Biliary atresia is the most common cause of neonatal cholestasis and 40–50% of liver transplantation in children is caused by this disease 
. Early intervention of Kasai portoenterostomy is very important for BA patients. When the surgery is performed before the age of 60 days, the success rate of establishing biliary drainage is higher than those performed later 
and the need for liver transplantations in infancy and childhood is greatly reduced 
In this study, we evaluated the six primary bile acids in dried blood spots of BA, neonatal jaundice and comparison infants obtained 3–4 days after birth. All the conjugated primary bile acids have already increased in BA newborns at 3–4 days of life which echoes the report that all these bile acids were elevated significantly in BA infants 7–10 days after birth 
. The results indicated that BA was present in the immediate newborn period or late pregnancy. This is also supported by a case report by Toubi et al., where a BA infants with disappeared gallbladder at age of 11 days was used to have normal gallbladder at the gestational age of 24 weeks 
. In our study, the increase of TC and GC was higher than TCDC and GCDC in early life of BA, while Mills et al. found that the later 2 bile acids increased more significant in cholestatic children at age of 3 weeks to 8 month 
. These findings may indicate that the liver damage has already begun but is still mild in BA infants within 3–4 days of age as greater rise of TCDC and GCDC meant severe liver damage 
. Furthermore, TC was found positively correlated with MCP-1, an HSC-responsive chemokine, in cholestatic rats and in cystic fibrosis liver disease 
. The early elevation of TC may indicate the early occurrence of fibrosis in BA patients.
According to previous research, two of the most promising screening methods for BA yet are early measurement of serum conjugated bilirubin and usage of stool color cards. In the test of serum conjugated bilirubin at 6–10 days after birth, the sensitivity and specificity reach 100% and 99.6% respectively 
. However, due to the light sensitivity of bilirubin, it is unsuitable to detect in dried blood spots, which are easy to obtain and deliver for newborn screening. Another method, the stool color card is widely used in Taiwan to screen BA patients. The sensitivity of this method was 97.1% in 2005 
. However the onset of pale stool in a large amount of cases usually occurs later than 30 days of age, which may delay the diagnosis and affect the outcome of treatment. Measurement of TC in dried blood spots by LC-MS/MS may be suitable for newborn screening due to the stability in dried blood spots and relatively simple detection technique, although the sensitivity and specificity are not as good as serum conjugated bilirubin and the stool color card. Furthermore, considering the effect of diurnal variations on the level of bile acids in blood 
, the detecting accuracy may increase if all blood spots were taken forenoon. After all, the elevated concentrations of TC and other bile acids in dried blood spots from BA infants have already indicted the potential screening strategy for the future study.
Interestingly, no significant difference of bile acids was observed between neonatal jaundice and comparison infants. This result was understandable. It is well known that genetic and environmental factors influence neonatal jaundice, such as ABO incompatibility, breastfeeding and the method of delivery 
. The infants with neonatal jaundice enrolled in this study have unconjugated hyperbilirubinemia which are unrelated with hepatobiliary disease.
The main shortage of this study is the small sample size of BA. Because of the scarcity of the disease, insufficient therapeutic effects and low levels of cooperation among pediatric hospitals in China, it is hard to obtain screening dried blood spots from BA patients. Furthermore, the detection methods need to be improved, which include but are not limited to the introduction of UPLC, more sensitive MS and proper sample pretreatment methods.
In conclusion, bile acids were elevated in BA infants at 3–4 days of age indicates the immediate occurrence of the disease at birth. The measurement of bile acids, especially TC, in dried blood spots from newborns could be the potential screening strategy for early detection of BA.