Design Verification HumaTex 1 Function... 2 2 Imprecision... 2 3 Sensitivity and Dynamic Range... 2 Preparation of Serum Control Panel... 2 Sensitivity Test Results... 2 Function test with Kit Controls... 2 Prozone Check... 3 4 Interferences... 3 Results... 3 5 Specificity and Sensitivity... 4 Results... 4 6 Stability... 5 Real-Time Stability... 5 QM-Element: ISO9001:2000 No. 7.3.3 1/5 valid of 08.01.2019
1 Function HumaTex ASO latex agglutination slide test has been designed as a qualitative and semi-quantitative test for the rapid determination of anti-streptolysin-o antibodies (ASO) in native undiluted serum samples. The test is based on latex agglutination technique, featuring polystyrene latex which has been coated with stabilized streptolysin-o. If ASO positive sera are reacted with the latex suspension, visible agglutinates will form within 2 minutes. Positive and negative controls are incorporated in the kit to perform function checks and to compare the results obtained with unknown samples. For semi-quantitative determinations the serum samples are diluted stepwise with glycine saline buffer. The last dilution step which give a positive result (agglutination) is used to calculate the ASO concentration in the sample. 2 Imprecision The within-run imprecision of HumaTex ASO latex agglutination slide test was calculated from 10-fold determinations. The day-to-day imprecision was calculated from the results obtained on 4 different dates. Positive and negative control sera were employed as sample materials. Within-run and day-to-day imprecision was found with an excellent CV. 3 Sensitivity and Dynamic Range Description of control materials In-house standards are employed which have been calibrated against a standard material (BioMerieux, #72271, equivalent to the WHO reference preparation). Standard dilutions are prepared by diluting the in-house standard with glycine buffered saline. The following standard dilutions are prepared: No. 1 2 3 4 5 6 7 ASO, IU/ml 0 50 100 150 200 300 400 The kit controls positive and negative are employed for function test. The positive control is typically adjusted to a ASO concentration of 400-600 IU/ml. Preparation of Serum Control Panel Sensitivity is determined with an ASO secondary international standard, calibrated by turbidimetry to the ASO International Standard (WHO). Dilutions are prepared with physiological saline and tested with 3 different ASO latex lots according to the recommendations in the package insert HumaTex ASO. Sensitivity Test Results All concentrations are tested in double. The difference in agglutination degree must not exceed one titer step. No. ASO, IU/ml Agglutination degree at 2 min. Lot 030100 Lot 171199 Lot 280999 1 0 - - - 2 50 - - - 3 100 - - - 4 150 5 200 1+ 1+ 1+ 6 250 2+ 2+ 2+ 7 400 3+ 4+ 3+ 8 500 4+ 4+ 4+ Intensity grades: 4+ (very strong agglutination); 3+ (strong agglutination); 2+ (medium agglutination); 1+ (visible); (borderline); - (no agglutination) Function test with Kit Controls Kit controls are employed for a function test of the latex reagent. Design Verification and Product Data for HumaTex ASO 2/5
Prozone Check Controls Agglutination degree at 2 minutes Positive control 4+ 1/2 diluted positive control 3+ 1/4 diluted positive control 1/8 diluted positive control - Negative control - For prozone check high concentrated ASO plasma (Biomedical Resources) is employed in the following concentrations for 3 different lots: No. ASO, IU/ml Agglutination degree at 2 min. Lot 280103 Lot 100203 Lot 170203 1 100 - - - 2 200 1+ 2+ 1+ 3 250 2+ 3+ 2+ 4 500 3+ 3+ 3+ 5 1000 4+ 4+ 4+ 6 1500 4+ 4+ 4+ 7 2000 4+ 4+ 4+ 8 2600 4+ 4+ 4+ Any prozone phenomenon can be excluded up to 2600 mg/l ASO. 4 Interferences Description of materials Several substances have been tested for potentially interfering effects: hemoglobin, bilirubin, lipids and rheumatoid factors (RF). Different concentrations of analytes are prepared in a human serum pool tested negative for ASO. Results Agglutination degree at 2 min., Lot 030100 Hemoglobin Bilirubin RF Lipids (g/l) (mg/dl) (IU/ml) (g/l) 0.63-0.5-0.5-0.63-1.25-2.5-2.5-1.25-2.5-5 - 5-2.5-5.0-10 - 10-5 - 10-20 - 20-10 - Control - Control - Control - Control - None of the analytes tested interfere with HumaTex ASO assay at the concentrations applied. Design Verification and Product Data for HumaTex ASO 3/5
5 Specificity and Sensitivity Description of method and specimen The specificity and sensitivity of HumaTex ASO test (Lot 30100, expiry date 2002/07) has been evaluated by method comparison against an independent latex slide test (BioSystems, Lot 048 A/B, expiry date 2000/02) and against a quantitative turbidimetric test method (Denka Seiken Lot 20619, expiry date 2001/02). 120 native sera, each 70 sera of healthy volunteers and 50 sera of rheumatoid patients (ASO positive), have been employed in this study (2 sera in each evaluation are excluded due to contamination). Results Latex slide test Detection limit of the competitor assay: 200 IU/ml. 48 of 49 positive sera were found positive and 67 of 69 negative sera were found negative with HumaTex ASO. From the above a diagnostic specificity for HumaTex ASO test resulted to 97%. The diagnostic sensitivity was found to be 98%. BioSystems Test Positive Negative HumaTex ASO Positive 48 2 Negative 1 67 Total 49 69 Turbidimetric test Cut off of the competitor assay: 200 IU/ml. The 50 positive sera were found positive with both HumaTex ASO and the competitor method. 64 of 66 negative sera were found negative with HumaTex ASO. From the above a diagnostic specificity for HumaTex ASO test resulted to 97%. Diagnostic sensitivity was found to be 100%. Denka Seiken ASO Positive Negative HumaTex ASO Positive 50 2 Negative 0 64 Total 50 66 Design Verification and Product Data for HumaTex ASO 4/5
6 Stability Real-Time Stability The stability of Humatex ASO latex agglutination slide test has been tested in real-time stability studies of both the ASO latex reagent and the positive control. ASO latex reagent Several dilutions of one standard were tested with 3 different lots according to the manufacturer's instruction. Results of the function tests are summarised below. ASO, IU/ml 1500 500 400 250 200 150 50 ASO lot 010797 31 months @ 2...8 C ASO lot 100697 32 months @ 2...8 C ASO lot 150497 35 months @ 2...8 C 4+ 4+ 4+ 4+ 4+ 2+ - 4+ 4+ 4+ 4+ 3+ - 4+ 4+ 4+ 3+ 2+ - The results show that the ASO latex reagent has a shelf life of 30 months when stored at 2-8 C. Positive control Three different lots of the positive control (ASOPOS020) were tested at different time points according to the manufacturer s instruction. Results of the function tests are summarised below. POSITIVE CONTROL ASOPOS020 lot 0032 36 months @ 2...8 C lot 0036 25 months @ 2...8 C lot 0034 31 months @ 2...8 C 3/4+ 4+ 4+ The results show that the positive control has a shelf life of 24 months when stored at 2-8 C. Conclusion: The results show that the shelf life of the ASO latex reagent considered alone is 30 months. The shelf life of the positive control is 24 months. The HumaTex ASO kit is stable for the claimed period of 24 months. Design Verification and Product Data for HumaTex ASO 5/5