Our studies were designed to evaluate the relationship between QA
, a gold standard measure of BBB function, and serum concentrations of monomeric TTR and S100B. S100B and monomeric TTR are found principally in astrocytes and CSF, respectively. Peripheral elevations of these proteins have been associated with disruption of BBB function in prior studies; however the evidence for this association is correlative (Marchi et al., 2003a
,b; Bertsch et al., 2001
; Kapural et al., 2002
). In the current study, BBB barrier function was determined in 10 subjects with moderate to severe TBI and nine subjects with benign headache. Elevated QA
, indicative of BBB dysfunction, was present in seven TBI subjects. In these subjects, QA
decreased with time ( and ). This temporal pattern is consistent with prior studies of QA
after severe TBI (Morganti-Kossmann et al., 1999
). The remaining three TBI subjects and all of the headache subjects had normal QA
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 (more ...)
Serum S100B concentrations correlated well with QA
, typically declining with time in the TBI patients with abnormal BBB function (). For our analysis of the relationship between S100B and QA
, we constructed ROC curves at three post-TBI time points as well as a combined ROC curve using data from all time points (). The combined analysis also accounted for correlation within the data from each individual TBI patient. A significant area under the curve was found at 12
h after TBI. At this time point, a serum S100B cutoff of 0.022
ng/ml is 80% sensitive and 90% specific for predicting an abnormal QA
. At 24 and 48
h, the area under the curve approached but did not reach statistical significance. However, at all three time points after TBI, a serum S100B concentration of greater than 0.027
ng/ml was 90% specific for elevated QA
(). At the later two time points, the sensitivity for S100B was poor. We did not find a relationship between QA
and monomeric TTR.
Although this study is limited by a small sample size, we believe the statistically significant relationship between 12-h QA and S100B to be a true positive finding. The probability that this result is a false positive (Type 1 error) is quite small (≤0.05) and is not related to our small sample size. We are less certain about our negative results, that is, the lack of a statistical relationship between S100B and QA at combined time points. The probability that this result is a false negative (Type 2 error) is indeed related to small sample size. With a larger sample size and more statistical power, we might have detected a statistically significant relationship.
Head CT findings from the TBI patients demonstrate typically heterogeneous injuries that include intraventricular, subarachnoid, epidural, subdural, and intraparenchymal hemorrhages as well as contusions. There was no injury type common in all patients (). Although diffuse axonal injury (DAI) is a common to all severities of TBI (Geddes et al., 2000
), it was not detectable in any of the TBI subjects on head CT. Head CT is notably insensitive for the detection of DAI (Mittl et al., 1994
), and it is likely that DAI was present but not detected in many of our TBI subjects. Our study design is limited in that it does not account for differences between specific sub-types of injury in the TBI subjects. Consequently, we cannot discern differences in S100B and QA
that are related to specific injury patterns. Our detection of a statistically significant relationship between S100B and QA
despite the heterogeneous makeup of our cohort suggests that this relationship is strong.
Interestingly, two of the three subjects with normal QA
values (TBI 4 and TBI 9), presented with epidural hematoma (EDH) and a clinical course consisting of a lucid presentation followed by rapid neurological deterioration. The lucid interval commonly seen in patients with EDH may be due to a rapidly expanding extra-axial hematoma caused by arterial bleeding compressing brain that is relatively uninjured. These patients have a better prognosis than patients with other types of traumatic extra-axial bleeding (Stieg and Kase, 1998
). While the two subjects with EDH in this study had other associated brain injuries identified on CT, it is possible that they did not have sufficient direct insult to the brain to cause BBB dysfunction. Although our study was not designed to differentiate between different sub-types of TBI, this finding may be an important consideration in the design of future studies that focus on specific TBI subtypes.
The third subject with normal BBB function (TBI 7) did not have EDH, and had a markedly unusual relationship between S100B and QA
relative to that observed in the remaining TBI subjects (). Despite normal QA
values, the serum concentrations of S100B increased at each successive time point for this subject. Furthermore, S100B concentrations were very high at all time points. At 48
h, the serum concentration of S100B in this subject was sevenfold higher than in any other subject at the same time point. Review of brain imaging results showed that this subject had hypo-attenuation in the left middle cerebral artery (MCA), distribution suggesting an infarction. This finding was not evident on the initial scan but was present on a subsequent CT at 16
h later. The temporal pattern of serum S100B concentrations in patients with MCA stroke closely parallels the observed S100B concentrations in this TBI subject (Büttner et al., 1997
Post-stroke BBB dysfunction occurs early after stroke and appears to evolve and worsen with time. BBB dysfunction is detectable as early as 3
h after stroke with the low molecular weight MRI contrast agent gadolinium (157
Da) (Hofmeijer et al., 2004
), whereas albumin, which weighs 68
kDa, does not cross the BBB until 3 days after the event (Gotoh et al., 1985
). S100B, which weighs 21
kDa, is intermediate in size between gadolinium and albumin, and is elevated in the systemic circulation at 6
h after stroke (Foerch et al., 2007
; Wunderlich et al., 2004
). It is possible that the S100B elevations seen in this subject are related to post-stroke BBB dysfunction that has not yet progressed sufficiently to allow albumin to cross into the CSF.
In the setting of trauma, late elevations in serum S100B are also associated with poor outcomes. Raabe et al. (1999b
) studied 84 patients with severe TBI and report that patients with poor outcomes had higher initial S100B levels that rose substantially over time in contrast to patients with good outcome who had lower initial serum S100B levels that fell with time. Similarly, Pelinka et al. (2003b
) report that non-survivors after severe TBI generally have elevated and sustained or rising plasma S100B levels lasting longer than 48
h, whereas survivors have S100B levels that fall to normal within 48
h. Within our study, two TBI subjects died as a result of their injuries. One, TBI 6, died early after enrollment and only data from the 12-h time are available. The other subject, TBI 7, did have elevated S100B concentrations that rose with time. It is possible that this late rise in S100B was due to brain trauma that was ultimately fatal.
Extra-cranial release of S100B in the setting of trauma has been identified in prior studies (Anderson et al., 2001a
; Savola et al., 2004
; Pelinka et al., 2003a
) and may have occurred in our subjects. Significant amounts of S100B are present in bone marrow, blood, and fat (Anderson et al., 2001b
). Fractures in particular are thought to be an important source of S100B after trauma. The timing of S100B release from extra-cranial tissue is variable and may depend on the specific injury. For isolated fracture in an animal model, plasma S100B was increased from 30 to 120
min following injury, after which levels were not significantly elevated above baseline (Pelinka et al., 2003a
). Others note that trauma patients with fractures or other injuries in the absence of TBI have elevated S100B concentrations the day after the injury (Anderson et. al., 2001a). Eight of 10 of our TBI subjects had extra-cranial injuries (). All of the patients with extra-cranial injuries had some type of fracture. Again, the small sample size limits our ability to discern relationships between specific injuries and serum S100B concentrations. While there were no obvious differences in serum S100B profiles between the two isolated TBI and the eight multi-system trauma subjects, it remains possible that some serum S100B may have originated from extra-cranial injuries.
Other known causes of peripheral S100B elevation include Alzheimer's disease, Down's syndrome, and schizophrenia (Wiesmann et al., 1999
; Adami et al., 2001
). Elevated levels have also be found in healthy individuals after distance running (Otto et al., 2000
; Hasselblatt et al., 2004
), and after playing soccer (Stalnacke et al., 2004
), basketball, or hockey (Stalnacke et al., 2003
). While the control patients were screened and excluded if they had any of these conditions or recent strenuous physical activity, the headache and TBI subjects were not. In retrospect, none of these subjects had Down's syndrome, schizophrenia, or clinically diagnosed dementia, although it is possible that some may have had preclinical Alzheimer's disease. We did not collect data regarding recent strenuous physical activity in any of these subjects.
Given the concerns regarding the specificity of S100B, we also investigated the effectiveness of serum monomeric TTR for the prediction of abnormal QA
. Monomeric TTR is found only in CSF and appears in serum after iatrogenic BBB disruption (Marchi et al., 2003a
). We did not find a relationship between monomeric TTR and QA
. Technical issues may have affected the TTR values obtained. Both serum and CSF specific forms of TTR share the same amino acid sequence and differ only in quaternary structure making the development of quantitative antibody-based tests (such as ELISA) difficult. Thus, we were limited to using the more error prone semi-quantitative technique of densitometry of Western blots. A further difficulty that may have affected the accuracy of our measurements is that the highly abundant serum tetramer may have broken down into monomer during sample processing. The development of an ELISA specific for monomeric TTR would address these technical issues. Other biomarkers with greater specificity for the CNS such as neurofilament proteins and UCHL-1 have recently been described (Anderson, 2008
; Papa, .
008) and could also be used in future studies of BBB dysfunction after trauma.
In summary, serum S100B is an accurate predictor of QA
h after TBI. These results reinforce findings in prior, correlative studies that relate serum S100B elevations with osmotic (Kapural et al., 2002
b; Kanner et al., 2003
; Marchi et al., 2003b
) and physical (Bertsch et al., 2001
) BBB disruption. The observed relationship between S100B and QA
became weaker with time as evidenced by smaller area under the curve values for each successive time point. The small sample size of our study and the very high S100B levels in subject TBI 7 at the 24- and 48-h time points may have obscured a significant relationship at these points. Future studies should include larger study populations to allow the identification of significant relationships between QA
and S100B at these time points as well. Furthermore, patients who may be suffering acute stroke as well as patients that die in the immediate post-injury period should also be excluded. Finally, further studies of other biomarkers with greater CNS specificity such as UCHL-1 and neurofilament proteins should be performed. Our current findings support the use of serum S100B within 12
h of TBI to determine the functional status of the BBB. This non-invasive tool should allow the design of improved studies to examine the relationship of BBB status to the diagnosis, prognosis, and treatment of TBI in clinical populations.