General histology techniques

Question 1

indications for frozen section

Answer 1

diagnosis (for intraop management)

tissue identification (eg parathyroid)

nature of a lesion (for special testing)

sufficiency of diagnostic material

identify metastatic disease

surgical margins

Question 2

frozen section procedure

Answer 2

identify sample/form and register

select portion of tissue (if possible reserve unfrozen tissue to avoid freezinf artefact)

freeze tissue in embedding medium

cut in cryostat

adhere to slide, fix in ethanol, rapid H+E stain

clear concise communication of result (esp answering question for which frozen was taken)

Question 3

accuracy of frozens?

Answer 3

depends on institution and types of cases (and experience)

overall >95% accuracy

discordance in 1-2%

deferral to paraffin in 1-4%

Question 4

sources of error in frozen section

Answer 4

1. errors of sampling: gross sampling error (not frozen), sectioning error (not faced)
2. errors of interpretation (esp due to artefact)
   nb: document any discrepancy, and alert surgeon if clinically significant

Question 5

considerations for taking tissue for banking

Answer 5

patient consent

adequate remaining tissue for diagnosis

should not include margin

should not include tissue near structures important for staging

Question 6

method for electron microscopy

Answer 6

fix in buffered gluteraldehyde 2-4%

dice into 1mm cubes

dehydrated and embedded in resin or plastic

semithin (1 um) sections stained with blue stain to select blocks

thin sections onto grid and further stains depending on tissue and question

Question 7

indications for EM

Answer 7

essential for medical renal, peripheral nerve/muscle disease, primary ciliary dyskinesia

metabolic and inherited diseases (tissue from prenatal, liver, lung or heart)

atypical or indeterminate tumours

infectious agents (rare)

Question 8

histology routine processing

Answer 8

formalin to ensure fixation (aids dehydration, prevents shrinkage, reduces artifacts)

graduated alcohol 70%-100% (removes water)

xylene or commercial substitue (removes alc and allows paraffin in)

heated paraffin (low melting point, but solid at RT)

Question 9

H&E staining procedure (regressive)

Answer 9

deparaffinised with xylene

xylene removed with alcohol

rehydrated

haematoxylin stain

‘differentiation’ using acid alcohol, then ‘blued’ using ammonia

eosin stain

resin and coverslip

Question 10

why H&E?

Answer 10

haematoxylin (binds chromatin)

eosin (binds + charge proteins at pH 4.6-5.0)

Question 11

PAS stain

Answer 11

oxidation of carbohydrates by periodic acid

coloured via Schiff’s reagent

can digest glycogen with diastase

Question 12

Congo red

Answer 12

stains amyloid in sheet-like fashion, giving apple-green birefringnce in polarized light

Question 13

reticulin stain

Answer 13

ionic silver added

reduced to metallic silver

silver replaced with gold

Question 14

Trichrome stain

Answer 14

iron haematoxylin: nuclear stain

acidic dye: cytoplasm

aniline blue: collagen

Question 15

gram stain

Answer 15

crytstal violet for G+ (retains dye in peptidoglycan coat)

basic fuschin for G-

picric acid yellow background

Question 16

AFB stain

Answer 16

carbol-fuschin solution, differentiated with acid alcohol

waxy bacteria resist decolorisation (‘acid fast’)

Question 17

Stains for iron, copper, calcium, bile, melanin?

Answer 17

iron: Perls’ Prussian blue (potassium ferrocyanide in acidic solution - tissue ferrous ions precipitate blue)
copper: rhodanine
calcium: von Kossa
bile: Fouchet’s reaction
melanin: Fontana-Masson (argentaffin staining)

Question 18

what components do you need for virtual microscopy in a lab?

Answer 18

scanner

file server and interface

image technologist

IT and LIS support

Question 19

uses for virtual microscopy

Answer 19

primary diagnosis

consultation

archiving

QA programs

education

MDM