Oxidase-based chemiluminescence assay of phagocytic leukocytes in whole blood and body fluids applicable to point-of-care (POC) diagnostic testing point-of-care (POC) measurement of absolute neutrophil function (ANF)

Abstract

To provide an oxidase-based chemiluminescence assay of phagocytic leukocytes in whole blood and body fluids applicable to a point-of-care (POC) diagnostic testing point-of-care (POC) measurement of an absolute neutrophil function (ANF).SOLUTION: A method for estimating a number of phagocytes in a body fluid of an animal includes the steps of: stimulating NADPH oxidase activity of the phagocytes; and quantifying a resulting reductive deoxygenation of a chemiluminigenic substrate by an emitted chemiluminescence of the chemiluminigenic substrate using an instrument capable of measuring light. The NADPH oxidase activity of the phagocytes is preferably stimulated by an immunologic or chemical capable of activating a respiratory burst by the phagocytes, and by a stimulus in solution or coated to a surface contacted by the phagocytes. The stimulus is preferably phorbol myristate acetate (PMA). The animal is preferably human, and the body fluid is preferably blood and / or spinal fluid.SELECTED DRAWING: None

Description

Technical Field

[0001] Technical Field The method quantifies the presence of phagocytic white blood cells (i.e., essentially neutrophils, but also including monocytes and eosinophils) in blood and body fluids in a manner applicable to clinical point-of-care diagnostic tests. Absolute neutrophil count (ANC) can be performed to assess bone marrow myelosuppression, usually in the context of chemotherapy or as a measure of inflammation. The methods disclosed herein can quantify phagocytes based on the activation of respiratory burst metabolism and the measurement of reductive dioxygenation of chemiluminescent production probes.

Background Art

[0002] Background Automated hematology analyzers are well-established instruments for the measurement of white blood cells, red blood cells, and platelets in whole blood. The white blood cell components of blood are morphologically and functionally distinct from red blood cells and platelets. Impedance, light scattering, and enzymatic and antigenic differences serve as the basis for counting and differentiating white blood cells by automated hematology analysis. These instruments are very complex and not suitable for the development of point-of-care (POC) assays.

[0003] Activation of phagocyte metabolism, i.e., the "respiratory burst," is characterized by a large increase in glucose metabolism (pentose pathway) via dehydrogenases of the hexose monophosphate shunt and a proportional increase in non-mitochondrial O2 consumption (Sbarra and Karnovsky 1959). Both activities reflect the activation of NADPH oxidase (NADPH:O2 oxidoreductase), an enzyme common to blood phagocytes (Rossi, Romeo et al. 1972). Activation of phagocyte NADPH oxidase drives a combustive dioxygenation reaction that produces natural chemiluminescence as an energy product (Allen et al 1972). Natural leukocyte chemiluminescence is O2-dependent and is directly proportional to hexose monophosphate shunt dehydrogenase activity. NADPH oxidase activation is HO2, O2- and results in the production of H2O2. The H2O2 produced serves as a substrate for secondary chemical reactions involving the MPO (H2O2: halide oxidoreductase) oxidation of chloride ions to hypochlorite ions (OCl - -) and the addition of H2O2 that generates singlet molecular oxygen ( 1 1O2*) (Allen, Yevich et al. 1974, Allen 1979). The halide-dependent haloperoxidase activity of isolated MPO also results in natural chemiluminescence as an energy product (Allen 1975, Allen 1975).

[0004] The chemiluminescence quantum yield, i.e., the ratio of photons emitted per oxygenation event, depends on the type and amount of oxygenating agent generated, as well as the nature and quantum efficiency of the substrate being oxygenated. Oxygenation of natural substrates is associated with relatively low chemiluminescence quantum yields, which vary with the nature of the substrate being oxygenated. Introduction of chemiluminescence-generating substrates (CLS) with high quantum yields overcomes problems of sensitivity and substrate variability. Addition of cyclic hydrazides (e.g., luminol) increases the yield of phagocyte luminescence by more than 1000-fold (Allen and Loose 1976). Acridinium salts such as lucigenin also greatly increase the yield of leukocyte luminescence (Allen 1981, Allen 1982).

[0005] Cyclic hydrazide and acridinium chemiluminescence can be chemically induced by exposure to H2O2 under alkaline conditions (Albrecht 1928, Totter 1964). However, these substrates do not produce CL under mildly acidic to neutral pH conditions of the physiological environment. Phagocyte CL activities dependent on luminol and lucigenin measure different oxygenation pathways (Allen 1982, Allen 1986). Luminol CL results from dioxygenation that produces electronically excited aminophthalate, i.e., luminol + O2 → aminophthalate + N2 + photons. In phagocytic white blood cells, such activity is strongly associated with MPO. Luminol CL results from non-reducing and simple dioxygenation. A small amount of luminol CL is observed in MPO-negative white blood cells, but MPO-positive white blood cells produce more than 100-fold greater luminescence (Allen 1986, Merrill, Bretthauer et al. 1996, Allen 2019).

[0006] Phagocyte NADPH oxidase catalyzes the monovalent reduction of O2 to HO2. In a neutral environment, HO2 dissociates to produce O2 - and H + , and O2 - and HO2 become unbalanced, producing H2O2. Under acidic to neutral conditions, divalent cationic lucigenin (N,N’-dimethyl-9,9’ biacridinium and bis-N-methylacridinium) can undergo a one-electron reduction to produce a monovalent cation radical (i.e., lucigenin ++ + e - → lucigenin + ). And this radical can react with O2 - to produce a dioxetane intermediate and ultimately two N-methylacridones and photons (i.e., lucigenin + + O2 - → lucigenin-O2 → 2 N-methylacridone + photons (Allen 1981, Allen 2019)).

Summary of the Invention

Means for Solving the Problems

[0007] Abstract The inventors have surprisingly found that the complex equipment required for impedance and flow cytometry measurements of phagocytic leukocytes can be rendered unnecessary by using a chemiluminescence approach that directly measures the functional activity of stimulated phagocytes present in diluted whole blood and body fluids (e.g., cerebrospinal fluid). The phagocytes per volume of blood or body fluid are determined by measuring the stimulated NADPH oxidase-dependent reductive dioxygenation of a chemiluminescent production substrate. Introduction of a stimulant (e.g., PMA) optimal for activation of phagocyte NADPH oxidase results in production of reductive deoxygenation activity.

[0008] The resulting reductive dioxygenation of lucigenin produces CL, which can be quantified by measuring the light emitted using a luminometer. This luminescence is proportional to the metabolic activity of phagocytes / neutrophils per volume of blood or body fluid examined. This absolute neutrophil function (ANF) assay is proportional to the absolute neutrophil count (ANC) and is applicable for assessing the clinical status of a patient with respect to inflammation / infection or chemotherapy-related myelosuppression. Such a function-based analysis provides an alternative and, albeit debatable, superior assay compared to the conventional ANC. The technical requirements of the ANF approach are applicable to point-of-care testing. As described in the examples herein, the ANF assay enables quantitative assessment of neutrophil count (ANC) and function in blood during the first 16-hour interval after venipuncture.

[0009] Further features and advantages are described in, and will be apparent from, the following detailed description and drawings. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for estimating the number of phagocytes in a body fluid of an animal, the method comprising: stimulating the NADPH oxidase activity of the phagocytes; and Quantifying the resulting reductive dioxygenation of the chemiluminescent generating substrate by the chemiluminescence emitted by the chemiluminescent generating substrate using a device capable of measuring light A method comprising: (Item 2) The method according to item 1, wherein the NADPH oxidase activity of the phagocyte is stimulated by an immunological or chemical substance capable of activating the respiratory burst by the phagocyte. (Item 3) The method according to item 1, wherein the NADPH oxidase activity of the phagocyte is stimulated by a stimulant in solution or coated on the surface with which the phagocyte comes into contact. (Item 4) The method according to item 3, wherein the stimulant is phorbol myristate acetate (PMA). (Item 5) The method according to item 1, wherein the animal is a human. (Item 6) The method according to item 1, wherein the body fluid is blood. (Item 7) The method according to item 1, wherein the body fluid is cerebrospinal fluid. (Item 8) The method according to item 1, wherein the phagocyte is a neutrophil. (Item 9) The method according to item 1, wherein the chemiluminescent generating substrate is lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate). (Item 10) The method according to item 9, wherein the lucigenin is in solution or coated on the surface with which the phagocyte comes into contact. (Item 11) The method according to item 1, comprising the step of diluting the body fluid to reduce the erythrocyte absorbance of chemiluminescence. (Item 12) The method according to item 6, comprising the step of diluting the blood to approximately at most 1 to 500 to reduce the erythrocyte absorbance of chemiluminescence. (Item 13) The method according to item 6, comprising the step of diluting the blood by up to about 1 to 1000 to reduce the chemiluminescent absorbance of red blood cells. (Item 14) The method according to item 7, comprising the step of diluting the cerebrospinal fluid by up to 1 to 100 to reduce the chemiluminescent absorbance of red blood cells. (Item 15) The method according to item 2 or item 3, comprising the step of aggregating or removing red blood cells from the body fluid using lectin to facilitate chemiluminescent detection. (Item 16) The method according to item 1, wherein the emitted chemiluminescence is measured by a portable or hand-held luminometer. (Item 17) The method according to item 1, wherein the components are pre-manufactured to facilitate point-of-care testing. (Item 18) The method according to item 1, further comprising the step of determining an absolute neutrophil count (ANC) using the estimated number of phagocytes. (Item 19) The method according to item 18, further comprising the step of evaluating myelosuppression in the animal using the ANC. (Item 20) The method according to item 19, wherein the myelosuppression is associated with a measure of chemotherapy or inflammation or infection. (Item 21) The method according to item 20, further comprising the step of treating the animal based on the evaluation of the myelosuppression. (Item 22) A method for inferring myelopoietic stimulation, the method comprising: Measuring the non-reducing dioxygenation-driven myeloperoxidase (luminol CL) activity and the reducing dioxygenation (lucigenin CL) activity of chemically activated blood neutrophils in an animal using a device capable of measuring light; and Calculating the ratio of the luminol CL activity to the lucigenin CL activity.

Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0016] Detailed Description General Embodiment

[0017] The present disclosure provides a method for estimating the number of phagocytes in an animal's body fluid, the method comprising: stimulating the NADPH oxidase activity of the phagocytes; and quantifying the resulting reductive deoxygenation of a chemiluminescence-generating substrate by the chemiluminescence emitted by the chemiluminescence-generating substrate using a device capable of measuring light.

[0018] In one embodiment, the NADPH oxidase activity of the phagocytes is stimulated by an immunological or chemical substance capable of activating the respiratory burst by the phagocytes.

[0019] In one embodiment, the NADPH oxidase activity of the phagocytes is stimulated by a stimulant in solution or coated on the surface with which the phagocytes are in contact. The stimulant can be phorbol myristate acetate (PMA).

[0020] In one embodiment, the animal is a human.

[0021] In one embodiment, the body fluid is blood.

[0022] In one embodiment, the body fluid is cerebrospinal fluid. The cerebrospinal fluid can be diluted up to 1 to 100 in order to reduce the erythrocyte absorbance of chemiluminescence.

[0023] In one embodiment, the phagocytes are neutrophils.

[0024] In one embodiment, the chemiluminescence-generating substrate is lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate). The lucigenin may be in solution or coated on the surface with which the phagocytes are in contact.

[0025] In one embodiment, the method includes diluting the body fluid to reduce the chemiluminescent absorbance of red blood cells.

[0026] In one embodiment, the method includes diluting the blood by up to about 1 to 500 to reduce the chemiluminescent absorbance of red blood cells.

[0027] In one embodiment, the method includes diluting the blood by up to about 1 to 1000 to reduce the chemiluminescent absorbance of red blood cells.

[0028] In one embodiment, the method includes using lectin to agglutinate or remove red blood cells from the body fluid to facilitate chemiluminescent detection.

[0029] In one embodiment, the emitted chemiluminescence is measured by a portable or hand-held luminometer. In one embodiment, the components are pre-manufactured to facilitate point-of-care testing.

[0030] In one embodiment, the method further includes determining an absolute neutrophil count (ANC) using the estimated number of phagocytes. The method may further include evaluating myelosuppression in the animal using the ANC, for example, myelosuppression is associated with measures of chemotherapy or inflammation or infection. In this regard, inflammation or infection typically increases myelopoietic activity, but neutrophil consumption in response to infection can actually decrease the neutrophil count. The method may further include treating the animal based on the evaluation of myelosuppression.

[0031] In another embodiment, a method for inferring bone marrow hematopoietic stimulation is provided. The method includes measuring the non-reducing dioxygenation-driven myeloperoxidase (luminol CL) activity and the reducing deoxygenation (lucigenin CL) activity of chemically activated blood neutrophils in an animal using a device capable of measuring light; and calculating the ratio of the luminol CL activity to the lucigenin CL activity.

[0032] Preferred Embodiment

[0033] The absolute neutrophil function (ANF) assay disclosed herein includes a highly sensitive chemiluminescence generation probe method for determining the number of neutrophils in diluted whole blood or body fluids (e.g., cerebrospinal fluid) with a minor contribution from functional phagocytes, i.e., monocytes and eosinophils. Specifically, phagocyte function is quantified by introducing lucigenin as a chemiluminescence generation probe and measuring the chemiluminescence product of stimulated NADPH oxidase reducing dioxygenation activity.

[0034] The method requires less than a drop of whole blood or body fluid. The small volume sample is diluted with an isotonic saline solution. The diluted sample is introduced into an environment containing a chemiluminescent probe sensitive to phagocyte NADPH oxidase-dependent respiratory burst metabolism and reducing dioxygenation, e.g., lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate), and a chemical stimulant (e.g., phorbol 12-myristate 13-acetate (PMA)) capable of activating it. Such contact activates the phagocyte NADPH oxidase-dependent reducing dioxygenation of lucigenin, which results in chemiluminescence that can be detected and quantified by luminescence spectrometry. Chemiluminescent probes (e.g., luminol) measure the non-reducing (simple) dioxygenation reactions of phagocytes, particularly those catalyzed by myeloperoxidase (MPO).

[0035] Under normal bone marrow hematopoietic conditions, the measured value of non-reducing dioxygenation is close to the existing neutrophil count. Inflammatory stimuli and g-CSF treatment expand the myeloid promyelocyte pool in the bone marrow, resulting in a several-fold increase in the MPO content per neutrophil. Non-reducing dioxygenation activity reflects the MPO content per phagocyte and the specific MPO content per phagocyte. Lucigenin's NADPH oxidase-dependent reducing dioxygenation is independent of MPO and, as a result, is directly proportional to the number of functional phagocytes in the specimen. The lucigenin-based ANF system quantifies the presence of phagocytes by measuring the reducing dioxygenation of stimulated phagocytes. In the absence of genetic, for example, chronic granulomatous disease or acquired neutrophil abnormalities, the lucigenin-dependent measurement of NADPH oxidase reducing dioxygenation activity is MPO-independent and very close to the neutrophil count of the specimen. The above ANC determines the number of neutrophils present in the specimen. The above ANF assay provides more clinically relevant information based on the quantification of the functional presence of neutrophils in the specimen. The above ANF assay has been shown to quantitatively reflect the total blood ANC during the first 16-hour interval after venipuncture and is applicable to point-of-care testing.

[0036] The NADPH oxidase activity per phagocyte is relatively constant, while the myeloperoxidase (MPO) activity per phagocyte varies with the state of myelopoietic stimulation. MPO and cationic proteases are lysosomal enzymes synthesized in the promyelocyte phase of development and stored in the azurophilic granules of neutrophils (Bainton 1999). The concentration of MPO per phagocyte depends on the degree of myelopoietic stimulation, i.e., activation by colony-stimulating factors (physiological or recombinant G-CSF or GM-CSF), and the number of mitoses during the myelocyte phase of neutrophil development (Allen, Stevens et al. 1997). For example, each division in the myelocyte phase dilutes the myeloperoxidase per neutrophil by half. The components of NADPH oxidase are synthesized in the myelocyte phase of development and are required for respiratory burst metabolism. As a result, the NADPH oxidase activity per phagocyte is relatively constant with respect to variations in myelopoietic activity. The specific NADPH oxidase activity per phagocyte remains relatively constant in various states of inflammation and myelopoietic stimulation or suppression.

[0037] Thus, the stimulated oxidase activity is very close to the phagocyte count, particularly the neutrophil count. Such measured values of activity are very close to the absolute neutrophil count per volume of blood or body fluid. The introduction of lucigenin as a chemiluminescence generation probe enables the sensitive quantification of the phagocyte NADPH oxidase reductive dioxygenation activity (Allen 1981, Allen 1982, Allen 1986). Simple or non-reductive dioxygenation is a reaction in which O2 is incorporated into the substrate (e.g., luminol + O2 → aminophthalate + N2 + photons (Allen and Loose 1976)). Reductive dioxygenation is a reaction in which O2 and two reducing equivalents (2 electrons + 2 protons) are incorporated (e.g., lucigenin + 2 electrons + 2 protons → 2 N-methylacridone + photons).

[0038] The NADPH oxidase-dependent reductive dioxygenation of lucigenin provides a measure of absolute neutrophil function (ANF) that is very close to the absolute neutrophil count (ANC). In addition to providing information that correlates with the ANC, the lucigenin chemiluminescence (CL) measurement of stimulated neutrophil oxidase activity provides useful clinical information and can be applied to point-of-care (POC) testing using a handheld luminometer.

[0039] Activity-based measurements of neutrophils provide additional information. The ANC quantifies the physical presence of neutrophils but not their function. Thus, disorders of phagocyte function, such as those associated with chronic granulomatous disease or any toxic effects of treatment on neutrophil function, are not detected. A functional measurement of the respiratory burst activity that drives reductive dioxygenation activity quantifies the microbicidal ability of neutrophils. By analogy, the antigenic detection of an enzyme does not provide information about its function. The enzyme may be antigenically present but not functional. The functional measurement of the enzyme provides more complete and clinically useful information; that is, the enzyme is present and functional.

[0040] When stimulated neutrophils are quantified by measuring the reductive dioxygenation of lucigenin, their CL response correlates with the neutrophil count. When stimulated neutrophils are quantified by measuring the non-reductive dioxygenation of luminol, their CL response shows little correlation with the neutrophil count. Chemiluminescence-generating probes (e.g., luminol) measure non-reductive dioxygenation or dioxygenation activity, particularly that catalyzed by MPO (Allen 2019). Under normal bone marrow hematopoietic conditions, the MPO content per neutrophil is stable, and the luminol dioxygenation activity is approximately close to the existing neutrophil count. However, in clinical conditions associated with increased bone marrow hematopoietic activity (e.g., G-CSF treatment and inflammatory stimuli), the myeloblast pool in the bone marrow is expanded, and there is little mitosis in the myelocyte pool of the bone marrow. MPO is synthesized only during the myeloblast phase of development. Inflammatory stimuli or therapeutic treatment with G-CSF stimulate and expand the myeloblast pool and reduce the number of divisions in the myelocyte pool. As a result, azurophilic granules containing MPO are not diluted by mitosis in the developing myelocyte phase. Neutrophils synthesized under such stimulated myelocyte conditions show a several-fold increase in MPO per neutrophil (Allen et al. 1997).

[0041] The ANF method disclosed herein is very sensitive and can be performed on less than 1 microliter of diluted whole blood. As shown in the following examples, the ANF chemiluminescence-generating technique for quantifying phagocytes in blood or body fluids provides a functionally equivalent amount of ANC. This lucigenin CL method is technically flexible and applicable to point-of-care testing (POCT) using portable or handheld luminometers for measurement. The acceptance and demand for POCT are continuing to increase (Asha, Chan et al. 2013, Schilling 2014). There is no handheld POCT method available for ANC (POCT).

Examples

[0042] Example

[0043] The following non-limiting examples provide scientific data that supports the concepts disclosed herein.

[0044] The human blood specimens examined were provided by a local clinical laboratory. Anonymized blood specimens were obtained along with the time of venipuncture of the subject, age (in years), sex, and a printout from an automated hematology analyzer (Advia 120, Siemens AG). Information regarding the reason for ordering the complete blood count and the medical condition of the subject was not provided.

[0045] The blood specimens were maintained at ambient temperature (22 ± 8 °C) until examined. Each of the 58 blood specimens was examined in triplicate. The mean (average) post-venipuncture age and standard deviation (SD) of the blood at the first examination were 4.1 ± 1.1 hours, and the median post-venipuncture age was 4.1 hours.

[0046] The specimens were examined again approximately 6 hours later; the mean post-venipuncture age and SD were 10.8 ± 2.0 hours, and the median was 10.4 hours. The blood was examined at later times post-venipuncture in an attempt to establish the limits of acceptance regarding the post-venipuncture age of the specimens.

[0047] Acceptably reproducible neutrophil functional activity was obtained over the first 16 hours post-venipuncture, and thus, the combined post-venipuncture mean (average) and SD of 7.4 hours ± 3.8, and median of 10.4 hours, were used for analysis. The subjects included 23 males and 35 females. The age range was 21 - 99 years, with a mean age of 69.4 and SD of 17.5, and a median age of 70.5 years. A total of 116 measurements were obtained.

[0048] The media used included dilution medium (DM), luminol buffered saline solution (LBSS), and lucigenin (dimethylviacridinium dinitrate) buffered saline solution (DBSS). DM contained the following: 5 mM 3-(N-morpholino)propanesulfonate (MOPS) buffered balanced salt solution (139 mEq / L Na+ , 5.0 mEq / L K + , 132 mEq / L Cl - , 0.8 mM H n PO4; pH 7.2; 290 ± 5 mOsmol / kg and endotoxin < 0.06 endotoxin unit (EU) / mL). LBSS contained the following: 139 mEq / L Na + , 5.0 mEq / L K + , 1.3 mM Ca 2+ , 0.9 mM Mg 2+ , 142 mEq / L Cl - , 0.8 mM H n PO4, and 5 mM MOPS-buffered salts solution containing 0.15 mM luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), and 5.5 mM D-glucose; pH 7.2; 290 ± 5 mOsmol / kg and endotoxin < 0.06 EU / mL. DBSS contained the following: 139 mEq / L Na + , 5.0 mEq / L K + , 1.3 mM Ca 2+ , 0.9 mM Mg 2+ , 142 mEq / L Cl - , 0.8 mM H n PO4, and 5 mM MOPS-buffered salts solution containing 0.2 mM lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate; also known as bis-N-methylacridinium nitrate), and 5.5 mM D-glucose; pH 7.2; 290 ± 5 mOsmol / kg and endotoxin < 0.06 EU / mL.

[0049] Figure 1 shows a plot of chemiluminescence intensity (rate), plotted as relative light units per second (RLU / sec) over a 29-minute interval against the time interval of the test for the first two subjects tested. Whole blood samples anticoagulated with potassium ethylenediaminetetraacetate (K3EDTA) were first diluted with DM and tested at a final dilution of 1:1200. The tests were performed in triplicate. An equivalent amount of 0.25 μL of blood was used per test. Activation of phagocyte respiratory burst metabolism was initiated by adding the diluted blood to microplate wells containing an equilibration salts solution coated with 0.5 nanomoles (nmol) of phorbol 12-myristate 13-acetate (PMA), i.e., the stimulant, and a chemiluminescence-generating probe, i.e., lucigenin or luminol. The final volume per well was 300 μL.

[0050] Measurements were taken at 37 °C at 2 minute 37 second intervals using an Orion II microplate luminometer (Titertek Berthold). For example, the absolute white blood cell counts (white blood cell count; WBC) of subjects 001 and 002 were 2475 / 0.25 μL and 350 / 0.25 μL, respectively, and the absolute neutrophil counts (ANC) of subjects 001 and 002 were 1,525 / 0.25 μL and 178 / 0.25 μL, respectively. The post-venipuncture ages of the blood samples from subjects 001 and 002 were 5 hours 40 minutes and 2 hours 49 minutes, respectively.

[0051] For each subject, 12 chemiluminescence (CL) intensity measurements were taken over a 28.9-minute interval. As shown in Figure 1, CL activity was measured as intensity, i.e., rate, and expressed as relative light units per second (RLU / sec). Thus, the plotted data illustrate the change in CL intensity (rate) over time. It is important to recognize that while the CL measurements are rate measurements, most techniques measure the accumulation of product or depletion of substrate, i.e., the integrated value.

[0052] For some comparisons, it may be appropriate to represent the CL as the accumulated RLU over a selected time interval of the assay, i.e., the integrated or sum value. The CL rate (RLU / sec) value can be converted to the integrated CL expressed as an RLU value by the sum of the areas under the RLU / sec plot over the assay interval.

[0053] Figure 2 shows the CL data of Figure 1 represented as the total or integrated RLU accumulated during the measurement time interval. The integrated representation of CL may be preferred when relating CL to other integrated values (e.g., the number of neutrophils present in the volume of blood assayed).

[0054] For comparison, a regression analysis of the integrated CL values was plotted against the types of white blood cells present in the assayed blood specimens as shown in Figures 3A, B, C, and D. As shown in Figure 3A, there is some correlation between luminol luminescence and the total white blood cell count (i.e., R 2 = 0.6782). Most of the white blood cells in the blood are phagocytes, and most of these phagocytes are neutrophils. The correlation between luminol CL and phagocyte and neutrophil counts is good (i.e., R 2 ) of 0.8336 and 0.8336, respectively). Luminol CL does not show a correlation with lymphocyte count (i.e., R 2 = 0.0053).

[0055] Luminol CL is a product of simple (non-reducing) dioxygenation activity. With respect to phagocytes, luminol measures oxidase-driven MPO activity or eosinophil peroxidase activity. Lucigenin measures oxidase-dependent phagocyte reductive dioxygenation activity, and its CL activity is haloperoxidase-independent. Measurement of phagocyte oxidase activity as lucigenin CL has advantages over luminol CL when the purpose is to quantify the phagocyte count present in blood or body fluids. The specific phagocyte NADPH oxidase activity, i.e., the oxidase activity per phagocyte / neutrophil, is relatively constant and independent of variations in the MPO concentration per neutrophil. The above MPO / neutrophil varies with respect to the number of mitoses in the myeloid phase of bone marrow hematopoiesis. Inflammatory or therapeutic increases in granulocyte colony-stimulating factor (G-CSF) expand the promyelocyte pool from which MPO is synthesized and decrease the mitotic activity of the myeloid pool. Each myeloid division halves the MPO per neutrophil. Thus, stimulation and expansion of the promyelocyte pool and a decrease in the number of divisions in the myeloid pool result in larger neutrophils with increased MPO (Allen, Stevens et al. 1997).

[0056] Activated phagocyte NADPH oxidase activity, which is responsible for reduced dioxygenation activity, is measured as lucigenin CL activity. This same oxidase activity drives haloperoxidase-dependent simple (non-reduced) dioxygenation activity. Phagocyte luminol-dependent CL activity reflects haloperoxidase, particularly MPO activity. The graphs and regression analyses of FIGS. 4A, B, C, and D show that luminol CL, which is MPO-dependent, is more variable than neutrophil-specific oxidase activity, which correlates with neutrophil count. Luminol CL per neutrophil varies with the state of host inflammation and with G-CSF or GM-CSF treatment (Allen, Stevens et al. 1997). The difference between simple (non-reduced) dioxygenation activity and reduced dioxygenation can be appreciated by comparing the integrated luminol CL regression analyses of FIGS. 4A-D with the integrated lucigenin CL regression analyses of FIGS. 3A-D. Reduced dioxygenation (lucigenin CL) plotted against neutrophils shows a greater correlation (i.e., R 2 = 0.7282) with phagocytes / neutrophils than simple (non-reduced) dioxygenation (luminol CL) plotted against neutrophils (i.e., R 2 = 0.8336). Similar to lucigenin CL, there is no correlation between luminol CL and lymphocyte count (i.e., R 2 = 0.0020).

[0057] In FIGS. 4B and C, it is noted that two subjects (the two points in the upper right of the graph) with the highest phagocyte / neutrophil counts also show high specific oxidase-driven MPO activity. High neutrophil counts and high specific MPO activity suggest that G-CSF-stimulated bone marrow hematopoiesis is usually in response to immune-physiological stimuli or therapeutic interventions (Allen, Stevens et al. 1997).

[0058] As shown in FIGS. 5A and B, plots of integrated luminol and lucigenin CL per neutrophil (i.e., specific activity / neutrophil) against post - venipuncture age of blood show that the CL activity per neutrophil remains relatively constant during the first 16 - hour interval after venipuncture, using either luminol or lucigenin as the chemiluminescence - generating probe. After this initial period, neutrophil oxidase and oxidase - driven MPO activity decrease exponentially. As previously described, luminol CL activity shows greater dispersion than lucigenin CL activity, regardless of post - venipuncture age. The R 2 values are 0.3434 and 0.6005, respectively. The lower R 2 observed for luminol CL is consistent with the previously described variation in MPO per neutrophil.

[0059] The functional lifespan after venipuncture is essentially the same whether neutrophil oxidase or oxidase - driven myeloperoxidase activity is measured. As shown in FIGS. 6A and B for individual subjects 036 and 037, both oxidase - driven MPO activity per neutrophil (i.e., luminol CL) and oxidase activity per neutrophil (i.e., lucigenin CL) show an exponential decrease with respect to the post - venipuncture age of blood neutrophils. The function of hexose monophosphate shunt enzymes that provide reducing equivalents to drive both NADPH oxidase activity and NADPH - oxidase - driven MPO activity is sensitive to age - related loss of function.

[0060] Averaging together the exponential relationships for each individual subject, the relationship of luminol CL to post - venipuncture age for 20 subjects with 4 complete measurements up to 60 hours was y = 59780e -0.026X and R 2 ± standard deviation = 0.9426 ± 0.0619. The relationship of lucigenin CL to post - venipuncture age for 24 subjects with 4 complete measurements up to 60 hours was y = 55404e -0.024X and R2 The standard deviation was 0.9038 ± 0.0938.

[0061] Finally, probing by chemiluminescence generation of phagocyte and particularly neutrophil NADPH oxidase activity can be applied to measure neutrophils in blood and body fluids. Oxidase function per neutrophil is best measured using lucigenin (DBSS: N,N'-dimethyl-9,9'-biacridinium dinitrate (lucigenin) equilibrated salts solution) as a chemiluminescence generation probe. Such lucigenin CL correlates with the number of phagocytes / neutrophils in an amount of whole blood or body fluid less than a microliter being examined. The functional activity of phagocytes in EDTA-anticoagulated blood is maintained relatively well during the first 16-hour period after venipuncture. After this initial period, the functional capacity of neutrophils (measured either as oxidase activity using lucigenin CL or oxidase-driven MPO activity using luminol CL) decreases exponentially with post-venipuncture age.

[0062] Luminol CL measures NADPH oxidase-driven MPO activity per neutrophil. The MPO content per neutrophil is variable and depends on the state of myelopoietic stimulation. Cell size, azurophilic granule content, and MPO per neutrophil increase after immunological production of G-CSF and therapeutic treatment with G-CSF (Allen, Stevens et al. 1997, Allen, Dale et al. 2000). As a result, the ratio of oxidase-driven MPO (luminol CL) activity to oxidase (lucigenin CL) activity provides useful information regarding treatment of neutrophil myelopoiesis or immuno-physiological stimulation.

[0063] Definition

[0064] As used herein, "about", "approximately" and "substantially" refer to a numerical range, e.g., a number within -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, and most preferably -0.1% to +0.1% of the referenced number.

[0065] Furthermore, all numerical ranges in this specification are to be understood to include all integers, integers or fractions within that range. Furthermore, these numerical ranges are to be construed as providing support for the claims for any number or subset of numbers within that range. For example, the disclosure of 1 to 10 should be construed as supporting ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0066] As used in this specification and the appended claims, the singular forms of the language include the plural unless the context clearly dictates otherwise. Thus, references to "a", "an", and "the" generally include the plural forms of their respective terms. For example, reference to "a stimulus" or "the stimulus" includes a plurality of such stimuli. The term "and / or" as used in the context of "X and / or Y" should be construed as "X" or "Y", or "X and Y". Similarly, "at least one of X or Y" should be construed as "X" or "Y", or "both X and Y".

[0001] Similarly, the terms "comprise", "comprises" and "comprising" are to be construed inclusively rather than exclusively. Similarly, the terms "including", "includes" and "or" are all to be construed as inclusive if such construction is not clearly prohibited by the context. However, embodiments provided by the present disclosure may lack any element not specifically disclosed herein. Accordingly, the disclosure of embodiments defined using the term "comprising" is also a disclosure of embodiments "consisting essentially of" the disclosed elements and embodiments "consisting of" the disclosed elements.

[0002] As used herein, the term "example" is merely illustrative and exemplary, particularly when a list of terms follows, and is not to be construed as exclusive or exhaustive. Any embodiment disclosed herein may be combined with any other embodiment disclosed herein, unless expressly stated otherwise.

[0003] "Animal" includes, but is not limited to, mammals, and mammals include, but are not limited to, rodents, aquatic mammals, domesticated animals (e.g., dogs and cats), livestock (e.g., sheep, pigs, cows and horses), and humans. When the terms "animal", "mammal" or their plurals are used, these terms apply to any animal that can be indicated by the context of the sentence or for which the intended effect is possible. As used herein, the term "patient" is understood to include an animal, e.g., a mammal, and preferably a human, that is receiving or intended to receive treatment (treatment as defined herein). The terms "individual" and "patient" are often used herein to refer to a human, but the present disclosure is not so limited. References

Table 1-1

Table 1-2

Claims

【Claim 1】 The invention described in the specification.

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