histo - trimming_fixation

PRECAUTIONS IN HANDLING, ACCEPTANCE & FIXATION OF SPECIMENS

Acceptance of Specimen

Important Responsibilities of a Med-Tech

Record pertinent patient data Evaluate and inspect request form Evaluate and inspect specimen


Request Form: Name of the patient, age, sex, room

number, address, civil status Type of specimen Examination desired Clinical diagnosis Requesting physician or surgeon


NUMBERING is the first and an important step in all histopathologic techniques,

Purpose: to identify properly all the specimens without writing the name of the patient to the accompanying tag of the specimen to be processed


Evaluate and inspect the request form and specimen Correct request form Written legibly Proper container – tight fitting &

transparent Proper fixative – volume Properly labeled

Fixation

First and most crucial step in histotechnology

AIM: To preserve the morphologic and chemical

integrity of the cell in as life-like manner as possible Stop all activities of the cell, such autolysis

To harden and protect the tissue from the trauma of further handling, so it may be easier to cut during gross examination Stabilize protein = mechanical strength

Fixation: Main Factors

Main factors Hydrogen Ion Concentration Temperature Thickness of section Osmolality Concentration Duration of fixation

Fixation: Effect in General

Effects of Fixatives in General Harden soft and friable tissues and make the

handling and cutting of sections easier. Make the cells resistant to damage and

distortion during the process Inhibit bacterial decomposition. Increase the optical differentiation of cells and

tissue components Act as mordents or accentuators Reduce the risk of infections during handling

and actual processing of tissues.

Fixation: Ideal Characteristics Cheap. Stable. Safe to handle. Kill the cell quickly

thereby producing minimum distortion of cell constituents.

Inhibit bacterial decomposition and autolysis.

Produce minimum shrinkage of tissue.

Permit rapid and even penetration of tissues.

Harden tissues Isotonic Make cellular components

insoluble to hypotonic solutions and render them insensitive to subsequent processing.

Permit the subsequent application of many staining procedures

NOTE: No single fixative posses all the qualities.

Fixation: Types – COMPOSITION

Simple Fixative

Compound Fixative

Fixation: Types – COMPOSITION

Simple Fixative Aldehydes

Formaldehyde Glutaraldehyde

Metallic Fixatives Mercuric chloride Chromate fixatives Potassium

dichromate Chromic acid

Metallic Fixatives Mercuric chloride Chromate fixatives Potassium dichromate Chromic acid

Lead fixatives Picric acid Acetic acid Alcohol Osmium tetroxide

(Osmic acid) Heat

Fixation: Types – ACTION

Microanatomical Fixatives Permit the general microscopic study of tissue structures

without altering the structural pattern and normal intercellular relationship of the tissues in question

10% Formol saline10% Neutral buffered formalinHeidenhain’s SusaFormol sublimate (Formol corrosive)Zenker’s solutionZenker-formol (Kelly’s solution)Bouin’s solutionBrasil’s solution


Cytological Fixatives Preserve specific parts and particular microscopic

elements of the cell itself. Nucler Fixatives

preserve the nuclear structures (e.g., chromosomes) Contain glacial acetic acid as their primary component

(pH <=

Flemming’s fluidCarnoy’s fluidBouin’s fluidNewcomer’s fluidHeidenhain’s Susa


Cytological Fixatives Preserve specific parts and particular microscopic

elements of the cell itself. Cytoplasmic Fixatives

Preserve cytoplasmic structures No glacial acetic acid (pH >= 4.6)

destroy mitochondria and Golgi bodies

Flemming’s fluid without acetic acidKelly’s fluidFormalin with “post-chroming”Regaud’s fluid (Muller’s fluid)Orth’s fluid


Histochemical Fixatives Preserves the chemical constituents of cells

and tissues10% Formal SalineAbsolute Ethyl AlcoholAcetoneNewcomer’s fluid

Fixation: Formaldehyde

A gas produced by the oxidation of methyl alcohol

Souluble in water (extent of 40% by weight) Routine: 10% (1:10 dilution) Duration: 12-24 Hours Turbid after prolonged storage (Formation of

paraformaldehyde) Removal of Formlin Pigments:

Karadeswitsch’s Method; Lillies’s Method; Picric Acid Method

Fixation: Formaldehyde

Cheap Readily available &

stable Penetrates tissue well Does not over

hardens tissue Preserves fat & mucin Best for nervous

tissue

Irritating Skin (allergic dermatitis) Eyes

Produce considereable shrinkage of tissue

Reduce both basophilic & eosinophilic staining cells

Forms abundant brown artifacts, pigments & granules

Advantage Disadvantage

Fixation: Simple

Fixation Best used Fixation Best used

Glutaraldehyde Small fragment; Small needle biopsies

Paraformaldehyde

Excellent for routine paraffin sections

Aldehyde

Metallic FixativesFixation Best used Fixation Best used

Mercuric chloride

Nuclei; CTReco: Renal, fibrin & CT

Fixation: Simple


Chromic fixatives

Carbohydrates Potassium dichromate

Certain lipids

Lead fixatives Mucopolysaccharides

Picric Acid Glycogen

Acetic Acid fixatives

Nucleoprotein Acetone fixatives

Enzyme studiesPhosphatesLipasesBrain tissue (rabies)

Alcohol fixatives

Small tissue fragemtsGlycogen

Osmium tetroxide fixatives

Fats & lipidsNuclear stainig

Fixation: Compound - Microanatomical


10% Formol saline

Nervous systemGeneral post mortem materials

10% Buffered formalin

Post mortem surgical research specimen

Heidenhein’s Susa solution

Skin biopsies Formol sublimate

Routine post mortem materials

Formol Saline sublimate

Post mortem materials

Zenker’s solution

Post mortem materials

Zenker’s Formol (Helly’s Fluid)

Pituitary tissue and bone

Bouin’s solution Embryos

Fixation: Compound - Cytological


Flemming’s solution

Nuclear structure

Carnoy’s fluid Chromosomes studyLympnodesUrgent studies – glycogen

Boiun’s fluid EmbryoGlycogen

Newcomer’s fluid

MucopoplysaccharidesNuclear proteinChromosomes

Fixation: Compound - Cytoplasmic


Flemming’s fluid w/o acetic acid

Cytoplasmic structures

Champy’s fluid MitochondriaGolgi elementsFats

Regaud’s fluid (Moller)

Mitochondria Orth’s fluid Early degenerative processTissue necrosis

FAULTS OCCURRING DURING TRIMMING/CUTTING OF PARAFFIN BLOCKSMary Christelle G. Aquitania

UST Medical Technology Intern

Trimming

Process wherein the paraffin block is exposed for actual cutting after when the wax is solidified and removed from the mold

Sides, top and bottom of the tissue block are trimmed until leveled perfectly and all sides are parallel to each other

Routine histologic procedures: Cut between 4-6μ.

Faults before Section-Cutting

FAULTS REASON REMEDY

Brittle or hard tissue

Prolonged fixation

Tissue may be softened by soaking in a small dish containing water with detergent, phenol

or Molliflex

Prolonged dehydrationProlonged clearingProlonged paraffin

infiltrationOverheated paraffin

ovenDrying out of tissue

before actual fixation

Clearing agent turns milky as soon as tissue is placed in it

Water not completely removed (incomplete

dehydration)

Repeat dehydration with absolute alcohol, then repeat clearing

Upon trimming, tissue smells of clearing agent

Clearing agent is not completely removed

due to insufficient impregnation

Blocked is trimmed down nearest to the

tissue. The remaining wax id melted on

embedding oven and paraffin impregnation is repeated, changing the paraffin at least once

before blocking.

Faults before Section-Cutting


Tissue is opaque, section cutting is difficult due to the presence of alcohol

Insufficient clearing

Repeat clearing; id object has already been embedded,

prolong oven and paraffin impregnations repeated, changing the paraffin at

least once before blocking

Tissue shrinks away from wax when trimmed

Insufficient dehydration, therefore incomplete

clearing and impregnationRepeat the whole procedure

Tissue is soft when block is trimmed

Incomplete impregnation Repeat whole procedure

Air holes on tissue during trimming

Incomplete impregnation Repeat impregnation

On trimming, wax appears crystalline

Contaminated waxRe-embed in freshly filtrated

wax

Block not cooled rapidly enough

Paraffin block, after cooling, is moist and crumbles

Insufficient paraffin impregnation

Repeat paraffin impregnation, then re-embed

Faults during Section-Cutting


Sections fail to form ribbons

Surfaces and edges of the block are not

parallelRe-trim the block

Horizontal surface of the block is not

parallel to the knife

Re-adjust and re-orient the block

Paraffin wax is too hard

Coat horizontal edges of the block with wax of lower melting point

Knife is tilted to much Reduce the tilt

Sections are too tickReadjust the thickness

of the sectionsHone and strop

Sections roll up on cutting so that they adhere and get broken against the knife edge

Knife is blunt Sharpen the knifeTilt of knife is too great Reduce the tile

Knife edge is dirty Clean the knife edge



Ribbon is curved, crooked or uneven instead of straight

Blunt of dull spot on the knife, producing an irregular knife edge

Adjust the knife so that the knife will present a uniformly sharp edge to

the block, or sharpenEdges of the block are not parallel but round wedge

shapeRe-trim the block

Knife is not parallel to the block

Readjust knife and block

Paraffin is impureRepeat impregnation

using pure wax

Sections are compressed, wrinkled or jammed

Knife is blunt or dull Re-sharpen the knifeParaffin block Is warm

and softCool the block on ice

water until firmKnife edge is coated with

paraffinClean the knife edge

Sections are too thinReadjust thickness of

sectionMicrotome set screw is

looseTighten the screw

Tilt of knife is too vertical Reduce the tilt


FAULTS REASON REMEDYSections are squashed (width of each section is less than that of block)

Bevel of knife is lost due to incorrect sharpening

Re-sharpen, using a knife back or automatic knife

sharpener

A hole is formed in the section

Bubble or dirt formed in the embedding medium

Re-embed in freshly filtered wax if necessary

Hard spot in tissue due to calcium

Once embedded in paraffin wax,

decalcification is impractical; use a base-sledge microtome with a

wedge knife

Section of unequal thickness are produced

Tilt of knife is too great or bevel is not cleared,

hence object is compressed against the

knife edge clamp set screw on knife

Reduce the tilt

Or blockholder is looseBlocks are too large

Tighten the screw

Block are too hardCut blocks into smaller

fragmentsSoften the blocks in detergent or phenol



Sections adhere to the knife or other parts of the microtome

Static electricity due to low atmospheric humidity

Breather out or blow gently on the block and knife to breakup static

electricity, or boil water in the room to increase the

humidityKnife edge is dirty Clean the knife edgeKnife edge is dull Sharpen the knife

Knife tilt is too great Reduce the tilt

Ribbon is split or lengthwise vertical scratches are seen on sections

Nicks or damage on the knife edge

Sharpen the knife

Dirty embedding Re-embed in filtered wax

Knife edge is dirtyClean knife edge with

xyleneTilt of knife is too great Reduce the tilt

Sections are lifted from the knife on upstrokes

Knife tilt is too great Reduce the tiltKnife is dull Sharpen the knife

Paraffin is too soft or room temperature is

warm

Cool paraffin wax in ice water



Resistance is felt on the lower part of the section during cutting

Tilt of knife is too small, paraffin block is therefore compressed against the

base of the knife towards the end of stroke

Increase the tilt

Horizontal or parallel lines or furrows across the section (“Chatters”) are seen, forming thin and thick zones

Knife edge vibrate due to hardness of tissue

Treat with phenol during processing or collodionize

Tilt of knife is too great Reduce the tilt

Section cut is sometimes thin, sometimes thick

Knife is blunt Sharpen knifeKnife is not clamped

properlyAdjust the knife

Tilt of knife is too great Reduce the tiltKnife or block holder is

looseTighten adjusting and

locking screwsKnife tilt is too small that block is compressed by bevel and section is not

cut

Increase the tilt



Knife makes a hard metallic scrapping or ringing sound on backstroke, when section is cut

Tilt of knife is too slanted or too big

Readjust the angulation of the knife

Tissue is too hardTake fresh block treated

with phenol during processing

Knife blade is too thin Change the knifeFrozen tissue crumbles and comes off the block holder when cut

Freezing is not adequate Refreeze the tissue block

Frozen tissue chips into fragments when cut

Tissue is frozen too hardWarm the tissue with

fingers

histo - trimming_fixation

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