U.S. patent application number 11/975895 was filed with the patent office on 2009-05-21 for apparatus and method to measure platelet contractility.
Invention is credited to Jennifer Orje, Enrique Saldivar.
Application Number | 20090130744 11/975895 |
Document ID | / |
Family ID | 32713805 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090130744 |
Kind Code |
A1 |
Saldivar; Enrique ; et
al. |
May 21, 2009 |
Apparatus and method to measure platelet contractility
Abstract
An apparatus and method for measuring blood platelet
contractility, hereinafter called a "retractometer" is disclosed.
Also disclosed is a system apparatus and method for automatically
measuring platelet contractility in a plurality of samples, having
an array of retractometer units and an electronic solenoid valve
controller to fully automate screening in clinical and research
situations and save costs in labor.
Inventors: |
Saldivar; Enrique; (Santee,
CA) ; Orje; Jennifer; (San Diego, CA) |
Correspondence
Address: |
FUESS & DAVIDENAS
6 ANTHONY LN
MABLEVALE
AR
72213
US
|
Family ID: |
32713805 |
Appl. No.: |
11/975895 |
Filed: |
October 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10632532 |
Aug 1, 2003 |
7335335 |
|
|
11975895 |
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Current U.S.
Class: |
435/286.5 |
Current CPC
Class: |
B33Y 80/00 20141201;
G01N 33/4905 20130101; G01N 33/00 20130101 |
Class at
Publication: |
435/286.5 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. A system apparatus for automatically measuring platelet
contractility in a plurality of samples, comprising: a pump;
mechanically connected to a pump motor; electronically connected to
a microprocessor having a plurality of pins a first pin to turn the
pump motor on a second pin to move fluid in the pump in one
direction a third pin to move fluid in the pump in an opposite
direction and at least one of the remainder of the plurality of
pins to activate each of an array of solenoid valves; a voltage
divider used to establish the position of the fluid in the pump; a
first fluid conduit; connecting the pump to a a hydraulic system
comprising a first manifold; connecting the pump to each of a
plurality of retractometers each activated by one of the solenoid
valves; a second fluid conduit manifold; connecting the
retractometers to a a pressure transducer; an analog to digital
(A/D) converter connected electronically to the transducer, the
pump motor and the microprocessor; and a computer wherein, a
readout position voltage from the voltage divider is entered
through the (A/D) converter to the microprocessor, which determines
the direction of flow in the pump and activates the pump fluid
pressure within the system, which pressure is then measured by the
pressure transducer connected electronically to the A/D converter
and a target pressure is registered in the microprocessor memory
and subsequently recorded and displayed by the computer.
11. The apparatus according to claim 10, wherein the pump moves
fluid with a sliding piston.
12. The apparatus according to claim 11, wherein the pump is a
syringe.
13. The apparatus according to claim 10, wherein the array of
solenoid valves comprises eight valves, each valve activating one
of eight retractometers.
14. The apparatus according to claim 10, further comprising a first
protection valve located at the entrance to the pressure transducer
to prevent damage to the system and a second protection valve to
control access to a fluid reservoir.
15. The apparatus according to claim 10, wherein the output voltage
of the pressure transducer is entered into the A/D converter and
subsequently to the microprocessor.
16. The apparatus according to claim 10, wherein subroutines are
burnt into the microprocessor.
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 121 of application Ser. No. 10/632,532, filed Aug. 1,
2003, which is incorporated by reference herein. This work was
supported by National Institute of Health Grant No.
R29-HL57430-01.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally concerns blood clotting
components and mechanisms.
[0004] The present invention particularly concerns new devices and
methods for studying clotting mechanisms and factors. More
specifically, an apparatus and method for measuring and monitoring
health and activity of platelets and other clotting factors are
described. Most specifically, a clot retractometer and its method
of use are provided to measure clot contractility forces as a means
to provide a single point "funnel detection" procedure useful in
aiding physiological and clinical research and patient diagnosis
and monitoring of many diseases, as well as screening
populations.
[0005] 2. Description of Related Art
INTRODUCTION
[0006] Everyone has seen a clot form as a result of injury to
tissue, such as, for example, a scrape, puncture or a bleeding
nose. However, the formation of a clot is a complex, cascading
process that is still not completely elucidated, either
physiologically or clinically. The clotting phenomenon, or lack
thereof, is manifest in numerous clinical conditions, and is
relevant to their prognosis.
[0007] In response to soft tissue injury, the haemostatic mechanism
is activated to stop bleeding and restore vascular integrity. Blood
protein and cellular interactions lead to the formation of a
platelet plug and ultimately generation of clot comprising
platelets and protein fibers. These reactions have to occur rapidly
because the amount of blood lost is dependent on the time required
to arrest the bleeding. Although rapid stoppage of blood loss is
critical in some cases, inappropriate induction of clotting can
have devastating effects such as decreased blood flow to the organs
and resultant ischemic damage, such as heart attacks and stroke if
the clot is not solubilised. To counterbalance these potentially
severe consequences the haemostatic system is uses certain clotting
inhibitors, and clot-dissolving enzymes.
[0008] Following vascular damage, the exposure of flowing platelets
to the subendothelial lining allows the establishment of adhesive
interactions with the immobilized surfaces. Platelets then become
activated due to contact with thrombogenic substrates and
stimulation by locally released or generated agonists. Subsequent
platelet deposition relies on the binding of plasma-soluble
adhesive molecules, and on the externalization of adhesive
molecules from the platelets' granular reservoirs, this process
conditions the newly recruited monolayer of platelets to become the
reactive surface for continuing platelet accrual.
[0009] Immediately after platelet arrest, the clotting process
begins by the participation of platelet released substances and
fluid phase coagulation factors. Initiation of the coagulation
cascade results in the conversion of prothrombin to thrombin (a
serine protease). Thrombin cleaves two pairs of peptides
(fibrinopeptides A and B) from the aminoterminal ends of the
A.alpha. and B.beta. chains of the fibrinogen molecule. Cleavage of
fibrinopeptide-A is sufficient to initiate clot assembly (4). The
monomer units formed initiate a self-assembly process of forming
protofibrils. Weak lateral interactions between protofibers
increase as the protofibers lengthen, resulting in their alignment
and coalescence, to ultimately yield fibers. This process leads to
the formation of a network composed of fibrin polymers and spaces
filled with fluid. Once the fibrin network is formed, the platelets
begin to contract, resulting in a pull on the strands of the fibrin
network. Platelet contraction requires active restructuring of the
platelet cytoskeleton.
[0010] Dynamic rearrangements in the cytoskeleton are crucial
during platelet activation in both, initial platelet adhesion to
surfaces (see FIG. 1) and platelet to platelet cohesion. Actin
polymerization in non-stimulated platelets is limited by
monomer-sequestering proteins such as thymosin .beta.4, profilin
and barbed end-capping proteins such as gelsolin (5-7). Under these
conditions, around 2,000 actin filaments are distributed in the
cytoskeleton and in the membrane skeleton right under the inner
surface of the plasma membrane (8). After the stimulation by strong
agonists, there is a rapid increase in actin polymerization, with
reorganization of the two actin networks, resulting in a change of
shape with formation of filopodia and lamellipodia at the cell
periphery. This is followed by redistribution of actin and other
cytoskeletal and signaling proteins form the membrane skeleton to
the cytoskeleton (9; 10). Platelet spreading is associated with the
appearance of actin stress fibers and focal-adhesion-like
structures that contain clusters of integrins and vinculin (11).
Small GTPases of the Rho family--such as cdc42Hs, Rac, and
Rho--have been implicated in the formation of filopodia,
lamellipodia and focal adhesion plaques in many cell types (12),
and the same may occur in platelets.
[0011] Under normal conditions, the coagulation system remains in a
fine balance. Pathologic alterations of the system may induce a
risk of hemorrhage or increase the potential for thrombosis. An
example of the former would be the bleeding disorder of hemophilia,
which results from a low activity of Factor VIII, a blood clotting
protein. An example of the latter would be recurrent venous
thrombosis in individuals who have decreased levels of the
coagulation inhibitor antithrombin III. Patients with decreased
ability to remove clots, decreased fibrinolytic potential, are also
at risk for thrombosis
[0012] To counterbalance the abovementioned mechanisms that
precipitate platelet activation, platelets are downregulated by the
anti-thrombotic potential of normal endothelial cells, in vivo, and
by substances produced by the activated platelets. The majority of
pathways that result in inhibition of platelet aggregation and
procoagulant activities act by increasing the internal level of
cyclic AMP, which activates the cyclic AMP-dependent
protein-kinase. This leads to serine-threonine phosphorilation of
an array of substrates.
[0013] Experimentally, the result of the platelet contraction and
the tension applied on the fibrin network strands is observed in
vitro as clot retraction. Macroscopically, clot retraction is seen
as a dramatic reduction in clot volume in a process that expels the
fluid trapped inside the clot. Although the physiological role of
clot retraction is not completely understood, it is assumed that it
helps in approximating the edges of a tissue defect and in
concentrating the clot in the area of injury (4). One issue that it
is clear in clot retraction is that in order for this process to
occur normally all haemostatic mechanisms must act in synchrony.
The two primary requirements for proper clot retraction to occur
are the formation of an appropriate fibrin network and healthy
platelets, capable of contracting and anchoring the fibrin network.
The structure and formation of the fibrin network are sensitive to
pH, ionic strength, calcium concentration, plasma proteins,
platelet release products, leukocyte materials, etc. (4).
[0014] Examples of pathological conditions that affect the
structure of the fibrin network are diabetes mellitus and multiple
myeloma (4; 13). Healthy platelets need to express the integrin
.alpha..sub.IIb.beta..sub.3 on their surface to properly anchor the
fibrin strands and they need to be metabolically fit for the task.
Examples of pathological conditions that affect platelet metabolism
are diabetes mellitus and uremia (14). Also, the age of platelets
affects their performance, this aspect is particularly important
for transfusion purposes.
[0015] The wide spectrum of processes involved in clot retraction,
including biochemical, biorheological and biomechanical mechanisms,
in conjunction with fine controlling and orchestration mechanisms,
makes clot retraction a very desirable focus point that represents
the well-being of all the steps required for this event to take
place. This approach of "funnel detection" yields excellent means
for population screening and individual patient monitoring for
clinical progress.
CURRENT STATE-OF-THE-ART
[0016] In clinical practice, the measurement of platelet viability
has been used mainly to set standards for appropriate storage and
handling of platelet concentrates. These techniques include
estimation of the life-span after storage with radiolabeling,
measuring the reduction of bleeding time, and semi-quantitative
estimation of platelets to form aggregates in vitro with the use of
platelet aggregometers. These techniques, however, are not
routinely used to evaluate platelet performance. A more practical
estimation of the capacity of platelets to function normally
appears to be retention of shape, ATP content and function in the
osmotic reversal reaction (15).
[0017] Methods Currently Used to Evaluate Clot Retraction
[0018] A common method utilized to evaluate clot retraction is
quantitation of the fluid volume expelled by the clot during
retraction, and estimation of the volume of the residual clot (16).
This is a qualitative essay that does not provide information about
the force generated during clot retraction.
[0019] Another known method involves the formation of cylinders or
strips of clots, which are then immobilized on one end and anchored
to a force transducer (17) on the other. This technique requires
mechanical manipulation of the sample and bathing of the clots in a
foreign substance that may alter the natural process of clot
retraction.
[0020] Yet another technique utilizes a rheometer to measure the
normal force development during clotting and retraction (18). An
important limitation of this technique is the high cost of the
equipment.
[0021] A method described by Carr in U.S. Pat. Nos. 4,986,964;
5,293,772 and 5,205,159 directly measures the force developed by
platelets during clot retraction. Carr's apparatus consists of a
cup in which the fluid sample (before clotting) is placed. The
opening of the cup is covered by an upper plate, which is coupled
to a steel arm attached to a force transducer. As the clot
retracts, the force generated is transmitted to the force
transducer, where it is measured (1;4; 13; 14). Although this is a
very reliable method, the cost per measurement is high, because
this method allows only the measurement of one sample at a time.
Also, this equipment has the added complication of the high
precision required for its alignment and setup.
[0022] Therefore, it would be advantageous to have a low-cost,
reliable method for the quantitation and monitoring of the force
developed during clot retraction. This would provide a simple way
to assess several variables of clinical relevance that converge
into one single measurable variable, i.e. using the abovementioned
funnel detection philosophy. Moreover, what is needed is an
easy-to-use and economical device to accurately measure the force
developed during clot retraction. This device should be
self-contained in order to minimize exposure to biohazardous
materials. Such a device would have a broad spectrum of clinical
applications, including, for example, patient evaluation and
population screening for pathological conditions.
SUMMARY OF THE INVENTION
[0023] The primary object of this invention is to provide a novel
platelet retractometer that will measure the force developed by
platelets during blood clot retraction.
[0024] Another object in accordance with the present invention is a
device that can automatically measure the force developed by
platelets during clot retraction, is easy and inexpensive to
operate, and provides no exposure of the operator to biohazardous
materials.
[0025] A further, most preferred object is to provide a method for
measuring the force developed by platelets during clot retraction,
as well as its clinical applications, in both research and patient
monitoring. The clinical applications comprise all conditions in
which platelet viability and platelet metabolism are impaired,
including, but not limited to, diabetes mellitus, chemotherapy, and
monitoring of platelet aging for blood transfusion. In experimental
applications, the device will increase the scope of study in
platelet biology by bringing a user-friendly ready-to-use method
useful to dissect the mechanisms involved in platelet contraction
in both, physiological and pathological conditions.
[0026] In accordance with these objects, this invention
contemplates an apparatus for measuring blood platelet
contractility, hereinafter called a "retractometer." The
retractometer has a spherical rigid chamber with an opening in its
dorsal aspect. Found inside this chamber is a smaller, spherical,
flexible membrane chamber concentrically aligned and isolated from
the larger rigid chamber, creating a void space between the walls
of the rigid and flexible chambers. The flexible membrane chamber
also has an opening in its upper aspect, smaller than and coaxial
to the opening in the rigid chamber. There is a tube attached at
the opening, leading out of the flexible chamber concentrically and
in perpendicular axis through the opening in the rigid chamber.
This concentric alignment of chambers creates a void space that is
isolated from the void space of the flexible inner chamber. A
second tubular passage is connected to the valve at one end and in
perpendicular alignment to the first passage. A pressure transducer
is connected to the distal end of this second tube. Thus, any force
exerted on the flexible chamber to alter its diameter would be
measured by the pressure transducer. The membrane chamber can be
manufactured from latex by dipping a mold and withdrawing a thin
spherical bag with an opening created by a shaft attached to a
spherical mold. The flexible membrane can be latex or any other
suitable material.
[0027] In further accordance with these objects, this invention
contemplates an alternative retractometer having similar spherical
rigid and flexible chambers, and openings in their upper aspect
isolating the two chambers from each other and creating a void
space between their walls. The variation from the abovedescribed
setup is that the tubular chamber leading out of the flexible
chamber concentrically and in perpendicular axis through the
opening in the rigid chamber, has both ends sealed. This creates a
void space that is isolated from the void space of the flexible
inner chamber. Through this tubular chamber, runs a glass capillary
tubing, coaxial to and longer than the tubular chamber, passing
through both ends of the sealed tubular chamber. This creates a
continuous passage from outside of the apparatus to the void space
of the inner flexible chamber. The distal opening of the capillary
tubing is plugged before directly reading the force applied by the
retracting clot as the height of the fluid column inside the
capillary. This plug can either be a removable type, like a cap or
stopper, or a sealed type that is opened by breaking the capillary
at an etched or scored point above the sealed tube.
[0028] The advantage of this embodiment is that direct readings can
be taken, with no need for electronic measuring equipment. The
disadvantage is that it does not readily lend itself to automation
except by optical readings.
[0029] A more specific and preferred embodiment of this invention
is an automated system for measuring blood platelet contractility
of a plurality of samples having an array of retractometer units
with valves as described hereinabove. Each unit retractometer is a
separate apparatus for measuring blood platelet contractility of a
single sample. As described above, it comprises a spherical rigid
chamber having an opening in its upper aspect, a smaller,
spherical, flexible membrane chamber placed concentrically within
the rigid chamber, creating a void space between the walls of the
rigid and flexible chambers, and having an opening in its upper
aspect that is smaller than and coaxial to the opening in the rigid
chamber. A first, attached contiguous tubular passage leads out of
the flexible chamber concentrically and in perpendicular axis
through the opening in the rigid chamber, creating a void space
that is isolated from the void space of the flexible inner chamber.
A two-way valve is attached to the distal end of the tubular
passage, which in turn, is connected to a second tubular passage.
The end distal to the valve is connected to a common pressure
transducer. The valves are operated automatically by solenoids,
energized and regulated by an electronic circuit. This circuitry is
programmed to choose and operate the solenoid valves in a
predetermined order written in software by the inventors.
[0030] An equally preferred embodiment in accordance with this
invention is an electronic solenoid valve controller to fully
automate a system comprising a number of retractometers and save
costs in labor. This embodiment is a system apparatus for
automatically measuring platelet contractility in a plurality of
samples. The system has a pump mechanically connected to a pump
motor, which in turn is electronically connected to a
microprocessor having a plurality of pins. One pin is used to turn
the pump motor on; a second pin to move fluid in the pump in one
direction; a third pin to move fluid in the pump in an opposite
direction; and at least one of the remainder of the pins to
activate each of an array of solenoid valves. The system also has a
voltage divider used to establish the position of the fluid in the
pump. There is a fluid conduit connecting the pump to a hydraulic
system having a manifold that connects the pump to each of a
plurality of retractometers controlled by solenoid valves. Each
retractometer communicates with one of the solenoid valves. A
pressure transducer reads each pressure and sends the reading to an
analog to digital (A/D) converter connected electronically to the
transducer, the pump motor, the microprocessor, and a computer.
[0031] Basically, sequence of events is as follows. A readout
position voltage from the voltage divider is entered through the
(A/D) converter to the microprocessor, which determines the
direction of flow in the pump and activates the pump to adjust the
fluid pressure within the system. The pressure is then measured by
the pressure transducer connected electronically to the A/D
converter, and a target pressure is registered in the
microprocessor memory, which is subsequently recorded and displayed
by the computer.
[0032] This apparatus embodiment may have a pump that moves fluid
with a sliding piston. Preferably, the pump is a syringe controlled
by a step motor with very fine gradations. Preferably, the
apparatus has an array of eight solenoid valves, each valve
communicating with one of eight retractometers. More preferably,
the system apparatus is expandable by addition of retractometers
and valves. Most preferably, the retractometers are packaged in a
cartridge, such that one cartridge is removed after sampling and
replaced with another having additional samples, and so on.
[0033] The apparatus is protected by a protection valve located at
the entrance to the pressure transducer to prevent damage to the
system and a second protection valve to control access to a fluid
reservoir. The subroutines in the analysis program are burnt into
the microprocessor.
[0034] A most preferred embodiment in accordance with this
invention is a method for measuring blood platelet contractility.
The method comprises the steps of preparing a retractometer
according to this invention by applying adhesive to the surface of
the inner flexible membrane to avoid slippage of clots. The
adhesive can be any suitable substance, for example, collagen Type
I suspension. The coated flexible membrane is then pressure
conditioned by mounting it on a rubber stopper pierced by a
hypodermic type needle attached to a two-way valve. A syringe is
attached to one opening of the valve and a second needle is
attached to a second opening of the valve, making certain that the
reach of the two needles is identical.
[0035] Next, the membrane chamber is slightly pressurized, the
valve to the syringe is closed, communication is opened to ambient
fluid. In this manner, the inner and ambient pressures are allowed
to equilibrate by siphoning. The fluid level inside the capillary
is adjusted to "zero pressure" level.
[0036] The second step involves loading of the sample into the void
created between the two chambers, surrounding and in contact with
the outside surface of the flexible membrane chamber. A small
amount of oil is added over the sample to avoid drying out. The
sample is then allowed, or induced, to clot and the force of the
clot retraction is measured in a pressure transducer and
recorded.
[0037] Also contemplated by this invention is a method for
automatically measuring a number of samples in a number of
retractometers to determine the strength of platelet contractility.
A first step requires calibration of the apparatus above. This
entails the microprocessor reading all initial pressures in all
retractometers sequentially by opening each solenoid valve, opening
the protection valve, measuring the voltage in the pressure
transducer and storing the measured value in the temporary memory
of the microprocessor. This process is repeated until all the
initial pressure values are registered as target values for each of
the retractometers. The second step adjusts the value of the
hydraulics by opening the protection valve only and activating the
pump until the target value is reached. The sample is then loaded
into the retractometers, and clot formation is induced. The third
step requires opening of the sample valve, measuring the pressure,
and closing the sample valve, in that sequence. The measured values
are then sent to a text file in a computer, and the new measured
value for each retractometer becomes the next target value. This
third step is repeated until all samples are measured. The entire
process of measuring the clotted samples takes less than one
minute.
[0038] The methods described here are useful in determining
platelet activity. The ability to determine platelet activity, and
contractile strength, is more specifically useful in determining
viability of stored blood products. Determining forces of
contractility is particularly useful in diagnosis or prognosis of
various diseases in patients. Because each of the components
associated with clotting is the result of a myriad of intermediate
steps, clot retraction is an excellent candidate for "funnel
detection" where with one simple measurement it is possible to
unmask a vast array of pathological stages. Funnel detection
methods are particularly important in population screening
studies.
[0039] Still further embodiments and advantages of the invention
will become apparent to those skilled in the art upon reading the
entire disclosure contained herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 shows Platelet interaction with surface immobilized
collagen type I. RICM images show platelets in a flow field. On a
gray scale, black indicates a distance from the surface of 4-12 nm;
white of >20-30 nm. The time after initiation of the experiment
is shown at the right side of each panel. For this experiment the
platelet count was reduced to 10,000 platelets per .mu.l.
[0041] FIG. 2 shows the evolution of an isolated thrombus at 100
s.sup.-1. For the experiment shown here, development of a single
thrombus was recorded. This image shows the thrombus once it is
developed (>10 minutes) to observe the grow changes. Each image
corresponds to the summation of a series of confocal images. Of
notice is the peculiar growth pattern of the thrombus. Platelet
deposition appears to occur in the downstream areas.
[0042] FIG. 3 is a graphic representation of the STL file
generation. For the experiment shown in this figure, an isolated
thrombus obtained with collagen spray was used. The wall shear rate
was 100 .sup.s-1 and the data presented correspond to images taken
after 10 minutes of flow. The geometry reconstructed here
corresponds to the thrombus shown in the early time of FIG. 2
[0043] FIG. 4 is a photograph of the sterolithography model. The
actual model was built to a scale of 300:1 and has a volume of 3.44
cm.sup.3.
[0044] FIG. 5 is a diagrammatic representation of two alternative
designs for the retractometer of this invention. Top panel (A) is a
design in which the individual retractometers are connected through
a system of communicating vessels sharing a common pressure
transducer. This design allows the simultaneous measurement of
several samples. Bottom panel (B), is an alternate design having a
clay plug closing the air filled capillary tube, which the operator
"snaps" by bending it around the etching before starting the
reading. The fluid inside the capillary then reaches the "zero
level" corresponding to the hydrostatic pressure of the system.
This design allows the direct measurement of the pressure without
the need of electronics.
[0045] FIG. 6 is a diagram showing the contracting element. The
upper panel of the figure represents the fibrin network before
platelet contraction.
[0046] FIG. 7 diagrammatically represents the origin of the forces
developed by platelet contraction within a retractometer of this
invention. When the fibrin network contracts, tension develops
along the surface of the element, due to the "pull" between the
contracting elements.
[0047] FIG. 8 is a force analysis of the retractometer. A is a
diametrical cross-section in a plane passing through the center of
the sphere. B is a geometrical representation of two arbitrary but
symmetrical vectors acting on the unit represented in A.
[0048] FIG. 9 shows the mechanical model comprising a cantilever
beam clamped at one end, subject to a constant bending moment.
[0049] FIG. 10 shows the calibration graph of a single cantilevered
transducer. Steps of 1 gram were used in the calibration. The means
of the experimental points are shown with their corresponding
standard error of the mean. The continuous line corresponds to the
linear regression of the measured points. The correlation
coefficient calculated is r.sup.2=0.9995. The force resolution of
the transducer is 2.85.times.10.sup.-4 gram force.
[0050] FIG. 11 Top: is a photograph of one embodiment of an
immersion mold. The prototype shown here was manufactured from
stainless steel. Bottom: is a prototype of the flexible membrane of
this invention.
[0051] FIG. 12 is a graphic representation of a method used to
pressure condition the membrane.
[0052] FIG. 13 depicts the results of a preliminary experiment to
show the feasibility of the proposed methodology. For the
experiment shown here the flexible membrane used was fabricated
with latex with a thickness of 150 .mu.m. Citrated blood (11 .mu.M
Sodium Citrate) was used.
[0053] FIG. 14 illustrates the change in shape of the clots when
separated from the membrane and cut open. The photograph shows a
petri dish with three sections of the clot. A digitally enhanced
magnification of the three samples shown is presented for better
appreciation of the process.
[0054] FIG. 15 is a schematic of the geometry of the cylindrical
clot during contraction. The contraction of the clot is considered
to be isotropic (17). The force F is measured directly by the force
transducer. The area A can be directly calculated from the
measurement of the clot diameter. The stress (F) can be estimated
from this simple model.
[0055] FIG. 16 is a comparison of the two methods used in this
description. Both experiments were performed using platelet rich
plasma.
[0056] FIG. 17 is a schematic diagram of an electronic solenoid
valve controller useful in simultaneous processing of many
samples.
[0057] FIG. 18 is a schematic representation of the fully automated
system apparatus that can greatly increase the speed and ease of
measuring platelet contractility in a number of retractometers,
each having the same or different sample. Such a system is highly
useful in screening populations and effectiveness of various
drugs.
[0058] It is stressed that the figures above represent only certain
fully tested working examples and do not limit the invention to
these precise illustrations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Introduction
[0059] After in vitro clot formation, the fibrin meshwork entraps
virtually all the serum and the cellular components of blood.
Within minutes to hours, the platelets in the clot contract,
expelling a very large fraction of the serum. This process is known
as clot retraction. Although the physiological relevance of clot
retraction is still not fully understood, the fact that platelets
are needed for this process to take place is well documented (1).
There is strong experimental evidence that suggests the
participation of an actinomyosin contractile mechanism as well as
the involvement of the platelet .alpha..sub.IIb.beta..sub.3 in the
process (2;3).
[0060] The present invention is based on the rationale that the
development of a reliable method for the study of clot retraction
will bring not only a useful tool to elucidate the mechanisms
involved in the physiological mechanisms, but also an important
tool for the monitoring of the overall well-being of the platelets
in a blood sample. This invention will potentially yield an
important diagnostic tool for the monitoring and detection of
pathological states, as well as an easy-to-use tool for the
monitoring of platelet viability for transfusion purposes.
Materials and Methods
[0061] In order to develop the instant invention, certain relevant
parameters had to be elucidated. In the examples hereinbelow, are
described determinations of platelet activation under certain
conditions, as well as manipulations of clot geometry.
Example 1
Perfusion Studies
[0062] Methods
[0063] Blood obtained from healthy volunteers was mixed with
D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone
dihydrocloride (PPACK, 93:M) to prevent clotting. The platelet
count was adjusted to 10,000/.mu.l to reduce the number of events
on the surface and facilitate image analysis. Perfusion experiments
were conducted in a parallel plate flow chamber at 37.degree. C.
(3) using type I collagen fibrils as reactive substrate onto glass
coverslips. The interaction of flowing platelets with the surface
was evaluated in real time by reflection interference contrast
microscopy (RICM) using a Zeiss Axiovert 135M microscope. In this
technique, interference colors indicate the distance between two
surfaces, such as cellular membranes and a substrate coated on
glass. On a gray scale, zero-order black indicates a separation of
4-12 nm, and white a distance>20-30 nm (15; 19). Experiments
were recorded on S-VHS videotape at the rate of 30 frames per
second and analyzed off-line with Metamorph (Universal Imaging)
software.
[0064] Results
[0065] Platelet Interaction with Surface Immobilized Collagen Type
I.
[0066] The top panel in FIG. 1 (0 minutes) shows the native
spheroidal shape of the platelets, before activation. A few seconds
after the initial platelet adhesion occurs, the first signs of
activation are seen as dramatic shape changes and subsequent
adhesion of the platelet membrane to the reactive surface. The
other panels (2 and 4 minutes) show the activation and spreading
that two single platelets undergo. Of notice is the large area that
a single platelet can cover. This experiment shows the large amount
of "membrane reservoir" contained by the relatively small
platelets. After activation, the platelets initiate contraction,
resulting in their deformation and clot formation. This phenomenon
is not observed in the photographs shown here because the platelets
are attached to a non-deformable surface (glass); however, the
platelets increase the tension on their membrane due to the
abovementioned cytoskeleton rearrangement.
Example 2
[0067] In order to demonstrate some of the technical capabilities
currently being developed in the Inventors' laboratory, a summary
of the development of a technique to create an upscale replica of
an actual thrombus is presented below. The geometrical data of the
thrombus are obtained with confocal microscopy while the blood is
continuously flowing as previously reported (20). This technique
was developed to study the flow field around a thrombus in an
upscale chamber. By matching the Reynolds number, it is possible to
determine the flow path in the microscopic realm, based on the
similarity principle.
[0068] The evolution of an isolated thrombus at 10 s.sup.-1 is
depicted in FIG. 2. For the experiment shown here, a single
thrombus was recorded from initiation of the flow. This image shows
the growth changes in the thrombus once it is developed (>10
minutes) to 30 minutes. Each image is derived from the summation of
a series of confocal image slices. Of note is the peculiar growth
pattern of the thrombus. Platelet deposition appears to occur in
the downstream areas.
Example 3
[0069] Below, Inventors describe their technical solution to create
an upscale three-dimensional (3-D) model of a thrombus based on the
information obtained with confocal microscopy.
[0070] As a first step, Inventors decided to investigate the
already available techniques for 3-D rapid prototyping. A commonly
used technique is stereolithography, which uses step-wise planar
buildup of the object, based on the solidification of a photoresin
by a laser beam. After each layer is cured, the object is lowered
into a fluid resin pool by a distance equal to the vertical
resolution of the system. This technique appears to be adequate in
view of the complex geometries that it can handle.
[0071] The main practical difficulty in merging confocal microscopy
and stereolithography is the lack of compatibility of the data.
Confocal microscopy renders the data in a series of images (TIFF
files in our system). These images are represented by a series of
pixels with a given grayscale value, and the images are separated
by the distance of the confocal sections (1 .mu.m in this case).
Stereolithography, on the other hand, uses ASCII files that contain
coordinates of the surfaces surrounding the object. This format has
the practical advantage of possessing the capability to rotate
objects to an orientation that facilitates the manufacturing
process. Inventors developed the series of steps that successfully
led to the generation of a Stereolithography file from the original
images of the experimental data.
Example 4
Generation of Stereolithography Files from Confocal Data
[0072] The confocal microscopy images obtained were preprocessed
with a 3.times.3 median filter, in order to minimize noise
originated by the flow. The images were then translated into a
voxel space with a software package written by Inventors
specifically for this purpose. This package maintains the relative
positions of the measured objects in a cartesian three-dimensional
space. The voxel representation of the microscopic field was then
used with AVS release 5.3 (Application Visual Systems, Inc.,
Waltham, Mass.) to render the three-dimensional field. This
software package allows the operator to interpolate a surface
(isosurface) between contiguous voxels with an intensity above a
preset threshold. This isosurface is comprised of a series of
triangles, each triangle being a "surface unit." The coordinates of
these surface units were then stored in a geometry information
file. The geometry file was then translated into an "STL" file
(standard input format for stereolithography) with a software
package written by Inventors specifically for this purpose. The STL
file contains the coordinates of the vortices of the surface units
and a normal vector pointing outside of the body of the object to
be materialized. During the fabrication process, a scale factor was
included to yield the desired dimensions. A graphic representation
of the file generation is shown in FIG. 3.
[0073] Graphic Representation of the STL File Generation.
[0074] For the experiment shown in this FIG. 3, an isolated
thrombus obtained with collagen spray was used. The wall shear rate
was 100 .sup.s-1 and the data presented correspond to images taken
after 10 minutes of flow. The geometry reconstructed here
corresponds to the thrombus shown in the early time of FIG. 2. The
upper left panel shows a summation of all the confocal images
obtained from a real thrombus. The upper right panel shows the
topographical representation of the thrombus in pseudocolor. The
bar on the right shows the color code for the height in
micrometers. The lower left panel shows the 3-D representation
rendered with AVS, as described above. Although it is possible to
orient the geometry to any position, a planar view was chosen for
easier comparison of the 3-D representation with the original
data.
[0075] The lower right panel shows a graphic representation of the
STL file. The graph shows a wire model of the file, with the
orientation identical to the previous panels. The "hedgehog"
appearance is due to normal vectors pointing outside of the body,
as described in the method. For clarity, the normal vectors are
shown in blue, and the wire model in white. This type of graphic
representation is not necessary for the actual fabrication process,
but it is useful for error detection in the creation of the files
and overall quality evaluation of the process. In the graph, the
complexity of the surface and the large number of triangles
necessary to reconstruct such a complex geometry can be
appreciated.
[0076] FIG. 4 shows the actual model described above built to a
scale of 300:1 and has a volume of 3.44 cm.sup.3. The running time
for this sample was 2.3 hours, using a FDM 2000 stereolithography
machine (Stratasys, Ontario CA). The orientation of this
photography is similar but not identical to the orientation shown
in FIGS. 2 and 3.
Example 4
Experimental Design of the Retractometer
[0077] Principle of Operation
[0078] The performance of the device of this invention is based on
the Laplace principle, in which the tension developed by the
contraction of a sheet of platelets during clot retraction is
transformed into an increase in pressure inside a semispherical
flexible membrane.
[0079] The geometry of the proposed device is the following. Let
r.sub.o be the radius of a spherical container and r.sub.i the
radius of a concentrical spherical membrane as shown in FIG. 5 (A
and B), r.sub.o>r.sub.i, h=r.sub.o-r.sub.i;
r.sub.o,r.sub.i>>h.
[0080] Two Alternative Designs for the Retractometer.
[0081] Two alternate embodiments of the retractometer of this
invention are shown in FIG. 5AB. Top panel (A), is a design in
which the individual retractometers can be connected through a
system of communicating vessels sharing a common pressure
transducer. This design allows the simultaneous measurement of
several samples. In Figure A, prior to clotting, a blood sample is
placed inside (2) the rigid reservoir (3). The thickness of the
sample at 2 is h=r.sub.o-r.sub.i. A thin layer of mineral oil
(light white oil, Sigma) is placed on top of the blood sample to
avoid evaporation. As tension along the wall of the flexible
membrane (4) increases due to clot retraction, the pressure inside
the tube (1) increases.
[0082] Bottom panel (B) is a design in which the operator "snaps"
the clay plug (7) of the air filled capillary tube, by bending it
around the etching (scoring) (6) before starting the reading. Then
the fluid inside the capillary reaches the "zero level"
corresponding to the hydrostatic pressure of the system (details of
the filling of the flexible membrane are given hereinbelow). The
filling fluid of the flexible membrane may contain a coloring agent
for easier visualization. The presence of the plug (7) prevents
both fluid evaporation and changes in the fluid level by
manipulation of the retractometer. This design allows the direct
measurement of the pressure without the need of electronics.
[0083] FIG. 6 is a diagrammatic representation of the contracting
element described in the body of the text. The upper object of FIG.
6 represents the fibrin network before platelet contraction. As an
example, FIG. 6 shows an isotropic retraction with a longitudinal
strain of -0.5. The strain g is defined as g=(L-L.sub.0)/L.sub.0,
where L is the length at the end of the deformation, and L.sub.0 is
the initial length. The bottom object shows the result of the
isotropic contraction.
[0084] An important consideration in the design and performance of
the retractometer device is that when the shell element described
in FIG. 6 contracts, the only changes that contribute to an
increase in the tension in the contracting element are L1 and L2. A
contraction in h does not contribute to development of tension on
the system described in FIG. 5. This concept is detailed in FIG.
7.
[0085] FIG. 7 is a diagram representing the origin of the forces
developed by platelet contraction in a retractometer. When the
fibrin network contracts, tension develops along the surface of the
element, due to the "pull" between the contracting elements.
Because the outer surface of the contracting element is free, the
contraction of the element along the thickness h results in a
decrease in volume and not a modification of tension on the
contracting element. In order to better represent this concept, the
diagram on the right shows a free body taken from the contracting
element. A change in radius of the cylindrical component shown
results in an increase in tension along the surface as shown, while
a change in height (h) does not modify the tension along the
surface of the plate.
[0086] In order to calculate the magnitude by which a variation in
the tension of the clot will result in a variation of internal
pressure in the device shown in FIG. 5, it is helpful to use a free
body diagram as shown in FIG. 8.
[0087] FIG. 8 represents a force analysis of the retractometer of
this invention. The flexible membrane of the retractometer is
modeled for this analysis as a perfect sphere. A is a diametrical
cross-section in a plane passing through the center of the sphere
as shown. The resultant inner pressure Pi is homogeneously
distributed on the inner surface of the retractometer. The origin
of all the vectors is the center of the sphere and all of them have
the same magnitude. The dotted line in this panel shows the
arbitrary cross-section where the analysis is performed.
[0088] B is a geometrical representation of two arbitrary but
symmetrical vectors acting on the unit represented in A. Notice
that the horizontal components (parallel to the cross-section shown
in A with the dotted line) of these vectors cancel each other, the
vertical component (normal to our arbitrary section shown in A)
does not cancel by any of the vectors acting on the lower half of
the sphere. Therefore, for the force analysis, the only vectoral
components of the force resultant of Pi acting on the surface are
normal to the cross section shown in A. These vectors act on the
area a.sub.1 shown in C, and a.sub.2 is the sectional area of the
wall of the sphere. D is a free body diagram of a thin slice of the
body shown in C cut by two parallel planes at a small distance
apart, one on each side of the center of the sphere. The
circumferential stress F is a stress acting on, and normal to, the
cross-sectional plane. <F> is the average value of F, which
is non-uniform across the thickness of the wall. The value of
<F> is computed hereinbelow. The vectors on the right side of
D show the condition of equilibrium. As explained above, the force
acting vertically and downwards (F.sub.2) is computed as the
pressure acting on a.sub.1, or Pi(a.sub.1) and
F.sub.2=Pi(a1)=Pi.pi.r.sub.i.sup.2. The area of the wall of the
contractile element is Br.sub.o.sup.2-Br.sub.i.sup.2. The resultant
tensile force due to clot retraction in this particular geometry is
F.sub.1=B(r.sub.o.sup.2-r.sub.i.sup.2)<F>. The balance of the
forces in equilibrium requires, therefore, that F1=F2, or:
.pi.(r.sub.o.sup.2-r.sub.i.sup.2).sigma.=.pi.r.sub.i.sup.2P.sub.i
(D:1)
[0089] or:
.sigma. = P i r i 2 r o 2 - r i 2 = r i 2 P i h ( r o + r i ) ( D :
2 ) ##EQU00001##
Therefore, the average tensile force can be easily calculated based
on the measurement of the hydrostatic pressure inside the
compartment defined by the flexible membrane.
[0090] This analysis is exact for a perfect sphere. Although the
retractometer deviates from this uniform stress field in the area
where the flexible membrane attaches to the capillary tube or
connecting tube, the stress analysis is an excellent approximation
at the operational level and it is valid for the purposes of the
design presented in this description.
[0091] In order to be able to calibrate their retractometer and
compare it with other known experimental models, Inventors decided
to implement a system described by others (17), in which a
cylindrical clot is immersed in ice-cold buffer to prevent platelet
contraction. The clots are then anchored and held vertically to the
bottom of the container at their lower end and to a force
transducer to the upper end of the clot.
[0092] Force Transducer:
[0093] An isotonic force transducer was implemented. The system is
based on the single supported beam principle. The mechanical model
is a cantilever beam clamped at one end, subject to a constant
bending moment. According to the following description:
The equation that dictates the behavior of the beam is:
2 y x 2 = 1 EI M ( x ) ( D : 3 ) ##EQU00002##
[0094] where: M is the bending moment imposed by the load on the
cantilevered beam, E is Young's modulus, I is a property of the
cross-sectional geometry of the beam, the term on the left is the
deflection of the beam (assuming a deflection much smaller than the
length of the beam).
[0095] The deflection y(x) can be calculated by using:
EI 2 y x 2 = M , EI 3 y x 3 = S ( D : 4 , 5 ) ##EQU00003## [0096]
where S is the transverse shear. The bending moment and the
transverse shear are related to the lateral load by:
[0096] M x = S , S x = w 2 y x 2 = 1 EI M ( x ) ( D : 6 , 7 )
##EQU00004##
[0097] Where w is the lateral load per unit length. [0098] Because
a small curvature is assumed, and the slope of the deflection is
finite, the equation to be used instead of D:3 is:
[0098] 2 y x 2 [ 1 + ( y x ) 2 ] - 3 2 = M ( x ) EI ( D : 8 )
##EQU00005## [0099] Integration of equation D:8 yields:
[0099] y ( x ) = M EI x 2 2 + Ax + B ( D : 9 ) ##EQU00006##
[0100] Therefore, for a load imposed at a fixed distance in x, for
a constant bending moment, and for a small deflection, the
deflection is a linear function of the moment.
[0101] For the implementation of the force transducer, Inventors
used a borosilicate glass rod, with a length of 15 cm and a
diameter of 1 mm. Due to the relative length of the rod,
deflections up to a maximum of 2 cm can be considered small.
[0102] The results of the calibration experiments are shown in FIG.
10. The range tested was from 0 to 5 gram force. This range proved
to be adequate for the experimental conditions. FIG. 10 shows the
calibration graph of a single cantilevered transducer that is part
of this invention. Steps of 1 gram were used in the calibration.
The means of the experimental points are shown with their
corresponding standard error of the mean. The continuous line
corresponds to the linear regression of the measured points. The
correlation coefficient calculated is r.sup.2=0.9995. The force
resolution of this transducer is 2.85.times.10.sup.-4 gram
force.
[0103] Results
Example 5
Fabrication of the Retractometer
[0104] Fabrication of the retractometer required research and
development in the following areas:
[0105] 1) Manufacturing of the flexible membranes. Which is
subsequently divided in two steps: [0106] a) Fabrication of a
suitable immersion mold [0107] b) Fabrication of the membranes
[0108] 2) Filling of the membranes. This step is necessary to
assure that the internal pressure of the membranes corresponds to
the hydrostatic pressure of the fluid around them, during
operation.
[0109] 3) Adjusting of the fluid level inside the capillary at
"zero pressure" level. This step is necessary for the operator to
see the fluid level above the capillary holders.
[0110] 4) Calibration of the system and comparison with an
alternative method. The alternative method will be to measure
directly the force developed by a cylindrical clot made with
platelet rich plasma of the same donor to serve as our "gold
standard."
[0111] Fabrication of a Suitable Immersion Mold
[0112] The first step in manufacturing of the flexible membrane is
the fabrication of a suitable immersion mold. In a preliminary
phase, Inventors fabricated a prototype mold from stainless steel.
Turning now to FIG. 11, the top panel is a photograph of the
stainless steel immersion mold. Although the embodiment shown here
was manufactured from stainless steel, it could likewise be made
from any other suitable material. The ball has a diameter of 9/16''
and the rod has a diameter of 3/32'', but these could be of any
suitable dimension. The bottom panel shows an embodiment of the
flexible membrane. This prototype was made to show the feasibility
of fabrication using the immersion mold shown on top. This
prototype embodiment was fabricated using urethane, but any other
suitable material could be used. Due to the transparency of the
material it is easy to study the thickness of the membrane. The
figure shows an even thickness of the material along the spherical
region of the membrane. However, this fabrication technique yields
an increase in thickness around the region of the neck (white
arrow). It can be concluded from the stress analysis shown in FIG.
8, that the stresses on the wall are uniformly distributed along
the flexible membrane, except around the point of insertion of the
capillary tube. Therefore, it is expected that this thicker region
will not have an important impact in the performance of the
retractometer.
[0113] Fabrication of the Membranes.
[0114] The membranes would preferably be fabricated by experts in
dip molding technology, for example by ACC Automation (Akron,
Ohio). For this application, their 4-axis dipping system would be
particularly suitable. Briefly, the system has 4 axis of operation
(vertical, horizontal/pallet rotate and form spin), allowing the
membrane coating to be uniform along the surface of the mold. The
equipment has a vertical stroke of 30 inches, a vertical axis speed
range of 0.01-12 inch/sec, with 0.001 inch/sec speed increments.
Rotate axis positional range is 1440 degrees in 1 degree positional
increments. Rotate axis speed range is 0.1-60 degrees/sec in 0.1
degree speed increments. Spin speed range is 10-100 RPM in 1 RPM
increments. Horizontal axis position is 18 inches in 0.01 inch
positional increments. Horizontal axis speed range is 0.01-4.0
inches/sec in 0.001 inches/sec/Maximum payload capacity of 10
pounds. The membranes are dried in an integrated force air
convection electric oven, with programmable temperature control up
to 200.degree. C. The forms are spun in the oven to increase drying
uniformity.
[0115] The membranes are fabricated using two coats of latex
without thickening agent, in a similar fashion to fabricating
condoms. Uniformity in thickness and mechanical properties of the
membranes are highly reproducible.
[0116] The second step is to develop a suitable method for the
filling of the membranes.
[0117] Preparation of the Membrane Prior to the Final Assembly of
the Retractometer.
[0118] In order to assure that the inner pressure of the membrane
is in equilibrium with the surrounding fluid, Inventors implemented
a simple technique shown in FIG. 12. In order to avoid slippage of
the clots over the membrane surface during contraction, the
membranes are coated with a suitable adhesive, for example, with a
bovine collagen type I suspension as described elsewhere (2.5 mg/ml
in 0.1 M acetic acid) (20). This method gave a firm adhesion of the
clot onto latex membranes. It is expected that membranes of
different materials may require other adhesives.
[0119] Membranes are pressure-conditioned as shown in FIG. 12. The
flexible membrane is mounted on a sealing rubber stop with a needle
inserted through it. The needle is connected to a two-way
stop-cock, which in turn is connected to a syringe and another
needle. The reach of both needles is the same. In a first step, the
syringe is used to slightly pressurize the flexible membrane. In a
second step, access to the syringe is closed and the two needles
are allowed to equilibrate the inner membrane pressure and the
ambient pressure by siphoning the fluids. This method is reliable
in giving zero pressure readings with the use of a pressure
transducer (Validyne DP15-22, controlled by a Validyne CD379)
immediately after inflation.
[0120] The third step is the adjusting of the fluid level inside
the capillary at "zero pressure" level. As seen in FIG. 5B, it
would be desirable to control the level of the column inside the
capillary to make the reading easier. The height of the column in a
capillary tube is dictated by the expression:
h = 2 .gamma. .rho. gr cos .theta. ( D : 10 ) ##EQU00007##
[0121] where, h is the height of the column, .gamma. is the surface
tension of the fluid, .rho. is the density of the fluid, r is the
radius of the capillary tube, and .theta. is the wetting angle. The
pressure inside the flexible membrane of the retractometer, can be
easily calculated with the expression. .DELTA.p=.rho.gh.
[0122] In order to make the retractometer more user-friendly, it is
necessary to have a good "zero pressure" level inside the
capillary, otherwise the reading error may increase. An easy way to
do this is by proper choice of the capillary radius. The
possibility of changing the angle of the meniscus .theta. in
equation D:10, with the following method (21).
[0123] Hydrophilic Modification of the Capillaries
[0124] The glass capillaries are immersed in a 1% (w/w) NaOH water
solution. The container is heated to near boiling (bubble formation
starting) (approx 90.degree. C.) and incubated for 10 minutes. The
solution is removed, and the capillaries are allowed to cool to
room temperature. The capillaries are then immersed in a 30% (w/w)
H.sub.2O.sub.2 solution, and heated to near boiling (approx
90.degree. C.) for one hour, washed five times with deionized,
demineralized water, tap dried and placed in a drying oven
(250.degree. C.) for 12 hours. Column heights were improved from 16
mm (with a 0.75 radius, untreated capillary) to 60 mm (with a 0.5
mm radius, hydrophilic modified capillary).
[0125] The fourth step is the calibration of the retractometer and
comparison of the results to an alternative, known method.
[0126] In order to explore the feasibility of the methodology in
the present invention, a prototype retractometer was implemented,
as detailed in FIG. 5. For the setup of these preliminary
experiments, it was decided to use a latex flexible membrane with a
thickness of 150 .mu.m. The pressure in both experiments was
continuously recorded using a pressure transducer (Validyne
DP15-22, controlled by a Validyne CD379). A citrated blood sample
was separated into two aliquots, one aliquot was used to prepare a
platelet-rich plasma sample by centrifugation at 150.times.g for 10
minutes. The other aliquot was used directly without enrichment.
Prior to beginning the experiment, a sample (platelet rich plasma
or blood) was supplemented with calcium to initiate coagulation. A
solution of 0.2 M CaCl.sub.2 at 42 .mu.l/ml of blood and 65
.mu.l/ml of platelet-rich plasma, the difference in volumes
accounts for the inert volume occupied by red blood cells in the
whole blood sample. During the experiment, samples were kept at
37.degree. C. The results of these experiments are shown in FIG.
13. As predicted, platelet contractility results in an increase in
the hydrostatic pressure inside the flexible membrane.
[0127] In order to demonstrate that the increase in hydrostatic
pressure shown in FIG. 13 was indeed due to an increase in the
tension on the fibrin network, the retractometer was disassembled
at the end of the experiment, the membrane and the clot were
immersed in a phosphate buffered saline solution to avoid drying of
the sample. The membrane and the attached fibrin clot were then
sectioned in rings parallel to the equator, the rings were attached
to ribbons and the clots were carefully separated from the latex
membrane. The rationale for the cuts was to unveil the residual
stresses in the clots. Cutting introduces new surfaces on which the
traction is zero. Cutting an unloaded body without residual stress
will not cause strain. If strain changes by cutting, there is
residual stress. The results shown in FIG. 13 demonstrate the
feasibility and validity of the principle of operation of the
methodology. The stresses along the thickness of the clot are not
uniform, due to the geometry of the retractometer. This lack of
uniformity in stresses must, therefore, result in "shearing strain"
across the thickness of the clot. This is seen macroscopically in
FIG. 14 as twisting of the clots. FIG. 14 shows a petri dish with
three sections of the clot. A digitally enhanced magnification of
the three samples shown is presented for better appreciation of the
process. It should be noted that the larger the deformation the
larger is the residual stress. The large twisting deformation is
due to the non-uniform increase in tension along the thickness of
the wall of the clots.
[0128] An Alternative Method and Comparison of Results
[0129] Inventors decided to implement a method described by others
(17) for calibration and comparison. The method is briefly
described below.
[0130] Cylindrical clots are obtained by pouring a human
platelet-rich plasma (PRP) suspension, immediately after thrombin
addition, into cylindrical plastic molds (6 mm diameter and 5 cm in
length). The molds are plugged at both ends with plastic plugs. The
sides of the molds are slit for easier clot extraction, but the
ends meet in a manner such that drying out is prevented. After 10
minutes, the clot cylinders are poured into a Petri dish containing
ice cold Tyrode solution to inhibit contraction. The clots are then
tied at one end with a cotton thread to a rigid stainless steel
support and the other end to a force transducer as described
hereinabove.
[0131] In order to compare the experimental results of clot
retraction with the two different setups, it is helpful to link the
two methods by the stress generated by platelet contraction.
[0132] FIG. 15 outlines schematically the geometry of the
cylindrical clot during contraction. The contraction of the clot is
considered to be isotropic (17). The force F is directly measured
by the force transducer. The area A can be directly calculated from
the measurement of the clot diameter. The stress (F) can be
estimated from this simple model.
[0133] Assuming that the stress generated by the platelets is the
same in the two retractometers, it follows from equation D:2 and
FIG. 15 that:
F A = P i r i 2 r 0 2 - r i 2 ( D : 11 ) ##EQU00008##
Regarding the units of both expressions: P.sub.i is given in cm
H.sub.20: 1 cm H.sub.20=1 gf/cm.sup.2.
[0134] Turning now to FIG. 16 for a comparison between the two
methods used. Both experiments were performed using platelet rich
plasma. In order to compare the results, data are presented in
terms of the stress as suggested by equation D:11. The solid line
shows the results obtained with the cylindrical clot and the
circles represent the experimental data points obtained with the
method of this invention.
[0135] The values calculated by equation D:11 are highly dependent
on the accurate measurement of the radii in both, the cylindrical
clot system and the retractometer of this invention.
[0136] Immediately after the mechanical test, the clots are fixed
in 1.25% (vol/vol) glutaraldehyde diluted in 0.1M phosphate buffer
(pH 7.2) for one hour at room temperature. The clots are then
postfixed in 1% (wt/vol) osmic acid containing 1.5% potassium
ferrocyanide for one hour at 4.degree. C. Subsequently, they are
dehydrated using graded alcohols and propylene oxide before being
embedded in Epon. We have successfully used this technique to
estimate the ultrastructure of fibrin clot deformation (22). This
step is done only for calibration purposes and it is not intended
to be used as a routine in the future.
Example 6
[0137] Described below is an electronic circuit designed to operate
the individual solenoid valves controlling the hydraulics of the
communicating vessels for the embodiment shown in FIG. 5 A.
[0138] The electronic solenoid valve controller circuit is shown in
FIG. 17. For the circuit shown here, a 10.times.16 array was
implemented. The circuit labeled digital row selection is meant for
an alternative computer control. When a word is written to the gain
select input of the CMOS circuit shown (analog to CD4066), a
voltage is generated at the output of the circuit, which is used to
select the row on the right hand circuits. In principle, the
voltage used for row selection can also be selected manually via a
potentiometer part of a voltage divider. Implementation of these
circuits allows the operator to select the row either manually or
via digital input. The operational amplifier (Row Gain) is intended
to give the maximal gain of the voltage divider for row selection.
The use of this is to select manually the maximal row number to be
read in a given cycle. This non-inverting input of the operation
amplifier (op amp) is amplified and sent to an A/D converter,
implemented by the comparators and the priority encoder. Should the
control be exclusively digital, this part of the circuit is
obsolete, in which case the already digital input should be sent
directly to the 1 of 10 decoder (10 is arbitrarily chosen in this
case, and the total number of rows can be different). The final
result of this architecture is that only one row is activated at a
time.
[0139] For column selection, Inventors chose to add a timer
assuming that the column selection is done in a continuous sweep.
The timer shown in the lower left corner of the diagram (FIG. 17)
has a feedback loop controlled by a potentiometer that allows the
operator to control the sweeping rate. The output of the timer
serves as the input for a 4-bit counter, the output of the counter
is input into a 1 of 16 decoder to select only one column at a
time.
Example 7
[0140] Another embodiment of an electronic solenoid valve
controller of this invention is shown in FIG. 18. For the example
shown here, an 8051 microprocessor is used. Three pins of the
microprocessor actuate a syringe pump. Pin one is used to turn the
pump on and the other two to move the pump piston either up or
down. The pump is directly connected to the hydraulics
(retractometers) of the system. The motor of the pump is connected
to a voltage divider that yields a voltage used to establish the
position of the piston of the pump. This readout position voltage
is entered through an analog to digital (A/D) converter to the
microprocessor. Other pins of the microprocessor are connected to
each one of the solenoid valves used in the array.
[0141] In this example, a series of eight valves are used to
measure each one of the retractometer samples and two others are
used to provide protection to the system. One of the protection
valves is located at the entrance of the pressure transducer, and
its role is to prevent damage to the system due to the operation of
the pump. The other valve is located to provide access to a fluid
reservoir. This valve is used in this example to fill the syringe
prior to the beginning of the experiments. The output voltage of
the pressure transducer is entered into the A/D converter and
subsequently to the microprocessor. For this example, the
subroutines were burnt into the microprocessor. In an initial stage
of operation, the microprocessor reads all initial pressures of all
the samples by opening each individual sample valve, followed by
opening of the protection valve. The voltage from the pressure
transducer is measured and stored in the temporary memory of the
microprocessor. This process is repeated until all the initial
pressure values are registered.
[0142] In the following cycles, the previous pressure value for the
valve that will be measured is taken as the target value. Then the
value of the hydraulics is taken, having only the protection valve
opened. The pump is then actuated (either up or down depending on
the relative value of the target pressure) until the target
pressure value is reached. The sample valve is then opened, the
pressure is measured, and the sample valve is closed. The measured
values are sent to a text file in a PC computer via a serial port.
The new measured value for each valve becomes the next target
value. These cycles are repeated until the end of the
experiment.
[0143] While the present invention has now been described in terms
of certain preferred embodiments, and exemplified with respect
thereto, one skilled in the art will readily appreciate that
various modifications, changes, omissions and substitutions may be
made without departing from the spirit thereof. It is intended,
therefore, that the present invention be limited solely by the
scope of the following claims.
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