Validation of serum markers for blood-brain barrier disruption in traumatic brain injury

Abstract

The blood-brain barrier (BBB), which prevents the entry into the central nervous system (CNS) of most water-soluble molecules over 500 Da, is often disrupted after trauma. Post-traumatic BBB disruption may have important implications for prognosis and therapy. Assessment of BBB status is not routine in clinical practice because available techniques are invasive. The gold-standard measure, the cerebrospinal fluide (CSF)-serum albumin quotient (Q(A)), requires the measurement of albumin in CSF and serum collected contemporaneously. Accurate, less invasive techniques are necessary. The objective of this study was to evaluate the relationship between Q(A) and serum concentrations of monomeric transthyretin (TTR) or S100B. Nine subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score < or =8) and 11 subjects with non-traumatic headache who had CSF collected by ventriculostomy or lumbar puncture (LP) were enrolled. Serum and CSF were collected at the time of LP for headache subjects and at 12, 24, and 48 h after ventriculostomy for TBI subjects. The Q(A) was calculated for all time points at which paired CSF and serum samples were available. Serum S100B and TTR levels were also measured. Pearson's correlation coefficient and area under the receiver operating characteristic (ROC) curve were used to determine the relationship between the serum proteins and QA. Seven TBI subjects had abnormal Q(A)'s indicating BBB dysfunction. The remaining TBI and control subjects had normal BBB function. No significant relationship between TTR and QA was found. A statistically significant linear correlation between serum S100B and Q(A) was present (r = 0.432, p = 0.02). ROC analysis demonstrated a significant relationship between Q(A) and serum S100B concentrations at 12 h after TBI (AUC = 0.800; SE 0.147, 95% CI 0.511-1.089). Using an S100B concentration cutoff of 0.027 ng=ml, specificity for abnormal Q(A) was 90% or higher at each time point. We conclude that serum S100B concentrations accurately indicate BBB dysfunction at 12 h after TBI.

Figures

FIG. 1.

Cerebrospinal fluid (CSF)/serum albumin quotients (QA) were calculated for headache and traumatic brain injury (TBI) subjects by measuring the concentrations of albumin in CSF and serum collected contemporaneously. The albumin quotient was then calculated by dividing the CSF albumin concentration by the serum albumin concentration. A QA value of 0.007 or less is considered normal. All headache subjects had normal QA values, with the exception of one whose QA was marginally elevated (0.0088 in HA 1). Seven TBI patients had abnormal QA values at all three time points (12, 24, and 48 h after TBI). The remaining three TBI subjects (TBI 4, TBI 7, and TBI 9) had normal QA values at all three time points. Mean QA values for headache subjects and for TBI subjects at 12, 24, and 48 h were 0.0055 (SD 0.0019), 0.0588 (SD 0.0747), 0.0727 (SD 0.1393), and 0.0156 (SD 0.013), respectively. The QA was significantly greater in TBI than in headache subjects (p = 0.02).

FIG. 2.

The monomeric form of transthyretin (TTR) is abundantly present in cerebrospinal fluid (CSF) and is normally absent in serum. The presence of monomeric TTR in serum has been reported to be a marker for blood-brain barriet (BBB) dysfunction. Monomeric TTR was measured in the serum of healthy control, headache, and traumatic brain injury (TBI subjects) by densitometry of Western blots. TTR concentrations were quite variable, with no significant differences between groups. Normal control and headache subjects had mean TTR concentrations of 0.089 (SD 0.089) and 0.055 (SD 0.060) ng/ml, respectively. TTR concentrations at 12, 24, and 48 h after TBI were 0.028 (SD 0.030), 0.102 (SD 0.235), and 0.071 (SD 0.072) ng/ml, respectively. There were no significant differences between any groups (p ≤ 0.98, analysis of variance [ANOVA]).

FIG. 3.

S100B is an astrocyte protein that has been noted to be released into serum under conditions of blood-brain barriet (BBB) compromise. We measured serum S100B concentrations in the serum of health control, headache, and traumatic brain injury (TBI) subjects by enzyme-linked immunosorbent assay (ELISA). Serum S100B concentrations were elevated in TBI subjects at all time points relative to healthy control and headache patients with a general trend towards decreasing S100B concentrations as time after injury increased. Normal control and headache subjects had mean serum concentrations of 0.0065 ng/ml (SD 0.0043) and 0.0089 ng/ml (SD 0.0101), respectively. There was no difference between these groups (p = 0.50). S100B concentrations at 12, 24, and 48 h after TBI were 0.0304 ng/ml (SD 0.0168), 0.0258 ng/ml (SD 0.0176), and 0.0168 ng/ml (SD 0.0195), respectively. S100B values were significantly elevated relative to headache (p < 0.02) and healthy control (p < 0.04) patients at 12 and 24, but not 48 h after TBI.

FIG. 4.

Seven traumatic brain injury (TBI) subjects had abnormal albumin quotients (QA) values. (A) For these subjects, peak S100B concentrations and (QA values occurred at the earliest time point, after which both values fell at each successive time point. Three TBI subjects had normal QA values. (B) Two of the subjects, TBI 4 and TBI 9, suffered from epidural hematoma and differed from the remaining TBI subjects in that they arrived at the emergency department lucid after which they had rapid deterioration. The third subject, TBI 7, had evidence of a stroke on CT and subsequently died before discharge from the hospital. Gray bars represent albumin quotients; black bars represent serum S100B concentrations.

FIG. 5.

In order to determine the relationship between the albumin quotient (QA) and serum S100B concentrations, receiver operating characteristic (ROC) curves were constructed for each traumatic brain injury (TBI) time point as well as for all TBI time points (A–C) taken together (D). Each of these analyses includes data from the headache subjects. The combined analysis accounts for intra-patient correlation between different time points. A significant relationship was found between QA and S100B at 12, but not at 24 or 48 h, after TBI. When data from all time points was considered together, the relationship between QA and S100B approached but did not reach significance.