Raising the Bar for Traumatic Brain Injury Biomarker Research: Methods Make a Difference.

Dear Editor: Research in blood-based traumatic brain injury (TBI) biomarkers has exploded over the last two decades and continues to grow at an unwavering pace. Commercialization of these biomarkers is ongoing, with a number of companies seeking United States Food and Drug Administration (FDA) approval to market their tests for clinical applications. Although there are an abundance of articles being published, many lack the rigorous methods and reporting required to adequately evaluate these markers for clinical use. Too often, there are inadequate sample sizes, inappropriate control groups and outcome measures, variable definitions of TBI, and differences in when samples are drawn relative to time of injury. Importantly, variability in how samples are processed and performance characteristics of the assays themselves are rarely known or incompletely described. It is time to raise the bar for how bloodbased TBI biomarker research is conducted, and to encourage researchers, laboratories, and companies (producing and/or running assays) to perform at a higher standard. In the recently published study by Posti and coworkers, the performance of glial and neuronal biomarkers glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) was compared between CT negative mild TBI patients (based on American Congress of Rehabilitation Medicine [ACRM] criteria) and orthopedic control patients. Proteomic analyses of GFAP and UCH-L1 were conducted at Randox Laboratories Ltd. (Crumlin, County Antrim, United Kingdom) with Randox Biochip technology. Blood samples were drawn, when available, on days 1, 2, 3, and 7 after patients’ admission to the hospital, and at a late time point somewhere between 3 and 10 months after the injury. The authors found no significant differences in the concentrations of GFAP or UCH-L1 between mild TBI patients with a negative CT (n = 55) and orthopedic control patients (n = 44). This is in contrast to the findings from previous studies comparing orthopedic controls to mild TBI patients (also based on ACRM definition) with and without intracranial lesions on CT. In a recently published study in JAMA Neurology, which enrolled 584 trauma patients within 4 h of injury, GFAP ( p < 0.001) and UCH-L1 ( p = 0.049) were both significantly higher in CT negative mild TBI patients than in patients with orthopedic injuries. Similar findings were published for GFAP in 2014. The question is why the study by Posti and coworkers did not show this difference using the same biomarkers. We will explore some possible explanations. In the study by Posti and coworkers there was significant variability in the timing of samples over the first 24 h of admission, with >40% of enrolled patients lacking blood samples for analysis within the initial post-injury period. Only 55/93 (56%) of the mild TBI and 44/73 (60%) of the orthopedic controls had samples available within 24 h. Compounding this, sample time was based on admission to hospital and not on time of injury (which may have occurred long before presenting to the hospital). These factors can have a significant impact on the performance of a biomarker whose peak is very early after injury. Previous work in a large cohort of trauma patients found that UCH-L1 peaked within 8 h of injury and that GFAP peaked within 20 h. Perhaps the samples were not drawn within the optimal window of detection. This may be particularly important in milder injuries where smaller amounts of the biomarkers may be released after injury. Understanding the temporal profile of a biomarker is crucial to measuring it at the appropriate time. With cases of mild TBI and orthopedic trauma, verification of symptoms and symptom burden must be carefully and prospectively performed by the study team/clinician (as done in other studies) and not simply obtained retrospectively from the patients’ medical records. It is not uncommon for patients with orthopedic injuries to have a concomitant undiagnosed mild TBI, particularly with upper limb fractures. If symptoms are not carefully and prospectively assessed, patients will not be correctly classified. Curiously, the MRI reports of 30% of the orthopedic controls in the Posti study showed some kind of abnormality. The authors reported the abnormalities as not related to the TBI. However, one subject had evidence of a previous contusion, and five others demonstrated nonspecific white matter lesions. The question arises as to whether these lesions could have affected the biomarker levels. Next, the question of whether CT negative mild TBI patients had their symptoms adequately verified and how significant these suspected brain injuries actually were, arises. There is no description or quantification of loss of consciousness,