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Normal Values for Cerebrospinal Fluid in Neonates: A Systematic Review

Background: The diagnosis of neonatal meningitis often rests on microscopic and biochemical findings in the cerebrospinal fluid (CSF). There is ongoing uncertainty about age-related normal values for CSF findings in neonates, and many previous studies have included infants in whom antibiotics were administered before lumbar puncture or in whom viral meningitis was not excluded. Methods: A systematic search was done using MEDLINE and EMBASE to identify original studies which investigated CSF normal values in either healthy neonates or febrile neonates in whom bacterial and viral meningitis were reliably excluded. Results: We identified seven studies investigating 270 term and 96 preterm neonates. There were minimal differences between preterm and term neonates in the CSF white blood cell (WBC) count and glucose concentration. In contrast, the CSF neutrophil count and protein concentration were influenced by gestational and chronological age. In the four studies that reported individual patient data, in 95% of cases the CSF WBC count was <12 cells/μL in preterm and <10 cells/μL in term neonates, the neutrophil count was <16 and 8 cells/μL, and the protein concentration was <210 and 110 mg/dL, respectively. Conclusion: The normal range for CSF parameters in neonates is different to that in older infants, and some parameters are influenced by gestational and chronological age. CSF parameters alone are not sufficiently reliable to exclude meningitis.


Introduction
The gold standard for diagnosing bacterial meningitis is the isolation of pathogens in the cerebrospinal fluid (CSF). However, the diagnosis often depends on the microscopic and biochemical findings in the CSF, including a Gram stain, white blood cell (WBC) count, and differential, together with protein and glucose concentrations. This is also the case when antibiotics are administered before lumbar puncture (LP). Even with all these parameters available, it is often difficult to diagnose meningitis and to differentiate bacterial from aseptic meningitis. Furthermore, there is ongoing uncertainty about age-related normal values of CSF parameters, especially in neonates. The blood-brain barrier in neonates differs to that in older children [1], and normal values in infants, particularly neonates, are therefore different from those in older children. Age-related normal values of CSF findings have previously been reviewed [2]. However, many studies included children in whom antibiotics were given be- Zimmermann fore LP or in whom viral meningitis was not excluded reliably [3][4][5][6][7]. Also, additional studies have been published in recent years. In this systematic review, we summarize studies which investigated CSF parameters in healthy neonates, including those born preterm, using stringent inclusion criteria to define normal neonatal CSF values more reliably.

Systematic Review Methods
To identify studies which investigated CSF normal values in healthy neonates or febrile neonates in whom meningitis was reliably excluded, MEDLINE (1946 to present) and EMBASE (1947 to present) were searched in August 2020 using the Ovid interface with the following search terms: (cerebrospinal fluid [analysis, chemistry, cytology, diagnosis, microbiology] OR meningitis [cerebrospinal fluid, diagnosis] OR spinal puncture OR lumbar puncture) AND (infant OR neonat* OR newborn) without any language limitations. Exclusion criteria were studies that: (i) did not include molecular diagnostics to exclude enterovirus infection; (ii) included neonates in whom antibiotics were administered before LP; (iii) included neonates with underlying disease; (iv) included neonates with CSF shunts, and (v) included neonates in whom CSF was collected by a route other than LP. References of retrieved articles were hand-searched for additional publications.
The following variables were extracted from the included studies: year of study, country, study design, number, and characteris-tics of participants and key findings including CSF WBC and CSF glucose and protein concentrations. Statistical analyses and graphs were done using R version 3.4.3. The Mann-Whitney U test was used to compare values between groups.

Systematic Review Results
Our search identified 1,530 studies. Of these, nine fulfilled the inclusion criteria ( Fig. 1) [8][9][10][11][12][13][14][15][16]. No additional relevant studies were identified by hand searching of references. Two studies reported the same group of neonates, one the cell counts and the other the biochemical findings, considered hereon in as one study [8,9]. Two other studies reported the same findings in different languages [10,11]. Therefore, 7 studies were included in the review, all of which were case series. These reported the results from 270 term (in 5 studies) [12][13][14][15][16] and 96 preterm infants (in 2 cohorts) [8][9][10][11]. The number of participants in each study ranged from 19 to 108 (mean 52, median 43). The studies were done in Brazil 4, USA 2, and Spain 1. The results of the studies are summarized in Table 1.

WBC Count
The two studies that investigated the CSF WBC count in preterm neonates, reported a range from 0 to Calculated from reported data. 23.0 cells/μL in the first week of life and from 1.0 to 13.0 cells/μL at 1 month of age [8,10]. In one of the studies in preterm neonates, the CSF WBC count decreased from a mean of 7.6 (range 1.0-23.0) cells/μL on day 7 of life to a mean of 3.9 (range 1.0-13.0) cells/μL on day 28 of life [8]. The other study in preterm neonates reported a mean of 4.0 (range 0-16.0) cells/μL on day 2 of life [10]. Four studies reported the CSF WBC count in term infants. This ranged from 0 to 130.0 cells/μL (though this upper value was deemed potentially indicative of meningitis by the authors) with means ranging from 1.5 to 7.3 cells/μL [12,[14][15][16]. There was no difference between the CSF WBC count on day 2 and 7 (p = 0.96) [15]. In one study there were 2 term neonates with very high WBCs (130.0 and 62.0 cells/μL) [14]. Apart from these two neonates, the highest WBC count in a term neonate was 28.0 cells/μL [14]. Table 2 and Figure 2 summarize the CSF WBC count for individual neonates in the four studies in which these data were reported [10,12,15,16].
The two studies which reported individual neutrophil values are summarized in Table 2 and Figure 3

Protein Concentration
Four studies (one in preterm neonates) investigated the protein concentration in CSF [10,[13][14][15]. The protein concentration was higher in preterm (n = 53) compared with term neonates (n = 79) (mean 144.6 mg/dL, range 52.0-300.0 vs. mean 61.0-71.4 mg/dL, range 8.0-140.0). In term neonates, no difference was found between the protein concentration on day 2 and day 7 of life (p = 0.66) in one study [15], while another found a higher protein concentration in week 1 and 2 compared to week 3 and 4 (p < 0.05) [14]. In the study in preterm neonates, there was no correlation between the red blood cell (RBC) count and protein concentration; therefore, the observed increase in CSF protein cannot be attributed to a higher RBC count (p = 0.21) [10].
The two studies which reported individual values are summarized in Figure 4

Discussion
The incidence of bacterial meningitis is highest in neonates, where it causes substantial morbidity and mortality. Despite an extensive literature review, we only found seven studies (reported in nine articles) investigating normal CSF values that were of sufficient quality to be included in this systematic review. The main reason for exclusion of studies was the absence of viral diagnostics to exclude aseptic meningitis (see online suppl. Table 1; see www. karger.com/doi/10.1159/000517630 for all online suppl. material) [5][6][7]. Even when viral culture is done, up to 35% of enteroviruses are not detected [39][40][41][42], and therefore polymerase chain reaction is necessary to reliably exclude viral meningitis. A further common exclusion reason was antibiotic administration before LP which can lead to false negative cultures and inclusion of neonates with meningitis [3,4,43,44]. The degree to which antibiotic administration changes CSF parameters is still unknown. One study found that protein concentrations were significantly higher in neonates who had received antibiotics before LP than those who had not (median 92 vs. 80 mg/dL, p = 0.02) but CSF WBC and glucose concentration did not differ between the two groups [3]. Comparing our findings with studies which included neonates who had received antibiotics before LP, the values for CSF glucose and protein concentrations (median and 95th percentile) and the median CSF WBC count in preterm and term infants were similar. However, the 95th percentile for CSF WBC was lower (12 vs. 16 cells/μL in term infants and 9 vs. 12 cells/μL in preterm term infants, respectively; and 16 and 78 cells/μL in the studies which did not specify separate values for preterm and term infants) [3,43,44].
Many guidelines or reviews recommend a cutoff value of 20-22 CSF WBCs/μL with a CSF neutrophil count of <2-8 cells/μL for the diagnosis of meningitis in neonates [2,[45][46][47]. However, these recommendations are based on studies that included a proportion of patients who had been administered antibiotics before LP [43,44] or which did not reliably exclude viral meningitis [6,7,19,26,28]. Consistent with this, a large study of neonatal meningitis including 9,111 neonates reported that a CSF WBC count of >21 cells/μL has a sensitivity of 79% and specificity of 81% for bacterial meningitis [48] and another study, including 3,467 neonates, reported a 95th percentile of 16.0 cells/μL and protein 118 mg/dL [43] (both studies did not meet the criteria for our review). In our review, we found that in 95% of cases, the CSF WBC count was <12.0 cells/ μL in preterm and <9.2 cells/μL in term neonates, and the CSF neutrophil count <16.0 and 8.0 cells/μL, respectively. However, these values are derived from only the 4 studies for which individual patient data were reported. Even though it has previously been claimed that the presence of even a single polymorphonuclear leucocyte in CSF is pathological, the data from the studies included in our review suggests this might not be the case in neonates.  CSF glucose and protein concentrations are highly variable in neonates [6,7,43,48]. However, a CSF protein concentration of <150.0 mg/dL in preterm and <100.0 mg/dL in term neonates, and a CSF glucose concentration of >30.0-36.0 mg/dL is often considered as normal [45,46]. In our review, in 95% of cases the protein concentration was <210.0 mg/dL in preterm neonates and 110.2 mg/dL in term neonates, respectively. In preterm neonates the 5th percentile for the CSF glucose concentration was 32.0 mg/dL, while there were not enough individual data to determine percentiles for term neonates. These values are also only derived from only the 3 studies for which individual patient data were reported.
We found minimal difference in the CSF WBC count or glucose concentration between preterm and term neonates [8][9][10][11][12][14][15][16]. However, the CSF neutrophil count and protein concentration were higher in preterm neonates than in term neonates [8,10,11,[13][14][15][16]. Consistent with this, one of the studies done in term neonates which analysed consecutive samples reported that there was no difference in the WBC count and glucose concentration between the first and the fourth week of life, but the protein concentration was higher in the first than in the fourth week [14]. This is consistent with animal studies, which found increased concentration of growth factors and other proteins in the CSF of preterm compared to term animals [49]. However, it is often stated that this difference is due to increased permeability of the blood-brain barrier in preterm infants [50], this remains unproven. Animal studies suggest that the blood-CSF tight junctions are functionally mature from early life and that proteins are actively transferred transcellularly [51][52][53].
Even a small amount of blood or bilirubin in CSF can significantly alter the protein concentration [54,55]. In neonates, it has been estimated that 1,000 RBCs/μL raises the protein concentration by 1.5-1.9 mg/dL [56,57]. However, in the study included in this review, there was no correlation between the RBC count and protein concentrations [10]; therefore, the observed increase in CSF protein is not attributable to a higher RBC count.
Unfortunately, none of the studies included in this review measured CSF lactate. Lactate is produced by anaerobic bacteria and not influenced by blood lactate concentration, which is an advantage compared to the CSF glucose concentration. A meta-analysis summarizing results from 30 studies in children and adults suggested that a CSF lactate concentration of 35 mg/dL could be used as a cutoff value to discriminate between viral and bacterial meningitis [58].
As we have previously shown, it is important to note that an initial CSF with a normal WBC count does not definitively exclude bacterial meningitis [59]. On the other hand, pleocytosis can be found in neonates without central nervous system infection, but with other infections, such as gastroenteritis [12] or urinary tract infections [60,61].
The main limitation of our review is that there were only a small number of studies of sufficient quality to include. However, the use of stringent inclusion criteria means the normal neonatal CSF values determined in our review are likely to be more reliable. Another potential limitation is that not all the studies included in our review used molecular diagnostics to exclude viruses other than enterovirus (e.g., parechovirus or herpes virus) as a cause of neonatal meningitis and that some studies included neonates with a high RBC count. The paradoxical finding in preterm infants of a higher upper limit of normal for CSF neutrophils than for WBC cells (16 vs. 12/µL) is a result of the small number of studies that provided differential CSF WBC counts. Furthermore, the small number of studies only enabled the influence of gestational age to be compared between preterm and term infants.

Conclusion
The interpretation of CSF in neonates can be challenging. The normal range for CSF parameters is different in neonates compared with older infants. Gestational and chronological age lead to only minor differences in CSF WBC and glucose concentrations, but have a greater influence on the CSF neutrophil count, RBC count, and protein concentration. CSF parameters alone are not sufficiently reliable to exclude meningitis. In clinical practice, CSF samples, regardless of cellularity and biochemistry, need to be subjected to culture and viral molecular diagnostics to reliably exclude meningitis.

Statement of Ethics
The paper is exempt from Ethical Committee approval as it is a review of previously published literature.