Introduction to Histology & Cytology II PDF

MLS 1004SEF Histology Laboratory Workflow Specimen reception & registration Introduction to Histology & Cytology II Technical staff Clerical staff Gross examination & block taking Common Equipment Laboratory Safety Quality Control Pathologist Technical staff Slide preparation Heidi Wong Tissue Proce...

MLS 1004SEF Histology Laboratory Workflow Specimen reception & registration Introduction to Histology & Cytology II Technical staff Clerical staff Gross examination & block taking Common Equipment Laboratory Safety Quality Control Pathologist Technical staff Slide preparation Heidi Wong Tissue Processing is designed to remove all extractable water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of tissue without damage or distortion. Content Steps in tissue processing: 1. Fixation – prevents autolysis and stabilizes tissue to maintain cellular structure 2. Dehydration – removes water and unbound fixative from the tissue 3. Clearing – displaces dehydrating solutions, making the tissue components receptive to the infiltrating medium 4. Infiltration – permeates tissue with a support medium Technical staff [email protected] Reporting Pathologist 1 5 9 13 Histology vs Cytology Factors influencing the rate of processing Histology is the study of the microscopic structure of the cells, tissues and organs. It enhances the understanding of the relationship between the structure and function. It is also called microanatomy or microscopic anatomy. https://www.youtube.com/watch?v=dPS6QZ-eoAA 6 2 Cytology is the study of the structure, function and pathology of cells under the microscope. It evaluates individual cells or cluster of cells to make diagnosis of certain diseases, including some forms of cancer. Specimen reception & registration Slide screening Incomplete dehydration  leaving the specimen soft and non-receptive to infiltration Pathologist 3 Dehydration Technical staff Reporting 14 removal of unbound water and aqueous fixatives from the tissue components should be accomplished slowly graded concentration: 70% ethanol 95% ethanol 100% ethanol Excessive dehydration  cell distortion  hard  brittle  shrunken Technical staff Clerical staff Slide preparation Viscosity Agitation Heat Vacuum & Pressure Processing solvent contamination 10 1. Automatic Tissue Processor Cytology Laboratory Workflow Specimens 1. 2. 3. 4. 5. Common Equipment 7 11 15 Routine Slide Preparation Histology Cytology Core biopsies Fine needle Cancer resection aspirates of specimens breast, thyroid, Excised skin salivary glands, lesions lymph nodes, Endoscopic lung biopsies Effusions Polyps Cervical smears Sputum Urine Cytology Fixation Pre-fixation 10% neutral buffered formalin 50% ethanol, PreservCyt, CytoRich Dehydration & Impregnation Cytopreparation Alcohol 4 Common dehydrating fluids Histology Xylene Paraffin Centrifuge, Cytospin, Pick&Smear, LBC system Embedding & Sectioning Post fixation Paraffin block and microtome 95% ethanol Staining Staining H&E Stain Papanicolaou Stain Dehydrate, Clear & Mount Dehydrate, Clear & Mount Ethanol Industrial methylated spirit (denatured alcohol) Methanol Isopropanol Acetone Embedding cassettes Loading the tissue cassettes into the chamber of tissue processor 8 12 16 Clearing a clearing reagent acts as an intermediary between the dehydration and infiltration solutions it should be miscible with both solutions Type of clearing agents  fast acting: xylene; toluene; benzene  intermediate acting: chloroform; methyl benzoate  slow acting: cedar wood oil; clove oil (for delicate tissue) Precautions during embedding No contamination of traces of clearing agent No dust particles present Correct labelling of specimens Avoid cross-contamination of tissues Correct orientation of tissue blocks Forceps must be kept warm Wax block must be cooled rapidly 6. Celloidin explosive in nature good elasticity limited tissue shrinkage useful in neurological specimens 17 21 Criteria for selecting clearing agent 2. Embedding Center 5. Gelatin m.p. lower than agar less satisfactory for double embedding commonly used for whole organ section, e.g Gough & Wentworth method used when frozen section of friable tissue is required Rapid removal of dehydrating agent Ease of removal by melted paraffin Minimal tissue damage Flammability Toxicity Cost A microtome is a mechanical instrument used for cutting biological specimens into very thin sections of uniform and predetermined thickness for microscopic examination. rapid process ribbon easily suitable for most tissues storage convenient many staining techniques can be used Metal embedding molds 18 Infiltration After clearing, tissue sections are infiltrated (impregnated) with embedding medium to support the tissue, allowing thin sections to be cut Infiltration must be sufficient to displace the clearant from the tissues, otherwise the wax will not harden properly and it interferes with microtomy Types of embedding medium Embedding cassettes 22 26 Paraffin wax - Disadvantages General Embedding Procedure cause tissue shrinkage most lipids are extracted unsuitable for electron microscopy 1. Open the tissue cassette and check with the worksheet to ensure the correct number of tissue pieces present 2. Select the mold in which sufficient space for the tissue with surrounding margin of wax 3. Fill the mold with paraffin wax 4. Use a warm forceps to take out the tissue gently from the cassette and then transfer to mold 5. Orient the tissue and firm it on the mold with warm forceps gently 19 23 3. Resins for electron microscopy 4. Agar used in double embedding with paraffin wax 20 30 27 31 Types of Microtome overnight processing is still considered to be the routine schedule in many laboratories rapid processing schedules for small biopsies or urgent specimens differences in the tissue types and size, number of cassettes, required turnaround time, processor models and choice of reagents all play a major role in determining an optimal processing schedule 2. Ester wax m.p. 46-48 oC Rotary Microtome Overview of Microtome components Processing schedule 1. Paraffin wax mixture of hydrocarbons m.p. 45-70 oC low m.p. – soft; high m.p. -- hard additives may be included to increase hardness. e.g beeswax, ceresin, rubber, plastic polymers, diethylene glycol distearate 29 3. Microtome Paraffin wax - Advantages  Xylene is the most widely used clearing agent in tissue processing. It is an excellent lipid solvent but has the negative characteristic of drying tissue specimens  Xylene is “practically insoluble” in water and therefore is capable of removing water from the tissue, especially in the longer clearing times needed for fatty tissue 25 1. Rotary microtome most commonly used also known as Minot’s Rotary microtome (invented by Minot in 1885) the basic mechanism requires the rotation of a fine advance hand-wheel by 360° degrees, moving the specimen vertically past the cutting surface and returning it to the starting position. Ideal for producing flat, serial sections, and ribbons of sections mainly used to section paraffin wax embedded tissue may be manual, semi-automated or fully automated 6. Place the labeled cassette base onto the mold 7. Fill the mold with paraffin wax 8. Cool the block on the cold plate 9. Remove the block from the mold 24 28 32 Principle of sample movement Microtome knife 2. Base sledge microtome the block is fixed in static position within a steel carriage the knife slides to and fro over the top of the block the block moves upwards automatically as each section is cut ideal for large tissue sample or hard tissue (large neuropathology and ophthalmology sections) Microtome knife is important to cut uniform and thin section of tissue Various types of knife profiles are available for different types of microtomes Made of steel Most commonly used knife profile is Profile C or wedge profile 33 37 Types of microtome knife 3. Ultramicrotome used to cut ultrathin sections for transmission electron microscopy sections are cut between 40 and 100 nanomicron thickness with the help of glass knife or diamond knife the tissue is trimmed to make small of 1 x 1 mm size, and then the section is cut by this ultramicrtome with the help of optical microscope The cut section is allowed to float on the water held by a boat and then finally picked up on a metal grid Manual staining When the sample has been moved down from position 1 to position 2, a section is cut by the knife at the pre-set thickness When the sample is at the lowest position 2, the sample holder will withdraw slightly away from the knife before moving upward The cut section remains on the knife At the highest point of the rotary motion (position 4), the sample holder is advanced by the pre-set thickness, allowing the next section to be made 41 Equipment required for paraffin section cutting Profile A Planoconcave Profile B Biconcave  Both surfaces are concave  Can be sharpened to give a very sharp edge  Cannot be used to cut sections of hard tissue  Used for sectioning both paraffin wax and celloidin embedded tissues 34 Eosin is an acidic and negatively charged dye Eosin is a counterstain which stains the basic structures It produces different varying shades     38 Profile D Tool edge  Both surfaces are flat but cutting edge is steep  Mainly used to cut the hard tissue such as decalcified bone  Difficult to sharpen and is not recommended currently 35 Advantage:  Rapid production of sections for Intraoperative diagnosis  Diagnostic and research enzyme histochemistry for labile enzymes.  Immunofluorescent methods  Immunohistochemistry techniques when heat and fixation may inactivate or destroy the antigens  Diagnostic and research non-enzyme histochemistry, e.g. lipids and some carbohydrates  Silver stains, particularly in neuropathology Disadvantage:  Need continuous supervision to maintain the temperature  Freezing artefact present  Very expensive instrument 36 39 Disposable microtome blade 46 Hematoxylin stains acid substances blue Eosin stains the basic compounds of the tissue pink Before the staining, sections should be treated for removing paraffin, and hydrated because both dyes are hydrosoluble.  The most common knife profile, with both surfaces flat  Used for production of celloidin, paraffin wax and frozen sections  Easy to sharpen A refrigerated cabinet, inside which is a modified rotary microtome All the controls of the microtome are operated from outside the cabinet The temperature inside the cabinet is adjustable between -5oC and -40oC or lower Tissues are hardened by freezing and cut frozen Red blood cells ---- bright red Collagen ---- pale pink muscle ---- deep pink Cytoplasm ---- light pink 42 4. Autostainer Profile C Wedge shaped 4. Cryostat Haematoxylin and eosin (H&E) Haematoxylin and eosin (H&E) is the most widely used stain in most histopathology labs Haematoxylin is a basic and positively charged dye Haematoxylin stains all the nucleic acids (DNA & RNA) bluish purple due to the presence of the phosphate backbones of nucleic acids which are negatively charged Flotation (water) bath Slide drying oven or hot plate Fine pointed or curved forceps painting brush Slide rack Clean slides Ice tray or cooling platform Chemical-resistant pencil or pen Electronic slide labeling instrument  One flat surface and one concave surface  Originally used for celloidin sectioning but can also be used for paraffin wax sections  Difficult to sharpen  Not widely used 45 43 47 Stain up to 200 slides per hour Easy to use menu-driven and microprocessor controlled Produces consistent, high-quality results for both routine and special stains Perform single or multiple protocols at the same time 12 Slide Racks 34 Reagent stations & 5 wash stations PC access for fault diagnosis Self contained fume control with replaceable filter Disposable blade is widely used to save time to sharpen Two type of disposable blades: Low-profile blade & high-profile blade Advantages:  Easy to replace  No need to sharpen Disadvantages:  Not very rigid like knife  Vibration effect may be present 40 44 48 LBC Systems with FDA Approval Papanicolaou Stain ThinPrep (Cytyc,Hologic, Marlborough, MA) was FDA approved on May 1996 SurePath (BD DiagnosticsTriPath, Burlington, NC) receiving FDA approval in June 1999 MonoPrep Pap (MonoGen, Lincolnshire, IL) tests approved on Mar 2006 ThinPrep (TP) and SurePath (SP) are most currently used liquid-based cytology preparations The Papanicolaou (Pap) stain is the most widely used stain in most cytology labs The Papanicolaou stain is a polychrome stain The Pap method involves nuclear stain & cytoplasmic counterstains Haematoxylin is the nuclear stain Orange 6 (OG6) and Eosin Azure (EA) are the two cytoplasmic counterstains 49 Cytoplasmic Stain Laboratory Safety 53 Principles of the preparation of LBC slides Orange 6 (OG 6) An acidic, monochromatic dye Stains keratin a bright and intense orange EA (Eosin Azure) A polychrome stain composed of three dyes: light green, eosin and Bismarck brown EA-36, EA-50, EA-65: the number denotes the proportion of the dyes Light green stains the cell that are metabolically active in blue colour like parabasal squamous cells, intermediate squamous cells & columnar cells Eosin gives a pink colour to cytoplasm of mature squamous cells, nucleoli, cilia and red blood cells Bismarck brown does not add a characteristic color to the cytoplasm and sometimes it is often omitted 58 62 Sharp        Glass slides Coverslips Glassware Knife Microtome blade Scalpel Needle Electrical equipment  Electrical shock  Risk of igniting flammable vapors  Refrigerators and freezers must never store highly flammable chemicals Burns  Hot surface 54 ThinPrep Preparation System 2. Chemical Safety ThinPrep5000 61 1. Physical Safety A sample of cells is collected e.g. from the cervix in the normal way using a spatula or broom sampling device The sample is transferred into a container of preservative/ transport medium The cell are dispersed in the fluid An aliquot of the suspension is selected for processing The cells are separated by centrifugation or filtration and deposited on a slide as a thin layer / monolayer by sedimentation or the application of pressure The slides are stained , mounted ready for microscopy 50 57 ThinPrep2000 Chemicals should be properly labelled Read the labels carefully Chemicals should be stored in appropriate cabinets  e.g For flammable and toxic and corrosive chemicals, etc. Material safety data sheet (MSDS) for chemicals  Provides chemical and safe handling information All chemicals must be stored in air-tight container Explosion proof refrigerators are used if storing volatile and flammable chemicals Chemical safety fume hood should be used ThinPrep slide Gynaecological specimen stained with Pap stain 51 55 59 ThinPrep Preparation Process 5. Liquid-based Cytology System General Chemical Safety Control Measures 1. Dispersion 1. Minimize all chemical exposure by taking precautions. 2. Laboratory staff should have information on correct handling and disposal. 3. Personal hygiene should be followed at all the times. (e.g no eating and drinking) 4. Personal protective equipment should be worn at all times. 5. Labels and signage should be evident wherever there are risks. 6. Ventilation should be adequate and monitored regularly. 7. First aid should be available and adequate. 8. Eye wash and shower facilities should be available. 9. Proper storage of hazardous chemicals (e.g acids) 10. Spill kits should be available. 11. Waste disposal and recycling provision should be provided and adequate. The Filter rotates within the sample vial, separating debris and dispersing mucus without adversely affecting the appearance of cells. Liquid based cytology (LBC) is a method of preparing cells for examination in a laboratory A small sample of cells are placed directly into a vial containing a preservative fluid Three liquid based Papanicolaou (Pap) tests are FDA approved for use in the United States as replacements for the conventional Pap smear 2. Cell Collection A gentle vacuum collects cells on the exterior surface of the Filter membrane. 3. Cell Transfer 52 After the cells are collected, the Filter is inverted and gently pressed against the ThinPrep Microscope Slide. Surface tension and air pressure cause the cells to adhere to the Slide, resulting in an even distribution within a circular area. 63 56 60 64 3. Biological Safety Histology specimens received in formalin should be handled on the Ventilated Specimen Table Histology specimens received in fresh should be handled in Biosafety Cabinet (BSC). Ventilated Specimen Table Biosafety Cabinet Sterilize surfaces with chlorine bleach or a suitable commercial disinfectant; avoid formaldehyde solutions as these present a chemical risk to the person doing the cleaning. Good-quality latex or nitrile surgical gloves are perfectly acceptable protective devices for biohazards. Goggles should be worn during grossing anyway to protect against chemical splashes, and will do double duty against infectious agents. A face shield may be warranted in some cases. Aprons or laboratory coats will keep clothes clean, but do not wear these used protective articles outside the laboratory (especially to the cafeteria!) 65 External Quality Assessment (EQA) A system of objectively checking laboratory results by means of an external agency Including comparison of a laboratory's result at intervals with those of other laboratories 69 73 Ventilated Specimen Table An EQA organizer provides surveys in which identical material will be tested by all participating laboratories Downdraft tables are workbenches with built-in ventilation to capture dust, smoke, and fumes and draw them away from the operator and the material being worked on. They typically consist of a perforated surface whose underside is connected to a ventilation or dust collection system, to draw material through the holes and away from the work. e.g. HKIMLS Quality Control Four runs per year  H&E  Special Stain  IHC  Cytology diagnosis Obtained HOKLAS accreditation 66 70 74 Handling Specimen Three potential routes of exposure: Quality is defined as a measure of how well a product or service does the job for which it is designed, i.e. conformity to specification. Quality control gives evidence that the tests being carried out are accurate and specific. This evidence may be provided internally within the organization by internal quality control (IQC) or by reference to an external body providing external quality assessment (EQA).  inhalation of aerosols  contact with non intact skin  contact with mucous membranes (eyes, nose and mouth) Practice universal precautions: handle every specimen as if it were infectious. Fresh specimens of human origin must always be considered potentially infectious. Most obvious source of biological risk is with fresh tissue and body fluids. Gross examination carries the highest risk of all histological activities. 67 Benefits: 1.Identify possible deficiencies in laboratory practice for improvement 2.Recognition of performance is comparable to peer participants 3.Improve laboratory performance by reference to the method of the best performer 4.Occasionally the organizer provides positive samples that are not easily obtainable 71 Internal quality control (IQC) Fixed specimens have a much-reduced risk because nearly all infectious agents are readily deactivated by fixation. Cryotomy presents special risks because tissue is usually fresh. Small dust-like particles generated from sectioning may become airborne, a risk vastly magnified with the use of cryogenic sprays. Do not clean the cabinet with a vacuum unless the device is equipped with a HEPA filter. 68 75 Reference Accurate in specimen registration Block and slide matched Routine H&E stain checked Microscopic checking  fixation ok?  section suitable?  deep enough? representative? any cutting defects?  staining quality good?  coverslipping ok?  presentation ok?  correct patient particulars on slide? Bancroft's Theory and Practice of Histological Techniques, 8th edition 2019 by S. Kim Suvarna, Christopher Layton and John D. Bancroft 72 76