PHILADELPHIA P210 Q - PCR Alert kit Ref. RTSG07-210

Transcription

1 ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: WEB site: PHILADELPHIA Q - PCR Alert kit Ref. RTSG Composto da: Composed by: Composé par: Compuesto por: Composta por: Komponiert von: PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliPROBE Q - PCR Alert AmpliMASTER RTSG P RTS000 RTSG07-210_Front

2 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX detection and measurement of the cdna of BCR- TE 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 3 OTHER PRODUCTS REQUIRED page 3 WARNINGS AND PRECAUTIONS page 3 SAMPLES AND CONTROLS page 5 PROCEDURE page 6 PROCEDURE LIMITATIONS page 12 PERFORMANCE CHARACTERISTICS page 13 REFERENCES page 15 TROUBLESHOOTING page 16 SYMBOLS page 17 INTENDED USE ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: C The «PHILADELPHIA Q - PCR Alert AmpliMIX» product is part of an amplification assay of nucleic acids for the detection of the cdna of the BCR- rearrangement, t(9;22) translocation, Philadelphia chromosome, variant () and for the measuring of the cdna of compared with the cdna of the gene () in the product of reverse transcription reaction obtained from total RNA extracted from peripheral blood samples collected in EDTA or citrate, medullary blood samples collected in EDTA or citrate, lymphomonocyte and leukocyte suspensions. The product is intended for use, alongside clinical data and other laboratory tests, in the diagnosis and monitoring of cases of chronic myeloid leukaemia (CML), acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) positive for this marker. The results obtained with this product can be aligned to the International Scale (IS) by a conversion factor that can be calculated using the product «PHILADELPHIA RNA Reference», manufactured by ELITechGroup S.p.A. and calibrated against the "1 st World Health Organization (WHO) International Genetic Reference Panel for quantitation of BCR- translocation by RQ-PCR". SCH mrtsg07210m_en 28/05/13 Review 04 Page 1/17 ASSAY PRINCIPLE For each sample, the assay involves a duplicate real-time amplification reaction for the target and a duplicate real-time amplification reaction for the control. The four reactions are conducted on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time). In the two first wells, the amplification reaction is carried out specific for a region of the mrna of, while in the other two wells, an amplification reaction is carried out specific for a region of the mrna of (internal suitability test of the sample and the reference system) using the cdna produced by the reverse transcription reaction of the RNA extracted from the test samples. In each amplification reaction a specific probe labelled with FAM fluorophore is activated when hybridized with the specific product of the amplification reaction. Fluorescent emission increases as the specific products of the amplification reactions increase and is measured and recorded by the apparatus. The processing of the data determines the presence and the titre of BCR- cdna in the starting sample compared to the reference cdna of. System standardization was carried out on Applied Biosystems 7000 series instruments. PRODUCT DESCRIPTION The product provides the following components: AmpliMIX A mixture of primer oligonucleotides which is specific for for real time amplification in a stabilizing solution, pre-dosed in aliquots into two disposable test tubes (WHITE cap). Each test tube contains 110 µl of solution, sufficient for 24 reactions. Primer oligonucleotides for are specific for a region of the mrna generated by BCR- rearrangement variant. AmpliMIX A mixture of primer oligonucleotides which is specific for for real time amplification in a stabilizing solution, pre-dosed in aliquots into two disposable test tubes. Each test tube contains 110 µl of solution, sufficient for 24 reactions.. The primer oligonucleotides are specific for a region of the mrna of the human gene codifying The product provides 24 duplicate determinations for the cdna of and 24 duplicate determinations for the cdna of, including standards and controls (e.g. 19 patients in one session). MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling AmpliMIX AmpliMIX primer oligonucleotides mixture, WHITE cap primer oligonucleotides mixture 2 x 110 µl 2 x 110 µl Oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X-100 Oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X-100 SCH mrtsg07210m_en 28/05/13 Review 04 Page 2/17 - -

3 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX 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. OTHER PRODUCTS REQUIRED The reagents for RNA extraction from the samples to be analysed, the reagents for RNA reverse transcription, the reagents optimized for amplification, the detection reagents (fluorescent probes) and the known-quantity DNA standard are not included in this kit. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended: «EXTRAzol» (code EXTR01), kit for RNA extraction from cellular and non-cellular samples; the kit enables 50 extractions. «RT - kit plus» (code BRK200), kit for reverse transcription of RNA with random primer ; the kit provides 50 reverse transcriptions. «Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification; the product enables 96 reactions. «PHILADELPHIA Q - PCR Alert AmpliPROBE» (code RTSG P), fluorescent probes for the real-time amplification of the cdna of and the cdna of ; the product provides 24 duplicate determinations for the cdna of and 24 duplicate determinations for the cdna of ; «PHILADELPHIA Q - PCR Standard» (code STDG07-210), known-quantity plasmid DNA to obtain the standard curve for cdna and cdna; the product is sufficient for 4 sessions. «PHILADELPHIA RNA Reference» (code SPG07-210), a solution of total RNA at Knownquantity intended to express the results in International Scale (IS) as in "1 st World Health Organization (WHO) International Genetic Reference Panel for quantitation of BCR- translocation by RQ-PCR"; the product is sufficient for 6 sessions. 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 potentially infective. 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. 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 does not carry risk phrases (R) and it carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. SCH mrtsg07210m_en 28/05/13 Review 04 Page 3/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 4/17

4 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX SAMPLES AND CONTROLS PROCEDURE Samples This product must be used with the product of the reverse transcription reaction (cdna) obtained from RNA extracted from the following biological samples: peripheral blood collected in EDTA or citrate, medullary blood collected in EDTA or citrate, suspensions of lymphomonocytes and suspensions of leukocytes. Peripheral blood collected in EDTA and medullary blood collected in EDTA or sodium citrate The peripheral blood collected in EDTA or medullary blood collected in EDTA or sodium citrate that are to be used in RNA extraction must be collected according to laboratory guidelines, transported at +2 / +8 C and stored at +2 / +8 C for a maximum of four hours. When starting with peripheral or medullary blood it is advisable to separate lymphomonocytes on Ficoll TM according to laboratory guidelines. The optimum quantity of leukocytes from which to extract total RNA is approximately 10,000,000 cells. Do not freeze peripheral or medullary blood in order to prevent degradation of RNA. Instructions for RNA extraction are contained in the instruction manual for the «EXTRAzol» product. Suspensions of lymphomonocytes or leukocytes. The suspensions of lymphomonocytes or leukocytes that are to be used for RNA extraction must be prepared according to laboratory guidelines, resuspended in sterile physiological solution or sterile PBS, counted and stored at +2 / +8 C for a maximum of four hours. The optimum quantity of lymphomonocytes and leukocytes from which to extract total RNA is approximately 10,000,000 cells. Do not freeze suspensions of lymphomonocytes or of leukocytes in order to avoid degradation of RNA. Instructions for RNA extraction are contained in the instruction manual for «EXTRAzol» kit. Interfering substances The cdna must be produced from RNA that does not contain heparin, haemoglobin or Ficoll TM to prevent the problem of inhibition and the possibility of frequent invalid results. There are no data available concerning inhibition caused by antibiotics, antiviral drugs, chemotherapeutic drugs or immunosuppressants. Amplification controls It is absolutely mandatory to validate each amplification session with a negative control reaction and a positive control reaction. For the negative control, use sterile bidistilled water (not supplied with the product) added to the reaction in place of the cdna obtained from the sample. For the positive control use the «PHILADELPHIA Q - PCR Standard» product. Quality controls It is recommended to validate the whole analysis procedure of each extraction, transcription and amplification session by processing a negative sample and a positive sample that have already been tested. Notes on reverse transcription reaction The «RT - kit plus» kit is a system for reverse transcription of RNA, which uses random primers to trigger the polymerization reaction and requires the addition of 10 µl of RNA extract to achieve a final volume of 25 µl. The optimum quantity of total RNA to be released into the reverse transcription reaction with a final volume of 25 µl is approx. 1 µg. 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 software and open an "absolute quantification" session; - referring to the instrument documentation, set the "detector" for the probe with the reporter as "FAM" and the "quencher" as " TAMRA " and name it ""; - referring to the instrument documentation, set the "detector" for the probe with the reporter as "FAM" and the "quencher" as " TAMRA " and name it ""; - 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, knownquantity standard DNA). 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. N.B.: in order to determine the cdna titre in the starting sample, set up two series of reactions with the Q - PCR standard (10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies) to obtain the two standard curves, one for and one for. N.B.: the wells of each pair of duplicates must be given the same name. Key: Below is an example of how the analysis of 3 samples can be organized. C1 C1 C2 C2 C3 C3 CN CN C1 C1 C2 C2 C3 C3 CN CN C1 - C5: reactions with the test samples; CN: reaction with the negative control; 10 2, 10 3, 10 4, 10 5, reactions with the DNA standard 10 2, 10 3, 10 4 and 10 5 copies. C1 - C5: reactions with the test samples; CN: reactions with the negative amplification control; 10 2, 10 3, 10 4, 10 5, reactions with the DNA standard 10 2, 10 3, 10 4 and 10 5 copies. SCH mrtsg07210m_en 28/05/13 Review 04 Page 5/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 6/17

5 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX - Referring to the instrument documentation, set the parameters of the thermal cycle on the thermalcycler setting 45 cycles and a reaction volume of 25 µl. When possible choose the "9600 emulation" option. Amplification thermal cycle Phase Temperature Timing Decontamination 50 C 2 min. Initial denaturation 95 C 10 min. 45 cycles 95 C 15 sec. 60 C 1 min. Amplification set-up (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. Mix gently and centrifuge the tubes to bring the contents to the bottom and keep in ice; - remove and thaw the AmpliMIX (WHITE cap tube) and AmpliMIX test tubes needed for the session, remembering that the contents of each test tube are enough for 24 reactions and that all the and reactions must be set up in duplicate. Mix gently and 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 (WHITE cap tubes) and of AmpliPROBE as the relevant AmpliMIX tubes. Mix gently and centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice; - remove the same number of test tubes of AmpliMASTER as the AmpliMIX (WHITE cap tubes). Write "" 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 the same number of test tubes of AmpliMASTER as the AmpliMIX tubes. Write "" and the date on the test tube label using indelible ink. Mix gently and centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice; - remove and thaw the Q - PCR Standard test tubes needed for the and reactions, which must be set up in duplicate. Mix gently and centrifuge the tubes 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 test tube labelled "". Mix well and pipette the volume of 100 µl three times into the mix. 2. Transfer 100 µl of AmpliPROBE to the AmpliMASTER test tube labelled "". 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. Repeat the procedure from points 1 to 4 by adding the AmpliMIX reagent and the AmpliPROBE reagent to the AmpliMASTER test tube labelled "". 6. Gently deposit 20 µl of the "" reaction mixture obtained in this way on the bottom of the "" wells in the amplification microplate as shown on the work sheet (remember that there must be two wells for each sample, two wells for the negative control and two wells for each Q - PCR Standard). 7. Gently deposit 20 µl of the "" reaction mixture obtained in this way on the bottom of the "" wells in the amplification microplate as shown on the work sheet (remember that there must be two wells for each sample, two wells for the negative control and two wells for each Q - PCR Standard). N.B.: If not all the reaction mixture is used store the volume left in the "" and "" test tubes in the dark at -20 C for a maximum of one month. Freeze and thaw the reaction mixture ONLY ONCE. 8. Gently deposit 5 µl of the cdna of the first sample in the two "" wells and the two "" wells in the amplification microplate as shown on the work sheet. Proceed in the same way for the cdna of the other samples. 9. Gently deposit 5 µl of sterile bidistilled water (not supplied with the product) for amplification negative control in the two "" wells and in the two "" wells of the amplification microplate, as previously established on the Work Sheet. 10. Gently deposit 5 µl of - Q - PCR Standard 10 2 copies in the two "" wells and in the two "" wells of the amplification microplate, as previously established on the Work Sheet Proceed in this way for the other - Q - PCR Standard (10 3, 10 4, 10 5 copies). 11. Carefully seal the amplification microplate using the amplification adhesive sheet. 12. Transfer the amplification microplate to the real time thermal cycler in the amplification / detection area of the amplification products and start the thermal amplification cycle. Qualitative analysis of the results The values of fluorescence emitted by the specific probe for (FAM detector ) in the amplification reactions and by the specific probe for (FAM detector ) in the amplification reactions must be analysed by the instrument software. 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*; *N.B.: in the case of a positive sample with a high titre of or, the FAM fluorescence of the specific probe for o may begin to grow before the 15th cycle. In this case the calculation range for the baseline must be set from cycle 6 to the cycle in which the FAM fluorescence starts to grow. - manually set the Threshold for the fluorescence emitted by the specific probe for (fluorescence FAM) to 0.2; - manually set the Threshold for the fluorescence emitted by the specific probe for (fluorescence FAM) to 0.2; The values of fluorescence emitted by the specific probes in the amplification reactions and the Threshold value of fluorescence are used to determine the Threshold Cycle (Ct), the thermic cycle at which the threshold fluorescence value is reached. In the reactions of the Q - PCR Standard 10 5 the Ct value is used to validate the PCR session as described in the following tables:: Reaction of Q - PCR Standards 10 5 (FAM ) Assay result Amplification / Detection Ct 25 POSITIVE CORRECT Reaction of Q - PCR Standards 10 5 (FAM ) Assay result Amplification / Detection Ct 25 POSITIVE CORRECT If the result of the Q - PCR Standard 10 5 amplification reaction is Undetermined or with a Ct > 25, it means that target DNA has not been detected. Problems have occurred during the amplification or detection phase (incorrect reaction mixture volumes, probe degradation, standard degradation, incorrect dispensing of the standards, incorrect standard position setting, incorrect thermal cycle setting) which may cause result errors. The session is invalid and must be repeated from the amplification phase. SCH mrtsg07210m_en 28/05/13 Review 04 Page 7/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 8/17

6 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX The Ct values of fluorescence emitted by the specific probes for and in the amplification reactions of Negative control and the Threshold value of fluorescence are used to validate amplification and detection as shown in the following table: Reaction of Negative Control (FAM ) Assay result Amplification / Detection Ct Undetermined NEGATIVE CORRECT Reaction of Negative Control (FAM ) Assay result Amplification / Detection Ct Undetermined NEGATIVE CORRECT If the result of the Negative control amplification reaction is anything other than Ct undetermined, it means that target DNA has been detected in the amplification reaction through determination of the cycle threshold (Ct), in which the threshold value of fluorescence has been reached. Problems have occurred during the amplification phase (contamination) which may cause incorrect results and false positives. The session is invalid and must be repeated from the amplification phase. The values of fluorescence emitted by the specific probes for and in the amplification reactions of each sample and the Ct values are used to detect the presence of target DNA and to validate the extraction, the reverse transcription, the amplification and detection. Please Note: Verify by using the software tools of the instruments (Results, Amplification Plot, delta Rn vs cycle) that the Ct is determined by a rapid and regular increase of fluorescence value and not by isolated peaks or background signal increases. This product is able to detect a minimum of 10 copies of cdna per amplification reaction (see paragraph on Performance Characteristics on page 13). The results for each sample are used to detect, as shown in the following table: (FAM "") Ct Undetermined Ct Determined Sample Reaction (FAM "") Ct > 29 or Undetermined Sample suitability Assay result cdna of not suitable invalid - Ct 29 suitable valid, negative NOT DETECTED Ct > 29 or Undetermined suitable* valid, positive PRESENT Ct 29 suitable valid, positive PRESENT If the result of the amplification reaction of a sample is Ct Undetermined for cdna and Ct > 29 or Ct Undetermined for cdna, this means that cdna was not detected efficiently. In this case, problems have occurred during the amplification phase (inefficient or invalid amplification) or in the reverse transcription phase (inefficient or invalid reverse transcription) or in the extraction phase (absence of DNA or presence of inhibitors) which may cause incorrect results and false negatives. 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 Ct Undetermined for the cdna and Ct 29 for the cdna, this means that cdna has not been detected in the cdna obtained from the sample but it is not possible to exclude the presence of cdna at a lower titre than the detection limit of the product (see paragraph on Performance Characteristics on page 13). In this case the result would be a false negative. 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. *Please Note: When cdna has been detected in the amplification reaction of a sample but the cdna amplification shows Ct > 29 or Ct Undetermined, this means that the sample is still suitable in terms of quality and the positive result of the assay is valid. In this case, however, it is not possible to use the data for the quantitative analysis of the results (see following pages). Quantitative analysis of the results After carrying out the procedure for qualitative analysis of the results it is possible to perform the quantitative analysis of the results of the positive samples. The Ct values of fluorescence emitted by the specific probe for and in the amplification reactions of the four Q - PCR Standard are used to calculate the two Standard Curves of the amplification session and to validate the amplification and detection as shown in the following table: (FAM "") Standard Curve Acceptance range Amplification / Detection Correlation coefficient (R2) R CORRECT (FAM "") Standard Curve Acceptance range Amplification / Detection Correlation coefficient (R2) R CORRECT If the Correlation coefficient value (R2) is not within the limits, it means that problem have occurred during the amplification or detection phase (incorrect preparation of the reaction mixture, incorrect dispensing of the reaction mixture or the standards, probe or standard degradation, incorrect standard position setting, incorrect thermal cycle setting) which may cause incorrect results. The session is invalid and must be repeated from the amplification phase. The Ct values of the specific probe for in the amplification reactions of each sample and the Standard Curve of the amplification session are used to calculate the Quantity of target DNA present in the amplification reactions of the samples. This product is able to measure from 1,000,000 to 10 copies of cdna per amplification reaction (see paragraph on Performance Characteristics on page 13) as shown in the following table. Result of the sample (FAM "") cdna of per reaction Quantity > 1 x 10 6 GREATER THAN 1,000,000 1 x 10 1 Quantity 1 x 10 6 = Quantity Quantity < 1 x 10 1 FEWER THAN 10 This product is able to measure from 1,000,000 to 100 copies of cdna per single amplification reaction (see paragraph on Performance Characteristics on page 13); however for the purposes of measuring, the useful interval is from 1,000,000 to c copies (corresponding to a Ct of around 29), as shown in the following table: Result of the sample (FAM "") Quantity > 1 x 10 6 cdna of per reaction GREATER THAN 1,000,000 COPIES ~ 1 x 10 3 Quantity 1 x 10 6 = Quantity Quantity < ~ 1 x 10 3 LOWER THAN ~ 1,000 COPIES (SAMPLE UNSUITE) SCH mrtsg07210m_en 28/05/13 Review 04 Page 9/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 10/17

7 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX Analysis of duplicates When this product is used for measuring of, the results (Quantity) of the amplification reactions on the samples are used to calculate the number of copies of the cdna of normalized to the total copies of the cdna of ( %) available in the starting sample, according to this formula: Quantity of cdna of % = x 100 Quantity of cdna of Before calculating % it is necessary to analyse the data obtained in the two repeat samples. The following table shows the different cases that might occur in one amplification session and the recommended approach to assess the data that must be interpreted considering all other clinical data and laboratory tests regarding the patient: Sample Sample suitability cdna of 1 st repeat suitable PRESENT 2 nd repeat suitable PRESENT 1 st repeat suitable NOT DETECTED 2 nd repeat suitable NOT DETECTED 1 st repeat suitable PRESENT (< 10 copies) 2 nd repeat suitable NOT DETECTED 1 st repeat suitable PRESENT (> 10 copies) 2 nd repeat suitable NOT DETECTED 1 st repeat not suitable 2 nd repeat suitable 1 st repeat not suitable 2 nd repeat not suitable PRESENT or UNDETECTED PRESENT or UNDETECTED PRESENT or UNDETECTED PRESENT or UNDETECTED Quantity of cdna of Average quantity of cdna 0 Quantity of cdna if present Retest the sample Retest the sample Retest the sample Quantity of cdna of Average quantity of cdna of Average quantity of cdna of Average quantity of AB cdna 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 With this product only use cdna obtained by reverse transcription from RNA extracted from the following human samples: peripheral blood collected in EDTA or citrate, medullary blood collected in EDTA or citrate, suspensions of lymphomonocytes and suspensions of leukocytes. With this product do not use cdna obtained by reverse transcription from RNA extracted from heparinized samples: heparin inhibits the reverse transcription and amplification reactions of nucleic acids and causes invalid results. With this product do not use cdna obtained by reverse transcription from RNA contaminated by haemoglobin or Ficoll TM : these substances may inhibit the reverse transcription reaction and amplification reactions of the nucleic acids and cause invalid results. There are no data available concerning inhibition caused by antibiotics, antiviral drugs, chemotherapeutic drugs or immunosuppressants. The results obtained with this product are subject to the correct collection, transport, storage and preparation of samples. To avoid incorrect results 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 nested amplification assay of nucleic acids used in this product is subject to contamination from clinical samples that are positive for, from positive controls and from the amplification reaction products themselves. Contamination leads to false positive 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 incorrect results. It is necessary to have separate areas for the extraction/preparation of amplification reactions and for the amplification/detection of amplification products to prevent false positive 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 false positive results. A negative result obtained with this product suggests that the cdna has not been detected in the product of the reverse transcription obtained from the RNA extracted from the sample, but it may also contain the cdna at a lower titre than the detection limit for the product (detection limit for the product, see paragraph on Performance Characteristics on page 13); in this case the result would be a false negative. 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 results, false positives and false negatives with this product. This residual risk cannot be eliminated or reduced any further. In particular situations such as emergency diagnoses, this residual risk can contribute to incorrect decisions with potentially grave consequences for the patient. SCH mrtsg07210m_en 28/05/13 Review 04 Page 11/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 12/17

8 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX Analytical sensitivity: detection limit for PERFORMANCE CHARACTERISTICS The analytical sensitivity for of this assay enables identification of approx. 10 target DNA molecules in 5 µl of cdna extract added to the amplification reaction. In terms of detection limit, the analytical sensitivity for of the assay was tested using plasmid DNA containing the amplification product whose initial concentration was measured by spectrophotometer. The plasmid DNA was diluted to a titre of 10 copies / 5 µl and has been used in 50 repeats for amplification with the ELITechGroup S.p.A. products (see paragraph on accessory products). The final results are summed up in the following table. Samples No. positive negative 10 copies plasmid DNA Analytical sensitivity: linear measuring range for In terms of linear measuring range, the analytical sensitivity for of this assay enables determination of a titre of between 1,000,000 and 10 target DNA molecules in 5 µl of cdna extract added to the amplification reaction. In terms of linear measuring range, the analytical sensitivity for of the assay was determined using a panel of dilutions (1 log10 between one dilution and the next) of plasmid DNA containing the amplification product, whose initial concentration was measured by spectrophotometer. The panel points from 10 7 molecules per reaction to 10 1 molecules per reaction were used in 9 repeats for amplification with ELITechGroup S.p.A. products. The analysis of the data obtained, performed via linear regression, demonstrated that the assay has a linear response for all the panel points (linear correlation coefficient greater than 0.99). The upper limit of the linear measuring range for was fixed at 10 6 molecules / 5 µl, within one logarithm of the highest concentration Q - PCR Standard amplification standard (10 5 molecules / 5 µl). The lower limit of the linear measuring range for was fixed at 10 molecules / 5 µl, within one logarithm of the lowest concentration Q - PCR Standard amplification standard (10 2 molecules / 5 µl). The final results are summed up in the following table. Upper limit Lower limit Analytical sensitivity for : Precision Linear measuring range 1,000,000 copies cdna of / reaction 10 copies cdna of / reaction The assay precision study for, that is the variability of the results of different repeats of a sample with the same concentration analysed during the same session, made it possible to determine a Coefficient of variation (CV %) of 24.9% within the linear measuring range of 10 6 molecules / 5 µl to 10 molecules / 5 µl. Analytical sensitivity for : Accuracy The assay accuracy study for, that is the difference between the mean results obtained in a single session on different repeats of a sample with the same concentration and the theoretical value of the concentration of the samples, made it possible to determine a mean percentage inaccuracy of 9.9% within the linear measuring range of 10 6 molecules / 5 µl to 10 molecules / 5 µl. Analytical sensitivity: linear measuring range for In terms of linear measuring range, the analytical sensitivity for of this assay enables determination of a titre of between 1,000,000 and 100 target DNA molecules in 5 µl of cdna extract added to the amplification reaction. In terms of linear measuring range, the analytical sensitivity of the assay was determined using a panel of dilutions (1 log10 between one dilution and the next) of plasmid DNA containing the amplification product, whose initial concentration was measured by spectrophotometer. The panel points from 10 7 molecules per reaction to 10 1 molecules per reaction were used in 9 repeats for amplification with ELITechGroup S.p.A. products. The analysis of the data obtained, performed via linear regression, demonstrated that the assay has a linear response for all the panel points between 10,000,000 and 100 molecules (linear correlation coefficient greater than 0.99). The upper limit of the linear measuring range for was fixed at 10 6 molecules / 5 µl, within one logarithm of the highest concentration Q - PCR Standard amplification standard (10 5 molecules / 5 µl). The lower limit of the linear measuring range for was fixed at c molecules / 5 µl, corresponding to a Ct of c. 29, inasmuch as this value represents the minimum useful value for this measuring. The final results are summed up in the following table. Upper limit Lower limit Analytical sensitivity for : Precision Linear measuring range 1,000,000 copies cdna of / reaction ~ 1,000 copies cdna of / reaction The assay precision study for, that is the variability of the results of different repeats of a sample with the same concentration analysed during the same session, made it possible to determine a Coefficient of variation (CV %) of 11.5% within the linear measuring range of 10 6 molecules / 5 µl to 100 molecules / 5 µl. Analytical sensitivity for : Accuracy The assay accuracy study for, that is the difference between the mean results obtained in a single session on different repeats of a sample with the same concentration and the theoretical value of the concentration of the samples, made it possible to determine a mean percentage inaccuracy of 11.8% within the linear measuring range of 10 6 molecules / 5 µl to 100 molecules / 5 µl. Diagnostic sensitivity: detection efficiency and quantification on polymorphisms The diagnostic sensitivity of the assay, that is the efficiency of detection and quantification on polymorphisms, was evaluated by comparison of sequences with nucleotide databases. The alignment test of the regions chosen for hybridization of the AmpliMIX primer oligonucleotides ( and ) and of the AmpliPROBE fluorescent probes ( and ) with the sequences available in the database of the BCR and human genes showed preservation and absence of significant mutations. SCH mrtsg07210m_en 28/05/13 Review 04 Page 13/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 14/17

9 PHILADELPHIA Q - PCR Alert AmpliMIX PHILADELPHIA Q - PCR Alert AmpliMIX Diagnostic sensitivity: positive samples The diagnostic sensitivity of the assay, confirming positive clinical samples, was evaluated by analysing a panel of positive samples that were positive for and was greater than 90.5%. The diagnostic sensitivity was tested using as the reference material 42 positive samples tested using real-time amplification by an external laboratory. Each sample was used to carry out the procedure for analysis, reverse transcription and amplification with ELITechGroup S.p.A. products. The final results are summed up in the following table. Samples No. positive negative Positive samples for The four samples expected to test positive were negative with the products of ELITechGroup S.p.A. and with the real-time amplification system of the external laboratory. Moreover, this is in line with the clinical datum since the samples belong to patients in major molecular remission. Analytical specificity: potential interference markers The analytical specificity of the assay, that is the cross-reactivity with other potential interference markers, was evaluated by comparison of sequences with nucleotide databases. The alignment test of the regions chosen for hybridization of the AmpliMIX primer oligonucleotides ( and ) and of the AmpliPROBE fluorescent probes ( and ) with the sequences available in the database of human genomic regions other than the BCR and human genes showed preservation and absence of significant homology. TROUBLESHOOTING Target DNA not detected in the Q - PCR Standard reaction or invalid correlation coefficient of the standard curve. 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 standard dispensed. Probe degradation. Degradation of standards. Instrument setting error. Target DNA present in the negative control reaction Use a new probe aliquot. Use a new aliquot of standard. Check the position settings of standard reactions on the instrument. Check the thermal cycle settings on the instrument. Diagnostic specificity: negative samples The diagnostic specificity of the assay, confirming negative clinical samples, was evaluated by analysing a panel of negative samples that were positive for and was greater than 94.7%. The diagnostic specificity was tested using as the reference material 20 negative samples tested using real-time amplification by an external laboratory. Each sample was used to carry out the procedure for analysis, reverse transcription and amplification with ELITechGroup S.p.A. products. The results are shown in the following table. Samples No. positive negative Negative samples for One sample was invalid. 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 "PHILADELPHIA Q - PCR Alert AmpliMIX" and "PHILADELPHIA Q - PCR Alert AmpliPROBE", FTP RTSG 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 controls and standards onto the microplate and comply with the work sheet. Check the position settings of the samples, negative controls and standards. Take care when sealing the microplate. Use a new aliquot of sterile water. Use a new aliquot of amplification mix. Clean surfaces and instruments with aqueous detergents, wash lab coats, replace test tubes and tips in use. REFERENCES J. Gabert et al. (2003) Leukemia 17: E. Beillard et al. (2003) Leukemia 17: High levels of background fluorescence in the reactions Baseline setting error. Possible causes Solutions Set baseline calculation interval within cycles where the background fluorescence has already stabilized (check the "component" recordings) and where the signal fluorescence has not started to grow yet, e.g. from cycle 9 to cycle 15. SCH mrtsg07210m_en 28/05/13 Review 04 Page 15/17 SCH mrtsg07210m_en 28/05/13 Review 04 Page 16/17

10 PHILADELPHIA Q - PCR Alert AmpliMIX 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. This product enables the purchaser to use it for the amplification and detection of nucleic acid sequences in order to provide in vitro human diagnostic services. The right to do so is only granted if the product is used together with the "Positive Control" or Q - PCR Standard licensed products by ELITechGroup S.p.A. No general right or other licence of any nature is conferred on the purchaser other than the specific right to use the product. SCH mrtsg07210m_en 28/05/13 Review 04 Page 17/17

11 WORK SHEET A B C D E F G H

12 PHILADELPHIA Q - PCR Alert AmpliPROBE RTSG P PHILADELPHIA Q - PCR Alert AmpliPROBE RTSG P TE 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 3 PROCEDURE page 4 REFERENCES page 4 SYMBOLS page 4 INTENDED USE The «PHILADELPHIA Q - PCR Alert AmpliPROBE» product is part of a quantitative amplification assay of nucleic acids for the detection of the cdna of the BCR- rearrangement, t(9;22) translocation, Philadelphia chromosome, variant () and for the quantitative measuring of the cdna of compared with the cdna of the gene () in the product of reverse transcription reaction obtained from total RNA extracted from peripheral blood samples collected in EDTA or citrate, medullary blood samples collected in EDTA or citrate, lymphomonocyte and leukocyte suspensions. The product is intended for use, alongside clinical data and other laboratory tests, in the diagnosis and monitoring of cases of chronic myeloid leukaemia (CML), acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) positive for this marker. The results obtained with this product can be aligned to the International Scale (IS) by a conversion factor that can be calculated using the product «PHILADELPHIA RNA Reference», manufactured by ELITechGroup S.p.A. and calibrated against the "1 st World Health Organization (WHO) International Genetic Reference Panel for quantitation of BCR- translocation by RQ-PCR". PRODUCT DESCRIPTION ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: C The product provides the following components: AmpliPROBE A fluorescent probe specific for for real-time amplification in a stabilizing solution, prealiquoted in two disposable test tubes (WHITE cap). Each test tube contains 110 µl of solution, sufficient for 24 tests. The probe for, labelled with FAM fluorophore and blocked with TAMRA fluorophore, is specific for a region of the mrna, which originates from the BCR- rearrangement, variant. SCH mrtsg07210p_en 28/05/13 Review 04 Page 1/4 AmpliPROBE A fluorescent probe specific for for real-time amplification in a stabilizing solution, prealiquoted in two disposable test tubes Each test tube contains 110 µl of solution, sufficient for 24 tests. The probe for, labelled with FAM fluorophore and blocked with TAMRA fluorophore, is specific for a region of the mrna which originates from the human gene codifying. The product provides 24 duplicate determinations for the cdna of and 24 duplicate determinations for the cdna of, including standards and controls (e.g. 19 patients in one session). MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling AmpliPROBE AmpliPROBE fluorescent probe labelled with FAM / TAMRA, WHITE cap fluorescent probe labelled with FAM / TAMRA 2 x 110 µl 2 x 110 µl Fluorescent oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X-100 Fluorescent oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X-100 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex 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 (thermal cycler for real time). OTHER PRODUCTS REQUIRED The reagents for RNA extraction from the samples to be analysed, the reagents for RNA reverse transcription, the reagents optimized for amplification, the primer reagents (oligonucleotides) and the knownquantity DNA standard are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended: «EXTRAzol» (code EXTR01), kit for RNA extraction from cellular and non-cellular samples; the kit enables 50 extractions. «RT - Kit plus» (code BRK200), product for reverse transcription of RNA with random primer ; the kit provides 50 reactions. «Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification; the product enables 96 reactions. «PHILADELPHIA Q - PCR Alert AmpliMIX» (code ), primer oligonucleotides for the real-time amplification of the cdna of and the cdna of ; the product provides 24 duplicate determinations for cdna and 24 duplicate determinations for cdna. «PHILADELPHIA Q - PCR Standard» (code STDG07-210), known-quantity plasmid DNA to obtain the standard curve for cdna and cdna; the product is sufficient for 4 sessions. «PHILADELPHIA RNA Reference» (code SPG07-210), a solution of total RNA at Knownquantity intended to express the results in International Scale (IS) as in "1 st World Health Organization (WHO) International Genetic Reference Panel for quantitation of BCR- translocation by RQ-PCR"; the product is sufficient for 6 sessions. SCH mrtsg07210p_en 28/05/13 Review 04 Page 2/4 - -

13 PHILADELPHIA Q - PCR Alert AmpliPROBE RTSG P PHILADELPHIA Q - PCR Alert AmpliPROBE RTSG P WARNINGS AND PRECAUTIONS PROCEDURE This product is exclusively for in vitro use. Warnings and general 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 potentially infective. 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. 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 does not carry risk phrase (R) and it carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. SCH mrtsg07210p_en 28/05/13 Review 04 Page 3/4 The «PHILADELPHIA Q - PCR Alert AmpliPROBE» product must be used with the products «Q - PCR Alert AmpliMASTER» and «PHILADELPHIA Q - PCR Alert AmpliMIX» to obtain the reaction mixture. AmpliPROBE and AmpliPROBE reagents are ready for use, hence must be added directly to the relevant reaction mixtures. The complete procedure involves preparation and execution of two real time amplification reactions in two different wells on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time) and is described in detail in the instruction manual enclosed with the «PHILADELPHIA Q - PCR Alert AmpliMIX» product. The performance characteristics and procedure limitations of the complete assay for detection and dosing of the cdna of BCR- are described in detail in the instruction manual enclosed with the «PHILADELPHIA Q - PCR Alert AmpliMIX» product. CONT Catalogue number. Upper temperature limit. Batch code. REFERENCES J. Gabert et al. (2003) Leukemia 17: E. Beillard et al. (2003) Leukemia 17: Use by (last day of month). 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. This product enables the purchaser to use it for the amplification and detection of nucleic acid sequences in order to provide in vitro human diagnostic services. The right to do so is only granted if the product is used together with the "Positive Control" or Q - PCR Standard licensed products by ELITechGroup S.p.A. No general right or other licence of any nature is conferred on the purchaser other than the specific right to use the product. SCH mrtsg07210p_en 28/05/13 Review 04 Page 4/4

14 Q - PCR Alert AmpliMASTER reagents optimized for real time amplification RTS000 RTS000/2 Q - PCR Alert AmpliMASTER reagents optimized for real time amplification RTS000 RTS000/2 TE 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 ACCESSORY PRODUCTS page 2 WARNINGS AND PRECAUTIONS page 3 PROCEDURE page 4 SYMBOLS page 4 INTENDED USE «Q - PCR Alert AmpliMASTER» is part of the quantitative amplification assay of nucleic acids for the detection and dosing of a target DNA with the real time amplification method in samples of DNA extract or cdna obtained from RNA extract and for allele determination with real time amplification method in samples of DNA extract. The product is an accessory of the products in the «Q - PCR Alert AmpliMIX», «Q - PCR Alert AmpliPROBE», «Q - PCR Standard» lines and in the «Positive Control» lines by ELITechGroup S.p.A. The product is intended for use in the preparation of diagnostic assays based on the real time amplification method. PRODUCT DESCRIPTION ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: +2 C +8 C In the complete size (RTS000) the product provides the mixture of reagents optimized for AmpliMASTER real time amplification in a stabilizing solution divided into aliquots in four ready-to-use test tubes, three amplification microplates with 96 wells and three adhesive sheets for amplification. In the reduced size (RTS000/2) the product provides the mixture of reagents optimized for AmpliMASTER real time amplification in a stabilizing solution divided into aliquots in two ready-to-use test tubes, two amplification microplates with 96 wells and two adhesive sheets for amplification. The reagent mixture provides the buffer system, magnesium chloride, triphosphate nucleotides, ROX fluorophore as the passive reference for the normalisation of the fluorescence, the enzyme Uracil N- glycosidase (UNG) to inactivate contamination from the amplification product, the enzyme Taq DNA polymerase for hot start. N.B.: the amplification microplates shouldn't be used with the Applied Biosystems 7000 series instruments, provided with "FAST" thermal block. In the complete size (RTS000) the product provides 96 determinations, including standards and controls. In the reduced size (RTS000/2) the product provides 48 determinations, including standards and controls. Complete size product, code RTS000 MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling AmpliMASTER optimized reagent mixture 4 x 340 µl Amplification microplate microplate with 96 x 0.2 ml wells TRIS base, TRIS hydrochloride, Glycerol, magnesium chloride, Deoxyribonucleotide triphosphates, ROX, Uracil-N-glycosidase, Taq DNA polymerase Amplification Sealing Sheet adhesive sealing sheet Reduced size product, code RTS000/2 Component Description Quantity Composition Labelling AmpliMASTER optimized reagent mixture 2 x 340 µl Amplification microplate microplate with 96 x 0.2 ml wells TRIS base, TRIS hydrochloride, Glycerol, magnesium chloride, Deoxyribonucleotide triphosphates, ROX, Uracil-N-glycosidase, Taq DNA polymerase Amplification Sealing Sheet adhesive sealing sheet 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 (thermal cycler for real time). ACCESSORY PRODUCTS The primer reagents (oligonucleotides), the detection reagents (fluorescent probes), the knownquantity DNA standard and the positive controls on the plasmid DNA, are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended: «Q - PCR Alert AmpliMIX» series, primer oligonucleotides for real time amplification. «Q - PCR Alert AmpliPROBE» series, fluorescent probes for real time amplification. «Q - PCR Standard» series, known-quantity plasmid DNA to obtain the standard curve. «Positive Control» series, positive control of plasmid DNA. - - SCH mrts000_en 28/05/13 Review 04 Page 1/4 SCH mrts000_en 28/05/13 Review 04 Page 2/4

15 Q - PCR Alert AmpliMASTER reagents optimized for real time amplification RTS000 RTS000/2 Q - PCR Alert AmpliMASTER reagents optimized for real time amplification RTS000 RTS000/2 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. 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. 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 AmpliMASTER does not carry risk phrases (R) and it carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. PROCEDURE The «Q - PCR Alert AmpliMASTER» product must be used with the products in the «Q - PCR Alert AmpliMIX» series and the «Q - PCR Alert AmpliPROBE» line to obtain the reaction mixture. AmpliMASTER is ready for use, hence must be used directly in the preparation of the reaction mixture. The complete procedure involves preparation and execution of a real time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time) and is described in detail in the instruction manual enclosed with the products in the «Q - PCR Alert AmpliMIX» series. The performance characteristics and procedure limitations of the assay including detection and dosing of the target DNA are described in detail in the instruction manual enclosed with the products in the «Q - PCR Alert AmpliMIX» series. CONT Catalogue number. Temperature limits. Batch code. Use by (last day of month). 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 mrts000_en 28/05/13 Review 04 Page 3/4 SCH mrts000_en 28/05/13 Review 04 Page 4/4