Patent Grant
7416891
The present invention relates to a reagent and a method for classifying cells, especially to a reagent and a method for classifying leucocytes in blood.
It is common and important to classify and count leucocytes to diagnose various diseases using the whole blood of patients in the field of a clinical test.
Up to now, many patents in regard to devices and methods for classifying leucocytes have been reported. For example, a method for dividing leukocytes into five types using two reagents has been disclosed in China Patent Application No. 95115317.X. Said two reagents both contain nonionic surfactant and buffer, and the first reagent further contains at least one ionic surfactant and one organic compound having an anionic group, to divide leukocytes into four groups, lymphocytes, monocytes, eosinophils and basophils plus neutrophils, and the second reagent further contains at least one ionic surfactant, to divide blood cells into basophils and other cells. Thus, Leukocytes are classified into five types by the combination of the two results. The measurement method used is laser scattering measurement.
Besides, U.S. Pat. Nos. 5,677,183, 5,618,733 and 5,731,206, and a Chinese Patent Application No. 88101677 also disclosed the method for dividing leucocytes into five types based on the signals of the scattered light from two channels.
It is found, however, that monocytes are usually classified as between the lymphocytes and neutrophils when classifying leucocytes using the conventional methods mentioned above. The clustering effect is not obvious, which results in an inaccuracy of classification.
An object of the present invention is to provide a reagent and a method for rapidly lysing erythrocytes and classifying leucocytes in one channel, increasing the clustering effect of monocytes, and improving the classification effect of monocytes, thereby improving the accuracy of classification.
To realize the above objects, the present invention adopts the following technical solutions with respect to an improved reagent for classifying leucocytes, comprising
a) a surfactant capable of lysing erythrocytes and partly damaging cell membrane of leucocytes, said surfactant is at least one of the surfactants selected from a cationic surfactant, an amphoteric surfactant, and the combination thereof;
b) an organic compound having phenyl or heterocyclic group, when said surfactant is a cationic surfactant, said organic compound is an organic compound having an anionic group; when said surfactant is an amphoteric surfactant or a combination of amphotheric and cationic surfactants, said organic compound is an organic compound having an anionic or cationic group;
c) a buffer for adjusting pH.
The above reagent may further comprise at least one of alcohols which is used as a cosolvent and can accelerate the reaction.
The above reagent may also further comprise at least one of acidic organic compounds and/or pigments.
Preferably, the buffer can adjust the range of pH between 2 and 6.
The cationic surfactant is preferably at least one of the quaternary ammonium salts represented by formula I,
wherein, R1 is C6-14 alkyl or C6-14 alkenyl, preferably hexyl, octyl, decyl, or dodecyl; R2 and R3 are independently C1-4 alkyl or C1-4 alkenyl, preferably methyl, ethyl, or propyl; R4 is C1-4 alkyl, C1-4 alkenyl, or benzyl; and X is a halogen atom.
The amphoteric surfactant is preferably at least one of the surfactants represented by formula II,
wherein, R5 is a C6-14 alkyl or C6-14alkenyl, preferably hexyl, octyl, decyl, or dodecyl; and R6 and R7 are independently C1-4 alkyl or C1-4 alkenyl, preferably methyl, ethyl, or propyl.
The present invention also relates to a method for classifying leukocytes using the reagent of the invention. The method comprises steps of adding said reagent to the whole blood sample for classifying leukocytes into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils, and counting each group by measuring the size and the shape thereof.
The measurement of the size is performed by using laser scattering with a low angle scattered light and the measurement angle ranges from 2 to 6 degree; and the measurement of shape is performed by using laser scattering with a high angle scattered light and the measurement angle ranges from 8 to 20 degree.
The advantages of the present invention are obvious through implementing the technical solution mentioned above, which lie in that:
The reagent and method of the present invention can not only classify the leukocytes into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils, but also can clearly differ monocytes from lymphocytes and neutrophils with obvious clustering effect. Moreover, various proportional monocytes can form individual scattered dot areas at stable locations and are not affected by the nucleus movement after the blood sample treated by reagents is measured by scattered light. Therefore, the resulting classification shows more accurately the real state of the blood.
The reagent and method of the present invention can not only divide leukocytes in the whole blood into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils, but also improve the accuracy of classification for the monocytes.
When adding the reagent of the present invention into the whole blood sample, the component of the reagent lyses erythrocytes rapidly and makes various types of leucocytes vary in size and morphology, thereby classifying leukocytes into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils. Then the laser scattering measurement is used, i.e., using the low angle scattered light to measure the size of the cells and the high angle scattered light to measure the morphological information of the cells, and thereby classifying and counting the leukocytes of four groups by detecting the scattered lights. The classified monocytes show obvious clustering effect and can be clearly differed from lymphocytes and neutrophils. Moreover, various proportional monocytes can form individual scattered dot areas at stable locations and are not affected by the nucleus movement after the blood sample treated by reagents is measured by scattered light. Therefore, the resulting classification shows more accurately the real state of the blood.
The main components of the reagent according to the present invention comprise:
a) a surfactant capable of lysing erythrocytes and partly damaging cell membrane of leucocytes, said surfactant is at least one of the surfactants selected from a cationic surfactant, an amphoteric surfactant, and the combination thereof;
b) an organic compound having phenyl or heterocyclic group, when said surfactant is a cationic surfactant, said organic compound is an organic compound having an anionic group; when said surfactant is an amphoteric surfactant or a combination of amphotheric and cationic surfactants, said organic compound is an organic compound having an anionic or cationic group;
(c) a buffer for adjusting pH.
The cationic surfactant is preferably at least one of quaternary ammonium salts represented by formula I,
Formula I represents a cationic surfactant having totally 9-26 carbon atoms, wherein, R1 is C6-14 alkyl or C6-14 alkenyl, preferably hexyl, octyl, decyl, dodecyl or tetradecyl, more preferably a linear alkyl comprising hexyl, octyl, decyl or dodecyl; R2 and R3 are independently C1-4 alkyl or C1-4 alkenyl, preferably methyl, ethyl, propyl, butyl or butenyl, more preferably methyl, ethyl or propyl; R4 is C1-4 alkyl, C1-4 alkenyl, or benzyl; and X is a halogen atom.
The amphoteric surfactant is preferably at least one of the surfactants represented by formula II,
wherein, R5 is C6-14 alkyl or C6-14 alkenyl, preferably hexyl, octyl, decyl, dodecyl or tetradecyl, more preferably a linear alkyl comprising octyl, decyl or dodecyl; R6 and R7 are independently C1-4 alkyl or C1-4 alkenyl, preferably methyl, ethyl, propyl, butyl, or butenyl, more preferably methyl, ethyl or propyl.
The surfactant is used in an amount sufficient to lyse erythrocytes and partly damage cell membranes of leucocytes. Typically, a suitable amount of the surfactant to be used in the improved reagent according to the invention is 50-6,000 mg/L, preferably 200-5,000 mg/L, more preferably 500-4,000 mg/L, though it can be suitably modified depending upon the type of surfactant used.
The reagent with suitable amount can completely separate monocytes from lymphocytes in size, and the monocytes have stable locations. Meanwhile, the monocytes can be easily differed from granulocytes in morphology, which benefits for cell counting.
Some surfactants and suitable amounts thereof according to the present invention are listed in Table 1.
A useful surfactant according to the present invention is capable of lysing the leucocytes to such an extent that it makes pores on cell membranes and the cytoplasm can overflow out from the leucocytes and an organic compound binding with a cationic group can enter, and the amount of the surfactant used is much less than that amount which is potent to make the cell nuclei naked. The effect of hemolysis for a surfactant is in proportion to the number of carbon atoms on the side chain thereof (i.e., R1 of cationic surfactant and R5 of amphoteric surfactant), the more the numbers of carbon atoms, the more the capacity of hemolysis, and further the required amount is lower.
Another main component of the reagent according to the present invention is organic compound having phenyl or heterocyclic group. When the used surfactant is a cationic surfactant, the organic compound is selected from an organic compound having an anionic group; when the surfactant used is an amphoteric surfactant or a combination of amphotheric and cationic surfactants, the organic compound is selected from an organic compound having an anionic group or a cationic group. The heterocyclic group is selected from those with 5 ring atoms or more.
An organic compound having an anionic or cationic group used in the present invention is capable of bonding with various ions in leukocytes, resulting in morphological differentiations among the various groups of leukocyte. Such organic compound contains phenyl or heterocyclic group having 5 or more ring atoms, and an anionic group (e.g., carboxyl or sulfonic group) or a cationic group (e.g., N+, or NH4+). A cationic organic compound may comprise pyridine, pyrrole, 2-pyridinyl thiourea, 2-naphthylamine, etc., and an anionic organic compound may comprise 1-naphthylacetic acid, naphthalene-1,4,5,8-tetracarboxylic acid, terephthalic acid, phthalic acid, chromotropic acid, naphthalene sulfonic acid and their derivatives and salts thereof. The amount of the organic compound in the reagent according to the present invention is typically 50-1000 mg/L, preferably 100-500 mg/L.
The reagent according to the present invention further comprises a buffer for adjusting pH. There are no particular limitations to the buffer. It may be a conventional buffer system, such as formic acid, phthalic acid, acetic acid, phosphoric acid, TRIS, boric acid, carbonic acid, etc. The range of pH has no essential influence on the classification of four groups, however the classification effect of monocytes can be improved by adjusting pH. In the present invention, a preferable pH of the reagent for classifying monocytes is 2-6, and the amount of buffer used in the reagent of the invention is 10-200 mM.
In addition, the reagent according to the present invention may also comprise an alcohol which is used as a cosolvent and can accelerate the reaction. There are no particular limitations to the alcohol used herein. It may be methanol, ethanol, iso-propanol or 2-phenoxyethanol, etc. The amount of an alcohol is 0.05-5% (v/v) relative to the total amount of the reagent of the present invention.
Further, to improve the separation effect of eosinophils from granulocytes, a certain amount of organic compounds or pigments can be added to the reagent of the present invention, such as acid blues, direct blue, acid greens, acid yellows, acid oranges, methyl reds, methyl orange, aniline blue or alizarin yellow, etc. The amount is 50-1000 mg/L, preferably 100-500 mg/L.
Further, a certain amount of NaCl can be added to the reagent of the present invention to adjust osmotic pressure of the solution to a suitable range.
The reagent of the present invention may be prepared as one reagent or a combination of two reagents. When using the combination of two reagents, the pHs of the two reagents can be the same or not.
The method of the present invention for classifying leukocytes comprises adding the reagent of the present invention to the whole blood sample and mixing for a period of time, detecting the size and morphological information of the various cells, thereby classifying leukocytes into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils, and one group corresponding to neutrophils and basophils according to the differences of the size and morphology of the cells, and meanwhile counting each group of cells.
The mixing ratio of the whole blood sample to the reagent of the invention has no particular limitation, and typically the ratio is 1:10-100. The measurement can be carried out after 30 seconds of the mixing.
The preferred method for measuring the size and morphology of cells is laser scattering measurement. The size is measured by the low angle scattered light with angle ranging from 2 to 6 degree, and the morphological information is measured by the high angle scattered light with angle ranging from 8 to 20 degree. A conventional photodiode sensor can be employed to detect the scattered light.
The following non-limiting examples illustrate the reagent and the method of the invention.
A reagent for dividing leukocytes into four groups□comprising:
The resultant reagent is adjusted to have pH of 3.8 and osmotic pressure of 135 mOsm.
Adding 0.98 mL of the above resultant reagent to 30 μL of blood sample A, and after mixing for 13 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
A reagent for dividing leukocytes into four groups comprising:
The resultant reagent is adjusted to have pH of 4.2 and osmotic pressure of 125 mOsm.
Adding 0.98 mL of the above resultant reagent to 30 μL of blood sample A, and after mixing for 13 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
A reagent for dividing leukocytes into four groups comprising:
The resultant reagent is adjusted to have pH of 2.5 and osmotic pressure of 100 mOsm.
Adding 2 mL of the above resultant reagent to 30 μL of blood sample B, and after mixing for 15 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
A reagent for dividing leukocytes into four groups comprising:
The resultant reagent is adjusted to have pH of 2.8 and osmotic pressure of 150 mOsm.
Adding 2 mL of the above resultant reagent to 30 μL of blood sample B, and after mixing for 15 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
A reagent for dividing leukocytes into four groups comprising:
The resultant reagent is adjusted to have pH of 3.5 and osmotic pressure of 136 mOsm.
Adding 2 mL of the above resultant reagent to 30 μL of blood sample C, and after mixing for 15 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
A reagent for dividing leukocytes into four groups comprising:
The resultant reagent is adjusted to have pH of 5.8 and osmotic pressure of 200 mOsm.
Adding 1.5 mL of the above resultant reagent to 30 μL of blood sample C, and after mixing for 18 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
The three blood samples A, B, and C employed in above examples contain monocytes with different proportions respectively. The results show that the leukocytes can be classified into four groups by the reagent and method of the present invention, and various proportional monocytes can form individual scattered dot areas at the stable locations which is not affected by the nucleus movement. Therefore, the result of classification is more accurately to show the real state of the blood.
Contrast Test
A reagent for dividing leukocytes into four groups comprising:
The resultant reagent is adjusted to have pH of 3.8 and osmotic pressure of 130 mOsm.
Adding 1.5 mL of the resultant reagent to 30 μL of blood samples A, B and C respectively, and after mixing for 15 seconds the leukocytes are measured by laser scattering measurement. The size information of cells is obtained by using the low angle scattered light with angle ranging from 2 to 6 degree, and the morphology information of cells is obtained by using the high angle scattered light with angle ranging from 8 to 20 degree.
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