Reagent And Method For Determining Leukocytes And Hemoglobin In The Blood

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.

Description

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 stromatolysing 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 chromogenforming reagents have been used in automated high speed hematology equipment. However, these reagents have been found to be unsatisfactory 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 Americam 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 hemoglobin embodies the use of Drabkin's reagent, which contains potassium ferricyanide, 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 methemoglobin. 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 instruments. 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 alkyl groups and one long chain alkyl group, and said ions being present in amounts sufficient to stromatolyse erythrocyte and platelet cells in the blood and to convert hemoglobin to a chromogen for 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 provides 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 general, 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.sub.1, R.sub.2, and R.sub.3, and the long chain alkyl group will be referred to as R.sub.4. Generally speaking, R.sub.1, R.sub.2, and/or R.sub.3 may have from 1 to 4 carbon atoms, as represented by methyl, ethyl, propyl, and butyl radicals. R.sub.4 may 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 percent, preferably about 1-5 percent, 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 amomonium 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.sub.4 carbon atoms, the hemolytic activity increases. Thus, for example, where R.sub.1, R.sub.2 and R.sub.3 are methyl, R.sub.4 may include from about 10 to 20 carbon atoms (C.sub.10 to C.sub.20) 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 percent and where R.sub.1, R.sub.2 and R.sub.3 are methyl, R.sub.4 should have at least about 14 carbon atoms to provide the necessary hemolytic activity. At a concentration of about 10 percent and where R.sub.1, R.sub.2 and R.sub.3 are methyl, R.sub.4 should have a maximum of about 16 carbon atoms, to provide the necessary solubility for the compound. As additional examples, where R.sub.4 is tetradecyl, R.sub.1, R.sub.2, and R.sub.3 may be methyl, ethyl, or propyl radicals at 0.5 to 10 percent concentration, while providing sufficient hemolytic activity and a complete solution. Where R.sub.4 is tetradecyl and R.sub.1 is methyl, R.sub.2 is methyl or ethyl, and R.sub.3 is butyl, the hemolytic activity is sufficient and the compound is soluble at 0.5 to 5 percent, but the compound is incompletely soluble at a concentration of 10 percent.

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.sub.4 is decyl, dodecyl, or tetradecyl, and R.sub.1, R.sub.2, and R.sub.3 are methyl, a solution of 5 percent concentration may be frozen and thawed with resulting formation of less than 200 particles in 0.5 ml., as determined using the Coulter Counter F.sub.N at red blood cell threshold settings. Where R.sub.4 is tetradecyl and R.sub.1, R.sub.2, and R.sub.3 are from methyl to propyl, a solution of 5 percent concentration may be frozen and thawed with resulting particle count of less than 200. On the other hand, where R.sub.4 is from C.sub.16 to C.sub.20, and R.sub.1, R.sub.2 and R.sub.3 are methyl, the particle count exceeds 200 after freezing and thawing a solution of 5% concentration. Likewise, where R.sub.4 is tetradecyl, R.sub.1 is methyl, R.sub.2 is methyl or ethyl, and R.sub.3 is butyl, the particle count exceeds 200 after freezing and thawing a solution of 5% concentration. Accordingly, it is preferred that R.sub.1, R.sub.2 and R.sub.3 each have from 1 to 3 carbon atoms, and that R.sub.4 have from 10 to 14 carbon atoms.

The concentration of cyanide ion preferably is selected to provide at least about 89 percent 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 (0.00385M) 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.

Claims

What is claimed is:

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 about 0.5 to 10 percent 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 percent 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 proportions:

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.