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Diagnostic methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments

Abstract

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices.

Impact

  • Key message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements.

  • What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood.

  • Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty.

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Fig. 1: Reference measurement system and metrological traceability chain for TSB.
Fig. 2: Total serum bilirubin measured in EQA samples by ~185 medical laboratories, common IVD manufacturers, and a JCTLM-endorsed reference laboratory.

References

  1. 1.

    Tiedemann, F. & Gmelin, L. In Die Verdauung nach Versuchen Ch. 10 (ed. Groos, K.) 79 (1826).

  2. 2.

    Frerichs, F. T. Klinik der Leberkrankheiten 1st edn, Vol. I of 2 volumes (1858).

  3. 3.

    Van den Bergh, A. A. H. & Snapper, J. Die Farbstoffe des Blutserums. Deut. Arch. Klin. Med. 110, 540–561 (1913).

    Google Scholar 

  4. 4.

    Malloy, H. T. & Evelyn, K. A. The determination of bilirubin with the photoelectric colorimeter. J. Biol. Chem. 119, 481–490 (1937).

    CAS  Article  Google Scholar 

  5. 5.

    Jendrassik, L. & Grof, P. Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins. Biochem Z. 297, 81–89 (1938).

    CAS  Google Scholar 

  6. 6.

    BIPM, IEC, IFCC, ILAC, IUPAC, IUPAP, ISO, OIML. The International Vocabulary of Metrology—Basic And General Concepts and Associated Terms (VIM) 3rd edn. https://www.bipm.org/utils/common/documents/jcgm/JCGM_200_2012.pdf (2012).

  7. 7.

    Ngashangva, L., Bachu, V. & Goswami, P. Development of new methods for determination of bilirubin. J. Pharm. Biomed. Anal. 162, 272–285 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Vreman, H. J. et al. Interlaboratory variability of bilirubin measurements. Clin. Chem. 42, 869–873 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9.

    Doumas, B. T. & Eckfeldt, J. H. Errors in measurement of total bilirubin: a perennial problem. Clin. Chem. 42, 845–848 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Cobbaert, C., Weykamp, C. & Hulzebos, C. V. Bilirubin standardization in the Netherlands: alignment within and between manufacturers. Clin. Chem. 56, 872–873 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Greene, D. N., Liang, J., Holmes, D. T., Resch, A. & Lorey, T. S. Neonatal total bilirubin measurements: Still room for harmonization. Clin. Biochem. 47, 1112–1115 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Kirk, J. M. Neonatal jaundice: a critical review of the role and practice of bilirubin analysis. Ann. Clin. Biochem. 45, 452–462 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Lano, I. M., Lyon, A. W., Wang, L., Ruskin, R. & Lyon, M. E. Comparative evaluation of neonatal bilirubin using radiometer whole blood co-oximetry and plasma bilirubin methods from Roche diagnostics and ortho clinical diagnostics. Clin. Biochem. 53, 88–92 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Wang, L. et al. Limitations and opportunities of whole blood bilirubin measurements by GEM premier 4000®. BMC Pediatr. 17, 92 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Kazmierczak, S. C. et al. Direct spectrophotometric method for measurement of bilirubin in newborns: comparison with HPLC and an automated diazo method. Clin. Chem. 48, 1096–1097 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. 16.

    Barko, H. A., Jackson, G. L. & Engle, W. D. Evaluation of a point-of-care direct spectrophotometric method for measurement of total serum bilirubin in term and near-term neonates. J. Perinatol. 26, 100–105 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Lo, S. F., Jendrzejczak, B. & Doumas, B. T. Laboratory performance in neonatal bilirubin testing using commutable specimens: a progress report on a college of american pathologists study. Arch. Pathol. Lab Med. 132, 1781–1785 (2008).

    PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Olusanya, B. O., Ogunlesi, T. A. & Slusher, T. M. Why is kernicterus still a major cause of death and disability in low-income and middle-income countries? Arch. Dis. Child 99, 1117–1121 (2014).

    PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Slusher, T. M. et al. Burden of severe neonatal jaundice: a systematic review and meta-analysis. BMJ Paediatr. Open 1, e000105 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Keahey, P. A. et al. Point-of-care device to diagnose and monitor neonatal jaundice in low-resource settings. PNAS 114, E10965–E10971 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Coda Zabetta, C. D. et al. Bilistick: a low-cost point-of-care system to measure total plasma bilirubin. Neonatology 103, 177–181 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Greco, C. et al. Diagnostic performance analysis of the point-of-care Bilistick system in identifying SNH by a multi-country approach. EClinicalMedicine 1, 14–20 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Greco, C. et al. Comparison between Bilistick System and transcutaneous bilirubin in assessing total bilirubin serum concentration in jaundiced newborns. J. Perinatol. 37, 1028–1031 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Thielemans, L. et al. Laboratory validation and field usability assessment of a point-of-care test for serum bilirubin levels in neonates in a tropical setting. Version 2. Wellcome Open Res. 3, 110 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Kamineni, B. et al. Accuracy of Bilistick (a point-of-care device) to detect neonatal hyperbilirubinemia. J. Trop. Pediatr. 66, 630–636 (2020).

    PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Rohsiswatmo, R. et al. Agreement test of transcutaneous bilirubin and bilistick with serum bilirubin in preterm infants receiving phototherapy. BMC Pediatr. 18, 315 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. 27.

    Boo, N.-Y. et al. The point-of-care Bilistick method has very short turn-around-time and high accuracy at lower cutoff levels to predict laboratory-measured TSB. Pediatr. Res. 86, 216–220 (2019).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  28. 28.

    Tabatabaee, R. S., Golmohammadi, H. & Ahmadi, S. H. Easy diagnosis of jaundice: a smartphone-based nanosensor bioplatform using photoluminescent bacterial nanopaper for point-of-care diagnosis of hyperbilirubinemia. ACS Sens. 4, 1063–1071 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Grohmann, K. et al. Bilirubin measurement for neonates: comparison of 9 frequently used methods. Pediatrics 117, 1174–1183 (2006).

    PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Lo, S. F. Laboratory accuracy in neonatal bilirubin: the search for truth in laboratory medicine. JAMA Pediatr. 170, 529–530 (2016).

    PubMed  Article  PubMed Central  Google Scholar 

  31. 31.

    Stepman, H. C. M. et al. Measurements for 8 common analytes in native sera identify inadequate standardization among 6 routine laboratory assays. Clin. Chem. 60, 855–863 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Vítek, L. Bilirubin as a predictor of diseases of civilization. Is it time to establish decision limits for serum bilirubin concentrations? Arch. Biochem. Biophys. 672, 108062 (2019).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  33. 33.

    Jones, G. R. & Jackson, C. The Joint Committee for Traceability in Laboratory Medicine (JCTLM) - its history and operation. Clin. Chim. Acta 453, 86–94 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Cobbaert, C., Smit, N. & Gillery, P. Metrological traceability and harmonization of medical tests: a quantum leap forward is needed to keep pace with globalization and stringent IVD-regulations in the 21st century! Clin. Chem. Lab Med. 56, 1598–1602 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Klauke, R. et al. Reference measurement procedure for total bilirubin in serum re-evaluated and measurement uncertainty determined. Clin. Chim. Acta 481, 115–120 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Fraser, C. G. & Peake, M. J. Problems associated with clinical chemistry quality control materials. CRC Crit. Rev. Clin. Lab Sci. 12, 59–86 (1980).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Vitek, L. Bilirubin as a signaling molecule. Med. Res. Rev. 40, 1335–1351 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Gazzin, S. et al. Bilirubin accumulation and Cyp mRNA expression in selected brain regions of jaundiced Gunn rat pups. Pediatr. Res. 71, 653–660 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Adachi, Y. et al. Clinical application of serum bilirubin fractionation by simplified liquid chromatography. Clin. Chem. 34, 385–388 (1988).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Muraca, M. & Blanckaert, N. Liquid-chromatographic assay and identification of mono- and diester conjugates of bilirubin in normal serum. Clin. Chem. 29, 1767–1771 (1983).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    Aziz, S. et al. Bilirubin-IX alpha and -IX beta pigments, coproporphyrins and bile acids in meconium and stools from full-term and preterm neonates during the first month of life. Acta Paediatr. 90, 81–87 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Spivak, W. & Yuey, W. Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease. Biochem. J. 234, 101–109 (1986).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Ostrea, E. M. Jr, Ongtengco, E. A., Tolia, V. A. & Apostol, E. The occurrence and significance of the bilirubin species, including delta bilirubin, in jaundiced infants. J. Pediatr. Gastroenterol. Nutr. 7, 511–516 (1988).

    PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Kuenzle, C. C., Sommerhalder, M., Ruttner, J. R. & Maier, C. Separation and quantitative estimation of four bilirubin fractions from serum and of three bilirubin fractions from bile. J. Lab Clin. Med. 67, 282–293 (1966).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Heirwegh, K. P., Fevery, J. & Blanckaert, N. Chromatographic analysis and structure determination of biliverdins and bilirubins. J. Chromatogr. 496, 1–26 (1989).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Zelenka, J. et al. Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 867, 37–42 (2008).

    CAS  Article  Google Scholar 

  47. 47.

    Jašprova, J. et al. A novel accurate LC-MS/MS method for quantitative determination of Z-lumirubin. Sci. Rep. 10, 4411 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. 48.

    Sawasaki, Y., Yamada, N. & Nakajima, H. Developmental features of cerebellar hypoplasia and brain bilirubin levels in a mutant (Gunn) rat with hereditary hyperbilirubinaemia. J. Neurochem. 27, 577–583 (1976).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Muchova, L. et al. Statin treatment increases formation of carbon monoxide and bilirubin in mice: a novel mechanism of in vivo antioxidant protection. Can. J. Physiol. Pharm. 85, 800–810 (2007).

    CAS  Article  Google Scholar 

  50. 50.

    Cuperus, F. J. et al. Beyond plasma bilirubin: the effects of phototherapy and albumin on brain bilirubin levels in Gunn rats. J. Hepatol. 58, 134–140 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Schreuder, A. B. et al. Albumin administration protects against bilirubin-induced auditory brainstem dysfunction in Gunn rat pups. Liver Int. 33, 1557–1565 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Schreuder, A. B. et al. Optimizing exchange transfusion for severe unconjugated hyperbilirubinemia: studies in the Gunn rat. PLoS ONE 8, e77179 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Bortolussi, G. et al. Life-long correction of hyperbilirubinemia with a neonatal liver-specific AAV-mediated gene transfer in a lethal mouse model of Crigler-Najjar syndrome. Hum. Gene Ther. 25, 844–855 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Vodret, S. et al. Albumin administration prevents neurological damage and death in a mouse model of SNH. Sci. Rep. 5, 16203 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Vodret, S., Bortolussi, G., Jasprova, J., Vitek, L. & Muro, A. F. Inflammatory signature of cerebellar neurodegeneration during neonatal hyperbilirubinemia in Ugt1 -/- mouse model. J. Neuroinflam. 14, 64 (2017).

    Article  CAS  Google Scholar 

  56. 56.

    Bockor, L. et al. Modulation of bilirubin neurotoxicity by the Abcb1 transporter in the Ugt1-/- lethal mouse model of neonatal hyperbilirubinemia. Hum. Mol. Genet. 26, 145–157 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Jasprova, J. et al. The biological effects of bilirubin photoisomers. PLoS ONE 11, e0148126 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  58. 58.

    Itoh, S., Isobe, K. & Onishi, S. Accurate and sensitive high-performance liquid chromatographic method for geometrical and structural photoisomers of bilirubin IX alpha using the relative molar absorptivity values. J. Chromatogr. A 848, 169–177 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  59. 59.

    Vitek, L., Kraslova, I., Muchova, L., Novotny, L. & Yamaguchi, T. Urinary excretion of oxidative metabolites of bilirubin in subjects with Gilbert syndrome. J. Gastroenterol. Hepatol. 22, 841–845 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  60. 60.

    Joerk, A. et al. Propentdyopents as heme degradation intermediates constrict mouse cerebral arterioles and are present in the cerebrospinal fluid of patients with subarachnoid hemorrhage. Circ. Res. 124, e101–e114 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  61. 61.

    Clark, J. F., Loftspring, M., Wurster, W. L. & Pyne-Geithman, G. J. Chemical and biochemical oxidations in spinal fluid after subarachnoid hemorrhage. Front. Biosci. 13, 1806–1812 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  62. 62.

    Konickova, R. Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds. Ann. Hepatol. 13, 273–283 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  63. 63.

    Ritter, M. et al. Pyrrolic and dipyrrolic chlorophyll degradation products in plants and herbivores. Chemistry 26, 6205–6213 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We greatly appreciate the help of T. van Wulfften Palthe in correcting the English grammar and language. The study was supported by grants NV18-07-00342 and RVO-VFN64165/2020 from the Czech Ministry of Health. The support of an intramural grant of Fondazione Italiana Fegato to Claudio Tiribelli is appreciated.

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Study concept and design, and retrieving and analyzing the literature: C.V.H., L.V., C.D.C.Z., C.C., and C.T. Drafting and critical revision of the manuscript for important intellectual content: C.V.H., L.V., C.D.C.Z., A.D., P.S., E.A.E.v.d.H., C.C., and C.T. All authors approved the final manuscript as submitted.

Corresponding author

Correspondence to Christian V. Hulzebos.

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Competing interests

Claudio Tiribelli is the President and Carlos D. Coda Zabetta is the CTO of Bilimetrix, the company responsible for the development of the Bilistick® System. The remaining authors declare no competing interests.

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Hulzebos, C.V., Vitek, L., Coda Zabetta, C.D. et al. Diagnostic methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments. Pediatr Res (2021). https://doi.org/10.1038/s41390-021-01546-y

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