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 1800’s, 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 1900’s, and the invention of automatic stainers by 1965.
Today, automated tissue processors, high-resolution microscopes, and other advanced laboratory equipment are available for use in the field of histopathology.
HISTOPATHOLOGY: THE PROCESS
To be able to study a tissue sample, it undergoes several processes to preserve and highlight the important components and structure of the sample.
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;
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;
FIXATIVE COMPOSITION AMOUNT
Zenker’s fluid distilled water 1000cc HgCl2 50gm K2Cr407 25gm Na2SO4 10gm
*add 5cc of glacial CH3COOHto
95cc of Zenker’s fluid before use
Bouin’s fluid
saturated picric acid 750cc 37-40%
formaldehyde 250cc
glacial CH3COOH 50cc
METHOD PROCEDURE SPECIMEN/INTENDED 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 acid-sodium 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
FIXATIVE COMPOSITION AMOUNT
10% formalin solution 37-40% formaldehyde 100cc tap water 900cc buffered neutral formalin solution 37-40% formaldehyde 100cc distilled water 900cc Na3PO4 monobasic 4gm Na3PO4 dibasic 6.5gm Carnoy’s fluid absolute alcohol 60cc chloroform 30cc glacial CH3COOH 10cc 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
• cooling and trimming
• microtomy - using a microtome, the block of paraffin-embedded specimen is cut into 5-15µm section
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
• 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
2. other tissue preparation techniques using different stains or dyes
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
microtome - a machine used to cut tissue sample to thinner sections, usually in µm
(micrometer)
ADHESIVE COMPOSITION AMOUNT
Mayer’s Egg Albumin egg white 50cc glycerin 50cc Egg Adhesive from Dried Albumin dried albumin 5.0 gm NaCl 0.5gm distilled water 100.0cc infiltration of paraffin - specimens are embedded
in melted paraffin before microtomy
specimen post-embedding and staining, ready for mounting and covering
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
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 x-ray 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
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 fetusesand 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 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 deathc. 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 areaSTAIN 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 selective when it comes to the visibility of the components of the sample basic dyes metachromasiaachieves
Schiff reagent helpful in demonstrating basement membranes
TECHNIQUE PROS CONS
Virchow excellent for demonstrating pathologic changes in organs sacrifices inter-organ relationships Rokitansky preserves inter-relationships of various organs some disadvantage in cases of aortic and neoplastic dissection
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
• needle biopsy - done by aspirating cells or particles of tissues from tumors 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 t e m p e r a t u r e s i n c e d e g e n e r a t i v e 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 takenBIBLIOGRAPHY
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.
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
TECHNIQUE PROS CONS
Ghon preserves inter-relationships of various organs some disadvantage in cases of aortic, esophageal, and neoplastic dissection Letulle
allows for rapid preparation for mortuary, excellent preservation of inter-relationships of various organs needs assistance to perform the technique
