Serum - Myconostica
Contents
1. creatinine dapsone dexamethasone sodium phosphate fluconazole meropenam metoclopramide hydrochloride paracetamol primaquine phosphate prednisone sodium phosphate prednisone prochlorperazine vancomycin and voriconazole The following were found to inhibit PCR reactions cefuroxime heparin methylpredisolone sodium succinate transaminase and urea When these inhibiting substances were added at clinically relevant levels to serum containing Aspergillus DNA and extracted with the modified High Pure kit no inhibition was observed However transaminase appeared to degrade the Aspergillus DNA prior to extraction as 25 of the replicates were negative for Aspergillus 030 177 Version 1 1 O5MAR12 English 26 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Analytical Specificity Analytical specificity was initially determined during the validation studies for use with respiratory samples and was not repeated Analytical specificity was tested using DNA extracted from 15 different Aspergilli species including several strains each of A fumigatus A niger A terreus and A nidulans Signals detected above the LoB were recorded as a positive result All of the 15 Aspergillus spp tested were positive with the assay In addition to those previously mentioned this includes A flavus A versicolor A glaucus A sclerotiorum A niveus A lentulus A unguis A candidus A wentii A tubi2. different Lot numbers Never pool reagents or controls from different tubes even if they are from the same Lot Never use the reagents or controls after their expiry date Reagents and controls should not be re frozen or re used after opening Wear protective clothing and disposable gloves while handling kit reagents Ensure all reagents not provided are free from fungal contamination To avoid contamination with Aspergillus or IAC amplicons do not open the reaction tubes after amplification Avoid microbial and deoxyribonuclease DNAse contamination of reagents when removing aliquots from tubes The use of sterile DNAse free low retention disposable filter tips or positive displacement pipette tips is recommended Use a new tip for each specimen or reagent Dispose of unused reagents and waste in accordance with country federal state and local regulations Additional controls may be tested according to guidelines or regulations of local state provincial federal or accrediting organisations 030 177 Version 1 1 O5MAR12 English 4 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Do not eat drink or smoke in areas where specimens or kit reagents are being handled Serum may be stored up to 48h in a refrigerator 2 8 C or freezer 15 to 25 C Low concentrations of DNA can be unstable if not stored correctly It is recommended that DNA extractions from clinical samples are
3. up Results from the entire run cannot be relied upon as accurate gt Repeat the entire run taking great care when adding the templates in particular the Positive Control Tube 4 to ensure that cross contamination does not occur gt Make sure that the work area and instruments are properly decontaminated before and after use The Negative Control was incorrectly positioned in the instrument gt Take care that the capillaries are annotated correctly within the software English 19 030 177 Version 1 1 05MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum 4 2 The Negative Control IAC Cp value is not within the acceptable range gt The PCR has been inhibited Ensure that the work area and instruments are thoroughly dry after the use of decontaminating agents prior to PCR set up The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with unexpired kit if necessary Either Tube 1 or 2 reagent was not added to the PCR or double the amount of Tube 2 was added Repeat the run taking care in the set up stage Such errors can be detected by seeing higher or lower levels of liquid in one reaction capillary compared to others The correct CC file was n
4. Cycler 2 0 Serum Exercise caution when handling Tube 4 This contains positive control DNA material and contamination could cause false positive test results Wear gloves at all times All reagent tubes must be capped following use and prior to disposal Accurately record the positions of all the capillaries within the 32 position carousel with their corresponding sample ID s on the experimental plan Accurate analysis of the data requires the application of a colour compensation file created using the Myconostica MycAssay CC kit Procedure for Use The procedure has 2 stages DNA extraction from serum followed by Real Time PCR DNA extraction is achieved using the High Pure PCR Template Preparation Kit High Pure kit The High Pure Kit is designed to purify nucleic acids from a variety of sample types The extraction protocol detailed in this IFU has been optimised to isolate Aspergillus spp DNA from serum and is suitable for use with the MycAssay Aspergillus Serum kit IMPORTANT NOTE The manufacturer s instructions have been modified to improve the yield of DNA recovered from a serum sample and to improve the sensitivity of the test Certain reagents detailed in steps 1 and 2 of Section 2 3 in the High Pure Kit IFU will be depleted before others and will need to be replaced During the validation process Proteinase K from Sigma Aldrich was used 030 177 Version 1 1 O5MAR12 English 8 For in vitro Di
5. For in vitro Diagnostic Use MycAssay Aspergillus Roche LightCycler 2 0 myconos ICa Serum MycAssay Aspergillus Roche LightCycler 2 0 Serum REF 080 045 Intended Use MycAssay Aspergillus is indicated for use by qualified laboratory professionals for the qualitative detection of Aspergillus spp genomic DNA extracted from serum as an aid to diagnosis of invasive aspergillosis MycAssay Aspergillus Serum has been validated for use with the Roche LightCycler 2 0 Summary and Explanation Aspergillus spp are ubiquitous opportunistic moulds which cause both allergic and invasive syndromes The genus is comprised of approximately 300 species of which 41 have been associated with human disease The majority of diseases are caused by A fumigatus A flavus A terreus and A niger less commonly A nidulans and other rarer species such as A sydowii A versicolor A lentulus and A pseudofischeri have been implicated Most diseases caused by Aspergillus spp affect the respiratory tract Invasive aspergillosis IA occurs in at risk patient groups including those having treatment for leukaemia and lymphoma haematopoetic stem cell HSCT and solid organ transplant patients as well as patients treated with corticosteroids and those with neutropenia or phagocyte dysfunction i e chronic granulomatous disease and HIV infection Invasive fungal disease IFD rates are nearly seven times higher in allogeneic HSCT pat
6. Root EC System Admin L Experiments Preferences rr 2 rete L Analysis T eae L Experiment Macros L Report Templates L Run Templates L Sample List Templates Name Macro MAA v 1 2 Cancel 2 4 Select the Run Macro option from the Toolbar 2y New i Run aAnalysis EQOpen amp Report 33 Template 2 5 Select the Macro MAA SERUM v1 ixo template file and press Open 2 6 Follow the wizard instructions Tick the Perform Self Test box if this is first run if a day x Welcome to the Experiment Kit Wizard The Experiment Kit Wizard helps you run a new experiment using the provided kit The first step is to create the new experiment To start the wizard and create a new experiment click Next If you don t want to create a new experiment at this time click Cancel Cancel 030 177 Version 1 1 05MAR12 English 14 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 z Select the instrument you want to run the experiment on If the instrument is not in the list click Search JLC_16997 LC 1 3 0S on COM1 LC_16937 7 Search M er lt Back Next gt Cancel 2 7 Name and save the run file in a desired location 2 8 Go to the Samples section by clicking the tab in the left of the screen Edit sample number in the Samples Count box and names in the Capillary view tab Name the samples as it states in your experimental plan accordi
7. agnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Extraction Protocol shaded areas identify those steps that are modified from manufacturer s instructions Add 400 uL of Binding buffer and 80 uL of Proteinase K mix o 5mL serum immediately incubate for 10 0 5 mL serum minutes at 70 C Add 200 uL of Isopropanol mix well and apply to High Pure filter tube by repeat addition centrifuge for 1 min at 8 000xg x Add 500 uL Inhibi K uL Inhibitor Discard flow through Centrifuge for 1 Removal Buffer and collection tube minute at 8 000xq xm and collection tube i and collection tube a Discard flow through Centrifuge for 1 retain collection tube minute at 10 000xg a Add new sterile 1 5 7 L tube and 65 uL Discard collection tub m E Centrifuge for 1 Elution Buffer 70 C minute at 8 000xg Purified DNA English 9 030 177 Version 1 1 05MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum 1 2 1 3 1 4 1 5 1 6 Real Time PCR Set Up To begin switch on the LightCycler 2 0 Real Time PCR System instrument associated computer and centrifuge and launch the relevant software Enter username and password as required and choose the Diagnostic database If this is the first run of a day perform an instrument Self Test first before starting a run Remember a colour compensation run must be completed prior to analysi
8. e baseline for some samples match the Negative control indicating no amplification has occurred However the software has reported out a positive Cp value as in figure below An output of a positive Cp for a negative amplification plot was seen only twice in 154 negative reactions performed during validation studies gt If this happens please repeat the sample s to confirm a Negative result Amplification Curves 1 37 1 s 2 no Cp reported a3 33 2 030 177 Version 1 1 05MAR12 English 22 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 4 8 4 9 When apply my CC object some of the data in the 560 IAC channel dips resulting in Cp values which are outside the acceptable range Amplification Curves Fhioeeacerce 540 This is entirely normal for reactions containing high concentrations of target DNA and will not interfere in the interpretation of patient results Follow the normal analysis you will see that for samples which are positive for Aspergillus the IAC result is not required for an outcome decision to be made for the patient There are no results for any channel with any samples or controls The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents
9. ed patients suspected of having invasive aspergillosis Principles of the Procedure Following mixing of the reagents in the MycAssay Aspergillus kit with a sample containing the Aspergillus target DNA sequence a section of the Aspergillus ribosomal 18S gene thermocycling will result in DNA amplification occurring The assay also contains an Internal Amplification Control IAC a DNA fragment not present in Aspergilli other fungal bacterial or human genomes to detect PCR inhibitory substances and confirm the functionality of the assay reagents The amplified DNA targets are detected using Molecular Beacon technology Molecular Beacons are single stranded oligonucleotide hybridisation probes that form a stem and loop structure The loop contains a probe sequence that is complementary to a target sequence and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence A fluorophore which fluoresces when excited by light of the appropriate wavelength is covalently linked to the end of one arm and a quencher which suppresses the fluorescence of the fluorophore when in close physical proximity is covalently linked to the end of the other arm Molecular Beacons do not fluoresce when they are free in solution However when they hybridise to a nucleic acid strand containing a target sequence they undergo a conformational change that physically separates the fluorophore and the quenc
10. he first capillary in the position 1 and continue in the ascending order leaving no gaps Push each capillary all the way down till it firmly rests in its place If not already spun down in 1 16 spin the samples using the LightCycler 2 0 carousel centrifuge Proceed to Section 2 promptly MycAssay Aspergillus reactions are stable on the bench for up to 60 minutes Following the PCR set up ensure the work area is thoroughly cleaned using DNA decontaminating reagents 030 177 Version 1 1 O5MAR12 English 12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum 2 Performing the run 2 1 Insert the MycAssay Aspergillus Myconostica Protocol CD ROM 2 2 Go to File select Import and select Object ixo files Import the Macro MAA SERUM v1 ixo file from the CD to your database File Edit Yiew Tools Window Help A New Ctrl N A Analysis EYopen Ctrl 0 Glose E Grits COF Std Curve Files Batch import Object ixo files Batch export a Report GENER Print Window Import File Lookin MA LC CC CD ROM arm My Documents File name Macro MAA CCo Files of type Object ixo files ixo English 13 030 177 Version 1 1 O5MAR12 MycAssay Aspe Lg For in vitro Diagnostic Use Roche LightCycler 2 Serum 2 3 Go to File select Save and save the macro in the desired location in your database A gt Save Experiment Kit ey a
11. her enabling them to fluoresce upon excitation The amount of fluorescence at any given cycle or following cycling depends on the amount of specific amplicons present at that time The Real Time PCR System simultaneously monitors the fluorescence emitted by beacons English 3 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum Precautions The kit is intended for use only by laboratory professionals Procedures are required for non aerosol manipulations of specimens Standard precautions and institutional guidelines should be followed in handling all samples A Material Safety Data Sheet is available from Myconostica Ltd This assay is for in vitro diagnostic use only In analytical validation studies levels of transaminase of 22 2 U per 0 5 mL serum were shown to have a possible degradation effect on Aspergillus DNA This assay has been evaluated with serum collected in Greiner Red Top serum collection tubes Other serum blood collection tubes may contain inhibiting or competing substances that have not been tested This assay has been validated for serum specimens Validation data are not available for plasma or whole blood This assay is for use with the Roche LightCycler 2 0 and LightCycler v4 1 software only Do not use reagents or controls if the protective pouches are open or broken when received Reagents and controls are not interchangeable between kits with
12. ients than in autologous transplant patients and invasive aspergillosis IA is Species Database in www aspergillus org uk English 1 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum responsible for approximately half of infections Aspergillosis is largely confined to the early post transplant neutropenic phase in autologous HSCT patients Allogeneic HSCT patients are at risk for much longer periods not only up to but also beyond 100 days owing to their more frequent GvHD and slow T cell recovery In patients receiving chemotherapy for acute leukaemia or salvage regimens for relapsed leukaemia or lymphoma IA is a leading cause of death Consensus definitions of Invasive Fungal Diseases have been revised and published by the European Organisation for Research Treatment Centre EORTC and the Mycoses Study Group MSG including defined criteria for diagnosis of proven probable and possible IA in patients with haematologic malignancy or following HSCT Currently the criteria for probable IA are defined as one host factor plus one clinical criterion plus one microbiological test Diagnosis of possible IA does not require a microbiology criterion The microbiological tests accepted in the probable IA criteria include a serum based ELISA test that detects the presence of galactomannan GM Two consecutive positive GM tests are recommended to improve diagno
13. lish 25 030 177 Version 1 1 05MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum The following species were specifically tested for potential presence in serum and did not report out a positive result Acinetobacter baumannii Aeromonas hydrophilia Burkholderia cepacia Citrobacter koseri Enterobacter cloacae Enterococcus faecium Klebsiella pneumoniae Morganella morganii Proteus mirabilis Salmonela enterica Serratia marcescens Stenotrophmonas maltophila Human genomic DNA does not report a positive result with this assay Limit of Detection This was determined to be to be lt 25 copies of target DNA using the AF293 strain of A fumigatus Interfering Substances contraindications for use The following compounds were tested at clinically relevant concentrations and found not to inhibit the assay acteylcysteine amphotericin beclometasone dipropionate budesonide colistimethate sodium fluticasone propionate formoterol fumarate dehydrate ipratropium bromide lidocaine mannitol salbutamol sulphate salmerterol sodium chloride sodium cromoglicate terbutaline tobramycin The following were tested for potential presence in serum Clinically relevant concentrations were tested and were found not to inhibit the PCR reaction Amoxicillin with clavulanic acid atovaquone azathioprine azotreonam ceftazidime ciproflaxicin chlorphenamine maleate clindamycin phosphate co trimoxazole
14. lus assay requires the application of a colour compensation file produced using the Myconostica MycAssay CC kit Once created the file can be applied to multiple runs on the same machine Please contact your local distributer for details Procedural Notes Read the entire protocol before commencing The entire MycAssay Aspergillus process including DNA extraction takes approximately 212 hours dependent on the number of samples tested Setting up of the test should be performed in a PCR workstation or pre PCR laboratory If a PCR workstation is not available then the test should be set up in a dedicated area of the laboratory separated from areas used for DNA extractions that is regularly cleaned with DNA decontaminating reagents However avoid using DNA decontaminating reagents when performing the Real Time PCR set up as they can inhibit the assay Use micropipettes for the transfer of fluids Dedicated micropipettes should be used for the set up of these reactions and they should be regularly decontaminated Low retention filtertips are recommended for use to ensure that no DNA is lost during the set up procedure T For example see Mifflin T E 2003 Setting up a PCR Laboratory In PCR Primer 2nd Ed eds Dieffenbach and Dveksler Cold Spring Harbour Laboratory Press Cold Spring Harbour NY USA English 7 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche Light
15. ng results for MycAssay Aspergillus on the LightCycler 2 0 However this does not have to be performed prior to using this product and can be carried out and applied to this run file later Ensure the work area has been cleaned using DNA decontaminating reagents and allowed to dry completely avoid use during assay set up as excess cleaning solution may inhibit the PCR A pouch contains one each of Tube 1 Tube 2 Tube 3 and Tube 4 There are sufficient reagents in one pouch to run 8 reactions At least one positive control and one negative control reaction must be performed per run where the reagents are from a single kit Lot One pouch therefore can analyse 6 patient samples If more than 6 samples need to be tested more than one pouch can be used if the pouches used are from the same kit However the LightCycler 2 0 can only hold up to 32 samples in a single run Therefore a maximum of 30 patient samples can be performed in a single run 4 pouches Calculate the number of reactions required referring to the table below Number of Pouches Maximum number of patient samples 6 Remove the appropriate number of pouches from the freezer Do not use any pouch that is no longer sealed If the patient samples were frozen after extraction also remove these from the freezer 030 177 Version 1 1 OS5MAR12 English 10 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 1 7 Tear open
16. ng to their position in the sample carousel Sample data sioCony Selected Channels 530 560 610 640 670 705 pin Samples Analysis Type_ amol Sample Count 16 LC Carousel ID MPLC Batch D Assay Lot No Color Comp ID Repl Of Sample Note Repl of Negative Control 3 Repl of Negative Control 1 4 Asp 10e3 copies _ 5 Repl of Asp 10e3 copies 4 l 6 Repl of Asp 1063 copies 4 7 Asp 10e4 copies Repl of Asp 10e4 copies 7 Repl of Asp 10e4 copies 10 Asp 10e5 copies 11 Repl of Asp 1065 copies 10 12 Repl of Asp 10e5 copies 10 os 13 Asp 1026 copies E 14 Repl of Asp 10e6 copies 13 115 Repl of Asp 1068 copies 3 lele lel elo aloe gt e gt lela loa na lalo fal fal eos al 16 Positive Control English 15 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum 2 9 Place the spun down sample carousel in the LightCycler 2 0 instrument Ensure that the notch below sample position 1 on the carousel locks into position with the pin on the thermal chamber Make sure that the carousel is inserted firmly in the chamber and close the lid 2 10 When finished press the Start Run button Make sure that the instrument has found all the capillaries in the carousel and the program has started Experiment Kit Wizard xj The experiment is ready to run Before starting edit the
17. ngensis and A foetidus Genomic DNA extracted from Penicillium spp also generated positive results This is due to the fact that the sequences of the molecular targets are highly conserved between Aspergillus and Penicillium Therefore it must be noted that a positive result with this assay may be the result of infection by Penicillium rather than Aspergillus Clinical Reporting The MycAssay Aspergillus kit is intended as an aid to diagnosis The results need to be taken in context of the clinical condition of the patient and other diagnostic test results The following are recommended reports each depending on the assay result interpretation Outcome No 1 Aspergillus spp not detected Outcome No 2 Aspergillus spp detected Positive result This assay also detects Penicillium spp Outcome No 3 Test failed inhibitors or other unknown substance present English 27 030 177 Version 1 1 OSMAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum Limitations of Procedure The principal limitation of this procedure relates to the quality of the primary sample If the levels of Aspergillus DNA in the serum are low extraction efficiency may impact the result and the test may give a false negative outcome Preliminary data indicate that freezing and storage of serum samples may affect the quantity of viable DNA available for assaying No data are available on the s
18. or Aspergillus DNA Check the test Sample Is the ASP Cp lt 38 0 Check the test Sample Negative for Aspergillus DNA Is the IAC Cp 30 3 33 9 IAC failure ACTION Repeat sample If the same result again suspect inhibitor present in sample 030 177 Version 1 1 OS5MAR12 English 18 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Sample Pathogen 530 IAC 560 Interpretation Further Action Cp Cp Negative Within 30 3 Negative Control Patient results are N Contro SEO or NOGP 33 9 acceptable valid N 7 r egatve 38 0 or No Cp lt 30 3 or gt 33 9 Failure in Negative Repeat entire run Contro Control Negative Within 30 3 R an Contro lt 38 0 33 9 Contamination Repeat entire run Positive Within 15 0 20 0 N A Positive Control Patient results are Contro acceptable valid Positi 7 7 ostve lt 15 0 or gt 20 0 N A Failure in Positive Repeat entire run Contro Control Patient Within 30 3 Negative for Report result gt Sample ee Oor No Op 33 9 Aspergillus Outcome 1 Patient Positive for Report result Sample 598 0 NA Aspergillus Outcome 2 Patient lt 38 00rNoCp lt 30 30r gt 33 9 IAC failure in sample ePeat sample Sample Outcome 3 See Clinical Reporting Outcome 1 2 or 3 Troubleshooting 4 1 The Negative Control has generated a positive signal in the ASP 530 channel Contamination occurred during the set
19. ot applied to the data Create a CC file using the Myconostica MycAssay CC kit and apply to the results and reanalyse See your local distributer for details of this kit 4 3 The Positive Control is negative gt The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with an unexpired kit if necessary An error occurred during step 1 11 1 13 and the Positive Control template Tube 4 was placed in the wrong reaction tube Repeat the run taking great care during the set up stage Such errors can be detected by seeing a higher level of liquid in one reaction and a lower level in another compared to normal Either Tube 1 or 2 reagent was not added to the reaction 030 177 Version 1 1 O5MAR12 English 20 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 gt Repeat the run taking care in the set up stage Such errors can be detected by seeing lower levels of liquid in this reaction capillary compared to others The Positive Control was incorrectly positioned in the instrument gt Take care that the capillaries are annotated correctly within the software 4 4 Patient sample s are negative and the IAC is out of range Outcome 3 4 5 gt It is likely tha
20. ot re frozen or re used at a later date Equipment Materials required but not provided Roche LightCycler 2 0 Real Time PCR system including User Manual attached computer and LightCycler Software v4 1 LightCycler 2 0 carousel centrifuge optionally capillary adaptors for mini centrifuge Sample carousel for 20 uL capillaries LightCycler 2 0 20 uL capillaries with caps Capillary rack holder Capillary releaser Capping tool Micro centrifuge Vortex mixer Micropipettes volumes required 7 5 uL 20 uL Sterile low retention filtertips Disposable gloves powderless Proprietary DNA decontaminating solution DNA isolation kit see below MycAssay CC kit see below 030 177 Version 1 1 OS5MAR12 English 6 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Specimen The specimen for the MycAssay Aspergillus assay is total genomic DNA extracted from serum samples The following DNA extraction kit and equipment used during validation is recommended for this purpose High Pure PCR Template Preparation kit Roche Diagnostics Cat No 11 796 828 001 Proteinase K solution Sigma Aldrich Chemicals Cat No P4850 5ML 2 Propanol Sigma Aldrich Chemicals Cat No 19516 25ML Vortex Genie 2 Scientific Industries Inc New York USA MycAssay Colour Compensation CC kit Accurate analysis of data produced using MycAssay Aspergil
21. sample information and run protocol as necessary Cancel 030 177 Version 1 1 05MAR12 English 16 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 3 Data Analysis and Interpretation 3 1 Remember a colour compensation object must be applied prior to analysing results for MycAssay Aspergillus on the LightCycler 2 0 If you have not yet created one please do so now before continuing with Data Analysis and Interpretation When the run has finished check for the contents of the popped up report and print it if desired 3 2 The Aspergillus results can be viewed in the ASP 530 analysis section and the IAC results in the IAC 560 analysis section 3 3 In both sections ASP 530 and IAC 560 select the correct Colour Compensation file MycAssay CC file to be applied to the experiment English 17 030 177 Version 1 1 O5MAR12 MycAssay Aspe aa For in vitro Diagnostic Use Roche LightCycler 2 Serum 3 4 Analyse each sample starting with the controls as shown in the flowchart below details can also be found in the table shown beneath the flowchart Check the Negative Control Is the ASP Cp 38 0 or recorded as No Cp Run is contaminated Check the Negative Control ACTION Repeat the run Is the IAC Cp 30 3 33 9 Run failure ACTION Repeat the run Run failure Check the Positive Control ACTION Repeat the run Is the ASP Cp 15 0 20 0 Positive f
22. see the kit box pouch label and repeat with an unexpired kit if necessary The equipment used is not functioning optimally Please check that your Real Time PCR instrument has an up to date service history and has been fully calibrated as described in its Installation and Maintenance Guide An incorrect protocol file was used during the software set up Please refer to Section 2 and choose the correct Protocol file as specified for each software type version from the Myconostica Protocol CD ROM Only English 23 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum the file appropriate to the software can be loaded Repeat the run using the correct protocol file If you have further questions or you experience any problems please contact Technical Support productsupport lab21 com 030 177 Version 1 1 OS5MAR12 English 24 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Performance Characteristics and Limitations The kit was initially validated for use with serum using the Cepheid SmartCycler Analytical sensitivity Limit of Blank was established on the LightCycler 2 0 platform using 20 uL glass capillaries Roche Cat 04929292001 or 11909339001 and is reported below Where the differences between platforms were not expected to affect the performance of the assay and therefore the performance claim the other s
23. stic accuracy A meta analysis by Mengoli et al reported on gt 10 000 blood serum and plasma samples from 1618 patients at risk for IA They calculated the sensitivity and specificity of a single PCR positive blood sample to be 88 95 C I 75 94 and 75 95 C I 63 84 respectively and the diagnostic odds ratio for proven and probable cases to be 16 41 95 C I 6 43 41 88 MycAssay Aspergillus is a molecular diagnostic kit for the detection of Aspergillus spp genomic DNA using Molecular Beacon Real Time PCR technology The whole test procedure including extraction of DNA from the clinical sample can be completed in approximately 2 2 hours compared to fungal culture which can take several days to produce positive results This assay offers advantages over currently available diagnostic methods for acute invasive and chronic pulmonary aspergillosis These advantages include faster detection of Aspergillus spp and the potential for increased sensitivity for a Kontoyiannis DP et al Clin Infect Dis 2010 50 8 1091 1100 3 Ascioglu S et al Clin Infect Dis 2002 34 7 14 Mengoli C et al The Lancet ID 2009 9 86 96 5 Tyagi S Kramer FR 1996 Molecular beacons Probes that fluoresce upon hybridization Nature Biotechnology 14 303 308 030 177 Version 1 1 O5MAR12 English 2 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 Aspergillus spp in highly immunocompromis
24. stored at 80 C to preserve their integrity Multiple rounds of thawing and refreezing should also be avoided whenever possible Kit Contents Description The kit consists of five 3 compartment sealed foil pouches each of which can be removed from the box and used separately Each pouch contains sufficient reagents for 8 reactions Volume Tube 1 dNTPs 66 uL Orange Cap MgCl Buffered solution of DNA Polymerase complex Tube 2 lt 0 01 Primers 66 uL Green Cap lt 0 01 Molecular Beacons lt 0 0001 Internal Amplification Control IAC The Internal Amplification Control is a recombinant DNA plasmid containing a non infective sequence unrelated to target Aspergillus sequence Tris HCl Buffer Tube 3 Negative Control 25 uL Clear Cap Water Tube 4 Positive Control 25 uL Black Cap lt 0 0001 Positive Control DNA The Positive Control molecule is a recombinant plasmid containing the Aspergillus target sequence Tris HCl Buffer English 5 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum The kit also contains MycAssay Aspergillus Myconostica Protocol CD ROM Instructions for Use Certificate of Analysis Storage The kit should be stored frozen 15 to 25 C until the expiry date indicated on the kit box label when it should be disposed of according to local regulations Once a pouch has been opened the contents must be used immediately n
25. t the patient sample s contain PCR inhibitors We recommend that DNA from samples is extracted using the High Pure kit following the modified procedure in Procedures for Use and not manufacturer s instructions for optimal DNA extraction Some collection tubes for serum may contain PCR inhibitors that have not been tested The Patient Sample is negative in the ASP 530 section and the IAC 560 plot drifts away significantly from the regular baseline as shown on the example picture below arrows indicate abnormal plots Amplification Curves 423456 7 8 5 10911213141516 17181920 21 222324 252527 28293031 32333435353738 3940 cycles The PCR reaction was inhibited English 21 030 177 Version 1 1 O5MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum gt Ensure that the work area and instruments are thoroughly dry after the use of decontaminating agents prior to PCR set up gt Run the patient sample again If the problem repeats the PCR inhibitor is present in the sample Report the sample as Undetermined Outcome 3 4 6 The results in the IAC 560 section almost exactly match the results in the ASP 530 section No Colour Compensation file or an incorrect Colour Compensation file was applied to the experiment results gt In both analysis sections check if the Colour Compensation is ON and that the same MycAssay CC file is applied in both channels 4 7 Th
26. tability of Aspergillus DNA in serum It is recommended therefore that samples are processed as quickly as possible after collection No data are available on the performance of serum collected in blood collection tubes other than the recommended Greiner Red Top serum collection tubes No data are available on the performance characteristics of the assay starting with Aspergillus DNA extracted from plasma or whole blood False positive results are possible if the infecting agent is Penicillium spp which cannot be differentiated from Aspergillus spp using this kit While the High Pure PCR Template preparation kit procedure may remove PCR inhibitors not all drugs or patient populations have been evaluated During analytical validation studies it was noted that transaminase at 22 2 U 0 5 mL serum may have caused Aspergillus DNA degradation prior to extraction During validation batches of Proteinase K were obtained and used that were subsequently found to be contaminated at source with Aspergillus Source all materials carefully and use recommended sources wherever possible False positive results may arise from external contamination of the original sample or test Such contamination could arise from Aspergillus contaminated air poor experimental technique with respect to the positive control or external especially pipettor contamination with Aspergillus DNA As a true positive result may be obtained from patients who are transien
27. the required number of pouches and remove the tubes If more than one pouch is being used but only one set of positive and negative controls are being run it is only necessary to remove Tubes 3 and 4 from one pouch Exercise caution when handling Tube 4 This contains positive control DNA material and contamination could cause false positive patient results 1 8 Allow the contents of the tubes to thaw by placing on the laboratory bench for 5 10 minutes ensuring that the contents of each tube are completely thawed before proceeding Vortex mix the contents of the tubes and the patient samples follow by a short spin in a microcentrifuge to ensure collection of all the contents at the base of the tubes before use 1 9 Place the required number of 20 uL capillaries in a capillary rack holder Take care not to leave any marks on the glass 1 10 Always set up the negative control first followed by the patient samples The positive control should always be set up last 1 11 Reagent and DNA volumes are shown in the table below Reaction Reagent Negative Patient Positive control samples control Tube 1 Orange cap 7 5 uL 7 5 uL 7 5 uL Tube 2 Green cap 7 5 uL 7 5 uL Tube 3 Clear cap 10 uL Patient Samples 10 uL Tube 4 Black cap Total volume 25 uL 1 12 Add reagents in the order shown in the table above Tube 1 then Tube 2 followed by the template Negative control Patient sample or Positive con
28. tly or persistently colonised by Aspergillus spp clinical judgment is required in interpretation of the test results in the context of disease 030 177 Version 1 1 OS5MAR12 English 28 For in vitro Diagnostic Use MycAssay Aspergillus Serum Roche LightCycler 2 0 LICENSING TopTaq Hot Start is provided by QIAGEN QIAGEN is a registered trade mark of Qiagen GmbH Hilden Germany This product is sold under license from the Public Health Research Institute Newark New Jersey USA and may be used under PHRI patent rights only for human in vitro diagnostics SmartCycler is a registered Trademark of Cepheid 904 Caribbean Drive Sunnyvale CA 94089 USA High Pure is a registered Trademark of Roche Diagnostics GmbH 68298 Mannheim Germany Part of this product is covered by an exclusive license to a patent application held by the Fred Hutchinson Cancer Centre Seattle USA LightCycler is a registered Trademark of Roche Diagnostics GmbH Lab21 184 Cambridge Science Park Cambridge CB4 0GA United Kingdom Telephone 44 0 1638 552 882 Facsimile 44 0 1638 552 375 Email productsupport lab21 com English 29 030 177 Version 1 1 05MAR12
29. trol Take care when taking aliquots from Tube 1 the liquid is viscous and can stick on the inner ridge of the tube If this happens re spin to collect the final contents in the base of the tube before attempting to remove the final aliquots English 11 030 177 Version 1 1 05MAR12 MycAssay Aspergillus For in vitro Diagnostic Use Roche LightCycler 2 0 Serum 1 13 Use a new pipette tip for every liquid transfer Re cap each reagent tube after use and immediately discard it and any remaining contents into a sealable clinical waste container Unused reagents cannot be saved for later use Take extra care when pipetting Tube 4 positive control DNA to ensure it does not contaminate any other reaction Capping all the other capillaries before opening Tube 4 can reduce the risk of cross contamination Carefully cap the capillaries with the caps provided in the capillary box using a capping tool Ensure the capillaries are firmly capped Capillaries can be capped once the template has been added to the reaction if desired to reduce the potential for cross environmental contamination If the LightCycler 2 0 carousel centrifuge is not available spin the samples down in a mini centrifuge using the capillary adapters provided with the capillary rack holder Otherwise proceed to 1 17 Very carefully transfer all the capillaries to the sample carousel in exactly the same order that they are in the capillary rack holder starting with t
30. tudies were not repeated These results obtained using the SmartCycler are considered transferable to the LightCycler 2 0 platform Analytical Sensitivity Using the LightCycler 2 0 protocol described above and PCR templates generated at Myconostica the LoB for the MycAssay Aspergillus was determined to be a Cp of 38 0 The following Performance Claims were established for serum using the Cepheid SmartCycler Analytical Selectivity Analytical selectivity was tested using DNA extracted from a variety of different fungal and non fungal species The following species were tested during the initial validation for respiratory samples and did not report out a positive result Alternaria alternata Blastomyces capitatus Candida albicans C glabrata C parapsilosis C tropicalis Cladosporium spp Cryptococcus neoformans Doratomyces microsporus Fusarium solani Histoplasma capsulatum Pneumocystis jirovecii Rhizomucor pusillus Rhodotonila rubra Saccharomyces cerevisiae Scedosporium apiosperinu S prolificans Sporothrix schenkii Trichosporon capitatu The following bacterial species did not report a positive result Bordetella pertussi Corynebacterium diphtheriae Escherichia coli Haemophilus influenza Lactobacillus plantarum Legionella pneumophila Moraxella catarrhalis Mycoplasma pneumonia Neisseria meningitides Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumonia S pyogenes S salivarius Eng
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