Fluorescent compounds and their use for measuring reticulocytes

Claim 2

A compound according to claim 1 wherein R 2 and R 3 are hydrogen atoms; R 4 is hydrogen atom, an acyl or a lower alkyl group; R 5 is hydrogen atom or an optionally substituted lower alkyl group.

Claim 3

A compound according to claim 1 wherein; R 1 is methyl, R 2 and R 3 are hydrogen atoms, R 4 and R 5 are hydrogen atoms, n = 1, and Z is sulfur atom.

Claim 4

A compound according to claim 1 wherein; R 1 is methyl, R 2 and R 3 are hydrogen atoms, R 4 is hydrogen atom, R 5 is hydroxymethyl, n = 1, and Z is sulfur atom.

Claim 5

A compound according to claim 1 wherein; R 1 is methyl, R 2 and R 3 are hydrogen atoms, R 4 is acetyl group, R 5 is hydrogen atom, n = 1, and Z is sulfur atom.

Claim 6

A compound according to claim 1 wherein; R 1 is methyl, R 2 and R 3 are hydrogen atoms, R 4 and R 5 are hydrogen atoms, n = 2, and Z is sulfur atom.

Claim 7

A reagent for dyeing reticulocytes comprising a compound represented by the formula (I) of claim 1.

Claim 8

A reagent for dyeing reticulocytes according to claim 7 further comprising a multivalent anion for inhibiting non-specific dyeing of erythrocytes and a buffer.

Claim 9

A reagent according to claim 8 wherein the multivalent anion is at least one selected from sulfuric ion, phosphoric ion and multivalent carboxylic acid ion.

Claim 10

A reagent according claim 7 which has a pH of 6.0 to 11.0.

Claim 11

A reagent according to claim 7 further comprising a cationic surfactant as a dyeing accelerator.

Claim 12

A reagent according to claim 11 in which the cationic surfactant has the formula: wherein R 6 is a C 8-12 alkyl group; R 7 , R 8 and R 9 are lower alkyl groups; X is at least one selected from the halogen group.

Claim 13

A reagent according to claim 11 wherein the cationic surfactant is at least one selected from lauryltrimetylammonium chloride, decyltrimetylammonium bromide and octyltrimetylammonium bromide.

Claim 14

A reagent according to claim 7 comprising (i) a fluorescent compound of the formula (I) in a concentration of 0.1 - 100mg/l, (ii) a multivalent anion providing 50% or more of the whole anions in the reagent, (iii) a buffer and (iv) an osmotic pressure compensating reagent in an amount sufficient for maintaining an osmotic pressure of 150 to 600m Osm/kg.

Claim 16

A method of measuring reticulocytes according to claim 15 comprising the steps of (i) preparing a sample to be measured by mixing a reagent for dyeing reticulocytes comprising a compound represented by the formula (I) and a hematological sample; (ii) introducing the sample to be measured into a flowing system of a flow cytometer provided with a light source emitting red wavelength light to form a sheath flow; (iii) irradiating the red wavelength light to cells in the sample flowing in the sheath flow; (iv) measuring scattered light and fluorescence light radiated from the cells to obtain the intensities thereof; (v) detecting platelets and erythroid cells by the scattered light intensity or by the scattered light intensity and the fluorescence intensity; (vi) detecting erythrocytes, reticulocytes and leukocytes by the fluorescence intensity or by the fluorescence intensity and the scattered light intensity; and (vii) classifying and counting the platelets, the erythrocytes, the reticulocytes and the leukocytes and calculating the percentages thereof.

Claim 17

A method of measuring reticulocytes according to claim 16 wherein the red wavelength light source is a He-Ne laser or a semiconductor laser emitting red light.

Claim 18

A method of measuring reticulocytes according to claim 16 further comprising calculating the ratio of reticulocytes having a fluorescence intensity within at least one specific range.