Hexagon PSA. Design Verification. Contents

Transcription

1 Design Verification Hexagon PSA Contents 1. Function Sensitivity, Dynamic Range and Traceability...2 Description of Control Materials...2 Analytical Sensitivity Clinical Evaluation...3 Evaluation of Sensitivity and Specificity Cross-reactivity, Interferences Reproducibility Stability...5 Accelerated Temperature Stress Studies...5 Real Time Stability Study...6 Form: /6 valid of

2 1. Function HEXAGON PSA has been developed for the rapid qualitative detection of prostate specific antigen in serum, plasma and whole blood. The test is based on a membrane immunochromatographic method featuring a combination of monoclonal antibodies. Sample brought into contact with the test causes re-dissolution of the reagents which are impregnated on the inner part of the device. As the sample flows through the conjugate pad, human PSA is bound by the anti-psa - dye conjugate to form an immunocomplex, which binds to anti-psa antibodies in the test line and produces a red test line. Excess conjugate reacts in the line with anti-mouse IgG antibodies, forming a second red line to demonstrate the correct function of the reagents. The test detects PSA in concentrations of 4 ng/ml and more. Depending on the PSA concentrations in the sample two red lines will appear within 10 minutes after start of test. Strong positive samples will develop a test line within a few minutes. 2. Sensitivity, Dynamic Range and Traceability Description of Control Materials WHO First International Standard Prostate specific antigen (90:10, NIBSC code 96/670) is used as positive to the sensitivity and function of the test. It is diluted with previously determined PSA negative serum, plasma and whole blood to the required concentrations of a dilution series. As negative sample phosphate buffered saline is used. Analytical Sensitivity All concentrations have been tested three-fold with three different lots (P2001, P2002, P2003, manufacturing date ) following the instruction for use. No significant difference could be observed between the individual results regarding intensities of test lines. The mean results are summarised in the following table: PSA Concentration (ng/ml). Results with Serum Results with EDTA Plasma Results with EDTA Whole Blood Test line Result Test line Result Test line Result 0 - Negative - Negative - Negative 1 - Negative - Negative - Negative 2 - Negative - Negative - Negative 4 + Positive + Positive + Positive 8 + Positive + Positive + Positive Positive ++ Positive ++ Positive Positive +++ Positive +++ Positive Positive +++ Positive - = negative; + = positive; ++ = strongly positive HEXAGON PSA showed an analytical sensitivity of 4 ng/ml. Design Verification and Product Data for HEXAGON PSA 2/6

3 3. Clinical Evaluation Evaluation of Sensitivity and Specificity A clinical evaluation was conducted using a total of 1,224 clinical specimens. HEXAGON PSA (lot X001, lot X002, lot X003; expiry date ; lot X8001, expiry date ) has been tested with residual samples comprising 252 serums (over 40 years old men and 86 female samples as negative samples) and 529 EDTA anticoagulated blood specimens (over 40 years old men). In case of whole blood and plasma samples, PSA concentration of equivalent serum specimens was determined. PSA quantitation of serum samples was performed with a quantitative PSA assay (ADVIA Centaur CP, Siemens, PSA Assay Analyzer). Results of evaluation were plotted regarding cut-off settings of PSA concentration of 4 ng/ml. Results 1. Tests with serum specimens HEXAGON PSA Positive 4 ng/ml PSA Negative 4 ng/ml PSA Total Positive Negative Total Relative Sensitivity: 100% (58/58) Relative Specificity: 97.2% (105/108) Overall Agreement: 98.2% (163/166) 2. Tests with EDTA plasma HEXAGON PSA Positive 4 ng/ml PSA Negative 4 ng/ml PSA Total Positive Negative Total Relative Sensitivity: 100% (23/23) Relative Specificity: 95.1% (481/506) Overall Agreement: 95.3% (504/529) 3. Tests with EDTA whole blood HEXAGON PSA Positive 4 ng/ml PSA Negative 4 ng/ml PSA Total Positive Negative Total Relative Sensitivity: 100% (23/23) Relative Specificity: 95.8% (485/506) Overall Agreement: 96.0% (508/529) With a cut-off set at 4 ng/ml, HEXAGON PSA showed a diagnostic sensitivity of 100 % and a diagnostic specificity in the range of % (plasma / whole blood / serum specimens). HEXAGON PSA detected all positive specimens, including specimens with a PSA concentration up-to 656 ng/ml, without any indication of a high dose hook effect. Design Verification and Product Data for HEXAGON PSA 3/6

4 3. Cross-reactivity, Interferences Potentially interfering substances (bilirubin, hemoglobin, triglycerides, EDTA, sodium citrate and heparin) have been added to PSA negative and to PSA positive plasma, serum and whole blood specimens, respectively. Substance Concentration PSA (ng/ml) Result PSA (ng/ml) Result Bilirubin 20 mg/dl 0 negative 4 positive Hemoglobin 250 mg/dl 0 negative 4 positive Triglycerides 1,250 mg/dl 0 negative 4 positive Human albumin 20,000 mg/dl 0 negative 4 positive EDTA 800 mg/dl 0 negative 4 positive Sodium citrate 500 mg/dl 0 negative 4 positive Heparin 6 IU/dl 0 negative 4 positive None of the potentially interfering substance caused any false negative or false positive result. To evaluate cross-reactivity with heterophilic antibodies a panel consisting of 48 panel members, positive for human anti-mouse antibodies (HAMA) up-to 780 ng/ml and positive for rheumatoid factor up-to 8,530 ng/ml has been tested. All 48 samples showed a correct result with HEXAGON PSA. HEXAGON PSA showed a very good specificity when tested with specimens containing potentially cross-reacting substances. 4. Reproducibility Samples WHO First International Standard Prostate specific antigen (90:10, NIBSC code 96/670) has been diluted with negative human serum, plasma and whole blood to a concentration of 4 ng/ml. Within-run PSA negative samples and samples containing 4 ng/ml PSA were tested in 20 replicates. Between-run PSA negative samples and samples containing 4 ng/ml PSA were tested on three different days by three different technicians in 20 replicates each. Lot-to-lot Three different lots pf HEXAGON PSA (X001, X002 and X003, manufacturing date ) were tested in 20 replicates with PSA negative samples and samples containing 4 ng/ml PSA. Results and conclusion: The agreement of all readings was 100%. There were no deviations between the runs, the lots or between the operators. Design Verification and Product Data for HEXAGON PSA 4/6

5 5. Stability Accelerated Temperature Stress Studies The stability of HEXAGON PSA was evaluated by an accelerated temperature stress study. Samples from three different lots (X001, X002, X003, manufacturing date ) were stored for up to 8 months in an incubator at 40 C. Testing at each time interval consisted of 5 replicates for each specimen, diluted standard material with 4 ng/ml PSA and a PSA negative. The tests have been done according to the instruction of use. The results are summarised in the following table. Results: Lot X001 Lot X002 Lot X003 Months Sample = negative; + = positive Over the testing period no significant change of the test line intensities could be observed. These data indicate a stability of at least 18 months. Design Verification and Product Data for HEXAGON PSA 5/6

6 Real Time Stability Study Samples from three different lots of HEXAGON PSA (X001, X002, X003, manufacturing date ) have been stored at room temperature and have been tested by using diluted standard material with 4 ng/ml PSA and PSA negative. Samples were run in five replicates. The tests have been done according to the instruction of use. The results are summarised in the following table. Results: Lot X001 Lot X002 Lot X003 Months Sample = negative; + = positive Real time stability data show a good stability for 24 months. Therefore, the stability was set to 18 monthsfrom the date of manufacturing. Design Verification and Product Data for HEXAGON PSA 6/6