Hologic MTHFR 1298 IVD Package Insert
Contents
1. Do not interchange components from their sources or from different lots Do not pool reagents from different lots or from different vials of the same lot Take reasonable precautions when handling reagents Use disposable gloves when handling suspected carcinogens or toxic materials Do not smoke eat or drink in areas where specimens or reagents are being handled Avoid contact of eyes and mucous membranes with reagents If reagents come in contact with sensitive areas wash with copious amounts of water Patient specimens and all materials coming into contact with them should be handled as if capable of transmitting infection and disposed of with proper precautions Never pipette by mouth and avoid contact of reagents and specimens with skin and mucous membranes Avoid microbial contamination of reagents as this could produce incorrect results Incubation times and temperatures other than those specified may give erroneous results The reagents have been optimally formulated and further dilution may result in loss of performance Do not use reagents after their expiration date Use fresh mineral oil for each reaction set up do not transfer these reagents back to the original container once they have been dispensed Provided genotype specific controls are in a blood like matrix and are not infectious Material can be used in a Bio Safety Level 1 laboratory IMPORTANT CONTAMINATION PRECAUTIONS This product generates amplified DNA targets2. F NOTE Product requires multiple storage temperatures for reagents Immediately upon receipt genotype specific controls are to be stored at 2 C to 8 C All other components of the kit should be stored between 30 C to 15 C in a non frost free freezer Prior to use allow reagents to equilibrate to room temperature excluding the Universal Enzyme Mix which should remain between 30 C to 15 C until just prior to use Minimize reagent exposure to light Do not subject the reagents to more than 15 freeze thaw cycles Indications of Instability When properly stored the reagents are stable through the dating indicated on the label There are no obvious signs to indicate instability of this product However genotype specific controls should be run simultaneously with unknown specimens Increase in non specific fluorescence signal may indicate reagent instability If this is observed contact Hologic Technical Support 888 898 2357 G Specimen Collection and Preparation for Analysis Clinical Specimens Human whole blood samples should be anti coagulated with potassium EDTA DNA extraction may be accomplished using commercially available DNA extraction chemistries capable of obtaining DNA concentrations greater than 5ng uL for use in the Invader MTHFR 1298 test Genotype Specific Control Samples Genotype specific i e WT HET MUT controls are provided with the test Genotype specific controls consist of synthetic DNA in
3. x 3 sites 270 9 samples Within Operator Within day x 10 day pairs x 4 2 reps per Within Operator day x 2 operators x 3 sites 2160 Between days 9 samples x 4 2 reps per operator x 25 day pairs between 2 operators x 3 sites 2700 2700 100 99 5 9 samples x 20 tests at site a x 20 tests at site b x 3 site pairs 10800 10800 100 99 5 b Lot to Lot Reproducibility Whole blood samples were extracted and subjected to bi directional DNA sequencing The same DNA samples were then analyzed using the Invader MTHFR 1298 test with three different lots of the reagents The observed agreement between all three lots of the Invader MTHFR 1298 test and bi directional DNA sequencing was 100 108 108 see Table 8 Across all genotypes tested across all three 3 lots the overall agreement with bi directional sequencing was 100 108 108 with 97 26 one sided lower 95 confidence limit Between operator within site Between Sites Table 8 Lot to LotAgreement between Invader MTHFR 1298 and Bi directional DNA Sequencing MTHFR 1298 Number of Replicates Correct Genotype Genotypes per Genotype Calls aea A ae a are ae bi directional Tested Sample in 1 Run g Invader MTHFR 1298 Homozygous Wild Type AA Heterozygous AC 12 3 Homozygous CC roa o o lele Mutant 3 4 12 12 12 0 0 Upper and Lower Limit
4. Hologic within one 1 year after the claim arises Customer shall be barred from instituting any legal action thereafter These remedies shall comprise Hologic s entire liability and Customer s exclusive remedy for breach of warranty and are in lieu of any other remedies at law or equity LIMIT OF LIABILITY HOLOGIC SHALL NOT BE LIABLE FOR ANY SPECIAL INCIDENTAL PUNITIVE EXEMPLARY OR CONSEQUENTIAL LOSSES DAMAGES OR EXPENSES INCLUDING BUT NOT LIMITED TO LOSS OF PROFITS DATA OR USE DIRECTLY OR INDIRECTLY ARISING FROM THE SALE HANDLING SERVICE OR USE OF PRODUCT ORDERED OR FURNISHED OR FROM ANY CAUSE RELATING THERETO UNLESS EXPRESSLY AGREED TO BY THE PARTIES IN WRITING EXCEPT FOR PERSONAL INJURY OR DEATH TO THE EXTENT RESULTING FROM HOLOGIC S NEGLIGENT OR INTENTIONALLY WRONGFUL ACTS OR OMISSIONS IN NO EVENT SHALL HOLOGIC BE LIABLE UNDER ANY LEGAL THEORY OR FOR ANY CAUSE WHATSOEVER WHETHER BASED UPON WARRANTY CONTRACT TORT NEGLIGENCE OR OTHER THEORY EVEN IF ADVISED OF THE POSSIBILITY THEREOF FOR ANY AMOUNT IN EXCESS OF THE PRICE FEE OR CHARGE THEREFORE RECEIVED BY HOLOGIC Cleavase Invader Invader Plus and Invader Call Reporter are registered trademarks of Hologic Inc All other Trademarks Registered Trademarks referenced within this product insert are the property of each of their respective companies Some components of nucleic acid analysis such as specific methods and compositions for manipulating or visualizin
5. a blood like matrix and are not infectious Genotype specific controls must be extracted prior to use and can serve as a DNA extraction control as well if prepared using the same method as the blood samples Prior to extraction genotype specific controls should be vortexed 30 60 seconds to re suspend the contents DNA Storage The purified DNA from samples and genotype specific controls can be used immediately or safely stored in elution buffer as per the DNA Extraction Kit manufacturer s recommendation DNA Preparation Extracted clinical specimen and genotype specific control DNA must be diluted 1 20 in nuclease free water just prior to running the Invader MTHFR 1298 test see Section IV A 3 The level of DNA present in the extracted genotype specific controls may not be detectable with certain quantitation methods and is not quantifiable by spectrophotometer measurements Page 2 of 9 lll SAFETY ISSUES A Safety and Handling Precautions 1 Universal safety precautions should be used when handling any human whole blood samples Specimens should be disposed of according to local requirements Product components product residuals and packaging can be considered laboratory waste Dispose of unused reagents and waste in accordance with applicable federal state and local regulations B Precautions h The Invader MTHFR 1298 test is intended for in vitro diagnostic use These components have been tested as a unit
6. be used within 30 minutes Note Controls must be placed in the correct wells for proper data interpretation Refer to Figure 2 Table 2 Invader MTHFR 1298 ReactionMix st Numb Component uLiwell erof 25 Overage tal Volume UL MTHFR 1298 Oligo Mix 7 5k 1 25 Universal Buffer 2 0 pL 15 2 0K 1 25 Universal Enzyme Mix 0 5 uL 1 25 0 5k 1 25 Total Mix Volume 10 uL 10k 1 25 7 Vortex the reaction mix thoroughly and spin down the contents in a microcentrifuge for 3 5 seconds Plate Set up 8 Reaction mix may be aliquoted into a 96 well plate to facilitate the use of a multi channel pipettor Page 3 of 9 9 Add 10 uL of reaction mix to the bottom of each control and sample well of the reaction plate 10 Dispense 10 uL of the appropriate control 1 Figure 2 or sample genotype specific controls and Plate all samples are diluted See Step 3 Position of Control 4 NDC is undiluted to bottom of Controls in the appropriate well of the reaction plate a 96 well See Figure 2 Mix by pipetting up and down 3 5 times upon addition to ensure plate reaction homogeneity Change pipette tips between every addition QOOOOOOC 0 SOOS0066 O0000000 11 Overlay all control and sample wells with 20 uL of mineral oil by dispensing along the side of the wells Change pipette tips between every addition QOOOOOO OQQQQQQQ OQQOQQQQQQ OQOQQQQQQ OQQQQQQQQ 12 Cover the reaction pla
7. plate Verify reactions are covered by 20 uL of mineral oil Verify the 96 well plate is firmly sealed with optical clear adhesive cover before incubating Verify thermal cycler heated lid is firmly closed If the thermal cycler requires a compression pad verify that the compression pad is seated properly on top of the 96 well plate Verify the correct Invader Reaction Program was used Section IV A Figure 3 Repeat sample test added to reaction Verify the 96 well plate is compatible with the thermal cycler is firmly seated in the thermal cycler and secured properly Store extracted DNA as indicated in the DNA extraction and purification protocol prior to the Invader test Troubleshooting Re test Procedure If the established criteria for an acceptable genotype call i e WT HET or MUT are not met by a given sample it is identified as either Low Signal or EQ and the sample s must be re tested A given extraction of a sample that has two EQ equivocal results in a row cannot be called by the Invader MTHFR 1298 test If a sample fails to produce the minimum fold over zero then the Invader test gives a Low Signal result and the sample must be re tested Figure 4 illustrates the recommended re test process for samples with Low Signal EQ or Invalid results Invader MTHFR 1298 Do Invader controls all generate valid results ReRun Invader test
8. reaction mix adding to the 96 well plate Visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate 20 uL of mineral oil Use DNase RNase free aerosol barrier tips at all times Do not allow pipette tips to touch any surface except the solution being pipetted Use sterile tubes for preparing reaction mixes Wear gloves at all times Verify control well location Section IV A Figure 2 Adjust gain setting so NDC is above 600 counts Extract genotype specific controls using standard laboratory method Verify reactions are covered by Table 9 Troubleshooting Guide Improper preparation of reaction mix Insufficient sample DNA used in the reaction Vortex each reagent before adding to reaction mix Verify correct reagent volumes were added to the reaction mix Verify all reagents were added to the reaction mix Vortex reaction mix before adding to the 96 well plate Visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate Verify concentration of at least 5ng uL prior to dilution and reaction set up Verify 1 20 dilution made correctly Sample Preparation section IV A If the DNA concentration is lt 5 ng uL pre dilution repeat the DNA extraction and purification protocol to obtain purified DNA at a higher concentration Repeat sample with Invader MTHFR 1298 te
9. results 30 If desired click the View Save PDF button located in the upper right corner of window Print the PDF and then close the PDF window Page 4 of 9 31 If desired click the Finish Active Assay button to delete run information when testing and analysis is completed B Quality Control Procedures Differences in blood processing and technical procedures in the user s laboratory may produce variability in results necessitating regular evaluation of laboratory designated controls in addition to the following procedures Prior to initial use of this test in the user s laboratory the performance of the test may be verified by testing a number of positive and negative samples with known characteristics These quality control tests should be repeated for each new lot or a change in test parameters Test verification on a daily basis may be accomplished through the proper use of the above mentioned laboratory designated controls as described in this section The No DNA Control C4 is used to establish the amount of signal generated in the absence of target Test runs are valid when the genotype specific controls yield the appropriate genotype results Table 4 If any of the genotype specific controls are called incorrectly or EQ equivocal the run is invalid and must be repeated A test run with invalid control results will fail to provide sample results In the event of a control failure all samples in the run sh
10. selected Label 1 Label 2 20 Place the 96 well plate to be analyzed onto the plate carrier with the A1 well oriented to the upper left corner of the plate carrier Do not remove the optically clear adhesive film from the surface of the plate Read the entire plate according to manufacturer s instructions Label Name NOTE If the No DNA Control NDC signal is not greater than 600 counts for FAM or Red fluorescence re read the plate adjusting the gain setting s accordingly so that each value is greater than 600 counts and the reader is in the linear dynamic range according to the manufacturer s instructions Data Analysis 21 Open the Invader Call Reporter software 22 Select the plate s to be analyzed by highlighting the appropriate row in the blue Active Assay field 23 Click the Load Selected button in lower left area of the window This should allow the Results tab to be selected 24 Click on the Results tab 25 Select the Raw Data File by clicking on the Select File button and select the appropriate file in the browser 26 Select the appropriate Worksheet from the available choices in the dropdown menu 27 Click the Import Raw Data button to populate data fields 28 If desired click View Save PDF button located in the upper right corner of window Print the PDF and then close the PDF window 29 Click on the Summary tab to view sample validity and genotype
11. 1298 WT Invader MTHFR 1298 HET Invader MTHFR 1298 MUT None No DNA Contro B Reaction Mix All of the Invader MTHFR 1298 reagents are supplied in concentrations ready for use The amount of reagents required for each reaction is summarized in Table 2 Make sure to mix reagents well prior to use C Other Materials Provided Invader Call Reporter software and Invader MTHFR 1298 Software Software User Manual for Invader MTHFR 1298 MAN 01690 Invader MTHFR 1298 Both software programs are provided along with the first order shipment of the Invader MTHFR 1298 test Contact Hologic Technical Support 888 898 2357 if an additional copy is needed D Materials and Reagents Needed But Not Provided Thermal cycler with heated lid capable of holding set temperatures within 1 C Multi well Fluorometer see Software User Manual for Invader MTHFR 1298 MAN 01690 for fluorometer software specifications Computer See Software User Manual for Invader MTHFR 1298 MAN 01690 for computer specifications Pipette tips filter barrier 96 well plates Optically Clear Adhesive Plate sealers Nuclease free water Mineral oil Microcentrifuge tubes Commercially Available DNA Extraction kit or validated in house laboratory method General laboratory equipment as needed tube racks micropipettors multichannel pipette microcentrifuge vortex mixer E Storage and Handling A
12. HOLOGIC Invader MTHFR 1298 95 455 144 tests or 95 459 1680 tests IVD In vitro diagnostic medical device 144 Contains sufficient reagents for 144 tests 1680 Contains sufficient reagents for 1680 tests 5 C 30 C Temperature limitation TABLE OF CONTENTS INDICATIONS AND USE MATERIAL AND METHODS SAFETY ISSUES INSTRUCTIONS FOR USE BIBLIOGRAPHY NOTICE TO RECIPIENT ABOUT LIMITED LICENSE LIMITED PRODUCT WARRANTY C Principles and Procedures The Invader MTHFR 1298 test utilizes the Invader Plus chemistry with DNA isolated from human whole blood for the detection of the targeted sequence polymorphism Specifically the Invader Plus chemistry utilizes a single tube two phase reaction including target amplification and signal generation mediated by Invader chemistry Invader Plus reaction mixes are assembled by combining the MTHFR 1298 Oligo Mix Universal Enzyme Mix and Universal Buffer In a 96 well plate reaction mix is combined with purified genomic DNA samples as well as four 4 controls included with the test The No DNA Control is used by the interpretive software to set the noise component of the run for signal to noise calculations The genotype specific controls WT HET MUT ensure reagents were assembled correctly and perform according to the specifications The 96 well plate is transferred to an appropriately programmed thermal cycler for target amplification and signal generation In the ta
13. When performing the test caution must be taken to prevent amplicon contamination of work areas Always use barrier pipette tips for pipetting procedures Perform the amplification set up in an isolated area with dedicated pipettes Use tips and tubes that are DNase RNase free C Toxicity of Invader Reagents The Invader MTHFR 1298 test reagents are not controlled as dangerous substances and no toxicity has been determined A Material Safety Data Sheet is available upon request Please call Hologic at 888 898 2357 for a copy if needed IV INSTRUCTIONS FOR USE A Invader Test Step by Step Procedure Software Set up 1 Open the Invader Call Reporter software and complete the testing information Details for using the software can be found in the software user manual Software User Manual for Invader MTHFR 1298 MAN 01690 a Enter the name of the operator b In the dropdown Menu Selection select the MTHFR 1298 test c Enter the number of samples to be tested in the space provided Invader MTHFR 1298 d Click the Proceed to Mix Preparation button located in the lower right corner of the window e On the Mix Preparation tab fill in the green shaded boxes for Lot Numbers and Expiration Dates for the reagents used during the testing f If desired click the View Save PDF button located in the upper right corner of the window Print the PDF and then close the PDF window g Onthe Sample Placemen
14. YOU MAY ALSO HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE These warranties do not apply to any item that is a repaired moved or altered other than by Hologic authorized service personnel b subjected to physical including thermal or electrical abuse stress or misuse c stored maintained or Operated in any manner inconsistent with applicable Hologic specifications or instructions or d designated as supplied subject to a non Hologic warranty or on a pre release or as is basis WARRANTY CLAIMS AND REMEDIES In the event of any warranty claim Hologic will replace with new or repaired items any Equipment part Component or Consumable Supply that is in breach of warranty and will use reasonable efforts to promptly fix or provide a workaround for any Software defect or bug which prevents operation in substantial conformity with functional specifications Alternatively Hologic may elect to repay or credit to Customer an amount equal to Invader MTHFR 1298 the purchase price of the defective Equipment component Software consumable supply or Service Items replaced shall become Hologic property All claims shall be initiated by contacting Hologic within the applicable warranty period and thirty 30 days after discovery of the breach or non conformity Hologic must be given reasonable access and an opportunity to inspect all associated materials If Hologic and Customer are unable to settle any claim and Customer has not notified
15. ed gain setting and read the whole plate again Partial plate reads are not allowed Fluorometer issues Incubation period was longer than specified length of time recommended Bubbles in reaction well High No DNA Control FAM or Red Signal Incorrect control volume or no control added to well No DNA Control is Invalid Result for one or more Genotype specific Control is Invalid Evaporation of reaction mix sample during run Evidence of contamination during genotype specific control preparation or reaction mix preparation Evidence of contamination during genotype specific control preparation or reaction mix preparation Controls in wrong location on plate Gain setting too low NDC value lt 600 counts No DNA Control is Invalid Result for one or more Genotype specific Control is Invalid Genotype specific controls not extracted Invader MTHFR 1298 See Invader Call Reporter Invader MTHFR 1298 Software User Manual troubleshooting guide Verify that the Invader Reaction Program Is as specified Section IV A Figure 3 Re run test taking care not to contaminate samples or reagents Remove bubbles e g centrifuge plate briefly and re read Remove bubbles e g centrifuge plate briefly Vortex each reagent before adding to reaction mix Verify correct reagent volumes were added to the reaction mix to the
16. g nucleic acids for analysis may be covered by one or more patents owned by other parties Similarly nucleic acids containing specific nucleotide sequences may be patented Making using or selling such components or nucleic acids may require one or more licenses Nothing in this document should be construed as an authorization or implicit license to make use or sell any so covered component or nucleic acid under any such patents 2011 Hologic Inc Part Number 15 3219 Revision 100 Page 9 of 9
17. iple rounds of Primary Probe cleavage for each DNA target resulting in an accumulation of the number of released 5 flaps The initial reaction the released 5 flap transiently hybridizes with a corresponding FRET cassette forming an overlapping structure that is recognized and the fluorophore is cleaved from the FRET cassette by the Cleavase enzyme The 5 flap is designed to have a melting temperature aligned with the Invader reaction temperature so that the 5 flaps cycle on and off of the corresponding FRET cassettes This allows for multiple rounds of FRET cassette cleavage for each 5 flap and an accumulation of released fluorophore When the FRET cassette is cleaved a fluorophore and quencher are separated generating detectable fluorescence signal The format uses two different discriminatory Primary Probes one for the mutant allele and one for the wild type allele Figure 1 Each Primary Probe is assigned a unique 5 flap and distinct FRET cassette with a spectrally distinct fluorophore By design the released 5 flaps will bind only to their respective FRET cassettes to generate a target specific signal linking the wild type allele with one fluorophore Fluorescence 1 FAM and the mutant allele with the second fluorophore Fluorescence 2 RED MATERIALS AND METHODS A Reagents Provided Table 1 Reagents Provided Reagent Vial Label Abbreviation Universal Buffer B Universal Enzyme Mix E Invader MTHFR
18. nts to the Product technology or any other commercial purpose Customer is not authorized to transfer this Product to any third party for any purpose whatsoever without the express written consent of HOLOGIC Except as otherwise stated in this paragraph no other license is granted expressly impliedly or by estoppel For information concerning the availability of additional licenses to practice the patented methodologies contact Legal Department Hologic Inc 502 South Rosa Rd Madison WI 53719 608 273 8933 U S Patent Nos 5 691 142 5 792 614 5 846 717 5 985 557 5 944 069 6 090 543 6 121 001 6 110 677 6 348 314 6 368 803 6 458 535 6 555 357 6 562 611 6 673 616 6 872 816 6 875 572 6 913 881 7 011 944 7 067 643 7 087 381 7 195 871 7 273 696 7 306 917 7 354 708 7 381 530 7 407 782 7 514 220 and any corresponding international equivalents VIII LIMITED PRODUCT WARRANTY WARRANTIES Equipment Supplies and Software are warranted to the original Customer to perform substantially in accordance with published Product Specifications for one 1 year starting from the date of Installation if applicable or from the date of Delivery whichever occurs first After sale options and accessories are warranted for six 6 months and x ray tubes are warranted on a straight line prorated basis as stated in the applicable Product Specification Warranty Period Replacement parts are warranted for the remainder of the Warra
19. nty Period or ninety 90 days from Delivery whichever is longer Consumable Supplies are warranted to conform to published specifications for a period ending on the expiration date shown on their respective packages Services are warranted to be supplied in a workman like manner Hologic does not warrant that use of Products will be uninterrupted or error free or that Products will operate with non Hologic authorized third party products HOLOGIC S ENTIRE WARRANTY RESPONSIBILITY IS EXPRESSLY LIMITED TO REPAIR OR REPLACEMENT AT HOLOGIC S OPTION AND IN THE FORM ORIGINALLY SHIPPED OF PRODUCT OR CORRECTION OF SERVICE SUBJECT TO ANY CLAIM OR AT HOLOGIC S ELECTION REPAYMENT OF OR CREDITING CUSTOMER WITH AN AMOUNT EQUAL TO THE HOLOGIC PRICE FEE OR CHARGE THEREFORE THE FOREGOING WARRANTIES ARE IN LIEU OF AND EXCLUDE ALL OTHER WARRANTIES NOT EXPRESSLY SET FORTH HEREIN WHETHER EXPRESS OR IMPLIED BY OPERATION OF LAW OR OTHERWISE INCLUDING BUT NOT LIMITED TO ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE SUCH LIMITED WARRANTY IS GIVEN SOLELY TO THE ORIGINAL CUSTOMER AND IS NOT GIVEN TO NOR MAY IT BE RELIED UPON BY ANY THIRD PARTY INCLUDING WITHOUT LIMITATION CUSTOMERS OF CUSTOMER THIS WARRANTY IS VOID UPON TRANSFER OF PRODUCT BY CUSTOMER TO ANY ENTITY WHO HAS LESS THAN FIFTY 50 PERCENT OWNERSHIP IN THE PRODUCT SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSIONS MAY NOT APPLY TO YOU
20. on controls and invalid samples Do samples all generate valid results Extract controls and samples Analyze Invader t Run Invader test gt test results A Do Invader controls all generate valid results Do samples all generate valid results Figure 4 Recommended testing process for samples producing Low Signal EQ or Invalid results with the Invader MTHFR 1298 test V BIBLIOGRAPHY 1 Patnaik M Dlott JS Fontaine RN Subbiah MT Hessner MJ Joyner KA et al Detection of genomic polymorphisms associated with venous thrombosis using the Invader biplex assay Mol Diagn 2004 6 137 144 2 Weisberg IS Jacques PF Selhub J et al The 1298A 3 C polymorphism in methylenetetrahydrofolate reductase MTHFR In vitro expression and association with homocysteine Atherosclerosis 2001 156 409 415 3 Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG et al A candidate genetic risk factor for vascular disease a common mutation in methylenetetrahydrofolate reductase Nat Genet 1995 10 111 113 4 Rozen R Genetic predisposition to hyperhomocysteinemia methylenetetrahydrofolate reductase MTHFR Thromb Haemost 1997 78 523 6 5 Wilcken DEL MTHFR 677C T mutation folate intake neural tube defect and risk of cardiovascular disease Lancet 1997 350 603 604 6 Rady PL Tyring SK Hudnall SD Vargas T Kellne
21. ould be re tested Unexplained discrepancies in control results should be referred to Hologic Technical Support 888 898 2357 See the Troubleshooting section of this package insert for additional information All quality control requirements should be performed in conformance with local state and or federal regulations or accreditation requirements C Interpretation of the Results Results from the Invader MTHFR 1298 test are reported to the user as a genotype call indicating which genotype was detected in the sample WT HET MUT The results also report sample validity and run validity Genotype call and corresponding nucleotides are shown in Table 4 Table 4 Interpretation of Results Genotype Invader MTHFR 1298 Genotype Nucleotides Homozygous Wild Type 1__ _ s__ Heterozygous Homozygous Mutant The Results in the Invader Call Reporter software display sample and control data If results are invalid or not displayed refer jo the Troubleshooting section of this package insert and the Software User Manual for Invader MTHFR 1298 MAN 01690 The Summary tab in the Invader Call Reporter software displays results for all samples and controls in a condensed format If results are invalid or not displayed refer to the Troubleshooting section of this package insert or in the Software User Manual for Invader MTHFR 1298 MAN 01690 If any of the controls are invalid sample results will not be displa
22. r LH Nitowsky H Matalon RK Methylenetetrahydrofolate reductase MTHFR the incidence of mutations C677T and A1298C in the Ashkenazi Jewish population Am Journal of Med Genet 1999 86 4 380 4 7 Peng F Labelle LA Rainey BJ Tsongalis GJ Single nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene are common in US Caucasian and Hispanic American populations Intl J of Mol Med 2000 8 5 509 1 8 Esfahani ST Cogger EA Caudill MA Heterogeneity in the prevalence of methylenetetrahydrofolate reductase gene polymorphisms in women of different ethnic groups Journal of the American Dietetic Association 2003 103 2 200 7 VI CONTACT INFORMATION deficiency of Manufactured and distributed by Hologic Inc Madison WI USA For further technical information or to order product contact Phone 608 273 8933 Toll free 1 888 898 2357 Hologic Inc 502 South Rosa Road Madison WI 53719 1256 Page 8 of 9 VII NOTICE TO RECIPIENT ABOUT LIMITED LICENSE Limited License The receipt of Product from HOLOGIC or its authorized distributor includes a limited non exclusive non transferable license under certain intellectual property rights held by HOLOGIC This license is only for the purpose of using the Product in the methods for which they were intended This limited license does not include a license to use the Product for new product research or development product manufacture reverse engineering improveme
23. rget amplification phase of the reaction amplification is carried out using two step cycling conditions i e denaturation amp annealing extension Following amplification Taq polymerase is inactivated by a 10 minute incubation at 99 C after which the thermal cycler proceeds to 63 C to initiate the signal generation Invader phase of the reaction see Figure 1 1a Structure Formation 2a Structure Formation Wildtype Specific Primary Probe Mutation Specific Primary Probe e a T a al a a wal alee D mert A Pip Wee E m gt A B i iy ia dii in f i TARGET A TARGET I TARGET TARGET i 1b Structure Recognition 2b Structure Recognition l INDICATIONS AND USE A Intended Use The Invader MTHFR 1298 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation A to C at position 1298 of the human 5 10 methylenetetrahydrofolate reductase MTHFR gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia Clinical Significance MTHFR is the metabolic enzyme involved in the conversion of homocysteine to methionine via the remethylation pathway The MTHFR A1298C mutation is a base change of an adenine A for a cytosine C resulting in a glutamate to an alanine change at amino acid 429 The MTHFR A1298C mutation is associated with a decrease in enzymatic activity 68 of the normal enzymatic activi
24. s of Detection Forty 40 replicates of genomic DNA samples representing the wildtype heterozygous and mutant MTHFR 1298 genotypes were tested at concentrations of both 5 ng uL and 80 ng uL prior to 1 20 dilution for the Invader reaction and the Invader results compared to bi directional sequencing For each concentration there was 100 120 120 agreement with bi directional sequencing Across all genotypes tested for a given DNA concentration the one sided lower 95 confidence limit was 97 53 Samples were also tested beyond the recommended concentrations of DNA at 10 fold extremes of the recommended range e g 0 5 ng uL and 800 ng uL At these extreme concentrations there was 89 17 107 120 agreement at 0 5 ng uL and 100 120 120 agreement at 800 ng uL concentrations Interfering Substances Heparin 1500 U dL human whole blood bilirubin 10 mg dL human whole blood cholesterol 300 mg dL human whole blood potassium EDTA 1 8 mg mL human whole blood hemoglobin up to 0 2 in human whole blood and ethanol based wash buffer 5 in DNA sample had no impact on Invader MTHFR 1298 performance Page 6 of 9 F Troubleshooting Table 9 Troubleshooting Guide Errors occur during data import Check FAM amp Red gain settings and read the whole plate again Partial plate reads are not allowed Check FAM gain setting and read the whole plate again Partial plate reads are not allowed Check R
25. st Remove bubbles e g Bubbles in reaction well centrifuge plate briefly and re read Repeat DNA extraction from specimen Result for sample is Low Signal EQ or Invalid Result for sample is Low Signal EQ or Invalid Refer to package insert performance characteristics Interfering substances Section IV E 4 Verify 1 20 dilution made correctly Sample Preparation section IV A Verify that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate Vortex each reagent before adding to reaction mix DNA sample inhibition Incorrect sample volume or no sample added to well Verify correct reagent volumes were added to the reaction Improper preparation of mix reaction mix Verify all reagents were added to the reaction mix Vortex reaction mix before adding to the 96 well plate Page 7 of 9 Result for sample is Low Signal EQ or Invalid Result for sample is Low Signal EQ or Invalid Table 9 Troubleshooting Guide Evaporation of reaction mix sample during run Insufficient DNA amplification 96 well plate incompatible with thermal cycler or positioned incorrectly within thermal cycler Sample DNA degradation DNA may degrade if stored at room temperature Visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the
26. t tab enter the Sample IDs into the list on the left side of window The Sample ID list runs down columns i e wells E1 through H1 followed by A2 through H2 and then A3 through H3 h Verify all samples are entered in the list and are in correct position of the sample grid i If desired click the View Save PDF button located in the upper right corner of the window Print the PDF and then close the PDF Window j Close the Invader Call Reporter software 2 Confirm the thermal cycler is programmed as stated in Figure 3 Sample Preparation 1 20 Dilution 3 Dilute extracted genotype specific controls and all extracted sample DNAs 1 20 using 5 uL of genotype specific control sample and 95 uL nuclease free water in a 0 5 mL tube or similar consumable Mix the diluted genotype specific controls samples thoroughly Do not dilute the No DNA Control Control 4 prior to use Mix Preparation 4 Remove the reagents Oligo Mix Universal Buffer No DNA Control from their respective storage locations and allow them to equilibrate to room temperature for approximately 30 minutes Do not remove the Universal Enzyme Mix from the 30 C to 15 C freezer until just prior to use 5 Vortex the components of the reaction mix thoroughly and spin down the contents in a microcentrifuge for 3 5 seconds 6 Combine the components of the reaction mix as shown in Table 2 in a microcentrifuge tube Note The prepared reaction mixture must
27. te with optically clear adhesive film Thoroughly secure the film to the surface of the plate 13 Visually confirm no bubbles exist in the reaction wells If bubbles are visible remove bubbles e g centrifuge plate briefly 14 Place the reaction plate in the thermal cycler 72 Annea Extera 2 15 Mint 72 Heat Inactivate 99 10 Minutes Polymerase Figure 3 Invader Reaction Program Invader Reaction 1 Minute 7 15 Start the Invader reaction program 16 When the reaction profile is complete the reaction plate can be held in the thermal cycler at 10 C or stored in a refrigerator 2 C to 8 C protected from light overnight Data Collection 17 Allow the reaction plate to equilibrate to room temperature on the bench top at least 1 minute prior to reading the plate 18 Visually confirm no bubbles exist in the reaction wells If bubbles are visible remove bubbles e g centrifuge plate briefly 19 Read the reaction plate on a multi well fluorometer according to manufacturers instructions Verify parameters match Table 3 Invader MTHFR 1298 Table 3 Recommended Multi well Fluorometer Settings Measurement 2 Red Fluorescence Top Reading Do not use Plate with cover option Setting Category Excitation Wavelength Bandwidth Emission wavelength Bandwidth Number of Reads Integration Time Lag Time Settle Time Multiple Reads per Well Not selected Not
28. ty Reduced levels of MTHFR activity lead to elevated plasma concentrations of homocysteine a condition referred to as hyperhomocysteinemia a recognized risk factor for thrombophilia Elevated plasma homocysteine has also been associated with an increased risk of cardiovascular disease and neural tube defects in fetuses of pregnant women The MTHFR A1298C allele frequency is about 27 in Ashkenazi Jews and 33 in US populations Invader MTHFR 1298 and Cleavage and Cleavage 1c Secondary Reaction FRET Cassette FAM FLUORESCENCE 1 FAM FLUORESCENCE 2 RED Figure 1 Invader Signal Generation Phase Page 1 of 9 During the signal generation phase a discriminatory Primary Probe transiently hybridizes to the amplified target sequence along with an Invader oligonucleotide to form an overlapping structure The 5 end of the Primary Probe includes a 5 flap that does not hybridize to the target DNA The 3 nucleotide of the bound Invader oligonucleotide overlaps the Primary Probe and does not hybridize to the target DNA The Cleavase enzyme recognizes this overlapping structure and cleaves off the unpaired 5 flap of the Primary Probe releasing it as a target specific product The Primary Probe is designed to have a melting temperature aligned with the Invader reaction temperature so that under the isothermal reaction conditions 63 C the Primary Probes cycle on and off the target DNA This allows for mult
29. uencing Number of Number of Valid MTHFR 1298 st Correct First Run Genotype MU NERC BESTE aP Genotype Calls Agreement Homozygous ie N 182 182 99 45 AC Homozygous N Genotype determined through am a DNA sequencing One sample failed to generate valid results This sample was reported as invalid EQ and no genotype call was assigned by the interpretive software The EQ result was used to calculate the First Run Agreement 99 71 2 Reproducibility a _Inter laboratory Reproducibility A multi center external study was conducted to determine the reproducibility of the Invader MTHFR 1298 test A single lot of the Invader MTHFR 1298 test was used to compare the test performance at three different study sites Aliquots of whole blood samples for each genotype were extracted at each site DNA from the samples underwent subsequent Invader analysis at each site on each of five 5 non consecutive days Results were obtained using the MTHFR 1298 Invader Call Reporter software see Tables 6 and 7 Page 5 of 9 Table 6 Inter laboratory Reproducibility of Invader MTHFR 1298 Test First Pass Samples No Calls Tested Invalid i EQ E E Tested 100 100 100 100 100 Final Following Single Retest Final Agreement One Sided Lower 95 Confidence Limit Percent Agreement Number of Agreements Number of Analyses y Comparisons 9 samples x 2 operators x 5 days
30. yed D Limitations General Limitations Invader MTHFR 1298 Reagents may demonstrate unexpected performance in previously untested samples The possibility of unexpected performance even in tested blood samples cannot be completely eliminated due to the biological variability of sample matrices Contact Hologic Technical Support 888 898 2357 with any documented unexpected result s Specific Limitations The MTHFR 1298 mutation is A1298C An additional mutation in the human 5 10 methylenetetrahydrofolate reductase MTHFR gene is T1317C This mutation has been shown to have no effect on the Invader MTHFR 1298 test s accuracy However it is recommended that the laboratory assess the possibility of any additional rare mutations that may generate false MTHFR 1298 results and report this as a limitation if applicable E Summary of Expected Results 1 Accuracy compared to bi directional DNA Sequencing Human whole blood samples n 348 underwent DNA extraction and subsequent bi directional DNA sequence analysis The same DNA samples were then analyzed using the Invader MTHFR 1298 test The observed agreement between the Invader MTHFR 1298 test and bi directional DNA sequencing was 100 347 347 The first run agreement with bi directional DNA sequencing was 99 71 347 348 with 98 41 one sided lower 95 confidence limit see Table 5 Table 5 Agreement between the Invader MTHFR 1298 Test and Bi directional DNA Seq
Download Pdf Manuals
Related Search