Serum markers may distinguish biliary atresia from other forms of neonatal cholestasis

Abstract

Background: Biliary atresia (BA) is the most serious liver disease in infants. Diagnosis currently depends on surgical exploration of the biliary tree. Noninvasive tests that distinguish BA from other types of neonatal liver disease are not available.

Patients and methods: To identify potential serum biomarkers that classify children with neonatal cholestasis, we performed 2-dimensional difference gel electrophoresis, statistical analysis, and tandem mass spectrometry using serum samples from 19 infants with BA and 19 infants with non-BA neonatal cholestasis.

Results: Eleven potential serum biomarkers were found that could in combination classify children with neonatal cholestasis.

Conclusions: Although no single biomarker or imaging test adequately distinguishes BA from other types of neonatal cholestasis, combinations of biomarkers, imaging tests, and noninvasive clinical criteria should be further explored as potential tests for rapid and accurate diagnosis of BA.

Figure 1

Workflow for biomarker discovery.

Figure 2

Representative 2-D and 3-D DIGE gels. (A,B) Identical images are shown (A) Highlighted spots are more abundant in BA than non-BA liver disease. (B) Highlighted spots are more abundant in non-BA than BA liver disease. Numbers beside spots correspond to numbers in Table 2. (C, D) Representative 2-D and 3-D images of single spots demonstrating quantitative differences between individuals. (C) Spot 4, Figure (A). (D) Spot 11, Figure (B).

Figure 3

Classifier Validation. (A) The relative abundance of 11 proteins in Table 2 correctly classified 9/10 of the children in the “test set”. (B) RDA algorithms are influenced by lambda and gamma parameters. Receiver operating characteristic (ROC) curve was generated by varying lambda and gamma, but maintaining the same 11 peptide spots as biomarkers. Sensitivity indicates the likelihood that the classifier generates an accurate classification. 1-specificity indicates the likelihood that the classifier generates an inaccurate classification.

Figure 4

Heat map. Relative abundance of potential biomarker proteins in infants with BA or non-BA cholestasis. Each column represents an individual child’s serum. Each row represents an individual protein. Colors show the protein abundance relative to that protein’s abundance in pooled samples. A scale bar is at the bottom. The abundance of single proteins cannot be used to classify serum samples, but trends in peptide abundance are evident. The top 7 proteins tend to be more abundant in serum of non-BA cholestatic infants than in children with BA. The bottom 4 proteins tend to be more abundant in children with BA than with non-BA liver disease. * Alpha-1 antitrypsin deficiency, # CMV IgM positive.