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George S. Abela [MEDLINE LOOKUP]Sections |
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| Abstract | TOP |
We evaluated a novel technique of laser-light scattering (LLS) to detect platelet-volume changes continuously, reflecting platelet aggregation in circulating fluid. Carotid arteries from 20 dogs were mounted in a dual perfusion chamber. Balloon angioplasty (BA) was performed and arteries perfused with platelet-rich plasma (PRP). A He-Ne laser beam was passed through cuvettes connected to tubing draining the arteries. From the angle of incidence, the average volume of aggregates was measured by the ratio of scattering light at 1 to 5 degrees' spread on the diode array of a multichannel analyzer. Platelet volume varied linearly with the scattered light ratio at 1 to 5 degrees (y = ¨C24.2 + 27.6 x [y = particle size, µm3; x = scattered light ratio at 1/5 degrees]). For comparison, we used an electronic particle counter (Coulter counter) to measure platelet volume. P-selectin expression was measured to confirm platelet activation. Comparing 10 uninjured and 10 BA-injured arteries, we found that platelet volume as measured with LLS increased from 21.6 ¡À 4.1 to 52.1 ¡À 12.5 µm3 (P < .003); as measured with the Coulter counter, it increased from 29.9 ¡À 2.4 to 62.3 ¡À 7.0 µm3 (P < .005). Six BA-injured arteries perfused with PRP and aspirin (0.2 mg/mL) were compared with six arteries treated with BA alone. The aspirin decreased platelet volume as measured with LLS from 56.2 ¡À 11.8 to 40.2 ¡À 12.7 µm3 (P < .01); the Coulter counter revealed a decrease from 51.9 ¡À 18.5 to 38.8 ¡À 14.2 µm3 (P < .001). Coulter counter and LLS results were correlated: r = 0.74, P < .05. The peak of P-selectin expression coincided with peak platelet volume. These data demonstrate that increases in circulating-platelet size stimulated by endovascular injury can be reliably and continuously monitored with the use of LLS. (J Lab Clin Med 2003;141:50-7)
| Abbreviations | ||||||||||||||
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| Methods | TOP |
Dogs
In this study, 20 male dogs
(8 mongrels and 12 beagles, 18-20 pounds) were used. While each dog was under
general anesthesia (sodium pentobarbital 65 mg/mL, 0.5 mL/kg IV), whole blood
(450 mL) was collected in 50 mL sodium citrate (150 mmol/L). We obtained PRP by
centrifuging whole blood at 150g in a swing-bucket rotor for 20 minutes
at 22¡ãC. The dog was then killed with a lethal-dose euthanasia solution, given
intravenously. Dogs were housed in accordance with National Institutes of Health
guidelines, and the study was conducted in accordance with the protocol approved
by the Michigan State University Animal Care and Use Committee.
Preparation of the carotid artery and
BA
Both carotid arteries from each dog were carefully
isolated, and all arterial branches were ligated with 4.0 silk suture. Two 5-cm
carotid-artery segments from each dog were mounted side by side in a dual
perfusion chamber immersed in oxygenated PBSat 37¡ãC (Fig 1).
| Fig. 1. Diagram of the
experimental setup, demonstrating the flow of PRP in the arteries of the
dual organ chamber. The He-Ne laser beam was split by a beam chopper to
enter the cuvettes and displayed the LLS on the diode array at 1 and 5
degrees. |
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The circulatory setup consisted of silicon tubing (3-mm internal diameter)
connected to and emerging from a plastic box with two parallel chambers
separated by a plastic divider. A roller pump was used to propel the fluid
through the closed circuit at a rate of 70 mL/min. We injured the intimal
surface of the carotid-artery segments with the use of a balloon catheter (3 ¡Á
30 mm) inflated to 10 atm at three different sites each for 1 minute, starting
most distally and moving proximally to avoid overlapping injury. During balloon
inflation, we wrapped surgical umbilical tape around the artery at the center of
the balloon to create a resistance between the balloon and the arterial wall to
ensure vascular injury. The total volume of PRP circulated in each chamber was
60 mL. Platelet concentration in the PRP averaged 5 ¡Á 107/mL. The
exterior of the arteries was bathed in oxygenated PBS. Both the bathing solution
and the circuit were kept at 37¡ãC.
Three groups were evaluated. Group 1
comprised 20 arteries: 10 uninjured arteries and 10 BA-injured arteries perfused
with PRP. Group 2 included 12 arteries: 6 BA-injured arteries perfused with PRP
but not treated with aspirin and 6 BA-injured arteries perfused with PRP and
treated with aspirin (0.2 mg/mL). Group 3 comprised 8 arteries: 4 BA-injured and
4 uninjured, perfused with PRP in an attempt to evaluate P-selectin
expression. Total perfusion time for each artery was 60 minutes.
Measurement of platelet volume with the LLS and
Coulter-counter methods
A He-Ne laser beam (633 nm) was split
and passed through two cuvettes interposed in the tubes draining the two
carotid-artery segments. The scattering light from particles in the cuvettes was
spread on the diode array of a multichannel analyzer (model ST-120; Princeton
Instruments Inc, Princeton, NJ). From the angle of incidence, the ratio of
scattering light at 1 to 5 degrees represented particle-size distribution. Small
particles have a higher distribution of scattered laser light at the wider angle
(5 degrees), whereas large particles have a greater distribution of scattered
light at the narrower angle (1 degree).1
We digitized the electronic signals from the multichannel analyzer using a
custom software package, after which we displayed the data graphically on a
computer screen and recorded them on a HP Color Pro printer (HP Corp, Palo Alto,
Calif) (Fig 1
). We carried out measurement of platelet-particle
volume as an index of aggregation with the Coulter counter (models Zf and MHR;
Coulter Electronics, Inc, Hialeah, Fla) by obtaining 50-µL samples of PRP
effluent at 5- to 10-minute intervals during PRP perfusion. We calibrated the
counter with particle-size latex beads (2, 3, and 5 µm in diameter; Coulter
Electronics). Calibration for the LLS method was carried out with the same size
latex beads, as well as canine RBCs (4.4 µm in diameter as determined with the
Coulter counter). LLS data were converted from the reflected light angle ratio
measurement at 1 and 5 degrees to a volumetric scale involving a standard curve.
This curve was generated from LLS of both the latex-particle standards and the
dog RBCs of known size (Fig 2).
Fig. 2. Graph of
callibration for LLS using standard particle sizes. The relationship was a
linear function represented by y = ¨C24.2 + 27.6x, where y is
particle size (cubic millimeters) and x is the scattered-light
ratio at 1 and 5 degrees. The third point on the graph ( ) represents the size of a dog RBC. |
) |
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To evaluate the effect of RBCs on the scattered light signal of the
PRP, we conducted optical measurements on PRP alone and with various numbers of
RBCs before and after adding ADP to aggregate the platelets. This allowed us to
determine what concentration of RBCs in the fluid would mask the ability of the
system to detect platelet aggregates. The concentrations of RBCs added ranged
from 1 ¡Á 105 to 108/mL; the PRP concentration was 1 ¡Á
108/mL platelets.
Flow cytometric analysis of P-selectin
expression
We confirmed platelet activation after vascular
injury by measuring the expression of canine platelet P-selectin. Because
P-selectin (CD62) is expressed on the surface of activated platelets
only, we assessed P-selectin expression on platelets used to perfuse
BA-injured and uninjured arteries. Detection of canine P-selectin was
performed with a fluorescein-labeled mouse monoclonal antibody, MDP-1, provided
by Dr. Samuel Burstein (University of Oklahoma, Oklahoma City, Okla). We labeled
the antibody with fluorescein using NHSF (Pierce Chemical Co, Rockville, Ill).
In brief, NHSF was dissolved in dimethylsulfoxide (1 mg/mL) and added in 20-fold
molar excess to 1 mg of MDP-1 and allowed to react on ice for 2 hours. We
removed unreacted NHSF by diluting the reaction solution with PBS and conducting
ultrafiltration through a Centricon-10 ultrafiltration device (Amicon Inc,
Beverly, Mass). Test platelets (100 µl) were labeled with 10 µg of
fluorescein-conjugated MDP-1 for 20 minutes at room temperature. The labeled
platelets were then diluted with PBS containing 0.025% NaN3 and
incubated at room temperature until they could be analyzed. Flow-cytometric
study was performed with a Vantage flow cytometer (Becton-Dickinson Co, San
Jose, Calif). Excitation was carried out at 488 nm, detection at 530 nm. We
analyzed data with Cell Quest software (Becton-Dickinson). All samples were
analyzed 2 to 3 hours after collection.
Histologic examination
We
evaluated thrombogenicity of treated sites by examining perfusion-fixed arterial
segments under light microscopy and SEM. Samples for SEM were taken from each of
the arterial segments after 1 hour's perfusion, then subjected to critical-point
drying in liquid carbon dioxide, mounted on stubs, and gold-coated in a sputter
coater. The SEM of the intimal surface was examined with a JEOL scanning
electron microscope (model JSM-6400V; JEOL Ltd, Tokyo, Japan), and
representative sections were photographed. The other arterial homografts were
embedded in paraffin and mounted on glass slides. Fixed, paraffin-embedded
tissues were cut into 7-µm-thick sections and stained with hematoxylin and eosin
and Masson's trichrome, after which they were examined with a light microscope.
Statistical analysis
Data are
reported as mean ¡À SD and were compared with the use of paired Student t
tests. We considered P values of less than .05 statistically significant.
SigmaStat statistical software was used to calculate statistical comparison
(Sigma, St. Louis, Mo). We conducted a correlation analysis of the
platelet-volume measurements obtained with the use of LLS and with the Coulter
counter.
| Results | TOP |
). Platelet volume was calculated from the
scattered-light ratio of 1 to 5 degrees as determined with the following
equation: y = ¨C24.2 + 27.6 x, where y is particle size as an indirect
measurement of platelet volume (cubic micrometer) and x is the
scattered-light ratio at 1 and 5 degrees. Non-activated platelets are discoid
and measure approximately 2 µm in diameter, with a volume of about 5
µm3. Because our measurement of platelet volume was 20 to 60
µm3, these data reflect measurements of platelet aggregates.
| Fig. 3. Graph of three
light-scattering patterns: (1) PRP at 1.2 ¡Á 108;
(2) PRP with RBCs at 2 ¡Á 106; (3) PRP and RBCs
after the addition of ADP to induce platelet aggregation. Graph 2 does not
mask graph 3, and the platelet-aggregate scattering pattern is not
blocked. |
) |
|
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The pattern was partially masked at 1 ¡Á 107/mL and obliterated by
1 ¡Á 108/mL.
Platelet-volume measurements obtained with the
use of LLS were compared with those obtained with the Coulter counter in groups
1 and 2. In group 1, platelets exposed to BA-injured arteries demonstrated a
significant increase in volume, which we considered to represent platelet
activation and formation of platelet aggregates. In group 2, inhibition of
platelet activation with aspirin significantly reduced the increase in platelet
volume (Fig 4).
| Fig. 4. A, Laser-light
scattering distribution at 1 and 5 degrees, emitted from the circulating
PRP effluent in a BA-injured artery. B, LLS distribution at 1 and 5
degrees, emitted from the circulating PRP effluent in a BA-injured artery
exposed to aspirin. C, Cumulative platelet volume over 60 minutes
from LLS data collected in A and B. |
) |
|
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These measurements were reported at the peak change in platelet volume that occurred 15 minutes after the initiation of PRP circulation. Also, the comparisons of LLS and Coulter-counter measurements for groups 1 and 2 reported in Figs 5 and 6 were made at the same peak effect.
| Fig. 5. Bar graphs of
platelet-volume measurements comparing the LLS and Coulter-counter methods
in normal and BA-injured arteries. BA injury significantly increased
platelet-aggregate volume compared with that in control uninjured arteries
with both methods. These measurements were obtained at the peak effects,
seen at 15 minutes. |
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| Fig. 6. Bar graphs of
platelet-volume measurements taken with the LLS and Coulter-counter
methods in BA-injured arteries with and without aspirin. Aspirin
significantly decreased platelet-aggregate volume compared with that seen
in the control injured arteries. These measurements were obtained at the
peak effects, seen at 15 minutes. |
) |
|
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In group 1, 20 arteries were studied: 10 uninjured controls and 10
with BA-induced injury. The platelet volume measured with LLS increased from a
control of 21.6 ¡À 4.1 to 52.1 ¡À 12.5µm3 after injury (P <
.005). Platelet volume as measured with the Coulter counter increased from a
control value of 29.9 ¡À 2.4 to 62.3 ¡À 7.0µm3 after injury (P
< .003; Fig 5).
In group 2, 12 BA-injured arteries were
studied: 6 perfused with PRP but not treated with aspirin and 6 treated with
aspirin. Aspirin treatment decreased platelet volume, a finding detected both
with LLS and with the Coulter counter. Platelet volume as measured with LLS
decreased from a control of 56.2 ¡À 11.8 to 40.2 ¡À 12.7 µm3 with
aspirin (P < .01). Platelet volume as measured with the Coulter
counter decreased from a control of 51.9 ¡À 18.5 to 38.8 ¡À 4.2 µm3
with aspirin (P < .01; Fig 6).
Comparison of findings obtained with
the LLS and Coulter counter methods showed a high correlation (r = .74,
P < .05). These data suggest that the LLS method can be used to
provide real-time continuous measurement of platelet-aggregate size in the
circulation after vascular injury.
To determine whether platelet volume
was reflective of platelet activation, we sampled platelets after circulation in
8 arteries (group 3; 4 injured and 4 uninjured) and measured platelet volume
with the Coulter counter and LLS. Using the monoclonal antibody MDP-1, we found
that the percent expression of P-selectin in the PRP used to perfuse the
arteries was consistently higher at 15 minutes than at the immediate baseline
(Fig 7).
| Fig. 7. Percent
P-selectin expression and perfusion time. This is an example of one
study demonstrating peak P-selectin expression at 15 minutes for
both small and large particles. This is coincident with the time for
aggregate peak detected both with LLS and the Coulter counter in Figs 3 through 6. |
) |
|
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These data demonstrate simultaneous platelet activation with increased LLS,
suggesting that platelets were activated as they traversed the circuit.
Light microscopy of BA-treated arterial segments showed medial
dissection and stretching of the arterial wall. These injuries were prominent at
sites where umbilical tape was used to create resistance to balloon inflation.
Also, platelet aggregates were seen attached to sites of disrupted endothelium
(illustration not shown).
SEM examination of the BA-treated arterial
segments showed wedge-shaped intimal-surface tears. Extensive platelet adhesion
was noted to cover the arterial surface at sites of intimal injury. Arteries
perfused with PRP and treated with aspirin demonstrated less platelet adhesion
compared with BA-injured controls (Fig 8).
| Fig. 8. SEM of canine
carotid arteries collected after 60 minutes' perfusion with PRP. A,
SEM of BA-injury site, demonstrating damage to the endothelial lining with
some platelet aggregates and scattered fibrin deposition. B,
Micrograph of BA-injury site of an artery perfused with PRP and aspirin.
The endothelial lining was disrupted, and only an occasional platelet was
noted. |
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Normal control arterial segments showed an intact endothelial surface
demonstrating a typical cobblestone appearance without platelets (illustration
not shown).
| Discussion | TOP |
. The measurements obtained with the
Coulter-counter technique, on the other hand, were performed after the
collection of samples at specific intervals; this may lead to greater
variability as a result of sample collection and handling. At the time of this
writing, the LLS technique is the only developed method of continuously
measuring platelet volume in circulating fluid in an in vitro setup.
We thank Dr Joel Eisenberg for reviewing the manuscript. Flow cytometry was carried out at the Michigan State University Flow Cytometry Facility. We also thank Ewa Danielewicz for technical support in SEM at the Center for Advanced Microscopy of Michigan State University.
| References | TOP |
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| Appendix | TOP |
The physical and chemical properties of a microscale aggregate such as blood platelets can be experimentally studied with the use of Mie scattering and Raman spectroscopy. Mie scattering can determine the average radius when the refractive index of the aggregate is known.
The typical Mie-scattering measurement on liquid suspensions employs a laser beam as the light source. This laser beam is scattered in all the directions at the suspension liquid. The measurement of the relative intensity at various scattering angles reveals the average radius and the relative refractive index of the microscale particles in suspension. Mie theorem describes the light scattering from homogeneous spheres. The authors argue that the Mie theorem is adequate for our work of measuring the degree of blood-platelet aggregation because the blood platelet aggregates roughly into spherical shapes.
A laser beam is a high-intensity coherent varying electromagnetic wave. When this electromagnetic wave is applied to a material, it interacts with the nuclei and electrons strongly, stimulating oscillations of nuclei and electrons in the material. The oscillating electrons and nuclei reradiate electromagnetic waves in all directions, resulting in scattered light. The theory on the scattering of electromagnetic wave by spherical objects was first developed by Mie in the early 1900s and is called Mie's scattering.21
A static electromagnetic wave has the form:
where 
is the refractive index of the material, H is the magnetic
field, E is the electric field, and 
is the frequency of the electromagnetic wave.
The incident electromagnetic field of the laser is taken as
a plane wave. When this electromagnetic wave is applied to a spherical
homogenous material, it is both reflected and refracted. The electromagnetic
field everywhere is the summation of the incident, reflected, and refracted
fields. The electromagnetic field should satisfy the above wave equation
everywhere. In the infinity limit, the total electromagnetic fields should be a
plane wave. The electromagnetic field should satisfy the boundary condition at
the surface of the sphere. From the above considerations, we can solve the
scattered electromagnetic wave equation as:
with
where
the relative refractive index is the ratio of refractive index of the sphere
over that of the solvent:
The intensity of the scattered light, which is the Pointing
vector is defined as:
Both the electric and magnetic fields are polynomial functions
of the scattering angle 
. The intensity also depends on the relative refractive index and the
radius of the sphere. The graph demonstrates the intensity as a function of
scattering angle with a fixed relative refractive index of 1.05 at various
sphere radii (Fig 9).22,23
Fig. 9. LLS for spherical
particles with refractive index of 1.05 and diameters ranging from 1 to
7.5 µm. These curves were derived by way of the Mie theory with MIEV
software.22,23
The curves demonstrate that smaller particles have a greater effect on
scattering light at wider angles.
)
| Publishing and Reprint Information | TOP |
Copyright © 2003 by Mosby, Inc. All rights reserved.