CyTOF Autosampler 5 User Manual

Transcription

1 CyTOF Autosampler 5 User Manual

2 CyTOF Autosampler 5 User Manual This is Class I, cord-connected equipment. For use in commercial, industrial or business enviroments. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of DVS Sciences Inc. or their respective owners DVS Sciences Inc. Revision 1, for SW v6.1.4; February 2014 Corporate Headquarters 639 N. Pastoria Ave, Sunnyvale, CA Canada 70 Esna Park Dr, Unit 12 Markham, ON, L3R 6E7 2

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4 Preface The CyTOF Autosampler 5 (AS 5) enables automated delivery of samples in 96 well plates to the CyTOF 2 Mass Cytometer. The AS 5 accommodates up to three shallow or deep well plates covered with X Pierce sealing film (Excel Scientific, Inc.). This manual describes installation, daily operation and maintenance of the AS 5. 4

5 Table of Contents PREFACE 4 CHAPTER CYTOF AUTOSAMPLER 5 OVERVIEW Overview of the Components 6 Fluidics System 11 CHAPTER INSTALLATION AND SET UP Safety 13 Waste Connections 13 Power Requirements 13 Connecting the Autosampler 14 Filling the Reservoir 15 CHAPTER CONFIGURATION AND OPERATION Switching to the Autosampler Configuration 16 Auto tuning 18 Batch Acquisition 22 Switching to Manual Configuration from the Autosampler Mode 28 CHAPTER MAINTENANCE Replacing the Peristaltic Pump Tubing 29 Cleaning the Bottle Tray 33 Cleaning the Covers 33 Replacing the Syringe 33 Parts Provided with the Autosampler 36 Instrument Specifications 37 Recommended Reagents and Solutions 38 5

6 Chapter 1 CyTOF Autosampler 5 Overview Overview of the Components The following are standard components of the CyTOF2 Autosampler: Figure 1 Front View 1) Autosampler Cover 2) Reservoir Assembly Three solution bottles on a tray (see Figure 5 below) 3) Syringe Pumps The Autosampler is a dual loop system with two syringe pumps that alternate wash and sample injection functions. 4) Sample trays For the 96 well microtiter plates to sit on the sample tray holders. 5) Sample Tray Holders Hold 96 well plates. 6) Probe Assembly Holds the sampling probe. 6

7 Figure 2 Autosampler Parts 7) System Fluid Port Connected to the System Fluid bottle on the Reservoir Assembly, this port is filled with System Fluid for cleaning the tip of sample probe. 8) Wash Solution Port Connected to the Wash Solution bottle on the Reservoir Assembly, it is filled with Washing Solution for cleaning the loops. 9) Waste Port Connected to the waste tubing which drains waste fluids to the drain vessel Figure 3 Bottom Rear View 10) Power Switch Controls power supply to all components of the Autosampler. 11) RS 232 Connector Communication Cable Serial I/O ports, connects to the computer. 7

8 12) Power supply connector (DIN type) connects to AC power supply adapter, provided with the system. Figure 4 Rear View 13) Opening for Waste tubing Waste tubing carries waste fluids from the waste port to the drain vessel. 14) Peristaltic Pump Compartment Contains the peristaltic pumps that pump the system fluid and wash solutions into and out of the ports through Tygon tubing. Figure 5 Reservoir Assembly 8

9 a. Carrier Fluid Bottle Contains sample carrier solution (high quality deionized water). b. System Fluid Bottle Contains System Fluid (high quality deionized water). c. Wash Fluid Bottle Contains CyTOF Washing Solution, E Pure. d. Bottle Tray Holds the three bottles in the reservoir assembly. Figure 6 Syringe Pump Assembly e. Syringe Pump 1 Alternates (with Syringe Pump2) between driving sample introduction and the wash cycle. f. Syringe Pump 2 Alternates (with Syringe Pump 1) between driving sample introduction and the wash cycle. g. Syringe (500 µl) h. Syringe (500 µl) i. Thumb Screws Hold the 500 µl syringe in place. j. M3 Screws Hold the syringe pump cover onto the syringe pump assembly. k. Syringe Pump Cover Covers the entire syringe pump assembly in its compartment. 9

10 Figure 7 Valve Assembly l. m. 4 Port Valve Controls fluidics associated with sample injection n. Valve Door (Figure 7) Provides the user with access to the valve assembly. Figure 8 Mount Block Assembly o. 10

11 p. Micro Switch Mount Block Holds the micro switch sensors. q. Return Spring Retracts the sampling probe after contacting a physical stop. r. Probe Guide Block Carries the sampling probe as it moves up and down. s. Probe Cap Holds the sampling probe to the probe guide block. t. Sampling Probe Picks up sample and wash solution. Figure 9 Peristaltic Pump Assembly u. Peristaltic Pump Green (RINSE IN) deionized water goes into the system v. Peristaltic Pump Red (RINSE OUT) deionized water comes out of the system w. Peristaltic Pump Yellow (WASH OUT) wash solution comes out of the system x. Peristaltic Pump Blue (WASH IN) wash solution goes into the system Fluidics System Similar to the CyTOF2 manual introduction, the Autosampler utilizes a dual loop sampling valve for sample introduction. It can be used for various liquid sample sampling applications with a number of user defined sample volumes ranging from 50µL to 900uL. The sampling probe and one of the syringe pumps re suspend the sample, then aspirate it from the desired well position into the available sample hold loop. The Carrier Fluid, the System Fluid, and Wash Solution bottles, 250 ml each, are housed within the Autosampler. Typically, the Carrier Fluid and System Fluid are Type I (ultrapure, deionized, 18.2MΩ) water (DIW). System Fluid DIW is used for cleaning the outer surface of the probe, CyTOF Washing Solution is used for washing the probe and the sample loops and Carrier Fluid DIW is used for rinsing the 11

12 probe, washing the sample loops and as a sample carrier. Washing Solution is fed from Washing Solution bottle to wash port and recycled using a pair of peristaltic pumps. Similarly, the deionized water used to rinse the probe is then fed from the System Fluid bottle to the rinse port and is also recycled using a pair of peristaltic pumps. To minimize carryover, the sampler is equipped with a wash function for washing in between the samples. The probe and sample hold loops are washed with washing solution and rinsed with carrier fluid (DIW). Note: Please refer to the CyTOF Washing Solution Material Safety Data Sheet (MSDS) for further information on handling. Figure 10 Schematic of the System. 12

13 Chapter 2 Installation and Setup Safety 1) Keep instrument covers closed when operating the CyTOF Autosampler 2) Use the AC Power Adaptor supplied with the device. Any substitutions may potentially damage the Autosampler unit. 3) Do not operate the instrument in the presence of flammable gases or fumes. Operation of any electrical instrument in such an environment constitutes a definite safety hazard. 4) Do not install substitute parts or make any unauthorized modification to the instrument. 5) Use caution when handling, testing and adjusting. Waste Connections 1) The CyTOF Autosampler 5 is supplied with a drain vessel that may be hooked up directly to the unit. 2) The drain vessel should sit in close proximity to the instrument to ensure smooth elimination of liquid waste from the system. 3) The drain vessel should be positioned below the instrument to allow gravity to drive the waste fluids down into the vessel. 4) Waste materials contain reagents and sample waste and should be treated according to the Laboratory Safety Guidelines of the facility. Power Requirements 1) The CyTOF Autosampler 5 should be in close proximity to a power supply adapter. 2) The connection between the power supply adapter and the AC outlet must be cleared from all obstructions at all times for quick disconnection if a hazardous situation arises. 3) The input power requirement for Autosampler unit is 24 VDC, 4.2A, 100W provided by the power supply adapter unit. 13

14 Figure 11 Electrical Connections with Power Supply Adapter Connecting the Autosampler 1) Ensure that power supply adapter is connected to a properly grounded (3 prong) receptacle as demonstrated in the figure above. 2) Connect the power supply for the Autosampler. 3) The communication port on the back of the Autosampler is immediately below the power switch. Connect the RS232 cable to the Autosampler and to the communication port labeled Autosampler on the right side of the CyTOF 2 instrument. 4) The sample line from the Autosampler also has to be connected to the sample capillary. Disconnect the existing sample line from the stainless steel bulkhead on the heater complex and connect the sample line from the Autosampler. 14

15 Filling the Reservoir 1) The CyTOF Autosampler 5 is equipped with three 250 ml reagent bottles. Carefully lift the reagent tray from the front and slide it out (see item d in Figure 5 in the Overview of the components section above). 2) Remove the snap caps of the reagent bottles and fill the bottles with their respective reagents remembering to replace the snap caps once filled. 3) The three solutions are as follows: a. Sample Carrier Fluid DIW b. Wash Solution CyTOF Washing Solution, E Pure labeled with Blue and Yellow Stickers on the tubing c. System Fluid DIW (rinse buffer) labeled with Red and Green stickers on the tubing 15

16 Chapter 3 Configuration and Operation Switching to Autosampler Configuration 1) Ensure that power to the sampler is on and that the communication cable has been connected to the instrument. 2) Disconnect the existing sample line from the stainless steel bulkhead on the heater complex and connect the sample line from the Autosampler. 3) Open CyTOF Software. 4) Close the Status panel and log into the Administrator mode. (Please refer to the User manual, Chapter 5 CyTOF 2 Operation for instructions) 5) The Control Panel button will appear in the Menu Bar. 6) Click on and go to the Devices Tab. 7) Under Sampler, Click on the dropdown and select DVSSampler3. 8) Enter COM5 in the Device field. 16

17 9) Click Set Configuration. When prompted with message below, click Yes. The configuration will be set to Autosampler mode. 11) You can verify by checking if Batch Acq is available in the Menu Bar. Removing Air from the Syringe pumps on the Autosampler 1) Ensure that the carrier reservoir is filled with DIW. 2) Click on Reload. The syringe pumps will recharge 3 4 times. 3) If the air bubbles persist, click on Reload again. 17

18 Auto Tuning 1) Fill enough wells with Tuning solution on a deep well plate. It is recommended to dedicate a plate for Tuning solution use only. 2) Insert plate with tuning solution onto one of the trays on the Autosampler with the letters facing inward. 3) It is crucial to make sure that the plate is placed properly onto the tray. The plate must be flat. Any misalignment may cause serious damage to the instrument and loss of samples. 4) Click on Acquisition Settings next to the Mass window and TOF window. 5) When the Data Acquisition Settings window is open, click on the Sample List. a. Select the appropriate tray holder. b. The Sample List has a tray type selection section for selecting the tray type and is by default set to 96 well microtiter 250uL plate. c. Each Sample well has user defined entries for specifying the location of sample that follows the syntax Tray# Row# Well#. d. There are three trays available, each numbered with I, II, or III. The location of rows and wells corresponds to the location as labeled on the plate with rows from A to H and wells from 1 to 12. e. For example, the top right well in the top tray is defined as I A1 and the bottom left well in the lowest tray is defined as III H12. Start setting up the Tuning solution run by selecting the correct tray size. For a deep well plate, select DVS T96 500uL. 18

19 6) Under Acquisition Time, enter the 600 for an acquisition time of 600 seconds. 7) Leave all other parameters default. 8) Once both the Acquisition Time and the Volume are filled in on the table, the row will become green (as shown above) indicating that the well is ready for acquisition. 9) Open Tuning, and select calibration parameters as in manual injection mode (See Chapter 5 for Auto Tuning details). Set Acquisition Delay to 80s and Sample Introduction Rate to ml/minute) 19

20 10) Click on RUN in the Control Tab when ready. 11) The Tuning control will wait for the Autosampler to deliver the sample before starting the calibration process. 20

21 12) Return to the Sample List tab in the Data Acquisition Settings window and click Start to start the Autosampler operation. 13) The Autosampler will begin initializing for ~ 5 minutes, and then inject and acquire the sample. 14) When the sample is introduced, the Tuning will resume with an Acquisition delay, followed by the calibrations selected. The Sample List will now show the sample as Loaded. Note: Initialization of the CyTOF Autosampler 5 will occur every time the Start button is clicked. Initialization involves the start up of the syringe pumps, washing of the loops with washing solution, rinsing the loops with carrier fluid, and cleaning the tip of the sample probe with system fluid. The initialization is approximately 4 5 minutes. 21

22 Batch Acquisition Before the start of a batch run, ensure all the reagent bottles are full. a. Full bottles of reagents will typically last the duration of all samples in one tray. b. Refills are necessary if running more than one tray of samples. 1) Open the Batch Acquisition window by clicking Batch Acq from the menu bar. 2) Enter path under Directory for the location where the output files (.imd,.txt, and.fcs) are to be saved. 3) Set up the order of samples in the Sample list: a. The default setting runs samples vertically down the columns of the plate. For instance, if all wells are set up to run the first sample to be run would be A 01, followed by B 01 until H 01 before it would move onto the next column starting at A 02. b. To run horizontally across the plate across the plate (From A 01 to A 12), click on Well and notice that the order of the column sort from A01, B01, C01 to A01, A02, A03. Select the entire sample list by clicking the upper left blank box (see image below), then right click and select Re number Selected samples starting from > 1 (move cursor to the 1 and hit enter on the keyboard). Now the sample order will change to running samples horizontally. 22

23 4) Then set up the experimental details using Acquisition and Analysis tabs as well as the sample list: a. These can be saved as templates with the Create Template based on current sample for future use b. To apply an existing template, choose Apply Template (F3) from the right click dropdown list. 23

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25 5) In the Acquisition Templates window, click on the Analysis Parameters Tab. 6) Click Select Template when done. 7) To apply a template for multiple wells, you can either batch select the rows of interest and right click to pull up the dropdown list, or copy with the Copy Parameters from Previous Sample (F6) option. The Sample section has user defined entries for the Well Address of a sample that follows the syntax Tray# Row# Well#. a. As described in the Auto Tuning section, there are three trays available, each numbered with I, II, or III. b. The location of rows and wells corresponds to the location as labeled on the plate with rows from A to H and wells from 1 to 12. c. The plate should be positioned in the tray such that the short edge with the letters (A to H) is furthest away from the user. d. For example, the top right well in the top tray is defined as I A1 and the bottom left well in the lowest tray is defined as III H12. Individual sample volume is can be programmed from 50 µl to 900µL. 8) Enter the name of the sample in the Sample Title field; this is the file name of the output file. a. Sample Title must therefore be unique to avoid file conflicts since files in the same batch are saved within the same folder. 9) The Add buffer checkbox is an option for pelleted samples. 25

26 a. When this is checked, carrier fluid will be dispensed into the sample well to suspend the sample. The amount of carrier fluid dispensed is defined by Sample Volume. b. If sample is prediluted in DIW or beads containing DIW, do not check Add buffer. 10) Enter an Acquisition time. a. The sample introduction speed for the Autosampler is ml/min, or 0.75 ul per second. The time it takes for the acquisition will be Sample Volume Sample introduction Speed b. For example, if the user has a 400 µl sample, the acquisition time should be = seconds. c. If a shorter acquisition time is desired than the amount of time it would take to finish the sample, users can also program the shorter acquisition time as well. 11) Enter the Volume (Sample Volume) desired to be injected and acquired. a. Always have 25uL extra volume in the well. For example, if 500uL of sample is to be acquired, ensure that the well has a minimum of 525uL and enter 0.5mL in Volume. b. If the sample is more than 900uL, split the sample into multiple wells. Select the wells and right click to Group Selected Samples. The resulting FCS file will be one that contains data from all the wells in the same group. c. To undo a Grouping, select the grouped samples and right click. Select Reset Select Samples Grouping. Then right click again and select Re Number Selected Samples. 12) Enter the desired amount of time for the washing cycles. a. The Wash Sol. Time and Buffer Wash Time are the parameters for the washes in between samples. b. The Wash Sol. Time specifies the amount of time it takes for the washing solution to pass through the nebulizer from the time of injection. c. The Buffer Wash Time controls the time it takes for the carrier solution to pass through the nebulizer from the time of injection. d. The Skip Washing checkbox allows users to select the samples where washes are not required before the sample acquisition. This is especially helpful when running samples that are split into multiple wells. 13) (Optional) Enter details of a batch experiment using the Title field, and Click Save Batch As to save the Batch file containing experiment for all the wells in this experiment for future use. 14) The Status column indicates the sample status when loaded or completed. 26

27 15) If the batch run is interrupted, the run can be continued on a different day by opening the saved Batch file. 16) The Replicate a batch file button gives users the option to save the input information in a separate folder for a separate batch acquisition. This clears the status for the new batch file so all the samples can be run as a new experiment. 17) When the experiment is set up and batch file is saved (optional), click Run under the Acquisition Control Tab. 27

28 18) The event log will be displayed at the bottom of the Batch Acquisition window. 19) There is the option to pause the acquisition with the Pause button or end the acquisition with the Stop button. Switching to Manual Configuration from the Autosampler Mode 1) With the Autosampler still on, click on and go to the Devices Tab. 2) Click on the Type dropdown menu under Sampler and hit Esc on your keyboard. 3) Click Set Configuration. 4) When prompted, click Yes. 5) Disconnect the sample line from the Autosampler and reconnect the blue Nebulizer line from the 8 port valve. 28

29 Chapter 4 Maintenance Replacing the Peristaltic Pump Tubing The peristaltic tubing will wear down under normal use and is usually replaced approximately every 6 months. 1) Turn off the power on the AS 5. 2) Remove the cover by unscrewing the six M4 screws holding the sides and back of the AS 5. 3) Open the right door, and then remove the entire cover by lifting it straight up. 29

30 4) Remove the three bottles for System Fluid, Carrier Fluid, and Washing Fluid from the system before you start the protocol. 5) Rotate the CyTOF Autosampler 5 to the backside so that the peristaltic pumps are visible at the lower right hand corner of the frame. 6) Remove each of the pump heads from the body by rotating them counter clockwise and pulling them straight out. 7) There will be residual fluid in the tubing lines, so ensure that there is a towel close by to soak up the liquid that will flow out when the tubing is unplugged. 30

31 8) Unplug the peristaltic pump Tygon (PharMed BPT ) tubing from each of the barbed tubing connectors. 9) Unclip and remove the tube holder from the bottom of the pump head. 10) Remove the rotor assembly from the cover and replace existing tubing with new PharMed BPT tubing. 11) Apply grease (provided with the Autosampler) onto the tubing, wrap tubing around rotor assembly and re insert into cover. 31

32 12) Re install the tube holder. A clicking sound will confirm that the tube holder has been properly installed. Reconnect the new tubing to the corresponding barbed tubing connectors. 13) Install the pump head back into the body by pushing it in and turning clockwise to lock. 14) Run the washing cycle maintenance (using the Service mode in the software; this requires remote access by DVS personnel) and check to ensure that there are no leaks in the system. Check that all four pumps are rotating and pushing fluids properly, that fluid is returning to bottles, and that no leaks are occurring in port collector. 32

33 Cleaning the Bottle Tray The bottle tray must be cleaned on a regular basis to ensure that liquids have not leaked from the bottles. 1) Remove the 3 bottles from the tray. 2) Clean the tray with a dry paper towel and replace the bottles. Cleaning the Cover The cover of the Autosampler should be cleaned occasionally using a damp cloth. Replacing the Syringe Contact DVS Sciences Inc. Technical Support to order the correct syringe and for assistance before replacing it. Note: If you do not install the syringe as recommended, the warranty on the CyTOF Autosampler 5 will be void. 4) Ensure that the Autosampler is turned off before replacing the syringe. To turn off the Autosampler press the power button located at the bottom rear of the instrument. 5) Set syringe pumps into service position. (This feature requires remote access by a DVS support personnel). 6) Remove syringe pump cover by removing thumb screws. 33

34 7) Unlock the syringes by loosening the thumb screws that are located at the bottom of the syringe pumps in a clockwise rotation. 8) Compress the piston and unscrew the syringes from the pumps in a clockwise rotation and remove. 9) Screw the new syringe into the pumps in a counter clockwise rotation, ensuring not to over tighten (finger tight only). 34

35 10) Extend the piston to the thumb screws and fasten them in a counter clockwise rotation. 11) Replace the syringe pump cover and replace the thumb screws back in the original location. 35

36 Parts Provided with the Autosampler Table 2. The table below summarizes the parts that are included in the shipment of the CyTOF Autosampler 5. Part Supplier Part number Quantities Supplied Washing Solution DVS Sciences Inc (250mL) 2 Bottles Uniplate 96 wells, 2mL, Round Bottom, Whatman Inc. VWR (Pack of 25) 1 Plate 96 well plate Shallow Greiner Bio one (Pack of 100) 2 Plates X Pierce TM Films (Non Sterile) Sigma Aldrich Z (Pack of 100) 3 Pieces RS232 Connector DVS Sciences Inc. Not for sale 1 Power Supply DVS Sciences Inc. Not for sale 1 Welco Tubing and Grease Kit DVS Sciences Inc Kit (includes4 sets of tubing and 4 grease vials 1L Drain Vessel DVS Sciences Inc. Not for Sale 1 36

37 Instrument Specifications Table 3. The following table summarizes the instrument specifications for the CyTOF Autosampler 5 including power specifications, dimensions and environmental conditions. Specifications Electrical Power Requirements AC Adaptor 4.2Amps max. Input: 100VAC to 240VAC, 50/60Hz Output: 4.2Amps max. AC adapter Input Plug US standard 3 prong plug (Nema 5 15) Dimensions Weight Environment conditions 390.5mm (width) x 365.9mm (height) x 240mm (depth) 10 kilograms Temperature range 18 C to 40 C Humidity Maximum relative humidity 80% for temperatures up to 31 C, decreasing linearity to 50% relative humidity at 40 C Communications RS232 Serial Port Instrument control CyTOF Software Safety features Tubing & connectors Fluidics Low operating voltages, Safety shield on syringe pumps CyTOF Autosampler Tubing/Connectors, or recommended sample loops, tubing/connectors only (contact DVS support for replacement) Injection speed: 0.045mL/min Sample volume: Variable from 50 µl to 500 µl in 1 µl increments Compatible Multi well microtiter plates 96 well microtiter plates 37

38 Specifications Sample capacity 3 x 96 well plates (MTP) Sample dead volume 25 µl Sample introduction delay 60 sec Recommended Reagents and Solutions The highest analytical grade reagents must be used in sample preparation in order to ensure the lowest contamination of samples. The CyTOF 2 Mass Cytometer is a very sensitive elemental detector with the capability to detect sub particle per trillion ultra trace impurities. Contamination of samples with metals such as barium (Ba), lead (Pb) or salts (> 0.1%) may jeopardize instrument response during analysis and may also shorten lifespan of the ion detector. DVS Sciences Inc. supplies the following ultra pure solutions required for the operation of the instrument and sample preparation MaxPar Cell Staining Buffer (Part number ) MaxPar Fix and Perm Buffer (Part number ) CyTOF Washing Solution, E Pure (Part number ) CyTOF Tuning Solution, E Pure (Part number ) CyTOF Calibration Beads, E Pure (Part number ) EQ TM Four Element Calibration Beads, E Pure (Part number ) Type I (Ultrapure, deionized, 18.2MΩ) water (DIW) must be used in sample preparation. 38