Histopathology

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.

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.

Zenker’s 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

Carnoy’s 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 Zenker’s fluid before use saturated picric acid

750cc

37-40% Bouin’s 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 Mayer’s 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 5-15µm 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 µm (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