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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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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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.

Thanks for reading and I welcome any comments and other tips.

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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.

I welcome any other tips and comments.

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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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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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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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The Need For Greater Education of Medical Scientists in Histopathology Laboratories

As you can tell from the title I am an advocate for the ongoing job-specific training of medical / biomedical scientists in pathology and in particular histopathology (as this is the area of which I am involved).

 

By “job-specific” I really do mean that. My experience with ongoing education of medical scientists within many laboratories involves mainly attending conferences which in fact do provide presentations on many interesting and varying medical subjects but do not translate into increased knowledge within the pathology sector that they are employed. Yet attendance at these conferences do fulfill the “ongoing education” condition of governing accreditation bodies. For example a conference attended by this author attended by many medical scientists of employed over all the different disciplines (eg. histology, microbiology, haematology), had many interesting talks (eg. the effect of a local major natural disaster and the providing of medical assistance from neighbouring countries), but he could not see how these talks would translate to increased laboratory knowledge beneficial to the conference attendee. 

It appears to this author that there is very limited opportunities offered to pathology laboratory employees which in turn is resulting in these employees not possessing an ever-growing knowledge base of their chosen discipline. Another example observed by this author is the huge majority of histopathology scientists not being able to recognise the simplest of skin tumours histologically (eg. BCC, SCC, melanoma), which is the ‘bread and butter’ of skin pathology.

What is the purpose of scientists being able to recognise tumours histologically I hear you say? If scientists can recognise these simplest of tumours, this talent can be put to a number of uses including cutting deeper levels on initial sections that are non-diagnostic before the initial sections are given to the pathologist thus increasing the efficiency of reporting. This example of course depends on the confidence of the pathologist to trust the scientist to recognise a case that requires deeper levels. 

Thank you very much for reading this post and hopefully you found it of interest.

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Sentinel Lymph Node Biopsy. Is it useful?

 I have recently read an article that caught my eye and somewhat relates to skin pathology.

The article was entitled  ‘Axillary Dissection vs No Axillary Dissection in Women With Invasive Breast Cancer and Sentinel Node Metastasis’ and was in the February 9th, 2011 issue of the Journal of the American Medical Association. Although this relates to patients with breast cancer I wonder if there will be any overflow of this argument into the ongoing debate over the use of sentinel lymph node biopsy (SLNB) with regards to patients with melanoma.

The results of the aforementioned study showed “among patients with limited sentinel lymph node (SLN) metastatic breast cancer treated with breast conservation and systemic therapy, the use of SLND (sentinel lymph node dissection) alone compared with ALND (axillary lymph node dissection) did not result in inferior survival.” This result has also been mirrored in multiple melanoma SLNB studies. Many people have and still continue to argue over the pros (eg. without a SLNB it is not possible to accurately stage melanoma according to international guidelines) and cons (eg. the impact of complete lymph node dissection (CLND) on a patient’s immune capability).

I wonder if in the future SLNB will remain standard practice or if it was a good idea at the time.

Many thanks for reading and hopefully this post will get some interesting comments and I am sure it will.

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PS. This is the link for the JAMA article (http://jama.ama-assn.org/content/305/6/569.abstract)