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Oxidase-based chemiluminescent assay of phagocytic leukocytes in whole blood and bodily fluids suitable for point-of-care (POC) measurements for point-of-care (POC) diagnostic testing of absolute neutrophil function (ANF)

A neutrophil-based, chemiluminescent technique for the point-of-care (POC) measurement of absolute neutrophil function (ANF) suitable for point-of-care (POC) diagnostic testing of phagocytic leukocytes in whole blood and body fluids In the field of chemiluminescent determination of oxidase, it can solve problems such as unsuitability and complicated instruments

Pending Publication Date: 2022-04-22
比纳瑞有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These instruments are complex and inherently unsuitable for point-of-care (POC) assay development

Method used

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  • Oxidase-based chemiluminescent assay of phagocytic leukocytes in whole blood and bodily fluids suitable for point-of-care (POC) measurements for point-of-care (POC) diagnostic testing of absolute neutrophil function (ANF)
  • Oxidase-based chemiluminescent assay of phagocytic leukocytes in whole blood and bodily fluids suitable for point-of-care (POC) measurements for point-of-care (POC) diagnostic testing of absolute neutrophil function (ANF)
  • Oxidase-based chemiluminescent assay of phagocytic leukocytes in whole blood and bodily fluids suitable for point-of-care (POC) measurements for point-of-care (POC) diagnostic testing of absolute neutrophil function (ANF)

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Embodiment approach

[0018]The present disclosure provides 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 chemiluminescence by emission of chemiluminescent substrates using an instrument capable of measuring light. Reductive deoxygenation of luminescent substrates.

[0019] In one embodiment, the NADPH oxidase activity of the phagocyte is stimulated by an immune or chemical agent capable of activating the respiratory burst of the phagocyte.

[0020] In one embodiment, the NADPH oxidase activity of the phagocyte is stimulated by a stimulus in solution or coated onto a surface that the phagocyte contacts. The irritant may be phorbol myristate acetate (PMA).

[0021] In one embodiment, the animal is a human.

[0022] In one embodiment, the bodily fluid is blood.

[0023] In one embodiment, the body fluid is spinal fluid. Spinal fluid can be diluted up to 1 / 100 to reduce...

Embodiment

[0044] The following non-limiting examples provide scientific data supporting the concepts disclosed herein.

[0045] The human blood samples tested were provided by local clinical testing laboratories. De-identified blood samples were obtained with time of venipuncture, subject's age in years, sex, and printout from an automated hematology analyzer (Advia 120, Siemens AG). Order full The reason for blood counts and information about the subject's medical condition is unknown.

[0046] Blood samples were kept at ambient temperature (22±8°C) prior to testing. Each of the 58 blood samples was tested in triplicate. The mean (mean) post-venipuncture blood age plus standard deviation (SD) of blood at the time of initial testing was 4.1 ± 1.1 hours and the median post-venipuncture blood age was 4.1 hours.

[0047] Samples were retested approximately 6 hours later; mean blood age plus SD after venipuncture was 10.8 ± 2.0 hours, with a median of 10.4 hours. Blood was also tested at ...

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Abstract

A method for estimating the number of phagocytes in a body fluid of an animal comprises stimulating the NADPH oxidase activity of the phagocytes; and quantifying the reductive deoxygenation of the chemiluminescent substrate by chemiluminescence of the emission of the chemiluminescent substrate using an instrument capable of measuring light. The NADPH oxidase activity of the phagocytes is preferably stimulated by immunity or a chemical substance capable of activating the respiratory burst of the phagocytes. The NADPH oxidase activity of the phagocytes is preferably stimulated by a stimulus in solution or coated onto the surface in contact with the phagocytes. The stimulant is preferably a phorbol myristic acid acetate (PMA). The animal is preferably a human, and the body fluid is preferably blood and / or spinal fluid. The phagocyte is preferably a neutrophil, and the chemiluminescent substrate is preferably a lucigenin (N, N '-dimethyl-9, 9'-bis acridinium dinitrate). A treatment method based on the estimation method is also provided.

Description

technical field [0001] The method quantifies the presence of phagocytic leukocytes (essentially neutrophils, but also monocytes and eosinophils) in blood and body fluids in a manner suitable for point-of-care diagnostic testing. Absolute neutrophil counts (ANC) can be performed to assess suppression of myelopoiesis usually associated with chemotherapy or as a measure of inflammation. The methods disclosed herein can quantify phagocytic cells based on activation of respiratory burst metabolism and measuring reductive dioxygenation of chemiluminescent probes. [0002] technical background [0003] Automated blood analyzers are well-established instruments for measuring 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 are the basis for counting and differentiating leukocytes b...

Claims

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Application Information

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IPC IPC(8): G01N21/76C12N9/02G01N33/52G01N33/53G01N33/50
CPCC12Q1/26G01N2333/90209G01N33/56972G01N33/582G01N33/5094G01N33/5038G01N33/5055G01N21/76G01N33/52G01N33/53C12Y106/03001G01N33/581
Inventor R·C·艾伦J·C·艾伦J·T·小斯蒂芬斯
Owner 比纳瑞有限责任公司
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