LECTURE 1

INSTRUCTOR: O.M.MUTUKU
INTRODUCTION TO HISTOPATHOLOGY

Histopathology refers to the microscopic examination of tissue in order to study the

manifestations of disease. Specifically, in Laboratory medicine, histopathology refers to the
examination of a biopsy or surgical specimen, after the specimen has been processed and
histological sections have been placed onto glass slides. Precisely, it is a branch of pathology
which deals with the study of disease in a tissue section. In contrast, cytopathology examines
free cells or tissue micro-fragments (as "cell blocks").

Surgical pathology includes gross and microscopic examination of resected specimens and
biopsies by histopathologists for tissue diagnosis. Several steps are followed to get the tissue in a
form, by which diagnosis can be made under light microscope. To achieve this it is important
that the tissue must be prepared in such a manner that it is sufficiently thick or thin to be
examined microscopically and all the structures in a tissue may be differentiated.

The term Histochemistry means study of chemical nature of the tissue components by
histological methods. The cell is the single structural unit of all tissues. The study of cell is called
cytology. A tissue is a group of cells specialized and differentiated to perform a specialized
function. Collection of different type of cells forms tissue.

Steps involved in the process of preparing the tissue for microscopic examination are:

1. Receipt of specimens (from surgical ward or autopsy room)

2. Grossing

3. Fixation

4. Tissue processing

5. Embedding

6. Section cutting

7. Staining and labelling

8. Dispatch of slides to pathologist for diagnosis

Once the diagnosis is made, the slides come back to the laboratory. After the reports are sent to
the surgeons either as soft copy or hard copy, the laboratory has to perform the following
functions:

1. Slides are filed for future reference or teaching/research for at least 10 years.
2. Requisition forms are filed and/or stored in digital form for at least 10 years.

3. Specimens may be divided according to their use;

(a) Well preserved specimens with representative lesion should be kept for:

 Teaching
 Research
 Museum

(b) For future reference (6 months to 1 year)

(c) Discard – The specimens which are not required or not useful for any of the above purpose
should be discarded.

Apart from these essential functions, various other procedures are performed in a surgical
pathology laboratory depending upon the requirement, feasibility and availability of instruments
and qualified personnel:

 Cryosections
 Histochemical stains
 Immunohistochemistry
 Electron microscopy
 Advanced techniques like in-situ hybridization, immunofluorescence

GROSSING

It is the process by which pathology specimens are inspected with bare eye to obtain diagnostic
information. Following points should be noted before the tissue is processed for microscopic
examination:

 Identification of the specimen-confirmation of patient and anatomical site from which the
specimen has been obtained
 Clinical details
 Gross description – written record of physical appearance of the specimen

Only a small portion from the large specimen can be subjected to microscopic examination,
hence gross examination should be done by a skilled person.

Only soft tissue can be cut into small blocks and processed directly. Bony specimens need to be
decalcified before processing. Stones and teeth also require special treatment.
Gross room

The size and features of surgical pathology gross room depend on the number of specimens,
number of staff pathologists and residents and type of institution. The room should be large
enough to permit the work to all the pathologists and other personnel simultaneously. The room
should be well illuminated, ventilated and with an exhaust fan to remove the formalin vapors.

Following items should be in a gross room.

(a) A cutting board. The fluid from the board must run directly into the sink.

(b) Shelves for specimen containers.

(c) Ready access to hot and cold water.

(d) Ready access to formalin.

(e) Box of instruments containing forceps of various size, scissors of various types and size,
probe, bone cutting saw or electric bone cutter, scalpel handle, disposable blades, long knife and
ruler to measure the size of lesion and specimens.

(f) Box with cassettes and labels.

Apart from these items a good gross room should also have -

(a) Large formalin container

(b) Other fixatives

(c) Refrigerator

(d) Photographic facility

(e) Balance for gross specimens

(f) X-ray view box

LABORATORY HAZARDS AND SAFETY MEASURES

Gross room

1. Formalin vapors are irritant to eyes and throat. Exhaust may be used as outlet for vapors.

2. One should always use mask, apron, eye glasses and gloves to protect oneself from:

 Infected material
 Formalin vapors
 Spilt blood or any other fluid
3. Keep the grossing table clean with antiseptic solution.

4. All specimens should be in container with 10% formalin and covered with lid.

5. After grossing specimen should be kept according to accession number.

HISTOPATHOLOGY LABORATORY

The laboratory should be large enough to accommodate various equipments and personnel to
work with ease. The equipments which are kept in this laboratory are:

 Tissue processor
 Tissue embedding table
 Microtome
 Tissue warming plate
 Tissue flotation bath
 Slide stainer or glassware for manual staining
 Table to label and dispatch the slides.

Laboratory hazards and safety measures in Histopathology laboratory

1. Most of the equipments present in this laboratory are functioning 24x7 days. Electrical
connections should be checked before leaving the laboratory every day.

2. Many chemicals are inflammable; hence care should be taken to avoid any fire hazard.

3. Fire extinguisher should always be available.

4. Minimum inflammable substances should be kept in the laboratory. Substances like wax,
xylene alcohol, and acetone should be stored at a separate place.

5. Some chemicals are carcinogenic or harmful to the skin. Therefore staining and other work
should be performed with the gloves on.

Type of material obtained in laboratory

The human tissue comes from the surgical ward and the autopsy room. From surgery two types
of tissue are obtained:

 Incisional biopsy – This is where a small piece of lesions or tumor is sent for diagnosis
before final removal of the lesion or the tumor.
 Excisional biopsy – This is when the whole of the tumor or lesion is sent for examination
and diagnosis by the pathologist.

Tissues from the autopsy are sent for the study of disease and its course, for the advancement of
medicine.
Types of Histological preparation

The histological specimen can be prepared as: Whole mount, Sections and Smears

Whole mounts- These are preparation entire animal

Sections- The majority of the preparations in histology are sections. The tissue is cut in about 3-
5 mm thick pieces processed and 5 microns thick sections are cut on a microtome. These are then
stained and permanently mounted. Microtomes are special instruments which have automatic
mechanism for cutting very thin sections. To cut the sections on the microtome; the tissue must
be made hard enough to not get crushed. There are 2 methods of hardening the tissues. One is by
freezing them and the other is by embedding them in a hard material such at paraffin wax or
gelatin.

Smears- Smears are made from blood, bone marrow or any fluid such as pleural or ascitic fluid.
These are immediately fixed in alcohol to presence the cellular structures are then stained.
Smears are also made by crushing soft tissue between two slides or an impression smear in made
by pressing a clean slide in contact with the moist surface of a tissue. By doing this, the cells are
imprinted on the slide and these may be stained for cytological examination.

Responsibility of a technician/technologist

The technician/technologist is responsible for:

1. Specimen preservation.

2. Specimen labeling, logging and identification.

3. Preparation of the specimen to facilitate their gross and microscopy.

4. Record keeping.

To obtain these aims the following point need consideration.

1. As soon as the specimen is received in the laboratory, check if the specimen is properly
labeled with the name, age, Hospital Registration No. and the nature of tissue to be examined
and the requisition form is also duly filled.

2. Also check if the specimen is in proper fixative. Fixative should be fifteen to twenty times the
volume of the specimen add fixative if not present in sufficient amount.

3. Check if the financial matters have been taken care off.

4. Make the entries in biopsy register and give the specimen a pathology number called the
accession number. Note this number carefully on the requisition form as well as the container.
This number will accompany the specimen everywhere.

5. If the specimen is large inform the pathologist who will make cut in the specimen so that
proper fixation is done. Container should be appropriate to hold the specimen without distorting
it.

6. Blocks of tissues taken for processing should be left in 10% formalin at 60°C till processing.
These would be fixed in 2 hours.

7. Slides should be released for recording after consultation with the pathologist.

8. Specimens should be kept in their marked container and discarded after checking with
pathologist.

9. Block must be stored at their proper number the same day. Note the blocks have to be kept
preserved for life long. Slides should be stored in their proper number after 3 days. It gives time
for the slides to be properly dried.