Dry Blood Spot Screening Test for Neonatal Cholestasis Patients
Dry Blood Spot Screening Test for Neonatal Cholestasis Patients
Cholestatic jaundice with multitude of causes affects approximately 1 in every 2,500 infants.
 Of the many conditions that cause neonatal cholestasis, the most commonly identifiable are
 biliary atresia (BA) (25%-35%). The incidence in Taiwan was 1.2 to 2.0 per 10,000 live birth.
 Prognosis and survival are improved if bile drainage is restored by a Kasai portoenterostomy.
 Stool color card was introduced to Taiwan in 2002 and national screening program was started
 in 2004. The rate of age at Kasai operation <60 days improved from 49.4% before year 2002 to
 65.7% after introducing nationwide stool color card screening in Taiwan. There was a great
 improvement in early diagnosis for biliary atresia after stool color card screening and there
 were still many researches tried to improve the timing of diagnosis. A prospective cohort
 observational study on neonates under 27 days old at London showed that serum conjugated
 bilirubin > 18 micromol/l in plasma measured at 6-10 days is a reliable marker for neonatal
 cholestasis liver disease with sensitivity 100%, specificity 99.59% (95% CI 99.5-99.67), PPV
 10.3% (95% CI 4.50-16.0).

 Our study aims to developed a screening test for neonatal cholestasis using dry blood spot to
 improve patient survival by early diagnosis of treatable neonatal cholestasis disease, such
 as biliary atresia and inborn error of bile acid metabolism. The diseases markers for
 neonatal cholestasis we aims including basic clinical blood test examination profiles,
 inflammatory markers, fibrosis markers, immune profiles. In phase I study we will enroll
 cholestasis patients at National Taiwan University Children Hospital. We will collect
 patients' blood on dry blood spot and measure the disease markers for cholestasis disease. In
 phase II study, we will enroll the cholestasis patients and healthy newborn without
 cholestasis. We will review their newborn screening at 3 days old. Newborn screening dry
 blood spot will be examined for the disease markers and compared with healthy control without
 cholestasis. ROC curve analysis will be performed to find the best cut-off to screen for
 neonatal cholestasis patients.

 The findings will aid early diagnosis in the patients and hence improve the survival.
Aims:

 1. Developed a screening test for neonatal cholestasis using dry blood spot

 2. Improve patient survival by early diagnosis of treatable neonatal cholestasis disease
 such as biliary atresia and inborn error of bile acid metabolism.

 Phase I - Validation of method for quantification of disease markers on dry blood spot.

 Pediatric children with cholestasis diagnose at National Taiwan University Hospital during
 admission or out-patient clinic will be collected for the Phase I study. 5ml of venous blood
 will be collected for measurement of disease markers during routine follow-up at National
 Taiwan University Hospital outpatient clinic. Dried blood spot (DBS) specimens are collected
 from patients by applying a whole blood, drawn by lancet from the finger, heel or toe, a
 provided filter paper. To evaluate the stability of disease markers on DBS, a total 10 blood
 spots (each blood spout contain 50 - 75 microL of blood) will be applied on the filter paper
 at day 0. The collected blood spots samples of a single patient at day 0 will be evaluated at
 the day of DBS collection and followed by day 3, day 7, 6 months and 1 year after sample
 collection. The purpose of repeat measurement of single patient sample at different time
 point is to evaluate the possible effect of time effect on the accuracy of the data. 5 blood
 spots are separated and protected from light with a light blocking envelope to compare the
 light effect on disease markers degradation (Table 1). Serum disease markers measured with
 conventional method will be served as reference and compared with DBS samples at different
 time points. 30 patients will be collected to validate the correlation between serum and DBS
 disease markers level.

 Phase II - Pilot study In the pilot study, we aim to develop dry blood spot method for the
 screening of neonatal cholestasis. Children with neonatal cholestasis disease previously
 diagnosed at Department of Pediatrics, National Taiwan University Children's Hospital from
 2007 or newly diagnosed children will be recruited retrospectively from chart review.
 Patients with indirect type hyperbilirubinemia and healthy neonates will be enrolled as
 control. We will analyze neonatal cholestasis and control patients dry blood spot screening
 test after birth to quantify disease markers level. ROC curve analysis will be conducted on
 bilirubin quantity to find the best cutoff to screen for neonatal cholestasis. The sample
 size of case and control study is 20 cases and 20 controls.
Inclusion Criteria:

 -  Patients with jaundice

 -  Healthy volunteers

 Exclusion Criteria:

 -  Subjects who do not agree with study protocol.