US3492095A - Product for and method of testing blood for the presence of hemoglobin s - Google Patents

Product for and method of testing blood for the presence of hemoglobin s Download PDF

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US3492095A
US3492095A US673071A US3492095DA US3492095A US 3492095 A US3492095 A US 3492095A US 673071 A US673071 A US 673071A US 3492095D A US3492095D A US 3492095DA US 3492095 A US3492095 A US 3492095A
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solution
blood
hemoglobin
test
testing
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US673071A
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Harold B Tillen
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HAROLD B TILLEN
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HAROLD B TILLEN
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    • 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

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  • a method of and product for testing for the presence of Hemoglobin S comprising the steps of preparing a buffer solution, preferably a phosphate butter and adding to the phosphate buffer a reducing agent such as sodium hydrosulfite. After dissolving the reducing agent in the butter solution an erythrocytic hemolysis agent, such as a 2 percent saponin solution is added and mixed with the buffer and reducing agent. The resultant solution is then placed in a test tube and a measured amount of whole blood is added. The test tube contents are mixed and within two minutes, the results can be determined. That is, if the resultant solution is cloudy or turbid and shows a precipitate, Hemoglobin S is present. If the solution is opalescent or translucent, with no preciptate, then the result is negative denoting the absence of Hemoglobin S.
  • a buffer solution preferably a phosphate butter and adding to the phosphate buffer a reducing agent such as sodium hydrosulfite.
  • Hemoglobin S is presently considered an inherited genetic trait which, when heterozygous gives little clinical evidence of its presence, but when homozygous results in profound anemia.
  • Sickle cell trait has been found predominantly in persons of the Negro race.
  • Homozygous Hemoglobin S results in an anemia whose symptoms include leg ulcers and acute attacks of pain.
  • The-homozygous condition is normally distinguished by the presence of peculiar sickle-shaped and oat-shaped red corpuscles.
  • the sickle cell trait may have been introduced into Africa from the northeast via the former land bridge between Egypt and Africa.
  • it appears that sickle-cell anemia presently is found more often in persons of the Negro race or persons having a mixture of Negro blood.
  • sickle cell anemia invariably shows more than 1 percent, usually between 30 and 60 percent sickled erythrocytes.
  • hemoglobin electrophoresis is the most specific method for determining the prescence of an abnormal hemoglobin, such as Hemoglobin S, and for distinguishing sickle cell anemia (homozygous) from sickle cell trait (heterozygous).
  • the supernatant is withdrawn and 10 ml. of saline is added to the packed cells.
  • the washed, packed cells are placed in graduated centifuge tubes and two volumes of barbital buifer pH 8.6 are added with gentle stirring, and the suspensions are then transferred to freezer storage tubes.
  • the samples frozen are maintained at least overnight and they may be kept in the frozen state until needed.
  • Hemolysis is effected by thawing.
  • One tube is thawed in the refrigerator or at room temperature.
  • the tube may be warmed at body temperature but should be cooled as 3 soon as all the ice has disappeared.
  • the tube cannot be placed in warm or hot water to thaw.
  • the sample is centrifuged for ten minutes and the clear hemolysate is then usable for further testing.
  • a phosphate buffer is then prepared by dissolving 16.9 grams of monobasic potassium phosphate (KH PO and 21.7 grams of dibasic potassium phosphate (K HPO or 17.7 grams of dibasic sodium phosphate (Na HPO in carbon dioxide-free distilled Water and the volume is djusted to 100 ml.
  • test tube is mixed and allowed to stand for fifteen minutes. A precipitate is formed which is then separated by filtering the solution through Whatman filter paper No. 5 or its equivalent.
  • 3.8 ml. of the phosphate buffer and mg. sodium hydrosulphite are measured into cuvettes. Then, .2 ml. f the hemoglobin filtrate is added and mixed by inverting the cuvettes twice. The absorbancy or optical density of the solution is then measured in a spectrophotometer at 415 mp Then, into a graduated cylinder of ml. capacity, 20 ml. of distilled water are added, and .1 ml. of hemoglobin solution is placed in the cylinder. Then the pipet is rinsed and the cylinder is mixed by inversion. About 4 ml. of the solution is transferred to a cuvette.
  • Another important problem with certain of the tests is that they require either large amounts of blood or they cannot use Whole blood. In those places where there are no elaborate facilities for processing blood, it may not be possible to utilize certain of the above tests and it may be necessary to send the blood, obviously in a large quantity, to a central testing laboratory where the test will be performed.
  • Solubility percent X 100 Summary of the invention
  • the present invention is intended to be a two-minute est o det sti g the p sence o H m g obi S.
  • the t 4 requires only 0.02 milliliter of whole blood and therefore, can be performed with a drop of blood. Further, it can be performed and evaluated by unskilled personnel.
  • Venous blood is not required and the test can utilize blood obtained from a pin prick.
  • This test utilizes the same phenomena noted in Goldbergs test, namely, that Hemoglobin S has a lower ferrohemoglobin solubility as distinguished from all other hemoglo'bins except perhaps Hemoglobin H.
  • Hemoglobin H is so rare a condition as to be of negligible importance.
  • the test is only used as a screening test, while this test shows Hemoglobin S to be present, it is normally followed by one of the quantitative tests discussed above and most probably electrophoresis, to determine the exact composition of the blood.
  • Hemoglobin S in its reduced form is insoluble in a phosphate buffer in the presence of sodium hydrosulfite.
  • a phosphate buffer is prepared, which in the preferred embodiment was formed from 16.9 grams of monobasic potassium phosphate, and 21.7 grams of dibasic potassium phosphate which are diluted with distilled water to a volume of 100 ml.
  • the phosphate buffer has a high hydrogen ion concentration and the pH of the solution is between 6.5 and 6.8.
  • To the phosphate buffer solution is added 6 grams of sodium hydrosulfite.
  • the sodium hydrosulfite is dissolved in the phosphate buffer solution by swirling or by a vortex method of mixing.
  • a rapid erythrocytic hemolyzing agent that is capable of inducing multiple lesions of the erythrocyte membrane is then added to the resultant solution.
  • 10 ml. of a 2 percent saponin solution in isotonic sodium chloride (NaCl) was added to the previously formed solution.
  • the resultant solution was then mixed and dispensed into 10 x mm. tubes with 2 ml. of the solution being placed in each tube.
  • the solution prepared in accordance with the above method would fill over 50 test tubes and thus 50 tests can be performed. It has been found that the solution with the erythrocytic hemolyzing agent can be kept at least six weeks under refrigeration and that the solution can be kept for even longer periods of time when the erythrocytic hemolyzing agent and reducing agent are kept separate.
  • the tests can be further modified by merely placing a drop of the buflfer, reducing agent, and hemolyzing agent solution on a glass plate and, then placing less than a drop of blood from an applicator stick in the solution.
  • the line in two minutes, will either be visible if the result is negative or will be blocked by the turbidity of the solution and blood mixture when Hemoglobin S is present. This would allow for mass screening of blood in the most simple and elemental form.
  • both of the tests outlined above can be performed by persons totally unskilled in the art of blood testing as they need merely drop .02 ml. of whole l o i to a ra -p pa d solut on and vis a y t m e.
  • FIGURE 1 is a front view of testing apparatus utilizing the principles of the present invention prior to the addition of whole blood.
  • FIGURE 2 shows the apparatus of FIGURE 1 after whole blood having Hemoglobin S therein has been added to the testing solution giving a positive result.
  • FIGURE 3 is a top plan view of a glass plate utilized for mass screening of bloods to determine the presence or absence of Hemoglobin S.
  • the basic invention is practiced by preparing a phosphate buffer solution discussed previously, that is, mixing 21.7 grams of dibasic potassium phosphate (K HPO with 16.9 grams of monobasic potassium phosphate (KH PO and diluting with distilled water to 100 ml.
  • a reducing agent in the form of 6 grams of sodium hydrosulfite is then mixed with the potassium phosphate butter and dissolved in the buffer by swirling or other suitable means.
  • the hemolytic agent is then added to the phosphate buffer and reductant solution.
  • Many types of hemolytic agents are available.
  • a preferred hemolytic agent is a 2 percent saponin solution in isotonic sodium chloride. This has been found to be especially effective for rapid hemolyzing.
  • Saponin C32H52017
  • saponin is a term applied to two groups of plant glucosides that have the ability to hemolyze red corpuscles at very great dilutions. The rapidity of hemolysis utilizing these saponins depends to a large extent on the plants fromwhich a particular saponin is produced, on the purity of the saponin and possibly even on the place where the particular plant was grown.
  • saponins manufactured by Glenwood Chemical Company, 83 Summit St., Tenafly, N.J., under the trademark Sapolysin and by Coulter Electronics Company of Hialeah, -Fla. are effective in the process of the present invention.
  • saponins manufactured by Glenwood Chemical Company, 83 Summit St., Tenafly, N.J., under the trademark Sapolysin and by Coulter Electronics Company of Hialeah, -Fla.
  • test tubes 10 with the test solution 12 as shown in FIG- URE 1 are placed in front of a test card 14.
  • the card 14 has a black line 16 running horizontally across the center thereof. As can be seen, the test line 16 will be visible through the solution 12 and the test tube 10.
  • a Sahli pipet (.02 ml.) 18 is utilized to supply .02 ml. of whole blood into the test tube 10 to mix with the solution 12. The blood from pipet 18 and the solution 12 are then mixed by vortex, lateral swirling, or by multiple inversions. Then the test tube is allowed to stand for approximately 2 minutes.
  • the solution 12' including the blood and the original test solution 12 is now cloudy, or turbid resulting from the precipitate caused by reason of the insolubility of Hemoglobin S in a phosphate buffer in the presence of sodium hydrosulfite, then the result is positive indicating the probable presence of Hemoglobin S.
  • the turbidity and cloudiness of the solution is easily seen by the fact that the line 16 is no longer visible through the new solution 12 and test tube 10. If the line 16 is visible,
  • FIGURE 3 A method of mass screening of blood samples for Hemoglobin S is shown in FIGURE 3.
  • a drop of the solution 12 of FIGURE 1 comprising the mixture of phosphate buffer, reducing agent, and hemolytic agent is placed in various spaces 20 on a glass or transparent plate 22. Then the glass plate 22 is placed on a background 24 having lines 26, 28 and 30 running horizontally along the width thereof. Samples of blood are then added by an application stick which has been dipped in blood and stirred in the solution on the positions 20. After 2 minutes, each of the positions 20 is observed. If the lines 26, 28 or 30 associated with a particular position 20 can be seen through the test solution blood mixture, then the test result is negative. If the particular line 26, 28 or 30 associated with a position 20 is not visible through the blood-test solution mixture, then the result is positive.
  • butter solution set forth above can be formed by dissolving 17.7 grams of dibasic sodium phosphate in carbon dioxide-free distilled water and the remainder of the process would be the same to produce similar results.
  • a method of testing blood for the presence of Hemoglobin S comprising the steps of providing a high ionic concentration buifer solution, adding a reducing agent to the butter, adding an erythrocytic hemolyzing agent to the buffer and reducing agent solution, and then adding a measured amount of blood to the buffer, reducing agent and hemolyzing agent solution after mixing the same, and, after a predetermined amount of time, observing the resultant solution to determine, by the turbidity of the resultant solution, either the absence or probable presence of Hemoglobin S in the blood.
  • test solution is formed in the same proportions as is achieved by providing ml. of a mixture of the phosphate buffer and reducing agent and 10 m1. of a saponin solution the concentration of which depends on its erythrocytic hemolyzing ability.
  • said buifer solution includes a mixture of dibasic potassium phosphate and monobasic potassium phosphate at a high hydrogen ion concentration.
  • a product for testing of whole blood for Hemoglobin S comprising a high ionic concentration buffer solution mixed with a reducing agent and an erythrocytic hemolyzing agent.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Urology & Nephrology (AREA)
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US673071A 1967-10-05 1967-10-05 Product for and method of testing blood for the presence of hemoglobin s Expired - Lifetime US3492095A (en)

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US (1) US3492095A (en:Method)
BE (1) BE712385A (en:Method)
CH (1) CH491386A (en:Method)
DE (1) DE1773363A1 (en:Method)
ES (1) ES352693A1 (en:Method)
FR (1) FR1559272A (en:Method)
GB (1) GB1213014A (en:Method)
IL (1) IL28990A (en:Method)
NL (1) NL6813544A (en:Method)
NO (1) NO127470B (en:Method)
SE (1) SE358029B (en:Method)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3847545A (en) * 1972-05-18 1974-11-12 Baxter Laboratories Inc Diagnostic test for sickle-cell
US3847482A (en) * 1972-07-10 1974-11-12 Bio Data Corp Apparatus for detecting a change in turbidity of a solution
US3877873A (en) * 1972-06-26 1975-04-15 Milton Winitz Test for metabolic conditions in blood or serum
US3918905A (en) * 1973-07-20 1975-11-11 Biolog Corp Of America Diagnostic test for the determination of sickling hemoglobinopathies
US4065383A (en) * 1975-04-14 1977-12-27 Helge Skare Procedure and means for collecting liquid containing radioactive tracer elements
US4336157A (en) * 1980-06-27 1982-06-22 Baxter Travenol Laboratories, Inc. Process for reclaiming biliverdin-containing fluids
US5064282A (en) * 1989-09-26 1991-11-12 Artel, Inc. Photometric apparatus and method for measuring hemoglobin
WO1992017787A1 (en) * 1991-04-08 1992-10-15 University Of South Alabama Method and apparatus for monitoring blood loss
US5231032A (en) * 1991-04-08 1993-07-27 University Of South Alabama Method of monitoring blood loss
US5514592A (en) * 1995-02-13 1996-05-07 Medicus Technologies, Inc. Method and composition for testing blood for hemoglobin S that incorporates mineral oil as an insoluble upper phase
US20070141709A1 (en) * 2005-12-16 2007-06-21 Artel Calibrating dispensing device performance for complex and/or non-aqueous liquids
US20070161114A1 (en) * 2006-01-06 2007-07-12 Artel. Inc. Method and apparatus for determining liquid volume
US20080070317A1 (en) * 2006-09-15 2008-03-20 Artel, Inc. Quantitative dual-dye photometric method for determining dilution impact
US20090251681A1 (en) * 2008-04-07 2009-10-08 Artel, Inc. System and method for liquid delivery evaluation using solutions with multiple light absorbance spectral features
US20120077218A1 (en) * 2010-09-24 2012-03-29 Saint Louis University Sickle confirm modified hemoglobin solubility test
US8404158B2 (en) 2008-04-07 2013-03-26 Artel, Inc. System and method for liquid delivery evaluation using solutions with multiple light absorbance spectral features
US10359431B2 (en) * 2014-08-29 2019-07-23 Map Ip Holding Limited Method for detecting abnormalities in hemoglobin

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2519997A (en) * 1945-11-10 1950-08-22 American Optical Corp Comparison method for measuring the hemoglobin content of blood
US3000836A (en) * 1958-09-02 1961-09-19 Ginsburg Ben Stabilized whole blood standard and method of making the same
US3374063A (en) * 1963-05-29 1968-03-19 Noller Hans Gunter Method of determining hemoglobin in blood
US3446751A (en) * 1965-11-29 1969-05-27 Brunswick Corp Hemolyzing composition

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2519997A (en) * 1945-11-10 1950-08-22 American Optical Corp Comparison method for measuring the hemoglobin content of blood
US3000836A (en) * 1958-09-02 1961-09-19 Ginsburg Ben Stabilized whole blood standard and method of making the same
US3374063A (en) * 1963-05-29 1968-03-19 Noller Hans Gunter Method of determining hemoglobin in blood
US3446751A (en) * 1965-11-29 1969-05-27 Brunswick Corp Hemolyzing composition

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3847545A (en) * 1972-05-18 1974-11-12 Baxter Laboratories Inc Diagnostic test for sickle-cell
US3877873A (en) * 1972-06-26 1975-04-15 Milton Winitz Test for metabolic conditions in blood or serum
US3847482A (en) * 1972-07-10 1974-11-12 Bio Data Corp Apparatus for detecting a change in turbidity of a solution
US3918905A (en) * 1973-07-20 1975-11-11 Biolog Corp Of America Diagnostic test for the determination of sickling hemoglobinopathies
US4065383A (en) * 1975-04-14 1977-12-27 Helge Skare Procedure and means for collecting liquid containing radioactive tracer elements
US4336157A (en) * 1980-06-27 1982-06-22 Baxter Travenol Laboratories, Inc. Process for reclaiming biliverdin-containing fluids
US5064282A (en) * 1989-09-26 1991-11-12 Artel, Inc. Photometric apparatus and method for measuring hemoglobin
WO1992017787A1 (en) * 1991-04-08 1992-10-15 University Of South Alabama Method and apparatus for monitoring blood loss
US5231032A (en) * 1991-04-08 1993-07-27 University Of South Alabama Method of monitoring blood loss
US5236664A (en) * 1991-04-08 1993-08-17 University Of South Alabama Apparatus for monitoring blood loss
US5514592A (en) * 1995-02-13 1996-05-07 Medicus Technologies, Inc. Method and composition for testing blood for hemoglobin S that incorporates mineral oil as an insoluble upper phase
US20070141709A1 (en) * 2005-12-16 2007-06-21 Artel Calibrating dispensing device performance for complex and/or non-aqueous liquids
US8003405B2 (en) 2005-12-16 2011-08-23 Artel, Inc. Calibrating dispensing device performance for complex and/or non-aqueous liquids
US20070161114A1 (en) * 2006-01-06 2007-07-12 Artel. Inc. Method and apparatus for determining liquid volume
US7772008B2 (en) 2006-01-06 2010-08-10 Artel, Inc. Method and apparatus for determining liquid volume
US20100290048A1 (en) * 2006-09-15 2010-11-18 Artel, Inc. Quantitative dual-dye photometric method for determining dilution impact
US7919327B2 (en) 2006-09-15 2011-04-05 Artel, Inc. Quantitative dual-dye photometric method for determining dilution impact
US7998747B2 (en) 2006-09-15 2011-08-16 Artel, Inc. Quantitative dual-dye photometric method for determining dilution impact
US20080070317A1 (en) * 2006-09-15 2008-03-20 Artel, Inc. Quantitative dual-dye photometric method for determining dilution impact
US20090251681A1 (en) * 2008-04-07 2009-10-08 Artel, Inc. System and method for liquid delivery evaluation using solutions with multiple light absorbance spectral features
US7791716B2 (en) 2008-04-07 2010-09-07 Artel, Inc. System and method for liquid delivery evaluation using solutions with multiple light absorbance spectral features
US8404158B2 (en) 2008-04-07 2013-03-26 Artel, Inc. System and method for liquid delivery evaluation using solutions with multiple light absorbance spectral features
US20120077218A1 (en) * 2010-09-24 2012-03-29 Saint Louis University Sickle confirm modified hemoglobin solubility test
US8623659B2 (en) * 2010-09-24 2014-01-07 Saint Louis University Sickle confirm modified hemoglobin solubility test
US10359431B2 (en) * 2014-08-29 2019-07-23 Map Ip Holding Limited Method for detecting abnormalities in hemoglobin

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Publication number Publication date
GB1213014A (en) 1970-11-18
CH491386A (fr) 1970-05-31
IL28990A (en) 1971-07-28
NO127470B (en:Method) 1973-06-25
SE358029B (en:Method) 1973-07-16
FR1559272A (en:Method) 1969-03-07
BE712385A (en:Method) 1968-07-31
DE1773363A1 (de) 1971-07-22
NL6813544A (en:Method) 1969-04-09
ES352693A1 (es) 1969-07-16

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