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Special techniques in
histopathology
Dr. A.A.F. Banjo, FMCPath
Associate Professor / Consultant
Department of Morbid Anatomy
College of Medicine/Lagos
University Teaching Hospital,
Idi-araba, Lagos.
Introduction.
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Special techniques are those that are used as
an adjunct to routine histopathological
techniques.
There are a wide variety of special studies
available to evaluate pathologic processes from
simple histochemical stains to the investigation
of gene expression
It is important that the pathologist is familiar with
the special studies available because the initial
handling of the gross specimen can limit the
types of studies that can be performed.
Routine histopathological
techniques
A.
B.
C.
D.
E.
F.
G.
H.
Request for pathologic evaluation
Specimen identification
Specimen Accessioning
Gross Examination
Fixation - types of fixatives
Tissue Processing
Sectioning
Staining
A. Request for pathologic
evaluation
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The pathologist plays an essential role in patient
care as a diagnostician, patient advocate and
the clinical teacher.
He examines all tissues submitted for
histopathological examination. These include
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All tissues. hair fingernails, toenails removed for
cosmetic reasons are not included unless there are
specific reasons
All products of conception
All medical devices implanted in the body and later
removed(exclude iv catheters, endotracheal tubes)
All foreign objects removed from the body including
objects introduced by trauma, and bullets.
Submitting specimen.
The following information most be provided
 Identification of the patient
 Identification of the individual making the
request
 Date of procedure and time if relevant
 Adequate clinical history
 Specimen identification,test required and any
special handling required
 Timely and appropriate transport to the
laboratory
 Instructions for disposal of the gross specimen.
Adequate clinical history.
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Purpose of removal of the specimen and
the type of specimen.
Gross appearance and locations of any
lesion present
Prior diagnosis
Prior or current treatment.
Specific purpose of consultations
Rush specimens
Fixation(1).
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Properties.
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Preserves tissue by preventing autolysis by
cellular enzymes and decomposition by the
actions of bacteria and molds
Hardens tissue to allow thin sectioning
Devitalize or inactivates infectious agents
exception CJD infections on glass slides
Stabilizes tissue components
Enhances avidity for dyes
Fixation (2).
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Undesirable effects
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Alteration of protein structure
Solubility of tissue components
Shrinkage of tissues
DNA and RNA degradation
Factors affecting fixation.
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Volume
Access of fixative to tissues
Time
Temperature
Buffer
pH
Fixation (3).
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Types of fixatives.
 Aldehydes
 Alcohols
 Mercurials
 Oxidizing agents
 Picrates.
Most commonly used fixative is 10% buffered formal saline or
Formalin
Boiun’s solution for testis small biopsies will also decalcify
Carnoy’s contains alcohol for rapid processing.Dissolves fat
good for identifying lymph node
B-5 for lymphoid tisues
Tissue processing.
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The tissue undergoes automatic processing
through three basic steps.
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Dehydration
Clearing
Infiltration
Problems with submitted tissue.
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Fatty tissues
Tissue too thick
Calcified tissues
Hair
Hard foreign material
Multiple small tissue fragments
Special techniques
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Histochemistry
Enzyme histochemistry
Immunohistochemistry
Flow cytometry
Cytogenetics
Molecular biology
Specimen radiography
Histochemistry.
Histochemistry.
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Almost all histochemical stains are
suitable for formalin fixed tissues
The commonly used ones are those for
the detection of carbohydrates, mucin
connective tissue, amyloid,lipids cells of
the neuro-endocrine system, microorganisms, and neuro-pathological
techniques
Stains for connective tissue.
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This is the name applied to the tissues that connect and
support the other tissues in the body.
It consist of a cellular portion in an enveloping framework of
non cellular substance
The ratio of cells to intercellular substance varies from one
type of connective tissue to other
The nature of the intercellular substance varies according to
function and it is classified into two main groups.
 Formed or fibrous type collagen, reticulin, elastic
 Amorphous or gel types glucosaminoglycans Structural
glycoproteins
Connective tissue
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Cells
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Fibroblast
Fat cells
Types of connective tissue
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Areolar
Adipose
Myxoid connective tissue
Dense connective tissue
Cartilage
Bone
Muscle
Connective tissue stains
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Trichrome stain collective name for
demonstration of muscle, collagen fibers, fibrin
and erythrocytes. Three stains are employed
one of which is a nuclear stain
Factors affecting trichrome stain.
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Tissue permeability and dye molecular size
Heat
pH 1.5-3.0
Effects of fixation. Sub optimal results with prolonged
fixation in formaldehyde. treat with picric acid or
mercuric chloride to enhance staining
Preferred fixative for trichrome
Connective tissues stains
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Trichrome stain
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Van Gieson technique buffered formalin satisfactory
Masson’s trichrome
Demonstration of fibrin.
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Masson’s trichrome stain older deposits of fibrin like
collagen.
Martius Scarlet Blue technique differentiates between
old and new fibrin.
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Early red,old yellow older blue
Elastic tissue
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verhoeff’s method stains fine fibers less satisfactory
Orcein simple
Reticulin stain
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Reticulin two types
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Those using dyes
Impregnation techniques
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Gordon and sweet, Gomori
Hematologica;l stains
Biologic Amines
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Cells that produce polypeptide hormones, active amines,
or amine precursors (epinephrine, norepinephrine) can
be found individually (Kulchitsky cell of GI tract) or as a
group (adrenal medulla).
Traditional classification of the staining patterns based
upon the ability of the cells to reduce ammoniacal silver
nitrate to metallic silver (black deposit in tissue section):
Chromaffin
Argentaffin
Argyrophil (pre-reduction step necessary)
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The distinction bw chromaffin and argentaffin is artificial.
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Depends upon the fixative used.
"Chromaffin" cells have cytoplasmic granules that appear brown
when fixed with a dichromate solution.
"Argentaffin" cells reduce a silver solution to metallic silver after
formalin fixation.
Either reaction can be produced depending upon which
fixative was used.
Chromaffin reaction - adrenal medulla or extraadrenal
paraganglion tissues (pheochromocytomas)
Argentaffin reaction-carcinoid tumors of the gut.
pre-reduction step -called "argyrophil"
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Types of stains for argentaffin include:
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Types of stains for chromaffin include:
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Diazo (diazonium salts)
Fontana-Masson
Schmorl's
Autofluorescence
Modified Giemsa
Schmorl's
Wiesel's
Types of stains for argyrophil include:
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Grimelius (Bouin's fixative preferred)
Pascual's
Amyloid.
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Congo red
Polarising microscope
Methyl voilet
Immunofluorescence using thioflavine for
cerebral plaques
Amyloid
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Pretreatment with
KMNO4 used to
differentiate between
AL and AA amyloid
Carbohydrates(1)
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There are a variety of mucin stains trying to
demonstrate one or more types of mucin
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Neutral mucins GI , prostate PAS, not with alcian blue
collidal iron, mucicarmine or metachromatic dyes
Acid simple or non sulphated mucin stain with PAS,
alcian blue at pH 2.5. Colloidal iron and
metachromatic dyes. Resist hyaluronidase digestion
Acid simple mesenchymal mucin contain hyaluronic
acid found in tissue stroma. Don't stain with PAS.
Stains with alcian blue at pH 2.5,colloidal iron and
metachromatic dyes digest with hyaluronidase
carbohydrate
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Acid complex or sulpahated
adenocarcinomas PAS + alcian blue at pH 1,
colloidal iron positive, mucicarcimine,
metachromatic stains resist digestion with
hyaluronidase.
Acid complex or connective tissue found in
tissue stroma, cartilage, bone . Include
chondroitin sulphate and keratin sulfate. PAS
+ alcian blue at pH 0.5
Mucin stains
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Most specific is murcicamine
Most sensitive is PAS
Alcian blue simple but has a lot of
background staining .
Colloidal iron unpredictable
Pigments
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Artefact pigments
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Formalin
Malarial
Mercury
dichromate
Endogenous pigments
Exogenous pigments
Artefact pigments
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Formalin formed in formalin with acid pH.
Occurs in blood rich tissues
Microcrystalline structure birefregrent in
polarized light
Remove from unstained sections by
treatment with picric acid
Best to prevent by using buffered formalin
fixative
Malarial pigments.
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Similar to formalin
Remove with treatment with saturated
alcoholic picric acid for longer periods.
Endogenous pigments
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Hemoglobin
Bile pigments
The lipofuscins
Melanin and pseudo-melanin
Chromaffin, argentaffin and argyrophyll
Hemosiderin and iron
Calcium
Cupper
Uric acid and urates.
Hemoglobin.
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Appears as vivid orange- red in H&E
Leuco-Patent blue V method for hemoglobulin
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Unreliable in tissues fixed for more than 36 hrs
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Hemoglobin dark blue
Oxidase dark blue
Nuclei acid red
Use
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Based on the demonstration of peroxidase activity
identification of cast in hemoglobinuria, GN
Simple method Dunn-Thompson picric acid and
acid fuschin technique neutral needs buffered
Bilirubin,biliverdin and
haemotoidin
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Fouchet –van Gieson technique
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Bile pigment blue green
Muscle yellow
Collagen red
Use controls
Treated with fouchet not counterstained with van
gieson
Gremelin is the only technique that will
demonstrate both liver bile, gall bladder bile and
hematoidin
Lipofusin
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Early lipofusin
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Staining characteristics of
lipids
Sudan black lipofuschin
black
Ziehl –Neelson magenta
Late lipofuschin
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Schmorl reaction
melanin,liver bile,
hematoidin
Varying shades of blue
melanin
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Masson’s fontana
Confirm using Mallory
bleach extraction
method
Iron
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Perls stain or
prussian blue rection
Always use a postive
control
Mercurial fixatives
best for bone marrow
Urates
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Present as sodium urates
in tissues
Tissue most be fixed iin
95% or absolute alcohol
to prevent leaching of
urates
Methamine silver stains
urates black
Polarisable yellow with
red plate, blue when axis
is aligned at 90o to wave
Cupper
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Stains: Rubeanic acid
and Rhodamine
Used to diagnose
Wilson's disease
which is a rare
disease transmitted
as an autosomal
recessive disorder
arising from def of
caeruloplasmin
calcium
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Only calcium that is bound to an anion can be
measured.
Alizarin red S method orange red at pH4.2
Useful to detect small amounts of calcium in
Michaelis-Gutman bodies
Can be used to measure serum calcium
photomerically
Von kossa demonstrates phosphates and
carbonates present with calcium
Exogenous pigments
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Asbestos ferruginous bodies
Silica polarized light
Carbon anthracotic pigments in lungs dd
melanin
Lead -Rhodizonate method
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lead salts red background green
Beryllium solochrome auzine method
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2 methods to differentiate between Al and Be
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Aluminuim and beryllium black
Beryllium blue black
Silver -Rhodamine method
Fat stains
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Oil red O to identify
neutral lipids and fatty
acids
Use fresh or formalin
fixed sections cut on
cryostat
Micro-organism
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Bacteria H&E blue
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Fungi
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Gram negatives don't stain well with gram
Brown and Brenn modification usually used
Blue with H&E. red with PAS most sensitive is the
Gomori methenamine silver stain or Gridley stain
Spirochetes Warthin-starry silver stain
AFB
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Zeihl Neelsen stain,
Fite farraco for m.leprae
most sensitive is auramine rhodamine stain
AFB
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Fat removed by
processing.
Auramine rhodamine
most sensitive
GMS for pneumocystis carinii
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Lots of background
artefacts.
PAS for candida
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PAS around purpose
stain
Immunohistochemistry.
Immunohistochemistry.
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Definition. It is a technique for the identification
of cellular or tissue constituents(antigens) by
means of antigen –antibody interactions, the site
of the antibody binding being identified either by
direct labeling of the antibody or by the use of a
secondary labeling method
There are two methods
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Direct
Indirect
Direct method
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Simplest and shortest.
The primary antiserum is conjugated directly
with a tracer molecule such as the horseradish
peroxidase
Least sensitive method
Antibody may need to be conjugated if not
commercially available and conjugate may be
low
Large aggregates of antibody may cause
background staining
Indirect method
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The primary uncongugated antibody is allowed
to bind to the antigen in tissue sections
A second tracer conjugated antibody raised in
another animal host and specific for the animal
and immunoglobulin class of the primary
antibody and allowed to bind to the primary
antibody
It is more sensitive than direct, rapid and
expensive
A common conjugate second antibody can be
used
Use of immunohistochemistry.
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Immunohistochemistry is used to accumulate
evidence for or against for or against specific
diagnostic possibilities
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Most be used in panels, and interpreted based on the
immunohistochemi9srty profile.
Slides most be pretreated
Factors affecting immunoreactivity.
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Type and duration of fixation
Prior decalcification
Temperature of baking the slide
Length of time glass was made
Antigen retival procedures
Type of antibody
Controls.
Positive and negative controls
Negative controls
Evaluation.
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Local of immunoreactivity
Identification of immunoreactive cells
Intensity of immunoreactivity
Number of immunoreactive cells
Common panels
Toxoplasma
The future-genomic
immunohistochemistry
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To date, immunohistochemistry has
largely focused on markers of cell type, as
an aid to the diagnosis of specific tumours.
More recently, it has been applied to a
limited number of markers of cell
proliferation, such as Ki-67 and PCNA,
The future-genogenic
immunohistochemistry
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To date, immunohistochemistry has
largely focused on markers of cell type, as
an aid to the diagnosis of specific tumours.
More recently, it has been applied to a
limited number of markers of cell
proliferation, such as Ki-67 and PCNA, as
prognostic factors in malignant tumours.
Genomic immunohistochemistry
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Documentation of loss of expression of a protein
due to specific mutations, usually truncation
mutations.
 Loss of expression of E-cadherin in lobular breast
cancer.
 Hereditary non-polyposis colon cancer arises
because of mutations in genes for DNA repair,
principally hMSH2 and hMLH1.
Identification of proteins expressed as a
consequence of specific translocations
 Expression of FLI-1 differentiates PNET/EWS from
other small blue round cell tumours
Genomic imunohistochmistry
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Identification of molecular targets for novel tumour
therapies
 Expression of c-kit (CD117) is highly characteristic of
gastrointestinal stromal tumours. The drug STI571
blocks the kinase pocket of c-kit and is highly
effective in the treatment of GISTs.
Identification of gene amplification and tumour
therapy
 Identification of overexpression of the HER-2/neu
protein to validate the use of trastuzumab
(HerceptinTM) in the treatment of breast cancer
patients. The HER-2/neu gene is overexpressed due
to gene amplification
Electron microscopy
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The only way to improve resolving power is to
reduce substantially the wavelength of the light.
This is achieved by the electromagnetic beam of
the electron microscope. The beam is focused
through the object suspended on its metal grid,
and is magnified before striking a fluorescent
screen to be transformed into a visible image
The resolutions so far achieved in biology with
transmission electron microscopy (TEM/EM) are
of the order of 1 nm at a magnification of X 2
000 000.
Electron microscopy.
Uses of electron microscopy.
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Diagnosis of child hood small round cells.
Diagnostic renal biopsies for glomerular disease
Difficult to classify tumours
Nerve and muscle biopsies
Bullous skin diseases
Ciliary dysmorphology
Fine needle aspirations
Endomyocardial biopsies(adriamycin toxicity,
amyloid)
Liver biopsies for microvesicular fat in acute fatty
liver of pregnancy
Method
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Ultra structural details of tissues are lost
rapidly so fresh tissue must be fixed
quickly and well
Fixative 2% paraformaldehyde and 2.5%
glutalraldehde in 0.1M cacodylate buffer at
pH 7.4
Minimal change Disease
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Effacement of foot
processes
Basement membrane
Red blood cells
Immunofluorescence
immunofluorescence
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Detects antigens in tissue.
Because signal is not amplified, it is better than
IM for the precise location of antigen antibody
complexes and for determining deposition
pattern of immune complexes
Uses immune complex deposition in glomerular
disease and bullous disease of the skin
Tissue may be snapped frozen or stored in
fixative for IF
Immunofluorescence.
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There are 2 methods
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Direct immunoflourescene antibodies to
detect antigen in patients tissue
Indirect uses control tissues to detect
antibodies in patients serum
Uses of immunoflorescence
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Skin biopsies e.g.. Lupus. Pemphigus,
pemphigoid, dermatitis herpetiformis
All diagnostic non transplant renal
biopsies
Some transplant renal biopsies
Evaluation of vasculitis in nerve biopsies
immunofluorescence
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The localization at the
dermal-epidermal
junction is typical, for
immune complexes tend
to be trapped along
basement membranes.
Diseases with this pattern
may include systemic
lupus erythematosus,
discoid lupus
erythematosus, and
bullous pemphigoid.
Tissue arrays
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Tissue microarrays are a method of re-locating tissue
from conventional histologic paraffin blocks such that
tissue from multiple patients or blocks can be seen on
the same slide.
This is done by using a needle to biopsy a standard
histologic sections and placing the core into an array on
a recipient paraffin block.
This technology should not be confused with DNA
microarrays where each tiny spot represents a unique
cloned cDNA or oligonucleotide.
In tissue microarrays, the spots are larger and contain
small histologic sections from unique tissues or tumors.
Advantages of tissue arrays
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amplification of a scarce resource,
experimental uniformity
decreased assay volume.
does not destroy original block for
diagnosis
Technique
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Robotic technology is employed in the
preparation of most arrays. The DNA
sequences are bound to a surface such as
a nylon membrane or glass slide at
precisely defined locations on a grid.
Using an alternate method, some arrays
are produced using laser lithographic
processes and are referred to as biochips
or gene chips.
Molecular genetic pathology.
Applications of Molecular genetic
pathology.
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Identification of Inherited diseases e.g. cystic
fibrosis, hemochromatosis, Factor V leden,
prothrombin 20210A, fragile X syndrome;
identification of genes conferring susceptibility to
diseases e.g. BRCA1
Detection of infectious diseases, identification of
specific organisms, quantitation of viral infection
Neoplasms. Identification of specific genetic
aberrations associated with tumours,
identification of clonality in hemato-lymphoid
proliferations, detection of minimal residual
diseases after treatment.
Techniques.
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Southern blotting
Polymerase chain reaction
Fluorescent in-situ hybridization
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The FISH technique is
shown with interphase
nuclei in three panels. A
specific DNA probe with a
fluorescent tag identifies
a specific region of a
chromosome. Different
colored fluorescent tags
for probes allow
identification of various
abnormalities.
DNA micro arrays-introduction
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Alterations in gene expression patterns or in a
DNA sequence can have profound effects on
biological functions. These variations in gene
expression are at the core of altered physiologic
and pathologic processes.
DNA array technologies provide rapid and costeffective methods of identifying gene expression
and genetic variations.
DNA array-technique
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DNA microarrays typically consist of thousands of
immobilized DNA sequences present on a miniaturized
surface the size of a business card or less.
Arrays are used to analyze a sample for the presence of
gene variations or mutations (genotyping), or for patterns
of gene expression, performing the equivalent of ca.
5,000 to 10,000 individual "test tube" experiments in
approximately two days of time.
Microarrays are distinguished from macroarrays in that
the DNA spot size is smaller, allowing the presence of
thousands of DNA sequences instead of the hundreds
present on macroarrays.
Types of DNA arrays
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The composition of DNA on the arrays is of two general
types:
Oligonucleotides or DNA fragments (approximately 2025 nucleotide bases). These arrays are frequently used
in genotyping experiments. The sequences of alternate
gene forms may be included for detection of mutations
or normal variants (polymorphisms).
Complete or partial cDNA (approximately 500-5,000
nucleotide bases). These arrays are generally used for
relative gene expression analysis of two or more
samples.
Applications of DNA micro arrays.
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Diagnostics
Custom Drug Selection
Discovery of Therapeutic Targets
Predict drug/toxin activity
Accelerate FDA approval
Determination of Pharmacologic
Mechanism
Future directions
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Custom designed DNA variation detection arrays will be
used to scan the genome and detect single nucleotide
polymorphisms (SNPs). The SNPs that are identified can
be used in designing further genotyping chips for
performing association and linkage analysis.
DNA arrays will assist in the formation of information
databases to assist in correlating genetic variation and
gene expression with patient status, prognosis and
responsiveness to treatment.
DNA array analysis provides an excellent tool to enlarge
our knowledge of genome function
Cytogenetics.
Indications.
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Soft tissue tumours
Mesotheliomas
Unusual tumours
Poorly differentiated tumours
Method of submitting tissue
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The tissue most be fresh, viable and
relatively sterile
If left overnight, mince and cover with
culture medium
Philadelphia chromosome
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"Philadelphia
chromosome" of
chronic myelogenous
leukaemia, which is
really a 9:22
translocation.
Flow cytometry
Introduction.
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Flow cytometry analysis disaggregated
cells as they pass by stationary detectors
It measures cell size, and cytoplasmic
granularity and DNA content
DNA content can be used to determine
number of cells in S-phase
Because cells are not visualized, it is
important to only submit lesional tissues
Indications for flow cytometry.
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Indications for ploidy and S phase analysis
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Hydatidiform mole
Carcinomas for prognosis
Indications for cell surface markers
analysis
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Lymphomas
leukemias
Tissue processing
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Submit single cell suspension
Tissue most be viable
Formalin fixed paraffin embedded tissues
can also be used for DNA ploidy analysis
but results are not satisfactory
Cytologic preparations from
surgical specimens.
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For intra operative diagnosis
Infectious disease
Neuropathology cases
Tumours
For special studies
Advantage. Nuclear not cut therefore intact
FISH and image analysis
Specimen radiography.
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Preferable to patient radiograph
A permanent record of the radiograph can be
kept with the a case
Reveals greater detail of the lesion
There might be a significant time interval
between removal of tissue and patient
radiograph
Indicates important sites to sample
To confirm that clinical lesion was removed
Indications for specimen
radiography
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Tumors of bone and cartilage
Tumours invading in to bone
Avascular necrosis of bone
All prosthetic heart valves to document the
degree of calcification
Breast biopsies or mastectomies
performed for mammographic lesions that
can not be located grossly
Microbiologic cultures and smears
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
Indications
 suspected infectious processes
 Suspected sarcoidosis as to exclude an infectious process
Types of culture
 Routine
 Bacteria(aerobic), mycobacteria, fungi
 Special
 Anaerobic, salmonella,norcardia, N.
gonorrhea.legionella, helicobacter
 Viral
 CMV,varicella-zooster,adenovirus herpes
 Influenza a B , RSV,para influenza require special
techniques