KR20230079276A - System for determining biological state and cartridge therefor - Google Patents

Abstract

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

Description

System for determining biological state and cartridge therefor

The presently disclosed subject matter relates to systems and methods for determining biological status, and more particularly to flow cytometry systems and methods for determining biological status.

There are many medical conditions that are most reliably diagnosed through laboratory tests on body specimens. Typically, these tests are performed in a laboratory facility remote from where the patient is receiving treatment.

The turnaround times of known diagnostic assays can range from 30 to 120 minutes. Oftentimes, wasting time waiting for lab results can make a patient's condition worse, sometimes even fatal. In some cases, doctors have to act without laboratory results. This can lead to providing inappropriate treatment to the patient. Thus, shortening the time required to detect biological conditions in samples taken from patients and providing a means to perform appropriate assays outside of laboratory facilities may allow physicians to administer more appropriate treatment and, in some cases, reduce mortality. .

One of the disadvantages of current analytical methods is that they require pre-treatment prior to introducing the sample into the analytical device for measurement. Such pretreatment requires reliable and precise training of laboratory personnel. In addition, such pretreatment is time consuming, requires additional laboratory equipment and tools, imposes a significant risk of biohazard and several sources of error in analytical results.

In one embodiment, the invention provides a method of analyzing a biological sample, the method comprising:

- inserting the biological sample into the cartridge;

- inserting the cartridge into the analysis instrument;

- starting the measurement;

- Getting results.

According to this aspect and in one embodiment, the method of the present invention eliminates the need for manual pretreatment. According to this aspect and in one embodiment, the pre-treatment is performed automatically within the cartridge.

The method of this invention provides a rapid, easy, safe, accurate and reliable means of diagnosis that can be performed at any location immediately following withdrawal of a blood sample from the patient, for example.

According to this aspect and in one embodiment, after withdrawal of a blood sample from a patient, the sample or portion thereof is injected into a cartridge, and the cartridge is conveniently inserted into the device. Using the touch display, measurements are started quickly and diagnostic results are obtained automatically.

According to this aspect and in one embodiment, no manual or semi-manual pre-treatment of the sample is required prior to introduction of the sample to the assay device for measurement. Therefore, field work by emergency medical assistants, medical technicians, etc. is possible because a high level of training of laboratory personnel is not required. The method of this invention eliminates the need for tedious pretreatment and thus saves valuable time. No additional pre-treatment laboratory instruments and tools are required, and the risk of biohazard from bench pre-treatment is eliminated. The method of this invention also eliminates sources of error arising from manual pre-processing.

In one embodiment, an opto-mechanical analysis instrument and a cartridge inserted therein are utilized to conduct an assay according to the method of the present invention (see FIG. 1). In one embodiment, the optomechanical device includes:

a. mechanical means operating on the cartridge to cause pre-treatment and treatment to occur inside the cartridge;

b. optical means for detecting an optical signal from the processed sample within the cartridge;

c. processing means for processing optical signals obtained from the sample;

d. Interface/display/in-out connection/remote communication features for operation and/or result display/result transmission.

In one embodiment, the processing of the optical data obtained from the sample includes computational processing. In one embodiment, computer processing includes the use of algorithms. In one embodiment, the computer processing/algorithm of this invention is novel and is designed to process the optical results to obtain various qualitative and/or quantitative information related to the biological sample. The information provided by the measurements of this invention can be tailored to a variety of user requirements. In embodiments of this invention the computer processing takes into account user requirements to obtain information relating to the identity of particular blood cells in a population, the percentage of particular blood cells from the total amount of cells, the ratio of two or more different cell types, the concentration of cells, etc. is designed by

In one embodiment, the novel cartridge of this invention is designed such that pretreatment and treatment are performed automatically within the cartridge using external mechanical means provided by the system. According to this aspect and in one embodiment, pre-treatment means mixing of cells and binding to antibodies, and treatment means dilution/lysis of the pre-treated sample. When the biological sample is inserted into the cartridge, no additional liquid is introduced into the cartridge for processing. In embodiments of the present invention, a feature that enables pretreatment within the cartridge is the presence of pre-packed dry reagents in channels/chambers within the cartridge. When the liquid sample is introduced into the cartridge and contacts the dry reagent, the dry reagent dissolves or disperses within the liquid sample. For example, when the dry reagent contains an antibody and the sample is a blood sample, the antibody dissolves into the blood sample and binds to the corresponding cells in the sample. Further processing within the cartridge may include dilution and/or lysis of the sample/cells. Dilution/dissolution entails liquid being placed in a chamber within the cartridge or in a blister connected to the cartridge beforehand, in some embodiments prior to sample insertion.

The dried reagent prepared in the cartridge may include fluorescent beads in addition to antibodies. Fluorescent beads are used for control and calibration. For example, in one embodiment, the optical fluorescence obtained from the bead represents the volume of the sample that is passed through the optical path. This can be used to assess the concentration of cells in an equal volume of sample. The amount of reference beads in the drying reagent can be controlled. Also in one embodiment, fluorescent reference beads may be used to test and calibrate optical signals obtained by optical detectors of the instrument's optical system.

In one embodiment, the cartridge includes the following features:

- sample handling elements for sample insertion and preparation;

- at least one bellows for causing fluid movement in, to or from the channel/chamber;

- at least one reactant chamber containing a liquid solution for treatment;

- at least one mixing chamber to facilitate mixing;

- at least one fluid path for conveying the processed sample to the optical analysis zone.

In one embodiment, the sample handling component includes:

- sample loading port; and

- Mixed channels.

In one embodiment, the sample loading port and/or mixing channel or portion thereof contains dry reagents. 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 partitioning and for transporting the sample to an irradiation zone for analysis, the cartridge being formed of a single cavity. become,

- two bellows each containing a flexible dome;

- a sample handling element configured to receive a biological sample therein;

- one or more mixing chambers;

- one or more reactant zones each containing a liquid reactant for selective delivery to one of the mixing chambers;

- an irradiation area comprising a reading area;

- a plurality of fluid passages each providing fluid communication between at least two other elements of the cavity; and

- comprises a vent comprising an opening to the environment;

When the sample handling element is in the sealed position, the cavity is in fluid communication with the environment only through the vent.

In one embodiment, the sample handling element includes a seal configured to move the sample handling element from an open position to a sealed position.

A sample handling unit may contain one or more dry reagents. Each of the dry reagents may include one or more reagent(s) selected from the group comprising antibodies and fluorescent beads.

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

In one embodiment, the antibody is a targeting antibody and is an antibody that specifically binds to a target antigen on a cell. In one embodiment, the antibody is linked to a colored moiety, 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 portion of the antibody functions as a cell 'stain' as its color/absorption/fluorescence signal can be optically monitored. In one embodiment, the dry reagent comprises a positive control identification antibody or a negative control identification detection moiety. In one embodiment, the drying reagent includes a chemical indicator and/or a biological indicator.

The sample handling unit may include a mixing channel having a serpentine passage.

The cartridge may include two of the mixing chambers, and each end of the mixing channel is connected to one of the mixing chambers by a fluid path spanning therebetween.

In one embodiment, each end of the mixing channels is directly connected to one of the mixing chambers without additional fluid pathways.

At least one of the bellows is connected to one of the mixing chambers by a fluid path spanning therebetween.

At least one of the bellows may be connected to the irradiation zone by a fluid path spanning therebetween.

Each of the reactant zones may include a blister pack containing a liquid reactant.

Each of the blister packs includes a flexible dome and an opposing rear backing, and each of the reactant zones includes a socket in which a corresponding one of the blister packs is sealedly received, the sockets having a rear backing. It includes a piercing element supported on the backing.

The cartridge is configured such that when the dome is pressed, the internal pressure of the blister pack rises and the rear backing is ruptured.

The liquid reactant may include one or more selected from the group consisting of a diluent, a solubilizer, and a reference substance. At least one of the fluid pathways may include a serpentine channel.

In order to better understand the subject matter disclosed herein, and to illustrate how it may be practiced, embodiments are described by way of non-limiting example only, with reference to the accompanying drawings.

1 schematically illustrates a system in accordance with the presently disclosed subject matter;
2A and 2B are front perspective and rear views, respectively, of an example of a cartridge of the system illustrated in FIG. 1;
Figure 3 is a front perspective view of another example of a cartridge of the system illustrated in Figure 1;
Fig. 4 is an example of an insert (plug) of a sample handling element, Fig. 4a is a front view, Fig. 4b is a back view, Fig. 4c isometric view, Fig. 4d is an assembled backbone;
5 is an example of an insert (plug) of a sample handling element, in which FIG. 5A is an exploded front view-isometric view and FIG. 5B is an exploded rear view-isometric view.
Figure 6 is a common partial backbone, Figure 6a is a common backbone, Figure 6b is a backbone with two insertion zones, each zone may contain a different element or no elements at all.
FIG. 7 is an embodiment of a backbone with two insertion sections, insertion section 1 (mixing channel) and insertion section 2 (loading port or sample handling element), FIG. 7A is a front view and FIG. 7B is a back view. ego;
Figure 8 is an embodiment of a backbone with two insertion zones, insertion zone 1 (loading port or sample handling element) and insertion zone 2 (blank), Figure 8a is a front view and Figure 8b is a back view. .
9A is a simplified three-dimensional interior front view of a system for detecting a biological state in accordance with an embodiment of the present invention;
9B is a simplified three-dimensional interior rear view of a system for detecting a biological state in accordance with an embodiment of the present invention.

The presently disclosed subject matter may be more readily understood with reference to the following detailed description, which forms a part of this disclosure. that the presently disclosed subject matter is not limited to the specific products, methods, conditions or parameters described and/or illustrated herein, and that the terminology used herein is intended to illustratively describe particular embodiments and presently disclosed subject matter; It should be understood that the subject matter is not intended to be limiting.

The presently disclosed subject matter provides systems, such as flow cytometry systems, and methods for biological analysis. The system provides rapid sample processing and accurate results, enabling accurate and rapid diagnosis of biological conditions. The system is compact and easily operated on-demand in the field. The system includes cartridges for initial sample processing and additional sample processing. The system, in some embodiments, further includes a spectrometer. The spectrometer device is configured to receive the cartridge and perform mechanical operations on its elements, thereby facilitating processing of the sample within the cartridge. The spectrometer is also configured to perform optical measurements of the processed sample. This spectroscopic device processes the data obtained by optical measurements and provides results indicative of the biological state. This result may be qualitative, quantitative, or both. Components of a sample analyzed in accordance with the presently disclosed subject matter may reflect the biological state of the subject.

Unless specifically defined herein, scientific and technical terms used in connection with the present application shall have meanings commonly understood by those skilled in the art. In addition, unless otherwise required by the context, singular terms include plural, and plural terms include singular terms. As employed above and throughout this disclosure, the following terms and abbreviations are to be understood to have the following meanings, unless specifically indicated.

In this disclosure, the singular forms “a” and “the” include the plural, and reference to a particular number includes at least that particular value unless the context clearly dictates 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. The term "plurality" as used herein means two or more. Where a range of values is expressed, other embodiments include from one particular value and/or to another particular value.

Likewise, when values are expressed as approximations using the term “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In the context of this disclosure, “about” a particular amount means that the amount is within ± 20% of the stated amount, preferably within ± 10% of the stated amount, or more preferably within ± 5% of the stated amount.

In some embodiments, the terms “subject” and “patient” are used interchangeably herein and refer to an animal, such as a human, being treated with a pharmaceutical composition according to the presently disclosed subject matter. In some embodiments, the terms “subject” and “patient” are used interchangeably herein, an animal from which a diagnostic test is performed and/or from which a sample is to be taken for a diagnostic test in accordance with the presently disclosed subject matter, e.g. , refers to a person The term “subject” as used herein refers to humans and non-human animals. The terms "non-human animal" and "non-human mammal" are used interchangeably herein, and include all vertebrates, including non-human primates (particularly higher primates), sheep, dogs, rodents (eg , mouse or rat), mammals such as guinea pigs, goats, pigs, cats, rabbits, cows, horses, and non-mammals (reptiles, amphibians, chickens, and turkeys, etc.). The formulations described herein can be used to diagnose/treat any suitable mammal, including primates (such as monkeys and humans), horses, cattle, cats, dogs, rabbits, and rodents (such as rats and mice). there is. In one embodiment, the mammal to be diagnosed/treated is a human. A person can be anyone of any age. In one embodiment, the person is an adult. In another embodiment, the person is a child. According to any method of the presently disclosed subject matter and in one embodiment, the subject is a 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 a dog, cat, cow, horse, lapine, or pig. In another embodiment, the subject is a mammal.

In one embodiment, the analytical instrument of the present invention is or comprises a spectroscopic device (SMD). In some embodiments, a spectrometer device is referred to as a 'system'. In another embodiment, a SMD is referred to as an optomechanical system or device, or an instrument or analytical instrument, or an optomechanical analytical instrument.

In some embodiments, pretreatment refers to mixing a sample with a dry reagent. In one embodiment, pretreatment refers to mixing the sample with antibodies/markers for binding to cells or other species within the sample. In an embodiment of this invention the treatment is any further treatment of the sample following the pretreatment step. For example, dilution/dissolution and mixing accompanying dilution/dissolution are considered treatments or additional treatments in embodiments of the present invention. This treatment is executed subsequent to the pretreatment step. In one embodiment, pretreatment and treatment together are referred to as treatment.

As illustrated in FIG. 1 , a system, generally denoted 10, is provided, which can be measured by a subject using, for example, flow cytometry and/or any other suitable method for performing one or more assays. for analyzing liquid biological samples from The system 10 includes a cartridge 12 for receiving a biological sample, and a spectroscopic instrument (SMD) 14 for facilitating manipulation and analysis of the biological sample within the cartridge.

A biological sample may include a bodily fluid sample such as blood, serum, plasma, urine, saliva, cerebrospinal fluid, serous fluid, peritoneal fluid, and/or synovial fluid. According to some embodiments, a biological sample may include a solid, such as a piece of hair, a piece of tooth, a piece of cartilage, a piece of skin, a piece of bone, and/or a piece of soft tissue.

Cartridge 12 is configured to receive therein a biological sample and optionally perform one or more processing steps on the biological sample, eg, on the pre-treated biological sample. and facilitating positioning of the processed biological sample at an interrogation point where the SMD 14 can perform the analysis. In this specification and appended claims, the term "biological sample" is used to refer to a biological sample at any stage of processing.

Thus, as illustrated in FIGS. 2A and 2B , the cartridge 12 includes a support panel 16 defining opposite front and rear surfaces 16a, 16b and 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, vent 25 , and an irradiation zone 26. (In this specification, a basic reference number may be used without a letter at the end to generically refer to all elements represented by it; thus, for example, the term “bellows 20” refers to the first and second bellows 20a , 20b)) In addition, various fluid paths are formed in the support panel 16, which fluidly connects elements of the support panel as will be described later.

As described above, at least some of the elements of the support panel 16, for example, the bellows 20, the mixing chamber 22, the irradiation zone 26, and the fluid path are press-fitting portions at the rear surface 16b. , which protrudes into the material of the support panel 16 towards its front face 16a.

The cartridge 12 further includes a backplate (not shown) disposed on the rear surface 16b of the support panel 16 . The backplate is positioned coincident with at least elements carried by the support panel 16, in particular elements projecting into the material of the support panel, as described above. It is sealingly attached to the rear surface 16b of the support panel 16, thereby fluidically isolating the element from the exterior of the cartridge 12 in the rearward direction. The backplate may be of any suitable design, including but not limited to rigid materials and adhesive films, etc., with the necessary modifications, without departing from the scope of the presently disclosed subject matter.

Support panel 16 has been described above as "bearing" the various elements of cartridge 12, but this is done for ease of explanation only, and terms used in this specification and appended claims should not otherwise be clear from context. It will be understood that it should be interpreted in its broadest sense as long as it exists, that is, including elements connected to it, elements formed therein, and the like. For example, the bellows 20 may include an element connected to the support panel 16 and the mixing chamber 22 may be formed by a structure formed in the material of the support panel.

The sample handling element 18 is configured to receive a biological sample therein and optionally to facilitate its pretreatment therein. Accordingly, it includes a mixing channel 28 formed across the first opening and the second opening 30a, 30b (the location of which is indicated and illustrated in FIG. 2B). The mixing channel 28 may include a serpentine passage 32 having a receiving socket 34 formed therein, for example for receiving a biological sample. Receiving socket 34 may be part of serpentine passage 32, but has a wider width, to ensure proper functioning of system 10 and/or to facilitate placement of a biological sample therein. It may indicate where the biological sample should be placed in order to

At least a portion of the top surface 36 of the sample handling element 18 is sealed thereto with a thin element having an adhesive surface, for example a seal (not illustrated in FIGS. 2A and 2B ), for example a sticker. It may be flat to facilitate attachment, thereby leaving the sample handling element 18 in a sealed condition. According to some embodiments, the continuous portion of the top surface 36 completely surrounding the tortuous passageway 32 may form a single plane, thereby facilitating contact therewith with the plane seal.

As illustrated in FIG. 3 , in accordance with some embodiments, the sample handling element 18 includes a sample port 34a for receiving a biological sample, and the cartridge 12 includes, for example, a support panel ( 16) and a corresponding plug 34b constituting a seal arranged to coincide with the sample port. The plug 34b is configured to sealably engage the sample port 34a, thereby bringing the sample handling element 18 into a sealed condition. According to these embodiments, mixing channel 28 is isolated from the environment other than through sample port 34a and vent 25 .

As best seen in FIG. 2B , the first opening 30a is in fluid communication with a first sample-mixing fluid passage 38a spanning between the sample handling element 18 and the first mixing chamber 22a, and The second opening 30b is in fluid communication with a second sample-mixing fluid pathway 38b spanning between the sample handling element 18 and the second mixing chamber 22b. Since the sample handling element 18 is disposed between the two mixing chambers 22, its serpentine passageway 32 can be utilized to mix the biological sample and reactants therein.

The sample handling element 18 may be provided with one or more reagents, eg, dry reagents disposed within the mixing channel 28 within the serpentine passage 32 . A reagent may be adapted for use in pretreatment of a biological sample. Thus, it may include an antibody, a target antibody, a positive control identification antibody, a negative control identification detection moiety, a target signal reference composition, a reference identification composition, a cell stain, a chemical indicator, a biological indicator, and/or any other suitable reagent. . Reagents can be provided on the surface of a reagent block, bound to a solid support, bound/deposited on a wall/surface of a channel, or provided by any other suitable means.

Although "reagent" and "reactant" may be used interchangeably, in this specification and the appended claims, the term "reagent" is used to refer to a substance disposed within the sample handling element 18, and the term "reactant" is used. It will be appreciated that it is used to refer to a material disposed within reactant zone 24. This distinction is only for clarifying the present disclosure and should not be regarded as limiting. In particular, the term “reactant” may include substances that those skilled in the art would classify as reagents, and vice versa. Moreover, a single substance may be considered both a “reactant” and a “reagent, depending on its location, for the purposes of this disclosure.

Each of the bellows 20 comprises a flexible dome 40 projecting from the front surface 16a of the support panel 16, for example in the form of a hemispherical or spherical cap, and a back plate constituting its planar base. . The dome can be formed so that it remains within its elastic limits through complete compression, ie it can be fully compressed and return to its original shape upon removal of the pressing force. Dome 40 may be formed as part of support panel 16 or may include 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 the second bellows and the irradiation zone (26). ) is in fluid communication with the bellows-irradiated fluid path 42b spanning between.

Each of the reactant zones 24 contains therein a blister pack 44 (one of which is illustrated in a separate configuration in FIG. 2A) and selectively delivers its contents to one of the mixing chambers 22. configured to facilitate Each of the blister packs 44 has a flexible dome 46 in the form of, for example, a hemispherical or spherical cap, and a rear backing (not shown) consisting of a thin layer of material on its opposite side, for example, a metal foil. city), including Each of the reactant zones 24 further comprises a socket 48 formed, for example, as a recess in the front surface 16a of the support panel 16, in which the base surface 50 is formed; It receives a corresponding one of the blister packs 44 in a sealed state therein, whereby the rear backing is spaced apart from the base face 50 . Each base face 50 includes a piercing element 52 projecting towards the front face 16a of the support panel 16 . Each socket 48 is formed with, for example, an outlet 53 including a through opening adjacent to its lower end. As best shown in FIG. 2B , each of the outlets 54 is in fluid communication with a reactant-mixing fluid pathway 56 spanning between each reactant zone 24 and one of the mixing chambers 22 .

Each of the blister packs 44 may contain one or more reactants, eg in liquid form, for delivery to the biological sample in one of the mixing chambers 22 . The reactants can be diluents, solubilizers, reference materials, or any other suitable reactants, and/or combinations thereof.

Each of the piercing elements 52 may be configured to be supported on the rear backing of a corresponding blister pack 44 when received within the socket 48, such that, for example, by pressing on the dome 46 The internal pressure of the blister pack rises, which prevents the piercing element from piercing the back backing until it causes rupture of the back backing. The support of piercing element 52 on the backing of blister pack 44 can create a rupture zone thereon, which is associated with an increased likelihood of rupture when dome 46 is depressed. Accordingly, the piercing element 52 may be positioned proximate to the corresponding outlet 54 such that reactants released from the blister pack 44 upon rupture may immediately enter the corresponding reactant-mixing fluid pathway 56. there is.

Vent 25 is in fluid communication with the environment and facilitates movement of the biological sample within the cavity when bellows 20 is depressed and/or released. Accordingly, it can be placed over the remaining elements of the cavity, i.e., in the orientation of cartridge 12 when inserted into SMD 14, and in fluid communication therewith, mixing via, for example, vent fluid pathway 25a. It is connected to one of the chambers 22.

As best seen in FIG. 2B , irradiation zone 26 is configured to facilitate analysis of a biological sample by SMD 14 . Thus, it includes a read fluid path 58 that includes a narrow read area 60 . The reading area 60 may be designed to be sufficiently wide to limit the number of particles passing through it simultaneously, for example, only one particle at a time for analysis by the SMD 14 . Furthermore, the material of the irradiation zone 26, particularly in the readout zone 60, may be made of a material that is transparent to the wavelength of light emitted by the SMD 14 when performing the analysis.

One end of the fluid readout path 58 is in fluid communication with the second bellows 20b via the bellows-irradiated fluid path 42b as described above. The other end of the fluid readout path 58 is in fluid communication with the second mix chamber 22b via a mix-irradiation fluid path 62. At least a portion of the mix-irradiate fluid path 62 may constitute a serpentine channel. It should be noted that the geometry of the irradiation fluid path can vary and can be tailored to any desired geometry to fit the design of a particular cartridge. For example, in one embodiment, fluid pathway 62 does not include tortuous channels.

When the sample handling element 18 is, for example, in the sealed position as described above, the elements of the aforementioned support panel 16 provide the necessary vents 25 to enable compression of the bellows 20. It will be appreciated that, except for , constitutes a closed system, i.e., constitutes a single cavity that is isolated from the environment, such that no further, including but not limited to air or other fluids, is introduced. Accordingly, movement of the biological sample therein may be performed entirely by mechanical interaction with elements of the support panel 16, including but not limited to acting on the bellows 20, as described below. can (The blister pack 44 can be considered a subchamber within the cavity as its contents are isolated from the environment and are intended to be introduced into the remainder of the cavity during operation.) Thus, to move a biological sample therein No external pump or other means for introducing air into the cavity is required. In one embodiment, no external air pump is used to force air directly into the cartridge.

SMD 14 is described in, for example, US 2013/0102087, US 2014/0170678, US 2014/0170680, US 2014/0287435, US 2015/0132776, US 2015/0293095, US 2015/0309049, US 2015 /0330971, Interface with cartridge 12 as described in one or more of US 2016/0146793, US 2017/0350888, US 2017/0370914, US 2018/0231532, US 2018/0299443, US 2018/0306698, and/or US 2020/0080928 Singh It may be any suitable device for doing so, the entire contents of which are incorporated herein by reference. In particular, SMD 14 is an element configured to selectively interact with bellows 20 and reactant zone 24, such as to press and/or release each when cartridge 12 is inserted. elements may be included. The SMD 14 includes, for example, an optics unit comprising a light source, a photon counter and/or an integrator configured to facilitate analysis of a portion of a biological sample within the reading area 60 of the irradiation zone 26. can include more.

SMD 14 may further include a processor for instructing its operation. One or more steps of the analysis, wherein the processor is configured to receive data relating to the multi-spectral luminescent signal, eg, detected by the optics unit, process the data, and output a result relating, eg, to a medical condition. can do more. Each of the multispectral luminescent signals may be associated with a biological marker.

In use, a biological sample is inserted into the receiving socket 34 formed in the sample handling element 18 and then sealed, for example by providing a seal thereon as described above. Cartridge 12 is inserted into SMD 14, for example, in a slot designed for this (see FIG. 9A, cartridge 910). SMD 14 selectively interacts with elements of the cartridge to facilitate selective pretreatment, processing, and analysis of the biological sample, for example, as described below.

In an optional pretreatment step, the first and/or second bellows 20a, 20b are selectively pressed or released by the SMD 14. Since the biological sample is in a closed system comprising the bellows 20, interaction with the bellows causes the biological sample to move within the tortuous passage 32 of the sample handling element 18, thereby causing the mixing channel ( 28), and the only opening in this closed system is the vent 25 required to press and release the bellows. The first and second bellows 20a, 20b are alternately pressed to move back and forth within the serpentine passage 32, thereby facilitating mixing with the dry reagent.

The recitation herein that the bellows 20 is "depressed" or "released" is not limited to fully depressed or fully released, and each of the bellows may be partially depressed or released to move the biological sample by a predetermined amount within the chamber. It will be understood that there is

The interaction of SMD 14 with bellows 20 may be performed to control the order of mixing of the biological sample with two or more reagents. For example, the first bellows 20a may be pressed to move a predetermined amount within the mixing channel 28 such that the biological sample reaches and dissolves the first dry reagent. The first and second bellows 20a, 20b are then pressed alternately so that the biological sample and the first reagent move back and forth within a portion of the serpentine passage 32 to allow sufficient mixing and time for the reagent to act on the biological sample. can be secured. Next, the first bellows 20a is depressed to move the biological sample further along the mixing channel 28, whereby the biological sample reaches and dissolves the second dry reagent. The order of interaction of the SMDs 14 with the bellows 20 can be implemented to achieve any suitable sequence, such as mixing of the biological sample with dry reagents within the sample handling element 18, with the necessary modifications. that will be understood

In one or more processing steps, the biological sample is selectively moved into one or both of the mixing chambers 22 . For example, to move the biological sample from the sample handling element 18 into the first mixing chamber 22a, the second bellows 20b is depressed, thereby creating a positive pressure behind it. The SMD 14 may selectively facilitate the transfer of the contents of one or more of the blister packs 44 into one of the mixing chambers 22 by depressing it, thereby, for example, in the back backing as described above. It can cause rupture to release the liquid reactants inside. The reactants released from the blister pack 44 upon rupture flow into the corresponding reactant-mixing fluid pathway and through it into the corresponding mixing chamber 22 .

In the irradiation step, the treated biological sample moves into the irradiation zone 26 by interaction of the SMD 14 with the bellows 20 . The SMD 14 can, if desired, adjust the amount of pressure applied on the bellows 20 to adjust the position of the biological sample within the reading area 60 of the irradiation area 26.

In some embodiments, any one of the two or more bellows may cause movement of the liquid sample between components of the cartridge. Each bellows can be depressed and released step by step to cause liquid movement back and forth through the channel, into/out of the chamber, or toward the optical irradiation zone. According to this aspect and in one embodiment, the cartridge includes only one bellows. In one embodiment, two bellows cooperate to drive liquid back and forth within the cartridge as described herein. In one embodiment, one bellows pushes the liquid in one direction and the other bellows pushes the liquid in the opposite direction. In one embodiment, one or two bellows drive the liquid to cause mixing, and only one of the two bellows drives the liquid towards the optical irradiation zone. In one embodiment, pressing and releasing one bellows is sufficient to move the liquid back and forth within the components of the cartridge. In one embodiment, alternating pressing and/or releasing of the two bellows is used to move the liquid back and forth within the components of the cartridge. In one embodiment, the degree to which liquid advances within a particular channel/component of the cartridge is determined by progressively depressing/releasing one or more bellows.

4 shows one embodiment of a cartridge in which a reagent sample plug (RSP) is connected to the cartridge after manufacture of the cartridge. This RSP is shown in Figures 4a (front view) and 4b (rear view). The sample loading chamber is shown in Figure 4a (Element 1). In one embodiment, reagents are dried within the sample loading chamber (1). In one embodiment, reagents are dried within the swollen region (shown on the left as part of a serpentine channel). The dried reagents may be dried within both regions, and in one embodiment within the serpentine channel. Any combination of locations for dry reagents within the sample handling element is included in embodiments of the present invention. Figure 4b shows an upper interface with the cartridge (element 2) and a lower interface with the cartridge backbone (element 3). Figure 4d shows the cartridge with the sample insert connected. According to this aspect and in one embodiment, the RSP is not part of the cartridge molding. Instead, it is added as an additional element and connected to the molded body. After connecting the two, the cartridge is ready for sample insertion. In this configuration, there is an external pump that aspirates the sample into the cartridge and, in some embodiments, effects mixing within the serpentine channel. In other embodiments, mixing or transporting the sample through the channel of the sample plug is described herein. This is achieved by at least one of the bellows as described. In an embodiment, the term 'backbone' is used for cartridges. It should be noted that the sample handling elements can be placed anywhere on the cartridge. It can be manufactured together with the other components of the cartridge in the molding step, or it can be added later and fixed to the cartridge. The sample handling element may be connected between the two mixing chambers, or may be connected to only one mixing chamber in a one or two mixing chamber configuration. 5A and 5B are front and rear exploded isometric views of the RSP showing the area to which the cartridge is connected, along with associated matching components. Figure 6 shows one embodiment of the invention in which the cartridge molded body is designed to have two zones. Each zone may contain different features or no features at all. Desired feature(s) may be included in the production of any shaped body as desired. 6A shows a common backbone that can have additional features in different regions. For example, FIG. 6B shows zone 1 and zone 2. In Zone 1, a sample handling element can be placed (when producing a molded body) or a mixing channel without a sample insertion port can be formed. In zone 2, sample handling elements can be formed (when producing molded bodies) or this zone can be left blank. It should be noted that additional zones can be formed as part of the shaped body. Features, such as sample handling elements, can be manufactured along with the molded body or added after fabrication of the molded body. 7A and 7B show rear and front views of a cartridge, where zone 1 contains the mixing channel and zone 2 contains the sample handling elements. Figure 8 shows one embodiment of a backbone with two insertion zones, insertion zone 1 (loading port or sample handling element) and insertion zone 2 (blank), Figure 8a is a front view and Figure 8b is a back view. It is also

Figure 9 shows one embodiment of the system (analysis instrument) of the present invention. In FIG. 9A, a diagram 920 on the left shows 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 forward scatter detector 930. In FIG. 9B , right side view 940 shows reader optics 942 , data collection board 944 and general electronics printed circuit board 946 .

In one embodiment, the present invention provides systems and methods for biological assays, comprising a cartridge as described herein for conducting an assay therein, the cartridge comprising at least one reagent adapted to react with a sample. It is configured to receive reagents. In one embodiment, the system of the present invention includes a mechanical controller comprising urging means configured to apply external forces to components of the cartridge, such as blisters and bellows. In one embodiment, a system includes an optical reader adapted to detect a sample, and a processor adapted to receive data from the optical reader and process the data to provide analytical results.

In one embodiment, the present invention provides a system for performing an assay on a biological sample, the system comprising:

- a cartridge as described herein for performing an assay therein, the cartridge comprising at least one reagent adapted to react with the sample;

— mechanical controls, including:

* at least one first urging means adapted to apply a force to the bellows to cause movement of the fluid in a first direction and/or a second direction;

* at least one second pressing means adapted to exert a force on the blister(s) to expel the diluent/dissolving solution;

- an optical reader adapted to detect the sample; and

- A processor adapted to receive data from the optical reader and process this data to provide analytical results.

Further, according to one embodiment of the present invention, the cartridge further comprises alignment means adapted to align the read channel on the cartridge with the optical component of the system for optical detection of a sample within the cartridge by an optical reader of the system.

In one embodiment, the method of the present invention comprises the following steps:

- loading the sample into the sample handling element;

- fixing the sample element to the cartridge;

- inserting the cartridge into the system;

- mixing the contents of the sample handling element (mixing the sample with dry reagents);

- extracting the sample from the sample element into the mixing chamber;

- Initiating processing and analysis.

The order of the steps may be varied or changed as described in the embodiments of the present invention. For example, in embodiments where the sample element is part of a cartridge molding, securing the element to the cartridge is unnecessary. In one embodiment, the mixing and extraction of samples are performed at least partially in parallel.

Other features of this system are described, for example, in WO2014/097287, the entire contents of which are incorporated herein by reference.

In one embodiment, the channel or fluid path of the cartridge has a cross-sectional dimension ranging from 200 μm to 1 mm. In one embodiment, the cross-section of the channel of the cartridge has a width ranging from 200 μm to 1 mm and a width ranging from 200 μm to 1 mm. It is a rectangle with depth. In one embodiment, the cross section of the channel has a width of 800 μm and a depth of 600 μm.

In one embodiment, reagents are dried inside the channel before the insert is connected to the cartridge. After drying, the insert is connected to the cartridge. All further operations such as sample insertion, flow of the sample through the channel with drying reagents, and processing are performed by the bellows. No external pump is required.

In one embodiment, the irradiation zone is an area comprising a readout channel, which is a channel through which fluorescently labeled cells (with or without additional fluorescent beads) pass. In one embodiment, the readout channel is the wavelength(s) of excitation It is transparent to light and is also transparent to the wavelength range of fluorescence emitted from cells. In one embodiment, being transparent at a particular wavelength is completely transparent. In one embodiment, the partial transparency of the channel at a particular wavelength is sufficient for emission/detection of a signal through the channel.

Processing in embodiments of the invention may in some embodiments relate to chemical/biological processing, and in some embodiments to computer processing of signals. In one embodiment, sample treatment refers to both chemical treatment and optical treatment. The meaning of the term processing is clear from the context of the embodiments described herein. In one embodiment, the read area of the cartridge is an optical read area. According to this aspect and in one embodiment, the sample in the readout zone is illuminated with light from a light source, in response to which the sample emits light towards the at least one detector. The optical (optical) signals obtained by the detector are processed to produce results representing the constituents of the sample. Such results may be qualitative, quantitative, semi-quantitative or any combination thereof.

In one embodiment, staining refers to mixing and/or binding of an antibody (or other complex not described herein) to a particle/species/fragment present in a sample. In one embodiment, staining refers to a pretreatment step or part thereof. In one embodiment, staining refers to the association of a fluorescent marker with a cell or other species in a sample. In one embodiment, staining or pretreatment refers to the process by which an antibody (or similar compound) that is already linked to a fluorophore attaches to or binds to cells (or other particles/species in a sample). According to this aspect and in one embodiment, a fluorescent antibody that binds to cells enables optical detection of cells.

In some embodiments, a sample handling element is called a sample handling unit. In some embodiments of the invention, zone, area, region, and channel are interchangeable and may refer to the same feature.

In one embodiment, instead of multiple fluid pathways each providing fluid communication between at least two other elements of the cavity, only one such fluid pathway is required in addition to the channel of the sample handling element. According to this aspect and in one embodiment, a fluid path connects the mixing chamber to the readout channel.

It should be noted that the orientation of components within a cartridge as shown in FIG. 1 is exemplary. The various components of the cartridge may be placed in many different orientations within the cartridge. Since the various components of the cartridge are connected by fluid pathways, the components can be assembled in many different configurations, and the fluid pathways (channels) can be designed to connect the components as needed. All of these orientations are included in the embodiments of the present invention.

In one embodiment, the system adjusts the time and/or rate of depression/release of the bellows 20 to adjust the time/rate of liquid flow within the elements of the cartridge.

In one embodiment, the cartridge does not include sample handling elements. According to this aspect and in one embodiment, the pretreatment is performed outside the cartridge, and the pretreated sample is directly injected into the mixing chamber or injected into a fluidic channel connected to the mixing chamber for further processing. According to this aspect and in one embodiment, the pretreatment comprises adding fluorescent beads to the sample.

It should be noted that the connection between the reactant zone and the mixing chamber as shown in the figure is exemplary. In embodiments, each reactant zone may be connected to each of the mixing chambers as needed for a particular application. In one embodiment, the cartridge contains 1 to 3 blisters. In one embodiment, the blister fill is for dilution/dissolution only. In one embodiment, the blister fill is not for antibody/reference beads. In one embodiment, where there are two or more blisters, different dilution ratios can be achieved by using different blisters/more than two blisters as needed. In one embodiment, two or more blisters contain the same solution, and in one embodiment, two or more blisters contain different solutions. In one embodiment, two or more blisters contain solutions of the same components but different concentrations of components.

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

The sample handling element insert is considered part of the cartridge in embodiments of the present invention.

In one embodiment, the sample loading area/port and serpentine dry reagent channels (sample handling elements) were fabricated as part of the cartridge mold. The sample handling element is part of the cartridge molded body according to the present embodiment.

The sample handling element insert is considered part of the cartridge in embodiments of the present invention.

In one embodiment, after introducing the sample into the sample handling element, the opening of the sample loading port is sealed using a sticker seal. In embodiments of the present invention, other sealing options known in the art are provided, such as, for example, the plug 34b of FIG. 3, plastic caps, screw caps, adhesives, rubber seals, and the like. In one embodiment, a 'co-molded plug' (such as element 34b in Figure 3) closes the opening (eg, element 34a) after the sample is inserted/loaded therethrough. used for sealing.

In one embodiment, there is a sample loading port on the dry reagent insert (eg, see the bulging region of element 1 in FIG. 4A). In one embodiment, the cartridge is referred to as the backbone. In one embodiment, the sample handling element is referred to as a reagent sample plug (RSP).

In one embodiment, an external pump draws the sample through a channel of the sample handling element for further processing. In another embodiment, an external pump is not necessary and the sample is drawn by bellows through a channel of the sample handling element into the cartridge for further processing.

In one embodiment, movement of liquid through the components of the cartridge is not effected by an external pump. In one embodiment, the cartridge does not include a valve. In one embodiment, the cartridge is valveless.

In one embodiment, movement of liquid through the components of the cartridge using bellows reduces the complexity of the system and allows for easy handling as external pumps are not connected to channels within the cartridge.

In one embodiment, reagents are dried inside the channels 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 aspiration, flow through the channels with drying reagents and processing) are carried out by bellows. No external pump is required. In one embodiment, staining is induced by bellows and an external pump is not required.

In one embodiment, the present invention provides an assay method comprising:

- providing a cartridge as described herein above;

- introducing the biological sample into the cartridge;

- inserting the cartridge into the optomechanical system described herein;

- actuating at least one bellows in the cartridge using at least one pressing means in the system to cause sample flow through the dry reagent channel of the sample handling element (18);

- actuating at least one bellows in the cartridge using at least one pressing means in the system to cause a flow of the stained (pretreated) sample from the sample handling element to the at least one mixing chamber;

- rupturing at least one blister in the cartridge using at least one compression means in the system to cause flow of the dilution/dissolution solution from the reactant zone to the at least one mixing chamber;

- operating at least one bellows in the cartridge using at least one pressing means in the system to cause mixing of the dilution/dissolution solution and the pretreated sample in the at least one mixing chamber;

- actuating at least one bellows in the cartridge using at least one pressing means in the system to cause a flow of the treated solution from the at least one mixing chamber to the irradiation zone comprising the reading channel;

- illuminating the readout channel using a light source of the system and detecting an optical signal obtained from the readout channel by at least one detector of the system;

- Obtaining analysis results by processing the detected optical signal using the system's processor.

Embodiments described herein for cartridges and systems are applicable to embodiments of the inventive method described herein.

In one embodiment, the system of the present invention does not include a heater. In one embodiment, the system of the present invention does not include a temperature control device. In one embodiment, the system of the present invention does not include a thermometer.

In addition to the antibodies (and optionally reference beads), the dry reagent(s) of the present invention may include salts, stabilizers, pH adjustment materials, ionic strength adjustment materials, and suitable and/or helpful agents to maintain the sample in a suitable state for processing and measurement. It may also include chemicals such as other compounds/materials. According to this aspect and in one embodiment, the dry reagent comprises a mixture.

In one embodiment, the sample comprises cells. In one embodiment, the cell is a blood cell. In one embodiment, the cell comprises red blood cells, white blood cells or a combination thereof. In one embodiment, the cells include stem cells. In one embodiment, the sample comprises cell fragments. In one embodiment, the sample includes particles. In one embodiment, the sample comprises an antigen. In one embodiment, the sample comprises cells, and the cells comprise cell surface markers. In one embodiment, a surface marker is or comprises an antigen. In one embodiment, in the cartridge assay of the present invention, an antigen in a sample (eg, an antigen on the surface of a cell) binds to an antibody in a dry reagent. In one embodiment, a fluorescent tag on the antibody is used to identify cells. Different cell surface markers may bind to different antibodies. In one embodiment, different antibodies include different fluorescent tags. According to this aspect and in one embodiment, certain cells bind different antibodies with different fluorescent tags. The fluorescence wavelength(s) and fluorescence intensity emanating from a particular cell indicates the type of cell in some embodiments.

In one embodiment, the assay of the invention is a flow cytometry assay. In one embodiment, determining/detection of a biological state is referred to as sample analysis, or analysis or determination of a medical state. In one embodiment, the reactant zone is referred to as a blister for simplicity. In one embodiment, the antibody is a fluorescent antibody. In one embodiment where a 'cell' is referred to, the cell is a biological cell. Embodiments that refer to cells may also refer to other particles, cell fragments, proteins, antigens, biomolecules, cellular components, viruses, bacteria, microorganisms or components thereof. All of these embodiments are included in the present invention. In one embodiment, the particle is a cell, bead, or any species described herein above.

In one embodiment, a reference bead is a fluorescent bead and may refer to a microsphere, nanosphere, fluorescent sphere or a combination of these terms. In one embodiment, phosphor and fluorochrome are interchangeable terms. In one embodiment, the fluorescence obtained from the cells/particles of the present invention is direct, indirect or any combination thereof. Embodiments of the invention described for treatment may also be referred to as diagnostic. In one embodiment, tags, probes, and markers are interchangeable. Mixing chambers of the present invention can take many shapes, sizes and geometries, all encompassed by embodiments of the present invention. In one embodiment, the present invention provides cell staining in an automated cartridge. In one embodiment, not all of the reactant zones contain blisters. In one embodiment, at least a portion of the reactant zone is free of blisters. In one embodiment, at least a portion of the reactant zone includes blisters. In one embodiment, the location of zones/chambers/elements/components on the cartridge can be varied as long as the required connections between any two or more zones/chambers/elements/components are maintained.

In one embodiment, the bulge area in the sample handling element is used for placement of dry reagents, introduction of sample, or both. In one embodiment, the assay of the present invention is a leukocyte assay. In one embodiment, the sample before treatment is a whole blood sample.

In one embodiment, an assay of the present invention is completed within 30 minutes or within 1 hour from the time of sample insertion into the sample handling element. In one embodiment, the assay of the present invention is completed within 30 minutes or within 1 hour from the time of cartridge insertion into the system.

Those skilled in the art to which this invention pertains will readily appreciate that many changes, modifications, and alterations may be made, incorporating necessary changes, without departing from the scope of the presently disclosed subject matter.

Claims (13)

A cartridge for preparing a biological sample for analysis and for transporting the sample to an interrogation zone for analysis, comprising:
The cartridge is formed of a single cavity, and the cartridge comprises:
- two bellows each containing a flexible dome;
- a sample handling element configured to receive the biological sample therein, the sample handling element comprising one or more dry reagents;
- one or more mixing chambers;
- one or more reactant zones each containing a liquid reactant for selective delivery to one of said mixing chambers;
- an irradiation area comprising a reading area;
- a plurality of fluid passages each providing fluid communication between at least two other elements of said cavity; and
- comprising a vent comprising an opening to the environment;
wherein when the sample handling element is in its sealed position, the cavity is in fluid communication with the environment only through the vent. According to claim 1,
wherein at least one of the dry reagents comprises an antibody. According to claim 2,
wherein the antibody binds to a fluorescent marker. According to claim 3,
The cartridge of claim 1, wherein the dry reagent comprises fluorescent beads. According to any one of claims 1 to 4,
wherein the sample handling element comprises a mixing channel having a serpentine passageway. According to claim 5,
A cartridge comprising two of said mixing chambers, wherein each end of said mixing channels is connected to one of said mixing chambers by a fluid path spanning therebetween. According to any one of claims 1 to 6,
and at least one of the bellows is connected to one of the mixing chambers by a fluid path intervening therebetween. According to any one of claims 1 to 7,
wherein at least one of the bellows is connected to the irradiation zone by a fluid path spanning therebetween. According to any one of claims 1 to 8,
wherein each of the reactant zones includes a blister pack containing the liquid reactant. According to claim 9,
each of the blister packs includes a flexible dome and an opposing rear backing, and each of the reactant compartments includes a socket in which a corresponding one of the blister packs is sealedly received; wherein the socket includes a piercing element supported on the back backing. According to claim 10,
The cartridge is configured such that pressing the dome increases the internal pressure of the blister pack to cause rupture of the rear backing. According to any one of claims 1 to 11,
The cartridge according to claim 1 , wherein the liquid reactant comprises at least one selected from the group consisting of a diluent, a solubilizer, and a reference substance. According to any one of claims 1 to 12,
wherein at least one of the fluid pathways includes a serpentine channel.

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