Lab. №.

1:
Introduction to pathology.
Tissue processing.
Obtaining specimens for pathologic examination:

1. cytology: obtaining group of cells to be examined. Eg. Fine

Needle Aspiration Cytology (FNAC) ,effusion fluid cyto.,
sputum cyto., Pap smear……etc.

2. Biopsy: piece of tissue or organ removed surgically from a

patient for examination. Types:
A. Incisional B.: removal of part of diseased tissue or
organ for examination.
B. Excisional B.: removal of whole diseased tissue or
organ for examination.
C. Laparoscopic / endoscopic B.
D. Frozen section biopsy.
Sending specimens for pathologic examination:
1. The Request form should be labeled with:
a) Patient’s information.
b) Hospital/health facility information.
c) Clinical notes / surgical findings.
d) Description of the organ , tissue or the site from where the specimen
was taken.

2. The Biopsy container should be labeled with the patient

name , hospital name, ward number, name of physician
and date.
The specimens should be put in fixative solution immediately after
removal the volume of which should be at least 10 times the size of the
specimens, 10% Formaldehyde is commonly used.
Methods to make pathologic diagnosis:

- Gross (macroscopic) appearance.

- Histological (microscopic) appearance.
- Immunohistochemical study.
- Electron microscopy (ultra structure) study.
- Genetic , chromosomal & molecular methods.
Gross examination:
It includes a gross
description of the specimen,
inking, dissection and taking
samples from the concerned
sites then it placed in a small
plastic cassette that are
labeled with patient & lab.
information and put in a
fixative.
These cassette retain the
tissue during further
processing to a paraffin block.
Tissue processing:

The most common method of tissue processing for the

routine histopathologic examination is the routine paraffin
wax method which include several steps:
1. Fixation:
Tissue is immersed in a fixative agent
to:
a. Preserve the protoplasm.
b. Avoid autolysis.
c. Prevent putrefaction by bacteria.

The choice of fixative depends on the

cellular component that you want to
preserve.

One of the best fixative solution for

routine light microscopy is a buffered
isotonic solution of 10%formaldehyde.

The amount of fixative should be 10

times the size of tissue.
Speed of fixation is 1mm/hour. After a few hours in formalin fixation..
2. Dehydration:
Extraction of water from cells by
bathing the tissue in increasing
concentrations of alcohol (mixtures
of ethanol & water usually from
70%-100% ethanol).

3. Clearing:
Making the tissue transparent by
removal of the dehydrant with a
substance that will be miscible with
the embedding medium (paraffin).
The commonest clearing agent is
xylene (xylol).

Automated tissue processor.

4. Embedding (paraffin
impregnation):
Bringing the tissues out of the
cassette & after been properly
aligned & oriented in the block,
molten paraffin (in the oven at
58-60 C ) is poured over them
the heat causes the solvent to
evaporate and the spaces within
the tissue will be filled with
paraffin.

The tissue together with its

impregnated paraffin hardens
after being taken out of the oven
to form paraffin block.
5. Sectioning:
The blocks containing the
tissue are cut into thin sections
(thickness of 1-10 micron) by
microtome’s steel blade.
These sections are floated on
water bath and then placed on
glass slides to be stained.

6. Rehydration:
Means reintroduce water into
the cells by using graded series of
ethanol in descending
concentration (i.e. 100% - 90% -
80% - 70% …). This step prepare
the tissue for staining.
7. Staining:
The most commonly used dye is
the combination of hematoxylin and
eosin (H&E).
Hematoxylin (basic) stains the cell
nucleus and other acidic structure (as
RNA- rich sites of the cytoplasm &
the matrix of hyaline cartilage) blue.
While eosin (acidic) stains the
cytoplasm and collagen pink.

8. Mounting (Cover slipping):

Stained sections on a slide covered
with a thin piece of glass to protect the
tissue , to better optical visualization
under the microscope and to preserve
permanently the tissue section.
End