Sample Preparation Equipment

Abstract

A sample preparation instrument with an integrated device for estimating the concentration of white blood cells in a sample is described. The sample preparation instrument receives a sample, which is then prepared for analysis. The integrated device can implement an optical method, an electrical resistance method, or a flow cytometry method to estimate, for example, the white blood cell concentration in the sample. For some types of analysis, the sample volume of the sample depends on the white blood cell concentration. Therefore, the sample preparation instrument can automatically determine and adjust the sample volume of the sample based on the estimated white blood cell concentration prior to adding additional reagents, such as a labeling reagent and / or a lysing reagent, to prepare the sample.

Description

[Background technology]

[0001] This application was filed as a PCT international patent application on December 18, 2020, and claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 954,299, filed December 27, 2019, the entire disclosure of which is incorporated by reference in its entirety.

[0002] To prepare a sample for analysis by an instrument such as a flow cytometer or other sample analysis instrument, a sample of the sample is stained with a labeling reagent that targets components in the sample and facilitates analysis of the targeted components. As an example, the target can be a subset of white blood cells in a blood sample, and the labeling reagent is an antibody that is attached to the subset of white blood cells. Staining of the sample with the labeling reagent is performed based on a certain number of white blood cells present in the sample. For example, if there is a normal white blood cell concentration of about 4,000 to about 10,000 cells / microliter (μL) in the sample, a specific volume of labeling reagent for staining is used. However, if the white blood cell concentration in the sample is abnormally high or low, the specific volume of labeling reagent will not be effective in targeting the white blood cells or will interfere with the analysis, respectively. Therefore, with respect to the white blood cell concentration in the sample to be obtained, it is desirable to confirm whether the white blood cell concentration is within an appropriate range for a specific volume of labeling reagent before the sample is prepared for analysis. Summary of the Invention [Means for solving the problem]

[0003] In general, the present disclosure is directed to a sample preparation device and methods implemented by the sample preparation device to prepare a sample of a specimen for analysis. In one possible configuration, as a non-limiting example, the white blood cell concentration in the sample is estimated, the volume of the sample to be prepared is determined based on the white blood cell concentration, and one or more reagents, including reagents that target components of the sample for analysis, are added to the sample to prepare the sample. Various aspects are described in the present disclosure, including, but not limited to, the following aspects:

[0004] One aspect is a sample preparation instrument. The sample preparation instrument can include an input station for receiving a sample, a cell concentration estimator for estimating a concentration of white blood cells in the sample, and a transfer station for dispensing a sample of the sample, the volume of the sample being based on the estimated concentration of white blood cells in the sample. The sample preparation instrument can also include a reagent station and a lysing station. The reagent station includes a labeling reagent that is added to the sample. The lysing station includes a lysing reagent that is added to the sample. The sample preparation instrument can further include an output station for providing a sample following addition of the labeling reagent and the lysing reagent to the sample.

[0005] Another aspect is a method that includes accepting a sample as an input, estimating a concentration of white blood cells in the sample, and dispensing a sample of the sample, the volume of the sample being based on the estimated concentration of white blood cells in the sample. The method also includes adding one or more labeling reagents to the sample, adding one or more lytic reagents to the sample, and providing the sample of the sample as an output following addition of the one or more labeling reagents and the one or more lytic reagents to the sample.

[0006] A further aspect is a sample preparation and analysis instrument that includes an input station that receives a sample, a cell concentration estimator that estimates a concentration of white blood cells in the sample, and a transfer station that dispenses a sample of the sample, the volume of the sample being based on the estimated concentration of white blood cells in the sample. The sample preparation and analysis instrument also includes a reagent station and a lysis station. The reagent station includes a labeling reagent that is added to the sample. The lysis station includes a lysis reagent that is added to the sample. The sample preparation and analysis instrument further includes a sample analyzer that performs an analysis on the sample following addition of the one or more labeling reagents and the one or more lysis reagents to the sample, and an output station that provides the results of the analysis. The present invention provides, for example, the following: (Item 1) A sample preparation device (100), comprising: an input station (124) for receiving a sample; a cell concentration estimator (106) for estimating a concentration of white blood cells in the sample; a transfer station (112) for dispensing a sample of the specimen, the volume of the sample being based on an estimated concentration of white blood cells in the specimen; a reagent station (108) containing a labeling reagent to be added to the sample; a lysis station (110) containing a lysis reagent that is added to the sample; an output station (140) for providing the sample following addition of the labeling reagent and the lysis reagent to the sample; A sample preparation device (100) comprising: (Item 2) The cell concentration estimator (106) a mixing chamber (400) configured to receive an aliquot of the sample; A counting chamber (402) connected to the mixing chamber (400); a fluid pump (406) configured to advance at least a portion of the aliquot from the mixing chamber (400) to the counting chamber (402); an optical system (404) configured to capture a plurality of images of at least a portion of the aliquot within the counting chamber (402); 2. The sample preparation apparatus (100) of item 1, comprising: (Item 3) The mixing chamber (400) comprises: A container (420); a cap (422) positioned over the open end of the vessel (420), the cap (422) including a plurality of input ports (428, 430, 432) and an opening (426) for a transfer station probe (114) that dispenses the sample into the vessel (420); a motor (424) for mixing the aliquot of sample with one or more reagents dispensed into the container (420) via the plurality of input ports (428, 430, 432), the one or more reagents including a lysis reagent and a diluent reagent; 3. The sample preparation apparatus (100) of item 2, comprising: (Item 4) 4. The sample preparation device (100) of claim 2 or 3, wherein the counting chamber (402) comprises one or more transparent flow cells. (Item 5) The sample preparation instrument (100) of any one of items 2-4, wherein the optical system (404) includes a camera (440), a light source (442), and a lens (444). (Item 6) 2. The sample preparation apparatus (100) of any one of the preceding items, further comprising a computing device (116) including a processing device (120) and a memory (118) coupled to the processing device (120). (Item 7) The memory (118) stores instructions that, when executed by the processing device (120), cause the processing device (120) to: receiving a plurality of images of the aliquots of the sample captured by an optical system (404) of the cell concentration estimator (106); determining (462) a total number of white blood cells in the plurality of images based on the number of white blood cells counted in each image; determining (464) a total volume of the subdivisions within the plurality of images; estimating a concentration of white blood cells based on the total number of white blood cells and the total volume of the aliquots in the plurality of images (466); 7. The sample preparation device (100) according to item 6, (Item 8) The memory (118) stores instructions that, when executed by the processing device (120), cause the processing device (120) to: receiving a selection of a panel (346) to be implemented; reading from the memory (118) a set of predefined rules for preparing the samples for the panel, the set of predefined rules including volumes of the samples corresponding to one or more ranges of white blood cell concentrations; determining a volume of the sample based on a predefined set of rules and an estimated concentration of white blood cells in the sample; 8. The sample preparation device (100) according to item 6 or 7, (Item 9) 9. The sample preparation device (100) of claim 8, wherein the set of predefined rules further comprises a minimum white blood cell concentration and a maximum white blood concentration, and wherein one or more ranges of the white blood cell concentrations are between the minimum white blood cell concentration and the maximum white blood cell concentration. (Item 10) The processing device (120) further comprises: the estimated leukocyte concentration for the sample is at or below the minimum leukocyte concentration; said estimated leukocyte concentration for said sample being at or above said maximum leukocyte concentration; 10. The sample preparation device (100) according to item 9, wherein processing of the sample is prevented in the event of one or more of the following: (Item 11) The display (122) further comprises: The range in which the estimated white blood cell concentration falls; and The volume of the sample dispensed; and An alert when the above sample is blocked and 4. The sample preparation instrument (100) of any one of the preceding claims, configured to provide a user interface including one or more of the following: (Item 12) The cell concentration estimator (106) a first chamber for receiving an aliquot of said sample suspended in an electrolyte solution; A second chamber; and an opening connecting the first chamber to the second chamber, through which an aliquot of the sample suspended in the electrolyte solution flows from the first chamber to the second chamber; at least two electrodes generating an electric field within the cell concentration estimator, and an estimate of the concentration of white blood cells in the sample is determined based on a change in electrical resistance detected across the aperture as an aliquot of the sample flows through the aperture; 2. The sample preparation apparatus (100) of item 1, comprising: (Item 13) 2. The sample preparation instrument (100) of item 1, wherein the cell concentration estimator (106) comprises a flow cytometer. (Item 14) The sample preparation device (100) of any one of the preceding items, wherein the input station (124) includes a rocking mechanism for rocking a cassette in which a tube containing the sample is received. (Item 15) Item 11. The sample preparation device (100) of any one of the preceding items, wherein the input station (124) includes a single tube loader (126). (Item 16) The sample preparation device (100) of any one of the preceding items, wherein the input station (124) includes a reader that scans a machine-readable code on a tube containing the sample, the machine-readable code identifying the sample. (Item 17) The sample preparation apparatus (100) of any one of the preceding items, wherein the reagent station (108) includes one or more of a device for storing a dry labeled reagent (132) and a device for storing a liquid labeled reagent (130). (Item 18) one or more probes (114) of said transfer station (112); A cell washer (128); A bead mixer (134); A plate mixer (136); a probe washing station (138); a tracking system including one or more code readers; 2. The sample preparation apparatus (100) of any one of the preceding claims, further comprising one or more of the following: (Item 19) The cell washer (128) comprises: a container including a body defining a cavity and a pocket, the pocket extending radially outward relative to an inner wall surface defining the cavity; an actuation device capable of spinning the container about an axis; Item 19. The sample preparation device (100) according to item 18, comprising: (Item 20) 20. The sample preparation apparatus (100) of claim 18 or 19, wherein the cell washer (128) washes the sample prior to addition of the labeling reagent. (Item 21) 21. The sample preparation apparatus (100) of any one of items 18-20, wherein the cell washer (128) washes the sample following addition of the lysis reagent. (Item 22) The sample preparation device (100) of any one of the preceding claims, wherein the cell concentration estimator (106) is removable from the sample preparation device (100). (Item 23) A method (386) comprising the steps of: Accepting a sample as an input (388); estimating the concentration of leukocytes in said sample (390); and Dispensing (392) a sample of the specimen, the volume of the sample being based on an estimated concentration of white blood cells in the sample; adding one or more labeling reagents to the sample (394); adding one or more lysis reagents to the sample (396); providing the sample as an output following addition of the one or more labeling reagents and the one or more lysis reagents to the sample (398); A method comprising: (Item 24) receiving an identifier for the sample; receiving a panel to be performed on said sample; The method according to Item 23 (386), further comprising: (Item 25) and retrieving a set of predefined rules associated with processing the sample based on the received panel, the set of predefined rules comprising: Whether the concentration of leukocytes in said sample is to be estimated; The lowest concentration of white blood cells, The highest concentration of white blood cells, a volume of said sample to be dispensed for one or more ranges of concentrations of leukocytes between said minimum concentration and said maximum concentration; Should the sample be diluted? 25. The method according to claim 24, comprising one or more of the steps of: (Item 26) 26. The method (386) of claim 25, further comprising determining to process (328) the sample when the estimated white blood cell concentration is within a range for processing the sample defined by the minimum concentration and the maximum concentration. (Item 27) determining that the sample should be diluted based on the set of predefined rules; Diluting the sample (336) The method according to item 25 or 26, further comprising: (Item 28) Estimating the concentration of white blood cells in the sample (390) capturing (460) a plurality of images as the lysed and diluted aliquot of the sample is advanced through a flow cell; determining (462) a total number of white blood cells in the plurality of images based on the number of white blood cells counted in each image; determining (464) a total volume of the subdivisions within the plurality of images; estimating a concentration of white blood cells based on the total number of white blood cells and the total volume of the aliquots in the plurality of images (466); 28. The method according to any one of Items 23 to 27, comprising the steps of: (Item 29) Estimating the concentration of white blood cells in the sample (390) suspending (602) an aliquot of the sample in an electrolyte solution; passing the suspended aliquot through an aperture through which a current flows (604); detecting (606) a change in electrical resistance across the aperture as the suspended aliquot flows through the aperture; estimating (608) a concentration of white blood cells in the sample based on the detected change; 28. The method according to any one of Items 23 to 27, comprising the steps of: (Item 30) Estimating the concentration of white blood cells in the sample (390) staining an aliquot of the sample with an antibody marker that targets leukocytes (692); analyzing the stained aliquots over a period of time using a flow cytometer (656); estimating (658) a concentration of leukocytes in the sample based on the number of leukocytes counted within the predetermined time period and the flow rate of the flow cytometer; 28. The method according to any one of Items 23 to 27, comprising the steps of: (Item 31) If the flow rate of the flow cytometer is not known, adding (654) a plurality of counting beads to the stained aliquot prior to analyzing the stained aliquot; estimating (658) a concentration of white blood cells in the sample based on the number of white blood cells counted and the ratio of the number of counting beads counted to the number of white blood cells; 31. The method of claim 30, further comprising: (Item 32) 32. The method (386) of any one of items 23-31, further comprising cell washing (228) of the sample prior to adding the one or more labeling reagents. (Item 33) 33. The method (386) of any one of items 23-32, further comprising cell washing (240) of the sample following the addition of the one or more lysis reagents. (Item 34) Following addition of the one or more labeling reagents, incubating the sample for a predetermined period of time (234); incubating (238) the sample for a predetermined period of time following the addition of the one or more lytic reagents; 34. The method according to any one of items 23-33, further comprising one or more of the following steps (386): (Item 35) A sample preparation and analysis instrument (150), comprising: an input station (124) for receiving a sample; a cell concentration estimator (106) for estimating a concentration of white blood cells in the sample; a transfer station (112) configured to dispense a sample of the specimen, the volume of the sample being based on an estimated concentration of white blood cells in the specimen; a reagent station (108) containing a labeling reagent to be added to the sample; a lysis station (110) containing a lysis reagent that is added to the sample; a sample analyzer (152) for performing an analysis on the sample following addition of the labeling reagent and the lysis reagent to the sample; an output station (140) for providing the results of said analysis; A sample preparation and analysis instrument (150). (Item 36) Item 36. The sample preparation and analysis instrument (150) according to item 35, wherein the sample analyzer (152) is a flow cytometer. (Item 37) 37. The sample preparation and analysis instrument (150) of claim 35 or 36, wherein the transfer station (112) comprises one or more probes (114) for transferring one or more of the sample, the labeling reagent, the lysis reagent, and the sample within the sample preparation and analysis instrument (150). [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 depicts an exemplary sample preparation device.

[0008] [Diagram 2] FIG. 2 is a block diagram of an exemplary sample preparation instrument.

[0009] [Diagram 3] FIG. 3 is a block diagram of the basic hardware components of an exemplary sample preparation instrument.

[0010] [Figure 4] FIG. 4 is a block diagram of additional hardware components of an exemplary sample preparation instrument.

[0011] [Diagram 5] FIG. 5 depicts the hardware components of the exemplary sample preparation instrument illustrated in FIGS.

[0012] [Figure 6] FIG. 6 is a block diagram of the basic hardware components of an exemplary sample preparation and analysis instrument.

[0013] [Figure 7] FIG. 7 is an exemplary computing device.

[0014] [Figure 8] FIG. 8 diagrammatically illustrates an exemplary sample preparation process for a specimen.

[0015] [Figure 9] FIG. 9 diagrammatically illustrates another exemplary sample preparation process for a specimen.

[0016] [Figure 10] FIG. 10 diagrammatically illustrates a further exemplary sample preparation process for a specimen.

[0017] [Figure 11] FIG. 11 diagrammatically illustrates a still further exemplary sample preparation process for a specimen.

[0018] [Figure 12] FIG. 12 is a process flow diagram illustrating a method for preparing a specimen sample for analysis.

[0019] [Figure 13] FIG. 13 is an exemplary sample loading user interface.

[0020] [Figure 14] FIG. 14 is an exemplary user interface for receiving a set of predefined rules for preparing a sample for analysis.

[0021] [Figure 15] FIG. 15 is a flow chart illustrating a method for preparing a sample of a specimen for analysis.

[0022] [Figure 16] FIG. 16 illustrates the hardware components of an exemplary cell concentration estimator.

[0023] [Figure 17] FIG. 17 is an exemplary mixing chamber of the cell concentration estimator.

[0024] [Figure 18] FIG. 18 is an exemplary counting chamber and optical system of a cell concentration estimator.

[0025] [Figure 19] FIG. 19 is a flow chart illustrating an optical method for estimating white blood cell concentration in a sample.

[0026] [Figure 20] FIG. 20 illustrates diagrammatically an optical method for estimating white blood cell concentration in a sample.

[0027] [Figure 21] FIG. 21 illustrates an image of an objective micrometer used to determine the total volume within multiple captured images.

[0028] [Figure 22] FIG. 22 illustrates the image analysis performed to determine the number of white blood cells in the image.

[0029] [Figure 23]FIG. 23 illustrates a graphical representation of the white blood cell count obtained during image analysis.

[0030] [Figure 24] FIG. 24 is a flow chart illustrating an electrical resistance method for estimating white blood cell concentration in a sample.

[0031] [Diagram 25] FIG. 25 is a flow chart illustrating a flow cytometry method for estimating leukocyte concentration in a sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description Various embodiments are described in detail with reference to the drawings, in which like reference numerals represent like parts and assemblies throughout the several views. Reference to various embodiments does not limit the scope of the claims appended hereto. Additionally, any examples described herein are not intended to be limiting, but merely describe some of the many possible embodiments of the appended claims.

[0033] FIG. 1 depicts an exemplary sample preparation instrument 100. The sample preparation instrument 100 can be used to prepare a sample of a specimen for subsequent analysis, such as for a flow cytometry panel. Exemplary samples can include whole blood, bone marrow, dissociated tissue, peripheral mononuclear cells, biopsies, cerebrospinal fluid, and other single cell suspensions. As an example, a laboratory providing flow cytometry services receives a whole blood sample to be prepared for a leukemia or lymphoma panel. Components of blood include plasma, red blood cells, white blood cells, and platelets. Leukemia or lymphoma affects white blood cells (e.g., causing an abnormally high white blood cell count), and thus, a subset of white blood cells is the component of blood that is targeted for analysis in a leukemia or lymphoma panel. To prepare a sample of blood for a panel, a labeling reagent, such as an antibody reagent, is used to stain the blood, which is attached to the white blood cells. For example, about 100 μL of blood is stained with about 20 μL of antibody reagent and incubated for about 10-20 minutes. The antibody reagent can include a fluorescent dye that allows for analysis of white blood cells, for example, by flow cytometry. In some examples, a cell wash is performed on the blood prior to adding the antibody reagent to remove plasma and associated proteins that interfere with staining (e.g., leaving primarily red and white blood cells). A lytic reagent is then added to the sample and incubated to destroy the red blood cells. In some examples, another wash can be performed to slow down the lytic activity, avoid destruction of white blood cells, and / or remove red blood cell debris. The sample can then be analyzed. For example, a leukemia or lymphoma panel can be performed by a flow cytometer.

[0034] The volume of antibody reagent added to the blood is based on the white blood cell concentration in the blood. For example, 20 μL of antibody reagent is added for a normal white blood cell concentration of about 4,000 cells / μL to about 10,000 cells / μL in the blood. However, if the patient has leukemia causing an abnormally high white blood cell concentration of 50,000 cells / μL, the 20 μL of antibody reagent will not be effective in targeting the white blood cells because it was dosed or administered for the lower normal range of white blood cell concentration. To account for this, the same volume of reagent may be added to a smaller volume of blood with a lower white blood cell count. For example, instead of staining about 100 μL of blood with about 20 μL of antibody, only 50 μL of blood is stained with about 20 μL of antibody reagent. Conversely, if a patient has an abnormally low white blood cell concentration of about 1,000 cells / μL, the antibody reagent, having been titrated or administered for a higher normal range of white blood cell concentration, would be excessive and interfere with the analysis. To account for this, the same volume of reagent may be added to a larger volume of blood with a higher white blood cell count. Thus, it is important that at least an estimate of the white blood cell concentration in the sample is obtained before the sample is prepared for analysis to ascertain whether the white blood cell concentration is within the appropriate range for the volume of antibody reagent, and if not, adjust the volume of blood added to the antibody reagent during preparation.

[0035] Currently, to obtain white blood cell concentration, the sample is first sent to a separate laboratory, such as a hematology laboratory, prior to preparing the sample for a flow cytometry panel. Depending on how busy the hematology laboratory is and / or how the hematology laboratory provides the results, it may take time to receive the concentration value. Alternatively, the concentration determination can be kept in the laboratory if the laboratory has a separate hematology analyzer or uses a microscopy technique to determine the concentration. However, having a separate hematology analyzer can be costly as another instrument is maintained that takes up valuable bench space, and using microscopy is time-consuming and labor-intensive. In addition, in each of the methods described above, manual adjustment of the volume of the specimen for sample preparation is required based on the obtained white blood cell concentration, which is time-consuming and prone to error.

[0036] Aspects of the present disclosure provide a more efficient sample preparation process and overcome the above-discussed disadvantages of current systems and methods. For example, the sample preparation instrument 100, described in detail in the following figures, includes a cell concentration estimator integrated therein that estimates the white blood cell concentration in a sample received for preparation. The sample volume of the sample to be prepared (e.g., also referred to as the "sample volume of the sample") is determined based on the estimated white blood cell concentration. The sample is then automatically prepared or processed with the determined sample volume of the sample and one or more other reagents, such as a labeling reagent, a lysis reagent, and / or a buffer. By integrating the cell concentration estimator into the sample preparation instrument 100, white blood cell concentration estimation is performed in a timely manner and without the need to maintain and make space for additional instruments. In addition, the sample volume of the sample is automatically adjusted within the sample preparation instrument 100, avoiding unnecessary errors caused by manual adjustment.

[0037] 2 is a block diagram of an exemplary sample preparation instrument 100. The sample preparation instrument 100 accepts as input 102 a sample, the sample of which is to be prepared for analysis. In some embodiments, the analysis targets a specific component of the sample. The sample preparation instrument 100 can estimate the concentration of the targeted component in the sample, prepare the sample, and thus provide the prepared sample as output 104. For example, the sample volume of the sample can be determined based on the estimated concentration and a set of predefined rules associated with the type of analysis for which the sample is being prepared. Reagents can then be added to the sample volume of the sample, which can include, for example, a labeling reagent to target components of the sample for analysis and a lysis reagent to remove interfering components of the sample.

[0038] 3 is a block diagram of the basic hardware components of an exemplary sample preparation instrument 100. The sample preparation instrument 100 includes at least a cell concentration estimator 106, a reagent station 108, a reaction station 109, and a lysis station 110.

[0039] The sample preparation instrument 100 accepts as input 102 a sample to be prepared for analysis. As an example, the sample is whole blood to be prepared for a leukemia or lymphoma panel performed by a flow cytometer. The leukemia or lymphoma panel can be performed to analyze subsets of white blood cells in the blood (e.g., the subsets of white blood cells can be targeted components). The cell concentration estimator 106 is a device or apparatus that can receive an aliquot of the sample and estimate the white blood cell concentration in the sample based on the estimated white blood cell concentration in the aliquot. The cell concentration estimator 106 can employ one or more of optical methods, electrical resistance methods, flow cytometry methods, and other similar methods known to those skilled in the art for estimating white blood cell concentration. The configuration of the cell concentration estimator 106 can depend on the type of method employed. Based on the estimated white blood cell concentration in the sample, a sample volume of the sample (e.g., the volume of the sample to be included in the sample prepared for analysis) is determined.

[0040] The reagent station 108, described in more detail with reference to FIG. 4 below, can include multiple labeling reagents, one or more of which are added to a determined sample volume of the sample. The one or more labeling reagents added can target specific components in the sample volume of the sample. For example, continuing with the above example, the reagent can be an antibody reagent with a fluorescent dye that attaches to a subset of white blood cells when performing a leukemia or lymphoma panel, allowing analysis of the subset of white blood cells by the flow cytometer.

[0041] In some embodiments, the labeled reagent and the sample volume of the sample are contacted at the reaction station 109. For example, the labeled reagent can be added to the sample volume of the sample in a well of a reaction plate located in the reaction station 109. As described in more detail with reference to FIG. 4 below, the reaction station 109 can also include a plate mixer that holds the reaction plate. In some embodiments, the reaction station 109 is a separate component of the sample preparation instrument 100. In other embodiments, the reaction station 109 can be integrated with the reagent station 108.

[0042] The lysis station 110, described in more detail with reference to FIG. 4 below, can include multiple lysis reagents, one or more of which are added to a determined sample volume of the sample. In some examples, the added lysis reagent can destroy red blood cells and prevent them from interfering with the analysis. As with the labeling reagent, the lysis reagent and the sample volume of the sample can come into contact in the reaction station 109. For example, the labeling reagent can be added to the sample volume of the sample in a well of a reaction plate located in the reaction station 109.

[0043] Following addition of the labeling reagent and lysing reagent, a sample comprising a sample volume of the specimen is provided as an output 104 of the sample preparation instrument 100 along with the added labeling reagent and lysing reagent. The sample can then be analyzed by a sample analyzer, such as a flow cytometer. In some embodiments, the sample analyzer is a separate component. In other embodiments, the sample analyzer can be integrated with the sample preparation instrument, as shown in FIG. 6.

[0044] Figure 4 is a block diagram of, and Figure 5 depicts, additional hardware components of, an exemplary sample preparation instrument 100. In addition to the cell concentration estimator 106, reagent station 108, reaction station 109, and lysis station 110 illustrated in Figure 3, the sample preparation instrument 100 can further include a transfer station 112 with one or more probes 114, a computing device 116 with memory 118 and at least one processing device 120, a touch display 122, an input station 124, a single tube loader 126, a cell washer 128, a liquid reagent housing 130 and a dry reagent carousel 132 in the reagent station 108, a bead mixer 134, a plate mixer 136 in the reaction station 109, a probe wash station 138, and an output station 140.

[0045] The probe 114 of the transfer station 112 can aspirate, transport, and dispense various materials between components of the sample preparation instrument 100. The materials can include samples, labeling reagents, lysis reagents, diluent reagents, and buffers, among other examples. The probe 114 can puncture capped or sealed tubes, vials, cartridges, bottles, or other similar containers and aspirate or be inserted into the materials in the open-top tubes, vials, cartridges, bottles, or other similar open-top containers.

[0046] The computing device 116, which is described in more detail in FIG. 7, is the controller and information processor of the sample preparation instrument 100. In some embodiments, the computing device 116 can store previously authored panels received from a remote computing device. For example, a user, such as a laboratory technician or analyst, can utilize a panel authoring and management application running on the remote computing device to author panels for different types of samples and different types of analyses for execution by the sample preparation instrument 100. The authored panels can then be transmitted to the sample preparation instrument 100 via a network or wired connection or a physical memory device, such as a USB drive. In other embodiments, the computing device 116 can run a panel authoring and management application to enable a user to author panels using the sample preparation instrument 100 (e.g., via a user interface provided via the touch display 122), and the authored panels are then stored by the computing device 116. Each panel can define a particular workflow to be performed when preparing samples of the specimens for the panel. For example, a panel may include a set of rules, hereafter referred to as a predefined set of rules, that defines a particular workflow associated with sample preparation.

[0047] The computing device 116 can provide the authored panels for display through a sample loading user interface, shown and described with reference to FIG. 13, and allow a user to select one or more of the panels to be paired with a received sample. In some embodiments, the sample loading user interface can be provided through the touch display 122 of the sample preparation instrument 100. The selected one or more panels can then be used to manage the operations of the sample preparation instrument 100 during sample preparation. For example, a set of predefined rules associated with each panel can provide, among other embodiments, the order of operations, the types and volumes of samples and reagents involved in each operation, the time associated with each operation, and the components of the sample preparation instrument 100 involved in each operation. Based on the one or more panels selected, the computing device 116 can generate signals for transmission to individual components of the sample preparation instrument 100 providing instructions for performing the operations.

[0048] Additionally, the computing device 116 can operate in conjunction with the cell concentration estimator 106 to determine the white blood cell concentration in the sample. Based on a set of predefined rules associated with one or more selected panels, the computing device 116 can further determine whether to process the sample, the sample volume of the sample based on the white blood cell concentration estimate, and whether the sample should be diluted, among other decisions discussed herein.

[0049] The touch display 122 provides a user interface that allows a user to interact with the sample preparation instrument 100. For example, one exemplary user interface is the sample loading user interface described above, through which a user can select one or more panels for which a sample volume of a sample is to be prepared. An exemplary user interface for selecting a panel is illustrated and described in more detail with reference to FIG. 13. In some examples, once the white blood cell concentration in a sample is estimated, the user interface can display the range in which the estimated white blood cell concentration falls, and the determined sample volume of the sample that will be prepared by the sample preparation instrument 100 based on the estimated white blood cell concentration. In addition, as the sample preparation instrument 100 processes the sample, the touch display 122 can display the status of the sample preparation instrument 100 components, including the amount of reagent or other similar solution remaining, and whether any errors have occurred throughout the sample preparation process. In some examples, the user interface is associated with a panel authoring and management application running on the computing device 116.

[0050] In some embodiments, the input station 124 accepts an input cassette that is inserted into the sample preparation instrument 100 by a user. The input cassette can hold one or more capped tubes containing a sample. In some embodiments, each tube contains a different sample (e.g., a sample from a different patient). The input station 124 can accept different types of cassettes corresponding to different types of tubes. The input station 124 includes a reader or scanner capable of reading machine-readable codes such as bar codes, QR codes, or radio frequency identification (RFID) tags, among other similar embodiments. In some embodiments, each tube has an associated code that is scanned by the reader or scanner to identify the sample in the tube.

[0051] The input station 124 may include a rocking mechanism for rocking the input cassette to maintain sample homogeneity prior to preparation. The input station 124 may further include a magnet-based mechanism to move the input cassette to a specific position such that the probe 114 of the transfer station 112 may puncture a given capped tube held by the input cassette and aspirate the sample from the tube. Once the sample of each sample in the tube of the input cassette has been processed or is otherwise designated as not to be processed, the input station 124 may provide the input cassette as an output cassette for removal from the sample preparation instrument by a user. In some embodiments, an opening on the front panel of the sample preparation instrument 100 provides continuous access to the input station 124. For example, a user may insert a new input cassette and / or remove an output cassette through the opening at any time, even when the sample preparation instrument 100 is in operation.

[0052] Optionally, the sample preparation instrument 100 also includes a single tube loader 126. The single tube loader 126 can be a component of the input station 124 or a separate component. The single tube loader 126 allows for the insertion of open-top tubes into the sample preparation instrument 100 and positioning such that the probe 114 of the transfer station 112 can aspirate a sample of the sample from the tube. In some embodiments, open-top tubes are utilized when pre-processing is performed manually by a user prior to insertion into the sample preparation instrument 100. Due to the tube having an open top, the single tube loader does not rock the tube to avoid sample spillage. Similar to capped tubes, open-top tubes can also include a machine-readable code that is read or scanned to obtain a sample identifier that identifies the sample. Alternatively, the sample identifier can be manually punched in when utilizing the single tube loader 126. In a further embodiment, the open-top tubes can be received by the input station 124 into an input cassette specifically designed for the open-top tubes, and machine-readable code on the cassette would instruct the input station 124 to disengage the rocking mechanism and prevent the input cassette with the open-top tubes from being rocked.

[0053] Based on the sample identifier, the computing device 116 can retrieve a set of predefined rules for the type of analysis. For example, the set of predefined rules can include whether the white blood cell concentration in the sample should be estimated by the sample preparation instrument 100. If the white blood cell concentration in the sample should be estimated by the sample preparation instrument 100, the probe 114 of the transfer station 112 can transfer an aliquot of the sample from the input station 124 or a tube in the single tube loader 126 to the cell concentration estimator 106. The cell concentration estimator 106, in conjunction with the computing device 116, can estimate the white blood cell concentration in the sample by employing one or more of optical methods, electrical resistance methods, flow cytometry methods, and other similar methods known to those skilled in the art for estimating white blood cell concentration. The aliquot, from which the white blood cell concentration is estimated, can then be discarded as waste.

[0054] Based on the estimated white blood cell concentration in the sample and the set of predefined rules, the computing device 116 can determine a sample volume of the sample to be prepared. For example, the set of predefined rules can also include a sample volume of the sample corresponding to a minimum white blood cell concentration, a maximum white blood cell concentration, and one or more ranges of white blood cell concentrations that fall between the minimum and maximum concentrations, respectively. Thus, the computing device 116 can determine a sample volume of the sample that corresponds to the range in which the estimated white blood cell concentration falls and generate a signal for transmission to the probe 114 of the transfer station 112. Based on the signal, the probe 114 can transfer the determined sample volume of the sample from a tube in the input station 124 or the single tube loader 126 and dispense into a well of a reaction plate or other similar container. In some examples, the reaction plate is a 48-well plate, with each well capable of holding approximately 5 milliliters (mL). In some examples, the reaction plate or other similar container can be in the reaction station 109. In other examples, the reaction plate or other similar container is in the reagent station 108.

[0055] Optionally, the probe 114 can first transfer a determined sample volume of the sample to the cell washer 128 to wash the sample volume of the sample prior to dispensing into a well of a reaction plate or other similar container. In yet other examples, a larger volume of the sample is washed prior to determining the sample volume, and depending on the determination, the probe 114 can then transfer a determined sample volume of the washed sample from the cell washer 128 to a well of a reaction plate. For example, when the sample is whole blood, the blood can contain red blood cells, white blood cells, platelets, plasma, and associated proteins. The cell washer 128 can perform cell washing to remove the plasma and associated proteins. The cell washer 128 can include a container that holds a sample volume of the sample and an actuation device capable of spinning the container about an axis. In some examples, the container includes a body that defines a cavity and a pocket, and the pocket extends radially outward relative to an inner wall surface that defines the cavity. As a result of this configuration, as the container is spun about its axis, the heavier blood cells in the sample diffuse toward the walls and are collected in the cavity defined by the pocket. In addition, as the container spins, a tube located in the body of the container aspirates the contents of the container that are not in the cavity (e.g., plasma and associated proteins). The actuation device can stop the spinning of the container about its axis to add buffer into the container and repeat the spinning and aspirating steps described above. These steps may be repeated multiple times, as desired. Additional details regarding the cell washer 128 and the process of cell washing are described in PCT Application No. PCT / US2017 / 066312, filed December 14, 2017, and entitled "CELL WASHING DEVICE AND METHOD," which is incorporated herein by reference in its entirety.

[0056] The liquid reagent housing 130 of the reagent station 108 stores a plurality of liquid labeled reagents in vials that are positioned in a carousel in an enclosed housing. In some embodiments, the enclosed housing can maintain a temperature of about 2° C. to about 8° C. to refrigerate the liquid labeled reagents. Different types of liquid labeled reagents can be stored. In addition, different vial types containing liquid labeled reagents can be positioned in the carousel. For example, vials with flat bottoms that need to be positioned at an angle, and vials with v-shaped bottoms that need to be positioned vertically.

[0057] In some examples, the vials have puncturable caps and the lid of the liquid reagent housing 130 (e.g., the lid of an enclosed housing) comprises two openings that are aligned with rows on the carousel such that the probe 114 of the transfer station 112 can enter the liquid reagent housing 130 through one of the openings and puncture a vial positioned in the carousel aligned with the opening. The probe 114 can transfer the liquid labeled reagent from the vial to a well in a reaction plate that comprises a sample volume of the sample.

[0058] The dry reagent carousel 132 of the reagent station 108 stores multiple cartridges, each with a dry labeled reagent sealed in an aluminum heat seal. Different dry labeled reagents can be stored in the cartridges. As an example, the sample preparation instrument 100 can include at least two dry reagent carousels 132, each with six or more cartridges per carousel, with 6-20 wells per cartridge. The probe 114 of the transfer station 112 pierces through the impermeable opaque seal of the cartridge and reconstitutes the dry reagent contained therein with a buffer. Once the dry reagent is reconstituted, the reconstituted reagent can be transferred by the probe 114 from the cartridge to a well in a reaction plate that comprises a sample volume of the sample. Alternatively, the sample volume of the sample can be transferred directly into a cartridge containing the dry labeled reagent, and the sample can be mixed with the dry labeled reagent inside the cartridge. The labeled sample can then be transferred to a well in a reaction plate for additional processing.

[0059] The lysis station 110 includes a carousel that holds vials with various types of lysis reagents. In some embodiments, the carousel consists of two half-moon adapters, each adapted to hold vials of variable sizes. The tops are not puncturable, so the user must remove the tops of the vials before placing the vials on one of the adapters in the lysis station 110. The probes 114 of the transfer station 112 can transfer the lysis reagents from the vials to wells in a reaction plate that contain the sample volumes of the specimens to which the labeled reagents have been added for incubation. In some embodiments, when multiple lysis reagents are required, incubations can be performed between the addition of each lysis reagent. In further embodiments, the cell washer 128 can perform another wash on the samples following the addition of the lysis reagent and the corresponding incubation time to slow down the lysis activity.

[0060] In some embodiments, the liquid reagent housing 130, the dry reagent carousel 132, and the lysis station 110 each include a reader or scanner to read machine-readable codes on the vials, cartridges, and bottles and adapters, respectively, and maintain an inventory (e.g., to determine when a particular cartridge has been reconstituted and aspirated, when a particular vial is low on a corresponding liquid labeled reagent, or when a bottle is low on lysis reagent). If one or more of the vials, cartridges, and bottles need to be replaced, a user can access each of the liquid reagent housing 130, the dry reagent carousel 132, and the lysis station 110 by opening the entire front panel of the instrument (e.g., the front panel with the access opening for the input station 124). For example, the front panel may slide upward. In some embodiments, the user must wait until operation has stopped to open the entire front panel. For example, a user can request the sample preparation instrument 100 to pause operation, the user can open the front panel and access and replace vials, cartridges, and / or bottles, and then the user can close the front panel and the sample preparation instrument 100 will resume operation.

[0061] In some examples, readers or scanners may be included among other hardware components, in addition to the liquid reagent housing 130, dry reagent carousel 132, and lysing station 110, to track the sample preparation process. Collectively, these readers or scanners may form a tracking system for the sample preparation instrument 100. For example, such tracking may form a chain of custody for a sample, whereby a user may then later precisely determine the components that should interact with the sample and details about the interactions (e.g., the amount and type of reagents added, and the duration of individual incubation periods, whether the specimen and / or sample was washed, etc.).

[0062] The sample preparation instrument 100 may optionally include a bead mixer 134. The bead mixer 134 may maintain the homogeneity of the counting bead suspension contained within the vial. In some sample preparation processes, a known concentration of counting beads may be added to the sample being prepared to provide a reference for determining the concentration of components in the sample. In some embodiments, the counting bead vial includes an evaporation cap to minimize evaporation of the counting bead suspension. The counting bead suspension vial may include a machine readable code that is scanned when utilized during the sample preparation process to facilitate tracking of the sample preparation process.

[0063] In some examples, the reaction station 109, where the labeling reagent and lysis reagent contact the sample volume of the sample (e.g., in the wells of a reaction plate), can include a plate mixer 136. The plate mixer 136 is, for example, an orbital shaker that can hold one or more plates. For example, the plate mixer 136 can hold a reaction plate with wells into which the sample volume of the sample, the labeling reagent, and the lysis reagent are dispensed. In some examples, the plate mixer 136 moves in a circular or elliptical motion to mix the sample volume of the sample and the labeling reagent, and mix the lysis reagent and the sample volume / labeling reagent of the sample during a discrete incubation period.

[0064] The probe washing station 138 washes the probe 114 of the transfer station 112 every time the probe 114 comes into contact with a sample, a labeling reagent, a lysis reagent, or any other type of liquid to prevent any contamination. To wash the probe 114, the probe 114 can be inserted into a well and a buffer can be added to surround the probe 114 within the well.

[0065] After the labeling and lysis reagents are added to the sample volumes of the samples in the wells of the reaction plate and incubated, the cell washer 128 can optionally perform another wash. Following incubation or optional wash, the output station 140 can receive the prepared samples and provide them as output 104. In some embodiments, the output station 140 includes an output carousel with multiple clean tubes, and the probe 114 of the transfer station 112 transfers the prepared samples from one of the wells in the reaction plate or the cell washer 128 to the clean tubes in the output carousel. The user can choose to remove the output carousel from the sample preparation instrument 100 when the output carousel is full or when the output carousel is partially full. For example, a sliding drawer on the left panel of the sample preparation instrument 100 can be utilized by the user to remove one or more tubes with prepared samples at any time, even when the sample preparation instrument 100 is in operation. For example, a user may request access and the sample preparation instrument 100 will then allow the sliding drawer to be accessed. In some cases, there may be a delay between the request and the time that access is granted, depending on operations currently ongoing within the sample preparation instrument 100. In other examples, the output station 140 includes a clean reaction plate and / or clean wells within a reaction plate into which prepared samples are transferred by the probe 114.

[0066] In addition to the hardware components illustrated in Figure 4, Figure 5 further depicts a waste container 142, a diluent reagent container 144, and a condensate collector 146. Waste associated with the sample preparation process, including sample waste from the cell concentration estimator 106, is dispensed into the waste container 142. The diluent reagent can be used at several steps throughout the preparation process, including to reconstitute the cell concentration estimator and dry reagents, among other examples. The condensate collector 146 can collect any condensate resulting from cooling of the labeled reagent (e.g., generated by the liquid reagent housing 130).

[0067] 6 is a block diagram of the basic hardware components of an exemplary sample preparation and analysis instrument 150. The sample preparation and analysis instrument 150 includes similar components as the sample preparation instrument 100, such as the cell concentration estimator 106, the reagent station 108, the reaction station 109, and the lysis station 110, which are described in detail above with respect to FIGS. 3-5. In addition, the sample preparation and analysis instrument 150 includes a sample analyzer 152 that accepts the prepared sample as an input, performs analyses on the prepared sample, and provides sample analysis results as output 154. In some embodiments, the sample analyzer 152 is a flow cytometer.

[0068] 7 is an example computing device 116 that may be used to implement aspects of the present disclosure. The computing device 116 may be used to execute operating systems, application programs, and software modules (including software engines) described herein.

[0069] Computing device 116, in some embodiments, includes at least one processing device 120, such as a central processing unit (CPU). Various processing devices are available from various manufacturers, e.g., Intel or Advanced Micro Devices. In this example, computing device 116 also includes a system memory 118 and a system bus 160 that couples various system components, including the system memory 118, to processing device 120. System bus 160 may be one of any number of types of bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures.

[0070] The system memory 118 includes a read-only memory 162 and a random access memory 164. A basic input / output system 166, containing the basic routines that act to transfer information within the computing device 116, such as during start-up, is typically stored in the read-only memory 162.

[0071] Computing device 116 also includes a secondary storage device 168, in some embodiments, such as a hard disk drive for storing digital data. The secondary storage device 168 is connected to system bus 160 by a secondary storage interface 170. The secondary storage device 168 and its associated computer-readable media provide non-volatile storage of computer-readable instructions (including application programs and program modules), data structures, and other data for computing device 116.

[0072] Although the exemplary environment described herein employs a hard disk drive as a secondary storage device, other types of computer-readable storage media are used in other embodiments. Examples of these other types of computer-readable storage media include flash memory cards, digital video disks, compact disks read-only memory, digital versatile disks read-only memory, random access memory, or read-only memory. Some embodiments include non-transitory media. In addition, such computer-readable storage media can include local storage or cloud-based storage.

[0073] A number of program modules may be stored in the secondary storage device 168 or system memory 118, including an operating system 172, one or more application programs 174, other program modules 176 (such as the software engines described herein), and program data 178. An exemplary application program includes a panel authoring and management application. The computing device 116 may be running an operating system such as Microsoft Windows, Google Chrome, or the like. TM Any suitable operating system may be utilized, such as OS, Apple OS, Unix, or Linux, and variants and any other operating system suitable for the computing device. Other examples may include Microsoft, Google, or Apple operating systems, or any other suitable operating system.

[0074] In some embodiments, a user provides input to the computing device 116 through one or more input devices 180. Examples of input devices 180 include a keyboard 182, a mouse 184, a microphone 186, and a touch sensor 188 (such as a touchpad or a touch-sensitive display). Other embodiments include other input devices 180. The input devices are often connected to the processing device 120 through an input / output interface 190, which is coupled to the system bus 160. These input devices 180 can be connected by any number of input / output interfaces, such as a parallel port, a serial port, a game port, or a universal serial bus. Wireless communication between the input devices and the input / output interface 190 is also possible, including infrared, Bluetooth® wireless technology, IEEE 802.11a / b / g / n, cellular, ultra-wideband (UWB), ZigBee®, LoRa, or other radio frequency communication systems in some possible embodiments.

[0075] In the exemplary embodiment, a display device 192, such as a monitor, liquid crystal display device, projector, or touch-sensitive display device, is also connected to the system bus 160 via an interface, such as a video adapter 194. In addition to the display device 192, the computing device 116 may include various other peripheral devices (not shown), such as speakers or printers.

[0076] When used in a local area networking environment or a wide area networking environment (such as the Internet), the computing device 116 is typically connected to the network through a network interface 196, such as an Ethernet interface. Other possible embodiments use other communication devices. For example, some embodiments of the computing device 116 include a modem to communicate across the network.

[0077] Computing device 116 typically includes at least some form of computer-readable media. Computer-readable media includes any available media that can be accessed by computing device 116. By way of example, computer-readable media include computer-readable storage media and computer-readable communication media.

[0078] Computer-readable storage media include volatile and nonvolatile, removable and non-removable media implemented in any device configured to store information, such as computer-readable instructions, data structures, program modules, or other data. Computer-readable storage media include, but are not limited to, random access memory, read-only memory, electrically erasable programmable read-only memory, flash memory or other memory technology, compact disc read-only memory, digital versatile disks or other optical storage devices, magnetic cassettes, magnetic tapes, magnetic disk storage devices or other magnetic storage devices, or any other medium that can be used to store the desired information and that can be accessed by the computing device 116.

[0079] Computer-readable communication media typically embodied computer-readable instructions, data structures, program modules, or other data in a modulated data signal, such as a carrier wave or other transport mechanism, and includes any information delivery media. The term "modulated data signal" refers to a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, computer-readable communication media includes wired media, such as a wired network or direct-wired connection, and wireless media, such as acoustic, radio frequency, infrared, and other wireless media. Combinations of any of the above are also included within the scope of computer-readable media.

[0080] The computing device 116 illustrated in FIG. 7 is also an example of a programmable electronic device, which may include one or more such computing devices, and when multiple computing devices are included, such computing devices may be coupled together using a suitable data communications network so as to collectively perform various functions, methods, or operations disclosed herein.

[0081] FIG. 8 diagrammatically illustrates an exemplary sample preparation process 200 for a sample. The sample preparation process 200 can be performed by the sample preparation instrument 100. Prior to the sample preparation instrument 100 performing the sample preparation process 200, a panel (e.g., a workflow defined for preparing samples of different types of samples for different types of analyses) is generated by a user through a panel authoring and management application running on a remote computing device, such as a laptop, tablet, or desktop, among other similar devices. The panel can then be transmitted to the sample preparation instrument 100 through a wired or wireless connection, such as via a USB connector, a local area network (LAN), or over the Internet. In some examples, the defined workflows of the panel each include a set of predefined rules associated with the preparation of the sample, including processing requirements such as sample volume, dilution requirements, labeling reagent volume, lysis reagent volume, incubation period, and other similar rules for the sample based on white blood cell concentration. The panel can be stored in the memory 118 of the computing device 116 of the sample preparation instrument 100.

[0082] The sample preparation process 200 can begin at operation 202, where a tube with at least one sample is received. In some embodiments, the tube is a capped tube that is received in an input cassette at the input station 124, which is then rocked via a rocking mechanism of the input station 124 to maintain a suspension of the sample within the tube. The input cassette can hold a plurality of capped tubes, where one or more of the capped tubes comprise different samples (e.g., each capped tube can include a sample associated with a different person or patient). In other embodiments, the tube is an open-top tube with a sample that is not rocked, either received in an input cassette at the input station 124 or received in the single tube loader 126. In some embodiments, the open-top tube is received when a user manually pre-processes a sample. In any of the above embodiments, the tube with the sample can include a machine-readable code that can be read by a scanner or reader of the input station 124 or the single tube loader 126. The machine readable code can identify the sample. Alternatively, the sample identifier can be entered manually, for example, via the touch display 122 user interface.

[0083] In operation 204, a panel selection can be received through a user interface of the touch display 122. For example, the user interface can display an identification (e.g., a sample identifier) ​​of the sample in the tube and one or more of the panels that can be selected for pairing with the sample. Once one or more of the displayed panels are selected for the sample in the tube, the sample preparation instrument 100 can process the sample of the sample according to a workflow defined for the panel, as described in 206-216. In some embodiments, the workflow for the panel, including a set of predefined rules, is retrieved from the memory 118 in response to receiving the panel selection in operation 204.

[0084] In operation 206, a sample volume of the sample is determined based on the white blood cell concentration in the sample and a set of predefined rules. For example, the set of predefined rules may include one or more ranges of white blood cell concentration values ​​and a corresponding sample volume of the sample for each range. The sample volume that corresponds to the range within which the white blood cell concentration in the sample falls is the determined sample volume of the sample.

[0085] In some examples, the cell concentration estimator 106 estimates the white blood cell concentration in the sample based on the estimated white blood cell concentration in the aliquot of the sample. For example, the probe 114 of the transfer station 112 punctures a capped tube containing the sample at the input station 124 or inserts into an open-top tube containing the sample at the input station 124 or single tube loader 126. The aliquot of the sample is aspirated by the probe 114 and transferred to the cell concentration estimator 106. The cell concentration estimator 106 can employ optical methods, electrical resistance methods, flow cytometry methods, or other similar methods known to those skilled in the art to estimate the white blood cell concentration in the aliquot, which collectively represents the estimated white blood cell concentration in the sample. The sample aliquot is then discharged from the cell concentration estimator 106 as waste. The processing device 120 can automatically determine the sample volume of the sample based on the estimated white blood cell concentration and a set of predefined rules.

[0086] In other examples, the white blood cell concentration in the sample used for the sample volume determination is received from a source external to the sample preparation instrument 100 and manually entered via a user interface of the touch display 122, or received via a laboratory information system (LIS) accessible by the sample preparation instrument 100. Alternatively, the sample volume determination is made by a user based on knowledge of the received white blood cell concentration and a set of predefined rules, and the sample volume can be manually entered via the user interface of the touch display 122.

[0087] In operation 208, one or more labeled reagents are added into the wells of a reaction plate or other similar container. The volume and type of labeled reagent to be added can be defined by a selected panel (e.g., within a set of predefined rules). The labeled reagent can target a particular component of the sample and facilitate analysis of that component. In some embodiments, the reaction plate is a component of the reagent station 108, in which the labeled reagent is stored. In other embodiments, the reaction plate is a component of a reaction station 109, which is separate from the reagent station 108.

[0088] In some embodiments, the labeled reagent is a liquid labeled reagent stored in a vial that is positioned in a row on a carousel. The carousel can be enclosed in a liquid reagent housing 130 that is maintained at a controlled temperature. To add the liquid labeled reagent to wells in a reaction plate, the carousel can be positioned such that a vial with the appropriate liquid labeled reagent is directly beneath an opening in the lid of the liquid reagent housing 130. A probe 114 of the transfer station 112 can enter the liquid reagent housing 130 through the opening, puncture the vial, aspirate the liquid labeled reagent from the vial, and dispense the liquid labeled reagent into the wells in the reaction plate.

[0089] Additionally and / or alternatively, the labeled reagent can be a dry labeled reagent stored in a cartridge of the dry reagent carousel 132 that is reconstituted into a liquid prior to addition to the wells in the reaction plate. For example, to add a liquid labeled reagent to a well, the probe 114 of the transfer station 112 can pierce through an impermeable opaque seal of the cartridge and reconstitute the dry reagent contained therein with a buffer. Once the dry reagent is reconstituted, the reconstituted reagent can be aspirated from the cartridge by the probe 114 of the transfer station 112 and dispensed into the wells in the reaction plate.

[0090] In some examples, a machine-readable code on a vial or cartridge from which liquid or reconstituted dried labeled reagent was aspirated can be scanned to facilitate tracking of the current sample preparation process and to provide inventory-related information (e.g., the amount of volume of labeled reagent remaining following aspiration).

[0091] Once the labeling reagent has been added to the wells in the reaction plate, in operation 208, the sample volume of the sample determined in operation 206 is added to the wells in the reaction plate in operation 210. For example, the probe 114 of the transfer station 112 can aspirate the sample volume of the sample from the input station 124 or a tube in the single tube loader 126. As an example, a 400 μL sample volume of the sample can be dispensed equally into four wells, where the labeling agent has already been dispensed into the wells.

[0092] In operation 212, the sample volume of the sample and the labeling reagent can be incubated in the wells of the reaction plate. The incubation can occur for a predetermined period of time defined, for example, by a panel. The period can be based on the type of labeling reagent. In some embodiments, the reaction plate is positioned on a plate mixer 136, which can move in a circular or elliptical motion to mix the sample volume of the sample and the labeling reagent for a portion of the incubation period.

[0093] In operation 214, one or more lysis reagents or buffers stored in the lysis station 110 are added into the wells of the reaction plate (e.g., into the wells with the sample volume of the sample and the labeling reagent). The volume and type of lysis reagent or buffer to be added can be defined by a panel (e.g., in a set of predefined rules). The lysis reagent can destroy certain components of the sample that may interfere with the analysis. For example, when the sample is whole blood and the analysis is a leukemia or lymphoma panel that focuses on a subset of white blood cells, a lysis reagent is added to destroy red blood cells that may interfere with the analysis of the white blood cell subsets during the panel.

[0094] To add lysis reagent into the wells of the reaction plate, the probe 114 of the transfer station 112 can enter an open bottle with the appropriate lysis reagent held by an adapter of the lysis station 110 and aspirate the lysis reagent. The lysis reagent is then dispensed into the wells of the reaction plate with the sample volume of the sample and the labeled reagent. In some examples, the bottle from which the lysis agent is aspirated and / or a machine-readable code on the adapter holding the bottle can be scanned to facilitate tracking of the current sample preparation process and provide inventory related information (e.g., the amount of volume of lysis reagent remaining in the bottle following aspiration). In other examples, a buffer is added rather than a lysis reagent based on the sample type and panel selected.

[0095] In operation 216, the sample from each well of the reaction plate, including the sample volume of the specimen, the labeling reagent, and the lysis reagent or buffer, can be dispensed for output. In some embodiments, the sample from each well is aspirated by the probe 114 of the transfer station 112 and dispensed into clean tubes arranged in an output carousel of the output station 140. In other embodiments, the sample from each well is aspirated by the probe 114 of the transfer station 112 and dispensed into clean wells of a multiwell plate at the output station 140. A user of the sample preparation instrument can then remove the individual tubes, the output carousel, or the multiwell plate from the output station 140.

[0096] Figure 9 diagrammatically illustrates another exemplary sample preparation process 220 for a sample. The sample preparation process 220 can be performed by the sample preparation instrument 100. Similar to operations 202, 204, and 206 of the sample preparation process 200, described in detail above in conjunction with Figure 8, the sample preparation process 220 begins with receiving a tube containing a sample, in operation 224, receiving a panel selection, in operation 224, and determining a sample volume for the sample, in operation 226, based on an estimated white blood cell concentration in the sample.

[0097] Once the appropriate sample volume of the sample is determined in operation 226, the probe 114 of the transfer station 112 can either puncture a capped tube containing the sample in the input station 124 or insert into an open-top tube containing the sample in the single tube loader 126 and aspirate the sample volume of the sample. The sample volume of the sample can be dispensed by the probe 114 into a receptacle of the cell washer 128, and the sample volume of the sample is washed in operation 228. Washing the sample volume of the sample can serve to remove components of the sample that are not necessary for analysis or that must be removed prior to it. For example, if the sample is whole blood, which includes red blood cells, white blood cells, platelets, plasma, and associated proteins, washing the sample volume of the sample can remove the plasma and associated proteins.

[0098] To wash the sample volume of the specimen, a wash fluid, such as a buffer solution, can be dispensed into the body of the vessel via a conduit extending into the vessel. In some embodiments, the vessel is spun about an axis in response to activation of the actuation device of the cell washer 128. As the vessel is spun about its axis, heavier blood cells in the sample volume of the specimen diffuse toward the walls of the vessel and are collected in cavities defined by pockets in the vessel, and vessel contents not in the cavities can be aspirated through the conduit. The actuation device can stop the spinning of the vessel about its axis to add more wash fluid into the vessel and suspend the cells collected in the pockets. These steps can then be repeated multiple times, as desired.

[0099] Similar to operations 208, 210, 212, and 214 of sample preparation process 200, described in detail above in conjunction with FIG. 8, sample preparation process 220 continues with the addition of one or more labeling reagents to the wells of the reaction plate in operation 230, the addition of a washed sample volume of the sample into the wells of the reaction plate containing the labeling reagent in operation 232, incubation of the washed sample volume of the sample with the labeling reagent in operation 234, and the addition of a lysis reagent into the wells of the reaction plate containing the washed sample volume of the sample and the labeling reagent in operation 236. In operation 238, the washed sample volume of the sample, the labeling reagent, and the lysis reagent can be incubated in the wells of the reaction plate. The incubation can be for a predetermined period of time defined, for example, by the panel. The period can be based on the type of lysis reagent. In some embodiments, the reaction plate is positioned on a plate mixer 136, which can move in a circular or elliptical motion to mix the washed sample volume of the specimen, the labeling reagent, and the lysis reagent and hold this mixture for an incubation period. In some embodiments, when multiple lysis reagents are required, the lysis reagents are added and incubated one at a time.

[0100] In operation 240, the sample, comprising the washed sample volume of the specimen, the labeling reagent, and the lysis reagent in each well, can be washed. For example, the probe 114 of the transfer station 112 can aspirate the sample from the well and transfer it to the cell washer 128 for washing. This operation 240 can be repeated for each sample-containing well, and the washing can be performed by the cell washer 128 similar to the washing performed in operation 228. The washing can help to slow down the lysis activity caused by the addition and incubation of the lysis agent in operations 236 and 238, respectively, to avoid destruction of components targeted for analysis (e.g., to avoid destruction of white blood cells). In addition, the washing can remove red blood cell debris. In other exemplary sample preparation workflows, a lysis-no wash protocol can be followed.

[0101] Similar to operation 216 of the sample preparation process 200, described in detail above in conjunction with FIG. 8, the sample preparation process 220 concludes in operation 242 with the sample from each well being dispensed for output.

[0102] FIG. 10 diagrammatically illustrates a further exemplary sample preparation process 250 for a sample. The sample preparation process 250 can be performed by the sample preparation instrument 100. Similar to operations 202 and 204 of the sample preparation process 200, described in detail above in conjunction with FIG. 8, the sample preparation process 250 begins with receiving a tube with a sample in operation 252 and receiving a panel selection in operation 254. In operation 256, a volume of the sample can be washed by the cell washer 128, similar to the washing performed in operation 228 of the sample preparation process 220, described in detail with reference to FIG. 9. In operation 258, a sample volume of the washed sample is determined based on an estimated white blood cell concentration in the sample, similar to the determination performed in operation 206, described in detail above in conjunction with FIG. 8. The volume of the sample washed in operation 256 is a volume larger than the potential sample volume, which can be determined in operation 258 to ensure that the volume of the washed sample exceeds the determined sample volume of the washed sample.

[0103] Similar to operations 208, 210, 212, and 214 of sample preparation process 200, described in detail above in conjunction with FIG. 8, sample preparation process 250 continues with the addition of one or more labeling reagents to the wells of a reaction plate in operation 260, the addition of a sample volume of a washed sample to the wells of the reaction plate containing the labeling reagent in operation 262, incubation of the sample volume of the washed sample with the labeling reagent in operation 264, and the addition of a lysis reagent or buffer into the wells of the reaction plate containing the sample volume of the washed sample and the labeling agent in operation 266.

[0104] 9, the sample preparation process 250 continues with incubation of the sample volumes of the washed samples, labeling reagent, and lysis reagent or buffer in the wells of the reaction plate in operation 268, and washing of the samples with the sample volumes of the washed samples, labeling reagent, and lysis reagent or buffer in each well in operation 270. The sample preparation process 250 then concludes with the sample from each well being dispensed for output in operation 272, similar to operation 216 of the sample preparation process 200, described in detail above in conjunction with FIG.

[0105] FIG. 11 diagrammatically illustrates a further exemplary sample preparation process 280 for a sample. The sample preparation process 280 can be performed by the sample preparation instrument 100. Similar to operations 202 and 204 of the sample preparation process 200, described in detail above in conjunction with FIG. 8, the sample preparation process 280 begins with receiving a tube with a sample in operation 282 and receiving a panel selection in operation 284. In operation 286, a volume of the sample is added into a first well (e.g., well A) in a reaction plate. In operation 288, one or more lysis reagents are added into a first well (e.g., a well that contains a volume of the sample). In operation 290, the volume of the sample and the lysis reagent can be incubated. In other examples, a buffer, rather than a lysis agent, can be added in operation 288 and incubated with the volume of the sample in operation 290.

[0106] In operation 292, the lysed volume of the sample may be washed by the cell washer 128, similar to the washing performed in operation 228 of the sample preparation process 220, described in detail above with reference to Figure 9. In operation 294, the sample volume of the washed, lysed sample is determined, similar to the determination performed in operation 206, described in detail above in conjunction with Figure 8.

[0107] In operation 296, one or more labeling reagents are added to a second well (e.g., well B) of the reaction plate. In operation 298, a sample volume of the washed, lysed sample is added to a second well (e.g., a second well containing a labeling reagent) in the reaction plate. The sample volume of lysed sample and labeling reagent added to the second well are then incubated in operation 300. The sample preparation process 280 then concludes with the sample from each well being dispensed for output in operation 302, similar to operation 216 of the sample preparation process 200, described in detail above in conjunction with FIG. 8.

[0108] 8, 9, 10, and 11 provide non-limiting examples of different sample preparation processes having various steps and their ordering. Other sample preparation processes comprising additional combinations of the steps or actions discussed herein are also possible.

[0109] 12 is a process flow diagram illustrating a method 310 for preparing a sample of a specimen for analysis. The method 310 can be performed by the sample preparation instrument 100. In some embodiments, the sample preparation instrument 100 is housed in a laboratory that provides sample analysis services, such as flow cytometry services. The method 310 begins with operation 312 after a sample is received at the input station 124 or single tube loader 126 of the sample preparation instrument 100. In some embodiments, the sample can be received in a tube that is labeled with a machine-readable code.

[0110] In operation 314, the sample identifier (ID) of the sample is retrieved. For example, a scanner or reader at the input station 124 or single tube loader 126 can scan or read the machine-readable code on the tube to retrieve the sample ID. The machine-readable code can be a barcode, a QR code, or a radio frequency identification (RFID) tag, among other similar embodiments. In other embodiments, the sample ID can be manually entered by a user through the user interface of the touch display 122. The user can select a panel to match or pair with the sample ID, where the panel includes a set of predefined rules and defines the workflow or actions for preparing the sample for a particular type of sample and the analysis to be performed on the sample. The set of predefined rules for the selected panel can be retrieved from the memory 118 of the computing device 116. Exemplary pre-defined rules may include, among other examples, one or more of whether a white blood cell concentration for the sample should be estimated by the sample preparation instrument 100, a minimum white blood cell concentration, a maximum white blood cell concentration, a sample volume of the sample for one or more ranges of white blood cell concentrations between the minimum and maximum concentrations, and whether the sample volume of the sample should be diluted.

[0111] Based on a set of predefined rules, a determination is made in decision 316 as to whether the concentration of white blood cells in the sample should be estimated by the sample preparation instrument 100. If the sample preparation instrument 100 does not need to estimate the white blood cell concentration, the sample volume of the sample is transferred by the probe 114 of the transfer station 112 from the input station 124 or single tube loader 126 in operation 318 for processing.

[0112] In some examples, the sample preparation instrument 100 does not need to perform a white blood cell concentration estimation because the white blood cell concentration for the sample has already been obtained, from which the sample volume for the sample can be determined. As one example, the laboratory may also house a hematology instrument, separate from the sample preparation instrument 100, which analyzes the sample and determines the white blood cell concentration. As another example, a laboratory technician may prepare a smear, count the number of white blood cells, and employ a microscopy method to estimate the concentration based thereon. As a further example, another laboratory, such as a hematology laboratory, may analyze the sample and provide the white blood cell concentration to the current laboratory housing the sample preparation instrument 100.

[0113] In the above examples, the acquired white blood cell concentration can then be manually entered into the sample preparation instrument 100 or can be accessed by the sample preparation instrument 100 through a laboratory information system (LIS). Once the white blood cell concentration is received by the sample preparation instrument 100, a sample volume of the specimen to be prepared for analysis can be determined. In some examples, the determination can be made based on a set of predefined rules. For example, the sample volume that corresponds to the range of white blood cell concentration values ​​into which the white blood cell concentration for the sample falls is determined to be the sample volume of the specimen. In alternative examples, the sample volume of the specimen, rather than the acquired white blood cell concentration, can be manually entered into the sample preparation instrument 100 by a laboratory technician (e.g., after the laboratory technician consults a set of predefined rules and compares it to the acquired white blood cell concentration).

[0114] Once the sample is processed in operation 320, the method ends in operation 322. Processing the sample in operation 320 includes adding at least one or more labeling agents and one or more lysis agents (or buffers) to the sample volume of the specimen. Processing may also involve one or more cell washes and incubation cycles. Exemplary processing steps are described in detail above in conjunction with Figures 8, 9, 10, and 11.

[0115] If a determination is made in decision 316 that the instrument needs to estimate a white blood cell concentration for the sample, an aliquot of the sample is transferred in operation 324 from the input station 124 or single tube loader 126 by the probe 114 of the transfer station 112 to the cell concentration estimator 106. The transferred aliquot is between about 10 microliters (μL) and about 50 μL of sample. The cell concentration estimator 106 can implement one or more of an optical method, an electrical resistance method, or a flow cytometry method, among other methods, in operation 326 to estimate the concentration of white blood cells in the aliquot, which represents the concentration of white blood cells in the sample. The sample is discharged from the cell concentration estimator 106 to a waste container, such as waste container 142.

[0116] The cell concentration estimator 106 provides the white blood cell concentration estimate to the computing device 116 of the sample preparation instrument 100, and the processing device 120 determines whether to process the sample based on the white blood cell concentration estimate and a set of predefined rules, at decision 328. If a decision is made at decision 328 that the sample should not be processed, the sample is not processed, at operation 330, and the method ends, at operation 322. In some examples, a decision is made not to process the sample if the white blood cell concentration estimate is at or below a minimum concentration set by the predefined rules, or if the white blood cell concentration estimate is at or above a maximum concentration set by the predefined rules.

[0117] If a determination is made in decision 328 that the sample should be processed, then a sample volume of the sample is aspirated, transferred, or dispensed by the probe 114 of the transfer station 112 from a tube in the input station 124 or single tube loader 126 for processing in operation 332. The sample volume of the sample is determined based on the white blood cell concentration estimate and a set of predefined rules. For example, the sample volume that corresponds to the range of white blood cell concentration values ​​that the estimate falls into is determined to be the sample volume of the sample.

[0118] In decision 334, a decision is made based on a set of predefined rules whether the sample volume of the sample should be diluted. If the sample volume of the sample should not be diluted, the sample is processed in operation 320 by adding at least, for example, one or more labeling agents and one or more lysing agents (or buffers) to the sample volume of the sample, and the method ends in operation 322. If the sample volume of the sample should be diluted, the sample volume of the sample is diluted in operation 336. In some examples, the sample volume of the sample is diluted to achieve a defined volume for labeling. To provide an example, based on the type of analysis, the defined volume of the sample for labeling is 200 μL. However, in a scenario where the white blood cell concentration estimate is high, the determined sample volume of the sample may be only 50 μL. Therefore, to maintain the defined volume of the sample at 200 μL, the sample volume of the sample is diluted with, for example, an additional 150 μL of buffer. Once the sample volume of the sample has been diluted in operation 336, the sample is processed in operation 320 by adding at least, for example, one or more labeling agents and one or more lysing agents (or buffers) to the sample volume of the sample, and the method ends in operation 322.

[0119] 13 is an exemplary sample loading user interface 340. The user interface 340 can be displayed on the touch display 122 of the sample preparation instrument 100. In some embodiments, a user can attach one or more sample tubes, each with a machine-readable code, to an input cassette. The user can then load the input cassette into the input station 124 of the sample preparation instrument 100 or hold the input cassette in his or her hand. The machine-readable code on the tube is scanned in the input station 124 or manually by the user to read the sample identifier from the machine-readable code, as described in operation 314 of method 310. The read sample ID is provided to the computing device 116 of the sample preparation instrument 100. The user can then load the input cassette into the input station 124 of the sample preparation instrument 100 if it is not already loaded.

[0120] The user interface 340 will display a sample identifier 342 for the scanned tube. In some embodiments, the user can use a menu 344 to assign one or more panels to the sample in the tube associated with the sample identifier 342. As shown, the menu 344 is a drop-down menu that includes a toggle that slides from one side to the other to assign a panel to a sample. Other types of menus and / or similar buttons or selection options can also be provided within the menu 344. The panels displayed within the menu 344 can include panels that have been previously generated using a panel authoring and management application. For example, the panel authoring and management application can be executed on a remote computing device separate from the sample preparation instrument 100, and any panels generated are transmitted to the sample preparation instrument via a wired connection, a wireless connection, or a physical memory device. The panel 346 selected from the menu 344 to pair with the sample is displayed adjacent to the sample identifier 342. In other embodiments, one or more panels to be paired with the sample are automatically assigned by the LIS, and the appropriate panel is automatically populated as the selected panel 346.

[0121] If one or more of the selected panels 346 require that a white blood cell concentration estimate of the sample be obtained prior to sample processing (e.g., because the sample volume of the sample to be prepared depends on the white blood cell concentration), the user interface 340 includes two additional options for selection. The first option 348 can be selected when the cell concentration estimator 106 of the sample preparation instrument 100 should estimate the internal white blood cell concentration. The second option 350 can be selected when an external source has already determined the white blood cell concentration. The white blood cell concentration can be manually typed into a text box 352 of the user interface 340 when the second option 350 is selected. Alternatively, the white blood cell concentration can be automatically populated into the text box 352 when the white blood cell concentration is received and downloaded from the LIS. In some examples, the user can approve or reject the automatically populated value.

[0122] This process can be repeated for each tube in the input cassette. For example, the machine readable code of each of the tubes comprising a sample in the input cassette can be scanned to retrieve a sample identifier associated with the individual sample. These sample identifiers 354 can be displayed in the user interface 340. Selected panels 356 assigned to the sample identifiers 354 can be displayed adjacent to the individual sample identifiers 354. The selected panels 356, similar to the selected panels 346, can be selected from a menu, similar to the menu 344, or automatically populated by the LIS. Additionally, either the first option 348 or the second option 350 is selected for each selected panel 356 that requires that the white blood cell concentration for the sample be determined.

[0123] Once the user has assigned a panel to each of the sample tubes in the input cassette and first option 348 or second option 350 has been selected for each applicable panel, the user may select a load control 358, which is displayed in user interface 340. In response to the selection of load control 358, the input cassette is now moved into input station 124 of sample preparation instrument 100 and ready for processing.

[0124] FIG. 14 is an exemplary user interface 360 ​​of a panel authoring and management application. In some embodiments, the panel authoring and management application can be executed on a remote computing device that is separate from the sample preparation instrument 100. The user interface 360 ​​can receive as input a set of predefined rules for preparing a particular type of sample for a particular type of analysis. The panels can be defined or generated based on the predefined rules and transmitted to the sample preparation instrument 100 via a wired connection, a wireless connection, or a physical memory device for storage. The panels can then be displayed in the user interface 340 of the touch display 122 of the sample preparation instrument 100 (e.g., in the menu 344 for pairing with a sample) for selection by a user, as discussed in detail above with reference to FIG. 13. In other embodiments, the panel authoring and management application can be executed on the sample preparation instrument 100 and the touch display 122 can be displayed on the user interface 360.

[0125] As one example, a user generating a panel can input a set of predefined rules for sample preparation via user interface 360. In some examples, the set of predefined rules can be specific to a type of sample and / or a type of analysis to be performed on the sample. As shown, user interface 360 ​​is provided to facilitate input of predefined rules associated with a sample volume of the sample to be contained within the sample. User interface 360 ​​can also facilitate input of other types of predefined rules, including labeling reagent volumes, lysis reagent volumes, incubation periods, and other similar rules.

[0126] To receive the rules associated with the sample volume of the sample, the user interface 360 ​​includes a menu 362 for selecting a basis for setting the sample volume of the sample. One exemplary basis can include setting the sample volume of the sample based on the white blood cell concentration in the sample. This basis can be selected, for example, for a panel that analyzes a subset of white blood cells, such as a leukemia or lymphoma panel. Another exemplary basis can include setting a fixed volume.

[0127] In response to a selection to set a sample volume of the sample based on the white blood cell concentration in the sample, the user interface 360 ​​may display a table 364. A range of white blood cell concentration values ​​and a corresponding sample volume may be entered into each row of the table 364. For example, each range may have a lower concentration value 366, an upper concentration value 368, and a corresponding sample volume 370. In some examples, one row of the table 364 (e.g., row 372) represents a lowest white blood cell concentration value rather than a range, and another row of the table 364 (e.g., row 374) represents a highest white blood cell concentration value rather than a range. For those rows representing the highest and lowest values, processing options 376 may be provided. For example, using the processing options 376, a user may define whether the sample should be processed based on the estimated white blood cell concentration in the sample (e.g., a user may define the rules used in decision 328 and operation 332). As an example, a sample is not processed when the white blood cell concentration is at or above a maximum concentration value, or when the white blood cell concentration is at or below a minimum concentration value.

[0128] Additionally, the user interface 360 ​​provides a dilution option 378 and a specific sample volume 380, indicating the amount of diluent to be added to reach the sample volume 380. For example, the user interface 360 ​​allows a user to define rules used in the decision 334 and operation 336 regarding whether to dilute the sample volume of the sample to maintain a defined volume of sample for labeling defined for the panel.

[0129] 15 is a flow chart illustrating a method 386 for preparing a sample for analysis. The method 386 is performed by the sample preparation instrument 100. In operation 388, a sample is received as an input 102 into the sample preparation instrument 100. The sample will be prepared for analysis, for example, by a sample analyzer. Exemplary samples can include whole blood, bone marrow, dissociated tissue, peripheral mononuclear cells, biopsies, cerebrospinal fluid, and other single cell suspensions. As an example, the sample is whole blood that is being prepared for a leukemia or lymphoma panel to analyze subsets of white blood cells.

[0130] In operation 390, the concentration of white blood cells in an aliquot of the sample is estimated, which represents the concentration of white blood cells in the sample. The concentration can be determined using optical, electrical resistance, or flow cytometry methods, among other methods. In operation 392, a sample of the sample is dispensed and the volume of the sample (e.g., also referred to as the "sample volume of the sample") can be determined based on the estimate and a set of predefined rules associated with the particular type of analysis (e.g., panel) for which the sample is being prepared. The set of predefined rules can include a minimum white blood cell concentration value, a maximum white blood cell concentration value, and one or more ranges of white blood cell concentration values ​​between the minimum and maximum values, each range having a corresponding sample volume of the sample. Thus, the determined sample volume of the sample is the sample volume that corresponds to the range of concentration values ​​within which the estimated value of the white blood cell concentration falls.

[0131] To process the sample, at least operations 394 and 396 are performed. In operation 394, one or more labeling reagents are added to the sample (e.g., added to a sample volume of the specimen). The sample volume of the sample and the labeling reagents can then be incubated for a predetermined period of time. The labeling agent targets specific sample components and facilitates analysis. For example, continuing with the above example, the labeling reagent can include an antibody reagent that targets a subset of white blood cells (e.g., adheres to the subset of white blood cells during the incubation period) and facilitates analysis of the subset of white blood cells during a leukemia or lymphoma panel.

[0132] In operation 396, one or more lysing reagents are added to the sample (e.g., added to the sample volume of the specimen). The sample volume of the sample and the lysing reagents may then be incubated for a predetermined period of time. Continuing with the above example, the lysing reagent may destroy red blood cells in the sample volume of the sample. In other examples, a buffer may be added to the sample volume of the sample rather than or in addition to the lysing reagent. In operation 398, the sample is provided as an output 104 of the sample preparation instrument 100 and the method 386 ends.

[0133] 16 illustrates the hardware components of an exemplary cell concentration estimator 106. The cell concentration estimator 106 is a component of the sample preparation instrument 100. In some embodiments, the cell concentration estimator 106 can be removable from the sample preparation instrument 100 for servicing. This particular configuration of the cell concentration estimator 106 can implement an optical method to estimate the concentration of white blood cells in a sample.

[0134] The cell concentration estimator 106 includes a mixing chamber 400, a counting chamber 402, an optical system 404 arranged relative to the counting chamber 402, a fluid pump 406, a plurality of reagent pumps 408, and one or more electronic boards 410. The mixing chamber 400 receives lysis and diluent reagents and mixes them with an aliquot of the sample using a motor of the mixing chamber 400. The lysed and diluted aliquot of the sample is advanced from the mixing chamber 400 to the counting chamber 402 by the fluid pump 406. As the lysed and diluted aliquot of the sample is advanced through the counting chamber 402, the optical system 404 captures an image of the sample. The electronic board 410 provides power and signals to the motor of the mixing chamber 400, the fluid pump 406, and the plurality of reagent pumps 408. The computing device 116 of the sample preparation instrument 100 provides signals to the electronics board 410 to control the timing, rate, and / or pattern of mixing performed by the motors, the timing and amount of volume of reagent pushed by each of the reagent pumps 408, and the timing and amount of volume pushed by the fluid pump 406.

[0135] 17 is an exemplary mixing chamber 400 of the cell concentration estimator 106. The mixing chamber 400 comprises a container 420, a cap 422 positioned on an open end of the container 420, and a motor 424. The container 420 holds an aliquot of the sample and one or more reagents that are received for mixing with the aliquot and / or for cleaning the cell concentration estimator 106 after obtaining the white blood cell concentration. As one non-limiting example, the maximum functional volume of the container 420 is 6.0 milliliters (mL).

[0136] The cap 422 includes an opening 426 for receiving the probe 114 of the transfer station 112, the probe 114 aspirating an aliquot of sample from a tube containing the sample at the input station 124 or single tube loader 126 and transferring it to the vessel 420. The cap 422 also includes multiple input ports 428, 430, and 432 for receiving one or more of a lysis reagent, a diluent reagent, and a cleaning reagent via one or more of a first line, a second line, and a third line. The reagents can be pushed through the lines and into the vessel 420 using a reagent pump 408. The lines entering the vessel 420 can be positioned for angular dispensing into the center bottom section of the vessel 420. In some embodiments, the position of the cap 422 is set during manufacture and remains constant during operation of the mixing chamber 400.

[0137] The motor 424 is configured to mix the contents of the mixing chamber 400. In some embodiments, the motor 424 is fitted with an eccentric and an arm 434 is attached to the center of the mixing chamber 400.

[0138] 18 is an exemplary counting chamber 402 and optical system 404 of the cell concentration estimator 106. The optical system 404 can be arranged and / or positioned relative to the counting chamber 402. In some embodiments, the counting chamber 402 is a flow cell chamber consisting of one or more transparent flow cells. An inlet port of the counting chamber 402 can be connected to the mixing chamber 400 via a first section of pump tubing. An outlet port of the counting chamber 402 can be connected to a fluid pump 406 via a second section of pump tubing. The fluid pump 406 advances at least a portion of the lysed and diluted aliquot of the sample from the mixing chamber 400 through the first section of pump tubing to the inlet port of the counting chamber 402 and continues to advance the aliquot through the counting chamber 402 and out the outlet port through the second section of pump tubing to be dispensed into a waste container, such as waste container 142. As the aliquot of the sample is advanced by the fluid pump 406 through the counting chamber 402 (e.g., as the cells move through the flow cell), the optical system 404 captures multiple images. In some embodiments, the fluid pump 406 momentarily stops the advancement of the aliquot so that the optical system 404 may capture images while the cells of the sample have stopped moving within the counting chamber 402. The fluid pump 406 can then resume until the next momentary stop, and this process continues until each image has been captured. As one non-limiting example, the fluid pump 406 can be set to 10 μL / push so that images are captured by the optical system 404 at a rate of 0.6 seconds / image.

[0139] The optical system 404 includes a digital camera 440, a light source 442, and a lens 444. In some examples, the optical system 404 implements a bright field imaging and illumination technique, where the sample is illuminated from below by the light source 442 and imaged from above by the camera 440. The sample illumination can be transmitted blue light, and contrast within the sample can be created by attenuation of the transmitted light within a circular area of ​​the sample. The lens 444 is used to image the cells in the camera 440. The camera 440 is communicatively connected to the computing device 116 of the sample preparation instrument 100 to facilitate capture and transmission of digitized images to the computing device 116 for processing and analysis.

[0140] In another embodiment, the optical system 404 includes a fluorescent microscope coupled with a digital camera, similar to camera 440, that implements fluorescent microscopy and is communicatively connected to the computing device 116 of the sample preparation instrument 100 and facilitates capture and transmission of digitized images to the computing device 116 for processing and analysis.

[0141] 19 is a flow chart illustrating an optical method 450 for estimating white blood cell concentration in a sample. The optical method 450 is performed by the cell concentration estimator 106 of the sample preparation instrument 100, which is described in detail in FIGS.

[0142] The method begins at operation 452, where a lysis reagent is dispensed into the mixing chamber 400. An aliquot of the sample is then dispensed into the mixing chamber 400 for mixing with the lysis reagent and incubation for a predetermined period of time, at operation 454. When the sample is whole blood, the lysis reagent lyses red blood cells in the sample. For example, the lysis reagent causes membrane disassembly and degradation of red blood cells, leaving behind red blood cell debris.

[0143] After a predetermined period of time for incubation of the sample aliquot and lysis reagent has elapsed, a diluent reagent is dispensed into the mixing chamber 400 to mix with the sample aliquot and lysis reagent in operation 456. The diluent reagent slows down the lysis activity, prevents destruction of white blood cells, and dilutes the sample to facilitate white blood cell counting. In operation 458, the fluid pump 406 advances at least a portion of the lysed and diluted aliquot of the sample from the mixing chamber 400 to the counting chamber 402.

[0144] In operation 460, multiple images of the lysed and diluted aliquot of the sample are captured by the optical system 404 as the aliquot flows through the counting chamber 402. For example, once the counting chamber 402 is filled with the aliquot, the fluid pump 406 will temporarily stop transferring fluid, stopping the movement of white blood cells in the sample. While the movement of the white blood cells is temporarily stopped, the optical system 404 will capture and digitize an image. Once an image has been captured and digitized, the fluid pump 406 will again advance the sample and then stop and capture another image. This process is repeated, capturing multiple images. For example, the process can be repeated until a sufficient number of images are captured to provide a statistically significant estimate of the number of white blood cells.

[0145] The multiple images are then transferred to the computing device 116 of the sample preparation instrument 100 for image processing and analysis. For example, in operation 462, a total number of white blood cells in the multiple images is determined based on the number of white blood cells counted in each image. In operation 464, a total volume of the aliquots in the multiple images is determined. For example, as described in more detail in FIG. 21, during manufacturing setup of the sample preparation instrument 100, an image of an objective micrometer captured by the optical system 404 is received and a horizontal length and a vertical length of the image are measured. The image volume can be determined based on the horizontal length, vertical length, and a known path length of the flow cell in the counting chamber 402. The image volume can be stored in the computing device 116. A total volume of the aliquots in the multiple images can then be determined based on the image volume and a number of images in the multiple images. In operation 466, a concentration of white blood cells is estimated based on the total number of white blood cells and the total volume of the aliquots in the multiple images.

[0146] 20 diagrammatically illustrates an exemplary optical method for estimating white blood cell concentration for a sample. The illustrated optical method can be similar to the optical method 450 described in FIG. 19 implemented by the cell concentration estimator 106 of the sample preparation instrument 100. For example, a lysis reagent pump 480 of the reagent pumps 408 aspirates lysis reagent from a container 482 that includes the lysis reagent. The lysis reagent pump 480 pushes the lysis reagent through a first line 484 and dispenses the lysis reagent into the vessel 420 of the mixing chamber 400 via one of the input ports (e.g., input port 428). In one example, a volume of 250 μL of lysis reagent is dispensed into the vessel 420.

[0147] The probe 114 of the transfer station 112 can then puncture a capped tube 486 with a sample, for example, held in an input cassette 488 at the input station 124. The probe 114 can aspirate, transport, and then dispense an aliquot of the sample into the container 420 of the mixing chamber 400. In one example, a volume of sample between 10 μL and 50 μL is dispensed into the container 420. The motor 424 of the mixing chamber 400 can be activated to provide mechanical mixing of the sample aliquot and lysis reagent. The sample aliquot and lysis reagent are allowed to mix and incubate for a predetermined period of time. As one example, the sample aliquot and lysis reagent can be mixed in a circular or elliptical pattern for 15 seconds. The mixing action is then stopped and allowed to incubate for an additional 9 seconds. The mixing is then resumed for an additional 9 seconds, and then stopped again to leave and incubate for an additional 9 seconds. The incubation is terminated with a 3 second mix.

[0148] The diluent reagent pump 490 of the reagent pump 408 can then aspirate the diluent reagent from a container 492 that comprises the diluent reagent. The diluent reagent pump 490 pushes the diluent reagent through a second line 494 and dispenses the diluent reagent into the vessel 420 of the mixing chamber 400 via another one of the input ports (e.g., input port 430). In some embodiments, the diluent reagent is further used by other components of the sample preparation instrument 100 during processing of the sample. The motor 424 of the mixing chamber 400 can again be activated to provide mechanical mixing of the sample aliquot, the lysing agent, and the diluent. In one embodiment, a volume of 900 μL of diluent reagent is dispensed and 3 seconds of mixing is performed.

[0149] The fluid pump 406 advances at least a portion of the lysed and diluted aliquot of the sample from the mixing chamber 400 to the counting chamber 402 through a first portion of the pump tubing 496. In one embodiment, an aliquot of the diluted and lysed sample of about 600 μL is primed by the fluid pump 406 through the pump tubing 496. After the counting chamber 402 is primed with the aliquot of the diluted and lysed sample, the fluid pump 406 temporarily stops the advancement of the aliquot, stopping the movement of the white blood cells in the sample. While the movement of the white blood cells is temporarily stopped, the optical system 404, consisting of the camera 440, the light source 442, and the lens 444, captures and digitizes an image 498. Once the image 498 has been captured and digitized, the fluid pump 406 will again begin advancing the aliquot, then stop and capture another image. This process is repeated to capture multiple images. As one non-limiting example, an image may be captured every 0.6 seconds until 50 images have been captured. However, some aliquot amounts may require up to 1,000 images to be captured. The multiple images are provided to the computing device 116 for processing and analysis to estimate the white blood cell concentration in the sample.

[0150] The fluid pump 406 advances the aliquot through the counting chamber 402 to the waste container 142 for disposal, such as in the waste container 142, via a second portion of the pump tubing 496. Once multiple images have been captured, the lysed and diluted aliquot of the remaining sample in the container 420 of the mixing chamber 400 can be advanced, if applicable, through the counting chamber 402 to the waste container 142. In some embodiments, a cleaning reagent pump 500 of the reagent pump 408 aspirates cleaning reagent from the container 502 comprising cleaning reagent through a third line 504 and dispenses into the container 420 of the mixing chamber 400. The cleaning reagent is then advanced by the fluid pump 406 from the mixing chamber 400 to the counting chamber 402 via a first portion of the pump tubing 496, and then from the counting chamber 402 to the waste container 142 via a second portion of the pump tubing 496, cleaning each component of the cell concentration estimator 106 in preparation for the next aliquot of sample to be received.

[0151] 21 illustrates an image of an objective micrometer used to determine the total aliquot volume within a number of captured images. During manufacturing setup of the sample preparation instrument 100, an image 520 of the objective micrometer (e.g., an image of a slide having a scale on its surface) can be captured and the horizontal and vertical lengths and image volume associated with the view of the image 520 can be determined. The determined lengths and / or image volumes can then be transmitted to the computing device 116 for storage and use in determining the total aliquot volume.

[0152] In some examples, a full field of view, hereafter referred to as a full view 522, can be measured from the image 520. For example, a horizontal length 524 and a vertical length 526 of the full view 522 can be measured to determine the area of ​​the full view 522. The depth of the full view 522 is based on a known measurement associated with the counting chamber 402, for example the known path length of a flow cell within the counting chamber 402. The area and depth are then multiplied to arrive at a full image volume for the full view 522. The full image volume can be stored in the computing device 116 of the sample preparation instrument 100.

[0153] As one non-limiting example, digital camera 440 may use a 1 / 3 inch format having 1,280 x 960 pixels and a pixel size of 3.75 mm horizontally by 3.75 mm vertically to provide a full image of area 4,800 (e.g., full image format 1280 x pixel size 3.75). The measured full image length is 1 mm for horizontal length 524 and 0.750 mm for vertical length 526, with 0.75 mm 2 The path length of the flow cell is 0.0853 mm, therefore the full image volume is 0.75 mm 2 ×0.0853mm or 0.064mm 3 It is.

[0154] Once the multiple images are captured by the optical system 404, the total aliquot volume within the multiple captured images can be determined by the computing device 116 based on the complete image volume. For example, the complete image volume may be in μL (e.g., 1 mm 3 = 1 μL) and multiplied by a number of images in the captured multiple images to obtain the total aliquot volume in the multiple images. For example, 50 images are captured, and a total aliquot volume of 0.064 mm is obtained. 3 If a full image volume of 100 μL was determined, the total aliquot volume in the multiple images would be 3.200 μL.

[0155] To obtain an estimate of the white blood cell concentration, the number of white blood cells counted in each image are added together to obtain a total number of white blood cells in the multiple images. The total number of white blood cells in the multiple images is then divided by the total aliquot volume in the multiple images to obtain an estimate of the white blood cell concentration (number of white blood cells / μL).

[0156] Additionally, in some embodiments, to avoid problems with identifying and counting white blood cells at the edge of the full view 522 of the digital camera 440, a counting boundary is introduced on at least two sides of the image 520. The boundary effectively reduces the number of pixels in the field of view. Thus, the field of view inside the counting boundary, hereafter referred to as the effective view 528, can be measured from the objective micrometer image. Continuing with the above example, a counting boundary set at 50 μm may reduce the image to 1,134×814 pixels, resulting in an effective image area of ​​3,600 microns (e.g., full image format 1134×pixel size 3.75). The measured effective image length is 0.889 mm for the horizontal length 530 and 0.639 mm for the vertical length 532, resulting in a 0.568 mm effective image area. 2 Therefore, the effective image volume is 0.568 mm 2 ×0.0853mm or 0.0485mm 3 It is.

[0157] The effective image volume may be stored in the computing device 116 of the sample preparation instrument 100, allowing the computing device 116 to determine the total aliquot volume within multiple images captured by the optical system 404. For example, the effective image volume may be in μL (e.g., 1 mm 3 = 1 μL) and multiplied by a number of images in the captured multiple images to obtain the total aliquot volume in the multiple images. Thus, 50 images are captured, yielding a volume of 0.0485 mm 3If an effective image volume of 100 μL is determined, then the total aliquot volume in the multiple images would be 2.4250 μL. To obtain an estimate of the white blood cell concentration, the number of white blood cells counted in each image (e.g., counted within the borders of the images) are added together to obtain a total number of white blood cells in the multiple images. The total number of white blood cells in the multiple images is then divided by the total aliquot volume in the multiple images to obtain an estimate of the white blood cell concentration (number of white blood cells / μL).

[0158] 22 illustrates image analysis performed to determine the number of white blood cells in an image. The image analysis can be performed by the computing device 116 of the sample preparation instrument 100. For example, the computing device 116 may be communicatively coupled to the optical system 404 of the cell concentration estimator 106 and receive a plurality of images of the sample captured by the optical system 404. In some embodiments, the computing device 116 and the optical system 404 can communicate via a wireless connection. In other embodiments, the computing device 116 and the optical system 404 can communicate via a wired connection.

[0159] The processing device 120 of the computing device 116 can analyze each image individually. When received, the image is in a raw image data format (e.g., raw image 550) that includes white blood cells 552, debris 554, and background 556. The debris 554 can include red blood cell debris resulting from mixing with a lysing reagent, among other debris. To increase contrast and facilitate counting the number of white blood cells 552, the background 556 can be subtracted to generate a subtracted image 558. For example, by subtracting the background 556, it may be easier to distinguish between the white blood cells 552 and the debris 554. In some examples, one or more of the white blood cells 552 or areas in the subtracted image 558 can be hovered or selected to see if each of the white blood cells 552 is analyzed (e.g., counted as a white blood cell). For example, white blood cells are analyzed and counted, and when a white blood cell or an area comprising white blood cells is hovered or selected, a symbol such as a cross can be displayed to indicate that the cells have been counted. In some embodiments, this function and display can be performed internal to the computing device 116 that performs the image analysis, and is not displayed to a user of the sample preparation instrument 100.

[0160] The computing device 116 can determine a variety of different values, including the white blood cell count per image, the average white blood cell count per image, and a cumulative white blood cell count for multiple images (e.g., the sum of the white blood cell counts per image). In addition, as part of the image analysis process, the computing device 116 can determine the total aliquot volume within the multiple images, as described in detail in FIG. 21 above. Thus, the computing device 116 can estimate the concentration of white blood cells based on the total white blood cell count and the total aliquot volume within the multiple images.

[0161] 23 diagrammatically illustrates a graphical representation 570 of a white blood cell count obtained during image analysis. For example, the graphical representation 570 is generated from data obtained during image analysis performed by the computing device 116 as described in FIG. 22 above.

[0162] The x-axis 572 of the graphical representation 570 can represent the number of images captured. For example, 50 images of a sample were captured by the optical system 404 (e.g., images 0-49). A first y-axis 574 on the left side of the graphical representation 570 represents the white blood cell count. A first line 576 represents the number of white blood cells counted per image. For example, 4 white blood cells are counted in a first image (e.g., image 0), 50 white blood cells are counted in a second image, 7 white blood cells are counted in a third image, 4 white blood cells are counted in a fourth image, 35 white blood cells are counted in a fifth image, etc. A second line 578 represents the average number of white blood cells counted per image. Continuing with the above example, if 4 white blood cells are counted in a first image and 50 white blood cells are counted in a second image, the average white blood cell count in the second image is the average of the counts in the first and second images, which is 27 (e.g., (4+50) / 2). Similarly, the average white blood cell count in a third image is the average of the counts in the first, second, and third images, which is approximately 20.

[0163] A second y-axis 580 on the right side of the graphical representation 570 represents the cumulative white blood cell count, and an associated line 582 represents the cumulative white blood cell count in each image. For example, as the white blood cell count for the next image is determined, the white blood cell count is added to the previous count of white blood cells. Continuing with the above example, after a first image, the total white blood cell count is 4, after a second image, the total white blood cell count is 54, after a third image, the total white blood cell count is 61, after a fourth image, the total white blood cell count is 65, after a fifth image, the total white blood cell count is 100, etc. The value of the cumulative white blood cell count in the final image (e.g., image 49) is the total number of white blood cells in the multiple images. Here, the total number of white blood cells in the multiple images is about 442 white blood cells. The concentration of white blood cells in the sample can then be estimated based on the total number of white blood cells in the multiple images and the total aliquot volume in the multiple images, which can be determined as part of the image analysis by the computing device 116, as described in detail in FIG. 22 above.

[0164] While this graphical display 570 can be generated by the computing device 116, in some embodiments the graphical display 570 is not provided externally to a user. For example, the graphical display 570 is not provided through the user interface of the touch display 122 of the sample preparation instrument 100. Instead, only the range within which the estimate of the white blood cell concentration falls is displayed to the user through the user interface of the touch display 122 of the sample preparation instrument 100. In further embodiments, if a decision is made based on the estimate that the sample should not be processed, the sample volume of the sample and / or an alert may also be displayed.

[0165] FIG. 24 is a flow chart illustrating an electrical resistance method 600 for estimating white blood cell concentration in a sample. The method 600 can be implemented by the cell concentration estimator 106 of the sample preparation instrument 100. However, the cell concentration estimator 106 can have different components than the cell concentration estimator 106 described in conjunction with the optical method of estimation 450 (e.g., different from the cell concentration estimator 106 described in FIGS. 16-23). ​​For example, the electrical resistance method 600 employed can be a Coulter principle, where the cell concentration estimator 106 is a counting chamber including at least a first chamber and a second chamber with an electrolyte solution, an aperture connecting the first chamber to the second chamber, and at least two electrodes. The electrodes are located at each end of the aperture and can generate an electric field (e.g., cause a current to flow through the aperture) that is applied through the aperture. In some embodiments, the aperture is a channel.

[0166] In operation 602, an aliquot of the sample is suspended in an electrolyte solution and dispensed into a first chamber of the counting cell. In some embodiments, the aliquot of the sample can also be dissolved and diluted in the counting cell. In operation 604, the suspension is passed through an aperture through which an electric current flows. For example, the suspension is advanced through the aperture from the first chamber to the second chamber. In some embodiments, the cell concentration estimator 106 also includes a pump to advance the suspension from the first chamber to the second chamber. In other embodiments, the suspension flows from the first chamber to the second chamber, for example, as a result of a pressure differential. As the cells of the sample in suspension flow through the aperture, the electrical resistance across the aperture changes.

[0167] A change in electrical resistance is detected in operation 606. The concentration of white blood cells in the aliquot, which represents the concentration of white blood cells in the sample, can then be estimated based on the detected change in electrical resistance in operation 608. In some embodiments, the electrical resistance change is detected and measured electronically as a series of pulses. The pulses can then be processed and counted by computing device 116, for example, to estimate the number of white blood cells per volume of the aliquot.

[0168] FIG. 25 is a flow chart illustrating a flow cytometry method 650 for estimating white blood cell concentration in a sample. Method 650 can be performed by cell concentration estimator 106 of sample preparation instrument 100. However, cell concentration estimator 106 can have different components than the cell concentration estimator 106 described in conjunction with optical method of estimation 450 (e.g., different from cell concentration estimator 106 described in FIGS. 16-23) and the cell concentration estimator 106 described in conjunction with electrical resistance method of estimation 600 in FIG. 24. For example, cell concentration estimator 106 can be a flow cytometer. In further embodiments, method 650 can be performed by sample preparation and analysis instrument 150, and cell concentration estimator and sample analyzer 152 can be the same component, such as a flow cytometer.

[0169] Method 650 begins at operation 652, where an aliquot of the sample is stained with an antibody marker that targets white blood cells. In some embodiments, the sample aliquot and the antibody marker are incubated. In addition, one or more lysis reagents can be added. If the flow rate of the flow cytometer is not known, a number of counting beads are also added to the stained aliquot of the sample in optional operation 654 to provide a reference for determining the volume of the sample.

[0170] In operation 656, the stained aliquot of the sample is analyzed over a predetermined period of time using a flow cytometer. The analysis includes counting the white blood cells over a predetermined period of time, where the antibody marker enables the white blood cells to be distinguishable from other cells in the sample. In addition, if counting beads were added in optional operation 654, the analysis can further include counting the counting beads.

[0171] In operation 658, the concentration of white blood cells in the aliquot, representing the concentration of white blood cells in the sample, is then estimated based on the number of white blood cells counted in the predetermined time period in operation 656 and the flow rate of the flow cytometer, if known. If the flow rate of the flow cytometer is not known, the concentration of white blood cells in the sample is estimated based on the number of white blood cells counted and the ratio of the number of counting beads to the number of white blood cells counted in operation 656.

[0172] The various examples and teachings described above are provided by way of illustration only and should not be construed as limiting the scope of the present disclosure. Those skilled in the art will readily recognize various modifications and changes that may be made without following the examples and applications shown and described herein and without departing from the true spirit and scope of the present disclosure.

Claims

1. A sample preparation device (100), comprising: a controller (116) including a processing device (120) and a memory (118) coupled to the processing device (120); an input station (124) for receiving a sample; a cell concentration estimator (106) for estimating the concentration of white blood cells in the sample; a transfer station (112) for dispensing a sample of the specimen, the controller (116) being configured to instruct the transfer station (112) to dispense a volume of the sample based on the estimated white blood cell concentration in the specimen; a reagent station (108) containing a labeling reagent to be added to the sample; a lysis station (110) containing a lysis reagent that is added to the sample; an output station (140) for providing the sample following addition of the labeling reagent and the lysis reagent to the sample; The cell concentration estimator (106) comprises: a mixing chamber (400) configured to receive an aliquot of the sample; a counting chamber (402) connected to said mixing chamber (400); a fluid pump (406) configured to advance at least a portion of the aliquot from the mixing chamber (400) to the counting chamber (402); an optical system (404) configured to capture a plurality of images of at least the portion of the aliquot in the counting chamber (402); and the controller (116) comprises: the estimated white blood cell concentration is equal to or less than a predetermined minimum white blood cell concentration; The estimated white blood cell concentration is equal to or greater than a predetermined maximum white blood cell concentration. the sample preparation device (100) being further configured to prevent processing of the sample if one or more of the following conditions are met:

2. The mixing chamber (400) comprises: A container (420); a cap (422) positioned over an open end of the vessel (420), the cap (422) including a plurality of input ports (428, 430, 432) and an opening (426) for a transfer station probe (114) that dispenses the sample into the vessel (420); a motor (424) for mixing the aliquot of the sample with one or more reagents dispensed into the vessel (420) via the plurality of input ports (428, 430, 432), the one or more reagents including a lysis reagent and a diluent reagent; The sample preparation instrument (100) of claim 1 comprising:

3. The sample preparation device (100) of claim 1 or claim 2, wherein the counting chamber (402) comprises one or more transparent flow cells.

4. The sample preparation device (100) of any one of claims 1 to 3, wherein the optical system (404) comprises a camera (440), a light source (442), and a lens (444).

5. The memory (118) stores instructions that, when executed by the processing device (120), cause the processing device (120) to: receiving the plurality of images of the aliquots of the sample captured by the optical system (404) of the cell concentration estimator (106); determining (462) a total number of white blood cells in the plurality of images based on the number of white blood cells counted in each image; determining (464) a total volume of the segments within the plurality of images; estimating a concentration of the white blood cells based on a total number of the white blood cells and a total volume of the aliquots in the plurality of images (466); The sample preparation instrument (100) of claim 1 ,

6. The memory (118) stores instructions that, when executed by the processing device (120), cause the processing device (120) to: Receiving a selection of a panel (346) to be implemented; retrieving from the memory (118) a set of predefined rules for preparing the samples for the panel, the set of predefined rules including volumes of the samples corresponding to one or more ranges of white blood cell concentrations; determining a volume of the sample based on the set of predefined rules and the estimated concentration of white blood cells in the sample; The sample preparation instrument (100) of claim 5,

7. 7. The sample preparation device of claim 6, wherein the set of predefined rules further comprises the predetermined minimum white blood cell concentration, the predetermined maximum white blood cell concentration, and one or more ranges of white blood cell concentrations, the one or more ranges of white blood cell concentrations being between the predetermined minimum white blood cell concentration and the predetermined maximum white blood cell concentration.

8. The display further comprises: a range within which the estimated white blood cell concentration falls; and The volume of the sample dispensed; and an alert when the processing of the sample is prevented; The sample preparation instrument (100) of any one of claims 1 to 7, configured to provide a user interface comprising one or more of the following:

9. The cell concentration estimator (106) a first chamber for receiving the aliquot of the sample suspended in an electrolyte solution; A second chamber; and an opening connecting the first chamber to the second chamber, through which the aliquot of the sample suspended in the electrolyte solution flows from the first chamber to the second chamber; at least two electrodes generating an electric field within the cell concentration estimator, and an estimate of the concentration of white blood cells in the sample is determined based on a change in electrical resistance detected across the aperture as the aliquot of the sample flows through the aperture; The sample preparation instrument (100) of claim 1 , comprising:

10. The sample preparation instrument of claim 1 , wherein the cell concentration estimator comprises a flow cytometer.

11. The sample preparation instrument (100) of any one of claims 1 to 10, wherein the input station (124) includes a rocking mechanism for rocking a cassette, in which a tube containing the sample is received.

12. The sample preparation instrument (100) of any one of claims 1 to 11, wherein the input station (124) comprises a single tube loader (126).

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