BACKGROUND OF THE DISCLOSURE

Field of the Disclosure

Environmentally-friendly, aqueous green concentrated sample reagent compositions are provided for use in suitable hematology analyzers for analyzing blood cells including for enumeration and sizing of blood cells, determination of hemoglobin parameters and differentiation of leukocyte subpopulations in a single blood cell sample.

Description of the Related Art

One problem with some hematology analyzers is the need to frequently replenish the sample diluent container. For example, when the sample diluent is manufactured and sold at working concentration, an ultra-high volume lab might need to make several changes in a high volume hospital in a single day of work. The storage space and the laboratory foot print are limited in many clinical labs making it challenging to keep large quantities of diluent at hand and/or store used containers until they can be removed from the lab. Another problem is that certain prior art blood sample diluents may form up to 150 ppm formaldehyde in the waste stream which may cause environmental concerns.

Attempts to simply prepare more concentrated forms of prior art sample diluents have been hampered by maximum concentration factors. For example, one commercially available blood diluent composition for use with certain hematology analyzers was found to have a maximum of 6× concentration factor because of limited component solubility.

In many cases diluents designed for different hematology analyzers may not be used interchangeably due to different hardware, chemistry volumes, reaction times, and software algorithms.

U.S. Pat. No. 5,935,857 discloses an isotonic blood diluent for use in blood cell analysis, the diluent containing EDTA, imidazole, alkaline metal chloride, alkaline metal sulfate and an antimicrobial.

U.S. Pat. No. 6,706,526 discloses a low formaldehyde aqueous blood diluent containing disodium EDTA, sodium chloride, sodium sulfate, procaine, N-(2-acetoamido)iminodiacetic acid, and various low formaldehyde producing antimicrobials.

U.S. Pat. No. 5,834,315 discloses a lytic reagent comprising a quaternary ammonium salt and/or a pyridinium salt in an amount effective to lyse erythrocytes for determining total hemoglobin in blood.

Stable aqueous concentrated reagents that exhibit low formaldehyde formation, both as manufactured and over time in waste stream effluent, are desirable, particularly for use as sample diluents in hematology analyzer instrumentation.

SUMMARY OF THE INVENTION

Environmentally-friendly, low-formaldehyde aqueous green concentrated aqueous reagent compositions and methods are provided.

An aqueous green concentrated sample reagent composition is provided which may be used, for example, as a body fluid sample diluent for use in a hematology analyzer or cellular analyzer, the concentrated reagent composition comprising a chelating agent, a hemoglobin ligand, a stabilizing agent, a buffer, and an antimicrobial agent.

An aqueous green concentrated sample reagent composition is provided which may be used, for example, as a body fluid sample diluent for use in a hematology analyzer or cellular analyzer, the concentrated reagent composition comprising a chelating agent, a hemoglobin ligand, a stabilizing agent, a buffer, and an antimicrobial agent, wherein the concentrated reagent composition remains clear and colorless in from a 10-fold to a 20-fold concentration factor compared to a working concentration reagent composition, when stored at a temperature from 2-30° C., for at least one year from date of manufacture. The aqueous green concentrated reagent composition comprises or produces less than about 2 parts per million (ppm) of formaldehyde over a period of time of at least 1 year when stored at ambient temperature. The aqueous green concentrated reagents may improve the efficiency of hematology analyzers by reducing storage requirements and shipping costs, and reducing environmental impact.

The aqueous green concentrated reagent compositions may include a chelating agent, for example, an EDTA sodium salt such as tetrasodium EDTA.

The aqueous green concentrated reagent compositions may include a hemoglobin ligand selected from the group consisting of imidazole, phenylimidazole, methylimidazole, ethylimidazole, and butylimidazole. The aqueous green concentrated reagent composition may include imidazole as the hemoglobin ligand.

The aqueous green concentrated reagent compositions may include one or more stabilizing agents selected from alkaline metal sulfates and/or alkaline metal halides, for example, alkaline metal sulfate stabilizing agents such as sodium sulfate and/or potassium sulfate, for example, comprising sodium sulfate and potassium sulfate in a weight ratio of from 0.5:1 to 3:1; or from 1:1 to 3:1.

The aqueous green concentrated reagent compositions may be diluted with water to provide a working concentration reagent composition, which may include from about 25 to about 75 mmol total stabilizing agent, or from about 30 to about 60 mmol total stabilizing agent.

The aqueous green concentrated reagent compositions may comprise a working concentration reagent composition from 25 to 75 mmol total sulfate ion.

The aqueous green concentrated reagent compositions may include a buffer comprising one or more of potassium sodium tartrate, potassium hydrogen phosphate, sodium hydrogen phosphate, or a hydrate thereof.

The aqueous green concentrated reagent compositions may include one or more antimicrobial agents selected from the group consisting of 5-fluorouracil, 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), 5-bromo-5-nitro-1,3-dioxane (Bronidox L), polyaminopropyl biguanide (Cosmocil CQ®), 1,2-benzisothiazolin-3-one (BIT), 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), 2-n-octyl-4-isothiazolin-3-one (OIT), ortho-phthaldialdehyde (OPA), and 1,2 dibromo-2,4 dicyanobutane (Bromothalonil).

The aqueous green concentrated reagent compositions may include a combination of antimicrobial agents including 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), 5-fluorouracil, and 5-bromo-5-nitro-1,3-dioxane.

The aqueous green concentrated reagent compositions may include one or more of an alkaline metal chloride (e.g., NaCl, KCl), a platelet stabilizing agent (e.g, procaine, tetracaine), and an osmotic stabilizing agent (e.g., glycerol).

The aqueous green concentrated reagent compositions may include an alkaline metal chloride selected from the group consisting of NaCl and KCl.

The aqueous green concentrated reagent compositions may include a platelet stabilizing agent selected from the group consisting of procaine and tetracaine.

The aqueous green concentrated reagent compositions may include glycerol as an osmotic stabilizing agent.

The aqueous green concentrated reagent compositions may be diluted with water by a dilution factor of 10-fold to 20-fold to form a working concentration reagent composition, such that the working concentration reagent compositions exhibit each of: i) pH in the range of from pH 6.90 to 7.30 at ambient temperature; ii) conductivity in the range of from 19 to 20 mS/cm at ambient temperature; and iii) osmolality in the range of from 315 to 360 mOsm/Kg at ambient temperature.

An aqueous green concentrated reagent composition is provided comprising 10 to 100 g/L tetrasodium EDTA; 20 to 50 g/L imidazole; 30 to 90 g/L sodium sulfate; 20 to 60 g/L potassium sulfate; 60 to 300 g/L sodium potassium tartrate; and an antimicrobial agent selected from the group consisting of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), 5-bromo-5-nitro-1,3-dioxane (Bronidox L), and 5-fluorouracil.

An aqueous green concentrated reagent composition is provided comprising 10 to 100 g/L tetrasodium EDTA; 10 to 50 g/L imidazole; 20 to 100 g/L sodium sulfate; 15 to 60 g/L potassium sulfate; 60 to 300 g/L sodium potassium tartrate; and an antimicrobial agent selected from the group consisting of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), 5-bromo-5-nitro-1,3-dioxane (Bronidox L), and 5-fluorouracil.

An aqueous green concentrated reagent composition is provided comprising 40 to 60 g/L tetrasodium EDTA; 20 to 45 g/L imidazole; 60 to 85 g/L sodium sulfate; 20 to 40 g/L potassium sulfate; 80 to 150 g/L sodium potassium tartrate, optionally further comprising one or more of 40 to 60 g/L sodium phosphate monobasic, 20 to 60 g/L sodium chloride, and 5 to 15 g/L 5-fluorouracil.

An aqueous green concentrated reagent composition is provided comprising 10 to 65 g/L tetrasodium EDTA; 15 to 50 g/L imidazole; 40 to 100 g/L sodium sulfate; 15 to 50 g/L potassium sulfate; and 80 to 180 g/L sodium potassium tartrate.

The aqueous green concentrated reagent compositions may include one or more antimicrobial agents selected from the group consisting of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), 5-bromo-5-nitro-1,3-dioxane (Bronidox L), and 5-fluorouracil.

A method is provided for analyzing a body fluid comprising: a) diluting an aqueous green concentrated reagent composition according to the disclosure from 10-fold to 20-fold to form a working concentration reagent composition; b) mixing a body fluid with the working reagent composition to form a diluted body fluid sample.

A method is provided for analyzing a blood sample containing blood cells comprising: a) diluting an aqueous green concentrated reagent composition according to the disclosure from 10-fold to 20-fold to form a working concentration reagent composition; b) mixing a blood sample containing blood cells with the working reagent composition to form a diluted blood sample, wherein the chelating agent and the hemoglobin ligand are in an amount effective to provide reproducible hemoglobin and cell volume measurements over a temperature range from 15° C. to 32° C.; and c) analyzing said diluted blood sample to determine a physical parameter of said blood cells.

A method is provided for analyzing a blood sample containing blood cells comprising: a) diluting an aqueous green concentrated reagent composition according to the disclosure from 10-fold to 20-fold to form a working concentration reagent composition; b) mixing a blood sample containing blood cells with the working reagent composition to form a diluted blood sample, c) mixing a lytic reagent with said diluted blood sample to lyse red blood cells prior to analyzing said diluted blood sample, wherein the chelating agent and the hemoglobin ligand are in an amount effective to provide reproducible hemoglobin and cell volume measurements over a temperature range from 15° C. to 32° C.; and d) analyzing said diluted blood sample with added lytic reagent to determine a physical parameter of said blood cells.

A method is provided for analyzing a blood sample containing blood cells comprising: a) diluting an aqueous green concentrated reagent composition according to the disclosure from 10-fold to 20-fold to form a working concentration reagent composition; b) mixing a blood sample containing blood cells with the working reagent composition to form a diluted blood sample, c) adding a lyse reagent to lyse the RBC releasing hemoglobin, wherein the chelating agent and the hemoglobin ligand are in an amount effective to provide reproducible hemoglobin and cell volume measurements over a temperature range from 23° C. to 32° C.; and d) analyzing said diluted blood sample with added lyse reagent to determine a physical parameter of said blood WBC cells and hemoglobin concentration on an automatic hematology analyzer.

In the methods, the blood cells may comprise red blood cells.

In the methods, analyzing a diluted blood sample to determine at least one physical parameter may comprise analyzing to determine mean cell volume of said red blood cells.

In the methods, the blood cells may comprise white blood cells.

In the methods, analyzing a diluted blood sample to determine at least one physical parameter of blood cells may comprise an automated differential analysis of white blood cells to determine at least three subpopulations of white blood cells.

In the methods, analyzing a diluted blood sample to determine at least one physical parameter of blood cells may comprise an automated differential analysis of white blood cells to determine at least five subpopulations of white blood cells.

In the methods, the lytic reagent may comprise an aqueous solution of at least one quaternary ammonium salt.

In the methods, the analyzing a diluted blood sample to determine at least one physical parameter of blood cells may comprise analyzing to determine the number of platelets.

In the methods, the analyzing a diluted blood sample to determine at least one physical parameter of blood cells may comprise analyzing to determine hemoglobin content.

