Histotechnology

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"You are your own raw material. When you know
what you consist of and what you want to make of
it, then you can invent yourself." - Warren B.
Bennis
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Man is the maker of his own happiness..
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PATHOLOGYIntroduction
Dr. Venkatesh M. Shashidhar. Senior Lecturer in
Pathology Fiji School of Medicine
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Study of Disease (Pathology)

• Epidemiology
• Etiology - Causes
• Pathogenesis - Evolution
• Morphology - Structural Changes
• Clinical Significance Functional Changes
• Management Medicine, Surg, Speciality
• Prognosis
• Prevention

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Four aspects of Pathology

• Etiology - Study of Causes
• Pathogenesis - Step by step evolution
• Morphology - Structural Changes
• Clinical Significance

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Branches of clinical Pathology

• Histopathology
• Cytopathology
• Haematology
• Forensic Pathology

• Immunology
• Chemical Pathology
• Genetics
• Toxicology
• Microbiology

Anatomic Pathology
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Learning Pathology

• General Pathology
• Common changes in any tissue.
• Systemic Pathology
• Specific changes in specific organs.

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Histo-Technology

• Dr. Venkatesh M. Shashidhar
• Associate Professor of Pathology
• Fiji School of Medicine

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HISTOTECHNOLOGY

• The technique of processing the tissues
  submitted for histopathological study until the
  preparation of the stained section on a glass
  microscopic slide ready for study is known as
  Histotechnology
• And persons specializing in this technique are
  known as Histotechnologists.

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Surgical Specimen

• Clinical Details
• Adequate specimen
• Proper Fixative
• 110 proportion
• 10 buffered Formalin

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Garbage in ! Garbage out !
Communication Team spirit is vital for the
patient !
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Gross Examination

• Description
• Specimen weight measurement (approx)
• Consistency
• Photo
• Cut section

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Taking Samples

• Edge of lesions.
• Wall of cysts.
• Include normal areas.
• Avoid necrotic area.
• Whole specimen if small.
• Direction, mark

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Inking the Margins

• To mark surgical margin.
• Spread of lesion
• Malignancy
• Adequacy of removal
• Different colors to identify margins

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Fixation

• Specimen bits are placed in porous cassettes
• Not more than 5mm thick
• In 10 formalin
• 1mm/hour fixation
• 6 hour

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Fixation function (Formalin)

• Preservative
• Provides stability
• Protects from infection
• Prevents autolysis
• Permits sectioning and staining

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Tissue Processing

• To make tissue firm for slicing with molten wax.
• Multiple stages - gradual change in conc.
• Water ? Formalin ? Alcohol ? Xylene ? Paraffin ?
  Slides ? Alcohol ? Water ? Stain.

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Automated Tissue Processor
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Embedding

• Paraffin block with embedded tissue
• consistency to cut
• Paraffin blocks taken for sectioning

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Sectioning

• Microtome
• 3-10 microns
• Ribbon of sections
• taken on hot water bath

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Picking up sections

• Floating sections onto slides
• Common float artefact

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Microscope slide preparation

• Taking the section onto slide
• Flat, no air bubbles, no stretch or breaks.

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Automated Tissue Processor
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Automatic slide stainer.
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Automated Staining

• Routine stain HE
• Hematoxylin (basic)
• Eosin (acidic)
• Nucleus is acidic and cytoplasm is relatively
  basic
• Special stains

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Coverslipping

• Clearing - xylene
• Thin glass coverslips to protect the section
• Using mounting media (Eg. DPX, Resins, Canada
  balsam etc.)

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Reporting

• Reporting typing dispatch. gt90. (3-5 days)
• lt10 need further..
• Additional sections
• Deeper / Thinner sect.
• Special Stains/tech.
• Repeat grossing.
• Reference..
• Discussions with Clin.

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Time line

• Histopath - Long and complex processing.
• 1st day receive, register, if early -
  grossing.
• 2nd day Grossing, blocks - fixation.
• 3rd day Processing cutting staining.
• 4th day slides seen by pathologist.
• 5th day reporting routine gt70
• 6th day - Secretery type and record.
• gt7 days Deeper, thinner, sp. Stain, repeat

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Nearly all men can stand adversity, but if you
want to test a man's character, give him
power..!  Abraham Lincoln
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Cytopathology

• No tissue processing.
• Fixation (alcohol) Staining - gt2 days.
• PAP stain Non hematologic cancer.
• Leishman stain Haematology.
• Not a confirmatory test.
• Exfoliative Non-exfoliative (FNAC)

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PAP HistLow grade squamous intraepithelial
lesion (LSIL)
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Low grade squamous intraepithelial lesion (LSIL)
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PAP Smear - Abnormal
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Frozen Section

• Rapid 5-15 minutes.
• Rapid Freezing(-20 to -70) to harden.
• Liquid nitrogen Freezing microtome.
• Cell architecture damage ice crystals.
• No thin section possible - gt5micron.
• Rapid H E stain.
• Imprint cytology enhances quality.

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Freezing Microtome.
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Rapid HE staining
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"Real knowledge is to know the extent of ones
ignorance." -- Confucius
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Special Techniques
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Light Microscopy

• Kohler Illumination
• Condenser
• Objectives
• 2 to 4x - Low power
• 100x lens Oil Imm.
• Eye piece of 10x and objective of 40x 400 times
  magnification.

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Normal Stomach
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Normal Skin
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Normal Skeletal Muscle
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Normal Kidney
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Summary

• Grossing
• Fixation
• Processing
• Embedding
• Sectioning
• Staining
• Mounting

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To recognize ones own weakness and errors and
draw back from them is the only way towards
perfection
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Some Special Techniques
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Frozen Sections

• Freezing acts as embedding agent by forming
  minute ice crystals within cells.
• More rapid (5min),
• Liquid nitrogen.

Freezing Microtome
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Immunohistochemistry

• Antigen antibody reaction
• Ab Tagged with marker
• Simple Dye
• Enzyme (peroxidase)
• Fluorescent Dye
• Radioactive Dye

Marker Sec. Antibody Pri. Antibody Tissue
Antigen
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Melanoma ve for HMB-45
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B cell Lymphoma CD20
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Breast Cancer Estrogen Receptor Antigen
Tamoxifen Sensitive
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Polarized Microscopy

• Under Polarized light, Some materials have the
  property of "birefringence" which is the ability
  to pass light in a particular plane.
• Eg. Crystals, fat, fibers. Amyloid etc.

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Cardiac Amyloidosis
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Urine Oval Fat Bodies
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Fluorescent Microscopy

• Property of materials that causes them to absorb
  light at a shorter (UV) wavelength, and to emit
  light at a higher (visible) wavelength
• Auto-Fluorescence
• Immuno-Fluorescence

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ANA Diffuse Pattern
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ANA Nucleolar Pattern
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Electron Microscopy

• Electron beam instead of light.
• Magnified images are typically from 1000X to
  50,000X. (Light microscope is 10-1000x).
• Gluteraldehyde fixative.
• Glass knives.
• Specimen is mounted on a metal grid.

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Membranous GN
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Minimal Change GN
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