Cellular Pathology Techniques 1

Question 1

Role of cellular pathology

Answer 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

Question 2

When are autopsies undertaken?

Answer 2

•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

Question 3

What happens in an autopsy

Answer 3

-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

Question 4

Histology/Histopathology

Answer 4

The microscopic examination of healthy or diseased tissue

Question 5

Cytology/Cytopathology

Answer 5

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

Question 6

Specimen Collection-Biopsy

Answer 6

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

Question 7

Endoscopic biopsy

Answer 7

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

Question 8

Punch Biopsy

Answer 8

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.

Question 9

Needle biopsy

Answer 9

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

Question 10

A Core needle biopsy

Answer 10

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

Question 11

Surgical Biopsy

Answer 11

•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

Question 12

Scraping cells

Answer 12

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

Question 13

Fluid collection

Answer 13

Urine
Sputum
Cerebral spinal fluid

Question 14

Why is tissue fixing important

Answer 14

•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

Question 15

Properties of Fixatives

Answer 15

•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

Question 16

List Common Fixatives

Answer 16

-Formaldehyde

-Alcohol

-glutaraldyhyde

Question 17

Advantages and disadvantages of using alcohol for fixing

Answer 17

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

Question 18

Advantages and disadvantages of using formaldehyde

Answer 18

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

-slow action
-toxic

Question 19

Advantages and disadvantages of using glutaraaldyhyde

Answer 19

+ Rapid action
Excellent ultrastructure morphology

-Poor penetration
-Only suitable for very small tissues
-Toxic
-Relatively expensive

Question 20

Sectioning

Answer 20

•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

Question 21

Processing

Answer 21

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

Question 22

Frozen Tissue

Answer 22

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

Question 23

sectioning

Answer 23

•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

Question 24

Staining

Answer 24

•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

Question 25

Formaldehyde as a fixative

Answer 25

• 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

Question 26

Glutaraldyhyde as a fixative

Answer 26

-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)

Question 27

Alcohol as a fixative

Answer 27

-Non-additive fixative
–Preserves chemical reactivity
–Tissue shrinks and becomes brittle
–More common for fixing smears or fresh sections

Question 28

Advantages of freezing tissues

Answer 28

-freezing water is quicker than replacing it
-it doesn’t involve treating with chemicals
-better survival of many antigens

Question 29

Disadvantages of frozen tissue

Answer 29

-poor morphology
-poor resolution at higher magnification
-cutting difficulties
-special storage

Question 30

Haemotoxylin and Eosin (H&E)

Answer 30

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

Question 31

Masson’s trichrome

Answer 31

Most recipes produce red keratin and muscle fibres, blue or green collagen and bone, light red/pink cytoplasm, and to black nuclei.

Question 32

Alcian blue

Answer 32

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

Question 33

Van Gieson

Answer 33

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.

Question 34

Luxol Fast Blue

Answer 34

•Attracted to bases found in lipoproteins of the myelin sheath.

Question 35

Silver Impregnation

Answer 35

•Variety of silver stains available to stain neurons
•Bielschowsky silver stain highlights tangles and plaques in Alzheimer’s disease

Question 36

Sudan Black

Answer 36

Stains triglycerides, lipids and lipoproteins
•Stains myeloblasts but not lymphoblasts

Question 37

Steps in staining

Answer 37

-Dewax
Clear
Rehydrate
Stain
Dehydrate
Clear
Mount

Question 38

Rehydration ,dyhydration

Answer 38

Less alcohol and more water
Less watere and more alcohol eg xylene

Question 39

Immunohistochemistry

Answer 39

Antibodies used to detect specific molecules (antigens)