Standard Operating Procedure: Liver Function Tests (LFTs) Panel

1. Purpose and Scope

To measure the full liver panel (ALT, AST, ALP, GGT, total/direct bilirubin, total protein, and albumin) for the detection, differentiation, and monitoring of hepatocellular injury, cholestasis, biliary obstruction, and impairment of hepatic synthetic function.

2. Specimen Requirements

• Type: Serum. Use a plain (no additive) tube or serum separator tube (SST).
• Volume: Minimum 1.0 mL per ordered analyte group.
• Hemolysis: Reject markedly hemolyzed samples for AST/ALT and bilirubin; hemolysis falsely raises these analytes.
• Light protection: Protect bilirubin samples from light after collection; place in amber containers or wrap immediately. Bilirubin is photosensitive.
• Stability: Separate serum from cells within 2 hours. ALT and AST are stable 24-48 h at 2-8°C; ALP and GGT are stable 2-3 days refrigerated; bilirubin is stable 24 h refrigerated if protected from light.
• Fasting: Fasting 8-12 hours is preferred for bilirubin, though non-fasting specimens are generally acceptable for ALT/AST.

3. Safety and Precautions

• Standard PPE (gloves, lab coat, eye protection). All blood samples are potentially infectious.
• Dispose of sharps and biohazard waste per laboratory policy.
• Do not pipette by mouth. Use mechanical pipetting devices.

4. Equipment and Reagents

• Automated chemistry analyzer (spectrophotometric, temperature-regulated to 37°C).
• ALT/AST: IFCC-recommended UV kinetic reagents (with or without pyridoxal phosphate), measuring NADH oxidation at 340 nm.
• ALP: Kinetic method using p-nitrophenyl phosphate (pNPP), measuring p-nitrophenol formation at 405 nm.
• GGT: Szasz method using gamma-glutamyl-3-carboxy-4-nitroanilide as substrate, measuring release of p-nitroaniline at 405 nm.
• Total and Direct Bilirubin: Diazo (Jendrassik-Grof) method; total bilirubin with accelerator, direct bilirubin measured after addition of diazo reagent without accelerator.
• Total Protein: Biuret method, measured at 540 nm.
• Albumin: Bromocresol green (BCG) or bromocresol purple (BCP) dye-binding method, measured at 600 nm.
• Two levels of quality control material for each assay.

5. Per-Analyte Procedures

5.1 ALT (Alanine Aminotransferase) Procedure

1. Reconstitute or load the IFCC ALT reagent (L-alanine, alpha-ketoglutarate, NADH, lactate dehydrogenase, pyridoxal phosphate) at 37°C.
2. Piper 200 µL of reagent and 20 µL of serum into the reaction cuvette; mix.
3. Monitor the decrease in absorbance at 340 nm (NADH oxidation) every 30 s for 3 min.
4. Calculate the rate; multiply by the instrument factor to express activity in U/L at 37°C.
5. Repeat for controls and report the mean of acceptable duplicate readings.

5.2 AST (Aspartate Aminotransferase) Procedure

1. Load the IFCC AST reagent (L-aspartate, alpha-ketoglutarate, NADH, malate dehydrogenase) at 37°C.
2. Piper 200 µL of reagent and 20 µL of serum; mix well.
3. Monitor the decrease in absorbance at 340 nm (NADH consumption) over 3 min, recording readings every 30 s.
4. Compute the linear rate of change; convert to U/L at 37°C using the instrument factor.
5. Note the expected hemolysis interference and reject grossly hemolyzed samples.

5.3 ALP (Alkaline Phosphatase) Procedure

1. Load the pNPP (p-nitrophenyl phosphate) kinetic reagent at 37°C.
2. Piper 200 µL of reagent and 20 µL of serum; mix.
3. Measure the increase in absorbance at 405 nm (formation of p-nitrophenol) at fixed intervals for 3 min.
4. Calculate activity in U/L from the change in absorbance per minute and the molar absorptivity.
5. Flag results above the linear range and dilute and re-run.

5.4 GGT (Gamma-Glutamyl Transferase) Procedure

1. Load the Szasz substrate (gamma-glutamyl-3-carboxy-4-nitroanilide with glycylglycine) at 37°C.
2. Piper 200 µL of reagent and 20 µL of serum; mix.
3. Monitor the increase in absorbance at 405 nm (release of p-nitroaniline) over 3 min.
4. Calculate the enzyme activity in U/L from the rate of absorbance increase.
5. Interpret with caution: GGT rises with alcohol and many hepatotoxic drugs; it is non-specific.

5.5 Total Bilirubin Procedure

1. Protect the sample and aliquot from light throughout.
2. Dilute serum 1:10 with distilled water or per kit instructions; add diazo reagent (sulfanilic acid and sodium nitrite, with caffeine/sodium benzoate accelerator).
3. Incubate for 10 min at room temperature to complete the reaction.
4. Measure absorbance at 540-546 nm against a blank; read azobilirubin chromogen.
5. Convert absorbance to mg/dL using a calibrator or the molar absorptivity.

5.6 Direct (Conjugated) Bilirubin Procedure

1. Protect serum from light.
2. Add diazo reagent to serum without the accelerator so that only conjugated (direct) bilirubin reacts.
3. Incubate for exactly 10 min at room temperature.
4. Measure absorbance at 540-546 nm and convert to mg/dL against the appropriate calibrator.
5. Compute indirect bilirubin as Total bilirubin - Direct bilirubin when required.

5.7 Total Protein Procedure

1. Piper 20 µL of serum into the biuret reagent (copper sulfate in alkaline tartrate/iodide).
2. Incubate for 10 min at 37°C (or per kit) to form the violet copper-protein complex.
3. Measure absorbance at 540 nm against a reagent blank.
4. Read concentration in g/dL from the linear calibration curve; multiply by 10 for g/L.
5. Account for lipemia by running a serum blank or diluting grossly lipemic samples.

5.8 Albumin Procedure

1. Piper 5 µL of serum into the dye-binding reagent (bromocresol green or bromocresol purple).
2. Incubate briefly (about 1 min) to allow dye-albumin complex formation.
3. Measure the change in absorbance at 600 nm (BCG) or 590 nm (BCP) against a blank.
4. Read albumin concentration in g/dL from the calibration curve.
5. Compute globulin as Total protein - Albumin when required for clinical interpretation.

6. Common Calibration and Quality Control

• Run two levels (normal and abnormal) of control for each assay daily, with every calibration, and with each new reagent lot.
• Enforce Levey-Jennings rules; investigate trends or shifts before reporting patient results.
• Participate in an external quality assessment (EQA) program for all panel analytes.
• Verify control values fall within acceptable target ranges before patient reporting.

7. Decision limits

Decision thresholds for the liver panel are interpretive patterns rather than single adult intervals; the canonical reference intervals are displayed in the Reference Ranges panel.

• Severe (massive) hepatocellular injury: AST and/or ALT above 10x the upper reference limit, often with ALT > 1000 U/L, suggests acute ischemic, toxic, or viral hepatitis; ALT > 1000 U/L is rarely caused by cirrhosis or obstruction alone.
• R ratio (DILI pattern): R = (ALT/ULN)/(ALP/ULN). R >= 5 = hepatocellular injury; R <= 2 = cholestatic; 2 < R < 5 = mixed.
• De Ritis ratio: AST/ALT > 2 is associated with alcohol-associated liver disease; AST/ALT < 1 is typical of most other hepatocellular conditions.
• ALP with normal GGT: Raised ALP with a normal GGT points toward non-hepatic sources (bone, placenta, pregnancy); a raised GGT with raised ALP confirms a hepatobiliary origin.
• Neonatal jaundice: Total bilirubin thresholds for phototherapy and exchange transfusion are age (hour-of-life) and risk-factor dependent; apply the AAP (Bhutani) nomogram for term and near-term infants.
• Albumin: Persistently low albumin (< 3.0 g/dL) with prolonged coagulation indicates reduced hepatic synthetic capacity in chronic liver disease; acute hepatitis commonly retains normal albumin synthesis.
• GGT as screening enzyme: GGT is non-specific and may rise with alcohol intake and many hepatotoxic medications; do not use an isolated GGT elevation in isolation for diagnosis.

Note on dietary and pre-analytical interference: bilirubin falls during prolonged fasting and is affected by food; grossly lipemic samples should be ultracentrifuged before analysis.