Lymphocyte Proliferation to Anti-CD3/Anti-CD28 and Anti-CD3/Interleukin-2 (IL-2) by Flow Cytometry
Method Description Describes how the test is performed and provides a method-specific reference
Peripheral blood mononuclear cells (1 x10 cells/mL) in RPMI 1640 medium supplemented with L-glutamine and 5% human AB serum are added in duplicate to 16 wells of a sterile, flat-bottom, 48-well culture plate that contains either medium plus 5% AB serum alone (unstimulated) or varying concentrations of anti-CD3 antibody (aCD3) or anti-CD3 plus anti-CD28 antibodies (aCD28) in a 1:2 concentration or anti-CD3 costimulated with exogenous interleukin-2 (IL2). Cells are incubated for 72 hours, after which EdU (thymidine analog) is added to all wells where it becomes incorporated into the synthesizing DNA during a second incubation of 18 to 24 hours. A daily experimental normal control is included with each batch of patient samples to serve as an internal control.
Following the second incubation, the duplicate wells are prepared separately, the first set for viability with viability stain 7-aminoactinomycin D (7-AAD), apoptosis stain annexin V, and lymphocyte marker CD45. The second set is stained for proliferation via a copper-catalyzed click chemistry reaction where the EdU, an alkyne, is covalently bonded to a fluorescent azide. Cells are also stained for the following markers: CD45+ lymphocytes, CD3+ T cells, and CD69+ activated cells. Results are reported for the percent viable lymphocytes on Day 0, as well as percent proliferating cells within each group of lymphocytes and T cells for each stimulant within the panel.(Package insert: 4, Invitrogen Click-iT-EdU; unpublished Mayo method)
Supplemental Report Indicates whether the report includes an additional document with charts, images or other enriched information
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday
Do not send specimen after Thursday.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
No specimen retention. Entire specimen is used in preparation of the assay.
Performing Laboratory Location The location of the laboratory that performs the test