Biomarkers of Liver Injury
Review Article
Received: 14 th March, 2019; Reviewed: 13 th June, 2019; Accepted :30th June ,2019.
Citation of article: Yogesh Waikar. Biomarkers of Liver Injury.New Indian Journal of Pediatrics, 2019; 8.2:
Review Article
Key words: biomarker, Cytokeratin – 18, Leucocyte cell derived chemokine2, Liver specific micro RNAs, Exosomes, M30/M65 antibody.
Abbreviations: SGOT: serum glutamic-oxaloacetic transaminase, SGPT: Serum glutamic pyruvic transaminase,GGT : gamma-glutamyl transferase ,HPC: hepatic progenitor cells,HSC: hepatic stellate cells,HMGB1: High mobility group box1,Mi RNA:microRNA,RNA: Ribonucleic acid,DNA: Deoxyribonucleic Acid,ACLF: acute on chronic liver failure,MELD: Model End Stage Liver Disease, ALF:Acute Liver failure.
Introduction:
Liver injury in children is common. It can present clinically as either subclinical hepatitis, acute hepatitis, chronic hepatitis, compensated liver chronic disease , decompensated liver cirrhosis , acute liver failure or acute on chronic liver failure. Routine biochemical parameters that are analysed as Liver Function test includes total bilirubin, direct bilirubin, SGOT, SGPT, albumin, pro-thrombin time & GGT.With recent advances and applications of technology newer biomarkers are now available . These biomarkers are earlier studied in animal models, later in humans. They do have diagnostic and prognostic value. They give an idea about histo-pathological status of liver. But they have their own limitations. This review of biomarkers of liver injury in children underscores newer possibilities and approaches to diagnosis of liver disease. Studies targeting individual biomarkers from therapeutic point can also be considered in near future.
Liver injury:
To understand the biomarker it’s important to understand pathogenesis and pathophysiology of liver disease in general. Detail histopathological marker analysis is beyond the scope of this article.
Liver consists of hepatocytes, cholangiocytes, kupffer cells, hepatic progenitor cells (HPC) hepatic stellate cells (HSC), bone marrow derived macrophages. It has been well studied now that different types of liver injury elicit different responses. Hepatocyte proliferation is seen in acute liver failure.(1) Hepatocyte progenitor cells (HPC) activity is higher in acute on chronic liver failure. HPC proliferation is comparatively more in acute on chronic liver failure as compared to chronic liver disease .More the HPC proliferation ,more is the component of inflammation & higher is the degree of liver necrosis and fibrosis.(1) Corresponding markers of liver pathology are expressed in the serum and liver tissue. This bio- phenomenon helps us to study biomarkers of liver injury.
Hepatic stellate cell (HSC) activation plays an important role in fibrogenesis. Activated HSCs are more in acute on chronic liver failure than in acute hepatitis vs chronic hepatitis. The number of HSC and HPC are proportionate to each other in the liver histopathology.(2) Biomarker that activates HSC and stimulates fibrosis e.g. HMGBI play a crucial role.(3) This can be studied and analysed depending on the grade and stage of liver disease.
HPC are bipotential cells.They are capable of forming both hepatocytes and cholangiocytes. During chronic hepatitis, HPCs are responsible for ductular reactions; increased number of ductules which when uncontrolled leads to hepatic fibrogenesis.(4) HPCs act as source of chemokine like chemokine ligand 2 & CX(3) CL1which stimulates macrophages. These chemokines are studied as biomarkers of liver injury and its response.(5)
Kupffer cells activation dominates as first line response while bone marrow derived macrophages act as emergency response team.(6)(7). Newer biomarkers like soluble CD163 are studied as response to macrophage activation in different liver diseases. Cell tracking, multi-omics phenotypes and single cell RNA sequencing help in analysis of these biomarkers.(6)(7) Chemokines like CXC and interleukin 1 L- 1B are secreted by activated kupffer cells which induce reactive oxygenated necrosis.(8) This biomarker’s application in clinical scenario needs further validation.
What is biomarker?
A biomarker is an objectively measurable indicator of biological process. It can be physiologic or pathological .It can predict outcome of the disease. (9) Newer biomarkers are now increasingly available from bench side to bedside. Initial enthusiasm is now faded and practical limitations of them are realized. Their proper application in clinical setting can help us in diagnosis and prognosis of children with liver diseases.
Biomarkers for liver injury are either biochemically measured like serum phosphorus, serum fibrinogen, alpha fetoprotein, CD markers of differentiation or genetically oriented like miRNAs. Various biomarkers are direct expression of liver cell injury or some are indirect by-products of cellular –intercellular reactions.
Cytokeratin – 18 measured by M30 antibody & M65 antibody. It is released after cellular hepatocyte injury. Hepatocyte mitochondrial protein biomarker molecule like carbamoyl phosphate synthase 1is studied and noted to be more specific than SGPT.(10) Other Mitochondrial damage markers of hepatocytes are mtDNA, glutamate dehydrogenase (GDM) , glycodeoxycholate.(11)
Leucocyte cell derived chemokine2 (Lect 2) is secreted into the serum by the liver. It plays the critical role in stimulation of liver regeneration. Lower the serum level of Lect 2 better is the prognosis.(11) Serum Lect 2 is inversely proportionate to SGPT and SGOT in acute liver failure.(12) The peripheral neutrophil lymphocyte ratio (NLR) is also proposed as biomarker. It is calculated from complete blood count .NLR > 5.7 correlates with poor prognosis.(13)
Right application of biomarker is important in any given clinical setting. Temporal association and rise – fall of biomarker should be analyzed and correlated with the stage and pattern of liver injury. Blind application of these biomarkers may prove counterproductive. Clinically important biomarkers are discussed here.
1. Cytokeratin 18 (K18).
Cytokeratin 18 is an intermediate filament protein highly expressed in epithelial cells.(14) When released into extracellular space, K18 can be used as biomarker. Apoptosis and necrosis are the two major approaches to hepatocyte cellular death. Caspase cleaved keratin 18 (CCK18) is considered to represent hepatocyte apoptosis measured by M30 antibody ELISA .Keratin 18 (cleaved & un-cleaved) represents necrosis measured by M65 ELISA antibody. M30: M65 ratio is proposed to differentiate apoptotic and necrotic cell death.
M65 Epideath is a modified version of total cell death indicator. It is predictive for mortality independent of age, gender, MELD Score, Child Pugh score and presence of complications of cirrhosis and infections.(15) Cleaved K18 is also studied marker in non alcoholic fatty liver disease.(16) In acute on chronic liver failure non apoptotic pattern of cell death is predominant .It is confirmed by cleaved K18: K18 ratio reduction.(17) M65 levels are higher in ALF as compare to ACLF. M30 / M65 ratio is noted to be useful in ACLF.(18) Mixed pattern of response sometimes do make picture difficult to analyse. Acute liver failure study group (ALFSG) demonstrated that M30 rather than M65 is an effective tool to predict patient that would require liver transplantation. A new ALFSG index is now used to prognosticate(19) these patients.
The limitation of this biomarker is the presence of cytokeratin in non hepatic epithelium. Extra hepatic epitheliolysis can also raise the biomarker levels.(20) In clinical settings, combined and mixed pattern of injury, association with sepsis make it complex scenario. Mixed patterns of cell deaths in which apoptosis and necrosis both take part in cellular injury by different molecular mechanisms can confabulate the interpretation of the biomarker.(21)
2. Liver specific micro RNAs(Mi RNA):
Mi RNAs are small RNA (Ribonucleic acid) molecules that are transcribed from genomic DNA. They do not code for protein. They regulate the transcript. In liver injury, circulated MiRNA are studied and are noted to be useful biomarker for determining the extent of liver damage.
MiRNA – 122 is the most abundant micro RNA in hepatocyte which is released into serum after liver injury.(22) In chronic liver disease, MiRNA – 122 correlate inversely with severity of liver fibrosis.(23) MiRNA – 122, MiRNA – 130, MiRNA – 183 , MiRNA – 196, MiRNA – 209 and MiRNA – 96 are the potential markers of liver injury. Blocking or stimulating these may unveil the therapeutic strategies in management of liver injury in future.(24)
Higher serum level or MiRNA-122, MiRNA-21, MiRNA – 221 are reported in the survivors of acute liver failure.(25) In patient experiencing acute rejection post transplant, serum MiRNA-122 levels are increased.(26) The release of MiRNA – 122 – 5P; MiRNA – 192 – 5P and MiRNA – 1224 – 5P from hepatocytes is generally attributed to oxidative stress.(27) MiRNA – 122 is proposed to predict graft outcome after liver transplant.(28). MiRNA – 122 inhibitors like Miravirsen and RG – 101 are studied in hepatitis C opening the therapeutic dimension for the future.(29)(30).
Unfortunately all these biomarkers take time for results by conventional quantitative RT – PCR or microarray. This time -lag to determine the outcome in condition like acute liver failure or post transplant rejection is an important limitation. Technological advances may reduce the time required for analysis.
3. Exosomes and Microparticles
Exosomes are endosomal derived small vesicles. They carry variety of cargos like MiRNAs , micro RNAs , non coding RNA s. All these convey cellular information and intercellular communications which enables them to be studied as biomarkers. Hepatocyte releases exosomes to neighbouring cells and communicate signal to regenerate, repair or necrose. CTGF exosome carrying fibrosis signal is now well studied in humans(31)(32) Exosomal MiRNA – 192 from damaged hepatocytes represents potential biomarker in NASH. It represents progression of liver histopathology from steatosis to hepatitis. MiRNA – 192 exosome is increased in patient with NAFLD advanced stage as compared to early stage.(33) Upon liver injury damaged hepatocyte produces exosomes which bind to toll like receptor – 3 ( TLR – 3 ) and activates HSC .This interaction enhances production of chemokines ( C – C Motif ) ,ligand 20 ( CCL20 ) and interleukin – 17A ( IL – 17A ) resultant in increase in liver fibrosis.(34) Mesenchymal stromal cells dervived exosome have been studied to have therapeutic value. MSC containing MiRNA 125 b reduces hepatic fibrosis by inhibiting hepatic stellate cell activation.(35) Further studies are needed for their clinical application.
4. Bile acid Conjugates:
Bile acid concentrations are increased in liver injury. The ratio of tauro conjugates to glyco-conjugates is increased in hepatic injury suggesting tauro – conjugates are more sensitive measures of liver injury.(36) The high values of conjugated bile acid like glycocholic acid , glycochenodeoxycholic acid are associated with disease likes paracetamol poisoning, chronic hepatitis and other primary liver disorders .Secondary bile acid conjugates like deoxycholic acid are increased in hepatic dysfunction secondary to cardio-vascular disease.(36) This can help in differentiation in co-morbid hepatocellular and cardiovascular pathologies.
In Obstructive cholestasis liver injury, studies done earlier reported hepatocellular death secondary to apoptosis.(37) Recent studies on humans have confirmed increase in M65 antibody secondary to acetylated HMIGB1 indicative of necrosis pattern in these patients.(38) High mobility group box1 ( HMGB1) is passively released from necrotic cells and after hyper-acetylation is secreted from inflammatory cells.(39) HMGB1 is thus an important biomarkers of necrotic cell death pattern .
5. Other Biomarkers:
APAP – protein adducts (related to paracetamol poisoning). GLDH, mt DNA, acylcarnitines , CPS – 1 , are shown to be increased in plasma after paracetamol hepato cellular failure.(40) Argininosuccinate synthetase levels can be detected even before rise in SGPT.(41)
Hypophosphatemia in liver failure is suggestive of good prognosis.Hyperphosphatemia is predictive of poor recovery. Hypophosphatemia may be the consequence of liver regeneration serum phosphorus level > 2. 9 mg / dl suggests massive hepatocyte necrosis and lack of liver regeneration.(42)(43)
Serum fibrinogen is also studied. Plasma fibrinogen level is significantly low in non survivor of acute on chronic liver failure patient compared to Survivor of acute on chronic liver failure; chronic hepatitis B and control patients.(44)
Serum alphafetoprotein is studied biomarker of liver failure. The alpha-fetoprotein ratio defined as day 3 alphafetoprotein level divided by that observed on day 1. The rise in alphafetoprotein value from day 1 to day 3 indicated better prognosis.(45) It reflects hepatic regeneration. (46) Collagen IV is proposed to be biomarker of liver fibrosis. Hyaluronic acid is also synthesized by fibroblast and is studied as a biomarker.
Collagens – 1, CK – 7, SMA are associated with increased mortality and need for liver transplant in cases of biliary atresia.(47) Increased expression of SHH (sonic hedgehog pathway) Gli- 2, collagen – 1, CK – 7, SMA, CD 34, TLR 317 and decreased expression of PXR and CAR (pregnone and receptor and constitutive androstane receptor) serve as markers of poor transplant free survival in billiary atresia patients.(48) Fibrotic gene signature than inflammatory gene signature is co-related to have lower transplant free survival(49).
Conclusion:
Biomarkers in pediatric liver disease require further validation. They vary depending on inflammatory, angiogenetic or fibrotic stage. The expression of biomarker needs to be co-related with grading and staging of liver injury. Early grades biomarker may not stay true for end stage liver disease. Inflammatory biomarkers tend to predominate depending on necrotic, cholestasis, drug induced or apoptotic liver injury pattern. Fibrotic biomarkers at end stage liver disease have different representation. Methodologically robust studies are need of hour. Therapeutic options targeting specific level of response / injury representative of biomarker is the logical next step in research. Understanding the pathogenesis and pathophysiology of liver injury by clinical biomarker signature would expand our vision in diagnostic, prognostic and therapeutic domains in liver Injury.
References:
1.Rastogi A, Maiwall R, Bihari C, Trehanpati N, Pamecha V, Sarin SK. Two-tier regenerative response in liver failure in humans. Virchows Archiv [Internet]. Springer Science and Business Media LLC; 2014 Mar 4;464(5):565–73. Available from: http://dx.doi.org/10.1007/s00428-014-1547-0
2.Rastogi A, Bihari C, Maiwall R, Ahuja A, Sharma MK, Kumar A, et al. Hepatic stellate cells are involved in the pathogenesis of acute-on-chronic liver failure (ACLF). Virchows Archiv [Internet]. Springer Science and Business Media LLC; 2012 Aug 11;461(4):393–8. Available from: http://dx.doi.org/10.1007/s00428-012-1291-2
3.He Y, Jin L, Wang J, Yan Z, Chen T, Zhao Y. Mechanisms of fibrosis in acute liver failure. Liver Int. 2015;35:1877–85.
4.Kaur S., Siddiqui H., Bhat M.H Hepatic Progenitor Cells in Action Liver Regeneration or Fibrosis? (2015) American Journal of Pathology, 185 (9) , pp. 2342-2350.
5.Viebahn CS, Benseler V, Holz LE, etal,: Invading macrophages play a major role in the liver progenitor cell response to chronic liver injury. J Hepatol 2010,53:500-507
6.Gronbaek H, Rodgaard-Hansen S, Aagaard NK, et al. CANONIC study investigators of the EASL-CLIF Consortium. Macrophage activation markers predict mortality in patients with liver cirrhosis without or with acute-on-chronic liver failure (ACLF). J Hepatol 2016;64:813-822
7.Zigmond E, Samia-Grinberg S, Pasmanik-Chor M,et al. Infiltrating monocyte-derived macrophages and resident kupffer cells display different ontogeny and functions in acute liver injury. J Immunol2014;193:344-353
8. Marra F, Tacke F: Roles for chemokines in liver disease. Gastroenterology 2014; 147: 577–594.e1
9.Strimbu K, Tavel JA: What are biomarkers? Curr Opin HIV AIDS 2010; 5: 463–466
10.Weerasinghe SV, Jang YJ, Fontana RJ, et al. Carbamoyl phosphate synthatase-1 (CPS1) is a rapid turnover biomarker in mouse and human acute liver injury. Am J Physiol Gastrointest Liver Physiol 2014;307:G355–G364
11.Slowik V, Borude P, Jaeschke H, et al. Leukocyte cell derived chemotaxin-2 (Lect2) as a predictor of survival in adult acute liver failure. Transl Gastroenterol Hepatol. 2019;4:17. Published 2019 Mar 20. doi:10.21037/tgh.2019.03.03
12. Sato Y, Watanabe H, Kameyama H, et al. Serum LECT2 level as a prognostic indicator in acute liver failure. Transplant Proc 2004;36:2359-61
13.J. Gong, W. Zhou, C. Xiao, Y. Jie, S. Zhu, J. Zheng, et al.A nomogram for predicting prognostic value of inflammatory biomarkers in patients with acute-on-chronic liver failure Clin. Chim. Acta, 478 (2018), pp. 7-12
14. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression innormal epithelia, tumors and cultured cells. Cell 1982; 31: 11–24
15.Waidmann O., Brunner F., Herrmann E., Zeuzem S., Piiper A., Kronenberger B.Cytokeratin 18-based cell death markers indicate severity of liver disease and prognosis of cirrhotic patients.Liver Int, 36 (2016), pp. 1464-1472
16.Vuppalanchi R, Jain AK, Deppe R, et al. Relationship between changes in serum levels of keratin 18 and changes in liver histology in children and adults with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2014;12(12):2121–30.e302. doi:10.1016/j.cgh.2014.05.010
17.Macdonald, S., Andreola, F., Bachtiger, P. et al. Cell death markers in patients with cirrhosis and acute decompensation. Hepatology. 2018; 67: 989–1002
18.D. Adebayo, V. Morabito, F. Andreola et al., “Mechanism of cell death in acute-on-chronic liver failure: a clinico-pathologic-biomarker study,” Liver International, vol. 35, no. 12, pp. 2564–2574, 2015.
19.Rutherford A, King LY, Hynan LS, et al. Development of an accurate index for predicting outcomes of patients with acute liver failure. Gastroenterology. 2012;143(5):1237–1243. doi:10.1053/j.gastro.2012.07.113
20.Ku NO, Strnad P, Bantel H, Omary MB. Keratins: Biomarkers and modulators of apoptotic and necrotic cell death in the liver. Hepatology. 2016;64(3):966–976. doi:10.1002/hep.28493
21.Eguchi A., Wree A., Feldstein A.E. Biomarkers of liver cell death (2014) Journal of Hepatology, 60 (5) , pp. 1063-1074.
22.Wang K, Zhang S, Marzolf B, et al. Circulating micro- RNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A 2009;106:4402–4407
23.Trebicka J, Anadol E, Elfimova N, et al. Hepatic and serum levels of miR-122 after chronic HCV-induced fibrosis. J Hepatol 2013;58:234-239
24.Ghulam Musaddaq, Naveed Shahzad, Muhammad Adnan Ashraf & Muhammad Imran Arshad (2019)Circulating liver-specific microRNAs as noninvasive diagnostic biomarkers of hepatic diseases in human,Biomarkers, 24:2, 103-109, DOI: 10.1080/1354750X.2018.1528631
25. John, K. , Hadem, J. , Krech, T. , Wahl, K. , Manns, M. P., Dooley, S. , Batkai, S. , Thum, T. , Schulze‐Osthoff, K. and Bantel, H. (2014), MicroRNAs play a role in spontaneous recovery from acute liver failure. Hepatology, 60: 1346-1355. doi:10.1002/hep.27250
26.Farid WR, Pan Q, van der Meer AJP, de Ruiter PE, Ramakrishnaiah V, de Jonge J, Kwekkeboom J, Janssen HL, Metselaar HJ, Tilanus HW, Kazemier G, van der Laan LJ. Hepatocyte-derived microRNAs as serum biomarkers of hepatic injury and rejection after liver transplantation. Liver Transpl. 2012;18(3):290
27.Matsuzaki J, Ochiya T. Extracellular microRNAs and oxidative stress in liver injury: a systematic mini review. J Clin Biochem Nutr. 2018;63(1):6–11. doi:10.3164/jcbn.17-123
28. Farid WRR, Verhoeven CJ, de Jonge J, Metselaar HJ, Kazemier G, van der Laan LJW. The ins and outs of microRNAs as biomarkers in liver disease and transplantation. Transpl Int Off J Eur Soc Organ Transplant. 2014 Dec;27(12):1222–32.
29. van der Ree MH, van der Meer AJ, van Nuenen AC, de Bruijne J, Ottosen S, Janssen HL, et al. Miravirsen dosing in chronic hepatitis C patients results in decreased microRNA-122 levels without affecting other microRNAs in plasma. Aliment Pharmacol Ther. 2016 Jan 1;43(1):102–13.
30. van der Ree MH, de Vree JM, Stelma F, Willemse S, van der Valk M, Rietdijk S, et al. Safety,tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: a phase 1B, doubleblind, randomised controlled trial. The Lancet. 2017 Feb;389(10070):709–17
31.Sung, S.; Kim, J.; Jung, Y. Liver-Derived Exosomes and Their Implications in Liver Pathobiology. Int. J. Mol. Sci. 2018, 19, 3715.
32.Gressner, A.M.; Yagmur, E.; Lahme, B.; Gressner, O.; Stanzel, S. Connective tissue growth factor in serum as anew candidate test for assessment of hepatic fibrosis. Clin. Chem. 2006, 52, 1815–1817
33. Lee YS, Kim SY, Ko E, et al. Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells. Sci Rep. 2017;7(1):3710. Published 2017 Jun 16. doi:10.1038/s41598-017-03389-2
34.Seo, W.; Eun, H.S.; Kim, S.Y.; Yi, H.S.; Lee, Y.S.; Park, S.H.; Jang, M.J.; Jo, E.; Kim, S.C.; Han, Y.M.; et al. Exosome-mediated activation of toll-like receptor 3 in stellate cells stimulates interleukin-17 production by gammadelta T cells in liver fibrosis. Hepatology 2016, 64, 616–631
35. Hyun J, Wang S, Kim J, Kim GJ, Jung Y. MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells. Sci Rep. 2015;5:14135. Published 2015 Sep 15. doi:10.1038/srep14135
36. Luo L, Aubrecht J, Li D, et al. Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans. PLoS One. 2018;13(3):e0193824. Published 2018 Mar 7. doi:10.1371/journal.pone.0193824
37. Malhi H, Guicciardi ME, Gores GJ. Hepatocyte death: a clear and present danger. Physiol Rev. 2010;90(3):1165–1194
38.Woolbright BL, Dorko K, Antoine DJ, Clarke JI, Gholami P, Li F, Kumer SC, Schmitt TM,Forster J, Fan F, Jenkins RE, Park BK, Hagenbuch B, Olyaee M, Jaeschke H. Bile acidinduced necrosis in primary human hepatocytes and in patients with obstructive cholestasis. Toxicol Appl Pharmacol. 2015;283(3):168–77
39. Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005;5:331–342
40. Bhattacharyya S, Yan K, Pence L, Simpson PM, Gill P, Letzig LG, Beger RD, Sullivan JE, Kearns GL, Reed MD, Marshall JD, Van Den Anker JN, James LP. Targeted liquid chromatography-mass spectrometry analysis of serum acylcarnitines in acetaminophen toxicity in children. Biomark Med. 2014; 8(2):147–59
41. McGill MR, Cao M, Svetlov A, Sharpe MR, Williams CD, Curry SC, Farhood A, Jaeschke H, Svetlov SI. Argininosuccinate synthetase as a plasma biomarker of liver injury after acetaminophen overdose in rodents and humans. Biomarkers. 2014a;19(3):222-30
42.Yeflim ÖZTÜRK1 , Sema BERKTAfi2 , Özlem B. SOYLU3 , et al Fulminant hepatic failure and serum phosphorus levels in children from the western part of Turkey. Turk J Gastroenterol 2010; 21 (3): 270-274
43.Baquerizo, Angeles, Anselmo, Dean,Shackleton, Christopher et al Phosphorus ans an early predictive factor in patients with acute liver failure Transplantation: June 27th, 2003 – Volume 75 – Issue 12 – p 2007-2014 doi: 10.1097/01.TP.0000063219.21313.32
44.Shao Z, Zhao Y, Feng L, Feng G, Zhang J, Zhang J. Association between plasma fibrinogen levels and mortality in acute-on-chronic hepatitis B liver failure. Dis Markers. 2015;2015:468596. doi:10.1155/2015/468596
45.Schiodt FV, Ostapowicz G, Murray N, et al. Alpha-fetoprotein and prognosis in acute liver failure. Liver Transpl. 2006;12(12):1776–81.
46.Varshney, A., Gupta, R., Verma, S. K., & Ahmad, S. (2017). Alpha-fetoprotein as a prognostic marker in acute liver failure: a pilot study. Tropical Doctor, 47(3), 202–205. https://doi.org/10.1177/0049475516653891
47.Santos JL, Kieling CO, Meurer L, Vieira S, Ferreira CT, Lorentz A, et al. The extent of biliary proliferation in liver biopsies from patients with biliary atresia at portoenterostomy is associated with the postoperative prognosis. J Pediatr Surg. United States; 2009 Apr;44(4):695–701.
48.Vibeke Brix Christensen, Lise Borgwardt, Allan Rasmussen, Olga Østrup, Mette Skalshøi Kjær , Prognostic molecular markers in pediatric liver disease – Are there any?. Bbadis (2018), https://doi.org/10.1016/j.bbadis.2018.12.01850.
49.Moyer K, Kaimal V, Pacheco C, et al. Staging of biliary atresia at diagnosis by molecular profiling of the liver. Genome Med. 2010;2(5):33. Published 2010 May 13. doi:10.1186/gm154.
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