Histopathologic Techniques - 01 Fixation

HISTOPATHOLOGIC AND CYTOGENETIC TECHNIQUES • FIXATION OUTLINE • • Introduction • Fixation o Review of Histopathologic Techniques o Fixation Methods o Factors Involved in Fixation/ Practical Considerations o Types of Fixative according to Mechanism of Action o Types of Fixative according to Action o Types of Fixative according to Active Component Substance o Metallic Fixatives o Other Fixatives • • INTRODUCTION • • • • • In histopathology, there are 3 specimens usually processed o Biopsy o Autopsy o Specimen for cytology 2 common specimen – biopsy & autopsy specimens Steps in Processing Specimen o Same between biopsy & autopsy Fixation is classically defined as the killing, penetration and hardening of tissues. Killing is a necessary part of fixation to stop the metabolic processes that continue to alter the state pf the tissue to be examined. Fixation is currently defined as the alteration of tissues by stabilizing protein so that the tissues become resistant to further changes. In histopathology, a tissue is extracted from the patient is examined to determine the disease process involved. therefore, it is important to preserve the tissue at the exact moment when it is removed from the body to accuratelt represent the diseased state. In other words, it is a snapshot of the diseased entity being diagnosed. Numbering o Includes entering the details of the specimen in a log book o Classification as to whether it is a surgical, autopsy or cytologic specimen is likewise indicated o Gross description is done by the pathologist FIXATION • FIXATION METHODS PHYSICAL METHOD • • • • • • • • Defined as the killing, penetration and hardening of tissues May also be defined as the alteration of tissues by stabilizing protein so that tissues become resistant to further changes Initial step in specimen processing Most important & most crucial step in tissue processing Must be carried out adequately o If not, all the other steps will be inadequate Primary goal: preserve cells and tissues as close to the original as possible Heat Fixation o Not usually carried out in histopathology ▪ Done in microbiology (fixing bacterial smear) o Done for frozen in histopath o Faster Microwave Technique - acetylcholine o Uncommon method o Increases movement of molecules thereby accelerating fixation, staining, decalcification, immunohistochemistry and EM o May be used for neurochemical substances in brain like acetylcholine CHEMICAL METHOD • • Immerse/subject specimens in chemical fixatives Fixative - Reagents used to fix specimen FACTORS INVOLVED IN FIXATION/ PRACTICAL CONSIDERATIONS REVIEW OF HISTOPATHOLOGIC TECHNIQUES FIXATION Secondary goals: o Harden tissues to facilitate easy cutting o Protect tissue from trauma of further handling ▪ In tissue processing, specimens are subjected to different reagents which may cause damage When specimen is removed immediately from the body, the tissue specimen should immerse immediately to the formalin – Primary Fixation o Blood supply is cut off – loss of oxygen – decomposition or autolysis o Autolysis – affect or alter cellular and tissue morphology/ loss of structure Steps In Tissue Processing: o Specimen delivered to the lab – accessioning or numbering o Pathologist – Gross examination – Dissection (small pcs of tissues) o Small pcs of tissues will be placed in tissue cassettes o Tissue cassettes with tissues – to be placed in beaker 1 with formalin Benefits Of Fixation/Effects Of Fixation o Prevents autolysis, reduce risk of infection o Allows thin sectioning o Acts as mordant thus facilitating staining • • • Ensures adequate fixation Temperature o Traditionally carried out at room temp o For EM & Histochemistry - 0-4C o Autotechnicon - to fix and process tissue (40C) o Formalin @ 60C - for rapid fixation of urgent biopsies o Formalin @ 100C - to fix tissues with tuberculosis Thickness/Size o Electron Microscopy (EM) - 1-2 mm2 o Light Microscopy (LM) - 2 cm2 o Tissues should not be more than 4-5 mm thick except in lung specimens (2 cm) ▪ Required is 1-2 cm; duration for primary fixation is 2-6 hours while continuing in the lab for 24 hours o • • • • • • Large solid tissues i.e. uterus must be opened or sliced thinly o Brain tissues must be fixed before grossing pH - 6.0-8.0 Osmolality o It must be slightly hypertonic (by the book) ▪ In practice, isotonic are used o Effect of hypertonic - cause cell shrinkage o Effect of hypotonic - cause cell swelling o Isotonic solution may be use as holding solutions for tissues to be transported to frozen sections or kidney biopsies for special processing Concentration o Concentrated solutions are never used ▪ Solutions are diluted o 10% Formalin o 3% glutaraldehyde - used for EM ▪ 0.25% - immunoEM Time and Duration of Fixation o Ideal time to perform fixation is 20-30 minutes following interruption of blood supply ▪ Specimens must be fixated immediately after removal from the body o Fixation time refers to the period the tissue is exposed to formalin o Fixation is retarded by: large tissues, presence of mucus, presence of blood, presence of fat and cold temperature - causes longer fixation time ▪ Can inactivate enzyme o Fixation is accelerated by smaller and thin tissues, agitation (continuous mixing), heat (37-56C only) causes short fixation time ▪ Beyond 56C can cause tissue damage Volume o For maximum effectiveness - required is 20x volume of specimen; can be 10-20x o Ratio of fixative to tissue - 20:1 o Formalin diffuses into the tissue at a rate of approximately (penetration rate) - 1mm/hour o If Osmium tetroxide (for EM) - required volume is 510x of specimen (due to cost) o For Museum preparations - not less than 50-100x the volume of specimen Other considerations: o Autopsy materials should be fixed as soon after death as possible. If not possible, body must be placed in a mortuary ref (temp 4C) o Hollow organs (stomach, intestine) should be packed with cotton soaked with fixative or completely open the specimen before immersing in adequate fixation solutions o Air filled lungs - may float on fixative ▪ To prevent floating, cover with layers of gauze to keep at the bottom of the container o Human Brain must undergo intravascular perfusion washing out of blood using Ringer’s lactate o Eyes should not be dissected before they are fixed o Hard tissues (cervix, uterine, fibroid etc) must undergo Lendrum’s Method ▪ Lendrum’s - immersing specimen in 4% aqueous phenol for 1-3 days EFFECTS OF FIXATIVES IN GENERAL • • • • Harden tissues Makes cells resistant to damage Increase optical differentiation of cells Acts as mordants or accentuators (facilitates easy staining) PROBLEMS IN FIXATION • • • • • Incomplete washing may lead to: presence of artefacts on tissues Overfixation will render the tissue brittle and hard, shrinkage & swelling Wrong choice of fixative can cause loss of substance soluble infixing agent Loss or inactivation of enzyme may result from wrong choice of fixative Inadequate washing can cause formation of artifacts FACTORS TO BE CONSIDERED WHEN CHOOSING THE RIGHT FIXATIVE • • • • Need for immediate examination o Urgency of cases o Eg. urgent biopsy - use rapid-acting fixative Type of specimen to be processed o Liver - formalin can be used but Zenker’s fluid is preferred Tissue structure to be studied o Preserving glycogen - use Picric acid fixative (Brasil’s) Staining technique to be applied o Fixative to be used must be compatible with the stain to be used o Formalin - routine fixative since it’s compatible with routine H&E stain TYPES OF FIXATIVE ACCORDING TO MECHANISM OF ACTION • • • Mechanism of action - manner of preserving tissue Additive fixatives - combines with the tissue, absorbed by the tissue o Stabilizes tissue proteins o All fixatives available are additive fixative except acetone and alcohol o Formaldehyde; Mercuric chloride; Chromium trioxide; Picric acid o Glutarladehyde ; Osmium tetroxide ; Zinc sulfate or chloride Non- additive fixatives - not absorbed/not taken in by tissue o Involves alteration of tissue components o Examples: acetone & alcohol TYPES OF FIXATIVE ACCORDING TO ACTION • • Action = purpose Microanatomical – allows the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues o 10% formol saline (formalin) o 10% neutral buffered formalin o Heidenhain’s Susa o Formol sublimate/formol corrosive o Zenker’s o Bouin’s o Brasil’s • • Cytological - used to preserve parts of the cell o Nuclear - preserves parts of the nucleus of the cell (nuclear chromatin; chromosomes); usually contains glacial acetic acid (pH <4.6) ▪ Flemming’s ▪ Carnoy’s ▪ Bouin’s ▪ Newcomer’s ▪ Heidenhain’s Susa o Cytoplasmic - preserves parts of the cytoplasm (Golgi bodies, mitochondria); do not usually contain glacial acetic acid (pH >4.6) ▪ Flemming’s without HAc ▪ Helly’s ▪ Formalin with post chroming ▪ Regaud’s / Moller’s ▪ Orth’s Histochemical - used to preserve the chemical components of tissues (enzymes) o 10% formol saline o Absolute ethyl alcohol o Acetone o Newcomer’s PICRIC ACID FIXATIVE • • • • • • • ALCOHOL FIXATIVES • TYPES OF FIXATIVE ACCORDING TO ACTIVE COMPONENT SUBSTANCE • Active component - main ingredient GLACIAL ACETIC ACID • • • • Not used as routine fixative Used in combination with nuclear fixative A compound fixative, recommended for nucleoproteins. Solidifies at 17C & contraindicated for cytoplasmic fixation • • • • TRICHLOROACETIC ACID • • • Not routinely used Used in combination with other fixative to produce compound fixative Used as fixative and decalcifying agent • ACETONE • • • • Can fix & dehydrate tissues at the same time It is used at ice cold temperatures (-5 - 4C) Recommended for preservation of enzymes (lipase) & for brain tissues (diagnosis of rabies) Disadvantages o Dissolves fats o Evaporates easily o Use as raw material in making shabu OSMIUM TETROXIDE • • • • • • Not commonly used o Expensive and slow acting Used for EM Used for 5-10x volume of specimen due to cost Used to fix myelin in peripheral nerves and processing neurological tissues Flemming’s – most common chrome osmium acetic acid fixative, excellent for nuclear structures Flemming’s without acetic acid – for cytoplasmic structures Act as fixative & can be used as stain or decalcifying agent Excellent for glycogen demonstration Major drawback: imparts a yellow color; can stain the tissue yellow Remedy: saturated solution of Lithium carbonate in 70% alcohol then washed with water. The tissue is then placed in 70% ethanol followed by sodium thiosulfate & washed with water Bouin’s solution o Recommended for fixation of embryos, pituitary biopsies and endometrial curettings. o NOT for kidneys & abolishes Feulgen reactions (demonstrate RNA & DNA) Brasil’s Alcoholic Picformol o Excellent for glycogen demonstration Hollande’s solution o For GIT biopsies & endocrine tissues • • Alcohol Fixation o Rapidly denatures and precipitates proteins o Can act both as fixative and dehydrating agent o Ideal for small tissue fragments o Causes: glycogen polarization Methyl alcohol (100%) - for fixing wet and dry smears, blood smears and BM tissues Isopropyl (95%) - for touch preparations Ethyl Alcohol (70-100%) – for blood, tissue films and smears Carnoy’s o Most rapid fixative; fast-acting fixative o Used for fixing chromosomes and lymph glands o Used with acetone for brain tissues for diagnosis of rabies o It contains absolute alcohol, glacial HAC and chloroform Newcomer’s o Classified both as nuclear and histochemical fixative o Used to preserve nuclear proteins o Used to preserve mucopolysaccharides Rossmann’s solution o CT mucins & umbilical cord Others: o Clarke’s solution o Methacarn METTALIC FIXATIVES • Mercuric Chloride o Most common metallic fixative o May form black mercury deposits, remedy: wash tissue with alcoholic iodine o Excellent for trichrome staining o Zenker’s Fluid ▪ Contains mercuric chloride and glacial acetic acid ▪ Recommended for fixing liver, spleen, connective tissue fibers, and nuclei o Zenker’s formol a.k.a. Helly’s fluid ▪ Contains potassium dichromate & 40% formaldehyde ▪ Preserves pituitary glands, bone marrow and other blood-containing organs Heidenhain’s Susa ▪ with TCA, Glac HAc & formalin ▪ for tumor skin biopsies o B5 ▪ with anhydrous sodium acetate ▪ For preserving bone marrow o Others: Schaudinn’s Ohlmacher’s, Carnoy – Lebrun solution Chromate Fixatives o Chromic acid (1-2%) - for preserving carbohydrates o Potassium dichromate (3%) – preserves lipids and mitochondria o Regaud’s aka. Moller’s fluid ▪ for Chromatin, mirochondria, mitotic figures, golgi bodies and RBC containing colloid tissues. o Orth’s fluid ▪ for Rickettsia and other bacteria ▪ for early degenerative processes and tissue necrosis Lead Fixatives o For acid mucopolysaccharides and tissue mucin Note: Lead, Mercuric Chloride & Chromate are METALLIC FIXATIVES ▪ o • • • • • ALDEHYDE FIXATIVES • Formaldehyde/Formalin o Used in routine tissue processing o Commercially available as 37-40% ▪ a.k.a 100% formalin o 10% formalin - routine tissue fixative ▪ Penetration rate of formalin - 1mm/hr ▪ To produce, 100 mL 37% formaldehyde + 900 mL distilled water o 10% formol saline – diluted with distilled water with sodium chloride ▪ Classified as histochemical fixative ▪ Used for general post mortem tissues (autopsy specimens) and for CNS tissues o 10% Neutral Buffered Formalin or Phosphate Buffered formalin - 10% formalin with phosphate buffer ▪ Recommended for fixing tissues with iron pigments and elastic fibers o Formol Corrosive ▪ A.k.a. formol sublimate ▪ Contains formaldehyde and mercuric chloride ▪ Recommended for lipids, neutral fats and phospholipids o Alcoholic Formalin ▪ A.k.a. Gendre’s solution ▪ Composition: 95% ethanol (ETOH) , Picric acid, Glacial Hac and formaldehyde; ▪ For sputum specimens and microincineration techniques (burn small tissues into ashes to identify mineral elements form ashes) o Advantages & Disadvantages of Formalin ▪ Advantages: Cheap, easy to prepare, readily available, stable. Recommended for mailing specimens (tolerant fixative) and for colored tissue photography Disadvantages: fumes are irritating, may cause allergic dermatitis on prolonged contact and may form brown pigment on blood containing tissues like spleen • Prolonged storage = precipitation of white paraformaldehyde (cause turbidity), remedy for this is adding 10% methanol o Methods of removing Formalin Pigments ▪ Kardasewitsch method - 70% ethanol, 28% ammonia water ▪ Lillie’s Method - Hydrogen peroxide, 28% ammonia water, acetone ▪ Picric acid Method - Saturated alcoholic picric acid ▪ KOH Method - 1% KOH in 80% alcohol Glutaraldehyde o Recommended for enzyme histochemistry and EM o 2.5% solution is for small tissue fragments o 4% solution is for larger tissues less than 4 mm thick o Karnovsky’s paraformaldehyde-glutaraldehyde solution and Acrolein are for EM Glyoxal o Supplied as a 40% aqueous solution o Smallest aldehyde fixative o Fast-acting fixative o Surgical specimens are fixed within 4-6 hours & small biopsy specimens within 45 min OTHER FIXATIVES FIXATIVES FOR ENZYME HISTOCHEMISTRY • • 4% Formaldehyde Formol saline FIXATIVE FOR EM • • • Glutaraldehyde Osmium tetroxide Paraformaldehyde FIXATIVE FOR ELECTRON HISTOCHEMISTRY AND ELECTRON IMMUNOCYTOCHEMISTRY • Karnovsky’s paraformaldehyde-glutaraldehyde SECONDARY FIXATION • • • Placing an already fixed tissue to a second fixative To improve demonstration of substance, ensure complete hardening and for special staining Not a mandatory step in processing POST CHROMATIZATION • Secondary fixation with the use of potassium dichromate/ chromate-containing fixative WASHING OUT • • Following fixation, specimen must be washing out Use tap water, alcoholic iodine, or 50-70% alcohol o In practice, tap water is usually used SUPPLEMENTAL NOTES FIXATION • • • • • • • • Specimen removed from the body Formalin = primary fixation o Specimens shall be immediately fixated o Prevents decomposition Blood supply is cutoff → loss of oxygen → decomposition/autolysis Autolysis → affect/alter cellular and tissue morphology/loss of structures Specimen delivered to the lab → accessioning/numbering Pathologist - grossing → dissection → small pieces of tissue Small pieces of tissue → will be place in tissue cassettes Tissue cassette with tissues → to be placed in beaker # 1 with formalin SPECIMENS EXCLUDED FROM MANDATORY SUBMISSION TO THE LABORATORY • • • • • • • • • • • • • • • • Bone donated to the bone bank Bone segments removed as part of corrective or reconstructive orthopedic procedures Cataracts removed by phacoemulsification Dental appliances and teeth with no attached soft tissues Fats removed by liposuction Foreign bodies such as bullets or other medicolegal evidence given directly to law enforcement personnel Foreskin from circumcision Middle ear ossicles Medical devices (catheters, gastrotomy tubes, stents, and sutures) that have not contributed to the patient’s illness, injury or death Orthopedic hardware and other radiopaque medical devices, provided that there is an alternative policy for documentation of their surgical removal Placentas that do not meet the institutionally specified criteria for examination Rib segments or other tissues removed only for the purpose of gaining surgical access, provided that the patient does not have any malignancy Saphenous vein segments harvested for coronary artery bypass Therapeutic radioactive sources Normal toenails and fingernails removed incidentally Skin or other normal tissues removed during a cosmetic or reconstructive procedure, provided it is not contiguous with a lesion or the patient does not have a history of malignancy ACCESSIONING • • • Specimens submitted to the histopathology laboratory must be entered into the surgical pathology database (in short enter sa logbook) Unlabeled specimens are absolutely unacceptable for accessioning Proper identification includes matching the data on the specimen container label and the accompanying surgical pathology request form. if there are inconsistencies between the two, the specimen and the request form are • • returned to the OR (operating room) for correction by the staff concerned. Incomplete specimens in which request form specifies a number of samples which do not correspond to the number submitted are likewise unacceptable Barcoding, which is a modern method of identification will greatly help in identification especially if handwritten requests and specimen labels are not clear because of illegible handwriting. GROSS EXAMINATION BY PATHOLOGIST • • • • • • is one of the most important processes in which the pathologist arrives at a diagnosis. The first step in specimen processing is the identification of specimen and all its components Specimen container must bear the same name and accession number as those in the requisition form All other components of the specimen sample container must be present: patient’s surname, name, birthdate and hospital number. When grossing, the pathologist will determine the WEIGHT of intact specimen. The weight is rounded to the nearest 0.1 gram. Gross weight may be important in arriving at a diagnosis DIMENSIONS are taken and rounded to the nearest 1.0 cm and the COLOR and CONSISTENCY of the specimen are likewise reported. CRITERIA FOR REJECTION OF GROSS SPECIMEN • • • • • • (hindi e-go-gross ng pathologist ang specimen if:) There are discrepancies between the request form and specimen label Specimens with no labels or mislabeled specimens Leaking specimen containers Absent clinical data or history Inappropriately identified specimens SPECIMENS FOR GROSS DESCRIPTION ONLY • • • • • • • • • • • • • Accessory digits Bunions and hammer toes Extraocular muscle from corrective surgical procedure Inguinal hernial sacs in adults Nasal bone and cartilage removed rhinoplasty Prosthetic breast implants Prosthetic cardiac valves without attached tissues Tonsils and adenoids from children Torn meniscus Umbilical hernial sacs in children Varicose veins These specimens, if received in the lab, after gross exam by the pathologist will not be processed anymore. In short hanggang gross lang. Not to be processed for microscopic examination