Application

This manual applies to the Isolated Heart apparatus Size 5 (IH5) Type 843 for the heart of rabbits up to a body weight of approximately 2.5kg. It can be operated both in the Langendorff mode (retrograde perfusion of the heart) and in the Working Heart mode. The size of heart which can be used is limited in the upward direction by the diameter of the tubes for inflow and outflow. This apparatus allows a maximum cardiac output of approximately 500 ml/min. 

In the Working Heart mode the heart actually performs pressure-volume work; i.e. the left heart chamber pumps liquid from the left atrium through the aorta cannula back into the apparatus. This is different from the usual Langendorff system where the heart is supplied with the perfusion solution through the coronaries; the heart does not perform any measurable volume work in the Langendorff mode. 

Operating Mode Working Heart

General Description:

The apparatus is designed mainly for use with hearts up to the size of rabbits with a weight of 2.5 kg. The following table summarizes the limits of the apparatus:

Operation:

Fig. 1 shows the principle of operation. 

With the exception of the solution reservoir and the thermostat, all parts illustrated are mounted or placed on a Plexiglass stand. Suitable stopcocks and movable connections are provided to permit and simplify the mounting of the organ, initially in the so-called Langendorff mode. 

Perfusate is supplied to the apparatus in excess. Pumps provide the organ with more solution than it needs. The excess solution not required by the organ is pumped back into the reservoir. The perfusate is aerated by bubbling inside the main reservoir and inside the preload reservoir. 

As shown in the schematic diagram, the perfusate ejected by the heart is returned to the large reservoir. By repositioning the return tubing, recirculation can also be arranged through the small preload reservoir or the apparatus can be operated without recirculation. 

Adjustment of the perfusion pressure (in Langendorff mode, during the preparation phase) and of the aortic pressure (during the normal working phase) is provided by the artificial flow resistor operated by a rotary nob. The pressure gauge permits a rough indication of the set pressure. The artificial flow resistor has a virtually linear characteristic and its resistance depends very little on the actual flow. The afterload pressure thus remains largely constant over a wide flow range.

Starting Up

The procedure suggested below has proved satisfactory in practical use. Depending on the type of experiment proposed it may be useful to proceed other than as described. The actual details of the experiment must therefore be left to the decision of the experimenter. Note however that the introduction of the heart into the apparatus must be carried out as rapidly and as gently as possible in order to avoid the possibility of working with a heart which right from the start is not fully functional. Carry out a check before the start of the experiment by using suitable functional tests (e.g. check Bayliss effect, note literature reference).

Note:

At the start of an experiment in the Working Heart mode (WH mode) the heart is initially being operated in the Langendorff mode and only switched over to the Working Heart mode when it has recovered from the stress of preparation. The flow through the coronaries is thus initially retrograde in the Langendorff mode and supplies the heart muscle without it having to perform any work. Pump (7) in conjunction with the afterload pressure regulator (2a) and (5) produces the necessary aortic pressure (e.g. 50 mm Hg). 

Later, after the recovery phase and when the atrium cannula has been bound in correctly, the atrial flow is opened up at stopcock (13d) and the heart takes over the function of pump (7). The pump is then switched off. The afterload pressure (aortic pressure) can be adjusted at the pressure regulator (5) exactly as before. The pressure gauge (5a) indicates the pressure setting. The aortic flow is measured with the flow transducer (3a).

Preparation

When starting up for the first time and also after each experiment, all parts in contact with the perfusate
have to be cleaned thoroughly. (Fill in hot cleaning solution and allow to stand e.g. overnight!).

Observe the cleaning specifications! 

Remove the cleaning solution from the apparatus and thoroughly rinse with distilled water. Do not
forget to rinse bubble trap and catheter tubing to remove all solution residues. 

Make up the perfusion solution, preferably fresh each day (filter it!). If possible warm it to the required
temperature e.g. 37° C in a warming cabinet, and place it into the reservoir (8).

Switch on the thermostat (37° C), allow the apparatus to warm up to the operating temperature.

Switch on the aeration and adjust the bubbling through the perfusate reservoir at the appropriate
needle valve (10).

Switch on the measuring instruments and recorder and after a warm-up period (approx. 15 min)
perform zero adjustment and calibration. 

Plug the preparation tubing with tubing adapter on to the connection for the aorta cannula (4d). Run the free end of the tubing back to the reservoir.

Filling the Aortic Block

Switch on pump (7) for Langendorff operation; ensure correct direction of rotation! Close the tubing pressure finger and adjust the correct pressure on the pump tubing. Note the information in the Operating Instructions of the pump. Set a low output, e.g. 10 ml/min.

On the pressure regulator close the vent valve (5b); using the control of the pressure regulator (5) set the afterload pressure controller to about 50 mm Hg. The pressure is indicated on the pressure gauge (5a).

If you are using a micro tip catheter pressure transducer (MTC) for later measurement of the LVP, release squeeze seal (4h) on the access port (4g) and insert the catheter. Advance it so far that it is located in the connection tube of the aorta cannula (4e) so that it is initially in a protected location. If you are not using a tip catheter, close the Luer taper (4g) with a Luer blind stopper.

Using the syringe (2h) and its shut-off stopcock (2g), allow the air vessel (2e) to fill about half full with perfusate. Filling the air vessel can also be achieved by a different method: remove syringe (2h) and open the corresponding stopcock (2g). Then on pump (7) press the "MAX“ key and at the same time prevent flow through the preparation tubing by squeezing it shut. The liquid level in the air vessel now rises rapidly. When the required level is reached (half full), close the open stopcock (2f) again, release the squeeze on the preparation tubing and release the "MAX“ key. The previously set low flow (10 m/min, see above) now flows again through the preparation tubing back to the reservoir. The pressure in the air vessel is then approx. 0 mm Hg

Filling the Atrium Supply

Close the Stopcock (13d)!

Switch on pump (11) for the atrium supply; note direction of rotation! Close the tubing pressure finger and adjust the correct pressure on the pump tubing. Set the flow rate higher than the expected atrial flow, e.g. 20 l/min. The preload vessel (13) fills itself up to the level of the draw-off tube (13b). If the pump tubing diameters have been chosen correctly the liquid level does not rise any higher. Check this condition before the vessel overflows!

Now vent the liquid system to the atrium head and the corresponding connecting tubing to remove all air from it. Carefully open the stopcock (13d) and wash out all air bubbles. Then set the stopcock (13d) so that there is a small perfusate flow out of the atrium cannula and fill the bubble trap in the atrium head (15) using the syringe (15g) and the corresponding shut-off stopcock (15f). To fill the tubing to the pressure transducer (17) and the dome itself free from air bubbles you close off the atrium cannula with the finger and open the venting stopcock on the dome.

Close the Stopcock 13D again!

Adjust the aeration in the reservoir (8) with the corresponding needle valve (10).

Test Run: Dry Run

After you have completed the preparations up to this point it is useful during the first start-up to carry out a few "dry runs“ without an organ, in order to become familiar with the behavior and function of the apparatus with its connected instrumentation and to practice its operation. In order to simulate the working heart you require a pump with a suitable output. If necessary you can use pump (7). The test run in the Langendorff mode should be carried out first since this involves the use of pump (7) in the apparatus. If you then carry out the test run in the Working Heart mode you use pump (7) as heart replacement. The connection (2a) must of course be closed with a suitable piece of tubing so that a pressure can build up in the air vessel (2e) through the pumping action of the replacement heart.

Test Run in Langendorff Mode

Place any aorta cannula on the aorta connection and close the outlet with a short piece of tubing (coronary replacement) which you partly close off with a hose clip during the test in order to simulate the coronary flow.

Check that the return tubing (8c) is not kinked and that the return flow is not impeded.

Switch on pump (7) and set a flow rate of approx. 50 ml/min.

Set the pressure regulator (5) for the adjustable flow resistor (2b) to 50 mm Hg [vent valve (5b) must be closed; the pressure setting is read on the pressure gauge (5a)].

The pressure in the air vessel (2e) should now rise to about 50 - 60 mm Hg. The rise in pressure is measured by the pressure transducer (6) and can be monitored on the pressure amplifier and the associated recorder.

Check all connections for leakage. Rectify any leakage before continuing.

Now change alternatively the pressure [by turning the pressure regulator (5)] and the output of pump (7) and note the corresponding changes in flow and pressure. When you increase the pump output you note an increase in pressure which you do not really expect. This pressure increase is caused by the flow resistance of the afterload pressure regulator (2b) and the return line (8c). 

Now check the effect of the air vessel (2e). Set the afterload pressure with the control of (5) to about 100 mm Hg, the pump (7) to a medium flow rate (e.g. 100 ml/min) and alter the filling level in the air vessel [use syringe (2h) and the corresponding stopcock (2g), remember the minimum filling level (=top edge of outlet bore!)]. On the recorder you can see the variation of the pressure pulsations caused by the pump. The pulsation amplitude which depends very much on the type of pump used, becomes larger as the air cushion is reduced, and vice versa. During the subsequent experiment you will observe the same effect. You have to select the air volume in the air vessel so that the recorded pressure curve has the most „physiological“ shape; then you have found the optimum air vessel adjustment.

Test Run Working Heart Mode

The description below assumes that the apparatus has been prepared according to Section 7.2. As mentioned earlier (Section 7.3) you require for this test run an additional pump as heart replacement. If you are using pump (7) for this test run you have to close the outlet (2a) with a short piece of tubing, for example.

Connect the additional pump as heart replacement into the apparatus between atrium cannula (16) and aorta cannula (4e).

Switch on pump (11) and set an output of approx. 200 ml/min.

Open the stopcock (13d) and switch on the heart replacement pump. Initially set a pump output of about 100 ml/min. The pump output is measured with the flow transducer (14) or (3a) and can be read on the corresponding flowmeter.

If you now set an afterload pressure of e.g. 50 mm Hg on the pressure regulator (5) [can be read on the pressure gauge (5a)], the aortic pressure should also increase due to the pump output. 

With data acquisition you can see the various measurement signals: 

• Aortic pressure [pressure transducer (6)]. Because of the additional hydrostatic pressure of the liquid column (air vessel - pressure transducer) the recorded pressure is slightly higher (about 8 mm Hg) than the value read on the pressure gauge (5a). Pulsation is more or less pronounced depending on the air cushion set in air vessel (2e).
• Atrial pressure [pressure transducer (17)]. The recorded atrial pressure may exhibit pronounced negative peaks, due to the (non-physiological) suction effect of the heart replacement pump used. Evaluation and comparison with the later experiment (with the heart bound in) is therefore possible only to a limited extent.
• Aortic flow [flow transducer (3a)]. The associated flowmeter should indicate the flow set on the heart replacement pump (here: 100 ml/min). With pulsatile recording a strong pulsation can be seen, due to the action of the mechanical pump
• Atrial flow [flow transducer in (14)]. The associated flowmeter should indicate the flow set on the heart replacement pump, as above (here: 100 ml/min). With pulsatile recording a strong pulsation can be seen, due to the action of the mechanical pump.
• Provided you have set the correct indication for zero flow and the calibration factors, the indications for atrial flow and aortic flow should differ very little (3 - 5%). If you find larger differences, then this may be due to different magnitudes of the flow pulsations at the two measuring points. The only remedy is the use of a heart replacement pump with less pulsation

Now vary the output of the heart replacement pump and observe the behavior of the different recorder traces. Aortic pressure increases slightly with an increase in flow, but not proportionately (see characteristic curves of the adjustable flow resistor [afterload] in Section 15). 

During the variation check for the maximum possible atrial flow. If you exceed the flow rate set on pump (11) (in this example the setting was 200 ml/min!), the reservoir (13) will obviously run dry. If you later use a heart with a large ejection capacity you have to set the output of pump (11) correspondingly large enough so that there is always sufficient perfusate available in the reservoir. You should however not set an excessively large flow rate in order to avoid unnecessary wear on the pump tubing.

If you have carried out the test procedures up to this point and have tried out the different settings of the apparatus, you should restore baseline settings.

Preparation and mounting of organ

The heart can now be prepared and placed into the apparatus. An extensive description of the actual preparation of the heart (for rats and guinea-pigs) will be found in the publication Biomesstechnik, V. "The isolated perfused heart after Langendorff," 

Before the start of the actual organ preparation the apparatus should be prepared as described above. Summarizing briefly, the apparatus should be in the following condition:

1. The reservoir is filled with fresh perfusate and is at the correct temperature (pre-warm the perfusate)
2. Thermostat is switched on, all parts of the apparatus at the correct temperature
3. Pump (11) is switched on, the required output is set
4. Aeration of perfusate is adjusted
5. Atrium head and all tubing and transducer domes are free of air bubbles
6. Shut-off stopcock (13d) is closed
7. Pump (7) is switched on, the required initial output is set (10 ml/min)
8. Air vessel (2e) is half full of perfusate
9. Afterload pressure is set on (5) to 50 mm Hg as read on pressure gauge (5a)
10. Preparation tubing is placed on aorta connector (4d) and filled free from air bubbles; the small perfusate flow (10 ml/min) provided by pump (7) drips off the free end of the preparation tubing and can initially drip into a container at the operating table
11. Measuring instruments and recorder switched on, calibration and zero adjustment have been completed
12. Tubing to the two pressure transducers (6) and (17) and the two domes filled free from of air bubbles.

Organ preparation:

Anesthetize the animal, perform tracheotomy and ventilate. Open the thorax.

Transfer the organ into the apparatus in two steps:

• Spray the heart with cold perfusate (spray bottle) to cool the heart. Place a suitable aortic cannula on the end of the preparation tubing, clamp off the vena cava, open the pulmonary artery, bind the cannula into the aorta and immediately raise the output of the pump (7) so that the aortic pressure of the about 50 mmHg is obtained. Blood is washed out, heart muscle color changes to light pink.
• Since the organ is now being supplied with oxygen and perfusate, the remaining preparation stages can be carried out less urgently. Take the heart out of the animal as it hangs from the preparation tubing, and remove any disturbing adhering tissue.

Remove the aorta cannula with the heart from the preparation tubing and mount it as rapidly as possible directly on the aorta connector (4d) in place of the preparation tubing. Ensure that no gas bubbles enter the aortic cannula during this operation. Position the cannula so that the left atrium points to the left. 

Allow the organ to stabilize and recovery (15 - 30 minutes).

The aortic flow sensed by the flow transducer (3a) corresponds to the coronary flow (in view of the flow direction the reading is negative).

Perform a test on the organ for perfect function (Bayliss effect, administer test substance, or similar).

Place a suitable atrium cannula (16) on the atrium head (15) and fill it with fresh solution free from air bubbles [open stopcock (13d) at little so that perfusate drips off the cannula (16)].

Bind the atrium cannula into the left atrium. Take care that as much of the atrial function as possible is retained in order to ensure good chamber filling.

Bind off open vessel stumps on the atrium. Now fully open the stopcock (13d) and open up the atrial flow. Check the atrium for any open vessel stumps and bind these off. 

The heart should now beat regularly and rhythmically and perform work. Perfusate is ejected at the aorta. The actual flow is measured by the flow transducer (3a) and can be read on the associated instrument. The polarity of the flow indication changes from negative to positive according to the flow direction in the aorta cannula. Now switch off the pump (7) and monitor the aortic pressure. If the heart ejection is too small so that no positive aortic flow is produced, the pump (7) must initially remain switched on to ensure an adequate supply to the heart. The problem must be rectified before switching off the pump. 

A few possible errors related to the organ:

Other possible errors not due to the organ:

When the aortic flow is large enough, you can switch off the pump (7). NOTE: do not release the tubing pressure finger on the pump, it must remain pressed down!

NOTE: the aortic pressure must not drop appreciably below 50 mmHg to ensure the supply to the heart muscle.

Adjust the control of the pressure regulator (5) to set the aortic pressure to the value you require and allow the organ to stabilize under the new conditions before starting the actual experiment.

If you are working with recirculation, remember that the test substances remain in the perfusion circuit and accumulate. When (or before) administering the test substance it may be useful not to return the perfusate discharge to the reservoir but to collect it in some other container.

If you are using a Micro Tip Catheter pressure transducer (MTC) for measuring the LVP you can now advance it into the left ventricle. The MTC is a sensitive and, as you must know, a very expensive item (price of SPR 407: DM 6000). Handle it with care and never use force when handling it. If it is damaged it is very rarely possible to have it repaired. You must then purchase a new one or abandon this excellent and precise method of measuring LVP (note Fig. 5 and the corresponding description).

• To insert the MTC, release the pressure piece (4h2) of the squeeze seal (4h) slightly until the MTC can be moved easily, and carefully move it forward. If the catheter is difficult to move in the squeeze seal you should apply a little Vaseline to it.
• Now advance it further until you can feel the movement of the aortic valve in your finger tips. Monitor the LVP trace on the appropriate recorder channel. Then quickly slide the catheter tip into the ventricle when the aortic valve is open during systole. The characteristic changes in the pressure curve show immediately that the catheter tip with its pressure sensor is in the ventricle. With a little practice you will find that inserting the MTC is a very simple and safe procedure.

The preparation for the experiment are now completed. Adjust the filling level of the air vessel (2e) so that the shape of the aortic pressure traces are as nearly physiological as possible. Do remember, however, that the rigid pipe system of the apparatus can never completely replace the elastic vascular walls in situ. The traces of the pressure and flow signals obtained on the apparatus can therefore never show an exactly "physiological“ form.

During the experiment you must monitor the bubble trap in the atrium head. Any gas bubbles washed in must be removed in good time before the trap is filled so far that gas bubbles can pass into the heart through the atrium cannula.

Now perform the experiment as planned: administer test substance as bolus or mixed into the perfusate, change perfusate, use a different aeration gas, alter temperature, or whatever.

Pressure Measurement

Pressure measurement is only correct and free from errors if certain preconditions are fulfilled. 

• Tubing and dome of the pressure transducer must be filled with liquid and free from bubbles, especially when the evaluation involves analyzing the trace (e.g. recognizing the notch in the trace in order to measure the heart ejection time) it is important that even the smallest bubble is removed so that steep parts of the trace can be measured accurately.
• The pressure measuring system must be calibrated (example instructions here). 
• Pressure transducers and connecting tubing must not move during measurement. Movement of the tubing or transducer produces movement artifacts which are superimposed on the measured pressure signal.
• The pressure transducer must be mounted at the level of the aorta or atrium.
• Perform zero adjustment correctly and in the correct order.

Performing zero adjustment:

• Allow instruments to war-up (at least 15 minutes).
• Move stopcock (6a) to position (6a2). IMPORTANT: stopcock (6a) must be mounted in such a position that he unused side connection is horizontal. The pressure in the dome is now atmospheric pressure.
• Now zero the pressure amplifier (e.g. bridge amplifier such as the TAMA).
• For measurement, move stopcock (6a) back to position (6a1). Stopcock (6b) remains continuously closed during zeroing [position 6b1]. It is opened only for filling the dome free from bubbles [position 6b2]. Stopcock position (6a3) serves for filling the connecting tubing free from gas bubbles. 

Flow Measurement

Two different principles can be used for flow measurement: electromagnetic measurement and ultrasonics (Transit Time Flow Meter TTFM). In general, good results can be obtained only if certain points are observed carefully. Electromagnetic flow measurement is generally more troublesome than ultrasonic measurement. If there are any problems, the Operating Instructions for the flowmeter should be consulted first. The notes below provide some additional information concerning the use of any flowmeters built into this apparatus.

IMPORTANT: fill flow transducer free from gas bubbles! Irrespective of the principle of your measuring system it is important to ensure that the lumen of the flow transducer (measuring head) is filled free from gas bubbles. If the lumen is not filled properly, the flow indication is either completely absent or greatly disturbed. Bubbles can be removed by producing a sudden movement of the liquid inside the lumen, using a syringe (not too small!). With ultrasonic flowmeter heads it is particularly important to ensure freedom from bubbles as these interfere with the measurement. If there are any air bubbles in the ultrasonic flow probe the meter indicates "Ac.Er“ (acoustic error). These bubbles, too, can readily be removed by producing a sudden movement of the liquid using a syringe. Use the same syringe as is used for adjusting the air vessel. (cf. Fig. 14). Air bubbles should be removed immediately on filling the apparatus and before the start of the experiment.

Check the calibration by volumetric measurement

You require a calibrated measuring cylinder and a stopwatch

• Set the factory calibration factor manually, using the information supplied, as described in the Operating Instructions of the flowmeter.
• Check the zero (clamp off tubing or close stopcock!).
• Pass flow through the transducer at a rate which is of the same order as the factory calibration factor and should be as constant as possible.
• Collect the perfusate flowing through in the measuring cylinder for exactly one minute. 
• Compare the volume obtained with the indicated measurement. If you find any deviation, correct the calibration on the instrument until the numerical indication agrees with the volume found. NOTE: in the case of the ultrasonic flowmeter (TTFM Type 700) the calibration of the measuring head is permanently programmed in the connector and can only be altered by the factory.
• Repeat the procedure, check the calibration found and note it in your records.

During the entire calibration procedure the set flow rate must of course remain unchanged and constant. The calibration factor found is valid only for the solution used for the calibration measurement. If you are using a different perfusate in your experiment you have to repeat the procedure described with this different perfusate.

Electromagnetic Flow Measurement

The following information concerns measurement with the electromagnetic flowmeter EFM Type 693 and the appropriate flow transducers. The first source of information for working with this instrument should be the Operating Instructions for the EFM which are supplied with the instrument.

Basics:

Electromagnetic flow measurement is based on the measurement of minute electrical voltages (a few µV) which are induced through the movement of the liquid in the magnetic field of the transducer. The measurement voltage is picked off through very small-area electrodes in the lumen of the transducer. Flow measurement is in general a difficult procedure and requires detailed adherence to certain rules if good results are to be obtained.

For successful flow measurements it is important that:

• Electrical interference is kept away from the flow measuring head
• Surfaces of the pick-up electrodes inside the measuring head are properly maintained so that they are kept "clean" (so the resistance stays low).

The first is achieved by carefully earthing the liquid column before and after the measuring head by means of metal tubes. It is important that the cross-section of the connecting cables is sufficiently large (at least 1.5 mm²) so that the so-called stray currents are efficiently discharged and kept away from the flowmeter head. In addition the earth socket (blank socket for 4 mm banana plug on the front panel of the PLUGSYS housing) must be used as the earth point.

When servicing the pick-off electrodes inside the measuring head you should observe the following points:

• Allow the measuring head lumen to dry out only after it has been thoroughly rinsed with distilled water.
• When the liquid is stationary (flow = 0) switch off the excitation current of the measuring head at the flowmeter to avoid unnecessary heating of the measuring head which results in deposits on the electrode surfaces.
• When starting up a dry measuring head, fill the lumen with perfusate free from bubbles and allow it to stand ("soak") for about 1/2 hr before starting the measurement.
• Clean dirty electrodes with a soap-free cleaning agent (a domestic detergent such as Ajax) using a suitable round brush or pipe cleaner. Take care that as little plastic as possible is "polished away" during this operation. Then thoroughly rinse with distilled water.

In principle: if the zero is unstable or the signal is noisy, the cause is virtually always a dirty electrode surface.

With trouble of this type the remedy is always the same: the measuring head must be cleaned, cleaned, and cleaned again ... (but do not polish away any plastic), or better still avoid any contamination in the first instance (see above)!

The points made in the introduction lead to the following additional requirements:

Plugs of measuring head and magnet/signal cable must be kept dry! Wet or moist plugs cause electrical shunting, leading to measuring errors or even to complete failure of the flow measurement.

Measuring head and magnet/signal cables must not move! Moving cables produce electrical artifacts which are superimposed on the measurement signal and produce errors. Therefore ensure that the cables are not arranged together with the "pulsating“ pump tubing.

Never arrange measuring head and/or magnet/signal cables close to a mains supply cable! The mains voltage and spikes superimposed on the mains voltage are some 100 000 times larger than the measuring voltage of the flowmeter head so that corresponding interference must be expected.

Calibration:

In order to achieve correct flow measurement the calibration factor of the measuring head used has to be set on the flowmeter.

The calibration factor of each measuring head is normally evaluated by the manufacturer and marked on the packaging together with the serial number.

Example: K - 5.0, Cal. Factor = 275 ml/min, Serial No: 2834

Serial number and lumen of the measuring head can be found on the connector (e.g. 2834 5.0) so that the transducer can always be clearly identified.

This value ("0275 ml/min“ in this example) must be set on the associated flowmeter. The measured flow is
then indicated directly in ml/min.

The calibration factor is measured by the manufacturer on the basis of a 0.9% saline solution. If you use a different solution or whole blood you have to check the calibration by the procedure described above and make an appropriate correction to the setting, since the sensitivity depends to a slight extent also on the liquid passing through.

Ultrasonic Flow Measurement

The following information concerns measurement with the ultrasonic Transit Time Flow Meter TTFM Type 700 or the Transonic Flowmeters T106 and T206, together with appropriate flow transducers. The first source of information for working with this instrument should be the Operating Instructions for the TTFM which are supplied with the instrument.

Basics:

Ultrasonic transit time flow measurement depends on measuring the transit time of ultrasound through the liquid. By measuring it in two opposite directions the movement of the liquid produces a displacement of the 
transit time which is converted by an integrating procedure into a flow in ml/sec or l/sec. Flow measurement by this principle is very simple and free from interference.

NOTE: The only requirement for obtaining good results is to ensure the probe is filled free from bubbles.

Any stray voltages which often cause problems in electromagnetic flow measurement do not influence the measurement. The flow probes are usually pre-calibrated. The zero can be corrected on the flowmeter.

In general, the following points have to be observed (as with electromagnetic flow probes):

• Allow the measuring head lumen to dry out only after it has been thoroughly rinsed with distilled water! This applies especially when working with whole blood or solutions containing erythrocytes.
• When starting up a dry measuring head, fill the lumen with perfusate free from bubbles and allow it to stand ("soak“) for about ½ hour before starting the measurement.

The points made in the introduction lead to the following additional requirements:

• Plugs of measuring head and extension cable must be kept dry! Wet or moist plugs cause electrical shunting, leading to measuring errors or even to complete failure of the flow measurement.
• Never arrange measuring head and/or extension cables close to a mains supply cable! The mains voltage and spikes superimposed on the mains voltage can influence the measurement.

Unlike in electromagnetic flow measurement, connecting the flow probe to the flowmeter results in automatic identification of the transducer type and calibration values.

The serial number of the measuring head is engraved on the connector so that it can be uniquely identified at all times.

The manufacturer's calibration has been determined based on 0.9% saline solution at 37°C. Calibration for whole blood is available upon request. 

Cleaning and Storage

After the experiment has been completed, drain any perfusate residue from the apparatus and rinse it thoroughly with distilled water. Use a short piece of tubing to link the atrium cannula (16) with the aorta cannula (4e), then fill the complete system with warm cleaning solution and allow the filled apparatus to stand overnight. Use only the permitted cleaning solutions!

NOTE: Do not keep the system filled with cleaning solution for more than 24hrs. If the solution is kept in the apparatus for longer than 24hrs then there is a danger that the cleaning solution diffuses into the tubing and plexiglass and won't be able to be removed upon rinsing. The cleaning solution should be removed at the latest after 24hrs and the system should be immediately rinsed through with distilled water.

Shutting Down

Short-term storage (< 1 week)

If you want to shut the apparatus down for a few days you should not allow it to dry out after cleaning but should let it stand filled it with distilled water. In order to avoid or at least reduce the growth of algae the apparatus should if possible not be placed in bright light and should be protected against the action of light (cover over with a dark cloth).

Long-term Storage (> 1 week)

If you plan for a longer shut-down period all liquids should be removed from the apparatus after cleaning and rinsing, and the apparatus should be allowed to dry out. Also drain the thermostat circuit and rinse it with distilled water. Ensure that no liquid residue remains inside the apparatus, if necessary by tilting it. Liquid residues form a good substrate to algal growth. When starting up again you may then have a great deal of (unnecessary) trouble to remove the undesirable algae from the apparatus.

Re-starting

When the apparatus has been kept dry and is being placed back in use after a longer period, it is best to proceed as if starting for the first time. As a first step carry out a thorough cleaning procedure. Check that all connections are tight. Replace any tubing which does not look good or is porous or brittle.

Measuring Coronary Flow and Oxygen Saturation

For accurate measurement of the coronary flow with an additional flowmeter it is necessary to cannulate the pulmonary artery. The perfusate outflow is measured with a flowmeter head. To measure also oxygen saturation, part of the coronary flow (approx. 1 ml/min) is pumped by a low-flow tubing pump in a bypass through a thermostated measuring chamber and evaluated there with an oxygen pO₂ electrode (ZABS No.1). Operation of the pO₂ electrode requires a suitable instrument, e.g. HSE PLUGSYS module OPPM Type 697.

When cannulating it is important that the pulmo-arterial outflow (= coronary flow) is not restricted (cannula lumen must be sufficiently large, avoid kinking the tubing) and that no suction is generated in the pulmonary artery through an outflow tubing hanging down low.

When measuring oxygen saturation it is also important to ensure that cannula and tubing are made from a material impervious to oxygen (stainless steel, thick-walled Tygon tubing) in order to prevent any oxygen exchange between the perfusate and the surrounding air. This would produce correspondingly large errors in oxygen measurement.

NOTE: Silicone tubing is not suitable for this application due to its high oxygen permeability. 

Technical Description

Figure 3. Functional diagram of the apparatus with item number for the legend below:

Diagram number	Item	Item Description
1	Mounted Isolated Heart	The heart is shown rotated out of position in situ. The left atrium which normally points towards the right (viewed from the front) is on the left.
2	Aortic Block	Central component, made from Plexiglass. It serves essentially as an artificial aorta. The block is supplemented by the flowmeter head adapter (3) and the stopcock (4), with the necessary connections and devices, in order to meet the various requirements of the different possible experiments.
2a	Connection	For feeding the perfusate to the aortic block (in the Langendorff Mode).
2b	Adjustable Flow Resistor	In the WH mode is serves as an adjustable artificial circulation resistance. In the Langendorff Heart mode it is used to set the required perfusion pressure. It consists essentially of an elastic diaphragm with clamping cover. The pressure set on the pressure regulator (5) acts on the cover side of the diaphragm. If the pressure on the perfusate side of the diaphragm is larger than the set pressure, the diaphragm lifts and the perfusate can flow through underneath the diaphragm and passes via the oscillation damper (2c) and the tubing (2d) back to the reservoir.
		TO REMOVE: In the WH mode is serves as an adjustable artificial circulation resistance. In the Langendorff Heart mode it is used to set the required perfusion pressure. It consists essentially of an elastic diaphragm with clamping cover. The pressure set on the pressure regulator (5) acts on the cover side of the diaphragm. If the pressure on the perfusate side of the diaphragm is larger than the set pressure, the diaphragm lifts and the perfusate can flow through underneath the diaphragm and passes via the oscillation damper (2c) and the tubing (2d) back to the reservoir.
	Note	the diaphragm thickness is essential for the correct functioning of the adjustable flow resistor. Cover and diaphragm are matched to each other. The correct diaphragm thickness in units of 0.01 mm is engraved on the cover (example: 50 = 0.5 mm). Fit only original diaphragms of the correct thickness!
2C	Oscillation Damper	The adjustable flow resistor (2b) is followed by an oscillation damper which serves to improve the dynamic characteristics of the system. The damper is mounted directly next to the flow resistor on the back of the aorta block (2) in order to achieve a favorable characteristic. The oscillation damper consists of an elastic diaphragm with clamping cover. With pulsing perfusate flow it reduces the resulting pulsation in the liquid system and dampens the reaction on the flow resistor (2b)
		TO REMOVE: The oscillation damper is mounted on the back of the aortic block (2) directly next to the flow resistor. After releasing the four fixing screws it can be removed, for example for cleaning. WARNING: do not damage the sealing edge in the cover! When re-assembling, ensure that the diaphragm is located accurately in the recess of the cover. Only tighten the fixing screws so far that the cover just rests on the aorta block without a gap; do not overtighten the screws!
	NOTES	Operating note: Proper operation of the oscillation damper is only ensured if there is no appreciable positive or negative pressure on the diaphragm to prestress it. The tubing to the connection (2d) must therefore not be kinked and should terminate approximately at the liquid level inside the aorta block. The difference in height should not exceed 50 cm.
		Cover and diaphragm are matched to each other. The correct diaphragm thickness in units of 0.01 mm is engraved on the cover (example: 50 = 0.5 mm). Fit only original diaphragms of the correct thickness!
2D	Outlet Connection	This tube connection (6 mm o.d.) forms the outflow of the oscillation damper (2c). In the Langendorff mode the excess volume supplied by pump (7) appears here. In the Working Heart mode the volume ejected by the heart is discharged from the aorta block through this connection and (during recirculating operation) back to the reservoir (8).
2E	Air Cushion of the Air Vessel	The upper volume of the aorta block (2) forms the air vessel. Its task is to reproduce as far as is possible the elastic properties of the aorta in situ. This desired replacement of the aorta can only be imperfect. For this reason the course of the aortic pressure trace measured on the apparatus approximates to the curve measured in situ only if the air cushion has optimum adjustment, i.e. in accordance with the actual situation. The volume of the air cushion determines the damping effect of the air vessel. A large air volume produces better damping of the pulsation than a small volume. The air volume is altered with the syringe (2h). The air volume is limited by the position of the outlet bore to the adjustable flow resistor (2b). With maximum air volume (approx. 15 ml) the perfusate level in the aorta block is approximately 2.5 cm high.
2F	Air Vessel Connection	A small tube with a female Luer taper is provided in the upper cover plate of the air vessel (2e). A syringe (2h) together with a shut-off stopcock (2g) is connected here. Syringe (2h) together with the shut-off stopcock serves to adjust the air cushion of the air vessel (2e).
2G	Shut-Off Stopcock
2H	Syringe	Example: 20ml
3	"Fitting Adapter" for Flowmeter Head	A suitable flowmeter head (option) can be fitted into this adapter to measure the coronary flow (in the Langendorff mode) or the aortic flow (in the WH mode). If no flowmeter is ordered, a blank piece with a through bore is fitted at this point. The adapter is mounted with 4 hexagon socket screws M3x12 each between aortic block (2) and stopcock block (4). After removing the 8 screws (use the screwdriver supplied!) the adapter can be dismantled. During re-assembly ensure that the O-ring seal (6x2 mm, Silicone) lies in the groove provided. This side must be towards the aortic block (2).
3A	Flowmeter Head (optional)	Mounted permanently inside the fitting adapter
4	Stopcock Block with Main Stopcock	The main stopcock (4a) is used to close the outflow from the air vessel. In the Langendorff mode it can be used e.g. to perform a total ischemia. Through the provision of a side access port (4b) the stopcock also permits feeding in a special solution, e.g. to perform cardioplegia experiments without mixing with the contents of the aorta block. The different stopcock plug positions and their relation to the plug handle are shown in Fig. 4. Dismantling: after releasing the 4 fixing screws the stopcock block can be removed from the intermediate adapter (3). During re-assembly it is important that the O-ring seal (6x2 mm, Silicone) is located in its groove. This side must be towards the adapter (3).
4A	Main Stopcock	For details of its function
4B	Side Access Port	Side access port to the main stopcock (4A)
4C	Connection	Connection for measuring the aortic pressure by a pressure transducer
4D	Connector for 4E	Connector for fitting the aortic cannula (4E). The connector carries 2 grooves to take 2 o-rings (7 x 1.2m), Silicone). These o-rings perform two tasks: they provide a seal and they retain the aortic cannula with the heart attached to it.
4E	Aortic Cannula	Aortic cannula. Several cannulae of different dimensions are supplied in the accessories to suit the heart size.
4F	Port for Micro-Tip Catheter	Insertion port for a micro-tip pressure transducer sizes 2F to 3F. The small tube is extended by a length of the thin guide tubing up to the systems' top plate. Here there is a metal luer connection (4G), female, wo which the necessary insertion adapter (4H) is connected.
4G	Connector for 4F	Connector to the insertion port (4F) - Female Luer Adapter
4H	Insertion Adapter	Insertion adapter for micro-tip catheter (MTC) pressure transducer MTC. This adapter allows the insertion of a micro-tip catheter (size 3French max) from above into the aortic cannula with a squeeze seal. The adapter is fixed with its male luer taper to the connector (4G). The insertion adapter (4H) consists of three parts.   The body (4H1) with luer taper, the silicone squeeze seal (4H3, 3mm OD, 1.6mm ID, 4mm long), and the pressure piece (4H2) with ring nut and anti-kink extension. The silicone seal is inserted into the body and compressed from above by the ring nut. This secures and seals the MTC passed through the adapter. To introduce the MTC (4H4) remove the pressure piece (4H2) with ring nut, apply a little Vaseline to the catheter tip and insert it a few centimeters (2-3 cm).  Then carefully screw on the thread of the pressure piece and tighten it so far that the catheter is just secured in position. To move the catheter, release the pressure piece (about half turn anticlockwise) so far that the MTC can be moved easily.
	WARNINGS	The MTC pressure transducers are very expensive items which are extremely sensitive to excessive mechanical force; when damaged they cannot be easily repaired.
	Handling Instructions	Read the care and handling instructions carefully
		Never press or squeeze the transducer tip or allow it to impact a hard object. Take care when removing from packaging.
		Do not kink the catheter or pull on it with any force
		If it is inserted in the apparatus but is not being used, it should be withdrawn into the guide tube (4F) in the stopcock block
		During insertion and removal, release the pressure piece (4H2) sufficiently, move it gently do not use force.
		When the tip catheter is not in use (e.g. when working only in Langendorff mode), store it in its original packaging.
4I	Shut-off Stopcock	Shut-off stopcock
4K	Syringe	Syringe for bolus drug administration
5	Pressure Regulator	Pressure regulator for generating the setting of the pressure in the adjustable flow resistor (2B). Rotation of the knob produces a corresponding movement of the plunger and the pressure is transmitted through the tubing connection to the diaphragm of the adjustable flow resistor. The pressure setting is indicated on the pressure gauge (5A). By opening the vent valve (5B) the pressure can be removed quickly and set to zero.
5A	Pressure Gauge	Pressure gauge to indicate the pressure set with the pressure regulator (5). Note that it indicates the pressure setting and not the actual aortic pressure. The aortic pressure is about 8 to 12mmHg higher, depending on the hydrostatic pressure.
5B	Vent Valve	Vent valve for rapidly removing the set pressure. To open it, turn it about half a turn anti-clockwise. To close it, tighten it gently clockwise.
6	Pressure Transducer for Aortic Pressure	Pressure transducer for measuring aortic pressure. The pressure transducer is mounted on the right side on the vertical column (below the pressure gauge 5A) on a holder with height adjustment. The connecting tubing is connected to the tube (4C) below the main stopcock (4A).
	NOTE	For correct pressure measurement the tubing and the dome of the transducer must be filled free from bubbles and the transducer must be set at the level of the aorta.
7	Peristaltic Pump	Roller pump for Langendorff mode. This pump is used to supply a retrograde flow to the heart in the Langendorff mode (during the preparation and recovery phases); the heart itself does not perform any pumping work.  Through the tubing (7A) the perfusate is drawn out of the reservoir at the suction tube (8B) and pumped through the heat exchanger (9) via the connection (2A) into the aortic block (2). The pump output must be set higher than the flow discharged through the coronaries of the heart. Excess pumped perfusate flows back through tubing (8C). The effective perfusion pressure is determined by the adjustable flow resistor (2B).
8	Perfusate Reservoir	The standard reservoir has a capacity of 6 liters and is a jacketed glass vessel. Thermostated water from the circulation thermostat required for the operation of the apparatus is passed through the reservoir jacket and warms the perfusate. The vessel cover lies loosely on the open top and ensures that the aeration gas introduced through the frit collects above the solution level and does not escape immediately to the surroundings. The vessel capacity of 6 liters is not large enough to perform a (non-recirculating) experiment on a rabbit heart for appreciably longer than 30 minutes. It is therefore necessary to provide continuous replacement of the used perfusate, for example by the arrangement shown in Fig. 6. In this system, satisfactory and uniform thermostating and gas saturation is ensured at all times. For obvious reasons it is at least questionable to refill the reservoir when there is only little perfusate left in the vessel. Thermostating and oxygen saturation of the perfusate would then drop suddenly and would only slowly return to normal levels. Depending on the experiment this would lead to greater or lesser instabilities in the course of the experiment. The cover of the reservoir carries the necessary tubing connections as adjustable tubes. Four connections are reserved for connecting the roller pumps (7) and (11), with the two longer tubes used as suction tubes. The two short tubes are used to connect up the return flow tubing. Aeration is provided using the glass frit (8a). The glass tube of the frit is connected by a short piece of tubing to the shortest (straight) metal tube. Aeration operates through small Silicone tubing (2 mm i.d.) which is connected to one of the two needle valves (10). The intensity of aeration is adjusted on the needle valve.
	NOTE	Do not fill the reservoir with cold (room-temperature) solution before the experiment! You will have to wait a long time before the solution has warmed to the temperature determined by the thermostat. It is advisable to pre-warm the solution to the required temperature (e.g. in a heating cupboard, monitored by a thermometer, preferably while bubbling carbogen gas through it).
	Refilling the system	The re-fill system consists of: Level probe (PTC) Power supply for submersible pump Submersible pump placed at the bottom in the low-level container The complete equipment for providing the perfusate is conveniently mounted in a separate mobile rack with three shelves (optional). The immersion level probe is mounted in the cover of the reservoir so that its sensing tip is at the height of the desired perfusate level. The sensor cable is connected to the control input of the Power Supply (5-pin plug). At the power supply output, "output to pump" (12V, 3A) the submersible pump is plugged in through a 2-pole extension cable. The connecting tubing is connected to the shortest bent tube on the reservoir.  It is essential to use the SHORTEST TUBE which is NOT immersed in the perfusate during operation. If you do not do this then the solution will be syphoned back from the reservoir into the low-level container when the pump is not working.    As the liquid level in the reservoir falls below the probe tip, the immersion pump is activated. It then pumps solution from the low-level container to the higher-level reservoir until the liquid level has risen far enough for the level probe to respond and switch off the pump. A switch on the power supply allows the immersion pump to be switched on manually at any time. The output of the immersion pump can be adjusted to suit requirements by altering the output voltage of the power supply. The voltage is adjustable over the range 0 to 12 V (normal setting 9V approximately).
	NOTE	The level probe works with a PTC resistor which is heated by the sensor current (approximately 60°C) and utilizes the heat dissipation to the surroundings for its measurement. The heat loss in air is appreciably less than in a liquid. The resulting different time constants for heating (slow) and cooling (rapid) lead to different times when the pump switches on and off. The pump switches off virtually immediately when the liquid level reaches the probe tip. When the level falls below the probe tip it takes a few seconds (5 - 10 sec) until the pump is switched on. The switch-off sensitivity can be adjusted with the "SENS" trimmer.
9	Heat Exchanger	The heat exchanger is used to compensate the heat loss as the perfusate flows through the pump tubing (7A).
10	Needle Valve	The needle valve is used for adjusting the aeration rate in the perfusate reservoir (8). It is mounted on the short vertical Plexiglass panel below the pressure gauge (5A). The tubing to the frit is connected to the small side tube on the valve body. The connection for the gas supply (carbogen) is accessible from back. The supply pressure should be limited to about 1 bar for safety reasons. The aeration rate is adjusted by turning the knurled knob on the needle valve; clockwise rotation reduces the aeration rate. The valve can be locked against unintentional movement by means of the small knurled nut (spindle lock).
	NOTE	Keep the needle valve dry, do not allow any perfusate to pass into it. It is subject to oxidation if it comes into contact with liquid.
11	2 Channel Peristaltic Pump	2-Channel Peristaltic Pump for atrium supply. This pump serves to provide perfusate in the preload vessel (13) at a constant level. Excess perfusate is supplied so that more perfusate is pumped in to the preload vessel than is required by the organ. The excess pumped volume is drawn off by a second pump channel [tubing (11b)] and pumped back.  In order that overflowing of the preload vessel is prevented under all operating conditions the two tubes in the two pump channels are of different size: the supply tubing (in 11a) should be slightly smaller than the suction return tubing (11b). When working with a rabbit heart, Silicone tubing of 4 mm ID and 5 mm ID. can be used for example.
	NOTE	On adjusting the pump output. As indicated above the outflow of pump (11) must always be larger than the atrial flow from the preload vessel. The pump should, however, not be run at maximum speed. Running at maximum speed will wear out tubing more quickly. It is better to adjust the pump so that some perfusate is always pumped back in the return tubing (11B).
12	Heat Exchanger	This heat exchanger compensates for the heat loss as the perfusate flows through the pump tubing (11A).
13	Atrium Reservoir (Preload Vessel)	This vessel provides the perfusate flowing into the atrium. The height difference between the liquid levels in the vessel and in the atrium (hydrostatic pressure) corresponds to the filling pressure of the atrium (= preload pressure). Excess perfusate solution is provided in the preload vessel. Pump (11) provides more solution than flows into the atrium of the attached heart. The excess perfusate pumped in at the inflow connection (13a) is drawn off by the second channel of the same pump through the suction tube (13b). [see also under (11)]. The filling level in the preload vessel can be adjusted by moving the suction tube (13b). Atrial pressures in the range of about 5 to 10 mm Hg are obtained depending on the adjustment. The short tube (13c) acts as a vent. The preload vessel is mounted on the upper horizontal Plexiglass plate to the left of the aorta block. After releasing the knurled screw and the two hexagon socket screws (M4x16) as well as the clamping mount (15a) for the atrium head (15) the complete atrium supply system with the preload vessel can be removed from the apparatus.
13A	Inflow Connection for Preload Vessel	Inflow connection for the preload vessel projects at the bottom edge of the side of the vessel (Plexiglass tube).
13B	Suction Tube	Suction tube (slide for adjustment), for adjusting the liquid level and maintaining a constant liquid level.
13C	Vent Tube	Vent Tube
13D	Control Stopcock for Atrium Supply	Control stopcock for the atrium supply. The positions of the stopcock plug and the corresponding handle positions are shown in Fig. 7. In addition to shutting off the atrium inflow (handle position A, upwards) and opening it up (handle position B, to the right) there is a further setting (handle position C, downwards) in order to relieve the load on the atrium by means of the tube (13e) when the atrium is cannulated and the heart is operated in the Langendorff mode. Slight return flow from the aorta to the left atrium may lead to an undesirable pressure increase inside the atrium if this relief position is not selected.
14	Fitting adapter for Flowmeter Head	For measuring the atrial flow, a suitable flowmeter head can be fitted into this adapter (optional). If no flowmeter was ordered a blanke piece with a through bore is fitted into the spot. The adapter is secured with 4 hexagon socket screws M3x12 to the underside of the preload vessel (13) after the stopcock (13D). After releasing the screws (use the screwdriver supplied) the adapter can be dismantled together with the connection plate (14A) with tubing connection. On re-assembly, please ensure that the O-ring seal (6x2 mm, silicone) is located in its groove. This side must be towards the stopcock (13D).
14A	Flowmeter Head (Optional)	Mounted permanently in the adapter (14).
15	Atrium Head	The atrium head contains all the components required for the functionally correct connection of the left atrium close to the atrium cannula (16). Connection (15d) (tube projecting inwards) is provided for atrial pressure measurement by the pressure transducer (17). Syringe (15g) is connected through the shut-off stopcock (15f) to the short tube which terminates at the inner top edge of the atrium head inner space. This syringe is used for removing any air/gas bubbles which may have been swept into the atrium head. The perfusate passes through a short piece of tubing (Tygon tubing 6.4 mm i.d.) from the tubing connection of the connection plate (14a) to the inlet connection (15d) of the atrium head. From there the solution passes through the connection (15b) to the atrium cannula (16). To improve filling of the atrium, the back side wall of the atrium head carries an elastic diaphragm whose outer surface is connected to atmospheric pressure [via support tube (15c)]
	NOTE	The free end of the support tube (15c) must not be closed and no liquid must enter it! The atrium head is mounted with a clamping fitting (15a) by the support tube (15c) on a rod (4 mm dia., 40 mm long) which is located at the side on the aorta block (2). After releasing the clamping fitting 15a) the atrium head can be adjusted as required.
15A	Clamp Fitting	Clamp fitting for supporting and adjusting the atrium head
15B	Connection	Connection for fitting the atrium cannula (16). 2 grooves to take two O-Rings (7 x 1.2mm, Silicone) are provided for sealing.
15C	Support Tube	Support tube for securing the atrium head.
	NOTE	The free end of the support tube (15c) must not be closed and no liquid must enter it!
15D	Connection (6.4mm ID)	Connection for feeding in the perfusate, suitable for Tygon tubing 6.4mm ID
15E	Metal Tube	Metal tube (1.3mm OD, short tube) for connecting the syringe (15G) via the stopcock (15F). Syringe (15G) serves to remove any bubbles which have been swept into the atrium head.
15F	Shut-off stopcock	Shut-Off Stopcock
15G	Syringe	Syringe (e.g. 10ml) for removing gas/air bubbles from the atrium head (15).
15H	Metal Tube (1.3mm OD)	Metal tube (1.3mm OD, longer tube projecting inwards) for connecting the pressure transducer (17) for measuring atrial pressure.
16	Cannula	Cannula for binding into the left atrium.
	NOTE	Atrium and aorta cannulae are interchangeable, they fit both here and on the connection (4) of the aorta block! Always use the largest possible cannula in order to achieve optimum atrium filling.
17	Pressure Transducer	Pressure transducer for measuring atrial pressure. The pressure transducer is mounted on a holder with vertical adjustment. The connection tubing is connected to the longer, inwards projecting tube (15D) of the atrium head (15).
	NOTE	For correct pressure measurement both the tubing and the transducer dome must be filled free from bubbles and the transducer must be adjusted to the level of the atrium
18	Thermostated Heart Chamber	Thermostated heart chamber, movable. The jacketed heart chamber serves to provide a moist and warm environment for the organ.
	Large Heart Chamber	The larger chamber (capacity approximately 1.3L) is required when MAP signals have to be picked off from the heart surface with a circular electrode arrangement, or when multi-electrode ECG recordings after Einthoven and Wilson are made using an "electrode basket."
	Small Heart Chamber	The smaller chamber (capacity approximately 0.6L) is recommended when neither of the above recordings are made.
		Both chambers are provided with an overflow tube. The smaller chamber (B) also has a drain connection so that the effluent dripping off can be discharged from the chamber without having to fill the chamber volume. The heart chamber is mounted on a platform whose height can be adjusted with a rotary control. The jacket of the vessel is included in the thermostating circuit of the apparatus so that thermostated water flows through it.
18A	Overflow Tube	Overflow tube. The perfusate overflowing here (coronary effluent) is usually discarded and not returned to the reservoir (8).
18B	Discharge Connection	Discharge connection for emptying the chamber (18). This connection is provided only on the small heart chamber (chamber B, see above).

Tubing and Connections on the apparatus, dimensions

The tubing diameters for perfusate transport as indicated in Fig. 8 apply for medium flow rates up to about 300 ml/min aortic flow. Under these conditions the dwell time in the tubing is short so that losses of heat and oxygen remain small. With low perfusate flow it may be useful to use smaller diameter tubing than those suggested here in order to reduce the dwell time of the perfusate in the supply tubing. Additionally it is then better to use Tygon tubing instead of Silicone. Apart from reduced heat losses on the way from the reservoir to the organ there is then also less reduction in oxygen saturation.

Figure 9 shows the tubing sections of the thermostating circuit. Silicone tubing only is suggested. The distribution block for the thermostated water is located on the back of the top Plexigalss plate. When connecting the individual jacketed vessels please ensure that the flow is from the bottom upwards where possible. The larger vessels (8) and (18) are connected through self-sealing quick-release couplings. They can readily be disconnected for cleaning without first having to drain their water jacket. 

NOTE: the reference numbers starting with R or S (e.g. R42005) are HSE part numbers. If you require any spares please quote where possible the part number indicated. 

Servicing and Maintenance

Perfusate Circuit

Before the first start-up, and daily after the experiment has been completed, the apparatus has to be cleaned. It is particularly important that the perfusion circuit and all parts in contact with the perfusate are kept clean.

WARNING: In order to avoid damage to the apparatus only the cleaning agents mentioned below may be used without special testing. 

Daily cleaning is important in particular because of bacteria. Apart from other effects, bacteria in the apparatus lead to early edema in the organ! Bacteria can grow overnight! Therefore leave the cleaning agent inside the apparatus overnight. 

For cleaning you first rinse the perfusate circuit thoroughly with distilled water. Use the pumps for the purpose. First connect the atrium cannula (16) to the aorta cannula (4e) with a short piece of tubing.

Then fill the various vessels (Preload Vessel (13) and aortic block (2)) with hot cleaning solution.

The apparatus filled with cleaning solution should be allowed to stand for a few hours or better still overnight. We do not recommend leaving the solution in the apparatus for more than 24hrs. 

NOTE: The longer the solution remains in the apparatus, the longer it takes until all solution residues have been washed out. 

If the apparatus is not in use over the weekend it should be filled with distilled water after cleaning and be kept in the dark (or covered against excessively bright light). 

With longer breaks in the experimental work it is better to keep the apparatus dry after cleaning; before the next experiment it has to be filled with cleaning solution which is allowed to act overnight. 

In the morning, switch on the thermostat in order to warm up the cleaning solution. Then remove the cleaning solution from the apparatus and rinse thoroughly with distilled water. Do not forget the side branches (tubing to the pressure transducer (6) and (17) and to the syringes (4k) and (15g)).

Alternatively:

• Rinse with 0.1N HCL (hydrochloric acid 0.1 normal)
• Rinse with distilled water
• Fill with 0.1N H₂O₂ (3% hydrogen peroxide)
• Leave overnight

Perfusate system servicing:

• Drain and remove the perfusate
• Rinse with distilled water
• Fill vessels with hot cleaning solution
• Allow to stand for a few hours or overnight (24hrs max)
• In the morning heat up the apparatus to 37°C with the thermostat
• Then remove cleaning solution
• Rinse with distilled water, do not forget side branches

Thermostatic Circuit

The thermostatic circuit must also be serviced regularly. If you are filling the thermostat with pure distailled water without any additives you have to replace the water every week to avoid algal growth inside the system. When adding Thermoklar or sodium azide (TOXIC) you should replace the contents at least once a month. 

• Thermostatic circuit servicing:
• Thermostatic circuit without additive, replace water weekly
• With additive, replace at least every month 
• Fill only with distilled water (with or without additives)
• Before filling, rinse with cleaning solution and clear water

Recommended cleaning agents

Use only the recommended cleaning agents. 

In order to avoid damage to the apparatus, only the cleaning agents listed below can be used without special testing. If you require a different cleaning agent for any particular reason you must first test it for compatibility before using it. In case of doubt contact the manufacturer of the apparatus.

Cleaning agent for Plexiglass surfaces:

WARNING: Not all cleaning agents commonly used in the laboratory are suitable for cleaning the apparatus. For example, Mucocit F is not compatible with plexiglass. 

Tubing Material 

The tubing used in the apparatus has to meet various requirements. It has to be selected so that its material properties best meets the requirements. In addition to chemical, mechanical and optical properties it is also necessary to account for the gas permeability of the various materials. The selection aid for certain materials as listed below is intended to do no more than provide assistance. The user must decide which type of tubing is most suitable for their particular application. If doubtful, take the necessary steps to test new tubing. 

When considering permeability to gas, the time the liquid remains in the tubing is important. Longer periods result in more gas exchange with the surrounding air than shorter periods. If doubtful it is necessary to carry out appropriate measurements. 

Silicone tubing:

Silicone is the tubing material most widely used in the laboratory. It is largely inert and resistant to most chemicals used in the laboratory. Natural Silicone is not glass clear but is sufficiently translucent so that the lumen can be examined visually. A further advantage is its favorable mechanical properties. When used in peristaltic pumps there is relatively little wear.

Silicone exhibits a high permeability to oxygen. When oxygen levels are important, Silicone tubing should be avoided. 

Tygon® Tubing (E-3603)

Tygon® is also frequently used in the laboratory. It is manufactured under strictly controlled conditions. It is largely inert and resistant to most chemicals used in the laboratory. Tygon is glass clear and permits optimum visual checking of the lumen. Tygon tubing is flexible and the remaining mechanical properties are also favourable. When used in roller pumps it suffers somewhat more wear than Silicone.

Tygon® has a low permeability to oxygen. It is therefore a better tubing material than Silicone when exchange of oxygen between the surroundings and the lumen has to be avoided. Thick-walled tubing is preferable to thin-walled tubing in this case.

Pharmed

PharMed™ tubing has been specially developed for medical applications and has only recently become available. Unfortunately only little information is available on this material. Chemical and mechanical properties are said to be excellent. Its permeability to oxygen is said to be practically zero. The disadvantage is that the material is not transparent. Visual examination of the lumen is not possible.