Histopathology Flashcards
Grossing
process whereby pathology specimens are inspected with the naked eye to obtain diagnostic info
Points to note before tissue processing for microscopic examination
- Identification of the specimen- confirmation of patient and anatomical site from which specimen was obtained
- clinical details
- gross description-written record of the physical appearance of the specimen
- only a small portion from a large specimen can be -subjected to microscopic examination hence during grossing a skilled person should do the examination
- only soft tissue can be cut into small blocks and processed directly
- bony specimen need to be decalcifies prior to processing
- bones and teeth require special treatment
Items that should be in a gross room
- cutting board fluid from board must run directly into a sink
- shelves for specimen containers
- ready access to hot and cold water
- ready access to formalin
- box 0f instruments containing forceps of various sizes, scissors of various types and sizes, a probe, a bone cutting saw or electric bone cutter, scalpel handle, disposable blades, long knife, ruler to measure size of lesions and specimens
- box with cassettes and labels
Lab hazards and safety measures
-formalin vapours are irritants affecting the eyes and throat. Exhaust may be used as outlet for vapours
-always use a mask, apron, aye glasses and gloves as protection from:
infected material
formalin vapours
spilt blood or any other fluid
-keep grossing table clean with antiseptic solution
-all specimen should be in a container with 10% formalin and covered with a lid
-after grossing specimens should be kept according to accession numbers
Histopathology lab
should be large enough to accomodate various types of equipment and must allow personnel to work with ease
Equipment kept in a histopathology lab
tissue processor tissue embedding table microtome tissue warming table tissue flotation bath slide stainer or glassware for manual staining table to label and dispatch the slides
Handling of specimen
specimen should be transported in a glass, plastic or metal container or in a plastic bag in 10% formalin. If formalin is not avail, place specimen in refrigerator at 4 degrees to slow autolysis. Container should have opening large enough so tissue can be removed after hardening by fixation
Fresh ,material is needed for
frozen section immunocytochemistry cytological examination microbiological sampling before histology chromosome analysis research purposes museum display
Principles of gross examination
proper identification and orientation of specimen
unlabelled specimen should never be processed
properly completed histopathology requisition form should contain patients name, sex, age, relevant clinical data, surgical findings, nature of operation and type of tissue submitted
careful search and examination of all the tissue in the order in which it was submitted
surgeon should be instructed to submit all the material he/she removed rather than selecting a portion from it
specimen should be placed on the cutting board in the correct anatomical position before recording type of specimen structure dimensions weight shape colour consistency surgical margin
measurements are usually given in cm unless specimen is very small in which mm can be used
endometrial and prostatic tissue should be measured by aggregate pieces in volume
endoscopic biopsies should be numbered
Sampling for histopathological examination
tissue submitted must not be more than 3mm thick and not larger than the diameter of the slides used
Histological technique
deals with preparation of tissue for microscopic examination. Aim is to preserve the microscopic anatomy of tissue and harden it so that a very thin section can be made.
Processes for the histological technique
fixation dehydration cleaning embedding cutting staining
Fixation
carried out as soon as possible after tissue removal to prevent autolysis. purpose is to preserve tissue constituents so they will withstand treatments with various reagents with min loss of architecture
expose tissue to chemical compounds called fixatives
Mechanism of action of fixatives
denature or precipitate proteins which then form a sponge or meshwork that tends to hold other constituents
important factors: fresh tissue, proper penetration of tissue by fixatives
correct choice of fixatives
no fixative will penetrate piece of tissue thicker than 1cm
How to deal with tissue thicker tan 1cm for fixation
solid organ: cut slices no thicker than 5mm
Hollow organ: either open or fill with fixative or pack lightly with wool soaked in fixative
large specimen which require dissection: inject fixative along vessels so that it reaches all parts of the organ
Properties of an ideal fixative
prevents autolysis and bacterial decomposition
preserves tissues in its natural state and fixes all components
makes cellular components insoluble to the reagent used in tissue processing
preserved tissue volume
avoids excessive hardness of tissue
allows enhanced staining of tissue
should be non-toxic non-allergic for user
should not be very expensive
Fixation temperature
can be carried out at room temp
tissue should not be frozen once it has been placed in fixative solution for a peculiar ice crystal distortion will result
Speed of fixation
almost 1mm/hour therefore fixation time of several hours is needed
Amount of fixation fluid
10-20 times volume of specimen
fixative should surround specimen on all side
Factors affecting fixation
size and thickness of piece of tissue
tissue covered by large amount of mucus’s fix slowly as does tissue covered by blood or organs containing very large quantities of blood
fatty and lipomatous tissue fixes slowly
fixation is accelerated by agitation and maintaining a temp of 60 degrees
Classification of fixatives
Tissue fixatives -buffered formalin -buffered glutaraldehyde -zenkers formal saline - Bowen's fluid Cytological fixatives -ethanol -methanol -ether Histochemical fixatives -formal saline -cold acetone-absolute alcohol
Routine formalin
Formalin is sold as 40% w/w solution of formaldehyde gas in water. It is used as 10% solution in water or normal saline. It does not precipitate protein but combined with the NH2 group to form an insoluble gel it preserves all elements including fats. It keeps phospholipids insoluble in fat solvents. Tissue can remain in it for prolonged periods without distortion. Compatible most special stains it is the cheapest and most popular fixative
Ethyl alcohol
used at 90-100% strength. It precipitates albumin but not nucleoprotein. It causes shrinkage and hardening of tissues and destroys mitochondrion. It preserves glycogen and is useful for the histochemistry of glycogen, uric acid and iron.
Tissue processing
process of getting tissue into paraffin wax
24 hours manually or mechanically
To cut thin sections of a piece of tissue it should have suitable hardness and consistency when presented to the knife edge. These properties can be imparted b y infiltrating and surrounding the tissue with paraffin wax, colloidin or low-viscocity nitrocellulose, various types of resins or by freezing.
Tissue processing is divided into
dehydration cleaning embedding impregnanting specimen should be labelled first shouldn't use a pen with ordinary ink, printed graphite pencil, written, typewritten or India ink-written labels
Transporting tissue through various steps of processing
the cut specimen are put in myelin cloth together with their labels and then transported from reagent to reagent in metal containers with perforated walls so reagent enters the tissue
Manual tissue processing
tissue is moved from one container of reagent to another by hand
Mechanical tissue processing
automatic tissue processors contain diff jars with reagents. These are arranged in a sequence. The tissue is moved from one jar to another by a mechanical device. Timings are controlled by a timer which can be adjusted to measure hours and or minutes. The temp is maintained at 60 degreed
Dehydration
water in tissue ir replaced by alcohol
tissue are dehydrated using strong alcohol e.g. 50% 70% 90% 100%
duration tissue are kept in each strength of alcohol depends on size of tissue, fixative used and the type of tissue. Delicate tissue will undergo a high degree of shrinkage in too great a concentration of alcohol therefore will start slowly at a lower % of alcohol
Volume of alcohol should be 50-100 times that of the tissue
Clearing
Alcohol is replaced by paraffin wax.
Parrafin is not alcohol soluble the alcohol must be replaced with a substance in which wax is soluble this step is called clearing
Tissue clearing is achieved by using any of these reagents
xylene ( commonly used small pieces of tissue are cleared in 0.5-1 hour while larger pieces in 2-4 hours) chloroform benzene carbon tetrachloride toluene
Impregnation with wax
occurs at melting point of paraffin wax which is at 54-60 degrees
Volume of wax should be about 25030 times the volume of tissue. Duration of impregnation depends on size and type of tissue and the clearing agents employed. Longer periods required for larger pieces and harder tissues like bone and skin. Using xylene is the easiest way to remove wax. IN total 4 hours is sufficient
Types of wax employed for impregnation
paraffin wax
water-soluble wax
other material like colloidin, gelatin, paraplast
Blocking aka ambedding
impregnated tissues are placed in a mould with their labels and then freshly melted wax is poured into the mould and allowed to settle and solidify. Once block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly. After the block has completely cooled down, it should be cut into individual blocks after which each must be trimmed. Labels must be made to adhere on the surface of the block by melting the wax with a metal strip which has been sufficiently warmed
Staining
process by which colour is given to a section
Classification of stains
Acid stain
Basic Stain
Neutral stain
Dye
all dye are composed of acid and base components
Dye is a compound which can colour fibres and tissue constituents
Acid dyes
basic component is coloured while the acid component is colourless. Acid dyes stain basic components. Colour imparted is a shade of red
Basic dyes
acid component colourless. Basic dyes stain acidic componets. Colour imparted in shade of blue
Neutral dyes
acid dye combined with basic dye
as it contains both coloured radicals, it gives different colours to each of the cytoplasm and nucleus. This is the basis of Leishman stain
Staining procedure
staining can be done manually or mechanically ( automatic )
Manual staining: method of choice in a small lab, few slides stained daily
time consuming and economical
diff reagents containers are placed in a special sequence and the slides are moved manually from one container to another
Automatic staining: in a busy histopathology lab where hundreds of slides are stained daily
uses diff containers of staining reagents arranged according to the desired sequence, Has a timer which controls period for which slides should remain in given container, A mechanical device shifts the slides from one container to the next after a specified period
Advantage of automated stainer
reduces manpower
accurately controls timing of staining
large number of slides can be stained simultaneously
lower quantities of reagents used
Haemotoxylin ( basic dye) and Eosin staining ( acid dye)
the most commonly used routine stains in any histopathology lab
Frozen sectioning
freezing makes tissue solid enough to section with microtome
tissue is frozen rapidly at -20 degreed and sections are cut and stained. In this way tissue can be examined microscopically within 5-10 min of its removal from the body. It reduces time of processing from 18 hours to five minutes. It has the disadvantage that only 8-16 micron thick sections can be cut and finer details of tissue cannot be examined. Performed on a machine called cryostat
Frozen sections helpful for
when rapid diagnosis regarding benign or malignant nature of a lesion is required to decide the extent of surgery while the patient is still on the operating table
when a study of fat proteins or antigenic markers is required as the routine processing of tissue destroys them
