Upload
mary-christelle
View
205
Download
1
Embed Size (px)
Citation preview
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
Acceptance of 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
Acceptance of Specimen
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
Acceptance of Specimen
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
Fixation: Types – ACTION
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
Fixation: Types – ACTION
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
Fixation: Types – ACTION
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
Fixation Best used Fixation Best used
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
Fixation Best used Fixation Best used
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
Fixation Best used Fixation Best used
Flemming’s solution
Nuclear structure
Carnoy’s fluid Chromosomes studyLympnodesUrgent studies – glycogen
Boiun’s fluid EmbryoGlycogen
Newcomer’s fluid
MucopoplysaccharidesNuclear proteinChromosomes
Fixation: Compound - Cytoplasmic
Fixation Best used Fixation Best used
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
FAULTS REASON REMEDY
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
FAULTS REASON REMEDY
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
Faults during Section-Cutting
FAULTS REASON REMEDY
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 during Section-Cutting
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
Faults during Section-Cutting
FAULTS REASON REMEDY
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
Faults during Section-Cutting
FAULTS REASON REMEDY
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
Faults during Section-Cutting
FAULTS REASON REMEDY
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