QUT - Lecture note 1-13 PDF

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Semester 2 2016...

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QUT

Histological Techniques LSB466

Week1: Histological Techniques What three clothing items are mandatory for entry into Histology laboratory classes? -

Laboratory coat Safety glasses Closed shoes

What two things must you do before leaving the Histology laboratory? -

Remove coat Wash hands

What should you do if you break or spill something in the laboratory? -

Report it to a demonstrator

How should xylene and alcohol be disposed of? -

They should not be disposed of by dispensing in a sink They need to be placed in a labelled drum Xylne = lid must always be on it

Where should cover-slipping be performed? -

Down-draft work station

How and when should slides be labelled? -

Graphite pencil label = before staining Paper label = after staining and mounting

Explain what is meant by the phrase…” take section to water”. -

This process involves the removal of paraffin This is done by the immersion in xylene followed by graded EtOH o Absolute (100%) o 90% o 70% o Water

What does the abbreviation “DCM” mean? -

D = dehydrate C = clear M = mount

Week 2: The Fundamentals of Histology: Fixation, Processing, Embedding and Microtomy Why does tissue need to be fixed? -

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This is because it prevents: autolysis and purification. o Autolysis = degradation of tissue post-mortem following release of lysosomal enzymes o Purification = this must be commenced immediately and for a sufficient length of time Helps in maintaining clear and consistent morphological features.

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Supports high quality and consistent staining with H&E. Preserves tissue structure through cross-linking or aggregation of molecules Proteins are normally the primary interest Numerous methods No one fixative is perfect o Therefore, often used in combo or sequentially  Post-fixation Can produce artefacts o Shrinking o Swelling o Hardening of tissue (can be a good thing) o Colour variations in staining o Components of tissue lost due to solubility The ideal fixative will hold these properties o Useful for a wide variety of tissues o Support histochemistry, immunohistochemistry and molecular techniques o Penetrate and fix rapidly o Have a shelf life i.e. 1 year o Be compatible with automated tissue processor o Be readily disposable or recyclable o Support long term storage of tissue o Give excellent microtomy of paraffin blocks o Cost effective o Safe – risk assessment

Which fixative is most commonly used? Formaldehyde

Which alternative fixatives might be used if a diagnosis is urgently required? Glutaraldehyde

What is the chemical basis for fixation with aldehydes? Gas + aqueous solution + polymer in powder form

Generally, what is the maximum thickness of tissue samples to be fixed ≤

4mm

What is the difference between formaldehyde, formalin and paraformaldehyde? -

Formaldehyde = gas Formalin = this is the trade name for 37% formaldehyde aqueous solution Paraformaldehyde = it’s a polymer in powder form o Converts to monomer in aqueous solution at 60deg with NaOH o Avoids impurities such as methanol and formic acid

Which two fixatives (other than formaldehyde) are especially important for electron microscopy? -

Glutaraldehyde Osmium tetroxide

Which fixative may also be used as a stain? -

Picric Acid o Post-fixation of tissue sections with this can enhance staining  Masson’s Trichrome

Describe the aims behind tissue processing and embedding? -

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Tissue processing o Replace water in fixed tissue with paraffin wax (but not 100%) o Water (formalin) and wax are not miscible o Transferred through a series of solution o Water  alcohol  xylene  wax Embedding o Mounting of tissue within paraffin block o Orientation of tissue within block is critical for accurate diagnosis

What considerations should be made when embedding pieces of tissue with respect to their orientation? -

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Tubular or hollow structures = oriented to enable transverse sectioning o Artery Layered or walled structures = oriented to show layers o Skin, intestine Ensure that tissue will be cut from side with least resistance first o Skin = knife should cut from dermis upwards o Also helps to cut at an angle More homogenous tissues = oriented flat o Liver, kidney, muscle

What is the approximate thickness of paraffin sections used in histology? 3um

When not sectioning, what two safety measures should always be in place? -

Blade guard in place when not cutting, especially when mounting blocks Fine feed hand-wheel lock: should also always be in locked position when not cutting

If sections appear crumpled, how might you correct this? Change the blade and possibly need to cool the block again on the ice block

Why is a small amount of gelatine added to the water bath? It helps assist with adhesion of sections to the slide

Week 3 – Principles of Histochemistry What is underlying histochemical basis? -

Enzyme histochemistry Biochemistry of disease processes Primary site determination of metastatic tumours Identification of non-pigmented melanomas Detection of surgically resected tumour margins Identification of microorganisms

Why do structures stain? -

Stains uptake due to dye-tissue or reagent-tissue affinities Affinity: attractive forces binding dye to tissue Stain-tissue, solvent-solvent, stain-solvent and stain-stain interactions

Why aren’t all structures stained? -

Stains have a higher affinity for some tissue elements depending on bonds formed

How is stain retained? -

Under alkaline conditions, solution proteins carry a negative charge and bind basic dyes

List three connective tissue fibres and give an example of where each may be found. -

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Reticular = identification is important in diagnosing liver cirrhosis and certain tumour types o Stains = any one of the modified silver stains  Gordon and Sweet’s  Gomori’s Collagenous = identification important for demonstrating overproduction of collagen in certain disease states and for diagnosing some tumours o Stains  Haematoxylin van Gieson (HVG) stain  Massons’ Trichrome  Picromallory Elastic = identification useful for identifying atrophy caused by arteriosclerotic changes, evidence of other vascular diseases and vessel invasion by tumours o Stains  Verhoeff’s van Gieson (VVG) = also demonstrates collagen

When is diastase digestion indicated in the PAS reaction? -

It is used to identify and diagnose some tumour types and distribution significant in identifying carbohydrate metabolism enzyme deficiencies

What is a good control for demonstrating diastase digestion? -

Best’s stain

Why are sections of stomach not a good control for alcian blue stains? -

Acidic mucin demonstrated using Alcian blue and Mucicarmine stains

Is the counterstain shown in this image considered good? Why/why not? Not a good stain as it is under differentiated

What might be the cause/s of the problem seen in this image? Over differentiated New batch of stains also may be required

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What is the problem seen in this image and how should it be corrected? -

Not dehydrated properly

Identify the problem shown in this image and give the possible cause/s -

Paraffin block not cold enough – needs to be very cold May also be a problem with the blade, change to a fresher one

What is the cause of the artefact seen in this image and how may it be corrected?

How can the artefact in this image be prevented? Air bubble with mounting medium

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How can the problem shown in this image be corrected? -

Block not cold enough

Identify the tissue in the image. Identify the substance stained black. Identify the problem with the slide.

What is the appropriate dimensions of a tissue sample during fixation and processing? 15mm x 20mm x 4mm

Briefly describe the histochemical basis behind the H&E staining technique -

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Fundamental histological stain Demonstrates enormous number of tissue structures Hx: blue/black/purple Eosin: pink/red/orange o Cytoplasm Used for all microscopic diagnoses: o Diseases o Cancer o Metabolic disorders o Identifying tissue types Haematoxylin must be oxidised to haematin Haematoxylin is not a dye o No chromophore Haematin exhibits indicator like properties o Blue and Less soluble in alkaline conditions o Red and more soluble in acidic conditions Requires a mordant to stain and introduce colour

Explain why hematoxylin must be oxidised prior to use -

To create haematin Alone it has no staining or dye properties By oxidising it, the haematin helps to indicate dye like characteristics o Blue = basic o Red = acid

Describe the role of “mordant” in staining with hematoxylin Hx requires a mordant to stain and introduce colour into the tissue

The two used for Hx are Aluminium and Iron

Name two commonly used Hematoxylin recipes and the mordants used. o

Commonly Al or Fe  Alum: Ehlrichs and Mayers  Fe: Weigerts

Define the terms: “bluing” and “differentiation”. -

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Bluing: by washing in tap water or alkaline solution o Weak ammonia o Saturated lithium carbonate o Scott’s tap water substitute Differentiation: destaining or decolorizing o Dilute mineral acids commonly used  HCl  Acid alcohol o Removes dye metal complexes from tissue o Good for:  Leave only desired element stained  To reduce background

Explain the differences between “progressive” and “regressive” staining procedures for hematoxylin. -

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Progressive staining o Hx with high alum: haematin ratios selectively stain nuclear chromatin slowly o Sections stained for predetermined time required to stain nuclei only  Mayer’s Regressive staining o Hx with low alum: haematin ratios colour all tissue components rapidly o Differentiation required as sections are overstained  Ehlrich’s

How do you determine that a slide has been adequately differentiated? -

Use microscope control Proper differentiation: o Distinguish between cytoplasm and different types of connective tissue

Week 4: Redox Stains What is the role of periodic acid in the PAS method? Oxidation

What does Schiff’s reagent react with? Aldehydes

How is colour developed using PAS method? Colour develops during washing in water

What structures are illustrated by PAS stain? -

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Neutral mucins o Found in goblet cell Polysaccharides o Glycogen deposits in liver and skeletal muscle Glycoproteins o Associated with extracellular matrix components including basement membranes Diagnosis of carb metabolism enzyme deficiencies Tumour differentiation Evaluation of basement membranes Diagnosis of fungal infections

What is the effect and role of diastase treatment? -

Can be performed as “control” Removes glycogen from section Saliva provides a good source

What chemical group is demonstrated by Schiff’s reagent? -

Detect aldehydes o Aldehydes produced by oxidation of glycol groups in saccharides

Why are gluteraldehyde or chromate-containing fixatives not suitable for tissue to be stained with PAS? -

Results in a weak stain They are not reactive to Schiff’s

What is the purpose of the G&S stain? -

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Used to demonstrate reticular fibres o Fine networks of ECM fibres o Predominantly collagen type III Based upon deposition of silver

In what ways is this stain similar to the PAS staining method? -

On the histochemical basis it is similar to PAS

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o Carbs involved Hexose groups associated with reticular fibres are first oxidized to form aldehydes Potassium permanganate traditionally used but PA can be used

In what form is silver applied to the slide? -

Freshly prepared ammoniacal silver solution

In what way is the process similar to developing a conventional B&W photograph? -

Argyrophilic process o Similar to processing and developing traditional photographs G&S: agryophilic method o A reducing agent must be added to assist deposition of silver  Aqueous formalin used for G&S as the reducing step

At what stages should contact with tap water be avoided? -

When using Schiff’s reagent o Only with deionized water before and after Schiff’s

Identify the staining technique Gordon and Sweets

Identify the problem shown List at least 2 possible causes for the problem

Which section is optimal for demonstrating renal pathology? Median or sagittal cut

Identify the tissue in this image -

skin

Identify the material stained black -

silver – black deposits

Identify the stain performed -

G&S

Week 5: Trichrome Stains in Histopathology Name the three staining mixtures that are employed in Masson’s trichrome method? -

Stain with Martius yellow Stain with Crystal red Differentiate with PTA Stain with methyl blue

What role does PMA play in this method? -

Phosphomolybdic Acid Used to differentiate Removes Masson’s Red from collagen prior to Methyl blue stain Only penetrates collagen

Describe how the molecular weights of dyes/PMA are exploited in this method. -

Weight of PA: 2880Da Weight of Methyl Blue: 800Da Weight of PMA: 1825Da

Which fixative is often employed in conjunction with this method? Picric acid helps with post-fixation

Compare the staining properties of muscle, collagen and red blood cells observed when using this method. -

Muscle: medium pores – red Collagen: largest pores – blue RBC: smallest pores – red

Which tissue components are typically demonstrated by trichrome stains? -

Collagen Muscle RBC Fibrin

Construct a table listing the three trichrome methods and their primary purposes Trichromes Stain

Purpose Collagen – red; lowest density - Muscle – yellow; medium density - RBC – yellow; highest density -

Van Gieson (VG or HVG)

Differentiates muscle and fibrin from collagen

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Masson Trichrome (MT)

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Martius Scarlet Blue (MSB)

Used to demonstrate fibrin - Martius Yellow - Brilliant Crystal Scarlet - Weigert’s Hx

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Describe the histochemical basis that is common to trichrome stains -

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Used to demonstrate connective tissue components o Muscle o Collagen o Fibrin o Erythrocytes Factors affecting Trichrome stains o Tissue permeability and dye size o Proteins exposed to fixatives o End results  Nature of proteins and fixative o Usually insoluble 3D network of proteins

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Different proteins  Different physical features RBC’s dense network  Small pores Collagen  Least dense  Open pores Small dye molecule  Penetrate all tissues Medium size  Muscle and collagen  Not RBCs Larger molecules  Only collagen Rules are not rigid Smaller dye  Penetrate and stains a tissue element Larger dye  Penetrate same tissue element and replace smaller dye Heat  Heat increasing rate of staining pH  low pH of stains = better results Nuclear stains  Iron Hx  Weigert’s preferred to Alum Hx  Due to acidity of dyes used Fixation  Formalin = not optimal results  Post-fixation  Picric acid: staining brighter and more saturated

Explain why Weigert’s Hx is used as a counterstain and why is should be left under-differentiated -

Preferred over the acidic nature of most alum Hx

Week 6: Elastin Stains What structures are selectively demonstrated using Weigert’s Resorcin Fuchsin method? -

Elastin fibres in tissue Stained purple

Name the dye mixture which is used to demonstrate these structures. -

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Mixture of three dyes: o Pararosaniline o Rosaniline o Magenta II Along with: o Resorcinol (resorcin)

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Ferric chloride

What other staining solution is often used in conjunction with this method? -

Van Gieson’s stain

Describe how the Weigert’s Resorcin Fuchsin mixtures should be applied to slides? -

Upside on petrie dish Using capillary motion

You should aim to be able to discuss the similarities and differences between WRF and VVG staining methods and their outcomes. -

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Similarities: o Counterstain Van Gieson o Application of mixture o Oxidation o Differences: o VVG:  Less complicated to prepare o WRF:  Elastin fibres

Week 8: Pimp My Slide H&Es Artefacts

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Prefixation artefacts o Heat damage  At surgical margins  Caused by laser surgical exicison  Strong acidophilic staining  General or local version caused by overheated forceps  Over hot wax during processing o Sutures  Silk sultures present with a strong birefringence under polarized light  Not of pathological significance  Damages knife and tears tissue  Should be removed at cut-up or embedding  Staples

Cellulose

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before processing Not pathologically significant Can cause tearing of tissue Recognized by plant cells

Commonly present in GIT  Not washed thoroughly

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Usually at surface of the tissue

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Gelfoam  A sponge or film made from gelatine  Used to control bleeding in surgery  Usually found on specimen surface  Gelatin walls of varying thickness with spaces containing blood or other cells  No tissue reaction

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Starch contamination  Powder used on surgical gloves  Occasionally associated with granulomas removed surgically  Theatre or lab  Difficult to see in H&E  Stain with PAS and Grocott’s methenamine silver for fungi  Birefringent with polarize light Catheterisation  Epithelium damaged or lost  By catheterization during surgery  Underlying tissue compressed  Prompt fixation

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Week 9: Principles of Microscopy Briefly describe the light path involved with bright field microscopy -

Light source o Halogen Light path o Emerges from light source  Directed with mirror o Focused onto specimen using condenser o Image magnified by lenses

Define the role of the condenser and explain the principle of Kohler illumination -

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Condenser o Must be focused and centered for best results o Correct alignment referred to as Kohler illumination Kohler Illumination o Reduces stray light o Ensures even light over all specimen o Restricts the exposure of the specimen to light to the observed area

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Reduces light exposure Minimizes photo-damage

Define the terms magnification and resolution -

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Magnification o Simply relates to how big something appears o But magnification alone does not imply a good quality image Resolution o Critical factor in light microscopy o Smallest distance between 2 particles at which they can still be distinguished from each other o

R=  

0.61 x λ NA λ = wavelength of light NA = numerical ape...