CA1076008A - Stabilized hematological reagent solutions - Google Patents
Stabilized hematological reagent solutionsInfo
- Publication number
- CA1076008A CA1076008A CA265,047A CA265047A CA1076008A CA 1076008 A CA1076008 A CA 1076008A CA 265047 A CA265047 A CA 265047A CA 1076008 A CA1076008 A CA 1076008A
- Authority
- CA
- Canada
- Prior art keywords
- solution
- sodium
- phenoxyethanol
- fluoride
- alkali metal
- 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
Links
- 230000002489 hematologic effect Effects 0.000 title claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 12
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 claims abstract description 39
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229960005323 phenoxyethanol Drugs 0.000 claims abstract description 23
- 239000011775 sodium fluoride Substances 0.000 claims abstract description 20
- 235000013024 sodium fluoride Nutrition 0.000 claims abstract description 20
- 229910001515 alkali metal fluoride Inorganic materials 0.000 claims abstract description 6
- -1 2-phenoxyethanol Chemical class 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 21
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 235000002639 sodium chloride Nutrition 0.000 claims description 11
- 239000001103 potassium chloride Substances 0.000 claims description 9
- 235000011164 potassium chloride Nutrition 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 150000003856 quaternary ammonium compounds Chemical class 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- 235000010288 sodium nitrite Nutrition 0.000 claims description 3
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims 3
- 235000019800 disodium phosphate Nutrition 0.000 claims 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims 2
- 229910052783 alkali metal Inorganic materials 0.000 claims 1
- 239000007864 aqueous solution Substances 0.000 claims 1
- 229940098773 bovine serum albumin Drugs 0.000 claims 1
- 235000011008 sodium phosphates Nutrition 0.000 claims 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical class [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 15
- 239000000022 bacteriostatic agent Substances 0.000 abstract description 7
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 6
- 239000003429 antifungal agent Substances 0.000 abstract description 5
- 230000002934 lysing effect Effects 0.000 abstract description 5
- 239000002245 particle Substances 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 239000003381 stabilizer Substances 0.000 abstract description 4
- 239000000654 additive Substances 0.000 abstract description 2
- 230000000996 additive effect Effects 0.000 abstract description 2
- 238000004140 cleaning Methods 0.000 abstract description 2
- 150000002989 phenols Chemical class 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 16
- 102000001554 Hemoglobins Human genes 0.000 description 9
- 108010054147 Hemoglobins Proteins 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 7
- 229960001484 edetic acid Drugs 0.000 description 7
- 210000000601 blood cell Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000006172 buffering agent Substances 0.000 description 3
- 239000002738 chelating agent Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 3
- GJMPSRSMBJLKKB-UHFFFAOYSA-N 3-methylphenylacetic acid Chemical compound CC1=CC=CC(CC(O)=O)=C1 GJMPSRSMBJLKKB-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- CMDLYHXLYMIGIH-UHFFFAOYSA-N C.S.S Chemical compound C.S.S CMDLYHXLYMIGIH-UHFFFAOYSA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 2
- 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 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- VUFOSBDICLTFMS-UHFFFAOYSA-M ethyl-hexadecyl-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)CC VUFOSBDICLTFMS-UHFFFAOYSA-M 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 2
- RBWSWDPRDBEWCR-RKJRWTFHSA-N sodium;(2r)-2-[(2r)-3,4-dihydroxy-5-oxo-2h-furan-2-yl]-2-hydroxyethanolate Chemical compound [Na+].[O-]C[C@@H](O)[C@H]1OC(=O)C(O)=C1O RBWSWDPRDBEWCR-RKJRWTFHSA-N 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229940117957 triethanolamine hydrochloride Drugs 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000001279 citrus aurantifolia swingle expressed oil Substances 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- PRZSXZWFJHEZBJ-UHFFFAOYSA-N thymol blue Chemical compound C1=C(O)C(C(C)C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C(=CC(O)=C(C(C)C)C=2)C)=C1C PRZSXZWFJHEZBJ-UHFFFAOYSA-N 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 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
-
- 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
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
STABILIZED HEMATOLOGICAL REAGENT SOLUTIONS
ABSTRACT
The conjoint use of an ethoxylated phenol such as 2-phenoxyethanol, as a bacteriostatic and fungistatic agent and an alkali metal fluoride such as sodium fluoride as a stabilizer in a system used for specified hematological reagents. Sodium fluoride is added as a counterbalancing agent to the action of 2-phenoxyethanol, and in some instances functions also as an anticorrosion agent for certain solutions. A solution employing 2-phenoxyethanol and sodium fluoride is described employed as a water bath additive, as a cleaning solution for electronic particle counting apparatus, as a lysing reagent and as an artificial plasma which can be used as a control in blood sample, analysis.
ABSTRACT
The conjoint use of an ethoxylated phenol such as 2-phenoxyethanol, as a bacteriostatic and fungistatic agent and an alkali metal fluoride such as sodium fluoride as a stabilizer in a system used for specified hematological reagents. Sodium fluoride is added as a counterbalancing agent to the action of 2-phenoxyethanol, and in some instances functions also as an anticorrosion agent for certain solutions. A solution employing 2-phenoxyethanol and sodium fluoride is described employed as a water bath additive, as a cleaning solution for electronic particle counting apparatus, as a lysing reagent and as an artificial plasma which can be used as a control in blood sample, analysis.
Description
10~6008 BACKGROUND OF THE INVENTION
This invention concerns the use of 2-phenoxyethanol as a bacteriostatic and fungistatic agent and sodium fluoride as a stabilization agent in reagents utilized in hematological studies utilizing electronic particle analysis apparatus of the COULTER type.
In Canadian Patent 1,a45,960, there was disclosed a multipurpose, electrolytic solution for use in hematological procedures principally, enumeration of blood cells and the determination of hemoglobin concentration and other important parameters of a blood sample by means of automated electronic particle analysis apparatus of the COULTER type. The solution was balanced osmotically for mean cell volume stability, was azide-free and utilized
This invention concerns the use of 2-phenoxyethanol as a bacteriostatic and fungistatic agent and sodium fluoride as a stabilization agent in reagents utilized in hematological studies utilizing electronic particle analysis apparatus of the COULTER type.
In Canadian Patent 1,a45,960, there was disclosed a multipurpose, electrolytic solution for use in hematological procedures principally, enumeration of blood cells and the determination of hemoglobin concentration and other important parameters of a blood sample by means of automated electronic particle analysis apparatus of the COULTER type. The solution was balanced osmotically for mean cell volume stability, was azide-free and utilized
2-phenoxyethanol as a bacteriostatic and fungistatic agent.
Sodium fluoride was employed to counterbalance the cell wall weakening activity of 2-phenoxyethanol and thus `
stabilize the mean cell volume of the red cells. Also, sodium fluoride served to aid in the conversion of hemoglobin to cyanmethemoglobin for the determination of hemoglobin colorimetrically.
The use of a bacteriostatic and fungistatic agent brings about heretofore undesirable variations in the mean cell volume of the red cells leading to the unstabilization of the mean red cell volume. In the desirability of utiliz-ing a bacteriostatic agent as a replacement for sodium azide,as explained in Canadian Patent 1,045,960, 2-phenoxyethanol was selected although it had never been employed heretofore in such capacity. However, the 2-phenoxyethanol may lead to the destruction of the cell walls or change of cell volume. In order to avoid the problems of hemolysis or crenation, a stabilizer was utilized in the resulting system. Although sodium fluoride had long been known to be a preservative, such as for preserving glucose in blood samples, its utility as a stabilizer with the resulting system was unexpected.
- 4 - `
~.~
SUMMARY OF THE INVENTION
The invention is directed to the combination of an ethoxylated phenol such as 2-phenoxyethanol and an alkali metal fluoride such as sodium fluoride as effective for use in other reagent solutions that are utilized for other purposes in hematoloqical analytical procedures, particularly those employing electronic particle study methods.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
I have discovered that 2-phenoxyethanol and sodium fluoride, could be utilized in a resulting system or combination in a red blood cell lysing reagent. Such a lysing reagent must be able to lyse the red cells quickly, destroying the red cell membrane to a particle size sufficient-ly low that white cells can be sensed and counted, for example.
Simultaneously, the lysing agent must release hemoglobin from the red cells, and the hemoglobin converted to cyanmethemoglobin so as to enable colorimetric determinations to proceed. The combination of the aforementioned two agents provided extremely active antibacterial and antifungal functions, particularly when combined with a quaternary ammonium compound. The use of sodium fluoride maintained the fluoride concentration and also aided in the conversion of hemoglobin to the cyanmethemoglobin.
The utilization of colorimetric hemoglobin de-terminations requires a calibrator solution which is utilized for calibrating the colorimeter. Specifically, a calibrator solution is prepared from fresh saline washed human red blood 2~ cells which are lysed to destroy the red blood cell walls.
After complete lysis of the cells, the released hemoglobin is converted to cyanmethemoglobin and diluted to the required concentration for use in colorimetric determinations. One problem arising is the rediness of the hemoglobin solution to grow bacteria, particularly bacteria of the type whose growth is encouraged in the presence of the cyanide ion, the same being required for the formation of cyanmethemoglobin.
107~;008 2-Phenoxyethanol and sodium fluoride can be utilized, the 2-phenoxyethanol serving as a very active bacteriostatic agent, particularly, in the presence of a quaternary ammonium compound.
- 5 Blood cells taken from humans or animals when suspended in a suitable medium or synthetic plasma are utilized as a "control" or "calibrator" in the operation of blood cell counting instruments. The blood cells are obtained from humans, clotting being prevented by employing anti- O
coagulants. Plasma is removed by centrifugation and the red cells washed under sterile conditions with sterile saline solution, the cells being later packed down for subsequent suspension in a preserving and stabilizing medium. Accordingly, such a medium as an artificial plasma herein is provided which is formulated for adding to such separated red blood cells. The medium includes a diluent such as described in Canadian Patent 1,045,960 and further includes bovine albumin, lactose, and additional 2-phenoxyethanol. The solution is mixed thoroughly with the osmolality adjusted with sodium chloride to approxi-mately 370 milliosmoles per kilogram water. After adjusting the osmolality, the solution is filtered and the filtrate stored in sterile glass bottles. Sodium fluoride is present in the solution functioning as a cell volume stabilizer. The addition of the bovine albumin and the lactose has been made to provide further physical-chemical balance of the passage ~ 7 --of water across the cell membrane to the inside of the cell.
2-Phenoxyethanol is used as a bacteriostatic and fungistatic agent. The medium or solution then can be added to washed blood cells as required to produce the desired count.
The use of the 2-phenoxyethanol and sodium fluoride agents as a blanking reagent in hematological determinations is contemplated.
Examples of preferred embodiments of the invention are as follows:
Approximate A. As a lysing reagent Unit Weight Example (1) Sodium chloride 7.936 grams/
li~er Potassium chloride 0.4 "
Sodium di-hydrogen phosphate 0.342 "
15 - Di-sodium phosphate 1.922 N
- Sodium Ethylenediaminetetracetic 0.3 "
acid (EDTA) - Sodium fluoride 0.5 "
2-Phenoxyethanol 3.3 "
Distilled Water quantity to produce one Quaternary Ammonium Compound, liter Bretol 10.0 grams/
liter Sodium Nitrite 0.20 "
Potassium Cyanide 0.6 "
Polyoxyethylated Alkylphenol 8.0 ml/liter Distilled water to 1000 ml Approximate B. As a blanking reagent Unit Weight (Example 12) Sodium chloride 7.847 grams/liter Potassium chloride 0.396 "
Mono sodium phosphate monohydrate 0.188 "
Di-sodium phosphate anydrons 2.071 "
Di-sodium EDTA, di-hydrate 0.376 "
2-Phenoxythanol 2.20 "
Sodium fluoride 0.495 "
Sodium nitroferricyanide 0.010 grams/liter Sodium nitrite 0.010 C.C.S.S. (Polyoxethylated Alkylphenol) General Analine 0.40 ml.
New York Film Bretol* (Fine Organics Co. New 0.500 grams/liter York) Distilled water to 1000 ml.
(1 gal.U.S. 3.785 Litre) - C. As a detergent cleaner having bacteriostatic, fungistatic and stabilizing characteristics Example (3) Sodium chloride 7.506 grams/liter Potassium chloride 0.379 "
Mono sodium phosphate mono-hydrate 0.180 n Di-sodium phosphate anydrons 1.846 grams/liter Di-sodium EDTA Di-hydrate 0.360 "
2-Phenoxyethanol (Dowanol*EPh) 2.10 "
Sodium fluoride 0.474 "
* Trade Mark _ g _ Approximate Unit Weight C.C.S.S.(Polyoxethylated Alkylphenol) 14.9 ml Lime Oil . 08 ml Green dye 0.56 ml Distilled water to 1000 ml (1 gal U.S. ~ 3.785 Litre) D. As a water bath additive Example (4) (a) As the Buffer system:
. 10 Triethanolamine Hydro-chloride 17.4 grams/
.liter Sodium Hydroxide,pellets 1.86 "
(b) As the anti-corrosion system and chelating agent:
Triethanolamine Hydro-- chloride (The amount indicated in ~a) above also functions for this purpose) Ethylenediamine tetracetic acid, tetrasodium salt4.00 n Sodium fluoride 20.00 "
(c) As the nonionic surfactant for cleaning (wetting agent) Pluronic*F-68, 20.00 "
(Manufactured by Wyn-dotte Chemicals Corp.) (d) pH indicator system:
Blue dye (French's Food 20.00 ml/
Dye) liter Bromo Cresol Purple 0.2 grams/
- liter Thymol Blue 0.4 "
(a), (b), (c) and (d) are mixed with enough distilled water to make 1000 ml and together when mixed added to the same volume (1000 ml) of 2-phenoxyethanol.
* Trade Mark i076008 The recommended volume for effective use is 1 volume of the resultant solution in 100 volumes distilled or deionized water.
During use, should there be a build-up of acid in the water bath, the following color changes take place as the pH falls:
pH Color Change 7.4 Definite blue 6.8 Greenish blue 6.5 Bluish green 6.0 Dark green 5.5 Light green E, As a control or calibrator solution;
Exa~ple (5~ ApproxL~ate Unit ~eight Sodium chloride 7,936 grams~liter - ~ Potassium chloride Q,4 ~' r Sodium di hydrogen phosphate Q,l9 Di~sodiu~ phosphate 1,922 n Sodium Ethylenediaminete-tracetic acid (EDTA) Q,3 Sodiu~ fluoride 0,5 2-phenpxyethan~l 3~3 ~' Distilled water quantity topro-duce one liter To 1000 ~1 of said solution is addedr Bovine albumin 3,4% by weight Lactose 4,3% ~
2-Phenoxyethanol to ~34%
Osmolality adjusted to approxi~ately 370 mosmols~kg water, ~076008 , .
The solutions specified above are adjusted to a pH of between 7 and 8, preferably, 7 2 - 7.5 by the suitable buffering agent, EDTA and phosphate salt. The osmolality of the solutions preferably is maintained at 320-340 milliosmoles. The concentration of sodium fluoride ranges fxom .03 to .1 percent by weight of total solution, with a preference of 0.05 percent, and the concentration of 2-phenoxyethanol ranges from 2 percent to 1.0 percent by weight of total solution, with a preference of ~34 percent in the calibrator or control solution, and 2 percent in the solutions used for other purposes.
The desirable characteristic of osmotic halance is procured through the judicious use of both sodium and potassium chloride. Buffering with use of sodium hydroxide is eliminated herein.
The use of osmotic balancing agents such as sodium chloride, potassium chloride and of buffering agents such as sodium hydroxide in such reagents is known. Also known ~as the use of a chelating agent which in cooperating with the phosphate salt employed served as buffering agents to achieve a desired pH range in which the diluent was operative. Such a chelating agent is ethylenediaminetetracetic acid (EDTA).
Preparation of the solutions do not require any special procedures or any special order of addition of ingredients to the water. Consequently, the invention does not concern any methodology in formulation of the diluent The mixture of ingredients is done mechanically by moderate stirring over a one to two hour period. The solution then is filtered through a 0.2 micron filter and storable in plastic containers directly.
. 1076008 ~ .~
Although preferred formulations have been specified above, the range of pH and osmolality may be broadened for useful purposes. Thus, the pH range may be maintained from between pH of 7.0 to 8Ø
Likewise, the useful range of osmolality can be between 300 to 380 milliosmoles. This can be accomplished by varying the amount of active ingredients.
It will be appreciated by the skilled artisan that the solutions described herein can be utilized in the same manner as previously available isotonic solution for the purposes described but avoids the adverse problems attendant in use of such prior solutions.
What I claim lS:
Sodium fluoride was employed to counterbalance the cell wall weakening activity of 2-phenoxyethanol and thus `
stabilize the mean cell volume of the red cells. Also, sodium fluoride served to aid in the conversion of hemoglobin to cyanmethemoglobin for the determination of hemoglobin colorimetrically.
The use of a bacteriostatic and fungistatic agent brings about heretofore undesirable variations in the mean cell volume of the red cells leading to the unstabilization of the mean red cell volume. In the desirability of utiliz-ing a bacteriostatic agent as a replacement for sodium azide,as explained in Canadian Patent 1,045,960, 2-phenoxyethanol was selected although it had never been employed heretofore in such capacity. However, the 2-phenoxyethanol may lead to the destruction of the cell walls or change of cell volume. In order to avoid the problems of hemolysis or crenation, a stabilizer was utilized in the resulting system. Although sodium fluoride had long been known to be a preservative, such as for preserving glucose in blood samples, its utility as a stabilizer with the resulting system was unexpected.
- 4 - `
~.~
SUMMARY OF THE INVENTION
The invention is directed to the combination of an ethoxylated phenol such as 2-phenoxyethanol and an alkali metal fluoride such as sodium fluoride as effective for use in other reagent solutions that are utilized for other purposes in hematoloqical analytical procedures, particularly those employing electronic particle study methods.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
I have discovered that 2-phenoxyethanol and sodium fluoride, could be utilized in a resulting system or combination in a red blood cell lysing reagent. Such a lysing reagent must be able to lyse the red cells quickly, destroying the red cell membrane to a particle size sufficient-ly low that white cells can be sensed and counted, for example.
Simultaneously, the lysing agent must release hemoglobin from the red cells, and the hemoglobin converted to cyanmethemoglobin so as to enable colorimetric determinations to proceed. The combination of the aforementioned two agents provided extremely active antibacterial and antifungal functions, particularly when combined with a quaternary ammonium compound. The use of sodium fluoride maintained the fluoride concentration and also aided in the conversion of hemoglobin to the cyanmethemoglobin.
The utilization of colorimetric hemoglobin de-terminations requires a calibrator solution which is utilized for calibrating the colorimeter. Specifically, a calibrator solution is prepared from fresh saline washed human red blood 2~ cells which are lysed to destroy the red blood cell walls.
After complete lysis of the cells, the released hemoglobin is converted to cyanmethemoglobin and diluted to the required concentration for use in colorimetric determinations. One problem arising is the rediness of the hemoglobin solution to grow bacteria, particularly bacteria of the type whose growth is encouraged in the presence of the cyanide ion, the same being required for the formation of cyanmethemoglobin.
107~;008 2-Phenoxyethanol and sodium fluoride can be utilized, the 2-phenoxyethanol serving as a very active bacteriostatic agent, particularly, in the presence of a quaternary ammonium compound.
- 5 Blood cells taken from humans or animals when suspended in a suitable medium or synthetic plasma are utilized as a "control" or "calibrator" in the operation of blood cell counting instruments. The blood cells are obtained from humans, clotting being prevented by employing anti- O
coagulants. Plasma is removed by centrifugation and the red cells washed under sterile conditions with sterile saline solution, the cells being later packed down for subsequent suspension in a preserving and stabilizing medium. Accordingly, such a medium as an artificial plasma herein is provided which is formulated for adding to such separated red blood cells. The medium includes a diluent such as described in Canadian Patent 1,045,960 and further includes bovine albumin, lactose, and additional 2-phenoxyethanol. The solution is mixed thoroughly with the osmolality adjusted with sodium chloride to approxi-mately 370 milliosmoles per kilogram water. After adjusting the osmolality, the solution is filtered and the filtrate stored in sterile glass bottles. Sodium fluoride is present in the solution functioning as a cell volume stabilizer. The addition of the bovine albumin and the lactose has been made to provide further physical-chemical balance of the passage ~ 7 --of water across the cell membrane to the inside of the cell.
2-Phenoxyethanol is used as a bacteriostatic and fungistatic agent. The medium or solution then can be added to washed blood cells as required to produce the desired count.
The use of the 2-phenoxyethanol and sodium fluoride agents as a blanking reagent in hematological determinations is contemplated.
Examples of preferred embodiments of the invention are as follows:
Approximate A. As a lysing reagent Unit Weight Example (1) Sodium chloride 7.936 grams/
li~er Potassium chloride 0.4 "
Sodium di-hydrogen phosphate 0.342 "
15 - Di-sodium phosphate 1.922 N
- Sodium Ethylenediaminetetracetic 0.3 "
acid (EDTA) - Sodium fluoride 0.5 "
2-Phenoxyethanol 3.3 "
Distilled Water quantity to produce one Quaternary Ammonium Compound, liter Bretol 10.0 grams/
liter Sodium Nitrite 0.20 "
Potassium Cyanide 0.6 "
Polyoxyethylated Alkylphenol 8.0 ml/liter Distilled water to 1000 ml Approximate B. As a blanking reagent Unit Weight (Example 12) Sodium chloride 7.847 grams/liter Potassium chloride 0.396 "
Mono sodium phosphate monohydrate 0.188 "
Di-sodium phosphate anydrons 2.071 "
Di-sodium EDTA, di-hydrate 0.376 "
2-Phenoxythanol 2.20 "
Sodium fluoride 0.495 "
Sodium nitroferricyanide 0.010 grams/liter Sodium nitrite 0.010 C.C.S.S. (Polyoxethylated Alkylphenol) General Analine 0.40 ml.
New York Film Bretol* (Fine Organics Co. New 0.500 grams/liter York) Distilled water to 1000 ml.
(1 gal.U.S. 3.785 Litre) - C. As a detergent cleaner having bacteriostatic, fungistatic and stabilizing characteristics Example (3) Sodium chloride 7.506 grams/liter Potassium chloride 0.379 "
Mono sodium phosphate mono-hydrate 0.180 n Di-sodium phosphate anydrons 1.846 grams/liter Di-sodium EDTA Di-hydrate 0.360 "
2-Phenoxyethanol (Dowanol*EPh) 2.10 "
Sodium fluoride 0.474 "
* Trade Mark _ g _ Approximate Unit Weight C.C.S.S.(Polyoxethylated Alkylphenol) 14.9 ml Lime Oil . 08 ml Green dye 0.56 ml Distilled water to 1000 ml (1 gal U.S. ~ 3.785 Litre) D. As a water bath additive Example (4) (a) As the Buffer system:
. 10 Triethanolamine Hydro-chloride 17.4 grams/
.liter Sodium Hydroxide,pellets 1.86 "
(b) As the anti-corrosion system and chelating agent:
Triethanolamine Hydro-- chloride (The amount indicated in ~a) above also functions for this purpose) Ethylenediamine tetracetic acid, tetrasodium salt4.00 n Sodium fluoride 20.00 "
(c) As the nonionic surfactant for cleaning (wetting agent) Pluronic*F-68, 20.00 "
(Manufactured by Wyn-dotte Chemicals Corp.) (d) pH indicator system:
Blue dye (French's Food 20.00 ml/
Dye) liter Bromo Cresol Purple 0.2 grams/
- liter Thymol Blue 0.4 "
(a), (b), (c) and (d) are mixed with enough distilled water to make 1000 ml and together when mixed added to the same volume (1000 ml) of 2-phenoxyethanol.
* Trade Mark i076008 The recommended volume for effective use is 1 volume of the resultant solution in 100 volumes distilled or deionized water.
During use, should there be a build-up of acid in the water bath, the following color changes take place as the pH falls:
pH Color Change 7.4 Definite blue 6.8 Greenish blue 6.5 Bluish green 6.0 Dark green 5.5 Light green E, As a control or calibrator solution;
Exa~ple (5~ ApproxL~ate Unit ~eight Sodium chloride 7,936 grams~liter - ~ Potassium chloride Q,4 ~' r Sodium di hydrogen phosphate Q,l9 Di~sodiu~ phosphate 1,922 n Sodium Ethylenediaminete-tracetic acid (EDTA) Q,3 Sodiu~ fluoride 0,5 2-phenpxyethan~l 3~3 ~' Distilled water quantity topro-duce one liter To 1000 ~1 of said solution is addedr Bovine albumin 3,4% by weight Lactose 4,3% ~
2-Phenoxyethanol to ~34%
Osmolality adjusted to approxi~ately 370 mosmols~kg water, ~076008 , .
The solutions specified above are adjusted to a pH of between 7 and 8, preferably, 7 2 - 7.5 by the suitable buffering agent, EDTA and phosphate salt. The osmolality of the solutions preferably is maintained at 320-340 milliosmoles. The concentration of sodium fluoride ranges fxom .03 to .1 percent by weight of total solution, with a preference of 0.05 percent, and the concentration of 2-phenoxyethanol ranges from 2 percent to 1.0 percent by weight of total solution, with a preference of ~34 percent in the calibrator or control solution, and 2 percent in the solutions used for other purposes.
The desirable characteristic of osmotic halance is procured through the judicious use of both sodium and potassium chloride. Buffering with use of sodium hydroxide is eliminated herein.
The use of osmotic balancing agents such as sodium chloride, potassium chloride and of buffering agents such as sodium hydroxide in such reagents is known. Also known ~as the use of a chelating agent which in cooperating with the phosphate salt employed served as buffering agents to achieve a desired pH range in which the diluent was operative. Such a chelating agent is ethylenediaminetetracetic acid (EDTA).
Preparation of the solutions do not require any special procedures or any special order of addition of ingredients to the water. Consequently, the invention does not concern any methodology in formulation of the diluent The mixture of ingredients is done mechanically by moderate stirring over a one to two hour period. The solution then is filtered through a 0.2 micron filter and storable in plastic containers directly.
. 1076008 ~ .~
Although preferred formulations have been specified above, the range of pH and osmolality may be broadened for useful purposes. Thus, the pH range may be maintained from between pH of 7.0 to 8Ø
Likewise, the useful range of osmolality can be between 300 to 380 milliosmoles. This can be accomplished by varying the amount of active ingredients.
It will be appreciated by the skilled artisan that the solutions described herein can be utilized in the same manner as previously available isotonic solution for the purposes described but avoids the adverse problems attendant in use of such prior solutions.
What I claim lS:
Claims (10)
1. An isotonic, asmotically balanced aqueous solution for use in or in connection with hematological determinations, which comprises:
(i) sodium chloride, potassium chloride and one or more sodium phosphates, in osmotically balanced proportions, (ii) an alkali metal fluoride, (iii) 2-phenoxyethanol, (iv) ethylenediaminetetraacetic acid or an alkali metal salt thereof, and one or more of the following:
(v) a polyoxethylated alkylphenol, (vi) a quaternary ammonium compound, potassium cyanide and sodium nitrite, and (vii) bovine serum albumin and lactose.
(i) sodium chloride, potassium chloride and one or more sodium phosphates, in osmotically balanced proportions, (ii) an alkali metal fluoride, (iii) 2-phenoxyethanol, (iv) ethylenediaminetetraacetic acid or an alkali metal salt thereof, and one or more of the following:
(v) a polyoxethylated alkylphenol, (vi) a quaternary ammonium compound, potassium cyanide and sodium nitrite, and (vii) bovine serum albumin and lactose.
2. A solution as defined in claim 1, in which the alkali metal fluoride is sodium fluoride.
3. A solution as defined in claim 1 wherein the alkali metal fluoride is present in a concentration of from 0.03 to 0.1% by weight.
4. A solution as defined in claim 1 wherein the alkali metal fluoride is sodium fluoride present in a concentration of from 0.03 to 0.1% by weight.
5. A solution as defined in any one of claims 1, 2 or 3 wherein the 2-phenoxyethanol is present in a concentration of from 0.2 to 1.0% by weight.
6. A solution as defined in claim 4 wherein the 2-phenoxyethanol is present in a concentration of from 0.2 to 1.0% by weight.
7. A solution as defined in any one of claims 1 to 3 wherein component (i) comprises sodium chloride, potassium chloride, sodium dihydrogen phosphate and disodium phosphate, in osmotically balanced proportions.
8. A solution as defined in claim 4 wherein component (i) comprises sodium chloride, potassium chloride, sodium dihydrogen phosphate and disodium phosphate, in osmotically balanced proportions.
9. A solution as defined in any one of claims 1, 2 or 3 which comprises components (i) to (iv) and component (v), and which also comprises a dispersed dye.
10. A solution as claimed in any one of claims 1, 2 or 3 having a pH of from 7.0 to 8.0 and an osmolality of from 300 to 380 milliosmoles.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/670,924 US4102810A (en) | 1975-01-13 | 1976-03-26 | Stabilized hematological reagent solutions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1076008A true CA1076008A (en) | 1980-04-22 |
Family
ID=24692448
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA265,047A Expired CA1076008A (en) | 1976-03-26 | 1976-11-05 | Stabilized hematological reagent solutions |
Country Status (18)
| Country | Link |
|---|---|
| JP (1) | JPS52117434A (en) |
| AU (1) | AU511980B2 (en) |
| BE (1) | BE849701A (en) |
| CA (1) | CA1076008A (en) |
| CH (1) | CH622619A5 (en) |
| DE (1) | DE2709576A1 (en) |
| DK (1) | DK62977A (en) |
| FI (1) | FI770587A7 (en) |
| FR (1) | FR2345164A1 (en) |
| GB (1) | GB1572509A (en) |
| HK (1) | HK90985A (en) |
| IL (1) | IL50858A (en) |
| IT (1) | IT1066691B (en) |
| MX (1) | MX3977E (en) |
| NL (1) | NL7613738A (en) |
| NO (1) | NO770897L (en) |
| SE (1) | SE7701509L (en) |
| ZA (1) | ZA771549B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4132598A (en) * | 1977-06-03 | 1979-01-02 | Modrovich Ivan Endre | Stabilized liquid phosphate containing diagnostic compositions and method of preparing same |
| 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 |
| US4506018A (en) * | 1982-12-30 | 1985-03-19 | Becton, Dickinson And Company | Blood diluent |
| DE4022878A1 (en) * | 1990-07-18 | 1992-01-23 | Boehringer Mannheim Gmbh | PRESERVATION OF DIAGNOSTIC TESTS |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR1493772A (en) * | 1965-07-28 | 1967-09-01 | Organon Nv | Method for the treatment of erythrocytes |
| 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 |
-
1976
- 1976-11-05 IL IL50858A patent/IL50858A/en unknown
- 1976-11-05 CA CA265,047A patent/CA1076008A/en not_active Expired
- 1976-11-15 IT IT52196/76A patent/IT1066691B/en active
- 1976-11-22 AU AU19871/76A patent/AU511980B2/en not_active Expired
- 1976-11-23 GB GB48832/76A patent/GB1572509A/en not_active Expired
- 1976-11-30 MX MX765179U patent/MX3977E/en unknown
- 1976-12-09 JP JP14720976A patent/JPS52117434A/en active Pending
- 1976-12-10 NL NL7613738A patent/NL7613738A/en not_active Application Discontinuation
- 1976-12-21 BE BE6045810A patent/BE849701A/en unknown
- 1976-12-31 FR FR7639783A patent/FR2345164A1/en active Granted
-
1977
- 1977-02-10 SE SE7701509A patent/SE7701509L/en not_active Application Discontinuation
- 1977-02-14 DK DK62977A patent/DK62977A/en not_active Application Discontinuation
- 1977-02-23 FI FI770587A patent/FI770587A7/fi not_active Application Discontinuation
- 1977-03-04 DE DE19772709576 patent/DE2709576A1/en not_active Ceased
- 1977-03-14 NO NO770897A patent/NO770897L/en unknown
- 1977-03-15 ZA ZA00771549A patent/ZA771549B/en unknown
- 1977-03-25 CH CH391377A patent/CH622619A5/en not_active IP Right Cessation
-
1985
- 1985-11-14 HK HK909/85A patent/HK90985A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IL50858A0 (en) | 1977-01-31 |
| AU1987176A (en) | 1978-06-01 |
| IT1066691B (en) | 1985-03-12 |
| FR2345164A1 (en) | 1977-10-21 |
| BE849701A (en) | 1977-04-15 |
| ZA771549B (en) | 1978-10-25 |
| IL50858A (en) | 1979-07-25 |
| NL7613738A (en) | 1977-09-28 |
| SE7701509L (en) | 1977-09-27 |
| DK62977A (en) | 1977-09-27 |
| NO770897L (en) | 1977-09-27 |
| AU511980B2 (en) | 1980-09-18 |
| CH622619A5 (en) | 1981-04-15 |
| FI770587A7 (en) | 1977-09-27 |
| GB1572509A (en) | 1980-07-30 |
| DE2709576A1 (en) | 1977-10-06 |
| JPS52117434A (en) | 1977-10-01 |
| HK90985A (en) | 1985-11-22 |
| MX3977E (en) | 1981-10-20 |
| FR2345164B1 (en) | 1982-11-05 |
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