MTAP - HP & MT LAWS - This is an MTAP exam reviewer on Histopathology and Medical Technology Laws PDF

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MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2

HISTOPATHOLOGY & MEDTECH LAWS Lecturer: Prof. Edmund Francisco, RMT, MLS (ASCPi) By: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King Squash preparation/ Crushing

HISTOPATHOLOGIC TECHNIQUES It deals with the preparation for microscopic examination It is accomplished by submitting the total or a selected part of the tissue presented for examination to a series of processes: “FDD-CIET-SSML” Fixation - Preservation Decalcification - Calcium or lime salts are removed from the tissues - Optional process: for calcified tissues only such as bones and teeth Dehydration - Desiccation - Removing intracellular and extracellular fluid/water Clearing - De-alcoholization - Removing alcohol used in dehydration Infiltration - Impregnation Embedding - Casting or Blocking Trimming - Removing excess wax from the tissue block - Optional process: not all tissue blocks have excess wax Sectioning - Section cutting - Cutting tissue blocks into uniformly thin slices Staining - Dyeing Mounting Labelling - Proper labelling Examination may be done on: 1. Fresh Tissues o usually examined when there is an immediate need for evaluation 2. Preserved Tissues o routinely done in the histopathology section. FRESH TISSUE EXAMINATION Advantage: Examined in the living state, thereby allowing protoplasmic activities such as: a. Motion (Cellular Movement) b. Mitosis (Cellular Reproduction) c. Phagocytosis (Cell-eating activity) d. Pinocytosis (Cell-drinking activity) Disadvantage: Its use has been limited, however, because of the fact that tissues examined in the fresh state are not permanent and therefore, are liable to develop the changes that have usually been observed after death. POST MORTEM CHANGES Autolysis

Putrefaction or Decomposition Degeneration

- the destruction of the tissues (breaking down of the protein of the cell) by enzymes which are produced by the tissues and eventually liquefy it. - It is the first to occur among all post-mortem changes - the decomposition of organic matter under the influence of microorganisms accompanied by the development of disagreeable odors. - a retrogressive pathologic process in cells in which the cytoplasm undergoes deterioration while the nucleus is preserved.

METHODS OF FRESH TISSUE EXAMINATION Teasing or Dissociation

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Process wherein selected tissue specimen is immersed in a watch glass containing isotonic salt solution (NSS or Ringer’s lactate), carefully dissected or separated and examined under the microscope, either unstained by Phase Contrast microscope or Bright-field microscope, or stained with differential dyes.

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Process where small pieces of tissue not more than 1mm in diameter are placed in a microscopic slide and forcibly compressed with another slide or with coverglass. Vital dyes are placed at the slide and coverslip junction and absorbed through capillary action. Normally utilized when a rapid diagnosis of the tissue in question is required, and especially recommended when lipids and nervous tissue elements are to be demonstrated. Useful in cytological examinations, particularly for cancer diagnosis.

Frozen section

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Smearing

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Smearing Technique a. Streaking

Material

Process/Important Notes

Applicator stick or platinum loop.

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b. Spreading technique

c. Pull-apart technique

d. Touch preparation or Impression smear

Applicator stick to tease the mucous strands to make a moderately thick film.

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Slides facing each other as a drop of secretion is sandwiched inbetween.

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One slide.

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Rapid and gentle direct or zigzag application to obtain uniform distribution. Too thick or too thin smears are unsuitable for examination. Little more tedious than streaking, but has advantage in maintaining the intercellular relationship. Especially recommended for fresh sputum, bronchial aspirates and thick mucoid secretions. The material disperses evenly over the surface of 2 slides. A single uninterrupted motion of pulling apart is applied. It is useful for serous fluids, concentrated sputum, enzymatic GIT lavage and blood smears Special method where slide surface is in contact and pressed on the site. Cells may be examined without destroying their actual intercellular relationship and without separating them from their normal surroundings.

STEPS IN PROCESSING PRESERVED TISSUES FIXATION AKA: Preservation The process by which the constituents of the cells, and therefore of the tissues are fixed in a physical, and partly also in a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss or significant distortion or decomposition. The first and most critical (important) step in histotechnology Why is it the most critical? Because it is the first procedure and is able to affect the subsequent procedures Primary aim of fixation  Preserve the morphologic and chemical integrity of the cell in as lifelike as possible Secondary goal of fixation  To harden and protect the tissue from trauma of further handling  For easy cutting during gross examination -

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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 Two Basic Mechanisms in Fixation Principle/ Notable Characteristics 1. Additive - The chemical constituent of the fixative is taken in and becomes part of the tissue through cross-link formation or molecular complexes. 2. Nonadditive

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The fixing agent is not incorporated into the tissue but alters the tissue composition and stabilizes it through water removal. New cross-links are formed preventing autolysis and bacterial decomposition.

Example Formaldehyde Osmium tetroxide/Osmic acid fixative Mercuric chloride Alcoholic

Main Factors Involved in Fixation 1. Hydrogen Ion - pH: 6.0-8.0 Concentration - Average: 7.0 (neutral pH) 2. Temperature - Traditional/usual: Room temperature (18-30oC) - Tissue processors: Autotechnicon (40-42 oC) - Electron Microscopy and Histochemistry: 0-4 oC o Mast cells for EM: Room temperature - Nucleic acids fixation: Rapid at higher temperature - Formalin heated to 60°C → rapid fixation of very urgent biopsy specimens - Formalin heated to 100°C → to fix tissues with tuberculosis 3. Thickness of - Small sections o Electron Microscopy: 1-2 mm2 o Light Microscopy: 2 cm2 - Thin o Light Microscopy: ≤0.4 cm or as prescribed by tissue processor manufacturer o Large solid tissue, such as uterus, should be opened or sliced thinly o Brain is usually suspended whole in 10% Neutral Buffered Formalin for 2-3 weeks. 4. Osmolality - Best results are obtained using slightly hypertonic solutions (400-450 mOsm) o Hypertonic solutions = Shrinkage o Isotonic (340 mOsm)/Hypotonic solutions = swelling and poor fixation - Added to Osmium tetroxide fixatives for EM: Sucrose 5. Concentration - Formaldehyde: 10% - Glutaraldehyde: 3% - Glutaraldehyde for Immunoelectron microscopy: 0.25% 6. Duration of - Most formalin fixatives: 24 hours (washed out) fixation - Buffered formalin: 2-6 hours up to 1 week - EM: 3 hours (New books: 0-4 hours; Average: 2 hours) - Prolonged fixation may cause shrinkage and hardening of tissue

Practical Considerations 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. o Fixation can kill microorganisms and prevent growth in culture o Fixatives can neutralize drugs and toxins 2. Penetration Formalin diffuses into the tissue at the rate of approximately 1mm/hr. Time of fixation varies with different types of tissue 3. Volume/Amount of Fixative Traditionally, the amount of fixative used has been 10-25x the volume of the tissue to be fixed. Recently, 20x is known as the maximum effective concentration for fixation Except when osmium tetroxide (5-10x) is used. o Because Osmium tetroxide is very expensive 4. Duration Some tissues take longer to fix than others, depending on their structures. Fibrous organs such as uterus and intestinal tract take longer. Fixation can be cut down by using Heat, Vaccum, Agitation or Microwave. IDEAL SIZE OF TISSUE TO BE FIXED: not more than 2cm2 in diameter not more than 4mm thick Ideal number of hours for fixation: 4-6 hours (New books: 6-18 hours)

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TYPES OF FIXATIVES

I. According to Composition SIMPLE FIXATIVES (made up of only 1 component substance)

A.

1. Aldehydes (Formaldehyde, Glutaraldehyde) 2. Metallic Fixatives (Mercuric chloride, Chromic acid, Lead) 3. Picric acid 4. Acetic acid 5. Acetone 6. Alcohol 7. Osmium tetroxide 8. Heat COMPOUND FIXATIVES (Made of up of 2 or more fixatives to obtain optimal results)

B.

II. According to Action A.

Microanatomical Fixatives: Permit the general microscopy study of tissue structures 1. 2. 3. 4. 5. 6. 7. 8.

10% Formol saline 10% NBF Heidenhain’s susa Formol sublimate (corrosive) Zenker’s solution Zenker-formol (Helly’s) Bouin’s solution Brasil’s solution

B.

Cytological Fixatives: Preserve specific parts and particular microscopic elements of the cell itself

Removal of formalin pigments: 1.

1.

Nuclear Fixatives Preserve nuclear structures (ex. chromosomes). They usually contain Glacial acetic acid as their primary component due to its affinity for nuclear chromatin. The pH is 4.6 a. b. c. d. e.

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2.

Helly’s Orth’s Regaud’s Flemming’s fluid w/o HAc Formalin w/ post-chroming

Histochemical Fixatives Preserve the chemical constituents of cells and tissues a. 10% Formol saline b. Absolute Ethyl alcohol c. Newcomer’s fluid (Both Nuclear and Histochemical fixative) d. Acetone ROUTINE FIXATIVES

I. ALDEHYDE FIXATIVES A. FORMALDEHYDE: FORMALIN -

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Commercial formaldehyde/Formalin A saturated solution of formaldehyde gas in water, approximately 35-40% gas by weight (Gregorios: 37-40% weight in volume). A mixture of 10 mL formalin with 90 mL of water/saline is known as 10% formalin (most widely used fixative of all) It is usually buffered to pH 7 with a phosphate buffer. Commercial formalin often becomes turbid, especially if stored in a very cold place, because of the formation of paraformaldehyde (whitish precipitate) → may be removed by filtration, but almost all commercial formaldehyde contains 10% methanol, which tends to inhibit the formation of paraformaldehyde. Formalin is the best fixative for the Nervous tissue

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

Lillies Method – involves placing formaldehyde fixed specimens in acetone, 28% ammonia water and hydrogen peroxide. It uses 70% alcohol as a rinsing agent. Kardasewitch’s Method – a method of formaldehyde clearance involving 70% ethanol and 28% ammonia water. Saturated Alcoholic Picric Acid

B. GLUTARALDEHYDE It is made up of two fomaldehyde residues, linked by three carbon chains. It is utilized for Light microscopy. Buffered Formaldehyde with secondary fixation in osmium tetroxide is satisfactory for Electron Microscopy. o 2.5% - for small tissue fragments and needle biopsies for 2-4 hours at room temperature. o 4% - large tissue fragments less than 4 mm thick for 6-8 hours up to 24 hours. - Fixation time: ½-2 hours - Recommended for histochemistry and electron microscopy. - The most effective aldehyde for protein cross-linking 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.

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II. METALLIC FIXATIVES 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. o Metal = Mercury o 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.

b.

c.

0.5% iodine in 70% ethanol (alcoholic iodine) for 5-10 minutes + water + 5% sodium thiosulfate (decolorized-5mins)-wash in running water. o Alcoholic iodine is used to remove excess mercury o 5% sodium thiosulfate is used to remove excess iodine Addition of saturated iodine solution in 96% alcohol (alcoholic iodine) and iodine decolorized with absolute alcohol in the subsequent stages of dehydration. 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 - Mercuric chloride + glacial acetic acid just before its use. o To prevent turbidity and formation of dark Fixation time: precipitates 12-24 hours - Good general fixative for all kinds of tissue - Recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei - May act as mordant to make certain special staining possible - Contains glacial acetic acid which makes the solution unstable 2. Zenker- Mercuric chloride + 40% formaldehyde just formol/ Helly’s before its use. Solution - It is an excellent microanatomic fixative for pituitary gland, bone marrow and bloodFixation time: containing organs such as the liver and spleen. 12-24 hours - Better nuclear fixations and staining than Zenker’s - It presevres cytoplasmic granules better than “Bloody Helly” Zenker’s - Disadvantage: Brown pigments are produced if tissues are allowed to stay in the fixative for more than 24 hours due to RBC lysis. - Remedy: immerse the tissue in alcoholic picric acid or sodium hydroxide 3. Heidenhain’s - Recommended for tumor biopsies especially of the Susa solution skin - It is an excellent Cytologic fixative Fixation time: - Produces minimum shrinkage and hardening of 3-12 hours tissues due to the counter-balance of the swelling effects of acid (trichloroacetic acid) and the shrinkage effect of a metal (mercury).

4.

5.

Schaudinn’s Solution/ Sublimated alcohol B-5 Fixative

Fixation time: 1 ½ to 2 hours (Rapid fixation) 6. CarnoyLebrun 7. Ohlmacher

- Counterbalance effect o Su (sublimat) = metal (mercury) → cell shrinkage o Sa (saure) = acid (trichloroacetic acid) → cell swelling - A solution of mercuric chloride, sodium chloride, alcohol, and glacial acetic acid - Used on wet smears for cytologic examinations. - Composed of mercuric chloride and sodium acetate. - Commonly used for bone marrow biopsies. - Just prior to use, add 1 mL of 40% formaldehyde to 10 mL of B5

B. CHROMATE A class of fixatives which are strong oxidizing agents used for precipitating proteins and preserving carbohydrates. Recommended for Chromaffin tissues, Adrenal medulla, Mitochondria. “CROP” CHROMATE FIXATIVES 1. Chromic acid Used in 1-2%, used as a constituent of a compound fixative. It precipitates all proteins and adequately preserves carbohdyrates. Formaldehyde must be added to chromecontaining tissues before use to prevent counteracting effects and consequent decomposition of solution upon prolonged standing. 2. Regaud’s Recommended for demonstration of Chromatin, Fluid/ Mitochondria, Mitotic figures, Golgi bodies, Moeller’s fluid RBC’s and colloid-containing tissues 3. Orth’s Fluid Recommended for study of early degenerative processes and tissue necrosis Fixation time: Demonstrates Rickettsia and other bacteria 36-72 hours 4. Potassium Preserves mitochondria (pH 4.5-5.2) dichromate Fixes lipids Used in 3% aqueous solution It fixes but does not precipitate cytoplasmic structures. C. LEAD FIXATIVES 1. Lillie’s alcoholic lead nitrate formalin 2. Lead subacetate Advantages: 1. It is recommended for acid mucopolysaccharides 2. It fixes connective tissue mucin Disadvantage: It takes up CO2 to form insoluble lead carbonate especially on prolonged standing. This may be removed by filtration or by adding acetic acid drop by drop to lower the pH and dissolve the residue. III. Picric Acid Fixatives 1. Excellent for glycogen demonstration 2. Normally used in strong saturated aqueous solution 3. It dyes the tissues yellow, thus preventing the tissue fragments from being overlooked. On the other hand, this hinders proper staining. 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.

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What must be done to remove this yellow color? o Add 50-70% ethanol o Acid dye/lithium carbonate o 70% ethanol → 5% sodium thiosulfate → Wash in running tap water “PBB” A. Bouin’s solution - Fixation of embryos and pituitary biopsies - It is an excellent fixative for preserving soft and Fixation time: delicate structures (endometrial curettings) 6-24 hours - Yellow stain is useful in fragmentary biopsies - Preferred fixative for Masson’s trichrome staining for collagen, elastic or connective tissue B. Brasil’s Alcoholic - Best routine fixative for glycogen Picroformol Fixative - It is better and less messy than Bouin’s solution

VI. Osmium Tetroxide/Osmic Acid 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)

IV. Glacial Acetic Acid 1. Flemming’s Solution 1. 2. 3. 4. -

It solidifies at 17ºC (New book: 16oC) Fixes and precipitates nucleoproteins It precipitates chrom...