Clinical diagnostic biochemistry - 10 Dr. Maha Al-Sedik 2015 CLS 334

Single-Stranded RNA Capsid

HAV-IgM:- Positive  Acute HAV infection. May be Negative in early infection within days after onset of symptoms. Present for 3 to 6 months after onset of infection.

HAV-IgG:- Positive  Past infection and immunity to HAV.

Hepatitis B surface antigen (HBsAg) Hepatitis B core antigen (HBcAg) Partially double- stranded DNA Polymerase Hepatitis B e antigen (HBeAg)

HighModerate Blood Serum Wound exudates Semen vaginal fluid saliva Urine Feces Sweat Tears Breast milk Low/Not Detectable

TIME HBs Ag HBe Ag HBc total Ab HBs Ab HBe Ab HBc IgM Ab Relative Concentration Duration Incubation Average 8-13 weeks Acute Infection (2 wek- 3mnth) Early Recovery ( 3- 6 mnth) Recovery 6-12mnth / Years symptoms

MeaningMarker PatientHBs Ag Not patient ( cured or immunized )HBs Ab Infectious ( replicating virus )HBe Ag Non infectious ( non replicating virus)HBe Ab Acute infectionHBc IgM If IgM is positive = acute If IgM is negative = chronic HBc total Positive VIP

Complete cure after infection Negative Positive HBs Ag HBc total Ab HBs Ab immune due to vaccination Negative Positive HBs Ag HBc total Ab HBs Ab How to differentiate between immunized individual and past infection?

Patient Acute infection Non infictious Positive Negative HBs Ag HBc IgM HBc total Ab HBs Ab Hbe Ag Patient Chronic infection Positive >6 ms Positive Negative HBs Ag HBc total Ab HBc IgM HBs Ab Patient Chronic Infectious patient with Active virus replication Positive Negative Positive Negative HBsAg HBc total Ab HBc IgM HBe Ag HBs Ab HBe Ab

Very early acute infection Positive Negative HBs Ag HBc total Ab HBe Ag HBs Ab HBe Ab more sensitive than ELISA. Confirming HBs Ag Monitor Therapy Qualitative Quantitative HBV Viral DNA PCR

HCV RNA characterized by rapid replication and high tendency to mutate forming multivariants.

Multiple measurements of ALT at regular intervals monthly or every 3 weeks give increasing results. HCV – Abs ELISA PCR qualitative and quantitative. Liver biopsy: can identify the type and degree of damage (and can determine the severity of the disease). The disease may gradually progress over a period of years.

Antibody to hepatitis C (Anti-HCV) Positive: Previous exp. but does not distinguish between an acute, chronic or resolved infection. False positive: little. results are found in patients with hypergammaglobulinemia. Positive ELISA for hepatitis C

Negative: Neither present infection nor past infection. False negative: immuno compromised ( HIV ). Negative ELISA for hepatitis C

T here is currently no vaccine for HCV. The difficulty in developing a vaccine is due to the mutability of the HCV genome. Vaccine for hepatitis C

(HBsAg) Hepatitis D antigen (HDAg) Single-stranded negative sense RNA HDV is a defective incomplete virus Cannot infect with its own, it needs the help of HBV to replicate by coating its RNA with HBs Ag

Patients with chronic hepatitis B and a positive HDV test are super-infected. Positive HDV tests in a patient with acute hepatitis B indicates HBV/ HDV Co-infection

Coinfection with HBV Positive HDV IgM HDV Ag HBs Ag HBc IgM Superinfection Positive Negative HDV IgM HBs Ag HBc Total HDV Ag HBC IgM

Single-stranded RNA

HEV-IgM: Positive  Acute HEV infection. HEV-IgG: Positive  Past infection and immunity to HEV.

Liver Enzymes

Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Gamma glutamyl transferase Lactate dehydrogenase

The five enzymes that are commonly measured and used in the diagnosis of liver disease: (1)Aspartate aminotransferase (AST) (2) Alanine aminotransferase (ALT) (3) Alkaline phosphatase (ALP) (4) Gamma glutamyl transferase (GGT) (5) lactate dehydrogenase (LD) is occasionally used.

 ALT and GGT are present in several tissues, but plasma activities primarily reflect liver injury, so they are more specific.  AST is found in liver, muscle (cardiac and skeletal), and to a limited extent in red cells.  LD has wide tissue distribution, and is thus relatively nonspecific.  ALP is found in a number of tissues, but in normal individuals primarily reflects bone and liver sources. Tissue Specificity

 Enzymes are found at different locations within cells.  AST, ALT, and LD are cytosolic enzymes. As such, they are released with cell injury, and appear in plasma relatively rapidly.  In the case of AST, there are both mitochondrial and cytosolic isoenzymes in hepatocytes, So ALT is mainly in acute diseases but AST is present also in chronic diseases.  In contrast, ALP and GGT are membrane-bound glycoprotein enzymes. The most important location of both enzymes is on the canalicular membrane of hepatocytes. Subcellular Distribution

Cytoplasmic : Acute Mitochondrial : chronic AST Cytoplasmic only : Acute ALT and LD canalicular membrane of hepatocytes : Obstructive diseases ALP and GGT

Mechanism of release  Cell injury is the simplest mechanism.  Alcohol appears to induce expression of mitochondrial AST on the surface of the hepatocyte.

 Clearance of liver enzymes from plasma occurs at variable rates.  The half-life of ALT is 47 hours, and half life of AST is 17 hours; thus although more AST is released from liver, the much longer half-life of ALT leads to higher activities of ALT than AST in most forms of hepatocellular injury.  The half-life of the liver isoenzyme of ALP has been variously reported as from 1 to 10 days.  The half-life of GGT has been reported as 4 days. Rate of clearance

De Rites ratio AST / ALT ratio : Normally it is nearly one. It increases in : chronic liver disease, malignancy, muscle disease and alcoholic liver. It decrease or inverted in: Acute hepatitis.

Laboratory finding in acute liver disease 1- Rapid increase in the AST and ALT followed by rapid fall. 2- Decrease or inverted De - Ritis ratio. 3- ALP is mildly elevated. 4- Bilirubin elevation in the direct type and indirect type. 5- Liver synthetic functions,like albumin and prothrombin time, are normal.

I Hemolytic anemia: jaundice + increased LD + increase in AST. The hyperbilrubinaemia is from the indirect type. LD increase is several times as the increase in AST. II Acute skeletal muscle injury: No hyper bilrubinemia. Increase in AST is more significant than ALT. DE Ritis ratio is more than 3 : 1 III Acute bile duct obstruction: Hyper bilrubinaemia is from the direct form. There is increase in ALP and Gama GT. Differential diagnosis of acute hepatitis:

Hemolytic jaundice Indirect Bilrubin Hepatic jaundice Direct and indirect Cholestatic ( post hepatic ) direct Jundice

1- Moderate to little increase in the AST and ALT. 2- The increase in AST is more significant than ALT. 3- Increased De Ritis ratio. 3- ALP is mildly elevated. 4- Bilirubin elevation in the direct type and indirect type. 5- Liver synthetic functions,like albumin and prothrombin time, are greatly affected. Laboratory finding in chronic liver disease

Cholestatic liver diseases:  Primary Biliary Cirrhosis.  Primary Sclerosing Cholangitis.  Drug-Induced Cholestasis.

1.Increase in plasma activity of canalicular enzymes ( ALP – GGT ). 2.Transient increase in ALT and AST. 3.Prolonged PT. Laboratory finding in Cholestatic liver disease

Abnormal liver function tests Hepatocellular If normal albumin ( Acute hepatitis ) If decreased albumin ( Chronic hepatitis ) Cholestatic If normal albumin ( Acute cholestasis ) If decreased albumin ( Chronic cholestasis )

Reference: Burtis and Ashwood Saunders, Teitz fundamentals of Clinical Chemistry, 4th edition, 2000.