WBC count is typically increased in inflammatory CNS diseases but also in leptomeningeal metastases. Cytological examination allows identification of the underlying cell type in the case of inflammation (e.g. mononuclear versus polymorphonuclear) as well as of tumour cells (Deisenhammer et al. 2006). Currently available guidelines established by a task force of the European Federation of Neurological Societies (EFNS) define a WBC count <5/μL as normal, indicating noninflammatory CSF. Cytological examination usually yields lymphocytes and monocytes at resting phase and occasionally ependymal cells (Deisenhammer et al. 2006). In recent years, there was an increasing interest in automated cell counting and cell sorting for CSF. Therefore, recommendations on at least advantages and disadvantages compared to the conventional methods of cell counting in a chamber under microscopy as well as performing cytology would be desirable. So far, studies comparing WBC count determined automatically and visually remain contradictory showing good overall correlation of results but still weak concordance rates at low WBC counts (Sandhaus et al. 2010; Zimmermann et al. 2011). The reliability of cell sorting is still equivocal at low WBC counts, and pathological cell types such as tumour cells are most likely only detected by microscopic cytological examination (Sandhaus et al. 2010; Zimmermann et al. 2011; Danise et al. 2013).

RBC count is essential for the diagnosis of SAH especially in patients with normal CT scan (refer to chapter 24.4.1) as well as to estimate whether blood contamination of CSF (by traumatic puncture) could affect the results of other CSF parameters. In general, the extent of interference with the analyte depends on its serum concentration, e.g. blood contamination can lead more easily to a false positive result of a CSF parameter in the case of high serum concentration. There are no recommendations at which RBC count a particular CSF parameter should not be determined or whether and how calculative corrections should be performed. Research recommendations consider CSF samples as blood contaminated if RBC count exceeds 500/μL; however, this cut-off value was based on proteomic data (Teunissen et al. 2009) and, thus, does not necessarily apply to routine CSF analysis.

Since the ratio of WBC to RBC in blood is roughly 1:750, some authors suggested to subtract 1–2 WBC per 1,000 counted RBC in CSF (Delank 1972; Olischer and von Suchodoletz 1972). Reske et al. recommended to correct 1 WBC per 666 RBC (Reske et al. 1981) and others even calculated a formula involving leukocytes and erythrocytes both in blood and CSF (WBC_CSF/RBC_CSF × RBC_blood/WBC_blood) in order to obtain the “true” WBC count in CSF (Pfausler et al. 2004). Concerning CSF total protein concentration, Reske et al. suggested the formula Protein_CSF (mg/L) − RBC_CSF (/μL)/333 × 5.25 (Reske et al. 1981), but the method used for the determination of CSF total protein has to be considered in this context (see below) (Boer et al. 2007). Although blood contamination in CSF also influences CSF Ig concentrations and calculated intrathecal Ig synthesis, there are so far no suggestions for a corrective approach.

Both measures, CSF total protein and CSF/serum albumin quotient (Q _alb), are increased in case of blood-CSF barrier (BCB) dysfunction, which is evident in different conditions such as meningitis, leptomeningeal metastases or inflammatory polyneuropathy (Reiber and Peter 2001). Q _alb should be preferred to CSF total protein concentration in order to assess BCB function (Deisenhammer et al. 2006). The superiority of Q _alb is based on the fact that it is not influenced by intrathecal Ig synthesis, is corrected for serum concentrations of albumin and is a technology-independent value (Deisenhammer et al. 2006). Although it is known that the protein concentration in CSF increases with age (Tibbling et al. 1977; Garton et al. 1991; Blennow et al. 1993; Eeg-Olofsson et al. 1981), no specific age-dependent upper normal limit for Q _alb has been recommended. Reiber et al. suggested the approximate formula “Age/15 + 4” (Reiber et al. 2001), but this still returns false positive/elevated results in roughly 15 % of patients without evidence of neurological disorders (ruled out by clinical, laboratory and imaging diagnostics) (Brettschneider et al. 2005).

Regarding CSF total protein, concentrations less than 0.45 g/L are considered as normal (Deisenhammer et al. 2006). However, there is no recommendation for age correction, and existing literature even provides evidence that the upper normal limit has to be corrected to levels ranging between 0.5 and 0.6 g/L (Tibbling et al. 1977; Garton et al. 1991; Ahonen et al. 1979; Gilland 1967; Dufour-Rainfray et al. 2013; Mertin et al. 1971; Breebaart et al. 1978). Comments on the methodology for CSF total protein measurement are lacking too (not relevant for Q _alb, as a ratio is dimensionless and method independent given that the same method is used for CSF and serum measurements).

In a subgroup of patients such as in SAH patients developing hydrocephalus and requiring ventricular drain, CSF samples are collected from the ventricular lumen (via the drain). The site of CSF collection (due to a rostrocaudal protein concentration gradient) has a significant impact on levels of CSF total protein concentration and CSF albumin (therefore also on Q _alb) with levels in lumbar CSF roughly 2.2 times higher than in ventricular CSF (Weisner and Bernhardt 1978). Cut-off values for CSF total protein or Q _alb in ventricular CSF have not been formally investigated; nevertheless, it is common practice to use cut-off values of lumbar CSF divided by 2.2.

CSF glucose and, thus, the CSF/S_Glu ratio are typically decreased in bacterial or fungal infectious CNS disease as well as in leptomeningeal metastases. According to EFNS guidelines, a CSF/S_Glu ratio between 0.5 and 0.6 is considered as normal and values below 0.4–0.5 are pathologic (albeit, e.g. the German Society of Neurology recommends to consider a CSF/S_Glu <0.3 for an accurate diagnosis of bacterial meningitis (Diener 2012)). It has to be stated that despite the long-term and widespread use of glucose measurement in routine CSF diagnostics, the level of evidence for the recommended cut-off is low (Deisenhammer et al. 2006). Although it is well known that CSF/S_Glu ratio decreases with increasing serum glucose level in a non-linear manner (Leen et al. 2012), proposed normal and cut-off values are not serum glucose adapted. A recent study linked CSF/S_Glu ratio to specific serum glucose concentrations and suggested CSF/S_Glu ratio >0.5 as normal for patients with serum glucose concentrations <1 g/L, >0.4 for those with a serum glucose level of 1 g/L up to 1.49 g/L and >0.3 for values exceeding 1.5 g/L (Hegen et al. 2014). Other factors which might influence CSF/S_Glu ratio such as age, WBC count and CSF total protein (i.e. lower CSF/S_Glu ratio with increase of these parameters) could be ruled out in this study by regression analysis revealing only serum glucose concentration as a significant covariant for CSF/S_Glu ratio (Hegen et al. 2014).

Intrathecal Ig production can be found in various, mainly inflammatory, conditions of the CNS (Deisenhammer et al. 2006), and specific Ig patterns are indicative for certain diseases, e.g. IgM dominance for neuroborreliosis or IgA dominance for neurotuberculosis and brain abscess (Reiber and Peter 2001). Evidence on the frequency of intrathecal IgG, IgA and IgM synthesis in different neurological diseases is summarised in Table 24.3. Nevertheless, existing guidelines conclude that there is no evidence to support the routine use of quantitative assessment of intrathecal Ig synthesis (Deisenhammer et al. 2006; Freedman et al. 2005). This might be due to several reasons: Calculation of intrathecal IgG synthesis, e.g. by non-linear formula such as Reiber hyperbolic function (Reiber 1994), has lower diagnostic accuracy in terms of sensitivity and specificity compared to oligoclonal band (OCB) evaluation for diseases such as multiple sclerosis (MS) (Reiber et al. 1998; Rudick et al. 1989). Regarding intrathecal IgM synthesis, there are reports of falsely positive results in patients with noninflammatory diseases without IgM oligoclonal bands in CSF (Sharief et al. 1990), and the same applies for IgA. Further ambiguities arise from recommendations such as those of the German Society for Cerebrospinal Fluid Diagnostics and Neurochemistry to consider intrathecal fractions of IgA and IgM, indicated by the percentage of intrathecally synthesised Ig, of less than 10 % as non-pathological (DGLN DGfLuKN 2004).

It is known that besides intact immunoglobulins, plasma cells secrete an excess of free light chains (FLC) (Nakano et al. 2011), which accumulate in the CSF in the case of intrathecal B-cell activity. Several studies have already indicated the potential diagnostic value of κFLC in MS (Rudick et al. 1989; Krakauer et al. 1998; Desplat-Jego et al. 2005; Senel et al. 2014; Duranti et al. 2013); however, there have been no recommendations on FLC as a measure for intrathecal B-cell activity.

Detection of OCB by isoelectric focusing (IEF) on agarose gels followed by immunoblotting is the gold standard method for the determination of an intrathecal IgG synthesis (Freedman et al. 2005). As mentioned above, intrathecal IgG synthesis is found primarily in inflammatory CNS diseases indicating a long-lasting B-cell activity (Deisenhammer et al. 2006), and its detection by OCB shows significantly higher sensitivity and specificity than IgG quantitation in CSF and serum followed by calculation of any formulae (e.g. Reiber IgG synthesis) (Reiber et al. 1998; Rudick et al. 1989). The incidence of OCB in different neurological diseases is shown in Table 24.4.

OCB are of high importance especially in the diagnosis of MS. A recent large meta-analysis showed that OCB (detected by IEF followed by immunofixation) were positive in roughly 90 % of more than 12,000 MS patients and 70 % of more than 2,600 patients with clinically isolated syndrome, the first manifestation of the disease (Dobson et al. 2013). In cases of primary progressive MS, the detection of intrathecal IgG synthesis (e.g. by OCB) is still included in the diagnostic criteria, as one of three criteria supporting evidence for dissemination in space (Polman et al. 2011). Although no longer required for diagnosis of relapsing MS (Polman et al. 2011), CSF analysis is still required for exclusion of differential diagnoses (Freedman et al. 2005; Polman et al. 2011; Miller et al. 2008; Tumani et al. 2011). Findings of mild lymphocytic pleocytosis and intrathecal IgG synthesis prove the inflammatory nature of the underlying condition in MS (the only test besides brain biopsy) (Freedman et al. 2005; Polman et al. 2011; Miller et al. 2008; Tumani et al. 2011).

As detection of OCB is a technically demanding method, clear recommendations exist on how to perform it. Freedman et al. provide a list of 12 red flags, which should be considered in order to achieve and guarantee high diagnostic accuracy of this method (Table 24.5) (Freedman et al. 2005). Furthermore, it is clearly defined how to interpret test results, classified into five different patterns (see chapter 10) (Freedman et al. 2005). In general, a positive result is found if two or more OCB are present in CSF but not in serum. The cut-off of two bands ensures the high specificity of the test for MS. In cases of a single detected band (most likely reflecting incomplete OCB in CSF but not in serum), if other (clinical or MRI) criteria clearly point to MS, CSF analysis might be repeated (after several months) for re-evaluation (Freedman et al. 2005; Davies et al. 2003).

Not a single CSF parameter but the integrative report allows reliable diagnosis making in a variety of neurological disorders. Here, we would like to highlight this special feature in CSF analysis by the following example: Whereas WBC count alone could not reliably distinguish between viral and bacterial meningitis (in a cohort of patients published by Spanos et al.), the combination of different parameters, i.e. of CSF glucose concentration <0.34 g/L, a CSF/S_Glu ratio <0.23, a CSF total protein concentration >2.2 g/L and a CSF WBC count of >2,000/μL, enables the correct assignment of patients with bacterial meningitis with ≥99 % certainty (Spanos et al. 1989). Table 24.6 summarises typical constellations of CSF results in different neurological conditions (Deisenhammer et al. 2006).