APPY1™Test Kit
Contents
1. Sample Parameters 1 2 3 4 5 APPY1 Test Score Mean 3 2 3 9 4 0 4 2 54 Repeatability within run van 2o14 2 012 oog ose a 6e Betweentin SD 0 004 0 010 0 019 0 017 0 049 CV 0 1 0 3 0 5 0 4 0 9 Bet d SD 0 005 0 014 0 010 0 012 0 036 SAMERNE OY CV 0 1 04 0 2 03 07 SD 0 003 0 005 0 011 0 010 0 030 Between operator CV 01 0 1 03 03 05 mee SD 0 015 0 026 0 054 0 054 0 182 Reproducibility total a 05 07 13 13 34 Reproducibility Testing MRP 8 14 Concentration Sample Parameters 1 5 3 7 5 MRP 8 14 pg mL Mean 0 10 0 15 0 25 0 34 0 59 i PP SD 0 003 0 010 0 029 0 026 0 656 Repeatability within run CV 29 66 115 77 11 Beiwearvun SD 0 002 0 005 0 009 0 008 0 013 CV 1 9 3 1 3 5 2 3 2 3 Betwe n da SD 0 003 0 007 0 006 0 007 0 017 y CV 2 6 4 7 24 2 0 3 0 SD 0 010 0 002 0 006 0 006 0 015 Between operator CV 9 6 1 6 2 5 1 7 2 5 PEN SD 0 009 0 013 0 031 0 029 0 071 Reproducibility total CV 86 88 12 6 34 120 Reproducibility Testing CRP Concentration Sample Parameters 1 z 3 J 5 CRP pg mL Mean 0 63 2 52 5 55 5 94 24 34 id Poe SD 0 039 0 166 0 309 0 568 3 375 Repeatability within run a 61 6 6 56 06 13 9 Bardecn tur SD 0 016 0 130 0 281 0 228 1 226 CV 2 5 5 2 5 1 3 8 5 0 Between da SD 0 012 0 116 0 065 0 132 0 743 y CV 19 46 1 2 22 31 SD 0 009 0 048 0 099 0 121 0 902 Between operator CV 14 1 9 1 8 2 0 3 7 AT SD 0 044 0 245 0 434 0 638 3 776 Rep2. Specificity 46 2 41 0 51 6 NPV 96 3 92 1 98 3 PPV 41 3 35 9 46 9 Positive Likelihood Ratio 1 78 1 60 1 99 Negative Likelihood Ratio 0 098 045 217 Analytical Performance Limit of Detection The Limit of Blank LOB Limit of Detection LOD and Limit of Quantitation LOQ were determined by 2 operators testing 3 lots of APPY1Test Kits against 6 plasma samples and blank samples of APPY1Test Buffer over 3 days with the samples randomized each day LOD LOB and LOQ Limit CRP pg mL MRP pg mL Blank 0 030 Detection 0 045 Quantitation 0 341 0 064 Linearity For each analyte linearity was demonstrated for an interval from approximately the LOD LOQ to above the upper truncation value of the assay The assay is linear for both analytes in the range of the clinical cut off value MRP assay is linear from 0 052 to 0 933 g mL CRP assay is linear from 0 187 to 51 4 ug mL Using CRP LOD as the low value the CRP assay is linear from 0 254 g mL to 51 4 ug mL MRP 8 14 Linearity 124 E B10 y 0 9998x 0 0062 a 7 2 R 0 9959 08 4 S o6 o oa 3 S 02 3 a ee 0 0 0 2 04 0 6 0 8 1 0 Estimated pg ml APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 8 CRP Linearity R 0 9915 Measured CRP pg mL 10 10 20 30 40 50 60 Estimated pg mL
3. Precision Precision performance of the APPY1 Test was evaluated in accordance with the CLSI guideline EP 5A2 Evaluation of Precision Performance of Quantitative Measurement Methods Approved Guideline Five plasma specimens between LOQ and the cut off APPY1 Test score of 4 0 at or close to the cut off and at intervals above the cut off were tested over 20 days 2 runs per day 2 replicates per run 1 operator Samples were randomized across 5 readers on each test day To complete the APPY1 Test score calculation a fixed WBC was assigned to each specimen Total percent coefficient of variation CV ranged from 0 0 to 2 8 for the APPY1 Test score 6 5 to 12 0 for MRP 8 14 and 4 8 to 15 1 for CRP Near the established cut off CVs for MRP 8 14 and CRP were lt 10 6 Precision Testing APPY1 Test Score with Fixed WBC APPY1 Within run Between run Total Test score Sample N Mean SD CV SD CV SD CV 1 80 3 2 0 000 0 0 0 000 0 0 0 000 0 0 2 80 3 9 0 000 0 0 0 000 0 0 0 000 0 0 3 80 4 0 0 050 1 2 0 000 0 0 0 050 1 2 4 80 41 0 037 0 9 0 027 0 7 0 046 1 1 5 80 5 2 0 143 2 8 0 000 0 0 0 148 2 8 Precision Testing MRP 8 14 Concentration S l Parameters 7 7 a e 7 5 MRP 8 14 pg mL Mean 0 10 0 15 0 24 0 31 0 52 di me SD 0 006 0 009 0 024 0 016 0 058 Repeatability within run CV 63 61 100 51 T3 Betweent n SD 0 000 0 007 0 002 0 012 0 000 CV 0 0 45 0 7 3 8 0 0 Tot
4. HIV 1 NAT ALT and syphilis by FDA approved methods Because no test method can offer complete assurance that infectious agents are absent these reagents should be handled as potentially infectious human plasma The use of Universal Precautions and Blood borne Pathogen Safety Rules is recommended Each laboratory should follow their established institutional guidelines for laboratory safety practices The test buffer contains 0 095 sodium azide as a preservative In case of skin contact flush with copious amounts of water Sodium azide may react with lead and or copper plumbing to form explosive metal azides flush with excess water upon disposal All patient specimens should be handled as though capable of transmitting disease The use of Universal Precautions and Blood borne Pathogen Safety Rules is recommended Each laboratory should follow their established institutional guidelines for laboratory safety practices Specimen Collection and Storage Patient blood should be drawn through routine venipuncture or an established IV line with flush into a K2 EDTA lavender top tube containing 1 8 mg of EDTA cc of blood After blood collection centrifuge the tube for 10 minutes at 1300 x g 100 RCF at ambient room temperature Centrifugation must start within one 1 hour of specimen collection time The centrifuge must have either a fixed rotor angle greater than 45 degrees or a swinging bucket rotor To calculate the conversion of RPM to R
5. Imaging for Pediatric Appendicitis 2005 2009 Trends and Outcomes Journal of Pediatrics 2011 MenochM etal Trends in Computed Tomography Utilization in the Pediatric Emergency Department Pediatrics 2012 129 e690 e697 Bealer J and Colgin M S100A8 A9 A potential New diagnostic Aid for Acute Appendicitis Academic Emergency Medicine 2010 17 333 336 Mills A et al Diagnostic Characteristics of S100A8 A9 in a Multicenter Study with Patients With Acute Right Lower Quadrant Abdominal Pain Academic Emergency Medicine 2012 19 48 55 Kharbanda A et al Novel Serum and Urine Markers for Pediatric Appendicitis Academic Emergency Medicine 2012 19 56 62 Thuijl G et al A Pilot study on potential new plasma markers for diagnosis of acute appendicitis 2010 Shindoh J et al Diagnostic Power of Inflammatory Markers in Predicting Severity of Appendicitis Hepato Gastroenterology 2011 58 2003 2006 Cole M Maldonado N Evidence Based Management of Suspected Appendicitis in the Emergency Department Emergency Medicine Practice 2011 13 1 32 Siddique K et al Diagnostic accuracy of white cell count and C reactive protein for assessing the severity of paediatric appendicitis Journal of the Royal Society of Medicine 2011 2 59 Doraiswamy NW Progress of acute appendicitis a study in children British Journal of Surgery 1978 65 877 9 Symbol Key Symbolschliissel Leyenda de los s mbolos L gende des symboles Descrizione dei simboli Ver
6. United States American Journal of Epidemiology 1990 132 910 25 Becker et al Atypical Clinical Features of Pediatric Appendicitis Academic Emergency Medicine 2007 14 124 129 Brennan G Pediatric Appendicitis Pathophysiology and appropriate use of diagnostic imaging Canadian Journal of Emergency Medicine 2006 8 425 432 Kharbanda et al A Clinical Decision Rule to Identify Children at Low Risk for Appendicitis Pediatrics 2005 116 709 716 Huckins DS et al A novel biomarker panel to rule out acute appendicitis in pediatric patients with abdominal pain American Journal of Emergency Medicine in press http dx doi org 10 1016 j ajem 2013 06 016 Cardall et al Clinical Value of the Total White Blood Cell Count and Temperature in the Evaluation of Patients with Suspected Appendicitis Academic Emergency Medicine 2004 11 1021 1027 Hennelly K and Bachur R Appendicitis Update Current Opinion Pediatrics 2011 23 1 5 Wan M et al Acute Appendicitis in Young Children Cost Effectiveness of US versus CT in Diagnosis A Markov Decision Analytical Model Radiology 2009 250 378 386 Glatter R What role do imaging studies play in diagnosing pediatric appendicitis in the ED MedScape Emergency Medicine Web MD June 18 2010 Available at http www medscape com viewarticle 723549 Accessed April 9 2012 Stoker J et al Imaging patients with acute abdominal pain Radiology 2009 253 31 46 Bachur R et al Advanced Radiologic
7. order to proceed with testing patient samples APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 6 APPY1 Control Kit Liquid quality controls are available as a procedural control for the APPY1 Test The established ranges for each control are provided with each kit lot Follow the Instructions for Use that come with each kit to run the controls as necessary Controls should be tested Atleast once a month With each new lot and new shipment of APPY1 Test Kits If there is any question regarding system integrity reagent storage conditions or the reliability of any test result In accordance with national country or local guidelines In accordance with your laboratory s QC procedures Limitations 1 A Negative Low Risk for Appendicitis APPY1 Test result does not preclude a diagnosis of acute appendicitis In the early stages of the acute phase response lt 24 hours from onset of symptoms or in focal appendicitis plasma levels of MRP 8 14 and CRP may not have reached clinically significant levels resulting in a false negative test result 2 AnInconclusive for Appendicitis APPY1 Test result should not be used as a diagnostic test for acute appendicitis or interpreted as positive as other inflammatory disorders or infectious disease may demonstrate increased plasma levels of MRP 8 14 and CRP and elevated WBC count 3 The APPY1 Test result is based on a score calculated by a p
8. will remain locked Contact Venaxis Technical Support at 1 303 794 2000 or your local distributor for assistance an Testing Patient Samples Prepare APPY1 Test Cassette 1 2 3 4 Ts Log in as an operator or change to operator mode Press to display the operator Main Menu screen Use and Y to highlight Run Test and press Select Enter the patient ID using one of the following methods Manually use the numeric keypad to enter the patient ID a Press Keypad to enter letters spaces and punctuation b When the keypad is displayed on the reader s screen use a v i 4 and gt to highlight the desired character c Press Confirm amp Next to add the highlighted character to the patient ID d Repeat Steps a and bas necessary e When the patient ID is complete press Back or select Done to exit the Keypad feature Using the optional external barcode reader scan the patient s barcode On the Enter Patient ID screen press Confirm If the patient WBC result is available enter it and press Confirm Otherwise press Skip If you press Skip you will be asked to enter the patient WBC after the test Remove test cassette from pouch and place it into the cassette drawer APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 5 Prepare Patient Plasma Specimen for Testing 1 Set up test buffer and test conjugate vials 2 Use anew disposable pipette tip for each transfer 3 Remo
9. APPY1 Test Kit English Intended Use The APPY1 Test is a qualitative In Vitro Diagnostic Multivariate Index Assay IVDMIA used to aid in the identification of patients at low risk for acute appendicitis The APPY1 Test measures the concentrations of the myeloid related protein heterocomplex MRP 8 14 calprotectin and C reactive protein CRP in EDTA plasma by lateral flow immunoassay The quantitative concentrations of MRP 8 14 and CRP in addition to an independently determined white blood cell WBC count are then computed by a preprogrammed proprietary algorithm to generate an APPY1 Test result Indications for Use The APPY1 Test is indicated for use in pediatric adolescent and young adult patients ages 2 through 20 years old with abdominal pain and other physical and clinical signs and symptoms suggestive of acute appendicitis For Professional Use Only Summary and Explanation Diagnosing patients presenting with abdominal pain remains one of the most common and challenging issues in emergency medicine Over 22 2 million patients visited European and U S hospital emergency departments ED in 2010 with the primary complaint of abdominal pain Appendicitis has the highest incidence in pediatric adolescent and young adult patients 10 19 years of age but the diagnosis of appendicitis in these patients remains challenging because children present with a wide variety of atypical clinical features Up to 50 of infants and young c
10. CF refer to the user manual for the centrifuge model or contact Venaxis Technical Support Test Procedure Reader Setup Follow reader setup as described in the APPYReader User Manual and or Start Up Guide Log into the reader using your assigned Operator ID Allow the reader to warm up for at least 10 minutes before running any patient samples Prepare QC and Test Reagents If performing liquid QC remove required number of Control 1 and Control 2 vials from the APPY1 Control Kit stored at lt 20 C Thaw at ambient room temperature while reader is warming up See APPY1 Control Kit Instructions For Use for more information Remove sufficient test cassettes test buffer vials and test conjugate vials from an APPY1 Test Kit stored at 2 8 C necessary to complete all testing QC and patient samples Allow components to reach ambient room temperature while reader is warming up APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 4 Run the QC Cassette Perform each day of patient testing and or before running Controls 1 and 2 Patient samples cannot be tested unless the QC cassette has a valid PASS result within the prior 24 hours IMPORTANT To maintain the expected useful life of the QC cassette avoid light exposure Store and reseal the QC cassette in its original foil pouch immediately after each use Do not discard the QC cassette until expiration date See APPYReader QC Casse
11. al SD 0 006 0 011 0 025 0 021 0 062 CV 6 5 7 1 10 6 6 9 12 0 Precision Testing CRP Concentration Sample Parameters 1 2 3 4 5 CRP pg mL Mean 0 58 2 31 5 06 5 25 18 88 u Si SD 0 027 0 126 0 341 0 364 2 953 Repeatability within run CV 46 55 67 69 156 Beiweantun SD 0 013 0 097 0 280 0 262 0 000 CV 2 3 4 2 5 5 5 0 0 0 Total SD 0 028 0 149 0 453 0 453 2 851 CV 48 6 5 8 6 8 6 14 APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 9 Reproducibility Reproducibility was evaluated in Venaxis laboratory by 3 operators each with a dedicated reader blinded to the other operators results An operator reader combination was designated as a separate site for a total of 3 sites Each operator tested 5 plasma specimens plus 2 controls for 3 days 2 runs per day 2 replicates per run The sample order was varied in each test run To complete the APPY1 Test score calculation a fixed WBC was assigned to each specimen For APPY1 Test scores the CV for within run repeatability was lt 3 4 the CV for between run was lt 0 9 the CV for between day was lt 0 7 and the CV between operator site was lt 0 5 Total imprecision reproducibility was lt 3 4 The results for APPY1 Test score MRP 8 14 and CRP are shown below Reproducibility Testing APPY1 Test Score with Fixed WBC
12. hildren with AA may present with atypical signs and symptoms gt which can delay diagnosis and lead to life threatening complications such as perforated appendix abdominal abscess peritonitis sepsis and shock Therefore it is important to identify diagnostic tools which aid in an objective timely and accurate diagnosis and risk stratification of patients with suspected acute appendicitis AA At present the ability to accurately diagnose appendicitis is limited to a collection of physical and clinical signs and symptoms and various diagnostic modalities Multiple clinical prediction tools using classic symptoms as data points have been developed to aid in the diagnosis of appendicitis The performance of these scoring systems is inconsistent and highly variable Moreover these systems are not utilized routinely by emergency departments A WBC count is routinely performed but has demonstrated limited utility as a single marker even when it is combined with signs and symptoms suggestive of AA 2 Depending on the physician s initial patient risk assessment diagnostic imaging including abdominal ultrasound US or computed tomography CT with or without contrast may be ordered in cases where the clinical presentation is equivocal and more information is needed While CT is accurate in the diagnosis of acute appendicitis when the appendix can be clearly visualized it carries significant risk from increased exposure to ionizing radiation thereb
13. klaring van symbolen Symbol EN Used for CE Mark Contents Symbol DE Verwendet fiir CE Zeichen Inhalt Simbolo ES Significa C Marca CE Contenido Symbole FR Utilis pour Marque CE Contenu Simbolo IT Spiegazione Marcatura CE Contenuto Symbool NL Gebruikt voor CE markering Inhoud Molenstraat 15 Castle Rock CO 80104 USA 2513 BH The Hague Venaxis Inc Emergo Europe 1585 South Perry Street www venaxis com The Netherlands Tel 1 303 794 2000 The APPY1 Test Kit is for U S Export Only Fax 1 303 798 8332 Venaxis is a registered trademark of Venaxis Inc APPY1 and APPYReader are trademarks of Venaxis Inc APPY1 Test Instructions for Use L10003 04 2014 Venaxis Inc Page 67
14. low levels of CRP were detected in this sample by immunoblot independent of the test pools If a cleaner source of RF is tested the difference shown below will be reduced A similar result was obtained with all levels of MRP 8 14 when testing the IgG interferent It was shown that the purified IgG is contaminated with MRP 8 14 and that if a cleaner source material is obtained the interference effects shown below should also be reduced Interference Testing APPY1Test Low Pool APPY1 Latin High Pool APPY1 Potential Interferent Concentration Test Score 3 1 3 6 Test Score 4 6 Difference from Control Sample or NSD Hemoglobin 2 g L 0 69 0 79 0 08 Bilirubin unconjugated 342 umol L 1 37 0 56 1 78 Bilirubin conjugated 342 umol L 0 28 1 52 1 80 Lecithin 11 1 mmol L 0 38 0 09 0 17 HAMA 153 3 ng mL 0 24 0 93 2 18 Rheumatoid Factor 60 IU mL 0 06 0 42 1 99 IgG 52 8 mg mL 5 55 7 06 4 30 APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 11 REFERENCES 1 2 12 13 14 15 17 18 19 20 21 22 EU Market Study by Venaxis Inc Benelux France Italy Germany UK 2012 Venaxis analysis of data from National Hospital Ambulatory Medical Care Survey 2010 Available at http www cdc gov nchs ahcd ahcd_questionnaires htm public_use Accessed November 2012 Addiss D et al The Epidemiology of Appendicitis and Appendectomy in the
15. n CRP is an acute phase protein that is elevated in various diseases and conditions associated with nonspecific acute inflammation Elevated concentrations of CRP in peripheral blood have been correlated with acute appendicitis 2 Analytical performance characteristics of the APPY1 Test CRP assay is consistent with that of a high sensitivity CRP assay hsCRP as described below Elevated total WBC counts are associated with acute appendicitis when symptoms are present for 24 hours or longer Leukocytosis is nonspecific for acute appendicitis as it is also seen in various other infectious and inflammatory disorders Studies suggest that a normal WBC count in conjunction with low levels of MRP 8 14 and CRP and other biomarkers decreases the probability that acute appendicitis is present Reagents and Materials APPY1 Test Kit 25 tests F10000 APPY1 Test Cassette pouched x 25 Nitrocellulose membrane striped with monoclonal antibodies specific for CRP and MRP 8 14 and an unrelated polyclonal goat anti chicken IgY as an internal control APPY1 Test Buffer 990 uL x 25 APPY1 Test Conjugate lyophilized cake x 25 Instructions for Use x 1 Materials Required but Not Provided APPYReader Kit F10008 APPYReader QC Cassette pouched x 1 REF F10004 APPY1 Control Kit 10 vials REF F10005 APPY1 Control Level 1 40 uL x 5 APPY1 Control Level 2 40 uL x 5 Centrifuge for plasma processing fixed a
16. ngle rotor 2 45 degrees or swing bucket rotor capable of achieving 1 300 x g 100 Relative Centrifugal Force RCF Calibrated micropipettes and disposable micropipette tips capable of dispensing 10 uL 70 uL and 150 uL Vortex mixer Mini centrifuge for pulse spins of Controls if necessary Personal protective equipment and biohazardous waste disposal containers Storage and Stability Upon receipt store the APPY1 Test Kit at 2 8 C 35 6 46 4 F away from direct light The kit components should be stored in the original packaging unopened until ready to use The APPY1 Test Kit is stable until the expiration date printed on the kit box and test components when stored as recommended The APPYReader Quality Control QC Cassette is stored at 15 30 C 59 86 F in the original packaging unopened until the first use The QC cassette is reusable and should be resealed in the original pouch immediately after each use Do not discard Protect from exposure to direct light The APPY1 Control Kit is stored in its original packaging at lt 20 C 4 F Do not store in a frost free freezer Only the number of required control vials should be thawed just prior to use and then discarded after testing The APPY1 Control 1 and Control 2 vials should not be refrozen after thawing APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 3 Precautions General Precautions For professional use
17. only ach test cassette is intended for single use with one patient specimen Test cassettes cannot be reused Do not mix components from different test kit lots as components are matched within each kit lot for optimum performance Donot remove test cassettes from their pouch until ready to use Do not remove test conjugate from the kit packaging until ready to use Store the APPYReader QC Cassette in its original light protective resealable pouch between each use After each use return the QC cassette to the original resealable pouch Do not discard Do not use APPY1 Control vials that have been thawed and refrozen Do not puncture or damage the test cassette during handling prior to use If punctured or damaged discard and use a new test cassette Do not use kit components beyond the printed expiration date on the kit box and component labels After loading the test sample into the sample port of the test cassette close the cassette drawer immediately to initiate test run Delay may result in an invalid test result A confirmed patient WBC value must be entered within 90 minutes of initiating test run to obtain a valid result All materials that have been in contact with human blood or its derivatives should be discarded as biohazardous waste The APPY1 Control Kit contains derivatives of pooled human source plasma Each individual donor unit has been tested and found negative for HBsAg HCV HIV 1 HIV 2 HIV 1Ag
18. press Confirm to view the test result on the Result List screen 3 Ifthe reader s automatic printing feature is enabled the result is printed automatically 4 Press to eject the drawer Remove the test cassette and dispose of in biohazardous waste container Interpretation of Results APPY1 TEST RESULT INTERPRETATION Below clinical decision point for the test NEGATIVE Low Risk ForAppendicitis Patient is at low risk for acute appendicitis PRT Above clinical decision point for the test INCONCLUSIVE For Appendicitis Inconclusive risk for acute appendicitis See Limitations below Repeat test If retest still results in Invalid Result or Error Message INVALID RESULT OR ERROR MESSAGE consult the APPYReader User Manual for troubleshooting instructions or contact Technical Support Quality Control Integrated Control Features The reader software includes an initialization self check that calibrates the optical motors checks the cassette transport system checks the optics receiver system and performs an integrity check of the reader settings Failure to meet pre programmed specifications will result in an Invalid Result or Error Message External Controls APPYReader QC Cassette The APPYReader QC Cassette provides an optical and system suitability check for proper reader operation and should be run daily before running liquid QC reagents or patient specimens The QC cassette must provide passing results in
19. rescently labeled MRP 8 14 and CRP complexes bind to the corresponding capture zone antibodies on the nitrocellulose membrane The test cassette is immediately inserted into the reader which measures the concentration of each analyte present in the sample based on the fluorescence intensity of each test line capture zone The concentration of MRP 8 14 and CRP present in the sample is calculated based preprogrammed calibration curves on RFID tags embedded within each individual test cassette The concentrations of the two markers in combination with WBC values obtained from a hospital s hematology analyzer and entered into the dedicated reader are used to generate the patient s APPY1 Test result utilizing a preprogrammed APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 2 proprietary algorithm The APPY1 System also includes electronic dry and procedural liquid quality controls to ensure the integrity of the results The total time required for sample processing and testing is approximately 20 minutes MRP 8 14 also known as 100A8 A9 or calprotectin is a calcium binding protein complex present in the cytoplasm of neutrophils that is associated with nonspecific acute inflammatory conditions MRP 8 14 has been found to be differentially elevated in the appendix tissue and peripheral blood in patients with acute appendicitis 518 C reactive protein CRP is produced by the liver and is associated with inflammatio
20. roducibility total MCV 70 97 73 10 7 155 APPY1 Test Instructions for Use L 0003EN 04 English 2014 Venaxis Inc Page 10 Interference Pooled plasma samples containing low medium and high concentrations of MRP 8 14 and CRP were evaluated for interference by hemoglobin bilirubin conjugated and unconjugated lecithin human anti mouse antibodies HAMA Rheumatoid Factor RF and Immunoglobulin G IgG Control values for each plasma pool L M H were established using the mean of 3 replicate measurements The effect of each interfering substance was assessed using the mean of 3 replicate measurements on each plasma pool Each plasma pool was assigned a fixed WBC value in order to calculate the APPY1 Test score The low plasma pool was assigned a WBC value of 4 the medium pool was assigned a WBC value of 7 and the high plasma pool was assigned a WBC value of 10 giving APPY1 Test scores of 3 1 3 6 and 4 6 respectively No significant interference was observed on the APPY1 Test score for any of the interferents all differences were below 10 However there was a potential interference detected at low levels of MRP 8 14 when challenged with unconjugated bilirubin at these test levels As demonstrated in the graph below this interference has very little effect on the APPY1 Test score Additionally there was interference detected at low levels of CRP when comparing the RF positive plasma sample with its control However
21. roprietary algorithm using multivariate inputs The score is then compared to a pre determined clinical decision point to arrive at the APPY1 Test result 4 MRP 8 14 CRP and WBC are up regulated nonspecifically in inflammatory conditions such as acute infections acute tissue injury and other inflammatory disorders Therefore a diagnosis of acute appendicitis should be made in the context of patient history and physical and clinical findings 5 Intra individual variations in CRP levels may range from 30 60 Variability in MRP 8 14 levels is also well characterized 6 Errors in specimen processing may result in false negative or false positive results Expected Results The algorithm and the clinical decision point cut off value were derived from a pilot clinical study of 503 patients Based on Receiver Operating Curve ROC analysis and clinical agreement with discharge diagnosis the clinical decision point for the APPY1 Test was established Patient test results below the cut off are considered NEGATIVE Low Risk for Acute Appendicitis and results above the cut off are considered INCONCLUSIVE Risk for Acute Appendicitis Clinical Performance The clinical validation study of the APPY1 System was conducted on archived frozen plasma samples Samples were collected during a previous multisite prospective open label observational study of the evaluation assessment triage and disposition of patients 2 20 years old presenting to emergenc
22. tte Instructions For Use for more information 1 2 3 4 5 7 8 Log in as an operator or change to operator mode Press to display the Main Menu screen Use and to highlight Maintenance and press Select Use and Y to highlight Check APPYReader and press Select Use and Y to highlight APPYReader QC Cassette and press Select The reader drawer will open and the Insert Cassette screen will be displayed The APPYReader QC Cassette screen is displayed when the test finishes To print the QC cassette result a Press Options b Use and Y to highlight Print Result and press Select To send the result to a Laboratory Information System refer to the APPYReader User Manual Press Back to return to the Check APPYReader screen or press Oto return to the Main Menu screen Expected Values a Ifthe relative fluorescence unit RFU values detected on the QC cassette are within the acceptable ranges programmed on the QC cassette RFID chip the test result is reported as PASS b If the RFU values detected on the QC cassette are outside the acceptable ranges programmed on the QC cassette RFID chip the test result is reported as FAIL and the operator is locked out from running patient samples Repeat the test with a different QC cassette If the result of the second QC cassette is PASS proceed to testing patient samples If a different QC cassette is not available or if the result of the second QC cassette is FAIL the reader
23. ve 10 uL of patient plasma specimen from the centrifuged K2 EDTA tube being careful not to disturb the buffy coat layer Transfer to the test buffer vial Cap and mix well by vortex or manually 4 Transfer 150 uL of the diluted patient plasma sample to the test conjugate vial Mix well by pipetting up and down 8 10 times ensuring all conjugate is resuspended Avoid introducing air to the mixture which will create foam Initiate Run 1 Immediately add 70 uL of the diluted patient sample antibody conjugate mixture to the test cassette sample port 2 Gently close the drawer The test will start automatically 3 The reader displays the status of the test on the Run Test screen 4 When the test is done you will be asked to enter the WBC or to confirm the WBC entered prior to the test The test cassette must remain in the reader during this time 5 Re enter the patient WBC and press Confirm Test Result screen will then be displayed 6 Press Next to display the Confirm Patient ID screen 7 Confirm that the patient ID displayed on this screen is correct View and Print Results 1 If the patient ID is correct press Confirm to view the test result on the Result List screen If the reader s automatic printing feature is enabled the result is printed automatically 2 Ifthe patient ID is incorrect Enter the appropriate patient ID and press Confirm to display the Patient ID does not match screen Re enter the patient ID and
24. y departments with symptoms suggestive of acute appendicitis Patients enrolled in the study had blood drawn processed to plasma and then frozen within 2 hours Specimens were stored on site at lt 70 C until shipment to Venaxis where they were archived and stored at lt 70 C until testing Blinded sample testing was conducted in Venaxis laboratory using the APPY1 System The APPY1 Test results were compared to the original discharge diagnosis Negative for acute appendicitis AA or histopathology Positive for acute appendicitis AA A total of 465 patient samples collected during previous pilot clinical studies and having sufficient volume for the complete analysis were used in the validation study Subjects ranged in age from 2 20 years old mean age 12 0 4 3 years Approximately 49 were male 52 female Caucasians were the predominant race at 70 Hispanic Latino 19 and Other 11 There was no significant difference in mean age between the AA cohort 12 5 3 8 and the AA cohort 11 9 4 5 APPY1 Test Instructions for Use L10003EN 04 English 2014 Venaxis Inc Page 7 APPY1 Test Results Compared to Clinical Diagnosis APPY1 Test Result N 465 AA AA Below cut off NEGATIVE Low Risk for AA 154 6 160 Above cut off INCONCLUSIVE for AA 179 126 305 333 132 465 Clinical Performance Data Measure Estimate 95 Cl AA Prevalence 28 4 24 5 32 6 Sensitivity 95 5 90 4 97 9
25. y increasing the reliance on less harmful tests 2 Recent reports have shown that the use of CT in the pediatric population has reached a plateau however the concern over CT exposure especially in young patients has not diminished 2 The combination of three biomarkers in the APPY1 Test rather than use of any individual marker improves the ability to identify patients at low risk The APPY1 Test used in conjunction with physical and clinical findings provides additional objective information to help guide clinical decision making in the risk stratification of patients presenting with abdominal pain suggestive of acute appendicitis Patients identified as low risk by the APPY1 Test may be managed more conservatively e g with minimal or no additional testing or imaging observation only or earlier discharge Principles of the Test The APPY1 System is comprised of the APPYReader Instrument the reader a single use APPY1 Test Cassette a single use vial of dilution buffer and a single use vial of lyophilized antibodies conjugated to fluorescently labeled microparticles The test procedure calls for the addition of patient plasma to the dilution buffer followed by reconstitution of the lyophilized conjugate with the diluted plasma buffer Available MRP 8 14 and CRP complexes in the diluted patient sample are bound by the reconstituted antibody conjugate An aliquot of this mixture is then applied to the test cassette where the now fluo
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