SHAPE Society

1. PresentationPresentation
Number:Number: 1054-191054-19Progress With the Calibration of a 3Fr Near Infrared Spectroscopy FiberProgress With the Calibration of a 3Fr Near Infrared Spectroscopy Fiber
Optic Catheter for Monitoring the pH of Atherosclerotic Plaque:Optic Catheter for Monitoring the pH of Atherosclerotic Plaque:
Introducing a Novel Approach for Detection of Active Vulnerable PlaqueIntroducing a Novel Approach for Detection of Active Vulnerable Plaque
Tania Khan, M.E.†
, Babs R. Soller, PhD†
; Peter Melling, PhD‡
; Mohammad Madjid, M.D.*; Ward Casscells, M.D.*;
Morteza Naghavi, M.D.*
†
Department of Surgery, University of Massachusetts Medical School, Worcester MA 01655 ; ‡
Remspec, Inc. Sturbridge MA; *Center for Vulnerable Plaque
Research, University of Texas-Houston, and Texas Heart Institute, Houston TX 77030
BACKGROUN
D
AAtherosclerotic plaques vary by
activityactivity and vulnerable plaques
are more active than others. Our
approach is based on identifying
the different metabolic activities
(e.g., tissue pH, temperature,
metabolite concentrations) of
atherosclerotic lesions using near
infrared spectroscopy. Previously
we reported pH heterogeneity in
human and rabbit atherosclerotic
lesions. Preliminary calibration
models were derived using a 3Fr
optical catheter prototype for
plaque tissue pH, using near-
infrared (NIR) spectroscopic
techniques in the 400-1100 nm
region. The tissue lactate
concentration, a by-product of
anaerobic glycolysis, is possibly
another useful vulnerability
parameter. Tissue lactate is
detectable in atherosclerotic
lesions through various
destructive methods. In addition,
the lactate molecule has known
absorbance bands in the near-
infrared at ~2250 and 2295 nm,
with a shoulder region at
approximately 2030 nm, which
may facilitate the calibration of
the NIR optical catheter.
In this study, we
propose a non-
destructive method
of assessing lactate
concentration in the
active, living
plaque, using a
near-infrared
spectroscopy based
optical catheter.
In addition,
macrophages
sustain activity
through anaerobic
metabolism and
lactate production.
The tissue lactate
concentration of
atherosclerotic
lesions may be an
additional indicator
orphology vs. Activity Imaging
Inactive and
non-inflamed
plaque
ctive and
inflamed
plaque
Appear Similar in
OCT MRI
w/o CM
Morphology
Show Different
Activity
Thermography, Spectroscopy,
immunoscintigraphy, MRI with
targeted contrast media…
TEXAS HEART INSTITUTE
CONCL
USIONS
RES
ULT
S
Figure 4: Changes in parameters over
~3.5 hrs of a representative plaque in
the plaque viability study. (m = media
values)
30
32
34
36
38
40
10:00:00 11:00:00 12:00:00 13:00:00 14:00:00
Temperature
7.00
7.10
7.20
7.30
7.40
7.50pH
temp pH
PlaqueViabilityStudy
0
10
20
30
40
50
60
10:00:00 11:00:00 12:00:00 13:00:00 14:00:00
PCO2
7.00
7.10
7.20
7.30
7.40
7.50
pH
PCO2 mPCO2 pH mpH
sensorin
0
100
200
300
400
500
10:00:00 11:00:00 12:00:00 13:00:00 14:00:00
Time
PO2
7.00
7.10
7.20
7.30
7.40
7.50
pH
PO2 mPO2 pH mpH
Figure 5: Box-whisker plots for
change in pH per hour and change in
temperature per hour (respectively)
for the control and test plaques in the
viability study. Rate of change in
controls are significantly different
than the test plaques.
Figure 6: Seventeen raw spectra
from 5 plaques are shown. The
best calibration results used
2030 –2330 nm data. This
wavelength region corresponds
to the theoretical lactate
absorbance spectrum.
Lactate Feasibility Study
HYPOTHESES
The excised human carotid
plaques can be maintained at
near in-vivo, physiologic
conditions using tissue culture
techniques for extended periods
during the measurements.
Using the same optical catheter
as for NIR pH determination,
NIR-measured lactate
concentration is feasible in
living, heterogeneous carotid
plaques.
MET
HOD
SPlaque Viability
Study
• Minimum
Eagle’s Med-
ium (MEM),
pH 7.4, 5.6
mM glucose,
26.2 mM
NaHCO3, with
non-essential
amino acids
was used
(Invitrogen).
• Media
equilibrated
with 75% O2 /
5% CO2 gas
mixture prior
to tissue
addition.
• Seven human
carotid plaques
were collected
and placed
immediately in
37°C media
enclosed in a
humidified
incubator at
37°C. Figure
1.
• Two plaques
that were not
placed in the
liquid media,
only in the
humidified air
of the
incubator,
served as
controls.
• Measurements
were taken
with a multi-
parameter,
intra-arterial
sensor placed
in the tissue
(Diametrics,
MN). Figure 2.
• Changes in
tissue pH,
temperature,
PO2 and PCO2
over time were
analyzed.
Lactate
Feasibility
Study
• Five additional
human carotid
plaques were
collected in a
similar manner.
3-4 areas of
each plaque
were randomly
chosen for total
of 17 points.
• Reflectance
spectra (1100 –
2500 nm) of
each area were
taken using
Nicolet FTIR
670
spectrometer
with a fiber
optic probe.
(Remspec,
MA) Figure 3.
• Tissue biopsies
of the same
area were taken
using a 4-mm
punch biopsy
and
immediately
frozen in liquid
nitrogen.
• Tissue lactate
(LA) was
assayed using
micro-
enzymatic
methods
(Sigma).
Values are
reported as
micromole LA
per gram wet
tissue.
• Matching
spectra and
tissue lactate
values modeled
by multivariate
calibration
techniques. R2
and root mean
squared
deviation
(RMSD) used
to assess model
accuracy.
Figure 2:
Plaque in media
with multi-
parameter
sensor in place.
The media is
oxygenated by
bubbling gas.
sens
or
Figure 1:
Experimental
setup for both
studies. Incubator
maintained at
37°C.
Figure 3: Fiber
optic probe used
for lactate
feasibility study.
The oxygenated
media environment
allowed the plaques
to remain in near in-
vivo status for the
duration of the data
collection.
The near infrared
(NIR) measurement
of tissue lactate in
living carotid
plaques is feasible
and well correlated
with the standard
destructive
measurement of
tissue lactate by
micro-enzymatic
assay.
This experimental
setup may allow us,
in the future, to
directly translate
calibration
equations generated
in-vitro, to in-vivo
validation studies.
Figure 7: Correlation between the
actual tissue lactate values and
the NIR determined values is
shown. The R2
of the
determination was 0.83. The
RMSD or estimated accuracy of
the model was 1.4 micromole
LA /gram tissue.
-2.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
-2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0
ActualLactateConcentration
(microle/gramtissue)
NIR-LactateConcentration(micromole/gramtissue)

2. BACKGROUND
AAtherosclerotic plaques vary by activityactivity and
vulnerable plaques are more active than others.
Our approach is based on identifying the
different metabolic activities (e.g., tissue pH,
temperature, metabolite concentrations) of
atherosclerotic lesions using near infrared
spectroscopy. Previously we reported pH
heterogeneity in human and rabbit
atherosclerotic lesions. Preliminary calibration
models were derived using a 3Fr optical
catheter prototype for plaque tissue pH, using
near-infrared (NIR) spectroscopic techniques
in the 400-1100 nm region. The tissue lactate
concentration, a by-product of anaerobic
glycolysis, is possibly another useful
vulnerability parameter. Tissue lactate is
detectable in atherosclerotic lesions through
various destructive methods. In addition, the
lactate molecule has known absorbance bands
in the near-infrared at ~2250 and 2295 nm,
with a shoulder region at approximately 2030
nm, which may facilitate the calibration of the
NIR optical catheter.

3. Morphology vs. Activity Imaging
Inactive and
non-inflamed
plaque
Active and
inflamed
plaque
Appear Similar in
IVUS OCT MRI
w/o CM
Morphology
Show Different
Activity
Thermography, Spectroscopy,
immunoscintigraphy, MRI with
targeted contrast media…

4. •In this study, we propose a non-
destructive method of assessing
lactate concentration in the active,
living plaque, using a near-infrared
spectroscopy based optical
catheter.
•Macrophages sustain activity
through anaerobic metabolism and
lactate production. The tissue
lactate concentration of
atherosclerotic lesions may be an
additional indicator of plaque
vulnerability.
GOALS

5. •The excised human carotid
plaques can be maintained at
near in-vivo, physiologic
conditions using tissue culture
techniques for extended periods
during the measurements.
•Using the same optical catheter
as for NIR pH determination,
NIR-measured lactate
concentration is feasible in
living, heterogeneous carotid
plaques.
HYPOTHESES

6. Plaque Viability Study
•Minimum Eagle’s Medium (MEM), pH 7.4,
5.6 mM glucose, 26.2 mM NaHCO3, with
non-essential amino acids was used
(Invitrogen).
•Media equilibrated with 75% O2 / 5% CO2
gas mixture prior to tissue addition.
•Seven human carotid plaques were collected
and placed immediately in 37°C media
enclosed in a humidified incubator at 37°C.
Figure 1.
•Two plaques that were not placed in the
liquid media, only in the humidified air of the
incubator, served as controls.
Figure 1: Experimental setup for both studies. Incubator maintained
at 37°C.
METHODS

7. • Measurements were taken with a multi-
parameter, intra-arterial sensor placed in
the tissue (Diametrics, MN). Figure 2.
• Changes in tissue pH, temperature, PO2
and PCO2 over time were analyzed.
sensor
Figure 2: Plaque in media with multi-parameter sensor in
place. The media is oxygenated by bubbling gas.
Plaque Viability Study (Con’t)

8. Lactate Measurement Study
•Five additional human carotid plaques were
collected in a similar manner. 3-4 areas of
each plaque were randomly chosen for total
of 17 points.
•Reflectance spectra (1100 – 2500 nm) of
each area were taken using Nicolet FTIR 670
spectrometer with a fiber optic probe.
(Remspec, MA) Figure 3.
Figure 3: Fiber optic probe used for lactate feasibility study.

9. • Tissue biopsies of the same area were
taken using a 4-mm punch biopsy and
immediately frozen in liquid nitrogen.
• Tissue lactate (LA) was assayed using
micro-enzymatic methods (Sigma).
Values are reported as micromole LA per
gram wet tissue.
• Matching spectra and tissue lactate values
modeled by multivariate calibration
techniques. R2 and root mean squared
deviation (RMSD) used to assess model
accuracy.
Lactate Measurement Study (Con’t)

10. Figure 4: Changes in parameters over ~3.5 hrs of
a representative plaque in the plaque viability
study. (m = media values)
30
32
34
36
38
40
10:00:00 11:00:00 12:00:00 13:00:00 14:00:00
Temperature
7.00
7.10
7.20
7.30
7.40
7.50
pH
temp pH
Plaque Viability Study
0
10
20
30
40
50
60
10:00:00 11:00:00 12:00:00 13:00:00 14:00:00
PCO2
7.00
7.10
7.20
7.30
7.40
7.50
pH
PCO2 m PCO2 pH m pH
sensor in
0
100
200
300
400
500
10:00:00 11:00:00 12:00:00 13:00:00 14:00:00
Time
PO2
7.00
7.10
7.20
7.30
7.40
7.50
pH
PO2 mPO2 pH m pH
RESULTS

11. Figure 5: Box-whisker plots for change in pH per hour and
change in temperature per hour (respectively) for the control
and test plaques in the viability study. Rate of change in
controls are significantly different from the test plaques.

12. Figure 6: Seventeen raw spectra from 5 plaques are shown. The best
calibration results used 2030 –2330 nm data. This wavelength region
corresponds to the theoretical lactate absorbance spectrum.
Lactate Measurement Study
-2.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
-2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0
Actual Lactate Concentration
(microle/gram tissue)
NIR-LactateConcentration
(micromole/gramtissue)
Figure 7: Correlation between the actual tissue lactate values and the
NIR determined values is shown. The R2
of the determination was
0.83. The RMSD or estimated accuracy of the model was 1.4
micromole LA /gram tissue.

13. The oxygenated media environment allowed
the plaques to remain in near in-vivo status for
the duration of the data collection.
The near infrared (NIR) measurement of
tissue lactate in living carotid plaques is
feasible and well correlated with the standard
destructive measurement of tissue lactate by
micro-enzymatic assay.
This experimental setup may allow us, in the
future, to directly translate calibration
equations generated in-vitro, to in-vivo
validation studies.
CONCLUSIONS