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Computer control of a modified Langendorff perfusion apparatus for organ preservation using cryoprotective agents
COMPUTERCONTROL OF A MODIFIEDLANGENDORFF PERFUSION APPARATUS FOR ORGAN PRESERVATION USING CRYOPROTECTIVE AGENTS C.G. Adem and J.B. Harness* ABSTRACT The construction of an isolated rat heart cryopreservation apparatus which is interfaced to a process-control computer is described. The flexibility of the equipment is demonstrated by the running of simultaneous dual sets of experiments under multiple and variable conditions. The computer-control program allows interactive calibration of the instruments, accurate
control of variables and modification of parameters during the experiments. By controlling the speed of the perfusate’s peristaltic pump, perfusion pressure was not allowed to exceed a present level. Experiments are reported which allow the storage of rat hearts at -22’C with the aid of the cryoprotective agent, ethylene glycol.
INTRODUCTION
THE PERFUSION APPARATUS
Clinical renal transplantation has become a routine procedure. However, heart transplantation is still at an experimental stage because of the organ’s sensitivity to anoxic damage and its uniqueness in the body. Many problems remain to be solved, foremost being that of long-term storage.
Experience gained by other workers in the field of organ preservation was on traditional equipment that suffered many of the drawbacks of the original Langendorff Column 1-3 . The isolated organ could not be cooled on the same appartus and the temperature of the organ fluctuated from the setpoint4y5. In the case of the isolated heart, ECG signals collected with high impedance probes were extremely susceptible to noise, pick up and are not repeatable. Accordingly a new method of recording the ECG was devised by Sahyoun and Hicks6, the column and organ chamber were redesigned by Bothamley, Gough and Harness' who replaced the gravity flow perfusion arrangement with peristaltic pumps.
This paper describes fully a modified computer-controlled Langendorff perfusion apparatus suitable for organ preservation using cryoprotective agents (C.P.A.) and its application to isolated rat hearts. Blood is now treated with C.P.A. and stored for long periods at liquid nitrogen temperatures (77K), it is hoped that the equipment described here will enable whole organs to be stored in refrigerated conditions for reasonable periods. Physiological systems involve many variables under complex conditions which require a great deal of skill for their control and the derivation of valid conclusions. Concepts and techniques imply synergetic solutions and computers may prove efficient, reliable and convenient as well as easy to manipulate, thus allowing experimentation under strictly controlled and repeatable conditions and the study of the response of an organ or organism to a specific stimulus. Designing and automating a process by which the function of the isolated rat heart under normal and abnormal conditions may be analysed is the main aim of the work presented in this paper. With suitable modifications the apparatus could be used in a variety of applications, of various organ storage and maintaining cancerous tissue. The development has been performed in a process computer laboratory of the University’s Control Engineering Department. Postgraduate School of Chemical Engineering, University Bradford, BD7 lDP, Yorkshire, UK *To whom all communications should be addressed
134
J. Corned. Engng. 1981, Vol. 3, April
of Bradford,
The glass perfusion column and organ chamber of the the modified Langendorff apparatus are shown diagramatically in Figure 1. The column consists of a glass coil surrounded by a jacket through which the bath liquid could be circulated. Provision is made for measurement of perfusion pressure and the insertion of thermocouples at various points. An ECG electrode is welded to a stainless steel cannula held by silicone tubing and a port on the side of the perfusion column permits the insertion of the other electrode in the perfusion bathing the heart. The organ chamber, acting as a shield, consists of a glass jacketed chamber, connected the same way as the column, in series with,the bath circulation. An overflow device was added so that the perfusate in the organ chamber may be kept at a constant level. If needed? the organ could be surrounded with perfusate and circulated by a pump to ensure a better heat transfer. Extra ports have been added so that pulmonary artery cannulation will facilitate effluent collection and the heart may be made to perform work in future experiments. Pulsatile outflow from the pump interfered with the normal functioning of the heart and this effect was
0141-5425/81/020134-06$02.00 0 1981 IPC Business Press
Computer
reduced by connecting a specially designed pulse suppressor/bubble trap to the pump outflow line before going into the heat exchanger. It was constructed from a small test-tube with a fine glass outlet and had on,the inlet a sphere containing the Dacron filter. A cannulation dish was designed to fit into the organ chamber; it had a built-in sintered glass oxygenator and allowed for the cannula to dip into the perfusate contained in the dish. Heart contact with the air was avoided, preventing any resulting emboli. Handling and damage were lessened and the ischaemic time was shortened. The experimental arrangement consisted of two modified columns mounted in parellel and connected to a Ferranti Argus 700E Computer. The flow diagram of the apparatue is shown in Figure 2 which allowed, through adequate positioning of piping clips, an increase in experimental efficiency and a multiplicity of conditions, Two isolated perfused hearts could be tested under four combinations: same or different temperatures with same or different perfusates. The apparatus is situated in a specially fitted laboratory next to the computer suite. The low-level analogue input signal are filtered for 50 Hz interference, then selected and sampled by the computer’s analogue-to-digital conversion system (ADC), at its most sensitive setting (-5 mV to + 5 mV). Random noise pick up is minimized further by taking an input average of 20 successive readings by software. The input reading is to a precision of 12 bits, including sign, which is equivalent to a resolution of 2 x 10e3mV on the range setting in use. A 600 Baud digital link is provided, linking
Coolant
i
Eiyt
Overflw
port
Coolant
-
Figure 1 Modified Langendorff column perfusion coil jacket and organ chamber and warming of isolated rat heart.
exchanger
with glass for cooling
control
of cryopreservation:
C.G.
Adem
and J.8.
Harness
the Ferranti Argus 700E to a Hewlett Packard 2100 A digital computer for the analysis and storage of data. The Ferrenti Argus 700E computer is used since it has the facility for multiplexing low voltage signals, so only one sophisticated amplifier is required for low voltage signals such as those from thermocouples. A remote ASR 33 teletype, located in the laboratory, allows for operator interaction, setting up of experimental conditions and their modification during runtime. It also displays the periodic logging of experimental variables. The program was written in Real Time Extended Basic (RTEB). This language has been developed at the Postgraduate School of Control Engineering at the University of Bradford8, in response to the need for a simple-to-use language for real-time computation. This allows the computer, through a self-contained timing device, to execute one or more program at preset intervals, thus enabling the implementation of complex control schemes, on-line program development, inspection of variables and their modification. RTEB has been constructed by enhancin the procedural language BASIC (BASIC is a subset o B RTEB) and by adding additional statements for real time. The Ferranti Argus 700E combined with RTEB is a powerful, reliable machine for experimental development of both program and rig before purchasing a dedicated computer. TEMPERATURE DEVICES
CONTROL
AND MEASUREMENT
The liquid circulating
in the Langendorff columns providing the temperature control originates from a fibre-glass Shandon insulated bath (30 x 20 x 19 cm) with a capacity of 10.5 1, is equipped with a lid, an.electrically driven stirrer and a pump to improve mixing very low temperatures render the circulating liquid highly viscous. The bath contains 10 turns, 3 m in length of 9 mm o/d x 8 mm i/d annealed copper tubing. This coil is fitted with an expansion valve (Danfoss Ltd) and connected to a commercial refridgeration unit (Frigidaire Ltd; A 55 0 - 2/H; ‘/2 HP) mounted on a wall bracket adjacent to the bath. A similar copper coil, fixed on top of the former, is linked to a ‘cryoflow’ submersible dewar pump (Cryoproducts Ltd; 12 V D C Type CS MPB) for transfer of small quantities of liquid nitrogen. A 3 kW domestic kettle heating element is immersed in the circulating fluid concentrically to the copper coil in the vessel. The power supply to the heating element is adjusted by thyristors in the inverse parallel configuration such that a computer signal in the range 2.5 V to 5 V corresponds to the power range 0.05 - 2.4 kW. The insulated vessel is filled with 40% w/w ethanediol to water mixture which can be varied depending on the experimental conditions. The liquid is recycled and circulated through the perfusion columns by a centrifugal pump (Stuart Turner (120/ 40 GPH)) mounted on the wall adjacent to the vessel to minimise vibration. Copper constant thermocouples, made from 42 SWG wire, were used for temperature measurement.
J. Biomed. Engng.
1981,
Vol.
3, April
135
Computer
control
of cryopreservaiion:
C.G. Adem
,.+43-l
CR 95%
0,
and J.6. Harness
I------
P FC
1 PR
RU
Figure 2 FC, jlow perfusion RJ, right
Flow diagram of the apparatus. CB, constant temperature vessel; CR, cryoprotectant reservoir; cuvette; FBT, filter and bubble trap; HE, heating element; LC, left organ chamber; LJ, left coil and jacket; LN, liquid nitrogen coil; PR, perfusion reservoir; RC, right organ chamber; perfusion coil and jacket; RU, refrigeration unit coil; VII, variable temperature vessel.
Temperature control depends upon the electrical heater output counteracting the cooling produced by the continuously running refrigeration unit. Switching on the liquid nitrogen pump gave the desired rates at low temperatures, where the refrigeration power of the Frigidaire unit was too small. A thermocouple at the aorta of the isolated heart generated the signal used to control the temperature of the perfusate. Preliminary tests showed that derivative control action produced an oscillatory response, so control was therefore limited to the proportional and integral modes. A signal U, was generated using the incremental form of the Z-term controller algorithm, at sample interval n, U, is given by:
u,
=zJn_l+Kc
((l+p)1,+ I,_,)
here In is the present and I,_, the past error signal, & is the proportional constant, T the sampling interval, Tc the intergral time constant and Td the differential time constant, which as stated above in this case is equal to zero. The control signal was set to a digital-to-analogue converter (DAC) which had an output range - 5 to 5 V, to a resolution of 1 part in 2.55. The analogue signal was used to drive the thyris tor ,unit which controlled the power to the 3 kW heater. The value of K, in Equation (1) was
136
J. Biomed. Engng. 1981, Vol. 3, April
The temperature at the aorta during normothermic (37°C) and hypothermic perfusions (15’) had a mean and maximal deviation from the setpoints as foIIows: Temperature,
37OC
l?C
Mean Deviation,
0.015”c
0.016”C
Maximal Deviation,
0260°C
0.171”c
During cooling and warming the following were obtained: Temperature
(1) (-1+2qj
derived theoretically by considering critical damping in a servo operation under proportional and integral control’.
Range,
results
3 7” C to -22°C
Rate of Change,
2’C/min
,
-l’C/min
Mean Deviation,
0.136’C
,
0.127”C
Maximal Deviation
0.358’C
,
0.209OC
Rates of change of 4’C/min and -3’C/min achieved with similar accuracy.
were
This control proved satisfactory for preliminary investigations into organ cryopreservation. The computer program allowed any controller settings to be selected at calibration time and changed during run-time. The thermocouple’s measurement error was smaller than 0.5”C. Its addition to the mean and maximum deviation values quoted above yielded the maximum error in temperature reading.
Computer
The process is a non-linear temperature control system; so the response to negative step changes will be slower than that to positive changes which is due to the difference in power output of the cooling and heating elements. The response was also dependent upon the temperature at which the change occurred. CONTROL OF PERFUSATEFLOW AND CPA CONCENTRATION IN THE PERFUSATE Watson-Marlow MHRE miniature flow inducers were used for perfusion and CPA addition. 2 mm x 6 mm (bore x external diameter) silicone elastomer tubings were fitted to the pumps with a maximal flow of 1.075 ml/min for the CPA pump and 4.800 ml/min for the perfusate’s. For applied voltages between 1 and 24 V and using 2 mm x 6 mm silastic tubing, the corresponding flows of the perfusion (Type MHRE 300 and CPA pumps (Type MHRE 7) were recorded. T h e perfusate could be delivered at constant or variable flows, thus either constant flow or constant pressure perfusion experiments could be performed. The physiological pressure measurement system used was of the Bell and Howell 4-327-L222 linear type. The pressure transducer, with a range O-750 mm Hg differential had a sensitivity of 2 pV/mm Hg at an excitation of 10 V d.c. For the ADC input range used, a pressure between 0 and 250 mm Hg could be measured to a resolution of 0.1 mm Hg. The perfusion pressure was logged on the remote teletype every 3 s. Once perfusate flow and molarity of the cryoprotectant were specified, the computer calculated and sent the voltage for the perfusion and CPA respectively and corrected the perfusate flow based on the feedback information from the pressure transducer while maintaining the preset CPA molarity. The direct voltages to drive the pumps were obtained from operational amplifiers powered by a 28 V (Farnell L30B) stabilized power supply.
controf
of cryopreservafion:
segment;
C.G.
Adem
and
1.6. Harness
after 10 s and every 10 s thereafter.
The temperature is read twenty times, averaged, converted into ‘C and printed out after control action. Once the controller settings are entered, the internal controller constants are calculated and control action takes place by sending an output in units through the appropriate DAC. Activation is periodic at 2 s intervals, which is the chosen sampling time. Task CHANGETEMP is invoked only when a certain temperature profile is needed. As it is run, the initialisation segment asks the user to input the number of changes required, followed by the desired setpoints, rates of change and durations at each specific temperature. Cooling and warming of the heart are initiated at the required time by CIHANGETE,MP activation segment. The setpoint is altered each sampling instant by the correct amount to achieve the desired rate of change of temperature. This and the logging of the setpoint and the temperature are carried out at 3 s, sampling time intervals. If the cooling rate is greater than - l*C/min the liquid nitrogen pump is activated through a relay by a signal from the digital output of the computer.
The program to control the cryopreservation apparatus consists of 3 tasks which may run simultaneously, interacting via global variables. The following describes the tasks in question - the name of each task is always written in capital letters.
Task PERFUSION, calibrates the pressure transducer, measures the vascular resistance and controls the CPA and perfusion pumps. The initialisation segment asks for the input of the sampling time, perfusate flow, CPA final molarity, its addition, holding and removal time, and the temperature at which addition and removal are to start. In case freezing is discontinued and resumed must be specified. In add discounted and resumed must be specified. In addition, the maximal pressure permitted is input, thus preventing the vascular resistance from exceeding it. It also allows for the calibration of the pressure transducer by reading the atmospheric pressure, converting it to mm Hg and printing it out on the remote teletype. A small alteration in the program would allow perfusion at constant vascular resistance. The initialisation segment then prints the total experimental time after cryoprotectant introduction and the molar volume of ethylene glycol. Linear addition and removal of CPA are initiated automatically on the specified temperature being reached during the cooling and warming regimes and holdtime terminated before removal is undertaken. If the perfusion experiment involves freezing, flow arrest and resumption occur at the preset temperatures respectively. Vascular resistance is read twenty times. The readings are averaged, converted to mm Hg and printed out. If the pressure exceeds a certain preset level, the perfusate flow is decreased gradually to maintain that level and the cryoprotectant flow is reduced correspondingly to preserve an accurate molarity. Constant pressure perfusion is also possible by varying the flow to maintain a constant vascular resistance pattern.
Task TEMPERATURE measures and maintains a preset temperature. The initialisation segment asks the user to input the controller settings, the sampling time, the heater output level and the setpoint. Initialisation is followed by a periodic activation
Logging of time, perfusion pressure, cryoprotectant’s actual and final molarities and perfusate flow are executed with the control action at the chosen sampling interval which is 3 s in this case. The pumps are switched off upon termination of the experiment.
The perfusion program calculated and delivered to the pump the required voltage, after the required molarity and perfusate flow rate were entered on the remote teletype. The keyboard input facility permitted changes to the appropriate equation required to calculate all the variables in this section. The maximal needed perfusate flow rate into the isolated rat heart was 3 ml/min and the maximal delivery of CPA was of 1.075 ml/min under the actual arrangements. The maximal CPA molarity obtained was 6.426 mol 1-i. THE CONTROL PROGRAM
J. Biomed. Engng.
1981, Vol. 3, April
137
Computer
control
of cryopreservafion:
C.G.
Adem
and
Computer assisted calibration of the instruments on the cryopreservation apparatus is a valuable facility enabling quick and accurate action. The following trol program.
are the facilities
offered
by the con-
(1) Computer
assisted calibration of instruments and setting-up of experimental conditions.
(2)Control
of heart temperature at any profile within the limitations of the apparatus. A
control signal is generated using an incremental form of the two-term control algorithm, proportional and integral modes being effected.
(3) Automatic
initiation of addition and removal of ethylene glycol. The concentration is controlled by setting the CPA pump rate to the correct value by means of a computer-generated voltage signal.
(4) Automatic
suspension and resumption of perfusion at the preset temperature during freezing experiments.
(5)Regular
data logging. Measured variables are logged out on the remote teletype at sampling time intervals. A complete operating record is therefore produced on the teletype, containing the pre-experimental set-up, calibration of measuring equipment, log of variables at regular intervals, and a record of all changes made via the keyboard input facility.
(6) Access to all variables
at any time.
(7) Perfusion required.
flow or pressure as
at constant
J.6.
Harness
the vascular resistance stabilized after 20 min. Normal ECGs reflected the absence of any damage to the heart. Figure 4 shows the changes in vascular resistance during a perfusion and freezing where the temperature is lowered to -22’C and then raised to 36’C. Ethylene glycol was introduced to the heart when the temperature was lowered to 19’C and reached 3 moles at that stabilized temperature. The perfusate was discontinued at -4°C and resumed as the heart was rewarmed at 9’C. The heart was held at 2 1.5’C for 12 min. that
It can be seen that the vascular resistance drops then increases when the glycol reaches the heart and drops as the CPA leaves the heart, then increases again. This can be explained by considering the binary diffusion processes of water and CPA” but because of the complicated flow patterns in the heart an estimate of the time process cannot be made. The results shown are for only short storage times at the lowest temperatures. Hearts were stored at the low temperatures for periods of up to 24 h. No difference was detected between the long and short storage times, as the damage to the heart is mainly during the heating and cooling periods. For the initial experiments, it was not thought necessary to hold all the hearts at the lowest temperatures for lengthy periods. CONCLUSIONS A modified Langendorff perfusion apparatus has been designed and constructed allowing simultaneperfusion or different in a shielded, temperature-controlled
by the
of ECG of the of special
(8) Full automation of the whole process including termination of the experiment. In addition the RTEB system allows variables examined and modified on-line, if so desired.
to be
RESULTS Changes in the mean vascular resistance during perfusion is shown in Fz+re 3 from which it can be seen
of the to -40°C, of perfusate of perfusate be recorded or analysed
. 20
:=r=:::=e,se Figure 3 Changes in mean vascular resistance during perfusion of isolated rat heart at 32’C. l, Pressure; 4 temperature; n, molarity. (Average of 5 experiments).
Engng.
80
DO
Tie (mid Ttme (mid
138 J. Biomed.
*. 40
1981, Vol. 3, April
Figure 4 Changes in vascular resistance at the isolated rat heart during, perfusion, freezing to -3’C and warming to 3 7 C., 0, Pressure; A, temperature; n, molarity. (Average of 6 experiments).
Computer
Experiments on isolated rat hearts have been performed, which have been cooled to -22°C with addition of ethylene glycol and rewarmed to 37’C successfully. This indicates that organ preservation experiments can now be easily performed without tiring manual control systems.
control
3
4
ACKNOWLEDGEMENTS
The authors are indebted to Drs Janice McCurrie, G. Irving and H.A. Sahyoun of the Postgraduate School of Pharmacology for assistance and patience, especially in teaching the surgical techniques. A special note of gratitude is due to Messrs I Dick and R. Butts for their precious help in the construction of the hardware and software. To Dr A. Mearns, Consultant at Bradford Royal Infirmary and Dr K. Hobbs, Professor of Surgery at the Royal Free Hospital, London, for many stimulating discussions. Lastly, but not least, many thanks are due to our typists and friends, Miss J.A. Squires and Mrs J.E. Steele.
5 6
7
8
REFERENCES 1
2
Langendorff, O., Untersuchungen am Ukerlaevenden Sangethierherzen, Pjlug. Arch. ges Physiol, 1895, 61, pp 291-332. Hobbs, K.E.F. and Ellis, M., ‘The Present Status of Sub-Zero Organ Preservation with Special Reference to the Rat Heart’, in Organ Preservation’ (Ed. D.E.
9
10
of cryopreservofion:
C.G.
Adem
and
J.B.
Harness
Pegg,)Churchill-Livingstone, London and Edinburgh, 1973. Armitage, W J. and Pegg, D.E., ‘An Evaluation of Colloidal Solutions for Normothermic Perfusion of Rabbit Heart: An improved Perfusate Containing Haemaccel’, Cryobiology, 1977, 14, 428-34. Pegg, D.E. and Green, C J., ‘Renal Preservation by Hypothermic Perfusion, IV. The Use of Gelatine Polypeptides as the Sole Colloid’, Cryobiology, 1978, 15, pp. 27-34. Jacobsen, A., Continuous Hypothermic Perfusion of Rabbit Kidneys’, Cryobiology, 1978, 15, 290-331. Sahyoun, H.A. and Hicks, R., ‘Electrocardiographic Recording of Normal and Infarct Bearing Rat Hearts in a Perfused Isolated Preparation’,]. Pharmac Methods 1978, 1, 351-600. Bothamley, P.E., Gough, N.E. and Harness, J.B., Computer Control of an Isolated Heart Preparation for Biomedical Research, in Digital Computer Applications to Process Control, (Ed. H.R. van Nauta Lemka, and H.B. Verbruggen,) North-Holland, Amsterdam, New York and London 1977. Butts, R., ‘Real Time Extended Basic Instruction Manual’, Published by Postgraduate School of Control Engineering, University of Bradford, Bradford, UK Ceaglske, N.H., ‘Automatic Process Control for Chemical Engineers’, Published by John Wiley and Sons Inc., New York. Harness, J.B., ‘An Apparent Permeability Coefficient for the Diffusion of Cryoprotective Agents into Red Blood Cells’. Inserm, 1976, 62, 135-138
J. Biomed. Engng. 1981, Vol. 3, April 139
control of variables and modification of parameters during the experiments. By controlling the speed of the perfusate’s peristaltic pump, perfusion pressure was not allowed to exceed a present level. Experiments are reported which allow the storage of rat hearts at -22’C with the aid of the cryoprotective agent, ethylene glycol.
INTRODUCTION
THE PERFUSION APPARATUS
Clinical renal transplantation has become a routine procedure. However, heart transplantation is still at an experimental stage because of the organ’s sensitivity to anoxic damage and its uniqueness in the body. Many problems remain to be solved, foremost being that of long-term storage.
Experience gained by other workers in the field of organ preservation was on traditional equipment that suffered many of the drawbacks of the original Langendorff Column 1-3 . The isolated organ could not be cooled on the same appartus and the temperature of the organ fluctuated from the setpoint4y5. In the case of the isolated heart, ECG signals collected with high impedance probes were extremely susceptible to noise, pick up and are not repeatable. Accordingly a new method of recording the ECG was devised by Sahyoun and Hicks6, the column and organ chamber were redesigned by Bothamley, Gough and Harness' who replaced the gravity flow perfusion arrangement with peristaltic pumps.
This paper describes fully a modified computer-controlled Langendorff perfusion apparatus suitable for organ preservation using cryoprotective agents (C.P.A.) and its application to isolated rat hearts. Blood is now treated with C.P.A. and stored for long periods at liquid nitrogen temperatures (77K), it is hoped that the equipment described here will enable whole organs to be stored in refrigerated conditions for reasonable periods. Physiological systems involve many variables under complex conditions which require a great deal of skill for their control and the derivation of valid conclusions. Concepts and techniques imply synergetic solutions and computers may prove efficient, reliable and convenient as well as easy to manipulate, thus allowing experimentation under strictly controlled and repeatable conditions and the study of the response of an organ or organism to a specific stimulus. Designing and automating a process by which the function of the isolated rat heart under normal and abnormal conditions may be analysed is the main aim of the work presented in this paper. With suitable modifications the apparatus could be used in a variety of applications, of various organ storage and maintaining cancerous tissue. The development has been performed in a process computer laboratory of the University’s Control Engineering Department. Postgraduate School of Chemical Engineering, University Bradford, BD7 lDP, Yorkshire, UK *To whom all communications should be addressed
134
J. Corned. Engng. 1981, Vol. 3, April
of Bradford,
The glass perfusion column and organ chamber of the the modified Langendorff apparatus are shown diagramatically in Figure 1. The column consists of a glass coil surrounded by a jacket through which the bath liquid could be circulated. Provision is made for measurement of perfusion pressure and the insertion of thermocouples at various points. An ECG electrode is welded to a stainless steel cannula held by silicone tubing and a port on the side of the perfusion column permits the insertion of the other electrode in the perfusion bathing the heart. The organ chamber, acting as a shield, consists of a glass jacketed chamber, connected the same way as the column, in series with,the bath circulation. An overflow device was added so that the perfusate in the organ chamber may be kept at a constant level. If needed? the organ could be surrounded with perfusate and circulated by a pump to ensure a better heat transfer. Extra ports have been added so that pulmonary artery cannulation will facilitate effluent collection and the heart may be made to perform work in future experiments. Pulsatile outflow from the pump interfered with the normal functioning of the heart and this effect was
0141-5425/81/020134-06$02.00 0 1981 IPC Business Press
Computer
reduced by connecting a specially designed pulse suppressor/bubble trap to the pump outflow line before going into the heat exchanger. It was constructed from a small test-tube with a fine glass outlet and had on,the inlet a sphere containing the Dacron filter. A cannulation dish was designed to fit into the organ chamber; it had a built-in sintered glass oxygenator and allowed for the cannula to dip into the perfusate contained in the dish. Heart contact with the air was avoided, preventing any resulting emboli. Handling and damage were lessened and the ischaemic time was shortened. The experimental arrangement consisted of two modified columns mounted in parellel and connected to a Ferranti Argus 700E Computer. The flow diagram of the apparatue is shown in Figure 2 which allowed, through adequate positioning of piping clips, an increase in experimental efficiency and a multiplicity of conditions, Two isolated perfused hearts could be tested under four combinations: same or different temperatures with same or different perfusates. The apparatus is situated in a specially fitted laboratory next to the computer suite. The low-level analogue input signal are filtered for 50 Hz interference, then selected and sampled by the computer’s analogue-to-digital conversion system (ADC), at its most sensitive setting (-5 mV to + 5 mV). Random noise pick up is minimized further by taking an input average of 20 successive readings by software. The input reading is to a precision of 12 bits, including sign, which is equivalent to a resolution of 2 x 10e3mV on the range setting in use. A 600 Baud digital link is provided, linking
Coolant
i
Eiyt
Overflw
port
Coolant
-
Figure 1 Modified Langendorff column perfusion coil jacket and organ chamber and warming of isolated rat heart.
exchanger
with glass for cooling
control
of cryopreservation:
C.G.
Adem
and J.8.
Harness
the Ferranti Argus 700E to a Hewlett Packard 2100 A digital computer for the analysis and storage of data. The Ferrenti Argus 700E computer is used since it has the facility for multiplexing low voltage signals, so only one sophisticated amplifier is required for low voltage signals such as those from thermocouples. A remote ASR 33 teletype, located in the laboratory, allows for operator interaction, setting up of experimental conditions and their modification during runtime. It also displays the periodic logging of experimental variables. The program was written in Real Time Extended Basic (RTEB). This language has been developed at the Postgraduate School of Control Engineering at the University of Bradford8, in response to the need for a simple-to-use language for real-time computation. This allows the computer, through a self-contained timing device, to execute one or more program at preset intervals, thus enabling the implementation of complex control schemes, on-line program development, inspection of variables and their modification. RTEB has been constructed by enhancin the procedural language BASIC (BASIC is a subset o B RTEB) and by adding additional statements for real time. The Ferranti Argus 700E combined with RTEB is a powerful, reliable machine for experimental development of both program and rig before purchasing a dedicated computer. TEMPERATURE DEVICES
CONTROL
AND MEASUREMENT
The liquid circulating
in the Langendorff columns providing the temperature control originates from a fibre-glass Shandon insulated bath (30 x 20 x 19 cm) with a capacity of 10.5 1, is equipped with a lid, an.electrically driven stirrer and a pump to improve mixing very low temperatures render the circulating liquid highly viscous. The bath contains 10 turns, 3 m in length of 9 mm o/d x 8 mm i/d annealed copper tubing. This coil is fitted with an expansion valve (Danfoss Ltd) and connected to a commercial refridgeration unit (Frigidaire Ltd; A 55 0 - 2/H; ‘/2 HP) mounted on a wall bracket adjacent to the bath. A similar copper coil, fixed on top of the former, is linked to a ‘cryoflow’ submersible dewar pump (Cryoproducts Ltd; 12 V D C Type CS MPB) for transfer of small quantities of liquid nitrogen. A 3 kW domestic kettle heating element is immersed in the circulating fluid concentrically to the copper coil in the vessel. The power supply to the heating element is adjusted by thyristors in the inverse parallel configuration such that a computer signal in the range 2.5 V to 5 V corresponds to the power range 0.05 - 2.4 kW. The insulated vessel is filled with 40% w/w ethanediol to water mixture which can be varied depending on the experimental conditions. The liquid is recycled and circulated through the perfusion columns by a centrifugal pump (Stuart Turner (120/ 40 GPH)) mounted on the wall adjacent to the vessel to minimise vibration. Copper constant thermocouples, made from 42 SWG wire, were used for temperature measurement.
J. Biomed. Engng.
1981,
Vol.
3, April
135
Computer
control
of cryopreservaiion:
C.G. Adem
,.+43-l
CR 95%
0,
and J.6. Harness
I------
P FC
1 PR
RU
Figure 2 FC, jlow perfusion RJ, right
Flow diagram of the apparatus. CB, constant temperature vessel; CR, cryoprotectant reservoir; cuvette; FBT, filter and bubble trap; HE, heating element; LC, left organ chamber; LJ, left coil and jacket; LN, liquid nitrogen coil; PR, perfusion reservoir; RC, right organ chamber; perfusion coil and jacket; RU, refrigeration unit coil; VII, variable temperature vessel.
Temperature control depends upon the electrical heater output counteracting the cooling produced by the continuously running refrigeration unit. Switching on the liquid nitrogen pump gave the desired rates at low temperatures, where the refrigeration power of the Frigidaire unit was too small. A thermocouple at the aorta of the isolated heart generated the signal used to control the temperature of the perfusate. Preliminary tests showed that derivative control action produced an oscillatory response, so control was therefore limited to the proportional and integral modes. A signal U, was generated using the incremental form of the Z-term controller algorithm, at sample interval n, U, is given by:
u,
=zJn_l+Kc
((l+p)1,+ I,_,)
here In is the present and I,_, the past error signal, & is the proportional constant, T the sampling interval, Tc the intergral time constant and Td the differential time constant, which as stated above in this case is equal to zero. The control signal was set to a digital-to-analogue converter (DAC) which had an output range - 5 to 5 V, to a resolution of 1 part in 2.55. The analogue signal was used to drive the thyris tor ,unit which controlled the power to the 3 kW heater. The value of K, in Equation (1) was
136
J. Biomed. Engng. 1981, Vol. 3, April
The temperature at the aorta during normothermic (37°C) and hypothermic perfusions (15’) had a mean and maximal deviation from the setpoints as foIIows: Temperature,
37OC
l?C
Mean Deviation,
0.015”c
0.016”C
Maximal Deviation,
0260°C
0.171”c
During cooling and warming the following were obtained: Temperature
(1) (-1+2qj
derived theoretically by considering critical damping in a servo operation under proportional and integral control’.
Range,
results
3 7” C to -22°C
Rate of Change,
2’C/min
,
-l’C/min
Mean Deviation,
0.136’C
,
0.127”C
Maximal Deviation
0.358’C
,
0.209OC
Rates of change of 4’C/min and -3’C/min achieved with similar accuracy.
were
This control proved satisfactory for preliminary investigations into organ cryopreservation. The computer program allowed any controller settings to be selected at calibration time and changed during run-time. The thermocouple’s measurement error was smaller than 0.5”C. Its addition to the mean and maximum deviation values quoted above yielded the maximum error in temperature reading.
Computer
The process is a non-linear temperature control system; so the response to negative step changes will be slower than that to positive changes which is due to the difference in power output of the cooling and heating elements. The response was also dependent upon the temperature at which the change occurred. CONTROL OF PERFUSATEFLOW AND CPA CONCENTRATION IN THE PERFUSATE Watson-Marlow MHRE miniature flow inducers were used for perfusion and CPA addition. 2 mm x 6 mm (bore x external diameter) silicone elastomer tubings were fitted to the pumps with a maximal flow of 1.075 ml/min for the CPA pump and 4.800 ml/min for the perfusate’s. For applied voltages between 1 and 24 V and using 2 mm x 6 mm silastic tubing, the corresponding flows of the perfusion (Type MHRE 300 and CPA pumps (Type MHRE 7) were recorded. T h e perfusate could be delivered at constant or variable flows, thus either constant flow or constant pressure perfusion experiments could be performed. The physiological pressure measurement system used was of the Bell and Howell 4-327-L222 linear type. The pressure transducer, with a range O-750 mm Hg differential had a sensitivity of 2 pV/mm Hg at an excitation of 10 V d.c. For the ADC input range used, a pressure between 0 and 250 mm Hg could be measured to a resolution of 0.1 mm Hg. The perfusion pressure was logged on the remote teletype every 3 s. Once perfusate flow and molarity of the cryoprotectant were specified, the computer calculated and sent the voltage for the perfusion and CPA respectively and corrected the perfusate flow based on the feedback information from the pressure transducer while maintaining the preset CPA molarity. The direct voltages to drive the pumps were obtained from operational amplifiers powered by a 28 V (Farnell L30B) stabilized power supply.
controf
of cryopreservafion:
segment;
C.G.
Adem
and
1.6. Harness
after 10 s and every 10 s thereafter.
The temperature is read twenty times, averaged, converted into ‘C and printed out after control action. Once the controller settings are entered, the internal controller constants are calculated and control action takes place by sending an output in units through the appropriate DAC. Activation is periodic at 2 s intervals, which is the chosen sampling time. Task CHANGETEMP is invoked only when a certain temperature profile is needed. As it is run, the initialisation segment asks the user to input the number of changes required, followed by the desired setpoints, rates of change and durations at each specific temperature. Cooling and warming of the heart are initiated at the required time by CIHANGETE,MP activation segment. The setpoint is altered each sampling instant by the correct amount to achieve the desired rate of change of temperature. This and the logging of the setpoint and the temperature are carried out at 3 s, sampling time intervals. If the cooling rate is greater than - l*C/min the liquid nitrogen pump is activated through a relay by a signal from the digital output of the computer.
The program to control the cryopreservation apparatus consists of 3 tasks which may run simultaneously, interacting via global variables. The following describes the tasks in question - the name of each task is always written in capital letters.
Task PERFUSION, calibrates the pressure transducer, measures the vascular resistance and controls the CPA and perfusion pumps. The initialisation segment asks for the input of the sampling time, perfusate flow, CPA final molarity, its addition, holding and removal time, and the temperature at which addition and removal are to start. In case freezing is discontinued and resumed must be specified. In add discounted and resumed must be specified. In addition, the maximal pressure permitted is input, thus preventing the vascular resistance from exceeding it. It also allows for the calibration of the pressure transducer by reading the atmospheric pressure, converting it to mm Hg and printing it out on the remote teletype. A small alteration in the program would allow perfusion at constant vascular resistance. The initialisation segment then prints the total experimental time after cryoprotectant introduction and the molar volume of ethylene glycol. Linear addition and removal of CPA are initiated automatically on the specified temperature being reached during the cooling and warming regimes and holdtime terminated before removal is undertaken. If the perfusion experiment involves freezing, flow arrest and resumption occur at the preset temperatures respectively. Vascular resistance is read twenty times. The readings are averaged, converted to mm Hg and printed out. If the pressure exceeds a certain preset level, the perfusate flow is decreased gradually to maintain that level and the cryoprotectant flow is reduced correspondingly to preserve an accurate molarity. Constant pressure perfusion is also possible by varying the flow to maintain a constant vascular resistance pattern.
Task TEMPERATURE measures and maintains a preset temperature. The initialisation segment asks the user to input the controller settings, the sampling time, the heater output level and the setpoint. Initialisation is followed by a periodic activation
Logging of time, perfusion pressure, cryoprotectant’s actual and final molarities and perfusate flow are executed with the control action at the chosen sampling interval which is 3 s in this case. The pumps are switched off upon termination of the experiment.
The perfusion program calculated and delivered to the pump the required voltage, after the required molarity and perfusate flow rate were entered on the remote teletype. The keyboard input facility permitted changes to the appropriate equation required to calculate all the variables in this section. The maximal needed perfusate flow rate into the isolated rat heart was 3 ml/min and the maximal delivery of CPA was of 1.075 ml/min under the actual arrangements. The maximal CPA molarity obtained was 6.426 mol 1-i. THE CONTROL PROGRAM
J. Biomed. Engng.
1981, Vol. 3, April
137
Computer
control
of cryopreservafion:
C.G.
Adem
and
Computer assisted calibration of the instruments on the cryopreservation apparatus is a valuable facility enabling quick and accurate action. The following trol program.
are the facilities
offered
by the con-
(1) Computer
assisted calibration of instruments and setting-up of experimental conditions.
(2)Control
of heart temperature at any profile within the limitations of the apparatus. A
control signal is generated using an incremental form of the two-term control algorithm, proportional and integral modes being effected.
(3) Automatic
initiation of addition and removal of ethylene glycol. The concentration is controlled by setting the CPA pump rate to the correct value by means of a computer-generated voltage signal.
(4) Automatic
suspension and resumption of perfusion at the preset temperature during freezing experiments.
(5)Regular
data logging. Measured variables are logged out on the remote teletype at sampling time intervals. A complete operating record is therefore produced on the teletype, containing the pre-experimental set-up, calibration of measuring equipment, log of variables at regular intervals, and a record of all changes made via the keyboard input facility.
(6) Access to all variables
at any time.
(7) Perfusion required.
flow or pressure as
at constant
J.6.
Harness
the vascular resistance stabilized after 20 min. Normal ECGs reflected the absence of any damage to the heart. Figure 4 shows the changes in vascular resistance during a perfusion and freezing where the temperature is lowered to -22’C and then raised to 36’C. Ethylene glycol was introduced to the heart when the temperature was lowered to 19’C and reached 3 moles at that stabilized temperature. The perfusate was discontinued at -4°C and resumed as the heart was rewarmed at 9’C. The heart was held at 2 1.5’C for 12 min. that
It can be seen that the vascular resistance drops then increases when the glycol reaches the heart and drops as the CPA leaves the heart, then increases again. This can be explained by considering the binary diffusion processes of water and CPA” but because of the complicated flow patterns in the heart an estimate of the time process cannot be made. The results shown are for only short storage times at the lowest temperatures. Hearts were stored at the low temperatures for periods of up to 24 h. No difference was detected between the long and short storage times, as the damage to the heart is mainly during the heating and cooling periods. For the initial experiments, it was not thought necessary to hold all the hearts at the lowest temperatures for lengthy periods. CONCLUSIONS A modified Langendorff perfusion apparatus has been designed and constructed allowing simultaneperfusion or different in a shielded, temperature-controlled
by the
of ECG of the of special
(8) Full automation of the whole process including termination of the experiment. In addition the RTEB system allows variables examined and modified on-line, if so desired.
to be
RESULTS Changes in the mean vascular resistance during perfusion is shown in Fz+re 3 from which it can be seen
of the to -40°C, of perfusate of perfusate be recorded or analysed
. 20
:=r=:::=e,se Figure 3 Changes in mean vascular resistance during perfusion of isolated rat heart at 32’C. l, Pressure; 4 temperature; n, molarity. (Average of 5 experiments).
Engng.
80
DO
Tie (mid Ttme (mid
138 J. Biomed.
*. 40
1981, Vol. 3, April
Figure 4 Changes in vascular resistance at the isolated rat heart during, perfusion, freezing to -3’C and warming to 3 7 C., 0, Pressure; A, temperature; n, molarity. (Average of 6 experiments).
Computer
Experiments on isolated rat hearts have been performed, which have been cooled to -22°C with addition of ethylene glycol and rewarmed to 37’C successfully. This indicates that organ preservation experiments can now be easily performed without tiring manual control systems.
control
3
4
ACKNOWLEDGEMENTS
The authors are indebted to Drs Janice McCurrie, G. Irving and H.A. Sahyoun of the Postgraduate School of Pharmacology for assistance and patience, especially in teaching the surgical techniques. A special note of gratitude is due to Messrs I Dick and R. Butts for their precious help in the construction of the hardware and software. To Dr A. Mearns, Consultant at Bradford Royal Infirmary and Dr K. Hobbs, Professor of Surgery at the Royal Free Hospital, London, for many stimulating discussions. Lastly, but not least, many thanks are due to our typists and friends, Miss J.A. Squires and Mrs J.E. Steele.
5 6
7
8
REFERENCES 1
2
Langendorff, O., Untersuchungen am Ukerlaevenden Sangethierherzen, Pjlug. Arch. ges Physiol, 1895, 61, pp 291-332. Hobbs, K.E.F. and Ellis, M., ‘The Present Status of Sub-Zero Organ Preservation with Special Reference to the Rat Heart’, in Organ Preservation’ (Ed. D.E.
9
10
of cryopreservofion:
C.G.
Adem
and
J.B.
Harness
Pegg,)Churchill-Livingstone, London and Edinburgh, 1973. Armitage, W J. and Pegg, D.E., ‘An Evaluation of Colloidal Solutions for Normothermic Perfusion of Rabbit Heart: An improved Perfusate Containing Haemaccel’, Cryobiology, 1977, 14, 428-34. Pegg, D.E. and Green, C J., ‘Renal Preservation by Hypothermic Perfusion, IV. The Use of Gelatine Polypeptides as the Sole Colloid’, Cryobiology, 1978, 15, pp. 27-34. Jacobsen, A., Continuous Hypothermic Perfusion of Rabbit Kidneys’, Cryobiology, 1978, 15, 290-331. Sahyoun, H.A. and Hicks, R., ‘Electrocardiographic Recording of Normal and Infarct Bearing Rat Hearts in a Perfused Isolated Preparation’,]. Pharmac Methods 1978, 1, 351-600. Bothamley, P.E., Gough, N.E. and Harness, J.B., Computer Control of an Isolated Heart Preparation for Biomedical Research, in Digital Computer Applications to Process Control, (Ed. H.R. van Nauta Lemka, and H.B. Verbruggen,) North-Holland, Amsterdam, New York and London 1977. Butts, R., ‘Real Time Extended Basic Instruction Manual’, Published by Postgraduate School of Control Engineering, University of Bradford, Bradford, UK Ceaglske, N.H., ‘Automatic Process Control for Chemical Engineers’, Published by John Wiley and Sons Inc., New York. Harness, J.B., ‘An Apparent Permeability Coefficient for the Diffusion of Cryoprotective Agents into Red Blood Cells’. Inserm, 1976, 62, 135-138
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