Cellular Pathology Techniques 1

• 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

• 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

-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

The microscopic examination of healthy or diseased tissue

• The microscopic examination of healthy or diseased cells • Usually split into gynaecological and non- gynaecological cytology

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

• Endoscope is used to remove tissue, such as from the stomach during a gastroscopy

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.

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

-larger hollow needed -solid sample of tissue is removed -assisted by ultrasound,pet,ct

• 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

Cell removed from the surface layer of tissue, such as from the inside of the mouth or from inside the cervix

Urine Sputum Cerebral spinal fluid

• 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

• 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

-Formaldehyde -Alcohol -glutaraldyhyde

+Preserves chemical reactivity +Rapid penetration +Relatively inexpensive -Renders tissue samples brittle -Shrinkage

+Penetrates tissues well +Suitable for large and small tissue samples +Relatively inexpensive +Provides ‘soft fixation’, good for dissection -slow action -toxic

+ Rapid action Excellent ultrastructure morphology -Poor penetration -Only suitable for very small tissues -Toxic -Relatively expensive

• 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

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

• Tissue is frozen quickly to reduce damage by ice crystals.Can be done by: • Chlorofluorocarbons (commercial spray) • Liquid CO2 • Liquid N2

• 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 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