Cellular Pathology Techniques 1 Flashcards
Role of cellular pathology
•Involves investigation of the aetiology and pathogenesis that result in the signs and symptoms of the patient
•Cell pathology looks at cells in tissue or fluid
•Cell arrangement, development and function is investigated to determine if a patient has a disease, inflammation, or cancer
•Multi-disciplinary teams are involved to decide an appropriate treatment plan or review how a treatment is progressing.
•Can be used to diagnose patients or determine cause of death
When are autopsies undertaken?
•At request of a coroner or hospital doctor
•Coroner-
•if death is sudden, suspicious, unexplained, violent, occurs soon after a hospital stay or occurs from accident or injury
•Doctor –
•To gain a better understanding of the illness
What happens in an autopsy
-First, the height, weight, age, and gender of the body should be noted and recorded.
-istinguishing characteristics like
-An external examination is performed.
-A blood sample for toxicology and biochemical analysis
-Internal examination:
-A large “Y” shaped incision is made from each shoulder across the chest to the brisket, then down to the belly button,The skin is then spread open to check to see if any ribs are broken.
-The ribcage is split open, and the internal organs examined
-Each organ is examined individually
-Organs are weighed and tissue samples taken and fixed for further examination
Histology/Histopathology
The microscopic examination of healthy or diseased tissue
Cytology/Cytopathology
•The microscopic examination of healthy or diseased cells
•Usually split into gynaecological and non- gynaecological cytology
Specimen Collection-Biopsy
Cells can be taken from tissue removed:
•During surgical procedures in theatres
•At outpatient clinics
•At GP clinics
•During post-mortem examination
Or can be collected from fluid
•Blood
•Urine
•CSF
•Sputum
Endoscopic biopsy
•Endoscope is used to remove tissue, such as from the stomach during a gastroscopy
Punch Biopsy
The lines of least skin tension are determined.
•Skin is stretched 90 degrees perpendicular to the lines of least skin tension
•The punch biopsy is performed.
•The instrument is pressed down and rotated clockwise and anti-clockwise, cutting down into the subcutaneous fat.
•The punch biopsy instrument is removed.
•The biopsy specimen is gently lifted with a needle to avoid crush artifact.
•Scissors are used to cut the specimen free at a level below the dermis.
Needle biopsy
Fine-needle aspirate
•Superficial lumps or masses
•Usually palpable
•A thin hollow needle is used
•Cells aspirated
-the needle is used to draw sample fluid and tissue from a lump to be studied
A Core needle biopsy
-larger hollow needed
-solid sample of tissue is removed
-assisted by ultrasound,pet,ct
Surgical Biopsy
•An excisional biopsy – where surgery is used to remove a larger section of tissue
•perioperative biopsy – if consent has been given, a perioperative biopsy can be carried out during surgery; the sample will be tested straight away so that the surgeon can be given the diagnosis and provide appropriate treatment
Scraping cells
Cell removed from the surface layer of tissue, such as from the inside of the mouth or from inside the cervix
Fluid collection
Urine
Sputum
Cerebral spinal fluid
Why is tissue fixing important
•Fixation is an attempt to preserve tissue as close to its natural state as possible
•Fixatives kill tissue/disable biomolecules (eg proteolytic enzymes) preventing autolysis and putrefaction
•Protects sample from extrinsic damage
•Fixatives are toxic to most common organisms preventing infection
•Preserve morphology
•Fixatives increase the strength and stability of the tissue
Crucial to prevent degradation/loss of integrity
Properties of Fixatives
•Penetrate tissue quickly and evenly
•Kill cells quickly and evenly
•Prevent autolysis
•Prevent putrifaction
•Not add extraneous material
•Not swell or shrink tissue
•Be compatible with later processing
•Prevent tissue from drying out
•Safe
•Cheap
•Convenient
List Common Fixatives
-Formaldehyde
-Alcohol
-glutaraldyhyde
Advantages and disadvantages of using alcohol for fixing
+Preserves chemical reactivity
+Rapid penetration
+Relatively inexpensive
-Renders tissue samples brittle
-Shrinkage
Advantages and disadvantages of using formaldehyde
+Penetrates tissues well
+Suitable for large and small tissue samples
+Relatively inexpensive
+Provides ‘soft fixation’, good for dissection
-slow action
-toxic
Advantages and disadvantages of using glutaraaldyhyde
+ Rapid action
Excellent ultrastructure morphology
-Poor penetration
-Only suitable for very small tissues
-Toxic
-Relatively expensive
Sectioning
•Fixed tissue is still quite soft and needs extra support to allow thin sections to be cut (usually 3-6µM)
•Hard embedding medium such as paraffin wax is therefore used to support the cells and keep the tissue firm
•Processing is the method by which tissues are converted into a block for microtomy
Processing
Paraffin wax impregnation
•Specimen cannot simply be immersed in wax, it must be impregnated
•Water must be replaced with wax
1)dehydration
2)clearing
3)imprergantion
4)removal of clearing agent
5)blocking out tissue
6)microtomy
Frozen Tissue
•Tissue is frozen quickly to reduce damage by ice crystals.Can be done by:
•Chlorofluorocarbons (commercial spray)
•Liquid CO2
•Liquid N2
sectioning
•Using a cryostat
•Deep freeze cabinet with microtome inside and controls outside
•Sections are cut from frozen tissue
•Sections are then picked up onto warm (room temp) slides
•Adhesion good due to sudden change in temperature
Staining
•Staining is required to provide contrast between different cell types and cell components
•Most staining methods rely on chemical principals, whereby specific chemical structures can be selectively stained
•Haematoxylin and eosin (H&E) is the most common staining method
Formaldehyde as a fixative
• Most commonly used fixative
• 40% solution in H20 called formalin
•Commonly used diluted 1:10 in PBS buffer called 10% neutral buffered formalin (10% NBF)
•Same as 4% Formaldehyde
•Best for good architecture
Glutaraldyhyde as a fixative
-Reacts more quickly than formaldehyde
–Difficult to remove from tissue
–Inactivates all enzymes preventing enzyme histochemistry
–Binds and masks many antigens
–Gives excellent morphology (widely used in EM)
Alcohol as a fixative
-Non-additive fixative
–Preserves chemical reactivity
–Tissue shrinks and becomes brittle
–More common for fixing smears or fresh sections
Advantages of freezing tissues
-freezing water is quicker than replacing it
-it doesn’t involve treating with chemicals
-better survival of many antigens
Disadvantages of frozen tissue
-poor morphology
-poor resolution at higher magnification
-cutting difficulties
-special storage
Haemotoxylin and Eosin (H&E)
Most common stain in histology
•Haemotoxylin (blue) is a basic dye and therefore stains acidic components eg DNA & RNA
•Eosin (Pink) is an acidic dye and therefore binds and stains basic components eg. Proteins in the cytoplasm, cytoplasmic filaments and extracellular fibres
Masson’s trichrome
Most recipes produce red keratin and muscle fibres, blue or green collagen and bone, light red/pink cytoplasm, and to black nuclei.
Alcian blue
A mucin stain that stains certain types of mucin blue. Cartilage is also stained blue. It can be used with H&E, and with van Gieson stains
Van Gieson
Stains collagen red, nuclei blue, and erythrocytes and cytoplasm yellow. It can be combined with an elastic stain that stains elastin blue/black. It is often used for blood vessels and skin.
Luxol Fast Blue
•Attracted to bases found in lipoproteins of the myelin sheath.
Silver Impregnation
•Variety of silver stains available to stain neurons
•Bielschowsky silver stain highlights tangles and plaques in Alzheimer’s disease
Sudan Black
Stains triglycerides, lipids and lipoproteins
•Stains myeloblasts but not lymphoblasts
Steps in staining
-Dewax
Clear
Rehydrate
Stain
Dehydrate
Clear
Mount
Rehydration ,dyhydration
Less alcohol and more water
Less watere and more alcohol eg xylene
Immunohistochemistry
Antibodies used to detect specific molecules (antigens)
