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Estimating cellular populations

A neutrophil and sample technology, applied in the field of biomarker expression level, can solve the problem of unsatisfactory sensitivity of sepsis markers

Pending Publication Date: 2019-07-02
THE MACFARLANE BURNET INSTITUTE FOR MEDICAL RESEARCH AND PUBLIC HEALTH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, nCD64 has suboptimal sensitivity as a marker of sepsis even among existing flow-based tests

Method used

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  • Estimating cellular populations
  • Estimating cellular populations
  • Estimating cellular populations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0258] Development of a point-of-care (POC) alternative to flow cytometry-based neutrophil activation assays. Immunoassay cutoffs were assessed using the CD64 to NNM ratio.

[0259] Recognizing that a POC test for measuring neutrophil activation (nCD64i) would be of great clinical utility, a novel method was devised as described herein that would allow the use of algorithms and independent adaptation to the POC measurement. Values ​​of the combination of markers were used to measure similar or equivalent values ​​of flow cytometry nCD64i values.

[0260] In some aspects, the methods of the invention take advantage of some of the inventors' experience in developing a lateral flow POC assay for measuring CD4 T cells (US Patent 8,409,818 and others).

[0261] According to the inventor's experience with CD4 POC test, it is known that the amount of CD4 per cell is constant, so the amount of CD4 is directly proportional to the number of CD4 T cells. In contrast, the amount of CD64...

Embodiment 2

[0277] Further developments, and in particular the utility of total CD64 levels and total NNM levels (ie internal and external levels) and different cut-off value determination methods in the diagnosis of sepsis are described.

[0278] In (FIG. 4C), it is illustrated that the amount of CD64 compared to NE (nCD64i measured by ELISA) is higher in samples with lower neutrophil / granulocyte counts, while novelly demonstrating the need for CD64i Variable cutoff value as a function of NE value to improve the use of CD64 to diagnose sepsis (see also Figure 5). It is worth noting that the results in that experiment were obtained using whole blood that had been monocyte / macrophage depleted using magnetic beads.

[0279] In further work these observations have been extended and a simplified method is described that only requires the use of NE and CD64 values ​​from monocyte / macrophage undepleted whole blood. Monocyte / macrophage depletion is an optional step, especially for those with lo...

Embodiment 3

[0289] To better understand the utility of the assays described herein, septic patients and a subset of healthy subjects were examined using the commercial Leuko64 assay, which measures cell surface expression of CD64 on neutrophils by flow cytometry.

[0290] The Leuko64 kit [see e.g. Beckman Coulter - see U.S. Patent Publication No. 2013 / 0230867 filed in the name of Trillium Diagnostics LLC] contains three species specific for surface CD64 (FITC conjugated) and surface CD163 (phycoerythrin conjugated). A mixture of monoclonal antibodies and a proprietary fluorescent bead suspension for instrument calibration and normalization of leukocyte CD64 and CD163 expression on human blood leukocytes. The kit also contains erythrocyte lysis solution concentrate. Variations of this kit are available, such as the Accellix CD64 cassette and reader (LeukoDx).

[0291] like Figure 13As shown in , Leuko64 showed highly significantly higher values ​​for sepsis patients compared to healthy ...

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Abstract

An immunoassay for assisting in the diagnosis of sepsis or severe infection in a patient / subject, the assay comprising the steps of: (i) optionally contacting a test sample comprising neutrophils fromthe patient with an agent that permeabilises or solubilises neutrophils; (ii) simultaneously with (i) or sequentially, contacting the sample with a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex a; (iii) simultaneously with (i) and / or (ii) or sequentially, contacting the sample with a second binding agent that binds specifically to a neutrophilnumber marker (NNM) in the sample and forms a neutrophil marker-binding agent complex b; (iv) employ the amount of complex a and complex b to determine the relative level of CD46 and of NNM in the sample.

Description

technical field [0001] The field of this specification relates broadly to diagnostics and the estimation of biomarker expression levels of biological populations of cells. More specifically, methods for identifying or monitoring neutrophil activation in subjects including neonatal subjects, pathophysiological conditions following neutrophil activation, and especially sepsis or severe Assays and kits for infection or risk of developing sepsis or severe infection. The assays of the present invention can be applied in a wide range of immunoassay formats and have been developed for algorithm-based diagnostic assays ranging from rapid point-of-care assays and devices to employing hardware and software for data input and processing ( Including algorithms to assess the statistical significance of altered biomarker levels) and more data-rich applications to develop output data that can be integrated with pathology platform systems. Background technique [0002] Bibliographic detai...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/53
CPCG01N2333/70535G01N2800/26G01N33/56972
Inventor 大卫·安德森里亚·帕尔乔杜里苏珊娜·克罗克洛维斯·帕默尔
Owner THE MACFARLANE BURNET INSTITUTE FOR MEDICAL RESEARCH AND PUBLIC HEALTH LTD
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