AU2021356770B2

AU2021356770B2 - System for determining a biological condition and cartridge therefor

Info

Publication number: AU2021356770B2
Application number: AU2021356770A
Authority: AU
Inventors: Zion Botesazan; Warren Edward FARNAM III; Ilan SEMMEL; Eric STIMPSON
Original assignee: Accellix Ltd
Current assignee: Accellix Ltd
Priority date: 2020-10-07
Filing date: 2021-10-07
Publication date: 2025-05-08
Anticipated expiration: 2041-10-07
Also published as: EP4225501A1; AU2021356770A1; WO2022074657A1; CN116438455A; JP2023545415A; CN116438455B; KR20230079276A; AU2025210862A1; EP4225501A4

Abstract

The presently disclosed subject matter relates to systems, cartridges and methods for determining a biological condition, and more specifically to flow cytometry systems and methods for determining a biological condition.

Description

SYSTEM FOR DETERMINING A BIOLOGICAL CONDITION AND CARTRIDGE THEREFOR TECHNOLOGICAL FIELD

[001] The presently disclosed subject matter relates to systems and methods for determining a biological condition, and more specifically to flow cytometry systems and methods for determining a biological condition. BACKGROUND

[002] There are numerous medical conditions which are most reliably diagnosed by a laboratory test performed on a bodily specimen. Typically, these tests are performed in a laboratory facility remote from that in which the patient is being treated.

[003] Turnaround times for known diagnostic assays can be as much as 30-120 minutes. Often, the time lost in waiting for laboratory results can lead to further deterioration of a condition in a patient, and sometimes death. In some cases, the physician has to act without having laboratory results. This can lead to providing the patient with an inappropriate treatment. Accordingly, decreasing the time necessary to detect a biological condition in a sample taken from a patient, as well as providing means to perform a suitable assay outside of a laboratory facility, would be associated with more appropriate treatments by doctors, and decreased mortality rates in some cases.

[004] One of the drawbacks of current assay methods is the need to pre-process the sample prior to introducing it to an analytical device for measurement. Such pre-processing requires reliable and accurate training of laboratory personnel. In addition, such pre-processing is often time consuming, it requires additional laboratory instruments and tools and it imposes significant biohazard risk and multiple sources for error in the assay result.

SUMMARY

[005] In one embodiment, this invention provides a method for analyzing a biological sample, the method comprising: . inserting a biological sample into a cartridge; • inserting the cartridge into an analytical instrument; • initiating measurement;

• obtaining a result.

[006] According to this aspect and in one embodiment, methods of the present invention eliminate the need for manual pre-processing. According to this aspect and in one embodiment, pre-processing is conducted automatically within the cartridge.

[007] Methods of this invention provides a fast, easy, safe, accurate and reliable diagnostic means that can be conducted at any location, and immediately following withdrawal of e.g. a blood sample from a patient.

[008] According to this aspect and in one embodiment, following withdrawal of a blood sample from a patient, the sample or a portion of it is injected to the cartridge, and the cartridge is conveniently inserted into the instrument. Using a touch display, a measurement is quickly initiated, and a diagnostic result is automatically obtained.

[009] According to this aspect and in one embodiment, no manual or semi-manual pre-processing of the sample is required prior to introducing it to an analytical device for measurement. Accordingly, there is no need for high-level training of laboratory personnel, thus enabling on-site operation by emergency medical assistants, medical technicians etc. Methods of this invention eliminate the need for tedious pre-processing, thus saving precious time. No additional pre processing laboratory instruments and tools are required and biohazard risk from bench pre processing is eliminated. Methods of this invention also eliminate the sources of error arising from manual pre-processing.

[0010] In one embodiment, an opto-mechanical analytical instrument and a cartridge inserted thereto are utilized for conducting an assay according to methods of this invention (see figure 1). In one embodiment, the optomechanical instrument comprise the following: a. mechanical means to operate on the cartridge such that pre-processing and processing are induced inside the cartridge; b. optical means to detect optical signals from the processed sample in the cartridge; c. processing means to process the optical signals obtained from the sample; d. interface/display/in-out connections/remote communication features for operation and/or for result display/result transfer.

[0011] In one embodiment, processing of optical data obtained from a sample comprises computer processing. In one embodiment, computer processes comprise the use of algorithms. In one embodiment, computer processing/algorithms of this invention are novel and are designed to process the optical result to yield various qualitative and/or quantitative information related to the biological sample. The information provided by measurements of this invention can fit various user requirements. Computer processing in embodiments of this invention is designed in view of user requirements to yield for example information with regards to identity of certain blood cells in a population, percentage of certain blood cells from the total amount of cells, quantity ratio of two or more different cell types, concentration of cells etc.

[0012] In one embodiment, novel cartridges of this invention are designed such that preprocessing and processing are automatically conducted within the cartridge using external mechanical means provided by the system. According to this aspect and in one embodiment, pre-processing refers to the mixing and binding of cells to antibodies, and processing refers to dilution/lysing of the pre processed sample. Once the biological sample is inserted to the cartridge, no additional liquids are introduced into the cartridge for processing. A feature that enables pre-processing within the cartridge in embodiments of this invention is the presence of a pre-packed dry reagent in a channel/chamber within the cartridge. Once the liquid sample is introduced to the cartridge and is brought into contact with the dry reagent, the dry reagent is dissolved or dispersed into the liquid sample. If for example the dry reagent comprises an antibody, and the sample is a blood sample, the antibody dissolves into the blood sample and binds to a corresponding cell in the sample. Further processing inside the cartridge may include dilution and/or lysing of the sample/cells. Dilution/lysing involves liquids that are placed in advance in chambers within the cartridge or in blisters connected to the cartridge in advance, and prior to sample insertion in some embodiments.

[0013] The dry reagent prepared in advance in the cartridge may include in addition to antibodies, fluorescence beads. The fluorescence beads serve for control and calibration. For example and in one embodiment, the optical fluorescence obtained from the beads indicate the volume of the sample transferred through the optical path. This in turn may be used to evaluate the concentration of cells in the same volume of sample. The amount of reference beads in the dry reagent can be controlled. In addition and in one embodiment, fluorescence reference beads may be used to test and calibrate the optical signal obtained by the optical detector of the optical system of the instrument.

[0014] In one embodiment, the cartridge includes the following features: • sample handling element for sample insertion and pre-processing; • at least one bellows to induce fluid movement in, to or from channels/chambers;

• at least one reactant chamber comprising a liquid solution for processing; • at least one mixing chamber to facilitate mixing; • at least one fluid path to convey processed sample to an optical analysis zone.

[0015] In one embodiment, the sample handling element comprises: • a sample loading port; and • a mixing channel.

[0016] In one embodiment, the sample loading port and/or the mixing channel or portion thereof comprise the dry reagent. In one embodiment, the sample loading port is connected to the mixing channel. According to one aspect of the presently disclosed subject matter, and in one embodiment, there is provided a cartridge for preparing a biological sample for an assay, and for bringing the sample to an interrogation zone for the assay, the cartridge being formed with a single cavity comprising: • two bellows, each of the bellows comprising a flexible dome; • a sample handling element configured for receiving therein the biological sample; • one or more mixing chambers; • one or more reactant zones, each comprising a liquid reactant for selective delivery to one of the mixing chambers; • interrogation zone comprising a reading region; • a plurality of fluid paths, each providing fluid communication between at least two other elements of the cavity; and • a vent comprising an opening to the environment; wherein when the sample handling element is in a sealed position, the cavity is in fluid communication with the environment only via the vent.

[0017] In one embodiment, the sample handling element comprises a seal configured to bring the sample handling element from an open position into a sealed position.

[0018] The sample handling unit may comprise one or more dry reagents. Each of the dry reagents may comprise one or more reagent(s) selected from the group including an antibody and fluorescence beads.

[0019] In one embodiment, the two bellows, are each disposed at one end of the cavity.

[0020] In one embodiment, the antibody is a target antibody, an antibody that binds specifically to a target antigen on a cell. In one embodiment, the antibody is connected to a colored moiety, to a fluorescent moiety or a combination thereof According to this aspect and in one embodiment, the antibody is a cell stain. In some embodiments the colored and/or fluorescent moiety on the antibody serves as a cell 'stain', as it's color/absorption/fluorescence signal can be monitored optically. In one embodiment, the dry reagent comprises a positive control identifying antibody or a negative control identifying detection moiety. In one embodiment, the dry reagent comprises a chemical indicator and/or a biological indicator.

[0021] The sample handling unit may comprise a mixing channel having a serpentine passage.

[0022] The cartridge may comprise two of the mixing chambers, each end of the mixing channel being connected to one of the mixing chambers by a fluid path spanning therebetween.

[0023] In one embodiment, each end of the mixing channel is being connected to one of the mixing chambers directly, without an additional fluid path.

[0024] At least one of the bellows may be connected to one of the mixing chambers by a fluid path spanning therebetween.

[0025] At least one of the bellows may be connected to the interrogation zone by a fluid path spanning therebetween.

[0026] Each of the reactant zones may comprise a blister pack containing the liquid reactant.

[0027] Each of the blister packs may comprise a flexible dome and a rear backing opposite thereto, wherein each of the reactant zones comprises a socket in which a corresponding one of the blister packs is sealingly received, the socket comprising a piercing element bearing upon the rear backing.

[0028] The cartridge may be configured such that depression of the dome increases the internal pressure of the blister pack such that a rupture of the rear backing occurs.

[0029] The liquid reactant may comprise one or more selected from the group including a diluent, a lysing agent, and a reference material. At least one of the fluid paths may comprise a serpentine channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0031] Figure 1 schematically illustrates a system according to the presently disclosed subject matter;

[0032] Figure 2 Figure 2A and Figure 2B are front perspective and rear views respectively of an example of a cartridge of the system illustrated in Figure 1;

[0033] Figure 3 is a front perspective view of another example of a cartridge of the system illustrated in Figure 1;

[0034] Figure 4 is an example of an insert (plug) of a sample handling element; Figure 4A Front view; Figure 4B Back view; Figure 4C isometric view; Figure 4D backbone assembled;

[0035] Figure 5 is an example of an insert (plug) of a sample handling element; Figure 5A front exploded view - isometric; Figure 5B back exploded view - Isometric.

[0036] Figure 6 is an example of a common partial backbone; Figure 6A common backbone; Figure 6B backbone with two insert zones, each zone can accommodate different or no elements.

[0037] Figure 7 is an example of backbone with two insert zones; insert zone 1 (mixing channel) and insert zone 2 (loading port or sample handling element); Figure 7A Front view and Figure 7B Back view respectively;

[0038] Figure 8 is an example of backbone with two insert zones; insert zone 1 (loading port or sample handling element) and insert zone 2 (blank); Figure 8A Front view and Figure 8B Back view.

[0039] Figure 9 Figure 9A is a simplified three-dimensional inner front view of a system for detecting a biological condition, in accordance with an embodiment of the present invention;

[0040] Figure 9B is a simplified three-dimensional inner rear view of a system for detecting a biological condition, in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION

[0041] The presently disclosed subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that the presently disclosed subject matter is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the presently disclosed subject matter.

[0042] The presently disclosed subject matter provides a system, such as a flow cytometry system, and methods for biological assays. The system provides fast sample processing and accurate result, thus enabling correct and fast biological condition diagnosis. The system is compact and is easily operated on-site on-demand. The system comprises a cartridge for initial sample handling and for further sample processing. The system further comprises in some embodiments, a spectral mechanical device. The spectral-mechanical device is configured to receive the cartridge and to perform mechanical operations on elements thereof, thus facilitating processing of the sample inside the cartridge. The spectral-mechanical device is further configured to perform optical measurement of the processed sample. The spectral-mechanical device processes the data obtained by the optical measurement and provides a result indicative of a biological condition. The result may be qualitative, quantitative, or both. The constituents of the sample assayed according to the presently disclosed subject matter may reflect the biological condition of the subject.

[0043] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

[0044] In the present disclosure, the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. The term "plurality," as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and/or to the other particular value.

[0045] Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In the context of the present disclosure, by "about" a certain amount it is meant that the amount is within 20% of the stated amount, or preferably within 10% of the stated amount, or more preferably within 5% of the stated amount.

[0046] In some embodiments, the terms "subject" and "patient" are used interchangeably herein, and refer to an animal, for example a human, to whom treatment with a pharmaceutical composition in accordance with the presently disclosed subject matter, is provided. In some embodiments, the terms "subject" and "patient" are used interchangeably herein, and refer to an animal, for example a human, for whom a diagnostic test is performed, and/or from whom a sample is taken for a diagnostic test in accordance with the presently disclosed subject matter. The term "subject" asused herein refers to human and non-human animals. Theterms "non-human animals"

and "non-human mammals" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys. The formulations described herein can be used to diagnose/treat any suitable mammal, including primates, such as monkeys and humans, horses, cows, cats, dogs, rabbits, and rodents such as rats and mice. In one embodiment, the mammal to be diagnosed/treated is human. The human can be any human of any age. In an embodiment, the human is an adult. In another embodiment, the human is a child. According to any of the methods of the presently disclosed subject matter and in one embodiment, the subject is human. In another embodiment, the subject is a non-human primate. In another embodiment, the subject is murine, which in one embodiment is a mouse, and, in another embodiment is a rat. In another embodiment, the subject is canine, feline, bovine, equine, lapine, or porcine. In another embodiment, the subject is mammalian.

[0047] In one embodiment, the analytical instrument of this invention is or comprises a spectral mechanical device (SMD). In some embodiments, the spectral-mechanical device is referred to as the 'system'. In other embodiment, the SMD is referred to as opto-mechanical system or device, or as the instrument or analytical instrument or as opto-mechanical analytical instrument.

[0048] In some embodiments, pre-processing refers to the mixing of the sample with a dry reagent. In one embodiment, pre-processing refers to the mixing of the sample with an antibody/marker for binding to cells or other species in the sample. Processing in embodiments of this invention is any further processing of the sample following the pre-processing step. For example dilution/lysing and mixing involved with dilution/lysing is considered processing or further processing in embodiments of this invention. Such processing occurs following the pre-processing step. In one embodiment, pre-processing and processing are referred to together as processing.

[0049] As illustrated in Figure 1, there is provided a system, which is generally indicated as 10, for analyzing a liquid biological sample from a subject, for example using flow cytometry, and/or by any other suitable method for performing one or more assays. The system 10 comprises a cartridge 12 for containing the biological sample, and a spectral-mechanical device (SMD) 14 for facilitating manipulation of the biological sample within the cartridge and for performing the analysis thereon.

[0050] The biological sample may comprise a bodily fluid sample, for example blood, serum, plasma, urine, saliva, cerebrospinal fluid, serous fluid, peritoneal fluid, and/or synovial fluid. According to some examples, the biological sample may comprise a solid, for example a piece of hair, a piece of tooth, a piece or cartilage, skin, a piece of bone, and/or a piece of soft tissue.

[0051] The cartridge 12 is configured for receiving the biological sample therein, and for facilitating optionally performing one or more pre-processing steps on the biological sample, performing one or more processing steps on the biological sample, e.g., on the pre-processed biological sample, and for positioning the processed biological sample at an interrogation point at which the SMD 14 may perform the analysis thereon. Herein the specification and appended claims, the term "biological sample" will be used to indicate the biological sample at any stage of processing.

[0052] Accordingly, as illustrated in Figure 2A and in Figure 2B, the cartridge 12 comprises a support panel 16 defining opposing front and rear sides 16a, 16b, and carrying a sample handling element 18, first and second bellows 20a, 20b, first and second mixing chambers 22a, 22b, first, second, and third reactant zones 24a, 24b, 24c, a vent 25, and an interrogation zone 26. (Herein the specification, base reference numerals may be used without trailing letters to collectively refer to all elements indicated thereby; accordingly, e.g., the term "bellows 20" may be used to collectively refer to the first and second bellows 20a, 20b, etc.) In addition, various fluid paths are formed in the support panel 16 fluidly connected between elements carried thereby, as will be described below.

[0053] At least some of the elements carried by the support panel 16 as described above, for example the bellows 20, mixing chambers 22, interrogation zone 26, and fluid paths may be formed as indentations in the rear side 16b, projecting into the material of the support panel 16 towards the front side 16a thereof.

[0054] The cartridge 12 further comprises a backplate (not illustrated) disposed on the rear side 16b of the support panel 16. The backplate lies in registration over at least the elements carried by the support panel 16 as described above, in particular those which are formed projecting into the material of the support panel. It is sealingly attached to the rear side 16b of the support panel 16, thereby fluidly isolating the elements from the exterior of the cartridge 12 in the rear direction. The backplate may be of any suitable design, including, but not limited to, a rigid material, and adhesive film, etc., without departing from the scope of the presently disclosed subject matter, mutatis mutandis.

[0055] It will be appreciated that while the support panel 16 is described above as "carrying" various elements of the cartridge 12, this has been done for ease of description only, and the term as used herein the specification and appended claims should be construed in its broadest sense, i.e., including elements which are connected thereto, formed therein, etc., unless otherwise clear from context. For example, the bellows 20 may include elements which are connected to the support panel 16, and the mixing chambers 22 may be defined by structures formed in the material of the support panel.

[0056] The sample handling element 18 is configured for receipt therein of the biological sample, and optionally for facilitating pre-processing thereof therein. Accordingly, it comprises a mixing channel 28 formed therein, spanning between first and second apertures 30a, 30b (the locations of which are indicated in Figure 2B, and which are illustrated in Figure 2B). The mixing channel 28 may comprise a serpentine passage 32, with a receiving socket 34 formed therein, e.g., for receipt of the biological sample. The receiving socket 34 may be a portion of the serpentine passage 32 but of a wider width, which may indicate the location at which the biological sample should be placed, for example to ensure proper functioning of the system 10, and/or to facilitate placement therein of the biological sample.

[0057] At least a portion of an upper surface 36 of the sample handling element 18 may be flat, for example to facilitate sealing attachment thereto of a seal (not illustrated in Figures 2A and 2B), for example a thin element with an adhesive side such as a sticker, thereby bringing the sample handling element 18 into a sealed position. According to some examples, a continuous portion of the upper surface 36, which completely surrounds the serpentine passage 32, may define a single plane, thereby facilitating contact therewith of a planar seal.

[0058] As illustrated in Figure 3, according to some examples, the sample handling element 18 comprises a sample port 34a for receipt of the biological sample, and the cartridge 12 comprises a corresponding plug 34b constituting a seal, e.g., formed with the support panel 16 and disposed so as to be brought into registration with the sample port. The plug 34b is configured to sealingly mate with the sample port 34a, thereby bringing the sample handling element 18 into a sealed position. According to these examples, the mixing channel 28 is isolated from the environment, except via the sample port 34a and the vent 25.

[0059] As best seen in Figure 2B, the first aperture 30a is in fluid communication with a first sample-mixing fluid path 38a, spanning between the sample handling element 18 and the first mixing chamber 22a, and the second aperture 30b is in fluid communication with a second sample mixing fluid path 38b, spanning between the sample handling element 18 and the second mixing chamber 22b. As the sample handling element 18 is disposed between the two mixing chambers 22, the serpentine passage 32 thereof may be utilized to mix the biological sample with reactants therewithin.

[0060] The sample handling element 18 may be provided with one or more reagents, for example dry reagents, disposed within the mixing channel 28, for example within the serpentine passage 32. The reagents may be configured for use in pre-processing the biological sample. Accordingly, it may comprise an antibody, a target antibody, a positive control identifying antibody, a negative control identifying detection moiety, a target signal reference composition, a reference identifier composition, a cell stain, a chemical indicator, a biological indicator, and/or any other suitable reagent. The reagent may be provided on the surface of a reagent block, bound to a solid support, bound to/deposited on the walls/surfaces of the channel, or by any other suitable means.

[0061] It will be appreciated that while "reagents" and "reactants" may be used interchangeably, herein the specification and appended claims the term "reagent" is used to indicate a substance which is disposed within the sample handling element 18, and the term "reactant" is used to indicate a substance which is disposed within the reactant zones 22. This distinction is for clarity of this disclosure only, and is not to be seen as limiting. In particular, substances which one skilled in the art would classify as a reagent may be included by the term "reactant," and vice versa. Moreover, a single substance may be, for the purposes of this disclosure, be considered both a "reactant" and a "reagent," depending on its location.

[0062] Each of the bellows 20 comprises a flexible dome 40 projecting from the front side 16a of the support panel 16, for example in the form of a hemisphere or spherical cap, and a portion of the backplate constituting a planar base thereof. The dome is formed such that it remains within the elastic limit through full depression, i.e., it may be fully depressed, and returns to its original shape upon removal of the depression force. The dome 40 may be formed as part of the support panel 16, or comprise a separate element connected thereto. The first bellows 20a is in fluid communication with a bellows-mixing fluid path 42a, spanning between the first bellows and the first mixing chamber 22a, and the second bellows 20b is in fluid communication with a bellows interrogation fluid path 42b, spanning between the second bellows and the interrogation zone 26.

[0063] Each of the reactant zones 24 comprises therein a blister pack 44 (one of which is illustrated in Figure 2A in a detached configuration) and is configured to facilitate selective delivery of its contents to one of the mixing chambers 22. Each of the blister packs 44 comprises a flexible dome 46, for example in the form of a hemisphere or spherical cap, and a rear backing (not illustrated) opposite thereto and made of a thin layer of material, for example a metallic foil. Each of the reactant zones 24 further comprises a socket 48 in the front side 16a of the support panel 16, for example as a depression formed therein defining a base surface 50 and sealingly receiving a corresponding one of the blister packs 44 therein, such that the rear backing is spaced therefrom the base surface 50. Each base surface 50 comprises a piercing element 52 projecting toward the front side 16a of the support panel 16. An outlet 54, comprising a through-going aperture, is formed in each socket 48, e.g., adjacent a bottom end thereof. As best seen in Figure 2B, each of the outlets 54 is in fluid communication with a reactant-mixing fluid path 56, spanning between the respective reactant zone 24 and one of the mixing chambers 22.

[0064] Each of the blister packs 44 may comprise one or more reactants, for example in liquid form, for delivery to the biological sample in one of the mixing chambers 22. The reactants may be a diluent, a lysing agent, a reference material, or any other suitable reactant, and/or a combination thereof.

[0065] Each of the piercing elements 52 may be configured to bear upon the rear backing of a corresponding blister pack 44 when received within the socket 48, such that it does not pierce it until the internal pressure thereof is increased, for example by depression of the dome 46, thereby causing a rupture of the rear backing. The bearing of the piercing element 52 on the rear backing of the blister pack 44 may give rise to a rupture zone thereon, which is associated with an increased likelihood of undergoing rupture when the dome 46 is depressed. Accordingly, the piercing element 52 may be disposed in proximity to its corresponding outlet 54, such that reactant released from the blister pack 44 upon rupture may flow immediately into the corresponding reactant mixing fluid path 56.

[0066] The vent 25 is in fluid communication with the environment, and facilitates movement of the biological sample within the cavity when the bellows 20 are depressed and/or released. Accordingly, it may be disposed above the rest of the elements of the cavity, i.e., in the orientation of the cartridge 12 when inserted in the SMD 14, and is in fluid communication therewith, e.g., connected to one of the mixing chambers 22, via a vent fluid path 25a.

[0067] As best seen in Figure 2B, the interrogation zone 26 is configured for facilitating analysis of the biological sample by the SMD 14. Accordingly, it comprises a reading fluid path 58, which comprises a narrow reading region 60. The reading region 60 may be designed to limit the number of particles passing therethrough simultaneously, for example being wide enough to permit only a single particle at a time to pass therethrough for analysis by the SMD 14. Moreover, the material of the interrogation zone 26, in particular in the area of the reading region 60, may be made of a material transparent to the wavelength of the light emitted by the SMID 14 in performing the analysis.

[0068] One end of the fluid reading path 58 is in fluid communication with the second bellows 20b via the bellows-interrogation fluid path 42b, as described above. The other end of the fluid reading path 58 is in fluid communication with the second mixing chamber 22b via a mixing interrogation fluid path 62. At least a portion of the mixing-interrogation fluid path 62 may constitute a serpentine channel 64. It is to be noted that the geometry of the interrogation fluid path can be varied and can be adjusted to any desired geometry that fits a certain cartridge design. For example and in one embodiment, fluid path 62 does not comprise a serpentine channel.

[0069] It will be appreciated that when the sample handling element 18 is in its sealed position, for example as described above, the elements of the support panel 16 described above constitute a single cavity constituting a closed system, i.e., isolated from the environment such that nothing further may be introduced therein, including, but not limited to, air or other fluids, with the exception of the vent 25, which is necessary to allow compression of the bellows 20. Accordingly, movement of the biological sample therewithin may be effected entirely by mechanical interactions with the elements of the support panel 16, including, but not limited to, acting upon the bellows 20, as will be described below. (The blister packs 44 may be considered as sub-chambers within the cavity, as their contents are isolated from the environment and are intended to be introduced into the rest of the cavity during operation.) Accordingly, no external pump or other means of introducing air into the cavity is necessary to move the biological sample therewithin. In one embodiment, no external air pump is used to directly push air into the cartridge.

[0070] The SMD 14 may be any suitable device for interfacing with the cartridge 12, for example as described in one or more of US 2013/0102087, US 2014/0170678, US 2014/0170680, US 2014/0287435, US 2015/0132776, US 2015/0293095, US 2015/0309049, US 2015/0330971, US 2016/0146793, US 2017/0350888, US 2017/0370914, US 2018/0231532, US 2018/0299443, US 2018/0306698, and/or US 2020/0080928, the full contents of which are incorporated herein by reference. In particular, the SMD 14 comprises elements which are configured to selectively interact with the bellows 20 and reactant zones 24, for example selectively depressing and/or releasing each one, when the cartridge 12 is inserted therein. The SMD 14 may further comprise an optics unit, for example comprising a light source, a photon-counter and/or an integrator, configured to facilitate performing the analysis of a portion of the biological sample within the reading region 60 of the interrogation zone 26.

[0071] The SMD 14 may further comprise a processor to direct operation thereof. The processor may further perform one or more steps of the analysis, for example being configured to receive data related to multi-spectral emission signals detected by the optics unit, to process the data, and to output a result, e.g., related to the medical condition. Each of the multi-spectral emission signals may be associated with a biological marker.

[0072] In use, a biological sample is inserted into the receiving socket 34 formed within the sample handling element 18, which is then sealed, for example by providing a seal thereon, e.g., as described above. The cartridge 12 is inserted into the SMD 14, for example within a slot designed therefor (see figure 9A, cartridge 910). The SMD 14 selectively interacts with elements of the cartridge in order to facilitate optional pre-processing, processing, and analysis of the biological sample, for example as described below.

[0073] In an optional pre-processing step, the first and/or second bellows 20a, 20b are selectively depressed and released by the SMD 14. As the biological sample is within a closed system which includes the bellows 20, the interactions with the bellows causes the biological sample to move within the serpentine passage 32 of the sample handling element 18, thereby coming into contact with the dry reagents within the mixing channel 28 and dissolving them; the only opening in this closed system is the vent 25, which is required in order to allow the bellows to be depressed and released. The first and second bellows 20a, 20b may be alternatingly depressed so as to move the biological sample back and forth within the serpentine passage 32, thereby facilitating mixing thereof with the dry reagents.

[0074] It will be appreciated that descriptions herein of the bellows 20 being "depressed" or "released" are not limited to a full depression or release; each of the bellows may be partially depressed or released in order to move the biological sample within the chamber a predetermined amount.

[0075] The interactions of the SMD 14 with the bellows 20 may be performed so as to control the sequence of mixing of the biological sample with more than one reagent. For example, the first bellows 20a may be depressed such that the biological sample is moved a predetermined amount within the mixing channel 28, such that it reaches a first dry reagent and dissolves it. The first and second bellows 20a, 20b may subsequently be alternately depressed such that the biological sample and first reagent move back and forth within a portion of the serpentine passage 32, thereby effecting sufficient mixing and time for the reagent to act upon the biological sample. Subsequently, the first bellows 20a may be depressed so as to move the biological sample further along the mixing channel 28, such that it reaches a second dry reagent and dissolves it. It will be appreciated that the sequence of interactions by the SMD 14 with the bellows 20 may be carried out in order to achieve any suitable sequence of mixing, etc., of the biological sample with the dry reagent within the sample handling element 18, mutatis mutandis.

[0076] In one or more processing steps, the biological sample is selectively moved into one or both of the mixing chambers 22. For example, in order to move the biological sample from the sample handling element 18 to the first mixing chamber 22a, the second bellows 20b is depressed, thereby inducing a positive pressure therebehind. The SMD 14 may selectively facilitate delivery of the contents of one or more of the blister packs 44 to one of the mixing chambers 22 by depressing it, thereby effecting a rupture in the rear backing thereof for example as described above, releasing the liquid reactants therein. The reactant released from the blister pack 44 upon rupture flows into the corresponding reactant-mixing fluid path and therethrough to the corresponding mixing chamber 22.

[0077] In an interrogation step, the processed biological sample is moved, by interactions of the SMD 14 with the bellows 20, into the interrogation zone 26. The SMD 14 may regulate the degree of depression on the bellows 20 in order to adjust the position of the biological sample within the reading region 60 of the interrogation zone 26 as necessary.

[0078] In some embodiments, either one of the two or more bellows can induce movement of the liquid sample between components of the cartridge. Each bellows can be pressed and depressed in a gradual manner to induce back and forth liquid movement through a channel, to/from a chamber or towards the optical interrogation zone. According to this aspect and in one embodiment, the cartridge comprises only one bellows. In one embodiment, two bellows work in concert to drive liquid back and forth in the cartridge as described herein. In one embodiment, one bellows pushes liquid in one direction and another bellows push liquid in an opposite direction. In one embodiment, one or two bellows drive liquid to induce mixing and only one of the two bellows drive the liquid toward the optical interrogation zone. In one embodiment, pressing and depressing one bellows is sufficient to move liquid back and forth in a component of the cartridge. In one embodiment, pressing and/or depressing two bellows alternately is used to move liquid back and forth in components of the cartridge. In one embodiment, gradual pressing/depressing one or more bellows, dictates how far liquid will proceed in a certain channel/component of the cartridge.

[0079] Figure 4 shows an embodiment of the cartridge wherein the reagent sample plug (RSP) is connected to the cartridge following cartridge production. Such RSP is shown in figure 4A (Front) and in Figure 4B (back). A sample loading chamber is shown in Figure 4A (element 1). In one embodiment, the reagent is dried within the sample loading chamber (1). In one embodiment, the reagent is dried within a bulging area (shown on the left side as part of the serpentine channel). The dried reagent may be dried in both areas and can also be dried within the serpentine channel in one embodiment. Any combination of locations for the dry reagent in the sample handling element are included in embodiments of this invention. In figure 4B, the upper interface with the cartridge (element 2) and the lower interface with the cartridge backbone (element 3) are shown. Figure 4D shows the cartridge with the sample insert connected to it. According to this aspect and in one embodiment, the RSP is not part of the cartridge mold. Instead, it is added as an additional element and connected to the mold. After connecting the two, the cartridge is ready for sample insertion. In this configuration, there is an external pump that draws the sample into the cartridge and affects mixing in the serpentine channel in some embodiments. In other embodiments, the mixing or sample conveyance through the channel of the sample plug is effected by at least one of the bellows as described herein. In embodiments, the term 'backbone' is used for the cartridge. It is to be noted that the sample handling element can be located anywhere on the cartridge. It can be produced with the other components of the cartridge at the mold stage or it can be added later and fixed to the cartridge. The sample handling element can be connected between the two mixing chambers or it can be connected to only one mixing chamber in a one- or two-mixing chambers configurations. Figure 5A and Figure 5B are front and back exploded views-isometric, showing the region where the RSP is being connected to the cartridge, with the relevant matching component. Figure 6 shows an embodiment of the invention wherein a cartridge mold is designed with two zones. Each zone can accommodate different features or no features. The desired feature(s) can be included for any mold production upon demand. Figure 6A shows the common backbone that can bear additional features in different zones. For example, Figure 6B shows zone 1 and zone 2. In zone 1, a sample handling element can be placed (when producing the mold) or a mixing channel with no sample insertion port can be formed. In zone 2, a sample handling element can be formed (when producing the mold) or this zone can be left blank. It is to be noted that additional zones can be formed as part of the mold. Features such as the sample handling element, can be produced with the mold or added on following mold production. Figures 7A and 7B shows the back and front of a cartridge wherein zone 1 comprises a mixing channel and zone 2 comprise a sample handling element. Figure 8 shows an example of backbone with two insert zones; insert Zone 1 (loading port or sample handling element) and insert zone 2 (blank); Figure 8A Front view and Figure 8B Back view.

[0080] Figure 9 shows an example of a system (analytical instrument) of the present invention. In Figure 9A, a left side view 920, showing an ITX computer, 922, a Galil motor controller, 924, an electronics power supply 926, a cartridge 910, inserted into a cartridge handling unit (CHU) 928 and a forward scatter detector 930. Figure 9B shows a right-side view 940 showing reader optics 942, a data acquisition board 944 and a general electronics printed circuit board 946.

[0081] In one embodiment, this invention provides systems and methods for a biological assay, the systems include a cartridge as described herein for performing the assay therein, the cartridge adapted to house at least one reagent adapted to react with a sample. In one embodiment, systems of this invention comprise a mechanical controller including an urging means adapted to apply a force externally onto components of the cartridge such as the blister(s) and the bellows. In one embodiment, the system comprise an optical reader adapted to detect the sample and a processor adapted to receive data from the optical reader and to process the data to provide an assay result.

[0082] In one embodiment, this invention provides a system for performing an assay on a biological sample, the system including; • a cartridge as described herein for performing the assay therein, the cartridge comprise at least one reagent adapted to react with a sample; • a mechanical controller including; o at least one first urging means adapted to apply a force on the bellows to induce fluidic movement in a first direction and/or in a second direction; o at least one second urging means adapted to apply a force onto the blister(s) to release dilution/lysing solution; • an optical reader adapted to detect the sample; and • a processor adapted to receive data from the optical reader and to process the data to provide an assay result.

[0083] Additionally, according to an embodiment of the present invention, the cartridge further includes an alignment means adapted to align a reading channel on the cartridge with optical components of the system, for optical detection of the sample in the cartridge by the system's optical reader.

[0084] In one embodiment, methods of this invention comprise the following steps: • load sample into the sample handling element; • fix sample element to cartridge; • insert cartridge into the system; • mix content of sample handling element (mix sample with dry reagent); • extract sample from sample element to a mixing chamber; • start processing and assay.

[0085] The sequence of steps can be changed or altered as described in embodiments of this invention. For example, in embodiments where the sample element is part of the cartridge mold, no fixing element to cartridge step is required. In one embodiment, the mixing and extraction of the sample occur at least partially in parallel.

[0086] Other features of the system are described for example in W02014/097287, the full contents of which are incorporated herein by reference.

[0087] In one embodiment, channels or fluid paths in the cartridge have a cross section dimension ranging between 200 pm and 1 mm. In one embodiment, the cross section of a channel in the cartridge is rectangular with a width ranging between 200 pm and 1 mm and a depth ranging between 200 pm and 1 mm. In one embodiment, cross section of a channel has a width of 800 pm and a depth of 600 pm.

[0088] In one embodiment, the reagent is dried inside the channel before the insert is connected to the cartridge. After drying, the insert is connected to the cartridge. All further operations (sample insertion, sample flow through the channel with dry reagent and processing are conducted by the bellows. No external pump is required.

[0089] In one embodiment, the interrogation zone is the region comprising the reading channel, the channel through which fluorescently labeled cells pass (with or without additional fluorescent beads). In one embodiment, the reading channel is transparent to the wavelength(s) of excitation and is also transparent in the wavelength ranges of fluorescence emitted from the cells. In one embodiment, transparent at a certain wavelength is a full transparency. In one embodiment, partial transparency of a channel at a certain wavelength is sufficient for emission/detection of signal through the channel.

[0090] Processing in embodiments of this invention may relate in some embodiments to chemical/biological processing and in some embodiment to computer processing of a signal. In one embodiment, sample processing refers to both chemical and optical processing. The meaning of the term processing is clear from the context of embodiments described herein. In one embodiment, the reading region of the cartridge is an optical reading region. According to this aspect and in one embodiment, a sample in the reading zone is irradiated with light from a light source, and in response, the sample emit light toward at least one detector. The light (optical) signals obtained by the detector are processed to yield a result indicative of the constituents of the sample. Such result may be qualitative, quantitative, semi-quantitative or any combination thereof

[0091] In one embodiment, staining refers to the mixing and/or binding of the antibody (or other binders as noted herein) to cells or to particles/species/fragments that are present in the sample. In one embodiment, staining refers to the pre-processing step or to a portion thereof. In one embodiment, staining refers to the association of a fluorescent marker with cells or other species in the sample. In one embodiment, staining or pre-processing refers to the process wherein an antibody (or similar compound) that is already connected to a fluorophore, is attached to or is binding to the cells (or to other particles/species in the sample). According to this aspect and in one embodiment, fluorescent antibodies that bind to the cells enable optical detection of the cells.

[0092] In some embodiments, the sample handling element is referred to as the sample handling unit. In some embodiments of this invention, the terms zone, area, region, channel are interchangeable, and may refer to the same feature.

[0093] In one embodiment, instead of a plurality of fluid paths, each providing fluid communication between at least two other elements of the cavity, only one such fluid path is required in addition to the channel of the sample handling element. According to this aspect and in one embodiment, the fluid path connects a mixing chamber to the reading channel.

[0094] It is to be noted that the orientation of the components in the cartridge as shown in the figures are examples. The various components of the cartridge can be located in many different orientations in the cartridge. Since various components of the cartridge are connected by fluidic paths, the components can be assembled in many different configurations, and the fluid path (channels) can be designed to connect components as required. All such orientations are included in embodiments of this invention.

[0095] In one embodiment, the system regulates the time and/or speed of pressing/depression of the bellows 20 in order to adjust the time/speed of liquid flow within elements of the cartridge.

[0096] In one embodiment, the cartridge does not comprise a sample handling element. According to this aspect and in one embodiment, the pre-processing is conducted outside the cartridge and the pre-processed sample is injected directly to the mixing chamber or to a fluid channel connected to the mixing chamber for further processing. According to this aspect and in one embodiment, pre-processing comprises adding fluorescent beads to the sample.

[0097] It is to be noted that the connection between the reactant zones and the mixing chambers as shown in the figures is an example. In embodiments, each reactant zone can be connected to each of the mixing chambers as required for a certain application. In one embodiment, the cartridge comprises 1-3 blisters. In one embodiment, the blister fill is only for dilution/lysing. In one embodiment, the blister fill is not for antibodies/reference beads. In one embodiment, when more than one blister is present, it is possible to use different blisters/more than one blister to achieve different ratios of dilution as required. In one embodiment, two or more blisters comprise the same solution and in one embodiment, two or more blisters comprise different solutions. In one embodiment, two or more blisters comprise a solution comprising the same constituents but at different constituent concentration.

[0098] In one embodiment, the insert for sample loading and serpentine dry reagent channel (sample handling element) was added to a cartridge after the cartridge was molded.

[0099] The sample handling element insert is considered part of the cartridge in embodiments of this invention.

[00100] In one embodiment, the sample loading area/port and serpentine dry reagent channel (sample handling element) was produced as part of the cartridge mold. The sample handling element is part of the cartridge mold according to this embodiment.

[00101] The sample handling element insert is considered part of the cartridge in embodiments of this invention.

[00102] In one embodiment, following sample introduction to the sample handling element, the opening of the sample loading port is sealed using a sticker seal. Other sealing options are provided in embodiments of this invention, for example, the plug 34b in Figure 3, plastic caps, screw caps, adhesives, rubber seals etc. as known in the art. In one embodiment, a 'co-molded plug' (such as element 34b in Figure 3) is used for sealing the opening after the sample is inserted/loaded through that opening (e.g. element 34a).

[00103] In one embodiment, there is a sample loading port in the dry reagent insert (see for example bulging area in figure 4A, element 1). In one embodiment, the cartridge is referred to as backbone. In one embodiment, the sample handling element is referred to as reagent sample plug (RSP).

[00104] In one embodiment, an external pump draws the sample through the channel of the sample handling element for further processing. In other embodiments, no external pump is required and the sample is drawn by the bellows through the channel of the sample handling element into the cartridge for further processing.

[00105] In one embodiment, moving liquids through components of the cartridge is not conducted by an external pump. In one embodiment, the cartridge does not comprise valves. In one embodiment, the cartridge is valveless.

[00106] In one embodiment, moving liquids through components of the cartridge using the bellows, reduce complexity of the system and allows facile processing as no external pump is connected to channels in the cartridge.

[00107] In one embodiment, the reagent is dried inside the channel of the sample handling element before the sample handling insert is connected to the cartridge. After drying, the insert is connected to the cartridge according to this embodiment. In one embodiment, all further operations (sample drawing, flow through the channel with dry reagent and processing are conducted by the bellows. No external pump is required. In one embodiment, the staining is induced by the bellows and no external pump is required.

[00108] In one embodiment, this invention provides an assay method comprising: • providing a cartridge as described herein above; • introducing a biological sample to the cartridge; • inserting the cartridge into an opto-mechanical system as described herein;

• using at least one urging means in the system to operate at least one bellows in the cartridge to induce sample flow through the dry reagent channel of the sample handling element; • using at least one urging means in the system to operate at least one bellows in the cartridge to induce flow of a stained (pre-processed) sample from the sample handling element to at least one mixing chamber; • using at least one urging means in the system to burst at least one blister in the cartridge to induce flow of a dilution/lysing solution from a reactant zone to at least one mixing chamber; • using at least one urging means in the system to operate at least one bellows in the cartridge to induce mixing of dilution/lysing solution with the pre-processed sample in at least one mixing chamber; • using at least one urging means in the system to operate at least one bellows in the cartridge to induce flow of processed solution from at least one mixing chamber to an interrogation zone comprising a reading channel; • irradiating the reading channel using a light source of the system and detecting light signal

obtained from the reading channel by at least one detector of the system. • processing the detected light signal using a processor of the system to obtain an analytical result.

[00109] Embodiments described herein for the cartridge and for the system are applicable to embodiments of methods of this invention as described herein.

[00110] In one embodiment, systems of this invention do not comprise a heater. In one embodiment, systems of this invention do not comprise a temperature control device. In one embodiment, systems of this invention do not comprise a thermometer.

[00111] Dry reagent(s) of this invention may include in addition to antibodies (and optionally reference beads) also chemicals such as salts, stabilizers, pH-adjusting materials, ionic strength adjusting materials and other compounds/materials that are compatible with and/or help to maintain the sample in a condition that fits processing and measurement. According to this aspect and in one embodiment, the dry reagent comprises a mixture.

[00112] In one embodiment, the sample comprises cells. In one embodiment, the cells are blood cells. In one embodiment, the cells comprise red cells, white cells or a combination thereof. In one embodiment, the cells comprise stem cells. In one embodiment, the sample comprises cell fragments. In one embodiment, the sample comprises particles. In one embodiment, the sample comprises antigens. In one embodiment, the sample comprises cells, the cells comprising cell surface markers. In one embodiment, the surface markers are or comprises antigens. In one embodiment, in cartridge assays of this invention, the antigens in the sample (e.g. antigens on the cell's surface) bind to antibodies in the dry reagent. In one embodiment, fluorescent tags on the antibodies are used to identify the cells. Different cell surface markers can be bonded to different antibodies. In one embodiment, different antibodies comprise different fluorescent tag. According to this aspect and in one embodiment, a certain cell will bind different antibodies with different fluorescent tags. The fluorescence wavelength(s) and fluorescence intensity coming from a certain cell indicate the type of cell in some embodiments.

[00113] In one embodiment, assay of this invention is a flow cytometry assay. In one embodiment, determination/detection of a biological condition is referred to as sample analysis or as sample assay, or as analysis or as assay or as determination of a medical condition. In one embodiment, the reactant zone is referred to as the blister for simplicity. In one embodiment, the antibody is a fluorescent antibody. In one embodiment, wherein reference is made to 'cells', the cells are biological cells. Embodiments that refer to cells, may also refer to other particles, to cell fragments, to proteins, to antigens, to biomolecules, to cell cmponents, to viruses, bacteria, microorganisms or components thereof All such embodiments are included in this invention. In one embodiment, a particle is a cell, a bead or any of the species described herein above.

[00114] In one embodiment, reference beads are fluorescent beads and may also be referred to as microspheres, nanospheres, fluorescent spheres or a combination of these terms. In one embodiment, fluorophore and fluorochrome are interchangeable terms. In one embodiment, fluorescence obtained from cells/particles of this invention is direct, indirect or any combination thereof. Embodiments of this invention that are described for treatment may refer also to diagnostics. In one embodiment, the terms tag, probe, marker are interchangeable. Mixing chambers of this invention may assume many shapes, sizes and geometries, all are included in embodiments of this invention. In one embodiment, this invention provides cell staining in an automated cartridge. In one embodiment, not all of the reactant zones comprise blisters. In one embodiment, at least some of the reactant zones do not comprise blisters. In one embodiment, at least some of the reactant zones comprise blisters. In one embodiment, the location of zones/chambers/elements/components on the cartridge may vary, as long as the connections required between any two or more of the zones/chambers/elements/components are maintained.

[00115] In one embodiment, bulging area in the sample handling element is used for placement of dry reagent, for introduction of the sample or for both. In one embodiment, assays of this invention are white blood cell assays. In one embodiment, the sample prior to processing is a whole blood sample.

[00116] In one embodiment, an assay of this invention is completed within 30 min or within 1 h from the time of sample insertion to the sample handling element. In one embodiment, an assay of this invention is completed within 30 min or within 1 h from the time of cartridge insertion to the system.

[00117] Those skilled in the art to which this invention pertains will readily appreciate that numerous changes, variations, and modifications can be made without departing from the scope of the presently disclosed subject matter, mutatis mutandis.

Claims

CLAIMS What is claimed is: 1. A cartridge assembly for preparing a biological sample for an assay, and for bringing said sample to an interrogation zone for the assay, the cartridge assembly comprising: a sample handling element configured for receiving therein the biological sample via a sample port, said sample handling element comprising one or more reagents; a plug configured to seal the sample port; a cartridge comprising: two bellows configured to induce controlled movement of liquid within the cartridge, each of said bellows comprising a flexible dome; one or more mixing chambers; one or more reactant zones, each comprising a liquid reactant for selective delivery to one of said mixing chambers, wherein the one or more mixing chambers are in fluid communication with the one or more reactant zones; s an interrogation zone comprising a reading region, configured to receive the biological sample, wherein the reading region is optically transparent and configured to allow the passage of light for a flow cytometry measurement; a plurality of fluid paths, each providing fluid communication between at least two other elements of said cartridge; and a vent in fluid communication with one of the mixing chambers, the vent comprising an opening to the environment; wherein when said sample handling element is in its sealed position, said cavity is in fluid communication with the environment only via the vent; an insert zone configured to receive the sample handling element.
2. The cartridge of claim 1, wherein at least one of said one or more reagents comprises an antibody.
3. The cartridge of claim 2, wherein said antibody is bonded to a fluorescent marker.
4. The cartridge of claim 3, wherein said one or more reagent comprises fluorescence beads.
5. The cartridge of any one of claims 1-4, wherein said sample handling element comprises a mixing channel having a serpentine passage.
6. The cartridge of claim 5, wherein each end of said mixing channel is connected to one of said mixing chambers by a fluid path spanning therebetween.

24 21679464_1 (GHMatters) P121482.AU
7. The cartridge of any one of claims 1-6, wherein at least one of said bellows is connected to one of said mixing chambers by a fluid path spanning therebetween.
8. The cartridge of any one of claims 1-7, wherein at least one of said bellows is connected to said interrogation zone by a fluid path spanning therebetween.
9. The cartridge of any one of claims 1-8, wherein each of said reactant zones comprises a blister pack containing said liquid reactant.
10. The cartridge of claim 9, wherein each of said blister packs comprises a flexible dome and a rear backing opposite thereto, wherein each of said reactant zones comprises a socket in which a corresponding one of said blister packs is sealingly received, said socket comprising o a piercing element bearing upon the rear backing.
11. The cartridge of claim 10, being configured such that depression of the dome increases the internal pressure of the blister pack such that a rupture of the rear backing occurs.
12. The cartridge of any one of claims 1-11, wherein said liquid reactant comprises one or more selected from the group including a diluent, a lysing agent, and a reference material.
13. The cartridge according to any one of claims 1-12, wherein at least one of said plurality of fluid paths comprises a serpentine channel.
14. The cartridge assembly according to any one of claims 1-13 wherein the one or more reagents are in dry form.
15. The cartridge assembly according to any one of claims 1-14 wherein the plug is selected !0 from: co-molded plug, sticker seal, plastic cap, screw cap, and rubber seal.

25 21679464_1 (GHMatters) P121482.AU

Figure 1

24c

16b 26 22b 30b 20b 36

16a

24b 16 50

32 40

18

34 28

52

40 54 24a 208 48

Figure 2A

22a

44

Figure 2B

12 208 428

56

54

388

54 no 308 380

56 300 220

25 20b 258 54

58 420

64 62

58 20 8

34a

34b 18 28

Figure 3

Front Back

2 1

3

Figure 4A Figure 4B

Backbone Isometric view Assembled

1

Figure 4C Figure 4D

Figure 5A

(a)

Figure 5B

Common Backbone

Figure 6A

Insert Zone 2

Insert Zone 1

Figure 6B

Insert Zone 2

Insert Zone 1

Figure 7A

runn $2.0

Insert Zone 1

Figure 7B

Figure 8A

Fluidics Side

Figure 8B

Figure 9B

944

942

940

930

928

910

Figure 9A

926 922

924 903