Biological sample preparation device and methods of using the same

Abstract

A microfluidic sample preparation device, a fully automated analysis pipeline, a method of conducting an immune cell activation and analytical test.

Description

DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION BIOLOGICAL SAMPLE PREPARATION DEVICE AND METHODS OF USING THE SAME CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The present application claims the benefit of priority to U.S. Provisional No. 63 / 551,998, entitled, “IMMUNE CELL ACTIVATION DEVICE AND METHODS OF USING THE SAME,” filed February 9, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number NIH Grant Nos. AI149277, EB030643 and AI104209. The United States government has certain rights in this invention. SEQUENCE LISTING STATEMENT

[0003] The Sequence Listing associated with this application is filed in electronic format via Patent Center and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is STFD-008-PCT_SL.xml. The size of the text file is 2,041,567 bytes and the .xml file was created on February 6, 2025. FIELD

[0004] The present disclosure generally relates to devices and systems for preparing biological samples for testing and associated methods of use. In particular, the present disclosure generally relates to devices and system for preparing whole blood samples for testing, and associated methods of use. BACKGROUND

[0005] Despite the increasing prevalence of food allergies,1–4their diagnosis remains difficult.5,6Conventional tests based on skin prick tests (SPTs) and allergen-specific IgE (sIgE) levels in blood only indicate sensitization to an allergen and cannot reliably predict a true food allergy.7Oral food challenge (OFC), the gold standard for food allergy assessment, exposes aDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION patient to anaphylaxis risk.8–12OFCs therefore necessitate close supervision by experienced allergists and are resource- and time-intensive.

[0006] The basophil activation test (BAT) has emerged as a powerful ex vivo functional assay for food allergy assessment.13–15The BAT has higher accuracy than SPTs and sIgE tests in assessing food allergies.16–24Despite its exceptional diagnostic performance, the clinical adoption of the BAT remains limited. Some challenges include: (1) The conventional BAT protocol can be time-consuming and prone to human error. (2) To preserve optimal basophil reactivity, the BAT requires fresh blood ideally within 24-48 hours from withdrawal,13,23necessitating express shipping of blood to a laboratory equipped to perform the BAT. (3) Flow cytometry analysis of basophil activation largely relies on separating cell populations through a series of hand-drawn gates. This process can be time-consuming (~1 hour per patient sample) and subjective, and can contribute to the variance in BAT results.25–27SUMMARY OF EMBODIMENTS

[0007] The present disclosure relates to sample preparation devices configured to receive a biological sample (e.g., whole blood), divide the biological sample into sub-samples, and stimulate cells in the sub-samples (e.g., via exposure to an antigen). In some examples the sample preparation device is further configured to stain (e.g., barcode, label, tag, etc.) cells in the sample. The sample preparation device may comprise a fluid circuit including one or more inlets, one or more outlets, and a plurality of reaction vessels, some or all in fluid communication with the at least one inlet via one or more fluid channels and some or all in fluid communication with the at least one outlet via one or more channels. The reaction vessels may comprise separate tubes or vessels (for example, as shown in the device of FIG. 19), or may comprise separate compartments defined by a shared housing (for example, as shown in the device of FIG.15). In any case, each of the reaction vessels is configured to receive a sub- sample.

[0008] The sample preparation devices disclosed herein may include one, two, three, four, five, six, seven, eight or more reaction vessels, each including a reagent for mixing with a biological sample (such as whole blood) received through the inlet(s) of the device. The reagent may be a stimulating agent, a staining agent, a stimulating and staining agent, a washing agent (such as a wash buffer), or a stabilizing agent. The stimulating agent may be an antigen (including any of the antigens disclosed herein), a positive control (such as any of the positive controls disclosed herein), or a negative control (such as any of the negative controls asDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION disclosed herein). Even though the negative control is designed to not induce a meaningful change in the cells of the sample, for ease of description a “stimulating agent” as used herein may include a negative control. The staining agent may be configured to confer a unique identifier to one or more cell surface proteins in the sample. In some embodiments, the staining agent comprises a two or more probes specific to an immune cell.

[0009] In some examples, each of the reaction vessels includes only a stimulating agent. In some embodiments, each of the reaction vessels includes both a stimulating agent and a staining agent. In yet other embodiments, the device includes a first reaction vessel containing a stimulating agent and a second reaction vessel containing a staining agent. The first and second reaction vessels occupy separate physical spaces but are in fluid communication with another such that a sub-sample can move from a first reaction vessel to a corresponding second reaction vessel. According to some examples, the device further includes third reaction vessels, each containing only a washing agent and / or a stabilizing agent.

[0010] Several embodiments of the present technology are directed to a sample preparation device that includes or is configured to be operably coupled to a pressure control device. The pressure control device is configured to generate pressure differentials between the inlet and reaction vessels, the reaction vessels and the outlet, and in some cases between first and second (and third, fourth, etc., if applicable) reaction vessels. The pressure control device may comprise a single pressure source or a plurality of pressure sources, and optionally a controller configured to automate activation / deactivation of the pressure source(s). In some examples, the sample preparation device and the pressure control device are integrated into a single device. In other embodiments, the reaction vessels, the inlet, and the outlet may comprise a cartridge and the pressure control device is a separate component configured to receive the cartridge. When in receipt of the cartridge, the pressure control device operably couples to the reaction vessels such that activation of the pressure control device causes fluid (e.g., the sample, a buffer solution, etc.) to move within the fluid circuit, for example between the inlet, the reaction vessels, and the outlet(s).

[0011] In some embodiments, the reaction vessels are connected in a fluid circuit. The fluid circuit may include reaction vessels containing at least two, three, four, five, six, seven or eight or more probes specific for the immune cells. In some embodiments, the biological sample (e.g., containing immune cells) is divided among and positioned within first, second, third, fourth, fifth, and sixth reaction vessels. In some embodiments, the sample preparation device may include more or fewer reaction vessels. Each reaction vessel may comprise at least one combination of two different probes specific for an immune cell and the combinations in eachDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION compartment may be different combinations. In some embodiments, the reaction vessels each comprise at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein.

[0012] Some embodiments of the device comprise (i) at least about six reaction vessels, each reaction vessel comprising a different pair of probes specific for an immune cell; and (ii) an antigen positioned in fluid communication with the reaction vessels. In some embodiments, the antigen is optionally positioned in one or a plurality of the reaction vessels. In some embodiments, the antigen is positioned in reaction vessels that comprise an experimental set of probes specific for an activated immune cell and a set of vessels comprising a control set of probes for unactivated immune cells are free of an antigen. In some embodiments, the antigen is desiccated or in solid form and, in a first operational mode, fluid from a sample is positioned within the fluid circuit capable of dissolving the solid form of the antigen upon exposure to the fluid. Similarly, in some embodiments, the probes are positioned in the reaction vessels in solid form, and in a first operational mode, fluid from a sample is positioned within the fluid circuit capable of dissolving the solid form of the probe upon exposure to the fluid.

[0013] In some embodiments, the present technology includes a device comprising a fluid circuit comprising an inlet, an outlet, and a plurality of reaction vessels. The plurality of reaction vessels are configured to be in fluid communication with the inlet and the outlet. In some embodiments, the device also comprises one or more valves positioned in the fluid circuit between the inlet and the reaction vessels. The one or more first valves are adjustable in an open and closed position. In the open position, the one or more first valves allow for fluid flow between the inlet and the reaction vessels. The device also comprises one or more second valves positioned in the fluid circuit between the outlet and the reaction vessels. The one or more second valves are adjustable between an open position and a closed position. In the open position, the one or more second valves allow for fluid flow between the outlet and the reaction vessels. In some embodiments, a single, adjustable valve allows for fluid flow between the inlet and reaction vessels, and the outlet and reaction vessels.

[0014] In some embodiments, each reaction vessel comprises a combination of at least two probes specific for an immune cell. In some embodiments the reaction vessel includes a reaction region and a reagent storage region, and the reagent storage region is in fluid communication with the reaction region.

[0015] In some embodiments of the device, in a first operational condition, the reagent storage region comprises a combination of at least two probes specific for an immune cell in a dried or powdered formulation. In some embodiments of the device, in a second operationalDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION condition, the reaction region comprises a combination of at least two probes specific for an immune cell, and one or a plurality of immune cells. In some embodiments, a first set of reaction vessels comprise probes specific for immune cells unexposed to the antigen and a second set of reaction vessels comprises probes specific for immune cells exposed to the antigen.

[0016] In some embodiments the device further comprises an inlet adaptor positioned proximate to and operably coupled to the inlet, such that the inlet adaptor receives an inlet vessel and aligns the inlet vessel to the inlet.

[0017] In some embodiments the device further comprises an inlet vessel in fluid communication to the inlet. The inlet vessel may contain a biological sample. The sample may be whole blood.

[0018] In some embodiments, the device further comprises an outlet adaptor positioned proximate to and operably coupled to the outlet and a storage vessel, such that the outlet adaptor is configured to receive the storage vessel and aligns the outlet to the storage vessel. In some embodiments, the storage vessel may be a 15-milliliter conical tube comprising protruding spiral threads around an opening of the tube. In some embodiments, the outlet adaptor may be a plastic protrusion comprising an inner and outer surface; wherein the inner surface protrudes around the outlet and comprises a circular or semicircular spiral inlay positioned around the outlet. In some further aspects, the tube and the outlet adaptor may define a joint positioned at the opening of the tube in fluid communication with the fluid circuit at the outlet.

[0019] In some embodiments the device further comprises a storage vessel in fluid communication with the fluid circuit and positioned proximate to the outlet of the fluid circuit.

[0020] In some embodiments the device further comprises an immune cell activator positioned within or proximate to the reaction vessels.

[0021] In some embodiments the device further comprises one or a plurality of immune cells chosen from one or a combination of: a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil, and / or platelets. In some embodiments the device further comprises a housing positioned over the fluid circuit, and the reaction vessels are channels positioned at or proximate to a first set of conduits connecting the inlet to the channels. In some embodiments, the fluid circuit comprises an inlet, a first set of conduits in fluid communication to the reaction vessels, and a second set of conduits connecting the reaction vessels to the outlet.

[0022] The device comprises first and second conduits which are parallel in a region but branched in some embodiments, and, in such embodiments, the paralleled, branched region ofDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION conduits intersect a valve channel that is positioned orthogonally or substantially orthogonally across the first and second sets of conduits at intersection points. Further, in some embodiments, the valve channel is positioned transverse to a width dimension of the housing. In some embodiments, the valve channel may comprise a valve element in operable contact with or even comprising a plurality of valves, wherein one valve is positioned at each intersection point of the fluid circuit. The valve element in the valve channel may comprise two or four total valves or be operably connected to two or four valves, and, in some embodiments, the valve element is adjustable and operably connected to the valves such that movement of the valve element opens and closes the valves at each intersection point. In some embodiments, the valve element is cylindrical or semicylindrical. In some embodiments, the valve element is rotatable about its longitudinal axis and has at least a first and a second operable position. In the first operable position and in some embodiments, a first valve is positioned at an intersection point proximate to the inlet is open and a second valve positioned at an intersection point proximate to the outlet is closed. In the second operable position and in some embodiments, the valve positioned at an intersection point proximate to the inlet is closed and the valve positioned at an intersection point proximate to the outlet is open. The valve element may be operably connected to a dial positioned on the side of the housing, the dial movable radially between the first and second operable position of the valve element.

[0023] The disclosure also relates to a device comprising a plurality of reaction vessels or a plurality of compartments, such as a first, second, third, fourth, fifth, sixth, seventh and seventh and eighth compartment; each compartment comprising a reaction region and a storage region. In some embodiments, the plurality of reaction vessels or the first, second, third, fourth, fifth, sixth, seventh and eighth compartments are uniformly cylindrical or rectangular in shape and in parallel orientation along their longitudinal axis; wherein each reaction vessel or compartment comprises at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein. In some embodiments, each reaction vessel or compartment comprises at least two probes specific for an immune cell forming a combination of probes; and wherein each combination of probes in each reaction vessel or compartment is different or independently selectable among a set of probes. In some embodiments, one or all of the plurality of reaction vessels or the compartments further comprises a plunger movable within a length of the reaction vessel or compartment. In some embodiments, the reaction vessels comprise a plunger configured for movement along a longitudinal plan across the reaction vessels. In some embodiments, each plunger may be mechanically connected to one or more handles positioned on the outside of the housing andDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION operably linked to the plungers, such that movement of the handle by an operator in a vector along a plane of the plungers also moves the plungers in the same vector. In some embodiments, where multiple handles connect at least two plungers in a reaction vessel or compartment, movement of the plungers operably connected to one of a plurality of handles is exacted by pulling the handle and independent of movement of the other handles. In some embodiments, where the device comprises a single handle, movement of the handle exacts movement of each plunger in parallel along a length of the reaction vessels that comprise a plunger. In some embodiments, movement of a single handle laterally from the exterior of the device may simultaneously slide each plunger along its respective longitudinal axis in the cylindrical or semicylindrical compartments or reaction vessels.

[0024] In some embodiments, the device comprises one or a plurality of adjustable valves positioned within the fluid circuit between the inlet and the reaction vessels; and / or between the reaction vessels and the outlet. In some embodiments, the adjustable valves are configured to open and close, such that, in a closed position, the valve prevents fluid flow to and from different regions of the fluid circuit. If, in some embodiments, the adjustable valve positioned between the inlet and the reaction vessels is open, fluid from a sample positioned at or proximate to the inlet can be drawn through the fluid circuit to the compartments or the reaction vessels, optionally through a first region of conduits. If, in some embodiments, the adjustable valve positioned between the reaction vessels and the outlet is open, fluid from a sample positioned in the reaction vessels can toward the outlet, optionally through a second region of conduits.

[0025] In some embodiments, the device further comprises a storage vessel attached to an outlet adaptor positioned at or around the outlet of the fluid circuit. The outlet is configured for fluid flow from the outlet into the storage vessel in an operable condition wherein one or a plurality of valves are open between the compartments and the outlet. In some embodiments, the storage vessel comprises one or a plurality of stabilizing agents.

[0026] The disclosure also relates to a system comprising a sample preparation device as disclosed herein. In some embodiments, the device comprises a fluid circuit comprising an inlet, an outlet, and a plurality of reaction vessels. The plurality of reaction vessels are in fluid communication with the inlet and the outlet. In some embodiments, the device also comprises first valves positioned in the fluid circuit between the inlet and the reaction vessels. The individual valves comprising the first valves may be the same or different types of valves. The first valves are adjustable between an open position and a closed position. In some embodiments in the open position, the first valves allow for fluid flow between the inlet andDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION the reaction vessels. In some embodiments, the device also comprises second valves positioned in the fluid circuit between the outlet and the reaction vessels. The individual valves comprising the second valves may be the same or different types of valves. The second valves can be adjustable between an open position and a closed position. In the open position, the second valves allow for fluid flow between the outlet and the reaction vessels.

[0027] In some embodiments, the system comprises a plurality of immune cells. In some embodiments, the immune cells are divided among and positioned within one or more reaction vessels. In some embodiments, the reaction vessels include first, second, third, fourth, fifth and sixth reaction vessels. More or fewer reaction vessels are possible. The reaction vessels may each comprise at least one reagent. The at least one reagent may comprise a stimulating agent, a staining agent, or both. In those embodiments including a staining agent, the staining agent may comprise at least about two probes, for example a first probe specific for a first immune cell surface protein and a second probe specific for a second immune cell surface protein.

[0028] In some embodiments, the system also comprises a heating element electrically connected to a battery source. In some embodiments of the system, the heating element is configured to receive at least a first region of the device comprising the reaction vessels, such that the heating element is proximate to one or more surfaces of the reaction vessels and at a distance sufficient to modulate the temperature around the reaction vessels. In some embodiments the reaction vessels are configured to dock with a heater, such that positioning of the reaction vessels of the device can be inserted into an opening on the surface of the heater, thereby positioning the reaction vessels at or near the heating element.

[0029] In some embodiments, the sample preparation device further comprises a processor operable to execute programs and a memory associated with the processor. In some embodiments, the system further comprises a computer program product on a non-transitory computer-readable software medium. In some embodiments, the computer program product may include instructions for conducting a method herein. In those embodiments in which the system comprises a cartridge and a separate pressure control device, the processor, memory, and / or computer program may be on the cartridge, the pressure control device, or both.

[0030] The disclosure further relates to a method of activating an immune cell or plurality of immune cells, wherein the method comprises exposing the immune cell to an antigen within the device disclosed herein. In some embodiments, the method comprises delivering the cells to a fluid circuit comprising an inlet, an outlet, and a plurality of reaction vessels, wherein the plurality of reaction vessels are in fluid communication with the inlet and the outlet. In some embodiments, the method further comprises exposing the cell or cells to an antigen to elicit anDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION immune response against the antigen, thereby activating the cell or cells. In some embodiments, the method comprises exposing the cell or cells to a combination of probes specific for the cell or cells after activating the cell or cells, then pooling the cell or cells in a storage vessel, and subsequently analyzing the cell or cell for the presence of a probe or plurality of probes. In some embodiments, analyzing the cell or cells comprises lysing the cells resulting in cell content, stabilizing the cell content and detecting the presence of a plurality of probes. Detection of the plurality of probes (corresponding to the particular reaction vessel of the device) corresponds to a probe signature. In some embodiments, movement of the cells through the device is accomplished by drawing a sample (such as whole blood comprising the cell or cells) through the inlet, through a first set of conduits and into the plurality of reaction vessels, wherein the cell or cells are exposed to an antigen and the probes. Drawing of the sample can be accomplished by movement of a handle and plungers positioned within the reaction vessels. After cells are drawn into the reaction vessels, the cells are divided among the reaction vessels. The reaction vessels may each comprise at least about two probes, a first probe specific for a first immune cell surface protein and a second probe specific for a second immune cell surface protein. In some embodiments, after the cell or cells are labeled with a probe and activated, the cells are pooled into a storage vessel or pooling vessel position at the outlet. Again, movement of the cells out of the reaction vessels and into the storage vessel can be accomplished by displacing the plungers in the direction opposite of the step of drawing, such that fluid from the sample moves from the reaction vessels, through a second region of conduits and through the outlet. In some embodiments, the method relates to introducing a plurality of immune cells into the device or composition disclosed herein, and exposing the cells to an antigen. In some embodiments, the cells are subsequently: exposed to probes such that the probes bind or associate to the cells, pooled into a storage vessel and analyzing for activation.

[0031] Some aspects of the disclosure relate to methods of cell sorting. Some methods comprise separating cells into the compartments within a sample preparation device, as disclosed herein. The method may optionally comprise exposing the cells to at least two probes, and pooling the cells in the pooling or storage vessel. In some embodiments, the composition comprises a plurality of immune cells, and the immune cells are divided among and positioned within a plurality of reaction vessels. In some cases, the immune cells are divided among and positioned within first, second, third, fourth, fifth, and sixth reaction vessels. In other embodiments, the immune cells are divided among and positioned within more or fewer than six reaction vessels. In some embodiments, each reaction vessel comprises at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION In some embodiments, the cells are introduced into a sample preparation device as disclosed herein, for example comprising a fluid circuit comprising an inlet, an outlet, and a plurality of reaction vessels in fluid communication with the inlet and the outlet. In some embodiments, the device also comprises a one or more first valves positioned in the fluid circuit between the inlet and the reaction vessels. The first valves may be adjustable between an open position and a closed position. In the open position, the first valves allow for fluid flow between the inlet and the reaction vessels. The device also comprises one or more second valves positioned in the fluid circuit between the outlet and the reaction vessels. The second valves may be adjustable between in an open position and a closed position. In the open position, the second valves allow for fluid flow between the outlet and the reaction vessels.

[0032] In some embodiments, a method comprises introducing the cells into a sample preparation device as disclosed herein, separating the cells into the reaction vessels, exposing the cells to the probes disclosed herein, pooling the cells and then counting or sorting the cells based upon detection of the probes. In some embodiments, at least a first and second probe form a pair corresponding to the pair of probes in the compartment or reaction vessels of the device. Upon detection of the pair of probes, cells exposed to the pair exhibit a unique cell signature corresponding to the pair of probes. This unique signature can be used to sort the cells. In some embodiments, the method of cell sorting comprises flow cytometry or mass cytometry.

[0033] The disclosure also relates to a point-of-care device and a system comprising a device, wherein the device comprises a compartment comprising: (i) one or a plurality of cells, such as immune cells; and (ii) at least about two probes specific for membrane proteins on the cells. In some embodiments, the compartment further comprises one or a plurality of barcodes specific to the compartment within which the cells are positioned. In some embodiments, the system or device comprises a first vessel and a second vessel, each vessel comprising (i) one or a plurality of cells, such as immune cells; and (ii) at least about two probes specific for membrane proteins on the cells, wherein the two probes in the first vessel are not identical to the two probes in the second vessel; or, wherein the two probes in the first vessel are capable of binding a membrane protein that is not identical to at least one of the membrane proteins in the second vessel.

[0034] The disclosure also relates to a method of analyzing a sample, the method comprising separating the cells into the compartments within a composition disclosed herein or the device herein. In some embodiments, the method also comprises exposing the cells to at least two probes, pooling the cells in the pooling or storage vessel, and analyzing the cells.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0035] In some embodiments, the disclosure relates to a computer program product. The computer program product comprises instructions for: (a) receiving detection data from a device disclosed above corresponding to the presence, absence or quantity of probes in each compartment; (b) quantifying the amount of probe in each compartment by normalizing the amount of probe in the compartment with the amount of probe in a control compartment; and (c) correlating the amount of probe in each compartment with the number of activated immune cells from a sample in the sample. In some embodiments, the amount of probe indicates the probability that a subject from which the sample is taken has an immune-related disorder.

[0036] In embodiments, the disclosure relates to a kit. The kit comprises device summarized above and a first container comprising at least one immune cell activator from Table 1 or an amino acid comprising at least about 75% sequence identity to an amino acid sequence from Table 1. BRIEF DESCRIPTION OF DRAWINGS

[0037] FIG.1A illustrates a schematic representation of a device workflow. The left panel illustrates a sample preparation device for inserting a blood tube, withdrawing blood into plungers and mixing with preloaded stimulation-stain cocktail, and after incubation in custom heater, ejecting barcoded sample into lyse / fix buffer. In embodiments, the process requires <2 minutes of active user involvement, and the sample can be stored for up to 7 days at 4°C. The right panel illustrates an automated sample analysis pipeline. Raw data are gathered by flow cytometry and are inputted to an automated gating pipeline that generates a dose response curve and other derived BAT metrics (e.g., baseline and maximum activation, area under the dose response curve (AUC), half maximal effective concentrations (EC50)). The process takes ~21 min: 15 minutes for washing and transferring cells into a FACS tube, 6 minutes for running the tube through a flow cytometer, and <1 second for automated data analysis.

[0038] FIG.1B illustrates a schematic representation of the conventional BAT workflow.

[0039] FIGS.2A–2H illustrates comparisons between automated and manual gating. FIG. 2A illustrates that each of eight stimulation conditions are associated with barcoded basophil population indices (BCI-1 to BCI-8), which consist of a unique combination of CD193 and CD123 markers (stain panel A of Table 1 is shown here). FIG. 2B illustrates a schematic representation of the automated gating pipeline. From raw FCS files, all events are classified as BCI-1 to BCI-8 or as non-basophils using feature set 1 (i.e., all flow cytometer parameters, stain panel ID, days stored, and incubation time). Basophils within each basophil populationDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION are then classified as CD63+ or CD63- using feature set 2 (i.e., same as feature set 1 but without CD193 and CD123 data) (Table 2). Next, basophils are parsed into their respective conditions and BAT metrics, such as %CD63+ and CD203 MFI, are extracted. FIG. 2C illustrates representative plots of test data from one device experiment showing classification of barcoded basophil populations (differentiated by different colors) plotted against combinations of CD193 and CD123 markers. Indices on CD193 and CD123 axis labels correspond to one of the two or four stains for CD193 or CD123 markers, respectively. FIG.2D illustrates representative plots of the fully- automated pipeline applied to test data from a sample storage experiment. The input of CD63+ / - classifier was barcoded basophil populations from the basophil classifier. BCI-1 and BCI-7 (RPMI and the 1,000 ng / mL anti-IgE dose, respectively) are shown for 0 days and 7 days of storage. The CD63+ / - classifier accounts for shifts in the baseline CD63 signal that are attributed to increased autofluorescence from storage in the lyse / fix buffer (see FIG.10B) Colored markers in the legend indicate events that were classified in agreement with manual gating, and white markers indicate events that were inconsistent with manual gating (1.2% of events on average across BCI-1 to BCI-8 and 2.1% events in the CD63 classifier). FIGS. 2E–2G: Test datasets were employed to compare the performance of manual gating vs. automated classifiers. CD203c MFI and total basophil counts obtained with the basophil classifier, as well as %CD63+ obtained with the CD63+ / - classifier, were highly correlated with values obtained with manual gating (see Fig. 26 for train / test data splits). FIG. 2H illustrates the correlation between %CD63+ AUCs calculated by the automated analysis pipeline, which consists of both basophil and CD63+ / - classifiers, vs. %CD63+ AUCs determined via manual gating. The automated analysis pipeline was used on all device data, i.e., no distinction between testing and training data, and manual gating only for conventional BAT data analysis. The R-values provided denote Pearson’s correlation coefficients for the least-squares regression lines applied to the scatter plots.

[0040] FIG.3 schematically depicts a sample preparation device configured in accordance with the present technology.

[0041] FIG.4 schematically depicts a sample preparation device configured in accordance with the present technology.

[0042] FIG.5 schematically depicts a sample preparation device configured in accordance with the present technology.

[0043] FIG.6 schematically depicts a sample preparation device configured in accordance with the present technology.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0044] FIG.7 schematically depicts a sample preparation device configured in accordance with the present technology.

[0045] FIG.8 schematically depicts a sample preparation device configured in accordance with the present technology.

[0046] FIGS.9A–9E illustrate a method for using the device shown in FIG.8.

[0047] FIG. 10 illustrates a sample preparation device and a process flow configured in accordance with the present technology.

[0048] FIG. 11 illustrates a sample preparation device configured in accordance with the present technology.

[0049] FIG. 12 illustrates a sample preparation device configured in accordance with the present technology.

[0050] FIG. 13 illustrates a sample preparation device configured in accordance with the present technology.

[0051] FIG. 14 illustrates a sample preparation device and a process flow configured in accordance with the present technology.

[0052] FIG. 15 illustrates a sample preparation device configured in accordance with the present technology.

[0053] FIG.16 illustrates a cross-section of the device of FIG.15.

[0054] FIG.17 illustrates a cross-section of the device of FIG.15.

[0055] FIG.18 illustrates a cross-section of the device of FIG.15.

[0056] FIG. 19 illustrates a sample preparation device configured in accordance with the present technology.

[0057] FIG.20 illustrates a cross-section of the device of FIG.19.

[0058] FIG.21 illustrates a cross-section of the device of FIG.19.

[0059] FIG. 22 illustrates another configuration of the sample preparation device of FIG. 18, in accordance with the present technology.

[0060] FIG.23 illustrates a heater for use with the sample preparation devices of the present technology.

[0061] FIG.24A illustrates a device coupled to a blood collection vial.

[0062] FIG.24B illustrates the device of FIG.24A after sample was drawn into the device from the vial by withdrawal of plungers on the device.

[0063] FIG. 24C illustrates the device of FIG. 24B upon combination of sample and reagents.

[0064] FIG.24D illustrates the device of FIG.24C inserted into a heater.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0065] FIG. 24E illustrates the device of FIG. 24D removed from the heater and coupled to a collection vial with plungers in a withdrawn position.

[0066] FIG.24E illustrates the device of FIG.24E with plungers in an inserted position.

[0067] FIG. 25 illustrates a representative example of the manual gating pipeline of barcoded basophils (stain panel A, Table 1) pooled into one tube for flow cytometry. All basophil barcoded populations share singlet, CD45+, CD203chigh / SSClow, and HLA-DR- gates. Each basophil barcode population is easily distinguishable from other barcodes on CD123 and CD193.

[0068] FIG.26 illustrates a flowchart outlining the machine learning pipeline for basophil and CD63+ / -classifications. The dataset consisted of approximately 77 million cells including 337 thousand basophils—all these cells were used for the basophil classifier with a 30 / 70 test / train split. 647 basophil populations with varying levels of activation were used for the CD63+ / -classifier with a 20 / 80 test / train split. Bayesian-optimized XGBoost models for basophil and CD63+ / -classification were trained separately. The performance of each classifier was evaluated with the test sets (solid arrows) (see FIGS.2C, 2E, 2F, and 2G), and the models were saved for later use. The fully automated pipeline was applied to all data (dotted arrows) to generate a population-level summary data frame (i.e., each row contains statistics of separate barcoded basophil populations). From this data frame, other BAT metrics were derived, e.g., area under the dose response curve (AUC) (FIGS.2D and 2H).

[0069] FIG.27 illustrates the influence of different input blood volumes, other than 50 µL as was used in other experiments, on the anti-IgE dose-response curves. By using blood volumes of 25 µL and 100 µL, the effective concentration of target anti-IgE was adjusted to 1.33 times and 0.67 times the intended doses, respectively. Following a post-hoc correction of anti-IgE concentrations, the dose-response data were better fitted by four-parameter logistic (4PL) regressions, as indicated by increased R-values compared with those from the original, non-adjusted dose responses.

[0070] FIG. 28 illustrates the average importance of features for basophil and CD63+ / -XGBoost classifiers. Weight describes the number of times a feature is used to split the data across all trees (i.e., count of how often a feature is used in the model). Gain describes the contribution of each feature to the model by considering the improvement in accuracy brought by a feature to the splits it is used in (i.e., how much a feature contributes to making more accurate predictions). High gain for a feature may be attributed to the brightness of the marker’s fluorophore which leads to more separation between the positive and negative populations for that marker, e.g., CD123-3 and CD193-1 were typically associated with PE and BV421,DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION respectively—two bright fluorophores. Cover describes the relative quantity of observations related to a feature (i.e., how many times on average a feature is used in a split across all trees, weighted by the number of training instances a node is responsible for). Despite the insignificant effect of days stored, the days stored feature in the CD63 classifier had high gain and cover indicating the importance of this feature in decision-making process of the classifier.

[0071] FIGS. 29A–29C illustrate comparisons between BAT with a device herein and conventional BAT for %CD63+and CD203c MFI activation markers. FIG. 29A illustrates turkey-style box and whisker plots show %CD63+and CD203c MFI activation measurements of all donors used for comparisons between the exemplified embodiment and conventional BAT (N=10 donors) at each dose and stimulation incubation time. Wilcoxon signed-rank tests were used to compare incubation times within BAT with a device herein or conventional BAT (‘ns’ spanning all incubation times), and to compare between BAT with a device herein and conventional BAT for each incubation time (annotated on conventional BAT plots). The Benjamini and Hochberg method was used to adjust P values (i.e., Q values) by correcting for false discovery rates during multiple hypothesis testing.28Values are given for Q < 0.05, **Q < 0.001, and ‘ns’ is not significant (Q > 0.05). FIG.29B illustrates representative dose response curves using %CD63+and CD203c MFI. Derived metric from the dose response curves (baseline, max, AUC, and EC50 values) are annotated. The shades regions on each trace represent the 95% confidence interval of activation across the three incubation times. FIG.29C illustrates comparisons between BAT with a device herein and conventional BAT of derived metrics from dose response curves of all incubation times combined (N=28 dose response curves). Wilcoxon signed-rank tests were used to show that all comparisons were significantly different with the exception of baseline %CD63+. *P < 0.01, **P < 0.001, and ‘ns’ is not significant (P > 0.05). For reference, use of the term “easyBAT” is synonymous with a device disclosed herein and exemplified.

[0072] FIGS. 30A–30D illustrate evaluation of the effects of samples stored in a lyse / fix buffer at 4°C. FIG. 30A: Non-allergic donor samples were either stimulated with anti-IgE, or with peanut extract to ensure that storage did not yield and false positive results. Data points represent the level of basophil activation at varying doses, with no significant changes observed except for CD203c ΔMFI after 7 days (N=49 data points for each storage time from 7 donors). FIG. 30B: Stored cells exhibited shifts in baseline CD63 and CD203c MFI at different times of storage (N=7 donors). FIG. 30C: Representative dose response curves from donor SBC12 for 0, 2, 4, and 7 days of storage. FIG.30D: A linear mixed-effects model was used on anti-IgE stimulated samples to quantify the minimal effect of storage on dose-dependent activation, asDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION indicated by low coefficient estimates for the storage duration variable, i.e., 0 to 7 days (N=112 data points from 4 donors fit to model). All pairwise comparisons in these plots were performed with Wilcoxon signed-rank tests and the Benjamini and Hochberg method was used to adjust P-values (i.e., Q values) by correcting for false discovery rates during multiple hypothesis testing.28Values given for Q < 0.05, *Q < 0.01, and ns is not significant (Q > 0.05). All stimulations were performed with 20 minutes of incubation.

[0073] FIG. 31 illustrates the dynamic range, i.e., absolute difference between the maximum and baseline expression levels, of BAT with a device herein and conventional BAT on %CD63+(mean values of 75.7% and 59.7%, respectively) and CD203c MFI (mean values 28.4^103and 16.4^103, respectively). Wilcoxon signed-rank tests were used to make pair- wise comparisons between at each incubation time. *P < 0.01 and **P < 0.001.

[0074] FIG.32 illustrates a flowchart of an model.

[0075] FIG.33 depicts a schematic of the automated gating pipeline embodiment.

[0076] FIG. 34 depicts representative plots of the fully automated pipeline applied to test data. The input of CD63+ / - gating routine was barcoded basophil populations from the basophil classifier. BCI-1 and BCI-7 (RPMI and the 1,000 ng / mL anti-IgE dose, respectively) are shown for 0 days and 7 days of storage. Because a negative control is always referenced for a set of experimental conditions, the CD63+ / - gating can account for shifts in the baseline CD63 signal that are attributed to increased autofluorescence from storage in the lyse / fix buffer. Colored markers in the legend indicate events that were classified in agreement with manual gating, and the white marker indicates events that were labelled as CD63+ with the automated gating pipeline and CD63- with manual gating.

[0077] FIG.35 depicts test data sets (not involved in training) were employed to compare the performance of manual gating with automated classifiers. (E) Basophil counts, and (F) %CD63+ obtained with the CD63+ / - gating routine (high baseline outliers omitted), were highly correlated with values obtained with manual gating. (G) The correlation between %CD63+ AUCs calculated by the full automated analysis pipeline, which consists of the basophil classifier and the CD63+ / - gating routine, and %CD63+ AUCs determined via manual gating. The R-values provided denote Pearson's correlation coefficients for the least-squares regression lines applied to the scatter plots.

[0078] FIG. 36 depicts a flowchart outlining the automated analysis pipeline for basophil classification and CD63+ / - gating. The dataset consisted of approximately 136 million cells including 540 thousand basophils. After a conservative thresholding routine onDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION CD45+ / SSClow / HLA-DR-, 76 million cells were used for the basophil classifier with a 30 / 70 test / train split. The performance of the Bayesian-optimized XGBoost model plus the CD63+ / - gating routine was evaluated with the test set. The fully automated pipeline was applied to all data (solid black arrows) to generate a population-level summary data frame (i.e., each row contains statistics of separate barcoded basophil populations). From this data frame, other BAT metrics were derived, e.g., area under the dose response curve (AUC).

[0079] FIG. 37 illustrates the average importance of features for the basophil classifier. Weight describes the number of times a feature is used to split the data across all trees (i.e., count of how often a feature is used in the model). Gain describes the contribution of each feature to the model by considering the improvement in accuracy brought by a feature to the splits it is used in (i.e., how much a feature contributes to making more accurate predictions). Cover describes the relative quantity of observations related to a feature (i.e., how many times on average a feature is used in a split across all trees, weighted by the number of training instances a node is responsible for).

[0080] FIG 38A-38C illustrate representative %CD63+ dose response curves for peanut- allergic (PA) and non-allergic (NA) donors with dose response curve metrics defined. (38A) Tukey-style box and whisker plots show %CD63+ activation measurements of all subjects in which paired comparisons on the same dose were made. Wilcoxon signed-rank tests were used for paired comparisons (brackets and black font). Mann-Whitney U-tests were used to compare between PA and NA. The Benjamini and Hochberg method was used to adjust P values (i.e., Q values) by correcting for false discovery rates during multiple hypothesis testing.50 *Q < 0.05, **Q < 0.01, and ns is not significant (Q > 0.05). FIG.38B illustrates there were no significant differences between the exemplified embodiment and conventional BAT for maximum activation, AUC and EC50 derived from %CD63+ dose response curves. FIG.29D show when using the exemplified embodiment and the automated analysis pipeline, differences between non-allergic (NA) and peanut-allergic (PA) were significant for %CD63+ maximum activation and AUC. Results are shown for the N=6 NA and N=20 PA patients. DETAILED DESCRIPTION OF EMBODIMENTS

[0081] Before the present systems and methods are described, it is to be understood that the present disclosure is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purposes of describing the particular versions or embodiments only, andDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the methods, devices, and materials in some embodiments are now described. All publications mentioned herein, including any references to sequences by way of GenBank Accession number, are incorporated by reference in their entireties. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0082] Definitions

[0083] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0084] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0085] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly oneDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0086] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[0087] The term “at least” prior to a number or series of numbers (e.g. “at least two”) is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context. When “at least” is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

[0088] As used herein, the term “animal” includes, but is not limited to, humans and non- human vertebrates such as wild animals, rodents, such as rats, ferrets, and domesticated animals, and farm animals, such as dogs, cats, horses, pigs, cows, sheep, and goats. In some embodiments, the animal is a mammal. In some embodiments, the animal is a human. In some embodiments, the animal is a non-human mammal.

[0089] As used herein, the terms “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0090] The term “diagnosis” or “prognosis” as used herein refers to the use of information (e.g., genetic information or data from other molecular tests on biological samples, signs and symptoms, physical exam findings, cognitive performance results, etc.) to anticipate the most likely outcomes, timeframes, and / or response to a particular treatment for a given disease, disorder, or condition, based on comparisons with a plurality of individuals sharing commonDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION nucleotide sequences, symptoms, signs, family histories, or other data relevant to consideration of a patient’s health status, including correlating the type, number or character of cells in a sample to the subject’s health status.

[0091] As used herein, the phrase “in need thereof” means that the animal or mammal has been identified or suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the subject in need thereof is a human seeking prevention or treatment of an allergy. In some embodiments, the subject in need thereof is a human diagnosed with an allergy. In some embodiments, the subject in need thereof is a human seeking treatment for an allergy-related disorder. In some embodiments, the subject in need thereof is a human undergoing treatment for an allergy-related disorder.

[0092] As used herein, the term “mammal” means any animal in the class Mammalia such as rodent (i.e., mouse, rat, or guinea pig), monkey, cat, dog, cow, horse, pig, or human. In some embodiments, the mammal is a human. In some embodiments, the mammal refers to any non- human mammal. The present disclosure relates to any of the methods or compositions of matter wherein the sample is taken from a mammal or non-human mammal. The present disclosure relates to any of the methods or compositions of matter wherein the sample is taken from a human or non-human primate.

[0093] As used herein, the term “predicting” refers to making a finding that an individual has a significantly enhanced probability or likelihood of benefiting from and / or responding to an immune-related disorder treatment. In some embodiments, the treatment is administration of an allergy modulating agent. In some embodiments, the treatment is administration of a vaccine. In some embodiments, the treatment is administration of an antigen or functional antigen fragment.

[0094] A “score” is a numerical value that may be assigned or generated after normalization of the value based upon the presence, absence, or quantity of deposition of a probe associated with a cell in the sample of a subject. In some embodiments, the score is normalized in respect to a control data value.

[0095] As used herein, the term “stratifying” refers to sorting individuals into different classes or strata based on the features of a neurological disease. For example, stratifying a population of individuals with an immune-related disorder involves assigning the individuals on the basis of the severity of the disease (e.g., mild, moderate, advanced, etc.).DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0096] As used herein, the term “subject,” “individual” or “patient,” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans. In some embodiments, the subject is a human seeking treatment for an immune-related disease. In some embodiments, the subject is a human diagnosed with an allergy to the antigen. In some embodiments, the subject is a human suspected of having an immune-related disorder. In some embodiments, the subject is a healthy human being.

[0097] As used herein, the term “threshold” refers to a defined value by which a normalized score can be categorized. By comparing to a preset threshold, a subject, with corresponding qualitative and / or quantitative data corresponding to a normalized score, can be classified based upon whether it is above or below the preset threshold.

[0098] As used herein, the terms “treat,” “treated,” or “treating” can refer to therapeutic treatment and / or prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes of the embodiments described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0099] As used herein, the term “therapeutic” means an agent utilized to treat, combat, ameliorate or improve an unwanted condition or disease of a patient.

[0100] A “therapeutically effective amount” or “effective amount” of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to treat, combat, ameliorate, prevent or improve one or more symptoms of an allergy-related disorder. The activity contemplated by the present methods includes both medical therapeutic and / or prophylactic treatment, as appropriate. The specific dose of a compound administered according to the present disclosure to obtain therapeutic and / or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. It will beDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION understood that the effective amount administered will be determined by the physician in the light of the relevant circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore the above dosage ranges are not intended to limit the scope of the present disclosure in any way. A therapeutically effective amount of compounds of embodiments of the present disclosure is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.

[0101] As used herein, the term “kit” refers to a set of components provided in the context of a system for delivering materials or diagnosing a subject with an allergy-related disorder. Such delivery systems may include, for example, systems that allow for storage, transport, or delivery of various diagnostic reagents (e.g., oligonucleotides, probes, extracellular matrix components etc. in appropriate containers) and / or supporting materials (e.g., buffers, media, cells, written instructions for performing the assay etc.) from one location to another. For example, in some embodiments, kits include one or more enclosures (e.g., boxes) containing relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a diagnostic assay comprising two or more separate containers that each contain a sub portion of total kit components. Containers may be delivered to an intended recipient together or separately. For example, a first container may contain a polystyrene plate or tube for use in a cell culture assay, while a second container may contain cells, such as control cells. As another example, the kit may comprise a first container comprising a device disclosed herein such a device with the disclosed reaction vessels and fluid circuit or a plurality of ligands with affinities to one or a plurality of probes disclosed herein and a second container comprising any one or plurality of reagents necessary for the detection and / or quantification of the amount of biomarkers (or probes) in a sample. The term “fragmented kit” is intended to encompass kits containing Analyte Specific Reagents (ASR’s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contain a sub-portion of total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all components in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0102] As used herein, “cell culture” means growth, maintenance, transfection, or propagation of cells, tissues, or their products. As used herein, "culture medium" refers to any solution capable of sustaining the growth of the targeted cells either in vitro or in vivo, or anyDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION solution with which targeted immune cells or exogenous nucleic acids are mixed before being applied to cells in vitro or to a patient in vivo. In some embodiments, culture medium means solution capable of sustaining the growth of the targeted cells either in vitro.

[0103] Any probes may be used in concert with any of the devices, systems, kits, or methods disclosed herein. As used herein, the term “probe” refers to any molecule that may bind or associate, indirectly or directly, covalently or non-covalently, to any of the amino acids expressed by the immune cells disclosed herein and whose association or binding is detectable using the methods disclosed herein. In some embodiments, the probe is a fluorogenic, fluorescent, or chemiluminescent probe, an antibody, or an absorbance-based probe. In some embodiments, an absorbance-based probe, for example the chromophore pNA (para- nitroanaline), may be used as a probe for detection and / or quantification of a toxin disclosed herein. In some embodiments, the probe comprises an amino acid sequence that is a natural or non-natural ligand of an toxin disclosed herein and / or an analog or salt thereof, including those analogs that comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to amino acids indicated in the Table 1 below. A probe may be immobilized, adsorbed, or otherwise non-covalently bound to a solid surface, such that upon exposure to an enzyme for a time period sufficient to associate with one or a plurality of amino acids expressed by immune cells disclosed herein. In some embodiments, association of the antigen to the amino acid sequence causes a biological change in the nature or chemical availability of one or more probes such that the biological change enables detection of the association event. For instance, if the step of detecting comprises use of FRET, cleavage of the amino acid compositions disclosed herein cause one of the chromophore to emit a fluorescent light under exposure to a wavelength sufficient to activate such a fluorescent molecule. The intensity, length, or amplitude of a wavelength emitted from fluorescent marker can be measured and is, in some embodiments, proportional to the presence, absence or quantity of antigen present in the reaction vessel, thereby the levels of activation can be determined from detection of the intensity of or fluorescence at a known wavelength of light.

[0104] An “activity-based probe,” as used herein, refers to a certain embodiment of probe comprising a small molecule that binds to or has affinity for a molecule such as an amino acid that binds an activated cell in the presence of such an amino acid sequence, such that its bound or unbound state confers an activity readout observable by a user. In some embodiments, the activity-based probe covalently or non-covalently binds to a toxin disclosed herein orDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION derivative herein. In some embodiments, the binding of the activity-based probe modifies the physical or biological activity of the toxin. In some embodiments, the activity-based probe can be fluorescent or chemiluminescent. In some embodiments, the activity-based probe has a measurable activity of one value if the enzyme is inactive and another measurable activity if in an activated state.

[0105] As used herein, the terms “fluorogenic” and “fluorescent” probe refer to any molecule (dye, quantum dot, peptide, or fluorescent marker) that emits a known and / or detectable wavelength of light upon exposure to a known wavelength of light. In some embodiments, the probes are covalently or non-covalently attached to a fluorogenic probe. In some embodiments, the attachment of the fluorogenic probe to the immune cells create a chimeric molecule or two associated molecules capable of a fluorescent emission or emissions upon exposure of the amino acid sequence a known wavelength of light, such that exposure to the wavelength of light creates a reaction product which is quantifiable in the presence of a fluorimeter. In some embodiments, light from the fluorogenic probe is fully quenched upon exposure to the known wavelength of light before association of the disclosed amino acid sequences to the fluorogenic probe emits a known wavelength of light, the intensity of which is quantifiable by absorbance readings or intensity levels in the presence of a fluorimeter. In some embodiments, the fluorogenic probe is a coumarin-based dye or rhodamine-based dye with fluorescent emission spectra measurable or quantifiable in the presence of or exposure to a predetermined wavelength of light. In some embodiments, the fluorogenic probe comprises rhodamine. In some embodiments, the fluorogenic probe comprises rhodamine-100. Coumarin-based fluorogenic probes are known in the art, for example in US Pat Nos.7,625,758 and 7,863,048, which are herein incorporated by reference in their entireties. In some embodiments, the fluorogenic probes are a component to, covalently bound to, non-covalently bound to, intercalated with one or a plurality of amino acid sequences or toxins disclosed herein. In some embodiments, the fluorogenic probes are chosen from ACC or AMC. In some embodiments, the fluorogenic probe is a fluorescein molecule. In some embodiments, the fluorogenic probe is capable of emitting a resonance wave detectable and / or quantifiable by a fluorimeter after exposure to one or a plurality of immune cells disclosed herein. “Fluorescence microscopy,” which uses the fluorescence to generate an image, may be used to detect the presence, absence, or quantity of a fluorescent probe. In some embodiments, fluorescence microscopy comprises measuring fluorescence resonance energy transfer (FRET) within a FRET-based assay.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0106] A “chemiluminescent probe” refers to any molecule (dye, peptide, or chemiluminescent marker) that emits a known and / or detectable wavelength of light as the result of a chemical reaction. Chemiluminescence differs from fluorescence or phosphorescence in that the electronic excited state is the product of a chemical reaction rather than of the absorption of a photon. Non-limiting examples of chemiluminescent probes are luciferin and aequorin molecules. In some embodiments, a chemiluminescent molecule is covalently or non-covalently attached to an immune biomarker disclosed herein, such that the excited electronic state can be quantified to determine directly to the amino acid sequences disclosed.

[0107] As used herein, an “enzyme” can be any partially or wholly proteinaceous molecule which carries out a chemical reaction in a catalytic manner upon exposure to a substrate. Such enzymes can be native enzymes, fusion enzymes, proenzymes, apoenzymes, denatured enzymes, famesylated enzymes, ubiquitinated enzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linked enzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurring or artificially-generated mutant enzymes, enzymes with side chain or backbone modifications, enzymes having leader sequences, and enzymes complexed with non- proteinaceous material, such as proteoglycans, proteoliposomes. Enzymes can be made by any means, including natural expression, promoted expression, cloning, various solution-based and solid-based peptide syntheses, and similar methods known to those of skill in the art. In some embodiments, signal peptides or probes bound to the amino acids or membrane-linked proteins on the surface of immune cells disclosed herein.

[0108] As used herein, "conservative" amino acid substitutions may be defined as set out in Tables A, B, or C below. Protein and antigen sequences disclosed herein may include those amino acid sequences that include or a plurality of conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the technology. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.

[0109] Table A -- Conservative Substitutions I Side Chain Characteristics Amino Acid Aliphatic Non-polar G A P I L V F Polar - uncharged CSTMNQDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Polar - charged D E K R Aromatic H F W Y Other NQDE

[0110] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77) as set forth in Table B.

[0111] Table B -- Conservative Substitutions II Side Chain Characteristic Amino Acid Non-polar (hydrophobic) Aliphatic: A L I V P. Aromatic: F W Y Sulfur-containing: M Borderline: G Y Uncharged-polar Hydroxyl: S T Y Amides: N Q Sulfhydryl: C Borderline: G Y Positively Charged (Basic): K R H Negatively Charged (Acidic): D E Alternately, exemplary conservative substitutions are set out in Table C.

[0112] Table C – Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Asp (D) Glu Cys (C) Ser Thr

[0113] As used herein, the term “sample” refers generally to a limited quantity of something which is intended to be similar to and represent a larger amount of that thing. In theDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION present disclosure, a sample is a collection, fluid, whole blood, plasma, swab, particular isolated cell type (such as peripheral blood mononuclear cells (PBMCs)) brushing, scraping, biopsy, removed tissue, surgical resection that is to be tested. In some embodiments, the sample is whole blood comprising an immune cell. As used herein, “control sample” or “reference sample” refer to samples with a known presence, absence, or quantity of substance being measured, that is used for comparison against an experimental sample. In some embodiments, the sample may be a tissue sample from a mammal or a human. In some embodiments, the human is exhibiting signs of allergy or suspected of having an allergic response in the presence of an antigen (and in some embodiments, the antigen used to activate or elicit an immune response in the device disclosed herein.

[0114] In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi- permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample of cells to techniques such as amplification or reverse transcription of mRNA, cytometry, isolation and / or purification of certain components, etc. in some embodiments, the methods disclosed herein do not comprise a processed sample.

[0115] In some embodiments, the sample is homogenized. It may be homogenized by any method known in the art. It may be homogenized with a mixer, a tissue grinder or a high- pressure homogenizer. Samples can be homogenized for any length of time necessary to create a uniform homogenate.

[0116] A filter as used herein may be any filter known in the art, including a cation exchange membrane filter, such as Nafion or similar perfluorinated ionomers. Anion exchange membranes may also be used, as well as various polymeric hydrogels such as acrylamide, poly(ethylene glycol) diacrylate, poly(2-hydroxylethyl methacrylate), or poly(vinyl alcohol). Additionally, other exemplary embodiments may include mechanisms for quantitative analysis of the color change by means of photodiodes and sensors or microfluidic devices that require smaller amounts of reagent and samples.

[0117] The term “sorting” as used herein means that, in respect to cells from a sample, cells are separated and / or unmixed in at least one vessel. In some embodiments, unmixing means that the cells mixed in at least one vessel are separated from one or a plurality of other cells in the sample. In some embodiments, unmixing means that the cells are unmixed in at least one vessel and manipulated by any means, such as labeling, separating, and / or unpooling. In someDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION embodiments, unmixing means that the cells mixed in at least one vessel are manipulated and separated during detection by labeling them with antibodies specific for proteins expressed on the cell surface, ejected from a device disclosed herein, and then exposed to flow cytometry based upon the presence or absence of the antibodies.

[0118] The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. "Identical" or "identity" as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast programDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001.

[0119] As used herein, “specific for” or “specifically binds to” means that the binding affinity of a probe to a specified target nucleic acid sequence or amino acid sequence, such as a tripeptidyl peptidase, is statistically higher than the binding affinity of the same substrate to a generally comparable, but non-target amino acid sequence. The substrate's Kd to each nucleotide sequence can be compared to assess the binding specificity of the substrate to a particular target nucleotide sequence.

[0120] As used herein, the terms “immune-related disorder” are those disorders associated with dysfunction of cell types identified in the disclosure. Immune-related disorder may be an allergy to an antigen identified herein, drug sensitivity (if the drug is the activation molecule or antigen), sepsis (if neutrophils are assayed and probed), and cancer, such as B cell precursor acute lymphoblastic leukemia (if B cells are activated by exposure to tumor associated antigens disclosed herein).

[0121] As used herein, the terms “unactivated” means that, in respect to a certain cell type, the cell is in a resting state, but capable of being activated by a stimulant, such as a ligand, small molecule, or ion, whose presence associates with a target protein or nucleic acid and confers a biological response. The term “unactivated” differs form the term “inactivated,” which means that the cell is biologically incapable of being stimulated, such as in the case of a terminally differentiated cell that is free of a biologically effective amount of a molecule that associates with a stimulant. Sample Preparation Devices

[0122] FIG. 3 schematically depicts a sample preparation device 200 (or “device 200”) configured in accordance with several embodiments of the present technology. The sampleDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION preparation device 200 is configured to prepare a biological sample, such as a sample of whole blood, for subsequent testing. In some embodiments, the device 200 is configured to divide a sample of whole blood into multiple smaller samples (also referred to herein as sub-samples) and expose each smaller sample to a different stimulating agent. The device 200 may be configured to divide the sample into sub-samples sequentially or substantially simultaneously. The stimulating agent can comprise, for example, an antigen and / or any other substance having the potential to cause a biological change in the blood sample. Once exposed to the stimulating agent, the device 200 may further be configured to dispense each of the smaller samples into corresponding storage vessels, which may be used to contain and / or transport the samples for further testing.

[0123] As shown in FIG.3, in some embodiments the device 200 comprises an inlet 204, a plurality of outlets 206, and a plurality of reaction vessels 208 configured to be in fluid communication with the inlet 204 and the outlets 206 via one or more channels 210. The device 200 can include one or more valves (not shown) disposed in the channels 210 and configured to control flow between the reaction vessels 208 and the inlet 204 and the reaction vessels and the outlets 206. The inlet 204 is configured to be detachably coupled (e.g., via an inlet adapter) to an inlet vessel (not shown) containing the blood sample for processing. Each of the outlets 206 are configured to be detachably coupled (e.g., via an inlet adapter) to a storage vessel (not shown), which receives the blood samples after processing within the reaction vessels 208.

[0124] While only two reaction vessels 208 are shown in FIG. 3, the device 200 may comprise additional reaction vessels 208, such as three, four, five, six, seven, eight, or more reaction vessels. Each of the reaction vessels 208 may comprise a sidewall defining a lumen, and each of the reaction vessels 208 includes a reagent disposed on an inner surface of the sidewall and / or within the reaction vessel lumen (e.g., a powder, a bead, a liquid, etc.). The reagent may be a stimulating agent 214, a staining agent, a stimulating agent and a staining agent, a washing agent (e.g., a washing solution, such as a wash buffer), or a fixation agent. In the example shown in FIG.3, one of the reaction vessels 208 includes a stimulating agent 214 (an “experimental reaction vessel”) and the other includes a stimulating agent in the form of a negative control 216 (a “negative control reaction vessel”).

[0125] In some embodiments, the device 200 comprises at least three reaction vessels: an experimental reaction vessel, a negative control reaction vessel, and a reaction vessel including a stimulating agent comprising a positive control (a “positive control reaction vessel”). In several embodiments, the device 200 does not include a positive control reaction vessel andDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION only includes a negative control reaction vessel and one or more experimental reaction vessels. In yet other embodiments, the device 200 does not include any control reaction vessels and only includes one or more experimental reaction vessels. In those embodiments in which the device 200 includes multiple experimental reaction vessels 208, each experimental reaction vessel 208 may contain a different stimulating agent. In some examples, two of more of the experimental reaction vessels 208 include the same stimulating agent.

[0126] Referring still to FIG. 3, the device 200 can include a pressure control device 212 configured to be operably coupled to the reaction vessels 208 to generate a pressure differential between the inlet 204 and the reaction vessels 208 (such that the blood sample moves from the inlet 204 into the reaction vessels 208, thereby separating the blood sample into a plurality of blood samples) and / or to generate a pressure differential between the reaction vessels 208 and the outlets 206 (such that the blood samples move from the reaction vessels 208 to the outlets 206).

[0127] In some examples, the pressure control device 212 comprises a single device configured to generate pressure (positive or negative) for moving blood through the inlet 204 to the reaction vessels 208 and pressure (positive or negative) for moving blood out of the reaction vessels 208 and through the outlets 206. The single device may comprise a pump operably coupled to each of the reaction vessels 208, a single plunger (e.g., comprising a shaft and seal at the distal portion of the shaft) disposed in a fluid channel common to all of the reaction vessels 208 (and thus configured to generate positive and negative pressure substantially simultaneously in each reaction vessel 208), or a plurality of plungers, each positioned in one of the reaction vessels 208. In the latter case, the plungers may be mechanically coupled to one another such that the plungers move as a single unit.

[0128] According to some embodiments, the pressure control device 212 can comprise multiple pressure control devices. For example, the pressure control device can comprise a first pressure control device configured to generate the pressure (positive or negative) for moving blood through the inlet 204 to the reaction vessels 208 and a second pressure control device configured to generate the pressure (positive or negative) for moving blood out of the reaction vessels 208 and through the outlets 206. In some examples, the pressure control device 212 comprises one or more pressure control devices for each reaction vessels 208, or for sub- combinations of reaction vessels 208 (e.g., a first pressure control device operably coupled to two of the three reaction vessels and a second pressure control device operably coupled to the remaining reaction vessel 208).DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0129] The pressure control device 212 can be manipulated by a user to generate pressure (positive or negative) or may be motorized. In the latter case, the device 200 can include an actuator (e.g., a button, a knob, etc.) operably coupled to the pressure control device 212 and configured to activate the pressure control device 212 when manipulated by a user. The device 200 may further include a controller configured to run the pressure control device 212 automatically to generate negative and positive pressure at predetermined time intervals and / or in response to one or more sensed parameters.

[0130] To use the device 200, a user may detachably couple an inlet vessel (such as a tube containing the sample) to the inlet 204, or otherwise place a sample source in fluid communication with the inlet 204. The user may then activate the pressure control device 212 to pull or push the sample (e.g., whole blood) from the inlet 204, through the channels 210 and into the reaction vessels 208. While moving the sample towards the reaction vessels 208, the device 200 divides the sample into smaller samples. In some embodiments, the device 200 is configured to divide the sample into smaller samples of substantially equal volume. Within the reaction vessels 208, the samples are exposed to the stimulating agent 214 (or control 216), which have the potential to cause a change to one or more cells present in the sample. In those embodiments used for basophil activation testing, the stimulating agent 214 (e.g., a suspected allergen) may activate one or more immune cells (e.g., basophils) in the sample. After mixing with the stimulating agent 214 (or control 216), the pressure control device 212 may be activated to push or pull the contents of the reaction vessels 208 through corresponding outlets 206 and into storage vessels (not shown).

[0131] As shown in FIG.4, in some embodiments one, some, or all of the reaction vessels 208 may include a staining agent 218 configured to confer a unique tag to the sample in the corresponding reaction vessels 208. The staining agent 218 may comprise, for example, one or more probes specific to an immune cell, as discussed herein. The staining agent 218 may be disposed on an inner surface of the sidewall of the corresponding reaction vessel 208 and / or within the reaction vessel lumen (e.g., as a powder, a bead, a liquid, etc.). Each reaction vessel 208 may contain a different staining agent 218.

[0132] As shown in FIG. 5, in some examples the device 200 includes first reaction vessels 208a, each containing a different stimulating agent 214, and second reaction vessels 208b, each containing a different staining agent 218. The first reaction vessels 208a can be fluidly coupled to the second reaction vessels 208b via corresponding channels 220. The channels 220 can include one or more valves (not shown) for controlling flow between the first and second reaction vessels 208a, 208b, and the channels 210 can include one or more valvesDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION for controlling flow between the first reaction vessels 208a and the inlet 204 and the second reaction vessels 208b and the outlets 206. The pressure control device 212 may comprise a single device operably coupled to both the first and second reaction vessels 208a, 208b and configured to generate pressure (positive or negative) for moving blood through the inlet 204 to the first reaction vessels 208, pressure (positive or negative) for moving blood from the first reaction vessels 208a to corresponding second reaction vessels 208b (or vice versa), and pressure (positive or negative) for moving blood out of the reaction vessels 208 and through the outlets 206. The single device may comprise any of the pressure control devices disclosed herein. Alternatively, the pressure control device 212 may comprise two or more pressure control devices. For example, the pressure control device 212 may comprise a first pressure control device operably coupled to the first reaction vessels 208a and a second pressure control device operably coupled to the second reaction vessels 208b.

[0133] In use, a user may detachably couple an inlet vessel (such as a tube containing the sample) to the inlet 204, or otherwise place a sample source in fluid communication with the inlet 204. The user may then activate the pressure control device 212 to pull or push the sample (e.g., whole blood) from the inlet 204, through the channels 210 and into the first reaction vessels 208a. While moving the sample towards the first reaction vessels 208a, the device 200 divides the sample into smaller samples then deposits each of the smaller samples into a corresponding reaction vessel. In some embodiments, the device 200 is configured to divide the sample into smaller samples of substantially equal volume. Within the first reaction vessels 208a, the samples are exposed to the stimulating agent 214 (or control 216), which have the potential to cause a change to one or more cells present in the sample. In those embodiments used for basophil activation testing, the stimulating agent 214 (e.g., a suspected allergen) may activate one or more immune cells (e.g., basophils) in the sample. After mixing the sample with the corresponding stimulating agent 214 (or control 216), the pressure control device 212 may be activated to push or pull the contents of the first reaction vessels 208a through corresponding channels 220 into corresponding second reaction vessels 208b. After mixing with the sample with the corresponding staining agent 218 in the second reaction vessels 208b, the pressure control device 212 may be activated to push or pull the contents of the second reaction vessels 208b through corresponding outlets 206 and into storage vessels (not shown).

[0134] According to some embodiments of the present technology, the device 200 may be configured to recombine the smaller samples after staining such that the device 200 dispenses a single, recombined sample through a single outlet. FIG. 6, for example, shows a device 200 having a single outlet 206. In use, after mixing the smaller samples with corresponding stainingDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION agents 218 in the second reaction vessels 208b, the pressure control device 212 may be activated to push or pull the contents of the second reaction vessels 208b through the outlet 206 and into a storage vessel (not shown).

[0135] As illustrated in FIG.7, the device 200 may be configured to wash the sample after staining. The device 200 may include third reaction vessels 208c fluidly coupled to the second reaction vessels 208b via channels 222. The channels 222 may include one or more valves (not shown) for controlling flow between the second reaction vessels 208b and the third reaction vessels 208c. Likewise, the device 200 may include one or more channels 226 extending between and fluidly coupling the third reaction vessels 208c and the outlet 206, and one or more valves disposed in the channels 226 and configured to control flow between the third reaction vessels 208c and the outlet 206. Each of the third reaction vessels 208c may comprise a washing solution, such as a wash buffer. In use, after mixing with the sample with the corresponding staining agent 218 in the second reaction vessels 208b, the pressure control device 212 may be activated to push or pull the contents of the second reaction vessels 208ba into the third reaction vessels 208c. After mixing the smaller samples with the corresponding washing solution in the third reaction vessels 208c, the pressure control device 212 may be activated to push or pull the contents of the third reaction vessels 208bc through a filter to remove excess washing solution before activating the pressure control device 212 to push or pull the contents of 208bc to the outlet 206 and into a storage vessel (not shown). In any of the embodiments disclosed herein, the storage vessel and / or outlet(s) may contain a lysis and / or a stabilizing agent (such as a fixation buffer) to preserve the cells.

[0136] Any of the devices disclosed herein (e.g., devices 200, devices 300, etc.) may optionally include a heating element configured to modulate the temperature around the reaction vessels 208. Additionally or alternatively, the device 200 may optionally include a mixing element configured to assist in mixing the contents of the reaction vessel 208. For example, the device 200 may include a mixing bead in some or all of the reaction vessels 208 (including first, second, and / or third reaction vessels 208a, 208b, 208c), as well as a magnet configured to move (manually or automatically) relative to the reaction vessels 208 to cause movement of the mixing bead. Other means for mixing the contents of the reaction vessels 208 are possible. Additionally or alternatively, any of the devices 200 can include an inlet, separate from inlet 204, configured to be fluidly coupled to a buffer source. The pressure control device 212 can be activated to move the buffer from the buffer source into the reaction vessels 208 (including first, second, and / or third reaction vessels 208a, 208b, 208c), for example to reconstitute a desiccated form of the stimulating agent 214 and / or a desiccated form of theDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION staining agent 218 prior to delivery of the sample to the corresponding first or second reaction vessels 208a, 208b. The buffer may additionally or alternatively be used during a washing step, as discussed herein.

[0137] FIG.8 shows an example sample preparation device 800 configured in accordance with the present technology. The device 800 includes a first pressure source 812 operably coupled to first reaction vessels 808a, a second pressure source 813 operably coupled to second reaction vessels 808b, a plurality of channels 810, a sample inlet 804, a buffer inlet 805, and an outlet 806. The device 800 may include a valve 819 that is adjustable between a first state in which fluid is allowed to flow into the channels 810 from the first inlet 804 (and blocked from flowing into and / or from the second inlet 805) and a second state in which the fluid is allowed to flow into the channels 810 from the second inlet 805 (and blocked from flowing into and / or from the first inlet 804). Each of the first reaction vessels 808a include a stimulating agent 814 in the form of a lyobead and each of the second reaction vessels 808b include a staining agent 818 in the form of a lyobead. Each of the first and second reaction vessels 808a, 808b further include a mixing bead 830, and the device 800 further includes a magnet 832 configured to move longitudinally along the first and section reaction vessels 808a, 808b to agitate the magnet 834 and stir the contents of the reaction vessels. In the embodiment shown in FIG.8, the first and second pressure sources 812, 813 comprise a plurality of plungers 817 disposed in corresponding reaction vessels. In some embodiments, the first and second pressure sources each comprise a pump, or the device 800 includes a single pump 800 configured to be operably coupled to both the first and second reaction vessels 808a, 808b.

[0138] FIGS.9A–9E illustrate a method for using the device 800. As shown in FIG.9A, a user may detachably couple an inlet vessel (shown schematically in FIG. 9A) to the second inlet 805, or otherwise place a buffer source in fluid communication with the second inlet 805. The user may then activate the second pressure control device 813 (e.g., by withdrawing the plungers 817 as a single unit, as indicated by arrow A1) with the valve 819 open to the second inlet 805 (and closed to the first inlet 804), thereby pulling the buffer B through the second inlet 805 and into the second reaction vessels 808b. While moving the buffer B towards the second reaction vessels 808b, the device 800 divides the buffer B into smaller buffer volumes. In some embodiments, the device 800 is configured to divide the buffer into smaller buffer volumes that are substantially equal. Within the second reaction vessels 808b, the buffer B mixes with the lyobead comprising the staining agent 818, thereby reconstituting the staining agent 818. In some embodiments, the magnet 832 may be activated to mix the buffer B and the staining agent 818.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0139] As shown in FIG. 9B, the user may activate the first pressure control device 812 (e.g., by withdrawing the plungers 817 as a single unit, as indicated by arrow A2) to pull the buffer B through the second inlet 805 and into the first reaction vessels 808a. While moving the buffer B towards the first reaction vessels 808a, the device 800 divides the buffer B into smaller buffer volumes. In some embodiments, the device 800 is configured to divide the buffer into smaller buffer volumes that are substantially equal. Within the first reaction vessels 808a, the buffer B mixes with the lyobead comprising the stimulating agent 814, thereby reconstituting the stimulating agent 814. In some embodiments, the magnet 832 may be activated to mix the buffer B and the stimulating agent 814.

[0140] As shown in FIG. 9C, the user may again activate the first pressure control device 812 (e.g., by further withdrawing the plungers 817 as a single unit, as indicated by arrow A3), this time with the valve 819 open to the first inlet 804 (and closed to the second inlet 805). As such, the first pressure control device 812 pulls the sample S through the first inlet 804 and into the first reaction vessels 808a. While moving the sample S towards the first reaction vessels 808a, the device 800 divides the sample S into smaller samples. In some embodiments, the device 800 is configured to divide the sample into smaller samples of substantially equal volumes. Within the first reaction vessels 808a, the sample S mixes with the reconstituted stimulating agent 814. In some embodiments, the magnet 832 may be activated to mix the sample S and the stimulating agent 814.

[0141] As shown in FIG.9D, the user may activate the second pressure control device 813 (e.g., by withdrawing the plungers 817 as a single unit, as indicated by arrow A4) and / or the first pressure control device 812 (e.g., by advancing the plungers 817 as a single unit, as indicated by arrow A5) to push and / or pull the samples S from the first reaction vessels 808a into the second reaction vessels 808b. In the second reaction vessels 808, the samples S mix with the staining agents 818. In some embodiments, the magnet 832 may be activated to mix the sample S and the staining agents 818.

[0142] As depicted in FIG. 9E, the second pressure source 813 can be activated (e.g., by advancing the plungers 817 as a single unit, as indicated by arrow A6) to eject the samples S from the second reaction vessels 808b. The samples are recombined into a recombined sample RS and dispensed into a storage vessel through outlet 806.

[0143] According to some embodiments of the present technology, the reaction vessels, inlet, and outlet(s) can be integrated into a single apparatus with the pressure control device 212. In other embodiments, the reaction vessels, inlet, and outlet(s) comprise a cartridge, and the pressure control device 212 comprises a separate apparatus configured toDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION receive, and thereby operably couple to, the cartridge. In several examples, the present technology includes a single pressure control device configured to receive multiple cartridges. Each of the cartridges, for example, can corresponding to a separate blood sample.

[0144] FIG. 10 illustrates an example sample preparation device 300 configured in accordance with several embodiments of the present technology. The device 300 includes a fluid circuit 310 comprising an inlet 320, an outlet 330 and a plurality of reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The plurality of reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h are in fluid communication with the inlet 320 and the outlet 330. The device 300 also includes at least one valve 350a positioned in the fluid circuit 310 between the inlet 320 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h and at least one valve 360a positioned between the outlet 330 and the reaction vessels340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The at least one valve is adjustable between a first and second state. The first state allows for fluid flow between the inlet 320 and the reaction vessels340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The second state allows for fluid flow between the outlet 330 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h.

[0145] In some embodiments, the at least one valve comprises first valves positioned in the fluid circuit between the inlet 320 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The first valves may be adjustable between an open position and a closed position. The open position allows for fluid flow between the inlet 320 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The device 300 may also include second valves positioned in the fluid circuit 310 between the outlet 330 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The second valves may be adjustable between an open position and a closed position. The open position allows for fluid flow between the outlet 330 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h.

[0146] Plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h are positioned within the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h in the device illustrated in FIG.10.

[0147] In the left-most panel of FIG. 10, the inlet 320 is in fluid communication with an inlet vessel (sample input) containing a sample. The sample may be whole blood. Further, the outlet 330 in the same panel is in fluid communication with an output vessel (collection output). The device may comprise at least one of the inlet vessel or the output vessel.

[0148] As illustrated in FIG.10, each of the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h comprises a reaction region 380 containing a reagent. In a first operational condition, the reaction region 380 contains a stimulating agent, a staining agent, and / or anotherDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION reagent. In some embodiments, the reaction region 380 includes a combination of at least two probes specific for an immune cell in a dried or powdered formulation. In a second operational condition, the reaction region 380 may comprise a combination of the stimulating agent, the staining agent, and / or another reagent, and the blood sample.

[0149] Still referring to FIG. 10, in some embodiments each reaction vessel 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h of the device 300 may comprise staining agent in the form of a combination of at least two probes specific for an immune cell. The combination may be different in one or more, or each of the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The device 300 may comprise a stimulating agent in the form of an immune cell activator positioned within or proximate to the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The device 300 may comprise one or a plurality of immune cells chosen from one or a combination of: a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil, and / or platelets.

[0150] The panels of FIG. 10 illustrate the device 300 in operation. The left-most panel illustrates the device 300 ready to receive sample, which may be whole blood. The second panel from the left illustrates the plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h being withdrawn with the first valves open such that the sample flows into the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The second panel from the right illustrates a stage when reagent is mixed with the sample, and optionally reconstituted. The right-most panel illustrates the device where the first valves are closed and the second valves are open. Further, the plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h are pushed into the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h such that reaction product is dispensed into the storage vessel.

[0151] FIG. 11 illustrates another example device 300 configured in accordance with the present technology. As depicted, the device 300 includes separate simulation and stain areas and is pressure controlled. The device 300 includes similar features to that of FIG. 10. Here, the at least one valve is a two-way valve, which depending on its state allows fluid flow through the fluid circuit from the inlet vessel (sample input) to the reagent vessels, or from the reagent vessels to the output vessel (collection output). Further, the reagent storage and reaction regions are split between stain and a stimulate subregions. Still further, the plungers are operably connected to a pressure source, which is operably connected to a pressure controller. The pressure controller is in turn controlled by a microcontroller in operably connection to a user interface.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0152] Referring to FIG. 12 illustrates another example device 300 configured in accordance with the present technology. The device 300 includes separate stimulation and stain chambers, similar to the device of FIG.11, with the addition of individual channel control. The device 300 illustrated also includes an addition buffer inlet connected to a two-way valve interposed in a fluid circuit connecting a buffer input, an inlet vessel (sample input), an outlet vessel (collection output), and the reaction vessels. Further, there are additional valves interposed between each staining region and each stimulant region allowing the individual channel control.

[0153] FIG.13 illustrates an automated version of the device 300 of FIG.12. As shown in FIG. 13, the device 300 may include a motor coupled to the pressure control device (in this instance, a plurality of plungers, though any pressure source may be used, including a pump). The motor may be a stepper motor and is coupled to the pressure control device via a lead screw. Other configurations are possible. The device 300 further includes a microcontroller in communication with the motor and configured to activate and deactivate the pressure control device. The device 300 may optionally include a user interface.

[0154] FIG. 14 illustrates another example device 300 configured in accordance with the present technology. The device 300 of FIG.14 can be generally similar to that of FIG.10, and the process flow in FIG.14 is similar to that illustrated in FIG.10. Here, however, a raw sample collection vessel 340i is added to the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, 340h. The plunger within the raw sample collection vessel 340i may be decoupled from the rest of the plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h such that the raw sample is retained in the raw sample collection vessel 340i when the remaining plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h are inserted back into the reaction vessels. Further, a valve is interposed between the raw sample collection vessel 340i and the rest of the fluid circuit such that raw sample may be retained in the raw sample collection vessel 340i and not mixed with reagent.

[0155] FIGS. 15–18 depict an example device 300 configured in accordance with the present technology. The device 300 can include a housing 317 supporting and / or enclosing and / or defining the fluid circuit. The reaction vessels 340 can comprise channels defined by the housing 317 and may be positioned at or near a first set of conduits connecting the inlet to the channels. The fluid circuit comprises an inlet, a first set of conduits in fluid communication to the reaction vessels, a second set of conduits connecting the reaction vessels to the outlet. The first and second conduits are branched, positioned parallel to one another and intersect a valve channel that is positioned orthogonally or substantially orthogonally across the first andDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION second sets of conduits at intersection points. The valve channel may be, as illustrated, positioned transverse to a width dimension of the housing. The valve channel may comprise a valve element in operable contact with a plurality of valves. One valve may be positioned at each intersection point. The valve element in the valve channel may comprise two or four total valves and the valve element is adjustable and operably connected to the valves such that movement of the valve element opens and closes the valves. The valve element may be cylindrical or semicylindrical. The valve element may be rotatable about its longitudinal axis and may have at least a first and a second operable position. In the first operable position, a valve positioned at an intersection region proximate to the inlet may be open and a valve positioned at an intersection region proximate to the outlet may be closed. In the second operable position, the valve may be positioned at an intersection region proximate to the inlet may be closed and a valve positioned at an intersection point proximate to the outlet may be open. The valve may be operably connected to a dial positioned on the side of the housing. The dial may be movable radially between the first and second operable positions of the valve.

[0156]

[0157] FIGS.19, 20, 21, and 22 illustrate an example device 300 configured in accordance with the present technology. The device 300 includes a fluid circuit 310, which includes an inlet 320 and an outlet 330. In the embodiment illustrated, the device includes eight reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h, and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h are in fluid communication with the inlet 320 and the outlet 330. In some embodiments, the device 300 can include more or fewer than eight reaction vessels. The reaction vessels 340a and 340h are labeled in FIG.19 and FIG.22, while the reaction vessels 340b, 340c, 340d, 340e, 340f, and 340g are unlabeled but between the reaction vessels 340a and 340h.

[0158] Referring to FIGS. 19–22, the device 300 can include a valve 311 (best shown in FIGS.20 and 21) positioned in the fluid circuit 310. The valve 311 can be positioned between the inlet 320 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. The valve 311 may also be positioned between the outlet 330 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. Further, the valve 311 can be adjustable between a first state (labeled Position 1, FIG.20) and second state (labeled Position 2, FIG.21). The first state allows for fluid flow between the inlet 320 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h, and the second state allows for fluid flow between the outlet 330 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. FIG. 19 illustrates an inlet vessel 391 (in the example illustrated, a blood container) positioned via anDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION inlet adaptor 390 around the inlet 320. FIG. 22 illustrates the inlet 320 and the inlet adaptor 390 without the inlet vessel 391. Both FIGS.19 and 22illustrate the outlet 330 encircled by an outlet adaptor 392, which includes a thread that may engage an storage vessel 393 (not illustrated in FIGS. 19, 20, 21, and 22 but see FIGS. 24E and 24F for an exemplary storage vessel 393 engaging an embodiment of a device herein).

[0159] Referring to FIG. 19, the device 300 is illustrated with plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h. Each of the plungers are mechanically connected to a handle 395. Still referring to FIG.19, the handle 395 is illustrated in a position distal to the inlet 320, while in FIG. 22, the handle 395 is illustrated in a position more proximal to the inlet 320. Likewise, FIG.19 illustrates the plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h in a position distal to the inlet 320, while FIG. 22 illustrates the plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h in a position more proximal to the inlet 320.

[0160] In operation, the valve may be set in the first state (Position 1 in FIG.20) while the inlet vessel 391 is in place at the inlet adaptor 392 and with the inlet 320 in contact with a sample therein. In the embodiment illustrated, the sample may be blood in the labeled blood container. Further, the handle 395 may be in the position illustrated in FIG. 22, being more proximal to the inlet. In this state, the fluid circuit 310 is open between the inlet 320 and the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. Movement of the handle 395 to the position distal to the inlet 320, as shown in FIG. 19, would then withdraw the plungers 394a, 394b, 394c, 394d, 394e, 394f, 394g, and 394h and sample within the inlet vessel 391 may flow into the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. A storage vessel 393 may be engaged with the outlet adaptor 392. The valve may then be set to the second state (Position 2, FIG. 21), and the handle 395 returned to the position more proximal to the inlet as shown in FIG. 22. With the valve in the second state, the fluid circuit 310 would be open between the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h and the outlet 330 and product would flow out of the outlet 330. When the storage vessel 393 is engaged with the outlet adaptor 392, the product would be collected in the storage vessel 393.

[0161] The device 300 illustrated in FIGS. 19–22 may comprise a reagent in each of the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. The reagent may comprise a stimulating agent, a staining agent, and / or another reagent. In some embodiments, the reagent may comprise a combination of at least two probes specific for an immune cell in each of the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h. The combination may be different in each of the reaction vessels 340a, 340b, 340c, 340d, 340e,DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 340f, 340g, and 340h. There may be a reaction region where when sample is drawn from the inlet 320 and into the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h, the sample reacts with the reagent. In the embodiment illustrated, the sample is blood and the blood has the potential to react with the reagent. There may be a reagent storage region where reagent is stored prior to use of the device. The reagent storage region may be a region within each of the reaction vessels 340a, 340b, 340c, 340d, 340e, 340f, 340g, and 340h and may be in fluid communication with the reaction region.

[0162] The reagent in the reagent storage region may comprise a stimulating agent, a staining agent, and / or another reagent. In some embodiments, each reagent storage region comprises a combination of at least two probes specific for an immune cell in a dried or powdered formulation. The reaction region may comprise a combination of at least two probes specific for an immune cell, and one or a plurality of immune cells. The device 300 may comprise an immune cell activator positioned within or proximate to the reaction vessels. The device 300 may comprise one or a plurality of immune cells chosen from one or a combination of: a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil, and / or platelets.

[0163] In some embodiments, the storage vessel (not shown) may be a tube (e.g., a 15- milliliter conical tube) comprising protruding spiral threads around an opening of the tube. Other storage vessels are possible. The outlet adaptor 392 can be configured to detachably coupled the storage vessel to the outlet. In some examples, the outlet adaptor 392 comprises a plastic protrusion comprising an inner and outer surface. The inner surface may protrude around the outlet 330 and comprise a circular or semicircular spiral inlay positioned around the outlet 330. The tube and the outlet adaptor 392 may define a joint positioning the opening of the tube in fluid communication with the fluid circuit 310 at the outlet 330. Other outlet adaptors are possible.

[0164] The storage vessel may be in fluid communication with the fluid circuit 310 and positioned proximate to the outlet 330 of the fluid circuit 310. In some embodiments, the storage vessel comprises one or more stabilizing agents. Further embodiments herein include devices with fewer numbers of reaction vessels, or greater number of reaction vessels. The number of plungers and the fluid circuit can be adapted to the number of reaction vessels. The number or reaction vessels may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

[0165] FIG. 23 illustrates a heater 400. A heater of embodiments herein may include a device insert slot into which a device herein may be inserted.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0166] FIGS. 24A–24F illustrate a process flow of a device 300 and a heater 400. The device may be an embodiment as illustrated in FIGS.19–22. However, other embodiments of a device herein may include a similar process flow. As illustrated in FIG.24A, a device 300 is engaged with an inlet vessel and the plungers are proximal to the inlet. FIG. 24E illustrates withdrawal of the plungers and entry of the sample into the reaction vessels. FIG. 24C illustrates further withdrawal of the sample such that it is entirely within the reaction vessels. FIG.24D illustrates insertion of the device containing sample and reagent into the heater 400, where application of heat facilitates the reaction. FIG.24E illustrates connection of the device 300 with an output vessel 393. FIG. 24F illustrates depression of the plungers and collection of reaction product in the output and / or storage vessel 393.

[0167] The embodiments illustrated in FIGS. 15–22 include eight reaction vessels, although more or fewer reaction may be used. FIG. 14 includes an additional raw sample collection vessel. A device of embodiments herein may have a different number of reaction vessels and corresponding plungers, valves, and the like. In an embodiment a device has a plurality of reaction vessels. In embodiments, the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 reaction vessels. Each reaction vessel may have a compartment. Each compartment across the reaction vessels may be uniformly cylindrical in shape and in parallel orientation to one another along their respective longitudinal axis. In some embodiments the plurality of reaction vessels includes a first, second, third, fourth, fifth, sixth, seventh and seventh and eighth compartment. Each compartment may comprise at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein. Each of the compartments may further comprises a plunger. Each plunger may be mechanically connected a movable handle positioned on the outside of the housing, such that movement of the handle laterally from the exterior of the device simultaneously slides the plungers along their longitudinal axis in the cylindrical or semicylindrical compartments. Each compartment may comprise an adjustable valve positioned opening and closing fluid flow from the compartments to the outlet. A device herein may comprise an inlet vessel and / or a storage vessel (also referred to herein as an “output vessel”). The inlet vessel may be attached to an adaptor at an inlet configured for flow of sample from the inlet vessel to the reaction vessels through a fluid circuit when valve(s) are open between the inlet vessel and the reaction vessels. The outlet may be attached to an outlet adaptor positioned at or around an outlet of the fluid circuit configured for fluid flow from the outlet into the storage vessel in an operable condition wherein one or a plurality of valves are open between the compartments and the outlet. The storage vessel may comprise one or a plurality of stabilizing agents.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0168] The disclosure relates to a sample preparation device which, in some embodiments, comprises a sample comprising cells from a subject. In some embodiments, the sample preparation device is stored at about 4ºC for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days. In some embodiments, the sample preparation device is stored at about 10ºC, 15ºC, 20º, or 25º for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days. In some embodiments, the sample preparation device is stored at about 4ºC for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days, wherein the device comprises cells positioned within at least one vessel. In some embodiments, the sample preparation device is stored at about 4ºC for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days, wherein the device comprises cells positioned within at least one vessel and are labeled by at least one probe. Methods of the disclosure include, in some embodiments, exposing the device disclosed herein to a sample from a subject comprising a plurality of cells by sliding one or a plurality of disclosed plungers in order to draw cells into the one or plurality of vessels, exposing the one or plurality of cells to one or plurality of probes in the one or plurality of vessels, and then storing the device at about 4ºC, 5ºC, 6ºC, 7ºC, 8ºC, 9ºC, 10ºC, 15ºC, 20º, or 25º for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days, In some embodiments, the method further comprises analyzing the cells by expelling the labeled cells through an outlet and interrogating the labeled cells for quantification, identification and / or cytometry by detection of the one or plurality of probes.

[0169] Embodiments herein can comprise a composition comprising a plurality of immune cells. The immune cells are divided among and positioned within one or a plurality of compartments or vessels within the composition. In some embodiments, In some embodiments, the composition comprises a solid support, such as a plastic or glass, comprising an upright, contiguous series of sided panels defining sidewalls around a bottom reaction surface. In some embodiments, the bottom reaction surface is the bottom region of a vessel or well. In some embodiments, the composition comprises a first, second, third, fourth, fifth and sixth compartment or vessel. The compartments can each comprise: at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein. The disclosure relates to a composition comprising a plurality of compartments for simultaneous barcoding or labeling of immune cells. In some embodiments, the composition comprises a plurality of compartments connected in a fluid circuit, the fluid circuit comprising at least two, three, four, five, six, seven or eight or more probes specific for the immune cells. In some embodiments, the immune cells are divided among and positioned within a first, second, third, fourth, fifth and sixth compartments or reaction vessels; wherein each compartment comprises at least one combination of two different probes specific for an immune cell and the combinations in each compartment being different combination. In some embodiments, theDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION first, second, third, fourth, fifth and sixth compartments each comprise at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein, respectively; wherein each compartment corresponds to a different pair of probes positioned in each compartment. Some embodiments of the composition include (i) at least about six reaction vessels, each reaction vessel comprising a different pair of probes specific for an immune cell; and (ii) an antigen positioned in fluid communication with the reaction vessels. In some embodiments, the antigen is optionally positioned in one or a plurality of the reaction vessels. In some embodiments, the antigen is positioned in reaction vessels that comprise an experimental set of probes specific for an activated immune cell and a set of vessels comprising a control set of probes for unactivated immune cells are free of an antigen. In some embodiments, the antigen is desiccated or in solid form and, in a first operational mode, fluid from a sample is positioned within the fluid circuit capable of dissolving the solid form of the antigen upon exposure to the fluid. Similarly, in some embodiments, the probes are positioned in the reaction vessels in solid form, and in a first operational mode, fluid from a sample is positioned within the fluid circuit capable of dissolving the solid form of the probe upon exposure to the fluid.

[0170] The first immune cell surface protein may be CD193 or a functional variant thereof. The second immune cell surface protein may be CD123 or a functional variant thereof.

[0171] The probes specific to CD193 may be chosen from: BV421, BV605 or derivatives thereof. The probes specific to CD123 may be chosen from: PE, PE / Cy7, APC, BV785 or a derivative thereof.

[0172] The composition may further comprise: (i) an inlet vessel in fluid communication with each of the plurality of compartments, preferably the first, second, third, fourth, fifth and sixth compartments; and (ii) immune cell activator positioned within or proximate to the inlet vessel or each compartment.

[0173] The immune cells may be chosen from one or a combination of: a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil and / or platelets. In some embodiments, the immune cells are isolated basophils, isolated prior to loading into the device. In some embodiments, the immune cells are isolated NK cells, isolated prior to loading into the device. In some embodiments, the immune cells are isolated B cells, isolated prior to loading into the device. In some embodiments, the immune cells are isolated T cells, isolated prior to loading into the device. In some embodiments, the immune cells are isolated PBMCs, isolated prior to loading into the device. In some embodiments, the immune cells are isolated eosinophils, isolated prior to loading into the device. In some embodiments, the immune cellsDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION are isolated macrophages, isolated prior to loading into the device. In some embodiments, the immune cells are isolated monocytes, isolated prior to loading into the device. In some embodiments, the immune cells comprise or consist of human immune cells. In some embodiments, the cells are non-human immune cells. In some embodiments, the immune cells are from a subject and the subject seeks a diagnosis for an unknown immune-related disorder, such as an allergy to exposure of one or a plurality of antigens.

[0174] Embodiments herein comprise a composition comprising a plurality of immune cells. The immune cells are divided among and positioned within a plurality of compartments, preferably a first, second, third, fourth, fifth and sixth compartment. The compartments each comprises: at least about two probes, a first probe specific for a first immune cell surface protein and a second probe specific for a second immune cell surface protein.

[0175] The first immune cell surface protein may be CD193 or a functional variant thereof. The second immune cell surface protein may be CD123 or a functional variant thereof.

[0176] The probes specific to CD193 may be chosen from: BV421, BV605 or derivatives thereof. The probes specific to CD123 may be chosen from: PE, PE / Cy7, APC, BV785 or a derivative thereof. In some embodiments, the immune cell surface protein comprises: CD63, CD203a, CD203b and CD203c.

[0177] The composition may further comprise: (i) an inlet vessel in fluid communication with each of the plurality of compartments, preferably the first, second, third, fourth, fifth and sixth compartments; and (ii) immune cell activator positioned within or proximate to the inlet vessel or each compartment.

[0178] The disclosure relates to a device comprising an antigen. In some embodiments, the antigen is dried and / or solid form. In some embodiments, the antigen is in liquid form. The antigen can be positioned at any place within the composition, device or system disclosed herein. In some embodiments, the antigen is chosen from Table 5 below or is an amino acid sequence comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any antigen sequence in Table 5. In some embodiments, the antigen is a functional fragment of the antigen sequence provided in Table 5. As used herein, the term "variants" is intended to mean substantially similar sequences. For polypeptide sequences, a variant comprises a polypeptide molecule having deletions (i.e., truncations) at the 5' and / or 3' end; deletion and / or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitution of at one or more sites in the native polypeptide. As used herein, a "native" nucleic acid molecule or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For nucleic acidDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION molecules, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the disclosure. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the disclosure. Generally, variants of a particular nucleic acid molecule or amino acid sequence of the disclosure will have at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.

[0179] Variants of a particular nucleic acid molecule of the disclosure (i.e., the reference amino acid sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant nucleic acid molecule and the polypeptide encoded by the reference nucleic acid molecule. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of nucleic acid molecule of the disclosure is evaluated by comparison of the percent sequence identity shared by the two polypeptides that they encode, the percent sequence identity between the two encoded polypeptides is at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the term "variant" protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and / or C-terminal end of the native protein; deletion and / or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a protein of the disclosure will have at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the disclosure may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 20, 15, 10, 9, 8, 7, 6, 5, as few as 4, 3, 2, or even 1 amino acid residue. The proteins or polypeptides of the disclosure may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations areDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION generally known in the art. For example, amino acid sequence variants and fragments of the proteins can be prepared by mutations in the nucleic acid sequence that encode the amino acid sequence recombinantly.

[0180] In some embodiments, the antigen sequence is a polypeptide variant of the protein sequence identified in Table 5 or disclosed herein. In some embodiments, the antigen is a polypeptide encoded by the nucleotide sequence set forth in Table 5 or disclosed herein or a functional variant of the polypeptide encoded by the nucleotide sequence set forth in Table 5 or disclosed herein comprising about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide encoded by the nucleotide sequence set forth in Table 5.

[0181] Table 5 – Antigen Sequences Allergen Antigen / Components / Proteins GenBank Nucleotide Seq. Protein Seq.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Allergen Antigen / Components / Proteins GenBank Nucleotide Seq. Protein Seq. Cor a 8 AF329829DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION In some embodiments, the antigen comprises a parvalubumins and tropomyosin from a fish. In some embodiments, the antigen is chosen from any sequence disclosed in https: / / www.allergen.org. or those sequence comprising about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequences disclosed on www.allergen.org. Sequences from Table 5. Wheregiven, a nucleic acid sequence can be inferred and vice versa. AAB00861 1 mrgrvsplml llgilvlasv sathaksspy qkktenpcaq rclqscqqep ddlkqkaces 61 rctkleydpr cvydprghtg ttnqrsppge rtrgrqpgdy dddrrqprre eggrwgpagp 121 rerereedwr qpredwrrps hqqprkirpe gregeqewgt pgshvreets rnnpfyfpsr 181 rfstrygnqn grirvlqrfd qrsrqfqnlq nhrivqieak pntlvlpkha dadnilviqq 241 gqatvtvang nnrksfnlde ghalripsgf isyilnrhdn qnlrvakism pvntpgqfed 301 ffpassrdqs sylqgfsrnt leaafnaefn eirrvlleen aggeqeergq rrwstrssen 361 negvivkvsk ehveeltkha ksvskkgsee egditnpinl regepdlsnn fgklfevkpd 421 kknpqlqdld mmltcveike galmlphfns kamvivvvnk gtgnlelvav rkeqqqrgrr 481 eeeededeee egsnrevrry tarlkegdvf impaahpvai nasselhllg fginaennhr 541 iflagdkdnv idqiekqakd lafpgsgeqv ekliknqkes hfvsarpqsq sqspsspeke 601 spekedqeee nqggkgplls ilkafn (SEQ ID NO: 1) AAA60336 1 mrgrvsplml llgilvlasv satqakspyr ktenpcaqrc lqscqqepdd lkqkacesrc 61 tkleydprcv ydtgatnqrh ppgertrgrq pgdydddrrq prreeggrwg paeprerere 121 edwrqpredw rrpshqqprk irpegregeq ewgtpgsevr eetsrnnpfy fpsrrfstry 181 gnqngrirvl qrfdqrskqf qnlqnhrivq iearpntlvl pkhadadnil viqqgqatvt 241 vangnnrksf nldeghalri psgfisyiln rhdnqnlrva kismpvntpg qfedffpass 301 rdqssylqgf srntleaafn aefneirrvl leenaggeqe ergqrrrstr ssdnegvivk 361 vskehvqelt khaksvskkg seeeditnpi nlrdgepdls nnfgrlfevk pdkknpqlqd 421 ldmmltcvei kegalmlphf nskamvivvv nkgtgnlelv avrkeqqqrg rreqeweeee 481 edeeeegsnr evrrytarlk egdvfimpaa hpvainasse lhllgfgina ennhriflag 541 dkdnvidqie kqakdlafpg sgeqveklik nqreshfvsa rpqsqspssp ekedqeeenq 601 ggkgpllsil kafn (SEQ ID NO: 2) AAK96887 1 makltilval alfllaahas arqqwelqgd rrcqsqlera nlrpceqhlm qkiqrdedsy 61 erdpyspsqd pyspspydrr gagssqhqer ccnelnefen nqrcmcealq qimenqsdrl 121 qgrqqeqqfk relrnlpqqc glrapqrcdl dvesgg (SEQ ID NO: 3) AAN77576 1 makltilval alfllaahas arqqwelqgd rrcqsqlera nlrpceqhlm qkiqrdedsy 61 grdpyspsqd pyspsqdpdr rdpyspspyd rrgagssqhq erccnelnef ennqrcmcea 121 lqqimenqsd rlqgrqqeqq fkrelrnlpq qcglrapqrc dlevesggrd ry (SEQ ID NO: 4) AAC63045DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 1 rqqpeenacq fqrlnaqrpd nrieseggyi etwnpnnqef ecagvalsrl vlrrnalrrp 61 fysnapqeif iqqgrgyfgl ifpgcprhye ephtqgrrsq sqrpprrlqg edqsqqqrds 121 hqkvhrfdeg dliavptgva fwlyndhdtd vvavsltdtn nndnqldqfp rrfnlagnte 181 qeflryqqqs rqsrrrslpy spyspqsqpr qeerefsprg qhsrreragq eeeneggnif 241 sgftpefleq afqvddrqiv qnlrgetese eegaivtvrg glrilspdrk rradeeeeyd 301 edeyeydeed rrrgrgsrgr gngieetict asakknigrn rspdiynpqa gslktandln 361 llilrwlgps aeygnlyrna lfvahyntna hsiiyrlrgr ahvqvvdsng nrvydeelqe 421 ghvlvvpqnf avagksqsen feyvafktds rpsianlage nsvidnlpee vvansyglqr 481 eqarqlknnn pfkffvppsq qsprava (SEQ ID NO: 5) AAD47382 1 makllelsfc fcflvlgass isfrqqpeen acqfqrlnaq rpdnrieseg gyietwnpnn 61 qefecagval srlvlrrnal rrpfysnapq eifiqqgrgy fglifpgcps tyeepaqqgr 121 ryqsqrpprr lqeedqsqqq qdshqkvhrf negdliavpt gvafwlyndh dtdvvavslt 181 dtnnndnqld qfprrfnlag nheqeflryq qqsrqsrrrs lpyspysphs rprreerefr 241 prgqhsrrer agqeeedegg nifsgftpef leqafqvddr qivqnlwgen eseeegaivt 301 vrgglrilsp dgtrgadeee eydedqyeyh eqdgrrgrgs rgggngieet ictacvkkni 361 ggnrsphiyd pqrwftqnch dlnllilrwl glsaeygnly rnalfvphyn tnahsiiyal 421 rgrahvqvvd sngnrvydee lqeghvlvvp qnfavagksq senfeyvafk tdsrpsianf 481 agensfidnl peevvansyg lpreqarqlk nnnpfkffvp pfqqsprava (SEQ ID NO:6) AAM46958 1 mgkllalsvc fcflvlgass isfrqqpeen acqfqrlnaq rpdnrieseg gyietwnpnn 61 qefecagval srlvlrrnal rrpfysnapq eifiqqgrgy fglifpgcps tyeepaqqgr 121 rhqsqrpprr fqgqdqsqqq qdshqkvhrf degdliavpt gvafwmyndh dtdvvavslt 181 dtnnndnqld qfprrfnlag nheqeflryq qqsrrrslpy spyspqtqpk qedrefsprg 241 qhgrreragq eqeneggnif sgftpeflaq afqvddrqil qnlrgenesd eqgaivtvrg 301 glrilspdrk rrqqyerpde eeeydedeye ydeeerqqdr rrgrgsrgsg ngieeticta 361 sfkknigrnr spdiynpqag slktanelql nllilrwlgl saeygnlyrn alfvphyntn 421 ahsiiyalrg rahvqvvdsn gdrvfdeelq eghvlvvpqn favagksqse nfeyvafktd 481 srpsianlag ensfidnlpe evvansyglp reqarqlknn npfkffvpps eqslrava (SEQ ID NO: 7) AAT39430 1 kllalslcfc vlvlgassvt frqggeenec qfqrlnaqrp dnrieseggy ietwnpnnqe 61 fqcagvalsr tvlrrnalrr pfysnaplei yvqqgsgyfg lifpgcpsty eepaqegrry 121 qsqkpsrrfq vgqddpsqqq qdshqkvhrf degdliavpt gvafwmynde dtdvvtvtls 181 dtssihnqld qfprrfylag nqeqeflryq qqqgsrphyr qisprvrgde qenegsnifs 241 gfaqeflqha fqvdrqtven lrgenereeq gaivtvkggl rilspdeede ssrsppsrre 301 efdedrsrpq qrgkydenrr gykngieeti csasvkknlg rssnpdiynp qagslrsvne 361 ldlpilgwlg lsaqhgtiyr namfvphytl nahtivvaln grahvqvvds ngnrvydeel 421 qeghvlvvpq nfavaakaqs enyeylafkt dsrpsianla gensiidnlp eevvansyrl 481 preqarqlkn nnpfkffvpp fdhqsmreva (SEQ ID NO: 8) AAD56337 1 ahasamrrer grqgdsssce rqvdgvnlkp ceqhimqrim geqeqydsyn fgstrssdqq 61 qrccdelnem entqrcmcea lqqimenqcd glqdrqmvqh fkrelmnlpq qcnfgapqrc 121 dldvsggrc (SEQ ID NO: 9) AAQ91847DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 1 mgvftfedei tstvppakly namkdadsit pkiiddvksv eivegnggpg tikkltived 61 getkfilhkv esideanyay nysvvggval pptaekitfe tklvegpngg sigkltlkyh 121 tkgdakpdee elkkgkakge glfraiegyv lanptqy (SEQ ID NO: 10) ABP97433 1 mgvhtfeees tspvppaklf katvvdgdel tpklipaiqs ieivegnggp gtvkkvtave 61 dgktsyvlhk idaideatyt ydytisggtg fqeilekvsf ktkleaadgg skikvsvtfh 121 tkgdaplpde vhqdvkqksq gifkaiegyv lsn (SEQ ID NO: 11) ABX56711 1 maslkfafvm lvcmamvgap mvnaiscgqv nsalapcipf ltkggapppa ccsgvrgllg 61 alrttadrqa acnclkaaag slrglnqgna aalpgrcgvs ipykiststn catikf (SEQ ID NO: 12) ABX75045 1 lscgqvnsal apcitfltkg gvpsgpccsg vrgllgaakt tadrqaacnc lkaaagslhg 61 lnqgnaaalp grcgvsipyk iststncati kf (SEQ ID NO: 13) P01005 1 mamagvfvlf sfvlcgflpd aafgaevdcs rfpnatdkeg kdvlvcnkdl rpicgtdgvt 61 ytndcllcay siefgtnisk ehdgecketv pmncssyant tsedgkvmvl cnrafnpvcg 121 tdgvtydnec llcahkveqg asvdkrhdgg crkelaavsv dcseypkpdc taedrplcgs 181 dnktygnkcn fcnavvesng tltlshfgkc (SEQ ID NO: 14) J00902 1 atctcaggag cagagcaccg gcagccgcct gcagagccgg gcagtacctc accatggcca 61 tggcaggcgt cttcgtgctg ttctctttcg tgctttgtgg cttcctccca gatgctgcct 121 ttggggctga ggtggactgc agtaggtttc ccaacgctac agacaaggaa ggcaaagatg 181 tattggtttg caacaaggac ctccgcccca tctgtggtac cgatggagtc acttacacca 241 acgattgctt gctgtgtgcc tacagcatag aatttggaac caatatcagc aaagagcacg 301 atggagaatg caaggaaact gttcctatga actgcagtag ttatgccaac acgacaagcg 361 aggacggaaa agtgatggtc ctctgcaaca gggccttcaa ccccgtctgt ggtactgatg 421 gagtcaccta cgacaatgag tgtctgctgt gtgcccacaa agtagagcag ggggccagcg 481 ttgacaagag gcatgatggt ggatgtagga aggaacttgc tgctgtgagt gttgactgca 541 gtgagtaccc taagcctgac tgcacggcag aagacagacc tctctgtggc tccgacaaca 601 aaacatatgg caacaagtgc aacttctgca atgcagtcgt ggaaagcaac gggactctca 661 ctttaagcca ttttggaaaa tgctgaatat cagagctgag agaattcacc acaggatccc 721 cactggcgaa tcccagcgag aggtctcacc tcggttcatc tcgcactctg gggagctcag 781 ctcactcccg attttctttc tcaataaact aaatcagcaa c (SEQ ID NO: 15) P02789 1 mklilctvls lgiaavcfaa ppksvirwct isspeekkcn nlrdltqqer isltcvqkat 61 yldcikaian neadaisldg gqafeaglap yklkpiaaev yehtegstts yyavavvkkg 121 teftvndlqg ktschtglgr sagwnipigt llhrgaiewe giesgsveqa vakffsascv 181 pgatieqklc rqckgdpktk carnapysgy sgafhclkdg kgdvafvkht tvnenapdqk 241 deyellcldg srqpvdnykt cnwarvaaha vvarddnkve diwsflskaq sdfgvdtksd 301 fhlfgppgkk dpvlkdllfk dsaimlkrvp slmdsqlylg feyysaiqsm rkdqltpspr 361 enriqwcavg kdekskcdrw svvsngdvec tvvdetkdci ikimkgeada valdgglvytDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 421 agvcglvpvm aeryddesqc sktderpasy favavarkds nvnwnnlkgk kschtavgrt 481 agwvipmgli hnrtgtcnfd eyfsegcapg sppnsrlcql cqgsggippe kcvassheky 541 fgytgalrcl vekgdvafiq hstveentgg knkadwaknl qmddfellct dgrranvmdy 601 recnlaevpt havvvrpeka nkirdllerq ekrfgvngse kskfmmfesq nkdllfkdlt 661 kclfkvregt tykeflgdkf ytvisslktc npsdilqmcs flegk (SEQ ID NO: 16) P00698 1 mrsllilvlc flplaalgkv fgrcelaaam krhgldnyrg yslgnwvcaa kfesnfntqa 61 tnrntdgstd ygilqinsrw wcndgrtpgs rnlcnipcsa llssditasv ncakkivsdg 121 ngmnawvawr nrckgtdvqa wirgcrl (SEQ ID NO: 17) Q28049 1 eqltkcevfr elkdlkgygg vslpewvcta fhtsgydtqa ivqnndstey glfqinnkiw 61 ckddqnphss nicniscdkf ldddltddim cvkkildkvg inywlahkal csekldqwlc 121 ekl (SEQ ID NO: 18) P02662 1 mklliltclv avalarpkhp ikhqglpqev lnenllrffv apfpevfgke kvnelskdig 61 sestedqame dikqmeaesi ssseeivpns veqkhiqked vpserylgyl eqllrlkkyk 121 vpqleivpns aeerlhsmke gihaqqkepm igvnqelayf ypelfrqfyq ldaypsgawy 181 yvplgtqytd apsfsdipnp igsensektt mplw (SEQ ID NO: 19) P02663 1 mkffiftcll avalakntme hvssseesii sqetykqekn mainpskenl cstfckevvr 61 naneeeysig ssseesaeva teevkitvdd khyqkalnei nqfyqkfpqy lqylyqgpiv 121 lnpwdqvkrn avpitptlnr eqlstseens kktvdmeste vftkktklte eeknrlnflk 181 kisqryqkfa lpqylktvyq hqkamkpwiq pktkvipyvr yl (SEQ ID NO: 20) AAM73729 1 pptkfsfslf lvsvlvlclg falakidpel kqckhqckvq rqydeqqkeq cvkecekyyk 61 ekkgrerehe eeeeewgtgg vdepsthepa ekhlsqcmrq cerqeggqqk qlcrfrcqer 121 ykkergqhny kreddedede deaeeedenp yvfededftt kvkteqgkvv llpkftqksk 181 llhalekyrl avlvanpqaf vvpshmdads iffvswgrgt itkilenkre sinvrqgdiv 241 sissgtpfyi anndenekly lvqflrpvnl pghfevfhgp ggenpesfyr afsweileaa 301 lktskdtlek lfekqdqgti mkaskeqvra msrrgegpki wpfteestgs fklfkkdpsq 361 snkygqlfea eridypplek ldmvvsyani tkggmsvpfy nsratkiaiv vsgegcveia 421 cphlssskss hpsykklrar irkdtvfivp aghpfatvas gnenleivcf evnaegniry 481 tlagkkniik vmekeakela fkmegeevdk vfgkqdeeff fqgpewrkek egrade (SEQ ID NO: 21) AAN76862 1 lsvcflilfh gclasrqewq qqdecqidrl dalepdnrve yeagtveawd pnheqfrcag 61 valvrhtiqp nglllpqysn apqliyvvqg egmtgisypg cpetyqapqq grqqgqsgrf 121 qdrhqkirrf rrgdiiaipa gvahwcyneg nspvvtvtll dvsnsqnqld rtprkfhlag 181 npkdvfqqqq qhqsrgrnlf sgfdtellae afqvderlik qlksednrgg ivkvkddelr 241 virpsrsqse rgseseeese dekrrwgqrd ngieetictm rlkenindpa radiytpevg 301 rlttlnslnl pilkwlqlsv ekgvlyknal vlphwnlnsh siiygckgkg qvqvvdnfgn 361 rvfdgevreg qmlvvpqnfa vvkrareerf ewisfktndr amtsplagrt svlggmpeevDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 421 lanafqisre darkikfnnq qttltsgess hhmrdda (SEQ ID NO: 22) AAL91665 1 makfllllsa favlllvana siyraiveve edsgreqscq rqfeeqqrfr ncqryvkqev 61 qrggrynqrq eslreccqel qevdrrcrcq nleqmvrqlq qqeqikgeev relyetasel 121 pricsispsq gcqfqssy (SEQ ID NO: 23) AF329829 1 atgggtagcc ttaagttggt atgcgcggtc ctcttgtgca tgatggtggc cgcacccgtt 61 gcccgggcgt ccctgacatg cccacagata aaaggcaacc tcacgccatg cgtgctctac 121 ctgaagaacg gcggcgttct tcctccctct tgctgcaagg gcgtcagggc tgtaaacgac 181 gcctccagga ccacgtccga ccgccagtcc gcttgcaact gcttgaaaga tacagccaaa 241 ggcatcgctg gcctcaaccc taatcttgct gctggcctcc ccggcaagtg tggtgtcaac 301 attccttaca agatcagccc ctccaccaac tgcaacaacg tgaagtga (SEQ ID NO: 24) AF449424 1 aaacaacatc agatatatat taccatggcc aaacttatct tggtctcttt ttctctgtgc 61 cttcttgtgc tcttcaatgg ctgcctgggt atcaatgtag gactaaggcg gcagcaacag 121 cgatattttg gcgagtgcaa cctcgacagg ctcaatgccc ttgaacccac aaaccgcatc 181 gaggctgaag cttgccagat cgagtcttgg gaccacaacg accagcaatt ccagtgcgct 241 ggggttgccg tcatccggcg aaccattgag cccaatggcc ttctcttgcc ccaatacagc 301 aatgctccag aactcatata cattgagaga ggtaggggaa tcaccggggt cctgtttcct 361 gggtgtcccg aaacatttga agatcctcaa caacaatctc aacagggaca gagacaggga 421 cagggacaga gccagaggtc tgaacaagac cggcatcaga agattcgaca cttccgagag 481 ggagacatca tcgcattgcc agctggagta gcccattggt gctataacga cggtgactcc 541 ccagttgtca cagtctctct ccttcacacc aacaactatg ctaaccagct tgacgagaac 601 cctagacact tctaccttgc cgggaaccca gacgacgagc atcagcgaca gggtcagcag 661 caattcgggc agcgtcgccg ccagcaacag catagccatg gtgagcaagg cgagcaagag 721 cagcagggcg aaggaaacaa cgtattcagt ggcttcgatg ctgagttttt ggcggacgcg 781 ttcaacgtgg atgttgacac ggccagaagg cttcagagca accaagacaa aaggaggaac 841 atcgtcaaag tggaaggcag gcttcaggtg gtgaggccgg aaaggtcgcg tcaggaatgg 901 gagcgacagg agagacaaga gagggagagt gagcaagagc gggaacgcca gcgtcgccag 961 ggaggacgtg gacgtgatgt caatggcttt gaggagacaa tatgcagctt gaggctcagg 1021 gaaaacatct gcacccgctc acgcgctgac atttacaccg aacaagtcgg tcgcatcaac 1081 accgtcaaca gcaacaccct cccagtcctc cgctggctcc agctcagcgc tgagagagga 1141 gatcttcaaa gggagggtct atatgtaccg cactggaacc tcaatgccca cagtgtggtg 1201 tatgccatac ggggtcgcgc ccgagttcag gtggtggacg acaacggtaa caccgtgttc 1261 gacgatgaac ttagacaggg tcaggtgttg accatccccc agaacttcgc ggtggcgaaa 1321 cgggcagaga gcgagggttt cgagtgggtg gccttcaaga ccaacgacaa cgcccaaatt 1381 agtcctcttg ccggacgaac ctcagccatc agggccttgc cggacgatgt ccttgccaac 1441 gcgtttcaga tttcaaggga ggaagccagg aggctgaaat acaacaggca ggagaccacc 1501 ttggttcgct caagcaggtc ttcatctgag aggaagagga ggtctgagtc tgagggcaga 1561 gctgaagctt aattgaagat caaaaaatgc taatgtaatg cggcacgtag ttttattttg 1621 tggttgctaa gcttagggtt tagggccatc actatgcaac agtccttcct aactagtata 1681 atctgagtgt aaaaatacgc acaagggcaa cctttactac aaagaataaa agtgaccgct 1741 ttttctaaaa aaaaaaaaaa aaaaaaa (SEQ ID NO: 25) FJ358504 1 atggccagac tcgcaacgct agcagctctt tttgcagccc ttttgttggt ggcgcatgcc 61 gctgccttcc gcaccaccat aacaaccgtg gacgtcgacg aggacattgt gaaccagcag 121 gggcggcgag gcgagagctg ccgcgagcag gctcagcgtc agcagaacct caaccagtgcDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 181 cagcggtaca tgaggcagca aagccagtac gggagttacg atggcagcaa ccagcagcag 241 cagcaggaac ttgagcagtg ctgccaacag ctgaggcaga tggacgagcg gtgccgatgt 301 gaggggttga ggcaggcggt aatgcagcag cagggtgaga tgcgaggtga ggaaatgagg 361 gaggttatgg agactgctag ggacttgcca aatcaatgcc gcctcagccc ccagcgctgt 421 gagattcgct ccgcaaggtt ctagaggaag ccatgcatgt gcacgatcta taataagggt 481 caccgctaga tataaatggt gactgggggg ctttcataag caagcacatc atcgtgtgta 541 gtaactagta tctactatgt agatatgagc agtttagttc gaggctttgt aggctgtctc 601 ttgaataaaa taaaatgctt cttagtttct taa (SEQ ID NO: 26) AAB41308 1 aallvallfv anaaafrtti ttmeidedid nprrrgegcr eqiqrqqnln hcqyylrqqs 61 rsggydednq rqhfrqccqq lsqmdeqcqc eglrqvvrrq qqqqglrgee meemvqsard 121 lpnecgissq rceirrswf (SEQ ID NO: 27) EU780670 1 atgactggct ccttggtcct taagctctca ggcatggtgc tgctgtgtat ggtggtggct 61 gcaccagttg cagaggcggt cataacatgt gggcaggtgg ctagcagcgt ggggagttgc 121 attggctacc tcaggggtac ggttcctaca gtccctccaa gctgctgcaa tggggtcaag 181 agcctcaaca aagcggccgc taccacagct gaccgccagg ccgcctgtga gtgcctgaaa 241 aagacttctg gttccatccc cggactcaac cctggtcttg ctgctggcct cccaggcaaa 301 tgtggtgtca gtgttcctta caagatcagc acctccacta actgcaaagc tgtgaaatga (SEQ ID NO: 28) CAA55009 1 makafvfslc lllvfngcla arqsqlspqn qcqlnqlqar epdnriqaea gqietwnfnq 61 gdfqcagvaa sritiqrngl hlpsysnapq liyivqgrgv lgavfsgcpe tfeesqqssq 121 qgrqqeqeqe rqqqqqgeqg rqqgqqeqqq erqgrqqgrq qqeegrqqeq qqgqqgrpqq 181 qqqfrqldrh qktrriregd vvaipagvay wsyndgdqel vavnlfhvss dhnqldqnpr 241 kfylagnpen efnqqgqsqp rqqgeqgrpg qhqqpfgrpr qqeqqgngnn vfsgfntqll 301 aqalnvneet arnlqgqndn rnqiiqvrgn ldfvqpprgr qereheerqq eqlqqerqqq 361 geqlmangle etfcslrlke nignperadi fspragrist lnshnlpilr flrlsaergf 421 fyrngiysph wnvnahsvvy virgnarvqv vnengdaild qevqqgqlfi vpqnhgviqq 481 agnqgfeyfa fkteenafin tlagrtsflr alpdevlana yqisreqarq lkynrqetia 541 lsssqqrrav v (SEQ ID NO: 29) CAA26478 1 mgkpftlsls slcllllssa cfaissskln ecqlnnlnal epdhrveseg gliqtwnsqh 61 pelkcagvtv skltlnrngl hspsyspypr miiiaqgkga lgvaipgcpe tfeepqeqsn 121 rrgsrsqkqq lqdshqkirh fnegdvlvip psvpywtynt gdepvvaisl ldtsnfnnql 181 dqtprvfyla gnpdieypet mqqqqqqksh ggrkqgqhqq eeeeeggsvl sgfskhflaq 241 sfntnediae klespdderk qivtveggls vispkwqeqq dededededd edeqipshpp 301 rrpshgkreq dededededk prpsrpsqgk rnktgqdede dededqprks rewrskktqp 361 rrprqeepre rgcetrngve enictlklhe niarpsradf ynpkagrist lnsltlpalr 421 qfqlsaqyvv lykngiysph wnlnansviy vtrgqgkvrv vncqgnavfd gelrrgqllv 481 vpqnfvvaeq ageqgfeyiv fkthhnavts ylkdvfraip sevlahsynl rqsqvselky 541 egnwgplvnp esqqgsprvk va (SEQ ID NO: 30) CAA37044DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 1 mgkpftlsls slcllllssa cfaissskln ecqlnnlnal epdhrvefeg gliqtwnsqh 61 pelkcagvtv skltlnrngl hlpsyspypr miiiaqgkga lqckpgcpet feepqeqsnr 121 rgsrsqkqql qdshqkirhf negdvlvipp gvpywtyntg depvvaisll dtsnfnnqld 181 qtprvfylag npdieypetm qqqqqqkshg grkqgqhqqe eeeeggsvls gfskhflaqs 241 fntnediaek lqspdderkq ivtvegglsv ispkwqeqqd ededededde deqipshppr 301 rpshgkreqd ededededkp rpsrpsqgkr eqdqdqdede dededqprks rewrskktqp 361 rrprqeepre rgcetrngve enictlklhe niarpsradf ynpkagrist lnsltlpalr 421 qfqlsaqyvv lykngiysph wnlnansviy vtrgqgkvrv vncqgnavfd gelrrgqllv 481 vpqnfvvaeq ageqgfeyiv fkthhnavts ylkdvfraip sevlahsynl rqsqvselky 541 egnwgplvnp esqqgsprvk va (SEQ ID NO: 31) BAA74953 mgkpftlsls slcllllssa cfaissskln ecqlnnlnal epdhrveseg gliqtwnsqh 61 pelkcagvtv skltlnrngl hlpsyspypr miiiaqgkga lgvaipgcpe tfeepqeqsn 121 rrgsrsqkqq lqdshqkirh fnegdvlvip pgvpywtynt gdepvvaisl ldtsnfnnql 181 dqtprvfyla gnpdieypet mqqqqqqksh ggrkqgqhqq eeeeeggsvl sgfskhflaq 241 sfntnediae klqspdderk qivtveggls vispkwqeqq dededededd edeqipshpp 301 rrpshgkreq dededededk prpsrpsqgk reqdqdqded edededqprk srewrskktq 361 prrprqeepr ergcetrngv eenictlklh eniarpsrad fynpkagris tlnsltlpal 421 rqfqlsaqyv vlykngiysp hwnlnansvi yvtrgqgkvr vvncqgnavf dgelrrgqll 481 vvpqnfvvae qageqgfeyi vfkthhnavt sylkdvfrai psevlahsyn lrqsqvselk 541 yegnwgplvn pesqqgsprv kva (SEQ ID NO: 32) BAD72975 1 mgkpftlsls slcllllssa cfaissskln ecqlnnlnal epdhrveseg gliqtwnsqh 61 pelkcagvtv skltlnrngl hlpsyspypr miiiaqgkga lgvaipgcpe tfeepqeqsn 121 rrgsrsqkqq lqdshqkirh fnegdvlvip pgvpywtynt gdepvvaisl ldtsnfnnql 181 dqtprvfyla gnpdieypet mqqqqqqksh ggrkqgqhqq eeeeeggsvl sgfskhflaq 241 sfntnediae klqspdderk qivtveggls vispkwqeqq dededededd edeqipshpp 301 rrpshgkreq dededededk prpsrpsqgk reqdqdqded edededqprk srewrskktq 361 prrprqeepr ergcetrngv eenictlklh eniarpsrad fynpkagris tlnsltlpal 421 rqfqlsaqyv vlykngiysp hwnlnansvi yvtrgqgkvr vvncqgnavf dgelrrgqll 481 vvpqnfvvae qageqgfeyi vfkthhnavt sylkdvfrai psevlahsyn lrqsqvselk 541 yegnwgplvn pesqqgsprv kva (SEQ ID NO: 33) KRH32185 1 mgkpftlsls slcllllssa cfaissskln ecqlnnlnal epdhrveseg gliqtwnsqh 61 pelkcagvtv skltlnrngl hlpsyspypr miiiaqgkga lgvaipgcpe tfeepqeqsn 121 rrgsrsqkqq lqdshqkirh fnegdvlvip pgvpywtynt gdepvvaisl ldtsnfnnql 181 dqtprvfyla gnpdieypet mqqqqqqksh ggrkqgqhqq eeeeeggsvl sgfskhflaq 241 sfntnediae klqspdderk qivtveggls vispkwqeqq dededededd edeqipshpp 301 rrpshgkreq dededededk prpsrpsqgk reqdqdqded edededqprk srewrskktq 361 prrprqeepr ergcetrngv eenictlklh eniarpsrad fynpkagris tlnsltlpal 421 rqfqlsaqyv vlykngiysp hwnlnansvi yvtrgqgkvr vvncqgnavf dgelrrgqll 481 vvpqnfvvae qageqgfeyi vfkthhnavt sylkdvfrai psevlahsyn lrqsqvselk 541 yegnwgplvn pesqqgsprv kva (SEQ ID NO: 34) CAB57802 1 dqtprvfyla gnpdieypet mqqqqqqksh ggrkqgqhqq eeeeeggsvl sgfskhflaq 61 sfntnediae klqspdderk qivtveggls vispkwqeqq dededededd edeqipshpp 121 rrpshgkreq dededededk prpsrpsqgk reqdqdqded edededqprk srewrskktq 181 prrprqeepr ergcetrngv eenictl (SEQ ID NO: 35)DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION AAO45103 1 qyghvrvlqr fnkrsqqlqn lrdyrilefn skpntlllph hadadylivi lngtailtlv 61 nnddrdsynl qsgdalrvpa gttyyvvnpd ndenlrmitl aipvnkpgrf esfflsstqa 121 qqsylqgfsk nileasydtk feeinkvlfg reegqqqgee rlqesvivei skkqirelsk 181 haksssrkti ssedkpfnlr srdpiysnkl gklfeitpek npqlrdldvf lsvvdmnega 241 lflphfnska ivvlvinege anielvgike qqqrqqqeeq plevrkyrae lseqdifvip 301 agypvvvnat sdlnffafgi naennqrnfl agskdnvisq ipsqvqelaf pgsakdienl 361 iksqsesyfv daqpqqkeeg nkgrkgplss ilrafy (SEQ ID NO: 36) CAA33215 1 maklvfslcf llfsgccfaf ssreqpqqne cqiqklnalk pdnriesegg lietwnpnnk 61 pfqcagvals rctlnrnalr rpsytngpqe iyiqqgkgif gmiypgcpst feepqqpqqr 121 gqssrpqdrh qkiynfregd liavptgvaw wmynnedtpv vavsiidtns lenqldqmpr 181 rfylagnqeq eflkyqqeqg ghqsqkgkhq qeeeneggsi lsgftlefle hafsvdkqia 241 knlqgenege dkgaivtvkg glsvikpptd eqqqrpqeee eeeedekpqc kgkdkhcqrp 301 rgsqsksrrn gidetictmr lrhnigqtss pdiynpqags vttatsldfp alswlrlsae 361 fgslrknamf vphynlnans iiyalngral iqvvncnger vfdgelqegr vlivpqnfvv 421 aarsqsdnfe yvsfktndtp migtlagans llnalpeevi qhtfnlksqq arqiknnnpf 481 kflvppqesq krava (SEQ ID NO: 37) CAA26723 1 maklvfslcf llfsgccfaf ssreqpqqne cqiqklnalk pgnriesegg lietwnpnnk 61 pfqcagvals rctlnrnalr rpsytngpqe iyiqqgkgif gmiypgcsst feepqqpqqr 121 gqssrpqdrh qkiynsregd liavptgvaw wmynnedtpv vavsiidtns lenqldqmpr 181 rfylagnqeq eflkyqqeqg ghqsqkgkhq qeeeneggsi lsgftlefle hafsvdkqia 241 knlqgenege dkgaivtvkg glsvikpptd eqqqrpqeee eeeedekpqc kgkdkhcqrp 301 rgsqsksrrn gidetictmr lrhnigqtss pdiynpqags vttatsldfp alswlrlsag 361 fgslrknamf vphynlnans iiyalngral iqvvncnger vfdgelqegr vlivpqnfvv 421 aarsqsdnfe yvsfktndtp migtlagans llnalpeevi qhtfnlksqq arqiknnnpf 481 kflvppqesq krava (SEQ ID NO: 38) AAA33966 1 maklvfslcf llfsgccfaf ssreqpqqne cqiqklnalk pdnriesegg lietwnpnnk 61 pfqcagvals rctlnrnalr rpsytngpqe iyiqqgkgif gmiypgcpst feepqqpqqr 121 gqssrpqdrh qkiynfregd liavptgvaw wmynnedtpv vavsiidtns lenqldqmpr 181 rfylagnqeq eflkyqqeqg ghqsqkgkhq qeeeneggsi lsgftlefle hafsvdkqia 241 knlqgenege dkgaivtvkg glsvikpptd eqqqrpqeee eeeedekpqc kgkdkhcqrp 301 rgsqsksrrn gidetictmr lrhnigqtss pdiynpqags vttatsldfp alswlrlsae 361 fgslrknamf vphynlnans iiyalngral iqvvncnger vfdgelqegr vlivpqnfvv 421 aarsqsdnfe yvsfktndtp migtlagans llnalpeevi qhtfnlksqq arqiknnnpf 481 kflvppqesq krava (SEQ ID NO: 39) BAC78522 1 maklvfslcf llfsgccfaf ssreqpqqne cqiqklnalk pdnriesegg lietwnpnnk 61 pfqcagvals rctlnrnalr rpsytngpqe iyiqqgkgif gmiypgcpst feepqqpqqr 121 gqssrpqdrh qkiynfregd liavptgvaw wmynnedtpv vavsiidtns lenqldqmpr 181 rfylagnqeq eflkyqqeqg ghqsqkgkhq qeeeneggsi lsgftlefle hafsvdkqia 241 knlqgenege dkgaivtvkg glsvikpptd eqqqrpqeee eeeedekpqc kgkdkhcqrp 301 rgsqsksrrn gidetictmr lrhnigqtss pdiynpqags vttatsldfp alswlrlsaeDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 361 fgslrknamf vphynlnans iiyalngral iqvvncnger vfdgelqegr vlivpqnfvv 421 aarsqsdnfe yvsfktndtp migtlagans llnalpeevi qhtfnlksqq arqiknnnpf 481 kflvppqesq krava (SEQ ID NO: 40) ACT53400 1 maklvfslcf llfsgccfaf ssreqpqqne cqiqklnalk pdnriesegg lietwnpnnk 61 pfqcagvals rctlnrnalr rpsytngpqe iyiqqgkgif gmiypgcpst feepqqpqqr 121 gqssrpqdrh qkiynfregd liavptgvaw wmynnedtpv vavsiidtns lenqldqmpr 181 rfylagnqeq eflkyqqeqg ghqsqkgkhq qeeeneggsi lsgftlefle hafsvdkqia 241 knlqgenege dkgaivtvkg glsvikpptd eqqqrpqeee eeeedekpqc kgkdkhcqrp 301 rgsqsksrrn gidetictmr lrhnigqtss pdiynpqags vttatsldfp alswlrlsae 361 fgslrknaee akakaeemal trpyap (SEQ ID NO: 41) ACT53401 1 maklvfslcf llfsgccfaf ssreqpqqne cqiqklnalk pdnriesegg lietwnpnnk 61 pfqcagvals rctlnrnalr rpsytngpqe iyiqqgkgif gmiypgcpst feepqqpqqr 121 gqssrpqdrh qkiynfregd liavpagvaw wmynnedtpv vavsiidtns lenqldqmpr 181 rfylagnqeq eflkyqqeqg ghqsqkgkhq qeeengggsi lsgftlefle hafsvdkqia 241 knlqgenege dkgaivtvkg glsvikpptd eqqqrpqeee eeeedekpqc kgkdkhcqrp 301 rgsqsksrrn gidetictmr lrhnigqtss pdiynpqags vttatsldfp alswlrlsae 361 fgslrknaee akakaeemal trpyap (SEQ ID NO: 42) BAN29067 1 arelnpsnke lqspqqsfsh qqqpfpqqpy pqqpypsqqp ypsqqpfptp qqqfpqqsqq 61 pftqpqqptp lqpqqpfpqq pqqpqqpfpq pqqpfpwqpq qpfpqtqqsf plqpqqpfpq 121 qpqqpfpqpq lpfpqqseqi ipqqpqqpfp lqpqqpfpqq pqqpfpqpqq pipvqpqqsf 181 pqqsqqsqqp faqpqqlfpe lqqpipqqpq qpfplqpqqp fpqqpqqpfp qqpqqsfpqq 241 pqqpfpqqpq qpfpqqpqqp fpqqpqqpfp lrpqqpfpqq pqqsqqsfpq pqpqqpqqps 301 ilqpqqplpq qpqqpfqqpq qqlsqqpeqt isqqpqqpfp qqphqpqqpy pqqqpygssl 361 tsiggq (SEQ ID NO: 43) AAK15088 1 makklalaav llvamvalas attytttvtt taiddeanqq sqqcrqqlqg rqfrscqryl 61 sqgrspygge edevlemstg nqqseqslrd ccqqlrnvde rcrceairqa vrqqqqeggy 121 qegqsqqvyq rardlprrcn mrpqqcqfrv ifv (SEQ ID NO: 44) AAD42943 1 marftivlav lfaaalvsas ahktvvttsv aeegeeenqr gcewesrqcq mrhcmqwmrs 61 mrgqyeesfl rsaeanqgqf ehfreccnel rdvkshcrce alrcmmrqmq qeygmeqemq 121 qmqqmmqylp rmcgmsypte crmrpifa (SEQ ID NO: 45) AAK15089 1 mscggrlclv lfalllasav vaseskdpel kqckhqckaq qqiskeqkea ciqackeyir 61 qkhqgehgrg ggdileeevw nrkspierlr ecsrgceqqh geqreeclrr cqeeyqrekg 121 rqdddnptdp ekqyqqcrlq crrqgegggf srehcerrre ekyreqqgre ggrgemyegr 181 ereeeqeeqg rgripyvfed qhfitgfrtq hgrmrvlqkf tdrsellrgi enyrvailea 241 epqtfivpnh wdaesvvfva kgrgtislvr qdrreslnik qgdilkinag ttaylinrdn 301 nerlvlakll qpvstpgefe lffgaggenp esffksfsde ileaafntrr drlqrifgqqDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 361 rqgvivkase eqvramsrhe eggiwpfgge skgtiniyqq rpthsnqygq lhevdasqyr 421 qlrdldltvs lanitqgamt aphynskatk ialvvdgegy femacphmsr srgsyqgetr 481 grpsyqrvas rltrgtvvii paghpfvava ssnqnlqvlc fevnannnek fplagrrnvm 541 nqlereakel afgmpareve evsrsqqeef ffkgprqqqq grada (SEQ ID NO; 46) XP_026772003 1 mafagvlnda diaaaldack adgsfdhkaf ftkvgltgks addvkkafsi idqdksgfie 61 edelklflqn fksdaraltd netkiflkag dtdgdgkigv defaslvka (SEQ ID NO: 47) XP_026803769 1 mamqdllkad dikkaldtfk vadtfdhkkf felvglkams aenvkkvfsv ldvdasgfie 61 edelkfvlkg fskdgrdltd ketkafltaa drdgdgkigi defeaivhq (SEQ ID NO: 48) CAC84590 1 meaikkkmqa mklekdnamd kadaleaqar danrkadkil eevqdlkkkp sqvetdfttt 61 kenlatankn ledkektltn tesevaslnr kvqmieenle rseerlgtal tklgeashaa 121 deasrmckvl enrsqqdeer mdqltnqlke armlaedadg ksdevsrkma qveddlevae 181 drvksgdski meleeelkvv gnslkslevs eekanqrvee ykrqiktltv klkeaearae 241 yaekyvkklq kevdrledel ginkdryral ademdqtfae lsgy (SEQ ID NO: 49) Computer Program Product

[0182] The disclosure relates to a computer program product with instructions for: (a) receiving detection data from the device disclosed herein corresponding to the presence, absence or quantity of probes in each compartment; (b) quantifying the amount of probe in each compartment by normalizing the amount of probe in the compartment with the amount of probe in a control compartment; and (c) correlating the amount of probe in each compartment with the number and percentage of activated immune cells from a sample in each compartment.

[0183] The computer program product, in some embodiments, further comprises step (d) determining whether a subject from which the sample is taken has an immune-related disorder if the number of activated immune cells in the sample is elevated as compared to the number of activated immune cells in a subject known not to have the immune disorder; wherein the subject has an increased probability of having the immune-related disorder if the number of activated immune cells are elevated in a compartment relative to a control.

[0184] In some embodiments, disclosed is a system comprising a disclosed computer program product, and one or more of: (i) a processor operable to execute programs; and (ii) a memory associated with the processor.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0185] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smart phone, or any other suitable portable or fixed electronic device.

[0186] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible format.

[0187] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks, or fiber optic networks.

[0188] A computer employed to implement at least a portion of the functionality described herein may include a memory, coupled to one or more processing units (also referred to herein simply as “processors”), one or more communication interfaces, one or more display units, and one or more user input devices. The memory may include any computer-readable media, and may store computer instructions (also referred to herein as “processor-executable instructions”) for implementing the various functionalities described herein. The processing unit(s) may be used to execute the instructions. The communication interface(s) may be coupled to a wired or wireless network, bus, or other communication means and may therefore allow the computer to transmit communications to and / or receive communications from other devices. The display unit(s) may be provided, for example, to allow a user to view various information in connection with execution of the instructions. The user input device(s) may be provided, for example, to allow the user to make manual adjustments, make selections, enter data or various other information, and / or interact in any of a variety of manners with the processor during execution of the instructions.

[0189] The various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. The disclosure also relates to a computer readable storage medium comprisingDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION executable instructions. Additionally, such software may be written using any of a number of suitable programming languages and / or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

[0190] In this respect, various inventive concepts may be embodied as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory medium or tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the technology disclosed herein. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present technology as discussed above. In some embodiments, the system comprises cloud-based software that executes one or all of the steps of each disclosed method instruction.

[0191] The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present technology.

[0192] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0193] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish aDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

[0194] Also, the disclosure relates to various embodiments in which one or more methods. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0195] Computer-implemented embodiments of the disclosure relate to methods of determining the count and proportion of immune cells and their activation levels in response to different stimulants or different concentrations of a single stimulant (simultaneously or in sequence with the same dataset of a sample), comprising steps of: (d) comparing the activation level relative to controls at one or more concentrations of the stimulant, the maximum activation level across all concentrations, the area under the dose response curve, the concentration eliciting the half-maximum activation (EC50), or other metrics; and (e) classifying the subject as having an immune-related disorder based upon results of comparing of step; (d) relative to a threshold; wherein each of steps (d) and (e) are performed after step (c).

[0196] In some embodiments, the disclosure relates to a system that comprises at least one processor, a program storage, such as memory, for storing program code executable on the processor, and one or more input / output devices and / or interfaces, such as data communication and / or peripheral devices and / or interfaces. In some embodiments, the user device and computer system or systems are communicably connected by a data communication network, such as a Local Area Network (LAN), the Internet, or the like, which may also be connected to a number of other client and / or server computer systems. The user device and client and / or server computer systems may further include appropriate operating system software. In some embodiments, the system comprises a processor comprising a computer program product for calculating the alignment of known telomeric repeat sequences to the data from a sample registered with the computer program product. In some embodiments, the system comprises a device that interacts with one or more communication channels or mediums or links, such that alignment processes for telomere sequences of a sample are compared to control sequences stored on a memory and shared with a network in operable communication with the device.

[0197] In some embodiments, components and / or units of the devices described herein may be able to interact through one or more communication channels or mediums or links, for example, a shared access medium, a global communication network, the Internet, the WorldDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Wide Web, a wired network, a wireless network, a combination of one or more wired networks and / or one or more wireless networks, one or more communication networks, an a-synchronic or asynchronous wireless network, a synchronic wireless network, a managed wireless network, a non-managed wireless network, a burstable wireless network, a non-burstable wireless network, a scheduled wireless network, a non-scheduled wireless network, or the like.

[0198] Discussions herein utilizing terms such as, for example, “processing,” “computing,” “calculating,” “determining,” or the like, may refer to operation(s) and / or process(es) of a computer, a computing platform, a computing system, or other electronic computing device, that manipulate and / or transform data represented as physical (e.g., electronic) quantities within the computer's registers and / or memories into other data similarly represented as physical quantities within the computer’s registers and / or memories or other information storage medium that may store instructions to perform operations and / or processes. In some embodiments, the

[0199] Some embodiments may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment including both hardware and software elements. Some embodiments may be implemented in software, which includes but is not limited to firmware, resident software, microcode, or the like.

[0200] Furthermore, some embodiments may take the form of a computer program product accessible from a computer-usable or computer-readable medium providing program code for use by or in connection with a computer or any instruction execution system. For example, a computer-usable or computer-readable medium may be or may include any apparatus that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

[0201] In some embodiments, the medium may be or may include an electronic, magnetic, optical, electromagnetic, InfraRed (IR), or semiconductor system (or apparatus or device) or a propagation medium. Some demonstrative examples of a computer-readable medium may include a semiconductor or solid-state memory, magnetic tape, a removable computer diskette, a Random Access Memory (RAM), a Read-Only Memory (ROM), a rigid magnetic disk, an optical disk, or the like. Some demonstrative examples of optical disks include Compact Disk- Read-Only Memory (CD-ROM), Compact Disk-Read / Write (CD-R / W), DVD, or the like.

[0202] In some embodiments, a data processing system suitable for storing and / or executing program code may include at least one processor coupled directly or indirectly to memory elements, for example, through a system bus. The memory elements may include, for example, local memory employed during actual execution of the program code, bulk storage,DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION and cache memories which may provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution.

[0203] In some embodiments, input / output or I / O devices (including but not limited to keyboards, displays, pointing devices, etc.) may be coupled to the system either directly or through intervening I / O controllers. In some embodiments, network adapters may be coupled to the system to enable the data processing system to become coupled to other data processing systems or remote printers or storage devices, for example, through intervening private or public networks. In some embodiments, modems, cable modems and Ethernet cards are demonstrative examples of types of network adapters. Other suitable components may be used.

[0204] Some embodiments may be implemented by software, by hardware, or by any combination of software and / or hardware as may be suitable for specific applications or in accordance with specific design requirements. Some embodiments may include units and / or sub-units, which may be separate of each other or combined together, in whole or in part, and may be implemented using specific, multi-purpose or general processors or controllers. Some embodiments may include buffers, registers, stacks, storage units and / or memory units, for temporary or long-term storage of data or in order to facilitate the operation of particular implementations.

[0205] Some embodiments may be implemented, for example, using a machine-readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, cause the machine to perform method steps and / or operations described herein. Such machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, electronic device, electronic system, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and / or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and / or storage unit; for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk drive, floppy disk, Compact Disk Read Only Memory (CD-ROM), Compact Disk Recordable (CD-R), Compact Disk Re- Writeable (CD-RW), optical disk, magnetic media, various types of Digital Versatile Disks (DVDs), a tape, a cassette, or the like. The instructions may include any suitable type of code, for example, source code, compiled code, interpreted code, executable code, static code, dynamic code, or the like, and may be implemented using any suitable high-level, low-level,DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION object-oriented, visual, compiled and / or interpreted programming language, e.g., C, C++, Java™, BASIC, Pascal, Fortran, Cobol, assembly language, machine code, or the like.

[0206] Many of the functional units described in this specification have been labeled as circuits, in order to more particularly emphasize their implementation independence. For example, a circuit may be implemented as a hardware circuit comprising custom very-large- scale integration (VLSI) circuits or gate arrays, off-the-shelf semiconductors such as logic chips, transistors, or other discrete components. A circuit may also be implemented in programmable hardware devices such as field programmable gate arrays, programmable array logic, programmable logic devices or the like.

[0207] In some embodiments, the circuits may also be implemented in machine-readable medium for execution by various types of processors. An identified circuit of executable code may, for instance, comprise one or more physical or logical blocks of computer instructions, which may, for instance, be organized as an object, procedure, or function. Nevertheless, the executables of an identified circuit need not be physically located together, but may comprise disparate instructions stored in different locations which, when joined logically together, comprise the circuit and achieve the stated purpose for the circuit. Indeed, a circuit of computer readable program code may be a single instruction, or many instructions, and may even be distributed over several different code segments, among different programs, and across several memory devices. Similarly, operational data may be identified and illustrated herein within circuits, and may be embodied in any suitable form and organized within any suitable type of data structure. The operational data may be collected as a single data set, or may be distributed over different locations including over different storage devices, and may exist, at least partially, merely as electronic signals on a system or network.

[0208] The computer readable medium (also referred to herein as machine-readable media or machine-readable content) may be a tangible computer readable storage medium storing the computer readable program code. The computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, holographic, micromechanical, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. As alluded to above, examples of the computer readable storage medium may include but are not limited to a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a portable compact disc read-only memory (CD-ROM), a digital versatile disc (DVD), an optical storage device, a magnetic storage device, a holographic storage medium, a micromechanical storage device, or any suitable combinationDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, and / or store computer readable program code for use by and / or in connection with an instruction execution system, apparatus, or device.

[0209] The computer readable medium may also be a computer readable signal medium. A computer readable signal medium may include a propagated data signal with computer readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated signal may take any of a variety of forms, including, but not limited to, electrical, electro-magnetic, magnetic, optical, or any suitable combination thereof. A computer readable signal medium may be any computer readable medium that is not a computer readable storage medium and that can communicate, propagate, or transport computer readable program code for use by or in connection with an instruction execution system, apparatus, or device. As also alluded to above, computer readable program code embodied on a computer readable signal medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, Radio Frequency (RF), or the like, or any suitable combination of the foregoing. In one embodiment, the computer readable medium may comprise a combination of one or more computer readable storage mediums and one or more computer readable signal mediums. For example, computer readable program code may be both propagated as an electro-magnetic signal through a fiber optic cable for execution by a processor and stored on RAM storage device for execution by the processor.

[0210] Computer readable program code for carrying out operations for aspects of the present disclosure may be written in any combination of one or more programming languages, including an object-oriented programming language such as Java, Smalltalk, C++ or the like and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable program code may execute entirely on a user's computer, partly on the user’s computer, as a stand-alone computer-readable package, partly on the user’s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user’s computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).

[0211] The program code may also be stored in a computer readable medium that can direct a computer, other programmable data processing apparatus, or other devices to function in a particular manner, such that the instructions stored in the computer readable medium produceDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION an article of manufacture including instructions which implement the function / act specified in the schematic flowchart diagrams and / or schematic block diagrams block or blocks. Turning to FIG.26, the disclosure relates to a computer program product. A computer program product may comprise instructions for: (a) receiving detection data from the device corresponding to the presence, absence or quantity of probes in each compartment; (b) quantifying the amount of probe in each compartment by normalizing the amount of probe in the compartment with the amount of probe in a control compartment; and (c) correlating the amount of probe in each compartment with the number of activated immune cells from a sample in each compartment.

[0212] The computer program product may further comprise determining whether a subject from which the sample is taken has an immune-related disorder if the number of activated immune cells in the sample is elevated as compared to the number of activated immune cells in a subject known not to have the immune disorder; wherein the subject has an increased probability of having the immune-related disorder if the number of activated immune cells are elevated in a compartment relative to a control. Methods Table 6: Assay Types for Activation of Immune Cells with Stimulants and Thresholds Assay Cell Type Markers Stimulant / Antigen Threshold Link Type w ur rt .DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Assay Cell Type Markers Stimulant / Antigen Threshold Link Type Neutrophils Surface markers: C3b, C3d, Stimulants for https: / / w i. .g / a 0 do .1 o. w i. .g / a 6 w o r 0. c 0 C w i. .g / a 9 doDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Assay Cell Type Markers Stimulant / Antigen Threshold Link Type CD59, CD88, CDw32, paclitaxel, org.stanf .o 1 0 1 w o g / S0 0) xt ta bi nt / a 0. 3 6-g embodiments, a basophil) for the detection and diagnosis of a subject’s immune sensitivity or allergy to an antigen. In some embodiments, the methods of the disclosure relate to a method of diagnosing a subject with an allergy to an antigen by loading a sample from the subject into the device or composition disclosed herein, exposing the sample to an antigen disclosed herein, exposing the sample (in some embodiments, in parallel fashion) to multiple probes specific for one or a plurality of immune cells and analyzing the immune cells to determine the presence, absence or quantity of immune cells in the sample. In some embodiments, the sample is whole blood. In some embodiments, the sample is isolated cells chosen from a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil and / or platelets. In some embodiments, the sample comprises cells chosen from a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil and / or platelets.

[0214] Methods of the disclosure also relate to methods of activating cells from a sample by loading the sample preparation device disclosed herein, exposing the cells to an antigen, labeling the cells with a probe and collecting the cells after performance of the aforementioned steps such that collection occurs in a vessel positioned proximate to the outlet of the sample preparation device and / or the vessel is detachably coupled to the device. In some embodiments, the vessel is equivalent to a pooling vessel or storage vessel disclosed herein. In someDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION embodiments, the storage vessel is coupled to the device via an outlet adapter, and, in some embodiments, the methods comprise expelling the labeled / probed samples by mechanically pushing the fluid out of the reaction vessels by depression of the one or plurality of plungers positioned in the reaction vessels. In some embodiments, the outlet adaptor by way of screwing the storage vessel to the outlet adaptor at a point proximate to the outlet. In some embodiments, the methods include a step of then analyzing the collected and labeled probe. Steps of analyzing can comprise steps of performing flow cytometry, mass cytometry, FACS or cell sorting based upon the presence of labels or probes, cell counting using microscopy (light, digital imaging or fluorescent), mass spectrometry, or combinations thereof.

[0215] Flow cytometry is generally a known method. But briefly, the flow cytometer can utilize a source of stream fluid to supply stream fluid to establish a sheath of fluid in which particles or cells can be suspended. The source of particles (in some embodiments, fluid or blood in the storage vessel) can insert the particles from time to time such that the particles or cells become suspended in the stream fluid and are hydrodynamically focused in the stream. A stream comprised of tile stream fluid and the particles or cells can then be established below a nozzle of the flow cytometer. A stream of fluid can be established in a steady state condition such that droplets are formed and break away from a contiguous part of the stream. When the stream is established in this steady state fashion, a stable break-off point. This stream can be strobed with a stroboscope to illuminate the stable stream. At the break-off point the stream breaks off into droplets with these droplets centered about the break-off point. The droplet break-off point is the point in a stream where a droplet separates from tile contiguous flow of the stream. For reference purposes, the center of tile droplet will be considered the droplet break-off point when it is necessary to define the point in such an exacting manner. Below the droplet break-off point a free fall zone can exist. This free fall zone embodies tile area where tile droplets move once they break away from the contiguous part of the stream. At the bottom of the nozzle a stream exit point is established. The exit point is the point in space where the fluid / cell / particle stream emerges from the flow cytometer. For example, the exit point on flow cytometer having a nozzle would exist where the stream exits from the nozzle. At this point the stream essentially emerges or is ejected from the flow cytometer. A droplet charging location can exist at a point along the stream. This droplet charging location can exist, for example, at the droplet break-off point. As a possible alternative, a charging ring call be used and positioned below the droplet break-off point such that the individual droplets can be charged. An oscillator can be used to perturb tile stream and to establish a steady state oscillation of the stream. It is preferred to use a piezoelectric crystal to accomplish this perturbation of the stream. TheDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION oscillator may have an adjustable oscillation frequency that can be adjusted to perturb the stream at different frequencies such that droplets are created at different rates. Furthermore, it can be used in conjunction with the stream pressure to establish the rate of droplet formation.

[0216] A detector such as a laser and receiver in combination, can be used to monitor the stream for a particle. The detector can detect the particle in the stream as the particle passes through, for example, a coherent beam of light aimed at the stream by the detector. When the coherent beam of light intercepts a particle in the stream, fluorescence or scattered light rays can then be emitted or deflected, respectively to a receiver of the detector. Alternative methods of detection are also well understood by those of ordinary skill in the art.

[0217] As the droplets fall in the free fall zone, they can pass through a sorting force generator such as electrostatic plates. If the droplets have been charged with a positive or negative charge, an electric field established between these electrostatic plates will deflect the charged droplets such that the trajectory of the droplets is changed. These droplets can then be deflected into a container which acts as a sample collector. Similarly, those droplets that are neutrally charged can fall into the container and droplets that are alternatively charged can fall into a third container. Furthermore, alternative techniques such as utilizing different quantities of charge can be used to accomplish an even greater deflection. A system comprising a flow cytometer can comprise a sensor. In some embodiments, the sensor can measure a property of the flow cytometer based on a captured image of the fluid stream. One such property that the sensor can measure is the speed of the stream at a point along the stream. Two points of particular interest are the speed of the stream at the area just below the exit point of the stream from the nozzle and at about the droplet break-off position of the stream.

[0218] The sensor can be oriented to measure a wavelength of the stream. In some embodiments, the methods of the disclosure relate to detecting the wavelength produced by the probe or probes associated with the cells upon excitation by a light source, such as a laser. Digital imaging routines can operate on the wave shape to measure the length of a standing wave, for example. Furthermore, the sensor can be oriented to measure a distance between droplets of the stream such that the distance and the oscillation frequency of the oscillator can be utilized to calculate the speed of the droplets at that point along the stream and / or the presence of one or a plurality of probes of the disclosure. The sensor can utilize a camera with a wide-angle lens that captures a large portion of the stream or multiple cameras where a camera captures an image of the stream at an exit point of the stream and camera captures an image at the droplet break-off point of the stream. In this fashion, a first camera and a second camera can serve as a first sensor and second sensor, respectively, for determining speeds at differentDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION points along the stream the camera can capture an image of the stream and display it on a monitor.

[0219] The camera can be oriented to capture various features of the stream. For example, the camera can be oriented to capture the width. Alternatively or additionally, the camera can be oriented to capture an image that permits determination of a speed of the stream. As discussed earlier, this may be accomplished by measuring the wavelength of the stream or measuring a distance between two droplets and utilizing the known oscillation frequency to calculate a speed for the stream at those two points. Namely, the product of the oscillation frequency and the distance between droplets (or the stream wavelength) would yield a stream velocity at that point.

[0220] Ini addition, the camera or sensor may be oriented to capture an image that permits determination of a change in stream position. This can be done by monitoring a first position of the stream, recording that position, and then monitoring the stream over time to see if the stream moves away from that previously determined position. The camera can capture an image of the stream at the break-off point as well as the stream exit point where the stream emerges from the cytometer. Essentially, the camera can be oriented in many different orientations to capture many views in order to determine information about the stream which consequently allows the sensor to determine information about the characteristics of the cytometer. The various cameras can be used to capture the image of the stream, however a camera can create a digital representation for the image of the stream and one such camera being one that uses a charge coupled device (CCD). A CCD can produce an output in a series of analog voltage pulses each of which corresponds to a light intensity received by a pixel of the CCD.

[0221] Once the sensor captures an image of the stream, the image can be displayed on a monitor. The monitor can then display the image of the stream to a user. Typically, a monitor will also be comprised of individual pixel elements that correlate to a digital embodiment of the image derived from the CCD. The CCD's pixel elements, as well as the monitor's embodiment) can be correlated with a physical distance, to arrive at an accurate determination of an actual dimension of the stream. The monitor can be positioned at the flow cytometer or even remotely from the rest of the system, if comprising necessary operable communication link to the interne. When remote access is desired to monitor the flow cytometer at a remote position, the monitor can be positioned at such a remote position.

[0222] Probes of the disclosure can be fluorescent molecules or biomolecules that fluoresce when exposed to a known wavelength of light that excites the probe. Detection of emitted light from the excitation step can occur through antibodies specific for the molecules to which theDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION probes bind. In some embodiments, these molecules are markers or biomarkers. In some embodiments, these molecules are those markers identified in Table 6. Probes specific to markers can be antibodies. Any probe disclosed herein may be an antibody. The term “antibody” as used herein refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. As used herein, a "targeted binding agent" is an antibody, or binding fragment thereof, that preferentially binds to a target site. In one embodiment, the targeted binding agent is specific for only one target site. In other embodiments, the targeted binding agent is specific for more than one target site. In one embodiment, the targeted binding agent may be a monoclonal antibody and the target site may be an epitope. “Epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody. "Binding fragments" of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counter-receptor when an excess of antibody reduces the quantity of receptor bound to counter-receptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay). An antibody may be oligoclonal, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody and antibodies that can be labeled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the technology; exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulphide stabilized variable region (dsFv). As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present technology, providing that the variations in the amino acid sequence maintain atDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION least 75%, more preferably at least 80%, 90%, 95%, and 99% sequence identity to the antibodies or immunoglobulin molecules described herein. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that have related side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are an aliphatic- hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding function or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three- dimensional structure are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated by reference in its entirety. In some embodiments, the antibody fragments, analogs thereof of the disclosure are antibodies, antibody fragments, analogs thereof bind a marker or immune cell protein disclosed herein. In some embodiments, the antibody fragments, analogs thereof of the disclosure are antibodies, antibody fragments, analogs thereof that bind to a marker or immune cell protein disclosed herein or portion thereof. In some embodiments, the antibody fragments, analogs thereof of the disclosure are antibodies, antibody fragments, analogs thereof that bind to a marker or immune cell protein disclosed herein. In some embodiments, the system comprises a solid substrate spotted in a patterned or non-patterned formation at discrete locations on the solid substrate by one or a plurality ofDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION substrates for a marker or immune cell protein disclosed herein. In other embodiments, the system comprises a solid substrate spotted in a patterned or non-patterned formation at discrete locations on the solid substrate by one or a plurality of substrates for a marker or immune cell protein disclosed herein; and the system further comprises one or a plurality of antibodies, antibody fragments, or analogs of the antibodies that bind to a reaction fragment resulting from the cleavage of a substrate exposed to marker or immune cell protein disclosed herein.

[0223] In some instances, it may be desired to modify the detection probes so that they are more readily able to bind to an analyte or a reaction product. In such instances, the detection probes may be modified with certain specific binding members that are adhered thereto to form conjugated probes. For instance, the detection probe may be conjugated with antibodies as specific to the markers identified in Table 6. The detection probe antibody may be a monoclonal or polyclonal antibody or a mixture(s) or fragment(s) thereof. In some embodiments, methods of the disclosure relate to a step of analyzing cells collected after performance of steps utilizing the device disclosed herein wherein the step of analyzing comprises detecting the presence or quantity of antibodies specific to the markers of Table 6. In some embodiments, methods of the disclosure comprise a step of analyzing a plurality of immune cells comprising exposing an antibody specific for a marker or immune cell protein (sometimes a surface protein) to the immune cells in the device, collecting the labeled cells and further exciting a fluorescent tag conjugated to the antibody such that emission wavelengths can be measured and correlated to the quantity of the immune cells in the system, composition or device.

[0224] Antibodies may generally be attached to the detection probes using any of a variety of well-known techniques. For instance, covalent attachment of the antibodies to the detection probes (e.g., particles) may be accomplished using carboxylic, amino, aldehyde, bromoacetyl, iodoacetyl, thiol, epoxy and other reactive or linking functional groups, as well as residual free radicals and radical cations, through which a protein coupling reaction may be accomplished. A surface functional group may also be incorporated as a functionalized co-monomer as the surface of the detection probe may contain a relatively high surface concentration of polar groups. In addition, although detection probes are often functionalized after synthesis, such as with poly(thiophenol), the detection probes may be capable of direct covalent linking with an antibody without the need for further modification. For example, in one embodiment, the first step of conjugation is activation of carboxylic groups on the probe surface using carbodiimide. In the second step, the activated carboxylic acid groups are reacted with an amino group of an antibody to form an amide bond. The activation and / or antibody coupling may occur in aDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION buffer, such as phosphate-buffered saline (PBS) (e.g., pH of 7.2) or 2-(N-morpholino) ethane sulfonic acid (MES) (e.g., pH of 5.3). The resulting detection probes may then be contacted with ethanolamine, for instance, to block any remaining activated sites. Overall, this process forms a conjugated detection probe, where the antibody is covalently attached to the probe. Besides covalent bonding, other attachment techniques, such as physical adsorption, may also be utilized in the present technology.

[0225] In one embodiment, the antibody may be detectably labeled by linking to an enzyme. The enzyme, in turn, when later exposed to a substrate or reaction product or enzyme disclosed herein, will react with a substrate or reaction product or enzyme disclosed herein in such a manner as to produce a chemical moiety which may be detected as, for example, by spectrophotometric or fluorometric means. Examples of enzymes which may be used to detectably label the antibodies as herein described include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

[0226] In some embodiments, in vivo or in vitro methods are performed to detect the presence, absence or quantity of one or a plurality of proteins expressed by the immune cells disclosed herein. In some embodiments, any of the disclosed methods or series of methods comprise exposing a sample or tissue in situ with one or a plurality of antibodies, optionally tagged with a visual detection agent such as a probe or fluorophore, which has binding affinity for one or a plurality of the immune cell proteins disclosed herein (such as the CD molecules disclosed on Table 5 or 6. Antibodies suitable for practicing the methods of the disclosure may be monoclonal and multivalent, and may be human, humanized or chimeric antibodies, comprising single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, and / or binding fragments of any of the above. In certain embodiments of the technology, the antibodies are human antigen-binding antibody fragments of the present technology and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, CH3 and CL domains. Also included in the disclosure are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CH1, CH2, CH3 and CL domains. Preferably, the antibodies are human, murine (e.g.,DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION mouse and rat), donkey, sheep, rabbit, goal, guinea pig, camelid, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries, from human B cells, or from animals transgenic for one or more human immunoglobulins. In some embodiments, the probes are specific to detection of the following sequences or those sequences comprising at least 75%, 80%, 85%, 90%, 95% sequence identity to those following sequences: Sequences from Table 6. Where protein sequence is given, a nucleic acid sequence can be inferred and vice versa. CD45 1 mtmylwlkll afgfafldte vfvtgqsptp sptglttakm psvplssdpl pthttafspa 61 stferendfs etttslspdn tstqvspdsl dnasafnttg vssvqtphlp thadsqtpsa 121 gtdtqtfsgs aanaklnptp gsnaisdvpg erstastfpt dpvspltttl slahhssaal 181 partsnttit antsdaylna setttlspsg savistttia ttpskptcde kyanitvdyl 241 ynketklfta klnvnenvec gnntctnnev hnltecknas vsishnscta pdktlildvp 301 pgvekfqlhd ctqvekadtt iclkwkniet ftcdtqnity rfqcgnmifd nkeiklenle 361 peheykcdse ilynnhkftn askiiktdfg spgepqiifc rseaahqgvi twnppqrsfh 421 nftlcyiket ekdclnldkn likydlqnlk pytkyvlslh ayiiakvqrn gsaamchftt 481 ksappsqvwn mtvsmtsdns mhvkcrpprd rngpheryhl eveagntlvr neshkncdfr 541 vkdlqystdy tfkayfhngd ypgepfilhh stsynskali aflafliivt siallvvlyk 601 iydlhkkrsc nldeqqelve rddekqlmnv epihadille tykrkiadeg rlflaefqsi 661 prvfskfpik earkpfnqnk nryvdilpyd ynrvelsein gdagsnyina syidgfkepr 721 kyiaaqgprd etvddfwrmi weqkatvivm vtrceegnrn kcaeywpsme egtrafgdvv 781 vkinqhkrcp dyiiqklniv nkkekatgre vthiqftswp dhgvpedphl llklrrrvna 841 fsnffsgpiv vhcsagvgrt gtyigidaml egleaenkvd vygyvvklrr qrclmvqvea 901 qyilihqalv eynqfgetev nlselhpylh nmkkrdppse pspleaefqr lpsyrswrtq 961 hignqeenks knrnsnvipy dynrvplkhe lemskesehd sdessdddsd seepskyina 1021 sfimsywkpe vmiaaqgplk etigdfwqmi fqrkvkvivm ltelkhgdqe icaqywgegk 1081 qtygdievdl kdtdksstyt lrvfelrhsk rkdsrtvyqy qytnwsveql paepkelism 1141 iqvvkqklpq knssegnkhh kstpllihcr dgsqqtgifc allnllesae teevvdifqv 1201 vkalrkarpg mvstfeqyqf lydviastyp aqngqvkknn hqedkiefdn evdkvkqdan 1261 cvnplgapek lpeakeqaeg septsgtegp ehsvngpasp alnqgs (SEQ ID NO: 50) HLADR 1 mgpgllcwvl lcllgagsve tgvtqspthl iktrgqqvtl rcssqsghnt vswyqqalgq 61 gpqfifqyyr eeengrgnfp prfsglqfpn ysselnvnal elddsalylc assfkglglp 121 srgyeqyfgp gtrltvtedl knvfpp (SEQ ID NO: 51) CD126 1 mlavgcalla allaapgaal aprrcpaqev argvltslpg dsvtltcpgv epednatvhw 61 vlrkpaagsh psrwagmgrr lllrsvqlhd sgnyscyrag rpagtvhllv dvppeepqls 121 cfrksplsnv vcewgprstp slttkavllv rkfqnspaed fqepcqysqe sqkfscqlav 181 pegdssfyiv smcvassvgs kfsktqtfqg cgilqpdppa nitvtavarn prwlsvtwqd 241 phswnssfyr lrfelryrae rsktfttwmv kdlqhhcvih dawsglrhvv qlraqeefgq 301 gewsewspea mgtpwtesrs ppaenevstp mqalttnkdd dnilfrdsan atslpvqdss 361 svplptflva ggslafgtll ciaivlrfkk twklralkeg ktsmhppysl gqlvperprp 421 tpvlvplisp pvspsslgsd ntsshnrpda rdprspydis ntdyffpr (SEQ ID NO: 52)DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION CD63 1 maveggmkcv kfllyvllla fcacavglia vgvgaqlvls qtiiqgatpg sllpvviiav 61 gvflflvafv gccgackeny clmitfaifl slimlvevaa aiagyvfrdk vmsefnnnfr 121 qqmenypknn htasildrmq adfkccgaan ytdwekipsm sknrvpdscc invtvgcgin 181 fnekaihkeg cvekiggwlr knvlvvaaaa lgiafvevlg ivfacclvks irsgyevm (SEQ ID NO: 53) CD203c 1 mestltlate qpvkkntlkk ykiacivlla llvimslglg lglglrklek qgscrkkcfd 61 asfrglencr cdvackdrgd ccwdfedtcv estriwmcnk frcgetrlea slcscsddcl 121 qrkdccadyk svcqgetswl eencdtaqqs qcpegfdlpp vilfsmdgfr aeylytwdtl 181 mpninklktc gihskymram yptktfpnhy tivtglypes hgiidnnmyd vnlnknfsls 241 skeqnnpaww hgqpmwltam yqglkaatyf wpgsevaing sfpsiympyn gsvpfeeris 301 tllkwldlpk aerprfytmy feepdssgha ggpvsarvik alqvvdhafg mlmeglkqrn 361 lhncvniill adhgmdqtyc nkmeymtdyf prinffymye gpaprirahn iphdffsfns 421 eeivrnlscr kpdqhfkpyl tpdlpkrlhy aknvridkvh lfvdqqwlav rsksntncgg 481 gnhgynnefr smeaiflahg psfkekteve pfenievynl mcdllriqpa pnngthgsln 541 hllkvpfyep shaeevskfs vcgfanplpt esldcfcphl qnstqleqvn qmlnltqeei 601 tatvkvnlpf grprvlqknv dhcllyhrey vsgfgkamrm pmwssytvpq lgdtsplppt 661 vpdclradvr vppsesqkcs fyladknith gflyppasnr tsdsqydali tsnlvpmyee 721 frkmwdyfhs vllikhater ngvnvvsgpi fdynydghfd apdeitkhla ntdvpipthy 781 fvvltscknk shtpencpgw ldvlpfiiph rptnvescpe gkpealwvee rftahiarvr 841 dvelltgldf yqdkvqpvse ilqlktylpt fetti (SEQ ID NO: 54) CD193 1 mttsldtvet fgttsyyddv gllcekadtr almaqfvppl yslvftvgll gnvvvvmili 61 kyrrlrimtn iyllnlaisd llflvtlpfw ihyvrghnwv fghgmcklls gfyhtglyse 121 iffiilltid rylaivhavf alrartvtfg vitsivtwgl avlaalpefi fyeteelfee 181 tlcsalyped tvyswrhfht lrmtifclvl pllvmaicyt giiktllrcp skkkykairl 241 ifvimavffi fwtpynvail lssyqsilfg ndcerskhld lvmlvtevia yshccmnpvi 301 yafvgerfrk ylrhffhrhl lmhlgryipf lpseklerts svspstaepe lsivf (SEQ ID NO: 55) CD163 1 msklrmvlle dsgsadfrrh fvnlspftit vvlllsacfv tsslggtdke lrlvdgenkc 61 sgrvevkvqe ewgtvcnngw smeavsvicn qlgcptaika pgwanssags griwmdhvsc 121 rgnesalwdc khdgwgkhsn cthqqdagvt csdgsnlemr ltrggnmcsg rieikfqgrw 181 gtvcddnfni dhasvicrql ecgsavsfsg ssnfgegsgp iwfddlicng nesalwnckh 241 qgwgkhncdh aedagvicsk gadlslrlvd gvtecsgrle vrfqgewgti cddgwdsyda 301 avackqlgcp tavtaigrvn askgfghiwl dsvscqghep aiwqckhhew gkhycnhned 361 agvtcsdgsd lelrlrgggs rcagtvevei qrllgkvcdr gwglkeadvv crqlgcgsal 421 ktsyqvyski qatntwlfls scngnetslw dcknwqwggl tcdhyeeaki tcsahreprl 481 vggdipcsgr vevkhgdtwg sicdsdfsle aasvlcrelq cgtvvsilgg ahfgegngqi 541 waeefqcegh eshlslcpva prpegtcshs rdvgvvcsry teirlvngkt pcegrvelkt 601 lgawgslcns hwdiedahvl cqqlkcgval stpggarfgk gngqiwrhmf hctgteqhmg 661 dcpvtalgas lcpseqvasv icsgnqsqtl sscnssslgp trptipeesa vaciesgqlr 721 lvngggrcag rveiyhegsw gticddswdl sdahvvcrql gcgeainatg sahfgegtgp 781 iwldemkcng kesriwqchs hgwgqqncrh kedagvicse fmslrltsea sreacagrle 841 vfyngawgtv gkssmsettv gvvcrqlgca dkgkinpasl dkamsipmwv dnvqcpkgpd 901 tlwqcpsspw ekrlaspsee twitcdnkir lqegptscsg rveiwhggsw gtvcddswdl 961 ddaqvvcqql gcgpalkafk eaefgqgtgp iwlnevkckg nesslwdcpa rrwghsecgh 1021 kedaavnctd isvqktpqka ttgrssrqss fiavgilgvv llaifvalff ltkkrrqrqr 1081 lavssrgenl vhqiqyremn sclnaddldl mnssenshes adfsaaelis vskflpisgmDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 1141 ekeailshte kengnl (SEQ ID NO: 56) CD61 1 mrarprprpl watvlalgal agvgvggpni cttrgvsscq qclavspmca wcsdealplg 61 sprcdlkenl lkdncapesi efpvsearvl edrplsdkgs gdssqvtqvs pqrialrlrp 121 ddsknfsiqv rqvedypvdi yylmdlsysm kddlwsiqnl gtklatqmrk ltsnlrigfg 181 afvdkpvspy myisppeale npcydmkttc lpmfgykhvl tltdqvtrfn eevkkqsvsr 241 nrdapeggfd aimqatvcde kigwrndash llvfttdakt hialdgrlag ivqpndgqch 301 vgsdnhysas ttmdypslgl mteklsqkni nlifavtenv vnlyqnysel ipgttvgvls 361 mdssnvlqli vdaygkirsk velevrdlpe elslsfnatc lnnevipglk scmglkigdt 421 vsfsieakvr gcpqekeksf tikpvgfkds livqvtfdcd cacqaqaepn shrcnngngt 481 fecgvcrcgp gwlgsqcecs eedyrpsqqd ecspregqpv csqrgeclcg qcvchssdfg 541 kitgkycecd dfscvrykge mcsghgqcsc gdclcdsdwt gyycncttrt dtcmssngll 601 csgrgkcecg scvciqpgsy gdtcekcptc pdactfkkec veckkfdrga lhdentcnry 661 crdeiesvke lkdtgkdavn ctykneddcv vrfqyyedss gksilyvvee pecpkgpdil 721 vvllsvmgai lliglaalli wkllitihdr kefakfeeer arakwdtann plykeatstf 781 tnityrgt (SEQ ID NO: 57) CD235a 1 mygkiifvll lseivsisas sttgvamhts tsssvtksyi ssqtndthkr dtyaatprah 61 evseisvrtv yppeeetger vqlahhfsep eitliifgvm agvigtilli sygirrlikk 121 spsdvkplps pdtdvplssv eienpetsdq (SEQ ID NO: 58) CD33 1 mplllllpll wagalamdpn fwlqvqesvt vqeglcvlvp ctffhpipyy dknspvhgyw 61 fregaiisrd spvatnkldq evqeetqgrf rllgdpsrnn cslsivdarr rdngsyffrm 121 ergstkysyk spqlsvhvtd lthrpkilip gtlepghskn ltcsvswace qgtppifswl 181 saaptslgpr tthssvliit prpqdhgtnl tcqvkfagag vttertiqln vtyvpqnptt 241 gifpgdgsgk qetragvvhg aiggagvtal lalclcliff ivkthrrkaa rtavgrndth 301 pttgsaspkh qkksklhgpt etsscsgaap tvemdeelhy aslnfhgmnp skdtsteyse 361 vrtq (SEQ ID NO: 59) CD16 (partial) 1 dlpkavvfle pqwyrvlekd svtlkcqgay spednstqwf hneslissqa ssyfidaatv 61 ddsgeyrcqt nlstlsdpvq levhigwlll qaprwvfkee dpihlrchsw kntalhkvty 121 lqngkgrkyf hhnsdfyipk atlkdsgsyf crglvgsknv ssetvnitit qglavstiss 181 ffppgyqvsf clvmvllfav dtglyfsvkt nirsstrdwk dhkfkwrkdp qdk (SEQ ID NO: 60) CD14 1 merascllll llplvhvsat tpepceldde dfrcvcnfse pqpdwseafq cvsaveveih 61 agglnlepfl krvdadadpr qyadtvkalr vrrltvgaaq vpaqllvgal rvlaysrlke 121 ltledlkitg tmpplpleat glalsslrlr nvswatgrsw laelqqwlkp glkvlsiaqa 181 hspafsceqv rafpaltsld lsdnpglger glmaalcphk fpaiqnlalr ntgmetptgv 241 caalaaagvq phsldlshns lratvnpsap rcmwssalns lnlsfagleq vpkglpaklr 301 vldlscnrln rapqpdelpe vdnltldgnp flvpgtalph egsmnsgvvp acarstlsvg 361 vsgtlvllqg argfa (SEQ ID NO: 61) CD64DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION 1 mwflttlllw vpvdgqvdtt kavitlqppw vsvfqeetvt lhcevlhlpg ssstqwflng 61 tatqtstpsy ritsasvnds geyrcqrgls grsdpiqlei hrgwlllqvs srvftegepl 121 alrchawkdk lvynvlyyrn gkafkffhwn snltilktni shngtyhcsg mgkhrytsag 181 isvtvkelfp apvlnasvts pllegnlvtl scetklllqr pglqlyfsfy mgsktlrgrn 241 tsseyqilta rredsglywc eaatedgnvl krspelelqv lglqlptpvw fhvlfylavg 301 imflvntvlw vtirkelkrk kkwdleisld sghekkviss lqedrhleee lkcqeqkeeq 361 lqegvhrkep qgat (SEQ ID NO: 62) CD206 1 mrlplllvfa svipgavlll dtrqfliyne dhkrcvdavs psavqtaacn qdaesqkfrw 61 vsesqimsva fklclgvpsk tdwvaitlya cdsksefqkw eckndtllgi kgedlffnyg 121 nrqeknimly kgsglwsrwk iygttdnlcs rgyeamytll gnangatcaf pfkfenkwya 181 dctsagrsdg wlwcgtttdy dtdklfgycp lkfegseslw nkdpltsvsy qinsksaltw 241 hqarkscqqq naellsitei heqtyltglt ssltsglwig lnslsfnsgw qwsdrspfry 301 lnwlpgspsa epgkscvsln pgknakwenl ecvqklgyic kkgnttlnsf vipsesdvpt 361 hcpsqwwpya ghcykihrde kkiqrdaltt crkeggdlts ihtieeldfi isqlgyepnd 421 elwiglndik iqmyfewsdg tpvtftkwlr gepshennrq edcvvmkgkd gywadrgcew 481 plgyickmks rsqgpeivev ekgcrkgwkk hhfycymigh tlstfaeanq tcnnenaylt 541 tiedryeqaf ltsfvglrpe kyfwtglsdi qtkgtfqwti eeevrfthwn sdmpgrkpgc 601 vamrtgiagg lwdvlkcdek akfvckhwae gvthppkptt tpepkcpedw gassrtslcf 661 klyakgkhek ktwfesrdfc ralggdlasi nnkeeqqtiw rlitasgsyh klfwlgltyg 721 spsegftwsd gspvsyenwa ygepnnyqnv eycgelkgdp tmswndince hlnnwicqiq 781 kgqtpkpept papqdnppvt edgwviykdy qyyfskeket mdnarafckr nfgdlvsiqs 841 esekkflwky vnrndaqsay figllisldk kfawmdgskv dyvswatgep nfanedencv 901 tmysnsgfwn dincgypnaf icqrhnssin attvmptmps vpsgckegwn fysnkcfkif 961 gfmeeerknw qearkacigf ggnlvsiqne keqafltyhm kdstfsawtg lndvnsehtf 1021 lwtdgrgvhy tnwgkgypgg rrsslsyeda dcvviiggas neagkwmddt cdskrgyicq 1081 trsdpsltnp patiqtdgfv kygkssyslm rqkfqwheae tycklhnsli asildpysna 1141 fawlqmetsn ervwialnsn ltdnqytwtd kwrvrytnwa adepklksac vyldldgywk 1201 tahcnesfyf lckrsdeipa teppqlpgrc pesdhtawip fhghcyyies sytrnwgqas 1261 leclrmgssl vsiesaaess flsyrveplk sktnfwiglf rnvegtwlwi nnspvsfvnw 1321 ntgdpsgern dcvalhassg fwsnihcssy kgyickrpki idakpthell ttkadtrkmd 1381 pskpssnvag vviivillil tgaglaayff ykkrrvhlpq egafentlyf nsqsspgtsd 1441 mkdlvgnieq nehsvi (SEQ ID NO: 63) CD123 1 mvllwltlll ialpcllqtk edpnppitnl rmkakaqqlt wdlnrnvtdi ecvkdadysm 61 pavnnsycqf gaislcevtn ytvrvanppf stwilfpens gkpwagaenl tcwihdvdfl 121 scswavgpga padvqydlyl nvanrrqqye clhyktdaqg trigcrfddi srlssgsqss 181 hilvrgrsaa fgipctdkfv vfsqieiltp pnmtakcnkt hsfmhwkmrs hfnrkfryel 241 qiqkrmqpvi teqvrdrtsf qllnpgtytv qirarervye flsawstpqr fecdqeegan 301 trawrtslli algtllalvc vfvicrrylv mqrlfpriph mkdpigdsfq ndklvvweag 361 kagleeclvt evqvvqkt (SEQ ID NO: 64) CXCR4 1 megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiifl tgivgnglvi 61 lvmgyqkklr smtdkyrlhl svadllfvit lpfwavdava nwyfgnflck avhviytvnl 121 yssvlilafi sldrylaivh atnsqrprkl laekvvyvgv wipallltip dfifanvsea 181 ddryicdrfy pndlwvvvfq fqhimvglil pgivilscyc iiisklshsk ghqkrkalkt 241 tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi 301 lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv stesesssfh ss (SEQ ID NO: 65)DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0227] The disclosure also relates to methods of activating immune cells comprising a step of exposing one or more immune cells to an antigen. In some embodiments, the step of activation occurs in the device at or proximate to the inlet and prior to the introduction of the cells into the reaction vessels. In some embodiments, probes are exposed to the cells in the reaction vessels prior to or contemporaneous to the step of labeling the cells.

[0228] The disclosure also relates to method of diagnosing a subject with an allergy or immune cell disorder comprising: (a) exposing immune cells from a subject to an antigen disclosed herein; (b) exposing the immune cells from the subject to a combination of probes, wherein the probes are specific to a protein expressed by the immune cells for form labeled or probed cells; (c) collecting or pooling the probed or labeled immune cells; (d) quantifying the number of labeled or probed cells; (e) correlating the number or presence of cells to a diagnosis of an immune-related disorder, such as an allergy to one or more antigens. In some embodiments, the step of quantifying the probes comprises normalizing a signal from the probes associated to the immune cell against a control amount of a probe known to be a positive or negative indication for the presence or respective absence of the disorder or disease. In some embodiments, the cells are used to diagnosis the disorder identified in the Assay Type column of Table 6, by isolating, probing or analyzing the cell type in Table 6 using probes specific for the markers in Table 6 and subsequently using the thresholds identified in Table to diagnose a subject from which the cells originated. As an example, in some embodiments, allergy to any listed activation agent or antigen listed in Table 6 can be diagnosed by taking blood from a subject, activating it with the listed antigen, labeling the cells with probes specific for the Markers identified in Table 6 and then quantifying the basophils labeled with the probes. If the normalized value exceeds the threshold identified in Table 6, the subject has an allergy to the antigen.

[0229] Methods of the disclosure also relate to a method of using a sample preparation device which, in some embodiments, comprises a sample comprising cells from a subject. In some embodiments, the methods results in collection of cells in a storage collection vessel or storage vessel. In some embodiments, after a step of mixing or sorting the cells in the disclosed devices, the cells are deposited in a storage vessel positioned at the outlet of the disclosed device. In some embodiments, the methods of the disclosure comprises a step of storing the storage vessel at about 4ºC for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days prior to a further step of analyzing the cells. In some embodiments, the methods of the disclosure comprises a stepDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION of storing the storage vessel at about 10ºC, 15ºC, 20º, or 25º for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days. In some embodiments, the methods of the disclosure comprises a step of storing the storage vessel at about 4ºC for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days, wherein the cells are labeled with one or more probes and the analyzing step comprises the step of detecting the presence, absence or quantity of an antibody or antibody fragment thereof In some embodiments, the methods of the disclosure comprises a step of storing the storage vessel at about 4ºC for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days, wherein the cells are labeled with one or more probes and the analyzing step comprises the step of detecting the presence, absence or quantity of an antibody or antibody fragment thereof and comprises characterizing the cells as expressing one or a plurality of biomarkers based upon the presence, absence or quantity of an antibody or antibody fragment thereof. Methods of the disclosure include, in some embodiments, exposing the device disclosed herein to a sample from a subject comprising a plurality of cells by sliding one or a plurality of disclosed plungers in order to draw cells into the one or plurality of vessels, exposing the one or plurality of cells to one or plurality of probes in the one or plurality of vessels; selectively ejecting a plurality of cells from one compartment or vessel into a storage vessel in fluid communication with the fluid circuit or ejecting all of the cells from the device into the storage vessel (depending upon the operable condition of the device and valve settings); detaching the storage vessel, and then storing the storage vessel at about 4ºC, 5ºC, 6ºC, 7ºC, 8ºC, 9ºC, 10ºC, 15ºC, 20º, or 25º for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days prior to a step of analyzing the cells, In some embodiments, the method further comprises analyzing the cells by expelling the labeled cells through an outlet and interrogating the labeled cells for quantification, identification and / or cytometry by detection of the one or plurality of probes. EXAMPLES Example 1

[0230] Embodiments herein include a device for BAT and / or sample preparation. The device may be an easy-to-use sample preparation device with a fully-automated data analysis workflow (FIG.1A). The device comprises: (1) a microfluidic device that performs all sample preparation steps of the BAT for eight stimulation conditions in parallel and stabilizes the stimulated blood prior to flow cytometry; (2) a barcoding method (FIG. 2A) that enables stimulated and stained basophils from the eight conditions to be pooled into a single tube; andDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION (3) a machine-learning-enabled data analysis pipeline for unpooling the eight barcoded basophil populations and determining their activation levels. Comparisons were made between basophil activation levels measured by BAT with the device (“the exemplified embodiment ”) vs. conventional BAT using blood from anonymous donors and evaluate the stability of blood prepared by the device.

[0231] Methods

[0232] The device performed concurrent basophil stimulation and staining for eight conditions in parallel. Barcoded staining of basophils allowed the pooling of all conditions into one lyse / fix buffer tube. An XGBoost-enabled analysis pipeline unpooled the eight conditions and generated basophil counts and activation levels. To characterize the device, nineteen blood samples from anonymous donors were stimulated with anti-IgE. A comparison was made between the exemplified embodiment and a conventional BAT protocol. The stability of stimulated samples stored in the lyse / fix buffer for 2- 7 days at 4°C was assessed.

[0233] Blood Samples

[0234] All blood samples were collected into heparinized tubes from the Stanford Blood Center (Palo Alto, CA) or through the Sean N. Parker Center for Allergy & Asthma Research (SNP) with informed consent under Stanford University's International Review Boards IRB #52850. They were stored at 4 °C for 4 to 6 hours before bringing them to room temperature prior to the start of experiments. Blood samples from N=19 anonymous donors were evaluated.

[0235] Reagents and material

[0236] Green-top, vacutainer sodium heparin blood collection tubes from BD Biosciences (catalog #368037, San Jose, CA) were acquired for blood collection. 5 mL round-bottom, polystyrene FACS tubes from Corning Falcon (Corning, NY) were used for flow cytometry, and 15 mL Nunc conical tubes from Thermo Fisher Scientific (catalog# 339650, Carlsbad, CA) were used for collecting fixed blood from the devices. Blood was introduced to the devices using BD Microtainers from BD Biosciences (catalog# 365965, San Jose, CA). RPMI-1640 medium was purchased from Gibco (Grand Island, NY). The two positive control stimulants were goat polyclonal anti-human IgE from Thermo Fisher Scientific (Carlsbad, CA) and N- Formylmethionyl-leucyl-phenylalanine (fMLP) from MilliporeSigma (St. Louis, MO). Peanut extract was prepared following an in-house protocol previously described.1

[0237] The barcode stain panel of this example consisted of antibody stains for CD45, HLA-DR, CD203c, CD193, CD123, and CD63. Various combinations of fluorophores were used for some markers, but the clones were the same across all stain panels (see Table 1). Stain cocktails were also supplemented with True-Stain Monocyte Blocker purchased fromDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION BioLegend (catalog# 426103, San Diego, CA) and BD Horizon Brilliant Stain Buffer (“BD buffer”) from BD Biosciences to mitigate interaction between BV stains (catalog# 566349, San Jose, CA).

[0238] The wash buffer consisted of 5% BSA and 2 mM EDTA in PBS (without calcium or magnesium). Anti-IgE, fMLP, and allergen extracts were stored at -80˚C and the rest of the reagents were stored at 4˚C. To lyse RBCs and fix the samples for storage, an RBC Lysis / Fixation Solution (“lyse / fix buffer”), purchased from BioLegend (catalog# 420302, San Diego, CA), was used.

[0239] Conventional BAT

[0240] Briefly, the conventional BAT sample preparation protocol comprised three steps each separated by a wash step (FIG.1B): (1) incubate 50 µL of whole blood with 50 µL of anti- IgE (10, 50, 100, 250, 500, or 1,000 ng / mL) or RPMI for 10, 20, or 30 minutes at 37˚C, (2) stain cells for CD45, HLA-DR, CD203c, CD193, CD123, and CD63 for 20 minutes on ice, and (3) lyse red blood cells and fix white blood cells in the dark for 30 minutes at room temperature.16,23,28,29

[0241] BATs were conducted with heparinized blood samples that were kept for 4 to 6 hours at 4˚C. Prior to beginning the BAT, the blood was brought to room temperature, as was the blood used for BAT with the devices.50 μL of blood was mixed with 50 μL of a dose of a stimulant in RPMI. Blood was stimulated with just RPMI (i.e., negative control), anti-IgE at 10, 50, 100, 250, 500, or 1,000 ng / mL. fMLP was used as a positive control in BAT experiments with the devices to indicate test validity and basophil responsiveness to the fMLP.

[0242] Blood was stimulated ex vivo for 10, 20, or 30 minutes. Samples were incubated at 37˚C in a water bath. Following the incubation period, basophils were quenched by adding 900 μL of ice-cold 2.5 mM EDTA in PBS, and the tubes were centrifuged at 500 G for 5 minutes at 4˚C. All centrifugations were performed with these settings unless specified otherwise. The supernatant was aspirated, the pellet was vortexed, and the antibody surface markers stains were added. Stains used were from the same panel as the exemplified embodiment experiments that were run in parallel with the conventional BAT controls. The stain cocktail included antibody labels for CD193, CD123, CD63, CD203c, HLA-DR, and CD45. The conventional BAT samples were not pooled, thus we only used one combination of CD193 and CD123 stains (e.g., anti-CD193 on BV421 and anti-CD123 on Alexa Fluor 700 or APC). Antibody stain cocktails contained 1 μL of each stain, 3 μL of monocyte blocker, and 25 μL of BD buffer (34 μL total per tube). Blood samples were stained for 20 minutes on ice, 3 mL of wash buffer was added, the tubes were centrifuged, and the supernatant aspirated. The pellet was vortexedDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION thoroughly prior to lysis and fixation to ensure all cells were resuspended and adequately exposed to lyse / fix buffer. 900 μL of room temperature 1x lyse / fix buffer was added, and the mixture was immediately mixed with a brief pulse on the vortex mixer. The tubes were left in the dark at room temperature for 30 minutes before topping them off ~4 mL with wash buffer. The tubes were centrifuged, the supernatant aspirated, and then the tubes were vortexed, This wash process was repeated after adding ~3 mL of wash buffer to the tubes. For flow cytometry, ~250 μL were left after the final aspiration step. In the experimenter’s hands, the total time to prepare these tubes for flow cytometry was ~3 hours.

[0243] Essential laboratory equipment includes an incubator, centrifuge, and vortex mixer. The duration of sample preparation and subsequent flow cytometry analysis is proportional to the batch size, e.g., processing eight tubes requires ~90 individual pipetting steps for exchange of buffer and addition of reagents. Data analysis, which includes manual gating and extracting basophil activation parameters to generate dose response curves and compute related metrics typically takes ~1 hour. The efficiency and accuracy of conventional BAT processes are heavily dependent on the skill and precision of the laboratory personnel.

[0244] Device experiments

[0245] Briefly, eight stimulation-stain cocktails each were prepared with a total volume of 50 µL. The same stimulation conditions were used (along with 1 µM N-Formylmethionyl- leucyl-phenylalanine (fMLP) as a positive control) and the stain panel as the conventional BAT was used, except (1) each stain panel had a unique combination of CD193 and CD123 fluorophores (Table 1), and (2) monocyte blocker was included to prevent the uptake of stain antibodies by monocytes. For a subset of experiments, blood was stimulated with peanut extract. Blood was mixed with stimulation-stain cocktails in the device and incubated at 37°C in a custom portable heater. Table 1 Stock Catalog StainDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION BV785 100 306032 A, B, D PE 50 306006A, B, C,DEantibody stains against a given marker always used the same clone even though the fluorophore differed across stain panels. Index values N=1-2 and N=1-4 were assigned to CD193 and CD123 stains, respectively, based on the index of the row in the look-up tables where barcodes and conditions were defined for each experiment. The complexity index of these stain panels ranged from 2.18 to 2.54, well within the acceptable range of complexity for a mixture of cells with 10 stains.29

[0246] More particularly, for the stain portion of the cocktail, 1 µL per stain was added for labelling CD193, CD123, CD63, CD203c, HLA-DR, and CD45 along with 3 µL of monocyte blocker and 25 µL of BD buffer. The volume of the stain portion in the stimulation-stain cocktails was 34 µL. The stimulation portion (anti-IgE, peanut extract, fMLP, or RPMI) comprised the remaining 16 µL and was added at concentrations that would yield the desired final concentration of the stimulation-stain cocktail before the addition of blood (e.g., 5 µL of 20,000 ng / mL anti-IgE diluted with 11 µL of RPMI added to 34 µL of stain for a final concentration of 2,000 µg / mL anti-IgE in the stimulation-stain cocktail). The concentrations of stimulants in the stimulation-stain cocktails were 20, 100, 200, 500, 1,000, or 2,000 ng / mL of anti-IgE, or 2 µM fMLP. In a subset of experiments the stimulant was 2, 20, 200, 2,000, or 20,000 ng / mL of peanut extract.

[0247] 50 µL of each stimulation-stain cocktail was loaded into individual syringes of the device (FIG. 2A) and allowed the cocktails to come to room temperature. For experiments to test a method herein of post-hoc concentration adjustment, 25, 50, or 100 µL of blood wasDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION stimulated per stimulation-stain condition. The syringe plungers were pulled in the device to withdraw blood into the syringes and the device was shaken gently to mix the blood with the stimulation-stain cocktails. Blood in the syringes was stimulated at 37˚C for 10, 20, or 30 min with a custom-made heater. Following stimulation, a 15 mL conical tube was loaded with 7.2 mL of lyse / fix buffer into the device’s collection port and ejected the blood into the lyse / fix buffer. Immediately after collection, we capped and inverted the tube to ensure adequate mixing of blood with the lyse / fix buffer. Samples were left in the lyse / fix buffer in the dark at room temperature for at least 30 min. For samples stored 2, 4, or 7 days, the lysed / fixed cell suspension from two device runs with 100 µL of blood per condition was split into fours tubes (one for day 0). Tubes were wrapped in foil and stored in a 4˚C fridge.

[0248] Before flow cytometry, tubes were topped off with ~6 mL of wash buffer, centrifuged for 10 min at 400 G (at room temperature or 4˚C), the supernatant aspirated to ~1 mL, and the pellet vortexed. Samples were transferred from the 15 mL tubes to FACS tubes and all cells were ensured to be collected by rinsing the 15 mL tube with ~3 mL of wash buffer. One final centrifugation of FACS tubes was performed for 5 min at 400 G, aspirated to ~500 µL, and vortexed the pellet.

[0249] Flow cytometry

[0250] All data were acquired with a Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA), a spectral flow cytometer which measures the entire emission spectrum of fluorophores rather than specific wavelengths like traditional flow cytometers.2Prior to each session with the Aurora, SpectroFlo QC beads from Cytek Biosciences (catalog# B7-10001, Fremont, CA) were run. The QC routine adjusted gains of each channel as needed to ensure day-to-day detector consistency. UltraComp eBeads from Thermo Fisher Scientific (catalog# 01-2222-42, Carlsbad, CA) were used as reference controls to provide the emission spectra of each fluorophore so that the Aurora can unmix raw data into distinct fluorescent intensities for each marker.

[0251] Fabrication of the device and heater

[0252] Stereolithographic printers were used to fabricate devices. Devices, consisting of the channel body and eight parallel syringe plungers (see, for example, the devices illustrated in FIGS. 15–#3H), were made from BioMed Amber resin on a Form 2 printer from Formlabs (Somerville, MA), from Onyxy Rigid Pro410 resin on a Saturn 2 from Elegoo (Shenzhen, Guangdong, China), or from KeySplint Hard resin on a Carbon M1 (Redwood City, CA). The syringes were made of 2-inch-long cast acrylic tubes, 1 / 8 inch ID and 1 / 4 inch outer diameterDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION (OD), from Emco Industrial Plastics (Cedar Grove, NJ) and were secured to the channel body with epoxy.

[0253] A heater was designed with heating pads that conformed to the shape of the syringes (see, for example, the heater illustrated in FIG.4). The heating pads were casted from lead-tin solder enclosed in a 3D printed case. The heat profile of samples in of our custom heater was comparable to samples warmed in a water bath.

[0254] Manual gating analysis and data annotation

[0255] All flow cytometry data was analyzed using FlowJo™ v10.8 Software (TreeStar, Ashland, OR). All workspaces shared common biex transformation parameters (negative decade and width of data compressed around zero) to ensure consistency in how data were represented and gated. The following parameters for each marker were used: negative = 1, width = -200 for CD63; negative = 1, width = -1000 for CD193, CD123, and CD203c; negative = 1, width = -500 for HLA-DR; and negative = 0, width = -200 for CD45. Basophils from singlets were gated as CD45+ / SSClowCD203chigh / HLA-DR- / CD123+CD193+(FIG.25),3–5and it was verified through backgating that no stained basophils were excluded from parent gates. The upper bound of CD63 expression was used in the negative control to establish the placement of the CD63+gate. Each specific experiment and condition assessed (i.e., BAT with a device, conventional BAT, 10 to 30 min incubation, 0 to 7 days stored) had its own negative control to reference. Manual gating provided the barcode index (BCI) and CD63+ / -labels required for training and testing automated gating models.

[0256] FlowKit was used to read Flow Cytometry Standard (FCS) files and loaded all data into Pandas DataFrames. To be consistent with how data was represented in FlowJo, the same biex transforms used FlowJo workspaces were applied to the FCS data frame using FlowKit’s WSPBiexTransform class. The membership of each event was determined to a specific barcoded basophil population (i.e., 1 if the event is in the gate, 0 if not) by analyzing manually gated FlowJo workspaces with FlowKit’s Workspace class.6Then, the membership of each event was encoded with a BCI: 0 for non-basophils and 1 to 8 for each barcode group. Similarly, basophils were labeled as CD63+ / -(1 if the basophils is CD63+, 0 if it is CD63-). All events were also annotated with a donor ID, stain panel ID, and various meta data features (i.e., BAT type, incubation time, days stored, blood volume). The barcode parser used these identifiers to reference look-up tables and assign a stimulation condition to each barcode index (BCI).

[0257] Automated gating pipeline

[0258] Briefly, we fed raw Flow Cytometry Standard (FCS) files as input into our gating pipeline, consisting of a basophil classifier, a CD63+classifier, and a barcode parser (FIG.2B).DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Both basophil and CD63+classifiers were built with XGBoost.30The basophil classifier unpooled the sample by labelling events with a barcode index (BCI) associated with each barcoded basophil population (1 to 8; 0 for non-basophils). The CD63+ / -classifier labelled activated basophils in barcoded basophil populations. Table 2 details the input features for each classifier. The barcode parser used predefined BCI look-up tables, defined for each sample, to assign a test condition (i.e., RPMI, a dose of anti-IgE or peanut, or fMLP) to each BCI to generate %CD63+ and CD203c mean fluorescent intensity (MFI) of each basophil population. All device results were generated with this pipeline, and conventional BAT results were generated with manual gating. See FIG.26 for additional details. Table 2 Input features Model Output nameFeatureFlow cytometer CategoricalclassificationPrecision Recallunpooling barcoded basophil populations and determining the activation status of basophils. Input features included flow cytometer parameters and categorical features describing the experiment. Indices on CD193 and CD123 parameters correspond to one of the two or four stains for CD193 or CD123 markers, respectively. The basophil classifier had nine output classifications, one for each barcoded population and one for non-basophils. The precision and recall were determined using test datasets according to Fig.26.

[0259] The automated gating pipeline was written in Python and consisted of an XGBoost barcoded basophil classifier, an XGBoost CD63+ / -classifier, and a data parser to assign conditions linked the BCIs of basophil populations and convert data of individual basophils to a population-level statistics data frame (i.e., calculate %CD63+and CD203c MFI) (FIG. 2B). First, the basophil classifier, an XGBoost model, unpooled the sample by labelling events as non-basophils or basophils from one of the eight barcoded populations. All flow cytometerDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION parameters, incubation time, stain panel ID, and days stored (Table 2) were used as input features to train and test the basophil classifier. Next, using the basophil populations from the basophil classifier as input, the CD63+ / -classifier, another XGBoost model, labelled activated basophils. The CD63+ / -classifier was supplied with all the same input features as the basophil classifier except for CD193 and CD123 to avoid introducing bias based on barcode-conditioncorrelations. The L2-norm of CD63 and CD203 fluorescent intensities (i.e.,√^^^^63ଶ + ^^^^203^^ଶ) were also supplied to the CD63+ / - classifier. Finally, the barcode parserused predefined BCI look-up tables, defined for each sample, to assign a test condition (i.e., RPMI, a dose of anti-IgE, or fMLP) to each BCI. The barcode parser generated %CD63+and CD203c mean fluorescent intensity (MFI) of each basophil population corresponding to a stimulation condition. See FIG. 26. After verifying that the automated gating pipeline was classifying basophils and CD63+ / -basophils accurately, the pipeline was relied on to generate results for device experiments in subsequent analyses. Across all experiments, a total of ~77 million events were collected, of which ~337,000 were basophils. From the manual gating analysis, each event was labelled with a BCI (1 to 8 and 0 for non-basophils) and a binary CD63+ / -label (1: CD63+, 0: CD63-). The basophils classifier was trained with 70% of all data (~54 million non-basophils, ~236,000 basophils). For the CD63+ / -classifier, populations of manually gated basophils (mean count of 521 ± standard deviation of 332) were partitioned and 80% (512) of these populations were used or training. There were ~63,000 CD63+and ~167,000 CD63- basophils represented in this training set.

[0260] The model hyperparameters of the basophil and CD63+ / -XGBoost classifiers were tuned using a Bayesian optimization method from BayesOpt which maximized the F1 score of model predictions. Both the basophil classifier and the CD63+ / -classifier used FSC-A, FSC-H, SSC, autofluorescence, CD45, HLA-DR, CD203c, CD63 (Table S2). For the basophil classifier, CD193-1, CD193-2, CD123-1, CD123-2, CD123-3, and CD123-4 were also included for the two CD193 stains and the four CD123 stains

[0261] Statistical Analysis

[0262] Wilcoxon signed-rank tests were used for all comparisons between BAT with a device and conventional BAT, incubation times, and samples stored for 0 and 2, 4, or 7 days. To control for false discovery rates during multiple hypothesis testing when comparing incubation times and day 0 samples with stored samples, the Benjamin and Hochberg method was used to compute adjusted P values (i.e., Q values). Also used were linear mixed-effects models with %CD63+or the change from baseline of CD203c MFI (CD203c ΔMFI) asDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION dependent variables to examine the effects of dose and sample storage period. P and Q values < 0.05 were considered statistically significant in all analyses. Results

[0263] The device performed sample preparation with <2 minutes of active user engagement. The analysis pipeline showed excellent agreement with manual gating analysis. Compared with conventional BAT, the exemplified embodiment exhibited similar baseline activation, higher maximum activation, higher area under the dose response curve (AUC), and lower EC50 values. Activation levels of basophils in samples stored in lyse / fix buffer for up to 7 days were similar to those analyzed on day 0.

[0264] The device simplifies sample preparation

[0265] Sample preparation in conventional BAT involved sequential stimulation, staining, and fixation (or red blood cell lysis), with each step separated by a wash (Fig. 1B).16,23,27,30,31Eight stimulation conditions took ~3.5 hours, during which active user engagement accounted for ~2 hours requiring ~90 pipetting and aspirating steps (Table S3). The device simplified the process, enabling (1) performance of concurrent stimulation and staining, and use of a lyse / fix buffer to both halt the stimulation and preserve the basophils with no wash steps, and (2) employing a staining method whereby the basophils in the eight stimulation conditions were “barcoded” with unique combinations of CD193 and CD123 stains (FIG. 2A). All stimulated and stained basophils were then pooled into a single tube with lyse / fix buffer. The eight conditions were unpooled during flow cytometry analysis

[0266] FIG. 33A and 33B illustrate a schematic representation of the automated gating pipeline.

[0267] FIG. 34 illustrates representative plots of the fully automated pipeline applied to test data. The input of CD63+ / - gating routine was barcoded basophil populations from the basophil classifier. BCI-1 and BCI-7 (RPMI and the 1,000 ng / mL anti-IgE dose, respectively) are shown for 0 days and 7 days of storage. Because a negative control is always referenced for a set of experimental conditions, the CD63+ / - gating can account for shifts in the baseline CD63 signal that are attributed to increased autofluorescence from storage in the lyse / fix buffer. Colored markers in the legend indicate events that were classified in agreement with manual gating, and the white marker indicates events that were labelled as CD63+ with the automated gating pipeline and CD63- with manual gating.

[0268] FIGS.35A-35B: Test data sets (not involved in training) were employed to compare the performance of manual gating with automated classifiers. (E) Basophil counts, and (F)DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION %CD63+ obtained with the CD63+ / - gating routine (high baseline outliers omitted), were highly correlated with values obtained with manual gating. (G) The correlation between %CD63+ AUCs calculated by the full automated analysis pipeline, which consists of the basophil classifier and the CD63+ / - gating routine, and %CD63+ AUCs determined via manual gating. The R-values provided denote Pearson's correlation coefficients for the least-squares regression lines applied to the scatter plots

[0269] FIG. 36 a flowchart outlining the automated analysis pipeline for basophil classification and CD63+ / - gating. The dataset consisted of approximately 136 million cells including 540 thousand basophils. After a conservative thresholding routine on CD45+ / SSClow / HLA-DR-, 76 million cells were used for the basophil classifier with a 30 / 70 test / train split. The performance of the Bayesian-optimized XGBoost model plus the CD63+ / - gating routine was evaluated with the test set. The fully automated pipeline was applied to all data (solid black arrows) to generate a population-level summary data frame (i.e., each row contains statistics of separate barcoded basophil populations). From this data frame, other BAT metrics were derived, e.g., area under the dose response curve (AUC).

[0270] FIG. 37 illustrates the average importance of features for the basophil classifier. Weight describes the number of times a feature is used to split the data across all trees (i.e., count of how often a feature is used in the model). Gain describes the contribution of each feature to the model by considering the improvement in accuracy brought by a feature to the splits it is used in (i.e., how much a feature contributes to making more accurate predictions). Cover describes the relative quantity of observations related to a feature (i.e., how many times on average a feature is used in a split across all trees, weighted by the number of training instances a node is responsible for).

[0271] FIGS. 38A-38C illustrates representative %CD63+ dose response curves for peanut-allergic (PA) and non-allergic (NA) donors with dose response curve metrics defined. (B) Tukey-style box and whisker plots show %CD63+ activation measurements of all subjects in which paired comparisons on the same dose were made. Wilcoxon signed-rank tests were used for paired comparisons (brackets and black font). Mann-Whitney U-tests were used to compare between PA and NA. The Benjamini and Hochberg method was used to adjust P values (i.e., Q values) by correcting for false discovery rates during multiple hypothesis testing.50 *Q < 0.05, **Q < 0.01, and ns is not significant (Q > 0.05). FIG. 29C illustrates there were no significant differences between the exemplified embodiment and conventional BAT for maximum activation, AUC and EC50 derived from %CD63+ dose response curves. FIG.29D show when using the exemplified embodiment and the automated analysis pipeline,DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION differences between non-allergic (NA) and peanut-allergic (PA) were significant for %CD63+ maximum activation and AUC. Results are shown for the N=6 NA and N=20 PA patients.

[0272] The automated gating pipeline was written in Python and consisted of an XGBoost barcoded basophil classifier, a data parser to assign conditions linked the BCIs of basophil populations, an algorithm to detect high CD63 baseline, and a CD63+ / - gating routine. First, the basophil classifier, an XGBoost model, unpooled the sample by labelling events as non- basophils or basophils from one of the eight barcoded populations. We used all flow cytometer parameters (FSC-A, FSC-H, SSC, autofluorescence, CD45, HLA-DR, CD203c, CD63, CD123, and CD193) and categorical features (incubation time, stain panel ID, and days stored) as input features to train and test the basophil classifier. After assigning BCIs the barcode parser used predefined BCI look-up tables, defined for each sample, to assign a test condition (i.e., RPMI, a dose of peanut extract or anti-IgE, or fMLP) to each BCI. Next, the RPMI negative control of each sample set was programmatically evaluated for high baseline activation using two criteria: 1) the z-score of the CD63 standard deviation exceeded a threshold of 2.5, or 2) the CD63 intensity distribution contained multiple peaks, with a second peak higher than the first. In cases of high baseline activation, all conditions associated with that RPMI control were manually gated. Next, parsed basophil data without instances of high baseline passed to the CD63+ / - gating routine which used the negative control within a set of test conditions to establish a cut-off for CD63+ / - basophils. This cut-off value was determined as the 97.5 percentile value of the negative control after outliers were removed, i.e., retaining basophils with CD63 fluorescent intensity < 75th percentile value + 1.5(interquartile range). The cut-off was applied to all test conditions within an experiment in the exemplified embodiment, including the negative control (with all basophils). The CD63 gating routine also converted data of individual basophils to population-level statistics data frames (i.e., calculate %CD63+).

[0273] Across all experiments, we collected a total of ~136 million events, of which ~540,000 were basophils. To reduce computational costs, we applied a conservative CD45+ / SSClow / HLA-DR- thresholding routine to reduce the data frame size to ~76 million cells. We trained and evaluated our models on all the data we collected, including experiments where we varied the blood volume and the storage time. We also supplemented our training and evaluation data with experiments where we incubated blood samples with anti-IgE or peanut extract for 5 to 30 minutes. From the manual gating analysis, each event was labelled with a BCI (1 to 8 and 0 for non-basophils) and a binary CD63+ / - label (1: CD63+, 0: CD63- ). We trained the basophils classifier with 70% of all data (~53 million non-basophils, ~378,000 basophils). We tuned the model hyperparameters of the basophil classifier using a BayesianDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION optimization method from BayesOpt which maximized the F1 score of model predictions. For the basophil classifier we also included CD193-1, CD193-2, CD123-1, CD123-2, CD123-3, and CD123-4 for the two CD193 stains and the four CD123 stains.

[0274] Table 3 Conventional BAT Device Sample preparation n ix) le ) erTable 3: Comparison of procedural differences for the exemplified embodiment and conventional BAT. The sample preparation comparison assumes eight stimulation conditions and focuses on the steps involved in sample preparation, specifically up to the stage, but before, the user would send the sample to a centralized lab for flow cytometry analysis. The flow cytometry comparison focuses on additional steps taken by a centralized lab to conduct run flow cytometry and conduct the data analysis. The approximate flow cytometry run times were extracted from meta data of FCS files.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0275] These modifications allowed design of simple microfluidic devices to perform all sample preparation steps prior to flow cytometry. A device comprised eight parallel syringes, one for each stimulation condition, to split whole blood equally among all syringes for the concurrent stimulation and staining for eight conditions in parallel without the need for lab equipment. Sample preparation using the device took <1 hour, during which active user engagement accounted for <2 minutes requiring a total of four manual steps only. Table 3 outlines the procedural differences between BAT with a device and conventional BAT.

[0276] The device was designed to generate a dose-response curve using 50 µL of whole blood per stimulation condition. However, in applications where pipettes are unavailable, achieving this precise volume may be difficult. Instead of engineering controls to meter exactly 50 µL per condition, correction for the potential variability in input blood volume was made with a post-hoc adjustment of the actual stimulant concentration. To test the feasibility of this method, 25 or 100 µL of blood was inputted for each condition and adjusted the stimulant concentration by a factor of 1.33 or 0.67, respectively. Although the broad range of stimulant doses (spanning three orders of magnitude) minimized the effect of varying blood volume, these post-hoc adjustments to the dose response curves resulted in a better four-parameter logistic regression fit to adjusted curves than non-adjusted curves (FIG.27).

[0277] Automated analysis is comparable to manual gating

[0278] The marker panel included HLA-DR, CD203c, CD193, and CD123, along with CD45 to exclude any unlysed red blood cells (Table 1). This panel was verified to identify basophils robustly.31–36For training and testing our model, manual gating was used to define barcode indices (BCIs) of basophil populations (belonging to one of the eight stimulation conditions) (FIG.2A, FIG.25), and their corresponding CD63+ / -labels.

[0279] When evaluated on the test datasets, the basophil classifier and the CD63+ / -classifier exhibited excellent performance, with 99% precision, 99% recall and 97% precision, 98% recall, respectively (Table 2). FIG. 28 shows the importance of each feature in the classification models. FIG.2C and FIG.2D show qualitative examples of the effectiveness of the basophil classifier in identifying each BCI from the test set, and of the fully-automated analysis pipeline in identifying CD63+ / -basophils, respectively. Excellent agreement (Pearson correlation coefficient, R > 0.989) was found in comparisons between metrics generated by individual classifiers and by manual gating (FIGS. 2E–2G) and between %CD63+area under the dose response curve (AUC) generated by the fully-automated pipeline and by manual gating (FIG. 2H). exemplified embodiment gives similar levels of activation compared with conventional BATo compare the performance of the exemplified embodiment withDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION conventional BAT, we generated dose response curves for %CD63+ basophils by stimulating whole blood from peanut-allergic (“PA”) donors (N=23) and from non-allergic (“NA”) donors (N=3) with 5 doses of peanut extract (1, 10, 100, 1,000, and 10,000 ng / mL) and 1 negative control (RPMI, i.e., peanut does = 0 ng / mL). We also included 2 positive controls in the exemplified embodiment to ensure the validity of the test: anti-IgE to indicate basophil responsiveness to IgE-mediated activation and fMLP to indicate test validity by stimulating non-specific activation pathways in leukocytes.

[0280] Compiling the BAT data from all subjects, we found no significant differences between the exemplified embodiment and conventional BAT in the median of %CD63+ except at peanut doses of 1,000 and 10,000 ng / mL in PA subjects. To gain a holistic interpretation of basophil response, we extracted dose response curve metrics, namely maximum activation, AUC, and the half maximal effective concentration (EC50) for %CD63+. We observed no significant differences between the exemplified embodiment and conventional BAT in the median of all these metrics. In particular, the similarity in EC50 indicates qualitative similarities in the shape of dose response curves generated by the exemplified embodiment and conventional BAT.

[0281] Finally, we show that the BAT prepared by the exemplified embodiment and conventional BAT distinguished between PA and NA samples. We found significant differences between PA and NA samples in median %CD63+ at each peanut dose, maximum activation, and AUC (P < 0.05).

[0282] We fed raw Flow Cytometry Standard (FCS) files as input into our gating pipeline, consisting of a conservative CD45+ / SSClow / HLA-DR- thresholding routine, a basophil classifier, an algorithm to detect high CD63 baseline, a CD63+ / - gating routine, and a barcode parser. The basophil classifier was built with XGBoost to unpool the sample by labelling events with a barcode index (BCI) associated with each barcoded basophil population (1 to 8; 0 for non-basophils).

[0283] Before applying the CD63+ / - gating routine, we implemented an algorithm to detect cases with high baseline activation, defined here to be %CD63+ >20% in the negative control. Previous studies have defined high baseline activation using varying criteria, ranging from a threshold of %CD63+ >5%, to %CD63+ / CD203c+ >20%. We defined high CD63 baseline using two criteria: 1) the z-score of the CD63 standard deviation exceeded a threshold of 2.5, or 2) the CD63 intensity distribution contained multiple peaks, with a second peak higher thanDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION the first. Implementing this high baseline detection algorithm avoided underestimating the actual %CD63+ in the stimulated conditions if we were to apply the CD63+ / - gating routine directly.

[0284] If the sample did not exhibit high baseline activation, we applied the CD63+ / - gating routine. The CD63+ / - gating routine used the negative control associated with each donor to determine a cut-off for the CD63+ / - gate. The cut-off was defined as the 97.5 percentile CD63 fluorescence intensity value of the negative control after removing outliers, defined as events with signal intensity that exceeded the third quartile by 1.5 times the interquartile range (Q3+1.5×IQR). To obtain the %CD63+ value for the negative control, outliers in the negative control were included when applying the CD63+ / - gate. The barcode parser used predefined BCI look-up tables, defined for each sample, to assign a test condition (i.e., RPMI, a dose of anti-IgE or peanut, or fMLP) to each BCI to generate %CD63+ of each basophil populationDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0285] An embodiment of the device has a higher dynamic range and sensitivity than conventional BAT

[0286] For both an exemplified embodiment and conventional BAT, no significant differences were found in %CD63+basophils and CD203c MFI, between 10, 20, and 30 minutes of incubation (FIG.8A). Qualitatively, an exemplified embodiment and conventional BAT dose response curves followed similar trends, but the an exemplified embodiment showed significantly higher levels of basophil activation, measured by %CD63+or CD203c MFI, compared with conventional BAT for all anti-IgE doses at each incubation time (Q < 0.01).

[0287] These differences were also reflected in the BAT metrics (defined in FIG. 28B). It was found that baseline activation on %CD63+was the only metric showing no significant difference between the exemplified embodiment and conventional BAT (P > 0.05). All other metrics showed significant differences (P < 0.01) between the exemplified embodiment and conventional BAT (FIG. 28C and Table 4). Baseline CD203c MFI was higher in the exemplified embodiment than in conventional BAT. Maximum activation and AUC on both %CD63+and CD203c ΔMFI were higher in the exemplified embodiment than in conventional BAT. The half maximal effective concentration (EC50) on both %CD63+and CD203c ΔMFI was lower in the exemplified embodiment than in conventional BAT. Reduced EC50 values obtained via the exemplified embodiment indicate enhanced sensitivity for detecting basophil reactivity at lower doses compared with conventional BAT. Both %CD63+and CD203c MFI measurements exhibited significantly higher dynamic ranges (i.e., absolute difference between maximum and baseline measurements) using the exemplified embodiment compared with conventional BAT (Fig.31, P < 0.01). Table 4 CD63 Baseline %CD63+Max %CD63+%CD63+AUC %CD63+EC50 AT3 7 ATDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION Table 4: Median values and median absolute deviation (MAD) of derived metrics half maximal effective concentrations (EC50), area under the dose response curve (AUC), baseline activation, and maximum activation for the exemplified embodiment and conventional BAT (conv. BAT). Wilcoxon signed- rank tests were used to calculate P values. These P values are represented in FIG.12.

[0288] Activated basophils remain stable in lyse / fix buffer.

[0289] Blood was stimulated with anti-IgE or peanut extract in the device, and a comparison made for samples with 0 days of storage with samples after 2, 4, or 7 days of storage at 4°C in the lyse / fix buffer prior to flow cytometry. The analysis showed no significant difference in %D63+levels across samples stored 0 to 7 days, and in CD203c ΔMFI across samples stored 0 to 4 days (FIG.30A). A significant decrease in CD203c ΔMFI was observed comparing samples stored 0 days vs.7 days (median values 7893.6 vs.7139.2, respectively).

[0290] Compared with day 0, an increase was observed in baseline MFI for CD63-FITC on days 2, 4, and 7, and for CD203c-BV510 on day 7 (FIG. 30B). However, this increase in baseline did not impact the performance of the CD63+ / -classifier, which was trained to learn these baseline shifts and adjust its classification outputs accordingly. Since the metrics %CD63+and CD203c ΔMFI were always calculated relative to the negative control (baseline activation) stored for the same number of days, these baseline shifts did not affect the activation measurements.

[0291] Representative dose response curves (FIG. 30C) illustrate the qualitative similarities between samples stored for 0 to 7 days. A linear mixed-effect model revealed that storage time (i.e., “days stored”) had a negligible effect on activation compared with anti-IgE dosage (FIG. 30D). The effect of days stored in the model was statistically insignificant for predicting %CD63+and CD203c ΔMFI (P = 0.57 and P = 0.35, respectively), indicating that the impact of storage duration was not greater than random variation.37DISCUSSION

[0292] This example demonstrates the potential of the device and the exemplified embodiment to broaden clinical access to embodiments of the disclosure by simplifying sample preparation to eliminate the requirement for laboratory equipment, stabilizing samples to remove the need for overnight blood shipping, and fully automating the data analysis pipeline.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0293] Embodiments of the disclosure involve a complex assay requiring fresh blood and 7–8 stimulation conditions. Conventional BAT has largely relied on express shipping of blood samples to a laboratory equipped to perform BAT within 24 hours.23,38A few studies explored performing sample preparation (i.e., activating, staining, and stabilizing basophils) in a laboratory close to the site of blood draw and shipping stabilized samples to a centralized laboratory for flow cytometry analysis.39,40The study of this example followed the latter approach, in part because the reliability of CD203c expression as an activation marker could drop in ~4 hours,41–44and basophil reactivity tends to diminish over time from its physiological levels once blood is drawn from the body.23,45With this approach, the ability to simplify sample preparation of embodiments of the disclosure is critical to increasing its accessibility. Ideally, this preparation could be performed at the point-of-care without requiring laboratory equipment and specialized personnel. Prior work aimed at streamlining the BAT has utilized liquid handing robots46or BAT kits.39,47–49Some commercial BAT kits combine stimulation and staining into a single step,50,51and basophil activation is halted by adding a lysis buffer to the blood directly. With these kits, sample preparation could be completed in ~1 hour for 4 stimulation conditions.51While BAT kits have fewer steps (4–6 pipetting steps per tube for each stimulation condition) than a conventional BAT (~11 pipetting steps per condition), they still require pipettes, incubator or water bath, vortex mixer, and trained personnel. The accessibility of BAT kits is therefore limited by the need for equipment that is unavailable outside of research laboratories.

[0294] In contrast, the device performed sample preparation (i.e., stimulate, stain, and lyse / fix blood) in four steps with <2 minutes of user involvement without the need for pipettes or laboratory-grade incubators or water baths. The total sample preparation time was ~42–62 minutes depending on the incubation time used (see details in Table 3). In this example, the cumulative time of active user involvement with the device utilized was only ~2.7 hours for a total of 648 individual stimulation conditions. The exemplified embodiment therefore achieved a ~60-fold and ~15-fold reduction in sample preparation time compared with conventional BAT and commercial BAT kit, respectively.50,51

[0295] An aspect of simplicity of its fluidic design, the device employed concurrent stimulation and staining as done in prior work.27,39,49In addition, the barcoding method using combinations of two fluorophores on CD193 and four on CD123 enabled sample pooling from eight separate stimulation conditions into one tube. This technique could be expanded to 16 conditions with two additional fluorophores on CD193. Barcoding and sample pooling also improved the efficiency of flow cytometry. The exemplified embodiment protocol reducedDOCKET NO. STFD-008-PCT PCT PATENT APPLICATION flow cytometry run time by >5-fold compared with using separate tubes for each condition (Table 3). For eight conditions, ~26 minutes of acquisition time was saved on the cytometer per donor sample, and a total reduction of ~35 hours across all 648 stimulation conditions.

[0296] The ability to stabilize blood after stimulation and staining is also an attractive aspect to the success of this example. A prior study showed that samples prepared using a commercial kit remained stable for up to 5 days at room temperature.39However, the details of the storage buffer was not described. Here it was shown that samples prepared with the device and stored in a commercially available lyse / fix buffer remained stable for up to 7 days at 4°C. This extended stability should reduce the need and the associated logistical hurdle for overnight shipping of fresh blood as required in conventional BAT.

[0297] In addition to simplifying the BAT sample preparation, a fully automated flow cytometry data analysis pipeline using XGBoost was demonstrated. SGBoost is a highly efficient ensemble-based supervised machine learning library.52Prior work on programmatic gating approaches for the BAT were only partially automated. They used a combination of approximate gate thresholds and k-means clustering on two-dimensional data. Manual inputs were required to define statistical cut-offs and correction factors to gate for basophils. One study estimated ~5 minutes of operator involvement per sample was necessary.26This work is the first application of XGBoost for the complete automation of the analysis of BAT flow cytometry data. The analysis took <1 second with no manual steps. The ensemble-based architecture of XGBoost, where each node of the decision trees evaluates a specific parameter with its own unique range of values, allowed us to combine flow cytometry features and non- flow cytometry features, such as incubation time, stain panel ID, and days stored.

[0298] The XGBoost models herein revealed the importance of each feature in the basophil and CD63+ / -classifiers across three metrics—weight, gain, and cover—each offering a different insight into feature importance (FIG. 28). Features with high weight, like CD203c, HLA-DR, and CD45 in basophil classification and autofluorescence in CD63+ / -classification, are frequently used for initial data splits, emulating initial manual gating steps (see FIG. 25). They are often located near the root of decision trees and have prominent effects on early split thresholds. Features with high gain, like CD123, CD193, HLA-DR and SSC in basophil classification and CD63 in CD63+ / -classification, provide the most improvement to the model’s accuracy. Features with high cover, like CD123 in basophil classification and non-cytometric parameters (i.e., stain panel ID, days stored) in CD63+ / -classification, affect a large portion of the dataset. They are typically found in the upper nodes of decision trees and influence how data is segmented.DOCKET NO. STFD-008-PCT PCT PATENT APPLICATION

[0299] A single study directly compared conventional BAT with BAT kits using concurrent stimulation and staining of basophils.27This prior study found similar levels of %CD63+but significantly lower CD203c MFI in the commercial BAT kit than their conventional “in-house” BAT method. This difference was attributed to the different labelling antibodies used in the two methods.27Under the conditions tested, the device achieved a higher mean anti-IgE-induced basophil activation (measured with both %CD63+and CD203c MFI), a higher dynamic range, and a higher sensitivity than did conventional BAT (FIGS. 29A–29C and FIG. 31). Because the same antibodies were used in the comparisons, it is speculated that these differences arose from concurrent stimulation and staining in the exemplified embodiment that permitted the labeling of activation markers as they were expressed. In contrast, staining after stimulation in conventional BAT might permit a slight down-regulation of activation markers before labelling. The baseline %CD63+(i.e., after RPMI incubation) was similar in the exemplified embodiment vs. conventional BAT. This result indicates that the device and process flow did not cause unintentional activation of basophils. Although the baseline CD203c MFI was higher in the exemplified embodiment than in conventional BAT, it did not impact other BAT metrics because CD203c activation is typically reported as CD203c ΔMFI after subtracting the baseline CD203c MFI (FIG.29C).

[0300] Both the embodiments of the disclosure and conventional BAT showed that anti- IgE-induced basophil activation was insensitive to the incubation time (from 10 to 30 minutes). This finding is consistent with prior work showing that Ca2+flux occurred in basophils within one minute of introducing anti-IgE.53However, due to differences between binding kinetics of anti-IgE and allergen peptides, it is expected that allergen-induced basophil activation will show increased sensitivity to incubation time.

[0301] Under the conditions tested, the levels of anti-IgE-induced and peanut-induced basophil activation in blood from anonymous donors were unaffected by storage for up to 7 days at 4°C for %CD63+and for up to 4 days for CD203c ΔMFI (FIG.30A). Consistent with prior work, shifts were observed in baseline fluorescence of the antibody stains after storing cells in a fixative (FIG.30B).54,55These shifts were likely due to increased autofluorescence or changes in the conformation of fluorophore molecules upon storage. Nevertheless, because basophil activation was measured relative to baseline fluorescence, the basophil populations and their activation levels remained distinguishable and accurately classified by our classifiers.

[0302] This study demonstrates the feasibility of a simplified workflow that has the potential to broaden the accessibility of the BAT for food allergy assessment.

Claims

AMENDED CLAIMS received by the International Bureau on 29 July 2025 (29.07.2025)1. A device for preparing a blood sample for testing, the device comprising: an inlet configured to receive a blood sample; an outlet; a plurality of reaction vessels, at least one of the reaction vessels including a stimulating agent, wherein each of the reaction vessels is configured to be in fluid communication with the inlet and the outlet; a pressure control device configured to: generate a pressure differential between the inlet and the reaction vessels such that the blood sample moves from the inlet into the reaction vessels, thereby separating the blood sample into a plurality of blood samples, generate a pressure differential between the reaction vessels and the outlet such that the blood samples move from the reaction vessels to the outlet, thereby recombining the plurality of blood samples into a recombined blood sample.

1. The device of Claim 1 , wherein the pressure control device is configured to distribute the blood sample into the reaction vessels in substantially equal volumes.

3. The device of Claim 1 or Claim 2, wherein the pressure control device is configured to generate a negative pressure to pull the blood sample from the inlet into the reaction vessels.

4. The device of Claim 1 or Claim 2, wherein the pressure control device is configured to generate a positive pressure to push the blood sample from the inlet into the reaction vessels.

5. The device of any one of Claims i to 4, wherein the pressure control device is configured to generate a negative pressure to pull the blood samples from the reaction vessels to the outlet.

6. The device of any one of Claims 1 to 4, wherein the pressure control device is configured to generate a positive pressure to push the blood samples from the reaction vessels to the outlet.

7. The device of any one of Claims 1 to 6, wherein the pressure control device comprises:a first pressure control device configured to create the pressure differential between the inlet and the reaction vessels, and a second pressure control device configured to create the pressure differential between the reaction vessels and the outlet.

8. The device of any one of Claims 1 to 7, wherein the pressure control device comprises a plurality of plungers, each disposed within a corresponding reaction vessel and configured to move along a longitudinal dimension of the corresponding reaction vessel.

9. The device of Claim 8, wherein the plurality of plungers are rigidly coupled to one another such that the plungers are configured to move within the reaction vessels as a single unit.

10. The device of Claim 8 or Claim 9, wherein the plungers are configured to move via manual actuation.

11. The device of Claim 8 or Claim 9, wherein the plungers are configured to move via activation of a motor.

12. T he device of any one of Claims 1 to 1 1, wherein the pressure control device comprises a single plunger disposed in a fluid path common to all of the reaction vessels such that movement of the plunger generates a pressure differential in each of the reaction vessels simultaneously.

13. T he device of Claim 12, wherein the plunger is configured to move via manual actuation.

14. The device of Claim 12, wherein the plunger is configured to move via activation of a motor.

15. The device of any one of Claims 1 to 7, wherein the pressure control device comprises one or more pumps.

16. The device of any one of Claims 1 to 15, wherein the stimulating agent is an antigen.

17. The device of any one of Claims 1 to 16, wherein the stimulating agent is a suspected allergen.

18. The device of any one of Claims 1 to 17, wherein the stimulating agent is a first stimulating agent, and wherein one of the reaction vessels has the first stimulating agent and another one of the reaction vessels has a second stimulating agent different than the first stimulating agent.

19. The device of any one of Claims 1 to 18, wherein the stimulating agent comprises a reconstituable powder.

20. The device of any one of Claims 1 to 18, wherein the stimulating agent comprises a liquid composition.

21. The device of any one of Claims 1 to 20, wherein the at least one reaction vessel further includes a staining agent.

22. The device of Claim 21 , wherein the staining agent is configured to confer a unique tag to the one of the plurality of blood samples in the at least one reaction vessel.

23. The device of Claim 21 or Claim 22, wherein the staining agent comprises a reconstituable powder.

24. The device of Claim 21 or Claim 22, wherein the staining agent comprises a liquid composition.

25. The device of any one of Claims 1 to 24, wherein the reaction vessels comprise first reaction vessels and second reaction vessels, and wherein: at least one of the first reaction vessels comprises a stimulating agent and at least one of the second reaction vessels comprises staining agent, and the pressure control device is configured to generate a pressure differential to move each of the blood samples in the first reaction vessels to a corresponding one of the second reaction vessels.

26. The device of Claim 25, wherein the first reaction vessels do not comprise a staining agent and the second reaction vessels do not comprise a stimulating agent.

27. The device of Claim 25 or Claim 26, wherein the pressure control device is configured to generate a pressure differential to move each of the blood samples in the second reaction vessels to the outlet.

28. The device of Claim 25 or Claim 26, wherein the reaction vessels comprise third reaction vessels, and wherein: the third reaction vessels contain a wash solution, and the pressure control device is configured to generate a pressure differential to move each of the blood samples in the second reaction vessels to a corresponding one of the third reaction vessels.

29. The device of Claim 28, wherein the pressure control device is configured to generate a pressure differential to move each of the blood samples in the third reaction vessels to the outlet.

30. The device of any one of Claims 1 to 29, further comprising a mixing element within the at least one reaction vessel.

31. The device of any one of Claims 1 to 30, further comprising a heating element configured to raise a temperature of a contents of the reaction vessels.

32. The device of any one of Claims 1 to 31 , wherein the inlet, the outlet, the reaction vessels, and the pressure control device are integrated into a single device.

33. The device of any one of Claims 1 to 31, wherein the inlet, the outlet, and the reaction vessels together comprise a first component, and the pressure control device comprises a second component configured to receive the first component.

34. The device of Claim 33, wherein receipt of the first component by the second copmonent operably connects the pressure control device to the reaction vessels.

35. A device for preparing a blood sample for testing, the device comprising: an inlet configured to receive a blood sample;a plurality of reaction vessels, at least one of the reaction vessels including a stimulating agent; a plurality of inlet channels, each extending between and fluidly connecting the inlet and a corresponding one of the reaction vessels; a plurality of outlet channels; an outlet, wherein each of the outlet channels extends between and fluidly connects a corresponding one of the reaction vessels to the outlet; and a pressure control device configured to: generate a pressure differential between the inlet and the reaction vessels such that the blood sample moves through the inlet channels and into the reaction vessels, thereby separating the blood sample into a plurality of blood samples; generate a pressure differential between the reaction vessels and the outlet such that the blood samples move from the reaction vessels into the outlet channels and through the outlet, thereby recombining the blood samples into a recombined blood sample.

36. A method for preparing a blood sample for testing, the method comprising: receiving a blood sample through an inlet of a sample preparation device; dividing the blood sample into a plurality of blood samples and moving each of the blood samples into a corresponding reaction vessel associated with the sample preparation device, at least one of the reaction vessels including a stimulating agent; and recombining the blood samples into a single recombined blood sample.

37. The method of Claim 36, wherein the stimulating agent is an antigen.

38. The method of Claim 36 or Claim 37, wherein each of the reaction vessels includes a staining agent.

39. The method of any one of Claims 36 to 38, wherein the reaction vessels are first reaction vessels, and wherein the method further comprises moving the blood samples from the first reaction vessels to second reaction vessels.

40. The method of Claim 39, wherein at least one of the first reaction vessels includes a stimulating agent and wherein each of the second reaction vessels include a staining agent.

41. The method of any one of Claims 36 to 40, wherein the blood samples are recombined into the single recombined blood sample within the sample preparation device.

42. The method of any one of Claims 36 to 41 , further comprising moving the single recombined blood sample through an outlet of the sample preparation device.

43. The method of any one of Claims 36 to 42, w herein the sample preparation device includes a pressure control device configured generate the motive force for dividing the blood sample and / or moving the blood samples into corresponding reaction vessels.

44. A device for preparing a blood sample for testing, the device comprising: an inlet configured to receive a blood sample; a plurality of outlets; a plurality of reaction vessels, at least one of the reaction vessels including a stimulating agent, wherein each of the reaction vessels is configured to be in fluid communication with the inlet and the outlets; and a pressure control device configured to: generate a pressure differential between the inlet and the reaction vessels such that the blood sample moves from the inlet into the reaction vessels, thereby separating the blood sample into a plurality of blood samples, generate a pressure differential between the reaction vessels and the outlets such that the blood samples move from the reaction vessels to the corresponding outlets.

45. The device of Claim 44, wherein the pressure control device is configured to distribute the blood sample into the reaction vessels in substantially equal volumes.

46. The device of Claim 44 or Claim 45, wherein the pressure control device is configured to generate a negative pressure to pull the blood sample from the inlet into the reaction vessels.

47. The device of Claim 44 or Claim 46, w-herein the pressure control device is configured to generate a positive pressure to push the blood sample from the inlet into the reaction vessels.

48. The device of any one of Claims 44 to 47, wherein the pressure control device is configured to generate a negative pressure to pull the blood samples from the reaction vessels to the outlets.

49. The device of any one of Claims 44 to 47, wherein the pressure control device is configured to generate a positive pressure to push the blood samples from the reaction vessels to the outlets.

50. The device of any one of Claims 44 to 49, wherein the pressure control device comprises: a first pressure control device configured to create the pressure differential between the inlet and the reaction vessels, and a second pressure control device configured to create the pressure differential between the reaction vessels and the outlets.

51. The device of any one of Claims 44 to 50. wherein the pressure control device comprises a plurality of plungers, each disposed within a corresponding reaction vessel and configured to move along a longitudinal dimension of the corresponding reaction vessel.

52. The device of Claim 51 , wherein the plurality of plungers are rigidly coupled to one another such that the plungers are configured to move within the reaction vessels as a single unit.

53. The device of Claim 51 or Claim 52, wherein the plungers are configured to move via manual actuation.

54. The device of Claim 51 or Claim 52, wherein the plungers are configured to move via activation of a motor.

55. The device of any one of Claims 44 to 50, wherein the pressure control device comprises a single plunger disposed in a fluid path common to all of the reaction vessels such that movement of the plunger generates a pressure differential in each of the reaction vessels simultaneously.

56. The device of Claim 55, wherein the plunger is configured to move via manual actuation.

57. The device of Claim 55 or Claim 56, wherein the plunger is configured to move via activation of a motor.

58. The device of any one of Claims 44 to 50, wherein the pressure control device comprises one or more pumps.

59. The device of any one of Claims 44 to 58, wherein the at least one reaction vessel further includes a staining agent.

60. The device of Claim 59, wherein the staining agent is configured to confer a unique tag to the one of the plurality of blood samples in the at least one reaction vessel.61 A method for preparing a blood sample, the method comprising: receiving a blood sample through an inlet of a sample preparation device; dividing the blood sample into a plurality of blood samples and moving each of the blood samples into a corresponding reaction vessel associated with the sample preparation device, at least one of the reaction vessels including a stimulating agent; and moving the blood samples out of the sample preparation device via corresponding outlets.

62. The method of Claim 61, wherein the sample preparation device includes a pressure control device configured generate the motive force for dividing the blood sample and / or moving the blood samples into corresponding reaction vessels.

63. A method of cell pooling or unpooling comprising: (a) mixing the cells in the compartments within the composition of claims 65 through 71 or the device of claims 72 through 97 or the device of claims 39 through 60.

64. The method of claim 63, further comprising (b) exposing the cells to at least two probes within the compartments; and (c) pooling the cells in a storage vessel.

65. A composition comprising a plurality of immune cells, the immune cells divided among and positioned within a first, second, third, fourth, fifth and sixth compartment; wherein the first, second, third, fourth, fifth and sixth compartments each comprise: at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein.

66. The composition of claim 65, wherein the first immune cell surface protein is CD193 or a functional variant thereof and the second immune cell surface protein is CD123 or a functional variant thereof.

67. The composition of claim 66, wherein the probes specific to CD 193 are chosen from: BV421 , BV605 or derivatives thereof; and wherein the probes specific to CD123 are chosen from: PE, PE / Cy7, APC, BV785 or a derivative thereof68. The composition of any of claims 65 through 67 further comprising: (i) an inlet vessel in fluid communication with each of the first, second, third, fourth, fifth and sixth compartments: and (ii) immune cell activator positioned within or proximate to the inlet vessel or each compartment.

69. The composition of any of claims 65 through 68, wherein the immune cells are chosen from one or a combination of: a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil and / or platelets.

70. The composition of any of claims 65 through 69 further comprising a seventh compartment and an eighth compartment; wherein the first, second, third, fourth, fifth, sixth, seventh and eighth compartments are uniformly cylindrical in shape and in parallel orientation along their longitudinal axis.

71. The composition of claim 70, 'wherein each of the compartments further comprises a syringe, each syringe mechanically connected a handle, such that movement of the handle simultaneously slides the syringes along the longitudinal axis of the compartments.

72. A device comprising:(i) a fluid circuit comprising an inlet, an outlet and a plurality of reaction vessels, the plurality of reaction vessels in fluid communication with the inlet and the outlet;(ii) a first set of valves positioned in the fluid circuit between the inlet and the reaction vessels, adjustable in an open and closed position; wherein an open position allows for fluid flow between the inlet and the reaction vessels;(iii) a second set of valves positioned in the fluid circuit between the outlet and the reaction vessels, adjustable in an open and closed position; wherein the open position allows for fluid flow between the outlet and the reaction vessels.

73. The device of claim 72, wherein each reaction vessel comprises a combination of at least two probes specific for an immune cell.

74. The device of claim 73, wherein the reaction vessel is separated into a reaction region and a reagent storage region; and wherein the reagent storage region is in fluid communication with the reaction region.

75. The device of claim 74, wherein, in a first operational condition, the reagent storage region comprises a combination of at least two probes specific for an immune cell in a dried or powdered formulation.

76. The device of claim 74, wherein in a second operational condition, the reaction region comprises a combination of at least two probes specific for an immune cell, and one or a plurality of immune cells.

77. The device of any of claims 72 through 7612 further comprising an inlet adaptor positioned proximate to and operably linked to the inlet, such that the inlet adaptor receives an inlet vessel and aligns the inlet vessel to the inlet.

78. The device of any of claims 72 through 77 further comprising an inlet vessel in fluid communication to the inlet.

79. The device of claim 78, wherein the inlet vessel comprises a sample.

80. The device of claim 79, wherein the sample is whole blood.

81. The device of any of claims 72 through 80, further comprising an outlet adaptor positioned proximate to and operably linked to the outlet and a storage vessel, such that the outlet adaptor is configured to receive the storage vessel and aligns the outlet to the storage vessel.

82. The device of claim 81, wherein the storage vessel is a fifteen milliliter conical tube comprising protruding spiral threads around an opening of the tube; wherein the outlet adaptor is a plastic protrusion comprising an inner and outer surface; wherein the inner surface protrudes around the outlet and comprises a circular or semicircular spiral inlay positioned around the outlet; and wherein the tube and the outlet adaptor define a joint positioning the opening of the tube in fluid communication with the fluid circuit at the outlet.

83. The device of claim 72 further comprising a storage vessel in fluid communication with the fluid circuit and positioned proximate to the outlet of the fluid circuit.

84. The device of any of claims 72 through 82 further comprising an immune cell activator positioned within or proximate to the reaction vessels.

85. The device of any of claims 72 through 84, further comprising one or a plurality of immune cells chosen from one or a combination of: a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil, and / or platelets.

86. The device of any of claims 72 through 85, further comprising a housing positioned over the fluid circuit; wherein the reaction vessels are channels positioned at or near a first set of conduits connecting the inlet to the channels.

87. The device of claim 86, wherein the fluid circuit comprises an inlet, a first set of conduits in fluid communication to the reaction vessels, a second set of conduits connecting the reaction vessels to the outlet.

88. The device of claim 87, wherein the first and second conduits are positioned are branched, positioned parallel to one another and intersect a valve channel that is positioned orthogonally or substantially orthogonally across the first and second sets of conduits at intersection points, and wherein the valve channel is positioned transverse to a width dimension of the housing.

89. The device of claim 88, wherein the valve channel comprises a valve element in operable contact with a plurality of valves, wherein one valve is positioned at each intersection point.

90. The device of claim 89, wherein the valve element in the valve channel comprises two or four total valves and the valve element is adjustable and operably connected to the valves such that movement of the valve element opens and closes the valves.

91. The device of claim 90 wherein the valve element is cylindrical or semicylindrical, the valve element is rotatable about its longitudinal axis and has at least a first and a second operable position; wherein, in the first operable position, a valve positioned at an intersection point proximate to the inlet is open and a valve positioned at an intersection point proximate to the outlet is closed; and, in the second operable position, the valve positioned the an intersection point proximate to the inlet is closed and a valve positioned at an intersection point proximate to the outlet is open.

92. The device of claim 91 wherein the valve element is operably connected to a dial positioned on the side of the housing, the dial movable radially between the first and second operable position of the valve element.

93. The device of any of claims 72 through 92 wherein the plurality of reaction surfaces comprises a first, second, third, fourth, fifth, sixth, seventh and seventh and eighth compartment; wherein the first, second, third, fourth, fifth, sixth, seventh and eighth compartments are uniformly cylindrical in shape andin parallel orientation along their longitudinal axis; and each compartment comprises at least about two probes specific for a first immune cell surface protein and a second immune cell surface protein.

94. The device of claim 93, wherein each of the compartments further comprises a syringe, each syringe mechanically connected a movable handle positioned on the outside of the housing, such that movement of the handle laterally from the exterior of the device simultaneously slides the syringes along their longitudinal axis m the cylindrical or semicylindrical compartments.

95. The device of any of claims 72 through 94, wherein each of the first, second, third, fourth, fifth and sixth compartments comprises an adjustable valve positioned opening and closing fluid flow from the compartments to the outlet.

96. The device of any of claims 72 through 95, further comprising a storage vessel attached to an outlet adaptor positioned at or around the outlet of the fluid circuit configured for fluid flow from the outlet into the storage vessel in an operable condition wherein one or a plurality of valves are open between the compartments and the outlet.

97. The device of claim 96, wherein the storage vessel comprises one or a plurality of stabilizing agents.98 A system comprising:(i) the device of claims 72 through 97 or the composition of any of cla ims 65 through 71 ; and(ii) a heating element electrically connected to a battery source.

99. The system of claim 98 wherein the heating element is configured to receive at least a region of the device or composition comprising the compartments, such that the heating element is proximate to one or more surfaces of the compartments100. The system of any of claims 98 or 99 further comprising:(c) a processor operable to execute programs; and(d) a memory associated with the processor.

101. The system of claim 100 further comprising a computer program product on a non- transitory computer-readable software medium, disclosed herein.

102. The system of claim 100 or claim 101 wherein the device or composition comprises one or more data communication interfaces configured for communicably connection by a data communication network.

103. The system of claim 102, wherein at least one data communication interface is a Bluetooth antenna positioned within the device configured to communicate to the internet and / or a computer-implemented data network in operably linked to a server.

104. A method activating an immune cell comprising:(a) exposing the plurality of immune cells to an antigen within the composition of claims 65 through 71 or the device of claims 72 through 97.

105. The method of claim 104, wherein the step of exposing the one or plurality of immune cells to an antigen is performed within at least one or a plurality of the compartments in the composition or the device.

106. The method of either of claims 104 or 105 further comprising:(a) separating the cells into the compartments;(b) exposing the cells to at least two probes within the compartments; and(c) pooling the cells in a storage vessel.

107. The method of claim 106 further comprising (e) identifying and / or sorting the cells based upon detection of at least two probes.

108. The method of any of claims 104 through 107 further comprising the step of:(f) incubating the cells in a heated condition from about 5 to about 30 minutes before or during the step (c).

109. The method of any of claims 104 through 108, wherein step (a) is performed at or about 37 degrees Celsius.1 10. The method of either of claim 107 or 109, wherein steps (b) and (d) are performed in no more than about 2 minutes.1 1 1. The method of any of claims 106 or 107 further comprising (g) lysing the cells, (h) fixing the cells and (i) quantifying the presence of the probes after pooling the cell in the storage vessel.1 12. The method of any of claims 104 through 1 1 1 , wherein the antigen is chosen from one or a combination of: solid or liquid nut extract.1 13. The method of any of claims 104 through 1 1 1, wherein the antigen is chosen from one or a combination of any antigen identified on Table 1 , Table 5 or Table 6 or a functional variant thereof comprising at least about 75% sequence identity to an amino acid sequence identified on Table 1 , Table 5 or Table 6.1 14. The method of any of claims 104 through 1 13, further comprisi ng drawing a sample of whole blood in an inlet vessel through the fluid circuit and into the compartments prior to the step of exposing.1 15. The method of any of claims 104 through 1 14, wherein the probes are chosen from one or a combination of probes on Table 2.1 16. The method of any of claims 104 through 1 15, wherein steps (a), (c), and (d) are performed after separating the cells into the compartments.1 17. A. method of cell sorting comprising:(a) separating the cells into the compartments within the composition of claims 65 through 71 or the device of claims 72 through 97;(b) exposing the cells to at least two probes;(c) pooling the cells in the pooling vessel.1 18. The method of claim 1 17 further comprising the step (d) of exposing the plurality of immune cells to an antigen to elicit an antigen-specific or non-specific immune response.1 19. The method of claims 1 17 or 1 18, wherein steps (b), (c), and (d) are performed after separating the cells into the compartments.

120. The method of claim 1 17, wherein the step of exposing I s performed m the compartments.

121. The method of any of claims 1 17 through 120, wherein steps (b) and (d) are performed in no more than about 2 minutes.

122. The method of any of claims 1 17 through 121 , further comprising (d) lysing the cells, and fixing the cells after step (c).

123. The method of any of claims 1 17 through 122 further comprising (f) quantifying the presence of the probes after step (c).

124. T he method of claim 1 18, wherein the antigen is chosen from one or a combination of: solid or liquid nut extract.

125. The method of claim 118, wherein the antigen is chosen from one or a combination of any antigen on Table 1, Table 5, Table 6 or a functional variant thereof comprising at least about 75% sequence identity to an amino acid sequence on Table 1, Table 5 or Table 6.

126. The method of any of claims 117 through 125 further comprising drawing a sample of whole blood in an inlet vessel through the fluid circuit and into the compartments prior to the step (a).

127. The method of any of claims 117 through 126, wherein the probes are chosen from one or a combination of probes on Table 2.

128. The method of any of claims 1 17 through 123 further comprising (g) identifying or sorting the cells based upon detection of at least two probes.

129. The method of any of claims 117 through 128 further comprising the step of:(h) incubating the cells in a heated condition from about 5 to about 30 minutes before or during the step (c).

130. A method of analyzing a sample comprising:(a) separating the cells into the compartments within the composition of claims 65 through 71 or the device of claims 72 through 97;(b) exposing the cells to at least two probes;(c) pooling the cells in the pooling vessel; and analyzing the cells.

131. The method of claim 130, wherein the step of analyzing comprises one or a combination of: (i) performing flow cytometry; (ii) performing fluorescent microscopy with or without digital imaging; (iii) performing light microscopy; (iv) counting the cells; (v) digital imaging of the cells; (vi) cell sorting with any computer program product disclosed herein; (vii) barcoding the nucleic acid sequences within the pooling vessel; (viii) sequencing or quantifying nucleic acid expression or amino acid expression of the sample; and (ix) mass cytometry.

132. The method of claim 130 further comprises step (e) of exposing the plurality of immune cells to an antigen to elicit an antigen-specific or non-specific immune response.

133. The method of any of claims 130 through 132, wherein steps (b), (c), and (d) are performed after separating the cells into the compartments.

134. The method of claim 132, wherein the step of exposing I s performed m the compartments.

135. The method of any of claims 130 through 134, wherein steps (b) and (d) are performed in no more than about 2 minutes.

136. The method of any of claims 130 through 135, further comprising (d) lysing the cells, and fixing the cells after step (c).

137. The method of any of claims 130 through 136 wherein the step of analyzing comprises quantifying the presence of the probes after step (c).

138. The method of claim 132, wherein the antigen is chosen from one or a combination of: solid or liquid nut extract.

139. The method of claim 132, wherein the antigen is chosen from one or a combination of any antigen on Table 1 , Table 5 or Table 6 or a functional variant thereof comprising at least about 75% sequence identity to an amino acid sequence on Table 1, Table 5 or Table 6.

140. The method of any of claims 130 through 139 further comprising drawing a sample of whole blood through the inlet and into the compartments prior to the step (a).

141. The method of any of claims 130 through 140, wherein the probes are chosen from one or a combination of probes on Table 2.

142. The method of any of claims 130 through 141 further comprising (g) identifying or sorting the cells based upon detection of at least two probes.

143. The method of any of claims 130 through 142 further comprising the step of:(h) incubating the cells in a heated condition from about 5 to about 30 minutes before or during the step (c).

144. A method of identifying a cell population and their activation status comprisingseparating the cells into the compartments within the composition of claims 65 through 71 or the device of claims 72 through 97; exposing the cells to at least two probes; pooling the cells in the pooling vessel; and analyzing the cells.

145. The method of claim 16, wherein the step of analyzing comprises one or a combination of: (i) performing flow cytometry; (ii) performing fluorescent microscopy with or without digital imaging; (iii) performing light microscopy; (iv) counting the cells; (v) digital imaging of the cells; (vi) cell sorting with any computer program product disclosed herein; (vii) barcoding the nucleic acid sequences within the pooling vessel; (viii) sequencing or quantifying nucleic acid expression or amino acid expression of the sample; and (ix) mass cytometry.

146. T he method of claim 17 further comprises step (e) of exposing the plurality of immune cells to an antigen to elicit an antigen-specific or non-specific immune response.

147. The method of any of claims 144 through 146, wherein steps (b), (c), and (d) are performed after separating the cells into the compartments.

148. The method of claim 147, wherein the step of exposing I s performed m the compartments.

149. T he method of any of claims 144 through 148, wherein steps (b) and (d) are performed in no more than about 2 minutes.

150. The method of any of claims 144 through 149, further comprising (d) lysing the cells, and (e) fixing the cells after step (c).

151. The method of any of claims 144 through 150 wherein the step of analyzing comprises quan tifying the presence of the probes after step (c).

152. The method of claim 145, wherein the antigen is chosen from one or a combination of: solid or liquid nut extract.

153. The method of claim 145 or 146, wherein the antigen is chosen from one or a combination of any antigen on Table 1, Table 5 or Table 6 or a functional variant thereof comprising at least about 75% sequence identity to an amino acid sequence on Table 1 , Table 5 or Table 6.

154. The method of any of claims 144 through 153 further comprising drawing a sample of whole blood through the inlet and into the compartments prior to the step (a).

155. The method of any of claims 144 through 154, wherein the probes are chosen from one or a combination of probes on Table 2 and probes specific to the markers on Table 5 or 6.

156. T he method of any of cla ims 81440 through 155 further comprising (g) identifying or sorting the cells based upon detection of at least two probes.

157. A method of preparing a sample of cells from a subject(a) separating the cells into the compartments within the composition of claims 65 through 71 or the device of claims 72 through 97;(b) exposing the cells to at least two probes;(c) pooling the cells in the pooling vessel; and(d) analyzing the cells.

158. The method of claim 157, wherein the step of analyzing comprises one or a combination of: (i) performing flow cytometry; (ii) performing fluorescent microscopy with or without digital imaging; (iii) performing light microscopy; (iv) counting the cells; (v) digital imaging of the cells; (vi) cell sorting with any computer program product disclosed herein; (vii) barcoding the nucleic acid sequences within the pooling vessel; (vi ii) sequencing or quantifying nucleic acid expression or amino acid expression of the sample; and (ix) mass cytometry.

159. The method of claim 158 further comprises step (e) of exposing the plurality of immune cells to an antigen to elicit an antigen-specific or non-specific immune response.

160. The method of any of claims 157 through 159, wherein steps (b), (c), and (d) are performed after separating the cells into the compartments.

161. The method of claim 159, wherein the step of exposing Is performed m the compartments.

162. The method of any of claims 157 through 161 , wherein steps (b) and (d) are performed in no more than about 2 minutes.163 The method of any of claims 157 through 162, further comprising (d) lysing the cells, and (a) fixing the cells after step (c).

164. The method of any of claims 157 through 163 wherein the step of analyzing comprises quantifying the presence of the probes after step (c).

165. The method of claim 159, wherein the antigen is chosen from one or a combination of: solid or liquid nut extract.

166. The method of claim 159 or 165, wherein the antigen is chosen from one or a combination of any antigen on f able 1, Table 5 or Table 6 or a functional variant thereof comprising at least about 75% sequence identity to an amino acid sequence on Table 1, Table 5 or Table 6.

167. The method of any of claims 157 through 166 further comprising drawing a sample of whole blood through the inlet and into the compartments prior to the step (a).

168. The method of any of claims 157 through 167, wherein the probes are chosen from one or a combination of probes on Table 2 and probes specific to the markers on Table 5 or 6.

169. The method of any of claims 157 through 168 further comprising (g) identifying or sorting the cells based upon detection of at least two probes.

170. A method of diagnosing a subject with an allergy-related disorder(a) exposing the plurality of immune cells in a sample from the subject to an antigen within the composition of claims 65 through 71 or the device of claims 72 through 97.

171. The method of claim 170, wherein the step of exposing the one or plurality of immune cells to an antigen is performed within at least one or a plurality of the compartments in the composition or the device.

172. The method of either of claims 170 or 171 further comprising:(a)separating the immune cells into the compartments;(b) exposing the cells to at least two probes within the compartments for a time period sufficient for the at least two probes to associate with the immune cells; and(c)pooling the immune cells in a storage vessel; and(d) quantifying the amount of probes associated to the cells; and(e) diagnosing the subject with an allergy-related disorder if the amount of probes associated to the cells is greater than the amount of probes associated to a control sample.

173. The method of claim 172, wherein step (e) comprising identifying and / or sorting the cells based upon detection of at least two probes.

174. The method of any of claims 170 through 173 further comprising the step of:(a) incubating the cells in a heated condition from about 5 to about 30 minutes before or during the step (c).

175. The method of any of claims 170 through 174, wherein step (a) is performed at or about 37 degrees Celsius.

176. The method of any of claims 170 through 173, wherein steps (b) and (d) are performed in no more than about 2 minutes.

177. The method of any of claims 106 or 107 further comprising (h) lysing the cells; and (i) fixing the cells after step (d); and step (e) is performed after pooling the cell in a storage vessel.

178. The method of any of claims 170 through 177, wherein the antigen is chosen from one or a combination of: solid or liquid nut extract.

179. The method of any of claims 170 through 177, wherein the antigen is chosen from one or a combination of any antigen on Table 1 or a functional variant thereof comprising at least about 75% sequence identity to an amino acid sequence on Table 1.

180. The method of any of claims 170 through 179 further comprising drawing a sample of whole blood from the subject in an inlet vessel through the fluid circuit and into the compartments prior to the step of exposing.

181. The method of any of claims 171 through 180, wherein the at least about two probes are specific for a first immune cell surface protein and a second immune cell surface protein.

182. The method of claim 181 , wherein the first immune cell surface protein is CD 193 or a functional variant thereof and the second immune cell surface protein is CD123 or a functional variant thereof.

183. The method of claim 64, wherein the probes specific to CD193 are chosen from: BV421 , BV605 or derivatives thereof; and wherein the probes specific to CD 123 are chosen from: PE, PE / Cy7, APC, BV785 or a derivative thereof.

184. The method of any of claims 170 through 183, wherein the immune cell is a B cell, T cell, natural killer cell, macrophage, monocyte, neutrophil, eosinophil, basophil, and / or platelet.

185. The method of any of claims 170 through 184, wherein the immune cell is a basophil and the immune-related disorder is an allergy to the antigen.

186. A computer program product comprising instructions for:(a) receiving detection data from the device corresponding to the presence, absence or quantity of probes in each compartment;(b) quantifying the amount of probe in each compartment by normalizing the amount of probe in the compartment with the amount of probe in a control compartment; and(c) correlating the amount of probe in each compartment with the number of activated immune cells from a sample in each compartment.

187. The computer program product of claim 186 further comprising step (d) determining whether a subject from which the sample is taken has an immune-related disorder if the number of activated immune cells in the sample is elevated as compared to the number of activated immune cells in a subject known not to have the immune disorder; wherein the subject has an increased probability of having the immune- related disorder if the number of activated immune cells are elevated in a compartment relative to a control.

188. A kit comprising:(a) the device of any of claims 72 through 97;(b) a first container comprising at least one immune cell activator or antigen from Table 1, Table 5, or Table 6 or an amino acid comprising at least about 75% sequence identity to an amino acid sequence from Table 1, Table 5 or Table 6.

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