https://www.slideshare.net/AdnanSami84/adnan-adil-physical-examination-of-urinepptx-260031620

3.  INTRODUCTION
 RECEIVING
 FIXATION
 DECALCIFICATION
 DEHYDRATION
 CLEARING
 IMPREGNATION
 EMBEDDING
 MICROTOMY
 SMEAR PREPARATION
 DEPARAFFINATION
 STAINNING
 MOUNTING

4. INTRODUCTION
 Tissue processing is the technique by which fixed tissues are made suitable
for embedding within a supportive medium such as paraffin.
 The aim of tissue processing is to embed the tissue in a solid medium firm
enough to support the tissue and give it sufficient rigidity to enable thin
sections to be cut, and yet soft enough not to damage the knife or tissue.
 There are three main steps in tissue processing, namely: 'dehydration',
'clearing', and ‘impregnation’.
 The tissue is removed from the body for diagnosis through the process of :-
o Biopsy – removing tissue from living body.
o Autopsy – removing tissue from dead body.

5. RECEIVING AND LABELING OF
SAMPLE
 Proper identification and orientation of the specimen.
 The tissue be properly labelled so avoid any confusion.
 When the specimen is received give a specific individual.
 Properly completed histopathology requisition from containing
 Patient name
 Age
 Sex
 Relevant
 Clinical data
 Surgical findings
 Name of tissue submitted

6. fixation
Fixation is important due to prevent the tissue from
autolysis and putrefication.
Fix the tissue so will not change their volume and shape
during processing.
The most common fixative use in lab is 10% formalin.
 Example:-formalin , Ethyl alcohol, picric acid ,acetic acid

7. GROSSING
The term grossing means inspecting the
specimens describe and measuring the tissue if
needed and sectioning the tissue to be
processed for diagnosis.(gross section that
containing or suspected tumor cells.)
After labelling with soft lead pencil tissue placed
in small plastic cassette which holds the tissue.

8. Decalcification
 Decalcification is a process of complete removal of calcium
salt from the tissue like bone , teeth and other calcified tissue
following fixation.
 The specimen is soft enough to allow cutting with the
microtome knife.
 Example
 NITRIC ACID 5%
 HCL
 FORMIC ACID 8%

9. DEHYDRATION
 Water is completely removed form fixed tissue because water is not
miscible in wax
 PROCEDURE
 Cassete are passed through a series of increasing asending order(form higher
to lower concentration) in alcohol.
 70% (15 min)
 80%(15 min)
 90%(15 min)
 100%(30 min)
 Other dehydrating agent such as acetone ,methyl alcohol, ethenol

10. CLEARING
 During dehydration water in tissue has been replaced by
alcohol that paraffin wax is not alcohol soluble.
 We replace alcohol with a substance in which wax is soluble
 Clearing is required when dehydrating agent is not miscible
with the impregnating medium
 Clearing agent :- xylene ,chloroform,
 Xylene 20min
 Xylene 20min
 Xylene 45min

11. IMPREGNATION
 Impregnation means diffuse wax in the tissue by replacing the
xylene
 Clearing agent is eliminate form the tissue by diffusion in the
surrounding melted wax
 PROCEDURE
 Replace xylene with paraffine
 2 bath in melted paraffin 1hr
 Temeprature of paraffin over should be b/w 50-60’c
 Remove all bubbles
 Amount of wax should be 25-50 times the volume of tissue

13. EMBEDDING
 The infiteration and impregnation tissue is placed in warm liquid paraffin which forms a trim block after
cooling.
 Leukharts L pieces these are two L pices which are made on metals usually brass.

14. MICROTOMY
 Microtome is the instrument used in histopath lab to cut the tissue block into extreme thin
slices and this process is called microtomy.
 size of tissue slice 3-5μm

15. SLIDE PREPARATION
 After microtomy the section are floated on the warm water bath for 15
min that remove wrinkles and section become flate.
 Then take a slide and place a drop of egg albumin and carefully take the
tissue on the slide
 After that perform the process of deparaffination ,place the slide into
the hot air over so the all wax get melted it is necessary before staining
process because wax is not miscible in staing

16. STAINING AND MOUNTING
Hematoxylin and eosin stain (or haematoxylin and
eosin stain or hematoxylin-eosin stain; often
abbreviated as H&E stain or HE stain) is one of the
principal tissue stains used in histology It is the most
widely used stain in medical diagnosis and is often
the gold standard.
H&E is the combination of two histological
stains: hematoxylin and eosin. The hematoxylin
stains cell nuclei a purplish blue, and eosin stains
cytoplasm pink

17. • Deparaffinization: flame the slide on burner and place in
the xylene. Repeat the treatment to remove the wax.
• Hydration: Drain xylene and hydrate the tissue section
by passing through decreasing concentration of alcohol
baths (100%, 90%, 80%, 70%) and water.
• Nuclear Staining: Stain in hematoxylin for 3-5
minutes.(purplish blue)
• Wash in running tap water until sections “blue” for 5
minutes or less.
• Differentiation: selective removal of excess dye from the
section). Dip in 1% acid alcohol (1% HCl in 70% alcohol)
for a few seconds.
• Blueing: Rinse in running tap water. Dip in ammonia

18. • Counterstain: Stain in 1% Eosin Y for 10
minutes.(pink)
• Wash in tap water for 1-5 minutes.
• Dehydration: Dehydrate in increasing
concentration of alcohols.
• Clearing: Put slides in two xylene baths for
clearing.
• Mounting: Mount in DPX or other mounting
media.
• Observe under compund microscope.

19. nucleus in purplish blue and cytoplasm in pink