US3663175A - Method of determining hemoglobin in blood - Google Patents
Method of determining hemoglobin in blood Download PDFInfo
- Publication number
- US3663175A US3663175A US78159A US3663175DA US3663175A US 3663175 A US3663175 A US 3663175A US 78159 A US78159 A US 78159A US 3663175D A US3663175D A US 3663175DA US 3663175 A US3663175 A US 3663175A
- Authority
- US
- United States
- Prior art keywords
- hemoglobin
- reagent
- moles
- acetone
- cyanmeth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
Definitions
- This invention relates to a method of determining hemoglobin in blood using Drabkins reagent.
- the reagent contains potassium ferricyanide (K Fe(CN) potassium cyanide (KCN), and sodium bicarbonate (NaI-ICO When the hemoglobin is added to this reagent, the potassium ferricyanide is reduced to potassium ferrocyanide (K,Fe(CN) and the hemoglobin is oxidized to methemoglobin which reacts with cyanide to produce to produce the stable chromogen, cyanmeth-hemoglobin.
- Drabkins reagent is not stable to low temperature. It has been found that Drabkins reagent, if exposed to freezing temperatures, loses its color with the subsequent loss of accuracy of the hemoglobin determinations made by using the reagent.
- a typical Drabkins reagent contains 61 X 10' moles of potassium ferricyanide, 77 X moles of potassium cyanide, and l 190 X 10 moles of sodium bicarbonate per liter. This reagent is very stable. It has been reported in the article by Crosby and Houchin published in the Journal, Blood, 12 1136 (1957), that the cyanmeth-hemoglobin when formed in this reagent shows only a 4 percent loss of color when stored in sealed amber bottles for a period of 3 years.
- the Drabkins reagent is made up and 0.2 to l0 weight percent of acetone is added as a loss of color inhibitor.
- the addition of acetone to the Drabkins reagent also results in attaining immediate recovery of the peak absorbance of cyanmeth-hemoglobin in using the test.
- the Drabkins reagent is made up in the conventional manner.
- a typical formulation contains 200 milligrams potassium ferricyanide, 50 milligrams of KCN, and 1 gram of sodium bicarbonate per liter.
- the hemoglobin determination is made by the observance of the height of the 540 millimicron peak in a typical spectrophotometer scan of the solution containing the blood sample.
- the concentration is computed from the following equation:
- C is the concentration of the unknown and C, is the concentration of the standard, in grams per 100 ml.
- A is the absorbance of the unknown and A, is the absorbance of the standard.
- Drabkins reagent was prepared by dissolving 200 milligrams of potassium ferricyanide, 50 milligrams of potassium cyanide, and 1 gram of sodium bicarbonate in a liter of water. The reagents were mixed thoroughly and when dissolved a quantity of acetone equal to 6 ml per liter of solution was added to one portion of this reagent. No acetone was added to the second portion. Both 'of the solutions were then exposed to the temperatures of -20 F for a period of about 96 hours. The solutions were then allowed to warm to room temperatures.
- test solutions were volumetrically dispensed in 13 X 50 mm. glass cuvettes known as Unitubes. All evaluations were performed using this container as the primary reaction cuvette. All cuvettes had a dispensed volume of 3.8 m1. of Drabkins solution. All whole blood samples were 13 microliters delivered via the Bio-Dynamics Automatic pipette. The cuvettes were then transferred to a model 124 Coleman Spectrophotometer with a 2 millimeter slit. The spectrophotometer was operated at 10 mv full scale. Absorbances of the reagent were measured at the 540 millimicron peak.
- the height of the peak was essentially the same as the height of the peak using fresh Drabkins solution when the solution used was the solution containing acetone.
- the height of the peak obtained when the solution used was the Drabkins solution that had been frozen but contained no acetone was only about 30 percent of the height obtained from the same test using fresh Drabkins reagent.
- EXAMPLE 2 In the process of evaluating the acetone modified reagent prepared according to the procedure described above, a spectral curve was run using a model 124 Coleman Spectrophotometer with a 2 millimeter slit. The spectrophotometer was operated at 10 millivolts full scale. Two separate tests were run. Blanks containing only the reagents were run as a reference. The first test was run using 3.8 ml of a Drabkins reagent that did not contain acetone. The second test was run using 3.8 ml. of Drabkins reagent containing 6 ml of acetone per liter. Both of these tests were run with 13 microliters of blood having normal hemoglobin with a fast scan at 10 minutes to ascertain if secondary reactions have occurred.
- the test run with the Drabkins reagent that contained no acetone had two characteristic peaks, one appearing at 570 millimicrons and one at 540 millicrons.
- the 570 millimicron peak is, of course, associated with oxy-hemoglobin.
- the 540 millimicron peak was obtained immediately. There was no initial peak at 570 millimicrons.
- the reagent containing the acetone was then run using a 10 minute scan. There was no interference, primary or secondary.
- the improvement comprising adding about 0.2 to 10 weight percent acetone to said solution to prevent loss of color in said solution on exposure of the solution to temperatures below 32 F and the resultant inaccuracies in the measurement.
- test solution contains about 61 X 10' moles of potassium ferricyanide, 77 X moles of potassium cyanide and l 190 X 10 moles of sodium bicarbonate per liter.
- the method of immediately attaining the cyanmeth hemoglobin peak in the spectrophotometric determination of hemoglobin using the cyanmeth-hemoglobin procedure which comprises adding from about 2% to 126 ml of acetone per liter to the reagent used in making the determination.
Abstract
Description
Claims (4)
- 2. The method according to claim 1 wherein the test solution contains about 61 X 10 5 moles of potassium ferricyanide, 77 X 10 5 moles of potassium cyanide and 1190 X 10 5 moles of sodium bicarbonate per liter.
- 3. The method of inhibiting loss of color in a hemoglobin test solution containing about 61 X 10 5 moles of potassium ferricyanide, 77 X 10 5 moles of potassium cyanide and 1,190 X 10 5 moles of sodium bicarbonate per liter, on exposure of said solution to temperatures below 32* F which comprises adding 0.2 to 10 weight percent acetone to said solution.
- 4. In the determination of the hemoglobin content of blood using a solution containing potassium cyanide, potassium ferricyanide and sodium bicarbonate in which the potassium ferricyanide is reduced to potassium ferrocyanide the improvement comprising adding from 0.2 to 10 weight percent acetone to said solution to assure immediate attainment of the peak absorbance of cyanmeth-hemoglobin in the spectrophotometric determination of the hemoglobin of blood sample.
- 5. The method of immediately attaining the cyanmeth hemoglobin peak in the spectrophotometric determination of hemoglobin using the cyanmeth-hemoglobin procedure which comprises adding from about 2 1/2 to 126 ml of acetone per liter to the reagent used in making the determination.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US7815970A | 1970-10-05 | 1970-10-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
US3663175A true US3663175A (en) | 1972-05-16 |
Family
ID=22142298
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US78159A Expired - Lifetime US3663175A (en) | 1970-10-05 | 1970-10-05 | Method of determining hemoglobin in blood |
Country Status (5)
Country | Link |
---|---|
US (1) | US3663175A (en) |
JP (1) | JPS5130796B1 (en) |
CA (1) | CA952799A (en) |
DE (1) | DE2149763C3 (en) |
GB (1) | GB1301635A (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3874852A (en) * | 1974-07-05 | 1975-04-01 | Coulter Diagnostics Inc | Reagent and method for determining leukocytes and hemoglobin in the blood |
US3950133A (en) * | 1971-10-20 | 1976-04-13 | Mallinckrodt, Inc. | Reagent formulations for assaying biological specimens and methods of preparing and using same |
US4077772A (en) * | 1977-05-31 | 1978-03-07 | Baxter Travenol Laboratories, Inc. | Method for the determination of hemoglobin in trace amounts |
US4349351A (en) * | 1980-07-01 | 1982-09-14 | Bayer Aktiengesellschaft | Reagent for the determination of haemoglobin |
US4526869A (en) * | 1980-09-24 | 1985-07-02 | Regents Of The University Of Minnesota | Method for quantitatively determining the concentration of hemoglobin in a biological sample |
US4543336A (en) * | 1982-02-12 | 1985-09-24 | Dragerwerk Aktiengesellschaft | Indicator for determining sulfur dioxide |
US4725555A (en) * | 1986-10-22 | 1988-02-16 | Helena Laboratories Corporation | Zinc protoporphyrin test system |
US5426032A (en) * | 1986-08-13 | 1995-06-20 | Lifescan, Inc. | No-wipe whole blood glucose test strip |
US6458326B1 (en) | 1999-11-24 | 2002-10-01 | Home Diagnostics, Inc. | Protective test strip platform |
US6525330B2 (en) | 2001-02-28 | 2003-02-25 | Home Diagnostics, Inc. | Method of strip insertion detection |
US6541266B2 (en) | 2001-02-28 | 2003-04-01 | Home Diagnostics, Inc. | Method for determining concentration of an analyte in a test strip |
US6562625B2 (en) | 2001-02-28 | 2003-05-13 | Home Diagnostics, Inc. | Distinguishing test types through spectral analysis |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS52141392A (en) * | 1976-05-21 | 1977-11-25 | Mitsui Miike Machinery Co Ltd | Process and apparatus for intermittent throwing of baits for fish culture |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2940448A (en) * | 1957-04-29 | 1960-06-14 | Jr Norman B Furlong | Disposable blood-gas analyzer |
US3427135A (en) * | 1966-07-11 | 1969-02-11 | Technicon Instr | Hematology apparatus |
US3519572A (en) * | 1966-10-03 | 1970-07-07 | Pfizer & Co C | Blood control |
US3546131A (en) * | 1968-07-05 | 1970-12-08 | Uni Tech Chem Mfg Co | Stabilized cyanmethemoglobin reagent containing ferricyanide,cyanide and polyvinylpyrrolidone |
-
1970
- 1970-10-05 US US78159A patent/US3663175A/en not_active Expired - Lifetime
-
1971
- 1971-09-16 CA CA123,049A patent/CA952799A/en not_active Expired
- 1971-09-17 GB GB4352071A patent/GB1301635A/en not_active Expired
- 1971-10-05 JP JP46078238A patent/JPS5130796B1/ja active Pending
- 1971-10-05 DE DE2149763A patent/DE2149763C3/en not_active Expired
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2940448A (en) * | 1957-04-29 | 1960-06-14 | Jr Norman B Furlong | Disposable blood-gas analyzer |
US3427135A (en) * | 1966-07-11 | 1969-02-11 | Technicon Instr | Hematology apparatus |
US3519572A (en) * | 1966-10-03 | 1970-07-07 | Pfizer & Co C | Blood control |
US3546131A (en) * | 1968-07-05 | 1970-12-08 | Uni Tech Chem Mfg Co | Stabilized cyanmethemoglobin reagent containing ferricyanide,cyanide and polyvinylpyrrolidone |
Cited By (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3950133A (en) * | 1971-10-20 | 1976-04-13 | Mallinckrodt, Inc. | Reagent formulations for assaying biological specimens and methods of preparing and using same |
US3874852A (en) * | 1974-07-05 | 1975-04-01 | Coulter Diagnostics Inc | Reagent and method for determining leukocytes and hemoglobin in the blood |
US4077772A (en) * | 1977-05-31 | 1978-03-07 | Baxter Travenol Laboratories, Inc. | Method for the determination of hemoglobin in trace amounts |
US4349351A (en) * | 1980-07-01 | 1982-09-14 | Bayer Aktiengesellschaft | Reagent for the determination of haemoglobin |
US4526869A (en) * | 1980-09-24 | 1985-07-02 | Regents Of The University Of Minnesota | Method for quantitatively determining the concentration of hemoglobin in a biological sample |
US4543336A (en) * | 1982-02-12 | 1985-09-24 | Dragerwerk Aktiengesellschaft | Indicator for determining sulfur dioxide |
US6268162B1 (en) | 1986-08-13 | 2001-07-31 | Lifescan, Inc. | Reflectance measurement of analyte concentration with automatic initiation of timing |
US6881550B2 (en) | 1986-08-13 | 2005-04-19 | Roger Phillips | Method for the determination of glucose employing an apparatus emplaced matrix |
US5563042A (en) * | 1986-08-13 | 1996-10-08 | Lifescan, Inc. | Whole blood glucose test strip |
US5843692A (en) * | 1986-08-13 | 1998-12-01 | Lifescan, Inc. | Automatic initiation of a time interval for measuring glucose concentration in a sample of whole blood |
US6887426B2 (en) | 1986-08-13 | 2005-05-03 | Roger Phillips | Reagents test strip adapted for receiving an unmeasured sample while in use in an apparatus |
US5426032A (en) * | 1986-08-13 | 1995-06-20 | Lifescan, Inc. | No-wipe whole blood glucose test strip |
US6858401B2 (en) | 1986-08-13 | 2005-02-22 | Lifescan, Inc. | Minimum procedure system for the determination of analytes |
US6821483B2 (en) | 1986-08-13 | 2004-11-23 | Lifescan, Inc. | Reagents test strip with alignment notch |
US20030073153A1 (en) * | 1986-08-13 | 2003-04-17 | Roger Phillips | Minimum procedure system for the determination of analytes |
US20030073151A1 (en) * | 1986-08-13 | 2003-04-17 | Roger Phillips | Minimum procedure system |
US4725555A (en) * | 1986-10-22 | 1988-02-16 | Helena Laboratories Corporation | Zinc protoporphyrin test system |
US6458326B1 (en) | 1999-11-24 | 2002-10-01 | Home Diagnostics, Inc. | Protective test strip platform |
US6979571B2 (en) | 1999-11-24 | 2005-12-27 | Home Diagnostics, Inc. | Method of using a protective test strip platform for optical meter apparatus |
US6562625B2 (en) | 2001-02-28 | 2003-05-13 | Home Diagnostics, Inc. | Distinguishing test types through spectral analysis |
US6541266B2 (en) | 2001-02-28 | 2003-04-01 | Home Diagnostics, Inc. | Method for determining concentration of an analyte in a test strip |
US6525330B2 (en) | 2001-02-28 | 2003-02-25 | Home Diagnostics, Inc. | Method of strip insertion detection |
US7390665B2 (en) | 2001-02-28 | 2008-06-24 | Gilmour Steven B | Distinguishing test types through spectral analysis |
Also Published As
Publication number | Publication date |
---|---|
GB1301635A (en) | 1973-01-04 |
DE2149763B2 (en) | 1973-03-01 |
CA952799A (en) | 1974-08-13 |
DE2149763C3 (en) | 1973-09-13 |
JPS5130796B1 (en) | 1976-09-02 |
DE2149763A1 (en) | 1972-04-06 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BOEHRINGER MANNHEIM DIAGNOSTICS, INC., 9115 HAGUE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:BIO-DYNAMICS, INC.;REEL/FRAME:004239/0504 Effective date: 19831028 |
|
AS | Assignment |
Owner name: BOEHRINGER MANNHEIM CORPORATION, A CORP. OF NEW YO Free format text: CHANGE OF NAME;ASSIGNOR:BOEHRINGER MANNHEIM DIAGNOSTICS, INC. A CORP. OF DE.;REEL/FRAME:004624/0055 Effective date: 19861008 |
|
AS | Assignment |
Owner name: BOEHRINGER MANNHEIM CORPORATION Free format text: MERGER;ASSIGNOR:BOEHRINGER MANNHEIM CORPORATION, A NY CORP.;REEL/FRAME:004912/0770 Effective date: 19870625 Owner name: BOEHRINGER MANNHEIM CORPORATION, A IN CORP. Free format text: MERGER;ASSIGNOR:BOEHRINGER MANNHEIM CORPORATION, A NY CORP.;REEL/FRAME:004912/0770 Effective date: 19870625 |