Clinical Assays Flashcards
Describe assay technique, how to interpret results, relevant controls, and utility of assay
Direct Coombs test
Clinical question: Is the patient making self Ab causing RBC’s to be destroyed [hemolytic anemia]?
Incubate patient’s RBC’s with AB’s to the Fc region of the Ab (ex. IgG) suspected of causing the hemolysis. If there is self-Ab bound to the RBC’s, the anti-human Ig will cross-link it
-Agglutination is a positive result; control could use known “clean” RBC’s, anti-nonhuman Ig, or saline
-If you don’t know what Ig to target, check for kappa and lambda chains generally first; then if there’s a positive
Describe assay technique, how to interpret results, relevant controls, and utility of assay
Immunofluorescence
Clinical question: is there anything at all here that fluorescent antibody tagging will reveal?
Wash a patient tissue sample with fluorescently tagged antibody either to an antigen [direct] or to antibody [indirect].
Describe assay technique, how to interpret results, relevant controls, and utility of assay
FACS
Clinical question: What types of cells do I have in the sample based on their surface markers? [and “I want to separate one subset out from the rest”]
Fluorescence activated cell sorting, a subset of flow cytometry. Cells are tagged with fluorophores (monoclonal antibodies) suspended in solution and passed in single file through a laser beam, the scatter from which can be analyzed by computer to ascertain identities (or characteristics) of cell populations within the sample
FACS is a subset of this which uses moment-to-moment analysis and charge assignment to sort populations of cells.
Describe assay technique, how to interpret results, relevant controls, and utility of assay
SPE
Clinical question: Does the overall plasma protein profile look right? And if not, is there overexpression of a single protein (monoclonal Ab in lymphoma, for example), or is expression of Ig’s broadly elevated?
serum protein electrophoresis
single spike, probably monoclonal
broad rise in gamma region, polyclonal
Describe assay technique, how to interpret results, relevant controls, and utility of assay
Complement dependent cytotoxicity assay
Clinical question: Does my patient already have antibody to a particular HLA [pregnancy, transfusion might make you suspect this particularly strongly]?
Put patient serum in wells containing lymphocytes with known HLA specificity. Give it time. Add complement. Watch for cell lysis. Lysis is a positive result for those anti-HLA Ab’s
Describe assay technique, how to interpret results, relevant controls, and utility of assay
Mixed lymphocyte culture
Clinical question: Is my patient going to reject this specific donor organ?
Get peripheral blood lymphocytes from donor and recipient. IRRADIATE [inactivate] donor cells. Mix in culture. Incubate 5 d. at 37C. Add radioactively labeled thymidine [DNA precursor] to culture. Incubate add’l 18h. Wash, check for uptake of labeled DNA in recipient cells.
High uptake of thymidine indicates high rate of proliferation - in response to donor MHC, especially HLA-D, especially DR
Describe assay technique, how to interpret results, relevant controls, and utility of assay
RAST
Radioallergosorbent Test
Clinical question: Does my patient have Ig-E to a particular allergen? [indicating an allergy to said allergen]
Bind allergen to insoluble material. Add patient serum. Wash. Add radiolabeled anti-human IgE. Wash. Read.
More radioactivity indicates greater expression of IgE and increases likelihood of allergic reaction to an allergen.`
Describe assay technique, how to interpret results, relevant controls, and utility of assay
Ouchterlony assay
aka agar gell immunoprecipitin assay
Clinical question: [?]
Punch wells into agar. Antigens placed into a set of wells. Antibodies (known) or patient serum (suspected antibodies) placed into opposing wells. Wait (allow proteins to diffuse through gel). If there is antibody to a particular antigen, a visible precipitation band will appear
