WO2014138196A2

WO2014138196A2 - Composition and method for calibrating or quality control testing a light scattering device

Info

Publication number: WO2014138196A2
Application number: PCT/US2014/020656
Authority: WO
Inventors: Edward R. Teitel; Philip C. Speros; Andrew T. YEE; Johnny Rhodes
Original assignee: Aggredyne, Inc.
Priority date: 2013-03-08
Filing date: 2014-03-05
Publication date: 2014-09-12
Also published as: WO2014138196A3

Abstract

The present disclosure is drawn toward a formulation suitable for use in a light scattering device for calibration or equipment testing, comprising a masking agent, a density modifier or a viscosity modifier, a detergent or surfactant at a concentration sufficient to prevent particles from sticking to surfaces of the device, and light scattering particles. A method of calibrating or testing a light scattering device can comprise using the composition described above in a light scattering device to calibrate or test the light scattering device.

Description

COMPOSITION AND METHOD FOR CALIBRATING OR QUALITY CONTROL TESTING A LIGHT SCATTERING DEVICE

BACKGROUND

A platelet aggregometer is an instrument that can assess certain aspects of platelet function. This device can be used by starting with a platelet suspension, such as blood or platelet rich plasma, which can be collected from a patient and dispensed into a disposable sample holder of the platelet aggregometer. A chemical stimulus, such as collagen, can be added to the platelet suspension in the sample holder, and subsequent agitation/mixing of the platelet suspension with the stimulus can cause the platelets to aggregate. The characteristics of this aggregation can be measured by various methods known by those skilled in the art, and the degree of aggregation measured can be directly related to the function of the platelets.

Currently available methods in the field of platelet

aggregometers include sample holders that provide thorough mixing and agitation of the platelet suspension to cause platelet

aggregation. However, most of these methods and devices create flow that is not conducive to enabling certain detection modalities of platelet aggregation, particularly for light scattering methods.

Many methods utilize mechanical mixing which often damages or otherwise alters fluid characteristics. For example, the use of a roller pump has been one proposed method for moving blood. However, the compression of a flow conduit containing blood by means of rollers often disfigures platelet aggregates, damages red cells, and alter their characteristics. Thus, the ergonomics of such designs can be undesirable, and loading of the blood sample and/or the chemical stimulus that causes platelet aggregation can be cumbersome. These limitations detrimentally influence the quality and consistency of platelet aggregation, which in turn adversely affects the reproducibility and reliability of the measurement of platelet function. Other methods include designs that present relatively good flow patterns for measurement using light scattering techniques. However, for these devices to be used properly and the results trusted, it would be advantageous to provide systems and methods for testing and/or calibration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of a fluid property

measurement system having a stenotic baffle system in accordance with an embodiment of the present disclosure.

FIG. 2 is a cross-sectional view of the embodiment shown in FIG. 1 having the rotor removed.

FIG. 3 is a top view of the embodiment shown in FIG. 1 having the cap removed.

FIG. 4 is a top schematic view of the device of the

embodiment shown in FIG. 1 having the stenotic baffle and the cap removed.

FIG. 5 is a graph that represents particle count (PAI) vs. relative particle concentration in EDTA-treated (platelet aggregation- blocked) human blood.

FIG. 6 is a graph that represents particle count (PAI) vs. relative concentration of particles in various concentrations (w/v) of titanium dioxide.

FIG. 7 is a graph that represents change in unadjusted particle count (PAI) with increased particle concentration.

The drawings are intended to illustrate several specific embodiments of the present invention and are not intended to be unnecessarily limiting. As such, departure may be had in dimensions, materials, and features while still falling within the scope of the invention. DETAILED DESCRIPTION

Reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used herein to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended.

Alterations and further modifications of the inventive features illustrated herein, and additional applications of the principles of the inventions as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention.

In describing and claiming the present invention, the following terminology will be used.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a rotor" includes reference to one or more of such structures, and reference to "a stimulus" includes reference to one or more of such factors.

As used herein, "recirculating," "recirculated," or "recirculation" refers to fluid flow along a path that is primarily related to a measuring region where fluid recirculation occurs, though

recircuation can also occur in other non-measuring regions. By recircuating in the measuring region, a better measurement sample over a predetermined time can be taken as to the properties of the fluid. For example, in one embodiment, recirculation can occur by circumferential recirculation.

As used herein, "fluid" refers to a flowable composition and can include liquid, gas, suspended solid or other flowable mass. Fluids can be in the form of suspensions, emulsions, solutions, mixtures, or the like.

As used herein, "mixing" refers to disturbed flow or separated flow of a fluid. As used herein, mixing does not include mixing that is merely the result of intermolecular, intercellular, or structural forces exerted within a fluid under substantially streamlined flow, or which is solely the result of diffusion due to concentration gradients.

As used herein, "streamlined" refers to a fluid flow state that is more streamlined than is present in a mixing region acting on the same fluid. Additionally, a streamlined flow is capable of providing fluid flow dynamics such that at least a substantially accurate measurement can be taken, such as by use of a light scattering device or other fluid property measuring device. Further, streamlined flow typically refers to minimally disturbed flow that can be predominantly laminar, including arcuate flow in case of a cylindrical container. Such flow is suitable for testing using methods such as light scattering, etc. Although a common definition of the term "streamlined" can define a path or paths characterized by a moving particle in a fluid such that the tangent to the path at every point is in the direction of the velocity flow, the term as used herein is intended to be broader in scope to include flow that is minimally disturbed such that more accurate readings using fluid measuring equipment can be used, e.g., light scattering particle detection devices.

As used herein, "free stream particulates" refers to masses which are non-liquid materials contained within a fluid which are not attached to a fixed structure such as a container wall or other solid member. Free stream particulates can include, but are not limited to, platelet aggregates, solid debris, air bubbles, clots, and the like. In accordance with calibration and/or testing systems described herein, the free stream particulates can be small synthetic beads, such as polystyrene beads.

As used herein, "stenotic" refers to any constriction or narrowing of a fluid flow path. Typically, stenotic baffles can have a gradually narrowing portion which leads to a flow path portion having substantially constant cross-sectional area, and a subsequent expanding portion where cross-sectional area gradually increases to an unobstructed flow. As used herein, the term "concentrated" when referring to streamlined flow, indicates that a greater number of streamlines per unit area are present than are present in other areas of the system in accordance with embodiments of the present invention. Areas outside of where there is "concentrated" streamline flow can be from streamlined (though less concentrated) to chaotic.

As used herein "fluid dynamic focus," "fluid dynamically focused," or the like, refers to fluid conditions where elements of the fluid are can become concentrated in a smaller cross-sectional area of controlled volume of flow.

Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.

In accordance with this, the present disclosure is drawn toward a formulation suitable for use in a light scattering device for calibration or equipment testing, comprising a masking agent, a density modifier or a viscosity modifier, light scattering particles, and a detergent or surfactant at a concentration sufficient to prevent the light scattering particles from sticking to surfaces of the device.

In another example, a method of calibrating or testing a light scattering device can comprise using a composition described above in a light scattering device to calibrate or test the light scattering device.

A device that can be used with the composition described herein and in carrying out the method described herein can include a fluid movement device positioned within a fluid container to cause fluid flow within the fluid container along a fluid flow path. The fluid movement device can often be a rotor although other devices can also be suitable. The system can further include a constricted region along the fluid flow path which generates a region of concentrated streamlined flow within the constricted region and mixing of the fluid outside of the constricted region. A property measuring device can also be functionally positioned with respect to the constricted region to measure fluid properties in the region of streamlined flow. In one detailed aspect of the present invention, the constricted region can be formed by a stenotic baffle system. The constricted region and fluid movement device can advantageously be configured to provide free stream aggregation of material such that measurement of aggregation can be based on free stream properties. Though this example is given as an acceptable device for use with the compositions and methods of the present disclosure, it is understood that other devices can likewise be used, such as other devices that can provide streamlined flow regions that are highly acceptable for light scattering analysis. The type of device is not really limited in this respect, as long as the compositions, systems, and methods can be effectively used or implemented.

Thus, as illustrated in FIG. 1 by way of example only, a fluid property measurement system 50 is shown. In this example, a fluid container 52 can include a fluid movement device such as a rotor 54 positioned within the fluid container. The fluid movement device can be configured to produce flow of fluid within the container along a desired fluid flow path, e.g., causing circumferential recirculation. The fluid flow path is an annular recirculating flow as the rotor spins within the fluid container. A cap 55 can be configured to fit over the rotor within the fluid container as shown to provide a seal and to prevent loss or contamination of the contents during use. The fluid container and cap can optionally include threaded surfaces to allow mating of the two pieces. Alternatively, the cap can be secured via an interference fitting, latch, snap, adhesive, seal, and/or other similar mechanism. A measuring region can be a constricted region where fluid flow passes through a volume having a smaller cross-sectional area than neighboring volumes along the fluid flow path. FIG. 2 is a cross-sectional view of the system 50 of FIG. 1 having the rotor removed so that other features can be viewed appropriately. A constricted region 56 can be formed which generates a region of streamlined flow within the constricted region, shown generally by three flow lines 57. Thus, the constricted region can cause the fluid to increase in velocity over a portion of the fluid flow path. An increase in fluid velocity can improve particulate measurement results based on light scattering. The constriction can also facilitate fluid dynamic focusing that concentrates particulates to be measured in the detection region. In addition, increased fluid velocity can reduce agglomeration or blockage of the fluid flow path. Such methods can benefit from increased resolution and decreased signal-to-noise ratio as fluid flow is increased. As the fluid exits the constricted region, at least some mixing occurs as the fluid expands outside of the constricted region into a mixing region 58.

Advantageously, the constricted region and subsequent expansion that occurs in the mixing region thus acts to provide streamlined flow and mixing using a single feature, e.g., stenotic baffle. Such expansion mixing also achieves some of the purposes of the present invention by reducing damage to platelets and other fragile materials.

The constricted region shown in FIG. 2 is a stenotic baffle system including a top baffle 60, which in this embodiment is positioned on the cap 55 to form an upper streamlined flow surface for the constricted region 56. A bottom baffle 64 can also be formed along a lower inner surface 66 of the fluid container to form a lower streamlined flow surface of the constricted region. In the

embodiment shown in FIG. 2, though the top baffle is positioned on the cap, the top baffle can alternatively be attached to or formed as in integral part of the fluid container. It should be noted that though not necessary, a secondary disruption member can also be used in conjunction the stenotic baffle embodiment described herein. The secondary disruption member can be positioned anywhere except in the constricted region (as it would disrupt the streamlined flow), but is typically positioned at or near the mixing region 58 following the streamlined stenotic baffle.

FIG. 3 is a top view of the fluid property measurement system 50 having the cap removed. From this perspective, the bottom baffle 64 can be seen having a width which covers nearly the entire fluid path width. In one embodiment, the distance between the rotor and the baffles can be carefully chosen in order prevent damage to fluid which passes therebetween while also maximizing the constricting affect of the stenotic baffle system. Typically, the stenotic baffles system can have a width from about 50% to about 95%, and preferably about 75% to about 95% of the shortest distance between the inner wall and the rotor. Further, the bottom baffle can be oriented adjacent a light transparent window 68 which is also placed along at least a portion of the constricted region. The constricted region, or measuring region, can allow the light transparent window to be used for measurement of aggregation or other particulates via light scattering devices or other property measurement devices as described above. The light transparent window can alternatively be translucent, as long as the wavelength of light used can pass through the window functionally. The constricted region can alternatively be formed by using a single stenotic baffle. In yet another aspect of the present invention, the constricted region can be formed having conically fluted entrance and exit points. In one embodiment, the baffle or baffle assembly design can produce three dimensional velocity vectors that result in radial, circumferential, and/or vertical mixing.

As alluded to, the fluid container includes a region(s) where the local flow patterns of the fluid are such that there is substantial mixing of the fluid. Further, the fluid container includes another region(s) separate from the mixing region(s) where the flow characteristics are substantially streamlined. Such streamlined flow is steady enough that the entities of interest in the fluid, e.g. platelet aggregates in a blood component-containing fluid, carried in it can be detected more accurately by certain detection methods, such as light scattering. As recirculation occurs in the measuring region, a more complete sample of fluid can also be measured. Moreover, the above mixing and streamlined flow characteristics are induced using methods that minimize damage to, or alteration of entities of interest, e.g., platelet aggregates, coagulated masses, or the synthetic compositions of the present disclosure. In addition, the present invention can be incorporated into a compact, disposable, and ergonomic design that further enables more reliable assessments of platelet function. The systems and devices of the present invention are designed to measure free stream particulates, such as platelet aggregates, and particularly in accordance with the present disclosure, synthetic composition that includes synthetic or polymeric beads.

Usable with these devices are methods, compositions, and systems as described herein. For example, a method of creating a calibration standard has been developed for this type or equipment and other similar equipment. For example, this calibration standard can be used with devices that count particles. The method comprises combining suitable components that can be selected to provide an appropriate background signal, masking of interfering signal arising from instrument components, appropriate and adjustable (intended) calibration signal, proper density, viscosity, surfactant and/or preservative properties. Optional components and characteristics facilitate formulation in a dry state, such as lyophilized or powdered form for reconstitution at the site of use.

In one particular use, a kit can be provided and used for any instrument that counts particles, including the instrument described in FIGS. 1 -3. In a more specific example, the kit can be used for instruments that count particles optically, such as UV, visible or IR light. Detection of particles can be based on any number of optical properties, including light scattering. In a more specific scope of use, the kit can be used for instruments that are used to observe particles in biological fluids, and specifically in one example, whole blood or blood derivatives (plasma, serum, etc.). The particle detection may be occurring against a background of other particles of differing size co-suspended in the sample of interest.

Furthermore, this method has been used to create several compositions that act as a quality control tool and calibration standard for instruments such as those described in FIGS. 1 -3 above.

In further detail regarding the equipment and the calibration process, a similar device is shown in FIG. 4, which includes a rotor 54, though it is noted that the baffle is not shown, but would typically be present. In some detail with respect to FIG. 4, in one example, the instrument sends an intense collimated beam of N IR (e.g., 810nm /780nm) light into a sample chamber 70. The incident light 72 penetrates the cassette face 74 and transits to some or all of the cassette sample chamber 76. At startup cassette validation, the incident light typically strikes the face of the rotor. During operation with a sample in the cassette sample chamber, light scattered from the sample components 78 is collected by optics in the instrument at a low angle to the incident light.

There are many sequential steps that can be carried out to evaluate the sample, calibrate the device, etc. For example, the instrument can evaluate the empty cartridge with a spinning rotor, the sample introduced into the cassette can also be evaluated using any of a number of techniques and fluid components, the instrument can establish baselines based on masking from backscattering or masking the rotor, the instrument can determining intensity of backscatter, the instrument can verifying that there is backscatter establishing that there are readable particles present, the instrument can detect motion or change of speed of particles, etc.

Calibration kits can be provided for a variety of purposes, including aiding the device in meeting the requirements of masking, reflectivity and variability, and allowing for a determined count of particles based on a known formulation. Reflectivity and rotor masking can be achieved by using a dilute suspension (e.g., 0.1 - 0.2% w/v) of titanium dioxide (TD). An alternate embodiment may be to use copper ion solutions, e.g., CuCI₂, CuS0₄, etc. that absorb strongly at the wavelength of a typical laser, e.g., 780nm, such that the movement of the rotor can be masked. Other possible masking agents include NIR dyes such as NIR783E and the protein cytochrome oxidase. Variability can be achieved by allowing a small amount of light come back from the rotor in "zero-signal" samples. In practice, a "blank" solution may not have sufficient variability to work appropriately. Thus, solutions of zero particle content may not be useful.

Thus, formulations can be prepared that work well in calibrating and quality control testing the equipment described herein in FIGS. 1 -4. The sample formulation can provide a signal that creates calibration levels due to particle-count events. The following components are provide by way of example, indicating optional ingredients that can be admixed together in preparing kit

formulations suitable for use as described herein. These

components are provided by way of example only, as follows:

- Microsphere particles of polystyrene, of diameter 106-125 μιη, added to the suspension allows the instrument to observe and achieve a count of particles. The resulting particle count readings vary proportionally to the concentration of particles.

- Detergent or other surfactants can be included to prevent the adherence of particles to working surfaces of the instrument or to one another. - Density-modifying agents can be included to provide that selected particles are near neutrally buoyant. Slight negative buoyancy can be useful so that the particles will not vacate the outer surface of the cartridge during spinning of the rotor.

- Cu salts, Cu ions, sugars (eg. Sucrose, mannose, trehalose), NaCI, or MgS0_4.

- Lower alcohols, oils, other low-density materials.

- Viscosity-modifying agents can be included to promote mechanical coupling of the particles to the rotor via a carrying fluid.

- Sugars, carbohydrates, polymer oils, gels, etc.

- Preservative components.

- Azides.

- Agents to facilitate dry formulation as in lyophilization, anti- caking agents for powdered forms, and agents to help in re- dissolution of powders.

- Agents to mask/block light from rotor, such as titanium dioxide (via light scattering), CuCI₂ and CuS0₄ (via absorbance at 780/810 nm), milk or powdered milk (via both scattering and absorbance), NIR dyes, e.g., QCR 782E, Cytochrome C, or polystyrene beads, e.g., about 5 μιη diameter.

With these components as a basis, formulations can be generated that work well for use with the devices described herein. A suitable composition, thus, may include (i) a masking agent, such as CuCI₂ , another copper salt, or another chromophore sufficient to cause an absorbance at 780 nm of 0.8 to 1.2, with a spectral bandwidth of 2nm and a pathlength of 1 cm; (ii) a density modifier, such as sugar, trehalose or NaCI, or a viscosity modifier, such as sugar or trehalose (note the density modify can be the same compound as the viscosity modifier); (iii) a detergent at a

concentration sufficient to prevent particles from sticking to surfaces of the device, such as Triton X-100, e.g., at about 0.1 wt%; and (iv) particles, such as polystyrene beads 106-125μιη in diameter with a specific gravity/density of about 1 .2. This specific composition description is not intended to be limiting, but is included to describe a specific composition that is suitable for use as part of the kit formulation described herein.

In further detail, a suitable calibration kit formulation has been created and successfully tested on an instrument shown and described FIGS. 1 -4 or other similar devices. This formulation typically meets nearly all of the desired characteristics set forth herein, namely that it provides appropriate background signal, masking of interfering signal arising from instrument components, appropriate and adjustable (intended) calibration signal, proper density, viscosity, surfactant and/or preservative properties.

A specific example of a suitable kit formulation is set forth below in Table 1 :

Table 1

*Note that in this example, the carrier is water, but other carrier solvents or fluids can likewise be used, such as glycerol, blood plasma, etc. Other formulations can likewise be prepared that are similar to the composition in Table 1 , as would be appreciated by on skilled in the art after considering the present disclosure. Key

characteristics of this and other similar formulations may include: stability in nominally high temperatures; formulations that are reasonably inert; formulations that are able to be re-suspended within 15 seconds of vortex mixing action; formulations that are able to generate functional responses in the instrument described in FIGS. 1 -4; formulations where responses are predictable and, after batch-calibration, suitable as a standard for instrument verification; formulations comprising readily and reproducible sources of material; and/or formulations having suitable ageing characteristics.

In further detail regarding the compositional components described herein, it is noted that the inter-relationships that govern formulation of standards can include:

- Use higher density beads with higher density masking agents.

- Use viscosity-increasing agents to slow bead movement and convert rotor motion to stirring more efficiently.

- Agents that can be formulated to freeze-dry and reconstitute appropriately

- High masking concentration that lead to low response to particles.

- High density composition floats beads over time, signal drifts downward during data collection.

- Use lower density masking agents (eg. NIR dye over Cu salts).

Using the formulation and the device of FIGS. 1 -4, calibration response curves have been recorded, and these curves, together with batch-specific determinations, allow measurements to be correlated between instruments, between batches of calibration material and across time. For example, initial batches of calibration material have been produced, including several 35 mL batches wherein various volumes were retained and various other volume samples were divided into aliquots for more immediate testing.

By way of Example, FIGS. 5-7 provide data tested using the formulations and techniques described herein. FIG. 5 provides data related to particle count (pai) vs. relative particle concentration in EDTA-treated (platelet aggregation- blocked) human blood. Thus, the linearity of particles in EDTA blood was evaluated. For example, human blood was treated with EDTA, preventing platelets from aggregating; polystyrene beads/particles were added to this blood in varying amounts; and particle count (PAI) signals were proportional to particles added.

FIG. 6 provides details regarding the monotonicity of particles in titanium dioxide. More specifically, this FIG. sets forth particle count vs. relative concentration of particles in various concentrations (w/v) of titanium dioxide. The dashed lines indicates the range of "valid" particle count (PAI) values, that is, the range of PAI values for which PAI scores are reported.

FIG. 7 provides details regarding the linearity of particles in CuCI_2. Specifically, this FIG., change in unadjusted particle count (PAI) on the instrument described herein with increased particle concentration is shown. Increasing volumes of 1 % (w/v) 106-125μιη particle suspension were added to 3.43 mL base solution of CuCI₂ as a masking agent and preservative, Trehalose as a density and viscosity modifier, NaCI as a density modifier, and Triton X-100 detergent as a surfactant.

The above description and examples are intended only to illustrate certain potential uses of this invention. It will be readily understood by those skilled in the art that the present invention is susceptible of a broad utility and applications. Many embodiments and adaptations of the present invention other than those herein described, as well as many variations, modifications, and equivalent arrangements will be apparent from or reasonably suggested by the present disclosure and description thereof without departing from the substance for scope of the present invention. Accordingly, while the present invention has been described herein in detail in relation to certain embodiments, it is to be understood that this disclosure is only illustrative and exemplary of the present invention and is made merely for purpose of providing a full and enabling disclosure.

Claims

What Is Claimed Is:

A formulation suitable for use in a light scattering device for calibration or quality control testing, comprising a masking agent, a density modifier or a viscosity modifier, a detergent or surfactant at a concentration sufficient to prevent particles from sticking to surfaces of the device, and light scattering particles.

The formulation of claim 1 , wherein the density modifier and the viscosity modifier are both present.

3. The formulation of claim 1 , wherein the masking agent is a copper salt.

4. The formulation of claim 1 , wherein the masking agent is a

chromophore sufficient to cause an absorbance at 780 nm of 0.8 to 1 .2 with a spectral bandwidth of 2 nm and a path length of 1 cm.

5. The formulation of claim 1 , wherein the density modifier and the viscosity modifier is the same compound or a different compound, and is selected from the group consisting of sugar and trehalose. 6. The formulation of claim 1 , wherein the density modifier is NaCI.

7. The formulation of claim 1 , wherein the detergent is a surfactant.

8. The formulation of claim 1 , wherein the light scattering particles include polystyrene beads having a particle size from 106-125μιη in diameter.

9. A method of calibrating or testing a light scattering device, comprising using a composition as in any one of claims 1 -8 in a light scattering device to calibrate or test the light scattering device.