What laboratory tests are used to diagnose hepatitis?

Laboratory Tests for Diagnosing Hepatitis

All patients presenting with biochemical evidence of hepatitis should undergo first-line testing for hepatitis A, B, C, and E simultaneously at initial presentation, as hepatitis E is now recognized as the most common cause of acute viral hepatitis in many developed countries. 1

Initial Serologic Panel

The following tests should be ordered together as the initial diagnostic workup:

Hepatitis A

• HAV IgM antibody (plasma or serum) - indicates acute infection 1
• HAV IgG antibody - indicates past infection or immunity 1
• HAV total antibodies can be used in asymptomatic patients with normal liver tests 1

Hepatitis B

• HBsAg (hepatitis B surface antigen) - primary marker for active infection; positivity >6 months defines chronic infection 1, 2
• Anti-HBc IgM - indicates acute or recent infection; positive during the serologic "window period" when HBsAg has disappeared but anti-HBs is not yet detectable 1, 2
• Anti-HBc total - indicates current or previous infection; persists for life 1, 2
• Anti-HBs (hepatitis B surface antibody) - indicates recovery and immunity from natural infection or vaccination 1, 2
• HBeAg - marker of high viral replication 1, 2
• Anti-HBe - usually indicates lower viral replication, though precore mutants can have high HBV DNA despite positive anti-HBe 1, 2
• HBV DNA quantification - essential for assessing viral replication, disease activity, and treatment decisions 2

Hepatitis C

• Anti-HCV antibody - screening test for HCV exposure 1, 3
• HCV RNA qualitative or quantitative (by PCR/NAT) - confirms active viremia; required because anti-HCV does not distinguish acute from chronic or past infection 1, 3, 4
• Serum aminotransferases must be >2.5 times upper limit of normal with negative HAV IgM and negative HBsAg/anti-HBc IgM for hepatitis C diagnosis 1

Hepatitis E

• HEV IgM antibody - appears 4-6 weeks after exposure, lasts 2-4 months 1
• HEV IgG antibody - detectable by 4 weeks after clinical presentation 1
• HEV RNA by NAT - required for diagnosis in immunosuppressed patients (organ transplant recipients) who may have delayed humoral response; essential for diagnosing chronic hepatitis E (≥3 months viremia) 1
• Combination of serology and NAT testing is recommended for optimal diagnosis 1

Hepatitis D (Delta)

• Anti-HDV - test in HBsAg-positive or anti-HBc IgM-positive patients, particularly those with injection drug use history or from endemic areas 1, 2

Liver Function Tests

These should accompany all viral hepatitis testing:

• ALT and AST - assess liver inflammation; ALT >2.5 times upper limit of normal required for hepatitis C diagnosis 1, 2, 5
• Total bilirubin - evaluates liver function and severity in acute injury 2, 5
• Alkaline phosphatase and GGT - additional markers of liver injury 2
• Albumin - assesses synthetic liver function 2
• Prothrombin time/INR - best indicator of severity in acute hepatic injury; evaluates coagulation and synthetic function 2, 5
• Complete blood count with platelets 6

Common Serologic Patterns for Hepatitis B

Understanding these patterns prevents diagnostic errors:

• Acute HBV: HBsAg positive, anti-HBc IgM positive 2
• Chronic HBV: HBsAg positive >6 months, anti-HBc total positive, anti-HBc IgM negative 2
• Past infection with immunity: HBsAg negative, anti-HBs positive, anti-HBc total positive 2
• Vaccine immunity: HBsAg negative, anti-HBs positive, anti-HBc total negative 2
• Window period: HBsAg negative, anti-HBs negative, anti-HBc IgM positive 2

Additional Testing for Autoimmune Hepatitis

If viral markers are negative, test for:

• ANA (antinuclear antibodies) 1
• SMA (smooth muscle antibodies) 1
• Anti-LKM-1 (liver/kidney microsome type 1) 1
• AMA (antimitochondrial antibodies) 1
• If conventional tests negative: anti-SLA/LP, F-actin, anti-LC1, atypical pANCA 1

Critical Pitfalls to Avoid

• Do not delay HEV testing until HAV, HBV, and HCV are negative - test all simultaneously as HEV is now the most common cause of acute viral hepatitis in many countries 1
• Do not miss the serologic "window period" in hepatitis B when both HBsAg and anti-HBs are negative; anti-HBc IgM will be positive during this time 1, 2
• Do not rely on anti-HCV alone - always confirm with HCV RNA testing as antibody tests cannot distinguish active from past infection 3, 4
• Do not overlook isolated anti-HBc positivity - requires follow-up testing 2
• Test for HEV in suspected drug-induced liver injury (DILI) - 13% of presumed DILI cases are actually acute hepatitis E 1
• In immunosuppressed patients with suspected HEV, use HEV RNA testing as antibody response may be delayed or absent 1
• Up to 20% of acute hepatitis C cases will be anti-HCV negative at initial presentation due to delayed seroconversion 1

Specimen Requirements

• Plasma: EDTA or plasma preparation tube, room temperature, <2 hours 1
• Serum: Clot or serum separator tube, room temperature, <2 hours 1
• Check with laboratory regarding minimum blood volumes for molecular platforms 1