Information about test and diagnosis of diseases
1. Purpose:
Estimation of total protein in serum/body fluids by Biuret method. Low protein levels are observed in malnutrition, acute or chronic liver diseases, nephrotic syndrome, water intoxication, salt retention syndromes, and massive intravenous infusions. Elevated protein levels are observed in dehydration due to vomiting, diarrhea, Addison’s disease and diabetic ketoacidosis. High protein levels of over 2 g/dL in body fluids are suggestive of inflammation or malignancy and are called exudates.
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2. Principle:
Peptide bonds of proteins in serum react with cupric ions in alkaline solutions to form a blue colored complex, the absorbance of which is measured at 578 nm. The intensity of the blue color is proportional to the amount of protein present. The reaction sequence employed in the assay of total proteins is as follows:
3. Performance specifications:
3.1. Linearity: Up to 12 g/dL
3.2. Measurement range: This method has a measurement range of 5.3–8.4 g/dL of total protein in serum and body fluids.
3.3. Sensitivity: The minimum detection limit by the kit is 5.3 g/dL.
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4. Primary sample:
4.1. Use serum/body fluids (Pleural, Pericardial, Ascitic fluid) as specimen for the test.
4.2. Collect blood sample in a red color vacutainer tube, separate serum within 30 minutes of collection.
4.3. Process the sample on the same day within 1 hour of collection. If analysis is done on the next day, separate the serum and store it at 2–8°C for up to 30 days.
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5. Type of container and additive:
Use plain vacutainer tubes for collecting samples. No additive/Preservative is needed to be added.
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6. Reagents/Consumables:
6.1. Biuret reagent.
6.2. Total protein standard.
7. Instrument: Semi-autoanalyzer
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8. Procedure:
8.1 Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes.
8.2. Press “PROC”. Different test procedures will be displayed.
8.3. Select the test to be processed by entering its number and then press “ENTER” key.
8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used.
8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, if the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm.
8.6. Then feed the test samples and record the values.
8.7. Check whether the sample is hemolyzed or icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10.
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Assay type: End point,
Wavelength: 546 nm.
Sample volume: 10 μL,
Reagent volume: 1000 μL
Incubation time: 20 min at RT, Temperature: 37oC
_______________Dr_Yousef______________
9. Interferences:
9.1. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, If the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm.
9.2. Keep the reconstituted reagent at 2–8ºC. Discard the same if it develops precipitate.
9.3. Highly hemolytic or icteric samples, prepare sample blank by adding 1 mL of 0–9% saline to 10 microliter samples. The value of the blank is subtracted from the corresponding sample value.
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10. Calculating results:
Adults: 6.6–8.4 g/dl.
12. Critical/Alert values:
Below 5.0 g/dL and above 9.0 g/dl.
13. Laboratory interpretation:
Increase of proteins in dehydration, multiple myeloma and chronic infections (gammopathy);
decrease in malnutrition, liver diseases, nephrotic syndrome.
14. Potential sources of variability:
14.1. The reagent is linear to 12.0 g/dL. Samples with values above 10 g/dL should be diluted 1:1 with 0.9% saline, re-run, and the result multiplied by two (2)
14.2 The biuret procedure is not sensitive at low ranges (< 1 g/dL). Do not use for urine or spinal fluid.
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BIBLIOGRAPHY
1. Henry J, Winkelman JW. Clinical Chemistry Principles and Technique. Harper and Row, 2nd edn 1974.
2. Stricklad RD, Freeman ML, Gurule FF. Copper Binding by proteins in alkaline solution. Anal Chem 1961;33.
Reference: Manual of Medical Laboratory Techniques
Produce by: Dr. Yousef Al Adbai
Laboratorist.
______________THANK_YOU______________
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