Reagent for measurement of leukocytes and hemoglobin in blood

Abstract: A reagent, satisfying the undermentioned conditions, for counting the number of leukocytes and measuring the hemoglobin concentration in the blood, said reagent not containing cyanides and being able to maintain hemoglobin in a stable state for a prolonged time but contains at least one cationic surfactant selected from the group consisting of a quaternary ammonium salt of a concentration capable of hemolyzing erythrocytes in the blood; oxidizing hemoglobin and a composition of pyridinium salts; at least one selected from the group consisting of cationic, nonionic and amphoteric surfactants; and at least one hemoglobin stabilizer is disclosed.

Description: This invention relates to a reagent for measuring leukocytes and hemoglobin in blood samples. Measurement of hemoglobin concentration and the number of leukocytes in a blood sample is crucial for clinical diagnoses of leukemia, anemia and other hematopathys. The basic method used for measuring or counting the number of leukocytes is a visual counting method with the aid of a microscope. However, with the visual counting method, the erythrocytes are hemolyzed by, for example, a Türk solution, while the leukocytes are stained and the number of leukocytes is counted one by one on a counting plate, which involves a time consuming and labor-intensive operation. Meanwhile, in counting the number of leukocytes in a blood sample, methods employing an automatic blood analyzer are frequently employed. In counting the number of leukocytes by means of an automatic blood analyzer, an erythrocytolytic agent including a surfactant is added to a blood sample for selective hemolyzation of erythrocytes in the blood sample to produce a sample for leukocyte measurement in which only leukocytes remain. This sample is passed through narrow paths or orifices provided in a detection section in an automatic blood analyzer, whereby electrical or optical signals are generated at the detection section, the number of these signals being a measure of the number of leukocytes. Recently, there has also evolved an apparatus in which the deformation which leukocytes undergo is reduced to a minimum through the use of improved erythrocytolytic agents and in which, during counting of the number of leukocytes, the leukocytes are classified into granulocytes, monocytes, lymphocytes and so on depending upon the difference in their signal intensities. With the use of this automatic blood analyzer, the leukocytes can be classified and counted far more easily than with the visual counting method. On the other hand, measurement of hemoglobin concentration is usually conducted by means of a cyanmethemoglobin method. With this method, an erythrocytolytic agent containing a nonionic surfactant or the like is added to the blood sample to elute hemoglobin contained in erythrocytes, the eluted hemoglobin is oxidize by the action of an oxidizing agent, such as potassium ferricyanate, to produce methemoglobin and ultimately the cyanide ions are added to methemoglobin for converting methemoglobin into cyanmethemoglobin (HiCN) to produce a stable hemoglobin measurement sample. The hemoglobin concentration of the produced sample is measured by measuring the absorbance at a predetermined wavelength of the sample. This method is accepted worldwide as the standard method. Although the sample prepared in this manner is an extremely stable substance, the hemoglobin is oxidized by using an oxidizing agent and it takes slightly longer than 10 minutes before oxidation is terminated. For this reason, with the automatic blood analyzer, the composition of the erythrocytolytic agent is improved to shorten the time necessary for oxidation, so that the reaction may be terminated in about 1 minute to enable hemoglobin measurement. In addition, it is possible to measure the number of leukocytes simultaneously. However, since toxic cyanides are used with the cyanmethemoglobin method reagent handling may endanger the operator. In addition, after measurement the waste liquor must be disposed of after cyanide decomposition with the use of, for example, sodium hypochlorite, entailing an extremely laborious operation. For this reason, there is also employed a method, known as an oxyhemoglobin method, whereby erythrocytes are hemolyzed only with nonionic surfactants for converting hemoglobin into oxyhemoglobin (HbO₂), without oxidizing hemoglobin to methemoglobin, for measuring the absorbance at a predetermined wavelength. With the oxyhemoglobin method, since cyanides are not used, there is no risk in handling the reagents, while there is no necessity of performing a troublesome operation of disposing of the waste liquor. However, with the oxyhemoglobin method, a problem is raised that a blood sample having a high methemoglobin content cannot be measured accurately, since methemoglobin then may not be converted to oxyhemoglobin. As an example, Table 1 shows comparison data of the measured results obtained with the Example 1 of the present invention and the oxyhemoglobin method for cases wherein the methemoglobin contents are changed with the use of control blood. The control blood, which is used as a substance for controlling the analytical accuracy of the automatic blood analyzer is usually stored in a cooled state, and may exhibit a stable hemoglobin concentration for a prolonged time. However, during storage at higher than ambient temperature, hemoglobin in the blood is gradually converted into methemoglobin. Therefore, when the control blood stored at 22°C is measured by the oxyhemoglobin method, as shown in Table 1, that portion of hemoglobin which has been converted into methemoglobin is unable to be measured such that the measured value of hemoglobin becomes gradually lower than the initial value over several days. As an example, Table 2 shows comparison data of measured results obtained with the Example 10 of the present invention and the oxyhemoglobin method for various values of the methemoglobin contents of the blood samples. With the oxyhemoglobin method, the hemoglobin concentration is lowered with an increase in the methemoglobin content. As a solution to this problem, a reagent for hemoglobin measurement comprised of dodecyl sodium sulfate or equal amounts of lauryl sodium sulfate (SLS), an anionic surfactant, and Triton X-100, a nonionic surfactant, in a neutral buffer (pH, 7.2), is taught by O shiro et al in Clinical Biochemistry, vol. 15, 83 (1982). With this method, erythrocytes are hemolyzed by the action of SLS and Triton X-100 and the eluted hemoglobin is converted into methemoglobin by the action of SLS at the same time that it is converted into SLS hemoglobin. With this method, the hemoglobin concentration in the blood may be measured without being affected by methemoglobin, while there is no necessity of disposing of the waste liquor because of the absence of cyanide contents. However, it is not possible with this method to measure leukocytes simultaneously with hemoglobin measurement. The Japanese Patent Public Disclosure (KOKAI) No. 61-148369 (1986) entitled "CYANIDE-FREE HEMOGLOBIN REAGENT" discloses a reagent for hemoglobin measurement with the aid of an ionic surfactant. However, it is similarly not possible with this reagent to measure the leukocytes simultaneously with the hemoglobin. As discussed hereinabove, it is not possible with the prior-art technology to measure hemoglobin and leukocytes simultaneously by a cyanide-free reagent or to make a correct measurement of hemoglobin in a blood sample with a high methemoglobin content. To solve the aforementioned problem, the present inventors have already proposed a reagent in Japanese Patent Application No. 1-275280 (1989). With this reagent, the types and concentrations of the cationic, nonionic or amphoteric surfactants used as the erythrocytolytic agents or the oxidants are strictly defined to provide for substantially instantaneous elution of hemoglobin and conversion thereof into methemoglobin to enable measurement of leukocytes simultaneously with hemoglobin. However, with this reagent, since there are no contents in the methemoglobin stabilizing component, a problem is raised that the hemoglobin concentration is changed slightly over a period of time after the preparation of the measurement samples, as shown in Comparative Example 4 of Table 3. This, however, is not detrimental to the effect of the invention of the aforementioned Japanese Patent Public Disclosure because the time which elapses since the sample preparation until the time of measurement is short and strictly defined with the fully automated system of sample preparation and measurement. There is also marketed a semi-automated system in which, for lowering the costs of the measurement system, sample preparation is made manually and only the measurement is performed by the system. With the semi-automatic system, the time which elapses since sample preparation until the time of measurement fluctuates in the range of several to several tens of minutes. For using the sample in the system, the hemoglobin concentration needs be stabilized at least for several tens of minutes after the time of sample preparation. WO-A-8 403 771 discloses a reagent for the differentiation of leukocytes which comprises an aqueous solution of quaternary ammonium salts and an additive which is at least one non-cationic surfactant, and wherein cyanide is added to the reagent in order to measure the concentration of hemoglobin. US-A-4 185 964 reveals a lysing agent for lysing erythrocytes being free from cyanides and comprising a quaternary ammonium salt and a polycarboxylic acid. The above reagent is used in a fully automated system. Finally, Chemical Abstracts, Vol. 102, No. 23, 1985, Abstr. No. 200716z, concerns a method for preventing the degradation of hemoglobin by preventing the release of iron from oxyhemoglobin, but does not refer to a technique for measurement of a concentration of hemoglobin. As discussed above, it is not possible with the prior-art technology to obtain a hemoglobin measurement sample which is cyanide-free and which will remain stable for a prolonged time and with which leukocytes may be measured simultaneously with hemoglobin and the hemoglobin contents in the blood sample with high methemoglobin contents may also be measured accurately. As a result of length research, however, the present inventors have arrived at the following reagent intended for the solution of the above-mentioned problems of the prior art. In accordance with the present invention, there is provided a reagent for use in counting the number of leukocytes and measuring the hemoglobin concentration in the blood, said reagent not containing cyanides and maintaining hemoglobin in a stable state for a prolonged time, comprising a solution with a pH ranging from 3.0 to 9.0 containing: i) at least one selected from the group consisting of quaternary ammonium salts and pyridinium salts of a concentration capable of hemolyzing erythrocytes in the blood and oxidizing hemoglobin, said quaternary ammonium salts having the formula: R₁: C₈ to C₂₀ alkyl, alkenyl or alkinyl group; R₂ to R₄: C₁ to C₈ alkyl, alkenyl or alkinyl group; X⁻: anionic group; the total concentration of said quaternary ammonium salts is in the range of from 0.1 to 15.0 g/l; and said pyridinium salts having the formula: n: an integer of from 7 to 19; X⁻: an anionic group; the total concentration of said pyridinium salts is in the range from 0.1 to 15.0 g/l; and ii) at least one selected from the group consisting of cationic, nonionic and amphoteric surfactants, said cationic surfactants having the formula: R₁: C₈ to C₂₀ alkyl, alkenyl or alkinyl group; R₂ to R₃: C₁ to C₈ alkyl, alkenyl or alkinyl group; X⁻: anionic group; the total concentration of said cationic surfactants is in the range of 0.1 to 15.0 g/l; and said nonionic surfactants having the formula: structure d R₁ - R₂ - (CH₂CH₂O) n - H R₁: C₈ to C₂₀ alkyl, alkenyl or alkinyl group; R₂: O, or COO; n: an integer of from 10 to 50; the total concentration of said nonionic surfactants is in the range of 0.1 to 15.0 g/l; and said amphoteric surfactants having the formula: R₁: C₈ to C₂₀ alkyl group; R₂ to R₃: C₁ to C₈ alkyl, alkenyl or alkinyl group the total concentration of said amphoteric surfactants is in the range of 0.1 to 15.0 g/l; and iii) at least one hemoglobin stabilizer selected from the group consisting of: (f) Tiron with a concentration of 0.1 to 1000 mg/ℓ: (g) 8-hydroxyquinoline with a concentration of 0.1 to 1000 mg/ℓ: (h) bipyridine with a concentration of 10 to 10000 mg/ℓ: (i) 1,10-phenanthroline and its derivatives with a concentration of 10 to 3000 mg/ℓ: R₁, R₄: -H, -CH₃; R₂, R₃: -H, phenyl group, -OH; (j) phenolic compounds with a concentration of 0.1 to 1000 mg/ℓ: R₁: -H, CH₂OH, -CHO, -COOH or -OH; R₂: -H, -COOH or -OH; R₃: -H or -COOH; (k) bisphenol A with a concentration of 0.1 to 1000 mg/ℓ: (l) pyrazole and its derivatives with a concentration of 0.1 to 10000 mg/ℓ: R₁, R₂, R₃: -H or C₁ to C₄ lower alkyl group; (m) phenyl 5-pyrazolone and its derivative with a concentration of 0.1 to 10000 mg/ℓ: R₁, R₂, R₃: -H or C₁ to C₄ lower alkyl group; (n) phenyl 3-pyrazolone with a concentration of 0.1 to 10000 mg/ℓ: (o) imidazole and its derivatives with a concentration of 1 to 20000 mg/ℓ: R₁, R₂: -H or C₁ to C₄ lower alkyl group; R₃: -H or C₁ to C₄ lower alkyl group or phenyl group. By suitably combining the components a to e in the reagent, leukocytes can be divided into two or three fractions, for example, into the fraction of lymphocytes, monocytes, eosinophilic leukocytes and basophilic leukocytes or neutrophilic leukocytes. According to a preferred embodiment, the reagent of the present invention contains at least two types of quaternary ammonium salts and a total concentration of said quaternary ammonium salts is in the range of 0.1 to 12.0 g/l, and said reagent is capable of dividing leucocytes into at least two fractions. According to a more preferred embodiment of the above reagent, said quaternary ammonium salts have the total concentration in the range of 2.2 to 5.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. Another embodiment of the inventive reagent contains at least one quaternary ammonium salt and a total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l, wherein it contains at least one nonionic surfactant and a total concentration of said nonionic surfactant or surfactants is in the range of 0.1 to 12.0 g/l, and said reaction is capable of dividing leukocytes into at least two fractions. According to a more preferred embodiment of the above said quaternary ammonium salt or salts have the total concentration in the range of 3.0 to 12.0 g/l, wherein said nonionic surfactant or surfactants have the total concentration in the range of 1.0 to 12.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. A further embodiment of the inventive reagent contains at least one quaternary ammonium salt and a total concentration of said quaternary ammonium salt or salts in the range of 0.1 to 12.0 g/l, wherein it contains at least one amphoteric surfactant and a total concentration of said amphoteric surfactant or surfactants is in the range of 0.1 to 12.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. According to a more preferred embodiment of the above said quaternary ammonium salt or salts have the total concentration in the range of 1.0 to 10.0 g/l, wherein said amphoteric surfactant or surfactants have the total concentration in the range of 0.1 to 10.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. Still a further preferred embodiment of the reagent of the present invention is characterized in that it contains at least one quaternary ammonium salt and a total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l, wherein it contains at least one cationic surfactant and a total concentration of said cationic surfactant or surfactants is in the range of 0.1 to 5.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. According to a still further preferred embodiment, the inventive reagent contains at least one quaternary ammonium salt and a total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l, wherein it contains at least one pyridinium salt and a total concentration of said pyridinium salt or salts is in the range of 0.3 to 10.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. A more preferred embodiment of the above is characterized in that said quaternary ammonium salt or salts have the total concentration in the range of 1.0 to 10.0 g/l, wherein said pyridinium salt or salts have the total concentration in the range of 1.0 to 5.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. Fig. 1 shows a correlation between the results of measurement of the number of leukocytes by the conventional method using an F-800 apparatus manufactured by Toa Medical Electronics Co., Ltd. and the results of measurement of the number of leukocytes with the use of the reagent of the Example 1; Fig. 2 shows a correlation between the results of measurement of the hemoglobin concentration by the conventional method, that is, the cyanmethemoglobin method, which is the international standard method, and the results of measurement of the hemoglobin concentration by the reagent of Example 1; Fig. 3 shows a correlation between the results of measurement of the number of leukocytes by the conventional method using an F-800 apparatus produced by Toa Medical Electronics Co., Ltd. and the results of measurement of the number of leukocytes with the reagent of Example 10; Fig. 4 shows a correlation showing the results of measurement of the hemoglobin concentration by the conventional method, that is, the cyanmethemoglobin method, which is the international standard method, and the results of measurement of the hemoglobin concentration with the reagent of Example 10; Figs. 5 to 7 are charts showing the leukocyte size distribution with the use of the reagents of the Examples 1, 14 and 15, respectively; and Figs. 8 to 10 are charts showing the leukocyte size distributions with the use of the reagents of the Examples 10, 16 and 17, respectively. In general, for measuring the hemoglobin concentration, the hemoglobin in erythrocytes is eluted by the action of a suitable erythrocytolytic agent, the hemeferrum in the eluted hemoglobin is oxidized by the action of the erythrocytolytic agent and the oxygen in the solution or by the action of a suitable oxidizing agent, the yielded methemoglobin is stabilized by the action of conventional methemoglobin stabilizers, such as cyanide ions, and the absorbance of the stabilized hemoglobin sample is measured, as described previously. However, the use of the conventional methemoglobin stabilizer has been the cause of the aforementioned problem of disposal of resulting waste liquor. The methemoglobin free of methemoglobin stabilizer remains temporarily unstable as compared to the aforementioned cyanmethemoglobin or oxyhemoglobin, while its absorbance is changed with the pH value of the solution, so that it has not been used for hemoglobin measurement. The present inventors recently proposed in Japanese Patent Application No. 1-275280 (1989) a methemoglobin stabilizing reagent which is free from the above-mentioned methemoglobin stabilizer. With this reagent, erythrocytes in the blood sample are selectively hemolyzed by the action mainly of quaternary ammonium salts or pyridinium salts for eluting hemoglobin in erythrocytes. The eluted hemoglobin is denatured, that is, modified in its steric structure, almost instantaneously, and hemeferrum in hemoglobin is oxidized by the oxygen dissolved in the reagent from the bivalent iron into trivalent iron to yield methemoglobin. If only one kind of the cationic surfactant is used, a problem similar to that encountered with methemoglobin prepared without methemoglobin stabilizer is presented. However, with the reagent of Japanese Patent Application No. 1-275280 (1989), suitable amounts of cationic, nonionic and amphoteric surfactants are added thereto to adjust the degree of denaturation of hemoglobin to yield stable hemoglobin. The effects of the pH may be eliminated by addition of a buffer as will be explained subsequently. Although the methemoglobin stabilizing mechanism has not been clarified, it may be presumed that the action of plural surfactants with different molecular structures on hemoglobin possibly results in fixing the degree of denaturation or change in steric structure into methemoglobin at a predetermined level. With the reagent of Japanese Patent Application No. 1-275280 (1989), the number of leukocytes can be measured simultaneously since the leukocytes remain unchanged. With the reagent of the present invention, the above-mentioned hemoglobin stabilizer is added to stabilize methemoglobin. The effect of the hemoglobin stabilizer is shown in Table 3. Although the operating mechanism of the hemoglobin stabilizer of the present invention has not been clarified, it may be presumed that lone electron pairs of nitrogen atoms in the molecular structure of the hemoglobin stabilizer or oxygen atoms in the phenolic hydroxy groups may chelate with hemeferrum in methemoglobin resulting in stabilizing methemoglobin. With the present reagent, the leukocytes may be divided into two or more fractions, subject to more strict limitations of surfactant concentrations. The following are some of the compositional examples of the reagent according to the present invention. Compositional Example 1 Compositional Example 2 Compositional Example 3 The reagents of the Compositional Examples 1 to 3 are prepared by hemolyzing and eluting erythrocytes by the action of surfactants such as quaternary ammonium salts or amphoteric surfactants to produce hemoglobin which is then converted into methemoglobin for stabilization and acted upon by Tiron for long term stabilization. With the Compositional Example 1, the combination of quaternary ammonium salts and amphoteric surfactants is used to enable measurement of the leukocytes; with the Compositional Example 2, the combination of quaternary ammonium salts and nonionic surfactants is used to enable the leukocytes to be divided into at least two fractions; and with the Compositional Example 3, the combination of two different kinds of quaternary ammonium salts is used to enable the leukocytes to be divided into at least three fractions. The term "total concentration of the quaternary ammonium salts" used in the claim means, in the case of Compositional Example 3, the concentration range of 0.31 to 12.0 g which is the sum of the concentration of lauryltrimethylammonium chloride and the concentration of cetyltrimethylammonium chloride. Compositional Example 4 Compositional Example 5 Compositional Example 6 With the Compositional Examples 4, 5 and 6, the combination of the quaternary ammonium salt, nonionic surfactant and 8-hydroxyquinoline, the combination of the quaternary ammonium salt, cationic surfactant and 1,10-phenanthroline and the combination of quaternary ammonium salt, pyridinium salt and 1-phenyl-3-pyrazolone, are used, respectively, for converting hemoglobin into methemoglobin for stabilization and the produced methemoglobin is then acted upon by a hemoglobin stabilizer or long-term stabilization. With the present reagent, by controlling the surfactant concentration more rigorously, the leukocytes may be fractionated into two or three groups. The following are further compositional examples of the reagent of the present invention. Compositional Example 7 Compositional Example 8 Compositional Example 9 With the Compositional Examples 7 to 9, the erythrocytes are hemolyzed and eluted by the action of surfactants such as the quaternary ammonium salt or amphoteric surfactants to produce hemoglobin which is then converted into methemoglobin for stabilization and subsequently acted upon by 4-phenylimidazole to provide for long-term stabilization of hemoglobin. With the Compositional Example 7, the combination of the quaternary ammonium salt and the amphoteric surfactant is used to enable measurement of the leukocytes; with the Compositional Example 8, the combination of the quaternary ammonium salt and the nonionic surfactant is used to enable the leukocytes to be divided into at least two fractions; and with the Compositional Example 9, the combination of two different types of the quaternary ammonium salts is used to enable the leukocytes to be divided into at least three fractions. Compositional Example 10 Hemoglobin is stabilized by the action of the quaternary ammonium salt and the nonionic surfactant and the produced methemoglobin is further acted on by imidazole to effect long-term stabilization. Meanwhile, it is possible, by adjusting the concentrations of the quaternary ammonium salt and the surfactants, to divide the leukocytes into two or more fractions. With the Compositional Example 1 to 10, the phosphate buffer is used to control the pH of the solution to be at an optional value within the range from 3.0 to 9.0. Thus, there is no limitation on the types of buffers, and any buffer other than a phosphate buffer, such as citrate, maleate or tris may be employed. The preferred pH range is from 5.0 to 8.0. If the pH value is 3.0 or less, damage to the leukocytes increases thus rendering measurement of the leukocytes difficult. If the pH value is 9.0 or more, the stability of hemoglobin deteriorates over time. Sodium chloride is used for adjusting electrical conductivity to a level able to be measured by an automatic blood analyzer and hence may be replaced by any inorganic or organic salts that may be dissociated into ions in an aqueous solution. The concentrations of the various ingredients defined in the claim mean the concentrations under the state in which measurement may be made only by adding the blood to the reagent without the necessity of using other diluents. However, in the case of a conventional apparatus in which the diluent and the lysing reagents are mixed together at a predetermined ratio for measuring hemoglobin or leukocytes, it is possible to change the concentration of the ingredients of the various surfactants as a function of the mixing ratio of the apparatus, in which case the concentrations of the various ingredients defined in the claim means the concentrations in the liquid mixture consisting of the diluent and the lysing reagent. In accordance with the present invention, i) hemoglobin concentration and leukocytes may be measured with a single reagent; ii) even a blood sample having a high methemoglobin content may be measured; iii) because cyanide is not contained, the troublesome operation of disposing of waste liquor may be eliminated; and iv) a reagent may be provided in which hemoglobin remains stable over a prolonged time. With the use of the present reagent in an automatic blood analyzer, since there is no necessity of separately providing a hemoglobin measurement sample preparating section and a leukocyte measurement sample preparating section, the analyzer may be simplified in structure enabling a lowering in costs. Examples The reagents of the following compositions were prepared, and the effects derived from addition of the hemoglobin stabilizer shown in Table 3 are shown. The changes in the hemoglobin concentration and the rates of change in Table 3 were computed by the following formulas: Amount of change in hemoglobin concentration = (hemoglobin concentration measured in 30 minutes after sample preparation) - (hemoglobin concentration measured in 20 seconds after sample preparation) Rate of change of hemoglobin = (amount of change of hemoglobin concentration)/hemoglobin concentration measured in 20 seconds after sample preparation) × 100 The F-800 measurement apparatus, produced by Toa Medical Electronics Co., Ltd., was used for all of the measurements. With the Comparative Example 1 of Table 3, measurement was made using a cyanmethemoglobin method (international standard method) and, since it takes a long time to oxidize hemoglobin, the hemoglobin quantity is drastically changed since 20 seconds until 30 minutes after the sample preparation, as shown in Table 3. With Comparative Example 2, an automated cyanmethemoglobin method used in the automatic blood analyzer is used, in which the amount of change is substantially less. With the Comparative Example 3, an example of hemoglobin measurement is shown, in which methemoglobin is produced using a sole surfactant (lauryltrimethylammonium chloride) excepting lauryldimethylaminoacetic acid betaine of the above composition. Since methemoglobin is not stabilized, the hemoglobin concentration is drastically changed since 20 seconds until 30 minutes after sample preparation. In the Comparative Example 4 in which lauryldimethylaminoacetic acid betaine, an amphoteric surfactant, is added to the composition of Comparative Example 3, that is, a compositional example shown in Japanese Patent Application No. 1-275280 (1989), the rate of change is decreased as compared to that of Comparative Example 3. With the compositions of the Examples 1 to 13 containing the hemoglobin stabilizer, the reagent of the present invention, the degree of change is further decreased as compared to the Comparative Example 4. Fig. 1 shows the correlation between the results of measurement of the number of leukocytes by the conventional method employing F-800 apparatus produced by Toa Medical Electronics Co., Ltd. and the results of measurement of the number of leukocytes with the use of the reagent of Example 1. The coefficient of correlation r = 0.999 and the regression line y = 1.009x + 0.179, indicating an extremely high correlation. Fig. 2 shows the correlation between the results of measurement of hemoglobin concentration by the conventional method, that is the cyanmethemoglobin method (international standard method), and the results of measurement of the hemoglobin concentration with the use of the reagent of Example 1. The coefficient of correlation r = 0.999 and the regression line y = 1.011x + 0.019, indicating an extremely high correlation. Fig. 3 shows the correlation between the results of measurement of leukocytes by the conventional method employing F-800 apparatus produced by Toa Medical Electronics Co., Ltd. and the results of measurement of the leukocytes with the use of the reagent of Example 10. The coefficient of correlation r = 1.000 and the regression line y = 1.001x + 0.331, indicating an extremely high correlation. Fig. 4 shows the correlation between the results of measurement of the hemoglobin concentration by the conventional cyanmethemoglobin method (international standard method) and the results of measurement of the hemoglobin concentration with the use of the reagent of Example 10. The coefficient of correlation r = 0.998 and the regression line y = 1.013x + 0.095, indicating an extremely high correlation. As shown in Table 1, even when the hemoglobin concentration as measured by the oxyhemoglobin method is lowered as a result of an increase in the quantity of methemoglobin, the measurement value of hemoglobin concentration as measured with the reagent of Example 1 is not changed but remains constant. Meanwhile, the difference between the initial value as obtained with the oxyhemoglobin method shown in Table 1 and the initial value as obtained with the reagent of the Example 1 is ascribable to the use of a control blood in which a predetermined quantity of hemoglobin in the blood has from the outset been converted into methemoglobin. Example 14 The reagent of the following composition was prepared. Example 15 The reagent of the following composition was prepared. Figs. 5 to 7 show the particle size distribution of the leukocytes with the use of the reagents of the Examples 1, 14 and 15, respectively. In these figures, the ordinate denotes the relative frequency of the blood cells, and the abscissa denotes the relative intensity of the blood cell signals, that is the size of the blood cells, obtained on measurement of the blood cells with the aid of the automatic blood analyzer. With the use of the reagent of Example 1, the leukocytes show a single-peak particle size distribution, as shown in Fig. 5. With the use of the reagent of the Example 14, the leukocytes show a particle size distribution composed of two regions A and B, as shown in Fig. 6. The region A stands for an aggregate of lymphocytes and the region B an aggregate of leukocytes other than the lymphocytes. With the use of the reagents of Example 15, the leukocytes show a particle size distribution composed of regions A, C and D, as shown in Fig. 7. The region A stands for an aggregate of lymphocytes, the region C an aggregate of monocytes, eosinophilic leukocytes and basophilic leukocytes and the region D stands for an aggregate of neutrophilic leukocytes. As shown in Table 2, even when the hemoglobin concentration as measured by the oxyhemoglobin method is lowered due to an increased methemoglobin contents, the hemoglobin concentration as measured by the reagent of Example 10 is not changed but remains constant. Example 16 The reagent of the following composition was prepared. Example 17 The reagent of the following composition was prepared. Figs. 8 to 10 show the particle size distribution of the leukocytes with the use of the reagents of the Examples 10, 16 and 17, respectively. In these figures, the ordinate denotes the relative frequency of the blood cells, and the abscissa denotes the relative intensity of the blood cell signals, that is the size of the blood cells, obtained upon measurement of the blood cells with the aid of an automatic blood analyzer. With the use of the reagent of Example 10, the leukocytes indicate a single-peak particle size distribution, as shown in Fig. 8. With the use of the reagent of Example 16, the leukocytes indicate a particle size distribution composed of two regions A and B, as shown in Fig. 9. The region A stands for an aggregate of lymphocytes and the region B an aggregate of leukocytes other than the lymphocytes. With the use of the reagents of Example 17, the leukocytes indicate a particle size distribution composed of regions A, C and D, as shown in Fig. 10. The region A stands for an aggregate of lymphocytes, the region C an aggregate of monocytes, eosinophilic leukocytes and basophilic leukocytes and the region D stands for an aggregate of neutrophilic leukocytes. From the foregoing, it is seen that, with the reagent of the present invention, not only the measured values which are the same as those of the prior art may be obtained, but i) the hemoglobin and leukocytes may be measured by one and the same sample, while the leukocytes may be fractionated; ii) correct measurement may be achieved even with blood samples with high methemoglobin contents; iii) since toxic substances, such as cyanides, are not used, it becomes unnecessary to dispose of waste liquor; and iv) the reagent may be supplied in which hemoglobin remains stable for an extended period of time.

Claims: A reagent for use in counting the number of leukocytes and measuring the hemoglobin concentration in the blood, said reagent not containing cyanides and being able to maintain hemoglobin in a stable state for a prolonged time comprising a solution with a pH ranging from 3.0 to 9.0 containing: i) at least one selected from the group consisting of quaternary ammonium salts and pyridinium salts; said quaternary ammonium salts having the formula: R₁: C₈ to C₂₀, alkyl, alkenyl or alkinyl group, R₂ to R₄: C₁ to C₈ alkyl, alkenyl or alkinyl group, X⁻: anionic group the total concentration of said quaternary ammonium salts is in the range of from 0.1 to 15.0 g/l, and said pyridinium salts having the formula: n: an integer of from 7 to 19, X⁻: an anionic group; the total concentration of said pyridinium salts is in the range of from 0.1 to 15.0 g/l; and ii) at least one selected from the group consisting of cationic, nonionic and amphoteric surfactants, said cationic surfactants having the formula: R₁: C₈ to C₂₀ alkyl, alkenyl or alkinyl group, R₂ to R₃: C₁ to C₈ alkyl, alkenyl or alkinyl group, X⁻: anionic group the total concentration of said cationic surfactants is in the range of from 0.1 to 15.0 g/l; and said nonionic surfactants having the formula: structure d R₁ - R₂ - (CH₂CH₂O) n - H R₁: C₈ to C₂₀ alkyl, alkenyl or alkinyl group, R₂: O, or COO, n: an integer of from 10 to 50 the total concentration of said nonionic surfactants is in the range of from 0.1 to 15.0 g/l; and said amphoteric surfactants having the formula: R₁: C₈ to C₂₀ alkyl group, R₂ to R₃: C₁ to C₈ alkyl, alkenyl or alkinyl group; the total concentration of said amphoteric surfactants is in the range of from 0.1 to 15.0 g/l; and iii) at least one hemoglobin stabilizer selected from the group consisting of: (f) Tiron with a concentration of 0.1 to 1000 mg/l: (g) 8-hydroxyquinoline with a concentration of 0.1 to 1000 mg/l: (h) bipyridine with a concentration of 10 to 10000 mg/l: (i) 1,10-phenanthroline and its derivatives with a concentration of 10 to 3000 mg/l: R₁, R₄: -H, CH₃; R₂, R₃: -H, phenyl group, -OH; (j) phenolic compounds with a concentration of 0.1 to 1000 mg/l: R₁: -H, CH₂OH, -CHO, -COOH or -OH; R₂: -H, -COOH or -OH; R₃: -H or -COOH; (k) bisphenol A with a concentration of 0.1 to 1000 mg/l: (l) pyrazole and its derivatives with a concentration of 0.1 to 10000 mg/l: R₁, R₂, R₃: -H or C₁ to C₄ lower alkyl group; (m) phenyl 5-pyrazolone and its derivative with a concentration of 0.1 to 10000 mg/l: R₁, R₂, R₃: -H or C₁ to C₄ lower alkyl group; (n) phenyl 3-pyrazolone with a concentration of 0.1 to 10000 mg/l: and (o) imidazole and its derivatives with a concentration of 1 to 20000 mg/l: R₁, R₂: -H or C₁ to C₄ lower alkyl group, R₃: -H or C₁ to C₄ lower alkyl group or phenyl group. The reagent according to Claim 1, wherein it contains at least two types of quaternary ammonium salts and the total concentration of said quaternary ammonium salts is in the range of 0.1 to 12.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. The reagent according to Claim 2, wherein said quaternary ammonium salts have the total concentration in the range of 2.2 to 5.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. The reagent according to Claim 1, wherein it contains at least one quaternary ammonium salt and the total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l, wherein it contains at least one nonionic surfactant and the total concentration of said nonionic surfactant or surfactants is in the range of 0.1 to 12.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. The reagent according to Claim 4, wherein said quaternary ammonium salt or salts have the total concentration in the range of 3.0 to 12.0 g/l, wherein said nonionic surfactant or surfactants have the total concentration in the range of 1.0 to 12.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. The reagent according to Claim 1, wherein it contains at least one quaternary ammonium salt and the total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l, wherein it contains at least one amphoteric surfactant and the total concentration of said amphoteric surfactant or surfactants is in the range of 0.1 to 12.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. The reagent according to Claim 6, wherein said quaternary ammonium salt or salts have the total concentration in the range of 1.0 to 10.0 g/l, wherein said amphoteric surfactant or surfactants have the total concentration in the range of 0.1 to 10.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions. The reagent according to Claim 1, wherein it contains at least one quaternary ammonium salt and the total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l wherein it contains at least one cationic surfactant and the total concentration of said cationic surfactant or surfactants is in the range of 0.1 to 5.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. The reagent according to Claim 1, wherein it contains at least one quaternary ammonium salt and the total concentration of said quaternary ammonium salt or salts is in the range of 0.1 to 12.0 g/l, wherein it contains at least one pyridinium salt and the total concentration of said pyridinium salt or salts is in the range of 0.3 to 10.0 g/l, and said reagent is capable of dividing leukocytes into at least two fractions. The reagent according to Claim 9, wherein said quaternary ammonium salt or salts have the total concentration in the range of 1.0 to 10.0 g/l, wherein said pyridinium salt or salts have the total concentration in the range of 1.0 to 5.0 g/l, and said reagent is capable of dividing leukocytes into at least three fractions.

Inventors: SAKATA TAKASHI, SAKATA, TAKASHI
Applicants: TOA MEDICAL ELECTRONICS, TOA MEDICAL ELECTRONICS CO., LTD.
Application year: 1990
Main classification: G01N33
IPC codes: G01N33