FACTOR V Q - PCR Alert kit Ref. RTSD01-V

Transcription

1 ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: WEB site: FACTOR V Q - PCR Alert kit Ref. RTSD01-V Composto da: Composed by: Composé par: Compuesto por: Composta por: Komponiert von: FACTOR V Q - PCR Alert AmpliPROBE Q - PCR Alert AmpliMASTER RTSD01-V-P RTS000 RTSD01-V_Front

2 ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: WEB site: NOTICE of CHANGE dated 28/10/13 IMPORTANT COMMUNICATION FOR THE USERS OF PRODUCT: «FACTOR V Q - PCR Alert Kit» Ref. RTSD01-V In the Instructions for Use Manual (IFU), SCH mrtsd01-v, some changes were introduced regarding: Product «FACTOR V Q - PCR Alert Kit», ref. RTSD01-V, can be used in association with the «ELITe STAR System» (ELITechGroup S.p.A.) for the automatic extraction of nucleic acids. Detailed instructions are reported in the enclosed Instructions for Use manual (IFU). The assay analytical principle and test reagents are unchanged. PLEASE NOTE LA REVISIONE DI QUESTO IFU E COMPATIBILE ANCHE CON LA VERSIONE PRECEDENTE DEL KIT (vedi Package Insert incluso nel kit) THE REVIEW OF THIS IFU IS ALSO COMPATIBLE WITH THE PREVIOUS VERSION OF THE KIT (see Package Insert included in the kit) CET IFU MIS A JOUR ANNULE ET REMPLACE ET EST PARFAITEMENT COMPATIBLE AVEC LA VERSION PRECEDENTE DU KIT (voir Package Insert inclus dans le kit) LA REVISIÓN DE ESTE IFU ES COMPATIBLE TAMBIÉN CON LA VERSIÓN ANTERIOR DEL KIT (véa Package Insert incluido en el kit) A REVISÃO DO ESTE IFU ÉTAMBÉM COMPATÍVEL COM A VERSÃO ANTERIOR DO KIT (ver Package Insert incluído no kit) DIE REVIEW VON DIESER IFU IST KOMPATIBLE MIT DER VORIGE VERSION VON DEM KIT (Sehen Sie Package Insert im Kit enthalten) Notice of Change nr. SCH mrtsd01-v_05_en dated 28/10/13

3 TABLE OF CONTENTS INTENDED USE page 1 ASSAY PRINCIPLE page 2 PRODUCT DESCRIPTION page 2 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 OTHER PRODUCTS REQUIRED page 3 WARNINGS AND PRECAUTIONS page 3 SAMPLES AND CONTROLS page 5 PROCEDURE page 6 PROCEDURE LIMITATIONS page 10 PERFORMANCE CHARACTERISTICS page 11 REFERENCES page 13 TROUBLESHOOTING page 14 SYMBOLS page 15 INTENDED USE ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: C is part of a qualitative amplification assay of nucleic acids for allele determination of the locus of coagulation Factor V (FV) for single nucleotide polymorphism (SNP) G1691A (R506Q, Leiden) in DNA samples extracted from whole blood collected in EDTA. The product is intended for use, alongside clinical data and other laboratory tests, in the assessment of risk of deep vein thrombosis. ASSAY PRINCIPLE The assay involves a real-time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time). In each well, an amplification reaction is carried out specific for the region of the human Factor V gene affected by SNP G1691A using the DNA extracted from the samples being tested. A specific probe for the normal FV 1691G allele (R506) labelled with FAM fluorophore is activated when hybridized with the normal product of the amplification reaction. Another specific probe for the mutated allele FV 1691A (Q506, Leiden) labelled with VIC fluorophore is activated when hybridized with the mutated product of the amplification reaction. Fluorescence emission increases as the specific products of the amplification reaction increase and is measured and recorded by the instrument. The processing of the data determines which alleles are present in the DNA extracted from the starting sample. System standardization was carried out on Applied Biosystems 7000 series instruments. PRODUCT DESCRIPTION The product supplies the mixture of AmpliMIX primer oligonucleotides for real-time amplification in a stabilizing solution, pre-dosed in aliquots into four disposable test tubes. Each test tube contains 110 µl of solution, sufficient for 24 tests. The primer oligonucleotides are specific for the region of the human Factor V gene affected by SNP G1691A (Leiden). The product provides 96 determinations, including controls. MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling FV AmpliMIX primer oligonucleotides mixture 4 x 110 µl Oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X-100 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex powder-free gloves or similar material. - Vortex mixer. - Bench microcentrifuge (12,000-14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement ( µl, 2-20 µl, 5-50 µl, µl, µl). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system. - SCH mrtsd01vm_en 28/10/13 Review 05 Page. 1/15 SCH mrtsd01vm_en 28/10/13 Review 05 page 2/15

4 OTHER PRODUCTS REQUIRED The reagents for DNA extraction from the test samples, the reagents optimized for amplification, the detection reagents (fluorescent probes) and heterozygous control DNA are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended: For manual nucleic acid extraction, it is recommended the use of generic products «EXTRAcell» (ELITechGroup S.p.A., code EXTD02), kit for DNA extraction from cellular samples; the kit enables 50 extractions. For automatic nucleic acid extraction, it is recommended the use of generic product by ELITechGroup S.p.A. «ELITe STAR 200 Extraction kit» (ElitechGroup S.p.A., code INT011EX), kit for extraction of DNA and RNA from non-cellular and cellular samples with «ELITe STAR instrument» (ElitechGroup S.p.A., code INT010), hereafter indicated as «ELITe STAR». «ELITe STAR 200 Extraction Kit» (ELITechGroup S.p.A., code INT011EX) and «ELITe STAR Instrument» (ElitechGroup S.p.A., code INT010) constitute «ELITe STAR System». «Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification and allele determination; the product provides 96 reactions. «FACTOR V Q - PCR Alert AmpliPROBE» (code RTSD01-V-P), fluorescent probes for allele determination; the product provides 96 reactions. «FACTOR V - Positive Control» (code CTRD01-V), heterozygous control of plasmid DNA; the product provides 25 reactions. When a 7500 Fast Dx Real-Time PCR Instrument is used, it is recommended the use of generic product: «Q - PCR Microplates Fast» (ELITechGroup S.p.A., code RTSACC02), microplates with 0.1 ml wells and adhesive sealing sheets for real time amplification. This product is exclusively for in vitro use. Warnings and general precautions WARNINGS AND PRECAUTIONS Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121 C for one hour before disposal. Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. Wear suitable protective clothing and gloves and protect eyes / face. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic products in the work areas. Wash hands carefully after handling samples and reagents. Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the product before running the assay. Follow the instructions provided with the product while running the assay. Do not use the product after the expiry date. Only use the reagents provided in the product and those recommended by the manufacturer. Do not mix reagents from different batches. SCH mrtsd01vm_en 28/10/13 Review 05 page 3/15 Warnings and precautions for molecular biology Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products. It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never introduce an amplification product in the area designed for extraction / preparation of amplification reactions. It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification / detection of amplification products to the area designed for the extraction / preparation of the amplification reactions. The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Test tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose. Warnings and precautions specific to components The test tubes containing AmpliMIX are disposable and therefore must be used once only in the preparation of the reaction mixture. AmpliMIX carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. SCH mrtsd01vm_en 28/10/13 Review 05 page 4/15

5 SAMPLES AND CONTROLS PROCEDURE Samples This product must be used with DNA extracted from whole blood collected in EDTA. The whole blood samples to be used for DNA extraction must be collected in EDTA according to laboratory guidelines, transported at +2 / +8 C an d stored at +2 / +8 C for a maximum of three days, otherwise they must be frozen and stored at -20 C f or a maximum of thirty days. It is advisable to split the samples that are to be stored frozen into aliquots in order to prevent repeated cycles of freezing and thawing. N.B.: When the manual nucleic acid extraction is carried out using «EXTRAcell» kit, please, carefully follow the instructions for use manual for pre-treatment of clinical samples. N.B.: When the nucleic acid extraction is carried out from whole blood sample with ELITe STAR System and software version please use the extraction protocol UUNI_E200S200_ELI and follow these directions: ELITe STAR is able to use a sample primary tube, the sample volume required for the extraction is 200 µl. The samples in primary tubes can be loaded directly on the ELITe STAR. It is always requested a minimum dead volume of µl depending on sample primary tube type. Further information and details on primary tubes and the extraction procedure can be found in the instruction for use of extraction kit. It is recommended to release c. 175 ng of DNA extract into the amplification reaction (equivalent to c cells). Do not release into the amplification reaction a higher quantity of DNA extract than 200 ng, in order to prevent the problem of non-specific hybridization or inhibition of the fluorescence emission. Interfering substances The DNA extracted from the starting sample must not contain heparin in order to prevent the problem of inhibition and the possibility of frequent invalid results. The DNA extracted from the starting sample must not contain haemoglobin in order to prevent the problem of inhibition of the amplification and fluorescence emission and the possibility of frequent invalid, undetermined or incorrect results. There are no data available concerning the problem of inhibition caused by drugs. Amplification controls It is absolutely mandatory to validate each amplification session with a positive control reaction and a negative control reaction. For the negative control, use sterile bidistilled water (not supplied with product) added to the reaction in place of the DNA extracted from the sample. For the positive control, use the DNA extracted from a heterozygous sample that has already tested positive, or «FACTOR V - Positive Control». Quality controls It is recommended to validate the whole analysis procedure of each extraction and amplification session by processing a normal sample, a mutated homozygous sample and a heterozygous sample that have already been tested with a different method and verify the obtained results were in accordance to this method. SCH mrtsd01vm_en 28/10/13 Review 05 page 5/15 Setting up the real-time amplification session (To be performed in the amplification / detection area of the amplification products) Before starting the session it is important to do the following: - referring to the instrument documentation, switch on the real-time thermal cycler, switch on the control computer, launch the special software and open an "absolute quantification" session; - referring to the instrument documentation, first set the detector for the "nor FV 1691G" normal allele probe with the "reporter" as "FAM" and the "quencher" as "none" (NFQ = non-fluorescent quencher); - referring to the instrument documentation, next set the detector for the "mu FV 1691A" mutated allele probe with the "reporter" as "VIC" and the "quencher" as "none" (NFQ = non-fluorescent quencher); - referring to the instrument documentation, for each well used in the microplate, set the "detector" (type of fluorescence that is to be measured), the "passive reference" as ROX (normalisation of measured fluorescence) and the type of reaction (sample, negative amplification control, positive amplification control). Add this information to the Work Sheet enclosed at the end of this instruction manual or print the microplate organisation. The Work Sheet must be followed carefully during the transfer of the reaction mixture and samples into the wells. Note: Below is an example of how the analysis of 6 samples can be organized. Key: S1 S2 S3 S4 S5 S6 NC PC S1 - S6: Samples to be analysed; NC: Negative amplification control; PC Positive amplification control. Referring to the instrument documentation, set the parameters of the thermal cycle on the thermal cycler, setting 30 cycles and a reaction volume of 25 µl. When a 7300 Real-Time PCR System instrument is used, set Run mode: standard When a 7500 Fast Dx Real-Time PCR Instrument is used, set Run mode: fast Amplification thermal cycle Phase Temperature Timing Decontamination 50 C 2 min. Initial denaturation 95 C 10 min. 30 cycles 95 C 15 sec. 60 C (fluorescence 1 min. acquisition) - save the settings of the "absolute quantification" session using an unambiguous and easy-torecognise label (e.g. "year-month-day-fv"). SCH mrtsd01vm_en 28/10/13 Review 05 page 6/15

6 Preparation of the real-time amplification (To be performed in the extraction / preparation area of the amplification reaction) Before starting the session it is important to do the following: - remove and thaw the test tubes containing the samples to be analysed. Centrifuge the tubes to bring the contents to the bottom and keep in ice; - remove and thaw the AmpliMIX test tubes needed for the session, remembering that the contents of each tube are sufficient for 24 reactions. Centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice; - remove and thaw the same number of test tubes of AmpliPROBE as the AmpliMIX tubes. Centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice; - remove and thaw the same number of test tubes of AmpliMASTER as the AmpliMIX tubes. Write "FV" and the date on the test tube label using indelible ink. Centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice; - remove and thaw the Positive Control test tubes. Centrifuge the tube for 5 seconds to bring the contents to the bottom and keep in ice; - if necessary, cut the Amplification microplate to separate the part that will be used in the session, being careful to handle it with powder-free gloves and not to damage the wells. 1. Transfer 100 µl of AmpliMIX to the AmpliMASTER tubes. Mix well and pipette the volume of 100 µl three times into the mix. 2. Transfer 100 µl of AmpliPROBE to the AmpliMASTER tube. Mix well and pipette the volume of 100 µl three times into the mix. 3. Vortex on a low setting for 5 seconds, avoiding the creation of foam. 4. Centrifuge the tubes for 5 seconds to bring the contents to the bottom. 5. Carefully deposit 20 µl of the reaction mixture obtained in this way on the bottom of the Amplification microplate wells, as previously established on the Work Sheet. N.B.: If not all the reaction mixture is used, store the remaining volume in the dark at -20 C for a maximu m of one month in the test tube labelled "FV". Freeze and thaw the reaction mixture only once. 6. Carefully deposit 5 µl of DNA extracted from the first sample in the reaction mixture in the corresponding well of the Amplification microplate, as previously established on the Work Sheet. Proceed in this way for all the other DNA extracts. 7. Carefully deposit 5 µl of sterile bidistilled water (not supplied with the product) in the reaction mixture in the well of the negative control Amplification microplate, as previously established on the Work Sheet. 8. Carefully deposit 5 µl of Positive Control in the reaction mixture in the well of the positive amplification control microplate, as previously established on the Work Sheet. 9. Carefully seal the Amplification microplate using the Amplification Sealing Sheet. 10. Transfer the Amplification microplate to the real-time thermal cycler in the amplification/detection area for amplification products and start the thermal amplification cycle. N.B.: At the end of the thermal cycle the Amplification microplate with the reaction products must be removed from the instrument and eliminated without producing environmental contaminations. In order to avoid the spilling of the reaction products, the Amplification Sealing Sheet must not to be removed from the Amplification microplate. Interpreting the results The values of FAM fluorescence emitted by the specific probe for the FV 1691G normal allele are identified by the detector "nor FV 1691G" (FAM). The values of VIC fluorescence emitted by the specific probe for the FV 1691A mutated allele are identified by the detector "mu FV 1691A" (VIC). These fluorescence values in the amplification reactions must be analysed by the software of the instruments. Before analysing, referring to the instrument documentation, it is necessary to: - manually set the calculation range for the fluorescence back ground level (Baseline) from cycle 6 to cycle 15; When a 7300 Real-Time PCR System instrument is used: - set manually the Threshold for the FAM detector nor FV 1691G to 0.2; - set manually the Threshold for the VIC detector "mu FV 1691A" to 0.2. When a 7500 Fast Dx Real-Time PCR Instrument is used: - set manually the Threshold for the FAM detector nor FV 1691G to 0.1; - set manually the Threshold for the VIC detector "mu FV 1691A" to 0.1. The values of fluorescence emitted by the specific probes for the FV 1691G normal allele and the FV 1691A mutated allele in the amplification reactions and the threshold value of fluorescence are used to determine the threshold cycle (Ct), the amplification cycle in which the value of fluorescence emitted by each probe has reached the threshold level. The Ct values for the specific probes for the normal allele and the mutated allele in the Negative control amplification reaction are used to validate amplification and detection as shown in the following table: Negative control amplification Ct FAM = Undetermined Ct VIC = Undetermined Amplification / Detection CORRECT CORRECT Where Ct FAM is the threshold cycle of the normal allele (FAM "nor FV 1691G") and Ct VIC is the threshold cycle of the mutated allele (VIC "mu FV 1691A"). If the result of the Negative control amplification reaction is other than Ct Undetermined, the target DNA has been detected in the amplification reaction. Problems have occurred during the amplification phase (contamination) which may cause incorrect results. The session is invalid and must be repeated from the amplification phase. The Ct values for the specific probes for the normal allele and the mutated allele in the Positive Control amplification reaction are used to validate amplification and detection as shown in the following table: Positive control amplification Amplification / Detection 20 Ct FAM or Ct VIC 25 CORRECT 0 Ct FAM - Ct VIC 1.5 CORRECT Where Ct FAM - Ct VIC is the absolute value of the difference between the threshold cycle of the normal allele (FAM "nor FV 1691G") and the threshold cycle of the mutated allele (VIC "mu FV 1691A"). If the result of the amplification reaction of the Positive Control is not within the limits, the target DNA has not been detected correctly in the amplification reaction. Problems have occurred during the amplification or detection phase (incorrect preparation of the reaction mixture, incorrect dispensing of the reaction mixture or the positive control, probe or positive control degradation, incorrect setting position of the positive control, incorrect setting of the thermal cycle) which may cause result errors. The session is invalid and must be repeated from the amplification phase. SCH mrtsd01vm_en 28/10/13 Review 05 page 7/15 SCH mrtsd01vm_en 28/10/13 Review 05 page 8/15

7 In the amplification reactions of each sample, the Ct values of normal and mutated probe are used to validate amplification and detection and for allele determination of the genotype. N.B.: Verify with the instrument software (Results > Amplification plot > delta Rn vs Cycle) that the Ct was determined by a fast and regular increase of the fluorescence values and not by peaks or an increase of the background (irregular or high background). This product is able to detect a minimal quantity of about 50 ng of human genomic DNA per reaction (see Performance Characteristics paragraph, page 11). The results as Ct of the amplification reactions of each sample are used as described in the following table: Amplification of the sample Sample suitability Assay result Genotype of the sample Ct FAM > 27.5 and Ct VIC > 27.5 not suitable invalid - Ct FAM 27.5 or Ct VIC 27.5 Ct FAM Undet. and Ct VIC 27.5 or Ct FAM - Ct VIC 2.5 with Ct FAM > Ct VIC Ct FAM 27,5 and Ct VIC Undet. or Ct FAM - Ct VIC 2.5 with Ct FAM < Ct VIC suitable suitable valid valid MUTATED HOMOZYGOUS NORMAL HOMOZYGOUS 2.0 < Ct FAM - Ct VIC < 2.5 not suitable valid UNDETERMINED 0 Ct FAM - Ct VIC 2.0 suitable valid HETEROZYGOTE If the result of the amplification reaction of a sample is <Ct FAM > 27.5 and Ct VIC > 27.5, this means that problems have occurred during the amplification phase (inefficient or invalid amplification) or in the extraction phase (absence of DNA or presence of inhibitors or insufficient number of cells in the starting sample) which may cause incorrect results. The sample is not suitable, the assay is invalid and must be repeated beginning with extraction of a new sample. If the result of the amplification reaction of a sample is absolute value of the difference between Ct FAM and Ct VIC from 2.0 to 2.5, this means that problems have occurred during amplification (inefficient amplification) or extraction (presence of inhibitors or excess number of cells in the starting sample) preventing allele determination of the genotype. The sample is not suitable, the assay is undetermined and must be repeated beginning with extraction of a new sample. The results obtained with this assay must be interpreted in consideration of all the clinical data and the other laboratory tests done on the patient. PROCEDURE LIMITATIONS Use only DNA extracted from the following human samples with this product: whole blood collected in EDTA Do not use a higher quantity of DNA extract with this product than the recommended amount: higher quantities of DNA extract could lead to incorrect or undetermined results. Do not use DNA extracted from heparinized samples with this product: heparin inhibits the amplification reaction of nucleic acids and causes invalid results. Do not use DNA extract that is contaminated with haemoglobin with this product: haemoglobin inhibits the amplification reaction of nucleic acids and may cause invalid results; haemoglobin interferes with the measuring of fluorescence and could cause undetermined or incorrect results. There are no data available concerning the problem of inhibition caused by drugs. The results obtained with this product are subject to the correct collection, transport, storage and preparation of samples. To avoid result errors it is therefore necessary to take particular care during these phases and to carefully follow the instructions provided with the products for nucleic acid extraction. Owing to its high analytical sensitivity, the real-time amplification assay of nucleic acids used in this product is subject to contamination from clinical samples, positive controls and the amplification reaction products themselves. Contamination leads to incorrect results. The product has been designed in such a way as to reduce contamination; nevertheless, this phenomenon can only be prevented by following good laboratory practices and by complying scrupulously with the instructions provided in this manual. This product must be handled by personnel trained in the processing of potentially infective biological samples and chemical preparations classified as dangerous to prevent accidents with potentially serious consequences for the user and other persons. This product requires the use of work clothes and premises that are suitable for the processing of potentially infective biological samples and chemical preparations classified as dangerous to prevent accidents with potentially serious consequences for the user and other persons. This product must be handled by personnel trained in molecular biology techniques, such as extraction, amplification and detection of nucleic acids, to avoid result errors. It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products to prevent incorrect results. This product requires the use of special clothing and instruments for extraction / preparation of amplification reactions and for amplification / detection of amplification products to avoid incorrect results. As with any diagnostic device, the results obtained with this product must be interpreted in consideration of all the clinical data and other laboratory tests done on the patient. As with any diagnostic device, there is a residual risk of obtaining invalid or incorrect results with this product. This residual risk cannot be eliminated or reduced any further. In particular situations, this residual risk can contribute to incorrect decisions with potentially grave consequences for the patient. SCH mrtsd01vm_en 28/10/13 Review 05 page 9/15 SCH mrtsd01vm_en 28/10/13 Review 05 page 10/15

8 Analytical sensitivity: detection limit PERFORMANCE CHARACTERISTICS In terms of the detection limit, the analytical sensitivity of this assay enables identification of approx. 14,000 target DNA molecules (equivalent to the genomes of 7,000 cells or 50 ng of human genomic DNA) in 5 µl of DNA extract added to the amplification reaction. The detection limit was tested using human genomic DNA whose initial concentration was determined by reading the absorption at 260 nm on the spectrophotometer. 50 ng / reaction of the diluted human genomic DNA was used in 50 repeats for the whole procedure of amplification and detection with ELITechGroup S.p.A. products. All repeats were valid and correctly determined. Sample No. correct incorrect invalid undetermined Human genomic DNA Analytical sensitivity: ability to identify the mutated allele In terms of its capacity to identify the mutated allele, the analytical sensitivity of the assay is higher than 99%. The capacity to identify the mutated allele (no. of correct results from valid results) was tested using a number of known-genotype heterozygous whole blood samples, a number of known-genotype mutated homozygous whole blood samples and a number of artificial DNA samples (a plasmid containing the region of interest) mimicking a mutated homozygous genotype. These samples were used for the entire analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products. Out of 222 determinations, 220 were valid (at least one of the Ct less than or equal to 27.5). Out of 220 valid determinations, 220 gave a correct result, no samples gave an incorrect result (no heterozygotes were mistaken for normal homozygotes and no mutated homozygotes were mistaken for heterozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5). Samples No. correct incorrect invalid undetermined Heterozygotes Mutated homozygotes (naturals) Mutated homozygotes (artificial) Analytical specificity: inability to identify mutated allele as normal In terms of inability to identify the normal allele as mutated, the analytical specificity of this assay is greater than 99%. The capacity to correctly identify the normal allele (no. of correct results from valid results) was tested using a number of known-genotype normal homozygous and heterozygous whole blood samples. These samples were used for the whole procedure of analysis, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products. Out of 222 determinations, 218 were valid (at least one of the Ct less than or equal to 27.5). Out of 218 valid determinations, 218 gave a correct result, no samples gave an incorrect result (no normal homozygotes were mistaken for heterozygotes and no heterozygotes were mistaken for mutated homozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5). Samples No. correct incorrect invalid undetermined Normal homozygotes Heterozygotes Robustness: Ct limit for validation The robustness of this assay enables a frequency of valid samples of 98.5% with a Ct limit for validation of The Ct limit was established using a number of known-genotype normal homozygous, heterozygous and mutated homozygous whole blood samples. These samples were used for the whole procedure of analysis, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products. Out of 275 determinations, 271 samples were valid (at least one of the Ct less than or equal to 27.5) and 4 samples were invalid (both Ct greater than 27.5). Samples No. correct incorrect invalid undetermined Normal homozygotes Heterozygotes Mutated homozygotes (natural) Robustness: difference between homozygote Ct values The robustness of this assay enables determination of homozygotes at a frequency greater than 99% with a Ct difference between the two alleles that is greater than or equal to 2.5. The Ct difference between the two alleles for homozygotes was established using a number of known-genotype normal homozygous whole blood samples, a number of known-genotype mutated homozygous whole blood samples and a number of artificial DNA samples (a plasmid containing the region of interest) mimicking a mutated homozygous genotype. These samples were used for the whole procedure of analysis, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products. Out of 224 determinations 222 were valid (at least one Ct less than or equal to 27.5). Out of 222 valid determinations, 222 gave a correct result, no samples gave an incorrect result (no normal homozygotes were mistaken for heterozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5). Samples No. correct incorrect invalid undetermined Normal homozygotes Mutate homozygotes (natural) Mutated homozygotes (artificial) Robustness: difference between heterozygote Ct values The robustness of this assay enables determination of heterozygotes at a frequency greater than 99% with a Ct difference between the two alleles of 0 to 2.0 The Ct difference between the two alleles for heterozygotes was established using a number of known-genotype heterozygous whole blood samples. These samples were used for the entire analysis procedure of analysis, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products. Out of 110 determinations 108 were valid (at least one Ct less than or equal to 27.5). Out of 108 valid determinations, 108 gave a correct result, no samples gave an incorrect result (no heterozygotes were mistaken for homozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5) Samples No. correct incorrect invalid undetermined Heterozygotes SCH mrtsd01vm_en 28/10/13 Review 05 page 11/15 SCH mrtsd01vm_en 28/10/13 Review 05 page 12/15

9 Reproducibility The reproducibility of this assay, in terms of its capacity to obtain the same results from the same samples in different sessions, is greater than 95%. The ability to obtain the same results from the same samples in different sessions was tested using a number of known-genotype normal homozygous and heterozygous whole blood samples. These samples were used for the entire analysis procedure of whole procedure of analysis, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products. Out of 3 allele determination sessions carried out on the same 21 samples on different days, 21 results were concordant. Samples No. sessions concordant discordant Normal homozygotes Heterozygotes Mutated homozygotes Diagnostic specificity The diagnostic specificity of this assay, as the ability to not identify as mutated the normal allele was tested on samples of archival blood from subjects with normal homozygous and known heterozygous genotype. The ability to correctly detect the normal allele was tested using 24 samples of whole blood with known normal homozygous genotype and 24 samples of heterozygous genotype from different donors. These samples were used for the whole procedure of analysis, automatic extraction with «ELITeSTAR System», amplification and detection with ELITechGroup S.p.A. products. Out of 48 determinations 48 have given a valid result. The results are summed up in the following table. Samples N concordant discordant Not valid Normal homozygous Heterozygous In this test the diagnostic specificity was equal to 100%. Diagnostic sensitivity The diagnostic sensitivity of this assay, as the ability to identify the mutated allele, was tested on samples of archival blood from subjects with known heterozygous genotype. The ability to correctly detect the mutated allele, even in single copy, was tested using 24 samples of whole blood with heterozygous genotype from different donors. These samples were used for the whole procedure of analysis, automatic extraction with «ELITeSTAR System», amplification and detection with ELITechGroup S.p.A. products. Out of 24 determinations 24 have given a valid result. TROUBLESHOOTING Target DNA present in the negative control reaction Possible causes Dispensing error on the microplate. Error while setting the instrument Microplate badly sealed. Contamination of the sterile bidistilled water. Contamination of the amplification mix. Contamination of the extraction / preparation area for amplification reactions. Solutions Avoid spilling the contents of the sample test tube. Always change tips between one sample and another. Take care when dispensing samples, negative and positive controls onto the microplate and comply with the work sheet. Check the position settings of the samples, negative and positive controls on the instrument. Take care when sealing the microplate. Use a new aliquot of sterile water. Use a new aliquot of amplification mix. Amplification absent or not efficient in the Positive Control reaction Clean surfaces and instruments with aqueous detergents, wash lab coats, replace test tubes and tips in use. Possible causes Solutions Check the volumes of reagent dispensed during Error in the preparation of the reaction mixture. preparation of the reaction mixture. Take care when dispensing reactions onto the microplate and comply with the work sheet. Dispensing error on the microplate. Check the volumes of reaction mixture dispensed. Check the volumes of positive control dispensed. Probe degradation. Degradation of the positive control. Instrument setting error. Use a new probe aliquot. Use a new aliquot of positive control. Check the position settings of positive control reaction on the instrument. Check the thermal cycle settings on the instrument. High frequency of invalid or undetermined results in the sample reactions The results are summed up in the following table. Samples N concordant discordant Not valid Heterozygous In this test the diagnostic sensitivity was equal to 100%. N.B.: The complete data and results of the tests carried out to evaluate the performance characteristics of the product are recorded in Section 7 of the Product Technical File "" and " FACTOR V Q - PCR Alert AmpliPROBE", FTP RTSD01-V. REFERENCES Voorberg, J. et al. (1994) The Lancet 343: Baker, R. et al. (1994) The Lancet 344: Possible causes DNA extract contaminated by haemoglobin. Excess DNA extract in the reaction. Problems with the optical system. Solutions Repeat the extraction avoiding contamination of the final product with haemoglobin. Assess the number of leukocytes in the sample from the blood count value; repeat the extraction from 500,000 cells. Do not add more than 200 ng of genomic DNA extract to the reaction. Check for contamination with the fluorophore of the thermal block or the lens. Check for obstacles in the optical path. 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10 SYMBOLS Catalogue number. Upper temperature limit. Batch code. Use by (last day of month). In vitro diagnostic medical device. In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic medical devices. Contents sufficient for "N" tests. CONT Contents. Please refer to the instructions for use. Manufacturer. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or "Q - PCR Standard". No general patent or other license of any kind other then this specific right of use from purchase is granted hereby. SCH mrtsd01vm_en 28/10/13 Review 05 page 15/15

11 WORK SHEET A B C D E F G H

12 Factor V Q - PCR Alert AmpliPROBE allele determination of Factor V SNP G1691A RTSD01-V-P ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling FV AmpliPROBE mixture of fluorescent probes labelled with FAM / MGB-NFQ and VIC / MGB-NFQ 4 x 110 µl fluorescent oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X FACTOR V Q - PCR Alert AmpliPROBE RTSD01-V-P -20 C MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex powder-free gloves or similar material. - Vortex mixer. - Bench microcentrifuge (12,000-14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement ( µl, 2-20 µl, 5-50 µl, µl, µl). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system TABLE OF CONTENTS INTENDED USE page 1 PRODUCT DESCRIPTION page 1 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 OTHER PRODUCTS REQUIRED page 2 WARNINGS AND PRECAUTIONS page 2 PROCEDURE page 4 REFERENCES page 4 SYMBOLS page 4 INTENDED USE «FACTOR V Q - PCR Alert AmpliPROBE» is part of a qualitative amplification assay of nucleic acids for allele determination of the locus of coagulation Factor V (FV) for single nucleotide polymorphism (SNP) G1691A (R506Q, Leiden) in DNA samples extracted from whole blood collected in EDTA. The product is intended for use, alongside clinical data and other laboratory tests, in the assessment of risk of deep vein thrombosis. PRODUCT DESCRIPTION The product supplies the mixture of AmpliPROBE fluorescent probes for hybridization in a stabilizing solution, pre-dosed in aliquots into four disposable test tubes. Each test tube contains 110 µl of solution, sufficient for 24 tests. The normal allele probe, labelled with FAM fluorophore and blocked by the MGB-NFQ group, is specific for the normal 1691G region (R506) of the human gene codifying Factor V. The mutated allele probe, labelled with VIC fluorophore and blocked by the MGB-NFQ group, is specific for the mutated 1691A region (Q506) of the human gene codifying Factor V. The product provides 96 determinations, including controls. OTHER PRODUCTS REQUIRED The reagents for DNA extraction from the test samples, the reagents optimized for amplification, the primer reagents (oligonucleotides) and the heterozygous control DNA are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended: For manual nucleic acid extraction, it is recommended the use of generic products «EXTRAcell» (ELITechGroup S.p.A., code EXTD02), kit for DNA extraction from cellular samples; the kit enables 50 extractions. For automatic nucleic acid extraction, it is recommended the use of generic product by ELITechGroup S.p.A. «ELITe STAR 200 Extraction kit» (ElitechGroup S.p.A., code INT011EX), kit for extraction of DNA and RNA from non-cellular and cellular samples with «ELITe STAR instrument» (ElitechGroup S.p.A., code INT010), hereafter indicated as «ELITe STAR». «ELITe STAR 200 Extraction Kit» (ELITechGroup S.p.A., code INT011EX) and «ELITe STAR Instrument» (ElitechGroup S.p.A., code INT010) constitute «ELITe STAR System». «Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents for real time amplification and allele determination; the product provides 96 reactions. «FACTOR V Q - PCR Alert Amplimix» (code ), primer oligonucleotides for allele determination; the product provides 96 reactions. «FACTOR V - Positive Control» (code CTRD01-V), heterozygous control of plasmid DNA; the product provides 25 reactions. This product is exclusively for in vitro use. Warnings and general precautions WARNINGS AND PRECAUTIONS Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121 C for one hour before disposal. SCH mrtsd01vp_en 28/10/13 Review 05 Page 1/4 SCH mrtsd01vp_en 28/10/13 Review 05 page 2/4

13 Factor V Q - PCR Alert AmpliPROBE allele determination of Factor V SNP G1691A RTSD01-V-P Factor V Q - PCR Alert AmpliPROBE allele determination of Factor V SNP G1691A RTSD01-V-P Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. Wear suitable protective clothing and gloves and protect eyes / face. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic products in the work areas. Wash hands carefully after handling samples and reagents. Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the product before running the assay. Follow the instructions provided with the product while running the assay. Do not use the product after the expiry date. Only use the reagents provided in the product and those recommended by the manufacturer. Do not mix reagents from different batches. Do not use reagents from other manufacturers' products. Warnings and precautions for molecular biology Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products. It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never introduce an amplification product in the area designed for extraction / preparation of amplification reactions. It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification / detection of amplification products to the area designed for the extraction / preparation of the amplification reactions. The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Test tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose. Warnings and precautions specific to reagents The test tubes containing AmpliPROBE are disposable and therefore must be used once only in the preparation of the reaction mixture. The AmpliPROBE carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. PROCEDURE The «FACTOR V Q - PCR Alert AmpliPROBE» product must be used with «Q - PCR Alert AmpliMASTER» and products to obtain the reaction mixture. AmpliPROBE is ready for use, hence must be added directly to the reaction mixture. The complete procedure involves preparation and execution of an amplification reaction and hybridization with fluorescent probes on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time) and is described in detail in the instructions manual enclosed with the kit. The performance characteristics and procedure limitations of the complete assay for allele determination of Factor V SNP G1691A are described in detail in the instructions manual enclosed with the kit. CONT Catalogue number. REFERENCES Voorberg, J. et al. (1994) The Lancet 343: Upper temperature limit. Batch code. Use by (last day of month). Baker, R. et al. (1994) The Lancet 344: In vitro diagnostic medical device. SYMBOLS In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic medical devices. Contents sufficient for "N" tests. Contents. Please refer to the instructions for use. Manufacturer. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or "Q - PCR Standard". No general patent or other license of any kind other then this specific right of use from purchase is granted hereby. SCH mrtsd01vp_en 28/10/13 Review 05 page 3/4 SCH mrtsd01vp_en 28/10/13 Review 05 page 4/4