MTLE - Histopathology & General Pathology PDF

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW - HTMLE HISTOPATHOLOGY & GENERAL PATHOLOGY Lecturer: Prof. Edmund Francisco, RMT, MLS (ASCPi) Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the G...

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW - HTMLE HISTOPATHOLOGY & GENERAL PATHOLOGY Lecturer: Prof. Edmund Francisco, RMT, MLS (ASCPi) Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King HISTOPATHOLOGIC TECHNIQUES METHODS OF FRESH TISSUE EXAMINATION - It deals with the preparation for microscopic examination - It is accomplished by submitting the total or a selected part of the tissue - Process wherein selected tissue specimen is presented for examination to a series of processes: immersed in a watch glass containing isotonic salt solution (NSS or Ringer’s lactate), carefully dissected Teasing or “FDD-CIET-SSML” or separated and examined under the microscope, Dissociation Fixation - Preservation; 1st and most critical step either unstained by Phase Contrast microscope or - Calcium or lime salts are removed from the tissues Bright-field microscope, or stained with differential Decalcification - Optional process: for calcified tissues only such as dyes. bones and teeth - Process where small pieces of tissue not more than - Desiccation 1mm in diameter are placed in a microscopic slide and Dehydration Squash forcibly compressed with another slide or with - Removing intracellular and extracellular fluid/water preparation/ coverglass. - De-alcoholization Crushing Clearing - Vital dyes/stains are placed at the slide and coverslip - Removing the alcohol used in dehydration junction and absorbed through capillary action. Infiltration - Impregnation - Normally utilized when a rapid diagnosis of the tissue Embedding - Casting or Blocking in question is required, and especially recommended Frozen section - Removing excess wax from the tissue block when lipids and nervous tissue elements are to be Trimming - Optional process: not all tissue blocks have excess wax demonstrated. - Section cutting - Useful in cytological examinations, particularly for Sectioning Smearing - Cutting tissue blocks into uniformly thin slices cancer diagnosis. Staining - Dyeing Mounting Smearing Material Process/Important Notes Labelling - Proper labelling Technique - Examination may be done on: - Rapid and gentle direct or zigzag Applicator stick application to obtain uniform 1. Fresh Tissues or platinum Streaking distribution. o usually examined when there is an immediate need for evaluation loop. - Too thick or too thin smears are 2. Preserved Tissues unsuitable for examination. o routinely done in the histopathology section. Applicator stick - Little more tedious than streaking, to tease the but has advantage in maintaining FRESH TISSUE EXAMINATION Spreading mucous strands the intracellular relationship. ADVANTAGE: Examined in the living state, thereby allowing protoplasmic technique to make a - Especially recommended for activities such as: moderately fresh sputum, bronchial aspirates a. Mitosis (Cellular Reproduction) thick film. and thick mucoid secretions. b. Motion (Cellular Movement) - The material disperses evenly Slides facing c. Phagocytosis (Cell-eating activity) over the surface of 2 slides. each other as a d. Pinocytosis (Cell-drinking activity) - A single uninterrupted motion of Pull-apart drop of pulling apart is applied. technique secretion is DISADVANTAGE: Its use has been limited, however, because of the fact that sandwiched in- - It is useful for serous fluids, tissues examined in the fresh state are not permanent and therefore, are liable to concentrated sputum, enzymatic between. develop the changes that have usually been observed after death. GIT lavage and blood smears - Special method where slide PRIMARY & SECONDARY SIGNS OF DEATH Touch surface is in contact and pressed preparation or on the site. PRIMARY (Occur during somatic death) Impression One slide. - Cells may be examined without 1. CNS Failure smear destroying their actual 2. Respiratory failure intercellular relationship and 3. Cardiovascular failure without separating them from their normal surroundings. SECONDARY (Occur after somatic death) 1. Algor mortis PRESERVED TISSUES  The cooling of the body (7oF/hr or 1-1.5oC/hr) - A better and more effective means of studying tissues whether normal or  1st demonstrable change abnormal is by examination of their sections and smears which have been 2. Rigor mortis permanently preserved, stained for the demonstration of specific structures  Stiffening of the skeletal muscles and mounted on glass slides with coverslips for permanent keeping. 3. Livor mortis - The aim of a good histopathological technique is to produce microscopic  Post-mortem lividity or post-mortem suggilation preparation of tissue, usually stained, that represent as closely as possible  Purplish discoloration their structure in life. 4. Post-mortem clot 5. Autolysis STEPS IN PROCESSING PRESERVED TISSUES 6. Putrefaction POST MORTEM CHANGES FIXATION - AKA: Preservation - The destruction of the tissues (breaking down of the - The process by which the constituents of the cells, and therefore of the protein of the cell) by enzymes which are produced by tissues are fixed in a physical, and partly also in a chemical state so that they Autolysis will withstand subsequent treatment with various reagents with minimum loss the tissues and eventually liquefy it. - It is the first to occur among all post-mortem changes or significant distortion or decomposition. - The decomposition of organic matter under the influence - The first and most critical (important) step in histotechnology Putrefaction or  Why is it the most critical? Because it is the first procedure/step and is of microorganisms accompanied by the development of Decomposition able to affect the subsequent procedures/steps disagreeable odors. - A retrogressive pathologic process in cells in which the - Primary aim of fixation Degeneration cytoplasm undergoes deterioration while the nucleus is  Preserve the morphologic and chemical integrity of the cell in as life-like preserved. a manner 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 4. Duration appearance) - Some tissues take longer to fix than others, depending on their structures. - Leaving the tissue in water (hypotonic solution) will cause the cell to: swell - Fibrous organs such as uterus and intestinal tract take longer.  If the environment is hypotonic, more water is outside the cell and the - Fixation can be cut down by using Heat, Vacuum, Agitation or Microwave. solute concentration inside the cell is high, the water would enter the cell and swells, bursts, or lyses (cytolysis) IDEAL SIZE OF TISSUE TO BE FIXED: - Leaving the tissue in strong salt (hypertonic solution) will cause the cell to: - not more than 2cm2 in diameter shrink - not more than 4mm thick  More solutes are outside the cell (strong salt solution) and water is less, - Ideal number of hours for fixation: 4-6 hours (New books: 6-18 hours) so water will move outside the cell and the cell shrinks CHARACTERISTICS OF A GOOD FIXATIVE: Two Basic Mechanisms in Fixation 1. Cheap Principle/ Notable Characteristics Example 2. Stable 1. Additive - The chemical constituent of the Formaldehyde 3. Safe to handle fixative is taken in and becomes Osmium tetroxide/ 4. Kill the cells quickly part of the tissue through cross-link Osmic acid fixative 5. Produce minimum shrinkage formation or molecular complexes. Mercuric chloride 6. Harden the tissues properly 2. Non- - The fixing agent is not incorporated Alcoholic 7. Inhibits bacterial decomposition and autolysis additive into the tissue but alters the tissue 8. Permit rapid and even tissue penetration composition and stabilizes it 9. The optimum osmolality for a specific tissue and fixative type through water removal. 10. Make cellular components insoluble - New cross-links are formed 11. Permit subsequent application of many staining procedures preventing autolysis and bacterial decomposition. “the fact that there many fixatives indicate that the ideal has not been found” Main Factors Involved in Fixation REVIEW!!! 1. Hydrogen Ion - pH: 6.0-8.0 1. Which of the following 7. What is the primary aim of fixation? Concentration - Average: 7.0 (neutral pH) shows the proper order of a. To facilitate easy cutting 2. Temperature - Traditional/usual: Room temperature (18-30oC) steps in tissue processing? b. To harden and protect the - Tissue processors (Autotechnicon): 40-42oC a. Fixation → tissue - Electron Microscopy and Histochemistry: 0-4oC Dehydration → c. To maintain the physical and o Mast cells for EM: Room temperature Decalcification → chemical characteristics of the Clearing → Infiltration tissue - Nucleic acids fixation: Rapid at higher temperature → Blocking d. To stabilize the protein - Formalin heated to 60°C → rapid fixation of very b. Fixation → through cross-links formation urgent biopsy specimens Decalcification → - Formalin heated to 100°C → to fix tissues with Dehydration → 8. In additive mechanism, the tuberculosis Infiltration → Clearing chemical constituent of the fixative 3. Thickness of - Small → Blocking is taken in and becomes part of the sections o Electron Microscopy: 1-2 mm2 c. Fixation → tissue. Which of the following o Light Microscopy: 2 cm2 Decalcification → fixative does not possess this - Thin Dehydration → property? o Light Microscopy: ≤0.4 cm (4mm) or as prescribed Clearing → Infiltration a. Formalin by tissue processor manufacturer → Blocking b. Osmium tetroxide o Large solid tissue, such as uterus, should be d. Fixation → c. Ethanol opened or sliced thinly Dehydration → d. Mercuric chloride Decalcification → o Brain is usually suspended whole in 10% Neutral Infiltration → Clearing 9. Best results in fixation are obtained Buffered Formalin for 2-3 weeks. → Blocking through what type of solutions? 4. Osmolality - Best results are obtained using slightly hypertonic a. Isotonic solutions (400-450 mOsm) 2. Destruction of tissues or b. Hypertonic o Hypertonic solutions = Shrinkage cells by self-produced c. Slightly hypotonic o Isotonic (340 mOsm)/Hypotonic solutions = enzymes: d. Slightly hypertonic swelling and poor fixation a. Putrefaction - Added to Osmium tetroxide fixatives for EM: Sucrose b. Decomposition 10. Which of the following is added to 5. Concentration - Formaldehyde: 10% c. Autolysis Osmium tetroxide fixatives for - Glutaraldehyde: 3% d. Degeneration electron microscopy? - Glutaraldehyde for Immunoelectron microscopy: 0.25% a. Glucose 6. Duration of - Most formalin fixatives: 24 hours (washed out) 3. Process where small pieces b. Sucrose of tissues are placed in a c. Levulose fixation - Buffered formalin: 2-6 hours up to 1 week microscopic slide and d. Fructose - EM: 3 hours (New books: 0-4 hours; Average: 2 hours) forcibly compressed with - Prolonged fixation may cause shrinkage and another slide: 11. The temperature utilized in hardening of tissue a. Teasing Autotechnicon: b. Crushing a. 0-4oC Practical Considerations of Fixation c. Frozen section b. 18-30oC d. Smearing c. 40-42oC 1. Speed d. 60-100oC - The specimen should be placed in fixative as soon as it is removed from the 4. A smearing technique body. recommended for fresh 12. The following are not characteristics sputum, bronchial aspirates, of a good fixative, except: - This is done to prevent autolysis and putrefaction/decomposition. and thick mucoid a. It should kill the cells gradually - If bacteriologic and toxicologic studies should be encouraged, fixation is not secretions: b. It should make cellular required. a. Streaking components soluble o Fixation can kill microorganisms and prevent growth in culture b. Spreading c. It should not be unstable o Fixatives can neutralize drugs and toxins c. Pull-apart d. It should enter the tissue d. Touch preparation slowly for even penetration 2. Penetration - Formalin diffuses into the tissue at the rate of approximately 1mm/hr 5. The process by which the 13. For fixation of tissues with - Time of fixation varies with different types of tissue tissues are fixed in a tuberculosis, formalin must be physical and partly also in a heated at what temperature? 3. Volume/Amount of Fixative chemical state: a. 100oC a. Dehydration b. 80oC - Traditionally, the amount of fixative used has been 10-25x the volume of the b. Preservation c. 60oC tissue to be fixed. c. Protection d. 50oC - Recently, 20x is known as the maximum effective concentration for fixation d. Conservation - Except when osmium tetroxide (5-10x) is used. o Because Osmium tetroxide is very expensive  6. The maximum effective 14. The concentration of formaldehyde PRINCIPLES AND PRECAUTIONS IN HANDLING concentration of a fixative used in routine fixation AND FIXATION OF SPECIMENS IN GENERAL a. 5x the volume of the a. 0.25% tissue to be fixed b. 3% 1. Autopsy materials should be fixed as soon after death as possible to prevent b. 10x the volume of the c. 5-7% tissue to be fixed d. 10% decomposition and autolysis due to deprived oxygen and metabolism. lf this c. 15x the volume of the is not possible, the body should be placed in mortuary refrigerator (4°C) or tissue to be fixed 15. The ideal number of hours in arterial embalming should be carried out without delay, provided that d. 20x the volume of the fixation: toxicological or microbiological examinations are not indicated. Such tissue to be fixed a. 24 hours embalming, well done, is most advantageous and gives excellent b. 36 hours preservation. c. 18 hours 2. Surgical specimens should be fixed as soon as possible after removal, or d. 48 hours refrigerated if fixation is to be delayed, to prevent drying of surface layers and ultimate tissue distortion. Answer key: C, C, B, B , B, D, C, C, D, B, C, C, A, D, C 3. The amount of fixative must be adequate, approximately 20 times the volume of the tissue specimen except in Osmium tetroxide which is very expensive, TYPES/CLASSIFICATION OF FIXATIVES requiring only 5-10 times that of tissue volume for fixation. 4. For prolonged fixation (e.g. Museum preparation) volume of fixing fluid I. According to Composition should not be less than 50-100 times that of the tissue. A. SIMPLE FIXATIVES (made up of only 1 component substance) 5. Hollow organs (e.g. Stomach and intestines) should be packed with cotton soaked in fixative or completely opened before being immersed in adequate 1. Aldehydes (Formaldehyde, Glutaraldehyde) fixing solution. 2. Metallic Fixatives (Mercuric chloride, Chromic acid, Lead) 6. Air-filled lungs may float on fixative. To avoid this, the organ may be covered 3. Picric acid with several layers of gauze to maintain it under surface. Note: normal lungs 4. Acetic acid will float while diseased lungs will sink. 5. Acetone 7. Eyes should not be dissected before they are fixed. Formol-alcohol must be 6. Alcohol injected before immersing the organ in the fixative. 7. Osmium tetroxide 8. Heat 8. Frozen sections may lead to formation of ice crystal artifacts. 9. Water should not be used for glycogen-containing tissues because glycogen B. COMPOUND FIXATIVES (Made of up of 2 or more fixatives to obtain is soluble in water. optimal results) 10. The choice of fixatives and mode of processing vary depending upon the following factors: II. According to Action a. Need for immediate examination b. Tissue structure or component to be studied A. Microanatomical Fixatives: Permit the general microscopy study of c. Type of tissue to be processed tissue structures d. Staining technique to be applied e. Type of section to be made, whether serial or individual 1. 10% Formol saline ROUTINE FIXATIVES 2. 10% Neutral Buffered Formalin (NBF) 3. Heidenhain’s Susa 4. Formol sublimate (corrosive) I. ALDEHYDE FIXATIVES 5. Zenker’s solution 6. Zenker-formol (Helly’s) A. FORMALDEHYDE: FORMALIN 7. Bouin’s solution - Commercial formaldehyde/Formalin 8. Brasil’s solution - A saturated solution of formaldehyde gas in water, approximately 35-40% gas by weight (Gregorios: 37-40% weight in volume). B. Cytological Fixatives: Preserve specific parts and particular microscopic - A mixture of 10 mL formalin with 90 mL of water/saline is known as 10% elements of the cell itself formalin (most widely used fixative of all) - It is usually buffered to pH 7 with a phosphate buffer. 1. Nuclear Fixatives o Calcium acetate can also be used as buffer but is prone to calcium - Preserve nuclear structures (ex. chromosomes) deposit formation. - They usually contain Glacial acetic acid as their primary - Commercial formalin often becomes turbid, especially if stored in a very cold component due to its affinity for nuclear chromatin. place, because of the formation of paraformaldehyde (whitish precipitate) → - The pH is 4.6 o Treated with: Saturated alcoholic picric acid or alcoholic KOH (1% KOH in 80% alcohol) a. Helly’s - Prolonged fixation may cause bleaching, fat dispersal (add cadmium or b. Orth’s cobalt salts) and dissolution or loss of glycogen, biurates of sodium crystal c. Regaud’s and uric acid. d. Flemming’s fluid w/o Acetic acid (HAc) Reagent-grade formaldehyde contains 10% methanol as a preservative to retard e. Formalin w/ post-chroming decomposition to formic acid. Prevents the formation of Paraformaldehyde. - Why should formic acid formation be avoided? 3. Histochemical Fixatives o Acidity reduces the quality of routine cytologic staining - Preserve the chemical constituents of cells and tissues o It leaches out hemosiderin o It can lead to formation of formalin pigments a. 10% Formol saline o To prevent pigmentation, a buffer can be used, such as a handful of b. Absolute Ethyl alcohol calcium carbonate or magnesium carbonate added to 10-15% formalin c. Newcomer’s fluid (Both Nuclear and Histochemical fixative) o Renders formalin unsuitable for the critical fixation required in electron d. Acetone microscopy and has denaturing effects on proteins - Formalin pigments – are also formed due to overfixation. - Fixation Time: 24 hours REMOVAL OF FORMALIN PIGMENTS: B. GLUTARALDEHYDE 1. Lillie’s Method - It is made up of two formaldehyde residues, linked by three carbon chains. o Involves placing formaldehyde fixed specimens in acetone, 28% - It is utilized for Light microscopy. ammonia water and hydrogen peroxide. - Buffered Formaldehyde with secondary fixation in osmium tetroxide is o It uses 70% alcohol as a rinsing agent. satisfactory for Electron Microscopy. 2. Kardasewitsch’s Method o 2.5% - for small tissue fragments and needle biopsies for 2-4 hours at o A method of formaldehyde clearance involving 70% ethanol and 28% room temperature. ammonia water. o 4% - large tissue fragments less than 4 mm thick for 6-8 hours up to 24 3. Saturated Alcoholic Picric Acid hours. - Fixation time: ½-2 hours ROUTINE FORMALIN FIXATIVES - The most effective aldehyde for protein cross-linking 1. 10% Formol- - Traditionally, it is the most commonly used fixative in Saline pathology ADVANTAGES: - Best fixative for central nervous tissues and general post- 1. Has more stable effect in tissues giving firmer and better tissue sections – Fixation time: mortem tissues for histochemical examination. CNS 24 hours at 2. Preserves plasma proteins better 35ºC Advantages: 1. Penetrates tissues evenly and preserves microanatomic 3. Produces less tissue shrinkage 48 hours at 20- 4. Recommended for Histochemistry and electron microscopy 25ºC and cytologic details with minimum shrinkage and distortion 5. More pleasant with less irritation to the nose 2. Should be changed every 3 months so large specimens 6. It does not cause dermatitis can be fixed DISADVANTAGES: 3. Preserves enzymes and nucleoproteins, and 1. It is more expensive. demonstrates fats and lipids 2. It reduces PAS positivity of reactive mucin. 4. Ideal for silver impregnation * How can this be prevented? → Immersion of Glutaraldehyde fixed tissues in a mixture of Concentrated Disadvantages: glacial acetic acid and aniline oil. *Similar to formaldehyde with the following additions 1. Slow and requires 24 hours or longer II. METALLIC FIXATIVES 2. It shrinks during alcohol dehydration Remedy: secondary fixation A. MERCURIC CHLORIDE 3. Amyloid metachromatic action is reduced - The most common metallic fixative 4. Reduced staining when fixed with mercuric chloride - Frequently used in aqueous saturated solutions of 5-7%. - The routine fixative of choice for preservation of cell detail in tissue 2. Calcium - It is used to preserve phospholipids photography acetate - Replaced formol-saline as the most commonly used - Tissues fixed with mercury chloride containing compounds produce black formalin fixative in pathology because: precipitates except Heidenhain’s Susa (Lillie’s o Simple to prepare - Satisfactory for immunoperoxidase techniques but ultrastructural preservation Fixative) o Buffered to pH 7 by acetate is poor. - Permits brilliant metachromatic staining 3. 10% Neutral - Best general tissue fixative - Excellent for Trichrome staining Buffered - Best fixative for frozen sections - Recommended for renal tissues, fibrin, connective tissues, and muscles Formalin/ - Recommended for surgical, post mortem and research Disadvantages: Phosphate specimens. 1. It causes marked shrinkage of cells. buffered - Prevents precipitation of acid formalin pigments. o How can one counteract this? Formalin - Best fixative for iron-containing pigments and elastic fibers.  By addition of an acidic solution such as glacial acetic acid (pH 7) Disadvantages:  Through secondary fixation 1. It is longer to prepare; time-consuming 2. Has black granular deposits and is extremely corrosive to metals. Fixation time: 4-24 hours 2. PAS positivity to mucin is reduced REMOVAL OF BLACK DEPOSITS CAUSED BY MERCURIC CHLORIDE: 3. Myelin reactivity to Weigert’s Iron hematoxylin is reduced a. 0.5% iodine in 70% ethanol (alcoholic iodine) for 5-10 minutes + water + 5% 4. Inert to neutral fats and phospholipids. sodium thiosulfate (decolorized-5mins)-wash in running water. o Alcoholic iodine is used to remove excess mercury 4. Formol - Recommended for routine post-mortem tissues. o 5% sodium thiosulfate is used to remove excess iodine corrosive/ - It is excellent for silver reticulum methods. b. Addition of saturated iodine solution in 96% alcohol (alcoholic iodine) and Formol- - It fixes lipids, especially neutral fats and phospholipids. iodine decolorized with absolute alcohol in the subsequent stages of sublimate/ Disadvantages: dehydration. Formol- 1. Tissue sections should not be more than 1cm thick (low c. Dezenkerization Mercuric penetration) - Bring slides to water Chloride 2. Forms mercuric/black deposits - Immerse in Lugol’s Iodine (5 minutes) 3. No frozen sections are made - Wash in running water (5 minutes) Fixation time: 4. Inhibits determination of tissue decalcification - Immerse in 5% Na thiosulfate (5 minutes) 3-24 hours - Wash in running water (5 minutes) 5. Alcoholic - Formula: 95% ethyl alcohol saturated with picric acid, - Proceed with required water soluble stain. Formalin/ strong formaldehyde solution, Glacial acetic acid Gendre’s - It enhances immunoperoxidase studies for EM if post-fixed Mercuric Chloride Fixatives fixative with phenol formalin for 6 hours or more. 1. Zenker’s - Mercuric chloride + glacial acetic acid just before its - It fixes and dehydrates at the same time and fixes sputum fluid use. - For sputum since it coagulates mucus o To prevent turbidity and formation of dark cytology Fixation time: precipitates Disadvantages: 12-24 hours - Good general fixative for all kinds of tissue 1. It produces gross tissue hardening and causes partial RBC - Recommended for fixing small pieces of liver, spleen, lysis connective tissue fibers and nuclei 2. Preservation of iron pigments is poor - May act as mordant to make certain special staining 3. It causes little cross-linking of proteins possible (ex. Brilliant staining) - Contains glacial acetic acid which makes the solution 6. SPECIAL - Cajol’s formol ammonium bromide - good fixative for unstable FORMALIN nervous tissue (astrocytes). - Recommended for Trichrome staining FIXATIVES: - Fixatives for acid mucopolysaccharides. 2. Zenker- - Formula: Mercuric chloride, potassium dichromate, - Baker’s formol calcium - used for the preservation of formol/ sodium sulfate, 40% formaldehyde, and distilled water lipids since most formalin fixatives are inert to lipids. Helly’s - Mercuric chloride + 40% formaldehyde just before its Solution use. - It is an excellent microanatomic fixative for pituitary Fixation time: gland, bone marrow and blood-containing organs 12-24 hours such as the liver and spleen. - Better nuclear fixation and staining than Zenker’s  - It preserves cytoplasmic granules better than 7. The use of this formaldehyde 16. Which of the following mercuric Zenker’s fixative can lead to formation chloride fixatives is commonly “Bloody Helly” - Disadvantage: Brown pigments are produced if of black precipitates used for bone marrow biopsies? tissues are allowed to stay in the fixative for more than a. Formol corrosive a. Helly’s 24 hours due to RBC lysis. b. 10% formol-saline b. Heidenhain’s Susa - Remedy: immerse the tissue in alcoholic picric acid c. Calcium acetate c. Zenker’s or sodium hydroxide formalin d. B-5 3. Heidenhain’s - Recommended for tumor biopsies especially of the d. Alcoholic formalin Susa skin 17. Tissues fixed with mercuric solution - It is an excellent Cytologic fixative 8. Best fixative for iron- chloride containing compounds - Produces minimum shrinkage and hardening of containing pigments and produce black precipitates Fixation time: tissues due to the counter-balance of the swelling elastic fibers except: 3-12 hours effects of acid (trichloroacetic acid) and the shrinkage a. 10% Formol saline a. Schauddin’s effect of a metal (mercury). b. 10% BNF b. Helly’s - Counterbalance effect c. Calcium acetate c. Heidenhain’s Susa o Su (sublimat) = metal (mercury) → cell shrinkage formalin d. Zenker’s o Sa (saure) = acid (trichloroacetic acid) → cell d. Formol corrosive swelling 4. Schaudinn’s - A solution of mercuric chloride, sodium chloride, 9. Which of the following Solution/ alcohol, and glacial acetic acid fixatives is considered as the Sublimated - Used on wet smears for cytologic examinations. best general tissue fixative alcohol and also used for frozen 5. B-5 Fixative - Composed of mercuric chloride and sodium acetate. sections? - Commonly used for bone marrow biopsies. a. Gendre’s Fixation time: - Just prior to use, add 1 mL of 40% formaldehyde to 10 b. 10% Formol-saline 1 ½ to 2 hours mL of B5 c. 10% BNF (Rapid fixation) d. Formol sublimate 6. Carnoy- Lebrun 7. Ohlmacher Answer key: C, B, A, D, C, B, A, B, C, C, C, B, C, B, B, D, C REVIEW!!! B. CHROMATE 1. The concentration of 10. Which of the following fixative is - A class of fixatives which are strong oxidizing agents used for precipitating formaldehyde used in used in sputum cytology? proteins and preserving carbohydrates. fixation is approximately __ a. Calcium acetate formalin - Recommended for Chromaffin tissues, Adrenal medulla, Mitochondria. weight in volume b. Formol sublimate a. 20% c. Alcoholic formalin CHROMATE FIXATIVES (“CROP”) b. 30% d. 10% BNF 1. Chromic acid - Used in 1-2%, used as a constituent of a compound c. 40% fixative. d. 50% 11. A formalin fixative used for - It precipitates all proteins and adequately preserves fixation of lipids since most carbohydrates. 2. Which of the following is not formalin fixatives are inert to - Formaldehyde must be added to chrome-containing a nuclear fixative? lipids: tissues before use to prevent counteracting effects a. Bouin’s a. 10% BNF and consequent decomposition of solution upon b. Orth’s b. 10% Formol-saline prolonged standing. c. Heidenhain’s Susa c. Formol calcium 2. Regaud’s Fluid/ - Recommended for demonstration of Chromatin, d. Carnoy’s d. Alcoholic formalin Moeller’s fluid Mitochondria, Mitotic figures, Golgi bodies, RBC’s and colloid-containing tissues 3. Which of the following 12. A fixative that is made up of two - Penetrates the tissues well fixatives is classified as both formaldehyde residues linked - It hardens tissue better and more rapidly than Orth’s nuclear and histochemical by three carbon chains: fluid. fixative? a. Formaldehyde - Low penetration and tends to produce precipitates a. Newcomer’s b. Glutaraldehyde of sub-oxides b. Carnoy’s c. Mercuric chloride - Prolonged fixation blackens the tissue pigments. c. Regaud’s d. Chromate - Remedy: wash in running tap water before d. Helly’s dehydration 13. Frequently used concentration 3. Orth’s Fluid - Recommended for study of early degenerative 4. Which of the following of mercuric chloride fixatives processes and tissue necrosis fixatives is classified as a. 35-40% Fixation time: - Demonstrates Rickettsia and other bacteria cytoplasmic fixative? b. 10% 36-72 hours - Preserves Myelin better than buffered formalin a. 10% Formol-saline c. 5-7% - Disadvantages: Same as that of Regaud’s b. Flemming’s d. 3% 4. Potassium - Preserves mitochondria (pH 4.5-5.2) c. Acetone dichromate - Fixes lipids d. Helly’s 14. The routine fixative of choice for preservation of cell detail in - Used in 3% aqueous solution 5. Paraformaldehyde deposits tissue photography: - It fixes but does not precipitate cytoplasmic are formed when formalin is a. Glutaraldehyde structures. stored for a long time in __ b. Zenker’s fluid temperature, but this can be c. Formol calcium C. LEAD FIXATIVES remedied by using __ d. Gendre’s 1. Lillie’s alcoholic lead nitrate formalin a. Warm; acid alcohol 2. Lead subacetate (4% aqueous solution) b. Cold; acid alcohol 15. An excellent microanatomic c. Cold; methanol fixative for pituitary gland, bone Advantages: d. Warm; methanol marrow, and blood-containing 1. It is recommended for acid mucopolysaccharides organs: 2. It fixes connective tissue mucin 6. Best fixative for nervous a. Zenker’s Disadvantage: tissue and ideal for silver b. Helly’s 1. It takes up CO2 to form insoluble lead carbonate especially on prolonged impregnation c. Heidenhain’s Susa standing. a. 10% BNF d. Schaudinn’s o This may be removed by filtration or by adding acetic acid drop by drop to b. 10% Formol-saline lower the pH and dissolve the residue. c. Formol corrosive d. Gendre’s  III. Picric Acid Fixatives (“PBB”) - It preserves Nissl granules (Tigroid bodies) 1. Excellent for glycogen demonstration and very suitable for curettings (small tissue 2. Normally used in strong saturated aqueous solution fragments). 3. It dyes the tissues yellow, thus preventing the tissue fragments from being - After fixation, tissues can be transferred overlooked. On the other hand, this hinders proper staining. directly to absolute alcohol, or an alcohol- o Can be used for fragmentary biopsies chloroform mixture (1:1) o What must be done to remove this yellow color? - It leads to polarization unless very cold  Add 50-70% ethanol temperatures (-70oC) are used  Acid dye/lithium carbonate 5. Alcoholic Formalin/ - Better preserves glycogen  70% ethanol → 5% sodium thiosulfate → Wash in running tap water Gendre’s - Also a formalin fixative 4. It is suitable for aniline stains and Trichrome method - Capable of coagulating mucus and is used as a fixative for sputum cytology - The chemical name for the general picric acid fixatives: 2,4, 6 - trinitrophenol 6. Newcomer’s Fluid - Formula: Isopropyl alcohol, propionic acid, - Picric acid is highly explosive when dry, and therefore must be kept moist with petroleum ether, acetone, and dioxane distilled water or saturated alcohol at 0.5 to 1% concentration during storage. Fixation time: - For fixing mucopolysaccharides and nuclear - Picrates are soluble in water and should be transferred directly from fixative 12-18 hours @ 3ºC proteins to 70% alcohol - It produces better reaction in Feulgen stain than Carnoy’s fluid 1. Bouin’s - Fixation of embryos and pituitary biopsies - It is both a nuclear and histochemical fixative. solution - It is an excellent fixative for preserving soft and delicate structures (endometrial curetting’s) VI. Osmium Tetroxide/Osmic Acid (“OFF”) Fixation time: - Produces minimal distortion (Picric - shrinking, - It is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form 6-24 hours Glacial HAc - swelling) strong oxidizing solution. - Yellow stain is useful in fragmentary biopsies - Adequately fixes materials for ultrathin sectioning in electron microscopy, - Preferred fixative for Masson’s trichrome staining for since it rapidly fixes small pieces of tissues and aids in their staining collagen, elastic or connective tissue - Also used for the preservation of glycogen Disadvantage: - Not suitable for fixing kidney structures, lipids, and - Inhibits Hematoxylin and makes counterstaining difficult mucus - Very expensive (less amount is required for fixation; 5-10x volume of fixative - It destroys cytoplasmic structures (ex. Mitochondria) used) 2. Brasil’s - Best routine fixative for glycogen - Formation of artifact pigments/black precipitate Alcoholic - It is better and less messy than Bouin’s solution - Prolonged exposure to acid vapors causes eye irritation(conjunctivitis) or Picroformol - Overnight tissue fixation by automatic processing black osmic oxide deposition in the cornea (blindness) Fixative technique may utilize 3-4 changes of Brasil’s fixative at ½ to 2 hours each, succeeded directly by absolute 1. Flemming’s - Formula: 1% Aqueous chromic acid, 2% aqueous alcohol. Solution osmium tetroxide, glacial acetic acid - The most commonly used Chrome-Osmium acetic IV. Glacial Acetic Acid Fixation time: acid, recommended for nuclear preparation of such 24-48 hours sections. - Excellent for nuclear structures such as 1. It solidifies at 17ºC (New book: 16oC) chromosomes 2. Fixes and precipitates nucleoproteins - Permanently fixes fats/lipids 3. It precipitates chromosomes and chromatin materials. 4. Causes tissues to swell specially those containing collagen. Disadvantages: 1. Very expensive (less amount is required for fixation) - Disadvantage: It is contraindicated for cytoplasmic fixation because it 2. Formation of artifact pigments/black precipitate destroys mitochondria and golgi elements. Remedy: wash in running tap water for 24 hours before dehydration V. Alcohol Fixatives 3. Prolonged exposure to acid vapors causes eye - Denatures and precipitates proteins irritation (conjunctivitis) or black osmic oxide - 70-100% concentrations are used; Less concentrations results to red blood deposition in cornea (blindness) cell lysis/hemolysis due to hypotonicity 4. Extremely volatile 5. It depresses the staining power of Ehrlich Advantages: hematoxylin 1. May be used both as a fixative and dehydrating agent 6. Flemming’s - Recommended for cytoplasmic structures such as 2. Excellent for glycogen preservation Solution mitochondria Disadvantages: without Acetic - The removal of acetic acid from the formula serves to 1. Causes RBC hemolysis and dissolves fats and lipids Acid improve the cytoplasmic detail of the cell. 2. Tissue shrinks on long usage 3. Polarization (major disadvantage); glycogenic granules moves towards the Fixation time: ends or poles of cells 24-48 hours General Rule: - Alcohol-containing fixatives are contraindicated when lipids are to be studied. VII. Trichloroacetic acid (TCA) 1. Methyl Alcohol - Used in Wright’s stain as a diluent 1. Sometimes incorporated into compound fixatives (Ex. Heidenhain’s Susa) 100% (CH3OH/ - Excellent for fixing wet and dry smears, blood 2. It precipitates proteins Methanol) smears and bone marrow tissues 3. Causes marked swelling effect on tissues 4. Can be used as a weak decalcifying agent. 2. Isopropyl Alcohol - It is used for fixing touch preparations 5. Has softening effect on dense fibrous tissue 95% (Isopropanol) (impression smears), although some are air 6. Suitable only for small pieces of tissues or bone because of its poor dried and not fixed, for certain procedures penetration such as Wright-Giemsa staining. 3. Ethyl Alcohol - Is used in 70-100% concentrations. VIII. Acetone (C2H5OH/ Ethanol) - Lower concentrations (70-80%) will cause RBC lysis and inadequate WBC preservation 1. It is used at ice cold temperature from -50C to 40C (New book: 0-4oC) Fixation time: - It fixes blood, tissue films and smears 2. Recommended for the study of water diffusible enzymes especially lipases 18-24 hours - Used for histochemistry especially for enzyme and phosphatases studies 3. Used in fixing brain tissues for diagnosis of rabies (Negri bodies). - Can be both used as a simple and compound 4. Used in freeze substitution techniques as a solvent for certain metallic salts fixative. 5. Evaporates rapidly 4. Carnoy’s Fluid - The most rapid tissue fixative - Recommended for fixing chromosome, lymph IX. Heat Fixation Fixation time: glands and urgent biopsies - This procedure involves thermal coagulation of tissue proteins for rapid 1-3 hrs. diagnosis, usually employed for frozen tissue sections and preparations of - It is used to fix brain tissue for rabies bacteriologic smears. diagnosis.  QUESTIONS! 4. Presence of - Tissues containing large amount of blood (e.g. 1. What are the two tissue constituents usually dissolved by heat fixation? blood Blood vessels and spleen) should be flushed out a. Glycogen with saline (arterial cannulization) before fixing. b. Starch 5. Cold - Inactivates enzymes 2. The 2 most commonly used fixatives for general use: temperature a. 10% formol saline (10% formalin) 6. Hot temperature - Denatures enzymes b. Zenker’s fluid 3. Best general tissue fixative B. Enhanced by: a. 10% Neutral Buffered Formalin 1. Size and - Smaller and thinner tissues require less fixative thickness and shorter fixation time - Cryostat or frozen sections should be used for 2. Agitation - Fixation is accelerated when automatic or demonstrating lipids in tissues mechanical tissue processing is used. - Mercuric chloride and potassium dichromate are used - Autotechnicon in cryostat sections 3. Moderate heat - Accelerates fixation but hastens autolytic - Aldehydes changes and enzyme destruction Lipid fixation - Baker’s formol calcium - Imidazole Osmium Tetroxide – improved IMPORTANT NOTES ON IMPROPER FIXATION ultrastructural demonstration of lipids 1. Failure to arrest early cellular Due to the failure to fix immediately by - Cholesterol may be fixed with Digitonin for autolysis which one first allowed the tissue to dry ultrastructural demonstration before fixing or insufficient fixative - Alcoholic fixatives are generally recommended for 2. Too brittle and too hard blocks Due to prolonged fixation Carbohydrate glycogen demonstration 3. Soft and feather-like tissues Due to incomplete fixation fixation - Alcoholic formaldehyde is a better fixative in human 4. Removal of fixative soluble skin compared with BNF substances Wrong choice of fixative Protein fixation/ - Neutral buffered formaldehyde or formaldehyde vapor 5. Enzyme inactivation and loss Amino acid are the most commonly used 6. Presence of artefact pigments Incomplete washing of fixative histochemistry on sections - Most useful fixatives are alcohol based such as 7. Shrinkage and swelling of cells Due to overfixation Glycogen Rossman’s fluid or Cold Absolute alcohol in tissue blocks fixation - Section is coated with Celloidin for better retention of Incomplete fixation → Improper and incomplete clearing and impregnation → glycogen incorrect sectioning and staining of pathologic slides MIXTURE OF FIXATIVES REVIEW!!! - Useful for Electron Microscopy (Mnemonic: KOGA) 1. Which fixative is 9. Which of the following fixatives o Karnovsky’s paraformaldehyde-glutaraldehyde solution recommended for the may cause blindness through its o Acrolein – mixture with glutaraldehyde or formaldehyde; simplest demonstration of Chromaffin deposition in the cornea if tissues, adrenal medulla, and exposure is prolonged? unsaturated aldehyde mitochondria? a. 10% Formalin o Other fixatives for Electron Microscopy: a. Mercuric chloride b. Osmic acid Glutaraldehyde b. Chromate c. Chromic acid Osmium tetroxide c. Picric acid d. Heidenhain’s Susa COMPATIBILITY AND INCOMPATBILITY d. Lead 10. Which of the following fixatives is - Few fixatives permit the use of all stains. 2. Which fixative is used in fixing brain tissues for - Some fixatives may act as a mordant for one group of dyes and an inhibitor recommended for the rabies diagnosis? for another set of stains. demonstration of acid SECONDARY FIXATION mucopolysaccharides? I. Acetone - The process of placing an already fixed tissue in a second fixative in order to: a. Picric acid II. Newcomer’s b. Mercuric chloride III. Carnoy’s a. To facilitate and improve the demonstration of particular substances c. Lead IV. Trichloroacetic acid b. To make special staining techniques possible (secondary fixative acting d. Chromate as mordant) a. I and II c. To ensure further and complete hardening and preservation of tissues 3. The recommended fixative for b. II and IV - Not recommended when primary fixation for specific observations is possible demonstration of c. I and III in the first instance mitochondria, chromatin, golgi d. II and III - This is done before dehydration and on deparaffinized sections before staining bodies, and mitotic figures: - 10% formol-saline/10% formalin: commonly used primary fixatives a. Regaud’s 11. Recommended fixative for b. Potassium dichromate cytoplasmic structures such as POST-CHROMATIZATION c. Chromic acid mitochondria: - AKA: Post-chroming or Post-mordanting d. Orth’s a. Bouin’s - A secondary fixation whereby a primary fixed tissue is placed in aqueous b. Brasil’s solution of 2.5 – 3% potassium dichromate for 24 hours, to act as mordant for 4. The recommended fixative for c. Flemming’s better staining effects and to aid in cytologic preservation of tissues. the study of early degenerative d. Flemming’s without acetic processes and tissue necrosis acid WASHING OUT and for demonstration of - The process of removing excess fixative from the tissue after fixation in order Rickettsia: 12. Fixatives used for electron to improve staining and remove artifacts from the tissue. a. Chromic acid microscopy Solutions that may be used: b. Moeller’s c. Orth’s I. Acrolein 1. Tap water – used to remove: d. Potassium dichromate II. Karnovsky’s paraformaldehyde a. Excess Chromates from tissues fixed in Helly’s, Zenker’s and Flemming’s glutaraldehyde mixture solutions 5. The recommended fixative for III. Osmium tetroxide b. Excess Formalin fixation of embryos and o 10% formalin is extracted more rapidly in 70% alcohol than in water pituitary biopsies: a. I and III c. Excess Osmic acid/Osmium tetroxide a. Bouin’s b. I and II 2. 50-70 % alcohol – used to wash out excess amount of picric acid b. Glacial acetic acid c. II and III 3. Alcoholic iodine – used to remove excessive mercury c. Brasil’s d. I, II, and III d. Lead subacetate SPECIAL FACTORS AFFECTING FIXATION 13. Which of the following is used to 6. Picric acid fixative: remove Osmium tetroxide black A. Retarded by: precipitate? 1. Size and - Larger and thicker tissues require more fixative I. Flemming’s a. Acid alcohol thickness and longer fixation time II. Bouin’s b. 50-70% alcohol 2. Presence of - Prevents complete penetration of fixative, hence, III. Flemming’s w/o acetic acid c. Alcoholic iodine mucus tissues that contain mucus are fixed slowly and IV. Brasil’s d. Cold water poorly - Excess mucus may be washed away with NSS. a. I and II b. III and IV 3. Presence of fats - Fatty tissues should be cut in thin sections and c. II and IV should be fixed longer. d. I and III  7. Which of the following is used 14. Which of the following factors can 2. Hydrochloric - Inferior compared to nitric acid (slower action and as a diluent for the Wright’s enhance fixation? acid/ greater distortion of tissue) stain and is excellent for fixing a. Large and thick tissues Muriatic acid - Good nuclear staining dry and wet smears? b. Presence of fats, mucus, - Recommended for surface decalcification of a. Methanol and blood tissue blocks if used in 1% solution with 70% b. Isopropanol c. Heat and agitation alcohol c. Glacial acetic acid d. Hot or cold temperature d. Picric acid Von Ebner’s fluid 15. Which of the following is the Formula: 36% Sat. Aq. NaCl, Conc. HCl, Distilled water 8. The most rapid fixative and is - Recommended for teeth and small pieces of possible cause of too hard or brittle specially recommended for bones tissue blocks? fixation of lymph glands, 3. Formic acid - Moderate-acting decalcifying agent chromosomes, and urgent a. Prolonged fixation b. Incomplete fixation - Recommended for routine decalcification of post- biopsies: a. Ethanol c. Wrong choice of fixative mortem research tissues b. Newcomer’s d. Overfixation Decalcification time: 2-7 days Disadvantage: Not suitable for urgent examination c. Carnoy’s a. Aqueous Formic Acid d. Gendre’s (Formic acid + 10% Formol saline) o Recommended for small pieces of bones and Answer key: B, C, A, C, A, C, A, C, B, C, D, D, D, C, A teeth o Suitable for most routine surgical specimen DECALCIFICATION and immunohistochemical staining. - Procedure whereby calcium or lime salts are removed from tissues (most b. Formic Acid – Sodium Citrate especially bones and teeth) (20% Aq. Sodium citrate + 45% Formic acid) - It is a form of ionization o Recommended for autopsy materials, bone - Done after fixation and before impregnation (before dehydration) marrow, cartilage and tissues studied for - Calcified tissues are harder to cut research purposes Recommended temperature: Room temperature (18-30oC) 4. Trichloroacetic - Does not require washing out Ideal time required for decalcifying tissue: 24-48 hours acid - Not recommended for urgent examinations (very Volume: 20x the volume of the tissue slow acting) 5. Sulfurous acid - Weakest decalcifying agent, suitable only for FACTORS INFLUENCING RATE OF DECALCIFICATION minute pieces of bone a. Structure = heavily calcified tissues take longer to decalcify 6. Chromic acid - Used as a fixative and decalcifying agent b. Temperature = heat hastens decalcification but increases the damaging effect - Caution: It is an environmental toxin, highly of acids on tissues corrosive to skin and mucous membrane and c. Concentration and Volume carcinogenic 7. Citric acid – - Does not produce cell or tissue distortion  too rapid removal of CA+2 salts may produce complete digestion of the tissue Citrate buffer specimen and poor staining capacity of the cell II. Chelating Agents DECALCIFYING AGENTS: - Substances which combine with calcium ions and other salts (iron and 1. Acids magnesium) to form weakly dissociated complexes and facilitate removal of 2. Chelating Agents Calcium salt 3. Ion Exchange Resins - The most common chelating agent: EDTA (Used as an anticoagulant and 4. Electrical Ionization / Electrophoresis water softener) o Commercial name: Versene, Sequestrene For routine purposes, only acids are recommended: EDTA + Ca2+ Insoluble non-ionized complex - Nitric acid - almost 2x as fast as formic acid (Removes Ca) - Formic acid - provides better tissue preservation and staining - The volume of acid decalcifying solution used should be at least 1 ounce per III. Ion-Exchange Resin gram of tissue; should be changed once or twice a day until decalcification is - Ion exchange resin (ammonium form of polystyrene resin) hastens completed decalcification - Removes calcium ions from formic acid, thereby increasing solubility from the I. Acid Decalcifying Agents tissue. 1. Nitric acid - The most common and fastest decalcifying agent used - Not recommended for fluids containing mineral acids such as nitric or so far hydrochloric acids. Recommended - Very rapid decalcifying agent, producing minimal IV. Electrical ionization (Electrophoresis) concentrations: distortion - Process whereby positively charged Calcium ions are attracted to a negative 5-10% - Recommended for routine purposes electrode (cathode) and subsequently removed from the decalcifying solution. - It undergoes spontaneous yellow discoloration owing to - The time required for decalcification is thereby shortened due to the heat and formation of nitrous acid (HNO2) electrolytic reaction produced in this process. o This accelerates decalcification - The principle applied is similar to that of chelating agents; with the main difference o Also stains and damages the tissues that this process utilizes electricity and is dependent upon a supply of direct current to remove calcium deposits. a. 10% aqueous - Rapid in action - Solutions used: Nitric Acid - Recommended for urgent biopsies and for needle 1. 88% Formic acid and small biopsy specimen 2. Concentrated HCl Decalcification time: - Used for large or heavily mineralized cortical bone 3. Distilled water 12-24 hours specimen b. Formol-Nitric - Less tissue destruction than 10% aqueous nitric 3 Ways to Measure the Extent of Decalcification acid 1. Physical or - By touching or bending the tissue with the fingers to acid Mechanical determine the consistency of tissues - For urgent biopsies with good nuclear staining. Method - By pricking the tissue with a fine needle/probe Decalcification time: - Yellow color imparted by nitrous acid is removed through neutralization with 5% sodium sulfate and 2. X-ray/ - Very expensive, although it is the most ideal, most 1-3 days Radiological sensitive and most reliable method running tap water for 12 hours method - Good but not always convenient o Yellow color formation is prevented by addition - Not recommended for mercuric chloride-fixed tissues of 0.1% urea to pure concentrated nitric acid (radio-opacity will interfere with the plate interpretation) - This should be used inside a fume hood 3. Chemical - Simple, reliable and convenient method recommended for c. Perenyi’s fluid - It is recommended for routine purposes. method/ routine purposes (still favored) - It decalcifies and softens tissues at the same time Calcium - Decalcifying fluid is changed every 24-48 hrs. Decalcification time: - Maceration is avoided due to the presence of Oxalate test 2-7 days chromic acid and alcohol Solutions used: d. Phloroglucin - Most rapid decalcifying agent, recommended for 1. Concentrated Ammonium Hydroxide Nitric Acid urgent works 2. Saturated Aqueous Ammonium Oxalate - Has poor nuclear staining - Detection of calcium in acid solutions by precipitation of Decalcification time: - Yellow color formation insoluble calcium hydroxide or calcium oxalate 12-24 hours - Presence of cloudiness indicates the presence of Ca2+ (incomplete decalcification) TISSUE SOFTENERS Commonly Used Dehydrating Agents - Unduly hard tissues which are reliable to damage the microtome knives may 1. Alcohol - Dehydration is best accomplished by the use of increasing require tissue softener, aside from decalcification. grades of alcohols, beginning with 70% 1. Perenyi’s fluid – act both as a decalcifying agent and tissue softener (70%→95%→100%→100%→100%) 2. 4% phenol (1-3 days) - Transfer of tissue directly from formalin to higher grades 3. Molliflex (tissues may appear swollen and soapy) of alcohol (85% - 95%) is risky, since it could lead to 4. 2% Hydrochloric acid distortion of tissues (shrinkage and hardening of tissues). 5. 1% HCl in 70% alcohol - Concentrated solutions (above 80%)