Biological sample delivery system and method

Abstract

The bioanalytical system includes a flow generator, a flow cell, a first flow path, and a second flow path. The flow generator is configured to provide a sheath fluid. The flow cell is configured to receive a biological sample. The first flow path is in fluid communication with the flow generator and configured to receive a first portion of the sheath fluid from the flow generator. The first flow path is configured to deliver the biological sample to the flow cell at a first flow rate using the first portion. The second flow path is in fluid communication with the flow generator and configured to receive a second portion of the sheath fluid from the flow generator. The second flow path is configured to deliver the second portion to the flow cell at a second flow rate. The second flow rate is different from the first flow rate.

Description

[Technical field]

[0001] Priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 294,648, entitled "Biological Sample Driving System and Method," filed December 29, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] In bioanalytical devices (e.g., body fluid analyzers and hematology analyzers), biological samples are delivered to a flow cell for analysis. These biological samples may include blood samples and / or other body fluid samples. The biological samples are typically surrounded by a sheath fluid to hydrodynamically focus the biological sample into a fine stream through the analysis region of the flow cell. The sheath fluid flow and the blood or other body fluid sample flow must be carefully calibrated and controlled to ensure that the flow cell analysis is accurate.

[0003] Some hematology systems use two separate chambers / reservoirs, each partially filled with liquid and pressurized with air. One chamber is used to obtain the desired sheath flow and the other chamber is used to obtain the desired sample flow. The chambers are set to different pressures and the chamber pressures are monitored by software. However, two-chamber systems are sensitive to variations in air pressure in the two chambers. As a result, two-chamber systems may incorporate specially sourced air pressure regulators and custom-designed sheath restrictors made of capillary glass that are susceptible to changes in temperature and fluid viscosity. This can affect the relative flow rates / fluid properties of the sheath and sample fluids separately.

[0004] Other hematology systems may use a two-chamber and two-pump driven sample flow system, where each of the first and second chambers is connected to its own pump, and each pump is used to generate a specific fluid delivery flow rate. For example, two high-precision syringe pumps may be used, where a first syringe pump is fluidly connected to a first liquid-containing chamber and drives the sheath flow, and a second pump is connected to a second liquid-containing chamber and drives the sample flow. Each of the high-precision syringe pumps operates at a different speed (flow rate) to pump the sheath fluid and sample into the flow cell. However, pump-driven systems utilize mechanical components and electric motor control to achieve a stable flow, which is susceptible to device-to-device variability. Additionally, the total amount of sheath fluid that can be delivered to the system is limited by the size of the syringe pump. Furthermore, these sample flow systems incorporating high-precision syringe pumps can be expensive.

[0005] The system may use quantitative and / or qualitative controls to assess system performance and obtain accurate and precise results. Quantitative controls use precise measurements in numerical form to control the system. Qualitative controls use features (e.g., image focus and sharpness) to assess system performance. For bioimaging systems, to ensure quantitative control, it is beneficial to image accurate and precise volumes of fluid. In other words, to ensure accurate counting, it is beneficial to control the volume of fluid analyzed in each separate image. For example, if the actual volume is smaller than the predicted volume, undercounting may occur. Conversely, if the actual volume is larger than the predicted volume, overcounting may occur. Volume is defined as the product of the imaging area of ​​the ribbon and the thickness of the ribbon. The imaging area of ​​the ribbon is determined by the optical system and is the product of the width and height of the field of view). It is beneficial to carefully control the thickness of the ribbon. The sheath and sample flow may be carefully controlled and calibrated to ensure quantitative control. This calibration and control can be useful in flow imaging systems where material is imaged as it flows through a flow cell where the material being analyzed (e.g., cellular material such as blood cells) is imaged in order to accurately classify and count the cells.

[0006] There is a need to maintain the sheath flow rate and sample flow rate, and the ratio of the two, to reduce or eliminate problems associated with existing systems. There is a need to ensure consistent and predictable delivery of sheath and sample fluids to the flow cell. There is also a need to allow careful calibration between the sheath flow and the sample flow to ensure accuracy in analytical flow instrumentation, e.g., flow imaging systems. [Brief description of the drawings]

[0007] [Figure 1]

[0013] Figure 1 is a schematic diagram of a first example of a bioanalytical system including a flow generating device. Figure 1A is a diagram depicting an alternative flow generating device in connection with the bioanalytical system of Figure 1. [Diagram 2] FIG. 2 is a perspective view of a schematic illustration of an exemplary flow cell for use with the bioanalytical system of FIG. 1. [Diagram 3] FIG. 3 is a top view of the flow cell of FIG. 2. [Figure 4] FIG. 3 is a side view of the flow cell of FIG. 2. [Diagram 5] FIG. 1 depicts an exemplary system for imaging particles. [Figure 6] 1 is a graph of exemplary sample flow rates, where the first plot includes a system with dual pumps and the second plot includes a system with a single pressurized chamber. [Figure 7] Figure 7A is a schematic diagram of a second exemplary bioanalytical system using the first exemplary circuit, and Figure 7B is a schematic diagram of the bioanalytical system of Figure 7A using the second exemplary circuit. [Figure 8] FIG. 2 depicts an exemplary method of analyzing a biological sample using the bioanalytical system of FIG. 1. Summary of the Invention

[0008] A bioanalytical system is described in various embodiments. In some embodiments, the bioanalytical system is used to analyze blood. In some embodiments, the bioanalytical system is used to analyze other bodily fluids, such as synovial fluid, cerebrospinal fluid, urine, bone marrow aspirate, or other bodily fluids / substances.

[0009] In some embodiments, the bioanalytical system includes a flow generator, a flow cell, a first flow path, and a second flow path. The flow generator is configured to provide a fluid. The flow cell is configured to receive a biological sample. The first flow path is in fluid communication with the flow generator and configured to receive a first portion of the fluid from the flow generator. The first flow path is configured to transfer the biological sample to the flow cell at a first flow rate using the first portion. The second flow path is in fluid communication with the flow generator and configured to receive a second portion of the fluid from the flow generator. The second flow path is configured to transfer the second portion to the flow cell at a second flow rate. The second flow rate is different from the first flow rate.

[0010] In some embodiments, the bioanalytical system includes a reservoir, a first flow path, a second flow path, a biological sample, and a flow cell. The reservoir contains a fluid. The first flow path is configured to receive a first portion of the fluid from the reservoir. The second flow path is configured to receive a second portion of the fluid from the reservoir. The biological sample is in fluid communication with the first flow path. The flow cell is configured for analysis of the biological sample. The flow cell includes a first passage and a second passage. The first passage is coupled to the first flow path such that a first portion of the fluid from the first flow path is configured to deliver the biological sample from outside the flow cell to the first passage of the flow cell. The second passage is coupled to the second flow path such that a second portion of the fluid from the second flow path is configured to be transmitted through the second passage of the flow cell.

[0011] In some embodiments, a blood analysis system includes a reservoir filled with a fluid, a first flow path, a second flow path, a blood sample, and a flow cell. The first flow path is configured to receive a first portion of the fluid. The second flow path is configured to receive a second portion of the fluid. The blood sample is in fluid communication with the first flow path. The flow cell is configured for analysis of the blood sample. The flow cell is in fluid communication with the first flow path such that a first portion of the fluid from the first flow path drives the blood sample from outside the flow cell into the flow cell. The flow cell is in fluid communication with the second flow path such that a second portion of the fluid is transmitted through the flow cell.

[0012] In some embodiments, the biological imaging system includes a sheath fluid, a first flow path, a second flow path, a biological sample, and a flow cell. The first flow path is configured to receive a first portion of the sheath fluid. The second flow path is configured to receive a second portion of the sheath fluid. The biological sample is in fluid communication with the first flow path. The flow cell is configured for imaging of the biological sample. The flow cell is in fluid communication with the first flow path such that a first portion of the sheath fluid from the first flow path drives a blood sample from outside the flow cell into the flow cell. The flow cell is in fluid communication with the second flow path such that a second portion of the sheath fluid is transmitted through the flow cell.

[0013] In some embodiments, the bioanalytical system includes a flow generator configured to provide a fluid, a first flow path in fluid communication with both the flow generator and the flow cell, and a second flow path in fluid communication with both the flow generator and the flow cell. The first flow path is configured to convey a biological sample through an analytical region of the flow cell at a first flow rate. The second flow path is configured to convey a sheath fluid through an analytical region of the flow cell at a second flow rate. The first and second flow rates, the first and second flow rates define a ratio, and the ratio is fixed.

[0014] In some embodiments, a method of analyzing a biological sample includes providing a sheath reservoir containing sheath fluid. The method also includes splitting the sheath fluid into first and second flow paths coupled to the sheath reservoir. The method also includes transmitting a first portion of the sheath fluid through the first flow path. The method also includes transmitting a second portion of the sheath fluid through the second flow path. The method also includes receiving a biological sample in fluid communication with the first flow path. The method also includes providing a flow cell configured for analysis of a biological sample, the flow cell including a first passage and a second passage, the first passage coupled to the first flow path such that a first portion of the sheath fluid delivers the biological sample from outside the flow cell into the first passage of the flow cell, and the second passage coupled to the second flow path such that a second portion of the sheath fluid is transmitted through the second passage of the flow cell.

[0015] In some embodiments, a method of analyzing a biological sample includes splitting a fluid obtained from a flow generating device into a first flow path containing a first portion of the fluid and a second flow path containing a second portion of the fluid. The method also includes using the first portion of the fluid to transmit the biological sample along the first flow path to a flow cell at a first flow rate. The method also includes transmitting a second portion of the fluid to the flow cell at a second flow rate, the second flow rate being different from the first flow rate. The method also includes analyzing the biological sample in the flow cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present disclosure relates to devices, systems, compositions, and methods for analyzing biological samples. Various exemplary bioanalytical systems (10, 310) are described in more detail with reference to Figures 1-4 and 7A-7B.

[0017] A. First Exemplary Bioanalytical System FIG. 1 illustrates a first exemplary bioanalytical system (10) including, among other components, a flow generator (11) and a flow cell (14). The flow generator (11) is illustrated as a pressure-based flow generator including a sheath reservoir (12) that is illustrated as being pressurized. Pressure-based flow generators may be sensitive to viscosity and pressure. The sheath reservoir (12) of the flow generator (11) is configured to provide sheath fluid at a bulk flow rate to the first and second flow paths (16, 18). The sheath reservoir (12) is illustrated and described as containing sheath fluid (20), although various fluids other than sheath fluid may be contained within the sheath reservoir (12), including, but not limited to, a diluent. The sheath reservoir (12) may be referred to as a single common reservoir / chamber, such as in the system (10) of FIG. 1, where a single sheath reservoir (12) supplies each of the first and second flow paths (16, 18) without additional sheath reservoirs.

[0018] In one variation, a float may be used to determine the volume of sheath fluid (20) in the sheath reservoir (12) (e.g., the sheath fluid (20) is filled up to the float). In other variations, the volume may be set electronically. In other variations, the volume may be customized depending on the sample analysis. For example, the volume of sheath fluid (20) in the sheath reservoir (12) may be adjusted to allow for extended imaging times, as desired for low density samples such as bodily fluids. Measurable cells in bodily fluids may be less dense than those in blood samples, and thus may be referred to as having a lower density. As such, a larger volume of the bodily fluid sample may be processed to determine cellular material located within the sample, which may entail longer / extended imaging times. In one variation, the volume of sheath fluid (20) in the sheath reservoir (12) may be about 100-200 mL, or about 150 mL. In one example, one cycle utilizes about 3 mL of sheath fluid, so a 150 mL reservoir of sheath fluid may last about 50 cycles (e.g., before the reservoir needs to be refilled via the sheath fluid supply - in a scenario where the reservoir is not being refilled as the sheath fluid is being pumped out). In this example, assuming a 1 minute cycle, the reservoir would last about 50 minutes. In some embodiments, the reservoir may be refilled with sheath fluid from the sheath fluid supply as the sheath fluid is being pumped out, for example, such that there is a relatively constant supply of sheath fluid in the reservoir, or such that the sheath fluid in the reservoir is refilled from the sheath fluid supply when it falls below a certain volume (e.g., 25 or 50 mL).

[0019] As shown in FIG. 1, the flow generator (11) additionally includes an air compressor (26), an air compressor valve (28), a vent (30), and a vent valve (32) in communication with a sheath fluid supply (34) containing sheath fluid (20). Examples of sheath fluids are described in U.S. Patent No. 9,316,635, entitled "Sheath Fluid Systems and Methods for Particle Analysis in Blood Samples," issued April 19, 2016, the disclosure of which is incorporated herein by reference in its entirety. The air compressor (26) is positioned upstream of the compressor valve (28), which is positioned upstream of the sheath fluid supply (34). In some variations, the pressure on the sheath reservoir (12) can be increased using air from the air compressor or using a pump to increase the amount of sheath fluid (20) contained within the sheath reservoir (12). The vent (30) is positioned upstream of a vent valve (32) that is positioned upstream of the sheath reservoir (12). A pump (17) connects the sheath fluid supply (34) with the sheath reservoir (12) for supplying fluid from the sheath fluid supply (34) to the sheath reservoir (12). A T-junction (15) is positioned between the outlet of the sheath reservoir (12) and the first and second flow path valves (36, 42).

[0020] The sheath reservoir (12) maintains the sheath fluid (20) at a desired pressure. For example, the sheath reservoir (12) may include a pressure tank into which gas (e.g., air) is selectively introduced or removed to maintain the pressure tank at the desired pressure. In some variations, the pressure of the sheath fluid (20) in the sheath reservoir (12) is between about 8 and about 30 pounds per square inch. In some variations, an optional pressure sensor (50) may be incorporated to monitor the pressure in the sheath reservoir (12). Software may monitor the pressure in the sheath reservoir (12) and / or the output flow through the first and second flow paths (16, 18) and adjust the flow rate by increasing or decreasing pressure to the sheath reservoir (12). For example, the pressure in sheath reservoir 12 may be increased by operating compressor 26 to open compressor valve 28, thereby allowing compressed air from compressor 26 to flow through compressor valve 28 and into sheath reservoir 12. Conversely, the pressure in sheath reservoir 12 may be decreased by opening vent valve 32, thereby allowing air from sheath reservoir 12 to flow out through vent 30.

[0021] FIG. 1A shows an example of an alternative flow generating device (11a) that does not utilize a sheath reservoir / tank (12). The flow generating device (11a) utilizes a pump or hydraulic flow generating device. The flow generating device (11a) includes a metering or gear pump (13) and a sheath fluid supply (34) that contains sheath fluid (20). Similar to FIG. 1, a T-junction (15) is positioned between the outlet of the pump (13) and the first and second flow path valves (36, 42). The pump (13) draws fluid from the sheath fluid supply (34) and provides sheath fluid (20) to the first and second flow paths (16, 18). Various other pump or hydraulic features may also be included.

[0022] The flow generators (11, 11a) are coupled (e.g., in fluid communication) with the first flow path (16) and the second flow path (18) such that the sheath fluid (20) passes through the first flow path (16) and the second flow path (18). In other words, a first portion of the sheath fluid (20) from the flow generators (11, 11a) is delivered to the first flow path (16) at a first pressure and a bulk flow rate. Similarly, a second portion of the sheath fluid (20) from the flow generators (11, 11a) is delivered to the second flow path (16) at a first pressure and a bulk flow rate. Splitting the output of the flow generators (11, 11a) into the first and second flow paths (16, 18) allows the flow generators (11, 11a) to create both a sample flow (using the first flow path (16)) and a sheath flow (using the second flow path (18)). The first flow path (16) and the second flow path (18) are disposed between the flow generator (11, 11a) and the flow cell (14).

[0023] The first flow path (16) is configured to deliver the biological sample using a first portion to the flow cell (14) at a first flow rate. The second flow path (18) is configured to deliver a second portion to the flow cell (14) at a second flow rate that is different from the first flow rate. In one variation, the bioanalytical system (10) is configured such that the sheath fluid flow rate through the second flow path (18) is faster or higher than the sheath flow rate through the first flow path (16).

[0024] The first flow path (16) includes a first flow path valve (36) positioned upstream of a first flow restrictor (38) positioned upstream of an optional first flow sensor (40). In other words, the first flow restrictor (38) and the first flow sensor (40) are positioned along the first flow path (16). Similar to the first flow path (16), the second flow path (18) includes a second flow path valve (42) positioned upstream of a second flow restrictor (44) positioned upstream of an optional second flow sensor (46). In other words, the second flow restrictor (44) and the second flow sensor (46) are positioned along the second flow path (18). In one example, the first and second flow path valves (36, 42) have an on / off or open / closed configuration that either permits or prevents flow therethrough. A variety of suitable valves may be used, including pinch valves and / or rocker valves. In one variation, the first and second flow path valves (36, 42) are configured to be in a closed position unless the analytical circuit / module is in an operating mode in which the flow path valves (36, 42) are in an open position.

[0025] The first and second flow restrictors (38, 44) maintain a constant ratio between the biosample flow rate and the sheath flow rate. This ratio is constant because the first and second flow restrictors (38, 44) are supplied by the same sheath fluid (20) from the same sheath source (e.g., sheath reservoir (12) in FIG. 1 or sheath fluid supply (34) / gear pump (13) in FIG. 1A). In other words, a single fluid source (i.e., sheath fluid (20)) drives the entire bioanalysis system (10), so any changes in pressure, temperature, or viscosity affect each flow path (16, 18) equally. In this manner, the sample to sheath flow rate ratio is constant and independent of pressure, temperature, and viscosity. This constant ratio can be beneficial in the context of an analysis system, including an imaging analysis system or a flow imaging analysis system that utilizes individual sample imaging. The constant ratio ensures a quantitative control over the bioanalysis system (10). For example, quantitative control of platelet count is beneficial in hematology applications. A constant ratio maintains a constant thickness of the sample ribbon / stream, which ensures the same imaging volume regardless of the actual flow rate (which may vary based on temperature, pressure changes, etc.). Variations in ribbon thickness may cause undercounting or overcounting.

[0026] The first flow restrictor (38) controls the sheath fluid flow rate through the first flow path (16) that delivers the sample flow to the flow cell (14). The second flow restrictor (44) controls the sheath fluid flow rate through the second flow path (18) that delivers the sheath fluid to the flow cell (14) and surrounds the sample flow through the analysis region of the flow cell (14). The first and second flow restrictors (38, 44) may be positioned at the input tubing to the flow cell (14). The first and second flow restrictors (38, 44) are selected based on the desired flow rate and the pressure of the sheath reservoir (12). For example, the first and second flow restrictors (38, 44) may have smaller inner diameters compared to the remaining output tubing. In other words, the maximum inner diameters of the first and second flow paths (16, 18) are smaller than the minimum inner diameters of the first and second flow paths (16, 18), respectively. In some variations, the first flow restrictor (38) may be about 2 inches to about 1 foot long and may have an inner diameter of about 0.002 to about 0.01 inches. In some variations, the second flow restrictor (44) may be about 1 foot to about 4 feet long and may have an inner diameter of about 0.01 to 0.04 inches. In one example, the first flow restrictor (38) may be about 6 inches long and may have an inner diameter of about 0.004 inches, and the second flow restrictor (44) may be about 2 feet long and may have an inner diameter of about 0.02 inches. In one variation, the components forming the first and second flow paths (16, 18) comprise polymeric (e.g., PEEK or PTFE) tubing. The first flow path (16) may comprise a polymeric tubing having an inner diameter of about 0.01 to 0.04 inches, or about 0.02. The first flow restrictor (38) may be configured as an inner tube configured within the first flow path (16) tubing, such as an inner tube that is coupled via threads or barbs within a portion of the first flow path (16). The second flow path (18) may comprise a polymeric tubing having an inner diameter of about 0.02 to about 0.06 inches, or about 0.04 inches. The second flow restrictor (44), similar to the first flow restrictor (38), may be configured as an inner tube, such as an inner tube that is coupled via threads or barbs within a portion of the second flow path (18).

[0027] The optional first and second flow sensors (40, 46) sense the first and second flow rates of the first and second flow restrictors (38, 44), respectively. The optional first and second flow sensors (40, 46) may also monitor and flag potential problems (e.g., air bubbles). Air bubbles reaching the flow cell (14) may obstruct the sample stream and affect image quality. The optional first and second flow sensors (40, 46) may monitor the sheath and sample flow rates in real time. The first and second flow sensors (40, 46) may include a variety of suitable flow sensors, one such suitable example being the SLF series commercially available from Sensirion. The first and second flow sensors (40, 46) may be located at different locations and may passively monitor the respective first and second flow paths (16, 18). For example, the first and second flow sensors (40, 46) are positioned relatively close downstream of the first and second flow restrictors (38, 44), but may be positioned anywhere downstream of the T-junction (15). As shown, the first and second flow sensors (40, 46) are positioned downstream of the first and second flow restrictors (38, 44).

[0028] Optionally, the controller (64) may be operatively connected to the bioanalytical system (10) to automatically control one or more aspects of the bioanalytical system (10). For example, as shown in FIG. 1, the controller (64) may be operatively connected to the first flow restrictor (38), the second flow restrictor (44), the first flow sensor (40), and / or the second flow sensor (46). In some variations, the controller (64) may be used to control the action of the first and second flow restrictors (38, 44) in an automated system. For example, the first and second flow restrictors (38, 44) may be configured as selectively and automatically adjustable to set specific flow rates (e.g., by programmable internal diameters, or local pumps at / near the flow restrictor areas) to dynamically set specific flow rates in each flow path (16, 18). For example, the controller (64) may monitor the sheath flow rate through each flow path (16, 18) and the associated ratio of sheath fluid in each flow path (16, 18) after passing through the restrictors (38, 44). The controller (64) may flag the sample or shut down operation if the ratio falls outside of a predetermined range (e.g., based on one or more signals from the first and second flow sensors (40, 46). In one example, this is performed in a dynamic manner where the flow rate is consistently monitored and adjustments are dynamically made to the system to maintain a particular optimal flow rate. The controller (64) may provide continuous data monitoring. In other variations, the first flow restrictor (38) and / or the second flow restrictor (44) are not controlled by the controller (64). Instead, the first flow restrictor (38) controls the flow rate of the first flow path (16) and the second flow restrictor (38) passively controls the flow rate of the second flow path (16) to the flow cell (14) without active control by the controller (64).

[0029] The bioanalytical system (10) is configured to analyze a biological sample (22). In variants where the bioanalytical system (10) images biological material, the bioanalytical system (10) may be referred to as a bioimaging system. For example, the bioanalytical system (10) may include an imaging device (24) configured to create an individual image of the biological sample (22) (e.g., blood cells) as the biological sample (22) passes through the flow cell (14). For example, the imaging device (24) may include a high-speed, high-resolution camera configured to take individual snapshots, as compared to a continuous detection system that may assess light scattering. In some variants, the biological sample (22) includes a blood sample. In variants where the bioanalytical system (10) analyzes a blood sample, the bioanalytical system (10) may be referred to as a blood analysis system. Although analysis and imaging of blood is shown and described herein, the bioanalytical system (10) may analyze (and optionally image) a variety of fluids, including, but not limited to, other bodily fluids, such as synovial fluid, urine, bone marrow aspirate, and the like.

[0030] The biological sample (22) is in fluid communication with the biological sample valve (53) and is configured to enter the first flow path (16) along the initial biological sample flow path (52) before being introduced into the first flow path (16). The biological sample valve (53), shown in one embodiment as a three-way valve, helps transfer the biological sample (22) from an area outside the flow cell circuit to the flow cell circuit, where the biological sample (22) can be transferred along a portion of the first flow path (16) by the sheath fluid (20). An external pressure source (e.g., air, a syringe pump, or other means) (not shown in FIG. 1 ) positioned upstream from the biological sample valve (53) can be configured to transfer the biological sample (22). Once the biological sample (22) has passed or is downstream of the biological sample valve (53), the sheath fluid (20) in the first flow path (16) directs the biological sample (22) toward the flow cell (14). The sheath fluid (20) is proximal to, or upstream from, the biological sample (22) and as such is configured to direct the biological sample (22) towards the flow cell (14).

[0031] The flow cell (14) utilizes the principle of fluid mechanics, where the biological sample (22) is accelerated and expanded into a thin stream to be analyzed in the analysis area (25) of the flow cell (14) and is surrounded by the sheath fluid (20) conveyed through the second flow path (18). The flow rate of the sheath fluid (20) is generally faster than the flow rate of the biological sample (22) because the cross section of the sheath channel is much larger than the sample / cannula. The linear velocity of the sheath fluid (20) may be the same as or higher than the flow rate of the biological sample (22) at the exit point (23) of the cannula (21) (see Figures 2-4). As shown in Figure 1, the sheath fluid (20) drives the flow of the biological sample (22) through the first flow path (16) into the cannula (21) positioned in the flow cell (14). The first flow restrictor (38) along the first flow path (16) is configured to induce a lower flow rate than the second flow restrictor (44) along the second flow path (18), such that a first flow rate of the sheath fluid (20) through the first flow path (16) is lower than a second flow rate of the sheath fluid (20) through the second flow path (18).

[0032] As shown generally in FIG. 1 and in more detail in FIGS. 2-4, the flow cell (14) is configured to analyze a biological sample (22). The flow cell (14) includes a first passage (54) and a second passage (56). The first passage (54) is coupled to the first flow path (16), and the second passage (56) is coupled to the second flow path (18). A second portion of the sheath fluid from the second flow path (18) may be referred to as a sheath source that surrounds the biological sample (22) passing through the flow cell (14). The first portion of the sheath fluid from the first flow path (16) may physically push the biological sample (22) such that the biological sample (22) is surrounded by the sheath source (e.g., the second portion of the sheath fluid) as the biological sample (22) exits the cannula (21). After exiting the cannula (21), the sample fluid stream merges and / or becomes surrounded by a sheath fluid stream in a pre-imaging region where the sample fluid stream becomes pinched by the sheath fluid stream before proceeding downstream to the analytical region (25) of the flow cell (14). The imaging device (24) may image the biological sample (22) within the analytical region (25) of the flow cell (14). For example, a blood sample may be surrounded by sheath fluid to ensure proper alignment of blood particles during imaging. Additional examples of flow cells and examples of hydrodynamic focusing (flow cell geometry narrowing the sample fluid stream and sheath fluid stream for imaging) are described in U.S. Pat. No. 9,316,635, issued April 19, 2016, entitled "Sheath Fluid Systems and Methods for Particle Analysis in Blood Samples," the disclosure of which is incorporated by reference in its entirety, and in U.S. Pat. No. 9,322,752, issued April 26, 2016, entitled "Flowcell Systems and Methods for Particle Analysis in Blood Samples," the disclosure of which is incorporated by reference in its entirety.

[0033] Alternate embodiments may utilize additional devices besides flow restrictors to create specific flow rates in each flow path. The inclusion of a common sheath reservoir (12) that provides a common sheath fluid to pump both the sample and sheath flows, as described above, results in a constant fixed ratio of sample to sheath flows.

[0034] B. Exemplary Systems for Imaging Particles FIG. 5 shows an embodiment of a system (110) for imaging particles in a blood fluid sample. The system (110) may be used in place of the imaging device (24) of FIG. 1. As shown, the system (110) includes a sample fluid injection system (112), a flow cell (114) that may be similar to the flow cell (14), and an image capture device (116), and a processor (118). The flow cell (114) provides a flow path (120) that carries a flow of sheath fluid, optionally in combination with the sample fluid. According to some embodiments, the sample fluid injection system (112) may include a cannula or injection tube (122) that is positioned before (i.e., upstream of) the image capture location (140). The sample fluid injection system (112) may be in fluid communication with the flow path (120) (e.g., via a sample fluid inlet (124) and may be operable to inject a sample fluid (126) through a distal exit port (130) of the injection tube (122) to provide a sample fluid stream (132), also referred to as a ribbon, and into the flowing sheath fluid (128) in the flow cell (114). For example, the processor (118) may include, or be operatively associated with, a storage medium having a computer application that, when executed by the processor, is configured to cause the sample fluid injection system (112) to inject the sample fluid (126) into the flowing sheath fluid (128).

[0035] As shown, sheath fluid (128) may be introduced into flow cell (114) by a sheath fluid injection system (134) (e.g., via a sheath fluid inlet (136). For example, processor (118) may include, or be operatively associated with, a storage medium having a computer application that, when executed by the processor, is configured to cause sheath fluid injection system (134) to inject sheath fluid (128) into flow cell (114). As depicted in FIG. 5, distal exit port (132) of injection tube (122) may be connected to a distal exit port (134) of injection tube (122). 0) may be positioned at a central location along the length of the narrowed transition region (138). In some cases, the distal exit port (130) may be positioned closer to the beginning (proximal portion) of the transition region (138). In some cases, the distal exit port (130) may be positioned closer to the end (distal portion) of the transition region (138). In some cases, the distal exit port (130) may be positioned entirely outside of the transition region (138) (when the distal exit port (130) is disposed proximal of the narrowed transition region).

[0036] Continuing to refer to FIG. 5, the sample fluid stream (132) has a first thickness T1 at the exit port (130). The flow channel (120) of the flow cell has a decrease in flow channel size such that the thickness of the sample fluid stream (132) decreases from an initial thickness T1 to a second thickness T2 adjacent the image capture location (140). The sample fluid (126) exits the exit port (130) where the sample fluid (126) merges and / or is surrounded by sheath fluid (128) in a pre-imaging region. In the pre-imaging region, the sample fluid stream (132) becomes sandwiched by the sheath fluid (128) before proceeding downstream to the imaging region, the image capture location (140). The image capture device (116) is aligned with the image capture location (140) to image a first plurality of particles from the first sample fluid at the image capture location (140) of the flow cell (114).

[0037] The processor (118) is coupled to the sample fluid injection system (112), the image capture device (116), and optionally the sheath fluid injection system (134). The processor (118) is configured to terminate the injection of the first sample fluid into the flowing sheath fluid (128) and to initiate the injection of a second sample fluid into the flowing sheath fluid (128). For example, the processor (118) may include or be operatively associated with a storage medium having a computer application that, when executed by the processor, is configured to cause the sample fluid injection system (112) to inject the second sample fluid into the flowing sheath fluid (128).

[0038] Additionally, the processor (118) is configured to initiate capture of an image of a second plurality of particles from the second sample fluid at the image capture location (140) of the flow cell (114) after the sample fluid transient. For example, the processor (118) may include, or be operatively associated with, a storage medium having a computer application that, when executed by the processor, causes the image capture device (116) to initiate capture of an image of a second plurality of particles from the second sample fluid at the image capture location (140) of the flow cell (114) after the sample fluid transient and imaging of the first plurality of particles.

[0039] Accordingly, an embodiment of the present invention encompasses a system (110) for imaging a plurality of particles in a sample fluid (126) having a sample fluid viscosity. The system (110) may include a flow cell (114) having a flow path (120) and an injection tube (122). The flow path (120) may have a reduction or narrowing transition area in the flow path size. Additionally, the system (110) may include a sheath fluid inlet (136) in fluid communication with the flow path (120) of the flow cell (114) to direct a flow of sheath fluid along the flow path (120) of the flow cell (114). The system (110) may also include a sample fluid inlet (124) in fluid communication with the injection tube (122) of the flow cell (114) to inject a sample fluid stream (132) of the blood fluid sample into the flowing sheath fluid in the flow cell (114). For example, sample fluid (126) may exit a distal exit port (130) of injection tube (122) into an envelope of flowing sheath fluid (128) forming a sample ribbon (132) therein.

[0040] The sheath fluid (128), along with a sample fluid ribbon (132) formed from the sample fluid (126), flows through a channel-sized transition area (138) toward a pre-imaging region and then to an imaging location / region (140). As shown, the system (110) also includes an imaging device (116) that images the plurality of particles at the imaging location (140).

[0041] C. Example graph of sample flow rate FIG. 6 shows an exemplary sample flow rate graph (210) including a first plot (212) for a system with a dual pump and a second plot (214) for a system with a single chamber (e.g., sheath reservoir (12)). An exemplary sample flow rate for a system with a single chamber (e.g., sheath reservoir (12)) may be about 6-24 microliters per minute, or about 10-14 microliters per minute. For example, this may correspond to the sheath flow rate downstream of the first flow restrictor (38) through the first flow path (16) as the circuit pumps the sample. As shown, there is greater consistency in the second plot (214) than in the first plot (212). It may be beneficial to reduce the variation within the first plot (212). For example, inconsistencies in the sample flow rate in the first plot (212), which represents a dual pump system, can cause data noise and can also cause problems in the analysis area (25) of the flow cell (14), for example, in a flow imaging system where changes in flow rate can adversely affect image focus (e.g., thicker ribbons can blur smaller particles such as platelets). In contrast, consistency in the sample flow rate in the second plot (214) can have advantages by minimizing noise and, for example, in a flow imaging system, by promoting constant image focus. As the flow rate varies, the ribbon thickness varies by the same relative amount. Conversely, the more stable the flow rate, the more constant the ribbon thickness, which improves quantitative results.

[0042] FIG. 6 shows an exemplary graph. In some examples, the sample flow rate is about 6-24 microliters / minute and the sheath flow rate is about 2600-3000 microliters / minute. In some variations, the analysis of white blood cells (WBC) and red blood cells (RBC) may utilize a sheath flow rate of about 3,000 microliters per minute. As described with reference to FIGS. 7A-7B, in some examples, the analysis system (10) utilizes a first analysis plan or circuit for red blood cells and a second analysis plan or circuit for white blood cells (e.g., the blood used for each analysis plan or circuit is stored in a different chamber to provide separate samples (22)), with different flow rates for each sample type. For example, the red blood cell sample may flow at about 6-12 microliters / minute and the white blood cell sample may flow at about 14-22 microliters / minute. In some examples, the ratio of the sheath flow rate to the sample flow rate (i.e., sheath flow rate divided by sample flow rate) is about 150-500, about 200-400, or about 200-250. The controller (64) may be utilized to monitor the sheath flow rate, the sample flow rate, and the ratio of the two and flag results if outside of a particular range (e.g., outside of a 5-10% threshold from a predefined ideal ratio). The ratios and corresponding thresholds may be different for different samples (e.g., red blood cell samples, white blood cell samples, body fluid samples). In some variants, analysis of the red blood cell sample may utilize a sample flow rate of about 8 microliters / minute and analysis of the white blood cell sample may utilize a sample flow rate of about 14 microliters / minute. The lower relative sample flow rate of the red blood cell sample compared to the white blood cell sample may be used to prevent the sample fluid containing the red blood cells from going out of focus when analyzing the red blood cell sample. For dilute body fluids, a longer collection duration may be used to acquire additional data.

[0043] D. Second Exemplary Bioanalytical System 7A-7B show a second exemplary bioanalytical system (310). The bioanalytical system (310) may include similar components as systems (10, 110). Although the bioanalytical system (310) is shown as including a flow generator including a sheath reservoir (312), the bioanalytical system (310) may include a pump-based flow generator similar to the flow generator (11a) shown in FIG. 1A. The sheath reservoir (312) may be similar to the sheath reservoir (12), the flow cell (314) may be similar to the flow cell (14), the first flow path (316) may be similar to the first flow path (16), the second flow path (318) may be similar to the second flow path (18), and the sheath fluid (not shown) may be similar to the sheath fluid (20). As will be described in more detail below, the fluid path of the first flow path (316) differs when comparing FIG. 7A to FIG. 7B. The bioanalytical system (310) includes a valve (328), which may be similar to valve (28), and a vent (330), which may be similar to vent (30).

[0044] A sheath fluid supply (334), which may be similar to the sheath fluid supply (34), supplies sheath fluid (20) to the sheath reservoir (312) using a pump (345). A pump (326), such as a peristaltic pump, and a pressure chamber (327) may pressurize the sheath reservoir (312). The sheath reservoir (312) is coupled (e.g., in fluid communication) with the first flow path (316) and the second flow path (318) such that sheath fluid (20) from the sheath reservoir (312) passes through at least the first flow path (316) and the second flow path (318). An air passage (329) is shown using a dashed line between the pressure chamber (327) and the sheath reservoir (312). In some embodiments, the air may be pressurized to allow pressure generated in the pressure chamber (327) to be transferred to the sheath reservoir (312). For example, the pressure in the air passage (329) may be about 8 psi of air pressure.

[0045] Downstream of the sheath reservoir (312), the bioanalytical system (310) includes a junction (335), valves (336a, 336b, 342), first, second, and third flow restrictors (338a, 338b, 344), optional first and second flow meters (340, 346), and a valve (353). The junction (335) may be used to split the sheath fluid. The first, second, and third flow restrictors (338a, 338b, 344) may be similar to the first and second flow restrictors (38, 44). Similarly, the optional first and second flow meters (340, 346) may be similar to the first and second flow sensors (40, 46), and the valve (353) may be similar to the valve (53). The bioanalytical system (310) includes first and second dilution chambers, shown as a red blood cell dilution chamber (366) and a white blood cell dilution chamber (368). The red blood cell and white blood cell dilution chambers (366, 368) are in communication with the biosample fluid path (352) using a three-way valve (348). Although not shown, fluid from the red blood cell and white blood cell dilution chambers (366, 368) may be advanced using a sheath fluid. The biosample fluid path (352) is introduced into the first flow path (316) using a valve (353). In some variations, the biosample fluid path (352) may terminate into the valve (353) with a separate fluid connection disposed between the valve (353) and the first passageway (354).

[0046] The flow cell (314) includes a first passage (354), which may be similar to the first passage (54), and a second passage (356), which may be similar to the second passage (56). The biosample fluid path (352) is shown using a dashed line that enters and terminates at a valve (353), which may be fluidly connected with the first passage (354) of the flow cell (314). Although not shown, the bioanalytical system (310) may include an imaging device or system (110), which may be similar to the imaging device (24). Downstream of the flow cell (314) is a waste (262), which may be similar to the waste (62). Although not shown, the bioanalytical system (310) may include an optional controller, similar to the controller (64) (see FIG. 1), for controlling one or more components of the bioanalytical system (310).

[0047] FIG. 7A shows a schematic diagram of a bioanalytical system (310) using a first exemplary circuit, and FIG. 7B shows a schematic diagram of the bioanalytical system of FIG. 7A using a second exemplary circuit. The difference between FIG. 7A and FIG. 7B is illustrated between the valve (336a) and the fitting (339) as well as between the valve (336b) and the fitting (339). As shown in FIG. 7A, the valve (336a) is closed and the valve (336b) is open. As a result, in FIG. 7A, the fluid in the first flow path (316) passes through the valve (336b), the second flow restrictor (338b), the fitting (339), and the optional first flow meter (340), and does not pass through the first flow restrictor (338a). As shown in FIG. 7B, the valve (336a) is open and the valve (336b) is closed. As a result, in Figure 7B, fluid in first flow path (316) passes through valve (336a), first flow restrictor (338a), fitting (339), and optional first flow meter (340), but does not pass through second flow restrictor (338b). The dimensions of the first and second flow restrictors (338a-b) may allow the flow rate of first flow path (316) in Figure 7A to differ from the flow rate of first flow path (316) in Figure 7B.

[0048] Each patient sample may be imaged using the first and second circuits of Figures 7A-7B. For example, the first circuit may include a red blood cell circuit (RBCC) and the second circuit of Figure 7B may include a white blood cell circuit (WBCC). For red blood cell imaging, the RBCC of Figure 7A may be utilized. Similarly, for white blood cell imaging, the WBCC of Figure 7B may be utilized. For example, using the RBCC, the sample may be imaged for as short as about 1 second. Similarly, for example, using the WBCC, the sample may be imaged for as short as about 10 seconds, or as short as about 5 seconds. A first flow rate of the first flow path (316) may be used for white blood cells received from the biological sample fluid path (352) and a second flow rate of the first flow path (316) may be used for red blood cells received from the biological sample fluid path (352). In Figure 7A, the first flow path (316) may have a flow rate of about 8 microliters per minute. In Figure 7A, the first flow path (316) can have a flow rate of about 14 microliters per minute. In Figures 7A-7B, the second flow path (318) can have a flow rate of about 3,000 microliters per minute.

[0049] E. Exemplary Methods of Analyzing Biological Samples In Figure 8, an exemplary method (410) of analyzing a biological sample (22) using a bioanalysis system (10, 310) is also described with reference to Figures 7A-7B. The method (410) may include steps (412, 414, 416, 418, 420, 422, 424). In step (412), the method (410) includes providing a flow generator (11, 11a, 311) configured to provide sheath fluid (20) at a bulk flow rate. The flow generator (11, 11a, 311) may include a pump (13) or a pressurized sheath reservoir (12, 312). In step (414), the method (410) includes splitting the sheath fluid (20) obtained from the flow generator (11, 11a, 311) into first and second flow paths (16, 18, 316, 318) coupled to the flow generator (11, 11a), the first flow path (16, 316) containing a first portion of the sheath fluid (20) and the second flow path (18, 318) containing a second portion of the sheath fluid (20).

[0050] In step (416), the method (410) includes conveying a first portion of the sheath fluid (20) through the first flow path (16, 316). For example, a biological sample can be conveyed to the flow cell (14, 314) along the first flow path (16, 316) at a first flow rate using the first portion of the sheath fluid (20). In step (418), the method (410) conveys a second portion of the sheath fluid (20) through the second flow path (18, 318) at a second flow rate different from the first flow rate. In some variations, the first flow rate can be obtained using a flow restrictor (38, 338a-b) positioned along the first flow path (16, 316). Similarly, in some variations, the second flow rate can be obtained using a flow restrictor (44, 344) positioned along the second flow path (18, 318).

[0051] At step (420), the method (410) includes receiving a biological sample in fluid communication with the first flow path (16, 316). At step (422), the method (410) includes providing a flow cell (14, 314) configured for analysis of a biological sample (22). The flow cell (14, 314) includes a first passageway (54, 354) and a second passageway (56, 356). The first passageway (54, 354) is coupled to the first flow path (16, 316) such that the sheath fluid (20) drives the biological sample (22) from outside the flow cell (14, 314) into the first passageway (54, 354) of the flow cell (14, 314). The second passage (56, 356) is coupled to the second flow path (18, 318) such that a second portion of the sheath fluid (20) is transmitted through the second passage (56, 356) of the flow cell (14, 314). In step (424), the method (410) includes analyzing the biological sample (22) in the flow cell (14, 314). In some variations, the analysis may include imaging the biological sample (22) in the flow cell (14, 314) using an imaging device (24) (see FIG. 1) or system (110) (see FIG. 5). The method may also include at least partially surrounding the biological sample (22) using the second portion of the sheath fluid (20).

[0052] All patents, patent publications, patent applications, journal articles, books, technical references, and the like, discussed in this disclosure are hereby incorporated by reference in their entirety for all purposes.

[0053] Different arrangements of the components depicted in the drawings or described above, as well as components and steps not shown and described, are possible. Likewise, some features and subcombinations are useful and may be used without reference to other features and subcombinations. The embodiments of the invention are described for purposes of illustration and not limitation, and alternative embodiments shall become apparent to the reader of this patent. In certain cases, method steps or operations may be performed or executed in a different order, or operations may be added, deleted, or modified. It should be understood that in certain aspects of the invention, a single component may be replaced with multiple components, and multiple components may be replaced with a single component, to provide an element or structure or to perform a given function(s). Except where such a substitution is not operable to practice a particular embodiment of the invention, such substitution is considered within the scope of the invention. Thus, the invention is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications may be made without departing from the scope of the following claims.

Claims

1. 1. A bioanalysis system comprising: a flow generating device configured to provide a fluid; a flow cell configured to receive a biological sample; a first flow path in fluid communication with the flow generating device and configured to receive a first portion of the fluid from the flow generating device, the first flow path configured to transfer the biological sample to the flow cell at a first flow rate using the first portion; a second flow path in fluid communication with the flow generating device and configured to receive a second portion of the fluid from the flow generating device, the second flow path configured to deliver the second portion to the flow cell at a second flow rate, the second flow rate being different from the first flow rate; A bioanalysis system comprising:

2. 2. The bioanalytical system of claim 1, wherein the flow generator is configured to provide the fluid at a bulk flow rate, the first flow path is configured to receive the first portion of the fluid at the bulk flow rate, and the second flow path is configured to receive the second portion of the fluid at the bulk flow rate.

3. The bioanalytical system of claim 1 , wherein the flow generator is a pressure-based flow generator including a pressurized reservoir configured to produce the bulk flow rate at a first pressure.

4. The bioanalytical system of claim 3 , wherein the pressure-based flow generator comprises a pressurized reservoir containing the fluid at the first pressure.

5. The bioanalytical system of claim 1 , wherein the flow generating device is a pump or a hydraulic flow generating device.

6. The bioanalytical system of claim 5 , wherein the pump or hydraulic flow generator comprises a metering pump or gear pump configured to create the bulk flow rate.

7. The bioanalytical system of claim 1 , further comprising a first flow restrictor along the first flow path configured to create the first flow rate.

8. 8. The bioanalytical system of claim 7, further comprising a junction or valve positioned between the flow generator and the first flow restrictor, the junction or valve configured to split the fluid into the first and second portions.

9. The bioanalytical system of claim 5 , further comprising a second flow restrictor along the second flow path configured to create the second flow rate.

10. The bioanalytical system of claim 1 , further comprising an imaging device configured to image the biological sample as it passes through the flow cell.

11. 1. A bioanalysis system comprising: a fluid generator configured to provide a fluid; a first flow path configured to receive a first portion of the fluid from the fluid generator; a second flow path configured to receive a second portion of the fluid from the fluid generator; a biological sample in fluid communication with the first flow path; a flow cell configured for analysis of the biological sample, the flow cell comprising a first passageway and a second passageway; Equipped with the first passage is coupled to the first flow path such that the first portion of the fluid from the first flow path is configured to direct the biological sample from outside the flow cell into the first passage of the flow cell; A bioanalytical system, wherein the second passage is coupled to the second flow path such that the second portion of the fluid from the second flow path is configured to be transmitted through the second passage of the flow cell.

12. The bioanalytical system of claim 11 , further comprising a first flow restrictor positioned along the first flow path and a second flow restrictor positioned along the second flow path.

13. The bioanalytical system of claim 11 , wherein the first and second flow rates define a ratio, and the ratio is fixed.

14. 12. The bioanalytical system of claim 11, wherein a fixed ratio between the first and second flow rates ensures that the bioanalytical system is quantitatively consistent in response to magnitudes of the first and second flow rates that vary due to at least one of viscosity or temperature.

15. The bioanalytical system of claim 11 , further comprising an imaging device configured to image the biological sample as it passes through the flow cell.

16. 1. A bioanalysis system comprising: a flow generating device configured to provide a fluid; a first flow path in fluid communication with both the flow generator and the flow cell; a second flow path in fluid communication with both the flow generator and the flow cell; Equipped with the first flow path is configured to convey a biological sample through an analytical region of the flow cell at a first flow rate; the second flow path is configured to convey sheath fluid through the analytical region of the flow cell at a second flow rate; The first and second flow rates define a ratio, the ratio being fixed.

17. 17. The bioanalytical system of claim 16, wherein the first flow rate and the second flow rate are increased or decreased by the same percentage.

18. 17. The bioanalytical system of claim 16, wherein a fixed ratio between the first and second flow rates ensures that the bioanalytical system is quantitatively consistent in response to magnitudes of the first and second flow rates that vary due to at least one of viscosity or temperature.

19. The bioanalytical system of claim 16 , wherein the flow generator is a pressure-based flow generator including a pressurized reservoir configured to produce the bulk flow rate at a first pressure.

20. The bioanalytical system of claim 16 , wherein the flow generating device is a pump or hydraulic flow generating device, the pump or hydraulic flow generating device comprising a metering pump or gear pump configured to create the bulk flow rate.

21. 1. A method for analyzing a biological sample, comprising: dividing a fluid obtained from a flow generating device into a first flow path containing a first portion of the fluid and a second flow path containing a second portion of the fluid; transmitting a biological sample at a first flow rate along the first flow path to a flow cell using the first portion of the fluid; conveying the second portion of the fluid to the flow cell at a second flow rate, the second flow rate being different from the first flow rate; analyzing the biological sample in the flow cell; The method includes:

22. The method of claim 21 , further comprising fixing a ratio of the first flow rate and the second flow rate.

23. 23. The method of claim 22, further comprising creating the first flow rate along the first flow path using a first flow restrictor.

24. 24. The method of claim 23, further comprising creating the second flow rate along the second flow path using a second flow restrictor.

25. 25. The method of claim 24, wherein fixing the ratio further comprises fixing a ratio of the first flow rate and the second flow rate using the first and second flow restrictors.

26. 23. The method of claim 22, wherein fixing the ratio of the first flow rate to the second flow rate ensures that the analysis is quantitatively consistent in response to changes in magnitude of the first and second flow rates due to at least one of viscosity or temperature.

27. 22. The method of claim 21, further comprising using the flow generating device to provide the fluid at a bulk flow rate prior to dividing the fluid.

28. 30. The method of claim 27, wherein providing the fluid further comprises providing the fluid at the bulk flow rate at a first pressure using a pressure-based flow generating device.

29. 30. The method of claim 28, wherein the pressure-based flow generator comprises a pressurized reservoir containing the fluid at the first pressure.

30. 30. The method of claim 27, wherein providing the fluid further comprises providing the fluid at the bulk flow rate using a pump or hydraulic flow generating device.

31. 22. The method of claim 21 , wherein splitting the fluid further comprises splitting the fluid obtained from the flow generating device into the first flow path containing the fluid at the bulk flow rate and the second flow path containing the fluid at the bulk flow rate.

32. 22. The method of claim 21, wherein dividing the fluid further comprises dividing the fluid using a junction or a valve.

33. 22. The method of claim 21, wherein splitting the fluid further comprises splitting the fluid using a three-way valve.

34. 22. The method of claim 21, wherein analyzing the biological sample further comprises imaging the biological sample using an imaging device as the biological sample passes through the flow cell.

Images