Devyser Thrombophilia Art. No.
Contents
1. 20 ccc cccecc ec cee ccc cececccccceececececeecseteeeeetseessteeseesseees 19 EN EA A A OO 19 Electrophoretic Artefacts 2 20 2 200 20 ccc ccc eee cece ccc cccccececccnceteeeceeseeseeseeneeseeseees 19 9 Performance Characteristics 0 00 0 0 0 cece cece cece ec ce cece cee cececeececceceeseceeeece 21 Sensitivity xo cot eel atte ek salen ath nei eds cas ed e aa A oral k re rr ice a ra 21 SDE CITI CI sosa d ci a Mais NADER ORSA ARE II S S E 21 Reproducibility 2 0 2 0 2 0 ccc ccc cece ccc ecceccecccccecceceeceeseecseneecseneeteenseestesetseeseesees 21 Within run reproducibility 0 20 2 00 20 c ccc cece cece ccc ccc ceccccceceecceceeseesetsseneeaees 21 Between run reproducibility 22 2200 00 00 cc oono cece cnccccceceeceesetstesseseeees 21 Clinical Evaluation osse sheen let tie 22 CrossiREAGUIVILY dde PARE e der e Sih SR BR BBB AAS e doo ein RER da BBE poo EG 23 10 Procedural Limitations 20 20 22 occ ccc cece cnc cccceccecceceeceeceesseetseeneensees 24 TTNotice to Purchaser ceci tocino msec ceria ddr tomos fuentes cero obe races eel Bess 25 12 RETErENCES asia 22256 A A 26 13 Contact Information occ ccoo c ence cece cenccncecceeceeseeseesetseteesseees 27 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 3 of 27 Devyser AB 2014 1 Introduction to Devyser Thrombophilia Intended Use The Devyser Thrombophilia kit is an in vitro diagnostic product for qualitative detection of genet2. Devyser p Devyser Thrombophilia Art No 8 A035 For in vitro Diagnostic Use Instructions for Use CE Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 1 of 27 O Devyser AB 2014 Table of Contents Table Of Contents ide 2 1 Introduction to Devyser Thrombophilia sssosooooosoooosssssoooosssssseooosssssseoooossseseoo ooo ena 4 INteNnded A 4 Included in the Kit 2 20 2 00 0 0 cece cece cece cece ec ceccecceceecceceeaceceecesceneetseeseteeeseess 4 ja ots tee seat g a Gece eae eee 4 Background cs nor dn ent A Gute este eR ee ee ae 4 Principle of the Procedure 2 2 2 0 2 0 cece ccc eccecceccecceccecceceeseeceesstseesetseeeeteeeees 5 2 Warnings and Precautions 22 2 0 20 0 e occ eee cece cece cece ececcececcececeececceceececeee 6 3 Symbols used on Labels 22 2 2 0 0 0 o occ c eee cece cee cececeececcececeeceeeeces 7 4 Required Material 2 2 2 0 2 0 0 0c ccc ccc cece ccc ec ccc cecceccecceeeeecesecscenettceteetetteseaes 8 4 1 Included in the kit 2 2 0 0 0 20 c cece cece ce cece cece ccecccceccececceeseeseeseeeseetnsetseneees 8 Configuration occ coco c cece ccc ec ccc ccccecccececccececeeteecseeseceeeeeteeesstsseteeseees 8 COMP OMENS ea sce se es te cee I EL NSA 8 4 2 Required but not Provided 0 20 2 c ccc cece cece ccc cccccccecencececsetceeseeseeseeseesees 8 Reagent Preparation 2 00 220 ccc ccc ence cece cece cece e eee n cece eee aaao aaan nnn 8 D
3. sample is to be considered heterozygously mutated in a certain locus if both a normal allele fragment and the corresponding mutation allele fragment are present Figure 8 2 Homozygous mutation The sample is to be considered homozygously mutated in a certain locus if the normal allele fragment is absent while the corresponding mutant allele fragment is present Figure 8 3 Peak cutoff signals rfu It is recommended that an instrument specific cutoff range is determined Do not to use lower cutoff values than 200 rfu ABI 310 3100 and 3130 and 500 rfu ABI 3730 3500 Do not anal yse samples where peak signals are saturated Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 16 of 27 Devyser AB 2014 Figure 8 1 Homozygous wildtype MTHFR 1298 1298A 180 200 220 180 200 220 Figure 8 2 Heterozygous MTHFR 1298 1298A 1298A gt C 180 200 220 MTHFR 12984 MTHFR 1298A C Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 17 of 27 Devyser AB 2014 Figure 8 3 Homozygous mutated MTHFR 1298 1298A gt C 180 200 220 MTHFR_12984 C Sizing of DNA fragments PCR fragments obtained using the Devyser Thrombophilia kit should be sized with the 560 SIZER ORANGE using a fragment analysis software e g GeneMapper Table 1 Normal alleles detected Normal alleles their expected PCR fragment length and dye colour ene Alle Size UI Colour Methyltetrahydrofolate Reductase MTHFR 1298A 215 Blu
4. stable at 2 8 C for at least 7 days and at below 18 C for at least 90 days Avoid repeated freeze thawing 7 2 Sample Preparation and PCR Amplification Sample Preparation It is recommended that alternative DNA extraction methods and sample materials are thor oughly evaluated with the Devyser Thrombophilia kit prior to the results being used for diag nostic use For the recommended PCR conditions and analysis settings see below results are consistently obtained at DNA concentrations between 25 and 150 ng PCR reaction 10 60 ng genomic DNA uL sample See also section 6 Addition of Sample Samples and controls should be added in a dedicated area separated from reagent preparation amplification and detection areas Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 12 of 27 Devyser AB 2014 1 Add 2 5 uL of clinical sample 10 60 ng genomic DNA uL sample to each PCR reaction tube containing activated Thrombo mix from step 7 1 2 Cap the tubes and centrifuge briefly to collect the content Amplification The Devyser Thrombophilia kit has been validated using Life Technologies ABl GeneAmp Sys tem 9700 and Life Technologies Veriti Thermal cycler Other PCR instruments should be tested and evaluated for optimal performance by the user before reporting results obtained with the Devyser Thrombophilia kit PCR instruments should be regularly calibrated and maintained to ensure accurate PCR performance For Life T
5. were tested using Devyser Thrombophilia All results obtained using Devyser Thrombophilia correlated with the previously obtained results Result gt 99 specificity Reproducibility Within run reproducibility Definition The degree of agreement between measurements conducted on replicate spec imens of the same measured where the measurements are carried out under unchanged conditions Experimental design Forty eight 48 replicates of a normal male sample was amplified in one run using identical reagent batches operator PCR and capillary electrophoresis instruments All 48 replicates gave the expected results Result gt 98 within run reproducibility Between run reproducibility Definition The degree of agreement between measurements conducted on replicate spec imens of the same measurand where the measurements are carried out under changed Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 21 of 27 Devyser AB 2014 conditions Experimental design One hundred and nine 109 replicates of a normal male sample were amplified on 3 different occasions using different reagent batches operators PCR and cap illary electrophoresis instruments All 109 replicates gave the expected results Result gt 99 between run reproducibility Clinical Evaluation One hundred and three 103 DNA samples previously characterised for at least one of the parameters analysed by Devyser Thrombophilia were tested blind Al
6. 7 Instructions for Use 7 1 Workflow Devyser Thrombophilia Each Devyser Thrombophilia kit art 8 A035 contains reagents for 48 samples The activated reaction mix should be prepared before preparing the DNA samples if the complete process is performed in one day The opposite order is advisable only if the DNA samples are prepared the day before amplification or earlier The activated reaction mix is prepared by adding the Thromb mix to the PCR Activator It is rec ommended that the activated reaction mix is dispensed into appropriate PCR reaction tubes after preparation Before dispensing ensure that the activated reaction mix is properly mixed see below Dispense in 10 uL aliquots and store at below 18 C Devyser Thrombophilia has been validated using a total PCR reaction volume of 12 5 uL Chang ing the reaction volume will compromise the kit performance Ensure that the Thromb mix is completely thawed before use Centrifuge each tube briefly to collect the content Do not vortex the tubes at this step Add 500 uL from the Thromb mix to the PCR Activator Carefully mix by pipetting several times from the bottom of each tube Vortex the activated reaction mix tube and centrifuge briefly to collect the content Add 10 uL of the activated reaction mix to separate PCR reaction tubes Cap the reaction tubes and centrifuge briefly to collect the contents Continue to step 7 2 NOUBWNPE The activated reaction mix is
7. NA Extraction 235300 ssustoslosos rbsrosdss de ssd ns area 8 Amplification 2202 00 00 epos died ccc cece ccc cncccccecccccececcecceeeeeetaceceetstteenseeseteeseenes 8 Detection oo cece cece cece eee e cece cece cee eeeeeeeeececeeeeeceeceeseeeeeeseeseeseeseeees 8 Size Standard 22 scc2e025 cccanbieeki ddd salgdc nens olle bedded a Eoi 8 4 3 Dye Set Calibration 22 20 2002 cocoon ccc cece ccc cc ccc cececeeececetcenseesteseeseesseneees 9 5 Storage and Handling Requirements 2 0 0 0 2 0 c cece ec cc ec e cee cececeececeececes 10 6 Sample Requirements coco coco ccoo aaao 1 cee cece aaan a aaeoa anann 11 Clinical Samples eee 11 DNA Extraction and Measurement soosssssssooooossssssooossssseeooosssssseoooossssseoooo soon nan 11 Procedure and Storage 2 0 ec ccc ccc ccc ence cece ccccceccececseeeeesecseceetseenetsetseeees 11 7 Instructions Tor Use coco ii dis 12 7 1 Workflow Devyser Thrombophilia oooooooococcccccccccccccccccccccccccccccncnncnncnnccnooo 12 7 2 Sample Preparation and PCR Amplification 0 0 00 0 0 0 0 c cece eee e ec eceecec ee ceceees 12 Sample Preparation 2 22 2 00 0 0c ccc cece cc cnccnccncceccececceececeeececseesstsseseteceneeseees 12 Addition of Sample 2 2 2 20 20 2002 2 cece cece cece ccc cccceccceceeccceeceteeeceeseeseeeteseseeneeaes 12 Amplification 2 0 20 0 00 20 ccc cece cece cece ccc ccccecccceeceeseesceecenseneeccecseeseeseeeeseeeess 13 A ees ee a 14 Sample Preparat
8. Thrombophilia Results obtained with Devyser Thrombophilia kit can only be directly applied to the tissue or spe cific sample material tested Only the following mutations are tested Factor V G1691A Factor V H1299R Prothrombin G20210A PAI 1 SERPIN1 4G 5G MTHFR C677T and MTHFR A1298C F Diagnostic errors can occur due to rare sequence variations Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 24 of 27 Devyser AB 2014 11 Notice to Purchaser Results from Devyser Thrombophilia should be interpreted with consideration of the overall pic ture obtained from clinical and laboratory findings Devyser AB will not accept responsibility for any clinical decisions taken LIZ Veriti and GeneAmp are registered trademarks of Life Technologies Corporation GeneS can M pop 41M pop 7 and Hi Di M are trademarks of Life Technologies Corporation Purchase of this product does not provide a license to perform PCR under patents owned by any third party Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 25 of 27 O Devyser AB 2014 12 References 1 Mitchell RS Kumar V Abbas AK Fausto N 2007 Chapter 4 Robbins Basic Pathology Eighth ed Philadelphia Saunders ISBN 1 4160 2973 7 2 Heit JA 2007 Thrombophilia common questions on laboratory assessment and man agement Hematology Am Soc Hematol Educ Program 2007 1 127 35 doi 10 1182 asheducation 2007 1 127 PMID 18024620 3 Kyrle PA Rosen
9. a is frequently caused by mutations in genes coding for coagulation fac tors such as factor V Leiden and prothrombin 1 4 5 Common conditions associated with thrombophilia are deep vein thrombosis DVT and pul monary embolism PE collectively referred to as venous thromboembolism VTE Throm bophilia has also been linked to recurrent miscarriage 6 7 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 4 of 27 Devyser AB 2014 Principle of the Procedure The Devyser Thrombophilia kit is based on multiplex allele specific PCR amplification for detec tion of normal non mutated and mutated alleles in the following loci Factor V Leiden Factor V R2 Prothrombin FIl 20210 MTHFR 677 MTHFR 1298 PAI 1 Serpin 1 4G 5G Allele specific PCR amplification generates fluorescently labelled fragments that are analysed by capillary electrophoresis CE on a Genetic Analyzer instrument Amplified fragments are identified based on size and fluorescent labels Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 5 of 27 Devyser AB 2014 2 Warnings and Precautions A Devyser Thrombophilia has been validated using a total PCR reaction volume of 12 5 uL Chang ing the reaction volume will compromise the kit performance B Avoid microbial contamination of ragents when removing aliquots from reagent vials The use of sterile disposable aerosol barrier pipette tips is recommended C Do not pool reag
10. daal FR Eichinger S December 2010 Risk assessment for recurrent venous thrombosis Lancet 376 9757 2032 9 doi 10 1016 S0140 6736 10 60962 2 PMID 21131039 4 Rosendaal FR 2005 Venous thrombosis the role of genes environment and behavior Hematology Am Soc Hematol Educ Program 2005 1 1 12 doi 10 1182 asheducation 2005 1 1 PMID 16304352 5 Crowther MA Kelton JG 2003 Congenital thrombophilic states associated with venous thrombosis a qualitative overview and proposed classification system Ann Intern Med 138 2 128 34 doi 10 7326 0003 4819 138 2 200301210 00014 PMID 12529095 Lay summary 6 Scarvelis D Wells PS October 2006 Diagnosis and treatment of deep vein thrombosis CMAJ 175 9 1087 92 doi 10 1503 cmaj 060366 PMC 1609160 PMID 17060659 7 Rai R Regan L August 2006 Recurrent miscarriage Lancet 368 9535 601 11 doi 10 1016 S0140 6736 06 69204 0 PMID 16905025 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 26 of 27 Devyser AB 2014 13 Contact Information Devyser AB Instrumentv gen 19 SE 126 53 H gersten SWEDEN Phone 46 8 562 15 850 Homepage www devyser com Technical Support Phone 46 8 562 15 850 E mail techsupport devyser com Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 27 of 27 Devyser AB 2014
11. dispenser with aerosol barrier tips or displacement tips Size Standard 560 SIZER ORANGE Devyser cat 8 A402 or GeneScan 600 LIZ Size Standard Life Tech nologies cat 4366589 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 8 of 27 Devyser AB 2014 4 3 Dye Set Calibration ABI 3100 3130 3730 Use DEV 5 Dye Set MultiCap kit Devyser cat 8 A401 in the Any5Dye Dye Set ABI 3500 Use DEV 5 Dye Set MultiCap kit Devyser cat 8 A401 and generate the DEV 5 Dye Set ABI 310 Matrix file generation Use DEV 5 Dye Set SingleCap kit Devyser cat 8 A400 Run with module file GS STR POP4 1 mL G5 md5 Detailed instructions for Dye Set calibration may be downloaded from the download section at http www devyser com af pcr accessories dev 5 calibrators Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 9 of 27 O Devyser AB 2014 5 Storage and Handling Requirements A Store all components below 18 C B The activated reaction mix prepared by addition of Thromb Mix to the PCR Activator tube may be stored at 2 to 8 C for at least 7 days and at below 18 C for at least 90 days Avoid repeated freeze thawing C Dispose of unused reagents and waste in accordance with country federal state and local reg ulations D Do not mix reagents from different kit lot numbers Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 10 of 27 Devyser AB 2014 6 Sample Requi
12. e Plasminogen Activator Inhibitor 1 PAI I SERPIN 1 sc 233 Blue MENE ASS E Reductase MTHFR 677C 295 Bue Factor II Prothrombin amos 312 Bue faoa EE ae Factor v eid a 7 ue Allele fragment lengths given in this table are based on average observed fragment lengths obtained using ABI3130 and ABI3500 POP 7 polymer and 560 SIZER ORANGE Allele fragment size ranges may vary depending on the instrument polymer type and size marker used during electrophoresis Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 18 of 27 Devyser AB 2014 Table 2 Mutant alleles detected Mutant alleles their expected PCR fragment length and dye colour gene ele ske bar Colour Factor V Leiden 1691G gt A Plasminogen Activator Inhibitor 1 PAI I SERPIN 1 Allele fragment lengths given in this table are based on average observed fragment lengths obtained using ABI3130 and ABI3500 POP 7 polymer and 560 SIZER ORANGE Allele fragment size ranges may vary depending on the instrument polymer type and size marker used during electrophoresis 8 3 Troubleshooting PCR Artefacts A peaks figure 8 4 are detected as extra peaks that is one base pair shorter than the full length A peak PCR product Figure 8 4 A and A peaks as indicated by the arrows Electrophoretic Artefacts Crosstalk bleed through between dye channels may occur during detection Figure 8 5 Cross talk appears as equally si
13. echnologies GeneAmp PCR System 9700 Set ramp speed to MAX For Life Technologies Veriti Thermal cycler In the Tools Menu select Convert a Method In the next step select 9700 Max Mode and then enter the PCR profile as outlined below Other thermal cyclers The following ramping rates must be applied heating 1 6 C s cooling 1 6 C s Amplification Area Program the Thermal Cycler for amplification according to the following thermal profile con sult the User s Manual for additional information on programming and operation of the ther mal cycler 95 C 15 min 94 C 0 5 min gt 62 C 1 min gt 72 C 1 min for 25 cycles 72 C 15 min 4 C FOREVER 49 C i fe a ale pe DISS dca ol E i 0 3 mn i TAA E Fen I 3 i i L E E i i Fl min 15 min A i i i y i x 6c FS i i mera i r i 1 min I 4 I I fen j A A E 4 C L x 25 ee T te Lia Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 13 of 27 Devyser AB 2014 1 Set reaction volume to 13 uL 2 Set the appropriate ramping rates heating 1 6 C s cooling 1 6 C 3 Start the amplification duration approximately 2 5 hrs 4 Following amplification remove the tubes containing completed PCR amplification reaction from the thermal cycler and place into a suitable holder Centrifuge briefly to collect the con tent Remove the caps carefully to avoid aerosol contamination Do not bring amplified mate rial
14. ents from different lots or from different vials of the same lot D Do not use a kit after its expiry date E Do not use opened or damaged kit reagent vials F Work flow in the laboratory should proceed in a unidirectional manner beginning in the rea gent preparation area and moving to the DNA extraction area and then to the amplification area and finally to the detection area Pre amplification activities should begin with reagent preparation and proceed to DNA extraction Reagent preparation activities and DNA extraction activities should be performed in separate areas Supplies and equipment should be dedicated to each activity and not used for other activities or moved between areas Gloves should be worn in each area and should be changed before leaving that area Equipment and supplies used for reagent preparation should not be used for DNA extraction activities or for pipetting or processing amplified DNA or other sources of target DNA Amplification and detection supplies and equipment should remain in the amplification and detection area at all times G Handling of kit components and samples their use storage and disposal should be in accord ance with the procedures defined by national biohazard safety guidelines or regulations H Wear powder free disposable gloves laboratory coats and eye protection when handling spec imens and kit reagents Wash hands thoroughly after handling specimens and kit reagents Devys
15. er Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 6 of 27 Devyser AB 2014 3 Symbols used on Labels LOT Lot or batch number Expiry date Number of tests Store below temperature shown Catalogue number Manufacturer sE gt lt J V In vitro diagnostic device Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 7 of 27 Devyser AB 2014 4 Required Material 4 1 Included in the kit Configuration The Devyser Thrombophilia test kit contains reagents for analysis of 48 samples Components 4 2 Required but not Provided Reagent Preparation Consumables for the Thermal Cycler Micropipette dispenser with aerosol barrier tips or displacement tips Disposable protective gloves powder free DNA Extraction Reagents and equipment according to manufacturer s instructions for use Micropipette multipipette with aerosol barrier tips Amplification Thermal Cycler Life Technologies ABI GeneAmp PCR System 9700 and Life Technologies Veri ti Thermal cycler For use of alternative thermal cyclers the following ramping rates must be applied heating 1 6 C s cooling 1 6 C s Micropipette dispenser with aerosol barrier tips or displacement tips Detection Life Technologies ABI Genetic Analyzer ABI 310 3100 3130 3500 3730 Performance optimized polymers POP 4 or POP 7 Hi Di Formamide Genetic Analysis Grade 1x Genetic Analyzer Buffer Micropipette multipipette
16. ic variants that may be associated with thrombophilia The test can distinguish between individuals who are heterozygote and homozygote for all tested genetic variants The following alleles are detected Factor V Leiden normal and mutated Factor V R2 normal and mutated Prothrombin Fll 20210 normal and mutated MTHFR 677 normal and mutated MTHFR 1298 normal and mutated PAI 1 Serpin 1 4G and 5G Included in the Kit Analysis of the different alles is performed using one single amplification mix Thromb Mix Test Procedure DNA extraction The Devyser Thrombophilia kit has been validated using QlAamp DNA Blood Mini Kit Qiagen cat 51104 for extraction of DNA from human whole blood Amplification The Devyser Thrombophilia kit has been validated using Life Technologies ABI GeneAmp System 9700 and Life Technologies Veriti Thermal cycler Detection Life Technologies ABI Genetic Analyzers ABI 310 3100 3130 3500 3730 that sup port detection of Devyser Dye Set DEV 5 Background Thrombophilia is caused by abnormalities in blood coagulation that increases the risk of throm bosis 1 2 These abnormalities are frequently seen in patients who have an episode of throm bosis that was not provoked by other causes 3 A significant proportion of the population has a detectable abnormality but most of these only develop thrombosis in the presence of an addi tional risk factor 2 Congenital thrombophili
17. ile GS STR POP4 1 mL G5 md5 Run Parameters Capillary length Run temperature Injection voltage Injection time Run voltage Run time ABI 3100 3130 ABI 3500 ABI 3730 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 15 of 27 O Devyser AB 2014 8 Data Analysis Background to data analysis An individual has two copies alleles of each of the investigated loci and is considered being homozygous for a given DNA sequence when both alleles have the same sequence An individ ual is considered being heterozygous for a given DNA sequence when the two alleles differ in sequence 8 1 The Devyser Thrombophilia kit The Devyser Thrombophilia kit is used for the qualitative analysis of mutations in the loci out lined in tables 1 and 2 Analysis of the different alleles is performed using one single ampli fication mix Thromb Mix All detected normal and mutant alleles are listed in tables 1 and 2 Normal alleles PCR fragments representing normal DNA sequences are detected in the blue channel as out lined in table 1 below Mutant alleles PCR fragments representing mutant DNA sequences are detected in the green channel as out lined in table 2 below 8 2 Data interpretation Normal non mutated alleles The sample is to be considered normal for a certain locus if a normal allele fragment is detected while the corresponding mutant allele fragment is absent Figure 8 1 Heterozygous mutation The
18. into the pre amplification areas Amplified material should be restricted to amplification and detection areas 7 3 Detection Sample Preparation Refer to the respective Life Technologies ABI Genetic Analyzers User Manual for instructions on maintenance and handling Prior to running the Devyser Thrombophilia kit the instrument must be spectrally calibrated to support detection of the Dye Set DEV 5 See section 4 3 for details Sample Preparation for Capillary Electrophoresis 1 Prepare a loading cocktail by combining and mixing 2 uL of the size standard e g 560 SIZER ORANGE with 100 uL Hi Di M Formamide sufficient mix for 6 wells tubes 2 Vortex for 15 seconds 3 Dispense 15 uL of the loading cocktail into the required number of wells of a microwell plate or into individual tubes to be placed on the Genetic Analyzer 4 Add 1 5 uL of the sample PCR product to the corresponding well tube containing loading cock tail 5 Seal the plate tubes Instrument Preparation Create a sample sheet using the data collection software with the following settings e Sample ID e Dye Set Any5Dye DEV 5 e Recommended run Module See below for different polymers and instruments Run Modules The amount of PCR product injected into the capillaries can be adjusted by increas ing decreasing the injection time and or injection voltage Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 14 of 27 O Devyser AB 2014 ABI 310 Run with module f
19. ion 2 22 2 00 00 20 ccc ccc cece cnceeccncceccececceececseesecseeestssesetseeneeseees 14 Sample Preparation for Capillary Electrophoresis 0 0 00 2 2ccccceeceeeeeeeeees 14 Instrument Preparation 2 0 2 2 200 22 cece cece ccc ce cece cece cece cece ecececeeeceeeeeeseeseees 14 Run Modules 22 20 2222 cece cc ccc cece ccc cccccececcecceeceeeecseeseeaeteetnetseeneeteseeeeeeeae 14 8 Data Analysis oia ta toa 16 Background to data analysis 2 2 0 2 00 2 cc ccc ecceccecceececceeceeeeecetceesetsteseseeees 16 8 1 The Devyser Thrombophilia kit 2 20 2 00 00 c cece cece cece ccc cecccccecceceeceeseeseeseeses 16 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 2 of 27 Devyser AB 2014 Normal alleles torrsmotrorna ro rod e od 16 AMA O E 16 8 2 Data interpretation a 16 Normal non mutated alleles coco coccion 16 Heterozygous Mutation 2 0 2 2 ooo cece cece e cece ence cece eee ececeeeceeeeeseesseeeees 16 Homozygous mutation 22 2 2 202 2 c cece eevee cece cece cece D DDD DLLD D L D r rr rron 16 Peak cutoffsignals o een tical et dea cero stele ins 16 Sizing of DNA fragments aaao aaa aaa ccc cece ence ccc cecceceececceeseeseeseesetseeneeees 18 Table 1 Normal alleles detected 0 2 2 00 o oo cece cece cece ec ceccccceceecceseeseeetesees 18 Table 2 Mutant alleles detected 222 0 0 cc cece cc cncccccecceceeceeseeseesseseesees 19 8 3 Troubleshooting 2 2 0 2
20. l samples were suc cessfully analysed Results are summarised in table 1 below Table 1 Results from the clinical evaluation of 103 previously characterised clinical samples Factor V 61691 Leiden A O EC Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 22 of 27 O Devyser AB 2014 Cross Reactivity No cross reactivities have been observed Devyser Thrombophilia CE IVD 7 AO30 EN v 4 2014 Page 23 of 27 Devyser AB 2014 10 Procedural Limitations A Use of this product should be limited to personnel trained in the techniques of PCR and capillary electrophoresis The Devyser Thrombophilia kit has been validated using Life Technologies ABI Thermal Cycler GeneAmp 9700 and Life Technologies Veriti Thermal cycler It is recommended that alter native thermocycler instruments are thoroughly evaluated with the Devyser Thrombophilia kit prior to the results being used for diagnostic use C The Devyser Thrombophilia kit has been validated using QlAamp DNA Blood Mini Kit for extrac tion of DNA from human whole blood Performance with other matrices and DNA extraction kits has not been validated and may result in false negative or false positive results D Devyser Thrombophilia kit should be used only for the detection of specific mutations according to the instructions for use Many other mutations are possible that may not be detected using Devyser Thrombophilia The assay has not been validated for diagnosis of
21. rements Clinical Samples The Devyser Thrombophilia kit is for use with human genomic DNA extracted from whole blood DNA Extraction and Measurement Preparation of DNA from whole blood samples Results are consistently obtained with DNA extracted from human whole blood using QlAamp DNA Blood Mini Kit Qiagen cat 51104 Follow the protocol starting with 200 uL fresh whole blood and elute in 200 uL elution buffer It is normally not necessary to determine the con centration of the purified DNA sample if this procedure is followed The purified DNA can be used directly for PCR without any further dilution It is recommended that alternative DNA extraction methods and sample materials are thor oughly evaluated with the Devyser Thrombophilia kit prior to the results being used for diag nostic use For the recommended PCR conditions and analysis settings see sections 7 2 7 3 results are consistently obtained at DNA concentrations between 25 and 150 ng PCR reaction 10 60 ng genomic DNA uL sample All DNA concentrations referred to in this handbook were determined using Qubit dsDNA HS Assay Kit Life Technologies cat Q32851 The DNA concentration determined in a sample using Qubit dsDNA HS Assay Kit may differ from the DNA concentration determined by other methods Procedure and Storage According to manufacturer s instructions for use Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 11 of 27 O Devyser AB 2014
22. zed peaks in neighbouring dye channels and should be excluded from the analysis Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 19 of 27 O Devyser AB 2014 Figure 8 5 Crosstalk peak from green to blue channel as indicated by the arrow Dye blobs may appear in the sample analysis range figure 8 6 In general dye blobs appear as broad undefined peaks of a single colour and tend to occur relatively early in the data Figure 8 6 Dye blob as indicated by the arrow 120 170 210 250 MTHFR_12934 Devyser Thrombophilia CE IVD 7 A030 EN v 4 2014 Page 20 of 27 Devyser AB 2014 9 Performance Characteristics Sensitivity Definition The proportion of subjects with a well defined genetic condition and whose test values are positive within the defined decision limit Experimental design One hundred 100 DNA samples previously characterised to carry at least one of the listed mutations chapter 8 table 2 were tested using Devyser Thrombophilia All results obtained using Devyser Thrombophilia correlated with the previously obtained results Result gt 99 sensitivitya Specificity Definition The proportion of subjects who do not have a well defined genetic condition and whose test results are negative or within the defined decision limit Experimental design One hundred one 101 DNA samples previously characterised to carry at least one copy of all the listed normal alleles chapter 8 table 1
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