CHT EXAM COMPLETE STUDY GUIDE Exam Questions And Answers

It is possible to convert the plasma into serum by recalcifying the plasma and allowing coagulation to occur. - ANS Calcium is added to the plasma to form a complex with the citrate present in the anti-coagulant. This restores a normal ionized calcium level allowing clotting to occur. Polyspecific antiserum can be______ with lymphocytes to determine if there is a single antibody or multiple antibodies. - ANS absorbed Another application of lymphocyte absorption is to use the patient's own cells to remove_____. Absorption can be performed at cold or warm temperatures depending on the type of antibody. - ANS autoantibody _________ are typically cold reacting (4oC) and________are typically warm reacting (22-37oC). - ANS Autoantibodies B cell antibodies Patient serum may contain autoantibodies and/or IgM antibodies that may interfere the binding of anti-HLA IgG antibodies to the antigen. In complement dependent assays (CDC), IgM antibodies can activate complement; potentially leading to ___________. - ANS false positive reactivity. DTT - ANS dithiothreitol, also known as Cleland's Reagent DTT inactivates IgM molecules by cleaving the _________ in the pentamer structure. - ANS disulphide bonds Heat inactivation works by cleaving the disulphide bonds in the pentamer structure, but is less specific than _____. - ANS DTT. ALA - ANS therapeutic antilymphocyte antibodies Thymoglobulin - ANS purified, pasteurized, gamma immune globulin, obtained by immunization of rabbits with human thymocytes OKT3 - ANS a murine IgG monoclonal antibody directed against CD3 If these therapeutic antilymphocyte antibodies are present in the patient serum, they can cause_______ reactivity in T cell cytotoxic assays. - ANS false positive ALA removal is accomplished by absorption using _____coated with anti-rabbit or anti-mouse immunoglobulin for Thymoblobulin or OKT3, respectively. - ANS magnetic beads serum preparations - ANS 1. Recalcificiation of plasma 2. Adsorption with lymphocytes 3. Inactivation of IgM antibodies a. DTT treatment b. Heat Inactivation 4. Depletion of therapeutic anti-lymphocyte antibodies from serum Why is calcium added to plasma to convert it to serum? - ANS Calcium is added to the plasma to combine with the citrate present in the anticoagulant. This restores a normal ionized calcium level and allows clotting to occur. At what temperature should serum absorption with lymphocytes be performed? - ANS Typically, auto-antibodies are cold reacting and should be absorbed at 4oC while alloantibodies are warm reacting and should be absorbed at 37oC. DTT treatment is more specific than heat treatment in removing IgM antibodies. However, DTT can also affect the _______ in IgG antibodies. - ANS disulfide bonds What happens if we don't remove OKT3 prior to cytotoxicity assays? - ANS OKT3 is a monoclonal antibody directed against the CD3 molecule. Therefore, the binding of OKT3 to any CD3+ cell will activate complement and lyse the target cell. This results in falsely positive reactivity. Lymphocytes have a density of_______ - ANS ~1.077 g/ml. Cells with a higher density than FH - ANS red cells and granulocytes cells of a lower density than lymphocytes - ANS platelets isolation of cells from lymph nodes or spleen is normally fast and simple and leads to greater _____ and ______ - ANS cell yield viability. Obtaining lymphocytes from spleen will require an additional FH step along with purification steps due to a large number of contaminating cells, such as _______ ,______ and _______. - ANS macrophages erythrocytes platelets. intrinsic cell properties - ANS cell size and granularity of the cell. One method of lymphocyte isolation (HP) utilizes an intrinsic cell property, __________, by using a density gradient medium. - ANS cell density extrinsic cell properties - ANS cell surface marker, phagocytic capabilities and adherence capabilities. Immunomagnetic bead separations use the extrinisic property of __________ as the isolation target. - ANS cell surface markers Nylon wool isolation incorporates the extrinsic property of ___________ - ANS adherence for B cells. Nylon wool isolation procedure: - ANS Lymphocytes are obtained and added to a column packed with nylon wool. B cells will adhere to the nylon wool at an optimal temperature of 37o C. The non-adherent T cells can be collected by washing the column several times with warm media. The B cells can then be eluted by plunging cold media through the column. Dead cells in a flow cytometry assay can lead to ____________ of monoclonal antibodies and/or fluorochromes. - ANS nonspecific binding Various cell purification methods are - ANS Percoll and Thrombin: remove granulocytes, platelets, and dead cells Lympho-kwik: isolate specific populations (T &/or B lymphocytes, lymphocytes and monocytes) centrifugation slow/short: remove platelets in the supernatant carbonyl iron: Granulocytes ingest altering density (heavier) Dnase: remove dead cells Lymphocyte interface is indistinctive or thin - ANS Hyperlipemic blood sample Centrifugation force is too low and time too short Adjust centrifugation to 2000 x g for 25 min Presence of RBC in the lymphocyte band - ANS Density of RBC altered due to disease Excessive harsh mixing of blood Low lymphocyte yield - ANS Low WBC of blood donor Buffy coat left on RBC. Collect buffy coat to extend to ¼ inch of RBC layer Cells left in interface