Total Protein Examination Procedure

1.Purpose of examination: Total Protein estimation from serum or plasma by Biuret Method.

2.Responsibility and Authority:

• Calibration: Technician
• Quality Control: Technician
• Routine operation: Technician
• Overall Monitoring: Quality Manager

3.Sample Details:

• Type of Sample: Serum, Plasma
• Type of container and additives: Plain without any additives
• Patient Preparation: As per Primary Sample Collection Manual sample_collection_manual
• Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
• Handling and transport: As per Primary Sample collection manual
• Storage: 24 hours at 2-8° C

4.Required Equipment: Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer

5.Required reagents: R1. copper sulphate pentahydrate (6.600 g/l)

6.Reagent Handling Remove any air bubbles present in the reagents with a new applicator stick, or allow the reagents to settle at the appropriate storage temperature to allow the bubbles to dissipate. To minimize volume depletion, do not use a transfer pipette to remove bubbles

7.Reagent Storage and stability: Unopened reagent stable at 15-30°C until expiration date. On board System temperature reagent is stable for 30 days Instability or deterioration should be suspected if there are precipitates, visible signs of leakage or contamination, turbidity, or if calibration or controls do not meet the appropriate criteria.

8.Calibration Procedure:

• Consolidated Chemistry Calibrator
• Frequency:
• Reagent lot change
• QC out of range
• After service or maintenance
• Replacement in any parts of Instrument

Procedure:

• Start the equipment.WDI abbotte fully.docx
• Calibrators are ready to use.
• Put calibrator 15-20 minutes at room temperature
• Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation.
• Take a 150 µl calibrator solution to separate aliquots.
• Go to the calibration and give the calibration order.
• Verify calibration with at least two levels of controls
• If control results fall outside acceptable ranges,root cause analysis or recalibration may be necessary.

9.Quality control Procedure:

Name: Bio Rad Level 1 & 2 Frequency: As per Quality Control Procedure

Procedure for Reconstitution of IQC

• Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
• Leave to stand for 30 min in the dark place.
• Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
• Prior to use, mix the contents by inverting the vial. Do not shake the vial as the information of foam should be avoided. Ensure that no lyophilized material remains un-reconstituted.
• Prepare aliquots of 150 µl from the reconstituted QC material.
• Store these aliquots at -15° C to -20° C.
• Prior to use, make sure that aliquots should be at room temperature for at least 15 min.

Procedure to run IQC

• Press Control order
• Select Assay /Panel, to be run.
• Select the control/s and its level/s
• Give Carrier Number and Position number
• Press F3 / Add order
• Put respected carrier in RSH rack
• Check IQC results, in case outliers call residents.

10.Principle of the procedure used for examinations:

• Polypeptides containing at least two peptide bonds react with the biuret reagent. In an alkaline solution, the cupric ion forms a coordination complex with protein-nitrogen with very little difference between albumin and globulin on a protein-nitrogen basis.

11.Sample Preparation:

• Required SampleVolume: 150 µl of the sample
• Temperature: 37°
• Take 150-200µl of the sample from Primary tube to the secondary aliquot. Write the sample ID on the aliquot.

12.Procedure to run Patient sample

1. Press Patient order
2. Select Assay /Panel, to be run.
3. Give Carrier Number and Position number
4. Press F3 / Add order
5. Put respected carrier in RSH rack.

13.Performance Characteristics:

• Linearity: 0.2 to 18.3 g/dL
• The limit of detection (LOD): 0.2 g/dL
• The limit of quantification(LOQ): 0.2 g/dL
• Unit: g/dL

Normal and critical ranges:

Protein total

14.Laboratory Clinical interpretation:

• Measurements of total protein are used in the diagnosis and treatment of a variety of diseases involving liver, kidney, lymph nodes, spleen, or bone marrow.
• High protein levels may be observed in cases of severe dehydration and disease states such as multiple myeloma. Changes in the proportions of the plasma proteins may occur in one or several of the protein fractions and often without alterations in the quantity of the total protein.
• Low protein levels may be caused by such conditions as nephrotic syndrome, extensive bleeding, sprue (deficient protein absorption), severe burns, salt retention syndromes, and Kwashiorkor (acute protein starvation).
• High or low total protein may lead one to suspect pathologic variation of individual proteins and may indicate additional testing including serum protein electrophoresis, hematocrit, electrolytes, testing for specific proteins and other organ or disease specific markers.

15.Interference and cross reaction: The Following analytes were tested up to the levels indicated at Direct Bilirubin concentrations of 0.14mg/dl and 5.03 mg/dl, and found not to interfere:

• No interference from Bilirubin up to30 mg/dL)
• No interference fromHemoglobin up to 250 mg/dL
• No interference from Human triglyceride up to 1000 mg/dl

16.Potential source of variation:

17.Turn around time (TAT):

Routine: 6.0 hours Urgent: 2.0 hours

18.Recording of observation:

• Software backup
• Machine raw data

19.Storage & Disposal of waste: Follow storage & discard procedure 20.Environmental & Safety control:

• For in vitro diagnostic use
• Do not use component before the expiration date
• Do not mix material from different kit lot number.

21.References:

1. US department of labor,Occupational safety and health administration.
2. US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories.
3. World health organization. Biosafety manual,3rd edition.
4. Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry
5. Kaplan LA,Pesce AJ editors. Clinical Chemistry Theory, Analysis, and Correlation, 3rd ed.