Staining Criteria Handbook General Pathology (Routine Histopathology) Neuropathology Edition 4 November 2015
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 2
Haematoxylin and Eosin Assessment Criteria 3
Haematoxylin and Eosin Assessment Criteria Pre Microtomy Microtomy Staining 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 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 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 4
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. 5
Special Stains A & B Assessment Criteria 6
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 Background Counterstain Nuclear Stain Intensity too strong Nuclear Stain Intensity too weak Nuclear Stain colour 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. 7
Amyloid (method for) Assessment Criteria Primary Stain - Bright field Congo red (stain) colour Intensity too strong Intensity too weak Background 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 Post staining Description of Staining Results 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 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. 8
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 Background Counterstain Nuclear Intensity too strong Nuclear Intensity too weak Nuclear Stain colour 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. 9
Copper Associated Protein (CAP) Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain colour Background Counterstain Intensity too strong Intensity too weak Stain colour 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. 10
Diastase/PAS Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain colour Background Residual Glycogen Counterstain Nuclear Stain Intensity too strong Nuclear Stain Intensity too weak Nuclear Stain colour 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. 11
Elastin van Gieson Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain Colour No contrast Fine Fibres Background 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 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. 12
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 Background Counterstain Intensity too strong Intensity too weak Stain colour 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. 13
Gram Stain Assessment Criteria Primary Stain Gram Positive Intensity too strong Gram Positive Intensity too weak Gram Positive Colour Background Low power visibility Counterstain Gram Negative Intensity too strong Gram Negative Intensity too weak Gram Negative Colour 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. 14
Grocott Assessment Criteria Primary Stain Organism Demonstration too strong Organism Demonstration too weak Organism Colour Background Non Specific Silver Precipitate Counterstain Intensity too strong Intensity too weak Stain colour 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. 15
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 Background Counterstain Nuclear Stain Intensity too strong Nuclear Stain Intensity too weak Nuclear Stain Colour 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. 16
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 Red blood cell colour Background Counterstain Nuclear Stain Intensity too strong Nuclear Stain Intensity too weak Nuclear Stain Colour 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. 17
Masson Fontana (for melanin) Assessment Criteria Primary Stain Counterstain Melanin Demonstration too strong Melanin Demonstration too weak Background Non Specific Silver Precipitate Incomplete Toning Intensity too strong Intensity too weak Stain colour 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. 18
Myelin (method for) Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain colour Myelin demonstration good Myelin demonstration poor Low power visibility Background Counterstain Intensity too strong Intensity too weak Stain colour 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. 19
Neurofibrillary Tangles Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain colour Tangle demonstration good Tangle demonstration poor Low power visibility Background Counterstain Intensity too strong Intensity too weak Stain colour 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. 20
Nissl Substance Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain colour Nissl demonstration good Nissl demonstration poor Low power visibility Background Counterstain Intensity too strong Intensity too weak Stain colour 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. 21
Perls Prussian Blue Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain Colour Background Unreacted iron Counterstain Intensity too strong Intensity too weak Stain colour 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. 22
Reticulin (silver method for) Assessment Criteria Primary Stain Counterstain Post staining Reticulin Demonstration too strong Reticulin Demonstration too weak Background Non Specific Silver Precipitate Incomplete Toning Intensity too strong Intensity too weak Stain colour 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. 23
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 Background Non Specific Silver Precipitate Incomplete Toning Counterstain Intensity too strong Intensity too weak Stain colour 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. 24
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 Red blood cell colour Background Counterstain Nuclear Stain Intensity too strong Nuclear Stain Intensity too weak Nuclear Stain Colour 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. 25
Ziehl Neelsen Assessment Criteria Primary Stain Bacilli stain Intensity too strong Bacilli stain Intensity too weak Bacilli Colour Background Low power visibility Counterstain Intensity too strong Intensity too weak Stain colour 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. 26
List of Assessment Criteria Haematoxylin and Eosin Special Stains 27
Haematoxylin and Eosin Assessment Criteria Pre Microtomy Microtomy Staining 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 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 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 28
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 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 29
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 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 Unreacted iron 30
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 Gram Negative Colour Gram Negative Intensity too strong Gram Negative Intensity too weak Intensity too strong Intensity too weak Low power visibility Nuclear Stain Colour Nuclear Stain Intensity too strong Nuclear Stain Intensity too weak Stain colour 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 31
Assessment Criteria Definitions Haematoxylin and Eosin Special Stains 32
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 33
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 34
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 35
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 36
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. 37
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 nonselective 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. 38
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. 39
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. - 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. 40
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. 41
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 - - 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. - 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 42