Apparatus and method for detecting coagulation in blood samples

The method and apparatus are useful for determining a propensity of a blood sample to change from a liquid state to a coagulated state and additionally for measuring the propensity of a coagulated blood sample to lyse. The method includes providing a porous sheet, at least one surface of which contacts an impervious layer; applying the blood sample to an exposed surface of the porous sheet so that the blood sample spreads through a part of the porous sheet; and after the applying of the blood sample to the porous sheet, measuring at least one of a spreading extent and a spreading rate of the blood sample in the porous sheet by measuring either an optical property, an electrical conductivity across the porous sheet, an electrical potential across the porous sheet and an electrical resistance of the porous sheet to determine the propensity of the blood sample to coagulate. The porous sheet can also be impregnated with a clotting agent or a lytic agent to affect the clotting or lysing of the blood sample.

BACKGROUND OF THE INVENTION 
The present invention relates to apparatus and methods for determining the 
susceptibility of certain liquids to coagulate and subsequently to lyse. 
The apparatus and methods of the present invention are particularly 
suitable for assessing blood coagulation status in a patient. These may 
also be adapted to assess fibrinolytic status in a patient. 
A wide variety of laboratory clotting tests are based on the phenomenon of 
measuring as an endpoint, a change of phase when a test solution changes 
from a liquid to a coagulated form. This change is due to the conversion 
of a soluble plasma protein fibrinogen to an insoluble one, fibrin by the 
action of the enzyme thrombin. The reverse change is also made use of in 
tests where one is measuring the lyric activity of a solution, whereby 
insoluble fibrin is broken down to soluble degradation products by the 
enzyme plasmin. 
For example, blood clotting tests are important to assess likelihood of 
bleeding in patients treated with anticoagulants or with haemostatic 
defects. The endpoint of the most frequently performed clotting test, the 
skin bleeding time, is the cessation of blood flow from a standardised 
skin incision. This is usually determined by the periodic blotting of the 
wound site until blood flow from the wound ceases. This test is 
particularly prolonged by platelet defects. Other clotting tests are 
usually carried out by mixing test plasmas with specific reagents and 
timing to an endpoint when the mixture suddenly clots. The clotting 
endpoint is usually determined physically, as in a tilt-tube, or optically 
by increased turbidity, as in a photoelectric clotting machine. 
This current innovation has been stimulated by a number of precursore 
developments. The first of these is the use of convenient dry cards such 
as the Nyco Card (Nyco Med Pharma) for rapidly testing antibody reactions 
(e.g. FDP, D-dimer). These devices comprise porous modules which are faced 
with an immunoreactive membrane. Test samples are applied to such surfaces 
and are drawn through by capillary action. Antigens or antibodies for test 
are quantitated by a color reaction after sequential washing and 
incubation steps. Secondly, small capillary coagulation testing devices 
have also recently been developed. These are based on blood or plasma 
drawn into a small capillary with coagulation detected using either a 
laser (Bio Track, Ciba-Corning) or pressure sensing systems (Nyco Med). 
One such device features dry coagulation reagents while the other relies 
on uncoated glass capillaries. 
Unfortunately, these prior art devices are quite complex requiring laser or 
pressure sensing equipment. 
It will be clear to persons skilled in the art that there is a need for 
simpler, more convenient and adaptable methods and apparatus for measuring 
the susceptability of liquids to coagulate or lyse over currently existing 
methods and apparatus. It is desirable to provide small testing modules 
which may be used at the bedside in a portable form or alternatively can 
be assembled together to process much larger numbers of samples in a 
central laboratory. 
Currently there is a significant market for rapid portable methods in the 
coagulation/lysis testing area. Larger laboratories usually purchase 
expensive sophisticated equipment for massive scale operation. Small 
inexpensive bedside testing units have an important role in a number of 
clinical situations especially where prompt interpretation of patient 
results is required and when screening tests are only rarely positive. 
SUMMARY OF THE INVENTION 
In order to ameliorate the disadvantages of the prior art it is proposed to 
provide a method and apparatus for determining the propensity of a test 
sample to change between a coagulated state and a liquid state which 
offers a choice over the prior art and which, at least in the preferred 
embodiments, is cheaper and easier to manufacture and use as compared to 
prior art devices. 
In a first aspect, the present invention provides a method for detecting 
the propensity of a blood sample to change from a liquid state to 
coagulated state comprising the steps of; 
(i) providing a porous sheet, at least one surface of the porous sheet 
being in contact with an impervious layer, 
(ii) applying the blood sample to an exposed surface of the sheet, 
(iii) allowing the sample to spread through a part of the sheet, and 
(iv) measuring a parameter indicative of the extent of spread of the sample 
in the sheet and/or the rate of spread of the sample in the sheet which is 
indicative of the propensity of the blood sample to coagulate. 
In a second aspect, the present invention provides an apparatus for 
detecting the propensity of a blood sample to change between a liquid 
state and a coagulated state comprising a porous sheet, an impervious 
layer in contact with one surface of the sheet and a coagulation/lysis 
detection means, the detection means being adapted to measure a parameter 
indicative of the extent of spread of the sample in the sheet and the rate 
of spread of the sample in the sheet. 
The present inventive method and apparatus operate as follows. As the blood 
sample is applied to the porous sheet, it disperses out from the 
application point across the area of the sheet. As this liquid disperses, 
the leading edge of the liquid continually comes into contact with the 
clotting agent. One or more clotting agents can be present in the porous 
phase in discrete zones allowing sequential mixing of test sample liquids 
with them. Eventually, this leading edge begins to coagulate reducing 
further spreading of the sample through the porous sheet. When the leading 
edge of the sample has fully coagulated, the rate of further spread of the 
sample in the porous sheet is virtually zero and the area of the spread 
sample is constant. It will be clear to persons skilled in the art, 
therefore, that by measuring a parameter indicative of the extent of 
spread of the sample in the porous sheet and/or the rate of spread of the 
sample in the porous sheet, we may obtain an accurate measurement of when 
coagulation has occurred. 
In one embodiment of the present invention, the parameter measured is the 
area covered by the spread sample or the rate at which this area grows. 
This parameter may be measured by any appropriate optical means even the 
human eye especially if whole blood samples are applied. If one uses such 
an optical measurement means with plasma samples it is preferred to 
include a dye in the test sample to assist visual detection or to 
impregnate a part of the porous sheet with a dye. 
Using such an optical measurement means, one may apply the test sample to 
the porous sheet and measure the extent of the area covered by the spread 
sample prior to coagulation giving a measurement of when coagulation 
occurred. Alternatively, an operator may monitor the rate of growth of the 
area of the spread sample. Graduations may be incorporated on at least one 
of the impervious layers of the inventive apparatus to assist in measuring 
the extent of sample spread. When the rate of growth of the area 
approaches zero, coagulation has occurred. 
In another embodiment of the present invention, the appropriate parameter 
for detecting coagulation may be measured by an electrical sensing means. 
For example, thin electrodes may be provided on either surface of the 
porous sheet. The conductivity or electrical impedance between these 
electrodes depends upon the wetted area between them. Prior to application 
of the test sample to the porous sheet, the impedance between the 
electrodes will approach infinity with conductivity at zero. As the test 
sample spreads through the sheet, it increases the wetted area between the 
electrodes leading to a decrease in impedance and increase in 
conductivity. As the test sample further spreads out through the sheet 
impedance will further reduce and conductivity will be further increased. 
It can therefore be seen that the impedance/conductivity between the 
electrodes is a measure of the extent of spread of the sample in the 
sheet. 
By examining the rate of change of the conductivity/impedance between the 
electrodes one may measure the rate of spread of the sample in the medium. 
As discussed above, when the leading edge of the sample coagulates further 
spread of the sample through the porous sheet is greatly reduced depending 
on the amount and permeability of the fibrin formed therein. Accordingly, 
when the leading edge of a normal sample coagulates there is virtually no 
increase in conductivity or corresponding decrease in impedance with time. 
By monitoring the change in conductivity/impedance between the electrodes 
one may detect when coagulation occurs since at this point the rate of 
change of conductivity/impedance will approach zero. 
If the fibrin formed in the sheet by clotting reactions is subsequently 
lysed by the fibrinolytic plasmin then further penetration of the liquid 
sample into the porous sheet will take place. Thus the time at which 
conductivity resumes its increase can be used to indicate fibrinolytic 
susceptability within a sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Turning firstly to FIG. 1, the inventive apparatus comprises a porous sheet 
1 sandwiched between surface electrodes 2 and 3 across which a small 
voltage is applied. A test sample 4 of a liquid may be applied to the top 
surface 6 of electrode 2 to be drawn down through aperture 5 and spread 
through sheet 1. 
Prior to application of the test sample, the resistance between surface 
electrodes 2 and 3 will approach infinity with conductivity at zero. If it 
is necessary to measure the time for coagulation of the test sample, 
application of the test sample 4 will automatically reduce the resistance 
between electrodes 2, 3 thereby indicating a "start" time on recorder 
device 8. As the test sample is drawn down and through sheet 1, it wets 
the area between electrodes 2 and 3 reducing resistivity and increasing 
conductivity. 
The test sample 4 will spread through sheet 1 as discussed above covering 
an ever increasing area. As the sample spreads its leading edge 
progressively mixes with the clotting agent impregnated through the porous 
sheet 1. Eventually the leading edge of the sample will coagulate and 
clots 7 will be formed. 
As will be clear to persons skilled in the art, the speed at which the 
sample coagulates is dependent on the properties of the sample itself as 
well as on the type and quantity of clotting agent or lytic agent in the 
sheet of absorbent medium. Subsequent lysis of the clot resulting in 
further dispersion of liquid into the sheet depends on the presence of 
fibrinolytic activator and its specific effect. 
Chart recorder 8 connected to electrodes 2 and 3 via leads 9 and 10 records 
the conductivity/resistivity between the electrodes for example as shown 
in FIG. 5. As discussed above, once clots 7 are formed the rate at which 
the sample 4 spreads is reduced leading to a corresponding reduction in 
the rate of increase of the conductivity between the electrodes. By 
monitoring the conductivity/resistivity or the change in 
conductivity/resistivity between the electrodes as a function of time an 
accurate indication may be obtained of when the sample coagulates. 
In preliminary studies, the porous sheet used comprised a thin filter paper 
which served to absorb and diffuse the plasma used in the test. It is 
preferred that the sheet 1 comprise a thin sheet so that even a small 0.05 
ml sample spreads into a large area. The material of the membrane should 
not absorb water from the blood or plasma samples as this leads to an 
unpredictable spreading pattern. Preferably, the sheet 1 includes small 
even pores so that spreading of the absorbed test sample is regular and 
susceptible to occlusion by fibrin and/or platelet aggregates. The sheet 
preferably contains dry stabilized reagent to clot the sample so that the 
fibrin formed and/or platelet aggregate significantly retard the 
subsequent spreading of the sample. 
Preferably clotting reagents which may be included within this medium sheet 
1 include tissue factor (thromboplastin), contact activator and 
phospholipid for use in a APTT-like test and various other coagulation 
activators including Russell's viper venom, contact product (activated 
factor XI) and platelet activating agents. Thrombin and activated factor X 
may be also applied with stabilizers to such papers for tests to be used 
for monitoring antithrombotic drugs such as low molecular weight heparin. 
Such tests yield prolonged clotting times relative to normal plasma and 
may be used to assess anticoagulant status. Fibrinogen levels may be 
interpreted from the rate of change of conductance after clotting by a 
reference thrombin like enzyme concentration has occurred. High fibrin 
content results in less rapid capillary flow and less change over time. 
Low fibrinogen levels yield more porous clot allowing more permeability 
through the clot. 
Specific assays for factor VIII are based on haemophilic (factor VIII 
deficient) plasma dried in a zone separate from an APTT reagent dried on 
the membrane so that it is dissolved by diluted test plasma first. Assays 
for other clotting factors including platelets may be developed similarly 
using specific deficient plasmas. The other component which may be 
included in medium 1 is calcium which will be applied as non-deliquescent 
salts in a zone furthest from the application point. The concentration of 
this will be adjusted to be about 0.025M and of the other reagents will be 
optimal for test sensitivity with zone spreading achieved in 30-60 sec 
from the time of sample application. 
Buffer additives and salts are required to yield physical conditions of 
high pH (in the range of 7.5-8.5) and ionic strength (in the range of 
0.1-0.2M [NaCl] under which fibrin gels are least permeable. 
Other main components which may be included in the medium are surfactants 
and hydrophilic polymers. Surfactants control the surface tension at the 
liquid interface and this is important in regulating the rate of 
spreading. Hydrophilic polymers in appropriate concentrations are required 
to control the rate of dissolution of dry components and the speed of 
transport of liquid through the porous medium. Gelatin coating of the 
porous phase enhances the adhesion of the fibrin clot. Specific polymers 
such as gum arabic also enhance the restrictive effects of fibrin (when it 
forms) on the rate of liquid transport. Typical hydrophilic colloids of 
use here include polyvinyl alcohol and sodium alginate at 1% 
concentration. Fibrinogen, bovine and/or modified covalently with polymers 
may be added to enhance fibrin gel strength in some tests. The 
concentrations of such polymers need to be controlled to avoid irregular 
spreading of liquid through the membrane due to high viscosity at the 
leading edge. Other dried components include well known protein 
stabilizers, lactose, antioxidants and sodium azide. 
In some circumstances two incompatible components (for example specific 
factor deficient plasmas and calcium) for a clotting test system may be 
introduced into a test module in two separate zones or on two separate 
over-lapping sheets. Thus different optimal conditions for each component 
can be maintained during storage and they will mix together only when 
wetted by test sample. For fibrinolytic studies, streptokinase, urokinase 
or tissue plasminogen activator will be included in the clotting reagent 
mixture. After clotting has occurred a subsequent increase in conductivity 
or decrease in impedance indicates the time required for fibrinolysis in 
individuals. This is often prolonged in patients resistant to 
fibrinolysis, for example those having antibodies against streptokinase. 
The present inventive method and device may also be applied to a skin 
bleeding time procedure in which blood issuing from a standardized wound 
is progressively quantitated by the increasing conductivity between a 
surface electrode and underlying skin. 
As shown in FIG. 4 the porous sheet 12 may be placed directly on the wound 
of a patient. This embodiment is particularly suitable for obtaining a 
skin bleeding test from a patient. In this embodiment, only one surface of 
the sheet 12 is covered by an impervious layer so that the blood emanating 
from the wound of the patient may be directly applied to the exposed side 
of sheet 12. 
In FIG. 4, the impervious layer on the exterior side of sheet 12 is 
provided by surface electrode 13. The combined sheet/electrode assembly is 
placed directly against the arm 14 of a patient. To obtain a sample of 
blood from the patient, a lancet 15 is forced through top layer 13 and 
porous sheet 12 into the arm 14 of the patient. 
The lancet has a standardised cutting surface to produce a standard size 
wound 17. As blood issues from the wound, it is drawn into and dispersed 
in sheet 12 as discussed above. Bottom electrode 18 is brought into 
contact with the skin of the-patient well removed from wound site 17. It 
is preferred that no coagulation influencing agent is included in sheet 12 
so as not to interfere with the patient's normal blood clotting. FIG. 4 
shows a typical arm incision. However, it may be preferable to apply such 
an improved device to a site on the lower leg to simplify the need for 
increased venous blood pressure. 
Skin bleeding time tests of this type are usually intended to measure 
procoagulant mechanism involving blood platelets, collagen and tissue 
factor released at the wound site. To achieve better specificity for 
platelet function, weak anticoagulants may be used in the absorbent medium 
sheet 12 so that platelet agglutination becomes relatively more important. 
Additionally platelet agonists such as ADP or collagen may be induced in 
the membrane. 
If it is desired to measure the coagulation properties of blood from a 
patient, it is preferred that the assembly is heated so the sample remains 
at a constant temperature, preferably 37.degree. C. i.e. the temperature 
of the human body. This may be accomplished by simply holding the 
inventive apparatus against the skin of the patient or alternatively a 
separate heating/cooling means may be provided. 
As discussed above, as the sample of blood from the patient spreads through 
porous medium 12 it may be monitored with chart recorder 8 set to measure 
electrical resistivity or conductivity between electrodes 18 and 13. 
It is preferred that very low voltage levels are used in the inventive 
device e.g. less than one volt, in order to avoid polarization at the 
electrode-liquid interface. The applied voltages may be constant or 
preferably pulsed or alternating. If pulsed voltages are used the 
responses may be analysed by an oscilloscope for additional impedance 
changes to confirm clotting. 
As shown in FIG. 3, individual modules comprising the absorbent medium 1 
with electrodes 2, 3 on either surface may be clamped to a flat 
thermostatted surface 11 to maintain the assembly and sample at. 
37.degree. C. This allows several modules to be used at the same time, 
each module being connected to chart recorder 8. Different modules may 
include a porous sheet 1 which has been treated with a different 
coagulation influencing agent. Application of identical samples to each of 
these modules allows an operator to ascertain the effectiveness of each 
coagulation influencing agent on identical liquid samples. 
In another embodiment electrodes 3 may be deleted with plate 11 acting as a 
common negative backing plate for several testing modules assembled 
thereon. 
It is also within the scope of the present invention to incorporate 
insulating films on the surface electrodes. These insulating portions may 
be oriented at particular positions on the surface electrodes in order to 
measure the extent of spread of the sample. To explain, as the sample 
spreads in the porous sheet it increases the conductivity and decreases 
the resistivity between the electrodes. When the spreading liquid reaches 
an insulating film there will be a temporary cessation of charge in 
conductivity indicating the position of the sample spread in the sheet. 
In another embodiment of the present invention if simple voltage analysis 
is preferred over conductivity/resistivity measurement, the surface 
electrodes may be made from non-inert dissimilar materials, for example 
aluminium and copper. Contact with the test sample, therefore will 
generate a small temporary voltage difference. This electric potential can 
directly be used to monitor the spread of sample liquid in the porous 
sheet. 
The electrical conductivity/resistivity measurements may be accumulated by 
a computer interface with subsequent analysis and/or conversion to 
conventional units e.g. seconds or ratios relative to normal through 
simple programming methods. It will be clear to persons skilled in the art 
that monitoring the resistivity or conductivity between electrodes over 
time will yield a more precise indication of clotting times than possible 
via subjective visual or optical analysis. 
As an example, the relationship between electrical resistance and amount of 
test sample within a 0.2 mm absorption medium sheet is shown in Table 1. 
TABLE 1 
______________________________________ 
Volume Plasma Absorbed 
Resistance .OMEGA. 
______________________________________ 
O.mu.l .infin. 
5 40 
10 25 
20 16 
50 12 
______________________________________ 
An alternative embodiment of the present invention is shown in FIG. 2 which 
includes an impervious transparent top sheet 2 allowing optical measuring 
of the extent of spread of the sample in the sheet and/or the rate of 
spread of the sample in the sheet. This embodiment may include one 
impervious layer, with the exposed side of the porous sheet being directly 
applied to a patient similar to the embodiment of FIG. 4. 
Alternatively, the porous medium 1 may be sandwiched between impervious 
layers one of which is transparent. 
This embodiment allows a person to simply view the extent of spread of the 
sample in the sheet and or the rate of spread of sample in the sheet. 
Graduations or reference points may be included on transparent top sheet 2 
to assist in optical measuring of the sample spread. It is also possible 
to view optical changes with thin electrode wires overlying each 
clot-sensing zone in strips of porous medium. 
In another embodiment of the present invention, as shown in FIG. 6, the 
porous sheet 1 is provided with a plurality of identical arms 25 radiating 
from a central sample application point 24. Once again, at least one 
surface of the sheet 1 is underlayed by an impervious conducting layer 26. 
Optional impervious layer 27 may also be included to contain discrete thin 
wire electrodes 28 overlying each clot-sensing zone leading to recorder 
connection 29. 
As previously discussed, when the sample is applied to the porous sheet it 
disperses throughout the sheet, in this case along each of the arms 25. 
Each arm preferably includes a different clotting/lysis agent with one arm 
acting as a control and having no clotting/lysis agents. 
With each arm 25 being impregnated with a different clotting/lysis agent, 
coagulation or lysis takes place at a different point along each arm 25. 
By monitoring the extent of spread of a sample in each arm and/or the rate 
of spread of the sample in each arm, the effectiveness of the various 
clotting or lysis agents may be determined. 
Once again the measurement of the extent of spread of the sample or rate of 
spread may be determined visually or electrically as discussed above. 
It will be clear to persons skilled in the art that the present inventive 
method and apparatus may equally be used to measure the propensity of a 
liquid to change from a coagulated state to a liquid state. As discussed 
briefly above, after clotting of the test sample has occurred the clot may 
become subject to lysis by a lytic agent present already in the clotting 
agent which returns the sample to a liquid state. Indeed, where the porous 
sheet is bounded on both surfaces by impervious layers, the clot formed 
may return to a liquid state over time without the addition of a lytic 
agent. 
It can therefore be seen that after clotting has occurred, one may 
determine the susceptability of a liquid to change from a coagulated state 
to a liquid state by measuring a parameter indicative of the speed of 
lysis. 
To explain, once the test sample changes from its coagulated state to its 
liquid state, the sample will begin to spread once more leading to an 
increase in the extent of spread of the sample and/or the rate of spread 
of the sample in the sheet. This further spreading of the sample may be 
noticed visually as discussed above or measured electrically since the 
further spreading of the sample will increase conductivity or decrease 
resistivity between the surface electrodes. 
The use of the porous sheet allows the inventive apparatus and method to be 
adapted to test for a large variety of individual clotting/lysis factors. 
A complete coagulation/lysis profile of an individual may be achieved 
quickly and cheaply by combining a panel of inventive test modules. 
The present invention allows an individual to purchase a number of modules 
bounded by one or two impervious sheets or surface electrodes. This person 
may then apply the test sample, e.g. blood to the porous sheet by direct 
contact with the skin or via an appropriate sampling means. 
Coagulation/lysis of the sample may be detected by measurement of an 
appropriate parameter indicative of the extent of spread of the sample in 
the sheet and/or the rate of spread of sample in the sheet. In the 
embodiments shown, visual detection or electrical measurement of the area 
of the spread sample is used however any other parameter is equally 
applicable. 
It is believed that the present invention will excel not only in the 
medical but also veterinary fields where simple inexpensive bedside 
testing units have an important role particularly where prompt 
interpretation and diagnosis of the patient is required.