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Biomarkers Improve Clinical Outcome Predictors of Mortality Following Non-Penetrating Severe Traumatic Brain Injury

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Abstract

Objective

This study assessed whether early levels of biomarkers measured in CSF within 24-h of severe TBI would improve the clinical prediction of 6-months mortality.

Methods

This prospective study conducted at two Level 1 Trauma Centers enrolled adults with severe TBI (GCS ≤8) requiring a ventriculostomy as well as control subjects. Ventricular CSF was sampled within 24-h of injury and analyzed for seven candidate biomarkers (UCH-L1, MAP-2, SBDP150, SBDP145, SBDP120, MBP, and S100B). The International Mission on Prognosis and Analysis of Clinical Trials in TBI (IMPACT) scores (Core, Extended, and Lab) were calculated for each patient to determine risk of 6-months mortality. The IMPACT models and biomarkers were assessed alone and in combination.

Results

There were 152 patients enrolled, 131 TBI patients and 21 control patients. Thirty six (27 %) patients did not survive to 6 months. Biomarkers were all significantly elevated in TBI versus controls (p < 0.001). Peak levels of UCH-L1, SBDP145, MAP-2, and MBP were significantly higher in non-survivors (p < 0.05). Of the seven biomarkers measured at 12-h post-injury MAP-2 (p = 0.004), UCH-L1 (p = 0.024), and MBP (p = 0.037) had significant unadjusted hazard ratios. Of the seven biomarkers measured at the earliest time within 24-h, MAP-2 (p = 0.002), UCH-L1 (p = 0.016), MBP (p = 0.021), and SBDP145 (0.029) had the most significant elevations. When the IMPACT Extended Model was combined with the biomarkers, MAP-2 contributed most significantly to the survival models with sensitivities of 97–100 %.

Conclusions

These data suggest that early levels of MAP-2 in combination with clinical data provide enhanced prognostic capabilities for mortality at 6 months.

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Conflict of interest

Drs. Gabrielli, Hannay, Heaton, Robertson, Robicsek, and Schmalfuss have no competing financial interests.

Fundings

This study was funded by NIH RO1 NS052831 “Biochemical Markers of Severe Traumatic Brain Injury”. Drs. Brophy and Papa are consultants of Banyan Biomarkers, Inc. but receive no stocks or royalties from the company and will not benefit financially from this publication. Dr. Hayes and Wang own stock, receive royalties from Banyan Biomarkers Inc., and as such may benefit financially as a result of the outcomes of this research or work reported in this publication.

Author information

Authors and Affiliations

  1. Department of Emergency Medicine, Orlando Regional Medical Center, 86 W. Underwood (S-200), Orlando, FL, 32806, USA

    Linda Papa

  2. Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA

    Claudia S. Robertson

  3. Department of Psychiatry, University of Florida, Gainesville, FL, USA

    Kevin K. W. Wang

  4. Department of Pharmacotherapy and Outcomes Science and Neurosurgery, Virginia Commonwealth University, Richmond, VA, USA

    Gretchen M. Brophy

  5. Department of Psychology, University of Houston, Houston, TX, USA

    H. Julia Hannay

  6. Department of Clinical and Health Psychology, University of Florida, Gainesville, FL, USA

    Shelley Heaton

  7. Department of Radiology, University of Florida and NF/SG Veterans Administration, Gainesville, FL, USA

    Ilona Schmalfuss

  8. Department of Anesthesiology, University of Florida, Gainesville, FL, USA

    Andrea Gabrielli & Steven A. Robicsek

  9. Banyan Laboratories, Banyan Biomarkers Inc., Alachua, FL, USA

    Ronald L. Hayes

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  1. Linda Papa
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  2. Claudia S. Robertson
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  3. Kevin K. W. Wang
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Corresponding author

Correspondence to Linda Papa.

Appendix 1

Appendix 1

See Table 7.

Table 7 Lower (LLoD) and upper (ULoD) limit of detection for all biomarkers
Full size table

Ubiquitin C-terminal Hydrolase (UCH-L1)

UCH-L1 sandwich ELISA (swELISA) was performed in accordance with previously published studies [29, 32–34, 58–60]. Both mouse monoclonal antibody (capture antibody) and rabbit polyclonal antibody (detection antibody) were made in-house at Banyan Biomarkers Inc. against recombinant human UCH-L1 full-length protein and protein A purified. Plates were coated with capture antibody in 0.05 M sodium bicarbonate, pH 9.6 overnight at 4 °C. Blocking and washing buffer was Tris buffered saline with 0.05 % Tween-20 (v/v) (TBST). Antigen standard (UCH-L1 standard curve: 0.78–200 ng/mL; unknown samples: 10 µL of CSF) were incubated with detection antibody overnight and then added to the plate for 2 h. After washing, secondary anti-rabbit-IgG HRP (GE Healthcare) was added and incubated for 1 h. Plates were developed with substrate solution Ultra-TMB ELISA (Pierce# 34,028), stopped with acidic solution and read at 450 nm with a spectrophotometer (Molecular Device SpectraMax 190). The interassay CV was 2–8 % while intraassay CV was 2–11 % within the dynamic range. The limit of detection (LOD) was 0.03 ng/mL.

αII-Spectrin Breakdown Products 150 kDa (SBDP150), 145 kDa (SBDP145), 120 kDa (SBDP120)

SBDP150, SBDP145 and SBDP120 swELISAs were constructed similarly to those described previously [29]. Briefly, a 96-well plate was coated with 100 μL/well capture antibody (5ug/ml purified goat polyclonal anti-SBDP150 [28, 61] or 10ug/ml rabbit anti-SBDP145 or 10ug/ml anti-SBDP120 [62] overnight at 4 °C. Antigen used was partially purified human brain αII-spectrin for SBDP150 or recombinant glutathione-S-transferase– αII-spectrin (including the SBDP145 cleavage site in repeat 13–18) fusion protein cleaved with either calpain-1 (1: 40 ratio for 10 min at 4C) for SBDP145 production or with caspase-3 (1 : 20 ratio for 4 h at room temperature) for SBDP120. After blocking buffer (Startingblock T20-PBS), SBDP150 calibrator (10 × dilution factor, 1.17 ng/ml–300 ng/ml), SBDP145 calibrator (10X dilution factor, 1–500 ng/ml) and SBDP120 Calibrators (3X dilution factor, 0.9–120 ng/mL) or samples were added (CSF, 10 μL for SBDP150, SBDP145; 34 uL for SBDP120) with diluent (total volume 100 uL) to the wells. After washing, plates were incubated with affinity purified detection antibody (mouse monoclonal anti-αII-spectrin antibody (Biomol FG6090 or equivalent). If amplification was needed, biotinyl-tyramide solution (Perkin Elmer Elast Amplification Kit) was added, washed and followed by Streptavidin-HRP (1:500) in PBS with 0.02 % Tween-20 and 1 % BSA. Lastly, the wells were developed with chemiluminescent substrate solution (SuperSignal ELISA Femto, Pierce) for 1 min and read by a luminescence microplate reader (GloRunner DXL Luminometer, Turner BioSystems). The interassay and intraassay CV were <3–14 % within the assay dynamic range. The LOD was 1.54 ng/mL for SBDP150, 0.98 ng/mL for SBDP145 and 0.474 ng/mL for SBDP120.

Microtubule Associated Protein 2 (MAP-2)

MAP-2 sandwich ELISA was performed using 10 uL CSF for quantitative determination. Mouse MAb anti-MAP2A/2B (clone M13, Zymed #13-1,500) was used as capture antibody (5 ug/well) to coat the plate. Biofluid samples (10 uL CSF, or recombinant antigen as GST-fusion protein with residue 1,078–1,551 of MAP-2 at 0.10–6.67 ng/mL) were added with diluent (100 uL total) to microtiter plate wells. After 2 h incubation and washing, HRP-labeled mouse monoclonal anti-MAP-2 (clone AP20; BD Bioscience; #552,320) antibody was added. After washing, plates were developed with substrate solution Ultra-TMB ELISA (Pierce# 34,028), stopped with acidic solution and read at 450 nm with a spectrophotometer (Molecular Device SpectraMax 190). The interassay and intraassay CV were <15 % within the assay dynamic range. Limit of detection (LOD) was determined to be 0.054 ng/mL.

S100B

S100B sandwich ELISA was performed using 5–10 uL CSF for quantitative determination. Mouse monoclonal anti-S100b was used as capture antibody (3ug/well) to coat the plate. After blocking buffer, biofluid samples (5–10 uL CSF) or standard protein (S100beta, human brain protein, Fitzgerald, at 0.0039 ng/ml–0.5 ng/ml) were added. After 30 min incubation and washing, detection polyclonal antibody was used and incubated for 1 h (1 ug/ml and 100 ul/well), followed by HRP-conjugated anti-rabbit-HRP (Jacksonville ImmunoResearch lab) for 30 min. After washing, plates were developed with substrate solution Ultra-TMB ELISA (Pierce# 34,028), stopped with acidic solution, and read at 450 nm with a microplate spectrophotometer (Molecular Device SpectraMax 190). The interassay and intraassay CV were <10 % within the assay dynamic range with a limit of detection (LOD).

Myelin Basic Protein (MBP)

MBP assay was based on commercial MBP ELISA for CSF (iPOC) according to manufacturer’s instructions. Briefly, 50 uL of calibrator (0.13–36 ng/mL) or 4 uL CSF samples with diluent (to 50 uL) was used and incubated with plate with capture antibody (goat polyclonal anti-MBP) for 2 h. After washing, 50 uL detection mouse monoclonal antibody to MBP was added (50 uL) and incubated for 30 min, followed by HRP-enzyme-conjugated secondary donkey anti- mouse IgG antibody. After washing, 50 uL of chromogenic TMB substrate was used for 15 min. 100 uL stop solution was added and absorbance at 450 nm was measured with a spectrophotometer (Molecular Device Spectramax 190). The interassay and intraassay CV were <10 % within the assay dynamic range. The limit of detection was determined to be 0.13 ng/ml.

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Papa, L., Robertson, C.S., Wang, K.K.W. et al. Biomarkers Improve Clinical Outcome Predictors of Mortality Following Non-Penetrating Severe Traumatic Brain Injury. Neurocrit Care 22, 52–64 (2015). https://doi.org/10.1007/s12028-014-0028-2

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