Neuronal and glial markers are differently associated with computed tomography findings and outcome in patients with severe traumatic brain injury: a case control study

Approval of this study was obtained from the local ethics committee of all the sites involved and from the Western Institutional Review Board and Human Research Protection Office. Eighty-one adult patients presenting to the University of Pécs (n = 30), University of Szeged (n = 21), and University of California Davis (n = 20), and from the University of Maryland Shock Trauma Center (n = 10) with severe head injury were included in this prospective multicenter study. Sixty-one patients presented with isolated head injury. Twenty severe TBI patients had minor concomitant injury of the thorax and⁄or abdomen and/or extremities. Multiple trauma were excluded (see exclusion criteria, below). Written informed consent was obtained from a next of kin because all eligible patients were in coma within 24 hours from the admission. Exclusion criteria were no informed consent, patients younger than 18 years of age, female patients that were or may have been pregnant, known history of neurological disease, and Injury Severity Score (ISS) greater than 15. Inclusion criteria were a Glasgow Coma Score (GCS) score of eight or less on presentation. Treatment of patients, according to international guidelines, was targeted at a normal ICP and maintaining cerebral perfusion pressure [9]. Initial CT scans obtained on admission were analyzed according to the classification of Marshall and colleagues [2]. CT scans were interpreted by a qualified neuroradiologist at a central location. For the purpose of our analysis, Marshall was further classified into two groups using dichotomized categories (diffuse injury versus focal mass lesion), as previously described [10]. Outcome was assessed as in-hospital mortality and at six months using the Glasgow Outcome Scale (GOS) [11].

In addition, 167 healthy blood donors were enrolled, during a one-year period, to establish normal serum UCH-L1 and GFAP levels.

Venous blood samples were taken at hospital admission (median seven hours) and every six hours thereafter. For the purpose of the analysis and to handle potential missing samples, time after injury for each sample was calculated and the samples were separated into 12 or 24-hour time period. Approximately 5 mL of serum was collected from each subject at each sample point. Samples were centrifuged for 10 minutes at 4,000 rpm and immediately frozen and stored at -70°C until the time of analysis. Samples were measured using a standard UCH-L1 sandwich ELISA protocol as described below. Reaction wells were coated with capture antibody (500 ng/well purified anti-rabbit UCHL1, made in-house) in 0.1 M sodium bicarbonate, pH 9 and incubated overnight at 4°C. Plates were then emptied out and 300 μl/well blocking buffer (Startingblock T20-TBS) was added and incubated for 30 minutes at ambient temperature with gentle shaking. This was followed by addition of antigen standard (UCHL1 standard curve: 0.05 to 50 ng/well) unknown samples (3 to 10 uL CSF) or assay internal control samples. The plate was incubated for two hours at room temperature then washed using an automatic plate washer (each well rinsed with 5 × 300 μl with wash buffer (TBST)). Detection antibody (anti-rabbit UCH-L1-HRP conjugation, made in-house at 50 μg/mL) in blocking buffer was then added to wells at 100 μl/well and the plates were further incubated for 1.5 hours at room temperature. After additional automatic washing, biotinyl-tyramide solution (Elast ELISA Amplification Kit, PerkinElmer, Inc., Waltham, MA, USA) was added and the plate was incubated for 15 minutes at room temperature followed by automatic washing. Addition of streptavidin-HRP (1:500, 100 ul/well) in PBS with 0.02% Tween 20 and 1% BSA for 30 minutes incubation at room temperature was followed by automatic washing. Finally, the wells were developed with substrate solution: Ultra-TMB ELISA 100 ul/well (Pierce# 34028) with incubation for 5 to 30 minutes and read at 652 nm with a 96- well spectrophotometer (Spectramax 190, Molecular Device, Sunnyvale, CA, USA). GFAP protein was analyzed using a commercially available (Biovendor Laboratory Medicine Inc., catalog n° rd192072200, Brno, Czech Republic) polyclonal two-side immunoluminometric assay according to the manufacturer's instructions. A standard curve was constructed by plotting absorbance values versus GFAP concentrations of calibrators.