Histotechnology Past Paper PDF

Learning Outcome 4: Perform special staining techniques to demonstrate cellular elements. Learning Step 4.1: Perform special staining techniques for collagen, reticulin and basement membrane. 4.1.1 Reading Assignment Read pages 164-167, 176-181 and 183-186 from the textbook, Histotechn...

Learning Outcome 4: Perform special staining techniques to demonstrate cellular elements. Learning Step 4.1: Perform special staining techniques for collagen, reticulin and basement membrane. 4.1.1 Reading Assignment Read pages 164-167, 176-181 and 183-186 from the textbook, Histotechnology, A Self-Instructional Text, Carson & Hladik, 5th ed. Image numbers from these pages are recorded throughout the learning step. Use these images as a visual reference while reading and studying the course manual. 4.1.2 Instruction Sheet Special Techniques for Collagen, Reticulin and Basement Membrane The H & E technique is used universally in all histotechnology laboratories to allow pathologists to view the general architecture of the tissue sample. The H & E technique demonstrates nuclei as blue/purple with the rest of the tissue components in varying shades of pink. This fact makes it difficult to specifically view certain tissue elements. Thus, special demonstration techniques have been developed using stains or other chemical solutions to allow the highlighting of special features in tissue, like connective tissue fibres, carbohydrates, pigments, bacteria, etc. The pathologist is now provided with more specific information about the pathology of the tissue which then leads to a definitive diagnosis. HSTC 210 Histotechnology 2- LO4 Page 63 Collagen Fibres Origin - produced by fibroblasts - over 27 types identified - type I is the most common in humans - reticulin is Type III collagen Structure - protein with carbohydrate side chains - may be fibrils (reticulin) or mesh-like (basement membrane) - birefringent under polarizing light - basic (cationic) Function - provides strength: the more collagen fibres, the stronger the tissue - important for mechanical function - provides strong harnessing (tendons and ligaments) Location - most common body protein - present in loose/dense fibrocollagenous connective tissue: o tendons o organ capsules o dermis of the skin Pathology - diseases associated with increased amounts of collagen (i.e. fibrosis, cirrhosis of the liver, collagen tumours) Demonstration - Masson trichrome method Page 64 HSTC 210 Histotechnology 2- LO4 Demonstration of Collagen A great many staining methods for collagen are characterized by the highly contrasting colour of collagen against the rest of the preparation. The methods usually involve a competition between two or more acid dyes whose molecular size and diffusion coefficients vary and will stain differentially. When basic tissues are exposed to two acid dyes whose characteristics vary, the smaller more diffusible one will attach to the denser structures while the larger dye molecule will go into the more loosely arranged, more accessible structures. (Molecular weight of the dye and porosity of the tissue.) Collagen fibre staining may also involve the use of phosphomolybdic or phosphotungstic acid or a combination of both. These acids are used to confine stains like aniline blue or light green to collagen only. It appears these acids may compete with the smaller dye molecules and replace them, hence decolourizing the collagen and subsequently allowing collagen fibres to be stained with another larger molecule, strong acid dye. Masson Trichrome Method A specialized technique which identifies increases in collagen fibres and also differentiates collagen fibres from smooth muscle fibres. Section thickness: 5 m Control: not required as most tissues have an internal control. If a control is desired, small intestine, uterus, appendix, or fallopian tube may be used. Principle: porosity of the tissue and molecular weight of the dye Fixative: 10% NBF with post-fixation in Bouin solution Method: Several different methods are available. Most contain the following steps: 1. Post fixation in Bouin solution. 2. Iron hematoxylin stain - e.g. Weigert’s stains nuclei black 3. Red cytoplasmic dye e.g. Biebrich scarlet and acid fuchsin 4. Differentiation with PTA/PMA solution 5. Collagen stain: Green - light green, or Blue - aniline blue 1. Post Fixation Step BOUIN SOLUTION Sections fixed in 10% NBF do not stain well, as formaldehyde masks chemical reactive groups that will bind the acid dyes used in this technique. Post-fixation with Bouin solution enhances the trichrome stain as it acts as a mordant between the dye and the targeted tissue components. HSTC 210 Histotechnology 2- LO4 Page 65 2. Nuclear Staining Step WEIGERT IRON-HEMATOXYLIN An iron hematoxylin must be used in this technique because it is not easily decolourized by the acidic solutions that follow in the staining procedure. Aluminum-mordanted hematoxylins are not useful in this technique since they would be decolourized by strong acid solutions. Weigert's hematoxylin is stored as two separate solutions. When mixed, they produce a strong iron hematoxylin with a black lake. Ferric chloride (FeCl3) acts both as an oxidizer and a mordant in this hematoxylin. Weigert's hematoxylin should be prepared just before use. Since no stabilizer is used in the solution, this hematoxylin will only remain stable for a few (3-4) days. This iron-mordanted hematoxylin combines with basic groups of the nuclei and stains them a black colour. 3. Cytoplasm Staining Step BIEBRICH SCARLET/ACID FUCHSIN This step uses a mixture of two acid dyes with small red molecules: a. Biebrich Scarlet acid dye, red in colour molecular weight of 556 has a high diffusion coefficient and will selectively stain muscle and cytoplasm which have a dense arrangement b. Acid Fuchsin acid dye, red in colour molecular weight of 625 has a lower coefficient of diffusion and will selectively stain the collagen which has a looser arrangement (i.e. more permeable) than cytoplasm or muscle Biebrich scarlet and acid fuchsin, both acid dyes, are applied simultaneously to the tissue section. This creates a competition between them for basic (or cationic) binding sites on the protein molecules of the tissue. Biebrich scarlet has slightly smaller, more rapidly diffusing ions which can penetrate quickly into tightly woven protein matrices e.g. cytoplasm, red blood cells, and muscle. It occupies their binding sites and excludes acid fuchsin whose dye particles are slightly larger. Acid fuchsin enters the loosely textured, more easily permeated parts of the tissue - especially collagen fibres. Page 66 HSTC 210 Histotechnology 2- LO4 4. Differentiation Step PMA/PTA Differentiation is controlled using strong acids, phosphotungstic acid (PTA) and/or phosphomolybdic acid (PMA). The PMA/PTA molecules are large and can penetrate only the collagen. (Use PMA/PTA with aniline blue; use PTA with light green) The green but clear PMA/PTA will differentiate the acid fuchsin molecules from the collagen while leaving the Biebrich scarlet molecules in the cytoplasm. The collagen will then become colourless. Thus, it is a differentiator in this technique. Many investigators also suggest that it accentuates or increases the final collagen dye intensity with light green. One theory is that PMA/PTA may act like a mordant or link with light green, but this has not yet been confirmed. 5. Collagen Staining Step LIGHT GREEN or ANILINE BLUE The collagen is stained by a large blue or green acid dye molecule. Ultimately, the dye that is used will depend upon the pathologist reading these slides. The choice is either light green (MW = 808) or aniline blue (MW = 737). These dyes, when applied after PMA/PTA, will selectively and brilliantly stain collagen a green or a blue colour. If there are only small amounts of collagen present, aniline blue is the better counterstain. If collagen is predominant, light green is preferable as a counterstain. 6. Removal of Excess Collagen Stain 1% ACETIC ACID 1% acetic acid sharpens the cytoplasmic stain by removing any excessive light green or aniline blue which could make cytoplasm and muscle appear muddy (the red is overlaid with green or blue.) Results: (page 166; [i8.4], [i8.5]; page 167: [i8.6]) Nuclei - black Cytoplasm, keratin, muscle fibres - red Collagen - green (if light green used) or blue (if aniline blue used) HSTC 210 Histotechnology 2- LO4 Page 67 Problem Solving for Masson's Trichrome Technique Problem Reason Correction 1. Collagen too pale. - post fixation in Bouin missed - post-fix in Bouin - time in acetic acid too long - shorten time in acetic acid 2. Cytoplasm muddy. - failure to rinse in acetic acid - include acetic acid rinse - time in acetic acid too short - lengthen time in acetic acid 3. Nuclei too pale. - Weigert's hematoxylin over-oxidized - make fresh - hematoxylin removed by acid dyes and - re-stain nuclei PMA/PTA - use of aluminum mordanted - use iron-mordanted hematoxylin hematoxylin Van Gieson Picric-Acid/Acid Fuchsin Method Although the van Gieson technique may be considered a primary connective tissue stain, it is not often performed that way. It is more frequently used as a counterstain for other methods, such as in Verhoeff's technique for elastic. connective tissue method - Weigert van Gieson elastic stain method - Verhoeff van Gieson (VVG) Section thickness: - 5 m Control: - not required as most tissues have an internal control. If a control is desired, small intestine, uterus, appendix, or fallopian tube may be used. Principle: - porosity of the tissue and molecular weight of the dye Fixative: - any fixative may be used Method: - Van Gieson’s Picric Acid/Acid Fuchsin Steps: 1. Nuclear Stain 2. Cytoplasmic Stain 1. Nuclear Stain WEIGERT'S IRON HEMATOXYLIN - used as in the Masson trichrome technique. Page 68 HSTC 210 Histotechnology 2- LO4 2. Cytoplasmic Stain VAN GIESON SOLUTION - picro-fuchsin (van Gieson solution) is a combination of picric acid (yellow) and acid fuchsin (red). - In addition to providing the acidic pH allowing for selective staining of collagen by acid fuchsin, picric acid also acts as a stain for muscle and cytoplasm Results: (page 170: [i8.9]) nuclei - black cytoplasm and muscle - yellow collagen - red Reticulin Fibres Origin - produced by fibroblasts Structure - type III collagen - fine, branching - smaller than collagen fibres Location - supporting framework for highly cellular organs i.e. - liver - lymph nodes - bone marrow - spleen - endocrine glands - often associated with collagen fibres - found in basement membrane in the fibroreticular lamina Function - supporting framework (stroma) Pathology - It is important to demonstrate reticulin fibres, and the amount present, in the following: - myelosclerosis in bone - differentiating certain carcinomas from lymphomas - cirrhosis of liver Demonstration - reticulin fibres are not seen on a routine H&E - can adsorb silver from solution, which can then be reduced to its visible metallic form. 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