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Methods and systems for measuring serotonin in a sample

A serotonin and sample technology, which is used in the field of measuring serotonin and systems in samples, can solve the problems of complex operation and the lack of popularization of platelet function assays, etc.

Active Publication Date: 2017-12-01
LAB OF AMERICA HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, functional assays utilizing washed platelets are not widely available due to the complexity of the procedure
Furthermore, serotonin release assays using washed platelets are usually performed using platelets that have been incubated with radiolabeled serotonin, with the attendant disadvantages associated with the use of radioactive substances

Method used

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  • Methods and systems for measuring serotonin in a sample
  • Methods and systems for measuring serotonin in a sample
  • Methods and systems for measuring serotonin in a sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1: Serotonin Release Assay (SRA) for Diagnosis of Heparin-Induced Thrombocytopenia (HIT)

[0159] Serotonin is measured by mass spectrometric detection after isotope dilution and chromatographic separation. A stable labeled isotope of serotonin is added to the sample aliquots as an internal standard. After adding the internal standard in the precipitation solution to the sample aliquots, the samples were mixed, centrifuged, further diluted with ethyl acetate, and then injected into the LC-MS / MS system. An MDS-Sciex API5500 triple quadrupole mass spectrometer operating in positive ion electrospray ionization mode was used for detection. Quantification of analytes and internal standards was performed in selected reaction monitoring mode (SRM). The back-calculated amount of serotonin in each sample was determined from a calibration curve generated by spiking known amounts of purified serotonin from 1-1000 ng / mL into blank charcoal-treated serum. Percent serotoni...

Embodiment 2

[0223] Example 2: Positive HIT Assay

[0224] Biological samples were tested for HIT according to the procedure in Example 1. This sample showed a 27% release rate of serotonin at 0.2 U / mL heparin relative to the total serotonin available in the platelets (100%), with inhibition of release present at 100 U / mL heparin (see image 3 ), the total serotonin available in the platelets is determined by analysis according to the method described herein without the addition of a biological sample. Such as image 3 As shown in the upper left panel, the endogenous serotonin level in the biological sample and buffer was 1.2 ng / mL. image 3 The upper middle panel shows biological samples and 0.2 U / mL heparin. Approximately 60.4 ng / mL of serotonin was measured in the sample. image 3 The upper right panel shows biological samples and 100 U / mL heparin. Approximately 0.2 ng / mL of serotonin was measured in the samples. The percent serotonin release was 27% for the sample and 0.2 U / mL he...

Embodiment 3

[0226] Example 3: Conversion Ratio Evaluation

[0227] By dividing the qualitatively transformed area response value in the sample by the quantitatively transformed area response value (i.e., the transformation ratio) and comparing this ratio to the average transformation ratio measured in calibrators from the same batch (excluding calibrator 1), comparison for conversion ratio evaluation. Compile the data for the conversion ratio assessment from the Quantitative Results table. Acceptance criteria established during validation were provided for the analytes (see Table 2). In cases where concentration-specific acceptance criteria cannot be used in an automated fashion, recommended acceptance criteria (i.e., concentration-independent acceptance criteria) for all samples are provided as a backup.

[0228] Table 2: Recommended acceptance criteria for conversion ratio monitoring

[0229]

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Abstract

Disclosed are methods and systems for measuring serotonin in a sample using liquid chromatography and mass spectrometry.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application No. 62 / 127,590, filed March 3, 2015, which is hereby incorporated by reference in its entirety. field of invention [0003] The present invention relates to methods and systems for the indirect measurement of heparin-induced autoantibodies by measuring serotonin in a sample. In certain embodiments, the present invention provides methods and systems employing liquid chromatography and mass spectrometry to measure serotonin released or stably labeled serotonin from donor platelets obtained from suspected heparin-induced Specimens from patients with acute thrombocytopenia (HIT) were challenged. Background technique [0004] Heparin-induced thrombocytopenia (HIT) is a potentially catastrophic, antibody-mediated complication of heparin therapy caused by immunity against platelet factor 4 (PF4) complexed with heparin or other polyanions. The HIT antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/94
CPCG01N33/942G01N33/6848G01N2800/222G01N2030/8813G01N33/49G01N2458/15
Inventor M·L·F·克劳弗德Y·Z·赖特R·P·格兰特
Owner LAB OF AMERICA HLDG
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