Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin

Abstract

Lymphocyte proliferation in response to mitogens, phytohemagglutinin (PHA), concanavalin A, pokeweed, and/or specific antigens has been the method of choice for in vitro diagnosis of cell-mediated immune dysfunction. Recently, an assay to measure intracellular adenosine triphosphate (ATP) production in response to PHA has been developed that requires a shorter, overnight incubation. We compared a standard 5- to 7-day lymphocyte mitogen stimulation assay utilizing tritiated thymidine (3H-thy) incorporation to one in which ATP production in response to PHA by CD4-positive cells is measured in a luminometer that requires only 18-24 hr. A total of 20 patient samples suspected of having decreased cell-mediated immunity submitted for mitogen induced lymphocyte proliferation and 21 normal controls were tested in both assays. A comparison of these two methods has demonstrated that the screening ATP assay has a sensitivity at 24 hr of 100% in detecting decreased PHA induced lymphocyte proliferation at 5 days and a specificity of 85% in the samples obtained from normal controls. The data indicate that the ATP assay may be a useful screening tool for more rapid detection of blood samples with decreased cell-mediated immune responses. However, a positive screen should always be confirmed by 3H-thy uptake using mitogens and recall antigens like candida and tetanus.

Immune response of young children using ATP-based Cylex® assay: A brief report

“…With increasing number of transplantations being performed in the pediatric age group, an ideal method of monitoring immune status of post‐transplant pediatric patients is still lacking. The Cylex assay, which requires a very small of amount of whole blood and has a rapid turn around time, has been proposed as an early measure of immune status in few studies (4, 6–8). Studies have shown that the immune system evolves over childhood and has different characteristics compared to adults (9).…”

Monitoring and long-term outcomes in vascularized composite allotransplantation

“…One assay that we use is the ImmunKnow assay (www.cylex.net). This is an assay that detects changes in CD4 cell ATP production, a known biomarker of global immune function [5,6], and is FDA approved to detect function of T cells in immunosuppressed populations. One drawback of this assay is that the blood must be tested within 30 h of drawing from the patient and many centers do not offer Cylex testing and the blood must be shipped to the testing center.…”

Chemically induced strong cellular hypertrophy often reduces the accuracy of cytotoxicity measurements obtained using the ATP assay

“…The ATP assay can be used to evaluate the functional integrity of living cells, since all cells need ATP for cellular functions. The ATP assay is especially useful, since its application is not limited to cancer cell lines (Augustine et al, 2007), and has a 100-fold higher sensitivity than that of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for counting the number of cells (Hunter et al, 1993;Petty et al, 1995). It has shown excellent linearity and a wide dynamic range (0-750,000 RLU) in RPMI-8226 cells (Sekhon et al, 2008).…”