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US 5,496,734 AGranted
Treatment method for blood analysis
AssigneeToa Medical Electronics Co., Ltd.
InventorTakashi Sakata
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Abstract
The present invention is a method for blood analysis which provides a rapid treatment of a blood sample so that it can be analysed for counting and classification of leukocytes. Leukocytes are permeablized by treatment with a surfactant solution and labeled, preferably with a fluorescent dye. The method provides labeled leukocytes that can be counted and classified by optical means, including flow cytometry by analysis of a fluorescence signal.
Claims14
A method for classifying and counting leukocytes by an optical means which comprises;
- (i) treating a blood sample containing leukocytes with an aqueous solution having a pH ranging from 5.0 to 11.0 and comprising an ionic surfactant and a fluorescent dye, to give an optical difference between eosinophils and other leukocytes, wherein
- the ionic surfactant is used at a concentration effective to not damage the whole of the cell membrane of leukocytes in the blood sample but effective to damage a part of the cell membrane so as to allow the fluorescent dye to enter the leukocytes and stain intracellular material, and wherein said ionic surfactant is at least one selected from the group consisting of:
- a) a quaternary ammonium salt of the following formula:
- ##STR7## wherein R.sub.1, R.sub.2 and R.sub.3 are independently a hydrogen atom, a C.sub.1 -C.sub.8 alkyl group or a C.sub.6 -C.sub.8 aralkyl group;
- R.sub.4 is a C.sub.8 -C.sub.18 alkyl group, a C.sub.8 -C.sub.18 alkenyl group or a C.sub.8 -C.sub.18 aralkyl group;
- and X.sup.- is an anion,
- b) a pyridinium salt surfactant of the following formula:
- ##STR8## wherein R5 is a C8-C18 alkyl group;
- and X.sup.- is an anion, and c) an amphoteric surfactant of the following formula:
- ##STR9## wherein R.sub.6 and R.sub.7 are independently a hydrogen atom or a C.sub.1 -C.sub.8 alkyl group;
- R.sub.8 is a C.sub.8 -C.sub.18 alkyl group;
- and n is 1 or 2;
- and (II) classifying and counting the leukocytes by measuring an optical difference between eosinophils and other types of leukocytes in the blood sample based on at least two kinds of light selected from the group consisting of forward scattered light, side scattered light, red fluorescence light and green fluorescence light.
10 more elements
A method of classifying and counting leukocytes by an optical means comprising;
- (i) treating a blood sample containing leukocytes with an aqueous solution having a pH ranging from 3.0 to 5.0 and containing an ionic surfactant, a nonionic surfactant, a water-soluble alcohol having at least two carbon atoms, and Acridine Orange 10 dodecyl bromide, to give an optical difference among groups of leukocytes comprising lymphocytes, monocytes, granulocytes and other types of leukocytes;
- wherein the ionic surfactant is used at a concentration effective to not damage the whole of the cell membrane of leukocytes in the blood sample but effective to damage a part of the cell membrane so as to allow the Acridine Orange 10 dodecyl bromide to enter the leukocytes and stain intracellular material and wherein the ionic surfactant is at least one quaternary ammonium salt of the following formula:
- ##STR11## wherein R.sub.1, R.sub.2 and R.sub.3 are independently a hydrogen atom, a C.sub.1 -C.sub.8 alkyl group or a C.sub.6 -C.sub.8 aralkyl group;
- R.sub.4 is a C.sub.8 -C.sub.18 alkyl group, a C.sub.8 -C.sub.18 alkenyl group or a C.sub.8 -C.sub.18 aralkyl group;
- and X.sup.- is an anion, and wherein the nonionic surfactant is of the following formula:
- R.sub.9 R.sub.10 (CH.sub.2 CH.sub.2 O).sub.n H
- wherein R.sub.9 is a C.sub.8 -C.sub.22 alkyl group or a C.sub.8 -C.sub.22 alkenyl group;
- R.sub.10 is ##STR12## n is an integer not less than 10;
- and (ii) classifying and counting the leukocytes by measuring an optical difference in at least two kinds of light selected from the group consisting of forward scattered light, side scattered light, red fluorescence light and green fluorescence light among groups of leukocytes comprising lymphocytes, monocytes, granulocytes and other types of leukocytes.
6 more elements
Dependencies are inferred from the claim text.