REVIEW LECTURE IN HISTOTECHNIQUE PDF

**REVIEW LECTURE IN HISTOTECHNIQUE** **[DOC J.E.M.]{.smallcaps}** HISTOLOGICAL TECHNIQUE A. FIXATION B. DEHYDRATION C. CLEARING D. EMBEDDING E. CUTTING F. STAINING SPECIMEN HANDLING AND IDENTIFICATION - The specimen container label and accompanying request from should include:...

**REVIEW LECTURE IN HISTOTECHNIQUE** **[DOC J.E.M.]{.smallcaps}** HISTOLOGICAL TECHNIQUE A. FIXATION B. DEHYDRATION C. CLEARING D. EMBEDDING E. CUTTING F. STAINING SPECIMEN HANDLING AND IDENTIFICATION - The specimen container label and accompanying request from should include: - **Patient's name** - **Age of Birth** - **A medical record number** - Label should be firmly attached to the body of the container \-\-- not the lid of the container methods of fresh tissue examination 1\. TEASING OR DISSOCIATION A process whereby a selected tissue specimen is immersed in a watched glass containing isotonic salt solution (NSS or Ringer's Lacate), carefully dissected or separated, and examined under the microscope. 2\. SQUASH PREPARATION AKA: CRUSHING A process whereby small pieces of tissue not more than 1mm in diameter are placed in a microscopic slied and forcibly compressed with another slide or with a cover glass  3\. SMEAR PREPARATION Useful in cytological examinations -- particularly in cancer diagnosis methods of smear preparation a. **STREAKING** -- applied in a **direct or zigzag line** throughout the slide, attempting to obtain a relatively uniform distribution of secretion. b. **SPREADING** -- a moderately thick film by teasing a mucous strands apart with an applicator stick. \- Little more tedious than streaking, but has advantage in maintaining the **intercellular relationship.** Recommended for smear preparation of: - **Fresh Sputum** - **Bronchial Aspirates** - **Thick Mucoid Secretions** c. **PULL-APART** -- useful for preparing a smears of thick secretions such as: - **Serous fluids** - **Concentrated sputum** - **Enzymatic Lavage Samples from GIT** - **Blood Smear** d. **TOUCH PREPARATION** -- **IMPRESSION SMEAR** - Advantage: Cells may be examined without destroying their actual intercellular relationship 4\. frozen section Normally utilized when a **[rapid diagnosis of the tissue in question is required]** Recommended when **LIPIDS** and **NERVOUS TISSUE** elements are being demonstrated - Very thin slices (**10-15u in thickness)** - **Cryostat --** temperature of -**10C to 20C** Applications: - Rapid pathologic diagnosis during surgery - Diagnostic and research enzyme histochemistry - Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates - Immunofluorescent and immunohistochemical staining - Some specialized silver stains -- particularly in **neuropathology** Tissue for freezing should be fresh, and freezing should be done as quickly as possible (not used if may fixative na) Slow freezing can cause distortion of tissue due to ice crystal artifacts. - since 70% of tissue are made with water - will make "Swiss cheese" appearance More commonly used methods of freezing include: **"LICA"** a. **LIQUID NITROGEN** -- generally used in histochemistry and during operative procedures. - **Most rapid of the commonly available freezing agent** b. **ISOPENTANE COOLED BY LIQUID NITROGEN** c. **CARBON DIOXIDE** d. **AEROSOL SPRAY** Processing of tissue Solid structures and tissues must be preserved and carefully processed in the following order: **"FD-CIETS-SML"** 1. **Fixation** 2. **Dehydration** 3. **Clearing** 4. **Infiltration** 5. **Embedding** 6. **Trimming** 7. **Section-Cutting** 8. **Staining** 9. **Mounting** 10. **Labelling** Histotechnology To produce microscopic preparations of tissue that represents as closely as possible their structure in life -- **AIM OF GOOD** To preserve the tissue -- **AIM OF IDEAL FIXATION** **FIXATION --** first and most critical step in histotechnology - Fixing or preserving fresh tissue for examination. - The quality of the section on the slide is only as good as the quality of the fixed tissue specimen. Underfixed -\> soft -\> mabubulok / difficult to cut Overfixation -\> harden -\> difficult to cut fixation - Tissues are fixed in a physical, and partly in a chemical state, so that they will withstand subsequent treatment with a various reagents with a minimum loss, significant distortion, or decomposition. - Fixation prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body. - To preserve the tissue -- by stopping all cellular activities - To prevent breakdown of cellular elements - To coagulate or precipitate protoplasmic substances (proteins) \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- **Primary aim of fixation** - Preserve the morphologic and chemical integrity of the cell in as life-like as possible **Secondary goal of fixation** - To harden and protect the tissue from trauma of further handling - For easy cutting during gross examination \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- The most important reaction in fixation: **Stabilization of proteins** - This can be achieved by: **Forming cross-links between proteins** Leaving a tissue specimen in air cause it to: **dry-out** (distortion of morphologic appearance) Leaving the tissue in water (hypotonic solution) will cause the cell to: **swell** - If the environment is hypotonic, more water is outside the cell and the solute concentration inside the cell is high, the water would enter the cell and swells, bursts, or lyses (cytolysis) Leaving the tissue in strong salt (hypertonic solution) will cause the cell to: **shrink** - More solutes are outside the cell (strong salt solution) and water is less, so water will move outside the cell and the cell shrinks 2 Basic mechanism involved in fixation main factors involved in fixation Practical consideration of fixation 1. speed - The specimen should be placed in fixative as soon as it is removed from the body. - This is done to prevent **autolysis** and **putrefaction/decomposition**. - If **bacteriologic and toxicologic studies** should be encouraged, therefore, fixation is not required. - Fixation can kill microorganisms and prevent growth in culture - Fixatives can neutralize drugs and toxins. 2. PENETRATION - Time of fixation varies with different types of tissue - Formalin diffuses into the tissues at a rate of **1mm/hour.** 3. volume/amount of fixative - **10-25x** the volume of the tissues to be fixed - **20x** -- maximum effectiveness of fixation - **OSMIUM TETROXIDE**: **5-10x** 4. duration - Fibrous organs such as uterus and intestinal track take longer. - Fixation can be cut down using **HEAT, VACUUM, AGITATION OR MICROWAVE** **IDEAL SIZE OF THE TISSUE TO BE FIXED** - Not more than **2cm^2^** in diameter - Not more than **4mm^2^** thick - Ideal number of hours for fixation: **4-6 hours** (New Book: **6-18 hours**) types of fixative According to composition A. simple fixative Made up of **only one component** substance 1. Aldehydes (Formaldehydes, Glutaraldehydes) 2. Metallic Fixatives \- Mercuric Chloride \- Chromate Fixative - Potassium dichromate - Chromic Acid \- Lead Fixative - Picric Acid - Acetic Acid - Alcohol - Osmium Tetroxide b\. Compound fixative **Two or more** fixatives According to action A. Microanatomical fixatives Tissues structures without altering the structural pattern and normal intercellular relationship of tissue 1. 10% Formol Saline 2. 10 % Neutral Buffered Formaline 3. Heidenhain's Susa 4. Formol Sublimate (Corrsive) 5. Zenker's Solution 6. Zenker Formol (Helly's) 7. Bouin's Solution 8. Brasil's Solution B. Cytological fixative Particular microscopic elements of the cell - Preserve specific parts and particular microscopic elements of the cell itself **TYPES OF CYTOLOGICAL FIXATIVES:** 1. **NUCLEAR FIXATIVES** - Preserves nuclear structure - **CONTAINS GLACIAL ACETIC ACID** (since DNA is acid) - have pH of **4.6 or less** a. Heidenhain's susa b. Bouin's fluid c. Newcomer's fluid d. Carnoy's Fluid e. Flemming's fluid 2. **CYTOPLASMIC FIXATIVE** - Must **NEVER CONTAIN GLACIAL ACETIC ACID** (it destroy cytoplasmic structures: Mitochondria and Golgi Bodies) - Have pH of **more than 4.6** a. Helly's fluid b. Orth's fluid c. Regaud's Fluid d. Flemming's fluid without acetic acid e. Formalin with post-chroming 3. **HISTOCHEMICAL FIXATIVE** - Preserves the chemical constituents of cell and tissues. a. 10% Formol Saline b. Absolute Ethyl Alcohol c. Newcomer's Fluid d. Acetone Fixation of tissues can be accomplished by: a. **Physical Method** - Heating - Freezing-drying - Mircrowaving b. **Chemical Method** - Coagulant Fixatives - Compound Fixatives - Cross-linking Fixatives Physical methods Heat fixation - Simplest form of fixation - Primarily used to accelerate other forms of fixation as well as the steps of tissue processing. microwave heating - From more than **12 hours to less than 20 minutes.** - Commercial glyoxal-based fixatives Freeze-drying and freeze substitution chemical methods Coagulant fixatives - Coagulate proteins making them insoluble. - Cytoplasmic flocculation **TYPES OF COGULANT FIXATIVES** 1. **DEHYDRANT COGUALANT FIXATIVE** **(Both Dehydrant and Fixative)** **- METHANOL -- 80% OR MORE** **- ETHANOL -- 50 TO 60%** **ALCOHOL AND ACETONE --** can react to polar and non-polar material - Alcohol denatures proteins differently depending on: - Concentration of Alcohol - Presence of organic(non-polar) and non-organic(polar) substances - pH - Temperature 2. **ACIDIC COAGULANTS** - **ACETIC ACID** - **PICRIC ACID OR TRINITROPHENOL** - Produces brighter staining - May cause hydrolysis and loss of nucleic acids -- due to low pH - Can cause yellow discoloration of tissue routine fixatives i\. aldehydes fixatives **A.** Formaldehyde FIXATION TIME: 24 hours - **AKA: FORMALIN / METHANAL** - Most widely used fixative - A saturated solution of formaldehyde: **35% - 40%** gas by weight (Gregorios: 37% - 40%) - **10% Formalin --** mixture of 10mL of Formalin with 90mL of water - It is usually buffered to **pH 7** with a [phosphate buffer] - The best fixative for **NERVOUS TISSUE** \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- PRECAUTIONS: - Concentrated solutions of formaldehyde must never be neutralized -- voilent explosion (do not add water; you add formalin to water) - At low temperature, Formalin becomes turbid -- due to the formation of **PARAFORMALDEHYDE** (whitish precipitate) REMEDY FOR PARAFORMALDEHYDE: - **Filtration** - Addition of **10% methanol** \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- **Disadvantages:** \- In tissues containing much blood (spleen), unbuffered formalin leads to the formation of **dark brown artifact pigment granules** → these granules consist of **acid formaldehyde hematin** and are doubly refractile. \- Prolonged fixation may cause [bleaching], [fat dispersal] and [dissolution or loss of glycogen], biurates of [sodium crystal] and [uric acid]. **\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--** **Reagent-grade formaldehyde** contains *10% methanol* as a preservative to retard decomposition to formic acid. Prevents the formation of Paraformaldehyde. \- Formalin pigments -- are also formed due to *overfixation*. \- Fixation Time: *24 hours* **Removal of formalin pigments:** 1\. **Lillies Method** -- involves placing formaldehyde fixed specimens in acetone, 28% ammonia water and hydrogen peroxide. It uses 70% alcohol as a rinsing agent. 2\. **Kardasewitch's Method** -- a method of formaldehyde clearance involving *70% ethanol* and *28% ammonia water*. 3. **Saturated Alcoholic Picric Acid** **\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--** ROUTINE FORMALIN FIXATIVE 1. 10% FORMOL-SALINE FIXATION TIME: 24 hours at 35C 48 hours at 20-25C - A simple microanatomical fixative saturated formaldehyde (40%) -\> 10% + sodium chloride. - Fixation of **CENTRAL NERVOUS TISSUES and GENERAL POST-MORTEM TISSUE for histochemical examination.** - **It is the most commonly used fixative in pathology** **FORMULA: FORMALDEHYDE, 40%** **NaCl** **DISTILLED WATER** 2. Calcium acetate formalin (Lillies fixative) - Used to preserved **PHOSPHOLIPIDS** - Replaced Formol-saline as the most commonly used fixative in pathology because: - Simple to Prepare - Buffered with **pH 7 by acetate** 3. 10% Neutral buffered formalin/ phosphate - buffered formalin\ fixation time: 4-24 hours - Recommended for preservation of and storage of **SURGICAL, POST-MORTEM AND RESEARCH** specimen - Best general tissue fixative - BEST FIXATIVE FOR: - **Frozen sections** - **Iron containing pigments and elastic fibers** - Prevents precipitation of acid formalin pigments **FORMULA:** **SODIUM DIHYDROGEN PHOSPHATE** **DISODIUM HYDROGEN PHOSPPHATE** **FORMALDEHYDE** **DISTILLED WATER** 4. Formol corrosive/ formol-sublimate/ formol-mercuric chloride Fixation time: 3-24 hours - Recommended for **ROUTINE POST-MORTEM TISSUES.** - Excellent for **silver reticulum methods.** - Fixes **lipids,** especially **neutral fats and phospholipids** - Forms [BLACK DEPOSITS] - No frozen sections are made 5. alcoholic formalin/ gendre's fixative - It contains ethyl alcohol saturated with [picric acid] - Enhances immunoperoxidase studies for EM if post-fixed with [phenol formalin] for 6 hours or more. - It fixes and dehydrates at the same time and fixes [sputum] since it coagulates mucus 6. special formalin fixatives: **CAJOL'S FORMOL AMMONIUM BROMIDE** - good fixative for nervous tissue (Astrocytes) - Fixatives for **acid mucopolysaccharide** **BAKER'S FORMOL CALCIUM** - Used for the **preservation of lipids** since most formalin fixatives are inert to lipids. \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- **B**. glutaraldehyde fixation time: ½ - 2 hours - Used form **electron microscopy** -- buffered glutaraldehyde then osmium tetroxide - Made up of two formaldehyde residues, linked by three carbon chains. - Recommended for **histochemistry** and **electron microscopy** - Most effective aldehyde [for protein cross-linking] - **2.5% solution** -- for small tissue fragments and needle biopsies fixed in **2-4 hours at room temperature.** - **4% solution** -- recommended for larger tissued **less than 4mm** thick fixed in **6-8 hours to 24 hours** **Disadvantages:** 1\. It is more expensive. 2\. It reduces PAS positivity of reactive mucin. \* How can this be prevented? → Immersion of Glutaraldehyde fixed tissues in a mixture of **Concentrated glacial acetic acid and aniline oil.** ii\. metallic fixative a\. mercuric chloride - The most common metallic fixative - Frequently used in aqueous saturated solutions of **5-7%.** - Permits **brilliant metachromatic staining** - The routine fixative of choice for preservation of cell detail in **tissue photography** - Tissues fixed with mercury chloride containing compounds produce [black precipitates] **except** **Heidenhain's SuSa** - Satisfactory for immunoperoxidase techniques but ultrastructural preservation is poor. **Disadvantages:** 1\. It causes marked shrinkage of cells. Metal = Mercury How can one counteract this? - By addition of an acidic solution such as **glacial acetic acid** - Through **secondary fixation** 2\. Has black granular deposits and is extremely corrosive to metals. **Removal of black deposits caused by Mercuric Chloride:** a. [0.5% iodine in 70% ethanol (alcoholic iodine)] for 5-10 minutes + water + [5% sodium thiosulfate] (decolorized-5mins)-wash in running water. - **Alcoholic iodine** is used to **remove excess mercury** - **5% sodium thiosulfate** is used to **remove excess iodine** b. Addition of s[aturated iodine solution in 96% alcohol (alcoholic iodine)] and iodine decolorized with [absolute alcohol] in the subsequent stages of dehydration. c. Dezenkerization - Bring slides to water. - Immerse in Lugol's Iodine (5 minutes) - Wash in running water (5 minutes) - Immerse in 5% Na thiosulfate (5 minutes) - Wash in running water (5 minutes) - Proceed with required water soluble stain. mercuric chloride fixatives 1. zenker's fluid Fixation time: 12-24 hours - Mercuric Chloride Stock Solution + GLACIAL ACETIC ACID -- added before its use - Good general fixative for adequate preservation of all kinds of tissues and give excellent staining results - Solutions must always be freshly prepared - Recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei. - Produces **Mercuric deposits** - **Remedy: Alcoholic Iodine** **Dezenkerization** 2. Zenker-formol / helly's solution Fixation time: 12-24 hours - Mercuric Chloride Stock Solution + 40% FORMALDEHYDE -- added before its use - Excellent microanatomic fixative for pituitary gland, bone marrow, and blood containing organs -- spleen and liver. "BLOODY HELLY" - Produces **brown pigments** - **Remedy:** immerse the tissue in **ALCOHOLIC PICRIC ACID or SODIUM HYDROXIDE** 3. heidenhain's susa Fixation time: 3-12 hours - For **TUMOR BIOPSIES ESPECIALLY OF THE SKIN** - An excellent cytologic fixative **FORMULA:** - Mercuric Chloride (shrinkage) - Sodium Chloride - Trichloroacetic acid (swelling) - Glacial Acetic Acid - Formaldehyde, 40% - Distilled Water - Counterbalance effect - Su (Sublimat) = metal (mercury) = shrink - Sa (Saure) = acid (tichloroacetic acid) = swell 4. B-5 fixative Fixation time: 1 ½ - 2 hours - For **BONE MARROW BIOPSY** **FORMULA:** - **Distilled Water** - **Mercuric chloride** - **Sodium Acetate** - **Just prior to use, add 1cc of 40% Formaldehyde for 10cc of B-5 fixative** **\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--** **B. chromate** - a class of fixatives which are strong oxidizing agents used for precipitating proteins and preserving carbohydrates. - recommended for **C**hromaffin tissue, **A**drenal Medulla, **M**itochondria Chromate fixatives **"CROP"** **1. CHROMIC ACID** - Used in **1-2%**, used as a constituent of compound fixative. - It precipitates all ***proteins*** and adequately preserves ***carbohydrates***. - ***Formaldehyde*** must be added to **chrome- containing tissues** before use to prevent counteracting effects and consequent decomposition of solution upon prolonged standing. **2. REGAUD'S FLUID/ MOELLER'S FLUID** **FIXATION TIME: 12-48 HOURS** - For Chromatin, Mitochondria, Mitotic Figures, Golgi Bodies, RBC and Colloid-containing tissues. **FORMULA:** - 3% Potassium dichromate - 40% Strong Formaldehyde 3\. **orth's fluid (Regaud's + sodium sulfate)** **Fixation time: 36-72 hours** - For study of **early degenerative processes** and **tissue necrosis** - **Myelin and Rickettsia** **FORMULA:** - 2.5% Potassium dichromate - Sodium sulfate (optional) - 40% Strong Formaldehyde \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- C. lead fixatives - Used in **4% aqueous solution** of basic **lead acetate** - Recommended for **acid mucopolysaccharides** **Disadvantage:** - It takes up CO2 to form **insoluble lead carbonate** especially on prolonged standing. - Remedy: **Filtration** or by adding ***acetic acid*** drop by drop to lower the pH and dissolve the residue. iii\. Picric acid fixatives - Excellent for **glycogen** demonstration - Normally used in strong saturated aqueous solution - It dyes the tissues **yellow**, thus preventing the tissue fragments from being overlooked. On the other hand, this hinders proper staining. - **REMEDY:** - Add **50-70% ethanol** - Acid dye/ **lithium carbonate** - 70% ethanol → 5% sodium thiosulfate → Wash in running tap water - Can be used for fragmentary biopsies - The chemical name for the general picric acid fixatives: *2,4, 6 - trinitrophenol* - Picric acid is highly **explosive when *dry***, and therefore must be kept moist with ***distilled water* or *saturated alcohol* at 0.5 to 1% concentration** during storage. **A. Bouin's solution** **Fixation time: 6-24 hours** - Embryos and pituitary biopsies - Preferred fixative for **Masson's trichrome staining** for collagen, elastic or connective tissue. **FORMULA:** - Saturated solution of Picric Acid - Strong formaldehyde 40% - Glacial acetic acid B. brasil's alcohol/ picrofromol fixative - Best routine fixative for **glycogen** **FORMULA:** - **Formaldehyde 37%** - Picric Acid - Ethanol or Isopropyl Alcohol - Trichloroacetic acid iV. glacial acetic acid - Solidifies at **17C** - Fixes and precipitates nucleoproteins - It precipitates chromosomes and chromatin materials. - Causes tissues to swell specially those containing collagen. **Disadvantage:** It is contraindicated for cytoplasmic fixation because it destroys *mitochondria* and *golgi elements*. v\. alcohol fixatives - Denatures and precipitates proteins - 70-100% concentrations are used; Less concentrations results to red blood cell lysis/ hemolysis due to hypotonicity **Advantages:** - May be used both as a fixative and dehydrating agent - Excellent for glycogen preservation **Disadvantages:** - Causes RBC hemolysis and dissolves fats and lipids - Tissue shrinks on long usage - Polarization (major disadvantage); glycogenic granules moves to towards the ends or poles of cells **General Rule:** - Alcohol-containing fixatives are contraindicated when lipids are to be studied. **A. methyl alcohol 100%** - Fixing **dry and wet smears**, **blood smears** and **bone marrow tissues.** - Fixes and dehydrates at the same time - Not to fix for more than 48 hours -- tissues may be overharden and difficult to cut - Used in WRIGHT'S STAIN as diluent **B. isopropyl alcohol 95%** - Used for fixing touch preparation (impression smear) - WRIGHT-GIEMSA STAIN C. ethyl alchol Fixation time: 18-24 hours - Used concentrations of 70% to 100% - Lower concentrations \-\-- RBC's become hemolyzed and WBC's are inadequately preserved. - Used as simple fixative D. carnoy's fluid Fixation time: 1-3 hours - Most rapid tissue fixative - Fixing chromosomes, lymph glands and urgent biopsies. - used to fix brain tissue for diagnosis of **RABIES** **FORMULA:** - Chloroform - Absolute Alcohol - Glacial Acetic acid E. newcomer's fluid - Mucopolysaccharide and nuclear proteins - Both a nuclear and a histochemical fixative **FORMULA:** - Isopropyl alcohol - Propionic Acid - Petroleum ether - Acetone - Dioxane vi\. Osmium tetroxide/ osmic acid - A poor penetrating agent ad very expensive - Should be kept in cool place or refrigerated to prevent deterioration - Extremely volatile - It is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form strong oxidizing solution. - Adequately fixes materials for **ultrathin sectioning in electron microscopy**, since it rapidly fixes small pieces of tissues and aids in their staining **Disadvantage:** - Inhibits Hematoxylin and makes counterstaining difficult - Very expensive (less amount is required for fixation; 5-10x volume of fixative used) - Formation of artifact pigments/black precipitate - Prolonged exposure to acid vapors causes eye irritation(conjunctivitis) or black osmic oxide deposition in the cornea (blindness) 1. flemming's solution fixation time: 24-48 hours - The most commonly used Chrome-Osmium - acetic acid, recommended for **nuclear preparation of such sections.** - Excellent for nuclear structures such as **chromosomes** - **Permanently fixes fats/lipids** **FORMULA:** - Aqueous chromic acid 1% - Aqueous osmium tetroxide - Glacial Acetic Acid 2. flemming's solution without acetic acid fixation time: 24-48 hours - Chromic and osmic acid - For cytoplasmic structures- mitochondria \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- vii\. trichloroacetic acid (TCA) - Sometimes incorporated into compound fixatives (Ex. Heidenhain's SuSa) - It precipitates **proteins** - Causes marked **swelling** effect on tissues - Can be used as a **weak decalcifying agent**. - Has softening effect on dense fibrous tissue - Suitable only for small pieces of tissues or bone because of its poor penetration viii\. acetone - It is used at ice cold temperature from -**5C to 4C** (New book: 0-4C) - Recommended for the study of water diffusible enzymes especially **lipases** and **phosphatases** - Used in fixing brain tissues for **diagnosis of rabies (Negri bodies)**. - **Used in freeze substitution techniques** as a solvent for certain metallic salts - Evaporates rapidly decalcification - Procedure whereby **calcium or lime salts are removed** from tissues (most especially **bones** and **teeth**) - It is a form of ionization - Done after fixation and before impregnation (before dehydration) - Calcified tissues are harder to cut - Tuberculous organs, arteriosclerotic vessels, bones, and teeth Recommended temperature: **Room temperature** (**18-30C**) Ideal time required for decalcifying tissue: ***24-48* hours** Volume: ***20x*** the volume of the tissue **Factors Influencing Rate of Decalcification** a. Structure b. Temperature c. Concentration and Volume \*too rapid removal of CA+2 salts may produce *complete digestion* of the tissue specimen and poor staining capacity of the cell **Decalcifying Agents:** 1. Acids 2. Chelating Agents 3. Ion Exchange Resins 4. Electrical Ionization / Electrophoresis For routine purposes, only **acids** are recommended: \- ***Nitric acid*** - almost 2X as fast as formic acid \- ***Formic acid*** - provides better tissue preservation and staining **I.** acid decalcifying agents   ii\. chelating agents - Substances which combine with calcium ions and other salts (*iron* and *magnesium*) to form weakly dissociated complexes and facilitate removal of Calcium salt - The most common chelating agent: *EDTA* (Used as an anticoagulant and water softener) - Commercial name: Versene, Sequestrene iii\. ion exchange resin - Ion exchange resin (ammonium form of polystyrene resin) hastens decalcification - **Removes calcium ions from formic acid**, thereby increasing solubility from the tissue. - Not recommended for fluids containing mineral acids such as **nitric or hydrochloric acids**. iv\. electrical ionization (electrophoresis) - Process whereby positively charged Calcium ions are **attracted to a negative electrode (cathode)** and subsequently removed from the decalcifying solution. - The time required for decalcification is thereby shortened due to the **heat and electrolytic reaction** produced in this process. - The principle applied is similar to that of chelating agents; with the main difference that this process **utilizes electricity and is dependent upon a supply of direct current** to remove calcium deposits. Solutions used: 1\. 88% Formic acid 2\. Concentrated H 3\. Distilled Water 3 ways to measure the extent of decalcification  Tissue Softeners \- Unduly hard tissues which are reliable to damage the microtome knives may require tissue softener, aside from decalcification. 1. **Perenyi's fluid** -- act both as a decalcifying agent and tissue softener 2. **4% phenol** (1-3 days) 3. **Molliflex** (tissues may appear swollen and soapy) 4. **2% Hydrochloric acid** 5. **1% HCl in 70% alcohol** dehydration - Process of removing intercellular and extracellular water from tissue following fixation and prior to wax impregnation - Many of these dehydrating agent are ALCOHOLS of increasing strengths to remove aqueous tissue fluids with little disruptions to the tissue. - Removal of water through osmosis. - 70% ethyl alcohol in water, 95% ethyl alcohol to 100% ethyl alcohol **GENERAL RULE: whatever dehydrating agent is used, the amount in each stage should not be less than 10 times** 1. Should dehydrate rapidly without producing considerable shrinkage or distortion of tissues. 2. Should NOT evaporate very fast. 3. Should be able to dehydrate even fatty tissue
 (Kasi ang alcohol ay may polar part) 4. Should NOT harden tissues excessively 5. Should NOT remove stain 6. Should NOT be toxic to the body 7. Should NOT be a fire hazard. **I.** alcohol a\. ethanol/ ethyl alcohol - For routine dehydration - Best dehydrating agent - A clear, colorless, flammable liquid - Fast-acting - Mixes with water and many organic solvents - Penetrates tissue easily - Not poisonous and not very expensive - Should be **at least 99.7**% pure **B. methanol/methyl alcohol** - Toxic dehydrating agent - For blood and tissue films and for smear preparation 3. butanol/ butyl alcohol - Slow dehydrating agent - Producing less shrinkage and hardening - Utilized for plant and animal microtechnique 4. isopropanol/ isopropyl ALCOHOL - Should be used id good-grade absolute ethyl alcohol is not easily available PROBLEM: Concentrated alcohols (95% or absolute) resulting in a relatively unequal impregnation of tissue - **REMEDY**: 70% or lower concentration of alcohol, gradually increased to 95% is used. **(IBABALIK MO LANG ULIT SA LOWER CONCENTRATION)** ii\. acetone - A cheap, rapid dehydrating agent utilized for most **urgent biopsies** - **DEHYDRATION TIME: ½ to 2 hours** - With water, ethanol, and most organic solvents iii\. dioxane/ diethylene dioxide - Excellent **DEHYDRATING AND CLEARING AGENT** miscible to water, melted paraffin, alcohol and xylol/xylene - Expensive and toxic and extremely dangerous iv\. cellosolve/ethylene glycol monoethyl ether - Dehydrates rapidly and not harmful to tissues - Toxic by inhalation, skin contact and ingestion (use propylene-based glycol esters) v\. Triethyl phosphate - Tissues can be transferred directly after fixation & washing - Produces very little distortion and hardening of tissue. - Soluble in alcohol, water ether benzene, chloroform, acetone and xylene. vi\. tetrahydrofuran (THF) - Both **DEHYDRATES AND CLEARS** tissue -- miscible in both water and paraffin. - It does not dissolve out aniline dyes - Less shrinkage and easier cutting of sections with fewer artifacts. - Toxic if ingested or inhales - An eye and skin irritant and prolonged exposure may conjunctivitis irritation - Vapor cause nausea, dizziness, headache and anesthesia clearing **AKA: DE-ALCOHOLIZATION** - Dehydrating agent (alcohol) is removed from the tissue - Replaced with a substance that will dissolve the wax (De-paraffin) - The tissue has translucent appearance -- "Clearing Agent" - Must be miscible with paraffin and alcohol - Microscopic tissue preparations **TRANSPRENT** due to their high index of refraction. - Not all clearing agents exhibit property of making tissues transparent - Miscible with **Canada balsam** and other resins that are used for mounting sections - **XYLENE** most commonly used - In frozen sections, **GLYCERIN and GUM SYRUP** are used when tissue is to be cleared directly from water\-\-- no de-alcoholization process involved. **CHARACTERISTICS OF A GOOD CLEARING AGENT:** - Miscible with **alcohol** to promote rapid removal of the dehydrating agent from the tissue. - Should be miscible with, and easily removed by **melted paraffin wax** and/or **mounting medium** to facilitate impregnation and mounting of sections. - Should NOT produce excessive shrinkage, hardening or damage of tissue - Should NOT dissolve out aniline dyes - Should NOT evaporate quickly in a water bath - Should make tissue transparent **I. xylene/xylol** clearing time: 30 mins to 1 hour - Most commonly used - Used for clearing and mounting procedures - Suitable for most routine histologic processing - **Rapid clearing agent and cheap** - Miscible with **absolute alcohol and paraffin** - For mounting procedures \-\--it does NOT dissolve celloidin and **can be used for celloidin sections** - Highly inflammable - Should not exceed 3 hours \-\--makes tissues excessively hard and brittle - NOT suitable for nervous tissues and lymph nodes - **MILKY** when an **incompletely dehydrated tissue**. ii\. toluene clearing time: 1-2 hours - Substitute for **xylene or benzene** - Acts fairly rapid and is recommended for routine purposes - Miscible with both **absolute alcohol and paraffin.** - Does not excessively make tissue brittle. - NOT carcinogenic - More expensive and relatively slower than benzene - Emit fumes -\> toxic upon prolonged exposure. iii\. benzene CLEARING TIME: 15 -- 60 MINUTES - Rapid acting - recommended for urgent biopsies (15 - 60 minutes) and routine purposes. - Volatilizes rapidly in paraffin oven - Miscible with **absolute alcohol** - Makes tissue transparent - Does NOT make tissue hard and brittle - Highly flammable - **APLASTIC ANEMIA** IV\. CHLOROFORM CLEARING TIME: 6 -- 24 HOURS - Slower action and causes less brittleness - Thicker blocks (**even up to 1 cm in thickness**) can be processed. - Do NOT become transparent. (Translucent only) - Recommended for routine work (**6 - 24 hours**) - Miscible with **absolute alcohol** - Tough tissues, nervous tissues, lymph nodes and embryos. - Large tissue specimens - Toxic to the liver - Does not make the tissues transparent - ↑SG = Tissue floats - Not very volatile in paraffin v\. cedarwood oil clearing time: 2-3 days celloidin: 5-6 days - clear both **paraffin and celloidin sections** during the embedding process - **central nervous system tissues** and **cytological studies - smooth muscles and skin**. - Least distortion - Clearing with this agent often improves cutting of the sections. - Very expensive and it requires *2* changes in clearing solution - Quality is not always uniform and good and is extremely slow - **It becomes *milky* on prolonged storage** vi\. aniline oil - Recommended for clearing **embryos**, **insects**, and **very delicate specimens** \-- due to its **ability to clear 70% alcohol without excessive tissue shrinkage and hardening.** vii\. carbon tetrachloride - Used in clearing tissues for embedding - Produces considerable tissue hardening - Dangerous to inhale on prolonged exposure due to its highly toxic effects. - Properties are very similar to chloroform but it is cheaper viii\. clove oil - It removes **aniline dyes** and **dissolves Celloidin**; - Tissues become brittle - Its quality is not guaranteed due to its **tendency to be *adulterated*** - Not suitable for routine purposes because it is expensive ix\. tetrahydrofuran (THF) - **Dehydrates and clears** at the same time since it is miscible in both water and paraffin x\. methyl benzoate/ mehyl salicylate - These are slow-acting clearing agents that can be used when **double embedding techniques** are required. impregnation **AKA: INFILTRATION** - The process whereby the clearing agent is completely removed from the will completely fill all the tissue cavities thereby: - firm consistency - easier handling - suitably thin sections without damage to tissues - Ideal volume: **at least *25x*** the volume of the tissue **3 Methods of Impregnation Used in Histology** **1. Paraffin wax Impregnation** - the simplest, most common, and by far the best for routine use - melting point is **54-58 C** **2. Celloidin wax/Collodion Impregnation** **3. Gelatin Impregnation** I. **3 Ways of Paraffin Wax Impregnation (and Embedding** 1. manual processing - Requires at least *4* changes of wax with *15* minutes interval - Total: 1 hour 2. automatic processing - Makes use of an automatic tissue processing machine (e.g. Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues, resulting in a more rapid diagnosis with less technicality - Autotechnicon/Elliot-Bench Type Tissue Processor: **Fixation, Dehydration, Clearing, Infiltration** 3. vacuum embedding - Impregnation under **negative atmospheric pressure** - Recommended for urgent biopsies and delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes spleen and CNS. - Reduces *25-75%* of impregnation time - Most rapid **NOTE:** Infiltration in overheated paraffin (above *60C*) produces shrinkage and hardening of tissues. Paraffin oven must be maintained at a temperature ***2-5°C* above the melting point of the wax.** II\. CELLOIDIN IMPREGNATION - **Celloidin** - purified form of **nitrocellulose** soluble in many solvents - suitable for specimens containing **large cavities or hollow spaces** which tend to collapse, for hard and dense tissues (bones and teeth) and **for large tissue sections of whole embryos**. - **Advantage:** Causes much less shrinkage and distortion than paraffin wax - **Disadvantage:** 1\. Slow and tedious 2\. Serial sections are difficult to prepare 3\. Very thin sections (\