Device and a method for measuring thrombus formation tendency

A device for in vitro measurement of a tendency to form thrombi in people and animals under simulated in vivo conditions. Blood is pumped at a constant flow through at least one flow channel. This flow channel can be coated or made of a thrombogenesis-promoting material. The pressure difference between the pressures upstream and downstream of the thrombogenesis unit, due to a thrombus formed in the flow channel, is measured. With this device, a method can measure the tendency to thrombogenesis.

FIELD OF THE INVENTION 
The invention concerns a device and a method for measuring the tendency to 
form blood clots in vitro by using non-anticoagulated venous blood which 
is pumped through one or more parallel synthetic channels with different 
diameters, "thrombogenesis units", which simulate in vivo bloodstream 
conditions, and where the fluid pressure is recorded before and after the 
thrombogenesis units, and the pressure drop is calculated as a function of 
the flow time, which will be an expression of how quickly a thrombosis is 
formed in the system. 
DESCRIPTION OF THE PRIOR ART 
The blood contains red and white blood corpuscles, plasma proteins and 
blood platelets and circulates in a closed circuit where the heart 
supplies the energy which impels the blood through the arterial part of 
the circuit. The consequences of clogging of this circuit vary in 
severity, depending on whether the peripheral circulation, cardial, 
pulmonary or cerebral circulation is affected. A considerable number of 
people in the industrialized part of the world are disabled or die due to 
circulatory disturbances and it is therefore vital from the social and 
economic points of view to be able to detect as early as possible the 
onset of possible circulatory problems, e.g. a thrombogenetic tendency, 
under different conditions and to monitor antithrombotic treatment. 
To transport blood through a blood vessel, from a point A to a point B, the 
pressure in A (P.sub.A) must be greater than the pressure in B (P.sub.B). 
The blood flow from A to B, Q, is dependent on P.sub.A -P.sub.B and the 
resistance R to the flow, according to formula I; 
##EQU1## 
In an undamaged blood vessel the blood flow will be laminar and in 
accordance with Poiseuille's law, expressed in formula II; 
##EQU2## 
where r=radius of the vessel 
l=length of the vessel 
.eta.=viscosity of the blood. 
It can be seen in formulae I and II that the resistance can thereby be 
expressed by III; 
##EQU3## 
This shows that the resistance to the flow is affected by characteristics 
of the blood vessel (length and radius) together with the viscosity of the 
blood. In a vessel the flow profile will describe a parabolic shape, the 
layers which are closest to the wall of the vessel will adhere to the 
wall, while towards the middle of the stream the liquid layers will slide 
towards one another and the viscosity alone affects the flow picture. Thus 
the shear rate will be highest at the wall (see below). 
With an increase in the speed of the blood the flow will remain laminar, 
and thus comply with Poiseuille's law, until a critical speed is reached, 
above which the flow becomes turbulent. In this state Osborne Reynolds 
demonstrated that the energy which drives the fluid is mainly used to 
create kinetic energy and the resistance to the flow will now depend on 
the density of the fluid instead of its viscosity, as was the case when 
the flow was laminar. The combination of turbulence and reduced vessel 
diameter will increase the resistance and lead to an increased drop in 
pressure over a vessel area in which there is a constriction. 
Thrombosis can arise as a result of activation of blood platelets with a 
resultant aggregation of these and/or coagulation of blood which includes 
blood platelet activation, the formation of prothrombin activator which in 
turn catalyzes the formation of thrombin from prothrombin. 
This thrombin thereafter catalyzes the conversion of fibrinogen into 
filaments of fibrin, which form a network and entrap blood platelets, 
blood cells and plasma thus forming a coagulum. Coagulation and blood 
platelet deposits are initiated amongst other things by damage to the 
vascular surface, collagen fibres in the vessel wall and damaged 
endothelial cells. The formation of thrombi, which is partly responsible 
for circulatory disturbances, often with disabling or fatal results, can 
be activated by an uneven endothelial surface, as in the case of 
arteriosclerosis, infections or trauma. Similarly, a very slow blood flow 
can be subject to coagulation since small amounts of thrombin and other 
procoagulants are constantly being formed. The thrombi, which are rich in 
blood platelets on the arterial side and rich in fibrin filaments and red 
blood corpuscles on the venous side, are most frequently formed on the 
vessel wall where they reduce the vessel's diameter. They can also be torn 
loose and carried with the blood to vital organs such as the lungs or 
brain, and form embolisms, with fatal results. 
On the other hand, if the tendency to coagulation is reduced and in many 
cases if the formation of thrombi is reduced, a person will be subject to 
numerous small haemorrhages which can cause anaemia or, in serious cases, 
fatal haemorrhages. 
Experiment had shown that many substances affect the formation of thrombi 
or thrombogenesis in flowing blood. These include synthetic polymers, such 
as, e.g., Dacron, biological materials, such as collagen fibrils, fibrin 
and other procoagulant protein-rich material (Baumgartner, H. R. 
Microvasc. Res. 5: 167, 1973; Baumgartner, H. R., Thromb. Haemostas. 37: 
1, 1977; Sakariassen, K. S., Aarts, P. A. M. M., de Groot, P. G., Houdijk, 
W. P. M., Sixma, J. J. J. Lab. Clin. Med., 102: 522, 1983. It is further 
demonstrated that it is not so much the rate of the flowing blood as the 
shear rate at the blood vessel wall or flow channel wall which is 
important for the deposits of blood platelets and activation of 
coagulation (Sakariassen, K. S., Joss, R., Muggli, R., Kuhn, H., Tschopp, 
T. B., Sage, H., Baumgartner, H. R. Arteriosclerosis, 10: 276-284, 1990; 
Sakariassen, K. S., Weiss, H., Baumgartner, H. R. Thromb. Haemostas, 65: 
596-600, 1991). Shear rates (.gamma.) in round channels are expressed by 
formula IV; 
##EQU4## 
where Q=blood flow in ml/sec 
r=radius in cm. 
In flow channels with a rectangular cross section the shear rate (.gamma.) 
at the wall is expressed by formula V; 
##EQU5## 
where Q=as above 
a=width of flow channel in cm 
b=height of flow channel in cm. 
There are several known types of perfusion chambers, used to study 
characteristic features of thrombi formed in flowing blood, where the 
shear rate can be varied and a procoagulating surface can be introduced 
into the flow (Baumgartner, H. R. Thromb. Haemostas. 37: 1, 1977; 
Sakariassen, K. S., Aarts, P. A. M. M., de Groot, P. G., Houdijk, W. P. 
M., Sixma, J. J. J. Lab. Clin. Med. 102: 522, 1983). In Norwegian patent 
application no. 92 2247 and international application no. PCT/N092/00117 
there is described a perfusion cheer where the flow channel cross section 
is constricted in order to simulate arteriosclerotic blood vessels and 
where the thrombi formed can be removed for closer inspection. These 
perfusion cheers are designed for studying the characteristic features in 
thrombogenic processes under simulated in vivo conditions, but they are 
not designed for mass study of the blood's tendency to form intravascular 
coagula or thrombi as a function of time. 
SUMMARY OF THE INVENTION 
To enable them to measure this, specialists use tests of bleeding time, by 
making an incision in, e.g., the ear or finger, coagulation time, where 
the time for coagulation is measured in a test tube which is shaken, and 
prothrombin time, where prothrombin is activated in a test tube and the 
time for coagulation is measured. There are, however, no specific tests 
for measuring the blood's tendency to form clots under standardised, 
reproducible circulatory conditions, which simulate the conditions in 
normal and pathological blood vessels, since the above-mentioned tests are 
based on a blood sample which is treated in a test tube. All known tests 
measure individual mechanisms in the complicated thrombogenesis. Thus, it 
is an object of the present invention to provide a device and method for 
measuring the total response in flowing blood. It is a further object to 
use native blood which has not been anti-coagulated. 
These objects are achieved by the present invention characterized by the 
claims presented. 
The present invention comprises an infusion set with a "Butterfly needle" 
where the needle is inserted into a suitable vein, e.g. a person's arm 
vein, and is connected via a mixing device, described in Norwegian patent 
application 92 2247 and PCT/N092/00117, or via an adaptor, with one or 
more parallel channel systems where at least the Walls consist of, or are 
internally coated with material which can activate or promote 
thrombogenesis. These materials may inclulde polymers, such as polyester, 
e.g. Dacron, polytetrafluorethylene (PTFE, Teflon) and Thermanox.RTM. 
plastic, and vessel wall components which promote thrombosis, e.g. 
collagen, fibronectin, von Willebrand factor, tissue factor and 
phospholipids or other thrombosis-promoting molecules. The channels have 
dimensions which give shear rates typical for arteries, veins and 
sclerotic arteries (formula IV), where the fluid pressure is measured by 
means of known per se methods upstream and downstream of the 
thrombogenesis unit or units, and where the blood is sucked through the 
thrombogenesis unit or units by means of a suction pump. The formation of 
clots or thrombi in the channel systems will alter the vessel dimensions 
and thereby affect the pressure drop over the thrombogenesis unit or 
units, the resistance to the flow being altered when the flow is kept 
constant (formulae I, II, III). 
The tendency to blood clot formation is measured as the change in the 
pressure drop (.DELTA.P) as a function of the flow time. 
Further scope of applicability of the present invention will become 
apparent from the detailed description given hereinafter. However, it 
should be understood that the detailed description and specific examples, 
while indicating preferred embodiments of the invention, are given by way 
of illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
A simple design of the device according to the invention, with one 
thrombogenesis unit, is illustrated in FIG. 1. More complicated designs 
are developed by combining this simple design in such a way that the 
system comprises several parallel-connected thrombogenesis units, with 
related pump devices and pressure sensors. The needle 1 in an infusion 
set, e.g. of the "Butterfly" type, is inserted into a suitable vein, e.g. 
a person's arm vein. The needle 1 is successively connected to different 
members beginning with suitable tubes 2 and then an adaptor 3-1. A T-tube 
4 with presure sensor 5-1 is next in line. The pressure sensor 5-1 
measures the fluid pressure before the thrombogenesis unit, e.g. 
thrombosis chamber 6. This chamber 6 has a through-flow 7. A T-tube 4 with 
pressure sensor 5-2 for measuring the fluid pressure after the 
thrombogenesis unit 6 is connected to the downstream side of unit 6. An 
adaptor 3-2 for the syringe 10 is then provided. The syringe 10 is in a 
device 9 which pulls the syringe out, such as a Harvard pump. The pressure 
sensors 5-1, 5-2 are electrically connected 11 with a suitable recording 
device 8, with an electronic device 12 for calculation and storage of the 
result sin the form of a pressure drop as a function of flow time, and a 
device 13 for displaying the results, such as an electronic screen device 
and/or paper printer device. 
The thrombosis chamber 6 can be in the form of a block, e.g. 2 cm long and 
1.2 cm wide and 1 cm thick, made of a synthetic material which 
activates/promotes thrombogenesis, such as polyester fibre, e.g. Dacron, 
or other suitable materials. In the longitudinal direction of the block 
there are drilled out one or more flow channels with a circular cross 
section and with a radius calculated according to which shear rates are 
desirable at the wall in the blood flow concerned. When using formula IV, 
for example, suitable shear rates will be calculated for a specific blood 
flow and the radius of the flow channel. When venous conditions are 
simulated, suitable shear rates are .ltoreq.100 sec.sup.-1, preferably 
20-100 sec.sup.-1, specially preferred 40-60 sec.sup.-1. Under arterial 
conditions the shear rate is between 100 and 1500 sec.sup.-1, preferably 
between 300 and 1000 sec.sup.-1, specially preferred 400-800 sec.sup.-1. 
The shear rate in sclerotic arteries is .gtoreq.1500 sec.sup.-1, 
preferably 1500-40 000 sec.sup.-1, specially preferred 4000-8000 
sec.sup.-1. The flow can vary between 0.1 and 10 ml/min. 
The thrombogenesis unit can also be in the form of a tube, with internal 
radii adapted to the flow in order to obtain the desired shear rates, made 
of a material which activates/promotes thrombogenesis, or where the length 
of the tube is adapted to give adequate sensitivity for recording a 
pressure drop over the thrombosis tube. 
A further design of the thrombogenesis unit comprises a perfusion device 
with a measuring chamber device according to Norwegian patent application 
no. 92 2247 and international patent application PCT/N092/00117, where the 
flow channel, which is drilled longitudinally through a hard plastic 
block, conveys the blood into a measuring chamber with a rectangular flow 
cross section, where the top/bottom is composed of a measuring chamber 
device. This top/bottom is inserted into the flow channel by means of a 
profile in the direction of flow, thus creating a unilateral constriction 
of the flow cross section, and in suitable grooves before, on and after 
the insertion there are fitted cover plates, uncoated or coated with 
biological material or chemical compounds which are suitable for 
activation of thrombogenesis. The bottom/top of this measuring chamber, 
opposite the bottom of the measuring chamber device, can also be inserted 
into the flow channel in order to create a bilateral constriction. The 
advantage of using this device is that the cover plate can be coated with 
any desired material, including native biological material from the person 
undergoing the measurement, and the thrombus created on the cover plate 
may be removed by removing the cover glass and subjected to closer 
inspection. 
According to a design of the device according to the present invention, 
adaptor 3-1 (FIG. 1) can be replaced by a mixing device as described in 
Norwegian patent application no. 92 2247 and international patent 
application PCT/N092/00117, when the effect on the tendency to blood 
clotting of added solutions requires to be studied. This mixing apparatus 
comprises a modified T-tube in which solutions, such as, e.g., 
thrombogenesis modifying medication, may be added through the side tube, 
while the flow channel is tapered slowly after the side tube to 
approximately half the initial diameter, and then abruptly expanded over a 
substantially shorter length to a diameter considerably larger than the 
flow channel's initial diameter, in order thereby create turbulent flow 
and thus cause the solution, added through the side tube, to be 
homogeneously mixed with the bloodstream. 
After a suitable needle has been inserted into a vein in a person or animal 
and connected to the device according to the invention for measuring the 
tendency to blood clotting, the pump 9, 10 is started up, which draws the 
venous blood from the vein, e.g. a person's arm vein, possibly through the 
mixing device or adaptor 3-1 and on through the T-tubes which contain the 
pressure sensors 5-1, 5-2 and the thrombogenesis unit's flow channel 7, 
with a flow which together with the diameter of the flow channel 7 gives 
the desired shear rate at the wall. Thrombosis or blood clots which occur 
in the flow channel 7 in the thrombosis chamber 6 constrict the flow 
channel cross section, increase the resistance (R) and thereby increase 
the pressure drop (.DELTA.P) from sensor 5-1 to sensor 5-2, according to 
formula I. Consequently sensor 5-2 will record lower fluid pressure than 
sensor 5-1, the signals from the pressure sensors are transmitted to the 
recording device 8, the pressure drop as a function of flow time is 
calculated by the electronic data processor 12 and stored, and the result 
is presented on a display device 13, such as an electronic screen device 
and/or a paper printer device. 
The entire measuring procedure can use under 20 ml, preferably 5-10 ml of 
blood, and the measuring can last for less than 15 minutes, preferably 
5-10 minutes. 
The concept of the invention comprises in vitro measuring of the tendency 
to form thrombosis under conditions which simulate in vivo circulatory 
conditions. A more complicated design comprises several parallel-connected 
systems with two or more thrombogenesis units, e.g. three, as illustrated 
in FIG. 2. There the blood's tendency to thrombogenesis as a function of 
flow time can be measured at the same time under conditions which simulate 
arterial (A), venous (V) and sclerotic arterial (SA) conditions. This is 
achieved by the use of three parallel thrombogenesis units, such as 
thrombosis chambers with one or more flow channels, thrombosis tubes 
and/or perfusion devices with measuring chamber devices, connected in 
parallel by replacing adaptor 3-1 with a branch unit. Each thrombogenesis 
unit is equipped with upstream and downstream pressure sensors (PV-1 and 
2, PA-1 and 2, PSA-1 and 2) and a multichannel, e.g. 3-channel recording 
device with corresponding multichannel electronic data processor and 
storage device, and display of all the results on an electronic computer 
screen and/or by means of a paper printer device. The blood is drawn 
through the entire parallel-connected system by means of, e.g. the 
insertion of three volume-divided syringes in the pump device, which 
thereby suck blood through the system with the same flow. This design will 
save time and blood volume, the blood being removed by a single needle 
inserted into a suitable vein. 
FIG. 3 illustrates the results from a possible mass study of the tendency 
to form thrombosis as a function of flow time with standardised shear 
rates in blood from healthy people (N), people with an increased bleeding 
tendency (B) and from people with an increased tendency to form thrombosis 
(T). This kind of figure is called a nomogram and is used in evaluating 
individual measurements performed by the method according to the invention 
and use of the device according to the invention. 
The invention will now be illustrated more clearly by means of an 
embodiment example. 
EXAMPLE 
Venous blood was removed from the arm vein of a healthy person and 
anticoagulated with Na.sub.3 -citrate. The blood was then drawn through a 
thrombogenesis unit in which the walls of the flow channel were made of 
Dacron. The start of the perfusion is illustrated by an arrow in FIG. 4. 
The flow was 1 ml/min. and 0.64 ml/min., and the radius of the flow 
channel was 0.14 mm and the length approximately 2 cm. Thus, the shear 
rate at the wall was (formula IV) .gamma.=4.965.103 sec.sup.-1 when the 
flow was 0.64 ml/min. and .gamma.=7.749.10.sup.3 sec.sup.-1 with a flow of 
1 ml/min. This corresponds to shear rates in sclerotic arteries. 
As illustrated in FIG. 4 the pressure difference (.DELTA.P) increased over 
the thrombogenesis unit to a constant value in the course of approximately 
the first 5 minutes of the perfusion. This increase in pressure difference 
shows that a thrombus has been formed in the thrombogenesis unit's flow 
channel. After a state of equilibrium has been achieved, FIG. 4 further 
illustrates that the drop in pressure increases with the shear rate and 
indicates that a greater part of the flow channel is occluded at higher 
shear rates. 
The device was connected as shown in FIG. 1, with the exception that the 
"Butterfly" needle was removed as venous blood was removed from the vein 
in advance and anticoagulated. 
Further scope of applicability of the present invention will become 
apparent from the detailed description given hereinafter. However, it 
should be understood that the detailed description and specific examples, 
while indicating preferred embodiments of the invention, are given by way 
of illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description.