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Staining Criteria Handbook
General Pathology (Routine Histopathology)
Neuropathology
Edition 4
November 2015
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
Edition 004
2
Index
Page
Haematoxylin and Eosin Assessment Criteria 3
Special Stains A & B Assessment Criteria 6
List of Assessment Criteria 26
• Haematoxylin and Eosin
• Special Stains
Assessment Criteria Definitions 30
• Haematoxylin and Eosin
• Special Stains
Appendix 40
• Haematoxylin and Eosin Model Description
• Scoring System
• Scoring based on criteria
• UK NEQAS CPT Stain Repertoire
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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Haematoxylin and Eosin
Assessment Criteria
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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 artefact
Water bath bubbles
Staining Haematoxylin Intensity too strong
Haematoxylin Intensity too weak
Haematoxylin colour not blue
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 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 extended description including pre Microtomy, Microtomy and post staining
please see model description in appendix.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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Special Stains A & B
Assessment Criteria
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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 Coloration
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 mucopolysaccharides should be coloured bright blue. Neutral mucopolysaccharides
should be bright magenta. Mixed mucins should be purple.
There should be 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 and should not mask or alter the primary stains. It should assist location.
Section quality and presentation should not impair the result.
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Amyloid (method for) Assessment Criteria
Primary Stain - Congo red (stain) colour
Bright field Intensity too strong
Intensity too weak
Not selective
Uneven Staining
Background
Deposit / precipitate present
Primary Stain - No green birefringence on cross polarisation
Cross polarised light Birefringence intensity too weak
Birefringence intensity too strong
Birefringence not selective
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
Amyloid: There is no amyloid specific dye. However, when performed correctly the alkaline alcoholic
Congo red method is highly selective for binding to amyloid, when sections are prepared at the
appropriate thickness (~5 10 µm) and staining is performed stringently. Use of other dyes may produce
sub optimal or erroneous results.
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 bright
green colour in cross polarised light (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 vivid white polarisation colours.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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
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
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
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Not selective
Uneven Staining
Background
Deposit / precipitate present
Counterstain Intensity too strong
Intensity too weak
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
All copper associated protein should be stained and clearly identifiable.
HepBSA and elastin, if stained, should be readily distinguishable from CAP. Background
should be negligible.
If a counterstain is used it should not mask or modify the primary stain. It should assist
location.
Section quality and presentation should not impair the result.
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Diastase/PAS Assessment Criteria
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Not selective
Uneven Staining
Background
Deposit / precipitate present
Residual Glycogen
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
The Schiff reaction is a histochemical method for aldehyde groups. Periodic acid oxidation
creates these groups on a variety of tissue structures notably glycogen, mucins, basement
membrane and fungi.
This modification uses the enzyme diastase (or amylase) to digest out any glycogen and is
normally used in conjunction with a straight PAS (not required for EQA unless requested).
Complete removal of glycogen and precise, complete demonstration of intestinal
mucopolysaccharide.
Membrane staining should not be confused with background, which should be minimal.
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
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
Elastin stains fall into two main groups, Haematoxylin based (e.g. Verhoeff) and
hydrophobic dye mixes (e.g. Miller’s and Weigert’s)
Precise staining of all coarse and fine elastin fibres.
The counterstain is a simple trichrome and there should be good colour separation
between muscle (yellow) and collagen (red).
Picro Sirius red is a suitable alternative, popular in the USA. The counterstain should
provide good colour contrast and assist location.
Section quality and presentation must not impair the result.
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Glial Fibres (method for) Assessment Criteria
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Glial Fibre demonstration good
Glial Fibre demonstration poor
Low power visibility
Not selective
Uneven Staining
Background
Deposit / precipitate present
Counterstain Intensity too strong
Intensity too weak
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
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 this 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
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
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 are based on the counterstain used and vary widely.
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
Uneven Staining
Background
Deposit / precipitate present
Non Specific Silver Precipitate
Counterstain Intensity too strong
Intensity too weak
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
There should be dark grey/black staining of all fungal spores and/or hyphae or pneumocysts.
Where appropriate, internal structures of the organisms should be visible.
Impregnation of other structures, particularly collagen, should be negligible. There should be
no 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.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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
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
HVG is perhaps the simplest of trichrome methods which are 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 but must not influence the cytoplasmic
colouration by being under differentiated.
Section quality and presentation should not impair the result.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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
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
MSB is a specialised trichrome method designed to demonstrate fibrin of various ages in
colours from orange to red. Early fibrin can also stain yellow and old fibrin blue.
All fibrin should be brightly coloured and distinguishable from other structures.
As with other trichromes, there should be good colour separation between the dyes best
seen in 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
Counterstain Intensity too strong
Intensity too weak
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
All melanin should be coloured black by silver impregnation.
The reaction product should be toned to distinguish it from unreacted melanin.
There should be no background staining arising from over impregnation and 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
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Myelin demonstration good
Myelin demonstration poor
Low power visibility
Not selective
Uneven Staining
Background
Deposit / precipitate present
Counterstain Intensity too strong
Intensity too weak
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
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.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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Neurofibrillary Tangles Assessment Criteria
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Tangle demonstration good
Tangle demonstration poor
Low power visibility
Not selective
Uneven Staining
Background
Deposit / precipitate present
Counterstain Intensity too strong
Intensity too weak
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
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
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Nissl demonstration good
Nissl demonstration poor
Low power visibility
Not selective
Uneven Staining
Background
Deposit / precipitate present
Counterstain Intensity too strong
Intensity too weak
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
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
Primary Stain Intensity too strong
Intensity too weak
Stain Colour
Not selective
Uneven Staining
Background
Deposit / precipitate present
Unreacted iron
Counterstain Intensity too strong
Intensity too weak
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
A histochemical reaction for ferric iron adapted for the demonstration of haemosiderin
Prussian Blue colouration of haemosiderin should not be modified by counterstain or
underlying, unreacted pigment.
The counterstain should provide good colour contrast and assist location.
Section quality and presentation should not impair the result.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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
Counterstain Intensity too strong
Intensity too weak
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
This method is primarily a low power scanning method so contrast is all important. There
are many methods, the most popular in the UK being Gordon and Sweet. In Europe and the
US Gomori is more popular. The latter impregnates nuclei which, when done well, renders
counterstaining unnecessary.
The main variation in method is toning. Without it, collagen would remain golden brown
and cell cytoplasm a pale yellow. Again, counterstaining is unnecessary if the rest of the
method is done well.
Reticulin should be black, under impregnation will result in incomplete staining, over
impregnation yields knobbly fibres. Fine reticulin fibres should be clearly seen at high
power.
There should be no non specific silver deposits and staining of cytoplasmic structures is
undesirable.
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.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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Senile Plaques (method for) Assessment Criteria
Primary Stain Intensity too strong
Intensity too weak
Stain colour
Plaque demonstration good
Plaque demonstration poor
Low power visibility
Not selective
Uneven Staining
Background
Deposit / precipitate present
Non Specific Silver Precipitate
Incomplete Toning
Counterstain Intensity too strong
Intensity too weak
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
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 neuritis 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.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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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
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
Trichrome methods are designed to give colour separation between smooth muscle and
collagen. There are a variety of methods; Masson’s being the most popular in the UK.
All muscle should be stained red, collagen blue or green and nuclei blue/black.
The collagen (milling) dye may be green or blue.
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”
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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
Counterstain Intensity too strong
Intensity too weak
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
A stain used for Mycobacterium tuberculosis and closely related organisms
All 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.
NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook
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List of
Assessment Criteria
• Haematoxylin and Eosin
• Special Stains
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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 artefact
Water bath bubbles
Staining Haematoxylin Intensity too strong
Haematoxylin Intensity too weak
Haematoxylin colour not purple blue
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 exposed
Water present
Contaminant on slide
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Special Stains Assessment Criteria
Primary Stain Alcian Blue Colour
Alcian Blue Intensity too strong
Alcian Blue Intensity too weak
Amyloid Bright field Background
Amyloid Bright field Deposit / Precipitate present
Amyloid Bright field Intensity too strong
Amyloid Bright field Intensity too weak
Amyloid Bright field Stain colour
Axonal Swelling demonstration good
Axonal Swelling demonstration poor
Bacilli Colour
Bacilli stain Intensity too strong
Bacilli stain Intensity too weak
Background
Birefringence intensity too weak
Birefringence colour
Birefringence not selective
Collagen Stain Colour
Collagen Stain Intensity too strong
Collagen Stain Intensity too weak
Cytoplasm Stain Colour
Cytoplasm Stain Intensity too strong
Cytoplasm Stain Intensity too weak
Deposit / precipitate present
Fibrin Demonstration too strong
Fibrin Demonstration too weak
Fine Fibres
Glial Fibre demonstration poor
Glial Fibre demonstration good
Gram Positive Colour
Gram Positive Intensity too strong
Gram Positive Intensity too weak
Incomplete Toning
Intensity too strong
Intensity too weak
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Primary Stain continued Low power visibility
Melanin Demonstration too strong
Melanin Demonstration too weak
Muscle Stain Colour
Muscle Stain Intensity too strong
Muscle Stain Intensity too weak
Myelin demonstration poor
Myelin demonstration good
Nissl demonstration good
Nissl demonstration poor
No green birefringence on cross polarisation
No Contrast
Non Specific Silver Precipitate
Not selective
Organism Colour
Organism Demonstration too strong
Organism Demonstration too weak
PAS Coloration
PAS Intensity too strong
PAS Intensity too weak
Plaque demonstration good
Plaque demonstration poor
Red blood cell colour
Residual Glycogen
Reticulin Demonstration too strong
Reticulin Demonstration too weak
Stain colour
Tangle demonstration good
Tangle demonstration poor
Uneven Staining
Unreacted iron
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Counterstain Collagen Stain Colour
Collagen Stain Intensity too strong
Collagen Stain Intensity too weak
Cytoplasm Stain Colour
Cytoplasm Stain Intensity too strong
Cytoplasm Stain Intensity too weak
Deposit / precipitate present
Gram Negative Colour
Gram Negative Intensity too strong
Gram Negative Intensity too weak
Intensity too strong
Intensity too weak
Low power visibility
Not selective
Nuclear Stain Colour
Nuclear Stain Intensity too strong
Nuclear Stain Intensity too weak
Stain colour
Uneven Staining
Post Staining Air bubbles
Air drying artefact
Excessive mountant
Mountant shrinkage
Residual wax
Section wiped / section off slide
Tissue exposed
Water present
Contaminant on slide
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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 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 - triangular 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 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.
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 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 variation in thickness causing stripes of alternate staining
intensity at right angles to direction of sectioning
Displacement - cells that become detached and move over other areas of section
Folds / creases - creases and folds in the section giving doubles layers that lift from the slide and
stain intensely
Knife back debris - accumulation of cells that are smeared on the edge of the knife and deposited
between alternate sections
Knife marks - scores and scratches at parallel to the direction of sectioning
Lifting - areas of section that lift and do not lie flat
Position on slide - section positioned appropriately on slide for full visualisation
Section too thick - unable to focus on a single cell layer
Section too thin - loss of stain intensity and contrast because of thinness of 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
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.
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 . Old dyes. Mixed incorrectly. Not evenly applied to slide. Uneven
washing. Dehydrated incorrectly.
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 - retractile areas and tissue damage
Excessive mountant - mountant outside the coverslip
Mountant shrinkage - areas of mountant have dried back from the coverslip edges
Residual wax - retractile 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 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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Special Stains Criteria Definitions
Primary Stain
Alcian Blue Colour - the acid mucin is not bright blue, and the mixed mucins are not purple. Old
dyes. Mixed incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly
Alcian Blue 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.
Alcian Blue 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.
Amyloid Bright field Background - Background staining present, reducing contrast of the nuclei or
obscuring detail or affecting colour of amyloid deposits. Caused by staining too long, not
differentiating enough or non-selective staining, or breakdown products in old stain.
Amyloid Bright field Deposit / Precipitate present - Random irregular or crystalline deposits above
and around amyloid deposits. Old or unfiltered dye.
Amyloid Bright field 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 Bright field Intensity too weak - Stain intensity is so weak that it is failing to demonstrate
amyloid deposits clearly.
Amyloid Bright field Stain colour – Amyloid is not stained the appropriate colour based on the
method employed and the expected staining results. Old dye. Mixed incorrectly. Not evenly
applied to slide. Uneven washing. Dehydrated incorrectly.
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. Old dyes. Mixed incorrectly. Not evenly
applied to slide. Uneven washing. Dehydrated incorrectly.
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. Old dye. Mixed
incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly.
Birefringence not selective – Birefringence not selective of different tissue components in cross
polarised light. Incorrect / non-stringent method. Old dye. Mixed incorrectly. Not evenly applied to
slide. Uneven washing. Dehydrated incorrectly.
Collagen Stain Colour - the collagen is not stained the appropriate colour based on the method
employed and the expected staining results . Old dyes. Mixed incorrectly. Not evenly applied to
slide. Uneven washing. Dehydrated incorrectly.
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- where the stain is too weak it will fail 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 . Old dyes. Mixed incorrectly. Not evenly applied to
slide. Uneven washing. Dehydrated incorrectly.
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.
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.
Fibrin Demonstration too weak- where the stain is too weak it will fail to allow the clear, selective
demonstration of fibrin and different components at low power the intensity must be appropriate to
the section thickness.
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Primary Stain continued
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. Old dyes. Mixed
incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly
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- where the stain is too weak it will fail 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- where the stain is too weak it will fail 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 . Old dyes. Mixed incorrectly. Not evenly applied to
slide. Uneven washing. Dehydrated incorrectly.
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.
Muscle Stain Intensity too weak- where the stain is too weak it will fail to allow the clear, selective
demonstration of muscle at low power the intensity must be appropriate to the section thickness.
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Primary Stain continued
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. Old or unfiltered dyes.
Not selective - Not selective of different cell types or tissue components. Old dyes. Mixed
incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly
Organism Colour - the organisms according to the staining results of the method employed. Old
dyes. Mixed incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly
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- where the stain is too weak it will fail to allow the clear,
selective demonstration of organisms at low power the intensity must be appropriate to the section
thickness.
PAS Coloration - the neutral mucins are not bright magenta, and the mixed mucins are not purple.
Old dyes. Mixed incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly
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- 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.
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.
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Primary Stain continued
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- where the stain is too weak it will fail 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 . Old dyes. Mixed incorrectly. Not evenly applied to slide. Uneven washing.
Dehydrated incorrectly.
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 . Old dyes. Mixed incorrectly. Not evenly applied to
slide. Uneven washing. Dehydrated incorrectly.
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- if the stain is too weak it will fail to allow the clear, selective
demonstration of collagen and different components at low power the intensity must be
appropriate to the section thickness.
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Counterstain continued
Cytoplasm Stain Colour - the cytoplasm is not stained the appropriate colour based on the method
employed and the expected staining results . Old dyes. Mixed incorrectly. Not evenly applied to
slide. Uneven washing. Dehydrated incorrectly.
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. Old or unfiltered dyes.
Gram Negative Colour - the gram negative organisms are not stained according to the colour of the
counterstain employed. Old dyes. Mixed incorrectly. Not evenly applied to slide. Uneven washing.
Dehydrated incorrectly
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- where the stain is too weak it will fail 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. Old dyes. Mixed
incorrectly. Not evenly applied to slide. Uneven washing. Dehydrated incorrectly
Nuclear Stain Colour – nuclei are not stained the appropriate colour based on the method employed
and the expected staining results . Old dyes. Mixed incorrectly. Not evenly applied to slide. Uneven
washing. Dehydrated incorrectly.
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
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Counterstain continued
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. 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 . Old dyes. Mixed incorrectly. Not evenly applied to slide. Uneven washing.
Dehydrated incorrectly.
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 - retractile areas and tissue damage
Excessive mountant - mountant outside the coverslip
Mountant shrinkage - areas of mountant have dried back from the coverslip edges
Residual wax - retractile 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
• Haematoxylin and Eosin Model Description
• Scoring guidelines
• Scoring based on criteria
• UK NEQAS CPT Stain Repertoire
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Haematoxylin and Eosin Model Description
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 complete 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
1 – Fail - No staining demonstrated based on the method employed and the expected staining
results
2 – Borderline Fail - unsatisfactory demonstration based on the method employed, with
expected staining results being inappropriate
3 – Pass - appropriate demonstration based on the method employed and the expected
staining results, although improvements need to be made in the staining.
4 – Good – good appropriate demonstration based on the method employed and the
expected staining results
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 a secondary assessor 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 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 must show appropriate colour spectrum.
• Counterstain intensity stain intensity 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, covering and labelling does 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.
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.
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• 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.
• Demonstration is appropriate based on the method employed and the expected staining
results.
• There may be some uneven staining or background staining but detail is visible in the
majority of the tissue.
• Suitable preparation to allow clear observation of the appropriate tissue constituents
depending on the method being employed.
• There may be some microtomy or processing irregularities, but the material is adequate to
assess.
• Dehydration, covering and labelling does 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.
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.
• Demonstration is appropriate based on the method employed and the expected staining
results.
• Staining may not be even across the preparation.
• Suitable preparation to allow clear observation of the appropriate tissue constituents
depending on the method being employed.
• There may be some microtomy or processing irregularities.
• The dehydration, covering and labelling may obscure visualisation of the tissue and its
components but there is sufficient visible to assess.
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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, covering and labelling may hinder tissue visualisation.
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, covering 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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UK NEQAS CPT Stain Repertoire
General Pathology Scheme
Haematoxylin and Eosin
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)
Trichrome (Not HVG)
Neuropathology Scheme
Haematoxylin and Eosin
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 Scheme Muscle Histochemistry Scheme
Haematoxylin and Eosin Haematoxylin and Eosin
Methenamine Silver NADH
PAS/dPAS Gomori
Elastin / van Gieson Cytochrome Oxidase
Non Gynae Diagnostic Cytology Scheme
Staining techniques: Specimen Types:
Papanicolaou Serous Fluid
Romanowsky Head and Neck
Respiratory
Urine
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