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Peripheral blood CD177+ cells as an early diagnostic marker for biliary atresia: A prospective multicentre study in pediatric patients with cholestasis

  • Author Footnotes
    # Co-first authors
    Ruizhong Zhang
    Footnotes
    # Co-first authors
    Affiliations
    Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China

    Department of Pediatric Surgery, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China
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  • Author Footnotes
    # Co-first authors
    Junyu Huang
    Footnotes
    # Co-first authors
    Affiliations
    Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China
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  • Jiarou Shan
    Affiliations
    Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China
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  • Yan Chen
    Correspondence
    Corresponding authors. Address: Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China. Tel.: +86 3807 6560, fax. +86 3836 7693.
    Affiliations
    Faculty of Medicine, Macau University of Science and Technology, Macau, 999078, China

    Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China
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  • Huimin Xia
    Correspondence
    Corresponding authors. Address: Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China. Tel.: +86 3807 6560, fax. +86 3836 7693.
    Affiliations
    Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China
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    # Co-first authors
Published:August 19, 2022DOI:https://doi.org/10.1016/j.jhep.2022.08.005

      Keywords

      To the Editor:
      The differential diagnosis of early-stage biliary atresia (BA) from other causes of infantile cholestasis is difficult with laboratory examinations, but is important for improving native liver survival and reducing postoperative complications.
      • Hopkins P.C.
      • Yazigi N.
      • Nylund C.M.
      Incidence of biliary atresia and timing of hepatoportoenterostomy in the United States.
      Gamma-glutamyltransferase (GGT) is commonly used to distinguish BA from non-BA with a cut-off value of 300 U/L, but it has a relatively low accuracy of 76% to 88%.
      • Chen X.
      • Dong R.
      • Shen Z.
      • Yan W.
      • Zheng S.
      Value of gamma-glutamyl transpeptidase for diagnosis of biliary atresia by correlation with age.
      Our recent study showed that a large number of CD177+ cells infiltrated the BA liver and damaged the bile ducts at an early stage,
      • Zhang R.
      • Su L.
      • Fu M.
      • Wang Z.
      • Tan L.
      • Chen H.
      • et al.
      CD177(+) cells produce neutrophil extracellular traps to promote biliary atresia [published online ahead of print, 2022 Jul 5].
      prompting us to investigate the potential of peripheral blood CD177+ cells as an early diagnostic indicator of BA.
      Patients from our own hospital were enrolled into a diagnostic cohort and a prospective validation cohort in two time periods. In addition, the patients collected from the other 4 hospitals were used as another prospective validation cohort. In each cohort, patients with cholestasis were separated into BA groups and non-BA groups and compared (Fig. S1 and Table S1). The diagnostic efficacy of CD177+ cells for BA was examined using the ratio of these cells to CD45+ cells in the peripheral blood in the diagnosis cohort. The results showed that the percentage of CD177+ cells in the group of patients with BA was significantly higher, with a median [IQR] level of 25.90 [19.75-31.10] compared to the non-BA group (8.56 [6.01-12.65]) and normal control group (7.15 [4.12-11.13]), respectively, p <0.0001. Compared to the non-BA group, the receiver-operating characteristic (ROC) curve analysis indicated a good diagnostic ability of CD177+ cells, with an area under the curve (AUC) of 0.9601 [95% CI 0.9218-0.9983]. The sensitivity was 0.8297, the specificity was 1.000, the accuracy was 0.9259, the positive predictive value was 1.000 and the negative predictive value was 0.8695. Meanwhile, the ability of the CD177+ cell proportion to discriminate between BA and non-BA was validated in two prospective validation cohorts with AUCs of 0.9482 [95% CI 0.9004-0.9959] and 0.9211 [95% CI 0.8529-0.9892], respectively. In the two prospective validation cohorts, the sensitivities of the proportion of CD177+ cells were 0.9844 and 0.9032; the specificities were 0.8776 and 0.8684; and the accuracies were 0.9345 and 0.8841, suggesting that the CD177+ cell ratio has good diagnostic performance for the differential diagnosis of BA (Fig. 1A and Table S2).
      Figure thumbnail gr1
      Fig. 1Diagnostic value of peripheral blood CD177+ cells for the differentiation of BA and non-BA patients.
      (A) Expression levels and ROC curves of CD177+ cells in the BA, non-BA and NC groups in the diagnostic cohort, prospective validation cohort 1 and cohort 2. Scatter plots (upper panel), ROC curves and AUCs (lower panel) are shown (B) CD177+ cells in the age subgroups compared between patients with BA (n = 136), those without BA (n = 127) and healthy controls (n = 72). Scatter plot (upper panel), ROC curve and AUC (lower panel) of the CD177+ cell proportion are shown (C) ROC curve of combined serum GGT and peripheral blood CD177+ cell proportions compared with the serum GGT alone in BA diagnosis. ROC curve and AUC of the BA and non-BA groups from 3 cohorts (upper panel), aged ≤45 days subgroups (middle panel) and age >45 days subgroup (lower panel). AUC, area under the curve; BA, bile acid; GGT, gamma-glutamyltransferase; NC, healthy control; ROC, receiver-operating characteristic.
      To further explore the potential of CD177+ cells as an early diagnostic indicator of BA, we performed a subanalysis of data collected within 45 days from across the three cohorts, including 21 infants in the BA group, 27 infants in the non-BA group, and 11 in normal controls (Table S3). The results showed that the median proportion of CD177+ cells in the peripheral blood of the patients in the non-BA subgroup (aged less than 45 days) was 11.30 [6.79-14.40], while it was significantly increased to 23.90 [19.75-32.10] (p <0.0001) in the BA group. The ROC analysis performed in this subgroup showed an AUC of 0.9189 (95% CI 0.8266-1.0000; p <0.0001), a sensitivity of 0.9048, a specificity of 0.9259, an accuracy of 0.9167, a positive predictive value of 0.9047, and a negative predictive value of 0.9310 (Fig. 1B), suggesting that CD177+ cells have the potential to differentiate between early-stage BA and non-BA.
      For analysis of the diagnostic value of the combination of peripheral blood CD177+ cells and serum GGT for BA, we grouped the three cohorts (a total of 335 individuals) and compared the diagnostic efficacy of BA between the GGT and CD177+ cell proportions by ROC curve analysis. The results showed that the AUC of GGT was 0.8319 (95% CI 0.7845-0.8793; p <0.0001) (Fig. 1C), which was significantly lower than the AUC of CD177+ cell proportion (0.9423; 95% CI 0.9124-0.9722; p <0.0001) (Fig. 1B). Furthermore, the composite model combining the value of CD177+ cells and GGT produced an AUC of 0.9558 (95% CI 0.9319-0.9797; p <0.0001), with a sensitivity of 0.8603 and specificity of 0.8898. In the subgroup of ≤45-day analysis, we also found that the AUC of GGT (0.6825; 95% CI 0.5305-0.8345) was significantly lower than that of CD177+ cells alone. Similarly, the combination of CD177+ cells and GGT showed good diagnostic performance for the subgroup (AUC 0.9121; 0.8186-1.000; p <0.0001) (Fig. 1C).
      Collectively, these data suggested that the diagnostic accuracy of CD177+ cells was significantly greater than that of GGT. Compared with CD177+ cells alone, adding the analysis of GGT to the analysis of CD177+ cells does not substantially increase the diagnostic accuracy for BA. Recently, increased expression of matrix metalloproteinase 7 (MMP-7) was suggested to be a potential biomarker of BA, and the combination of MMP-7 and GGT has a relatively stable diagnostic ability with an AUC of 0.94.
      • Lertudomphonwanit C.
      • Mourya R.
      • Fei L.
      • Zhang Y.
      • Gutta S.
      • Yang L.
      • et al.
      Large-scale proteomics identifies MMP-7 as a sentinel of epithelial injury and of biliary atresia.
      However, whether it has diagnostic value for early BA is unknown. Additional investigation of the early age subgroup analysis of MMP-7 and in combination with CD177+ cells and GGT tests is worth performing in the future.

      Financial support

      National Natural Science Foundation of China (81974056) to RZ, Science and Technology Planning Project of Guangdong Province (No. 2019B020227001) and Science and Technology Planning Project of Guangzhou (No. 202206080002) to HX.

      Authors’ contributions

      R.Z. designed the experiments and recruited patients. J.Y. was responsible for samples collection and flow analysis. J.R. performed statistical analysis. Y.C. and H.X. wrote the letter and supervised the project.

      Clinical trial registration

      Peripheral blood CD177+ cell as early diagnostic markers for biliary atresia: multicenter study (ChiCTR1800015717).

      Ethical statement

      The study was approved by the Ethics Committee of Guangzhou Women and Children's Medical Center, China.

      Conflicts of interest

      Authors declare no conflicts of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      The authors would like to thank the Clinical Biological Resource Bank of Guangzhou Women and Children’s Medical Center for providing the clinical samples. Thank Yanlu Tong and Hezhen Wang for sample collection and flow analysis.

      Supplementary data

      The following are the supplementary data to this article:

      Appendix

      Collaborators: Wenxian Ouyang1, Xiaomei Qin1, Shangjie Xiao2, Bin Wang3, Hongguang Shi.4
      1Department of Liver Disease Center, Hunan Children's Hospital, Hunan, 410007, China.
      2Department of Neonatal Surgery, Guangdong Women and Child Hospital, Guangzhou, 511442, China.
      3Department of General Surgery, Shenzhen Children's Hospital, Shenzhen, 518034, China.
      4Department of Pediatric Surgery, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.

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