Histopathology

Question 1

What is a histopathologist?

Answer 1

They deal with tissues and cross-examine sections noting the architecture of tissue and what it tells us about a condition

Question 2

What is a cytopathologist?

Answer 2

They deal with cells

They’re often taking cells from a patient, preparing them for examination and then delivering diagnosis

Question 3

What tissue samples does a histopathologist work with?

Answer 3

Biopsies
Resection specimens
Frozen sections
Post-mortems

Question 4

What are biopsies?

Answer 4

Small sections of tissue removed from the patient and placed in formalin solution which preserves the tissue by cross-linking proteins
They’re embedded in paraffin wax to allow thin sections to be cut by a microtome
These are mounted onto a glass microscope slide for further preperation

Question 5

What questions can microscopic examination of the biopsy answer?

Answer 5

Is the tissue normal?
Is the tissue inflamed and what’s the likely cause?
Is the tissue cancerous and if it is what type of cancer?

Question 6

What is the importance of chemical stains?

Answer 6

Often employed with the tissue section to aid the identification of cells within the biopsy

Question 7

What are examples of stains and what are their use?

Answer 7

Haemotoxylin and Eosin (H&E) staining can be used to identify the nuclei and cytoplasmic granules of leukocytes within tissues
Ziehl- Neelsen stain will stain acid-fast bacteria red aiding diagnosis of Tb

Question 8

What are resection specimens?

Answer 8

Taken from tissue thats been removed as part of surgical procedure and can be processed for a biopsy
Resections are mostly used to look at stage of disease

Question 9

What questions can be answered with a resection?

Answer 9

For cancer:
How far is the disease progressing?
Has the cancer penetrated the bowel wall, spread to lymph nodes or liver?

Question 10

How else can tissue from resection specimens be used?

Answer 10

Can be donated to biobanks and used to perform genomic studies of disease process

Question 11

What are frozen sections?

Answer 11

Taken during surgical procedures and are examined in real time while patient is being operated upon
Freshly taken tissue is frozen by machine called cryostat, cut then mounted onto glass slide and stained for biopsy
procedure can give rapid diagnosis in minutes
Tissue must be fresh and free of preservatives such as formalin

Question 12

What questions can we answer from frozen sections?

Answer 12

Is the tissue cancerous?
Has all the cancerous tissue been removed?
Is there another pathological process going on?

Question 13

What are fine needles used for and how?

Answer 13

Used for fine needle aspirates
Fine needle can be used to get into lesson and suck out (aspirate) the cells which can then be analysed for a smear
Very powerful technique as needle can penetrate relativeness inaccessible tissues e.g. thyroid nodule and assess suspect mass without need of surgery

Question 14

What is a downside to fine needle aspirates?

Answer 14

Cytopathologists only look at cells and are unable to look at architecture of tissue

Question 15

What is the timescale for a result for different histopathological samples?

Answer 15

Frozen section: 30 mins
Biopsies: 2-3 days
Resection specimens: 5-7 days

Question 16

What can immunohistochemistry be used for?

Answer 16

Can be used to identify key molecules within tissue

Question 17

How can we identify an endothelial tumour?

Answer 17

An antibody recognising endothelial cell marker CD31 on surface of endothelial cells allows us to identify the biopsy as an endothelial tumour

Question 18

What can high levels of antibodies recognising dsDNA be associated with?

Answer 18

System lupus erythematosus
SJrogen’s syndrome
Rhematoid arthritis

Question 19

What do manufactured antibodies do?

Answer 19

Can be used to specifically detect molecules in process of immunohistochemistry
Attachments (conjugates) to Fc region of antibody can be made permitting a wide variety of uses

Question 20

What are examples of antibody conjugates?

Answer 20

Enzymes: peroxidase, alkaline phosphatase

Fluorescent probes: allow rapid measurement of levels of molecules in sample. Multiplexing (using several antibodies with different fluorescents) can allow measurement of several molecules in single sample

Magnetic beads: e.g. purification of cell types. With use of magnets, cells can be depleted of contaminants

Drugs: the biological kadcyla, an anti-HER2 antibody linked to cytotoxic chemical emtansine

Question 21

How are antibodies used as a diagnostic tool?

Answer 21

Unique specificity of antibodies for their target is beneficial for testing

Antibodies can be used to detect proteins and other biological molecules such a s carbohydrates and lipids

This can be direct detection of antigen or via detection of unlabelled antibody by secondary antibody conjugated with a fluorescent probe or enzyme

Question 22

What are examples of antibody uses for diagnostic tools?

Answer 22

Blood group serology
Immunoassay e.g. detection of hormones, circulating antibody/antigen
Immunodiagnosis e.g. infectious diseases and antibody levels

Question 23

What is an ELISA test?

Answer 23

Enzyme linked immunosorbant assay

1. Clinical samples are allowed to adhere to a plastic plate
2. Then they’re probed with a specific antibody raised against the molecule of interest
3. Antibody is conjugated with an enzyme which can turn a colourless substance a particular colour

Question 24

What is flow cytometry?

Answer 24

Allows detection of specific cells, most notably lymphocyte subpopulations using fluorescent conjugated specific antibodies

Question 25

What lymphocyte populations may be detected by flow cytometry?

Answer 25

Anti- CD3+ = pan T cell markers
Anti - CD4+ = T helper cells
Anti- CD8+ = Cytotoxic T cells
Anti- CD19+ = B cells
anti- CD56+ = NK cells

Question 26

How does flow cytometry work?

Answer 26

1. Cells are labelled with a different conjugated antibody
2. Then run as a stream of single cells through a laser beam
3. Colour of light emitted and the forward or side scatter of the laser beam can tell us the identity of the cell surface molecule expressed and size and granularity of the cells