The Coagulation Analysis of Reveals Novel Biomarkers for Neonatal Hyperbilirubinemia in Full Term Newborn

Background: The coagulation test is used to evaluate in various diseases. Little is known about correlation between the coagulation test and neonatal hyperbilirubinemia. The aim of the study was to determine the neonatal coagulation analysis value in infants with neonatal hyperbilirubinemia as reliable markers. Methods: All newborns were born and inpatients in the newborn wards of the Chengdu Second People’s Hospital. Measurement of serum bilirubin was done to diagnose hyperbilirubinemia. All neonates involved were full term of either gender without any signicant illness or major congenital malformations. Data were collected from newborns who had neonatal hyperbilirubinemia and healthy in Chengdu Second People’s Hospital. Prothrombin time(PT), Thrombin time(TT), Fibrinogen(Fbg), activated partial thromboplastin time(APTT), and calculated international normalized ration(INR) values were recorded. Linear relationship between INR and level of total bilirubin and indirect bilirubin were analyzed by linear regression. The receiver operating curve(ROC) and the area under the curve(AUC) were made evaluated. Results: In this case-control study, the mean PT, APTT level were signicant higher in the infants with hyperbilirubinemia group compared to the healthy infants. We found the positive correlation between INR and total bilirubin(R=0.3327; P<0.0001). Furthermore, there was also signicant positive correlation between INR and indirect bilirubin(R=0.3402; P<0.0001). Meanwhile, INR in neonates with hyperbilirubinemia signicantly achieved an AUC of 0.800(95% CI=0.6288-0.9712, cutoff value 1.060, specicity71.43%, and sensitivity 80.00%). Conclusions: PT, APTT were signicant high in neonatal hyperbilirubinemia group. Correlation were observed between the INR and neonatal hyperbilirubinemia. Importantly, the study revealed INR as novel biomarkers for diagnosis of neonatal hyperbilirubinemia in full term newborn.


Introduction
Neonatal hyperbilirubinemia was a common problem in newborns [1]. Hyperbilirubinema was a major risk factor for brain damage, bilirubin-induced neurologic dysfunction [2]. It was manifested through yellowish change of skin and mucous membrane [3].
And neonatal jaundice induced blood incompatibility [4]. Some studies have shown the correlation of neonatal hyperbilirubinemia include G6PD de ciency, serum vitamin D level, ASO haemolytic disease [4][5][6]. Some causes of signi cant hyperbilirubinemia were relation with erythrocyte membrane defects, red blood cell enzyme de ciencies [7,8]. For coagulation system, the interaction of coagulation factors has been found to go beyond their traditional effect and to affect some diseases [9,10]. So we appreciated the correlation between hyperbilirubinemia and coagulation system.
The present study was therefore to determine the relationship between PT, PATT, Fbg, TT and hyperbilirubinemia in full term neonates. Through the coagulation analysis, we reveals novel biomarkers for the diagnosis of neonatal hyperbilirubinemia in full term newborn.

Material and methods
This study was conducted in Chengdu, Sichuang Province, China, from October 2019 through September 2020. All newborns were born and inpatients in the newborn wards of the Chengdu Second People's Hospital. The birth weight of full term neonates were included in the study. All neonates involved were full term of either gender without any signi cant illness or major congenital malformations. The neonates with conditions that could aggravate hyperbilirubinemia, such as sepsis, respiratory distress syndrome, asphyxia, diabetic mothers, or intrauterine growth retardation, were excluded from the study. In this casecontrol study, 40 eligible infants with signi cant hyperbilirubinemia were compared with 30 healthy infants. Additionally, written consent was obtained from all of the parents before any procedures were performed. Hyperbilirubinemia was determined by testing serum bilirubin and clinical manifestation on the second day before they were discharged from the hospital. Increasing of the total bilirubin level more than 34umol/L was considered as hyperbilirubinemia. Venous blood samples were collected and sent for prothrombin time(PT), Thrombin time(TT), Fibrinogen(Fbg), activated partial thromboplastin time(APTT), and calculated international normalized ratio(INR).

Statistical analysis
Statistical analysis Data was entered and analysed using GraphPad Prism 8. Mean and standard deviation were used for the analysis of the data. The unpaired test was used to compare the two groups and line regression to compare the correlation in terms of the Pearson's Correlation Coe cient. For interpretation of results, signi cance was adopted at P < 0.05.

Clinical characteristics
In the study, we selected 70 newborns to evaluate in two groups, including 40 neonatal hyperbilirubinemia infants in the case group and 30 healthy neonatal infants in the control group. No asphyxia and in ammation occurred in 70 newborns. Baseline clinical characteristics were similar in the two groups (Table 1). But there was signi cant difference in WBC, PLT, HGB in the two groups( Table 1). The mean WBC in hyperbilirubinemia group (12.98 ± 4.67×10 9 /L) was signi cantly higher than that of the control group(6.55 ± 1.63×10 9 /L)(P < 0.0001). The mean PLT in hyperbilirubinemia group(329.25 ± 76.77×10 9 /L) was signi cantly higher than that of the control group(255.13 ± 38.11×10 9 /L)(P < 0.0001). The mean HGB in hyperbilirubinemia group(162.15 ± 17.41g/L) was signi cantly higher than that of the control group(134.33 ± 11.25g/L)(P < 0.0001).  Table 2). The mean PT in hyperbilirubinemia group(12.54 ± 1.39 second) was signi cantly higher than that of the control group(11.3 ± 0.63 second)(P < 0.0001). The mean INR in hyperbilirubinemia group(1.14 ± 0.13 ) was signi cantly higher than that of the control group(1.03 ± 0.06)(P = 0.0001). The mean APTT in hyperbilirubinemia group(47.1 ± 8.89 second) was signi cantly higher than that of the control group(27.67 ± 1.97 second)(P < 0.0001). Table 2. Comparison of various characteristics between the signi cant neonatal hyperbilirubinemia groups and control groups the relationship was observed between INR and level of total bilirubine As shown, the relationship was observed between INR and level of total bilirubine(R = 0.3327;P < 0.0001) (Fig. 1). As shown in Fig. 2, the relationship was also observed INR and level of indirect bilirubin(R = 0.3406; P < 0.0001) (Fig. 2). The e cacy of detection of INR was reported using the ROC AUC phenomenon in neonates with hyperbilirubinemia. INR in neonates with hyperbilirubinemia signi cantly achieved an AUC of 0.800(95% CI = 0.6288-0.9712, cutoff value 1.060, speci city71.43%, and sensitivity 80.00%) (Fig. 3).

Discussion
The study found the relationship between coagulation test and hyperbilirubinemia in full term infants. The mean PT, APTT level were signi cant higher in the infants with hyperbilirubinemia group compared to the healthy infants. At the same time, the mean serum PCT level was higher in the infants with hyperbilirubinemia group. In our study, the WBC, PLT and HGB level in hyperbilirubinemia group were higher than the healthy group(P < 0.0001). We found there is the correlation between INR and total bilirubin(R = 0.3327; P < 0.0001). There is also correlation between INR and indirect bilirubin(R = 0.3406; P < 0.0001).
Hyperbilirubinemia often occurred in infants without apparent reason [11]. The causes of hyperbilirubinemia and effective prevention strategies have recommended as clinical guidelines [12]. The American Academic of Pediatrics(APP) recommends a follow-up visit after 48-72 h of the newborns discharged [13]. For the main cause of neonatal hyperbilirubinemia, a large of study have received developments [14]. Compared with other study, we used INR to evaluate the correlation between hyperbilirubinemia and coagulation. The coagulation system have easily activated by in ammation, severe disease in human [15]. Therefore, in this study, we evaluated the hyperbilirubinemia and health groups, PT and APTT were signi cantly prolonged in infants with hyperbilirubinemia than health control and there was signi cant difference(P < 0.0001).
The e cacy of detection of INR was reported using the ROC AUC phenomenon in neonates with hyperbilirubinemia. INR in neonates with hyperbilirubinemia signi cantly achieved an AUC of 0.800(95% CI = 0.6288-0.9712, cutoff value 1.060, speci city71.43%, and sensitivity 80.00%).

Conclusion
In Conclusion, our study suggested that hyperbilirubinemia in infants may induce coagulation system to change. Maybe, the change of coagulation system may be a cause of early signi cant hyperbilirubinemia. Moreover, it appears to have a pathogenic potential in newborns, high PLT may be a potential problem. Importantly, we may consider coagulation system when hyperbilirubinemia was observed. We recommend the INR as novel biomarkers for diagnosis of neonatal hyperbilirubinemia in full term newborn.