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Manual Tissue Processing Guide

This document outlines the key steps and considerations for histopathologic tissue processing techniques, including specimen accessioning, gross examination, tissue fixation using various chemicals like aldehydes and alcohols, dehydration, clearing, impregnation using paraffin wax, embedding, sectioning, and staining. It discusses factors that affect fixation such as pH, buffer used, penetrability, volume of fixative, temperature, and concentration.

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Kim Ruiz
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250 views48 pages

Manual Tissue Processing Guide

This document outlines the key steps and considerations for histopathologic tissue processing techniques, including specimen accessioning, gross examination, tissue fixation using various chemicals like aldehydes and alcohols, dehydration, clearing, impregnation using paraffin wax, embedding, sectioning, and staining. It discusses factors that affect fixation such as pH, buffer used, penetrability, volume of fixative, temperature, and concentration.

Uploaded by

Kim Ruiz
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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HISTOPATHOLOGIC TECHNIQUES

MANUAL TISSUE PROCESSING


HANDLING BIOPSY
‣ SPECIMEN ACCESSIONING
‣ GROSS EXAMINATION
‣ TISSUE FIXATION
‣ DEHYDRATION
‣ CLEARING
‣ IMPREGNATION
‣ EMBEDDING
‣ SECTIONING
‣ STAINING
SPECIMEN
ACCESSIONING
S - 18 - 2201
S - 18 - 2201 A

TISSUE SPECIMENS
P - 18 - 2201
P - 18 - 2201 A

FLUID SPECIMENS
GROSS
EXAMINATION
TISSUE FIXATION
ALDEHYDES
MERCURIALS
ALCOHOLS
OXIDIZING AGENTS
PICRATES
‣ FORMALDEHYDE AND GLUTARALDEHYDE
‣ CROSS-LINKING OF PROTEINS TO
PREVENT DENATURATION
‣ GOOD FOR IHC TECHNIQUES
‣ PENETRATES TISSUE WELL
‣ A BUFFER IS USED

ALDEHYDES
‣ CONTAINS MERCURIAL CHLORIDE
‣ B-5 AND ZENKER’S
‣ PENETRATE RELATIVELY POORLY AND
CAUSE SOME TISSUE HARDNESS
‣ FAST AND GIVE EXCELLENT NUCLEAR
DETAIL
‣ HEMATOPOIETIC AND
RETICULOENDOTHELIAL TISSUES

MERCURIALS
‣ METHANOL OR ETHANOL
‣ PROTEIN DENATURANTS
‣ TOO MUCH BRITTLENESS AND
HARDNESS
‣ VERY GOOD FOR CYTOLOGIC SMEARS
AND GIVE NUCLEAR DETAIL
‣ SPRAY CANS

ALCOHOLS
‣ PERMANGANATE FIXATIVES
‣ DICHROMATE FIXATIVES
‣ OSMIUM TETROXIDE
‣ CROSS-LINK PROTEINS, BUT CAUSE
EXTENSIVE DENATURATION

OXIDIZING AGENTS
‣ PICRIC ACID: BOUIN’S SOLUTION
‣ SAME WITH MERCURIALS BUT DOES
NOT CAUSE AS MUCH HARDNESS
‣ EXPLOSION HAZARD
‣ STAINS SPECIMENS YELLOW

PICRATES
FACTORS AFFECTING FIXATION
‣ NEUTRAL PH: 6-8
‣ PHOSPHATE, BICARBONATE, CACODYLATE,
AND VERONAL
‣ PHOSPHATE: PH 7

BUFFERS
‣ DIFFUSIBILITY OF FIXATIVES
‣ FORMALIN AND ALCOHOL: BEST
‣ GLUTARALDEHYDE: WORST
‣ 2-3 MM

PENETRATION
‣ 10:1
‣ AGITATION OF THE SPECIMEN IN THE
FIXATIVE WILL ALSO ENHANCE
FIXATION

VOLUME
‣ INCREASE TEMPERATURE: INCREASE
SPEED OF FIXATION

TEMPERATURE
‣ FORMALIN: 10%
‣ GLUTARALDEHYDE: 0.25% TO 4%

CONCENTRATION
COMMON USES OF FIXATIVES
FORMALIN
‣ ROUTINE SURGICAL PATHOLOGY AND
AUTOPSY TISSUES
ZENKER'S FIXATIVES
‣ FIXES NUCLEI VERY WELL AND GIVES
GOOD DETAIL
‣ MERCURY DEPOSITS MUST BE
REMOVED
BOUIN’S SOLUTION
‣ TESTIS, GI TRACT, AND ENDOCRINE
TISSUE
ALCOHOLS
‣ CYTOLOGIC SMEARS
‣ ETHANOL
DEHYDRATION
70% 95% 100%
ACETONE
‣ VERY FAST
‣ FIRE HAZARD
DIOXANE
‣ DIOXANE CAN BE USED WITHOUT
CLEARING
‣ HAS TOXIC FUMES.
CLEARING
XYLENE
‣ MOST COMMON CLEARING AGENT
TOLUENE
‣ WORKS WELL
‣ MORE TOLERANT OF SMALL
AMOUNTS OF WATER LEFT IN TISSUES
CHLOROFORM
‣ MAY BE USED
‣ HEALTH HAZARD
‣ SLOW CLEARING AGENT
METHYL SALICYLATE
‣ RARELY USED
IMPREGNATION
PARAFFIN
‣ PARAPLAST: CONTAINS ADDED
PLASTICIZERS THAT MAKE THE
PARAFFIN BLOCKS EASIER FOR SOME
TECHNICIANS TO CUT
EMBEDDING
METHYL METHACRYLATE
‣ VERY HARD
‣ THEREFORE GOOD FOR EMBEDDING
UNDECALCIFIED BONE
GLYCOL METHACRYLATE
‣ MOST WIDESPREAD
‣ EASIEST TO WORK WITH
ARALDITE
‣ REQUIRES A MORE COMPLEX
EMBEDDING PROCESS
EPON
‣ ROUTINELY USED FOR ELECTRON
MICROSCOPY WHERE VERY THIN
SECTIONS ARE REQUIRED.

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