To design a Standard Operating Procedure that describes the procedure for operation and maintenance of the Plethysmometer.
2.0 SCOPE
This Standard Operating Procedure (SOP) is applicable for the operation, and maintenance of plethysmometer Pharmacology studies conducted in the in-vivo laboratory.
3.0 RESPONSIBILITY
3.1 All personnel in the Pharmacology Department shall be responsible for implementation and compliance with the SOP.
3.2 Head/Designees/Scientists/Study Directors of the Pharmacology department shall be responsible to ensure the implementation and compliance with the SOP.
4.0 DISTRIBUTION
4.1 The quality assurance department is responsible to keep SOP ‘Master Copy’ approved through the Quality Assurance Head.
4.2 The copy of the ‘Control Copy’ of SOP is being distributed in the user department and placed near related Equipment/ Instrument as ‘Display Copy’.
5.0 DEFINITION(S)
5.1 Definition(s)
5.1.1 The Digital Plethysmometer is an instrument used to determine the variation in the volume of rodent limbs, measuring the variation in fluid level on inserting the limb into a tank.
6.0 ABBREVIATION(S)
6.1.1 SOP: Standard Operating Procedure
7.1 Introduction
The plethysmometer (paw Edema) test serves to measure the effectiveness of anti-inflammatory agents to reduce endemic conditions. In use, the paw is interested in water, contained in a special water cell of which the resistance is changed due to immersion of the animal's paw. This resistance change is calibrated in ml and shown on the electronic monitor.
Fig 1 plethysmometer
7.2 Principle
The Digital Plethysmometer is an instrument used to determine the variation in the volume of rodent limbs, measuring the variation in fluid level on inserting the limb into a tank. The introduction of a tissue (or object) into the container changes the level of fluid and conductivity between two platinum electrodes introduced beforehand into the container. This change is expressed in milliliters (mL) with a resolution of 0.01 Ml
7.3 Instrument description
The Plethysmometer basically consists of the following elements which are described below:
7.3.1 The Electronic Unit 7141
7.3.1.1 The Plethysmometer Electronic Unit is lodged in a resilient aluminum cylindrical vessel.
7.3.1.2 The 7141 is a microprocessor-controlled unit, which has the capability to store a great number of measurements and features a direct connection to the PC for data transfer via the data acquisition software provided as standard.
7.3.1.3 The experimental data are shown on the multifunction graphic display, in four digits, with 0.01 ml resolution.
7.3.1.4 All connections are located on the back of the electronic unit, except the connector to the Mini Printer, which is positioned on the bottom of the vessel.
7.3.2 The water cell
The standard package 37140 includes a Standard Water Cell for Rat, diam. 1.8 cm, provided with an extra paw tube diam. 1.3 cm, for measuring the mouse paw volume.
The Plethysmometer can be ordered with a different Water Cell. The additionally available configurations are:-
7.3.2.1 37140-25, which includes a 2.5cm diam. Water Cell
7.3.2.2 37140-35, which includes a 3.5cm diam. Water Cell
37140-25 & 37140-35 include all the Plethysmometer standard accessories.
7.3.3 Calibration volumes
Each cell is complete with a Calibration Kit, including the following Calibration Volumes:
7.3.3.1 0.1ml (yellow)
7.3.3.2 0.2 ml (orange)
7.3.3.3 0.5 ml (pink)
7.3.3.4 1 ml (grey)
7.3.3.5 2 ml (blue)
7.3.3.6 4 ml(green)
7.4 Operations
7.4.1 Switching On
7.4.2 Switch on; the graphic display lights and shows for some seconds the logo “UGO BASILE” and the firmware version which is actually installed.
7.4.3 After some seconds the display shows the configuration of the previous experiment. The operator can maintain it, if convenient, without the need for a fresh setting.
7.4.4 A warm-up period of 2-3 minutes is recommended, in order to improve the measurement precision and minimize drift.
Figure 2 “Top Panel”
7.4.2 “FAULT” Message
In case the “FAULT” caption
appears on the display, this may be due to:
7.4.2.1
Transducer
not properly connected.
7.4.2.2
Empty
cell.
7.4.2.3
Either
too low or too high solution molarity.
7.4.2.4
Solution
level outside the two red marks.
Figure 3: Fault Message
7.
4.2.5 The last inconvenient (i.) can be fixed immediately. Once the solution level is within the correct range, Depress the ZERO key.
7.4.2.6 When the display shows the caption FAULT the operation of the instrument is inhibited; the fault must be detected and its causes removed.
7.4.3 “MEMORY LOSS!” Message
7.4.3.1 In case the caption “MEMORY LOSS!! BATTERIES!” appears on the display, it means that the internal watch-type batteries (non-rechargeable), which maintain the memory, are down and consequently have to be replaced.
7.4.3.2 The data saved in the memory are lost. Once the batteries have been replaced, the operator has to set again the date, time, and experiment configuration including a sequence number, animal number, etc.
Figure 4: Memory Loss
7.4.4 Graphic Display
The graphic display is basically divided into three levels of characters.
7.4.4.1 The upper level shows the volume in ml, in large characters.
7.4.4.2 The intermediate level, generates “information strings” which monitor the status of the instrument and supplies date, time, animal sequence, volume (in ml) sent to the PC, etc.In medium size characters on a single line or in small characters on two lines.
7.4.4.3 The lower level, separated by a horizontal line, indicates in square brackets the function of the keys F1, F2, F3, F4.
Fig 5 graphic display
7.4.5 The function keys
7.4.5.1 The function keys or “soft Keys” F1, F2, F3, and F4 form the top row of the keyboard; the function of these keys is different, according to what the corresponding display section is showing.
7.4.5.2 Each function menu has a different subroutine software; in order to enter a fresh software level, the corresponding function key should be depressed. To leave a menu and go back to the previous software level, depress the F4 (ESC) key.
7.4.5.3 If a key is kept depressed for more than one second, the command is repeated at a higher speed. This helps to introduce numerical data.
7.5 F4 Key
7.5.1 The F4 key, which has the function of the ESCAPE key, enables the operator to go back to the previous menu.
7.5.2 If F4 is depressed in sequence from the main menu, the intermediate level of the display shows:
7.5.3 Date and time
7.5.4 Data-output printing format with sequence number, if preset, etc.
Figure 6: f4 key display
7.6 The zero key
7.6.1 The ZERO key has a kind of “right of way”, in the sense that it zeroes the measure irrespective of the subroutine which manages the instrument at the moment.
7.6.2 By depressing the ZERO key the meter is automatically zeroed, provided the water is in the marked range (red lines on the cell tube) and the instrument warmed up.
7.6.3 The display shows:
+/- 00.00 or +/- 00.01
7.6.4 Function Key Abbreviations:
7.7 Calibration
7.7.1 When the 37140 is put in operation for the first time, it requires calibration via a volume probe.
7.7.2 Calibration is also necessary if the instrument has been off duty for a long time or after having changed the solution, replaced the cell, replaced the transducer, etc.
7.7.3 Use the calibration probes provided with the standard package.
7.2.1.1 0.1ml (yellow)
7.2.1.2 0.2 ml (orange)
7.2.1.3 0.5 ml (pink)
7.2.1.4 1 ml (grey)
7.2.1.5 2 ml (blue)
7.2.1.6 4 ml(green)
7.7.4 To calibrate, depress the F1 (CAL) key, from the main menu; the display is automatically zeroed and shows:-
7.7.5 Select the calibration volume by the F1 (UP ARROW) key, choosing the value in the sequence loop 0.5, 1, 2, 4.0 ml.
7.7.6 Confirm the choice by depressing the F2 (OK ) key.
The display shows:-
7.
7.7 Dip the selected calibration probe into the cell, wait for a few seconds until the value indicated on the display is stable, and then depress the F1 (ENTER) key.
7.7.8 The software updates the display with the correct value and then goes back to the main menu.
7.7.9 This operation may take 1-2 seconds.
7.7.10 Also space the measures by at least 10-second intervals, either when calibrating or evaluating the edema. This will let the water film formed on the tube wall by the water column oscillations settle down and will give time to the electrodes/solution interface to recover steady conditions.
7.8 Quick Calibration
7.8.1 Before each working session, it is advisable to check the calibration: simply zero the display and dip the suitable calibration probe into the cell, then read the value shown by the instrument.
7.8.2 If the “quick calibration” is not successful, it is possible that the solution is somewhat degraded.
7.8.3 Never use a solution older than 2-3 days.
7.9 Maintenance
7.9.1 Unplug the mains cord before carrying out any maintenance job
7.9.2 Electronic Block
7.9.3 The 7141 Electronic Block has no mechanical or moving parts and requires practically no maintenance. The very reliable electronics are assembled on two compact plugin boards, one of them containing the display.
7.9.4 In the remote event of some malfunction, these can be replaced en block.
7.9.5 Replacing the Fuses
7.9.5.1 To inspect and/or replace the fuses, disconnect the main cable first! Insert a miniature screwdriver in the slot indentation and snap out the slide which houses the fuses.
7.9.5.2 It is recommended to use fuses type T800 (800 mA); spares are provided with the Standard Package.
7.9.5.3 Make sure that only fuses with the required rated current and of the specified types are used for replacement. The use of repaired fuses and the short-circuiting of the fuse holders must be avoided.
7.9.5.4 Snap in the fuse slide: the mechanical “click” ensures that it is locked. Check the voltage flag before applying electrical power.
7.9.6 Battery Replacement
7.9.6.1 The 7141 memory is supported by two watch-type batteries Silver 1.55V SR43W. Always use the same type of batteries for replacement.
7.9.6.2 When the batteries are down, the display shows:-
7.
9.6.3 Switch off the unit and disconnect the main cable. Remove the 3 screws positioned on the upper rim of the cylinder, which fasten the display & PCB to the vessel.
7.9.6.4 Extract the panel/circuit block carefully. The cables connecting the panel to the electronics are long enough to enable the drawing of the board, without disconnecting them.
7.9.6.5 The batteries are located on the electronic board; replace them. Reposition the board, fix the screws.
7.9.6.6 At this point, set again DATE and TIME, and carry out the CALIBRATION procedure.
7.10 Cell
7.10.1 To clean the cell, remove the transducer first, by lifting its plastic head, after having freed the connection cable from its clamp.
7.10.2 Avoid the use of organic solvents as they are liable to damage the Perspex surface. Use tap water, a test tube brush, and a soapy solution or a mild detergent.
7.10.3 The large and small tubes of all cell models are detachable. Close tolerances, dimensionally stable Perspex, and a carefully designed O-ring seal provide mechanical stiffness and perfect water tightness without any clamping or fastening device.
7.10.4 To detach the tubes, in case of replacement becomes necessary, exert pull and rotation as when uncorking a bottle, see paragraph 12.2-Other Available Water Cells and Accessories for catalog numbers.
7.10.5 To insert new tube/s, some drops of a soapy solution or silicone oil on the O-Ring may ease insertion.
7.11 Transducer
7.11.1 The 7153 Conductance Transducer provides years or trouble-free operation if not mechanically damaged by accidental knocks.
7.11.2 Handle it with care and never poke with the nail or some improvised “tool” (pencil, lab. spatula, etc.) to “feel” the wires, this may just lead to spoiling their parallelism which is pivotal for the linearity of the instrument.
7.11.3 Grease spots caused by finger contact when handling the transducer and/or traces of organic wastes dropped by paws and diffused through the water, in the long run, may form a film over the wires, which may decrease the transducer efficiency.
7.11.4 To clean the transducer, keep its plastic head between forefinger and thumb and shake it a Becker vessel, filled with alcohol. Keep the transducer head and cable dry. To remove particles, use a small soft water-color type brush. Make a final quick rinsing, using acetone, and let the transducer dry in air before repositioning it in the cell.
7.12 Long inactivity
7.12.1 The instrument does not require any particular maintenance after long inactivity, except cleaning.
8.0 PRECAUTIONS
8.1 All PPEs like apron, gloves, mask, head caps, shoes cover should be worn before using the instrument.
8.2 Press Keys softly.
8.3 Clean the equipment with a dry piece of robe or a lightly moistened one, do not use organic solutions.
8.4 Never use abrasive products or dissolvents.
8.5 NEVER pour water or liquids on the equipment.
8.6 Keep the device dry. ln case it wets, please wipe away the water in no time and dry it. The liquid including minerals may corrode the circuits inside.
8.7 Keep it clean on its surface; you can usually clean the surface of the device with a wet cloth. Do not use chemical reagents, cleansers or strong scour to clean.
.9.0 REFERENCES AND ATTACHMENT
9.1 Reference
9.1.1 F. Vincenzi et alia: “A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats” PLoS ONE 8(1): e54195, 2013.
9.1.2 T. Bertaim et alia: “Dose and Administration Schedule Effect of Tiludronate on Joint Damage in the Model of Complete Freund Adjuvant Induced Monoarthritis in Rats” Open Journal of Rheumatology and Autoimmune Diseases 3: 18-25, 2013.
9.1.3 E. Borbély et alia: “Role of Tachykinin 1 and 4 Gene-Derived Neuropeptides and the Neurokinin 1 Receptor in Adjuvant-Induced Chronic Arthritis of the Mouse” PLoS ONE 8(4): e61684, 2013.
9.1.4 R. Korhonen et alia: “Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase-1” Br. J. Pharmacol. 169 (7): 1525-1536, 2013.
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