Download - LFT - web.medicine.kln.ac.lk
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Biochemical Investigations in Liver Disease
Dr Roshitha de SilvaDepartment of Pathology
Faculty of MedicineUniversity of Kelaniya
Biochemical markers
• Albumin
• ALP
• ALT, AST
• Gamma-glutamyl transpeptidase (γGT)
• Serum bilirubin
• Urine bilirubin, urobilinogen
• Alpha feto protein
Serum albumin
• Albumin is the major protein in the human body – TP 6-8 g/dL– Alb 3.5-5.5 g/dL
• Representing about 50% of the total protein content• Its main function is to regulate the colloidal osmotic
pressure of blood• Serves as a carrier for molecules of low water
solubility: – lipid soluble hormones (thyroxine)– bile salts– unconjugated bilirubin– cations (Ca2+, K+)
Serum albumin
• Half life of albumin in serum is 20 days
• Decreased serum albumin levels are seen in chronic liver failure
• The serum albumin levels are normal in diseases like
– acute viral hepatitis
– drug related hepatotoxicity
– obstructive jaundice
Serum albumin
• Only 20-30% of hepatocytes are committed to the daily production of albumin
Low Serum albumin
• Not specific for liver disease• Low levels of albumin may be the result of
– Inadequate production– Inadequate intake
• Protein malnutrition
– Excessive loss
• Nephrotic syndrome
• Protein losing enteropathies
• Burns
• Hyperalbuminaemia
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Albumin/globulin ratio
• Calculated value
• It is important to look at changes in the individual as well as the ratio
• The albumin/globulin ratio remains if both proteins change together– Loss of both albumin and globulin
• Acute blood loss
• Exudative skin disease
• Gastrointestinal disease
– Dehydration (increased albumin and globulin)
Reversed albumin/globulin ratio
• Decrease in albumin
– Decreased production
• Severe liver disease (hyperglobulinemia)
• Inflammation (negative acute phase protein)
• Increased globulins
– Infection
– Chronic inflammation
– Multiple myeloma
Alkaline Phosphatase
• Produced both from liver & bone
• Increased in any form of biliary tree obstruction
• Extrahepatic > intrahepatic obstruction (x 3)
• Degree of elevation does not help to distinguish between the two
Alkaline Phosphatase Cont.
• Billiary obstruction• Iry/IIry hepatic cancer• Infectious hepatitis • Drugs
• Verapamil• Carbamazepine, Phenytoin• Erythromycin• Allopurinol• Ranitidine
• Pregnancy, Children• Bone disorders- Paget disease, osteomalacia• Transient ALP increase in children
ALP & Pregnancy
• Levels of 2-4 times normal in late in pregnancy
– Negative Pregnancy Adult: 33 - 96 U/L
– Pregnancy Trimester One: 17 - 88 U/L
– Pregnancy Trimester Two: 25- 126 U/L
– Pregnancy Trimester Three: 38 - 229 U/L
Transient hyperphosphatasemia
• Marked elevation of serum ALP in the absence of detectable liver or bone disease
• Recognition of this phenomenon avoids unnecessary procedures and concerns
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Transient hyperphosphatasemia
• Increased ALP activity in vary from 3 to 50 times URL of adults
– variability may be attributed to the timing of the ALP sample in relation to the peak ALP activity
• Serum ALP levels gradually return to normal within two to three months but have persists as long as six months in other cases
Transient hyperphosphatasemia
• Observed in infants and children younger than 5 years of age
• THP is usually diagnosed by excluding other diseases
• Workup
– LFT, Isoenzymes
– Ca, PO4, Vit D, Cr
Diagnosing BTH
• Age < 5 years
• No clinical evidence of bone or liver disease
• No Lab evidence of bone or liver disease
• Serum ALP isoenzymes elevated in both bone and liver
• Normalisation of serum ALP levels within 4 months of onset
Alkaline Phosphatase Cont.
• Reference intervals
Specimen
• The test should not be done on plasma if citrate, oxalate or EDTA were used as anticoagulants
• They form a complex with zinc and the alkaline phosphatase, causing irreversible enzyme inactivation
• ALPs are zinc-containing metalloenzymes
• Three metal ions including two Zn2+ and one Mg2+ in the active site are essential for enzymatic activity
Characterization of the ALPs
• Three general methods to separate different ALP:
– thermostability studies
– differential inhibition
– immunologic methods
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Types of isozymes
• Human ALPs can be classified tissue-specific forms or isozymes:
• ALP – liver
• ALP – bone
• ALP – placental
• ALP – intestinal
ALP electrophoresis
Placental Alkaline Phosphatase
• Placental ALP is a heat stable enzyme present at high levels in the placenta.
• A trace amount of this isoenzyme can be detected in normal sera
• The placental ALP gene can be re-expressed by cancer cells as the Regan isoenzyme
REGAN ISOENZYME
• Isolated from the sera of patients with neoplasms
• Regan has been isolated from patients with lung cancer, breast cancer, ovarian cancer, and carcinoma of the colon
NAGAO ISOENZYME
• A variant of Regan isoenzyme that migrates in the same position as Regan on cellulose acetate.
• It has been isolated in metastatic carcinoma to the pleural surfaces and in adenocarcinoma of the pancreas or bile duct
Transaminases
• Aspartate aminotransferase (AST or SGOT)
– Liver, skeletal muscle, cardiac muscle, kidneys, rbc
• Alanine aminotransferase (ALT or SGPT)
– 1° in liver
• Sensitive indicators of liver cell injury
– Even very mild liver abnormalities can cause slightly elevated AST/ALT
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Reference range Obesity & transaminases
• AST and ALT values are higher in obese patients
• ALT levels have been noted to decline with weight loss
Macro-AST
• Rarely individuals can have chronically elevated AST levels
• Macro-AST must be considered when the AST level is chronically high without any liver, cardiac, or muscle disease
AST
ALT
Release of AST/ALT from Liver Cells during Acute Hepatocellular Injury
Liver zones Zone 3
• Zone 3 of the acinus is more vulnerable to both hypoxic and toxic (hepatocytes are richer in microsomal enzymes) damage
• Zone 3 of the hepatic acinus has a higher concentration of AST, and damage to this zone, whether ischemic or toxic, may result in greater alteration to AST levels.
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Clearance
• Aminotransferase clearance is carried out within the liver by sinusoidal cells
• The half-life in the circulation is about 47 hours for ALT, about 17 hours for total AST
• Most liver diseases are associated with greater elevation of ALT than AST
AST/ALT Ratio
• AST:ALT <1
– Viral hepatitis
• AST:ALT >1
– Alcoholic steatosis
– Alcoholic hepatitis
– Alcoholic cirrhosis
Aminotranferases
• Convenient to measure
• Present in liver cells in large amounts
• “quantitative” assessment of ongoing hepatocyte necrosis
• Blood level roughly proportional to the number of hepatocytes that died recently (hours-days)
Problems with using transaminases to assess liver injury
• Only assess injury over the past 1-2 days
• Magnitude of elevation does not necessarily correlate with extent of liver function or dysfunction.
• AST and ALT = rate of destruction of hepatocytes
• Liver function = number of functional hepatocytes left
Gamma glutamyl transferase
• GGT is an enzyme found in cell membranes of many tissues:
– Liver, kidney, pancreas, intestine
• The highest concentration is in the kidney, but the liver is considered the source of normal enzyme activity
• Intracellular antioxidant (involved in gluthathione metabolism)
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GGT
• GGT is not very useful in the differential diagnosis of liver disease
• The highest GGT levels are seen in cholestaticliver disease and rise in parallel with alkaline phosphatase
GGT
• Isolated elevation in alkaline phosphatase
• Monitor the cessation of alcohol consumption in patients with chronic alcoholism
Hepatic causes of high GGT
• Hepatitis (acute and chronic)
• Cirrhosis
• Liver metastasis and carcinoma
• Cholestasis
• Alcoholic liver disease
• Primary biliary cirrhosis
• Sclerosing cholangitis
Non-hepatic causes of high GGT
• Pancreatitis
• Carcinoma of prostate
• Carcinoma of breast and lung
• Systemic lupus erythematosus
• Alcoholism
• Medications- carbamazepine, furosemide, heparin, phenobarbital, phenytoin, valproicacid
GGT and alcohol
• Chronic ethanol consumption is known to readily induce a rise in serum GGT
• Indicate continuous, rather than episodic drinking
• Used as a biomarker for heavy drinking
GGT and alcohol
• GGT elevation in serum is primarily due to hepatic enzyme induction, rather than to liver cell injury.
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GGT
• Why do you need GGT when you have ALP?
• GGT is used in different diagnostic algorithms
• It is a powerful predictor of presence of liver disease in patients discovered to have first time abnormal LFT’s
ALFIE
• Large study helped to develop diagnostic algorithm for patients who had abnormal LFT’s
• Patients followed up 15 yrs to see subsequent liver disease or mortality
• They concluded GGT is a significant predictor of liver disease and it should be included in the LFT in primary care
NAFLD
• GGT is more sensitive detector of NAFLD than ALT
• FLI (fatty liver index)
– Used for the diagnosis of NAFLD
– Calculated using a formula, includes:
• BMI, waist circumference, TG, GGT
– FLI shows good accuracy in detecting NAFLD and avoids the need of USS abdomen
NAFLD
• Liver biopsy is considered as the gold standard for assessing NAFLD
• Non-invasive markers of fibrosis (FibroTest), steatosis (SteatoTest) and steato-hepatitis (NashTest, ActiTest)
• Steato test– Measures/predicts hepatic steatosis
– This use 9 B/C markers to calculate• α2 macroglobulin, haptoglobin, GGT, FPG, apolipoprotein A1,
total bilirubin, TG, TC, ALT
Gamma-glutamyl transpeptidase
• Elevated in most subjects with liver disease regardless of the cause
• May be elevated with even minor, sub-clinical levels of liver dysfunction
• Most sensitive, but least specific of all LFTs
• Helpful in identifying the cause of an isolated elevation in ALP
Serum bilirubin
• The byproduct of Hb breakdown
• Total bilirubin
– indirect bilirubin (when > 80% of total bilirubin is unconjugatedindirect hyperbilirubineamia)
– direct bilirubin
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Hyperbilirubinemia
• Conjugated hyperbilirubinemia– Cholestasis/biliary obstruction– Hepatitis– Rare disorders of canalicular secretion of
conjugated bilirubin - DJ, Rotor• Unconjugated hyperbilirubinemia
– Massive overproduction - acute hemolysis– Impaired conjugation
common: Gilbert's syndrome (mild)rare: Crigler-Najjar syndrome (severe)
Gilbert's syndrome
• Benign, familial disorder
• Present in 5% of the population
• Genetic origin-reduction in the glucuronidation activity of UGT
• UGT activity is decreased to 30%
• Reduced UGT activity due to
– reduced number of enzyme molecules ?
– qualitative enzyme defect ?
Gilbert's syndrome
• By definition, bilirubin levels in GS are <6 mg/dL, but most patients show levels <3 mg/dL
• Bilirubin levels occasionally may be normal
• Precipitated by – Dehydration
– Fasting
– Menstruation
– Causes of stress• Intercurrent illness
• Vigorous exercise
Fasting test for Gilbert's syndrome
• Plasma bilirubin concentrations rise after fasting• Day 1: take blood for total and unconjugated plasma
bilirubin estimations: begin calorie restriction to 300 kcal per day (approximately 2 biscuits)
• Day 2: take blood for total and unconjugated plasma bilirubin estimations– Total bilirubin concentrations rise in normal individuals by
60% after 48h fasting – whereas in subjects with Gilbert’s syndrome
• total bilirubin concentration rises by 90% after 24h• unconjugated bilirubin concentration rises by more than 110%
after 24h
Urine Bilirubin/Bile
• Not present in the urine of healthy individuals
• Bilirubin leaks back into the blood stream and is excreted in urine in
– Obstructive jaundice
– Hepatitis
• Fouchet’s test
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Urine Urobilinogen
• Conjugated bilirubin is excreted via bile salts to intestine
• Bacteria in the intestine break down bilirubinto urobilinogen for excretion in the feces
• Normally there are mere traces of urobilinogen in the urine
• Ehrlich’s test
Condition Urine Bilirubin Result Urine Urobilinogen
Result
Hemolytic disease Negative Increased
Hepatitic disease Positive or negative Increased
Biliary obstruction Positive Negative
Cirrhosis
Suspected Disorder Confirmatory Investigations
Iry biliary cirrhosis •Antinuclear antibodies (ANAs)•immunoglobulin levels (mainly IgM)
Haemachromatosis •Serum ferritin•Transferrin saturation
Wilsons disease •Caeruloplasmin•Serum copper
Alpha 1 antitrypsin deficiency •Alpha1 antitrypsin
Cystic fibrosis •Sweat test
AFP
• Indications
• Pregnancy screening for congenital defect
– Triple Screen
• Tumor Marker
– Hepatocellular carcinoma
– Nonseminomatous germ cell tumor
AFP
• Sites of synthesis
– Embryonic yolk Sac
– Developing gastrointestinal tract
– Liver
• AFP is major protein in developing fetus
– Decreases to undetectable level after birth?
– Decreased by 50% in 1 month
– After 2 years - normal adult levels
Increased AFP in Non-Pregnant States
• Benign causes– Cirrhosis
– Acute or chronic active Viral Hepatitis
– Pregnancy
• Malignant causes– Hepatocellular Carcinoma (usually AFP >1000 ng/ml)
– Non-seminomatous germ cell tumor– Embryonal carcinoma
– Yolk sac carcinoma
– Choriocarcinoma
– Teratoma
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Non-pregnant
• Normal AFP <5.4 ng/ml
• AFP >500 ng/ml unlikely to be from benign cause
HCC
• Serum AFP levels are often elevated in HCC, but this is not always the case.
• AFP levels may be elevated initially in the early stages of HCC and then drop or even normalize before rising again as disease progression occurs
• In general, consistently elevated serum AFP levels greater than 500 ng/mL are indicative of HCC
• Lower serum concentrations which are only transient in nature are more often present in benign liver disease
HCC
• Increased in 80% of HepatocellularCarcinoma case: others-highly dedifferentiated anaplastic tumors
• Over 1000 ng/ml in 40% of HCC
• Sensitivity and specificity of the test depends significantly on the cut-off range
HCC vs benign
• Hepatitis and cirrhosis cases serum AFP level is lower than 50 ng/ml while about 15% of patients demonstrate moderate AFP levels (>500 ng/ml)
• AFP produced by nonmalignant hepatocytescan be distinguished from detection of AFP glycoforms
AFP glycoforms
• Total AFP can be divided into three different glycoforms:– AFP-L1– AFP-L2– AFP-L3
• AFP is fractionated by affinity electrophoresis based on the reactivity with lectin Lens culinaris agglutinin (LCA)
• AFP-L1 - does not react with LCA : non-malignant liver diseases
• AFP-L2 (intermediate reactive) detected– In maternal serum during pregnancy – In patients with yolk sac tumors
• AFP-L3 - highly LCA-reactive fraction : malignant liver tumors.
Triple Screen
• Done in the second trimester to detect for chromosomal abnormalities and neural tube defects
• Screening for Downs, trisomy 21, open neural tube defects
• Maternal serum – AFP
– Oestriol
– hCG
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Thank you