Test ID: LPMGF    
Lymphocyte Proliferation to Mitogens, Blood

Peripheral blood mononuclear cells (500,000 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 mitogens pokeweed (PWM) (0.5, 5, and 10 micrograms/mL) and phytohemagglutinin (PHA) (0.5, 2.5, and 5 micrograms/mL). 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-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, CD69+ activated cells, and CD19+ B cells (PWM only). Results are reported for the percent viable, dead, and apoptotic lymphocytes, as well as percent proliferating cells within each group of lymphocytes, T cells, and B cells (PWM only) (Package insert: 4, Invitrogen Click-iT-EdU; unpublished Mayo method)

Monday through Friday

Do not send specimen after Thursday.