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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