Abstract
OBJECTIVE: Cerebrospinal fluid (CSF) white blood cell (WBC) counts for neonates and young infants are usually interpreted on the basis of values reported in reference texts or handbooks; however, current reference texts either present normal CSF parameters without citation or cite studies with significant limitations. The objective of this study was to determine accurate, age-specific reference values for CSF WBC counts in a large population of neonates and young infants.
METHODS: This cross-sectional study included patients who were aged ≤56 days and had a lumbar puncture performed in the emergency department from January 1, 2005, to June 30, 2007. Patients were excluded from analysis for conditions that are suspected to cause CSF pleocytosis, including traumatic lumbar puncture, serious bacterial infection, congenital infection, seizure, and presence of a ventricular shunt. Children who tested positive for enterovirus (EV) in the CSF by polymerase chain reaction were also excluded. Two-sample Wilcoxon rank-sum tests were used to compare median CSF WBC values of those who had negative EV testing with those who did not have EV testing.
RESULTS: A total of 380 (36%) of 1064 patients met inclusion criteria; 54% were male, 15% were preterm, and 39% presented during EV season. The median CSF WBC count was significantly higher in infants who were aged ≤28 days (3/μL, 95th percentile: 19/μL) than in infants who were aged 29 to 56 days (2/μL, 95th percentile: 9/μL; P < .001). In both age groups, infants with a negative EV PCR had a higher upper bound of the 95% confidence interval of the mean values compared with infants who did not have EV testing performed.
CONCLUSIONS: We determined age-specific CSF WBC reference values in a large cohort of neonates and young infants that can be used to interpret accurately the results of lumbar punctures in this population.
WHAT'S KNOWN ON THIS SUBJECT:
CSF reference values for neonates and young infants are commonly reported in textbooks of generalist, hospitalist, and subspecialty pediatrics. Previous studies contained important methodologic limitations, and most were conducted before the widespread use of molecular tools.
WHAT THIS STUDY ADDS:
This study provides age-specific CSF WBC count reference values that can be used to interpret accurately results of lumbar puncture. The 95th percentile value is 19/μL for infants aged ≤28 days and 9/μL for infants aged 29 to 56 days.
Lumbar puncture and analysis of cerebrospinal fluid (CSF) aids clinicians in identifying patients with meningitis or encephalitis and, therefore, requires knowledge of accurate reference values for white blood cell (WBC) counts. Determining normal CSF values for young infants and neonates is challenging. It is unethical to evaluate healthy children with a painful, potentially harmful procedure at a time when they cannot provide either written consent or verbal assent; therefore, reference values must be based on children who are not truly normal; generally, values are determined from infants who undergo lumbar puncture for suspicion of meningitis.
Reference literature is widely available to guide clinicians' interpretation of the results of a lumbar puncture. Textbooks and handbooks of general pediatrics,1,–,3 infectious diseases,4,5 hospital medicine,6,–,8 emergency medicine,9,10 neonatology,11 and neurology12,13 outline normal CSF parameters. These parameters are based on other reference texts or on studies with important limitations. Previous studies determined reference values on the basis of children who were considered healthy after initial evaluation for infection of the central nervous system (CNS); however, few patients aged ≤56 days have been studied, and different ranges of reference values have been established because authors have used varying exclusion criteria. These past studies included children with traumatic lumbar puncture,14,–,20 seizures,17 sepsis,21 congenital infections,19,22 and very low birth weights.23
Most previous work was also conducted at a time when molecular tools were not routinely used in clinical practice.15,–,26 Polymerase chain reaction (PCR) tests to detect viral genomes allow more accurate identification of neonates and young infants who have infection. Only 1 study excluded children with CSF positive for enterovirus (EV) by PCR.27 The objective of our study was to determine clinically relevant CSF WBC reference values in a large cohort of children aged ≤56 days with strict exclusion criteria and the incorporation of results of PCR tests.
METHODS
Study Design and Setting
This cross-sectional study was performed at the Children's Hospital of Philadelphia (Philadelphia, PA), an urban, tertiary care children's hospital. The Committees for the Protection of Human Subjects of the Children's Hospital of Philadelphia approved this study with a waiver of informed consent.
Review of Literature
A systematic review of literature defining normal CSF WBC counts was conducted through an Ovid Medline search of all work published before September 1, 2008. The initial search term was “cerebrospinal fluid,” which was then combined with “white blood cell” or “leukocytosis” and either “neonate,” “infant,” or “newborn.” Limits included “humans” and “English language.” Titles and abstracts of 300 articles were reviewed for relevance: 5 studies were found to be relevant.17,21,23,25,27 Nine additional studies were identified during review of the references of textbooks and published studies.14,–,16,18,–,20,22,24,26
Study Population
Infants who were aged ≤56 days were eligible for inclusion when they had a lumbar puncture performed as part of their emergency department (ED) evaluation between January 1, 2005, and June 30, 2007. Children in this age range were selected because they routinely undergo lumbar puncture when presenting with fever at our institution.28,29 Patients who were undergoing lumbar puncture in the ED were identified by using 2 data sources to ensure accurate identification of all eligible infants: (1) ED computerized order entry records identified all infants with CSF testing (including CSF Gram-stain, culture, cell count, glucose, or protein) performed during the study period and (2) clinical virology laboratory records identified all infants on whom CSF herpes simplex virus or EV PCR testing was performed. Medical records of infants who were identified by these 2 sources were reviewed for determination of study eligibility.
Figure 1 outlines major exclusion criteria that were used to derive the reference group. Patients were excluded sequentially when the lumbar puncture was traumatic or a condition that is known or suspected to cause CSF pleocytosis was present. In a traumatic lumbar puncture, the presence of red blood cells (RBCs) in the CSF alters WBC counts, and adjustment formulas cannot reliably approximate the actual values.30,–,33 Conditions that are known or suspected to cause CSF pleocytosis include stroke, hydrocephalus, seizure on presentation, ventricular shunt or previous intracranial infection, congenital infection, herpes simplex virus meningoencephalitis, and bacterial meningitis.34,–,36 Patients with serious bacterial illness including bacteremia, urinary tract infection, osteomyelitis, septic arthritis, pneumonia, and bacterial gastroenteritis were also excluded because studies have identified CSF pleocytosis with non-CNS infections.36,–,38 Infants may have met >1 of the exclusion criteria.
Infants who were aged ≤56 days and presented to an ED with an indication for lumbar puncture and indication for exclusion from the study population.
The remaining infants were categorized on the basis of whether testing for EV was performed in the CSF by PCR and, when performed, whether the test result was positive or negative. Details of our approach to EV PCR testing have been published previously.39 Because viral meningitis can cause CSF pleocytosis, patients with a positive CSF EV PCR result were excluded from the reference group.40,41 Whereas previous studies have examined preterm infants separately from term infants, CSF WBC counts are influenced by postnatal rather than postgestational age.42 Our primary analysis, therefore, combined preterm and term infants into a single group.
Study Definitions
Preterm was defined as a gestational age of <37 weeks. Fever was defined as presence of temperature of ≥38.0°C. “History of fever” was defined as report of tactile temperature or recorded temperature >38.0°C at home. Traumatic lumbar puncture was defined as the presence of ≥500 RBCs/μL in the CSF.43 Seizure was defined by clinical description of the event within 48 hours of presentation or by an electroencephalogram demonstrating epileptiform activity. Bacterial meningitis was defined as the isolation of a bacterial pathogen from the CSF. Because CSF sterilization may occur after antibiotic administration,44 when antibiotics were administered before lumbar puncture, bacterial meningitis was defined as bacteria on CSF Gram-stain with low CSF glucose or elevated CSF protein values. Bacteremia was defined as isolation of a known bacterial pathogen from blood culture; isolates that reflected commensal skin flora such as coagulase-negative staphylococci were considered contaminants.
Urinary tract infection was defined as growth of a single known pathogen that met 1 of 3 criteria: (1) ≥1000 colony-forming units (CFU)/mL for urine cultures that were obtained by suprapubic aspiration; (2) ≥50 000 CFU/mL from a catheterized specimen; or (3) ≥10 000 CFU/mL from a catheterized specimen in association with a positive urinalysis result.45 Positive urinalysis result was defined as ≥1 of the following: (1) trace or greater result for leukocyte esterase and/or nitrite on dipstick; (2) >9 WBC per high-power field on standard microscopic examination of centrifuged urine; or (3) >10 WBCs/μL by hemocytometer count of uncentrifuged urine.46,47
Osteomyelitis was defined as the growth of pathogenic bacteria from blood, bone, or subperiosteal aspirate culture in a patient with fever and localized tenderness, edema, or erythema overlying the suspected site of bony infection and compatible radiologic findings. Septic arthritis was defined as the growth of pathogenic bacteria from synovial fluid or blood culture in a patient with purulent joint fluid or positive results of Gram-staining of joint fluid.48 Bacterial pneumonia was defined as a new, discrete infiltrate on chest radiograph with growth of a respiratory bacterial pathogen from blood culture.28 EV season was defined as June 1 through October 31 in each year.
Data Collection
Data were abstracted from the medical records of study patients and entered onto a standardized data collection form. Information collected included demographics, vital signs, clinical and historical findings on presentation, birth history, empiric antibiotic use, imaging studies performed, and results of laboratory testing.
Data Analysis
Data were analyzed by using Stata/SE 10 (Stata Corp, College Station, TX). The primary variable considered was CSF WBC count. Continuous variables were described by using mean, median, interquartile range (IQR), and 90th and 95th percentile values and compared by using the Wilcoxon rank-sum test.
RESULTS
Of 1064 infants identified for the study, 380 (36%) met inclusion criteria (Fig 1). Most (54%) patients were male; 15% were preterm, 39% presented during EV season, and 80% had fever on presentation. Forty-three (11.3%) infants received antibiotics before lumbar puncture. The median length of hospital stay was 2 days (Table 1).
Characteristics of Eligible Infants
Infants who were aged 0 to 28 days had a median CSF WBC of 3/μL with a 95th percentile value of 19/μL, whereas infants who were aged 29 to 56 days had a statistically lower median CSF WBC count of 2/μL with a 95th percentile value of 9/μL (P < .001; Table 2). Within age groups, we subsequently compared patients who tested negative for EV by PCR with those who did not have EV PCR testing performed. In the infants who were aged 0 to 28 days, those who had negative CSF EV PCR had a median CSF WBC of 4/μL (Table 3). This value was significantly higher than for those who did not have EV testing performed either within or outside EV season (Fig 2A). In the infants who were aged 29 to 56 days, those who had their CSF tested for EV and had a negative result had a median CSF WBC of 2.5/μL (Table 3). This value was higher than the median CSF WBC count of those who did not have EV testing performed within EV season, although the differences were not statistically significant (Fig 2B). In both age ranges, there were no significant differences between infants who were evaluated within or outside EV season when EV CSF PCR testing was not performed.
CSF WBC Counts
CSF WBC Counts Stratified by EV Testing and Season for Patients Aged 0 to 28 Days and 29 to 56 Days
(A) Patients who were aged 0 to 28 days and stratified by CSF enterovirus (EV) PCR status and season. Group I represents neonates who tested negative for EV by PCR during all seasons. Group II represents neonates who were not tested for EV during EV season. Group III represents neonates who were not tested for EV outside EV season. On each box plot, the box spans the interquartile range (IQR), the horizontal line through the box is the median value, and the whiskers denote outlying values, defined by convention as 1.5 IQRs lower than the first quartile and 1.5 IQRs higher than the third quartile. Dots represent individual patient values that fall beyond 1.5 IQRs. Data from 2 patients with CSF WBC values of 320/μL and 198/μL are not shown for clarity of presentation. (B) Patients who were aged 29 to 56 days and stratified by CSF EV PCR status and season. Group I represents infants who tested negative for EV by PCR during all seasons. Group II represents infants who were not tested for EV during EV season. Group III represents infants who were not tested for EV outside EV season. On each box plot, the box spans the interquartile range (IQR), the horizontal line through the box is the median value, and the whiskers denote outlying values, defined by convention as 1.5 IQRs lower than the first quartile and 1.5 IQRs higher than the third quartile. Dots represent individual patient values that fall beyond 1.5 IQRs. Data from 2 patients with CSF WBC values of 34/μL and 43/μL are not shown for clarity of presentation.
We also compared CSF WBC counts between infants with and without fever. Among infants who were aged ≤28 days, differences in the median CSF WBC count values between infants with fever (median: 3/μL; 95th percentile: 19/μL) and without fever (median: 4/μL; 95th percentile: 18/μL) were not statistically significant (P = .27, Wilcoxon rank-sum test). Among infants who were aged 29 to 56 days, the median CSF WBC count was lower in infants with fever (median: 2/μL; 95th percentile: 9/μL) compared with infants without fever (median: 3/μL; 95th percentile: 20/μL; P = .04, Wilcoxon rank-sum test), although the latter group included only 25 infants.
Several infants had modestly elevated CSF WBC counts without an identifiable cause. A patient with a CSF WBC count of 320/μL did not have sufficient CSF obtained for EV PCR but had a mononuclear cell predominance (ie, 40% lymphocytes and 33% monocytes) and was discharged with a diagnosis of probable aseptic meningitis. A patient with a CSF WBC count of 198/μL had a negative CSF EV PCR result and was treated for presumed bacterial meningitis even though all cultures were negative. Two patients with CSF WBC counts of 43/μL and 34/μL had negative CSF EV PCR and were discharged from the hospital after 2 days.
The 57 preterm infants had a median CSF WBC count of 3/μL. Those aged 0 to 28 days (n = 22) had a median CSF WBC count of 3/μL and a 95th percentile of 19/μL, which was not statistically different (P = .58, Wilcoxon rank-sum test) than the comparable term group. Those aged 29 to 56 days (n = 35) had a median CSF WBC count of 2/μL with a 95th percentile of 7/μL, which was not statistically different from term 29- to 56-day-old infants (P = .43, Wilcoxon rank-sum test).
DISCUSSION
Our study establishes reference values for CSF WBC counts in neonates and young infants to bring literature up to date at a time when molecular tools are commonly used in clinical practice. Previous studies have based reference ranges on infants with an indication for a lumbar puncture because it is not ethical to test healthy infants; however, those studies had small sample sizes and included patients with conditions that often are associated with CSF pleocytosis, including traumatic lumbar puncture, seizure, and congenital infections.
Our study was designed to address the limitations of previous studies that are frequently cited by reference texts. Sarff et al18 enrolled 87 term children who were younger than 10 days; although all of the infants had sterile urine, blood, and CSF, the exclusion criteria for a traumatic lumbar puncture was defined as “gross blood.” CSF RBC counts up to 45 000/μL are documented by Sarff et al, obscuring the validity of those reference values. Portnoy and Olson17 enrolled 131 children who were younger than 3 months; although enrolled infants had negative CSF bacterial and viral cultures, infants with bacteremia, urinary tract infections, and seizures were included. In addition, traumatic lumbar puncture was not specified as an exclusion criterion. Bonadio et al24 applied strict exclusion criteria and included only infants with negative blood, urine, and CSF cultures yet had a small population: only 75 infants who were younger than 8 weeks were enrolled. Ahmed et al27 also used strict exclusion criteria and were the first study to incorporate PCR testing but enrolled only patients who were younger than 30 days. Our approach overcomes many of the limitations of previous studies to establish reference values for infants who routinely undergo lumbar puncture. The values established in this study are based on the largest population of neonates and young infants identified to date, and we incorporated results of CSF EV PCR testing to define accurately an upper limit of normal for use by clinicians.
For infants who were aged 0 to 28 days, the reference values that we observed are a median value of 3/μL with a 90th percentile value of 12/μL. In this age range, Bonadio et al24 documented median values of 8.5/μL with a 90th percentile value of 22/μL. The values proposed by Bonadio et al may be higher as a result of a smaller sample size, the inability to integrate PCR testing, and use of a higher CSF RBC count cutoff value (>1000 RBCs/μL) to define traumatic lumbar puncture.24 Our work confirms the values found by Ahmed et al27 in 108 infants who were younger than 30 days, which determined a median CSF WBC count of 4/μL with a 90th percentile value of 11/μL. The 95th percentile value in our study was 19/μL; Ahmed et al did not report comparable data.
Because CSF WBC counts are age dependent,24 it is imperative to examine each age group independently. Infants who are aged 29 to 56 days must also have age-dependent reference values, yet reference CSF WBC counts for this population have been specifically studied only once in a cohort of 40 patients with a median CSF WBC value of 4.5/μL and a 90th percentile value of 15/μL.24 Viral PCR was not used at that time, and the sample size was insufficient to guide practice. The values established in our study can now be used for this patient population.
The observation that patients who tested negative for EV by PCR had consistently higher median CSF WBC values than those who were not tested was unforeseen. Ideally, those with negative EV testing represent the noninfected neonates and most closely approximate normal; however, because this was a retrospective study, those with EV testing may merely represent a group of patients who had a CSF WBC count on the higher end of normal and whose CSF was sent for EV PCR testing because of physician concern for infection. Alternatively, the higher CSF WBC counts of these patients may in fact represent viral infections, such as parechovirus, that are not yet part of routine testing.49 As our understanding of viral pathogens evolves, our value for the upper limit of normal for CSF WBC counts may also change. Finally, although a statistical difference exists between infants with negative EV testing and infants who did not have testing done, the differences are small and the clinical management of both groups is likely the same.
Several patients had CSF WBC counts far outside the range that most clinicians would consider “normal.” Although we assume that CSF WBC counts such as 320/μL and 198/μL represent pathology, we chose to include these patients because the aim of our study was to define ranges of normal and none of these patients had identifiable conditions that warranted exclusion. With that in mind, outliers were included in analysis and the 90th and 95th percentile values are documented to guide physicians in using these reference values.
This study had several limitations. First, not all young infants and neonates had EV testing, and viral culture of the CSF was not performed; therefore, patients without testing may have viral meningitis, and those with negative testing may have another virus not identified with PCR tests that routinely are used at our institution. The misclassification of infants with aseptic meningitis as uninfected may lead to an overestimation of the upper bound of the reference values determined in this study; however, many infants in the first few months of life with documented viral infection of the CNS lack CSF WBC abnormalities,41,50,51 mitigating the impact of such misclassification. Second, certain patients received antibiotics before lumbar puncture, leaving open the rare possibility that a child with bacterial meningitis was included in the study population; this approach could falsely elevate our reference values. This possibility is unlikely because bacterial meningitis is rare and we incorporated results of CSF Gram-stain, glucose, and protein into our definition of meningitis in patients who received antibiotics before lumbar puncture.
Third, this was an observational study, so physicians selected which infants underwent lumbar puncture. Because it is routine practice in our ED to perform a lumbar puncture on all febrile infants, it is likely that most febrile infants who were aged ≤56 days had a lumbar puncture regardless of presentation, and our population does not represent only ill-appearing patients. Fourth, this is a single-center study. If different viral pathogens have a different propensity to cause CSF WBC elevations, then generalizability of our study results may be influenced by the variability of causative viral pathogens by region. Fifth, this study includes preterm infants who are categorized by postnatal age; although these infants demonstrate an age-dependant decline in CSF WBC counts that is not statistically different from term infants, it is not known whether changes in their physiology are identical to term infants. Finally, although our study objective was to define age-specific reference values for CSF WBC counts in neonates and young infants, we recognize that infants with CSF WBC counts within the reference ranges defined in this study may still have serious infection.
CONCLUSIONS
CSF WBC counts are routinely measured in ill neonates. The age-dependent reference values presented in this study serve to guide clinicians in determining when the CSF WBC count of an ill neonate is above the upper limits of normal.
Footnotes
- Accepted August 7, 2009.
- Address correspondence to Samir S. Shah, MD, MSCE, Children's Hospital of Philadelphia, Division of Infectious Diseases, Room 1526 (North Campus), 34th Street and Civic Center Boulevard, Philadelphia, PA 19104. E-mail: shahs{at}email.chop.edu
FINANCIAL DISCLOSURE: Dr Kestenbaum has received support from the Clinical Neurosciences Scholars Track at the University of Pennsylvania School of Medicine; Dr Shah has received support from the National Institute of Allergy and Infectious Diseases (K01 AI73729) and the Robert Wood Johnson Foundation under its Physician Faculty Scholar Program; the other authors have no financial relationships relevant to this article to disclose.
- CSF =
- cerebrospinal fluid •
- WBC =
- white blood cell •
- CNS =
- central nervous system •
- PCR =
- polymerase chain reaction •
- EV =
- enterovirus •
- ED =
- emergency department •
- RBC =
- red blood cell •
- CFU =
- colony-forming units
REFERENCES
Math Scores Being Left Behind With Five Years to Go: It is interesting that since the passage of the “No Child Left Behind” law in 2001, math scores on student achievement tests have increased only marginally for eighth graders and not at all for fourth graders. According to an article by S. Dillon in The New York Times (October 14, 2009), only 39% of fourth graders and 34% of eighth graders scored at or above a level of proficiency in the most recent administration of the National Assessment of Educational Progress given to more than 329 000 fourth and eighth graders. Ironically student achievement scores were rising much faster before the federal law was passed when states were overseeing educational policy. With only 5 years left on the goal set by Congress 9 years ago of bringing proficiency in math and reading to 100% of students by 2014, the test scores are not adding up to a successful solution.
Noted by JFL, MD
- Copyright © 2010 by the American Academy of Pediatrics
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