gms | German Medical Science

Patients with a CSF leak are at risk: in 40% of patients with a known CSF fistula, Streptococcus pneumoniae or Haemophilus influenzae meningitis occurred. The cumulative risk to develop meningitis exceeded 85% after 10 years in 160 patients with traumatic CSF leaks. The mortality rate of bacterial meningitis was described to be as high as 25–50% in spite of excessive antibiotic treatment. Once detected, it is generally accepted that surgical repair is the therapy of choice to avoid all the complications. At the anterior skull base, surgical repair predominantly by the endonasal approach is nowadays a modern and safe procedure.

CSF leaks always require precise diagnostic in form of (a) identification of CSF, (b) localization and (c) visualization before surgery. Ideally, the methods should be non-invasive, non-radiation, easy to perform everywhere with routine laboratory equipment and require only small quantities of specimen. Only laboratory tests using CSF markers meet these requirements for identification.

Beta-trace protein (ß-TP) is the most abundant protein of human cerebrospinal fluid (CSF) and produced within the central nervous system. ß-TP shows a CSF/serum ratio of 33, which is the highest of the CSF specific proteins known today. ß-TP is not only contained in CSF but in aqueous humor, urine, sperm and inner ear fluids too. For the detection of CSF fistulas a high sensitivity and specificity of ß-TP was shown by clinical experience.

For quantitative determination of ß-TP, a newly developed nephelometric research assay, N-latex-ß-trace-protein N-Latex-ß-TP (Dade Behring, 65835 Liederbach, Germany) is the new standard today. N-Latex-ß-TP is a lyophilized reagent for the Behring Nephelometer Analyzer (BNA). Polystyrene particles coated with immunoaffinity-purified polyclonal antibodies from rabbit against human ß-TP were agglutinated in the presence of ß-TP. The increase in light scattering caused by agglutination is measured by laser absorption. The minimal volume for a sample is 5 µL. The analytical imprecision of the assay is 2.3 to 6.5%. The detection limit for the undiluted sample is 2.5 µg/L. The cut-off of ß-TP values was defined to 1.31 mg/l in accordance to our and other recent studies on comparing ß-TP with ß₂-transferrin in diagnosing liquorrhea.

To investigate the reliability and the sensivity of ß-trace protein in the diagnostic of CSF-leaks, in the period between 1989 and 1995 154 samples of 92 patients with a suspected fistula of the anterior or lateral skull base from have been investigated for ß-trace protein.

41 probes were positive and 64 negative. In correlation with the clinical findings there was no false positive result. In isolated cases a CSF fistula could not be excluded by a negative result. Between 1994 and 2000 another 84 patients were investigated for (ß-TP) by nephelometry with an positive predictive value of .971 to 1.

69 Patients were selected between 1998 and 2000. Average age was 47.9 (standard deviation 13.0) years and gender relation was 27.5:72.5. Indication for surgery was chronic paranasal sinus disease with or without nasal polyps. All patients underwent endoscopic endonasal sinus surgery. 13 subjects underwent unilateral surgery and 56 subjects underwent bilateral procedures. Patients with previous surgery and patients with fungal paranasal sinus disease were included. Patients with a clear CSF leakage during surgery were not included.

112 fluid samples were collected after endoscopic paranasal sinus surgery. At the end of the surgical procedure Raucocel (Rauscher, Germany) sinus packs were placed at the ethmoid roof. In unilateral disease, only one sinus pack was used. After the Raucocel packs were inserted, they were soaked with doxycycline solution to prevent local postoperative infection. At the first day after the operation, a serum sample was taken and at the second day, the Raucocel sinus packs were removed. Fluid samples were obtained by pressing the Raucocel sinus packs and both, the Raucocel and the serum samples were centrifugated at 4000 R/min for 5 min. The samples were kept at –40°C before investigating for ß-TP nephelometry. In accordance to recently published data samples with a ß-TP concentration of 6 mg/L or higher were reported as “positive”, indicating the presence of CSF.

A result lower than 3 were defined as negative for CSF traces. A result between 3 and 6 mg/L was suggestive of CSF. Patients with a positive or a suggestive ß-TP result were re-investigated with nasal endoscopy and a repeat Raucocel tamponade for ß-TP analysis. Re-investigation was performed at least 6 months after surgery: Under local anesthesia, Raucocel paranasal sinus packs were situated at the ethmoid roof on both sides. The sinus packs were left in place for several hours and fluid samples, obtained from the Raucocel packs, were analyzed for ß-TP.

The average value of ß-TP in serum was 0.69 ± 0.33 mg/L. The samples from the ethmoid roof showed elevated ß-TP results in two patients. Also, when taking into account that up to 30% of ß-TP was bound to the Raucocel tamponade, the results of the sinus packs showed presence of CSF in these two casesCalculating the incidence in regard to 69 subjects, we found an occult CSF fistula in 2.9%. When calculating on the basis of 112 samples the incidence of an occult CSF fistula was 1.78%.

The proof of CSF in patients with a suspected skull base defect is still a challenging task, because CSF leaks do not regularly cause clinical symptoms, because minute amounts of CSF can flow intermittently through a small fistula. Furthermore, CSF flowing down the Eustachian tube or the posterior area of the paranasal sinuses might constantly be transported to the choana and swallowed. Therefore, sensitive, cheap – and if possible non-invasive – methods to detect traces of CSF are warranted. Once a CSF leak is diagnosed, otorhinological or neurosurgical approaches offer the closure of the fistula with high success rates 21-27. Methods with high predictive values might help to reduce the rate of meningitis.

As another immunological method for CSF examination Meurman et al. developed the ß₂-transferrin assay. This method takes about 5 hours until a result is obtained. The ß₂-transferrin assay is performed by a two-dimensional immunoelectrophoresis or by immunofixation. Body fluids with a protein content of 5 g/L or higher have to be precipitated with ammonium sulphate prior to ß₂-transferrin immunoelectrophoresis. Furthermore, contamination with blood requires a column chromatography in order to eliminate hemoglobin. ß₂-transferrin is absent in serum and has been identified so far in CSF, perilymph and aqueous humor. In subjects with cirrhosis or genetic protein variants, however, ß₂-transferrin has been detected in serum.

In comparison to the ß₂-transferrin test, the ß-TP assay has shown its superiority. The ß-TP test can be done within 20 minutes using routine laboratory equipment and the costs are reasonable (about 20–30 USD/test). In addition, the ß-TP test showed higher predictive values. The CSF/serum ratio of ß-TP is the highest of all CSF specific proteins. Since the protein is stable, the specimens can be send for investigation by surface mail. The clinical experience revealed that smallest traces of CSF (<5%) can be detected by ß-TP. Also in other studies excellent analytical sensitivity was stated [Ref. 1]. In this study, CSF dilutions of 1:80 (1 mL–79 mL), corresponding to 1–2% CSF in nasal secretions, were reliably detected. From our results, unobserved, occult CSF fistulas occur during endoscopic sinus surgery with an incidence of 2.9% and the experience of the surgeon does not seem to correlate with this type of complication. In contrast, experienced surgeons operated both patients with an occult CSF fistula. Furthermore, the results of the presented study indicate that an intra-operative occult CSF fistula might close spontaneously during the post-operative healing process.

Furthermore it demonstrates that the investigation using ß-TP is not invasive and this method is well suitable for screening CSF leaks. In recently done experiments, we could demonstrate that ß-trace protein can also be found in perilymph, which will facilitate perilymphatic leakage diagnostics in case of head trauma and disturbance of inner ear function [Ref. 4].