# US 8,569,010 B2 · Mass spectrometric diagnosis of septicemia > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,569,010 B2 - Document ID: US8569010B2 - Status: Granted - Kind: B2 - Application number: 13/322,363 - Filed: 2010-07-14 - Published: 2012-05-10 - Granted: 2013-10-29 - Earliest claimed: 2009-07-16 - Assignees: Bruker Daltonik GmbH - Applicants: Thomas Maier - Inventors: Thomas Maier - Web page: https://patentgrep.com/patent/US8569010B2 ## CPC - C12Q 1/24: Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms - C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} - G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry} - G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material - G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes - G01N 2800/26: Infectious diseases, e.g. generalised sepsis - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The invention mainly relates to the mass spectrometric identification of pathogens in blood cultures from blood-stream infections (septicemia). The invention provides a method with which microbial pathogens can be separated in purified form from blood after a relatively brief cultivation in a blood culture flask, without any interfering human proteins or any residual fractions of blood particles such as erythrocytes and leukocytes, and can be directly identified by mass spectrometric measurement of their protein profiles. The method is based on the use of relatively strong tensides to destroy the blood particles by dissolving the weak cell membranes and most of the internal structures of the blood particles; in spite of the fact that tensides are regarded as strong ionization inhibitors in MALDI and other ionization processes required for mass spectrometric measurements. This method allows unknown pathogens to be obtained in their pure form by centrifuging or filtration and to be identified on the taxonomic level of species or subspecies. Problems with DNA from high levels of leukocytes can be resolved by special measures. After sufficient cultivation, the identification in a mass spectrometric laboratory takes only half an hour. ## Claims ### Claim 1 A method for the mass spectrometric identification of microbes comprising bacteria and yeast in blood in which the microbes are deposited by centrifuging or by filtration, after which a mass spectrum is acquired from the microbes and said microbes are identified by the mass spectrum, characterized by dissolving the blood particles of a blood sample from a blood culture by adding a tenside prior to the deposition of the microbes. ### Claim 2 A method for the mass spectrometric identification of microbes comprising bacteria and yeast in blood in which the microbes are deposited by sedimentation or by micro-filtration, after which a mass spectrum is acquired from the microbes and said microbes are identified by the mass spectrum, characterized by dissolving the blood particles of a blood sample from a blood culture by adding a tenside prior to deposition of the microbes. ### Claim 3 A method for the mass spectrometric identification of microbes comprising bacteria and yeast in blood in which the microbes are pelleted by centrifuging or by filtration, after which a mass spectrum is acquired from the microbes and said microbes are identified by the mass spectrum, characterized by dissolving the blood particles of a blood sample from a blood culture by adding a tenside prior to pelleting of the microbes. ### Claim 4 (depends on 1) The method of claim 1, wherein sufficient numbers of microbes in the blood are produced by cultivation. ### Claim 5 (depends on 1) The method of claim 1, wherein the microbes deposited by centrifugation are washed and again centrifuged. ### Claim 6 (depends on 1) The method of claim 1, wherein the tenside is added as a solution. ### Claim 7 (depends on 1) The method of claim 1, wherein an anticoagulant is added to the blood sample. ### Claim 8 (depends on 1) The method of claim 1, wherein one or more nucleases are added to the blood sample. ### Claim 9 (depends on 1) The method of claim 1, wherein the blood for the blood sample is diluted with distilled water by a dilution factor between about 1:2 and 1:10. ### Claim 10 (depends on 1) The method of claim 1, wherein the blood sample is centrifuged and the supernatant is removed before the tenside solution is added. ### Claim 11 (depends on 1) The method of claim 1, wherein the deposited microbes are disintegrated by sonication or mechanical treatment. ### Claim 12 (depends on 1) The method of claim 1, wherein the deposited microbes are disintegrated by solution containing formic acid or trifluoroethanoic acid and acetonitrile. ### Claim 13 (depends on 1) The method of claim 1, wherein the preparation of measurement samples comprises transferring some of the microbes of the deposit onto a mass spectrometric sample support where a solution with a matrix substance for matrix assisted laser desorption/ionization is added. ### Claim 14 (depends on 5) The method of claim 5, wherein sodium dodecyl sulfate (SDS) is used as the tenside. ### Claim 15 (depends on 6) The method of claim 6, wherein the tenside solution contains a defoamer. ### Claim 16 (depends on 5) The method of claim 5, wherein the solution is about a 5 to 20% aqueous sodium dodecyl sulfate (SDS) solution. ### Claim 17 (depends on 9) The method of claim 9, wherein the blood for the blood sample is diluted with distilled water by a dilution factor of about 1:5. ### Claim 18 (depends on 12) The method of claim 12, wherein the preparation of measurement samples on a mass spectrometric sample support plate is performed using a matrix substance for matrix assisted laser desorption/ionization in whose crystals the soluble proteins of the disintegrated microbes are embedded. ### Claim 19 (depends on 18) The method of claim 18, wherein the mass spectrometric measurement is performed in a time-of-flight mass spectrometer with ionization by matrix assisted laser desorption (MALDI).