A hemolytic agent for blood cell analysis

Abstract

The present invention relates to the field of blood cell analysis, specifically to a hemolytic agent for blood cell analysis. The hemolytic agent for blood cell analysis includes a cationic surfactant, a nonionic surfactant, a preservative, a soluble salt, an organic acid, and a buffer solution; the pH value range of the hemolytic agent is 3.0 - 5.0; wherein, the concentration range of the cationic surfactant is 1.00 g / L - 10.00 g / L; the concentration range of the nonionic surfactant is 1.00 g / L - 5.00 g / L; the concentration range of the preservative is 0.50 mL / L - 2.00 mL / L; the concentration range of the soluble salt is 5.00 g / L - 20.00 g / L; the concentration range of the organic acid is 1.00 mL / L - 10.00 mL / L; the concentration range of the buffer solution is 0.50 g / L - 3.00 g / L. The hemolytic agent for blood cell analysis provided by the embodiments of the present invention has easily obtainable raw materials, low cost, and short detection time; at the same time, under the action of the pH value, it can accurately classify monocytes, lymphocytes, neutrophils, and eosinophils normally, keeping white blood cells in their original state and having high selectivity during the classification process.

Description

Technical Field

[0001] The present invention relates to the field of blood cell analysis, and particularly to a hemolytic agent for blood cell analysis. Technical Background

[0002] White blood cell classification can be divided into five types: monocytes, lymphocytes, neutrophils, eosinophils, and basophils. Among them, an increase in monocytes is commonly seen in chronic myeloid leukemia, Hodgkin's disease, and lead poisoning; an increase in lymphocytes is commonly seen in allergic reactions, after surgery, and burns; an increase in neutrophils is commonly seen in acute suppurative infections, after surgery, uremia, and acidosis; an increase in eosinophils is commonly seen in whooping cough, tuberculosis, and hepatitis; an increase in basophils is commonly seen in typhoid fever, kala-azar, and monocytic leukemia. Therefore, in the field of medical clinical tests, the determination of white blood cell classification in a patient's blood has important reference significance for the diagnosis of blood diseases and subsequent treatment plans.

[0003] In the blood test industry, the use of manual microscopy is the "gold standard" for white blood cell classification technology. However, the manual microscopy method is time-consuming, laborious, and cumbersome. Therefore, various hemolytic agents for blood cell analysis have emerged, but the raw materials for the current hemolytic agents are not easily obtained and the cost is relatively high, requiring a large amount of manpower and material resources. Summary of the Invention

[0004] An embodiment of the present invention provides a hemolytic agent for blood cell analysis, which can solve the technical problems of difficult raw material acquisition, high cost, time-consuming, and laborious detection.

[0005] In order to solve the above technical problems, a technical solution adopted in an embodiment of the present invention is:

[0006] An embodiment of the present invention provides a hemolytic agent for blood cell analysis, and the hemolytic agent for blood cell analysis includes a cationic surfactant, a nonionic surfactant, a preservative, a soluble salt, an organic acid, and a buffer solution. Among them, the concentration range of the cationic surfactant is 1.00 g / L - 10.00 g / L, the concentration range of the nonionic surfactant is 1.00 g / L - 5.00 g / L, the concentration range of the preservative is 0.50 mL / L - 2.00 mL / L, the concentration range of the soluble salt is 5.00 g / L - 20.00 g / L, the concentration range of the organic acid is 1.00 mL / L - 10.00 mL / L, and the concentration range of the buffer solution is 0.50 g / L - 3.00 g / L.

[0007] Optionally, the cationic surfactant includes octyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, cetyltrimethylammonium bromide, or a combination thereof.

[0008] Optionally, the concentration range of octyltrimethylammonium chloride is 0.50 g / L - 2.00 g / L, the concentration range of decyltrimethylammonium chloride is 0.20 g / L - 1.50 g / L, the concentration range of dodecyltrimethylammonium chloride is 0.10 g / L - 0.50 g / L, the concentration range of cetyltrimethylammonium chloride is 0.10 g / L - 0.50 g / L, the concentration range of dodecyltrimethylammonium bromide is 0.20 g / L - 1.50 g / L, and the concentration range of cetyltrimethylammonium bromide is 0.10 g / L - 0.15 g / L.

[0009] Optionally, the non-ionic surfactant includes polyoxyethylene (10) oleyl ether, polyethylene glycol 2000, 1,2-propanediol, or a combination thereof.

[0010] Optionally, the concentration range of polyoxyethylene (10) oleyl ether is 0.50 g / L - 2.00 g / L, the concentration range of polyethylene glycol 2000 is 0.50 g / L - 1.50 g / L, and the concentration range of 1,2-propanediol is 0.10 mL / L - 1.00 mL / L.

[0011] Optionally, the preservative includes isothiazolinone, formaldehyde, gentamicin sulfate, phenoxyethanol, or a combination thereof.

[0012] Optionally, the concentration range of isothiazolinone is 0.50 g / L - 2.00 g / L, the concentration range of formaldehyde is 0.50 mL / L - 1.50 mL / L, the concentration range of gentamicin sulfate is 0.50 g / L - 1.00 g / L, and the concentration range of phenoxyethanol is 0.50 g / L - 2.00 g / L.

[0013] Optionally, the soluble salt includes sodium chloride, 1,3-dimethylurea, sodium N-hydroxymethylglycinate, or a combination thereof.

[0014] Optionally, the concentration range of sodium chloride is 5.00 g / L - 20.00 g / L, the concentration range of 1,3-dimethylurea is 1.00 g / L - 5.00 g / L, the concentration range of 1,3-dimethylurea is 1.00 g / L - 5.00 g / L, and the concentration range of sodium N-hydroxymethylglycinate is 0.05 g / L - 0.30 g / L.

[0015] Optionally, the organic acid includes formic acid, acetic acid, or a combination thereof.

[0016] Optionally, the concentration range of formic acid is 1.50 mL / L - 10.00 mL / L, and the concentration range of acetic acid is 0.50 mL / L - 5.00 mL / L.

[0017] Optionally, the buffer solution includes Tris, potassium hydrogen phthalate, disodium hydrogen phosphate, citric acid, or a combination thereof.

[0018] Optionally, the concentration range of the Tris is 0.50 g / L - 3.00 g / L, the concentration range of the potassium hydrogen phthalate is 0.05 g / L - 0.50 g / L, the concentration range of the disodium hydrogen phosphate is 0.10 mol / L - 1.00 mol / L, and the concentration range of the citric acid is 0.10 moL / L - 0.50 mol / L.

[0019] Optionally, the pH value range of the hemolytic agent is 3.0 - 5.0.

[0020] Preferably, the cationic surfactant includes octyltrimethylammonium chloride with a concentration of 1.50 g / L, dodecyltrimethylammonium chloride with a concentration of 0.25 g / L, cetyltrimethylammonium chloride with a concentration of 0.15 g / L, or a combination thereof.

[0021] Preferably, the nonionic surfactant includes 1,2 - propanediol with a concentration of 0.30 mL / L.

[0022] Preferably, the preservative includes phenoxyethanol with a concentration of 1.50 mL / L.

[0023] Preferably, the soluble salt includes 1,3 - dimethylurea with a concentration of 2.00 g / L, sodium chloride with a concentration of 7.50 g / L, sodium N - hydroxymethylglycinate with a concentration of 0.15 g / L, or a combination thereof.

[0024] Preferably, the organic acid includes formic acid with a concentration of 6.50 mL / L.

[0025] Preferably, the buffer solution includes disodium hydrogen phosphate with a concentration of 0.20 mol / L, citric acid with a concentration of 0.10 mol / L, or a combination thereof.

[0026] Compared with the prior art, the beneficial effects of the embodiments of the present invention are as follows: The hemolytic agent for blood cell analysis provided by the embodiments of the present invention has easily obtainable raw materials, low cost, and short detection time; it can accurately classify monocytes, lymphocytes, neutrophils, and eosinophils normally. At the same time, under the action of the pH value, it can not only correctly distinguish basophils, but also keep white blood cells in their original state during the process of lysing red blood cells, and has high selectivity during the classification process. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0028] Figure 1 It is a flowchart for preparing a hemolytic agent for blood cell analysis provided by an embodiment of the present invention. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] An embodiment of the present invention provides a hemolytic agent for blood cell analysis. The hemolytic agent for white blood cell analysis includes a cationic surfactant, a non-ionic surfactant, a preservative, a soluble salt, an organic acid, and a buffer solution; the pH value range of the hemolytic agent is 3.0 - 5.0.

[0031] Among them, the concentration range of the cationic surfactant is 1.00 g / L - 10.00 g / L, the concentration range of the non-ionic surfactant is 1.00 g / L - 5.00 g / L, the concentration range of the preservative is 0.50 mL / L - 2.00 mL / L, the concentration range of the soluble salt is 5.00 g / L - 20.00 g / L, the concentration range of the organic acid is 1.00 mL / L - 10.00 mL / L, and the concentration range of the buffer solution is 0.50 g / L - 3.00 g / L.

[0032] The preparation process of the above-mentioned hemolytic agent for blood cell analysis is as Figure 1 shown:

[0033] 100. First, add the above components into a beaker and dissolve them with a certain amount of pure water.

[0034] 101. Secondly, transfer the solution from the beaker to a volumetric flask with a glass rod.

[0035] 102. Then use a dropper to make up the volume to 1 liter with pure water.

[0036] 103. After making up the volume, shake it well.

[0037] In some embodiments, the cationic surfactant includes octyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, cetyltrimethylammonium bromide or a combination thereof.

[0038] In some embodiments, the concentration range of octyltrimethylammonium chloride is 0.50 g / L - 2.00 g / L, the concentration range of decyltrimethylammonium chloride is 0.20 g / L - 1.50 g / L, the concentration range of dodecyltrimethylammonium chloride is 0.10 g / L - 0.50 g / L, the concentration range of cetyltrimethylammonium chloride is 0.10 g / L - 0.50 g / L, the concentration range of dodecyltrimethylammonium bromide is 0.20 g / L - 1.50 g / L, and the concentration range of cetyltrimethylammonium bromide is 0.10 g / L - 0.15 g / L.

[0039] Quaternary ammonium salt surfactants are a type of cationic surfactant formed by completely substituting the four hydrogen atoms of the ammonium ion [NH4 + . Quaternary ammonium salt cationic surfactants are different from general ammonium salts. The former can also exhibit good stability in alkaline aqueous solutions, and no amine is released.

[0040] In some embodiments, the cationic surfactant includes octyltrimethylammonium chloride with a concentration of 1.50 g / L, dodecyltrimethylammonium chloride with a concentration of 0.25 g / L, cetyltrimethylammonium chloride with a concentration of 0.15 g / L, or a combination thereof.

[0041] In some embodiments, the non-ionic surfactant includes polyoxyethylene (10) oleyl ether, polyethylene glycol 2000, 1,2-propanediol, or a combination thereof.

[0042] In some embodiments, the concentration range of polyoxyethylene (10) oleyl ether is 0.50 g / L - 2.00 g / L, the concentration range of polyethylene glycol 2000 is 0.50 g / L - 1.50 g / L, and the concentration range of 1,2-propanediol is 0.10 mL / L - 1.00 mL / L.

[0043] In some embodiments, the non-ionic surfactant includes 1,2-propanediol with a concentration of 0.30 mL / L.

[0044] In some embodiments, the preservative includes isothiazolinone, formaldehyde, gentamicin sulfate, phenoxyethanol, or a combination thereof.

[0045] The bactericidal mechanism of isothiazolinone preservatives is mainly electrophilic active bactericides. Isothiazolinone preservatives have the advantages of strong antibacterial ability, small application dosage, good compatibility, low toxicity, etc., and they have a strong antibacterial effect on a variety of bacteria and fungi. At the same time, isothiazolinone preservatives have high efficiency, good compatibility, a wide pH application range, and can be naturally biodegradable.

[0046] Both formaldehyde and gentamicin sulfate can be used to replace the commonly used highly toxic antibacterial preservative sodium azide. Among them, formaldehyde not only has an antiseptic effect but also has the effect of fixing blood cells, can maintain the morphology and structure integrity of blood cells to a certain extent, and can also increase the effect of blood cells against mechanical damage.

[0047] Phenoxyethanol has antibacterial efficacy (usually used together with quaternary ammonium salts), has low toxicity, and is often used as a substitute for highly toxic sodium azide in biological buffer solutions.

[0048] In some embodiments, the concentration range of the isothiazolinone is 0.50 g / L - 2.00 g / L, the concentration range of the formaldehyde is 0.50 mL / L - 1.50 mL / L, the concentration range of the gentamicin sulfate is 0.50 g / L - 1.00 g / L, and the concentration range of the phenoxyethanol is 0.50 g / L - 2.00 g / L.

[0049] In some embodiments, the preservative includes phenoxyethanol with a concentration of 1.50 mL / L.

[0050] In some embodiments, the soluble salt includes sodium chloride, 1,3-dimethylurea, sodium N-hydroxymethylglycine, or a combination thereof.

[0051] In some embodiments, the concentration range of the sodium chloride is 5.00 g / L - 20.00 g / L, the concentration range of the 1,3-dimethylurea is 1.00 g / L - 5.00 g / L, the concentration range of the 1,3-dimethylurea is 1.00 g / L - 5.00 g / L, and the concentration range of the sodium N-hydroxymethylglycine is 0.05 g / L - 0.30 g / L.

[0052] In some embodiments, the soluble salt includes 1,3-dimethylurea with a concentration of 2.00 g / L, sodium chloride with a concentration of 7.50 g / L, sodium N-hydroxymethylglycine with a concentration of 0.15 g / L, or a combination thereof.

[0053] In some embodiments, the organic acid includes formic acid, acetic acid, or a combination thereof.

[0054] In some embodiments, the concentration range of the formic acid is 1.50 mL / L - 10.00 mL / L, and the concentration range of the acetic acid is 0.50 mL / L - 5.00 mL / L.

[0055] In some embodiments, the organic acid includes formic acid with a concentration of 6.50 mL / L.

[0056] In some embodiments, the buffer includes Tris, potassium hydrogen phthalate, disodium hydrogen phosphate, citric acid, or a combination thereof.

[0057] In some embodiments, the concentration range of Tris is 0.50 g / L - 3.00 g / L, the concentration range of potassium hydrogen phthalate is 0.05 g / L - 0.50 g / L, the concentration range of disodium hydrogen phosphate is 0.10 mol / L - 1.00 mol / L, and the concentration range of citric acid is 0.10 moL / L - 0.50 mol / L.

[0058] In some embodiments, the buffer solution includes disodium hydrogen phosphate with a concentration of 0.20 mol / L, citric acid with a concentration of 0.10 mol / L, or a combination thereof.

[0059] The following provides multiple embodiments to illustrate in detail the detection effect and accuracy of a hemolytic agent for blood cell analysis provided by the embodiments of the present invention.

[0060] Example 1: A hemolytic agent 1 for blood cell analysis:

[0061] In the hemolytic agent for blood cell analysis provided in this embodiment, each liter of the hemolytic agent includes a cationic surfactant, a nonionic surfactant, a preservative, a soluble salt, an organic acid, and a buffer solution. Among them, the cationic surfactant is 1.50 g of octyltrimethylammonium chloride and 0.15 g of cetyltrimethylammonium chloride, the nonionic surfactant is 0.30 mL of 1,2 - propanediol, the preservative is 1.50 mL of phenoxyethanol, the soluble salt is 7.50 g of sodium chloride and 2.00 g of 1,3 - dimethylurea, the organic acid is 6.50 mL of formic acid, and the buffer solution is 0.20 mol of disodium hydrogen phosphate and 0.10 mol of citric acid. The pH of this hemolytic agent is 4.0.

[0062] Example 2: A hemolytic agent 2 for blood cell analysis:

[0063] In the hemolytic agent for blood cell analysis provided in this embodiment, each liter of the hemolytic agent includes a cationic surfactant, a nonionic surfactant, a preservative, a soluble salt, an organic acid, and a buffer solution. Among them, the cationic surfactant is 0.25 g of dodecyltrimethylammonium chloride and 0.15 g of cetyltrimethylammonium chloride, the nonionic surfactant is 0.30 mL of 1,2 - propanediol, the preservative is 1.50 mL of phenoxyethanol, the soluble salt is 7.50 g of sodium chloride and 0.15 g of N - hydroxymethylglycine sodium, the organic acid is 6.50 mL of formic acid, and the buffer solution is 0.10 mol of citric acid. The pH of this hemolytic agent is 4.0.

[0064] Example 3: Repeatability of test results:

[0065] Using the hemolyzing agents for blood cell analysis provided in Embodiment 1 and Embodiment 2 of the present invention, whole blood samples were respectively detected and classified on the Jinrui KT-6610 blood cell analyzer. A total of 5 samples were detected, and the same whole blood sample was repeatedly tested 10 times on the blood cell analyzer with different hemolyzing agents for blood cell analysis.

[0066] The test results of the hemolyzing agent for blood cell analysis in Embodiment 1 on the Jinrui KT-6610 blood cell analyzer are presented in Table 1;

[0067] The test results of the hemolyzing agent for blood cell analysis in Embodiment 2 on the Jinrui KT-6610 blood cell analyzer are presented in Table 2;

[0068] Normal reference range for differential count of white blood cells in healthy human blood:

[0069] Neutrophils: 40%-75%;

[0070] Lymphocytes: 20%-40%;

[0071] Monocytes: 3%-8%;

[0072] Eosinophils: 0.5%-5%.

[0073] Among them: Neu refers to neutrophils, Lym refers to lymphocytes, Mon refers to monocytes, and Eos refers to eosinophils; Neu% refers to the percentage of neutrophils, Lym% refers to the percentage of lymphocytes, Mon% refers to the percentage of monocytes, and Eos% refers to the percentage of eosinophils.

[0074] Table 1 Repeatability test results (Embodiment 1)

[0075]

[0076] Note: The absolute deviation d represents the difference between the maximum or minimum value and the average value of 10 test data.

[0077] Table 2 Repeatability test results (Embodiment 2)

[0078]

[0079] Note: The absolute deviation d represents the difference between the maximum or minimum value and the average value of 10 test data.

[0080] As can be seen from Table 1 and Table 2, the test results of the hemolyzing agents for blood cell analysis in Embodiment 1 and Embodiment 2 on the Jinrui KT-6610 blood cell analyzer have good repeatability, and the obtained absolute deviations are all within the acceptance criteria for the repeatability test of blood white blood cell differential count.

[0081] Embodiment 4: Accuracy comparison test:

[0082] The lysing agents of Example 1 and Example 2 were respectively used. According to the counting method of the lysing agent described in the present invention, 20 healthy fresh whole blood samples were detected using the Jinrui KT-6610 hematology analyzer. Each sample was tested three times, and the value with the largest deviation from the test result (target value) of the microscopy method among the three test results was selected as the test value of the sample (as shown in Table 3 below), and its test result was respectively compared and evaluated with the test result (target value) of the microscopy method for accuracy.

[0083] Table 3 Comparison of accuracy test results (Jinrui KT-6610)

[0084]

[0085]

[0086]

[0087] Note: The absolute deviation d is the difference between the maximum or minimum value and the fixed value (target value). Here, the target value refers to the result measured by the microscopy method.

[0088] According to the test results in the above table, it can be seen that when using the lysing agent provided by the present invention, there is no obvious difference between the experimental results of neutrophils, lymphocytes, monocytes, and eosinophils tested on the Jinrui KT-6610 and the results detected by the microscopy method. Therefore, the lysing agent provided by the present invention has high accuracy in the classification parameters tested on the Jinrui KT-6610.

[0089] In summary, compared with the prior art, the lysing agents for blood cell analysis provided in Example 1 and Example 2 of the present invention are easy to obtain raw materials, have low costs, and short detection time; they can accurately classify monocytes, lymphocytes, neutrophils, and eosinophils. At the same time, under the action of the pH value, they can not only correctly distinguish basophils, but also keep white blood cells in their original state during the process of lysing red blood cells, and have high selectivity during the classification process. In Example 3 and Example 4, the lysing agents for blood cell analysis of Example 1 and Example 2 were used to detect and classify whole blood samples, and the test results had good reproducibility and high accuracy.

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hemolytic agent for blood cell analysis, characterized in that, The hemolytic agent for blood cell analysis comprises a cationic surfactant, a nonionic surfactant, a preservative, a soluble salt, an organic acid, and a buffer solution; The pH value range of the hemolytic agent is 4.0; Among them, in each liter of the hemolytic agent for blood cell analysis, the cationic surfactant is 1.50 g of octyltrimethylammonium chloride and 0.15 g of cetyltrimethylammonium chloride, the nonionic surfactant is 0.30 mL of 1,2-propanediol, the preservative is 1.50 mL of phenoxyethanol, the soluble salt is 7.50 g of sodium chloride and 2.00 g of 1,3-dimethylurea, the organic acid is 6.50 mL of formic acid, and the buffer solution is 0.20 mol of disodium hydrogen phosphate and 0.10 mol of citric acid.

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