Hematocrit determination

Abstract

Methods and devices for determining the hematocrit of a blood sample. One aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample. The method 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 to the hematocrit of the blood sample. Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample. The method includes lysing a 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 to the hematocrit of the blood sample.

Description

[Technical Field]

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

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to determining hematocrit in a blood sample. [Background technology]

[0003] background Hematocrit is the percentage of blood volume that is composed of red blood cells and is expressed as HCT %. In healthy adult individuals, red blood cells comprise approximately 40–48% of blood volume, whereas newborns can have a hematocrit of up to 60%. Clinically, hematocrit measurement may be requested when a patient is suspected to be anemic or suffering from dehydration, bleeding, or other medical and surgical conditions. A low hematocrit reflects a low number of circulating red blood cells and is an indicator of reduced oxygen-carrying capacity or overhydration. Examples of conditions that cause low hematocrit (anemia) include internal or external bleeding, chronic renal failure and / or complications of renal disease, pernicious anemia (vitamin B12 deficiency), and hemolysis (often associated with transfusion reactions). Low hematocrit can also be found in autoimmune diseases and bone marrow failure. High hematocrit can reflect an absolute increase in red blood cell count or a decrease in plasma volume in conditions such as severe dehydration, polycythemia (excessive red blood cell production), polycythemia vera (an abnormal increase in blood cells), and hemochromatosis (a hereditary disorder of iron metabolism). High hematocrit is also used as an indicator of excessive uptake of exogenous erythropoietin (EPO), which stimulates red blood cell production.

[0004] Conventionally, HCT % is often determined by centrifuging a liquid whole blood sample to separate the liquid whole blood components based on density and determining the percentage of packed red blood cell volume relative to the total blood volume. However, this method is not useful for low volume samples or dried blood samples. Therefore, the present inventors have identified a need in the art for efficiently determining hematocrit from such samples. Summary of the Invention [Means for solving the problem]

[0005] Abstract One aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method including the steps of 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 to the hematocrit of the blood sample.

[0006] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method including 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 to the hematocrit of the blood sample.

[0007] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method including the steps of 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 is directed to a method for determining the hematocrit of a blood sample, the method including the steps of 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 is directed to a method for determining the hematocrit of a blood sample, the method comprising extracting a predetermined amount of dried blood 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 to the hematocrit of the blood sample.

[0010] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method including extracting a predetermined amount of dried blood from the blood sample on a blood absorbing device, measuring the amount of reduction products of a tetrazolium dye in the extracted dried blood, and correlating the amount of reduction products of the tetrazolium dye to the hematocrit of the blood sample.

[0011] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising extracting a predetermined amount of dried blood 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 to the hematocrit of the blood sample.

[0012] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising extracting a predetermined amount of dried blood 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 to the hematocrit of the blood sample.

[0013] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising extracting a predetermined amount of dried blood 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 to the hematocrit of the blood sample.

[0014] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method including extracting a predetermined amount of dried blood from the blood sample on a blood absorbing device, measuring the amount of reduction products of a tetrazolium dye in the extracted dried blood, and correlating the amount of reduction products of the tetrazolium dye to the hematocrit of the blood sample.

[0015] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising extracting a predetermined amount of dried blood 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 to the hematocrit of the blood sample.

[0016] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising extracting a predetermined amount of dried blood 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 to the hematocrit of the blood sample.

[0017] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising the steps of 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 to the hematocrit of the blood sample.

[0018] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising the steps of receiving from a blood collector a dried blood sample from an animal on a blood collection device, extracting a predetermined amount of dried blood from a blood collection card, measuring the amount of tetrazolium dye in the extracted dried blood, and correlating the amount of tetrazolium dye to the hematocrit of the blood sample.

[0019] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising the steps of receiving a dried blood sample from an animal on a blood collection device 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 to the hematocrit of the blood sample.

[0020] Another aspect of the present disclosure is directed to a method for determining the hematocrit of a blood sample, the method comprising the steps of receiving from a blood collector a dried blood sample from an animal on a blood collection device, extracting a predetermined amount of dried blood from a blood collection card, measuring the activity of glutathione reductase in the extracted dried blood, and correlating the activity of glutathione reductase to the hematocrit of the blood sample.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are included to provide a further understanding of the present disclosure, and which are incorporated in and constitute a part of this specification, illustrate embodiments of the present disclosure and, together with the detailed description, serve to explain the principles of the present disclosure. No attempt is made to show structural details of the present disclosure in more detail than may be necessary for a fundamental understanding of the disclosure and the various ways in which it may be practiced. [Brief explanation of the drawings]

[0022] [Figure 1]Figures 1A and 1B are linear plots of the correlation between hematocrit (HCT %) and absorbance at 340 nm, 405 nm, and 578 nm for lysed blood samples made from either 3 μL or 6 μL of liquid whole blood, respectively.

[0023] [Figure 2] Figure 2A is a plot of UV-visible absorption spectra from lysed red blood samples obtained from dried blood spots containing different hematocrits, and Figure 2B shows the correlation between hematocrit (HCT %) and absorbance at 340 nm, 405 nm, and 578 nm for lysed blood samples obtained from dried blood spots.

[0024] [Figure 3] Figure 3A is a plot of UV-visible absorption spectra from three samples: a lysed red blood sample without tetrazolium dye (MTT) and without single-electron transfer catalyst (phenazine methosulfate), a lysed red blood sample with tetrazolium dye (MTT) and single-electron transfer catalyst (phenazine methosulfate), and tetrazolium dye (MTT) and single-electron transfer catalyst (phenazine methosulfate) in Tris buffer. Figure 3B shows the correlation between hematocrit (HCT%) and absorbance at 578 nm for the lysed blood samples.

[0025] [Figure 4] FIG. 4 is a plot of the change in absorbance at 340 nm over time for lysed red blood samples containing different hematocrits (HCT %).

[0026] [Figure 5] Figures 5A and 5B are linear plots of the correlation between hematocrit (HCT %) and the rate of NADPH formation over periods of 10-15 minutes and 0-45 minutes for lysed red blood samples made from either 3 μL or 6 μL of liquid whole blood, respectively.

[0027] [Figure 6] FIG. 6 is a mass spectrometry chromatogram of 6-phosphogluconolactonate (PGA) from a lysed blood sample.

[0028] [Figure 7] Figure 7 is a linear plot of the correlation between hematocrit (HCT %) of lysed red blood samples containing different hematocrits (HCT %) and the area of each mass spectrometry chromatogram of 6-phosphogluconolactonate (PGA).

[0029] [Figure 8] FIG. 8 shows mass spectrometry chromatograms of glutathione disulfide (GSSG) from lysed red blood samples containing different hematocrits (HCT %).

[0030] [Figure 9] FIG. 9 is a scatter plot of the correlation between hematocrit (HCT %) of dried blood samples containing different hematocrits (HCT %) and the area of each mass spectrometry chromatogram of glutathione disulfide (GSSG). DETAILED DESCRIPTION OF THE INVENTION

[0031] explanation In various aspects, the present disclosure relates to methods for determining the hematocrit of a blood sample. The methods include measuring the amount of one or more analytes and / or the activity of an enzyme in the blood sample and correlating the measurements to the amount of hematocrit. The 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.

[0032] The methods described herein provide an efficient method for determining the hematocrit of dried blood samples, as well as blood samples that require smaller volumes (e.g., less than 100 μL), and are more accurate than conventional methods. The methods can use conventional analytical laboratory equipment, and can be easily incorporated into the routine workflow of analytical laboratories.

[0033] Blood samples for use in the methods disclosed herein can be collected using conventional and proprietary devices and methods, hi some embodiments, blood can be collected with absorbent blood collection devices, microneedles, or devices that combine both of these technologies.

[0034] definition

[0035] Before describing this disclosure in further detail, a number of terms will be defined:

[0036] The blood sample may include liquid whole blood or dried blood. Liquid whole blood includes red blood cells, white blood cells, platelets, and plasma and may be collected from humans or any other animal species. For example, in some embodiments, the whole blood sample is selected from the group including dog blood, avian blood, cat blood, mouse blood, horse blood, and human blood. Dried blood spots (DBS) are formed from liquid whole blood and dried onto a blood collection implement or other suitable collection device (e.g., a blood-absorbing implement such as a card, pad, or rigid foam).

[0037] Lysed blood samples are produced by treating liquid whole blood or dried blood samples with a lysis process. Lysing a blood sample can be achieved by any means known in the art. For example, lysing a blood sample can be achieved by mixing the blood sample with a lysis buffer.

[0038] NADP + is nicotinamide adenine dinucleotide phosphate, and NADPH is NADP +It is the reduced form of

[0039] NAD + is nicotinamide adenine dinucleotide, and NADH is NAD + It is the reduced form of

[0040] G6PDH is the enzyme glucose-6-phosphate dehydrogenase, which oxidizes D-glucose-6-phosphate to 6-phosphogluconolactone and NADP + It catalyzes the reduction of NADPH to NADPH.

[0041] PGA is 6-phosphogluconolactone, the oxidized form of D-glucose-6-phosphate.

[0042] GR is glutathione reductase, an enzyme that catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH).

[0043] LCMS is liquid chromatography mass spectrometry.

[0044] The term "analyte," as used herein, generally refers to a substance or set of substances present in a sample. An analyte may be inherent in the composition of a sample or may be added to the sample. Visible dyes and fluorescent dyes may be considered examples of analytes that are added to a sample. When an analyte is added to a sample, the measurement of the analyte may be related to the hematocrit as a result of a reaction between the added analyte and other inherent components in the sample.

[0045] The term "enzyme activity," as used herein, generally refers to a reaction that catalyzes the conversion of a substance in a sample to produce a reaction product, such as a redox product. For example, NADPH and PGA are redox products of G6DPH.

[0046] method

[0047] In various aspects, the present disclosure relates to a method for determining 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 to 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 measuring the analyte in a standard blood sample having a known hematocrit and creating a standard curve by relating the hematocrit of the standard sample to the amount of analyte in the standard sample. The amount of 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 analyte in a standard blood sample having a known hematocrit. If a lysed blood sample is used to determine the hematocrit, the standard curve may be created using the lysed blood sample.

[0048] In one embodiment, the analyte can 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 to the hematocrit in the blood sample. The method can include measuring the amount of NADPH in standard blood samples having known hematocrits 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.

[0049] 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 can be measured by measuring the activity of NADP + For example, G6PDH and GR depend on NADP+ to NADPH.

[0050] The method may 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 of the blood sample based on the activity of the enzyme.The activity of the enzyme in the lysed blood sample may be determined by measuring the amount of the enzyme's redox product.For example, if the enzyme is G6PDH, the redox product may be 6-phosphogluconolactone (PGA) and NADPH.

[0051] In one embodiment of the present disclosure, NAD + or NADP + is added to the lysed blood sample before measuring the activity of G6DPH in the lysed blood sample. + or NADP + The addition of NADPH may allow for the detection of greater amounts of the redox product, NADH or NADPH, than would normally be inherently present in the sample. For example, in some embodiments, NAD + and NADP + is added to the lysed blood sample prior to measuring the activity of G6PDH in an amount ranging from about 1% weight (mg) / volume (mL) to 30% weight (mg) / volume (mL), from about 5% weight (mg) / volume (mL) to 25% weight (mg) / volume (mL), from about 10% weight (mg) / volume (mL) to 20% weight (mg) / volume (mL), or from about 10% weight (mg) / volume (mL) to 15% weight (mg) / volume (mL). In certain embodiments, NAD +is added to the lysed blood sample before measuring the activity of G6PDH. In another embodiment, glucose-6-phosphate (G6P) is added to the lysed blood sample before measuring the activity of G6PDH in the lysed blood sample. For example, in some embodiments, G6P is added in an amount ranging from about 1% weight (mg) / volume (mL) to about 20% weight (mg) / volume (mL), from about 5% weight (mg) / volume (mL) to about 15% weight (mg) / volume (mL), or from about 5% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL). In another embodiment, a small amount of G6PDH is added to the lysed blood sample before measuring the activity of G6PDH in the lysed blood sample. For example, in some embodiments, G6PDH is added in an amount ranging from about 0.5×10 -5 % Weight (mg) / Volume (mL)~Approx. 5.0×10 -5 % weight (mg) / volume (mL) range, approximately 1.5 x 10 -5 % Weight (mg) / Volume (mL)~Approx. 3.5×10 -5 % weight (mg) / volume (mL) range, or approximately 2.0 x 10 -5 % Weight (mg) / Volume (mL)~Approx. 3.0×10 -5 % weight (mg) / volume (mL) range.

[0052] 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 of the blood sample. The standard curve can be created by measuring the amount of PGA in standard blood samples with known hematocrit.

[0053] In certain aspects of the present disclosure, one exemplary method of the present disclosure relates to measuring the activity of GR in a blood sample and correlating the activity of GR in the blood sample with the hematocrit of 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 with a standard curve to determine the hematocrit of the blood sample. The standard curve can be created by measuring the amount of GSSG in standard blood samples with known hematocrit.

[0054] In some embodiments, the method also includes adding a GSSG internal standard solution to the blood sample, determining the GR activity in the blood sample, and correlating the GR activity with the standard curve to determine the hematocrit in the blood sample. For example, in some embodiments, the amount of 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 (g) / volume (mL), about 1% weight (mg) / volume (mL) to about 15% weight (mg) / volume (mL), about 1% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL), or about 1% weight (mg) / volume (mL) to about 7% weight (mg) / volume (mL).

[0055] In some embodiments of the present disclosure, a method for 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 redox products of the visible dye in the lysed blood sample, and comparing the amount of redox products of the visible dye in the blood sample to the hematocrit of the blood sample. The visible dye is intended to react with other components specific to the sample to provide a signal related to the amount of the specific components in the sample.

[0056] The method may also include measuring the redox product of the visible dye in standard blood samples of 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 to the hematocrit of the blood sample by comparing the amount of the redox product of the visible dye to the standard curve. The method may also include determining the hematocrit in the blood sample based on the amount of the redox product of the visible dye using the standard curve. The method may also include lysing the blood sample by mixing the blood sample with a lysis buffer.

[0057] The visible dye is not particularly limited, and may be NAD + and / or NADP + Any visible dye that changes color in the presence of NAD may be suitable. For example, the visible dye may be + and / or NADP + In some embodiments, the visible dye may be added in a range of about 0.1% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL), about 0.2% weight (mg) / volume (mL) to about 7.5% weight (mg) / volume (mL), about 0.5% weight (mg) / volume (mL) to about 5.0% weight (mg) / volume (mL), or about 1.0% weight (mg) / volume (mL) to about 2.0% weight (mg) / volume (mL).

[0058] In one example, the visible dye is NAD + and / or NADP +The tetrazolium dye may be selected from tetrazolium dyes having suitable redox ability to reduce . For example, the tetrazolium dye may be selected from 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) (MTS), nitroblue tetrazolium chloride (NBT), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In certain embodiments, the tetrazolium dye is 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide (MTT). In some embodiments, the tetrazolium dye may be added in a range of about 0.1% weight (mg) / volume (mL) to about 10% weight (mg) / volume (mL), about 0.2% weight (mg) / volume (mL) to about 7.5% weight (mg) / volume (mL), about 0.5% weight (mg) / volume (mL) to about 5.0% weight (mg) / volume (mL), or about 1.0% weight (mg) / volume (mL) to about 2.0% weight (mg) / volume (mL).

[0059] In some embodiments of the present disclosure, the method for measuring the redox product of a visible dye may also include adding a mediator to facilitate single electron transfer. Suitable mediators include NAD + and / or NADP +Examples of suitable mediators include single-electron transfer mediators that promote the reduction of phenazine methosulfate. For example, phenazine methosulfate is a suitable mediator that can be added to a lysed blood sample to promote single-electron transfer. In some embodiments, the mediator can be added in a range of about 0.05% weight (mg) / volume (mL) to about 2.0% weight (mg) / volume (mL), about 0.2% weight (mg) / volume (mL) to about 1.5% weight (mg) / volume (mL), or about 0.5% weight (mg) / volume (mL) to about 1.0% weight (mg) / volume (mL).

[0060] measurement

[0061] In some embodiments, the activity of an analyte or an enzyme can be measured by absorbance spectroscopy. In certain embodiments, the analyte, or the redox product of an enzyme, or the redox product of a visible dye has electronic absorption in the visible range of the electromagnetic spectrum. In certain embodiments, the analyte, or the redox product of an enzyme or a visible dye can be detected by UV-visible spectroscopy. In some embodiments, the amount of the analyte, or the redox product of an enzyme or a visible dye in a blood sample can be measured by absorbance spectroscopy.

[0062] For example, the amount of an analyte can be measured by UV-visible spectroscopy. In particular, the amount of an analyte in a blood sample can be measured between about 200 nm and 800 nm, between about 300 nm and 700 nm, or between about 330 nm and 650 nm. In some embodiments, the amount of an analyte is measured at about 340 nm, 415 nm, 540 nm, and 575 nm. In some embodiments, the amount of an analyte is measured at about 340 nm. In some embodiments, the amount of an analyte is measured at about 415 nm. In some embodiments, the amount of an analyte is measured at about 540 nm. In some embodiments, the amount of an analyte is measured at about 575 nm. For example, the amount of NADPH is measured in the range of about 300 nm to about 600 nm, or in the range of about 330 nm to about 350 nm. In other embodiments, the amount of NADPH is measured at about 340 nm.

[0063] In particular, the amount of the redox product of an enzyme or a visible dye can be measured between about 200 nm and 800 nm, or between about 300 nm and 700 nm, or between about 300 nm and 600 nm, or between about 330 nm and 650 nm. In some embodiments, the amount of the redox product of an enzyme is measured at about 340 nm. In particular, the amount of the reduction product of a tetrazolium dye is measured in the range between about 570 nm and 650 nm, or between about 570 nm and 600 nm, or between about 615 nm and 645 nm. For example, the amount of the reduction product of a tetrazolium dye is measured at 578 nm.

[0064] 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 the rate of formation of NADPH based on the change in absorption between the second absorption and the first absorption, and comparing the rate of formation to a standard curve to determine the hematocrit of the blood sample.

[0065] In one embodiment of the present disclosure, measuring the amount of redox product of a visible dye (e.g., the amount of reduction product of a tetrazolium dye) includes reacting a lysed blood sample for a predetermined reaction time, measuring a first absorbance of the lysed blood sample at the start of the 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 the redox product of the visible dye based on the change in absorbance between the second absorbance and the first absorbance, and comparing the formation rate to a standard curve to determine the hematocrit of the blood sample.

[0066] In certain embodiments, the predetermined reaction time begins when cells are lysed or when additional reagents (e.g., a visible dye) are added to the reaction mixture. For example, the predetermined reaction time begins at about 0 minutes, 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 certain embodiments, the end of the 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 start of the predetermined reaction time can be at least about 0 minutes, at least about 5 minutes, at least about 10 minutes, or at least about 15 minutes, and the end of the 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 certain embodiments, the start of the predetermined reaction time is at least about 0 minutes, and the end of the predetermined reaction time is at least about 45 minutes. In certain embodiments, the start of the predetermined reaction time is at least about 10 minutes and the end of the predetermined reaction time is at least about 15 minutes.

[0067] In certain embodiments, the rate of formation of NADPH or the rate of formation of a redox product of a visible dye (e.g., the amount of a reduction product of a tetrazolium dye) is calculated based on the change in absorption between the second absorption and the first absorption. For example, the second absorption is subtracted from the first absorption and divided by a predetermined reaction time. Alternatively, the method may include calculating the rate of formation of NADPH or the rate of formation of a redox product of a visible dye (e.g., the amount of a reduction product of a tetrazolium dye) in standard blood samples with known hematocrits to create a standard curve, and comparing the rate of formation of NADPH or the rate of formation of a redox product of a visible dye (e.g., the amount of a reduction product of a tetrazolium dye) with the standard curve to determine the rate of formation of NADPH or the rate of formation of a redox product of a visible dye (e.g., the amount of a reduction product of a tetrazolium dye) relative to the hematocrit of the blood sample. In certain embodiments, the rate of formation of NADPH is inversely related to the hematocrit in the blood sample.

[0068] In some embodiments, when the first and second absorptions are measured as described herein, the first and second absorptions can be measured in the UV-visible spectrum. For example, the first and second absorptions can be measured in the range of 200 nm to 800 nm, or in the range of 300 nm to 700 nm, or in the range of 330 nm to 650 nm, or in the range of about 330 nm to about 350 nm. In some embodiments, the first and second absorptions can be measured in the range of 570 nm to 650 nm, or in the range of 570 nm to 600 nm, or in the range of 615 to 645 nm. In particular, in some embodiments, the first and second absorptions are measured at about 340 nm.

[0069] In some embodiments, the activity of an analyte or an enzyme is measured by mass spectrometry. In other embodiments, the amount of an enzyme's redox product can be measured by mass spectrometry. The mass spectrometry instrument 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 photo-ionization 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.

[0070] 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 performance liquid chromatography (UHPLC) or high performance liquid chromatography (HPLC) with a normal-phase or reverse-phase column. In a specific example of the present disclosure, reverse-phase UHPLC is used. The mass spectrometry instrument 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 GSSG in a blood sample. The amount of PGA or GSSG can be determined by a common LCMS quantification method known in the art.

[0071] Sample collection and preparation

[0072] In some embodiments of the present disclosure, the blood sample is a liquid whole blood sample or a dried blood spot. Lysing the blood sample can be accomplished by any method known in the art. For example, a lysed blood sample can be formed by mixing the blood sample with a lysis buffer (e.g., a known detergent-based buffer). A commercially available buffer (e.g., 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) can be used.

[0073] Several blood collection devices are known, including blood collection tubes and absorbent blood collection devices. The shape, form, or composition of the blood absorption device is not limited, but the device should be sufficient to receive and retain a blood sample and allow for efficient extraction of the sample. For example, a blood sample may be collected on a blood absorption device (e.g., a pad, foam, or card) made from a porous hydrophilic polymeric material (e.g., plastic, paper, or fabric). When a collection card is used, the blood sample may be obtained by extracting dried blood from a predetermined area of the dried blood spot on the blood collection card.

[0074] The predetermined area of the dried blood spot is typically defined by the desired sample size required for use in the assay and may range from about 1 mm to 5 mm, about 1.5 mm to 4.5 mm, about 2 mm to 4 mm, or about 2.5 mm to 3.5 mm. In certain embodiments, the predetermined area of the dried blood spot is about 3 mm.

[0075] In some embodiments, collecting a blood sample involves applying a device containing microneedles to the animal's skin. In some embodiments, collecting a blood sample involves applying to the animal's skin an empty absorbent collection device (e.g., a blank collection card) and a device further containing microneedles. For example, the empty absorbent collection device and the microneedles are in fluid communication with the empty absorbent collection device, and the absorbent collection device containing absorbed blood can be removed from the device.

[0076] The laboratory may measure the amount of NADPH in a 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 a blood sample, or the activity of GR in a blood sample. The dried blood sample may be extracted from the device using a lysis buffer or other suitable extraction reagent.

[0077] In certain aspects of the present disclosure, one method of the present disclosure involves providing a blank collection card or other collection device to a blood collector who collects a blood sample on the blood collection card or device, dries the blood sample, and delivers the blood collection card / device to a laboratory for analysis according to the methods of the present disclosure. In some embodiments, the blood collector delivers the blood collection card / device to the laboratory by mail or commercial delivery service.

[0078] device

[0079] Blood samples for use in the methods disclosed herein can be collected by any method that allows for analysis of analytes. In some embodiments, blood is collected with a blood collection device that includes a blood absorption device (e.g., a blood collection card or other absorbent device that allows for efficient extraction of the sample). In some embodiments, the device includes a microneedle array that includes a plurality of microneedles. The device can also include a blood storage layer, such that the microneedle array transfers the blood sample from the animal to the blood storage layer.

[0080] In some embodiments, the microneedle array includes multiple microneedles. Without being bound by theory, each microneedle can draw a predetermined volume of blood over a predetermined amount of time. Thus, the number of microneedles in the microneedle array can be selected to draw a predetermined amount of blood over a predetermined amount of time, where the predetermined amount of blood is sufficient to perform one or more blood tests. These collection times can range from 30 seconds to 5 minutes, depending on, among other factors, the number of microneedles, the type of animal, and / or the amount of blood to be drawn.

[0081] In some examples, the blood storage layer is made of an absorbent material structurally configured to store dried blood, defines a chamber designed to store liquid blood, or is a combination of the two. For example, blood may be drawn by and through the microneedle array due to one or more factors, including the animal's blood pressure, which pumps blood through an appendage into which the microneedle array is inserted, osmotically by the absorbent material, and / or other factors.

[0082] In some embodiments, the blood collection device includes, among other possibilities, a peel-to-expose package, a clamping device, and / or a wearable sleeve, or any suitable combination thereof, to allow a user associated with and / or responsible for the animal to more easily collect a blood sample from the animal using the array of microneedles. For example, a peel-to-expose package can be used to selectively cover the microneedle array and blood storage layer so that the microneedle array and / or blood storage layer are exposed for use of the device and resold when collection is complete. In some embodiments, the blood collection device can include a clamping device that deflects the microneedle array into the animal's appendage with a biasing member and secures itself to the appendage. In some embodiments, the blood collection device includes a wearable sleeve, where the microneedle array is accessible from and located on the outer surface of the wearable sleeve.

[0083] 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 includes a blood collection card, and the microneedles are in fluid communication with the blood collection card. In certain embodiments, the blood collection tool is removable from the device.

[0084] 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 tool to a laboratory. More specifically, the laboratory extracts the dried blood and measures the amount of NADPH in the blood sample, the amount of a redox product of a visible dye (e.g., the amount of a reduction product of a 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 tool to the laboratory by mail or commercial delivery service. [Example]

[0085] Example The examples that follow are illustrative of specific embodiments of the present disclosure. They are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0086] Example 1: Preparation of whole blood standards and dried blood spot standards

[0087] To prepare liquid whole blood samples, 50 mL of canine whole blood was centrifuged at 1500 × g at room temperature to separate the blood from the plasma. The plasma was then drawn off, and appropriate volumes of blood and plasma were then transferred using a positive displacement pipette to create blood samples with various hematocrit levels. Table 1 describes the preparation of blood samples with various hematocrit levels. [Table 1]

[0088] Each prepared sample was analyzed using commercially available (Sysmex XN series hematology analyzers and / or IDEXX ProCyte DX) (登録商標) Hematocrit levels were confirmed by testing with a hematocrit measurement device (hematology analyzer). To prepare DBS samples, 3 μL of each liquid whole blood sample was spotted onto a DBS card and allowed to dry overnight. DBS cards were stored in dry plastic bags with desiccant until needed.

[0089] Example 2: NADPH as a measure of hematocrit in liquid whole blood samples

[0090] Lysed blood samples containing different hematocrits (HCT%) were assayed for the amount of NADPH present in the blood. To perform the assay, 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) was added to a microcentrifuge tube. Liquid whole blood samples with 0%, 36.5%, 44%, 52%, or 70% hematocrit were added to the microcentrifuge tube in volumes of 3 μL, 6 μL, or 12 μL. The solutions were vortexed briefly (approximately 10 seconds) and then incubated at room temperature for 15 minutes. The electronic absorption spectrum of each sample was measured, tracking absorbance at 340 nm, 405 nm, and 578 nm. Table 2 reports the absorbance at 340 nm, 405 nm, and 578 nm across different lysed blood samples for initial liquid whole blood sample volumes of 3 μL and 6 μL. [Table 2]

[0091] 1A and 1B plot the absorbance at each specified wavelength as a function of the percent hematocrit present. From FIGS. 1A and 1B, a linear correlation is observed between the measured absorbance and the hematocrit present. By using this linear relationship, the amount of NADPH present in another blood sample can be used to determine the hematocrit of the blood sample.

[0092] Example 3: NAPDH as a measure of hematocrit in dried blood spots (DBS)

[0093] The NADPH present in blood was determined in dried blood spots (DBS) obtained from whole blood samples with different hematocrits (HCT%). To generate DBS samples, hematocrit whole blood standards (as prepared in Example 1) were dried onto DBS cards. A 3 mm punch from 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 seconds. Electronic absorption for each sample was obtained and the absorbance was tracked at 340 nm, 415 nm, 540 nm, and 575 nm. Table 3 reports the absorption across the different DBS samples. [Table 3]

[0094] Figure 2A plots the electronic absorption spectrum of each measured DBS card solution. Figure 2B plots the absorbance at each specific wavelength as a function of the hematocrit present in the DBS hematocrit standard. Figure 2B shows a linear correlation between the measured absorbance and the hematocrit present in the whole blood sample, demonstrating that rehydrating DBS with lysis buffer can provide a rapid and simple method for directly measuring the absorption from endogenous NADPH or hemoglobin. This assay also provides a method for measuring the hematocrit present in dried blood samples by correlating the amount of NADPH present in the sample to the hematocrit.

[0095] Example 4: Tetrazolium dye kinetic assay

[0096] To assay hematocrit in liquid whole blood samples or dried blood spots (DBS), 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 rate of reduction of the tetrazolium dye by promoting single electron transfer.

[0097] Lysed blood samples are prepared by either the method described in Example 2 for liquid whole blood samples or in Example 3 for DBS samples for use in the tetrazolium dye assay. Once lysed, 50 μL of red blood sample solution (from either liquid whole blood or DBS) was pipetted in duplicate into two wells of a 96-well plate (Corning - Costar, UV-transparent flat-bottom plate, non-sterile, acrylic).

[0098] For the assay, a 1.5 mM working solution of MTT (Molecular Probes, ref. M6494, mw 414.32 g / mol) in DI water and a 75 μM working solution of PMS (Sigma-Aldrich, PN: P9625-10G, mw 306.34 g / mol) in DI water were prepared. The MTT and PMS working solutions were mixed in a 1:1 ratio. 100 μL of the MTT / PMS solution was added to each well containing a blood sample. The plate was immediately transferred to a plate reader, and the absorbance of the reduced MTT dye was measured.

[0099] A Biotek Synergy 4 Microplate Reader was used to monitor the absorption spectra. The temperature of the plate reader was set to 37°C with the shaker speed set to medium. The electronic absorption of the reduced dye was measured continuously at the desired wavelength over a set reaction time. For the MTT dye assay, absorption was measured every minute at 578 nm over a 5-minute period. However, when using other tetrazolium dyes, different wavelengths and reaction times may be used. Figure 3A shows the absorption spectrum of the MTT dye / sample mixture over a 30-minute reaction time and compares it to the spectra of the sample without dye and the dye alone.

[0100] At the end of the 5-minute reaction period, data from the Biotek plate reader was exported for analysis. The absorbance at 578 nm 5 minutes after dye addition was plotted as shown in Figure 3B. By plotting absorbance against the hematocrit present in the lysed samples, a linear correlation is observed.

[0101] Example 5: G6PDH kinetic assay

[0102] The formation rate of NADPH was determined in lysed blood samples containing different hematocrits (HCT%). Lysed blood samples were prepared by the method described in Example 2. Lysed blood samples could also be obtained from DBS by the method described in Example 3. Once lysed, 50 μL of 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).

[0103] 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, mw 663.43 g / mol), and a 12 mM working solution of G6P (Sigma-Aldrich, PN: G7879-25G, mw 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 the dark until needed to protect it from any light.

[0104] The G6P and NAD working solutions were mixed in a 1:1 ratio. 100 μL of the G6P / NAD mixture was added to each well. 50 μL of the G6PDH working solution was added to each well containing a blood sample. The plate was immediately transferred to a plate reader to measure the absorbance of the NADPH dye.

[0105] A Biotek Synergy 4 Microplate Reader was used to monitor absorbance. The temperature of the plate reader was set to 37°C with the shaker speed set to medium. The electronic absorbance of the samples was measured continuously at the desired wavelength for the set reaction time. For the G6PDH assay, absorbance was measured every minute at 340 nm over a 45-minute period. Figure 4 illustrates the change in absorbance at 340 nm over time for the different hematocrit standards assayed.

[0106] At the end of the 45-minute reaction period, data from the Biotek plate reader was exported for analysis. The change in absorbance at 340 nm was calculated as the difference in absorbance between 0 and 45 minutes and between 10 and 15 minutes. The reaction rate based on these changes in absorbance was also calculated. The results are reported in Tables 4 and 5, respectively, for assays of an initial 6 μL of liquid whole blood with different hematocrits. [Table 4] [Table 5]

[0107] A linear correlation was observed by plotting the reaction rate against the hematocrit present in the lysed blood sample. Figures 5A and 5B show this correlation for an initial 3 μL sample of liquid whole blood and an initial 6 μL sample of liquid whole blood. As can be seen, the 6 μL liquid whole blood sample reacted for 45 minutes produced a good correlation between enzyme activity and hematocrit percentage. The correlation previously observed between decreasing enzyme activity and increasing hematocrit percentage was observed. Without being bound by theory, it is believed that the decrease in enzyme activity is due to the addition of NADP to the reaction mixture. + Not NAD + This functional assay is hypothesized to be due to the addition of NAD + The addition of NADP + This confirms that the enzyme activity is indeed inhibited in the presence of G6PDH. Furthermore, the G6PDH assay provides a measurement of hematocrit in a blood sample based on the rate of NADPH formation. The rate of NADPH formation can be used to determine the hematocrit in other blood samples based on the correlation shown in Figures 5A and 5B.

[0108] Example 6: LCMS assay of G6PDH activity as a marker for hematocrit

[0109] Another type of assay that can be used to determine hematocrit in blood samples is a liquid chromatography-mass spectrometry (LCMS) assay. Just like the electroabsorption assay of Examples 4 and 5, this assay can be performed on either liquid whole blood or dried blood spots. For this assay, the production of the G6PDH redox product, 6-phosphogluconolactone (PGA), in dried blood samples was monitored by LCMS and correlated with the hematocrit present in whole blood.

[0110] To generate lysed blood samples for measurement, the same lysis procedure was used as described in Example 3 for DBS samples. Lysed blood samples could 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 plate, non-sterile, acrylic).

[0111] For the assay, a 10 ng / mL G6PDH working solution was prepared by diluting a 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, mw 663.43 g / mol) and a 12 mM working solution of G6P (Sigma-Aldrich, PN: G7879-25G, mw 282.12 g / mol) were prepared in deionized water. All solutions were stored at 4°C. The NAD working solution was stored in the dark to protect it from any light. The G6PDH working solution was used within 30 minutes of preparation.

[0112] 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 well plate was gently vortexed. 600 μL of methanol was then added to each well. The samples were then centrifuged at 3000 × g for 10 minutes at room temperature. The resulting supernatant was transferred to a new 96-well plate for analysis by LCMS. The LCMS parameters used are reported in Table 7. [Table 7-1] [Table 7-2]

[0113] A representative MS chromatogram of 6-phosphogluconolactone (PGA) is shown in Figure 6. The area of the MS chromatogram of PGA (proportional to the amount of PGS present) was calculated for four different samples containing different levels of hematocrit. This is reported in Table 8 relative to the theoretical hematocrit (HCT %). The MS chromatogram area of PGA is plotted as a function of the actual HCT % in Figure 7, reflecting a good linear correlation between the MS chromatogram area of PGA and the hematocrit present. The amount of PGA present can be used to determine the hematocrit in other blood samples based on the correlation shown in Figure 7. [Table 8]

[0114] Example 7: LCMS assay of glutathione reductase activity as a marker for hematocrit

[0115] Another LCMS assay that can be used to determine hematocrit in blood samples measures the production of oxidized glutathione (i.e., glutathione disulfide). As with the other examples, this assay can be performed on either liquid whole blood or dried blood spots.

[0116] Liquid whole blood samples were prepared in the same manner as described in Example 6. To prepare samples for analysis by LCMS, a 3 mm DBS punch of a blood spot or 3 μL of liquid whole blood was placed in a tube (or well). 50 μL of a 200 μg / mL glutathione disulfide (GSSG) internal standard solution (made in DI water) was added. The solution was then sonicated for 30 minutes and subsequently centrifuged at 3500 × g for 10 minutes at room temperature. 150 μL of methanol was added to the solution, and the solution was again sonicated for 30 minutes and then centrifuged at 3500 × g for 10 minutes at room temperature. To generate the final solution to be analyzed by LCMS, the centrifuged solution was filtered through a 0.2-0.4 μm filter / filter plate (GHP or Teflon®). LCMS parameters are shown in Table 9. [Table 9-1] [Table 9-2]

[0117] This LCMS assay was used to determine the hematocrit of seven different samples containing different hematocrit levels. A representative MS chromatogram of GSSG is shown in Figure 8. The area of the MS chromatogram of GSSG was used to calculate the amount of GSSG. Based on the amount of GSSG present, the hematocrit was calculated for each of the samples. The average hematocrit content for each level tested is reported in Table 10. The calculated HCT% is plotted as a function of the reference HCT% in Figure 9, reflecting a good linear correlation between the calculated HCT% and the reference HCT%. The amount of GSSG present can be used to determine the hematocrit in other blood samples based on the correlation shown in Figure 9. [Table 10]

Claims

1. 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; correlating the amount of NADPH in the lysed blood sample to the hematocrit of the blood sample; A method that encompasses

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

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

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

7. 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 activity of G6PDH in the lysed blood sample; correlating the activity of G6PDH in the lysed blood sample to the hematocrit of the blood sample; A method that encompasses

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

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

10. NAD ＋ or NADP ＋ to the lysed blood sample prior to measuring the activity of the G6PDH; determining the activity of G6PDH in the lysed blood sample; The method of claim 7 further comprising:

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

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

13. 11. The method of 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. 14. The method of claim 13, wherein the G6PDH redox product is selected from 6-phosphogluconolactone and NADPH.

15. The step of 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 rate of formation of NADPH based on the change in absorbance between the second absorbance and the first absorbance; and correlating the rate of formation with a standard curve to determine the hematocrit of the blood sample; The method of claim 7 or claim 10, comprising:

16. 16. The method of claim 15, wherein the steps of measuring the first absorption and measuring the second absorption comprise measuring UV-visible absorption.

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

18. 18. The method of claim 17, wherein the first absorption and the second absorption are measured at 340 nm.

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

20. The step of 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; comparing the amount of PGA in the lysed blood sample to a standard curve to determine the hematocrit of the blood sample; The method of claim 7, comprising:

21. 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 activity of glutathione reductase in the blood sample; correlating the activity of glutathione reductase with the hematocrit in the blood sample; A method that encompasses

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

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

24. 1. A method for 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 reduction products of the tetrazolium dye in the lysed blood sample; correlating the amount of reduction product of the tetrazolium dye in the blood sample to the hematocrit of the blood sample; A method that encompasses

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

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

29. 28. 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. 25. The method of claim 24, wherein 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. measuring the amount of the reduction product of the tetrazolium dye; 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 rate of formation of a redox product of the tetrazolium dye based on the change in absorption between the second absorption and the first absorption; and correlating the rate of formation with a standard curve to determine the hematocrit of the blood sample; 25. The method of claim 24, comprising:

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

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

34. 32. The method of claim 31, wherein the steps of measuring the first absorption and measuring the second absorption comprise measuring UV-visible absorption.

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

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

39. 39. The method of any one of claims 24 to 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. 40. The method of 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 device.

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

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

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

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

46. 1. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood from said blood sample on a blood collection card; Measuring the amount of NADPH in the extracted dried blood; and correlating the amount of NADPH to the hematocrit of the blood sample; A method that encompasses

47. 1. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood from the blood sample on a blood absorbing device; measuring the amount of a reduction product of the tetrazolium dye in the extracted dried blood; and correlating the amount of the reduction product of the tetrazolium dye to the hematocrit of the blood sample; A method that encompasses

48. 1. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood from said blood sample on a blood collection card; Measuring the activity of G6PDH in the extracted dried blood; and correlating the activity of G6PDH to the hematocrit of the blood sample; A method that encompasses

49. 1. A method for determining the hematocrit of a blood sample, comprising: extracting a predetermined amount of dried blood from said 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 to the hematocrit of the blood sample; A method that encompasses

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

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

51. The method of claim 50, wherein each microneedle is capable of collecting up to about 20 μL of blood.

52. 52. The method of claim 51, wherein the blood absorbing device is a blood collection card.

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

54. 53. The method of claim 52, wherein a blank collection card is provided to a blood collector who collects the blood sample on the blood collection card, dries the blood sample, and delivers the blood collection card to a laboratory where the amount of NADPH is measured.

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

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

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

58. 58. The method of any one of claims 54-57, wherein the blood collector delivers the blood collection card to the laboratory by mail or by commercial delivery service.

59. 1. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from the animal on a blood collection card from a blood collector; extracting a predetermined amount of dried blood from said blood collection card; Measuring the amount of NADPH in the extracted dried blood; and correlating the amount of NADPH to the hematocrit of the blood sample; A method that encompasses

60. 1. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from the animal on a blood collection device from a blood collector; extracting a predetermined amount of dried blood from said blood collection card; measuring the amount of tetrazolium dye in the extracted dried blood; and correlating the amount of tetrazolium dye to the hematocrit of the blood sample; A method that encompasses

61. 1. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from the animal on a blood collection device from a blood collector; extracting a predetermined amount of dried blood from said blood collection card; Measuring the activity of G6PDH in the extracted dried blood; and correlating the activity of G6PDH to the hematocrit of the blood sample; A method that encompasses

62. 1. A method for determining the hematocrit of a blood sample, comprising: receiving a dried blood sample from the animal on a blood collection device from a blood collector; extracting a predetermined amount of dried blood from said blood collection card; measuring the activity of glutathione reductase in the extracted dried blood; and correlating the activity of glutathione reductase to the hematocrit of the blood sample; A method that encompasses

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

64. 64. The method of claim 63, wherein the device is a blood collection card.

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

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

67. 63. The method of any one of claims 59 to 62, wherein said step of collecting said blood sample is accomplished for a time period of up to about 2 minutes.

68. 63. The method of any one of claims 59 to 62, wherein said step of collecting said blood sample is accomplished for a period of about 1 to 2 minutes.

69. 64. The method of claim 63, wherein the device is provided to a blood collector who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory that extracts the dried blood and measures the amount of NADPH.

70. 64. The method of claim 63, wherein the device is provided to a blood collector who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory that extracts the dried blood and measures the amount of the reduction product of the tetrazolium dye.

71. 64. The method of claim 63, wherein the device is provided to a blood collector who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory that extracts the dried blood and measures the activity of G6PDH.

72. 64. The method of claim 63, wherein the device is provided to a blood collector who collects the blood sample, dries the blood sample, and delivers the blood collection card to a laboratory that extracts the dried blood and measures the activity of glutathione reductase.

Images