haemtoxylin for approx. 15 secs.

7. Differentiate (if necessary) and blue.

8. Dehydrate, clear and mount.

TIPS

- Periodic acid and Schiff’s reagent are easily available commercially prepared, the technique for self-made Schiff’s reagent is arduous by comparison but can be found.

- Keep your Schiff’s reagent out of UV light and refrigerated when not in use. Failure to do so will result in the loss of sulphur dioxide in your Schiff’s reagent leading to the solution turning from colourless to a magenta colour resembling the original basic fuchsin colour. When this happens replace your solution. Also keep your periodic acid solution refrigerated when not in use.

- The purpose of washing in running tap water after exposing the sections to Schiff’s is to intensify the magenta colour. This author has found that when the water has runs from a magenta colour to a clear colour the colour isn’t going to intensify any further therefore the washing in running water can be ceased. This may vary from lab to lab.

- There are numerous variations of the PAS technique (eg. PAS + diastase, PAS + Alcian Blue). This will be discussed in a further blog post.

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Alcian Blue Stain For Acidic Mucins – Method and Tips

The alcian blue stain is this author’s preferred stain for the demonstration of acidic mucins. The dye was originally used for the dyeing of cotton before being discovered as a by Steedman in 1950.

 

The alcian blue itself is a cationic copper phthalocyanine dye which stains mucopolysaccharides and glycosaminoglycans a bluish colour. Within skin, acidic mucins can be found in many differing conditions such as a mucinoma, lupus and alopecia mucinosa.

Below is the preferred alcian blue method of this author

Solutions

1g alcian blue in 3% acetic acid (check pH = 2.5)

1% safranin

Method

1. Take sections to water

2. Cover slide with FILTERED alcian blue solution and leave for 20 minutes

3. Rinse in tap water

4. Counterstain with FILTERED 1% safranin for 10-15 seconds

5. Rinse in tap water

6. Dehydrate quickly, clear and mount.

Tips

- this author prefers an alcian blue staining time of about 20 minutes but can be done within the range of 10-30 minutes if desired.

- by reducing the pH to 0.2 the stainer can select for only strongly sulphated mucins. A pH of 1.0 stains both weak and strongly sulphated mucins. If using a lower pH method be sure no to rinse in tap water between the steps for too long as this can affect the alcian blue staining.

- this author prefers safranin as a counterstain due to its crisper staining, but safranin leeches out quickly in the dehydrating alcohols therefore blot dry after counterstaining and quickly dehydrate through the alcohols.

- neutral red can also be used as a counterstain but this author prefers safranin as it stains quicker and has a greater contrasting colour.

- the alcian blue staining solution expires after approximately 6 months.

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Ziehl–Neelsen Stain For Acid-Fast Organisms – Method and Tips

The Ziehl–Neelsen (ZN) stain is a common standard stain which is readily performed in a majority of histopathology laboratories around the world. It was first described by Dr. Franz Ziehl and Dr Friedrich Neelsen, a German bacteriologist and a German pathologist respectively. The ZN stain is mostly used to identify acid-fast mycobacteria, the most important of which is Mycobacterium Tuberculosis, the organism responsible for tuberculosis (TB). The ZN stain also stains other organisms such as Nocardia.

 

As Mycobacterium are unable to be visualised on standard haematoxylin and eosin (H+E) and gram stains, the ZN stain was developed. It is based on the tubercle bacilli having a lipid-rich cell wall that takes up phenol-dye solutions (eg. carbol fuchsin, the main dye used in the ZN stain) and after subsequent differentiation, retains the phenol-dye.

Below is the method used by this author.

Solutions

Carbol fuchsin – (1g basic fuchsin in 10ml ethanol) + (5g phenol in 100ml distilled water), then filter.

Methylene Blue – 0.2% methylene blue

Method

1. Take sections to water.

2. Cover section with filtered carbol fuchsin for 20 minutes.

3. Wash well in tap water.

4. Differentiate in 1% acid alcohol until section is a very pale pink.

5. Wash well in tap water.

6. Stain with methylene blue for 1 minute.

7. Dehydrate, clear and mount.

Tips

- Before covering section with carbol fuchsin try covering the section with a little filter paper to reduce precipitate on the slide.

- Some methods still say to the slide to steaming temperature after covering it with carbol fuchsin. This author has found this of no use and is an unnecessary extra step, plus removes the hazard of using a naked flame.

- Before differentiation with acid alcohol wash slide with 70% alcohol for about 1 minute to remove a majority of the stain. This will reduce your differentiation time.

- Blot dry your slide after washing in water after the methylene blue counterstain. This will reduce your dehydration time and therefore result in less leaching of the methylene blue counterstain from the section.

- Some tap water contaminants have been described that stain with carbol fuchsin and are resistant to differentiation. These appear on a different focal plane to true acid-fast organisms within the section.

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Cutaneous Squamous Cell Carcinoma – Overview

Squamous cell carcinoma (SCC) is defined by the World Health Organisation as ‘a malignant neoplasm of epidermal (and mucous membrane) keratinocytes in which the component cells show variable squamous differentiation.’

 

Most SCCs appear on the areas of the skin which get the most sun exposure though this is not the only place which the can arise. SCCs can also arise on mucosal areas such as on the lip. Patients who have a pale complexion and those who do not tan readily are at a greater risk. SCC is very uncommon in the Black population.

The most important causative agent is sun exposure, more correctly UVB radiation. Others factors that have been incriminated include human papilloma virus (HPV) infection, ulcers, immunosuppression and radiotherapy. Patients with organ transplants are also at a greater risk. SCC can be fatal in some cases (most commonly found in Australia) giving rise to the notion that sun exposure, which causes DNA damage and also suppresses the skin immune system, plays a lead role in the cause of aggressive SCCs. 

As sun exposure is the major cause factor of SCC, it is no surprise that the forehead, ears, scalp, face, neck, back of the hands and lips are the most common places to find SCCs on the human body.

SCCs commonly appear as plaques/nodules with an elevated/indurated, crusty surface. The areas immediately surrounding the SCC show the typical signs of sun damage.

I have previously blogged about the prognostic factors of SCC, please click on the link to see more (Prognostic Factors of Cutaneous Squamous Cell Carcinoma)

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Superficial Basal Cell Carcinoma – A closer look

Superficial basal cell carcinoma (SBCC) is a growth pattern of basal cell carcinoma (BCC) which accounts for approximately 10-30% of all BCCs.

CLINICAL

SBCC presents typically as a reddish (erythematous) patch ranging in size from approximately 3 to >10 mm. They often have a fine pearly border with central superficial erosions. Pale areas within the lesion can be a clue to regression and the lesion may have a history of bleeding. SBCCs are most commonly found on the trunk.

HISTOPATHOLOGY

SBCC is seen as superficial collections of atypical basaloid cells originating from the bottom layer (basal layer, stratum basale) of the epidermis and projecting down into the papillary dermis. These collections are typically surrounded by a loose myxoid (mucin-like) stroma. Although histologically they have an apparent multifocal appearance recent 3D imaging techniques have found that a huge majority of these foci are truly interconnected and therefore not multifocal. Remember SBCC is also usually seen in conjunction with other BCC growth patterns such as nodular and infiltrative.

PROGNOSIS AND TREATMENT

Due to its superficial nature, SBCC has a very good prognosis and there is a wide range of treatments available. SBCC does have a high recurrence rate due to its margins being difficult to assess (this is attributed to its apparent multifocal appearance histologically). Treatments include topical chemotherapy (eg. aldara), photodynamic therapy (PDT) and curettage.

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Solar Keratosis and Its Histological Subtypes

Solar keratosis is defined by the World Health Organization as ‘a common intraepidermal neoplasm of sun-damaged skin characterized by variable atypia of keratinocytes.’

Subtypes that are recognised are hypertrophic, atrophic, acantholytic, pigmented. lichenoid and bowenoid. All subtypes usually display the common features of hypogranulosis, parakeratosis along with keratinocyte atypia confined to the bottom two layers of the epidermis (basal and spinous). Below is some of the histological features commonly seen in the subtypes apart from the features mentioned above.

HYPERTROPHIC

This variant exhibits hyperkeratosis, acanthosis, papillomatosis, rete ridge elongation, telangiectasia and parakeratosis. The parakeratosis can be seen alternating with the hyperkeratosis.

ATROPHIC

This variant exhibits epidermal atrophy, basal epidermal budding with adnexal extension.

LICHENOID

This variant exhibits exocytosis, keratinocytic vacuolation, keratinocytic apoptosis, colloid bodies, band-like superficial dermal lymphocytic infiltrate and pigment incontinence.

ACANTHOLYTIC

This variant exhibits acantholysis (with possible extension down adnexae), suprabasal clefting and dyskeratosis.

PIGMENTED

This variant exhibits increased pigmentation of atypical keratinocytes with associated dermal melanophages.

BOWENOID

Although most pathologists consider this Bowen’s disease, some say bowenoid solar keratosis exhibits less than full thickness atypia and sparing of follicles.

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Basal Cell Carcinoma and It’s Histological Growth Types

Basal cell carcinoma (BCC) is the most common skin malignancy and it’s incidence is on the increase. Below is a description of the four main different histological growth types and what is features are associated with each of them.

Superficial

Superficial BCC presents as a scaly, reddish patch ranging in size from a few mm to over 100mm. Due to this clinical appearance there is often confusion with psoriasis. Superficial BCCs are most commonly found on the trunk and account for 10-30% of all BCCs. Histologically they are characterised by superficial collections of atypical basaloid cells projecting from the epidermis or from the sides of adnexal structures such as hair follicles or eccrine ducts. Due to the 2 dimensional processing of histology specimens most superficial BCCs appear multifocal but recent studies using digital imaging techniques show that the tumours nests are actually all interconnected. Truly multifocal superficial BCCs do occur but these are less common. 

Nodular

Nodular BCC most commonly appear as pale, pearly nodules often with macroscopically visible dilated blood vessels coursing over the top of the lesion. Nodular BCCs are most often found on the more sun exposed areas of the body (eg. face and neck). Histologically they are characterised by large, solid lobules of atypical basaloid cells exhibiting a peripheral palisade and often invading as far as the reticular dermis.  Other commons features including the classical BCC retraction artefact and tumour cystic degeneration.

Micronodular

Micronodular BCC most often present as slightly elevated/flat pale lesions. They are most commonly found on the back. Histologically, micronodular BCC appears as an invasive BCC with the tumour islands between 3-10 cells in width (approximately the size of a hair bulb). These smaller tumour islands commonly exhibit perineural invasion. Compared to nodular BCC, the excision margins of micronodular BCC can be more commonly underestimated leading to a higher recurrence rate. 

Infiltrating

Infiltrating BCC presents most commonly as an indurated, pale lesion whose clinical margins appear poorly demarcated. They are mostly found on the face and upper trunk. Histologically they appear as diffuse cords, strands, columns of atypical basaloid cells infiltrating deep into the dermis and that rarely exhibit a retraction artefact or peripheral palisade. Due to the highly diffuse infiltrating nature of this tumour perineural invasion is extremely common therefore recurrences are common. 

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Prognostic Factors of Cutaneous Squamous Cell Carcinoma

Below is a brief description of the prognostic factors involved with cutaneous squamous cell carcinoma, ie. SCC of the skin.

Site

The actual site of the SCC provides much prognostic information, most importantly that SCCs from sites such as the ear, lip and sole have an increased rate of metastasis.

Size

The physical size of the tumour is also related to prognosis, generally speaking the larger the tumour the poorer the prognosis.

Invasion

If the SCC is seen to be invasive this reflects a poorer prognosis. The actual depth of invasion is again prognostic, ie. the deeper the invasion, the poorer the prognosis.

Tumour differentiation

Histologically SCCs are graded for their differentiation with them being either well, moderately or poorly differentiated. The poorer the tumour differentiation, the poorer the prognosis.

Other

There are many other factors which can affect the prognosis of cutaneous squamous cell carcinoma including immune status of the patient, HPV infection status, age and genetic predisposition.

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Non-melanoma skin cancer (NMSC) treatments

Below are the most common treatments available, plus a brief description, once a NMSC has been diagnosed (usually a basal cell carcinoma or a squamous cell carcinoma).

Surgical excision (standard) – the most common and preferred form of treatment. Good for nodular tumours with a sharply demarcated border. Has a high cure rate and is dependent on the closeness of tumour to the resection margin in regards to the pathology (ie. the closer the tumour is to the margin the more likely it is to recur). One let down is the bread loafing technique which results in only approximately 5% of the actual margins being visualised by the pathologist for assessment of complete excision.

Moh’s /  Moh’s Micrographic Surgery – form of treatment with the highest reported cure rate (~97-99%). This technique results in the entire peripheral margins and the deep margins being visualised and assessed for completeness of excision (this is why the cure rate is so high). One let down is the time-consuming nature of the technique and the specialised training that is involved.

Topical chemotherapy – the most common available topical chemotherapy agents include 5-fluorouracil (5-FU) and 5% imiquimod. Generally speaking 5-FU works by inhibiting DNA replication therefore the growth of the tumour and imiquimod works by modifying the local tumour immune response of the patient. Advantages include the non-invasiveness topical therapy. Disadvantages are that, used alone, they can only be used on superficial tumours and not invasive tumours. Experimentation of their use in conjunction with other treatments (eg. curettage then topical treatment, or topical use to reduce tumour size before excision) have resulted in reports of higher cure rates.

Curettage +/- Electrodissection – put simply the tumour is physically scrapped away then the treated area is exposed to an electrical current which results in the softening of the skin and the procedure is repeated until the treating physician is satisfied excision is complete. The curettage portion technique can be applied alone without the electrodissection. This technique is usually reserved for site which are cosmetically unimportant (eg back). The cure rate is dependent on how aggressive the technique is applied (ie. the more the aggressive the higher the cure rate) and the growth type of the tumour being treated (ie. the more invasive the tumour the lower the cure rate).

Cryotherapy – one of the older treaments for NMSC which involves treatment of the tumour most commonly with liquid nitrogen. Cure rate can be high but there is reduced tumour margin control resulting in a higher recurrence rate.

Photodynamic Therapy (PDT) – fairly new technique which involves applying a topical photosensitizer to the target tumour and then exposure of the target area to light. This results in the production of aggressive chemicals which damage the cell causing death. Disadvantages include the ineffectiveness on invasive and thicker tumours due to lack of light penetration, and high cost. Advantages include the non-invasiveness of the technique.

Radiotherapy – usually reserved for older patients or where the surgical removal of the tumour is not a viable option. Has a reported cure rate of approximately 80-95%. Tumours recurring after radiotherapy are generally more aggressive and can become radiotherapy resistant.

I welcome any comments or other therapies you have encountered.

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Melanoma – Prognostic Factors – Quick Overview

Listed below are the most important prognostic factors with regard to cutaneous melanoma.

AGE – the older the patient, the poorer the prognosis.

SEX – generally females have a better prognosis than males.

BODY SITE – melanomas on the extremities (eg. legs and arms) have a better prognosis than those on the neck, trunk and face.

INVOLVEMENT OF LYMPH NODES – presence of tumour lymph node involvement has a poorer prognosis. Generally the more nodes involved the poorer the prognosis.

TUMOUR THICKNESS – the thicker the melanoma the poorer the prognosis.

ULCERATION – the presence of ulceration indicates a poorer prognosis.

MITOTIC RATE – the higher the mitotic rate the poorer the prognosis.

REGRESSION – presence of regression in thin melanomas indicates a poorer prognosis.

There are a few other prognostic factors such as Clarks level, tumour-infiltrating lymphocytes, BRAF mutations and LDH (lactate dehydrogenase) serum level.

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Special Stains for Amyloid - Method and Tips

Amyloid is a common substance found in the skin, in association with a number of disorders including, lichen amyloidosis, macular amyloidosis and also basal cell carcinoma. Amyloid results from the death of cells (apoptosis).

The presence of amyloid maybe diagnostic (as in the case with lichen amyloidosis) or coincidental (as in basal cell carcinoma where its has no prognostic significance). Since the presence of amyloid is sometimes coincidental there is no need to do a special amyloid stain as it would not add any diagnostic value.

Amyloid can sometimes be easily recognised on a standard H+E stain as amorphous eosinophilic material, especially if it is ubiquitous in the sample. If only small amounts of ?amyloid are present (as may be in the case of lichen amyloidosis) this is where the amyloid special stain can come into play. Since the presence of amyloid can make or break the diagnosis the scientist needs to ensure his/her method and technique is up to scratch.

The Congo Red method, which requires light polarizing equipment, seems to remain the gold standard amongst most laboratories with the thioflavin method also popular. I have included below my favoured method for amyloid as is it very quick, only needs light microscopy and produces a very good visual result.


Crystal Violet
1. Sections to water
2. Stain with crystal violet solution (same as one used in Gram stain) for 2 – 3 mins.
3. Wash in water then diff in very weak (~0.2%) acetic acid for about 5 secs.
4. Wash in water and mount using aqueous mounting media
5. If wanted seal coverslip around the edges with nail varnish.

This is a metachromatic stain with the amyloid appearing pink/purple and the surrounding tissue staining purple.

I would invite anyone to submit their favoured amyloid staining technique along with its advantages and disadvantages.

 

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Cutaneous Spindle Cell Tumour Immunohistochemistry Panel

This post is about cutaneous spindle cell tumours and their immunohistochemistry (IHC) profiles. I get many questions from laboratory staff about why we perform certain IHC and in what situations are they used. Since spindle cell tumours require IHC for their correct specific diagnosis I thought I would do a post on them (plus I was also involved in a recent published journal article about this very subject).

Cutaneous spindle cell tumours include atypical fibroxanthoma (AFX), spindle cell melanoma, leiomyosarcoma and spindle squamous cell carcinoma. Why can’t the pathologist diagnose these tumours simply on H+E without the need for IHC? Well, cutaneous spindle cell tumours look extremely similar on H+E and the effects of misdiagnosing a spindle cell melanoma (which obviously is extremely serious) as, for example, an AFX (which has a rather benign, indolent clinical course despite it’s alarming histopathology appearance) can be diastrous for the patient. 

Below is a table which provides an example panel leading to the diagnosis of AFX and the reasons for the use of each particular antibody.

S100 – Negative. Essential to exclude melanoma.  Also highlights Langerhans cells, which can be prominent in some AFX.

Melan A - Negative    Optional, if S100 is negative.

HMB45 – Negative    Optional, if S100 is negative.

Broad spectrum cytokeratin (e.g. MNF116, AE1 & 3) – Negative   Beware of included normal adnexal structures or hyperplastic epidermal downgrowths.

34betaE12 – Negative    Highlights some squamous cell carcinomas more prominently than broad spectrum cytokeratins.

Smooth muscle actin – Positive    ~75% of AFX tumors are positive.

Desmin – Negative    Useful to exclude leiomyosarcoma in actin positive cases.

CD68 – Positive    ~90% of AFX tumors are positive.

CD10 – Positive    Although most AFX are positive, the specificity of this antibody is low, making it of limited discriminatory value.

At a minimum an S100 ( to exclude melanoma), CD68 (positive for AFX), a keratin (preferably 34betaE12 as this stains most spindle squamous cell carcinomas) and desmin (positive for leiomyosarcoma), should be performed on all cutaneous spindle cell tumours. 

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