Immune Function Assay Enters the Clinical Laboratory

Abstract

Methods for assessing a patient’s state of immune responsiveness has traditionally involved the use of radioactive markers to purified populations of lymphocytes after they have been stimulated. Briefly, a lymphocyte stimulant or mitogen such as phytohemagglutinin (PHA) is added to isolated lymphocytes. Lymphocytes in the peripheral blood express receptors on the cell surface, which bind to mitogens or specific antigens presented in conjunction with the major histocompatibility complex (MHC). Exposure to the mitogen or antigen results in activation and expansion of the lymphocyte subpopulation reactive to the antigen or mitogen. The suspension can then be labeled with a radioactive marker such as 3 H thymidine where DNA synthesis is measured on a scintillation counter. The drawbacks of this assay include a long turnaround time (72 hours) and the use of radioactive materials. A new assay, Immuknow by Cylex (Columbia, MD) was FDA approved in 2002 for measuring global cell mediated immunity. Heparinized whole blood is diluted in sample diluent and stimulated with the mitogen PHA. After an overnight incubation period at 37°C in 5% CO 2 , the CD4 lymphocyte population is isolated using a magnetic separation device and monoclonal antibodies to CD4. After a series of washes, the target cell population is lysed releasing cellular adenosine triphosphate (ATP), which is measured using a microplate luminometer and the luciferase-luciferin enzyme system. In the presence of luciferin and Mg-ATP, luciferase catalyzes a reaction producing oxyluciferin and light which is directly proportional to the ATP concentration 1 in the patient sample. An unstimulated control of patient sample is run in parallel, and the results are expressed as the change or delta between the stimulated and non-stimulated wells. A standard curve utilizing sequential ATP concentrations is incorporated in the test system. This assay has a 24-hour turnaround time, utilizes whole blood, and requires no radioactive materials.