Anatomical pathology

H&E

What it highlights = nuclei and cytoplasm

Primary stain colour and how = haematoxylin + scotts tap water blueing makes purple

counter stain colour = eosin makes pink

PAS

What it highlights = Fungi, glycogen, BM, neutral and acid mucins

How:

1. Periodic acid oxidises the carbohydrate by cleaving C-C in between and removes H+ from OH groups forming adjacent aldehydes

• Periodic acid is gentle enough to not cause oxidation into carboxylic acid

2. Colourless Schiff's reagent reacts with the aldehyde to bind to the carbohydrate and reconstitute its chromophore, turning red

Alcian blue

Acetic acid reduces the pH to ionize acidic groups enabling for them to bind to the alcian blue

ZN

Carbol-fuchsin stains acid fast bacteria red and acid alcahol is used to decolourize the non-bacterial tissue followed by counterstaining with methylene blue

Gram Twort

Crystal violet stains GP bacteria purple and iodine acts as a trapping agent, acetone decolourises everything else and twoets stain is applied as a coutnerstain, highlighting gram negative bacteria pink and background tissue green.

GMS

Primary = fungi, black

How = fungi are oxidised in chromic acid to form aldehydes, the slide is bleached than, the aldehydes reduce silver hexamine into metallic precipitate through an argentaffin reaction. gold chloride is used to tone and sodium thiosulphate removes any unreeduced silver complexes from the tissue. followed by light green cvounterstaining

Perls

Primary = haemosiderin, dark blue

How = Hydrochloric acid is used to release ferric iron from iron stores which than reacts with Potassium ferrocyanide to form ferric ferrocyanide (prussian blue. and counterstained with nuclear fast red.

Amyloid/Congo red

Primary = amyloid, red or apple-green birefringence under polarised light

How = Congo red stain involves anionic dye molecules that fit in between the beta pleated sheets and attach via H-bonding. The alignment of the dye molecules cause apple-green birefringenence when viewed under polarised light.

Counterstain = Haematoxylin, purple

Reticulin

sample is oxidised using acidified potassium permanganate producing aldehydes folllowed by bleaching with oxalic acid followed by sensistsation using ferric ammonium sulphate as a mordant, the addition of the sliver through wilders solution and reduction through an agryophillic reaction requiring 10% formalin, followed by sodium thiosulphate treatment to reduce unreduced silver complexes

Masson trichrome

Dye by permeation involving secondary fixation by Bouin’s fluid to reverse formalin masking and improve dye affinity followed by acid resistant iron haematoxylin staining and small red acid dye for cytoplasm and muscle followed by phosphotungstic acid decolourisation of lose molecules followed by large blue acid dye for collagen

Gomoris aldehyde fucshin stain

Initially stained in aldehyde-fichsion to highlight elastic fibresm mucins and beta cells purple followd by rinsing in alcahol and permeation staining in indigo carmine picric acid to stain collagen fibres blue green and muscle yellow followed by quick dehydration in albsolute alchohol and clearing in xyelen and mounting

HRP visualisation

HRP (horse radish protein) can be used to oxidise DAB monomers peroxide into water which causes it to form an insoluble brown polymer/chromogen or to oxidise AEC into an insoluble red coloured chromagen (soluble in alcohol - no alcohol dehydration ie air drying).

IHC workflow

Cutting of sections/baking etc - cut small onto postively charged slides + baking to prevent slipping of

• Antigen retrieval - unmasking of epitopes masked by formalin cross-linking

• Blocking - prevent endogenous enzymes and non-specific binding of antibodies

• Primary antibody

• Secondary antibody

• Visualisation

Lung cancer IHC maerks

Adenocarcinoma = TTF-1 (lung), Napsin A (adenocarcinoma) and CK7 (glandular)

Squamus cell = P40 (squamous) and CK 5/6 (squamous)

Small cell = Synaptophysin, INSM1, CD56, Chromogranin A = Neuroendocrine

Breast cancer IHC markers

Oestrogen receptor = nuclear receptor, predictive for targetted endocrine therapy by oestrogen receptor blockers or oestrogen reducers

Progesterone receptor = nuclear receptor, predictive for targetted endocrine therapy

Her2 receptor = membrane receptor, prognostic for increased aggression, predictive for her2 targetted therapy

Fish, Cish and DISHH

FISH = fluorescent ISH - fade overtime and can overlap

CISH = chromogenic ISH - stable and permanent

DISH = dual ISH - different dna tags targetted at once

ISH workflow

Pretreatment

Formalin fixed paraffin embedded tissue

Cytology smears/cytospins

Denaturation

Heat or chemical treated to disrupt hydrogen bonds into ssDNA for probe binding

Probe hybridisation

Probe added to bind to complimentary sequence and form a hybrid

Washing

Done to remove unbound probes

Signal detection

Detection system applied e.g FISH fluorescence microscopy or CISH enzyme labelled antibodies

Controls

Pos = contains target sequence to confirm probe, reagent and procedure validity

Con = non targeting/omitted probe to identify non-specific signal or background

HER 2 cancer determination

IHC assesses overexpression based on faint/moderate/intesnse staining in less/more than 10% of tumour cells

DISH assesses amplifciation based on Her2:chromosome 17 ratio

Adenocarcinoma detectoin

EGFR = mutation leading to chroncic activation promoting cell proliferation, survivial, differentiation and migration, detecterd using PCR or NGS

ALK = gene rearrangmenet causing abnormal fusion protein production which activates cell proliferation and survival, detected using IHC for ALK fusion protein and confirmed using break apart Dual-Fish based on signal closeness or just PCR/NGS

ROS1 = mutation causing permanent ocnoogenic signalling, detected using IHC for ROS1 receptor and confrimed using FISH, PCR or NGS