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.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Hematology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (1)

Publications (1)

Family

ID=22142298

Family Applications (1)

Country Status (5)

Cited By (12)

Families Citing this family (1)

Citations (4)

• 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 DE DE2149763A patent/DE2149763C3/de not_active Expired
  • 1971-10-05 JP JP46078238A patent/JPS5130796B1/ja active Pending

Patent Citations (4)

Also Published As

Similar Documents

Legal Events