IL46137A - Reagent and method for determining leukocytes and hemoglobin in the blood - Google Patents
Reagent and method for determining leukocytes and hemoglobin in the bloodInfo
- Publication number
- IL46137A IL46137A IL46137A IL4613774A IL46137A IL 46137 A IL46137 A IL 46137A IL 46137 A IL46137 A IL 46137A IL 4613774 A IL4613774 A IL 4613774A IL 46137 A IL46137 A IL 46137A
- Authority
- IL
- Israel
- Prior art keywords
- ion
- quaternary ammonium
- reagent
- hemoglobin
- cyanide
- Prior art date
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 38
- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 30
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 30
- 210000000265 leukocyte Anatomy 0.000 title claims abstract description 24
- 210000004369 blood Anatomy 0.000 title claims abstract description 18
- 239000008280 blood Substances 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims description 7
- 125000001453 quaternary ammonium group Chemical group 0.000 claims abstract description 17
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 claims abstract description 16
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 13
- YAGKRVSRTSUGEY-UHFFFAOYSA-N ferricyanide Chemical compound [Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] YAGKRVSRTSUGEY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 20
- 125000000217 alkyl group Chemical group 0.000 claims description 13
- 125000004432 carbon atom Chemical group C* 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 9
- -1 trimethyl tetradecyl ammonium halide Chemical class 0.000 claims description 7
- 150000002500 ions Chemical class 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 229910052783 alkali metal Inorganic materials 0.000 claims description 4
- 239000004615 ingredient Substances 0.000 claims description 4
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 4
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims 1
- CEYYIKYYFSTQRU-UHFFFAOYSA-M trimethyl(tetradecyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCC[N+](C)(C)C CEYYIKYYFSTQRU-UHFFFAOYSA-M 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 12
- 230000002934 lysing effect Effects 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 6
- 230000002949 hemolytic effect Effects 0.000 description 6
- 230000008014 freezing Effects 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 125000001204 arachidyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001767 cationic compounds Chemical class 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 229910001412 inorganic anion Inorganic materials 0.000 description 1
- 229910001411 inorganic cation Inorganic materials 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 230000000063 preceeding effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
- BYGOPQKDHGXNCD-UHFFFAOYSA-N tripotassium;iron(3+);hexacyanide Chemical compound [K+].[K+].[K+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] BYGOPQKDHGXNCD-UHFFFAOYSA-N 0.000 description 1
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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Ecology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
Leukocytes and hemoglobin in the blood are determined in vitro with a reagent which comprises a ferricyanide ion-free aqueous solution containing a quaternary ammonium ion and cyanide ion in amounts sufficient to stromatolyse erythrocyte and platelet cells and to convert hemoglobin to a chromogen for the determinations.
[US3874852A]
Description
REAGENT AND ME HOD FOR DETERMINING LEUKOCYTES AND HEMOGLOBIN IN THE BLOOD / REAGENT AND METHOD FOR DETERMINING LEUKOCYTES AND HEMOGLOBIN IN THE BLOOD ABSTRACT OF THE DISCLOSURE Leukocytes and hemoglobin in the blood are determined in vitro with a reagent which comprises a ferrlcyanide ion-free aqueous solution containing a quaternary ammonium ion and cyanide ion in amounts sufficient to stromatolyse erythrocyte and platelet cells and to convert hemoglobin to a chromogen for the determinations .
BACKGROUND OF THE INVENTION This invention relates to a reagent and a method for determining leukocytes and hemoglobin in the blood.
More particularly, the invention relates to an In vitro diagnostic reagent for high speed erythrocyte stromatolyslng with rapid conversion of hemoglobin to a chromogen, for use in the determination of leukocytes and hemoglobin in the blood .
The introduction of high speed automated hematology instruments such as the Coulter Counter Model "S" has resulted in a need for high speed erythrocyte-stromatolysing and chromogen-forming reagents which give a clear, stable, reproducible solution whose optical density is directly proportional to the hemoglobin concentration. In an instrument of this type, blood is mixed with a conventional diluent, to provide a first dilution, and then mixed with a lysing agent, to provide a second dilution. The respective first and second dilutions may be, for example, 224:1 and 250:1. The mixture remains in the lysing chamber for a short but sufficient amount of time for the erythrocytes or red blood cells to be disintegrated and release their hemoglobin. The resulting suspension is passed into tubes in a leukocyte aperture counting bath, wherein the leukocytes or white blood cells are counted electronically. With the released hemoglobin contained in the leukocyte count suspension, the hemoglobin concentration is determined spectrophotometrically in the same bath. Inasmuch as the ratio of erythrocytes to leukocytes in normal blood is in the vicinity of 1000:1, the erythrocytes must be reduced rapidly to very small fragments, to avoid interference with leukocyte counting. At the same time, leukocytes must not be destroyed, and it is necessary to convert hemoglobin to a form suitable for accurate photometric determination.
Heretofore, lysing and cyanmethemoglobin chromogen-forming reagents have been used in automated high speed hematology equipment. However, these reagents have been found to be unsatisf ctory as respects their stability.
It has been known also that a quaternary ammonium salt advantageously is employed as a stromatolysing agent, with the virtually instantaneous destruction of erythrocytes to a level avoiding interference with leukocyte estimation, and providing a relatively stable reagent (see, for example, The American Journal of Clinical Pathology, Vol. 36, No. 3, Pages 220-223, Sept., 1961). The quaternary ammonium ion in the salt is of the type having three short chain alkyl groups and one long chain alkyl group attached to nitrogen.
The standard method of determining hemoglobi embodies the use of Drabkin's reagent, which contains potassium ferri-cyanide, potassium cyanide, and sodium bicarbonate. When hemoglobin is added to this reagent, the potassium ferricyanide is reduced to potassium ferrocyanide, and the hemoglobin is oxidized to methemo lobin. The latter reacts with the cyanide ion to produce the stable chromogen, cyanmethemoglobin, which may be measured photometrically. Drabkin's reagent is not stable upon exposure to freezing temperatures.
Ferricyanide ion has a tendency to form insoluble complexes with quaternary ammonium compounds. Thus, a mixture of the prior quaternary ammonium compound reagent with Drabkin's reagent may be unreliable, since in the event of precipitation, the precipitate, no matter how slight, would falsely raise the leukocyte count.
It would be desirable to provide an accurate, stable high speed stromatolysing and chromogen-forming reagent for use in high speed automated hematology instru ments. It would be preferable to provide a reagent which also is not adversely affected by exposure to freezing temperatures .
SUMMARY OF THE INVENTION The reagent of the invention comprises a ferricyanide ion-free aqueous solution containing a quaternary ammonium ion and cyanide ion, the quaternary ammonium ion having attached to nitrogen three short chain alk l groups and one long chain alkyl group, and said ions being present in amounts suf icient to stromatolyse erythrocyte and platelet cells in the blood and to convert hemoglobin to a chromogen or leukocyte and hemoglobin determinations. In the method of the invention, the foregoing reagent is reacted with a blood sample for the determination of leukocytes and hemoglobin in the blood.
The reagent of the invention is a stable composition that ovides leukocyte and hemoglobin determinations with the necessary diagnostic accuracy. More particularly, the reagent reduces erythrocytes to very small fragments within a short period of time, without destroying leukocytes. The reagent forms no precipitate which would interfere with leukocyte determination.
The reagent converts hemoglobin to a stable chromogen having spectral characteristics resembling cyanmethemoglobin, which is suitable for photometric analysis with a high degree of accuracy: A clear, stable and reproducible solution is provided for analysis, the optical density of which is directly proportional to the hemoglobin concentration.
The invention embodies the discovery that ferricyanide ion need not be employed, and only cyanide ion need be employed together with a quaternary ammonium ion to convert hemoglobin to a stable chromogen suitable for analysis. Consequently, there is no risk of complex formation caused by the presence of ferricyanide ion. The reagent is a colorless solution.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The reagent of the invention is an aqueous solution free of ferricyanide ion and containing as the active ions a quaternary ammonium ion and cyanide ion. Each of the respective ions may be provided in the form of a suitable salt, with an ion of opposite sign. The quaternary ammonium ion preferably is supplied in the form of a salt with an inorganic anion, especially as a halide, such as chloride or bromide, a sulfate, a phosphate, or a nitrate. The cyanide ion preferably is supplied in the form of a salt with an inorganic cation, especially an alkali metal cation such as sodium or potassium. The solution may consist essentially of such salts dissolved in water.
In gene al; the quaternary ammonium ion is of the type previously employed in the art as a stromatolysing agent, i.e., having attached to nitrogen three short chain alkyl groups and one long chain alkyl group. For convenience, the short chain alkyl groups will be referred to hereinafter at times by the symbols R^, R^, and ^, and the long chain alkyl group will be referred to as R4. Generally speaking, Rl* R2' and/°r R3 mav n»ve from 1 to 4 carbon atoms, as represented by methyl, ethyl, propyl, and butyl radicals. 4 nay vary in the range of about 10 to 20 carbon atoms, ranging from decyl to eicosyl.
The quaternary ammonium ion is provided by a quaternary ammonium salt incorporated in aqueous solution in a concentration within the overall range of about 0.5 to 10%, preferably about 1-5%, by weight of the solution.
A solution of such concentration is employed as the lysing agent, and is mixed with blood previously diluted with standard diluent, in a ratio of about 1:8.6, lysing agent to dilute sample, as described above. It will be understood that different strength lysing agent may be employed where the initial dilution of the blood sample differs from that described above, in order to provide the same ultimate concentration of reactive ion.
The specific quaternary ammonium ion and its concentration are selected to provide the necessary hemolytic activity and solubility of the quaternary ammonium compound. In general, with increasing number of carbon atoms, the solubility of the compound decreases, and with increasing number of R. carbon atoms, the hemolytic activity increases. Thus, for example, where R^, R2 and Rg are methyl, R4 may include from about 10 to 20. carbon atoms (C1Q to C2Q) to provide sufficient hemolytic activity at a concentration of 5% when employed in the above-described analytical method.
At a concentration of about 0.5% and where R^, R2 and R^ are methyl, R4 should have at least about 14 carbon atoms to provide the necessary hemolytic activity. At a concentration of about 10% and where R1, 2 and 3 are methyl, R4 should have a maximum of about 16 carbon atoms, to provide the necessary solubility for the compound. As additional examples, where R4 is tetradecyl, R^, R2, and R^ may be methyl, ethyl, or propyl radicals at 0.5 to 10% concentration, while providing sufficient hemolytic activity and a complete solution. Where R4 is tetradecyl and ^ is methyl, R2 is methyl or ethyl, and R3 is butyl, the hemolytic activity is sufficient and the compound is soluble at 0.5 to 5%, but the compound is incompletely soluble at a concentration of 10% It is a feature of the invention that the reagent may be compounded to allow for freezing and thawing of the solution without formation of excessive particulate matter, i.e., such as would exceed the accuracy of the instrument and interfere with an accurate leukocyte count. The above-identified instrument has an accuracy limit of about 200 cells. For example, when R^ is decyl, dodecyl, or tetradecyl, and R^, R2, and R3 are methyl, a solution of 5% concentration may be frozen and thawed with resulting formation of less than 200 particles in 0.5 ml., as determined using the Coulter Counter FM at red blood cell threshold settings. Where R^ si 4 is tetradecyl and R^, Rg, and Rg are from methyl to propyl, a solution at 5% concentration may be. frozen and thawed with resulting particle count of less than 200. On the other hand, where R. is from C,„ to C__, and R-, , R„ and R are methyl, the 4 16 20 1 2 3 * ' particle count exceeds 200 after freezing and thawing a solution of 5% concentration. Likewise, where R^ is tetradecyl, R^ is methyl, 2 is methyl or ethyl, and R3 is butyl, the particle count exceeds 200 after freezing and thawing a solution of 5% concentration. Accordingly, it is preferred that ^, Rg and 3 each have from 1 to 3 carbon atoms, and that R have from 10 to 14 carbon atoms.
The concentration of cyanide ion preferably is selected to provide at least about 89% conversion of hemoglobin to a chromogen. Thus, for example, in providing a solution for use in the above-described manner, the cyanide ion preferably is present in a concentration in the range of about 0.0006 to 0.005 molar, and preferably in excess of about 0.001 molar. A salt furnishing the cyanide ion is employed in a molarity to provide the desired cyanide ion concentration, being the same molarity in the case of a salt with a monovalent cation such as an alkali metal. Conversions to a chromogen of up to about 96% of the hemoglobin may be obtained. The chromogen is analyzed spectrophotometrically at 540 nanometers.
The following is a specific example of a reagent according to the invention. It will be understood that the example is only illustrative, and various other ingredients and proportions may be employed, in accordance with the preceeding disclosure.
EXAMPLE The following composition may be employed as a lysing and chromogen-forming reagent in the Coulter Counter Model "S", following the procedure described hereinabove: Ingredient Proportion Water 1 liter Potassium cyanide 0.25 gram (0.00385 ) Trimethyl tetradecyl > ammonium chloride 35 grams (3.38%) The composition has a pH of approximately 9. When employed for analysis with the commercial buffered blood diluent Isoton and following said procedure, the pH of the final solution is approximately 7.6.
What is claimed is :
Claims (8)
1. A reagent for use in the determination of leukocytes and hemoglobin in the blood, which comprises a ferricyanide ion-free aqueous solution containing a quaternary ammonium ion and cyanide ion, said quaternary ammonium ion having attached to nitrogen three short chain alkyl groups and one long chain alkyl group, and said ions being present in amounts sufficient to stromatolyse erythrocyte and platelet cells and to convert hemoglobin to a chromogen for said determinations.
2. A reagent according to claim 1 wherein said quaternary ammonium ion is provided by a quaternary ammonium salt incorporated in said solution in a concentration in the range of ab t. 0,5 to 10% by weight of said solution, and said cyanide ion is present in a concentration in the range of about 0.0006 to 0.005 molar.
3. A reagent according to claim 2 wherein said salt is a trimethyl tetradecyl ammonium halide, sulfate, phosphate, or nitrate, and said cyanide ion is provided by an alkali metal cyanide incorporated in said solution.
4. A reagent according to claim 1 wherein said short chain alkyl groups each have from 1 to 3 carbon atoms and said long chain alkyl group has from 10 to 14 carbon atoms .
5. A reagent according to claim 4 wherein said quaternary ammonium ion is provided by a quaternary ammonium salt incorporated in said solution in a concentration in the range of about 0.5 to 10% by weight of said solution, and said cyanide ion is provided by an alkali metal cyanide incorporated in said solution in a concentration in the range of about 0.0006 to 0.005 molar.
6. A reagent according to claim 5 wherein said salt is a halide, sulfate, phosphate, or nitrate.
7. A reagent according to claim 1 comprising a composition having the following ingredients and relative n *—rn-nA-n—rt—ln-n■s · Ingredient Proportion Water 1 liter Potassium cyanide 0.25 gram Trimethyl tetradecyl ammonium chloride 35 grams
8. In a method of determining leukocytes and hemoglobin in the blood wherein a reagent is reacted with a blood sample to stromatolyse erythrocyte and platelet cells and to convert hemoglobin to a chromogen for said determinations, the improvement which comprises employing as said reagent a ferricyanide ion-free aqueous solution containing a quaternary ammonium ion and cyanide ion, said quaternary ammonium ion having attached to nitrogen three short chain alkyl groups and one long chain alkyl group.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US485931A US3874852A (en) | 1974-07-05 | 1974-07-05 | Reagent and method for determining leukocytes and hemoglobin in the blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IL46137A0 IL46137A0 (en) | 1975-02-10 |
| IL46137A true IL46137A (en) | 1977-02-28 |
Family
ID=23929974
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL46137A IL46137A (en) | 1974-07-05 | 1974-11-27 | Reagent and method for determining leukocytes and hemoglobin in the blood |
Country Status (15)
| Country | Link |
|---|---|
| US (1) | US3874852A (en) |
| JP (1) | JPS616341B2 (en) |
| BE (1) | BE824206A (en) |
| CA (1) | CA1024868A (en) |
| CH (1) | CH588078A5 (en) |
| DE (1) | DE2501855C3 (en) |
| ES (1) | ES433929A1 (en) |
| FR (1) | FR2277344A1 (en) |
| GB (1) | GB1489303A (en) |
| IL (1) | IL46137A (en) |
| IT (1) | IT1029631B (en) |
| MX (1) | MX4259E (en) |
| NL (1) | NL164669B (en) |
| SE (1) | SE415609B (en) |
| ZA (1) | ZA747574B (en) |
Families Citing this family (40)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3962125A (en) * | 1975-01-13 | 1976-06-08 | Coulter Diagnostics, Inc. | Multi-purpose diluent for use in blood analysis by electronic instrumentation of the coulter type |
| US4141856A (en) * | 1976-05-24 | 1979-02-27 | Dorwart Jr William V | Reference material for establishing anion concentrations in analytical chemistry tests |
| US4185964A (en) * | 1977-02-08 | 1980-01-29 | Central Laboratories of Associated Maryland Pathologists, Ltd. | Lysing reagent |
| US4219440A (en) * | 1979-06-06 | 1980-08-26 | Coulter Electronics, Inc. | Multiple analysis hematology reference control reagent and method of making the same |
| DE2905531A1 (en) * | 1979-02-14 | 1981-01-08 | Boehringer Mannheim Gmbh | DIAGNOSTIC AGENT FOR DETECTING LEUCOCYTES IN BODY LIQUIDS |
| US4455376A (en) * | 1979-09-17 | 1984-06-19 | R. J. Harvey Instrument Corp. | Photometric methods for counting the particulate components of blood |
| US4286963A (en) * | 1979-11-23 | 1981-09-01 | Coulter Electronics, Inc. | Differential lymphoid-myeloid determination of leukocytes in whole blood |
| US4346018A (en) * | 1980-06-16 | 1982-08-24 | Coulter Electronics, Inc. | Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes |
| US4521518A (en) * | 1980-06-16 | 1985-06-04 | Coulter Electronics, Inc. | Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes |
| US4962038A (en) * | 1980-06-16 | 1990-10-09 | Coulter Electronics, Inc. | Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes |
| DE3024835A1 (en) * | 1980-07-01 | 1982-01-28 | Bayer Ag, 5090 Leverkusen | REAGENT FOR HEMOGLOBIN DETERMINATION |
| 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 |
| DE3204938C2 (en) * | 1982-02-12 | 1985-01-17 | Drägerwerk AG, 2400 Lübeck | Indicator for the determination of sulfur dioxide |
| US4528274A (en) * | 1982-07-06 | 1985-07-09 | Coulter Electronics, Inc. | Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes |
| US4485175A (en) * | 1983-01-03 | 1984-11-27 | Coulter Electronics, Inc. | Method for three-volume differential determination of lymphocyte, monocyte and granulocyte populations of leukocytes |
| US4529705A (en) * | 1983-06-06 | 1985-07-16 | Coulter Electronics, Inc. | Reagent for combined diluting and lysing whole blood |
| US4637986A (en) * | 1983-08-22 | 1987-01-20 | Ortho Diagnostic Systems, Inc. | Flow cytometry lysing reagent with leukoprotective agent for producing a 3-part WBC count |
| JPS6073356A (en) * | 1983-09-29 | 1985-04-25 | Toa Medical Electronics Co Ltd | Reagent for blood analysis |
| AU4406185A (en) * | 1984-05-31 | 1985-12-31 | Coulter Electronics Inc. | A reagent system and method for identification, enumeration and examination of classes and subclasses of blood leukocytes |
| FR2582103B1 (en) * | 1985-03-19 | 1989-05-05 | Canavaggio Michel | METHOD FOR DETERMINING A BIOLOGICAL SUBSTANCE IN A SAMPLE |
| US4745071A (en) * | 1985-09-05 | 1988-05-17 | Sequoia-Turner Corporation | Method for the volumetric differentiation of blood cells types |
| US4725555A (en) * | 1986-10-22 | 1988-02-16 | Helena Laboratories Corporation | Zinc protoporphyrin test system |
| IL85532A (en) * | 1987-03-13 | 1992-03-29 | Coulter Electronics | Method and lytic reagent system for isolation,identification and/or analysis of leukocytes from whole blood samples |
| US5155044A (en) * | 1987-03-13 | 1992-10-13 | Coulter Electronics, Inc. | Lysing reagent system for isolation, identification and/or analysis of leukocytes from whole blood samples |
| US4853338A (en) * | 1987-05-20 | 1989-08-01 | Technicon Instruments Corporation | Cyanide-free hemoglobin reagent |
| US5045474A (en) * | 1987-05-28 | 1991-09-03 | Sequoia-Turner Corporation (Unipath) | Semi-automatic process for white cell differential count |
| US5008202A (en) * | 1988-11-29 | 1991-04-16 | Sequoia Turner Corporation | Blood diluent for red blood cell analysis |
| CA2016699C (en) * | 1989-05-15 | 2003-11-18 | Paul N. Marshall | Lytic agents and uses thereof |
| US5284940A (en) * | 1990-11-14 | 1994-02-08 | Hri Research, Inc. | Preparation for nucleic acid samples |
| US5620852A (en) * | 1990-11-14 | 1997-04-15 | Hri Research, Inc. | Nucleic acid preparation methods |
| US5654179A (en) * | 1990-11-14 | 1997-08-05 | Hri Research, Inc. | Nucleic acid preparation methods |
| WO1996002841A1 (en) | 1994-07-14 | 1996-02-01 | Abbott Laboratories | Methods and reagents for cyanide-free determination of hemoglobin and leukocytes in whole blood |
| US5834315A (en) * | 1994-12-23 | 1998-11-10 | Coulter Corporation | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentitation |
| US5858794A (en) * | 1997-05-13 | 1999-01-12 | Bayer Corporation | Cyanide-containing hemoglobin reagent composition and method providing acceptable precision, accuracy and freedom from white cell interference on automated hematology analyzers |
| US20020098589A1 (en) * | 2001-01-19 | 2002-07-25 | Crews Harold Richardson | Multi-purpose reagent system and method for enumeration of red blood cells, white blood cells and thrombocytes and differential determination of white blood cells |
| US9133024B2 (en) * | 2003-09-03 | 2015-09-15 | Brigitte Chau Phan | Personal diagnostic devices including related methods and systems |
| US9091677B2 (en) | 2010-08-09 | 2015-07-28 | Beckman Coulter, Inc. | Isotonic buffered composition and method that enables counting of cells |
| EP2749882B1 (en) | 2011-08-24 | 2017-05-03 | Eiken Kagaku Kabushiki Kaisha | Leukocyte measurement device and reagent kit |
| IN2012DE02074A (en) | 2012-07-03 | 2015-08-14 | Srivastava Ambar | |
| CN113959911B (en) * | 2020-07-20 | 2024-06-18 | 深圳迈瑞生物医疗电子股份有限公司 | Detection method, reagent and application of antiplatelet aggregation interference |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3546131A (en) * | 1968-07-05 | 1970-12-08 | Uni Tech Chem Mfg Co | Stabilized cyanmethemoglobin reagent containing ferricyanide,cyanide and polyvinylpyrrolidone |
| US3607695A (en) * | 1969-02-14 | 1971-09-21 | Pfizer & Co C | Hemoglobinopathy controls |
| US3663175A (en) * | 1970-10-05 | 1972-05-16 | Bio Dynamics Inc | Method of determining hemoglobin in blood |
| BE790042A (en) * | 1971-10-14 | 1973-04-13 | Coulter Electronics | METHOD FOR CLASSIFYING PARTICLES |
-
1974
- 1974-07-05 US US485931A patent/US3874852A/en not_active Expired - Lifetime
- 1974-11-22 NL NL7415233.A patent/NL164669B/en not_active IP Right Cessation
- 1974-11-27 ZA ZA00747574A patent/ZA747574B/en unknown
- 1974-11-27 IL IL46137A patent/IL46137A/en unknown
- 1974-11-28 IT IT54278/74A patent/IT1029631B/en active
- 1974-11-29 CH CH1585074A patent/CH588078A5/xx not_active IP Right Cessation
- 1974-12-28 JP JP754195A patent/JPS616341B2/ja not_active Expired
-
1975
- 1975-01-07 CA CA217,470A patent/CA1024868A/en not_active Expired
- 1975-01-08 BE BE6044886A patent/BE824206A/en not_active IP Right Cessation
- 1975-01-08 FR FR7500452A patent/FR2277344A1/en active Granted
- 1975-01-10 SE SE7500245A patent/SE415609B/en not_active IP Right Cessation
- 1975-01-15 MX MX754018U patent/MX4259E/en unknown
- 1975-01-17 DE DE2501855A patent/DE2501855C3/en not_active Expired
- 1975-01-17 ES ES433929A patent/ES433929A1/en not_active Expired
- 1975-01-17 GB GB2184/75A patent/GB1489303A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| SE7500245L (en) | 1976-01-07 |
| SE415609B (en) | 1980-10-13 |
| JPS616341B2 (en) | 1986-02-25 |
| JPS516788A (en) | 1976-01-20 |
| AU7572574A (en) | 1976-05-27 |
| IT1029631B (en) | 1979-03-20 |
| CH588078A5 (en) | 1977-05-31 |
| US3874852B1 (en) | 1989-07-11 |
| US3874852A (en) | 1975-04-01 |
| ZA747574B (en) | 1976-07-28 |
| DE2501855B2 (en) | 1980-02-07 |
| FR2277344B1 (en) | 1977-11-18 |
| CA1024868A (en) | 1978-01-24 |
| IL46137A0 (en) | 1975-02-10 |
| BE824206A (en) | 1975-07-08 |
| DE2501855A1 (en) | 1976-01-22 |
| DE2501855C3 (en) | 1980-10-16 |
| MX4259E (en) | 1982-03-08 |
| FR2277344A1 (en) | 1976-01-30 |
| GB1489303A (en) | 1977-10-19 |
| ES433929A1 (en) | 1976-11-16 |
| NL164669B (en) | 1980-08-15 |
| NL7415233A (en) | 1976-01-07 |
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