The ImmuKnow assay – Does it really put us in the know about the immune system?

For decades, early recognition and treatment of acute rejection have been top priorities for transplant professionals. In solid organ transplantation, less invasive biomarkers, such as serum creatinine in kidney transplant recipients, have traditionally been used to predict the risk of acute rejection. Yet such markers were highly imperfect and represented graft function, not the degree of immune system suppression (1). The ImmuKnow assay was therefore developed by Cylex, Inc. (Columbia, MD, USA) as a means of globally assessing the alloreactivity of the host immune system, based on the concept that the CD4 T cell was a central player in alloreactivity and that metabolic activity of the cell (via ATP production) could represent T-cell activation better than clonal proliferation or cytokine production. Initial development of the test (2) was performed in 1995–1996 through a Small Business Innovation Grant from the US Department of the Army to develop a test for the efficacy of a Q fever vaccine, which provided the fundamental proof of principle for the ImmuKnow technology in its current form. In 1998, the first patent for the assay, then called the Luminetics Assay, was filed with the US Patent & Trademark Office titled, “Methods for Measurement of Lymphocyte Function” (US 6 630 316). FDA approval of the test Immune Cell Function Assay (another precursor name) was granted in 2002 and the product name changed to ImmuKnow. Readers should note here that FDA approval of a test (versus a drug) is based more on reproducibility of test results rather than clinical utility. European Union CE marking was obtained in 2004, and a new Class One CPT code was obtained in 2010. ImmuKnow and the assets of Cylex, Inc., were acquired by Viracor-IBT Laboratories, Inc. (Lee’s Summit, MO, USA) in February, 2013. Since its development and release, several studies have investigated the clinical utility of ImmuKnow. What became apparent was that a high ATP level, thought to represent under-immunosuppression, did not associate well with acute rejection events. In fact, the association was stronger at the opposite end, with an early meta-analysis showing a 12-fold higher risk for infection with very low ATP levels (3). This result should not be counterintuitive, as viral infection defense mechanisms also utilize the same pathways as allorecognition. Later studies and metaanalyses have added to the conflicting results even for viral infection (4), but each study is heterogeneous from the other, differing in characteristics such as type of organ transplant, immunosuppression protocols, timing of ImmuKnow assay to event, and the measure of the event. In this issue of the journal, Ryan et al. (5) investigated the association of ImmuKnow with a rigorous measure of acute rejection, surveillance kidney transplant biopsies, and something not attempted previously in children. They show that the ImmuKnow level did not associate well with surveillance biopsy acute rejection. These findings are similar to the findings obtained by Helantera et al. (6) in adult patients, despite the lower zones of immune response in children (Fig. 1). The ImmuKnow level also did not associate with viremia, often a precursor to systemic symptomatic viral illness. The limitation of this study is the small sample size of 42 samples from 31 subjects, with only five surveillance biopsies