«ELITe GALAXY 300 Extraction Kit» code INT021EX

Transcription

1 ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: WEB site: NOTICE of CHANGE dated 18/06/15 IMPORTANT COMMUNICATION FOR THE USERS OF PRODUCT: code In the Instruction for Use Manual (IFU), SCH m, some changes were introduced regarding the classification and labeling of substances / mixtures contained in the kit in accordance with Regulation n. 1272/2008 (CLP) and subsequent amendments. All changes are reported in detail in the enclosed Instructions for Use (IFU) and in the Material Safety Data Sheets related to the product. 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 TEST-KIT (Sehen Sie Package Insert im Kit enthalten) Instruments accessories and kits Specifics accessories and/or kits are intended to be used in association with instruments for automatic extraction and amplification as indicated in the User s manual, in Other products required section. Accessories and/or kits requirement must be expressly indicated in the purchase order of present amplification kit. Notice of Change nr. SCH m_03_en dated 18/06/15

2 TABLE OF CONTENTS INTENDED USE page 1 ASSAY PRINCIPLES page 2 MATERIALS PROVIDED IN THE KIT page 3 MATERIALS REQUIRED BUT NOT PROVIDED IN THE KIT page 3 OTHER PRODUCTS REQUIRED page 4 WARNINGS AND PRECAUTIONS page 5 SAMPLES AND CONTROLS page 9 PROCEDURE page 10 PROCEDURE LIMITATIONS page 33 PERFORMANCE CHARACTERISTICS page 34 TROUBLESHOOTING page 36 SYMBOLS page 37 NOTICE TO PURCHASER: LIMITED LICENSE page 37 INTENDED USE ELITechGroup S.p.A. Corso Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: C +25 C The is a nucleic acid, DNA and RNA extraction kit for purification of human genomic DNA, bacterial DNA and viral DNA and RNA from fluid samples using the «ELITe GALAXY» automated liquid handling workstation. (ELITechGroup S.p.A., code ), «ELITe GALAXY» instrument (ELITechGroup S.p.A., code INT020) and «ELITe GALAXY Software» (version or later equivalent versions, ELITechGroup S.p.A. code INT023SW) constitute the ELITe GALAXY System. The nucleic acid isolation protocol is based on magnetic beads and designed for automated large scale preparation of highly pure DNA and RNA from fresh or frozen whole blood samples collected in EDTA or citrate and plasma samples collected in EDTA or citrate, cerebrospinal fluid,, urine or clarified stool. The procedure has been optimized for the isolation of nucleic acids from 300 µl of sample. The product is intended for use by professionals such as technicians, physicians and biologists trained in molecular biological techniques. It can be used with downstream assays based on Nucleic Acid Amplification Technologies (NAT assay). The use of this product in association with any downstream diagnostic assay must be validated. Any diagnostic results generated using the extracted nucleic acids in association with any downstream diagnostic assay should be interpreted taking into account other clinical or laboratory findings. Adequate controls for downstream assays should be used in order to mitigate risk of incorrect diagnostic results. ASSAY PRINCIPLES The is the reagent set for automated DNA extraction and purification from fresh or frozen cellular fluid samples (e.g. Whole Blood) or DNA and RNA extraction and purification from fresh or frozen non cellular fluid samples (e.g. Plasma) in association with the ELITe GALAXY. The nucleic acid isolation process is based on reversible adsorption of nucleic acids to magnetic beads under appropriate buffer conditions. The Magnetic beads are highly reactive, superparamagnetic beads. Their binding capacity is approximately 0.4 µg of genomic DNA / µl of bead suspension containing about 130 µg of Magnetic Beads. The ELITe GALAXY automatically performs sample dispensing from primary tubes. The nucleic acid purification procedure is carried out without user involvement, except the initial loading of the instrument, thus allowing safe handling of potentially infectious samples. Sample cross-contamination and reagent cross-over is effectively reduced. The use of a unique barcode for each sample and for the elution plate avoids unwanted transpositions. The ELITe GALAXY uses a specific magnetic separator to collect the magnetic beads through the various extraction phases: nucleic acid binding, washing and elution. The ELITe GALAXY combines it with a microplate heater-shaker that allows optimal resuspension of the Magnetic Beads during the washing and elution steps. For fully-automated use, the ELITe GALAXY is provided with a gripper tool: the plate is transferred to the magnetic separator for collection of the Magnetic Beads and transferred to the heatershaker for their resuspension. In this way a high automation level provides a reliable and robust technique. The sample is lysed using Lysis Buffer and proteinase K (PK reconstituted in PK Buffer). A carrier RNA (Carrier reconstituted in Carrier Buffer) and an Internal Control template (CPE) are dispensed in lysed samples. After the lysis step, the magnetic bead suspension (Magnetic Beads) is added. Binding conditions, which allow nucleic acids to bind to the magnetic beads, are adjusted by addition of Binding Buffer 1. After magnetic separation and removal of the supernatant, the magnetic beads are washed twice with Wash Buffer 2 and once with Wash Buffer 4 (80% Ethanol, not provided in the Kit) to remove contaminants and salt. A last wash with Wash Buffer 3 and a drying step remove ethanol from previous wash steps. Finally highly purified nucleic acids are eluted with low-salt Elution Buffer. The complete process on 48 samples takes approximately 2 hours and 40 minutes. The purified nucleic acids are ready to use for downstream assays based on Real-time PCR or on reverse transcription reactions followed by Real-time PCR. Otherwise, the purified nucleic acids can be stored at-20 C or -70 C for subsequent use. The kit provides reagents for 100 extractions (5 runs x 20 samples), including positive and negative controls. Note: The minimum number of samples to be processed per run with the ELITe GALAXY is 1, the maximum number is 48. SCH m_en 28/05/15 Review 03 Page 1/28 SCH m_en 28/05/15 Review 03 Page 2/37

3 MATERIALS PROVIDED IN THE KIT Component Quantity Description PK 3 vials x 50 mg Lyophilized proteinase K PK Buffer 3 bottles x 3.6 ml Buffer to reconstitute proteinase K Magnetic Beads 2 bottles x 3 ml Magnetic bead suspension Carrier 3 vials x 300 µg Lyophilized carrier RNA Carrier Buffer 3 tubes x 500 µl Buffer to reconstitute carrier RNA Lysis Buffer 2 bottles x 10 ml Reagent for the lysis of biological samples Binding Buffer 1 2 bottles x 40 ml Wash Buffer 2 2 bottles x 100 ml Washing solution Wash Buffer 3 1 bottle x 125 ml Washing solution Reagent for optimal adsorption of nucleic acids to the magnetic beads Elution Buffer 1 bottle x 30 ml Buffer for nucleic acid elution Processing Plate 5 plates Disposable square-well plate for the lysis of samples and washing Elution Plate 1 bag x 5 plates Microplate for the elution of nucleic acids Lid 5 lids Cover for the Elution Plate Plate Sealing Foils 1 bag x 15 sealing foils Protection foil for the Elution Plate 12 ml Tubes 3 bags x 5 tubes 100 ml Troughs 2 ml Tubes Material Storage Disposable tubes for the Lysis Buffer and the Elution Buffer 4 bags x 6 troughs Disposable trough for Binding Buffer 1 and the Wash Buffers 2, 3, 4 1 bag x 20 tubes Disposable tubes for PK, Magnetic Beads and IC + Carrier Solution All reagents and components of the should be stored at room temperature (+18 / 25 C). For the expiration date, please refer to the product label. Material Quality Controls ELITechGroup S.p.A. (EGSpA) guarantees the performance characteristics of the for applications as described in the manual. In accordance with the EGSpA certified Quality Management System, the has been tested against established acceptance criteria to ensure consistent product quality. MATERIALS REQUIRED BUT NOT PROVIDED IN THE KIT Sample tubes are not provided with the kit. The user should directly use the non-capped primary tubes listed below. If the samples are collected in different tubes, the user should use one of the secondary tubes listed below. Sample input tube types are grouped into different classes. In one run, only tubes from the same class can be used. The user has to select the correct tube class during the extraction set-up. The following tube classes have been defined: Tube Class Class 1: C1: 13x75/100mm U-bottom tube Class 2: C2: 12x80mm V-bottom tube Class 3: C3: 16x100mm V-bottom tube Class 4: C4: 16x100mm U-bottom tube Tubes admitted BD 3.0 ml Vacutainer, 13 x 75mm (e.g. BD #368856) BD 4.0 ml Vacutainer, 13 x 75mm (e.g. BD #368861) BD 6.0 ml Vacutainer, 13 x 100mm (e.g. BD #367864) Sarstedt 5 ml tube, 13 x 75mm (Sarstedt # ) Copan UTM 1 ml tube, 12 x 80 mm (Copan #359C) Copan UTM 1 ml tube, 12 x 80 mm (Copan #361C) Copan UTM 3 ml tube, 16 x 100mm (Copan #346C) BD 10.0 ml Vacutainer, 16 x 100mm (e.g. BD #367525) Sarstedt 13 ml tube, 16 x 100mm (Sarstedt #55.459) Class 5: C4: 16x100mm U-bottom tube Eppendorf 1.5 ml tube (Eppendorf # ) Class 6: C6: 2mL V-bottom tube Sarstedt 2.0 ml tube skirted screw-cap (Sarstedt # ) Disposable filter tips and solid waste bags are not supplied within the kit. The required consumables are reported below and can be ordered individually from EGSpA. Component Code Quantity Description Standard Volume Tips (300 µl) High Volume Tips (1000 µl) box x 60 racks with 96 tips box x 40 racks with 96 tips Standard Volume Tips (300 µl) with filter conductive tips High Volume Tips (1000 µl) with filter conductive tips Waste bags bags / pack Disposable plastic containers OTHER PRODUCTS REQUIRED This product must be used in association with the instrument «ELITe GALAXY» (EGSpA code INT020), automated liquid handling workstation. The template for the Extraction and Inhibition Internal Control, is not included in this kit. The generic product «CPE - Internal Control» (EGSpA code CTRCPE), which provides the Internal Control templates of a stabilised plasmid solution containing two plasmid DNAs and genomic RNA of MS2 phage for nucleic acid extraction from cellular and non-cellular samples, is recommended for use with this product (used as template DNA). The following equipment and reagents are not provided with the kit: - Disposable powderfree gloves in latex or similar material. - Laminar airflow hood. - Micropipettes and sterile tips with aerosol filter or sterile positive displacement tips. - Vortex mixer. - Bench microcentrifuge (12,000-14,000 RPM). - Bench centrifuge (3,000 RPM). - Absolute ethanol ( 99.8% ethanol, ACS reagent or equivalent). SCH m_en 28/05/15 Review 03 Page 3/37 SCH m_en 28/05/15 Review 03 Page 4/37

4 The same consumables provided in the kit can be also ordered separately as «ELITe GALAXY Consumable Set» (EGSpA code INT ). The list of included consumables is reported below. Component Quantity Description Processing Plate 5 plates Disposable square-well plate for the lysis of samples and washing Elution Plate 1 bag x 5 plates Microplate for the elution of nucleic acids Lid 5 lids Cover for the Elution Plate Plate Sealing Foils 1 bag x 15 sealing foils Protection foil for the Elution Plate 12 ml Tubes 3 bags x 5 tubes 100 ml Troughs 4 bags x 6 troughs 2 ml Tubes 1 bag x 20 tubes Disposable tubes for the Lysis Buffer and the Elution Buffer Disposable trough for Binding Buffer 1 and Wash Buffers 2, 3, 4 Disposable tubes for PK, Magnetic Beads and IC + Carrier Solution WARNINGS AND PRECAUTIONS This product is exclusively designed for in-vitro use. General warnings and precautions Handle and dispose of all biological samples as if they were able to transmit infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. All materials that come into contact with the biological samples must be treated for at least 30 minutes with 3% sodium hypochlorite or autoclaved for one hour at 121 C before disposal. Handle and dispose of all reagents and all materials used to carry out the assay as if they were able to transmit infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be handled and disposed of in compliance with adequate safety standards. Disposable combustible material must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. After receiving the kit, check the kit components for damage. If reagent bottles are damaged, contact the EGSpA Technical Services or your local distributor. In the case of liquid spillage, refer to Warnings and precautions for specific components (page 6) and to the appropriate Material Safety Data Sheets (MSDS). The chemicals and plastic parts are for laboratory use only; they have to be stored in the laboratory and are not to be used for purposes other than intended. Wear suitable protective clothes and gloves and protect eyes and face. Discard gloves if they get contaminated. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic products in the work areas. Carefully wash hands after handling samples and reagents. Dispose of leftover reagents and waste in compliance with the regulations in force. Carefully read all the instructions provided in the product before running the assay. While running the assay, follow the instructions provided in the product. Do not use the product after the indicated expiry date. Do not use damaged kit components. Only use the reagents provided in the product and those recommended by the manufacturer. Do not use reagents from different batches. Do not use reagents from other manufacturers. Warnings and precautions for molecular biology Molecular biology procedures, such as nucleic acid extraction, amplification and detection, require qualified and trained staff to avoid the risk of erroneous results, especially due to the degradation of nucleic acids contained in the samples or contamination of the samples by amplification products. It is necessary to have available 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 designated for extraction / preparation of amplification reactions. It is necessary to have available lab coats, gloves and tools which are exclusively used for the extraction / preparation of the amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designated for the amplification / detection of amplification products to the area designated for the extraction / preparation of the amplification reactions. The samples must be exclusively used for this type of analysis. Samples must be handled under a laminar airflow hood. Pipettes used to handle samples must be exclusively used for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips used must be both DNase and RNase free, and DNA and RNA free. The reagents must be handled in order to avoid any contamination. The pipettes used to handle the reagents must be exclusively used for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips used must be free from DNAse and RNAse, and free from DNA and RNA. Warnings and precautions specific for the components The following components of the contain hazardous reagents. European Community Risk and Safety phrases and GHS Hazard and Precautions phrases applied to those components are listed below. Please, note that hazard labeling is not necessary if quantity per bottle is below 125 g or 125 ml. PK Contains lyophilized Proteinase K. Danger GHS07 GHS08 H315: Causes skin irritation. H319: Causes serious eye irritation. H334: May cause allergy or asthma symptoms or breathing difficulties if inhaled. H335: May cause respiratory irritation. P261: Avoid breathing dust/fumes/gas/mist/vapours/spray. P271: Use only outdoors or in a well-ventilated area. P280: Wear protective gloves/protective clothing/eye protection/face protection. P284: [In case of inadequate ventilation] wear respiratory protection. P302+P352: IF ON SKIN: Wash with plenty of water and soap. P304+P340: IF INHALED: Remove person to fresh air and keep comfortable for breathing. P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do. Continue rinsing. P312: Call a POISON CENTER/ doctor, if you feel unwell. P321: Specific treatment. P332+P313: If skin irritation occurs: Get medical advice/attention. P337+P313: If eye irritation persists get medical advice/attention. P342+P311: If experiencing respiratory symptoms: Call a POISON CENTER/doctor. P362+P364: Take off contaminated clothing and wash it before reuse. P403+P233: Store in a well ventilated place. Keep container tightly closed. P405: Store locked up. P501: Dispose of contents/container in conformity to national regulation. SCH m_en 28/05/15 Review 03 Page 5/37 SCH m_en 28/05/15 Review 03 Page 6/37

5 Carrier Buffer Contains guanidine thiocyanate 30 60%. Binding Buffer 1 Contains sodium perchlorate 20 40% and ethanol 35 55%. Warning GHS07 H302: Harmful if swallowed. H312: Harmful in contact with skin. H332: Harmful if inhaled. H412: Harmful to aquatic life with long-lasting effects. P261: Avoid breathing dust/fumes/gas/mist/vapours/spray. P264: Wash hands thoroughly after handling. P270: Do not eat, drink or smoke when using this product. P271: Use only outdoors or in a well-ventilated area. P273: Avoid release to the environment. P280: Wear protective gloves/protective clothing/eye protection/face protection. P : IF SWALLOWED: Call a POISON CENTER/doctor, if you feel unwell. P302+P352: IF ON SKIN: Wash with plenty of water and soap. P304+P340: IF INHALED: Remove person to fresh air and keep comfortable for breathing. P312: Call a POISON CENTER/ doctor, if you feel unwell. P321: Specific treatment. P330: Rinse mouth. P362+P364: Take off contaminated clothing and wash it before reuse. P501: Dispose of contents/container in conformity to national regulation. EUH032: Contact with acids liberates very toxic gas. Lysis Buffer Contains guanidine hydrochloride %. Warning GHS07 H302: Harmful if swallowed. H315: Causes skin irritation. H319: Causes serious eye irritation. P264: Wash hands thoroughly after handling. P270: Do not eat, drink or smoke when using this product. P280: Wear protective gloves/protective clothing/eye protection/face protection. P : IF SWALLOWED: Call a POISON CENTER/doctor, if you feel unwell. P302+P352: IF ON SKIN: Wash with plenty of water and soap. P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do. Continue rinsing. P312: Call a POISON CENTER/ doctor, if you feel unwell. P321: Specific treatment. P330: Rinse mouth. P332+P313: If skin irritation occurs: Get medical advice/attention. P337+P313: If eye irritation persists get medical advice/attention. P362+P364: Take off contaminated clothing and wash it before reuse. P501: Dispose of contents/container in conformity to national regulation. Warning GHS07 GHS02 H226: Flammable liquid and vapour. H302: Harmful if swallowed. P210 Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking. P233: Keep container tightly closed. P240: Ground/bond container and receiving equipment. P241: Use explosion-proof electrical/ventilating/light equipment. P242: Use only non-sparking tools. P243: Take precautionary measures against static discharge. P264: Wash hands thoroughly after handling. P270: Do not eat, drink or smoke when using this product. P280: Wear protective gloves/protective clothing/eye protection/face protection. P : P303+P361+P353: P305+P351+P338: IF SWALLOWED: Call a POISON CENTER/doctor, if you feel unwell. IF ON SKIN (or hair): Take off immediately all contaminated clothing. Rinse skin with water/ shower. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do. Continue rinsing. P330: Rinse mouth. P370+P378: In case of fire: Use foam, water spray, powder, CARBON DIOXIDE to extinguish. P403+P235: Store in a well ventilated place. Keep cool. P501: Dispose of contents/container in conformity to national regulation. Wash Buffer 2 Warning GHS02 H226: Flammable liquid and vapour. P210 Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking. P233: Keep container tightly closed. P240: Ground/bond container and receiving equipment. P241: Use explosion-proof electrical/ventilating/light equipment. P242: Use only non-sparking tools. P243: Take precautionary measures against static discharge. P280: Wear protective gloves/protective clothing/eye protection/face protection. P303+P361+P353: IF ON SKIN (or hair): Take off immediately all contaminated clothing. Rinse skin with water/ shower. P370+P378: In case of fire: Use foam, water spray, powder, CARBON DIOXIDE to extinguish. P403+P235: Store in a well ventilated place. Keep cool. P501: Dispose of contents/container in conformity to national regulation. For further information, please, see Material Safety Data Sheets. No other component of the contains hazardous reagents and need European Community Risk and Safety phrases and GHS Hazard and Precautions phrases. SCH m_en 28/05/15 Review 03 Page 7/37 SCH m_en 28/05/15 Review 03 Page 8/37

6 SAMPLES AND CONTROLS For reproducible and high yields of extraction, appropriate sample collection, transport and storage is essential. Yields may vary from sample to sample depending on factors such as the patient, the sample age and the kind of sample extracted. Whole Blood collected in EDTA or citrate Whole blood samples (peripheral and from bone marrow) for DNA extraction must be collected in EDTA or citrate according to laboratory guidelines and transported at +2 / +8 C. For short term storage, samples should be stored at +2 / +8 C for a maximum of two days. For long term storage, we recommend to freeze the samples at -20 C (up to 30 days) or -70 C for longer periods. Avoid multiple freeze-thaw cycles of the sample. The whole blood samples do not require pre-treatment and may be directly extracted. Different primary tubes (see Materials required but not provided in the kit, page 3) and anticoagulants (EDTA, citrate, but not heparin) can be used to collect blood samples to be used with the ELITe GALAXY System. Plasma collected in EDTA or citrate After collection in EDTA or citrate and centrifugation, according to laboratory guidelines, plasma for DNA and RNA extraction must be transported at +2 / +8 C and stored at +2 / +8 C for a maximum of 4 hours. For long term storage, we recommend freezing samples in aliquots at -20 C (up to 30 days storage) or at -70 C for longer periods. Multiple freeze-thaw cycles before isolating the DNA and RNA should be avoided. Doing so, causes denaturation and precipitation of proteins, resulting in reduced viral titers. The plasma samples do not require pre-treatment and may be directly extracted. Cerebrospinal fluid (CSF) The CSF samples for nucleic acid extraction must be collected according to laboratory guidelines avoiding contamination by patient blood, transported at +2 / +8 C and stored at +2 / +8 C for a maximum of four hours, otherwise they must be frozen and stored at -20 C for a maximum of thirty days or at -70 C for longer periods. It is recommended to split the samples to be frozen into aliquots in order to prevent repeated cycles of freezing and thawing. Urine The urine samples for nucleic acids extraction must be collected in preservative-free containers according to laboratory guidelines, transported at room temperature (+18 / +25 C) and stored at room temperature (+18 / +25 C) for a maximum of four hours, otherwise they must be frozen and stored at -20 C for a maximum of thirty days or at -70 C for longer periods. It is recommended to split the samples to be frozen into aliquots in order to prevent repeated cycles of freezing and thawing. N.B.: Freezing of urine samples often causes the formation of precipitates that may compromise subsequent stages of the assay: thaw urine samples carefully, dissolving all possible precipitates, only use the supernatant for the extraction. Clarified Stool The stool samples, intended for DNA extraction, should be collected following standard stool collection and handling procedures and identified according to laboratory guidelines. Raw stool should be sealed in a sterile screw-cap container that can be adequately sealed to prevent accidental discharge of the contents, and must be transported following all applicable regulations for the transport of etiologic agents. Store samples refrigerated (+2 / +8 C) for up to 24 hours before processing. If stool clarification cannot be performed within 24 hours of collection, store samples at -70 C. Store purified nucleic acids at +2 / +8 C if they will be used on the same day they were extracted or at -20 C for long term storage. ELITechGroup will not take responsibility if sample types other than the ones described above are used or if the recommended procedures are modified. Interfering substances Whole blood samples and plasma samples must not contain heparin, as it is a powerful inhibitor of DNA polymerase enzymes (such as thermostable DNA polymerases and reverse transcriptase) and leads to invalid or incorrect results in downstream assays performed on the extracted DNA and RNA. Any inhibitory effect caused by drugs that may be contained in the starting sample will have to be evaluated each time by the user in account of downstream assays performed on the extracted DNA and RNA. Extraction quality controls It is recommended to validate the whole procedure of each extraction session by using one Positive Specimen Processing Control and one Negative Specimen Processing Control. As a Negative Specimen Processing Control, use a negative sample that has already been tested with the downstream assay or carry out a simulated extraction using molecular biology grade water in place of the sample. As a Positive Specimen Processing Control, use a positive sample that has already been tested with the downstream assay or a certified reference material. PREPARATION OF WORKING SOLUTIONS (To be performed in the extraction area). PROCEDURE Before starting an extraction run, prepare the following reagents: Lysis Buffer, Binding Buffer 1, Wash Buffer 2, Wash Buffer 3 and Elution Buffer are ready to use. Magnetic Beads need to be resuspended before removing them from the storage bottle. Vortex storage bottle briefly until a homogenous suspension is formed, then transfer the required volume to the 2 ml Tube. PK Solution must be reconstituted as follows: spin down the lyophilized PK, resuspend it by adding 2.4 ml of PK Buffer, mix thoroughly and store PK Solution at -20 C for a maximum of 6 months. Carrier Solution must be reconstituted as follows: spin down the lyophilized Carrier, resuspend it by adding 450 µl of Carrier Buffer, mix thoroughly and store Carrier Solution at - 20 C for a maximum of 6 months. Note: Carrier (a synthetic RNA) enhances the recovery of viral nucleic acids, especially if there are very few target molecules in the sample. Furthermore, the addition of large amounts of Carrier reduces the chance of viral nucleic acid degradation. IC + Carrier Solution must be prepared as follows: Calculate the Internal Control (IC) template (e.g. CPE Internal Control) and the Carrier Solution volumes needed according to the following rules: Volume of IC = Number of samples x 10 µl µl, Volume of Carrier Solution = Number of samples x 5 µl + 50 µl. Thaw the necessary tubes of IC and Carrier Solution (already reconstituted). Mix the content gently and spin it down for 5 seconds. Mix the IC and the Carrier Solution in a 2 ml Tube. Vortex and spin down the IC + Carrier Solution. The solution cannot be stored or frozen. It has to be used on the same day as preparation. SCH m_en 28/05/15 Review 03 Page 9/37 SCH m_en 28/05/15 Review 03 Page 10/37

7 Wash Buffer 4 (not provided in the kit) must be prepared just before use as follows: add 8 parts of absolute ethanol to 2 parts ultrapure water and mix thoroughly. Note: Using different amount of reagents than those recommended during working solution preparation may lead to incorrect results and/or invalid results. PREPARATION OF SAMPLES When whole blood collected in EDTA or citrate, plasma collected in EDTA or citrate, cerebrospinal fluid, and urine are used for extraction of DNA and RNA with the ELITe GALAXY Extraction System, the sample can be processed directly without any pre-treatment. When stool are used for extraction of DNA and RNA with the ELITe GALAXY Extraction System, the sample have to be pre-treated before loading on the instrument. Keep the raw stool on ice throughout the process. Prepare two labeled 1,5 ml tubes for each raw stool, and dispense 0.4 ml of S.T.A.R. buffer into one tube. Vortex the raw stool, and then use a pipettor with an aerosol resistant tip to transfer approximately 100 µl (use a wide bore tip or plastic spatula as necessary for thick stool samples) of the raw stool into the 1.5 ml tube containing the S.T.A.R. buffer. Cap the tube securely, and then vortex the tube to homogenize mixture (20-30 sec). Centrifuge the homogenized solution at g (RCF) for 1 minute to clarify the sample. Carefully transfer the clarified stool supernatant into the second labeled tube, being careful not to disturb the pelleted fecal material. Store the clarified stool in at +2 / +8 C for up to 7 days before proceeding with the extraction. N.B.: The S.T.A.R. Buffer must be stored at room temperature. White precipitates may form when the buffer is stored below room temperature. Before beginning the extraction procedure check if any precipitates have formed, and if so warm the solution to C in a water bath or incubator until the precipitates have dissolved. Note: Samples have to be pipettable : please, check that there are no clots or other solid materials. If you use primary tubes that are completely filled, please mix the sample to ensure a homogenous solution is formed before loading onto the instrument. Minimum volume of samples in primary or secondary tubes The procedure of the ELITe GALAXY Extraction System is optimized for the isolation of DNA and RNA from 300 µl of sample. However, due to sample tube type, an adequate sample volume is needed to prevent pipetting errors. The minimum volume of samples required are shown in the table below. Tube Class Dead-volume Minimum volume of sample Tube Class 1: C1: 13x75/100mm U-bottom tube 250 µl 550 µl Tube Class 2: C2: 12x80mm V-bottom tube 300 µl 600 µl Tube Class 3: C3: 16x100mm V-bottom tube 600 µl 900 µl Tube Class 4: C4: 16x100mm U-bottom tube 350 µl 650 µl Tube Class 5: C4: 16x100mm U-bottom tube 100 µl 400 µl Tube Class 6: C6: 2mL V-bottom tube 100 µl 400 µl When the available sample volume is lower than the minimum, please adjust sample volume with Saline or Phosphate Buffered Saline (PBS). Note: If the ELITe GALAXY detects a low sample volume, it skips the sample and flags in the Result Report. Sample Barcode Specifications: The system is able to read sample tubes with the following barcode standards: Code 128 (subset B and C) Code 39 Codabar Code 2 of 5 Interleaved ISBT Standard JAN/EAN 8 UPC A/E The label with the barcode must meet the following specifications: Length of string: Maximum 20 characters excluding start, stop and check characters, depending on the code length (see label dimensions). Code Density, Tolerance: Minimum module width (X dimension) including a print tolerance: " ( mm); Maximum module width (X dimension) including a print tolerance: 0.02" (0.508 mm); Best reading performance with X dimension between ( mm) and 0.01" (0.254 mm). Check character: Code 128: One character; Code 39: None; Codabar: None. Quiet Zone: 10 times the X dimension, but at least 3 mm. Print quality: The quality of barcode print must be high. A printed barcode with an ISO/EC15416 grade 4 (A) or 3 (B) is required. Offset, typographic, intaglio and flexographic printing are suitable. Mechanical dot matrix and thermo matrix printing are not suitable. The surface may be treated, sealed or plastic-coated. Figure 1: Example and specification of sample barcode. Barcode Label Positioning: Dimension Min. Max. A Label length - 80 mm B Code length - 74 mm C Quiet Zone 3 mm - D Label width 12 mm - E Code width 12 mm - F Distance from code to label edge - 1 mm The label must be attached between 20 mm and 100 mm from the bottom of the tube. The label must fit tightly at an angle of approximately 90 to the tube and over its whole length. Figure 2: Example of barcode label position. SCH m_en 28/05/15 Review 03 Page 11/37 SCH m_en 28/05/15 Review 03 Page 12/37

8 DESCRIPTION OF THE EXTRACTION PROCEDURE The procedure is performed automatically by the ELITe GALAXY. Briefly, the procedure comprises of the following steps: 1. Lysis step Samples are lysed in the presence of chaotropic salt and proteinase K: Dispense 30 µl of PK solution into each well of a Processing Plate. Transfer 300 µl of sample to each well of the Processing Plate. Add 120 µl of Lysis Buffer to each sample and mix by pipetting up and down. Add 15 µl of IC + Carrier solution previously prepared to each sample and mix by shaking. 2. Binding step The DNA and RNA is bound to the surface of the Magnetic Beads after the addition of Binding Buffer 1 and Magnetic Beads to the lysate. Add 25 µl of Magnetic Beads to each sample. Add 450 µl of Binding Buffer 1 to each sample and mix by pipetting up and down and by shaking. Separate the Magnetic Beads against the bottom of the wells by placing the Processing Plate on the magnetic separator. Remove and discard the supernatant. 3. Washing step Salt, contaminants and enzyme inhibitors are efficiently washed away by Wash Buffer 2, Wash Buffer 3 and Wash Buffer 4 while the DNA and RNA remains bound to the Magnetic Beads Washing with Wash Buffer 2. Place the Processing Plate on the heater-shaker. Add 800 µl of Wash Buffer 2 to each well and resuspend the bead / nucleic acid complex by shaking. Separate the Magnetic Beads by placing the Processing Plate on the magnetic separator. Remove and discard supernatant Washing with Wash Buffer 2 Place the Processing Plate on the heater-shaker. Add 800 µl of Wash Buffer 2 to each well and resuspend the bead / nucleic acid complex by shaking. Separate the Magnetic Beads by placing the Processing Plate on the magnetic separator. Remove and discard supernatant Washing with Wash Buffer 4. Place the Processing Plate on the heater-shaker. Add 800 µl of Wash Buffer 4 to each well and resuspend the bead / nucleic acid complex by shaking. Separate the magnetic beads by placing the Processing Plate on the magnetic separator. Remove and discard supernatant Washing with Wash Buffer 3. Leave the Processing Plate on the magnetic separator. Add 700 µl of Wash Buffer 3 to each well while the Magnetic Beads are still attracted to the magnet. Remove and discard supernatant Drying of Magnetic Beads. Place the Processing Plate on the heater-shaker and incubate at 60 C to evaporate all ethanol. 4. Elution step The purified DNA and RNA is finally eluted in Elution Buffer. Add 200 µl of Elution Buffer (alternatively, 100 µl can be used) to each well and resuspend the bead / nucleic acid complex by shaking. Separate the Magnetic Beads by placing the Processing Plate on the magnetic separator. Transfer the supernatant containing the purified DNA and RNA to the Elution Plate. DESCRIPTION OF EXTRACTION SESSION (To be performed in the extraction area) General overview of the ELITe GALAXY The ELITe GALAXY is an automatic and CE-IVD marked instrument for nucleic acids extraction and PCR set-up. The platform performs both pipetting operations on liquids in containers and the transport of plates placed on its work surface. The association of the use of advanced liquid handling technologies and the decades of experience in providing diagnostic solutions, allowed ELITechGroup to develop a system for laboratory routine with high productivity and allow users to set up several assays simultaneously. The ELITe GALAXY instrument and ELITe GALAXY Software has been developed and validated for specific IVD applications by ELITechGroup S.p.A. in combination with IVD extraction kits and IVD Real-Time PCR kits. Figure 3: Front view of the ELITe GALAXY. The instrument deck is divided into equal tracks for loading carriers in predetermined positions. The labware carriers are adapted to those partitions. SCH m_en 28/05/15 Review 03 Page 13/37 SCH m_en 28/05/15 Review 03 Page 14/37

9 Login of the system Warning: Before switch on the ELITe GALAXY check that the waste bag is empty and correctly positioned. If not, replaced it by moving the Modular Arm from the right site of the instrument to the left one. Move the Autoload Barcode Reader from the right site of the instrument to the left one. Remove the frame that holds the waste bag with used tips, close it and discard in the laboratory contaminated waste. Put the frame back in place. Warning: Before switch on the ELITe GALAXY check also the liquid waste bottle: if the waste bottle is not empty, please discard the liquid in the laboratory contaminated waste. Note: Follow local regulations for waste disposal. Switch on the ELITe GALAXY using the power switch located on the front left side of the instrument. Switch on the PC connected to the Instrument. After operative system booting and operator login, the desktop of Microsoft Windows will be automatically shown. Check of Extraction Sample List Before starting the Extraction session, check the Extraction Sample List file generated by the LIMS and saved into the MethodInput directory (C:\MethodInput) by the Microsoft Office Excel Viewer software. The Extraction Sample List is a Microsoft Excel file and it must have the following name format and extension: [name].ext.xls. The list must contain at least the sample ID, the sample barcode information and the elution volume. An existing Extraction Sample List can be modified by the user through the ELITe GALAXY software (see next points). A new Extraction Sample List can be created by the user through the ELITe GALAXY software (see next points). Figure 5: Desktop of Microsoft Windows and its icons. The ELITe GALAXY Extraction & PCR Setup window will be shown. Start the Extraction session clicking on the Extraction button. Note: Before starting the extraction session, manually unload all of the Carriers on the loading shelf. Figure 6: Main menu of the ELITe GALAXY software. Figure 4: Example of Extraction Sample List. Note: When Sample Tubes without barcodes are used in the extraction session, the Sample Tubes have to be put in the sample Carrier(s) in the same order indicated on the Extraction Sample List to assure the traceability of results. Please print the Extraction Sample List before starting the Extraction session. Start the Extraction session To start the ELITe GALAXY software click on ELITechGroup Extraction and PCR Setup icon. Loading of Extraction Sample List created by LIMS The Extraction setup window will be shown. Load the Extraction Sample List file clicking on Import button. SCH m_en 28/05/15 Review 03 Page 15/37 SCH m_en 28/05/15 Review 03 Page 16/37

10 Figure 7: Screen of the Extraction session. The folder MethodInput will be shown. Choose and select the correct Extraction Sample List from the folder MethodInput and click on Open button. Figure 9: Sample selection. Creating an Extraction Sample List The Extraction setup window will be shown. Use the keyboard to input the first sample information in the first row of the Extraction Sample List shown in the Extraction setup window. Note: The list must contain at least the sample ID, the sample barcode information and the elution volume. The ELITe GALAXY Software will automatically show a second row in the Extraction Sample List shown in the Extraction setup window. Use the keyboard to input, individually, information about all of the samples in the rows in the Extraction Sample List shown in the Extraction setup window. Figure 8: MethodInput Folder of the Extraction session. The Extraction Sample List will be automatically loaded in the Extraction setup window. Select the samples to be processed by tagging boxes on the left side of the sample list. On the right side of the window the number of selected samples is shown in the Number of selected samples area. Note: The minimum number of samples to be processed per run is 1, the maximum number is 48. Figure 10: Screen of the Extraction session. Select the samples to be processed by tagging boxes on the left side of the sample list. On the right side of the window, the number of selected samples is shown in the Number of selected samples area. Note: The minimum number of samples to be processed per run is 1, the maximum number is 48. SCH m_en 28/05/15 Review 03 Page 17/37 SCH m_en 28/05/15 Review 03 Page 18/37

11 Figure 11: Samples selection. Figure 12: Example of input of extraction session information. Record of extraction session information On the right side of the Extraction setup window three boxes are shown. 1. Run information box: User ID: fill in the field by keyboard; Tube type: pull down the menu to select the tube type used in the Extraction session (the six tube classes are described in the section Other products required page.4). Note: A description of the six tube classes is shown when the cursor is placed on the Information icon (see fig.13). step. 2. Extraction Kit Details box: Kit barcode /Ref. (input by manual barcode reader or by keyboard); Kit Lot Number (input by manual barcode reader or by keyboard); Kit Expiry Date (input by manual barcode reader or by keyboard). 3. Internal Control Details box: IC barcode /Ref. (input by manual barcode reader or by keyboard); IC Lot Number (input by manual barcode reader or by keyboard); IC Expiry Date (input by manual barcode reader or by keyboard). Click on the start button ( ) on the left side of the Extraction setup window to proceed to the next Figure 13: List of tube classes in the information window. Note: the ELITe GALAXY Extraction setup window is replaced by the Hamilton Run Control Extraction window. The Start Extraction window will be shown. Check the accuracy of information in the Start Extraction window. The Extraction Status window will also be shown on the right-hand bottom corner. The steps of extraction method will be displayed on this window. Click on the OK button in the Start Extraction window to proceed to the next step. Figure 14: Check of extraction session information. SCH m_en 28/05/15 Review 03 Page 19/37 SCH m_en 28/05/15 Review 03 Page 20/37

12 Loading of waste containers Warning: Possible Biohazard. Before working on the instrument deck and with clinical samples, wear gloves and laboratory coats. An Information window will be shown. Check the Solid waste bag is empty and placed in the correct position. Check the Liquid waste bottle is empty and placed in the correct position. Click on the OK button in the Information window to proceed to the next step. The instrument automatically loads the sample Carrier(s) and reads the barcodes. Figure 15: Reminder about waste containers. Loading of barcoded Sample Tubes The Loading of Sample Tubes window will be shown. The following message appears on the left side: Please put the Carrier(s) with the Sample Tubes on the loading shelf. Location: track Note: The samples have to be prepared according to the paragraph Preparation of samples (page 10). Make sure that the sample tubes belong to the same tube class. Make sure that at least the minimum sample volume requested in the paragraph "Minimum volume of samples in primary or secondary tubes" (page 11) is present in the sample tubes. When barcoded Sample Tubes are used, the position of the Sample Tubes on the Sample Carrier is not important. For the correct reading of the Sample Tube barcode, the unique barcode must face the barcode scanner located on the right-hand side of the loading area. table. Place the sample tubes without caps in the correct sample Carrier(s) as illustrated in the following Tube Class Sample Carrier ID Sample Carrier code Tube Class 1 and 2 SMP_CAR_32C12_A Tube Class 3 and 4 SMP_CAR_24_A Tube Class 5 and 6 SMP_CAR_32C12_A00 (Carrier) SMP_INS_2ML_32 (insert) Note: The Sample Carrier allows for the loading of 24 Sample Tubes. One or two Sample Carrier(s) can be used in a session depending on the Sample Tube numbers. Insert the sample Carrier(s) into tracks 22 and / or 23 until they touch the stop hooks on the far side of the loading shelf. Click on the OK button on Loading of Sample Tubes window to proceed to the next step. Figure 16: Loading of sample Carrier(s). Note: In case of unsuccessful sample identification by barcode reader, the sample Carrier(s) is (are) unloaded. Check the barcode orientation and reinsert the sample Carrier(s). The instrument automatically reloads the carrier(s) slowly. Loading of Sample Tubes without barcode The Loading of Sample Tubes window will be shown. The following message appears on the left side: Please put the Carrier(s) with the Sample Tubes on the loading shelf. Location: track Note: The samples have to be prepared according to the paragraph Preparation of samples (page 10). Make sure that the sample tubes belong to the same tube class. Make sure that at least the minimum sample volume requested in the paragraph "Minimum volume of samples in primary or secondary tubes" (page 11) is present in the Sample Tubes. Make sure that the Extraction Sample List is printed (see paragraph Check of Extraction Sample List, page 14). table. Put the Sample Tubes without caps in the correct sample Carrier(s) as illustrated in the following Tube Class Sample Carrier ID Sample Carrier code Tube Class 1 and 2 SMP_CAR_32C12_A Tube Class 3 and 4 SMP_CAR_24_A Tube Class 5 and 6 SMP_CAR_32C12_A00 (Carrier) SMP_INS_2ML_32 (insert) Put the Sample Tubes in the same order indicated in the Extraction Sample List. The position in the Sample Carrier of the Sample Tubes is very important for the traceability of results. Note: The Sample Carrier allows for the loading of 24 Sample Tubes. One or two Sample Carrier(s) can be used in a session depending on the Sample Tube numbers. Insert the sample Carrier(s) into the tracks 22 and 23 until they touch the stop hooks on the far side of the loading shelf. Click on the OK button on Loading of Sample Tubes window to proceed to the next step. The instrument automatically loads the sample Carrier(s) and tries to read the barcodes. The Load Carrier - Error window will be shown. Click on the Barcode button in the Load Carrier - Error window. SCH m_en 28/05/15 Review 03 Page 21/37 SCH m_en 28/05/15 Review 03 Page 22/37

13 Figure 17: Load Carrier - Error window. The Insert Barcode window will be shown. Use the keyboard to input the sample barcodes in Enter barcode and Confirm barcode boxes for each sample loaded. Input the sample barcode in the same order as indicated in the Extraction Sample List. Click on the OK button on Insert Barcode window to proceed to the next sample. Figure 19: Fixed load carrier error after manual barcode input. Loading of Tips: Warning: After sample loading change gloves. The Loading of Tips window will be shown. The following message appears on the left side: Please put the carriers with the tips on the loading shelf. Location: starting at track -1. Note for the required number of Standard volume tips (300 µl), see Quantities in the table. Note for the required number of High volume tips (1000 µl), see Quantities in the table. Insert the first Carrier (TIP_CAR_480_A00, code ) into the tracks -1 6 and the second one (TIP_CAR_480_A00, code ) into the tracks 7-11 until they touch the stop hooks on the far side of the loading shelf. Click on the OK button on Loading of Tips window to proceed to the next step. Note: The tip racks must be present in all positions even if they are empty. Figure 18: Manual sample barcode input. When all sample barcodes are inserted, red dots become green in the Load Carrier - Error window. Click on the Execute button in the Load Carrier - Error window. Figure 20: Tip loading. The Edit Tip Count window will be shown. Check the number of the Standard volume tips (300 µl) at positions 1 and 2 on the first Carrier. If the Remaining Standard volume tips (300 µl) are less than the Quantities needed, add at least the the requested Standard volume tips (300 µl) in positions 1 and 2 on the first Carrier. SCH m_en 28/05/15 Review 03 Page 23/37 SCH m_en 28/05/15 Review 03 Page 24/37

14 Note: For correct reading of the Tip rack barcode, the barcode must face the barcode scanner located on the right side of the loading area. Input the updated tip number (by selecting with cursor) or positions (by keyboard) of Standard volume tips (300 µl) in the First and Last boxes of the Edit Tip Count window. Click on the OK button on Edit Tip Count window to proceed to the next step. Figure 21: Standard volume tips (300 µl) count update. The Edit Tip Count window will be updated. Check the number of the High volume tips (1000 µl) at positions 3 to 10 on the first and the second Carrier. If the Remaining High volume tips (1000 µl) are less than the Quantities needed, add at least the minimum number of the requested High volume tips (1000 µl) in positions 3 to 10 on the first and the second Carrier. Note: For correct reading of the Tip rack barcode, the barcode must face the barcode scanner located on the right side of the loading area. Input the updated tip number (by selecting with cursor) or positions (by keyboard) of High volume tips (1000 µl) in the First and Last boxes of the Edit Tip Count window. Click on the OK button on Edit Tip Count window to proceed to the next step. The instrument automatically loads the Tip Carriers and reads the barcodes. Loading of 2 ml Tubes with reagents, Processing Plate and Elution Plate with Lid: The Loading of Processing Plate, Elution Plate and Reagents window will be shown. The following message appears on the left side: Please put the Carrier with the processing plate, the elution plate and the 2 ml tubes with reagent on the loading shelf. Location: tracks The required amount of extraction reagents is displayed in a table in the window. Dispense the required amount of Magnetic Beads and reconstituted PK in 2 ml Tubes as indicated in the table. Warning: Magnetic Beads must be re-suspended before transferring them from the storage bottle. Vortex the storage bottle briefly until a homogenous suspension has been formed. Note: PK has to be reconstituted by the user (see Procedure page 10) in the storage bottle. Thaw the solution if needed. Vortex the storage bottle briefly and spin down the reconstituted PK. Transfer the solution avoiding bubbles. Prepare the required amount of IC + Carrier Solution in 2 ml Tubes as indicated in the table. Note: IC + Carrier Solution has to be prepared by the user (see Procedure page 10). Put the 2 ml Tubes with reagents without caps in the correct positions in the Reagent cold block as shown in the window. Put the Processing Plate and Elution Plate with a Lid in the correct positions on the Carrier. Note: For the correct reading of the Elution Plate barcode, the unique barcode must face the barcode scanner located on the right side of the loading area. Put the Processing Plate with well A1 on the upper left side in position 5 of the Carrier as shown in the window. Insert the Carrier into the tracks until it touches the stop hooks on the far side of the loading shelf. Click on the OK button on the Loading of Processing Plate, Elution Plate and 2 ml Tubes with reagents window to proceed to the next step. The instrument automatically loads the Carrier and reads the barcodes. The instrument automatically checks the reagent volumes. If an insufficient volume is detected, the Carrier is unloaded allowing the user to fix the issue. Figure 23: Loading of 2 ml Tubes with reagents, Processing Plate and Elution Plate with Lid. Figure 22: High volume tips (1000 µl) count update. SCH m_en 28/05/15 Review 03 Page 25/37 SCH m_en 28/05/15 Review 03 Page 26/37

15 Loading of 100 ml Troughs with reagents The Loading of 100 ml trough Reagents window will be shown. The following message appears on the left side: Please put the Carrier with the 100 ml Troughs on the loading shelf. Location: track 20. The required amount of extraction reagents is displayed in a table in the window. Dispense the required amount of Binding Buffer 1, Wash Buffer 2, Wash Buffer 3, Wash Buffer 4 in 100 ml Troughs as indicated in the table. Note: Wash Buffer 4 (80% Ethanol Solution) is not provided in the kit and has to be prepared by the user (see Procedure page 10). Put the 100 ml Troughs in the correct positions on the Carrier. Insert the Carrier into the track 20 until it touches the stop hooks on the far side of the loading shelf. Click on the OK button on Loading of 100 ml Reagent Troughs window to proceed to the next step. The instrument automatically loads the Carrier and reads the Carrier barcode. The instrument automatically checks the reagent volumes. If an insufficient volume is detected, the Carrier is unloaded allowing the user to fix the issue. Figure 24: Loading of 100 ml Troughs with reagents. Loading of 12 ml Tubes with reagents The Loading of Reagent Tubes window will be shown. The following message appears on the left side: Please put the Carrier with the 12 ml tubes on the loading shelf. Location: track 21. The required amount of extraction reagents is displayed in a table in the window. Dispense the required amount of Lysis Buffer and Elution Buffer in 12 ml Tubes as indicated in the table. Note: Transfer the Lysis Buffer avoiding bubbles. Put the 12 ml Tubes without caps in the correct positions in the Carrier. Insert the Carrier into the track 21 until it touches the stop hooks on the far side of the loading shelf. Click on the OK button on Loading of Reagent Tubes window to proceed to the next step. The instrument automatically loads the Carrier and reads the Carrier barcode. The instrument automatically checks the reagent volumes. If an insufficient volume is detected, the Carrier is unloaded allowing the user to fix the issue. Figure 25: Loading of 12 ml Tubes with reagents. End of Loading The Information window will be shown. The following message appears: Loading is complete. To start the extraction run, click OK. This will take about hh:mm. Please, note the expected time of the extraction run is displayed in the window. Click on the OK button on Information window to start the extraction run. Note: The instrument automatically performs the extraction method. The status of the extraction run is displayed in the Extraction Status window. No further user action is required. In case of error, the instrument stops, a warning window appears on the screen, an acoustic alarm starts and an is sent to a user-defined address. When an error occurs, the ELITe GALAXY software could ask the user for actions. A trouble shooting guide is provided in the ELITe GALAXY Instructions for use (recovery code paragraph). The instrument associates a specific code to each kind of error and saves it in the Report Files. A cross reference list for decoding these is provided in the ELITe GALAXY Instructions for use (list of main error codes paragraph). Figure 26: Information at the end of loading. SCH m_en 28/05/15 Review 03 Page 27/37 SCH m_en 28/05/15 Review 03 Page 28/37

16 Unloading When the ELITe GALAXY has completed the extraction method without any errors, the Run finished successfully window will be shown. The following message appears: The extraction run was finished successfully at hh:mm. Click OK to unload all carriers. The time of the end of extraction is displayed in the window. Click on the OK button in the Run finished successfully window to proceed to the next step. The instrument automatically unloads the Carriers on the loading shelf. The Samples, Materials and Reagents Unloading window will be shown. In this window a table lists the instructions needed to unload the Carriers and store or discard samples, materials and reagents. Please carry out these actions immediately. The table also lists the instructions for cleaning the work area. Please note for the required actions that must be carried out after the Extraction run has finished. Note: The Elution Plate containing eluted samples must be closed off with a Plate Sealing Foil under the Lid. The extracted DNA or RNA may be stored at -20 C for a maximum of thirty days or at -70 C for longer periods. Freeze/Thawing cycles of extracted DNA or RNA must be limited to five times in order to avoid titre loss. Close off the sample tubes using the correct caps and store them as described in Sample and Controls paragraph, (page 9). As with other diagnostic equipment, all waste products (liquids, tips, troughs, tubes and Processing Plate) should be treated as potentially dangerous bio-hazardous waste and discarded accordingly. The used Processing Plate contains DNA or RNA and can contaminate subsequent extraction sessions. Do not reuse and adequately discard the used Processing Plate. Click on the OK button on Samples, Materials and Reagents Unloading window to proceed to the next step. Figure 27: Message at the end of extraction run. When the ELITe GALAXY finishes the extraction session with errors, the Run finished with error window is shown. The following message appears: The extraction run was finished with error at hh:mm. Please check the output files for detailed information: C:\MethodOutput\[DateAndTime]_[ElutionPlateBarcode]. Click OK to unload all carriers. The time of the extraction end is displayed in the window. Click on the OK button in the Run finished with error window to proceed to the next step. The instrument automatically unloads the Carriers on the loading shelf. Figure 29: Unloading of carriers. Report creation The Hamilton Run Control Extraction window is closed. The ELITe GALAXY Extraction window is shown in grey for a few seconds while the ELITe GALAXY software creates and saves the reports. Warning: Do not close the ELITe GALAXY Extraction window until the software completes the reporting activities. Closing the program will cause the loss of the extraction run data and results. The corresponding extracted samples will not be available for downstream assays. The Information window will be shown. The following message appears: The extraction run has finished. All files have been saved in: C:\MethodOutput\[DateAndTime]_[ElutionPlateBarcode]. Please note for this path and file name in order to find the extraction output reports. Click on the OK button in the Information window. Figure 28: Message at the end of extraction run with errors. SCH m_en 28/05/15 Review 03 Page 29/37 SCH m_en 28/05/15 Review 03 Page 30/37

17 Note: Extraction output files are created in the Microsoft Excel.xls format during the extraction run and they have the following naming conventions: General report about extraction session with data records: [DateAndTime]_[ElutionPlateBarcode]_EG_EXT_General.xls, Report about samples processed in the extraction session, their position in the Elution Plate and information about pipetting status: [DateAndTime]_[ElutionPlateBarcode]_EG_EXT_Samples.xls, Report with technical level information about pipetting tracking: [DateAndTime]_[ElutionPlateBarcode]_EG_EXT_PipettingTracking.xls, Technical report about samples processed in the extraction session and Elution Plate used by instrument for PCR setup input: [DateAndTime]_[ElutionPlateBarcode]_EG_PCRSampleList.SMP.xls. Figure 30: Report creation and end of extraction method. Closing ELITe GALAXY Software The ELITe GALAXY Extraction window is shown. Close the ELITe GALAXY software by clicking window closing button. The desktop of Microsoft Windows will be automatically shown. Figure 31: ELITe GALAXY software closing. Cleaning: After any session follow the instructions shown in the table in Samples, Materials and Reagents Unloading window (paragraph Unloading, page 26) for work area cleaning. Warning: Possible Biohazard. Before working on the instrument deck, wear gloves and laboratory coats. Warning: Carry out the cleaning of the instrument after switching off the instrument. Move the Modular Arm from the right site of the instrument to the left one. Move the Autoload Barcode Reader from the right site of the instrument to the left one. Remove the frame that holds the waste bag with used tips, close it and discard in the laboratory contaminated waste. Put the frame back in place. Check also the liquid waste bottle: if the waste bottle is not empty, please discard the liquid in the laboratory contaminated waste. Note: Follow local regulations for waste disposal. Clean the tip waste sink by pouring in about 50 ml of water. Remove the tip eject plate of the tip waste station and clean it with disinfectant and then water. Put the tip eject plate back in place. Treat the work area with UV light for 5 minutes (see UV decontamination paragraph at page 29). Note: Do not use disinfectant materials which contain hypochlorite or bleaching fluids. Use cleaning, disinfecting and decontaminating fluid in accordance with manufacturer s instructions (see the manual MICROLAB STAR IVD / STARlet IVD OPERATOR S MANUAL ). If any parts of the instrument, e.g. carriers or tip eject plate (outer part of the pipetting channels), are contaminated, the weekly maintenance procedure must be performed (see Maintenance paragraph, page 29). For the complete cleaning procedure see the manual ELITe GALAXY Instructions for use. Maintenance: Periodic maintenance routines need to be run in order to ensure safe and reliable operation of the ELITe GALAXY and the accessories. The maintenance routines are mandatory. The following maintenance is to be undertaken periodically: Daily maintenance: before ELITe GALAXY start-up or shut-down; Weekly maintenance: at the end of the week before ELITe GALAXY shut-down; Six-monthly maintenance: preventive service maintenance carried out by technical service. Note: If the operator decides not to run either daily or weekly maintenance before shut-down, these routines must be executed at the next start-up. For the maintenance procedure see the manual ELITe GALAXY Instructions for use (UV decontamination paragraph). UV Decontamination The UV light is used for the daily decontamination of the ELITe GALAXY deck. This is done when no other protocol is run on the system and uses a dedicated method. Due to the UV radiation, the ELITe GALAXY is equipped with UV resistant covers and a front panel has to be placed on the loading shelf opening. The UV decontamination is only for the deck of the ELITe GALAXY and is not meant to be used in combination with the different carriers. Warning: UV radiation is harmful. It causes serious burns to the skin and leads to permanent damage to the eyes and skin. Ensure that no laboratory personnel are exposed to direct UV light. Always keep the instrument front door and the front panel closed during the decontamination process. The Instrument is equipped with an internal UV lamp that should be used daily either at the end of the working day or in the morning before any run starts. The suggested decontamination time is about 5 min. To start the UV decontamination method click on Decontaminate STARlet icon on the desktop of Microsoft Windows. When the UV decontamination is finished the device is decontaminated and can be either switched off or used for sample processing. For the UV decontamination procedure, see the manual ELITe GALAXY Instructions for use (UV decontamination paragraph). SCH m_en 28/05/15 Review 03 Page 31/37 SCH m_en 28/05/15 Review 03 Page 32/37

18 PROCEDURE LIMITATIONS PERFORMANCE CHARACTERISTICS Only use the following clinical samples with this product: whole blood (peripheral and from bone marrow) collected in EDTA or citrate, plasma collected in EDTA or citrate, cerebrospinal fluid, urine and clarified stool. The kit validation is limited to the matrices mentioned in the intended use, other matrices leads to loss of compliance with EU Directive 98/79/EC for the respective process. No guarantee is issued with differing sample type or change in the procedure. The user is responsible to validate the performance of the product for use with different assays from the validated EGSpA reported in this instruction for use. EGSpA does not provide validation of performance characteristics of the product regarding these applications. The product may be used in clinical laboratory if the laboratory diagnostic system has been validated as per EN ISO in European countries or equivalents in other countries. Do not use whole blood and plasma samples collected in heparin with this product. Heparin inhibits DNA polymerase enzymes (such as thermostable DNA polymerases) and leads to invalid or incorrect results in subsequent steps of the analysis performed on the extracted nucleic acids. Any inhibition phenomena from drugs that may be contained in the starting sample is evaluated from time to time as they appear according to the use of the extraction product. The results obtained with this product are subject to the correct identification, 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. This product must be handled by personnel qualified and trained in the processing of potentially infective biological samples and dangerous chemical preparations to prevent accidents with potentially serious consequences for the user or other persons. This product requires the use of work clothes and areas that are suitable for the processing of potentially infective biological samples and dangerous chemical preparations to prevent accidents with potentially serious consequences for the user or other persons. This product must be handled by personnel qualified and trained in molecular biology techniques, such as extraction, amplification and detection of nucleic acids, to avoid incorrect results in subsequent steps of the analyses performed on the extracted nucleic acids with potentially serious consequences for the patient. This product must be handled in separate areas for extraction / preparation of amplification reactions and for amplification / detection of amplification products to avoid false positive results in subsequent steps of the analyses performed on the extracted nucleic acids with potentially serious consequences for the patient. 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 in subsequent steps of the analysis performed on the extracted nucleic acids with potentially serious consequences for the patient. Yield and quality of genomic DNA from Blood The amount of purified DNA by the from whole blood depends on the leucocyte content, the sample source, transport, storage and age. The kit provides reagents for the purification of pure genomic DNA from 300 µl whole blood with an ABS 260 / ABS 280 ratio The concentration depends on the health status of the blood donor and the elution volume used, concentrations of ng / µl can be obtained. Please keep in mind that the amount of Carrier in the eluted sample will increase the spectrophotometer measure of DNA content. Yield and quality of pathogen DNA/ RNA The amount of purified DNA and RNA by the from plasma and whole blood depends on the sample type, the pathogen content, sample source, transport, storage and age. Yield and quality of isolated pathogen DNA or RNA are suitable for downstream assays based on Nucleic Acid Amplification Technologies. The diagnostic tests should be performed according to manufacturers specifications. Eluted samples contain pathogen DNA or RNA and Carrier (a synthetic RNA). Please keep in mind that amount of Carrier will greatly exceed amounts of pathogen nucleic acids and bias measure by spectrophotometer. Extraction efficiency The efficiency of the extraction method, in terms of recovery of the sample DNA, was checked during validations of ELITechGroup S.p.A. products using negative samples and positive samples for HHV6 DNA. Tests with whole blood collected in EDTA The test was carried out with whole blood samples from 37 donors negative for HHV6 DNA. 29 of these samples were spiked to a nominal titre of about HHV6 copies / ml using the calibrated and certified reference material QCMD 2009 Human Herpes Virus 6 EQA Panel (Qnostics, Scotland,UK). The whole blood samples were tested by carrying out the whole analysis procedure, extraction and amplification, by ELITechGroup S.p.A. products. The results are summed up in the following table. Whole blood, HHV6 ELITe MGB Kit code RTS036PLD Samples Expected result Obtained result Positive/Replicates Log copies / ml Log geq / ml ± SD IC Ct ± SD Negative NA 3 / 37 NA ± 0.62 HHV6 Spiked / ± ± 0.75 Legend: geq genome Equivalents; SD Standard Deviation; Ct threshold Cycle; IC Internal Control; NA Not Applicable. Three negative whole blood samples reported positive results with a very low viral titre (about 17 geq / ml). Discordant results may be explained considering that HHV6 is a virus largely widespread in the population and latent infection is common. The titre is so low that it could randomly give either negative or positive results in different testing sessions. SCH m_en 28/05/15 Review 03 Page 33/37 SCH m_en 28/05/15 Review 03 Page 34/37

19 Tests with Plasma collected in EDTA The test was carried out with plasma samples from 34 donors negative for HHV6 DNA. 29 of these samples were spiked to a nominal titre of about HHV6 copies / ml using the calibrated and certified reference material QCMD 2009 Human Herpes Virus 6 EQA Panel (Qnostics, Scotland,UK). The plasma samples were tested by carrying out the whole analysis procedure, extraction and amplification, by ELITechGroup S.p.A. products. The results are summed up in the following table. Plasma, HHV6 ELITe MGB Kit code RTS036PLD Samples Nominal titre Obtained result Positive/Replicates Log copies / ml Log geq / ml ± SD IC Ct ± SD Negative NA 0 / 34 NA ± 0.30 HHV6 Spiked / ± ± 0.75 Legend: geq genome Equivalents; SD Standard Deviation; Ct threshold Cycle; IC Internal Control; NA Not Applicable. N.B.: The complete data and results of the tests carried out to evaluate the product performance characteristics are recorded in the Section 7 of the Product Technical File "ELITe GALAXY 300 Extraction Kit", FTP. TROUBLESHOOTING Problem Probable cause Comments and suggestions Pipetting distribution errors. Samples transfer failed / The sample tube must contain at incomplete. least µl of sample. Be sure that no blood clots are present. Low concentration or purity of extracted DNA and RNA Degraded or sheared DNA and RNA Reagent less concentrated Make sure that you have reconstituted the lyophilized reagents in the correct buffer and volume. Blood components settled Carefully premix the sample tube before inserting it into the sample carrier. No / too much ethanol added to Wash Buffer 4 IC less concentrated Incorrect storage of samples Ensure that the Wash Buffer 4 has been prepared with 80% of ethanol. Make sure that you have mixed the correct volume of IC with reconstituted Carrier. Ensure that the storage of starting material was correct. Avoid multiple freeze-thaw cycles of samples. Old samples Old samples often contain degraded DNA and RNA. When possible, start from fresh material or material that has been stored under appropriate conditions. Avoid multiple freeze-thaw cycles of the material. SCH m_en 28/05/15 Review 03 Page 35/37 SCH m_en 28/05/15 Review 03 Page 36/37

20 SYMBOLS Catalogue Number. Temperature limits. Batch code. Use by (last day of month). In vitro diagnostic medical device. Fulfilling the requirements of the European Directive 98\79\EC for in vitro diagnostic medical device. Contains sufficient for "N" tests. Attention, consult instructions for use. CONT Contents. Keep away from sunlight. Manufacturer. NOTICE TO PURCHASER: LIMITED LICENSE The kit is in compliance with EU Directive 98/79/EC on in vitro medical devices. In-vitro diagnostic use of the kit in countries where the EU Directive 98/79/EC is not recognized may be subjected to the fulfillment of registration procedures according to local competent authorities prescriptions. MICROLAB is a registered trademark of Hamilton Bonaduz AG. Microsoft is a registered trademark of Microsoft Corporation. Windows is a registered trademark of Microsoft Corporation. Excel is a registered trademark of Microsoft Corporation. SCH m_en 28/05/15 Review 03 Page 37/37