1. Name, Age, Sex, Date of Birth 2. Hospital or Lab Accession 3. Specimen Type/Source; Clinical Impressions 4. Pertinent History, Operative Findings 5. Test Requested, Procedure performed 6. Date & Time of Request, Collection, and Transport 7. Requesting Physician

PRELIM LAB: THE HISTOPATHOLOGY LABORATORY Flashcards by Chin Lys

Study of tissues affected by disease

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HISTOPATHOLOGY

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Useful in making a diagnosis and in determining the severity and progress of a condition

HISTOPATHOLOGY

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Duration of specimen storage:

at least 1-2 weeks

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obsolete, not often used
nowadays

Fresh tissue examination

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should be stored in the
lab forever

Autopsy specimen

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head of the laboratory

Pathologist

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supports the pathologist

Associate Pathologist

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The medical technologist
Provides slides that are properly labeled,
processed, stained, mounted, and
sequenced

Histotechnologist/Histotechnician

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Ensures that formalin and other agents are
fresh and in good working quality
Maintains equipment in high-quality condition
Performs preventing maintenance, as well as
troubleshooting procedures

Histotechnologist/Histotechnician

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Sections large and hollow organs to allow fixation
Examines the tissue sections, cytologic slides
under the microscope
Monitors staff performance
Pinpoint problematic situations and find
solutions

Pathologist and Assistant Pathologist

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Set of procedures or technical activities on fulfilling
quality

QUALITY CONTROL

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Ensuring that everything is right (test, time,
specimen, patient, diagnosis, and price)
Includes availability of reagents, supplies, preventive maintenance and monitoring of equipment and evaluation of the quality of services

QUALITY ASSURANCE

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TYPES OF HISTOPATHOLOGIC PROCESS

Tissue Processing
Frozen Biopsy
Special Staining
Immunohistochemistry

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HISTOPATHOLOGIC TECHNIQUES

Includes all activities done in the laboratory in order to produce a suitable specimen side for viewing by the pathologist

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Proper specimen collection and processing of results and documentation
High quality of reagents and equipment
Continuous professional education of staff

QUALITY MANAGEMENT SYSTEMS

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Set of coordinated activities to regulate a lab in order to continually improve its performance
Skilled personnel
Considers pre-analytic, analytic, and post-analytic phase

QUALITY MANAGEMENT SYSTEMS

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from receiving of specimen to
encoding of patient information

Pre-analytic

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Skilled histotechnologist/histotechnician
which are responsible for:

Proper specimen collection
Proper processing of specimen
Efficient processing of results

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tissue processing phase

Analytic

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To ensure an effective QMS, the histopathology laboratory should have the following:

Skilled histotechnologist/histotechnician

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reading of slides and final
diagnosis

Post-analytic

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Numerically, alphabetically, or chronologically arranged

DOCUMENTS

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Request Form

Name, Age, Sex, Date of Birth
Hospital or Lab Accession #
Specimen Type/Source; Clinical Impressions
Pertinent History, Operative Findings
Test Requested, Procedure performed
Date & Time of Request, Collection, and
Transport
Requesting Physician

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Request Form info + Diagnosis and Gross/Microscopic findings + Name of pathologist

Patient Report

TYPES OF RESULTS

1. Surgical Pathology 2. Cytopathology 3. Autopsy Report

# TURNAROUND TIME (TAT) OF RESULTS Surgical Pathology and Cytology

2 days

# TURNAROUND TIME (TAT) OF RESULTS Frozen Sections

5-15 minutes

# TURNAROUND TIME (TAT) OF RESULTS Autopsy Report

7 days

preliminary diagnosis

Telephone Report

status of results 48-74 hours from receiving

Prelim Report

conveys results after test is completed

Final Report

documents occurrence of problems

Incident Report

Tissues for examination are usually obtained through biopsy or autopsy. They range from whole organs or very large specimens to tiny fragments of tissue

EXAMINATION OF TISSUES IN HISTOPATHOLOGY LABORATORY

● AKA Necropsy; Thanatopsy ● Post-mortem examination of tissues

AUTOPSY

AUTOPSY PURPOSES:

● Determine cause of death and extent of injury ● Uncovering existence of an undetected disease

TYPES OF AUTOPSY ACCORDING TO:

* PURPOSE * COMPLETENESS * MANNER OF INCISION

# TYPES OF AUTOPSY ACCORDING TO: PURPOSE performed on a patient who dies in a hospital during course of treatment

Medical/Hospital

# TYPES OF AUTOPSY ACCORDING TO: ● Partial ● Complete

COMPLETENESS

# TYPES OF AUTOPSY ACCORDING TO: ● Y-shaped ● Straight Cut (I-shaped)

MANNER OF INCISION

# TYPES OF AUTOPSY ACCORDING TO: PURPOSE generates evidentiary document that forms a basis for opinions rendered in a criminal trial, civil suit, and the like

Medico-legal

DISSECTION/EVISCERATION TECHNIQUES

* VIRCHOW * ROKITANSKY * GHON * LETULLE

# DISSECTION/EVISCERATION TECHNIQUES ● One by one removal of organs ● MOST WIDELY USED

VIRCHOW

# DISSECTION/EVISCERATION TECHNIQUES ● “In situ” (in place) dissection, followed by en bloc removal

ROKITANSKY

# DISSECTION/EVISCERATION TECHNIQUES ● “en bloc” removal ● Organs of same group/cavity/region are removed at the same time

GHON

# DISSECTION/EVISCERATION TECHNIQUES ● “en masse” removal of organs ● All organs are removed at the same time, then dissected by blocks

LETULLE

PREREQUISITES TO PERFORMING AUTOPSY

1. Written or informed consent from the legal next-of-kin - Order of priority: spouse, adult child, either parent, adult sibling, grandparent, guardian 2. Medical abstract or clinical data 3. Autopsy Request (suspicious evidence of foul play)

# PREREQUISITES TO PERFORMING AUTOPSY spouse, adult child, either parent, adult sibling, grandparent, guardian

Order of priority

PERSONNEL

1. Coroner 2. Prosector 3. Diener

a public official who is empowered to order an inquest into the manner or cause of death

Coroner

pathologist who performs the dissection

Prosector

comes from German word “leichendiener” meaning “servant of the dead”; assists during autopsy, and assumes many and varied responsibilities in the autopsy laboratory

Diener

Organ block removed from the body cavity should be thoroughly washed of blood using ___________ to minimize the blood staining of organs. NEVER USE HOT WATER.

cool or cold water

Organ blocks are placed in a large enameled potmcontaining fixative, filled to about __________.

⅓ capacity

Tissues should not be pressed against each other ormthe bottom or walls of the container. TRUE OR FALSE?

TRUE

Lesions that are encountered during dissection should be _____________ before organ is fully incised.

obtained early and placed in fixative

● Ante-mortem examination of tissues (ante=before; mortem=death) ● Examination of tissue sample from the living

BIOPSY

TYPES OF BIOPSY

1. FINE NEEDLE ASPIRATION (FNA) 2. CORE NEEDLE 3. INCISIONAL 4. EXCISIONAL 5. PUNCH 6. SHAVE 7. CURETTAGE

Simplest, LEAST INVASIVE Uses very thin needle attached to syringe to take out small amount of fluid and tissue from area

FINE NEEDLE ASPIRATION (FNA)

Surgical; small part of a large lesion or tumor is taken

INCISIONAL

Uses slightly larger needle Remove small column of tissue (1/16 inch in diameter, ½ inch long)

CORE NEEDLE

Surgical; entire affected area is taken

EXCISIONAL

For skin; uses circular blade to obtain deeper skin sample that removes a short cylindrical core of tissue (“apple core”)

PUNCH

For skin; small fragments of out layers of skin are “shaved” or scraped

SHAVE

Tissues are removed from body cavity (or canals) using a curette (instrument with a tip shaped like a small scoop or hook)

CURETTAGE

METHODS OF EXAMINATION OF BIOPSY SPECIMENS

1. FRESH 2. PRESERVED

Allows examination of cells in their living state

FRESH TISSUE EXAMINATION

FRESH TISSUE EXAMINATION DISADVANTAGES

Subject to ischemia, therefore not permanent, and liable to changes

FRESH TISSUE EXAMINATION ADVANTAGES

View protoplasmic activities (motion, mitosis, phagocytosis, pinocytosis)

METHODS FOR FRESH TISSUE EXAMINATION

1. Teasing 2. Squash Preparation 3. Smear a. Streaking b. Spreading c. Pull-apart d. Touch Prep 4. Frozen Sections

AKA Dissociation

TEASING

Selected tissue is immersed in petri dish/watch glass containing isotonic solution, and then carefully dissected and separated using needle or applicated stick

TEASING

Tissue is then transferred to slide and examined under the microscope (phase contrast or bright field)

TEASING

May be stained using supravital dyes

TEASING

AKA Crushing

SQUASH PREPARATION

Small pieces of tissue with diameter <1mm are compressed between two slides

SQUASH PREPARATION

May be stained using supravital dye

SQUASH PREPARATION

Method depends on nature of material to be examined

SMEAR

Useful for cytological examinations

SMEAR

Cellular materials are spread over a slide using wire loop, applicator stick or slide

SMEAR

Vital stains may also be applied

SMEAR

May be made permanent by fixing them

SMEAR

SMEAR TYPES:

a. Streaking b. Spreading c. Pull-apart d. Touch Prep

Rapid, but gentle zigzag application of the material throughout slide

Streaking

Must have a relatively uniform distribution

Streaking

Material is gently spread onto the slide, and the mucus strands are teased apart using an application stick

Spreading

ADVANTAGE: Maintains intercellular relationships

Spreading

For fresh sputum, bronchial aspirates, and thick mucoid secretions

Spreading

Slides are then pulled apart with a single, uninterrupted movement in opposite directions

Pull-apart

A drop of the material is placed into a clean slide and covered with another clean slide. Material is allowed to disperse evenly

Pull-apart

Surface of a freshly cut tissue is pressed to a clean slide

Touch Prep

AKA Impression Smear

Touch Prep

For Phase-Contrast Microscopy

Touch Prep

ADVANTAGE: Maintains intracellular relationships

Touch Prep

For rapid diagnosis of tissue

FROZEN SECTIONS

Requested during intra-operative procedures to help surgeon in choosing his next plan of action

FROZEN SECTIONS

FROZEN SECTIONS DISADVANTAGE:

relatively poor quality of the final slide; expensive

Fresh tissues are frozen using a

cryostat or freezing microtome

Recommended for demonstration of lipids and nervous tissue

FROZEN SECTIONS

FROZEN SECTIONS ADVANTAGE:

rapid processing time with less equipment requirement and less need for ventilation

Accomplished by fixing the tissues and carefully processing them to preserve their structures, then impregnating them with hardening substance to permit making thin slices suitable for staining and microscopic evaluation

FIXED TISSUE EXAMINATION

● END GOAL: produce a tissue section of good quality that allows for adequate interpretation of microscopic cellular changes for diagnosis

FIXED TISSUE EXAMINATION

# ``` Various steps required to take the tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome

TISSUE PROCESSING

Preservation of tissue constituents in a life-like manner as possible

Fixation

● Remove of calcium or lime salts from bones following fixation ● For ease of cutting

Decalcification (optional)

Removal of water

Dehydration

Removal of the dehydrating agent with the use of a clearing agent (a solvent miscible with both the dehydrant and impregnating material)

Clearing/Dealcoholization

● Replacement of clearing material with impregnating material ● Gives firm consistency to tissue for ease of handling and sectioning

Infiltration/Impregnation

Placing the infiltrated tissue into a mold filled with molten wax to form a solid tissue block

Embedding

Removal of excess wax into fine tissue sections with the use of a microtome

Trimming

Cutting of tissues into fine tissue sections with the use of a microtome

Section-Cutting/Microtomy

Application of dyes to sections for visualization

Staining

Use of media to mount a coverslip for ease of handling, storage, and protection of sectioned tissue

Mounting

Before tissues are mounted on slides, they should undergo different processes. Each step in the process necessitates the use of various specific components.

INSTRUMENTATION

Indicating of accession number and year for proper identification

Labelling

GROSS TABLE:

● Forceps ● Scalpel ● Chopping board ● Weighing balance ● Ruler ● Orientation markers ● Watch glass/petri dish ● Tissue cassettes ● Diamond pen, pencils

AUTOMATED TISSUE PROCESSORS (FDCI)

● Paraffin oven ● Embedding center ● Refrigerator: 40C ○ Freezer: 20C

Wax is heated at ____ in the embedding machine

60C

Processes that an automated machine can perform

FDCI (Fixation, Dehydration, Clearing, Infiltration)

- For unstained and transparent samples - Only oblique rays hit the object - Samples are made brightly lit against dark background

Dark Field Microscope

- Phase shifts in light passing through transparent and colorless specimen are converted into brightness (contrast) changes in the specimen, making them visible - does not require staining

Phase Contrast Microscope

for tissue and cell visualization

Microscope

simplest and most popular

Bright Field Microscope

- Designed to examine birefringent properties of anisotropic specimens - Birefringence: splitting of one ray of light into two - Anisotropism: substances that exhibit different properties when measured in different directions - NOTE: Amyloid in Congo Red has Apple Green birefringence

Polarizing Microscope

- Uses ability of substance to exhibit fluorescence - Fluorescence: emission of low frequency light of substances when they are illuminated with high energy light - Only allows observation of the specific structures which have been labeled for fluorescence - NOTE: Acridine Orange stains ssDNA – green fluorescence, and ssDNA or RNA (cytoplasm) – red fluorescence;

Fluorescence Microscope

- Uses a beam of accelerated electrons as source of illumination - Has higher resolving power than light microscopes and can reveal the structure of smaller objects

Electron Microscope

For producing tissue ribbons or sections

Microtome

KINDS OF MICROTOME

* Rotary * Cryostat * Sliding * Freezing * Rocking * Ultrathin

Microtome Main Parts:

a. Block Holder b. Knife Holder c. Pawl and Feedwheel Mechanism

MOST COMMONLY USED; for routine and serial (continuous) sections; knife is stationary

Rotary

Rotary Thickness:

3-5 micrometers

Rotary Inventor:

Minot

Rotary Microscope:

Light Microscope

Rotary Embedding material:

Paraffin

Sliding Types:

Base-Sledge and Standard Sliding

consists of a microtome (usually rotary), kept inside a cold chamber maintained at -5C to -30C (avg: -20C); capable of freezing fresh tissues, thus used for STAT diagnosis

Cryostat

knife is moving

Sliding

invented by Queckette (1848)

Freezing

SIMPLEST

Rocking

Sliding Inventor:

Addams (1783)

60-100 nm thickness of tissue - Mostly used for tissues embedded in epoxy resin

Ultrathin

best for antigen preservation

Microwave Oven

used in epitope retrieval for immunohistochemistry

microwave oven

eliminate the need for the laborious manual staining

automated stainers

agitation and heating will increased fixation rate

microwave oven

- In situ hybridization and enzyme reactions - Thermal Requirements: 37C

Incubator Oven

may operate through: - dipping the slides into the stain; and/or - applying the stain to the slides

automated stainers

Eliminate the need for the laborious manual staining

automated stainers

slide driers thermal requirement:

5-10C HIGHER than the melting point of paraffin

removing water collected during sectioning (water from floatation bath)

slide dryers

causes uneven staining, artifacts formation and tissue destruction

Overheating

Fishing out of tissue section; keeps sections from wrinkling

FLOTATION BATH

Has “black” interior, for easy visualization of sections

FLOTATION BATH

FLOTATION BATH Thermal requirement:

5-10C LOWER than the melting point of paraffin

System designed for paraffin embedding

EMBEDDING CENTERS

EMBEDDING CENTERS Thermal requirement:

2-4C HIGHER than the melting point of the paraffin

MOST COMMON EMBEDDING CENTERS

PAPER BOAT

EMBEDDING CENTERS Components:

○ Paraffin dispenser and reservoir ○ Orientation stage ○ Chilling plate ○ Warm storage (for embedding mold)

● May be stationary or moving ● Equipped with alarm and warn technicians if high temperature ● Often use vacuum in heat to speed up procedures

AUTOMATED TISSUE PROCESSOR

AUTOMATED TISSUE PROCESSOR MAIN TYPES:

* Tissue-Transfer Machine (dip and dunk) * Fluid-Transfer Machine (enclosed)

specimens are transferred from container to container

Tissue-Transfer Machine (dip and dunk)

specimens are held in a single chamber, and then fluids are pumped in and out as required

Fluid-Transfer Machine (enclosed)

For gross examination and dissection of submitted specimens

GROSS TABLE

GROSS TABLE It should have:

○ Sink ○ Table Top ○ Water Supply ○ Irrigation System ○ Fume Ventilation ○ Waste Disposal Unit

Coplin jars, staining racks, staining dishes (all three are for staining) and other glass and plastic materials (for storage and preparation of solutions)

Wares

measurement of solution and buffers

pH Meters

for gross examination

Grossing Tools

Freezer Thermal requirement:

-20C

Refrigerators Thermal requirement:

– liver tissues and renal tissues | Tissue cassette

YELLOW

routine tissues (fallopian tube, cervix, gallbladder, appendix) | Tissue cassette

GREEN

BONE SAMPLE | Tissue cassette

WHITE

SAMPLE IS SKIN | Tissue cassette

GRAY

lymph node | Tissue cassette

PINK

PRELIM LAB: THE HISTOPATHOLOGY LABORATORY Flashcards

- HISTPATHOLOGY LABORATORY, EXAMINATION OF TISSUES IN HISTOPATHOLOGY LABORATORY, FIXED TISSUE EXAMINATION