Apparatus for high throughput assessment of cell properties, fragility or deformability

Abstract

The disclosure provides apparatus and methods related to the assessment of the fragility and / or deformability of cells, comprising applying negative pressure to an interior volume defined by a hollow member that comprises a fenestration, wherein the negative pressure is sufficient to(1 ) partially aspirate a cell into the interior volume of the hollow member through the fenestration at a time to, and (2) following to, rupture the cell at a time tr, resulting in the complete aspiration of the cell into the interior volume of the hollow member, and measuring a time to rupture of the cell (T) between tr and to. The apparatus and methods provided herein use small sample volumes and provide improved sensitivity and reproducibility over existing methods of assessing cell fragility and / or deformability. The apparatus and methods provided herein are useful for, e.g., evaluating the quality of stored blood to facilitate decisions regarding transfusion and diagnosing a disease, disorder, or condition.

Description

APPARATUS FOR HIGH THROUGHPUT ASSESSMENT OF CELL PROPERTIES, FRAGILITY OR DEFORMABILITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority under 35 U.S.C. § 119(e) to U.S. Provisional Application U.S.S.N. 63 / 575,472, filed April 5, 2024, the entire contents of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under HL 154150 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] The mechanical properties of cells are integral to their functions. Cell mechanics contribute to homeostasis and development, while aberrant mechanics contribute to various diseases.

[0004] Mechanical integrity in the face of stresses and strains is important for cells in tissue and cells undergoing migration and transport, while deformation and flexibility allow cells to adapt their shape based on conditions. For example, deformability and fragility of both RBCs and white blood cells (leukocytes) are relevant in view of the differences between the average size of human erythrocytes (~8 pm) and leukocytes (-10-25 pm) compared to the size of smallest capillaries (-4-5 pm) and splenic interendothelial slits (0.5-1.0 pm)

[0032] ,

[0005] Changes in the mechanical properties of cells are also implicated in disease progression. For example, leukocyte fragility occurs in chronic lymphocytic leukemia (CLL)

[0033] , As another example, blood cell disorders, including red blood cell (RBC) disorders, such as those that involve hemolysis, are often closely linked to the blood cell’s mechanical or osmotic fragility. A wide range of causes of hemolytic anemia have been documented, which include some genetic disorders (e.g., hereditary spherocytosis, sickle cell anemia, and G6PD deficiency), some parasites (e.g., Plasmodium), many Gram-positive bacteria (e.g., Streptococcus, Enterococcus, and Staphylococcus), some autoimmune disorders (e.g., drug- induced hemolytic anemia), or blood with too low a solute concentration (hypotonic to cells) [1], In addition, fragility of human blood platelets can be used as a biomarker

[0034] ,

[0006] Hemolysis during blood storage and / or after transfusion has been a major issue of concern in transfusion research [2-7], Within the FDA approved blood storage time of 42days, some studies show deteriorating RBC properties as RBCs age with longer storage days (storage lesion) and raise safety concerns of using long-storage RBCs in transfusion [3-7], Many studies show hemolysis of 5-10% of transfused RBCs within the first 24 hours [8], and the percentage of hemolyzed RBCs can increase with storage time to 25% or more [8, 9], Hemolysis of transfused RBCs can release cell-free hemoglobin in plasma, resulting in nitric oxide scavenging, vasoconstriction, and vascular injuries [2, 7],

[0007] Susceptibility of RBCs to hemolysis can be evaluated by testing RBCs’ mechanical fragility (MF) [10-13] or osmotic fragility (OF) [13, 14], but currently available assays have many limitations. For example, OF is used for diagnosing hereditary spherocytosis, but the current testing is difficult to standardize and not highly reproducible

[0014] , There is no standardized MF test yet, although MF tests using solid beads are used for calibration purposes

[0013] , There is also no single-cell based RBC fragility test available so far, and the existing methods for assessing fragility generally require large amounts (3-20 mL) of blood [10, 13], Accordingly, there is a need for methods of assessing cell fragility and / or deformability that improve sensitivity and reproducibility and require smaller sample volumes (e.g., of blood).SUMMARY

[0008] In one aspect, provided herein is an apparatus for assessing the fragility or deformability of a cell, comprising: a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member; a negative pressure means for applying negative pressure to the interior volume of the hollow member, wherein the negative pressure means, when operating, applies a negative pressure to the interior volume of the hollow member sufficient to (1) partially aspirate the cell into the interior volume of the hollow member through the fenestration at a time to; and (2) following to, rupture the cell at a time , resulting in the complete aspiration of the cell into the interior volume of the hollow member; a sensor for measuring the pressure of the interior volume of the hollow member; at least one processor; and a memory coupled to the at least one processor, wherein the memory comprises a plurality of instructions executable by the processor, and wherein the instructions comprise instructions for measuring a time to rupture of the cell (7) between and to.

[0009] In another aspect, provided herein is an apparatus for assessing the fragility or deformability of a cell, comprising: a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member; a negative pressure means for applying negative pressure to the interior volume of the hollow member, wherein the negative pressure means, when operating, applies a negative pressure to the interior volume of the hollow member sufficient to (1) partially aspirate the cell into the interior volume of the hollow member through the fenestration at a time to; and(2) following to, rupture the cell at a time , resulting in the complete aspiration of the cell into the interior volume of the hollow member; a sensor for measuring the pressure of the interior volume of the hollow member; and at least one processor operatively connected to a memory, the processor when executing configured to: measure a time to rupture of the cell (7) between and to.

[0010] In another aspect, provided herein is a method of assessing the fragility or deformability of a cell, the method comprising:(1) applying a negative pressure to a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member;(2) partially aspirating the cell into the interior volume of the hollow member through the fenestration at a time to,'(3) rupturing the cell and completely aspirating the cell into the interior volume of the hollow member at a time tr following to; and(4) measuring the time to rupture of the cell (7) between and to.

[0011] In another aspect, provided herein is a method of transfusing blood into a patient in need thereof, the method comprising:(1) providing a population of exogenous blood cells;(2) assessing the fragility or deformability of a plurality of the population of exogenous blood cells according to a method provided herein, and(3) transfusing at least a portion of the remaining population of exogenous blood cells to the patient if the average, median, or mode time to rupture of the plurality of the population of exogenous blood cells is similar to or greater than the average, median, ormode time to rupture for a population of blood cells determined to have sufficient fragility or deformability properties for transfusion to the patient.

[0012] In another aspect, provided herein is a method of transfusing blood into a patient in need thereof, the method comprising:(1) providing a candidate population of exogenous blood cells;(2) assessing the fragility or deformability of a plurality of the candidate population of exogenous blood cells according to a method provided herein,(3) providing a control population of fresh blood cells;(4) assessing the fragility or deformability of a plurality of the population of fresh blood cells according to a method provided herein,(5) calculating the difference in average, median, or mode time to rupture of the plurality of the candidate population from the average, median, or mode time to rupture of the plurality of fresh blood cells, and(6) transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than one standard deviation.

[0013] The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims. It should be understood that the aspects described herein are not limited to specific embodiments, methods, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the present disclosure and together with the description, provide nonlimiting examples of the disclosure. The figures are exemplary and do not limit the scope of the present disclosure.

[0015] FIGs. 1 A-1C show illustrations and applications of an exemplary Fragility Test Assay disclosed herein. FIG. 1 A shows a schematic of an exemplary experimental setup with computer controlled negative pressure pulling RBCs into the micropipette. FIG. IB shows an illustration of the RBC rupture process, where a red blood cell is partially aspirated into a glass micropipette using a controlled constant negative pressure and stopped at the entrance of the micropipette at time to. The time to rupture, Z, is defined as the duration from themoment the RBC is initially aspirated (to) to the time of its rupture (tr), where T= tr- to. Post-rupture, the RBC is completely drawn into the micropipette. FIGs. 1C-1F show sequential still images of a healthy RBC being partially aspirated into a micropipette and ruptured / lysed during an exemplary fragility test under constant negative pressure at the tip of a 1.5 pm micropipette: 5827 ms (FIG. 1C), 5835 ms (FIG. ID), 5871 ms (FIG. IE), and 5879 ms (FIG. IF). While FIGs. 1 A-1C depict RBCs, the methods and apparatus may be applied to other cell types, such as leukocytes, white blood cells, and platelets. FIGs. 1A-1B depict a micropipette, but other hollow members may be used.

[0016] FIG. 2 illustrates changes in RBC fragility when exposed to two drugs, Phenylhydrazine (PHZ) and Diamide as measured by an exemplary apparatus and method disclosed herein, each of which lead to a decrease in fragility. RBCs studied in FIG. 2 were tested in PBS with 1% BSA.

[0017] FIGs. 3A-3B show box plot analyses of time to rupture for populations of RBCs at varying storage times under 375 mbar (FIG. 3A) and 435 mbar (FIG. 3B) pressure. Statistically significant reductions in the time to rupture are observed and marked with asterisks. Significance levels between two time points are indicated by the number of stars, with no star signifying non-significance, one star for p < 0.05, and five stars for p < 0.00001. All RBCs studied in FIGs. 3 A-3B were tested in SAGM medium.

[0018] FIGs. 4A-4B show RBC fragility test results on Day 6 (FIG. 4A) and Day 20 (FIG. 4B) after blood draw on Day 0. The results show an exponential decay of the average time to rupture for RBCs versus the increasing aspiration pressure. Comparing the results between Day 20 with Day 6, significantly decreased time to rupture under the same reduced pressure (i.e., increased fragility) can be observed with longer storage time. Error bars are standard deviations. Significance levels at each time point are indicated by the number of stars, with one star signifying for p < 0.05, two stars for p < 0.01, two stars for p < 0.001, four stars for p < 0.0001, and five stars for p < 0.00001. All RBCs studied in this FIGs. 4A-4B were tested in SAGM medium.

[0019] FIG. 5 shows a chart of shear modulus in red blood cells over a 30-day storage period, using the same RBC sample set as in FIGs. 3A-3B and FIGs. 4A-4B. Shear modulus measurements were performed employing the conventional micropipette aspiration technique described in Robert, M. H., Journal of Biomechanics 33, 15-22, 2000, the contents of which are hereby incorporated by reference in their entirety. The results show a 20% increase in shear modulus within the first 9 days, starting from the baseline established on day 3. Error bars represent standard deviation. All RBCs studied in FIG. 5 were tested in SAGM medium.

[0020] FIG. 6 is a block diagram of an example special purpose computer system improved by the functions and / or processes disclosed herein.

[0021] FIGs. 7A and 7B show single-cell RBC fragility tests comparing blood samples from healthy controls with diabetic blood samples. FIG. 7A shows comparison test results conducted under a suction pressure of 40 mbar and using a micropipette tip radius of 1.2 pm. Error bars represent standard deviation. FIG. 7B shows a summary of RBC fragility test results comparing 3 diabetic patient samples and 3 healthy controls using a micropipette tip radius of 1.4-1.8 pm across multiple suction pressures between 30 and 250 mbar.DEFINITIONS

[0022] The term “blood” refers to whole blood or one or more components prepared from blood (e.g., packed red blood cells). In some embodiments, the term “blood” refers to whole blood. In some embodiments, the term “blood” refers to packed red blood cells.

[0023] The term “white blood cell” refers to leukocytes. Specific populations of white blood cells include, for example, all white blood cells, monocytes, neutrophils, lymphocytes, eosinophils, basophils, or any subgroup thereof or any combination thereof.

[0024] The term “platelets” refers to thrombocytes or non-nucleated blood cells.

[0025] The term “red blood cell” refers to erythrocytes that contain hemoglobin and deliver oxygen to body tissues. In some embodiments, the red blood cell is a human red blood cell. Red blood cells are shaped as a biconcave discs having a diameter of about 6-10 pm and a thickness of about 0.8-2.5 pm. In some embodiments, the red blood cell has an average volume of about 90 fL. In some embodiments, the red blood cell has a surface area of about 136 pm2. The red blood cell comprises a three-layer membrane comprising a glycocalyx, a lipid bilayer, and a membrane skeleton. The membrane is flexible to allow the cell to travel through capillaries. A red blood cell with a stable membrane can circulate without damage, whereas a red blood cell with a fragile membrane is more likely to suffer hemolysis or plug capillaries in vivo.

[0026] The term “blood transfusion” or “transfusion” refers to the intravenous infusion of whole blood or one or more components prepared from blood (e.g., packed red blood cells) to a subject. In some embodiments, the term “blood transfusion” or “transfusion” refers to the transfer of blood or one or more components prepared from blood from a donor to a recipient. In some embodiments, the term “blood transfusion” or “transfusion” refers to the transfer of blood or one or more components prepared from blood from a subject to the subject at a later time. In some embodiments, transfused blood is screened for bloodborne diseases, includingbut not limited to one or more of Hepatitis B, Hepatitis C, HIV, syphilis, Chagas disease, and malaria. In some embodiments, the blood type of the transfused blood is identified. In some embodiments, the blood type of the transfused blood is identified and matched to that of the recipient’s blood. In some embodiments, the transfused blood is stored following collection from a donor. In some embodiments, the transfused blood is subjected to processing after it is collected. In some embodiments, the transfused blood has been collected and separated into components by centrifugation (e.g., red blood cells, plasma, platelets, albumin protein, clotting factor concentrates, cryoprecipitate, fibrinogen concentrate, and / or immunoglobulins).

[0027] The term “deformability” refers to a red blood cell’s propensity to deform (e.g., stretch) in response to an applied stress. Reduced deformability in RBC affects posttransfusion survival time in the bloodstream. Reduced deformability in RBC affects red blood cells’ ability to traverse the capillary network. More rigid red blood cells can significantly alter pulmonary hemodynamics, resulting in increased vascular resistance and may disappear from circulation more rapidly.

[0028] The term “fragility” refers to a red blood cell’s propensity for hemolysis under an applied stress. In some embodiments, the stress applied to test fragility is higher intensity than that applied to test deformability.

[0029] The terms “rupture” or “cell rupture” refer to breaking the cell wall and / or membrane. In some embodiments, the terms “rupture” or “lysis” are used interchangeably.

[0030] The phrase “following to, rupture the cell at a time resulting in the complete aspiration of the cell into the interior volume of the hollow member” and the phrase “rupture the cell and completely aspirate the cell into the interior volume of the hollow member at a time tr following to” are used interchangeably. As used herein, refers to the time of rupture of the cell.

[0031] The term “fenestration” refers to an opening, such as an opening or penetration hole to a hollow member.

[0032] The term “hollow member” refers to an element with a hollow interior. In some embodiments, a hollow member is a tubular element with a hollow interior. In some embodiments, a hollow member is a channel (e.g., a microfluidic channel).

[0033] The term “negative pressure” refers to a pressure below ambient atmospheric pressure.

[0034] A “subject” refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-agedadult, or senior adult)) or non-human animal. The term “patient” refers to a human subject in need of treatment of a disease or medical intervention.

[0035] The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In some embodiments, the biological sample is blood.

[0036] A “hematological disease” includes a disease which affects a hematopoietic cell or tissue. Hematological diseases include diseases associated with aberrant hematological content and / or function. Examples of hematological diseases include diseases resulting from bone marrow irradiation or chemotherapy treatments for cancer, diseases such as pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HTV, hepatitis virus or other viruses, myelophthisic anemias caused by marrow deficiencies, renal failure resulting from anemia, anemia, polycythemia, infectious mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, Wilm’s tumor, Ewing’s sarcoma, retinoblastoma, hemophilia, disorders associated with an increased risk of thrombosis, herpes, thalassemia, antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical trauma to red blood cells such as micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, infections by parasites such as Plasmodium, chemical injuries from, e.g., lead poisoning, and hypersplenism.

[0037] An “autoimmune disorder” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g, Goodpasture’s disease which may affectthe basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g, medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture’s syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, , psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g, Wegener’s granulomatosis, microscopic polyangiitis), uveitis, Sjogren’s syndrome, Crohn’s disease, Reiter’s syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, and cardiomyopathy.

[0038] A “diabetic condition” refers to diabetes and pre-diabetes. “Diabetes” refers to a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger). There are several types of diabetes. Type I diabetes results from the body’s failure to produce insulin, and presently requires the person to inject insulin or wear an insulin pump. Type II diabetes results from insulin resistance a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. Gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes develop a high blood glucose level. Other forms of diabetes include congenital diabetes, which is due to genetic defects of insulin secretion, cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of monogenic diabetes, e.g., mature onset diabetes of the young (e.g., MODY 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Pre-diabetes indicates a condition that occurs when a person’s blood glucose levels are higher than normal but not high enough for a diagnosis of diabetes. All forms of diabetes increase the risk of long-term complications. These typically develop after many years, but may be the first symptom in those who have otherwise not received a diagnosis before that time. The major long-term complications relate to damage to blood vessels. Diabetes doubles the risk of cardiovascular disease and macrovascular diseases such as ischemic heart disease (angina, myocardial infarction), stroke, and peripheral vascular disease. Diabetes also causes microvascular complications, e.g., damage to the small blood vessels. Diabetic retinopathy, which affects blood vessel formation in the retina of the eye, can lead to visual symptoms, reduced vision, and potentially blindness. Diabetic nephropathy, the impact of diabetes on the kidneys, can leadto scarring changes in the kidney tissue, loss of small or progressively larger amounts of protein in the urine, and eventually chronic kidney disease requiring dialysis. Diabetic neuropathy is the impact of diabetes on the nervous system, most commonly causing numbness, tingling and pain in the feet and also increasing the risk of skin damage due to altered sensation. Together with vascular disease in the legs, neuropathy contributes to the risk of diabetes-related foot problems, e.g., diabetic foot ulcers, that can be difficult to treat and occasionally require amputation.

[0039] When a range of values is listed, it encompasses each value and sub-range within the range. A range is inclusive of the values at the two ends of the range unless otherwise provided.

[0040] Other than in the examples, or where otherwise indicated, all numbers expressing quantities used herein should be understood as modified in all instances by the term “about.” “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, or more typically, within 5%, 4%, 3%, 2%, or 1% of a given value or range of values.

[0041] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0042] The present disclosure describes an apparatus and method for quantifying the mechanical properties e.g., fragility and / or deformability) of one or more cells. The described apparatus and methods provide new modalities for assessing important properties of cells that have potential impact on a variety of therapeutic and diagnostic applications.

[0043] The methods and apparatus presented herein provide several advantages over other methodologies for assessing the mechanical properties of cells. First, in certain embodiments, the method is a single-cell assay. To the Applicant’s knowledge, no reliable, single-cell fragility test is publicly available. Second, in certain embodiments, the method is high-throughput, allowing for testing of at least 1-10 cells per second. Third, in certain embodiments, the methods and apparatus provided herein require minimal sample volume. The methods and apparatus provided herein may be performed using only a single drop of sample, which is much less than, for example, the 3-20 mL of sample required for conventional fragility tests.

[0044] In the context of analyzing blood cells, the apparatus and method are useful in a clinical setting to provide clinicians and physicians with data on blood cell quality when making decisions regarding blood storage and transfusion (e.g., duration of storage, whether to give a transfusion, which units to use for transfusion, how many units to give via transfusion).

[0045] The methods and apparatus provided herein have been utilized to quantify a previously unidentified increased fragility within the first week of blood storage due to their improved sensitivity. This is consistent with anecdotal evidence amongst doctors that fresher stored blood lowers the danger to those patients who are severely ill and need transfusion. The disclosed apparatus and methods accordingly provide for a substantial patient benefit, particularly for severely ill patients in need of high-quality blood transfusions.

[0046] The apparatus and methods are further useful for capturing patient-specific changes in blood cell fragility. In certain embodiments, the methods and apparatus provided herein are capable of capturing patient-specific changes in blood cell fragility (e.g., RBC fragility) in diabetic patients.

[0047] In one aspect, provided herein is an apparatus for assessing the fragility or deformability of a cell, comprising: a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member; a negative pressure means for applying negative pressure to the interior volume of the hollow member, wherein the negative pressure means, when operating, applies a negative pressure to the interior volume of the hollow member sufficient to (1) partially aspirate the cell into the interior volume of the hollow member through the fenestration at a time to; and (2) following to, rupture the cell at a time , resulting in the complete aspiration of the cell into the interior volume of the hollow member; a sensor for measuring the pressure of the interior volume of the hollow member; at least one processor; and a memory coupled to the at least one processor, wherein the memory comprises a plurality of instructions executable by the processor, and wherein the instructions comprise instructions for measuring a time to rupture of the cell (7) between and to.

[0048] In another aspect, provided herein is an apparatus for assessing the fragility or deformability of a cell, comprising:a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member; a negative pressure means for applying negative pressure to the interior volume of the hollow member, wherein the negative pressure means, when operating, applies a negative pressure to the interior volume of the hollow member sufficient to (1) partially aspirate the cell into the interior volume of the hollow member through the fenestration at a time to; and(2) following to, rupture the cell at a time , resulting in the complete aspiration of the cell into the interior volume of the hollow member; a sensor for measuring the pressure of the interior volume of the hollow member; and at least one processor operatively connected to a memory, the processor when executing configured to: measure a time to rupture of the cell (7) between and to.

[0049] In another aspect, provided herein is a method of assessing the fragility or deformability of a cell, the method comprising:(1) applying a negative pressure to a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member;(2) partially aspirating the cell into the interior volume of the hollow member through the fenestration at a time to,'(3) rupturing the cell and completely aspirating the cell into the interior volume of the hollow member at a time tr following to; and(4) measuring the time to rupture of the cell (7) between and to.

[0050] In another aspect, provided herein is a method of transfusing blood into a patient in need thereof, the method comprising:(1) providing a population of exogenous blood cells;(2) assessing the fragility or deformability of a plurality of the population of exogenous blood cells according to a method provided herein, and(3) transfusing at least a portion of the remaining population of exogenous blood cells to the patient if the average, median, or mode time to rupture of the plurality of the population of exogenous blood cells is similar to or greater than the average, median, or mode time to rupture for a population of blood cells determined to have sufficient fragility or deformability properties for transfusion to the patient.

[0051] In another aspect, provided herein is a method of transfusing blood into a patient in need thereof, the method comprising:(1) providing a candidate population of exogenous blood cells;(2) assessing the fragility or deformability of a plurality of the candidate population of exogenous blood cells according to a method provided herein,(3) providing a control population of fresh blood cells;(4) assessing the fragility or deformability of a plurality of the population of fresh blood cells according to a method provided herein,(5) calculating the difference in average, median, or mode time to rupture of the plurality of the candidate population from the average, median, or mode time to rupture of the plurality of fresh blood cells, and(6) transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than one standard deviation.

[0052] In some embodiments, the cell is a blood cell. In some embodiments, the cell is a red blood cell, a white blood cell, or a platelet. In some embodiments, the cell is a red blood cell. In some embodiments, the cell is a white blood cell. In some embodiments, the cell is a platelet. In some embodiments, the blood cell is a red blood cell, white blood cell, or platelet.

[0053] In some embodiments, the at least one processor is configured to control operation of the negative pressure means based on communicated control signals. In some embodiments, the at least one processor is configured to communicate control signals to the negative pressure means to generate the negative pressure. In some embodiments, the at least one processor is configured to communicate control signals to the negative pressure means to adjust the negative pressure. In some embodiments, the at least one processor is configured to communicate control signals to the negative pressure means to release the negative pressure. In some embodiments, the at least one processor is configured to determine the time at which the rupture of the cell occurs ( / ,). In some embodiments, the at least one processor is configured to determine the time at which the rupture of the cell occurs based on image analysis of captured visual data. In some embodiments, the at least one processor is configured to control capture of the visual data via an imaging means. In some embodiments, the at least one processor is configured to determine the time at which the rupture of the cell occurs based presence or change of an electrical signal. In some embodiments, the electrical signal is impedance or resistance.

[0054] In some embodiments, the hollow member comprises a micropipette, a microfluidic channel, or a microtube. In some embodiments, the micropipette is glass, metal, or plastic. In some embodiments, the micropipette is transparent.

[0055] In some embodiments, the hollow member comprises a channel having a largest interior width of about 0.5 microns to about 3.0 microns. In some embodiments, the hollow member comprises a channel having a largest interior width of about 1.5 microns to about 2.0 microns.

[0056] In some embodiments, the negative pressure means comprises a vacuum pump. In some embodiments, the negative pressure means comprises a syringe pump. In some embodiments, the negative pressure means comprises a feedback pressure sensor to adjust the negative pressure.

[0057] In some embodiments, the measured time to rupture of the cell (7) correlates to the membrane fragility or deformability of the cell.

[0058] In some embodiments, the cell is a member of a population of cells, and the measured time to rupture of the cell (7) correlates to the membrane fragility or deformability of the remaining population of cells. In some embodiments, the apparatus is configured to assess the fragility or deformability a plurality of cells. In some embodiments, the instructions further comprise instructions for calculating an average, median, or mode time to rupture from multiple T measurements determined for a plurality of cells. In some embodiments, the cell is a member of a population of cells, and the average, median, or mode time to rupture from multiple T measurements correlates to the membrane fragility or deformability of the remaining population of cells.

[0059] In some embodiments, steps (1) through step (4) of the method are performed using an apparatus provided herein

[0060] In some embodiments, the method further comprises a preliminary step of isolating the cell from whole blood. In some embodiments, the isolating comprises centrifuging whole blood, removing the buffy coat, and pelleting the cell. In some embodiments, the method further comprises washing the pelleted cell to obtain a packed cell. In some embodiments, the method further comprises incubating the packed cell.

[0061] In some embodiments, the cells are incubated with an additional agent. In some embodiments, the agent is a blood storage medium. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a toxin, an oxidant, or a pollutant.

[0062] In some embodiments, the method comprises assessing the fragility or deformability a plurality of cells. In some embodiments, the method comprises assessing the fragility ordeformability of at least 1 cell per second. In some embodiments, the method comprises assessing the fragility or deformability of at least 2 cells per second. In some embodiments, the method comprises assessing the fragility or deformability of at least 5 cells per second. In some embodiments, the method comprises assessing the fragility or deformability of at least 10 cells per second.

[0063] In some embodiments, the method comprising calculating an average, median, or mode time to rupture from multiple T measurements determined for a plurality of cells. In some embodiments, the method comprises assessing the fragility or deformability of a population of cells.

[0064] In some embodiments, the population of cells is derived from about 1 drop of blood. In some embodiments, the population of cells is derived from about 0.5 pL to about 1 pL of blood.

[0065] In some embodiments, the control population of fresh blood cells was obtained from a subject within 42 days. In some embodiments, the control population of fresh blood cells was obtained from a subject within 7 days. In some embodiments, the control population of fresh blood cells was obtained from a subject within 1 day. In some embodiments, the control population of fresh blood cells was obtained from a subject within 8 hours. In some embodiments, the control population of fresh blood cells was obtained from a subject within 2 hours.

[0066] In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than three standard deviations. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than two standard deviations. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than one standard deviation. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than half of a standard deviation. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than a quarter of a standard deviation. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than an eighth of a standard deviation. In some embodiments, the method comprises transfusing at least a portion of the remainingcandidate population of exogenous blood cells to the patient if the difference is not statistically significant.

[0067] In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 75%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 60%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 50%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 40%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 30%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 25%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 20%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 15%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 10%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 9%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 8%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 7%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 6%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 5%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 4%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference isless than 3%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 2%. In some embodiments, the method comprises transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than 1%.

[0068] In certain embodiments, the cell is present in vitro. In certain embodiments, the cell is present ex vivo.

[0069] In some embodiments, the fenestration has a largest interior dimension that is dimensioned to partially aspirate a target cell into the hollow member, and to completely aspirate the cell only after its rupture.

[0070] In some embodiments, the fenestration has a largest interior dimension of about 5 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 4 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 3.5 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 3.0 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 2.5 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 2.4 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 2.3 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 2.2 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 2.1 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 2.0 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 1.9 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 1.8 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 1.7 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 1.6 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 1.5 microns or less. In some embodiments, the fenestration has a largest interior dimension of about 1.0 microns or less.

[0071] In some embodiments, the fenestration has a largest interior dimension of at least 0.5 microns. In some embodiments, the fenestration has a largest interior dimension of at least 0.6 microns. In some embodiments, the fenestration has a largest interior dimension of at least 0.7 microns. In some embodiments, the fenestration has a largest interior dimension of at least 0.8 microns. In some embodiments, the fenestration has a largest interiordimension of at least 0.9 microns. In some embodiments, the fenestration has a largest interior dimension of at least 1.0 microns. In some embodiments, the fenestration has a largest interior dimension of at least 1.1 microns. In some embodiments, the fenestration has a largest interior dimension of at least 1.2 microns. In some embodiments, the fenestration has a largest interior dimension of at least 1.3 microns. In some embodiments, the fenestration has a largest interior dimension of at least 1.4 microns. In some embodiments, the fenestration has a largest interior dimension of at least 1.5 microns.

[0072] In some embodiments, the fenestration has a largest interior dimension of about 0.1 microns to about 3.5 microns. In some embodiments, the fenestration has a largest interior dimension of about 0.5 microns to about 3.0 microns. In some embodiments, the fenestration has a largest interior dimension of about 0.5 microns to about 1.0 microns. In some embodiments, the fenestration has a largest interior dimension of about 1.0 microns to about 1.5 microns. In some embodiments, the fenestration has a largest interior dimension of about 1.5 microns to about 2.0 microns. In some embodiments, the fenestration has a largest interior dimension of about 2.0 microns to about 2.5 microns. In some embodiments, the fenestration has a largest interior dimension of about 2.5 microns to about 3.0 microns.

[0073] In some embodiments, the hollow member comprises a channel that is dimensioned to partially aspirate a target cell into the hollow member, and to completely aspirate the cell only after its rupture. In some embodiments, the hollow member comprises a channel that is dimensioned to partially aspirate a target cell into the hollow member, and to completely aspirate the cell only after damage to the cell membrane. In some embodiments, the hollow member comprises a channel that is dimensioned to partially aspirate a target cell into the hollow member, and to completely aspirate the cell only after deformation to the cell. In some embodiments, the hollow member is mechanically robust. In some embodiments, the hollow member can withstand high pressure. In some embodiments, the hollow member is a microfluidic channel or microtube.

[0074] In some embodiments, the hollow member comprises a channel having a largest interior width of about 5 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 4 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 3.5 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 3.0 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 2.5 microns or less. In some embodiments, the hollow member comprises a channel having a largest interiorwidth of about 2.4 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 2.3 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 2.2 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 2.1 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 2.0 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 1.9 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 1.8 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 1.7 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 1.6 microns or less. In some embodiments, the hollow member comprises a channel having a largest interior width of about 1.5 microns or less.

[0075] In some embodiments, the sensor for measuring the pressure of the interior volume of the hollow member is capacitive, inductive, reluctive, piezoelectric, strain gauge, vibrating element, or potentiometric.

[0076] In some embodiments, the negative pressure means comprises a pump or withdrawal configured to reduce the pressure of the interior volume of the hollow member.

[0077] In some embodiments, the negative pressure is about 1 mbar to about 1,000 mbar. In some embodiments, the negative pressure is about 10 mbar to about 1,000 mbar. In some embodiments, the negative pressure is about 50 mbar to about 1,000 mbar. In some embodiments, the negative pressure is about 100 mbar to about 1,000 mbar. In some embodiments, the negative pressure is about 250 mbar to about 1,000 mbar. In some embodiments, the negative pressure is about 1 mbar to about 750 mbar. In some embodiments, the!9rocessl9e pressure is about 1 mbar to about 500 mbar. In some embodiments, the negative pressure is about 50 mbar to about 750 mbar. In some embodiments, the negative pressure is about 50 mbar to about 550 mbar. In some embodiments, the negative pressure is about 50 mbar to about 250 mbar. In some embodiments, the negative pressure is about 200 mbar to about 650 mbar. In some embodiments, the negative pressure is about 250 mbar to about 550 mbar. In some embodiments, the negative pressure is about 300 mbar to about 500 mbar. In some embodiments, the negative pressure is about 350 mbar to about 450 mbar. In some embodiments, the negative pressure is about 375 mbar to about 435 mbar.

[0078] In some embodiments, the negative pressure is constant. In some embodiments, the negative pressure is approximately constant. In some embodiments, the negative pressure varies by less than 20% throughout the duration of its application. In some embodiments, the negative pressure varies by less than 15% throughout the duration of its application. In some embodiments, the negative pressure varies by less than 10% throughout the duration of its application. In some embodiments, the negative pressure varies by less than 5% throughout the duration of its application. In some embodiments, the negative pressure varies by less than 2% throughout the duration of its application. In some embodiments, the negative pressure varies by less than 1% throughout the duration of its application. In some embodiments, the negative pressure is varied. In some embodiments, the negative pressure is increased over a measurement period. In some embodiments, the negative pressure is decreased over a measurement period. In some embodiments, the negative pressure is released.

[0079] In some embodiments, the apparatus is configured to assess the fragility or deformability of at least 1 cell per second. In some embodiments, the apparatus is configured to assess the fragility or deformability of at least 2 cells per second. In some embodiments, the apparatus is configured to assess the fragility or deformability of at least 5 cells per second. In some embodiments, the apparatus is configured to assess the fragility or deformability of at least 10 cells per second.

[0080] In some embodiments, the apparatus is configured to assess the fragility or deformability of up to 20 cells per second. In some embodiments, the apparatus is configured to assess the fragility or deformability of up to 15 cells per second. In some embodiments, the apparatus is configured to assess the fragility or deformability of up to 10 cells per second.

[0081] In some embodiments, the apparatus further comprises a means for counting cells. In some embodiments, counting is performed by visually counting observed cells. In some embodiments, counting is performed by automated technologies. In some embodiments, the counting is performed using a high-speed camera. In some embodiments, the counting is performed using light microscopy. In some embodiments, the counting is performed using light microscopy fluorescence. In some embodiments, the counting is performed using an image analysis algorithm. In some embodiments, the image analysis algorithm uses artificial intelligence or machine learning. In some embodiments, the counting is performed using electrical impedance or resistance or changes thereof. In some embodiments, the cells are labeled (e.g., with a fluorescent tag or radio-label) to facilitate counting.

[0082] In some embodiments, the instructions comprise at least one mathematical process selected from a group consisting of mathematical, statistical, and numerical processes. In some embodiments, the instructions comprise generating one or more numerical values representing red blood cell suitability for transfusion.

[0083] Additionally, an illustrative implementation of a special purpose computer system 100 that may be specially programmed to be used in connection with any of the embodiments of the disclosure provided herein is shown in FIG. 6. The computer system 100 may include one or more processors 110 and one or more articles of manufacture that comprise non- transitory computer-readable storage media (e.g., memory 120 and one or more non-volatile storage media 130). The processor 110 may control writing data to and reading data from the memory 120 and the non-volatile storage device 130 in any suitable manner, as the aspects of the apparatus described herein are not limited in this respect.

[0084] To perform any of the functionality described herein (e.g., generate system control signals for respective system components (e.g., negative pressure means, imaging means (e.g., camera, high speed camera, digital camera, video camera, etc.)), perform analysis (e.g., image analysis, image segmentation, object identification, object boundary identification, comparison of objects, comparison of object boundaries, comparison of object segmentation, etc.), generating timing information, generate time measurements, etc.), among other options), the processsor 110 may execute one or more processor-executable instructions stored in one or more non-transitory computer-readable storage media (e.g., the memory 120), which may serve as non-transitory computer-readable storage media storing processorexecutable instructions for execution by the processor 110.

[0085] In some embodiments, the apparatus further comprises any other processor, controller or control unit needed to route data, perform computations, perform I / O functionality, etc. In some embodiments, the apparatus further comprises any number and type of input functionality to receive data and / or may include any number and type of output functionality to provide data, and may include control apparatus to perform I / O functionality.

[0086] The terms “program” or “software” or “app” are used herein in a generic sense to refer to any type of computer code or set of processor-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the disclosure provided herein need not reside on a single computer or processor, but may bedistributed in a modular fashion among different computers or processors to implement various aspects of the disclosure provided herein.

[0087] Processor-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0088] Also, data structures may be stored in one or more non-transitory computer-readable storage media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a non-transitory computer-readable medium that convey relationships between the fields. However, any suitable mechanism may be used to establish relationships among information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationships among data elements.

[0089] Also, various inventive concepts may be embodied as one or more processes, of which examples have been provided. The acts performed as part of each process may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0090] In some embodiments, an imaging means is a device capable of capturing visual data. In some embodiments, an imaging means is a device capable of capturing and / or relaying a still or moving image. In some embodiments, the imaging means is a camera. In some embodiments, the imaging means is a high-speed camera. In some embodiments, the imaging means is a digital camera. In some embodiments, the imaging means is a video camera. In some embodiments, the imaging means is coupled to a microscope (e.g., light microscope, fluorescence microscope).

[0091] In certain embodiments, the patient is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the patient described herein is a human. In some embodiments, the patient is an adult human. In some embodiments, the patient is an adult human of 18 years of age to 65 years of age. In some embodiments, the patient is a healthy adult human. In some embodiments, the patient has a disease, disorder, or condition. In some embodiments, the patient has been diagnosed with or is at risk of a disease, disorder, or condition. In some embodiments, the patient is a patient ina medical procedure. In some embodiments, the medical procedure is a blood transfusion, a surgical procedure, or a dental procedure. In some embodiments, the patient has been diagnosed with or is at risk of a hematological disease. In some embodiments, the patient has been diagnosed with or is at risk of a bleeding disorder. In some embodiments, the patient has been diagnosed with or is at risk of an illness that causes reduced or poor-quality RBCs. In some embodiments, the patient has been diagnosed with or is at risk of an illness that causes reduced or poor-quality leukocytes. In some embodiments, the patient has been diagnosed with or is at risk of kidney failure. In some embodiments, the patient has been diagnosed with or is at risk of anemia, leukemia, or kidney disease. In some embodiments, the patient has been diagnosed with or is at risk of leukemia. In some embodiments, the patient has experienced blood loss. In some embodiments, the patient has experienced blood loss due to injury. In some embodiments, the patient has experienced blood loss due to childbirth. In some embodiments, the patient has experienced blood loss due to surgery. In some embodiments, the patient is undergoing or has undergone chemotherapy. In some embodiments, the patient has been diagnosed with or is at risk of diabetes or pre-diabetes. In some embodiments, the diabetes is Type I diabetes, Type II diabetes, gestational diabetes, congenital diabetes, cystic fibrosis-related diabetes, steroid diabetes, or monogenic diabetes. In some embodiments, the diabetes is Type I diabetes. In some embodiments, the diabetes is Type II diabetes. In some embodiments, the diabetes is gestational diabetes.

[0092] In certain embodiments, the patient is a non-human animal. In certain embodiments, the patient is a mammal. In certain embodiments, the patient is a non-human mammal. In certain embodiments, the patient is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the patient is a companion animal, such as a dog or cat. In certain embodiments, the patient is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the patient is a zoo animal. In another embodiment, the patient is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the patient is a fish or reptile.

[0093] In some embodiments, the additional agent is a therapeutic agent. In some embodiments, the therapeutic agent is a pharmaceutical agent. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations(CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing a disease. Pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti-inflammatory agents, immunosuppressants, antibacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, antidiabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti-pyretics, hormones, and prostaglandins. In some embodiments, the agent is diamide. In some embodiments, the agent is phenylhydrazine. In some embodiments, the agent is penicillin. In some embodiments, the agent is a blood thinner or anti-coagulant. In some embodiments, the agent is apixaban, dabigatran, dalteparin, edoxaban, enoxaparin, fondaparinux, heparin, rivaroxaban, or warfarin. In some embodiments, the agent is aspirin, cilostazol, clopidogrel, dipyridamole, eptifibatide, prasugrel, ticagrelor, tirofiban, or vorapaxar.

[0094] In some embodiments, the agent is an oxidant. In some embodiments, the agent is a reactive oxygen species. In some embodiments, the reagent is a toxin. In some embodiments, the toxin is a hemolytic toxin.

[0095] In some embodiments, the agent is a pollutant. In some embodiments, the agent is an environmental pollutant. In some embodiments, the agent is an air pollutant. In some embodiments, the pollutant is particulate matter. In some embodiments, the agent is ozone. In some embodiments, the pollutant is a microplastic. In some embodiments, the pollutant is an organic pollutant or derivative thereof. In some embodiments, the pollutant is a halogenated organic pollutant or derivative thereof. In some embodiments, the pollutant is an aromatic organic pollutant or derivative thereof. In some embodiments, the pollutant is a heavy metal.

[0096] In some embodiments, the method further comprises assessing or predicting the impact of a medical device or treatment on a patient’s blood based on membrane fragility or deformability. In some embodiments, the method further comprises diagnosing a disease, disorder, or condition based on membrane fragility or deformability. Increased fragility of blood cells (e.g., red blood cells) is correlated with various hematological diseases, includingbut not limited to those that involve hemolysis. In some embodiments, the method further comprises diagnosing a hematological disease. In some embodiments, the method further comprises diagnosing anemia. In some embodiments, the method further comprises diagnosing hemolytic anemia. In some embodiments, the method further comprises diagnosing hereditary spherocytosis, sickle cell anemia, or G6PD deficiency. In some embodiments, the method further comprises diagnosing a disorder associated with a parasite (e.g., Plasmodium) or a Gram-positive bacteria (e.g., Streptococcus, Enterococcus, and Staphylococcus'). In some embodiments, the method further comprises diagnosing an autoimmune disorder. In some embodiments, the method further comprises diagnosing drug- induced hemolytic anemia. In some embodiments, the method further comprises diagnosing or identifying blood with too low a solute concentration. Increased fragility of leukocytes is correlated with various types of leukemia (e.g., chronic lymphocytic leukemia). In some embodiments, the method further comprises diagnosing leukemia based on membrane fragility or deformability. In some embodiments, the method further comprises diagnosing chronic lymphocytic leukemia based on membrane fragility or deformability. Changes in RBC deformability are correlated with diabetes. Williams, A. el al. Pathophysiology 2023, 30(3), 327-345. In some embodiments, the method further comprises diagnosing diabetes or pre-diabetes.

[0097] In some embodiments, the method further comprises managing blood unit inventory based on erythrocyte membrane fragility. In some embodiments, the method further comprises evaluating blood storage methods. In some embodiments, the method further comprises evaluating blood handling methods.

[0098] Those skilled in the art will be able to assess the appropriate level of fragility or deformability for a particular application (e.g, blood transfusion) based on their experience, the patient, and the circumstances.EXAMPLES

[0099] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.Example 1

[0100] Presented herein is an exemplary high-throughput single-cell fragility assay that assesses red blood cell membrane integrity, which is sensitive enough to detect reductions in membrane robustness within the first week of blood storage. The test is a high-throughput method enabling testing of at least 1-10 cells per second and needs only a drop of blood to perform the test.Experimental setup and results

[0101] The measurement method presented herein employs a glass micropipette with an inner diameter of less than 2 microns. As illustrated in FIGs. 1A-1B, a controlled high negative pressure was applied through a syringe attached to one end of the micropipette, while the other end, featuring a less than 2-micron opening, was used to aspirate RBCs individually. The pressure for aspirating the RBCs into the micropipette remained constant. This led to partial entry of the RBC into the pipette, while the rest of the cell remained outside (FIGs. 1C-1E). Over time, the sustained high pressure caused the RBC to rupture, resulting in the complete intake of the cell into the micropipette (FIG. IF).

[0102] A parameter of this measurement is the time to rupture, T, defined as the interval between the time of initial aspiration of the RBC (to) and the time of its eventual rupture ( / r), where T= tr- to. This duration is a direct indication of the RBC membrane’s robustness and is inversely correlated to the RBC fragility, i.e., the less time it takes for the RBC to rupture, the higher the fragility of the RBC. The method is a single-cell assay, and the average time to rupture provides a measure of the average RBC fragility for a population of RBCs. As shown in FIG. 2, the average time to rupture exhibited an exponential decay relationship with pressure.

[0103] When assessing RBCs treated with two drugs, diamide and phenylhydrazine (PHZ), known for changing RBC cytoskeleton properties, a significant decrease in RBC fragility was observed, as shown in FIG. 2. While diamide and PHZ increase RBC rigidity [15, 16], they also lead to a decrease in fragility. This highlights the distinct effects of these drugs on RBC cytoskeletal properties (reflected in rigidity) and membrane integrity (reflected in fragility).

[0104] Additionally, RBC fragility was evaluated following the standard blood bank storage procedure. As indicated in FIGs. 3A-3B, RBC fragility significantly increased (i.e., the time to rupture decreased eight-fold) after 8 days of storage (Day 0 is the day of blood draw). The same trend was observed in FIGs. 4A-4B, which also show an exponential decayof the average time to rupture for RBCs versus the increasing aspiration pressure, illustrating the fragility response of RBCs to varying pressures.

[0105] The change in fragility versus storage time is much more pronounced compared to the changes in RBC shear modulus, which is a conventional method for assessing RBC mechanical properties that reflect the cytoskeleton characteristics beneath the RBC membrane. As shown in FIG. 5, the shear modulus exhibited no obvious changes after 8 days of storage. The comparison between the fragility test presented herein and the traditional shear modulus test underscores the sensitivity and effectiveness of the RBC fragility measurement presented herein capturing subtle changes in RBC mechanical properties, especially during the first week of storage.

[0106] This improved sensitivity and effectiveness presents a major advantage over previous methods and apparatus (e.g., shear modulus or osmotic measurements) unable to distinguish small changes in fragility and / or deformation. In addition, this single-cell high- throughput assay requires small sample volumes, which presents a significant advantage over methods requiring larger samples (e.g., numbers of cells, volume).

[0107] The single-cell fragility assay was used to examine RBC fragility between diabetic patients and healthy controls. FIGs. 7A and 7B show results from such tests. As shown in FIG. 7A, the average time to rupture was 0.07 0.07±0.03 s for healthy sample #1 and 0.91 0.91 ±0.48 s for diabetic sample #1. Diabetic sample #1 showed a significantly reduced fragility (p<0.00001). As shown in FIG. 7B, RBCs from diabetic patients generally had reduced fragility compared to RBCs from healthy subjects, although there was significant heterogeneity between different diabetic patients. The observed trend was consistent with the results reported using osmotic gradient ektacytometry

[0035] ,Sample Preparation MethodsComparative measurement of RBC fragility influenced by drug treatments using diamide and phenylhrdrazine (PHZ).

[0108] Whole blood was centrifuged at 1000 g for 10 minutes at room temperature. After removal of the buffy coat, RBCs were pelleted and washed 3 times with phosphate buffered saline (PBS) to obtain packed RBCs.

[0109] Control RBCs. 9 mL PBS was added to a 15 mL conical tube. 1 mL washed, packed RBCs were added to the conical tube and the tube was inverted to mix. Then, RBCs were incubated for 2 hours at 37 °C.

[0110] Diamide treatment of RBCs. A 20 mM stock solution of diamide (Sigma catalog # D3648) was prepared by dissolving 3.44 mg diamide per 1 mL PBS. 8 mL PBS was added to a 15 mL conical tube. 1 mL washed packed human RBCs was added to the conical tube and the tube was and inverted to mix (not vortexed). 1 mL 20 mM diamide (final concentration 2 mM) was added to the conical tube and the tube was and inverted to mix. RBCs were incubated with diamide for 2 hours at 37 °C. After incubation, the RBCs were washed twice with PBS.

[0111] PHZ treatment of RBCs . A 20 mM stock solution of PHZ (Sigma catalog # P26252) was prepared by dissolving 2.16 mg PHZ per 1 mL PBS. RBCs were suspended at 20% hematocrit in PBS glucose and incubated at 37 °C in 1 mM PHZ for 2 hours. After incubation, the RBCs were washed twice with PBS.

[0112] After diamide or PHZ treatment and washing, the collected packed RBCs were then resuspended in PBS containing 1% Bovine Serum Albumin (BSA) for fragility testing. The prepared RBC suspension was utilized for fragility experiments on the same day of the blood draw.Measurement of RBC fragility changes due blood storage time.

[0113] 11 mL of whole blood was centrifuged at 2500 g for 15 minutes. After centrifugation, the bottom 4 mL of packed RBCs were aspirated into a new tube. The 4 mL sample was then washed with PBS. Following the wash, the sample was centrifuged again at 2500 g for 15 minutes. Post-centrifugation, the sedimented RBCs at the bottom of the tube were carefully aspirated. These RBCs were then resuspended in SAGM (Saline, Adenine, Glucose, Mannitol) medium. The resultant RBC suspension was evenly distributed into 16 separate tubes. For each subsequent experiment, corresponding to the intended storage days, one tube was utilized. The fragility tests were performed in SAGM medium.Comparative measurement of RBC fragility between diabetic patients and healthy controls

[0114] Whole blood was centrifuged at 1000 g for 10 minutes at room temperature. After removal of the buffy coat, RBCs were pelleted and washed 3 times with phosphate buffered saline (PBS) to obtain packed RBCs.

[0115] For both healthy and diabetic RBCs, 9 mL PBS were added to a 15 mL conical tube. 1 mL washed packed RBCs were added, and the tube was inverted to mix.

[0116] The collected packed RBCs were then resuspended in PBS containing 1% Bovine Serum Albumin (BSA) for fragility testing. The prepared RBC suspension was utilized for fragility experiments on the same day of the blood draw.References:[1] Kaushansky K, A. LM, Prchal JT, Levi MM, Press OW, Burns LJ, Caligiuri M. Williams Hematology, 9th ed. New York, NY: McGraw-Hill Education, 2016.[2] Muenster S, Beloiartsev A, Yu B, Du E, Abidi S, Dao M, Fabry G, Graw JA, Wepler M, Malhotra R, Fernandez BO, Feelisch M, Bloch KD, Bloch DB, Zapol WM. Exposure of Stored Packed Erythrocytes to Nitric Oxide Prevents Transfusion-associated Pulmonary Hypertension. Anesthesiology 2016; 125(5):952-963.[3] Koch CG, Li L, Sessler DI, Figueroa P, Hoeltge GA, Mihaljevic T, Blackstone EH. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med 2008; 358(12): 1229-1239.[4] Baek JH, D'Agnillo F, Vallelian F, Pereira CP, Williams MC, Jia Y, Schaer DJ, Buehler PW. Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy. J Clin Invest 2012; 122(4): 1444-1458.[5] Hod EA, Spitalnik SL. Stored red blood cell transfusions: Iron, inflammation, immunity, and infection. Transfus Clin Biol 2012; 19(3):84-89.[6] Hess JR, Greenwait TG. Storage of red blood cells: new approaches. Transfus Med Rev 2002; 16(4):283-295.[7] Wang D, Cortes-Puch I, Sun J, Solomon SB, Kanias T, Remy KE, Feng J, Alimchandani M, Quezado M, Helms C, Perlegas A, Gladwin MT, Kim-Shapiro DB, Klein HG, Natanson C. Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia. Transfusion 2014; 54(7): 1712-1724.[8] Bosman G. Survival of red blood cells after transfusion: processes and consequences. Frontiers in Physiology 2013; 4(1):376.[9] Luten M, Roerdinkholder-Stoel winder B, Schaap NP, de Grip WJ, Bos HJ, Bosman GJ. Survival of red blood cells after transfusion: a comparison between red cells concentrates of different storage periods. Transfusion 2008; 48(7): 1478-1485.

[0010] Ziegler LA, Olia SE, Kameneva MV. Red Blood Cell Mechanical Fragility Test for Clinical Research Applications. Artificial organs 2016; 41(7):678-682.

[0011] Baskurt OK, Meiselman HJ. Red blood cell mechanical stability test. Clinical hemorheology and microcirculation 2013; 55(1): 55-62.

[0012] Sethu P, Anahtar M, Moldawer LL, Tompkins RG, Toner M. Continuous flow microfluidic device for rapid erythrocyte lysis. Analytical chemistry 2004; 76(21):6247-6253.

[0013] Gu L, Smith WA, Chatzimavroudis GP. Mechanical Fragility Calibration of Red Blood Cells. ASAIO Journal 2005; 51(3): 194-201.

[0014] Paleari R, Mosca A. Controversies on the osmotic fragility test. Enerca News 2008; (9): 1-3.

[0015] Safeukui I, Buffet PA, Deplaine G, Perrot S, Brousse V, Sauvanet A, Aussilhou B, Dokmak S, Couvelard A, Cazals-Hatem D, Mercereau-Puijalon O, Milon G, David PH, Mohandas N. Sensing of red blood cells with decreased membrane deformability by the human spleen. Blood Adv 2018; 2(20):2581-2587.

[0016] Ramot Y, Koshkaryev A, Goldfarb A, Yedgar S, Barshtein G. Phenylhydrazine as a partial model for beta-thalassaemia red blood cell hemodynamic properties. Br J Haematol 2008; 140(6):692-700.

[0017] Bow, H.; Pivkin, I.; Diez-Silva, M.; Goldfless, S. J.; Dao, M.; Niles, J. C.; Suresh, S.; Han, J. A microfabricated deformability-based flow cytometer with application to malaria. Lab Chip 2011, 77, 1065-1073.

[0018] Chien S. Blood Cells. 1977, 3, 71-95.

[0019] Kim, D.-H.; wong, P. K.; Park, J.; Levchenko, A.; Sun, Y. Microengineered Platforms for Cell Mechanobiology. Annu. Rev. Biomed. Eng. 2009, 77, 203-233.

[0020] Li, X.; Chen, W .; Li, Z.; Li, L.; Gu, H.; Fu, J. Emerging microengineering tools for functional analysis and phenotyping of blood cells. Trends Biotechnol. 2024, 32, 586-594.

[0021] Nash G.; O’Brien, E.; Gordon-Smith, E. C.; Dormandy, J. A. Abnormalities in the Mechanical Properties of Red Blood Cells Caused by Plasmodium Falciparum. J. Blood. 1989, 74, 855-861.

[0022] Nash, G. B.; Meiselman, H. J. Red Cell and Ghost Viscoelasticity. Biophys. J. 1983, 43, 63-73.

[0023] Hochmuth, R. M. Micropipette aspiration of living cells. J. Biomech., 2000, 33, 15-22.

[0024] Zheng, Y.; Nguyen, J.; Wang, C.; Sun, Y. Electrical measurement of red blood cell deformability on a microfluidic device. Lab Chip 2013, 13, 3275-3283.

[0025] U.S. Patent No. 8,026,102

[0026] U.S. Patent No. 8,263,408

[0027] U.S. Patent No. 8,268,244

[0028] U.S. Patent Application Publication No. 2011 / 0300574 Al

[0029] U.S. Patent Application Publication No. 2012 / 0178121 Al

[0030] International Patent Application Publication No. WO 2010 / 090848 A2

[0031] Chinese Patent Application Publication No. CN114295533 A

[0032] Scott, M. D.; Matthews, K.; Ma, H. Assessing the Vascular Deformability of Erythrocytes and Leukocytes: From Micropipettes to Microfluidics. In Current and Future Aspects of Nanomedicine IntechOpen, 2018. DOI: 10.5772 / intechopen.90131.

[0033] Gudgeon P.; Pereira M. Pseudohyperkalemia in chronic lymphocytic leukemia. CMAJ. 2023, 195, El 580.

[0034] Gurevitch, J.; Nelken, D. Osmotic Fragility of Human Blood Platelets. Blood 1956, 11, 924-928.

[0035] Ebenuwa, I.; Violet, P.-C.; Tu, H.; Lee, C.; Munyan, N.; Wang, Y.; Niyyati, M.; Patra, K.; Wilkins, K. J.; Parrow, N.; Levine, M. Altered RBC deformability in diabetes: clinical characteristics and RBC pathophysiology. Cardiovasc. DiabetoL 2024, 23, 370.EQUIVALENTS AND SCOPE

[0117] All definitions, as defined and used herein, should be understood to control over dictionary definitions, and / or ordinary meanings of the defined terms. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.

[0118] This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0119] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multipleelements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0120] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed. Such terms are used merely as labels to distinguish one claim element having a certain name from another element having the same name (but for use of the ordinal term).

[0121] The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing”, “involving”, and variations thereof, is meant to encompass the items listed thereafter and additional items.

[0122] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0123] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is / are referred to as comprisingparticular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0124] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0125] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. An apparatus for assessing the fragility or deformability of a cell, comprising: a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member; a negative pressure means for applying negative pressure to the interior volume of the hollow member, wherein the negative pressure means, when operating, applies a negative pressure to the interior volume of the hollow member sufficient to (1) partially aspirate the cell into the interior volume of the hollow member through the fenestration at a time to; and (2) following to, rupture the cell at a time , resulting in the complete aspiration of the cell into the interior volume of the hollow member; a sensor for measuring the pressure of the interior volume of the hollow member; at least one processor; and a memory coupled to the at least one processor, wherein the memory comprises a plurality of instructions executable by the processor, and wherein the instructions comprise instructions for measuring a time to rupture of the cell (7) between and to.

2. An apparatus for assessing the fragility or deformability of a cell, comprising: a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member; a negative pressure means for applying negative pressure to the interior volume of the hollow member, wherein the negative pressure means, when operating, applies a negative pressure to the interior volume of the hollow member sufficient to (1) partially aspirate the cell into the interior volume of the hollow member through the fenestration at a time to; and (2) following to, rupture the cell at a time , resulting in the complete aspiration of the cell into the interior volume of the hollow member; a sensor for measuring the pressure of the interior volume of the hollow member; and at least one processor operatively connected to a memory, the processor when executing configured to: measure a time to rupture of the cell (7) between and to.

3. The apparatus of any preceding claim, wherein the cell is a red blood cell, a white blood cell, or a platelet.

4. The apparatus of any preceding claim, wherein the cell is a red blood cell.

5. The apparatus of any preceding claim, wherein the at least one processor is configured to control operation of the negative pressure means based on communicated control signals.

6. The apparatus of any preceding claim, wherein the at least one processor is configured to communicate control signals to the negative pressure means to generate the negative pressure.

7. The apparatus of any preceding claim, wherein the at least one processor is configured to communicate control signals to the negative pressure means to adjust the negative pressure.

8. The apparatus of any preceding claim, wherein the at least one processor is configured to communicate control signals to the negative pressure means to release the negative pressure.

9. The apparatus of any preceding claim, wherein the at least one processor is configured to determine the time at which the rupture of the cell occurs ( / ,).

10. The apparatus of any preceding claim, wherein the at least one processor is configured to determine the time at which the rupture of the cell occurs based on image analysis of captured visual data.

11. The apparatus of any preceding claim, wherein the at least one processor is configured to control capture of the visual data via an imaging means.

12. The apparatus of any preceding claim, wherein the at least one processor is configured to determine the time at which the rupture of the cell occurs based presence or change of an electrical signal.

13. The apparatus of any preceding claim, wherein the electrical signal is impedance or resistance.

14. The apparatus of any preceding claim, wherein the hollow member comprises a micropipette, a microfluidic channel, or a microtube.

15. The method of any preceding claim, wherein the micropipette is glass, metal, or plastic.

16. The method of the preceding claim, wherein the micropipette is transparent.

17. The apparatus of any preceding claim, wherein the fenestration has a largest interior dimension of about 3.0 microns or less.

18. The apparatus of any preceding claim, wherein the fenestration has a largest interior dimension of about 2.0 microns or less.

19. The apparatus of any preceding claim, wherein the fenestration has a largest interior dimension of about 0.5 microns to about 3.0 microns.

20. The apparatus of any preceding claim, wherein the fenestration has a largest interior dimension of about 1.5 microns to about 2.0 microns.

21. The apparatus of any preceding claim, wherein the hollow member comprises a channel having a largest interior width of about 3.0 microns or less.

22. The apparatus of any preceding claim, wherein the hollow member comprises a channel having a largest interior width of about 2.0 microns or less.

23. The apparatus of any preceding claim, wherein the hollow member comprises a channel having a largest interior width of about 0.5 microns to about 3.0 microns.

24. The apparatus of any preceding claim, wherein the hollow member comprises a channel having a largest interior width of about 1.5 microns to about 2.0 microns.

25. The apparatus of any preceding claim, wherein the negative pressure means comprises a liquid withdrawal pump or vacuum pump.

26. The apparatus of any preceding claim, wherein the negative pressure means comprises a syringe pump.

27. The apparatus of any preceding claim, wherein the negative pressure means comprises a feedback pressure sensor to adjust the negative pressure.

28. The apparatus of any preceding claim, wherein the negative pressure is about 50 mbar to about 550 mbar.

29. The apparatus of any preceding claim, wherein the negative pressure is about 50 mbar to about 250 mbar.

30. The apparatus of any preceding claim, wherein the negative pressure is about 250 mbar to about 550 mbar.

31. The apparatus of any preceding claim, wherein the negative pressure is about 375 mbar to about 435 mbar.

32. The method of any preceding claim, wherein the negative pressure is constant.

33. The apparatus of any preceding claim, wherein the measured time to rupture of the cell (7) correlates to the membrane fragility or deformability of the cell.

34. The apparatus of any preceding claim, wherein the cell is a member of a population of cells, and the measured time to rupture of the cell (7) correlates to the membrane fragility or deformability of the remaining population of cells.

35. The apparatus of any preceding claim, wherein the apparatus is configured to assess the fragility or deformability a plurality of cells.

36. The apparatus of claim 35, wherein the apparatus is configured to assess the fragility or deformability of at least 1 cell per second.

37. The apparatus of claim 35, wherein the apparatus is configured to assess the fragility or deformability of at least 10 cells per second.

38. The apparatus of any preceding claim, wherein the instructions further comprise instructions for calculating an average, median, or mode time to rupture from multiple T measurements determined for a plurality of cells.

39. The apparatus of claim 38, wherein the cell is a member of a population of cells, and the average, median, or mode time to rupture from multiple T measurements correlates to the membrane fragility or deformability of the remaining population of cells.

40. A method of assessing the fragility or deformability of a cell, the method comprising:(1) applying a negative pressure to a hollow member defining an interior volume and comprising a fenestration, wherein the fenestration is dimensioned to allow for partial aspiration of the cell into the interior volume of the hollow member;(2) partially aspirating the cell into the interior volume of the hollow member through the fenestration at a time to,'(3) rupturing the cell and completely aspirating the cell into the interior volume of the hollow member at a time tr following to; and(4) measuring the time to rupture of the cell (7) between and to.

41. The method of any preceding claim, wherein steps (1) through step (4) of the method are performed using the apparatus of any preceding claim.

42. The method of any preceding claim, wherein the measured time to rupture of the cell (7) correlates to the membrane fragility or deformability of the cell.

43. The method of any preceding claim, further comprising a preliminary step of isolating the cell from whole blood.

44. The method of claim 43, wherein the isolating comprises centrifuging whole blood, removing the huffy coat, and pelleting the cell.

45. The method of claim 44, further comprising washing the pelleted cell to obtain a packed cell.

46. The method of claim 45, further comprising incubating the packed cell.

47. The method of claim 46, wherein the cells are incubated with an additional agent.

48. The method of claim 47, wherein the agent is a blood storage medium.

49. The method of claim 47, wherein the agent is a therapeutic agent.

50. The method of claim 47, wherein the agent is a toxin, an oxidant, or a pollutant.

51. The method of any preceding claim, wherein the negative pressure is about 50 mbar to about 550 mbar.

52. The method of any preceding claim, wherein the negative pressure is about 50 mbar to about 250 mbar.

53. The method of any preceding claim, wherein the negative pressure is about 250 mbar to about 550 mbar.

54. The method of any preceding claim, wherein the negative pressure is about 375 mbar to about 435 mbar.

55. The method of any preceding claim, wherein the negative pressure is constant.

56. The method of any preceding claim, wherein the fenestration has a largest interior dimension of about 3.0 microns or less.

57. The method of any preceding claim, wherein the fenestration has a largest interior dimension of about 2.0 microns or less.

58. The method of any preceding claim, wherein the fenestration has a largest interior dimension of about 0.5 microns to about 3.0 microns.

59. The method of any preceding claim, wherein the fenestration has a largest interior dimension of about 1.5 microns to about 2.0 microns.

60. The method of any preceding claim, wherein the hollow member comprises a channel having a largest interior width of about 3.0 microns or less.

61. The method of any preceding claim, wherein the hollow member comprises a channel having a largest interior width of about 2.0 microns or less.

62. The method of any preceding claim, wherein the hollow member comprises a micropipette.

63. The method of any preceding claim, wherein the micropipette is glass, metal, or plastic.

64. The method of any preceding claim, wherein the cell is a member of a population of cells, and the measured time to rupture of the cell (7) correlates to the membrane fragility or deformability of the remaining population of cells.

65. The method of any preceding claim, the method comprising assessing the fragility or deformability a plurality of cells.

66. The method of any preceding claim, the method comprising assessing the fragility or deformability of at least 1 cell per second.

67. The method of any preceding claim, the method comprising assessing the fragility or deformability of at least 10 cells per second.

68. The method of any preceding claim, the method comprising calculating an average, median, or mode time to rupture from multiple T measurements determined for a plurality of cells.

69. The method of any preceding claim, wherein the cell is a member of a population of cells, and the average, median, or mode time to rupture from multiple T measurements correlates to the membrane fragility or deformability of the remaining population of cells.

70. The method of any preceding claim, comprising assessing the fragility or deformability of a population of cells.

71. The method of any preceding claim, wherein the population of cells is derived from about 1 drop of blood.

72. The method of any preceding claim, wherein the population of cells is derived from about 0.5 pL to about 1 pL of blood.

73. The method of any preceding claim, wherein the cell or population of cells is derived from a patient.

74. A method of transfusing blood into a patient in need thereof, the method comprising:(1) providing a population of exogenous blood cells;(2) assessing the fragility or deformability of a plurality of the population of exogenous blood cells according to the method of any preceding claim, and(3) transfusing at least a portion of the remaining population of exogenous blood cells to the patient if the average, median, or mode time to rupture of the plurality of the population of exogenous blood cells is similar to or greater than the average, median, or mode time to rupture for a population of blood cells determined to have sufficient fragility or deformability properties for transfusion to the patient.

75. A method of transfusing blood into a patient in need thereof, the method comprising:(1) providing a candidate population of exogenous blood cells;(2) assessing the fragility or deformability of a plurality of the candidate population of exogenous blood cells according to the method of any preceding claim,(3) providing a control population of fresh blood cells;(4) assessing the fragility or deformability of a plurality of the population of fresh blood cells according to the method of any preceding claim,(5) calculating the difference in average, median, or mode time to rupture of the plurality of the candidate population from the average, median, or mode time to rupture of the plurality of fresh blood cells, and(6) transfusing at least a portion of the remaining candidate population of exogenous blood cells to the patient if the difference is less than one standard deviation.

76. The method of any preceding claim, wherein the blood cell is a red blood cell, white blood cell, or platelet.

77. The method of any preceding claim, wherein assessing cell fragility or deformability is performed using the apparatus of any preceding claim.

78. The method of any preceding claim, wherein the control population of fresh blood cells was obtained from a subject within 42 days.

79. The method of any preceding claim, wherein the control population of fresh blood cells was obtained from a subject within 7 days.

80. The method of any preceding claim, wherein the control population of fresh blood cells was obtained from a subject within 1 day.

81. The method of any preceding claim, wherein the control population of fresh blood cells was obtained from a subject within 8 hours.

82. The method of any preceding claim, wherein the control population of fresh blood cells was obtained from a subject within 2 hours.

83. The method of any preceding claim, wherein the method further comprises diagnosing a disease, disorder, or condition based on membrane fragility or deformability.

84. The method of any preceding claim, wherein the patient has been diagnosed with or is at risk of a disease, disorder, or condition.

85. The method of any preceding claim, wherein the disease, disorder, or condition is diabetes or pre-diabetes.

Images