Histopathology Flashcards
What is histopathology?
Deals with tissues, examining the architecture of the tissue; observations used in diagnosis, as well as towards informing the efficacy of a particular treatment
What are tissue samples?
Biopsies, resection specimens, frozen sections, and post-mortems
What are biopsies?
Concerns small sections of tissues removed from the patient; immersed in a formalin solution that preserves the tissues by cross-linking proteins.
What is formalin?
Preserves tissues by cross-linking proteins, samples are embedded in paraffin wax, enabling thin sections to be cut by a microtome.
Sample mounted on a microscope slide for preparation prior to analysis
What is the main function of a biopsy?
As a diagnostic tool
Which chemical stains are used for cells in the biopsy section?
Haematoxylin and Eosin staining used to identify nuclei and cytoplasmic granules of leukocytes
Which type of stain will stain acid-fast bacteria red, aiding in diagnosis for TB infection?
Ziehl-Neelsen stain
What are resection specimens?
Sourced from tissue removed from surgical procedure, processed as for a biopsy
What are resections used for?
To identify the stage and progression of the disease (cancers, diagnostic too for further treatment)
Why are tissues donated to biobanks?
Used to inform genomic studies of the disease process
Which sample is taken during surgical procedures and examined by pathologists in real time?
Frozen sections
How are sections frozen?
By cryostat, cut then mounted on glass slide and stained for biopsies
Why are frozen sections used?
Rapid diagnosis, relayed back to the surgeon to inform the surgery. Establishes presence & quantity of cancerous tissue, as well as identifying other pathological processes
Fresh tissue sample w/o formalin
How long are frozen sections stored and used for?
30 minutes
What is the timescale for biopsies?
2-3 days
What is the timescale for resection specimens?
5-7 days
What is cytopathology?
The study of disease at a cellular level.
Cytopathologists identify cells, collected and smeared onto a microscope slide
The slide is stained and examined.
What is used to analyse for a smear when inserted into a lesion & aspriate?
Fine needle aspirates
Why are fine needle aspirates used?
The needle penetrates inaccessible tissue (thyroid nodule), and assess the suspect mass without the surgical requirement
What is the disadvantage with fine needle aspirates?
Cytology concerns individual cells, and thereby unable to determine the architecture of the tissue and cellular influences on adjacent cells.
What does brown coloration indicate during a skin biopsy?
Antibody recognizing the endothelial cell marker CD31 certifies the biopsy as an endothelial cell tumor.
Combining biopsies with analysis of fine needle aspirate taken from an enlarged lymph node, a diagnosis of reactive lymphadenopathy can be established due to mixed cell population observed
What is used in immunohistochemistry?
Antibodies can be used to specifically detect molecules in the process of immunochemistry.
Which region of the antibody is used to attach with markers?
Fc region.
What are the four main attachments to antibody Fc regions?
Enzymes
Fluorescent probes
Magnetic beads
Drugs
Which enzymes are attached to Fc regions of antibodies?
Peroxidases, and alkaline phosphatase used in conjunction with a colorless substrate to produce a colored product- CD31 staining; in addition to the detection of estrogen receptors on breast cancer tissue biopsies.
Why are fluorescent probes used in antibody diagnostics?
Rapid measurement of the levels of molecules within a sample.
Multiplexing allows a measure of several molecules ina single clinical sample
What is multiplexing?
Several antibodies with different fluorescence
Why are magnetic beads used in antibody diagnostics?
Cells can be depleted of containment, Anti-CD3 antibodies used to deplete bone marrow of T cells prior to use in bone marrow grafts
When are antibody diagnostics used?
Blood group serology, immunoassays (detection of hormones) circulating antibodies/antigens
Immunodiagnosis - presence of circulating anti-HIV antibodies suggests infection with HIV
High circulating levels of antibody suggest myeloma
IgE classes suggestive of the allergic phenotype
What does ELISA stand for?
Enzyme-linked immunosorbent assay
What is ELISA?
Serum samples allowed to adhere to a plastic plate; probed with a specific antibody raised against the molecule of interest
Antibody conjugated with an enzyme catalyses colourless substrate into a colour product
Reference to a standard curve, relatively absorbance of the solution can be used to determine precise levels of the molecule being studied
What is the first stage of ELISA?
Antibody coating: Specific capture antibody is immobilized on high-protein binding plates, incubated
Plates are blocked with irrelevant protein (alubmin)
What is protein capture during ELISA?
Samples and standard dilutions are added to the wells, and will be captured by the bound antibodies.
What is a detection antibody during ELISA?
Specific biotinylated detection antibody inserted to wells, enables detection of the captured protein, Wash unbound detection antibodies.
Which enzyme is conjugated with the wells?
Strepavidin-enzyme conjugate with alkaline phosphates is added to the wells and will bind to the biotinylated antibody
What happens upon addition of substrate to ELISA?
Colorimetric substrate is added to the wells, and will form a coloured solution when catalysed by the enzyme
How is the absorbance measured during ELISA?
By ELISA reader = amount of protein established.
What is flow cytometry?
Allows detection of specific cells using fluorescently conjugated specific antibodies that associate to CD specific receptor sites on the lymphocyte cell surface membrane
How does flow cytometry work?
Cells labeled with differently conjugated antibodies, run as a stream of single cells through a laser beam. Colour of emitted light and forward/side scatter denotes the identity of the cell surface molecules expressed, the size and granularity of cells.
