Elucigene® QST*R-PL (Pregnancy Loss) Assay Instructions for Use
Contents
1. 2 copies of chromosome 3 can therefore be used as a reference peak to assist in the determination of the number of X chromosomes present 121bp peak Analysed in combination with Amelogenin and the other sex chromosomes markers it is particularly useful in the detection of sex chromosome aneuploidy In a normal female the markers should fall within a ratio window of 0 8 to 1 4 In a normal male the markers will give a ratio 21 8 Further details on the interpretation of the TAF9L marker can be found in the Guide to Interpretation 3 The Y specific marker SRY will give a single peak in normal males and will not amplify in normal females AN6XYR1EN 001 OCT 2014 Page 12 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use APPENDIX 1 Dye Labels The markers are labeled as follows AMEL TAF9L SRY D22S686 D22S689 D16S2621 D21S1411 D15S659 D13S628 AN6XYR1EN 001 OCT 2014 Page 13 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use APPENDIX 2 Table of Maker location Observed Heterozygosity allele size range D13S305 13q13 3 0 79 453 504 green D13S325 13q14 11 0 80 292 343 green D13S634 13q21 33 0 84 396 453 blue D13S628 13q31 1 0 75 451 499 yellow D15S822 15q12 0 86 247 308 red D15S659 15q21 1 0 86 395 441 yellow FESFPS 15q25 2 0 61 202 243 red D15S1515 15q26 3 0 81 1812. in the thermal cycler block Initiate the 95 C activation program followed by the amplification program see step 1 On completion of the amplification program the samples may be stored at room temperature overnight or at 2 8 C for up to 7 days before analysis by capillary electrophoresis AN6XYR1EN 001 OCT 2014 Page 7 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Capillary Electrophoresis It is recommended that each user ensure that the chosen equipment is used according to the manufacturer s instructions and is compatible with this test In this context the key parameters are the polymer and the capillary array Optimal results can be obtained using the following capillary electrophoresis conditions on an ABI3130 or ABI3500 Genetic Analyzer 1 Combine 6 85ul of size standard with 250ul Hi Di Formamide and mix thoroughly sufficient mix for 16 wells Dispense 15pul of the mix into the required number of wells of a 96 well optical plate 2 Add 3ul of test sample PCR product to the size standard mix from step 1 already dispensed into the plate and mix using the pipette Seal the plate 3 Denature the PCR product dispensed into the optical plate on a thermal cycler using the following parameters 94 C for 3 minutes linked to 4 C for 30 seconds 4 Centrifuge the plate at 1 000g for 10 seconds to remove any bubbles in the wells and load onto the Genetic Analyzer Note t is essential that unu
3. obtained post miscarriage or whole blood of fetal origin The target population is individuals who have experienced a spontaneous miscarriage The results obtained from QST R PL kit can help determine the aneuploidy status of the fetus This Product is for Research Use Only RUO Summary and Explanation Statistically 10 20 of all pregnancies end in spontaneous abortion miscarriage the majority of which occur towards the end of the first trimester Of these over 50 of cases have been shown to be caused by a chromosome abnormality 1 primarily aneuploidy the most commonly noted are trisomies which account for 60 of all chromosome abnormalities in miscarriage The most frequent trisomy found in products of conception POC is trisomy for chromosome 16 however trisomies for chromosomes 13 15 18 21 and 22 are also amongst the most common Other aneuploidies commonly seen include monosomy X and triploidy which account for approximately 20 and 15 of all abnormalities respectively These data are represented in Figure 1 below 2 Autosomal Trisomy Figure 1 Showing the chromosomal findings in products of conception with 46N representing normal results AN6XYR1EN 001 OCT 2014 Page 2 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Principles of the procedure The method employed by Elucigene QST R kits uses the QF PCR Quantitative Fluorescence Polymerase Chain Reaction technique 3 6 Using PCR amp
4. the factors mentioned in step 6 8 To interpret a result as normal at least two informative markers consistent with a di allelic genotype are required with all other markers being uninformative A normal result indicates the normal complement of two for the chromosome tested AN6XYR1EN 001 OCT 2014 Page 11 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use 9 Peak area ratios that fall between the normal and abnormal ranges are classed as inconclusive Inconclusive results may be resolved by using the single chromosome kits 10 If both normal and abnormal allele patterns are obtained for a single chromosome then it is recommended that follow up studies are carried out to identify the reason for the discrepant results prior to any conclusions being reached 11 In rare cases allele size ranges for markers may overlap If this is suspected analysis with the single chromosome kits may resolve this Analysis of Sex chromosome markers AMEL TAF9 and SRY 1 The AMEL marker amplifies non polymorphic sequences on the X 104bp and Y 110bp chromosomes and can be used to determine the presence or absence of a Y chromosome and represents the relative amount of X to Y sequence Please note that on rare occasions amplification failure due to mutation of the AMEL Y sequence has been reported 2 TAF9L is an invariant paralogous marker with sequences on chromosomes 3 and X The chromosome 3 specific peak 116bp representing
5. 209 green D16S753 16p11 2 0 77 442 477 red D16S2624 16q22 3 0 71 110 140 blue D16S2621 16q23 2 q24 2 0 79 268 308 yellow D16S539 16q24 1 0 76 130 166 green D18S1002 18q11 2 0 76 343 380 blue D18S819 18q11 2 0 73 401 435 red D18S535 18q12 3 0 77 462 503 blue D18S386 18q22 1 0 92 353 440 green D21S11 21q21 1 0 82 234 283 blue D21S1437 21q21 1 0 76 291 332 blue D21S1442 219q21 3 0 85 328 394 red D21S1411 219q22 3 0 83 319 389 yellow D22S686 22q11 2 0 69 146 199 yellow D22S685 22q11 23 0 77 222 264 green D22S689 22q12 1 0 74 209 254 yellow D22S683 22q12 3 0 87 152 223 blue AMEL Xp22 22 Yp11 2 n a 104 110 yellow TAF9 3p24 2 Xq21 1 n a 116 121 yellow SRY Yp11 31 n a 124 145 yellow Observed heterozygosities are based on number of alleles observed with Elucigene Diagnostics testing panel These figures may therefore differ from published data and may also vary according to the population being tested AN6XYR1EN 001 OCT 2014 Page 14 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use APPENDIX 3 GeneMapper Profile GeneMapper normal male profile showing relative positions of the markers detected by QST R PL AN6XYR1EN 001 OCT 2014 Page 15 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Limitations to the Procedure This test is designed to detect specific chromosomal trisomies and sex chromosome aneuploidies as detailed in the Instructions for Use It may not de
6. 5 65 Gardner RJM Sutherland GR 2004 Chromosome Abnormalities and Genetic Counselling New York Oxford University Press 339 360 Mansfield E S Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms Human Molecular Genetics 1993 2 1 43 50 Mann K Fox SP Abbs SJ Yau SC Scriven PN Docherty Z Mackie Ogilvie C Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis The Lancet 2001 358 9287 1057 1061 Mann K Donaghue C Fox SP Docherty Z Mackie Ogilvie C Strategies for the rapid prenatal diagnosis of chromosome aneuploidy European Journal of Human Genetics 2004 12 907 915 Mackie Ogilvie C Donaghue C Fox SP Docherty Z Mann K Rapid prenatal diagnosis of an aneuploidy using Quantitative Fluorescence PCR QF PCR Journal of Histochemistry and Cytochemistry 2005 53 3 285 288 Deutsch S Choudhury U Merla G Howald C Sylvan A Antonarakis SE Detection of aneuploidies by paralogous sequence quantification Journal of Medical Genetics 2004 41 908 915 ELUCIGENE and QST R are trademarks of Delta Diagnostics UK Ltd GENEMARKER is a trademark of Softgenetics Corporation GENEMAPPER GENESCAN NED VIC PET POP 7 LIZ and HI DI are trademarks of Life Technologies Corporation Note to purchaser Limited License This product is
7. Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Elucigene Diagnostics Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Cat Code AN6XYR1 25 tests Research Use Only The performance characteristics of this product have not been established and must not be used for diagnostic purposes till ienutactures by Elucigene Diagnostics Greenheys House Pencroft Way Manchester Science Park Manchester M15 6JJ For Sales Customer Service and T echnical Support T 44 0 161 669 8122 F 44 0 161 669 8129 E enquiries elucigene com E techsupport elucigene com Elucigene Diagnostics is the trading name of Delta Diagnostics UK Limited a company registered in England and Wales registration number 8696299 AN6XYR1EN 001 OCT 2014 Page 1 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Elucigene QST R PL Intended Use The QST R PL assay can be used for the detection of the six most common autosomal trisomies associated with pregnancy loss trisomy 13 Patau syndrome trisomy 15 trisomy 16 trisomy 18 Edwards syndrome trisomy 21 Down syndrome and trisomy 22 The kit also includes X and Y chromosome markers and the TAF9L marker for the determination of sex status The method employed by the Elucigene QST R PL kit is the QF PCR Quantitative Fluorescence Polymerase Chain Reaction technique The assay can be used on DNA extracted from either fetal material
8. LYZER A QST R Instrument Protocol needs to be created which can then be used for each QST R run Create the QST R Instrument Protocol through the 3500 Instrument Protocols library Ensure the following are selected e Run Module FragmentAnalysis50_POP7 Enter the settings detailed in the image below E Edit Instrument Protocol QSTR Setup an Instrument Protocol Application Type Fragment X Dye Set 65 Instrument Protocol Properties Run Module FragmentAnalysis50_POP7 Capillary Length Protocol Name QSTR Description Oven Temperature C 60 Run Voltage kVolts 19 5 Run Time sec 1300 PreRun Time sec 180 gt Advanced Options Close AN6XYR1EN 001 PreRun Voltage kVolts Injection Time sec 15 OCT 2014 50 v cm Polymer Pop 5 Injection Voltage kVolts 3 0 Data Delay sec 1 Page 9 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use To run the samples create a sample plate by clicking on Create Plate from Template in the Dashboard ensure the correct Instrument Protocol for QST R has been assigned see above Analysis of Results General Interpretation of Results Best practice guidelines for QF PCR have been documented by the UK s Association for Clinical Genetic Science and are available for reference at www acgs uk com PCR products are observed as a 5 dye labelled system using filter set G5 Filt
9. R vials in 10ul volumes and frozen at 20 C Ensure that vial contents are thoroughly thawed and mixed before dispensing The Control DNA should be frozen at 20 C Materials required but not provided General Laboratory consumables gloves screw capped microfuge tubes 0 2ml PCR vials or microtitre plates recommended by the manufacturer of the thermal cycler used pipette tips Laboratory equipment precision pipettes 2 sets 1 for pre amplification and 1 for post amplification handling preferably positive displacement pipettes protective clothing vortex mixer microfuge 96 well microtitre plate centrifuge PCR Amplification Thermal cycler to accommodate 96 well microtitre plates or 0 2ml vials with a temperature accuracy of 1 C between 33 C and 100 C and static temperature uniformity of 1 C Capillary Electrophoresis Capillary Electrophoresis GeneScan 500 LIZ size standard ABI Cat No 4322682 POP 7 Polymer ABI Cat No 4352759 DS 33 dye set G5 matrix standard ABI Cat No 4345833 10x Genetic Analyzer Buffer ABI Cat No 402824 and Hi Di Formamide ABI Cat No 4311320 Applied Biosystems ABI 3130 and 3500 Genetic Analyzers with GeneMapper software 36cm capillary array 50cm capillary array for 3500 Genetic Analyzer 96 well optical plates 96 well septa 96 well cassettes AN6XYR1EN 001 OCT 2014 Page 5 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Data Analysis O
10. ctious No test method can offer complete assurance that HBV HCV HIV or other infectious agents are absent 3 Handling of samples and test components their use storage and disposal should be in accordance with the procedures defined by the appropriate national biohazard safety guideline or regulation 4 In line with current good laboratory practice laboratories should process their own internal QC samples of known type in each assay so that the validity of the procedure can be assessed 5 If kit box is damaged there may be damage to the contents do not use the kit contact Customer Service AN6XYR1EN 001 OCT 2014 Page 3 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Symbols used on labels The symbols used on all labels and packaging conform to the harmonised standard ISO 15223 Manufacturer Number of tests eH lt I S See Instructions for Use Store below temperature shown KI Use before date shown Catalogue code a m TI Lot or batch number m OT AN6XYR1EN 001 OCT 2014 Page 4 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Materials Provided Store all components below 20 C The Elucigene QST R PL RUO kit contains 450048 1 x 250ul Reaction Mix TA 404485 1 x 50ul Control DNA DC Sufficient for 25 tests Kit Preparation and Storage Upon opening the kit it is recommended that the reaction mix be dispensed into 0 2m PC
11. er set G5 detects the 6 FAM blue VIC green NED yellow and PET red labelled fragments plus the Size Standard marker labelled with LIZ orange on an electrophoretogram and in the GeneMapper or GeneMarker program A Guide to Analysis Software for GeneMarker and GeneMapper is available from the Elucigene website www elucigene com Please note that IVD labelling on these QST R guides does not apply to QST R PL Important Note different combinations of instrument polymer and size standard may cause the size calling to vary slightly During validation of the kit users should check that the default bin settings result in accurate peak labelling and adjust if necessary In case of any difficulty please contact Technical Support techsupport elucigene com for advice General analysis guidelines for all QST R kits 1 The negative control should show no sharp peaks within the read range of 100 to 510bp 2 The positive control must show the expected results and all peaks must meet the criteria below 3 For analysis of DNA samples at least 1 peak should be observed for each marker tested The acceptable range for marker peaks analysed on the 3130 Genetic Analyzer is between 50 and 6000 relative fluorescent units rfus and for the 3500 Genetic Analyzers is between 175 and 32000 rfus Peak heights falling outside this range must not be analysed 4 Electrophoretograms of poor quality due to excessive bleed through between dye colours al
12. lification fluorescent dye labelled primers target highly polymorphic regions of DNA sequence called short tandem repeats STRs that are located on the chromosomes of interest Each targeted STR marker is specific to the chromosome on which it is located thus the copy number of the STR marker can be diagnostic of the copy number of the chromosome Informative STR markers have been selected that exhibit a high heterogeneity so that copy number can be easily determined A normal diploid sample has the normal complement of two of each of the somatic chromosomes thus two alleles of a chromosome specific STR are determined by the QF PCR technique as two peaks in a 1 1 ratio The observation of an extra STR allele as either a three peak pattern in a 1 1 1 ratio or two peak pattern in a 2 1 or 1 2 peak ratio is diagnostic of the presence of an additional sequence which in turn may represent an additional chromosome as in the case of a trisomy Amplified products of the QF PCR technique are analysed quantitatively on a capillary electrophoresis Genetic Analyzer to determine the copy number of the analysed STR markers Warnings and Precautions 1 The normal DNA Control provided in the kits has been independently tested and found to be negative for Hepatitis B Virus HBV Hepatitis C Virus HCV and Human Immunodeficiency Virus HIV 1 and 2 2 Care should be taken when handling material of human origin All samples should be considered potentially infe
13. ne of the following data analysis software packages is required GeneMapper 3 7 Applied Biosystems Inc or above or GeneMarker 1 65 SoftGenetics LLC or above Additional Elucigene QST R Documentation These Instructions for Use include a basic section on interpretation of the results obtained A supplemental Guide to Interpretation with examples and glossary and a Guide to Analysis Software are available from the Elucigene website www elucigene com DNA Extraction A DNA Extraction method to yield PCR amplifiable quality DNA at a concentration of 0 5ng ul to Ang ul DNA Concentration Using the recommended PCR conditions and sample injection settings stated in the capillary column run module pages 10 and 11 acceptable results are consistently obtained with input DNA amounts of 1 25ng to 10ng Note sample injection settings can be modified to suit the amount of amplicon produced during the PCR reaction which can vary due to amount of input genomic DNA added Less amplicon can be applied to the column for analysis by reducing time of injection Conversely more amplicon can be applied to the column for analysis by increasing either time or voltage of injection Previously amplified samples can be re injected multiple times for re analysis AN6XYR1EN 001 OCT 2014 Page 6 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Test Protocol Amplification Procedure Note to minimise the risk of contamination ste
14. ps 3 5 must be carried out in an area free from DNA Steps should also be taken to avoid contamination with PCR product 1 Program the thermal cycler for a single step cycle to activate the DNA polymerase at 95 C for 15 minutes linked to an amplification cycling program of 30 seconds at 95 C denaturation 1 minute and 30 seconds at 59 C annealing and 1 minute and 30 seconds at 72 C extension for 26 cycles This should be linked to a 30 minutes time delay file at 72 C extension on the final cycle Enzyme Activation Cycling Final Extension 15 min 30 secs 72 C 30 secs 30 min Ambient 1 min 30 secs Temperature q 26 Cycles D A negative water control must be included in each PCR run It may also be considered appropriate to include other controls e g positive normal DNA control supplied and positive trisomy control DNA not supplied Thaw sufficient vials of pre aliquoted QST R reaction mix for the number of samples and controls to be run see note under Materials Provided and centrifuge the vials at 12 000g for 10 seconds Using separate pipette tips add 2 5ul of test DNA to a sample vial containing 10ul QST R reaction mix and mix by pipetting up and down Do this for all samples to be tested Do not add DNA to the PCR vial for the negative control instead add 2 5ul of sterile distilled water Briefly centrifuge the vials until all liquid is at the bottom of each vial Place all vials firmly
15. r many of these Homozygous markers are uninformative since a ratio cannot be determined 7 To interpret a result as abnormal i e trisomy present at least two informative markers consistent with a tri allelic genotype are required with all other markers being uninformative It is not recommended to interpret a result as abnormal based on information from only one marker If required follow up testing with the single chromosome kits i e Elucigene QST R 13 Elucigene QST R 18 Elucigene QST R 21 may provide sufficient information for interpretation Trisomy is determined by either 7 1 Two peaks of uneven height due to one of the peaks representing two alleles which are common to one or both parents In this case the ratio between the two peaks will be classed as 2 1 or 1 2 such that A1 A2 will give a result in the region of 1 8 to 2 4 when the peak representing the shorter length allele is greater in area than the peak representing the longer length allele or where A1 A2 will give a result in the region of 0 45 to 0 65 when the peak representing the shorter length allele is smaller in area than the peak representing the longer length allele 7 2 Three peaks of comparable height present The ratio of the peaks will be classed as 1 1 1 and their values fall within the normal range of 0 8 1 4 although for alleles separated by more than 24bp an allele ratio of up to 1 5 is acceptable If this does not occur then it may be due to one of
16. sed wells i e wells in which No DNA sample is loaded are still loaded with Hi Di Formamide to ensure that the capillaries do not dry out Post PCR Data Analysis ABI3130 GENETIC ANALYZER Create a sample sheet using the 3130 data collection software with the following settings e Sample Name this must be the same sample specific name or number e Run owner select the default owner for lab e Run Protocol QSTR contains QST R 3130 run module see below Note It is necessary to create a run module detailing the instrument settings and subsequently assign this to a Run protocol in which Dye set G5 has been selected For more information on creating run modules please refer to your instrument user manual AN6XYR1EN 001 OCT 2014 Page 8 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use 3130 RUN MODULE FOR POP7 POLYMER 36cm Capillary Module QSTR tt Parameter Name Value Range 1 Oven Temperature 60 int 18 65 Deg C 2 Poly_fill_ Vol 6500 6500 38000 steps 3 Current Stability 5 0 int 0 2000 uAmps 4 PreRun_Voltage 15 0 0 15 kvolts 5 Pre_Run_Time 180 1 1000 sec 6 Injection_Voltage 3 0 1 15 kvolts 7 Injection_Time 15 1 600 sec 8 Voltage _Number_of Steps 20 1 100 nk 9 Voltage_Step_Interval 15 1 60 sec 10 Data_Delay_Time 60 1 3600 sec 11 Run_Voltage 15 0 0 15 kvolts 12 Run_Time 1200 300 14000 sec ABI3500 GENETIC ANA
17. so known as pull up or electrophoretic spikes sharp peaks present in more AN6XYR1EN 001 OCT 2014 Page 10 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use than one dye should not be interpreted The PCR products should be re injected and re analysed 5 Analysis is performed by assessment of peak ratios A1 A2 where A1 is the peak area of the shorter length fragment and A2 is the peak area of the longer length fragment The resulting ratio is indicative of locus copy number For disomic chromosomes heterozygous markers should show two peaks with similar heights A complete analysis of chromosome copy number status is performed by comparison of peak area ratios 6 Heterozygous di allelic i e two alleles markers should fall within a ratio window of 0 8 to 1 4 However for two alleles separated by more than 24bp in size a ratio of up to 1 5 is acceptable Any values falling within this region are referred to as having a ratio of 1 1 If the ratio balance falls out of this window then it may be due to a number of factors such as Whole chromosome trisomy Partial chromosome trisomy including sub microscopic duplications Mosaicism Contaminating second genotype e g maternal twin external Stutters causing skewing Preferential amplification of one allele causing skewing Primer site polymorphisms Somatic microsatellite mutations The Guide to Interpretation gives examples of typical profiles fo
18. sold pursuant to an agreement with Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use only the purchased amount of the product and its components for Research Use Only solely for the indication described in the accompanying instructions for use For information on obtaining additional rights to use this product or its components please contact outlicensing lifetech com Copyright 2014 Delta Diagnostics UK Ltd AN6XYR1EN 001 OCT 2014 Page 17 of 17
19. tect structural rearrangements involving the chromosomes tested and will not detect abnormalities in any other chromosomes Mosaicism for the chromosomes tested may not be detected A QST R PL result can only be directly applied to the tissue tested and may not represent the fetal karyotype Maternal cell contamination MCC and confined placental mosaicism CPM may result in discrepancies between the QST R PL and karyotype results Note Heterozygosities of the markers used were derived from a random set of samples submitted for routine analysis from a predominantly Northern European Caucasian population Any calculations using these heterozygosities strictly only apply to the population from which the samples were taken A small study using locally derived samples may be carried out as part of a validation study to establish heterozygosities in the population to be tested It is not expected that population variation will significantly alter the overall informativeness of the assay Disclaimer For Research use only RUO the performance characteristics of this assay have not been established Not for In Vitro Diagnostic Use Further details of Elucigene QST R products are available at www elucigene com AN6XYR1EN 001 OCT 2014 Page 16 of 17 Elucigene QST R PL Pregnancy Loss Assay Instructions for Use References Goddijn M Leschot NJ Genetic aspects of miscarriage Baillieres Best Pract Res Clin Obstet Gynaecol 2000 Oct 14 5 85
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