tissue stains (h&e) (pap) · 2020-06-03 · pap stain continue procedure 2 (modified pap...
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Tissue Stains
(H&E) (PAP)
Prepared by M Hlasek March 2017
Reviewed February 2020
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Aims of staining
Commonly used medical process in the medical diagnosis of tumors
Technique used to enhance contrast in samples
Make the cell structure visible
Show variation in structure
Indicate the chemical nature of tissue entities
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Staining methods
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Haematoxylino Three main types:
Alum
Iron
Tungsten
o Other:
Lead
Molebdenum
Haematoxylin without mordant
These haematoxylins are named as such because of the mordant that is used.
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Alum Haematoxylin
The mordant contains aluminium egPotassium aluminium sulphate in Mayer’s Haematoxylin.
Disadvantage: very sensitive to acid solutions
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Iron HaematoxylinHere the mordant is an iron salt eg ferric chloride or ferric ammonium. These salts acts also as the oxidising agent.
Disadvantage: Over oxidise and “ripen” very quickly
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Tungsten HaematoxylinThe haematoxylin can be ripened chemically with potassium permanganate or left to ripen in sunlight.
Disadvantage: only Mallory’s phosphotungstic acid haematoxylin is the only one widely used
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MordantIs a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material/tissue section.
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Ionic bonding / Coulombic attractions
Acid and basic dyes, and other ionic reagents, including inorganic salts
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Hydrogen bonding
Is a dye-tissue attraction arising when a hydrogen atom lies between two
electronegative atoms
(e.g. oxygen or nitrogen)
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Van der Waals forces
Intermolecular attractions as dipole-dipole, dipole-induced dipole and dispersion forces. These occur between all reagents and tissue
substrates.
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Van der Waals Forces cont.
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Covalent bonds Between tissue and stain also occurs, which bonds may be regarded merely as another source of stain-tissue affinity
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Covalent bonds cont.
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Hydrophobic interaction Staining systems using aqueous solutions of dyes or other organic reagents; enzyme substrates for example
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Hydrophobic interaction
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Histology classification of dyes
Basic dyes – these are cationic dyes and will stain anionic or acidic materials (e.g. the phosphates in nucleic acid) in the tissue e.g. Methylene Blue, Safranin, Basic fuchsin.
Acidic substances that stain with basic dyes are termed basophilic
Acidic dyes - these are anionic dyes and will stain cationic or basic groups in tissue such as amino groups e.g. Picric acid, Eosin, Acid fuchsin.
Substances that stain with acid dyes are called acidophilic
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Histology classification of dyes continue
Neutral Dyes- simply compounds of basic and acidic dyes. Such dye complexes will stain both nucleus and cytoplasm e.g. Romanowsky stains, EosinY or Eosin B
Amphoteric dyes - have both anionic and cationic groups, but on the same ion. Such dyes stain either the nucleus or the cytoplasm if conditions are appropriate e.g. Celestine blue B, Acid fuchsin
Natural dyes – dye substances extracted from natural sources e.g. Haematoxylin from the logwood of a tree.
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Important dyes in histology Nuclear stains
Haematoxylin
Carmine and Carminic acid
Methylene blue
Neutral Red and Safranine O
Methyl green
Toluidine blue
Cytoplasmic stains
Eosin
Methyl blue and aniline blue
Fast green FCF and Light green SF
Orange G
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Routine stains
Haematoxylin and Eosin (H &E Stain) for histology
Papanicolaou stain (PAP Stain) for cytology
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Principle of the H&E Stain The staining method is based on the
chemical attraction between tissue and dye. Charges on the dye and tissue are opposite and therefore attract (Van der Waals forces or Ionic bonding)
The Nuclei is acidic (-ve charges) and reacts with haematoxylin a basic dye
The cytoplasm or connective tissue are basic (+ve charges) components reach with Eosin an Acid dye.
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Method H&E Staina. Scott’s Tap Water Sodium hydrogen carbonate 3.5gm/17.5gm Magnesium sulphate 20gm/100 gm
Tap water 1000ml/ 5000mlL
b. 1% Acid alcohol Ethanol 70 ml
Distilled water 30 ml Use 99ml of above, add HCL 1ml
OR Ethanol 3500ml
Distilled water 1500ml Use 4950ml of above, add HCL 50ml
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H&E stain continuec. Eosin/Phloxine solution
1% Eosin Stock Solution
Eosin Y 1gm
Distilled water 100ml
1% Phloxine Stock Solution
Phloxine B 1gm
Distilled water 100ml
Eosin/Phloxine Working Solution
1% Eosin stock solution 80ml/380ml
1% Phloxine solution 40ml/160ml
Distilled water 120ml/480ml
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H&E continueMethod
Dewax and hydrate sections to distilled water
Stain nuclei with the alum haematoxylin for 5-6 minutes
Rinse in running tap water
Differentiate with 1.0% acid alcohol for ±2 seconds (if staining regressively)
Rinse in tap water
Blue in Scott's tap water for 1minute OR blue in running tap water for 5 minutes
Rinse well in tap water
Counterstain with Eosin for 1- 2 minutes
Rinse well in tap water
Dehydrate, clear and mount.
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H&E results
Collagen Pale pink
Muscle Deep pink
Acidophilic cytoplasm Red
Basophilic cytoplasm Purple
Nuclei Blue
Erythrocytes Cherry red
Calcium deposits Blue
Bacteria Blue
Mucin Pale blue/Grey
Cartilage Pale blue/grey
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H&E results continue
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H&E Troubleshoot
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Special stains vs H&E
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Papanicolaou Stain
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Principle of the PAP stain
The pap stain is a special stain for keratin that is capable of detecting minimal or focal evidence of squamous cell differentiation.
As cells keratinized, the glassy cytoplasm progress from blue-green (abundant free ribosomes and prekeratin) to intensely orange (true keratin).
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Principle of the PAP stain
Papanicolaou stain (also Pap stain) is a multi-chromatic staining histological technique histological technique.
Pap stain involves five dyes in three solutions:
A nuclear stain, haematoxylin, is used to stain cell nuclei.
OG-6 counterstain. The Orange G is used to stain keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.
EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dye e.g. Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.
Light Green SF yellowish stains the cytoplasm of all other cells.
Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.
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Method of PAP staina. Scott’s tap water
Sodium Hydrogen Carbonate 3.5gm OR 17.5gm
Magnesium Sulphate 20gm OR 100gm
Tap Water 1000ml OR 5000ml
b. 95% Ethanol
Ethanol 50ml OR 5ml
DDW 950ml OR 95ml
c. Solutions OG-6 and EA-50 (commercial)
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PAP stain continueProcedure 2 (Modified Pap Procedure): Fix in 95% Ethanol 15 minutes Rinse in 2 changes DDW - 10 dips each Stain in Gill’s Haematoxylin for 2 minutes Rinse in DDW - 10 dips Blue in Scott's tap water for 1 minute Rinse in 2 changes 95% Ethanol - 10 dips Stain in OG-6 stain for 5-7 minutes Rinse in 3 changes 95% Ethanol - 10 dips Stain in EA-50 (or EA-65 stain) for 6-10 minutes Rinse in 3 changes 95% Ethanol - 20-30 dips Rinse in Absolute ethanol - 10 dips Clear in xylene, mount
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Results
Cell nuclei Crisp blue to black
Cells (high content of keratin) Yellow/orange
Glycogen stains Yellow
Superficial cells Pink to orange
Intermediate & para-basal cells Turquoise green to blue
Metaplastic cells Blue/green and pink at once.
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Results continue
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Results continueA. REGRESSIVE - Differentiator
Haematoxylin stain is applied Tissue is overstained with haematoxylin Differentiator is used to aggressively remove excess haematoxylin Stain procedure continues on with counterstain
B. PR0GRESSIVE - No differentiator Haematoxylin stain is applied Tissue is stained with haematoxylin only to a point Traditionally no differentiator is used to removeexcess haematoxylinStain procedure continues on with counterstain
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PAP Stain Troubleshoot
Causes of inconsistent staining
Varying thickness of material on slide
Type of fixative used
Inadequate filtering of stain solutions
Age of staining solution Degree of usage of staining solutions
Use of chlorinated tap water
pH of water can effect nuclear staining
Temperature of water and reagents
Insufficient rinsing after acid
Speed of dipping slides in reagents - agitation
NB Improper draining of slides during staining.
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PAP Stain Troubleshoot
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PAP Stain Troubleshoot
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References John D. Bancroft, Christopher Layton and
S.Kim Suvarna, (2013), Bancroft’s Theory and Practice of Histological Techniques, 7thEdition, Elsevier, China
3.J.A.Kiernan,(2015)Histological and Histochemical Methods, 5th Edition, Scion, UK
Pathcare Academy Notes