SEPAX Cell Processing System Operator s Manual

Transcription

1 SEPAX Cell Processing System Operator s Manual

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3 SEPAX Cell Processing System Operator's Manual OM-114-EN Date of release: 17 th January 2008 OM EN

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5 TABLE OF CONTENTS 1. PREFACE SEPAX S-100 MAIN UNIT SINGLE-USE SEPARATION KITS...3 A. USER INSTRUCTIONS CS-470.x... 3A B. USER INSTRUCTIONS CS B C. USER INSTRUCTIONS CS-490.x... 3C D. USER INSTRUCTIONS CS D E. USER INSTRUCTIONS CS E F. USER INSTRUCTIONS CS-530.x... 3F G. USER INSTRUCTIONS CS-600.x...3G H. USER INSTRUCTIONS CS-900.x... 3H I. USER INSTRUCTIONS CS-540.x...3I Z. PREPARATION OF THE CRYOSC CRYOBAG FOR CRYOPRESERVATION.. 3Z 4. THE SEPARATION PROTOCOL...4 A. USER INSTRUCTIONS PBSC... 4A B. USER INSTRUCTIONS UCB-HES... 4B C. USER INSTRUCTIONS UCB... 4C D. USER INSTRUCTIONS UCB-LBC... 4D E. USER INSTRUCTIONS PBSC POST THAWING WASHING... 4E F. USER INSTRUCTIONS UCB- POST THAWING WASHING... 4F G. USER INSTRUCTIONS GENERIC VOLUME REDUCTION...4G H. USER INSTRUCTIONS DENSITY GRADIENT BASED SEPARATION... 4H Z. USER INSTRUCTIONS PURGE... 4Z 5. ACCESSORIES MAINTENANCE ERROR MESSAGES...7 OM EN

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7 1. PREFACE 1.1 Introduction to the SEPAX system and its features The SEPAX cell processing system uses a rotating syringe technology that provides both separation through rotation of the syringe chamber (centrifugation) and component transfer through displacement of the syringe piston. The SEPAX system allows the automated processing of blood component in a functionally-closed and sterile environment. The separation protocol offers similar cell processing performance to the widely accepted manual separation procedure while ensuring a high level of reproducibility. The by-products of the blood separation (plasma and red-blood cells) are collected in standard blood bags while the output product (buffy coat or BC) is collected in a cryogenic (or freezing) storage bag. All bags are connected to the SEPAX single-use kits and form part of the sterile environment. The SEPAX system consists of: SEPAX Main Unit The S-100 main (processing) unit provides centrifugal and axial displacement drive to the chamber on the single-use separation kit, as well as drive to the directional valves. The main unit can be used with the AS-610 Traceability Kit that includes a bar code reader and thermal printer. It can also be used with the SepaxNet SN-100 system that is a powerful traceability tool that transfers procedure data presently stored in the Sepax and Coolmix devices to a centralized standard SQL database. Single-use kit Processing kits contain the blood in a sterile environment during the complete operation valves control the flow of blood components to the correct bag. 1.2 Indication for use The SEPAX system is a blood cell processing system intended for laboratory use in exclusive combination with a compatible single-use separation kit supplied by Biosafe. The blood to be processed has been previously collected and transported to the laboratory by other means. The Sepax system allows the fast, automated and reproducible separation of blood in a closed and sterile environment. The SEPAX system is not intended for use in transfusion applications at bedside, where blood circulates directly between a patient and the SEPAX unit. Important note: Sepax Single-use Kits have been sterilized by Ethylene Oxide (EtO). Patients with hypersensitivity to EtO should not receive cord blood processed with these kits. Anaphylactic reactions can occur from hypersensitivity. 1.3 Manufacturer Biosafe SA is an ISO 9001 / ISO certified company, working under a number of national and regional directives. The SEPAX technology is protected by patents EUR and US Other patents are pending Address Preface OM

8 Biosafe SA Route du Petit-Eysins Eysins Switzerland Telephone: Fax: [email protected] 1.4 How to use this manual This operating manual is organized in functional sections to provide easy access to the information concerning the SEPAX equipment. Prior to using the SEPAX system this manual should be read in its entirety. The Table of Contents can be used to search for specific information Proprietary clause The content of this manual is the exclusive property of Biosafe SA. It is strictly forbidden to reproduce and/or disseminate any of the contents without the prior written permission of Biosafe SA. 1.5 Warnings and Precautions The SEPAX system in general To ensure safe and effective use of the SEPAX system operation should only be entrusted to trained personnel. The SEPAX equipment and single-use kits have not been designed for any modification by the end-user or third party. Intervention such as modification, revision, maintenance or repair should only be performed by approved Biosafe technicians. Prior to using any part of the SEPAX system, including the traceability kit or the SepaxNet, the operator should read all of instructions in this manual. In addition, the operator/user must check that the equipment functions safely and ensure that it is in proper working condition before being used. The separate manuals for the SepaxNet, the barcode reader, printer and power supply should also be read prior to using the SEPAX system. A Biosafe representative should be contacted if any doubt exists concerning the use of the SEPAX system. Biosafe is not liable for any injury or damage resulting from use of the SEPAX system that does not conform to the indications in this Operator s Manual Biosafe cannot be held responsible for the quality and subsequent effects of products processed on the SEPAX system that have undergone subsequent post-processing The single-use separation kit The SEPAX system should be used exclusively with SEPAX separation kits, which are supplied sterile and are for single use only. Each kit should be disposed of in the appropriate manner following blood processing. Biosafe shall not be held responsible for any consequences of single-use kits other than those specified in this document being used with the SEPAX system. Preface OM

9 1.5.3 Blood manipulations If blood spillage or leakage occurs, the product should be discarded (see Chapter 6 for cleaning procedures). Gloves and protective clothing should be worn for all blood handling operations Electro-Magnetic Compatibility Recommendations as required by IEC Medical Electrical Equipment needs special precautions regarding EMC and need to be installed and put into service according to the EMC information provided in this accompanying document. Portable and mobile RF communications equipment can affect Medical Electrical Equipment. All staff involved has to receive an explanation of the ESD warning symbol and training in ESD precautionary procedure, and that the users hand should be discharged by earth bonding. (Refer to 1.8 Symbol chart and abbreviations). The Sepax should not be used adjacent to or stacked with other equipment. If adjacent or stacked use is necessary, the Sepax should be observed to verify normal operation in the configuration in which it will be used. Tables below help to determine such conditions. Preface OM

10 Table 201 Guidance and manufacturer s declaration electromagnetic emission for all EQUIPMENT AND SYSTEMS (see a) 3)) Guidance and manufacturer s declaration electromagnetic emission The Sepax is intended for use in the electromagnetic environment specified below. The customer or the user of the Sepax should assure that it is used in such an environment. Emissions test Compliance Electromagnetic environment - guidance RF emissions CISPR 11 Group 1 The Sepax uses RF energy only for its internal function. Therefore, its RF emissions are very low and are not likely to cause any interference in nearby electronic equipment. RF emissions Class A CISPR 11 Harmonic emissions IEC Voltage fluctuations / flicker emissions IEC Class A Complies The Sepax is suitable for use in all establishments other than domestic and those directly connected to the public-voltage power supply network that supplies buildings used for domestic purposes. Table 202 Guidance and manufacturer's declaration electromagnetic immunity for all EQUIPMENT and SYSTEMS (see a) 6)) Guidance and manufacturer s declaration electromagnetic immunity The Sepax is intended for use in the electromagnetic environment specified below. The customer or the user of the Sepax should assure that it is used in such an environment. Immunity test Electrostatic discharge (ESD) IEC Electrical fast transient / burst IEC Surge IEC Voltage dips, short interruptions and voltage variations on power supply input lines IEC test level ± 6 kv contact ± 8 kv air ± 2 kv for power supply lines ± 1 kv for input/output lines ± 1 kv differential mode ± 2 kv common mode < 5 % U T (>95 % dip in U T ) for 0,5 cycle 40 % U T (60 % dip in U T ) for 5 cycles Compliance level ± 6 kv contact ± 8 kv air ± 2 kv for power supply lines ± 1 kv differential mode ± 2 kv common mode < 5 % U T (>95 % dip in U T ) for 0,5 cycle 40 % U T (60 % dip in U T ) for 5 cycles for 230V only Electromagnetic environment - guidance Floors should be wood, concrete or ceramic tile. If floors are covered with synthetic material, the relative humidity should be at least 30 %. Mains power quality should be that of a typical commercial or hospital environment. Mains power quality should be that of a typical commercial or hospital environment. Mains power quality should be that of a typical commercial or hospital environment. If the user of the Sepax requires continued operation during power mains interruptions, it is recommended that the Sepax be powered from an uninterruptible Preface OM

11 IEC % U T (30 % dip in U T ) for 25 cycles 70 % U T (30 % dip in U T ) for 25 cycles power supply or a battery. Power frequency (50/60 Hz) magnetic field IEC NOTE < 5 % U T (>95 % dip in U T ) for 5 sec 3 A/m 3 A/m < 5 % U T (>95 % dip in U T ) for 5 sec U T is the a. c. mains voltage prior to application of the test level. Power frequency magnetic fields should be at levels characteristic of a typical location in a typical commercial or hospital environment. Preface OM

12 Table 204 Guidance and manufacturer s declaration electromagnetic immunity for EQUIPMENT and SYSTEM that are not LIFE-SUPPORTING (see b)) Equipment with which the Sepax has been tested in stacked of adjacent configuration and with which stacked or adjacent use is permitted: Guidance and manufacturer s declaration electromagnetic immunity The Sepax is intended for use in the electromagnetic environment specified below. The customer or the user of the Sepax should assure that it is used in such an environment. Immunity test IEC test level Compliance level Electromagnetic environment - guidance Portable and mobile RF communications equipment should be used no closer to any part of the Sepax, including cables, than the recommended separation distance calculated from the equation applicable to the frequency of the transmitter. Conducted RF 3 Vrms 3 V Recommended separation distance c IEC khz to 80 MHz d = 1, 2 P 80 MHz to 800 MHz Radiated RF IEC V/m 80 MHz to 2,5 GHz 3 V/m d = 2, 3 P 800 MHz to 2,5 GHz where p is the maximum output power rating of the transmitter in watts (W) according to the transmitter manufacturer and d is the recommended separation distance in metres (m). b Field strengths from fixed RF transmitters, as determined by an electromagnetic site survey, a should be less than the compliance level in each frequency range. b Interference may occur in the vicinity of equipment marked with the following symbol: NOTE 1 At 80 MHz and 800 MHz, the higher frequency range applies. NOTE 2 These guidelines may not apply in all situations. Electromagnetic is affected by absorption and reflection from structures, objects and people. Preface OM

13 a Field strengths from fixed transmitters, such as base stations for radio (cellular/cordless) telephones and land mobile radios, amateur radio, AM and FM radio broadcast and TV broadcast cannot be predicted theoretically with accuracy. To assess the electromagnetic environment due to fixed RF transmitters, an electromagnetic site survey should be considered. If the measured field strength in the location in which the Sepax] is used exceeds the applicable RF compliance level above, the Sepax should be observed to verify normal operation. If abnormal performance is observed, additional measures may be necessary, such as reorienting or relocating the Sepax. b Over the frequency range 150 khz to 80 MHz, field strengths should be less than 3 V/m. Recommended separation distances between portable and mobile RF communications equipment and the Sepax The Sepax is intended for use in an electromagnetic environment in which radiated RF disturbances are controlled. The customer or the user of the Sepax can help prevent electromagnetic interference by maintaining a minimum distance between portable and mobile RF communications equipment (transmitters) and the Sepax as recommended below, according to the maximum output power of the communications equipment Separation distance according to frequency of transmitter m Rated maximum output of transmitter W 150 khz to 80 MHz d = 1, 2 P 80 MHz to 800 MHz d = 1, 2 P 800 MHz to 2,5 GHz d = 2, 3 0,01 0,12 0,12 0,23 0,1 0,37 0,37 0,74 1 1,17 1,17 2, ,69 3,69 7, ,67 11,67 23,33 For transmitters rated at a maximum output power not listed above the recommended separation distance d in metres (m) can be estimated using the equation applicable to the frequency of the transmitter, where P is the maximum output power rating of the transmitter in watts (W) according to the transmitter manufacturer. NOTE 1 At 80 MHz and 800 MHz, the separation distance for the higher frequency range applies. NOTE 2 These guidelines may not apply in all situations. Electromagnetic propagation is affected by absorption and reflection from structures, objects and people. P 1.6 Warranty Biosafe s products are designed and manufactured to provide reliable performance when properly maintained and used in accordance with the instructions provided in this manual. Each unit is carefully inspected and tested before shipping. In case of equipment failure or malfunction, Biosafe will replace or repair the concerned equipment according to the agreement in place. Preface OM

14 Equipment failure or malfunction for reasons other than a manufacturing defect (such as improper handling of the machine or non-compliance with the Operator s Manual) is not covered under the Biosafe warranty program and such equipment will be replaced or repaired at the charge of the end-user. Biosafe shall under no circumstances be liable for consequential or economical damage that may be an indirect or direct consequence of a defective part. 1.7 Customer support All SEPAX equipments supplied with a copy of the Operator s Manual. In addition, competent technical staff will provide end-user training prior to use and will always be available for specific questions or clarifications For assistance in technical or application issues, please contact your local representative or call Biosafe Customer Service in Switzerland Symbol chart and abbreviations Operating instructions must be observed by user Class I Equipment energized from an external electrical power source Type B equipment providing a degree of protection against electrical shocks particularly regarding allowable leakage current 0123 CE marking of the SEPAX instrument and kits ~ Alternating current O I Power off Power on STERILE EO Sterilization with ethylene oxide Expiry date Not reusable, for single use only. Date of manufacture REF LOT Product number Batch designation Refer to chapter 6.2 Pins of connectors identified with the ESD warning symbol should not be touched. Connections should not be made to these connectors unless the users hand is discharged by earth bonding. Preface OM

15 2. SEPAX S-100 MAIN UNIT 2.1 General description The SEPAX S-100 main unit that provides centrifugal and axial displacement drive to the chamber on the single-use separation kit, as well as drive to the directional valves. The main components of the SEPAX S-100 are: Centrifuge motor and cabinet including separation chamber pit (the actual separation chamber is part of the single-use separation kit) Pneumatic pump system providing positive and negative pressure to displace the separation chamber piston - the unit is equipped with a line pressure monitor Charge-Coupled Device (CCD) to allow precise measurement of the volume in the separation chamber Rotary pins drives to position the stopcocks on the single-use separation kit Optical line sensor to monitor the different components passing through the tubes. Computer system (CPU) for controlling the automated process, including memory card and RS-232 communication port User interface with liquid crystal display (LCD), membrane keypad and speaker The machine OM

16 2.2 Operating and storage requirements Please read the following instructions carefully before installing the SEPAX equipment Environmental specifications The SEPAX system must only be operated under the following environmental conditions: Mode Operation Storage & Transport Temperature +7 C to +27 C 0 C to 50 C Relative humidity Maximum Altitude 30% to 75%, non-condensing 20% to 75%, non-condensing 2000m 2000m The SEPAX S-100 should:. only be operated on a flat, stable, horizontal and clean surface. be used in an open environment to allow sufficient ventilation. cleaned regularly (see Chapter 6 for cleaning instructions). kept upright during transport.. be connected to an earthed power supply directly (no adapters or extension leads) The SEPAX S-100 should not be exposed to: - direct sunlight or strong light sources - liquids or corrosive substances - physical shocks or vibrations. - heavy weights - Other equipment that contain magnets or that generate magnetic or electromagnetic fields (such as mobile/cellular phones). The machine OM

17 2.3 SEPAX S-100 Main Processing Unit Components 1. Keyboard 2. Optical line sensor 6. Rotary pins 3a. Separation chamber pit covers 3b Separation chamber pit 7. Line pressure sensor 4. Air filter 10. Input product bag support 9. Handles 8b. Collection bag hook 11. Bubble chamber support 8a. By-product bag hooks 12. Data card slot 15. Serial ports 13. Power switch 14. Cable socket 16. IBM PS2 port The machine OM

18 1. Keyboard Keyboard, with 6 keys that allow the operator to select options, such as traceability parameters (see Key Board). 2. Optical line sensor The equipment uses the different light absorbance of the separated components to manage the automated separation procedure. 3. Separation chamber pit & covers The separation chamber pit is where the single-use kit separation chamber is installed. The separation chamber pit covers are closed around the separation chamber rotary seal to ensuring stability during the procedure the SEPAX S-100 informs the operator if the pit covers are not closed. The SEPAX S-100 provides both electrical centrifugal drive and pneumatic axial drive to the separation chamber for separation of the input product and transfer of each component to the appropriate bag/container. A Charge-Coupled Device detector (CCD), situated in the pit and positioned alongside the separation chamber, monitors the position of the chamber piston allowing real-time volume measurements. 4. Air filter The air filter prevents dust from entering the machine. 5. LCD display The display provides continuous information concerning progress of the separation procedure and any procedure anomalies. 6. Rotary pins Two rotary pins position the stopcocks on the single-use separation kit, to direct flow of the separated components to the appropriate bag. 7. Line pressure sensor The line pressure sensor monitors pressure in the single-use separation kit tubing, to avoid overpressure. 8. Bag hooks Hooks are provided on which the collection and by-product bags are hung during the separation procedure. 9. Handle Handles on each side of the SEPAX-100 allow easy transport - the SEPAX S-100 only weighs 13kg (29lbs). 10. Input product bag support Support to hang the input (or source) bag. 11. Bubble chamber support Support to hang the bubble chamber. 12. Data card slot The machine OM

19 PCMCIA card slot onto which is down-loaded procedure and input product data. 13. Power switch On/off button. 14. Cable socket Socket for the power supply cable. 15. Serial ports 2 x serial ports, one for connection to thermal printer or the connection boxes of the SepaxNet SN-100 system, the second for use by service technician. 16. IBM PS2 port Serial interface connector for connection to barcode reader Keyboard Functions The keyboard has six keys with the following functions: KEY ACTION START START - Print barcode hardcopy of manually-entered traceability references STOP - Emergency STOP. Stops the procedure MENU Yes - Moves the display cursor up No ENTER - Moves the display cursor down ENTER - Validates selection MENU - Displays the menu. The machine OM

20 2.4 Technical specifications of the SEPAX Dimensions (approximate): Width: 29 cm (11.4 ) Length: 36 cm (14.2 ) Height: 37 cm (14.6 ) Weight: 13kg (29lbs) Power: Use only the original certified cable for the power supply. The SEPAX equipment should always be connected to an Uninterruptible Power Supply (UPS) Input range: 100 to 240 VAC Input frequency: Hz Consumption: 200 VA Leakage current: < 500 μa (The SEPAX S-100 automatically adjusts for the local supply voltage) Fuse: T2AH Centrifuge: Max speed: Speed range: Over speeds protection: 8000 rpm rpm 8800 rpm The machine OM

21 3. THE SINGLE-USE SEPARATION KIT 3.1 General description The separation kits are composed of a proprietary separation chamber, tubing and collection/by-product bags. The separation chamber is a syringe pump that enables both the centrifugation of the processed sample and the transfer of the sample from the initial bag to the various collection and by-product bags in a closed and sterile environment the separation chamber is the same for all separation kits The kits are individually packed and sterilized, and have a shelf life of 2 years. Only separation kits provided by Biosafe for specific protocols (applications) should be used with the Sepax system 3.2 Possible kits for the different protocols Kits Protocols UCB UCB-HES UCB-LBC PBSC PBSC-CW UCB-CW Vol. Red. Ficoll CS-470.x 1 X X CS X X X X CS-490.x X X X X X CS X 2 X 2 X 2 X 2 X 2 CS X 2 X 2 X 2 X 2 X 2 CS-530.x X X X CS-600.x X X CS-900.x CS-540.x 4 X X X CS-430.x X X CS-570.x 4 X X 1 Works only with SEPAX machines equipped with a stopcock blocker. 2 Up to a total volume of 70 ml (CS-500) or 20 ml (CS-510). 3 Discountinued 4 Functionnaly closed Only the separation kits CS-530.x and CS-540.x and the Protocol UCB-HES are available on the US market. X 3.3 Storage requirements The separation kit must be stored in a clean and dry place, free from biological contaminants and chemical vapours for storage conditions see Section The separation kit OM

22 3.4 Single-use Kit components Each kit includes a separation chamber and associated harness. The harness includes: Bubble chamber with a micro aggregate filter Set of tubing lines with roller and slide clamps Stopcock manifold Collection/by-product bags Line pressure monitor with a micro-filter Spike for connecting the sample bag to the kit (Biosafe advises use of a sterile connection device Cord blood collection bag The SEPAX system is compatible with standard input bags. 20cm of tubing should be left on the input bag at the time of collection to facilitate connection of the bag to the single-use separation kit. If a spike is to be used, the input bag must have a standard blood bag membrane port (septum) with a diameter of 4-6 mm. 3.5 Instructions for Use (IFU) Each box of 6 single-use separation kits includes Instructions for Use corresponding to that particular kit. Instructions for Use should be read carefully prior to opening the kit blister pack. Verify expiry date and check the general condition of the kit / blister before use DO NOT use kits that shown sign of damage or mishandling DO NOT twist or rotate harness connections as this may compromise integrity The separation kit OM

23 SEPAX cell separation kit CS Configuration E C D F B A A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock D: Bubble chamber E: Spike to source bag F: Buffy coat collection vial with injection site and microbial filter. Warning: this is not a cryovial! CS-470.x OM 3A 04 3A - 1

24 CS-470.x OM 3A 04 3A - 2

25 SEPAX cell separation kit CS-480 (Discontinued) Configuration C F E D B A G A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Spike to buffy-coat collection bag CS-480 OM 3B 02 3B - 1

26 CS-480 OM 3B 02 3B - 2

27 SEPAX cell separation kit CS E C F D I H B A F G A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Spike to output bag H: Luer lock to output bag I: Injection site The tube between the injection site (I) and the spike (G) is double-coated type and may be difficult to seal with certain types of sealers. That portion of tube is not compatible with sterile connection device (SCD). CS-490.x OM 3C 05 3C - 1

28 CS General Notes: The CS is a separation kit conceived for GVR and PBSC protocols in cellular products processing. - The CS kit can be connected to the cellular product input bag with a sterile connection device: to use a sterile connection device, the input bag must have at least 20cm of available tubing to be inserted in the sterile connection device with the tubing between the spike port and the bubble chamber in the input bag line ( E in figure n.1). This operation must be done under LAMINAR FLOW. - If 20cm (8 ) of tubing is not available, the connection of the input bag will require use of the spike connector pre-installed on the CS kit: this operation must be done under LAMINAR FLOW. STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit with AS-610 traceability kit Cellular product input bag (not supplied by Biosafe) Output Bag. (not supplied by Biosafe) A freezing bag or a transfer bag can be used. Be sure that the bag has enough capacity to contain your final product CS single-use kit Figure 1 Sterile Connection Device if necessary (not supplied by Biosafe) Tubing sealer (not supplied by Biosafe) Tubing stripper (not supplied by Biosafe) All laboratory material related with the cell count operations STEP 2: Sampling of Cellular product Input bag Prior connection of the initial bag to the CS kit, follow your validated procedure to perform initial cell count. CS-490.x OM 3C 05 3C - 2

29 STEP 3: CS kit sterility verification Before opening the kit blister pack under laminar flow, ensure that the sterility indicator on the Tyvek cover is brown, indicating that the kit is sterile. If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 4: CS kit integrity verification Under laminar flow spread the kit out in order to identify the tubing lines and components as in figure 1. Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 5: Closing of the roller clamp Close ONLY the red roller clamp placed on the input line. Leave the other clamps open. STEP 6: Output bag connection Figure 2 There are three ways to connect the output bag. The output bag will contain the final product, TNC concentrate also called buffy coat, at the end of the procedure. If you have a sterile connection device, you can use it to connect the output bag to the kit, the output bag needs a 10 cm tubing so to use with the SCD (see Instructions for Use supplied with the sterile connection device). The sterile connection should be made between the stopcock and the luer lock. This operation must be done under LAMINAR FLOW. If a sterile connection device is not available, you can use a spike. First connect the spike to output bag to the kit by using the luer lock (figure 3). The CS kit can then be connected to the output bag using spike connection in the output bag line. This operation must be done under LAMINAR FLOW. It s possible to connect a bag directly on the luer-lock of the output line. In that case there is no sampling port available. This operation must be done under LAMINAR FLOW. Remark: with a SCD or luer lock connection, you will loose the sampling port on the output line. Be sure that a sampling port on your output bag is available. CS-490.x OM 3C 05 3C - 3

30 Figure 3 STEP 7: Input Bag connection There are two ways to connect the input bag. If you have a sterile connection device, you can use it to connect the input bag to the kit (see Instructions for Use supplied with the sterile connection device). The sterile connection should be made between the spike pre-installed spike connector and the bubble chamber in the input bag line, as shown in figure 4. This operation must be done under LAMINAR FLOW. Spike If a sterile connection device is not available, the CS kit can be connected to the input bag using the pre-installed spike connector in the input bag line ( E in figure n.1). This operation must be done under LAMINAR FLOW conditions. SCD Connection Figure 4 Once the input and the output bags have been connected, you kit should be similar to the one on the figure 5. In that example, the output bag has been connected via spike. Figure 5 CS-490.x OM 3C 05 3C - 4

31 STEP 8: CS kit installation on the Sepax For instructions on installation of the CS kit on the Sepax S-100 main unit and launching of the automated separation procedure, refer to the Operator s Manual (Section 4.6) provided with the Sepax S-100. The GVR protocol will perform automatically the priming of the lines. If you are using the GVR protocol, go directly to step 10. If you are using the PBSC protocol, the priming of the lines should be performed manually, in that case go to step 9. STEP 9: Manual Priming of the line (PBSC protocol only) The tubing line between the source bag and the white stopcock has to be primed manually with blood. The display shows Prime Bubble Chamber (Figure 6) Figure 6 Squeeze the bubble chamber, without opening the roller clamp, and release it 2-3 times until the bubble chamber is half-filled with blood (Figure 7) then press Enter. Figure 7 Lower the input bag position by adjusting the holder position. This adjustment will decrease the flowing speed of the blood during the priming (Figure 8). Figure 8 CS-490.x OM 3C 05 3C - 5

32 Press the up-key (Figure 9): the blood flows towards the stopcock; press a second time the upkey when the blood is between the blue and the white stopcocks: the blood stops flowing (Figure 10). Figure 9 Figure 10 If the priming succeeded, select START PBSC in the main menu, Figure 11. If the blood did not reach the blue stopcock, you can redo the priming by selecting REDO PRIMING in the Menu, Figure 11. If the blood went too far, figure 12, select REDO PRIMING, and use the gravity force to draw back the blood to the initial bag (figure13. After that by selecting REDO PRIMING again, prime again the tubes by bringing the blood between the blue and white stopcock. Figure 11 Figure 12 CS-490.x OM 3C 05 3C - 6

33 Figure 13 AUTOMATED PROCEDURE Warning: Avoid touching the machine and the kit: Moving bag, tubes, stopcock and covers may cause errors. STEP 10: Mixing of the input bag GVR protocol only That step is only true for the GVR protocol. If you are running the PBSC protocol, go directly to step 11. After each sedimentation cycle the procedure will pause and will begin to beep. The operator should then mix the input bag and press on the ENTER key so to resume the procedure (Figure 14 and 15). Figure 14 Figure 15 AUTOMATED PROCEDURE CS-490.x OM 3C 05 3C - 7

34 STEP 11: Post Procedure Remove Bags and Filter At the end of the automated procedure, the message REMOVE BAGS AND AIR FILTER - ENTER appears on the display. Please do so, as shown on the figures 16 and 17 and press ENTER : Figure 16 Figure 17 STEP 12: Post Procedure Line Stripping The message STRIP BC LINE - ENTER appears on the display. Strip the BC line as shown in figure 18. Then Press Enter. The STRIP RBC LINE - ENTER message will appear on the Sepax display. Strip the RBC line as shown in figure 19. Then Press Enter. The STRIP PLASMA LINE - ENTER message will appear on the Sepax display. Strip the Plasma line as shown in figure 20. Then Press Enter Figure 18 Figure 19 CS-490.x OM 3C 05 3C - 8

35 Figure 20 STEP 13: Post Procedure Close Clamps and remove kit The message CLOSE ALL CLAMPS- ENTER appears on the display. Close all the four clamps, one for each line, then press ENTER. The message REMOVE KIT- ENTER appears on the display Dismount the kit (figure 21) and press enter. The protocol will exit and the display shows the Main Menu. You can then start a new procedure. Figure 21 Please note that the RBC bag remains empty for the PBSC protocol. CS-490.x OM 3C 05 3C - 9

36 STEP 14: Buffy-coat sampling Prior sample taking, the final product (buffy-coat) must be mixed in order to obtain a homogeneous and representative sample. If you used the spike to connect your output bag to the kit, it s possible to take the sample, under laminar flow, by using the dedicated syringe port placed on the Y connector for needle insertion, as shown in figure 22, and then follow your validated procedure. If you used A SCD or the luer lock to connect your output bag to the kit, you will need to use a port of the bag so to take your sample. Figure 22 CS-490.x OM 3C 05 3C - 10

37 SEPAX cell separation kit CS-500 (Discontinued) Configuration C F E D B F A G A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Buffy-coat collection bag CS-500 OM 3D 03 3D - 1

38 CS-500 OM 3D 03 3D - 2

39 SEPAX cell separation kit CS-510 (Discontinued) Configuration C F D B F G A E A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Buffy-coat collection bag CS-510 OM 3E 02 3E - 1

40 CS-510 OM 3E 02 3E - 2

41 SEPAX cell separation kit CS / CS E Configuration C H D J B K F A I Fig. 1 A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Bubble chamber E: Spike to input product bag F: Output bag with 25 ml nominal volume G: letter not used for this kit H: 500ml PVC by-product (plasma) bag I: Luer port for AK-10 accessory J: DMSO extension line K: AK-10, DMSO Vial Spike CS-530.x OM 3F 08 3F - 1

42 CS General Notes: NOTE : THE INSTRUCTION FOR USE IS THE SAME FOR THE CS-530.0, CS KITS The CS is a separation kit conceived for UCB -HES and UCB protocols in Umbilical Cord Blood processing. - The CS kit can be connected to the UCB input bag with a sterile connection device: to use a sterile connection device, the UCB input bag must have at least 20cm of available tubing to be inserted in the sterile connection device with the tubing between the spike port and the bubble chamber in the UCB input bag line ( E in figure n.1). This operation must be done under LAMINAR FLOW. - If 20cm (8 ) of tubing is not available, the connection of the UCB input bag will require use of the spike port pre-installed on the CS kit: this operation must be done under LAMINAR FLOW. STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit with AS-610 traceability kit UCB input bag (not supplied by Biosafe) If UCB-HES protocol is used, Hydroxy-Ethyl-Starch HES solution (HES %) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant) CS single-use kit Sterile connection device (not supplied by Biosafe) Sterile tubing sealer (not supplied by Biosafe) Tubing stripper (not supplied by Biosafe) All laboratory material related with the cell count operations STEP 2: Sampling of UCB input bag Prior connection of the initial UCB bag to the CS kit, follow your validated procedure to perform initial cell count. STEP 3: CS kit sterility verification Before opening the kit blister pack, ensure that the sterility indicator on the Tyvek cover is brown, indicating that the kit is sterile. If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 4: CS kit integrity verification Spread the kit out in order to identify the tubing lines and components as shown above. Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question. CS-530.x OM 3F 08 3F - 2

43 STEP 5: CS kit preparation Close ONLY the red roller clamp placed in the input UCB line and the two blue clamps on the DSMO extension line ( J in figure n.1). Leave the other 2 white clamps open. STEP 6: If UCB-HES protocol is used: Hydroxy-Ethyl-Starch (HES) preparation If you are not using the UCB-HES protocol, go directly to step 8. In accordance with your internal validation procedure, prepare a Hydro-Ethyl-Starch (HES) solution (HES %) corresponding to 20% of the UCB input volume (UCB + CPD anticoagulant). For example, if the volume of the UCB input bag is 100ml including CPD, you should prepare 20ml of HES solution. Fill a syringe with the prepared HES, wait for the HES to reach room temperature. STEP 7: If UCB-HES protocol is used: HES injection in the UCB input bag Under LAMINAR FLOW conditions, the operator should insert the syringe s needle in the dedicated port of the UCB input bag, and then manually agitate the UCB input bag with one hand while injecting the HES with the other hand: this ensures a homogenous HES dispersion in the bag. Inject the HES into the UCB input bag at a rate of approximately 2 seconds per ml. STEP 8: UCB input bag connection Connect the UCB input bag to the CS kit with a sterile connection device (see Instructions for Use supplied with the sterile connection device). The sterile connection should be made between the spike pre-installed spike connection and the bubble chamber in the UCB input bag line ( E in figure n.1). If a sterile connection device is not available, the CS kit can be connected to the UCB input bag using the pre-installed spike connection in the UCB input bag line ( E in figure n.1) under LAMINAR FLOW conditions. STEP 9: CS kit installation and UCB or UCB-HES automated procedure For instructions on installation of the CS kit on the Sepax S-100 main unit and launching of the automated separation procedure, refer to the Operator s Manual (Section 4.6) provided with the Sepax S-100. Once the procedure is complete, the Sepax S-100 guides the operator through a manual stripping of the buffy-coat (BC) bag line. CS-530.x OM 3F 08 3F - 3

44 STEP 10: Post -procedure actions At the end of the automated procedure, with the kit still installed on the S-100, the STRIP BC LINE ENTER message will appear on the S-100 display and the stopcock valves will be in the position shown in Fig. 2 and 3. Before pressing ENTER, manually squeeze the air from the BC bag until there is no air in the bag, as shown in Fig. 3. While holding the BC bag in this position with one hand, press the ENTER button on the S-100 with the other hand. The BC bag can then be released as the stopcock on the S-100 has been closed preventing return of the air. Fig. 2 Fig. 3 Continue following the instructions on the S-100 display panel until instructed to DISMOUNT KIT. STEP 11: Removal of the CS kit from the Sepax S-100 Close the WHITE clamp above the Y connector and as CLOSE to the Y-connector as possible. Seal 3 times above the white clamp. Sealing 3 times and cutting out the line in the middle seal ensures safety in case of imperfect seal. After sealing, detach the BC bag line with its components as shown in Fig. 4. Fig. 4 CS-530.x OM 3F 08 3F - 4

45 STEP 12: Buffy-coat sampling Prior sample taking, the buffy-coat must be mixed in order to obtain a homogeneous and representative sample: press manually 30 times the small (5ml) section of the buffy-coat bag. To take the sample, under laminar flow use the dedicated syringe port placed on the Y connector for needle insertion and then follow your validated procedure. STEP 13: DMSO preparation Preparation of the DMSO solution for cryopreservation must be performed following your validated procedure. The quantity of DMSO solution to be injected in the BC cryobag is 5ml or your validated volume. Note: If you inject DMSO by syringe pump, you will need to add an extra volume of 1.5 ml. (ex: If you need to add 5 ml, you put ml = 6.0 ml in the syringe). This volume corresponds to the dead volume that remains in the tube. If adding DMSO by syringe pump, go to step 14A. If adding DMSO by gravity feed, go to step 14B. STEP 14A: DMSO injection into the BC bag by automated Syringe pump Connect one end of the DMSO extension line to the luer-lock syringe containing the DMSO cryosolution and the other end to the luer-lock on the kit. Check that the blue clamp on the DMSO extension line is closed. Install cryobag on the Coolmix device, install the syringe in the syringe pump device and select the flow rate following the User Instructions of the syringe pump device. Inject DMSO with the syringe pump. If you are using the Coolmix automated cooling and mixing device, refer to the Coolmix Operator Manual, for display settings and automated procedure. The Coolmix is not available on US market STEP 14B: DMSO injection into the BC bag by Gravity feed Insert the DMSO Vial Spike (AK-10) in the DMSO vial and place the spike/vial in the DMSO Vial Adapter. Please check that you are using the correct DMSO vial adapter. If you use the CS and the 7ml DMSO vial you need the DMSO vial adapter with the green spot. If you use the 8ml DMSO vial, use the DMSO vial adapter with the red spot. See fig 5. with 7ml DMSO vial with 8ml DMSO vial CS-530.x OM 3F 08 3F - 5

46 use DMSO Vial Adapter 530 for 7ml vial use DMSO Vial Adapter 530 for 8ml vial Fig 5. Connect one end of the DMSO extension line to the DMSO Vial Spike (AK-10) and the other end to the luer-lock on the kit. Install the kit and DMSO vial assembly on the Coolmix - see fig. 6. If you are using the Coolmix automated cooling and mixing device, refer to the Coolmix Operator Manual, for display settings and automated procedure. Fig. 6 The Coolmix is not available on US market After the kit and DMSO vial assembly installation on the Coolmix, open: the air vent on the DMSO Vial Spike the DMSO extension line BLUE clamp the BLUE clamp on the kit where the DMSO extension line is connected Strip gently and carefully the DMSO extension line until the DMSO flow reaches the Coolmix cover. Press Start Mix and Start Timer on Coolmix - the DMSO will be mixed with the BC as it flows into the BC bag. After the end of the DMSO injection, strip once again the DMSO extension line and close the DMSO extension line clamps (WHITE and BLUE). CS-530.x OM 3F 08 3F - 6

47 SEPAX cell separation kit CS E Configuration C D J B H I F A Fig. 1 A: Separation chamber 200ml B: Line pressure monitor luer with 0.2µm filter C: Stopcock manifold D: Bubble chamber E: Spike to input product bag F: Biosafe CryoSC cryobag (storage capacity 10-30ml, DMSO included) G: letter not used for this kit H: 500ml PVC by-product (plasma) bag I: Luer port J: DMSO extension line CS-530.x OM 3F 08 3F - 7

48 CS General Notes: The CS is a single-use separation kit conceived for UCB-HES and UCB protocols in Umbilical Cord Blood processing. - The CS kit can be connected to the UCB input bag with a sterile connection device (SCD): to use a sterile connection device, the UCB input bag must have at least 20cm of available tubing. This tubing needs to be placed in the SCD device with the tubing of the CS kit located between the spike port and the bubble chamber in the UCB input bag line ( E in figure n.1). This operation must be done under LAMINAR FLOW. - If 20cm (8 ) of tubing is not available, the connection of the UCB input bag will require use of the spike port pre-installed on the CS kit: this operation must be done under LAMINAR FLOW. STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit UCB input bag (not supplied by Biosafe) If UCB-HES protocol is used, Hydroxy-Ethyl-Starch HES solution (HES %) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant) CS single-use kit Sterile connection device SCD (not supplied by Biosafe) Sterile tubing sealer, also compatible with EVA tubing (not supplied by Biosafe) Tubing stripper (not supplied by Biosafe) All laboratory material related with the cell count operations STEP 2: Sampling of UCB input bag Prior connection of the initial UCB bag to the CS kit, follow your validated procedure to perform initial cell count. STEP 3: CS kit sterility verification Before opening the kit blister pack, ensure that the sterility indicator on the Tyvek cover is brown, indicating that the kit is sterile. If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 4: CS kit integrity verification Spread the kit out in order to identify the tubing lines and components as shown above. Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question. CS-530.x OM 3F 08 3F - 8

49 STEP 5: CS kit preparation Close ONLY the red roller clamp placed in the input UCB line and the two blue clamps on the DSMO extension line ( J in figure n.1). Leave the other 2 white clamps open. STEP 6: If UCB-HES protocol is used: Hydroxy-Ethyl-Starch (HES) preparation If you are not using the UCB-HES protocol, go directly to step 8. In accordance with your internal validation procedure, prepare a Hydroxy-Ethyl-Starch (HES) solution (HES %) corresponding to 20% of the UCB input volume (UCB + CPD anticoagulant). For example, if the volume of the UCB input bag is 100ml including CPD, you should prepare 20ml of HES solution. Fill a syringe with the prepared HES solution; wait for the HES solution to reach room temperature. STEP 7: If UCB-HES protocol is used: HES injection in the UCB input bag Under LAMINAR FLOW conditions, the operator should insert the syringe s needle in the dedicated port of the UCB input bag, and then manually agitate the UCB input bag with one hand while injecting the HES with the other hand: this ensures a homogenous HES dispersion in the bag. Inject the HES into the UCB input bag at a rate of approximately 2 seconds per ml. STEP 8: UCB input bag connection Connect the UCB input bag to the CS kit with a sterile connection device (see Instructions for Use supplied with the sterile connection device) under LAMINAR FLOW conditions. The sterile connection should be made between the spike and the bubble chamber in the UCB input bag line ( E in figure n.1). If a sterile connection device is not available, the CS kit can be connected to the UCB input bag using the pre-installed spike connection in the UCB input bag line ( E in figure n.1) under LAMINAR FLOW conditions. STEP 9: CS kit installation and UCB-HES automated procedure For instructions on installation of the CS kit on the Sepax S-100 main unit and launching of the automated separation procedure, refer to the Operator s Manual (Section 4.6) provided with the Sepax S-100 device. Once the procedure is complete, the Sepax S-100 guides the operator through a manual stripping of the buffy-coat (BC) bag line. CS-530.x OM 3F 08 3F - 9

50 STEP 10: Post -procedure actions At the end of the automated procedure, with the kit still installed on the S-100, the STRIP BC LINE ENTER message will appear on the S-100 display and the stopcock valves will be in the position shown in Fig. 2 and 3. Before pressing ENTER, manually squeeze the air from the BC bag until there is no air in the bag, as shown in Fig. 3. While holding the BC bag in this position with one hand, press the ENTER button on the S-100 with the other hand. The BC bag can then be released as the stopcock on the S-100 has been closed preventing return of the air. Fig. 2 Fig. 3 Continue following the instructions on the S-100 display panel until instructed to DISMOUNT KIT. STEP 11: Removal of the CS kit from the Sepax S-100 Close the WHITE clamp above the Y connector and as CLOSE to the Y-connector as possible. Seal 3 times above the white clamp. Sealing 3 times and cutting out the line in the middle seal ensures safety in case of imperfect seal. After sealing, detach the BC bag line with its components as shown in Fig. 4. Fig. 4 CS-530.x OM 3F 08 3F - 10

51 STEP 12: Buffy-coat sampling Prior sample taking, the buffy-coat must be mixed in order to obtain a homogeneous and representative sample: press manually 30 times the small (5ml) section of the buffy-coat bag. To take the sample, under laminar flow use the dedicated syringe port placed on the Y connector for needle insertion and then follow your validated procedure. STEP 13: DMSO preparation Composition and preparation of the DMSO solution for cryopreservation must be performed following your validated procedure. The quantity of DMSO solution to be injected in the BC CryoSC cryobag is indicated in the table below or follow your validated volume. To inject DMSO by syringe pump, you will need to prepare a syringe filled with with an extra 1.5 ml to the volume wanted in order to compensate the dead volume that remains in the tubing (DMSO extension line) after DMSO injection. (ex: If you need to add 5 ml, you put ml = 6.0 ml in the syringe). Output volume [ml] DMSO solution* [ml] Total volume [ml] 16 min max 6 30 * 55% w/v DMSO and 5% w/v Dextran 40 Note: Minimum output volume for UCB -HES and UCB protocols is 20ml and 10 ml, respectively. In order to use the Coolmix for an efficient mixing, the minimum volume before DMSO addition should be 16 ml (see table above). Note: Addition of DMSO by gravity feed is not possible STEP 14: DMSO injection into the BC bag by automated Syringe pump Connect one end of the DMSO extension line to the luer-lock syringe containing the DMSO cryosolution and the other end to the luer-lock on the kit. Check that the blue clamp on the DMSO extension line is closed. Install CryoSC cryobag on the Coolmix device. Prime manually the DMSO extension line tubing by pushing the syringe until the DMSO solution reaches the cover of the Coolmix. Then install the syringe in the syringe pump device and select the flow rate following the User Instructions of the syringe pump device. Start injection of the DMSO solution with the syringe pump device. If you are using the Coolmix automated cooling and mixing device, refer to the Coolmix Operator Manual, for display settings and automated procedure. CS-530.x OM 3F 08 3F - 11

52 CS-530.x OM 3F 08 3F - 12

53 SEPAX cell separation kit CS Configuration D C E H I G B F A Fig. 1 A: Separation chamber 200ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Double input line with female luer lock connectors and clamps E: Washing solution input with spike and clamp. F: 1000ml PVC waste collection bag G: Double output line with spike, injection site and clamps. H: 2 x extensions for input bag connection with double spike, male luer lock connector and clamps. For use with cryobag requiring a double connection during the thawing process. I: 2 x extensions for input bag connection with a single spike, male luer lock connector and clamp. For use with cryobag with a single connection during the thawing process. CS General Notes: The CS is a separation kit conceived for PBSC Washing and UCB Washing protocols to perform routine wash of thawed PBSC or cord blood products. Two different working procedures are described. One requires use of a Sterile Connection Device (SCD) to perform sterile connections outside the laminar flow. The other describes when no SCD is available and connections must be performed under controlled environment (laminar flow). It is strongly recommended to use the SCD to minimize the time between thawing and the beginning of the washing procedure. Note for PBSC Washing: the input bag volume capacity should be at least the double of the initial volume of the product to be washed. The UCB Washing and the PBSC Washing are using the same kit, the CS But the protocols are different. Thus we describe the handling of the kit for each protocol in 2 different parts. Please refer to the protocol that you are using. CS-600.x OM 3G 04 3G - 1

54 SEPAX cell separation kit CS with PBSC Washing Washing several bag - recommendations The PBSC Washing protocol allows the washing of 1 or 2 bags per washing cycle. It s possible to have 2 washing cycles per procedure. It means that with the same kit, it s possible to wash from 1 to 4 bags. If you wash several bags, be sure to wash bags containing product from the same patient. If you perform a 2 washing cycle procedure, it s easier to have a sterile connection device to connect the bags to the kit. If you don t have a SCD, you will have to dismount the kit from the machine and connect the bag(s) for the second cycle under sterile conditions. STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit with AS-610 traceability kit One or several input bags with product from the same patient (not supplied by Biosafe) One or two output bags (not supplied by Biosafe) 2 CS single-use kit, 1 for backup washing solution (not supplied by Biosafe), please check the chapter 4E to estimate the volume needed Sterile connection device recommended (not supplied by Biosafe) Tubing sealer (not supplied by Biosafe) Tubing stripper (not supplied by Biosafe) Orbital Shaker (not supplied by Biosafe) All laboratory material related with the cell count operations Material related to the thawing of the product STEP 2: Sepax parameter setting In order to reduce the time between the thawing of the input product and the beginning of the washing procedure, we recommend setting the parameters of the protocol ready. Please check the chapter 4E of the operator manual for more details about parameter selection. Select the PBSC washing protocol on the main menu and check the parameters. NOTE: The dilution ratio should be set to 1.0. Do not change the dilution ration parameter without validation. Fig. 2 It is recommended to set the output volume to at least 100ml. The input volume represents the volume processed by cycle. If 2 bags are processed in parallel (during the same cycle), enter the sum of their volume. CS-600.x OM 3G 04 3G - 2

55 STEP 3: CS kit sterility verification Before opening the kit blister pack under laminar flow, ensure that the sterility indicator on the Tyvek cover is brown, indicating that the kit is sterile. If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 4: CS kit integrity verification Under laminar flow spread the kit out in order to identify the tubing lines and components as in figure 1. Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 5: CS kit preparation under laminar flow Under laminar flow, close ALL the clamps of the single-use kit, the clamps on the extension lines included. That protocol can wash from 1 to 4 bags of PBSC product in two sequential cycles. Always wash bags containing the product from the same patient. Usually when performing 2 washing cycles, 1 or 2 output bags will be used. If you do only one cycle, you will use one output bag. Connect the output bag(s) to the CS kit (spike of line G in Fig. 1). You can connect up to two output bags as there are two spikes available. If you connect only one output bag, seal and detach the unused spike. Connect the washing solution bag to the washing solution spike (spike of line E in Fig. 1, see Fig. 3). Use 2.5% albumin in isotonic solution. Please refer to the Operator s Manual chapter 4E provided with the Sepax S-100 to see the quantity of washing solution needed. Fig. 3 CS-600.x OM 3G 04 3G - 3

56 DO NOT THAW the unit before it is necessary. You can connect 1 or 2 input bags that you will process in one washing cycle. The CS kit is provided with 2 different input bag lines (H and I on fig. 1). Choose the one corresponding to your input bag. Take the mono-spike line (I) if you wash one bag per cycle, shown on fig. 4. Take the double-spike line (H) if you wash two bags per cycle, shown on fig. 5. Fig. 4 Fig. 5 STEP 6A: Connection of the Input Bag if SCD is available Do that step only if a sterile connection device is available. After having selected the input bag line that you will use, go directly to step 7. DO NOT THAW THE PRODUCT at this stage. Leave the extension lines (H and I) under the laminar flow. You will use them later. STEP 6B: Connection of the Input Bag if SCD is not available Do that step only if a sterile connection device is not available. Prior connection of the initial bag to the CS kit, follow your validated procedure to thaw and then perform initial cell count of the PBSC product. Connect the chosen input bag(s) line to the input bag with the spike and then connect the line to the kit with the luer-lock connection. This operation must be done under LAMINAR FLOW. If you plan to do only one washing cycle, seal and detach the unused tube (Fig. 6). Seal here the unused tube Fig. 6 CS-600.x OM 3G 04 3G - 4

57 STEP 7: CS kit installation on the Sepax For instructions on installation of the CS kit on the Sepax S-100 main unit, parameter settings and launching of the automated separation procedure, refer to the Operator s Manual (Section 4.6) provided with the Sepax S-100. If your input bag(s) is already connected to the kit (SCD not available) put it/them on an orbital shaker (fig. 7). Do not switch on the shaker at this stage. Washing solution Connection without SCD: input bag is placed on a orbital shaker Fig. 7 Output bag(s) STEP 8: Automatic kit test and priming Start kit test by pressing Enter (Fig. 8). Fig. 8 When asked for, open washing solution line clamp, the waste line clamp and the output bag line clamp (Fig. 9). Press Enter. If you plan to do 2 washing cycles, open only one output bag clamp. The second output bag will be used for the second washing cycle. CS-600.x OM 3G 04 3G - 5

58 Fig. 9 Select either Standard wash or High wash and then press Enter. For details, please check chapter 4E. The machine then primes the kit and the washing solution for primary dilution is prepared in the separation chamber. Once the priming is finished, the Sepax displays (Fig. 10): Fig. 10 If your input bag is already connected to the kit with the spike, press Enter* and go to step 10. If connecting the input bag with an SCD device, go to step 9. STEP 9: input bag connection WITH SCD device Prior connection of the initial bag to the CS kit, follow your validated procedure to thaw and then perform initial cell count of the PBSC product. This operation must be done under LAMINAR FLOW. If you plan to perform two sequential washing cycles, only thaw the bag(s) that will be processed during the first cycle (1 or 2 bags). Connect the input bag(s) to the chosen input bag line by using the spike. This operation must be done under LAMINAR FLOW (fig. 11). Fig. 11 CS-600.x OM 3G 04 3G - 6

59 Outside of laminar flow connect the input bag(s) to the kit by using a sterile connection device. The sterile connection should be made on the input extension line and the kit. (Fig. 12) Sterile connection Fig. 12 Put the input bag(s) on the orbital shaker (fig. 7) Once the sterile weld done and the weld opened, press Enter. STEP 10: PBSC Washing automated procedure The display shows the following message (Fig. 13): Fig. 13 Open the input bag clamp, there are two or three clamps to open, then press Enter (Fig. 14). Open these two clamps Fig. 14 CS-600.x OM 3G 04 3G - 7

60 The display shows: Fig. 15 You can now start the orbital shaker and press Enter The washing solution will be extracted into the input bag to dilute the initial product, and then an osmolarity balancing step will be done. If you wash 2 bags during the same cycle, ensure that the washing solution is equally portioned in the 2 bags by mixing both bags together. After osmolarity balancing time, Sepax will emit tones asking to hang initial bag: Fig. 16 Remove the input bag from the orbital shaker and install in on the holder, then press Enter (Fig. 17). Switch off the orbital shaker. Fig. 17 AUTOMATED WASHING PROCEDURE Warning: Avoid touching the machine and the kit: Moving bag, tubes, stopcock and covers may cause errors. CS-600.x OM 3G 04 3G - 8

61 STEP 11: Post -procedure actions At the end of the washing procedure the following message appears (Fig. 18): Fig. 18 Close all clamps and press Enter. The display shows then: Fig. 19 If you want to perform a second washing cycle, go to step 12. If the washing process is finished, select End washing and go to step 13. STEP 12: Second Washing cycle Note: Do NOT wash different patient s bag with the same kit. Do NOT perform more than 2 washing cycles with the same kit. The second washing process with the same kit must be completed within 3 hours. Select Wash another bag by pressing Enter (Fig. 20) Fig. 20 You have then to confirm/change the parameters to process the second bag (refer to step 2 for more details). The procedure will then restart from step 6A. If you want to perform a second washing cycle, repeat all the steps from step 6A CS-600.x OM 3G 04 3G - 9

62 Note: check that there is enough washing solution left for the second washing cycle. When you perform 2 washing cycles with 2 different output bags, please seal and disconnect the first output bag and open the clamp of the 2 nd output bag. STEP 13: Removal of the kit If the washing process is finished, select the END WASHING and press Enter (Fig. 21) Follow the instructions of the display (Fig. 22). Fig. 21 Fig. 22 Open the clamps of the washing solution clamp, the waste bag clamp and the output bag clamps. Then press Enter. Remove the bags and filter, press Enter. Fig. 23 CS-600.x OM 3G 04 3G - 10

63 Then strip the cells line (Fig 24) Press Enter. Then strip the waste line (Fig 25) Fig. 24 Finally, you can dismount the kit (Fig.26): Fig. 25 Fig. 26 The quality of the cell washed must be controlled prior to the transplant, following your validated procedure. Transplant the output product using a transfusion filter. If blood spillage or leakage occurs during the process, the head of the hematology department should decide the final use of the product. CS-600.x OM 3G 04 3G - 11

64 SEPAX cell separation kit CS with UCB Washing STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit with AS-610 traceability kit One input bag with product (not supplied by Biosafe) One output bag (not supplied by Biosafe) 2 CS single-use kit, 1 for backup washing solution (not supplied by Biosafe), please check the chapter 4F to estimate the volume needed. Sterile connection device recommended (not supplied by Biosafe) Tubing sealer (not supplied by Biosafe) Tubing stripper (not supplied by Biosafe) All laboratory material related with the cell count operations Material related to the thawing of the product STEP 2: Sepax parameter setting In order to reduce the time between the thawing of the input product and the beginning of the washing procedure, we recommend setting the parameters of the protocol ready. Please check the chapter 4F of the operator manual for more details about parameter selection. Select the UCB washing protocol on the main menu and check the parameters NOTE: The dilution ratio should be set to 1.0. Do not change the dilution ration parameter without validation. Fig. 27 It is recommended to set the output volume to at least 75ml. STEP 3: CS kit sterility verification Before opening the kit blister pack under laminar flow, ensure that the sterility indicator on the Tyvek cover is brown, indicating that the kit is sterile. If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question. CS-600.x OM 3G 04 3G - 12

65 STEP 4: CS kit integrity verification Under laminar flow spread the kit out in order to identify the tubing lines and components as shown above. Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 5: CS kit preparation under laminar flow Under laminar flow, close ALL the clamps of the single-use kit, the clamps on the extension lines included. Connect the output bag to the CS kit (spike of line G in Fig. 1), seal and detach the unused spike. Connect the washing solution bag to the washing solution spike (spike of line E in Fig. 1, see Fig. 28). Use 2.5% albumin in isotonic solution. Please refer to the Operator s Manual chapter 4F provided with the Sepax S-100 to see the quantity of washing solution needed. Fig. 28 DO NOT THAW the unit before it is necessary. The CS kit is provided with 2 different input bag lines (H and I on fig. 1). Select the one corresponding to your input bag. Use the mono-spike line (I), shown in fig 29, for long spiking port, i.e. Baxter Cryocite. Take the double-spike line (H), shown in fig. 30, for bags like the CryoSC or Pall cryobags. Fig. 29 Fig. 30 CS-600.x OM 3G 04 3G - 13

66 STEP 6A: Connection of the Input Bag if SCD is available Do that step only if a sterile connection device is available. After having selected the input bag line that you will use, do nothing and go directly to step 7. DO NOT THAW THE PRODUCT at this stage. Leave the extension line (H and I) under the laminar flow. You will use them later. STEP 6B: Connection of the Input Bag if SCD is not available Do that step only if a sterile connection device is not available. Prior connection of the initial bag to the CS kit, follow your validated procedure to thaw and then perform initial cell count of the UCB product. Connect the chosen input bag line to the input bag with the spike and then connect the line to the kit with the luer-lock connection. This operation must be done under LAMINAR FLOW. Seal the unused input tube (Fig.31). Seal here the unused tube Fig. 31 STEP 7: CS kit installation on the Sepax For instructions on installation of the CS kit on the Sepax S-100 main unit, parameter settings and launching of the automated separation procedure, refer to the Operator s Manual (Section 4.6) provided with the Sepax S-100. STEP 8: Automatic kit test and priming Start kit test by pressing Enter (Fig. 32). Fig. 32 CS-600.x OM 3G 04 3G - 14

67 Then open washing solution line clamp, the waste line clamp and the output bag line clamp (Fig. 33). Press Enter. Fig. 33 Select either Standard wash or High wash and then press Enter (Fig. 34). For details, please check chapter 4F. Fig. 34 The machine then primes the kit and the washing solution for primary dilution is prepared in the separation chamber. Once the priming is finished, the Sepax displays (Fig. 35): Fig. 35 CS-600.x OM 3G 04 3G - 15

68 If your input bag is already connected to the kit with the spike, press Enter* and go to step 10. If connecting the input bag with an SCD device, go to step 9. STEP 9: input bag connection WITH SCD device Prior connection of the initial bag to the CS kit, follow your validated procedure to thaw and then perform initial cell count of the PBSC product. This operation must be done under LAMINAR FLOW. Connect the input bag to the chosen input bag line by using the spike(s) (fig 36). This operation must be done under LAMINAR FLOW. Fig. 36 Outside of laminar flow connect the input bag to the kit by using a sterile connection device. The connection should be made on the input extension line and the kit. Once the sterile weld done and the weld opened, press Enter. CS-600.x OM 3G 04 3G - 16

69 STEP 10: UCB Washing automated procedure The display shows the following message (Fig. 37): Fig. 37 Open the input bag clamp, there are two or three clamps to open, then press Enter (Fig. 38). Open these two clamps Fig. 38 AUTOMATED WASHING PROCEDURE Warning: Avoid touching the machine and the kit: Moving bag, tubes, stopcock and covers may cause errors. STEP 11: Post -procedure actions Remove the bags and filter, press Enter. Fig. 39 CS-600.x OM 3G 04 3G - 17

70 Then strip the cells line (Fig. 40) Press Enter. Then strip the waste line (Fig. 41) Fig. 40 Fig. 41 Finally, close all clamps and dismount the kit (Fig. 42): Fig. 42 The quality of the cell washed must be controlled prior to the transplant, following your validated procedure. Transplant the output product using a transfusion filter. If blood spillage or leakage occurs during the process, the head of the hematology department should decide the final use of the product. CS-600.x OM 3G 04 3G - 18

71 SEPAX cell separation kit CS Configuration D C E I B H F A G Fig. 1 A: A-200/F separation chamber (210 ml). B: Line pressure monitor luer with microbial filter. C: Stopcock manifold. D: Input line with drip chamber and clamp. E: Washing solution input with spike and clamp. F: Re-suspension media line with spike and clamp. G: 1000ml PVC density gradient media& waste bag. H: Luer lock input for density gradient media injection. I: Exit line with spike, injection site and clamp. A unique bar code placed on the rotary seal of the A-200/F separation chamber identifies this special kit, ensuring its traceability (see figure below). CS-900.x OM 3H 03 3H - 1

72 CS General Notes: The CS is a separation kit conceived for DGBS protocol to perform routine separation of blood products (blood, blood components or bone marrow) with density gradient technique. - The CS kit can be connected to the input bag with a sterile connection device: to use a sterile connection device, the input bag must have at least 20cm of available tubing to be inserted in the sterile connection device with the tubing between the spike port and the bubble chamber in the input bag line ( D in Fig.1). This operation must be done under LAMINAR FLOW. - If 20cm (8 ) of tubing is not available, the connection of the input bag will require use of the spike port pre-installed on the CS kit: this operation must be done under LAMINAR FLOW. STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit with AS-610 traceability kit Input bag with product (not supplied by Biosafe) One output bag with ml volume capacity (not supplied by Biosafe) CS single-use kit ml density gradient solution, 100ml recommended (not supplied by Biosafe) 500ml or 1000ml washing solution (not supplied by Biosafe) 50ml cell re-suspension media if needed (not supplied by Biosafe) Sterile connection device if needed (not supplied by Biosafe) Tubing sealer (not supplied by Biosafe) Tubing stripper (not supplied by Biosafe) All laboratory material related with the cell count operations STEP 2: Sampling of input product Prior connection of the initial bag to the CS kit, follow your validated procedure to perform initial cell count. STEP 3: CS kit sterility verification Before opening the kit blister pack under laminar flow, ensure that the sterility indicator on the Tyvek cover is brown, indicating that the kit is sterile. If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question. STEP 4: CS kit integrity verification Under laminar flow spread the kit out in order to identify the tubing lines and components as shown above. Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question. CS-900.x OM 3H 03 3H - 2

73 STEP 5: CS kit preparation Close ALL the clamps of the single-use kit. All the handling of step 5 must be done under laminar flow. Connect first the output bag to the CS kit (spike of line I in Fig. 1). Connect then the 500ml washing solution bag to the washing solution spike (spike of line E in Fig. 1, see Fig. 2). As general rule use a 500 ml washing solution bag for a two cycles washing process and a 1000 ml bag for a three cycles washing procedure (albumin is added to this volume). Use 2.5% human albumin in isotonic solution. Fig. 2 If the washing solution and the cell re-suspension media used are the same, seal and detach the re-suspension media line with spike and clamp from the rest of the kit ( F in the Fig. 1). When a different media than the washing solution is used to re-suspends the cells at the end of the procedure, prepare and connect a bag containing 50 ml of re-suspension media spike of line ( F in Fig. 1, see Fig.3). Fig. 3 CS-900.x OM 3H 03 3H - 3

74 STEP 6: Fill in the Density Gradient Solution/Waste bag Under laminar flow, prepare a syringe filled with 100 ml of density gradient solution (density gradient media). Wait for the solution to reach room temperature. Unscrew the cap on the injection port (Fig. 4). Open the clamp and inject the density gradient solution into the bag by using the injection port, no need to use a needle. Close the clamp and seal the tubing (Fig. 5) and remove the syringe and the sealed tubing from the kit. open this clamp use this port to inject the density gradient solution in the bag Fig. 4 STEP 7: input bag connection Fig. 5 seal the tube and remove the syringe Connect the input bag to the kit with a sterile connection device under laminar flow conditions (see Instructions for Use supplied with the sterile connection device). The sterile connection should be made between the pre-installed spike and the bubble chamber of the input bag line (Fig.6). SCD connection : connect here Fig. 6 CS-900.x OM 3H 03 3H - 4

75 If a sterile connection device is not available, the CS kit can be connected to the input bag using the pre-installed spike on the input bag line (Fig. 7)) under laminar flow conditions. Connect the input bag with this spike Fig ml washing solution 50ml cell resuspension media 100 ml density gradient media ml initial product empty output bag Fig. 8: CS ready to be installed on the Sepax STEP 8: Parameter setting and CS kit installation Select the DGBS protocol on the main menu and check the parameters (Fig. 9). Fig. 9 CS-900.x OM 3H 03 3H - 5

76 NOTE: The Density gradient media volume parameter can be set from ml, but should be left at default value of 90 ml (Fig. 10). Remember if you select 90 ml in the parameters, you need to add 100 ml in the bag. Fig. 10 Set the pause switch bag parameter to 1 if the re-suspension media is different from the washing solution (Fig.11) 0 if washing solution is the same as the re-suspension media 1 if washing solution is NOT the same as the re-suspension media Fig. 11 For more details on the parameters initial volume and washing cycle number, please refer to the Operator Manual, chapter 4H. For instructions on installation of the CS kit on the Sepax S-100 main unit, parameter settings and launching of the automated separation procedure, refer to the Operator s Manual (Section 4.6) provided with the Sepax S-100. Once the parameters set, install the kit on the Sepax (Fig.12) input product washing solution cell re-suspension solution (if needed) ouput bag Bag with density gradient media Fig. 12 CS-900.x OM 3H 03 3H - 6

77 STEP 9: DGBS automated procedure: manual priming Once the kit installed on the Sepax and the procedure launched, the device performs an automated kit test. When the Input done, continue displays (Fig.13), press Enter Fig. 13 The tubing line between the source bag and the white stopcock has to be primed manually with blood. The display shows Prime Bubble Chamber (Fig. 14) Fig. 14 prime bubble chamber Squeeze the bubble chamber and release it 2-3 times until the bubble chamber is half-filled with blood (Fig.14) then press Enter. Fig. 15 squeeze the bubble chamber then press Enter CS-900.x OM 3H 03 3H - 7

78 Open all clamps except the clamp for the re-suspension solution if applicable. Press Enter. Fig. 16: open all clamps then press Enter Lower the input bag position by adjusting the holder position. This adjustment will decrease the flowing speed of the blood during the priming. Fig. 17: lower the holder Press the up-key (Fig. 18): the blood flows towards the stopcock; press a second time the up-key when the blood is between the blue and the white stopcocks: the blood stops flowing (Fig. 19). Fig. 18: press the up-key CS-900.x OM 3H 03 3H - 8

79 Fig.19: the blood flows towards the stopcock until the white stopcock NOTE: The line is open towards the plasma bag during this operation so if the blood goes beyond the white stopcock it will be directed towards the density gradient solution bag (avoid blood reaching the density gradient solution bag). In this case, hang down the initial bag, select Redo priming by pressing Enter. The display will show (Fig 20): Fig. 20 Press the upkey while lifting the tubing on the left of the red stopcock: your product will flow back towards the bubble chamber. Press again on the upkey to close the red clamp. You can redo the priming again. When the priming is properly done, press Enter to start the procedure. The automated procedure starts. Fig. 21: start procedure Warning: Avoid touching the machine and the kit: Moving bag, tubes, stopcock and covers may cause errors. CS-900.x OM 3H 03 3H - 9

80 STEP 10: Cell resuspension media Note: You need to do this step only if the parameter pause switch bag was activated (value = 1) in step 8: the re-suspension media you use is different from the washing solution At the end of the washing procedure, the cells are contained in the separation chamber. A resuspension and extraction are performed to put the cells into the final product bag. The resuspension could be done using the washing solution or a special buffer (culture media ). In this case, Switch solution bag using clamps will be displayed (Fig.22): Fig. 22 Switch solution bag using clamps Close the clamp of the washing media and open the clamp of the re-suspension media (Fig. 23). washing solution Cell re-suspension media Close first the clamp of the washing solution bag Press Enter to confirm (Fig. 24). Fig. 23 Then open the clamp of the cell re-suspension bag Fig. 24 CS-900.x OM 3H 03 3H - 10

81 The following message will then appear (Fig. 25). The cell re-suspension media will go into the separation chamber and the final product extraction into the output bag will start. STEP 11: Post-procedure actions Fig. 25 At the end of the automated procedure, with the kit still installed on the S-100 (Fig. 26), the End of process message appears on the display (Fig. 27) empty input bag output product Fig. 26 waste bag Press Enter Fig. 27 Fig. 28 Remove the bags and the filter, press Enter. CS-900.x OM 3H 03 3H - 11

82 Then strip the cells line (Fig. 29) Press Enter. Then strip the waste line (Fig. 30) Fig. 29 Fig. 30 Close all clamps, press enter, remove the kit first and then press Enter again (Fig.31). Fig. 31 CS-900.x OM 3H 03 3H - 12

83 STEP 12: Final sample Under laminar flow, the final sample can be taken using the injection port (Fig. 32). Use this injection port to take a sample of your final product Fig. 32 CS-900.x OM 3H 03 3H - 13

84 CS-900.x OM 3H 03 3H - 14

85 SEPAX cell separation kit CS Configuration E A 2 D 2 3 C 6 B Fig. 1 A UCB input bag line B Buffy-coat (BC) bag line C Pressure monitoring line D Separation chamber line E Plasma bag line 1 Sampling chamber 2 Needleless port 3 HES filter 4 DMSO filter 5 DMSO extension line 6 Air filter CS-540.x OM 3I 05 3I - 1

86 General Notes: The CS is a functionally-closed separation kit when used with a sterile connection device for initial sampling and connection of UCB input bag To use a sterile connection device the UCB input bag must have at least 20cm of available tubing, as shown in Fig. 2. Use of luer-lock syringes are recommended as they are safer. Fig. 2 If 20cm (8 ) of tubing is not available, the initial sampling and connection of the UCB input bag will require use of a standard syringe and the spike port pre-installed on the CS kit, respectively - both operations must be done under LAMINAR FLOW WARNING: ONLY REMOVE SPIKE CAP UNDER LAMINAR FLOW IF KIT IS BEING CONNECTED USING A STERILE CONNECTION DEVICE THEN LEAVE SPIKE CAP IN PLACE. STEP 1: Required material Prior to starting the procedure, ensure that the following material is within easy reach: Sepax S-100 main processing unit with AS-610 traceability kit Coolmix device equipped with DMSO vial extension rod. UCB input bag (not supplied by Biosafe) If you se he UCB-HES protocol, Hydroxy-Ethyl-Starch HES solution - HES % (not supplied by Biosafe) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant) CS single-use kit CS accessories blue and red Sample Calibrators AK-100 Sampling Line accessory DMSO single-use vial (not supplied by Biosafe), if injection by gravity feed AK-10 DMSO Vial Spike accessory DMSO vial adaptor Sterile connection device (not supplied by Biosafe) Sterile tubing sealer (not supplied by Biosafe) Tubing manual stripper (not supplied by Biosafe) Luer-lock syringes as required (not supplied by Biosafe) STEP 2: Sampling of UCB input bag with AK-100 Sampling Line Remove the AK-100 sampling line from its sterile pack and avoid tampering with the end cap. CS-540.x OM 3I 05 3I - 2

87 STEP 2A: Connection of the AK-100 accessory to the UCB input bag. Fig. 3a Fig. 3b Close the BLUE clamp on the AK-100 accessory approximately 2cm (1 ) from the sampling chamber. Connect the AK-100 accessory to the UCB input bag with a sterile connection device see Fig. 3a & 3b. (Refer to the Instructions for Use supplied with the sterile connection device). If a sterile connection device is not available, the AK-100 accessory can not be connected to the UCB input bag. In this case you should take the sample from the UCB input bag in sterile conditions with a needle-syringe, following your validated procedure. NOTE: FOR THE CS KIT TO BE CONSIDERED FUNCTIONALLY CLOSED THE INITIAL SAMPLE MUST BE TAKEN USING THE AK-100 SAMPLING LINE ACCESSORY. THE AK-100 ALSO ELIMINATES THE USE OF A NEEDLE SYRINGE IN THE SAMPLING PROCESS. STEP 2B: sample taking Once the sterile connection is complete, ensure the integrity of the tubing connection, then open the BLUE clamp on the AK-100 sampling line. Fig. 4a Fig. 4b Fig. 4c Fig. 4d Take the BLUE Sample Calibrator supplied with the CS accessory kit. Insert the sampling chamber in the Biosafe Sample Calibrator, as shown in Figs. 4a, 4b, 4c & 4d, then close and release the Sample Calibrator to draw product from the UCB input bag, as shown in Figs. 5a & 5b. CS-540.x OM 3I 05 3I - 3

88 Fig. 5a Fig. 5b STEP 2C: Sample line flushing After taking the sample, position the UCB input bag and the sampling chamber as shown in Figs. 6a & 6b. and press on the sample chamber to push the UCB remaining in the tubing back to UCB input bag. This procedure is performed with the thumb and index and does not require use of the Sample Calibrator. Fig. 6a Fig. 6b Seal the tubing 3 times near the sampling chamber; seals should be made with a standard tubing sealer (see Biosafe representative for suggested sealing equipment). The middle seal should then be cut to remove the sampling chamber the other 2 seals ensure sterility in case of an incomplete seal. Detach the sampling chamber. The sample can then be transferred to a luer-lock syringe under laminar flow and tested following your validated procedure (Fig. 7). Fig. 7 STEP 3: CS kit sterility verification Ensure that the Sterility Indicator on the single-use kit blister is BROWN if the indicator is BLUE then put the kit aside and inform your Biosafe representative. CS-540.x OM 3I 05 3I - 4

89 STEP 4: CS kit integrity verification Remove the single-use kit from the blister and lay it out flat to check that there are no kinks or other visible damage. STEP 5: CS kit preparation Close all BLUE clamps (total of 5 including separate DMSO extension line) DO NOT CLOSE WHITE CLAMPS Please close the blue clamp of the entry line in the middle of the line (between the Y connector and the bubble chamber, to avoid kink formation). STEP 6: If you use UCB-HES protocol, Hydroxy-Ethyl-Starch (HES) preparation In accordance with your internal validation procedure, prepare a Hydro-Ethyl-Starch (HES) solution (HES %) corresponding to 20% of the UCB input volume (UCB + CPD anticoagulant). For example, if the volume of the UCB input bag is 100ml including CPD, you should prepare 20ml of HES solution. Fill a luer-lock syringe with the prepared HES, wait for the HES to reach room temperature and avoid having air in the syringe (it may block the HES filter of the kit). STEP 7: UCB input bag connection Connect the UCB input bag to the CS kit with a sterile connection device (see Instructions for Use supplied with the sterile connection device). Fig. 8 If a sterile connection device is not available, the CS kit can be connected to the UCB input bag using the pre-installed spike connection (UCB input bag line A ) under LAMINAR FLOW conditions. With SCD: DO NOT REMOVE THE CAP OF THE SPIKE and connect the input bag to the kit at the location shown in Fig. 8. Without SCD: Under laminar flow, remove the cap of the spike and connect the spike to the input bag. NOTE: FOR THE CS KIT TO BE CONSIDERED FUNCTIONALLY CLOSED THE CONNECTION TO THE UCB INPUT BAG MUST BE PERFORMED WITH A STERILE CONNECTION DEVICE AND NOT THE SPIKE CONNECTION. CS-540.x OM 3I 05 3I - 5

90 STEP 8: If you use UCB-HES protocol, HES injection After preparation of the luer-lock syringe with the required volume of HES (refer to STEP 6), connect the HES syringe to the filter (there is no cap on the HES filter) and then open ONLY the HES clamp immediately downstream of the HES filter. Fig. 9 Note: If by error, the blue clamp nearest to the HES filter is not closed prior to connecting the UCB input bag, the cord blood may flow to the HES filter. The subsequent impregnation of the HES filter does not affect sterility in any way. Inject the HES with one hand while mixing the bag thoroughly with the other - injection flow rate should be approximately 0.5ml per second. Close the HES filter clamp as close as possible to the Y connector and then using a tubing sealer, seal off the HES filter upstream of the HES clamp (with syringe attached) and remove syringe/filter for disposal - the HES syringe/filter must be removed to avoid any tubing kinks. Note: The HES between the connector and the HES filter can not be recovered. This dead volume corresponds to approximately 1 ml. STEP 8: If you use UCB protocol, HES filter removal Close the HES filter clamp as close as possible to the Y connector and then using a tubing sealer, seal off the HES filter upstream of the HES clamp and remove filter for disposal.. STEP 9: CS kit installation and UCB-HES automated procedure Select UCB-HES protocol on the Sepax and choose/confirm parameters. Install CS kit on the Sepax and position the Buffy-Coat (BC) cryobag as shown in Fig.10. This position minimizes the risk of generation of kinks in the output BC line. CS-540.x OM 3I 05 3I - 6

91 Fig. 10 Launch procedure as described in the Sepax Operator s Manual. AUTOMATED PROCEDURE At the end of the automated procedure, follow the instructions indicated on the Sepax display - strip the RBC line if and when indicated to do so (this may or may not be required depending on the volume being processed). STEP 10: Post-procedure actions Strip BC line when Strip BC Line message appears - DO NOT PRESS ENTER AT THIS TIME Note: If Enter is pressed at this time, the blue stop-cock valve will close, preventing the removal of air from the bag. In this case, carefully lift the stopcock manifold, turn the blue stopcock back to the open position as shown in Fig. 11a and replace the stopcock manifold on the Sepax. From To Fig. 11a Connect an empty 20ml or 30 ml syringe to the kit air filter as shown in Fig. 11b. CS-540.x OM 3I 05 3I - 7

92 Fig. 11b CS-540.x OM 3I 05 3I - 8

93 Using the air syringe remove all of the air from the BC cryobag until the BC reaches the bag/tubing transition as shown in Fig. 12 (DO NOT PRESS ENTER BEFORE REMOVING ALL THE AIR) to Fig. 12 Once the air is removed, press Enter and then manually mix the BC bag contents STEP 11: Removal of the CS kit from the Sepax S-100 Close the WHITE clamp above the double-y connector and as close to the Y-connector as possible (see red arrow, shown in Fig. 13). Press Enter. Detach the Input line, the plasma bag and the separation chamber. You can detach it by sealing the tubes at 2 cm of the stopcock manifold (see red arrows, shown in Fig. 14). Fig. 13 Fig. 14 CS-540.x OM 3I 05 3I - 9

94 STEP 12: Buffy-coat sampling Prior sample taking, the buffy-coat must be mixed in order to obtain a homogeneous and representative sample: press manually 30 times the small (5ml) section of the buffy-coat bag. Fig. 15 Fig. 16 To take the sample, open only the BLUE clamp on the sampling chamber line (see red arrow, fig. 16). In order to obtain precise and repeatable sample volumes of 0.3 ml, the RED Sample Calibrator accessory shown in Fig. 17 must be used. The Sample Calibrator should be used as shown in Figs. 17a, 17b, 17c. Fig. 17a Fig. 17b Fig. 17c Press and hold firmly the sampling calibrator with the sampling chamber positioned straight and centrally in it. Incorrect positioning, of the sample chamber as shown in Figs. 18a, 18b and 18c can adversely affect sampling volumes. Fig. 18a Fig. 18b Fig. 18c CS-540.x OM 3I 05 3I - 10

95 Position the buffy-coat bag line upside down as shown in Fig. 19. Fig. 19 shows exactly how to handle the BC bag, the line and the Sample Calibrator, holding the buffy-coat bag with one hand (avoid applying pressure to the bag) and holding firmly the sample calibrator with the other hand. Fig. 19 Release the Sample Calibrator to draw product from the BC bag, as shown in Figs. 20a, 20b and 20c. Fig. 20a Fig. 20b Fig. 20c Having extracted a 0.3ml sample to the sampling chamber, the buffy-coat remaining in the tubing between the buffy-coat bag and the sampling chamber should be returned to the BC bag. To do this, place the BC bag and the sampling chamber in the position shown in Figs. 21a, 21b and 21c. Fig. 21a Fig. 21b Fig. 21c Press the sampling chamber until only air remains in the tubing see Fig. 21c. CS-540.x OM 3I 05 3I - 11

96 STEP 13: Detach sampling chamber Fig. 22a Fig. 22b Close the BLUE clamp of the sampling chamber line (see red arrow) and seal 3 times; sealing 3 times and cutting out the line in the middle seal ensures safety in case of imperfect seal. Detach the sampling chamber from the buffy-coat bag line - see Fig. 22a. The sample should then be tested according to your validated sampling procedure. Note The CS sampling chambers are designed to connect to luer-lock syringes under LAMINAR FLOW see Fig. 22b. STEP 14: DMSO preparation Preparation of the DMSO solution for cryopreservation must be performed following your validated procedure. The quantity of DMSO solution to be injected in the BC cryobag is 5ml or your validated volume. You need a syringe pump to inject the DMSO Note: You need to add an extra volume of 1.5 ml. (ex: If you need to add 5ml, you put ml = 6.5ml in the syringe). This volume corresponds to the dead volume that remains in the DMSO filter and the tube. STEP 15: DMSO injection into the BC bag by automated Syringe pump Connect one end of the DMSO extension line to the luer-lock syringe containing the DMSO cryosolution and the other end to the DMSO filter on the kit (Fig. 22c) Fig. 22c CS-540.x OM 3I 05 3I - 12

97 Install the kit and the syringe assembly on the Coolmix. Install the syringe on the syringe pump device and select the flow rate following the User s Instructions of the syringe pump device. Proceed to injection of DMSO with the syringe pump. Please refer to the Coolmix Operator s Manual, for display settings and automated procedure. STEP 16: Stripping of DMSO line Close the DMSO filter pinch clamp (BLUE). Remove the kit from the Coolmix, place it on the bench. Open the white clamp above the double-y connection and turn the blue stopcock to the position shown in fig. 23. From To Fig. 23 By using the air that is in the syringe attached to the air filter, push the DMSO remaining in the tubing line into the BC bag, ensuring that no DMSO remains in the tubing (Fig. 24a), and then use the same syringe to remove all the air from the BC bag (Fig. 24b, 24c). Fig 24a Fig. 24b Fig. 24c IT IS VERY IMPORTANT THAT ALL OF THE AIR IS REMOVED UP TO THE BC BAG/TUBING TRANSITION (SEE FIG. 28a) TO AVOID ANY BUBBLES FORMING DURING THE NEXT STEP. DO NOT FILL THE TUBING WITH ANY PRODUCT AS THIS PART OF THE PRODUCT WILL NOT BE MIXED IN THE NEXT STEP. To prepare the product for storage, go to step 17 To take an additional sample, go to step 19 CS-540.x OM 3I 05 3I - 13

98 STEP 17: Ensuring a homogeneous product for storage With the BC bag full and no product in the tubing (Fig. 25a), mix carefully the bag manually to ensure a homogeneous product. Once the product is homogeneous, draw the buffy-coat up to the beginning of the Y-connector using the syringe (Fig. 25b). Fig. 25a Fig. 25b Note: Even if any product remaining in the tubing flows up to the air filter, the system still remains closed. STEP 18: preparing BC bag for storage Close the white clamp above the Y-connector and turn the blue stopcock to the position shown in Fig. 26. From To Fig. 26 The BC bag tubing can now be sealed and segments defined as per your validated procedure and the BC bag prepared for cryopreservation. END OF THE PROCEDURE Fig. 27 CS-540.x OM 3I 05 3I - 14

99 STEP 19: Taking sample after DMSO addition and preparation for storage Once the product is homogeneous, use the syringe to draw the buffy-coat up past the Y- connector to obtain the required sample volume (Fig. 28a-28b). Seal the cryobag segments as per your validated procedure (Fig. 28c). To retrieve sample, refer to Fig. 28d. Note: The maximum attainable volume for this sample is 2ml. DO NOT DRAW THE PRODUCT UP TO THE STOPCOCK MANIFOLD. Fig. 28a Fig. 28b Fig. 28c Fig. 28d: retrieve sample END OF PROCEDURE CS-540.x OM 3I 05 3I - 15

100 LEGEND Press Product flow direction Release Weld 3x Clamp is closed Air flow direction DMSO flow direction Clamp is open TIP Before mixing, always purge the air from the Pall bag to avoid creation of bubbles and cell loss v + mix = x + mix = BUBBLES = CELL LOSS! CS-540.x OM 3I 05 3I - 16

101 Biosafe CryoSC cryobag Preparation for Cryopreservation Biosafe CryoSC Cryobag Latex free EVA material Fits into existing standard canister Compatible also with BioArchive system Biosafe CryoSC Cryobag Related Accessories Product# #3001 #7014 DMSO solution injection Coolmix Device v.1.5 Syringe Pump Device Cryobag accessories New version compatible with Biosafe CryoSC and Pall freezing bags Automated DMSO solution injection for one or two syringes #4085 #7008 #7012 #7000 Overwrap Bag Overwrap Sealing Device Storage Canister Canister Opening Tool Elastic, tough and extreme low temperature resistant protection Sealer for overwrap bag provided with with air-purger tool Canister for efficient cryopreservation, also Pall bag compatible Canister opening tool before thawing procedure #7010 #7011 Traceability Label Printer & Software Labels Printer for traceability labels with software ISBT128 compliant Sets of labels for cryopreservation CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 1

102 Biosafe CryoSC cryobag General Notes: The present document describes Biosafe CryoSC cryobag preparation prior to cryopreservation. The Biosafe CryoSC cryobag are available only pre-connected to Biosafe singleuse kits, they can not be sold independently. Description A B C STEP 1: Required material allows safe and efficient cryopreservation of blood derivatives and cellular product in liquid and/or vapor phase nitrogen 10ml to 30ml product can be stored (cryopreservant included) single-compartment CryoSC cryobag with integrated sampling tube (A) Enhanced traceability with a batch number (B) hermetically sealed membrane port for standard spike (C) compatible with Coolmix device version 1.5. Prior to starting the procedure, ensure that the following material is within easy reach: Equipment Sterile tubing sealer, compatible with EVA polymer material (not supplied by Biosafe) Equipment and materials related with cell count operations (not supplied by Biosafe) Equipment and materials related with cryopreservation (not supplied by Biosafe) Canister opener tool Coolmix 1.5 version or above Traceability accessories Biosafe labels Barcode reader Label printer Cryopreservation accessories Biosafe overwrap bag for CryoSC cryobag (Product # 4085) Biosafe storage canister (Product # 7012) Rack for canister (not supplied by Biosafe) CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 2

103 STEP 2: CryoSC cryobag traceability Prior to starting the Sepax procedure, scan the CryoSC cryobag label barcode with the barcode reader. The printer automatically issues a set of labels. An example of a set of labels: Fig. 1 Fig. 2 label for Biosafe cryobag label for canister label for canister seal Fig. 3 Stick the label on the CryoSC cryobag, ensure that the label is centered. Fig. 4 STEP 3: Sepax processing and DMSO addition with Coolmix Follow Operator Manual for processing procedure with Sepax Follow Operator Manual for processing procedure with Coolmix CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 3

104 STEP 4: CryoSC cryobag air purge and aliquot sample segments sealing Extract air contained in the CryoSC cryobag and the tubing. You can either use the injection port of the kit under laminar flow conditions or use the pressure line air filter of the kit with a syringe. No air in the cryobag and tubing Fig. 5 Seal the CryoSC cryobag tubing as shown in (Figs 6 and 7) close to the kit connector. Biosafe recommends the Sebra sealer (standard model ref 1105). Cut carefully this seal in the middle with a scissor (Fig. 8). Do not tear the seal apart. to the kit seal here Fig. 6 Fig. 7 Fig. 8 Make then the other seals to form the number of aliquot sample segments requested. Maximal number of segments is 3, with 120 microliters volume each. Seal towards the CryoSC cryobag to avoid pressure building in the segments. Fig. 9 Fig. 10 CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 4

105 STEP 6: Overwrap bag and sealing the overwrap bag The CryoSC cryobag is stored in an overwrap bag to minimize potential cross contamination during the storage. Simply place the CryoSC cryobag in the overwrap bag (Figs. 11) Fig. 11 To seal the overwrap bag with the CryoSC cryobag in it, place the overwrap bag in the Air-purger tool and close the cover to remove the air comprised in the overwrap bag (Figs. 12, 13 and14). Fig. 12 Fig. 13 Fig. 14 Press the pedal to seal the overwrap bag. The sealing takes 6-7 seconds (Fig.15). You do not need to keep the pedal pressed. When the jaw of the sealer opens, remove the overwrap bag from the Air-purger tool. Visually inspect the seal (Figs. 16 and 19). Never re-seal on an existing seal or never seal twice on the same seal. Fig. 15 CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 5

106 Parameter setting range for the overwrap bag sealer Temperature Sealing time Cooling time C seconds seconds seal Fig. 16 Fig. 17 STEP 7: Placing the CryoSC cryobag in the canister For standard cryopreservation system Place the overwrap bag containing a CryoSC cryobag in the canister as shown. (Figs. 18and 19) Close the canister and stick the traceability label on the canister (Fig. 20). Stick the canister seal label (safety closure) (Fig. 21). Fig. 18 Fig. 19 CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 6

107 For BioArchive System Fig. 20 Fig. 21 Place the CryoSC cryobag in its sealed overwrap bag in the BioArchive canister exactly as shown in Figs. 22 and 23. The positioning is important as it avoids misplacement and pinching of the overwrap bag when the canister is closed. Then stick the traceability labels on the canister (Figs. 24 and 25). Fig. 22 Fig. 23 Fig. 24 Fig. 25 STEP 8: Transfer to the controlled rate freezer and cryotank Please follow your validated SOP s. CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 7

108 General notes for thawing procedure Please follow your own validated SOP for thawing procedure of the CryoSC cryobag. During thawing procedure, handle with care the Biosafe CryoSC cryobag in its overwrap bag. Do not drop or bend the CryoSC cryobag at any time. Do not use sharp instruments/tools. Notes for product retrieval after thawing procedure To collect the cells for use after thawing you need to cut the protection of the membrane connector (Figs. A and B). This must be done under laminar flow. You can then use a sampling site and connect it to the spike connection port of the CryoSC cryobag by piercing carefully the membrane. Fig. A Fig. B CryoSC Cryopreservation Preparation OM 3Z 01 3Z - 8

109 4. THE PROTOCOLS 4.1 General description (see flowchart on the following page) A typical separation procedure starts by filling the separation chamber. The product from the input (source) bag is aspirated into the chamber by a downward movement of the piston. The filling phase ends when the optical sensor detects that the supply tube is empty or when the chamber is full. At the beginning of the separation process the chamber starts rotating along its axis. The centrifugation lasts for a few minutes and results in separation of the product components. Finally, in the extraction and collection phase, the separated product components are distributed into the respective collection bags. The procedure is complete when the piston has reached the top of the chamber. The product in the output bag (BC bag for UCB procedures) is then ready for further processing. 1. Chamber filling 2. Sedimentation 3. Plasma Buffy-Coat RBC extraction 4.2 Starting the SEPAX S-100 Before turning on the SEPAX S-100, ensure that no kit has been installed Switch on the SEPAX S-100 by using the main switch on the rear panel. Diagnostic tests will then be carried out (informative messages will appear on the display panel no action is required). Three beeps and the sequential display of digits from 0 to 9 on the LCD-display indicate that the test is complete. Do not press any keys during this test. At the end of the test, the rotary pins are initialised and the software version is indicated on the LCD display during 2 seconds. A menu is then displayed enabling the protocol to be initiated or the application to be terminated and the SEPAX S-100 turned off. At this stage, various settings can be selected prior to choosing the required separation protocol. If the settings need to be changed once the protocol has been selected, it is sufficient to press the Menu key to display the options. The protocols OM

110 4.3 Start-up procedure Appropriate protective gloves and clothing should be worn when operating the SEPAX system. UP and DOWN keys (arrows) are used for scrolling through the different menus ENTER validates the current selection (first line on the screen, high-lighted by an arrow) "MENU" brings up the menu options STOP key should only be used in case of emergency, to stop the S-100 and restart the procedure. 4.4 Protocol selection Use the UP and DOWN scroll keys to choose between the different software protocols. The arrow on the right hand side of the protocol name indicates the current selection. Only the first two lines are visible on the display to see more options use the UP and DOWN arrow keys scroll through the entire menu list. Message on the display PURGE MODE UCB-HES (Other protocol) QUIT MENU Yes No ENTER Action Go to previous option Go to next option ENTER Validate selection To launch the protocol, first select it and then press ENTER. The protocol is then loaded, it take few seconds. In some protocol (i.e. UCB-HES) the output volume is displayed while the protocol is loaded. 4.5 Connecting the input product bag to the Separation Kit Refer to specific Instructions for Use for kit preparation indications. 4.6 Separation Kit installation Before installing the separation kit: Message on the display Action START PROCEDURE CHANGE PARAMETERS MAIN MENU CONFIGURE TRACE ID MENU Yes No ENTER Go to previous option Go to next option ENTER Validate marked option The protocols OM

111 Before starting the kit test, install the kit as follows: 1. Open the two separation chamber pit covers 2. Install the chamber into the separation by pushing it down firmly - ensure that the separation chamber is well inserted Important note: Do not close separation chamber pit cover until indicated 3. Insert the separation chamber tubing line into the optical sensor - ensure that the tubing is correctly inserted. 4. Verify that all stopcocks on the separation kit are aligned as shown 5. If your Sepax is equipped with a stopcock holder go directly to point 6. If your Sepax is not equipped with a stopcock holder, install the stopcocks on the SEPAX S-100 rotary drive pins by pushing them down firmly. Go directly to point Open the stopcock holder by pushing down the two levers, place the stopcock in right position and close the holder by pushing up the levers. OPEN CLOSE 7. Connect the pressure sensor line (luer connector / filter) to the pressure sensor port on the top surface of the S-100. Tighten the luer lock firmly. The protocols OM

112 8. Hang the by-product bags on the hooks provided on the SEPAX S-100. If you use a single-use kit with the Biosafe CryoSC cryobag, let it hang on the front panel of the device. If you use a single-use kit with the Pall Bag, be aware of installing the bag in the right position. If it s is not installed in a proper way, there is a risk of kink between the bag and the line. Please look carefully at the pictures: RIGHT WRONG 9. Close the centrifuge cover and tighten the centrifuge cover lock (screw). 10. Press "ENTER" to validate and begin kit testing. Do not close the separation chamber pit covers until the kit has been positioned in the separation chamber pit and the tubing placed in the optical sensor. To avoid any risk of leakage, the tubing connector on top of the separation chamber should not be rotated once the pit covers have been closed. The protocols OM

113 4.7 Automatic Single-Use Kit test During or on completion of the separation kit test, one or more of the following messages may be displayed: Message Signification Action KIT OK Correct message: The kit is OK to use The procedure will automatically go to the next step OPEN COVER The S-100 pit covers need to be opened to check the cover sensors Open both covers CLOSE COVER The S-100 pit covers need to be closed Close both covers and tighten locking screw VERIFY LINE IN OPTICAL SENSOR The line may be wrongly inserted or is not empty Correct and press ENTER CHAMBER NOT EMPTY, DO PURGE MODE ENTER CHAMBER NOT READY, REDO TESTKIT ENTER CHAMBER NOT READY, REDO TESTKIT ENTER CHAMBER NOT DETECTED REDO TESTKIT ENTER The piston is not at the top of the chamber, which means there is product (or air) in the chamber The chamber is defective or the kit test was not done correctly The chamber is defective or the kit test was not performed correctly The kit is not installed or not installed correctly Press ENTER to execute Purge Mode protocol to empty chamber the separation procedure should then be restarted with the Separation Kit Test Press "ENTER" to acknowledge and reinstall the separation chamber the separation procedure should then be restarted from with the kit test Press "ENTER" to acknowledge then reinstall the kit and restart the kit test as explained in 4.6 and 4.7. If the same reoccurs, replace the kit and return defective kit to Biosafe for exchange TEST FAILURE REDO TESTKIT ENTER The chamber is defective or the kit test was not done correctly The protocols OM

114 4.8 Warning / Error Messages See Section 7 for Error and Warning messages. 4.9 End of procedure At the end of the procedure, the tubing lines still contains some of their respective products and must be stripped in order to optimize cell recovery. Message on the display REMOVE BAGS AND AIR FILTER ENTER ENTER Action Go to next step Remove the bags from their hooks and lay them on the operating table next to the SEPAX unit. Disconnect the pressure sensor (to the left of optical sensor) and then press "ENTER". Strip the lines corresponding to each bag one at a time as prompted. Press the "ENTER" key after each line has been stripped. Ensure that all the clamps are closed and replace caps on spikes (if used). The separation chamber can now be removed from the pit and the single-use kit totally separated from the SEPAX S-100 unit. Message on the display END OF PROCESS CLOSE ALL CLAMPS ENTER Action Exit and go to main menu Message on the display REMOVE KIT ENTER Action Exit and go to main menu To remove the kit from the SEPAX S-100: 1) Remove the kit the stopcock valves from the rotary drive pins 2) Take the line out of the optical sensor 3) Open the separation chamber pit covers 4) Remove the chamber out of the pit 5) Dispose of the kit in a manner that is appropriate for blood products and in accordance with regulations in place in your region At the end of the procedure, the protocol automatically exits and returns to the main menu. All valves are set to their initial position and the SEPAX S-100 is then ready for a new run or to be switched off. If product spillage or leaking occurs the product should be discarded. All parts of the separation procedure should be performed in accordance with the single-use separation kit Instructions for Use (see below). The protocols OM

115 Switching off the SEPAX S-100 In the main menu, select the quit option. Message on the display QUIT UCB HES PURGE MODE (Other protocols) No ENTER ENTER C MENU Yes Action Go to "QUIT" Select "QUIT" When the following message appears on the display, the SEPAX S-100 can be switched off: 4.10 Emergency stop Message on the display TURN OFF SEPAX In an emergency, the procedure can be stopped at any time by pressing the "STOP" key. Message on the display ANY MESSAGE STOP Action To exit procedure press STOP When stopped, the centrifuge drive and the pneumatic systems are switched off. The SEPAX machine goes back to the main menu where PURGE MODE can be selected Other Functions Service, Settings and Data functions can be accessed by pressing the MENU key when MAIN MENU is displayed on the screen. Message on the display PURGE MODE UCB-HES QUIT MENU Action Press MENU key to enter the sub-menu Different functions can be chosen from the sub-menu: The protocols OM

116 Message on the display Action SERVICE SETTINGS SAVE PROTOCOLS DATA SAVE SUMMARY DATA PRINT SUMMARY DATA MENU Yes No ENTER ENTER Go to previous option Go to next option Validate marked option Pressing he MENU key returns the display to the MAIN MENU and the START KIT TEST option Service function The service sub-menu is only to be used by Biosafe qualified service technicians. A four digit pin code is required to access the service options. Select the Service function by pressing ENTER while the arrow is on the Service option. PASSWORD will then be displayed and should be validated by again pressing ENTER. Message on the display PASSWORD ENTER Action Press the ENTER key to enter password Input the service pin code using the UP and DOWN scroll arrows to set the value and ENTER to move the cursor horizontally to the next character. Message on the display Action PASSWORD CODE :???? MENU Yes No ENTER Change digit value ENTER Validate and set next digit The service options are: Message on the display PROTOCOL ACTIVATION ENABLE PRINTER MENU Yes Action Go to previous option The protocols OM

117 No ENTER Go to next option ENTER Select option 4.13 Settings function Message on the display TIME HH:MM:SS DATE DD/MM/YYYY LANGUAGE AUTO PRINT DATA a TRACEABILITY SETUP MENU Yes No ENTER Action Go to previous option Go to next option ENTER Select option To change date and time Press ENTER to select the first character. The selected character will then be displayed between square brackets. Use UP and DOWN to set the value and ENTER to move the cursor horizontally to the next character. To leave the character unchanged, press ENTER only. Message on the display TIME [ HH ]:MM DATE DD/MM/YYYY LANGUAGE MENU Yes No ENTER Action Increase selected digit Decrease selected digit ENTER Validate and / or go to next digit The protocols OM

118 To change language Enter the language menu and select the appropriate option. Message on the display ENGLISH FRENCH (other languages) MENU Yes No ENTER Action Go to previous option Go to next option ENTER Validate marked option To enable automatic printout To have an automatic printout after each procedure, activate the by pressing ENTER, to disable the option place the symbol after the text. Message on the display Action AUTO PRINT DATA ENTER Enable or disable function To enable the traceability option The SEPAX S-100 can be used with 2 different traceability systems. The first option is the a Traceability Kit AS-610 that allows both procedure and protocol traceability and an option to allow the operator to modify a limited number of parameters The printer supplied as part of the Traceability Kit can print protocol and summary procedure data and full protocol and procedure data can be downloaded to the PCMCIA card integrated into the SEPAX S-100. The components of the Traceability Kit are described in Chapter 5. The second traceability is the SepaxNet SN-100 system. SepaxNet is a powerful traceability tool that transfers procedure data presently stored in the Sepax and Coolmix devices to a centralized standard SQL database. Compatible with all new and existing devices, SepaxNet can network up to 20 devices using the latest communication technologies. Data transmission is either by Ethernet cable or WiFi wireless with ISBT 128 compliance. All procedure data is simultaneously sent and stored in the PC database, which are both included in the package. The components of the SepaxNet system are described in Chapter 5. If you are using the SepaxNet SN-100 system, please refer to it s Operator Manual, the OM Enabling of the traceability option requires a password. Biosafe provides traceability option passwords to approved operators. The protocols OM

119 Once the traceability option has been enabled, the Manual ID Input option can also be activated allowing operators of that unit to add additional traceability parameters or skip existing parameters. Enabling of the traceability parameter and the decision to activate Manual ID Input should only be entrusted to administrator operators who fully understand the end-user s internal tracability procedure. Following confirmation of the traceability options, any subsequent change requires the password to be entered again. General rules on traceability Ensure that the traceability option password remains confidential. To ensure high levels of traceability only prepare one procedure at a time do not place barcodes, worksheets or input product collection bags corresponding to different procedures in the same work area Traceability setup step-by-step Message on the display TIME HH:MM:SS DATE DD/MM/YYYY LANGUAGE AUTO PRINT DATA a TRACEABILITY SETUP ENTER Action Select option Select Traceability Setup and press ENTER Message on the display PASSWORD ENTER Action Press ENTER key to enter password Input password provided by Biosafe Use the UP and DOWN arrow keys to set the value, and ENTER to move the cursor horizontally to the next character. Message on the display Action PASSWORD CODE :???? MENU Yes No ENTER Change digit value ENTER Validate and set next digit Press ENTER and the following options will be displayed. The protocols OM

120 Message on the display Action ENABLE TRACE ID MANUAL ID INPUT a a MENU Yes Go to previous option No ENTER Go to next option ENTER Activate or deactivate ENABLE TRACE ID a If athe ENABLE TRACE ID is selected, traceability is enabled and the operator can only add protocol data using the barcode reader. MANUAL ID INPUT a If athe MANUAL ID INPUT is activated, the operator can: - Enable or disable the different protocol data traceability IDs using CONFIGURE TRACE ID. - Skip any of the proposed IDs - Enter the ID on the keyboard - Print a barcode for an ID by pressing START when the ID is displayed the barcode is printed on the thermal printer included in the Traceability Kit. The following flow-chart describes how the traceability option functions within the protocol The protocols OM

121 67$57830( &2/ ',63/$<35272&2/1$0( 0$,10( &2/ &+$1*(3$5$0(7( 56 &21),* 85(75$&(,' 67$57352&( '85( 75$&( $%,/,7<(1$%/(' 75$&( $%,/,7<',6$%/(' (17( 575$&(,' 23(1,1387%$*&/ $03 $8720$7,&.,77( 67 $8720$7,&352&( '85( 35,175$ Configure traceability ID The traceability parameters to be used during the procedure are displayed following the kit test at the beginning of a procedure. Message on the display Action BC BAG PLASMA BAG a a MENU Yes Go to previous option RBC BAG OPERATOR CENTER a a No ENTER ENTER Go to next option Activate or deactivate The protocols OM

122 a : ID activated. : ID not activated. Press the MENU key to return to the main menu Edit traceability ID Allows traceability IDs values to be modified during a protocol Traceability IDs can only be modified if in SETTINGS/TRACEABILITY configuration the traceability was enabled and Manual ID Input selected: ENABLE TRACE ID a MANUAL ID INPUT a Following the kit test the SEPAX S-100 will require that each selected parameter (ID) be completed either manually or using the barcode reader : Manual Input: Once ENTER has been pressed two brackets [ ] will appear, between which the required ID characters should be inserted using UP and DOWN arrows to select the required digit. To confirm press ENTER. Message on the display BC BAG [A] MENU Yes Action Increase selected digit No ENTER Decrease selected digit The protocols OM

123 ENTER Validate and / or go to next digit Barcode Reader Input: The barcode reader inserts the traceability ID automatically and there is no need to press ENTER to confirm. Once a traceability ID has been entered, use the UP and DOWN scroll keys to move to the next traceability parameter. To skip a traceability ID without entering a value, press the DOWN key. Note: To print a traceability barcode, press START when the traceability ID is displayed. A maximum barcode of seven characters can be printed. To return to the main menu: Place ENTER = CONTINUE then ENTER : ENTER = CONTINUE OPERATOR S ID The IDs have now been entered and the separation procedure can be started Saving sub-menu data options (protocol) The protocol data sub-menu contains statistical information on the procedure, including:. date and time of processing. input and final unit volumes. line pressure maximum and minimum values These values ensure that the separation process has been performed correctly and that the product has suffered no extreme conditions. The protocol data sub-menu provides more technical information, including:. sensors and actuator recordings (every 200 ms). valve positions. power supply data These files are used by Biosafe technicians to diagnose the cause of malfunctions or low performance procedures. On the rear panel of the SEPAX S-100 is a data card port that enables the downloading of procedure and/or protocol data to a PCMCIA memory card. The PCMCIA card can store up to 50 procedure files (CB unit data) and 8 protocol files (Sepax operating data). Protocol files only need to be archived if an incident occurred during processing this file can be sent to Biosafe for analysis. The download procedure is explained below. Ensure that a PCMCIA data card is inserted before starting the procedure Introduce the memory card in the PCMCIA slot. Correct insertion of the PCMCIA card is indicated by the release of a black clip on the card slot. Ensure that this clip is released before continuing the procedure. The protocols OM

124 Once either SAVE PROTOCOL DATA or SAVE SUMMARYDATA has been chosen from the main menu, a list of files is displayed along with two options: "SAVE ALL FILES" and "SAVE SELECTED FILES". The files are identified by the time and date they were created (corresponding to the end of the procedure). All files can be saved simultaneously, using the SAVE ALL FILES option, Message on the display HH:MM DD/MM/YYYY HH:MM DD/MM/YYYY SAVE ALL FILES SAVE SELECTED FILES No ENTER ENTER C MENU Yes Action Go to "save all files" Select current file Alternatively, individual files can be saved by confirming SAVE SELECTED FILES and pressing ENTER when the arrow is next to the desired file (an asterisk is displayed to identify the chosen file). Message on the display HH:MM DD/MM/YYYY HH:MM DD/MM/YYYY SAVE ALL FILES SAVE SELECTED FILES MENU Yes No ENTER Action Go to previous file Go to next file ENTER C Select current file Print summary data option The most recent protocol file can be printed to the supplied printer by pressing ENTER when the message PRINT SUMMARY DATA is on the first line of the display. To enable automatic printouts after each procedure, refer to Settings Reminder: If the printer is not installed, these options are not displayed Software update/upgrade Updating and/or upgrading the SEPAX S-100 with new protocol revisions are performed using a PCMCIA card supplied by Biosafe. This operation should only be performed by an approved Biosafe technician. The protocols OM

125 PERIPHERAL BLOOD STEM CELL VOLUME REDUCTION PROTOCOL (PBSC) PBSC OM 4A 07 4A - 1

126 USER INSTRUCTIONS: Peripheral Blood Stem Cell Volume Reduction Protocol General description The "PBSC - Volume Reduction" protocol is designed for volume reduction by plasma depletion of peripheral blood stem cell (PBSC) preparations after leukapheresis. The hematopoietic stem cells can then be further processed for freezing and storage until required for human transplantation. The "PBSC - Volume Reduction" protocol allows for the processing of samples from ml using multiple cycles if required and aims for maximal volume reduction (typically in the order of 70-80%) with minimal cell loss. The final volume is a function of the cell concentration and the volume of the initial sample, and can be selected between ml. At the end of the program, the separation chamber is rinsed with plasma in order to maximize cell recovery. Centrifugation speed (rpm) Chamber filling Acceleration Sedimentation Deceleration Plasma extraction Cells extraction Chamber rinsing Time (min) Process diagram Specification Final product volume Accuracy Duration of the process Adjustable from 20 to 150 ml +/- 2 ml Volumes up to 220 ml: 1 cycle 15 min Volumes between 220 and 440 ml: 2 cycles 20 min Volumes between 440 and 660 ml: 3 cycles 25 min PBSC OM 4A 07 4A - 2

127 Flow chart for the SEPAX PBSC protocol START PROCEDURE PARAMETER MODIFICATION FINAL VOLUME SETTING KIT INSTALL ATION & TEST CHAMBER FILLING SEDIMENTATION Repeated cycle if volume > 220 ml PLASMA EXTRACTION CELLS EXTRACTION CHAMBER RINSING DISPLAY: FINAL VOLUME = xx ml END OF PROCESS PBSC OM 4A 07 4A - 3

128 Step 1: Connecting the PBSC unit to the Biosafe kit For the PBSC bag connection to the input line, refer to chapter 4.5. Step 2: Parameter modification and trace ID configuration The device should now be switched on, the machine settings chosen and the desired protocol selected. If these steps have not been completed, please go back to the beginning of chapter 4. If you would like to set a desired final volume, choose CHANGE PARAMETERS in the menu displayed. If you would like to continue without changing the parameter you should go to step 3: Kit installation without pressing any buttons. Message on the display START KIT TEST CHANGE PARAMETERS MAIN MENU CONFIGURE TRACE ID MENU Ye s No ENTER Action Go to previous option Go to next option ENTER Validate marked option Final volume parameter The final volume represents the volume that shall be collected as the final product in the cryobag. This value depends on the initial cell number, i.e. the volume and the cell concentration of the initial sample, and the final cell concentration after processing. The recommended value of this parameter can be found in the table on page 4A-5. The value can be chosen between 20 and 150 ml; the default value is 50 ml. Using a value above the recommended value will produce a less concentrated final product. If the real WBC volume happens to be larger than the chosen value, the machine will automatically collect the whole WBC product, modifying the final volume. PBSC OM 4A 07 4A - 4

129 Use the UP and DOWN arrow keys to select the parameter to change, then ENTER to enter edit mode. The first character will be displayed between square brackets. Use "UP" and "DOWN" arrow keys to set the value, and "ENTER" to move the cursor horizontally to the next character. Pressing "ENTER" alone leaves the value unchanged. If a value exceeds the upper or lower limits, the maximum/minimum value is automatically selected. Message on the display Action MENU Yes Increase selected digit FINAL VOLUME [0]50 ml No ENTER ENTER Decrease selected digit Validate and / or move to next digit MENU Cancel your input or exit the submenu When the parameter has the desired value, press return to the kit installation. MENU to exit the submenu and Remark: to end parameter s validation, always get with the cursor to the last character before pressing MENU to exit. Configure trace ID If you have chosen the traceability option, refer to chapter to select which ID to use. PBSC OM 4A 07 4A - 5

130 Minimum recommended volume for PBSC- Volume reduction protocol Calculated for a maximum cell concentration of WBC/ml Minimum recommended volume (ml) Input volume (ml) Input concentration (*10 6 WBC/ml) [WBC] in Vol in (ml) *10 6 /ml *10 6 /ml *10 6 /ml *10 6 /ml *10 6 /ml *10 6 /ml *10 6 /ml Warning: The cell concentration used to establish this table, was defined by the engineers to achieve perfect functionality of the machine and it is not related to the maximum cell concentration accepted by clinicians. PBSC OM 4A 07 4A - 6

131 Step 3: Kit installation For the kit installation procedure, refer to chapter 4.6. After the kit installation, the machine performs an automated kit test, as explained in chapter 4.7. Step 4: Enter the traceability IDs If you have chosen the traceability option, enter the IDs manually or with code bar reader as explained in chapter Step 5: Kit priming For proper operation, the tubing line between the source bag and the stopcocks has to be primed with the PBSC product. For priming the line is open towards the plasma bag; thus, in case the blood goes too far, it will be collected in the plasma bag. Message on the display PRIME BUBBLE CHAMBER ENTER Action Squeeze the bubble chamber until it is half full. ENTER Go to next step Message on the display OPEN INPUT BAG CLAMP ENTER Action Open the roller clamp so the blood can flow towards the stopcocks. ENTER Go to next step Message on the display Action PRIMING UP KEY TO BEGIN MENU Ye s Starts priming Pressing the "UP" arrow key once opens the left and right stopcocks so that blood can flow along the tubing and fill it. Message on the display Action PRIMING UP KEY TO STOP MENU Ye s Stops priming When blood arrives at the level of the left stopcock, press UP again to close the line and stop the priming. PBSC OM 4A 07 4A - 7

132 If the blood goes further than the middle stopcock, it is possible to push it back into the initial bag manually. Lower the initial bag by hanging it down, lift up the valve and turn the stopcocks in the direction which permits the blood to flow back into the initial bag; push back the blood and go back to the main menu. Step 6: Automated procedure Avoid touching the machine and the kit during automated procedure. Moving bags, tubes, stopcocks and covers may cause errors requiring the process to be restarted. Next step is to confirm successful priming and start the protocol. If you would like to continue priming, choose REDO PRIMING. Message on the display Action START PBSC REDO PRIMING MAIN MENU MENU Ye s No ENTER Go to previous option Go to next option ENTER Validate marked option The automated procedure has now started. During the protocol, the current phase is shown on the first line of the display. The PBSC protocol goes through the following phases: 1. CHAMBER FILLING VOLUME =... ml 3. SEDIMENTATION 5. PLASMA EXTRACTION VOLUME = 4 ml 7. DECELERATING VOLUME =... ml 9. CELLS EXTRACTION VOLUME =... ml 11. MIXING 2. ACCELERATING VOLUME =... ml 4. DECELERATING VOLUME =... ml 6. PLASMA EXTRACTION VOLUME =... ml 8. MIXING 10. CHAMBER FILLING VOLUME =... ml 12. CHAMBER RINSING VOLUME =... ml Remark: If the initial volume is larger than 220 ml, steps 1-7 will be repeated until the initial bag is empty. At the end of the procedure, the Sepax emits a series of tones, indicating the end of the procedure. PBSC OM 4A 07 4A - 8

133 Step 7: End of procedure To follow all the steps for the end of the procedure and to exit protocol, refer to chapter 4.9. PBSC OM 4A 07 4A - 9

134 PBSC OM 4A 07 4A - 10

135 4B. UMBILICAL CORD BLOOD WITH HES PROTOCOL (UCB-HES) UCB-HES OM 4B 10 4B - 1

136 USER INSTRUCTIONS : UCB-HES PROTOCOL General description and performance The HES protocol is designed for routine processing of umbilical cord blood (UCB) to isolate the buffy-coat fraction enriched in haematopoietic stem cells and requires the addition of HES (hydroxyethyl starch). The protocol allows processing of initial CB unit volumes above 35ml and below 320ml (anti-coagulant included, after HES addition) with optimal performance with units of between 80ml and 180ml. The volume reduction of UCB is performed in 25 to 35 min and results in a predetermined volume between 20 and 50 ml. Initial product inclusion criteria Optimal results can be expected with UCB units meeting the criteria listed below: - UCB units should be less than 96 hours old - no visible major clots in UCB unit to be processed - UCB unit at room temperature before connection to the single-use kit and processing Step 1: HES addition Refer to chapter 3 to find the instructions for the kit you are using. Step 2: Connecting the cord blood input bag to the single-use kit Refer to Section 4.5 of this document. If the kit you are using doesn t have a cryobag, you should connect one in sterile condition. Close the clamp on the DMSO injection line and between the cryogenic and transfer bag (if applicable). Step 3: Parameter modification and trace ID configuration Configure traceability ID Refer to Section to select which ID to use. Parameter Modification Certain non-critical parameters of the procedure can be modified by the operator prior to the KIT TEST. To modify parameters select CHANGE PARAMETERS. Use the UP and DOWN arrow keys to select the parameter to change, then press ENTER to edit the value(s). The first character will be displayed between square brackets. Use "UP" and "DOWN" arrow keys to set the value then press "ENTER" to move the cursor horizontally to the next character. To leave a value unchanged, press "ENTER". If no parameter needs modifying go directly to Step 4 KIT INSTALLATION without pressing any keys. UCB-HES OM 4B 10 4B - 2

137 Message on the display Action START PROCEDURE CHANGE PARAMETERS MAIN MENU CONFIGURE TRACE ID MENU Yes No ENTER Go to previous option Go to next option ENTER C Validate marked option Final volume parameter Parameter value can be set between 20 and 50 ml with a default value of 20 ml. If a value exceeds the upper or lower limits, the maximum/minimum value is automatically selected. Message on the display Action MENU Yes Increase selected digit FIXED VOLUME [2]0.00 ml No ENTER ENTER C Decrease selected digit Validate and / or move to next digit PRG Cancel your input or exit the submenu Once the desired parameter value is indicated press MENU to exit the submenu and return to the kit installation step. Note: to complete validation of a parameter the cursor must be to on the last character before pressing MENU to exit. HES percentage parameter HES percentage can be indicated between 20% and 40% - the default value is 20%. UCB-HES OM 4B 10 4B - 3

138 RBC extraction flag parameter The operator can choose whether the red blood cells (RBC) should be returned to the UCB input bag or remain in the separation chamber at the end of the procedure. With the RBC extraction flag set to 1 the RBCs are transferred to the UCB input bag. If the flag is set to 0, the RBCs will remain in the chamber in the chamber. Small Volume Factor parameter In the parameters list you can set the Small Volume Factor between 0.0 and 1.0, the default value is 0.8. That factor triggers the small volume strategy. When the total volume of cells is smaller than SmallVolumeFactor * FinalVolume, the SmallVolume strategy occurs. The default value of 0.8 for the SmallVolumeFactor gives the best theoretical results; it is advised not to change it. The influence of that parameter on the performances of the procedure is described in the next paragraph. UCB-HES OM 4B 10 4B - 4

139 Small Volume Strategy The Small Volume (SV) strategy consists of the extraction of the whole cellular part of the initial unit (after concentration) to the BC bag and the subsequent rinsing of the separation chamber. That implies that the cellular part of the initial product has to be lower that the final volume. The SV parameter defines the minimum amount of plasma used for the rinsing of the chamber. The following chart indicates the theoretical maximum cell recovery and final HCT for a given cellular volume (in the initial unit). Please note that there are 4 ml that stay into the separation chamber, so the volume represented is up to 24 ml (for a standard extracted volume of 20 ml). The chart shows that by increasing the value of the parameter, the range for the small volume strategy is increased but the haematocrit of the final product is also increasing and is higher than the physiological range. Final HCT [%] HCT R e c o v e r y Volume of Cells [ml] Theoretical performances for the Small Volume strategy Cell Recovery [%] Note: The small volume strategy is applied when the total volume of the cells (white + red) is smaller than the product of the SmallVolumeFactor and the Output Volume. The plasma is extracted and to the initial input bag and all the volume remaining in the chamber (buffy-coat/red cell mix) is sent to the buffy-coat bag. There will be no red cells returned to the initial input bag and the plasma will remain in the initial input bag. A small amount of plasma will be used to rinse the chamber and ensure that a maximum number of cells are collected in the BC bag. UCB-HES OM 4B 10 4B - 5

140 Normal strategy Small volume strategy Input/RBC bag Plasma bag Input/RBC Input/Plasma bagbag Plasma Empty Bag bag BC bag BC bag The small volume strategy is triggered automatically without any user intervention. When that strategy is applied, the plasma is extracted to the input bag where a small quantity of red cells remains. It means it s not possible to get a pure plasma sample. To avoid adoption of the Small Volume strategy set the SmallVolumeFactor to 0.1. Step 4: Kit installation For the kit installation procedure, refer to chapter 4.6 and 4.7. After the kit installation, select in the menu the entry START PROCEDURE and press ENTER. Step 5: Enter the traceability IDs Refer to Section Step 6: Input bag clamp opening Following input of the traceability IDs, the system requires the operator to open the input bag clamp. Message on the display OPEN INPUT BAG CLAMP ENTER Action Open the input bag clamp so the UCB can flow towards the stopcocks. ENTER Start automated kit test After pressing ENTER the automated procedure begins with the automated kit test. Step 7: Automated kit test An automatic kit test is performed. It will test the kit and it s proper installation on the device. In case of error, the device will beep and the operator should check the message on the display so to fix the error. At the end of the test, the protocol waits 12 seconds before beginning the priming. That delay allows the device to stabilize the pressure in the chamber. UCB-HES OM 4B 10 4B - 6

141 Step 8: Automated priming To ensure optimum evacuation of the air from the system, automated priming is an integrated part of the protocol. The system will prime the bubble chamber, the line and the separation chamber automatically before starting the separation process. It will also empty the air from the BC Bag Approximately 35 ml of the input product will be drawn into the separation chamber and then returned to the UCB input bag. Step 9: Automated procedure Avoid touching the SEPAX S-100 during processing. Do not place any other equipment near the SEPAX S-100 that may cause vibration this will affect the quality of separation Avoid moving bag, tubes, stopcocks and covers during the procedure as this may cause an error and require the procedure to be re-started. Note: The purge mode allows an aborted procedure to be restarted and the cord blood unit reprocessed one time with minimal loss of cells. After the automated priming, the SEPAX S-100 display will indicate the processing step that is being performed at any given time. The processing steps for the UCB HES protocol are listed below: 1) Automated priming 2) Chamber filling 3) Sedimentation 4) Plasma extraction 5) Buffy coat extraction 6) RBC extraction 1 7) Chamber/Plasma filling 1 8) Sedimentation 1 9) Plasma extraction 1 10) Buffy coat extraction 1 11) RBC extraction 2 1 Depending on the total input volume these steps may be eliminated. This will not affect the efficacy of the separation procedure. 2 If RBC extraction flag is set to 1 If the input volume (volume of CB in the collection bag) is greater than 220ml (the volume of the separation chamber), 2 cycles are performed. During the 1st cycle the product remaining in the input bag may begin to sediment and so the operator will be required to mix the input bag contents before the 2nd cycle can begin. At the end of the 1st cycle, an alarm will sound requiring the operator to press validate (by pressing ENTER) that the input bag has been mixed to a homogeneous state. The 2nd cycle of the protocol will then begin and separation process will be completed with no further operator intervention. At the end of the procedure, the SEPAX S-100 will sound an alarm a number of times to indicate the end of the procedure. UCB-HES OM 4B 10 4B - 7

142 Step 10: End of procedure Refer to chapter 4.9 of the Operator s Manual. Warning / Error messages Refer to Section 7 of Operator s Manual. UCB-HES OM 4B 10 4B - 8

143 4C. UMBILICAL CORD BLOOD PROTOCOL (UCB) UCB OM 4C 10 4C - 1

144 USER INSTRUCTIONS : UCB PROTOCOL General description and performance The UCB protocol is designed for routine processing of umbilical cord blood (UCB) to isolate the buffy-coat fraction enriched in haematopoietic stem cell. That protocol does not require the addition of any sedimentation agent. The protocol allows processing of initial UCB unit volumes above 35ml and below 320* ml (anti-coagulant included). The volume reduction of UCB is performed in 25 to 35 min and results in a predetermined volume between 10 and 50 ml. Initial product inclusion criteria Optimal results can be expected with UCB units meeting the criteria listed below: - UCB units should be less than 72 hours old - no visible major clots in UCB unit to be processed - UCB unit at room temperature before connection to the single-use kit and processing - >= 20 ml of output volume Step 1: Connecting the cord blood input bag to the single-use kit Refer to Section 4.5 of this document. If the kit does not have an integrated cryobag, one should be connected under sterile conditions. Close the clamp on the DMSO injection line between the mixing and cryobag (if applicable). Step 2: Parameter modification and trace ID configuration Configure traceability ID Refer to Section to select which ID to use. Parameter Modification Certain non-critical parameters of the procedure can be modified by the operator prior to the START PROCEDURE. To modify parameters select CHANGE PARAMETERS. Use the UP and DOWN arrow keys to select the parameter to change, then press ENTER to edit the value(s). The first character will be displayed between square brackets. Use "UP" and "DOWN" arrow keys to set the value then press "ENTER" to move the cursor horizontally to the next character. To leave a value unchanged, press "ENTER". If no parameter need modifying go directly to Step 4 KIT INSTALLATION without pressing any keys. UCB OM 4C 10 4C - 2

145 * Depending on initial haematocrit Message on the display Action START PROCEDURE CHANGE PARAMETERS MAIN MENU CONFIGURE TRACE ID MENU Yes No ENTER Go to previous option Go to next option ENTER C Validate marked option Final volume parameter Parameter value can be set between 10 and 50 ml with a default value of 20 ml. If a value exceeds the upper or lower limits, the maximum/minimum value is automatically selected. Message on the display Action MENU Yes Increase selected digit BUFFYCOAT VOLUME [2]0.00 ml No ENTER ENTER C Decrease selected digit Validate and / or move to next digit PRG Cancel your input or exit the submenu Once the desired parameter value is indicated press MENU to exit the submenu and return to the kit installation step. Note: to complete validation of a parameter the cursor must be to on the last character before pressing MENU to exit. UCB OM 4C 10 4C - 3

146 Reduced Kit Flag parameter The Reduced Kit Flag parameter must be set to 1 if you use a reduced kit, like the CS-470.x or CS-570.x otherwise it must be set to 0. RBC extraction flag parameter The operator can choose whether the red blood cells (RBC) should be returned to the UCB input bag or remain in the separation chamber at the end of the procedure. With the RBC extraction flag set to 1 the RBCs are transferred to the UCB input bag. If the flag is set to 0, the RBCs will remain in the chamber. When the Reduced Kit flag and the RBC extraction flag are both set to 1, then the plasma and the RBCs are extracted in the initial bag. Sensitivity That protocol is equipped with a smart BC detection feature which sensitivity can be set do different levels. The higher the sensitivity is, the higher the TNC recovery (mainly GRC) but also resulting in a higher final HCT. This parameter also permits to use this protocol to process other products, like bone marrow. This parameter can be set to: 1: Low (lower HCT) 2: Medium (default) 3: High (higher HCT) Small Volume Factor parameter In the parameters list you can set the Small Volume Factor between 0.0 and 1.0, the default value is 0.8. That factor triggers the small volume strategy. When the total volume of cells is smaller than SmallVolumeFactor * FinalVolume, the SmallVolume strategy occurs. The default value of 0.8 for the SmallVolumeFactor gives the best theoretical results; it is advised not to change it. The influence of that parameter on the performances of the procedure is described in the next paragraph. UCB OM 4C 10 4C - 4

147 Small Volume Strategy The Small Volume (SV) strategy consists of the extraction of the whole cellular part of the initial unit (after concentration) to the BC bag and the subsequent rinsing of the separation chamber. That implies that the cellular part of the initial product has to be lower that the final volume. The SV parameter defines the minimum amount of plasma used for the rinsing of the chamber. The following chart indicates the theoretical maximum cell recovery and final HCT for a given cellular volume (in the initial unit). Please note that there are 4 ml that stay into the separation chamber, so the volume represented is up to 24 ml (for a standard extracted volume of 20 ml). The chart shows that by increasing the value of the parameter, the range for the small volume strategy is increased but the haematocrit of the final product is also increasing and is higher than the physiological range. Final HCT [%] HCT R e c o v e r y Volume of Cells [ml] Cell Recovery [%] Theoretical performances for the Small Volume strategy Note: The small volume strategy is applied when the total volume of the cells (white + red) is smaller than the product of the SmallVolumeFactor and the Output Volume. The plasma is extracted and to the initial input bag and all the volume remaining in the chamber (buffy-coat/red cell mix) is sent to the buffy-coat bag. There will be no red cells returned to the initial input bag and the plasma will remain in the initial input bag. A small amount of plasma will be used to rinse the chamber and ensure that a maximum number of cells are collected in the BC bag. UCB OM 4C 10 4C - 5

148 Normal strategy Small volume strategy Reduced Kit Input/By-product bag 1 Plasma bag 4 Input/By-product bag 1 Empty Bag 4 Input/By-product bag 1 2 BC bag 2 BC bag 2 BC bag The small volume strategy is triggered automatically without any user intervention. When that strategy is applied, the plasma is extracted to the input bag where a small quantity of red cells remains. It means it s not possible to get a pure plasma sample. To avoid adoption of the Small Volume strategy set the SmallVolumeFactor to 0.1. Step 3: Kit installation For the kit installation procedure, refer to chapter 4.6 and 4.7. After the kit installation, select in the menu the entry START PROCEDURE and press ENTER. Step 4: Enter the traceability IDs Refer to Section Step 5: Input bag clamp opening Following input of the traceability IDs, the system requires the operator to open the input bag clamp. Message on the display OPEN INPUT BAG CLAMP ENTER Action Open the input bag clamp so the UCB can flow towards the stopcocks. ENTER Start automated kit test After pressing ENTER the automated procedure begins with the automated kit test. Step 6: Automated kit test An automatic kit test is performed. It will test the kit and its proper installation on the device. In case of error, the device will beep and the operator should check the message on the display so to fix the error. UCB OM 4C 10 4C - 6

149 Step 7: Automated priming To ensure optimum evacuation of the air from the system, automated priming is an integrated part of the protocol. The system will prime the bubble chamber, the line and the separation chamber automatically before starting the separation process. It will also empty the air from the BC Bag Approximately 35 ml of the input product will be drawn into the separation chamber and then returned to the UCB input bag. Step 8: Automated procedure Avoid touching the SEPAX S-100 during processing. Do not place any other equipment near the SEPAX S-100 that may cause vibration this will affect the quality of separation Avoid moving bag, tubes, stopcocks and covers during the procedure as this may cause an error and require the procedure to be re-started. Note: The purge mode allows an aborted procedure to be restarted and the cord blood unit reprocessed one time with minimal loss of cells. After the automated priming, the SEPAX S-100 display will indicate the processing step that is being performed at any given time. The processing steps for the UCB protocol are listed below: 1. Automated priming 2. Chamber filling 3. Sedimentation 4. Plasma extraction 5. Buffy coat extraction 6. Chamber Rinsing 3 7. RBC extraction 1 8. Chamber/Plasma filling 1 9. Sedimentation Plasma extraction Buffy coat extraction RBC extraction 2 1 Depending on the total input volume these steps may be bypassed. This will not affect the automated separation procedure. 2 Depending on the RBC extraction flag 3 Only in case of SmallVolume strategy, there is a chamber rinsing step, and then the procedure ends At the end of the procedure, the SEPAX S-100 will sound an alarm a number of times to indicate the end of the procedure. UCB OM 4C 10 4C - 7

150 Step 9: End of procedure Refer to chapter 4.9 of the Operator s Manual. Warning / Error messages Refer to Section 7 of Operator s Manual. UCB OM 4C 10 4C - 8

151 UCB LOW BUFFY-COAT VOLUME PROTOCOL (UCB-LBC) UCB-LBC OM 4D 05 4D - 1

152 USER INSTRUCTIONS: UCB-LBC General description This protocol is used for processing of umbilical cord blood in order to isolate the buffy-coat fraction enriched in hematopoietic stem cells. This protocol has been specially developed to collect an output volume of 8 ml. Step 1: Connecting cord blood unit to the Biosafe kit For the cord blood bag connection to the input line, refer to chapter 4.5. Open the filter cap on the collection vial and verify that the clamp on the collection vial line is open. Label the collection vial in order to prevent sample loss. Step 2: Parameter modification and trace ID configuration If you would like to set a desired final volume, choose CHANGE PARAMETERS in the menu displayed. If you would like to continue without changing the parameter go to Step 4: KIT INSTALLATION without pressing any buttons. Message on the display START KIT TEST CHANGE PARAMETERS CONFIGURE TRACE ID MAIN MENU MENU Yes No ENTER Action Go to previous option Go to next option ENTER Validate marked option Choose the parameter to change using the UP and DOWN arrow keys. Press ENTER to select the first character, which will be displayed between square brackets. Use "UP" and "DOWN" to set the value, and "ENTER" to move the cursor horizontally to the next character. Pressing "ENTER" alone leaves the value unchanged. UCB-LBC OM 4D 06 4D - 2

153 Message on the display Action FINAL VOLUME [0]8.50 ml MENU Yes Increase selected digit No ENTER Decrease selected digit ENTER Validate and / or move to next digit PRG Cancel your input Setting the final volume The final volume represents the volume that shall be collected as the final product in the collection vial. The value can be chosen between 7.00 and 20.00ml, the default value is 8.50 ml. The protocol was designed to collect 8.5 ml final product. Increasing too much the final volume could lead to an increment of product haematocrit that might put at risk the cells during freezing procedure. Setting the output tubing volume The output tubing volume represents the volume contained into the tubing between the stopcock and the collection vial/bag. This volume is a rejected volume and by default is adapted to Biosafe s kit CS-470.x. Please call Biosafe to know the adapted parameter to enter in this field in the case you are using a different kit. The value can be chosen between 1.0 and 9.9 ml, the default value is 1.3 ml. Setting the smart BC detection parameter This protocol is equipped of a special detection function that automatically increases the buffy-coat volume in case of a low output haematocrit (and NC recovery). This increment is done by step of 4 ml starting from the selected output volume. The sensitivity of the optical detection regulates the start/end extraction of the buffycoat. Sensitivity can be chosen between 3 pre-regulated values. Set this parameter to 0 to deactivate this function or 1 to 3 to activate it with following options: 1. permissive 2. medium 3. strict The permissive level will admit a lower output haematocrit than the strict level. In the other hand, the output volume will be increased more often using the strict level setting than using the permissive level setting. Users can set one of these 3 levels according to their specific needs. By default this function is activated in permissive mode (1). UCB-LBC OM 4D 06 4D - 3

154 Setting the RBC extraction parameter This protocol is equipped of an option which permits to extract the RBC fraction contained into the chamber after the separation process. Set 1 to extract the RBC fraction to the initial bag (the erythrocytes are mixed with the plasma) or 0 to keep it into the separation chamber. By default this function is deactivated (0). When the parameters have the desired values, press and return to the kit installation option. PRG to exit the submenu Configure trace ID If you have chosen the traceability option, refer to the chapter to select which ID to use. Step 3: Kit installation For the kit installation procedure, refer to chapter 4.6. Enter the traceability IDs: if you have chosen the traceability option: enter the IDs manually or with code bar reader as explained in chapter After the kit installation, the machine performs an automated kit test, as explained in chapter 4.7. Step 4: Automated procedure Avoid touching the machine and the kit during automated procedure. Moving bag, tubes, stopcocks and covers may cause errors and the process must be restarted. After the successful test of kit, the automated procedure will start by itself and will go through the following phases: 1. PRIMING VOLUME = ml 3. ACCELERATING VOLUME =... ml 5. DECELERATING VOLUME =... ml 7. BC EXTRACTION VOLUME =... ml 9. DECELERATING VOLUME =... ml 2. CHAMBER FILLING VOLUME =... ml 4. SEDIMENTATION 6. PLASMA EXTRACTION VOLUME =... ml 8. RBC EXTRACTION VOLUME =... ml UCB-LBC OM 4D 06 4D - 4

155 The phase 8 is optional and can be deactivated. See Step 2: Parameter modification and trace ID configuration for more details. Step 5: End of procedure When the process is terminated, the following message with an approximate value of the final volume is displayed: Message on the display PRODUCT VOLUME = 8ml REMOVE KIT. ENTER ENTER Action Exit from protocol At the end of the procedure you need to separate the collection vial from the rest of the kit in order to continue with the freezing procedure. To do that, close all clamps and seal 3 times the collection vial tubing near the vial. Cut the tubing on the central sealing. The product still contained in the tubing has been taken into account for the volume calculation. It is not necessary to purge or strip the tube. After sealing, dismount the rest of the kit as follows: 1) Take down the bag and place it on the operating table. 2) Remove the filter that connects to the pressure sensor (to the left of the optical sensor). 3) Lift the stopcock valve from the rotary pin. 4) Take the line out of the optical sensor. 5) Open the covers. 6) Lift the chamber out of the centrifuge pit. 7) Place the kit in a bin suitable for blood products. Mix well the product in the collection vial and take a sample for quality control under aseptic conditions (laminar flow) following your validated procedure. Always following your validated procedure and under aseptic conditions, add slowly DMSO into the collection vial. Make sure that the product temperature is maintained (for example place vial in ice). After the cryoprotectant addition, aspirate the product from the vial and place it in the freezing vial(s). Freeze samples in the shortest time possible (sample with DMSO should not wait more than 10 minutes before being frozen). The quality of the collected buffy coat must be controlled prior to freezing and transplantation, following a validated procedure. If blood spillage or leaking occurs, the product should be discarded. The collection vial included into the Biosafe kit CS-470.x is NOT adapted to be frozen. NEVER use it for cell conservation. To follow the other steps for the end of the procedure and to exit protocol, refer to chapter 4.7. UCB-LBC OM 4D 06 4D - 5

156 UCB-LBC OM 4D 06 4D - 6

157 PERIPHERAL BLOOD STEM CELL POST THAWING WASHING PROTOCOL (PBSC WASHING) PBSC Washing OM 4E 06 4E - 1

158 USER INSTRUCTIONS: Peripheral Blood Stem Cell Washing Protocol Post Thawing General description The cell washing protocol is designed for the routine wash of thawed PBSC products. It performs a supernatant extraction and re-suspension of the cells with the help of a washing solution (also called buffer). It is based on the manual washing method proposed by Pablo Rubinstein et al. (Proc Natl Acad Sci U S A Oct 24; 92(22): ). Two different working procedures are described. The first one requires the use of a Sterile Connection Device (SCD) to perform sterile connections outside the laminar flow. The second describes what happens when no SCD is available and connections must be performed under controlled environment (laminar flow). It is strongly recommended to use procedure with the SCD to minimize the time between thawing and the beginning of the washing procedure. Workflow with SCD (recommended) BEGIN 1. Prepare washing solution Workflow without SCD 2. Prepare kit 3. Parameters modification 5. Kit installation 4. Thawing of the unit* 6. Automated kit test 7. Automated kit priming 8. Thawing of the unit* 4. Thawing of the unit* 9. Automated washing 3. Parameters check Another bag? 3. Parameters check Second washing cycle 10. Dismount kit END * The thawing of the unit is performed when the kit is already primed (if a SCD is used) or before kit installation (if the initial bag is connected under the laminar flow) The protocol accepts initial volumes of up to 440 ml and provides a final volume between 50 ml and 200 ml. (adjustable by the user). The kit designed for this protocol allows the connection of up to 2 input and 2 output bags. The bags will be processed sequentially (one after the other). Before beginning a washing procedure, be sure that all material is available. Also be ready for a possible manual recovery procedure of the unit. DO NOT thaw the unit before it is necessary (refer to the chart on this page).only users trained on this protocol by BIOSAFE or by an official trainer are authorized to perform the washing procedure. PBSC Washing OM 4E 06 4E - 2

159 Step 1: Preparing washing solution bags To prepare the washing solution, some details about the process need to be known: number of bags to process (maximum 2 per kit), volume in each bag, desired final volume, dilution ratio and number of output bags (maximum 2 per kit). Client s head of process has the responsibility for the choice of the washing solution and its validation. The following graph shows the volume of washing solution per bag needed to perform a washing procedure. Simply chose the input volume on the horizontal scale and intercept the line of the corresponding output volume and application (standard or high wash). For example to wash a 150 ml input bag and obtain 100 ml output product after a high wash cycle, it is necessary to prepare 500 ml of washing solution (1 liter for 2 bags). Washing solution volume required per bag [ml] High w as h Vout = 150ml Vout = 100ml Vout = 50ml 500 Standard w ash Vout = 150ml Vout = 100ml Vout = 50ml Input volume [ml] This graph is applicable for the most common cases. The dilution factor used is 1 and it indicates the ratio between the initial volume and the washing solution volume used for the first dilution. When 1 is selected the volume used for the first dilution will be the same as the input volume. Alternatively, the following formulas can be used (washing solution volume per bag): Vwashing _ solution = Vinput Dilution + Voutput + 30ml for standard wash Vwashing _ solution = Vinput Dilution + Voutput + 250ml for high wash PBSC Washing OM 4E 06 4E - 3

160 Where: Vwashing_solution: washing solution volume required Vinput: total product volume to be washed Dilution: dilution factor Voutput: desired final product volume All these values are intended per bag. Multiply by the number of bags washed with the same kit. Step 2: Connecting washing solution bags to the Biosafe kit Unwrap the Biosafe kit in aseptic conditions (under laminar flux). Close all clamps (including the two-connection tubes). Connect the washing solution bag to the spike on the upper right line. Connect the number of output bags you will need (minimum one and generally the same number as the input bags; ensure the bags have sufficient capacity). Open the waste line clamp (lower right) and only one output line clamp (with an empty output bag connected - lower left). Seal tubes that are not used. Inspect the installed Sepax kit for any ruptures. Be sure that there are no kinks, twists or flattened areas in the tubes that might obstruct the flow. In this case kit should be discarded and returned to Biosafe. Keep protection covers on unused spikes and luer lock connectors. Close clamps on lines not in use. Step 3: Parameter modification and trace ID configuration This is a very important step for the successful processing of a unit. There are three parameters to check in order to ensure the correct procedure: 1) Input initial volume. Total volume to be washed. Selectable between 50 and 440 ml. 2) Dilution ratio. Indicates the ratio between initial volume and dilution volume. It is not recommended to change this parameter. Selectable between 0.5 and 1.5. DO NOT change the dilution ratio parameter without validating the new value. 3) Final volume. Biosafe recommends using at least 100 ml (selectable between 50 and 200 ml). To change/check parameters select CHANGE PARAMETERS in the first menu that appears after having selected a protocol. PBSC Washing OM 4E 06 4E - 4

161 Message on the display Action START KIT TEST CHANGE PARAMETERS MAIN MENU MENU Yes No ENTER Go to previous option Go to next option ENTER C Validate marked option Use the UP and DOWN arrow keys to select the parameter to change, then ENTER to enter edit mode. The first character will be displayed between square brackets. Use "UP" and "DOWN" arrow keys to set the value, and "ENTER" to move the cursor horizontally to the next character. Pressing "ENTER" alone leaves the value unchanged. If a value exceeds the upper or lower limits, the maximum/minimum value is automatically selected. Message on the display Action FINAL VOLUME [1]00.00 ml MENU Yes Increase selected digit No ENTER Decrease selected digit ENTER C Validate and / or move to next digit PRG Cancel input or exit the submenu When the parameter has the desired value, press return to the kit installation. PRG to exit the submenu and Remark: to end parameter s validation, always get with the cursor to the last character before pressing MENU to exit. Configure trace ID If you have chosen the traceability option, refer to chapter to select which ID to use. PBSC Washing OM 4E 06 4E - 5

162 Step 4: Thawing - When a SCD is not available This section is applicable only if working without a SCD. When SCD is used, go directly to step 5. When it is not possible to use a SCD to perform a sterile connection outside the laminar flow, you should connect the input bag to the kit under aseptic conditions, which means before installing the kit on the machine. In this case, complete steps 1, 2 and 3 and prepare all the material you need to continue the procedure. Thaw the unit to be washed following your internal validated thawing procedure. For each unit use an overbag, to avoid any product loss in case of unit s rupture during thawing or handling. Connect, under aseptic conditions (laminar flow), one of the connection tubes delivered with the kit to the thawed unit. Connect the bag to the main part of the kit using the luer lock connectors and continue immediately with point 5. Skip step 8. Step 5: Kit installation The kit should be installed when the following message appears on the display (before or after parameter change/check): Message on the display START KIT TEST CHANGE PARAMETERS MAIN MENU MENU Yes Action Go to previous option No ENTER Go to next option ENTER C Validate marked option Before starting the kit test, install the kit as follows: 1 Open the two covers of the centrifuge pit. 2 Install the chamber into the centrifuge by pushing it down firmly. Ensure the separation chamber is well inserted. 3 Insert the effluent line into the optical line sensor. Ensure the tubes are well inserted. PBSC Washing OM 4E 06 4E - 6

163 4 Verify that all stopcocks are well aligned (Position : ). 5 Install the stopcocks on the rotary pins by pushing them down gently. 6 Connect the pressure sensor line (luer connector / filter) to the pressure sensor port on the top-deck. Tighten this luer lock firmly. 7 Hang the waste bag and the output bag on the pins located on the side of SEPAX. 8 Hang the washing solution bags on the rear stand. 9 Close the centrifuge cover. Tighten the centrifuge cover lock (screw). After the kit installation, the machine performs an automated kit test, as explained in chapter 4.7. Enter the traceability IDs: if you have chosen the traceability option: enter the IDs manually or with code bar reader as explained in chapter Step 6: Kit priming With this operation the kit is primed and the washing solution for primary dilution is prepared in the chamber. To start follow the instructions as described. Message on the display OPEN BUFFER, WASTE & OUTPUT BAG CLAMPS Action Open/check washing solution line clamp, waste line clamp and output bag line clamp ENTER Go to next step When ready select one of these options from the menu: STANDARD WASH for a standard procedure with one washing cycle HIGH WASH for a procedure with 2 washing cycles Message on the display STANDARD WASH HIGH WASH MAIN MENU MENU Yes No ENTER Action Go to previous option Go to next option ENTER C Validate marked option PBSC Washing OM 4E 06 4E - 7

164 Once the priming is finished, the machine will beep and displays: Message on the display THAW AND CONNECT INPUT BAG ENTER ENTER Action Validate message and continue with washing cycle. Input bag must be connected before validation. STOP Stops the procedure. USE ONLY IN CASE OF EMERGENCY!? Any other key: stops beep Remark: When this message appears use any key except ENTER or STOP to stop the beep. Step 7: Thawing and connecting unit Thaw the unit to be washed following your internal validated thawing procedure. For each unit use an overbag, to avoid any product loss in case of unit s rupture during thawing or handling. Under aseptic conditions (laminar flow), connect one of the connection tubes (see drawing below) delivered with the kit to the thawed unit. Connect the unit thanks to this connection tube to the main kit already mounted on the machine using a Sterile Connection Device. Step 8: Automated washing cycle After connection push ENTER key to validate message and display the following message: Message on the display OPEN INPUT BAG CLAMP ENTER Action Open all clamps except those not on used lines ENTER Begin washing procedure DO NOT open clamps or caps on lines directly exposed to an external environment. Avoid touching the machine keyboard or the kit during the automated procedure. Moving bags, tubes, stopcocks and covers may cause errors and the process must then be restarted. PBSC Washing OM 4E 06 4E - 8

165 The washing protocol for PBSC product goes through the following phases: 1) Primary dilution (typically 1volume for 1volume) into initial bag (slow extraction). 2) Osmolarity balancing time (about 1 ). 3) Chamber filling. 4) First sedimentation and bag rinsing. 5) Supernatant product extraction. 6) In case of an HIGH WASH process: chamber refilling with washing solution and back to point D. 7) Cell resuspension. 8) Cell extraction. 9) Chamber rinsing (0 2 cycles). During primary dilution and osmolarity balance time, the operator should gently mix the initial bag or put on a rocking shaker. After osmolarity balancing time, Sepax will emit tones asking to hang initial bag and validate with ENTER key to start washing automated part. Message on the display HANG BAG ENTER Action Hang input bag on Sepax s rear holder ENTER C Go to next step Wash another bag? At the end of the procedure, Sepax emits a series of tones, requesting all clamps to be closed (validate with ENTER when done) and show: Message on the display Action WASH ANOTHER BAG END WASHING MENU Yes Go to previous option No ENTER Go to next option ENTER C Validate marked option PBSC Washing OM 4E 06 4E - 9

166 No If the washing process is finished, select the END WASHING option and go to step 9 for kit de-installation. The quality of the cell washed must be controlled prior to the transplant, following your validated procedure. Transplant the output product using a transfusion filter. If blood spillage or leakage occurs during the process, the head of the hematology department should decide the final use of the product. Yes If there is a second bag to wash, select the WASH ANOTHER BAG option. DO NOT wash different patient s bags with the same kit. DO NOT wash more than 2 bags with the same kit. The second washing process with the same kit has to be finalised within the maximum time limit of 3 hours. When using a new kit, select the END WASHING option, dismount the kit and restart the procedure from the beginning. When you decide to wash another bag with the same kit, it is advisable to seal and disconnect the first output bag. Once selected the WASH ANOTHER BAG option, you have to confirm/change volume parameters to process the second bag (refer to step 3 for more details). The procedure will then restart from step 6. When SCD is not available This section is applicable only to the procedure without SCD. When a SCD is used, go back directly to step 7. When it is not possible to use a SCD to perform a sterile connection outside the laminar flow, the second bag should be connected under aseptic conditions. The kit should therefore be dismounted from the Sepax and returned to the laminar flow. Thaw and connect the second bag as in step 4. Re-install the kit on the machine and immediately continue with step 6. Skip step 7. Step 9: End of procedure To follow all the steps for the end of the procedure and to exit protocol, refer to chapter 4.9. PBSC Washing OM 4E 06 4E - 10

167 UMBILICAL CORD BLOOD STEM CELL POST THAWING WASHING PROTOCOL (UCB WASHING) UCB Washing OM 4F 02 4F - 1

168 USER INSTRUCTIONS: Umbilical Cord Blood Stem Cells Washing Protocol Post Thawing General description The cell washing protocol is designed for the routine wash of thawed cord blood products. It performs a supernatant extraction and resuspension of the cells with the help of a washing solution (also called buffer). Two different working procedures are described. The first one requires the use of a Sterile Connection Device (SCD) to perform sterile connections outside the laminar flow. The second describes what happens when no SCD is available and connections must be performed under controlled environment (laminar flow). It is strongly recommended to use the work procedure with the SCD to minimize the time between thawing and the beginning of the washing procedure. Workflow with SCD (recommended) BEGIN 1. Prepare washing solution Workflow without SCD 2. Prepare kit 3. Parameters modification 5. Kit installation 4. Thawing of the unit* 6. Automated kit test 7. Automated kit priming 8. Thawing of the unit* 9. Automated washing 10. Dismount kit END * The thawing of the unit is performed when the kit is already primed (if a SCD is used) or before kit installation (if initial bag is connected under the laminar flow) The protocol accepts initial volumes of up to 50 ml and provides a final volume of about 100 ml (adjustable by the user) Before beginning a washing procedure, make sure that all material is available. Also be ready for a possible manual recovery procedure of the unit. DO NOT thaw the unit before it is necessary (refer to the chart on this page). ONLY users trained on this protocol by BIOSAFE or by an official trainer are authorized to perform the washing procedure. UCB Washing OM 4F 03 4F - 2

169 Step 1: Preparing washing solution bags To prepare the washing solution, you need to know the volume of the bag and the desired final volume The unit head of processing is responsible for choosing and validating the washing solution. The following graph helps to choose the volume per bag of washing solution. Simply choose the input volume on the horizontal scale and intercept the line of the corresponding output volume and application (standard or high wash). For example to wash a 40 ml input bag and obtain 100 ml output product after a standard wash cycle, it is necessary to prepare 300 ml of washing solution Washing solution volume required [ml] High wash Vout = 150ml Vout = 100ml Vout = 50ml Input volume [ml] Standard wash Vout = 150ml Vout = 100ml Vout = 50ml This graph is applicable for the most common cases. Alternatively, the following formulas can be used (washing solution volume per bag): Vwashingso lution = ml V i n p u t + V o u t p u t for standard wash Vwashingso lution = ml V i n p u t + V o u t p u t for high wash Where: Vwashingsolution: washing solution volume required Vinput: total product volume to wash Voutput: desired final product volume UCB Washing OM 4F 03 4F - 3

170 Step 2: Preparing the Biosafe CS kit Unwrap the Biosafe CS kit in aseptic conditions (under laminar flux). Close all clamps (including the connection extensions). Connect the washing solution bag to the spike on the upper/right line (red stopcock valve). Connect the output bag to one of the spikes on the output line (left/lower, blue stopcock valve ensure the bag has sufficient capacitance). Seal the unused output line and only one input line (upper/right, blue stopcock valve). Open the waste line clamp (lower/right) and the output line clamp (lower/left). Keep protections on unused spikes and luer lock connectors. Close clamps on lines not in use. Inspect the installed Sepax kit for any ruptures. Be sure that there are no kinks, twists or flattened areas in the tubing that might obstruct the flow. In this case, the kit should be discarded and returned to Biosafe. Step 3: Parameter modification and trace ID configuration This is a very important step for the successful procedure of a unit. There are three parameters to check in order to ensure the correct procedure: 1) Input initial volume. Total volume to wash. Selectable between 10 and 100 ml. 2) Dilution ratio. Indicates the ratio between initial volume and dilution volume. It is not recommended to change this parameter. Selectable between 0.5 and 2. DO NOT change the dilution ratio parameter without validating the new value. 3) Final volume. Biosafe recommends using at least 75 ml (selectable between 50 and 150 ml) to optimize the cell recovery. UCB Washing OM 4F 03 4F - 4

171 To change/check parameters select CHANGE PARAMETERS in the first menu that appears after having selected a protocol. Message on the display Action START KIT TEST CHANGE PARAMETERS MAIN MENU MENU Yes Go to previous option No ENTER Go to next option ENTER C Validate marked option Use UP and DOWN arrow keys to select the parameter to change, then ENTER to enter edit mode. The first character will be displayed between square brackets. Use "UP" and "DOWN" arrow keys to set the value, and "ENTER" to move the cursor horizontally to the next character. Pressing "ENTER" alone leaves the value unchanged. If a value exceeds the upper or lower limit, the maximum/minimum value is automatically selected. Message on the display Action FINAL VOLUME [1]00.00 ml MENU Yes Increase selected digit No ENTER Decrease selected digit ENTER C Validate and / or go to the next digit PRG Cancel your input or exit the submenu When the parameter has the desired value, press to exit the submenu and return to the kit installation. PRG Remark: to end parameter s validation, always get with the cursor to the last character pressing ENTER. Press MENU to exit. Configure trace ID If you have chosen the traceability option, refer to chapter to select which ID to use. UCB Washing OM 4F 03 4F - 5

172 Step 4: Thawing - When a SCD is not available This section is applicable only if working without a SCD. When SCD is used, go directly to step 5. When it is not possible to use a SCD to perform a sterile connection outside the laminar flow, you should connect the input bag to the kit under aseptic conditions, which means before installing the kit on the machine. In this case, complete steps 1, 2 and 3 and prepare all the material to continue the procedure. Thaw the unit to be washed following your internal validated thawing procedure. For each unit use, it is recommended always to use an overbag, that may avoid any cell loss in case of cryobag s rupture. Connect, under aseptic conditions (laminar flow), the Y connection extension delivered with the kit to the thawed unit (drawing on right). Connect the extension to the kit using the luer lock connectors and immediately continue with point 5. Skip step 8. Step 5: Kit installation The kit should be installed when the following message appears on the display (before or after the parameter change/check): Message on the display Action START KIT TEST CHANGE PARAMETERS MAIN MENU MENU Yes Go to previous option No ENTER Go to next option ENTER C Validate marked option Before starting the kit test, install the kit as follows: 1) Open the two covers of the centrifuge pit. 2) Install the chamber into the centrifuge by pushing it down firmly. Ensure the separation chamber is well inserted. 3) Insert the effluent line into the optical line sensor. Ensure the tubing is well inserted. 4) Verify that all stopcocks are well aligned (Position : ). 5) Install the stopcocks on the rotary pins by pushing them down gently. UCB Washing OM 4F 03 4F - 6

173 6) Connect the pressure sensor line (luer connector / filter) to the pressure sensor port on the top-deck. Tighten this luer lock firmly. 7) Hang the waste bag and the output bag on the pins located on the side of SEPAX. 8) Hang the washing solution bags on the rear stand. 9) Close the centrifuge cover. Tighten the centrifuge cover lock (screw). After the kit installation, the machine performs an automated kit test, as explained in chapter 4.7. Enter the traceability IDs: if you have chosen the traceability option: enter the IDs manually or with code bar reader as explained in chapter Step 6: Automated kit priming With this operation the kit is primed and the washing solution for primary dilution is prepared in the chamber. To start follow the instructions as described: Message on the display OPEN BUFFER, WASTE & OUTPUT BAG CLAMPS Action Open/check washing solution line clamp, waste line clamp and output bag line clamp ENTER Go to next step When ready select one of these options from the menu: STANDARD WASH for a standard procedure with one washing cycle HIGH WASH for a procedure with 2 washing cycles Message on the display Action STANDARD WASH HIGH WASH MAIN MENU MENU Yes Go to previous option No ENTER Go to next option ENTER C Validate marked option UCB Washing OM 4F 03 4F - 7

174 Once the priming is finished, the machine will beep and display the following message: Message on the display THAW AND CONNECT INPUT BAG. ENTER ENTER? Action Validate message and continue with washing cycle. Input bag must be connected before validation. Any other key: stops beep, except ENTER or STOP. When this message appears use any key except ENTER or STOP to stop the beep. Step 7: Thawing and connecting unit Thaw the unit to wash following your internal validated thawing procedure. For each unit, it is recommended always to use an overbag, that may avoid any cell loss in case of cryobag s rupture. Under aseptic conditions (laminar flow), connect the Y connection extension delivered with the kit to the thawed unit (picture on right). Using this connection tube, connect the unit to the main kit already mounted on the machine with a Sterile Connection Device. Step 8: Automated washing cycle After connection push ENTER key to validate message and display the following message: Message on the display OPEN INPUT BAG CLAMP ENTER Action Open all clamps except those not on used lines ENTER Begin washing procedure DO NOT open clamps or caps on lines directly exposed to an external environment. Avoid touching the machine, keyboard or the kit during the automated procedure. Moving bags, tubes, stopcocks and covers may cause errors and the process must then be restarted. UCB Washing OM 4F 03 4F - 8

175 The washing protocol for the UCB stem cells goes through the following phases: 1) Primary dilution (typically 1volume per 1volume) into initial bag and chamber (slow extraction). 2) Osmolarity balancing time (about 5 ). The product is mixed into chamber and input bag 3) Chamber filling (product and washing solution) 4) First sedimentation and bag rinsing (2 cycles). 5) Supernatant product extraction. 6) In case of an HIGH WASH process: chamber refilling with washing solution and back to point D. 7) Cell resuspension. 8) Cell extraction. 9) Chamber rinsing (0 3 cycles). The quality of the cell washed must be controlled prior to the transplantation following your validated procedure. Transplant the output product using a transfusion filter. If blood spillage or leakage occurs during the process, the head of hematology department should decide of the final use of the product. Warning and error messages During primary dilution, should a high pressure appear, a correction menu will be displayed after warning validation. HIGH PRESSURE ENTER CONTINUE DILUTION STOP DILUTION In such a case, verify that the input bag is not (excessively) full. If this is the case, choose STOP DILUTION on the menu. If the input bag can accept more washing solution, verify that all clamps leading to the input bag are open and that there are no twisted or kinked tubes. When the problem has been corrected choose CONTINUE DILUTION from the menu. For other error messages please go to chapter 4.8 or to chapter 7. Step 9: End of procedure To follow all the steps for the end of the procedure and to exit protocol, refer to chapter 4.9. UCB Washing OM 4F 03 4F - 9

176 UCB Washing OM 4F 03 4F - 10

177 GENERIC VOLUME REDUCTION PROTOCOL Generic Volume Reduction OM 4G 04 4G - 1

178 USER INSTRUCTIONS Generic Volume Reduction Protocol General description This protocol is used to reduce the volume of a generic product (peripheral whole blood, cord blood, ) ranging from 30 to more than 800ml. The volume collected in the buffy-coat bag can be chosen in two ways: Proportional: Fixed: The final volume corresponds to a predetermined percentage of the total volume remaining in the processing chamber after the plasma fraction has been extracted (volume of cells). The final volume corresponds to a predetermined amount selected between 1 and 400 ml. Step 1: Connecting the input unit to the Biosafe kit In case of maximum input volume (880 ml) it is strongly recommended that the input unit has a haematocrit at least of 38% For the input bag connection, refer to chapter 4.5. Step 2: Parameter modification and trace ID configuration Parameters modification To change/set/verify a specific parameter, choose CHANGE PARAMETERS in the menu displayed. Message on the display START KIT TEST CHANGE PARAMETERS MAIN MENU CONFIGURE TRACE ID MENU Ye s No ENTER Action Go to previous option Go to next option ENTER Validate marked option Press UP and DOWN keys to navigate the parameters. Once the wanted parameter is displayed, press the ENTER key to select the first character, which will be displayed between square brackets. Use the "UP" and "DOWN" arrow keys to set the value, and "ENTER" to move the cursor horizontally to the next character. Pressing "ENTER" alone leaves the value unchanged. Generic Volume Reduction OM 4G 04 4G - 2

179 Message on display Action MENU Ye s Increase selected digit FIXED VOLUME [0]30 ml No ENTER ENTER Decrease selected digit Validate and / or move to next digit PRG Cancel input or exit the submenu When all the parameters have been set/verified, press and to return to the kit installation. PRG to exit the submenu Remark: to end the parameter s validation, go to the last character with the cursor pressing ENTER. Press MENU to exit. Setting the final volume Proportional: The final volume corresponds to a predetermined percentage of the total volume remaining in the processing chamber after the plasma fraction has been extracted. The value can be chosen between 1 and 100%, the default value is 80%. Fixed: The final volume represents the volume that shall be collected as the final product in the cryobag. The value can be chosen between 1 and 400 ml, the default value is 30 ml. Wanted plasma vol: The wanted plasma volume parameter is the amount of pure plasma contained into the final volume (useful to decrease the final HCT). This volume is part of the volume set with the fixed volume (e.g. a fixed volume of 30 ml and a wanted plasma vol of 5 ml correspond to 25 ml BC plus 5 ml of pure plasma). Setting the initial volume Enter here the volume that should be processed with the Sepax. The number of cycles is limited to 4, so the input volume should not exceed 880ml (in best conditions). This volume could be lower depending to the Reprocessed BC volume and Reprocessed Plasma volume. An automatic check is done at the beginning of the process to verify the coherence of these inputs. Reprocessed BC Reprocessed Plasma Maximal Input volume ml ml ml ml ml ml Generic Volume Reduction OM 4G 04 4G - 3

180 Belonging to an approximate input/output volumes ratio, some values for Reprocessed BC and Reprocessed Plasma are suggested: Input/Output vol. ratio Reprocessed BC Reprocessed Plasma approx. 5 0 ml 0 ml approx ml 20 ml Setting the reprocessed volumes These parameters are useful to improve cell recovery performances in case of a high volume reduction ratio (e.g. 15 times from 450ml to 30ml). Reprocessed BC: Reprocessed Plasma The amount of volume after the BC extraction is extracted back to the input bag and reprocessed at the next cycle (only active if the initial volume is processed in more than one cycle). User selectable from 0 to 50 ml, default 30 ml. The amount of plasma extracted back to the initial bag in order to equilibrate the HCT increasing due to the Reprocessed BC parameter. To be used in conjunction with the over indicated parameter. User selectable from 0 to 50 ml, default 30 ml. Use typically the same volume than the Reprocessed BC parameter. Configure trace ID If you have chosen the traceability option, refer to chapter to select which ID to use. Step 3: Kit installation and automated kit test For the kit installation procedure, refer to chapter 4.6. After the kit installation, the machine performs an automated kit test, as explained in chapter 4.7. Step 4: Enter the traceability IDs If you have chosen the traceability option, enter the IDs manually or with code bar reader as explained in chapter Generic Volume Reduction OM 4G 04 4G - 4

181 Step 5: Kit priming Avoid touching the machine and the kit during automated procedure. Moving bags, tubes, stopcocks and covers may cause the process to be interrupted. To ensure a maximum evacuation of the air from the system, automated priming is integrated to the automated procedure. After the introduction of the traceability IDs, the system asks to open the roller clamp. Message on the display OPEN INPUT BAG CLAMP ENTER Action Open the roller clamp so the blood can flow towards the stopcocks. ENTER Start automated priming The system will prime the bubble chamber, the line and the chamber automatically before starting the separation process. About 35 ml of the product is aspired into the chamber and then extracted back to initial bag. Step 6: Automated procedure Avoid touching the machine and the kit during automated procedure. Moving bags, tubes, stopcocks and covers may cause the process to be interrupted. Start the protocol by choosing the wanted volume calculation (fixed or proportional). Only the corresponding parameter (explained in 2) will be taken into account. Message on the display FIXED VOLUME PROPORTIONAL VOL. MAIN MENU MENU Ye s No ENTER Action Go to previous option Go to next option ENTER Validate marked option Generic Volume Reduction OM 4G 04 4G - 5

182 The automated procedure has now started. During the protocol, the current phase is shown on the display. The generic volume reduction protocol goes through the following phases: 1. ACCELERATING VOLUME =... ml 3. ACCELERATING VOLUME =... ml 5. DECELERATING VOLUME =... ml 7. BC EXTRACTION VOLUME =... ml 9. RBC EXTRACTION VOLUME =... ml 2. CHAMBER FILLING VOLUME =... ml 4. SEDIMENTATION 6. PLASMA EXTRACTION VOLUME = ml 8. DECELERATING VOLUME =... ml These phases will be repeated until the initial bag is completely empty (maximum 4 cycles). At the end of the procedure, the Sepax emits a series of tones, indicating the end of the procedure. Step 7: End of procedure To follow all the steps for the end of the procedure and to exit protocol, refer to chapter 4.9. Generic Volume Reduction OM 4G 04 4G - 6

183 DENSITY GRADIENT BASED SEPARATION PROTOCOL Density Gradient based separation OM 4H 04 4H - 1

184 General description USER INSTRUCTIONS: DENSITY GRADIENT BASED SEPARATION The Density Gradient based separation protocol was designed especially to perform routine separation of blood with density gradient technique. That completely automated protocol replaces the current manual technique, permitting to obtain better repeatability in a functionally closed system. BEGIN 1. Prepare kit 2. Parameters modification 3. Kit installation 4. Automated kit test 5. Kit priming 6. Ficoll based separation 7. Automated washing 8. Cell re-suspension 9. Dismount kit END The separation chamber can process up to 210 ml total volume (blood and ficoll). ONLY users trained on this protocol by BIOSAFE or by an official trainer are authorized to use this application. Density Gradient based separation OM 4H 05 4H - 2

185 Step 1: Prepare the kit Unwrap the Biosafe kit in aseptic conditions (under laminar flux). Close all clamps. The CS kit was especially designed for the Density Gradient based separation application. A unique bar code placed on the rotary seal of the A-200/F separation chamber identifies this special kit, ensuring its traceability. C E D F A B To prepare the kit following operations needs to be done: Connection of the output bag (B) Connection of the washing solution bag (C) Connection of the re-suspension media bag (D) Filling of the Ficoll/Waste bag with the ficoll (F) Connection of the initial product bag (A) Close all clamps prior to perform the connections. Connection of the output bag Connect a 150 ml transfer bag to the output spike (B). Connection of the washing solution bag Prepare and connect the bag containing the washing solution to the washing solution spike (C). Use 2.5% albumin in isotonic solution. As general rule use a 500 ml isotonic solution bag for a two cycles washing process and a 1000 ml bag for a three cycles washing procedure (albumin is added to this volume). For more detailed volumes, refer to the table below. Density Gradient based separation OM 4H 05 4H - 3

186 Connection of the re-suspension media bag When a different media than the washing solution is used to re-suspends the cells at the end of the procedure, prepare and connect a bag containing 50 ml of re-suspension media to the re-suspension media spike (D). Set the Pause switch bag parameter to 1 if needed. Seal this line when unused. Filling the Ficoll/waste bag with ficoll The waste bag is used at the beginning of the procedure as Ficoll bag. Inject the needed amount of Ficoll through the female luer lock connector (E) located near the Ficoll/waste bag (F). Always use 100 ml of ficoll. The chosen Ficoll is very important for a successful processing. Please ask Biosafe or its representative to be sure to use the best available solution. Connection of the initial product bag Connect the initial product bag to the input spike (A). Step 2: Parameter modification That is a very important step for a successful process of a unit. There are four parameters to check in order to assure a correct procedure: Input product volume: Indicates the approximate volume to the machine. Selectable from 30ml to 120ml, default 50ml. It will be used to compute rotation speed in order to have a constant G force on the Ficoll / Product interface. Ficoll volume. Ficoll used for the procedure. Selectable from 50ml to 150ml, default 90ml. The Ficoll bag will contain 10 ml more than that value (see Filling the Ficoll/waste bag with Ficoll in Step1) Washing cycles. Number of washing cycles performed in the washing process. Selectable from 2 to 3, default 2. The change of this parameter will affect the needed washing solution volume (see Connection of the washing solution bag in Step1) Pause switch bag. Permits to insert a pause into the protocol to switch the washing solution and the re-suspension media bags. Selectable 0 (no pause) or 1 (pause), default 0. Density Gradient based separation OM 4H 05 4H - 4

187 To change/check parameters select CHANGE PARAMETERS in the first menu that appears after having selected a protocol. Message on display START KIT TEST CHANGE PARAMETER MAIN MENU MENU Yes Action Go to previous option No ENTER Go to next option ENTER C Validate marked option Use UP and DOWN keys to select the parameter to change, then ENTER to enter edit mode. The first figure will be displayed between square brackets. Use "up" and "down" to set the value, and "Enter" to go to the next figure. "Enter" alone leave the value unchanged. If a value exceed upper or lower limit, the maximum respectively minimum value is automatically selected. Message on display FICOLL VOLUME [0]90.00 ml MENU Yes Action Increase selected digit No ENTER Decrease selected digit ENTER C Validate and / or go to the next digit PRG Cancel your input When the parameter has the desired value, press submenu and return to the kit installation. PRG to exit the Configure trace ID If you have chosen the traceability option, refer to chapter to select which ID to use. Density Gradient based separation OM 4H 05 4H - 5

188 Step 3: Kit installation For the kit installation procedure, refer to chapter 4.6. After the kit installation, the machine performs an automated kit test, as explained in chapter 4.7. Step 4: Enter the traceability IDs If you have chosen the traceability option: enter the IDs manually or with code bar reader as explained in chapter Step 5: Kit priming For proper operation, the tubing line between the source bag and the stopcocks has to be primed with blood. For priming the line is open towards the plasma bag. Thus in case the blood goes too far, it will be collected in the plasma bag. Message on the display PRIME BUBBLE CHAMBER ENTER Action Squeeze the bubble chamber until it is half full. ENTER Go to next step Message on the display OPEN INPUT BAG CLAMP ENTER Action Open the roller clamp so the blood can flow towards the stopcocks. ENTER Go to next step Message on the display Action PRIMING UP KEY TO BEGIN MENU Yes Starts priming Pressing "up" once opens the left and right stopcocks so that blood can flow along the tubing and fill it. Message on the display Action PRIMING UP KEY TO STOP MENU Yes Stops priming When the blood arrives at the level of the left stopcock, press again on "up" to close the line and stop the priming. Density Gradient based separation OM 4H 05 4H - 6

189 If the blood goes further than the middle stopcock, it is possible to push it back into the initial bag manually. Hang down the initial bag, lift up the valve and turn the stopcocks in the right direction, push back the blood and go back to the main menu. Step 6: Density Gradient based separation Avoid touching the machine and the kit during automated procedure. Moving bag, tubes, stopcocks and covers may cause errors and the process must be restarted. Next step is to confirm successful priming and start the procedure. If you would like to prime more, chose Redo priming. Message on the display START PROCEDURE REDO PRIMING MAIN MENU Action MENU Yes Go to previous option No ENTER Go to next option ENTER Validate marked option The automated procedure is now started. The Ficoll separation process goes through the following phases: 1. ACCELERATING VOLUME = 3. ACCELERATING VOLUME = 5. REACHING SPEED VOLUME = 7. SEDIMENTATION 9. BC EXTRACTION VOLUME = ml ml ml ml 2. PRIMING VOLUME = 4. PRIMING VOLUME = 6. CHAMBER FILLING VOLUME = 8. DECELERATING VOLUME = ml ml ml ml 10. WASTE EXTRACTION VOLUME = ml The cells obtained with this phase are not ready to use. The washing process will follow automatically to wash and prepare the cells. Density Gradient based separation OM 4H 05 4H - 7

190 Step 7: Automated washing The washing procedure will perform following operations: remove the residues of red cells into the separation chamber fill the chamber rinse the cell bag wash 2 or 3 times (depending on what was set by the user) the cells to remove the Ficoll The following messages are displayed on the screen: 1. FICOLL CHAMBER RINSING 3. FICOLL SEDIMENTATION 5. FICOLL SEDIMENTATION 2. FICOLL CHAMBER FILLING 4. FICOLL RINSE BAG 6. FICOLL WASTE EXTRACTION Messages from 2 to 6 are repeated 2 or 3 times, depending on the number of washing cycles. Step 8: Cell re-suspension At the end of the washing procedure, the cells are contained into the separation chamber. A re-suspension and extraction are performed to put the cells into the final product bag. The re-suspension could be done using the washing solution or a special buffer (culture media ). To change the bag containing the re-suspension solution set the parameter PAUSE SWITCH BAG to 1 (see 2); this message will be displayed: Message on the display SWITCH SOLUTION BAG USING CLAMPS ENTER Action ENTER Continue with re-suspension At this moment the operator should switch between washing and re-suspension bags and validate the message to continue the process. If the PAUSE SWITCH BAG parameter is set to 0, this message will be bypassed. The re-suspension process goes through the following phases: 1. FICOLL PRODUCT DILUTION 2. FICOLL CELLS EXTRACTION 3. FICOLL CHAMBER RINSING Step 9: End of procedure To follow all the steps for the end of the procedure and to exit protocol, refer to chapter 4.9. Density Gradient based separation OM 4H 05 4H - 8

191 Annex 1: Input product guidelines Introduction The Density Gradient protocol can be used with input products that may or may not have been pre-processed (this is no the case for the UCB/UCB-HES cord blood protocols where the input product is always in its collected state).pre-processed products can vary significantly and may affect the performance, or even the successful outcome, of the Density Gradient procedure. To assist the end-user in preparing for a successful procedure, Biosafe has prepared the following guidelines for using the Density Gradient protocol. The important parameters are: Haematocrit Nucleated Cell concentration Viscosity of the product Clots (any type of cell aggregate) Presence of tissue debris (bone, etc.) Product transparency Age of the product Haematocrit and nucleated cells concentration To obtain a reasonable sedimentation the amount of supernatant in the input product should be sufficient. The Density Gradient protocol has been developed to process product possessing certain physiological characteristics and, in general, the following criteria should be considered a general rule: Haematocrit should not exceed 55 % Total cell volume (TNC and RBC) should not exceed 60 % of the initial volume Initial TNC count should not exceed 100*10 6 / ml If any one these criterion is not met, an adjustment will be required (usually a dilution will be sufficient). Product viscosity The product viscosity may affect: The chamber filling / emptying process cell sedimentation If the input product has a high viscosity, the product should be adapted accordingly (again, a dilution should be sufficient). Clots, cell aggregates and tissue debris Clots, cell aggregates and tissue debris may adversely affect the: sedimentation process optical detection pressure detection fluid flow (especially around the stopcock valves) Such product should be filtered before the use of the Density Gradient protocol. The filter should then be rinsed to prevent cell losses. Density Gradient based separation OM 4H 05 4H - 9

192 Product transparency A product that is too transparent (low concentration of TNC and RBC) may lead to premature empty bag detection only one part of the input product being processed.. If such a product requires processing, please contact Biosafe support ([email protected]) for instructions on how to by-pass the optical sensor. Age of the unit An old input product (collection time > 72 hrs or a product that has been inappropriately stored may be not produce representative cell recovery and cell depletion results (particularly for GRC depletion). Processing of units larger than 120 ml The maximum input volume for the density gradient protocol is 120 ml. It is possible to process a higher input volume unit, by performing a pre-process volume reduction. Biosafe recommends the use of the Sepax Generic Volume Reduction (GVR) protocol to perform this step. The following conditions should be observed: The output product should meet all other guidelines indicated in this document The pre-process volume reduction should be a validated process Only the Sepax GVR protocol is recommended for pre-process volume reduction Generic Volume Reduction (GVR) protocol parameters For a GVR volume-reduced product to be compatible with the Density Gradient protocol, the GVR protocol parameters should be set as follows: Volume reduction ratio (V initial / V final ) should not exceed 6 The Wanted Plasma volume should be set to 35 40% of the final volume (for convenience the fixed volume strategy may be used). This is done to balance the final haematocrit. The Reprocessed BC and Reprocessed plasma parameters should be set to a minimum of 20 ml If in doubt, do not hesitate to contact BIOSAFE support ([email protected] or ) prior to commencing a procedure. Density Gradient based separation OM 4H 05 4H - 10

193 Annex 2: Recovery procedure Case 1: Separation by density gradient not finished 1. Close all clamps. The product has to be washed prior to a new density gradient separation procedure. The washing procedure can be done with the same single-use kit CS In the main menu select the program PURGE MODE. Follow the instructions. The content of the chamber will be put back into the input product bag. 3. If product is in the output bag, remove the stopcock ramp from the device, and by turning the stopcock purge manually the entire output bag by putting all the products back to the input bag. 4. Dilute the product in the input bag with the washing product used for the density gradient separation procedure to a volume up to 300 ml. Check that the capacity of the initial bag is big enough. 5. Close all clamps and turn the stopcock into the initial TTT position. 6. In the main menu, select the program PBSC WASHING. 7. In the protocol menu select CHANGE PARAMETERS. 8. Set FINAL VOLUME to the volume of the input product for the aborted gradient separation procedure (i.e. 50ml.) 9. Set INITIAL VOLUME to 150 ml and DILUTION to Check that the washing solution remaining in the washing bag is enough; it means the set final volume ml (i.e = 270ml). 11. Install the kit as usually with the stopcocks in TTT position. Do not select START KIT TEST, but select with the arrow down button the entry RESUME AFTER DILUTION and press enter. 12. The kit is tested. It will be necessary to put a clean line into the optical line sensor instead of the usual line that is filled with product. It s a way to cheat the kit test. Check that you put the right line back into the line captor after the kit test. 13. Open all clamps. Attention: the message OPEN INPUT CLAMPS doesn t appear. 14. Select HIGH WASH which corresponds to two washing cycles like in the density gradient procedure. 15. Wait until the end of the procedure. The washed product is in the output bag. 16. Begin a new density gradient separation procedure with a new CS singleuse kit by using the washed product as input product. Density Gradient based separation OM 4H 05 4H - 11

194 Case 2: The red cells have already been extracted into the by-product bag (separation by density gradient already done) 1. Close all clamps. The product should be washed. 2. Purge the product in the chamber into the output bag. The PURGE MODE can be used. Attention: the stopcock ramp should be removed and the stopcock turned manually so to connect the chamber to the output bag. 3. Remove the output bag in sterile condition and dilute the product to 300ml with the washing solution that was prepared for the density gradient separation procedure. Change the bag if it s too small to contain all the volume. 4. Take a new single-use kit, a CS can be used if available. And connect it to the bag containing the product as input bag. 5. Connect an empty bag (if available with a volume of 150 ml) as output bag. 6. Connect to the kit a bag containing the washing solution (it is possible to use the same as for the density gradient separation procedure. Check that there is enough volume left: For 2 washing cycles (High wash): Volume washing solution = 270 ml For 1 washing solution (Standard wash): Volume washing solution = 270 ml Check point 14 for more details about which washing strategy to select. 7. In the main menu select the protocol PBSC WASHING 8. In the protocol menu select CHANGE PARAMETERS. 9. Set FINAL VOLUME as the volume of the input product for the aborted gradient separation procedure (i.e. 50ml.) 10. Set INITIAL VOLUME to 150 ml and DILUTION to Install the kit as usually with the stopcocks in TTT position. Do not select START KIT TEST, but select with the arrow down button the entry RESUME AFTER DILUTION and press enter. 12. The kit is tested. 13. Open all clamps. Attention: the message OPEN INPUT CLAMPS doesn t appear. 14. Select High Wash if no washing cycle occurred during the density gradient separation procedure. In case of doubt select High Wash. Select Standard Wash if one washing cycle already occurred during the aborted density gradient separation procedure. 15. Wait until the end of the procedure. 16. You will find the washed cells in the output bag. Density Gradient based separation OM 4H 05 4H - 12

195 4Z. PURGE MODE Purge Mode OM 4Z 05 4Z - 1

196 USER INSTRUCTIONS : PURGE MODE The purge chamber mode can be accessed through the main menu and allows the user to empty the separation chamber. The following instructions explain how to return the product from the separation chamber back into the input bag. If this mode has been activated during a protocol and the kit has not been removed from the machine, go directly to step 2. The purge mode allows an aborted procedure to be restarted and the unit reprocessed one time with minimal loss of cells. Step 1: Kit installation Install single-use chambers and close separation chamber pit covers. Verify that all the stopcocks are aligned as shown (Position: ) Message on the display Action INSTALL KIT ENTER ENTER Go to next step Press ENTER to confirm installation. Step 2: Open clamps Verify that the clamp between the separation chamber and the input bag is open and that all the other clamps are closed. Ensure that the S-100 has turned the stopcocks to the following position: Message on the display Action OPEN ROLLER CLAMP ENTER ENTER Go to next step Press ENTER to confirm. Purge Mode OM 4Z 05 4Z - 2

197 Step 3: Automated procedure Message on the display PURGE CHAMBER MODE WAIT END EXTRACTION Once the ENTER key has been pressed, the SEPAX S-100 will begin extracting the volume contained in the separation chamber to the input bag. When the chamber is completely purged and the product is again in the input bag the purge process is complete. Message on the display END OF PROCESS CLOSE ALL CLAMPS The S-100 will then require the operator to close all clamps and press ENTER once all clamps are closed. The SEPAX S-100 will then return to initial start-up mode and the operator can choose to either remove the kit or re-run the procedure. 2-3 ml of product will remain in the chamber and in the tubing between the chamber and the initial input bag. This product will return to the chamber once the procedure is re-initiated. Step 4: End of procedure The separation can now be re-initiated and will start with the Separation Kit test - refer to chapter 4.7. Warning / Error messages Refer to Section 7 of Operator s Manual. Purge Mode OM 4Z 05 4Z - 3

198 Purge Mode OM 4Z 05 4Z - 4

199 5. ACCESSORIES 5.1 Cover A protective cover is provided to protect the SEPAX S-100 when not in use. 5.2 Data card A PCMCIA data storage card is supplied with the SEPAX S-100 and should be inserted in the corresponding slot on the rear panel of the unit. Up to 20 procedure log-files can be down-loaded to the PCMCIA card. The PCMCIA slot is also used to install software updates such updates must only be performed by approved Biosafe technicians. Only PCMCIA cards provided by Biosafe article number 1025 should be used. PCMCIA cards provided by Biosafe should not be used for applications other than the SEPAX S Pneumatic kit Biosafe supplies a pneumatic kit with the S- 100 that can be used for manual extraction of the separation chamber contents by applying pressure under the piston. This manual procedure can replace the Purge Mode in case of complete failure of the SEPAX S-100 or in the event of a power loss. The manual procedure is performed as follows: 1. Before removing the single-use kit from the SEPAX S-100, close all clamps. 2. Remove the single-use separation kit. 3. Clip the pneumatic kit chuck (see arrow on the picture) into the base of the separation chamber. 4. Manually position the stopcocks on the single-use kit to ensure a path from the separation chamber to the initial unit input bag. 5. Gently press the syringe to transfer the separation chamber contents back to the initial unit input bag. 6. Once the separation chamber piston has reached the top of the chamber, all the clamps should be closed and the stop-cocks returned to the (TTT) position). Accessories OM

200 5.4 SepaxNet, Printer and code bar reader A barcode reader (scanner) and thermal printer are supplied with the SEPAX S-100 as part of the Traceability Kit see In order to use a SEPAX S-100 device with the SepaxNet System SN-100 you need: A bar code reader Gryphon D100 (AS-665). That barcode reader is connected to the SEPAX S-100 by the PS2 port on the rear panel of the device. A WiFi (AS-660-W) or an Ethernet (AS-660-E) connection box with its power supply. That box is connected to the SEPAX S-100 by the serial port on the rear panel of the device. The MAP software with a version 2.64 or higher. Accessory specifications: List of all cables and other accessories with which do not affect compliance with the electrical and EMC requirements. The use of accessories, transducer or cable with equipment other than specified may result in increased emissions or decreased immunity of the system (refer to Electro-Magnetic Compatibility) as required by IEC Article no. Description Manufacturer 6059 Printer: Martel MCP 8810, with Egston P 2 EFMW3 12W 12V or Egston N 2 EFSW 12W 12V Printer method: Direct linear thermal 6059b Printer Martel power cable Egston length 3,5 m 6059c Printer serial cable Martel MGK 20 length 2,5 m 6057 Barcode reader Gryphon D-100 (scanner) Reader method: Remote 6057c Barcode Reader Gryphon PS/2 cable Cable PS2 compatible length 2,5 m 6080 Power cable Medical Grade Nema 5.15p (IEC/EN /C13 North America cord type SJT). Manufacturer Schurter, length 3,1 m 6046 Protective cover Manufacturer: Sarna Plastec AG Warnings and precautions - Do NOT direct the barcode reader at human eyes. - Only printers and barcode readers supplied by Biosafe should be used with the SEPAX S The separate manuals for the barcode reader, printer and power supply should also be read prior to using the SEPAX system. Any manual of additional connected device should be read as well. - The printer and barcode reader should not be used in a patient environment according to CEI / When the barcode reader is not being used, the barcode reader data port on the rear panel of the SEPAX S-100 should be protected with the supplied cover. Accessories OM

201 - When the printer is not being used, the printer data port on the rear panel of the SEPAX S-100 should be protected with the supplied cover. - If the printer power cable is not connected to the printer, the supplied protective cover should be placed over the printer power cable socket. - Barcode reader, printer and associated power cables should be cleaned using an anti-static cloth and an antibacterial solution to disinfect the surfaces (such as Meliseptol ). Additional equipment connected to medical electrical equipment must comply with the respective IEC or ISO standards (e.g. IEC for data processing equipment). Furthermore all configurations shall comply with the requirements for medical electrical systems (see IEC or clause 16 of the 3Ed. of IEC , respectively). Anybody connecting additional equipment to medical electrical equipment configures a medical system and is therefore responsible that the system complies with the requirements for medical electrical systems. Attention is drawn to the fact that local laws take priority over the above mentioned requirements. If in doubt, consult your local representative or the technical service department. 5.5 Tubing Stripper The tubing stripper is NOT supplied by Biosafe but is required to complete the procedure. Any stripper designed for standard tubing lines (4.1mm outer diameter) will be suitable for stripping the different tubing line. It is highly recommended to use the stripper at the end of the procedure to reduce cell loss. Accessories OM

202 Accessories OM

203 6. MAINTENANCE The SEPAX system is designed to require minimal maintenance. The only maintenance the operator has to provide consists of cleaning the outer surfaces of the SEPAX S-100, the Traceability Kit components and the associated power cables Any other maintenance or technical intervention should only be performed by Biosafe approved technicians. The SEPAX equipment should be serviced every 6 months by a Biosafe approved technician. The elements that require servicing by the Biosafe approved technician are described in the Service Manual. 6.1 Cleaning The Sepax S-100 should be cleaned on a regular basis or after any incident such as product leakage. The following cleaning procedure should be used for cleaning: 1) Switch off the SEPAX S-100 before beginning the cleaning procedure to prevent risk of electrical shock. 2) Rubber gloves and a protective gown should be worn to prevent direct skin contact with any spilled cord blood during the cleaning procedure. 3) With Wet a gauze bandage or a soft paper soaked with warm water and clean the blood away Sepax S-100 circuit. Dry the damp surface with soft paper and repeat as necessary to ensure the surface is clean. 4) Use an antibacterial solution to disinfect the surface - such as Meliseptol. As with any electrical equipment, fluid entering the SEPAX S-100 can adversely affect its performance. Do not attempt to clean any internal part of the Sepax S-100 this should only be done by a Biosafe approved technician. If in doubt, contact Biosafe for advice. 6.2 Waste management After processing, discard the parts of the separation kit that are not to be stored, according to your internal procedures. This product is subject to Directive 2002/96/EC of the European Parliament and the Council of the European Union on waste electrical and electronic equipment (WEEE) and, in jurisdictions adopting that Directive, is marked as being put on the market after August 13, 2005, and should not be disposed of as unsorted municipal waste. Please utilize your local WEEE collection facilities in the disposition of this product and otherwise observe all applicable requirements. Maintenance OM

204 6.3 Warranty Biosafe products are designed and manufactured to provide reliable, trouble free performance when properly maintained and used in accordance with the present Operator s Manual. Biosafe warrants to the original purchaser that the unit has been be fully tested and delivered according internal Biosafe procedures. Maintenance service and, if required, repairs are free of charge for one year from the date of shipment. Equipment failure due to reasons other than manufacturing defects such as accidents, misuse or failure to perform scheduled maintenance is excluded from warranty coverage. Maintenance OM

205 7. WARNING & ERROR MESSAGES 7.1 General Warnings and errors are signalled by an alarm. Warning messages allow the procedure to continue once the problem has been rectified. Error messages do not allow the procedure to continue and will require the operator to re-start the procedure. Prior to re-starting the procedure, the SEPAX S-100 provides the option to return the contents of the separation chamber to the input bag (see Purge Mode protocol). When an Error message appears, you are asked to follow these steps: 1. Press ENTER STOP to turn off the alarm. 2. Read and write down the error message. 3. Take out the tube from the optical sensor (if noted on the screen) and press ENTER. If the error does not permit you to continue even after restarting the procedure, you will be asked to restart the Sepax S-100. When an error message appears, please take a few minutes to fill in the report (see below) and fax it to Biosafe on Start-up warning messages Startup warning messages Continuous beeps Description The cover closing screw is closed but not the covers. Close the covers and the screw correctly. 7.3 Start-up error messages Startup Error messages CHECK CHAMBER PRESS CHECK LINE PRESSURE REMOVE OPTICAL LINE 5 beeps every 10 seconds Description Incorrect start-up chamber pressure or chamber pressure sensor failure. Incorrect start-up line pressure or line pressure sensor failure. Start-up optical line sensor failure. The rear fan has a problem and can not assure normal system cooling. You should reduce the machine usage to the minimum and call for service maintenance as soon as possible. Error messages OM

206 7.4 Warning messages during the procedure Warning messages PRESSURE WARNING WARNING COVERS OPEN 4 beeps every 15 seconds 3 beeps every 15 seconds Description Line pressure reached the vacuum/pressure warning limit. Centrifuge covers were opened during the procedure not during centrifugation Casing temperature over 50 C. End procedure if it is still running. Verify air circulation and wait a few minutes before restarting. Cover temperature over 55 C. End procedure if it is still running. Excessive centrifuge usage? Leave the machine in stand by mode or turn it of. 7.5 Error messages during the procedure Error messages CENTRI OPENED CENTRI OVERSPEED CENTRIFUGE BLOCKED CH. OVERPRESSURE CH. UNDERPRESSURE CHECK CENTRI SPILL CHECK LEFT STOPCOCK CHECK RIGHT STOPCOCK EMERGENCY STOP FAN IS DEFECTIVE Description Centrifuge covers were opened during centrifugation. Centrifuge speed was too high. Centrifuge rotor locked. Check inside the centrifuge cabinet. The pressure under the piston is too high. Verify that all clamps are open and stopcocks well positioned. The vacuum under the piston is too high. Verify that clamps are open and stopcocks well positioned. Chamber spill detected. If leakage has occurred see chapter 6 otherwise clean spill sensor inside the centrifuge cabinet. The left stopcock was manually rotated during the procedure. The right stopcock was manually rotated during the procedure. STOP key was pressed. Press enter to restart when ready. Rear panel fan failure. Error messages OM

207 LEFT STOPCOCK BLOCKED RIGHT STOPCOCK BLOCKED PISTON OVERSPEED CASE HIGH TEMP COVER HIGH TEMP INVALID MEDIA CHANGE LINE OVERPRESSURE LINE UNDERPRESSURE INPUT VOLUME TOO LOW MEDIA ACCESS ERROR PISTON IS UNSTABLE PISTON POSITION PNEUMATIC CIRCUIT UNABLE TO OPEN FILE SYSTEM ERROR INCORRECT SHUTDOWN Unable to move the stopcock electrical problem or obstacle blocking the motor. Unable to move the stopcock electrical problem or obstacle blocking the motor. Problem with the chamber, verify kit. Risk of overheating. Verify that rear fan is not obtrude, switch machine OFF and restart later. Risk of product damage (overheating). Switch machine OFF and restart later. Wrong memory card inserted. Change memory card. Line overpressure. Verify that clamps are open and stopcocks well positioned. Line under-pressure. Verify that clamps are open and stopcocks well positioned. The input volume is lower than the output volume. The protocol stops, does a purge of the chamber and asks the user to check the input/output volumes. The memory card is not inserted correctly. Failure during piston stabilization. Too much air could have been aspirated into the chamber. Restart and check the optical line sensor. Incoherent piston position. Check that the inside of the centrifuge cabinet is clean. The pneumatic system is defective. System failure (corrupted files). Reinstall the program on the machine using the install memory card. System failure, restart the machine and inform Biosafe. Indicates incorrect shutdown of the machine in the previous procedure: to avoid this message, always use QUIT in the main menu before switching off the machine. Error messages OM

208 Biosafe SA SEPAX observations Date : Centre : Contact person : Telephone number : Machine number : Kit lot: 1. Problem description (Please be as specific as possible; all your observations are very important to us.) At what stage of the protocol did the problem occur? How were the stopcocks positioned? Did the beeps-100 alarm sound? Were all the clamps open? Which message appeared on the display? What did you do to solve the problem? Were there any irregularities with the procedure (noises, etc.)? Other comments: Action to be taken: Send this sheet to Biosafe by fax: Save the protocol data from the SEPAX S-100 and send the LOG file to Biosafe by at [email protected] If the problem is related to the chamber, please send the chamber only (not the rest of the harness) in appropriate packaging, to Biosafe by post to Biosafe SA, Route d Eysins 1, 1262 Eysins, Switzerland. We would like to thank you for communicating this problem, which will help us to improve our products and we apologize for any inconveniences caused. If you have any questions, please contact Biosafe on telephone #: or [email protected] Error messages OM