Determination of hematocrit

Abstract

Methods and devices for determining the hematocrit of a blood sample.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 389,487, filed on July 15, 2022, which is incorporated by reference in its entirety.

[0002] The present disclosure generally relates to the determination of hematocrit in a blood sample. Background Art

[0003] Hematocrit refers to the ratio of volume occupied by red blood cells in the blood, and is expressed as HCT%. In healthy adult individuals, red blood cells account for about 40-48% of the blood volume, while newborns can have up to 60% hematocrit. Clinically, when it is suspected that the patient is anemic or suffers from dehydration, bleeding or other medical and surgical conditions, hematocrit measurement may be required. Low hematocrit reflects a low number of circulating red blood cells and is an indicator of decreased oxygen carrying capacity or water intoxication. Examples of conditions that lead to low hematocrit (anemia) include internal or external bleeding, complications of chronic renal failure and / or kidney disease, pernicious anemia (vitamin B12 deficiency) and hemolysis (usually associated with transfusion reactions). Low hematocrit may also be found in autoimmune diseases and bone marrow failure. A high hematocrit may reflect an absolute increase in the number of erythrocytes or a decrease in plasma volume in conditions such as severe dehydration, polycythemia (excessive production of red blood cells), polycythemia vera (an abnormal increase in red blood cells), and hemachromatosis (an inherited disorder of iron metabolism). A high hematocrit is also used as an indicator of excessive intake of exogenous erythropoietin (EPO), which stimulates red blood cell production.

[0004] Conventionally, HCT% is usually determined by centrifuging a liquid whole blood sample to separate the components of the liquid whole blood based on density and determining the volume of packed red blood cells as a percentage of the total blood volume. However, this method cannot be used for low volume samples or dried blood samples. Therefore, the inventors have identified a need in the art to effectively determine the hematocrit from such samples. Summary of the invention

[0005] One aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes lysing the blood sample to provide a lysed blood sample, measuring the amount of NADPH in the lysed blood sample, and correlating the amount of NADPH in the lysed blood sample with the hematocrit in the blood sample.

[0006] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes lysing the blood sample to provide a lysed blood sample, measuring the activity of G6PDH in the lysed blood sample, and correlating the activity of G6PDH in the lysed blood sample with the hematocrit in the blood sample.

[0007] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes lysing the blood sample to provide a lysed blood sample, measuring the activity of glutathione reductase in the blood sample; and correlating the activity of glutathione reductase with the hematocrit in the blood sample.

[0008] Another aspect of the present disclosure relates to a method of determining the hematocrit of a blood sample, the method comprising lysing the blood sample to provide a lysed blood sample; adding a tetrazolium dye to the lysed blood sample; measuring the amount of a reduction product of the tetrazolium dye in the lysed blood sample, and correlating the amount of the reduction product of the tetrazolium dye in the blood sample to the hematocrit of the blood sample.

[0009] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from a blood sample on a blood collection card, measuring the amount of NADPH in the extracted dried blood, and correlating the amount of NADPH with the hematocrit of the blood sample.

[0010] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from the blood sample on a blood absorbing material, measuring the amount of a reduction product of a tetrazolium dye in the extracted dried blood, and correlating the amount of the reduction product of the tetrazolium dye with the hematocrit of the blood sample.

[0011] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from a blood sample on a blood collection card, measuring the activity of G6PDH in the extracted dried blood, and correlating the activity of G6PDH with the hematocrit of the blood sample.

[0012] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from a blood sample on a blood collection card, measuring the activity of glutathione reductase in the extracted dried blood, and correlating the activity of glutathione reductase with the hematocrit of the blood sample.

[0013] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from a blood sample on a blood collection card, measuring the amount of NADPH in the extracted dried blood, and correlating the amount of NADPH with the hematocrit of the blood sample.

[0014] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from the blood sample on a blood absorbing material, measuring the amount of a reduction product of a tetrazolium dye in the extracted dried blood, and correlating the amount of the reduction product of the tetrazolium dye with the hematocrit of the blood sample.

[0015] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from a blood sample on a blood collection card, measuring the activity of G6PDH in the extracted dried blood, and correlating the activity of G6PDH with the hematocrit of the blood sample.

[0016] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes extracting a predetermined amount of dried blood obtained from a blood sample on a blood collection card, measuring the activity of glutathione reductase in the extracted dried blood, and correlating the activity of glutathione reductase with the hematocrit of the blood sample.

[0017] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes receiving a dried blood sample from an animal on a blood collection card from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the amount of NADPH in the extracted dried blood, and correlating the amount of NADPH with the hematocrit of the blood sample.

[0018] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes receiving a dried blood sample from an animal on a blood collection material from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the amount of tetrazolium dye in the extracted dried blood, and correlating the amount of tetrazolium dye with the hematocrit of the blood sample.

[0019] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes receiving a dried blood sample from an animal on a blood collection material from a blood collector; extracting a predetermined amount of dried blood from a blood collection card; measuring the activity of G6PDH in the extracted dried blood, and correlating the activity of G6PDH with the hematocrit of the blood sample.

[0020] Another aspect of the present disclosure relates to a method for determining the hematocrit of a blood sample. The method includes receiving a dried blood sample from an animal on a blood collection material from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the activity of glutathione reductase in the extracted dried blood, and correlating the activity of glutathione reductase with the hematocrit of the blood sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings (which are included to provide a further understanding of the present disclosure) are incorporated into and constitute a part of this specification, illustrate embodiments of the present disclosure, and together with the detailed description are used to explain the principles of the present disclosure. No attempt has been made to show the structural details of the present disclosure in more detail, except as may be necessary for a basic understanding of the present disclosure and its various embodiments.

[0023] Figure 1A and Figure 1B Linear plots of the correlation between the hematocrit (HCT %) and the absorbance at 340, 405 and 578 nm for lysed blood samples prepared from 3 μL or 6 μL of liquid whole blood, respectively.

[0024] Figure 2A is a graph of UV-visible absorption spectra from lysed red blood samples obtained from dried blood spots containing different hematocrits. Figure 2B The correlation between the hematocrit (HCT%) and the absorbance at 340, 405, 578 nm of lysed blood samples obtained from dried blood spots is shown.

[0025] Figure 3A The graph is a graph of the UV-visible absorption spectra of the following three samples: a lysed red blood sample without tetrazolium dye (MTT) and without a single electron transfer catalyst (phenazine methosulfate), a lysed red blood sample with tetrazolium dye (MTT) and a single electron transfer catalyst (phenazine methosulfate), and a tetrazolium dye (MTT) and a single electron transfer catalyst (phenazine methosulfate) in tris buffer. Figure 3B The correlation between the hematocrit (HCT %) and the absorbance at 578 nm of the lysed blood samples is shown.

[0026] Figure 4 is a graph showing the change in absorbance at 340 nm over time for lysed red blood samples containing different hematocrits (HCT %).

[0027] Figure 5A and Figure 5BLinear plots of the correlation between hematocrit (HCT %) and NADPH formation rate over time periods of 10 to 15 minutes and 0 to 45 minutes for lysed red blood samples prepared from 3 μL or 6 μL of liquid whole blood, respectively.

[0028] Figure 6 is a mass spectrometric chromatogram of 6-phosphogluconate (PGA) of a lysed blood sample.

[0029] Figure 7 is a linear plot of the correlation between the hematocrit (HCT%) and each mass spectrometry chromatogram area of ​​6-phosphogluconate (PGA) of lysed red blood samples containing different hematocrits (HCT%).

[0030] Figure 8 is a mass spectrometry chromatogram of glutathione disulfide (GSSG) from lysed red blood samples containing different hematocrits (HCT%).

[0031] Fig. 9 It is a scatter plot of the correlation between the hematocrit (HCT%) and each mass spectrometry chromatogram area of ​​glutathione disulfide (GSSG) of dried blood samples containing different hematocrits (HCT%). DETAILED DESCRIPTION OF THE INVENTION

[0033] In various aspects, the present disclosure relates to methods for determining the hematocrit of a blood sample. These methods include measuring the amount of one or more analytes and / or the activity of an enzyme in the blood sample, and correlating these measurements with the amount of hematocrit. These methods may also include correlating the amount of the analyte or the activity of the enzyme with a standard curve to determine the hematocrit of the blood sample.

[0034] The methods described herein provide an effective method to determine the hematocrit of dried blood samples and blood samples requiring smaller volumes (such as less than 100 μL), which is more accurate than traditional methods. These methods can use conventional analytical laboratory equipment that can be easily integrated into the conventional workflow of an analytical laboratory.

[0035] Conventional and unique devices and methods can be used to collect blood samples for use in the methods disclosed herein. In some embodiments, blood can be collected using absorbent blood collection materials, microneedles, or a device that combines both technologies.

[0036] definition

[0037] Before describing the present disclosure in further detail, some terms are defined:

[0038] Blood samples can include liquid whole blood or dried blood. Liquid whole blood contains red blood cells, white blood cells, platelets and plasma, and can be collected from humans or any other animal species. For example, in some embodiments, the whole blood sample is selected from the group comprising: dog blood, avian blood, cat blood, mouse blood, horse blood and human blood. Dried blood spots (DBS) are formed by drying liquid whole blood on a blood collection material or other suitable collection device (e.g., a blood absorbing material such as a card, a pad or a rigid foam).

[0039] The blood sample of cracking is produced by treating liquid whole blood or dried blood sample with cracking process. The cracking blood sample can be completed by any means known in the art. For example, the cracking blood sample is completed by mixing the blood sample with cracking buffer.

[0040] NADP + is nicotinamide adenine dinucleotide phosphate, and NADPH is NADP + The restored form.

[0041] NAD + is nicotinamide adenine dinucleotide, and NADH is NAD + The restored form.

[0042] G6PDH is an enzyme, glucose-6-phosphate dehydrogenase. G6PDH catalyzes the oxidation of D-glucose-6-phosphate to 6-phosphogluconolactone and NADP + Reduction to NADPH.

[0043] PGA is 6-phosphogluconolactone and is the oxidized form of D-glucose-6-phosphate.

[0044] GR is an enzyme, glutathione reductase, which catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH).

[0045] LCMS is liquid chromatography mass spectrometry.

[0046] As used herein, the term "analyte" generally refers to a substance or group of substances present in a sample. The analyte may be inherent to the composition of the sample or added to the sample. Visible dyes and fluorescent dyes may be considered examples of analytes added to the sample. When an analyte is added to a sample, the measurement of the analyte may be correlated with the hematocrit due to the reaction between the added analyte and other inherent components in the sample.

[0047] As used herein, the term "enzyme activity" generally refers to a reaction that catalyzes the conversion of substances in a sample to produce a reaction product such as a redox product. For example, NADPH and PGA are redox products of G6DPH.

[0048] method

[0049] In various aspects, the present disclosure relates to a method for determining the hematocrit in a test blood sample. The method may include measuring an analyte in the blood sample, and correlating the amount of the analyte in the blood sample with the hematocrit in the blood sample. The method may include lysing the blood sample and measuring the analyte in the lysed blood sample. The method may also include creating a standard curve by measuring the analyte in a standard blood sample with a known hematocrit, and correlating the hematocrit of the standard sample with the amount of the analyte in the standard sample. The amount of the analyte in the test blood sample may be compared to the standard curve to determine the hematocrit of the test sample. The standard curve may be created by measuring the amount of the analyte in the standard blood sample with a known hematocrit. When a lysed blood sample is used to determine the hematocrit, the standard curve may be created using the lysed blood sample.

[0050] In an example embodiment, the analyte may be NADPH. According to this example, the method of the present disclosure includes lysing a blood sample to provide a lysed blood sample, measuring the amount of NADPH in the lysed blood sample, and correlating the amount of NADPH in the lysed blood sample with the hematocrit in the blood sample. The method may include measuring the amount of NADPH in a standard blood sample with a known hematocrit to create a standard curve, and comparing the amount of NADPH in the lysed blood sample to the standard curve to determine the hematocrit of the lysed blood sample.

[0051] In another aspect of the present disclosure, an exemplary method measures the activity of an enzyme in a blood sample (e.g., a lysed blood sample). The activity of the enzyme can be correlated to the hematocrit of the blood sample. For example, the enzyme can be selected from glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR). In some embodiments, the activity of the enzyme depends on NADP + For example, G6PDH and GR convert NADP + Catalyzed to NADPH.

[0052] The method can include measuring the activity of the enzyme in a standard blood sample with a known hematocrit to create a standard curve, and comparing the activity of the enzyme in the lysed blood sample with the standard curve to determine the hematocrit in the blood sample based on the enzyme activity. The activity of the enzyme in the lysed blood sample can be determined by measuring the amount of the redox product of the enzyme. For example, when the enzyme is G6PDH, the redox product can be 6-phosphogluconolactone (PGA) and NADPH.

[0053] In one embodiment of the present disclosure, before measuring the activity of G6DPH in the lysed blood sample, NAD +or NADP + Add to lysed blood samples. Add NAD to samples + or NADP + This can enable the detection of a greater amount of the redox product NADH or NADPH than would normally be present in the sample. For example, in some embodiments, prior to measuring the activity of G6PDH, NAD + and NADP + The amount of NAD is added to the lysed blood sample in the range of about 1% weight (mg) / volume (mL) to 30% weight (mg) / volume (mL), in the range of about 5% weight (mg) / volume (mL) to 25% weight (mg) / volume (mL), in the range of about 10% weight (mg) / volume (mL) to 20% weight (mg) / volume (mL), or in the range of about 10% weight (mg) / volume (mL) to 15% weight (mg) / volume (mL). In a specific embodiment, NAD is added to the lysed blood sample before measuring the activity of G6PDH. + Add to the lysed blood sample. In another embodiment, before measuring the activity of G6PDH in the lysed blood sample, glucose-6-phosphate (G6P) is added to the lysed blood sample. For example, in some embodiments, G6P is added in the following amounts: in the range of about 1% weight (mg) / volume (mL) to about 20% weight (mg) / volume (mL), in the range of about 5% weight (mg) / volume (mL) to about 15% weight (mg) / volume (mL), or in the range of about 5% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL). In another embodiment, before measuring the activity of G6PDH in the lysed blood sample, a small amount of G6PDH is added to the lysed blood sample. For example, in some embodiments, G6PDH is added as follows: in the range of about 0.5x10 -5 % weight (mg) / volume (mL) to about 5.0x10 -5 % weight (mg) / volume (mL) range, about 1.5x10 -5 % weight (mg) / volume (mL) to about 3.5x10 -5 % weight (mg) / volume (mL) range, or about 2.0x10 -5 % weight (mg) / volume (mL) to about 3.0x10 -5 % weight (mg) / volume (mL) range.

[0054] In another embodiment, the activity of G6PDH is determined by measuring the amount of PGA in a blood sample and comparing the amount of PGA in the lysed blood sample with a standard curve to determine the hematocrit in the blood sample. A standard curve can be created by measuring the amount of PGA in a standard blood sample with a known hematocrit.

[0055] In certain aspects of the present disclosure, exemplary methods of the present disclosure involve measuring the activity of GR in a blood sample and correlating the activity of GR in the blood sample with the hematocrit in the blood sample. For example, in some embodiments, the activity of GR is determined by measuring the amount of GSSG in the blood sample and comparing the amount of GSSG to a standard curve to determine the hematocrit in the blood sample. A standard curve can be created by measuring the amount of GSSG in a standard blood sample with a known hematocrit.

[0056] In some embodiments, the method further comprises adding a GSSG internal standard solution to the blood sample, determining the activity of GR in the blood sample, and correlating the GR activity with a standard curve to determine the hematocrit in the blood sample. For example, in some embodiments, the amount of the GSSG internal standard solution added to the blood sample is in the range of about 0.5% weight (mg) / volume (mL) to about 20% weight (mg) / volume (mL), in the range of about 1% weight (mg) / volume (mL) to about 15% weight (mg) / volume (mL), in the range of about 1% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL), or in the range of about 1% weight (mg) / volume (mL) to about 7% weight (mg) / volume (mL).

[0057] In some embodiments of the present disclosure, a method of determining the hematocrit of a blood sample includes lysing the blood sample to provide a lysed blood sample, adding a visible dye to the lysed blood sample, measuring the amount of a redox product of the visible dye in the lysed blood sample, and comparing the amount of the redox product of the visible dye in the blood sample to the hematocrit of the blood sample. The visible dye is intended to react with other intrinsic components in the sample to provide a signal related to the amount of the intrinsic components in the sample.

[0058] The method may also include measuring the redox product of the visible dye in a standard blood sample with a known hematocrit to create a standard curve. The method may also include correlating the amount of the redox product of the visible dye in the blood sample with the hematocrit of the blood sample by comparing the amount of the redox product of the visible dye with the standard curve. The method may also include determining the hematocrit in the blood sample using the standard curve based on the amount of the redox product of the visible dye. The method may also include lysing the blood sample by mixing the blood sample with a lysis buffer.

[0059] The visible dye is not particularly limited, and any + and / or NADP + A visible dye that changes color in the presence of may be suitable. For example, the visible dye may be selected from a group having a color suitable for reducing NAD + and / or NADP + In some embodiments, the visible dye can be added in a range of about 0.1% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL), in a range of about 0.2% weight (mg) / volume (mL) to about 7.5% weight (mg) / volume (mL), in a range of about 0.5% weight (mg) / volume (mL) to about 5.0% weight (mg) / volume (mL), or in a range of about 1.0% weight (mg) / volume (mL) to about 2.0% weight (mg) / volume (mL).

[0060] In one example, the visible dye can be selected from a group having a suitable + and / or NADP +For example, the tetrazolium dye can be selected from 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) (XTT), iodonitrotetrazolium chloride (INT), triphenyltetrazolium chloride (TTC), 3-(4,5-dimethylthiazol-2-yl)-5 -(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS), nitro blue tetrazolium chloride (NBT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In a specific embodiment, the tetrazolium dye is 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide (MTT). In some embodiments, the tetrazolium dye can be added as follows: in the range of about 0.1% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL), in the range of about 0.2% weight (mg) / volume (mL) to about 7.5% weight (mg) / volume (mL), in the range of about 0.5% weight (mg) / volume (mL) to about 5.0% weight (mg) / volume (mL), or in the range of about 1.0% weight (mg) / volume (mL) to about 2.0% weight (mg) / volume (mL).

[0061] In some embodiments of the present disclosure, the method of measuring the redox product of the visible dye may further include adding a mediator to facilitate single electron transfer. Suitable mediators include NAD + and / or NADP + Reduced single electron transfer mediator. For example, phenazine methyl sulfate is a suitable mediator that can be added to the lysed blood sample to promote single electron transfer. In some embodiments, the mediator is added as follows: in the range of about 0.05% weight (mg) / volume (mL) to about 2.0% weight (mg) / volume (mL), in the range of about 0.2% weight (mg) / volume (mL) to about 1.5% weight (mg) / volume (mL), or in the range of about 0.5% weight (mg) / volume (mL) to about 1.0% weight (mg) / volume (mL).

[0062] Measurement

[0063] In some embodiments, the activity of analyte or enzyme can be measured by absorption spectroscopy. In specific embodiments, analyte, or the redox product of enzyme, or the redox product of visible dye has electronic absorption in the visible range of electromagnetic spectrum. In specific embodiments, the redox product of analyte or enzyme or visible dye can be detected by UV-visible spectroscopy. In some embodiments, the amount of the redox product of analyte or enzyme or visible dye in blood sample can be measured by absorption spectroscopy.

[0064] For example, the amount of analyte can be measured by UV-Visible spectroscopy. In particular, the amount of analyte in a blood sample can be measured between about 200nm and 800nm, or between about 300nm and 700nm, or between about 330nm and 650nm. In some embodiments, the amount of analyte is measured at about 340nm, 415nm, 540nm and 575nm. In some embodiments, the amount of analyte is measured at about 340nm. In some embodiments, the amount of analyte is measured at about 415nm. In some embodiments, the amount of analyte is measured at about 540nm. In some embodiments, the amount of analyte is measured at about 575nm. For example, the amount of NADPH can be measured in the range of about 300nm to about 600nm, or in the range of about 330nm to about 350nm. In other embodiments, the amount of NADPH is measured at about 340nm.

[0065] In some embodiments, the amount of the redox product of enzyme or visible dye can be measured between about 200nm and 800nm, or between about 300nm and 700nm, or between about 300nm and 600nm, or between about 330nm and 650nm.In some embodiments, the amount of the redox product of enzyme is measured at about 340nm.Especially, in the scope between about 570nm and 650nm, or in the scope between about 570nm and 600nm, or in the scope between about 615 and 645nm, the amount of the redox product of tetrazolium dye is measured.For example, the amount of the redox product of tetrazolium dye is measured at 578nm.

[0066] In one embodiment of the present disclosure, the activity of G6PDH is measured by: reacting a lysed blood sample for a predetermined reaction time, measuring a first absorption of the lysed blood sample at the beginning of the predetermined reaction time, measuring a second absorption of the lysed blood sample at the end of the predetermined reaction time, calculating a formation rate of NADPH based on an absorbance change between the second absorption and the first absorption; and comparing the formation rate with a standard curve to determine the hematocrit in the blood sample.

[0067] In one embodiment of the present disclosure, measuring the amount of the redox product of the visible dye (e.g., the amount of the reduction product of the tetrazolium dye) includes: reacting the lysed blood sample for a predetermined reaction time, measuring a first absorption of the lysed blood sample at the beginning of the predetermined reaction time, measuring a second absorption of the lysed blood sample at the end of the predetermined reaction time, calculating a formation rate of the redox product of the visible dye based on an absorption change between the second absorption and the first absorption, and comparing the formation rate with a standard curve to determine the hematocrit of the blood sample.

[0068] In specific embodiments, once cell has been cracked or once extra reagent (for example, visible dye) has been added in reaction mixture, predetermined reaction time begins.For example, predetermined reaction time begins at about 0 minute, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes or at least about 15 minutes.In specific embodiments, the end of predetermined reaction time can be at most about 60 minutes, at most about 45 minutes, at most about 30 minutes or at most about 15 minutes.In some embodiments, the beginning of predetermined reaction time can be at least about 0 minute, at least about 5 minutes, at least about 10 minutes or at least about 15 minutes, and the end of predetermined reaction time is at most about 60 minutes, at most about 45 minutes, at most about 30 minutes or at most about 15 minutes.In specific embodiments, the beginning of predetermined reaction time is at least about 0 minute and the end of predetermined reaction time is at least about 45 minutes.In specific embodiments, the beginning of predetermined reaction time is at least about 10 minutes and the end of predetermined reaction time is at least about 15 minutes.

[0069] In a specific embodiment, the formation rate of NADPH or the formation rate of the redox product of the visible dye (such as the amount of the reduction product of tetrazolium dye) is calculated based on the absorption change between the second absorption and the first absorption. For example, the second absorption is subtracted from the first absorption and divided by the predetermined reaction time. The method can also include calculating the formation rate of NADPH or the formation rate of the redox product of the visible dye (such as the amount of the reduction product of tetrazolium dye) in the standard blood sample with known hematocrit to create a standard curve, and by comparing the formation rate of NADPH or the formation rate of the redox product of the visible dye (such as the amount of the reduction product of tetrazolium dye) with the standard curve, the formation rate of NADPH or the formation rate of the redox product of the visible dye (such as the amount of the reduction product of tetrazolium dye) is determined to the hematocrit of the blood sample. In a specific embodiment, the formation rate of NADPH is inversely correlated with the hematocrit in the blood sample.

[0070] In some embodiments, as described herein, in the case of measuring the first absorption and the second absorption, the first absorption and the second absorption can be measured in the UV-visible spectrum. For example, the first absorption and the second absorption can be measured in the range of 200nm to 800nm, or in the range of 300nm to 700nm, or in the range of 330nm to 650nm, or in the range of about 330nm to about 350nm. In some embodiments, the first absorption and the second absorption can be measured in the range of 570nm to 650nm, or in the range of 570nm to 600nm, or in the range of 615 to 645nm. In particular, in some embodiments, the first absorption and the second absorption are measured at about 340nm.

[0071] In some embodiments, the activity of the analyte or enzyme is measured by mass spectrometry. In other embodiments, the amount of the redox product of the enzyme can be measured by mass spectrometry. The mass spectrometer used can be any instrument known in the art and is not particularly limited. For example, the ion source can be selected from an electrospray ionization source, an atmospheric pressure chemical ionization source or an atmospheric pressure photoionization source, and the mass analyzer can be selected from a quadrupole analyzer, a time-of-flight analyzer, an ion trap analyzer and a hybrid analyzer. In a specific example of the present disclosure, an electrospray ionization source and a quadrupole mass analyzer are used.

[0072] For example, in some embodiments, the amount of the redox product of the enzyme can be measured by LCMS. The LCMS method used is not particularly limited and can be any method known in the art. For example, LCMS can use ultra-high pressure liquid chromatography (UHPLC) or high pressure liquid chromatography (HPLC) with a normal phase or reverse phase column. In a specific example of the present disclosure, reversed-phase UHPLC is used. The mass spectrometer used can be any instrument known in the art and is not particularly limited. For example, LCMS can be used to determine the amount of PGA or the amount of GSSG in a blood sample. The amount of PGA or the amount of GSSG can be determined by the common quantitative methods of LCMS known in the art.

[0073] Sample collection and preparation

[0074] In some embodiments of the present disclosure, the blood sample is a liquid whole blood sample or a dried blood spot. The lysis blood sample can be completed by any method known in the art. For example, the lysis blood sample can be formed by mixing the blood sample with a lysis buffer (such as a known surfactant-based buffer). Commercially available buffers such as RIPA lysis and extraction buffer (G BioSciences, 25mM Tri-HCl, 150mM NaCl, 1% Np-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.8, CAT#786-490) can be used.

[0075] A variety of blood collection devices are known, including blood collection tubes and absorbent blood collection materials. Although the shape, form or composition of the blood absorbing material is not limited, the material should be sufficient to receive and secure the blood sample and allow for efficient extraction of the sample. For example, the blood sample can be collected onto a blood absorbing material such as a pad, foam or card made of a porous hydrophilic polymer material (e.g., plastic, paper or fabric). When a collection card is used, the blood sample can be obtained by extracting dried blood from a predetermined area of ​​a dried blood spot on the blood collection card.

[0076] The predetermined area of ​​the dried blood spot is generally determined by the desired sample size for the assay and may be in the range between about 1 mm and 5 mm, in the range between about 1.5 mm and 4.5 mm, in the range between about 2 mm and 4 mm, or in the range between about 2.5 mm and 3.5 mm. In a specific embodiment, the predetermined area of ​​the dried blood spot is about 3 mm.

[0077] In some embodiments, collecting a blood sample comprises applying a device comprising microneedles to the skin of an animal. In some embodiments, collecting a blood sample comprises applying a device to the skin of an animal, the device further comprising an empty absorbent collection material (e.g., a blank collection card) and microneedles. For example, the empty absorbent collection material and the microneedles are in fluid communication with the empty absorbent collection material, and the absorbent collection material with absorbed blood can be removed from the device.

[0078] The laboratory can measure the amount of NADPH in the blood sample, the amount of redox products of a visible dye (e.g., the amount of reduction products of a tetrazolium dye), the activity of G6PDH in the blood sample, or the activity of GR in the blood sample. The dried blood sample can be extracted from the material using a lysis buffer or other suitable extraction reagent.

[0079] In certain aspects of the present disclosure, an exemplary method of the present disclosure includes providing a blank collection card or other collection material to a blood collector, the blood collector collecting a blood sample on the blood collection card or material, allowing the blood sample to dry, and delivering the blood collection card / material to a laboratory for analysis according to the methods of the present disclosure. In some embodiments, the blood collector delivers the blood collection card / material to the laboratory via postal mail or a commercial delivery service.

[0080] Device

[0081] Blood samples for use in the methods disclosed herein can be collected by any method that allows analysis of analytes. In some embodiments, blood is collected using a blood collection device that includes a blood absorbing material, such as a blood collection card or other absorbent material that allows for efficient extraction of a sample. In some embodiments, the device includes a microneedle array that includes a plurality of microneedles. The device may also include a blood storage layer such that the microneedle array delivers a blood sample from an animal to the blood storage layer.

[0082] In some embodiments, the microneedle array includes a plurality of microneedles. Without being bound by theory, each microneedle can draw a predetermined volume of blood within a predetermined time. Thus, the number of microneedles in the microneedle array can be selected to draw a predetermined amount of blood within a predetermined amount of time, the predetermined amount of blood being sufficient to perform one or more blood tests. These collection times can range from 30 seconds to 5 minutes, depending on factors such as the number of microneedles, the type of animal, and / or the amount of blood to be drawn.

[0083] In some examples, the blood storage layer is made of an absorbent material that is structurally configured to store dried blood, the blood storage layer defines a chamber designed to store liquid blood, or a combination of both. For example, blood can be drawn from and through the microneedle array by one or more of the following factors, including the blood pressure of the animal pumping blood through the appendage inserted into the microneedle array, by osmotic action of the absorbent material, and / or other factors.

[0084] In some embodiments, the blood collection device includes a peel-to-expose package, a clamping device, and / or a wearable sleeve to make it easier for a user associated with and / or responsible for an animal to collect a blood sample from the animal using a microneedle array or any suitable combination thereof (as well as other possible means). For example, a peel-to-expose package can be used to selectively cover the microneedle array and the blood storage layer so that the microneedle array and / or the blood storage layer can be exposed for use of the device and resalable when the collection is completed. In some embodiments, the blood collection device can include a clamping device that biases the microneedle array into the appendage of the animal and secures itself to the appendage via a biasing member. In some embodiments, the blood collection device includes a wearable sleeve, wherein the microneedle array can be contacted from and positioned on the outer surface of the wearable sleeve.

[0085] In some examples, each microneedle contains up to about 100 μL, or up to about 80 μL, or up to about 60 μL, or up to about 40 μL, or up to about 20 μL of blood. In another embodiment, the device further comprises a blood collection card and the microneedles are in fluid communication with the blood collection card. In certain embodiments, the blood collection material can be removed from the device.

[0086] In certain embodiments, the device is provided to a blood collector who collects a blood sample, dries the blood sample, and delivers the blood collection material to a laboratory. More particularly, the laboratory extracts the dried blood and measures the amount of NADPH in the blood sample, the amount of the redox product of the visible dye (e.g., the amount of the reduction product of the tetrazolium dye), the activity of G6PDH in the blood sample, or the activity of GR in the blood sample. In some embodiments, the blood collector delivers the blood collection material to the laboratory via postal mail or a commercial delivery service. Example

[0087] The following embodiments are specific embodiments of the present disclosure that are illustrative. They are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0088] Example 1: Preparation of whole blood standards and dried blood spot standards

[0089] To prepare liquid whole blood samples, 50 mL of canine whole blood was centrifuged at 1500 x g at room temperature to separate blood from plasma. The plasma was decanted, and then appropriate volumes of blood and plasma were transferred using a positive displacement pipette to prepare blood samples with different hematocrit levels. Table 1 describes the preparation of blood samples with different hematocrit levels.

[0090] Table 1

[0091]

[0092] Each prepared sample was run on a commercially available (Sysmex XN series hematology analyzer and / or IDEXX The test was performed on a hematocrit measurement device (hematology analyzer) to confirm the hematocrit level. To prepare the DBS sample, 3 μL of each liquid whole blood sample was spotted on a DBS card and allowed to dry overnight. The DBS card was stored in a dry plastic bag with desiccant until needed.

[0093] Example 2: NADPH as a measure of hematocrit in liquid whole blood samples

[0094] The amount of NADPH present in the blood in the lysed blood samples containing different hematocrits (HCT%) was determined. For determination, 200 μL RIPA lysis and extraction buffer (G BioSciences, 25 mM Tri-HCl, 150 mM NaCl, 1% Np-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.8, CAT#786-490) was added to a microcentrifuge tube. Liquid whole blood samples with 0%, 36.5%, 44%, 52% or 70% hematocrit were added to a microcentrifuge tube in an amount of 3 μL, 6 μL or 12 μL. The solution was briefly vortexed (for about 10 seconds) and then incubated at room temperature for 15 minutes. The electronic absorption spectrum of each sample was measured, tracking the absorption at 340, 405 and 578 nm. Table 2 reports the absorbance at 340, 405 and 578 nm between different lysed blood samples for 3 μL and 6 μL initial liquid whole blood sample volumes.

[0095] Table 2

[0096]

[0097] Figure 1A and Figure 1B The absorbance at each specified wavelength is plotted as a function of the percent hematocrit present. Figure 1A and 1B It can be observed that there is a linear correlation between the absorbance measured and the hematocrit present. By using this linear relationship, the amount of NADPH present in other blood samples can be used to determine the hematocrit of that blood sample.

[0098] Example 3: NAPDH as a measure of hematocrit in dried blood spots (DBS)

[0099] The presence of NADPH in the blood was determined in dried blood spots (DBS) obtained from whole blood samples with different hematocrits (HCT%). To generate DBS samples, the hematocrit whole blood standard prepared in Example 1 was dried on DBS cards. The 3 mm punch of each card was then mixed with 200 μL of RIPA lysis and extraction buffer (G BioSciences, 25 mM Tri-HCl, 150 mM NaCl, 1% Np-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.8, CAT# 786-490). The DBS card solution was vortexed for approximately 10 seconds and mechanically mixed with a pipette tip to improve solubility. The DBS card solution was then incubated at room temperature for 15 minutes. Electronic absorbance was obtained for each sample, tracking the absorption at 340, 415, 540, and 575 nm. Table 3 reports the absorbance between different DBS samples.

[0100] Table 3

[0101]

[0102] Figure 2A The measured electronic absorption spectra of each DBS card solution are plotted. Figure 2B The absorbance at each designated wavelength is plotted as a function of the hematocrit present in the DBS hematocrit standard. Figure 2B A linear correlation between the measured absorbance and the hematocrit present in the whole blood sample is shown, and reflects that rehydrating DBS with lysis buffer can provide a rapid and simple method to directly measure the absorbance from endogenous NADPH or hemoglobin. This assay also provides a method to measure the hematocrit present in a dried blood sample by correlating the amount of NADPH present in the sample with the hematocrit.

[0103] Example 4: Tetrazolium dye kinetics assay

[0104] To measure the hematocrit in liquid whole blood samples or dried blood spots (DBS), the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), or other tetrazolium dyes can be used with phenazine methosulfate (PMS) as a mediator. Phenazine methosulfate improves the reduction rate of tetrazolium dyes by promoting single electron transfer.

[0105] Lysed blood samples were prepared for tetrazolium dye assays by the methods described for liquid whole blood samples in Example 2 or for DBS samples in Example 3. Once lysed, 50 μL of red blood sample solution (from liquid whole blood or DBS) was pipetted in duplicate into two wells of a 96-well plate (Corning-Costar, UV-transparent flat bottom plates, non-sterile, acrylic).

[0106] To perform the assay, prepare a 1.5 mM working solution of MTT (Molecular Probes, No. M6494, molecular weight 414.32 g / mol) in DI water and a 75 μM working solution of PMS (Sigma-Aldrich, PN: P9625-10G, molecular weight 306.34 g / mol) in DI water. Mix the MTT working solution and the PMS working solution in a 1:1 ratio. Add 100 μL of the MTT / PMS solution to each well containing a blood sample. Immediately transfer the plate to a plate reader to measure the absorbance of the reduced MTT dye.

[0107] The absorbance spectra were monitored using a Biotek Synergy 4 microplate reader. The temperature of the plate reader was set to 37 °C with the oscillator speed set to medium. The electronic absorption of the reduced dye was measured continuously at the desired wavelength during the set reaction time. For the MTT dye assay, the absorbance was measured every minute at 578 nm for 5 minutes. However, if other tetrazolium dyes are used, different wavelengths and reaction times can be used. Figure 3A The absorbance spectrum of the MTT dye / sample mixture at 30 minutes reaction time is shown and compared to the spectra of the sample without dye and the dye alone.

[0108] At the end of the 5 minute reaction time, the data from the Biotek plate reader were exported for analysis. The absorbance at 578 nm 5 minutes after dye addition was plotted as Figure 3B By plotting the absorbance versus the hematocrit present in the lysed sample, a linear correlation was observed.

[0109] Example 5: G6PDH kinetics assay

[0110] The rate of NADPH formation in lysed blood samples containing different hematocrits (HCT%) was determined. Lysed blood samples were prepared by the method described in Example 2. Lysed blood samples can also be obtained from DBS by the method described in Example 3. Once lysed, 50 μL of the red blood sample solution was pipetted in duplicate into two wells of a 96-well plate (Corning-Costar, UV transparent flat bottom plate, non-sterile, acrylic).

[0111] For the assay, a 10 ng / mL working solution of G6PDH (Sigma-Aldrich, PN: G8529-10KU, 2 mg / mL), an 8 mM working solution of NAD (Sigma-Aldrich, PN: N7004-10G, molecular weight 663.43 g / mol), and a 12 mM working solution of G6P (Sigma-Aldrich, PN: G7879-25G, molecular weight 282.12 g / mol) were prepared. All these solutions were prepared in 100 mM tris-HCl buffer (pH 8.0) and stored at 4°C. The NAD working solution was stored in a dark place until needed to protect from any light.

[0112] Mix G6P and NAD working solutions at a 1:1 ratio. Add 100 μL of the G6P / NAD mixture to each well. Add 50 μL of G6PDH working solution to each well containing a blood sample. Immediately transfer the plate to a plate reader to measure the absorbance of the NADPH dye.

[0113] The absorbance was monitored using a Biotek Synergy 4 microplate reader. The temperature of the plate reader was set to 37°C with the oscillator speed set to medium. During the set reaction time, the electronic absorption of the sample was continuously measured at the desired wavelength. For the G6PDH assay, the absorbance was measured every minute at 340 nm for 45 minutes. Figure 4 The change in absorbance at 340 nm over time for the different hematocrit standards measured is illustrated.

[0114] At the end of the 45 minute reaction time, the data from the Biotek plate reader was exported for analysis. The absorbance change at 340 nm was calculated as the difference in absorbance between 0 and 45 minutes and the difference in absorbance between 10 and 15 minutes. The reaction rate based on these changes in absorbance was also calculated. The determination of the initial 6 μL liquid whole blood with different hematocrits is reported in Tables 4 and 5, respectively.

[0115] Table 4

[0116]

[0117] Table 5

[0118]

[0119] By plotting the reaction rate versus the hematocrit present in the lysed blood sample, a linear correlation was observed. Figure 5A and Figure 5B This correlation is shown for an initial 3 μL sample of liquid whole blood and an initial 6 μL sample of liquid whole blood. It can be seen that the 6 μL sample of liquid whole blood that was reacted for 45 minutes produced a good correlation between enzyme activity and hematocrit percentage. Previously, a correlation was observed between decreased enzyme activity and increased hematocrit percentage. Without being bound by theory, it is assumed that the decrease in enzyme activity is due to the addition of NAD to the reaction mixture. + Instead of NADP + This functional assay confirmed that in the presence of NADP + When present, NAD was added + In fact, the enzyme activity is inhibited. In addition, the G6PDH assay provides a measurement of the hematocrit in a blood sample based on the rate of NADPH formation. Using the NADPH formation rate, it is possible to Figure 5A and Figure 5B The correlations shown in determine the hematocrit in other blood samples.

[0120] Example 6: LCMS determination of G6PDH activity as a hematocrit marker

[0121] Another assay that can be used to determine the hematocrit in a blood sample is a liquid chromatography mass spectrometry (LCMS) assay. As with the electron absorption assays of Examples 4 and 5, this assay can be performed with liquid whole blood or dried blood spots. For this assay, the production of the G6PDH redox product 6-phosphogluconate (PGA) in a dried blood sample is monitored by LCMS and correlated with the hematocrit present in whole blood.

[0122] To generate lysed blood samples for measurement, the same lysis procedure as described for DBS samples in Example 3 was used. Lysed blood samples can also be obtained by the method described in Example 2. Once lysed, 50 μL of the red blood sample solution was pipetted in duplicate into two wells of a 96-well plate (Corning-Costar, UV transparent flat bottom plates, non-sterile, acrylic).

[0123] For the assay, a 10 ng / mL G6PDH working solution was prepared by diluting G6PDH (50 μg / mL stock solution of G6PDH (Sigma-Aldrich, PN: G8529-10KU, 2 mg / mL) in 10 mM ammonium bicarbonate buffer (Sigma-Aldrich, PN: A6141, pH 8.5). An 8 mM working solution of NADP (Sigma-Aldrich, PN: N7004-10G, molecular weight 663.43 g / mol) and a 12 mM working solution of G6P (Sigma-Aldrich, PN: G7879-25G, molecular weight 282.12 g / mol) were prepared in deionized water. All solutions were stored at 4°C. The NAD working solution was stored in a dark place to protect from any light. The G6PDH working solution was used within 30 minutes of preparation.

[0124] To the wells containing the lysed blood samples, 50 μL of G6P working solution, 50 μL of NADP working solution, and 50 μL of G6PDH working solution were added. The wells were gently vortexed. Then, 600 μL of methanol was added to each well. The samples were then centrifuged at 3000 x g for 10 minutes at room temperature. The resulting supernatant was transferred to a new 96-well plate for analysis by LCMS. Table 7 reports the parameters of the LCMS used.

[0125] Table 7

[0126]

[0127]

[0128]

[0129] Figure 6A representative MS chromatogram of 6-phosphogluconate (PGA) is shown in . The area of ​​the MS chromatogram of PGA for 4 different samples containing different hematocrit levels (which is proportional to the amount of PGS present) was calculated and reported in Table 8 against the theoretical hematocrit (HCT%). Figure 7 The MS chromatogram area of ​​PGA was plotted as a function of actual HCT%, Figure 7 This reflects the good linear correlation between the area of ​​the MS chromatogram of PGA and the hematocrit present. Using the amount of PGA present, it is possible to Figure 7 The correlations shown in determine the hematocrit in other blood samples.

[0130] Table 8

[0131]

[0132] Example 7: LCMS Assay of Glutathione Reductase Activity as Hematocrit Marker Another LCMS assay that can be used to determine hematocrit in a blood sample measures the production of oxidized glutathione (i.e., glutathione disulfide). As with the other examples, this assay can be performed with liquid whole blood or dried blood spots.

[0133] Liquid whole blood samples were prepared in the same manner as described in Example 6. In order to prepare samples for analysis by LCMS, 3 mm DBS punched blood spots or 3 μL liquid whole blood were placed in a tube (or well). 50 μL of 200 μg / mL glutathione disulfide (GSSG) internal standard solution (prepared in DI water) was added. The solution was then ultrasonically treated for 30 minutes, followed by centrifugation at room temperature for 10 minutes at 3500 x g. 150 μL of methanol was added to the solution, and the solution was ultrasonically treated again for 30 minutes, and then centrifuged at room temperature for 10 minutes at 3500 x g. In order to produce a final solution to be analyzed by LCMS, the centrifuged solution was filtered through a 0.2-0.4 um filter / filter plate (GHP or Teflon). The parameters of LCMS are shown in Table 9.

[0134] Table 9

[0135]

[0136]

[0137] This LCMS assay was used to determine the hematocrit of seven different samples containing different hematocrit levels. Figure 8A representative MS chromatogram of GSSG is shown in . Using the area of ​​the MS chromatogram of GSSG, the amount of GSSG was calculated. The hematocrit of each sample was calculated based on the amount of GSSG present. The mean hematocrit information for each level tested is reported in Table 10. Fig. 9 The calculated HCT% is plotted as a function of the reference HCT%, Fig. 9 This reflects the good linear correlation between the calculated HCT% and the reference HCT%. Using the amount of GSSG present, it is possible to Fig. 9 The correlations shown in determine the hematocrit in other blood samples.

[0138] Table 10

[0139] HCT Level HCT% Reference Standard N Calculated HCT% (average) 1 10 3 10.4 2 20 3 19.0 3 30 3 30.7 4 40 3 40.5 5 50 3 49.9 6 60 3 59.4 7 70 3 70.3

Claims

1. A method for determining the hematocrit of a blood sample, the method comprising: lysing the blood sample to provide a lysed blood sample, measuring the amount of NADPH in the lysed blood sample, The amount of NADPH in the lysed blood sample is correlated to the hematocrit of the blood sample.

2. The method of claim 1, wherein said measuring the amount of NADPH in said lysed blood sample comprises measuring UV-visible absorbance.

3. The method of claim 2, wherein the amount of NADPH is measured in the range of about 300 nm to about 600 nm.

4. The method of claim 3, wherein the amount of NADPH is measured in the range of about 330 nm to about 350 nm.

5. The method of claim 4, wherein the amount of NADPH is measured at about 340 nm.

6. The method of claim 1, wherein said correlating the amount of NADPH in the lysed blood sample with the hematocrit of the blood sample comprises comparing the amount of NADPH to a standard curve.

7. A method for determining the hematocrit of a blood sample, the method comprising: lysing the blood sample to provide a lysed blood sample, measuring the activity of G6PDH in the lysed blood sample; The activity of G6PDH in the lysed blood sample is correlated with the hematocrit of the blood sample.

8. The method of claim 7, wherein said measuring the activity of G6PDH in said lysed blood sample comprises measuring UV-visible absorbance.

9. The method of claim 7, wherein said measuring the activity of G6PDH in said lysed blood sample comprises measuring mass spectrometry.

10. The method according to claim 7, further comprising: Before measuring the activity of the G6PDH, NAD + or NADP + adding to the lysed blood sample; The activity of G6PDH in the lysed blood sample was determined.

11. The method of claim 1 or claim 7, wherein said lysing said blood sample comprises mixing the blood sample with a lysis buffer.

12. The method of claim 7 or claim 10, wherein said correlating the activity of G6PDH in the lysed blood sample with the hematocrit of the blood sample comprises comparing the activity of G6DPH to a standard curve.

13. The method according to claim 7 or claim 10, wherein the activity of G6PDH in the blood sample is determined by measuring the amount of G6PDH redox products in the blood sample.

14. The method of claim 13, wherein the G6DPH redox product is selected from the group consisting of 6-phosphogluconolactone and NADPH.

15. The method according to claim 7 or 10, wherein said measuring the activity of G6PDH comprises: Allowing the lysed blood sample to react for a predetermined reaction time; measuring a first absorbance of the lysed blood sample at the beginning of a predetermined reaction time; measuring a second absorbance of the lysed blood sample at the end of the predetermined reaction time; calculating a formation rate of NADPH based on a change in absorbance between the second absorbance and the first absorbance; and The rate of formation is correlated to a standard curve to determine the hematocrit of the blood sample.

16. The method of claim 15, wherein said measuring a first absorption and said measuring a second absorption comprises measuring UV-visible absorption.

17. The method of claim 16, wherein the first absorption and the second absorption are measured in a range of about 330 nm to about 350 nm. The method of claim 17 , wherein the first absorption and the second absorption are measured at 340 nm.

19. The method of any one of claims 15-18, wherein the rate of formation of NADPH is inversely correlated with the hematocrit in the blood sample.

20. The method according to claim 7, wherein said measuring the activity of G6PDH comprises: lysing the blood sample to provide a lysed blood sample, measuring the amount of 6-phosphogluconolactone (PGA) in the blood sample; The amount of PGA in the lysed blood sample is compared to a standard curve to determine the hematocrit of the blood sample.

21. A method of determining the hematocrit of a blood sample, the method comprising: lysing the blood sample to provide a lysed blood sample, measuring the activity of glutathione reductase in the blood sample; The activity of the glutathione reductase is correlated to the hematocrit in the blood sample.

22. The method of claim 21, further comprising: adding an internal standard solution of GSSG to the blood sample; determining the activity of glutathione reductase in the blood sample; The glutathione reductase activity was correlated to a standard curve to determine the hematocrit of the blood sample.

23. The method according to claim 21 or 22, further comprising: measuring the amount of GSSG in the blood sample; The amount of GSSG was compared to a standard curve to determine the hematocrit of the blood sample.

24. A method of determining the hematocrit of a blood sample, the method comprising: lysing the blood sample to provide a lysed blood sample; adding a tetrazolium dye to the lysed blood sample; measuring the amount of the reduction product of the tetrazolium dye in the lysed blood sample, The amount of reduction product of the tetrazolium dye in the blood sample is correlated to the hematocrit of the blood sample.

25. The method of claim 24, wherein said measuring the amount of reduction product of the tetrazolium dye in the lysed blood sample comprises measuring UV-visible absorbance.

26. The method of claim 25, wherein the amount of the reduction product of the tetrazolium dye is measured in the range between about 570 nm and 650 nm.

27. The method of claim 26, wherein the amount of the reduction product of the tetrazolium dye is measured in the range between about 570 nm and 600 nm.

28. The method of claim 27, wherein the amount of the reduction product of the tetrazolium dye is measured at 578 nm.

29. The method of claim 27, wherein the amount of the reduction product of the tetrazolium dye is measured in the range between about 615 nm and 645 nm.

30. The method of claim 24, wherein said correlating the amount of the reduction product of the tetrazolium dye in the blood sample to the hematocrit comprises comparing the amount of the reduction product of the tetrazolium dye to a standard curve.

31. The method of claim 24, wherein measuring the amount of the reduction product of the tetrazolium dye comprises: Allowing the lysed blood sample to react for a predetermined reaction time; measuring a first absorbance of the lysed blood sample at the beginning of a predetermined reaction time; measuring a second absorbance of the lysed blood sample at the end of a predetermined reaction time; calculating a formation rate of a redox product of the tetrazolium dye based on a change in absorbance between the second absorbance and the first absorbance; and The rate of formation is correlated to a standard curve to determine the hematocrit of the blood sample.

32. The method of claim 24 or 31, further comprising adding phenazine methosulfate to the lysed blood sample.

33. The method of claim 24 or 31, wherein lysing the blood sample comprises mixing the blood sample with a lysis buffer.

34. The method of claim 31, wherein said measuring said first absorption and said measuring said second absorption comprises measuring UV-visible absorption.

35. The method of claim 34, wherein the first absorption and the second absorption are measured in the range between 570 nm and 650 nm.

36. The method of claim 35, wherein the first absorption and the second absorption are measured in the range between 570 nm and 600 nm.

37. The method of claim 36, wherein the first absorption and the second absorption are measured at 578 nm.

38. The method of claim 34, wherein the first absorption and the second absorption are measured in a range between 615 nm and 645 nm.

39. The method of any one of claims 24-38, wherein the tetrazolium dye is selected from 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide or 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.

40. The method of any one of claims 1 to 39, wherein the blood sample is a liquid whole blood sample.

41. The method according to any one of claims 1 to 39, wherein the blood sample is obtained by extracting dried blood from a predetermined area of ​​dried blood on a blood collection material.

42. The method of claim 40, wherein the dried blood spot is obtained by obtaining whole blood from an animal using a microneedle.

43. The method of claim 40 or 42, wherein the whole blood sample is selected from the group consisting of canine blood, avian blood, feline blood, mouse blood, horse blood, and human blood.

44. The method of claim 41, wherein the predetermined area is between approximately 1 mm and 5 mm.

45. The method of claim 41, wherein the predetermined area is approximately 3 mm.

46. ​​A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood obtained from the blood sample on the blood collection card, measuring the amount of NADPH in the extracted dried blood, and The amount of NADPH is correlated to the hematocrit of the blood sample.

47. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood obtained from the blood sample on the blood absorbing material, measuring the amount of reduction product of the tetrazolium dye in the extracted dried blood, and The amount of the reduction product of the tetrazolium dye is correlated to the hematocrit of the blood sample.

48. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood obtained from the blood sample on the blood collection card, measuring the activity of G6PDH in the extracted dried blood, and The activity of the G6PDH is correlated to the hematocrit of the blood sample.

49. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood obtained from the blood sample on the blood collection card, measuring the activity of glutathione reductase in the extracted dried blood, and The activity of the glutathione reductase is correlated to the hematocrit of the blood sample.

50. The method of any one of claims 46 to 49, wherein the blood sample is collected using a device comprising a microneedle array, the microneedle array comprising a plurality of microneedles.

51. The method of claim 50, wherein the device further comprises a blood absorbing material.

52. The method of claim 50, wherein each microneedle can collect up to about 20 μL of blood.

53. The method of claim 51, wherein the blood absorbing material is a blood collection card.

54. The method of claim 52, wherein the blood collection card is removable from the device.

55. The method of claim 52, wherein a blank collection card is provided to a blood collector, who collects a blood sample on the blood collection card, allows the blood sample to dry, and delivers the blood collection card to a laboratory that measures the amount of NADPH.

56. The method of claim 52, wherein a blank collection card is provided to a blood collector, who collects a blood sample on the blood collection card, allows the blood sample to dry, and delivers the blood collection card to a laboratory that measures the amount of the reduction product of the tetrazolium dye.

57. The method of claim 52, wherein a blank collection card is provided to a blood collector, who collects a blood sample on the blood collection card, allows the blood sample to dry, and delivers the blood collection card to a laboratory that measures the activity of G6PDH.

58. The method of claim 52, wherein a blank collection card is provided to a blood collector, who collects a blood sample on the blood collection card, allows the blood sample to dry, and delivers the blood collection card to a laboratory that measures the activity of glutathione reductase.

59. The method of any one of claims 54 to 57, wherein the blood collector delivers the blood collection card to the laboratory via postal mail or a commercial delivery service.

60. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from an animal on a blood collection card from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the amount of NADPH in the extracted dried blood, and The amount of NADPH is correlated to the hematocrit of the blood sample.

61. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from an animal on a blood collection material from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the amount of tetrazolium dye in the extracted dried blood, and The amount of the tetrazolium dye is correlated to the hematocrit of the blood sample.

62. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from an animal on a blood collection material from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the activity of G6PDH in the extracted dried blood, and The activity of the G6PDH is correlated to the hematocrit of the blood sample.

63. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from an animal on a blood collection material from a blood collector; extracting a predetermined amount of dried blood from the blood collection card; measuring the activity of glutathione reductase in the extracted dried blood, and The activity of the glutathione reductase is correlated to the hematocrit of the blood sample.

64. The method of any one of claims 59 to 62, wherein the blood collector collects the blood sample using a device comprising: (i) a microneedle array comprising microneedles; and (ii) the blood collection material.

65. The method of claim 63, wherein the material is a blood collection card.

66. The method of claim 64, wherein the blood collection card is removable from the device.

67. The method of claim 64, wherein each microneedle contains up to about 20 μL of blood.

68. The method of any one of claims 59-62, wherein said collecting a blood sample is completed in up to about 2 minutes.

69. The method of any one of claims 59-62, wherein said collecting a blood sample is completed within about 1 to 2 minutes.

70. The method of claim 63, wherein the device is provided to the blood collector, who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory where the dried blood is extracted and the amount of NADPH is measured.

71. The method of claim 63, wherein the device is provided to the blood collector, who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory where the dried blood is extracted and the amount of the reduction product of the tetrazolium dye is measured.

72. The method of claim 63, wherein the device is provided to the blood collector, who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory where the dried blood is extracted and the activity of G6PDH is measured.

73. The method of claim 63, wherein the device is provided to the blood collector, who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory where the dried blood is extracted and the activity of glutathione reductase is measured.

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