GB2059051A - An apparatus for measuring the aggregation of dispersed particles - Google Patents

Abstract

Particles distributed in a liquid or gas flow are directed against a wall (4), the particles (5) adhering to the wall and/or to one another are illuminated by means of an optical illuminating device (3, 14 or 2, 13) and the scattered, reflected or diffused light (6) is directed against a detector (10) by means of an objective lens (7), the detected signal then being evaluated. The flow 12 may be of platelet-rich blood plasma. <IMAGE>

Description

SPECIFICATION Apparatus for measuring the aggregation of particles The present invention relates to an apparatus for measuring the aggregation of dispersed particles at or on the surface of a wall to which they adhere or of dispersed particles distributed in a liquid or gas flow whereby this liquid or gaseous multiphase system is directed against the wall.

The tendency of these particles to form aggregates is of major interest in diagnostic medicine since thromboses in blood vessels and blood vessel prosthesis are caused by the aggregation of blood cells and components of the plasmatic coaguiating system originating from the blood stream. To gauge the tendency of the system to coagulate, the tendency of the individual components of the system, such as thrombocytes, must be taken in account. Medicai workers generaily use so-called aggregometers to effect such measurements. A stem-point aggregometer can be used to determine the adhesion of, amongst other things, cellular blood components which are subject to the influence of predetermined hydrodynamic forces.Rotationally symmetrical stagnation point flows of suspensions of blood cells produce mechanical reciprocating actions between the cellular blood components themselves and between the components and the wall of the vessel in which they are located. The reciprocal effect may be quantitatively expressed in the form of hydrodynamic measurement values, particularly contact frequency (collision rate) and contact pressures.

Various blood cell aggregometers are already known. The most frequently used methods utilise the increase in light transmission from suspensions of blood cells which is dependent upon the aggregation of the blood cells.

In the known aggregometer developed by G.V.R. Born (J. of Physiol. (Lond.) 162, 67 (1962)), a platelet rich plasma is maintained constantly in a convection flow by means of a magnetic stirring device. The flow conditions such as the velocity field within the measurement chamber are not known. The flow conditions, amongst other things, are standardised, however because the speed of rotation of the plastics coated magnetic core of the stirrer is set at a constant value. The aggregation of the cells is triggered by a definite stimulation, for example, by the addition of ADP. At the same time the light transmission is measured by means of a photoelectric cell. If the blood cells aggregate, the light transmission increases. The amplitude and rate of increase in the photoelectric current are used as an empirical measurement of the tendency of the cells to aggregate.

In the rheoaggregometer of Scymid-Schoenbein et al (Thromb. Res. 7, 261 (1975) the aggregation of a suspension of blood cells flowing into a cone plate rheometer is measured. The advantage of this system is that the flow conditions in the measurement cell are accurately known since the same but adjustable velocity gradient prevails at all points within the cell. The measurement is effected as in the Born aggregometer. The application of this instrument is, however, restricted to research use.

In a method described by Marx et al (Blood 3, p. 247 (1975)), a platelet-rich plasma having a defined volumetric flow is forced through a capillary. The number of thrombocytes adhering to the capillary wall is calculated by measuring the difference in the thrombocyte concentration before and after passage of the plasma through the capillaries by counting microscopically.

Moreover, a further method is known (Baldauf, W. et al, Paths, Res. Pract. 163, 9-33 (1978)) in which the blood particles are deposited on a glass wall by a stagnation point flow.

The particles adhering to the wall are photographed and counted. This method misses platelet wall aggregations or adhesions under defined flow conditions.

The first two known methods described hereinbefore cover only particle/particle aggregation, but do not take into account the particle/wall adhesion. The aggregation cannot be ascertained quantitatively, since the absorbance is dependent both on the number of particles and the size of the particles. The two methods supply quantitative values only when either the number of particles or the particle size is measured independently utilising other measuring methods.

In the third above-mentioned known apparatus, the rate of transportion of the thrombocytes towards the capillary wall is not clearly one-valued, because the radial movement of the blood cells only occurs due to diffusion. The diffusion of thrombocytes is very small and may fluctuate by as much as several powers of ten.

The disadvantage of the final prior art method described herein before is the requirement of large samples, of the order of 500 ml of blood, and the cumbersome and time consuming methods of evaluation. It is, therefore, suitable for use in experimental research but is not appropriate for use as a routine operation in a clinical laboratory.

The present invention seeks to provide an apparatus in which the particle/wall and particle/particle aggregation, and thus adhesion, can be measured wherein small samples may be used and the particle/particle collision rate may be accurately determined. Finally, the invention seeks to provide an apparatus which permits the calculations to be made as simpiy as possible and to provide an interchangeable wall so that it may be used as any object support for the adhering aggregate and fixes the aggregate.

In accordance with the present invention, there is provided an apparatus for measuring the aggregation of dispersed particles with a wall surface (adhesion) or the aggregation together of dispersed particles, the particles being distributed in a liquid or gas flow, whereby the multiphase liquid or gaseous system is directed against the wall, wherein the particles adhering to the wall and/or to one another are illuminated by means of an optical illuminating device, such illumination being by incident light or transmitted light, and that the scattered, reflected light or light diffused by absorption is directed against a detector by means of an objective lens, the results.obtained then being evaluated.

The apparatus of the present invention permits walls or rather natural blood vessels to be tested with standardised particles.

Preferably, the illuminating device comprises an annular capacitor or condenser.

The wall may be a glass plate, be formed from a plastics material or from living or synthetic organic material. The illuminating device desirably includes a source of infra-red light.

The invention will be described further, by way of example, with reference to the accompanying drawings, in which: Figure 1 is a schematic view of an apparatus in accordance with the present invention; Figure 2 shows a plot of photo-voltage against time obtained for tungstic acid particles; and Figure 3 is a plot of photo-voltage against time for thrombocytes.

An apparatus in accordance with the invention is shown in Fig. 1. The apparatus comprises two walls 4 and 4' between which is defined a chamber in which there prevails a rotationally symmetrical stagnation point flow 11, 1 2. Particles 5 are transported towards the wall 4, the equation 1 approximately applies:

where rp is the particle radius, y = shear rate at location R, R being the distance from the stagnation point centre, C is the concentration of the particles and t is time. The validity of the equation 1 has been experimentally verified.

The shear rate y is measured as a function of R for the geometry of the stagnation point flow, which is dependent upon the shape of the chamber and the flow volume. The quantity of particles 5, such as thrombocytes, deposited on the wall 4 is measured by using a diffusion light technique. The incident flow from the suspension against the wall 4, which may be a cover glass or slide is illuminated by incident or transmitted dark-field light utilising capacitor 1 3 shown in broken lines and beams 2, and the intensity of the diffused light 6 is measured with a photo-detector 10.

To estimate the number of thrombocytes it is assumed that: (1) the thrombocyte concentration C and the volume V of the individual thrombocytes for all slides 4 of a sample are substantially identical, (2) the refractive index nT and the concentration of the colloidal dispersion of intra-cellular proteins, which act as diffusion centres, are identical in all thrombocytes in a sample and (3) the diameter of the diffusing proteins is smaller than the wave length -A of the illuminating light 2 or 3.

In such a case, as a first approximation, Rayleigh's diffraction theory may be applied as set out in equation 2.

where IRa is the intensity of diffusion radiation 6 of one platelet, lo is the intensity of the incident light 2 or 3, A is the wave length of the incident light 2 or 3, R is the spacing of the objective from diffusion centre 18, V is the thrombocyte volume, nT is the refractive index of the intracellular proteins, na is the diffraction index of the electrolyte solution and a is the angle between the dipole-oscillation and the direction of observation.If the dilution of the particles 5 is sufficiently great, so that neither the primary illumination 2 or 3 not the diffusion illumination 6 are mutually overshadowed, the number (N) of deposited thrombocytes can be calculated according to equation 3;

where 1G is the measured entire diffused light intensity and 1Unl is the diffusion light of the background caused by plasma proteins and reflections from the glass 4.

The liquid or gaseous multiphase system 11, 12, such as platelet-rich plasma, flows out from a nozzle 1 9 of a static tube 1 and is directed perpendicularly against the glass wall 4 of the cuvette 4, 4' (stagnation point flow). If the adhesion between the particles 5 and the glass wall 4 exceeds a minimum value and if suitable flow velocities occur, the stagnation point flow leads to a deposition of the particles on the glass wall 4, and indeed, in a surface region 1 5 which corresponds to the flat projection of the aperture of the nozzle 1 9 on the glass wall 4.The transmitted-light capacitor 1 3 (shown in broken lines) or the incident light capacitor 14 focusses the light 2 or 3 of a powerful light source, such as a xenon lamp, but which is not shown in detail at the level of the boundary surface between the wall and the fluid. An objective lens 7 receives the light 6 diffused by the deposited particles 5 and produces an image of the particles 5 in the plane of a central slit 8. The light 20 passing through the central slit 8 is focused at 21 by a field lens 9 so as to fall onto the photocathode of a photoelectron multiplier 1 0. The output signal thereof is amplified and is recorded, as a function of the elapsed time, by means not shown.

The illumination components 2, 3, 13, 14, the image forming system 6, 7, 8, 9, and the photocathode 10 are conjugated with each other in such a manner that only the diffused light 6 formed in the plane of the slit 8 produces a photoelectric signal (a reduction of the depth of the focal field of 1 ym to 50 yam). Since the central aperture 8 is centered on the stagnation point 15, there prevails identical amounts of speed components everywhere in the annular measurement plane 1 6 because of the rotationally symmetrical flow 11, 12, (flow rates and constant shear rates).

Fig. 2 shows the time-aggregation graph of an aqueous suspension of approximately 3 y size tungstic acid platelets, corresponding to a particle concentration of 50,000 particles/mm3. The first section of the graph (0-3.2 min) shows the photoelectric signal oscillations of the flowing stable initial suspension. The photovoltage (photodiode 10) on an average amounts to substantially 21 V. The fluctuations therein can be traced back to static concentration fluctuations of the particles 5 in the measurement field 1 6.

If the sodium chloride concentration of the suspension 5 is increased by the addition (see arrow) of a concentrated sodium chloride solution of up to 0.9 M Mol, the dispersion becomes unstable. Some particles 5 adhere to the wall and to one another, and the particle/wall and particle/particle adhesion is increased. The aggregation operation during this period leads, as can be seen from Fig. 2, to an almost linear rise in the photo voltage V, with respect to time.

As the microscope photmeter, a Leitz MPV device (300 V operating voltage) is used. A Leitz Ultropak U-O 22 with incident light capacitor 22-100 is used as the capacitor and a xenon lamp (25 A direct current operation) is used as the light source. In the present experiment, the outlet diameter of the nozzle 19 is 0.5 mm, the wall spacing from the nozzle 19 is 0.5 mm and the flow volume in the stagnation point aggregometer is 0.06 mm/s.

Fig. 3 shows the results obtained when platelet-rich cattle plasma flows through the stagnation point aggregometer. The thrombocyte activity is stimulated by the addition of 3.5 x 10-6 M ADP to the plasma. In the first part of the graph (0-32 mins), a slow rise of light diffusion 6 occurs with respect to time. This is caused by the adhesion of individual thrombocytes to the wall 4 which can be proved by the finding of individual deposits by conventional microscopic investigations. The wall shearing stress amounts to 0.7 dyne/cm2 at the measuring point. From this graph section, the thrombocyte-wall adhesion may be ascertained. If the wall shearing stress is increased to 3 dyne/cm2, the blood platelets 5, which slide into the proximity of the wall in the flow, are deposited on the already deposited platelets.

A steep rise then occurs in the diffusion light intensity 6. The second section of the graph (32-40 mins) represents the platelet-platelet adhesion, which can be confirmed microscopically by the finding of polycellular deposits.

A Leitz UO 52 W objective was used and the particle concentration was 50,000 platelets/mm3.

Claims (9)

1. An apparatus for measuring the aggregation of dispersed particles with a wall surface (adhesion) or the aggregation together of dispersed particles the particles being distributed in a liquid or gas flow, whereby the multi-phase liquid or gaseous system is directed against the wall, wherein the particles adhering to the wall and/or to one another are illuminated by means of an optical illuminating device, such illumination being by incident light or transmitted light, and that the scattered, reflected light or light diffused by absorption is directed against a detector by means of an objective lens, the results obtained then being evaluated.

2. An apparatus as claimed in claim 1, wherein the illuminating device comprises an annular capacitor or condenser.

3. An apparatus as claimed in claim 1 or 2, wherein the wall is a glass plate.

4. An apparatus as claimed in claim 1 or 2, wherein the wall is formed of a plastics material.

5. An apparatus as claimed in claim 1 or 2, wherein the wall is formed of a living or synthetic organic material.

6. An apparatus as claimed in any preceding claim, wherein the illuminating device includes a source of infra-red light.

7. An apparatus as claimed in any preceding claim, wherein a central slit is located in the image plane of the objective lens.

8. An apparatus as claimed in any preceding claim, wherein the plate and the particles are capable of being used for subsequent microscopic, examination.

9. An apparatus for measuring the aggregation of particles constructed and arranged to operate substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.