[Histonet] HPS stain

[Sharon Allen]

Hi,
I would like to know if there are many labs out there doing the
"Haematoxylin, Phloxine, Saffron stain, routinely & how they like it.
We have been doing it & it is lovely when it works but can be very
unpredictable. I am aware of its hydrophobic properties & do what I can
to accommodate this trait. It is mainly the saffron part that is
temperamental.
Thanks
Sharon Allen
***@hsc.mb.ca

[Hermina Borgerink]

Hi Sharon,

During the sixties and seventies the HPS stain was our routine diagnostic stain. We never had problems with the saffron, but we did take care keeping it free from all moisture, including the alcohols prior to and right after the staining with saffron. Method below.

HPS – hematoxylin, phloxine, saffron

Any fixative. Paraffin.

1. Deparaffinize and bring to running water. Wash for 5 minutes.
2. Stain in Harris Hematoxylin for 5 minutes - stains nuclei
3. Wash in running water for 5 minutes.
4. Differentiate in 1% acid alcohol – removes excess hematoxylin
5. Wash in running water for 5 minutes.
6. Blue in ammonia water – blues nuclei
7. Wash in running water for 5 minutes.
8. Stain in 2% Phloxine B for 5 minutes – stains cytoplasm and connective tissue
9. Wash in running water for 5 minutes – removes excess phloxine from connective tissue
10. Differentiate through 1 change of 95% alcohol – finishes removal of phloxine from connective tissue
11. Continue dehydration through 4 changes of 100% alcohol – saffron is hydrophobic so all water must be removed from the section
12. Stain in Saffron for 5 – 10 minutes – stains collagen and connective tissue
13. Remove excess saffron through 4 changes of 100% alcohol.
14. Clear through 4 changes of xylene.
15. Mount sections with permount.


RESULTS:
Nuclei – dark blue
Cytoplasm – shades of red
Connective tissue – yellow

Hematoxylin
Distilled water - 500ml
Potassium aluminum sulphate - 60gm
Hematein - 1.25gm
Glacial acetic acid - 5ml
Make up potassium alum by adding 60gm to 500ml of distilled water. Heat and let come to a boil. Add 1.25gm of hematein (remove from flame when adding) and continue boiling gently for 10 minutes. Cool, while shaking occasionally. Add 5 ml acetic acid when cool. Filter and add 1 crystal of thymol to prevent bacteria. Store in a dark bottle.


2% Phloxine B
Distilled water – 500ml
Phloxine B - 10gm
Add Phloxine to water and dissolve. Add 1 crystal of thymol to prevent bacteria. Store in a dark bottle.


Saffran du Gatinais
Absolute alcohol - 1000ml
Saffron du Gatinais – 30gm
(or dried Spanish saffron in small amounts)
Add 30gm of saffron to 500ml of absolute alcohol and let ripen in a 60ºC oven for 48 hours. Pour off the alcohol and store in a dark bottle. Add another 500ml of absolute alcohol to the saffron and again let ripen for 48 hours in a 60ºC oven. Add to the first batch obtained. Keep away from all moisture.

Hermina

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu [mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Sharon Allen
Sent: Monday, February 09, 2009 9:56 AM
To: ***@pathology.swmed.edu
Subject: [Histonet] HPS stain

Hi,
I would like to know if there are many labs out there doing the "Haematoxylin, Phloxine, Saffron stain, routinely & how they like it.
We have been doing it & it is lovely when it works but can be very unpredictable. I am aware of its hydrophobic properties & do what I can to accommodate this trait. It is mainly the saffron part that is tem

[Gagnon, Eric]

Hi Sharon,

HPS is our routine stain (instead of H&E, which 98% of labs use). Not sure what part of the stain you are having trouble with, but most unpredictability usually comes in the phloxine/saffron balance.

Some things to watch for are:
-keeping your saffron covered, and I would suggest parafilm-ing it when not in use
-keep the alcohols before the saffron rigorously free of phloxine-contaminated alcohol, thus not allowing tainted alcohol to contaminate/dilute the saffron during staining
-getting all the background hematoxylin and phloxine out, which can be checked as you go along when you validate your method
-be diligent about the preparation of the saffron. In addition to Hermina's suggested method, we microwave the dry saffron, grind it, then boil the alcoholic mixture to remove as much moisture as possible

What do you use the stain for, i.e. differentiating muscle and collagen? As a routine stain, I understand its use in Canada is limited to a few laboratories in the east such as the Ottawa, Kingston, Sudbury areas. This is often because pathologists who trained here at Queen's Pathology like to take the stain with them. Having said that, many of our residents request slides stained with H&E, to familiarize themselves with that stain before exams.

Good luck, hope this helps...

Eric Gagnon MLT
Histology Laboratory,
Kingston General Hospital
Kingston, Ontario, Canada

[Robert Richmond]

The hematoxylin-phloxin- saffron stain, which supposedly originated at
the Montreal Neurological Institute, migrated from there to
Columbia-Presbyterian Hospital in New York City, where it was used as
the general oversight stain in surgical pathology at least into the
1960's. When I saw it in use there in 1966 they didn't seem to be
having trouble with it.

On the other side of Central Park, New York Hospital (Cornell Medical
Center) had used a Light Green trichrome stain as a general oversight
stain. I think they abandoned it in favor of H & E after Chandler
Foot's retirement in 1948.

Saffron is a natural dye derived from the stigmas of the saffron
crocus, Crocus sativus. Although it was (and is) extremely expensive,
it was used both as a textile dye and as a spice (in paella, for
example). It's sometimes seen (and smelled) as a component of
expensive curry powders - my wife and I have been experimenting with
Penzey's Maharajah curry powder, which is 2% saffron. The immense hand
labor of extracting all those stigmas from the flowers makes saffron
"the most expensive spice in the world".

Saffron as a histologic dye has traditionally been specified as
"safran du Gâtinais" - from a particular region of France - though I
am not certain that such a product still actually exists. I would
think that any good quality saffron would suffice (don't substitute
safflower, 'dyer's saffron'). You'll pay at least ten dollars a gram
for saffron, at Penzeys anyway.

Some recipes specified as many as seven changes of hot alcohol to
extract the dye, and some people used a reflux condenser for the
extraction. The alcohol extract has a strong medicinal smell which
some people find quite unpleasant.

(I have no connection with Penzeys.com, except that my wife and I have
gotten addicted to their shipments of very high quality spices.)

Bob Richmond
Samurai Pathologist
Knoxville TN