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Some Applications and Limitations of Electromagnetic Blood Flow Measurements in Chronic Animal Preparations
Vol. 52, No.2, Part 2 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1967 by The Williams & Wilkins Co.
SOME APPLICATIONS AND LIMITATIONS OF ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS IN CHRONIC ANIMAL PREPARATIONS ALVIN
F.
SELLERS, PH.D., V.M.D. AND ALAN DOBSON, PH.D.
Department of Physiology, New York State Veterinary College, Cornell University, Ithaca, New York
The ruminoreticular compartment of the gut of the cow is a large vat of about 100liter capacity, in which the animal ferments the grass it ingests. The animal derives its nutrients from the products of this fermentation, and the major fraction, about 75%, of the energy requirement is absorbed from this organ. About 50 moles of the short chain fatty acids, acetic, propionic, and butyric, are absorbed here each day. The contents are mixed by the contractions of a powerful smooth musculature, its activity depending on vagal innervation. The establishment of a large permanent fistula allows access to the ruminoreticulum. Through such a fistula, the right ruminal artery, which supplies major portions of the ventral and posterior rumen compartments, can be readily palpated. vVe were interested in studying effects on blood flow of feeding, psychic, and special reflexes, such as rumination and eructation. Since these are all abolished by anesthesia, the chronic unanesthetized animal must be used. This preparation had the additional advantages of avoiding the variables superimposed by surgical trauma and allowing repeated observations on the same animal. The gated sine wave flowmeter of Kolin and Kado l was used for these experiments with the probes implanted under paralumbar anesthesia through an incision in the right flank. 2 , 3 Our primary interest was in mean blood flow. Observations were usually begun about a week after implantation and continued, usually, for about a month. Feeding effect. Stable flows were attained during an initial period. The animal was then allowed to see and smell its ration, which resulted at times in an increase in blood flow of about 20%. A
further increase to about 140% of the initial blood flow, together with an increased rate of the rumen contraction, was observed when the cow was eating. The increase in blood flow was maintained for several hours after feeding ceased, while the contraction rate tended to return to the prefeeding value within about 30 to 45 min. Removal of the ingesta resulted in a decrease in both the blood flow and the contraction rate. Replacement of the ingesta confined in a plastic bag gave an increase in contraction rate, with no marked change in blood flow. Puncturing the bag markedly stimulated the blood flow without affecting the contraction rate. This indicated that stimulatory substances within the rumen contents were important in determining blood flow, but there was no indication that rumen volume or contraction rate had any effect on this function. Since concomitant observations on other branches of the posterior abdominal aorta showed no increase in blood flow during feeding, it was assumed that the increase in flow seen in the right ruminal artery was due to local vasodilation in this organ. Further analysis of this situation was possible by substituting artificial solutions for natural rumen contents. It was apparent that the products of fermentation, particularly carbonic, butyric, and propionic acids strongly enhanced the blood flow when in concentrations normally found in the ingesta. The pH of the rumen contents is normally between 5 and 7. A low pH within the rumen did not necessarily stimulate blood flow, since solutions of lactate, phosphate, and citrate at pH 5 gave a similar blood flow to sodium chloride at pH 7. The effective stimuli with carbon dioxide and the short chain fatty
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, ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS
acids appeared to be the un-ionized fractions, which penetrate the epithelium much more rapidly than the corresponding anions. Thus, blood flow was stimulated by a low pH in the presence of the fatty acids. It was tentatively concluded that the sustained effect of feeding on blood flow was associated with the production of these substances within the rumen. Feeding in the absence of fermentation, i.e., when the rumen was filled with a solution of 150 mM sodium chloride, was associated with an increase in blood flow only during the act of feeding. This, with suitable controls, indicated reflex stimulation of blood flow from mouth, pharynx, or esophagus. Thus, initial characterization of the regulation of blood flow changes during normal feeding appeared to involve both local chemical stimuli and reflex effects from other organs. Auxiliary Techniques Used with Blood Flow Measurements
Since our main concern has been with measurement of mean flow under chronic conditions, we will confine our discussion to this topic. We would particularly like to draw attention to the reviews of Wyatt4 and Bergel and Gessner 5 for more complete di scus~ions of theory and limitations. A list of errors likely to be encountered under conditions of chronic implantation is given in table 1. During observations on right ruminal artery blood flow, doubts arose as to how closely the measurements made corresponded to the true blood flow, and different techniques were employed in order to examine aspects of this problem. Gate setting. In order to set the gate to see only that component of the amplifier output which is a function of blood flow, we started by adjusting the phase so that the signal with flow occluded coincided with the signal with no magnet current. This technique was soon abandoned, since it led to a drift in phase setting with time and, indeed, occasionally gave a phase setting where the net flow appeared to be in the opposite direction to what one would expect from the pulse shape. In a second
TABLE
1. Errors of electromagnetic blood flowmeter Source
Electrode and amplifier impedances Wall conductivity Hematocrit Nonaxial flow Incorrect gating Electrical eddy currents Poor magnet-electrode insulation Frequency response Phase response Poor fit of probe to artery
Effect
Sensitivity Sensitivity Sensitivity Sensitivity Sensitivity and zero Zero Zero
Reference
7
8,9
5, 7 10
11,12 14
Dynamic char15, 16 acteristics ?
method, a g ate setting was sought where the pulse appeared minimal. The phase was then switched accurately through 90° in order to record blood flow. This method proved somewhat insensitive in chronic preparations, and since the transformer component of the output signal could alter with the pulse, it was capable of giving an erroneous setting at times. A more reliable method depends on varying the transformer coupling between the magnet current and the electrode leads. When the gate is set so that variation in coupling cannot be seen, the meter is correctly phased. A variable air core transformer can be used to facilitate this.6 Distortion. Some early observations indicated that overloading of the amplifier output could easily occur by that fraction of the signal which the gate does not see. A monitor oscilloscope was, therefore, always used on the output of the amplifier to keep a continuous check on the whole signal amplified. Gain. To ensure that changes in the amplifier gain were not overlooked, e.g., in tube failure, a calibrating box actuated from the measured magnet current source was incorporated. This also allowed a check on the linearity of the system. Electrode currents. In the chronic preparations, drift in signal over several days was observed where no change in blood flow would be expected. One possible source of change in sensitivity would be change in
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impedance between the electrodes. Since conventional methods for measuring impedance employed currents which polarized the electrodes, a technique was developed whi ch allowed the measurement of this impedance to be made in vivo under the operating conditions of the flowmeter.7 Systematic losses of signal from this source of up to 25 % were encountered under the conditions used. By measuring the impedance, allowance may be made for these losses. In addition, the impedance gives some indication of the fit of the probe to the artery. In chronic preparations, encapsulation quickly ensures some stabilization of geometry. Only once have we observed the e lectrical impedance to vary with the pulse. Pro be insulation. The insulation resistance between magnet coils and electrodes has been suspected as a source of zero error. This has been routinely examined by applying 22 volts dc between magnet and ground and measuring the current that flows . Validation of the Blood Flow Technique in Vivo
Several sources of error in calibration of electromagnetic blood flowmeters have
FIG. 1. Cannulated carotid loop; cuff-type blood flow probe implanted on carotid just ant erior to first rib; terminals held in skin incisions with threaded nylon washers.
Vol. 52, No.2, Pm·t 2
been described (table 1) . An a llowance for some of these errors can be made, but for others the quantitative contribution in vivo is unknown. Doubtless, some errors still remain to be discovered. Thus, the flowmeter needs calibration under conditions as realistic as possible, using a technique with systematic errors that are fully known. Since the situation of the right ruminal artery makes it difficult to use for this purpose, a preparation using the carotid artery of the sheep was developed. 6 The carotid artery was first exteriorized within a loop of skin. After this had healed, a flow probe was implanted on the same artery in the neck between the loop .and the bifurcation of the common carotid artery. During this procedure, all branches of the artery between the implant and the loop were ligated. This allowed a valid zero flow to be attained by occlusion at least 10 cm from the probe. AT-cannula was then inserted in the artery within the loop. After recovery from anesthesia, a known volume of blood could be passed through the probe to and from a syringe attached to the side arm of this cannula, while the artery cranial to the cannula was occluded (fig. 1). This allowed the calibration of the flowmeter in the conscious sheep, together with measurements of zero offset under realistic conditions for periods up to 3 weeks after cannulation. At all times other than during calibration, a normal flow of blood through the artery could be maintained. Sensitivity. The sensitivity of the probes in vivo showed systematic trends in either direction with time. In addition, the sensitivity on the live artery using blood was less than that measured in vitro without an artery, using saline. This was predicted from the shorting effect of the artery wallS, 9 and depends on the relative conductivities and diameters of the artery wall and the lumen fluid. In practice, in the living animal the loss of signal varied from 7 to 23% during five implants. The signal lost from dead arteries was small since they were equilibrated with the same physiological saline solution as was used for calibration. The equilibration is necessary to achieve
February 1967
ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS
stable sensitivity. 7 The sensitivity to flow in either direction was not always the same. The difference was small, but significant. No persistent pattern of this difference was apparent. This could probably be attributed to changes in sensitivity with nonaxial flow, which might vary with small changes in geometry.lO In the same animals, the flow probe was calibrated with blood post moriem. Although we have too few comparisons to allow generalization, it is of interest that in one case an apparently technically competent calibration post mortem showed a drop in sensitivity amounting to about 60% of the preceding calibration in vivo. In this case, the application of the calibration post mortem to results in vivo on the same day would have led to considerable systematic error. The hematocrit largely determines the conductivity of whole blood. An effect of hematocrit on sensitivity can, therefore, be predicted from the change of relative conductance of vessel wall and lumen contents.9 The relevance of this has clearly been demonstrated in vitro.; In vivo, changes in lumen conductivity correspondin g to a change in the hematocrit from 30 to 40 would give rise to alterations of about 2% in the sensitivity.6 This effect is s o sma n that it could easily be overlooked, unless corrections for changes in electrode impedance were made. We would suggest that the disagreements observed in the effect of hematocrit5 are partly due to the failure to correct the measured sensitivities for changes in impedance between the electrodes. Zero error. This appears as a considerable error in those probes so far examined in chronic preparations. These probes were obtained commercially. The zero offset varied from time to time and from probe to probe. The best probe we have seen so far had an offset corresponding to a blood flow of 50 ml per min. Since the normal mean flow through this vessel was about 250 ml per min, the offset in zero contributed a 20% error. In four other implants, the error varied by at least 100 ml per min during the period of implantation, and
377
in two of these the zero error was of similar magnitude to the blood flow signa l. Direct electrical leakage between the magnet and electrodes is a possible source of zero error, The resistance between the magnet and ground has shown a decline with time, either in vivo or in 150 mM NaCI at body temperature. For example, although the resistance initially has been in the neighborhood of 1010 to 10 12 ohms, over the course of a few days this commonly declined by two orders of magnitude and, in extreme cases, reached 106 ohms. In our situation, it has been calculated that with a leakage of 1010 ohms a zero error of 50 ml per min is possible. 6 Another source of zero error appears to be related to the situation during implantation. That is, probes which gave substantial zero error in chronic experiments showed no zero error · when soaked for 21 days in saline before implantation or after removal from the animal. It is possible that electrical eddy currents in the vicinity of the electrodes would give rise to errors of this type. l1 , 12 Difficulties with zero error in vivo do not appear to be confined to the sine wave electromagnetic flowmeter. For instance, Lydtin and H amilton 13 using a square wave flowmeter said, "\Ve were unable to quantify flow through the renal artery because the recording of zero shifted when the artery was occluded by the nylon snare." Of course, it is possible to offset a ze ro error by suitable injection of signal. This, however, is merely a palliative, since variation in zero error with time would require a comparable variation of inj ected signal to annul the error. Our experience with a device of this sort led us to conclude that, even a t its best , it could do no more than provide a means of offsetting one unknown source of error by another.6 Conclusions
The signal of an electromagnetic flowmeter under conditions of chronic implantation gives a measure of mean blood flow with appreciable systematic error, both in sensitivity and in zero setting. It goes with-
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out saying that the importance of the errors of any technique depends upon the use to which the results are put. In the light of these observations on the characteristics of the flowmeter with chronically implanted probes, we would now wish that the figures we previously published had not been given as flow rates, since we were extrapolating from the postmortem ca libration with the animals' own blood. In addition, some of the variability in changes of relative blood flow with different procedures probably arose from changes in zero error. In our preparation using the right ruminal artery of the cow, occlusion was possible in order to check the zero error. In this situation, one can either occlude some distance from the probe, with the fair certainty of missing a side branch, or close to the probe, in which case the geometry is altered. We tended to be more tolerant of apparently small changes in zero error t han we would be now, because of the difficulty of attaining a satisfactory zero. In the past, a considerable effort has been made to improve the electronic part of the apparatus, e.g., by using different modes of actuation of the magnet . It has yet to be demonstrated that a m arked improvement in performance in chronic preparations has ensued. At this stage, improvements in probe materials and construction are indicated. Weare not aware of a basis for predicting or circumventing the errors we have observed from measurements made in vitro, at necropsy, or in vivo. Since, however, a method of measuring these errors exists, some advance toward a more accurate t echnique is now possible.
Addendum The authors wish to direct attention to: Wyatt, D. G. 1966. Baseline errors in cuff electromagnetic flowmeters. Med. BioI. Engng. 4: 17-45. This article was overlooked in the preparation of our manuscript. Wyatt distinguishes 10 sources of zero error and predicts their magnitude in model situations.
Vol. 52, N o.2, Part 2 REFERENCES
1. Kolin, A., and R. T. Kado. 1959. Miniaturization of the electromagnetic blood flowmeter and its use for the recording of circulatory responses of conscious animals to sensory stimuli. Proc. Nat. Acad. Sci. U. S. A. 40: 1312-1321. 2. Sellers, A. F ., C. E. Stevens, A. Dobson, and F. D . McLeod. 1964. Arterial blood flow to the ruminant stomach. Amer. J. Physiol. 207: 371-377. 3. Selle rs, A. F. 1965. Blood flow in the rumen vessels, p. 171-184. In R. W. Dougherty [ed.], Physiology of digestion in the ruminant. Butterworth Inc., Washington. 4. Wyatt, D. G. 1961. Problems in the measurement of blood flow by magnetic induction. Phys. Med. BioI. 5: 289-352. 5. Bergel, D. H., and V. Gessner. 1966. III. The electromagnetic flowmeter, p. 70-82. In Methods in medical research, Vol. 11. Year Book Publishers, Inc., Chicago. 6. Dobson, A., A. F. Sellers, and F. D. McLeod. 1966. The performance of a cuff-type blood flowmeter in vivo. J. Appl. Physiol. 21: 16421648. 7. Dobson, A., and F. D. McLeod. 1963. The cuff-type electromagnetic blood flowmet er, p. 73-78. Proceedings of San Diego Symposium for Biomedical Engineering. 8. Hill, W. S. 1960. On the theoretical basis of electromagnetic methods to measure rates of flow. Bol. Fac. Ing. Agri-Mensura Montevideo 7: 295-320. 9. Gessner, V. 1961. Effects of the vessel wall on electromagnetic flow measurement. Biophys. J. 1: 627-637. 10. Goldman, S. C., N. B. Marple, and W. L. Scolnick. 1963. Effects of flow profile on electromagnetic flowmeter accuracy. J . Appl. Physiol. 18: 652-657. 11. Colvin, A., and A. Warnick. 1962. A newly recognized source of zero instability in the A. C. electromagnetic flowmeter, p. 1. In Digest of the Fifteenth Annual Conference on Engineering in Medicine and Biology. Chicago, Illinois. 12. Wyatt, D. G. 1964. Electromagnetic flowmeter for use with intact vessels. J. Physiol. 173 :
8P 13. Lydtin, H., and W. F. Hamilton. 1964. Effect
of acute changes in left atrial pressure on urine flow in unanesthetized dogs. Amer. J. Physiol. 207 : 530-536.
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ELECTROMA GNETIC BLOOD FLOW MEASUREMENTS
14. Spencer, M. P ., C. A. Barefoot, C. D. McNeil, and A. B. Denison, Jr. 1963. Stability of zero flow reference in electromagnetic flowmeters. Fed. Proc. 22: 522. 15. Gessner, V., and D. H. B erg~l. 1964. Fre-
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quency response of electromagnetic flowmeters. J . Appl. Physiol. 19: 1209-1211. 16. O'Rourke, M . F. 1965. Dynamic accuracy of the electromagnetic flowmeter. J. Appl. Physiol. 20: 142-147.
DISCUSSION OF "SOME APPLICATIONS AND LIMITATIONS OF ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS IN CHRONIC ANIMAL PREPARATIONS" Alexander Kolin, Ph.D.
In the introduction of a new method into a field of research, two types of contributions are of comparable importance: (a) the paving of new avenues of approach to the solution of scientific problems by pointing out the possibilities of the new method and (b) critical contributions assessing the limitations of the method, safeguarding investigators against pitfalls which could lead to gross errors. The paper by Sellers and Dobson is a thorough and helpful contribution of the latter kind. It is especially to be hoped that, by pointing out specific limitations of commercially available flow transducers, the authors may stimulate efforts toward much needed improvements. It is certain that the procedures outlined by the authors will be of great help to other investigators, and their review of sources of error is apt to promote more reliable and more critical publications in this field. The authors devote themselves mainly to four problems: (a) adjustment of the gating so as to reject the unwanted quadrature voltage, (b) magnitude and stability of the zero offset voltage, (c) stability of the sensitivity in vitro and in vivo, and (d) validity of calibration obtained in vitro in the chronic animal preparation. In the method employed by Sellers and Dobson, the magnetic field varies sinusoidally with time. The electromagnetic force (EMF) induced in the blood traversDr. Kolin is from the Department of Biophysics and Nuclear Medicine, UCLA School of Medicine, Los Angeles, California.
ing this magnetic field is a sine function of time precisely in phase with the magnetic field. The induced voltage is the desired flow signal. In addition, there is an undesired signal present, owing to a flowindependent EMF induced in transformer fashion in the electrode leads circuit and in the blood and artery wall, enclosed in the transducer, giving rise to eddy currents. This "transformer EMF" is represented by a cosine function being proportional to the time rat e of change of the magnetic field. Thus, it is zero precisely at the moment when the magnetic field and the flow signal reach their maximum. The purpose of gating is to look at the flow signal at the precise moment when the unwanted transformer EMF is exactly zero. Lack of precision in this adjustment leads to a nonzero reading at zero flow. The zero pulsation method, recommended in the original description of the gated sine wave flowmeter used by the authors, employed an indirect method to adjust for the zero of the cosine curve. This method adjusts for the zero of the sine curve by watching the flow signal and gating for its disappearance. The gate is then shifted through 90 and is thus synchronized with the zero point of the cosine signal (transformer EMF). This step introduces a n error, since the precision of the 90 shift and its long-term stability are limited. The authors introduce a new method of gate adjustment based on direct determination of the zero point of the cosine signal, which avoids the necessity for 0
0
GASTROENTEROLOGY
Copyright © 1967 by The Williams & Wilkins Co.
SOME APPLICATIONS AND LIMITATIONS OF ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS IN CHRONIC ANIMAL PREPARATIONS ALVIN
F.
SELLERS, PH.D., V.M.D. AND ALAN DOBSON, PH.D.
Department of Physiology, New York State Veterinary College, Cornell University, Ithaca, New York
The ruminoreticular compartment of the gut of the cow is a large vat of about 100liter capacity, in which the animal ferments the grass it ingests. The animal derives its nutrients from the products of this fermentation, and the major fraction, about 75%, of the energy requirement is absorbed from this organ. About 50 moles of the short chain fatty acids, acetic, propionic, and butyric, are absorbed here each day. The contents are mixed by the contractions of a powerful smooth musculature, its activity depending on vagal innervation. The establishment of a large permanent fistula allows access to the ruminoreticulum. Through such a fistula, the right ruminal artery, which supplies major portions of the ventral and posterior rumen compartments, can be readily palpated. vVe were interested in studying effects on blood flow of feeding, psychic, and special reflexes, such as rumination and eructation. Since these are all abolished by anesthesia, the chronic unanesthetized animal must be used. This preparation had the additional advantages of avoiding the variables superimposed by surgical trauma and allowing repeated observations on the same animal. The gated sine wave flowmeter of Kolin and Kado l was used for these experiments with the probes implanted under paralumbar anesthesia through an incision in the right flank. 2 , 3 Our primary interest was in mean blood flow. Observations were usually begun about a week after implantation and continued, usually, for about a month. Feeding effect. Stable flows were attained during an initial period. The animal was then allowed to see and smell its ration, which resulted at times in an increase in blood flow of about 20%. A
further increase to about 140% of the initial blood flow, together with an increased rate of the rumen contraction, was observed when the cow was eating. The increase in blood flow was maintained for several hours after feeding ceased, while the contraction rate tended to return to the prefeeding value within about 30 to 45 min. Removal of the ingesta resulted in a decrease in both the blood flow and the contraction rate. Replacement of the ingesta confined in a plastic bag gave an increase in contraction rate, with no marked change in blood flow. Puncturing the bag markedly stimulated the blood flow without affecting the contraction rate. This indicated that stimulatory substances within the rumen contents were important in determining blood flow, but there was no indication that rumen volume or contraction rate had any effect on this function. Since concomitant observations on other branches of the posterior abdominal aorta showed no increase in blood flow during feeding, it was assumed that the increase in flow seen in the right ruminal artery was due to local vasodilation in this organ. Further analysis of this situation was possible by substituting artificial solutions for natural rumen contents. It was apparent that the products of fermentation, particularly carbonic, butyric, and propionic acids strongly enhanced the blood flow when in concentrations normally found in the ingesta. The pH of the rumen contents is normally between 5 and 7. A low pH within the rumen did not necessarily stimulate blood flow, since solutions of lactate, phosphate, and citrate at pH 5 gave a similar blood flow to sodium chloride at pH 7. The effective stimuli with carbon dioxide and the short chain fatty
374
February 1967
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, ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS
acids appeared to be the un-ionized fractions, which penetrate the epithelium much more rapidly than the corresponding anions. Thus, blood flow was stimulated by a low pH in the presence of the fatty acids. It was tentatively concluded that the sustained effect of feeding on blood flow was associated with the production of these substances within the rumen. Feeding in the absence of fermentation, i.e., when the rumen was filled with a solution of 150 mM sodium chloride, was associated with an increase in blood flow only during the act of feeding. This, with suitable controls, indicated reflex stimulation of blood flow from mouth, pharynx, or esophagus. Thus, initial characterization of the regulation of blood flow changes during normal feeding appeared to involve both local chemical stimuli and reflex effects from other organs. Auxiliary Techniques Used with Blood Flow Measurements
Since our main concern has been with measurement of mean flow under chronic conditions, we will confine our discussion to this topic. We would particularly like to draw attention to the reviews of Wyatt4 and Bergel and Gessner 5 for more complete di scus~ions of theory and limitations. A list of errors likely to be encountered under conditions of chronic implantation is given in table 1. During observations on right ruminal artery blood flow, doubts arose as to how closely the measurements made corresponded to the true blood flow, and different techniques were employed in order to examine aspects of this problem. Gate setting. In order to set the gate to see only that component of the amplifier output which is a function of blood flow, we started by adjusting the phase so that the signal with flow occluded coincided with the signal with no magnet current. This technique was soon abandoned, since it led to a drift in phase setting with time and, indeed, occasionally gave a phase setting where the net flow appeared to be in the opposite direction to what one would expect from the pulse shape. In a second
TABLE
1. Errors of electromagnetic blood flowmeter Source
Electrode and amplifier impedances Wall conductivity Hematocrit Nonaxial flow Incorrect gating Electrical eddy currents Poor magnet-electrode insulation Frequency response Phase response Poor fit of probe to artery
Effect
Sensitivity Sensitivity Sensitivity Sensitivity Sensitivity and zero Zero Zero
Reference
7
8,9
5, 7 10
11,12 14
Dynamic char15, 16 acteristics ?
method, a g ate setting was sought where the pulse appeared minimal. The phase was then switched accurately through 90° in order to record blood flow. This method proved somewhat insensitive in chronic preparations, and since the transformer component of the output signal could alter with the pulse, it was capable of giving an erroneous setting at times. A more reliable method depends on varying the transformer coupling between the magnet current and the electrode leads. When the gate is set so that variation in coupling cannot be seen, the meter is correctly phased. A variable air core transformer can be used to facilitate this.6 Distortion. Some early observations indicated that overloading of the amplifier output could easily occur by that fraction of the signal which the gate does not see. A monitor oscilloscope was, therefore, always used on the output of the amplifier to keep a continuous check on the whole signal amplified. Gain. To ensure that changes in the amplifier gain were not overlooked, e.g., in tube failure, a calibrating box actuated from the measured magnet current source was incorporated. This also allowed a check on the linearity of the system. Electrode currents. In the chronic preparations, drift in signal over several days was observed where no change in blood flow would be expected. One possible source of change in sensitivity would be change in
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impedance between the electrodes. Since conventional methods for measuring impedance employed currents which polarized the electrodes, a technique was developed whi ch allowed the measurement of this impedance to be made in vivo under the operating conditions of the flowmeter.7 Systematic losses of signal from this source of up to 25 % were encountered under the conditions used. By measuring the impedance, allowance may be made for these losses. In addition, the impedance gives some indication of the fit of the probe to the artery. In chronic preparations, encapsulation quickly ensures some stabilization of geometry. Only once have we observed the e lectrical impedance to vary with the pulse. Pro be insulation. The insulation resistance between magnet coils and electrodes has been suspected as a source of zero error. This has been routinely examined by applying 22 volts dc between magnet and ground and measuring the current that flows . Validation of the Blood Flow Technique in Vivo
Several sources of error in calibration of electromagnetic blood flowmeters have
FIG. 1. Cannulated carotid loop; cuff-type blood flow probe implanted on carotid just ant erior to first rib; terminals held in skin incisions with threaded nylon washers.
Vol. 52, No.2, Pm·t 2
been described (table 1) . An a llowance for some of these errors can be made, but for others the quantitative contribution in vivo is unknown. Doubtless, some errors still remain to be discovered. Thus, the flowmeter needs calibration under conditions as realistic as possible, using a technique with systematic errors that are fully known. Since the situation of the right ruminal artery makes it difficult to use for this purpose, a preparation using the carotid artery of the sheep was developed. 6 The carotid artery was first exteriorized within a loop of skin. After this had healed, a flow probe was implanted on the same artery in the neck between the loop .and the bifurcation of the common carotid artery. During this procedure, all branches of the artery between the implant and the loop were ligated. This allowed a valid zero flow to be attained by occlusion at least 10 cm from the probe. AT-cannula was then inserted in the artery within the loop. After recovery from anesthesia, a known volume of blood could be passed through the probe to and from a syringe attached to the side arm of this cannula, while the artery cranial to the cannula was occluded (fig. 1). This allowed the calibration of the flowmeter in the conscious sheep, together with measurements of zero offset under realistic conditions for periods up to 3 weeks after cannulation. At all times other than during calibration, a normal flow of blood through the artery could be maintained. Sensitivity. The sensitivity of the probes in vivo showed systematic trends in either direction with time. In addition, the sensitivity on the live artery using blood was less than that measured in vitro without an artery, using saline. This was predicted from the shorting effect of the artery wallS, 9 and depends on the relative conductivities and diameters of the artery wall and the lumen fluid. In practice, in the living animal the loss of signal varied from 7 to 23% during five implants. The signal lost from dead arteries was small since they were equilibrated with the same physiological saline solution as was used for calibration. The equilibration is necessary to achieve
February 1967
ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS
stable sensitivity. 7 The sensitivity to flow in either direction was not always the same. The difference was small, but significant. No persistent pattern of this difference was apparent. This could probably be attributed to changes in sensitivity with nonaxial flow, which might vary with small changes in geometry.lO In the same animals, the flow probe was calibrated with blood post moriem. Although we have too few comparisons to allow generalization, it is of interest that in one case an apparently technically competent calibration post mortem showed a drop in sensitivity amounting to about 60% of the preceding calibration in vivo. In this case, the application of the calibration post mortem to results in vivo on the same day would have led to considerable systematic error. The hematocrit largely determines the conductivity of whole blood. An effect of hematocrit on sensitivity can, therefore, be predicted from the change of relative conductance of vessel wall and lumen contents.9 The relevance of this has clearly been demonstrated in vitro.; In vivo, changes in lumen conductivity correspondin g to a change in the hematocrit from 30 to 40 would give rise to alterations of about 2% in the sensitivity.6 This effect is s o sma n that it could easily be overlooked, unless corrections for changes in electrode impedance were made. We would suggest that the disagreements observed in the effect of hematocrit5 are partly due to the failure to correct the measured sensitivities for changes in impedance between the electrodes. Zero error. This appears as a considerable error in those probes so far examined in chronic preparations. These probes were obtained commercially. The zero offset varied from time to time and from probe to probe. The best probe we have seen so far had an offset corresponding to a blood flow of 50 ml per min. Since the normal mean flow through this vessel was about 250 ml per min, the offset in zero contributed a 20% error. In four other implants, the error varied by at least 100 ml per min during the period of implantation, and
377
in two of these the zero error was of similar magnitude to the blood flow signa l. Direct electrical leakage between the magnet and electrodes is a possible source of zero error, The resistance between the magnet and ground has shown a decline with time, either in vivo or in 150 mM NaCI at body temperature. For example, although the resistance initially has been in the neighborhood of 1010 to 10 12 ohms, over the course of a few days this commonly declined by two orders of magnitude and, in extreme cases, reached 106 ohms. In our situation, it has been calculated that with a leakage of 1010 ohms a zero error of 50 ml per min is possible. 6 Another source of zero error appears to be related to the situation during implantation. That is, probes which gave substantial zero error in chronic experiments showed no zero error · when soaked for 21 days in saline before implantation or after removal from the animal. It is possible that electrical eddy currents in the vicinity of the electrodes would give rise to errors of this type. l1 , 12 Difficulties with zero error in vivo do not appear to be confined to the sine wave electromagnetic flowmeter. For instance, Lydtin and H amilton 13 using a square wave flowmeter said, "\Ve were unable to quantify flow through the renal artery because the recording of zero shifted when the artery was occluded by the nylon snare." Of course, it is possible to offset a ze ro error by suitable injection of signal. This, however, is merely a palliative, since variation in zero error with time would require a comparable variation of inj ected signal to annul the error. Our experience with a device of this sort led us to conclude that, even a t its best , it could do no more than provide a means of offsetting one unknown source of error by another.6 Conclusions
The signal of an electromagnetic flowmeter under conditions of chronic implantation gives a measure of mean blood flow with appreciable systematic error, both in sensitivity and in zero setting. It goes with-
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out saying that the importance of the errors of any technique depends upon the use to which the results are put. In the light of these observations on the characteristics of the flowmeter with chronically implanted probes, we would now wish that the figures we previously published had not been given as flow rates, since we were extrapolating from the postmortem ca libration with the animals' own blood. In addition, some of the variability in changes of relative blood flow with different procedures probably arose from changes in zero error. In our preparation using the right ruminal artery of the cow, occlusion was possible in order to check the zero error. In this situation, one can either occlude some distance from the probe, with the fair certainty of missing a side branch, or close to the probe, in which case the geometry is altered. We tended to be more tolerant of apparently small changes in zero error t han we would be now, because of the difficulty of attaining a satisfactory zero. In the past, a considerable effort has been made to improve the electronic part of the apparatus, e.g., by using different modes of actuation of the magnet . It has yet to be demonstrated that a m arked improvement in performance in chronic preparations has ensued. At this stage, improvements in probe materials and construction are indicated. Weare not aware of a basis for predicting or circumventing the errors we have observed from measurements made in vitro, at necropsy, or in vivo. Since, however, a method of measuring these errors exists, some advance toward a more accurate t echnique is now possible.
Addendum The authors wish to direct attention to: Wyatt, D. G. 1966. Baseline errors in cuff electromagnetic flowmeters. Med. BioI. Engng. 4: 17-45. This article was overlooked in the preparation of our manuscript. Wyatt distinguishes 10 sources of zero error and predicts their magnitude in model situations.
Vol. 52, N o.2, Part 2 REFERENCES
1. Kolin, A., and R. T. Kado. 1959. Miniaturization of the electromagnetic blood flowmeter and its use for the recording of circulatory responses of conscious animals to sensory stimuli. Proc. Nat. Acad. Sci. U. S. A. 40: 1312-1321. 2. Sellers, A. F ., C. E. Stevens, A. Dobson, and F. D . McLeod. 1964. Arterial blood flow to the ruminant stomach. Amer. J. Physiol. 207: 371-377. 3. Selle rs, A. F. 1965. Blood flow in the rumen vessels, p. 171-184. In R. W. Dougherty [ed.], Physiology of digestion in the ruminant. Butterworth Inc., Washington. 4. Wyatt, D. G. 1961. Problems in the measurement of blood flow by magnetic induction. Phys. Med. BioI. 5: 289-352. 5. Bergel, D. H., and V. Gessner. 1966. III. The electromagnetic flowmeter, p. 70-82. In Methods in medical research, Vol. 11. Year Book Publishers, Inc., Chicago. 6. Dobson, A., A. F. Sellers, and F. D. McLeod. 1966. The performance of a cuff-type blood flowmeter in vivo. J. Appl. Physiol. 21: 16421648. 7. Dobson, A., and F. D. McLeod. 1963. The cuff-type electromagnetic blood flowmet er, p. 73-78. Proceedings of San Diego Symposium for Biomedical Engineering. 8. Hill, W. S. 1960. On the theoretical basis of electromagnetic methods to measure rates of flow. Bol. Fac. Ing. Agri-Mensura Montevideo 7: 295-320. 9. Gessner, V. 1961. Effects of the vessel wall on electromagnetic flow measurement. Biophys. J. 1: 627-637. 10. Goldman, S. C., N. B. Marple, and W. L. Scolnick. 1963. Effects of flow profile on electromagnetic flowmeter accuracy. J . Appl. Physiol. 18: 652-657. 11. Colvin, A., and A. Warnick. 1962. A newly recognized source of zero instability in the A. C. electromagnetic flowmeter, p. 1. In Digest of the Fifteenth Annual Conference on Engineering in Medicine and Biology. Chicago, Illinois. 12. Wyatt, D. G. 1964. Electromagnetic flowmeter for use with intact vessels. J. Physiol. 173 :
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of acute changes in left atrial pressure on urine flow in unanesthetized dogs. Amer. J. Physiol. 207 : 530-536.
February 1967
ELECTROMA GNETIC BLOOD FLOW MEASUREMENTS
14. Spencer, M. P ., C. A. Barefoot, C. D. McNeil, and A. B. Denison, Jr. 1963. Stability of zero flow reference in electromagnetic flowmeters. Fed. Proc. 22: 522. 15. Gessner, V., and D. H. B erg~l. 1964. Fre-
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DISCUSSION OF "SOME APPLICATIONS AND LIMITATIONS OF ELECTROMAGNETIC BLOOD FLOW MEASUREMENTS IN CHRONIC ANIMAL PREPARATIONS" Alexander Kolin, Ph.D.
In the introduction of a new method into a field of research, two types of contributions are of comparable importance: (a) the paving of new avenues of approach to the solution of scientific problems by pointing out the possibilities of the new method and (b) critical contributions assessing the limitations of the method, safeguarding investigators against pitfalls which could lead to gross errors. The paper by Sellers and Dobson is a thorough and helpful contribution of the latter kind. It is especially to be hoped that, by pointing out specific limitations of commercially available flow transducers, the authors may stimulate efforts toward much needed improvements. It is certain that the procedures outlined by the authors will be of great help to other investigators, and their review of sources of error is apt to promote more reliable and more critical publications in this field. The authors devote themselves mainly to four problems: (a) adjustment of the gating so as to reject the unwanted quadrature voltage, (b) magnitude and stability of the zero offset voltage, (c) stability of the sensitivity in vitro and in vivo, and (d) validity of calibration obtained in vitro in the chronic animal preparation. In the method employed by Sellers and Dobson, the magnetic field varies sinusoidally with time. The electromagnetic force (EMF) induced in the blood traversDr. Kolin is from the Department of Biophysics and Nuclear Medicine, UCLA School of Medicine, Los Angeles, California.
ing this magnetic field is a sine function of time precisely in phase with the magnetic field. The induced voltage is the desired flow signal. In addition, there is an undesired signal present, owing to a flowindependent EMF induced in transformer fashion in the electrode leads circuit and in the blood and artery wall, enclosed in the transducer, giving rise to eddy currents. This "transformer EMF" is represented by a cosine function being proportional to the time rat e of change of the magnetic field. Thus, it is zero precisely at the moment when the magnetic field and the flow signal reach their maximum. The purpose of gating is to look at the flow signal at the precise moment when the unwanted transformer EMF is exactly zero. Lack of precision in this adjustment leads to a nonzero reading at zero flow. The zero pulsation method, recommended in the original description of the gated sine wave flowmeter used by the authors, employed an indirect method to adjust for the zero of the cosine curve. This method adjusts for the zero of the sine curve by watching the flow signal and gating for its disappearance. The gate is then shifted through 90 and is thus synchronized with the zero point of the cosine signal (transformer EMF). This step introduces a n error, since the precision of the 90 shift and its long-term stability are limited. The authors introduce a new method of gate adjustment based on direct determination of the zero point of the cosine signal, which avoids the necessity for 0
0