Towards improved neonatal care: Developing reference intervals for biochemical parameters in umbilical cord blood: An Indian study

Abstract

BACKGROUND

The reference ranges for biochemical parameters can fluctuate due to factors like altitude, age, gender, and socioeconomic conditions. These values are crucial for interpreting laboratory data and guide clinical treatment decisions. Currently, there is no established set of reference intervals for cord blood biochemical parameters of newborns in India, particularly in Mumbai.

AIM

To create cord blood biochemical parameters reference intervals specifically for Mumbai, India.

METHODS

A cross-sectional study was conducted in an Indian tertiary care hospital. This study focused on healthy newborns with normal birth weight, born to pregnant mothers without health issues. Cord blood samples, approximately 2-3 mL in volume, were collected from 210 term neonates. These samples were divided into fluoride (glucose) and clot activator (serum) tubes and were subsequently analyzed in the institute's biochemical laboratory. The data obtained from the analysis was then subjected to statistical analysis. The result of the Shapiro-Wilk test suggested non-normality in the data distribution. Consequently, non-parametric statistics were utilized for analysis. The Mann-Whitney U test was utilized to compare parameter distributions among different factors, including the infant’s sex, delivery method, maternal age, and obstetric history. A significance level of P < 0.05 was considered to indicate statistical significance.

RESULTS

The following represent the median figures and central 95 percentile reference intervals for biochemical parameters in umbilical cord blood of newborns: Serum direct bilirubin = (0.1–0.55) mg/dL, indirect bilirubin = (0.64–2.26) mg/dL, total bilirubin = (0.62–3.14) mg/dL, creatinine = (0.27–0.76) mg/dL, sodium = (128.19–143.26) mmol/L, chloride = (100.19–111.68) mmol/L, potassium = (1.62–9.98) mmol/L and plasma glucose = (24.75–94.23) mg/dL. Statistically significant differences were observed in serum sodium, potassium, and plasma glucose levels when comparing delivery modes.

CONCLUSION

This is the pioneering study in which first time, the biochemical reference intervals in cord blood for newborns are established in western India. The values are applicable for newborns from this area. Larger study throughout the country is required.

Key Words: Biochemical Parameters; Reference interval; Umbilical cord blood; Peripheral venous blood; Neonates; India

Core Tip: The reference ranges for biochemical parameters can fluctuate due to factors like altitude, age, gender, and socioeconomic conditions. These values are crucial for interpreting laboratory data and guide clinical treatment decisions. Currently, there is no established set of reference intervals for cord blood biochemical parameters of newborns. This study seeks to create these reference intervals.

INTRODUCTION

A reference interval is defined as a range that, when applied to the population served by a laboratory, appropriately includes the majority of individuals who share similar characteristics with the reference group, while excluding others[1]. Reference intervals are among the most commonly utilized tools in clinical decision-making, aiding physicians in diagnosing medical conditions and identifying diseased individuals. Hence, it is essential for the laboratory community to dedicate sufficient resources to the proper establishment of these intervals. However, due to challenges in recruiting an adequate number of reference individuals, there is a scarcity of studies focused on reference intervals for biochemical analytes in neonates. As a result, existing reference intervals often reflect values derived from healthy adults. This study, therefore, aims to establish reference intervals for commonly tested biochemical analytes specifically in neonates.

To prevent misunderstanding, it is important to acknowledge that the term “normal” can carry various interpretations. Rather than using outdated terms like “normal values” or “normal ranges” as substitutes for reference range or reference intervals (RIs), it is advisable to use the more accurate and widely accepted terms “reference range” or “reference interval”[1]. These terms have been broadly adopted in medical practice to reduce ambiguity and promote clarity.

Interpretation of laboratory data is essential in patient management, including evaluating whether a patient’s results fall within the normal reference interval and determining suitability for clinical and vaccine trials. The application of RIs is central to these processes[2]. These RIs can vary due to several factors such as age, sex, race, geographical location, and dietary habits. Numerous studies worldwide have demonstrated age-related variations in biochemical parameters. Infants, in particular, show significant quantitative and qualitative differences in these parameters compared to older children and adults[3]. Therefore, it has been concluded that using adult reference ranges to assess pediatric blood samples is highly inappropriate.

Using reference intervals derived from a different geographical region or age group for a specific target population can lead to suboptimal patient management and inefficient resource utilization[4]. Furthermore, when individual laboratories establish their own RIs, discrepancies often arise in the resulting reference intervals that are not justified by factors affecting analysis or differences specific to population[5]. Recent studies have developed neonatal RIs for biochemical analytes based on local populations. In this context, our study aimed to establish reference intervals for common biochemical analytes—plasma glucose, serum bilirubin, serum creatinine, and serum electrolytes such as sodium, potassium, and chloride—in term newborns from India.

The biochemical profile of neonates can be affected by various factors, including the mother’s lifestyle choices—such as smoking and alcohol consumption—and her medical history, including conditions like diabetes mellitus, eclampsia, and hypertension[6-9]. Furthermore, factors such as the mode of delivery, number of previous pregnancies, and obstetric and abortion history can also influence neonatal biochemical parameters. These types of variations in reference intervals—arising from geographical location, lifestyle, and other aforementioned factors—highlight the critical need to establish reference intervals that take such variations into account.

Umbilical cord blood collection (UCBC) is a safe and non-invasive method. Neither mother nor fetus get harmed in this. Apart from minor risks associated with delayed cord clamping and cord milking—which may influence certain biochemical parameters[10]—UCBC remains a relatively simple and safe technique for obtaining blood samples. Moreover, studies have demonstrated that UCBC offers higher sensitivity and accuracy in predicting disease outcomes compared to peripheral venous blood collection, supporting its reliability as a predictive tool[11]. Therefore, for the present study, the cord blood collection method was employed. This study aimed to establish reference intervals for cord blood biochemical parameters in a tertiary care hospital and teaching institute in western India.

MATERIALS AND METHODS

Study design and setting

A cross-sectional study was conducted after due approval from the ICEC in a tertiary care hospital attached to a medical college in western part of Maharashtra, India. The study was conducted from October 2022 to December 2022. Study participants were recruited from mothers coming to the hospital to get delivery service.

Sample size determination and sampling technique

Cross-sectional study was carried out among healthy, term neonates (37 to 42 weeks of gestational age) with normal birth weight, born to apparently healthy mothers and receiving delivery services at the institution. Clinical Laboratory Standard Institute (CLSI) guideline C28-A3 recommended at least 120 samples per group for estimating the 95% reference interval[12]. Strategy used was ‘a priori selection’ in which eligible mothers were selected in the age group of 18 to 45 years. Total 210 subjects were recruited in this study.

Mothers were excluded if they had a medical disorder like infectious diseases (Hepatitis B, human immunodeficiency virus, Syphilis), chronic diseases (insulin-dependent diabetes mellitus), complications during pregnancy (preeclampsia) or parturition, psychiatric illness, or addictions (e.g., smoking, excessive alcohol intake). Exclusion was established by a combination of diagnostic tests— such as neonatal screening tests (Perkin Elmer system), thyrotropin, Biotinidase, immunoreactive Trypsinogen determination, results from ultrasound, and clinical history.

The inclusion criteria of mothers were hemoglobin 12.0 g/dL or higher[13], and an inter-pregnancy interval of 18 months or longer, as defined by the World Health Organization[14]. Furthermore, posteriori selection strategy was used to select eligible neonates—i.e., with a gestational age of 37 to 42 weeks and with a 5^th-minute Apgar score of 7 or greater. Neonates with respiratory distress, meconium aspiration, apparent congenital anomalies (e.g., Down syndrome, cystic fibrosis, neural tube defects, or congenital hypothyroidism), or true knots of umbilical cords were not included in the study.

Data collection procedure

Prior to initiating data collection, all involved professionals were briefed on the study objectives, the criteria for participant selection, data confidentiality and safety precautions related to collection, transport, and storage of cord blood samples. To gather demographic details and a brief medical history, a structured questionnaire was administered to mothers who met the inclusion criteria and provided their consent to participate.

Due to the physically demanding nature of childbearing, the attending midwives obtained informed written consent in line with the standards set by the Helsinki Declaration and assisted the consenting women to complete the questionnaire before being admitted to the labor room. Cord blood sampling was performed only after the placenta was fully delivered and separated from the mother, and the umbilical cord was cut following clamping. The cord was clamped within one minute after birth, and 4-5 mL of blood was drawn from the umbilical vein using a sterile needle and syringe. The operation room (OR) nurse collected the samples into vacutainers (BD vacutainers)— one containing sodium fluoride for blood glucose measurement, and plain vacutainers for analyzing serum electrolytes, bilirubin, and creatinine. The samples were sent immediately to the biochemistry laboratory for evaluation.

Screening tests

As per the protocol of routine prenatal care, participants of the study were tested for retro viral disease via rapid diagnostic tests, Australian antigen test and Syphilis using venereal disease research laboratory test. Ultrasonography was done to rule out any gross congenital defects.

Quality assurance

Both external and internal quality control measures were conducted in a timely manner. Internal quality control was performed daily—prior to the test run, intermittently during the run, and upon completion. Cord blood sample collection was carried out by experienced OR nurses in accordance with established guidelines for cord blood collection. All procedures were performed in strict adherence to the respective standard operating procedures.

Biochemical investigations

Over the course of 3 months, 210 blood samples were collected by operation theater staff from umbilical vein of the expelled placenta, into appropriate vacutainers (gray for estimating sugar and plain vacutainer for serological parameters) and sent to laboratory immediately for analysis. Namely plasma glucose, serum creatinine, serum electrolytes and serum bilirubin were estimated using XL- 640 Erba system packs. All the methods on this machine were calibrated by using XL multical solution. Plasma glucose was measured by glucoses oxidase – peroxidase method. Serum electrolytes were estimated using ion selective electrode, creatinine by Jaffe’s reaction and bilirubin by diazo method.

Statistical analysis

After meticulously reviewing all data collected from the questionnaire and laboratory results for completeness and accuracy, we inputted the data into MS Excel and performed further analysis using IBM^® SPSS^® Statistics. As per the CLSI recommendation, we calculated the 2.5^th and 97.5^th percentiles for biochemical parameters in 210 neonates[10], To compare the distribution of parameters across sex, modes of delivery (excluding assisted deliveries), age of the mother, and obstetric history (including prior pregnancies, miscarriages, etc.), the non-parametric Mann–Whitney U test was employed. Descriptive statistics were presented as percentages, ranges, and visualized using charts and graphs. A P value of less than 0.05 was considered statistically significant.

Data were assessed for outliers using the sorting method, and identified outliers were removed using the trimming method. The Shapiro–Wilk test was applied to check whether data is normally distributed or not.

RESULTS

210 full-term, healthy neonates were enrolled in the study, with 112 (53.5%) males and 98 (46.5%) females. Approximately 81.3% of the mothers were in the age group of 18–30 years, and 12% resided outside the metropolitan area. Among the 210 mothers, 27.56% were primiparous. The relatively high proportion of caesarean sections observed in the study may be attributed to a substantial number of referrals from peripheral centers, as the study site serves as the only public-sector tertiary care hospital within a 50-kilometer radius. Complete sociodemographic and clinical characteristics of study participants are presented in Table 1 and Figure 1.

Figure 1 Sociodemographic and clinical characteristics of study participants.

Table 1 Sociodemographic and clinical characteristics of study participants.

Information		Number of subjects	Percentage
Resident of	Mumbai	185	88.2
	Outside Mumbai	25	11.8
Parity	Non–primipara	152	72.4
	Primipara	58	27.6
Obstetric history1	Abortions	40	18.9
	No abortions	170	81.1
Delivery mode	Vaginal delivery	38	18.1
	C-Section delivery2	172	81.9
Sex of neonate	Male	112	53.5
	Female	98	46.5
Weight of neonate	2.5–3.0 kg	164	78
	Above 3 kg	46	22
Maternal age	< 35 years	199	95
	> or = 35 years	11	5
Gestational age of infant	Term (38-42 weeks)	170	81
	Before term	40	19

¹Abortion history implies spontaneous abortions only.

²C-Section delivery includes only elective or planned surgeries.

No statistically significant differences in any of the biochemical parameters were found with respect to the mother's obstetric history (primiparous vs multiparous) or the infant's sex (male vs female) (P > 0.05). An independent Mann–Whitney U test revealed a statistically significant difference in the measured parameters between the different delivery modes (P < 0.05) (As displayed in Table 2).

Table 2 Independent (2 Groups) Mann-Whitney U test (P values less than 0.05 determines the significance level) of selected Biochemical parameters by sex of infant, mode of delivery, age of mother and obstetrics history along with Median and P value.

Parameters	P value			Median
	By sex	Mode of delivery	Obstetric history
Serum bilirubin (T)	0.9278	0.1305	0.1800	1.9
Serum bilirubin (D)	0.5564	0.2016	0.9971	0.3
Serum bilirubin (I)	0.3965	0.1461	0.5466	1.4
Serum creatinine	0.2804	0.7301	0.5292	0.56
Serum sodium	0.4030	0.0128	0.7638	136.3
Serum potassium	0.5892	0.0411	0.7086	5.085
Serum chloride	0.7847	0.1800	0.6612	106
Plasma glucose	0.8293	0.0039	0.8404	57.2

A comprehensive summary of the upper and lower reference intervals for biochemical analytes in neonatal umbilical cord blood has been provided in Table 3. Except serum sodium, potassium and plasma glucose, there was no statistical significance observed in case of mode of delivery.

Table 3 Reference intervals for umbilical cord blood biochemical parameters by sex of the baby.

Parameter	Sex	N	Median	Mean	Max	Min	Reference interval
							Lower limit	Upper limit
Serum bilirubin (T)	Male	112	1.85	1.824	3.2	0.25	0.509	3.139
	Female	98	1.8	1.834	3.17	0.3	0.687	2.981
	Combined	210	1.9	1.879	3.24	0.25	0.618	3.14
Serum bilirubin (D)	Male	112	0.3	0.318	0.6	0.1	0.095	0.541
	Female	98	0.3	0.326	0.6	0.1	0.095	0.565
	Combined	210	0.3	0.354	1.22	0.1	0.097	0.55
Serum bilirubin (I)	Male	112	1.16	1.159	1.8	0.2	0.7276	2.036
	Female	98	1.2	1.334	1.9	0.8	0.7234	1.944
	Combined	210	1.4	1.453	2.5	0.8	0.644	2.262
Serum creatinine	Male	112	0.55	0.492	0.65	0.32	0.248	0.736
	Female	98	0.545	0.515	0.67	0.31	0.282	0.748
	Combined	210	0.56	0.511	0.72	0.31	0.2677	0.755
Serum sodium	Male	112	137	136.075	142.3	137	129.392	142.76
	Female	98	136.2	135.51	142.6	124	127.03	143.99
	Combined	210	136.3	135.72	142.6	124	128.19	143.26
Serum potassium	Male	112	5.31	5.77	9.89	3.9	2.16	9.37
	Female	98	4.955	5.592	9.6	3.9	1.952	9.233
	Combined	210	5.085	5.797	15.41	3.9	1.62	9.98
Serum chloride	Male	112	106.1	105.94	113.3	97	99.65	112.23
	Female	98	106.65	106.08	111	97.8	100.65	111.52
	Combined	210	106	105.94	113.3	97	100.19	111.68
Plasma glucose	Male	112	55.6	59.74	91.8	33.4	17.85	101.62
	Female	98	59	59.073	94.8	42.1	34.742	83.4036
	Combined	210	57.2	59.49	94.8	31.6	24.75	94.23

The combined median values and central 95^th percentile reference intervals for cord blood analytes are presented in Table 3. We also have included a comparative analysis between the existing reference intervals of cord blood and those provided by Sysmex for neonates aged 0–24 hours, along with reference data from previously published studies. As most previous studies have reported their findings as mean ± SD, the comparisons in the present study were performed accordingly and are presented in a similar format in Table 4.

Table 4 mean ± SD or median comparison with company derived values, other published reference values and peripheral venous blood with current study results.

Parameter	PVB[19]	Current Study			China[17]	Korea[16]
	RI	RI	Median	Mean	Median	Mean
Direct bilirubin (mg/dL)	0.33–0.71	0.1-0.55	0.3	0.354	NA	NA
Indirect bilirubin (mg/dL)	NA	0.64-2.262	1.4	1.453	NA	NA
Total bilirubin (mg/dL)	0.19–16.6	0.62-3.14	1.9	1.879	NA	0.5
Creatinine (mg/dL)	0.32–0.92	0.27-0.76	0.56	0.511	NA	0.38
Chloride (mEq/L)	102–111	100.19-111.68	106	105.94	101	NA
Potassium (mEq/L)	3.7–5.9	1.62–9.98	5.085	5.797	4.05	NA
Sodium (mEq/L)	133–146	128.19-143.26	136.3	135.72	135	NA
Glucose (mg/dL)	30–60	24.75–94.23	57.2	59.49	NA	96.5

PVB: Peripheral venous blood; NA: Not available; RI: Reference interval.

A complete summary of sex specific upper and lower margins for the biochemical parameters’ reference ranges of from umbilical cord blood is provided in Table 3. The combined median, mean and central 95 percentiles reference interval of cord blood parameters are also shown in Table 3.

DISCUSSION

Establishing reference intervals for neonates, infants, and children is demanding task as their physiological attributes undergo constant changes during their development[15]. During the initial hours and days after birth, there are notable alterations in the blood, particularly in newborns[16]. Furthermore, the majority of research studies have been performed on hospitalized individuals instead of healthy neonates, resulting in limited evidence for establishing reference intervals for neonates[15]. Consequently, it is crucial to establish these reference intervals specifically for healthy neonates.

There was no previously reported statistical significance in the mode of delivery, sex of the infant or the obstetrics history for serum creatinine which is in accordance with the study conducted in Korea[16].

There was no statistical significance in the mode of delivery for serum sodium, potassium and chloride levels which is in accordance to the result obtained from a study conducted in China[17], it is important to note that this study describes not cord blood parameters but postnatal (day 1-3) levels of concerned parameters. Although a statistical significance was obtained in case of mode of delivery for serum levels of potassium in this study.

This study also found narrowed reference interval for serum levels of total bilirubin, sodium whereas widened interval is seen in serum direct bilirubin, chloride, potassium and plasma glucose.

The high glucose levels in the cord blood of Indian neonates might be associated with the higher prevalence of gestational diabetes mellitus among pregnant women in India. Another reason might be the lower insulin sensitivity and higher fat mass in Indian babies which could lead to higher glucose levels. A higher glucose level in Korean neonates may be associated with their higher levels of insulin resistance and lower insulin secretion capacity in comparison to Indian neonates[18].

A higher prevalence of maternal anemia in the Indian population can lead to fetal hypoxia and acidosis which may be associated with the high levels of potassium in the fetal bloodstream. Stress due to labor may also be a cause for these high levels. After comparing the two studies, it was realized that Indian cord blood has elevated potassium levels as compared to Chinese cord blood. This may be associated with differences in fetal-maternal circulation as well as difference in maternal nutrition. The high level of potassium due to hemolysis is ruled out as hemolysis would result in evident elevation of other electrolytes as well.

Lowered serum sodium in Indian cord blood in comparison to peripheral venous blood may be due to high prevalence of malnutrition. Another cause may be difference in environmental factors such as temperature and climate. Indian samples showed higher sodium levels than Chinese which may be associated with dietary preferences in India i.e. higher salt intake.

Deviations seen in Indian levels of bilirubin maybe associated with different dietary preferences, different environmental factors, and genetic variabilities.

CONCLUSION

Establishing reference intervals is essential for interpreting biochemical variations in neonatal care. However, to date, there are only few published reference intervals for cord blood biochemical markers in neonates from India. The current study provides various statistical parameters (i.e., Reference interval, mean, median etc.), that enables the reader to develop a comprehensive understanding of the available literature. Although the study was conducted on a smaller population in Mumbai, its applicability is broad due to the diversity of the participants' backgrounds. Nevertheless, confirmation of the results is necessary through larger sample sizes from different regions of the country to ensure wider utilization.

Limitations

A key limitation of this study is the absence of stratified reference intervals based on variables such as ethnicity, gestational age, and mode of delivery. Ideally, separate reference ranges accounting for these factors would enhance clinical applicability; however, this study provides generalized intervals without such subgroup differentiation.

ACKNOWLEDGEMENTS

The authors would like to acknowledge study participants for their kind collaboration, Rajiv Gandhi Medical College and Chhatrapati Shivaji Maharaj Hospital for the material support, labor room and operation theatre nurses, and lab technicians of the hospital for the unreserved support.