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Serologic Tests 

Enzyme Immunoassay
Anti-HCV is detected by enzyme immunoassay (EIA). The third-generation  test (EIA-3) used today is more sensitive and specific than previous  ones. However, as with all enzyme immunoassays, false-positive results are occasionally a problem with the EIA-3. Additional or confirmatory testing is often helpful.

The best approach to confirm the diagnosis of hepatitis C is to test for HCV RNA using a sensitive polymerase chain reaction (PCR) assay. The presence of HCV RNA in serum indicates an active infection. Testing for HCV RNA is also helpful in patients in whom EIA tests for anti-HCV are unreliable. For instance, immunocompromised patients may test negative for anti-HCV despite having HCV infection because they may not produce  enough antibodies for detection with EIA. Likewise, patients with acute hepatitis may test negative for anti-HCV when the physician first tests. Antibody is present in almost all patients by 1 month after onset of acute illness; thus, patients with acute hepatitis who initially test negative may need followup testing. In these situations, HCV RNA is usually present and confirms the diagnosis.

Recombinant Immunoblot Assay 
Immunoblot assays are used to confirm anti-HCV reactivity, too. These tests are also called "Western blots"; serum is incubated on nitrocellulose strips on which four recombinant viral proteins are blotted. Color changes indicate that antibodies are adhering to the proteins. An immunoblot is considered positive if two or more proteins react and is considered indeterminate if only one positive band is detected. In some clinical situations, confirmatory  testing by immunoblotting is helpful, such as for the person with anti-HCV detected  by EIA who tests negative for HCV RNA. The EIA anti-HCV reactivity could  represent a false-positive reaction, recovery from hepatitis C, or continued virus infection with levels of virus too low to be detected (the last occurs only rarely when sensitive PCR assays are used). If the immunoblot test for anti-HCV is positive, the patient has most likely recovered from hepatitis C and has persistent antibody without virus. If the immunoblot test is negative, the EIA result was probably a false positive. Immunoblot tests are routine in blood banks when an anti-HCV-positive sample is found by EIA. Immunoblot assays are highly specific and valuable in verifying anti-HCV reactivity. Indeterminate tests require further followup testing, including attempts to confirm the specificity by repeat testing for HCV RNA.

PCR Amplification
PCR amplification can detect low levels of HCV RNA in serum. Testing for  HCV RNA is a reliable way of demonstrating that hepatitis C infection is  present and is the most specific test for infection. Testing for HCV RNA by PCR is particularly useful when aminotransferases are normal or only slightly elevated, when anti-HCV is not present, or when several causes of liver disease are possible. This method also helps diagnose hepatitis C in people who are immunosuppressed, have recently had an organ transplant, or have chronic renal failure. At present, however, there are no PCR assays approved by the Food and Drug Administration for general use, although commercial test systems are available. Many commercial laboratories offer their own PCR assays, which are not subject to strict independent quality controls. Thus, the reliability and specificity of the PCR technique are not standardized. In addition, it is expensive and prone to technical or laboratory error. When ordering HCV RNA testing by PCR, the physician should use a high-quality  laboratory willing to document standardization of the test.

Biochemical Indicators of Hepatitis C Virus Infection
•In chronic hepatitis C, increases in the alanine and aspartate aminotransferases range from 0 to 20 times (but usually less than 5 times) the upper limit of normal. 

•Alanine aminotransferase levels are usually higher than aspartate aminotransferase levels, but that finding may be reversed in patients who have cirrhosis. 

•Alkaline phosphatase and gamma glutamyl transpeptidase are usually normal. If elevated, they may indicate cirrhosis.

•Rheumatoid factor and low platelet and white blood cell counts are frequent in patients with cirrhosis, providing clues to the presence of advanced disease.

•The enzymes lactate dehydrogenase and creatine kinase are usually normal.

•Albumin levels and prothrombin time are normal until late-stage disease. 

•Iron and ferritin levels may be slightly elevated. 

Quantification of HCV RNA in Serum
Several methods are available for measuring the titer or level of virus in serum, which is an indirect assessment of viral load. These methods include a quantitative PCR and a branched DNA (bDNA) test. Unfortunately, these assays are not standardized, and different methods from different laboratories can provide different results on the same specimen. In addition, serum levels of HCV RNA can vary spontaneously by 3- to 10-fold over time. Nevertheless, when performed carefully, quantitative assays provide important insights into the nature of hepatitis C. 
Viral load does not correlate with the severity of the hepatitis or with a poor prognosis (as it seems to in HIV infection); but viral load does correlate with the likelihood of a response to antiviral therapy. Rates of response to a course of alpha interferon and ribavirin are higher in patients with low levels of HCV RNA. There are several definitions of a 
"low level" of HCV RNA, but the usual definition is below 2 million copies per milliliter (mL).

In addition, monitoring viral load during the early phases of treatment may provide early information on the likelihood of a response. Yet because of the shortcomings of the current assays for HCV RNA level, these tests are not reliable guides to therapy. More sensitive and reliable methods of quantitating HCV RNA in serum are needed. Until that time, these tests should not be routinely used in practice.

 
http://www.niddk.nih.gov/health/d  

Liver Function Tests:

Liver function tests represent a broad range of normal functions performed by the liver. The diagnosis of liver disease depends upon a complete history, complete physical examination, and evaluation of liver function tests and further invasive and noninvasive tests. Many patients become confused regarding the meaning of a liver function test. This section is designed to describe the basic liver function tests and the meaning for patients.

The hepatobiliary tree represents hepatic cells and biliary tract cells. Inflammation of the hepatic cells results in elevation in the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and possibly the bilirubin. Inflammation of the biliary tract cells results predominantly in an elevation of the alkaline phosphatase. In liver disease there are crossovers between purely biliary disease and hepatocellular disease. To interpret these, the physician will look at the entire picture of the hepatocellular disease and biliary tract disease to determine which is the primary abnormality.

Alanine Aminotransferase (ALT):

ALT is the enzyme produced within the cells of the liver. The level of ALT abnormality is increased in conditions where cells of the liver have been inflamed or undergone cell death. As the cells are damaged, the ALT leaks into the bloodstream leading to a rise in the serum levels. Any form of hepatic cell damage can result in an elevation in the ALT. The ALT level may or may not correlate with the degree of cell death or inflammation. ALT is the most sensitive marker for liver cell damage.

Aspartate Aminotransferase (AST):

This enzyme also reflects damage to the hepatic cell. It is less specific for liver disease. It may be elevated and other conditions such as a myocardial infarct (heart attack). Although AST is not a specific for liver as the ALT, ratios between ALT and AST are useful to physicians in assessing the etiology of liver enzyme abnormalities.

Alkaline Phosphatase:

Alkaline phosphatase is an enzyme, which is associated with the biliary tract. It is not specific to the biliary tract. It is also found in bone and the placenta. Renal or intestinal damage can also cause the alkaline phosphatase to rise. If the alkaline phosphatase is elevated, biliary tract damage and inflammation should be considered. However, considering the above other etiologies must also be entertained. One way to assess the etiology of the alkaline phosphatase is to perform a serologic evaluation called isoenzymes. Another more common method to asses the etiology of the elevated alkaline phosphatase is to determine whether the GGT is elevated or whether other function tests are abnormal (such as bilirubin)

Alkaline phosphatase may be elevated in primary biliary cirrhosis, alcoholic hepatitis, PSC, gallstones in choledocholithiasis.

Gamma Glutamic Transpeptidase (GGT):

This enzyme is also produced by the bile ducts. However, it is not very specific to the liver or bile ducts. It is used often times to confirm that the alkaline phosphatase is of the hepatic etiology. Certain GGT levels, as an isolated finding, reflect rare forms of liver disease. Medications commonly cause GGT to be elevated. Liver toxins such as alcohol can cause increases in the GGT.

Bilirubin:

Bilirubin is a major breakdown product of hemoglobin. Hemoglobin is derived from red cells that have outlived their natural life and subsequently have been removed by the spleen. During splenic degradation of red blood cells, hemoglobin (the part of the red blood cell that carries oxygen to the tissues) is separated out from iron and cell membrane components. Hemoglobin is transferred to the liver where it undergoes further metabolism in a process called conjugation. Conjugation allows hemoglobin to become more water-soluble. The water solubility of bilirubin allows the bilirubin to be excreted into bile. Bile then is used to digest food.

As the liver becomes irritated, the total bilirubin may rise. It is then important to understand the difference between total bilirubin, which has undergone conjugation (that is hepatic cell metabolism), and at portion of bilirubin which has not been metabolized. These two components are called total bilirubin and direct bilirubin. The direct bilirubin fraction is that portion of bilirubin that has undergone metabolism by the liver. When this fraction is elevated, the cause of elevated bilirubin (hyperbilirubinemia) is usually outside the liver. These types of causes are typically gallstones. This type of abnormality is usually treated with surgery (such as a gallbladder removal or choleycystectomy).

If the direct bilirubin is low, while the total bilirubin is high, this reflects liver cell damage or bile duct damage within the liver itself.

Albumin:

Albumin is the major protein present within the blood. Albumin is synthesized by the liver. As such, it represents a major synthetic protein and is a marker for the ability of the liver to synthesize proteins. It is only one of many proteins that are synthesized by the liver. However, since it is easy to measure, it represents a reliable and inexpensive laboratory test for physicians to assess the degree of liver damage present in the in any particular patient. When the liver has been chronically damaged, the albumin may be low. This would indicate that the synthetic function of the liver has been markedly diminished. Such findings suggest a diagnosis of cirrhosis. Malnutrition can also cause low albumin (hypoalbuminemia) with no associated liver disease.

Prothrombin time (PT):

Another measure of hepatic synthetic function is the prothrombin time. Prothrombin time is affected by proteins synthesized by the liver. Particularly, these proteins are associated with the incorporation of vitamin K metabolites into a protein. This allows normal coagulation (clotting of blood). Thus, in patients who have prolonged prothrombin times, liver disease may be present. Since a prolonged PT is not a specific test for liver disease, confirmation of other abnormal liver tests is essential. This may include reviewing other liver function tests or radiology studies of the liver. Diseases such as malnutrition, in which decreased vitamin K ingestion is present, may result in a prolonged PT time. An indirect test of hepatic synthetic function includes administration of vitamin K (10mg) subcutaneously over three days. Several days later, the prothrombin time may be measured. If the prothrombin time becomes normal, then hepatic synthetic function is intact. This test does not indicate that there is no liver disease, but is suggestive that malnutrition may coexist with (or without) liver disease.

Platelet count:

Platelets are cells that form the primary mechanism in blood clots. They're also the smallest of blood cells. They derived from the bone marrow from the larger cells known as megakaryocytes. Individuals with liver disease develop a large spleen. As this process occurs platelets are trapped with in the sinusoids (small pathways within the spleen) of the spleen. While the trapping of platelets is a normal function for the spleen, in liver disease it becomes exaggerated because of the enlarged spleen (splenomegaly). Subsequently, the platelet count may become diminished.

Serum protein electrophoresis:

This is an evaluation of the types of proteins that are present with in a patient's serum. By using an electrophoretic gel, major proteins can be separated out. This results in four major types of proteins. These are 1) albumin, 2) alpha globulins, 3) beta globulins and 4) gammaglobulins. This test is useful for evaluation of patients who have abnormal liver function tests since it allows a direct quantification of multiple different serum proteins. If the gamma globulin fraction is elevated, autoimmune hepatitis may be present. In addition a deficiency in the alpha globulin fraction can result in the diagnosis, or a clinical clue, to A. alpha-1 antitrypsin deficiency. This is a simple blood test that is commonly performed by hepatologists.

http://www.gastromd.com/education/hepatitisc2.html 

Biochemical Indicators of Hepatitis C Virus Infection

• In chronic hepatitis C, increases in the alanine and aspartate aminotransferases range from 0 to 20 times (but usually less than 5 times) the upper limit of normal.
  
   
• Alanine aminotransferase levels are usually higher than aspartate aminotransferase levels, but that finding may be reversed in patients who have cirrhosis.
  
   
• Alkaline phosphatase and gamma glutamyl transpeptidase are usually normal. If elevated, they may indicate cirrhosis.
  
   
• Rheumatoid factor and low platelet and white blood cell counts are frequent in patients with cirrhosis, providing clues to the presence of advanced disease.
  
   
• The enzymes lactate dehydrogenase and creatine kinase are usually normal.
  
   
• Albumin levels and prothrombin time are normal until late-stage disease.
  
   
• Iron and ferritin levels may be slightly elevated.
   

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