histology - the study of tissues, comprising their

cellular structure and function; the major application

of histology is in the diagnosis of disease
histopathology - a branch of histology concerned
with the effects of disease on the microscopic
structure of tissues
HISTORY OF HISTOPATHOLOGY
In the early 1800s, pathology was introduced in
Germany. The combining of the already existing
histology and pathology gave birth to a new field,
histopathology. Johannes Muller, the father of
histopathology, along with other researchers, such
as Rudolph Virchow, Jacob Henle, Karl
Reichert, and Karl Kuppfer, pioneered in the
advancement of the aforementioned field.
Malphigi, the founder of pathology, used
different methods of fixation, one of which is heat
fixation, to preserve tissues and other samples.
Formalin, the most popular fixative today, was
discovered by Blum in 1893.
Microtomes were then used to cut up different
tissue samples for both plants and animals to
avoid destroying the samples.
Paraffin wax as embedding media soon
became popular, followed by the discovery of
various stains throughout the early 1900s, and the
invention of automatic stainers by 1965.

FIXATIVE

To be able to study a tissue sample, it

undergoes several processes to preserve and
highlight the important components and structure
of the sample.

Zenkers
fluid

FIXATIVE

COMPOSITION

AMOUNT

10%
formalin
solution

37-40% formaldehyde

100cc

tap water

900cc

37-40% formaldehyde

100cc

distilled water

900cc

buffered
neutral
formalin
solution

Carnoys
fluid

Na3PO4 monobasic

4gm

Na3PO4 dibasic

6.5gm

absolute alcohol

60cc

chloroform

30cc

glacial CH3COOH

10cc

1000cc

HgCl2

50gm

K2Cr407

25gm

Na2SO4

10gm

*add 5cc of glacial CH3COOH to

95cc of Zenkers fluid before use
saturated picric acid

750cc

37-40%
Bouins fluid formaldehyde

250cc

glacial CH3COOH

50cc

b. decalcification - using a decalcifying

machine which is similar in mechanism to that
of a centrifuge, specimens are submerged in
solutions to remove calcium and other minerals
present in the specimen, then spun around to
speed up and make decalcification more
efficient. Decalcification is most often done to
bone tissues;
METHOD

PROCEDURE

INTENDED
SPECIMEN/
QUALITY

nitric acid
method

decalcify sections in
5% aqueous nitric
acid solution for 1-4
days, wash in
running water for
24hrs, neutralize in
10% formalin, wash
in running water for
24-48hrs, dehydrate,
clear, and embed

for rapid
processing
of small
pieces of
bone
specimen

formic acidsodium
citrate
method

decalcify sections in
formic acid-sodium
citrate solution, wash
in running water for
48hrs, dehydrate,
clear, and embed

produces
better
staining
quality than
the nitric
acid method

electrolytic
method

decalcify sections
using an electrolytic
apparatus with formic
acid-hydrochloric
acid for 1-4hrs, wash
in running water for
24hrs, dehydrate,
clear, and embed

fastest
decalcifying
method

1. preparation of specimen
a. fixation - process of killing and hardening,
stops cell metabolism and preserves tissue
structure for other treatments with the use of a
fixative, which is chosen by the purpose of
which the tissue is to be stained or preserved;

AMOUNT

distilled water

Today, automated tissue processors, highresolution microscopes, and other advanced

laboratory equipment are available for use in the
field of histopathology.
HISTOPATHOLOGY: THE PROCESS

COMPOSITION

decalcifying
machine specimens are
submerged in a
decalcifying
solution to
eradicate
minerals

c. embedding - infiltration with an embedding

medium, usually paraffin, for sectioning or
cutting; involves six processes;
washing - done after decalcification to
get rid of the excess formalin
dehydration - using different solutions of
alcohol with ascending concentrations up
to 100%
dissolving of alcohol - using xylol or
toluol, to remove the excess alcohol

- xylol and toluol are miscible in both

alcohol and paraffin

infiltration of paraffin - done to allow

uniform sectioning of the specimen

rehydration - using different solutions of

alcohol with descending concentrations
staining with hematoxylin in water
- hematoxylin(primary stain) is soluble in
water
dehydration - using different solutions of
alcohol with ascending concentrations up to
100%
staining with eosin in alcohol
- eosin(counterstain) is soluble in alcohol
dissolving of alcohol - using xylol or
toluol, to remove the excess alcohol
mounting and covering - using a
mounting medium, usually variants of
albumin, the stained specimen is covered
with a coverslip, thus completing the process
ADHESIVE
Mayers
Egg
Albumin

COMPOSITION

AMOUNT

egg white

50cc

glycerin

50cc

dried albumin
Egg
Adhesive NaCl
from Dried
Albumin distilled water

5.0 gm
0.5gm
100.0cc

infiltration of paraffin - specimens are embedded

in melted paraffin before microtomy
cooling and trimming
microtomy - using a microtome, the
block of paraffin-embedded specimen is
cut into 5-15m section

specimen post-embedding and staining, ready

for mounting and covering
2. other tissue preparation techniques using
different stains or dyes
microtome - a machine used to cut tissue
sample to thinner sections, usually in m
(micrometer)
d. staining - the colourless paraffin sections are
stained with hematoxylin and eosin to make it
visible under a light microscope; involves seven
processes
dissolving of paraffin - using xylol or
toluol, to remove the surrounding paraffin
and expose the specimen

a. acidic dyes - done to attract acidophilic

intracellular and extracellular components,
such as cytoplasmic filament, intracellular
membranous component, and extracellular
fibre
b. basic dyes - done to attract basophilic
intracellular and extracellular components and
achieve metachromasia
metachromasia - occurs when tissue
components shift from their normal colour
blue to red or purple upon absorption of
a basic dye

c. Schiff reagent/bleached basic fuchsin used to demonstrate glycogen in cells, cell and
tissue mucous, and basement membranes
basement membranes - membranes that
lie under the epithelial tissues, and some
connective tissues
STAIN

PROS

CONS

hematoxylin
and eosin

display general
structural features
of the specimen

many
components
are lost in the
preparation of
the specimen

acidic dyes

attracts
cytoplasmic
filament,
intracellular
membranous
component, and
extracellular fibre

basic dyes

achieves
metachromasia

Schiff
reagent

helpful in
demonstrating
basement
membranes

and newborn babies. This technique

searches for malformation in the body that
results to some syndromes. For fetuses, the
placenta and umbilical cord needs to be
autopsied while in infants, the whole chest
cavity may be opened underwater to test if
the infants have pneumothorax or not. This
type of autopsy needs Letulle type of
technique

postoperative autopsy - this type of

autopsy identifies possible medico legal

implications such as complication of
surgical intervention in anesthesia or drug
administration

immediate autopsy program - this type of

selective when
it comes to the
visibility of the
components of
the sample

3. other tissue preparation techniques using

radiography
a. autoradiography - uses a photographic
emulsion over a section to localise radioactive
material within tissues
b. historadiography - the production of an xray photograph or microradiograph of a
specimen on a slide
APPLICATION OF HISTOPATHOLOGY
1. Autopsy - derived from the Greek words auto
meaning self and opsis meaning view;
a. three kinds of seeing in autopsy
careful examination of the exterior of the
body
dissection and examination of the major
organs
examination of the tissues extracted from
the major organ

autopsy is a tool for investigating if the body

experiences physiologic effects like trauma,
shock, and metabolic diseases. In this
technique, the tissues that will be processed
for autopsy must be obtained immediately
after the somatic death

c. autopsy techniques

R. Virchow Technique - organs are

removed one by one; the first step in this

technique is to expose the cranial cavity,
then the spinal cord, followed by the
thoracic, cervical and abdominal organs

C. Rokitansky Technique - this technique is

characterised by in situ dissection; the
organs here are removed in en bloc manner

- in situ dissection - removal of organs

from their original places

- en bloc removal - organs are

removed all at the same time

A. Ghon - the easiest autopsy technique, this

technique may be done by only one person.

In this technique, the thoracic, cervical,
abdominal, and urogenital organs are
removed en bloc

M. Letulle - the thoracic, cervical,

abdominal, and pelvic organs are removed
en masse and subsequently dissected into
organ blocks

- en masse - organs are removed by

groups, usually by system or area

TECHNIQUE

PROS

CONS

Virchow

excellent for
demonstrating
pathologic
changes in
organs

sacrifices
inter-organ
relationships

b. four types of autopsies

adult autopsies - used for adults wherein

after the external description (weight and

height of the body), a Y incision is being
made. At this point, the kind of technique
may be varied according to lesion; en
masse removal is used if the pathologist
needs the diaphragmatic level such as
dissecting aortic aneurysms. Otherwise,
the technique of Virchow is used

pediatric autopsies - focuses on external

examination of children specifically fetuses

Rokitansky

some
preserves
disadvantage
interin cases of
relationships of
aortic and
various organs
neoplastic
dissection

TECHNIQUE

Ghon

Letulle

PROS

CONS

preserves interrelationships of
various organs

some
disadvantage
in cases of
aortic,
esophageal,
and neoplastic
dissection

allows for rapid

preparation for
mortuary,
excellent
preservation of
inter-relationships
of various organs

needs
assistance to
perform the
technique

2. Biopsy - the process of removing small pieces of

tissue from the body and the examination of these
tissues microscopically, biopsies are done on a
living subject to determine the presence or
extent of a disease
a. types of biopsies
surgical or section biopsy - done by
obtaining surgical sections of tissues for
histological diagnosis

b.

functions of biopsies

to establish a particular diagnosis and

exclude other disorders

to assess the extent of the disease

to assess prognosis and to determine
the treatment of choice

to assess the results of treatment

c. proper handling of biopsy specimens

do not leave it lying around at room

temperature since degenerative
changes may take place which may lead
to difficulty in diagnosis

if it is not sent to the laboratory right

away, place it in a refrigerator at 4
degree celsius or be placed in a suitable
fixative

proper labelling is essential, this

includes:

patient's full name

sex
age
ward
type of biopsy
surgeon or physician
date and time it was taken

BIBLIOGRAPHY
punching - a type of section biopsy which involves
the literal punching of a hole on the skin for the
cut area to serve as the specimen
needle biopsy - done by aspirating
cells or particles of tissues from tumors
for histological diagnosis

Mukherjee, KL., Ghosh, S. Medical Laboratory

Technology: A Procedure Manual for Routine
Diagnostic Tests, 2nd edition, volume 3. New
Delhi: Tata McGraw-Hill Education Private
Limited; 2010.1009p.
Ocampo, AM. Laboratory Guide in Histopathology.
Manila: UST Printing Office; 1975. 1-2p.
Read, AEA. Biopsy Procedures in Clinical Medicine.
Bristol: John Wright; 1968.
Sood, R. Medical Laboratory Technology: Methods
and Interpretations, 6th edition, volume 2. New
Delhi: Jaypee Brothers Medical Publishers; 2009.
1413p.
Trump, BF., et. al. Principles of Autopsy Techniques:
The Immediate Autopsy Program.

HISTOPATHOLOGY
Basco, Pauline Anne
Graycochea, Valerie Arianne
Ocampo, Miguel Joaquin
Tuana, Hazel
1B - MT