Histology Apology

Chaotic Neutral Arcane Lab Science Study Blog

[histologyapology]

Any old fool can cut themselves on a blade. Now, maiming yourself on the blade holder? That takes true grit!

[biomedicool]

Lymph Nodes

Lymph nodes are organs with a collagenous capsule, which are frequently found within adipose tissue.

They are strategically placed along the lymphatic network. Here they can trap foreign material (antigens), which are presented to the lymphocytes by antigen-presenting cells, to initiate an immune response.

The cortex contains primary lymphoid follicles and secondary follicles, which contain  densely packed lymphocytes. They have a distinct mantle zone and a paler-staining germinal centre.

The tissue is organised both to facilitate the interactions needed to generate an immune response against the antigen, and to promote rapid division of the responding lymphocytes.

Nodes vary in size (from a few millimetres to 1–2 cm)

Distributed in different areas of the body.

Linked in chains by lymphatic ducts, so that fluid flowing out of one lymph node via the efferent lymphatic vessel becomes the inflow to the next in line, via the afferent lymphatics.

Fluid = lymph,  derived from the tissue that carries cells and foreign material to the lymph nodes. 

Lymph returns to the bloodstream via one of the body’s two major lymphatic ducts.

Can be considered guard posts that are strategically placed to intercept any infectious agent that enters an area of the body. 

So, for example, the lymph nodes in the axilla of the arm (the armpit) will intercept infections which enter that part of the body.

Lymphocytes located in the local lymph nodes are responsible for the initial recognition of the infection and the development of the immune response. 

Once the immune response has developed, cells will migrate out from the lymph node into the blood 

move to the site of infection to combat the pathogen there.

Lymph nodes have a well defined structure with different sub-regions. 

Antigens and cells enter the node through afferent lymphatics

cells and fluid leave through the efferent lymphatic. 

Cells (lymphocytes) can also enter the node from the blood by migrating across the specialised high endothelial venules. 

Within the node, cells distribute themselves to distinct zones. 

B cells proliferate and develop within the follicles of the cortex,

T cells are primarily located in the paracortex. 

The capsule, medullary cords and hilus are fixed structural elements of the tissue.

[badgerofshambles]

There is one other Nerd™️ in my lab and yesterday I blew up a rubber glove and balloon-animal’d three of the fingers into a shape and gave it to him like look!!! I made an sp2 hybrid orbital glove turnkey!!! And the look of utter disappointment he gave me was just. Life changing. I will be chasing that level of condemnation for the rest of my life, believe you me

[ihearthisto]

🔬 Histological Holiday Family Fun! 🍗

You’ll be the envy of the party if you bust out this board game after the turkey dinner!

Player 1: Is your tissue pink? Player 2: duh..yes ๏̯͡๏  Player 2: Does your tissue contain cells that release matrix vesicles from their surface which aid in the formation of hydroxyapatite crystal between extracellular type I collagen fibrils? Player 1: 😲

 i♡histo 

[specialmemefresh-deactivated202]

[histologyapology]

When you find out that today is your last day of class even tho you checked and were previously told you had to come in for two more weeks

[histologyapology]

Hhhhhhhhhenzymes

[histologyapology]

Quiz #????: Lipids and Oil Red O stains

This is a fairly simple stain but the very last step (coverslipping) is probably one of the most nerve wracking things i’ve done in the lab so far this year. Read on to find out why I almost pass out working on these bad boys on a daily basis!

So the human body is made up of a surprising amount of fat. for the purpose of this stain, we consider a lipid to be any substance that is soluble in organic solvents like xylene. We can separate lipids into three classes:

Simple lipids

what you typically think of as ‘fat’; adipose deposits that act as stored energy.

Compound lipids

lipids that are or were incorporated into larger complexes and are usually assoicated with the nervous system. examples include phospholipids, glycolipids and sulpholipids. 

Derived lipids

lipids that result from the breakdown of other lipid types. Examples include cholesterol and bile acids. 

Why/how are fats clinically significant? 

lipids show up in many metabolic diseases affecting the nervous system and may also show up in muscle biopsies. The location of the fat within the tissue is critical to making a diagnosis; as such, it is important to never ever press down on the coverslip when sealing the slide, since it can displace the lipids and ruin the sample. More on this in a second. 

Fixation:

Fats are soluble in anything non-polar covalent, so organic solvents like xylene, acetone, alcohol, chloroform and paraffin cannot be used for the oil red O stain. Most settings will only deal with cytospins of different bodily fluids (I see a lot of cerebrospinal fluid spins for leukemia progression along the spinal column in the pediatric rotation i’m on now). Some pathologists will also order oil red O’s on frozen  muscle biopsies. Most control slides used for oil red O’s are frozen muscle sections. For cytospins, the first step in fixation is usually to hydrate with PBS, and then fix for a short period in formaldehyde. Frozen sections can go directly into formaldehyde. 

The Reagents:

Oil Red O solution:

flammable 

slight skin irritant 

just wear gloves and a lab coat and you’ll be fine

ok to dispose of down the skin just flush with a lot of water

alum hematoxylin

slightly flammable 

skin, eye and inhalation hazard

gets everywhere, seriously wear a lab coat and change your gloves

check with the water authority first but should be okay to flush down the drain

How’s it work:

ORO is a physical stain. The dye is soluble in lipids, and will appear on the slide like small red bubbles within the fats of the tissue. You cannot overstain with ORO; obviously don’t leave it in the solution for like a week, but doing 25 minutes instead of twenty isn’t going to kill anyone. 

Tissue and Control:

cut frozen sections at 10 microns. Control tissue can be anything with fat in it. 

Nobody cares about procedure pbbbbbt

Results:

Fat- stains red

nuclei- stains blue/purple (hematoxylin)

Okay so the coverslipping thing. This is probably the one stain where you have one and only one shot at coverslipping it correctly. What i mean is, you can’t use  paramount or anything like that because it’s an organic solvent, so you have to hand-coverslip using this aqueous coverslipping media that has a thicker consistency, like honey. The downside to this is that it loves to catch bubbles as you drop the slip onto the slide. On a cytospin, every single cell counts; a bubble over the countable area could mean that a painful procedure has to be repeated, and we don’t want to compromise patient care like that. So what can you do to decrease your chances of bubbles? some tricks:

put two to three drops leading from the center of the cytospin area out towards the edge of the slide, with the tails of each drop connecting them to the last. This makes a big difference in flow/spread as the slip comes down. 

after applying the aqueous coverslipping media, dunk the slide in a coplin jar of DI water and remove smoothly. this will thin out the media a bit so it spreads nicer. 

If you’ve REALLY hecked it up, place the slide with the coverslip still on into a coplin jar of di water and let it slide off. Don’t push, pull or shake the slide; pick it up out of the water and see if it falls off. If it doesn’t, put it back in. This shouldn’t damage or displace the lipids as long as you keep an eye on the coverslip and make sure it doesn’t scratch the cytospin area. 

If you’re like me, you have a bit of a resting tremor that can cause bubbles during coverslipping. The best way to counter this is to take a minute before starting to coverslip, deep breathe like you’re about to dive under water, and then hold your breath until the cover slip is safely adhered to the cytospin area. Then go call Lecia and ask them when they’re going to make a robot to do this for you cause. damn. 

alright, that’s all for now. I’ll be on a lot more this week as I start cramming for finals/HTL. bye for now!

[histologyapology]

okay but in all seriousness. I’ve scheduled my HTL test for July 19th, so starting tomorrow we’re eight weeks out. I’ll make a rolling checklist tomorrow, get organized and start really digging by this weekend. 

[histologyapology]

“fleaker” my ass, that’s an inkwell. @corning what sort of shit are you trying to pull here

[histologyapology]

you look me in the eye and tell me these aren’t just glass inkwells. you come tell me to my face that some harry potter LARPer isn’t going to come to my lab and steal all of these out of my glassware cabinet so they can use them with a giant quill pen. it’s a fucking liability, my guy. 

[histologyapology]

“fleaker” my ass, that’s an inkwell. @corning what sort of shit are you trying to pull here

[histologyapology]

Quiz Review #???: Masson Trichrome

Alrighty so after today’s misdaventures at pep boys I’m finally ready to study a thing. This week’s stain is the Masson trichrome, a stain for connective tissue. heeeere we go.

Now I’ve been doing massons since the first week at my current rotation and they’re pretty fun. A little time consuming, but very pretty results. You can use a bunch of different dyes to perform a trichrome, but they all have a few things in common:

youre always going to use some combination of three dyes with a gradient of molecular weights

theyre going to deferentially stain for muscle, collagen fibers and nuclei (aka connective tissue)

Now what’s connective tissue? Basically it’s not an organ, and it provides some kind of structural support to things that are organs. Connective tissues are made of fibers, cells and amorphous ground substance which is the sickest band name I’ve ever heard. Some examples of connective tissue include cartilage, bone and blood (which gives me the same sense of doom as referring to butter as a milkshake but, I’m not in charge of these things I guess). When you do a trichome, you are looking for the following connective tissue fibers:

collagen fibers-the most plentiful connective tissue in the body. They provide strenght to tissues and are found nearly everywhere in the body. 

elastic fibers-allow for flexibility; you’ll find them in areas where tissues need to constantly stretch and snap back into shape, ie in the respiratory, circulatory and integumentary systems. 

Reticular fibers-these are mainly found within cellular organs such as the spleen, liver, and lymph nodes.

Important note: do not confuse the masson trichrome with an elastic trichrome! they are different stains!!! I keep doing this and my director keeps yelling at me. Don’t be like me kids. stay in school. 

Appearance:

Now what do collagen fibers look like under the scope? They have a threadlike structure and are very acidophilic/eosinophilic/stain pink on H&E. They are also birefringent under polarized light. If you’re doing a masson you’re likely looking at slides of muscle, specifically cardiac or involuntary smooth muscles in the blood vessels, bronchioles and digestive tract. 

Pathology:

We care about what collagen looks like/how much of it there is in a given area because it’s a factor in several disease processes. An increase in collagen in many tissues indicates scarring; in the liver, for example, high amounts of collagen would indicated cirrhosis, and in the kidneys clusters of collagen around the glomeruli would signal renal diseases. Once upon a time collagen was used to distinguish tumors originating from muscle cells from other tumor types, but we now do this using IHC. 

Fixation- so ideally you’d put your masson-slated tissues in bouin’s, but formalin will do fine for most routine purposes as long as you mordant in bouin’s afterwards. As with any formaldehyde based fixative you don’t want to leave the tissue in there for too long as you’ll get lots of cross-linked proteins and the steric hindrance from this will stop your dyes from binding. 

Reagents & hazards:

Bouins

corrosive, toxic and irritant

contains picric acid; WILL explode if you let it dry out and then apply some sort of friction or shock (ie, WATCH THE THREADING ON YOUR COPLIN JARS!!! keep the edges moist!!!)

do NOT leave the bouin’s in the oven for more than an hour or so at a time, it will dry out and explode 

very noxious smelling, kind of sickly sweet; open and handle under a fume hood whenever possible

do not pour down drain; collect for disposal by licensed waste hauler 

Wigert’s iron hematoxylin

corrosive and flammable

inhalation and skin irritant 

must be made fresh (carson disputes this but everyone I’ve worked with so far won’t make it more than 2-3 hours before use so, do with that what you will)

licensed waster hauler blah blah

Biebrich Scarlet-Acid fuchsin

skin, eye, inhalation hazard

use under fume hood

licensed haulter etc 

Phosphomolybdic-phosphotungstic acid

strong oxidizing agent, corrosive 

skin, eye and inhalation hazard

store in the fridge 

use under chemical hood

liscendsed haulter jsjfsld

Aniline blue

skin, eye and inhalation hazard

use under fume hood

collect for disposal via slkdfjldskjflsdkjf

1% acetic acid

corrosive and combustable 

respiratory irritant; use under hood

at this low of a concentration it’s not that bad if you get it on your skin but it’ll def sting if you get it in a paper cut you know?

acid into water is the way the you gotter

neutralized with 1M sodium carbonate to pour down the drain 

Masson Trichrome- How’s it Work?

We just don’t know, basically. The working theory for now is that different tissue structures have different levels of permeability after fixation; some will accept dyes of any molecular size, some will accept only the smallest of the three you choose to apply. Fixed tissue is full of ‘pores’ and some tissue types have more than others. Muscle, for example, has very few pores, while collagen has many. Erythrocytes have the fewest pores of any connective tissue. We apply the hematoxylin first, as it is the smallest in molecular weight and binds to the erythrocytes; it will also bind to collagen, but because those pores are so large and numerous it will rinse right out in the next steps. Beibrich scarlet will fit within the pores of muscle, and aniline blue fits within collagen. 

I’m leaving out the procedures from now on because I find there’s never any test questions on them but I’ll still include cautions and notes and stuff

Cut the slides at 4 microns. Use something with muscle (small intestine is good) as a control. Follow the procedure. Your nuclei will stain black ( from the iron in your hematoxylin), your cytoplasm, keratin and muscle fibers should stain red (beibrich scarlet) and your collagen fibers, mucins and cartilage should stain blue (aniline blue). You can also used light green to stain the collagen but note that it has a larger molecular weight than aniline blue and may not work well in all cases. In a similar vein, you could also use another aluminum hematoxylin but iron is preferred because it will not decolorize when it comes in contact with the beibrich scarlet or anilaine blue. 

If you’re having issues with weak staining, the most likely culprit is water carryover from DI rinsed slides. this is especially common in labs that do large quantities of stains each week and re-use the aniline blue and beibrich scarlet each day. it’s also possible you’ve over-differentiated with acid, but as long as you stay within the 3-5 minute range with your final acetic acid solution this shouldn’t cause you trouble. 

Alright, that’s all for now. G’night!

-Reby

[histologyapology]

Well darn

Missing class/test today because of this son of a gun right here.

Currently studying at pep boys, as one does. This is what I get for driving around the city all the time I guess. It’s a small hole, hopefully they can just patch it. Anyhow. This week’s guide is on the Masson trichrome, hopefully I’ll have it up by tonight.

[badgerofshambles]

Me, slip slidin around the lab in my new shoes with mesh tops and no tread on the soles:

[histologyapology]

Quiz Review series #4: Amyloid, congo red and thio T

alright well i’ve already lost this post once to my computer crashing so let’s see what i can get through before it goes again! Today’s sponsor is Amyloid, Congo Red and Thioflavine T! Party under the cut, leggo

What is Amyloid?

protein with a teensy bit of carbohydrate stuck to it

not all pathologies lead to amyloid production but amyloid is always a sign of some sort of disease; you don’t find health tissue with amyloid in it

got a lot of amyloid? that’s called amyloidosis. you get big ol deposits of the stuff that slowly takes over and crowds out other cellular components, and if you get enough of it you end up with organ failure and you die probably. you see amyloidosis in rheumatoid arthritis diabetes and Alzheimer's disease.

We used to classify amyloid into four categories but that’s outdated and nobody cares. Now we know there’s 20 types of amyloid, they have fun little naming conventions but again if you wanted to know this you should have gone to PA school. The big one you’re going to see/test for in the US is immunoglobulin light chain amyloid, or AL.

Fixation for amyloid: you want to use either alcohol, carnoys or just plain NBF. Like many carbohydrate-containing things you should avoid letting it sit in formalin for too long because amyloid is water soluble and can leach out of the tissue, causing false negatives/weak staining.

Congo Red:

Reagents and their hazards:

alkaline salt solution:

corrosive

respiratory irritant

use typical ppe, dispose of by waste hauler

Congo Red Stain soln

flammable

skin, eye and respiratory irritant

use ppe dispose of by waste hauler

Alum Hematoxylin

slighly flammable

skin, eye, respiratory irritant

ppe waste hauler blah blah blah

How it works:

you use the alkaline salt soln to open the hydrogen bonds in tissue’s proteins. This reduces nonspecific staining and improves the selectivity for amyloid. Then, the congo red dye allows azo and amine groups to form hydrogen bonds with the hydroxyl radicals of amyloid.

Method: you want thick sections for amyloid, 8-10 microns. Cut your controls the same day that you intend to use them, since amyloid degrades rapidly on the slide even after fixation. to stain, start with the alum hematoxylin, wash in tap, treat with alkaline salt soln for 20 minutes. DONT RINSE. put directly into congo red for 20 minutes. do a fast rundown (like 10 dips max in each alcohol, less if you see it start to bleed) clear in xylene and mount with synthetic media.  you should get a slide with pink/red amyloid that produces bright apple green birefringence under polarized light. Under normal light the nuclei will be blue.

important notes: we prefer the congo red to thio T because congo red slides have a longer shelf life and it’s also the most specific histochemical method for amyloid at this time. any issues with stain quality are going to come from section thickness issues; if you’re too thin, you’ll get no birefringence and faint red positive controls.If you’re too thick, you’ll get yellow birefringence instead of green. If you’re still not getting birefringence, try using a stronger light source with a polarizing filter.

Thioflavin T Stain for amyloid

reagents and their concerns:

1% thioflavin t solution

pretty safe, just don’t lick it or whatever

dispose of via licensed waste hauler

1% acetic acid

corrosive and combustible

respiratory, eye and skin irritant at higher concentrations

do any measuring/pouring of acetic acid under a fume hood, literally if that mess breaks it’ll eat your lungs, do you like having lungs??? okay good me too, carry your glacial acetic in a carry bucket, only open it under the hood and lock it up when you’re done

acid into water is the way that you otter

mix with 1M sodium carbonate, flush down the drain with lots of water

Alum hematoxylin

slightly flammable (alcohol)

skin, eye and respiratory hazard

normal ppe, licensed waste handler etc

How it works: This isn’t a terribly popular stain so there isn’t too much info on how the chemistry of the stain works. Basically all you need to know is that it’s a fluorescent dye that attaches to amyloid  and makes it birefringent. The hematoxylin is used to cover up any background autofluorescence.

procedure: cut sections at 8-10 microns. Cut controls on the day of, so the amyloid doesn’t degrade before you get it stained. run down your sections, re hydrate to water and then stain with the hematoxylin. Wash in DI for five minutes. Stain in filtered thio T stain for 3 minutes, rinse in DI again and differentiate using acetic acid for 20 mins. Wash in DI for 2 minutes and coverslip with aqueous mounting medium (this is probably another reason nobody likes this stain, aqueous mounting media is messy and dumb). Amyloid will glow yellow to yellow green under fluorescent light.

Special notes:

Thio T requires a UG1 or UG2 exciter filter for your fluorescent scope, which not everyone has as standard equipment so just do a congo red for gods sake. Thio T also fades within a month of sealing the slide, so if you need to examine the slide after that good luck.

Alright that’s all for now. ttfn!

[biomedicool]

Cytology

Investigation of cervical smears or nongynaecological preps, e.g. pleural fluids, aspirations, sputum, urine, imprints.

Cytology vs Histology?

Advantages

Non invasive and easily done in day clinic 

Lends itself to screening, particularly the ‘well’ population 

Speed of preparation and doesn’t usually involve formalin

 Disadvantages 

No tissue architecture - difficult to evaluate whether tumour is ‘invasive’

Less morphological detail(?) 

Limited material - limits resources for retrospective studies, range of tests 

Alcohol based fixation - not always good for ICC or molecular

Specimens are usually exfoliated or aspirated.

Exfoliated Samples 

Brushings, scrapes and body fluids 

tissue biopsies are more common, but often individual cell morphology is the basis of diagnosis 

malignant cells in exfoliated samples may be very few in number 

may also be obscured by elements such as polymorphs or mucus 

minute screening of the entire sample may be required to identify sparse numbers of relevant cells on which to make the diagnosis 

Aspiration Cytology

FNA = fine needle aspiration

very cellular preparation may be obtained with thousands of tumour cells - rapid diagnosis 

diagnostic cells may be aspirated with other elements which can make screening difficult, eg such as normal tissue or necrotic debris - difficult to detect and evaluate 

[badgerofshambles]

“I need to alert the scientific community that the periodic acid schiff stain is, clinically speaking, both righteous and tubular”-Peggy, apparently