Rod and cup assembly for measuring viscoelastic properties of blood
The present invention relates to a rod and cup assembly, particularly for use in an apparatus for measuring viscoelastic properties of blood. Background Viscoelastic analysis of clotted whole blood, i.e. blood with all types of blood cells present that has been allowed to coagulate, is a difficult task. The problems arise from the fact that the blood cells, particularly the thrombocytes, will activate their cytoskeleton and, unless restricted, reduce the volume of the coagulum. This phenomenon, known as clot- retraction, will tend to detach the coagulum from the surfaces of the rheometer introducing detrimental artifacts into the analysis. The phenomenon even compromises the rational procedure to use rheological methods to determine the coagulation time, an important measure in laboratory medicine. The above has lead to over-utilization of less rational optical procedures, including the favoring of analysis of plasma, as plasma in contrast to blood, is infinitely better as light conductor. Viscoelastic analysis of blood coagulum is intuitively believed to supply much valuable clinical information but has not attained the position it deserves much because of the tensions that are built up by the contracting thrombocytes (an interesting phenomenon in its own right with the proposed function of proximating the edges of a wound and facilitating proper healing) which dislodge the coagulum from the surfaces of the rheometers with which the analysis is to be performed. There are several candidate techniques with which the changes in viscoelastic properties could be measured as the blood coagulates and the rigidity of the coagulum develops. Most of these techniques will encompass at least two surfaces which move relative to each other and with which forces exerted by the coagulum, a reaction to the distortion of coagulum shape induced by the movement, is measured. Clot retraction and detachment of the coagulum has been a powerful obstacle for development in this promising direction. What is needed to advance viscoelastic analysis in laboratory medicine is a means of preventing the coagulum from detaching from the surfaces in a rheometer that move relative to each other. One promising method to determine viscoelastic properties in liquids and gels is free oscillation rheometry (FOR). It may be selected in advance of other techniques because it requires small sample volumes, uses low-cost disposables, determines viscosity of the magnitude displayed by water and can deliver correct viscoelastic information for biological gels, e.g. jams, creams, cheesed milk and plasma coagulum. Viscoelastic analysis of blood coagulum by FOR had been straightforward if only the attachment problem was solved.
In FOR the sample is set into free oscillation and the damping and frequency of the oscillation are measured. In its advanced form, which includes precise determinations of viscoelastic properties, the method was invented by Leif Bohlin ( Bohlin, L.Method for measuring rheological properties and rheometer for carrying out the method. WO 94/08222. 1994 14.04.) A convenient way to practice this mvention is a torsion pendulum where the sample is set into oscillation around its axis. The frequency shift and damping are influenced by the rheological properties of the sample and are recorded as a function of time by repeated initiations of the oscillation. FOR is particularly good at measuring viscosity in sample volumes of less than 1 mL, and in water-like fluids (Bohlin, ibid.). As mentioned, FOR is well suited for determinations of viscoelastic properties. Most conveniently this is accomplished with a bob (a device placed in the central part of the sample defining it as stationary) and at conditions under which the oscillation propagates throughout the gap between the cup and the bob.
FOR has been shown to perform well for the monitoring of blood plasma coagulation ( Ranby, M., Gustafsson, K. M. and Lindahl, T. L., 1999. Laboratory diagnosis of thrombophilia by endothelial cell modulated coagulation. Blood Coagul Fibrinolysis 10, 173- 179.) and is particularly suitable for studies on coagulation of whole blood, since the method is not restricted by sample turbidity and allows measurements in the presence of blood cells (Ramstrδm, S., Ranby, M. and Lindahl, T., 1999. Iso-citrate is a more gentle anticoagulant than citrate. Thromb Haemost 82 suppl, 292-293.)
However, due to experimental difficulties, the viscoelastic properties of blood coagulum have defied FOR studies. During coagulation, platelets retract the coagulum from the surfaces in the sample cup, limiting the real time for meaningful measurement of the viscoelastic properties of blood coagulum that is formed. Description of the invention
The present invention enables measurement of viscoelastic properties of blood coagulum in an apparatus comprising a rod and a cup that move relative to each other, for a considerably longer time, until the full rigidity of the blood coagulum has developed, than was previously possible. It has surprisingly been found that the material coming into contact with the blood coagulum during measurement of its viscoelastic properties is of crucial importance for the result that is obtained.
Thus, one aspect of the invention relates to a rod and cup assembly for use in an apparatus for measuring viscoelastic properties of blood, especially blood coagulum, which
apparatus comprises a rod and a cup that move relative to each other, wherein the rod surface and/or the cup surface which will come into contact with blood are of a noble metal or noble metals.
The term "rod" is used to cover any suitable device that is used for immersion into a liquid sample placed in a cup in an apparatus for viscoelastic measurements, and the rod is e.g. in the form of a stick or a bob on a bob-shaft.
The term "cup" is used to cover any suitable vessel that is used for a liquid sample in an apparatus for viscoelastic measurements.
In an embodiment of the invention the noble metal is selected from the group consisting of gold, silver, palladium, ruthenium, iridium, titan and platinum.
It is conceived, as a working hypothesis, that any metal that allows protein components of the blood direct access to, i.e. to come in direct contact with, the delocalised •electrons of the metal, or other electrically conducting material, will allow use of the present invention. Therefore, in the spirit of the invention, noble metals are; all metals or other electrically conducting material that either lack a surface film of non-conducting matter, e.g. a film of oxides of sulphides, or which have such a film but of such making that it is dislodged in the presence of blood, e.g. pushed aside by the plasma protein, giving the plasma protein direct contact with delocalised electrons.
In a presently most preferred embodiment of the invention the noble metal is gold. In a specific example of the invention the noble metal surface is a coating applied by physical vapour deposition onto a core material or materials of which the rod and/or the cup is (are) made.
In a further embodiment of the invention the core of the cup is made of a polymeric material. Injection moulding is a suitable method for manufacturing of cups and rods, using any suitable plastic material that can be injection moulded. However, any other forming method may as well be utilized, e.g. turning. The rods and cups may also be made of thermosetting plastics, rubber or metal.
In a preferred embodiment of the invention the polymeric material is selected from the group consisting of polyamides, polycarbonates, polystyrenes, polyacrylonitriles, polyacrylates and polyesters. In addition to these plastic materials, the rod and the cup may be made of polyolefins, POM, SAN and other suitable thermoplastics.
In yet another embodiment of the invention the relative movement between the cup and the rod are either oscillating around a common axis or lateral along the same axis.
In a preferred embodiment of the invention the apparatus for measuring viscoelastic properties is selected from the group consisting of rheometers or elastometers.
Another aspect of the invention relates to a rod coated with a noble metal for use in an apparatus for measuring viscoelastic properties of blood, which apparatus comprises a rod and a cup that move relative to each other.
Yet another aspect of the invention relates to a cup having at least the inner surface coated with a noble metal, for use in an apparatus for measuring viscoelastic properties of blood, which apparatus comprises a rod and a cup that move relative to each other.
The invention will now be illustrated by description of experiments and drawings, but it should be understood that the scope of the invention is not limited to the disclosed specific embodiments. Description of the drawings
Figure 1 :1 shows a rod and cup assembly 1 according to the present invention, wherein the rod is in the form of a stick 2, which is immersed in a blood sample 4 in a cup 6. Figure 1:2 shows a rod and cup assembly 1 according to the present invention, wherein the rod is in the form of a bob 8 and a bob-shaft 10, which is immersed in a blood sample 4 in a cup 6.
Figures 2:1-2:4 show the analytical results of Example 1 (frequency and damping as a function of time for different combinations of gold-coating surfaces on the bob and the rod). Figure 2:1 shows the results for PA (polyamide-6) bob and gold-coated cup.
Figure 2:2 shows the results for PA bob and PA cup. Figure 2:3 shows the results for gold-coated bob and gold-coated cup. Figure 2:4 shows the results for gold-coated bob and PA cup.
Figure 3 shows a the analytical results of Example 2, where curve A represents the combination of a gold-coated rod and a gold-coated cup, curve B represents the combination of a PA rod and a gold-coated cup and curve C represents the combination of a PA rod and a PA cup.
Figure 4 shows the analytical results of Example 3, where the curve A represents a cup of polypropylene, curve B represents a cup of polycarbonate, curve C represents a cup of polyester and curve D represents a cup of a copolymer of styrene and acetonitrile. Description of experiments
Materials and Methods
Blood was collected from the cubital vein of a healthy volunteer into an evacuated plastic container (S-Monovette neutral, Sarstedt, Nurnbrecht, Germany). A 21 gauge
hypodermic needle was used and the draw into each container was 4.5 mL. There was no anticoagulant added and the analysis was commenced within 5 minutes of blood collection. The temperature of the blood was maintained at about 37°C. The volunteer, a member of the laboratory staff, was informed of the nature of the study. The study was approved by the local ethical committee of the University Hospital of Linkδping, Sweden.
Thromboplastin from acetone-dried rabbit brain was obtained from Global Hemostasis Institute MGR AB (GHI), Linkδping, Sweden, product GHI 113, which is derived by physiological saline extraction and washing. The particle size is in the range of 0.1 to 1 μm. The concentration, corresponding to the 0.3 g of dried brain powder per milliliter, was about 3000 arbitrary units (AU) per milliliter, by definition that 1 AU/mL induces coagulation in plasma in 2 minutes under standardized conditions. To obtain thromboplastin solutions of desired potency, the product GHI 113 was diluted in 150 mM NaCl containing 4 mM of sodium azide as anti-microbial agent.
Analysis was performed by opening the collection container and withdrawing 1 mL of blood with a disposable syringe. The blood was mixed with 10 μL of 10 AU/mL of thromboplastin in a 5 mL polystyrene tube and again drawn into the 1 mL disposable syringe. The mixture of blood and thromboplastin was injected into the sample cup of a ReoRox ® 4 sample free oscillation rheometer, Global Hemostasis Institute MGR AB, Linkδping, Sweden. The cup was set into oscillation every 2.5 seconds around its vertical axis, and the damping and the frequency of the oscillation were recorded. The frequency of the oscillation was about 10 Hz and the amplitude about 0.04 radians (2.3 degrees). The first data was obtained in about 20 seconds of the mixing which in turn was within 5 minutes of drawing the blood. First signs of blood coagulation, an increase in the damping, significative of increase in blood viscosity, could be observed within 25 seconds and clotting had occurred some 25 seconds later. In the absence of thromboplastin, blood coagulation occurred in about 30 minutes. As described by Bohlin (ibid.), the visco-elasticity of blood prior to clotting can be estimated under the assumption of surface loading, i.e. the penetration depth of the oscillation wave into the blood is small compared to the 3 mm gap between the cup and the bob, and can be estimated after clotting under the assumption of gap loading, i.e. the oscillation wave propagates throughout the gap. Naturally, conditions for gap loading and other reasonable assumptions for rheological studies of a blood coagulum, requires that the coagulum is in contact with the rheometer surfaces. In an analysis, not troubled by detachment and ruptures, the dynamic viscosity (loss modulus, G") and the storage module (G') can be estimated by
the following formulae: G',:=kι(d-do) and G'=k2(ω2-ω0 2) where ki and k2 are constants to be determined by calibration and d and d0 are the logarithmic damping with and without sample and ω and ω0 are the respective angular velocities (2π times the frequency). In the present examples the appropriate calibrations were not performed, but irrespectively of this, the formulae given by Bohlin gives a notion of how changes in dynamic viscosity and the elasticity are reflected in changes in damping and frequency.
In other experiments, blood coagulation was initiated with the snake venom Reptilase® , Pentapharm AG, Basel, Switzerland, lot 1742/108. Prior to filling the rheometer cups, 1 mL of blood was mixed with 20 μL of Reptilase® which induced coagulation in about 3 minutes. One experiment was formed with plastic cups molded from various plastic materials, polypropylen (PP), polycarbonate (PC), polyester (PE) and co-polymer of styrene and acrylonitrile (SAN). In all cases the bob was coated with gold.
In all experiments, the rheometer cup was cylindrical in shape with an inner diameter of 12 mm and 18 mm perpendicular walls. The bob was 6 mm in diameter and 8 mm in height. When filled with 1 mL of blood, the bob was totally submerged in blood with only the shaft of the bob protruding through the blood surface which was located about 3 mm below the rim of the cup. A schematic drawing of the cup with the bob is given in Figure 1 :2. The cup and the bob were of polyamide-6. The cup was injection molded, product GHI 206, and the bob lathed from a rod shaped blank. The bob holder was lathed from a polyoxy- methyl blank. In some experiments the surfaces in contact with the blood of either the cup or the bob or both were coated with gold. In others they were naked plastic. The naked cups were as they appeared from the mold, used without washing. Prior to use, naked bobs were soaked in 50 mM NaOH for a few minutes, rinsed extensively in deionized water and allowed to dry. The gold coating was by physical vapor deposition, also known as sputtering, as performed by the Impact Coating AB, Linkδping, Sweden. In short, cups and bobs were placed in an evacuated chamber on a slowly rotating drum about 200 mm away from a bar shaped piece of gold. At a pressure of about 10"6 torr, argon to a partial pressure of 1 to 10 fold 10" torr was admitted into the chamber and exposed to an electrical field of about 400 V with the gold as cathode causing, first spontaneously occurring and at steady state β-radiation induced, argon ions to hit the gold surface expelling gold atoms and electrons. The gold atoms were allowed to diffuse across the 200 mm gap to hit the plastic pieces at thermal velocities and coat these. Excess electrons were lured away from the area of interest by a strong
magnetic field (a magnetron). At the completion of the process, the layer of gold covering the inside of the cups and the outside of the bobs was estimated to be about 100 nm thick.
Sodium chloride, sodium azide and deionized water were all of high analytical grade. Example 1. Four experiments were performed as described under Materials and Methods using blood from one blood sampling. The experiments were performed in the four analysis channels of a ReoRox® 4 rheometer. In channels 1, 2, 3 and 4 bobs with a surface of PA (polyamide),PA, gold and gold, respectively, were used together with cups with surfaces of gold, PA, gold and PA, respectively. The analytical results are shown in Figures 2:1, 2:2, 2:3 and 2:4.
The experiment depicted in Figure 2:3, performed with gold coated bob and cup, displayed a smooth increase in frequency once the coagulum had formed, i.e. after five minutes. The frequency increases from about 10.5 Hz to reach a maximum level of 12.5 Hz at about 30 minutes. From the maximum the frequency decreases slowly to reach 12.2 Hz at about 60 minutes. The progression of the curve was at all times smooth and at the end of the experiment, it could be verified by inspection, the coagulum was firmly attached to the rheometer surfaces.
The experiment performed with a gold coated bob and a naked PA cup, Figure 2:4, showed a similar pattern as in the experiment above where all surfaces in contact with the blood were gold coated. The coagulum appeared sufficiently well attach, also to the PA of the cup, to allow viscoelastic analysis of the blood coagulum Inspection of the assembly at the end of the run indicated attachment, but not as strong as after the run made with gold on both bob and cup.
In the two experiments performed with bob of naked PA, Figures 2:1 and 2:2, a totally different picture is displayed. During the time period in which the blood coagulates, up to 1 minute and for some two minutes afterwards, the tracings are close to identical with those obtained with gold coated bob or with gold coated bob and cup. At 1 minute, the frequency, which then has reached about 11 Hz levels off abruptly and turns rapidly downwards. This is interpreted as a detachment of the coagulum from the rheometer surfaces. Once detached, meaningful interpretation of the rheometer tracings cannot be made. There can be seen attempts to interpret the patterns, or signatures as they can be called, from analysis where the blood coagulum is detached or semi-detached from the surfaces of rheometers but such attempts are launches of imagination that leave classical rheology behind.
Example 2.
Blood was mixed with the procoagulant snake venom bathroxobin, commercialized as Reptilase®, and injected into three of the measuring channels of a free oscillation rheometer, ReoRox®4, A, B and C. The channels were fitted with various rod and cup assemblies made of plastic. Two of these, A and B, were according to the invention. In A, all surfaces in contact with the blood were coated with gold. In B, only the cup was coated. In C, all surfaces were of naked plastic. The rods, in the shape of a bobs, as well as the cups were of polyamide-6 (6-nylon). The rods were lathed out of a nylon blank and the cups were injection molded. Only the frequency tracing from the rheometer is displayed in the figure 3 as this suffices to detect detachment of the blood coagulum from the rheometer surfaces. In all of the three parallel runs, coagulation occurs at about three minutes as indicated by a notch in the originally flat tracing. Following the coagulation a step increase in free oscillation frequency is observed. The three tracing are near super-imposable for about ten minutes following coagulation. In C, a sharp fall in frequency is observed which is interpreted as a detachment of the coagulum from the naked plastic surfaces of the rheometer. At 20 minutes this tracing reaches the original level and remains there for the duration. Inspection at the end revealed detachment of the coagulum. In A, which is according to the invention, no such detachment is suspected from the smooth progression of the frequency tracing. A maximum of about 13.3 Hz is reached at about 22 minutes followed by a decline, first appreciable and then more gradual. At 100 minutes, about 12 Hz is recorded. Inspection revealed no detachment. The rod-cup assembly was glued together by the coagulum and the whole assembly could readily be removed from the rheometer by pulling the rod shaft upwards. In B, also according to the invention, no detachment of the coagulum is evident during the first 50 minutes of analysis. Later, small notches or discontinuities indicate semi-detachments. The later course of tracing B also displays a more rapid decline in frequency as compared to the tracing A which is a departure from the mirrored trajectories along which the two were progressing. At the end of the run, it could be found that the rod and cup were less firmly glued to each other than in A, still it was possible to lift the assembly by the shaft of the bob (in C there was no gluing at all). To summarize, run A appeared flawless for the whole 100 minute duration whereas B generated reliable data for the 50 minutes allowing the analysis of the entire build-up of rigidity in the coagulum. Both A and B, which were according to the invention, are deemed to supply good information about the viscoelastic properties of a blood coagulum. Run C, which is according to prior art, leaves the experimenter with little clue as to how much
rigidity the coagulum will display. The modest difference in the trajectories of tracings A and B are attributed experimental imprecision. See Materials and Methods for further experimental details. Example 3. Blood was mixed with Reptilase® and injected into four channels of a free oscillation rheometer, much as in Example 2. All rod and cup assemblies were according to the mvention. All rods were coated with gold and all cups were naked plastic. The results are presented in figure 4. The difference between the four runs, A, B, C and D, was that the cups were injection molded from four different thermo-plastics. They were of polypropylene, polycarbonate, polyester and co-polymer of styrene and acetonitrile, respectively. In all cases the frequency tracings were smooth and without notches and discontinuities, indicative of absence of detachments. This was true for the duration of all runs. At the end of the runs, all rod and cup assemblies were glued together by the blood coagulum but not as firmly as in run A of Example 2, more like run B in the same example. There were differences in the initial frequencies. This is the fault of the experimenter. The channels were calibrated with the same cup and then used with cups of different plastic material of different specific weights. Hence, as all cups were made by injections into the same mold, they weighed different. The cups made from plastic with high specific weight were heavier and showed a lower initial frequency that those with low specific weight - correctly predicted by models for the resonance frequency of a torsion pendulum. Three of the runs, A, C and D show close to identical frequency tracings. The tracing of run B mirrored this common trajectory but at a slightly lower lever. Again, as in Example 2, the difference is attributed experimental imprecision.
To summarize, practice of the invention, as described in the example, shows that there are no obvious restrictions regarding the type of plastic that can be used to make one of the parts of the rod and cup assembly.