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1
Investigating Liver Disease
Prof Anil Dhawan MD FRCPCH
Director – Paediatric Liver GI and Nutrition Center
Director –Child Health
Remit of the talk
• Biochemical tests of liver function
• Radiology
• Endocopy
• Histology
• Immunopathology
• Genomics/Proteomics/Glycomics/Metabolo
mics
Assessment of Liver Function
• Hepatobiliary cell injury• Biochemical tests
• Immunological tests
• Imaging studies
• Histology
• Synthetic function• Non specific proteins
• Disease related
Biochemical Tests
Predominantly Hepatocellular
– Alanine aminotransferase (ALT)
– Aspartate aminotransferase (AST)
– Bilirubin (Unconjugated, Conjugated,other)
Predominantly Biliary Epithelium
– Gamma glutamyl transferase (GGT)
– Alkaline phosphatase (ALP)
– 5- nucleotidase (5-NT)
– Bilirubin
– Bile acids blood and urine
– Urine urobilinogen
AST ALT
Cytosol and Mitochondria
liver
Heart
Skeletal muscle
Kidney
Pancreas
Red cells
Cytosol
Liver
Muscle
Very little in other
tissues
AST/ALT
Co enzyme –pyridoxal phosphate (B6)• Low values in B6 deficiency
• Uremia
AST/ALT
Clinical clues
Very high values in ,000s
Herpes hepatitis/other viruses
Paracetamol OD
Ischaemia/shock
Macro AST
Metabolic disorders not very high values (WD, Tyrosenemia)
No correlation to severity of disease or liver necrosis
Myopathies!!!
Biliary Enzymes
• Alkaline phosphatase• Group of isoenzymes that hydrolyze organic phosphate esters
at alkaline pH producing inorganic phosphate and organic radical
• Predominantly elevated in extra or intarhepatic biliary pathologies
• Bone isoenzymes
• 20% of patients with hepatitic illnesses have >4 times elevation of ALP
• In children ALP values are higher(bone isoenzyme)
• Low in Zinc def.
• Measure an another enzyme like GGT to differentiate
Biliary Enzymes
• GGT
– Epithelium of small bile ductules and canaliculi
– Also renal tubules,pancreas,brain , breast and
small intestine
– Elevated in – chronic alcoholism,pancreas
disease,myocardial infarction, renal
failure,COPD, diabetes and anticonvulsants
(valproic acid ? true toxicity)
Biliary Enzymes
• GGT
– Levels in newborns normal X 5-8 times of
adults but reach adult values by 9 months.
– Most sensitive test for hepatobiliary disease
– Can not differntiate between extra or
intrahepatic biliary disease
GGT and PFIC
• High GGT cholestasis
– MDR 3 def
– Neonatal sclerosing cholangitis
– Others
• Low GGT cholestasis
– BRIC
– PFIC 1
– PFIC 2
– ARC complex
Bilirubin
• Conjugated ( normally none, >15% of total)
– Hepatocellular and biliary disease
– Not neurotoxic
– Renal failure or haemolysis leads to high serum levels
• Unconjugated (almost all bili is unconjugated)
– Newborn (important neurotoxicity)
– Crigler Najjar Syndrome (neurotoxicity)
– Gilbert’s Syndrome
Bile acids
• Serum bile acids invariably elevated in liver
disease (not much clinical application)
• Urine bile acid (mass spectrometery) to
diagnose disorders of bile acid synthesis
Synthetic Function
• Prothrombin time
• Albumin
• Ammonia
• Cholesterol
Synthetic Function
Prothrombin time
Extrinsic pathway of Coagulation
Prothrombin (FcII) thrombin
Thromboplastin
calcium
activated Fc V, VII, and X
expressed as seconds or ratio of plasma PT to control
INR
When is Vitamin K helpful ?
Role of Vitamin K
Factors II, VII, IX and X
1 mg /year max 5 mg
Deficiency corrected in 4-6 hrs
Ammonia
• Ammonia produced in gut and kidneys
• Converted to urea in the liver
• Elevated in liver failure, PS shunting, drugs
like Valproate (induction of renal
production), large protein load, Gi bleed
• No correlation with severity of liver disease
Case study• 4 year ole male
• Doctor visit for abdominal pain
• Liver palpable 1 cm
• Spleen not enlarged
• Liver tests
– AST 750, ALT 570
– GGT , 37, ALP 267, Bilirubin 16
– CK 12,560
– USS normal
Case 1
What is the diagnosis ?
Case 2
• 9 yr old boy
• Pain abdomen for 1 month, tiredness
• Examination
– BMI 27
– Few spider angiomas and telengectatsia
– Mild jaundice ( Bili 180/congugated 106
– Liver 3 cm , Spleen 4 cm
– KF rings Negative
Case 2
• Investigations
• AST/ALT/GGT 157/179/101
• INR 1.9
• Hepatitis markers B and C negative
• Caeruloplasmin 0.1
• ANA 1:20. ASM 1;40
• Cholestrol/TG mildly elevated
• 24 hr Urine copper 4.6 mmol/24hrs
Case 2
• Ultrasound
• coarse liver , normal blood vessles
• Spleen 14 cms
• No ascites
Differential Diagnosis
• Wilson Disease
• Autoimmune Liver Disease
• NAFLD
• Venous outflow obstruction
• Storage disorder
Copper Associated Protein,steatosis
Liver Copper 870mcg/gm
dry wt
Genetics Homozygous for
WD
6 weeks old baby with jaundice
and pale stools
Which one test will you order ?
• Bilirubin
• AST/ALT
• GGT/ALP
• PT
• USS/CT/MRI
• Liver Biopsy
Radiology
• Portal Hypertension
• Cholestasis
• Liver mass
normal
reversed end diastolic flow
DISIDA Scan 2 wks after
Tx
DISIDA Scan 1 Year post Tx
Abdominal Distension
Cardiac Failure
Consumptive Coagulopathy
Hypthyroidism
Steroids, VCR, Propranalol
Embolisation, HA Ligation
Resection, Transplantation
Handling Liver Bx Specimen
• Formalin
• Snap Freeze
• Electron Microscopy
• Special studies
– Mitochondrial enzyme estimation
– GSD 1 b
– Fructosaemia
Histology
• Indications
– Decreasing with availabilty of non invasive
tests
– Institutional preference
– Prognostic value
– Special staining that can be diagnsotic
– Liver transplatation
– Investigation of liver masses
Liver Biopsy
Contraindications
Percutaneous
Prolonged INR >1.3
Low platelet count <70
Ascites
HCC
Liver biopsy
• Percutaneous needle biopsy
• Transjugular
• Laproscopic
• Laparotoamy /open
Giant cells
Biliary Features on Biopsy
Cirrhosis
Serology
• Autoimmune Liver Disease
• Viral serology (Hep A,B,C,D, E)
• Molecular diagnostics DNA/RNA
quantitative estimation
Autoimmune liver diseaseDiagnostic autoantibodies
Anti-nuclear antibody = ANA Anti-smooth muscle antibody = SMA
Autoimmune hepatitis type 1Autoimmune sclerosing cholangitis
Autoimmune liver diseaseDiagnostic autoantibodies
Liver kidney microsomal type1
= LKM1
Autoimmune hepatitis type 2Autoimmune sclerosing cholangitis
Portal Hypertension
• USS
• Endoscopy (Diagnostic and therapeutic)
• MRV
• CT
• Angiography/ Pressure studies
Investigating Metabolic Liver Dis
Metabolic Liver Disease
Presentation
• Infantile Cholestasis
• Acute liver failure
• Neonatal Ascites
• Hepatomegaly/splenomegaly
• Hyperammonemia/Acidosis
Alagille syndrome
paucity of intrahepatic bile ducts
cardiac anomalies (peripheral pulmonary
stenosis)
posterior embryotoxon
characteristic
facies
butterfly
vertebrae
Autosomal
dominant
Metabolic Liver DiseaseCommon Features
• Positive family history
• Unexplained neonatal deaths/multiple miscarriages
• Consanguinity
• Recurrent episodes of unexplained vomiting, encephalopathy
• Developmental delay/regression
• Dysmorphism
Metabolic Liver DiseaseClinical Clues
• Coarse facies-– Gangliosidosis, MPS, Sialidosis
• Macroglossia-– GM1 Gangliosidosis
• Diarrhea-– Wolman’s Disease, Cystic fibrosis
• Mild Lymphadenopathy-– Wolman’s Disease, Gaucher’s Disease
• Upward gaze palsy,opisthotonous-– Gaucher’s Disease
• Cherry red spot-– Niemann-Pick,GM1Gangliosidosis
Metabolic Liver DiseaseClinical Clues
abnormal odor
• Sweaty feet
– Glutaric acidemia, Isovaleric acidemia
• Rancid, fishy, or cabbage like
– Tyrosinemia
Metabolic Liver Disease
Radiologic Clues
• Adrenal Calcification
– Wolman’s Disease
• Stippled Epiphysis
– Zellweger’s Syndrome, GM1 Gangliosidosis
• Rickets
– Tyrosinemia
Metabolic Liver DiseaseJaundice in Neonate
• Unconjugated Jaundice
– Crigler Najjar Syndrome
– Galactosemia, hypothyroidism initially
– HLH
Metabolic Liver DiseaseInfantile Cholestasis
• Galactosemia
• Tyrosinemia
• Hypo/hyperthyroidism
• Hypopituitarism(SOD)
• Bile acid defects
• Gaucher’s disease
• Fructosemia
• NN hemochromatosis
• Alpha-1antitrypsin def
• PFIC
• Cystic fibrosis
• HLH
• Peroxisomal disorders
• NiemannPick Band C
• Wolman’s Disease
• Organic acidemia
Metabolic Liver Disease
Infantile cholestasis Investigations
• Blood sugar q 6 hrs
• Lactate, pyruvate
• Blood gas
• Uric acid
• Ammonia
• CPK
• Transferrin
Electrophoresis
• Save Blood sample
• Urinary Inv
• Reducing sub
• amino acids
• Organic acids
• Bile acids
• Electrolytes
Metabolic Liver Disease
Infantile Cholestasis Specific Investigations
• Alpha-1 antitrypsin def
• Galactosemia
• Tyrosinemia
• Bile acid defects
• Cystic fibrosis
• Hypothyroidism
• Hypopituitarism
• Phenotype (ZZ) level?
• Gal-1 PUT levels
• Urine succinyl acetone
• Serum and urine bile acid
spectroscopy
• IRT/Sweat test
• TSH and T4
• Cortisol/ synecthin
Metabolic Liver Disease
Infantile cholestasis Tests on White blood cells or
cultured fibroblats
• Wolman’s disease
• Gaucher’s disease
• Sialidosis type II
• GM 1 Gangliosidosis
• MPS VII
• Acid lipase levels
• B-glucocerebrosidase
• Neuraminidase levels
• Acid B-galactosidase
• B-glucronidase def
Liver Biopsy
Metabolic liver disease
presenting as acidosis
Pyruvate DH
deficiency
Normal
(<20)
Mitochondrial
disorders
Increased
(>20)
L/P ratio
Prop.acidaemia
MM acidaemia
IV acidaemia
Glu.acidaemia
Organic
acidaemias
MCAD
LCAD
LCHAD
GAII
Fatty ac ox
defects
Acidosis & ketosis
Urea cycle defects
Normal or alkalosis
Blood pH
Muscle Bx
Liver Bx
Resp chain enzyme
Mitoch DNA
Metabolic liver disease
presenting as lactic acidosis
Resp chain dis
Ms & liver Bx
Ketosis
L/P >30
3-OH-B/AA >2
Pyruvate carbo def
Mulitple carbo def
Ketosis
L/P <20
3-OH-B/AA <1.5
Encephalopathy
Glycogenosis II
Glycogen synth def
Hepatomegaly
During feeding
Glyconeogenesis def
Glycogenosis I
*Prominent
Hepatomegaly
Hypoglycaemia
Ketosis
Fatty ac ox def
(SCAD, MCAD
LCAD, LCHAD)
Moderate
Hepatomegaly
Hypoglycaemia
No ketosis
During fasting
Go to *
Lactic acidosis >10
Ketosis
Encephalopathy
Hepatomegaly
Organic aciduria
(Methyl malonic,
proprionic, glutaric
isovaleric)
Lactic ac. 5 - 10
Ketosis
High ammonia
Urea cycle defect
Lactic ac. <5
No ketosis
Resp. alkalosis
High ammonia
Permanent
Lactic acidosis
Investigating Cholangiopathies
• Liver enzymes
• USS
• MRCP
• CT
• ERCP
Evolution from Autoimmune Hepatitis to
Sclerosing Cholangitis
At diagnosis
8 years later
Evolution from Autoimmune Hepatitis to
Sclerosing Cholangitis
Non Invasive estimation of Fibrosis
Fibrosis...the final common pathway
Biliary atresia
Autoimmune liver disease
Viral hepatitis
Post-transplant
Alagille and other
cholestasis syndromes
Wilson disease
NAFLD
α1 anti-trypsin
deficiency
Fibrosis
Cirrhosis HCC
How it happens
Wieckowska et al Hepatology 2007
How is fibrosis evaluated?
• Liver biopsy is the accepted standard for
evaluating fibrosis
• Scoring systems to attempt comparability
between centres
Direct serum biomarkers
Index Parameters Liver disease PPV/NPV AUC
MP3 P3NP, MMP1 HCV 66/99% 0.88
ELF HA, P3NP, TIMP1 HCV/NAFLD 75/91% 0.82
FSII a2M, HA, TIMP HCV 74/76%
Serum biomarkers in paediatrics
AUROC ≥F1 0.92 (9.28), ≥F2 0.98 (10.8), ≥F3 0.99 (10.51)
As the liver becomes more fibrotic it becomes lesselastic. A probe with ultrasonic transducer induces avibration of low frequency to liver which induces anelastic shear wave which propagates through the liver.The velocity of the ultrasonic wave correlates withliver stiffness.
FibroScan℮
FibroScan℮: results
50 studies, F4 versus rest AUROC 0.94(0.93-0.95), adjusted AUROC of 0.99Optimal cut off 13.01kPa
≥F2 versus <F2 AUROC 0.84 (0.82, 0.86) adjustd AUROC 0.91 cut off 7.65kPa
MR based techniques
MR Elastography
Modified phase contrast imaging to image the
propagation of shear wave through the liver
Expensive, time consuming
High throughput techniques
•Microarray: identification of susceptibility genes eg.
PNPLA3
• Proteomics: proteins associated– can go onto validate
markers- lumican FABP, VDBP
• Glycomics: glycosylation of proteins – Glycofibro-test /
Glycocirrho-test
EX 1 2 3 4
Missense Nonsense Insertion Deletion
> 500 ATP7B Gene Mutations: ATP7B Mutation Negative in Patient
6
9
19 20
8
EX 21
5
11
1514
7 13
18
10 12
1716
Del15bp
SardinianPatients
Promoter
Diane Cox:
http://www.wilsondisease.med.ualberta.ca/database.asp
•Cost of sequencing a genome has outpaced Moore’s law•2001 $100M to sequence 1 human genome, in 2013 its $10,000• Whole exome sequencing $1,000
Exome = Looking at Exon’s in DNA
>85% human disease-causing mutations reside in coding regions
of genes (i.e., exons)
Conclusions
• Investigation liver disease is complex and
expensive
• We recommend a step wise and multidisciplinary
approach
• Keep an open mind particularly when investigating
metabolic liver disease
• Rare things are rarely correct so please concentrate on
diagnosing treatable conditions and not waste time and money
on uncommon untreatable conditions