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Instructions for Use suPARnostic® TurbiLatex Reagents INTERPRETATION OF suPAR suPAR level Clinical decision soluble urokinase Plasminogen Activator Receptor <3 ng/mL Supports the decision of discharge. T005 The underlying health condition is good, and the prognosis for surviving is very high. suPARnostic® and the ViroGates A/S ViroGates logo are Banevaenget 13 3-6 ng/mL Some disease activity or co-morbidity is present. registered trademarks of Birkeroed 3460, Some readmissions and an elevated mortality are ViroGates A/S Denmark. Denmark expected after 6 months follow-up. ©2008 ViroGates. Tel: +45 2113 1336 All rights reserved. www.virogates.com >6 ng/mL Attention is needed. High risk of mortality.
Further tests are needed to find the presence of severe disease. Table 1: Simplified suPAR clinical decision scheme (data from Danish hospital)3.
Interpretation of results must be made considering the patient´s clinical history and results of other diagnostic tests. The decision to discharge This product is protected by one or more US, European, and/or foreign can, therefore, not be performed solely based on the patients suPAR patents. value.
The T005 product is validated on the automated Roche Cobas c701/2 PRINCIPLES OF ASSAY PROCEDURE ® instrument (Cobas is a trademark of Roche), and this instruction for use The suPARnostic® TurbiLatex test is a turbidimetric immunoassay is dedicated for the Roche cobas c701/c702 biochemistry analyser. that quantitatively determines suPAR in human plasma samples. The first stage of testing is an incubation of human origin specimen (EDTA- Refer to the webpage http://www.virogates.com for instructions for use or Heparin plasma) with the R1 reagent. After 5 minutes of incubation, for other biochemistry analysers and other languages. Alternatively, the R2 reagent is added, and the reaction starts. The reaction buffer R2 contact your local distributor for instructions in your language. is a suspension of latex particles coated with rat and mouse monoclonal antibodies to suPAR. After the R2 addition the process of suPAR INTENDED PURPOSE aggregation begins, the level of accumulation is determined by the For Professional Laboratory Use amount of scattered light during measurement of light absorption. The ® The suPARnostic TurbiLatex Reagents are used for automated linear calibration curve, created before the start of the test, is used to determination of soluble urokinase Plasminogen Activator Receptor determine the concentration of suPAR in human plasma samples. (suPAR) in human EDTA- or Heparin plasma in ng/mL on Roche cobas c701/2 analyzers. REAGENTS AND MATERIALS suPARnostic® is a prognostic biomarker of chronic inflammation and Reagents Provided; immune activation across diseases. suPARnostic® can be used for risk stratification of acute medical patients. • Reagent 1 – Dilution Buffer. • Reagent 2 – Latex Particle Reagent. suPAR IS A MARKER OF DISEASE PROGRESSION • Instruction for use. suPAR is the soluble form of urokinase Plasminogen Activator Receptor (uPAR). The amount of suPAR is a measure of immune activation and This kit contains ready-to-use R1 Dilution Buffer and R2 Solution of inflammation.1 suPAR is a biomarker which is increased by the presence Latex particles coated with anti-suPAR antibodies. The volume is and severity of disease. suPAR has a high negative predictive value for enough for a full cassette for the c701/2. ruling out disease progression. This means that patients with a low (<3 ng/mL) suPAR level have a good prognosis and a low risk of re- Product/Buffer R1 in mL R2 in mL No. tests admission and mortality3, supporting the decision of discharge of the T005 (c701/2) 75 28 420* patient. A high suPAR level (< 6 ng/ml) is a strong measure of chronic *no. of tests refer to the actual number of tests in the bottle, including dead volume inflammation and the underlying risk of negative outcomes, including but excluding 12 tests for calibration, when all reagent is transferred to the empty 2 short term mortality (in hospital, 30 days, or 90 days) supporting the cassette. decision of further examination of the patient. Material required but not provided The use of suPAR in clinical routine adds significant additional • suPARnostic® TurbiLatex Calibrators T002 knowledge to the standard assessment based on early warning score • suPARnostic® TurbiLatex Controls T003 systems and standard parameters in pre-admission of acute medical • Clinical chemistry analyser patients. Hence, suPAR is broadly applicable, e.g. in the Emergency • Adjustable pipette with tips, 20 200 Department, especially in relation to discharge decisions of patients, as μL – μL well as for identifying non-diagnosed inflammatory disease. • Disposable gloves A cluster-randomised interventional study showed that up- or down- triaging of patients based on suPAR levels increased the number of NUMBER OF DETERMINATIONS patients to be discharged (low risk) with 34%4 and reduced the number 1 mL of R2 reagent and 3 mL of R1 reagent is sufficient for 20 tests of hospital bed days5. when measured on Roche Cobas® c701/2 analyser. This does not include dead volume in the bottles. EXPECTED VALUES All individuals have a measurable suPAR level, and in healthy blood LOADING OF REAGENTS donors (N=9305) the median suPAR level for men age 18-65 years old The Reagents shall be transferred to the appropriate cassettes from is 2.22 ng/mL (25-75% interval from 1.76-2.90 ng/mL)6, for women 18- 65 years old is 2.56 ng/mL (25-75% interval from 2.05-3.23 ng/mL)6, Roche before they can be used on the analyser. patients attending Emergency Departments around 3-6 ng/mL2,3,7, and R1 reagent is transferred to position B and R2 into position C. For more in patients with severe disease and organ failure, suPAR is often in the information refer to the Roche Cobas c MULTI pack IFU. double-digits8, 9. The higher the level, the higher the risk of disease progression and the worse the prognosis. Note: suPARnostic® TurbiLatex Reagent 2 is on the Roche Cobas platform identified as R3.
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STORAGE AND HANDLING • Do not mix plasma samples from different patients or different Store at 2-8°C. blood samplings of the same patient. • Human samples may be contaminated with infectious agents. Do The suPARnostic Reagents is produced with 12 month shelf life from not ingest, expose to open wounds, or breathe aerosols. Wear protective gloves and dispose of biological samples properly. production date. Before use, check the expiry date on the label. • Be aware of possible dilution of suPAR in the case of a transfusion, The reagents have 8 weeks of onboard stability when kept at 2-8°C and infusion, or similar. monthly calibration performed. If not stored correctly the stability of the reagent may be affected and PERFORMANCE CHARACTERISTICS might not be efficient and misleading results may be obtained. Results presented below were obtained with the use of suPARnostic® TurbiLatex Reagents on the Roche Cobas® c702 analyser. The data SPECIMEN COLLECTION AND HANDLING shown is only valid for Roche Cobas c701/c702. Sample Type Sample Requirement Plasma sample 10 µL RESULTS Results are calculated by linear regression. If a different calculation SAMPLE COLLECTION AND STORAGE method than linear regression is used, the method must be validated. Collecting blood samples should be performed by trained and qualified Control the curve fitting by using suPARnostic® TurbiLatex Controls and staff in antiseptic conditions using approved venepunctures techniques. undertake corrective actions if results exceed the upper and lower limit.
To prepare plasma samples, whole blood is drawn into a blood collection tube containing EDTA or Heparin anticoagulant. Centrifuge the blood at MEASURING RANGE 3,000 x g between 1 - 10 minutes until blood cells and plasma have The measuring range of the suPARnostic® TurbiLatex test is 1.8 ng/mL separated. to 16.0 ng/mL on a Cobas® c701/c702 analyser. It is not recommended to dilute samples above measuring range. Do not use hemolysed, contaminated, or hyper-lipemic sample specimens. ANALYTICAL LIMITS
SAMPLE STABILITY LIMIT OF BLANK (LOB) It is preferred to analyse the samples as soon as possible, but EDTA- Three human plasma samples, diluted in 1:16 factor with water, were and Heparin- plasma samples are stable for: measured 20 times. In total, 60 blank measurements for 1 day.
• 24 hours at room temperature (20-25°C). LIMIT OF DETECTION (LOD) • 3 days at 2-8°C. Five low concentration samples were tested in 12 replicates for 2 days. • -20°C for longer storage.
Five freeze/thaw cycles over 5 days do not have an impact on suPAR The LOD for suPAR was determined in accordance with the guidelines concentration in a sample. of CLSI (Clinical and Laboratory Standard Institute)10 protocol and with
proportions of false positives (α) less than 5% and false negatives (β) CALIBRATION less than 5%; based on 120 determinations, with 60 blank and 60 low- Together with suPARnostic® TurbiLatex Reagents, the suPARnostic® level samples. TurbiLatex Calibrators T002 should be used for calibration. The laboratory shall work out their frequency of calibration, but it is LIMIT OF QUANTIFICATION (LOQ), LOQ=LOD recommended to repeat the calibration at least once a month, and it is Acceptable Total Error = 30% to suPARnostic® ELISA. required to recalibrate when a new batch of TurbiLatex Reagents are LOB LOD LOQ used. Result 1.0 ng/mL 1.3 ng/mL 1.3 ng/mL
Limit Result REAGENT PRECAUTIONS AND RECOMMENDATIONS No interference was observed below the following concentrations For professional laboratory use. • Do not use kit components beyond the indicated kit expiration date. Substance Concentration • Do not switch caps on reagent containers as it may cause Bilirubin 350 µmol/L contamination or mix-up. Haemoglobin 1.4 g/L • Do not mix reagents from different kit lots. Triglycerides 3.3 g/L • Do not freeze any of the kit components. The interference studies were performed using a modified CLSI EP7- • Do not mouth pipette or ingest any of the reagents. A2 protocol. 10 • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled. PI 175 Edition 1.0 June 2020 3 Other interference studies were performed, and no interference was Bias and correlation calculations toward suPARnostic® ELISA was observed below the following concentrations: carried out to estimate the TurbiLatex ability to quantify suPAR in patient samples. 103 samples were measured with one batch of Turbidimetry Substance Concentration reagents, results were compared to suPARnostic® ELISA results. Rheumatoid Factor >440 IU/mL HAMA Titer >640* Results: Sample No. of Slope y-Intercept Pearson Range *>640 times more activity than a known negative. These tests were Type pairs correl. value performed on Roche Cobas® c111. 1.8-15.7 Plasma 103 1.0957 -0.1036 0.959 ng/mL Rheumatoid Factor and HAMA solutions were prepared by adding X = suPARnostic® ELISA Y = suPARnostic® TurbiLatex concentrated rheumatoid and HAMA solutions to human plasma pools. ASSAY VARIATION The suPARnostic® TurbiLatex is calibrated against an internal control In very rare cases, gammopathy, especially type IgM (Waldenström's verified with suPARnostic® ELISA. The maximum allowed variation macroglobulinemia), may produce inaccurate results. Patients with a between suPARnostic® ELISA and TurbiLatex is 15% and between lots confirmed diagnosis of anti-TPO or other autoimmune-related diseases of TurbiLatex it is 10%. have shown to interfere in a few cases. WASTE HANDLING Although precautions have been taken to minimise interference caused Discard unused reagents and waste in accordance with country, federal, by heterophilic antibodies, erroneous results can be observed. state, and local regulations. Therefore, any suPAR value above 10 ng/mL should be carefully investigated, and unusually high results, e.g. above 20 ng/mL may be false-positive results caused by interference. Catalogue no. Contains sufficient for Use by For diagnostic purposes, the results should always be compared to the LINEARITY In vitro diagnostic Temperature Limits LOT no. (Batch The suPARnostic® TurbiLatex test is linear from 1.8 ng/mL to 26.5 medical device No.) ng/mL. REFERENCES HOOK EFFECT 1. Desmedt S, et al. The intriguing role of soluble urokinase receptor in The suPARnostic® TurbiLatex showed no prozone effect in inflammatory diseases. Crit Rev Clin Lab Sci. 2017 Mar;54(2):117-133 2. Rasmussen LJH, et al. Combining National Early Warning Score with concentrations below 47.5 ng/mL (this was the highest tested suPAR Soluble Urokinase Plasminogen Activator Receptor (suPAR) Improves Risk concentration). Prediction in Acute Medical Patients: A Registry-Based Cohort Study. Crit Care Med. 2018 Dec;46:1961-8. 3. Rasmussen LJ, et al. Soluble urokinase plasminogen activator receptor PRECISION PERFORMANCE (suPAR) in acute care: a strong marker of disease presence and severity, Low, medium and high samples were measured with two replicates in re-admission and mortality. A retrospective cohort study. Emerg Med J. 2016 two separate runs per day for 20 days. Nov;33:769-75. 4. Schultz et al. Availability of suPAR in emergency departments may improve risk stratification: a secondary analysis of the TRIAGE III trial Scandinavian Factors which were changed during Intermediate precision study: Journal of Trauma, Resuscitation and Emergency Medicine BMC (2019) · Operators (3 different) 27:43 · Reagent Lots (3 batches) 5. Schultz, M. Early Discharge from the Emergency Department Based on · Calibrations (with 2 different Calibrator batches) Soluble Urokinase Plasminogen Activator Receptor (suPAR) Levels: A · Days (20) TRIAGE III Substudy, Hindawi, Disease Markers, Volume 2019 6. Haastrup E, Grau K, Eugen-Olsen J, Thorball C, Kessing LV, Ullum H: Soluble urokinase plasminogen activator receptor as a marker for use of Inter- Mean Repeatability Between Between Within- antidepressants. PLoS One 2014, e110555. mediate ng/mL CV runs CV Day CV Device 7. Raggam RB, Wagner J, Pruller F, Grisold A, Leitner E, Zollner-Schwetz I, et Precision CV al: Soluble urokinase plasminogen activator receptor predicts mortality in Low 3.7 3.5% 1.0% 10.8% 11.4% patients with systemic inflammatory response syndrome. J Intern Med 2014, Medium 6.9 3.5% 2.4% 4.6% 5.8% 276(6):651-8 High 9.9 4.1% 1.6% 4.5% 6.1% 8. Koch A, Zimmermann HW, Gassler N, Jochum C, Weiskirchen R, Bruensing 4 Replicates pr. day, for 20 days. J, et al.: Clinical relevance and cellular source of elevated soluble urokinase plasminogen activator receptor (suPAR) in acute liver failure. Liver Int ACCURACY (METHOD COMPARISON) 2014;34:1330–1339. The suPARnostic® TurbiLatex is calibrated against an internal control 9. Donadello K, Scolletta S, Taccone FS, Covajes C, Santono-cito C, Cortes DO, et al: Soluble urokinase-type plasminogen activator receptor as a verified with suPARnostic® ELISA. The maximum allowed variation prognostic biomarker in critically ill patients. J Crit Care. 2014 Feb;29(1):144- between suPARnostic® ELISA and TurbiLatex is 15% and between lots 9. of TurbiLatex it is 10%. 10. Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline. EP17-A, Vol. 24 No. 34 Replaces EP17-P, Vol. 24 No. 10. https://www.clsi.org/ PI 175 Edition 1.0 June 2020 APPLICATION PARAMETERS Analyse Calib. Range Other Assay/Time/Point 2Point End 10 22 37 0 0 Wavelength (2./Prl.) 800 570 Sample volume Cassette configuration Norm. 10.0 0.0 0 code ####### Dec. 10.0 0.0 0 Expiration Days 60 inc. 10.0 0.0 0 Reagent volume Dilution R1 150 0 Inactive Water R2 0 0 Inactive Diluent R3 50 0 Inactive Linearity Limit % % Prozone-Limit -32000 32000 Inside 0 0 Abs. Limit 32000 Increase Cell Detergent Stirring Level 4 Stirring Setting M1 M2 M3 UP Stirring LOW Stilling Stirring Stirring Analyse Calib. Range Other Calibration type Linear Auto-calibration Timeout cassette Point 6 Span 3 cassette Active Weight 0 30 Day Changeover Update Type Ingen 0 0 cassette Cancel QC-Violation SD-Limit 999 Method Blank Rule 1s Duplicate Limit 99 % 32000 Abs. Control1 None Control2 None Sensitivity Limit -99999 99999 Control3 None S1 Abs. Limit -32000 32000 Auto masking AN 205 Edition 1.1 June 2019 Analyse Calib. Range Other Applications code ### Expected Values Unit ng/ml Male Report Name suPAR -99999 999999 Data mode Active 99 Year -99999 999999 Automatic Rerun 100 Year -99999 999999 Technical Limit 1.8 16.0 Repeat Limit -9999 99999 Female Control interval Time 0 -99999 999999 Automatic QC on-board-stability 1 99 Year -99999 999999 100 Year -99999 999999 Quality (1) 0 L 0 Default (2) 0 H 0 Sex (3) 0 I 0 Male Female (4) 0 Interval (5) 0 Range 1 Range 2 Range 3 (6) Analyse Calib. Range Other Standards (1) (2) (3) (4) (5) (6) Calibrator code *1 *1 *1 *1 *1 *1 Concentra- *2 *2 *2 *2 *2 *2 Rack-nr. pos. *3 *3 *3 *3 *3 *3 Sample Volume 10.0 10.0 10.0 10.0 10.0 10.0 Diluted Sample volume 0.0 0.0 0.0 0.0 0.0 0.0 Diluent Volume 0 0 0 0 0 0 Cassette type for cobas® 6000 (c701) Cassette type for cobas® 8000 (c702) Type A Type A Bottle Bottle a N/A N/A N/A a N/A N/A N/A b R1 434 75 b R1 434 75 c R3 434 28 c R3 434 28 *1: User defined value *2: Batch-specific concentrations - see CoA *3: User defined value AN 205 Edition 1.1 June 2019