Independent Study 1 Flashcards
- Types of leukocytes in human peripheral blood from most to least (5) 2. Describe why morphology alone can not be used to assess the numbers and types of leukocytes in the peripheral blood (which cells can you differentiate with morphology? Which can’t you?)
- Neutrophils > lymphocytes (T, B, NK cells) > monocytes > eosinophils > basophils 2. First know the 2 classes of leukocytes - granulocytes (neutrophils, eosinophils, basophils) and mononuclear cells (lymphocytes and monocytes) A. Granulocytes can be differentiated from one another by morphology (neutrophil - clear nucleus, eosinophil - red, basophil - blue) B. Monocytes can be differentiated from lymphocytes based on nuclear morphology C. YOU CANNOT DIFFERENTIATE LYMPHOCYTES (B cells, T cells, NK cells, CD4 and CD8) - NO GRANULES
Explain how one could separate peripheral blood leukocytes from red blood cells and platelets for immunologic analysis 1. Mononuclear cells from whole blood; 2. To study subset of mononuclear cells; 3. To distinguish different mononuclear cells (e.g?) 4. To study granulocytes (e.g?) 5. **Distinguish T cell subsets from B cells, monocytes and NK cells (similarity vs diff)
- Mononuclear cells from whole blood; PURIFICATION by used of density gradient purification step 2. To study subset of mononuclear cells; pull off Buffy coat layer - wash it in sterile saline - count the cells (assessing both number and viability) 3. To distinguish different mononuclear cells (monocytes vs lymphocytes); additional analyses required 4. To study granulocytes (neutrophils, basophils etc); other density gradients can be used to purify them to allow functional studies. 5.*Similarity; nuclear to cytoplasmic ratio, nuclear shape Diff; cell surface molecule expression to differentiate the cells
- What is used to discriminate cells, molecules and pathogens? - what cells make antibody? What is antibody? 2. Discuss how the specificity of the antibody can be utilized for diagnostic purposes - serum has lots of? Referred to as? 3. use of mAB?
- Monoclonal antibodies (mAb) - B cells make antibodies - proteins that recognize and bind to different biomolecules in a very specific manner. 2. -Serum has thousands of antibodies all with different specificities. Pool of antibodies is “polyclonal” - mABs have been used to differentiate essentially every cell in the body. Combinations of antibodies can DISTINGUISH SUBSETS of CELLS 3. - each antibody defines a particular antigen found on a cell (leukocytes, tumor, somatic cells) - some antibodies bind to cytokines, growth factors, toxins, bacteria, virus etc - Antibodies can also be made against other antibodies
- Identify the following - binding patterns of the antibodies 2. What is most important to recognize about above? 3. Identify the various cell types (what does + or - indicate) - CD3+CD45+CD4+CD8- - CD3+CD45+CD4-CD8+ -CD3+CD45+CD4+CD25+FoxP3+ - CD3-CD45+CD4-CD8-CD16+CD56+ - CD3-CD45+CD4-CD8-CD19+CD22+
- Clusters of differentiation (CD markers) - once the antibody is obtained, extensive functional and binding analyses are required before the antibody gets a CD designation and is accepted by specific community 2. Recognize that they define not only leukocyte types but essentially many types of cells in the body. 3. - Helper T cells - Cytotoxic T cells - Suppressor cell (Treg) - NK cell - B cell **+ or - indicate the presence or absence of CD marker in particular cell.
- Describe what CD designations are and identify the following cells using only CD nomenclature: helper T cell, cytolytic T cell, suppressor cell (Treg), Natural Killer Cell and a B cell. 2. What are common markers, what markers allow you to distinguish one from the other?
- Binding patterns of antibodies are called clusters of differentiation or CD Helper T cell; CD3+CD45+CD4+CD8- Cytolytic T cell; CD3+CD45+CD4-CD8+ Suppressor T cell; CD3+CD45+CD4+CD25+FoxP3+ NK cell; CD3-CD45+CD4-CD8-CD16+CD56+ B cell; CD3-CD45+CD4-CD8-CD19+CD22+ 2. Common markers; CD3+, CD3-, CD45+ Distinguishing marker; CD4+, CD4-, CD25+FoxP3+,CD19+CD22+, CD16+CD56+
Flow cytometry is one of the uses of? 1. Describe in general the principles behind flow cytometry and 2. how data obtained from the cytometry lab be utilized to gain insight on a patients clinical status?
Uses of mAb; flow cytometry 1. Flow cytometry allows us to enumerate/list leukocytes or any cell using florescent labelled antibodies against different CD antigens - fist incubate the cells with antibody of interest - each antibody is stained with a different color fuorochrome - after washing steps, cells are passed through the flow cytometer and different lasers identify the different colors - each color is coupled to the antibody defining the CD marker - cells pas thorough in single file - cells are quantitative and can be isolated sterilely as individual cells for further analysis (STERILE SORTING)
- How can one purify specific subsets of leukocytes utilizing monoclonal antibodies?
MAGNETIC BEAD SEPARATION - mAb can also e used to separate cells using magnetic beads - mAb are coated with magnetic beads and then incubated with mixed cell population - antibody binds to specific cell in presence of magnet (all non bound cells are washed out) - after washing, remove column from magnetic field and release the coupled cells - bound cells can be used in functional assays.
Identify all functions on monoclonal antibodies (6)
- Used to discriminate cells, molecules and pathogens 2. Defines and binds to a particular antigen found on a cell (not only leukocytes, but any tumor or outer somatic cell) - give it a CD marker 3. Antibodies can bind to cytokines, growth factors, toxins, bacteria, virus etc 4. Antibodies can be made against other antibodies 5. Used in flow cytometry; give different fluorescent color to see what antigen it binds to. Cells are quantitative and can be isolated sterile for further analysis (sterile sorting) 6. Magnetic bead separation; bind to antigen, purify and use in functional assay
Due to improved techniques in molecular biology and gene cloning techniques, large quantity of what is now available for clinical use?
Certain recombinant CYTOKINES
Describe how alpha and beta interferon clinical use 1. - treatment of infectious autoimmune disorders (MS) 2. - approved to treat viral hepatitis (Hep C); it can prevent infection if treatment is immediate 25% cure rate - certain cancers like melanoma
- Beta interferon 2. Alpha interferon
Describe clinical use of cytokines
- Cytokine genes have been cloned and produced in large quantities under GMP lab conditions 2. Testes in Phase I to phase III clinical trials are are used routines in clinical practice (immunotherapy?) 3. FDA approval to used in humans **Interferons used against viral infections **Interleukins (various purposes - lymphocyte activation, inflammation, antiinflammation etc) **TNF - inflammation (induce apoptosis) **CSF - in bone marrow
- Describe mechanism of action of IL2 2. How is it used in cancer therapy 3. Does it work?
- MoA - stimulate the host’s anti-tumor and anti-viral response by increasing the number of effector T cells (lymphocytes, proliferation) - both helper T cells (cytokine release) and killer T cells, CTL traffic to site of infection and provide the effector function - IL2 bind to IL2 receptor which is assembled on the T cell surface following activation by a dendritic cell 2. IL2 is the proliferative signal for activated T cells - Used in vitro and also in Vivo to treat cancer 3. Yes, it is FDA approved
Difference between cytokines GCSF and GMCSF - mechanism of action - clinical use 3. CSF general action - stimulate what? 4. Produced by?
- GCSF - cause proliferation and terminal differentiation of granulocytes 2. GMCSF - cause proliferation and terminal differentiation of granulocytes and monocytes (myeloid) 3. Colony stimulating factor (CSF) - glycoprotein that stimulate bone marrow to make new WBCs to provide adequate protection against infection - esp in cancer patients in chemotherapy (to replenish bone marrow) 4. Produced by monocytes, granulocytes fibroblasts and endothelial cells
Identify 1. What is produced by body in response to virus, bacteria, parasite and tumor cells 2. Differentiate passive with active immunotherapy (e.g)
- Interferons (a,b,y) 2. Passive - clinical use of interferons Active - Vaccines
- How do interferons work? Main effect - MoA 2. Other effects (4)
- Infection (viral - Hep C for INF a, MS for INF b) induce the release of interferons into the infected cell - bind to receptors expressed on most cell types - induce healthy cells to manufacture enzymes that help fight viral infection - result in production of over 30 diff proteins in target cell that aid in inhibiting viral replication (ribonuclease - degrade viral mRNA, kinase - phosphorylate initiation factor and inhibit protein synthesis) 2. - modulate and regulate immune response - alter surface antigen expression; MHC class I and II proteins - increased phagocytic activity of macrophages and ability of macrophages to kill intracellular bacteria - augment cytotoxicity of NK cells for viral infected target cells
