A Pilot Study to Evaluate the Utility of Placental/Umbilical Cord Blood (PUCB) in Early Onset Neonatal Sepsis (EONS) in Very Low Birth Weight Infants
Phase: NA
Conditions: Preterm Infant, Early-Onset Neonatal Sepsis, Umbilical Cord
Sponsor: The University of Texas Medical Branch, Galveston

OFFICIAL TITLE: “A Pilot study to Evaluate the Utility of placental/Umbilical Cord 
Blood (PUCB) in Early Onset Sepsis in Very Low Birth Weight Infants 
 
VERSION NUMBER AND DATE: Version 2 May 31/2019 
 
PI:  Sunil K. Jain, MD 
 
 
 
 
NCT NUMBER: NCT03694613 
 
DOCUMENT DATE: May 31, 2019 
 
 

Version 2  May 31/2019 
Page 1 of 12 
 
 
“A Pilot study to Evaluate the Utility of placental/Umbilical Cord Blood (PUCB) in Early 
Onset Sepsis in Very Low Birth Weight Infants” 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PI: Sergio Lerma 
301 University Blvd, Galveston, TX 77450 
 
 
 
 
 
 
 
 
Supervisor: Sunil Jain, M.D.,  
Professor,  
Department of Pediatrics 
UTMB Galveston 
 
 
 
 
 
 
 
 
Sub-investigators: 
 
Joan Gonzalez 
 
 
 
 
 
 

Version 2  May 31/2019 
Page 2 of 12 
 
 
 
Table of contents: 
 
 
Study Schema: 
 
1. Introduction and purpose 
 
2. Background 
 
2.1 CRP 
2.2 IL 6 
2.3 Combination of biomarkers 
2.4 Reducing blood withdrawals in preterm infants 
 
3. Concise summary of project  
 
4. Study procedures 
 
5. Criteria for inclusion of subjects 
 
6. Criteria for exclusion of subjects 
 
7. Source of research material 
 
8. Recruitment methods and consenting process 
 
9. Potential risks 
 
10. Subject safety and Data monitoring  
 
11. Procedures to maintain confidentiality  
 
12. Potential benefits  
 
13. Biostatistics 
 
14. References 
 
 
 
 
 
 
 
 
 

Version 2  May 31/2019 
Page 3 of 12 
 
 
 
 
 
1. Introduction and Purpose: 
 
 
Sepsis is the leading cause of neonatal mortality and every year three million infants die 
worldwide due to neonatal sepsis (1). In the US, despite significant advances in care, sepsis 
remains a significant cause of neonatal morbidity and mortality, particularly among Very Low 
Birthweight (VLBW) preterm infants (PI) (2).   
 
Neonatal early onset sepsis (EOS) is defined as onset of signs and symptoms of sepsis with 
positive culture <72 hours of age.  Compared to term infants, the incidence of EOS is 11 times 
higher in preterm infants with a mortality rate ranging from 1.5% in term infants to almost 40% 
in VLBW preterm infants (2).  
 
The diagnosis of neonatal sepsis is challenging, and the prognosis depends on early detection 
and treatment. The current “gold standard” to diagnose sepsis is a positive blood culture, but 
there is a variable sensitivity mostly due to inadequate sample volume, transient bacteremia, 
intrapartum antibiotics and administration of antibiotics before blood sample collection (3,4,5). 
Although the gold standard for diagnosis of bacterial sepsis is blood culture, only in 25%-40% 
of blood culture pathogens can be detected (6). Serum markers as IL-6 and CRP have shown 
to be suitable markers for detecting early onset neonatal sepsis, with a high degree of 
sensitivity and specificity (18). 
 
In VLBW infants, almost 3 ml of blood is drawn to evaluate EOS for blood culture, CBC and 
blood group and cross match. This baseline work-up laboratory may represent almost 5-10% 
total blood volume of a VLBW infant. This iatrogenic acute blood loss may lead to hemodynamic 
instability and early red blood transfusion in the preterm infant when they are at the highest 
period risk for intra ventricular hemorrhage (IVH) (23).  
 
The average infant’s blood volume is 80ml/kg, while the average blood in the placenta and 
umbilical cord is15 to 34 ml/kg which is discarded in the current practice (29). EOS evaluation 
which includes blood culture, CBC, IT ratio along with biomarkers of EOS like CRP and IL-6 
could be done by using placenta/umbilical cord blood (PUCB). PUCB collection is painless, 
easy and adequate volume may be collected for blood culture. This procedure could decrease 
iatrogenic blood losses of VLBW infants (23). Currently, there is inadequate published data to 
support the routine use of PUCB in the evaluation of EOS in VLBW preterm infants.  
 
The purpose of the present study is to evaluate the utility of placenta/umbilical cord blood 
(PUCB) to perform the baseline workup testing (CBC, I/T ratio, blood culture) along with CRP 
and IL-6 levels in the evaluation of EOS in VLBW infants. 
 
 
 
 
2. Background: 

Version 2  May 31/2019 
Page 4 of 12 
 
Early and timely diagnosis of EOS is very important but challenging at the same time as clinical 
signs are nonspecific. Routine Laboratory tests such as complete white blood count (WBC) 
with differential count and immature/total neutrophil (I/T) ratio are routinely used to assist in the 
diagnosis of sepsis in neonates; however, multiple studies have determined that the WBC and 
immature-to-total neutrophil (I/T) ratio have low sensitivities and specificities (7). The I/T ratio 
has been found elevated in a quarter to half of the presumptively in infants without sepsis (8,9) 
with low positive predictive values (7). Consequently, just CBC indices in the evaluation of EOS 
in neonates is not very helpful, hence use of empiric antibiotic therapy is used in a large number 
of neonates with clinical suspicion of EOS (8,9). 
 
The “Gold Standard,” blood cultures are not always positive because of low blood volumes are 
often put in the blood culture bottles and prenatal administration of antibiotics (6, 27). Using 
biomarkers of sepsis to distinguish septic from non-septic neonates may allow judicious 
discontinuation of antibiotics, consequently preventing prolonged use of antibiotics and 
avoiding the appearance of antibiotic-resistant bacteria. Besides, appearance of antibiotic-
resistant bacteria, prolonged empiric antibiotic use has been associated with increased risk of 
necrotizing enterocolitis (NEC), late onset sepsis (LOS), and even increased mortality (10).  
 
Recent studies have shown improved sensitivity and negative predictive value (NPV) of various 
biomarkers of sepsis compared to CBC indices (11,12). Acute phase proteins, components of 
the complement system, chemokines, cytokines, adhesion molecules, and cell surface markers 
have all been investigated as biomarkers of neonatal sepsis (12). The most widely studied and 
most promising markers include C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-8 (IL-
8), procalcitonin (PCT), and tumor-necrosis-factor-alpha (TNF-alpha). 
 
2.1 C-Reactive Protein (CRP) 
 
CRP is one of the most sensitive acute-phase reactants for inflammation. CRP is synthesized by 
the liver. The CRP response frequently precedes clinical symptoms of sepsis. CRP acts within the 
humoral immune system, particularly in the detection and clearance of bacterial 
microorganisms. CRP level increases within 6-18 hours after a stimulus and peaks 8-60 hours 
later (13). The fetus and newborn synthesize CRP; its level is not affected by gestational age, 
and CRP does not cross the trans-placental passage of maternal CRP (14).   
 
CRP elevations are nonspecific and may be useful for the detection of systemic inflammatory 
processes; to assess treatment of bacterial infections with antibiotics. In neonates, CRP has been 
shown to be more sensitive and specific in diagnosing sepsis than the more commonly used 
CBC indices (total neutrophil count and I/T ratio) (15). Joram at al found that CRP (cut-off point 
of 5 mg/dl) measured in umbilical cord blood has a sensitivity, specificity, positive and negative 
predictive values are respectively 50%, 97%, 67% and 94%.  Besides, the same study found 
that the positive and negative likelihood ratios were 16.7 and 0.5 for CRP (28). 
 
 
 
2.2 IL-6 
 
IL-6 is a pro-inflammatory cytokine produced by mononuclear phagocytes, endothelial cells, 
and fibroblasts in response to inflammation. IL-6 levels peaks 2-3 hours after inflammatory 

Version 2  May 31/2019 
Page 5 of 12 
responses, and returns to baseline after 6 to 8 hours (16). IL-6 is the primary inducer of hepatic 
acute-phase protein synthesis, including CRP and fibrinogen (17). IL-6 levels are not influenced 
by gestational age and has been shown to have high sensitivity in diagnosing EOS (16,18). 
 
Messer et al. in a prospective study, measured IL-6 levels (n=157) upon admission to the NICU 
and the in umbilical cord blood (n=131) (18). The sensitivity and NPV of IL-6 in the diagnosis 
of blood culture positive and clinical sepsis was 100% in those infants who had an early 
measurement of IL-6 at birth (umbilical cord blood) or blood drawn from the infant within one 
hour after birth. Moreover, they found high sensitivity and NPV persisted until the twelfth hour 
of age. Krueger et al. found umbilical cord blood IL-6 levels have high sensitivity (87%) and 
specificity (90%) in 71 term and 100 preterm infants. When only preterm infants were included 
in the analysis, IL-6 sensitivity and specificity increased to 96% and 94% (19). 
 
2.3 Combination of Biomarkers 
 
Because single biomarker cannot be reliably used to diagnose EOS , many investigators have 
used combinations of biomarkers to improve the sensitivity and specificity in diagnosing EOS. 
Buck et al. (20) found the sensitivity of IL-6 in CRP-negative newborns and the sensitivity of 
CRP in IL-6 negative newborns on admission to be 100% in newborns with blood-culture 
positive or clinical sepsis. They concluded that the combination of IL-6 and CRP is the best 
combination of biomarkers of sepsis in diagnosis of EOS. Doellner et al. (21) also found the 
combination of IL-6 and CRP is superior in diagnosing neonatal sepsis than either parameter 
alone. Laborada et al. also concluded that the combination of IL-6 and CRP is the best 
diagnostic test for the detection of EOS sepsis in preterm infants (22). 
 
 
2.4 Reducing blood withdrawals in preterm infants  
 
Placental/umbilical cord blood (PUCB) could be used in the evaluation of EOS in PI. (23,26). 
Christensen found reduced iatrogenic blood loss using PUCB sample during the first day of life 
(p < 0.007), and for the first week of life (p <0.001) in contrast with infant blood sample controls 
(23). This postpone or eliminating blood transfusion in the VLBW preterm infant (23,26). 
 
PUCB for blood cultures could be a convenient alternative to ensure an adequate volume of 
blood (24). Meena at al. compared PUCB with blood from infants, (n=40) in the evaluation of 
sepsis. They found PUCB showed 100% sensitivity and 95% of specificity compared with 100% 
of sensitivity and 74% of specificity using blood from infants.  Beeram et al found that the 
utilization of umbilical blood samples for CBC and blood cultures are a reliable alternative for 
sepsis workup in neonates (25). 
 
In the current study, we want to find out the efficacy of PUCB in the evaluation of EOS in 
preterm infants compared to blood collected from the infant (24). 
 
 
3. Concise Summary of Project: 
 
Hypothesis: Placental/umbilical cord blood can be used to do early onset sepsis evaluation in 
low birth weight infants.  
 

Version 2  May 31/2019 
Page 6 of 12 
Objective: To compare placental/umbilical cord blood and blood from the infant in the 
evaluation of EOS in VLBW infants. 
 
Specific Aim 1: To compare blood culture, CBC and I/T ratio results of blood from the infant 
and placental/umbilical cord blood (PUCB). 
Objective: All VLBW infants receive empirical broad-spectrum antibiotics after collecting blood 
for CBC, I/T ratio, and blood culture. We will compare the results of PUCB and blood from the 
infants, especially comparing white cell count, band count, I/T ratio and if the blood culture is 
positive or negative. If the blood culture is positive, then we will find out if the micro-organism 
is the same or not. 
 
Specific Aim 2: To evaluate the role of adding IL-6 and CRP in the evaluation of EOS in VLBW 
infants. 
Objective: We will collect 1 mL of PUCB which will be centrifuged at -40C and serum will be 
stored at -80 degrees. We will use ELISA to measure IL-6 and CRP. A combination of IL-6 and 
CRP has been shown to be very helpful in the diagnosis of EOS in VLBW infants. We will also 
calculate sensitivity, specificity, negative predictive and positive predictive values of IL-6 and 
CRP individually, as well as, a combination of IL-6 and CRP in the diagnosis of EOS in VLBW 
infants. We will also find out the duration of antibiotics used in all subjects and find out from IL-
6 and CRP data if the antibiotic use was warranted. In future, this may be very useful 
information in deciding antibiotic duration which will help in avoiding side effects of the 
unnecessary use of broad-spectrum antibiotics. At present, in our NICU, IL-6 and CRP are not 
part of EOS evaluation in VLBW infants. 
 
Clinical team will be blinded to the PUCB sepsis evaluation results. In the present study, IL-6 
and CRP will not be used clinically but only for research purpose to find out if the addition of 
IL-6 and CRP help in the management of EOS in VLBW infants. 
All VLBW infants get CBC done at admission before starting antibiotics as part of EOS work 
up. After CBC is done, left over blood sample is saved in the hematology laboratory for 24 
hours. We will collect the left-over sample and collect serum after centrifugation. The serum 
will be saved at -800C for future measurement of IL-6 and CRP. We will compare IL-6 and CRP 
from infant and PUCB.   
 
Routine management of VLBW infants in NICU at UTMB Galveston:  
 In NICU at UTMB Galveston, routinely, all VLBW infants admitted to NICU get sepsis 
evaluation by collecting blood from infant for CBC, I/T ratio, and blood culture followed by a 
course of empiric antibiotics for 2 – 3 days. This requires almost 2 – 3 mL of blood volume 
which may be up to 5% blood loss in an extremely premature infant which may lead to 
hemodynamic instability and anemia, resulting in the requirement for blood transfusion. If the 
sepsis evaluation is not conclusive, then the course of antibiotics may be extended to 3 to 7 
days. Addition of CRP and IL-6 in EOS evaluation may be helpful in determining the duration 
of antibiotics. However, in NICU at UTMB Galveston, CRP and IL-6 are not part of EOS 
evaluation.  
 
Placenta/Umbilical Cord blood (PUCB):   
In the current research project, we want to use PUCB to do sepsis evaluation in VLBW infants. 
There is approximately 15 – 20 mL/kg blood in the placenta and umbilical cord. That means in 
VLBW infants (<34 w) there will be approximately 15 – 20 mL blood in the placenta and 
umbilical cord which is fetal blood hence it can be utilized to do EOS sepsis evaluation. This 

Version 2  May 31/2019 
Page 7 of 12 
will save blood in VLBW infants.  As blood in PUCB is nothing but fetal blood. There is a 
misconception that fetal and maternal blood is mixed in the placenta. Actually, there is no 
mixing of maternal and fetal blood in the placenta. After birth, the placenta and umbilical cord 
are discarded along with the blood inside. We want to utilize fetal blood in the placenta/umbilical 
cord for the evaluation of EOS in VLBW infants.  
 
The current research project will evaluate the utility of PUCB to assess early onset sepsis 
(EOS) in VLBW infants (<34 weeks). This study will not affect the management of the infants 
at all. The lab tests will be done in blocks and hence the results will not be available in real 
time. The clinical management of infants will be decided by the neonatal team taking care of 
the infants. The neonatologists will continue to treat infants as per the NICU protocol.  
 
We will compare blood culture, CBC, and I/T ratio results from the infant and PUCB. We will 
calculate the sensitivity, specificity, positive and negative predictive value of these tests in the 
evaluation of EOS.  
 
During hospital stay, all VLBW infants get CBC done immediately after birth. After CBC is done, 
the left-over sample is stored in the hematology laboratory for 24 hours. After 24 hours, this 
blood sample is discarded. We will collect left over blood from the hematology laboratory to 
extract serum by centrifuging the blood sample. The serum will be stored at -800C for future 
measurements of CRP and IL-6. We will also compare CRP and IL-6 levels in 
placenta/umbilical cord blood and discarded infant blood. Additionally, we will evaluate the role 
of adding IL-6 and CRP in the evaluation of EOS in VLBW infants. 
 
Power analysis:  
In order to do a power analysis to determine sample size, an estimate of effect size was based 
upon the Rashawn et al (30) study which found significant correlations of CRP with other 
biomarkers. 
In G*Power (31) the following was used: 
• Effect size of r=.4 
• Alpha=.05 
• Power (1-alpha)=.95 
• Yielding a sample size of 63 
To accomplish this project, a cohort of preterm infants will be recruited from the infants admitted 
to the NICU at UTMB, Galveston. We routinely admit around 80 infants/year who are  <34 
weeks’ gestation. We expect to recruit about 50 – 60% VLBW infants born in UTMB, Galveston 
in the current study.  Therefore, we hope to recruit our desired sample size (n=63) in about 18 
months.     
 
Clinical Data Collection:   
We will use electronic medical records to collect medical data from mother’s antenatal, delivery 
and post-delivery information and detailed medical information of the infants.  Data derivated 
from blood test analysis in placenta/umbilical blood cord and blood from the infants will be used 
to assess the accuracy of placenta/umbilical blood cord samples. We will also find out 
specificity, sensitivity, positive and negative predictive values of CBC, I/T ratio, blood culture, 
CRP and IL-6 in the evaluation of EOS in ELBW infants. All data will be de-identified but the 
clinical data will be linked to the discarded samples.  
 

Version 2  May 31/2019 
Page 8 of 12 
 
4. Study Procedures: 
 
Placenta/Umbilical cord blood (PUCB) sample collection:  
 
After infant is delivered, placenta along with clamped umbilical cord Blood will be obtained from 
the ObGyn team.  One umbilical clamp will be placed at the umbilical end, and the other clamp 
will be placed on the placental end of the umbilical cord. Then the umbilical cord will be cut 
between the clamps. The umbilical cord will be cleaned three times with 2% chlorhexidine, plus 
70% isopropyl alcohol under sterile conditions (sterile gloves). Cord blood samples will be 
collected using vacutainer blood collecting system with a sterile 22-gauge needle. We will 
collect 3 - 4 ml of PUCB. 
 
Discarded blood sample from infants:  
 
During hospital stay, all VLBW infants get CBC done immediately after birth. After CBC is done, 
the left-over sample is stored in the hematology laboratory for 24 hours. After 24 hours, this 
blood sample is discarded. We will collect left over blood from the hematology laboratory to 
extract serum by centrifuging the blood sample at 40C for 10 minutes. The serum will be stored 
at -800C for future measurements of CRP and IL-6.  
 
PUCB culture:  
 
After UCB collection, the needle will be replaced by another sterile needle and 1 ml of UCB will 
be transferred to the blood culture bottle. The culture bottle will be labeled with a unique 
identification number and immediately will be sent to the microbiology laboratory at UTMB 
Galveston for blood culture. On identification of positive blood culture, the blood culture bottle will be 
removed from the machine and will be Gram stained to make sure there are organisms seen and then 
sub-cultured on blood agar and Mac-Conkey agar plates. The organisms isolated on the culture plates 
will be identified by VITEK MS (BioMerieux). 
 
Complete blood count and I/T ratio:  
 
After PUCB culture, approximately 1 ml of whole blood will be transferred in an EDTA vial which 
will be labeled with a unique identification number. The blood sample will be sent to the 
hematology laboratory at UTMB Galveston for CBC and differential count. I/T ratio will be 
calculated from the differential count. 
 
 
 
5. Sub-Study Procedures: 
 
IL-6 and CRP measurement:  
 
The remainder of the PUCB will be transferred to a heparinized container. PUCB will be 
centrifuged at 40C for 10 minutes to collect supernatants (serum). Serum will be divided into 
aliquots of 200 microliters each and stored at -800C for future IL-6 and CRP measurements. 
We will measure IL-6 by enzyme-linked immunosorbent assay (ELISA) for human IL-6. 
 
 

Version 2  May 31/2019 
Page 9 of 12 
6. Criteria for Inclusion of Subjects: 
 
Inclusion criteria:  Subjects who will fulfil following criteria will be included in the study -  
1.     Infants  < 34 weeks’ gestational age, born at UTMB, Galveston  
 
 
7. Criteria for Exclusion of Subjects: 
 
Exclusion criteria: Subjects meeting any of the exclusion criteria at baseline will be excluded 
from study participation.  
1.    Known congenital or chromosomal anomalies 
2.    Congenital heart disease (other than PDA, PFO or ASD) 
3.    Multiple pregnancy 
 
 
8. Sources of Research Material: 
 
We are going to use the discarded placenta/Umbilical Cord unit and as the resource of PUCB 
samples and left-over blood sample after CBC is done as blood from infant.  
 
 
9. Recruitment Methods and Consenting Process: 
 
Recruitment 
All eligible infants born at UTMB Galveston will be recruited in the study. We expect 50 - 60% 
success in the recruitment after accounting for exclusion criteria and timing of delivery. If the 
infant is born after hours, the research team may not be available to collect the PUCB sample. 
We admit around 80 infants/year who are <34 weeks’ gestation.  We hope to recruit our desired 
sample size (n=63) in 18 months.  
 
Consenting process 
In the current research protocol, we will collect PUCB after birth, which is considered as 
discarded tissue. We will not collect any blood or any other body fluid from the subjects. This 
will be less than minimal risk to the subjects. Therefore, consent is not required.  
 
 
10. Potential Risks: 
There is no potential risk to the subjects since PUCB will be collected after birth and placenta 
and umbilical cord is considered discarded. There will not be any direct interaction with the 
subjects.  
 
 
11. Subject Safety and Data Monitoring: 
 
Subject’s safety will not be affected as we will be collecting the blood samples from the 
placenta/umbilical cord after birth which is discarded material. All the data will be collected and 
saved in a password protected computer. Only PI and research team will have access to the 
computer and the data.  
 

Version 2  May 31/2019 
Page 10 of 12 
 
12. Procedures to Maintain Confidentiality: 
 
No information regarding the study or the clinical data will be released to any unauthorized third 
party without the prior written approval of the principal investigator. The investigators are 
committed to maintaining the confidentiality of the subjects through the proper use and 
disclosure of patients’ personal health information (PHI). Study records may be made available 
for internal compliance reviews and quality assurance representatives. 
 
 
13. Potential Benefits: 
 
There will be no direct benefit to the subjects. But the results of this study may help us to find 
out if PUCB could be utilized to evaluate EOS in VLBW infants. The results of this study will 
provide understanding of the utility of PUBC samples to evaluate EOS in VLBW preterm infants. 
Moreover, this alternative modality of blood analysis could contribute to the prevention of early 
hemodynamic instability and early blood transfusion in a very high-risk population of VLBW 
preterm infants. Furthermore, an additional test for sepsis screening as CRP and IL 6, might 
improve the accuracy for EOS evaluation, and may avoid or prevent unnecessary and prolong 
antibiotic use in ELBW infants. 
 
 
14. Biostatistics:  
 
Statistical analyses will be performed using SPSS 25 for Windows (SPSS Inc., Chicago, IL, 
USA). Demographic and clinical characteristics will be compared using the non-parametric 
Mann-Whitney U test, and the results will be presented as medians (range). Categorical 
variables will be compared using the linear-by-linear association Chi-squared test and will be 
presented as percentages (%). Receiver operator curve (ROC) analysis will be employed to 
display the relationship between sensitivity and false positive (FP) rate (1-specificity) and to 
select the best cut-off value for umbilical cord IL-6 and CRP to predict EOS. Data will be as 
adjusted by gestational age at delivery, administration of prenatal corticosteroids and 
antibiotics. Differences will be considered statistically significant at a confidence level of p <0.05 
with two-sided alternative hypotheses 
 
 
 
 
 
 
 
 
 
 
References 
 
 
1. Qazi SA, Stoll BJ. Neonatal sepsis: a major global public health challenge.  Pediatric 
infectious Dis J. 2009: 28:1-2. 

Version 2  May 31/2019 
Page 11 of 12 
2. Weston EJ, Pondo T, Lewis MM, Martell-Cleary P, Morin C, Jewell B, et al. The burden 
of invasive early-onset neonatal sepsis in the United states, 2005-2008.  Pediatric 
Infectious Disease J. 2011; 30(11): 937-41. 
3. Polin RA.  Committee on Fetus and Newborn.  Management of neonates with suspected 
or proven early onset bacterial sepsis; Pediatrics. 2012; 129:1006-15 
4. Brown DR,  Kutler D, Rai B, Can T, Cohen M. Bacterial concentration and blood volume 
required for positive blood culture. J Perinatology.  1995;157-9 [PubMed:7595778] 
5. Neal PR, Kleiman MB, Reynolds JK, Allen SD, Lemons JA, Yu PL.  Volume of blood 
submitted for culture from neonates.  Clin Microbiol.  196; 1986;24:353-6  
6. Lukacs SL, Schrag SJ. Clinical sepsis in neonates and young infants, United States, 
1988-2006. J Pediatr. 2012;160(6):960-965 e961. 
7. Hornik CP, Benjamin DK, Becker KC, et al. Use of the complete blood cell count in early-
onset neonatal sepsis. Pediatr Infect Dis J. 2012;31(8):799-802 
8. Hornik CP, Benjamin DK, Becker KC, et al. Use of the complete blood cell count in early-
onset neonatal sepsis. Pediatr Infect Dis J. 2012;31(8):799-802. 
9. Hornik CP, Benjamin DK, Becker KC, et al. Use of the complete blood cell count in late-
onset neonatal sepsis. Pediatr Infect Dis J. 2012;31(8):803-807. 
10. Cotten CM, Taylor S, Stoll B, et al. Prolonged duration of initial empirical antibiotic 
treatment is associated with increased rates of necrotizing enterocolitis and death for 
extremely low birth weight infants. Pediatrics. 2009;123(1):58-66. 
11. Malik A, Hui CPS, Pennie RA, Kirpalani H. Beyond the complete blood cell count and 
C-reactive protein: a systematic review of modern diagnostic tests for neonatal sepsis. 
Arch Pediatr Adolesc Med. 2003;157:511-516. 
12. Ng PC. Diagnostic markers of infection in neonates. Arch Dis Child Fetal Neonatal Ed. 
2004;89:F229-F235. 
13. Hofer N, Zacharias E, Muller W, Resch B. An update on the use of C-reactive protein in 
early-onset neonatal sepsis: current insights and new tasks. Neonatology. 
2012;102(1):25-36. 
14. Martin RJ, Fanaroff AA, Walsh MC. The Immune System. Fanaroff and Martin's 
Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant. Vol 2. 9th ed. St. Louis, 
Mo: Mosby/Elsevier; 2011. 
15. Da Silva O, Ohlsson A, Kenyon C. Accuracy of leukocyte indices and C-reactive protein 
for diagnosis of neonatal sepsis: a critical review. Pediatr Infect Dis J. 1995;14:362–366. 
16. Weitkamp JH, Aschner JL. Diagnostic Use of C-Reactive Protein (CRP) in Assessment 
of Neonatal Sepsis. NeoReviews. 2005;6(11):e508-e515. 
17. Chirico G, Loda C. Laboratory aid to the diagnosis and therapy of infection in the 
neonate. Pediatr Rep. 2011;3(1):e1. 
18. Messer J, Eyer D, Donato L, Gallati H, Matis J, Simeoni U. Evaluation of interleukin-6 
and soluble receptors of tumor necrosis factor for early diagnosis of neonatal infection. 
J Pediatr. 1996;129:574-580. 
19. Krueger M, Nauck MS, Sang S, Hentschel R, Wieland H, Berner R. Cord blood levels 
of interleukin-6 and interleukin-8 for the immediate diagnosis of early-onset infection in 
premature infants. Biol Neonate. 2001;80:118-123. 
20. Buck C, Bundschu J, Bartmann P, Pohlandt F, Gallati H. Interleukin-6: A sensitive 
parameter for the early diagnosis of neonatal bacterial infection. Pediatrics. 1994;93:54-
58. 
21. Doellner H, Arntzen KJ, Haereid PE, Aag S, Austgulen R. Interleukin-6 concentrations 
in neonates evaluated for sepsis. J Pediatr. 1998;132:295-299. 

Version 2  May 31/2019 
Page 12 of 12 
22. Laborada G, Rego M, Jain A, et al. Diagnostic value of cytokines and C-reactive protein 
in the first 24 hours of neonatal sepsis. Am J Perinatol. 2003;20(8):491-501 (28) 
23. Christensen RD, Lambert DK, Baer VL, et al. Postponing or eliminating red blood cell 
transfusions of very low birth weight neonates by obaining all baseline laboratory blood 
tests from otherwise discarded fetal blood in the placenta. Transfusion. 2011; 
Feb;51(2):253-8. 
24. Meena J, Charles MVP, Kali A, Ramakrishnan S, Gosh S, et al. Utility of cord blood 
culture in early onset neonatal sepsis. Australian Medical Journal. 2015;8(8):263-267 
25. Beeram MR, Loughran C, Cipriani C, Govande V. Utilization of Umbilical Cord Blood for 
the Evaluation of Group B streptococcal sepsis screening. Clinical Pediatrics. 2012 
May;51(5):447-53. 
26. Baer VL, Lambert DK, Carrol PD, Gerday E, Christensen RD. J Perinatol.  2013 
May;33(5):363-5.  Epub 2012 Oct 4.  
27. Buttery JP. Blood cultures in newborns and children: optimising an everyday test. Arch 
Dis Child Fetal Neonatal Ed 2002; 87(1):f25-f28 
28. Joram N, Boscher C, Denizot S at al. Umbilical cord blood procalcitonin and C reactive 
protein concentrations as markers for early diagnosis of very early onset neonatal 
infection.  Arch Dis Child Fetal Neonatal Ed 2006;91:F65-F66 
29. Carroll PD, Widness JA. Nonpharmacological, blood conservation techniques for 
preventing neonatal anemia – effective and promising strategies for reducing 
transfusion.  Semin Perinatol. 2012;36:232-243 
30. Rashwan NI, et al., Validity of biomarkers in screening for neonatal sepsis: A single 
center hospital based study, Pediatrics and Neonatology (2018). 
31. Faul, F., Erdfelder, E., Buchner, A., & Lang, A.-G. (2009). Statistical power analyses 
using G*Power 3.1: Tests for correlation and regression analyses. Behavior Research 
Methods, 41, 1149-1160.