Method of preparing specimen for counting subpopulations of leukocytes and erythroblasts

Claim 1

A method for preparing a specimen for classifying and counting blood corpuscles into at least eight groups, namely, two comprising immature granulocytes group 1 and immature granulocytes group 2, one comprising erythroblasts, one comprising basophils, one comprising eosinophils, one comprising lymphocytes, one comprising monocytes and one comprising neutrophils, by assaying a single specimen with a flow cytometer, which comprises the following steps: (1) a step for eliminating influences of erythrocytes from a hematological sample without changing the leukocytes morphology comprising: i) fragmentizing said erythrocytes contained in said hematological sample by adding a first aqueous solution of a low osmotic pressure to said sample to form a mixture, said aqueous solution comprising a buffer for maintaining the mixture within an acidic pH range of the hematological sample and for damaging the cell membranes of erythroblasts only; ii) adding to said mixture of hematological sample and first aqueous solution obtained in i) a second solution comprising an osmolarity compensating agent for maintaining the morphology of leukocytes and a buffer for neutralizing the first aqueous solution and for adjusting the pH to a level suitable for staining; and (2) a step for staining said blood corpuscles contained in said hematological sample with at least four dyes specified below: i) Astrazon Yellow 3G capable of differentially staining at least basophils and immature granulocytes; ii) Neutral Red capable of differentially staining at least eosinophils; iii) a dye capable of differentially staining at least either or both of nuclei and cytoplasm of leukocytes; and iv) a fluorochrome capable of staining exclusively nuclei of the damaged erythroblasts.

Claim 2

A method for preparing a specimen for classifying and counting blood corpuscles into at least eight groups, namely, two comprising immature granulocytes group 1 and immature granulocytes group 2, one comprising erythroblasts, one comprising basophils, one comprising eosinophils, one comprising lymphocytes, one comprising monocytes and one comprising neutrophils, by assaying a single specimen with a flow cytometer, which comprises the following steps: (1) a step for eliminating influences of erythrocytes from a hematological sample without changing the leukocytes morphology comprising: i) fragmentizing said erythrocytes contained in said hematological sample by adding a first aqueous solution of a low osmotic pressure to said sample to form a mixture, said aqueous solution comprising a buffer for maintaining the mixture within an acidic pH range of the hematological sample and for damaging the cell membranes of erythroblasts only; ii) adding to said mixture of hematological sample and first aqueous solution obtained in i) a second solution comprising an osmolarity compensating agent for maintaining the morphology of leukocytes and a buffer for neutralizing the first aqueous solution and for adjusting the pH to a level suitable for staining; and (2) a step for staining said blood corpuscles contained in said hematological sample with at least four dyes specified below: i) Astrazon Yellow 3G capable of differentially staining at least basophils and immature granulocytes; ii) Neutral Red capable of differentially staining at least eosinophils; iii) a dye capable of differentially staining at least either or both of nuclei and cytoplasm of leukocytes; and iv) a fluorochrome capable of staining exclusively nuclei of the damaged erythroblasts.

Claim 5

A method for preparing a specimen for classifying and counting blood corpuscles into at least nine groups, namely, two comprising immature granulocytes group 1 and immature granulocytes group 2, one comprising erythroblasts, one comprising blasts, one comprising basophils, one comprising eosinophils, one comprising lymphocytes, one comprising monocytes and one comprising neutrophils, by assaying a single specimen with a flow cytometer, which comprises the following steps: (1) a step for eliminating influences of erythrocytes from a hematological sample without changing the leukocytes morphology comprising: i) fragmentizing said erythrocytes contained in said hematological sample by adding a first aqueous solution of a low osmotic pressure to said sample to form a mixture, said aqueous solution comprising a buffer for maintaining the mixture within an acidic pH range of the hematological sample and for damaging the cell membranes of erythroblasts only; ii) adding to said mixture of hematological sample and first aqueous solution obtained in i) a second solution comprising an osmolarity compensating agent for maintaining the morphology of leukocytes and a buffer for neutralizing the first aqueous solution and for adjusting the pH to a level suitable for staining; and iii) lysing the erythrocytes fragmentized in the above i) or ii) with a nonionic surfactant; iv) adding a salt, which dissociates into ions in aqueous solutions of said salt to thereby maintain electrical conductivity of such solution at a suitable level, to the specimen to be finally prepared so as to control the electrical conductivity of said specimen to a level suitable for measuring with a device provided with an electrical resistance assay system, thus making it possible to accurately determine cell volume; and (2) a step for staining said blood corpuscles contained in said hematological sample with at least four dyes specified below: i) Astrazon Yellow 3G capable of differentially staining at least basophils and immature granulocytes; ii) Neutral Red capable of differentially staining at least esoinophils; iii) a dye capable of differentially staining at least either or both of nuclei and cytoplasm of leukocytes; and iv) a fluorochrome capable of staining exclusively nuclei of the damaged erythroblasts.

Claim 6

A method for preparing a specimen for classifying and counting blood corpuscles into at least nine groups, namely, two comprising immature granulocytes group 1 and immature granulocytes group 2, one comprising erythroblasts, one comprising blasts, one comprising basophils, one comprising eosinophils, one comprising lymphocytes, one comprising monocytes and one comprising neutrophils, by assaying a single specimen with a flow cytometer, which comprises the following steps: (1) a step for eliminating influences of erythrocytes from a hematological sample without changing the leukocytes morphology comprising: i) fragmentizing said erythrocytes contained in said hematological sample by adding a first aqueous solution of a low osmotic pressure to said sample to form a mixture, said aqueous solution comprising a buffer for maintaining the mixture within an acidic pH range of the hematological sample and for damaging the cell membranes of erythroblasts only; ii) adding to said mixture of hematological sample and first aqueous solution obtained in i) a second solution comprising an osmolarity compensating agent for maintaining the morphology of leukocytes and a buffer for neutralizing the first aqueous solution and for adjusting the pH to a level suitable for staining; and iii) lysing the erythrocytes fragmentized in the above i) or ii) with a nonionic surfactant; iv) adding a salt, which dissociates into ions in aqueous solutions of said salt to thereby maintain electrical conductivity of such solution at a suitable level, to the specimen to be finally prepared so as to control the electrical conductivity of said specimen to a level suitable for measuring with a device provided with an electrical resistance assay system, thus making it possible to accurately determine cell volume; and (2) a step for staining said blood corpuscles contained in said hematological sample with at least four dyes specified below: i) Astrazon Yellow 3G capable of differentially staining at least basophils and immature granulocytes; ii) Neutral Red capable of differentially staining at least esoinophils; iii) a dye capable of differentially staining at least either or both of nuclei and cytoplasm of leukocytes; and iv) a fluorochrome capable of staining exclusively nuclei of the damaged erythroblasts.