Devyser Extend ART.No. 8-A015

Transcription

1 Devyser Extend ART.No. 8-A015 For in vitro Diagnostic Use Instructions for Use Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 1 of 24

2 Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 2 of 24

3 Table of contents Page 1. Introduction to Devyser Extend 4 2. Warnings and Precautions 5 3. Symbols used on Labels 6 4. Required Material Included in the kit Required but Not Provided 7 5. Storage and Handling Requirements 8 6. Sample Requirements 8 7. Instructions for Use Workflow Devyser Extend Sample Preparation and PCR Amplification Detection Results and Analysis Procedural Limitations References Notice to Purchaser Contact Information 24 Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 3 of 24

4 1. Introduction to Devyser Extend Intended use Included in the kit Test procedure The Devyser Extend kit is an in vitro diagnostic product for diagnosis of whole chromosome aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X and Y The Devyser Extend kit contains ready-to-use reagents for PCR amplification of genetic markers. DNA extraction The Devyser Extend kit has been validated using QIAamp DNA Blood Mini Kit (Qiagen, cat#51104) for extraction of DNA from human whole blood and amniotic fluid (AF); QIAamp DNA Mini Kit (Qiagen, cat# 51304) for extraction of DNA from chorionic villus (CV) biopsies. Amplification The Devyser Extend kit has been validated using ABI GeneAmp System 9700 with a PCR profile temperature tolerance of ± 1 C. Detection compatible for use with Applied Biosystems Genetic Analyzers (ABI PRISM 310, 3100, 3130, 3700) that support dye set D. Data Analysis GeneScan and GeneMapper. Principle of the Procedure QF-PCR analysis includes amplification, detection and analysis of short tandem repeat (STR) markers and non-variable markers. Fluorescently labeled primers are used for amplification of chromosome specific markers and thus the copy number of each marker is indicative of the copy number of the chromosome. The resulting PCR products are separated and analyzed using an automated genetic analyzer. The relative amount of each allele is quantified by calculating the ratio of the peak heights or peak areas. A normal diploid sample has the contribution of two of each of the somatic chromosomes. Two alleles of a chromosome specific STR marker are detected as two peaks in a 1:1 ratio when the marker is heterozygous and as one peak when the marker is homozygous. The detection of an additional allele as three peaks in a 1:1:1 ratio or as two peaks in a 2:1/1:2 ratio indicates the presence of an additional STR sequence possibly corresponding to an additional chromosome, as in the case of trisomy. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 4 of 24

5 2. Warnings and Precautions A. B. C. D. E. F. G. H Devyser Extend has been validated using a total PCR reaction volume of 25 µl. Changing the reaction volume will compromise the kit performance. Avoid microbial contamination of reagents when removing aliquots from reagent vials. The use of sterile disposable aerosol barrier pipette tips is recommended. Do not pool reagents from different lots or from different vials of the same lot. Do not use a kit after its expiry date. Do not use opened or damaged kit reagent vials. Work flow in the laboratory should proceed in a uni-directional manner, beginning in the reagent preparation area and moving to the DNA extraction area and then to the amplification area and finally to the detection area. Pre-amplification activities should begin with reagent preparation and proceed to DNA extraction. Reagent preparation activities and DNA extraction activities should be performed in separate areas. Supplies and equipment should be dedicated to each activity and not used for other activities or moved between areas. Gloves should be worn in each area and should be changed before leaving that area. Equipment and supplies used for reagent preparation should not be used for DNA extraction activities or for pipetting or processing amplified DNA or other sources of target DNA. Amplification and detection supplies and equipment should remain in the amplification and detection area at all times. Handling of kit components and samples, their use, storage and disposal should be in accordance with the procedures defined by national biohazard safety guidelines or regulations. Wear powder free disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Wash hands thoroughly after handling specimens and kit reagents. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 5 of 24

6 3. Symbols used on Labels Lot or batch number Expiry date Manufacturer Number of tests In vitro diagnostic device Store below temperature shown 4. Required Material Kit Product code Content Devyser Extend 8-A015 Devyser Extend Mix1 (4-A025) Activator Mix1 (4-A030) Devyser Extend Mix2 (4-A028) Activator Mix2 (4-A031) Devyser Extend Mix3 (4-A029) Activator Mix3 (4-A032) Devyser Extend M1 8-A015-M1 Devyser Extend Mix1 (4-A025) Activator Mix1 (4-A030) Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 6 of 24

7 4. Required Material (contd). 4.1 Included in the kit Configuration Components The Devyser Extend kit contains reagents for analysis of maximum 25 samples. It is recommended that the activated reaction mix is dispensed into appropriate PCR reaction vials after preparation. Before dispensing ensure that the activated reaction mix is properly mixed (see section 7.1). Dispense in 20 µl aliquots and store at below 18 C. Avoid repeated freeze-thawing. Cap Colour Tube Colour Label Art.Nr. Kit Content Yellow Amber Devyser Extend Mix1 4-A025 1x25 Test White Amber Devyser Extend Mix2 4-A028 1x25 Test Red Amber Devyser Extend Mix3 4-A029 1x25 Test Orange Clear Activator Mix1 4-A030 1x25 Test Orange Clear Activator Mix2 4-A031 1x25 Test Orange Clear Activator Mix3 4-A032 1x25 Test 4.2 Required but Not Provided Reagent Preparation DNA Extraction Amplification Detection Consumables for the Thermal Cycler. Micropipette/dispenser with aerosol barrier tips or displacement tips (20 ul). Disposable protective gloves (powder free). Reagents and equipment according to manufacturer instructions for use. Micropipette/multipipette with aerosol barrier tips (5 ul). Thermal Cycler: ABI GeneAmp PCR System 9600/9700/2720 or Eppendorf Mastercycler or MJ Research/Bio-Rad PTC200 with 96-well alpha unit. Applied Biosystems Genetic Analyzer (ABI PRISM 310, 3100, 3130, 3700). Performance optimized polymers: POP-4, POP-6 or POP-7 Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 7 of 24

8 4.2 Required but Not Provided (contd.) Detection (contd.) DS-30 (Dye set D) Matrix Standard Kit (ABI cat.# ). Gene-Scan-500 ROX Size Standard (ABI cat.#401734/ # ). Hi-Di Formamide, Genetic Analysis Grade (ABI cat.# ). 1x Genetic Analyzer Buffer Micropipette/multipipette/dispenser with aerosol barrier tips or displacement tips (1,5 ul, 15 ul). Dye Set Calibration: ABI3100/3130/3700: DS-30 (Dye set D) Matrix Standard Kit (ABI cat.# ). ABI310: Use: 6FAM (a), HEX (a), ROX (a) and NED (b). Run with module file GS STR POP4 (1 ml) D.md4 (a): Fluorescent Amidite Matrix Standards [6FAM, TET, HEX, TAMRA, ROX ] (ABI cat# ) (b) NED Matrix Standard (ABI cat # ) 5. Storage and Handling Requirements A. B. C. D. Store all components below -18 C. Reaction Master Mix may be stored at +2 to +8 C for at least 7 days and at below -18 C until the expiry date. Avoid repeated freeze-thawing. Dispose of unused reagents and waste in accordance with country, federal, state and local regulations. Do not mix reagents from different kit lot numbers. 6. Sample Requirements Clinical Samples Procedure & Storage The Devyser Extend kit is for use with genomic DNA extracted from whole blood, amniotic fluid and chorionic villus biopsy samples. According to manufacturer instructions for use. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 8 of 24

9 7. Instructions for Use Run Size Each Devyser Extend kit contains reagents for 25 reactions which may be performed separately or simultaneously. It is recommended that a non-template control and positive control is included in each test run. To avoid contamination always use un-opened vials. Any reagents left in opened vials should be discarded Workflow Devyser Extend The Reaction Master Mixes should be prepared before preparing the samples, if the complete process is performed in one day. Only if the samples are prepared the day before amplification or earlier, the opposite order is advisable. The Reaction Master Mix is prepared by addition of the appropriate Devyser Extend PCR mix to the dedicated Activator Mix (see step 2 below). Devyser Extend has been validated using a total PCR reaction volume of 25 µl. Changing the reaction volume will compromise the kit performance. Reagent Preparation Area: 1. Centrifuge each tube briefly to collect the content. Do not vortex the tubes at this step! 2. Add 500 µl of the appropriate Devyser Extend Mix to the dedicated Activator Mix (Extend Mix 1 to Activator Mix 1, Extend Mix 2 to Activator Mix 2 and Extend Mix 3 to Activator Mix 3). NOTE! >>>>> 3. Mix manually by pipetting several times from the bottom of the tube. Vortex the Reaction Master Mix vial and centrifuge briefly to collect the content. The Reaction Master Mix is stable at +2-8 C for at least 7 days. 4. Add 20 µl of each Reaction Master Mix to separate PCR reaction tubes. 5. Cap the reaction tubes and centrifuge briefly to collect the contents. 6. Each Reaction Master Mix, prepared by addition of Devyser Extend Mix (1, 2 and 3) to Activator Mix (1, 2 and 3), may be stored at +2 to +8 C for at least 7 days and at below 18 C until the expiry date. Avoid repeated freezethawing. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 9 of 24

10 7.2 Sample Preparation and PCR Amplification DNA Extraction According to manufacturer instructions for use. It is recommended that alternative DNA extraction methods and sample materials are thoroughly evaluated with the Devyser Extend kit prior to the results being used for diagnostic use. For recommended PCR conditions and analysis settings (see below), optimal results are obtained at DNA concentrations between 10 and 20 ng/pcr. Addition of Sample Samples should be added in a dedicated area separated from reagent preparation, amplification and detection areas. 1. Add 5 µl of clinical sample (2-4 ng/µl) and appropriate controls to dedicated PCR reaction tubes containing the reaction mixes (step 4 page 9). 2. Cap the tubes and centrifuge briefly to collect the content. Amplification Turn on the Thermal Cycler at least 30 minutes prior to amplification.» For Eppendorf Mastercycler use CNTRL TUBE mode» For GeneAmp System 9700 set ramp speed to 9600 mode.» For MJ Research/Bio-Rad PTC200 with 96-well alpha unit, use Calculated vial temperature and MAX ramping mode. Amplification Area: Program the Thermal Cycler for amplification according to the following Thermo Profile (consult the User s Manual for additional information on programming and operation of the thermal cycler): 95 C 15 min 94 C 30 sec; 58 C 90 sec; 72 C 90 sec for 26 cycles 72 C 30 min 4 C FOREVER 1. Set reaction volume to 25 µl. 2. Start the amplification (duration approximately 2hrs 45mins). Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 10 of 24

11 7.2 Sample Preparation and PCR Amplification (contd.) Amplification (contd.) 3. Following amplification, remove the tubes containing completed PCR amplification reaction from the thermal cycler and place into a suitable holder. Centrifuge briefly to collect the content. Remove the caps carefully to avoid aerosol contamination. Do not bring amplified material into the preamplification areas. Amplified material should be restricted to amplification and detection areas. Pre-Denaturation 95 C 94 C 15 min 30 sec Cycling 58 C 90 sec Final Extension 72 C 72 C 90 sec 30 min Ambient 26 cycles 4 C 7.3 Detection Sample preparation Refer to the respective ABI PRISM Genetic Analyzers User Manual for instructions on maintenance and handling. Prior to running the Devyser Extend kit, the instrument must be spectrally calibrated to support dye set D with the polymer used. Sample Preparation for ABI 310/ 3100/ 3130/ Prepare a loading cocktail by combining and mixing 9 µl of the size standard (Gene-Scan-500 ROX) with 300 µl Hi-Di Formamide (sufficient mix for 18 wells/tubes). 2. Vortex for 15 seconds. 3. Dispense 15 µl of the loading cocktail into the required number of wells of a microwell plate or into individual tubes (ABI310) to be placed on the Genetic Analyzer. 4. Add 1,5 µl of the sample PCR product to the corresponding well/tube containing loading cocktail. 5. Seal the plate/tubes. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 11 of 24

12 7.3 Detection (contd.) Instrument Preparation Create a sample sheet using the data collection software with the following settings:» Sample ID.» Select size standard: Ensure that GS500 (-250)ROX is identified as the size standard.» Dye Set: D.» Recomended run Module: See below for different polymers and instruments. Run Modules ABI 310/3700 Run Parameters POP-4 (ABI310) POP-6 (ABI310) Capillary length 47 cm 47 cm Run temperature Injection voltage Injection time Run voltage Run time 40 min 65 min POP-6 (ABI3700) 50 cm ,5 5000s ABI 3100/3130 Run Parameters POP-4 POP-6 POP-7 (ABI3130) Capillary length 36 cm 36 cm 36 cm Run temperature Injection voltage 2,5 2,5 2,5 Injection time Run voltage Run time 1700 s 3200s 1700 s The amount of PCR product injected into the capillaries can be adjusted by increasing/decreasing the injection voltage and/or injection time. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 12 of 24

13 8. Results and Analysis Principles of QF- PCR Chromosome-specific, repeated DNA sequences known as small tandem repeats (STRs) are amplified by PCR. By using fluorescently labelled primers visualisation and quantification of the fluorescently labelled PCR products may be performed. Quantification can be achieved by calculating the ratio of the specific peak areas of the respective STR using an automated DNA sequencer. STRs vary in size between subjects, depending on the number of times the tri-, tetraor penta-nucleotides are repeated. DNA amplified from normal subjects who are heterozygous (have alleles of different lengths) for a specific STR marker is expected to show two peaks of different length with the same peak areas (figure 8.1). Figure 8.1. Heterozygous marker (area ratio 1:1) DNA amplified from subjects who are trisomic will exhibit either an extra peak (being tri-allelic) with the same area (figure 8.2), or only two peaks (being di-allelic), one of them with twice as large area as the other (figure 8.3 and 8.4). Figure 8.2. Trisomic tri-allelic marker (area ratio 1:1:1) Figure Trisomic di-allelic marker (8.3: area ratio 2:1; 8.4: area ratio 1:2) Subjects who are homozygous (have alleles of same length) or monsosomic will display only one peak (figure 8.5). Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 13 of 24

14 8. Results and Analysis (contd.) Principles of QF- PCR (contd.) Figure 8.5. Homozygous/monosomic marker The inability of STR analysis to distinguish subjects who are homozygous or monosomic is a major shortcoming when testing for sex chromosome abnormalities. When STRs specific for chromosome X are used, some samples from normal XX females may show homozygous QF PCR patterns, indistinguishable from those produced by samples with a single X, as in Turner syndrome. Incorporating additional X-chromosome STR markers into the analysis will reduce but not eliminate the likelihood of homozygosity. To facilitate the detection of monosomy X, Devyser Extend Mix3 includes the T2 marker for relative quantification of chromosome X and 2 as well as the 7X marker for relative quantification of chromosome 7 and X. In a normal female T2 and 7X area ratios of 1:1 are expected (figure 8.6). In normal males and females with monosomy X a 1:2 ratio is expected for marker T2 and a 2:1 ratio is expected for marker 7X (figure 8.7). Figure 8.6: Normal female with T2 and 7X area ratios of 1:1 Figure 8.7: Normal Male with T2 ratio 1:2 and 7X ratio 2:1. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 14 of 24

15 8. Results and Analysis (contd.) Marker overview: Devyser Extend Mix 1 Marker ID Location Marker size range (bp)* Dye Colour 15 A 15q15.1** Green 15 B 15q Green 15 C 15q12** Yellow 15 D 15q Blue 16 C 16q24.1** Yellow 16 D 16p Green 16 E 16q13** Blue 16 F 16q11.2** Blue 16 G 16q Yellow 22 A 22q Green 22 B 22q Blue 22 C 22q Green 22 D 22q Blue 22 E 22p Blue *Based on observed marker sizes using ABI3130 and POP7 and calculated marker sizes. Marker peaks with sizes outside given ranges may appear. **According to the NCBI website Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 15 of 24

16 8. Results and Analysis (contd.) Marker overview: Devyser Extend Mix 2 Marker ID Location Marker size range (bp)* Dye Colour 13A 13q Green 13B 13q Blue 13C 13q Yellow 13D 13q Green 18A 18q Green 18B 18q Yellow 18C 18q Blue 18D 18q Green 18M 18p Yellow 21A 21q Blue 21B 21q Blue 21C 21q Yellow 21D 21q Blue *Based on observed marker sizes using ABI3130 and POP7 and calculated marker sizes. Marker peaks with sizes outside given ranges may appear. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 16 of 24

17 8. Results and Analysis (contd.) Marker overview: Devyser Extend Mix 3 Marker ID Location Marker size range (bp)* Dye Colour X1 Xq Green X2 Xq Green X3 Xq Yellow X4 Xq Blue X5 Xq Green X6 Xq Blue X8 Xq Blue Y1 Yp (+/- 3bp) Blue Y2 Yq Blue XY1 Xp22.22 Yp11.2 X = 105 Y = 111 (+/- 2,5 bp) Green XY2 Xq21.3 Yp Blue 7X 7q34 Xq13 7 = 182 X = 202 (+/- 3 bp) Green T2 Xq23 2p23.2 X= = 118 (+/- 3 bp) Yellow *Based on observed marker sizes using ABI3130 and POP7 and calculated marker sizes. Marker peaks with sizes outside given ranges may appear. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 17 of 24

18 8. Results and Analysis (contd.) Performing analysis When performing manual analysis the marker peaks in the electrophoretograms should be identified according to the specific marker size ranges presented in the marker overview (page 15-17). Please note that marker size ranges varies depending on the polymer used for electrophoresis. The analysis of di-allelic markers is performed by calculation of peak area ratios (peak1/ peak2). Peak1 is the peak area of the shorter length fragment and peak2 is the peak area of the longer length fragment. See ratio criteria below for interpretation. For tri-allelic markers the ratio is always calculated starting with the area of the shortest length fragment (peak1). i.e. peak1/ peak2 and peak1/peak3, respectively. Homozygous markers are considered non-informative. Ratio Criteria (RC) Ratio criteria (RC) 1 is used when the peaks distance is <24bp Ratio criteria (RC) 2 is used when the peaks distance is 24bp Di-allelic markers Ratio 1:2 Inconclusive 1:1 Inconclusive 2:1 RC 1 <0,65 0,65-0,74 0,75-1,44 1,45-1,80 >1,80 RC 2 <0,65 0,65-0,74 0,75-1,54 1,55-1,80 >1,80 Tri-allelic markers Ratio Inconclusive 1:1:1 Inconclusive RC 1 <0,74 0,75-1,44 >1,45 RC 2 <0,74 0,75-1,54 >1,55 Height ratio Height ratio may be calculated when an area ratio is classified as "inconclusive". The same ratio criteria (RC) are applied as for area ratio calculation Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 18 of 24

19 8. Results and Analysis (contd.) Results interpretation To interpret a result as normal, at least two informative markers consistent with a di-allelic genotype are required with all other markers being non-informative. To interpret a result as abnormal (trisomic), at least two informative markers consistent with a tri-allelic genotype are required with all other markers being non-informative. Di-allelic pattern is determined by: Marker showing two peaks of similar height/area and the peak ratio is classified as 1:1. Tri-allelic pattern is determined by: a) Marker showing two peaks of differing height/area and the peak ratio is classified as 2:1 or 1:2. b) Three peaks of similar height/area and the peak ratio is classified as 1:1:1 Monosomy X pattern is determined by: a) All X and XY markers showing homozygous allelic pattern. b) The XY1 peak2 (green, bp) and the Y1 peak (blue, bp) are not detected. c) Marker 7X showing two peaks of differing height/area and the peak ratio is classified as 2:1. d) Marker T2 showing two peaks of differing height/area and the peak ratio is classified as 1:2. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 19 of 24

20 8. Results and Analysis (contd.) Results interpretation (contd.) If a marker displays inconclusive results or is not detected a number of reasons are possible:» Mosaicism» Partial chromosome trisomy» Stutter phenomenon» -A peak phenomenon» Crosstalk between dye-channels» Electrophoretic spike» Preferential amplification causing skewing» Contaminating DNA: second genotype, PCR amplicons.» Primer site polymorphism/alteration.» DNA concentration too high or too low.» DNA used in PCR is degraded.» Submicroscopic duplication of individual markers. In rare cases, amplification failure due to mutation of the primer site has been reported for the AMEL-Y (XY1-Y) sequence. In rare cases, amplification failure due to mutation of the primer site has been reported for the 7X-X sequence. If both normal and abnormal allele patterns are obtained for a single chromosome, it is recommended that follow-up studies are performed to identify the reason. If electrophoretograms are of poor quality (extensive crosstalk/cross-talk between dye-channels or electrophoretic spikes) the data should not be interpreted. The PCR product may be re-injected and re-analyzed. Fluorescence citeria The acceptable range for marker peak height is between 50 and 6000 relative fluorescent units. Peaks outside this range should not be analysed Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 20 of 24

21 8. Results and Analysis (contd.) PCR Artefacts Stutter peaks (figure 8.8) are detected as extra peaks that are one repeat or a multiple of repeats smaller than the actual STR allele. Stutter peaks may be included in the ratio calculation. The stutter peak area is typically less than 15% of the corresponding STR peak area. Figure 8.8. Stutter peak as indicated by the arrow. -A peaks (figure 8.9) are detected as extra peaks that is one basepair shorter than the fullength (+A peak)pcr product. A peaks may be included in the ration calculation. Figure 8.9. A and +A peaks as indicated by the arrows. +A peak -A peak Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 21 of 24

22 8. Results and Analysis (contd.) Electrophoretic/ Detection Artefacts Crosstalk between dye-channels may occur during detection (Figure 8.10). Crosstalk peaks should be excluded from the analysis. Figure Crosstalk peak (from green to blue channel) as indicated by the arrow. Electrophoretic spikes (figure 8.11) may occur during detection as sharp peaks present in several dye-channels. Peaks resulting from electrophoretic spikes should be excluded from the analysis. Figure Electrophoretic spike as indicated by the square Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 22 of 24

23 9. Procedural Limitations A. B. C. D. Use of this product should be limited to personnel trained in the techniques of PCR. Devyser Extend has been validated using the ABI Thermal Cycler GeneAmp 9700 with a temperature tolerance of ±1 C. It is recommended that alternative thermocycler instruments are thoroughly evaluated with the Devyser Extend kit prior to the results being used for diagnostic use. The Devyser Extend kit has been validated using QIAamp DNA Blood Mini Kit for extraction of DNA from human whole blood and amniotic fluid; QIAamp DNA Mini Kit for extraction of DNA from chorionic villus biopsies. Performance with other matrices and DNA extraction kits has not been validated and may result in false negative or false positive results. Devyser Extend should be used only for the detection of specific chromosomal aneuploidies according to the instructions for use. The assay has not been validated for detection of structural rearrangements, mosaicisms nor abnormalities in any other chromosome. Results obtained with Devyser Extend can only be directly applied to the tissue or specific sample material tested. Contamination by maternal cells or placental mosaicism may result in discepancies between results obtained with Devyser Extend and karyotyping results. 10. References 1. CMGS Best Practice Guidelines for QF-PCR for the diagnosis of aneuploidy 2. Hulthén, M et al. Reproduction (2003) 126, Rapid and simple prenatal diagnosis of common chromosome disorder: Advantages and disadvantages of the molecular methods FISH and QF-PCR. 3. Nicolini, U et al. Human Reproduction Update. Vol 10, no.6 pp. 5, The introduction of QF-PCR in prenatal diagnosis of fetal aneuploidies: Time for reconsideration. Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 23 of 24

24 11. Notice to Purchaser Results from Devyser Extend should be interpreted with consideration of the overall picture obtained from clinical and laboratory findings. Devyser AB will not accept responsibility for any clinical decisions taken. Purchase of this product does not provide a license to perform PCR under patents owned by any third party. 12. Contact Information Devyser AB Instrumentvägen 19 SE Hägersten SWEDEN Phone: Homepage: Technical Support Phone: techsupport@devyser.com Devyser Extend, Instructions for Use, 7-A015-EN, Jun page 24 of 24