Reagents and methods for classifying leukocytes
View Patent ↗The present invention relates to a reagent for classifying leukocytes including (a) at least one surfactant capable of lysing erythrocytes and partly damaging the cell membrane of leukocytes, (b) at least one organic compound bearing an anionic group capable of binding to the cationic component present in the leukocytes to provide morphological differences between the leukocytes, and (c) a buffer for adjusting pH to 2˜8. Also disclosed is a method for classifying leukocytes into four groups.
1. A reagent for classifying leukocytes into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils, the reagent comprises:
(a) at least one surfactant for lysing erythrocytes and partly damaging the cell membrane of leukocytes, the at least one surfactant is selected from the group consisting of:
one or more cationic surfactants;
one or more anionic surfactants;
one or more ampholytic surfactants;
a combination of one or more cationic surfactants and one or more ampholytic surfactants; and
a combination of one or more anionic surfactants and one or more ampholytic surfactants;
(b) at least one organic compound bearing an anionic group for binding with a cationic component present in the leukocytes to give morphological differences between the leukocytes; and
(c) a buffer for adjusting pH into a range of 2˜8;
wherein the reagent is free from nonionic surfactants.
2. The reagent according to claim 1 , further comprising an alcohol as a solubilizer.
3. The reagent according to claim 1 , wherein the cationic surfactant is a quaternary ammonium salt represented by formula II:
wherein R 3 is a C 6 -C 14 alkyl or alkenyl group, R 4 and R 5 each independently is a C 1 -C 4 alkyl or alkenyl group, R 6 is a C 1 -C 4 alkyl or alkenyl group or a benzyl group; and X is a halogen.
4. The reagent according to claim 1 , wherein the anionic surfactant is sodium laurylsulfonate or a anionic surfactant represented by formula III:
R 7 —R 8 —(CH 2 CH 2 O) n —Y III
wherein R 7 is a C 8 -C 22 alkyl, alkenyl or alkynyl, R 8 is —O— or —COO—, n is an integer of 8-30, and Y is —SO 3 Na, —COONa, —OSO 3 Na or —ONa.
5. The reagent according to claim 1 , wherein the ampholytic surfactant is represented by formula IV:
wherein R 9 is a C 6 -C 14 alkyl or alkenyl, R 10 and R 11 each independently is a C 1 -C 4 alkyl or alkenyl.
6. The reagent according to claim 1 , wherein the organic compound bearing an anionic group is an acidic dye.
7. A method for classifying leukocytes with the reagent according to any of claims 1 ˜ 6 , comprising the steps of: adding the regent to a whole blood sample to classify the leukocytes into four groups consisting of three groups corresponding to lymphocytes, monocytes and eosinophils and one group corresponding to neutrophils and basophils, and classifying and counting each group by measuring cell size information and information on morphological features.
8. The method according to claim 7 , wherein the classification is carried out at a temperature in the range of 10˜40° C.
9. The method according to claim 8 , wherein the classification is carried out at 35° C.
10. The method according to claim 8 , wherein a laser light scattering method with low angle scattered light of 2°˜6° is used to measure the cell size information, and a laser light scattering method with high angle scattered light of 8°˜20° is used to measure the information on morphological features.