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Technical Briefs |
1 Department of Laboratory Medicine, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan;
2
Department of Laboratory Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan;
3
Department of Medical Informatics, Yamaguchi University School of Medicine, Ube, Yamaguchi 755-8505, Japan;
4
International Clinical Pathology Center, Setagaya-ku, Tokyo 154-0003, Japan
aauthor for correspondence: fax 81-42-995-0633, e-mail yutakemu{at}interlink.or.jp
C-Reactive protein (CRP) is a well-known indicator of inflammation. Advances in laboratory technology have enabled its quantification at lower concentrations by an automated analyzer (1)(2)(3). Because an increase in serum CRP concentration, even within the reference intervals of conventional analytic methods, has been related to increases in the risk of atherothrombotic events (4)(5)(6)(7), efforts for CRP quantification are currently directed toward healthy, elderly individuals or populations at risk of atherosclerotic diseases. When measured with a high-sensitivity analytic method, CRP may have diagnostic value in neonatal infection because newborns are unable to produce sufficient amounts of acute-phase proteins (8) and respond to infection with a smaller increase in CRP compared with that in adults (9).
The utility of CRP for the diagnosis of neonatal infection has been the subject of lengthy controversy because of its unsatisfactory sensitivity for early-onset neonatal infection (10)(11)(13). The CRP concentration increases physiologically in newborns within the first few days after birth (9)(13). This increment seems to be related to the low diagnostic accuracy of CRP measurements in neonatal infection, particularly when measured shortly after birth. Similarly, other indicators of inflammation, such as procalcitonin and interleukin-6, demonstrate increases in noninfected babies within a few days after birth (14)(15)(16). In addition, very low CRP concentrations are found in the cord blood and sera of neonates in the immediate postnatal period (17). Therefore, the postnatal physiologic changes in CRP remain poorly understood. In this context, elucidation of serum CRP kinetics and an understanding of the mechanism(s) that cause its increase in the immediate postnatal period in noninfected babies would improve its diagnostic accuracy for early-onset neonatal infection. Using a high-sensitivity analytic method, we analyzed the serial changes in serum CRP concentrations in healthy newborns immediately after birth based on different delivery conditions.
Serum CRP concentrations were measured by a particle-counting immunoassay (PACIA) (18), a high-sensitivity CRP (hsCRP) assay based on the agglutination of antibody-sensitized latex particles and subsequent counting of these agglutinated particles by laser beam in a PAMIA-20 automated analyzer (Sysmex). This system requires only 40 µL of serum, results are available in 15 min, and the lower limit of detection is 0.001 mg/L. The within- and between-run CVs are 0.96–2.6% at 0.081–1.52 mg/L and 2.5–3.4% at 0.16–85.5 mg/L, respectively (19).
The present study included 110 neonates born in the Obstetrics Unit of Keio University Hospital during the period June 1992 to December 1992. Although we reported preliminary results on the methodologic precision of the assay based on analysis of serum samples collected prospectively (19), the perinatal clinical data were incompletely assembled and remained to be analyzed. To complete the study, we again closely reviewed the medical records of mother–baby pairs for the retrospective clinical data collection. Blood samples were obtained from babies whose parents were informed by their baby’s pediatrician and who gave consent to the study. Samples for which the pediatricians had ordered routine CRP measurements were analyzed by the conventional method (a latex photometric immunoassay) and the PACIA. The samples were collected serially between the time of birth (cord blood) and the ninth day of age and analyzed immediately after collection. Clinical data analyzed included maternal perinatal conditions (age, preexistent or pregnancy-associated diseases, history of preceding pregnancy/delivery, mode of delivery, interval between rupture of the membranes and delivery, and length of active labor) and neonatal characteristics (gestational age, birth weight, Apgar scores, poor activity, respiratory distress, and other clinical signs that might suggest an infection). Because our earlier study had demonstrated that the increases in serum bilirubin concentrations had no influence on the hsCRP values measured by the PACIA (19), infants with neonatal jaundice who required phototherapy were not excluded from the study.
The distribution of hsCRP values was positively skewed, and log-transformed values were approximately normally distributed when the number of samples was sufficiently large. Therefore, all comparisons of hsCRP results and multiple regression analyses were performed after the raw values were transformed to logarithms; the reported mean values are geometric means with 95% confidence intervals (CIs). Differences in the highest hsCRP values between neonatal groups (e.g., vaginal delivery vs cesarean section babies) were examined by the Wilcoxon rank-sum test. Multiple linear regression analyses were performed to explore the associations between the highest hsCRP values within 48 h after birth and the following variables: gestational age, birth weight, mode of delivery, interval between rupture of the membranes and delivery, length of active labor, and Apgar scores at 1 and 5 min. All tests were considered significant at P <0.05.
Serum hsCRP concentrations were measured serially up to the ninth day after birth in 83 babies who were delivered vaginally and 27 babies who were born by cesarean section. The study included four babies who were born before 35 weeks of gestation and eight low-birth-weight babies (birth weight <2500 g). Twenty newborns with incomplete hsCRP data and 5 babies who were treated with antimicrobial agent(s) because of a definite (culture-positive) or suspected infection were excluded from the analyses of postnatal hsCRP kinetics. The geometric mean hsCRP in the cord blood was 0.033 mg/L (95% CI, 0.012–0.320 mg/L; n = 25). Fig. 1
illustrates the serum hsCRP kinetics immediately after birth in the representative 37 babies born by vaginal delivery and the 10 babies born by cesarean section for whom hsCRP was measured three or more times and the peak values were clearly identified. In the analysis of newborns delivered vaginally (n = 64), hsCRP concentrations increased exponentially after birth, reached a peak concentration within 48 h, and then decreased gradually. Median hsCRP results and the 25th–75th percentiles at various times after birth were as follows: 12 h, 1.46 mg/L (1.09–2.48 mg/L); 24 h, 4.28 mg/L (1.91–8.46 mg/L); 36 h, 5.63 mg/L (1.83–13.70 mg/L); 48 h, 3.61 mg/L (1.97–5.68 mg/L); 60 h, 1.69 mg/L (0.81–3.63 mg/L); 72 h, 2.65 mg/L (1.47–3.26 mg/L); 96 h, 1.29 mg/L (0.90–2.29 mg/L); and 120 h, 0.52 mg/L (0.34–1.13 mg/L). Respective median values and the 25th–75th percentiles were obtained from data points within ± 6 h of each time.
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The highest hsCRP concentrations varied the most among neonates born under different conditions, from 0.11 mg/L to 64.0 mg/L. hsCRP concentrations returned to baseline by the sixth or seventh day after birth in healthy babies (0.42 mg/L; 95% CI, 0.08–2.32 mg/L; n = 17). The maternal and neonatal characteristics were analyzed to identify those that influenced the magnitude of the increases in hsCRP, and the results are shown in Table 1
. The highest hsCRP concentrations within 48 h after birth were significantly lower in babies born by cesarean section (n = 21) than in those born by vaginal delivery (n = 64; median values, 1.80 vs 5.18 mg/L, respectively; P = 0.0015). hsCRP values increased immediately after birth in the cesarean section babies as well, but the peak values were smaller and demonstrated delayed increases in some cases compared with those of babies born by vaginal delivery. The median hsCRP values and the 25th–75th percentiles obtained from all of the cesarean section babies analyzed at the following times after birth were as follows: 12 h, 1.45 mg/L (1.20–2.20 mg/L); 24 h, 1.39 mg/L (0.59–4.62 mg/L); 36 h, 0.41 mg/L (0.17–2.74 mg/L); 48 h, 1.09 mg/L (0.93–3.39 mg/L); 60 h, 3.12 mg/L (1.90–3.28 mg/L); 72 h, 2.64 mg/L (0.96–3.27 mg/L); and 96 h, 0.91 mg/L (0.50–1.80 mg/L). Other variables that revealed significant associations with the highest hsCRP values within 48 h after birth in the nonparametric statistical analysis (Wilcoxon rank-sum test) are presented in Table 1
. In multiple linear regression analyses, the highest hsCRP values within 48 h were positively associated with mode of delivery (P = 0.0005) and next with the baby’s birth weight (P = 0.019).
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The present study used a hsCRP method to elucidate serum CRP kinetics in individual healthy babies immediately after birth. Recently, Chiesa et al. (16) analyzed serum CRP concentrations in 148 healthy babies and attempted to establish the upper limits of normal for CRP in the immediate postnatal period. They reported that only 19 (13%), 54 (36%), and 55 (37%) of the neonates had detectable concentrations of CRP (>4 mg/L) at birth (cord blood), at 24 h, and at 48 h, respectively, as measured by a rate nephelometric assay that had conventional sensitivity for CRP determinations. Thus, median values were not determined, and changes could not be demonstrated in most individual babies in that study. In contrast, a hsCRP method was able to quantify CRP at much lower concentrations, even in babies delivered by cesarean section. These different methods may have produced the different results and conclusions reached by Chiesa et al. (16) for identifying factors that influenced the increase in CRP during the postnatal period.
The cesarean section babies had significantly lower peak hsCRP concentrations than did the babies delivered vaginally, which implies that the physical stress on babies during delivery may be related to the magnitude of the increases in hsCRP. In fact, the significance was much greater when babies born by elective cesarean section (n = 16) were compared with babies delivered vaginally (P = 0.00079). Similarly, birth immediately after rupture of the membranes led to significantly lower hsCRP peaks. Because 9 of the 14 babies who experienced very short intervals (<0.1 h) between rupture of the membranes and delivery were born by cesarean section, this finding might confound the small hsCRP increases in cesarean section babies. In contrast, a longer duration of active labor (
20 h) paradoxically led to significantly lower peak hsCRP concentrations within 48 h. One possible explanation for this unexpected finding is weaker maternal uterine contractions and, subsequently, less physical stress on the baby during delivery. Neonatal characteristics such as lower gestational age (<38 weeks) and lower birth weight (<2500 g) were also associated with significantly smaller hsCRP increases compared with those in babies with a higher gestational age (38 weeks) and higher birth weights (2500 g). However, perinatal distress as estimated by the Apgar scores did not influence the hsCRP increases in our study.
To elucidate possible confounding variables positively associated with the hsCRP increase in univariate analyses, we subsequently performed multiple linear regression analyses. Both mode of delivery and birth weight were independently associated with hsCRP increases in the multivariate analyses. The mode of delivery may reflect the intensity of physical stress on the baby during delivery, whereas low birth weight may be related to possible immature liver function and the inability of the liver to produce sufficient amounts of some proteins, such as CRP.
Increases in hsCRP values in the first few days of life differed greatly among babies born under different conditions. Accordingly, increases in hsCRP concentrations do not necessarily indicate an infected status in the immediate postnatal period. This might lead to heterogeneity in the diagnostic accuracy of CRP in those studies that use this protein for determination of neonatal infection (11). Different investigators have proposed different cutoff points for the upper limits of normal for CRP, based on their measurement methods, to distinguish infected babies from those with typical, physiologic increases in CRP. Nevertheless, introduction of a certain cutoff value at a specified time in the immediate postnatal period may cause some infected babies to be misdiagnosed, because CRP values do not necessarily increase to the cutoff point in some neonates, even in some with an infection, because of immaturity of the liver cells. Therefore, some studies suggest that the pattern of CRP changes be determined by serial measurements rather than a single assessment at one time with a certain cutoff value for the diagnosis of neonatal infection (9)(20)(21)(22)(23)(24). One large study, however, demonstrated that the pattern of CRP changes had a high specificity (>95%) and negative predictive value (98%) but low sensitivities (75% and 61.5% for term and preterm babies, respectively) (9). The unsatisfactory sensitivity of CRP pattern recognition for neonatal infection might be related to the insensitive analytic method used in that study because one-fourth of the babies with a definite infection did not have a peak CRP value >10 mg/L. Therefore, analysis of serum CRP kinetics with sequential measurements and a high-sensitivity assay method is likely to improve the diagnostic accuracy of CRP for early-onset neonatal infection.
Acknowledgments
We thank Teruko Ohtake and Yoshiko Inose (Keio University Hospital, Tokyo, Japan) for assistance with the sample analyses and data collection. We are grateful to Masaki Kobayashi (Sysmex, Kobe, Japan) for technical assistance with the analyzer operation and supply of reagents. We thank Dr. Tsunekazu Kita (National Defense Medical College, Tokorozawa, Japan) for valuable comments on this study.
References
The following articles in journals at HighWire Press have cited this article:
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  J.-H. Weitkamp and J. L. Aschner Diagnostic Use of C-Reactive Protein (CRP) in Assessment of Neonatal Sepsis NeoReviews, November 1, 2005; 6(11): e508 - e515. [Full Text] [PDF]
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 K. Skogstrand, P. Thorsen, B. Norgaard-Pedersen, D. E. Schendel, L. C. Sorensen, and D. M. Hougaard Simultaneous Measurement of 25 Inflammatory Markers and Neurotrophins in Neonatal Dried Blood Spots by Immunoassay with xMAP Technology Clin. Chem., October 1, 2005; 51(10): 1854 - 1866. [Abstract] [Full Text] [PDF]
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C. Chiesa, A. Panero, J. F. Osborn, A. F. Simonetti, and L. Pacifico Diagnosis of Neonatal Sepsis: A Clinical and Laboratory Challenge Clin. Chem., February 1, 2004; 50(2): 279 - 287. [Full Text] [PDF]
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