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Technical Briefs |
Dept. of Clin. Chem., Leiden Univ. Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
a author for correspondence: fax + 31/71/5266753, e-mail roelandse{at}rullf2.medfac.leidenuniv.nl
Leakage of cerebrospinal fluid (CSF) from the subarachnoid space
into the nasal or aural cavity creates a pathway for life-threatening
central nervous system infection. Detection of the leakage can be
cumbersome (1). In former days the identification and
localization of CSF leakage was almost entirely dependent upon the
clinical history and methods involving intrathecal injection of
radioactive material or a dye. Furthermore, various chemical tests,
such as determination of glucose, protein, and electrolytes, performed
on the secreted material have been advocated for the differentiation
between CSF leakage and other secretions (2). The
injection methods are risk-bearing for the patient and the above
methods yield a considerable chance of false-positive or false-negative
results (3). Nowadays O-sialotransferrin, also
named ß2-transferrin, asialotransferrin, or the 
fraction, is an accepted marker protein for the detection of CSF in
excretions from the nose or the ear, or from head or neck wounds.
O-Sialotransferrin is not strictly unique to CSF. The
protein can also be demonstrated in human aqueous humor
(4) and in perilymph fluid (5)(6)(7). It is also
found in the blood of patients with chronic liver diseases
(8)(9), with an inborn error of glycoprotein
metabolism (10), and with a genetic variant of transferrin
(8)(11)(12). The protein can be
detected by direct immunofixation of transferrins after agarose
electrophoresis (high-resolution electrophoresis, HRE)
(3)(13) or after blotting
(1)(14)(15). The method we used up
to now was the agarose/immunofixation method according to Zaret et al.
(3). We used the Paragon High Resolution Electrophoresis
kit (HRE kit, Beckman Instruments) and an anti-transferrin reagent
(Beckman Array® System, Beckman Instruments). One
disadvantage of this fast method is the high protein concentration that
is necessary for use: The secretion extract must be concentrated to a
protein amount of 10 g/L. Therefore small samples cannot be analyzed.
Furthermore, the location of the O-sialotransferrin band in
secretions can be displaced, compared with the location of the
O-sialotransferrin band in a control CSF. This phenomenon
hampers detection. An admixture of large quantities of blood
(hemoglobins) disturbs the pattern because of
ß1-transferrin (3).
Our currently described method improves the detection of O-sialotransferrin in the case of minor CSF leakages and even in small blood-containing samples. The described method is not meant to be a stat method. O-Sialotransferrin (pI 5.9) and other transferrin isoforms, such as monosialo- (pI 5.8), disialo- (pI 5.7), and trisialotransferrin (pI 5.6), are easily recognized. The method is suited for small samples, requires less protein, is sensitive, and gives more information compared with the agarose electrophoresis methods. Because in rare cases O-sialotransferrin can occur in blood, the necessity of involving a serum sample from the same patient for comparison is obvious.
In detail, the procedure is as follows: Nose and ear secretions from patients suspected of having a CSF leakage are absorbed with Merocel® tampons, cotton swabs, or gauze. Next the material is extracted with a small amount of distilled water. After centrifugation of the extract at 1900g for 10 min at room temperature, the total protein concentration of the supernatant is measured (Hitachi 911 analyzer, Boehringer Mannheim). Blood samples to prepare serum are obtained through venipuncture. After centrifugation at 1900g for 10 min at room temperature, the total protein concentration of the serum is measured (Hitachi 747 analyzer, Boehringer Mannheim). Control CSF consists of a CSF sample that shows a clear fraction of the O-sialotransferrin by isoelectric focusing (IEF). Extract supernatant, serum, and control CSF are stored at -20 °C before analysis. If required, extract supernatant and control CSF are concentrated in Fugisep® centrifuge concentrators (type PES 10, Intersep Filtration Systems) to a total protein amount of 250 mg/L. Serum and extract supernatant with a total protein amount of >250 mg/L are diluted to 250 mg/L with a 1:2 mixture of saline and distilled water. pH focusing experiments are performed on a Multiphor® II electrophoresis kit with a 2103 power supply (Pharmacia-LKB) equipped with a Kryostat WK5 cooling unit (Colora) adjusted to 8 °C. Polyacrylamide gels are ready-to-use Ampholine® PAG plates for IEF, pH range 3.5–9.5 (Pharmacia Biotech). Sample volume is 20 µL. Samples of patients' sera are placed adjacent to the prepared extract. Control CSF is incorporated in each assay. The power supply is limited to a maximum of 1500 V and 50 mA. Total focusing time is 90 min. After pH focusing the PAG plate is (gel-side down) placed on top of a 1-mm layer of a 50-fold dilution in distilled water of transferrin antibody (A0061, Dako Corp.) in a plexiglass tray. Incubation is for 60 min at 37 °C. After the incubation several washings with distilled water are applied (overnight). Silver staining is performed at room temperature with a 1 g/L silver nitrate solution as coloring reagent for 30 min, a developer reagent containing 30 g of sodium carbonate and 500 µL of formalin per liter for 4–10 min (dependent on the visual band intensity/background ratio), a 480 g/L citric acid solution as stopping reagent for 10 min, and a 50 g/L glycerol solution as preserving reagent for 30 min (all chemicals for the silver staining are purchased from Merck). Finally the PAG plate is covered with a cellophane preserving sheet (Pharmacia Biotech), which is fixed with glass strips on a glass plate. The PAG plate is dried for 5 h at 60 °C. This method gives excellent results in recognizing O-sialotransferrin in secretions.
In Fig. 1
a comparison with the agarose/immunofixation assay is
demonstrated for five patients. All samples were extractions from
cotton swabs in which the secretion was collected. All the specimens
were contaminated with blood. Patient 1 shows a positive finding for
O-sialotransferrin with the currently described method, but
the test is negative with the agarose/immunofixation assay. Patients 2
and 4 show positive findings with both methods. Patient 3 is clearly
positive with the currently described method, but only slightly
positive with the agarose/immunofixation assay (hardly visible in the
Figure
). Patient 5 is negative with both methods.
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Of 43 samples, we found 11 to be positive and 26 to be negative with both the agarose/immunofixation assay and with the IEF method; 6 samples were positive with the IEF method, but negative with the agarose/immunofixation assay. None of the samples was positive with the agarose/immunofixation assay when a negative result was obtained with the IEF method. From these data we can conclude that the currently described method is more sensitive than the agarose/immunofixation assay: With the proposed method we found 17 samples positive for CSF, whereas with the former method only 11 samples were positive. Not only O-sialotransferrin is seen, but also other transferrin isoforms such as monosialo-, disialo-, and trisialotransferrin. This low sialic acid transferrin pattern is characteristic for CSF (admixture). The method is selective and sensitive. For this purpose we tested mixtures of CSF and distilled water (data not shown). A CSF admixture of 2% is detectable, vs 10% in our agarose/immunofixation assay. Normansell et al. were able to detect ß2-transferrin (O-sialotransferrin) in CSF that had been diluted up to eightfold (12.5% of CSF) with other fluids (1).
A great advantage of silver staining is that only 5 µg of total protein is necessary in the electrophoretic procedure (vs 50 µg in the agarose method). Silver staining is easy to perform, reproducible, and inexpensive compared with commercially available kits. The drying procedure of the gel results in perfectly dry gels without folds and wrinkles. Dried gels are tenable and do not fade in daylight. We estimate the costs per result to be $18.00, working hours included. A study at our department is currently ongoing and involves a comparison of clinical findings with both the currently described IEF method with immunofixation and the previously used method of agarose electrophoresis (HRE) with immunofixation.
Acknowledgments
We thank the staff of the department for their cooperation in the production of this manuscript.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Warnecke, T. Averbeck, U. Wurster, M. Harmening, T. Lenarz, and T. Stover Diagnostic Relevance of {beta}2-Transferrin for the Detection of Cerebrospinal Fluid Fistulas Arch Otolaryngol Head Neck Surg, October 1, 2004; 130(10): 1178 - 1184. [Abstract] [Full Text] [PDF]
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 E. Arrer, C. Meco, G. Oberascher, W. Piotrowski, K. Albegger, and W. Patsch {beta}-Trace Protein as a Marker for Cerebrospinal Fluid Rhinorrhea Clin. Chem., June 1, 2002; 48(6): 939 - 941. [Full Text] [PDF]
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