Patent Application
20050220668
The field of the invention is microvolume in vitro test kits.
The analytical and diagnostic test markets need rapid, inexpensive, disposable, microvolume devices and test methods. Clinical, pharmaceutical and biotechnology laboratories are adopting rapid microvolume testing methods. These types of tests are commonly referred to as “lab on a chip” (LOC) or “point of care” (POC) tests.
These rapid, microvolume in vitro diagnostic tests are based on test methods that use whole blood, urine, saliva or other unprocessed body fluids as the test specimen. The tests are packaged as a disposable device containing the necessary reagents. The specimen may be transported within the test cartridge by wicking membranes (lateral flow), capillary action, vacuum or pneumatic pressure. The test results may be determined either visually or with a small instrument. The iterations, classifications and complexity of these devices are varied.
The drawbacks to the existing rapid clinical diagnostic test methods are cost, poor sample quality, inadequate sample volume, inaccurate sample and reagent mixing, poor correlation with standard laboratory tests performed on serum, plasma samples, or other body fluids. Sample variability and interfering substances often cause these conditions. Nevertheless, these methods have been adopted because the market is demanding rapid test results to support immediate medical or other decisions and there are no existing acceptable or proven alternative technologies or products.
In any test, accuracy and precision are critical to performance. The elements for an accurate and precise test or analysis are:
An example of current methodology is International Technidyne Corporation's products that utilize whole blood specimens and methods for reagent and sample mixing. Its patents include: U.S. Pat. Nos. 6,451,610; 5,731,212; and 5,372,946. In these devices the whole blood sample is a continuous stream. The sample is moved into a chamber that contains a dried reagent and is moved in and out of that chamber through an orifice that causes the mixing of the sample and reagent. This method has shortcomings: the sample and reagent ratio (volumes) are not accurately controlled; the sample is a continuous stream of which the reagent can diffuse; throughout the entire volume the sample flow over the reagent is laminar, therefore the mixing is not turbulent chaotic or consistent; and the reaction is only partially controlled, limiting the test accuracy, precision, and reproducibility.
The apparatus and method according to the invention provide the control, precision and accuracy of the core laboratory analyzer test methodology in a simple disposable device that provides rapid, accurate, reliable, microvolume tests. These tests produce immediate and reliable information and eliminate the requirement for special skills or training of the operator.
A sample testing device comprises a volume chamber that separates a known volume of a sample from a remaining sample through the introduction of a fluid between the known volume of the sample and the remaining sample wherein the introduction of the fluid is through a fluid inlet port that has an open and closed state. The device further comprises a passage including a mixing chamber connected to the volume chamber and adapted to mix the sample; a test chamber connected to the mixing chamber and adapted to perform a test on the sample; and a vent port that has an open and a closed state. When the fluid inlet and vent ports are in the open state, the introduction of a pressurized fluid into the fluid inlet port drives the sample from the volume chamber, into one or more mixing chambers, and then into the test chamber.
The foregoing summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments, which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:
a-c and 20a-c show common cell varied configurations; and
The present invention has several advantages over sampling devices in the prior art.
1. Sample Volume Measurement
Sample volume for analysis is precisely measured. The measured sample is then moved discretely through the device to the reagent chamber and then to the test chamber. This provides accurate and reproducible control of the sample and reagent concentrations or ratio. Variations in the sample and reagent ratio will effect the reaction or analysis. Variations in volume as little as 5% can significantly alter the test result.
2. Sample and Reagent Mixing
Static mixing caused by flowing the sample through the dried reagent can be enhanced by two methods. The method used depends on the materials to be mixed, dissolved or re-hydrated by the sample and how vigorous the mixing must be to ensure complete mixing of the reagents. These two methods are direct mixing and diverter mixing.
In direct mixing, a magnetic component, cylinder, ball or other shape is placed in the reagent chamber. When the sample is moved into the chamber, the magnet is moved from one end of the chamber to the other and back one or more times. This motion is driven by electromagnetic fields produced by a moving magnet or an inductor. This motion causes the sample to flow around the magnet against the interior chamber walls, causing higher flow and shear rates, and “washes” the reagent adhered to the walls off and into the sample. The shape of the magnet will affect the mixing dynamics it imparts to the materials. The force of the magnet motion, the frequency of the motion and the duration of the mixing are all individually and precisely controlled and can be programmed for each reagent or test method.
In diverter mixing, a mixing chamber with one or more flow diverters and a full volume passageway causes the sample that has passed over the reagent to be divided, brought back together, and in the process, mixed by turbulent flow. The mixture may be moved back through the mixing chamber several times, as required, for complete dissolution and mixing. The shape of the diverter will affect the mixing dynamics it imparts to the materials. Diverters that are round shaped are preferred, while other shapes such as ovals, rectangles or other shapes are also effective. The force of the fluid motion, the frequency of the motion and the duration of the mixing are all individually and precisely controlled and can be programmed for each reagent or test method.
3. Routine Assay Methods
The test methods can be the same as those used in routine assays in the clinical or other laboratories. This provides direct correlation of results and consistent diagnosis and management of the patient. The current use of whole blood as a specimen yields results that are mathematically manipulated to correlate to the standard laboratory test methodology. Point of care (POC) test results are useful in the area where they are performed, but when the testing is moved to a central laboratory and the test method is changed, the patient result history is often discarded due to differences in the results. The users of POC tests must also be taught to understand the meaning of the various results, which may not fall within normal or expected ranges creating a risk of the results being misleading. This closed assay system eliminates any operator influence that may affect the test results and minimizes biohazardous exposure.
4. Reagents Contained Within the Test Device
Once reconstituted, many reagents have a limited time during which they may be used. This limited stability causes poor, marginal, or variable results over time or reagent waste, because the reagents must be removed and disposed of after the specified time. The device eliminates the need to prepare the reagents, i.e. reconstitution and loading into the device , because the device physically contains the reagents.
Another result of incorporating the reagents within the device and the sample processing and measurement within the device, is the elimination of robotic fluid handling systems that require mechanisms, precision pumps and rinsing or cleaning solutions. This significantly reduces costs and complexity of the analyzer, cost of the rinse solution, and the cost and hazards of the waste disposal.
5. Monitors Sample Quality
In the prior art, there is not any measurement for poor sample quality or imprecise volume, and reagent or mixing issues that affect the test result. In the inventive method, in contrast, the sample quality can be measured when the sample is in the volume chamber by color or turbidity and the sample/reagent mixture optical transmission is measured when the mixture enters the reaction chamber. These measurements are compared to a pre-determined optical transmission level for that test type. This level can have multiple stages such as a warning stage and an abort stage. If the measurement is beyond the limits of a preset range, the test is identified as questionable, initiating examination and thereby minimizing reporting errors.
6. Microfiltration Sample Preparation
Microfiltration sample separation produces plasma, serum or other fluids and eliminates the normal centrifugation process and related artifactual errors, greatly simplifying the test process and reducing the time required to obtain a result by a factor of ten or more, as discussed in U.S. Pat. No. 6,398,956. Using plasma or serum sample test methods, instead of whole blood methods, eliminates interferences from the cellular matter in the whole blood and allows the use of accepted clinical laboratory test methods. The cellular components of the whole blood preclude the use of optical and calorimetric test methods, which are the traditional laboratory methods. The cellular component also adds additional variables to the assay. The rapid test results provide direct correlation to results of the main laboratory that provide for consistent diagnosis and management of the patient. This design will function in a similar manner when the sample is prepared by other methods such as centrifugation.
Certain terminology is used in the following description for convenience only and is not limiting. The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made. The words “inwardly” and “outwardly” refer to directions towards and away from, respectively, the geometric center of the disposable test device in accordance with the present invention, and designated parts thereof. The terminology includes the words noted above as well as derivatives thereof and words of similar import.
Referring to
For ease of use, the device may have identification features (not shown) that identify the test type such as notches, holes, barcodes, colored areas, or writing.
In a preferred embodiment, as shown in
As shown in
With reference to the figures, the major steps in using the device, (1) sample measurement, and (2) mixing will now be described.
The accuracy of any analysis depends on having an acceptable sample quality, as well as an accurate and reproducible sample volume. The apparatus provides a volumetric measurement of the sample 20 in the volume chamber 12. A volume of sample 20 is moved into the chamber 12 until a volume sensor 24 indicates that the chamber 22 has been filled. At a fixed position along the chamber 12, a connecting passage to an air inlet 26 is provided. This air inlet 26 remains sealed to prevent the sample 20 from flowing into the passage 20. When the sensor 24 senses the presence of the sample 20, the connecting air inlet passage 26 is opened and air, or a compatible liquid at a low pressure, enters through this passage and separates a sample 20 of known volume from the remaining sample, and moves this sample 20 of known volume along the chamber 12.
As shown in
First, the sample may be directed into one of several adjoining passages 28 as shown in
Second, the sample may be directed into an open well 30 as described in the U.S. Pat. No. 6,398,956 and shown in
Third, with reference to
Obtaining a homogenous mixture is critical to stoichiometric reactions and accurate, precise and reproducible analysis. The sample 20 and reagent 14 must be precisely measured and fully mixed to initiate consistent reaction rates and complete the reaction between the sample 20 and reagent 14. The nature of the materials will define the amount of physical mixing required. Some materials, such as inorganic salts, readily diffuse into solution. Other materials, such as cellular samples, require low shear, gentle mixing. Still other materials require intense physical action to achieve complete mixing. Finally, in many applications, mixing must take place within a fixed time period, at a controlled temperature, as the reactions are usually time and temperature dependent.
When a liquid flows through a passage, a flow pattern described as laminar flow occurs. The liquid near the walls flows at a lesser rate than the liquid at the center because of the drag or friction imparted on the liquid by the surface of the walls. The use of a restriction in the passage or chamber causes some turbulence that enhances the mixing process. The effectiveness of this is dependent on the liquid materials.
As shown in
Although the flow pattern splits the mixture and recombines it in a larger area, the basic flow pattern is laminar, which has minimal turbulence resulting in ineffective mixing and an incomplete reaction with variable results. Other methods of inducing mixing include modifying the surface with grooves or steps to disrupt the laminar flow patterns. These methods appear to enhance the mixing on a micro basis. For more on this, see Stroock et al, published in Science vol. 295, 25 Jan. 2002, page 647-651.
Stationary flow disruption mixing is a known method for mixing two materials. In this design, the mixing is performed using restrictors and obstructions to cause turbulence. An unwanted by-product is often shear stress that can cause physical damage to biological materials which may contain large proteins or cellular material. Therefore the flow must be smooth and turbulent so as not to induce high shear stresses.
Direct disruptive mixing is another method that can be used in the device 10. As shown in
A moving magnet located outside of the device 10 can be used instead of the inductors 56 in order to move the magnet 54. As this motion is performed, the sample flows around the magnet 54 against the chamber walls and “washes” the reagent 14 adhered to the walls off and into the sample 20. The passages connected to the chamber must be sealed to prevent the sample 20 from being pushed back into the passages. The chamber passage design is such that the mixing magnet 54 cannot obstruct the flow of the sample into or the sample/reagent mixture 21 out of the chamber. Another advantage of this method is that the flow passages of the cell may be shorter, thus allowing for a smaller cell. The reagent 14 is mixed in the mixing chamber 16 and the mixture 21 does not have to flow out of and back into the chamber 16.
The flow paths through the device 10 may have other shapes than linear, as shown, and in fact could incorporate many variations to perform a particular analysis. For example,
Having described sample measurement and mixing, several embodiments of the invention, with some variations thereon, will now be described.
(3) Test Method Variations:
The described methods may be altered in at least the following ways.
In all of the embodiments of the device, preferably all liquid passages have smooth radii or tapered transitions because sharp corners damage cells, trapped air and cause dead areas without mixing.
Plasma volume measurements are set to account for losses that occur in the transport from the measuring position to the first reagent position in the mixing chamber 16.
Each system specimen volume will be determined by the sample requirements. Typically, the maximum sample volume is equal to 30% of the specimen volume. Lower percentage i.e. 20% provides better sample quality. Both being of better analytical quality than otherwise available for LOC POC tests.
Volumes are specimen type dependent, previous volumes are for Plasma and are about the worse case.
The disposable test device can include an analyzing device that has several functions: filtering the sample from the specimen; incubating the test unit to the required temperature; controlling sample volume independently for each test type; controlling sample reagent mixing actions independently for each test type, measuring optical transmission of the sample / reagent mixture to verify the quality of the sample; analyzing by optical (turbidity nepherometry or calorimetric), electrical (conductive, impedance, inductance, etc.) or other methods.
The analyzer can be configured to: perform direct or indirect analysis such as optical density measurements, immunoassays or calorimetric assays, and allow additional test components (reagents, diluents) that cannot be incorporated within the device to be added.
This description is based on applications in medical diagnostics using whole blood as the specimen and plasma or serum as the test sample. The invention should not however be limited to these specimens or samples, and can include any body fluid (urine, spinal fluid, saliva and so forth) or any liquid sample as used in pharmaceutical, biotechnology or other industrial laboratories (i.e. cell culture or fermentation samples).
This application claims the benefit of U.S. provisional application No. 60/559,907 filed Apr. 6, 2004, which is incorporated by reference as if fully set forth.
| Number | Date | Country | |
|---|---|---|---|
| 60559907 | Apr 2004 | US |
