Hologic FV IVD Package Insert
Contents
1. Warranty Period Replacement parts are warranted for the remainder of the Warranty 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 ALLO2. DVT Study Arch Intern Med 1991 151 933 938 4 Spector EB Grody WW Matteson CJ et al Technical standards and guidelines venous thromboembolism Factor V Leiden and prothrombin 20210G gt A testing a disease specific supplement to the standards and guidelines for clinical genetics laboratories Genet Med 2005 7 6 444 53 5 Ridker PM Miletich JP Hennekens CH Buring JE Ethnic distribution of factor V Leiden in 4047 men and women Implications for venous thromboembolism screening JAMA 1997 277 16 1305 7 VI CONTACT INFORMATION 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 VII NOTICE TO RECIPIENT ABOUT LIMITED LICENSE The receipt of this product from Hologic Inc or its authorized distributor includes a limited non exclusive license under patent rights held by Hologic Inc Acquisition of this product constitutes acceptance by the recipient of this limited license Recipients unwilling to accept the limited license must return the product for a full refund Such license is solely for the purposes of using this product to detect a specific analyte For avoidance of doubt the foregoing license does not include rights to use this product for agriculture or veterinary medicine applications The foregoing license does not include a license to
3. If results are invalid or not displayed refer to the Troubleshooting section of this package insert or in the Software User Manual for Invader Factor V MAN 01688 If any of the controls are invalid sample results will not be displayed D Limitations General Limitations Reagents may demonsirate 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 Factor V Leiden FVL mutation is G1691A Additional rare mutations in Factor V include A1692C G1689A and A1696G It is recommended that the laboratory assess the possibility of rare Factor V mutations to generate false FVL results and report this as a limitation if applicable E Summary of Performance Characteristics 1 Accuracy compared to bi directional DNA Sequencing Human whole blood samples n 352 underwent DNA extraction and subsequent bi directional DNA sequence analysis The same DNA samples were then analyzed using the Invader Factor V test The observed agreement between the Invader Factor V test and bi directional DNA sequencing was 100 352 352 The overall agreement with bi directional sequencing was 100 352 352 with 99 15 one sided lower 95 confidence limit see Table 5 Page 5 of 9
4. Table 5 Agreement between the Invader Factor V Test and Bi directional DNA Sequencing Number of Correct 1 Run Genotype Calls Agreement on 1 Run Run Homozygous ie aoe 100 Table 7 Invader Factor V Test Summary of Agreement Data for all Three Sites Number of Valid Factor V Number Tested Results on 1 One Sided Genotype Analv es Number of Number of Percent Lower 95 y Comparisons Agreements Agreement Confidence x 25 day pairs aoe pee omocygous x 3 sites 2700 9 samples a a ee 100 Between Sites x 20 tests at site Genotype determined through bi directional DNA 2 se b BEN x 3 site pairs 2 Reproducibility 10800 a Inter laboratory Reproducibility A multi center external study was conducted to b Lot to Lot Reproducibility Whole blood samples were extracted and subjected determine the reproducibility of the Invader Factor V test A single lot of the to bi directional DNA sequencing The same DNA samples were then analyzed Invader Factor V test was used to compare the test performance at three using the Invader Factor V test with three different lots of the reagents The different study sites Blood samples for each genotype were extracted at each observed agreement between all three lots of the Invader Factor V test and bi site DNA from the samples underwent subsequent Invader analysis at each directional DNA sequencing was 100 60 60 see Table 8 Across all site on each of fiv
5. control and centrifuge plate briefly sample of the reaction plate Q Or Q QQOQOOOOOO 19 Read the reaction plate on a multi well fluorometer according to the manufacturer s instructions Verify the parameters match Table 3 10 Dispense 10 uL of the appropriate control or sample genotype specific controls and all samples are diluted See step 3 Control 4 NDC is undiluted to bottom of the appropriate well of the reaction plate See QQQOOOOG QQQOOOOO QQOOOOOCO QQOOOOOOC QQOQQOOOOG QOQOOOOOO QQOQOOOOOO OOO00000 Table 3 Recommended Multi well Fluorometer Settings Figure 2 Mix by pipetting up and down 3 5 Setting Category Measurement 1 FAM Measurement 2 Red times upon addition to ensure reaction homogeneity Change pipette tips between Mode Fluorescence Top Reading Fluorescence Top Reading every addition Do not use Plate with cover option 11 Overlay all control and sample wells with 20 uL of fresh mineral oil by dispensing along the side of the wells Change pipette tips between every addition archer aaa ak 485 20 nm 560 20 nm 12 Cover the reaction plate with optically clear adhesive film Thoroughly secure the n film to the surface of the plate Emission Wavelength Bandwidth 13 Visually confirm no bubbles exist in the reaction wells If bubbles are visible remove bubbles e g centrifuge plate briefly Number of Reads or flashes 10 10 14 Place the
6. use the product for new product research or development product manufacture or any reverse engineering purposes The purchaser of this product is not authorized to transfer this product to any third party for any purpose without the express written consent of Hologic Inc Except as expressly provided 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 Page 8 of 9 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 Vill 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
7. HOLOGIC Invader Factor V 95 453 144 tests or 95 457 1680 tests IVD SG In vitro diagnostic medical device 144 Contains sufficient reagents for 144 tests 1680 Contains sufficient reagents for 1680 tests 15 C Temperature limitation C Principles and Procedures The Invader Factor V 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 Factor V 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 target amplification phase of the reaction amplification is carried out using two step cycling conditions i e denaturation amp annealing extension Following amplification Taq polymeras
8. IMPORTANT CONTAMINATION PRECAUTIONS This product generates amplified DNA targets 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 Factor V 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 Technical Support at 888 898 2357 for a copy if needed Invader Factor V IV INSTRUCTIONS FOR USE A Invader Test Step by Step Procedure Software Set up 1 2 O 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 Factor V MAN 01688 Enter the name of the operator In the dropdown Menu Selection select the Factor V test Enter the number of samples to be tested in the space provided Click the Proceed to Mix Preparation button located in the lower right corner of the window On the Mix Preparation tab fill in the green shaded boxes for Lot Numbers and Expiration Dates for the reagents used during the testing If desired click the View Save PDF button located in the upper right corn
9. W THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSIONS MAY NOT APPLY TO YOU 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 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 a
10. able 2 or print out from Mix Preparation tab of software in a microcentrifuge tube Page 3 of 9 E Note The prepared reaction mixture must be used within 30 minutes Note Controls must be placed in the correct wells for proper data interpretation Step Description Temperature C Time Cvcles Refer to Figure 2 p p C a T A 4 Table 2 Invader Factor V Reaction Mix Factor V Oligo Mix 7 5 uL C Ameal Extend E a a 1 5 Minutes Polymerase Figure 3 Invader Reaction Program 7 5k 1 25 Universal Bute a 2 0K1 25 Universal Enzyme Mix 0 5 uL pk as 0 5k 1 25 Total Mix Volume tom K 128 012 7 Vortex the reaction mix thoroughly and spin down the contents in a 15 Start the Invader reaction program microcentrifuge for 3 5 seconds 16 When the Invader reaction program 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 Plate Set up 1 Figure 2 8 Reaction mi peaini nioa oee Plate Data Collection e eaction mix may be aliquoted into a 96 we a plate to facilitate the use of a multi channel Position of 17 Allow reaction plate to equilibrate to room temperature on the bench top at least 1 pipettor aera minute prior to reading plate 9 Add 10 uL of reaction mix to the bottom of plate 18 Visually confirm no bubbles exist If bubbles are visible remove bubbles e g each well designated for each
11. ded with the test Genotype specific controls consist of synthetic DNA in 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 Page 2 of 9 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 Factor V 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 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 A s The Invader Factor V test is int
12. e 5 non consecutive days Results were obtained using the genotypes tested across all three 3 lots the overall agreement with bi Factor V Invader Call Reporter software see Tables 6 and 7 directional sequencing was 100 60 60 with 95 13 one sided lower 95 Table 6 a Reproducibility of Invader Factor V Test confidence limit Final Following Single Retest Final Table 8 Lot to Lot Agreement between Invader Factor V Test and Bi directional DNA Samples __ Calls No Calls Agreement Operator Sous Correct EQ Samples Tested ene 0 Sequencing Site 100 Correct Invalid calls in z genotype calls Cran 100 in 1 run Percent 100 100 Agreement 100 98 89 99 81 Eighteen 18 of these No Call results were due to an Invalid Control result on a single run Upon an Invalid Control result the call reporting software automatically prevents the display of all sample genotypes which resulted in 18 No Call samples Upon retraining of the Operator and retesting see Figure 4 of the run all controls reported Valid and all 18 samples were found to be in agreement with sequencing Upon re extraction and re testing this sample was found to be in agreement with sequencing Factor V Genotype bi directional sequencing Number of Replicates Genotypes per tested sample Homozygous Wild Type GG G n Table 7 Invader Factor V Test S
13. e 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 INDICATIONS AND USE MATERIAL AND METHODS SAFETY ISSUES INSTRUCTIONS FOR USE BIBLIOGRAPHY CONTACT INFORMATION 1a Structure Formation Wildtype Specific Primary Probe 2a Structure Formation Mutation Specific Primary Probe Uia NOTICE TO RECIPIENT ABOUT LIMITED LICENSE LIMITED PRODUCT WARRANTY l INDICATIONS AND USE A Intended Use The Invader Factor V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation G to A at position 1691 of the human Factor V gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia Clinical Significance The Factor V Leiden FVL mutation refers to a base change from guanine G to adenine A at position 1691 in the gene coding for the Factor V protein an amino acid substitution that eliminates one of three activated protein C cleavage sites in Factor V As a result Factor V is inactivated to a lesser extent and persists for a longer period in the circulation leading to more thrombin generation In the United States a single FVL allele is present in about 5 2 2 and 1 2 percent of the Caucasian Hispanic and African American populations respectively overall about 1 in 5 000 individuals i
14. ended for in vitro diagnostic use These components have been tested as a unit 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 The 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
15. er of the window Print the PDF and then close the PDF window On the Sample Placement 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 Verify all samples are entered in the list and are in correct position of the sample grid 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 Close the Invader Call Reporter software 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 DNA 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 Vortex the components of the reaction mix thoroughly and spin down the contents in a microcentrifuge for 3 5 seconds Combine components of reaction mix as shown in T
16. erol 300 mg dL human whole blood potassium EDTA 1 8 mg mL human whole blood hemoglobin up to 0 2 in whole blood and ethanol based wash buffer 5 in DNA sample had no impact on Invader Factor V performance 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 Red gain setting and read the whole plate again Partial plate reads are not allowed See Invader Call Reporter Invader Factor V software User Manual troubleshooting guide Fluorometer issues Verify that the Invader Reaction Program is as specified Section IV A Figure 3 Re run test taking care not to Contamination contaminate samples or reagents Remove bubbles e g Bubbles in reaction well centrifuge plate briefly and re read l Remove bubbles e g 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 Incubation period was longer than specified length of time recommended High No DNA Cont
17. ick the 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 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 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 equiv
18. isually 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 Factor V test Remove bubbles e g Bubbles in reaction well centrifuge plate briefly and re read Repeat DNA extraction from specimen Refer to package insert performance characteristics Interfering substances Section IV E 4 Verify 1 20 dilution made DNA sample inhibition 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 Insufficient sample DNA used in the reaction Result for sample is Low Signal EQ or Invalid DNA sample inhibition Result for sample is Low Signal EQ or Invalid Incorrect sample volume or no sample added to well Page 7 of 9 Table 9 Troubleshooting Guide Improper preparation of reaction mix Evaporation of reaction mix sample during run Result for sample is Low Signal EQ or Invalid Insufficient DNA amplification 96 well plate incompatib
19. le with thermal cycler or positioned incorrectly within thermal cycler Sample DNA degradation DNA may degrade if stored at room temperature Troubleshooting Re test Procedure Vortex each reagent before adding to reaction mix Verify correct reagent volumes were added to the reaction AT 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 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 Verify all reagents have been 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 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
20. nalysis such as specific methods and compositions for manipulating or visualizing 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 3217 Revision 100 Page 9 of 9
21. ocal 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 should 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 Invader Factor V Homozygous Mutant 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 Factor V 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 calls and corresponding nucleotides are shown in Table 4 Table 4 Interpretation of Results Nucleotides at Genotype Invader Factor V Genotype Call Position 1691 Homozygous WildType wr n e l ee AA 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 Factor V MAN 01688 Heterozygous The Summary tab in the Invader Call Reporter software displays results for all samples and controls in a condensed format
22. pettors multichannel pipettor microcentrifuge vortex mixer E Storage and Handling 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 Ina 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 included on each run as an 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 Factor V test Genotype Specific Control Samples Genotype specific i e WT HET MUT controls are provi
23. re unable to settle any claim and Customer has not notified 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 Invader Factor V 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 a
24. reaction plate in the thermal cycler Integration Time 20 us 20 us Lag Time 0 us O us Settle Time Multiple Reads per Well Not selected Not selected Label Name Label 1 Label 2 Invader Factor V Page 4 of 9 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 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 j NOTE If the No DNA Control NDC signal is not greater than 600 counts for 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 in the raw data file from the available choices in the dropdown menu 27 Click the Import Raw Data button to populate data fields 28 If desired cl
25. rol 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 Verify reactions are covered by 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 Evidence of contamination during genotype specific control preparation or reaction mix preparation No DNA Control is Invalid Result for one or more Genotype specific Control is Invalid Use sterile tubes for preparing reaction mixes Wear gloves at all times Invader Factor V Table 9 Troubleshooting Guide Controls in wrong location on plate Verify control well location Section IV A Figure 2 Gain setting too low NDC Adjust gain setting so NDC is value lt 600 counts above 600 counts Genotype specific controls not quae clash asaracied controls using standar laboratory method Vortex each reagent before adding to reaction mix No DNA Control is Invalid Result for one or more Genotype specific Control is Invalid Verify correct reagent volumes were added to the reaction mix Improper preparation of reaction mix Verify all reagents were added to the reaction mix Vortex reaction mix before adding to the 96 well plate V
26. 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 Factor V 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 sample re test process for samples with Low Signal EQ or Invalid results Invader Factor V Do Invader controls all generate valid results ReRun Invader test on controls and invalid samples Do samples all generate valid results Extract controls Run Invader test Analyze Invader and samples 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 Factor V test V BIBLIOGRAPHY 1 Lee R Factor V Leiden a clinical review Am J Med Sci 2001 322 88 102 2 Kujovich JL Factor V Leiden Thrombophilia GeneReview on GeneTests Last Updated 2 17 07 htto www ncbi nim nih gov bookshelf or fcgi book gene amp part factor v leiden 3 Anderson FA Jr Wheeler HB Goldberg RJ et al A population based perspective of the hospital incidence and case fatality rates of deep vein thrombosis and pulmonary embolism The Worcester
27. s homozygous for the mutation The FVL mutation has been estimated to be present in 15 to 20 percent of patients with first venous thromboembolism VTE and is the most common heritable prothrombotic risk factor in the United States In population based studies FVL increases the risk of a first VTE 4 to 7 fold in heterozygous individuals and 40 to 80 fold in homozygous individuals Invader Factor V X X A aiian mmu Yi anii o gt B abaia mu Yi Kii aid it Seem wre i l J i TARGET i TAREE a 1 TARGET A TAREE 1b Structure Recognition and Cleavage 2b Structure Recognition and Cleavage FLUORESCENCE 2 FAM FLUORESCENCE 1 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 condi
28. tions 63 C the Primary Probes cycle on and off the target DNA This allows for multiple rounds of Primary Probe cleavage for each DNA target resulting in an accumulation of the number of released 5 flaps 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 RED and the mutant allele with the second fluorophore Fluorescence 2 FAM ll MATERIALS AND METHODS A Reagents Provided Table 1 Reagents Provided Reagent Vial Label Abbreviation Fac
29. tor V Oligo Mix None Universal Buter B Universal Enzyme Mix Invader Factor V WT None Invader Factor V HET None Invader Factor V MUT None No DNA Control B Reaction Mix All of the Invader Factor V 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 Invader Factor V Other Materials Provided Invader Call Reporter software and Invader Factor V software Software User Manual for Invader Factor V MAN 01688 Both software programs and the software user manual are provided along with the first order shipment of the Invader Factor V test Contact Hologic Technical Support 888 898 2357 if an additional copy is needed 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 Factor V MAN 01688 for fluorometer software specifications Computer See Software User Manual for Invader Factor V MAN 01688 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 micropi
30. ummary of Agreement Data for all Three Sites 3 Upper and Lower Limits of Detection One Sided Lower 95 Confidence Limit Forty 40 replicates of genomic DNA samples representing the wildtype and heterozygous Factor V 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 80 80 agreement with bi directional sequencing 9 samples Across all genotypes tested for a given DNA concentration the one sided lower 95 Within Operator x 2 operators 270 100 99 0 confidence limit was 96 32 Samples were also tested beyond the recommended Within day x 5 days ij concentrations of DNA at 10 fold extremes of the recommended range e g 0 5 ng uL and 800 x 3 sites 270 ng uL At these extreme concentrations there was 100 80 80 agreement at 0 5 ng uL and 100 80 80 agreement at 800 ng uL concentrations Percent Agreement Number of Agreements Number of Analyses y Comparisons 9 samples x 10 day pairs Between days x 4 2 reps per a Within Operator day 2160 100 99 6 x 2 operators x 3 sites 2160 Between 9 samples operator x 4 2 reps per 2700 100 99 6 within site paisa Invader Factor V Page 6 of 9 4 Interfering Substances Heparin 1500 U dL human whole blood bilirubin 10 mg dL human whole blood cholest
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