HCV Real TM Quant L - bio
Contents
1. 15 PROTOCOL FOR ROTORGENE 6000 a ai 16 PROTOCOL FOR SaCycler 96 Real Time PCR system ccccccssseccccesssccccescceeeeeceeeeseceeeeueceseuaeceteeneceesenens 21 TROUBLE SH eonl cm 23 NAME HCV Real TM Quant Dx INTRODUCTION Hepatitis C virus HCV is a single stranded RNA virus that belongs to the family Flaviviridae In 1974 HCV was initially recognized as non A non B hepatitis virus NANBH until 1989 when the etiologic agent was cloned It is estimated by the World Health Organization that worldwide there are 170 million HCV infected persons The primary mode of HCV transmission is the exposure to infected human blood via intravenous drug use or unscreened transfusions Nosocomial HCV transmission during dialysis colonoscopy and surgery has also been reported Perinatal and sexual transmission of the virus is inefficient but occurs more frequently if the HCV infected mother or sexual partner is also infected with human immunodeficiency virus type 1 Most people with acute HCV infection are asymptomatic or have mild symptoms fatigue nausea jaundice but they are unable to clear the virus and in approximately 80 of cases this leads to chronic infection In 15 to 20 of patients chronic HCV infection progresses at a variable rate to cirrhosis with a 1 to 4 annual risk of d2. f 299 0000000 8 ARAARSRSAASRAL 4 4 amp 4 44 Fo Clicking Apply will bring to the Start Program window EXE The thermalcycling program will be the following 1 500 C 15 00 2 950 15 00 60 0 C 00 20 3 950 C 00 05 x5 7207 19 15 60 0 C 00 30 990 4 950 C 00 05 x 40 Z2U0 C 10 15 Click Open block insert PCR tubes into the instrument click Close block and then click Start program to start the PCR amplification program After the run is complete click OK to interpret the results select analysis type Multiplex detection Threshold Ct method and Quantitative from results table Analysis type Multiplex detection Results Additional standards Method Threshold Ct Quantitative Click on parameter of data analysis icon select Normalization data option and click Apply 4 Nomalization data Apply Adjust the threshold line using mouse drag and drop Suggested values for Threshold line are 40 for HEX channel 25 for FAM channel slightly adapt threshold level according to your result curves Viral load results will appear in the Results column for HEX channel Final results are already expressed in IU ml To import standard curve from other run click on Additional standards tab and select Z the run file containing standards Then come back to Results tab to see viral load results NOTE for IVD version
3. Blender Forma Auto Find Threshold 7 HCVG10 03 2500 It Unknown 2079 2079 8 HCVG10 03 12501 Unknown 21 07 21 07 log Cond Curve 9 HCVG10 03 82510 Unknown 2084 2084 lions Ne 10 HCVG10 03 31210 Unknown 20 60 2060 11 HCVG10 05 5000 I Unknown 2078 20 78 From External Source Pre 6 0 Standard Curve Format 12 HCVG10 05 2500 It Unknown 2144 2114 x Conc 10 j m cT Reset 13 HCVGIO 05 1250 It Unknown 21 00 21 00 Boe x 14 HCVG10 05 625 IU Unknown 20 88 20 88 Eficiency Imported Settings 18 HCVG10 05 312 IU Unknown 21 48 21 48 16 negexrRV Unknown 21 59 21 58 13 19 Data Analysis IC amplification analysis Cycling A Green Fam T Press Analysis and then select Quantitation gt Cycling A Green Cycling A Fam Show 2 Std Curves Other Hide Auto shrink window Turn off the automatic option Threshold Activate the Dynamic tube and Slope Correct buttons in the main window menu Quantitation analysis 4 Quantitation Analysis Cycling A Green Page 1 EEBESE i Outlier Removal bed Save Defaults In the table of results QuantitationAnalysis select More settings and set NTC Threshold to 10 select Threshold 0 03 In the table of results QuantitationResults appear the values of Ct Threshold cycle which should be lt
4. 2003 38 645 652 Terrault NA Pawlotsky JM McHutchisonJ et al Clinical utility of viral load measurements in individuals with chronic hepatitis C infection on antiviral therapy J Viral Hepat 2005 12 465 472 Rotor Gene Technology is a registered trademark of Qiagen SaCycler is a registered trademark of Sacace Biotechnologies SacaceBiotechnologiesSrl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com 24
5. 30 see Internal Control of the IC Fam Green channel HCV amplification analysis Cycling A Yellow Joe 1 Ol Press Analysis and then select Quantitation gt Cycling A Yellow Cycling Show 2 Std Curves Rel Other sf Cycling Green Page 1 Hide Auto shrink window Turn off the automatic option Threshold Press the buttons Dynamic Tube Slope Correct 4 Quantitation Analysis Cycling A Yellow Page 1 lt Dynamic lt Slope Correct lt Ignore First Z Outlier Removal bd Save Defaults In the table of results QuantitationAnalysis select More settings and set NTC Threshold to 10 Select Threshold 0 03 In the table of results QuantitationAnalysis appear the values of Ct Threshold cycle In the new window QuantitationResults appear values of HCV RNA IU ml PROTOCOL FOR SaCycler 96 Real Time PCR system Recommended settings 1 2 Double click on the RealTime PCH software icon e Select Device handling Select the plugged device and click Connect Wo Z Create Edit Test Gy group of tests Select from Menu Test gt Create Edit test Click Create new test button and enter name HCV Real TM Quant DX click OK Select Analysis Type Multiplex detection and Method Threshold Standard Quantity 2 and copy
6. If these solutions come into contact rinse immediately with water and seek medical advice immediately This product does not contain potentially infectious components Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of amplification Workflow in the laboratory must proceed in a uni directional manner beginning in the Extraction Area and moving to the Amplification Area Do not return samples equipment and reagents in the area where you performed previous step Personnel should be using proper anti contamination safeguards when moving between areas STORAGE AND SHIPPING INSTRUCTIONS All components of the HCV Real TM Quant Dx PCR kit must be be stored at 2 8 C when not in use All components of the HCV Real TM Quant Dx PCR kit are stable until the expiration date on the label The shelf life of reagents before and after the first use is the same unless otherwise stated Ship HCV Real TM Quant Dx PCR kit at 2 8 C STABILITY HCV Real TM Quant Dx Test is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results Whe
7. Repeat the RNA extraction with the new set of reagents 5 Any signal with Negative PCR Control e Contamination during PCR preparation procedure All samples results are invalid gt Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive controls at the end gt Repeat the PCR preparation with the new set of reagents REFERENCES World Health Organization Hepatitis C Global prevalence update WklyEpidemiol Rec 1999 74 425 427 Alter 1999 Discovery of non A non B hepatitis and identification of its etiology Am J Med 107 Suppl 6B 16S 20S Bendinelli M M Pistello F Maggi and M Vatteroni 2000 Blood borne hepatitis viruses hepatitis B C D and G viruses and TT virus p 306 337 In S Specter R L Hodinka and S A Young ed Clinical virology manual 34 ed ASM Press Washington D C Choo Q L G Kuo A J Weiner L R Overby D W Bradley and M Houghton 1989 Isolation of a cDNA clone derived from a blood borne non A non B viral hepatitis genome Science 244 359 362 Lauer G M and B D Walker 2001 Hepatitis C virus infection N Engl J Med 345 41 52 De Medina M Schiff ER Hepatitis C diagnostic assays Sem Liver Dis 1995 1 33 40 Kao J H M Y Lai Y T Hwang P M Yang P J Chen J C Sheu T H Wang H C Hsu and D S Chen 1996 Chronic hepatitisC without anti hepatitis C antib
8. at all 15 PROTOCOL FOR ROTORGENE 6000 Q 1 Switch on instrument by pressing the main switch start Rotor Gene software 2 Start a New Run Quick Stat Advanced Perform Last Rur 3 Empty Three Step with Melt 29 Two Step TT Other Runs 8 Instrument Maintenance Open Template In Another Folder Show This Screen When Software Opens Figure 1 Start New Run 3 Select the appropriate rotor check the Locking Ring Attached box and click Next T c wan Welcome to the Advanced Run Wizard Rotor Type 72 Well Rotor Rotor Disc 72 Rotor Disc 100 Figure 2 Rotor and Locking Ring 4 Input operators name notes and reaction volume 50 Subsequently press Next New Run Wiza d y hb Enter any additional notes for the run in his text box This screen displays miscellaneous options for the run Complete the fields clicking Next when you are ready to move to the next page Operator Notes HCV Real TM Quant Dx Reaction 50 Volume HL i Sample Layout Skip Wizard lt lt 3 Figure 3 Reaction Volume Sacace HCV Real TM Quant Dx 06 06 2013 5 Click the Edit Profile and program the temperature profile Temperature Profile This box displays help on elements in the wizard For help on an item hover A p DUI icon combo
9. obtained from the European Directorate for the Quality of Medicine amp HealthCare EDQM They did not generate any signal with the specific HCV primers and probes The specificity of the kit HCV Real TM Quant Dx was 100 GENOTYPE DETECTION The detectability of all relevant subtypes and genotypes has thus been ensured All tested HCV RNA genotypes give positive results with HCV Real TM Quant Dx kit CROSS REACTIVITY The potential cross reactivity of the kit HCV Real TM Quant Dx was tested against the group control listed in the following table It was not observed any cross reactivity with these pathogens Table 1 Testing the specificity of the kit with other pathogens Control group Results HCV Results IC Joe Yellow channel Fam Green channel Cytomegalovirus CMV Epstein Barr virus EBV Hepatitis B virus HBV Hepatitis D virus HDV Hepatitis A virus HAV Parvovirus B19 Herpes Zoster Virus VZV Human immunodeficiency vius T CO CAO 0 07 Humanhepesvius Humanhepesvirus8 West Nile Virus WNV EE Tick borne encephalitis virus TBEV m Adenwmuslppe2 S Adeowmsyped Rotavirus sd Escherichia coli E coli a ae Staphylococcus aureus O O O OOo o o oS S S Smeplococuspyogenes Streptococcus agalactiae ROBUSTNESS The robustness of the assay is a measure of its capacity to remain unaffected by small and accidental variatio
10. of SaCycler since the HCV Real TM DX protocol is already inserted in the PC supplied with the instrument ignore points 4 8 and just follow instructions from point 9 TROUBLESHOOTING 1 Weak Ct gt 30 or exceeding the established range signal see Internal Control of the IC Fam Green channel retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended RNA extraction method and follow the manufacturer s instructions e The reagents storage conditions didn t comply with the instructions Check the storage conditions e he PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol he IC was not added to the sample during the pipetting of reagents Make attention during the RNA extraction procedure 2 The correlation coefficient is less than 0 9 retesting of all samples is required 3 The calculated concentrations of CONTROL 1 and or CONTROL 1are different from given control concentrations reported in the Data Sheet retesting of all samples is required 4 Any signal on the Joe HEX Yellow channel with Negative Control of extraction e Contamination during RNA extraction procedure All samples results are invalid gt Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips among tubes
11. 3 total 6 standards 2 positive controls 1 negative control FAM IC and R6G s 50 ul of Mixture volume 5 Mixture volume 50 3 md i ma po Positive K 2 ju m Type Multiplex detection v Een DUET Fam 9 RG Rx v Q5 Cy55 Method Threshold Ct Negative KJ 1 t3 Ic v 5 v Y v v Under Amplification Program click to create a new program Select Preliminary heating and choose the template below Click Apply then program the instrument as follows Click and save the amplification program file in a convenient folder Then click OK in the previous window Click add test button then select HCV Real TM Quant DX from dropdown menu Test indicate the number of click first Add then OK Sacace HCV Real TM Quant Dx 06 06 2013 21 10 11 12 15 14 15 16 17 18 19 Use the default Autofill positioning of PCR tubes into the plate the Free Filling to chose manually Concentration the position of the tubes In the Concentration column click the green circle and insert the HCV standard values reported in the DataCard see example below Protocol number 50 Operator name Sample_1 Standard 1 Standard 2 Rea T K HCV Rea T K HCV Rea T G HCV Rea T AutoFill
12. RES RENS VERON EE 7 STORAGE AND SHIPPING 5 8 IPIE ep c H 8 8 SAMPLE COLLECTION STORAGE AND TRANSPORT ssssscccccccseessesececccceceeeesseeecceeessueaueeeeesessugusseeeseeees 8 INTERFERING SUBSTANCE 5 cT 9 REAGENT PREPARATION m 9 RNA TOTON ates E M 9 INTERNAL CONTRO TT 10 RSSAY CALUBRA TION T 10 ASSAT PROTOCOL 11 QUALITY CONTROL PROCEDURE 12 RESULT INTERPRETA TION 12 PERFORMANCE CHARACTERISTICS sciisdesaesuvetvne E UC Fo SR RETI V HE Un CES V ERE R UTR OU SFE NEN Gd u dU 13 Map e SEN TV quu 13 alzidsclm 13 SPECIFICI T ses 14 GENOFF PE DETECTION rr 14 eec E E A 14 ROBU TNES oen 14 PRECISION E E E E E E A E E E E 15 CROSS
13. The recombinant immunoblot assay RIBA was used extensively to confirm the presence or the absence of antibody to HCV epitopes In RIBA recombinant or peptide HCV antigens are blotted as separate bands onto a nitrocellulose strip flanked by a weak positive Level and a moderately positive Level Il strip control Since ELISA and RIBA are antibody tests the positivity of either one or both does not necessarily indicate current HCV infection as patients who have recovered from infection may remain anti HCV positive for many years Conversely during seroconversion antibody tests may be negative The direct molecular biology detection of HCV RNA by reverse transcriptase polymerase chain reaction RT PCR is considered the gold standard for the diagnosis of HCV infection 4 INTENDED USE HCV Real TM Quant Dx Kit is a Real Time Amplification test for the Quantitative detection of Hepatitis C Virus in human plasma and the simultaneous detection of a HCV specific Internal Control IC by dual color detection Quantitative HCV RNA testing provides prognostic information regarding likelihood of treatment response to interferon monotherapy interferon plus ribavirin combination therapy and peginterferon plus ribavirin combination therapy Sustained virological response is defined as testing negative for HCV RNA 6 months after cessation of therapy Recent studies suggest that the rate of response to therapy is also important For istance con
14. _ sacace BIOTECHNOLOGIES u HCV Real TM Quant Dx Handbook Real Time PCR Kit for the Quantitative detection of Hepatitis C Virus in human plasma 1023 For Quantitative inVitro Diagnostic Use V1 96 3FRT 96 For use withRotor Gene 6000 Q Qiagen For use with SaCycler 96 Sacace Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy 2l 8 L lt gt EA KEY TO SYMBOLS USED List Number Lot Number For inVitro Diagnostic Use Temperature limitations Manufacturer Consult instructions for use Expiration Date Caution gt N ONTROL IN lt ONTROL ONTROL N ONTROL I 2 C gt r Contains sufficient for lt n gt tests Version Internal control High Positive Control Low Positive Control Negative Control Standard 1 Standard 2 TABLE OF CONTENTS nn 4 DOCG IN mc 4 INTENDED Ui 5 dul eMe won 5 MATERIALS de dpzpm 6 MATERIALS REQUIRED BUT NOT PROYVIDEDL ccr trn EE ku Eve ERE ERE EE VEF PE ERE YE Hen rea VER baa ke ue ant UE IS 6 WARNINGS AND PRECAUTIONS iota bk
15. act lenses in laboratory work areas Do not pipette by mouth Do not use a kit after its expiration date Do not mix reagents from different kits To reduce the risk of nucleic acid contamination due to aerosols formed during pipetting pipettes with aerosol barrier tips must be used Change aerosol barrier pipette tips between all liquid transfers During preparation of samples compliance with good laboratory practices is essential to minimize the risk of cross contamination between samples as well as the inadvertent introduction of nucleases into samples during and after the extraction procedure Proper aseptic technique should always be used when working with RNA or DNA Use only powder free gloves Dispose all specimens and unused reagents in accordance with local regulations Heparin has been shown to inhibit reaction The use of heparinized specimens is not recommended opecimens may be infectious Use Universal Precautions when performing the assay opecimens and controls should be prepared in a laminar flow hood Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of specimens or controls Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Follow by wiping down the surface with 7096 ethanol Avoid contact of specimens and reagents with the skin eyes and mucous membranes
16. box ta display help about its available settings Edit Profile 1 Channel Setup 470nm 10 530nm 10 585nm 625nm 680nm 2 Skip Wizard lt lt Back Next gt gt Figure 4 Edit Profile 8 a New Open Save Click on a cycle below to modify it Insert after Insert before Remove Hold Temperature 5p deg Hold Time 15 mins 0 secs Figure 5 Hold Reverse transcription step Y New Open SaveAs Help The run will take approximately 109 minute s to complete The graph below represents the run to be performed M Click on a cycle below to modify it Insert after Insert before Remove Hold Temperature 95 deg Hold Time 15 mins 0 secs Figure 6 Hold 2 Enzyme activation step Sacace HCV Real TM Quant Dx 06 06 2013 17 Open SaveAs Help The run will take approximately 109 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Insert after Insert before Cycling 2 Remove This cycle repeats 5 time s ick on one of the steps below to modify it or press or to add and remove steps for this cycle Timed Step 60 deg 95 deg for 5 secs 20 seconds using Long Range Od 20 Touchdown EO ONE TI EU SUY 72 deg for 15 secs New Open SaveAs Help The run will take approximate
17. e HCV Real TM Quant Dx assay is 13 IU mL with the 1 0 mL sample preparation procedure The LOD was determined by testing dilutions of the 4th WHO International Standard for Hepatitis C Virus for Nucleic Acid Amplification Techniques NIBSC code 06 102 15 prepared in HCV negative human plasma The resultswere determined by Probit analysis LINEAR RANGE Linear range or Linearity of an assay is its ability to obtain test results that are directly proportional to the concentration of the nucleic acid In other words it is the interval between the upper and the lower concentration where test results are directly proportional to the concentrations The upper limit of quantitation for the HCV Real TM Quant Dx assay is 100 million IU mL and the lower limit of quantitation is equal to the Limit of Detection 13 IU mL with the 1 0 mL sample preparation procedure The linear range of the HCV Real TM Quant Dx kit was determined by analyzing a dilution series from 8 00 log IU ml to 1 00 log IU ml of an HCV synthetic quantitative standard calibrated against the 4th WHO International HCV RNA Standard The results representative of the HCV Real TM Quant Dx assay linearity areshown in Figure 1 d n 0 163 E i IE 24 O e N U A u DN WD oO HCV Real TM Quant Dx Conc log IU ml 4 6 Target Concentration log IU ml SPECIFICITY The analytical specificity of the primers and the probes was validated with 100 negative plasma samples
18. e the tubes and transfer them into the Real Time PCR instrument Create a temperature profile on your Real time instrument as follows Fluorescence detection Cycle repeats Home 1 Osm 1 Ca l oe m 20 s using Rotor Gene 6000 Qinstrument 5 9 9 9 C 0 o 5 72 5 72 QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC is introduced into each sample at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition One replicate each of the Negative Control CONTROL the HCV High Control CONTROL 1 and the HCV Low Control CONTROL 2 must be included in each test run If the controls are out of their expected range see Data Sheet all of the specimens and controls from that run must be processed beginning from the sample preparation step RESULTS INTERPRETATION The Internal Control IC is detected on FAM Green channel and HCV RNA on Joe Yellow HEX channel Each DNA amplification is associated with generation of a fluorescence signal measurable in FAM Green channel for IC or in Joe Yellow HEX channel for HCV RNA resulting in a sigmoid growth curve log scale The data analysis is performed according to manufacturer s instructions Operation manual Rotor Gene 6000 Q Qiagen and Instrument Manual SaCycler 96 Sacace using the respective software and conside
19. eveloping hepatocellular carcinoma The discovery of HCV genome in 1989 by Choo et al paved the way for the development of serological and molecular assays for viral hepatitis C In the first generation of an enzyme linked immunosorbent assay ELISA wells of microtitre plates were coated with purified recombinant antigen c100 3 which was derived from the non structural 4 NS4 region of the HCV genome However ELISA 1 was associated with a high percentage 50 to 70 of false positive results among low risk blood donors and in the presence of hyperglobulinemia Thus second generation anti HCV ELISAs were developed ELISA 2 by Ortho Diagnostics contained recombinant antigens from the core c22 3 NS3 region c33c and NS4 region c100 3 as well as a part of c100 3 named 5 1 1 Third generation anti HCV ELISA was introduced in Europe in 1993 and in the USA in 1996 In addition to the antigens of ELISA 2 third generation anti HCV ELISA uses a NS5 region antigen of the viral genome However synthetic peptide antigens c22 and c 100 replaced recombinant antigens of ELISA 2 Other manufactures for example Abbott Diagnostics used recombinant antigens derived from the same regions of HCV genome Despite the increased sensitivity and specificity with each generation of ELISA false positive antibody results continue to be observed particularly among low risk blood donors Thus supplemental or confirmatory assays were developed in parallel with ELISA
20. fter the real time amplification Internal Control IC serves as an extraction and an amplification control for each individually processed specimen and to identify possible inhibition IC is detected in a channel other than the HCV RNA HCV IC L is a lyophilized Internal Control and represents recombinant RNA containing structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification detection The presence of HCV Rec IC allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the RNA during extraction procedure thus enabling to calculate precisely the HCV viral load The assay is standardized against the 4th WHO International Standard for Hepatitis C Virus for Nucleic Acid Amplification Techniques NIBSC code 06 102 15 and results are reported in International Units mL IU mL The target sequence for the HCV Real TM Quant Dx assay is in the 5 utr region of the HCV genome This region is specific for HCV and is highly conserved MATERIALS PROVIDED Sacace HCV Real TM Quant Dx Amplification Reagent Kit RT PCR REAGENT PACK 96 vials 0 2 ml with lyophilized amplification reagents Sacace HCV Real TM Quant Dx Control Kit Q ONTROL INT 4 vials with lyophilized reagent HCV IC L ONTROL 1 4 vials with lyophilized reagent HCV Pos1 L C Sacace HCV Real TM Quant High Positive Control ONTROL 2 4 vials with l
21. ibration run A defined consistent quantity of IC 10 ul is introduced into each specimen calibrator and control at the beginning of sample preparation and measured on the real time pcr instrument to demonstrate proper specimen processing and assay validity The median amplification cycle at which the IC target sequence fluorescent signal is detected during the calibration procedure two calibrators are run in replicates of three establishes an IC Ct validity range to be met by all subsequent processed specimens The established range of samples IC Ct Medium IC Ct of 6 calibrators 2 cycles Specimens whose IC Ct value exceeds the established range must be retested starting from sample preparation ASSAY CALIBRATION A calibration curve is required to quantitate the HCV RNA concentration of specimens and controls Two assay calibrators are run in replicates of three to generate a calibration curve HCV concentration versus the threshold cycle Ct at which a reactive level of fluorescent signal is detected Thecalibration curve slope and intercept are calculated and stored on the instrument The concentrationof HCV RNA in a sample is calculated from the stored calibration curve The quantitative standards CAL1 and CAL2 must be treated in the same way as patients specimens Before the first use of a new lot of HCV Real TM Quant Dx 6 calibrators run must be performed beginning from RNA extraction procedure to generate a calibration curve tw
22. ly 109 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Insert after Insert before Remove This cycle repeats 40 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle 95 deg for 10 secs 2 deg for 15 secs on Green Yellow Long Rares 60 deg for 20 secs Touchdown Figure 8 Cycling 2 Amplification and Detection Step 6 Click Gain Optimisation in the New Run Wizard menu and set the following parameters pg Auto Gain Optimisation etup Optimisation e Auto Gain Optimisation will read the fluoresence on the inserted sample at gt different gain levels until it finds one at which the fluorescence levels are acceptable The range of fluorescence you are looking for depends on the chemistry you are performing Set temperature to H degrees Optimise All Optimise Acquiring Perform Optimisation Before 1st Acquisition Perform Optimisation At 60 Degrees At Beginning Of Run Channel Settings Green SF 10 10 Yellow 5FI 10 10 Figure 9 Gain Optimisation Sacace HCV Real TM Quont Dx 06 06 2013 7 Place PCR tubes carefully onto RotorGene 6000 Q rotor close lid and start run Green Gain 10 Yellow Gain 10 Auto Gain Optimisation Before First Acquisi
23. n a positive or negative control value is out of the expected range it may indicate deterioration of the reagents Associated test results are invalid and samples must be retested Assay recalibrationmay be necessary QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality SAMPLE COLLECTION STORAGE AND TRANSPORT Note Handle all specimens as if they are potentially infectious agents Current studies refer to EDTA or citrate plasma as the most suitablesample materials for HCV detection Therefore we recommend the use of thesematerials with the HCV Real TM Quant Dx The internal validation of the HCV Real TM Quant Dx assay has been performedusing human EDTA plasma samples Other sample materials are not validated HCV Real TM Quant Dx assay 15 only for use with human plasma specimens 1 EDTA tubes may be used with the HCV Real TM Quant Dx Follow sample tube manufacturer s instructions 2 Whole blood collected in EDTA should be separated into plasma and cellular components by centrifugation at 800 1600 x g for 20 min within six hours The isolated plasma has to be transferred into a sterile polypropylene tube Plasma may be stored at 2 8 C for an additional 3 days Alternatively plasma may be stored at 18 C for up to one month or 1 year when stored at 70 C Do not freeze whole blood Specimens anti c
24. ns in method parameters and provides an indication of its reliability during normal usage As HCV Real TM Quant Dx kit contains RT PCR REAGENTS PACK 96 vials with ready to use lyophilized reagents the only probable variation during normal usage of the kit could be the different volume of added eluted samples obtained from RNA purification step The robustness of HCV Real TM Quant Dx kit was evaluated adding different volume of eluted sample from 46 to 52 ul 20 HCV RNA negative plasma pools spiked with HCV RNA to a final concentration of 3 times 95 per cent cut off value were added with small variations from 46 to 52 ul to the vials containing lyophilized reagents All tested samples were found positive for HCV RNA 14 PRECISION The precision of the HCV Real TM Quant Dx kit was evaluated for the manual sample preparation using the SaCycler 96 instruments The precision data of the HCV Real TM Quant Dx kit allow the determination of the total variance of the assay The total varianceconsists of the intra assay variability variability of multiple results of samples ofthe same concentration within run the inter assay variability variability of multiple results of the assay generated on different instruments and different days and the inter lot variability variability of multiple results of the assay using various lots The data obtained were used to determine the standard deviation and the coefficient of variation for the specific pa
25. ntitative results To do this click Import Curve select From Other Run option choose the Calibration file to be imported and Import Joe Yellow Channel SS Page Page Quantitation Analysis Cycling A Green Page 1 amp 8 amp Quantitation Analysis Cycling A Yellow Page 1 e Tae Std Curve 7 Results lt Dynamic Tube lt Slope Correct Ignore First El Outlier Removal bd Save Defaults 51 O Results Dynamic Tube lt Slope Correct lt Ignore First 2 Outlier Removal bd Save Defaults 12 10 08 ico 04 7 of 02 E 5 10 15 20 25 30 35 40 Cycle Bank On Bank Ott Adjust Scale Auto Scale Default Scale Log Scale j iene Cle ll NamedOn AllOn d Current Run Edit Samples E Quant Results Cycling Page 1 eme RY From Other Run 23 CT Calculation No Name Type e Given Conc Cale Conc Copie Var Rep Ct Rep Ct Std Rep Ct 857 Channels D 1 HCVG10 01 50001 Unknown 2027 2027 Invert Raw Data 2 HCVG10 01 2500 Il Unknown 21 06 21 06 Threshold 005 3 HCVG10 01 1250 Il Unknown 21 46 21 46 4 HCVG10 01 625 IU Unknown 21 09 21 09 F iminel 088 1 5 HCVG10 01 312 IU Unknown 20 77 2077 6 HCVG10 03 5000 Il Unknown 2064 2064 Frei Eitemal
26. o calibrators are run in replicates of three Prepare 3 sample preparation tubes for CAL 1 tubes for CAL2 for each calibration run e Add 10 ul of CONTROL INT to each tube AddCAL1 and CAL2 to the appropriate tubes inthe quantity indicated in the manual of RNA purification kit for example 1000 ul using Magno Virus kit Sacace REF K 2 16 Follow the samplepreparation procedure described in the Handbook of used RNA purification kit ASSAY PROTOCOL Note Reaction Mix volume 501 No attempt should be made to weigh out any portion of the freeze dried material prior to reconstitution REAL TIME PCR CALIBRATION PROCEDURE 1 Prepare 6 reactiontubes with lyophilized reagents to perform PCR of extracted calibrators 2 Add 50ul of eluted samples obtained from RNA purification of 6 calibrators Follow the procedure for amplification and detection as described in this manual Once an HCV Real TM Quant Dx calibration run is accepted and stored it may be used until the lot in use expires During this time all subsequent samples may be tested without further calibration by importing the experiment with Calibration Curve in the experiment with clinical samples using the same HCV Real TM Quant Dx lot number REAL TIME PCR SAMPLE PROCEDURE 1 Prepare requested quantity of reactiontubes with lyophilized reagents to perform PCR of extracted samples and controls 2 Add 50yl of eluted samples obtained from RNA purification step Clos
27. oagulated with heparin are unsuitable for this test Thaw frozen specimens at room temperature before using ae w Whole blood must be transported at 2 25 C and processed within 6 hours of collection Plasma may be transported at 2 8 C or frozen 7 Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Serum can be used also as starting material on some occasions In this cases the analytical sensitivity of the kit HCV Real TM Quant Dx is the same but the clinical sensitivity may be significantly decreased because of the precipitation of viral particles during the clot retraction phase of serum preparation 8 INTERFERING SUBSTANCES Elevated levels of bilirubin 215 mg dl and lipids 2800 mg dl do not influence the system Heparin 210 IU ml affects the PCR Samples that have been collected in tubes containing heparin as ananticoagulant should not to be used Also samples of heparinized patients mustnot be used REAGENTS PREPARATION Before starting any HCV Real TM Quant Dx protocol prepare the following reagents 1 Choose the requested quantity of lyophilized controls and calibrators and centrifuge briefly 2 Add Negative Control CONTROL as for table below Lyophilized reagent CONTROL ul CAL 1 1100 CAL 2 1100 CONTROL 1 1100 CONTROL 2 1100 CONTROL INT 3 Close the tubes and incubate all tubes for 2 min at room tem
28. odies by second generation assay a clinicopathologic study and demonstration of the usefulness of a third generation assay Dig Dis Sci 41 161 165 Schiff ER de Medina M Kahn RS New perspectives in the diagnosis of hepatitis C Sem Liver Dis 1999 19 3 15 Lok ASF Gunaratnam NT Diagnosis of hepatitis C Hepatology 1997 26 Suppl 1 48 569 Pawlotsky J M Bouvier Alias M Hezode C Darthuy F Remire J Dhumeaux D Standardization of hepatitis C virus RNA quantification Hepatology 2000 32 654 659 Manns MP McHutchisonJG GordonSC etal Peginterferonalfa 2bplusribavirincompared with interferonalfa 2bplusribavirinforinitialtreatmentofchronichepatitisC a randomized trial Lancet 2001 358 958 965 Weiland O Braconier JH Fryden A Norkrans Reichard O Uhnoo Influence of pretreatment factors on outcome of interferon alpha treatment of patients with chronic hepatitis C Scand J Infect Dis 1999 31 115 118 Fried MW Shiffman ML Reddy KR et al Peginterferon alfa 2a plus ribavirin for chronic hepatitis C virus infection NEngl J Med 2002 347 975 982 Jensen DM Morgan TR Marcellin P et al Early identification of HCV genotype 1 patients responding to 24weeks peginterferon alpha 2a AOkd ribavirin therapy Hepatology 2006 43 954 960 Davis GL Wong JB McHutchison JG Manns MP Harvey J Albrecht J Early virologic response to treatment with peginterferonalfa 2b plus ribavirin in patients with chronic hepatitis C Hepatology
29. perature Vortex periodically 4 Centrifuge the tubes for 5 sec 5 Dissolved reagents must be stored at 2 8 C and always protected from light up to 30days do not freeze RNA ISOLATION The following isolation kitis recommended Magno Virus Sacace REF K 2 16 sample volume 1000 yl SaMag 24 Sacace REF SM102 automated nucleic acid purification system SaMag Viral Nucleic Acid Extraction Kit sample volume 400 ul Please carry out the RNA extraction according to the manufacturer s instructions Add 10ul of CONTROL INTduring the RNA isolation procedure directly to the sample lysis mixture in all samples controls calibrators see Internal Control High and Low Positive Controls CONTROL 1 and CONTROL 2 as well as Neg Control CONTROL must be always used with patients specimens during RNA isolation procedure These controls must be treated in the same way as patients specimens followingthe Handbook of used RNA purification kit INTERNAL CONTROL HCV IC Lrepresents recombinant RNA containing structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification detection The presence of HCV IC Lallows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the RNA during extraction procedure thus enabling to calculate precisely the HCV viral load An IC threshold cycle Ct assay validity parameter is established during a cal
30. ring the recommendations of thehandbook Check the obtained results to ensure that the run is valid and to interpret results HCV RNA is determined basingon CT values and astandard curve resulting from analysis of quantitation standards RNA concentration is expressed in IU ml If the result is more than 100 000 000 IU ml then it will be displayed as the result more than 100 000 000 IU HCV ml If the result is more than the linear measurement range the sample can be analyzed after 10x dilution and the obtained result should be multiplied by 10 If the result is less than 13 IU ml in case of extraction from 1 ml then it will be displayed as the result less than 13 IU HCV ml PERFORMANCE CHARACTERISTICS The HCV Real TM Quant Dx Kit was evaluated according to the common technicalspecifications CTS for in vitro diagnostic medical devices 2009 108 EC and European Pharmacopoeia 7 0 p 2 6 21 Nucleic acid amplification techniques 07 2010 20621 ANALYTICAL SENSITIVITY The analytical sensitivity or Limit of Detection LOD is defined as the smallest amount of HCV RNA that can be precisely detected with a probability of 95 or greater The LOD in consideration of RNA purification from the sample is determined using HCV positive clinical specimens in combination with a particular extraction method HCV RNA was extracted with Magno Virus purification kit Sacace REF K 2 16 according to the manufacturer s instructions he LOD of th
31. thogen and the internal control The total variability calculated was mean SD Log Ct HCV mean SD Log Calc Conc log IU ml mean SD Log Ct IC 0 0045 0 0608 0 0046 mean CV Ct HCV mean CV 96 Calc Conc IU ml mean CV Ct IC 1 05 14 68 1 07 Intra assay variability SD log Ct HCV SD log Calc Conc log IU ml SD log Ct IC 0 004 0 004 CV Ct HCV CV Calc Conc IU ml CV Ct IC 0 86 15 70 0 81 Inter assay variability SD log Ct HCV SD log Calc Conc log IU ml SD log Ct IC 0 006 0 050 0 005 CV Ct HCV CV 96 Calc Conc CV 96 Ct IC 1 371 11 607 1 086 Inter lot variability SD log Ct HCV SD log Calc Conc log IU ml SD log Ct IC 0 004 0 069 0 006 CV 96 Ct HCV CV 96 Calc Conc CV 96 Ct IC 0 925 16 738 1 319 CROSS CONTAMINATION The cross contamination of the HCV Real TM Quant Dx kit was evaluated for the manual sample preparation using RotorGene Qinstrument To demonstrate absence of cross contamination a panel of 20 samples consisting of alternate samples of negative plasma spiked with high concentration of HCV and negative plasma samples was tested All the positive samples were correctly detected and gave a fluorescence positive reaction with good and clear sigmoid shaped fluorescence curves and a Cycle threshold Ct less than 35 both in HCV channel and in Internal Control channel All the negative samples gave no fluorescence signal in the HCV channel and the Internal Control channel showing no cross contamination
32. tion Rotor 36 VY ell Rotor Sample Layout ae oe Reaction Volume in microliters 50 Once you ve confirmed that your run settings are correct click Start Run to Save Template begin the run Click Save Template to save settings for future runs Skip Wizard lt lt Back Figure 10 Starting the run 8 Enter sample positions and names for samples Negative Control and Positive Control for controls If the kit calibration run is performing use name standard for CAL1 and CAL2 and enter the concentrations of the Quantitative Standards reported on HCV Real TM Quant Dx Data Card X Quantitation Analysis Cycling A Yellow Page 1 Bank On Bank Off Named AllOn All Off Edit Samples CT Calculation 15 20 25 2 Cycle Adjust Scale Auto Scale Default Scale Log Scale Invert Raw Data Cycling A Yellow Page 1 Threshold 0 03 Standard 12 70 3 500 000 R 0 99973 Elimi ate Cycles Standard 12 47 3 500 000 R 2 0 99945 i Standard 12 48 3 500 000 2 aaa M 3 293 Standard 22 39 3 500 d B 34 098 Auto Find Threshold Standard 22 59 3500 Efficiency 1 01 d Standard 22 30 3500 v 1 1 Concentration Standard Curve conc 10 0 304 CT 10 354 CT 3 293 og conc 34 098 A 9 User must import the calibration experiment with Standard Curves to subsequent experiments with clinical samples to have qua
33. version to an HCV RNA negative test result after 4 weeks of therapy constitutes a rapid virological response and it is a strong predictor of treatment success Patients who have not had an early virological response defined as at least a 2 log decline in HCV RNA after 12 weeks of therapy are unlikely to respond with an additional 36 weeks of therapy and should stop therapy HCV Real TM Quant Dx Real Time HCV assay is not for screening blood plasma serum or tissue donors for HCV or to beused as a diagnostic test to confirm the presence of HCV infection PRINCIPLE OF ASSAY HCV Real TM Quant Dx Kit is a Real Time test for the Quantitative detection of Hepatitis C Virus in human plasma HCV RNA is extracted from plasma amplified using real time amplification and detected using fluorescent reporter dye probes specific for HCV or HCV IC During each round of thermal cycling amplification products dissociate to single strands at hightemperature allowing primer annealing and extension as the temperature is lowered Exponentialamplification of the product is achieved through repeated cycling between high and lowtemperatures resulting in a billion fold or greater amplification of target sequences Amplificationof both targets HCV and IC takes place simultaneously in the same reaction Monitoring the fluorescence intensities during Real Time allows the detection and quantification of the accumulating product without having to re open the reaction tube a
34. yophilized reagent HCV Pos2 L C Sacace HCV Real TM Quant Dx Low Positive Control lt mr 2 4 0 vials 4 0 ml per vial with Negative Control Sacace HCV Real TM Quant Dx Calibrator Kit AL 1 4 vials with lyophilized reagent HCV Quantitative Standard 1 AL 4 vials with lyophilized reagent HCV Quantitative Standard 2 N Standards and controls concentrations are specific for every lot must be used during the sample preparation procedure see RNA isolation MATERIALS REQUIRED BUT NOT PROVIDED RNA isolation kit see RNA isolation Desktop microcentrifuge for eppendorf type tubes Vortex mixer Disposable gloves powderless Biohazard waste container Refrigerator Freezer Real Time Thermal cycler Biological safety cabinet approved for working with infectious materials Pipettes adjustable Sterile pipette tips with filters Tube racks WARNINGS AND PRECAUTIONS 10 11 12 13 14 15 16 17 18 In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only Read the instructions in this package insert carefully before processing samples Strict compliance with the user manual is required for optimal PCR results Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Use routine laboratory precautions Do not eat drink smoke apply cosmetics or handle cont
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