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 leukocytes (i.e., essentially neutrophils, but also including monocytes and eosinophils) in blood and body fluids in a manner applicable to point-of-care diagnostic testing. Absolute neutrophil counts (ANCs) can be performed to assess myelopoiesis suppression, usually in the context of chemotherapy or as a measure of inflammation. The methods disclosed herein can quantify phagocytes based on measuring the activation of respiratory burst metabolism and the reductive dioxygenation of a chemiluminescent-generating probe. [Background technology]

[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 component of blood is morphologically and functionally distinct from red blood cells and platelets. Impedance, light scatter, and enzymatic and antigenic differences serve as the basis for counting and differentiating white blood cells by automated hematology analysis. These instruments are highly 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 hexose monophosphate shunt dehydrogenase 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 generates natural chemiluminescence as an energy product (Allen et al. 1972). Natural leukocyte chemiluminescence is O2-dependent and directly proportional to hexose monophosphate shunt dehydrogenase activity. NADPH oxidase activation increases the production of HO2, O2, and- and H2O2 is produced. The H2O2 produced is converted from chloride ions to hypochlorite ions (OCl - ) and singlet molecular oxygen ( 1 The halide-dependent haloperoxidase activity of isolated MPO also produces 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 oxygenated agent produced, as well as the nature and quantum efficiency of the substrate being oxygenated. Oxygenation of natural substrates is associated with a relatively low chemiluminescence quantum yield, which varies with the nature of the substrate being oxygenated. The introduction of high quantum yield chemiluminescence-generating substrates (CLS) overcomes the problems of sensitivity and substrate variability. The 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 the mildly acidic to neutral pH conditions of the physiological environment. Luminol- and lucigenin-dependent phagocyte CL activity measures different oxygenation pathways (Allen 1982, Allen 1986). Luminol CL results from dioxygenation, which generates electronically excited aminophthalate: luminol + O2 → aminophthalate + N2 + photon. In phagocytic leukocytes, this activity is strongly associated with MPO. Luminol CL results from a nonreductive, simple dioxygenation. While little luminol CL is observed in MPO-negative leukocytes, MPO-positive leukocytes produce 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 and O2 - and H + produces O2 - and HO2 become disproportionate, yielding HO2. Under acidic to neutral conditions, divalent cationic lucigenins (N,N'-dimethyl-9,9' biacridinium and bis-N-methylacridinium) can undergo one electron reduction to yield monovalent cation radicals (i.e., lucigenin ++ +e - → Lucigenin + ) and this radical is O2 - to give a dioxetane intermediate and ultimately two N-methylacridones and a photon (i.e., lucigenin). + +O2 - → lucigenin-O2 → 2 N-methylacridone + photon (Allen 1981, Allen 2019)). Summary of the Invention [Means for solving the problem]

[0007] Abstract The inventors have surprisingly found that the complex instrumentation required for impedance and flow cytometry measurements of phagocytic leukocytes can be obviated by using a chemiluminescent approach to directly measure the functional activity of stimulated phagocytes present in diluted whole blood and body fluids (e.g., spinal fluid). Phagocytic cells per volume of blood or body fluid are determined by measuring stimulated NADPH oxidase-dependent reductive dioxygenation of a chemiluminescent-generating substrate. The introduction of an optimal stimulator of phagocyte NADPH oxidase activation (e.g., PMA) results in the generation of reductive deoxygenation activity.

[0008] The resulting reductive dioxygenation of lucigenin produces CL, which can be quantified by measuring the emitted light 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 to assessing a patient's clinical status with respect to inflammation / infection or chemotherapy-related myelosuppression. Such function-based analysis provides an alternative, and arguably superior, assay compared to traditional ANC. The technical requirements of the ANF approach make it applicable to clinical point-of-care testing. As described in the Examples herein, the ANF assay allows for quantitative assessment of blood neutrophil count (ANC) and function during the first 16-hour interval after venipuncture.

[0009] Additional features and advantages are described in, or are apparent from, the following detailed description and drawings. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for estimating the number of phagocytes in a body fluid of an animal, said method comprising: stimulating NADPH oxidase activity of the phagocyte; and quantitating the resulting reductive dioxygenation of the chemiluminescence-generating substrate by the released chemiluminescence of said chemiluminescence-generating substrate using an instrument capable of measuring light. A method that encompasses (Item 2) 2. The method of claim 1, wherein the NADPH oxidase activity of the phagocyte is stimulated by an immunological or chemical substance capable of activating a respiratory burst by the phagocyte. (Item 3) 2. The method of claim 1, wherein the NADPH oxidase activity of the phagocytes is stimulated by a stimulator in solution or coated on a surface with which the phagocytes contact. (Item 4) 4. The method of claim 3, wherein the stimulatory agent is phorbol myristate acetate (PMA). (Item 5) Item 10. The method of item 1, wherein the animal is a human. (Item 6) Item 10. The method of claim 1, wherein the body fluid is blood. (Item 7) Item 10. The method of claim 1, wherein the body fluid is spinal fluid. (Item 8) 2. The method of claim 1, wherein the phagocyte is a neutrophil. (Item 9) 2. The method according to item 1, wherein the chemiluminescence-generating substrate is lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate). (Item 10) 10. The method of claim 9, wherein the lucigenin is in solution or coated on a surface that comes into contact with the phagocytes. (Item 11) 2. The method according to item 1, comprising diluting the body fluid to reduce chemiluminescent red blood cell absorbance. (Item 12) 7. The method of claim 6, comprising diluting the blood up to about 1:500 to reduce chemiluminescent red blood cell absorbance. (Item 13) 7. The method of claim 6, comprising diluting the blood up to about 1:1000 to reduce chemiluminescent red blood cell absorbance. (Item 14) 8. The method of claim 7, comprising diluting the spinal fluid up to 1:100 to reduce chemiluminescent red blood cell absorbance. (Item 15) 4. The method according to item 2 or 3, comprising using a lectin to agglutinate or remove red blood cells from the body fluid to facilitate chemiluminescent detection. (Item 16) 2. The method of claim 1, wherein the emitted chemiluminescence is measured by a portable or handheld luminometer. (Item 17) 2. The method of claim 1, wherein the components are pre-manufactured to facilitate point-of-care testing. (Item 18) 2. The method of claim 1, further comprising determining an absolute neutrophil count (ANC) using the estimated number of phagocytes. (Item 19) 19. The method of claim 18, further comprising assessing myelopoietic suppression in the animal using the ANC. (Item 20) 20. The method of claim 19, wherein the myelopoiesis suppression is associated with chemotherapy or a measure of inflammation or infection. (Item 21) 21. The method of claim 20, further comprising treating the animal based on the assessment of myelopoiesis suppression. (Item 22) 1. A method for predicting myelopoietic stimulation, said method comprising: Measuring non-reductive dioxygenation-driven myeloperoxidase (luminol CL) activity and reductive dioxygenation (lucigenin CL) activity of chemically activated blood neutrophils of the animal using an instrument capable of measuring light; and calculating the ratio of said luminol CL activity to said lucigenin CL activity. A method that encompasses [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the chemiluminescence intensity (rate) for the example experiments disclosed herein, expressed as relative luminescence units per second (RLU / s) plotted against time in minutes. A 0.25 μL equivalent of blood was tested for the first two subjects enrolled, subjects 001 (circles) and 002 (diamonds). CL activation occurred upon mixing the 0.25 μL equivalent of blood with 0.5 nmol PMA in balanced salt medium containing lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate) as the chemiluminescence-generating probe. Measurements were performed in triplicate, and the average rate is indicated by the larger open symbol, along with the standard deviation (indicated by the small dots).

[0011] [Figure 2] 2 shows the integrated chemiluminescence for the example experiments disclosed herein, expressed as accumulated relative luminescence units (RLU) plotted against time in minutes. The integrated CL measurements (RLU) are calculated from the intensity measurements shown in FIG.

[0012] [Figure 3A] Figures 3A, B, C, and D are plots of integrated lucigenin-dependent CL activity versus leukocyte, phagocyte (neutrophil, monocyte, and eosinophil), neutrophil, and lymphocyte counts, respectively, for the example experiments disclosed herein. The abscissa represents cell counts per 0.25 μL of blood plotted against oxidase-dependent reductive dioxygenation activity, measured as PMA-stimulated lucigenin CL. Linear regression analysis and coefficient of determination (R2) are shown for each plot. Leukocyte counts were per 0.25 μL of blood tested. DBSS indicates the medium was N,N'-dimethyl-9,9'-biaclidinium dinitrate (lucigenin) balanced salt solution. Blood age ranged from 1 to 16 hours after venipuncture. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above.

[0013] [Figure 4A] Figures 4A, B, C, and D are plots of integrated luminol-dependent CL activity versus leukocyte, phagocyte (neutrophil, monocyte, and eosinophil), neutrophil, and lymphocyte counts, respectively, for the example experiments disclosed herein. The abscissa represents cell counts per 0.25 μL of blood; the ordinate represents oxidase-driven MPO-dependent (non-reductive) dioxygenase activity, measured as PMA-stimulated luminol CL. Other than the chemiluminescence-generating probe, i.e., LBSS (luminol-balanced salt solution), conditions were as described for Figures 3A-D. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above.

[0014] [Figure 5A] Figures 5A and 5B are CL activity plots for an example experiment disclosed herein. Figure 5A is a plot of integrated luminol-dependent CL activity per neutrophil versus age of blood (in hours) after venipuncture. Figure 5B is a plot of integrated lucigenin-dependent CL activity per neutrophil versus age of blood (in hours) after venipuncture. The integrated test interval was 28.9 minutes. [Figure 5B] Same as above.

[0015] [Figure 6A] Figures 6A and 6B are CL activity plots for example experiments disclosed herein. Figure 6A is a plot of integrated luminol-dependent CL activity per neutrophil versus age of blood post-venipuncture for subjects 036 and 037. Figure 6B is a plot of integrated lucigenin-dependent CL activity per neutrophil versus age of blood post-venipuncture for subjects 036 and 037. The integrated test interval was 28.9 minutes. [Figure 6B] Same as above. DETAILED DESCRIPTION OF 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 NADPH oxidase activity of the phagocytes; and quantifying the resulting reductive deoxygenation of a chemiluminescence-generating substrate by the released chemiluminescence of the chemiluminescence-generating substrate using an instrument capable of measuring light.

[0018] In one embodiment, the NADPH oxidase activity of the phagocyte is stimulated by an immunological or chemical agent that can activate a respiratory burst by the phagocyte.

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

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

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

[0022] In one embodiment, the bodily fluid is spinal fluid, which may be diluted up to 1:100 to reduce red blood cell absorbance of chemiluminescence.

[0023] In one embodiment, the phagocyte is a neutrophil.

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

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

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

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

[0028] In one embodiment, the method includes using a 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 handheld luminometer. In one embodiment, the components are pre-manufactured to facilitate point-of-care testing.

[0030] In one embodiment, the method further comprises determining an absolute neutrophil count (ANC) using the estimated number of phagocytes. The method may further comprise using the ANC to assess myelopoiesis suppression in the animal, for example, myelopoiesis suppression correlated with chemotherapy or a measure of inflammation or infection. In this regard, inflammation or infection typically increases myelopoiesis activity, but neutrophil consumption in response to infection may actually decrease neutrophil count. The method may further comprise treating the animal based on the assessment of myelopoiesis suppression.

[0031] In another embodiment, a method for predicting myelopoiesis stimulation is provided, comprising measuring the non-reductive dioxygenation-driven myeloperoxidase (luminol CL) activity and the reductive deoxygenation (lucigenin CL) activity of chemically activated blood neutrophils of an animal using an instrument capable of measuring light; and calculating the ratio of the luminol CL activity to the lucigenin CL activity.

[0032] Preferred Embodiments

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

[0034] The method requires less than a drop of whole blood or body fluid. A small volume of the specimen is diluted with a balanced salt solution. The diluted specimen is introduced into an environment containing a chemical irritant (e.g., phorbol 12-myristate 13-acetate (PMA)) that can activate phagocyte NADPH oxidase-dependent respiratory burst metabolism and a chemiluminescence-generating probe sensitive to reductive dioxygenation, such as lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate). Such contact activates the phagocyte NADPH oxidase-dependent reductive dioxygenation of lucigenin, producing chemiluminescence that can be detected and quantified by luminometry. The chemiluminescence-generating probe (e.g., luminol) measures phagocyte nonreductive (simple) dioxygenation reactions, particularly those catalyzed by myeloperoxidase (MPO).

[0035] Under normal bone marrow hematopoietic conditions, measurements of non-reductive dioxygenation closely approximate the neutrophil count present. Inflammatory stimuli and g-CSF treatment expand the bone marrow promyelocyte pool, resulting in a several-fold increase in MPO content per neutrophil. Non-reductive dioxygenation activity reflects the MPO content per phagocyte and reflects the specific MPO content per phagocyte. Lucigenin-dependent NADPH oxidase-dependent reductive dioxygenation is MPO-independent and, as a result, directly proportional to the number of functional phagocytes in a specimen. The lucigenin-based ANF system quantifies the presence of phagocytes by measuring the reductive dioxygenation of stimulated phagocytes. In the absence of genetic disorders, such as chronic granulomatous disease or acquired neutrophil disorders, lucigenin-dependent measurements of NADPH oxidase reductive dioxygenation activity are MPO-independent and closely approximate the neutrophil count of the specimen. The ANC determines the number of neutrophils present in the specimen. The ANF assay provides more clinically relevant information based on the quantification of functional neutrophil presence in a specimen. The ANF assay has been shown to quantitatively reflect whole blood ANC during the first 16-hour interval after venipuncture, making it applicable to clinical point-of-care testing.

[0036] While NADPH oxidase activity per phagocyte is relatively constant, myeloperoxidase (MPO) activity per phagocyte fluctuates with the state of myelopoietic stimulation. MPO and cationic proteases are lysosomal enzymes synthesized during the promyelocytic phase of development and stored in 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 mitotic divisions during the myeloid phase of neutrophil development (Allen, Stevens et al. 1997). For example, each division during the myeloid phase dilutes the myeloperoxidase per neutrophil by half. Components of NADPH oxidase are synthesized during the myeloid phase of development and are required for respiratory burst metabolism. As a result, NADPH oxidase activity per phagocyte is relatively constant with respect to fluctuations in myelopoietic activity. The specific NADPH oxidase activity per phagocyte remains relatively constant in various states of inflammation and myelopoiesis stimulation or suppression.

[0037] Thus, stimulated oxidase activity closely approximates phagocyte counts, particularly neutrophil counts. Such activity measurements closely approximate absolute neutrophil counts per volume of blood or body fluid. The incorporation of lucigenin as a chemiluminescence-generating probe allows for sensitive quantification of phagocyte NADPH oxidase reductive dioxygenation activity (Allen 1981, Allen 1982, Allen 1986). Simple or nonreductive dioxygenation is a reaction in which O2 is incorporated into a substrate (e.g., luminol + O2 → aminophthalate + N2 + photon (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 + photon).

[0038] NADPH oxidase-dependent reductive dioxygenation of lucigenin provides a measure of absolute neutrophil function (ANF) that closely approximates the absolute neutrophil count (ANC). In addition to providing information consistent with the ANC, lucigenin chemiluminescence (CL) measurements of stimulated neutrophil oxidase activity provide useful clinical information and can be applied to point-of-care (POC) testing using handheld luminometers.

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

[0040] When stimulated neutrophils are quantified by measuring the reductive dioxygenation of lucigenin, the CL response correlates with neutrophil count. When stimulated neutrophils are quantified by measuring the nonreductive dioxygenation of luminol, the CL response correlates poorly with neutrophil count. Chemiluminescence-generating probes (e.g., luminol) measure nonreductive dioxygenation or dioxygenation activity, particularly that catalyzed by MPO (Allen 2019). Under normal myelopoietic conditions, the MPO content per neutrophil is stable, and luminol dioxygenation activity roughly approximates the neutrophil count present. However, in clinical conditions associated with increased myelopoietic activity (e.g., g-CSF treatment and inflammatory stimuli), the bone marrow promyelocyte pool is expanded, and mitosis in the bone marrow myeloid pool is rare. MPO is synthesized only during the promyelocytic phase of development. Inflammatory stimuli or therapeutic treatment with G-CSF stimulate and expand the promyelocyte pool and reduce the number of divisions in the myelocyte pool. As a result, MPO-containing azurophilic granules are not diluted by mitosis during the myelocyte phase of development. Neutrophils synthesized under such stimulated myelocyte conditions exhibit a several-fold increase in MPO per neutrophil (Allen et al. 1997).

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

[0042] Example

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

[0044] The human blood specimens tested were provided by a local clinical laboratory. Anonymized blood specimens were obtained along with the subject's time of venipuncture, age (in years), sex, and an automated hematology analyzer (Advia 120, Siemens AG) printout. Subjects were blinded to the reason for ordering the complete blood count and information regarding the subject's medical condition.

[0045] The blood specimens were maintained at ambient temperature (22 ± 8°C) until testing. Each of the 58 blood specimens was tested in triplicate. The average (mean) age and standard deviation (SD) of blood at the time of initial testing was 4.1 ± 1.1 hours, with a median age of 4.1 hours.

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

[0047] Acceptably reproducible neutrophil function activity was obtained over the first 16-hour period after venipuncture; therefore, a combined venipuncture mean (mean) and SD of 7.4 hours ± 3.8 hours and a median of 10.4 hours were used for analysis. The subjects included 23 men and 35 women. The age range was 21-99 years, with a mean age of 69.4 years and SD of 17.5 years, and a median age of 70.5 years. A total of 116 measurements were obtained.

[0048] The media used included dilution medium (DM), luminol balanced salt solution (LBSS), and lucigenin (dimethylbiacridinium dinitrate) balanced salt solution (DBSS). DM contained: 5 mM 3-(N-morpholino)propanosulfonate (MOPS) buffered balanced salt solution (139 mEq / L Na+ , 5.0mEq / LK + , 132 mEq / L Cl - , 0.8 mM H n PO4; pH 7.2; 290 ± 5 mOsmol / kg and endotoxin <0.06 endotoxin units (EU) / mL). LBSS contained: 139 mEq / L Na + , 5.0mEq / LK + , 1.3 mM Ca 2+ , 0.9mM Mg 2+ , 142 mEq / L Cl - , 0.8 mM H n PO4, and 0.15 mM luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), 5.5 mM D-glucose; pH 7.2; 290 ± 5 mOsmol / kg and endotoxin <0.06 EU / mL. DBSS contained: 139 mEq / L Na + , 5.0mEq / LK + , 1.3 mM Ca 2+ , 0.9mM Mg 2+ , 142 mEq / L Cl - , 0.8 mM H n PO4, and 0.2 mM lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate; also known as bis-N-methylacridinium nitrate), in 5 mM MOPS-buffered saline solution, 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), expressed as relative luminescence units (RLU / sec), over a 29-minute interval plotted against the test time interval for the first two subjects tested. Whole blood specimens anticoagulated with potassium ethylenediaminetetraacetic acid (K3EDTA) were initially diluted in DM and tested at a final dilution of 1:1200. Tests were performed in triplicate. The equivalent of 0.25 μL of blood was used per test. Activation of phagocyte respiratory burst metabolism was initiated by adding diluted blood to microplate wells coated with 0.5 nanomolar (nmol) phorbol 12-myristate 13-acetate (PMA), a stimulator, and containing balanced salt solution with a chemiluminescence-generating probe, i.e., lucigenin or luminol. The final volume per well was 300 μL.

[0050] Measurements were taken at 2 minute 37 second intervals using an Orion II microplate luminometer (Titertek Berthold) at a temperature of 37°C. For example, the absolute white blood cell counts (WBC) for subjects 001 and 002 were 2475 / 0.25 μL and 350 / 0.25 μL, respectively, and the absolute neutrophil counts (ANC) for subjects 001 and 002 were 1,525 / 0.25 μL and 178 / 0.25 μL, respectively. The ages of the blood specimens from subjects 001 and 002 after venipuncture 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., velocity, and expressed as relative luminescence units per second (RLU / sec). Thus, the plotted data illustrates the change in CL intensity (velocity) with respect to time. It is important to recognize that CL measurements are velocity (rate) measurements, whereas most techniques measure product accumulation or substrate depletion, i.e., integrals.

[0052] For some comparisons, it may be appropriate to express CL as the accumulated RLU, i.e., integrated or summed value, over a selected time interval of the test. CL rate (RLU / sec) values ​​can be converted to integrated CL, expressed as an RLU value, by summing the area under the RLU / sec plot over the interval of the test.

[0053] Figure 2 shows the CL data from Figure 1 expressed as total or integrated RLU accumulated over the time interval of measurement. An integrated representation of CL may be preferable when relating CL to other integral values ​​(e.g., the number of neutrophils present in the blood volume examined).

[0054] For comparison, a regression analysis of the integrated CL values ​​was plotted against the type of leukocyte present for the blood specimens examined, as illustrated in Figures 3A, B, C, and D. As shown in Figure 3A, there was some correlation between lucigenin luminescence and total leukocyte count (i.e., R 2 =0.6782). The majority of white blood cells in the blood are phagocytes, and the majority of these phagocytes are neutrophils. The correlation between lucigenin CL and phagocyte and neutrophil counts is good (i.e., R = 0.8336 and 0.8336, respectively). 2 Lucigenin CL does not correlate with lymphocyte counts (i.e., R 2 =0.0053).

[0055] Luminol CL is a product of simple (non-reductive) dioxygenase activity. In phagocytes, luminol measures oxidase-driven MPO activity or eosinophil peroxidase activity. Lucigenin measures oxidase-dependent phagocyte reductive dioxygenase activity, whereas its CL activity is haloperoxidase-independent. Measurement of phagocyte oxidase activity as lucigenin CL has advantages over luminol CL when the goal is to quantify phagocyte counts in blood or body fluids. Specific phagocyte NADPH oxidase activity, i.e., oxidase activity per phagocyte / neutrophil, is relatively constant and independent of fluctuations in MPO concentration per neutrophil. The MPO / neutrophil ratio fluctuates with the number of mitoses during the myeloid phase of bone marrow hematopoiesis. Inflammatory or therapeutic increases in granulocyte colony-stimulating factor (G-CSF) expand the promyelocytic pool from which MPO is synthesized and decrease the mitotic activity of the myeloid pool. Each myelocyte division removes half the MPO per neutrophil, so stimulation and expansion of the promyelocyte pool and a reduction in the number of divisions in the myelocyte pool will result in larger neutrophils with increased MPO (Allen, Stevens et al. 1997).

[0056] Activated phagocyte NADPH oxidase activity is responsible for reductive dioxygenation activity and is measured as lucigenin CL activity. This same oxidase activity drives haloperoxidase-dependent simple (non-reductive) dioxygenation activity. Phagocyte luminol-dependent CL activity reflects haloperoxidase, specifically MPO, activity. The graphs and regression analysis in Figure 4A, B, C, and D show that unlike neutrophil specific oxidase activity, which correlates with neutrophil count, luminol CL, which is MPO-dependent, is more variable. Luminol CL per neutrophil varies with the inflammatory state of the host and with G-CSF or GM-CSF treatment (Allen, Stevens et al. 1997). The difference between simple (non-reductive) dioxygenation activity and reductive dioxygenation can be appreciated by comparing the integrated luminol CL regression analysis in Figures 4A-D with the integrated lucigenin CL regression analysis in Figures 3A-D. Reductive dioxygenation (lucigenin CL) plotted against neutrophils is shown in R 2 = 0.7282. 2 =0.8336). As with lucigenin CL, there is no correlation between luminol CL and lymphocyte count (i.e., R 2 =0.0020).

[0057] It is noted in Figures 4B and C that the two subjects with the highest phagocyte / neutrophil counts (the two points at the top right of the graph) also exhibited high specific oxidase-driven MPO activity. High neutrophil counts and high specific MPO activity suggest that G-CSF-stimulated myelopoiesis is normally responsive to immuno-physiological stimuli or therapeutic intervention (Allen, Stevens et al. 1997).

[0058] As illustrated in Figure 5A and B, plots of integrated luminol and lucigenin CL per neutrophil (i.e., specific activity / neutrophil) versus age after venipuncture of blood show that 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 exponentially decline. As previously described, luminol CL activity exhibits greater variance than lucigenin CL activity, regardless of age after venipuncture. The R of combined luminol and lucigenin CL activity per neutrophil measurements 2 are 0.3434 and 0.6005, respectively. The lower R observed for luminol CL 2 is consistent with previously described variations in MPO per neutrophil.

[0059] The post-venipuncture functional lifespan is essentially the same whether neutrophil oxidase or oxidase-driven myeloperoxidase activity is measured. As illustrated in Figures 6A and 6B 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 decline with post-venipuncture age of blood neutrophils. The function of the hexose monophosphate shunt enzyme, which provides the reducing equivalents that drive both NADPH oxidase activity and NADPH oxidase-driven MPO activity, is susceptible to age-related loss of function.

[0060] When the exponential relationships for each individual subject were averaged together, the relationship of luminol CL to age after venipuncture for 20 subjects with four 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 age after venipuncture for 24 subjects with four complete measurements up to 60 hours was y = 55404e -0.024X and R2 ±standard deviation = 0.9038 ± 0.0938.

[0061] Finally, chemiluminescence-generated probing of phagocyte and especially 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) balanced salt solution) as a chemiluminescence-generated probe. Such lucigenin CL correlates with the number of phagocytes / neutrophils in submicroliter volumes of whole blood or body fluids being tested. The functional activity of phagocytes in EDTA-anticoagulated blood is relatively well maintained during the first 16 hours after venipuncture. After this initial period, neutrophil functional capacity (measured either as oxidase activity using lucigenin CL or oxidase-driven MPO activity using luminol CL) decreases exponentially with age after venipuncture.

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

[0063] definition

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

[0065] Furthermore, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions within that range. Furthermore, these numerical ranges should be interpreted as providing claim support for any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges of 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 terms include the plural unless the context clearly dictates otherwise. Thus, references to "a," "an," and "the" generally include the plural of the respective term. For example, reference to "a stimulus" or "the stimulus" includes a plurality of such stimuli. The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y," or "X and Y." Similarly, "at least one of X or Y" should be interpreted as "X" or "Y," or "both X and Y."

[0001] Similarly, the words "comprise," "comprises," and "comprising" should be interpreted inclusively rather than exclusively. Similarly, the terms "including," "including," and "or" should all be interpreted inclusively unless such construction is clearly prohibited by context. However, embodiments provided by the present disclosure may lack any element not specifically disclosed herein. Thus, disclosure of embodiments defined using the term "comprising" is also a disclosure of embodiments "consisting essentially of" and "consisting of" the disclosed components.

[0002] As used herein, the term "example," particularly when followed by a list of terms, is illustrative and illustrative only and should not be construed as exclusive or exhaustive. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein, unless expressly stated otherwise.

[0003] "Animal" includes, but is not limited to, mammals, including, but not limited to, rodents, aquatic mammals, domesticated animals (e.g., dogs and cats), livestock (e.g., sheep, pigs, cattle, and horses), and humans. When "animal," "mammal," or their plural forms are used, these terms apply to any animal capable of the effect indicated or intended to be indicated by the context of the text. As used herein, the term "patient" is understood to include animals, e.g., mammals, and preferably humans, undergoing or intended to undergo treatment (treatment as defined herein). Although the terms "individual" and "patient" are often used herein to refer to humans, the present disclosure is not so limited. References [Table 1-1] [Table 1-2] The invention is claimed as follows:

Claims

[Claim 1] A method for predicting myelopoietic stimulation, said method comprising: obtaining a whole blood specimen; diluting the whole blood specimen 1:1200 with dilution medium; adding a volume of the diluted whole blood to a microplate well coated with phorbol 12-myristate 13-acetate and containing balanced salt solution; adding a chemiluminescence generating probe, which is lucigenin, to the microplate wells and measuring the emitted chemiluminescence of lucigenin using a handheld or portable light measuring instrument, wherein the lucigenin chemiluminescence measures neutrophil NADPH oxidase activity; adding a chemiluminescence generating probe, luminol, to the microplate wells and measuring the released luminol chemiluminescence using a handheld or portable light measuring instrument, wherein the luminol chemiluminescence measures NADPH oxidase-driven myeloperoxidase activity per neutrophil; and calculating the ratio of the oxidase-driven myeloperoxidase luminol chemiluminescent activity to the oxidase lusigenin chemiluminescent activity, said ratio providing information regarding the immuno-physiological stimulation of neutrophil myelopoiesis. A method that encompasses

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