SOP NO.

: TNT/CB/53

TITLE: QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM

ON COBAS 6000
ISSUE NO.: 01
ISSUE DATE: 04-03-2021
VALID UPTO: 03-04-2024

NAME DESIGNATION FUNCTION SIGNATURE DATE

Mr. K Bheem Reddy Biochemist Preparation 04-04-2023

Dr. Abdur Rehman Asif Consultant Biochemist Review 04-04-2023

Mr. Syed Basheer Uddin Quality Manager Review 04-04-2023

Dr. Sreedevi D Lab Director Approval 04-04-2023

RELEASE OF AUTHORIZATION
This document is issued under the authority of Dr. Sreedevi D, Lab Director. Do not share this document either verbally or by any other means with out
the written consent of the Lab Director.
 QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM ON COBAS 6000
SOP NO. TNT/CB/53

DISTRIBUTION LIST
The following are the details of holders of controlled/uncontrolled of this document.

Copy No. Designation of the copy holder Type of copy Signature

Master Copy Lab Director Master

01 Quality Manager Controlled

02 Biochemist Controlled

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 QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM ON COBAS 6000
SOP NO. TNT/CB/53

AMENDMENT SHEET

S. Page Cl. Review date Date of Amendment Reasons Signature Signature

No. No. No. Amendment made Quality Director
Manager
1 All - 04-04-2023 04-04-2023 To include Internal
pages new lab decision
director
signature
and To
include
equipment
backup
policy.
2
3
4
5
6
7
8
9
10

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 QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM ON COBAS 6000
SOP NO. TNT/CB/53

1.0 PURPOSE AND SCOPE: This procedure is intended for the quantitative estimation of total
protein Cobas 6000. Measurement of total protein is used in the diagnosis and treatment of a
variety of diseases involving the liver, kidney or bone marrow as well as metabolic or
nutritional disorder.

2.0 DEFINITIONS & ABBREVIATIONS:

2.1 TNT: Tenet Diagnostics
2.2 CB: Clinical Biochemistry
2.3 TP: Total Protein
2.4 SOP: Standard Operating Procedure
2.5 QA: Quality Assurance
3.0 RESPONSIBILITY: It is the responsibility of the Lab Technologist to follow and implement
this procedure. Section Head shall ensure implementation and QA shall monitor compliance of
the same.
4.0 PROCEDURE:
4.1 Method: Biuret (Colorimetric assay)
4.2 Principle: Divalent copper reacts in alkaline solution with protein peptide bonds to form
the characteristic purple-colored biuret complex. Sodium potassium tart rate prevents the
precipitation of copper hydroxide and potassium iodide prevents auto reduction of copper.

Alkaline
Solution

Protein + Cu2+ Cu- Protein Complex

The color intensity is directly proportional to the protein concentration which can be
determined photo metrically.
4.3 Primary Sample Collection:
4.3.1 Collect blood in a yellow or red top vacutainer and allow it to clot and separate the
serum by centrifugation. Centrifuge samples at 2500 – 3500 rpm for 10 – 15 minutes.
4.3.2 To prevent formation of fibrin in serum samples, complete clot formation should take

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 QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM ON COBAS 6000
SOP NO. TNT/CB/53
place before centrifugation. Specimens should be free of particulate matter.

4.3.3 Sample Type: Serum

4.3.4 Storage &Stability:
 Room temperature (20-25°C): 6 days
 Refrigerated (4-8°C):4 weeks
 Frozen (-20°C): 1 year
4.3.5 Shipment condition: As per cl. 4.3.3
4.3.6 Sample rejection criteria:
 Insufficient sample quantity
 Inadequately labeled specimens
 Incomplete requisitions
 Hemolysed specimens
 Improper transportation
 Wrong collection container
4.3.7 All the samples rejected are recorded in the Sample Rejection Record
(TNT/CB/SRR).
4.4 Equipment:
4.4.1 Name of the Equipment: Cobas 6000
4.4.2 Location: Clinical Biochemistry
4.4.3 Operating temperature: 15 – 35o C
4.4.4 Relative humidity: Max 80 % (Non condensing)
4.5 Reagents Required:
4.5.1 Reagents –
R1 Sodium hydroxide: 400 mmol/L; potassium sodium tartrate: 89 mmol/L
R2 potassium iodide: 61 mmol/L; copper sulfate: 24.3 mmol/L
R1 is in position B and R2 is in position C.
4.5.2 Reagent Storage and Stability:
 Reagent packs are ready for use as supplied. Stable up to the expiry date
mentioned on the reagent cartridges when stored at 2 – 8 o C.

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 QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM ON COBAS 6000
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 On-board in use and refrigerated on the instrument are stable for 8 weeks.

 Reagents are stored at appropriate temperature as specified by the manufacturer.

 All unopened reagents are stable until the expiration date printed on the product
label.
 Reagent details are recorded in Kit-In Use Record (TNT/CB/KUR-RB).
4.5.3 Controls: Bio-Rad assayed chemistry Levels 1 & 2
4.5.4 Storage and Stability of Controls:
 The product is stable until the expiry date when stored unopened at 2 – 8 o C. Once
the control is reconstituted all analytes will be stable for 7 days when stored tightly
capped at 2 – 8 o C except acid phosphatase and prostatic acid phosphatase. These
two analytes will be stable for 3 days when stored tightly at 2 – 8 o C.
 After reconstituting and freezing the controls the analytes will be stable for 30 days
when stored tightly capped at – 10 to – 20 o C with the following exceptions –
Tobramycin will be stable for 20 days when the control is stored tightly capped at
– 10 to - 20 o C .
 Once thawed do not refreeze the control, discard remaining material.
 Do not use the control material past the expiry date.
4.5.5 Reconstitution of controls:
 Reconstitute each vial with 5 ml of distilled or de ionized water using a
micropipette. Replace the stopper and allow the control to stand for 20 minutes,
swirling occasionally.
 Mix for homogeneity and make control aliquots as required. If there is evidence of
microbial contamination or excessive turbidity in the reconstituted control, discard
the vial.
 These controls cannot be used as a standard.
4.5.7 TP Calibrator contents: S1: H2O
S2: C.F.A.S

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 QUANTITATIVE ESTIMATION OF TOTAL PROTEIN IN HUMAN SERUM ON COBAS 6000
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o
4.5.8 Storage and Stability of calibrator: The product should be stored at 2 – 8
C .Unopened products are stable up to the expiration date mentioned on the product
label. Assigned values are stable 8 hrs. After reconstitution when vials are tightly

closed and stored at 2 – 8 o C.

4.5.9 Reconstitution of calibrator:
 Remove the vial from the refrigerator and allow it to stand at room temperature for
10 - 15 minutes.
 Remove the stopper and add 2 ml of regent grade water using a micropipette. The
water should be at room temperature.
 Replace the stopper, and let stand for 5 minutes. Do not invert the vials.
 Swirl vials for 60 seconds, then gently invert 20 times.
 Let vials stand for 30 minutes, then invert 20 times & swirl gently.
 Let vials stand on bench top for 30 minutes. Then invert 20 times and swirl gently.
 Use immediately or refrigerate at 2 – 8 o
C for future use. Prior to use, visually
inspect vials for un-dissolved material. If present position vial to fully immerse un-
dissolved material in liquid. Let stand for 10 minutes. Then invert 20 times and
swirl gently.
4.6 Calibration Frequency:
4.6.1 Calibration is performed once in every 4 weeks
4.6.2 When reagent lot changes
4.6.3 After major equipment breakdown or maintenance performed
4.6.4 When internal QC is repeatedly out of range
4.6.5 Calibration details are recorded in the Calibration Details Record (TNT/CB/CDR-
RB)
4.7 Additional Requirements: Calibrated pipette, distilled or de ionized water, 1 g/L sodium
hypo chlorite, Diluent NaCl 9%, tissue rolls etc.
4.8 Process:
 The system is switched on, maintenance performed as per schedule and the details are
recorded in the maintenance forms.

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 Reagents sufficient for sample processing are verified and replenished.
 Run two levels of QC (Normal & Abnormal) daily before testing patient samples.

 Internal quality control is run after each new calibration and after every major
equipment breakdown or maintenance.
 QC results are entered into Unity software and verified for acceptability before reporting
patient results. The same results are also entered into IQC Record (TNT/CB/IQCR-RB).
 If QC outlier is observed, appropriate corrective and preventive action is taken and
recorded in the IQC Corrective Action Record (TNT/CB/IQCCAR-RB).
 Controls are tested in the same manner and by the same personnel testing patient
samples.
 Patient samples are processed after review of QC results by the Section Head.
 The instrument will be operated only by a trained Technician.
 The samples are processed directly by programming on the screen or by a bar code
system.
 The sample can be processed while the instrument is in standby or add samples while
the system is in running status or the sample details can be directly downloaded from an
LIS.
 Program all the samples and load as per the wok order. Ensure all the lids are closed and
press RUN on the key board. If a single sample is processed at a time select PROCESS
SINGLE SAMPLE from the Enter Sample Data screen.
 If bar coded samples are processed the patient and test details will be automatically
downloaded from the LIS.
 For more details on sample order and processing refer to the Cobas 6000 operator
manual.
 In case of breakdown of the instrument, samples shall be processed on the back-up
Cobas 8000 Pro
4.9 Repeat & Recheck Criteria:
 When the results are beyond the reference ranges of the laboratory.

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 When the instrument displays error flags.
 When the results do not correlate with the clinical condition of the patient/ with the other
clinical results of the patient.

 Samples having higher concentration are diluted in 1:3 dilution and rerun function are
automatically multiplied by a factor of 3.
 Results less than 0.2 g/dL should be reported as “< 0.2 g/dL.”
 Repeat values are recorded in Repeat Tests Record (TNT/CB/RTR).
 Repeat testing is performed after informing the Section Head.
4.10Biological Reference Intervals: Normal value: 6.6 – 8.7 g/Dl
 Expected values according to Josephson
w Adults: 66-87 g/L (6.6-8.7 g/dL)
 Expected values according to Tietz
w Umbilical cord: 48-80 g/L (4.8-8.0 g/dL)
w Premature: 36-60 g/L (3.6-6.0 g/dL)
w Newborn: 46-70 g/L (4.6-7.0 g/dL)
w 1 week: 44-76 g/L (4.4-7.6 g/dL)
w 7 months-1 year: 51-73 g/L (5.1-7.3 g/dL)
w 1-2 years: 56-75 g/L (5.6-7.5 g/dL)
w >3 years: 60-80 g/L (6.0-8.0 g/dL)
w Adults (ambulatory): 64-83 g/L (6.4-8.3 g/dL)
4.11 Critical/Panic Values: < 3.4 & > 9.5 g/dL
4.12 Performance Characteristics:
4.12.1 Uncertainty of Measurement: 2.13 % CV
4.12.2 Accuracy:- NA
4.12.3 Limit of Quantification (LOQ):-0.2 g/dL
4.12.4 Limit of Detection (LOD): 0.2 g/dL
4.12.5 Analytical Measurement Range (AMR): 0.2 – 12.0 g/dL
4.12.6 Clinical Reportable Range (CRR): 0.2 – 12.0 g/dL
4.12.7 Sensitivity: 0.2 g/dL

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4.12.8 Specificity: Refer to cl. 4.14
4.13Results and interpretation:
 Low serum protein levels are observed in malnutrition, acute or chronic liver diseases,
and nephrotic syndrome

 Elevated protein levels are seen mainly in dehydration

4.14 Interferences:Criterion: Recovery within ± 10 % of initial value at a total protein
concentration of 66 g/L (6.6 g/dL).
4.14.1 Icterus: No significant interference up to an I index of 20 for conjugated bilirubin
and 20 for unconjugated bilirubin (approximate conjugated bilirubin concentration:
20 mg/dL or 342 μmol/L, approximate unconjugatedbilirubin concentration: 20
mg/dL or 342 μmol/L).
4.14.2 Hemolysis: No significant interference up to an H index of 1000 (approximate
hemoglobin concentration: 622 μmol/L (1000 mg/dL)).
4.14.3 Lipemia (Intralipid): No significant interference up to an L indexof 2000. There is
poor correlation between the L index (correspondsto turbidity) and triglycerides
concentration.
4.14.4 Dextran up to concentrations of 30 mg/mL does not interfere.Drugs: No
interference was found at therapeutic concentrations using common drug panels.
In very rare cases, gammopathy, in particular type IgM
(Waldenström’smacroglobulinemia), may cause unreliable results. For diagnostic
purposes, the results should always be assessed in conjunction with the patient’s
medical history, clinical examination and other findings.
4.15 Trouble Shooting: NA
4.16 Safety Precautions:
 Handle all samples as potentially infectious.
 Handle all reagents with care and avoid contact with eye mouth and skin.
 Disinfect work surfaces when procedures are completed at the end of each working day.
An effective multipurpose disinfectant is a 1% hypochlorite solution.
 Discard all reagents and samples as per disposal procedure.

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5.0 REFERENCES
5.1 Product Roche Cobas kit insert
5.2 Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics, Carl A. Burtis, David
E. Bruns and Roche Cobas kit literature
6.0 APPENDICES & FORMS: None

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