Final Practical

The purpose of running the controls
ensure the accuracy of both the procedure, and the instrumentation used in the method. The level 1 and level 2 controls must both fall within their expected value range in order for the patient results to be considered accurate and reportable.
Globulin Calculation
Globulin = TP Concentration – Albumin Concentration
5 disease states that could cause an increased total protein
1. Chronic infection
2. Liver dysfunction
3. Dehydration
4. Leukemia
5. Multiple myeloma
5 disease states that could cause a decreased total protein concentration
1. Malnutrition/malabsorption
2. Terminal liver failure
3. Diarrhea
4. Severe burns – protein lost through skin
5. Renal disease – protein lost in urine
If the patient result is outside the limit of linearity
you should dilute the patient sample, re-run the procedure and multiple the results by the dilution factor to obtain your final result.
Three additional methods for measurement of total protein
Kjeldahl technique, a refraction technique or a UV absorption technique.
The biuret reaction for the analysis of serum protein depends on
number of peptide bonds
What is the basis for the Kjeldahl technique for the determination of serum total protein
quantification of nitrogen content in protein
Which total protein method requires copper sulfate, potassium iodide in sodium hydroxide, and potassium sodium tartrate in its reagent system
Biuret
The biuret method is performed by
labeling test tubes blank, C1, C2, standard and Patient. Then you add 1mL of total protein reagent into each test tube, followed by 10µL of specified liquid. You then cover each tube with parafilm and mix, and then incubate at room temperature for 5 minutes. You then use the blank to zero the spectrophotometer to 550nm, and then record the absorbances of each reaction. You then calculate the concentration of each reaction using beer’s law.
The interfering substances in this method
hemolysis due to lack of prompt removal of the clot from the specimen, or a lipemic sample which cannot be tested.
The principle of the biuret method is
dependent upon the amount of peptide bonds in the proteins. The Cu2+ ions in the reagent are in an alkaline solution, when they encounter at least two peptide bonds they form a color complex which can be measured spectrophotometrically. The intensity of the color is directly related to the amount of peptide bonds and in turn is related to the amount of protein in the sample.
General causes of abnormal total protein values (2)
Change in volume of plasma water or Change in concentration of one or more of specific proteins
2 assumptions in total protein measurement
A. All protein molecules are pure polypeptide chains containing 16% nitrogen by weight
B. Each protein reacts alike chemically
Biuret Total Protein Reagent Composition (4)
1. Copper sulfate supplies Cu2+ ions
2. Sodium hydroxide to create alkaline solution
3. Potassium sodium tartrate to keep the copper in solution
4. Potassium iodide to prevent oxidation of copper
Specimen
serum
Fibrinogen in plasma
gives elevated results
Total protein stable in serum for
1 week at room temperature and 30 days refrigerated
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