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Study of Disease: (Pathology)

 Epidemiology
 Etiology - Causes
 Pathogenesis - Evolution
 Morphology - Structural Changes
 Clinical Significance – Functional Changes
 Management
 Complications
 Prevention

Four aspects of Pathology:

 Etiology - Study of Causes


Important.!
 Pathogenesis - Step by step evolution
 Morphology - Structural Changes
 Clinical Significance*


Branches of clinical Pathology:
 Histopathology  Immunology
Anatomic
 Cytopathology  Chemical
Pathology
 Haematology Pathology
 Forensic  Genetics

Pathology  Toxicology

 Microbiology


Learning Pathology:

 General Pathology
 Common changes in tissues.
 Systemic Pathology
 Specific changes in organs.


Techniques in Pathology:
 Gross Pathology:
 Light Microscopy:
 Histopathology, Cytology, Autopsy
 Histochemistry, Biochemical
 Immunohistochemistry
 Electron Microscopy
 Cell Cultures, Medical Microbiology
 Molecular Pathology

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.


Surgical Specimen

 Clinical Details
 Adequate specimen
 Proper Fixative
 10% buffered
Formalin


Gross Examination:

Description:
 Specimen weight &
measurement (approx)
 Consistency
 Photo *
 Cut section


Taking Samples:

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


Inking the Margins*
 To mark surgical
margin.
 Spread of lesion
 Malignancy
 Adequacy of removal
 Different colors to
identify margins


Fixation:

 Specimen bits are


placed in porous
cassettes
 Not more than 5mm
thick
 In 10% formalin
 1mm/hour fixation
 ~ 6 hour

Fixation:

 After fixation is
Replacing aqueous
formalin with alcohol
in gradual sequence
(70, 95, 100%) to
make way for
paraffin.


Clearing:

 Removal of alcohol
with “xylene” that will
be miscible with the
embedding medium
(paraffin)
 Impregnating with
paraffin.


Embedding:
 Paraffin block with
embedded tissue
 consistency to cut
 Paraffin blocks taken
for sectioning


Tissue Processing:
 Preservative
 Provides stability
 Protects from infection
 Prevents autolysis
 Permits sectioning and staining


Sectioning:
 Microtome
 3-10 microns
 Ribbon of sections
 taken on hot water bath


Picking up sections:

 Floating
sections onto
slides
 Common
“float” artefact


Microscope slide preparation:
 Taking the section onto
slide
 Flat, no air bubbles, no
stretch or breaks.


Automated Staining:

 Routine stain H&E


 Hematoxylin (basic)
 Eosin (acidic)
 Nucleus is acidic and
cytoplasm is relatively
basic
 Special stains


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


Reporting:
 Additional sections
 Deeper / Thinner sect.
 Special Stains/tech.
 Reference..
 Discussions with Clin.
 Diagnosis
 Report Typing
 Despatch.
 (>3-5 days)


Cytopathology:
 Cytopathology is study of cells in diagnosis
of disease.
 Exfoliative & Non-Exfoliative - cytology.
 Exfoliative: Cell samples are collected
from normally shedding tissues like
epithelium. Spatula or brush to enhances
collection.
 Non-Exfoliative: Cells samples collected by
needles with suction pressure. (FNAC)

PAP* Smear Normal:


PAP Smear - Abnormal:


Special Techniques:


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.

Normal Stomach


Normal Skin


Normal Skeletal Muscle


Normal Kidney


Summary

1. Grossing
2. Fixation
3. Processing
4. Embedding
5. Sectioning
6. Staining
7. Mounting •
Some
Special
Techniques

Frozen Sections:

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

Immunohistochemistry
 Antigen antibody
reaction
Marker  Ab Tagged with
Sec. Antibody marker
Pri. Antibody  Simple Dye
 Enzyme (peroxidase)
Tissue Antigen
 Fluorescent Dye

 Radioactive Dye

Melanoma +ve for HMB-45


B cell Lymphoma – CD20


Breast Cancer
Estrogen Receptor Antigen
Tamoxifen Sensitive


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.

Cardiac Amyloidosis


Urine Oval Fat Bodies


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

ANA – Diffuse Pattern


ANA – Nucleolar Pattern


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.

Membranous GN


Minimal Change GN

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