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Technical Briefs |
1
Neuroradiology and
2
Transfusion Medicine, Klinikum Grosshadern, Ludwig Maximilian University, 81377 Munich, Germany;
3
Departments of Immunology and Transfusion Medicine, University of Luebeck, School of Medicine, 23566 Luebeck, Germany;
a address correspondence to this author at: Abteilung fuer Neuroradiologie, Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany, fax 0049 (89) 7095 2509, e-mail wiesmann{at}ikra.med.uni-muenchen.de
Glial fibrillary acidic protein (GFAP), found only in glial cells of the central nervous system (CNS) (1)(2), was first isolated by Eng et al. (1) in 1971. GFAP is a monomeric molecule with a molecular mass between 40 and 53 kDa (3)(4) and an isoelectric point between 5.7 and 5.8 (2). GFAP represents the major part of the cytoskeleton of astrocytes. DeArmond et al. (5) reported on the spontaneous degradation of GFAP in vitro and in vivo; under physiological conditions, GFAP polymerizes spontaneously to astrofibrils with a length of 0.8–1.06 µm.
Numerous reports in the literature document the usefulness of measuring GFAP in cerebrospinal fluid (CSF) as a specific indicator of CNS pathology (6)(7)(8)(9)(10)(11)(12)(13). Measuring GFAP in blood would have clinical advantages over CSF measurements; however, to our knowledge, the only method attempted to date to measure GFAP in blood (14) was not successful.
We describe the results of measuring the concentrations of both GFAP and S-100 protein in the blood of patients with acute severe head trauma and healthy controls, using the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system for detection of GFAP (DELFIA can also be used to measure the concentration of GFAP in CSF).
Blood samples were obtained from 25 patients (19 males and 6 females;
ages, 16–72 years; mean age, 38.6 ± 19.0 years) who were
admitted <24 h after severe head trauma (a Glasgow Coma Scale score of
6 on admission). Three samples were obtained from each patient: the
first sample was obtained on admission to the hospital, the second was
obtained 24 h after the head injury, and the third was obtained
48 h after the injury.
Blood samples were also obtained from 70 healthy blood donors between 20 and 65 years of age (35 males and 35 females; mean age, 38.6 ± 13.2 years) who had no history of previous neurological deficit or any other serious disorder. The healthy donors were receiving no medications, and the results of physical examinations and routine laboratory tests were within health-related reference intervals.
The study protocol was in accordance with the current revision of the Helsinki Declaration, and the healthy blood donors and the patients, or the patients' relatives, gave informed consent.
For each assay, a microtiter plate (Maxisorp F96®;
Nunc) was coated with an affinity-purified goat anti-mouse
immunoglobulin antibody (400 ng/well; Medac) in 0.05 mol/L carbonate
buffer, pH 9.6 (200 µL/well). The plate was then coated with a
monoclonal mouse anti-GFAP antibody (600 ng/well; Sigma Chemical Co.)
in 200 µL of 0.05 mol/L Tris buffer containing 0.15 mol/L NaCl, 5 g/L
bovine serum albumin, 0.5 g/L bovine 
-globulin, 0.15 mmol/L
NaN3, and 100 µL/L Tween 20 (chemicals from Merck,
proteins from Sigma). As the detection antibody, we used a polyclonal
rabbit anti-GFAP antibody (Dako), which was biotinylated as described
previously (15). Streptavidin (Biomol) was labeled with the
Europium Labeling Reagent® from Pharmacia, according to
the procedure recommended by the manufacturer. Purified GFAP (Progen)
diluted in pooled human plasma was used as the calibration reagent and
internal control. All incubations were performed at room temperature,
and all measurements were performed in duplicate.
A plate washer (Wallac) was used to wash the coated microtiter plates twice with 5 mL/well of washing buffer (0.05 mol/L Tris, 0.15 mol/L NaCl, 0.1 mL/L Tween 20, pH 7.5). A 100-µL aliquot of calibrator solution and a control or sample was then added to each well. Assay buffer (100 µL/well) was added, and the plate was placed on a microtiter plate shaker (Heidolph) for incubation at 200 rpm.
After 1 h, the plate was washed three times with washing buffer, and 200 µL of biotin-labeled anti-GFAP antibody diluted to a final concentration of 2 µg/L in Tris-NaCl-albumin buffer containing 500 mL/L newborn calf serum (Life Technologies) was added. The plate was then incubated for 1 h.
After incubation, the plate was washed three times with washing buffer, and 200 µL of streptavidin-europium in Tris-NaCl-albumin buffer was added to each well, and the plate was incubated for 30 min. The final concentration of the streptavidin-europium was 25 µg/L. After incubation, the plate was again washed three times with washing buffer. We then added 200 µL/well enhancement solution (0.01 mol/L acetic acid, 38 mg/L tri-n-octyl phosphine oxide, 1.3 g/L potassium phthalate, 222 mg/L thenoyltrifluoroacetone, and 2 mL/L Triton X-100) and incubated the plate for 20 min.
The resulting fluorescence was measured using a DELFIA® 1234 fluorometer (Wallac). Measurements were subjected to log/log transformation before analysis by a modified spline function in the MultiCalc® data reduction program of the DELFIA 1234 system.
S-100 protein measurements were performed as described previously (16).
Results were analyzed by group and are reported as mean ± 1 SD unless stated otherwise. The significance of the differences between groups was examined using the Mann–Whitney U-test. Correlations between variables were calculated using the Spearman rank-order correlation coefficient. P <0.05 was considered significant.
The lower detection limit of the method was <0.01 µg/L GFAP (zero calibrator + 3 SD; n = 33). The calibration curve was from 0.01 to 12.5 µg/L. Analytical recovery was studied by the addition of two different definite amounts of purified GFAP to 10 patient samples. The recovery of GFAP added to 10 serum samples was 68–102% (mean, 88%; n = 10) for an expected concentration of 0.475 µg/L and 60–95% (mean, 77%; n = 10) for an expected concentration of 4.75 µg/L. Linearity of the assay was confirmed by serial dilution of three human serum samples after addition of GFAP (measured GFAP, 8.08, 3.63, and 8.24 µg/L, respectively; regression coefficient after five serial dilutions, r2 = 1.0, 0.98, and 1.0, respectively). When patient samples with previously measured GFAP content were mixed 1:1 (by volume), the measured values were 91–110% (mean, 99.6%; n = 21) of the calculated means for concentrations between 0.04 and 0.12 µg/L. The intraassay imprecision (CV) was 4.7% at a mean concentration of 0.2 µg/L (n = 33), 4.2% at 1.96 µg/L (n = 24), and 3.5% at 9.6 µg/L (n = 32). The total imprecision for 43 assays on 15 days was 8.8% at 0.74 µg/L and 10.4% at 7.71 µg/L. We observed no interferences from hemolysis or lipemia. No "high-dose hook" effect was observed up to a concentration of 500 µg/L GFAP (response for 12.5 µg/L calibrator, 1 530 235 counts; response for 500 µg/L GFAP, 4 231 950 counts). To assess the stability of GFAP in blood over time, we added measured amounts of GFAP to five samples and measured them immediately and after 1, 2, and 3 days of storage at 4 °C. Differences between measured GFAP concentrations did not reach statistical significance. Freezing and thawing for up to four cycles did not influence the measured concentration of GFAP in the samples.
GFAP was detectable in 10 of 70 serum samples from healthy blood donors (range, 0.002–0.049 µg/L; median, 0.004 µg/L; 97.5 percentile, 0.033 µg/L).
Six patients were admitted within 3 h after head injury, 9 patients were admitted 4–6 h after the injury, and 10 patients were admitted 7–16 h after the injury. Serum samples were available from all 25 patients on admission and at 24 h after the injury. Samples from only 16 patients were available at 48 h because 9 patients had died or been discharged. GFAP was considered increased at concentrations >0.033 µg/L, representing the 97.5 percentile in healthy subjects.
Increased GFAP was found in 12 of 25 patient samples obtained at admission, in 1 of the 25 samples obtained 24 h after the injury, and in 1 of the 16 samples obtained 48 h after head trauma. The mean GFAP concentrations were 0.10 ± 0.18 µg/L on admission, 0.012 ± 0.026 µg/L 24 h after the injury, and 0.017 ± 0.052 µg/L 48 h after the injury. The sooner an admission sample was obtained after the injury, the higher the probability was that the GFAP concentration would be increased. Increased GFAP was found in 6 of 6 samples obtained within 3 h after the injury, in 5 of 9 samples obtained 4–6 h after the injury, and in only 1 of 10 samples obtained 7–16 h after the injury.
We had measured a mean S-100 plasma concentration of 0.059 ± 0.038 µg/L in 200 healthy subjects previously (16). S-100 concentrations >0.135 µg/L (mean + 2 SD) were found in 24 of the 25 samples obtained on admission, in 23 of 25 samples obtained 24 h after the head trauma, and in 10 of 16 samples obtained 48 h after the head trauma. The mean S-100 concentration was 1.50 ± 1.65 µg/L on admission, 1.24 ± 1.92 µg/L 24 h after the head trauma, and 1.49 ± 2.15 µg/L 48 h after the head trauma.
In the 15 patients admitted within 6 h after severe head trauma,
serum GFAP (y) and plasma S-100 (x) were
significantly correlated (r2 = 0.781;
y = 0.666 + 8.034x; P <0.05;
Fig. 1
).
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To the best of our knowledge, this is the first report on the measurement of GFAP in human blood. Our data indicate that GFAP is released into the blood very soon after brain injury; thus, measurement of the GFAP concentration in blood, similar to measurement of the concentration of S-100 protein in blood, could provide an early indication of the volume of brain parenchyma involved in head trauma. However, we found differences that need to be taken into account in the profiles of GFAP and S-100 protein concentrations after head trauma.
First, the absolute amounts of GFAP released into the blood are 10-fold lower than those of S-100 protein. A possible explanation for this difference is that the molecular mass of GFAP (40–53 kDa) is twice that of S-100 protein, which means that S-100 protein passes through the blood-brain barrier more easily than GFAP. On the other hand, neuron-specific enolase, with a molecular mass (78 kDa) nearly twice that of GFAP, is easily detectable in peripheral blood after brain lesions. Enzymatic degradation of GFAP in blood would be another explanation for this phenomenon.
Second, the temporal profile of GFAP concentration after head trauma seems to differ from that of S-100 protein. We have shown that the concentration of S-100 protein after ischemic stroke or subarachnoid hemorrhage is a quantitative indicator of the degree of damage to the CNS (17)(18). The data we report here regarding concentrations of S-100 protein after head trauma are consistent with those reported by other investigators after minor or major head trauma (19)(20)(21), and in the present study we found good correlation between GFAP and S-100 concentrations in blood for the first 6 h after head trauma. After the first 6 h following head trauma, however, the concentration of GFAP in blood drops rapidly, whereas the concentration of S-100 protein in blood may remain increased for days. Therefore, the concentration of GFAP in blood may be a suitable marker for acute CNS damage only.
The likelihood that GFAP may indicate only early CNS damage may not be a hindrance to its usefulness in clinical prognostication. To our knowledge, GFAP is not found in tissues outside the CNS. Thus, in contrast to other markers of CNS damage (neuron-specific enolase, S-100 protein, and myelin basic protein), GFAP seems to be strictly specific for damage to tissues in the CNS. Additional studies are needed to confirm our findings and to evaluate GFAP blood concentrations in patients with CNS damage attributable to causes other than acute head trauma. In addition, because of the very low concentrations of GFAP in human blood, the lower detection limit and the analytical sensitivity of our method should be improved. Nevertheless, measurement of GFAP concentrations in blood does appear to have possibilities as a relatively noninvasive means to identify early acute damage to the CNS.
References
The following articles in journals at HighWire Press have cited this article:
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  C. S. Jung, C. Foerch, A. Schanzer, A. Heck, K. H. Plate, V. Seifert, H. Steinmetz, A. Raabe, and M. Sitzer Serum GFAP is a diagnostic marker for glioblastoma multiforme Brain, December 1, 2007; 130(12): 3336 - 3341. [Abstract] [Full Text] [PDF]
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 J Steiner, H Bielau, H-G Bernstein, B Bogerts, and M T Wunderlich Increased cerebrospinal fluid and serum levels of S100B in first-onset schizophrenia are not related to a degenerative release of glial fibrillar acidic protein, myelin basic protein and neurone-specific enolase from glia or neurones. J. Neurol. Neurosurg. Psychiatry, November 1, 2006; 77(11): 1284 - 1287. [Abstract] [Full Text] [PDF]
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C Foerch, I Curdt, B Yan, F Dvorak, M Hermans, J Berkefeld, A Raabe, T Neumann-Haefelin, H Steinmetz, and M Sitzer Serum glial fibrillary acidic protein as a biomarker for intracerebral haemorrhage in patients with acute stroke J. Neurol. Neurosurg. Psychiatry, February 1, 2006; 77(2): 181 - 184. [Abstract] [Full Text] [PDF]
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 P. E. Vos, K. J.B. Lamers, J. C.M. Hendriks, M. van Haaren, T. Beems, C. Zimmerman, W. van Geel, H. de Reus, J. Biert, and M. M. Verbeek Glial and neuronal proteins in serum predict outcome after severe traumatic brain injury Neurology, April 27, 2004; 62(8): 1303 - 1310. [Abstract] [Full Text] [PDF]
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 J. Vaage and R. Anderson Biochemical markers of neurologic injury in cardiac surgery: The rise and fall of S100{beta} J. Thorac. Cardiovasc. Surg., March 1, 2003; 125(90030): S31 - 33. [Full Text] [PDF]
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A. Petzold, M. J. Eikelenboom, D. Gveric, G. Keir, M. Chapman, R. H. C. Lazeron, M. L. Cuzner, C. H. Polman, B. M. J. Uitdehaag, E. J. Thompson, et al. Markers for different glial cell responses in multiple sclerosis: clinical and pathological correlations Brain, July 1, 2002; 125(7): 1462 - 1473. [Abstract] [Full Text] [PDF]
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J. Vaage and R. Anderson Biochemical markers of neurologic injury in cardiac surgery: The rise and fall of S100{beta} J. Thorac. Cardiovasc. Surg., November 1, 2001; 122(5): 853 - 855. [Full Text] [PDF]
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 M. Herrmann, P. Vos, M. T. Wunderlich, C. H. M. M. de Bruijn, and K. J. B. Lamers Release of Glial Tissue-Specific Proteins After Acute Stroke : A Comparative Analysis of Serum Concentrations of Protein S-100B and Glial Fibrillary Acidic Protein Stroke, November 1, 2000; 31(11): 2670 - 2677. [Abstract] [Full Text] [PDF]
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