# US 10,246,733 B2 · Assay and method for quantitating carbonic anhydrase activity and assessing red blood hemolysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,733 B2 - Document ID: US10246733B2 - Status: Granted - Kind: B2 - Application number: 15/174,445 - Filed: 2016-06-06 - Published: 2016-12-08 - Granted: 2019-04-02 - Earliest claimed: 2015-06-04 - Assignees: Case Western University - Applicants: Case Western Reserve University - Inventors: Ryan R. Geyer; Pan Zhao; Mark D. Parker; Walter F. Boron - Web page: https://patentgrep.com/patent/US10246733B2 ## CPC - C12Q 1/527: involving lyase - C12Y 402/01001: Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase - G01N 2333/988: Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase - 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 - C12Y: ENZYMES - 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 A method and assay for using carbonic anhydrase (CA), particularly CA-I or CA-II, as a biomarker of hemolysis. The method and assay detect hemolysis by determining a percentage erythrocyte hemolysis in a specimen or sample of blood based upon quantification of carbonic anhydrase present in the extracellular portion of the blood. The method and test serve to optimize therapeutic efficacy for treatments of hemolysis. ## Claims ### Claim 1 A method of measuring the amount or activity of carbonic anhydrase enzyme, in a bodily sample, the method comprising: mixing a bodily sample including RBC lysate with a first physiological CO2/HCO3− solution, the first physiological CO2/HCO3− solution having a pH of about 7; mixing the first physiological CO2/HCO3− solution, which includes the bodily sample, with a second CO2/HCO3− solution, which includes NaHCO3, having a basic dissimilar pH; and measuring the rate at which the pH of the newly mixed solution equilibrates under the influence of the enzyme to determine carbonic anhydrase activity. ### Claim 2 (depends on 1) The method of claim 1, wherein the first physiological CO2/HCO3− solution and the second CO2/HCO3− solution are mixed in a stopped flow device. ### Claim 3 (depends on 1) The method of claim 1, wherein the rate at which the pH equilibrates is measured by adding a fluorescent pH indicator dye to the first physiological CO2/HCO3− solution and/or the second physiological CO2/HCO3− solution and measuring a change of fluorescence of the dye. ### Claim 4 (depends on 3) The method of claim 3, wherein the pH indicator dye is pyranine. ### Claim 5 (depends on 1) The method of claim 1, wherein the bodily sample includes red blood cells and an increase in enzyme or enzyme activity is indicative of increased red blood cell hemolysis. ### Claim 6 (depends on 5) The method of claim 5, wherein the red blood cells are from stored blood. ### Claim 7 (depends on 1) The method of claim 1, wherein the first physiological CO2/HCO3− solution is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffered solution. ### Claim 8 (depends on 1) The method of claim 1, wherein the second physiological CO2/HCO3− solution has a pH of about 8.4 prior to mixing. ### Claim 9 (depends on 8) The method of claim 8, the first physiological CO2/HCO3− solution and the second physiological CO2/HCO3− solution having a temperature of about 10° C. ### Claim 10 A method for detecting red blood cell hemolysis in a bodily sample, the method comprising: mixing a bodily sample including red blood cells (RBC) and/or RBC lysate with a first physiological CO2/HCO3− solution, the first physiological CO2/HCO3− solution having a pH of about 7; mixing the first physiological CO2/HCO3− solution, which includes the bodily sample, with a second CO2/HCO3− solution, which includes NaHCO3, having a basic dissimilar pH; and measuring the rate at which the pH of the newly mixed solution equilibrates under the influence of the carbonic anhydrase enzyme in the bodily sample to determine red blood cell hemolysis in the bodily sample. ### Claim 11 (depends on 10) The method of claim 10, wherein the first physiological CO2/HCO3− solution and the second CO2/HCO3− solution are mixed in a stopped flow device. ### Claim 12 (depends on 10) The method of claim 10, wherein the rate at which the pH equilibrates is measured by adding a fluorescent pH indicator dye to the first physiological CO2/HCO3− solution and/or the second physiological CO2/HCO3− solution and measuring a change of fluorescence of the dye. ### Claim 13 (depends on 12) The method of claim 12, wherein the pH indicator dye is pyranine. ### Claim 14 (depends on 12) The method of claim 12, wherein the bodily sample includes red blood cells from stored blood. ### Claim 15 (depends on 10) The method of claim 10, wherein the first physiological solution is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) solution. ### Claim 16 (depends on 10) The method of claim 10, wherein the second physiological CO2/HCO3− solution has a pH of about 8.4 prior to mixing. ### Claim 17 (depends on 16) The method of claim 16, the first physiological CO2/HCO3− solution and the second physiological CO2/HCO3− solution having a temperature of about 10° C. ### Claim 18 A method for determining red blood cell hemolysis in a bodily sample, the method comprising: mixing a bodily sample including red blood cells (RBC) and/or RBC lysate with a first physiological CO2/HCO3− solution, the first physiological CO2/HCO3− solution having a pH of about 7; mixing the first physiological CO2/HCO3− solution with a second CO2/HCO3− solution having a pH of about 8.4; measuring the rate at which the pH of the newly mixed solution equilibrates under the influence of the carbonic anhydrase enzyme in the bodily sample to determine red blood cell hemolysis in the bodily sample.