HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES PDF

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WEEK 2 – INTRODUCTION TO HISTOPATHOLOGYHISTOPATHOLOGIC TECHNIQUE HISTOPATHOLOGY – basic component of tertiary hospital laboratory where human tissues and body fluids are processed into slides for microscopic examination by the anatomic pathologist. (Lo, et. al. Basic Histopathologic Techniques) CYTO...

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY c.

HISTOPATHOLOGIC TECHNIQUE HISTOPATHOLOGY – basic component of tertiary hospital laboratory where human tissues and body fluids are processed into slides for microscopic examination by the anatomic pathologist. (Lo, et. al. Basic Histopathologic Techniques)

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The speed of decomposition appears to be proportional to the natural metabolic activity of the tissue. o

CYTOPATHOLOGIC TECHNIQUE - Preparation and examination of cells collected by fine needle aspiration, bronchial washings, and other techniques that will aid in the diagnosis of diseases.

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Action of various enzymes which leads to autolysis

Rapid decomposition occurs in the following organs: a. Kidney b. Liver c. Pancreas

HISTOLOGICAL TECHNIQUE • •

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Deals with the preparation of tissues for microscopic examination. Accomplished by submitting the total or a selected part of the tissue presented for examination to a series of processes: a. Fixation b. Dehydration c. Clearing d. Embedding e. Cutting f. Staining As soon as a tissue is removed from the body or cut off from its blood supply, tissue degradation starts or decompose Reasons: ✓ Lack of oxygen → series of biochemical changes eventually lead to poor tissue quality ✓ Initial anoxic insult suffered by tissues – Warm ischemia Cold ischemia – lack of oxygen once tissue sample is removed from the patient’s body until all metabolic functions are stopped by fixation. Delay in the fixation (specimen left on the OR tray until the surgery is done) extends the cold ischemia time which results in poor tissue fixation. Further aggravated if the tissue is la large rge and not sectioned at regular intervals before immersing in the fixative.

Rapid degradation – brain & pancreas To preserve the natural state of tissue cells → it is essential to check these processes of decomposition with a minimum of delay.

SPECIMEN HANDLING AND IDENTIFICATION • •

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Identification of the specimen and all of its components – first step in specimen processing. Each laboratory has its own way of specimen identification. ✓ Giving the tissue a unique accession number ✓ Date and time the specimen is received The specimen container must bear the same name and accession number as those in the acquisition form. If multiple specimens are received on the same patient from the same operation/procedure → the specimen may be given the same number followed by a numerical or alphabetical designation.

Decomposition results from: a. b.

Deprivation of oxygen and essential metabolites Accumulation of carbon dioxide and other products of cell metabolism

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The specimen container label and the accompanying request form should include: a) Patient’s name (Includes maiden or middle name)

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY b) c) d)

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Age or birth date A medical record or hospital number Bar codes are also frequently used by clinical laboratories. Label should be firmly attached to the BODY of the container not to the lid of the container.

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The following can gross spec specimens: imens: a. Pathologist b. Resident c. Physician assistant d. Histotechnologist e. Biomedical scientists Gross descriptions are important The type of biopsy and the number of fragments received should be documented. Most laboratories have developed standardized formats that describe the gross examination and processing of specimen.

Materials used for gross examination: a) Cutting tools – standard fits/sets ✓ Scissors ✓ Forceps ✓ Blades ✓ blade holders b) Measuring tool c) Casette

DESCRIBING SPECIMENS 1. • • •

The request form should have a provisional diagnosis and brief clinical d details etails Any discrepancies in specimen identification or labeling should be resolved prior to processing. Incorrect identification of any specimen results in wrong diagnosis and incorrect treatme treatment nt

Criteria for rejecting of gross specimens: 1. Discrepancies between the requisition specimen labels 2. No labels or mislabeled specimens 3. Leaking specimen containers 4. Absent clinical data or history 5. Inappropriately identified specimens

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GROSSING GROSS ROOM – Specimen Reception Laboratory → Is where tissue specimens from the operating theaters and clinics are received.

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Markers for orientation: o Some surgeons use orientation markers such as inks, nicking and suturing which are also specified in the requisition form. o One or 2 ink colors maybe used to identify and orient the specimen’s components. o Nicking is done for indicating laterality. o Sutures attached may be represented as: LL – long lateral SS – short superior Weight of intact specimen is rounded to the nearest 0.1g – In some specimens, the weight maybe more important than the histopathologic characteristics in arriving at a diagnosis. – Example: Hyperplastic thymic tissue Dimensions are rounded to the nearest 1 cm Color Consistency • Minute specimens are inked to ensure that the entire face of the specimen is present in the slide.

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY MAJOR COMPONENTS IN GROSSING A SPECIMEN 1. 2. 3. 4. 5. 6. 7.

Reliable and rapid transfer of the specimen from surgery to pathology Accurate identification of the specimen Description of additional specimens received from the same patient Gross description of the specimen’s normal and abnormal features Recording the sites from which blocks of tissue are taken Recording markers that help with the correct orientation Identifying special studies requested and/or needed.

SPECIMEN FROM DERMATOLOGY • •

Often small and can be excisional, shave, core, or reexcisional biopsies. Each type of biopsy is handled differently and is often processed separately from other tissues.

SMALL SPECIMENS ➢ Should NOT be cut, bisected, or inked while fresh and infixed. ➢ Are processed in cassettes either with a fine mesh, in lens paper, or in a “tea bag”. ➢ Use of spo sponges nges is an alternative → but may dry out, harden and may stick to sponge.

CORE BIOPSIES ➢ Usually taken with a l arger lesion or of a generalized inflammatory or other disease process. ➢ Should be taken with the lesion at its center ➢ Larger core biopsies (4mm) → should be bisected eccentrically and embedded with cut surfaces down. ➢ Small core biopsies (2mm (2mm)) → should be embedded totally without cutting it.

SHAVE BIOPSIES OF SKIN ➢ Depending upon the size of the biopsy, it may be bisected, trisected, or cut into sections. ➢ Most specimens of skin or other epithelial surfaces should be cut → all aliquots are embedded on edge. ➢ Care should be taken with any pigmented lesions of the skin. EXCISIONAL BIOPSY ➢ Method of cho choice ice for surgical removal of melanomas but may be sometimes removed by shaving ➢ Biopsies of skin are examined to ensure that the lesion has been completely removed and the original clinician’s diagnosis was correct. ➢ Can be oriented using sutures or dyes.

RE-EXCISION SPECIMENS RE-EXCISION ➢ Original site of a lesion may need to be re-excised if: ▪ The margins are invaded by tumor ▪ Too close to the tumor → melanoma or basal cell carcinoma. ❖ ❖

For skin specimens, vertical orientation of the specimen should always be maintained at all times. Objective of the pathologist is to visualize the epidermis in a perpendicular section.

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY PEDIATRIC SPECIMEN Gross examination and processing of pediatric biopsies requires special care due to diagnostic difficulties of pediatric lesions/diseases. • Pediatric tumors are rare → often necessary to use immunohistochemistry, electron microscopy, flow cytometry, cytogenetics, and molecular genetics to confirm diagnosis. fresh, frozen or other • These studies may require fresh specifically processed tissues. •

PUNCH BIOPSIES: ➢ 3 mm or less are submitted as a whole. ➢ 4 mm or more are bisected or trisected depending on the size.

FRESH TISSUE EXAMINATION Methods of tissue examination may vary ac according cording to to: a. Structural and chemical components of the cells to be studied b. Nature and amount of the tissue to be evaluated c. Need for an immediate examination of a tissue structure Examination may be d one on fresh or preserved tissues tissues** ❑

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For skin ellipses, the entire specimen is serially cut along the short axis at 2 to 3 mm intervals. The two tips are submitted in separate cassettes. And the remainder are submitted in one or more cassettes.

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Excisional biopsies Operative specimens → tumors, unidentifiable inflammatory masses, tissues removed prior to transplantation, traumatic, congenital malformations, or cosmetic surgical specimens. All specimens must be examined carefully → may harbor unsuspected malignant tumors

Important determinants of n neoplastic eoplastic specimens: 1. Overall size of the tumor 2. Depth of invasion into or through the tissue walls 3. Involvement of margins and lymph nodes

Advantage: For the study of protoplasmic activities such as: 1. Motion 2. Mitosis 3. Phagocytosis 4. Pinocytosis Disadvantage Disadvantage:: Not permanent and tissues develop changes that are observed after death (decomposition).

METHODS OF FRESH TISSUE EXAMINATION 1. TEASING or DISSOCIATION ➢ A process whereby a selected tissue specimen is watch glass containing isotonic immersed in a watch salt solution solution, carefully d issected or separated, and examined under the microscope. 2. SQUASH PREPARATION (Crushing (Crushing)) ➢ small pieces of tissue not more than 1 mm in diameter are placed in a microscopic slide and forcibly compressed with another slide or with a cover glass glass.

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY 3. SMEAR PREPARATION ➢ The process of examining sections or sediments, wherein cellular materials are spread lightly over a slide by a wire loop or applicator, or by making an apposition smear with another slide. ➢ Useful in cytological examinations → particularly for cancer diagnosis

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STREAKING o with an applicator stick or platinum loop, the material is rapidly and gently applied in a d irect or z igzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion.

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SPREADING o a selected portion of the material is transferred to a c lean slide and gently spread into a moderately th thick ick film by teasing the mucous strands apart with an applicator stick o Little more tedious but with an advantage of maintaining cellular interrelationships of the materials to be examined. o Recommended for smears preparations of fresh sputum and bron bronchial chial aspi aspirates rates and for thick mucoid secretions.

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PULL-APART o done by placing a drop of secretion or sediment upon one slide and facing it to another clean slide. o The material disperses evenly over the surface of two slides. o Slight movement of the two slides in opposite directions may be necessary to initiate the flow of materials.

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The two slides are then pulled apart with a single uninterrupted motion, and the specimen placed under microscope for immediate examination, or applied with vital stains. Useful for preparing smears of thick secretions such as serous fluid, concentrated sputum, enzymatic lavage samples from GIT, and blood smear.

TOUCH PREPARATION – Impression smear o A special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean glass slide, allowing the cells to be transferred directly to the slide for examination. o Advantage Advantage: Cells may be examined without destroying their actual intercellular relationship

4. FROZEN SECTION ➢ is normally utilized when a rapid diagnosis of the tissue in question is required.

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY ➢ ➢

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Especially recommended when lipids and nervous tissue elements are to be demonstrated. Very thin slices (10 10-15u in thickness) are cut from fresh tissue frozen on a microtome with CO2 or on a Cryostat. CRYOSTAT – a cold chamber kept at an atmospheric temperature of -10C to --20C 20C Frozen sections are then transferred to a slide, and processed for light microscopic study.

Applications Applications: a. Rapid pathologic diagnosis during surgery b. Diagnostic and research enzyme histochemistry c. Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates d. Immunofluorescent and immunohistochemical staining e. Some specialized silver stains → particularly in neuropathology ➢ ➢ ➢

Tissue for freezing should be fresh, and freezing should be done as quickly as possible. Slow freezing can cause distortion of tissue due to ice crystal artifacts More commonly used methods of freezing include: a) Liquid nitrogen b) Isopentane cooled by liquid nitrogen c) Carbon dioxide d) Aerosol sprays LIQUID NITROGEN ✓ generally used in histochemistry and during operative procedures ✓ Most rapid of the commonly available freezing agents Disadvantage Disadvantage: o Soft tissue is liable to crack due to rapid expansion of the ice within the tissue → producing ice crystals or freeze artifacts

Overcools urgent biopsy blocks → causing damage to both block and blade if sectioning is done at -70°C or below. Tissue must be allowed to equilibrate to cryostat chamber temperature before sectioning is attempted. Non-fatty unfixed tissues are sectioned well at temperatures between -10°C and -25°C o

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PROBLEM PROBLEM: Use of liquid nitrogen can cause a vapor phase to form around the tissue → acting as an insulator that causes uneven cooling of tissue (particularly in muscle biopsies) and making diagnostic interpretation difficult. o Can be overcome by freezing the tissue in Isopentane, OCT, or Freon 2.2 → has a high thermal conductivity.

PROCESSING OF TISSUES •

Solid structures and tissues must be preserved and carefully processed in the following order: 1. Fixation 2. Dehydration 3. Clearing 4. Infiltration 5. Embedding 6. Trimming 7. Section-cutting 8. Staining 9. Mounting 10. Labeling

HISTOTECHNOLOGY •

Is the art and science performed by the histotechnologist to produce a tissue section of good quality that will enable the pathologist to diagnose the presence or absence of disease.

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FIXATION ➢ first and most critical step in histotechnology ➢ Fixation is classically defined as the killing, penetration and hardening of tissues. ✓ alteration of tissues by stabilizing proteins so that the tissues become resistant to further changes.

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY ✓ ✓

Fixing or preserving fresh tissue for examination. The quality of the section on the slide is only as good as the quality of the fixed tissue specimen.

FIXATION – also the process by which the constituents of the cells and tissues are fixed in a physical, and partly in a chemical state, so that they will withstand subsequent treatment with various reagents with a minimum of loss, significant distortion, or decomposition

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Aim of good histopathological techniq technique: ue: ✓ To produce microscopic preparations of tissue that represent as closely as possible their structure in life. Aims of ideal fixation: I. Primary aim: To preserve the morphologic and chemical integrity of the cell as it is in the living state.

FIXATION 2 BASIC MECHANISMS INVOLVED IN FI FIXATION: XATION: 1.

ADDITIVE FIXATION ➢ The chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to protein. ➢ Example: a) Formalin b) Osmium tetroxide c) Mercuric Cl d) Chromium trioxide e) Glutaraldehyde f) Picric acid g) Zinc Sulfate of chloride

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NON-ADDITIVE FIXATION ➢ The fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to Hbonds. ➢ E.g. Alcoholic fixatives

With fixation, the shape, structure, intercellular relationship and chemical constituents of tissues are preserved.

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Preserve the tissue by preventing the breakdown of cellular elements by stopping all cellular activities so that cells can be viewed under the microscope as if they are still in their original living state.

Fixation prevents autolysis by inactivating the lysosomal enzymes, or by chemical altering, stabilizing, and making the tissue components insoluble. o Fixation also protects the tissue from further decomposition after death due to bacterial or fungal colonization and overgrowth. To coagulate or precipitate protoplasmic substances Do not leave the tissue specimen in air for prolonged period of time → dry out and will result in distortion of its morphologic appearance Leaving the tissue in w ater → causes the cell to swell while a strong salt → causes the cell to shrink.

II. Secondary aim aim: To harden and protect the tissue from trauma of handling, so it is easier to cut during gross examination

Inadequate or poor fixation: poorly processed tissue and pathologist will have a difficult time in giving a proper diagnosis.

Prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body.

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MTY1216 – HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LAB) WEEK 2 – INTRODUCTION TO HISTOPATHOLOGY MAIN FACTORS INVOLVE INVOLVED D IN FIXATION 1.

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Hydrogen Ion Concentration ➢ Satisfactory fixation occurs between pH 6 and 8 ➢ Outside this range → changes may occur that are detrimental to preservation of tissue. Temperature ➢ Traditionally carried out at room temperature ➢ Many laboratories use tissue processors that work at 40°C ➢ Electron microscopy and some histochemistry → ideal temperature is 04°C ➢ Formalin heated to 60 °C is sometimes used for the rapid fixation of very urgent biopsy specimens → increased risk of tissue distortion ➢ Formalin at 100 °C can be used to fix tissues with tuberculosis ➢ Thickness of section ➢ Large solid tissue should be opened or sliced thinly. ➢ Brain is usually suspended whole in buffered formalin for 2-3 weeks Osmolality ➢ Best results are usually obtained using slightly hypertonic solutions (400 (400-450 -450 mOsm) ➢ The vehicle osmolality is generally important than the total osmolality of the fixative, and ideally should be more or less isotonic with tissues in their normal living environment. Concentration ➢ 10% Formaldehyde and 3% glutaraldehyde is normally used. ➢ Low concentrations of glutaraldehyde have been found to be an ideal concentration for immunoelectron microscopy. Duration of fixation ➢ Buffered formalin – 2 to 6 hours during the day the specimen is obtained ➢ ...