This is about General Histopathology and its techniques.

1. •

2. GENERAL
HISTO-
PATHOLOGY
PREPAREDBY
FAZALABBAS
GCUF

3. • Introduction to histology?
• What is histopathology?
• Surgical Specimens or biopsies?
• Collection of biopsy specimens?
• Merits and demerits of different types of biopsies?
• What are common histological techniques?
LearningObjectives:

4. • Histology is the study of
tissues and their
structure.
• Disease processes affect
tissues in distinctive
ways, which depend on
the type of tissue, and the
disease itself.
Histology
“Histo” means “tissue”
&
“logy” means “study”

5. • Histopathology refers to the
examination of a biopsy or
surgical specimen by
a pathologist to diagnose any
histological disease.
• Histopathological examination
of tissues starts with the
surgery, biopsy,or autopsy.
• The tissue is removed from
the body or plant, and then,
often following expert dissection
in the fresh state, placed in
a fixative for specific time
interval and then examined
under microscope.
Histopathology:

6. Biopsy
• A biopsy is a small piece
of tissue removed
primarily for the
purposes of surgical
pathology analysis,
most often in order to
render a definitive
diagnosis.
Surgical
Resection
• Surgical resection specimen
are obtained by therapeutic
surgical removal of an
entire diseased area or
organ (and multiple
organs). These procedures
are often intended for
surgical treatment of a
disease in which the
diagnosis is already known.
SurgicalSpecimens:
Surgical specimens mostly of two types:

7. Fixation
Chemical Fixation
Storage
Jars with screws
Labelling
Accurate labelling
CollectionofBiopsyspecimens:
There are three steps involved:

8. • Surgical specimens after
removal should be placed in
an adequate quantity of
fixative as soon as possible.
• For optimal fixation a piece of
tissue should be immersed in
at least 10 times its own
volume of fixative.
• Mostly the fixative used for
surgical specimen or biopsy is
10 % Formal Saline.
1.Fixation:

9. • Jars or bottles with screw
tops and of suitable
capacity should be used.
• Large specimens should
not be squeezed into a
smaller container.
• If a specimen is too large
to fit easily into the
largest size of container,
it should be brought as
such to the laboratory
without delay in a bucket.
• The specimen should
never be put into water or
normal saline because this
will hasten autolysis.
2.Storage:

11. • It is the responsibility of the
Medical Officer to see that the
specimens are correctly
labelled.
• It includes the:
Name of the patient
ward of patient
Hospital
Date and time of obtaining
the specimen
3.Labelling:

12. Request form:
If more than one biopsy is taken
from the same patient at same
time, a single request form will
suffice for all those specimens.
If two or more apparently
unrelated pathological lesions are
biopsied from the same patient,
separate forms should accompany
each specimen.
The clinical data must include any
specific information contributing
or relating to the present illness.

13. Renal Biopsy
The specimen of renal
biopsy for immuno-
fluorescence should be
submitted fresh in
normal saline.
collected in 10% formal
saline
Liver Biopsy
Liver biopsy specimen
for the diagnosis of
storage disorders
should be collected in
absolute alcohol.
collected in 10% formal
saline
Bone marrow Biopsy
Information regarding age,
sex of the patient, site of
biopsy, clinical history and
x-ray with radiologists
opinion are required for
reporting on bone
specimens
collected in 10% formal
saline
SeveralBiopsyspecimens:

14. • Major types of biopsy are following:
1. Core needle biopsy
2. Fine needle biopsy
3. Incisional biopsy(except core biopsies)
4. Excisional biopsy
5. Frozen section biopsy
• Others:
1. Punch biopsy
2. Brush biopsy
Differenttypesofbiopsies:

15. • Mostly done for breast lesion
investigation
• Needle ,with larger bore than
fine needle, are used to extract
sample.
• Sometimes under the guidance
of radiological techniques such
as ultrasound, CT scan,
or magnetic resonance
imaging.
1.CoreNeedleBiopsy:

16. Merits or Advantages
• Relatively easily procedure.
• Done with local anesthesia.
• Can detect angio or neural
invasion.
• Preserves tissue architecture.
• Low rate of complications.
• More accurate in diagnosis of
specific lesions.
• Higher costs (equipments and
histopathology procedures).
• Therapeutic aspiration is not
possible.
• Longer turnaround time.
• Require extensive care standards.
• Operator dependent (so still a
chance of sampling error).
Demerits or Drawbacks
1.CoreNeedleBiopsy:

17. • Fine Needle biopsy (FNB) is a
minimally invasive technique
which is suitable for those
sensitive areas where an
incisional biopsy is
contraindicated or is not
possible.
• It is used in conjunction to
the clinical and radiological
findings to rapidly provide
the best possible initial
assessment.
FineNeedleBiopsy:

18. Merits or Advantages
• Safe and easy procedure.
• Done without anesthesia.
• Lower cost, suitable for low
income people.
• Shorter turnaround time.
• Low risk of infection.
• Small sized needle doesn’t
damage vital structure.
• High diagnostic accuracy.
• Loss of tissue architecture.
• Capsular and lymphovascular
invasions can’t be detected.
• Difficulty is some specific
diagnosis e.g. papillary invasion,
radial scar etc.
• Require extensive care standards
and experienced operator.
• False positive or negative results
may obtained.
Demerits or Drawbacks
2.FineNeedleBiopsy:

19. • Involves complete excision of
affected lesion for both
diagnostic and therapeutic
purposes.
• This type of biopsy is mostly
recommended in those cases
in which the size of biopsy is
small.
• It is mostly done for skin
lesions.
3.ExcisionalBiopsy:

20. Merits or Advantages
• Excisional biopsy is more accurate
ad gives few false negative
results.
• It gives all the information about
invasion including tumor size,
tumor type, tumor grade etc.
• Because entire tumor is removed,
so it helps histopathalogist to
recognize cancer cells in lesion.
• Most appropriate for small
peripheral benign lesions.
• Excisional biopsy is more invasive.
• It has longer, more uncomfortable
recovery time.
• It has a higher risk of infection and
bruising.
• Removing the whole tissue leaves
a noticeable scar.
• Difficult to perform in large
lesions.
Demerits or Drawbacks
3.ExcisionalBiopsy:

21. • In order to make a definite
diagnosis, removal of
representative sample of the
lesion and normal adjacent
tissue is done.
• If the lesion is extensive,
different samples should be
obtained, placing each of the
in a separate and adequately
identified container.
• Necrotic tissue must be avoided.
4.IncisionalBiopsy:

22. Merits or Advantages
• Only a small fragment of tissue is
required.
• Can be done in cases of suspected
malignancy and premalignancy.
• It is also used in establishing the
diagnosis in a systemic and
autoimmune disease process.
• Useful in the cases in which is
difficult to excide the lesion due to
large size.
• May increase the risk of
metastasis of malignant lesions.
• Must be avoided in vascular cases
as it may cause profuse bleeding.
• Splits and hemorrhage are
artifacts mostly found in the
incisional biopsies.
Demerits or Drawbacks
4.IncisionalBiopsy:

23. • For rapid diagnosis during
intra-operative period, the
sampled material is
processed without fixation,
frozen with dry ice.
• A specimen processed in
this manner is not
satisfactory for detailed
study of the cells, but it
gives the surgeon
immediate information
regarding the malignancy
of a piece of tissue.
• Frozen sections can be
fixed, stained and mounted
for permanent reference.
5.FrozenSectionBiopsy:

24. Merits or Advantages
• Fastest of all methods.
• Differentiate between benign and
malignant state of tumor.
• Excellent for IHC (Immuno-
histochemistry), IF (Immuno-
fluorescence), ISH (In situ
hybridization).
• For generating reports during
intra-operative sites.
• Poor morphology is obtained.
• Not suitable for hard tissue
biopsies.
• Also not suitable for extensive
complex lesions.
• Risk of freezing artifact-must be
snap frozen.
Demerits or Drawbacks
5.FrozenSectionBiopsy:

25. • Histological technique deals
with the preparation of
tissues for microscopic
examination.
• The aim of good histological
technique is to preserve
microscopic anatomy of the
tissues, and make them
hard, so that very thin
sections (5 micron) can be
made.
• This is achieved by passing
the total or selected part of
the tissue through a series
of processes.
HistologicalTechniques:

26. Fixation Dehydration Clearing
Embeddin
g Sectioning Staining
Stagesofahistologicaltechnique:
Mounting/Coverslipping

27. • This is the process by which the
constituents of cells and tissues are
fixed in a physical and partly also in
a chemical state so that they will
withstand subsequent treatment
with the various reagents with
minimum loss of architecture.
• There are five major groups of
fixatives, classified according
to mechanism of action:
1.Aldehydes
2.Mercurials
3.Alcohols
4.Oxidizing agents
5.Picrates
1.Fixation:

29. • Wet fixed tissues (in aqueous
solutions) cannot be directly
infiltrated with paraffin.
• First, the water from the tissues
must be removed by the process of
dehydration.
• This is usually done with a series
of alcohols, say 70 to 95% to 100%.
• For routine, sections no thicker
than 7 µm, following scheme may
be followed:
1. 70% alcohol - Methylated spirit
for 1 hour
2. 90% alcohol - Rectified spirit 2
changes for 2 hours each
3. 100% alcohol - Absolute alcohol
2 changes for 2 hours each
2.Dehydration:

30. • The next step is called ‘clearing’
and consists of removal of the
dehydrant with a substance that
will be miscible with the
embedding medium.
• The most common clearing
agent is xylene.
• Other clearing agents are also
available in market like,
1. Toluene
2. Chloroform
3. Benzene
4. Carbon tetrachloride
3.Clearing:

31. • Finally, the tissue is infiltrated with
the embedding agent.
• Embedding consists two steps.
• Impregnation with wax:
• This is allowed to occur at melting
temperature of wax, which is 54-
60°C.
• Volume of wax should be about
25-30 times the volume of tissue.
• Types of waxes employed for
impregnation are:
1. Paraffin Wax: It is used
routinely. 3-4 µ section obtained.
2. Water soluble wax: Tissue can
be directly placed in it, without
dehydration and clearing.
4.Embedding:

32. • Embedded tissues are placed in a
mould, which may be metal or
plastic with their label and then
fresh molten wax is poured in it
and allowed to settle and solidify.
• After the block has completely
cooled it is cut into individual
blocks and each is trimmed.
• This is done with the help of a
microtome.
• Microtomes have a mechanism
for advancing the block across the
knife. Usually this distance can be
set, for most paraffin embedded
tissues at 4–6 microns.
5.BlockingandSectioning:

33. • Once sections are cut, they are
floated on a warm water bath
that helps to remove wrinkles.
Then they are picked up on a
glass microscopic slide and after
this, stained.
• Staining is a process by which a
colour is imparted to sectioned
tissue. Specially manufactured
dyes are used for this purpose.
• Generally the stains are classified
as:
• Acid stains (eosin stain)
• Basic stains (haematoxylin)
• Neutral stains (Romanowsky
stain)
6.Staining:

34. •
CommonHistologicalTechniques:

35. • This technique can be used to sample superficial and subcutaneous
lesions in breast, thyroid, lymph node, salivary gland and superficial
abdominal masses.
• Procedure:
• To avoid any mishap during procedure, the patient should be explained
and assured about the procedure.
• A disposable syringe with 21-25 gauge needle can be used for this
purpose (5-10 ml).
• The area is cleaned thoroughly with a spirit swab.
• The needle is introduced into the mass, negative pressure is applied by
retracting the plunger and mass is probed in several directions.
• Prior to withdrawal of needle, the plunger is released allowing
equalization of pressure.
FineNeedleAspirationTechnique:

36. • Slide Preparation:
• After aspiration the syringe is detached from the needle and filled
with air.
• The syringe is reattached to the same needle and plunger is pushed to
gently express the material onto glass slides.
• The material is gently spread on the slides by using spreader slide.
• Smear Fixation:
• The slides are allowed to air dry or fixed wet in solution containing
ether and alcohol in equal proportions or 95% alcohol.
• Staining:
• The slides can be stained by Papanicolaou (PAP), Haematoxylin and
Eosin (H&E), Leishman and modified Giemsa stains.
• Microscopic examination:
• Slides are then placed under microscope to examine the lesions.

37. • Procedure:
• Frozen sections are performed
with an instrument called a
cryostat.
• The cryostat is just a refrigerated
box containing a microtome.
• The piece(s) of tissue to be studied
are snap frozen in a cold liquid or
cold environment, e.g. in
isopentane (OCT), which is cooled
to -150°C by immersing in liquid
nitrogen.
• This causes rapid freezing of tissue
and prevents the formation of ice
crystals within the cell.
FrozenSectionTechnique

38. • Slide Preparation:
• The tissue sections are cut and picked up on a glass slide.
• It is done by placing a room temperature slide above the frozen section
that causes adherence of the tissue to slide because of force of attraction.
• The sections are then ready for staining.
• Staining:
• The most common staining technique used for frozen section is Rapid
Haematoxylin and Eosin Staining.
• Tissue section are placed in xylol and then absolute alcohal for 3mins.
Then placed in rectified spirit(80%) and then methylated spirit for 2 min.
• Wash the slide in running water for 1 min and put it in Harris
haematoxylin for 3-5 min.
• Excessive dye washed with water, then acid alcohol and then again water.
• Then counter stain with eosin for 2-3 min and wash in running tap water
for 30 seconds.
• Now the slides are dehydrated and examined under microscope.

39. • Exfoliative cytology is microscopic
study of shed or desquamated cells
from epithelial surface, by scraping
the tissue surface or from body
fluids (sputum, saliva etc).
• Procedure:
• The smears are collected from the
buccal mucosa before and after a
toluidine blue rinse.
• The oral cavity is made to rinse with
water and then a smear is taken
using a cytobrush.
• Mouth is then rinsed with 10ml of
1% acetic acid for 30 secs, followed
by 10ml of toluidine blue rinse and
finally repeated with 1% acetic acid
and another smear is taken.
ExfoliativeCytology:

40. • Slide Preparation and Staining:
• Both the smears are spread on to a clean microscopic glass slide,
fixed and stained using standard PAP(Papanicolaou) staining
protocol.
• The polychromatic PAP stain involves 5 dyes in 3 solutions.
• (Haematoxylin, Orange green 6, Eosin Azure, Light green SF,
Bismarck Brown Y).
• Microscopic Examnation:
• The smears are then observed under 40X & 100X magnifications.

41. • IHC is a technique for identifying cellular or tissue constituents (antigens)
by means of antigen-antibody interaction.
• The selection of antibodies for the IHC testing is made on the basis of
their tumor specificity and fact that they will react with tumor or not.
• IHC studies are most often performed on specimen fixed in neutral
buffered formalin.
• Traditional Direct Technique:
• The primary antibody is conjugated directly to the label.
• The conjugate may be either a fluorochrome (more commonly) or an
enzyme.
• The labeled antibody reacts directly with the antigen in the histological
or cytological preparation.
• Then smear is microscopically examined.
Immunohistochmistry(IHC):

42. • Ground Section Technique:
• A section of bone or tooth prepared for histological study by polishing
until thin enough for microscope viewing.
• Teeth are first soaked in 20% formaldehyde for 24 hours, washed in
water and sectioned to any thickness by using Ultra-microtomes.
• Tooth grinding is done manually or automatically by using machines and
then finally ground tooth section examined under microscope.
• Tissue Microarrays:
• It consist of paraffin blocks in which up to 1000 separate tissue cores are
assembled in array fashion to allow multiplex histological analysis
OtherTechniques: