Validated age-specific reference values for CSF total protein levels in children

Accepted Manuscript Validated age-specific reference values for CSF total protein levels in children V. Kahlmann, J. Roodbol, N. van Leeuwen, C.R.B. Ramakers, D. van Pelt, R.F. Neuteboom, C.E. Catsman-Berrevoets, M.C.Y. de Wit, B.C. Jacobs PII:

S1090-3798(16)30280-X

DOI:

10.1016/j.ejpn.2017.03.006

Reference:

YEJPN 2199

To appear in:

European Journal of Paediatric Neurology

Received Date: 11 January 2017 Revised Date:

21 February 2017

Accepted Date: 19 March 2017

Please cite this article as: Kahlmann V, Roodbol J, van Leeuwen N, Ramakers CRB, van Pelt D, Neuteboom RF, Catsman-Berrevoets CE, de Wit MCY, Jacobs BC, Validated age-specific reference values for CSF total protein levels in children, European Journal of Paediatric Neurology (2017), doi: 10.1016/j.ejpn.2017.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Validated age-specific reference values for CSF total protein levels in children V. Kahlmann1,2*, J. Roodbol1,2*, N. van Leeuwen4, C.R.B. Ramakers5, D. van Pelt1,2, R.F.

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Neuteboom2, C.E. Catsman-Berrevoets2, M.C.Y. de Wit2, B.C. Jacobs1,3

Department of Neurology (1), Paediatric Neurology (2), Immunology (3), Public Health (4),

Center Rotterdam, The Netherlands.

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* These authors contributed equally to the manuscript.

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and Clinical Chemistry (5) Erasmus MC – Sophia Children’s Hospital, University Medical

Corresponding author: M.C.Y. de Wit, MD, PhD

Department of Pediatric Neurology, Erasmus MC Sophia, P.O. box 2060, 3000CB Rotterdam,

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The Netherlands. T: +31107036341, F: +31107036345, E-mail: [email protected] E-mail addresses other authors:

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V. Kahlmann: [email protected], J. Roodbol: [email protected], N. van Leeuwen: [email protected], C.R.B. Ramakers: [email protected], D.

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van Pelt: [email protected], R.F. Neuteboom: [email protected], C.E. Catsman-Berrevoets: [email protected], B.C. Jacobs: [email protected]

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ACCEPTED MANUSCRIPT Abstract Objective: To define age-specific reference values for cerebrospinal fluid (CSF) total protein levels for children and validate these values in children with Guillain-Barré syndrome (GBS), acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS).

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Methods: Reference values for CSF total protein levels were determined in an extensive cohort of diagnostic samples from children (<18 year) evaluated at Erasmus Medical Center / Sophia Children’s Hospital. These reference values were confirmed in children diagnosed

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with disorders unrelated to raised CSF total protein level and validated in children with GBS, ADEM and MS.

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Results: The test results of 6145 diagnostic CSF samples from 3623 children were used to define reference values. The reference values based on the upper limit of the 95% CI (i.e. upper limit of normal) were for 6 months-2 years 0.25 g/L, 2-6 years 0.25 g/L, 6-12 years 0.28 g/L, 12-18 years 0.34 g/L. These reference values were confirmed in a subgroup of 378

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children diagnosed with disorders that are not typically associated with increased CSF total protein. In addition, the CSF total protein levels in these children in the first 6 months after birth were highly variable (median 0.47g/L, IQR 0.26-0.65). According to these new

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reference values, CSF total protein level was elevated in 85% of children with GBS, 66% with ADEM and 23% with MS.

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Conclusion: More accurate age-specific reference values for CSF total protein levels in children were determined. These new reference values are more sensitive than currently used values for diagnosing GBS and ADEM in children.

Keywords: Multiple sclerosis, Acute disseminated encephalomyelitis, Guillain-Barré syndrome, Pediatric and Cerebrospinal Fluid

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ACCEPTED MANUSCRIPT 1. Introduction Cerebrospinal fluid (CSF) total protein level is frequently tested in the diagnostic work-up of immune-mediated neurological disorders, including Guillain-Barré syndrome (GBS), acute

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disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS). In the acute phase, the diagnosis GBS is mainly based on the patient’s medical history and neurological examination and can be difficult especially in children.(1, 2) Misdiagnosis can lead to life threating

complications. The presence of an elevated CSF total protein level can be used to support the

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diagnosis and is one of the key features of the current diagnostic criteria for GBS(3, 4). Past

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studies have shown a large variation in CSF total protein level in adult GBS patients and in children with ADEM and MS.(5-8) The variation in CSF total protein level in children with GBS is still unknown. CSF total protein level is not only influenced by the underlying disease but also by the age of the patient. Multiple studies have found a physiological CSF total protein level that slowly rises with age (see supplementary table 1).(9-11)

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The optimal cut-off value for CSF total protein levels in the diagnostic work-up therefore should depend on the age of the patient. Unfortunately, age-dependent and validated reference

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values for CSF total protein levels in children are currently not available. Instead many clinicians use one cut-off point for all ages, or reference values based on relatively small

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populations of local controls.

The aim of this study was to define age-specific reference values for CSF total protein level in children and validate these values in children with GBS, ADEM and MS.

2. Methods 2.1. Study design, settings and patients A retrospective single center study was performed, on children (<18 years) previously evaluated for different purposes at Sophia Children’s hospital of the Erasmus Medical Center 3

ACCEPTED MANUSCRIPT in Rotterdam, the Netherlands, a tertiary care hospital. All protein concentrations used in our study were determined by the quality controlled routine diagnostic test used in our hospital based on the turbidimetric benzethonium method. In short, a diagnostic sample is

detected by turbidimetry after adding benzethonium chloride.

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preincubated with an alkaline solution containing EDTA, which denatures the protein and is

To evaluate which reference values for CSF total protein are used for current clinical practice in children, all eight university children’s hospitals in the Netherlands were asked for their

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local standards.

New reference values for CSF total protein were determined by two independent

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analyses. First, the reference values for CSF total protein were determined using the Bhattacharya method,(12-14) a standard procedure conducted by clinical chemists to establish reference values based on previously acquired diagnostic data.(15, 16) In short, the Bhattacharya method identifies a Gaussian distribution in a large dataset of unselected patients without

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having to exclude pathological samples prior to analysis. The Bhattacharya method was applied to the CSF samples from all children aged between six months and 18 years who underwent a lumbar puncture (LP) between September 1996 and April 2013 in Sophia

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Children’s hospital, irrespective of the diagnosis. Second, we confirmed the reference values for CSF total protein level found using the

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Bhattacharya method in a subgroup of children with diseases unlikely to influence the CSF total protein level aged 0-18 years. Patients were divided in those with non-inflammatory neurological disorders and those without a neurological disorder. All test results used in this study were based on children who had a diagnostic LP between September 2006 and April 2013. In this study, the children were divided in seven age-categories (1-4 weeks, 1-3 months, 3-6 months, 6 months-2 years, 2-6 years, 6-12 years, and 12-18 years). In each age category, at least 15 patients were included. Data were collected on age, gender, indication to perform

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ACCEPTED MANUSCRIPT LP, diagnosis and various CSF test results: protein concentration (g/L), white blood cell count (/µL) (WBC), red blood cell count (/µL) (RBC). In case of multiple procedures, test results of the first LP were used. Excluded were samples of a traumatic lumbar puncture (> 500 red blood cells/µL), pleiocytosis (> 50 white blood cells/µL), prematurity (age at birth < 37 weeks

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and age under one year) or patients with confirmed diagnosis associated with increased protein level. The reference values that were found using the Bhattacharya method and

reanalysed in a cohort of children with a confirmed diagnosis, were then validated in children

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with GBS, ADEM and MS (age 0-18 years) who were seen between June 1987 and March 2013. The same age-categories were used as for the patients in the first subgroup. Samples

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obtained by traumatic LP were excluded. RBC was unknown in some patients with GBS, ADEM and MS; comparative analyses were done to investigate if there was an influence on protein concentration in these cases. The study was approved by the Medical Ethics

2.2. Statistical analysis

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Committee of the Erasmus MC.

Data were analysed using SPSS statistics 20. Continuous data were described using means

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and standard deviations (SD) when normally distributed, and using medians and interquartile ranges (IQR) when not normally distributed. Categorical data were described using

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frequencies and percentages. Continuous variables were analysed using Kolmogorov-Smirnov test and Mann-Witney U test. Correlation between continuous variables was tested using Pearson correlation coefficient. A two sided p-value of <0.05 was considered to be statistically significant. To determine the diagnostic value of CSF total protein for GBS, ADEM and MS patients we created receiver operating characteristic (ROC) curves for all three diseases. An ROC curve evaluates the overall test performance of a certain measurement or test, independently

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ACCEPTED MANUSCRIPT of the ultimately chosen cut-off value. For each disease a specific cut-off was defined with the combination of highest sensitivity and specificity.

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3. Results We performed an inventory of the CSF total protein levels for children in the eight academic children’s hospital in the Netherlands. Six hospitals used one of two sets of age-specific

reference values for the CSF total protein levels in children, with different age categories and

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cut-off levels for a raised protein levels, The other two used no age-specific reference values

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both with a different cut-off level.

In the current study, the Bhattacharya method was applied on the diagnostic test results of 6145 CSF samples collected from 3623 children aged 6 months to 18 years (median age 6 years, IQR 3-12 year) admitted at Sophia Children’s Hospital. Included were 2025 boys and 1598 girls. CSF total protein reference values could not be calculated in children aged

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younger than 6 months due to an insufficient number of CSF samples. The median CSF total protein concentration in the group of children older than 6 months was 0.19 g/L (IQR 0.15 -

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0.3). The calculated reference values based on the upper limit of the 95% CI (i.e. upper limit of normal) are: 6 months - 2 years 0.25 g/L, 2-6 years 0.25 g/L, 6-12 years 0.28 g/L, 12-18

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years 0.34 g/L (see table 1).

Excluding the samples from a traumatic LP and/or with a pleiocytosis did not

influence the observed upper limit of the 95% CI in each of the age categories used in the Bhattacharya analysis (data not shown). The reference values for CSF total protein level reported above were confirmed in a subgroup of children. This subgroup consisted of 378 children with a confirmed diagnosis that are not typically associated with increased CSF total protein level. Of this group 274 (72%) children had a neurological disorder and 104 (28%) children had a non-neurological disorder. Patients 6

ACCEPTED MANUSCRIPT with the following diagnoses were included in the neurological reference group: epilepsy, movement disorder, psychomotor retardation, previous history of asphyxia, papilledema with or without hydrocephalus, idiopathic intracranial hypertension, headache, vascular malformation, mitochondrial disorder, stroke, non-inflammatory polyneuropathy and other

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neurological disorders. Included in the non-neurological reference group were patients with the following diagnosis: non-CNS infectious diseases, psychiatric disorder, immunological disorders and other diagnosis (see supplementary table 2). Included were 184 (48.7%) girls

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and 194 (51.3%) boys. The median age was 3 years, (IQR: 0.49 – 9.94). The median CSF total protein concentration was 0.28 g/L, (IQR: 0.16 – 0.29, range: 0.08 – 2.25). The relation

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between the defined age-categories and protein concentration is shown in table 1 and figure 2. Median WBC was 3.0/µL, (IQR: 1.0 – 5.0, range: 0 - 46). There was a weak correlation between WBC and CSF total protein level with a correlation coefficient of 0.242 (p < 0.001). There was no relation between CSF total protein level and gender or diagnostic subgroup.

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The newly determined reference values for CSF total protein level were validated in a second subgroup of children with GBS, ADEM or MS. In 58 children with GBS, 6 (10.3%) patients had a traumatic LP and were excluded. RBC count was <500 in 20 liquor samples (34.5%)

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and was unknown in 32 (55.2%). Included were 52 children, of which 29 boys (55.8%). Median age was 5.0 years (IQR 3.0-10.0). Median protein concentration was 0.74 g/L (IQR:

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0.41-1.76, range 0.13-3.60). The ROC-analysis for GBS resulted in an AUC of 0.93 (95% CI 0.88 -0.98). The optimal CSF total protein cut-off was 0.315 g/L with a sensitivity of 85% and a specificity of 93% (Supplementary figure 1). A sensitivity analysis in which we only included patients with an established low RBC (n = 20) resulted in an AUC of 0.91 (95% CI 0.81-1.00). The optimum cut-off point remained at 0.315 g/L with a sensitivity of 85% and a specificity of 93%. Based on the new determined reference values for CSF total protein 85% of the children with GBS had an elevated protein level (See table 2).

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ACCEPTED MANUSCRIPT There were 44 CSF samples of ADEM patients, 3 were excluded because of a traumatic LP (6.8%). RBC count was unknown in 10 patients. Comparative analyses showed a significant difference between the group of patients with an atraumatic LP and with unknown RBC (p = 0.001). 41 patients were included (16 boys; 39.0%). Median age was 3.9

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years (IQR 2.7-5.9). Median protein concentration was 0.32g/L (IQR 0.24-0.47, range 0.132.90). The ROC-analysis resulted in an AUC of 0.81 (95% CI 0.73-0.89). The optimal CSF total protein cut-off was 0.235g/L with a sensitivity of 76% and a specificity of 78%

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(Supplementary figure 1). The sensitivity analysis, in which only patients without a traumatic LP were included, resulted in a AUC of 0.81 (95% CI 0.72-0.90). The optimum cut-off point

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was 0.265 with a sensitivity of 65% and a specificity of 88%. Based on our new determined reference values for CSF total protein 66% of the children with ADEM had an elevated protein level (see table 2).

CSF samples from 43 children diagnosed with MS were eligible for inclusion, 4

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(9.3%) traumatic lumbar punctures were excluded. RBC count was unknown in 13 liquor samples (30.2%). Comparative analyses showed a significant difference between the group of patients with an atraumatic LP and with unknown RBC (p = 0.018). Included were 39

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children of which 18 were boys (46.2%). Median age was 15.1 years (IQR 13.0-16.1). Median protein concentration was 0.28 g/L (IQR 0.24-0.34, range 0.16-0.75). The ROC-analysis for

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MS resulted in an AUC of 0.84 (95% CI 0.78-0.89). The optimal CSF total protein cut-off was 0.255g/L with a sensitivity of 72% and a specificity of 83%. The ROC-analysis in which only patients were included without a traumatic LP resulted in a AUC of 0.82 (95% CI 0.750.89). The optimal cut-off point was 0.1835 with a sensitivity of 96% and a specificity of 54%. Of the 39 children with MS 23% had an elevated protein level based on our new determined reference values (See table 2).

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ACCEPTED MANUSCRIPT The ROC analysis was also performed for the combined population of 145 patients with GBS, MS or ADEM. 13 patients were excluded due to a traumatic LP and in 55 patients RBC count was unknown. The ROC analyses in which we included all patients resulted in an optimum cut-off point of 0.265 (sensitivity 75%, specificity 85%). The AUC was 0.87(95%

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CI 0.83-0.91) (Supplementary figure 1). After excluding all patients in who RBC count was unknown the optimum cut-off point was 0.265 (sensitivity 69% and specificity 85%). The

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AUC was 0.84 (95%CI 0.78-0.89).

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4. Discussion

Currently there is no consensus on the use of age-specific reference values for protein levels in children. An inventory of the reference values for CSF total protein level for children used in the eight academic children’s hospitals in the Netherlands showed that four different reference values are used at present of which two are using adult reference values. Our study

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shows that the highest total protein levels and highest variation in total protein levels were observed in the first six months after birth. These high CSF total protein levels in neonates have previously been attributed to birth trauma, permeability of the immature brain barrier

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and the CSF flow rate.(17-19) We conclude that because of the large variation, CSF total protein

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level measurement has little value in diagnosing neurological diseases in this age category. After six months, the variation in CSF total protein level is considerably smaller, with the lowest values between the age of 2 and 6 years. From 6 to 18 years there is a gradual increase in CSF total protein level with age. The protein level at 18 years is still lower than the cut-off value that is used for adult patients in our center and other reference values described in the literature.(20, 21)Based on these results, we propose new reference cut-off values for children, which were validated in two independent sets of patient subgroups. These new reference values are more sensitive than those currently in use for diagnosing GBS, MS and ADEM in 9

ACCEPTED MANUSCRIPT children, although the CSF total protein level could not be used to differentiate between these neuro-immunological diseases. Studies in animal models showed a similar age-dependent effect on CSF total protein level as seen in humans. Both in rat and in sheep the turnover of CSF is higher in

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young then in older animals, which may increase the ability to clear proteins from the CSF and results in lower levels of protein in the CSF from young animals.(22), (23, 24) A similar

mechanism may explain the lower CSF total protein levels in children compared to adults.

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Our findings are in line with previous studies reporting lower CSF total protein levels in

children than in adults and a difference in these levels in different age categories in children

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(see supplementary table 1).(9-11) The references values reported by previous studies are close to the values we determined in the current study. An important limitation of previous studies however is that patients with neurological disorders were excluded and large age categories were used, which results in reference values that are less applicable to the patient population

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presenting with neurological signs or symptoms. To make our reference values clinically useful, we did include patients with neurological symptoms, used small age categories and applied two distinct methods of data analysis. Both methods resulted in very similar age-

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specific reference values for children. In the first analysis, an unselected and extensive set of 6145 CSF samples was analyzed using the Bhattacharya method. This method is frequently

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used by clinical chemists to validate diagnostic tests and provides the opportunity for each local center to define local cut-off values.(12) A possible disadvantage of this method is the absence of information on diagnosis, and the inclusion of cases with elevated CSF total protein levels, however we found that this inclusion had little influence on the generated reference values. In the second method, we defined reference values in a subset of 378 children with a confirmed diagnosis unrelated to increased CSF total protein levels, including neurological disorders. Here we found cut off values (95% CI) that were even lower than

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ACCEPTED MANUSCRIPT those determined using the first method. In the future, more investigation is necessary to confirm how these new reference values perform in clinical practice. The age-categories for which we identified reference values are frequently used in clinical pediatrics. They reflect the stages of development of the nervous system in infants,

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preschool children, school age children and adolescents. The period of maximal brain growth and myelination is up to 2 years. Preschoolers go through the common childhood infections, School age children have a mostly mature brain and spinal cord. The bodily changes of

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puberty separate adolescents from the children. The first results showed a large variation in CSF total protein concentration in the newborns, therefor we decided to create extra

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categories in the age range of 0 to 6 months old.

Based on the reference values determined in our study the CSF total protein level was elevated in 85% of GBS, 66% of ADEM, 23% of MS patients and 9% of the control group (see table 2). These new reference values are more sensitive to identify these inflammatory

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neurological diseases than the old reference values. The increase in the proportion of patients with an elevated CSF total protein level according to the new reference values in the ‘negative control group’ however also indicates that this CSF marker has limited specificity. Children

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with an elevated protein level in this group had different diagnosis including epilepsy, small fiber neuropathy, migraine, idiopathic intracranial hypertension and developmental delay of

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unknown cause. A lower specificity is accompanied with a higher risk of unnecessary additional tests. However we considered that the consequences of missing a diagnosis of a neuro-inflammatory disease usually outweigh the impact of an additional test on the child. The two most frequently used sets of diagnostic criteria for GBS developed by the NINDS (1992) and the Brighton collaboration (2011) respectively use an elevated protein level in CSF as a method to support the diagnosis.(3, 4) Such a diagnostic biomarker may be especially relevant in young children were the clinical examination is more complex and the

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ACCEPTED MANUSCRIPT diagnosis of GBS is frequently delayed.(2) Despite the fact that CSF total protein level is of importance in diagnosing GBS in children, the protein level can be affected by various factors such as the timing of the LP. The CSF may show normal protein levels, especially in patients who undergo the LP shortly after developing weakness.(6) The fact that current reference

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values show 20% of children with GBS to have normal CSF total protein levels could in part be related to the fact that the current reference values are too high, but also the patients with a normal protein in CSF had their LP shortly after onset of weakness (median time between

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onset of weakness and LP was 2,5 days). The diagnosis ADEM is based on clinical and

radiological features.(25, 26) In a majority of these patients analyses of CSF show a pleiocytosis

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and elevated CSF total protein. However, these factors are not included in the diagnostic criteria. In this study CSF total protein level was elevated in 66% of the ADEM patients. In the case of MS, the diagnoses is also based on clinical and radiological features and can be supported by specific CSF abnormalities, the presence of oligoclonal bands and elevated IgG

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index being the most important.(27, 28) An increase in CSF total protein has been described in only a minority of MS patients.(5, 8) The current diagnostic criteria for MS indicate that a markedly elevated CSF total protein is a “red flag” finding that suggest an alternative

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diagnosis should be considered.(29) Our study showed that these reference values for an elevated CSF total protein cannot be used to differentiate between these three neuro-

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inflammatory disorders. The CSF total protein level is only one of the diagnostic tests for these disorders and should be interpreted in light of the clinical symptoms and other diagnostic tests.

The specificity of the CSF analysis may increase when adding tests for albumin, immunoglobulin subclasses and clonality and/or other biomarkers,(30) not examined systematically in the current study. The CSF tests results used in the current diagnostic criteria for GBS, however, are only based on the total protein level and cell count. At present it is

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ACCEPTED MANUSCRIPT unknown if information on the CSF protein composition or blood- CSF barrier function would add to the accuracy of the diagnosis GBS. These additional tests in CSF more frequently are performed in patients suspected with a central demyelination disorder (like MS or ADEM).

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In conclusion, diagnostic value of CSF total protein level in children can be

improved by using validated age-specific reference values. Below the age of 6 months, CSF total protein levels are highly variable and have no diagnostic value for neuro-inflammatory

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disorders. For children over the age of 6 months, we recommend the use of the newly

developed age-specific reference values that are considerable lower than those currently used

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in clinical practice. Future studies are required to define if the usage of age-specific reference values in clinical practice result in a more accurate diagnosis. While these reference values are more sensitive for differentiating between some neuroinflammatory and nonneuroinflammatory disorders, a higher CSF total protein level cannot help to differentiate

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between the neuroinflammatory diseases: GBS and ADEM.

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ACCEPTED MANUSCRIPT Competing interests Drs. V. Kahlmann has no disclosures to report. Drs. J. Roodbol has no disclosures to report. N. van Leeuwen has no disclosures to report.

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C.R.B. Ramakers has received honoria from Roche Diagnostics for participating in a discussion panel and presenting at a conference. Drs. D. van Pelt has no disclosures to report.

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Dr. R.F. Neuteboom has no disclosures to report. Dr. C.E. Catsman-Berrevoets has no disclosures to report.

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Dr. M.C.Y. de Wit received honoraria paid to her institution by Novartis for serving on a steering committee and presenting at a conference, and has received research funding from the Nationaal Epilepsie Fonds (Dutch epilepsy fund), Hersenstichting and Sophia Foundation. Prof. dr. B.C. Jacobs has received research funding from the Netherlands Organization for

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Health Research and Development, Erasmus MC, Prinses Beatrix Spierfonds, Stichting

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Spieren voor Spieren, GBS-CIDP Foundation International, CSL-Behring and Grifols.

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ACCEPTED MANUSCRIPT References

6.

7. 8. 9.

10.

11.

12. 13. 14. 15.

16.

17.

18. 19.

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SC

5.

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4.

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3.

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2.

Schessl J, Koga M, Funakoshi K, et al. Prospective study on anti-ganglioside antibodies in childhood Guillain-Barre syndrome. Arch Dis Child. 2007; 92: 48-52. Roodbol J, de Wit MC, Walgaard C, de Hoog M, Catsman-Berrevoets CE, Jacobs BC. Recognizing Guillain-Barre syndrome in preschool children. Neurology. 2011; 76: 807-810. Asbury AK, Cornblath DR. Assessment of current diagnostic criteria for GuillainBarre syndrome. Ann Neurol. 1990; 27 Suppl: S21-24. Sejvar JJ, Kohl KS, Gidudu J, et al. Guillain-Barre syndrome and Fisher syndrome: case definitions and guidelines for collection, analysis, and presentation of immunization safety data. Vaccine. 2011; 29: 599-612. Dale RC, de Sousa C, Chong WK, Cox TC, Harding B, Neville BG. Acute disseminated encephalomyelitis, multiphasic disseminated encephalomyelitis and multiple sclerosis in children. Brain. 2000; 123 Pt 12: 2407-2422. Fokke C, van den Berg B, Drenthen J, Walgaard C, van Doorn PA, Jacobs BC. Diagnosis of Guillain-Barre syndrome and validation of Brighton criteria. Brain. 2014; 137: 33-43. Marchioni E, Tavazzi E, Minoli L, et al. Acute disseminated encephalomyelitis. Neurol Sci. 2008; 29 Suppl 2: S286-288. Pohl D, Rostasy K, Reiber H, Hanefeld F. CSF characteristics in early-onset multiple sclerosis. Neurology. 2004; 63: 1966-1967. Biou D, Benoist JF, Nguyen-Thi C, Huong X, Morel P, Marchand M. Cerebrospinal fluid protein concentrations in children: age-related values in patients without disorders of the central nervous system. Clin Chem. 2000; 46: 399-403. Renuart AJ, Mistry RD, Avery RA, et al. Reference range for cerebrospinal fluid protein concentration in children and adolescents. Arch Pediatr Adolesc Med. 2011; 165: 671-673. Wong M, Schlaggar BL, Buller RS, Storch GA, Landt M. Cerebrospinal fluid protein concentration in pediatric patients: defining clinically relevant reference values. Arch Pediatr Adolesc Med. 2000; 154: 827-831. Bhattacharya C. A simple method of resolution of a distribution into gaussian components. Biometrics. 1967; 23: 115-135. Oosterhuis WP, Modderman TA, Pronk C. Reference values: Bhattacharya or the method proposed by the IFCC? Ann Clin Biochem. 1990; 27 ( Pt 4): 359-365. Vogt MH, Bons JA, Janssen MJ, et al. A multicenter evaluation of dysthyroxinemia in a defined patient cohort. Clin Chem Lab Med. 2014; 52: 1153-1158. Hoffmann J, J,M,L., van den Broek N, M, A., Curvers J. Reference intervals of reticulated platelets and other platelet parameters and their associations. Arch Pathol Lab Med. 2013; 137: 1635 -1640. Schellenberg F, Wielders JP. Evaluation of capillary electrophoresis assay for CDT on SEBIA's Capillarys System: intra and inter laboratory precision, reference interval and cut-off. Clin Chim Acta. 2010; 411: 1888-1893. Berger R, Bender S, Sefkow S, Klingmuller V, Kunzel W, Jensen A. Peri/intraventricular haemorrhage: a cranial ultrasound study on 5286 neonates. Eur J Obstet Gynecol Reprod Biol. 1997; 75: 191-203. Reiber H. Proteins in cerebrospinal fluid and blood: barriers, CSF flow rate and source-related dynamics. Restor Neurol Neurosci. 2003; 21: 79-96. Saunders NR, Liddelow SA, Dziegielewska KM. Barrier mechanisms in the developing brain. Front Pharmacol. 2012; 3: 46.

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27.

28.

29. 30.

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25.

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24.

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23.

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22.

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Lott JA, Warren P. Estimation of reference intervals for total protein in cerebrospinal fluid. Clin Chem. 1989; 35: 1766-1770. Tibbling G, Link H, Ohman S. Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scand J Clin Lab Invest. 1977; 37: 385-390. Chen CP, Chen RL, Preston JE. The influence of cerebrospinal fluid turnover on agerelated changes in cerebrospinal fluid protein concentrations. Neurosci Lett. 2010; 476: 138-141. Serot JM, Bene MC, Faure GC. Choroid plexus, aging of the brain, and Alzheimer's disease. Front Biosci. 2003; 8: s515-521. Preston JE. Ageing choroid plexus-cerebrospinal fluid system. Microsc Res Tech. 2001; 52: 31-37. Ketelslegers IA, Catsman-Berrevoets CE, Neuteboom RF, et al. Incidence of acquired demyelinating syndromes of the CNS in Dutch children: a nationwide study. J Neurol. 2012; 259: 1929-1935. Ketelslegers IA, Neuteboom RF, Boon M, Catsman-Berrevoets CE, Hintzen RQ, Dutch Pediatric MSSG. A comparison of MRI criteria for diagnosing pediatric ADEM and MS. Neurology. 2010; 74: 1412-1415. Krupp LB, Tardieu M, Amato MP, et al. International Pediatric Multiple Sclerosis Study Group criteria for pediatric multiple sclerosis and immune-mediated central nervous system demyelinating disorders: revisions to the 2007 definitions. Mult Scler. 2013; 19: 1261-1267. van Pelt ED, Neuteboom RF, Ketelslegers IA, et al. Application of the 2012 revised diagnostic definitions for paediatric multiple sclerosis and immune-mediated central nervous system demyelination disorders. J Neurol Neurosurg Psychiatry. 2014; 85: 790-794. Rubin JP, Kuntz NL. Diagnostic criteria for pediatric multiple sclerosis. Curr Neurol Neurosci Rep. 2013; 13: 354. Reiber H. Flow rate of cerebrospinal fluid (CSF)--a concept common to normal bloodCSF barrier function and to dysfunction in neurological diseases. J Neurol Sci. 1994; 122: 189-203.

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20.

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ACCEPTED MANUSCRIPT Table 1: CSF total protein levels (g/L) in children and proposed reference values Derivation cohort (N=6145)1

Age

Validation cohort with confirmed diagnosis (N=378)2

95% CI (N)

Median

95 % CI (N)

(g/L)

(g/L)

(g/L)

(g/L)

0–1 wk

-

-

0.76

1–4 wk

-

-

0.54

1–3 m

-

-

0.28

3–6 m

-

-

0.20

*

0.18

0.18 – 0.22 (73)

*

0.17

0.16 – 0.18 (76)

0.06 – 0.25 (925)

0.69 – 0.97 (32) 0.49 – 0.67 (18)

0.27 – 0.37 (20) 0.21 – 0.31 (25)

SC

6m-2 yrs

0.21

RI PT

Median

0.17

0.05 – 0.25 (1896)

6–12 yrs

0.19

0.05 – 0.28* (1725)

0.17

0.17 – 0.21 (53)

12–18 yrs

0.24

0.06 – 0.34* (1599)

0.21

0.21 – 0.25 (81)

1 2

M AN U

2–6 yrs

Based on diagnostic CSF samples in which reference values were determined by Bhattacharya method.

(12)

Based on diagnostic CSF samples from patients who were diagnosed with disorders not associated with

TE D

increased CSF total protein levels.

CI = confidence interval, N = number of infants, wk = weeks, m = months, yrs = years *

= The values in bold are the upper limit of the 95% CI and the cut-off values for an elevated protein level in that

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EP

age category.

17

ACCEPTED MANUSCRIPT Table 2: Sensitivity and specificity of the currently used and new reference values for CSF total protein in the three disease groups. GBS (n= 52) ADEM (n=41) MS (n=39) Sensitivity

81 (68 - 90)

46 (31 - 63)

10 (3 - 24)

(%, 95% CI)

Specificity 97 (94 - 99)

97 (94 - 99)

97 (94 - 99)

New reference values (Bhattacharya

Sensitivity

85 (72 - 93)

66 (49 - 80)

23 (11 - 39)

method) (%, 95% CI)

Specificity 91 (87 - 94)

91 (87 - 94)

91 (87 - 94)

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EP

TE D

M AN U

SC

RI PT

Currently used reference values

18

ACCEPTED MANUSCRIPT Figure 1: Age-related protein levels in diagnostic CSF samples from children (N=6145).

250

100

200

Number of patients

Number of patients

80 60 40 20

150 100 50 0

M AN U

0.05 0.13 0.21 0.29 0.37 0.45 0.53 0.61 0.69 0.77 0.85 0.93 1.00

0.04 0.12 0.20 0.28 0.36 0.44 0.52 0.60 0.68 0.76 0.84 0.92 1.00

0

Protein concentration g/L

Protein concentration g/L

C. Children aged 6-12 years

200

100 80

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60 40

120 100 80 60 40 20

0

0 0.03 0.11 0.19 0.27 0.35 0.43 0.51 0.59 0.67 0.75 0.83 0.91 1.00

20

0.02 0.10 0.18 0.26 0.34 0.42 0.50 0.58 0.66 0.74 0.82 0.90 1.00

Number of patients

120

140

Number of patients

140

TE D

160

D. Children aged 12-18

160

EP

180

SC

120

RI PT

B. Children aged 2-6 years

A. Children aged 0.5-2

Protein concentration g/L

Protein concentration g/L

19

ACCEPTED MANUSCRIPT Figure 2: Age-related protein levels in CSF samples from children with a confirmed diagnosis (N=378)

RI PT

1.5

yr s

yr s

2

-1 8

12

yr s

-1

-6

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6

m

-2

yr s

m -6

m 3

-3 1

-4 1

0

-1

w

w k

k

0.0

6

0.5

M AN U

SC

1.0

2

Protein concentration g/L

2.5

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EP

Age

20

ACCEPTED MANUSCRIPT Supplementary table 1: CSF total protein levels in children reported in previous studies.

Previous studies

Number of

Age

children

Biou et al, 2000(9)

(g/L)

(g/L)

92

1 – 9 yr

0.22

0.22 ± 0.08

118

10 – 18 yr

0.33

0.33 ± 0.1

99

0–2m

0.60

0.59 ± 0.21

36

2- 4 m

0.37

0.37 ± 0.15

77

4 m -14 yrs

0.15

0.17 ± 0.06

13

14-18 yrs

0.28

0.26 ± 0.06

26

1–8d

0.71

-

76

8 – 30 d

0.59

-

1–2m

0.47

-

2–3m

0.35

-

3–6m

0.23

-

6 m – 10 yrs

0.18

-

10 - 16 yrs

0.22

-

155 115 66 599 37

RI PT

Wong et al, 2000(11)

Mean ± SD

SC

2011

(10)

M AN U

Rennuart et al,

Median

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EP

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d = day(s), wk, = week(s), m = month(s), yr = year(s).

21

ACCEPTED MANUSCRIPT Supplementary table 2: Diagnosis of patients in the subgroup analysis.

N=274Nnnhgnjf N= 274 101

Movement disorder

9

Psychomotor retardation

56

Previous history of Asphyxia

7

Stroke

14

Non inflammatory polyneuropathy

3

Mitochondrial disorder

9

SC

Epilepsy

Vascular malformation

4

12

Hydrocephalus

M AN U

Headache

RI PT

Neurological reference group

5

Idiopathic intracranial hypertension

15

Papilledema with or without hydrocephalus

6

Other neurological diagnosis

33

Non-neurological reference group

N=104

69

Psychiatric disorder

12

Immunological disorders

5

Other diagnosis

18

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EP

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Non CNS infectious diseases

22

ACCEPTED MANUSCRIPT Supplementary figure 1: ROC analysis of CSF total protein concentration in children with GBS, ADEM and MS. ADEM

GBS/ADEM/MS

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EP

TE D

MS

M AN U

SC

RI PT

GBS

23

ACCEPTED MANUSCRIPT Highlights CSF protein levels in children are normally lower than in adults.

•

Improved age-dependent reference values for CSF protein in children were defined.

•

New reference values increase sensitivity to diagnose pediatric GBS, ADEM and MS.

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EP

TE D

M AN U

SC

RI PT

•