Chapter 1: FRESH TISSUE EXAMINATION

1. Chapter 1: FRESH TISSUE EXAMINATION Learning Objectives: 1. To be able to differentiate histotechnology and histotechnologist. 2. To be able to differentiate the advantages and disadvantages of fresh tissue examination. 3. To be able to know the basis for method used in fresh tissue examination. 4. To be able to name and differentiate the methods of fresh tissue examination. 5. To be able to understand the fresh frozen method.

2. Chapter 1: FRESH TISSUE EXAMINATION Histotechnology - is the art and science performed by the histotechnologist to produce a tissue section of good quality that will enable the pathologist to diagnose the presence of disease. The tissue may be done fresh or preserved. Advantage of fresh tissue examination: The specimen may be in living state, therefore may observe protoplasmic activities (motility, mitosis, phagocytosis, pinocytosis). Disadvantage: not permanent, and therefore liable to changes

3. Chapter 1: FRESH TISSUE EXAMINATION The choice of tissue examination method would depend on the following conditions; • Cell Structure and chemical components • Amount and nature of tissue • Urgency of result

4. Chapter 1: FRESH TISSUE EXAMINATION The methods of fresh tissue examination: Teasing or dissociation – selected tissue is in isotonic solution, dissected and examined under the microscope. Squash Preparation (Crushing) – less than 1 mm tissue pieces are compressed (stained when necessary), and examined under the microscope. Smear preparation – spread cellular materials (secretions, sediments) are examined. useful for cytologic examinations Streaking – using an applicator stick by direct or zigzag spread Spreading – teasing (sputum, bronchial aspirates, thick mucoid secretions) by applicator stick and spread in circular spread. Pull-Apart – thick secretions (gastric lavage, serous fluids, blood) are dispersed evenly on two slide surfaces. Touch Preparation (Impression) - freshly cut tissue surface is brought into contact to slide. Cells are examined in their actual intercellular relationship. Frozen Section – for rapid diagnosis during surgery, and demonstration of lipids and nervous tissue elements.

6. Chapter 2: Fixation and Fixatives Learning objectives: To understand the process of autolysis To determine the contributing factors in autolysis. To discuss why certain tissues are affected severely by autolysis. To know the objectives of fixation and qualities to serve its objective. To understand the factors involve in fixation. To know the types of fixative. To understand the properties of formaldehyde.

7. Fixation process • Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. • Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. • For example, immunohistochemistry utilizes antibodies which bind to a specific protein target. • Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used.

8. Chapter 2: Fixation and Fixatives Once tissues are removed from the body, they undergo a process of self-destruction or autolysis. Soon after tissue death, the intracellular enzymes break down the protein and then the cell eventually undergo liquefaction. – Autolysis. Properties of Autolysis: independent of a bacterial action retarded by cold accelerated at 30 degree C temperature, inhibited at 50 degrees Celsius more severe in tissue which are rich in enzymes (e.g. liver, brain, and kidney) and less rapid in elastic and collagen tissues.

9. Chapter 2: Fixation and Fixatives Autolysed tissue presentations: “washed-out” appearance with swelling of cytoplasm, eventually to a granular, homogenous mass which fails to take up stains. The nuclei of autolytic cells may show some of the changes including condensation (pyknosis), fragmentation (karyorrhexis) and lysis (karyolysis). There may be diffusion of intracellular importance like glycogen. The epithelium may desquamate from its basement membranes. Dead tissue may allow bacteria to proliferate. When these bacteria decompose, it can mimic those of autolyzed tissue.

10. Chapter 2: Fixation and Fixatives The objective of fixation; To preserve cells and tissue constituents in as close to life-like state. To ready for further processing without or minimal tissue change. Fixative should have the following qualifications to serve its objective; Arrest autolysis and bacterial decomposition Stabilizes cellular and tissue constituents. Preservation of tissue substances and proteins (for Immunohistochemisatrytechniques to augment diagnosis) Fixation is therefore the first step and the foundation in a sequence of events that culminate in the final examination of ta tissue section. Each fixative has specific properties and disadvantages. Their varied effects to tissues would require careful selection of fixative when specific cellular substances is to be studied.

11. Fixation In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. • Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. • Purposes of fixation 1- To prevent or arrest autolysis and bacterial decomposition and putrefaction. 2- To coagulate the tissues as to prevent loss of easily diffusible substances 3-To fortify the tissue against the deleterious effects of the various stages of tissue processing 4- To leave the tissues in a condition which facilitate differential staining with dyes and other reagents

12. Chapter 2: Fixation and Fixatives Factors involve in Fixation: 1. Temperature – standardized fixation is carried out at room temperature. However, for electron microscopy and some histochemical procedure, fixation is usually at 0 – 4 degrees Celsius. Lower temperatures autolysis is slowed down allowing a more life-like appearance of tissues. 2. Size of specimen – the penetration of fixative is inversely directly relative to size. In which, larger tissue size will be fixed slowly. Thus, large specimens are opened and washed of contaminant or sliced thinly. . The fixative should be at least 20 times the volume of the specimen. Cut sections should be 1-4 mm Thickness 3. Change in volume – the volume changes in tissue with fixation, as the tissue shrinks by 33%. This is evident as the tissue show larger nuclei and cells in frozen sections (unfixed). 4. pH and Buffers – The hydrogen ion concentration varies between fixatives, Should be kept in the physiological range, between pH 4-9. The pH for the ultra structure preservation should be buffered between 7.2 to 7.4 The buffer systems maintain this physiological range of pH level (e.g.; phosphate, acetate, bicarbonate). The buffer should have the following properties; a. do not interfere with fixative b. do not inhibit enzymes 5. Osmolality – the fixative solutions; osmotic pressure may be affected by the addition of buffer. The best result is obtained by using slightly hypertonic solutions (isotonic solutions adjusted by using sucrose). Hypertonic solutions give rise to cell shrinkage. Hypotonic solutions result in cell swelling and poor fixation. 6. Concentration of fixative 7. Duration of fixation - As a general rule 1hour per1mm

13. Types of fixationThere are generally three types of fixation process: • Heat fixation: After a smear has been allowed to dry at room temperature, the slide is gripped by tongs or a clothespin and passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the slide 2. Perfusion: Fixation via blood flow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms)

14. 3. Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 20 times greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account.

15. Chemical Fixation • Process whereby Structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. • This requires a chemical fixative which can stabilize the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble. Types of Chemical Fixatives 1. Cross linking fixatives – ALDEHYDES • Cross linking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. • By far the most commonly used fixative in histology is formaldehyde. It is usually used as a 10% Neutral Buffered Formalin (NBF). • Formaldehyde is a gas. Formalin is formaldehyde gas dissolved in water. • Paraformaldehyde is a polymerized form of formaldehyde, usually obtained as a fine white powder, which depolymerises back to formalin when heated.

16. Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. • Its effects are reversible by excess water and it avoids formalin pigmentation. Other benefits include: Long term storage and good tissue penetration. • It is particularly good for immunohistochemistry techniques. • Also the formaldehyde vapour can be used as a fixatives for cell smears.

17. 2. Glutaraldehyde. • causing deformation of the alpha-helix structures in proteins. - larger molecule, and may not penetrate thicker tissue specimens as effectively as formaldehyde Thus small blocks of tissue are required. - may offer a more rigid or tightly linked fixed product—its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules. • It causes rapid and irreversible changes, fixes quickly, is good for electron microscopy, fixes well at 4oC, and gives best overall cytoplasmic and nuclear detail. • However it is not ideal for immunohistochemistry staining • Some fixation protocols call for a combination of formaldehyde and glutaraldehyde, so that their respective strengths complement one another.

18. Precipitating fixatives : Alcohols Precipitating (or denaturing) fixatives • act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions which give many proteins their tertiary structure. • The alcohols, by themselves, are known to cause considerable shrinkage and hardening of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology • The most common precipitating fixatives are ethanol and methanol. They are commonly used to fix frozen sections and smears. • Acetone is also used and has been shown to produce better histological preservation than frozen sections when employed in the Acetone Methylbenzoate Xylene (AMEX) technique. • The protein denaturants - methanol, ethanol and acetone - are rarely used alone for fixing blocks unless studying nucleic acids. • Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives.

19. . Oxidising agents The oxidising fixatives can react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks which stabilize tissue structure. However they cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary fixatives. • Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. (It is not used for light microscopy as it penetrates thick sections of tissue very poorly.) • Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.

20. MERCURIALS Mercurials and Zenker'shave an unknown mechanism which increases brightness of staining along with giving excellent nuclear detail. Despite being fast, mercurialspenetrate poorly and produce tissue shrinkage. • Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Also note that since they contain mercury care must be taken with disposal PICRATES Picrates penetrate tissue well to react with histones and basic proteins in order to form crystalline picrateswith amino acids and precipitate all proteins. • It is a good fixative for connective tissue, preserves glycogen well and extracts lipids in order to give superior results to formaldehyde in immunostaining of biogenic and polypeptide hormones • However it causes a loss of basophilia unless the specimen is thoroughly washed following fixation.

21. . • HOPE Fixative Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) gives formalin-like morphology, excellent preservation of protein antigens for immunohistochemistry and enzyme histochemistry, good RNA and DNA yields.

22. Chapter 2: Fixation and Fixatives Types of fixative according to action: A. Microanatomical fixatives – are those the permit the general microscopic study of tissues structures without altering the structural pattern and normal intercellular relationship of the tissues in question. 10% formol saline 10% neutral buffered formalin Zenkers solution Bouin’s solution B. Cytological fixatives – are those that preserve specific parts and particular microscopic elements of the cell itself. 1. Nuclear fixative – are those that reserve nuclear structure (e.g. chromosome) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin. Usually have pH of 4.6 or less Flemmings Carnoys Newcomers Heidenhain’s susan 2. Cytoplasmic – are those that preserve cytoplasmic structures in particular. They never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm. Ph of more than 4.6. Kelly flemmings Regauds’s fluid Orth’s fluid C. Histochemical fixative - preserve chemical constituents of cells and tissues. Formol saline 10% Absolute ethyl alcohol Acetone Newcomer’s fluid

23. Chapter 2: Fixation and Fixatives Formaldehyde (Formalin) Properties: The most widely used and routine fixative particularly for paraffin embedded sections. It is a gas with a very pungent odor. The commercially available solution of formaldehyde (formalin) contains 35 – 40% gas by weight. However, pure stock solution of 40% formalin is unsatisfactory. Formaldehyde is commonly used as 4% solution, giving 10% formalin for fixation. Therefore, it should be diluted 1:10. Formaldehyde is usually buffered to pH 7 with phosphate buffer. It is thought that formaldehydes form cross-links between proteins, creating a gel, thus retaining cellular constituent. It is a forgiving fixative – requires a relatively short fixation time (24 hours) but can be used for long term usage with no deleterious effects on tissue. Prepared by adding 100 ml of 40% formaldehyde to 900 ml distilled water with 4 g sodium phosphatase (monobasic) and 6.5 g sodium phosphate (dibasic anhydrous).

24. COMPOUND FIXATIVES 1. Formalaldehyde fixatives • 10% formal saline • Formalin 100 ml • Sodium chloride 8.5 grams • Tap water 900 ml 2. Buffered 10% formalin Formalin 100 ml Acid sodium phosphate monohydrate 4 g Anhydrous disodium phosphate 6.5 g Tap water 900 ml ALCOHOLIC FIXATIVES 1. Carnoy’s fixatives Absolute alcohol 60 ml Chloroform 30 ml Glacial acetic acid 10 ml 2. Picric acid fixatives Bouin’s fluid Rossman’s fluid Gendre’s fluid 3. Mercuric chloride containing fixatives Formal sublimate Zenker’s solution Susa fluid Helly’s fluid