Tissue Processing: Fixation PDF 2021-2022

TISSUE PROCESSING: FIXATION LEC 2021-2022 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES 2nd Semester Instructor: Prof. Ki...

TISSUE PROCESSING: FIXATION LEC 2021-2022 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES 2nd Semester Instructor: Prof. Kimberly Pulga, RMT, MPH WEEK 8 HPCT311 LEC Date: March 3, 2022 TOPIC OUTLINE A. Fixation * When you receive a sample in the laboratory it's either the a. Principles sample is already in fixative or the sample specimen is fresh b. Objectives from the operating room. So, if the sample specimen is c. Basic mechanisms of action fresh kailangan malagay na sya agad sa fixatives, if the d. Benefits request is preserved tissue. Kasi kung hindi naman fresh e. Practical considerations f. Main factors involved tissue exam no need (to place it in fixative). g. Effects * *Kapag nareceive na yung specimen it should be placed h. Characteristics of good fixatives immediately into the fixative to prevent the decomposition. i. Types of fixatives i. According to composition Fixation is done asap: ii. According to action o After removal of tissue in case of surgery j. Secondary fixation o or after death in case of autopsy k. Washing out l. Difficulties caused by improper fixation Dehydration, clearing, impregnation, embedding, m. Artifacts microtomy and staining affected if inadequate fixation n. Lipid fixation = difficult diagnosis o. Carbohydrate fixation * There will be difficulty in the diagnosis if fixation p. Protein fixation will not be properly done. Inadequate fixation can affect the dehydration, clearing, impregnation, Learning Objectives: embedding, microtomy and staining. Almost all the At the end of the session, the students are expected to be steps are affected if inadequate or improper able to: fixation. Identify which of the fixative will be used for a given sample specimen. Objectives: Employ the significance of using specific fixative for a 1. Preserve the tissue specific tissue specimen. by stopping all cellular activities: Analyze the importance of tissue fixation immediately If left exposed to air for prolonged period: dry out after removing it from human body If left in hypotonic solution: swell If left in hypertonic solution: shrink Fixation * These three can lead to distortion to the morphologic First and most critical step in histotechnology appearance of the tissue. 2 important goals: o Preserve the morphology and chemical integrity of the 2. Prevent breakdown of cellular elements cell and tissue in a life-like manner removal of tissue from body leads to deprivation to o Harden and protect the tissue from trauma of further nutrients, oxygen = cell injury/ death. (Worst is the cell handling death) * If the tissue did not hardened properly or the fixation * To prevent these from happening kailangan ma- was not successfully performed. Then, it will be difficult preserve na natin, malagay na natin agad sa fixative. for us to perform the proceeding steps in tissue * We will prevent the autolysis and putrefaction. processing. Autolysis: inactivating the lysosomal enzymes * The quality of the fixed tissue is equal to the quality of Putrefaction: prevent the microorganism the tissue slide that will be made (output). colonization. * If the fixation is improperly performed then the quality of the picture under the microscope of the tissue slide 3. To coagulate or precipitate protoplasmic substances is also affected. By that it will be difficult for us to view Methods: and diagnose, and also narrow down the disease a) Physical present in the patient. * Tissue is harden to easily cut into section, or ease in heating, microwaving, cryo-preservation section cutting/microtomy * Take note here in physical this is NOT FOR TISSUES and it can only be done for microorganisms. The physical methods are for Principles: microorganisms. Terminate any ongoing biochemical reaction b) Chemical * This is only possible for the fresh tissue examination, Immersion fixation but if it's not then you don’t need to terminate it. Perfusion fixation Prevents autolysis and putrefaction Vapor-fix-freeze-dried fixation: use of * Prevent the lysosomal enzymes and microbial paraformaldehyde or Osmium tetroxide (also colonization of the microorganisms or the decay of the called as Osmic acid) tissue sample. Increase mechanical strength. Basic Mechanisms of action for fixative/fixation: Stabilize fine structure making structure resistant to 1. Additive fixation dissolution by water and other liquids. Tissue is incorporated and becomes part of the Inhibit growth of bacteria and molds (microorganisms). tissue * The fixative becomes part of the tissue. The M. LIONG & J. LABASTIDA– TRANSCRIBERS  fixative is incorporated to the tissue it becomes part of the tissue. * It will happen because of the formation of the Sufficient time for penetration*of fixative: rate varies per cross-links or complexes. fixative Giving stability to protein by cross-linking * Formalin (commonly used): 1mm/hr macromolecules Prolonged fixation: difficult to reverse *the step* & loss of * These cross-links or complexes that were formed immunohistochemical antigenicity will give stability to the tissue proteins. In additive Small tissue blocks allow adequate permeation fixation, e fixative becomes part of the tissue and * Tissue Length: 2.5 cm; Width: 2 cm; Thickness: 0.5 cm it stabilizes the tissue proteins because of the or 5 mm formation of the cross-links or complexes. Correct orientation as tissue hardens upon fixation Examples of additive fixatives are: Formalin, * So, it's not only in embedding that the tissue needs to Mercury, and Osmium tetroxide (or Osmic acid) be oriented but also in fixation. * You need to orient it in a tissue cassette or before 2. Non-additive fixation placing it into fixatives. Not incorporated into the tissue * It should be properly oriented since it can also affect * The fixative will not become part of the tissue. The the quality of tissue slide. fixative will not be incorporated into the tissue. But the tissue proteins here can still be stabilized by Main Factors Involved: removing the bound water. 1. Volume Giving stability by removing bound water attached to H-bonds within protein molecules 10-20x greater than that of tissue sample * The fixative stabilizes the tissue by removing the * Because the fixatives will be reduced once it will bound water. Removal of water will result to the bind to the tissue therefore you will need large formation of new cross-links within the tissue so amount of fixative therefore the tissue proteins will still be stabilized. Principle: depletion of fixative molecules upon binding * When using non-additive fixative there will be to tissue alterations in the tissue composition. Avoid exhaustion of fixative by: changing solutions at Removal of water: forms new cross-links within interval tissue components Examples: Alcohols or alcoholic fixatives 2. pH * Fixation is best carried out in neutral pH Benefits: Neutral pH: 6-8 Hypoxia in tissue lowers pH; need for buffering activity 1. Thin sections in fixative * It allows us to have thin sections. During the section cutting we can adjust the thickness and it Acidity → heme-formalin complex→ black polarizable will be possible for us to have thinner sections if deposits in tissue we properly performed the fixation in the tissue. * For example, if you use formalin and mixed it with 2. Prevents autolysis water the solution will become acidic. Then it can 3. Prevent putrefaction form a heme-formalin complex, it will form formalin * Except for Prions pigments a black polarizable deposit in tissue. 4. Improves cell avidity to stains When you view it under microscope you can see several black dots and this occurrence can affect the diagnosis. Practical considerations: * To prevent it from happening we need to add a * First is the speed, once you received the specimen and you buffer such as sodium hydrophosphate/ Sodium know that request is for preserved tissue examination then dihydrophosphate/ sodium dihydrogen phosphate. you need to place into the fixatives. And you will now have 10% neutral buffered Specimens should be transferred to fixative within 1 hour formalin once you added the sodium dihydrogen after surgery phosphate. Fixative to tissue ratio: 10:1 or 20:1 (maximum Example: Phosphate, Bicarbonate, Cacodylate, and effectiveness) Veronal * 20:1 will give maximum effectiveness that is why most Formalin (commercial) contains phosphate at pH 7 of the time it is the used ratio Remove anatomical barriers 3. Temperature * If you receive the specimen you have to consider Regular tissue processing: 40 C removing the anatomical barriers: Electron microscopy and histochemistry: 0 to 4 C o Fascia, bone, feces, thick and large tissue: Fixation: Room temperature sectioned o Lungs: infiltrated Refrigeration o GIT: cleaned o Used to slow down tissue decomposition when it needs to be photographed or can’t be Pinning of tissue to corkboard or inserting paper/ gauze fixed immediately. “wick” into tubular structures * If you will not be fixing the specimen Contaminated fixatives (feces, blood, bile or any body immediately you can put it into the refrigerator fluids): replaced it immediately or ref temp. Because refrigeration can slow * If you see small drop of blood in fixative then you need down the tissue decomposition. to replace it. Brain cells: decomposed very quickly * Contaminated fixative cannot be reuse since it can affect the quality of tissue slide and the diagnosis. M. LIONG & J. LABASTIDA– TRANSCRIBERS  staining * If the request is preserved tissue as much as o Formaldehyde: intensifies stain possible you need to preserve it immediately o Osmium tetroxide: inhibits hematoxylin staining or placed it in fixative immediately. * If you are going to use hematoxylin stain, we Bone Marrow: undergo mitosis up to 30 mins after cannot use osmium tetroxide or osmic acid as death when refrigerated fixative because it will inhibit the hematoxylin * Within 30 minutes makapagdecide na if it stain. should be placed in fixative or not. Characteristics of a Good Fixative: Increase temperature = increase rate of fixation Cheap * If you want a rapid fixation then set the * It should be cheap since we will be using the temperature to 60C fixative multiple times * It needs to be cheap because we are using or we 4. Thickness of section are needing large amount of fixative in the Electron microscopy: 1-2 mm laboratory Light microscopy: 2 cm x 0.4 cm and the thickness * Fixatives are used in large amount. should be no more than or less than 4 mm * You need around 10-20x fixative for each sample * Usually used thickness in laboratory is not more that will be processed than 4 mm still depends on the laboratory. * Example: * If lung tissue specimen is received the Sample: 25mL thickness can be 1-2 cm Fixative: 20x more = you need 50 mL for one In general: most tissue can be cut and trimmed without sample only prior fixation * What if you have 20 samples then you need a liter * or tissue is placed first in fixative before cutting it of a fixatives. Therefore, the fixative should be Brain tissue: placed in 10% formalin first for 2-3 weeks cheap and affordable but it should not compromise before cutting and grossing the quality of the fixative syempre dapat maganda Rate of penetration for aldehyde: 2-3 mm/hr kahit mura lang kasi malulugi ang hospital. Solid material (liver): no more than 10-15 mm * It is not a cost efficient if you will buy an expensive fixative because you will be using a large amount 5. Osmolality of it. Best: slightly hypertonic solutions 400-450 mOsm Stable * It needs to be stable because it may affect Hypertonic: shrinking of cells subsequent steps in tissue processing Hypotonic and isotonic: swell and bursting of tissues * To maintain the cellular morphology Agent (best): Normal Phosphate Buffered Saline * It should be stable because it will preserve our tissue and it may affect the other steps in 6. Concentration processing Should be adjusted to its lowest level possible Safe to handle Formaldehyde: 10% Kill cell quickly Glutaraldehyde: 3% (immuno-electron microscopy: * It should not be cancerous or carcinogenic 0.25%) Inhibit bacterial decomposition & autolysis Produce minimum cell shrinkage 7. Duration of fixation Permit rapid and even penetration Formaldehyde: 2-6 hours * To speed up the fixation process Prolonged fixation Harden tissue o cause shrinkage and hardening of tissue * Needs to be hardened because we are going to o inhibit enzymatic activity and immunological use it in the preceding steps activity Isotonic o resolve: washed with running water * Why isotonic? Because isotonic solution or Electron microscopy: 3 hours then placed in holding isotonic fixative can cause minimal, physical and buffer chemical alterations of the cells and their constituents. But this is not true all the time. 8. Time interval * In fact, in our main factors involve the best It should be fixed immediately after removal or death solution is slightly hypertonic solution * The recommended is isotonic that is around 340 Effects: mOsm/L o Glacial acetic acid (Hypotonic solution) + Reduce risk of infection during handling and actual Picric acid (Hypertonic solution) processing * Hypotonic can produce swelling o Because we already prevent the microbial growth * Picric acid is a hypertonic that can cause or inhibit the microbial colonization shrinkage of the cell Harden soft and friable tissue--- easy section cutting (part * But if you combine the glacial acetic acid of the main goal is to harden the tissue) which is hypotonic and picric acid which is a Makes cell resistant to damage and distortion during hypertonic solution it can give optimal effect processing on the tissue structure Inhibit bacterial decomposition Make cellular constituents insoluble to subsequent Increase optical differentiation --- easy visualization during hypotonic solutions examination Permit subsequent application of stains Acts as mordants or accentuators --- promotes and hasten M. LIONG & J. LABASTIDA– TRANSCRIBERS Types of Fixative: Primary component: Glacial acetic acid due to 4 major groups: affinity to chromatin. Aldehydes pH: < 4.6 o Formaldehyde Examples of nuclear fixatives: o Glutaraldehyde They act by o Flemming’s fluid cross-linking Oxidizing agents proteins o Carnoy’s fluid o Osmium tetroxide o Bouin’s fluid o Potassium permanganate o Newcomer’s fluid Alcohol based o Heidenhain’s Susa o Methanol o Ethanol 2. Cytoplasmic fixatives o Acetic acid Preserve cytoplasmic structures o Protein denaturing * Preserves the cytoplasm as well as the organelles Metallic group No glacial acetic acid: destroys mitochondria and Golgi o Mercuric chloride apparatus o Picric acid * Cytoplasmic fixative should never contain glacial o Forming insoluble metallic precipitates acetic acid because it can destroy the mitochondria and Golgi apparatus Types according to: Composition pH: > 4.6 A. SIMPLE o Flemming’s fluid without acetic acid * 1 component substance o Kelly’s fluid Aldehyde o Formalin with “post-chroming” (secondary o Formaldehyde fixative for post-chromatization) o Glutaraldehyde o Regaud’s fluid (Muller’s fluid) Metallic fixative o Orth’s fluid o Mercuric chloride RNA: precipitant fixatives ethanol and acetone (best o Chromate fixatives quantitative result using frozen tissue) for rapid Picric acid diagnosis or cryostat Acetic acid 3. Histochemical fixatives Acetone Preserve chemical constituents Alcohol Examples of histochemical fixatives: Osmium tetroxide o 10% formal saline o Absolute ethanol B. COMPOUND o Acetone 2 or more component substance o Newcomer’s fluid Added together to obtain optimal combined effect * Just like in glacial acetic acid and picric acid, when Secondary Fixation being combined they give optimal effect to tissue structures. Placing the fixed tissue to second fixative Purpose: Types according to: Action o Improved demonstration of particular substances A. Microanatomical o Special staining techniques (mordant) Permit general microscopic study tissue structures o Complete hardening & preservation of without altering the structural pattern and normal tissue intercellular relationship. Done: Examples of microanatomical fixatives: o Before dehydration o 10% formal saline o Before staining (deparaffinized sections) o 10% neutral buffered formalin Primary fixative: 10% formalin or 10% formal saline o Heidenhain’s Susa Example of secondary fixatives: o Formal sublimate or formal corrosive o 10% Neutral formalin ---- Zenker’s solution o Zenker’s solution o ---- Masson’s trichrome (for connective tissue) o Zenker-formal or Kelly’s solution o ---- Mallory’s aniline blue (for collagen) o Bouin’s solution o ----Phosphotungstic acid hematoxylin (PTAH) o Brasil’s solution stain (for striated muscle) B. Cytological Post-chromatization Preserve specific parts and particular microscopic o Form of 2° fixation in aqueous solution of 2.5%-3% elements potassium dichromate for 24 hrs. 1. Nuclear fixatives o Acts as mordant Preserve nuclear structures o Better staining * Preserves the nucleus or the chromatin o Aid in cytologic preservation of tissue materials M. LIONG & J. LABASTIDA– TRANSCRIBERS Washing Out Artifacts: Removal of excess fixative from tissue after fixation Formalin pigment To improve staining and remove artifacts from the * Example: Formalin and when you mix it with water tissues. the solution will be acidic. Formalin pigment (black deposits) can be produced. 1. Tap water o Produce under acid conditions o Excess chromates from tissue fixed with Kelly’s, o Reduced or eliminated: Phenol formalin or 10% Zenker’s & Flemming’s solution Neutral Buffered Formalin o Excess formalin * Phenol formalin or 10% Neutral buffered formalin o Excess osmic acid or osmium tetroxide can completely stop the formation of formalin pigment 2. 50-70% alcohol o Excess picric acid Crush artifact o Found in liver biopsies associated with intense 3. Alcoholic iodine eosinophilic staining (or hypereosinophilia) at the o Excess mercuric fixatives center of the tissue in H&E stain o Due to: Difficulties caused by Improper Fixation: ▪ Partial coagulation of partially fixed protein by ethanol ▪ Incomplete wax infiltration 1. Failure to arrest early autolysis Failure to fix immediately Insufficient fixative * If kulang na yung fixative natin so instead of 50mL for a 2.5 x 2 x 0.5 measurement of tissue or volume tissue or tissue sample and ang ginamit niyo lang ay 10mL. It is insufficient yan and hindi niyo pa na fix immediately so it will lead to failure to arrest early autolysis * Our goal is to prevent autolysis * Black arrow: are the crush artifacts and caused by a needle used for marking the location of the samples. 2. Removal of substances soluble in fixing agent * REMEMBER: crush artifacts can simulate cellular Wrong choice of fixative atypia, cytoplasmic, hypereosinophilia, and nuclear * Very important that we have an initial diagnosis or pleomorphism. primary diagnosis to have an idea na ito yung gagamitin natin na fixative. Lipid Fixation * For example: Carbohydrate fixation kasi feeling In general: aldehydes niyo may something doon na gusto nyo makita so o Disadvantage: reacts with unsaturated fats -- ito yung gagamitin niyo na fixative. -- less lipid demonstrated * So kapag mali yung napili ninyo na fixative then Baker’s formal-calcium: preserve phospholipids chances are the substance that you will need will Imidazole osmium tetroxide: improve ultrastructural be removed so naging soluble na sila don sa demonstration of lipids fixative or fixing agent. Digitonin: improve ultrastructural demonstration of cholesterol 3. Presence of artifact pigments on tissue sections Cryostat or frozen section: mercuric chloride and Incomplete washing of fixative potassium Dichromate * Presence of the artifact pigments, like the formalin * Process use if you want rapid diagnosis and pigments or the crush artifacts. also we are using this process for the demonstration of lipids, nervous tissues 4. Soft and feather-like consistency of tissue elements and also the enzymes. Incomplete fixation Carbohydrate Fixation * It will be hard for us to cut it using a microtome because friable or brittle or mabilis na siyang Recommended for glycogen: alcohols masisira if incomplete fixation. o Rossman’s fluid o Cold absolute alcohol 5. Loss or inactivation of enzymes needed for study Human skin: alcoholic formaldehyde Better retention of glycogen: tissue coated with Wrong choice of fixative celloidin 6. Shrinkage and swelling of cells and tissue structure Protein Fixation Over fixation Neutral buffered formal saline or formaldehyde vapor. 7. Tissue blocks are brittle and hard Prolonged fixation References * It will be difficult to cut if the tissues are brittle and Prof. Kim Pulga’s ppt and notes too hard. Gregorios Histopathologic Techniques: Chapter 6 pg.82 M. LIONG & J. LABASTIDA– TRANSCRIBERS