IMTEC-ANA-LIA MAXX. Design Verification

Transcription

1 Design Verification IMTEC-ANA-LIA MAXX CONTENTS 1 Intended Use Diagnostic Sensitivity and Specificity Interferences Imprecision Within-Run Imprecision Between-Run Imprecision Lot-to-Lot Imprecision Stability Accelerated Stress Test Open Vial Stability Test Real Time Stability Form: /27 valid of

2 1 Intended Use IMTEC-ANA-LIA Maxx is an indirect membrane based enzyme immunoassay (LIA) for the qualitative measurement of IgG class autoantibodies against nuclear antigens in human serum. The assay is intended for in vitro diagnostic use only as an aid in the diagnosis of connective tissue diseases. Nuclear and associated cytosolic antigens are applied as lines on a nitrocellulose membrane: antigens dsdna nucleosomes histones SmD1 PCNA P0 SS-A/Ro60kD SS-A/Ro52kD SS-B/La CENP-B Scl70 U1-snRNP AMA M2 Jo-1 PM-Scl Mi2 Ku identity native native native peptide synthetic recombinant recombinant native recombinant recombinant recombinant recombinant recombinant native recombinant recombinant recombinant recombinant The nitrocellulose membrane is blocked to prevent unspecific reactions. During incubation of a blotting strip with diluted patient serum autoantibodies present in the sample will bind to the antigens on the strip. For the detection of the antibodies bound to the strip a horseradish peroxidase-labelled secondary antibody (goat) is used that is directed against human IgG. After of a substrate solution comprising precipitating 3,3,5,5 tetramethylbenzidine and of stop solution the bound autoantibodies are visualised as brown lines. Comparison with the evaluation template provided with each kit enables the identification of the patient's autoantibody specificities. 2 Diagnostic Sensitivity and Specificity Diagnostic sensitivity and diagnostic specificity were calculated according to following formula: True positive results (TP) Sensitivity = ---- x 100 [%] true positive results (TP) + false negative results (FN) true negative results (TN) Specificity = ---- x 100 [%] true negative results (TN) + false positive results (FP) Sera from antibody positive patients in IIF were tested to study the diagnostic sensitivity of the IMTEC-ANA-LIA Maxx. The diagnostic specificity was determined using serum samples from apparently healthy blood donors: Design Verification and Product Data for Imtec-ANA-LIA Maxx 2/27

3 Immunofluorescence HEp-2 IMTEC- ANA-LIA Maxx Positive Negative Cumulative Positive Result Negative 1 49 dsdna Positive 15 1 Negative Nucleosomes Positive 18 0 Negative Histones Positive 13 0 Negative SmD1 Positive 15 0 Negative PCNA Positive 8 0 Negative P0 Positive 6 0 Negative SS-A 60 / Ro 60 Positive 30 0 Negative SS-A 52 / Ro 52 Positive Negative SS-B / La Positive 20 0 Negative CENP-B Positive 18 0 Negative Scl70 Positive 13 0 Negative U1-snRNP Positive 12 0 Negative AMA M2 Positive 14 0 Negative Jo-1 Positive 9 0 Negative PM-Scl Positive 7 0 Negative Design Verification and Product Data for Imtec-ANA-LIA Maxx 3/27

4 Mi2 Positive 14 0 Negative Ku Positive 7 0 Negative Relative Sensitivity 110/ % Relative Specificity 49/ % 3 Interferences Positive and negative samples are tested prior and after of interfering substances with lot # Every sample is tested in duplicate. Data interpretation and calculation: Index values of HumaScan and classification of line intensity according to HumaScan. Acceptance criteria: Negative samples are also negative after of an interfering substance. Positive samples are positive also with interfering substances. The intensity of the positive samples varies not more than one level within the intensity classification scheme (e.g. from + to ++). Interfering substances Bilirubin: Hemoglobin: Triglycerides: Final conc. in serum 40 mg/dl 500 mg/dl 2500 mg/dl Results of positive sample w/o interfering substances dsdna Nucleosomes Histones SmD1 PCNA Index Intensity bilirubin hemoglobin triglycerides w/o interfering substances P0 SS-A/Ro60 SS-A/Ro52 SS-B/La CENP-B Index Intensity bilirubin hemoglobin triglycerides Design Verification and Product Data for Imtec-ANA-LIA Maxx 4/27

5 w/o interfering substances Scl70 U1-snRNP AMA M2 Jo-1 PM-Scl Index Intensity bilirubin hemoglobin triglycerides w/o interfering substances Mi-2 Ku Index Intensity Index Intensity bilirubin hemoglobin triglycerides Results of negative sample dsdna Nucleosomes Histones SmD1 PCNA w/o interfering substances Index Intensity bilirubin hemoglobin triglycerides w/o interfering substances P0 SS-A/Ro60 SS-A/Ro52 SS-B/La CENP-B Index Intensity bilirubin hemoglobin triglycerides Design Verification and Product Data for Imtec-ANA-LIA Maxx 5/27

6 w/o interfering substances Scl70 U1-snRNP AMA M2 Jo-1 PM-Scl Index Intensity bilirubin hemoglobin triglycerides w/o interfering substances Mi-2 Ku Index Intensity Index Intensity bilirubin hemoglobin triglycerides Conclusion: All antigens on the LIA strip are within the acceptance criteria. Bilirubin, hemoglobin and triglycerides do not interfere with the IMTEC-ANA-LIA Maxx in the tested concentrations. 4 Imprecision The imprecision of the test was established by testing three serum samples with different autoantibody concentrations. 4.1 Within-Run Imprecision Three patient samples are tested with 20 repetitions each. The 20 repetitions are executed within one run. Lot # was used therefore. Data interpretation and calculation: Index values of HumaScan and classification of line intensity according to HumaScan. Acceptance criteria: Negative samples are negative at all repetitions. Positive or borderline samples show a coefficient of variation lower than 20%. Classification of line intensity: Index (HumaScan) Line intensity Interpretation negative o borderline weak positive mid-level positive strong positive Design Verification and Product Data for Imtec-ANA-LIA Maxx 6/27

7 dsdna Nucleosomes Index Intensity Index Intensity 1.57* n/a * n/a CV [%] *first strip with sample-1 not valid Histones SmD1 Index Intensity Index Intensity 1.08* n/a * n/a o CV [%] Design Verification and Product Data for Imtec-ANA-LIA Maxx 7/27

8 PCNA Index Intensity Index Intensity 0.40* n/a 0.98 o * n/a 1.07 o o o o o o o o o o o o o CV [%] *first strip with sample-1 not valid SS-A/Ro60 P0 SS-A/Ro52 Index Intensity Index Intensity 0.62* n/a * n/a CV [%] Design Verification and Product Data for Imtec-ANA-LIA Maxx 8/27

9 SS-B/La CENP-B Index Intensity Index Intensity 0.49* n/a * n/a o o CV [%] *first strip with sample-1 not valid Scl70 U1-snRNP Index Intensity Index Intensity 0.92* n/a * n/a o o o o o CV [%] Design Verification and Product Data for Imtec-ANA-LIA Maxx 9/27

10 AMA M2 Index Intensity Index Intensity 1.13* n/a * n/a CV [%] *first strip with sample-1 not valid PM-Scl Index Intensity Index Intensity 1.43* n/a * n/a CV [%] Jo-1 Mi-2 Design Verification and Product Data for Imtec-ANA-LIA Maxx 10/27

11 Ku Index Intensity Index Intensity Index Intensity Sample 1 Sample 2 Sample * n/a CV [%] *first strip with sample-1 not valid Conclusion: All parameters meet the acceptance criteria with all three samples. The parameters show an appropriate intra-assay precision. 4.2 Between-Run Imprecision The between-run precision was determined with IMTEC-ANA-LIA Maxx lot # Three patient samples are tested in duplicates each. This testing scheme is repeated 10 times on different days. Data interpretation and calculation: Index values of HumaScan and classification of line intensity according to HumaScan. Acceptance criteria: Negative samples are negative at all repetitions. Positive or borderline samples show a coefficient of variation lower than 30%. Classification of line intensity: Index (HumaScan) Line intensity Interpretation negative o borderline weak positive mid-level positive strong positive dsdna Nucleosomes Run Index Intensity Index Intensity Design Verification and Product Data for Imtec-ANA-LIA Maxx 11/27

12 o o o o o o o o o 0.80 o o CV [%] Histones SmD1 Run Index Intensity Index Intensity o o o o o o o o o Design Verification and Product Data for Imtec-ANA-LIA Maxx 12/27

13 o CV [%] o PCNA P0 Run Index Intensity Index Intensity o 0.90 o o 0.97 o o 0.95 o o 0.85 o o o 0.91 o o o o o o o 1.05 o o o o o o o o CV [%] o o SS-A/Ro60 SS-A/Ro52 Run Index Intensity Index Intensity Design Verification and Product Data for Imtec-ANA-LIA Maxx 13/27

14 CV [%] SS-B/La CENP-B Run Index Intensity Index Intensity o 1.09 o o 1.02 o o o o o 0.91 o o o 0.86 o o o o o 0.93 o o o 0.91 o o o 0.94 o CV [%] 1.14 o o Scl70 U1-snRNP Run Index Intensity Index Intensity Design Verification and Product Data for Imtec-ANA-LIA Maxx 14/27

15 o o o o o o o o o o CV [%] AMA M2 Jo-1 Run Index Intensity Index Intensity Design Verification and Product Data for Imtec-ANA-LIA Maxx 15/27

16 CV [%] PM-Scl Mi-2 Run Index Intensity Index Intensity o o o o o o CV [%] Ku Sample 1 Sample 2 Sample 3 Run Index Intensity Index Intensity Index Intensity o o Design Verification and Product Data for Imtec-ANA-LIA Maxx 16/27

17 o o o CV [%] Conclusion: All parameters meet the acceptance criteria with all three samples. The parameters show an appropriate intrer-assay precision. 4.3 Lot-to-Lot Imprecision Three patient samples are tested in triplicates each. This testing scheme is repeated for three lots (# 15002; 15003; 15004). Data interpretation and calculation: Index values of HumaScan and classification of line intensity according to HumaScan. Acceptance criteria: Negative samples are negative at all repetitions. Positive samples are positive at all repetitions. Weak positive samples ("+") can have borderline results ("o"). Classification of line intensity: Index (HumaScan) Line intensity Interpretation negative o borderline weak positive mid-level positive strong positive dsdna Nucleosomes Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three Nucleosomes and dsdna samples are within the acceptance criteria. Histones SmD1 Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three Histones and SmD1 samples are within the acceptance criteria. PCNA P0 Design Verification and Product Data for Imtec-ANA-LIA Maxx 17/27

18 Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three P0 samples are within the acceptance criteria. The PCNA negative sample is negative with all three lots. The positive samples both had very weak to borderline specification. Thus, two LIA batches show weak positive results, the third batch is high negative with these samples. This result is accepted since the batch to batch variation range of the index values corresponds to the findings with other antigens with higher absolute values. SS-A/Ro60 SS-A/Ro52 Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three SS-A/Ro52 and SS-A/Ro60 samples are within the acceptance criteria. SS-B/La CENP-B Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three SS-B/La samples are within the acceptance criteria. All CENP-B results are within the acceptance criteria exept one sample with batch This originally weak positive sample shows high negative results with the mentioned LIA lot. The overall lot to lot variation of CENP-B is acceptable. Scl70 U1-snRNP Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three Scl70 samples are within the acceptance criteria. U1-snRNP meets the acceptance criteria. Only the weak positive/borderline sample 2 reacts strongerwith lot All other values are within the specification. The RNP antigen shows a good lot to lot precision. AMA M2 Jo-1 Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three Jo-1 and AMA M2 samples are within the acceptance criteria. PM-Scl Mi-2 Lot Mean Index Mean Index Mean Index Mean Index Mean Index Mean Index The results of all three Mi-2 and PM-Scl samples are within the acceptance criteria. Design Verification and Product Data for Imtec-ANA-LIA Maxx 18/27

19 Ku Sample 1 Sample 2 Sample 3 Lot Mean Index Mean Index Mean Index The results of all three Ku samples are within the acceptance criteria. 5 Stability The stability of the IMTEC-ANA-LIA Maxx has been evaluated by accelerated temperature stress of all test kit components and real-time studies. The kit has been tested at the date of manufacture and re-tested after the indicated periods. Interpretation of test results was performed by using HumaScan Software. 5.1 Accelerated Stress Test LIA Kits (lot # 15002) are stored at 37 C. They are tested after the storage periods given below. Four patient samples (two positive and two negative) are tested in duplicates each at every time point. Data interpretation and calculation: Index values of HumaScan and classification of line intensity according to HumaScan. Acceptance criteria: Negative samples are negative over the tested period. The intensity of the positive samples varies not more than one level within the intensity classification scheme over the tested period (e.g. from "++" to "+" or from "+" to "o"). Classification of line intensity: Index (HumaScan) Line intensity Interpretation negative o borderline weak positive mid-level positive strong positive dsdna Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day Nucleosomes Sample-1 Sample-2 Sample-3 Sample-4 Day Day Design Verification and Product Data for Imtec-ANA-LIA Maxx 19/27

20 Day Histones Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day SmD1 Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day PCNA Sample-1 Sample-2 Sample-3 Sample-4 Day Day o 0.81 o o Day o 1.00 o o The PCNA results are within the acceptance criteria except one high negative value with sample-2. This sample is only weak positive. All other single PCNA measurements of all samples meet the specification, also at the last evaluation time after 10 days. Therefore this negative outlier result is accepted for the test. P0 Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day SS-A/Ro60 Sample-1 Sample-2 Sample-3 Sample-4 Design Verification and Product Data for Imtec-ANA-LIA Maxx 20/27

21 Day Day Day SS-A/Ro52 Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day SS-B/La Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day o CENP-B Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day Scl70 Sample-1 Sample-2 Sample-3 Sample-4 Day Day o Day The Scl70 results are within the acceptance criteria except one borderline value with sample-2 on day 7. This sample is generally weak to mid-level positive. All other single Scl70 measurements of all samples meet the Design Verification and Product Data for Imtec-ANA-LIA Maxx 21/27

22 specification, also at the last evaluation time after 10 days. Therefore this single outlier result is accepted for the test. U1- snrnp Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day AMA M2 Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day Jo-1 Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day PM-Scl Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day Mi-2 Sample-1 Sample-2 Sample-3 Sample-4 Day Design Verification and Product Data for Imtec-ANA-LIA Maxx 22/27

23 Day Day Ku Sample-1 Sample-2 Sample-3 Sample-4 Day Day Day Conclusion: The results are within the acceptance criteria. For all parameters a stability of 15 months at 2-8 C can be assumed. 5.2 Open Vial Stability Test LIA Kits (lot # 15002) are stored at 2-8 C. They are tested after 2, 4, 6 and 8 weeks with patient samples in duplicates. Data interpretation and calculation: Index values of HumaScan and classification of line intensity according to HumaScan. Acceptance criteria: Negative samples are negative over the tested period. The intensity of the positive samples varies not more than one level within the intensity classification scheme over the tested period (e.g. from "++" to "+" or from "+" to "o"). Results of positive sample dsdna Nucleosomes Histones SmD1 PCNA P0 Index Intensity Index Intensity * o o o o SS-A/Ro60 SS-A/Ro52 SS-B/La CENP-B Scl70 U1-snRNP Index Intensity Index Intensity Design Verification and Product Data for Imtec-ANA-LIA Maxx 23/27

24 o o o o o o o o o * o * *one high negative result accepted for weak positive/borderline RNP sample; PCNA and SS-B/La see remark in the conclusion of this stability test AMA M2 Jo-1 PM-Scl Mi-2 Ku Index Intensity Results of negative sample dsdna Nucleosomes Histones SmD1 PCNA P0 Index Intensity Index Intensity Design Verification and Product Data for Imtec-ANA-LIA Maxx 24/27

25 SS-A/Ro60 SS-A/Ro52 SS-B/La CENP-B Scl70 U1-snRNP Index Intensity Index Intensity AMA M2 Jo-1 PM-Scl Mi-2 Ku Index Intensity Conclusion: The results are within the acceptance criteria. During eight weeks, the signals of all antigens are comparable. The positive samples for PCNA and SS-B/La have only very weak to borderline intensities. Due to the variation of the method they show high negative results with two LIA strips in the middle of the evaluation period. This is no sign of decreasing stability after openig the kit. The stability of the IMTEC-ANA LIA Maxx after opening the reagents is at least 6 weeks. Design Verification and Product Data for Imtec-ANA-LIA Maxx 25/27

26 5.3 Real Time Stability Lot: Storage: C Date of Production: Date of 1 st Re-Testing: Duration: over 16 month 2-8 C Result fresh over 16 months Positive control positive positive passed Negative control negative negative passed Lot: Storage: C Date of Production: Date of 2 nd Re-Testing: Duration: over 21 month 2-8 C Result fresh over 21 months Positive control positive positive passed Negative control negative negative passed Lot: Storage: C Date of Production: Date of 1 st Re-Testing: Duration: over 15 months 2-8 C Result fresh over 15 months Positive control positive positive passed Negative control negative negative passed Lot: Storage: C Date of Production: Date of 2 nd Re-Testing: Duration: over 20 months 2-8 C Result fresh over 20 months Positive control positive positive passed Negative control negative negative passed Lot: Storage: C Date of Production: Date of Re-Testing: Duration: over 15 months Design Verification and Product Data for Imtec-ANA-LIA Maxx 26/27

27 2-8 C Result fresh over 15 months Positive control positive positive passed Negative control negative negative passed No visible decrease in the positive control could be observed after 15 months (and later) from the date of production. Equally no false positive reaction could be seen with negative controls. Conclusion: The stability data support the shelf life claim of 15 months after production. Design Verification and Product Data for Imtec-ANA-LIA Maxx 27/27