HISTOPATHOLOGY

SUBMITTED BY:-
MONIKA SHARMA
B.Sc.MLT (6SEM)
1640991109
 DEFINITION
• It is the branch of science which deals with the
gross and microscopic study of tissue affected
by the disease.
 WORKFLOW
• ACCESSIONING.
• GROSSING.
• TISSUE PROCESSING.
• EMBEDDING.
• SECTION CUTTING.
• STAINING.
• MOUNTING.
 ACCESSIONING
 All the specimens are received ,sorted and
entered into the laboratory information system ,
labelled with barcode.
 All the biopsies are given biopsy number and
labelled with it.
 Biopsy form and worksheet are attached and
biopsy number is written on it.
 GROSSING
• Grossing involves a careful examination of
specimen to ensure that the proper area is
chosen.
• Large specimen may require further dissection
for selecting appropriate area.
• The tissue selected for processing are placed
in cassettes labelled with biopsy number.
 TISSUE PROCESSING
• Tissue processing involves the steps required
from fixation to embedding of tissue.
 EMBEDDING
• In this step, tissue is transferred form
cassettes to a mould filled with melted
paraffin wax.
• Then it is allowed to solidify by placing the
moulds on cooling center.
• Embedding media used is paraffin.
 TRIMMING
• After embedding , extra wax is removed from
the blocks .
• Wax is removed from the four corners with
the help of a sharp blade.
• All the blocks are rough cut and placed in
nitric acid for proper section cutting.
 MICROTOMY
• This step is also known as section cutting.
• It is a procedure in which blocks are cut or
sectioned in thin ribbons of varying thickness
are prepared.
• The instrument by which section cutting is
done is known as microtome.
• Rotary microtome is used.
 ROTARY MICROTOME
• It is the most commonly used microtome.
• In this , the block holder moves up and down
while the knife remains fixed.
• It is suitable for cutting of small tissues and
serial sections .
• Parts of rotary microtome:-
• Block holder.
• Knife clamps.
• Knife clamps screw.
• Block adjustment.
• Thickness gauge.
• Operating handle.
• After section cutting , sections are lifted from
floatation bath (temp-50 degree) using slides
covered with adhesive (egg albumin).
• After lifting , slides are placed on hot plate
(temp-70 degree).
 STAINING
• The most commonly used stain for routine
studies is haematoxylin and eosin stain.
• Principle:-
• H and E stain contains acidic and basic dye.It is
used to differentiate nucleus and cytoplasm .
Haematoxylin is basic dye and it will stain
acidic part (i.e-nucleus) and eosin is an acidic
dye and it will stain basic part (i.e-cytoplasm).
 Procedure
• Deparafinize the slides by placing the slides in
xylene 1 (10 minutes) and xylene 2 (4dips).
• Rehydration using absolute alcohol 1,absolute
alcohol 2 ,70% alcohol (decreasing order)
(2dips each).
• Stain with haematoxylin for 6 minutes.
• Wash under running tap water.
• Fast dip in 1%acid alcohol.
• Wash under running tap water.
• Stain with eosin for 2 minutes.
• Rehydrate by using absolute alcohol 1
,absolute alcohol 2, 70% alcohol (2 dips each).
• 4 dips in each xylene 1 and xylene2.
 INTERPRETATION
• Nucleus stains blue.
• Cytoplasm stains pink.
 MOUNITNG
• It is done to preserve the morphology of cells
of tissue.
• Mounting media used is DPX
• Refractive index = 1.515.