US4567148A
Expired / Lapsed
US
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Method for quantitatively determining the amount of hemoglobin in a biological sample
Assignee: University of Minnesota System
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Filing: 1982-09-13
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Granted: 1986-01-28
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Expires: 2002-09-13
Abstract
Abstract A method of quantitatively determining the amount of hemoglobin in a biological material which includes the steps of preparing a test sample of the biological material by removing contaminating elements such as sources of interfering fluorescence, converting the hemoglobin in the test sample to porphyrin, performing an extraction step for the purpose of separating the porphyrins derived from hemoglobin from various contaminants and determining the level of porphyrin in the test sample by means such as fluorescence assay.
Inventors
- 👤 Samuel Schwartz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 18:41
CPC Classifications
A; A61; A61B; A61B10/00; A61B10/0038; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/72; G01N33/721; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/145555; Y10T436/146666; Y10T436/25; Y10T436/25375; Y10T436/255
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
162 words
A method of quantitatively determining the amount of hemoglobin in a biological material comprising the steps of: preparing a test sample of said biological material; converting the hemoglobin in said test sample to hemoglobin derived porphyrins; performing an extraction procedure for the purpose of separating the hemoglobin derived porphyrins from various contaminants in said test sample including mixing said test sample with a first extraction reagent comprising an organic solvent effective to separate the hemoglobin derived porphyrins from iron; allowing said test sample and first extraction reagent mixture to separate into an aqueous phase containing iron and a supernatant containing hemoglobin-derived porphyrins; mixing said supernatant with a second extraction reagent comprising an alkaline solvent containing a soluble salt effective to separate the hemoglobin derived porphyrins from naturally occurring porphyrins; allowing said supernatant and second extraction reagent mixture to separate into a phase containing naturally occurring porphyrins and a second supernatant containing hemoglobin derived porphyrins; and determining the amount of hemoglobin derived porphyrins.
Claim 2
Independent Claim
162 words
A method of quantitatively determining the amount of hemoglobin in a biological material comprising the steps of: preparing a test sample of said biological material; converting the hemoglobin in said test sample to hemoglobin derived porphyrins; performing an extraction procedure for the purpose of separating the hemoglobin derived porphyrins from various contaminants in said test sample including mixing said test sample with a first extraction reagent comprising an organic solvent effective to separate the hemoglobin derived porphyrins from iron; allowing said test sample and first extraction reagent mixture to separate into an aqueous phase containing iron and a supernatant containing hemoglobin-derived porphyrins; mixing said supernatant with a second extraction reagent comprising an alkaline solvent containing a soluble salt effective to separate the hemoglobin derived porphyrins from naturally occurring porphyrins; allowing said supernatant and second extraction reagent mixture to separate into a phase containing naturally occurring porphyrins and a second supernatant containing hemoglobin derived porphyrins; and determining the amount of hemoglobin derived porphyrins.
Claim 3
Dependent Claim
35 words
The method of claim 1 wherein said organic solvent is selected from the group consisting of: ethyl acetate, butyl alcohol, amyl alcohol, chloroform, perchlorethylene, carbon tetrachloride, cyclohexanone, tributyl phosphate, toluene, acetic acid, and ethylene chloride.
Claim 4
Dependent Claim
16 words
The method of claim 1 wherein said first extraction reagent includes an ethyl acetate:acetic acid mixture.
Claim 5
Dependent Claim
13 words
The method of claim 1 wherein said second extraction reagent includes potassium hydroxide.
Claim 6
Dependent Claim
16 words
The method of claim 1 wherein said second extraction reagent includes potassium hydroxide and potassium acetate.
Claim 7
Dependent Claim
13 words
The method of claim 1 wherein said second extraction reagent includes butyl alcohol.
Claim 8
Dependent Claim
15 words
The method of claim 1 wherein said biological material is feces, urine or gastric juice.
Claim 9
Dependent Claim
17 words
The method of claim 1 including determining the level of hemoglobin derived porphyrin via a fluorescence assay.
Claim 10
Dependent Claim
21 words
The method of claim 1 wherein said first extraction reagent includes an effective quantity of a suitable salt to decrease acidity.
Claim 11
Dependent Claim
12 words
The method of claim 9 wherein said suitable salt is potassium acetate.
Claim 12
Dependent Claim
30 words
The method of claim 1 wherein said extraction further includes mixing said second supernatant with a third extraction reagent comprising a strong acid effective to separate the hemoglobin derived porphyrins.
Claim 13
Dependent Claim
18 words
The mixture of claim 11 wherein said strong acid comprises a mixture of phosphoric acid and acetic acid.
Claim 14
Independent Claim
92 words
A method of quantitatively determining the amount of hemoglobin in a biological material comprising the steps of: preparing a test sample of said biological material; converting the hemoglobin in said test sample to hemoglobin derived porphyrins; performing an extraction procedure for the purpose of separating the hemoglobin derived porphyrins from various contaminants in said test sample including combining said test sample with an extraction reagent comprising an alkaline solvent containing a soluble salt effective to separate the hemoglobin derived porphyrins from naturally occurring porphyrins; and determining the level of hemoglobin derived porphyrins.
Claim 15
Independent Claim
92 words
A method of quantitatively determining the amount of hemoglobin in a biological material comprising the steps of: preparing a test sample of said biological material; converting the hemoglobin in said test sample to hemoglobin derived porphyrins; performing an extraction procedure for the purpose of separating the hemoglobin derived porphyrins from various contaminants in said test sample including combining said test sample with an extraction reagent comprising an alkaline solvent containing a soluble salt effective to separate the hemoglobin derived porphyrins from naturally occurring porphyrins; and determining the level of hemoglobin derived porphyrins.
Claim 16
Dependent Claim
29 words
The method of claim 13 wherein said extraction procedure for separating hemoglobin derived porphyrins from naturally occurring porphyrins includes combining of quantity of butyl alcohol with said test sample.
Claim 17
Dependent Claim
12 words
The method of claim 13 wherein said alkaline solvent includes potassium hydroxide.
Claim 18
Dependent Claim
12 words
The method of claim 15 wherein said soluble salt includes potassium acetate.
Claim 19
Independent Claim
221 words
A method of analyzing a test sample of feces to determine information regarding the total amount of hemoglobin originally in said test sample and the location of the source of such hemoglobin in the gastrointestinal tract, in which said test sample is expected to contain currently existing hemoglobin and porphyrins derived from the conversion of previously existing hemoglobin as a result of exposure to various naturally occurring converting substances in the gastrointestinal tract, said method comprising the steps of: preparing a test sample of feces; converting substantially all of the currently existing hemoglobin in said test sample to hemoglobin derived porphyrins by combining said test sample with a converting reagent; determining the total amount of hemoglobin derived porphyrins in said test sample resulting from combining said test sample with said converting reagent and its exposure to naturally occurring converting substances in the gastrointestinal tract; preparing a duplicate test sample of feces; determining the amount of hemoglobin derived porphyrins in said duplicate test sample resulting from exposure to naturally occurring substances in the gastrointestinal tract; and determining the source of the hemoglobin in the test sample by comparing the total amount of hemoglobin derived porphyrins in said test sample with the amount of hemoglobin derived porphyrins in said duplicate test sample resulting from exposure to naturally occurring substances in the gastrointestinal tract.
Claim 20
Independent Claim
221 words
A method of analyzing a test sample of feces to determine information regarding the total amount of hemoglobin originally in said test sample and the location of the source of such hemoglobin in the gastrointestinal tract, in which said test sample is expected to contain currently existing hemoglobin and porphyrins derived from the conversion of previously existing hemoglobin as a result of exposure to various naturally occurring converting substances in the gastrointestinal tract, said method comprising the steps of: preparing a test sample of feces; converting substantially all of the currently existing hemoglobin in said test sample to hemoglobin derived porphyrins by combining said test sample with a converting reagent; determining the total amount of hemoglobin derived porphyrins in said test sample resulting from combining said test sample with said converting reagent and its exposure to naturally occurring converting substances in the gastrointestinal tract; preparing a duplicate test sample of feces; determining the amount of hemoglobin derived porphyrins in said duplicate test sample resulting from exposure to naturally occurring substances in the gastrointestinal tract; and determining the source of the hemoglobin in the test sample by comparing the total amount of hemoglobin derived porphyrins in said test sample with the amount of hemoglobin derived porphyrins in said duplicate test sample resulting from exposure to naturally occurring substances in the gastrointestinal tract.
Claim 21
Dependent Claim
36 words
The method of claim 17 wherein the step of determining information regarding the amount of hemoglobin includes combining said duplicate test sample with a blank reagent which does not convert significant amounts of hemoglobin to porphyrin.
Claim 22
Dependent Claim
14 words
The method of claim 18 wherein said blank reagent is comprised of citric acid.
Claim 23
Dependent Claim
48 words
The method of claim 17 wherein the step of determining information regarding the amount of hemoglobin includes separating the total amount of hemoglobin derived porphyrins from naturally occurring porphyrins in said test sample and separating the hemoglobin derived porphyrins from naturally occurring porphyrins in said duplicate test sample.
Claim 24
Dependent Claim
53 words
The method of claim 20 wherein the step of separating the hemoglobin derived porphyrins from the naturally occurring porphyrins in each of said test sample and said duplicate test sample includes mixing said test sample and said duplicate test sample, respectively, with an extraction reagent comprising an alkaline solvent containing a soluble salt.
Claim 25
Dependent Claim
12 words
The method of claim 21 wherein said extraction reagent includes potassium hydroxide.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-01-27 | 1989-07-29 | 1990-01-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-01-27 | 1993-07-29 | 1994-01-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-01-27 | 1997-07-29 | 1998-01-28 | $7700.00 | Window Closed / Lapsed |