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UK NEQAS Cellular Pathology Technique

Providing worldwide external quality assessment and proficiency testing for all aspects of tissue diagnostics

Staining Criteria Handbook

General Pathology (Routine Histopathology)

Neuropathology

Edition 5

October 2017

NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook

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Index

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Haematoxylin and Eosin Assessment Criteria 5

Special Stains A & B Assessment Criteria 9

Assessment Criteria Definitions 33

Haematoxylin and Eosin

Special Stains

Appendix 46

Haematoxylin and Eosin Model Description

Scoring Guidelines

Scoring Based on Criteria

UK NEQAS CPT Stain Repertoire


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Haematoxylin and Eosin Assessment Criteria


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Haematoxylin and Eosin Assessment Criteria

Pre Microtomy Insufficient cellular features for assessment Crush artefacts

Foam inset artefact Red blood cell lysis Poor chromatin detail Cracking

Nuclear bubbling Nuclear meltdown Incorrect orientation suspected Incorrect trimming suspected

Microtomy Chatter / vibration Displacement Folds / creases Knife back debris Knife marks

Lifting Position on slide Section too thick Section too thin Section thickness variable Squames / floaters / fibres

Trimming artefacts Water bath bubbles

Staining Haematoxylin intensity too strong Haematoxylin intensity too weak Haematoxylin colour Haematoxylin background staining

Eosin intensity too strong Eosin intensity too weak

Eosin colour Eosin not selective

Uneven staining Stain deposit present

Post Staining Air bubbles Air drying artefact Excessive mountant

Mountant shrinkage Residual wax Section wiped / section off slide Tissue damage Tissue exposed

Water present Contaminant on slide


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Description of Staining Results

Nuclei must be stained purple blue with haematoxylin. The intensity must be strong

enough allow clear demonstration of nuclear detail at a medium power, but not too

strong to cause a loss of the chromatin granularity or excessive cytoplasmic or

connective tissue staining.

Where the haematoxylin has been differentiated out, minimal cytoplasmic or

connective tissue background staining with haematoxylin must remain. This

background if present must not reduce the effectiveness of the nuclear demonstration

or affect the colour and selectiveness of the eosin.

The eosin should be selective enough to demonstrate different cellular components

such as collagen, cytoplasm, red blood cells, cellular granules, amyloid etc.

The intensity must be appropriate to the section thickness and the haematoxylin

intensity. Where the eosin is too weak it will fail to allow selective demonstration of

different components at low power. If the eosin intensity is too strong the colour and

detail of the nuclear stain will be obscured and selectivity will be reduced.

For an extended description including pre Microtomy, Microtomy and post staining

please see the model description in the appendix.


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Special Stains A & B Assessment Criteria


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Alcian Blue/PAS Assessment Criteria

Primary Stain Alcian Blue intensity too strong Alcian Blue intensity too weak Alcian Blue colour PAS intensity too strong

PAS intensity too weak PAS colour Not selective Uneven staining

Background Deposit / precipitate present

Counterstain Nuclear stain intensity too strong Nuclear stain intensity too weak

Nuclear stain colour Not selective

Uneven staining Deposit / precipitate present

Post staining Air bubbles Air drying artefact Excessive mountant

Mountant shrinkage Residual wax Section wiped / section off slide Tissue damage Tissue exposed Water present Contaminant on slide

Description of Staining Results Acid mucins (also called mucopolysaccharides) should be coloured bright blue. Neutral mucins should be bright magenta. Mixed mucins should be purple. There should be a clear distinction between acid, neutral and mixed mucins. Background staining should be negligible. A counterstain (typically haematoxylin) is considered optional. If present, it should be light and even to assist location. It should not mask or alter the primary staining. Section quality and presentation should not impair the result.


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Amyloid (method for) Assessment Criteria

Primary Stain - Bright field Intensity too strong

Intensity too weak Not selective

Stain colour Uneven Staining Background Deposit / precipitate present

Primary Stain - Cross polarised light

No green birefringence on cross polarisation Birefringence intensity too weak Birefringence colour Birefringence not selective

Counterstain Nuclear stain intensity too strong

Nuclear stain intensity too weak Nuclear stain colour

Not selective Uneven staining

Deposit / precipitate present




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Description of Staining Results

Amyloid: There is no amyloid specific dye. However, Congo red is highly selective for amyloid when

staining is performed stringently/correctly by the alkaline alcoholic Congo red method, and when

sections are prepared at the appropriate thickness (~5 to 10µm). Use of other dyes or methods may

produce sub-optimal or erroneous results. Assessment is conducted in brightfield and cross polarised

light, with a 10x objective, on microscopes equipped with a high intensity light source, polarisation

filters and colour corrected and strain free optics. Sections that are too thin or too thick may not show

the green birefringence of amyloid-Congo red complexes in cross polarised light.

Brightfield: Amyloid should stain pink to red (congophilic). Stain intensity is seldom homogeneous and

may differ in different tissues reflecting the deposition, abundance and underlying organisation of the

amyloid fibrils. Collagen and elastin may be weakly coloured. Background staining must be negligible.

Haematoxylin counterstain should be light, even and not mask or alter the primary stain; it should assist

location.

Cross Polarised Light: Amyloid stained pink / red in brightfield microscopy must also exhibit a green

colour in cross polarised light (strong green birefringence). Denser deposits of amyloid may exhibit

yellow green or bright yellow birefringence. The colour and/or intensity of the primary stain must not

mask the green birefringence of amyloid in cross polarised light. Collagen and elastin usually give pale

yellow to bright white polarisation colours.


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Axonal Swelling (method for) Assessment Criteria

Primary Stain Intensity too strong Intensity too weak

Stain colour Axonal Swelling demonstration good Axonal Swelling demonstration poor

Low power visibility Not selective Uneven staining

Background Deposit / precipitate present

Counterstain Nuclear intensity too strong Nuclear intensity too weak





Description of Staining Results Axonal swelling may be demonstrated by silver impregnation methods such as Bielschowsky or using antibodies such as amyloid precursor protein (APP). Swellings should be clearly stained, visible at low power, and discernible from other structures. Immunohistochemical methods require a light nuclear counterstain. Section quality and presentation should not impair the result.


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Copper Associated Protein (CAP) Assessment Criteria


Stain colour Not selective

Uneven staining Background Deposit / precipitate present

Counterstain Intensity too strong Intensity too weak





Description of Staining Results All copper associated protein should be stained and clearly identifiable. Hepatitis B surface antigen and elastin, if stained, should be readily distinguishable from CAP. Background staining should be negligible. If a counterstain is used it should not mask or modify the primary stain, and should assist location. Section quality and presentation should not impair the result.


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Diastase/PAS Assessment Criteria



Uneven staining Background Deposit / precipitate present Residual glycogen






Description of Staining Results The Schiff Reaction is a histochemical test method for aldehyde groups. Periodic acid oxidation creates these groups on a variety of tissue structures notably glycogen, mucins, basement membranes and fungi. This modification uses the enzyme diastase (or amylase) to digest and remove any glycogen present in the section. There must be complete removal of glycogen and precise, complete demonstration of intestinal mucopolysaccharides. Membrane staining should not be confused with background staining, which should be minimal. The counterstain (typically haematoxylin) should provide good colour contrast and assist location. Section quality and presentation must not impair the result.


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Elastin Van Gieson Assessment Criteria

Primary Stain Intensity too strong

Intensity too weak Stain colour No contrast

Fine fibres Not selective Background Uneven staining Deposit / precipitate present

Counterstain Collagen stain intensity too strong

Collagen stain intensity too weak Collagen stain colour

Cytoplasm stain intensity too strong Cytoplasm stain intensity too weak Cytoplasm stain colour Not selective




Description of Staining Results Elastin is the main constituent of elastic fibres and its staining falls into two main groups: Haematoxylin based (e.g. Verhoeff) and hydrophobic dye mixes (e.g. Miller or Weigert) There must be precise staining of all coarse and fine elastic fibres.

The Van Gieson counterstain is a simple trichrome and should demonstrate good colour

separation between muscle (yellow) and collagen (red).

Picro Sirius Red is a suitable alternative counterstain, popular in the USA.

The counterstain should provide good colour contrast and assist location.


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Glial Fibres (method for) Assessment Criteria


Stain colour Glial Fibre demonstration good Glial Fibre demonstration poor

Low power visibility Not selective







Description of Staining Results Glial fibres may be demonstrated using tinctorial methods such as PTAH or using antibodies such as GFAP. Astrocytic fibres should be clearly stained against a pale or clear background. With PTAH, cell nuclei and myelin will also stain, thus requiring no further counterstaining. Immunohistochemical methods require a light nuclear counterstain. Section quality and presentation must not impair the result.


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Gram Stain Assessment Criteria

Primary Stain Gram positive intensity too strong Gram positive intensity too weak

Gram positive colour Not selective

Uneven staining Background Deposit / precipitate present Low power visibility

Counterstain Gram negative intensity too strong Gram negative intensity too weak

Gram negative colour Not selective

Uneven staining Deposit / precipitate present Low power visibility



Description of Staining Results Crystal Violet staining should render all Gram positive organisms blue/black without over or under differentiation. There should be no violet background. The counterstain serves to demonstrate all Gram negative organisms in a contrasting colour and to assist location. Most variations of this technique are based upon varied counterstains. Section quality and presentation must not impair the result.


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Grocott Assessment Criteria

Primary Stain Organism demonstration too strong Organism demonstration too weak

Organism colour Not selective


Non-specific silver precipitate






Description of Staining Results There should be dark grey or black staining of all fungal spores, hyphae or pneumocystis as appropriate. Where appropriate, internal structures of the organisms should be visible. Impregnation of other structures, particularly collagen, should be negligible. There should be not be any non-specific silver precipitation. The counterstain should provide good colour contrast and assist location, but must not mask any delicately impregnated fungi. Section quality and presentation must not impair the result.


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Haematoxylin / Van Gieson Assessment Criteria

Primary Stain Cytoplasm stain intensity too strong Cytoplasm stain intensity too weak Cytoplasm stain colour Collagen stain intensity too strong Collagen stain intensity too weak

Collagen stain colour Not selective







Description of Staining Results HVG is a simple trichrome method designed to give colour separation between smooth muscle and collagen. The emphasis is on collagen, all of which should be stained bright red. Muscle, erythrocytes and general cytoplasm (including hepatocytes) should be yellow. Nuclear staining should be dark blue or black and must not influence the cytoplasmic colouration by being under differentiated. Section quality and presentation should not impair the result.


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Martius Scarlet Blue Assessment Criteria

Primary Stain Fibrin demonstration too strong Fibrin demonstration too weak

Muscle stain intensity too strong Muscle stain intensity too weak

Muscle stain Colour Collagen stain intensity too strong

Collagen stain intensity too weak Collagen stain colour

Not selective Uneven staining

Red blood cell colour Background Deposit / precipitate present



Uneven ctaining Deposit / precipitate present



Description of Staining Results MSB is a specialised trichrome method designed to demonstrate fibrin of various ages in colours from orange through to red. Note that early fibrin can also stain yellow and old fibrin blue. All fibrin should be brightly coloured and distinguishable from other structures. As with other trichrome stains, there should be good colour separation between the dyes i.e. red blood cells (yellow), muscle (red) and collagen (blue). Nuclear staining should be dark blue or black. Section quality and presentation should not impair the result.


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Masson Fontana (for melanin) Assessment Criteria

Primary Stain Melanin demonstration too strong Melanin demonstration too weak Not selective

Uneven Staining Background Deposit / precipitate present

Non-specific silver precipitate Incomplete toning






Description of Staining Results All melanin should be coloured black by silver ammonical silver reduction. The reaction product should be toned to distinguish it from unreacted melanin. There should not be any background staining arising from over impregnation and there should be no non-specific silver precipitation. The counterstain should provide good colour contrast and assist location. Section quality and presentation should not impair the result.


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Myelin (method for) Assessment Criteria


Stain colour Myelin demonstration good Myelin demonstration poor








Description of Staining Results Axonal myelin sheaths may be demonstrated by tinctorial methods such as Luxol Fast Blue (LFB) or using antibodies such as myelin basic protein. All myelin should be clearly stained against a pale/clear background. Fine fibres should be discernible. LFB should be counterstained, traditionally with Cresyl Fast Violet. Section quality and presentation should not impair the result.


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Neurofibrillary Tangles Assessment Criteria


Stain colour Tangle demonstration good Tangle demonstration poor








Description of Staining Results Neurofibrillary Tangles may be demonstrated quite specifically using silver impregnation methods such as Gallyas or using antibodies such as Tau or Ubiquitin All tangles should be clearly stained, visible at low power, and discernible from other structures. Immunohistochemical methods require a light nuclear counterstain. Section quality and presentation should not impair the result.


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Nissl Substance Assessment Criteria


Stain colour Nissl demonstration good Nissl demonstration poor








Description of Staining Results All Nissl granules in neuronal cell bodies should be clearly stained purple / blue. Cell nuclei should also be demonstrated. There should be no negligible background and no stain precipitate. This method may also be employed as a counterstain for other techniques. Section quality and presentation should not impair the result.


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Perls’ Prussian Blue Assessment Criteria



Uneven staining Background Deposit / precipitate present Unreacted iron






Description of Staining Results A histochemical reaction for ferric iron adapted for the demonstration of haemosiderin. All pigment with iron containing haemosiderin should be demonstrated without stain precipitate. The Prussian Blue colouration of haemosiderin should not be modified by the counterstain or underlying, unreacted pigment. The counterstain should provide good colour contrast and assist location. Section quality and presentation should not impair the result.


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Reticulin (silver method for) Assessment Criteria

Primary Stain Reticulin demonstration too strong Reticulin demonstration too weak Not selective

Uneven Staining Background Deposit / precipitate present

Non-specific silver precipitate Incomplete toning






Description of Staining Results This method for demonstrating reticular fibres is visualised foremost at a low power scanning, so contrast is all important. There are many methods, the most popular in the UK being Gordon and Sweet or Foots. In Europe and the US Gomori is more popular. The latter impregnates nuclei which, renders counterstaining unnecessary. Toning is optional but must be complete when used. Untoned collagen remains golden brown and cell cytoplasm pale yellow. Reticulin should appear black. Under impregnation results in incomplete staining, and over impregnation yields knobbly fibres. The finer reticulin fibres should be clearly seen at high power. There should be no non-specific silver deposit or precipitate. Counterstaining, if used, should provide good contrast and assist location. Gomori and its variants may render toned collagen rose-pink. Section quality and presentation should not impair the result.


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Senile Plaques (method for) Assessment Criteria


Stain colour Plaque demonstration good Plaque demonstration poor


Uneven staining Background Deposit / precipitate present Non-specific silver precipitate

Incomplete toning






Description of Staining Results Plaques may be demonstrated quite specifically using silver impregnation methods such as Haga or by virtue of their amyloid component using antibodies such as β amyloid A4. All plaques should be clearly stained against a pale or clear background and be visible at low power. The dystrophic neurites of neuritic plaques may also be demonstrated. Silver methods generally create a yellow background which requires no further counterstaining. Immunohistochemical methods require a light nuclear counterstain Section quality and presentation should not impair the result.


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Trichrome (not HVG) Assessment Criteria

Primary Stain Muscle stain intensity too strong Muscle stain intensity too weak

Muscle Stain Colour Collagen stain intensity too strong

Collagen stain intensity too weak Collagen stain Colour

Not selective Uneven staining Red blood cell colour Background Deposit / precipitate present


Nuclear stain Colour Not selective




Description of Staining Results Trichrome methods are designed to give colour separation between smooth muscle and collagen. There are a variety of techniques; Masson’s Trichrome being the most popular method. All muscle should be stained red, and collagen blue or green. The collagen may be stained green or blue, depending on the dye used. Nuclear staining should be dark blue/black. Section quality and presentation should not impair the result. NB: HVG is assessed separately and will not be accepted if submitted as a “standard” trichrome stain.


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Ziehl – Neelsen Assessment Criteria

Primary Stain Bacilli stain intensity too strong Bacilli stain intensity too weak

Bacilli colour Not selective

Uneven staining Background Deposit / precipitate present Low power visibility






Description of Staining Results A stain used for acid-fast organisms, mainly Mycobacterium tuberculosis and other closely related organisms, such as Mycobacterium leprae. All acid-fast mycobacteria, including isolated bacilli, should be sharply stained magenta/red by carbol fuchsin with minimal background staining. The counterstain should be light, even and predominantly nuclear. It should provide good colour contrast assist location. There should be no dye precipitation and the tissue should not be damaged by heating. Section quality and presentation should not impair the result.


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Assessment Criteria Definitions

Haematoxylin and Eosin

Special Stains


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Haematoxylin and Eosin Criteria Definitions

Pre Microtomy The material supplied must be paraffin wax processed and have sufficient variety of cellular features, in particular nuclei, to allow a meaningful assessment. Insufficient cellular features for assessment - the material supplied does not have sufficient variety of cellular features, in particular nuclei to allow assessment Artefacts, such as crush effects and foam insert impressions should not be introduced during the laboratory handling and preparation stages. Crush artefacts - distortion of nuclei to give a thin elongated appearance because of crushing prior to fixation Foam inset artefact – apparent holes on the outer surface of biopsies produced by the surface of foam cassette inserts The tissue must show no evidence of being inadequately fixed or having had delayed fixation and there must be a clear demonstration of the chromatin detail within the nuclei at medium and low power and little cell lysis. Red blood cell lysis - loss of structure and shape and fusion of red cells Poor chromatin detail - insufficient chromatin clumping, granularity or perinuclear chromatin There must be no evidence of poor processing. Epithelial cells groups and connective tissue components must not appear to be separated by cracking. There should be no disruption of the nuclear membrane resulting in pale, fused nuclear staining (nuclear meltdown). The appearance of bubble like artefacts over the nuclei will be noted but no marks will be deducted. Cracking - excessive separation of epithelial cells, lymphoid cells or connective tissue Nuclear bubbling - the appearance of a vacuole or bubble shape over and within nuclei Nuclear meltdown - loss of a distinct nuclear membranes and pale staining in nuclei

If the biopsy is suspected of being orientated the block in a way that fails to demonstrate the cell types or layers appropriately this will be noted but no deduction will be made from the score. Similarly if it is felt that the tissue has not been trimmed to full face, or has been trimmed past full face this will be noted but no deduction will be made. Incorrect orientation - suspected lack of all the cell layers expected in a tissue type Incorrect trimming - suspected lack of areas of epithelium or other which could be expected


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Microtomy The section must be of a thickness appropriate to the tissue type. The section is too thick if it is difficult to focus on a single layer of nuclei, or too thin if staining intensity is compromised. It must be free from variations in thickness, creases, folds, scores and contaminants. No accumulation or fragments of tissue should be seen over or between sections. The section should be flat, with no lifted areas. There should be no holes, tearing or damage either as a result of trimming or sectioning or cover-slipping. The section must be positioned on the slide so that microscopy is not compromised. Chatter / vibration - very closely placed variations in thickness causing stripes of alternate staining intensity across the direction of sectioning Displacement - cells that become detached and move over other areas of the section Folds / creases - creases and folds in the section giving double layers that lift from the slide and stain intensely Knife back debris - accumulation of cells smeared on the edge of the knife and are deposited between alternate sections Knife marks - scores and scratches parallel / in line with the direction of sectioning Lifting - areas of the section that lift and do not lie flat Position on slide - section positioned inappropriately on the slide for full visualisation Section too thick - unable to focus on a single layer of cells Section too thin - loss of stain intensity and contrast due to the thinness of the section Section thickness variable - varying thickness in different areas of the section, recognised by varying dye intensity Squames / floaters / fibres -contamination by material that is not in the block, usually above the section Trimming artefact - tears and rough edged holes, often with lifted edges, within the section Water bath bubbles - circular areas of lifting, often cracked and intensely stained


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Staining Nuclei must be stained purple/ blue with haematoxylin. The intensity must be strong enough allow clear demonstration of nuclear detail at a medium power, but not too strong to cause a loss of the chromatin granularity or excessive cytoplasmic or connective tissue staining. Where the haematoxylin has been differentiated out, minimal cytoplasmic or connective tissue background staining with haematoxylin must remain. This background if present must not reduce the effectiveness of the nuclear demonstration or affect the colour and selectiveness of the eosin. Haematoxylin intensity too strong - a loss of the chromatin granularity or excessive cytoplasmic or connective tissue staining Haematoxylin intensity too weak - intensity must be strong enough to allow clear demonstration of nuclear detail at a medium power Haematoxylin colour not purple blue - nuclei must be stained purple/ blue with haematoxylin Haematoxylin background staining - where the haematoxylin has been differentiated out, minimal cytoplasmic or connective tissue background staining with haematoxylin must remain. If present , it must not reduce the effectiveness of the nuclear demonstration or affect the colour and selectiveness of the eosin

The eosin should be selective enough to demonstrate different cellular components such as collagen, cytoplasm, red blood cells, cellular granules, and amyloid etc. The intensity must be appropriate to the section thickness and the haematoxylin intensity. Where the eosin is too weak it will fail to allow selective demonstration of different components at low power. If the eosin intensity is too strong the colour and detail of the nuclear stain will be obscured and selectivity will be reduced. Eosin Intensity too strong - the intensity must be appropriate to the section thickness and the haematoxylin intensity. If the eosin intensity is too strong the colour and detail of the nuclear stain will be obscured and selectivity will be reduced Eosin Intensity too weak - the intensity must be appropriate to the section thickness and the haematoxylin intensity. Where the eosin is too weak it will fail to allow selective demonstration of different components at low power Eosin colour - the cytoplasm is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, stain not evenly applied to slide, uneven washing or incorrect dehydration Eosin not selective - the eosin should be selective enough to demonstrate different cellular components such as collagen, cytoplasm, red blood cells, cellular granules, amyloid etc

All staining should be even across the section and there should be no dye deposits or precipitate. Uneven staining - all staining should be even across the section Stain deposit present -dye deposits or precipitate


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Post Staining Coverslipping must be free from air bubbles, air drying artefact or drying back. There should be no excessive mountant, the sections must be totally covered and there must be no evidence of incomplete dewaxing or dehydration. The difficulties of processing and sectioning bony tissue will be taken into account when assessing. Air bubbles - air bubbles within the mountant Air drying artefact - refractile areas and tissue damage Excessive mountant - mountant outside the coverslip Mountant shrinkage - areas of mountant dried back from the coverslip edges Residual wax - refractile areas of undissolved wax are visible Section wiped / section off slide - section scratched by hand or off edge of slide Tissue exposed - the section is not covered by the coverslip Water present - droplets of water under the coverslip Contaminant on slide - Contamination by non biological or biological material, excluding dye / reagent deposit, usually above the tissue section between the section and the coverslip. For example, squames / floaters / fibres/ pencil or ink deposits


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Special Stains Criteria Definitions

Primary Stain Alcian Blue colour - the acid mucins are not bright blue, and the mixed mucins are not purple. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Alcian Blue intensity too strong - stain intensity is so strong that it is obscuring or interfering with the nuclear stain or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. Alcian Blue intensity too weak - the stain intensity is too weak and is failing to show the clear, selective demonstration of different components at low power at the intensity appropriate to the section thickness. Amyloid Brightfield background - background staining present, reducing contrast of the nuclei or

obscuring detail or affecting colour of amyloid deposits. Caused by staining too long, insufficient

differentiation, non-selective staining, or breakdown products in old stain.

Amyloid Brightfield deposit / precipitate present – random, irregular or crystalline deposits above

and around amyloid deposits, possibly due to old or unfiltered dye.

Amyloid Brightfield intensity too strong - stain intensity is so strong that it obscures / interferes

with the nuclear counterstain, or staining areas that should be another colour. Other detail may be

missing or selectivity could be reduced.

Amyloid Brightfield intensity too weak - stain intensity is so weak that it is failing to demonstrate

amyloid deposits clearly.

Amyloid Brightfield stain colour – amyloid is not stained the appropriate colour based on the

method employed and the expected staining results. May be caused due to old dyes, incorrect

mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Axonal Swelling demonstration good – swellings should be clearly stained, visible and discernible from other structures. Axonal Swelling demonstration poor - ineffective demonstration prevents successful visualisation. Bacilli Colour - the bacilli are not stained magenta / red. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Bacilli stain Intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of bacilli or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. Bacilli stain Intensity too weak - where the stain is too weak it will fail to allow the clear, selective demonstration of different components at low power the intensity must be appropriate to the section thickness.


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Primary Stain continued

Background - background staining present, reducing contrast of the nuclei or obscuring detail or affecting colour of another feature. Caused by staining too long, not differentiating enough or non-selective breakdown products in old stains.

Birefringence intensity too weak – Birefringence intensity is so weak in cross polarised light that it is

failing to demonstrate amyloid deposits clearly.

Birefringence colour – Birefringence is not the appropriate colour based on the method employed and the expected results in cross polarised light. Incorrect / non-stringent method. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Birefringence not selective – Birefringence not selective of different tissue components in cross polarised light. Incorrect / non-stringent method. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Collagen stain colour - the collagen is not stained the appropriate colour based on the method

employed and the expected staining results . May be caused due to old dyes, incorrect mixing, not

evenly applied to slide, uneven washing or incorrect dehydration

Collagen stain intensity too strong - stain intensity is so strong that it is obscuring / interfering with demonstration of collagen and different components or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. Collagen stain intensity too weak- the stain is too weak and is failing to allow the clear, selective demonstration of collagen and different components at low power. The intensity must be appropriate to the section thickness.

Cytoplasm stain colour - the cytoplasm is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Cytoplasm stain intensity too strong - stain intensity is so strong that it is obscuring/ interfering with demonstration of different cytoplasmic components. Other detail may be missing or selectivity could be reduced. Cytoplasm stain intensity too weak- where the stain is too weak it will fail to allow the clear, selective demonstration of different cytoplasmic components at low power. The intensity must be appropriate to the section thickness.

Deposit / Precipitate present - random irregular or crystalline deposits above and around the cells, due to old or unfiltered dyes.

Fibrin demonstration too strong - stain intensity is so strong that it is obscuring/ interfering with demonstration of fibrin and different components or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced.


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Primary Stain continued Fibrin demonstration too weak - the stain is too weak and is fail to allow the clear, selective demonstration of fibrin and different components at low power the intensity must be appropriate to the section thickness. Fine fibres – effective demonstration allows fine fibres to be visualised successfully. Glial Fibre demonstration good – fibres should be clearly stained against a pale or clear background, and visible and discernible from other structures.

Glial Fibre demonstration poor - ineffective demonstration prevents successful visualisation. Gram positive colour - the gram positive organisms are not stained blue / black. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Gram positive intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of bacilli or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. Gram positive intensity too weak - the stain is too weak and is failing to allow the clear, selective demonstration of bacilli at low power the intensity must be appropriate to the section thickness. Incomplete toning – colouration impairs clear visualisation / demonstration. Intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the nuclear stain or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced.

Intensity too weak - stain intensity is so weak that it is failing to demonstrate tissue components clearly

Low power visibility - where it is effective and precise to allow the clear, selective demonstration of different components at low power. The intensity must be appropriate to the section thickness.

Melanin demonstration too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of melanin or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced.

Melanin demonstration too weak - the stain is too weak and is failing to allow the clear, selective demonstration of melanin at low power the intensity must be appropriate to the section thickness.

Muscle stain colour - the muscle is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Muscle stain intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of muscle or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced.


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Muscle stain intensity too weak - the stain is too weak and is failing to allow the clear, selective demonstration of muscle at low power the intensity must be appropriate to the section thickness. Myelin demonstration good – myelin should be clearly stained against a pale or clear background, and discernible from other structures. Myelin demonstration poor - ineffective demonstration prevents successful visualisation.

Nissl demonstration good – myelin should be clearly stained against a pale or clear background, and discernible from other structures. Nissl demonstration poor - ineffective demonstration prevents successful visualisation.

No green birefringence on cross polarisation - Amyloid deposits that are stained pink/red in

brightfield microscopy must also exhibit green birefringence (strong) in cross polarised light

No contrast – colouration impairs clear visualisation / demonstration of target cells, tissue component or organism. Non-specific silver precipitate - deposit / precipitate present as deposits above and around the cells. Due to old or unfiltered dyes. Not selective - Not selective of different cell types or tissue components. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Organism colour - the organisms according to the staining results of the method employed. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Organism demonstration too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of organisms or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced.

Organism demonstration too weak - the stain is too weak and is failing to allow the clear, selective demonstration of organisms at low power the intensity must be appropriate to the section thickness.

PAS colour - the neutral mucins are not bright magenta, and the mixed mucins are not purple. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

PAS intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the nuclear stain or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. PAS intensity too weak - the stain is too weak and is failing to allow the clear, selective demonstration of different components at low power the intensity must be appropriate to the section thickness.


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Plaque demonstration good – plaques should be clearly stained against a pale or clear background, and discernible from other structures. Plaque demonstration poor - ineffective demonstration prevents successful visualisation.

Red blood cell colour – red blue cells must be stained according to the staining results of the method employed. Residual glycogen – residual glycogen present in tissue components after digestion, where glycogen should no longer be demonstrated. Reticulin demonstration too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of thick and / or fine reticulin fibres or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. Reticulin demonstration too weak - the stain is too weak and is failing to allow the clear, selective demonstration of different thick and / or fine at low power the intensity must be appropriate to the section thickness. Stain colour – is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Tangles demonstration good – neurofibrillary tangles should be clearly stained against a pale or clear background, and discernible from other structures. Tangles demonstration poor - ineffective demonstration prevents successful visualisation. Uneven staining - varying intensity of staining in different parts of the slide preparation. Dyes applied but not spread evenly. Uneven dehydration.

Unreacted iron– a significant amount of haemosiderin has not reacted with the Perls’ reagent and remains brown in colour.

Counterstain Collagen stain colour - the collagen is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Collagen stain intensity too strong - stain intensity is so strong that it is obscuring/ interfering with demonstration of collagen and different components or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced.


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Counterstain continued

Collagen stain intensity too weak - the stain is too weak and is failing to allow the clear, selective demonstration of collagen and different components at low power the intensity must be appropriate to the section thickness.

Cytoplasm stain colour - the cytoplasm is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Cytoplasm stain intensity too strong - stain intensity is so strong that it is obscuring/ interfering with demonstration of different cytoplasmic components. Other detail may be missing or selectivity could be reduced. Cytoplasm stain intensity too weak- if the stain is too weak it will fail to allow the clear, selective demonstration of different cytoplasmic components at low power. The intensity must be appropriate to the section thickness. Deposit / Precipitate present - Deposit / precipitate present - random irregular or crystalline deposits above and around the cells. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Gram Negative colour - the gram negative organisms are not stained according to the colour of the counterstain employed. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Gram Negative intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the clear, selective demonstration of bacilli or staining areas that should be another colour. Other detail may be missing or selectivity could be reduced. Gram Negative intensity too weak - the stain is too weak and is failing to allow the clear, selective demonstration of bacilli at low power the intensity must be appropriate to the section thickness. Intensity too strong - stain intensity is so strong that it is obscuring/ interfering with the nuclear stain or staining areas that should be another colour. Other detail may be missing. Intensity too weak - stain intensity is so weak that it is failing to demonstrate tissue components clearly. Low power visibility - where it is effective and precise to allow the clear, selective demonstration of different components at low power. The intensity must be appropriate to the section thickness. Not selective - Not selective of different cell types or tissue components. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.

Nuclear stain Colour – nuclei are not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration.


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Counterstain continued

Nuclear stain Intensity too strong - Obscuring chromatin detail or staining non-nuclear features. Usually caused by under differentiation/ or staining for too long. If so there may also be background staining of cytoplasm. Nuclear stain intensity too weak - Failing to clearly show chromatin detail. Nuclei don’t stand out well on low power and are feint relative to the background. May be caused by old dyes or too short staining time or over differentiated. Stain colour – is not stained the appropriate colour based on the method employed and the expected staining results. May be caused due to old dyes, incorrect mixing, not evenly applied to slide, uneven washing or incorrect dehydration. Uneven staining - varying intensity of staining in different parts of the slide preparation. Dyes applied but not spread evenly. Uneven dehydration.

Post Staining Air bubbles - air bubbles within the mountant.

Air drying artefact - refractile areas and tissue damage.

Excessive mountant - mountant outside the coverslip.

Mountant shrinkage - areas of mountant have dried back from the coverslip edges.

Residual wax - refractile areas of un-dissolved wax are visible.

Section wiped / section off slide - section scratched by hand or off edge of slide .

Tissue exposed - the section is not covered by the coverslip.

Water present - droplets of water under the coverslip.

Contaminant on slide - Contamination by non biological or biological material, excluding dye / reagent deposits, usually above the tissue section between the section and the coverslip. For example, squames / floaters / fibres/ pencil or ink deposits.


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Appendix 1


Scoring guidelines

Scoring based on criteria



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The material supplied must be paraffin wax processed and have sufficient variety of cellular features, in

particular nuclei to allow assessment. Artefacts, such as crush effects and foam insert impressions should

not be introduced during the laboratory handling and preparation stages.

The tissue must show no evidence of being inadequately fixed or having had delayed fixation and there

must be a clear demonstration of the chromatin detail within the nuclei at medium and low power and

little red cell lysis.

There must be no evidence of poor paraffin processing. Epithelial cells groups and connective tissue

components must not appear to be separated by cracking. There should be no disruption of the nuclear

membrane resulting in pale, fused nuclear staining (nuclear meltdown). The appearance of bubble like

artefacts over the nuclei will be noted but no marks will be deducted.

If the biopsy is suspected of being orientated in a way that fails to demonstrate the cell types or layers

appropriately this will be noted but no deduction will be made from the score. Similarly if it is felt that the

tissue has not been trimmed to full face, or has been trimmed past full face this will be noted but no

deduction will be made.

The section must be of a thickness appropriate to the tissue type. The section is too thick if it is difficult to

focus on a single layer of nuclei, or too thin if staining intensity is compromised. It must be free from

variations in thickness, creases, folds, scores and contaminants. No accumulation or fragments of tissue

should be seen over or between sections. The section should be flat, with no lifted areas. There should

be no holes, tearing or damage either as a result of trimming or sectioning or coverslipping. The section

must be positioned on the slide so that microscopy is not compromised.

Nuclei must be stained purple blue with haematoxylin. The intensity must be strong enough allow clear

demonstration of nuclear detail at a medium power, but not too strong to cause a loss of the chromatin

granularity or excessive cytoplasmic or connective tissue staining. Where the haematoxylin has been

differentiated out, minimal cytoplasmic or connective tissue background staining with haematoxylin must

remain. This background if present must not reduce the effectiveness of the nuclear demonstration or

affect the colour and selectiveness of the eosin.

The eosin should be selective enough to demonstrate different cellular components such as collagen,

cytoplasm, red blood cells, cellular granules, amyloid etc. The intensity must be appropriate to the

section thickness and the haematoxylin intensity. Where the eosin is too weak it will fail to allow selective

demonstration of different components at low power. If the eosin intensity is too strong the colour and

detail of the nuclear stain will be obscured and selectivity will be reduced.

All staining should be even across the section and there should be no dye deposits or precipitate.

Coverslipping must be free from air bubbles, air drying artefact or drying back. There should be no

excessive mountant, the sections must be totally covered and there must be no evidence of incomplete

dewaxing or dehydration. The difficulties of processing and sectioning bony tissue will be taken into

account when assessing.


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Scoring Guidelines

Each pair of assessors completes an assessment form and data from these forms are converted into

a mark out of 5, from each assessor. The mark out of 5 from each assessor is based on the criteria

for a given method.

Guide lines for individual assessors mark (out of 5)

where 0 – non submission, 1- Fail, 2 - Borderline Fail, 3 - Pass, 4 – Good, 5 - Excellent

0 – non submission

Score 1 Fail

No staining demonstrated based on the method employed and the expected staining results

Score 2 Borderline Fail

Unsatisfactory demonstration based on the method employed, with expected staining

results being inappropriate

Score 3 Pass

Appropriate demonstration based on the method employed and the expected staining

results, although improvements need to be made in the staining.

Score 4 Good

Good appropriate demonstration based on the method employed and the expected staining

results

Score 5 Excellent

Excellent demonstration based on the method employed and the expected staining results

Each assessor submits their mark out of 5 based on the criteria for a given method, giving a total

score for the submitted slide out of 10.

Guide lines for total score (out of 10)

Score <5 a score of less than 5 / 10 is given for poor staining, where the participant has failed to clearly demonstrate the expected results.

Score 5/6 a score of 5 or 6 / 10 is a pass. Whilst the staining appropriately demonstrates the expected staining results, staining is suboptimal and improvements are still required overall.

Score 7/8 a score of 7 or 8 / 10 shows good appropriate demonstration of the expected results, and an acceptable level of quality.

Score 9/10 a score of 9 or 10 / 10 shows excellent appropriate demonstration of the expected results, and a high level of quality.

NB. Any slides which score a mark of 4 or below are passed to secondary assessors for further assessment before a final score is issued. If there is a discrepancy of 2 between the assessing pair e.g. 3 & 5, the slide will be passed for secondary assessing. If there is a discrepancy of pass / fail between the assessing pair, the slide will also be passed for secondary assessing.


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Scoring based on criteria Below is a generic ideal score for Haematoxylin & Eosin, and special stains.

It is intended purely as a guide for laboratories and assessors. Slides may not achieve all of the points

listed and may have elements which span several score boundaries.

Score 5 - Excellent Excellent nuclear and tissue constituent staining in a suitable preparation allowing full visualisation of nuclear and component features within the tissue.

Nuclear staining intensity which demonstrates the chromatin detail clearly.

Staining colour, intensity and balance allows clear distinction between nuclear detail and other non-nuclear features.

Primary stain and counterstain demonstrates the appropriate tissue constituents depending on the method being employed.

The cytoplasm shows the appropriate colour spectrum.

Counterstain intensity stain does not obscure / interfere with the nuclear stain or staining areas that should be another colour. Intensity is such that it is allows clear demonstration of tissue components.

Demonstration is appropriate based on the method employed and the expected staining results.

Even staining across the tissue section, with minimal background staining and no dye deposits or precipitate.

Suitable preparation to allow clear observation of the appropriate tissue constituents depending on the method being employed.

There are no microtomy or processing irregularities.

Dehydration, coverslipping and labelling do not impair the visualisation of the tissue and its components.

The overall preparation and staining is excellent and allows full visualisation of the tissue and its components.


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Score 4 - Good Good nuclear and tissue constituent staining in a suitable preparation to

allow visualisation of nuclear and component features within the tissue.

Nuclear Staining intensity which demonstrates the chromatin detail clearly.

Staining colour, intensity and balance may not be consistent across the preparation but allows clear distinction between nuclear detail and other non-nuclear features.

The tissue constituents may lack appropriate colour spectrum in some areas.

Primary and counterstain intensity may be weak or intense but does not obscure detail.


There may be some uneven staining or background staining but detail is visible in the majority of the tissue.


There may be some microtomy or processing irregularities, but the material is adequate to assess.

Dehydration, coverslipping and labelling do not impair the visualisation of the tissue and its components.

The overall preparation and staining is good and allows full visualisation of the tissue and its components. Loss of staining or preparation quality is not detrimental to the identification of tissue constituent details.


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Score 3 - Pass Adequate nuclear and tissue constituent staining in a suitable preparation

to allow visualisation of nuclear and component features within the tissue.

There may be alteration to nuclear and non-nuclear intensity and colour, but most nuclear

detail is visible.

The tissue constituents do not show full colour spectrum. Nuclear staining may be under or over stained.

Primary and counterstain may be weak or intense but detail is still visible. Some background staining.


Staining may not be even across the preparation.


There may be some microtomy or processing irregularities.

The dehydration, coverslipping and labelling may obscure visualisation of the tissue and its components but there is sufficient visible to assess.

Score 2 - Borderline Fail Suboptimal nuclear and or tissue constituent staining which does not

allow full visualisation of nuclear and or component features. The

preparation quality or method does not allow full observation within the

tissue.

There may be alteration to nuclear and non-nuclear staining intensity and colour, but some nuclear detail is visible.

The tissue constituents do not show full colour spectrum. Nuclear and / or tissue component staining may be under or over stained.

Primary and counterstain may be intense and / or background staining may obscure detail.

Demonstration is inappropriate based on the method employed and the expected staining results.

Staining may not be even across the preparation.

The preparation quality may be suboptimal obscuring some tissue detail. There may be areas of microtomy or processing irregularities.

The dehydration, coverslipping and labelling may hinder tissue visualisation.


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Score 1- Fail The nuclear and or tissue constituent staining does not allow visualisation

of nuclear and / or component features or the preparation quality does

not allow clear observation within the tissue.

Alteration to nuclear and non-nuclear staining intensity and colour which obscures nuclear detail.

Background staining which obscures nuclear and tissue constituent detail.

Demonstration is inappropriate based on the method employed and the expected staining results.

Uneven or patchy staining amounting to loss of nuclear and tissue constituent detail in the majority of the tissue.

Preparation is not suitable to allow clear observation of tissue components.

The preparation quality may be suboptimal obscuring some tissue detail.

The dehydration, coverslipping and labelling obscures tissue visualisation.

Score 0 - Non submission No slides submitted for assessment or slides returned late without

contacting the Scheme Manager.


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General Pathology (Routine Histopathology) Stains assessed: Selected / In-house Material Haematoxylin and Eosin (H&E) stained sections (all 6 runs) Distributed Material Special A Special B Diastase / PAS Alcian blue / PAS Elastin / Van Gieson Amyloid (method for) Gram Grocott Perls’ Prussian blue Haematoxylin / Van Gieson Reticulin (silver method for) Masson-Fontana Ziehl Neelsen Martius Scarlet Blue (MSB)

Copper Associated Protein (method for)

Neuropathology Stains assessed: Selected/In-house Material Haematoxylin and Eosin (H&E) stained sections (all 6 runs) Distributed Material Special A Special B

Diastase/PAS Axonal swelling (method for) Elastin/Van Gieson Glial fibres (method for) Gram Myelin (method for) Perls’ Prussian blue Neurofibrillary tangles Reticulin (silver method for) Nissl substance Ziehl Neelsen Senile plaques (method for)

For neuropathology, the methods in list B may include immunocytochemical techniques where that is the department’s method of choice.

Renal Biopsy Pathology Stains assessed: Selected/In-house Material (all 6 runs) Haematoxylin and Eosin (H&E) stained sections Methenamine silver Periodic Acid Schiff Elastin Van Gieson


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Muscle Histochemistry Stains assessed: Selected/In-house Material (all 6 runs) Haematoxylin and Eosin (H&E) stained sections Gomori Trichrome NADH Cytochrome Oxidase (COx) One of the following stains will also be requested, in addition to H&E, Gomori, NADH and COx*; Acid Phosphatase Lipid PAS Primary fibre typing Succinate Dehydrogenase (SDH)

For muscle histochemistry, the methods in list bold are requested on a rotational basis and

may include immunocytochemical techniques where that is the department’s method of choice.

Diagnostic Non Gynaecological Cytology Selected/In-house Material (all 6 runs) Stains assessed: Specimen Types: Papanicolaou Serous Fluid Romanowsky Head and Neck Respiratory Urine

Bone Marrow Trephine Biopsy

Selected / In-house Material (all runs) Stains assessed: Haematoxylin and Eosin (H&E) stained sections Reticulin (silver method for)

Mohs’ Procedure

Selected / In-house Material (all runs) Stains assessed: Specific Site/Tissue Composition*: Haematoxylin and Eosin (H&E) stained sections Cutaneous Toluidine Blue Mucosal Hair Bearing

Cartilaginous


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