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GE Analytical Instruments ©2006

DLM 14291 Rev. A

iii

PRINTING HISTORY

The information contained in this document may be revised without notice.
Sievers Instruments makes no warranty of any kind with regard to this material.
Sievers Instruments shall not be liable for errors contained herein or for incidental,

or consequential damages in connection with the furnishing, performance or use of

this material.

No part of this document may be photocopied or reproduced without the prior

written consent of Sievers Instruments, Inc.

Initial Printing

December 2000

DLM 14290-01 Revision A

March 2001

DLM 14291 Revision A

May 2006

Printed in USA

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Содержание MOA 280i

Страница 1: ...alyzer NOA 280i Operation and Maintenance Manual Firmware Version 3 00 and later 6060 Spine Road Boulder CO 80301 USA phone 800 255 6964 303 444 2009 fax 303 444 9543 DLM 14291 Rev A www geinstruments com Printed in USA 2006 ...

Страница 2: ...ng numbers as they are listed on the identification labels located on the back panel of the NOA and the front of the vacuum pump Analyzer serial no Pump serial no Warranty Start Date Date of receipt Installation Trademark of General Electric Company may be registered in one or more countries ...

Страница 3: ...erial Sievers Instruments shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material No part of this document may be photocopied or reproduced without the prior written consent of Sievers Instruments Inc Initial Printing December 2000 DLM 14290 01 Revision A March 2001 DLM 14291 Revision A May 200...

Страница 4: ...e support 1 800 255 6964 Monday Friday from 8 00 a m to 5 00 p m Mountain Time excluding all company and legal holidays Telephone support is provided for troubleshooting and determination of parts to be shipped from GE Analytical Instruments to the customer in order to return the product to operation If telephone support is not effective the product may be returned to GE Analytical Instruments for...

Страница 5: ...stated shelf life provided these items are maintained within the stated environmental limitations Warranty claims for consumables and verification standards are limited to the replacement of the defective items prorated from the time of claim to the expiration of shelf life Shipping A Repair Authorization Number RA must be obtained from the Technical Support Group before any product can be returne...

Страница 6: ...GE Analytical Instruments 2006 DLM 14291 Rev A vi ...

Страница 7: ...TRAP 1 7 ELECTRONICS 1 7 ANALOG PRINTER AND RS 232 OUTPUTS 1 8 EXHALATION PRESSURE TRANSDUCER 1 9 THERMAL MASS FLOW METER 1 9 2 SPECIFICATIONS 2 1 3 MENUS AND CONTROL OVERVIEW 3 1 MAIN MENU 3 1 STATUS SCREEN 3 1 ANALYSIS 3 2 MEASUREMENT MENU 3 3 MAIN MENU OPTIONS 3 6 Control 3 6 Calibration 3 7 Messages 3 7 Maintenance 3 8 TIME OUT FUNCTION 3 8 4 INSTALLATION 4 1 LOCATION 4 1 POWER REQUIREMENTS 4 ...

Страница 8: ...NU OPTIONS 4 14 Com Port 4 14 Pressure Units 4 15 SETTING THE CONSUMABLES INSTALLATION DATA 4 16 START UP 4 16 5 INSTALLATION AND SETUP GAS PHASE MEASUREMENTS 5 1 INSTALLATION OF GAS SAMPLING PACKAGE 5 1 INSTALLATION OF THERMAL MASS FLOWMETER 5 2 NOA SETUP FOR GAS PHASE MEASUREMENTS 5 4 Exhalation Mode 5 4 Nitric Oxide Mode 5 7 6 CALIBRATION 6 1 ZERO GAS CALIBRATION 6 1 Calibration with Zero Air F...

Страница 9: ...ing 7 10 Drying 7 10 CHECKING THE INSPIRATORY GAS FILTER 7 11 8 OFF LINE EXHALED NITRIC OXIDE BAG SAMPLING 8 1 ASSEMBLY OF VITAL CAPACITY BAG COLLECTION KIT 8 1 ASSEMBLY OF DEADSPACE DISCARD BAG COLLECTION KIT 8 2 CLEANING THE BAGS 8 5 COLLECTING THE SAMPLES VITAL CAPACITY BAG KIT 8 6 Connecting the bag to the filler 8 7 Instructing the Subject and Collecting the Samples 8 7 Disconnecting the bag ...

Страница 10: ... NITRIC OXIDE 10 1 RECOMMENDED SETUP 10 1 PERFORMING THE MANEUVER 10 2 NOA SETUP 10 3 11 INSTALLATION AND SETUP LIQUID MEASUREMENTS 11 1 SUPPLIES 11 1 Gases 11 1 Reagents 11 1 Lab Equipment 11 1 SETUP OF PURGE VESSEL 11 2 Connections of tubing to glassware 11 3 Procedure for Tightening Swagelok Fittings 11 4 DILUTION OF ANTI FOAMING AGENT 11 7 NOA SETUP FOR LIQUID MEASUREMENTS 11 8 DEPROTEINIZATIO...

Страница 11: ...SEL AND BUBBLER 12 16 13 MEASUREMENT OF NITRATE NITRITE AND NITRIC OXIDE IN LIQUID SAMPLES 13 1 APPARATUS FOR NITRATE REDUCTION 13 1 Preparation of the Nitrate Reducing Agent 13 2 Preparation of 1M NaOH 13 2 Startup Procedures for Nitrate Reduction 13 3 ADJUSTMENT OF PURGE GAS FLOW RATE 13 4 LEAK CHECK FOR PURGE VESSEL 13 5 ADJUSTMENT OF LIQUID LEVEL 13 5 Preparation of Stock Solution 13 6 Prepara...

Страница 12: ...mples 14 3 MEASUREMENT OF IRON BOUND NO 14 3 HEADSPACE MEASUREMENT OF NITRIC OXIDE 14 4 Apparatus for Headspace Analysis 14 5 Sample Collection 14 5 Preparation of Standards for Headspace 14 6 DYNAMIC HEADSPACE ANALYSIS 14 7 15 MAINTENANCE 15 1 CHANGING THE VACUUM PUMP OIL 15 1 CHANGING THE HOPCALITE TRAP 15 4 CLEANING THE CHEMILUMINESCENCE REACTION CELL 15 5 VACUUM TEST 15 10 RESET THE CELL CLEAN...

Страница 13: ...W 16 Cooler Needs to be Serviced Soon 16 5 CLEARING THE ERROR AND WARNING STACKS 16 6 START UP TESTS 16 6 Vacuum Pump 16 7 Cooler Temp 16 7 Supply Pressure 16 7 PMT Signal 16 7 TROUBLESHOOTING THE NOA 16 8 No Power to NOA 16 9 No Display 16 10 CELL PRESSURE TOO HIGH OR TOO LOW 16 11 GAS SAMPLING PROBLEMS 16 11 HIGH BACKGROUND NO AFTER CALIBRATION 16 12 LIQUID MEASUREMENTS PROBLEMS 16 12 Low Sensit...

Страница 14: ...GE Analytical Instruments 2006 DLM 14291 Rev A xiv ...

Страница 15: ...is 1 picomole In biological systems nitric oxide is produced from the enzymatic oxidation of arginine Three isoforms of the enzyme nitric oxide synthase NOS have been identified in many cell types endothelial NOS neuronal NOS and inducible NOS The biological functions of NO include action as a vasodilator neurotransmitter cytotoxic agent inhibit of platelet aggregation and activator of smooth musc...

Страница 16: ...e power cord before removing the cover WARNING This is a safety Class I product provided with a protective earthing ground incorporated into the power cord The mains plug shall only be inserted in a socket outlet provided with a protective earth contact Any interruption of the protective conductor inside or outside the instrument is likely to make the instrument dangerous Intentional interruption ...

Страница 17: ...ilas de litio de la RAM Card deshechadas deben seguir las regulaciones medioambientales locales FRANÇAIS ATTENTION Chaque opération à l intérieur de l appareil peut causer du préjudice Afin d éviter un shock qui pourrait être dangereux disconnectez l appareil du réseau avant de l ouvrir ATTENTION Afin de protéger l appareil continuellement contre l incendie échangez le fusible par un fusible du mê...

Страница 18: ...Inc USA le protezioni di cui esso è dotato potrebbero essere alterate ATTENZIONE Le batterie al Litio sulla RAM CARD quando sono esaurite devono essere gettate secondo le regolamentazioni vigenti localmente DUTCH OPGELET Iedere handeling binnenin het toestel kan beschadiging veroorzaken Om iedere mogelijk gevaarlijke shock te vermijden moet de aansluiting met het net verbroken worden vóór het open...

Страница 19: ...le Ozone Generator Chemiluminescence Reaction Chamber and Optical Filter Photomultiplier Tube and Cooled Housing Vacuum Pump and Chemical Trap Front Panel Display Four Button Keypad PMT Amplifier Power Supply Analog to Digital Converter Microprocessor and Output Electronics Analog Printer and RS 232 Outputs Exhalation Pressure Transducer Optional Thermal Mass Flowmeter Sample Inlet Systems The NOA...

Страница 20: ... flow rate of 200 mL min and restrictors for other flow rates are available from Sievers Instruments Ozone Flow Control Module The connection for the gas supply for the ozone generator oxygen or 95 O2 5 CO2 is made using Teflon tubing and a Swagelok bulkhead connector The gas must be a regulated supply from an external cylinder lecture bottle or house oxygen The flow rate of gas into the ozone gen...

Страница 21: ...he main power switch is on The temperature of the cooled housing is measured using a K type thermocouple and monitored on the front panel display Vacuum Pump and Ozone Destruction Trap A vacuum pump is used to draw the sample into the NOA and maintain the reaction cell at low pressure The exhaust from the reaction cell exits the analyzer at the rear of the instrument using a metal tube connected t...

Страница 22: ...output range is 0 10V The amplifier is also connected to the ADC board which also monitors three pressure transducers cell supply and exhalation the cooler thermocouple and the thermal mass flowmeter The microprocessor and firmware calculate gas concentration monitor the performance of the analyzer keep track of maintenance items and control the output of data The power supply board and 24V power ...

Страница 23: ...dures Chapter 5 contains the installation and setup for gas phase measurements Chapter 6 describes how to calibrate the NOA for gas phase measurements Chapter 7 describes on line measurement of NO in exhaled breath Chapter 8 describes off line measurement of exhaled NO Chapter 9 describes breath by breath and chamber sampling for exhaled NO measurements Chapter 10 describes measurement of nasal ni...

Страница 24: ......

Страница 25: ...s nanomolar to millimolar Response Time Electronics 67 msecs to 90 full scale Lagtime 1 second Repeatability Gas 5 Liquid 5 10 Sample Size Gas 10 300 mL min Liquid 0 001 5 mL Display Back lit LCD screen ppb ppm or mV Outputs Analog 0 1V 0 10 V Digital RS 232 9600 38 4K baud Printer parallel port Data Sampling Rate 0 002 32 samples second ...

Страница 26: ...kg Vacuum Pump with installed trap Height14 5 in 37 cm Width 7 5 in 19 cm Length 19 in 48 cm Weight 47 lbs 21 5 kg Operating Environment Ambient Temperature 32 F to 86 F 0 C to 30 C Relative Humidity 0 to 90 Fuse Requirements Main Fuse 100 VAC model T 10A 250V 120 VAC model T 5A 250V 230 VAC model T 5A 250V Ozone Fuse 100VAC model T 200mA 250V 120 VAC model T 200mA 250V 230 VAC model T 100mA 250V ...

Страница 27: ...Button is also used to display the Status Menu from the Main Menu select the Main Menu from the Measurement Menu or to return to the Measurement Menu from the Main Menu A cursor is used for selection of the menu options and the location of the cursor is indicated by a highlighted menu option Main Menu There are five options in the Main Menu Analysis Control Calibration Messages and Maintenance A t...

Страница 28: ...re starting the NOA the status screen should be checked to confirm that the cooler temperature cell and supply pressures are within the specifications required for the start up tests Pressing either the ENTER or CLEAR button will return to the Main Menu Analysis The Analysis option is used to start and stop the NOA The NOA has three modes of operation Start Stand by and Stop From the Main Menu pre...

Страница 29: ...ne supply pressure is 4 psig 6 psig recommended Turn on the PMT and record an ozone off baseline signal Turn on the ozone generator and wait for an increase in the PMT signal due to the background chemiluminescence from ozone Display the PMT signal in the Measurement Display If any of the above conditions are not met the start up screen will FAILED for that test Pressing ENTER from the Start up Sc...

Страница 30: ...ing the ENTER button Pressing CLEAR returns to the Measurement Menu The firmware keeps track of usage and when maintenance is required a WARN shortcut is displayed in the Measurement Menu The UP or DOWN Arrow buttons can be used to scroll between DATA and WARN With the WARN shortcut highlighted pressing the ENTER button will display the Warning Menu with a list of the current warnings see Chapter ...

Страница 31: ...ill display two options Stand by and Stop The Stand by option is always available from the Analysis Menu If the analysis has been started selecting the Stand by option will turn off the PMT and ozone generator but leave the vacuum pump on If the analysis was stopped selecting the Stand by option will turn on the vacuum pump ...

Страница 32: ... pump for 2 minutes to clear residual ozone from the system Turn off the vacuum pump Main Menu Options The other options from the Main Menu are used to configure the NOA for the different applications perform the calibration for gas phase measurements view warnings and errors and view and install consumables Control The Control Menu is used to setup the NOA for liquid or gas phase measurements set...

Страница 33: ... the calibrations for gas phase measurements and can be used to view and print the gas and pressure transducer calibration values Messages The Messages Menu is used to view warnings and errors Warnings indicate that maintenance is required and a WARN shortcut is displayed in the Measurement Menu Errors indicate that the cooler temperature or the supply or cell pressures are out of range When an er...

Страница 34: ...ance The Maintenance Menu is used to setup the security view and install consumable and delete old warnings and errors Time out Function The NOA s firmware monitors keyboard activity and if no button pushes are detected for 10 minutes the firmware will return to the Main Menu if the NOA is in the Stop or Standby Mode or the Measurement Menu if the NOA is in the Start Mode ...

Страница 35: ...minimum height clearance of 14 5 37 cm Pump weight is 47 lbs 21 5 kg The analyzer and pump can also be placed on a cart for mobile operation The cart should have a bottom shelf with enough clearance for the vacuum pump and should be sturdy enough to support the total weight of the analyzer and pump 82 lbs 38 kg plus any additional equipment or gas cylinders that will be placed on the cart Power Re...

Страница 36: ...ing regulators to gas cylinders Phillips head Screw Driver Hexdriver or regular screwdriver Gases A cylinder of oxygen or 95 oxygen 5 CO2 equipped with a two stage regulator is required for the ozone generator House oxygen and a flow controller may also be used If oxygen is not available air can be used however the NOA will need to recalibrated for gas measurements if air is used for the ozone gen...

Страница 37: ...tical Instruments 2006 DLM 14291 Rev A 4 3 to digital converter For the printer output any Centronics style printer can be used Contact Sievers Instruments with any questions regarding data collection equipment ...

Страница 38: ...sories kit pump fittings Tygon tubing hose clamps and mounting bracket is shipped in a separate box Step 1 Add Oil to the pump Remove one of the two oil fill plugs from the top of the pump see Figure 4 1 and add oil until the oil level just reaches the MAX mark on the pump at the top of the sight glass DO NOT OVERFILL Step 2 Install Pump Inlet Fitting Remove the plastic caps from inlet and outlet ...

Страница 39: ...ing nut finger tight Step 3 Install the Chemical Trap Mounting Bracket Locate the Allen wrench black Allen screws and trap mounting bracket in the pump accessories kit Use the Allen wrench to remove the two screws on the rear of the top plate of the pump Insert the black Allen screws through the holes in the bracket and connect to the pump with bracket s clip on the same side as the pump inlet Fig...

Страница 40: ...ength of Tygon tubing over the barbed outlet fitting and place the other end of the tubing in a fume hood To install the charcoal trap locate the Allen wrench and mounting bracket in the charcoal trap kit Use the Allen wrench to remove the two screws on the front of the top plate of the pump Insert the screws through the holes in the bracket and connect to the pump with bracket s clip on the same ...

Страница 41: ...GE Analytical Instruments 2006 DLM 14291 Rev A 4 7 on the straight end of the Hopcalite trap Use a 5 16 hexdriver or a regular screwdriver to tighten the hose clamp ...

Страница 42: ...GE Analytical Instruments 2006 DLM 14291 Rev A 4 8 Figure 4 3 NOA Back Panel ...

Страница 43: ... the power cord from the vacuum pump into the socket labeled Vacuum Pump on the back of the NOA In the center of the back panel is a protective cap on the exhaust of the NOA The cap protects the photomultiplier tube from exposure to light during shipping and anytime the vacuum hose is removed from the analyzer Remove the protective cap from the exhaust port of the NOA Anytime the vacuum hose is re...

Страница 44: ...TER The cell pressure will be displayed Reaction Cell Pressure Vacuum Test _____________________ torr If the vacuum pump connections are tight the reaction cell pressure should decrease to 1 2 torr within 10 minutes If the pressure decreases to 2 torr the connections are tight If the pressure is higher check the connections to the vacuum pump retighten the hose clamps and the connection of the vac...

Страница 45: ...g to the bulkhead fitting Connect the other end of the tubing to the adapter on the regulator Use a 7 16 wrench to tighten the nuts 1 4 turn past finger tight Turn on the main valve on the oxygen tank and adjust the outlet pressure on the regulator to 10 psig 700 mBar 520 torr or 0 7 kg cm2 The flow of gas through the ozone generator is 30 mL min and is controlled by a pressure regulator located o...

Страница 46: ... from coming off of the barbed adapters Frit Restrictor The flow into the NOA is controlled by a metal frit restrictor in an adapter attached to the Sample Inlet bulkhead fitting on the back panel of the NOA Figure 4 3 The standard restrictor has a flow rate of 200 mL min and restrictors with other flow rates are available Remove the protective cap and locate the frit restrictor stainless steel 1 ...

Страница 47: ...ers the cable is a 9 pin male to 8 pin DIM ACH 14000 Connect the 9 pin male end to the NOA port and secure with a screwdriver Connect the DIM end to either the printer or modem port on the Macintosh Newer Macs do not have serial ports and either a USB to serial adapter or a serial port card is required Contact Sievers Instruments for more information on these devices Printer a Centronics 25 pin pr...

Страница 48: ...s ENTER then use the Arrow buttons to change the minutes and press ENTER to save After setting the time press CLEAR to return to the Configuration Menu Configuration Menu Options The Configuration Menu contains two other menu options Com Port and Pressure Units These options along with the clock and only be changed when the NOA is in the Stop or Standby mode Com Port The Com Port option is used to...

Страница 49: ...D E L E T E WA R N E R R C O N S UMA B L E S V I E W I N S T A L L To change the Baud Rate From the Configuration Menu select Com Port Select Baud Rate and use the Arrow buttons to scroll to the desired rate Press ENTER to display the Confirmation screen not shown and press ENTER to save the new rate Press CLEAR to return to the Configuration Menu Pressure Units The units for the cell and supply p...

Страница 50: ...ly Press Units Menu use the Arrow buttons to scroll to the desired units and press ENTER From the Confirmation Screen press ENTER to save the new units Press CLEAR five times to return to the Main Menu Setting the Consumables Installation Data To set the installation dates From the Main Menu select Maintenance From the Maintenance Menu select Consumables From the Consumables Menu select Install Fr...

Страница 51: ... installation start the NOA to verify proper operation To Start the NOA From the Main Menu press CLEAR to display the Status Screen Verify that the Cooler Temperature is 12 0 2 C the Supply Pressure is 6 0 2 psig and the Cell Pressure is 300 torr Press ENTER or CLEAR to return to the Main Menu With the Analysis option highlighted press ENTER From the Analysis Menu select Start The Startup screen i...

Страница 52: ......

Страница 53: ...To install the gas sampling package Connect the 1 8 Swagelok nut on the Nafion drier tubing with outer mesh to the frit restrictor on the sample inlet on the NOA s back panel It is not necessary to use a wrench to tighten the nut finger tight is acceptable Connect the white Teflon filter to the female Luer fitting on the Nafion drier Connect the NO sample line clear PVC tubing without the white Lu...

Страница 54: ...ion flow rate can be measured using the optional Thermal Mass Flowmeter To install the flowmeter Locate the flow meter cable with 8 pin mini DIN connectors on both ends On the connector is a small positioning arrow Hold the connector with the positioning arrow on top and plug the cable into the Flowmeter connector on the NOA s back panel see Figure 4 3 Hold the flowmeter with the serial number tag...

Страница 55: ...nstall the flow meter in the clamp Locate the plastic cable clamp for the flowmeter cable Remove the adhesive backing and secure the cable clamp to the NOA s top cover near the back below the gas sampling clamp Secure the flow meter cable using the clamp ...

Страница 56: ...the Nitric Oxide or Exhalation Modes In the exhalation mode the NOA outputs the signal from the breath pressure transducer and the thermal mass flowmeter The Exhalation Mode must be used for on line and breath by breath NO measurements The Nitric Oxide Mode is used for measurements that do not require the flowmeter or breath pressure such as off line exhaled NO measurements Exhalation Mode To setu...

Страница 57: ...briefly to indicate that the Mode has been changed The Exhalation Mode has three additional parameters that are set in the Modes Setup Menu Pressure Units Desired Pressure and Display Filter Pressure Units determines the units for the Breath Pressure transducer in the Com Port output Desired Pressure and Display Filter only effect the bar graph in the Measurement Menu Desired Pressure sets the val...

Страница 58: ...the Arrow buttons to select the desired value and press ENTER A confirmation screen is shown press ENTER to change the value The exhalation pressure is shown in the Measurement Menu in bar graph form and updated four times a second The pressure can be displayed as unfiltered pressure or the value filtered to produce a smoother signal The default setting is unfiltered To change the Display Filter F...

Страница 59: ...tances such as measurement of a sample near 1 ppm or for integration of a signal it may be desirable to force the NOA into high or low sensitivity The interval determines how often data is outputted to the computer and or printer The recommended Com Port intervals depend on the application On line Exhaled NO 1 16 or 1 8 sec Off line Exhaled NO 1 2 or 1 4 sec Breath by breath 1 32 or 1 16 sec The p...

Страница 60: ...GE Analytical Instruments 2006 DLM 14291 Rev A 5 8 From the Operation Setup Menu select Units From the Units Menu select Concentration The Sensitivity can be set to High Low or Auto as described above ...

Страница 61: ...A must be in the Start Mode to perform the Calibration Zero Gas Calibration There is always a background signal from the PMT and the ozone in the NOA This background or offset is measured by running a gas that contains 1 ppb NO and is subtracted from the PMT signal for standards and samples Air containing 1 ppb NO can be produced using the Zero Air Filter from Sievers Instruments Sievers Part No A...

Страница 62: ...e NOA to draw ambient air through the filter for 5 minutes Calibrating with Zero Air Cylinder The setup for sampling gas from a pressurized gas source is shown in Figure 7 1 The Luer adapter tee included in the gas sampling package is connected to the outlet of the regulator on the gas cylinder the PVC gas sampling line connected to one leg of the tee and the remaining leg is open to the atmospher...

Страница 63: ...n flowing into the NOA for 5 minutes press ENTER to start the zero gas calibration The analyzer switches displays and shows a moving average of the PMT signal in millivolts with the amplifier in the low sensitivity range The moving average starts at 0 mV and slowly increases To ensure a accurate calibration the firmware compares the most recent measurement of the ozone background signal to the mov...

Страница 64: ...zero calibration exceeded the recommended limits This usually indicates that the gas being used for the zero calibration contains 1 ppb NO or there is a light leak or other cause of the high background When the offsets are out of range a warning message will be displayed Press ENTER to view the out of range offsets values values may be rejected from the Accept Menu or press CLEAR to return to the ...

Страница 65: ...ining 45 ppm NO in N2 and regulator is available from Sievers Connect the gas sampling line to one leg of the tee see Figure 7 1 and adjust the gas flow rate to provide 200 mL min so that excess gas flows out the open leg of the tee For the Sievers calibration gas install the tee on the Luer adapter on the outlet of the regulator and open the valve on the cylinder The flow rate is set at 300 mL mi...

Страница 66: ...e menu will change to indicate that the concentration value was accepted and then will display the concentration and units Press ENTER to accept the concentration Press CLEAR to reject the concentration and return to the Calibrations Menu Select PPM PPB to calibrate both ranges and press ENTER to start the calibration The display changes and shows the moving average for the PMT signal in millivolt...

Страница 67: ...ocedure for checking the flow through the frit restrictor and cleaning the reaction cell and frit An out of range message can also mean the flow of the NO calibration gas is 200 mL min so that ambient air is being drawn into the tee and the concentration of NO reaching the detector is less than the entered concentration If the NO standard is older than 12 months then the actual concentration of NO...

Страница 68: ...nsitivity and less than 500 ppm for low sensitivity The maximum signal for the ppb range can be calculated using the formula 4095 ppb offset X ppb scaling When the signal from the PMT exceeds the maximum signal the NOA will display the greater than sign and the current maximum value For example if the ppb offset is 20 and the ppb scaling is 24 the maximum value that can be measured is 978 ppb If t...

Страница 69: ... level Calibration Chemiluminescence has been conclusively shown to provide a linear response over 6 orders of magnitude 0 5 ppb to 500 ppm for the NOA This means that the error associated with measurement of ppb levels of NO when calibrating with a ppm level gas will be low for the NOA typically 5 at 10 ppb While gas standards are commercially available in the 100 500 ppb range they are expensive...

Страница 70: ... is 175 to 250 mL min Over this flow range the response of the NOA is independent of flow into the analyzer 2 deviation from maximum and small changes in flow rate will not cause changes in the response of the NOA At higher flow rates sensitivity of the NOA decreases due to quenching at higher reaction cell pressures At lower flow rates sensitivity also decreases as fewer NO molecules enter the re...

Страница 71: ... a lower flow rate remove the standard flow restrictor Install the new flow restrictor and recalibrate the NOA at the lower flow rate Calibration at Lower Flow Rates Since the response of the NOA at flow rates below 200 mL min will be lower than with the standard flow rate the analyzer must be recalibrated for the lower flow rates In most cases high voltage for the PMT will need to be adjusted to ...

Страница 72: ......

Страница 73: ... measurements is 0 05 L s 10 at Body Temperature and Pressure Saturated with water vapor BTPS For adults the duration of exhalation should be at least 6 seconds in order to obtain a stable NO value for 3 seconds Repeated reproducible exhalations are performed until three NO values are obtained that agree within 10 of the mean value Exhaled NO FENO 0 05 is the mean of these three values Instantaneo...

Страница 74: ...ans Rudolph non rebreathing valve from its package Unscrew the white mouthpiece from the non rebreathing valve Install the white Sievers mouthpiece with two Luer ports by screwing the mouthpiece into the valve clockwise until secure Unscrew the clear exhaust port fitting from the valve making sure the spiral diaphragm valve remains inside the non rebreathing valve Install the Sievers exhaust port ...

Страница 75: ...rrow on the flowmeter will point towards the non rebreathing valve Rotate the flowmeter or valve so that the signal cable from the flowmeter to the NOA is aligned with the Luer fittings on the mouthpiece of the valve Connection of Gas Sampling and Pressure Tubing Connect the male Luer fitting on the gas sample line to one of the female Luer ports on the mouthpiece of the valve To avoid kinks in th...

Страница 76: ...O air an inspiratory gas filter and a 15 L Tedlar bag and adapter Inspiratory Gas Filter This filter is part of the Bag collection and sampling kit but can also be used with the Accurate NO Breath Kit It consists of two parts a charcoal filter AFL 01410 and an adapter MTM 01461 To install the filter Screw the male threaded end of the adapter into the charcoal filter Connect the female end of the a...

Страница 77: ...mL s at 760torr the target pressure is 16 cm H2O It is usually necessary to have the subject practice a few times before good exhalations can be achieved Subjects that are used to spirometry usually will exhale at too high of flow rate as the on line measurement uses a very slow exhalation When a subject exhale too fast there is a tendency to stop exhaling rather than continuing to exhale but at a...

Страница 78: ...ect inhaled through the breath kit and they must inhale through the breath kit if the inspiratory gas filter or Tedlar bag is being used Have the subject inhale to TLC and immediately exhale while maintaining the pressure at 16 cm H2O should be between 13 19 cm H2O adjusting the force of exhalation as required to maintain the desired pressure flow After the NO levels have reached a stable plateau ...

Страница 79: ...ues should not differ by more than 1 ppb The REB program uses these criteria to select the value for the NO plateau Three exhalation profiles can be observed constant NO versus time increasing NO versus time and decreasing NO versus time For increasing NO plateaus the end NO minus the beginning NO divided by the beginning NO must be 10 the NO during the interval must be less than the end NO and th...

Страница 80: ...estrictors Restrictor Target Flow Required Pressure 760 torr Atmospheric Pressure cm H2O torr mbar 1 30 mL s 8 9 6 8 9 2 50 mL s 16 12 16 17 3 100 mL s 11 12 8 9 11 12 4 150 mL s 12 13 9 10 13 5 200 mL s 12 8 9 12 13 6 250 mL s 13 14 9 10 13 14 Models of Nitric Oxide Production in the Airways Several groups have published mathematical models of NO production in the airways By measuring the concent...

Страница 81: ...e restrictor and tubing are outlined below Consult your institution s infection control procedure for recommended frequency For the valve and flowmeter avoid using denatured alcohol or alcohol based solutions as they may cause cracking Also avoid temperatures in excess of 45 C 113 F Autoclaving pasteurization and ethylene oxide gas sterilization are not recommended Do not try to clean or sterilize...

Страница 82: ... recommended by the valve s manufacturer Cidex Cidex 7 Surgikos Inc Metricide Metricide 28 and ColdSpor Metrex Research Corp Glutarex 3M Company Follow the manufacturer s instructions for sterilization Glutaraldehyde is also available in spray form for spot decontaminations Rinsing After sterilization rinse all parts thoroughly with warm water Drying A thorough drying is a necessity using a heated...

Страница 83: ...valve and tubing are cleaned Checking the Inspiratory Gas Filter The lifetime of the inspiratory filter will depend on the levels of NO in ambient air and the frequency of use The performance of the filter to remove NO should be tested periodically to determine when it is no longer removing NO The NOA must be in the Start Mode and should be calibrated before performing this test To test the filter...

Страница 84: ......

Страница 85: ... Discard Bag Collection and Sampling Kit BSK 01400 permits collection of exhaled breath at the same flow rate 0 05 L s employed for on line measurements A divert value in the filler permits discarding of the first portion of the exhalation physiological deadspace and then a small sample 300 mL of exhaled breath is collected in a 1 5 L Mylar bag equipped with a self sealing valve Assembly of Vital ...

Страница 86: ... concentration of nitric oxide in the inspired air and the frequency of use A reasonable estimate is three months from the date it is installed Calculate three months from the day the filter is installed and write that expiration date in the space provided on the filter Screw the filter onto the threaded end of the body The Bag Kit is now ready for use Assembly of Deadspace Discard Bag Collection ...

Страница 87: ... 14291 Rev A The bag collection kit must be assembled before use refer to Figure 8 2 To assemble the kit Remove all packages from the shipping container Remove the Body of the Sampler the valves pin screw and adapters are already installed ...

Страница 88: ...pressure gauge into the bag filler body Teflon tape is not required Inspect the pressure gauge If the needle does not read 0 cm H2O unscrew the clear plastic cover of the gauge and adjust the screw at the base of the gauge with a screwdriver until the needle points to 0 cm H2O Replace the clear plastic cover of the gauge ...

Страница 89: ... gases is 5 ppb The gas is again removed using the vacuum pump on the NOA Usually a single flush with low NO gas is sufficient to remove any NO absorbed on the bag s walls although multiple flushes with low NO gas can also be performed The flow rate on the NOA 280i is limited to 200 mL min by the frit restrictor This means a full bag 12 L will take about 60 minutes to empty If the frit restrictor ...

Страница 90: ... necessary to completely fill the bag Reconnect the bag to the 3 way valve Turn the valve so that OFF points toward the zero air filter Allow the NOA to completely evacuate the contents of the bag After the bag is evacuated turn the 3 way valve so that OFF points toward the sampling line connected to the NOA This will allow gas from the zero air filter to be drawn into the Mylar bag and bring the ...

Страница 91: ...fully clockwise until the o ring seals against the base of the bag filler Rotate the bag so that there are no folds or restrictions at the end of the tube Open a new viral and bacterial filter and connect the filter to the tapered end on the top of the filler Connect a sterile mouthpiece to the filter Instructing the Subject and Collecting the Samples To perform the maneuver Have the subject comfo...

Страница 92: ...n the bag WARNING IF THE FILLER IS NOT HELD LEVEL THE BAG MAY FOLD AGAINST THE END OF THE INTERNAL TUBE RESTRICTING THE FLOW OF AIR INTO THE BAG Disconnecting the bag from the filler and sealing the bag After the subject has completed the maneuver Stand the bag filler on the inspiratory filter Grasp the Luer fitting and tube on the bag rotate counterclockwise to disconnect the bag from the filler ...

Страница 93: ...ed exhalation to collect the sample Disconnecting the bag from the filler and sealing the bag Connecting the bag to the filler To prepare the filler for sample collection Stand the filler on the inspiratory filter Push the female Luer adapter on the bag onto the male Luer fitting on the filler and secure the bag by rotating the nut on the filler The male Luer fitting on the filler must contact and...

Страница 94: ...y filter to total lung capacity Have the subject to immediately exhale while watching the pressure gauge and adjusting the force of exhalation to maintain a pressure of 5 cm H2O for a flow of 50 mL s BTSP After exhaling for 4 6 seconds have the subject push the divert valve to the in position to start collecting gas in the bag The subject must continue exhaling while maintaining a pressure to 5 cm...

Страница 95: ...ter should be discarded while the mouthpiece can be reused after sterilization Analyzing the Samples The same procedure is used to analyze samples collected using either the vital capacity or deadspace discard bag kits The NOAnalysis Bag program is designed for collecting data during analysis of these samples consult the NOAnalysis manual for instructions Alternatively the NOA s front panel displa...

Страница 96: ... CALIBRATION prior to measurement Start the measurements with the three way valve positioned so that OFF points toward the bag or open leg of the valve so that zero air is drawn into the NOA Connect the first bag to the three way valve making sure that the Luer connection between the valve and bag is secure Turn the three way valve so that OFF points toward the zero air filter Gas from the bag is ...

Страница 97: ...it Consult Figure 8 1 for disassembly of the bag filler To disassemble the filler Unscrew the small knurled fitting opposite the pressure gauge and remove the small spiral check valve Remove the Inspiratory Filter and pressure gauge from the bag filler Unscrew the large knurled fitting and remove the large spiral check valve from the body Clean the two knurled fittings the check valves and the bod...

Страница 98: ...ert valve can now be removed from the filler body Clean the knurled fitting check valve divert valve pin setscrew and the body using your institution s approved cold sterilization procedure for respiratory equipment After sterilization and drying reassemble the body Insert the divert valve fully in the body Discard position in Figure 8 4 Use forceps to insert the pin in the hole of the body Place ...

Страница 99: ...ata For the Vital Capacity bag kit flows ranging from 200 to 475 mL s are obtained over this pressure ranges For the Deadspace Discard kit flows range from 50 to 125 mL s for these pressures Stability of NO in Mylar Bags It is best to analyze the samples as soon after collection as possible In the ATS guidelines it is reported that the concentration of NO in new Mylar bags is stable for up to 48 h...

Страница 100: ...nterquartile range 2nd and 3rd quartiles and includes half of the measurements The hinges are 1 5 times the interquartile range and outliers are identified using asterisks outside of 1 5 times the interquartile range or open circles outside of 3 times the interquartile range There are a couple of reasons for this observed increase Bags filled with NO free air show a small increase 1 ppb in 24 hour...

Страница 101: ...fline Collection of Exhaled Nitric Oxide Chest 1999 116 754 759 and Canady et al Vital Capacity Reservoir and Online Measurement of Childhood Nitrosopnea Are Linearly Related Am J Respir Crit Care Med 1998 159 311 314 but the values are not interchangeable Testing Bags for Pinhole Leaks Mylar bags have been reused over 50 times without problems The most common problem in reusing the bags is the de...

Страница 102: ...in the concentration of NO over time If ambient NO is higher than the NO concentration in the bags an increase in NO above the small increase normally observed with the bags will be detected If ambient NO is lower than the NO concentration in the bag a decrease in NO can be observed Discard any bags that show large changes in NO concentration ...

Страница 103: ...que is a simple and rapid method for measuring exhaled NO from small animals e g rats and mice using a head out double chamber plethysmograph The head chamber is flushed with NO free air then sealed for a few seconds to allow exhaled breath to accumulate the contents of the chamber are then flushed into the NOA for measurement Breath by Breath Measurements Spontaneously Breathing Subjects A face m...

Страница 104: ...o the circuit Since the NOA using the standard frit restrictor is drawing 200 mL min of gas from the circuit the ventilator must be set so that the product of minute ventilation times tidal volume is greater than 200 mL min Lower flow frit restrictors can be used if the ventilator cannot be adjusted to provide 200 mL min of gas The inspired gas must be low in nitric oxide preferably 1 ppb In most ...

Страница 105: ... syringe AAK 01410 The NO and Pressure lines are connected to the syringe the syringe is filled with NO calibration gas pressurized and then injected The program then determines the difference 1 second between the NO and pressure signals Humidified Circuits When the NOA is used to measure exhaled NO in humidified circuits water will condense in the NO sample line and can block the Teflon filter re...

Страница 106: ...ng with male Luer fittings A tee is installed on the zero air inlet to the chamber The flow rate of zero air is adjusted so that a small excess of gas exits the open leg of the tee and the vacuum pump on the NOA draws the gas through the chamber The a head out double chamber plethysmograph is preferred to a single chamber system as the head out configuration helps to minimize NO from urine and fec...

Страница 107: ...h ambient air when the animal is inserted or removed from the chamber sufficient time to thoroughly flush the chamber before collection is required The NOA can be used to determine when all of the NO has been removed from the chamber The duration of the collection period will depend on the size of the chamber the size of the animal and the tidal volume respiratory rate of the animal Since the cham...

Страница 108: ...e used for viewing the concentration of NO in the samples NOA Setup For the off line NO measurements the NOA can be in either the Exhalation Mode or the Nitric Oxide mode with the Units set to Concentration Sensitivity should be set to Auto and the Com Port Interval set to 1 2 or 1 4 second See Chapter 5 INSTALLATION AND SETUP GAS PHASE MEASUREMENTS for configuring the NOA The NOA should be calibr...

Страница 109: ... Care Med Vol 160 pp 2104 2117 1999 The recommended procedure uses two nasal olives with a central lumen that are securely placed in the nares The subject inserts a mouthpiece with expiratory resistance inhales to total lung capacity and then orally exhales while maintaining a mouth pressure of 10 cm H2O While the oral exhalation is occurring air is aspirated at a flow rate of 3 L min via one of t...

Страница 110: ...sting the flow The nasal olives should be composed of a soft non traumatizing material Soft rubber one hole stoppers have also been used in place of nasal olives Performing the Maneuver Connect the vacuum line to one leg of the Luer tee connect the NOA s gas sampling line to another leg and the line to the nasal olive the final leg of the tee Adjust the flow rate into the vacuum source to give 3 L...

Страница 111: ...etween replicate measurements The ATS recommendations do not specify how many replicates should be performed or what repeatability should be achieved NOA Setup For the nasal NO measurements the NOA should be in the Exhalation Mode with the Sensitivity should be set to Auto and the Com Port Interval set to 1 16 or 1 8 second See Chapter 5 INSTALLATION AND SETUP GAS PHASE MEASUREMENTS for configurin...

Страница 112: ...GE Analytical Instruments 2006 10 4 DLM 14291 Rev A target exhalation pressure of 10 cm H2O See the NOAnalysis manual for operation of these programs ...

Страница 113: ... The following items are required to perform liquid measurements Gases A cylinder of Helium Nitrogen or Argon equipped with a two stage regulator is required for the purge vessel The outlet pressure of the regulator is 5 psi typically 1 2 psi Reagents Deionized water containing 1 µM nitrite nitrate Nitrite Glacial acetic acid sodium or potassium iodide and sodium or potassium nitrite Nitrate Conce...

Страница 114: ...he tap water is not cold Setup of Purge Vessel The Purge Vessel consists of five components Gas inlet line ASM 00430 Purge vessel with heating jacket and condenser ASM 03292 NaOH gas bubbler trap with Teflon sleeve ASM 04000 Connection tubing from the purge vessel to the gas bubbler ASM 00440 Outlet tubing with IFD filter ASM 00450 There are two different configurations of the purge vessel dependi...

Страница 115: ...and the needle valve The purge vessel should be high enough that a small beaker can be placed under the drain stopcock The gas bubbler NaOH trap consists of three pieces with a ground glass joint Install the Teflon sleeve in the base of trap insert the top piece and secure with the plastic clamp Support the gas bubbler NaOH trap on the same ring stand using a clamp Connections of tubing to glasswa...

Страница 116: ... one end and an 1 8 nut and ferrules on the other end Install the Purge Line by loosening the 1 4 nut and carefully sliding the 1 4 nut and reducing union containing the Teflon ferrules over the 1 4 OD glass on the gas inlet of the purge vessel see Figure 11 2 In some cases the OD of the glass is slightly larger than 1 4 and it may necessary to disassemble the 1 4 nut and ferrules and slip the nut...

Страница 117: ...e NaOH trap for nitrate or the outlet of the purge vessel for nitrite and tighten the nut fingertight Connect the 1 8 Teflon tubing from the outlet of the IFD to the inlet of the frit restrictor on the Sample Inlet of the NOA and tighten fingertight The purge vessel requires an inert gas supply helium nitrogen or argon from a gas cylinder The cylinder must be equipped with a two stage regulator a ...

Страница 118: ...GE Analytical Instruments 2006 11 6 DLM 14291 Rev A ...

Страница 119: ...ing provided with the purge vessel to make the connections between the water bath and the barbed fittings on the purge vessel Tygon tubing expands when heated and is not recommended Connect a piece of silicon tubing from the outlet of the water bath place a hose clamp over the tubing and connect the tubing to the bottom barbed fitting on the purge vessel heating jacket Wetting the barbed fitting o...

Страница 120: ...d 3 mL of deionized water to a vial falcon tube or other container Use a pipette to withdraw 100 µL of the antifoaming agent Since the material is very viscous allow sufficient time for the agent to be drawn into the pipette Add the agent to the water rinsing the pipette tip with water to completely transfer the agent The diluted anti foaming agent is stable for several months at room temperature ...

Страница 121: ...A G E c r n t C O N C E N T R A T I O N S E N S I T I V I T Y L OW H I G H c u r r e n t L OW From the Control Menu select Setup From the Setup Menu select Change From the Login menu select Operator From the Change Setup Menu select Analysis From the Analysis Setup Menu select Modes From the Modes Menu select Select From the Select Modes Menu if not already select select Nitric Oxide From the Conf...

Страница 122: ...es Slower intervals 1 2 or 1 second will not provide sufficient resolution to accurately fit the peak and should only be used if the computer cannot collect data at 1 4 second To set the COM port interval Press CLEAR to return to the Analysis Setup Menu Select Interval From the Reporting Interval Menu select COM PORT Use the Arrow buttons to scroll to 1 4 second and press ENTER to save The printer...

Страница 123: ... at 0 C for 30 minutes Centrifuge at 14 000 RPM for 5 minutes Remove the supernatant for analysis Remember to account for 3 fold dilution in the calculations Zinc Sulfate Sodium Hydroxide precipitation Rinse all glassware and tubes with deionized water to remove any residual nitrate Add 200 µL of sample to microcentrifuge tube Add 400 µL of 0 5 N NaOH and 400 µL of 10 by weight aqueous ZnSO4 Vorte...

Страница 124: ......

Страница 125: ...2I2 H2O For most applications 5 mL of the reducing agent is prepared in the purge vessel and this volume is sufficient for measurement of 20 50 samples depending on the volume of sample injected As the reagent is depleted the solution will turn yellow due to formation of I3 Apparatus for Nitrite Reduction Figure 12 1 shows the setup of the purge vessel for nitrite reduction Since acetic acid is vo...

Страница 126: ...d it may be necessary to experiment to determine the best volume of acid to use in the purge vessel Adjust the outlet pressure on the inert gas cylinder s regulator to 1 2 psi With the needle valve on the purge vessel fully closed open any shutoff valves on the inert gas regulator and open the gas inlet stopcock on the purge vessel Slowly open the needle valve on the purge vessel to allow gas to f...

Страница 127: ...g in a high background signal If the flow rate is too high NO from the samples can leak out into the atmosphere causing reduced sensitivity and poor repeatability To adjust the flow rate Disconnect the 1 8 stainless steel nut on the IFD filter line from the frit restrictor on the NOA s sample inlet With the frit restrictor open to the atmosphere and the NOA in the Start mode record the cell pressu...

Страница 128: ...d water through the septum until the level is near the top Water acetic acid mixtures will foam even with the antifoaming agent and this foaming should be near the top of the purge vessel but not into the condenser If the liquid level is too high close the outlet stopcock while continuing to allow gas to flow into the purge vessel Once the bubbling slows close the gas inlet stopcock Release any pr...

Страница 129: ...solid dissolves Transfer the stock solution to air tight storage container Calculate the concentration of the stock solution For NaNO2 the concentration is Conc of Stock mgNaNO2 69 mg mmol 10 mL x 1000 mL L Use this stock 100 mM standard to prepare dilutions to construct a standard curve for the NOA The concentrations of the dilutions will depend on the nitrite levels in the samples Prepare diluti...

Страница 130: ...GE Analytical Instruments 2006 12 6 DLM 14291 Rev A the standard curve may be extended to include standards with nitrite concentrations greater than 100 µM ...

Страница 131: ... accordingly Using a new pipette tip transfer 100 µL of the 10 mM standard to the next tube mix thoroughly and label the tube 1 mM Using a new pipette tip transfer 100 µL of the 1 mM standard to the next tube mix thoroughly and label the tube 100 µM Using a new pipette tip transfer 100 µL of the 100 µM standard to the next tube mix thoroughly and label the tube 10 µM Repeat these steps to prepare ...

Страница 132: ...5 µL is a good injection volume and will allow construction of calibration curves down to 0 2 µM For best results using the 10 µL syringes injection volumes should be between 2 to 8 µL For measurement of lower concentrations injection of 10 to 100 µL will be required using larger syringes For a 100 µL injection calibration curves down to 10 nM can be constructed Water Blanks Before analyzing the s...

Страница 133: ... purge vessel it helps use to one hand to help guide the needle through the septum on the purge vessel Push the syringe all the way down until the barrel rests on the top of the screw cap The end of the needle should be in the liquid reducing agent Rapidly depress the plunger to transfer the liquid into the purge vessel Withdraw the syringe wipe the needle to remove any reducing agent and then rin...

Страница 134: ...ill automatically multiply the signals collected in the Low sensitivity by 500 to account for the difference in the PMT amplifier gain However Sensitivity cannot be changed while collecting data with the Liquid program and then have the program correct for the gain difference Stop the acquire program and collect data in either the High Sensitivity setting or the Low Sensitivity setting Repeatabili...

Страница 135: ...he water Most laboratory water systems can produce water with 1 µM nitrite if properly maintained Ion exchange resins must be replaced at regular intervals and the manufacturer s recommendations should be followed to ensure high purity water is produced Water exposed to the atmosphere can absorb nitric oxide which will be converted to nitrite It is best to use water fresh from the purification sys...

Страница 136: ...lter into the reaction chamber If there is liquid in the filter stop the purge vessel clean and replace the reducing agent and clean the filter as described below see page 12 16 Foaming problems can also be minimized by keeping the injection volumes small The amount of protein injected into the purge vessel will be the same for one injection of 100 µL or 100 injections of 1µL thus more samples can...

Страница 137: ...nt from the vessel it is necessary to bring the pressure in the purge vessel back to near atmospheric pressure using the procedure below To open the purge vessel Close the outlet stopcock on the purge vessel Continue purging the solution with the inert gas until the flow of gas as indicated by the gas bubbles has stopped or has slowed considerably Close the gas inlet stopcock to stop the flow of g...

Страница 138: ...he old septum from the screw cap and discard Install the new septum with the Teflon side facing the reducing agent The lifetime of the septum can be extended by varying the location on the septum when injecting Cleaning the Purge Vessel After running samples follow the procedure below to clean the purge vessel By following this procedure each time the purge vessel is used clogging of the glass fri...

Страница 139: ...e vessel Use a wash bottle or pipette to fill the glass tube between the needle valve connection and the frit with water Replace the needle valve and open the gas inlet stopcock to allow the gas to force the water through the frit Once the water has been passed through the frit turn off the gas inlet stopcock remove the needle valve and add more water to the tube Replace the needle valve and turn ...

Страница 140: ...ressure and then remove the nut and tubing from the outlet of the filter Remove the cap on the Luer port and use a syringe 10 50 cc with a Luer adapter to draw the liquid out of the filter Use a wash bottle filled with water to rinse the filter by squirting water in the port where the tubing is connected letting the water flow out the Luer port After rinsing use the syringe to remove the remaining...

Страница 141: ...hat cannot be rinsed from the purge vessel Turn off the inert gas supply and remove the 1 4 to 1 8 reducing unions on the gas inlet and the outlet of the purge vessel Remove the needle valve from the purge vessel and the three stopcocks Soak the purge vessel needle valves and stopcocks in warm detergent for several hours Use an ultrasonic cleaner to assist in cleaning Rinse the purge vessel thorou...

Страница 142: ......

Страница 143: ... vapor a gas bubbler filled with aqueous sodium hydroxide is installed between the purge vessel and the NOA Vanadium III HCl will also convert nitrite and S nitrosocompounds to NO Nitro compounds such as nitroarginine sodium nitropruside nitroglycerin are slowly converted to NO by the reagent This conversion is not quantitative but will result in an elevated baseline as the nitro group is slowly c...

Страница 144: ...repare small volumes of the reagent to ensure that the reagent is active Some researchers report that the reagent may be stored with refrigeration for 2 4 weeks To prepare 100 mL of the reagent Use a balance to weigh approximately 0 8 grams of VCl3 into a clean dry 100 mL flask When water is added to solid VCl3 an exothermic reaction occurs and the glassware will get hot Use caution when rinsing t...

Страница 145: ... supply to the condenser and check for any leaks If water is leaking reposition the tubing and tighten the hose clamps to stop the leak Open the Gas Bubbler and add 15 mL of 1 M NaOH to the bubbler base The gas bubbler has a Teflon sleeve that prevents the ground glass joint from being etched by the NaOH Make sure the Teflon sleeve is in place and there are no holes in the sleeve Replace the bubbl...

Страница 146: ...wly open the outlet stopcock on the gas bubbler while keeping an eye on the level of NaOH in the bubbler When opened slowly the NaOH will not bump as the gas is added If the gas is added too fast the NaOH will bump into the IFD filter Gas should now be bubbling through the purge vessel and gas bubbler Adjustment of Purge Gas Flow Rate The flow rate of inert gas into the purge vessel must be carefu...

Страница 147: ...best way to ensure that all of the connections to the purge vessel and gas bubbler are tight is to see if the VCl3 HCl reagent will vacuum distill when the gas flow is turned off The water bath should be at 95 C for this test To perform the leak check Close the needle valve on the purge vessel to stop the flow of gas After a few seconds the VCl3 HCl reagent should begin to vacuum distill as indica...

Страница 148: ...uid level is too high close the outlet stopcock while continuing to allow gas to flow into the purge vessel Once the bubbling slows close the gas inlet stopcock Release any pressure in the purge vessel by slowly opening the screw cap and then drain some of the reagent using the drain stopcock WARNING THE VCl3 HCl REAGENT IS HOT Preparation of Nitrate Standard Solutions An analytical balance capabl...

Страница 149: ... the nanomolar to micromolar range Analysis of standard solutions containing 10 nM 50 nM 100 nM 1 µM 5 µM 10 µM 50 µM and 100 µM will allow construction a calibration curve suitable for most samples For urine and other samples that contain high levels of NO the standard curve may be extended to include standards with nitrate concentrations greater than 100 µM Preparation of Dilute Standards The be...

Страница 150: ... plus 100 µL of nitrate free water Set aside or discard the two most concentrated standards 10 mM and 1 mM as these will not be used WARNING ALWAYS USE NEW PIPETTE TIPS FOR EACH DILUTION SOME NITRATE REMAINS IN THE TIP AND WILL CONTAMINATE SUBSEQUENT DILUTIONS The 100 mM nitrate standard is stable for several weeks if stored in an air tight container refrigerated 4 C or lower and not exposed to li...

Страница 151: ...ections should be made until small repeatable peaks are obtained Injection Technique For best results develop a standard procedure for the injections One approach that works well is Rinse the syringe once or twice with the sample and then pump the syringe several times in the sample to remove any air bubbles Draw the plunger up to the desired volume remove the needle from the sample and then draw ...

Страница 152: ...les are injected When the foaming becomes severe as indicated by foam exiting the condenser replace the reagent and clean the purge vessel The reagent should be replaced before the foaming reaches the NaOH If the reagent foams into the gas bubbler the gas bubbler should be cleaned and fresh NaOH added to the bubbler The samples can also be deproteinized as described on page 11 10 Serum and Plasma ...

Страница 153: ...is nitrate contamination in the water used to prepare the standards Check for contaminated water simply by injecting a sample of the water Most laboratory water systems can produce water with 1 µM nitrate if properly maintained Ion exchange resins must be replaced at regular intervals and the manufacturer s recommendations should be followed to ensure high purity water is produced It is best to us...

Страница 154: ...pcock on the purge vessel is opened rapidly the NaOH will bump into the IFD filter If the stopcock is opened slowly the NaOH will not bump Slowly open the outlet stopcok while watching the level of the NaOH The NOA signal will initally increase with fresh VCl3 HCl but the signal will return to baseline after a few minutes Opening the Gas Bubbler The gas bubbler containing NaOH is also under a vacu...

Страница 155: ...an be used to tell when it is time to replace the septum With a new septum some force is required to insert the needle but after many punctures the septum loses its integrity and the needle can be inserted without force To replace the septum follow the procedures to open the purge vessel then remove the old septum from the screw cap and discard Install the new septum with the Teflon side facing th...

Страница 156: ...annot be totally cleaned by rinsing loosen the clamp so that the purge vessel can be tilted Fill the purge vessel with water then replace the screw cap a few turns so that it is not tightly sealed and will allow air to escape Tilt the purge vessel to fill of the condenser with water then return to the upright position Repeat until all of the contamination has been removed To clean the frit turn of...

Страница 157: ...ver the VCl3 HCl reagent gets into the bubbler To clean the bubbler Follow the procedures listed above to open the gas bubbler and remove the Swagelok unions on the inlet and outlet of the bubbler Remove the green clamp and twist the bubbler s top to open the bubbler Discard the NaOH and rinse the bubbler bottom thoroughly with water To clean the frit close the bubbler stopcock fill the bubbler bo...

Страница 158: ...the NaOH in the gas bubbler gets too high it will be drawn into the tubing and blocked by the IFD filter Most often this is as a result of injection of a sample containing protein causing the VCl3 reagent to foam into the bubbler and subsequent foaming of the NaOH While running samples observe the IFD filter to check if liquid is present in the filter While the filter will initially block liquid f...

Страница 159: ...s AFL 01400 and also available from most laboratory supply houses Two styles of IFD filters are sold by the supply houses the polypropylene filter used in the NOA and a nylon filter Always use the polypropylene filter with the NOA Long term maintenance of the purge vessel and bubbler Cleaning the purge vessel and bubbler after use will help to prevent clogging of the frits or contamination of the ...

Страница 160: ...GE Analytical Instruments 2006 13 18 DLM 14291 Rev A ...

Страница 161: ...m 1999 252 535 540 have reported the use of copper I ions in combination with cysteine for the conversion of low molecular weight nitrosothiols to NO using the purge vessel While the efficiency of conversion is high for lower molecular weight compounds such as S nitroso glutathione lower efficiencies were reported for high molecular weigh species such as S nitroso albumin This regent was specific ...

Страница 162: ... Iodine Reagent Marley and co workers Marley R Feelisch M Holt S and Moore K A Chemiluminescence based Assay for S nitroso albumin and Other Plasma S nitrosothiols Free Rad Res 2000 32 1 1 9 have reported a technique for measurement of S nitroso albumin and other plasma nitrosothiols based on alkylation of free thiols with N ethylmaleimide removal of nitrite by reaction with acidified sulfanilamid...

Страница 163: ...ide final concentration 5 mM After centrifugation the samples are treated with 0 5 sulfanilamide in 0 1 M HCl to remove the nitrite A similar method has been developed by Gladwin and co workers Gladwin M T Shelhamer J H Schechter A N Pease Fye M E Waclasis M A Panza J A Ognibene F P and Cannon III R O Role of circulating nitrite and S nitrosohemoglobiun in the regulation of regional blood flow in ...

Страница 164: ...cal conditions nitric oxide is a gas with limited solubility in water In cell cultures some of the NO produced will diffuse from the liquid into the gas above the media Analysis of the NO in the gas above cultured cells so called headspace can be used as an alternative to measuring nitrite nitrate in the media In headspace analysis a gas tight syringe is used to collect the gas above the media and...

Страница 165: ... concentration After the equilibration period a gas tight syringe is used to withdraw the gas sample for injection into the purge vessel The sample sizes can be adjusted to achieve the desired sensitivity but typically 0 1 to 5 cc of gas can be removed from the sample container The best procedure is to first fill the gas tight syringe with a volume of NO free gas The syringe is then inserted into ...

Страница 166: ...ion gas By injecting different volumes of the calibration gas a calibration of peak area versus moles NO injected can be prepared The amount of NO injected is calculated from the volume injected the concentration of the standard and the ideal gas law This technique allows quantitation of the amount of NO in the headspace above the samples but cannot be used to determine the concentration of NO in ...

Страница 167: ...uipped with a gas inlet and outlet is required for dynamic headspace In this technique NO can either be continuously monitored or the sample allowed to equilibrate and then the entire gas sample is injected into the NOA For this technique the purge vessel is not used instead the outlet of the apparatus is connected directly to the gas sampling package of the NOA A source of NO free gas is connecte...

Страница 168: ......

Страница 169: ...zer is serviced and the timer is reset The maintenance schedule is 900 hours for pump oil chemical trap and reaction cell cleaning Service on the cooler is required after one year of operation The pump oil timer is on whenever the vacuum pump is on The timers for the reaction cell cleaning and Hopcalite trap are on whenever the NOA is in the Start mode Changing the Vacuum Pump Oil A flat head scre...

Страница 170: ... trap to the NOA and remove the tubing from the trap Figure 15 1 shows the oil fill and oil drain plugs on the vacuum pump To change the oil Open the oil fill cap and set aside Position the waste oil container and funnel under the oil drain plug Use a screwdriver to slowly open the oil drain plug by turning the screwdriver counterclockwise When the plug is removed the oil will immediately flow out...

Страница 171: ...n plug and reinstall the plug using a screwdriver to secure the plug Fill the pump with 1 2 quart of fresh Edwards oil View the oil level in the window on the pump Add enough oil so that the level is above the minimum level mark but below the maximum level mark Replace the oil fill cap If the vacuum hose was disconnected reconnect the hose and secure with the hose clamp Follow the procedures below...

Страница 172: ...ontact your safety or environmental health department with any questions on disposal of the trap Remove the plastic caps from the ends of the new chemical trap Hold the trap vertically and gently tap on the trap to remove any dust Turn the trap upside down and repeat the process to remove any dust from the other end of the trap Place the hose clamp over the short length of clear Tygon tubing and c...

Страница 173: ...cell and reduce sensitivity In order to restore sensitivity it is necessary to clean the optical filter and reaction cell The recommended schedule is 900 hours of operation with the ozone generator on However it may be necessary to clean the cell more frequently particularly if any liquid is drawn into the analyzer If measuring gas phase NO a calibration outside recommended limits may also indicat...

Страница 174: ...m to room temperature Make sure the power cord is disconnected and then remove the NOA cover by removing the phillips head screws on the side of the analyzer Carefully lift the cover off the NOA Remove the two coaxial cables connected to the PMT socket In order to minimize exposure of the PMT to light turn off the room lights before removing the PMT Unscrew the PMT socket counterclockwise until th...

Страница 175: ... the cooler toward the front of the NOA The optical filter is not attached at this point and may fall out of the cooler if the cooler is moved too rapidly The cooler should slide far enough forward to give access to the filter and the reaction cell With the filter still in the cooler use an alcohol wipe or wet a clean cloth with methanol or ethanol and wipe the deposits from the surface of the red...

Страница 176: ...licone vacuum grease can be applied to the o rings to help secure them Be sure to wipe any excess grease from the o rings before replacing the cooler After cleaning the filter and reaction cell carefully slide the cooler being sure that the o rings and red filter remain in place Slide the cooler back until it contacts the o rings on the reaction cell Insert one of the Allen screws into the back pa...

Страница 177: ...e wires on both sides of the analyzer Position the back of the cover on top of the back panel of the NOA Position the front of the cover so that it rests on the front panel of the NOA Move the front and rear panels a little to help align the cover With the cover resting on top of both the front and rear panels position the front panel to align the front right hand screw holes in the front of the a...

Страница 178: ...top or Stand by mode From the Main Menu use the Arrow Buttons to scroll to Maintenance and press ENTER The Maintenance Menu is displayed Use the Arrow Buttons to scroll to Consumables and press ENTER The display will change giving the option to view or install the consumables Select Install and press ENTER to display the Consumables Menu Select the Cell option and press ENTER to display the Reacti...

Страница 179: ...ill a light leak after tightening the Allen screws turn off the NOA remove the top cover remove the PMT and check the o rings in the reaction cell Reconnect the cooler to the reaction cell install the PMT and top cover and repeat the light leak test Cooler Maintenance Over time the cooler will pick up water from the atmosphere that reduces its efficiency After one year of operation it is necessary...

Страница 180: ...abs on the top and bottom of the connector and pulling the connector off the board Feed the thermocouple connector and the cooler power connector over to the left side of the NOA Lift the cooler from the chassis It may be necessary to move the cooler slightly towards the back to the NOA to align the cooler with the holes in the chassis Place the cooler in a drying oven set to 90 C for at least 2 h...

Страница 181: ...stall and press ENTER to reset the cooler timer After the PMT has cooled to 12 C perform a vacuum test and light leak test as described in the cell cleaning sections Testing and Cleaning the Flow Restrictor Frit Over time the metal frit in the flow restrictor at the back of the NOA can become clogged reducing the flow into the NOA The flow can be checked using a flow meter or by simply monitoring ...

Страница 182: ...estrictor Reconnect the restrictor to the NOA and repeat the flow check In some cases sonication will not remove the blockage and the restrictor will have to be replaced Gas Sampling Particle Filter The filter in the gas sampling package will get clogged over time and needs to be replaced The filter should be replaced when it becomes visibly dirty or wet The cell pressure can be used to test if th...

Страница 183: ...GE Analytical Instruments 2006 15 15 DLM 14291 Rev A NOA delete methods clear error and warning stacks etc Use of these features is not recommended ...

Страница 184: ......

Страница 185: ...d the NOA will indicate the problem by activating the Warning shortcut in the Measurement Menu Use this shortcut to view the warning and when appropriate take corrective action The NOA will also monitor certain parameters during the initial startup and if there is a problem will report that the instrument has failed a startup test without reporting an error The cause of the problem must be determi...

Страница 186: ...Pressure was Above the Limit When the NOA is running this error is reported if the cell pressure rises above 100 torr Make sure that the main power switch on the vacuum pump is in the on position the power cord from the pump is securely plugged into the rear of the NOA and the vacuum hose connections to the NOA and Hopcalite trap are secure Select the Analysis Stand by Mode to start the vacuum pum...

Страница 187: ... cooler need maintenance see Cooler Maintenance page 15 11 This error can also be reported if the cooler power cable is not connected to the Power Supply Board the thermocouple is not connected to the Analog Board or if the fan is not powered Check all of the electrical connections and if the connections are OK contact Sievers Warnings Less serious problems with the NOA are indicated by Warnings U...

Страница 188: ...e lifetime of the oil is reached W 11 Hopcalite Needs to be Replaced The ozone destruction trap has exceeded its recommended lifetime Replace the trap and reset the timer This warning will be repeated each day until the trap is replaced W 12 Hopcalite Needs to be Replaced Soon The ozone destruction trap has reached 90 of its lifetime This warning will be reported each day until the trap is replace...

Страница 189: ...s been exceeded The warning will be repeated each day until the service is performed and the counter reset W 16 Cooler Needs to be Serviced Soon The counter for PMT cooler service has reach 90 of the recommended interval This warning will be repeated each day until the service is performed and the counter reset or the interval is exceeded ...

Страница 190: ...ing can remain in the stack The next time the error or warning is detected it will be activated as indicated by the asterisks the last occurrence date updated and the count incremented Active errors or warnings are always listed on the top row so it is not necessary to scroll down to see active errors or warnings To clear the error or warning stack select the Maintenance Menu scroll to Delete Erro...

Страница 191: ...n E size cylinder and flow controller are being used make sure the tank is on and the flow controller is set to a non zero flow Also check the tubing from the oxygen tank to the oxygen inlet at the rear of the NOA and check that the oxygen supply regulator at the rear of the NOA is adjusted PMT Signal There are several reasons why the NOA may fail the PMT test including a light leak a blown fuse f...

Страница 192: ... replace the top cover plug in the power cord and turn the Main Power Switch ON If the ozone fuse is OK then the problem may be a clogged restrictor There are two restrictors in the ozone flow path a pre restrictor before the ozone generator and a post restrictor that delivers the ozone to the reaction cell To test for a clogged restrictor From the Main Menu press ENTER to display the Status Scree...

Страница 193: ... main fuse for the NOA is located in the power entry module at the rear of the NOA Remove the power cord from the analyzer to open the power entry module To check the main fuse on the NOA Remove the power cord from the analyzer Open the power entry module by inserting a small screwdriver behind the slot at the top of the power entry module and lifting the cover on the power entry module back away ...

Страница 194: ...se is blown then the resistance will be infinite If a fuse is blown replace it with one of identical type and current rating Refer to the System Specification section of this manual for a listing of fuse types and ratings appropriate the NOA 280i To check the Cables Remove the power cord from the analyzer and remove the NOA top cover A ribbon cable connects to the LCD display and the Digital Board...

Страница 195: ...If the Cell Pressure is 4 torr the restrictor may be clogged See Testing and Cleaning the Flow Restrictor Frit page 15 13 for assistance Gas Sampling Problems The most common problem encountered in gas phase NO measurements is out of range calibration The ppm response factor should be in the range 0 09768 0 1465 and the ppb response factor should be in the range 0 1953 0 293 If the response factor...

Страница 196: ... high background will be observed and a loner time required to return to the background levels The most common problem is a dirty Teflon filter Try replacing the filter and see if this solves the problem Liquid Measurements Problems The most common problems observed in liquid measurements are low sensitivity and poor repeatability Both of these problems can be caused by leaks in the purge system a...

Страница 197: ... inside the reducing union Another common location for leaks when using the gas bubbler is at the Teflon sleeve Make sure the connection for the bubbler is tight Leaks can also occur at the septum Try replacing the septum to see if this solves the problem Low Conversion for Nitrate If the VCl3 HCl reagent is oxidized or the temperature of the water bath and purge vessel is too low the conversion o...

Страница 198: ...n from the barrel of the syringe Try using a new syringe to see if this solves repeatability problems Foaming of theVCl3 Reagent When samples containing protein are injected the reagent may foam into the condenser cooling the reagent When the reagent stops foaming and returns to the heated portion of the purge vessel residual nitrate will be converted to NO The result of this foaming is multiple p...

Страница 199: ...hout any reagents in the purge vessel or by trying another purge gas cylinder The most common cause of increasing baseline signals over time is injection of materials that are slowly converted to nitric oxide such as L NAME and other nitro containing compounds If possible use other NOS inhibitors such as methyl arginine or other non nitro containing compounds contact Sievers for additional informa...

Страница 200: ...GE Analytical Instruments 2006 16 16 DLM 14291 Rev A ...

Страница 201: ...gent 11 7 12 3 13 4 Stability 11 8 Apparatus for Nitrate Reduction 13 1 Apparatus for Nitrite Reduction 12 1 Assembly of the Accurate NO Breath Kit 7 2 ATS guidelines 8 15 ATS Recommendations 7 5 7 7 8 1 Auto sensitivity 5 7 Available Flow Restrictors 6 12 B Background Nitrite 12 12 bacterial filter 5 1 Bag Collection and Sampling Kit 1 7 Bar graph in the Measurement Menu 5 5 baud rate 4 16 Breath...

Страница 202: ...he Accurate NO Breath Kit and Flowmeter Disassemble the Valve 7 9 Drying 7 10 Prewash the Components 7 10 Sterilization 7 10 Cleaning the Accurate NO Breath Kit and Flowmeter 7 9 Cleaning the Accurate NO Breath Kit and FlowmeterRinsing 7 10 Cleaning the Bag Kits 8 13 Cleaning the Bags 8 5 Cleaning the Bubbler Tubing 13 15 Cleaning the Chemiluminescence Reaction Cell 15 5 Light Leak Test 15 10 Rese...

Страница 203: ...6 2 E 03 Ozone Supply Pressure was Below the Limit 16 2 E 05 Cooler Temperature Above the Limit 16 2 Electronics 1 9 24V Power Supply 1 10 ADC Board 1 10 microprocessor 1 10 PMT amplifier 1 9 Power Supply Board 1 10 ENTER Button 3 1 Entering the units and concentration for calibration gas 6 6 Environmental Considerations 4 2 Errors E 01 Setup Data Corrupted Check Before Running 16 2 E 02 Cell Pres...

Страница 204: ...e ozone generator 1 7 gas units menu 6 7 Gases 4 2 H Headspace Measurement of Nitric Oxide 14 4 Apparatus 14 4 Sample Collection 14 5 Standards 14 6 High sensitivity 5 7 Hopcalite 1 9 I IFD Filter 11 5 12 1 12 16 Independent Calibration of ppb and ppm Range 6 9 Injection Technique 12 9 13 9 Inspiratory Gas Charcoal Filter Inspiratory Gas Connections 7 4 Inspiratory Gas Filter 7 4 Adapter 7 4 Insta...

Страница 205: ...d Measurements 13 10 Adjustment of Liquid Level 12 3 13 5 Adjustment of Purge Gas Flow Rate12 3 13 4 Analysis of Samples and Standards 13 10 Anti foaming Agent 11 7 Apparatus for Nitrate Reduction 13 1 Apparatus for Nitrite Reduction 12 1 Background Nitrite 12 12 Cell Pressure 12 3 Cleaning the Bubbler Tubing 13 15 Cleaning the gas Bubbler 13 15 Cleaning the Purge Vessel 12 14 COM Port interval 11...

Страница 206: ...lute Standards 13 7 Stability 13 8 Stock Solution 13 6 Preparation of Nitrite Standard Solutions 12 5 Dilute Standards 12 7 Stability 12 8 Stock Solution 12 5 L NAME 13 1 Login menu 5 4 6 4 11 9 Login Menu 4 15 Long term maintenance of the purge vessel and bubble 12 16 13 16 Low sensitivity 5 7 Luer adapter tee 6 3 Luer tee 5 2 M main fuse 16 8 Main Menu 3 1 displaying Measurement Menu 3 5 Main Me...

Страница 207: ...Contamination 12 10 NO Calibration Gas 6 5 recommended concentration 6 6 No Display 16 9 Blown Fuse 16 9 Contrast Adjustment 16 9 Loose Cables 16 9 NO is lower than the zero gas warning 6 9 No Power to NOA 16 8 NO sample line 5 1 NO Pressure Offset Calibration syringe 9 3 NOA Back Panel 4 9 NOA displays 0 0 ppm or 0 0 ppb 6 9 NOA displays ppb 6 9 NOA Setup for Gas Phase Measurements 5 4 NOA Setup ...

Страница 208: ... 6 Selection of NO Plateau 7 6 typical exhalations 7 6 Opening the Gas Bubbler 13 12 Operation Setup Menu 5 7 5 8 11 9 Optical Filter 1 6 1 8 optimum flow rate into the analyzer 6 11 Oxygen tubing 4 13 Ozone Destruction Trap 1 8 Ozone Flow Control Module 1 6 1 7 pressure transducer 1 8 regulator 1 7 ozone fuse 16 6 Ozone Generator 1 6 1 8 P part per million ppm 5 7 parts per billion ppb 5 7 Photom...

Страница 209: ...ecommended limits possible causes 6 8 response factors out of range warning 6 8 response factors too low possible causes 6 8 S SAFETY WARNINGS 1 3 Sample Flow Control Device 1 6 1 7 4 13 frit restrictor 1 7 Sample Inlet System 1 6 Accurate NO Exhaled Breath Kit 1 7 Bag Collection and Sampling Kit 1 7 Gas Sampling Kit 1 6 Purge vessel 1 7 Sampling Inlet Systems 1 6 Schematic of the 280i NOA 1 6 Scr...

Страница 210: ...16 11 Liquid Measurements Problems 16 11 Contaminatio 16 13 Foaming of theVCl3 Reagent 16 13 Ghost Peaks 16 14 Injection Technique 16 13 Leaks in Purge System 16 12 Low Conversion for Nitrat 16 12 Low Sensitivity 16 11 Poor Repeatability 16 12 Syringe Problems 16 13 Liquid Measurements Problems High background Signal and Rising Baselines 16 14 main fuse 16 8 No Display 16 9 Blown Fuse 16 9 Contras...

Страница 211: ...s to be Replaced Soon 16 3 W 11 Hopcalite Needs to be Replaced 16 4 W 12 Hopcalite Needs to be Replaced Soon 16 4 W 13 Reaction Cell Needs to be Cleaned 16 4 W 14 Reaction Cell Needs to be Cleaned Soon16 4 W 15 Cooler Needs to be Serviced 16 4 Warning Shortcut 3 5 3 8 Warnings 3 8 16 3 Clearing the Error and Warning Stacks 16 5 W 09 Pump Oil Needs to be Replaced 16 3 W 09 Pump Oil Needs to be Repl...

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