#pasd

Alright so sometimes in the course of a patient’s disease, they’re either going to have carbohydrates end up in tissues that aren’t meant to store them or have too few carbohydrates in places where they ought to be. A handy test for this is the Periodic Acid-Schiff test and/or the Periodic Acid Schiff with Diastase digestion. This isn’t to say that the PAS/D is a strict presence/absence test but it gets used that way fairly often depending on the case. 

First things first: What’s a carbohydrate? Well that’s a big chemistry question and if we wanted to deal with that mess we’d have gone to grad school. For the purposes of the HTL this post, carbohydrates are organic compounds such as sugars, starches, cellulose and polymers that are typically bound to proteins found in tissue. When we eat food, we break the complex sugars down into simple sugars such as glucose. What we don’t immediately use we store as glycogen. This glycogen *generally* should be found within liver and skeletal muscle tissue, specifically within the cytoplasm of those cells.

 Disease and uses- A healthy liver should be able to store glycogen up and release it as the body’s demand for it changes. We can use the PAS/D to look at how much glycogen the liver has and where that glycogen is within the liver.  Most of the disease you’ll see in medical histology regarding glycogen storage disorders are congenital and fatal. A good example is Pompe’s disease, in which the  patient has a mutation that causes them to be unable to convert glycogen back into glucose, causing an over-accumulation of glycogen within the skeletal muscle, cardiac muscle and liver; most people born with Pompe’s disease will die in less than a year. Other illnesses that effect glycogen storage are diabetes (low glycogen in the liver) and certain wasting disease. There are also some applications for differentiating tumor types on the oncology end of things; for instance you can use the PAS to differentiate secreting adenocarcinomas (should be PAS positive) from undifferentiated squamous cell carcinomas (should be PAS negative). I am told that these days we would use an immuno stain to answer this question but, you know, half of histology is actually Arcane Medical History so whatever. My favorite application of the PAS, however- one that could have saved my undergrad research project had I known what the hell I was doing but that’s another topic for another day- is its ability to stain for fungal cell walls. Fungal cell walls are made of chitin, a complex carbohydrate that stains nicely with PAS. Some of the techs in my current lab will automatically order PAS along with any silver stains they get on a suspected fungal case, just because it’s a nice way of double checking (especially if you’re looking at tissues with lots of argentaffin/argyrophil structures where the fungi could be obscured other things). 

Fixation- now if you’re in a hospital, chances are you’re just going to throw the tissue in question into a bucket for formalin and be done with it. And that’s not necessarily bad; tissue slated for PAS can be fixed in either aqueous or alcoholic solutions. Just remember that structurally, the carbohydrates you’re testing for are bound to the tissue proteins, so the faster you preserve those proteins the more honest and indicative the results of your PAS will be. If you know that this tissue is going to have to wait a while before going to processing, go for alcohol if you can. Formalin and most other aqueous fixatives will slowly leach glycogen from tissue because glycogen is water soluble. Of some note to pathologists, glycogen degrades rapidly postmortem, so get that liver out of there and into some sort of fixative asap of you’ll be missing that particular piece of the puzzle. The only real fixatives to avoid are those containing chromate, since these may over oxidize the tissue and result in a weak stain or cause resistance the diastase digestion in the PASD. 

Reagents and their purposes- Fairly straight forward. 

Periodic acid, aqueous, 0.5%. This will act as your oxidizer. It opens up ‘certain tissue elements’ (nice dodge, Frieda) and exposes aldehydes such as 1,2 glycol groups. At this concentration periodic acid probably will not burn you to death if you spill it on yourself but you should be extremely careful when preparing it from concentrate (under a hood, wearing goggles and gloves, full weenie regalia required, this is not fun stuff to get in your eyes). Remember: acid into water is the way that you oughta. Dispose of with lots of water down the sink. 

Schiff Reagent. This stuff is N A S T Y but you gotta use it. It binds to the aldehyde groups exposed by the periodic acid  and forms an intermediate colorless compound known as leucofuchsin. Schiff’s contains sodium metabisulfite, hydrochloric acid and basic fuchsin. Basic fuchsin is classified as a known carcinogen by OSHA so you need to make and use schiff’s under a fume hood whenever possible; most labs i have been in are not good about this and some even heat their shiff’s in a microwave, which causes techs to breath in even more vapors. Most labs need to dispose of used solution via a licensed waste hauler; do not flush down the sink. 

Sodium metabisulfite, aqueous, 10%. This will remove any excess schiff’s that’s hanging onto the tissue and prevent overstaining. At 10% it’s not horribly dangerous but it is a strong reducing agent and if you’re preparing it from powder you should do your mixing under the hood. dispose of down the sink with lots of water. 

Alum hematoxylin (counterstain). This is just hematoxylin that contains alcohol. Normal alcohol precautions apply (ie it’s slightly flammable and skin/eye irritant; normal ppe is fine). Dispose of down the sink. 

How it works, step by step:

First- are you doing a PAS or a PASD? If you’re doing a PAS, start at step 2. For a PASD, start here. You need some way to chop up the glycogen in your section, and that’s a job for an enzyme. Once upon a time we used malt disatase to do this, which is a mix of a bunch of different amylases. If possible, you should use alpha amylase because it’s the most specific enzyme for glycogen, aka your target. Now, if you’re anything like my lab, you have chill pathologists and cheap administrators, so what you gotta do is think real hard about sour candy and go ahead and just spit on the slide. I’m dead serious. You have perfectly good amylase in your spit, and on the off chance that you have free cells in your mouth the pathologist should be able to figure out what doesn’t belong to the patient tissue; it’s a thing that’s done. The first time I saw my instructor do it I almost walked out of the lab but. you know. When in Rome. We usually let ours digest for about 20 minutes, rinse real well in DI and then add the slides to the PAS group for the rest of the procedure.

oxidize your tissue with periodic acid. We use periodic acid because it’s not going to over-oxidize the aldehyde groups it creates when it opens the c-c bonds in the sugars. 

add schiff reagent. it will bind to the aldehyde groups creating an unstable, colorless compound (leucofuchsin) that resembles a partially opened ring. When you run warm water over the slide, the ring will close and this creates the colored reaction product (pink). 

rinse in sodium metabisulfate. some labs omit this step to save time/money and some labs do get good results just by rinsing for extra time in water but you’re better safe than sorry. Sodium metabisulfate should remove any excess schiff reagent. 

counterstain with hematoxylin. My instructor told me that some labs use fast green instead. I’ve never seen it done before but it strikes me as a good idea (who wants to look at a bunch of pink/purple on top of the schiff reaction? not me). 

Other important details:

Controls should be kidney (specifically the golmeruli and basement membranes) if you’re doing PAS and Liver if you’re doing PASD. 

Good results should show glycogen, neutral mucosubstances, epithelial sulfomucin and sialomucins, colloid, basment membranes and fungal cell walls. They will appear ‘bright rose’ (aka pink). 

A PAS and a PASD  of the same tissue  slides run in the same batch should be loose negative images of eachother; this is to say, the PAS will show the presence of glycogen in the section, while the PASD will show what the tissue looks like once that glycogen is digested away. This is not to say that a PASD is necesarily PAS negative, but rather that a PASD will show any PAS positive substances that are NOT glycogen. 

Before you use your schiff reagent you want to test it for freshness. Do this by pouring a small beaker (10ml should do) of formalin. Drip a few drops of schiff into the beaker. If it’s good to use, you’ll see a red to purple color. If it’s degraded, you’ll get a delayed reaction and an eventual dark purple to deep blue color. This a good test but remember: when it doubt, throw it out. It’s not worth having to repeat a day’s worth of PAS/Ds just because you don’t want to take ten minutes to make your reagents properly. 

Calling back to last week’s quiz on water quality, PAS procedures have been known to fail if the water you’re using to rinse is heavily chlorinated, since the chlorine can oxidize the schiff reagent and turn it back into basic fuchsin, giving you a false negative. I’m not sure what constitutes ‘heavily chlorinated’ but I figure you’d probably smell it and say, you know, maybe i shouldn’t put this precious sample into this nasty water and go talk to my supervisor before i ruin this poor sick person’s life. 

What-if list:

no PAS/D reaction occurs at all, including control- check your periodic acid. If oxidation fails, so does everything after it. 

faint of pale staining-you probably cut the tissue too thin. This is a little counter-intuitive but the thicker the section is, the less time you will need to stain it for in PAS/D. My theory is that staining time is strictly is a function of quantity of glycogen, and quantity of glycogen should go up with section thickness. There’s biochemistry afoot here but my ochem-failing-self is ill-equipped to suss this one out. have at it, you nerds. 

you still see glycogen on a PASD- give it more time in amylase and keep it around 37C. If you’re heating your slide too high you can degrade your enzymes (remember, most enzymes used in the clinical setting work at body temperature. Don’t go above that if you can help it). 

you have weird glycogen looking artifacts in places where glycogen 100% can’t be-should have used some kind of sulfurous rinse. this is what you get for cutting corners and being a cheapskate. are you happy now. look at your life, look at your choices. 

Alright that’s all for now. My instructor is traveling this week so I don’t know if I’ll have a quiz next monday but I’ll keep you posted. 

Peace!