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Alcian Blue/PAS Assessment Criteria Staining Criteria Handbook General Pathology (Routine Histopathology) Neuropathology Edition 5 October 2017 UK NEQAS Cellular Pathology Technique Providing worldwide external quality assessment and proficiency testing for all aspects of tissue diagnostics NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 2 Index Page Haematoxylin and Eosin Assessment Criteria 5 Special Stains A & B Assessment Criteria 9 Assessment Criteria Definitions 33 Haematoxylin and Eosin Special Stains Appendix 46 Haematoxylin and Eosin Model Description Scoring Guidelines Scoring Based on Criteria UK NEQAS CPT Stain Repertoire NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 3 Haematoxylin and Eosin Assessment Criteria NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 4 NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 5 Haematoxylin and Eosin Assessment Criteria Pre Microtomy Insufficient cellular features for assessment Crush artefacts Foam inset artefact Red blood cell lysis Poor chromatin detail Cracking Nuclear bubbling Nuclear meltdown Incorrect orientation suspected Incorrect trimming suspected Microtomy Chatter / vibration Displacement Folds / creases Knife back debris Knife marks Lifting Position on slide Section too thick Section too thin Section thickness variable Squames / floaters / fibres Trimming artefacts Water bath bubbles Staining Haematoxylin intensity too strong Haematoxylin intensity too weak Haematoxylin colour Haematoxylin background staining Eosin intensity too strong Eosin intensity too weak Eosin colour Eosin not selective Uneven staining Stain deposit present Post Staining Air bubbles Air drying artefact Excessive mountant Mountant shrinkage Residual wax Section wiped / section off slide Tissue damage Tissue exposed Water present Contaminant on slide NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 6 Description of Staining Results Nuclei must be stained purple blue with haematoxylin. The intensity must be strong enough allow clear demonstration of nuclear detail at a medium power, but not too strong to cause a loss of the chromatin granularity or excessive cytoplasmic or connective tissue staining. Where the haematoxylin has been differentiated out, minimal cytoplasmic or connective tissue background staining with haematoxylin must remain. This background if present must not reduce the effectiveness of the nuclear demonstration or affect the colour and selectiveness of the eosin. The eosin should be selective enough to demonstrate different cellular components such as collagen, cytoplasm, red blood cells, cellular granules, amyloid etc. The intensity must be appropriate to the section thickness and the haematoxylin intensity. Where the eosin is too weak it will fail to allow selective demonstration of different components at low power. If the eosin intensity is too strong the colour and detail of the nuclear stain will be obscured and selectivity will be reduced. For an extended description including pre Microtomy, Microtomy and post staining please see the model description in the appendix. NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 7 Special Stains A & B Assessment Criteria NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 8 NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 9 Alcian Blue/PAS Assessment Criteria Primary Stain Alcian Blue intensity too strong Alcian Blue intensity too weak Alcian Blue colour PAS intensity too strong PAS intensity too weak PAS colour Not selective Uneven staining Background Deposit / precipitate present Counterstain Nuclear stain intensity too strong Nuclear stain intensity too weak Nuclear stain colour Not selective Uneven staining Deposit / precipitate present Post staining Air bubbles Air drying artefact Excessive mountant Mountant shrinkage Residual wax Section wiped / section off slide Tissue damage Tissue exposed Water present Contaminant on slide Description of Staining Results Acid mucins (also called mucopolysaccharides) should be coloured bright blue. Neutral mucins should be bright magenta. Mixed mucins should be purple. There should be a clear distinction between acid, neutral and mixed mucins. Background staining should be negligible. A counterstain (typically haematoxylin) is considered optional. If present, it should be light and even to assist location. It should not mask or alter the primary staining. Section quality and presentation should not impair the result. NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 10 Amyloid (method for) Assessment Criteria Primary Stain - Bright field Intensity too strong Intensity too weak Not selective Stain colour Uneven Staining Background Deposit / precipitate present Primary Stain - Cross polarised light No green birefringence on cross polarisation Birefringence intensity too weak Birefringence colour Birefringence not selective Counterstain Nuclear stain intensity too strong Nuclear stain intensity too weak Nuclear stain colour Not selective Uneven staining Deposit / precipitate present 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 NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 11 Description of Staining Results Amyloid: There is no amyloid specific dye. However, Congo red is highly selective for amyloid when staining is performed stringently/correctly by the alkaline alcoholic Congo red method, and when sections are prepared at the appropriate thickness (~5 to 10µm). Use of other dyes or methods may produce sub-optimal or erroneous results. Assessment is conducted in brightfield and cross polarised light, with a 10x objective, on microscopes equipped with a high intensity light source, polarisation filters and colour corrected and strain free optics. Sections that are too thin or too thick may not show the green birefringence of amyloid-Congo red complexes in cross polarised light. Brightfield: Amyloid should stain pink to red (congophilic). Stain intensity is seldom homogeneous and may differ in different tissues reflecting the deposition, abundance and underlying organisation of the amyloid fibrils. Collagen and elastin may be weakly coloured. Background staining must be negligible. Haematoxylin counterstain should be light, even and not mask or alter the primary stain; it should assist location. Cross Polarised Light: Amyloid stained pink / red in brightfield microscopy must also exhibit a green colour in cross polarised light (strong green birefringence). Denser deposits of amyloid may exhibit yellow green or bright yellow birefringence. The colour and/or intensity of the primary stain must not mask the green birefringence of amyloid in cross polarised light. Collagen and elastin usually give pale yellow to bright white polarisation colours. NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 12 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. NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 13 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. Hepatitis B surface antigen and elastin, if stained, should be readily distinguishable from CAP. Background staining should be negligible. If a counterstain is used it should not mask or modify the primary stain, and should assist location. Section quality and presentation should not impair the result. NEQMANMA003 General Pathology and Neuropathology Staining Criteria Handbook Edition 005 14 Diastase/PAS Assessment Criteria Primary Stain Intensity too strong Intensity too weak Stain colour Not selective Uneven staining Background Deposit / precipitate present
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