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Clin. Cardiol. 4, 180-187 (1981)
0 G. Witzstrock Publishing House, Inc.
Preliminary Report
PISA: A Noninvasive Method in Detection and Quantification of
Acid-induced Myocardial Infarction in Dogs
K. PRASAD. M.D.,PH.D., M. M. GUPTA, PH.D.*
Department of Physiology, College of Medicine, and *Systems Science Research Laboratory, College of Engineering,
University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Summary: Phase invariant signature algorithm (PISA), a
new noninvasive technique, was used in the detection and
quantification of acid-induced myocardial damage in anesthetized dogs. The diagnostic capabilities of this method were
compared with those of conventional electrocardiogram and
biochemical markers, MB-creatine phosphokinase (MBCPK) and lactic dehydrogenase (LDH,). Myocardial
damage of varying degree was induced by injecting diluted
sulphuric acid (0.01 to 0.10 ml) into the free wall of the left
ventricle. Conventional ECG, wideband ECG for PISA
analysis, blood samples for LDH I , and MB-CPK were taken
before and after 15,30,60, and 90 min of acid injection. The
heart was removed at the end of 90 min for estimation of
myocardial damage. PISA Index increased within 10-15 min
of acid injection and remained elevated for the duration of
the experiment (90 min). The increase in the PISA index was
directly related to the extent of myocardial damage and the
amount of acid injected. Although the conventional electrocardiogram detected large myocardial damage, it was
unable to detect small myocardial damage. Also, most of
initial changes in conventional ECG with large myocardial
damage disappeared within 90 min, while the PISA index was
still elevated to the maximum level. The MB-CPK was not
detected before or after myocardial damage. There was no
significant change in the LDHl at any time after myocardial
infarction. These results suggest that the PISA technique is
superior to the conventional ECG and the biochemical
markers and would be a valuable diagnostic tool in the detection and quantification of incipient as well as advanced
myocardial infarction.
Address for reprints:
K. Prasad, M.D.
Department of Physiology
College of Medicine
Received: January 29, 1980
Accepted: February 1 I , 1981
Key words: noninvasive PISA system, myocardial infarction,
MB-CPK, lactic dehydrogenase, conventional ECG, wideband ECG, detection and quantification of myocardial infarction
Introduction
Early and accurate identification of ischemic heart disease
is an important clinical challenge. Because of certain limitations the diagnostic techniques currently available are incapable of detecting ischemic heart diseases in their early
stages. They also present with some difficulties in quantification. Radionuclide imaging has been used extensively in
the detection and quantification of myocardial infarction
(Holman, 1976; Holman et al., 1978; Parisi et al., 1976).
However, quantification by this method is difficult and needs
elaborate equipment (Holman et al., 1978; Parisi et al.,
1976). Many biochemical markers have been used in the
detection and quantification of myocardial infarction. The
MB isoenzyme of creatine phosphokinase (MB-CPK) which
has been widely used in recent years does not give an accurate
measurement of infarct size. Also, detectable amounts appear
too late for early diagnosis (Ahumada et al., 1976; Bleifeld
et al., 1976; Shell et a1.;1973). The popularity of ST-segment
mapping for estimating infarct size is attributed to its noninvasive approach and simply applied rules in interpretation.
The reliability of information obtained by this process in
estimating changing size of infarction is controversial
(Holland and Brooks, 1977; Henning et al., 1978; Surawicz,
1972). ST-segment mapping provides a qualitative index of
size of infarction and can signal directional changes in size
of evolving infarct, but it cannot quantitate the degree of
necrosis (Maroko et al., 1972). Currently, no technique can
accurately measure the infarct size during the early period,
4-8 h, after onset of symptoms. Creatinine kinase curves
require 10-1 2 h before accurate sizing is possible (Sobel and
Shell, 1972).
K. Prasad and M.M. Gupta: PISA
A diagnostic technique which is noninvasive, and therefore
widely applicable and of low risk, and which can detect and
quantify at a very early stage of the disease, would be a tremendous advantage. Present studies using the statistical
processing and pattern recognition aspects of wideband
electrocardiogram have led to the development of a new algorithm called the phase-invariant signature algorithm
(PISA) for detection and quantification of early as well as
advanced cardiac disorders that introduce phase-locked
perturbations in the ECG (Prasad and Gupta, 1978; 1979;
1980; Prasad el al., 1978a,b; Roberts et al., 1975). Our
previous studies (Prasad and Gupta, 1980; Prasad et al.,
1978b) in dogs have shown that the PISA index increased
with coronary ligation and returned to control value after
some time, which might be due to the existence of an extensive collateral circulation. However, it was difficult to demonstrate this theory.
The present investigation was, therefore, undertaken to
study the effect of localized acid-induced cardiac damage in
dogs on the PISA index, conventional ECG, biochemical
markers (MB-CPK and LDHl), and histologic changes. The
diagnostic capabilities of these techniques were compared
and are reported in this paper.
Methods
The Methods section has been divided into two parts, (a)
the basic concept of PISA method, and (b) the data acquisition and signal processing.
PEA Concept
The basics of PISA concept have been reported in detail
elsewhere (Prasad and Gupta, 1980; Prasad et al., 1978b).
A summary of the concept is given here.
The ECG is a measurement of electrical activity of the
cardiac system, which is a cyclic process and can be regarded
as a function of time (t) from 0-T s or equivalently as a
function of phase (I$) from 0-27r radians. The period T may
vary from beat to beat, but the cyclic process will always go
through the same phase over 0-27r radians. Thus in the
phase-domain, the ECG can be regarded as a periodic or
phase-invariant process. The hypothesis made in this PISA
technique is that the ECG of a diseased heart is composed of
three main components:
s(4) = a hypothetical ECG signal generated by a healthy
heart, under noise-free conditions;
n(4) = the background measurement noise component uniformly distributed over (0,27rr) which occurs during
the transmission and measurement of ECG and
which is not phase-locked to the cardiac cycle;
p(4) = the phase-locked wideband perturbation due to a
disorder in the heart.
The measured ECG, m(I$), is therefore a function [f(.)] of
s(I$), n(4), and p(@),and is expressed as:
181
m(I$) = fls(I$); n(I$)*P(4)L
where I$ is the cardiac phase, I$E (0,27rr).
The detection of cardiac disorders using the PISA technique is therefore the detection of p(4) in the presence of s(4)
and n(4). The signal [s(&)] is a stationary process and,
therefore, is distinguishable from perturbations [p(#)] and
noise[n($)]. By employing a phase-locked statistical demodulation technique, p(4) can be identified. Wideband ECG
is recorded on an FM tape. Fifty cycles of ECG are digitized
and converted from the time-domain to the phase-domain.
Using the standard signal averaging technique the stationary
component [s(4)] is obtained by averaging out the random
components [n(4), p($)] from the measurement [m(#)]. The
power in the random component is then averaged over many
heart beats and plotted as a function of cardiac phase I$. The
power distribution of noise component [n(I$)] which is independent of phase, is uniformly distributed over the phase
I$ E (0,27rr). The power distribution of random component
of the perturbation [p(d)] which is phase-locked to the cardiac cycle, appears as peaks or spikes, occurring at a particular phase of the cardiac cycle. The graphic representation
of the characteristic properties of cardiac system processed
by the PISA method is called the PISA signature. Since the
phase-locked perturbation component [p(4)] is absent in a
healthy heart, the signature is a straight line (phase-invariant), and thus this processing algorithm is called the phaseinvariant signature algorithm (PISA) (Fig. 1). However, for
a diseased heart the signature is phase-invariant and the
signature has spikes (Figs. 2 and 3). In order to make the
PISA signature information more objective, the signature
is quantified. This quantification is called the PISA index,
which is a measure of the extent of the cardiac disorder. A
low PISA index is a sign of a healthy heart, while a high PISA
index denotes a cardiac disorder.
Data Acquisition and Signal Processing
Experiments were carried out in eight dogs weighing 14- 18
kg under pentobarbital anesthesia (35 mg/kg, i.v.). The
trachea was intubated, and the dogs were ventilated with
room air using a Harvard respiratory pump with a volume
of 20 ml/kg and a respiratory rate of 15-20 min. The heart
was exposed through left fifth intercostal space thoracotomy,
and the pericardium was removed.
Various grades of myocardial damage were produced by
injecting 0.01-0.10 ml of diluted sulfuric acid (1:l) in the left
ventricular wall. Only one dose was used in each dog. Conventional ECG, wideband ECG for PISA analysis, and blood
samples for LDHl and MB-CPK were taken before and after
15, 30, 60, and 90 min of acid injection. The heart was removed at the end of 90 min for morphologic and histologic
studies.
Conventional ECG and wideband ECG for PISA analysis
were recorded on an FM tape. The computer analysis of the
records was made to determine the PISA signature and PISA
index.
Total CPK was determined on an Abbott ABA-100 using
Clin. Cardiol. Vol. 4, July/August
182
TIME MSEC
@)
,o.L...-I
!. .
.O
80.0
180.0
2 1.0
320.0
400.0
40.0
110.0
200.0
280.0
360.0
TIME MSEC
.04
.O
45.0
(C)
FIG. 1 (a) Conventional ECG, (b) ensemble average of wideband
ECG, (c) corresponding PISA signature from a normal healthy
1
I_
I
I
!
!
1-4--4+
210.0
360.0
450.0
180.0
135.0
225.0
315.0
405.0
TIME MSEC
90-0
'
(d
dog.
FIG.2 (a) Conventional ECG. (b) ensemble average of wideband
ECG, (c) corresponding PISA signature from a small myocardial
infarct in a dog with normal conventional ECG.
Warthington Statzyme CPR-N-1 reagent and the methods
of Oliver (1955) and Rosalki (1967). CPK Isoenzymes were
separated with agarose gel electrophoresis using a Corning
ACI system. Fluorometric quantification of isoenzyme bands
was made with the use of a Helena Fluro-vis scanner. Total
LDH determination was made on an Abbott ABA-100 using
Warthington Statzyme LDH (L-P) reagent and the methods
of Wacker et al. (1956) and Amador et al. (1963). The LDH
isoenzymes were separated with agarose gel electrophoresis
using a Corning ACI system. Fluorometric quantification
of isoenzyme bands was made by using a Helena fluoro-vis
scanning densitometer.
Histologic studies were carried out by the method described earlier by Prasad and Gupta (1980). A large piece
of muscle including necrosed, ischemic, and normal tissue,
was dissected from the left ventricle and fixed in Boun's fluid.
The tissue was kept in fixative for 1 week and then washed
with a mixture of 50% alcohol in water. The tissue was then
dehydrated and embedded in paraffin at 57OC. Seven-micron
thick sections were cut and stained with hematoxylin/eosin
for histological studies.
Results
Myocardial Damage and PISA Index
Sulphuric acid (50% diluted) was injected into the left
ventricular myocardium in various volumes (0.01-0.10 ml)
and determination of PISA index and recording of conventional electrocardiogram were simultaneously made at various intervals in eight dogs. The myocardium was removed
at the end of the experiment for histologic and naked eye
examination. Sulfuric acid produced a volume-dependent
damage of the myocardium (Figs. 4 and 5). The extent of
myocardial damage with various volumes of acid injected into
K. Prasad and M. M. Gupta: PISA
183
TIME MSEC
@)
FIG.4 Histological changes in myocardium in (a) dog no. 97 and
(b) dog no. 73. Dog no. 97 received 0.03 ml,and dog no. 73 received
0.10 ml, of sulfuric acid. Note the myocardial damage.
TIME MSEC
(C1
FIG.3 (a) Conventional ECG, (b) ensemble average of wideband
ECG, (c) corresponding PISA signature from a relatively large
myocardial infarct in a dog where there was an obvious change in
the conventional electrocardiogram.
the myocardium is summarized in Fig. 5. The PISA index
was directly related to the extent of myocardial damage. The
results from 3 such dogs are shown in Fig. 6. The PISA index
increased within 10 min and remained elevated to the end of
the experiment.
When two methods, i.e., conventional electrocardiogram
and the PISA technique, were compared for their capability
in detecting cardiac damage, it was observed that in the
presence of definite myocardial damage, even though there
was no observable change in the conventional electrocardiogram, there was a significant increase in the PISA index.
For example, the conventional electrocardiogram of dog #61
(Fig. 7) did not show any observable change although there
was definite myocardial damage (Fig. 5 ) and a marked in-
crease in the PISA index (Fig. 6). Conventional electrocardiogram of dog # 80 shows a marked elevation of ST segment
within 10 min which progressively subsided, and there was
only T-wave inversion a t the end of 90 min (Fig. 8). The
PISA index in this dog increased significantly within 10-1 5
min and remained elevated despite improvement in the
ST-segment changes (Fig. 6). These results indicate that the
PISA method was not only able to detect a small myocardial
infarct but was also able to quantify when the conventional
ECG was unable to do so.
Correlation between PISA Index and Amount of Acid
Injected
The results of the acid-induced changes in the PISA index
are summarized in Fig. 9. The P E A index increased within
10-1 5 min after acid injection and remained elevated to the
end of the experiment. There was a direct relationship between the amount of acid injected and the increase in the
PISA index. It is apparent from the graph that two similar
doses produced similar changes in the PISA index.
Clin. Cardiol. Vol. 4, July/August
184
__
Dog No.
91
Dog No.
97
Dog No.
61
Dog No.
Q Smm
Im
I
-1 0 m m d
1m
€T
-I
Dog No.
80
30mm ___c(
Dog No.
002
20 mm
1M
IM
NIM
IM
+11mm
t-9mm-i
Dog No.73
Dog No.
mm
4
NIM
NIM
NIM
FIG.5 Extent of myocardial damage observed with naked eye in 8 dogs with varying amounts of id injection. The extent of damage
was related to the amount of acid iniected.
Abbreviations: IM, intramural damage; NIM, nonintramural (epicardial or endocardial damage included).
0
10
t
a
MJ. (acid)
60
40
90
OL,
0
10
I
30
1
Time
do
'(min)'
do
FIG.6 Sequential changes in the PISA index in 3 dogs. Also the
relation between PISA index and the extent of myocardial damage
is shown. Note that PISA index increased within 10 min and remained elevated for the duration of the study. Also note that the
increase in the PlSA index is related to the extent of myocardial
damage. Symbols: 0 , dog no. 80; 0, dog no. 91; A,dog no. 61.
4-4-4-
No. 61
FIG. 7 Sequential changes in the conventional electrocardiogram
after 0.01 ml of sulfuric acid injection into the myocardium. 0,
Control ECG; arrow MI (Acid), myocardial damage was produced
by injecting 0.01 ml of diluted sulfuric acid. The numbers above each
tracing show the time in minutes after acid injection. Note that there
is no observable change in the ECG.
K. Prasad and M. M. Gupta: PISA
Acid-induced Myocardial Damage and Serum Enzymes
Blood samples were collected before and a t intervals after
intramyocardial injection of acid for estimation of creatine
phosphokinase (CPK) and lactic dehydrogenase (LDH) and
their isoenzymes. The results are summarized in Table I. It
is apparent that the total CPK increased after acid injection.
The MM-CPK was the largest component of the total CPK.
The MB-CPK was not detectable either before or any time
after acid-induced myocardial damage. Total LDH increased
at 60 and 90 min after acid injection. There was no significant
U
change in the LDHI. These results indicate that although
there was an increase in the total CPK and LDH heart specific MB-CPK did not appear and LDHl did not change after
acid-induced myocardial damage.
Discussion
The purpose of this investigation was to use the PISA
method, and compare it with the conventional ECG and
biochemical markers (MB-CPK and LDHI) in the detection
10
0
L
I85
L
t
M.I. (acid)
60
30
90
No. 80
04
FIG.8 Sequential changes in the conventional ECG after 0.1 ml
of intramyocardial sulfuric acid injection. Arrow indicates where
acid was injected to produce myocardial damage (MI). The other
notations are the same as Fig. 7. Observe the elevation of the ST
segment 10 min after acid injection. Also observe that the ST segment slowly returned to normal level, and at the end of 90 min only
the T wave was inverted.
30
eb (min)
Acid-induced myocardial damage
15
Q
FIG. 9 Effects of intramyocardialinjection of acid on PISA index
in six dogs. Note the dose-dependent changes in the PISA index.
Symbols and corresponding doses of acid (ml): 0 ,dog no. 0061,0.01
ml acid; 0 ,dog no. 0097,0.03 ml of acid; X, dog no. 0002,0.05 ml
of acid; A,dog no. 0080,O.lO ml of acid; A,dog no. 9815,0.08 ml
of acid; 0 ,dog no. 0073,O.lO ml of atid.
TABLEI Serum enzyme level before and at various time intervals ( h i n ) after intramyocardial injection of diluted sulfuric acid in 8
dogs
Serum
enzymes
IU/L
Total CPK
MMCPK%
BBCPK%
MBCPK%
Total LDH
LDH,%
LDH2%
LDH3%
LDH&
LDH&
0
15
30
60
90
218f23
80- 100
trace to 20
285f20
80- 100
trace to 20
319f37
80- 100
trace to 20
442433
80-100
trace to 20
420437
80- 100
trace to 20
235f96
208f61
Not detected
4
148f28
greater
LDH'
than
L%5nd
149f24
1
J
i
134f18
w
Insignificantchange
b
Slight increase
w
The results are expressed as mean fSE. lsoenzymes of CPK (MMCPK, BBCPK, and MBCPK) have been expressed as percent of total
CPK.
Abbreviations: CPK, creatine phosphokinase; LDH, lactic dehydrogenase.
186
Clin. Cardiol. Vol. 4, July/August
and quantification of induced myocardial damage. The
myocardial damage was induced by injecting diluted sulfuric
acid into the free wall of the left ventricle. In previous studies
(Prasad and Gupta, 1980; Prasad et al., 1978b), it was observed that coronary ligation produced an initial increase in
the PISA index followed by a return to the control value. The
return to control value could have been due to the presence
of an extensive collateral circulation in dogs (Cohen et al.,
1974; Schaper, 197 1). It was therefore decided to produce
irreversible and definite myocardial damage by injecting
diluted (1 :1) sulfuric acid into the free wall of the left ventricle. One could argue that sulfuric acid would circulate in
the blood and produce blood pH changes. The acid destroys
the tissue immediately, and hence there is very little chance
that the acid would circulate in the blood. Various amounts
were injected to produce various degrees of myocardial
damage.
The myocardial damage was determined with naked-eye
observation. Thus the estimate of the extent of the myocardial
damage is not truly representative of the myocardial damage.
However, this gives a rough correlation between the amounts
of acid injected and the extent of myocardial damage.
The present investigation shows that P E A index increased
within 10-15 min and remained elevated for the duration of
the experiment. Also the increase in the PISA index was related to the extent of myocardial damage or to the amount
of acid injected. For the reason given above the PISA index
correlated better with the amount of acid injected than with
the extent of myocardial damage.
Significant and definite changes were observed in the
conventional electrocardiogram with large myocardial
damage induced by a large amount of acid. However, no
ECG changes were observed with small myocardial damage
induced by a small amount of acid. Most of the initial changes
in the ECG disappeared 90 min after acid injection.
Total CPK before acid-induced myocardial infarction was
higher than reported normal values in dogs (Carlson et al.,
1978; Shell, et al., 1971). This high level of CPK might be
due to the fact that a blood sample was taken after chest
surgery to expose the heart and after exposure of the femoral
artery and vein for cardiac catheterization. These surgical
procedures did produce tissue injury. It is known that a
marked rise in CPK activity occurs relative to tissue trauma
and postoperative injury (Dixon et al., 197 1). Use of electrocautery also makes CPK levels rise (Mostert, 1970).
Further increase in CPK after acid-induced myocardial infarction might be due to a progressive increase in the noncardiac tissue damage. It has been shown that total CPK rises
progressively and reaches peak at approximately 24-48 h
after operation (Dixon et al., 1971). Although total CPK
increased, the MB-CPK which is a specific indicator of
myocardial infarction did not appear in the blood in any
animal serum sample before acid-induced myocardial
damage. This is to be expected since MB-CPK makes up a
much smaller fraction of total myocardial CPK activity in
dogs than it does in man (Roberts et al., 1975). The MBCPK was not present in a detectable amount after acid-induced myocardial damage. It is possible that the amount of
damage could not release enough MB-CPK to be detected
in the blood. Also it is possible that appearance of MB-CPK
is time-dependent, and 90 min was not enough time for the
release of MB-CPK to be detected in the blood. It has been
reported that the appearance of a detectable amount of
MB-CPK in the blood is too late for early diagnosis of myocardial infarction (Ahumada et al., 1976; Bleifeld et al.,
1976; Sobel and Shell, 1972). The other possibility is that
sulfuric acid destroyed the enzyme, so it could not appear in
the blood. However, the cell surrounding the complete necrotic area must be partially alive to release enzymes.
Total LDH increased but this increase could have been due
to tissue damage (Dixon et al., 1971). There was no significant change in LDHl after acid-induced myocardial
damage.
These studies indicate that the PISA method has the
ability to detect and quantify small as well as large myocardial damages. The conventional electrocardiogram was able
to detect myocardial damage only when it was large. Biochemical markers failed to detect any of the acid-induced
myocardial damage present. Where there was extensive
myocardial damage the configuration of the conventional
electrocardiogram changed with time, and at the end of 90
min the ECG appeared normal except the T wave was inverted. However, the PISA index increased within 10-15 min
and remained elevated for the duration of the experiment.
This was expected because whatever damage is produced will
stay there.
It appears that the PISA technique is superior to biochemical markers and conventional ECG in detecting and
quantifying the myocardial damage. It is envisioned that this
method will be a valuable noninvasive diagnostic tool for the
detection and quantification of incipient as well as advanced
cardiac disorders.
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