Hologic CF IVD Package Insert
Contents
1. 88 7 In the initial study four 4 samples confirmed by sequencing as 7T 9T were called 7T 7T by the InPlex CF Molecular Test Upon retesting all four 4 samples gave the correct 7T 9T result once per day for five consecutive days Table 10 Reproducibility of the InPlex CF Molecular Test Between Days Within B ii bat etween Operators Within Genomic Samples Mutations and Variants detected Within Operator Within Day Operator Agreement Site Agreement Between Sites Agreement by bi directional seqgeuncing Agreement 23 calls per sample x 40 4 for each 23 calls per sample x 23 Samples x 23 calls per sample x 400 run pairs i 23 calls per sample x 5 days x 2 of the 10 day pairs from days 1 2 f all other loci were normal Negative operators 1 3 4 4 1 5 2 3 2 4 2 10 runs Paes 10 runs for each site pair 5 3 4 3 5 4 5 x 2 operators Sample Genotype Sample 40 loci tested for each Variants sample CF1 AI507 HET CF2 CF3 CF4 CF5 CF6 CF7 CF8 CF9 CF10 CF11 CF12 CF13 CF14 CF15 CF16 CF17 1717 1G gt A HET CF18 CF19 5T 9T CF20 CF21 Agreements between calls Normal HET MUT for each Analyte on the InPlex CF Molecular Test 99 1 100 100 99 6 99 6 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 102. Volume Total Volume for Reaction Component per Card including 25 overage Cleavase Enzyme 60uL DNA Reaction Buffer 1mL See Mix Worksheet printout Total volume is determined by the number of samples and controls entered on Mix Worksheet 12 6 Open the individual card packets Each card will contain an individual lot ID number Mark the backside of the cards 1 2 3 etc 12 7 Vortex the Invader Reaction Mix for 5 seconds 12 8 Transfer to a 25mL reagent reservoir 12 9 Remove film seal from the amplification sample plate 12 10 Using a single channel or an 8 channel multi channel pipette add 105yuL column wise of Invader Reaction Mix to each well containing the amplified sample and mix carefully by pipetting up and down seven 7 times 12 11 Transfer 105uL of Invader Reaction amplified sample mix from column one of the amplified sample plate into the InPlex card labeled 1 as indicated on the Call Reporting Software Sample Placement worksheet print out 12 12 Using clean tips transfer 105uL of Invader reaction amplified sample mix from column two of the amplified sample plate into the InPlex card labeled 2 Repeat this step for all cards 12 13 Centrifuge each card 2 times at 1 200 rpm for 1 minute 2 separate spins in a Centrifuge with appropriate clips bucket and rotor IMPORTANT Two 2 separate 1 minute centrifuge spins are necessary to adequately distribute sample to all cha
3. 100 100 Hologic InPlex CF Molecular Test Page 9 of 14 3 Limit of Detection DNA samples within the concentration range from 10 50ng ul can be used in the InPlex CF Molecular Test and produce accurate genotype calls however using a DNA concentration at 15ng uL total input DNA of 75ng is recommended 4 Interfering Substances The performance of the InPlex CF Molecular Test was evaluated using eight CF positive samples from peripheral whole blood in the absence and presence of potential interfering substances These substances included compounds that are endogenous to the blood sample matrix or associated with blood collection as well as those that may result from sample preparation solutions Depending on the nature of the substance compounds were added to the blood sample or to purified genomic DNA Compounds added to the blood sample included bilirubin triglycerides and potassium EDTA blood collection and anti coagulant Compounds added to the purified genomic DNA included Qiagen Buffer AW2 and hemoglobin The results of this study showed that the InPlex CF Molecular Test achieved a 100 agreement between the genotypes of the samples containing potential interfering substances and bi directional sequencing 5 Lot To Lot Reproducibility A study was executed to test the equivalency between three lots of the InPlex CF Molecular Test Based on the results of this study the three lots of InPlex CF Molecular T
4. assay reaction Verify that the incubation time is 40 minutes Remove cards from incubator promptly after 40 minutes Problem Potential Cause Possible Solution 21 Equivocal call for Oligonucleotides used to Dilute the sample to a lower concentration AF508 in con detect the AF508 and 10ng uL input DNA concentration and retest junction with a AI507 HET or MUT Al507mutations have similar sequences For incubation verify temperature is set to 63 C and holds at 63 C 1 C Ensure that the correct temperature is maintained for 40 minutes The above actions may not alleviate this problem in all cases due to the sequence similarity and Product Limitations amp Precautions described in section F 3 18 Same as 2 Incubator or Oven Incubation time at 63 C is less than 40 minutes for the Invader Assay Reaction due to temperature decreases when door is opened to place cards in incubator or oven 1 Verify temperature is 63 C after closing incubator or oven door and that the unit is holding 63 C 1 C 2 If not document the time it takes for temperature to return to 63 C 3 Ensure that the correct temperature is maintained for 40 minutes 19 Same as 2 Incubator or Oven e Incubation time at 63 C is less than 40 minutes for the Invader Assay Reaction due to incubator or oven being opened multiple times during incubation Place all cards in incubator or ove
5. control results If results are invalid or not displayed refer to the Troubleshooting section of the Call Reporting Software User Manual The Executive Summary in the Call Reporting 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 pack insert or in the Call Reporting Software User Manual e f any No DNA Control fails sample results are not displayed e f all mutation s or variants are Normal for a sample Normal is displayed as the Result for the sample Page 6 of 14 e If NIC Result is Increase gain for a sample only the NIC Result is displayed If any results are invalid EQ Low Signal or INVALID only the invalid results are displayed Refer to the Troubleshooting section of this pack insert and in the Call Reporting Software User Manual e f any results are positive HET or MUT only the results for the positive mutation s are displayed If R117H is positive IVS8 5T 7T 9Tvariant results are also displayed e Failure to maintain consistent temperature throughout the incubator can result in hot spots resulting in miscalls particularly with IVS8 Genotype results indicating that a given sample is positive for more than two 2 mutant alleles e g homozygous mutant for R117H and heterozygous for 711 1G gt T or three 3 heterozygous results may be the result of sample contamination
6. SOME STATES DO NOT ALLOW 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 Hologic InPlex CF Molecular Test 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 oppor
7. Specification 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
8. evaluated for suitability prior to using the InPlex CF Molecular Test The InPlex CF Molecular Test reagents have been optimally diluted and further dilution may result in loss of performance Do not use the InPlex CF Molecular Test reagents or any components of the kit past the expiry date indicated on the outer box label Each Master Kit Lot contains a set of reagents Do not mix components from one Master Kit Lot with those of another Hologic InPlex CF Molecular Test Vil 10 Vill 12 The methodology and instructions for use detailed in this package insert as well as the Call Reporting Software instructions must be followed as written with these reagents and consumables Any deviation from these methodologies instructions for use may result in aberrant results BIBLIOGRAPHY Watson MS Cutting GR Desnick RJ Driscoll DA Klinger K Mennuti M Palomaki GE Popovich BW Pratt VM Rohlfs EM Strom CM Richards CS Witt DR Grody WW Cystic fibrosis population carrier screening 2004 revision of American College of Medical Genetics mutation panel Genet Med 6 387 91 2004 Mennuti M Lights Camera Action Obstetrics and Gynecology Vol 97 No 4 539 541 2001 Watson M Puryear M Mann M Rinaldo P Howell RR Newborn screening panel and system Genet Med Vol 8 No 5 Supplement 13s 252s Amos J Feldman G Grody W Monaghan K Palomaki G Prior T Richards CS Watson M Technical Standards and Guidelines for CFTR Muta
9. of the most common mutations in the non Hispanic Caucasian and Ashkenazi Jewish populations due to the high incidence of disease in these groups The 23 mutations occur with frequencies of at least 0 1 in clinically diagnosed patients The InPlex CF Molecular Test includes the 23 recommended mutations plus the variants IVS8 5T 7T 9T The IVS8 5T 7T 9T testing is a reflex test for individuals positive for the R117H mutation as recommended by ACMG 2183AA gt G F508C IVS8 5T IVS8 9T Page 1 of 14 ACMG mutations are shaded Required for use in the interpretation of the 2184delA mutation only V Variant Poly T variants are assessed for use when the test is positive for the R117H mutation F508C is assessed for use in the interpretation of Al507 or AF508 The following events mark the clinical significance of CF screening 1997 National Institutes of Health convene a Consensus Development Conference on Cystic Fibrosis resulting in a recommendation that genetic screening for CF mutations should be offered to identify carriers among adults with a positive family history of CF partners of individuals with CF couples currently planning a pregnancy and couples seeking prenatal care The American College of Medical Genetics ACMG American College of Obstetricians and Gynecologists ACOG and National Human Genome Research Institute form a Steering Committee to coordinate the implementation of population based
10. total of steps 5 through 7 9 99 15 10 10 Hold Total time 1 hour and 45 minutes 11 14 Start program 11 15 When amplification program is complete the amplification plate can be stored in the thermal cycler or at 2 8 C overnight 11 16 Remove the plate s from the thermal cycler or storage and centrifuge for 1 minute at 1 200rpm Visually confirm again that no volume discrepancies exist in the sample reagent plates by viewing the bottom side of the plate If a discrepancy is found the source of the discrepancy must be identified and wells with low volume must be repeated InPlex CF Molecular Test Invader Reaction Procedure 12 1 Preheat incubator or oven to 63 C Incubator or oven must hold 63 C 1 C 12 2 Remove Invader DNA Reaction Buffer from storage Thaw on bench top for 30 minutes 12 3 Remove InPlex card s and Cleavase enzyme from storage Allow 10 min to thaw Leave cards in the individual sealed packets until just prior to use 12 4 Vortex Invader reagents each individually for 5 seconds Pulse spin the tubes for 10 seconds to remove liquid from the lid prior to pipetting 12 5 Prepare the Invader Assay Reaction Mix using the volumes calculated in the Call Reporting Software Mix Worksheet printout Table 7 lists the volume of Cleavase enzyme and DNA Reaction Buffer to be used on a per card basis Hologic InPlex CF Molecular Test 13 Table 7 Invader Assay Reaction Mix
11. universal fluorescence resonance energy transfer FRET system combined with interpretative software and third party thermal cycler and multi well fluorometer instrumentation Invader is the term used to generically refer to the patented chemistry on which the InPlex CF Molecular Test is based In the Invader reactions of the InPlex CF Molecular Test two oligonucleotides a discriminatory Primary Probe and an Invader Oligo hybridize in tandem to the target DNA to form an overlapping structure The 5 end of the Primary Probe includes a 5 flap that does not hybridize to the target DNA Figure 1 The 3 nucleotide of the bound Invader Oligo overlaps the Primary Probe but need 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 close to the reaction temperature Therefore under the isothermal assay conditions Primary Probes which are provided in excess cycle on and off the target DNA This allows for multiple rounds of Primary Probe cleavage for each target DNA and accumulated increase of the number of released 5 flaps In a secondary reaction each released 5 flap can serve as an Invader oligo on a fluorescence resonance energy transfer FRET cassette by creating another overlapping structure that is recognized and cleaved by t
12. 0 54 9 A455E 123 4 119 4 119 0 100 97 6 100 97 5 100 47 3 G85E 123 3 120 3 120 0 100 97 6 100 97 5 100 36 8 2184delA 123 2 121 2 121 0 100 97 6 100 97 6 100 22 4 1898 1G gt A 123 3 120 3 120 0 100 97 6 100 97 5 100 36 8 IVS8 5T 7T 9T Variant t 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 Total calls 2952 144 2808 144 2807 1 99 96 99 9 99 96 99 9 100 97 9 t For the purpose of the IVS8 5T 7T 9T Variant Positive samples are regarded as those that have at least one copy of the 5T allele while Negative samples are regarded as having only the 7T and or 9T allele In the initial study four 4 samples confirmed by sequencing as 7T 9T were called 7T 7T by the InPlex CF Molecular Test Upon retesting all four 4 samples gave the correct 7T 9T result once per day for five consecutive days Hologic InPlex CF Molecular Test Page 8 of 14 Table 9b Accuracy of the InPlex CF Molecular Test IVS8 5T 7T 9T 9 IVS 8 5T 7T 9T Calls per Sequencing Calls InPlex Calls am Agreements 95 Cl Variant Mutation iti i iti i Positive Negative Positive Negative Negative Overall Neg Pos 5T 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 7T or No 5T 123 116 7 116 7 0 100 97 6 100 65 2 100 97 5 9T or No 5T 123 63 60 59 60 4 96 74 94 1 100 95 1 93 65
13. 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 CF22 100 100 100 100 100 100 100 100 100 100 100 100 CF23 AF508 2184delA HET 100 100 100 100 100 100 100 100 100 100 100 100 SITE TOTALS 99 980 100 000 100 000 99 962 100 000 100 000 99 962 100 000 100 000 99 981 99 981 100 000 TOTALS ACROSS ALL SITES 99 994 99 987 99 987 1 The calls on Sample CF1 1507 del HET were accurate and in 100 agreement for 1507 throughout However for Sample CF1 Site1 Operator 1 on Day 2 the AF508 genotype results were equivocal for both replicates Both equivocal results were treated as discordants for reproducibility purposes 99 6
14. 1 2 1 3 1 4 1 5 only or 40 of the total numbers of comparisons Between operators within site agreement was 99 987 and was assessed by calculating the between runs agreement for any two runs done by the two different operators from the same site There were 23 calls per sample x 23 samples x 10 runs from operator 1 x 10 runs from operator 2 52 900 comparisons for each site for a total of 158 700 comparisons across the 3 sites Between sites agreement was 99 987 and was assessed by calculating the between runs agreement for any two runs done by two different sites for all possible pairings There were 23 calls per sample x 23 samples x 400 run pairs for each site pair 211 600 comparisons per site pair for a total of 634 800 comparisons over 3 site pairs Summary Out of 31 740 calls generated 23 samples x 23 calls x 2 replicates x 2 operators x 5 days x 3 sites there were two calls at one site that yielded equivocal results on a single mutation for both replicates of the same sample The overall accuracy rate was 31 738 31 740 99 994 99 986 1 sided lower 95 Confidence Limit The results of this study are summarized in Table 10 The only discordant results were the 2 equivocal results noted above and also noted in Table 10 for Site 1 Operator 1 Sample CF1 on Day 2 where values for deltaF508 were equivocal for both replicates Page 7 of 14 Table 9a Accuracy of the InPlex CF Mo
15. 5 MHz equivalent or higher and 36 MB RAM Microsoft Windows 95 98 NT 4 0 SP3 or later Microsoft Excel 2000 C Storage and Handling Reagents should be stored at 30 to 15 C when not in use Do not store in a FROST FREE freezer Allow reagents to equilibrate to room temperature prior to use and minimize reagent exposure to light Do not subject the InPlex cards Amplification Reagents and Invader Reaction reagents to more than 8 freeze thaw cycles Hologic InPlex CF Molecular Test Vi D 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 positive and negative controls should be run simultaneously with unknown specimens If unexpected or invalid results are obtained with the control samples and results cannot be addressed in the Troubleshooting section of this document or the Call Reporting Software User Manual contact Hologic Technical Support 888 898 2357 E Specimen Preparation Blood samples extracted using four commonly used commercially available kits have provided sufficient purity and quantities of DNA to be compatible with the InPlex CF Molecular Test kit Although internal studies have shown the InPlex CF Molecular Test performs consistently with input DNA concentrations ranging from 10 50ng uL 50 500ng total input DNA an input DNA concentration of 15ng
16. CF carrier screening March April 2001 ACMG Statement Laboratory standards and guidelines for population based cystic fibrosis carrier screening is published ACMG recommended a panel of 25 mutations for population based cystic fibrosis carrier screening These 25 mutations were recommended on the basis that they have an allele frequency of at least 0 1 among CF patients in the US pan ethnic population Recommendations were also made for reflex testing for IVS8 5T 7T 9T and codon 506 507 and 508 variants e October 2001 ACOG recommends offering CF carrier screening to individuals with a positive family history of CF reproductive partners of people who have CF couples in whom one or both members are non Hispanic Caucasian and are planning a pregnancy or seeking prenatal care and that screening be made available to all couples in other ethnic groups who seek preconception or prenatal care 2003 The Centers for Disease Control CDC and the Cystic Fibrosis Foundation CFF convene a workshop and generate a resulting in a report supporting newborn screening for cystic fibrosis As of November 27 2006 36 state newborn screening programs report offering or implementing newborn screening for CF e 2004 Based on further data ACMG revises the mutation panel recommendations for reflex testing were not revised The 2004 ACMG CFTR mutation panel is detailed in Table 1 e 2006 The Maternal Child Health Bureau MCHB and ACMG
17. HOLOGIC HOLOGIC InPlex CF Molecular Test 96 Tests Catalog No 95 432 For In Vitro Diagnostic Use INDICATIONS FOR USE Intended Use Intended Use InPlex CF Molecular Test is an in vitro diagnostic device used to simultaneously detect and identify a panel of mutations and variants in the cystic fibrosis transmembrane conductance regulator CFTR gene in genomic DNA samples isolated from human peripheral whole blood specimens The panel includes mutations and variants recommended by the 2004 American College of Medical Genetics ACMG The InPlex CF Molecular Test is a qualitative genotyping test that provides information intended to be used for cystic fibrosis carrier screening as recommended by ACMG and the 2005 American College of Obstetricians and Gynecologists ACOG for adults of reproductive age as an aid in newborn screening for cystic fibrosis and in confirmatory diagnostic testing for cystic fibrosis in newborns and children The test is not indicated for use in fetal diagnostic or pre implantation testing This test is also not indicated for stand alone diagnostic purposes and results should be used in conjunction with other available laboratory and clinical information SUMMARY AND EXPLANATION A Clinical Description The InPlex CF Molecular Test is designed to detect genetic mutations and variations in the CFTR gene associated with cystic fibrosis to establish CF carrier status or to establish the diag
18. In the event this outcome is encountered testing should be repeated using a newly acquired patient sample e g either repeat blood draw or DNA extraction D Summary of Expected Results Accuracy Compared to Bi directional Sequencing Accuracy and repeat rate of the InPlex CF Molecular Test was determined by comparing genotyping results from a panel of 123 unique genomic DNA gDNA samples to bi directional DNA sequence analysis The panel tested consisted of gDNA samples isolated from human blood and commercially available cell lines A total of 23 CFTR mutations and the IVS8 5T 7T 9T variants for R117H positives were detected in this study Genotype calls were compared between DNA sequencing results and the InPlex CF Molecular Test results in the calculation of overall agreement accuracy In addition percent agreement for each mutation was calculated Repeat rate was determined by the number of samples which gave an invalid genotype call for one or more mutations with the InPlex CF Molecular Test on the first attempt This study was performed on a total of 123 unique samples containing 144 positive CFTR calls and 2808 normal negative CFTR calls The results of this study showed that the InPlex CF Molecular Test achieved 99 96 overall agreement accuracy 99 90 1 sided lower 95 Confidence Limit 100 positive agreement 97 9 1 sided lower 95 Confidence Limit 99 96 negative agreement 99 90 1 sided lower 95 C
19. NVALID the run is invalid and must be repeated An assay run with invalid control results may fail to provide accurate sample results All samples in an invalid run should be retested in the InPlex CF Molecular Test Refer to the Troubleshooting section of this pack insert and in the Call Reporting Software User Manual Prior to initial use of this product in the user s laboratory the specificity of the assay should be verified by testing a number of positive and negative samples with known performance characteristics Assay verification on a daily basis may be accomplished through the proper use of the positive and negative controls as described in this section All quality control requirements should be performed in conformance with local state and or federal regulations or accreditations requirements Interpretation of the Results InPlex CF Molecular Test Call Reporting Software provides the data analysis in determining the CFTR result s based on the mutations and variants detectable by this kit Interpretation of the results is described in detail in the Call Reporting Software User Manual provided on the software CD ROM Results from the InPlex CF Molecular Test Call Reporting Software are reported as Result e g HET or MUT for each mutation or variant e g AF508 N1303K etc in samples The results also report sample validity and run validity The Sample Summary in the Call Reporting Software displays sample and
20. S25 S33 S41 S49 S57 S65 S73 S81 S89 1 B S02 S10 S18 S26 S34 S42 S50 S58 S66 S74 S82 S90 2 C S03 S11 S19 S27 S35 S43 S51 S59 S67 S75 S83 S91 3 D S04 S12 S20 S28 S36 S44 S52 S60 S68 S76 S84 S92 4 E S05 S13 S21 S29 S37 S45 S53 S61 S69 S77 S85 S93 5 F S06 S14 S22 S30 S38 S46 S54 S62 S70 S78 S86 S94 6 G S07 S15 S23 S31 S39 S47 S55 S63 S71 S79 S87 S95 7 No H S08 S16 S24 S32 S40 S48 S56 S64 S72 S80 S88 DNA 8 Control Card Card Card Card Card Card Card Card Card Card Card Card 1 2 3 4 5 6 7 8 9 10 11 12 great care to ensure the correct samples are dispensed in the appropriate f IMPORTANT Due to repeated pipetting steps the operator should use wells 11 3 Prepare the Amplification Reaction Mix as indicated on the Call Reporting Software Mix Worksheet printout Table 4 lists the reagent volume per well 11 4 Vortex the Amplification Reaction Mix for 5 seconds Pulse spin the Mix for 10 seconds to remove liquid from the lid prior to pipetting 11 5 Add 10uL of amplification reaction mix to each sample well of the plate according to the plate layout 11 6 Vortex samples and controls and aliquot 5 uL of each including one No DNA Control Nucleas
21. TANT Use only RNase DNase free aerosol barrier tips and sterile tubes throughout the InPlex CF Molecular Test set up and procedure Test Procedures Follow a DNA extraction and purification protocol to obtain purified DNA It is recommended that the DNA concentration be adjusted to 15ng yL with 10mM Tris 0 10mM EDTA pH 8 0 See Section IV E Specimen Collection amp Preparation for more information on ranges and methodologies tested Page 3 of 14 10 Open the InPlex CF Molecular Test Call Reporting Software For initial use follow the installation instructions in the InPlex CF Call Reporting Software User Manual Confirm Microsoft Excel Tools Toolbar Macro Safety default is set to either Medium or Low Open the InPlex CF Molecular Test Call Reporting Software Detailed instructions are provided in the Call Reporting Software User Manual on the software CD ROM If a macro warning appears click on the Enable Macros button or Yes button On the Mix Worksheet tab enter the appropriate information in the green shaded boxes and print the Mix Worksheet for use in preparing reaction mixes Remember to include the number of controls in the total number of samples On the Sample Placement tab enter the sample ID into the plate layout for each sample to be tested To enter the sample ID of a No DNA Control use the drop down menu found by right clicking on the box in the sample plate layout Print the Sample Pl
22. acement worksheet for use in pipetting samples and controls in the 96 well plate and InPlex cards Save the file by clicking on the File menu and selecting Save As Name the file and click OK Perform the InPlex CF Molecular Test Amplification and Invader Reaction Procedures amplification procedures caution must be taken to prevent contamination of M IMPORTANT CONTAMINATION PRECAUTIONS When performing the 11 the lab and equipment with amplicons 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 Amplification of Samples 11 1 Remove samples and amplification reagents from storage and allow 20 minutes to thaw 11 2 Vortex the amplification primer mix and the amplification buffer and not the amplification enzyme each for 5 seconds Pulse spin the primer and buffer tubes for 10 seconds to remove any liquid from the lid prior to use Table 4 Amplification Reaction Mix Volume Total Volume for Reaction Component per well including 25 overage 5uL See Mix Worksheet printout Total volume is determined by the number of samples and controls Amplification Enzyme entered on Mix Worksheet Amplification Buffer Hologic InPlex CF Molecular Test Table 5 Sample Plate Layout for Amplification Lane 1 2 3 4 5 6 7 8 9 10 11 12 A S01 S09 S17
23. e Free Ultra Pure Water into appropriate wells of the sample plate layout Pipette up and down two times upon adding 11 7 Once the Amplification Reaction Mix has been prepared the unused reagents should be returned to the freezer immediately 11 8 If needed store the remaining DNA samples at 2 8 C 11 9 Seal the plate with an optical adhesive cover Thoroughly secure to the plate by using the plate sealing spatula 11 10 Visually confirm that no volume discrepancies exist in the sample reagent plates by viewing the bottom side of the plate s If residual liquid is observed on the sides of the wells centrifuge the plate at 1 200rpm for 10 seconds 11 11 After the plate s has been sealed it should be moved to the post amplification area Page 4 of 14 12 A IMPORTANT CONTAMINATION PRECAUTION The Invader Reaction Procedure is to be performed in a separate area away from the amplification area to prevent contamination with extraneous DNA 11 12 Place the plate in a licensed thermal cycler Orient the compression pad properly on top of the amplification plate Close and tighten the thermal cycler lid 11 13 Before performing the amplification program set the thermal cycler settings as indicated in Table 6 Table 6 Thermal Cycler settings Step Temp C Time min 1 95 3 2 55 1 5 3 72 2 5 4 2 cycles total of steps 1 through 3 5 95 0 5 6 55 1 5 7 72 2 5 8 12 cycles
24. est produced genotype calls that were 100 concordant to known genotyping of the sample test panel which consisted of 23 gDNA samples representing the ACMG recommended panel E Troubleshooting If the established criteria for an acceptable genotype call e g AF508 N1303K etc are not met by a given sample it is identified as either Low Signal or Equivocal and the sample Validity is considered Invalid and the sample must be retested A sample that has two Invalid results in a row cannot generate a result by the InPlex CF Molecular Test Each assay has a required minimum fold over zero FOZ that must be generated for one or both alleles If a sample fails to produce the minimum FOZ then the InPlex CF Molecular Test gives a Low Signal result and the sample must be retested Assays that generate a Valid FOZ minimum of one or both alleles generate a FOZ ratio which are required to be within a specified range If a sample s fails to produce a ratio within the specified ratio range then the InPlex CF Molecular Test gives an Equivocal result and the sample must be retested A software specific troubleshooting guide is located in the Call Reporting Software User Manual to supplement Table 11 that follows Hologic InPlex CF Molecular Test Table 11 Amplification Reaction amp Invader Assay Reaction Troubleshooting Problem Potential Cause Possible Solution 1 No DNA Con
25. he Cleavase enzyme Figure 1 When the FRET Cassette is cleaved a fluorophore F1 or F2 and quencher Q are separated generating detectable fluorescence signal Similar to the initial reaction the released 5 flap and the FRET Cassette cycle resulting in amplified fluorescence signal The initial and secondary reactions run concurrently in the same well The format of this test utilizes two different discriminatory Primary Probes each with a unique 5 flap and two different FRET Cassettes each 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 Figure 1 Invader Reaction 1a Structure Formation f 2a Structure Formation Mutation Specific Primary Probe Normal Primary Probe T it kS The oN Ere a gt gt ry l TEF z TARGET s x TARGET 5 x TARGET s j TARGET s 4 4 1b Structure Recognition i 2b Structure Recognition and Cleavage and Cleavage NN i a ma A Ps Ay gt gt a j S i gt P N Feral tye a Fertai i Va TARG er TARG or s i k 1c Secondary Reaction H 2c Secondary Reaction Ree TS FRET Cassette FAM FRET Cassette RED aD ts 3 ef Se ee i gt Neos Oe 5 see LUORESCENCE 1 FAM wee FLUORESCENCE 2 RED Page 2 of 14 MATERIALS AND METHODS A Reagents Provided InPlex CF Micro fluidic Cards 12 cards e Amplification Primer Mix 2
26. hree nucleotides Although the deletions are distinct A1507 occurs immediately upstream of AF508 amino acid 507 and amino acid 508 and consequently the oligonucleotides used to detect these mutations have similar sequences Internal studies have shown that samples positive for AI507 may result in an equivocal call for AF508 due to this sequence similarity Please refer to the Troubleshooting section 21 of this package insert if it should occur This test should not be used alone to diagnose cystic fibrosis Avoid contamination of reagents as this could produce aberrant results Separate areas must be used for pre and post amplification steps Clean gloves must be worn in each area and must be removed before leaving that area Incubators or ovens that do not hold 63 C at 1 C can cause aberrant results Failure to maintain consistent temperature throughout the incubator can result in hot spots resulting in miscalls particularly with IVS 8 Reagents may demonstrate unexpected reactions in previously untested samples The possibility of unexpected reactions even in tested blood samples cannot be completely eliminated due to biological variability of sample matrices Contact Hologic Technical Support 888 898 2357 for assistance with documented unexpected reaction s The performance of the InPlex CF Molecular Test was established using DNA extracted from four commonly used commercially available kits Extraction methods should be
27. ion 100 Page 14 of 14
28. l Reporting Software User Manual on the software Data Import 14 1 14 2 14 3 14 4 14 5 14 6 14 7 14 8 14 9 Detailed instructions for importing data from software files are included in the Call Reporting Software User Manual Open the InPlex CF Molecular Test Call Reporting Software file saved previously If the macro warning appears click on the Enable Macros button or Yes button On the SamplePlacement worksheet click on the Import Raw Data for All Cards button Navigate to the location of the saved file from the fluorometer reads Select the file to import and click Open The software will prompt you to confirm the correct file name card number worksheet name and SamplelDs for each card Click on the OK button at each prompt to proceed with import Click on the Cancel button at any prompt to abort import When all cards are imported click on Display Results button in the Executive Summary worksheet Save the file by clicking on the File menu and selecting Save or Save As Print Sample Summary and Executive Summary worksheets for patient results B Quality Control Procedures Differences in blood processing and technical procedures in the user s laboratory may produce significant variability in results necessitating regular evaluation of in house controls in addition to the following procedures Hologic InPlex CF Molecular Test Positive Co
29. lecular Test Calls per Sequencing Calls InPlex Calls Agreements 95 Cl eee Mutation Positive Negative Pos Neg Indet Overall Negative Positive delta F508 123 38 85 38 84 1 99 2 97 9 98 8 96 9 100 92 8 G542x 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 W1282X 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 G551D 123 7 116 7 116 0 100 97 6 100 97 5 100 65 2 621 1G gt T 123 8 115 8 115 0 100 97 6 100 97 4 100 68 8 N1303K 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 R553X 123 5 118 5 118 0 100 97 6 100 97 5 100 54 9 delta 1507 123 3 120 3 120 0 100 97 6 100 97 5 100 36 8 3849 10kbC gt T 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 3120 1G gt A 123 3 120 3 120 0 100 97 6 100 97 5 100 36 8 R117H 123 5 118 5 118 0 100 97 6 100 97 5 100 54 9 1717 1G gt A 123 6 117 6 117 0 100 97 6 100 97 5 100 60 7 2789 5G gt A 123 4 119 4 119 0 100 97 6 100 97 5 100 47 3 R347P 123 4 119 4 119 0 100 97 6 100 97 5 100 47 3 711 1G gt T 123 3 120 3 120 0 100 97 6 100 97 5 100 36 8 R334W 123 3 120 3 120 0 100 97 6 100 97 5 100 36 8 R560T 123 4 119 4 119 0 100 97 6 100 97 5 100 47 3 R1162X 123 4 119 4 119 0 100 97 6 100 97 5 100 47 3 3659delC 123 5 118 5 118 0 100 97 6 100 97 5 10
30. lume added in the amplification reaction was 10L Problem Potential Cause Possible Solution Prior to amplification visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate 11 Same as 2 Amplification Reaction Sample DNA degradation DNA may degrade if stored at room temperature Store extracted DNA as indicated in the DNA extraction and purification protocol prior to the amplification reaction 7 Same as 2 Amplification Reaction e Evaporation of amplification reaction mix sample during amplification Confirm the 96 well plate is firmly sealed with optical adhesive cover before amplification Confirm thermal cycler top is firmly closed Confirm that the compression pad is in the correct orientation when placed on top of the amplification plate 12 Same as 2 Invader Assay Reaction e Sample amplicon degradation Amplicons degrade if stored at room temperature Store amplicons in the 96 well plate at 2 8 C after the amplification reaction and before the Invader assay reaction 8 Same as 2 Thermal Cycler Insufficient or excessive DNA amplification 1 Verify the correct thermal cycler settings were used for the amplification reaction Thermal Cycler settings Step Temp C Time min 1 95 3 2 55 1 5 3 72 2 5 4 Repeat 1 3 for 2 cycles to
31. mbers of the cards 12 14 Visually inspect the cards to ensure that all wells are filled with liquid and there is excess liquid remaining in the ports If air bubbles are visible repeat the 1 minute centrifuge spin until they are not visible 12 15 Seal the wells and ports by placing the InPlex card in the micro fluidic card sealer and moving the carriage over the card as indicated on the sealer 12 16 Cut off the loading port section of the card with scissors so that the card is flush with the carrier 12 17 Place cards into preheated incubator s or oven s at 63 C unit s must hold this temperature 1 C Turn rotor on and incubate for 40 minutes If oven temperature has dropped below 63 C for any reason do not start the 40 minute incubation timer until the oven has returned to 63 C and is holding this temperature 1 C 12 18 After incubation remove cards from incubator s or oven s lie out individually and let cool at room temp for at least 2 minutes Do not stack the cards while cooling Data Collection 13 1 After cooling lock each card securely in place on an InPlex Card Holder Visually confirm that the card is seated flush on the holder 13 2 Load the card holder assembly onto the plate carrier of the multi well fluorometer Confirm that the card is oriented such that well A1 is in the upper left hand corner Page 5 of 14 13 3 Read the card s on the multi well fluorometer using the settings indica
32. n at the same time Coordinate time of incubation so that incubator or oven door is not opened repeatedly by others or for other procedures 20 Same as 2 Incubator or Oven e Variance in incubation temperature during the Invader assay reaction due to stacking cards Do not stack cards in the incubator or oven Hologic InPlex CF Molecular Test Page 12 of 14 F 1 10 11 Product Limitations amp Precautions This product is an in vitro diagnostic device and is for use in CLIA high complexity laboratories only Prior to mitigation of hazards failure of or a latent design flaw in the InPlex CF Molecular Test could result in an erroneous patient genotype result The InPlex CF Molecular Test is not indicated for stand alone diagnostic purposes prenatal diagnostic pre implantation or population screening Results from the InPlex CF Molecular Test should be used only in the context of other available laboratory findings and the total clinical status of the patient The presence of rare mutations in the CFTR gene not tested by the InPlex CF Molecular Test may result in false results miscalls The InPlex CF Molecular Test is designed to detect the presence of 23 cystic fibrosis mutations and sequence variants including both the Al507 and AF508 mutations when present in a patient sample Both the Al507 and AF508 result in the deletion of one amino acid caused by the deletion of t
33. nctions as a regulated chloride channel in epithelia Mutations at the gene level can affect the CFTR protein quantitatively qualitatively or both More than 1 000 CFTR mutations have been identified with AF508 accounting for 48 88 of mutant alleles across most ethnic groups The carrier frequency of any given CFTR mutation or variant as well as birth incidence varies between ethnic groups See Tables 1 2 Hologic InPlex CF Molecular Test Table 1 2004 ACMG CF Mutation Panel Frequencies Among Individuals with Clinically Diagnosed CF By Racial Ethnic Group in US Population Non Hispanic Hispanic African Asian Mutation Reference Caucasian Caucasian American American Ashkenazi Pan Ethnic asos 1 za sas mor sos sa oest R560T 1 0 38 0 0 17 0 0 0 3 N1303K 1 1 27 1 66 0 35 0 76 2 78 1 27 seaortorbcar 1 oss ts omr s531 477 oss 6551D 1 2 25 0 56 1 21 3 15 0 22 1 93 2184delA A455E 1 0 34 0 05 0 0 0 0 26 711 G gt T 1 0 43 0 23 0 0 0 1 0 35 staorrera 1 ow ow os o or os 3659delC 1 0 34 0 13 0 06 0 0 0 28 1717 G gt A G85E 1 0 29 0 23 0 12 0 0 0 26 Total 88 29 71 72 64 46 48 93 94 04 83 59 Table 2 Carrier Rates and Birth Incidence by Racial Ethnic Group Ethnic Group Carrier Rates Ashkenazi Jewish 1 23 African American 1 61 C Clinical Significance The 2004 ACMG recommended panel includes 23
34. nosis of CF in an individual Cystic fibrosis CF one of the most common life limiting autosomal recessive diseases in Caucasians is caused by mutations in the CFTR gene on chromosome 7q31 2 4 As defined in the 2006 ACMG Technical Standards and Guidelines for CFTR Mutation Analysis CF is characterized by viscous mucus in the lungs with involvement of digestive and reproductive systems as well as sweat glands Pulmonary disease is the most important factor in prognosis and recurrent pulmonary infections are frequent and result in respiratory failure Pancreatic insufficiency occurs in 85 of affected individuals Neonatal meconium ileus occurs in 10 to 20 of newborns with CF Other manifestations include chronic sinusitis nasal polyps liver disease pancreatitis and congenital absence of the vas deferens The overall average survival of CF patients including those with milder presentation is approximately 30 years Treatment for CF patients is palliative and includes antibiotics bronchodilators anti inflammatory agents mucolytic agents and chest physiotherapy CF currently affects approximately 30 000 children and adults in the United States and occurs in 1 in every 2 500 to 3 300 live births among Caucasians B CFTR Mutations and Incidence Cystic fibrosis transmembrane conductance regulator CFTR encodes a 1 480 amino acid integral membrane protein that is a member of the ATP binding cassette family of transporter proteins and fu
35. ntrols Hologic recommends that the user run CFTR mutation positive controls when performing the InPlex CF Molecular Test Method according to ACMG Standards and Technical Guideline recommendations The CFTR mutation AF508 which accounts for 48 88 of mutant alleles across most ethnic groups is recommended and is readily available commercially Please contact Hologic Technical Support at 888 898 2357 regarding commercially available CFTR genomic reference materials No DNA Control Hologic requires that at least one No DNA Control be included in each set of card runs Run validity is based on the results for No DNA Control s A valid run indicates that no contamination occurred during the amplification process The No DNA Control should be located in the well below the last sample well to detect contamination Nuclease Free Ultra Pure Water is recommended for use as the No DNA Control s Unexplained Discrepancies Unexplained discrepancies in control results should be referred to Hologic Technical Support 888 898 2357 If quality control results do not meet specifications patient results are invalid See troubleshooting section of this insert and the CRS User Manual for additional information Assay Verification Assay runs are valid when all controls yield correct results for all mutations If any No DNA Control is invalid OR any result for Positive Control s is invalid incorrect result based on known genotype EQ Low Signal or I
36. oducibility Table 10 summarizes the results for all the analyses described below Within operator within day agreement was 99 994 and was assessed by calculating the same day between replicate agreement among all possible comparisons For each site there were 23 calls per sample x 23 samples x 5 days x 2 operators for a total of 5 290 comparisons The total number of comparisons from the 3 sites was 15 870 Between days within operator agreement was 99 987 and was assessed by calculating the between runs agreement for any two runs done on two separate days by the same operator for all possible pairings There were 21 160 comparisons per operator i e 23 calls per sample x 23 samples x 40 4 for each of the 10 day pairs from days 1 2 1 3 1 4 1 5 2 3 2 4 2 5 3 4 3 5 4 5 Combined over 6 operators there were a total of 126 960 comparisons For unbiased estimates of between days reproducibility all possible comparisons were used in calculating percent agreements as described above However all comparisons were not independent For example if days 1 2 and days 1 3 comparisons both resulted in agreements for a particular call it implied day 2 and day 3 results would also agree for this call For this reason when calculating the 1 sided 95 confidence lower limits the sample sizes were conservatively adjusted to numbers of independent comparisons e g the numbers from day pairs
37. ol to obtain purified DNA at a higher concentration 4 Repeat sample with InPlex CF Molecular Test 4 Same as 2 Amplification Reaction Incorrect sample volume or no sample added to well in the amplification reaction Verify sample volume added in the amplification reaction was 5uL Prior to amplification visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate 5 Same as 2 Amplification Reaction Improper preparation of amplification reaction mix 1 Vortex each reagent before adding to amplification reaction mix 2 Verify correct reagent volumes were added to the amplification reaction mix 3 Verify all reagents were added to the amplification reaction mix 4 Vortex amplification reaction mix before adding to 96 well sample plate 5 Prior to amplification visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate Page 10 of 14 Table 11 Amplification Reaction amp Invader Assay Reaction Troubleshooting Problem Potential Cause Possible Solution Table 11 Amplification Reaction amp Invader Assay Reaction Troubleshooting 6 Same as 2 Amplification Reaction Incorrect volume of amplification reaction mix or no amplification reaction mix was added to well in the amplification reaction Verify amplification reaction mix vo
38. onfidence Limit The overall agreement of 99 96 is derived using initial test results only No repeat testing was done to support this study Table 9a Of the 123 unique samples in the initial study four 4 samples negative for R117H were confirmed by sequencing as 7T 9T but miscalled as 7T 7T by the InPlex CF Molecular Test resulting in an initial miscall rate of 0 14 Upon retesting all four 4 samples gave the correct 7T 9T result on each of the five consecutive days tested Table 9b Out of the 123 samples tested there was 1 sample with an Invalid call resulting in a repeat rate of 0 8 Inter laboratory Reproducibility In a multi center reproducibility study the InPlex CF Molecular Test s overall accuracy rate was 99 994 1 sided lower 95 Confidence Limit 99 986 The Hologic InPlex CF Molecular Test percent agreement for the use of the device ranges from 99 962 to 100 at each of the three sites and ranged from 99 987 to 99 994 across all three sites This performance was established by testing the same panel of samples representing the ACMG recommended panel of mutations at each site The samples consisted of 22 blinded CFTR Positive gDNA samples one CFTR Positive gDNA control plus one No DNA Control Each operator ran these samples in duplicate on 5 non consecutive days The InPlex CF Molecular Test results were then compared to those from the other investigational sites to establish assay repr
39. quately disperse sample to all chambers of the cards 3 Confirm card was sealed 4 Visually inspect the cards to ensure that all wells are filled with liquid and there is excess liquid remaining in the ports 10 Same as 2 Thermal Cycler 96 well plate compatibility or positioning in thermal cycler Confirm that the 96 well plate is compatible with the thermal cycler is firmly seated in the thermal cycler and secured properly 15 Same as 2 Invader Assay Reaction e Incorrect sample Invader assay reaction mix volume or no sample Invader assay reaction mix added to lane in card Verify 105uL of sample Invader assay reaction mix added to each lane in the cards Hologic InPlex CF Molecular Test Page 11 of 14 Table 11 Amplification Reaction amp Invader Assay Reaction Troubleshooting Table 11 Amplification Reaction amp Invader Assay Reaction Troubleshooting Problem Potential Cause Possible Solution 16 Same as 2 Incubator or Oven Incorrect incubation temperature for the Invader assay reaction Use calibrated heating units for incubation 1 Verify temperature is set to 63 C and holds at 63 C 1 C 2 Confirm temperature periodically by using a thermocouple thermometer equipped with a probe traceable to NIST standards 17 Same as 2 Incubator or Oven Incorrect incubation time for the Invader
40. recommends newborn screening for cystic fibrosis lll PRINCIPLES AND PROCEDURES A Device Description InPlex CF Molecular Test utilizes a limited cycle multiplex amplification reaction to enrich specific regions of the CFTR gene in genomic DNA gDNA extracted from human peripheral whole blood Each amplified DNA sample is subsequently mixed with Cleavase enzyme and buffer then added to a loading port on an InPlex micro fluidic card An InPlex card contains eight 8 sample loading ports each connected to 48 independent reaction chambers Twenty eight of these reaction chambers contain dried assay mixes for reporting the 23 ACOG CFTR mutations and variants The remaining chambers consist of a No Invader Control an independent quality control and several unused chambers After an InPlex card is loaded the channels are mechanically sealed using a micro fluidic card sealer isolating each individual reaction chamber from all other chambers The card is then incubated to allow individual Invader reactions to occur Following incubation the card is read in a multi well fluorometer and the raw signal data are Hologic InPlex CF Molecular Test imported into the InPlex CF Molecular Test Call Reporting Software for final result analysis B Invader Chemistry The InPlex CF Molecular Test is an in vitro diagnostic test which utilizes sequence specific Invader DNA probes a structure specific cleavage enzyme and a
41. tal 5 95 0 5 6 55 1 5 7 72 2 5 8 Repeat 5 7 for 12 cycles total 9 99 15 10 10 Hold Total time 1 hour and 45 minutes 2 Repeat sample with InPlex CF Molecular Test 13 Same as 2 Invader Assay Reaction Improper preparation of Invader assay reaction mix 1 Vortex each reagent before adding to Invader assay reaction mix 2 Verify correct reagent volumes were added to the Invader assay reaction mix 3 Verify all reagents were added to the Invader assay reaction mix 4 Vortex Invader assay reaction mix before adding to 96 well sample plate 5 After adding Invader assay reaction mix to amplification reaction mix sample pipette up and down 7 times 14 Same as 2 Invader Assay Reaction Incorrect volume of Invader assay reaction mix or no Invader assay reaction mix was added to well in the 96 well amplified sample plate 1 Verify 105uL of Invader assay reaction mix added to each well in the 96 well amplified sample plate 2 Prior to transferring from the 96 well plate to the cards visually confirm that no volume discrepancies exist in the 96 well plate by viewing the bottom side of the plate 9 Same as 2 Invader Assay Reaction e Sample Invader assay reaction mix not distributed in all wells in cards 1 Verify 105yL of sample Invader assay reaction mix added to each lane in the cards 2 Two 2 separate 1 min centrifuge spins are necessary to ade
42. ted in Table 8 for commonly used fluorometers Detailed instructions for fluorometer settings for software are included in the Call Reporting Software User Manual Table 8 Multi well Fluorometer Specifications Settings Multi Labeling Measurement Parameters Measurement 1 Measurement 2 Read mode Top Top Excitation wavelength 485 20 nm 562 10 nm Emission wavelength 535 25 nm 635 35 nm Gain Manual 48 102 90 152 Number of flashes 10 10 Integration time lt 20 us lt 20 us 14 13 4 13 5 After the card has been read confirm that the NIC raw signal values FAM and Red are greater than 600 wells A11 C11 E11 G11 111 K11 M11 O11 in the raw data file After importing the raw data into the CRS in step 14 7 below the NIC result will be Increase gain for any Sample or No DNA Control having a NIC value less than 600 and no other analyte results will be displayed for a sample with NIC result of Increase gain If NIC values are less than 600 increase gain setting s and repeat plate reading Read each card Data for each card will be in a different worksheet but in the same file If this is not the case the import function may not work properly Renaming the resulting Excel sheet tab with the barcode number on the InPlex card is recommended Detailed instructions for saving software files are included in the Cal
43. tion Testing 2006 Edition Published on ACMG website www acmg net Accessed 12 15 2006 Grody WW Cutting GR Klinger KW Richards CS Watson MS Desnick RJ Subcommittee on Cystic Fibrosis Screening Accreditation of Genetic Services Committee ACMG American College of Medical Genetics Laboratory standards and guidelines for population based cystic fibrosis carrier screening Genet Med 2001 Mar Apr 3 2 149 54 Kerem B Zielenski J Markiewicz D Bozon D Gazit E Yahav J Kennedy D Riordax JR Collins FS Rommens JM Tsui L Identification of mutations in regions corresponding to the two putative nucleotide binding ATP binding folds of the cystic fibrosis gene PNAS 87 8447 8451 1990 http consensus nih gov 1997 1997GeneticT estCysticFibrosis106html htm Accessed 12 10 2006 http genes r us uthscsa edu nbsdisorders htm Accessed 12 10 2006 http Awww genet sickkids on ca cftr MutationDetailPage external sp 245 246 Accessed 12 15 2006 www labtestsonline Accessed 12 15 2006 CONTACT INFORMATION Manufactured and distributed by Hologic Inc Madison WI USA For further technical information or to order product contact Hologic Inc Phone 608 273 8933 502 South Rosa Road Toll free 1 888 898 2357 Madison WI 53719 1256 www hologic com NOTICE TO RECIPIENT ABOUT LIMITED LICENSE The receipt of Product from Hologic or its authorized distributor includes a limited non exclusive non transferable license under certain intellec
44. trol is invalid Result for one or more mutation is Invalid Control Amplification Reaction or Invader Assay Reaction Evidence of contamination during sample preparation or amplification reaction mix preparation Use sterile tubes for preparing reaction mixes Wear gloves at all times Use DNase RNase free aerosol barrier tips at all times Do not allow pipette tips to touch any surface except the solution being pipetted 2 Result for sample is Low Signal or EQ or INVALID in the Sample Summary and Executive Summary Amplification Reaction or Invader Assay Reaction Reagent Degradation Store all InPlex CF Molecular Test reagents at the temperature indicated in the package insert Do not use InPlex CF Molecular Test reagents past the Expiration Date Do not freeze thaw InPlex CF Molecular Test reagents more than 8 times 3 Same as 2 Amplification Reaction Insufficient or excessive sample DNA used in the amplification reaction 1 Concentration of at least 10ng uL and not more than 50 ng uL prior to the amplification reaction 2 If DNA concentration is gt 50 ng uL then dilute the DNA to 10 50ng yL with 10mM Tris 0 10mM EDTA pH 8 0 Note that 15ng uL is the recommended concentration Dilute according to DNA extraction purification kit protocol 3 If the DNA concentration is lt 10 ng yuL repeat the DNA extraction and purification protoc
45. tual 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 Page 13 of 14 reverse engineering improvements 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 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 6 562 611 6 673 616 6 872 816 6 875 572 6 913 881 7 067 643 7 087 381 7 122 364 7 273 696 7 306 917 7 312 033 7 354 708 7 381 530 7 407 782 7 514 220 and any corresponding international equivalents 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
46. tunity to inspect all associated materials If Hologic and Customer are 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 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 Hologic Cleavase CF InPlex and Invader are trademarks and or registered trademarks of Hologic Inc and or its subsidiaries in the United States and or other countries 2010 Hologic Inc Part Number 15 3122 Revis
47. uL 75ng total is recommended NOTE It is recommended that DNA extraction methods be evaluated before use with the InPlex CF Molecular Test V SAFETY ISSUES A Precautions e The InPlex CF Molecular Test kit is intended for in vitro diagnostic use Do not smoke eat or drink where specimens or reagents are being handled e Avoid contact of eyes and mucous membranes with reagents If reagent comes in contact with sensitive areas wash with copious amounts of water e According to Biosafety in Microbiological and Biomedical Laboratories BMBL 4th Edition Biosafety Level 2 is appropriate when handling any materials of human origin 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 Gloves and protective eyewear should be worn at all times Never pipette by mouth and avoid contact of reagents and specimens with skin and mucous membranes Refer also to U S Department of Labor Occupational Safety and Health Administration 1991 Occupational Exposure to Blood borne Pathogens Final Rule Fed Register 56 64175 64182 B Toxicity of Invader Reagents The InPlex CF Molecular Test components 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 INSTRUCTIONS FOR USE A IMPOR
48. vials at 320uL each e Amplification Buffer 2 vials at 270uL each e Amplification Enzyme 2 vials at 72uL each DNA Reaction Buffer 2 vials at 6 3mL each Cleavase Enzyme 2 vials at 450uL each e Other Materials Provided Call Reporting Software User Manual CD ROM containing InPlex CF Molecular Test Call Reporting Software This CD ROM is provided with the first order shipment of the InPlex CF Molecular Test Contact Hologic Technical Support 888 898 2357 if an additional copy is needed InPlex Card Holders 12 each B Materials and Reagents Needed But Not Provided e Thermal Cycler with heated lid Micro fluidic card sealer Hologic p n 12 170 Centrifuge with appropriate clips buckets and rotor Incubator or oven capable of reaching and holding 63 C 1 C with carousel or air circulation Multi well fluorometer See Table 8 Optical adhesive plate seal 15mL conical tubes RNase DNase free pipette tips 10uL filter barrier RNase DNase free pipette tips 200uL filter barrier RNase DNase free pipette tips 1000uL filter barrier e 96 well PCR cycling plates Plate compression pads e Troughs reagent reservoirs 1 7mL tubes or 2mL screw cap tubes Nuclease Free Ultra Pure Water 10mM Tris 0 10mM EDTA pH 8 0 DNA quantification kit e 8 channel pipettes sizes P20 and P200 e Vortex Marker Personal computer with Processor 7
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