Overview
Liver function tests are a group of tests done to assess the functional capacity of the liver and to detect cellular damage to hepatocytes. They evaluate synthetic ability (via plasma proteins like albumin and prothrombin, and lipids), and secretory/excretory ability (via serum bilirubin).
Interpreting the pattern of abnormalities — bilirubin fractions, liver enzymes, and synthetic markers together — allows the laboratory to differentiate pre-hepatic, hepatic and post-hepatic causes of liver dysfunction.
Learning Objectives
After this lesson you will be able to…- Enumerate the various tests undertaken to measure liver function and damage.
- Differentiate conjugated (direct) and unconjugated (indirect) bilirubin.
- Classify jaundice as pre-hepatic, hepatic, or post-hepatic based on bilirubin patterns.
- Describe the role of albumin, globulin, and prothrombin time as synthetic function markers.
- Explain the significance of AST, ALT, ALP and GGT in hepatocellular versus cholestatic injury.
Clinical Story
Why This MattersA 40-year-old woman presents with yellowing of the eyes and dark urine. Her doctor orders a full liver function test panel. The lab technologist must determine whether the rise in bilirubin is pre-hepatic, hepatic, or post-hepatic by correlating conjugated and unconjugated bilirubin fractions with enzyme patterns — a decision that directly shapes the clinical workup that follows.
Core Concepts
Bilirubin is derived from haem breakdown. Unconjugated bilirubin is water-insoluble and travels bound to albumin to the liver, where glucuronyl transferase conjugates it with glucuronic acid, making it water soluble and excretable into bile. A rise above 2 mg/dL causes clinical jaundice. Pre-hepatic jaundice (e.g. haemolysis) raises unconjugated bilirubin predominantly. Hepatic jaundice (e.g. hepatitis, cirrhosis) raises both direct and indirect bilirubin due to hepatocellular dysfunction. Post-hepatic (obstructive) jaundice (e.g. gallstones, tumour) raises conjugated bilirubin due to outflow obstruction.
Serum albumin is synthesized exclusively by the liver and maintains plasma oncotic pressure, so it directly reflects hepatic synthetic function. The normal albumin:globulin (A:G) ratio is 1.2 to 1.5. In hepatic dysfunction this ratio falls toward or below 1, since albumin synthesis falls while globulins may rise due to inflammatory/infective processes.
Prothrombin (clotting factor II) is synthesized in the liver, so prothrombin time reflects hepatic synthetic function. Because the test result varies by analytical method, the International Normalized Ratio (INR) standardizes reporting using the International Sensitivity Index (ISI) of the reagent kit. Normal INR (not on warfarin) is 0.8–1.2; the therapeutic target on warfarin is 2.0–3.0.
Transaminases (AST/SGOT, ALT/SGPT) leak from damaged hepatocytes; ALT is more liver-specific since AST is also found in cardiac and muscle tissue. Alkaline phosphatase (ALP) is found in cells lining the biliary system, bone, and placenta, and rises with cholestasis or biliary obstruction. Gamma glutamyl transpeptidase (GGT) is a highly sensitive marker specific to the biliary tree, rising even in minor, subclinical biliary damage.
Laboratory Principle
Serum bilirubin is measured by the Diazo method using diazotized sulfanilic acid, forming coloured azobilirubin read colorimetrically at 550–580 nm; conjugated bilirubin reacts directly (direct method) while unconjugated bilirubin requires dissolution in DMSO (indirect method, measuring total bilirubin). Albumin is measured by the Bromocresol green method (read at 630 nm) and total protein by the Biuret method (cupric ions forming a violet complex, read at 540 nm). Transaminases are measured by a UV kinetic method coupling their reaction to NADH consumption, read at 340 nm. ALP uses p-nitrophenylphosphate as substrate, measured at 405 nm.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Diazotized sulfanilic acid | Diazo reagent kit | Bilirubin estimation (direct & total) | 2–8°C, protect from light |
| DMSO (Dimethyl sulfoxide) | Solvent grade | Solubilizes unconjugated bilirubin for indirect method | Room temperature |
| Bromocresol green reagent | Acidic buffer, pH ~4.2 | Serum albumin estimation | 2–8°C |
| Biuret reagent | Copper (II) sulfate, alkaline | Total serum protein estimation | Room temperature |
| Sodium citrate anticoagulant | 3.2% citrate tube | Prothrombin time / INR sample collection | Room temperature, use within 4 hrs |
Step-by-Step Procedure
Collect a fasting venous blood sample in a plain (serum) tube, protected from light, for bilirubin, protein, albumin and enzyme assays. Collect a separate citrate tube for PT/INR.
Centrifuge the clotted sample to separate serum. Process bilirubin samples promptly, as bilirubin degrades on exposure to light.
React serum with diazotized sulfanilic acid to form azobilirubin for direct (conjugated) bilirubin; add DMSO for the indirect method to measure total bilirubin. Calculate indirect (unconjugated) bilirubin as Total minus Direct.
Measure total protein by the Biuret method at 540 nm and albumin by the Bromocresol green method at 630 nm. Calculate globulin as Total protein minus Albumin, then compute the A:G ratio.
Measure AST, ALT via UV kinetic NADH-coupled reactions at 340 nm; measure ALP colorimetrically at 405 nm. Run the citrated plasma sample on a coagulation analyzer for PT, then calculate INR using the kit's ISI value.
Flow Diagram
Quality Control
Run normal and pathological control sera for bilirubin, enzymes, and total protein with every batch. Verify PT/INR controls fall within the manufacturer's acceptable range before reporting patient values, since PT results guide critical clinical decisions such as anticoagulation dosing.
Enrol in external proficiency testing schemes for clinical chemistry and coagulation to ensure inter-laboratory comparability of bilirubin, enzyme and INR results.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| ↑ Indirect bilirubin, normal enzymes | Pre-hepatic jaundice — commonly haemolytic disorders | Order reticulocyte count, LDH, haptoglobin |
| ↑ Direct and indirect bilirubin, ↑ AST/ALT | Hepatic (hepatocellular) jaundice — hepatitis, cirrhosis | Viral hepatitis markers, imaging, hepatology referral |
| ↑ Direct bilirubin, ↑ ALP/GGT | Post-hepatic (obstructive) jaundice — gallstones, tumour | Abdominal ultrasound, MRCP |
| A:G ratio < 1 | Reduced hepatic synthetic function or chronic inflammation | Correlate with albumin trend and clinical picture |
Common Errors & How to Avoid Them
Cause: Bilirubin photodegrades rapidly when exposed to light.
Prevention: Use amber tubes or wrap samples in foil, and analyse promptly.
Cause: Red cells release AST and LDH on lysis, falsely elevating enzyme results.
Prevention: Use careful venipuncture technique and reject grossly haemolysed samples.
Cause: Under-filled citrate tubes alter the blood-to-anticoagulant ratio, skewing results.
Prevention: Fill citrate tubes to the exact mark and test within 4 hours of collection.
Laboratory Tips from the Bench
ALT is more liver-specific than AST — use the AST:ALT ratio to help distinguish causes; a ratio greater than 2 suggests alcoholic liver disease.
GGT rises earlier and more sensitively than ALP in biliary tract disease, and is useful to confirm that a raised ALP is hepatic rather than bony in origin.
Remember "SGPT stays in the liver, SGOT goes everywhere" — ALT (SGPT) is liver-specific, AST (SGOT) is also found in heart and muscle.
Important Notes
Clinical jaundice becomes visible only once serum bilirubin exceeds approximately 2 mg/dL, so subclinical liver dysfunction can exist without visible jaundice.
Because prothrombin time varies by reagent and instrument, INR (using the kit's ISI) allows results to be compared consistently across laboratories, especially important for monitoring warfarin therapy.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgePresents with yellowing of the eyes, dark urine, pale stools, and severe right upper quadrant pain for one week. No history of alcohol use.
Markedly raised direct bilirubin and ALP with only mild ALT elevation, alongside pale stools and dark urine, points to biliary obstruction rather than hepatocellular injury.
- →Pale stools and dark urine are classic clues to conjugated hyperbilirubinaemia.
- →A disproportionately raised ALP relative to ALT favours a cholestatic/obstructive pattern.
- →Imaging (ultrasound/MRCP) is essential to confirm and localize biliary obstruction.
Frequently Asked Questions
ALT is found predominantly in the liver, while AST is also present in cardiac muscle, skeletal muscle, and red blood cells, so AST elevation can occur without liver disease.
ALP is also found in bone and placenta, so it alone cannot confirm liver origin. Since GGT is specific to the biliary tree, a simultaneous rise in both strongly suggests hepatobiliary disease.
Raw PT values vary between reagents and instruments, making cross-laboratory comparison unreliable. INR standardizes this using the reagent's International Sensitivity Index.
Quick Revision
10-Minute ReviewKey Takeaways
- Liver function tests assess synthetic capacity and secretory/excretory capacity of the liver.
- Total, direct and indirect bilirubin patterns classify jaundice as pre-hepatic, hepatic, or post-hepatic.
- Serum albumin and the A:G ratio reflect hepatic protein synthesis.
- Prothrombin time/INR reflects clotting factor synthesis by the liver.
- ALT and AST indicate hepatocellular injury; ALP and GGT indicate cholestatic/biliary injury.
- Correlating bilirubin fractions with enzyme patterns is key to correctly classifying liver disease.
Competency Checklist
Track Your MasteryReferences
- NIOS Biochemistry Course Material, Lesson 17: Liver Function Tests.
- Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, latest edition.
- Harper's Illustrated Biochemistry, latest edition.