Histopath LAB Complete Notes PDF

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MLS-LAB 410 | BSMLSLESSON 1: INSTRUMENTATION IN HISTOPATHOLOGY(Receiving)  Gross  Fix  Decal  Dehy  Clea  Infil  Embd  Sect  Stain  MountUsually... - Fixating media – formalin - Embedding media – paraffin Thus... - Other name for this tissue processing: FFPE (Formalin Fixed Paraffin Embedd...

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MLS-LAB 410 | BSMLS LESSON 1: INSTRUMENTATION IN HISTOPATHOLOGY

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(Receiving)  Gross  Fix  Decal  Dehy  Clea  Infil  Embd  Sect  Stain  Mount

Usually… - Fixating media – formalin - Embedding media – paraffin Thus… - Other name for this tissue processing: FFPE (Formalin Fixed Paraffin Embedded) tissue Whole turn-around time (TAT)… - 7 days (normal) - Some laboratories with less samples: possibly 5 days - Some laboratories with more samples: possibly 1 month Gross Examination - Macroscopic examination of the tissue sample - Without the use of the microscope, with the use of the naked eye - Cutting materials: o Dissecting scissors, scalpel, saw (for bones – scroll saw, hacksaw, diamond band pathology saw), knife, chopping board o Forceps o Ruler or measuring tape, weighing scale o PPE, apron o Pencil o Tissue cassettes Tissue Processing (From Fixation to Infiltration) - Manual Tissue Processing o Beakers (especially useful for Infiltration: stainless steel beaker because of the use of melted paraffin wax) o Hot plate o Forceps (for picking up the tissue cassettes) - Automated Tissue Processor o Aka Autotechnicon o A machine that processes the tissue from fixation to infiltration o Decreases time and labor needed for tissue processing o Constant tissue agitation ensures consistency and improves tissue penetration o

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E.g. Leica “dip and dunk” Automated Tissue Processor, also known as Elliot Bench-Type Tissue Processor Other examples: Leica Automated Vacuum Tissue Processor (vacuum – for embedding); Yu Shuo Da Linear Automated Tissue Processor

Embedding - Paraffin Wax Dispenser o Maintains the paraffin wax in liquid form aids in dispensing wax into molds and casts o Used in embedding o Paraffin wax is kept in liquid form by having a temp. of 5-10 degrees above the melting point of paraffin wax (56-57°C: used in the Histopath Lab; but it still depends on the type of paraffin wax) o Paraplast – a type of paraffin wax; combination of paraffin and plastic polymers; 56-57°C melting point

Embedding Center o Complete system designed for embedding tissue in paraffin o Combination of paraffin wax dispenser, hot plate, and cooling plate o Provides controlled heated environment (paraffin kept at 2-4 degrees above MP) for the processed cassettes and eliminates xylene contamination o Also provides cooling plate for solidifying paraffin blocks o Embedding molds alternative: Paper Boat **usually made with magazine paper

Sectioning - Cutting of the tissue sample - Microtome o Precision instruments designed to cut uniformly thin sections of a variety of material for detailed microscopic examination o Importance of THIN section: no overlapping cells/one layer of cells - Types of Microtome o Rotary Microtome o Sliding Microtome o Rocking Microtome o Freezing Microtome o Cryostat o Ultramicrotome 1. Rotary Microtome - Generally used for cutting semi-thin to thin sections (3-5μm) of paraffin wax-embedded material for light microscopy - Operation is based on the rotary motion of a hand wheel activating the advancement of a block towards a rigidly held knife o Tissue block moves up and down a vertical plane in relation to the knife and cuts the tissue in flat sections

2. Sliding Microtome - Designed for cutting large blocks of paraffin and resin-embedded material (including whole organs) for light microscopy - Knife-holding clamps allow the knife to be offset to one direction, a major advantage when sectioning large, hard blocks - Not suitable for cutting very hard resins (like araldi because of the risk of vibration) 3. -

Rocking Microtome (Cambridge Microtome) not anymore used murag bomba sa tubig Razor is fixed and the specimen to be sliced passes up and down in an arc of a circle across the razor in a rocking motion

4. Freezing Microtome - Used for cutting thin to semi-thin (8-12 μm) sections of fresh, frozen tissue o Fresh Section Biopsy (FSB) – using a tissue that is not chemically treated with fixative o Formalin Fixed Paraffin Embedded (FFPE) – unfixed - Equipped with a stage upon which tissue can be quickly frozen using either liquid CO2 from a cylinder, or a low temperature recirculating coolant  Some cooling systems also allow the knife to be cooled at the same time - Knife is moved while the tissue block remains stationary - Consistent, high-quality thin-sections are very difficult to obtain with this type of microtome 5. -

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Cryostat (Cryo-freezing, Stat-madalian) For FSB (TAT: 15-30 minutes) Primarily used for cutting sections of frozen tissue Consists of a microtome (usually rotary microtome*) contained within a refrigerated chamber (temperature maintained at a preset level) Block, blockholder, and knife all at the same temperature o Sections as thin as 1 micron are possible More superior than freezing microtome Can go as thin as 1 μm

6. Ultramicrotome - Used to prepare ultrathin sections for light and electron microscopy - Very small samples of tissue are embedded in hard resin before cutting - Cutting stroke is motor-driven to provide regular, smooth motion for sections of even thickness and constant reproducibility - Can cut sections as 0.5 μm thin - Knives are usually made from glass, diamond, or sapphire - The block is brought to the knife edge under microscopic control - Sections are floated on to a water bath adjacent to the knife edge - Has ocular lens **A brush made of hair should be used to brush of unnecessary paraffin shavings

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Flotation Bath (or Water Bath) o Used for floating the paraffin ribbon o Recommended temperature: 5-10°C below the melting point of paraffin used before embedding

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Slide Dryers o Used for drying the water that is collected during sectioning of tissue section o Temperature: 5-10°C above the melting point of paraffin o To melt the paraffin or “deparaffinization” o Slides are left to dry 15-20 mins after they have been appropriately drained o Alternative: oven (but it is more superior)

Staining - Microwave Oven o Used to heat and speed up some procedures o Some special stains are performed in microwave oven  Heat-induced epitope retrieval in IHC (Immunohistochemistry) - Automated Stainers o Linear types transfer slides from one container to the next (with the same time allowed in each container) o Revolving types are similar to the linear stainer but the time in each container can be varied o Robotic types are programmable; allows continuous loading and the use of the same solutions at different timings Mounting - For the protection of the tissue section - After staining, put the cover slip with the adhesive or the mounting medium - Applicator sticks - Glass slides - Cover slips Cytocentrifuge - Aka Cytospin - Not used in routine tissue processing - Used in cytology (study of cells), specifically exfoliative cytology (the cells that we study are the products of desquamation) - Specialized centrifuge used to concentrate cells in fluid specimens directly onto a microscope slide so that they can be stained and examined Miscellaneous Appliances - Automated voltage regulator (AVR) – for regulating the voltage in cases of fluctuations in electricity - Uninterrupted power supply (UPS) – to protect the tissues being processed from power interruptions; will give you a 10-minute bridge, enough time for a backup power of a hospital to be turned on Video Playlist: https://bit.ly/BongHPLabPlaylist

MLS-LAB 410 | BSMLS THE PROPER PROCESSING OF FRESH TISSUE SAMPLES FOR MICROSCOPIC EXAMINATION Histopathologic Examination - Offers beneficial diagnostic information - Fresh or fixed (preserved) state - Factors affecting the method to be used: o Structures or inclusions (e.g. lipid deposits, glycogen, carbohydrate deposits – fresh state should be used) o Amount and nature of the tissue (e.g. small amount – preserved state should be used) o Urgency of investigation (e.g. if you want to have a result within half an hour – fresh tissue examination or frozen section biopsy) Fresh Tissue Examination - For rapid microscopic analysis while surgery is taking place - A STAT procedure - In the laboratory, the surgery department must coordinate with the laboratory department to ensure that the pathologist is present while the surgery is being done because if the surgeon asks for a fresh tissue examination or frozen section biopsy (FSB), the nurse will immediately take the tissue sample down to the laboratory and the histotechnician will process it WITHOUT FIXATIVES. Within 15 minutes, the pathologist should be able to read the result and tell whether the sample that was taken out is malignant or not. - Uses CRYOSTAT - Done while the patient is OPEN (char) - Advantage: o Tissues are examined in a living state (but not for a long time since they are being cut off with the blood supply from the body of the patient and the first effect of this is hypoxia) o Protoplasmic activities may be observed (e.g., motion, mitosis, phagocytosis, pinocytosis) - Disadvantage: o Tissues examined in fresh state are not permanent o Tissues develop changes that are usually observed after death (ex: hypoxia) Fixed Tissue Processing - Preferred in many conditions for more accurate diagnosis - FFPE - Disadvantage: it takes a very long time before it is being done

Methods of Preparing Fresh Tissue Mounts - Teasing or Dissociation - Squashing or Crushing - Smearing - Frozen Section a. Teasing or Dissociation - Tissue specimen is immersed in a watch glass containing isotonic solution, carefully dissected or separated, and examined under the microscope - Done by carefully separating very thin tissue slices with dissecting needles

STEPS: 1.From the frozen block of tissue, cut several small, thin slices of tissue using a scalpel. 2.Immerse the tissue slices in a petri dish filled with saline solution. 3.Using a dissecting needle, carefully tease the tissue slices until they unfold and spread out. 4.Carefully mount a teased slice on a slide and cover it with a coverslip b. Squashing or Crushing - Done by simply crushing small pieces of tissue (≤ 1 mm in diameter) between two glass slides or between a glass slide and a cover slip Squash Preparation STEPS th **From the 4 step of the previous procedure: 1.Add 1-2 drops of methylene blue(!!) stain directly on the tissue slice on the glass slide. 2.Cover the stained tissue with a cover slip or another glass slide. 3.Applying enough pressure to crush the tissue flat. 4.Label the slide.

c. Smearing - Method of choice for examining sediments (e.g. fluids) - Cellular materials are spread lightly over a slide with: o A wire loop or an applicator stick; or o Another glass slide - Smear Preparation o Centrifuge body fluid samples and decant the supernatant o Avoid making smears that are too thin or too thick since this will render your smears unsuitable for examination

Streaking o Materials is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion STEPS IN STREAKING: -

1. Pour a small amount of the sediments on one end of a clean glass slide. 2. Using an applicator stick, streak the sediments in a straight or zigzag line throughout the remaining length of the slide. If a wire loop is used instead, collect a loopful of the material and smear the material on the slide. A relatively uniform distribution of the materials should be obtained. Cover it with a cover slip. 3. Label the slide.

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Spreading o Main advantage: maintains cellular interrelationships o Portion of a material is gently spread into a moderately thick film by teasing the strands apart with an applicator stick o Recommended for smear prep of fresh sputum, bronchial aspirates, and thick mucoid secretions STEPS IN SPREADING: -

1. Transfer a small amount of the sediments on one end of a clean glass slide. 2. Using an applicator stick, spread the sediments throughout the slide (similar to how butter is spread on bread). Another technique is to place the drop of sediments at the center of the slide. Then with a circular motion, gently spread it into a moderately thick film. 3. Label the slide. -

Pull-apart o Two-slide preparation o Placing a drop or secretion of sediment upon one slide and facing it to another clean slide o Material disperses evenly over the surface of two slides, then slides are pulled apart with a single uninterrupted motion

Touch preparation (Impression smear) o Surface of a freshly cut piece of tissue is brought into contact and pressed into the surface of a clean glass slide o Cells are transferred dire ctly to the slide for examination STEPS IN IMPRESSION SMEAR: -

1. Retrieve the block of tissue from the first procedure. Cut the block in half, exposing a fresher surface 2. Allow the freshly cut surface to come in contact with the surface of a clean glass slide . Apply gentle pressure, allowing the cells to be transferred directly to the slide. Cover the smear with a cover slip. 3. Label the slide.

Frozen Section (Cryosection) Rapid diagnosis Uses freezing microtome or cryostat Tissue should be fresh, and freezing be done as quickly as possible o Liquid N2 (around -70°C), isopentane, CO2, aerosol sprays (most widely used) Commonly used for: o Rapid pathologic dx during surgery o Dx and research for enzyme histochemistry o Dx and research demonstration of soluble substances such as lipids and carbohydrates o Immunofluorescent and immunohistochemical staining o Some specialized silver stains (silver impregnation is the correct term), particularly in neuropathology Cryostat o 5-10 μm, frozen rapidly to about -20 to -30°C o Embedded in a gel like medium consisting of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) – counterpart of paraffin in FFPE

MLS-LAB 410 | BSMLS LESSON 3 & 4: SPECIMEN ACCESSIONING, GROSS EXAMINATION, FIXATION AND DECALCIFICATION Hispathology Laboratory WORKFLOW 1. Lab Reception - Samples are received and registered - Protocol upon receiving the specimen: It must be documented in the receiving logbook and all the necessary information must be written - The liable person should also affix his/her name and signature in the said logbook - Specimen accession follows 2. Specimen Preparation - Specimen type with correct number of cassette slides and stains - Labeling of cassettes 3. Grossing - Pathologists cut off the specimen - Dictate macrodescription of the specimen - Photograph and annotate sections 4. Automatic Tissue Processor - Tissues pass through various chemicals for fixation, dehydration, clearing, and infiltration 5. Embedding & Microtomy - Specimen from the autotechnicon will immerse into a mold with liquid paraffin wax - This is for external support so that the tissue will not crumble during microtomy - Sectioning will follow by cutting of the embedded tissues into thin slices using the microtome 6. Staining - The thin tissue slices are added with dyes for enhanced visualization and differentiation of cellular structures 7. Mounting - After staining, mounting of slides will follow in which the stained slides will be coated with transparent mounting medium 8. Pathologist Reporting - The slides will be examined under the microscope by the pathologist 9. Secretary office - Describing the results of the examined slides - Creation of final report signed by the pathologist SPECIMEN ACCESSIONING - Tissue specimen received in the laboratory have a request form that lists the patient information, history, and description in the site of origin - The specimens are accessioned by giving them a serial number that will identify each specimen for each patient. Specimen Accessioning Proces 1. Make sure all the important forms are completed such as the Histopath Request Form, Receipt of transaction and other pertinent documents. 2. Check the specimen and make sure that it was prefixed with formalin. 3. Accession of the specimen by writing the serial number (if multiple samples received from one patient follow the correct format)  Common format: Type of Specimen-Last 2 digits of the Year-Specimen control number  Example: SP-20-143

AP – Autopsy Pathology; SP – Surgical Pathology o “2019” – 19; “2020” – 20 rd o 143 specimen of the year  If more than one specimen in 1 patient: SP-20143-A A. Right Ovary B. Left Ovary C. Intestine implant D. Bladder E. Endocervical tissues 4. Write all the details in the logbook (serial number, Px’s name, age, gender, room, specimen, name of pathologist who will examine, date specimen received, due date) 5. Fill up the surgical pathology worksheet (name of the patient, accession number, additional test/s e.g. immunohistochemical testing, age and gender of the patient, due date, pathologist, specimen label, annotations after gross examination, microscopic description, diagnosis) 6. Attach all documents in one folder o

Policy - All specimens must be delivered to the laboratory immediately (within 1 hour maximum) with a completed requisition form. - Specimen information must be written legibly and completely - Incomplete labeling sample container and inadequate information in the request form will not be accepted - If no label  subject for REJECTION Requisition Form 1. Name of Patient 2. Age, Date of Birth, Sex 3. Medical Record Number 4. Date and Time of Collection 5. Type of Specimen (anatomic site) 6. Location of the Patient 7. Clinical history of the patient 8. Pre/post operation diagnosis 9. The requesting physician’s name 10. Physician or authorized person’s signature on the request form 11. Name and address of physician (for referral laboratories) Specimen Container 1. Full name of the patient 2. Medical record number 3. Age and sex of the patient 4. Type of specimen (Anatomic site) 5. Date and time of collection 6. Location of the patient Specimen Rejection Criteria: 1. Unlabeled or wrong label on the specimen 2. Incomplete patient information/clinical history 3. Left unfixed or unrefrigerated for an extended period 4. Putrefied or autolyzed specimens 5. Damaged specimen or broken slides 6. Insufficient sample for processing 7. Spilled or contaminated specimens 8. Failure of the requesting physician to enter the request in the computer

9. Empty containers without the specimen or form 10. Referral pathology consultation material without Histopathology report of referring hospital GROSS EXAMINATION - Important stage in Histopathology - Examined by Pathologist, pathology assistant, or pathology resident - It involves:  Accurate naked eye description of intact specimen  Correct method of sectioning  Gross examination of cut surface  Selection of proper tissue blocks for microscopy  Instructions for embedding and block making - Histopathology specimens are vital in patient care since not only they establish tissue diagnosis, but also they are crucial in clinical management and provide important prognostic data - Gross is an art  Specimens, large or small, are laid out on these benches, waiting for an artist to come along and perfect their craft  A knowledge of what needs to be taken for microscopic is crucial for final diagnosis - Grossing Area  Must be well illuminated and properly ventilated  The cutting board placed inside a metal box is designated in such a fashion that all the fluids flow directly into the sink  The shelves for specimen containers must be comparted  Must have a ready access to a sink with hot and cold water  Must have a ready access to formalin  Must have a box of instruments  Must have a box of cassettes  Must have a large formalin container  Must have photographic facilities  Must have a large table with sink for large specimens and a central table for multiple purposes  Most important feature: must have an exhaust fan to eradicate the strong fumes of formalin - Instruments used in Grossing  Large scissors  Dissecting scissors  Scalpel  Saw  Blade  Forceps  Ruler  Gloves  Cutting board  Cassettes Inking - When a malignancy is suspected, the specimen is often covered with ink in order to mark the margins of the specim...