Overview of

Histotechnology
CECILIA LEKPOR

OVERVIEW OF HISTOTECHNOLOGY 1
LEARNING OBJECTIVES
• At the end of this course students should be able to
explain in detail the following:
• Sample reception and Grossing
• Fixation
• Tissue processing
• Embedding
• Microtomy
• Staining

OVERVIEW OF HISTOTECHNOLOGY 2
 INTRODUCTION
• Histopathology- the branch of biology which deals
with the study of diseased tissue under the
microscope
• Histology- the study of normal tissue
• The purpose of its study is to compare normal tissue
from abnormal tissue
• Pathologist/histotechnologist understands the
different between normal and abnormal tissue based
on their:
• Size, shape and nucleus to cytoplasmic ratio
OVERVIEW OF HISTOTECHNOLOGY 3
INTRODUCTION-CONT
❖Also use for the purpose of the diagnosing
➢ Cancer stage
➢ Precancerous stage
➢ Other inflammatory disease
❑COMMON TERMINOLOGIES
➢ Biopsy
➢ Autopsy
➢ Autolysis
➢ Putrefaction

OVERVIEW OF HISTOTECHNOLOGY 4
 How to Prepare Tissue
for Light Microscope

• Tissue specimen received in the

pathology Lab be accompanied
with a request form that lists:
• Name, Age, Sex clinical history, site
of origin, Type of operation,
clinician’s name, date, phone
number.

OVERVIEW OF HISTOTECHNOLOGY 5
 • Tissue specimens received in the surgical pathology
laboratory have a request form that lists the patient
information and history along with a description of
RECEPTION & the site of origin
SPECIMEN
ACCESSIONING
• Request card & specimen container are cross checked
to verify the information provided

• Information received are logged

manually/electronically

• The specimens are accessioned by giving them a

number that will identify each specimen for each
patient
OVERVIEW OF HISTOTECHNOLOGY 6
GROSS EXAMINATION

• During grossing, tissue removed from the body for diagnosis 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

OVERVIEW OF HISTOTECHNOLOGY 7
GROSS EXAMINATION-CONT

• Only soft tissue can be cut into small blocks and processed directly

• Bony specimens need to be decalcified before processing

• Stones and teeth require special treatment

• After grossing, specimen are kept according to accession number

OVERVIEW OF HISTOTECHNOLOGY 8
AFTER GROSS EXAMINATION
❖Well preserved specimens with representative lesion are kept for
• Teaching
• Research
• Museum
• For future reference, they are kept from 6 months to 1 year
• The specimens which are not required or not useful for any of the
above purpose should be discarded

OVERVIEW OF HISTOTECHNOLOGY 9
Decalcification
• Bone specimens as well as calcified tissues need to be decalcified
before processing

• The calcium must be removed before embedding to allow sectioning

• A variety of reagents have been used to decalcify tissue such as

mineral acids, organic acids, EDTA, and electrolysis

OVERVIEW OF HISTOTECHNOLOGY 10
DECALCIFICATION-CONT….
• Strong mineral acids such as nitric and HCl can be used

• Strong acids will remove large quantities of calcium at a rapid rate,

but they will cause damage of cellular morphology

• Organic acids such as acetic and formic acid act more slowly on
dense cortical bone

• EDTA can remove calcium safely, it works slowly, it penetrates tissue

poorly, but it is expensive in large amounts
OVERVIEW OF HISTOTECHNOLOGY 11
HOW IS MICROSCOPIC EXAMINATION OF
TISSUES DONE???
❖To observe the tissue under the microscope the following steps are followed:
• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding
• Sectioning
• Staining
• Mounting
OVERVIEW OF HISTOTECHNOLOGY 12
FIXATION
• The purpose of fixation is to preserve tissues permanently in as life-
like state as possible

• Fixation should be carried out as soon as possible after removal of the

tissues to prevent autolysis

• Formaldehyde is the most commonly used fixative

• However, a variety of fixatives are available for use, depending on the

type of tissue present and features to be demonstrated
OVERVIEW OF HISTOTECHNOLOGY 13
 FIXATION-CONT….
• The technique of getting fixed tissue into paraffin is called tissue processing
• The main steps in this process are dehydration and clearing

1. Dehydration: Gradual removal of water from the tissue using ascending

grades of ethanol to prevent tissue shrinking

2. Clearing: Replacement of alcohol in tissue by clearing fluid like xylene,

benzene
3. Infiltration and Embedding:
- Tissues are impregnated in paraffin
OVERVIEW OF HISTOTECHNOLOGY 14
• THE PROCESS

OVERVIEW OF HISTOTECHNOLOGY 15
SECTIONING
• Once the tissues have been embedded, they must be cut into very
thin sections that can be placed on a slide
5. Paraffin block are sectioned by microtome using metal knife, into
thin sections ~ 3-5µ(microns)

• The important thing for proper sectioning is to use a very sharp

knife/blade

OVERVIEW OF HISTOTECHNOLOGY 16
CONT….

6. Mounting/picking of sections:
- Sections are mounted on glass slides, allow to drain,
placed on a hot plate/ hot oven until ready for staining
7. Staining:
- Haematoxylin and Eosin (H&E) stain
- Variable stains are used for specific tissues.

OVERVIEW OF HISTOTECHNOLOGY 17
Mounting/coverslipping- stained sections are mounted with DPX and
cover slip

OVERVIEW OF HISTOTECHNOLOGY 18
STAINING
• The embedding process must be reversed in order to get the paraffin
wax out of the tissue and allow water soluble dyes to penetrate the
sections

• Therefore, before any staining can be done, the slides are

"deparaffinized" by running them through xylene then, to alcohols
and lastly to water

• There are no stains that can be done on tissues containing paraffin

OVERVIEW OF HISTOTECHNOLOGY 19
Frozen Sections
• Frozen sections are performed with an
instrument called a cryostat.
• The cryostat is just a refrigerated box
containing a microtome.
• The temperature inside the cryostat is
about -20 to -30 C.
• The tissue sections are cut and picked up
on a glass slide.
• The sections are dried and then stained.

OVERVIEW OF HISTOTECHNOLOGY 20
Automated stainer
• Frozen sections are stained by hand,
because this is faster for one or a few
individual sections

OVERVIEW OF HISTOTECHNOLOGY 21
Cover slipping
The stained section on the slide must be
covered with a thin piece of glass to protect
the tissue from being scratched, and to
preserve the tissue section and for long
storage without any harm

OVERVIEW OF HISTOTECHNOLOGY 22
OVERVIEW OF HISTOTECHNOLOGY 23