ISSUE NO.

: 04
SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

A) PURPOSE OF THE EXAMINATION:

This electrochemiluminescence binding assay is intended for the quantitative determination of

total prostate specific antigen (tPSA) in human serum and plasma on cobas e immunoassay
analyzer.

B) PRINCIPLE AND METHOD OF THE PROCEDURE USED FOR

EXAMINATION:

It is based on the “Sandwich principle”

1st incubation : 20 µl of sample, a biotinylated monoclonal PSA- specific antibody and a

monoclonal PSA- specific antibody labeled with a ruthenium complex react to form a sandwich
complex.

2nd incubation: After addition of streptavidin – coated microparticles, the complex becomes
bound to the solid phase via interaction of biotin and streptavidin.

The reaction mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are then removed
with Procell. Application of a voltage to the electrode then induces chemiluminescent emission
which is measured by photomultiplier.

Results are determined via a calibration curve which is instrument specifically generated by 2-
point calibration and a master curve provided via the reagent barcode.

C) PERFORMANCE CHARACTERISTICS :

Precision –Lab will calculate its own CV% as per daily internal quality control program.

Analytical sensitivity or lower detection limit is 0.003 ng /mL, Linearity –100 ng /mL

Measuring range is 0.003-100 ng /mL

Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

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 ISSUE NO.: 04
SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

D) TYPE OF SAMPLE:
Serum should be obtained aseptically by venipuncture.

Storage and stability:

Samples are stable for 14 days at 2-8O C and for 6 months at -20 oC. Centrifuge samples
containing precipitates before performing the assay.

E) PATIENT PREPARATION

No specific instructions for patient preparation.

F) TYPE OF CONTAINERS AND ADDITIVES

Yellow top vacutainer, Red top acceptable

Separated serum sample in transfer vial aaceptable
Sample rejection:

If sample is inadequate
If grossly hemolysed and lipemic sample
If sample and request form is not labeled properly.

G) REQUIRED EQUIPMENT AND REAGENT:

Centrifuge: After collecting blood sample let it clot and centrifuge at 3000 rpm for 10 min.

Roche cobas e immunoassay analyzer.

Reagents:

M- Streptavidin –coated microparticles.

R1- Anti PSA Ab ~ Biotin

R2- Anti PSA Ab ~ Ru(bpy)32+

Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

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 ISSUE NO.: 04
SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

Storage and stability:

Shelf life at 2-8oC up to the stated expiration date.

Open reagents are stable for 12 weeks at 2-8oC, onboard 4 weeks

H) ENVIRONMENTAL AND SAFETY CONTROLS

This kit is for in vitro diagnostic use only. All human material should be considered potentially
infectious. Exercise the universal precaution for handling of all laboratory reagents. For details
refer to precaution and warnings of reagent kit inserts. Disposal of all waste material should be
as per defined guidelines. Refer material safety data sheet (MSDS) for any accidental exposure.
For detail refer to laboratory safety manual.

I) CALIBRATION PROCEDURE (METROLOGICAL TRACEABILITY)

Traceability: This method has been standardized against the Stanford reference standard / WHO /
96/670.

Calibration frequency:

After reagent lot change, after one month when using the same reagent lot.

As required following quality control procedure

Load the rack and request calibration: Calibrator and controls are loaded as per assigned rack.
Calibrator and controls of same lot is requested for calibration on the equipment. Calibration is
performed automatically by the equipment after respective scanning of the control and calibrator.
Performance of the calibration and control is checked for acceptance.

J) PROCEDURE

Pre-start physical inspection

1) Check the water level

2) All surfaces are clean and clear of loose articles and debris.
3) Probes and mixer paddles are not bent.

Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

Page 3 of 7
 ISSUE NO.: 04
SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

4) Pippeter syringe, associate tubings and reaction cells are free from bubbles and no
leaking is there.
5) Check the reagent

Switch the analyzer on:

1. Switch on the circuit breakers located on the back side of the analyzer and operation
switch located on left side of the analyzer.
2. Turn the CPU and monitor on, Log on to the system
3. After initialization is complete ensure stand by status is displayed.
4. Check that the printer paper supply is adequate.
5. Clear sample data
6. Check equipment alarm if any
7. Print/check the reagent load list
8. Replace required reagents
9. Empty solid and liquid waste container
10. Maintenance is given as per instructions

Assay

After keeping the reagent cassette at room temperature for 15 minutes. Place the reagent cassette
in the cassette reagent disc of the analyzer. The system automatically regulates the temperature of
the reagent and the opening/closing of the bottles. Place the sample in the assigned position after
selecting the test and press start.

K) QUALITY CONTROL PROCEDURE

Quality Assurance: Quality assurance is ensured as per defined Quality control procedure. The
frequency of control is two levels once daily and after every calibration. Internal controls are as
follows:

Preci Control Tumor marker TM1 & TM2

QC involves two steps: Run the control and check the result as follows:

To run the control:

1) Place the control cups in assigned rack at assigned position as displayed on screen.
2) Before start up is given make sure that number on the rack reflects similar to the currently
loaded.
Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

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SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

3) Start up given after selecting the parameter of control

4) Remove the controls from the control rack when sampling is completed.

To check control results:

Value obtained should fall with in established limits as per quality control procedure. If the
values obtained of quality control are not in range, error should be checked followed by
appropriate corrective action.

EQAS: Performed monthly from BIO RAD.

L) INTERFERENCE AND CROSS REACTION

BILIRUBIN : Presence of Bilirubin in concentrations up to 200 mg/L has no effect on result

within the precision of the assay.
HEMOLYSIS : Presence of packed red blood cell concentrations upto 65l/ml has no effect on
results, within the precision of the assay.
LIPEMIA : Highly lipemic sample may interfere with the assay.
In patients receiving therapy with high biotin doses, no sample should be taken until at least 8
hours after the last biotin administration.
No interference was observed from rheumatoid factors up to a
concentration of 1500 IU/ml.

M) PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS

The analyzer automatically calculate the analyte concentration.

Conversion factor: µIU/mL x 0.047 =ng/mL

N) BIOLOGICAL REFERENCE INTERVAL

< 4.0 ng/ ml

O) REPORTABLE INTERVAL FOR EXAMINATION RESULT

0.003-5000 ng/mL

P) PROTOCOL TO DETERMINE QUANTITATIVE RESULTS WHEN VALUE IS

NOT WITHIN MEASUREMENT INTERVAL
Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

Page 5 of 7
 ISSUE NO.: 04
SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

If result > 100ng/mL

Determine sample with higher concentration via re-run function or autodilution function
in the ratio of 1:50 dilution. Results automatically multiplied by factor of 50.

P) CRITICAL VALUE

Not applicable

Q) LABORATORY CLINICAL INTERPRETATION:

For monitoring, successful radiation or antiandrogen therapy reduces PSA in patients with
residual disease. Increasing levels after treatment indicate relapse after many months before
clinical symptoms appear.
Failure of radiation therapy to decrease PSA < 1 ng/ml means likely hood of recurrence: Some
reports suggest that a value of 0.5 ng/ml predict long term disease free interval.
After removal of all tumor, PSA >2 ng/ml indicates recurrence.

R) POTENTIAL SOURCE OF VARIATION

Potential source of variations are: Incorrect sampling, insufficient reagent, fibrin clot in sample,
insufficient sample volume. Precaution should be taken while preparing the reagent,
reconstituting controls & when reconstituting calibrators. Other sources of variations are
discussed under the headings of interferences and cross reactions.

S) REFERENCES:

 Teitz NW, ed. Fundamentals of Clinical Chemistry. Philadelphia , 5th edition

 Kit insert and manual of cobas e immunoassay analyzer.

PERSON (S) RESPONSIBLE:

Technician is responsible for performing the test/control as per written procedure. Senior
technician is responsible to supervise and check the clerical labeling/ technical error and sort out
problem with staff and in machine and is responsible for initial review of the result of
examination. Section head is responsible for confidentiality, accuracy and review of all
procedure. Quality manager is responsible for restricted access to the procedures and proper

Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

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 ISSUE NO.: 04
SOP NO. IMM /QM/11 ISSUE DATE : 15April 2015
SBAMI REV. NO.: 00
PSA (TOTAL) REV. DATE: 00

storage of all electronic data. Annual review of the procedure to be conducted by the section
head.

RIVIEW

Procedures must be reviewed and approved prior to method change.

Procedure must be reviewed annually.

Prepared by (Section Head, Biochemistry) Approved by (HOD, Lab)

Dr. D. K. Yadav Dr. Aruna Sud

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