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36.2. CSF FLCs as a marker of intrathecal immunoglobulin synthesis

Chapter 36

A number of researchers have investigated FLCs in CSF as alternative markers of intrathecal inflammation [844][845][846][847][848][886][933][934]. The detection methods for CSF FLCs have included isoelectric focusing, quantitation by enzyme/radio-immunoassays and nephelometry but results have not led to routine incorporation of the test. However, the development of Freelite® nephelometric assays provoked renewed interest in the measurement of FLCs in CSF. Indeed, CE-marked Freelite assays intended for the measurement of FLCs in CSF (in addition to serum and urine) are now available for Binding Site SPAPLUS® and Siemens BN™II instruments.

Fischer et al. [843] studied CSF/serum pairs from 95 patients who had been investigated for intrathecal immunoglobulin synthesis. Oligoclonal immunoglobulin synthesis was identified in 71/95 patients, including 49 with multiple sclerosis and 22 with other neurological diseases. The median κ FLC concentrations in the CSF from both groups of patients with neurological diseases were higher than non-diseased samples (Figure 36.1) but λ FLC concentrations were found to be uninformative. When samples with increased albumin leakage were excluded, there was no overlap between normal and disease groups (cut-off level of κ FLC concentrations: 0.5 mg/L). This indicated that determination of κ FLC concentrations in CSF provided information similar to that of oligoclonal band measurements. As an alternative strategy for eliminating the influence of impaired blood-CSF barrier function, a κ FLC index was constructed ([CSF κ FLC/serum κ FLC]/[CSF albumin/serum albumin]). An empirically defined, non-linear κ FLC index threshold line provided good separation of patients with and without intrathecal immunoglobulin synthesis; only two normal samples were misclassified (Figure 36.2). The authors stated that a major advantage of the use of κ FLC CSF measurements in MS diagnosis was the availability of the Freelite assay on automated nephelometric analysers (Chapter 37).

A number of other groups have reported a strong correlation between elevated κ FLC CSF concentrations and positive oligoclonal bands and/or the diagnosis of MS [849][850][851][887][888][889][898][911]. Calculation of a κ FLC index (as above) has been shown to improve the diagnostic sensitivity and specificity [849][850][851][898]. Presslauer et al. [852] studied the use of a κ FLC index to diagnose MS in 438 subjects who underwent lumbar puncture. This included 41 patients with MS, and 29 patients with a CIS suggestive of MS. A control group (n=45) comprised individuals with normal pressure hydrocephalus, undefined dementia, or a primary suspected but unconfirmed subarachnoid bleed (and with no sign of inflammation). For the control group FLC CSF concentrations were low: κ FLC CSF median value 0.18 mg/L (range 0.13 - 0.22 mg/L); λ FLC CSF median value 0.16 mg/L (range 0.13 - 0.2 mg/L). Serum FLC (sFLC) concentrations were within the normal range, resulting in low κ and λ FLC indices (Figure 36.3), λ FLC index not shown). Similar findings were also reported by Arneth and Birklein [850].

Conversely, in the MS group, κ FLC CSF concentrations were typically high (median 4.12 mg/L, range 1.4 – 8.77 mg/L), whilst λ FLC concentrations were only moderately elevated (median 0.67 mg/L, range 0.25 - 1.54 mg/L) [852]. Patients with other diseases associated with intrathecal infection (e.g. meningitis/encephalitis) or inflammation (e.g. Guillain-Barré syndrome), typically had only moderate increases in both κ and λ FLC CSF concentrations and indices (Figure 36.3, λ FLC index not shown). Similar findings were also reported by Arneth and Birklein [850].

Presslauer and colleagues [852] also compared the diagnostic performances of the κ FLC index, κ FLC CSF concentration, λ FLC index and IgG index using receiver operating characteristic (ROC) analysis. The test with the best performance (and highest area under the ROC curve) was the κ FLC index. Using a cut-off value of 5.9, the diagnostic sensitivity of the κ FLC index was 96%. Only 3 patients (1 MS and 2 CIS suggestive of MS) had a κ FLC index below this level (these patients were also negative for oligoclonal bands and had a normal IgG index). By comparison, the diagnostic sensitivity of oligoclonal bands and the IgG index (≥0.6) was 91% and 80%, respectively. The κ FLC index for the MS patients was significantly higher than those for the other disease groups (meningitis/encephalitis: p=0.003; neuroborreliosis: p=0.001; and Guillain-Barré syndrome: p=0.009, Figure 36.3) [852]. Fifty patients without MS also had an elevated κ FLC index, resulting in a diagnostic specificity (for MS) of 86%; this was lower than the specificity of oligoclonal bands (92%), but distinctly higher than that of the IgG index (77%). Presslauer et al. [852] concluded that the κ FLC index should be interpreted alongside clinical findings together with other CSF analyses (including λ FLC concentrations and the λ FLC index). The cut-off for the FLC index (5.9) was subsequently validated in a multicentre study by the same authors [889] and an Austrian group [934], but alternative cut-offs have been suggested by others [933][898]. Zeman et al. [933] compared cut-offs for a number of quantitative FLC assays and concluded that method-specific cut-offs should be used (Chapter 8).

Presslauer et al. [853] refined their cut-off for the κ FLC index by expanding the number of samples included in defining the normal range . CSF samples were collected from 861 patients who underwent lumbar puncture and after exclusion of patients with contaminated samples or possible inflammatory conditions, 420 control samples remained. κ FLC CSF measurements in controls were used to define the upper limit of normal for the κ FLC ratio (CSF κ FLC/serum κ FLC) under different blood-CSF barrier conditions [853]. Briefly, controls were divided into 23 subgroups based on blood-CSF barrier function (defined by the [CSF albumin/serum albumin] ratio). For each subgroup, the mean value of the κ FLC ratio + 3SD was plotted against the mean albumin ratio, to produce a κ FLC index threshold line (Figure 36.4). The upper limit of the threshold line included 98% of control patients (i.e. the diagnostic specificity was 98%). Intrathecal immunoglobulin synthesis (defined by a κ FLC index above the threshold line) was detected in 97% of MS patients (n=65, Figure 36.5A) and 97% of CIS patients (n=69, Figure 36.5B) [853]. In both MS and CIS, the diagnostic sensitivity of the κ FLC index was superior to that of oligoclonal band detection or the IgG index (Table 36.1). In the subgroup of MS or CIS patients who were negative for oligoclonal bands, 76% had elevated κ FLC index values (Figure 36.5C). This suggests that κ index can detect very low plasma cell activity in the CSF, beyond the analytical sensitivity of IEF. Of the patients who were positive for oligoclonal bands, 98% had elevated κ FLC index values (Figure 36.5D). The authors concluded that the measurement of κ FLCs in CSF should become a first-line screen in diagnostic algorithms for MS and CIS [853].

Brivio et al. [854] recently validated the use of the κ FLC index threshold line in a study of 59 patients with MS and 31 controls (with a non-inflammatory disease of the CNS). The threshold line provided good separation of the two populations, with superior performance compared to that of oligoclonal band detection. The κ FLC index threshold line has also been validated by a multicentre study conducted in Austria and Germany [889].

Disease Oligoclonal bands IgG index κ FLC index (threshold value)
MS (n=65)
94%
85%
97%
CIS (n=69)
81%
74%
97%

Table 36.1. Diagnostic sensitivity of oligoclonal bands, IgG index and κ FLC index in MS and CIS [853].

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