Automated Sample Preparation Platform for Cellular Analysis

Abstract

The present disclosure relates, inter alia, to an automated flow cytometry method and system for analyzing and enumerating at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells. The newly developed AQUIOS STEM System and method described herein are the first in vitro diagnostic (IVD) solution for CD34+ enumeration that addresses these important aspects. Based on the automated AQUIOS Flow Cytometry System and method described herein, we provide a complete solution for automated CD34+ enumeration and on-demand CD3+ enumeration on samples, resulting in a workflow that minimizes the need for human intervention.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 050,637, filed July 10, 2020, which is incorporated by reference as if fully set forth herein. [Background technology]

[0002] background Cell analysis instruments using flow cytometers are known in the art. See, for example, U.S. Patent Application Publication No. 2008 / 0010019, which is incorporated by reference as if fully set forth herein. A flow cytometer directs a stream of particles through a sensing zone where the particles can be excited by a light beam. The light beam causes the particles to fluoresce and / or scatter light, and the emitted light is separated by filters into portions of the electromagnetic (EM) spectrum. By examining the filtered EM spectrum, an analysis of the cellular contents can be performed and certain properties and values ​​can be reported. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2008 / 0010019 Summary of the Invention

[0004] Abstract However, to date, no flow cytometers exist that enable (i) automated preparation of blood samples (e.g., umbilical cord blood samples), including hematopoietic stem cell, progenitor cell, or T cell samples, with optimal incubation and volume details preprogrammed; and (ii) analysis and enumeration of at least one of hematopoietic stem cells, hematopoietic progenitor cells, T cells, and even white blood cells in the same instrument in which these samples are prepared. Furthermore, no flow cytometers exist that, in addition to (i) and (ii), allow analysis to be performed on a single sample with or without a negative control, or on replicate samples (e.g., duplicate samples) with or without a negative control. The present disclosure describes such instruments. The present disclosure also provides high-performance instruments whose performance is improved, at least in part, by their ability to accurately and precisely transfer various liquids (e.g., specimen types) with suspended solids (e.g., cells) for accurate particle counting. CD34+ stem and progenitor cell enumeration is one of the most highly regulated tests in clinical flow cytometry laboratories. There are four main factors that contribute to the criticality of this test.

[0005] (1) Before transplantation, donors typically undergo chemotherapy and / or radiation therapy to eliminate the patient's hematopoietic system. Consequently, patient recovery depends on transplanting a sufficiently large number of stem cells to reconstitute hematopoiesis. Therefore, accurate counting of CD34+ cells is essential.

[0006] (2) Regulatory frameworks for CD34+ enumeration vary by region, and currently available quantification methods approved for IVDs do not provide the flexibility needed to accommodate these differences, forcing laboratories to self-validate laboratory-developed tests on scarce and precious specimen types to meet the requirements of local governing bodies.

[0007] (3) Allogeneic (non-autologous) transplants contain residual immunocompetent CD3+ T cells that have the potential to cause graft-versus-host disease (GvHD), a potentially life-threatening complication in stem cell transplantation regimens. Therefore, it is essential to count the number of CD3+ T cells in these samples. However, currently, there are no IVD kits on the market that allow for the parallel counting of CD34+ stem cells and CD3+ T cells in samples, and similarly, laboratories are forced to work with user-defined tests.

[0008] (4) Currently available CD34+ enumeration procedures are highly manual, creating room for human error and resulting in the need for sample reruns, delays in reporting results, or patient / donor discomfort if additional samples need to be collected. Furthermore, these samples often arrive at laboratories as urgent (STAT) samples, disrupting laboratory workflow and forcing laboratory staff to deprioritize the analysis of other samples. Both factors have created a demand for an automated solution for CD34+ enumeration.

[0009] The newly developed AQUIOS STEM System and the method described herein are the first in vitro diagnostic (IVD) solution for CD34+ enumeration that addresses these critical aspects. Based on the automated AQUIOS Flow Cytometry System and method described herein, we provide a complete solution for automated CD34+ enumeration and on-demand CD3+ enumeration on samples, resulting in a workflow that minimizes the need for human intervention. Samples are loaded into the system by an operator, and sample preparation and data analysis are performed automatically by the analyzer. This reduction in hands-on time provides a smooth workflow in the laboratory and minimizes the number of manual, and therefore potentially error-prone, steps. This also means that CD34+ enumeration can be performed by non-flow cytometry specialists, enabling laboratories to offer CD34+ enumeration outside of regulated laboratory hours, such as during night shifts or weekends. Both aspects enhance the level of patient care and reduce the time to results for this time-critical application.

[0010] Additionally, the AQUIOS STEM System and methods described herein provide analytical options that comply with distinct regulatory requirements, such as the International Society for Hematotherapy and Graft Engineering (ISHAGE) Guidelines and the European Pharmacopoeia for CD34+ enumeration, which have different requirements for duplicate runs and the use of negative controls. This flexibility, in that the AQUIOS STEM System offers a total of six analytical options to choose from as part of an IVD solution, also allows for parallel enumeration of CD3+ T cells along with CD34+ stem and progenitor cell populations. This is unique in the market and eliminates the need for time-consuming self-validation of laboratory-developed tests.

[0011] The level of automation offered by the AQUIOS STEM System is achieved by using a combination of premixed, ready-to-use reagents compatible with automation, while ensuring the highest level of data traceability. Currently available manual test kits require daily manual preparation from concentrates and use a solution for red blood cell lysis (an essential step in sample preparation) that requires samples to be stored on ice prior to actual analysis due to its adverse effect on cell viability. In contrast, the AQUIOS STEM System uses a gentler red blood cell lysis reagent that is ready to use and can be used at room temperature, enabling automation of CD34+ and CD3+ enumeration in a manner known for other automated flow cytometry applications but not for stem cell enumeration. All test-critical reagent vials are barcoded with individual identifiers and essential quality control parameters, such as reagent type, reagent lot, date of first use, and expiration date, and are stored in a single database along with sample information, providing the level of data traceability required by today's accreditation bodies.

[0012] The AQUIOS STEM System has been clinically validated against its underlying method, the Stem-Kit for the FC500 Flow Cytometer, which has been recognized as the "gold standard" in the market for clinical CD34+ enumeration and used as the benchmark against which other manufacturers' manual CD34+ enumeration solutions are measured. The AQUIOS STEM System takes the "gold standard" to the next level by offering a unique new solution for CD34+ stem and progenitor cell enumeration that takes the burden of stem cell enumeration out of the clinical laboratory. [Brief explanation of the drawings]

[0013] These and other features, aspects and advantages of the present technology will be better understood in connection with the following drawings.

[0014] [Figure 1-1]Figure 1A is a checklist of features of a solution for the identification and quantification of CD34+ hematopoietic progenitor cells that addresses the requirements of a clinical laboratory.

[0015] [Figure 1-2] FIG. 1B is a flow diagram of a method described herein for analyzing and enumerating at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells.

[0016] [Figure 2] FIG. 2 is a perspective view of an example of a diagnostic instrument, the instrument shown coupled to a specimen autoloader, and including a flow cytometer.

[0017] [Figure 3] FIG. 3 is an enlarged perspective view of a portion of the diagnostic instrument shown in FIG.

[0018] [Figure 4] FIG. 4 is a front perspective view of the diagnostic instrument of FIGS. 2-3, showing the instrument in operation.

[0019] [Figure 5] FIG. 5 is an enlarged view of a portion of a diagnostic instrument that can sample a single specimen tube at a time.

[0020] [Figure 6] FIG. 6 is a front perspective view of the outer housing of the proposed diagnostic instrument shown in FIGS.

[0021] [Figure 7] FIG. 7 is a front perspective view of another example outer housing with the sample autoloader removed and sample tubes inserted through the front door.

[0022] [Figure 8] FIG. 8 is an example of a software component system that can be used in the methods and systems described herein.

[0023] [Figure 9] FIG. 9 is a table showing panel options for clinical quantification of CD34+ HPCs with or without analysis of residual T cells.

[0024] [Figure 10] Figures 10A-10C are plots showing the equivalence of three CD34+ assay options for absolute CD34+ counts (cells / µL): STEM Panel (duplicate, tested with negative control), STEM Duplicate (duplicate, tested without negative control), and STEM Single (single assay). These three assay options are relative to the CD34+ assay.

[0025] [Figure 11] 11A-11C are plots showing the equivalence of three CD34+ plus CD3+ assay options for CD34+ absolute counts (cells / μL): STEM ALLO Panel (duplicate, tested with negative control), STEM ALLO Duplicate (duplicate, tested without negative control), and STEM ALLO Single (single assay). These three assay options are for tests that include CD3.

[0026] [Figure 12] 12A-12C are plots showing the equivalence of three CD34+ plus CD3+ assay options for CD3+ absolute counts (cells / μL): STEM ALLO Panel (duplicate, tested with negative control), STEM ALLO Duplicate (duplicate, tested without negative control), and STEM ALLO Single (single assay). These three assay options are for tests that include CD3.

[0027] [Figure 13] FIG. 13 is a representative manual flow cytometry workflow described in Example 2.

[0028] [Figure 14] FIG. 14 is a representative AQUIOS CL Flow Cytometry System workflow as described in Example 2.

[0029] [Figure 15] Figures 15A-15B are plots of sample processing time and operator hands-on time for a single CD34+ sample or a batch of 10 CD34+ samples on the Stem-Kit and FC500 (alternative method, open bars) and AQUIOS STEM System (AQUIOS, hatched bars).

[0030] [Figure 16] Figures 16A-16B are plots of process times and operator hands-on time for quality control (QC) procedures on the Stem-Kit and FC500 (alternative method, gray bars) and AQUIOS STEM System (AQUIOS, red bars) per work day (Figure 16A) and 5-day work week (Figure 16B). DETAILED DESCRIPTION OF THE INVENTION

[0031] explanation Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0032] With the increasing use of mobilized peripheral stem and progenitor cells (PBSCs) for transplantation, researchers, in collaboration with the International Society for Hematotherapy and Graft Engineering (ISHAGE), described a set of criteria for CD34+ enumeration in 1996, intending to provide a simple, sensitive method allowing for a high degree of accuracy and interlaboratory reproducibility. The resulting "ISHAGE Guidelines" soon became the gold standard for enumeration of hematopoietic CD34+ progenitor cells by flow cytometry. In 1998, one research group published a modified version of the 1996 guidelines by introducing beads for absolute enumeration, adding 7-aminoactinomycin D (7-AAD) as a viability dye to exclude dead cells, and adding a lysis reagent lacking fixatives such as formaldehyde. These modifications transformed the basic protocol into a single-platform method, and the resulting "ISHAGE Guidelines with Viability Dye, Single-Platform" have remained largely unchanged for over 20 years.

[0033] A notable feature of the ISHAGE Guidelines is the sequential gating strategy to derive the number of CD34+ cells from viable leukocytes. The Guidelines require that the tests be performed in duplicate with negative controls to account for test variability and nonspecific cell and fluorescent dye binding.

[0034] Due to the selectivity of sequential gating, the ISHAGE Guidelines made the use of negative controls optional. In contrast, the European Pharmacopoeia (Ph.Eur.) requires the use of controls. In contrast to the ISHAGE Guidelines, the standards set out in the Ph.Eur. are legally binding, as defined by the Council of Europe Convention on the Details of the European Pharmacopoeia and EU and national pharmaceutical legislation.

[0035] Currently available software solutions that are part of in vitro diagnostic (IVD) systems do not offer the flexibility to run panels for CD34+ enumeration either with or without a negative control while maintaining IVD status. Furthermore, not all CD34+ enumeration reagent kits contain a negative control reagent.

[0036] While the sequential ISHAGE gating strategy significantly facilitates accurate quantification of rare CD34+ cell populations, its endpoint is that all counted CD34+ cells are automatically viable, leading to the fact that it can be difficult to directly calculate the percentage of viable CD34+ cells among all CD34+ cells. Furthermore, it can be difficult to read out the total number of CD45+ leukocytes from the applied gating strategy to assess overall specimen viability.

[0037] For cord blood-derived CD34+ cells, the recently published 7th edition of the “NetCord-FACT International Standards for Cord Blood Collection, Banking, and Release for Administration” requires that both total CD34+ and total viable CD34+ counts be determined for cord blood samples after processing and before cryopreservation, and that CD34 viability be assessed before release of cord blood units into clinical programs.

[0038] When CD34+ quantification is performed for quality control purposes, laboratories do not run a "full" ISHAGE panel consisting of duplicate tests plus a negative control, but rather require the ability to run a single test, especially when very small sample volumes are available for analysis. Current acquisition software does not offer the flexibility to adapt the panel as part of an IVD solution accordingly. While an IVD system is not necessarily required for QC purposes, most laboratories that perform these tests are highly regulated and therefore refrain from validating a separate user-defined test solely for this purpose.

[0039] In the case of hematological diseases or non-malignant dysfunction of the hematopoietic system, CD34+ cells are collected from the peripheral blood (PB), bone marrow (BM), or umbilical cord blood (CB) of non-autologous (allogeneic) donors. Today, mobilized PB is the most commonly used source for CD34+ stem and progenitor cells in allogeneic transplantation schemes, as well as in the autologous situation where donor and recipient are the same person.

[0040] The success of allogeneic hematopoietic progenitor cell (HPC) transplantation depends on various factors, including the availability of a suitable donor, human leukocyte antigen (HLA) compatibility, and the ability to balance the patient's immune response while maintaining the graft-versus-leukemia / tumor effect of the transplant. The use of CD34+ cells from mobilized peripheral blood has the advantage of a relatively rapid recovery of hematopoiesis after transplantation, but carries an increased risk of acute graft-versus-host disease (aGvHD) due to the higher number of circulating T cells. Acute and chronic GvHD develop in approximately 30–40% of patients undergoing allogeneic transplants. Because donor T cells are recognized to play a central role in mediating aGvHD, counting CD3+ T cells in the graft along with CD34+ cells has become standard practice in many laboratories. Because no commercially available kits currently exist that allow IVD analysis of CD34 and CD3 in a single test, laboratories must rely on user-defined test validation for this purpose.

[0041] Laboratories performing CD34+ HPC enumeration are highly regulated in terms of data traceability and must establish extensive QC systems, especially if they are accredited. Some important aspects of these controls include: (a) proper identification of all samples to avoid sample mix-ups throughout the process; (b) adequate provisions for monitoring the reliability, accuracy, precision, and performance of testing procedures and equipment; (c) functional checks of equipment and reagents; (d) use of appropriate reference materials and documentation of ongoing proficiency testing; (e) procedures to prevent the use of expired reagents and supplies; and (f) mechanisms that allow for linking lot numbers, expiration dates, and supply and reagent manufacturers to each specimen.

[0042] In particular, aspects (e) and (f) can be challenging because reagent and specimen documentation is often unrelated and occurs "offline," i.e., not present on the platform used for data acquisition and analysis.

[0043] In hematology and oncology laboratories, HPC samples often arrive at the laboratory as urgent (STAT) samples, requiring immediate attention and disrupting routine workflow. Any issues with these samples result in duplicated effort, increasing the potential for human error. For these laboratories, analysis of CD34+ HPCs is time-critical, so ideally, HPC samples should be integrated into the regular workflow to minimize the risk of sampling errors or other issues. For laboratories specializing in CD34+ enumeration, such as cord blood centers, greater automation can help manage the larger number of samples to be analyzed while providing a high level of traceability, as outlined herein.

[0044] Current IVD solutions for CD34+ enumeration by flow cytometry cannot be automated, primarily due to the use of red blood cell lysis reagents that adversely affect cell viability and therefore require prepared samples to be kept on ice before analysis. The ISHAGE Guidelines suggested the use of ammonium chloride because it was the only lysis reagent available at the time that was suitable for a lysis / no-wash approach without requiring additional fixatives and without altering the scatter characteristics of the cell population of interest. While the use of ammonium chloride is well established in essentially all commercially available CD34+ enumeration kits for flow cytometry, its effect on cell viability and the need to prepare working dilutions fresh daily preclude the performance of CD34+ testing on automated sample preparation and flow cytometry platforms.

[0045] Fixative-free, ready-to-use red blood cell lysis reagents are now available that specifically lyse red blood cells without significantly affecting white blood cell viability during sample preparation, allowing CD34+ enumeration to be performed on automated flow cytometry systems.

[0046] An ideal CD34+ quantification kit for flow cytometry would combine the benefits of established standards and protocols with sufficient flexibility to adapt the reagents and software tools to the needs of the clinical laboratory. This would include acquisition and analysis panels for different sample types, quality control mechanisms that meet the requirements of highly regulated work environments, and a high degree of automation, eliminating the need to prepare user-defined tests in parallel with the IVD solution. (See Figure 1A.)

[0047] The methods described herein for analyzing and enumerating at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells are designed to take the so-called gold standard to the "next level." The methods described herein embody, among other things, a modular approach for automated analysis of CD34+ hematopoietic stem and progenitor cells. In one example, the methods described herein include software and a reagent kit for CD34+ enumeration; software and a reagent kit for simultaneous enumeration of CD3+ T cells and CD34+ cells in sample material from an allogeneic donor; and CD34 control cells (two levels), as described in Tables 1 and 2 herein. [Table 1] [Table 2] In the table, "CD45-FITC" generally refers to a fluorescein-conjugated antibody manufactured by Immunotech SAS (Beckman Coulter Company), Marseille, France, which allows for the analysis and enumeration of cell populations expressing the CD45 antigen present in a human biological sample using flow cytometry; "CD34-PE" generally refers to a phycoerythrin-conjugated antibody manufactured by Immunotech SAS (Beckman Coulter Company), Marseille, France, which allows for the analysis and enumeration of cell populations expressing the CD34 antigen present in a human biological sample using flow cytometry; and "CD3-PC7" generally refers to a phycoerythrin cyanine 7-conjugated antibody, which allows for the analysis and enumeration of cell populations expressing the CD3 antigen present in a human biological sample using flow cytometry. Although fluorophores such as FITC, PE, and PC7 are specified herein, any suitable fluorophore-conjugated antibody that allows for the analysis and enumeration of cell populations expressing CD45, CD34, and CD3 antigens can be used, as long as the fluorophore can be detected within the detection capabilities of the instrument.

[0048] The methods described herein can use CD34 control cells, a liquid preparation of stabilized human leukocytes, to validate the parameters CD34, CD45, and CD3 as part of the systems described herein. In one example, the kit contains two levels of CD34: approximately 10 CD34+ cells / µL (Level 1) and approximately 30 CD34+ cells / µL (Level 2). Assay values ​​can be entered into the system by scanning the barcode on the control cell assay sheet.

[0049] As used herein, the term "hematopoietic stem cell" or "HSC" generally refers to cells that have both pluripotency, which allows them to differentiate into functional mature cells such as granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages), and the ability to regenerate (self-renewal) while maintaining their pluripotency.

[0050] As used herein, the term "hematopoietic progenitor cell" or "HPC" generally refers to a cell that has the potential to differentiate into functional mature cells such as granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages).

[0051] HSCs and / or HPCs can be obtained from the body or organs containing cells of hematopoietic origin, as needed. Such sources include unfractionated bone marrow, umbilical cord blood, and peripheral blood. All of the above unpurified or unfractionated blood products can be enriched for cells with hematopoietic stem cell characteristics using methods known to those skilled in the art.

[0052] During hematopoiesis, HSCs first branch into the myeloid and lymphoid lineages at the progenitor stage, then differentiate into myeloid stem cells (mixed colony-forming units, CFU-GEMM) and lymphoid stem cells, respectively. Furthermore, myeloid stem cells differentiate into erythrocytes via burst-erythroid (BFU-E) and erythroid colony-forming cells (CFU-E), platelets via megakaryocyte colony-forming cells (CFU-MEG), monocytes, neutrophils, and basophils via granulocyte-macrophage colony-forming cells (CFU-GM), and eosinophils via eosinophil colony-forming cells (CFU-Eo). Lymphoid stem cells differentiate into T cells via T lymphocyte progenitors and B cells via B lymphocyte progenitors. These myeloid stem cells and the various hematopoietic progenitors derived from them are identified by the characteristics of the colonies they form in soft agar, semi-solid methylcellulose medium, or other media in the presence of various cytokines.

[0053] In the example shown in Table 2, for the analysis of residual CD3+ T cells in samples of allogeneic origin, the AlloCD3 Kit can be combined, if desired, with the Base Kit described in Table 1. The combination of both kits provides a validated solution for integrated quantification of CD34 and CD3 in a single run, with the ability to choose between the three panel options described herein.

[0054] With this framework in mind, the present disclosure provides an automated flow cytometry method, such as that shown in FIG. 1B, for the analysis and enumeration of at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, comprising: placing precise volumes of one or more blood samples (e.g., blood aliquots from the same patient blood sample) into one or more receptacles of a sample plate located in a sample preparation area of ​​the flow cytometer; treating the one or more receptacles with at least one reagent to obtain at least one first composition, wherein at least one of the at least one reagent is at least one imaging reagent comprising a recognition element specific for a marker on at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells; incubating the at least one first composition for a period of time sufficient for binding of at least one imaging reagent comprising a recognition element specific for a marker on at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells to provide at least one second composition; treating the at least one second composition with a lytic reagent to provide at least one third composition; analyzing the at least one third composition by flow cytometry to obtain at least one of viable and total hematopoietic stem cell counts, viable and total progenitor cell counts, and viable and total T cell counts in the one or more blood samples; The present invention relates to an automated flow cytometry method, comprising: performing precise addition of counting beads; and then analyzing at least one third composition by flow cytometry to obtain at least one of viable and total hematopoietic stem cell counts, viable and total progenitor cell counts, and viable and total T cell counts in one or more blood samples. The first composition may further comprise a viability dye as an example of the at least one reagent. As discussed herein, one example of a viability dye is 7-aminoactinomycin D (7-AAD).

[0055] As discussed herein, the methods described herein can be performed using duplicate blood aliquots from various sources described herein. Alternatively or additionally, the methods described herein can be performed with (or without) a negative control. See, for example, the test panels in Tables 1 and 2. The negative control can be for CD34. Alternatively, the negative control can be for CD3. Also, as described herein, the marker is at least one of CD45 (e.g., the CD45 antigen expressed on certain cell populations, including hematopoietic stem cells), CD3 (e.g., the CD3 antigen expressed on certain cell populations, including T cells), and CD34 (e.g., the CD34 antigen expressed on certain cell populations, including hematopoietic stem cells). The negative control can be an isotype control or an isoclonal control. In an isoclonal control, cells are stained in the presence of an excess of the same unlabeled antibody. The unlabeled antibody occupies all binding sites, preventing the labeled antibody from specifically binding. Therefore, all detected signal should be due to nonspecific binding.

[0056] The at least one imaging reagent can be any suitable imaging agent, including imaging agents that include a fluorescent reporter. Thus, for example, imaging agents contemplated herein can be antibodies conjugated to fluorescent reporters, including, but not limited to, FITC, PE, PC7, etc.

[0057] The methods described herein can provide at least one of viable and total hematopoietic stem cell counts, viable and total progenitor cell counts, and viable and total T cell counts.

[0058] The methods described herein also include an automated flow cytometry method for the analysis and enumeration of at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, comprising: placing a first blood sample in a first receptacle of a sample plate; treating the first receptacle with at least one reagent to obtain a first composition f1, and incubating the first composition f1 to obtain a second composition s1, wherein at least one of the at least one reagent is at least one imaging reagent comprising a recognition element specific for a marker on at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells; While the first composition f1 is being incubated, placing a second blood sample in a second receptacle of the sample plate, treating the second receptacle with at least one reagent to obtain a first composition f2, at least one of the at least one reagent being at least one imaging reagent comprising a recognition element specific for a marker on at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, and incubating the first composition f2 to obtain a second composition s2; treating the second compositions s1 and s2 with a lytic reagent to provide third compositions t1 and t2; analyzing the third compositions t1 and t2 by flow cytometry to obtain at least one of viable and total hematopoietic stem cell counts, viable and total progenitor cell counts, and viable and total T cell counts; The present invention also includes an automated flow cytometry method, comprising:

[0059] In some examples, as described herein, the methods can further include preparing a negative control. The negative control can be prepared before the first composition f1, the negative control can be prepared after the first composition f1, or the negative control can be prepared after the first composition f2.

[0060] The systems and methods described herein have various advantages. For example, they streamline operations by incorporating automated loading, sample preparation, reagent management, and barcode scanning and patient tracking, as well as data analysis and bidirectional LIS connectivity, into a single platform. Furthermore, the systems and methods described herein allow for the addition of CD34+ enumeration to existing software, thereby automating most steps of the methods described herein. This is achieved through the use of a red blood cell lysis reagent that meets the criteria of the ISHAGE Guidelines (e.g., no washes, no addition of fixatives, no changes in the scattering properties of the target population), yet can be stored and handled at room temperature, is ready to use without the need for daily preparation of working dilutions from a stock solution, and is gentle on the population of interest. Examples of suitable lysis reagents include, but are not limited to, the VersaLyse Lysing Solution manufactured by Immunotech SAS (a Beckman Coulter Company), Marseille, France. See, for example, U.S. Patent No. 7,300,797, which broadly describes lysis reagents and is incorporated by reference as if fully set forth herein.

[0061] The bead solution used for absolute counting (see Tables 1 and 2) has a carefully balanced buoyancy that does not require individual mixing before each pipetting step, and the bottle containing the beads has a special lid that avoids evaporation.

[0062] The samples are then loaded using either a cassette specimen autoloader or a single-tube loader, with STAT samples taking priority over other samples in the queue. This ensures a smooth workflow without the need to interrupt ongoing processes. Sample preparation can be performed in 96-well deep-well plates or any suitable sample plate, where the samples are not only prepared but also processed in a processing step performed by an automated pipette configured to deliver a predetermined volume of at least one reagent. Sample preparation can be performed using separate probes for sample preparation and analysis, so that both steps occur in parallel and the samples are analyzed immediately upon sample preparation completion.

[0063] Reagents used in the systems and methods described herein include unique barcode identification for tracking expiration dates, on-board expiration dates, lot and container numbers. Reagent consumption and plate usage are monitored by the systems described herein as samples are processed. A barcode reader scans the reagent or plate upon first use to ensure that "consumable" information is error-free or at least substantially error-free. The systems described herein assume that a reagent container or plate is at full capacity the first time it is "seen" by the system. The systems described herein can be equipped with a reagent tracking system, such as the AQUIOS Smart Track reagent tracking system present in the AQUIOS CL flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN), to provide real-time consumable tracking to ensure that properly dated reagents are used and that sufficient reagent is present for each sample, eliminating the risk of having to rerun samples due to insufficient or missing reagents. For example, the systems described herein will not run a test unless they find the required antibody vial.

[0064] The systems described herein have additional advantages. For example, the systems described herein can include a tube barcode reader that can automatically read specimen barcodes without the use of a handheld barcode scanner. The system aligns the barcodes on the tubes with LIS requirements. To prevent misidentification, specimen IDs are recorded before specimen aspiration, and specimen IDs are automatically tracked throughout the run.

[0065] Examples of systems capable of implementing the methods described herein include the system shown in FIGS. 2-7 in the form of a diagnostic instrument 10. In the illustrated example, an autoloader section 12 can be seen with multiple specimen cassettes 14 loaded thereon. In such an example, the cassettes 14 can be loaded with multiple identical specimen tubes or vials 16 (hereinafter referred to as "tubes"), a variety of specimen tubes 16, or only a single specimen tube 16. The cassettes are then loaded into the top of the autoloader section 12 and processed in the order received. Alternatively, if, for example, more rapid single-sample processing is desired, as shown in FIG. 4, a sample tube can be inserted directly into a separate specimen entry, e.g., door 18 (visible in FIG. 5), and processed ahead of all waiting cassettes 14. This, along with the ability to immediately run the test, allows immediate access to the test by the clinician, thereby interrupting (but not adversely affecting) the testing of other specimen tubes when desired by the clinician. Additionally, specimen tubes with damaged or no barcodes (discussed below) can be manually inserted.

[0066] As described in detail herein, the diagnostic instrument 10 illustratively performs the following steps upon receipt of a specimen tube 16 (or specimen tube cassette 14). It is contemplated that such steps are performed by the instrument 10 without intervention by a clinician, and that these steps may be modified, added, or eliminated depending on the particular test to be performed. While blood tubes are discussed throughout this disclosure, it should be understood that other types of bodily fluids and samples are within the scope of this disclosure and may be analyzed in the proposed instrument 10. For example, bone marrow, serum, urine, synovial, spinal, peritoneal, pleural, and other types of fluids or samples may be tested and analyzed substantially as described below.

[0067] Steps that may be performed by instrument 10 include mixing (e.g., agitating) the sample while in the specimen tube 16 (e.g., while in an autoloader); piercing the lid of the specimen tube 16 and sampling the required amount of specimen; reading the barcode (or any other form of marking / identification) to verify sample / patient ID and / or to verify tube type / size; matching the ID, test to be performed, and required reagents and assigning a serial number for computerized tracking; placing the specimen / sample into a selected empty tube or well in storage area 20 (e.g., shown as a microtiter plate in Figures 2-4) for further processing; mixing the reagents once added and the number of tests to be performed. reacting the sample with the reagents for a predetermined incubation time (which may vary based on the reagent); dividing the sample into multiple tubes / wells in the storage area 20 (if desired or required by the test); tracking all samples, cassettes, reagents and associated locations via bar codes or other types of tracking devices (e.g., RFID); aspirating the prepared sample / reagent combination from the storage area in a timely manner and analyzing the prepared sample / reagent combination via a flow cytometer (while preparing subsequent samples); and automatically verifying the results or withholding the results for review depending on clinician-driven decision rules.

[0068] Device 10 provides, among other things, automated and integrated specimen sampling, meaning that each of the above steps (if required by a particular test) can be performed within and by device 10 without the use of additional diagnostic equipment. Moreover, if desired by a clinician, such steps can be performed without any interaction from the clinician. However, it should be understood that device 10 can be configured to alert the clinician in the event of a fault or other problem.

[0069] In the illustrated example, instrument 10 uses a single-axis probe carrier 22 that allows probe carrier 22 to perform various functions while moving along single-axis track 24. For example, probe carrier 22 (and, therefore, probe 26) can be positioned to retrieve a sample from tube 16 when probe carrier 22 is in position A, to deposit a sample in storage area 20 at position B, and to sample reagents at position C. When a sample is placed at any point in pivotable tray 36 (e.g., for immediate processing of the sample), instrument 10 can sense the presence of the sample and insert it ahead of any samples awaiting processing in autoloader 12. Probe carrier 22 can then be moved to position D so that probe 26 can retrieve a sample from a tube placed in pivotable tray 36. Reagents can be placed in storage area 20 either before or after (or both before and after) the sample is deposited for reaction with the sample, as required by the particular test to be performed, and can track themselves as discussed herein.

[0070] The steps may be performed in the following order: However, it is contemplated that certain tests may skip one or more steps or modify the steps to achieve the best test results for the desired blood test.

[0071] First, the sample tubes 16 can be loaded into a pre-configured cassette 14 appropriate for the particular sample tubes 16 to be used. For example, the sample tubes 16 can be the commonly found size of 13 mm by 75 mm sample tubes, in which case the five-tube cassette 14 shown in FIGS. 2 and 4 can be used. However, it should be understood that various sizes and types of sample tubes 16 can be used and the cassette 14 can be designed accordingly. The cassette 14 can even be configured to hold a variety of sample tubes 16. As described herein, sample tubes 16 of various sizes can also be individually inserted through a door 18 shown in FIG. 6.

[0072] If the sample tube 16 has a lid 32, the sample tube (held by cassette 14) can be rocked so that the blood inside the tube is agitated and more homogenous (for more accurate sampling). Such rocking can occur at station A, where cassette 14 can be seen in its rocked position in FIG. 4.

[0073] While cassette 14 is rocking, probe carrier 22 can be instructed to move to station C and begin sampling the appropriate portion of reagent 34 for the test to be performed. However, if the test does not require reagent 34 to be placed on storage area 20 prior to the blood sample, probe carrier 22 may perform such a step after sampling blood from tube 16.

[0074] As can be seen at position C, reagents 34 (e.g., those described in Tables 1 and 2) may be held in vials. However, reagents may alternatively or additionally be held in reservoirs located elsewhere, such as on plate base 30 (shown in FIGS. 2 and 3) or in other areas (not visible), which may be plumbed directly to probe 26, for example.

[0075] As described herein, the diagnostic instrument 10 also contemplates that a clinician may insert specimen tubes 16 through an exterior door 18. To accommodate this, the illustrated instrument 10 is provided with a tube receiver 38 that can accommodate various types of specimen tubes 16, including pediatric tubes, as seen in FIGS. 3-5. In the illustrated example, the specimen tubes 16 may be held by a rotatable tray 36, which allows for easy access and retrieval of the specimen tubes 16. Alternatively, as shown in FIG. 4, the specimen tubes 16 may be held by a rotatable cassette 40.

[0076] Between and after sampling of specimens and / or reagents 34, the probe carrier 22 can be moved to a probe washing station 28 so that the probes 26 can be washed. Washing the probes 26 prevents cross-contamination and therefore inaccurate test results.

[0077] After sufficient mixing of the specimen in the tube (e.g., at station A), the specimen is sampled by probe 26 and deposited into a designated well or tube in storage area 20. Depending on the test to be performed, such as analysis and enumeration of at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells using the methods described herein, specimen samples can be placed in two or more wells or tubes, and corresponding amounts of specimen (e.g., blood) can be pre-aspirated. Probe 26 is then washed in washing station 28, as described herein.

[0078] Depending on whether reagents are added to the specimen sample after it is placed in the storage area 20, the probe carrier 22 may be moved to station C for sampling of the appropriate reagent 34. Again, if more than one reagent is required, the probe 26 may be washed in wash station 28 between each reagent 34 sampling and after the final reagent 34 sampling.

[0079] Plate base 30 may be positioned on a rotational axis so that, depending on the point of rotation of plate base 30, each well or tube may be presented to probe 26 for placement of specimen samples and reagents within each well or tube of storage area 20. Such a configuration and rotational movement of plate base 30 is disclosed in U.S. Patent No. 7,832,292, which is incorporated herein by reference.

[0080] Although multi-axis probe carriers can also achieve these goals, single-axis devices offer certain advantages: they require fewer parts and less programming, result in a smaller instrument 10 footprint, are easier to align, are more reliable, are more stable, and ultimately are faster to move between stations.

[0081] After placement in the wells or tubes, the specimen sample is allowed to react with the reagents for a specific amount of time (depending on the reagents and the test to be performed) and then processed through a flow cytometer for analysis. It is contemplated that other testing devices could also be incorporated, such as devices that use electronic volumetrics for cell size sorting and differentiation, or hemoglobin measurements using absorbance.

[0082] Advantageously, storage area 20 serves as a common interface between sample preparation and analysis. Additionally, storage area 20 can include fixed or removable, and / or disposable or reusable components, allowing the clinician to choose to discard the entire interface (e.g., microtiter plate) after use. By serving as a common interface between the preparation arm and the analysis arm, storage area 20 provides a system that is less prone to error and less exposed to external or environmental influences.

[0083] A processor and a software scheduler (not shown) configured to run on the processor are also incorporated into the disclosed system. The software scheduler can be programmed to, for example, recalculate available windows for fixed reaction kinetics (optimizing throughput while maintaining reproducible reaction kinetics) (e.g., antibody incubation, RBC lysis time, reaction quench time, etc.). An example of a software component system that can be used in the methods and systems described herein is shown in FIG. 8.

[0084] Additionally, as discussed herein, numerous items can be barcoded and tracked during operation. Such barcoding and tracking can be registered by a software scheduler. For example, barcodes can be assigned to reagent vials 34, specimen tubes 16 (with different barcodes for different patients and / or sizes), sheath fluid, common interfaces (e.g., storage areas 20), prepared reagents, bead reagents, cassettes 14, etc. By barcoding these various items, various important information can be tracked, such as reagent usage / consumption, the number of tests remaining in each reagent bottle, expiration dates for open containers, expiration dates for closed containers, assay values, etc.

[0085] The software scheduler can be configured to perform the steps described in FIG. 8 and / or the following steps: determine whether it is acceptable to add a new sample at this time, and queue a door or multiloader (random access) if another activity needs to take priority; minimize the impact of sample door 18 unavailability by adjusting non-kinetic reactions, if any, or kinetic reactions with a wider acceptable window; optimize throughput by minimizing collision effects and defining an acceptable window for each kinetic reaction; ensure that analyses take a predetermined amount of time (scheduled stop / constant volume sample); use predetermined times for each cycle (blood acquisition, reagent addition including mixing, analysis) so that all activities can be scheduled appropriately; take into account all scheduled sample time windows when determining whether it is acceptable to add a new sample to the schedule, and schedule such a new sample so that all its activities occur at the predetermined time; and take into account hardware resources and physical hardware conflicts when determining whether the schedule can be achieved.

[0086] Using the instrument 10 in combination with the software scheduler disclosed herein, the time to first result (TFR) can be less than one hour, with subsequent results reported about every 30 minutes or less. Throughput can be greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 samples per day, with much faster and earlier results reported, significantly increasing laboratory capacity.

[0087] In the illustrated example, the analysis of the data to obtain reportable results is automated (e.g., setting gates, regions, and cursors, and warning or notification of questionable results). The warning / notification aspect can be referred to as an automatic verification function in the system.

[0088] Because all sample preparation and analysis are fully integrated into one instrument 10, clinicians do not have to perform slow and tedious "batch processing," where samples are collected and processing begins once a sufficient number have been collected, i.e., stepping through each step of blood processing for an entire group of samples. In contrast, because instrument 10 is configured to automatically prepare patient samples within storage area 20, there are no daughter tubes to label and track, and significantly less blood and reagents are required. Samples can be loaded into the system at any time, and in one example, each is automatically processed and exits the system pipeline in less than an hour, such as less than 30 minutes, less than 20 minutes, or less than 15 minutes. Subsequent samples can exit the system pipeline at intervals of approximately 30 minutes or less, although the exact time varies depending on the tests to be performed and the required sample preparation time.

[0089] A key benefit is cost savings for the laboratory. Not only can more samples be processed in a single day, but using one system also results in lower system costs, lower reagent costs, and less manual labor. Thus, the overall cost of owning and operating the instrument 10 is significantly lower.

[0090] Furthermore, prior art methods and systems, with their multiple modules and computer screens, occupy 10 to 13 feet of valuable bench space. In contrast, the diagnostic instrument 10 is compact, measuring only 31 inches wide, including the autoloader portion 12. In the example shown in FIG. 6, where the autoloader portion is absent, the required footprint is even smaller. A touchscreen computer / screen (not shown) can also be conveniently located on top of the system, maintaining a small footprint and preserving valuable laboratory space.

[0091] It is contemplated that the proposed system can also be used for contract research, drug development, and clinical researchers running one or more fixed immune monitoring panels for research at academic medical centers and reference laboratories. In addition to the described tests for analyzing and enumerating stem cells and T cells, it is further contemplated that existing standardized immune monitoring panels can be used to monitor immune deficiencies (HIV-AIDS), autoimmune diseases, organ transplant responses, infectious diseases, oncology, etc. Both applications can be run in parallel on the present system.

[0092] Generally, two characteristics contribute to performance: resolution (the ability to measure two particles with the same amount of fluorescence and assign them the same value) and sensitivity (the ability to distinguish between dim particles and slightly brighter particles).To measure these characteristics, "microspheres" or "beads" can be used herein.These microspheres can be made, for example, from fluorophore-labeled materials with known fluorescence values.When these microspheres are passed through a flow cytometer, certain tests have been carried out that reflect the resolution and sensitivity values ​​measured by the flow cytometer.

[0093] Other tests can be performed after the bead test to ensure that the reagents used are functioning properly. A user trained in the field of flow cytometry will make a judgment, based primarily on experience or their own (variable) insight, as to whether the flow cytometer is sufficiently "optimized" to perform the required diagnostic tests that day.

[0094] Diagnostic tests performed by flow cytometers may have minimum resolution and sensitivity needs that differ from industry-dominant bead and reagent tests. The bead and reagent test may indicate to the clinician that the flow cytometer is not optimized, when in fact the flow cytometer may be operating satisfactorily to perform the required diagnostic test and simply did not pass the hypothetical bead and reagent test.

[0095] Therefore, it may be desirable to utilize a known patient sample, such as a blood processing control, so that a lack of resolution or sensitivity can be narrowed down to the performance of the reagents and instrument. When a known patient sample, such as a blood control, is used, only the performance of the reagents and instrument can affect the resolution and sensitivity results of the test.

[0096] In some instances, a known patient sample can be used as a control / initial test sample. The known patient sample is characterized by having identifiable populations, e.g., at least two cell types, that are similar or identical to the population to be diagnosed by the device. In the example of a diagnostic device running a standardized immune monitoring panel, such as those shown in Figures 2-7, the known sample has cellular content that includes the cell type (e.g., CD34+ hematopoietic stem cells) to be analyzed by the device 10.

[0097] According to this example, when a known patient sample is passed through the instrument 10 undergoing evaluation, the results should indicate whether the instrument 10 was able to detect multiple cell populations. If the resolution and sensitivity of the instrument are optimized, distinct cell populations should be indicated in the results. Software can be used to calculate off-light scatter, ECV, and / or fluorescence data as described herein.

[0098] The disclosed examples can also be used to measure the resolution and sensitivity of the instrument 10 for a particular parameter in a particular test and derive statistics to quantify it in terms of sufficiency for performing the test. Such statistics can then be used to determine whether the materials used in the test are appropriate. For example, one can define the minimum resolution and sensitivity needs from a cytometer / reagent package and then use the statistics to analyze whether the cytometer / reagent package is functioning properly to perform the test on a given patient. The statistics can also be used to determine whether data from previous patient samples should be accepted as accurate. The final result can also be a numerical measure that qualifies the performance of the test. [Example]

[0099] Example Example 1 Enumeration of CD34+ Hematopoietic Progenitor Cells and Residual CD3+ T Cells by the System Described Herein: Verification of the Comparability of the Test Protocol background With the increasing use of mobilized peripheral stem and progenitor cells (PBSCs) for transplantation purposes, Sutherland et al., in collaboration with the International Society for Hematotherapy and Graft Engineering (ISHAGE), described a set of criteria for CD34+ enumeration in 1996, with the intention of providing a simple, sensitive method that would allow a high degree of accuracy and interlaboratory reproducibility. The resulting "ISHAGE Guidelines" soon became the gold standard for enumeration of hematopoietic CD34+ progenitor cells by flow cytometry.

[0100] A notable feature of the ISHAGE Guidelines is the sequential gating strategy for deriving the number of CD34+ cells from viable leukocytes. The Guidelines require that tests be performed in duplicate with a negative control to account for test variability and nonspecific cell and fluorescent dye binding. Due to the selectivity of sequential gating, the use of a negative control was made optional by the authors of the ISHAGE Guidelines in subsequent years, but the European Pharmacopoeia (Ph.Eur.) now mandates its use. In contrast to the ISHAGE Guidelines, the standards set forth in the Ph.Eur. are legally binding, as stipulated in the Council of Europe Convention on the Details of the European Pharmacopoeia and in EU and national pharmaceutical legislation.

[0101] Currently available software solutions that are part of IVD systems do not offer the flexibility to run panels for CD34+ enumeration either with or without a negative control while maintaining IVD status. Furthermore, not all CD34+ enumeration reagent kits contain a negative control reagent. Therefore, laboratories must validate their own user-defined tests.

[0102] When stem and progenitor cells are transplanted in an allogeneic (non-autologous) setting, it is recommended to quantify not only the number of CD34+ progenitor cells but also the number of immunocompetent CD3+ residual T cells in the graft to predict and manage potential graft-versus-host disease. To date, no commercially available IVD kits are available that allow for the simultaneous analysis of CD34+ progenitor cells and CD3+ residual T cells, so laboratories must again rely on the validation of user-defined tests for this application.

[0103] AQUIOS STEM System The system described herein intends to overcome these limitations by, among other things, offering a total of six different acquisition panels for clinical CD34+ enumeration (Figure 9). All protocols follow the sequential gating strategy of the ISHAGE Guidelines, and the panels give the choice to run either the "full" panel of three tests (duplicate + negative control) recommended by ISHAGE and mandated by Ph.Eur., an optional ISHAGE panel without a negative control, or a single test that can be used for QC purposes for rare specimen types. All panel combinations are part of an IVD solution, eliminating the need for user-defined tests.

[0104] For analysis of residual CD3+ T cells in samples of allogeneic origin, the optional AQUIOS STEM Allo-CD3 Kit can be combined with the base AQUIOS STEM Kit. The combination of both kits provides a validated solution for integrated quantification of CD34 and CD3 in a single run, also with the ability to choose between the three panel options described herein in Figure 9.

[0105] Test Method The AQUIOS CL Flow Cytometer is one system that can be used to implement the methods described herein. It is a quantitative automated analyzer that performs STEM diagnostic applications with a "no-wash" sample preparation process. This system is referred to as a "Load & Go" flow cytometer because it is intended to be an automated analytical device with automated sample processing from specimen introduction to result reporting. The AQUIOS System Software and AQUIOS STEM Tests and Quality Control Reagents do not require user verification of light scatter and fluorescence intensity normalization or verification of color compensation settings.

[0106] An automated run is initiated by creating a request and loading a cassette containing multiple specimen tubes in the Autoloader or a single specimen tube in the Single-Tube Loader. Samples are automatically processed according to these requests. Samples are stained and incubated, and red blood cells are lysed using AQUIOS STEM Lysing solution. White blood cells are analyzed on the AQUIOS CL Flow Cytometry System using AQUIOS STEM Tests. STEM sample preparation is optimized to work with barcoded 96-deep-well plates with deep, conical wells. Each well holds up to 600 μL.

[0107] The AQUIOS STEM System is available with up to two kits: the AQUIOS STEM Kit alone or in combination with the AQUIOS STEM ALLO-CD3 Kit. The AQUIOS STEM-Kit reagents consist of a CD45-FITC / CD34-PE mouse monoclonal antibody reagent, a corresponding negative control (CD45-FITC / CD34-CTRL), an absolute count reagent (AQUIOS STEM-Count Fluorospheres), a cell viability reagent (7-AAD), and a ready-to-use lysis reagent. The AQUIOS STEM Allo-CD3 Kit is an optional reagent kit for simultaneously enumerating CD3+ T cells and CD34+ cells in allogeneic donor-derived sample material. The kit contains CD3-PC7 and the appropriate negative control.

[0108] AQUIOS STEM Tests use AQUIOS STEM Kit reagents containing monoclonal antibodies for the simultaneous identification and enumeration of viable absolute counts / μL of CD34+ HPCs, viable absolute counts / μL of CD45+ HPCs, and percentage of viable CD34+ HPCs. Three AQUIOS STEM tests are available as part of the AQUIOS STEM menu: Stem Panel: Run tests in duplicate + negative control (3 wells); Stem Duplicate: Run tests in duplicate (no negative control; 2 wells); and Stem Single: Single test (1 well) (Figure 9).

[0109] The AQUIOS STEM ALLO Tests use the AQUIOS STEM Kit reagents in combination with the AQUIOS STEM ALLO-CD3 Kit reagents. The two kit combinations contain monoclonal antibodies and allow for simultaneous identification and enumeration of the absolute number of viable CD34+ HPCs / μL, the absolute number of viable CD45+ HPCs / μL, the absolute number of viable CD3+ HPCs / μL, and the percentage of viable CD34+ HPCs. Three AQUIOS STEM ALLO tests are available: STEM ALLO Panel: Tests run in duplicate + negative control (3 wells); STEM ALLO Duplicate: Tests run in duplicate (no negative control; 2 wells); and STEM ALLO Single: Single test (1 well) (Figure 9).

[0110] All tests use 43 μL of sample stained with 13 μL of each reagent (monoclonal antibody and viability dye). After a 15-minute incubation, the sample is lysed using 430 μL of Lysing Reagent, and then AQUIOS STEM-Count is added after approximately 15 minutes of lysis incubation. The sample is then mixed and aspirated for analysis. For the AQUIOS STEM / STEM ALLO Duplicate or AQUIOS STEM / STEM ALLO Panel, two or three wells are used, respectively.

[0111] Research objectives and scope As part of product characterization, this study was conducted to compare the equivalent performance of viable CD34+ absolute counts and CD3+ T cell absolute counts across the assay options summarized in Figure 9.

[0112] Forty fresh apheresis samples were collected and analyzed. To assess the comparability between the test options for CD34+ enumeration, the analytical option "STEM Panel" (CD34+ negative control in duplicate) was used as a reference against two additional analytical options: "STEM Duplicate" (CD34 in duplicate, no negative control) and "STEM Single" (single test).

[0113] The same testing scheme was performed for CD34+ and CD3+ enumeration, with the analytical option being able to include CD3 as an additional marker by using the analytical protocol "STEM ALLO Panel" (CD34 and CD3+ negative control in duplicate) as a reference for two additional analytical options "STEM ALLO Duplicate" (CD34 and CD3 in duplicate, no negative control) and "STEM ALLO Single" (single test for CD34 and CD3).

[0114] The study was performed in accordance with CLSI EP09c: Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guidelines - Third Edition. Samples were analyzed using an AQUIOS CL flow cytometer equipped with AQUIOS STEM Software and AQUIOS STEM System reagents.

[0115] Test Design Daily start-up and shutdown of the AQUIOS CL device used in this study was performed according to the manufacturer's instructions. Maintenance activities were recorded in a maintenance log.

[0116] Daily quality control (QC) and process control runs were performed using Flow-Check Fluorospheres and AQUIOS STEM CD34 Control Cells to confirm instrument calibration and ensure operation within assigned assay values.

[0117] Apheresis samples used in this study were collected using ACD-A as the anticoagulant and stored at 2–8°C until use. Samples were used undiluted if the white blood cell count was less than 30,000 cells / µL; otherwise, they were diluted in PBS / 6% bovine serum albumin (BSA) to a white blood cell concentration not exceeding 30,000 cells / µL. Individual aliquots of each apheresis sample were used for a total of 12 runs following the testing options summarized in Figure 1. Hemolyzed specimens and specimens containing visible clots were excluded.

[0118] Data analysis was performed according to CLSI EP09c by using weighted Deming regression. Upper and lower 95% confidence limits of the bias estimate were calculated based on the standard error of bias and compared with the manufacturer's acceptance limits (specifications).

[0119] result Equivalence of AQUIOS STEM System Testing Options for Absolute CD34+ Count The outcome of hematopoietic progenitor cell transplantation depends on the successful infusion of sufficient numbers of viable CD34+ cells to reconstitute hematopoiesis in the recipient, and therefore, accurate determination of CD34+ cells by flow cytometry is a key element in the transplant procedure.

[0120] In contrast to other solutions for CD34+ enumeration, the AQUIOS STEM System offers a total of six different protocols for CD34+ enumeration, either with or without optional analysis of CD3+ T cells (Figure 9). To demonstrate the comparability between these different testing options, aliquots from the same sample were run with the different options and analyzed for correlation.

[0121] In the first series of experiments, absolute numbers of CD34+ cells / µL were assessed by running apheresis samples using three analytical protocol options (STEM Panel, STEM Duplicate, and STEM Single) that did not include CD3 to demonstrate the equivalence of CD34 counts across the three options (Figures 10A-10C). For comparisons of analytical options in which tests were performed in duplicate (STEM Panel vs. STEM Duplicate, Figure 10A), the average of both runs was used for analysis. When comparing test options involving duplicate runs to single-run test options (STEM Panel vs. STEM Single, Figure 10B, and STEM Duplicate vs. STEM Single, Figure 10C), the results of the first run were used as the reference. For all regression pairs analyzed, equivalent performance of absolute viable CD34+ cell counts across analytical options was demonstrated to be within the established acceptance criteria.

[0122] In a second series of experiments, the number of CD34+ cells was analyzed by using three analytical protocols intended for parallel enumeration of CD34+ and CD3+ cells, while all other criteria were kept identical (Figures 11A-11C). Again, for all regression pairs analyzed, equivalent performance of absolute viable CD34+ cell counts across analytical options was demonstrated to be within the defined acceptance criteria, which were also the criteria for the analytical protocol using CD3+.

[0123] In summary, all six analysis options for CD34+ enumeration, either with or without CD3, gave comparable results for the correlation pairs analyzed.

[0124] Equivalence of AQUIOS STEM System test options for enumeration of residual CD3+ T cells In the case of hematological diseases or non-malignant dysfunction of the hematopoietic system, CD34+ cells are collected from the peripheral blood (PB), bone marrow (BM), or umbilical cord blood (CB) of non-autologous (allogeneic) donors. Today, mobilized PB is the most commonly used source for CD34+ stem and progenitor cells in allogeneic transplantation schemes, as well as in the autologous situation where donor and recipient are the same person.

[0125] The use of CD34+ cells from mobilized peripheral blood has the advantage of a relatively rapid recovery of hematopoiesis after transplantation, but is associated with an increased risk of acute graft-versus-host disease (aGvHD) due to the higher number of circulating T cells. Acute and chronic GvHD develop in approximately 30–40% of patients undergoing allogeneic transplants. Because donor T cells are recognized to play a central role in mediating aGvHD, counting CD3+ T cells in the graft along with CD34+ cells has become standard practice in many laboratories.

[0126] In addition to the standard testing protocol for CD34+ enumeration, the AQUIOS STEM System offers the option to analyze CD3+ T cells along with CD34+ T cells in a single run. To ensure equivalence of testing options for CD3+ enumeration as well as CD34+ analysis, a third series of experiments was performed comparing CD3+ enumeration results for the three analysis options described herein (Figures 12A-12C). With regard to CD34+, equivalent performance of absolute viable CD3+ cell counts across analysis options was demonstrated to be within the defined acceptance criteria for all regression pairs analyzed.

[0127] conclusion The AQUIOS STEM System is a modular approach for the automated analysis of CD34+ hematopoietic stem and progenitor cells on the AQUIOS CL Flow Cytometer, intended to overcome the limitations of currently available solutions regarding panel and assay flexibility.

[0128] This study addressed the question of to what extent the testing options offered by the system yield equivalent data for CD34+ and CD3+ absolute counts, to eliminate potential bias from selecting one option over the other.

[0129] In summary, it was possible to demonstrate data equivalence for all regression pairs analyzed, and equivalent performance of absolute viable CD34+ cell count and absolute viable CD3+ count across analysis options was demonstrated to be within the defined acceptance criteria.

[0130] Example 2: Comparison of workflow between the AQUIOS STEM System and its underlying method, the FC500 Stem-Kit background Today, over 50,000 hematopoietic stem and progenitor cell (HPC) transplants are performed annually worldwide for the treatment of several hematologic malignancies as well as for non-hematologic indications.

[0131] Clinical laboratories rely on commercially available IVD solutions for CD34+ HPC enumeration to avoid time- and resource-consuming user-defined test validation. Most reagent kits and software packages were developed in response to the 1996 and 1998 ISHAGE Guidelines but have not since been updated to meet the evolving demands of diagnostic laboratories, particularly with regard to automation capabilities. In hematology and oncology laboratories, HPC samples often arrive at the laboratory as urgent (STAT) samples, requiring immediate attention and disrupting routine workflow. Any issues associated with these samples result in duplicated efforts, increasing the potential for human error. For these laboratories, time is of the essence when analyzing CD34+ HPC, and ideally, HPC samples should be integrated into the regular workflow to minimize the risk of sampling errors or other issues.

[0132] This example compares the newly developed AQUIOS STEM System workflow for automated CD34+ enumeration to the method on which it is based, the Stem-Kit for the FC500 flow cytometer (both Beckman Coulter, Inc.), in terms of turnaround time and operator interaction time.

[0133] Overview of the traditional flow cytometry workflow Many conventional flow cytometry solutions for CD34+ enumeration, such as the Beckman Coulter Stem-Kit for the FC500, require manual sample preparation, manual creation of worklists, manual data review, and manual compilation of numerical data. This workflow can result in longer run times, longer hands-on time, and requires more experienced operators.

[0134] To perform a CD34+ enumeration test using the underlying method (Stem-Kit on FC500), the operator must manually check the instrument adjustments, prepare controls to verify and adjust (compensate) for fluorochrome spillover, prepare and run process controls, and finally prepare and run the actual patient / donor samples. The resulting data must be verified and manually transferred to the Laboratory Information System (LIS). Reagent and quality control (QC) logs are typically kept manually. See Figure 13 for a typical manual flow cytometry workflow.

[0135] AQUIOS CL Flow Cytometer Workflow Overview The AQUIOS CL is an automated system that performs most of the preparation steps that result in test results, thereby eliminating most manual preparation steps.

[0136] With the AQUIOS STEM System for CD34+ enumeration on AQUIOS, the operator needs to load reagents onto the system once, start the instrument, run a QC sample, and then be ready to load and analyze patient samples throughout the rest of the workday (Figure 14).

[0137] Comparison Protocol The objective of this case study was to evaluate the AQUIOS CL Flow Cytometer design, specifically for CD34+ enumeration, and to model the resulting workflow through comparison with the Beckman Coulter Stem-Kit on the FC500 (the underlying method). This case study estimates the following workflow parameters:

[0138] Turnaround Time - For alternate systems, the time period begins with the first sample preparation step and ends with the completed test result. For the AQUIOS CL system, the time period begins with the sample placed in the autoloader and ends with the completed test result for the last sample displayed. For both systems, the time does not include QC.

[0139] Operator Time - The time required by the user to physically perform the steps listed in the provided test procedure according to the manufacturer's instructions.

[0140] Time points were captured by video recording the sample preparation and analysis process and modeled using in-house workflow analysis software. result

[0141] Sample Processing Time and Operator Hands-On Time This test case consisted of scenarios using either a single sample or a batch of 10 samples of mobilized peripheral blood prepared in duplicate + negative control. The AQUIOS STEM System was compared directly to its underlying method, the Stem-Kit on FC500. Data is shown for a study consisting of duplicate runs + negative control.

[0142] For single-analyte samples, the time to result from sample preparation to result generation did not show significant differences (56:19 min for the underlying method and 53:54 min for AQUIOS; Figure 15A), as the process steps were primarily determined by the incubation times with the antibody and red blood cell lysis reagent.

[0143] However, the actual operator time was reduced by 95%, from 6:23 min for the base method to 0:20 min for AQUIOS (Figure 15A).

[0144] For batches of 10 specimens (average daily workload at a medium-sized stem cell transplant center), these differences become even more pronounced: the time to result, from specimen preparation to result generation, was reduced from 2:40:29 hours for the base method to 2:03:54 hours for the AQUIOS (Figure 15B), and the operator hands-on time was reduced from 1:01:13 hours for the base method to 3:20 minutes for the AQUIOS STEM System (Figure 15B).

[0145] Even for manual processes, certain steps can be performed in parallel (such as pipetting the next sample during the incubation time of the first sample), and the total time for 10 samples is less than the sum of 10 times that for one sample.

[0146] Assuming 10 samples per workday over a yearly period, the AQUIOS STEM System will save laboratories hundreds of valuable technical hours each year. Additionally, the faster time to results when running an average workload of 10 samples per day is a boost for time-critical assays such as CD34+ enumeration.

[0147] Process times and operator hands-on time for quality control (QC) procedures In a regulated environment, system performance must be controlled by appropriate quality control and process controls for both the instrument settings and the tests being performed. For both the Stem-Kit and AQUIOS systems on the FC500, optical alignment of the flow cytometer and daily verification of the fluidics system are performed using Flow-Check Fluorospheres, an assayed suspension of fluorospheres (fluorescent microspheres). While the underlying method uses lyophilized CD34+ cells (Stem-Trol Cells) spiked into normal blood samples as process controls to verify analytical parameters of interest, the AQUIOS STEM System uses a liquid preparation of stabilized human white blood cells (lymphocytes, monocytes, and granulocytes) and red blood cells (AQUIOS STEM CD34 Control Cells) with lysis, light scattering, antigen expression, and antibody staining characteristics representative of those found in human whole blood specimens.

[0148] The total time required for QC procedures associated with CD34+ enumeration was reduced from 2:02:22 hours for the base method to 1:09:44 hours for AQUIOS. Notably, QC had to be completed before patient / donor samples could be run, resulting in a time savings of 52:38 minutes per day (Figure 16A). When working five days per week, this totals approximately 4.5 hours for QC procedures alone (Figure 16B).

[0149] The operator's hands-on time for the QC procedure was reduced from 12:22 minutes (based method) to 1:51 minutes (AQUIOS) per day (Figure 16A), resulting in a total hands-on time savings of 52 minutes per week for the QC process (Figure 16B).

[0150] conclusion In all test case scenarios, the AQUIOS STEM System required significantly less hands-on time than the method on which it was based. In a typical five-day week of operation with 10 specimen samples per day, the AQUIOS STEM System reduced manual labor by approximately six hours, allowing laboratories to allocate resources more efficiently. Additionally, the AQUIOS STEM reduced the overall turnaround time from sample preparation to patient results, which is essential for time-sensitive tests such as CD34+ enumeration.

[0151] The AQUIOS STEM System is a quantitative, automated solution for enumeration of CD34+ hematopoietic stem and progenitor cells with a "no-wash" sample preparation process. The system is called a Load&Go flow cytometer because it is intended to be an automated analyzer with automated sample processing from specimen introduction to result reporting. These automated features distinguish the AQUIOS STEM System from alternative methods (including the method on which it is based, the FC500 with Stem-Kit), which require many process steps to be performed manually. The AQUIOS STEM System's all-in-one approach is intended to help save time and increase workflow efficiency in modern laboratories.

[0152] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​expressly stated as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly stated. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the stated range. The statement "about X to Y" is equivalent to "about X to about Y" unless otherwise specified. Similarly, the statement "about X, Y, or about Z" is equivalent to "about X, about Y, or about Z" unless otherwise specified.

[0153] As used herein, the terms "a," "an," or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to indicate a non-exclusive "or" unless otherwise indicated. Furthermore, expressions or terms used herein and not otherwise defined should be understood to be for descriptive purposes only and not for limiting purposes. The use of all section headings is intended to aid in the reading of this specification and should not be construed as limiting. Furthermore, the information associated with a section heading may be located within or outside of that particular section. Furthermore, all publications, patents, and patent documents mentioned herein are incorporated by reference in their entirety, as if individually incorporated by reference. In the event of a conflict in usage between this document and a document so incorporated by reference, the usage in the incorporated reference should be considered supplementary to the usage in this document, and in the event of any irreconcilable discrepancy, the usage in this document will control.

[0154] In the methods described herein, steps may be performed in any order without departing from the principles of the present disclosure, unless a temporal or operational order is explicitly recited. Furthermore, specified steps may be performed simultaneously, unless express claim language recites that the specified steps be performed separately. For example, a claimed step of performing X and a claimed step of performing Y may be performed simultaneously in a single operation, and the resulting method would fall within the literal scope of the claimed method.

[0155] As used herein, the term "about" may allow for some variation in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or the limits of a stated range.

[0156] As used herein, the term "substantially" refers to a majority or majority, such as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0157] As used herein, the term "substantially free" refers to less than about 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.001%, or less than about 0.0005% or less, or about 0% or 0%.

[0158] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Accordingly, the present description is not intended to be, and should not be construed as, limited to the given examples, and the full scope of protection conferred by the appended claims and their equivalents should be accorded. Moreover, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Thus, the foregoing description or exemplary embodiments are provided for the purpose of illustrating the principles of the present disclosure and are not intended to limit the present disclosure, and may include modifications to and variations of the present disclosure. In particular embodiments, for example, the following items are provided: (Item 1) 1. An automated flow cytometry method for analyzing and enumerating at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, comprising: placing one or more blood samples into one or more receptacles of a sample plate located in a sample preparation area of ​​the flow cytometer; treating the one or more receptacles with at least one reagent to obtain at least one first composition, wherein at least one of the at least one reagent is at least one imaging reagent comprising a recognition element specific for a marker on at least one of the hematopoietic stem cells, the hematopoietic progenitor cells, and T cells; incubating said at least one first composition for a period of time sufficient for binding of said at least one imaging reagent comprising a recognition element specific for said marker on said at least one of said hematopoietic stem cells, hematopoietic progenitor cells, and T cells to provide at least one second composition; treating said at least one second composition with a lytic reagent to provide at least one third composition; analyzing the at least one third composition by flow cytometry to obtain at least one of a viable and total hematopoietic stem cell count, a viable and total progenitor cell count, and a viable and total T cell count in the one or more blood samples; 1. An automated flow cytometry method comprising: (Item 2) 2. The method of claim 1, wherein the method is performed using duplicate blood samples. (Item 3) 3. The method of claim 2, wherein the method is performed using a negative control. (Item 4) 4. The method of item 3, wherein the negative control is against CD34. (Item 5) 4. The method of item 3, wherein the negative control is against CD3. (Item 6) The method of any of the preceding items, wherein the marker is at least one of CD45, CD3, and CD34. (Item 7) The method of any of the preceding items, wherein the at least one imaging reagent comprises a fluorescent reporter. (Item 8) 8. The method of any one of items 1 to 7, wherein the at least one imaging reagent comprises a FITC, PE, or PC7 fluorescent reporter. (Item 9) 10. The method of any of the preceding items, wherein the first composition further comprises a viability dye. (Item 10) 10. The method of claim 9, wherein the viability dye is 7-aminoactinomycin D (7-AAD). (Item 11) The method of any of the preceding items, wherein viable and total hematopoietic stem cell counts, viable and total progenitor cell counts, and viable and total T cell counts are obtained. (Item 12) 10. The method of any of the preceding items, wherein the sample plate is a 96-well microtiter plate. (Item 13) 10. The method of claim 1, wherein treating the one or more receptacles with at least one reagent to obtain at least one first composition is performed by an automated dispenser configured to deliver a predetermined volume of the at least one reagent. (Item 14) The method of any of the preceding items, wherein the incubating is automated to incubate the at least one first composition for a predetermined period of time sufficient for binding of the at least one imaging reagent comprising a recognition element specific for the at least one of the hematopoietic stem cells, hematopoietic progenitor cells, and T cells to provide at least one second composition. (Item 15) 1. An automated flow cytometry method for analyzing and enumerating at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, comprising: placing a first blood sample in a first receptacle of a sample plate; First composition f 1 treating the first receptacle with at least one reagent to obtain a second composition s 1 a first composition f 1 wherein at least one of said at least one reagent is at least one imaging reagent comprising a recognition element specific for a marker on at least one of said hematopoietic stem cells, hematopoietic progenitor cells, and T cells; First composition f 1 While the first composition f is being incubated, a second blood sample is placed in a second receptacle of the sample plate and 2 treating the second receptacle with at least one reagent, at least one of the at least one reagent being at least one imaging reagent comprising a recognition element specific for a marker on at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, to obtain a second composition s 2 a first composition f 2 and incubating the Third composition t 1 and t 2 a second composition s 1 and s 2 treating the sample with a lysis reagent; and a third composition t to obtain at least one of viable and total hematopoietic stem cell count, viable and total progenitor cell count, and viable and total T cell count. 1 and t 2 and analyzing by flow cytometry; 1. An automated flow cytometry method comprising: (Item 16) 15. The method of claim 14, further comprising preparing a negative control. (Item 17) The negative control is the first composition f 1 Item 16. The method according to Item 15, wherein the composition is prepared before (Item 18) The negative control is the first composition f 1 Item 16. The method according to item 15, wherein the compound is prepared after (Item 19) The negative control is the first composition f 2 Item 16. The method according to item 15, wherein the compound is prepared after

Claims

1. 1. An automated flow cytometry method for the analysis and enumeration of at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, comprising: placing one or more blood samples into one or more receptacles of a sample plate located in a sample preparation area of ​​the flow cytometer; treating said one or more receptacles with at least one reagent to obtain at least one first composition, at least one of said at least one reagent being at least one imaging reagent comprising a recognition element specific for a marker on at least one of said hematopoietic stem cells, said hematopoietic progenitor cells, and T cells; incubating said at least one first composition for a period of time sufficient for binding of said at least one imaging reagent comprising a recognition element specific for said marker on said at least one of said hematopoietic stem cells, hematopoietic progenitor cells, and T cells to provide at least one second composition; treating the at least one second composition with a lytic reagent to provide at least one third composition; analyzing the at least one third composition by flow cytometry to obtain at least one of a viable and total hematopoietic stem cell count, a viable and total progenitor cell count, and a viable and total T cell count in the one or more blood samples; Including, the marker is at least one of CD45, CD3, and CD34; the first composition further comprises a viability dye; and The method uses a control that is a first liquid preparation of human white blood cells and red blood cells having a first concentration of CD34+ cells and a second liquid preparation of human white blood cells and red blood cells having a second concentration of CD34+ cells; the first concentration and the second concentration are different; both said first and second liquid preparations have lysis, light scattering, antigen expression, and antibody staining properties representative of those found in a human whole blood biopsy; An automated flow cytometry method, wherein said first and second liquid preparations are not diluted with a normal blood sample prior to use.

2. 10. The method of claim 1, wherein the method is performed with duplicate blood samples.

3. The method of claim 2, wherein the method is performed with a negative control.

4. The method of claim 3, wherein the negative control is against CD34.

5. The method of claim 3, wherein the negative control is for CD3.

6. The method of any one of claims 1 to 5, wherein the at least one imaging reagent comprises a fluorescent reporter.

7. The method of any one of claims 1 to 6, wherein the at least one imaging reagent comprises FITC, PE or PC7 fluorescent reporter.

8. The method according to any one of claims 1 to 7, wherein the viability dye is 7-aminoactinomycin D (7-AAD).

9. The method of any one of claims 1 to 8, wherein viable and total hematopoietic stem cell counts, viable and total progenitor cell counts and viable and total T cell counts are obtained.

10. The method of any one of claims 1 to 9, wherein the sample plate is a 96-well microtiter plate.

11. 11. The method of any one of claims 1 to 10, wherein treating the one or more receptacles with at least one reagent to obtain at least one first composition is performed by an automated pipette configured to deliver a predetermined volume for the at least one reagent.

12. 12. The method of any one of claims 1 to 11, wherein said incubating is automated to incubate said at least one first composition for a predetermined period of time sufficient for binding of said at least one imaging reagent comprising a recognition element specific for said at least one of hematopoietic stem cells, hematopoietic progenitor cells and T cells to provide at least one second composition.

13. 1. An automated flow cytometry method for the analysis and enumeration of at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, comprising: placing a first blood sample in a first receptacle of a sample plate; First composition f １ The first receptacle is treated with at least one reagent to obtain a second composition s １ A first composition f １ wherein at least one of said at least one reagent is at least one imaging reagent comprising a recognition element specific for a marker on at least one of said hematopoietic stem cells, hematopoietic progenitor cells, and T cells; First composition f １ While the first composition f is being incubated, a second blood sample is placed in a second receptacle of the sample plate and the first composition f is added to the second receptacle of the sample plate. ２ and treating the second receptacle with at least one reagent, at least one of the at least one reagent being at least one imaging reagent comprising a recognition element specific for a marker on at least one of hematopoietic stem cells, hematopoietic progenitor cells, and T cells, to obtain a second composition s. ２ A first composition f ２ incubating the Third composition t １ and t ２ A second composition s １ and s ２ with a lysis reagent; and a third composition t to obtain at least one of viable and total hematopoietic stem cell count, viable and total progenitor cell count, and viable and total T cell count. １ and t ２ analyzing the sample by flow cytometry; Including, the marker is at least one of CD45, CD3, and CD34; the first composition further comprises a viability dye; and The method uses a control that is a first liquid preparation of human white blood cells and red blood cells having a first concentration of CD34+ cells and a second liquid preparation of human white blood cells and red blood cells having a second concentration of CD34+ cells; the first concentration and the second concentration are different; both said first and second liquid preparations have lysis, light scattering, antigen expression, and antibody staining properties representative of those found in a human whole blood biopsy; An automated flow cytometry method, wherein said first and second liquid preparations are not diluted with a normal blood sample prior to use.

14. The method of claim 13, further comprising preparing a negative control.

15. The negative control is the first composition f １ The method of claim 14, wherein the composition is prepared prior to the step of

16. The negative control is the first composition f １ The method of claim 14, wherein the composition is prepared after

17. The negative control is the first composition f ２ The method of claim 14, wherein the composition is prepared after

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