JP2005534896A - Disposable cartridge for characterizing particles suspended in liquid - Google Patents

Abstract

The present invention relates to a disposable cartridge for characterizing particles suspended in a liquid, particularly a self-contained disposable cartridge for single use analysis (eg, single use analysis of small amounts of whole blood). Self-contained disposable cartridges facilitate a simple and clear inspection procedure that can be performed by most people without any special education. In addition, the device used to test with this cartridge is simple, light weight, maintenance free, and portable.

Description

  The present invention relates to a disposable cartridge for characterizing particles suspended in a liquid, and particularly to a self-contained disposable cartridge for single use analysis (eg, single use analysis of small amounts of whole blood). This self-contained disposable cartridge facilitates a simple and clear inspection procedure that can be performed by most people without any special education. Furthermore, the device used to perform the inspection with this cartridge can be simple, lightweight, and maintenance-free, and thus can be completely portable, providing a wide range of implementations to the user. The present invention provides a process for pre-analysis handling of samples such as red blood cell hemolysis and inactivation of coagulation.

  Current devices for particle characterization such as counting and sizing are quite expensive, difficult to move and require operation by trained personnel. As a result, many devices are located in dedicated laboratories operated by specialized personnel. In addition, the sample to be analyzed must be transported to this laboratory and the results returned to the requesting person for reporting.

  WO 01/11338 (incorporated herein by reference) discloses an apparatus for characterizing particles suspended in a liquid, the apparatus for removably receiving a disposable cartridge and cartridge Equipped with a docking station. The cartridge passes through a first collection chamber, delimited by a wall containing an orifice for the passage of particles and having an inlet / outlet for connection to a source of positive or negative pressure, and an orifice A housing is provided that includes components of the particle characterization device for characterizing particles (connectable from outside the housing). The docking station has a port for connection to a source of positive or negative pressure and for forming a gas connection with the inlet / outlet when the cartridge is received in the docking station, and the cartridge is a docking station Means for functional connection with the components of the particle characterization device when received in the device.

  WO 02/089670 (incorporated herein by reference) discloses a device for sampling small volumes and precise volumes of liquid. The device comprises a movable member having a cavity for capturing and moving an accurate portion of the liquid sample.

  It is a disadvantage of these prior art devices that some devices are used to perform analyzes (eg, analysis of whole blood samples). Sample collection is performed on a separate device, and the sample must be transferred to another device for sample preparation before it is finally transferred to the analytical sensor.

  WO 99/01742 discloses a disposable sampling device for an apparatus for counting particles contained in a liquid. This sampling device can be connected to the apparatus at a defined location. The device comprises a means for introducing a sample therein, a means for metering a defined volume of sample, a means containing a defined volume of dilution liquid, a dilution chamber, a defined volume for obtaining a diluted sample in the dilution chamber. Means for simultaneously directing the sample and a defined volume of dilution liquid to the dilution chamber, means for directing at least a portion of the diluted sample through the particle counting means and the signaling means connected to the particle counting means, and a housing define Having terminal means located at the outer boundary of the housing at a position corresponding to the position of the terminal means of the apparatus when connected to the apparatus in position.

  During blood analysis using the device described in WO 99/01742, the blood sample is pumped back and forth many times for dilution, mixing and analysis, and the flow system is The pressure in the system is closed so that during the sample movement, it rises above atmospheric pressure and decreases below atmospheric pressure. In addition, sample collection requires pumping with a membrane or other flow actuator that causes blood entry into the flow system of the device. Thus, the disclosed flow system is rather complex.

The particle count is performed in an open end tube as described in WO 99/01742, so that the volume of diluted sample through the particle count sensor is very small.
In order for blood analysis to be accurately recorded by the detection principle, as described in WO 99/01742, it can be seen that different types and concentrations of particles may require separation, degradation, staining or labeling prior to analysis. Not considered.
A blood test sequence as described in WO 99/01742 does not take into account that a user who has not received prior education should be able to learn how to perform this test itself (ie before analysis). The dilution step should not be required).

Thus, it allows sample collection, sample preparation, and particle characterization so that analysis can be performed in a liquid that can be performed in one device without the need for sample handling and sample transfer to another unit. It is an object of the present invention to provide a cartridge for characterizing turbid particles.
It is a further object of the present invention to provide a cartridge that is adapted for single use to be discarded after analysis of one liquid sample.
It is another object of the present invention to provide a cartridge having a simple flow system.
It is yet another object of the present invention to provide a flow system in a cartridge that communicates with the surrounding environment such that the pressure in the flow system is maintained substantially constant at atmospheric pressure.

  In accordance with the present invention, the above and other objects are directed to a first mixing chamber and a first that are separated by a wall that includes an orifice for passage of particles between the first mixing chamber and the first collection chamber. This is achieved by a cartridge for characterizing particles suspended in a liquid comprising a housing with a collection chamber. Particle characterization means are provided for characterizing particles passing through the orifice.

Sample collection can be done through a bore in the outer surface of the housing for entry of a liquid sample. The housing further includes a sampling member positioned movably within the housing. The sampling member has a first cavity for receiving and holding a small, precise volume of liquid. At the first position of the sampling member, the first cavity is in communication with the hole for entry of the liquid sample into the first cavity, and at the second position of the sampling member, the first cavity is In communication with the inlet to the first mixing chamber.
Thus, the sampling member operates to receive and hold a precise volume of liquid sample and to move the sample to the inlet of the first mixing chamber.

Preferably, the liquid to be sampled enters the cavity by capillary attraction causing liquid flow. The use of capillary forces simplifies the flow system, as opposed to WO 99/01742, since no pumps, membranes, syringes or other means of flow generation are required to take a sample.
Thus, the hole may constitute a first capillary tunnel for entry of a liquid sample by capillary attraction. The capillary tunnel is dimensioned such that upon contact between the hole and the liquid to be sampled, a sample of the liquid is drawn into the hole by capillary attraction.
Further, the first cavity may constitute a second capillary tunnel adapted to draw a liquid sample into the first cavity by capillary attraction. Preferably, the first and second capillary tunnels have the same diameter. Also, at the first position, the first and second capillary tunnels preferably extend along substantially the same longitudinal central axis.

Preferably, the sampling member is rotatable about an axis of rotation substantially perpendicular to the long axis of the first cavity.
Additionally or alternatively, the sampling member may move in a direction substantially perpendicular to the long axis of the first cavity.

The surfaces of the inner walls of the first and second capillary tunnels may be hydrophilic, which facilitates capillary attraction of the liquid sample. For example, the tunnel inner wall may be made of a polymer such as glass or polystyrene.
Alternatively, the capillary tunnel wall may be made from another type of material or coated covalently or non-covalently with a hydrophilic material such as a polymer or reagent.
The capillary tunnel may also include one or more reagents attached or chemically bound to the tunnel inner wall. These reagents serve the purpose of further promoting the capillary attraction of the sample, and optionally also causing a chemical reaction in the liquid sample (for example, the purpose of introducing anticoagulant activity in the blood sample). Such reagents may include heparin, EDTA salts, and the like.
Preferably, the sampling member is made from a polymer.

According to a further aspect of the invention, an apparatus for characterizing particles suspended in a liquid is provided. The apparatus comprises a cartridge as disclosed herein, and a docking station that removably receives the cartridge, the docking station functioning with the particle characterization means when the cartridge is received in the docking station. A connector for general connection is provided.
The cartridge may further comprise a cartridge port in communication with the first collection chamber to cause liquid flow through the orifice, and the docking station is configured to dock the cartridge for application of pressure that causes liquid flow through the orifice. It may further comprise a corresponding port for making a gas connection with the cartridge port when received in the station.

  The particle characterization means may comprise a first electrode in the first mixing chamber and a second electrode in the first collection chamber. Each electrode is electrically connected to its own terminal member accessible on the outer surface of the cartridge for functional connection with its own connector when the cartridge is received in the docking station. In general, all necessary electrical and fluid connections to the cartridge are preferably established by snapping the cartridge into the docking station, preferably by simply pushing.

  The first and second electrodes may facilitate particle characterization utilizing well-known Coulter impedance principles, for example for blood cell counting and measurement. This method has become a globally accepted method and is used in most hematology analyzers. Thousands of particles per second can be characterized with a high degree of precision and accuracy using this principle.

With electrical impedance techniques, it is possible to determine the particle volume from the measured values. By maintaining a constant current across the orifice, the recording voltage pulse from the particle replaced by the electrolyte at the orifice has a height proportional to the volume of the particle. This can be considered that the particles are non-conductive compared to the electrolyte, and the electric field (DC or RF) at the center of the orifice is homogeneous (this is usually the diameter D is the length of the orifice). Should be considered small (d <0.2 ^* D) compared to the diameter of the orifice, only one particle passes at a time, This is because the particles pass through the orifice along the length or depth of the orifice.
Typically, such devices are operated so that the flow through the orifice is into the first collection chamber.

Preferably, the length of the orifice is 1-1000 μm, for example about 50 μm. Desirably, the length of the orifice is selected such that there is only one particle in the orifice when detecting particles of 0.1-100 μm diameter. However, considering the homogeneity of the electric field in the orifice, an orifice length that is longer than or equal to the diameter may be required. The counts (some of which can be simultaneous counts of two particles) can be mathematically calibrated by making statistical estimates. The aspect ratio (length or depth / diameter) of the orifice is preferably 0.5: 1 to 5: 1, more preferably 1: 1 to 3: 1.
Preferably, the maximum cross-sectional dimension of the orifice is 5 to 200 μm, such as 10 to 50 μm.

  As explained above, the present invention provides, in a preferred aspect, a sensor based on a film made, for example, on a polymer sheet by laser ablation. The membrane has an orifice positioned relatively in the center of the membrane. This orifice can be used for aspiration of particles suspended in the liquid since the sensor is submerged in the liquid. This method of transporting particles to the measurement region is known for electrical characterization of particles by the Coulter principle (V. Kachel, "Electrical Resistance Pulse Sizing: Coulter Sizing", Flow Cytometry and Sorting, 2. ed., Pp. .80, 1990 Wiley-Liss, Inc.).

  The cartridge may further comprise an inlet / outlet of an intake / exhaust device in communication with the ambient environment for storing substantially ambient atmospheric pressure within the cartridge flow system for facilitating liquid flow through the orifice.

  Preferably, the cartridge is designed to be disposable after a single use. It is desirable that after use, the device does not need to be cleaned before it can be used in a new assay procedure using a new cartridge. Accordingly, liquid leakage from the cartridge upon entry into the docking station should be avoided. For this purpose, the arrangement of the orifices with respect to the inlet / outlet of the inlet / outlet, the second chamber inlet / outlet and the particle characterization device component is preferably such that the liquid has a volume sufficient for the desired particle characterization. The arrangement is such that it can be drawn or pumped through the orifice without passing out of the housing. In general, it should be possible for a given volume, at least 0.1 ml to 10 ml, for example 0.5 ml, of liquid to pass through the orifice while making particle characterization measurements without the liquid leaving the cartridge.

The cartridge may comprise volume metering means to determine the beginning and end of the period when a predetermined volume of liquid has passed through the orifice.
Preferably, the volume metering means comprises a volume metering chamber having an input in communication with the first collection chamber and an output. Here, the presence of liquid is detected at each of the input and output.
For example, the presence of liquid may be detected optically by a change in optical properties of the channel configuration from when it is filled with air to when it is filled with liquid. This can be configured as reflectance or transmittance detection from the surface. Here, the incident light is reflected from the empty channel and transmitted through the filled channel, thus producing a clear shift in the detected reflected or transmitted light.
The input and output of the metering chamber is preferably formed by a narrow channel for accommodating only a small volume of liquid compared to the capacity of the metering chamber. As a result, the actual placement of the capacity metering means (eg optical reflectance detection) in the channel does not substantially affect the accuracy of the measured value of the capacity metering means.
The first mixing chamber or the first collection chamber may constitute a volumetric chamber. However, it is preferred to provide a separate volumetric chamber that facilitates the placement of volumetric metering means (eg optical reflectance detection).
The volume metering means may be arranged to detect when the liquid in the metering chamber is at or above the respective level of the volume metering chamber.
Volumetric metering means may be used to detect when the level of liquid is such that each metering means is filled or unfilled with liquid, so that a fixed volume of liquid passes through the orifice May work to determine the beginning and end of that period. For example, particle characterization may begin when the liquid level just rises above the level of the metering means and end when the liquid level just rises above the second metering means. The volume of liquid passing through the orifice during this period is defined by the separation of the respective metering means.
If the end point of the passage of the defined volume of liquid is that one chamber is effectively emptied below the level of the orifice, then each of the collection chamber and the first mixing chamber (or at least the chamber from which the liquid leaves) Preferably having a cross-sectional area at an orifice level substantially smaller than the cross-sectional area of the chamber over a substantial portion above the high orifice

According to a further aspect of the present invention, there is provided a method of operating a particle characterization device comprising a cartridge as disclosed herein that is removable from the device. The method comprises: sampling a liquid containing particles with a cartridge through a hole having a sampling member in a first position; placing the cartridge in the apparatus; moving the sampling member to a second position; Pumping the liquid in the storage chamber with the liquid sample through the first mixing chamber, making particle characterization measurements, removing the cartridge from the device, and discarding the cartridge.
In general, in all embodiments, it is preferred that all components that wet the sample in use are disposable and that all non-disposable components are reusable without purification.
It is an important advantage of the present invention that the means for liquid sample preparation and analysis is incorporated into a disposable cartridge. For example, the analysis step includes sampling a precise amount of blood, diluting that amount of blood, and finally mixing the blood with a diluent to form a homogeneous solution. The analysis can include spectrophotometric analysis of the liquid.
Thus, in accordance with the present invention, a means is provided for unambiguously performing blood analysis (eg, counting blood cells in a small amount of blood from capillary blood droplets). Means are provided for collecting an accurate amount of blood sample, reagents present in the diluent may be added, for example for dilution and / or chemical preparation of the sample, and mixed sample and diluent Flows through the sensor for analysis of individual blood cells and measurement of the volume of the small amount of liquid analyzed.
As a supplement, spectrophotometric measurements are carried out, for example to quantify the content of hemoglobin.

The cartridge may comprise the following parts:
1. 1. Liquid storage chamber 2. Blood sampling device First mixing chamber 4. 4. Flow-through sensor mechanism First collection chamber 6. 6. A volumetric meter consisting of a chamber and two connected flow channels Hydraulic connection to move liquid through the cartridge

The concept of a disposable unit can be further combined with the following additional parts.
A. B. Optical structure for optical liquid level measurement Electrode for liquid level measurement C.I. Anticoagulation treatment of surface D. For example, reagents in diluents for the modification of blood cells Mixing free (flee) or baffle to aid mixing
F. Multi-capacity metering mechanism for changing capacity G. B. Covering tape covering sample inlet before use Waste chamber for waste / overflow A valve to prevent liquid from exiting through the exhaust pipe. Built-in piston or membrane replaced with external pressure source Windows for spectrophotometric measurements

  The liquid storage chamber (Part 1) holds the required amount of diluent used for blood analysis. When blood is sampled into the cartridge, the diluent is flushed through the capillary to wash out the sampled blood and dilutes the blood as needed for the test. A dilution ratio of 100 to 100,000 is considered to be a normal rating, and a dilution ratio of 500 to 10,000 is preferred. The liquid storage chamber should preferably be constructed to facilitate complete drainage of the chamber. This is accomplished by having a slope at the bottom of the chamber.

  The sampling unit (part 2) may comprise a capillary tube that extends through a movable rod located in a tight-fitting support body. The movable rod is used to capture a precise amount of blood sample. As the blood fills the capillary with capillary forces, the rod rotates and / or moves from the initial position of the support body, thus separating the portion of the capillary that extends through the rod.

  After the support body rod has moved to the second position, the capillary tube forms a liquid path between the liquid storage chamber and the first mixing chamber (part 3). When low pressure is applied to the first mixing chamber, the diluent and blood sample are pushed into the first mixing chamber, where mixing is effected by convection or subsequently by blowing bubbles into the mixing chamber.

  The flow-through sensor mechanism (Part 4) consists of a small orifice in the membrane that establishes a liquid path from the first mixing chamber to the first collection chamber. On each side of the membrane (in the first mixing chamber and in the first collection chamber), electrodes are placed in contact with the liquid.

  The first collection chamber (Part 5) forms a liquid priming function at the rear of the sensor system.

  A volumetric metering system (Part 6) is required for cell concentration measurements. This comprises a volumetric chamber of a known volume with two relatively thin channels connecting the bottom inlet and the top outlet. Detection of liquid at the inlet and outlet can be applied by optical or electrical means.

  The outlet of the volumetric system is connected through a channel (part 7) to a pressure source for moving liquid through the cartridge.

Additional parts to the concept are further described below:
Addition A: Optical detection due to a change in the optical properties of the channel (e.g. a change in reflectivity or transmittance due to air being replaced by liquid in the channel). The top surfaces of the volumetric cell inlet and outlet should be structured to optimize the coupling of the light into the channel. The presence of liquid in the permeable polymer channel results in signal transmission as opposed to reflection in the absence of liquid. This can be recorded by an optical sensor.

  Add B: Two electrodes for liquid level measurement are connected through the body of the cartridge to the inlet and outlet of the volumetric cell, respectively. The electrode is short-circuited with the saline disposed in the electrode disposed in the first collection chamber. This can be recorded through an external electrical mechanism.

  Addition C: Anticoagulation treatment of the surfaces of the sampling structure can be achieved by attaching or chemically bonding selected compounds to these surfaces. Examples of such compounds are heparin and EDTA salts.

  Add D: Reagent in diluent for example for modification of blood cells. This reagent can consist of one or several compounds capable of lysing red blood cells. In addition, to stabilize white blood cells and / or platelets, adjust pH values and osmotic pressure, minimize bacterial growth, modify existing hemoglobin, and minimize batch-to-batch variability In addition, other compounds may be added. The following examples have been included to provide information on the subject of problems related to the performance of self-contained test cartridges.

  Examples of compounds capable of selectively lysing red blood cells include fourth examples as described in, for example, U.S. Pat. A mixture of quaternary ammonium salts.

  Examples of compounds capable of stabilizing white blood cells during hemolysis of red blood cells include N- (1-acetamido) iminodiacetic acid, such as procaine hydrochloride as described in US Pat. No. 4,485,175, and, for example, US 1,3-dimethylurea as described in Japanese Patent No. 4,745,071. In addition, N- (1-acetamido) iminodiacetic acid further assists the quaternary ammonium salt in minimizing residues derived from hemolyzed red blood cells as described, for example, in US Pat. No. 4,962,038, And has been proposed to adjust the pH value (see below).

  Examples of compounds added to adjust the pH value and, more importantly, the osmotic pressure of the diluent include, for example, N- (1-acetamido) iminodiacetic acid, sodium chloride, such as US Pat. No. 4,485,175 and the like. Sodium sulfate as described in US Pat. No. 4,962,038.

  Examples of compounds that can minimize bacterial growth are 1,3-dimethylolurea and chlorhexidine diacetate as described, for example, in US Pat. No. 4,962,038.

  Examples of compounds added to convert hemoglobin species into a final product suitable for spectrophotometric analysis include potassium cyanide and WO 99 /, as described, for example, in U.S. Pat. Tetrazole or triazole as described in 49319.

  Examples of particles or compounds that can be added to introduce tools to minimize variation between different batches of disposable devices are known size latex beads and known size glass beads.

  Addition E: If auxiliary mixing is required, the first mixing chamber may optionally be equipped with mixing free or baffles. Magnetic free may be used to generate convection through a magnetic field moved from outside. The baffle may be used to mechanically agitate the liquid when moved by an externally connected mechanical device. This may be required if mixing with bubbles (eg, bubbles blown into the sample by the sensor) is not appropriate or possible.

  Additional F: A multi-capacity metering mechanism can be included continuously when the test must handle different concentrations of different particles.

  Additional G: A lid or coated tape may be used to cover the sample inlet prior to use. This ensures a clean sampling area at the starting point of the inspection.

  Additional H: A waste chamber may be applied to the outlet of the volumetric cell for liquid disposal or overflow.

  Additional I: A small valve can be incorporated in any connection port (eg, connection port to a pressure source) to prevent liquid from leaking out of the cartridge.

  Addition J: A piston or membrane can be incorporated into the cartridge to include a pressure source for moving the liquid. The piston or membrane can be moved by a mechanical force supplied by the device.

  Additional K: An optical window can be incorporated into the cartridge to perform optical measurements such as spectrophotometric detection of hemoglobin content in a blood sample.

  The described methods can be combined to give the best solution for the final application. Disposable sensors require portable, inexpensive, simple or flexible devices, for example, in small laboratories, for field measurements, or as “care place” (“by the patient”) diagnostic tool It is particularly useful when

  When using the Coulter principle, the diluent for use in the device according to the invention may comprise an inorganic salt that makes the liquid highly conductive. If the sample is applied to an electrolyte, the electrolyte to sample volume should preferably be higher than 10. Sample preparation should result in preferably between 1,000 and 10,000,000 particles / ml, more preferably between 10,000 and 100,000 particles / ml. Mixing of the sample after addition of electrolyte is recommended. The particle size should preferably be within 1-60% of the orifice diameter, more preferably between 5-25% of the orifice diameter. The volume flow rate should preferably be between 10 μl / min and 10 ml / min, more preferably between 100 μl / min and 1 ml / min. For the measurement, a constant current of about 1-5 mA is preferably applied. The current source preferably has a signal to noise ratio (S / N) better than 1,000. The response from the electrode can be electrically filtered by a bandpass filter.

  According to yet another aspect of the invention, a first mixing chamber and a first mixing chamber separated by a wall including a first orifice for the passage of particles between the first mixing chamber and the first collection chamber. A cartridge comprising a housing having one collection chamber, first particle characterization means for characterizing particles passing through the first orifice, and a hole in the outer surface of the housing for entry of a liquid sample Provided. The hole communicates with a first sampling member located in the housing for sampling the liquid sample and having a first cavity for receiving and holding the liquid sample. The first sampling member is in a first position and the first cavity is in communication with the hole for entry of the liquid sample into the first cavity, and in the second position, the first cavity is in the first position. It is movably located in relation to the housing such that it is in communication with the first mixing chamber for the discharge of the liquid sample into the one mixing chamber.

  The cartridge includes a second mixing chamber and a second collection chamber separated by a second wall including a second orifice for passage of particles between the second mixing chamber and the second collection chamber; Second particle characterization means for characterizing particles passing through the second orifice may further be provided.

  In one embodiment of the invention, the first cavity has a first cavity for liquid entry from the first mixing chamber into the first cavity when the first sampling member is in the first position. In communication with the mixing chamber and at a third position of the first sampling member, the first cavity is a second mixing chamber for the discharge of liquid in the first cavity to the second mixing chamber. Is in communication with

  In another embodiment of the invention, the cartridge is positioned in the housing for sampling a small, precise volume of liquid from the first mixing chamber and has a second cavity for receiving and holding the sampled liquid. And a second sampling member. The second sampling member is in a first position, wherein the second cavity is in communication with the first mixing chamber for entry of liquid from the first mixing chamber to the first cavity, In position, movable relative to the housing such that the second cavity is in communication with the second mixing chamber for the discharge of the sampled liquid in the second cavity to the second mixing chamber Located in.

The cartridge may further comprise a reagent chamber located adjacent to the first mixing chamber to hold a reagent to enter the first mixing chamber.
Preferably, the cartridge further comprises a breakable seal separating the reagent chamber and the first mixing chamber.

In this embodiment, different chemical treatments of different parts of the liquid sample may be performed.
In this embodiment, further dilution of the liquid sample may also be performed.

The invention will be further described and explained with reference to the accompanying drawings.
FIG. 1 shows a side sectional view of the components of a disposable unit 85 (referred to as a cartridge).
FIG. 2 shows the concept of a flow-through sensor.
FIG. 3 comprises a device based on a disposable cartridge, a docking station 66 and a reader 74.
FIG. 4 shows a cartridge with a built-in piston.
FIG. 5 schematically illustrates the sampling procedure.
FIG. 6 is a plot of the results obtained in Example 1.
FIG. 7 is a plot of the results obtained in Example 2.
FIG. 8 is a plot of the results obtained in Example 3.
FIG. 9 is a plot of the results obtained in Example 4.
FIG. 10 is a plot of the results obtained in Example 5.
FIG. 11 is a schematic illustration of the cartridge and hydraulic connection of Example 6.
FIG. 12 is a plot of the process described in Example 7.
FIG. 13 is a plot of the process described in Example 8.
FIG. 14 schematically shows a second embodiment of the cartridge.
FIG. 15 schematically shows a third embodiment of the cartridge.
FIG. 16 shows a device according to the invention in perspective.

FIG.
A disposable cartridge having a housing 85 for blood analysis is located in the liquid storage chamber 1 containing the liquid diluent 11, in the housing 85 for sampling the blood sample 8 and for receiving and holding the blood sample 8. A first sampling member 2 having 10 is provided. The member 2 is in a first position where the cavity 10 is in communication with the hole 90 for entry of the blood sample 8 into the cavity 10 by capillary forces, and in the second position, the cavity 10 is in contact with the liquid diluent 11. Is movably located in relation to the housing 85 so as to be in communication with the liquid storage chamber 1 and the mixing chamber 3 for the discharge of the blood sample 8 diluted by The mixing chamber 3 is separated from the collection chamber 5 by a wall containing an orifice 59 for the passage of the blood sample 8 between the mixing chamber 3 and the collection chamber 5. The wall containing the orifice 59 forms part of the flow-through sensor 4.

  The volume metering mechanism is connected to the collection chamber and has two connection channels 12, 13 with a relatively small internal volume for recording the entry and exit of liquids by optical or electrical means to measure during the measurement. A volume metering chamber 6 having a power capacity size is provided. A channel 7 is drawn from the volumetric chamber to a connection port 67 where pressure can be applied.

FIG.
The flow-through sensor 4 has a partition wall 94 having a relatively narrow passage 59 for the passage of particles suspended in the liquid. The orifice serves as a sensing area for the detection and measurement of individual cells. The orifice in the sensor may be formed as a counting orifice for counting and measuring particles by an impedance method known as Coulter counting. The particles can be drawn through the orifice by a flow driven to pressure in either direction. When saline or other electrolyte solution is added to the chamber, the two chambers are electrically isolated from each other except for a current path provided by a passage through the orifice.

FIG.
The chamber on each side of the flow-through sensor may have electrodes 34, 35 extending from the external terminals 61, 62 through the base wall 63 of the disposable unit and facing the inside of the chamber. To perform the inspection, the cartridge is placed in the docking station 66 of the portable device. The docking station 66 has a cup-shaped housing having a base 70 and a peripheral side wall 71. The base 70 has its own spring-loaded electrical connectors 64, 65 for automatically contacting the cartridge terminals 61, 62 when the cartridge is received in the docking station as a push fit. There is also a conduit 68 that passes through the base wall 70 in alignment with the conduit 67 of the cartridge. The conduit 67 has a seal 69, such as an O-ring, at the opening to the top surface of the wall 70 to form an airtight connection with the bottom surface of the base wall 63 of the cartridge. A vacuum pump 72 connects to the lower end of conduit 68 by line 73. In a variation of this device, the vacuum pump 72 can be reversed to apply a positive pressure to the conduit 68. Shown schematically at 74 is a further conventional component of the Coulter counter, including all electronic circuitry and display necessary for the operation of the device.

FIG.
As an alternative to a gas pump, a piston 9 can be provided in the cartridge for direct application of negative or positive pressure.

FIG.
FIG. 5 schematically illustrates the blood sampling operation. The depicted portion of the cartridge 2 includes a liquid storage chamber 83 for storing a diluent for diluting the sample and a first mixing chamber 77 for mixing the sample 84 and the diluent. This figure schematically illustrates a device for sampling small and accurate amounts of liquid according to the present invention. Device 10 has a first opening 87 for entry of the liquid sample into hole 75 in first member 86 and a second opening 76 for outputting the liquid sample from hole 75. A first member 86 is provided. The hole 75 forms a capillary tunnel. The first opening 87 of the first member 86 can be brought into contact with the liquid 8 (shown in FIG. 1), 84 to be sampled, so that the liquid 84 is brought into contact with the first opening by capillary attraction. Flow through 87 into hole 75 and out of second opening 76. The device 12 further comprises a sampling member 78 having a first cavity 82 for receiving and holding a liquid sample 84 and having a third opening 88 in communication with the first cavity 82. The first cavity forms a capillary tunnel having substantially the same diameter as the hole 75. The sampling member 78 is a cylindrical body arranged so as to be movable relative to the first member 86. During liquid sampling, the sampling member 78 is located in a first position illustrated relative to the first member 86. In this position, the second opening 76 is in communication with the third opening 88, so that the sampled liquid passes through the second opening 76 and the third opening 88 to the second. It can flow into one cavity 82 by capillary attraction. The third opening 88 may be disconnected from the second opening 76 at a second position of the sampling member 78 relative to the first member 86 and, as a result, included in the first cavity 82. The liquid sample 84 to be connected is disconnected from the hole 75.

  Sampling member 78 is inserted into third cavity 34 of first member 86 to receive and receive a portion of sampling member 78. The sampling member 78 moves between a first position and a second position along the long axis of the sampling member 78 (which is also substantially perpendicular to the long axis of the first cavity 82). May be. Sampling member 78 may also be rotatable about a longitudinal axis that is substantially perpendicular to the long axis of first cavity 82. In the first position, the first capillary tunnel 75 and the second capillary tunnel 82 extend along substantially the same central longitudinal axis.

  In the illustrated embodiment, the first member 86 is symmetric, has a fourth cavity 80 with openings 81, 79 opposite the hole 75, and the sampling member 78 faces the opening 88. And, as a result, in the first position, the capillary tunnel extends through the first member 86 and the second member 78 and communicates with the surrounding environment through the openings 87, 79. . Thus, air can escape from the capillary tunnel through the opening 79. Further, in the first position, a portion of the liquid that enters the first cavity 82 exits the cavity 82 through the opening 89. This ensures that cavity 82 is completely filled with liquid during liquid sampling and eliminates the risk of sampling with reduced sample volume leading to less accurate sampling.

  FIG. 5a illustrates a device 2 prepared for receiving a liquid. In FIG. 5b, the sample has entered the capillary tunnel 82. In FIG. 5c, the sampling member 78 has been rotated to the second position for separation of the correct volume sample 84. Finally, FIG. 5d shows that the sample 84 has been washed out from the capillary tunnel 82 into the first mixing chamber 77 with diluent.

Example: The capillary tunnel constituting the first cavity 82 may have a length of 8 mm and a diameter of 0.9 mm to contain 5.089 μL of blood sample.
Example: The capillary tunnel that constitutes the first cavity 82 may have a length of 5 mm and a diameter of 0.5 mm to contain a 0.982 μL blood sample.
Example: The capillary tunnel that constitutes the first cavity 82 may have a length of 3 mm and a diameter of 0.3 mm to contain 0.212 μL of blood sample.

FIG.
Example 1 Measurement of Polymer Beads A mixture of 5 μm particles and 10 μm particles suspended in an electrolyte was aspirated through the orifice of the apparatus shown in FIG. The number of detected particles and the size of each detected particle were recorded. A bimodal distribution of detected particle sizes is clearly seen in FIG.

FIG.
Example 2 Counting of Red Blood Cells Blood cells were measured and the results are shown in FIG. As can be understood from FIG. 7, the red blood cells are usually around 5 to 7 μm in diameter and are present in the whole blood with the highest frequency. The distribution is a Gaussian curve, as expected. Blood cell count can be used for clinical diagnosis. It is considered a basic parameter for hematology (see "Fundamentals of Clinical Haematology", Stevens, WB Saunders Company, ISBN 0-7216-4177-6). It is quite easy to count thrombocyte)

FIG.
Example 3 White Cell Count Material Using Diluent Containing Reagent Composition Selected to Store All Blood Cells Cartridge and Device with Functions as Described in the Present Invention Sodium Chloride 7.9 g / L, including potassium chloride 0.4 g / L, disodium hydrogen phosphate 1.9 g / L, sodium dihydrogen phosphate 0.2 g / L, 2 sodium-EDTA 0.4 g / L and sodium fluoride 0.3 g / L, Beckman Coulter (product number 24655)
Vacutainer (K3E, Becton & Dickinson, product number 367652)
Bayer, ADVIA-120 equipment

The complete flow of the execution procedure is as follows:
-Collect venous blood sample in Vacutainer tube-Leave sample for 3 hours to allow the sedimentation process to proceed-Extract plasma phase containing most of buffy coat segment-Bayer to obtain comparative value for leukocyte content Perform analysis using the Advia 120 instrument − Add 5.00 ml of isotonic solution as diluent to the chamber of the test rig − Add 10.0 μl of sample to the chamber − Mix the liquid in the chamber − Test with the computer Start the sequence (start pumping and prepare for sampling)
-Start sampling when liquid reaches the first level electrode-Stop sampling when liquid reaches the second level electrode-Save the sampled value to a file-Analyze the data and red blood cells Open the file using “pulse-viewer” to calculate the results using a calculation method that includes subtraction of overlapping white blood cells.

result
Bayer Advia-120: 11.96 × 10 ⁹ leukocytes / L
Inspection rig: 11.92 × 10 ⁹ leukocytes / L
Difference in accuracy: (11.96−1.92) /11.96=0.33%

FIG.
Example 4 White Blood Cell Separation Material Using Diluent Containing Reagent Composition Selected to Hemolyze Red Blood Cells Mainly Cartridge and Device with Functions as Described in the Present Invention Procaine Hydrochloride 0.10 g / L, 1 , 3-Dimethylolurea 0.90 g / L, N- (1-acetamido) iminodiacetic acid 1.28 g / L, dodecyltrimethylammonium chloride 7.51 g / L and sodium chloride 0.03 g / L
Vacutainer (K3EDTA, Becton & Dickinson, product number 367652)

The complete flow of the execution procedure is as follows:
-Collect venous blood sample in Vacutainer tube-Leave sample for 3 hours to proceed with sedimentation process-Extract plasma phase containing most of buffy coat section-Dilution of 2.000 ml as above into test rig chamber Add agent-Add 4.0 μl sample to chamber-Mix liquid in chamber-Start test sequence on computer (start pumping and prepare for sampling)
-Start sampling when the liquid reaches the first level electrode-Stop sampling when the liquid reaches the second level electrode-Save the sampled value to a file-Analyze the data and Open the file using "pulse-viewer" to create it.

Results As can be seen in the histogram of FIG. 6, the particle population corresponding to the white blood cells is easily identified in the absence of red blood cells.

FIG.
Example 5-Somatic Cell Count Milk quality is important for farmers, daily producers and consumers. Farmers must deliver milk of a certain quality. Quality is controlled by the so-called somatic cell count (SCC). In the examination of milk quality, somatic cells in milk are counted to determine infection (clinical mastitis). For daily resale, farmers must meet the limit of 400,000 cells / ml. Dietary changes, stress or mastitis lead to higher SCC levels, thus reducing milk quality and consequently lower price per unit volume. Inexpensive cell counters will help farmers and daily growers to monitor SCC levels.

FIG.
Example 6 Hematology Diagnostic System This is an example of a three part discriminating white blood cell count (monocytes, lymphocytes, granulocytes), platelets and hemoglobin measurements and corresponding devices and cartridges realized through the present invention.
A three-part discriminating white blood cell, platelet counter with hemoglobin measurement can be achieved with dedicated components.

  A lysis reagent for selectively lysing red blood cells is added to the diluent in the storage chamber 1. As whole blood 8 is added to the opening 58 of the first capillary compartment 15, blood is drawn into the capillary and through the middle compartment 10 and the final compartment 14 of the capillary. The final compartment of the capillary connects to the filling chamber 43 for visual confirmation of fullness. The filling chamber 43 connects to open air through a conduit 44.

  The intermediate section filled with capillary blood is moved to the second position to connect the two ends of the capillary to the other two conduits (the conduit 45 connecting to the storage chamber 1 and the first mixing chamber 3, respectively). It is the part of the knob 2 that can be connected to the second conduit 40). A third conduit 39 leads from the first mixing chamber to the port opening 42 of the cartridge. The port openings connect to the three position valve 51 through the tubing 46 through the pair of port openings 37 of the device and through the second tubing 55 through the two positions of the valve, or are directed to open air, or It is guided to the suction port of the membrane pump 47 through the third pipe 50.

  When diluent containing blood and reagents is aspirated into the first mixing chamber, the blood can be mixed by blowing bubbles through the orifices of the sensor 4. Air pressure is applied through the collection chamber 5 through the fourth conduit 12A, the small volume chamber 6A, the fifth conduit 12B, the large volume chamber 6B and the sixth conduit 7 which are directed to the cartridge opening port 41. The device pair port 36 connects to a second three-position valve 52 through a fourth tubing 48. This three-position valve leads both to the vacuum port through the fifth tubing 56 to the suction port of the membrane pump or through the third two-position valve 53 and the sixth tubing 49 to the exhaust system of the membrane pump. Has the position of The third valve has two positions for connection and for directing pump exhaust to open air through the seventh tubing 54, respectively.

  Diluted and lysed blood (with red blood cells removed) is ready for measurement after mixing. The first mixing chamber connects to open air through a first valve and the collection chamber connects to the pump suction port through a second valve. The exhaust device of the membrane pump is connected to open air through a third valve. As blood and diluent flow from the first mixing chamber to the collection chamber, an electrical connection between the counter electrodes 34 and 35 disposed in each chamber is established through the liquid. Cells are counted and differentiated by size according to the Coulter principle. Depending on the size of the cells, the cells can be discriminated and classified into different groups containing certain types of cells. Thus, white blood cells (leukocytes) can be distinguished into granulocytes, lymphocytes and monocytes. Furthermore, platelets can be distinguished from leukocytes as well. In order to determine the concentration, the volume of diluted blood (counted) must be known. Since platelets are about 10 times as many as white blood cells, it may be necessary to measure two different volumes. Platelets are counted according to a small volume chamber 6A located between the collection chamber and the larger volume. A counting period is provided by recording the entry and exit of the liquid at the entrance and exit of the small volume chamber, respectively. Liquid level recording is preferably done by optical reflectance measurements at the inlet 33 and outlet 32. The outlet of the small volume chamber is also the inlet of the large volume chamber 6B. This chamber is used in connection with the white blood cell count. At the exit of the large volume chamber, a third optical reflectance measurement 31 is performed to record the liquid exiting from this chamber.

  After counting both white blood cells and platelets, the hemoglobin content can be measured, preferably by optical spectrophotometry through the middle compartment of the large volume chamber 30.

Procedure for Inspection (Example 6)
1) Use a lancet device to draw blood 2) Collect blood droplets by touching blood to the cartridge inlet 3) Mount the cartridge on the device (the device starts the test and does it)
4) Read the result from the display 5) It can be characterized that the cartridge is removed and discarded.

FIG.
Example 7 Photolithography Orifices can be suitably formed in a photoreactive polymer by photolithography and subsequent development. Thus, a free standing sheet of a polymer of the type traditionally used as a photoresist material is exposed to define an orifice to make the spot soluble (or to make the non-spot forming area insoluble), Subsequently, the material is removed by developing with a solvent to form an orifice. Usually, multiple count wafers, each containing an orifice, are made simultaneously on one sheet. Suitable photoresist polymers are described in M. Madou “Fundamentals of Micro fabrication, CRC Press LLC, 1997, ISBN 0-8493-9451-1. These include AZ-5214E, SU8, polyamide, and the like.
Alternatively, the photoresist polymer may be used as a protective layer on a substrate, such as silicon, where an orifice is formed by etching the exposed areas by developing the photoresist. The etched substrate, if it is conductive, may be insulated before use by forming a suitable insulating layer thereon. Photoresist polymers may be used as such layers.

  A lithographically produced count wafer may be used in all forms of apparatus and methods according to the present invention. FIG. 12 shows one process for making a count wafer: (a) Application of a thin sheet of photoresist; (b) Mask development; (c) Deep Reactive Ion Etching (DRIE, M. Madou “Fundamentals of Micro Fabrication, CRC Press LLC, 1997, ISBN 0-8493-9451-1).

FIG.
Example 8 Orifices Made by Laser Micromachining Orifices for coulter counting can be made by laser micromachining of polymers, leading to a simple way to make and assemble orifices for cartridges. A series of 50 μm small orifices have been manufactured with UV lasers. Holes are created in less than 10 ms in a 50 μm polymer sheet. The uniformity of the holes is very high and the smoothness of the orifice entrance is unique. FIG. 13 shows the process of laser micromachining of the orifice. The laser cuts into a circle through the polymer foil, thus defining the size of the orifice.

FIG.
FIG. 14 schematically shows a preferred embodiment of a cartridge according to the invention. The illustrated cartridge has a first member 104 for sampling blood. The member 104 has three positions, a first position for blood sampling, a second position connecting the first storage chamber 103 to the first mixing chamber 112, and a second storage chamber 105 in the second position. A third position connected to the mixing chamber 110 is movably positioned in relation to the housing 100. Blood is passed through the hole 122 to the first cavity of the member 104 by capillary force or by depressurization at the end of the sampling channel 111. A liquid shut-off valve 116 is placed after the first sampling member to prevent blood from passing through the channel. After blood sampling, the sampling member switches to the second position and the sample is swept into the first mixing chamber 112 by the liquid in the first storage chamber 103. In the first mixing chamber 112, the sample is diluted 1: 200 with the liquid in the first storage chamber 103 and a fraction is flowed back into the first cavity of the sampling member 104. The sampling member 104 switches to the third position so that the diluted sample is swept into the second mixing chamber 110 by the liquid in the second storage chamber 105. In the second mixing chamber 110, the sample is further diluted 1: 200 with the liquid in the second storage chamber 105 to a total dilution ratio of 1: 40,000. The hemolytic reagent is injected into the first mixing chamber 112 by the piston 115. The piston breaks the seal 118 between the reagent chamber 119 and the first mixing chamber 112. After hemolysis, the 1: 200 diluted sample is ready to count non-hemolyzed white blood cells and to measure hemoglobin by spectroscopic measurement. White blood cells are counted by passing through the first orifice 113 and measuring the response by impedance cell count at the first electrode pair 117,120. A fixed volume is counted by a first volume metering mechanism 107 connected to the first collection chamber 114. A first overflow capacity 106 is disposed after the first capacity metering mechanism 107. White blood cells can be distinguished by the volume after adding a lysis reagent to the blood. White blood cells can be grouped into granulocytes, monocytes and lymphocytes by volume. The three groups together add up to the total number of white blood cells.

  In the second mixing chamber 110, red blood cells and platelets are counted. Red blood cells and platelets are counted by passing the second orifice 109 and measuring the response by impedance cell count at the second electrode pair 106,121. A fixed volume is counted by a second volume metering mechanism 101 connected to the second collection chamber 108. A second overflow capacity 102 is arranged after the second capacity measuring mechanism 101.

  This embodiment may further comprise an additional optical detector for spectroscopic measurement of hemoglobin content. When simply referring to “total hemoglobin”, this test involves lysing red blood cells and thus creating a solution with uniform distribution of hemoglobin in the sample. Hemoglobin is chemically converted to a methemoglobin triazole-complex that is more stable and easily measured. This is a colored compound that can be measured by colorimetry, and this concentration is calculated from the amount of light absorption using Beer's law. This method requires a measurement of hemoglobin at about 540 nm with high absorption and a turbidity calibration measurement at 880 nm with low absorption.

Figure 15
FIG. 15 schematically shows another preferred embodiment of a cartridge according to the present invention. The illustrated cartridge has a first member 104 for sampling blood. The member 104 is movable in relation to the housing 100 between two positions, a first position for blood sampling and a second position connecting the first storage chamber 103 to the first mixing chamber 112. Placed in. The blood sample is passed through the hole 122 to the first cavity of the member 104 by capillary force or by depressurization at the end of the sampling channel 111. A liquid shut-off valve 116 is placed after the first sampling member to prevent blood from passing through the channel. After blood sampling, the sampling member switches to the second position and the sample is swept into the first mixing chamber 112 by the liquid in the first storage chamber 103. In the first mixing chamber 112, the sample is diluted 1: 200 with the liquid in the first storage chamber 103.

  The cartridge is located in the housing 100 for sampling a small, precise volume of liquid from the first mixing chamber 112 and has a second cavity 123 for receiving and holding the sampled liquid. The sampling member 123 is further provided. The member 123 is in a first position with the second cavity 123 in communication with the first mixing chamber 112 for entry of diluted sample from the first mixing chamber 112 into the first cavity 123, At position 2, the second cavity 123 is in communication with the second mixing chamber 110 so that the diluted sample is swept into the second mixing chamber 110 by the liquid in the second storage chamber 105. In addition, it is arranged to be movable in relation to the housing 100. In the second mixing chamber 110, the sample is further diluted 1: 200 with the liquid in the second storage chamber 105 to a total dilution ratio of 1: 40,000. A hemolysis reagent is injected into the first mixing chamber 112 by the piston 115. The piston breaks the seal 118 between the reagent chamber 119 and the first mixing chamber 112. After hemolysis, the 1: 200 diluted sample is ready to count non-hemolyzed white blood cells and to measure hemoglobin by spectrophotometry. White blood cells are counted by passing through the first orifice 113 and measuring the response by impedance cell count at the first electrode pair 117,120. A fixed volume is counted by a first volume metering mechanism 107 connected to the first collection chamber 114. A first overflow capacity 106 is disposed after the first capacity metering mechanism 107. White blood cells can be distinguished by the volume after adding a lysis reagent to the blood. White blood cells can be grouped into granulocytes, monocytes and lymphocytes by volume. The three groups together add up to the total number of white blood cells.

  In the second mixing chamber 110, red blood cells and platelets are counted. Red blood cells and platelets are counted by passing the second orifice 109 and measuring the response by impedance cell count at the second electrode pair 106,121. A fixed volume is counted by a second volume metering mechanism 101 connected to the second collection chamber 108. A second overflow capacity 102 is arranged after the second capacity measuring mechanism 101.

  This embodiment may further comprise an additional optical detector for spectrophotometric measurement of hemoglobin content. When simply referring to “total hemoglobin”, this test involves lysing red blood cells and thus creating a solution with uniform distribution of hemoglobin in the sample. Hemoglobin is chemically converted to a methemoglobin triazole-complex that is more stable and easily measured. This is a colored compound that can be measured by colorimetry, and this concentration is calculated from the amount of light absorption using Beer's law. This method requires a measurement of hemoglobin at about 540 nm with high absorption and a turbidity calibration measurement at 880 nm with low absorption.

A side sectional view of the components of a disposable unit 85 (referred to as a cartridge) is shown. The concept of a flow-through sensor is shown. A device based on a disposable cartridge, a docking station 66 and a reader 74 is provided. Fig. 2 shows a cartridge with a built-in piston. The sampling procedure will be schematically described. 2 is a plot of the results obtained in Example 1. 3 is a plot of results obtained in Example 2. 4 is a plot of results obtained in Example 3. 10 is a plot of results obtained in Example 4. 10 is a plot of results obtained in Example 5. 7 is a schematic illustration of a cartridge and hydraulic connection of Example 6. 10 is a plot of the process described in Example 7. 9 is a plot of the process described in Example 8. Fig. 2 schematically shows a second embodiment of the cartridge. Figure 3 schematically shows a third embodiment of the cartridge. 1 shows a device according to the invention in perspective.

Claims (28)

A first mixing chamber and a first collection chamber separated by a wall including a first orifice for passage of particles between the first mixing chamber and the first collection chamber A first particle characterization means for characterizing particles comprising a housing having a hole in the outer surface of the housing for entry of a liquid sample;
A hole is located in the housing for sampling the liquid sample and has a first cavity for receiving and holding the liquid sample, where the first cavity is the liquid sample into the first cavity In relation to the housing so as to be in communication with the hole for entry of the liquid and in communication with the first mixing chamber for discharge of the liquid sample into the first mixing chamber at the second position. Communicating with a first sampling member movably located;
A cartridge for characterizing particles suspended in a liquid sample. Second mixing chamber and second collection chamber separated by a second wall including a second orifice for the passage of particles between the second mixing chamber and the second collection chamber Second orifice A second particle characterization means for characterizing particles passing through the first cavity, wherein the first cavity is for the liquid to enter the first cavity from the first mixing chamber at the second position. In communication with the first mixing chamber, and in a third position, the first cavity is in communication with the second mixing chamber for release of liquid in the first cavity to the second mixing chamber. The cartridge according to claim 1. A second mixing chamber and a second collection chamber separated by a second wall including a second orifice for passage of particles between the second mixing chamber and the second collection chamber;
A second particle characterization means for characterizing the particles passing through the second orifice, and a housing located to receive a sampled liquid for sampling a small and precise volume of liquid from the first mixing chamber; A second cavity for holding, and in the first position, the second cavity is in communication with the first mixing chamber for entry of liquid from the first mixing chamber to the first cavity; And in a second position, the housing such that the second cavity is in communication with the second mixing chamber for the discharge of the sampled liquid in the second cavity to the second mixing chamber. The cartridge according to claim 1, further comprising a second sampling member that is movably disposed in the relationship.   4. A cartridge according to any of claims 1 to 3, further comprising a reagent chamber located adjacent to the first mixing chamber for holding a reagent to be entered into the first mixing chamber.   The cartridge of claim 4, further comprising a breakable seal that separates the reagent chamber from the first mixing chamber.   At least one of the first and second particle characterization means has a first electrode in each of the first and second mixing chambers and a second in each of the first and second collection chambers. The cartridge according to claim 1, further comprising an electrode, wherein each electrode is electrically connected to each terminal member accessible on the outer surface of the cartridge.   The housing further comprises a first liquid storage chamber for holding liquid, wherein the first liquid storage chamber is a first position of the first sampling member and the liquid is first from the first liquid storage chamber. 7. A cartridge according to any one of the preceding claims, in communication with the first cavity so that it can be discharged with the liquid sample through the first cavity of the sampling member into the first mixing chamber.   The housing further comprises a second liquid storage chamber for holding liquid to be discharged from the second liquid storage chamber to the second mixing chamber along with the liquid sample through respective chambers of the first and second cavities. The cartridge according to any one of 1 to 7.   9. A cartridge according to any one of the preceding claims, comprising volume metering means for determining the beginning and end of a period during which a predetermined volume of liquid has passed through at least one of the first and second orifices.   12. A cartridge according to claim 11, wherein the volume metering means comprises a volume metering chamber having an input and an output in communication with the respective collecting chamber, wherein the presence of liquid is detected at each of the input and output.   13. A cartridge according to claim 12, wherein the presence of liquid is detected at a second electrode located at the input and a further second electrode located at the output.   14. A cartridge according to claim 13, wherein the presence of liquid is detected optically.   13. Each of the mixing chamber and collection chamber has a cross-sectional area at the level of its own orifice that is substantially less than the cross-sectional area of its own chamber over a substantial portion of the chamber height above its own orifice. The cartridge described.   The cartridge according to any one of claims 1 to 13, wherein the surface defining the first cavity of the first sampling member has an anticoagulant reagent.   15. A cartridge according to any one of the preceding claims, wherein the first liquid storage chamber holds a chemical reagent for blood sample modification.   The cartridge according to claim 1, wherein the mixing member is disposed in at least one of the mixing chambers.   The cartridge according to claim 18, wherein the mixing member is of a magnetic type.   The cartridge according to claim 1, further comprising a sensor for characterizing the liquid.   21. A cartridge according to claim 20, wherein the sensor for liquid characterization is adapted for spectrophotometric characterization of the liquid.   20. A cartridge as claimed in claim 1-19, further comprising a pump chamber having a pump actuator in which the housing communicates with one of the first and second collection chambers and causes fluid flow through the respective orifice.   The cartridge of claim 22, wherein the pump actuator is a piston.   The cartridge of claim 22, wherein the pump actuator is a membrane. Sampling a liquid containing particles through a hole using a first sampling member in a first position with a cartridge;
Placing the cartridge in the device,
Moving the sampling member to the second position;
Pumping the liquid in the first storage chamber with the liquid sample through the second cavity into the first mixing chamber;
Making particle characterization measurements,
23. A method of operating a particle characterization device comprising a cartridge according to any of claims 1-22, comprising removing the cartridge from the device and discarding the cartridge. Sampling a liquid containing particles through a hole using a first sampling member in a first position with a cartridge;
Placing the cartridge in the device,
Moving the first sampling member to the second position;
Pumping the liquid in the first storage chamber with the liquid sample through the first cavity into the first mixing chamber;
Sampling a liquid sample from the first mixing chamber with a second sampling member in a first position;
Moving the second sampling member to the second position pumping the liquid in the second storage chamber with the liquid sample through the second cavity into the second mixing chamber;
Making particle characterization measurements,
25. A particle comprising a cartridge according to claim 3 or any of claims 4 to 24 dependent on claim 3, comprising removing the cartridge from the device and discarding the cartridge, wherein the cartridge is removable from the device A method of operating a characterization device. 25. A cartridge according to any of claims 1 to 24, and a connector for removably receiving the cartridge for functional connection with the particle characterization means when the cartridge is received in a docking station. An apparatus for characterizing particles suspended in a liquid comprising a docking station.   The cartridge further comprises a first port in communication with the first collection chamber to cause liquid flow through the first orifice, and the docking station is adapted to apply pressure to cause liquid flow through the orifice. 28. The apparatus of claim 27, further comprising a port for forming a gas connection with the cartridge port when received in the docking station.   25. A cartridge according to any one of claims 2 to 24, wherein the docking station further comprises a connector for functional connection with the second particle characterization means when the cartridge is received in the docking station. 29. Apparatus according to claim 27 or claim 28.   The cartridge further comprises a second port in communication with the second collection chamber to cause liquid flow through the second orifice, the docking station for applying pressure to cause liquid flow through the second orifice 30. The apparatus of claim 29, further comprising a second port for forming a gas connection with the second cartridge port when the cartridge is received in the docking station.

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