AlphA-fetoprotein As A biochemicAl diAgnostic And prognostic mArker for prolonged jAundice in newborns

Prolonged jaundice of newborns is a common pathology during the neonatal period. recently, there has been a tendency toward an increased number of newborns with prolonged jaundice with duration longer than 14 days of life. according to the polyetiologic nature of neonatal jaundice, it is necessary to investigate new diagnostic signs that can predict the development of prolonged jaundice in newborns and, allow identification of new methods of differential diagnosis of neonatal jaundice, as well as decrease the frequency of this pathology. the parameters of serum alpha-fetoprotein (aFP) content in newborns with prolonged jaundice were studied. the content of aFP in blood serum was 1.7 times higher in newborns with prolonged jaundice than in newborns with jaundice for up to 14 days of life: Me = 671.1 [Q1 = 479.9; Q3 = 815.03] ng/ml and Me = 401.0 [Q1 = 284.9; Q3 = 684.0] ng/ml, respectively (P < 0.05). the content of blood serum aFP in newborns with prolonged jaundice was higher for total serum bilirubin greater than 250 μmole/l: Me = 626.2 [Q1 = 454.7; Q3 = 793.2] ng/ml which was confirmed by a strong direct correlation relationship (rxy = 0.64, P < 0.05). The results of the ROC analysis confirmed that AFP ≥ 571.7 ng/ml can identify newborns with prolonged jaundice with a sensitivity of 74.2% and a specificity of 74.5%. The area under the curve (AUC) was 0.870 (95% CI 0.804, 0.937), which confirms the good quality of the AFP model ≥ 571.7 ng/ml.

eonatal jaundice occurs in 50% of term and 80% of preterm newborns all over the word [1]. Jaundice is one of the most common causes of neonatal readmission to hospital, especially during the first 2 weeks after birth. The icteric color in most newborns may accompany postnatal adaptation, but may also be a manifestation of the pathological accumulation of serum bilirubin.
Of particular note are jaundices with a prolonged course, when term newborns remains clinically jaundiced at 14 days of postnatal life or after 21 days in premature newborns. Up to 30-40% of breastfed newborns remain jaundiced for the first 3 to 4 weeks of life [2][3]. The most common cause is breast milk jaundice which is characterized by a satisfactory state of newborns, normal physical development and in most cases does not require medi-cal intervention. This type of jaundice is diagnosed by excluding other pathological conditions and, a number of disease processes including haemolysis, sepsis, hypothyroidism, cystic fibrosis, metabolic disease and liver disease (mainly congenital hepatitis B/C or biliary atresia) [4][5][6].
The risk of the development of indirect hyperbilirubinemia of prolonged genesis is necessary to predict, since newborns are discharged from the hospital at the peak of physiological jaundice. Prolonged hyperbilirubinemia of greater than 2 to 3 weeks duration requires additional investigation [7][8][9][10].
Excessive accumulation of unconjugated bilirubin in the blood can potentially lead to acute and chronic bilirubin encephalopathy (kernicterus) or can lead to long-term complications such as cerebral palsy or neuropathy of the auditory analyzer [11,12]. There is also evidence that excessive accumulation of unconjugated bilirubin in the blood can lead to disturbances of intestinal microbiocynosis, which is due to the anatomical and physiological features of the digestive tract of newborns: the absence of specific microflora contributing to the conversion of indirect bilirubin to sterkobilin, as well as the connection of the hepatobiliary system and the intestine (enterohepatic circulation) [13][14][15][16].
An analysis of the foreign medical literature indicates that the severity and duration of indirect hyperbilirubinemia is determined by etiological and pathogenetic features and requires a differentiated approach to the diagnosis and treatment of neonatal jaundice, as well as optimization of the prevention of adverse factors that can lead to a decrease in the frequency of this pathology [17].
Foreign studies have shown ambiguous effects of serum alpha-fetoprotein (AFP) on the development of prolonged jaundice in newborns. AFP, by its physical and chemical properties, is similar to the main protein of blood serum, namely albumin. AFP has long been used as a marker in prenatal diagnosis for the detection of developmental fetal defects and as a cancer marker in malignant neoplasms [18].
However, the mechanisms of a postnatal increase in AFP remain unexplained, but possibilities include an increased rate of synthesis in the liver, or prolonged half-life due to altered clearance of blood. During the first year of life, AFP gradually ceases to be synthesized by the liver and is completely eliminated from the body of the child by two years. An increase in the AFP content by 95.0% or prolonged preservation of its synthesis after birth can indicate liver damage. Determination of the content of AFP would allow establishment of the mechanisms of the prolonged jaundice in the early stages of its development and would broaden the thinking of pediatricians, neonatologists, and family physicians about the peculiarities of the course of prolonged jaundice in newborns [19].
Thus, the purpose of this study was to determine the role of serum AFP levels in newborns with prolonged jaundice.

materials and methods
This prospective study was performed in the neonatology unit of the Regional Children's Clinical Hospital in Vinnytsia, Ukraine. The study methodology was approved by the Ethics Committee of the Vinnytsya National Pirogov Memorial Medical University (Protocol №4). Informed consent was obtained from the parents of all newborns for their blood sampling.
In total, 65 newborns were enrolled in this study. Thirty-five of the 65 newborns (examination group) had unexplained prolonged indirect hyperbilirubinemia. The remaining 30 newborns (control group) were healthy neonates and had physiological jaundice during their follow-up period.
Study inclusion criteria were: breastfed newborns with duration of jaundice longer than 14 days of life, gestational age 37-41 weeks, body weight > 2500 g, and total serum bilirubin concentration above 205 μmolе/l (12 mg/dl).
Study exclusion criteria were as follows: blood group incompatibilities, haemolytic anemia, absence of congenital anomalies, hepatobiliary diseases, congenital and acquired infectious diseases including hepatitis, congenital hypothyroidism, congenital metabolic disorders, or delayed intrauterine develop ment, as well as gestational period less than 37 weeks.
None of the newborns in the examination group or control group had a history of any important medi cal problem that developed in the postneonatal period. All newborns were breastfeeding.
For each newborn, the following records were reported: gestational age, mode of delivery (cesarean section versus vaginal delivery), Apgar score at birth, sex, anthropometric characteristics (body weight and body length), age, and clinical examination.
Blood samples for serum (AFP) measurement in the examination group were obtained when blood was drawn from the newborn for other tests necessary for evaluating the cause of jaundice: general blood test, biochemical (direct [dBil] and indirect [iBil]) bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Blood samples from the control group were conveniently available from newborns who were admitted to our neonatology unit due to the puncture of a cephalohematoma. All blood samples were obtained by venipuncture and collected in sterile tubes, placed on ice, and immediately sent to the laboratory. After centrifugation of blood (1000 g for 30 min at 4 °C), the plasma supernatant fluid was separated and stored at -20 °C until analyzed further.
Serum AFP measurements were made using an AccuBind® ELISA Test System (Monobind Inc., Lake Forest, USA). The quantitative determination of serum AFP concentration was made by a colorimetric microplate enzyme immunoassay. In the wells of the plates (on the walls of which were adsorbed antibodies to AFP), 25 μl of standard solutions were added (AFP at 0; 5; 25; 50; 250; 500 ng/ ml) along with 25 μl of blood serum samples and 100 μl of anti-AFP enzyme reagent. The wells were stirred, covered with adhesive film, and incubated for 60 min at 25°C to form the AT-AG-AT-enzyme complex on the solid phase. The wells were rinsed to remove unbound reagents, and then 100 μl of the chromogenic substrate was added. The wells were stirred again and incubated for 15 min at 25 °C. The reaction was stopped with 50 μl of stop solution and photometrated at 450 nm (differential filter of 630 nm) on the STAT FAX 303/PLUS automatic analyzer which is an open system (USA). The sensitivity of the kit was 1 ng/ml AFP, with a variation factor <10%.
Statistical analysis was performed using the software package STATISTICA 6.0 (StatSoftInc., 2001, USA), "Microsoft Excel". Indicators that charac terize the normal data distribution are presented as mean ± standard deviation (SD). Nonparametric data are presented as the median (Me) and boundaries of the interquartile segment [Q1; Q3] (quartile). The Kolmogorov-Smirnov criterion was used to estimate the probability of differences between independent statistical groups with nonparametric distribution. The exact method of Fisher was used for the relative values (percentage). The differen ce in parameters was considered statistically significant at P < 0.05. The Spearman rank correlation was used as a criterion for the statistical depen dence between the parameters. The receiver operating characteristic (ROC) curve was plotted to determine the predictive value of serum AFP in newborns with prolonged jaundice. The results represent the value of the area under the curve (AUC) of the ROC curve based on the values of the sensitivity and specificity of the test, and indicate a 95% confidence interval (CI).

results and discussion
The gestational age, age at hospital admission, sex, and anthropometric characteristics of the newborns in the two study groups (examination and control) are presented in Table 1.
The mean gestation ages in the examination group and the control group were not statistically significantly different (38.9 ± 0.2-versus 39.2 ± 0.2 weeks, respectively, (P > 0.05)). The mean age of the examination group at the time of hospital admission was significantly greater than that of the control group (20.1 ± 0.7 versus 10.1 ± 1.7 days, (P < 0.05)).
Among the newborns of both groups, there were significantly more boys: 26 boys (74.3%) and 9 girls (25.7%) in the examination group, and 24 boys (80.0%) and 6 girls (20.0%) in the control group (P < 0.05). According to percentile tables for normal growth, the physical development indicators were within the 10-90 percentiles. In newborns of the examination group, the mean body weight at birth was 3312.1 ± 58.1 g and the length was 51.6 ± 0.4 cm. In the control group, the mean body weight was 3349.7 ± 100.1 g and the length was 51.7 ± 0.4 cm. There were no significant differences in mean birth weight or mean length between the two groups (P > 0.05).
In most of the 35 newborns of the examination group, the state of adaptation after birth was conside red satisfactory according to the Apgar score: 30 newborns (85.7%) had > 8 points at the first and fifth minutes of life. The other 5 newborns (14.3 %) had 6 points at the first minute and 8 points at the fifth minute. t a b l e 1. characteristics of the two study groups (mean ± SD), % *a significant difference versus the control group (P < 0.05); # a significant difference versus the girls of the two study groups (P < 0.05)

Characteristics
Examination In the examination group, 25 newborns (71.4%) had been transferred to the neonatology unit by the referral of a family physician. The reason for hospitalization was prolonged jaundice which had no tendency to decrease. The other 10 newborns (28.6%) were transferred from the maternity hospital.
In 29 newborns (83.0%) of the examination group, jaundice appeared on the 3 rd day of life, and in the other 6 newborns (17.0%) on the 4 th day of life. In all newborns of the control group, jaundice appeared on the 3rd day of life.
The risk factors for the development of prolonged jaundice in newborns are complications during pregnancy and childbirth, the presence of gynecological and extragenital pathology in mothers , and onerous obstetric history, as well as prematurity, asphyxia, and cerebral ischemia [20][21][22]. The maternal age, weight, body mass index (BMI), numbers of pregnancies and prolonged delivery were significantly associated with bilirubin levels [23][24][25].
In order to determine the possible influence of perinatal factors on the development of prolonged jaundice in newborns, we investigated the features of pregnancy in the mothers of the newborns in this study. We found that the majority of newborns (21 newborns, 60.0%, P < 0.05) in the examination group were born from mothers who had a complicated pregnancy. Among the most frequent complications of pregnancy, were a threat of interruption in 11 mothers (52.3%), acute respiratory viral infection in 6 (28.5%), and anemia in 4 (19.2%) (P < 0.05).
In the examination group, 30 newborns (85.7%) were born by vaginal delivery and 5 newborns (14.3%) by caesarean section. All newborns of the control group were born by vaginal delivery (P > 0.05).
The neonatal period in all newborns of the examination group was complicated only by hyperbilirubinemia. Activity and congenital reflexes were present, and all newborns actively sucked their mother's breasts. Except for jaundice, all patients had no other pathological symptoms. The dimensions of parenchymal organs (liver and spleen) were normal for the age, according to clinical and ultrasound data. The color of urine and feces had a physiological character was physiologically normal.
Thus, the risk factors for the development of prolonged jaundice of the newborn, according to our data are male sex and complicated obstetric history.
The hematological and biochemical parameters of the study groups are shown in Table 2.
Analysis of the content of serum AFP showed that this indicator was 1.  Table 3).

Indicators
Examination group, (n = 35) Control group, (n = 30) P value Hemoglobin (g/l) 172. Correlation analysis established a strong direct correlation between the content of AFP and total serum bilirubin >250 µmole/l in newborns of the examination group (r xy = 0.64, P < 0.05). Also in the examination group, there was a somewhat weaker direct relationship between AFP and total serum bilirubin in the range of 201-250 µmole/l (r xy = 0.34, P < 0.05).
In order to assess the prognostic value of a diag nostic method for assessing the risk of developing prolonged jaundice in newborns, ROC analysis was performed (Fig.).
The results of the ROC analysis confirmed that AFP ≥ 571.7 ng/ml can identify newborns with prolonged jaundice with a sensitivity of 74.2% and a specificity of 74.5%. The AUC was 0.870 (95% CI Our data suggest that in newborns with jaundice lasting longer than 14 days of life there were higher levels of serum AFP in comparison with nonjaundiced newborns. The mechanism by which postnatal AFP levels are elevated in jaundiced newborns remains to be clarified. We can speculate, that the increased content of serum AFP competes with albumin, displacing it from the binding zones with bilirubin and as a result of such competition, increases the concentration of indirect bilirubin which diffuses into body tissues, as well as into the enterohepatic circulation, which increases the load on the enzymatic and excretory capacity of the liver [26]. The practical focus of our work is to include an assessment of the serum content of AFP in the evaluation of newborns with duration of jaundice more than 14 days, that will differentiate the mechanisms that lead to a prolonged course of neonatal jaundice and start treatment in a timely manner.