Compositions and methods for whole blood preservation

Abstract

Systems and methods for the storage of small volumes of whole blood are provided. Whole blood can be stored and preserved for extended periods at room temperature. The stored whole blood can be used in single-cell-based applications. The above method includes the step of contacting a whole blood sample with a solution containing a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group to obtain a whole blood solution, wherein the ratio (v / v) of the solution to the whole blood sample is equal to or greater than 20.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit and priority based on U.S. Provisional Patent Application No. 63 / 489,172, entitled "Compositions and Methods for Whole Blood Preservation," filed on March 8, 2023. The disclosure of U.S. Provisional Patent Application No. 63 / 489,172 is incorporated by reference in its entirety for all purposes.

[0002] (Technical Field) This disclosure is directed to compositions and methods for preserving whole blood; more particularly, to compositions and methods for preserving whole blood for single - cell nucleic acid processing.

Background Art

[0003] (Background) Whole blood contains red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes) suspended in plasma. Blood cells contain various biomolecules (e.g., proteins and nucleic acids). Proteins (e.g., hemoglobin in red blood cells) transport oxygen from the lungs to all parts of the body. Many tests have been designed to determine the number of red and white blood cells in the blood, along with the volume of red blood cells, sedimentation rate, and hemoglobin concentration (hematology). Additionally, certain tests are used to classify blood according to specific red blood cell antigens or blood types. Other tests can elucidate the details of the shape and structure of blood cells as well as hemoglobin and other blood proteins. Blood can also be analyzed to determine the activity of various enzymes or protein catalysts, either bound to blood cells or found free in plasma.

[0004] Blood can also be analyzed based on its characteristics (e.g., total volume, circulation time, viscosity, clotting time and clotting abnormalities, acidity (pH), oxygen and carbon dioxide levels, and clearance rates of various substances). Tests based on the presence of substances characteristic of specific infections in the blood (e.g., serological tests for syphilis, hepatitis, and human immunodeficiency virus (HIV)) also exist.

[0005] Whole blood is typically stored at low temperatures to preserve biomolecules for downstream applications. However, when cells undergo the cooling process for low-temperature storage, their gene expression can change, leading to different expression states after extraction. Furthermore, prolonged exposure to low temperatures can result in cell death. Low-temperature storage can also generate extra costs and complex logistics for the supply chain. Methods for preserving whole blood without using low-temperature storage and transport could extend blood-based analytical technologies to distributed consumers. [Overview of the Initiative] [Means for solving the problem]

[0006] (Summary) Compositions and methods for preserving whole blood, as well as their components, are described.

[0007] Some embodiments of the present invention include a method for preserving whole blood, comprising the steps of: contacting a whole blood sample with a solution containing a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group to obtain a whole blood solution, wherein the ratio (v / v) of the solution to the whole blood sample is equal to or greater than 20; and adding a quencher to the whole blood solution.

[0008] In some embodiments, the whole blood sample has a volume of 20 μL to 1 mL.

[0009] In some embodiments, whole blood samples are collected using a capillary blood collection procedure.

[0010] In some embodiments, the molecular compound containing the aldehyde functional group is formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, or formalin.

[0011] In some embodiments, the molecular compound containing the aldehyde functional group has a concentration in the range of 0.5% to 5.0% (%w / v).

[0012] In some embodiments, the sugar-epichlorohydrin copolymer has a molecular weight of 60,000 Da to 500,000 Da.

[0013] In some embodiments, the sugar-epichlorohydrin copolymer is a sucrose-epichlorohydrin copolymer.

[0014] In some embodiments, the sucrose-epichlorohydrin copolymer is selected from the group consisting of polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol, Ficoll®, Ficoll® 70, and Ficoll® 400.

[0015] In some embodiments, the sugar-epichlorohydrin copolymer has a concentration of 2% to 20% (%w / v).

[0016] In some embodiments, the solution is dissolved in a solution selected from the group consisting of distilled water, deionized water, phosphate buffer, phosphate buffer solution, physiological saline, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, and any combination thereof.

[0017] In some embodiments, the contacting step is carried out at a temperature of 20 °C to 25 °C.

[0018] In some embodiments, the quenching step is carried out 20 to 60 minutes after the contacting step has been carried out.

[0019] In some embodiments, the quencher comprises a molecular compound containing an amine functional group dissolved in a solution containing a sucrose-epichlorohydrin copolymer.

[0020] In some embodiments, the molecular compound containing an amine functional group is provided as a Tris buffer, a glycine buffer, or a combination thereof; the Tris buffer comprises a Tris base selected from the group consisting of tris(hydroxymethyl)aminomethane, Trizma (登録商標) base, amino-2-(hydroxymethyl)-1,3-propanediol, 2-,2-amino-2-(hydroxymethyl)-1,3-propanediol, THAM, tris(hydroxymethyl)aminomethane, and tromethamine buffer.

[0021] In some embodiments, the quencher has an equal or excess molar concentration relative to the molecular compound containing an aldehyde functional group.

[0022] In some embodiments, the quencher is provided in a quenching solution at a ratio (v / v) equal to or greater than 2 with respect to the solution.

[0023] In some embodiments, the quencher completes quenching within a period of 5 to 60 minutes.

[0024] Some embodiments of the present invention include a step of contacting a whole blood sample with a solution containing a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group to obtain a whole blood solution, wherein the ratio (v / v) of the fixing solution to the whole blood sample is equal to or greater than 20: A step of adding a quencher to the whole blood solution: A step of enriching nucleated cells from a whole blood solution; and A step of performing a single cell assay on the enriched nucleated cells A method for preserving whole blood for single cell applications, including the above steps.

[0025] In some embodiments, the whole blood sample has a volume of 20 μL to 1 mL.

[0026] In some embodiments, the whole blood sample is collected using a capillary blood collection procedure.

[0027] In some embodiments, the molecular compound containing an aldehyde functional group is formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, or formalin.

[0028] In some embodiments, the molecular compound containing an aldehyde functional group has a concentration in the range of 0.5% to 5.0% (% w / v).

[0029] In some embodiments, the sugar-epichlorohydrin copolymer has a molecular weight of 60,000 Da to 500,000 Da.

[0030] In some embodiments, the sugar-epichlorohydrin copolymer is a sucrose-epichlorohydrin copolymer.

[0031] In some embodiments, the sucrose-epichlorohydrin copolymer is selected from the group consisting of polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol, Ficoll®, Ficoll® 70, and Ficoll® 400.

[0032] In some embodiments, the sugar-epichlorohydrin copolymer has a concentration of 2% to 20% (%w / v).

[0033] In some embodiments, the solution is dissolved in a solution selected from the group consisting of distilled water, deionized water, phosphate buffer, phosphate buffer solution, physiological saline, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, and any combination thereof.

[0034] In some embodiments, the contact step is carried out at a temperature of 20°C to 25°C.

[0035] In some embodiments, the quenching step is performed 20 to 60 minutes after the contact step.

[0036] In some embodiments, the quencher comprises a molecular compound containing an amine functional group, dissolved in a solution containing a sucrose-epichlorohydrin copolymer.

[0037] In some embodiments, molecular compounds containing amine functional groups are provided as Tris buffer, glycine buffer, or a combination thereof; Tris buffer is Tris(hydroxymethyl)aminomethane, Trizma (登録商標) The solution contains a Tris base selected from the group consisting of a base, amino-2-(hydroxymethyl)-1,3-propanediol, 2-,2-amino-2-(hydroxymethyl)-1,3-propanediol, THAM, tris(hydroxymethyl)aminomethane, and trometamol buffer.

[0038] In some embodiments, the quencher has a molar concentration equivalent to or exceeding that of the molecular compound containing the aldehyde functional group.

[0039] In some embodiments, the quencher is provided in a quenching solution in a ratio (v / v) equal to or greater than 2 with respect to the solution.

[0040] In some embodiments, the quencher completes the quenching within a period of 5 to 60 minutes.

[0041] Some embodiments further include the step of storing the quenched whole blood solution at a temperature of 20°C to 25°C for at least 24 hours.

[0042] In some embodiments, nucleated cells include PBMCs.

[0043] Some embodiments further include the step of isolating PBMCs from a quenched whole blood solution using a method selected from the group consisting of centrifugation, density gradient centrifugation, cell preparation tubes, PBMC isolation tubes, magnetic cell separation kits, immunoprecipitation, immunoseparation, and column purification.

[0044] Some embodiments further include the step of storing the isolated PBMCs in a freezer or using a cryogenic liquid.

[0045] In some embodiments, the single-cell assay includes single-cell RNA sequencing (scRNA-seq).

[0046] In some embodiments, scRNA-seq is performed on the 10X Genomics® scRNA-seq platform.

[0047] Some embodiments include a solution for preserving whole blood, comprising a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group, dissolved in a solution.

[0048] In some embodiments, the molecular compound containing the aldehyde functional group is formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, or formalin.

[0049] In some embodiments, the molecular compound containing the aldehyde functional group has a concentration in the range of 0.5% to 5.0% (%w / v).

[0050] In some embodiments, the sugar-epichlorohydrin copolymer has a molecular weight of 60,000 Da to 500,000 Da.

[0051] In some embodiments, the sugar-epichlorohydrin copolymer is a sucrose-epichlorohydrin copolymer.

[0052] In some embodiments, the sucrose-epichlorohydrin copolymer is selected from the group consisting of polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol, Ficoll®, Ficoll® 70, and Ficoll® 400.

[0053] In some embodiments, the sugar-epichlorohydrin copolymer has a concentration of 2% to 20% (%w / v).

[0054] In some embodiments, the solution is selected from the group consisting of distilled water, deionized water, phosphate buffer, phosphate buffer solution, physiological saline, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, and any combination thereof.

[0055] Additional embodiments and features are shown in part in the following description, some of which will be apparent to those skilled in the art by examining the specification, or may be acquired by practicing the disclosed subject matter. A further understanding of the nature and merits of this disclosure may be achieved by referring to the remainder of the specification and the drawings that form part of this disclosure.

[0056] This description will be better understood with reference to the following drawings, which are presented as exemplary embodiments of the invention and should not be construed as a complete enumeration of the scope of the invention. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 shows a process for preserving whole blood according to one embodiment.

[0058] [Figure 2] Figure 2 shows a process for preserving whole blood for single-cell analysis according to one embodiment.

[0059] [Figure 3] Figure 3 shows various cell types derived from whole blood preserved using a preservation process according to one embodiment.

[0060] [Figure 4] Figure 4 shows a comparison between PBMC storage using an aldehyde solution and PBMC storage using a storage solution according to a certain embodiment.

[0061] [Figure 5] Figure 5 shows MALAT1 gene expression at various time points in cells isolated from preserved whole blood according to one embodiment.

[0062] [Figure 6] Figure 6 shows the unique molecular identifier counts of cells isolated from stored whole blood at various time points according to one embodiment.

[0063] [Figure 7] Figure 7 shows the median values ​​for various cell types according to one embodiment.

[0064] [Figure 8] Figure 8 shows the gene abundance correlation for CD4 T cells between 24 hours and 72 hours, according to one embodiment.

[0065] [Figure 9] Figure 9 shows the gene abundance correlation for CD8 T cells between 24 hours and 72 hours, according to one embodiment.

[0066] [Figure 10] Figure 10 shows the gene abundance correlation for CD14 monocytes between 24 hours and 72 hours, according to one embodiment.

[0067] [Figure 11] Figure 11 shows the gene abundance correlation for CD16 monocytes between 24 hours and 72 hours, according to one embodiment.

[0068] [Figure 12] Figure 12 shows the gene abundance correlation for NK cells between 24 hours and 72 hours, according to one embodiment.

[0069] [Figure 13] Figure 13 shows the gene abundance correlation for B cells between 24 hours and 72 hours, according to one embodiment.

[0070] [Figure 14] Figure 14 shows the correlation of cell percentages for CD4 T cells, CD8 T cells, NK cells, B cells, and monocytes between 24 hours and 72 hours, according to one embodiment. [Modes for carrying out the invention]

[0071] (terminology) The following definitions are provided to assist those skilled in the art in understanding the detailed description.

[0072] The term "peripheral blood mononuclear cell (PBMC)" refers to any peripheral blood cell that has a round nucleus. PBMCs consist of lymphocytes (T cells (CD4+, CD8+), B cells, natural killer cells) and monocytes (CD14+, CD16+). Red blood cells and platelets do not have a nucleus, while granulocytes (neutrophils, basophils, and eosinophils) have a multi-lobed nucleus.

[0073] The term "capillary blood" refers to a blood sample collected by pricking the skin of a finger, heel, deltoid muscle, or another area of ​​the body with a sharp needle or lancet. Capillary blood is not obtained from a vein or by venipuncture.

[0074] The term "preservation" refers to the preservation of a biological sample from decay by terminating any ongoing biochemical reactions.

[0075] The terms "preservatives" and "preservatives" refer to chemical substances and formulations used in the preservation process.

[0076] The term "RNA sequencing (RNA-seq)" refers to a sequencing technique that uses next-generation sequencing (NGS) to determine the quality of RNA and gene expression in biological samples.

[0077] The term "room temperature" is used interchangeably with ambient temperature and refers to a temperature between approximately 20°C and 25°C (68°F and 77°F), including deviations between approximately 15°C and 30°C (59°F and 86°F).

[0078] The term "aldehyde" refers to an organic compound containing a functional group with the structure R-CH=O, where R is the side chain.

[0079] (Detailed explanation) The diagrams present a description of a system and method for whole blood preservation for single-cell application. An individual's immune system is driven by both time-varying genetic and environmental factors. Single-cell analysis platforms (e.g., multiple single-cell RNA sequencing (scRNA-seq) using blood) are being developed to better understand the transient and inter-individual variability of gene expression in different immune cell types. For example, performing scRNA-seq on capillary blood can identify genes exhibiting diurnal behavior in subpopulations of cells, potentially revealing target-specific immune-related gene signatures. (See, for example, MWThomson et al., U.S. Patent Application Publication 2021 / 0324447, the disclosure of which is incorporated herein by reference in its entirety).

[0080] Small volumes of blood are increasingly used in point-of-care testing because they require only a small sample. Capillary blood sampling is typically used to collect small volumes of blood due to its simplified and cost-effective collection process compared to venous puncture. While out-of-hospital capillary blood extraction reduces the physical and economic burden on patients, storing and transporting small volumes of whole blood requires storage at low temperatures (e.g., approximately -10°C to approximately 10°C) and rapid processing time to preserve gene expression information. Blood left at room temperature, and even at low temperatures, for extended periods can cause cells to alter their gene expression. Single-cell gene expression and high-plex plasma proteomics provide evidence that cells exhibit time-dependent changes that distort the underlying biology. These changes are extensive and dynamic, complicating the technical analysis of scRNA-seq data and inferences of in vivo physiological function from ex vivo assays. (See, for example, AKSavage, et al., iScience, 24, 5, 102404, 2021; its disclosure is incorporated herein by reference). This alteration of gene expression and tendency toward apoptosis can shift cell type distributions, masking true biological signals intended to be captured. Attempts to preserve immune cells are limited to hospitals and mobile venotomies, constrained by the high costs of cryogenic transport and personnel distribution.

[0081] Various systems and methods for preserving whole blood, according to many embodiments of this disclosure, can preserve whole blood (including cells and their biomolecules) at room temperature for a certain period of time. The preservation process can preserve cells and other components derived from the whole blood (including, but not limited to, nucleated cells, red blood cells (erythrocytes), platelets, pathogens, and biomolecules (including, but not limited to, nucleic acids (DNA and RNA), proteins, polysaccharides, and lipids)) while simultaneously reducing and / or minimizing artifacts during the storage period. In some embodiments, the whole blood is fixed at a specific time (e.g., at or near the time of extraction) so that the biological activity of the blood cells is preserved from the time of fixation. In some embodiments, the preserved whole blood may be used immediately after fixation (e.g., within 1 hour). In some embodiments, the preserved whole blood may be stored at room temperature for a long period of time (e.g., at least 24 hours). In some embodiments, the storage of the preserved whole blood may be carried out at room temperature without any additional steps (e.g., heating or cooling). The procedures for collecting whole blood at room temperature, storing it, and keeping it are easy to follow, enabling distributed users to collect blood samples themselves, safely and properly carry out the storage process, and properly store and / or return the blood samples.

[0082] Various systems and methods preserve nucleated cells in whole blood for downstream single-cell analysis. These preserved nucleated cells can be stored at room temperature for extended periods before single-cell application is performed. Analysis of genomics, transcriptomics, toproteomics, metabolomics, and intercellular interactions at the single-cell level can help elucidate intercellular variability and identify unique cells within cell populations. In some embodiments, whole blood storage enables the analysis of biomolecules (including, but not limited to, nucleic acids, proteins, and metabolites) within nucleated cells in various single-cell applications. Examples of single-cell assays compatible with stored whole blood include, but are not limited to, single-cell nucleic acid sequencing, single-cell DNA sequencing, single-cell genome analysis, single-cell RNA sequencing, single-cell transcriptome analysis, single-cell gene expression assays, single-cell proteomics analysis (e.g., by mass spectrometry), single-cell immunodetection assays, single-cell metabolomics analysis (e.g., by mass spectrometry), single-cell chromatin analysis, single-cell methylome sequencing, single-cell bisulfite sequencing, single-cell immunoprecipitation assays, and single-cell chromatin immunoprecipitation sequencing.

[0083] Certain embodiments preserve nucleated cells. Nucleated cells may include any nucleated cells that may originate from whole blood, and may include cells having multilobed nuclei. Nucleated cells that may be preserved include, but are not limited to, peripheral blood monocytes (PBMCs), granulocytes, hematological cancer cells, circulating tumor cells, and pathogens. PBMCs include, but are not limited to, lymphocytes, monocytes, T cells, B cells, natural killer cells, and dendritic cells. Granulocytes include, but are not limited to, neutrophils, basophils, and eosinophils. Hematological cancer cells include, but are not limited to, leukemia cells, lymphoma cells, and myeloma cells. Circulating tumor cells are cells that have entered the bloodstream from a tumor (primary or otherwise) and therefore may originate from any solid tissue. Pathogens may include any pathogens that can be found in the bloodstream (e.g., bloodborne pathogens, bloodstream infections) (including intracellular and extracellular pathogens). Examples of pathogens include (but are not limited to) hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma. Nucleated cells may be enriched or isolated from whole blood for downstream application. In one example, collected nucleated cells may be used for single-cell RNA-seq for immunoprofiling.

[0084] Small volumes of blood refer to whole blood samples having a volume equal to or less than approximately 1 mL. Small volumes of blood can be collected by capillary blood sampling, venous sampling, and / or arterial sampling. Systems and methods for preserving capillary whole blood enable dispersed users to collect their own capillary blood and send it to facilities for single-cell processing and analysis. Preservation according to many embodiments can maintain the biomolecules of cells in a fixed and stable state at ambient temperature for extended periods. Methods for long-term preservation of whole blood at room temperature can minimize the complex and costly logistics associated with cryogenic transport. Fixation and preservation solutions can adequately preserve biomolecules while reducing and / or minimizing artifacts during fixation, preservation, and storage.

[0085] (cell preservation) The storage and preservation of whole blood is crucial for biological research and clinical evaluation to minimize intracellular contamination and genetic alteration of biological samples. Traditionally, cryopreservation (where cells and tissues are stored in a low-temperature environment) is used to preserve cells isolated from whole blood. Standard cryopreservation techniques require the isolation of nucleated cells from whole blood before cryopreservation. Cell isolation (which typically requires laboratory techniques such as gradient buffering and centrifugation) prevents dispersed users lacking both the necessary skills and laboratory access from preserving whole blood samples at home by cryopreservation. The ability to store whole blood at low temperatures allows for the transport of biological samples and extends the shelf life for completing safety and quality control tests. However, cryopreservation typically requires pre-freezing, introduction of cryopreservation solutions, and low-temperature storage to execute the freezing protocol. Low-temperature processes can be difficult and expensive to implement due to the cost of maintaining and storing liquid nitrogen and the extreme conditions involved.

[0086] Cell fixation preserves the morphology, integrity, and structure of cells by fixing biomolecules in space and time, thereby preventing the process of decay (cellular decay). Biological samples may be fixed immediately after extraction to limit autolysis and decay. Common cell fixation reagents include precipitation and crosslinking agents. Organic solvents (e.g., methanol, acetone, and picric acid) can act as strong dehydrating agents and precipitate cellular proteins. While these fixatives are effective in preserving cellular structure, they can remove soluble low-molecular-weight molecules and lipids.

[0087] Aldehydes (e.g., formalin, glutaraldehyde, paraformaldehyde, or formaldehyde) are fixatives that crosslink proteins via free amine groups, forming intermolecular crosslinks. Biological samples can be incubated at room temperature with a solution of formalin, glutaraldehyde, paraformaldehyde, or formaldehyde to carry out the crosslinking reaction.

[0088] (Preservation of whole blood) Many embodiments of this disclosure relate to various preservation processes for preserving small volumes of whole blood. These preservation processes can preserve single-cell biomolecules within whole blood for various downstream applications, including, but are not limited to, single-cell nucleic acid sequencing, single-cell DNA sequencing, single-cell genome analysis, single-cell RNA sequencing, single-cell transcriptome analysis, single-cell gene expression assays, single-cell proteomics analysis (e.g., by mass spectrometry), single-cell immunodetection assays, single-cell metabolomics analysis (e.g., by mass spectrometry), single-cell chromatin analysis, single-cell methylome sequencing, single-cell bisulfite sequencing, single-cell immunoprecipitation assays, and single-cell chromatin immunoprecipitation sequencing.

[0089] Figure 1 shows a whole blood storage process according to one embodiment of the present disclosure. Method 100 may begin with a step (101) of obtaining a small volume of whole blood sample. The small volume of whole blood sample may be collected using any blood collection method, such as capillary blood sampling, venous sampling, and / or arterial sampling. In one example, a small volume of whole blood sample may be collected by a capillary blood collection procedure. Capillary whole blood may be collected by pricking the skin of any part of the body (such as a finger, arm, leg, heel, toenail, or deltoid muscle). Often, capillary sampling is performed in a skin area containing a large number of capillaries. Various methods may be used to collect capillary whole blood, including but not limited to needles, microneedles, and / or lancets. The blood may be collected in a container (such as but not limited to pipettes, tubes, vials, or microcontainers). The container may be manufactured from a non-reactive material (including, but not limited to, glass, plastic, rigid plastic, resin, or polymer). Examples of commercially available containers for collecting capillary whole blood include, but are not limited to, the TAP Touch Activated Phlebotomy® device, the TAP Blood Collection® device, and the Tasso+® device.

[0090] In some embodiments, a small volume of whole blood sample may be collected after a venous puncture procedure in which blood is collected from a vein. The blood may be drawn from a vein in the elbow or hand (but not limited to these). A needle may be inserted into the vein, and the blood may be collected in an airtight vial or syringe. In some embodiments, a small volume of whole blood sample may be collected after an arterial sampling procedure. A small volume of blood sample derived from venous puncture sampling and / or arterial sampling may be taken using a variety of methods (including, but not limited to, pipetting).

[0091] In some embodiments, a small volume of whole blood sample has a volume of approximately 20 μL to approximately 1 mL. In various embodiments, a small volume of whole blood has a volume of approximately 20 μL to approximately 30 μL; a small volume of whole blood has a volume of approximately 30 μL to approximately 40 μL; a small volume of whole blood has a volume of approximately 40 μL to approximately 50 μL; a small volume of whole blood has a volume of approximately 50 μL to approximately 60 μL; a small volume of whole blood has a volume of approximately 60 μL to approximately 70 μL; a small volume of whole blood has a volume of approximately 70 μL to approximately 80 μL; a small volume of whole blood has a volume of approximately 80 μL to approximately 90 μL; a small volume of whole blood has a volume of approximately 90 μL to approximately 100 μL. A small volume of whole blood has a volume of approximately 100 μL to 110 μL; a small volume of whole blood has a volume of approximately 110 μL to 120 μL; a small volume of whole blood has a volume of approximately 120 μL to 130 μL; a small volume of whole blood has a volume of approximately 130 μL to 140 μL; a small volume of whole blood has a volume of approximately 140 μL to 150 μL; a small volume of whole blood has a volume of approximately 150 μL to 200 μL; a small volume of whole blood has a volume of approximately 200 μL to 250 μL; a small volume of whole blood has a volume of approximately 250 μL to 300 μL It has volume; a small volume of whole blood has a volume of approximately 300 μL to approximately 350 μL; a small volume of whole blood has a volume of approximately 350 μL to approximately 400 μL; a small volume of whole blood has a volume of approximately 400 μL to approximately 450 μL; or a small volume of whole blood has a volume of approximately 450 μL to approximately 500 μL; or a small volume of whole blood has a volume of approximately 500 μL to approximately 550 μL; or a small volume of whole blood has a volume of approximately 550 μL to approximately 600 μL; or a small volume of whole blood has a volume of approximately 600 μL to approximately 650 μL; A small volume of whole blood has a volume of approximately 650 μL to 700 μL; or a small volume of whole blood has a volume of approximately 700 μL to 750 μL; or a small volume of whole blood has a volume of approximately 750 μL to 800 μL; or a small volume of whole blood has a volume of approximately 800 μL to 850 μL; or a small volume of whole blood has a volume of approximately 850 μL to 900 μL; or a small volume of whole blood has a volume of approximately 900 μL to 950 μL; or a small volume of whole blood has a volume of approximately 950 μL to 1 mL.

[0092] The small volume of whole blood sample may be treated with a preservation solution (102). The preservation solution can fix the whole blood and its components to preserve nucleated cells, biomolecules, or other components within the whole blood for downstream analysis (particularly analysis against single cells). In many embodiments, the preservation solution comprises an aldehyde and a polymeric crowder. Examples of polymeric crowders include (but are not limited to) polyethylene glycol, dextran, and / or sugar-epichlorohydrin copolymers. In many embodiments, the preservation solution comprises an aldehyde and a sugar-epichlorohydrin copolymer (e.g., sucrose-epichlorohydrin copolymer).

[0093] The polymeric crowder in the preservation solution can reduce cell aggregation during the time cells are exposed to the aldehyde, inhibiting intercellular contact and preventing cells from clumping together and becoming fixed. In embodiments utilizing sugar-epichlorohydrin copolymers, the sugar may be any monosaccharide, disaccharide, oligosaccharide, or polysaccharide that can act as a polymeric crowder when present together with epichlorohydrin in the copolymer. Examples of sugar-epichlorohydrin copolymers that can be used include, but are not limited to, sucrose-epichlorohydrin copolymers. In an example of a sucrose-epichlorohydrin copolymer, (molecular formula C 12 H 22 O 12Organic compounds containing sucrose-(molecular formula C3H5ClO)epichlorohydrin copolymer may be used. In some embodiments, the sucrose-epichlorohydrin copolymer may have a molecular weight in the range of about 60,000 Da to about 500,000 Da. In various embodiments, the sucrose-epichlorohydrin copolymer has a molecular weight of about 60,000 Da to about 70,000 Da; the sucrose-epichlorohydrin copolymer has a molecular weight of about 70,000 Da to about 80,000 Da; the sucrose-epichlorohydrin copolymer has a molecular weight of about 80,000 Da to about 90,000 Da; the sucrose-epichlorohydrin copolymer has a molecular weight of about 90,000 Da to about 100,000 Da Sucrose-epichlorohydrin copolymers have a molecular weight of approximately 100,000 Da to approximately 200,000 Da; sucrose-epichlorohydrin copolymers have a molecular weight of approximately 200,000 Da to approximately 300,000 Da; sucrose-epichlorohydrin copolymers have a molecular weight of approximately 300,000 Da to approximately 400,000 Da; or sucrose-epichlorohydrin copolymers have a molecular weight of approximately 400,000 Da to approximately 500,000 Da. Examples of sucrose-epichlorohydrin copolymers include (but are not limited to) polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol, Ficoll®, Ficoll® 70, and Ficoll® 400.

[0094] Sucrose-epichlorohydrin copolymers of various concentrations can be used in storage solutions. In some embodiments, the sucrose-epichlorohydrin copolymer may have a concentration of about 2% to about 20% (%w / v). In various embodiments, the sucrose-epichlorohydrin copolymer has a concentration of about 2% to about 3%; the sucrose-epichlorohydrin copolymer has a concentration of about 3% to about 4%; the sucrose-epichlorohydrin copolymer has a concentration of about 4% to about 5%; the sucrose-epichlorohydrin copolymer has a concentration of about 5% to about 6%; the sucrose-epichlorohydrin copolymer has a concentration of about 6% to about 7%; the sucrose-epichlorohydrin copolymer has a concentration of about 7% to about 8%; the sucrose-epichlorohydrin copolymer has a concentration of about 8% to about 9%; the sucrose-epichlorohydrin copolymer has a concentration of about 9% to about 10%; the sucrose-epichlorohydrin copolymer has a concentration of about 10% to about 11%; sucrose Sucrose-epichlorohydrin copolymers have a concentration of approximately 11% to 12%; sucrose-epichlorohydrin copolymers have a concentration of approximately 12% to 13%; sucrose-epichlorohydrin copolymers have a concentration of approximately 13% to 14%; sucrose-epichlorohydrin copolymers have a concentration of approximately 14% to 15%; sucrose-epichlorohydrin copolymers have a concentration of approximately 15% to 16%; sucrose-epichlorohydrin copolymers have a concentration of approximately 16% to 17%; sucrose-epichlorohydrin copolymers have a concentration of approximately 17% to 18%; sucrose-epichlorohydrin copolymers have a concentration of approximately 18% to 19%; or sucrose-epichlorohydrin copolymers have a concentration of approximately 19% to 20%.

[0095] The preservation solution may contain molecular compounds containing aldehyde functional groups to fix cells in whole blood. The aldehyde can crosslink biomolecules via free amine groups, forming intermolecular crosslinks and networks of linked molecules. Any aldehyde-containing organic compound may be used for fixation in the preservation solution according to some embodiments. Examples of aldehyde compounds include, but are not limited to, formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, formalin, and combinations thereof. As can be easily understood, any of the various aldehyde compounds may be used in the preservation solution in a manner suitable for the requirements of specific applications according to various embodiments of the present invention.

[0096] The concentration of aldehyde molecules in the preservation solution can vary. Higher concentrations of aldehyde may increase the amount of crosslinking of biomolecules, which may better preserve those biomolecules but may interfere with downstream applications. Therefore, the appropriate concentration of aldehyde and treatment time can be optimized based on the needs of the specific downstream application being carried out. Aldehydes can have concentrations (%w / v) of approximately 0.5% to 5%; or approximately 0.5% to 1.0%; or approximately 1.0% to 1.5%; or approximately 1.5% to 2.0%; or approximately 2.0% to 2.5%; or approximately 2.5% to 3.0%; or approximately 3.0% to 3.5%; or approximately 3.5% to 4.0%; or approximately 4.0% to 4.5%; or approximately 4.5% to 5.0%.

[0097] In some embodiments, the preservation solution also includes a permeation surfactant. For example, Triton® X-100, Tween®-20, saponins, or other permeation agents may be used.

[0098] The preservation solution can be prepared using an aqueous solution (e.g., water, deionized water, filtered water, or reverse osmosis water). In some embodiments, the preservation solution includes a buffer. Examples of buffers include (but are not limited to) phosphate buffer (PBS), HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, physiological saline, phosphate-buffered physiological saline, and any combination thereof. The preservation solution can be pre-filled in a container for whole blood sample collection or added to a container containing the collected whole blood sample. In some embodiments, the preservation solution is brought into contact with the whole blood sample in a large ratio of preservation solution to whole blood. In various embodiments, the ratio of the preservation solution to whole blood is greater than or equal to about 10:1 (v / v), greater than or equal to about 15:1 (v / v), greater than or equal to about 20:1 (v / v), greater than or equal to about 25:1 (v / v), greater than or equal to about 30:1 (v / v), greater than or equal to about 35:1 (v / v), or greater than or equal to about 40:1 (v / v).

[0099] The contact time between whole blood and the preservation solution can range from approximately 20 minutes to approximately 80 minutes. In various embodiments, the contact time between whole blood and the preservation solution is approximately 20 minutes to approximately 30 minutes; approximately 30 minutes to approximately 40 minutes; approximately 40 minutes to approximately 50 minutes; approximately 50 minutes to approximately 60 minutes; approximately 60 minutes to approximately 70 minutes; and approximately 70 minutes to approximately 80 minutes. In some embodiments, the contact time between whole blood and the preservation solution is greater than or equal to approximately 45 minutes. In some embodiments, the contact time between whole blood and the preservation solution is less than approximately 80 minutes. In some embodiments, the contact time between whole blood and the preservation solution is approximately 45 minutes to approximately 80 minutes. In certain embodiments, a contact time between whole blood and the preservation solution of less than approximately 45 minutes may result in a larger amount of cellular debris, which may result in a poor PBMC yield.

[0100] Contact between the preservation solution and whole blood can be carried out at room temperature. Contact between the preservation solution and whole blood can be carried out without heating, heat treatment, cooling, or refrigeration.

[0101] Once storage is complete, the storage can be quenched to inactivate aldehyde 103 (103). Contact with a quencher may enable long-term storage of the stored whole blood without removing the aldehyde from the whole blood solution. In some embodiments, the quencher is a molecular compound containing an amine (or amino functional group). In some embodiments, the quencher is provided in a quenching solution; the quenching solution is used to contact the storage solution to inactivate the aldehyde. In some embodiments, the quenching solution comprises a sugar-epichlorohydrin copolymer (such as, but not limited to, sucrose-epichlorohydrin copolymer) and a quencher (e.g., a molecular compound containing an amine (or amino functional group)). The amine functional group may react with the aldehyde group to quench its crosslinking reaction. Examples of molecular compounds containing amine functional groups include (but are not limited to) Tris, Tris buffer, glycine, glycine buffer, and any combination thereof. Tris buffer contains Tris base dissolved in distilled or deionized water and has a pH in the range of about 6.0 to about 8.5; or about 6.0 to about 6.5; or about 6.5 to about 7.0; or about 7.0 to about 7.5; or about 7.5 to about 8.0; or about 8.0 to about 8.5. Tris base is also known as Tris(hydroxymethyl)aminomethane, Trizma (登録商標)It is also known as Tris-glycine buffer, amino-2-(hydroxymethyl)-1,3-propanediol, 2-,2-amino-2-(hydroxymethyl)-1,3-propanediol, THAM, Tris(hydroxymethyl)aminomethane, and trometamol buffer. Tris-glycine buffer contains Tris base and glycine dissolved in distilled or deionized water and has a pH in the range of about 6.0 to about 8.5; or about 6.0 to about 6.5; or about 6.5 to about 7.0; or about 7.0 to about 7.5; or about 7.5 to about 8.0; or about 8.0 to about 8.5. The quenching solution may be dissolved in a buffer solution (such as phosphate buffer, phosphate buffer, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, physiological saline, phosphate-buffered physiological saline, water, deionized water, filtered water, and any combination thereof, but not limited to these).

[0102] In some embodiments, ethylenediaminetetraacetic acid (EDTA) may be added to the quenching buffer solution. In certain embodiments, the EDTA in the quenching buffer may provide additional protection against nuclease activity. In some embodiments, the concentration of EDTA in the quenching buffer may be in the range of about 1 mM to about 10 mM; about 1 mM to about 2 mM; or about 2 mM to about 3 mM; or about 3 mM to about 4 mM; or about 4 mM to about 5 mM; or about 5 mM to about 6 mM; or about 6 mM to about 7 mM; or about 7 mM to about 8 mM; or about 8 mM to about 9 mM; or about 9 mM to about 10 mM.

[0103] In some embodiments, the amine in the quencher has a molar concentration equal to that of the aldehyde in the fixative solution. In some embodiments, the amine in the quencher has an excess molar concentration relative to the free aldehyde in the fixative solution. In certain embodiments, the ratio of glycine to aldehyde may be about 1:2 or greater than 1:2.

[0104] In some embodiments, the ratio of quenching solution volume to storage solution volume is approximately equal to or greater than or equal to 2:1 (v / v); or approximately equal to or greater than or equal to 3:1 (v / v); or approximately equal to or greater than or equal to 4:1 (v / v); or approximately equal to or greater than or equal to 5:1 (v / v); or approximately equal to or greater than or equal to 6: It is equal to or greater than 1(v / v), or equal to 1(v / v), or the ratio of quenching solution volume to storage solution volume is approximately equal to or greater than 7:1(v / v), or equal to 8:1(v / v), or equal to 8:1(v / v), or equal to 9:1(v / v), or equal to 9:1(v / v), or equal to 10:1(v / v), or equal to 10:1(v / v).

[0105] The quenching time for capillary whole blood in the quenching solution may range from approximately 60 minutes (approximately 1 hour) to approximately 240 minutes (approximately 4 hours); or approximately 60 minutes to approximately 90 minutes; or approximately 90 minutes to approximately 120 minutes; or approximately 120 minutes to approximately 150 minutes; or approximately 150 minutes to approximately 180 minutes; or approximately 180 minutes to approximately 210 minutes; or approximately 210 minutes to approximately 240 minutes.

[0106] The quenching time for capillary whole blood in the quenching solution may range from approximately 5 minutes to approximately 60 minutes; or approximately 5 minutes to approximately 10 minutes; or approximately 10 minutes to approximately 15 minutes; or approximately 15 minutes to approximately 20 minutes; or approximately 20 minutes to approximately 30 minutes; or approximately 30 minutes to approximately 40 minutes; or approximately 40 minutes to approximately 50 minutes; or approximately 50 minutes to approximately 60 minutes.

[0107] In some embodiments, stirring may be applied to inactivate aldehydes during the quenching process. In some embodiments, gentle, periodic stirring may be applied to quench aldehyde activity during the quenching process. In certain embodiments, stirring (e.g., stirring, shaking, rapid shaking, mixing, and / or mixing) may be applied manually or by instrument or device. The periodicity and / or intensity of stirring may be optimized to adequately inactivate aldehyde activity while preserving the whole blood sample.

[0108] The stored whole blood can be used for various downstream applications (104). In some embodiments, the stored blood sample is used immediately after quenching (e.g., within 1 hour). In some embodiments, the stored whole blood is stored for a longer period, which can be done at room temperature. In various embodiments, the stored whole blood is stored at room temperature for about 2 hours or more; the stored whole blood is stored at room temperature for about 4 hours or more; the stored whole blood is stored at room temperature for about 6 hours or more; the stored whole blood is stored at room temperature for about 8 hours or more; the stored whole blood is stored at room temperature for about 12 hours or more; the stored whole blood is stored at room temperature for about 16 hours or more; the stored whole blood is stored at room temperature for about 24 hours or more; the stored whole blood is stored at room temperature for about 30 hours or more; the stored whole blood is stored at room temperature for about 36 hours or more or stored at room temperature for the same amount of time; stored whole blood is stored at room temperature for more than approximately 42 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 48 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 72 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 96 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 120 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 144 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 168 hours or the same amount of time; or stored whole blood is stored at room temperature for more than approximately 336 hours or the same amount of time.

[0109] Various processes for preserving whole blood for downstream applications are described above with reference to Figure 1, but any type of process utilizing a preservative to preserve a small volume of whole blood may be used as appropriate to the requirements of specific applications according to various embodiments of the present invention. For illustrative purposes, specific examples of preserving a small volume of whole blood for single-cell applications according to various embodiments of the present invention are further discussed below.

[0110] (Storage of small volumes of whole blood for single-cell application) Many embodiments perform various preservation processes to fix and store nucleated cells derived from small volumes of whole blood for single-cell applications. These preservation processes allow for the storage of whole blood and the single cells within it for analysis after long-term storage.

[0111] Figure 2 shows a method for preserving whole blood for single-cell application according to one embodiment of the present invention. Method 200 may begin with a step (201) of obtaining a small volume of whole blood sample to be preserved. The small volume of whole blood sample may first be collected using a blood collection method (including, but not limited to, capillary blood sampling, venous puncture sampling, and arterial sampling). In some embodiments, the small volume of whole blood sample is preserved as shown in Figure 1.

[0112] In some embodiments, a small volume of whole blood sample has a volume of approximately 20 μL to approximately 1 mL. In various embodiments, a small volume of whole blood has a volume of approximately 20 μL to approximately 30 μL; a small volume of whole blood has a volume of approximately 30 μL to approximately 40 μL; a small volume of whole blood has a volume of approximately 40 μL to approximately 50 μL; a small volume of whole blood has a volume of approximately 50 μL to approximately 60 μL; a small volume of whole blood has a volume of approximately 60 μL to approximately 70 μL; a small volume of whole blood has a volume of approximately 70 μL to approximately 80 μL; a small volume of whole blood has a volume of approximately 80 μL to approximately 90 μL; a small volume of whole blood has a volume of approximately 90 μL to approximately 100 μL. A small volume of whole blood has a volume of approximately 100 μL to 110 μL; a small volume of whole blood has a volume of approximately 110 μL to 120 μL; a small volume of whole blood has a volume of approximately 120 μL to 130 μL; a small volume of whole blood has a volume of approximately 130 μL to 140 μL; a small volume of whole blood has a volume of approximately 140 μL to 150 μL; a small volume of whole blood has a volume of approximately 150 μL to 200 μL; a small volume of whole blood has a volume of approximately 200 μL to 250 μL; a small volume of whole blood has a volume of approximately 250 μL to 300 μL It has volume; a small volume of whole blood has a volume of approximately 300 μL to approximately 350 μL; a small volume of whole blood has a volume of approximately 350 μL to approximately 400 μL; a small volume of whole blood has a volume of approximately 400 μL to approximately 450 μL; or a small volume of whole blood has a volume of approximately 450 μL to approximately 500 μL; or a small volume of whole blood has a volume of approximately 500 μL to approximately 550 μL; or a small volume of whole blood has a volume of approximately 550 μL to approximately 600 μL; or a small volume of whole blood has a volume of approximately 600 μL to approximately 650 μL; A small volume of whole blood has a volume of approximately 650 μL to 700 μL; or a small volume of whole blood has a volume of approximately 700 μL to 750 μL; or a small volume of whole blood has a volume of approximately 750 μL to 800 μL; or a small volume of whole blood has a volume of approximately 800 μL to 850 μL; or a small volume of whole blood has a volume of approximately 850 μL to 900 μL; or a small volume of whole blood has a volume of approximately 900 μL to 950 μL; or a small volume of whole blood has a volume of approximately 950 μL to 1 mL.

[0113] Small volumes of whole blood samples can be preserved using a preservation solution. In some embodiments, the preservation solution contains a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group. Fixation of whole blood intended for single-cell application requires that the cells in the sample remain separated from each other. The sugar-epichlorohydrin copolymer in the fixation solution can prevent cells from being fixed together by reducing cell aggregation and inhibiting intercellular contact when exposed to aldehydes. Preservation of whole blood intended for single-cell RNA sequencing also requires prevention of RNA degradation by ribonucleases (RNA degradation proteins). The aldehydes in the fixation solution can inactivate ribonucleases.

[0114] The preservation of small volumes of whole blood can be carried out at room temperature. This preservation process can be performed without heating, heat treatment, cooling, or refrigeration.

[0115] A quencher may be added to inactivate the aldehyde. The addition of a quencher allows whole blood to be preserved without removing the aldehyde from the preserved whole blood solution. The quencher may be a molecular compound containing an amine (or amino) functional group. The amine functional group can react with the aldehyde group to quench its crosslinking reaction. Examples of molecular compounds containing an amine functional group include (but are not limited to) Tris, Tris buffer, glycine, glycine buffer, and any combination thereof.

[0116] In some embodiments, the stored blood sample is used immediately after quenching (e.g., within 1 hour). In some embodiments, the stored whole blood may be stored for a longer period (202), which may be done at room temperature. In various embodiments, the stored whole blood is stored at room temperature for about 2 hours or more; the stored whole blood is stored at room temperature for about 4 hours or more; the stored whole blood is stored at room temperature for about 6 hours or more; the stored whole blood is stored at room temperature for about 8 hours or more; the stored whole blood is stored at room temperature for about 12 hours or more; the stored whole blood is stored at room temperature for about 16 hours or more; the stored whole blood is stored at room temperature for about 24 hours or more; the stored whole blood is stored at room temperature for about 30 hours or more; the stored whole blood is stored at room temperature for about 36 hours or more. or stored at room temperature for the same amount of time; stored whole blood is stored at room temperature for more than approximately 42 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 48 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 72 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 96 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 120 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 144 hours or the same amount of time; stored whole blood is stored at room temperature for more than approximately 168 hours or the same amount of time; or stored whole blood is stored at room temperature for more than approximately 336 hours or the same amount of time.

[0117] In some embodiments, the stored blood samples may be transported to a processing facility by various means (including, but not limited to, transportation, delivery, and collection by courier services). The transport of the blood samples may be carried out at room temperature without heating, heat treatment, cooling, or any other cooling treatment. Transport may be carried out immediately after quenching or after a period of storage at room temperature. At the processing facility, the blood samples may be processed immediately upon receipt or stored at room temperature for a period of time.

[0118] Nucleated cells can be enriched from stored whole blood (203). Nucleated cells (e.g., PBMCs, granulocytes) and / or multilobed nuclei cells (e.g., neutrophils) can be isolated and collected from stored whole blood. Nucleated cells can be enriched by many methods (e.g., centrifugation, density gradient centrifugation, cell preparation tubes, PBMC isolation tubes (e.g., SepMate®, Vacutainer®), magnetic cell separation kits (e.g., EasySep®), immunoprecipitation, immunoseparation, and column purification). In one example, density gradient centrifugation is performed by adding density gradient buffer to the blood sample. Centrifugation can separate components by their density. Plasma can be removed by pipetting. Any remaining residue can be removed by pipetting. Isolated nucleated cells can be collected using pipettes and / or Pasteur pipettes. To further remove any remaining red blood cells, erythrocyte lysis buffer may be added to the enriched nucleated cells. These nucleated cells may then be washed and centrifuged. The isolated and collected nucleated cells may be counted for preparation for single-cell applications. In some embodiments, the enriched nucleated cells may be stored for extended periods in a freezer or using a cryogenic source (e.g., liquid nitrogen). If cryopreserved, the nucleated cells may be thawed when ready for use in downstream applications.

[0119] Single-cell analysis may be performed on the nucleated cells (204). Examples of single-cell assays compatible with conserved whole blood include, but are not limited to, single-cell nucleic acid sequencing, single-cell DNA sequencing, single-cell genome analysis, single-cell RNA sequencing, single-cell transcriptome analysis, single-cell gene expression assays, single-cell proteomics analysis (e.g., by mass spectrometry), single-cell immunodetection assays, single-cell metabolomics analysis (e.g., by mass spectrometry), single-cell chromatin analysis, single-cell methylome sequencing, single-cell bisulfite sequencing, single-cell immunoprecipitation assays, and single-cell chromatin immunoprecipitation sequencing.

[0120] In one example, nucleated cells are used for single-cell RNA-seq. Conserved and enriched nucleated cells derived from whole blood are compatible with a variety of single-cell RNA-seq platforms (including, but not limited to, droplet-based single-cell RNA-seq). Examples of droplet-based single-cell RNA-seq platforms include, but are not limited to, 10x Genomics®, 10x Genomics® Chromium, Bio-Rad Laboratories' ddSEQ®, 1CellBio's InDrop®, Dolomite Bio / Blacktrace Holdings' μEncapsulator®, Fluent BioSciences' PIPseq®, Parse Biosciences' Evercode®, and Scale Biosciences' ScaleBio®.

[0121] Various processes for preserving whole blood for single-cell applications are described above with reference to Figure 2, but any type of process utilizing a preservative to preserve a small volume of whole blood may be used as appropriate to the requirements of the specific applications according to the various embodiments of the present invention. [Examples]

[0122] The following examples are provided to give a complete disclosure and explanation of how the present invention is manufactured and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments are all experiments performed or that these are the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into consideration.

[0123] (Example 1: Capillary whole blood preservation using formaldehyde and Ficoll®) Ficoll® can be combined with formaldehyde to preserve capillary whole blood for the preservation and isolation of PBMCs for downstream applications. A Ficoll® solution can be prepared by dissolving Ficoll® powder in PBS. Dissolving Ficoll® in water may result in hemolysis when added to formaldehyde-fixed solutions and blood, possibly due to low volume molar osmotic concentrations. The combination of Ficoll® and formaldehyde in the preservation solution yields better yields of enriched PBMCs from the preserved whole blood.

[0124] To maintain a preserved blood sample at room temperature for at least 24 hours without removing the preservation solution from the blood cells, the formaldehyde in the solution is inactivated with a quenching solution at least four times the volume of the preservation solution. The quenching solution may contain Ficoll®, glycine / Tris buffer, and an additional Tris buffer in equimolar amounts to the final amount of formaldehyde, in a PBS solution.

[0125] While preservation solutions containing only aldehyde compounds may be able to fix capillary whole blood, robust enrichment of PBMCs after fixation cannot be achieved. Direct use of formaldehyde for preservation results in the fixation of all cells and debris derived from whole blood to such an extent that PBMCs cannot be recovered and isolated in quantities suitable for high-quality analysis.

[0126] In one experiment, various concentrations of formaldehyde were added to capillary whole blood samples at final concentrations of approximately 1%, 2%, and 4%. All three experiments showed that formaldehyde fixed all cells and debris from the whole blood that were fixed together, and that PBMC recovery could not be achieved. After the experiment, the capillary whole blood samples were diluted with PBS, and then formaldehyde was added. Tenfold and twentyfold dilutions of the whole blood samples in PBS did not improve PBMC recovery after formaldehyde fixation.

[0127] (Example 2: Cell preservation using capillary whole blood) To test the ability to preserve cells within whole blood, capillary whole blood was extracted and then processed according to the method described herein. Briefly, capillary whole blood was preserved using a solution containing an aldehyde and a sugar-epichlorohydrin copolymer. The aldehyde reaction was quenched using a glycine solution. The blood was analyzed immediately after quenching (0 hours) and at 4 hours. After quenching, the whole blood was further processed and single-cell RNA sequencing was performed. Known cell type markers were used to identify which blood cells were preserved (Figure 3). As can be seen from these data results, B cells, CD4 T cells, CD8 T cells, monocytes, and NK cells each had a similar percentage of cells at 4 hours post-preservation compared to 0 hours. These results suggest that no single cell type was independently affected in terms of cell preservation.

[0128] (Example 3: Cell isolation from capillary whole blood after storage) To test the effect of preservation solutions on cell isolation from whole blood, capillary whole blood was extracted and then treated according to the methods described herein. Briefly, capillary whole blood was preserved using a solution containing an aldehyde and a sugar-epichlorohydrin copolymer. As a control, cells treated with aldehyde alone were also prepared. The aldehyde reaction was quenched using a glycine solution. These two preservation techniques were compared in terms of their ability to isolate PBMCs using a commercially available PBMC isolation kit (STEMCELL Technologies, Vancouver, CA) (Figure 4). As can be seen from these data results, PBMCs were better isolated when preserved using a solution containing an aldehyde and a sugar-epichlorohydrin copolymer. These results suggest that preservation solutions containing an aldehyde and a sugar-epichlorohydrin copolymer result in better cell isolation.

[0129] (Example 4: Preservation of MALAT1 gene expression) To test the ability to preserve gene expression in cells within whole blood, capillary whole blood was extracted and then processed according to the method described herein. Briefly, capillary whole blood was preserved using a solution containing an aldehyde and a sugar-epichlorohydrin copolymer. The aldehyde reaction was quenched using a glycine solution. MALAT1 gene expression was analyzed immediately after quenching (0 hours) and at 72 hours. After quenching, the whole blood was further processed and fluorescence in situ hybridization using a MALAT1 probe was performed (Figure 5). As can be seen from these data results, the amount of MALAT1 in the sample preserved for 72 hours was similar to that in the sample analyzed immediately. These results suggest that this preservation method protects RNA for extended storage periods.

[0130] (Example 5: Storage of RNA transcripts) To test the ability to preserve RNA transcripts in whole blood, capillary whole blood was extracted and then processed according to the method described herein. Briefly, capillary whole blood was preserved using a solution containing an aldehyde and a sugar-epichlorohydrin copolymer. The aldehyde reaction was quenched using a glycine solution. Blood was analyzed immediately after quenching (0 hours) and 4 hours later. After quenching, the whole blood was further processed and single-cell RNA sequencing was performed. Unique molecular identifiers (UMIs) were counted from all cell types to determine the ability to preserve the entire unique RNA transcript within a single cell (Figure 6). As can be seen from these data results, the number of UMIs was similar between whole blood analyzed after 4 hours of preservation and whole blood analyzed immediately afterward. These results suggest that this cell preservation protocol can preserve the entire cellular transcriptome for extended periods.

[0131] (Example 6: Storage of RNA transcripts over various periods) To test the ability to preserve RNA transcripts in whole blood over various periods, capillary whole blood was extracted and then processed according to the methods described herein. Briefly, two blood samples were collected simultaneously from two individuals. The sample from individual 1 was divided into two aliquots, and the sample from individual 2 was divided into three aliquots. All samples were fixed immediately after collection. Whole blood samples were preserved using a solution containing an aldehyde and a sugar-epichlorohydrin copolymer. The aldehyde reaction was quenched using a glycine solution. After quenching the reaction, aliquots from individual 1 were maintained at room temperature for approximately 0 hours (0-hour sample) and approximately 24 hours (24-hour sample). After quenching the reaction, aliquots from individual 2 were maintained at room temperature for approximately 0 hours (0-hour sample), approximately 24 hours (24-hour sample), and approximately 72 hours (72-hour sample).

[0132] After quenching and waiting for various periods, each aliquot was further processed and single-cell RNA sequencing was performed. PBMC isolation was performed on each aliquot. After isolation, each aliquot could be used immediately or transferred to -80°C storage. These samples could be recovered from -80°C storage. Sample barcoding was performed on them. The 0-hour and 24-hour samples were further divided into four aliquots each. Subsequently, all samples were subjected to 10x Genomics® Flex kit probe hybridization and single-cell sequencing protocols and processed in a 2-lane 10x Genomics® Chip Q.

[0133] The DNA library was sequenced. Using 10x Genomics® Cell Ranger software on the resulting FASTQ files, probe reads were mapped onto relevant genes, and the number of each gene for each cell and sample barcode was tallied. Cells in the resulting gene count matrix were grouped together by performing Leiden clustering in the latent space of a variational autoencoder (scVI) trained to encode the gene count matrix.

[0134] From this data, clusters were assigned to known cell types based on the relative expression of RNA of known cell type marker genes. From these cells assigned to cell types, the total number of UMIs (detected transcripts) could be calculated for each aliquot of each sample. Figure 7 shows the median UMI counts for various cell types derived from samples of subject 2. UMIs were counted from all cell types to determine the ability to preserve the entirety of unique RNA transcripts within a single cell. Figure 7 shows an initial decrease in UMI between 0 hours and 24 hours, but no significant decrease in UMI in the 72-hour samples compared to the 24-hour samples. This means that RNA degradation or other causes of decreased transcript detection stabilize for some time before 24 hours and remain stable between that point and 72 hours, suggesting that 72-hour fixed samples can certainly be equivalent to samples fixed for only 24 hours.

[0135] The relative abundance of gene numbers is investigated. Some embodiments provide a strong correlation between the cumulative relative abundance of all cells of a certain type between 24-hour and 72-hour samples. Figure 8 shows the gene abundance correlation between 24-hour and 72-hour samples for CD4 T cells. The correlation is approximately 0.97 between non-mitochondrial genes and mitochondrial genes. Figure 9 shows the gene abundance correlation between 24-hour and 72-hour samples for CD8 T cells. The correlation is approximately 0.97 between non-mitochondrial genes and mitochondrial genes. Figure 10 shows the gene abundance correlation between 24-hour and 72-hour samples for CD14 monocytes. The correlation is approximately 0.89 between non-mitochondrial genes and mitochondrial genes. Figure 11 shows the gene abundance correlation between 24-hour and 72-hour samples for CD16 monocytes. The correlation is approximately 0.91 between non-mitochondrial genes and mitochondrial genes. Figure 12 shows the correlation of gene abundance between 24-hour and 72-hour samples for NK cells. The correlation between non-mitochondrial genes and mitochondrial genes is approximately 0.96. Figure 13 shows the correlation of gene abundance between 24-hour and 72-hour samples for B cells. The correlation between non-mitochondrial genes and mitochondrial genes is approximately 0.96. Figures 8 to 13 show a strong correlation of gene abundance between 24-hour and 72-hour samples for all of these cell types. Therefore, 72-hour fixed samples are equivalent to 24-hour fixed samples.

[0136] The cell type abundances were further investigated. Subsequent experiments were conducted to replicate the fixation protocol described above with three samples, comparing 24-hour and 72-hour intervals. Cells were assigned to their individual clusters using the calculation method described above. It was confirmed that cell types maintained their relative abundances between 24-hour and 72-hour intervals for all involved samples. Figure 14 shows the correlation of cell percentages between 24-hour and 72-hour samples for CD4 T cells, CD8 T cells, NK cells, B cells, and monocytes.

[0137] (The doctrine of equality) As can be inferred from the above discussion, the above concepts can be implemented in various configurations of the embodiments of the present invention. Therefore, although the present invention has been described in a particular manner, many additional modifications and variations are obvious to those skilled in the art. Accordingly, it should be understood that the present invention can be implemented in ways different from those specifically described. Therefore, the embodiments of the present invention should be interpreted as illustrative rather than restrictive in all respects.

[0138] As used herein, the singular forms “a,” “an,” and “the” may refer to multiple objects unless otherwise specified by the context. A singular reference to an object is intended to mean “one or more,” and not “only” unless explicitly stated otherwise.

[0139] Where used herein, the terms “approximately” and “about” are used to describe and consider small variations. When used in conjunction with an event or situation, these terms may refer to the event or situation being exactly as it is, as well as being approximately as it is. When used in conjunction with a numerical value, these terms may refer to a range of variation of that value that is less than or equal to ±10% (for example, less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%).

[0140] Furthermore, quantities, ratios, and other numerical values ​​may sometimes be presented herein in range form. Such range forms are used for convenience and conciseness, and it should be understood flexibly that such range forms include not only numerical values ​​explicitly identified as limits of a range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly identified. For example, a ratio in the range of about 1 to about 200 should be understood to include not only the explicitly identified limits of about 1 and about 200, but also individual ratios such as about 2, about 3, and about 4, as well as subranges such as about 10 to about 50 and about 20 to about 100.

Claims

1. A step of obtaining a whole blood solution by contacting a whole blood sample with a solution containing a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group, wherein the ratio (v / v) of the solution to the whole blood sample is equal to or greater than 20; and The step of adding the quencher to the whole blood solution. A method for preserving whole blood, including...

2. The method according to claim 1, wherein the whole blood sample has a volume of 20 μL to 1 mL.

3. The method according to claim 1, wherein the whole blood sample is collected using a capillary blood collection procedure.

4. The method according to claim 1, wherein the molecular compound containing the aldehyde functional group is formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, or formalin.

5. The method according to claim 1, wherein the molecular compound containing the aldehyde functional group has a concentration in the range of 0.5% to 5.0% (% w / v).

6. The method according to claim 1, wherein the sugar-epichlorohydrin copolymer has a molecular weight of 60,000 Da to 500,000 Da.

7. The method according to claim 1, wherein the sugar-epichlorohydrin copolymer is a sucrose-epichlorohydrin copolymer.

8. The method according to claim 7, wherein the sucrose-epichlorohydrin copolymer is selected from the group consisting of polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxan-3,4,5-triol, Ficol®, Ficol® 70, and Ficol® 400.

9. The method according to claim 1, wherein the sugar-epichlorohydrin copolymer has a concentration of 2% to 20% (% w / v).

10. The method according to claim 1, wherein the solution is dissolved in a solution selected from the group consisting of distilled water, deionized water, phosphate buffer, phosphate buffer solution, physiological saline, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, and any combination thereof.

11. The method according to claim 1, wherein the contact step is carried out at a temperature of 20°C to 25°C.

12. The method according to claim 1, wherein the quenching step is performed 45 to 80 minutes after the contact step is performed.

13. The method according to claim 1, wherein the quencher comprises a molecular compound containing an amine functional group, dissolved in a solution containing a sucrose-epichlorohydrin copolymer.

14. The molecular compound containing an amine functional group is provided as a Tris buffer, a glycine buffer, or a combination thereof; the Tris buffer is Tris(hydroxymethyl)aminomethane, Trizma （登録商標） The method according to claim 13, comprising a Tris base selected from the group consisting of a base, amino-2-(hydroxymethyl)-1,3-propanediol, 2-,2-amino-2-(hydroxymethyl)-1,3-propanediol, THAM, tris(hydroxymethyl)aminomethane, and trometamol buffer.

15. The method according to claim 13, wherein the quencher has a molar concentration equivalent to or exceeding that of the molecular compound containing the aldehyde functional group.

16. The method according to claim 13, wherein the solution further comprises ethylenediaminetetraacetic acid.

17. The method according to claim 1, wherein the quencher is provided in a quenching solution in a ratio (v / v) equal to or greater than 2 with respect to the solution.

18. The method according to claim 1, wherein the quencher completes the quenching within a period of 60 to 2400 minutes.

19. A step to obtain a whole blood solution by contacting a whole blood sample with a solution containing a sugar-epichlorohydrin copolymer and a molecular compound containing an aldehyde functional group, wherein the ratio (v / v) of the fixation solution to the whole blood sample is equal to or greater than 20: Steps to add the quencher to the whole blood solution: A step of enriching nucleated cells from the whole blood solution; and The process of performing a single-cell assay on the enriched nucleated cells. A method for preserving whole blood for single-cell application, including...

20. The method according to claim 19, wherein the whole blood sample has a volume of 20 μL to 1 mL.

21. The method according to claim 19, wherein the whole blood sample is collected using a capillary blood collection procedure.

22. The method according to claim 19, wherein the molecular compound containing the aldehyde functional group is formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, or formalin.

23. The method according to claim 19, wherein the molecular compound containing the aldehyde functional group has a concentration in the range of 0.5% to 5.0% (% w / v).

24. The method according to claim 19, wherein the sugar-epichlorohydrin copolymer has a molecular weight of 60,000 Da to 500,000 Da.

25. The method according to claim 19, wherein the sugar-epichlorohydrin copolymer is a sucrose-epichlorohydrin copolymer.

26. The method according to claim 19, wherein the sucrose-epichlorohydrin copolymer is selected from the group consisting of polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxan-3,4,5-triol, Ficol®, Ficol® 70, and Ficol® 400.

27. The method according to claim 19, wherein the sugar-epichlorohydrin copolymer has a concentration of 2% to 20% (% w / v).

28. The method according to claim 19, wherein the solution is dissolved in a solution selected from the group consisting of distilled water, deionized water, phosphate buffer, phosphate buffer solution, physiological saline, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, and any combination thereof.

29. The method according to claim 19, wherein the contact step is carried out at a temperature of 20°C to 25°C.

30. The method according to claim 19, wherein the quenching step is performed 45 to 80 minutes after the contact step.

31. The method according to claim 19, wherein the quencher comprises a molecular compound containing an amine functional group, dissolved in a solution containing a sucrose-epichlorohydrin copolymer.

32. The molecular compound containing an amine functional group is provided as a Tris buffer, a glycine buffer, or a combination thereof; the Tris buffer is Tris(hydroxymethyl)aminomethane, Trizma （登録商標） The method according to claim 31, comprising a Tris base selected from the group consisting of a base, amino-2-(hydroxymethyl)-1,3-propanediol, 2-,2-amino-2-(hydroxymethyl)-1,3-propanediol, THAM, tris(hydroxymethyl)aminomethane, and trometamol buffer.

33. The method according to claim 31, wherein the quencher has a molar concentration equivalent to or exceeding that of the molecular compound containing the aldehyde functional group.

34. The method according to claim 31, wherein the solution further comprises ethylenediaminetetraacetic acid.

35. The method according to claim 19, wherein the quencher is provided in a quenching solution in a ratio (v / v) equal to or greater than 2 with respect to the solution.

36. The method according to claim 19, wherein the quencher completes the quenching within a period of 60 to 240 minutes.

37. The method according to claim 19, further comprising the step of storing the quenched whole blood solution at a temperature of 20°C to 25°C for at least 24 hours or at least 72 hours.

38. The method according to claim 19, wherein the nucleated cells include PBMCs.

39. The method according to claim 38, further comprising the step of isolating PBMCs from the quenched whole blood solution using a method selected from the group consisting of centrifugation, density gradient centrifugation, cell preparation tubes, PBMC isolation tubes, magnetic cell separation kits, immunoprecipitation, immunoseparation, and column purification.

40. The method according to claim 38, further comprising the step of storing the isolated PBMC in a freezer or using a cryogenic liquid.

41. The method according to claim 19, wherein the single-cell assay comprises single-cell RNA sequencing (scRNA-seq).

42. The method according to claim 41, wherein the scRNA-seq is executed on the 10X Genomics® scRNA-seq platform.

43. Dissolved in the solution, Sugar-epichlorohydrin copolymer and Molecular compounds containing aldehyde functional groups, A solution containing whole blood for preservation.

44. The solution according to claim 43, wherein the molecular compound containing an aldehyde functional group is formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, or formalin.

45. The solution according to claim 43, wherein the molecular compound containing the aldehyde functional group has a concentration in the range of 0.5% to 5.0% (% w / v).

46. The solution according to claim 43, wherein the sugar-epichlorohydrin copolymer has a molecular weight of 60,000 Da to 500,000 Da.

47. The solution according to claim 43, wherein the sugar-epichlorohydrin copolymer is a sucrose-epichlorohydrin copolymer.

48. The solution according to claim 47, wherein the sucrose-epichlorohydrin copolymer is selected from the group consisting of polysucrose, polysucrose 400, 2-(chloromethyl)oxirane; (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxan-3,4,5-triol, Ficol®, Ficol® 70, and Ficol® 400.

49. The solution according to claim 43, wherein the sugar-epichlorohydrin copolymer has a concentration of 2% to 20% (% w / v).

50. The solution according to claim 43, selected from the group consisting of distilled water, deionized water, phosphate buffer, phosphate buffer solution, physiological saline, HEPES buffer, MES buffer, MOPS buffer, PIPES buffer, potassium phosphate, sodium phosphate, TAPS, citrate-sodium citrate buffer, bicarbonate buffer, and any combination thereof.

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