Histopathological techniques

Special histo-chemical stains

ImmunoHistoChemistry
CLINICAL (DIAGNOSTIC) CYTOLOGY)

Assistant Professor Dr. Rafal Abdulrazaq

M. B. Ch. B. F.I.C.M.PATH.
Histopathological techniques
Tissues from the body taken (whether biopsies
removed at surgery, or tissues from autopsy) for
diagnosis of disease processes must be processed
in the histology laboratory to produce microscopic
slides that are viewed under the microscope by
pathologists..
Biopsy:
Is a piece of tissue or organ removed surgically from a patient
for histopathological examination.
Types of biopsy:
1. Incisional biopsy: means removal of part of diseased tissue
or organ for histopathological examination.
2. Excisional biopsy: means removal of whole diseased tissue
or organ for histopathological examination.
3-Endoscopic biopsy: means removal of small piece of tissue
during endoscopic examination for histopathological study.

examination.
• Requesting “Quick-frozen section" diagnosis is
sometimes desirable
- In determining nature of a lesion
- Evaluating margins of excised cancer
- Permits histologic evaluation within minutes
 How to send specimen for
histopathological examination
1. Special form that contain the following informations:
a. name of patient, age and sex.
b. name of hospital, name and number of ward where
the patient is present.
c. name and signature of the physician whose
responsible for the patient.
d. date of sending the biopsy.
e. diagnosis of the disease and summary about patient’s
clinical sign, symptoms and physical finding.
f. name of the organ or the tissue that the biopsy is taken
from.
Tissue specimens received
in the surgical pathology
laboratory have a request
form that lists the patient
information and history
along with a description
of the site of origin. The
specimens are
accessioned by giving
them a number that will
identify each specimen for
each patient.
2. The biopsy should be put in fixative solution immediately
after removal of the tissue .

3. Name of patient, name of hospital , name and number of

ward, name of physician and date should be written on the
biopsy container.
The most common method used for tissue processing is
the Routine Paraffin Wax method.
This method include several steps:
1. Fixation.
2. Dehydration.
3. clearing.
4. Paraffin wax impregnation.
5. TISSUE EMBBEDING
6. Cutting with microtome.
7. Staining.
8. Microscopical examination.
Fixation:
aims of fixation
1- To avoid autolysis (i.e. digestion by enzymes present
within the cells).
2- Prevent putrefaction by bacteria.
3- To preserve the structure & molecular composition of
the tissue.
4-To harden the tissue to facilitate handling and
sectioning.
- Amount of fixative should be 10 times the size of tissue.
- Speed of fixation is 1mm/hour.
- Fixative: 10% buffered formalin ,for 18 - 24 hrs.
- Bouin's fixative used for fixation of testicular, renal and
bone marrow biopsies.
Surgery & sampling

Grossing & Fixation

 Gross examination

Tissues removed from the body for diagnosis arrive in the Pathology Department and are
examined by a pathologist, pathology assistant, or pathology resident. Gross examination
consists of describing the specimen and placing all or parts of it into a small plastic cassette
which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes
are placed into a fixative.
Tissue processing
 Dehydration:
means extraction of water from cells by bathing
the tissue in a graded series of mixtures of ethanol
& water (usually from 70%-100% ethanol).
 Clearing:
The ethanol in the tissue then replaced by a
solvent miscible with the embedding medium. The
most common clearing solution used is Xylene. It
also clear the tissue and make it transparent.
Paraffin impregnation:
Once the tissue is impregnated with the solvent it is placed
in melted paraffin in the oven typically at 58-60 C . The heat
causes the solvent to evaporate and the spaces within the
tissue become filled with paraffin.

TISSUE EMBBEDING: 
Tissues that come off the tissue processor are still in the cassettes 
and must be manually put into the blocks by a technician who
must pick the tissues out of the cassette and pour molten paraffin
over them. This "embedding" process is very important, because
the tissues must be aligned, or oriented, properly in the block of
paraffin.
Tissue embedding
Cutting by microtome:
The blocks containing the tissue are then taken to a
microtome and are sectioned by the microtome’s steel
blade to a thickness of 1-10 micron.
The sections are floated on water and transferred to
glass slides to be stained.
 Sectioning
Once the tissues
have been
embedded, they
must be cut into
sections that can be
placed on a slide.
This is done with a
microtome.
Staining:
Of all dyes the combination of hematoxylin and eosin
(H&E) is the most commonly used.
Hematoxylin stains the cell nucleus blue.
While eosin stains the cytoplasm pink.
The de-waxed slides are brought to water through
decreasing concentration of alcohol , then put in
haematoxylin. This is followed by washing in tap water,
differentiation in acid alcohol and rewashing in tap
water. The section are then stained by eosin, dehydrated
in rising concentrations of alcohol until (100%), cleared
by xylene and mounted by glass cover-slides using
"Canada Balsam" or "DPX".
 Staining

The staining process makes use of a variety of dyes that have been chosen for t
stain various cellular components of tissue. The routine stain is that of hematoxylin and
eosion (H and E). Other stains are referred to as "special stains" because they are
employed in specific situations according to the diagnostic need.
 Coverslipping

The stained section on the slide must be covered with a thin piece plastic or glass to protect the
tissue from being scratched and to provide better optical quality for viewing under the
microscope .
 Special histo-chemical stains:

These are done when indicated for improving 

histpathological diagnosis
E.g. 
Periodic acid Schiff stain  Demonstrate mucin 
Masson Fontantana stain  Melanin 
 ImmunoHistoChemistry
Detection of antigens by antibodies and
visualized in a microscope.
This involves the detection of cell products or
surface markers by monoclonal antibodies. The
binding of antibodies can be detected by
fluorescent labels or chemical reactions that
result in the generation of a colored product.
Antigens everywhere !
This technique is useful in: 
 Categorization of undifferentiated malignant tumors e.g.,
cytokeratin in carcinoma and desmin in tumors of muscle.
 Classification and categorization of leukaemias and
lymphomas. B and T cell lymphomas .
 Determination of the site of origin of metastasis using
antibodies against tissue specific antigens. E.g.,
thyroglobulin and PSA in thyroid and prostatic neoplasms,
respectively.
 Detection of molecules that have therapeutic or prognostic
significance e.g., estrogen and progestrone receptors in
breast cancer. Products of certain cancer suppressor genes
(e.g.,p53) .
 Accurate identification of infectious micro-organisms in
tissues.


HER2/NEU receptor (is a cell membrane protein is 
closely related to EGFR )
is amplified in 25%-30% of breast cancers.
High level of HER2/NEU protein in breast cancer
cells is associated with poor prognosis.
Blocking this receptor with anti-HER2/NEU
antibodies using the drug trastuzumab (Herceptin)
is clinically beneficial because it prevents
HER2/neu receptors activation.
Immunohistochemistry-detected targets for
cancer treatment

* HER2/NEU Breast carcinoma

Herceptin

* ER/PR Breast carcinoma

Anti-hormonal (Tamoxifen)

* CD117 GIST (gastrointestinal stromal

tumor ) Gleevec
 Cancer classification
Cancers are classified by the type of cell that resembles
the tumor and, therefore, the tissue presumed to be the
origin of the tumor. Is that easy?

Carcinoma? Lymphoma ? Sarcoma ? Mesothelioma ?

Rabbit ? Duck ?
Dubbit ?
Morphology
Clinical informations
Laboratory studies
Ancillary techniques

Immunohistochemistry
* Electron microscopy
Glioma ?
* Molecular genetics Metastatic carcinoma ?
Malignant lymphoma?
Other ?
“Primary Panel”

Pan-CK S-100

CD45 VIM
Differences between histopathology 
and cytology
Clinical cytology 
1. Deals with the structural changes in the 
nucleus & cytoplasm.
2. Evaluation requires cells only. 

Histopathology 
1. Deals with the form & the structure of 
the tissue.
2. Evaluation usually begins with biopsy. 
Squamous cell carcinoma
 Collection of the materials for cytology:

1. From normal (physiological) desquamation 

products:
Examples include : Sputum, Urine, Vaginal, 
cervical , endometrial secretion and Nipple
discharge.
2. By superficial scraping of the lesion 
(artificial mechanical desquamation) including
- Cervical scraping or smear 
- Buccal mucosal smear 
- Direct imprint of a tumor 
- Skin lesion scraping 
3. FNA (fine needle aspiration) including 
FNA of:
a- Pathologic mass (tumor or tumor-like 
mass) for example nodules of the thyroid and
breast masses; this is to help differentiating
between benign and malignant lesions.
b- Accumulated fluid within anatomical 
cavity, for example the pleural , peritoneal and
pericardial aspirates .
c- Enlarged lymph nodes 
(lymphadenopathy)
FNA technique
In general, the definitive diagnosis of a tumor mass 
like a breast lump can be established by open biopsy,
tissue core biopsy (Tru-cut) & Fine needle Aspiration
(FNA) .
Compared to FNA, Tru-cut biopsy is a more 
traumatic procedure which should be performed
under local anesthesia. It requires more time &
special equipments that are more expensive. Pain,
discomfort & bleeding are common complications.
FNAC, on the other hand, provides many advantages 
to the surgeons. It is an easy, non-invasive, reliable,
cost effective diagnostic technique that can give
rapid results. The procedure could be performed in
an office setting without anesthesia. It is usually not
more painful than a venipuncture & can be repeated
immediately if the acquired material is inadequate.
Pitfalls to Reliable Results from FNAC 
Failure of good localization of especially a small mass 
may lead to a false negative diagnosis. This can be
reduced by doing FNA under ultrasound guidance.

Densely fibrotic masses and hemorrhagic masses may 

result in scanty informative cellular elements interfering
adequate diagnosis.
4. Brush cytology (during fiber-optic endoscope); 
this is considered as mechanical exfoliation
(desquamation).
The clinical uses of cytologic examination: 
1-Evaluation of the gonad’s hormonal activity 
e.g. in vaginal smears the desquamated cells are
under the influence of ovarian hormones .

2-Detection of inflammatory lesion. 

3- Detection of precancerous cellular changes. 

No tumor occurs suddenly; there are precancerous
steps that are manifested by the presence of
dysplastic cells. Dysplasia precede invasive cancer
and may be graded as mild, moderate, & severe. The
diagnosis of cancer at an early or in pre-invasive
stage can help in its radical treatment.
4-Identification of neoplastic cells in primary, 
metastatic (secondary) or recurrent tumors:
Detection of primary cancers e.g. sputum containing 
blood (hemoptysis) could be due to for e.g. T.B. or cancer.
Cytological examination can differentiate between these
two.
Secondary (metastasis) for e.g. The fluid in pleural 
cavity after breast cancer removal may contain metastatic
cancerous cells in the pleural fluid .
Recurrent tumor. It is important to follow-up the patient 
after the removal of his cancer through the checking the
local area from which the tumor has been removed.
Patients with carcinoma breast may develop nodules at the
site of mastectomy scar. FNA of the nodule may reveal
cancerous cells and in such a case signifies recurrence.
5-The identification of sex chromosome 
Sometimes a baby is born with ambiguous genitalia 
and one can not tell whether the sex is male or
female; an important point to detect in a newborn
child.
- The presence of a dark dot attached to the nuclear 
membrane from inside (Barr body +ve) in squamous
cells of a buccal smear indicates that a sex
chromosome is present i.e. the genotype of the
individual is XX (♀). The bar body represent an
inactivated X chromosome.
- Conversely the absence of the spot (Barr body) 
indicates that there is no X chromosome i.e. the
newborn is genotypically a male so (XY; ♂).
Barr Bodies
ADVANTAGES OF CYTOLOGICAL TECHNIQUES: 
1-Simple to apply. 
2-Produce no or minimal side effects. 
3-Cheap. 
4-Provide rapid diagnosis. 
5-Repeatable. 
6-In (EXPERIENCED HANDS) they are very 
sensitive and highly specific for the diagnosis of
malignancy.
Cytology techniques 
- The materials obtained from the previous 
techniques are smeared on microscopic slide.

- Smears fixed in 95% ethanol are stained with 

hematoxylin and eosin stains.

- Body fluids (Ascitic, pleural, etc.) or cyst aspirates 

have to be centrifuged first and the deposit smeared
on clean glass slides
Sample Processing--Smears 
Push smears ,like making a blood smear. 
Make sure to label slides: Name, date ,no. 
Evaluating Sample Quality : 

Inadequate samples 
No or only rare cells 
Smears too thick 
Formalin exposure 
No. of slides 
Step 1: Low power examination. 

Describe what you see at low power 

Quality of slide 
Background material: mucus and blood 
Cells: Degree of cellularity 
Are cells alone or in clusters? 
Step 2: High power exam 

Describe what you see at high power . 

Are the cells inflammatory cells, tissue cells, or both? 

Type of cells :Epithelial, mesenchymal 

Features of cells ,Cell margins, cytoplasm, nucleus 
Benign or Malignant 
Inflammatory cells
Cytologic Criteria of 
Malignancy:

-Pleomorphic cells 
-Large cell size 
-Increased/abnormal 
mitotic figures 
What is the aim of cervical screening?
To reduce the incidence of cervical cancer
To reduce the mortality from cervical cancer 

What is cervical screening? 

-Cervical screening is a method of preventing cancer by 
detecting and treating pre-cancerous changes in the cervix
-The cervical cytology( PAP smear) test is the first stage in 
cervical screening
Risk factors for cervical cancer

-High risk HPV , more than 90% of cervical cancer 

cases linked with certain types of HPV
-Early marriage (Young age at first intercourse ) 
-Multiparity 
-Smoking 
-History of CIN (Cervical intraepithelial neoplasia) 
Taking the cervical smear
Conventional smear 
Small brush or spatula used to collect cells from 
the cervix
Specimen sent to the laboratory where cells are 
examined and reported
Squamous cell carcinoma