A TBI Study: Porcine Cross-Reactivity in Human Assays

The Quanterix Simoa HD-1 Analyzer provides an unparalleled ability to compute low abundant brain injury proteins in human circulation. A Yorkshire swine experimental traumatic brain injury (TBI) model that is used to investigate innovative neuroprotective therapies for TBI offers the perfect opportunity for exploring the kinetics of blood biomarkers of brain injury.

This article covers the potential porcine cross-reactivity in human assays for glial fibrillary acidic protein (GFAP), Neurofilament light (NF-light), cardiac Troponin I (cTnI), and neuron-specific enolase (NSE).

The Simoa assays listed here were selected because raised levels of GFAP have been observed to relate to many diseases, like TBI,1 brain tumors,2 etc., and can polymerize with neurofilament proteins, for example, NF-light.3 Neurofilaments are vital components of the neuronal cytoskeleton and it has also been established that they are related to TBI and several other neurodegenerative diseases.4–6

Despite the fact that the presence of NSE in erythrocytes can greatly elevate serum levels with even mild hemolysis,7 it is well defined as a TBI maker in the initial 12 hours after injury.8 Finally, cTnI is well established as a marker of cardiac dysfunction, including cardiac injury, myocardial infarction, acute coronary syndrome,9 etc. However, recent studies have revealed that TBI exhibits certain potential as a good indicator of the seriousness of the injury and the result.10

Study

Plasma and serum samples from four experimental TBI porcine animals were examined. Two were treated with Valproic acid (VPA) and two control animals were treated with standard saline. Examined adult anesthetized and intubated swine with instrumentation for vascular access went through computer-controlled cortical impact TBI. The test models were maintained in an unresuscitated state for 120 minutes after TBI and hemorrhagic shock, to which all were simultaneously hemorrhaged with 40% of their total blood volume.

In treated as well as control groups, blood samples were taken at baseline (0 hours) and 2, 4, and 8 hours after injury. Plasma and serum samples were stored at a temperature of −80 °C and transported to Quanterix for investigation.

Porcine samples were evaluated in duplicate on human Simoa assay kits NSE, NF-light, GFAP, and cTnI. Samples were diluted in accordance with the package insert directions as such: 50x dilution for NSE and 4x dilution for NF-light, GFAP, and cTnI. Calibration curves used in this research were derived from the human recombinant source and were prepared consistent with the package insert exclusive to each kit.

Results

As indicated in Figure 1, porcine samples could be detected in human Simoa assays NF-light, NSE, GFAP, and cTnI: 100% of the samples measured above the assay-specific limit of detection (LOD) for NSE, NF-light, and GFAP; and 84% measured above cTnI LOD. Dilutional linearity evaluated on NF-light 2x and 4x beyond the minimum required dilution (MRD) of 4x confirmed measured readings were precise with a mean recovery of 116% (range: 95%–136%) for four porcine samples (two plasma, two serum).

Concentration (pg/mL) values shown for baseline and post-TBI from porcine plasma and serum for (A) NSE, (B) NF-light, (C) GFAP, and (D) Troponin (cTnI). Error bars depict median with interquartile ranges. NF-light, NSE, and GFAP levels were all above the assay LOD; 84% of samples measured above the LOD (depicted as a horizontal line) of cTnI.

Figure 1. Concentration (pg/mL) values shown for baseline and post-TBI from porcine plasma and serum for (A) NSE, (B) NF-light, (C) GFAP, and (D) Troponin (cTnI). Error bars depict median with interquartile ranges. NF-light, NSE, and GFAP levels were all above the assay LOD; 84% of samples measured above the LOD (depicted as a horizontal line) of cTnI.

Figure 1 shows that the TBI state’s median concentrations over the baseline in plasma revealed a major increase in circulating levels of NF-light (baseline 15.3 pg/mL; post-TBI 44.0 pg/mL), NSE (baseline 521 pg/mL; post-TBI 3556 pg/mL), and GFAP (baseline 6.39 pg/mL; post-TBI 275 pg/mL) with a p value of ≤0.001.

Over plasma baseline, cTnI levels in plasma TBI were not statistically significant, but there was a small increase in the TBI state (0.131 pg/mL) over baseline (0.062 pg/mL).

Levels of circulation of GFAP and NSE in serum were analogous to that noticed in plasma; however, NF-light and cTnI measured 33% and 32% higher signal in plasma than serum. Moreover, cTnI baseline levels in plasma could be 100% detected (above the functional LOD of 0.052 pg/mL) while serum was only 25% detectable. Therefore, further analysis concentrated solely on plasma.

A large increase in the levels of circulation of GFAP and NSE was noticed within the first two hours after TBI and continued until 8 hours after injury for GFAP (see Figure 2C) and NSE after a small decrease at the 4-hour time point (refer Figure 2A). The levels of NF-Light consistently increased above baseline from 2 to 8 hours post-injury (refer Figure 2B).

Plasma concentrations (pg/mL) in (A) NSE, (B) NF-light, (C) GFAP, and (D) cTnI measured at baseline (before TBI; 0 hours) and 2, 4, and 8 hours post-injury in four porcine animals. Treated animals were dosed with VPA, whereas control animals were treated with normal saline. Mean of duplicate readings shown.

Figure 2. Plasma concentrations (pg/mL) in (A) NSE, (B) NF-light, (C) GFAP, and (D) cTnI measured at baseline (before TBI; 0 hours) and 2, 4, and 8 hours post-injury in four porcine animals. Treated animals were dosed with VPA, whereas control animals were treated with normal saline. Mean of duplicate readings shown.

As seen in Figure 2D, a single TBI animal treated with standard saline exhibited a substantial increase in cTnI over baseline (0.06 to 3.49 pg/mL) at 8 hours after injury. A spike in cTnI circulating levels in humans has been noticed after acute TBI, where the level of the spike was correlated to the seriousness of the injury.10 No major variances in biomarker levels were noticed between animals treated with valproic acid and those treated with standard saline; however, the study was not driven to detect this variance.

Conclusion

To sum up, human NSE, GFAP, NF-light, and cTnI cross-react with porcine and are established as good indicators of TBI in the swine model. While GFAP and NSE measured similarly in serum and plasma, higher circulating levels of cTnI and GFAP were measured in plasma than serum and were more detectable in cTnI baseline.

Moreover, the TBI state exhibited substantial increases in NSE, GFAP, and NF-light (TBI biomarkers that have been documented well) and was demonstrated to have an increase in cTnI for a single animal signifying cTnI as a marker of the gravity of TBI.

References

  1. Schiff L, Hadker N, Weiser S, et al. A Literature Review of the Feasibility of Glial Fibrillary Acidic Protein as a Biomarker for Stroke and Traumatic Brain Injury. Mol Diagn Ther 2012 16:79.
  2. Gullotta F, Schindler F, Schmutzler R, et al. GFAP in brain tumor diagnosis: possibilities and limitations. Pathol Res Pract 1985 Jul;180(1):54–60.
  3. Reeves SA, Helman LJ, Allison A, et al. Molecular cloning and primary structure of human glial fibrillary acidic protein. Proceedings of the National Academy of Sciences of the United States of America. Jul 1989 86(13):5178–82.
  4. Meeter LH, Dopper EG, et al. Neurofilament light chain: a biomarker for genetic frontotemporal dementia. Ann Clin Transl Neurol. 2016 3(8):623–36.
  5. Eikelenboom MJ, Petzold A, et al. Multiple sclerosis: Neurofilament light chain antibodies are correlated to cerebral atrophy. Neurology. 2003 60(2):219–23
  6. Gaiottino J, Norgren N, Dobson R, et al. Increased Neurofilament Light Chain Blood Levels in Neurodegenerative Neurological Diseases. PLOS One 2013 8(9): e75091.
  7. Lima JE, Takayanagui OM, Garcia LV, Leite JP. Use of neuron-specific enolase for assessing the severity and outcome in patients with neurological disorders. Braz J Med Biol Res 2004; Jan;37(1):19–26.
  8. Cheng F, Yuan Q, Yang J, Wang W, Liu H. The Prognostic Value of Serum Neuron-Specific Enolase in Traumatic Brain Injury: Systematic Review and Meta-Analysis. PLoS One 2014 Sep 4;9(9):e106680.
  9. Mair J, Genser N, Morandell D, et al. Cardiac troponin I in the diagnosis of myocardial injury and infarction. Clin Chim Acta 1996; 245:19–38.
  10. Salim A, Hadjizacharia P, Brown C, et al. Significance of Troponin Elevation After Severe Traumatic Brain Injury. J of Trauma Injury Infect Crit Care 2008 Jan;64(1):46–52.

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Last updated: Aug 13, 2019 at 3:53 AM

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