# US 10,308,976 B2 · Selective lysis of cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,976 B2 - Document ID: US10308976B2 - Status: Granted - Kind: B2 - Application number: 13/514,734 - Filed: 2010-12-07 - Published: 2013-07-04 - Granted: 2019-06-04 - Earliest claimed: 2010-12-07 - Assignees: Koninklijke Philips N.V. - Applicants: Bart Edward Jozef Gusta Van Meerbergen; Oana Mihaela Piciu; Ron Gill; Kristiane Anne Schmidt; Sieglinde Neerken; Marc Wilhelmus Gijsbert Ponjee; Zeynep Seflek; Roel Penterman; Paul Arnold Van De Wiel - Inventors: Bart Edward Jozef Gusta Van Meerbergen; Oana Mihaela Piciu; Ron Gill; Kristiane Anne Schmidt; Sieglinde Neerken; Marc Wilhelmus Gijsbert Ponjee; Zeynep Seflek; Roel Penterman; Paul Arnold Van De Wiel - Web page: https://patentgrep.com/patent/US10308976B2 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12N 1/06: Lysis of microorganisms - C12Q 1/689: for bacteria - C12Q 1/6895: for plants, fungi or algae - C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis} - C12M 47/06: {Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003)} - G01N 33/48: Biological material, e.g. blood, urine (G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence); Haemocytometers (counting blood corpuscules distributed over a surface by scanning the surface G06M11/02) - 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 - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - 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 Methods and devices for the selective lysis of cells in a sample comprising micro-organisms such as bacteria are provided. The selective lysis is obtained by incubating the sample in a non-ionic detergent under alkaline conditions. ## Claims ### Claim 1 A method for detecting the presence of micro-organisms contained or suspected to be contained within a mammalian blood sample, the method comprising the steps of: providing the mammalian blood sample containing or suspected to contain the micro-organisms, wherein: said mammalian blood sample comprises whole blood that has not been cultivated, said whole blood including blood plasma and eukaryotic blood cells, said mammalian blood sample has a volume between 1 ml and 20 ml, said micro-organisms are selected from the group consisting of bacteria and fungi, and there are less than 10,000 micro-organisms per milliliter of the mammalian blood sample, adding a selective lysis buffer at a selected concentration to the mammalian blood sample to obtain a final solution having a pH level of around 9.5 or higher, said selective lysis buffer including (i) a non-ionic detergent and (ii) an alkaline buffer, said non-ionic detergent present in a concentration of between 0.1% and 5% (w/v % or v/v %), wherein the selected concentration of the selective lysis buffer in the final solution is a concentration that achieves said pH level of around 9.5 or higher for the final solution, with a volume of the selective lysis buffer that is between 200% and 10% of a volume of the mammalian blood sample; incubating the final solution in the presence of the selective lysis buffer for a time period sufficiently long to selectively lyse the blood cells in the mammalian blood sample while keeping the micro-organisms intact, thereby forming a lysed sample, wherein the percentage of micro-organism cells that remain intact in the lysed sample is at least two times higher than the percentage of blood cells that remain intact in the lysed sample; and detecting the presence of the micro-organisms without cultivation of the micro-organisms within said mammalian blood sample. ### Claim 2 (depends on 1) The method according to claim 1, wherein said time period for the incubating step is between 30 seconds and 10 minutes. ### Claim 3 (depends on 1) The method according to claim 1, wherein the non-ionic detergent is selected from the group consisting of Nonidet P40, deoxycholate, lgepal CA 630, Triton-X 100, and any combinations thereof. ### Claim 4 (depends on 1) The method according to claim 1, further comprising centrifuging the incubated final solution and isolating the micro-organisms. ### Claim 5 (depends on 1) The method according to claim 1, further comprising filtering the incubated final solution on a filter with a pore size that retains micro-organisms on the filter. ### Claim 6 (depends on 1) The method according to claim 1, further comprising lysing the micro-organisms. ### Claim 7 (depends on 6) The method according to claim 6, further comprising using a nucleic acid based molecular assay on the lysed micro-organisms. ### Claim 8 (depends on 1) The method according to claim 1, wherein the method further comprises: adding an acid or acidic buffer to neutralize the lysed sample. ### Claim 9 (depends on 8) The method according to claim 8, wherein the lysed sample is neutralized to a pH in a range between 7 and 9.