A method of separating target cells from non-target cells in a liquid biopsy sample, and a kit therefor

Abstract

Methods are provided to separate target cells from non-target cells in a liquid biopsy sample, based on the cells' sedimentation properties. One method involves providing a container comprising a volume Vd of a density medium and then adding a volume Vb of a liquid biopsy sample on top of the density medium; the volume Vb of the liquid biopsy sample is a sum of the volume of a liquid fraction Vp and the volume of cells Vc in the liquid biopsy sample. Next, the non-target cells in the liquid biopsy sample are allowed to sediment in a sedimentation layer at the bottom of the container; the sedimentation layer comprises a liquid volume Vremaining between the sedimented cells. Lastly, the supernatant comprising target cells is collected. In this method, the density of the density medium is higher than the density of the liquid biopsy sample but lower than the density of the non-target cells and the volume Vd of the density medium is at least equal to Vremaining. A method is provided to separate target cells from non-target cells in a liquid biopsy sample, based on the cells' sedimentation properties The method involves first providing a container comprising a volume Vd of a density medium. Next, a volume Vb of a liquid biopsy sample is added on top of the density medium. The non-target cells in the liquid biopsy sample are then allowed to sediment in the density medium. Next, the supernatant comprising target cells is collected. The volume Vb of the liquid biopsy sample is a sum of the volume of a liquid fraction Vp and the volume of cells Vc in the liquid biopsy sample, the volume Vd of the density medium is at least equal to the Vp. A kit to separate target cells from non-target cells in a liquid biopsy sample includes a container that contains a volume Vd of a density medium, and instructions to carry out the method.

Description

[0001] A METHOD OF SEPARATING TARGET CELLS FROM NON-TARGET CELLS IN A LIQUID BIOPSY SAMPLE, AND A KIT THEREFOR

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties. Also, a related kit for separating target cells from non-target cells in the liquid biopsy sample is disclosed.

[0004] BACKGROUND

[0005] Sepsis is a severe medical condition characterized by a systemic host inflammation response to infection. Sepsis has an incidence of approximately 50 million cases annually, causing a significant healthcare burden. The mortality of diagnosed patients is between 25% and 40%. An estimated 25-30% of sepsis cases involve bloodstream infections (BSI).

[0006] The rapid and identification of causative organisms in patient blood are important for determining effective antibiotic treatment. The low microbial loads in the bloodstream of patients, as low as one to ten colony-forming units (CFUs) per ml of blood, require large sample volumes of blood, which is particularly challenging for pediatric patients. Furthermore, the microorganisms typically need several hours to days of culture before the presence and identification of the pathogens can be confirmed. The latter is problematic, considering the 8% drop in survival rate per hour delayed treatment for patients suffering septic shock.

[0007] The urgency of the condition, coupled with the latency of the aforementioned diagnosis methods, means that combination therapy with broad-range antibiotics is often prescribed as first-line therapy immediately after the blood drawing. This praxis results in suboptimal treatment, contributes to the increase of antibiotic resistance, and has also been shown to increase liver toxicity compared to targeted antibiotic monotherapy.

[0008] The state-of-the-art method for detecting bacteria from sepsis patients is by culturing the blood, followed by bacterial identification with genotypic (e.g., polymerase chain reaction), phenotypic (e.g., subcultures), or mass spectrometry methods (e.g., matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF)). While genotypic methods can identify bacteria at low concentrations without culturing the blood, they do not suit drug profiling, limiting clinical impact. Phenotypic and proteomic approaches typically require a higher bacterial load, necessitating culture and therefore long processing time. Single-cell phenotypic methods, which specifically bypass the requirement for blood culture, offer a quicker alternative but necessitate the initial removal of blood cells. Isolation of bacteria from whole blood has been accomplished with inertial and elastoinertial microfluidics, sedimentation velocity-based separation, filtration, chemical capture, magnetic bead-based separation, dielectrophoresis, or acoustic separation.

[0009] However, most of these isolation methods suffer limited throughput and low blood cell rejection rates or have only been demonstrated for bacteria concentrations of 1000 CFU / ml or above, thus necessitating time-consuming bacterial preculture.

[0010] There is a need for a rapid and high-throughput assay for isolating bacteria from whole blood with high efficiency and to allow integration with microfluidic trapping and rapid microscopy detection. There is a need for a method with potential for rapid identification and drug profiling of bacterial agents in the blood of septic patients within 3-4 hours, as opposed to the current several days. Such capability promises timely and appropriate antibiotic treatment and could greatly impact the sepsis patients’ survival rates.

[0011] The need for a high-throughput assay for isolating is not limited just to bacteria, but also to other target cells, generally microbial cells, specifically fungal and viral cells.

[0012] SUMMARY

[0013] Preferred embodiments of the present invention preferably seek to mitigate, alleviate or eliminate one or more deficiencies, disadvantages or issues in the art, such as the aboveidentified, singly or in any combination by providing a method, according to the appended dependent patent claims.

[0014] It is an object of the present invention to provide a method to separate target cells from nontarget cells in a liquid biopsy sample, based on the cells’ sedimentation properties.

[0015] According to a first aspect of the present disclosure, there is provided a method to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties. The method involves providing a container comprising a volumedof a density medium and then adding a volume Vbof a liquid biopsy sample on top of the density medium, wherein the volume Vbof the liquid biopsy sample is a sum of the volume of a liquid fraction Vpand the volume of cells Vcin the liquid biopsy sample. Next, the non-target cells in the liquid biopsy sample are allowed to sediment in a sedimentation layer at the bottom of the container, wherein the sedimentation layer comprises a liquid volume Vremaining between the sedimented cells. Lastly, the supernatant comprising target cells is then collected. In this method, the density of the density medium is higher than the density of the liquid biopsy sample but lower than the density of the non-target cells and the volume Vdof the density medium is at least equal to Vremaining .

[0016] According to a second aspect of the present disclosure, there is provided a method to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties. The method involves first providing a container comprising a volumedof a density medium. Next, a volume Vbof a liquid biopsy sample is added on top of the density medium. The non-target cells in the liquid biopsy sample are then allowed to sediment in the density medium. Next, the supernatant comprising target cells is collected. In the method, the volume Vbof the liquid biopsy sample is a sum of the volume of a liquid fraction Vpand the volume of cells Vcin the liquid biopsy sample, the volume Vdof the density medium is at least equal to the Vp.

[0017] According to a third aspect of the invention, a kit to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties is provided. The kit comprises a container that contains a volume Vdof a density medium, and instructions to carry out the method according to any one of the method claims.

[0018] It should be emphasized that the term “comprises / comprising” when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0019] Further features of, and advantages with, the present invention will become apparent when studying the appended claims and the following description. The skilled person realizes that different features of the present invention may be combined to create embodiments other than those described in the following, without departing from the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above, as well as additional objects, features and advantages of the present invention, will be better understood through the following illustrative and non-limiting detailed description of exemplary embodiments of the present invention, wherein:

[0021] Fig. 1A -1D show flowchart illustrations of different embodiments of the methods to separate target cells from non-target cells in a liquid biopsy sample.

[0022] Fig. 2 shows a graphical representation of the method steps.

[0023] Fig. 3 shows a workflow for bacterial detection from blood samples in four steps: 1) isolation using smart centrifugation; 2) selective blood cell lysis; 3) volume reduction; and 4) detection in microfluidic traps. BCM is blood culture medium.

[0024] Figs. 4A-4C show bacterial isolation from blood by smart centrifugation. Fig. 4A. Illustration of liquid and cell movement. The left and right tubes illustrate the positions of sample liquid, density medium, red blood cells and bacteria before and after smart centrifugation. The middle graph qualitatively illustrates the trajectories (solid lines) of bacteria (green) and red blood cells (red) during centrifugation, from a mixed state (left brackets) to a separated state (right brackets). The slopes of the lines are the particle sedimentation speeds, vRBC and vbac, and liquid interface, vint, and sedimentation interface velocity, vsed, derived in SI. Fig. 4B. shows blood cell removal efficiency, meaning the fraction of blood cells removed from the supernatant after centrifugation relative to the initial number of blood cells in the sample. Fig.4C. shows bacterial isolation efficiency, meaning the number of colony-forming units in the supernatant after centrifugation relative to the initial number of colony-forming units in the spiked sample. Bar heights are mean; error bars are sd; n.s. and ***indicate significance levels p > 0.05 and p < 0.001 , respectively; the bacterial concentration C refers to the CFU / ml in the blood sample.

[0025] Figs. 5A-5D show microtrapping and bacterial detection. Fig.5A. Filtrate flow rate for E. coli spiked sample, where the solid line is the mean :low through the microtraps relative to the sample input :low and the shaded area indicates one standard deviation (n=5). Fig. 5B. shows trapping efficiency for E. co / / -spiked sample, which is the number of bacteria-containing traps after 70 min relative to the colony forming units in the sample after smart centrifugation (bar height is mean, error bar is sd). Fig.5C. shows the overall assay performance, where dots represent the number of positive microtraps detected, / V, for various bacterial concentrations in the blood sample, C, and lines depict the least square Titting linear calibration curves / V = rj.C. Fig. 5D shows timelapse images of bacterial capture and growth in a single microtrap, showing the capture of one bacterium of K. pneumoniae 40 min after sample addition, followed by bacterial cell division.

[0026] Fig. 6 shows the isolation efficiency of bacteria from blood by smart centrifugation in different concentration ranges, n.s. and * indicate significance levels p > 0.05 and p < 0.05, respectively.

[0027] Fig. 7 shows the bacterial isolation efficiency in conventional centrifugation of blood.

[0028] Fig. 8 shows the isolation efficiency of S. aureus from blood with and without argatroban and the same density control liquid in smart centrifugation. *** indicates significance levels p < 0.001.

[0029] Fig. 9 shows the distribution of bacteria in supernatant and pellet after Smart centrifugation. *** indicates significance levels p < 0.001 .

[0030] Fig. 10 shows that filtrate and retentate flows are smoother and more reproducible when using high density media in the volume reduction step.

[0031] Fig. 11 shows a comparison of E. coli isolation efficiency after volume reduction with and without diluted percoll at the bottom, ns means not significant.

[0032] Fig. 12 shows the detection rate, meaning the number of bacteria-containing traps after 70 min onchip sample flow relative to the number of colony-forming units spiked in the blood, ns and * indicate significance levels p > 0.05 and p < 0.05, respectively.

[0033] DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS OF THE INVENTION

[0034] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness. Like reference character refer to like elements throughout the description.

[0035] Specific embodiments of the invention now will be described with reference to the accompanying drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the detailed description of the embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention. In the drawings, like numbers refer to like elements. The following description focuses on an embodiment of the present invention applicable to a blood sample and in particular to a blood sample containing bacterial cells. However, it will be appreciated that the invention is not limited to this application but may be applied to many other liquid biopsy samples such as, for example, stool samples, urine samples, and wound swabs.

[0036] As used herein, the term “supernatant” is understood to refer to a liquid portion on top of the non-target cells, in particular, plasma and excess density medium.

[0037] As used herein, the term target cells refers to microbial cells or tumor cells. Examples of microbial cells include bacterial, fungal, parasitic and viral cells.

[0038] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms "includes," "comprises," "including" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other element or intervening elements may be present. Furthermore, "connected" or "coupled" as used herein may include wirelessly connected or coupled. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0039] The present invention has been described above with reference to specific embodiments. However, other embodiments than the above described are equally possible within the scope of the invention. Different method steps than those described above, performing the method by hardware or software, may be provided within the scope of the invention. The different features and steps of the invention may be combined in other combinations than those described. The scope of the invention is only limited by the appended patent claims.

[0040] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used.

[0041] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0042] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.

[0043] Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0044] In a first aspect of the invention, an improved method to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties will now be described.

[0045] As shown in Fig. 1 A, this method involves i) providing a container comprising a volumedof a density medium 100 and then ii) adding a volumebof a liquid biopsy sample on top of the density medium, wherein the volume Vbof the liquid biopsy sample is a sum of the volume of a liquid fraction Vpand the volume of cells Vcin the liquid biopsy sample 110. Next, in step iii) the non-target cells in the liquid biopsy sample are allowed to sediment in a sedimentation layer at the bottom of the container, wherein the sedimentation layer comprises a liquid volume remaining between the sedimented cells 120. In step iv), the supernatant comprising target cells is then collected 130. In this method, the density of the density medium is higher than the density of the liquid biopsy sample but lower than the density of the non-target cells and the volume Vdof the density medium is at least equal to Vremaining.

[0046] This method enables the volume of liquid biopsy sample or plasma that is "trapped" between cells to be replaced by density medium. This way, target cells that would thus also be trapped are released and therefore detected when the supernatant is analyzed. Advantageously, this method provides improved detection of target cells, e.g. bacteria for sepsis. In one embodiment, the ratio Vdover Vremaining may be less than 2. In preferred embodiments, the ratio Vdover Vremainin may be less than 1.5, less than 1 .2 or less than 1.1.

[0047] In a second aspect of the invention, an alternative improved method to separate target cells from non-target cells in a liquid sample, based on the cells’ sedimentation properties are described.

[0048] As shown in Fig. 1 B, this method comprises i) providing a container comprising a volume Vdof a density medium 100, then ii) adding a volume Vbof a liquid biopsy sample on top of the density medium 111 , next iii) allowing the non-target cells in the liquid biopsy sample to sediment in the density medium 121 , and iv) collecting the supernatant comprising target cells 130. In this method, the volume Vbof the liquid biopsy sample is a sum of the volume of a liquid fraction Vpand the volume of cells Vcin the liquid biopsy sample. Also, the volume Vdof the density medium is at least equal to the volume Vp.

[0049] In some embodiments, the volume Vdof the density medium can be 5- 60 % of the volume Vbof the liquid biopsy sample.

[0050] The liquid biopsy sample can comprise a blood sample, a cerebrospinal fluid sample, a wound swab sample, a urine sample or a stool sample. In preferred embodiments the blood sample may be whole blood or blood in culture medium. In some embodiments, the liquid biopsy sample may be diluted prior to step i) of the method. The liquid biopsy sample may be diluted with a liquid medium of a different density than that of the liquid biopsy sample.

[0051] In some embodiments the density of the liquid biopsy sample may be altered prior to step i) of the method.

[0052] In some embodiments where the liquid biopsy sample may comprise a blood sample, the density may be adjusted to be as low as possible but high enough to allow sedimentation of more than 98% of red blood cells, more than 80% of white blood cells and more than 50% of platelets. The concentrations of the blood cells in whole blood before dilution and in the supernatant after smart centrifugation may be measured using a haematology analyzer (Swelab Alfa Plus, Boule Diagnostics, Sweden).

[0053] In this method, the container may have a tubular shape; however, other types of shapes may also be used. Advantageously, in step iii), a centrifugal force may be applied to the container. This centrifugal force may be between 100g and 3000g. In a preferred embodiment the centrifugal force may be 600 g.

[0054] One embodiment, shown in Fig. 1 B, can comprise step v) bringing together a selective lysis agent and the supernatant to remove remaining non-target cells from the supernatant 140. In preferred embodiments, a selective lysis agent may be added to the supernatant; the supernatant may be added to the selective lysis agent; and / or the supernatant may be added on top of higher density lysis agent followed by sedimentation.

[0055] In another embodiment, wherein the liquid biopsy sample may comprise whole blood, the whole blood sample may be diluted with a liquid of volume in blood: dilutant 3:1 volume ration, a 1 :1 volume ratio, a 1 : 10 volume ratio, 1 : 5 volume ratio, 1 : 4 volume ratio, 3:10 volume ratio, 2: 3 volume ratio, 1 :2 volume ratio, or 3: 4 volume ratio, prior to step i).

[0056] In yet another embodiment, the density medium may be selected from a blood culture medium, a nutrient-rich liquid medium, a medium used for the growth and cultivation of microorganisms, a lysogeny broth, medium of polysucrose and sodium diatrizoate, a high- molecular-weight branched polysaccharide, a density gradient medium, a density gradient medium composed of an iodinated compound, a density gradient medium based on a solution of iodixanol, a density gradient medium specifically formulated for the isolation of polymorphonuclear leukocytes (PMNs) from human blood, a radiopaque contrast medium, a density gradient medium composed of a tri-iodinated benzoic acid derivative, a colloidal silica-based density gradient medium, an aqueous solution, a salt solution, or mixtures thereof.

[0057] The blood dilution medium for diluting the blood sample may be selected from a blood culture medium, nutrient-rich liquid medium, a medium used for the growth and cultivation of microorganisms, a lysogeny broth, a medium of polysucrose and sodium diatrizoate, a high- molecular-weight branched polysaccharide, a density gradient medium, a density gradient medium composed of an iodinated compound, a density gradient medium based on a solution of iodixanol, a density gradient medium specifically formulated for the isolation of polymorphonuclear leukocytes (PMNs) from human blood, a radiopaque contrast medium, a density gradient medium composed of a tri-iodinated benzoic acid derivative, a colloidal silica-based density gradient medium, aqueous solution, a salt solution, or mixtures thereof.

[0058] The density of density media may be substantially 1025-1055 Kg / m3. Preferably, the density of density media may be selected from substantially 1052, 1038, and 1030 Kg / m3.

[0059] In preferred embodiments, when using conventional blood collection tubes, the volume of density media may be optimized to be substantially 1 ml and a total volume of the density liquid and the diluted blood is substantially 4 ml.

[0060] In embodiments, sedimentation can be effected by centrifuging the container. The container may be a standard or conventional tube, such as a standard centrifuge tube as used in clinical or microbiology settings.

[0061] In some embodiments, the method may further include reducing the volume of the supernatant sample with a less than 40 %, or less than 20% loss of target cells, or without a decrease in the target cells.

[0062] In a preferred embodiment, the volume of the supernatant sample may be reduced such that the remaining volume of the supernatant sample may be 50% or less, 30% or less, or 15% or less, compared to the original volume of the supernatant sample.

[0063] In a preferred embodiment, the volume of the supernatant sample may be reduced such that the remaining volume of the sample is 3ml or less, 1 ml or less, or 0.5 ml or less. In preferred embodiments, the volume reduction may be achieved by centrifugation on top of a cushion fluid, such as Percoll, and subsequent removal of the supernatant. In a preferred embodiment, cells are not exposed to sedimentation forces against the container wall.

[0064] In certain embodiments, as shown in Fig. 1 D, the method may further comprise the step of vi) analyzing and / or quantifying the target cells 150. In preferred embodiments, after step v) and before step vi), the concentration of target cells in the supernatant may be increased. In other embodiments, the volume of the supernatant sample may be reduced after placing the sample on top of a density medium such that either more target cells may be recovered in the reduced sample or such that the flow rate of the sample may increases when flowing the sample in a microfluidic device in a subsequent operation.

[0065] In an alternative embodiment, the volume of the supernatant sample may be reduced without loss of target cells on top of a density medium; the flow rate on chip may be improved, such density medium may be diluted, and the supernatant may be removed.

[0066] In certain embodiments, the container may be incubated to culture cells prior to step i). In other embodiments, the container may be incubated to culture cells between any of the steps, for example, steps i) and ii), and / or between steps ii) and iii), and / or between steps iii) and iv), and / or between steps iv) and v).

[0067] In preferred embodiments, one or several additional container incubations to culture cells may be performed between the steps, wherein the container incubations are performed for 15 min - 12 hours, preferably for 30 min, 60 min, 90 min or 120 min. The additional container incubations may provide target cells, such as bacteria, extra time to grow, either at room temperature, at an optimal bacterial growth temperature of 37 deg C, or at any other temperature most suited. Including said additional container incubations steps may result in that the bacteria have time to increase in number and thereby improve the detection limit of the method and system.

[0068] In some embodiments, after step iii) target cells, for example microbial cells or tumor cells, may be isolated using filtration and / or density separation. In preferred embodiments, the supernatant may be filtered through a membrane filter to remove any remaining non-target cells; the membrane may have a pore size of 3 pm. In some embodiments, as shown in Fig. 1C, step vi) may comprise further processing the solution. In one preferred embodiment, step vi) may comprise layering the filtered solution over a volume of density gradient medium, for example 650 pl, with a density of about 1080 kg / m3, and centrifuging the filtered supernatant / solution at 2000g for 15 minutes.

[0069] In one embodiment, step vi) may further comprise flowing the solution in a microfluidic device, wherein the microfluidic device is configured to filter the solution.

[0070] In one embodiment , the microfluidic device may comprise microtraps.

[0071] In one embodiment, step vi) may further comprise reseeding target cells trapped in at least one of the microtraps to other microtraps. In other preferred embodiments, the microtraps may be configured to filter out the non-target cells from the target cells after reducing the nontarget cells from liquid biopsy sample. In other preferred embodiments, the microtraps are configured to further filter out the non-target cells from the target cells after up-concentration of the target cells and / or selective lysis of the non-target cells.

[0072] In a preferred embodiment the microtraps may be used as culture chambers for the target cells.

[0073] In one embodiment, the method may further comprise dynamically changing the flow or flow direction in the microtraps such that trapped bacterial cells may leave the microtrap after which they enter different microtraps.

[0074] A target cell, for example, a bacterial cell, inside a microtrap may increase the fluidic resistance of such microtraps. During any seeding of bacteria into microtraps, target cells bacteria will preferentially flow into microtraps with the lowest fluidic resistance. Therefore, flowing several target cells or bacteria from one microtrap into the common channel upstream from the microtrap, followed by reseeding, may result in each target / bacterial cell preferentially entering a microtrap that is not yet occupied by other bacterial cells. For example, if a microtrap contains 7 cells, such reseeding will typically lead these 7 target / bacterial cells to each enter an individual microtrap. After trapping the target / bacterial cells in microtraps, the cells start dividing.

[0075] Indeed, as indicated in Figure 5, trapping one target cell / bacteria leads the microtrap to fill up with several target cells / bacteria. Reseeding these bacteria to other microtraps can make microscopy detection easier or allow easier bacterial growth on-chip or allow testing the susceptibility of the bacterial strain by flowing specific antibiotic agents with specific antibiotic concentrations through specific traps, thereby exposing bacteria in the different microtraps to different antibiotic agents or antibiotic agents with different concentrations.

[0076] The method may further comprise, after step v), performing an antibiotic susceptibility test (AST).

[0077] In another embodiment the method may involve adding a first compound, the first compound being configured to prevent bacteria from binding to the target cells / blood cells. The first compound may be Argotroban.

[0078] This first compound may be added prior to step i; between steps i and ii; or between steps ii and Hi. Said preventing the target cells / bacteria from binding to the blood cells increases the bacterial isolation rate, because the bacterial cells are no longer dragged into the blood cell sediment during centrifugation.

[0079] In one embodiment, the method may involve adding a adding a second compound, the second compound being configured to break down any lysate or debris remaining in the sample after the non-target cell lysis. The second compound may be an enzyme, for example DNase. The second compound may be added prior to step i; between steps i and ii; between steps ii and Hi; or between steps Hi and iv. Said breaking down lysate of debris remnant in the sample may lead to reduced clogging of the microfluidic system.

[0080] In any of the embodiments, the target cells may be bacterial cells, fungal cells or viral particles.

[0081] In one embodiment, the microfluidic device may contain microtraps of different sizes.

[0082] In a preferred embodiment, step vi) may further comprise adding fluorescence markers for on-chip target cell detection or identification or bacterial antibiotic susceptibility detection. The fluorescence markers may comprise, FISH makers, for easier on-chip target cell / bacterial detection or target-cell / bacterial species identification or bacterial antibiotic susceptibility detection. In one embodiment, such markers may be added after trapping the bacteria on- chip. In another embodiment, such markers may be added to the sample prior to loading the sample to the microfluidic chip. After said trapping the target cell / bacteria on-chip, they can be identified by fluorescence in-situ hybridization (FISH) and AST, in 3-4 hours. Rapid identification and drug profiling within 4 hours instead of the current several days means that septic patients can get appropriate antibiotics, which has great power to impact the survival rate of the patients positively.

[0083] In one embodiment, the method may be used for detection of fungal cells or viral particles. In one embodiment, the microfluidic chip can contain microtraps of a size large enough to contain at least one fungal cell. In one embodiment, such microfluidic chip may contain microtraps of different sizes. In one embodiment, such microfluidic chip contains both 1) microtraps of size adapted for bacteria, i.e., with a minimal cross-sectional geometry in the range 1-2 micrometer, and 2) microtraps of size adapted for containing fungal cells, i.e., with a minimal cross-sectional geometry equal or larger than 2 micrometer. In one embodiment, such microtraps may be of a size large enough to contain at least one fungal cell are placed downstream from the microtraps with size adapted for bacteria.

[0084] In one embodiment, the method and system are adapted to detect or analyze both bacteria and fungi.

[0085] In one embodiment, the first three steps of the method may be concatenated with other detection or analysis methods. In one preferred embodiment, the first three method steps may be followed by steps or detection methods in the current workflow of bloodstream infection diagnosis in clinical microbiology. In one preferred embodiment, early isolation of the bacteria from the immune system components present in blood enhances their growth rate, which reduces the time needed for blood culture. Concatenation of these steps with current identification (MALDI-TOF or PCR) or antimicrobial susceptibility testing (AST) techniques would thus speed up time-to-answer.

[0086] In one embodiment, the method wherein the different steps may be integrated or automated.

[0087] In one embodiment, such integration or automation may reduce the reliance on trained human operators or reduce the cost of diagnosis or increases the repeatability of the method.

[0088] The method may be used for detecting bacteria in blood culture medium bottles. The method may be used for separating or isolating cells from blood. Additionally, the method may be used for isolating tumor cells from blood.

[0089] In one embodiment, WBCs and platelets form a buffy layer above the density media and most of the bacteria settle at the bottom or in the density liquid. In one embodiment, the buffy layer is removed. In one embodiment of the method bacteria are isolated from the blood cells using a selective cell lysis step.

[0090] In a third aspect of the invention, a kit to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties, is provided. The kit (not shown) comprises a container that contains a volumedof a density medium, and set of instructions to carry out the method according any of the previous embodiments.

[0091] In one embodiment, the different steps may be integrated or automated. Such integration or automation can reduce the reliance on trained human operators or reduce the cost of diagnosis or increase the repeatability of the method.

[0092] EXPERIMENTAL DATA

[0093] Resu / fs

[0094] Over-all workflow

[0095] The method concatenates four steps to isolate and detect bacteria from blood: smart centrifugation, selective blood cell lysis, volume reduction, and microscopy-based detection in microfluidic traps (Fig. 3). For ethical reasons and to allow for repeatability during technical development, this study was conducted with healthy human EDTA donor blood, where it was ensured all the blood parameters were within the normal count. The blood was spiked with bacteria at concentrations, C, in the range 4-4000 CFU / ml. To facilitate development and quantification, a fluorescent lab strain of E. coll in the stationary phase (after overnight culture) was used. This choice facilitated easy detection and allowed for the monitoring of individual cell transport, minimizing the interference from daughter cells. Furthermore, fluorescence facilitated the easy distinction between the E. coll test strain and potential contaminating bacteria, none of which were observed. Method evaluation was performed with clinical isolates of K. pneumoniae, E. faecalis, and S. aureus in their exponential growth phase. Smart centrifugation

[0096] The first step, called smart centrifugation, removes most of the blood cells while recovering most of the bacteria in the supernatant. This step is essential to avoid downstream clogging of the microfluidic device. During blood centrifugation, bacteria are enriched into the supernatant (see SI for a detailed description of bacterial cell trajectories during blood sedimentation). However, when centrifuging pure whole blood, a large fraction of the bacteria become trapped in the remaining plasma inside the blood cell sediment. The strategy was to avoid such bacterial loss is to layer the sample on top of a density medium with a higher density and with a volume sufficient to replace all plasma in the sediment. The volumes and densities of both the sample and density medium were adjusted for optimal bacterial isolation efficiency with minimal dilution. To tune the densities of the blood sample and density medium, blood culture medium (BCM) to support bacterial growth throughout the assay was used. The density medium was adapted for an as high as possible density, to increase sedimentation differentiation, with a limit determined by the lowest particle density, to enable the sedimentation of all blood cells into the density medium. The densities of RBCs, WBCs and platelets are in the ranges (1.086 -1.122 g / ml), (1.057 - 1.092 g / ml) and (1.072 - 1.077 g / ml), respectively; as density medium, a 2:1 volumetric mixture of Lymphoprep and BCM, which has a density of 1.051 g / ml was chosen. The volume of the density medium was experimentally tuned to be minimal but sufficient to replace all plasma remaining between the sedimented cells. Diluting the blood sample with 25% BCM provided minimal reduction of the bacterial concentration while ensuring a sample density below that of the density medium. Centrifuging time and force were experimentally tuned for optimal enrichment of E. coli in the supernatant while removing at least 99.8 % of the RBCs. Optimal conditions involved layering 3 ml of BCM-diluted spiked blood on top of 1 ml density medium and centrifuging for 5 min at 600g in a hanging bucket centrifuge. The relative movements of the RBCs, bacteria and liquid in such a system are described with a linear model in and illustrated in Fig. 4A. After centrifugation, approximately 2.5 ml of clear supernatant containing most bacteria (Fig. 9) could be removed for further processing.

[0097] Blood cell counting of the supernatant showed the removal of 99.82 ± 0.04% of the RBCs, 95 ± 4% of the WBCs, and 63 ± 2% of the platelets (mean ± sd, n=3; Figure 4B). Agar plate culturing of the supernatant revealed the isolation of 65 ± 16% of E. coli, 95 ± 17% of K. pneumoniae, 64 ± 24% of E. faecalis, or 8 ±7% of S. aureus (mean ± sd, n=10-26; Fig. 5C). Recovery exceeding 100% can be attributed to the low plate count numbers, leading to considerable variations. Selective blood cell lysis

[0098] The second assay step removes the remaining blood cells in the sample using a selective lysis mixture of sodium chocolate hydrate and saponin. Around 2.5 ml supernatant from the smart centrifugation was mixed with 1 ml of the selective lysing solution and kept in a shaking incubator at 37C for 10 min, completely lysing remaining RBCs, WBCs and platelets while not affecting bacterial viability.

[0099] Volume reduction

[0100] The third step enriches the sample and removes the excess lysing buffer in a second centrifugation step. The sample-lysate mixture was layered on top of 0.3 ml high-density liquid, consisting of a 1 :2 volumetric mixture of percoll and BCM, and centrifuged for 13 min at 1000g to sediment the bacterial cells on the liquid interface. The supernatant was removed to withhold approximately 0.5 ml liquid containing sedimented bacterial cells, as well as some blood lysate. Sedimenting into a higher density liquid provided a smoother liquid flow in the microfluidic chip during downstream processing (Fig. 10), while not significantly affecting the bacterial isolation efficiency (p-value >0.05) (Fig. 11)

[0101] Detection in microfluidic traps

[0102] In the last step, we processed the resuspended sample through a microfluidic device with bacterial traps for 70 min while imaging by microscopy. The chip design introduced by Baltekin et al. [1] was used for cross-flow filtration of the bacteria into individual filter traps. The chips contained in total 8000 microtraps of length 50 pm and width 1 .25 pm with a 0.95 pm restriction at their end. Each microtrap could capture bacterial cells and function as a culture chamber. The microtraps were monitored with fluorescence microscopy to detect E. coir, alternatively phase contrast microscopy to detect the clinical isolates of K. pneumoniae, E. faecalis and S. aureus. The 0.5 ml bacterial sample was loaded into the inlet at 1 bar pressure, keeping all outlets at atmospheric pressure. For E. co / / -spiked sample, approximately 30% of the sample flowed through the traps with a typical filtrate flow rate of 1- 2 pl / min (Figs. 5A and 9). The trapping efficiency, meaning the number of positive traps, N, relative to the number of CFUs after smart centrifugation, was 29 ± 6% (mean ± sd, n=5; Figure 5B).

[0103] Overall assay performance The entire assay was evaluated starting from blood spiked with E.coli, K. pneumonia, and E. faecalis, respectively. All experiments resulted in the successful trapping and subsequent detection of bacteria. The lowest bacterial concentrations, C, tested and detected were 9, 7 and 32 CFU / ml, respectively (Fig. 5C). The least mean square linear curve fit, N = q.C, to the data allows estimating the overall assay sensitivity, q. It can be inferred that the limits of detection, which are the bacterial concentrations for which it is expected that A / = 1 positive trap, C / V=1 = 1 / rj, are in the range 1-10 CFU / ml. After trapping, the bacterial cells divide inside the microchannels, while the sample is still being loaded (Fig. 5D).

[0104] Due to the poor isolation during smart centrifugation, S. aureus (n=2) could not be detected. Discussion

[0105] The unique isolation and concentration of three sepsis-causing bacterial species from blood, coupled to their detection by microscopy in microfluidic traps within 2 h were demonstrated. These pathogens cover approximately 20% of the bacterial species causing BSIs in the European Union and 35% of those in Japan. The estimated detection limit in the range 1-10 CFU / ml is clinically relevant. Compared to previous work, the inventors’ approach does not require blood culture and allows the detection of bacteria phenotypically at clinically relevant concentrations.

[0106] Smart centrifugation is a simple, inexpensive, high-throughput, and easily parallelizable method to isolate bacteria from whole blood rapidly with a high bacterial isolation efficiency and high blood cell rejection. The method’s compatibility with standard lab equipment supports translation to the clinical setting. The bacteria are isolated in a small volume of liquid, similar to the original plasma volume, which facilitates downstream processing, bacterial enrichment in the supernatant during centrifugation results from two effects. First, the Stokes terminal velocity of bacteria is a factor a - 1 / 30 times that of blood cells, allowing efficient terminal velocity-based differentiation. Second, the high hematocrit results in downward sedimenting RBCs causing a backflow of the plasma, thereby dragging bacteria upward and further increasing differentiation between the two. Smart centrifugation enhances the separation by extending the sedimentation path of particles through the density medium. For comparison, inventors experimentally optimised isolation of E. coli from spiked whole blood in conventional centrifugation, i.e., without sample dilution or density medium, and achieved 34% ±7% isolation efficiency in the supernatant (Fig. 7). Smart centrifugation thus provides a 65% 134% = 1 .9 times improved isolation efficiency for E. coli for a comparable blood cell rejection efficiency (Table A1). Even though the method was optimised for E. coli, inventors achieved even higher isolation e]iciency of K. pneumoniae. Isolation efficiency of S. aureus, however, was much lower. Isolation efficiency of E. coli and E. faecalis were not significantly different. Inventors found no significant differences in bacterial isolation efficiencies between samples spiked with the same strain at different concentrations (Fig. 6).1 Several factors may cause the recovery differences between the species, as well as the large variation in recovery between different measurements with the same strain. These factors include differences in Stokes radius between the bacterial species, differences in interaction between the blood cells and the bacteria, differences in viscosity and hematocrit between the blood samples, variations in the immune response of the patient blood to the bacteria (reducing the bacterial count), or variations in bacterial growth phase (compared to K. pneumonia, E. coli were spiked in stationary phase) or growth rate (E. faecalis have a slower growth rate than K. pneumonia). It is speculated that the low isolation efficiency of S. aureus is caused by their binding to blood cells or plasma protein, leading to their rapid sedimentation. Indeed, spiking S. aureus in a sample liquid with the same density as diluted blood - but without blood cells - resulted in 54 ± 6% (n=6) isolation efficiency by smart centrifugation, compared to 8 ±7 % (n=10) isolation efficiency from spiked blood (Fig. 8).

[0107] Based on the literature, smart centrifugation has superior recovery and throughput for E. coli, data for which are presented for most tests (Table 1).

[0108] Table 1 Key performance parameters of smart i cul ' ibiyil i"ii methods for E. coli.

[0109] The concentrated bacterial sample was loaded onto the microfluidic chip at 1000 mbar. At higher pressures, bacterial loss through the 300 nm constrictions of the microtraps was observed.

[0110] Sedimenting into a higher-density liquid during the prior volume reduction step reduced the compression of bacterial cells and lysate and allowed more homogeneous resuspension, leading to lower on-chip clogging. The trapping of bacterial cells and blood cell debris in the traps increases the fluidic resistance of the filter and decreases the throughfilter flow over time. The chips enabled processing 0.5 ml sample within 70 to 90 min, during which 30%of the sample flows through the microtraps (filtrate flow) and the rest flows past the filter directly to the waste outlet (retentate flow). The low filtrate flow rate is thus a major contributor to bacterial loss between isolation and detection.

[0111] In summary, the invetors addressed the critical limitation in state-of-the-art BSI diagnosis: the long blood culture time to reach positive culture. The detection of bacteria in microtraps was demonstrated without blood culture at clinically relevant bacterial concentrations in 2 h. Since the cells were living it was possible to do phenotypic AST or species ID with FISH.

[0112] Methods

[0113] Blood Sample Preparation

[0114] Blood in EDTA tubes from healthy donors was purchased from the blood bank (Blodcentralen, Stockholm, Sweden) or Uppsala University Hospital (Akademiska Sjukhuset, Uppsala, Sweden). The blood samples were stored at 4 °C, and used for experiments no later than two days after collection. Samples presenting a milky supernatant (<10%), i.e., indicating abnormally high concentrations of triglycerides in the blood, were discarded. Before processing the samples, they were put at room temperature, and blood samples were spiked with respective bacteria concentrations of approximately 103, 102, and 10 CFU / ml. The concentration of the spiking solution was quantified by plate counting (n=3). The spiking volume was always less than 5% of the total blood solution volume.

[0115] Bacterial Strains

[0116] We used three clinical isolates that were randomly collected from a clinical microbiology laboratory in Sweden, covering both gram negative and gram-positive species. As grampositive representatives, S. aureus (DA70300) and E. feacalis (DA70208) were used, and as gram-negative, K. pneumoniae (DA72206). We also used E. coli (EL3041) cells harbouring a plasmid expressing mVenusNB fluorescence proteins. Bacteria were stored for long term at - 80 °C in standard glycerol solution and were incubated overnight at 37 °C in BD BACTEC Plus Aerobic medium (BD, USA), referred to as Blood Culture Medium (BCM), prior to use, and later diluted to approximately 104, 103, and 102 CFU / ml and used to spike blood with different concentrations and quantified for isolation. For detection experiments with E. coli cells harbouring a plasmid expressing mVenusNB fluorescence proteins, the above mentioned protocol was followed. For detection experiments with clinical isolates, 2 pL of bacteria from an overnight culture were spiked in 2 ml of BD BACTEC Plus Aerobic Medium, and cultured for 2-4 h depending on the species. They were then diluted to approximately 104, 103, and 102 CFU / ml and used to spike blood with different concentrations.

[0117] Smart Centrifugation

[0118] Lymphophrep (STEMCELL Technologies, Canada), a density medium with 1.077 g / ml density, was mixed with BD BACTEC Plus Aerobic medium (BD, USA) in a volumetric ratio of 2:1 , and 1 ml of the solution, with 1.051 g / ml density, was poured into a 15 ml Falcon centrifuge tube. 3 ml of spiked blood mixed with BD BACTEC Plus Aerobic medium (BD, USA) (3:1 Blood:BD BACTEC Plus Aerobic medium) was gently placed over the density media. The tube was centrifuged at 600g for 5 min in a hanging bucket centrifuge. The supernatant was removed and mixed with the lysing solution.

[0119] Conventional centrifugation for bacteria isolation

[0120] To establish a control measurement, 4 ml of whole blood was centrifuged without any dilution and density media. The sample, after being spiked, underwent centrifugation at 500g for 4 min, resulting in the separation of clean plasma at the top. This process effectively settled most of the blood cells.

[0121] Selective Lysing Solution

[0122] The selective lysing solution was prepared by mixing sodium chocolate hydrate (Sigma- Aldrich, USA) and saponin (Sigma-Aldrich, USA). Concentrations of 2% (W / V) sodium cholate hydrate and 1% (W / V) saponin were prepared by dissolving the chemicals in BD BACTEC Standard Aerobic medium.

[0123] Microfluidic Platform Fabrication

[0124] The microfluidic chip design and fabrication were previously reported. A silicon wafer mold was fabricated according to design specifications by the company Conscience AB, Sweden. The wafer was once silanized for 30 min prior to polydimethylsiloxane (PDMS; Sylgard 184, DOW, USA) replication. Subsequently, a 10:1 w / w PDMS:curing agent mixture was poured on the wafer and baked at 80°C overnight. Holes on the PDMS ports 2.0, 2.1 , 2.2, 5.1 and 5.2 were made using a 0.5 mm puncher. Then, the PDMS stamps were cleaned in IPA before covalent bonding to a glass coverslip (No. 1.5, Menzel-Glaser, Germany) after plasma treatment, followed by heat curing for 1 h at 80°C. Microfluidic Flow Control

[0125] The microfluidic device was mounted on the microscope and connected to the sample reservoirs with flexible plastic tubing (TYGON, Saint-Gobain, North America). OB1 CONTROLLER (Elveflow, France) flow control units were used to pressurize reservoirs. The reservoirs connected to ports 2.1 , 2.2, 5.1 and 5.2 were first pressurized at 500 mbar for chip priming with water infused with 0.085 g / l of Pluronic F108 (Sigma-Aldrich, USA) and was decreased to 0 mbar. Then the reservoir connected to inlet port 2.0 was pressurized at 500 mbar for priming and at 1000 mbar for sample loading.

[0126] Volume reduction

[0127] Percoll (Sigma-Aldrich, USA), a density medium having a density of 1.1251.135 g / ml was mixed with BD BACTEC Plus Aerobic medium (BD, USA) in a volumetric ratio of 1 :2, and 0.3 ml of the solution, with approximately 1.04 g / ml density, was poured into a 15 ml Falcon centrifuge tube. The supernatant infused with the lysing solution was gently placed over the density medium. The tube was centrifuged at 1000g for 13 min in a fixed rotor centrifuge. All excess liquid above 0.5 ml was removed, without removing the pellet.

[0128] Optical setup

[0129] Images of all the microtraps contained in the microfluidic device were acquired using a Nikon Ti, inverted microscope. For imaging experiments containing clinical strains, a CFI plan Apo lambda 100x (1.45 NA, oil) objective was used. Phase contrast images were acquired by using DMK 38UX304 (the imaging source) camera, with an exposure time of 20 ms with an interval of 10 min, for a maximum of 90 min. Cells expressing mVenus fluorescence proteins were captured using a filter cube consisting of a Di02-R488 (Semrock, USA) dichroic mirror, a FF02- 482 / 18 (Semrock, USA) excitation filter, and a FF01 -524 / 24 (Semrock, USA) emission filter and CFI Plan Apo lambda DM Ph2 20x objective. Each fluorescence image was acquired with an exposure time of 500 ms with an interval of 5 min, for a maximum of 90 min. A constant temperature of 37°C was maintained during the measurements using a temperature controllable unit (Okolab, Italy). The imaging setup was operated by micromanager 1 .4 version software.

[0130] Bacteria Quantitation

[0131] All bacterial sample quantitation was performed by plate counting after plating a sample aliquot on agar plates and overnight culture at 37C in the incubator. The agar plates were prepared by dissolving LB broth with agar (Miller) (Sigma-Aldrich, USA) in deionized water at 40 g / L concentration followed by autoclaving and placing in Petri dishes. For on-chip bacteria quantitation of the clinical isolates, the inventors employed Imaged software image analysis of phase-contrast microscopy images. Each microtrap was analyzed manually and determined positive if filled with at least one bacterium. The same analysis pipeline was performed for fluorescence microscopy images of the E. coli cells expressing mVenus fluorescence proteins.

[0132] Blood Cell Quantitation

[0133] The concentrations of the blood cells in whole blood before dilution and in the supernatant after smart centrifugation were measured using a haematology analyzer (Swelab Alfa Plus, Boule Diagnostics, Sweden).

[0134] Flow characterization

[0135] The ports 2.1 and 2.2 were coupled together, and ports 5.1 and 5.2 were coupled together, using a PEEK union T-connector (Thermo Fisher Scientific, USA). Flow measurements were taken from both couplers using a SLI Liquid Flow Sensor (80 pL / min) (Sensirion, Switzerland). Two independent flow measurements were acquired with a sampling time of 1 s, for a maximum time of 90 min, using the Sensor Viewer Software (Sensirion, Switzerland). The data was analyzed using MATLAB.

[0136] Statistical analysis

[0137] Data analyses were performed with R. The data samples were tested for normality using the Shapiro-Wilk test; variance between normal populations was studied using the F-test; and statistically significant differences between groups were studied using a t-test, for samples following a normal distribution, or a Mann Whitney test for samples not following a normal distribution. A two-sided p-value of < 0.05 was considered statistically significant.

[0138] Cell and fluid movement during smart centrifugation

[0139] The movement of blood cells during centrifugation has been studied extensively. To understand the displacement of bacteria during centrifugation, inventors considered a linear model for the simultaneous sedimentation of two types of particles, bacteria and RBCs, through two types of density-layered liquids, diluted plasma and density medium, in which particle-particle interactions were neglected. The indices bac, RBC, pl, DM, int, and sed, are used to indicate bacteria, RBCs, plasma, density medium, liquid: liquid interface and sediment, respectively. The theoretical stokes terminal velocity, vst, of particles in blood can be deduced from Stoke’s law, which describes the unhindered sedimentation velocity of a single particle in a standing fluid. The stokes velocity depends linearly on the density difference between the particle and the medium, Ap, quadratically on the particle size, and inversely linear on the viscosity, p. The stokes velocities of a given particle in the two liquids thus relate as

[0140] Because the densities of bacteria and RBCs are in the same range, the ratio of their stokes velocities can be considered a constant, a, independent from the liquid medium: a has been estimated to be typically 1 / 30.

[0141] In regions with a large local volume fraction of RBCs, h, the RBC sedimentation causes a significant upward backflow of liquid medium. Considering RBCs incompressible, the local backward liquid velocity, v / , can be deduced from mass conservation, i.e., the downward flux of RBCs must equal the backward flux of liquid:

[0142] (Here a negative sign is used for velocities to indicate an upward movement direction opposite to that of downward sedimenting RBCs.) Where the sedimenting RBCs cross the liquid interface between the sample and density medium, mass conservation demands that the local liquid velocities across the interface, and the velocity with which the interface itself moves backward, are all equal. This equality implies a relation between the difference in local RBC fractions, hpl and hDM, across the liquid interface:

[0143] The effective sedimentation velocity of the particles equals thesum of the local liquid velocity and their theoretical Stokes velocity. For RBCs, the resulting elective sedimentation velocity can be expressed as i.e., VRBC.pl - (1 - hpi).Vst,pl,RBC 3r\d VRBC.DM - (1 - hoM).Vst,DM,RBC.

[0144] The values for Vst.RBc can be derived relative to those of blood using equation 1 . The value of hpl can be estimated from the hematocrit value while accounting for the 25 % blood sample dilution with BCM. The value of hDM can then be derived from hpiusing equation 4.

[0145] During sedimentation, bacteria can assume any of four velocities, depending on their location in either the plasma or density medium, and on the local RBC fraction being zero or not. = - (ft .fn. ‘ i - a, . (0)

[0146] In the supernatant and in density medium depleted from RBCs, h= 0 and vpi= 0 and bacteria reach their stokes terminal velocity vbac= Vst,Pi,bac- Importantly, and perhaps counterintuitively, bacteria move upwards where bacterial fractions h > a. The speed at which the sedimentation layer grows depends on the liquid fraction, / 3, remaining between the RBCs in the sediment: v = w(1 +B (7)

[0147] The values of vint, vbac, VRBC and vsedare visualised as the slopes of the position-versus-time trajectories indicated in Figure 4A.

[0148] Statistical Analysis of Smart Centrifugation of Samples with Different Bacterial Concentration The variation in isolation efficiency of bacteria by smart centrifugation was studied between the different concentration ranges< 20, 20-100 and >100 CFU / ml for three bacterial species (Figurel). For K. Pneumoniae and E. Faecalis no significant difference was found. For E. coll there was a significant difference only in between the concentration ranges < 20 CFU / ml and 20-100 CFU / ml.

[0149] Isolation of bacteria by conventional centrifugation of undiluted blood without density medium The bacterial isolation efficiency for (E. co / / ) has also been studied for conventional centrifugation, i.e., centrifugation without sample dilution or density medium, of whole blood. 4 ml of whole blood was centrifuged at 500g for 4 min. 4 min was sufficient for most of the blood cells to settle. We recovered 34 ± 7 % (mean ± sd; n=3) of the bacteria in the supernatant (Figure 7), removing 99.957 ± 0.007% of the RBCs, 98.9 ± 0.6 % of the WBCs, and 65 ± 2 % of the platelets (mean ± sd; n=3) (Table A1). TSable Al Data points for % rejection of blood cells in conventional centrifugation.

[0150] Blood cell rejection in smart centrifugation

[0151] Hable A2 Data points for % rejection of blood cells in smart centrifugation.

[0152] Low isolation efficiency of S. aureus

[0153] It was investigated whether the poor isolation efficiency of S. aureus is caused by their interaction with the blood cells or plasma protein, as reported by many, or whether the poor performance is intrinsic to smart centrifugation. The spiked blood in smart centrifugation was replaced with a control liquid having the same density and volume as the diluted sample, and smart centrifugation was performed by placing it over 1 ml density media. After 5 min of centrifugation, the top 2.5 ml of liquid was removed and plated on an agar plate for bacteria quantification. Approximately 55% of the bacterial cells were recovered, whereas the isolation efficiency from blood was less than 10% (Figure 8). This difference indicates that the interaction between S. aureus and blood cells is stronger than the fluid drag, transporting the bacteria into the sediment.

[0154] Distribution of bacteria in the supernatant and pellet after smart centrifugation

[0155] The distribution of E. coli in the pellet and the supernatant after smart centrifugation were investigated. After the centrifugation step, the supernatant was removed and the pellet was resuspended in 2 ml of BCM to make plate counting more accurate as a very high number of interfering blood cells can decrease the accuracy of plate counting. Almost all (> 90%) of the spiked bacteria were recovered. 30% of the bacteria were found in the pellet. Fig. 9 shows the distribution of bacteria in supernatant and pellet after smart centrifugation.

[0156] Volume reduction with and without diluted percoll as sedimentation interface Placing a bottom layer of diluted percoll (1 :2; percoll:BCM) prevents the aggregation of cells and lysate, resulting in more stable and reproducible flow rates. Figure 10 shows how the filtrate and retentate flow in the presence or absence of high-density media (n=5). The use of high-density media in the volume reduction step resulted in better resuspension of the lysate and cells, thus ensuring smoother and more reproducible flow. Additionally, how the presence of a density media at the bottom affected the bacteria isolation efficiency during volume reduction was tested. The isolation efficiency with percoll was found 15% higher than without the percoll, although this difference was not significant. Fig. 11 shows a comparison of E. coli isolation efficiency after volume reduction with and without diluted percoll at the bottom.

[0157] Overall detection rates

[0158] The measurements plotted in Fig. 12 are the same as in Fig. 5, but now shown as detection rate, meaning the number of bacteria containing traps after 70 min on-chip sample flow relative to the number of colony-forming units spiked in the blood, ns and * indicate significance levels p > 0.05 and p < 0.05, respectively.

[0159] REFERENCES

[0160] [1] Baltekin, O., Boucharin, A., Tano, E., Andersson, D.I., Elf, J.: Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging. Proceedings of the National Academy of Sciences of the United States of America 114(34), 9170-9175 (2017). https: / / doi.Org / 10.1073 / pnas.1708558114

Claims

CLAIMS1. A method to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties, the method comprising the steps of i. providing a container comprising a volume Vdof a density medium, ii. adding a volume Vbof a liquid biopsy sample on top of the density medium, wherein the volume Vbof the liquid biopsy sample is a sum of the volume of a liquid fraction Vpand the volume of cells Vcin the liquid biopsy sample,Hi. allowing the non-target cells in the liquid biopsy sample to sediment in a sedimentation layer at the bottom of the container, wherein the sedimentation layer comprises a liquid volume Vremaining between the sedimented cells, iv. collecting the supernatant comprising target cells; wherein the density of the density medium is higher than the density of the liquid biopsy sample but lower than the density of the non-target cells, and wherein the volume Vdof the density medium is at least equal to Vremaining.

2. A method to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties, the method comprising the steps of i) providing a container comprising a volume Vdof a density medium, ii) adding a volume Vbof a liquid biopsy sample on top of the density medium, iii) allowing the non-target cells in the liquid biopsy sample to sediment in the density medium, iv) collecting the supernatant comprising target cells; wherein the volume Vbof the liquid biopsy sample is a sum of the volume of a liquid fraction Vpand the volume of cells Vcin the liquid biopsy sample, and wherein the volume Vdof the density medium is at least equal to the Vp.

3. The method according to any of the previous claims, wherein the volume Vd of the density medium is 5- 60% of the Vb.

4. The method according to claim 1 , wherein the ratio Vdover Vremaining less than 2.

5. The method according to any of the previous claims, wherein the liquid biopsy sample comprises a blood sample, a cerebrospinal fluid sample, a wound swab sample, a urine sample or a stool sample.

6. The method according to claim 5, wherein the blood sample is whole blood.

7. The method according to claim 5, wherein the blood sample is blood in blood culture medium.

8. The method according to any one of the preceding claims, wherein the liquid biopsy sample is diluted prior to step i) of the method.

9. The method according to any one of the preceding claims, wherein the density of the liquid biopsy sample is altered prior to step i) of the method10. The method according to any one of the preceding claims, wherein the container is tubular.

11. The method according to any one of the preceding claims, wherein in step iii), a centrifugal force is applied to the container.

12. The method according to claim 11 , wherein the centrifugal force is between 600g and 3000g.

13. The method according to claim 12, wherein the centrifugal force is 600 g.

14. The method according to any one of the preceding claims, further comprising step v) bringing together a selective lysis agent and the supernatant to remove remaining nontarget cells from the supernatant.

15. The method according to claim 6, wherein prior to step i), the whole blood sample is diluted with a liquid of volume in a blood:dilutant 3:1 volume ratio, 1 :1 volume ratio, a 1 : 10 volume ratio, 1 : 5 volume ratio, 1 : 4 volume ratio, 3:10 volume ratio, 2: 3 volume ratio, 1 :2 volume ratio, or 3: 4 volume ratio.

16. The method according to anyone of the preceding claims, wherein the density medium is selected from a blood culture medium, a nutrient-rich liquid medium, a medium used for the growth and cultivation of microorganisms, a lysogeny broth, medium of polysucrose and sodium diatrizoate, a high-molecular-weight branched polysaccharide, a density gradient medium, a density gradient medium composed of an iodinated compound, a density gradient medium based on a solution of iodixanol, a density gradient medium specifically formulated for the isolation of polymorphonuclear leukocytes (PMNs) fromhuman blood, a radiopaque contrast medium, a density gradient medium composed of a tri-iodinated benzoic acid derivative, a colloidal silica-based density gradient medium, an aqueous solution, a salt solution, or mixtures thereof.

17. The method according to anyone of the preceding claims, wherein the blood dilution medium for diluting the blood sample is selected from a blood culture medium, nutrientrich liquid medium, a medium used for the growth and cultivation of microorganisms, a lysogeny broth, a medium of polysucrose and sodium diatrizoate, a high-molecular-weight branched polysaccharide, a density gradient medium, a density gradient medium composed of an iodinated compound, a density gradient medium based on a solution of iodixanol, a density gradient medium specifically formulated for the isolation of polymorphonuclear leukocytes (PMNs) from human blood, a radiopaque contrast medium, a density gradient medium composed of a tri-iodinated benzoic acid derivative, a colloidal silica-based density gradient medium, aqueous solution, a salt solution, or mixtures thereof.

18. The method according to any of the preceding claims, wherein, the density of density media is substantially 1025-1055 Kg / m3.

19. The method according to claim 18, wherein the density of density media is selected from substantially 1052, 1038, and 1030 Kg / m3.

20. The method according to any of the previous claims, wherein the volume density media is optimized to be substantially 1 ml and a total volume of the density liquid and the diluted blood is substantially 4 ml.

21. The method according to anyone of the previous claims, where sedimentation is effected by centrifuging the container.

22. The method according to claim 14, the method further comprising the step of: vi) analyzing and / or quantifying the target cells.

23. The method according to claim 22, wherein after step v) and before step vi), the concentration of target cells in the supernatant is increased.

24. The method according to anyone of the previous claims, wherein, the container is incubated to culture cells prior to step i).

25. The method according to anyone of the preceding claims, wherein the container is incubated to culture cells between any of the steps.

26. The method according to claim 25 wherein one or several additional container incubations to culture cells are performed between the steps, wherein the container incubations are performed for 15 min - 12 hours, preferably for 30 min, 60 min, 90 min or 120 min.

27. The method according to any one of the previous claims, wherein, after step iii) target cells are isolated using filtration and / or density separation.

28. The method according to claim 27, wherein, after step iv), the supernatant is filtered through a membrane filter to remove any remaining non-target cells.

29. The method according to claim 22, wherein step vi) comprises layering the filtered solution over a density gradient medium with a density of about 1080 kg / m3, and centrifuging the filtered supernatant / solution at 2000g for 15 minutes.

30. The method according to claim 22 or 29, wherein step vi) further comprises flowing the solution in a microfluidic device, wherein the microfluidic device is configured to filter the solution.

31. The method according to claim 30, wherein the microfluidic device comprises microtraps.

32. The method according to claim 31 , wherein the microtraps are configured to filter out the non-target cells from the target cells after reducing the non-target cells from liquid biopsy sample.

33. The method according to claim 32, wherein the microtraps are configured to further filter out the non-target cells from the target cells after up-concentration of the target cells and / or selective lysis of the non-target cells.

34. The method according to any one of claims 31-33, wherein the microtraps are used as culture chambers for the target cells.

35. The method according to any one of claims 31-34, wherein step vi) further comprises reseeding target cells trapped in at least one of the microtraps to other microtraps.

36. The method according to any of the previous claims, further comprising adding a first compound, the first compound being configured to prevent bacteria from binding to the blood cells.

37. The method according to claim 36, wherein the first compound is added: prior to step i; between steps i and ii; or between steps ii and Hi.

38. The method according to any of the previous claims, further comprising adding a second compound, the first compound being configured to break down any lysate or debris remaining in the sample after the non-target cell lysis.

39. The method according to claim 38, wherein the second compound is an enzyme.

40. The method according to claim 38 or 39, wherein the second compound is added: prior to step i; between steps i and ii; between steps ii and Hi; or between steps Hi and iv.

41. The method according to any of the previous claims, wherein the target cells are bacterial cells, fungal cells or viral particles.

42. The method according to any one of claims 31-35, wherein the microfluidic device contains microtraps of different sizes.

43. The method according to any one of claims 22, 29, 30, or 35, wherein step vi) further comprises adding fluorescence markers for on-chip target cell detection or identification or bacterial antibiotic susceptibility detection.

44. The method according to any one of the previous claims for detecting bacteria in blood culture medium bottles.

45. The method according to any one of the previous claims for separating / isolating cells from blood.

46. The method according to any one of the previous claims for isolating tumor cells from blood.

47. A kit to separate target cells from non-target cells in a liquid biopsy sample, based on the cells’ sedimentation properties, the kit comprising- a container that contains a volumedof a density medium, and- instructions to carry out the method according to any one of the claims 1-46.

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