Application Note for Gentian Cystatin C Immunoassay on. Cobas c501 1 (Optimised Volumes II), Roche Diagnostics 1 v06-september 2010
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1 Page of 6 Application Note for Gentian Cystatin C Immunoassay on For in vitro diagnostic use only Cobas c50 (Optimised Volumes II), Roche Diagnostics v06-september 200 General Information Intended Use The Gentian Cystatin C Immunoassay is an in vitro diagnostic test for quantitative determination of cystatin C in human serum and plasma. The measurement of cystatin C is used in the diagnosis and treatment of renal disease. Measuring range The overall measuring range of cystatin C concentration for Gentian Cystatin C Immunoassay on the Cobas c50 analyzer is mg/l. Reference intervals Gentian followed the CLSI Guideline, C28-A2 to determine the transferability of the reference interval. The reference interval was determined from a population of ostensibly healthy subjects with no history of renal disease. A total of 36 samples obtained from 58 males and 78 females ranging in age from 20 to 80 years were tested. The reference interval was calculated non-parametrically and was determined to be mg/l. This represents the central 95 % of the population. It is recommended that every laboratory should determine a local reference interval since values may vary depending on the population tested. GFR prediction calculation Several cystatin C based prediction equations for calculation of GFR for adults and children have been published. It should noted that these formulas were evaluated with different Cystatin C assays (particle-enhanced nephelometric immunoassay PENIA or particle enhanced turbidimetric immunoassay PETIA) and may reveal inaccurate GFR results if an inappropriate combination of formula and assay is used. For calculation of GFR from cystatin C values measured with the Gentian assay the following prediction equation is recommended using mg/l as the unit factor [4]: The equation is valid for persons above 4 years. GFR [ml/min/.73 m ] = Cystatin C (mg/l).4389 Materials Provided by Gentian AS Gentian Cystatin C Kit (REF0): Gentian Cystatin C Immunoparticles, bottle of 0 ml,ref04 Gentian Cystatin C Assay Buffer, bottle of 58 ml, REF007 Gentian Cystatin C Calibrator: vial of ml, REF02 Gentian Cystatin C Calibrator Kit: 6 levels, vials of ml, REF05 Gentian Cystatin C Control Kit: 2 vials of ml, REF09 Gentian Cystatin C Control Kit: 2 vials of 5 ml, REF026 All materials are ready to use. stable for 4 days at room temperature (8-25 C) and for 2 days if stored at 2-8 C, otherwise samples can be stored frozen at below -20ºC for 3 months. Mix samples well before analysing. Calibrator The calibrator is ready to use and a dilution series for calibration curve establishment is prepared automatically by the instrument. Assay Buffer (R) The Gentian Cystatin C Assay Buffer is the Reaction Buffer R. The buffer is ready for use. Immunoparticles (R3) The Gentian Cystatin C Immunoparticles are ready for use. All reagents are stable until the expiry date given on the labels when stored at 2-8ºC. Calibrator Standardisation An international cystatin C calibrator is being prepared by a working group formed by the IFCC/EU. The University of Lund, a leading partner in the working group, has validated Gentian calibration and calibrators against the coming IFCC standard. The Gentian Cystatin C calibrator is already standardised against the international cystatin C calibrator and is compatible with existing calibrators. See for more details. Calibration Stability The established calibration curve is stable for 4 weeks on Cobas c50. When reagent lots are changed or measured control values are outside the assigned range given in the Analytical Value Sheet, a recalibration is recommended. Onboard Stability In appropriate bottles, Gentian Cystatin C Immunoparticles and Assay Buffer are stable for at least 9 weeks when stored onboard the analyser. Assay Procedure Application Notes/Assay Installation An Instrument Parameter list is available in the section Instrument Settings below. The Application Note is also available at the following link: Instrument set up, maintenance, operation and precautions must be handled in accordance with the instrument manuals. Reagents Preparation Reagent is supplied ready to use. Use a suitable pipette to transfer the calculated volumes (see table below) of Reagent and Reagent 3 into the appropriate compartments of the Open Channel Reagent Cassette (cobas c pack MULTI ). Mix well before transfer. Use care to avoid contamination. Reagents should be stored capped at 2-8 C when not in use. Materials required but not provided Diluent : Saline, see Instrument settings below. Specimen Required sample material is human serum or EDTA / Heparinised plasma. It is recommended to analyse the samples as fresh as possible. Sample stability testing showed that cystatin C in serum and plasma samples are (35 tests) Cystatin C Kit Cystatin C Reagent Buffer Cystatin C Immunoparticles Compartment A 25.5 ml - Compartment B ml Application Note for Gentian Cystatin C Immunoassay on Cobas c50, Roche v06-september 0
2 Page 2 of 6 Establishment of the Calibration Curve Use Gentian Cystatin C Calibrator, REF02 to establish a 6- point standard curve as defined in the instrument manuals. Calibrator values are lot dependent and a new calibration must be performed whenever a new calibration lot is used. The calibrator s assigned values are given on the analytical value sheet provided with the calibrator. QC Controls The controls low and high must be assayed each day before any samples are assayed in order to validate the calibration curve. The controls have an assigned value range that must be met before measuring samples. The assigned values are given in the Analytical Value sheet included with the Gentian Cystatin C Control Kit (REF09). If the control values are not valid, repeat the control measurements. If the calibration cannot be performed without error, or valid control values cannot be reproduced, contact the manufacturer for support. Measuring Patient Samples When a valid calibration has been performed and the control values are within the valid range, serum or plasma samples may be measured. Check that minimum volume of sample is present and assay the samples according to the instructions given in the Instrument Manuals. Results The results are calculated automatically by the Cobas c50 instrument. The results are presented in mg/l. Performance Characteristics All results refer to the validation of Gentian Cystatin C Immunoassay on Cobas c50. Detection Limit Limit of Quantification is defined as: The lowest actual amount of an analyte that can be reliably detected and at which the total error meets the requirements for accuracy. Gentian Cystatin C Immunoassay has a Limit of Quantitation of 0.30 mg/l. Precision The Gentian Cystatin C Immunoassay was used in a 5-day precision study designed in accordance with CLSI protocol EP5-A. 4 human serum pools and 2 control levels were measured on the Cobas c50. Sample Serunm pool Mean value (mg/l) Within-Run CV (%) Total CV (%) Low High Medium Medium Low Control High Control Linearity Gentian Cystatin C Immunoassay on Cobas c50 is linear in the range of mg/l. Concentrations outside this range have not been tested. Interference No interference is detected with Triglycerides (2.7 mmol/l) Haemoglobin (8.0 g/l), Intralipid (4 g/l) or Bilirubin (600 mg/l) on Cobas c50. There is no interference detected with the drugs tested on recommendation from Sonntag and Scholer []. The interference study was designed in accordance with the protocol EP7-A from CLSI [2]. There is no RF interference present in the Gentian Cystatin C Immunoassay because the antibodies are made from avian (chicken) [3]. Comparison between applications Results from Passing-Bablok regression comparison between ) The first generation Gentian Cystatin C Immunoassay on Cobas c50 and the Gentian Cystatin C Immunoassay on Hitachi 97, and 2) between The second (optimised) and first generation Gentian Cystatin C Immunoassay on Cobas c50. 2 Methods Cobas st gen vs Hitachi 97 Cobas 2 nd vs st gen N Range specimen (mg/l) Term Intercept Slope Intercept Slope Coefficie nt % CI of Coefficient Shipping Damage Please notify your distributor if this product is received damaged. Symbols Key Lot number Temperature limitations In Vitro Diagnostic Medical Device Expiration date Consult Instructions for Use Manufacturer Catalogue Number References [] Sonntag O, Scholer A: Ann Clin BIochem 200; 38: [2] CLSI; Document EP7-A; Interference Testing in Clinical Chem.; Approved Guideline [3] Larsson A et al: Poultry Science 993;72: [4] Flodin M et al: Scand J Clin Lab Invest 2007;67: Manufacturer: Gentian AS, PO BOX 733, N-509 Moss, Norway EC REP Authorized European Representative in EU: Gentian AS, PO BOX 733, N-509 Moss, Norway TEL: FAX: Application Note for Gentian Cystatin C Immunoassay on Cobas c50, Roche v06-september 0
3 Page 3 of 6 Instrument Settings for Gentian Cystatin C Immunoassay on For in vitro diagnostic use only Cobas c50 (Optimised Volumes II), Roche Diagnostics ANALYSE Assay/Time/Point 2 point end Wavelength (2 nd /primary) N/A 546 S.vol (Normal) S.vol (Decrease) N/A 0 0 S.vol (Increase) N/A 0 0 Diluent: Saline Reagent (R) T x 0 Reagent (R2) T x 0 Reagent (R3) T x 0 Reagent (R4) T x 0 Abs. Limit Increase Prozone Limit Cell Detergent Detergent Application Note for Gentian Cystatin C Immunoassay on Cobas c50, Roche v06-september 0
4 Page 4 of 6 CALIBRATION Calibration Type Spline Point 6 Span Point 4 Weight 0 Auto calibration: Time out Change over Blank: 0 Lot 0 Span: 0 Bottle 0 2Point 0 Full 0 SD Limit 999 Duplicate Limit 99% Abs Sensitivity Limit S Abs Limit Application Note for Gentian Cystatin C Immunoassay on Cobas c50, Roche v06-september 0
5 Page 5 of 6 RANGE Application Code: * Unit: mg/l Report name: CysC Data Mode: On Board Control Interval: 0 Instrument Factor (Y=ax + b):.0 0 Technical Limit: Repeat Limit: Expected Values: (Male) 00 Y Y (Female) 00 Y Y (Default) Male Range Application Note for Gentian Cystatin C Immunoassay on Cobas c50, Roche v06-september 0
6 Page 6 of 6 OTHERS Standard: Calib Code 0* 0* 0* 0* 0* 0* Concentration *CV *CV 0.46*CV 0.348*CV 0.62*CV.0*CV Position 0** 0** 0** 0** 0** 0** CV: assigned Calibrator Value, see analytical value sheet enclosed with the calibrator * User defined ** Calibrator Position chosen by user Sample Volume: Diluted S.Volume: Diluent Volume: Application Note for Gentian Cystatin C Immunoassay on Cobas c50, Roche v06-september 0
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