Final Practical

Explain the differences in methodology between total and direct bilirubin.
The differences in methodology between total bilirubin and direct bilirubin is that in the total bilirubin methodology acid caffeine sodium benzoate is used as an accelerator to help unconjugated bilirubin become water soluble to react with diazo reagent. The direct bilirubin methodology does not use an accelerator.
Briefly explain the bilirubin metabolism process in the body (include conjugated and unconjugated).
When blood cells hemolyze they release their contents into the blood. One of the products that they release is heme and when heme breaks down it becomes unconjugated bilirubin. Unconjugated bilirubin enters the liver where it is conjugated to glucaronic acid by uridine diphosphate glucuronyl transferase. It then leaves the liver and enters the biliary system where it is excreted
List three other common liver function tests?
Three other common liver function tests are (1) ALT enzyme test (2) AST enzyme test and (3) ? glutamyl transpeptidase enzyme test.
Explain why elevated bilirubin values frequently occur in premature infants
Bilirubin is usually high in premature infants because they were still relying on their mothers liver to conjugate bilirubin, when they were born their liver was not functioning at an optimal level.
Name three causes of increased bilirubin.
Three causes of increased bilirubin are (1) hemolytic processes (2) ineffective erythropoiesis and (3) cirrhosis.
Perform the Jendrassik Grof method for determining total bilirubin via a manual kit by Stanbio.
Label tubes and add total bilirubin reagent. Add DI water to blank and appropriate specimen to other tubes. Add one drop of oxidant to each tube. Mix and incubate at RT for 5 minutes. Read and record results at 540 nm. Calculate concentration via beer’s law.
Perform the Jendrassik Grof method for determining direct bilirubin via a manual kit by Stanbio.
Label tubes and all direct bilirubin reagent to each tube. Add DI water to blank, and appropriate specimen to other tubes. Add one drop of oxidant to one tube and read absorbance 3 minutes later at 540nm. Repeat with all other tubes, one at a time. Calculate concentration using beer’s law.
Identify interfering substances Jendrassik Grof method
gross hemolysis, drugs, turbid or lipemic serum
The reference values for total bilirubin for adults
0.2-1.2 mg/dL
The reference values for total bilirubin for newborns
<0.3 mg/dl
Principle of the Jendrassik Grof method
The principle of this method is a classic diazo reaction which combines sulfanilic acids in dilute HCL with sodium nitrite to form nitrous acid. Nitrous acid forms the basis for the diazized sulfanilic acid. Direct bilirubin combines with the sulfanilic acid to form a blue color at a alkaline pH. Indirect bilirubin is diazotized in the presence of an accelerating agent. The blue produced is from both direct and indirect bilirubin. Final color is measured at 540 nm and bilirubin concentration is calculated using Beer’s law.
Total Bilirubin calculation
Au/As x 10
Direct Bilirubin calculation
(As-Ab)/ As x 10
x

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