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.

Thanks for reading and I welcome any comments.

Follow me on twitter (@skinpathology)

Keep an eye out for my website (www.skinpathonline.com)

Feel free to email me with any questions or queries on feedback@skinpathonline.com 

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.

Many thanks for reading and please leave any comments you wish.

Keep an eye out for my website coming soon www.skinpathonline.com

Follow me on twitter as well (@skinpathology)

My email is feedback@skinpathonline.com for any questions or queries.

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.

 

Thanks for reading and I invite any questions or comments, email me them if you want (feedback@skinpathonline.com)

Keep an eye out for my up and coming skin pathology website (www.skinpathonline.com)

Follow me on twitter (@skinpathology)

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. 

Thanks for reading and I invite any questions or comments, email me them if you want (feedback@skinpathonline.com)

Keep an eye out for my up and coming skin pathology website (www.skinpathonline.com)

Follow me on twitter (@skinpathology)

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.

PS. keep a look out for www.skinpathonline.com (up and running soon)

Follow me on twitter (@skinpathology) 

9ENPHVAHPJP9

Any questions / queries email me feedback@skinpathonline.com