Reagent for blood analysis and method of use thereof

• US 8,940,499 B2
• Filed: 10/16/2009
• Issued: 01/27/2015
• Est. Priority Date: 10/17/2008
• Status: Active Grant

• Current Assignee: Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Excelsior Union Ltd.)
• Original Assignee: Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Excelsior Union Ltd.)
• Inventors: Kuang, Yuji, Zhang, Baohua, Xu, Bing, Shao, Jianhui, Lei, Ting, Zhang, Li
• Primary Examiner(s): Desai, Rita
• Assistant Examiner(s): Michelson, Ben S
• Application Number:
  US12/580,474
• Publication Number:
  US 20100267080A1
• Time in Patent Office: 1,929 Days
• Field of Search: None
• US Class Current: 435/39
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1011 Condensed systems
• CPC Class Codes: C09K 2211/1029 containing one nitrogen atom as the heteroatom
• CPC Class Codes: C09K 2211/1037 with sulfur
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1402 Data analysis by thresholding or gating operations performed on the acquired signals or stored data
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/80 involving blood groups or blood types or red blood cells white blood cells G01N33/56972

Abstract

The present disclosure provides a reagent for blood analysis which may include: (1) a compound having the general formula I as a fluorescent dye, wherein n, X, R₁, R₂, R₃, R₄, R₅ and Y⁻ are as defined in the specification; (2) a surfactant selected from cationic surfactants, zwitterionic surfactants and anionic surfactants. The present disclosure also provides a method to perform blood analysis including the following steps of: (a) mixing the blood sample with the reagent for blood analysis disclosed to form a cell suspension; (b) detecting the scattered light signals and fluorescence signals from the cells; and (c) differentiating and counting the cells in the blood in terms of the scattered light signals and fluorescence signals.

Claims

1. A reagent for blood analysis, said reagent comprising:

(1) a compound having the general formula I as the fluorescent dye;

2. The reagent for blood analysis according to claim 1, wherein R₁ and R₂ are each independently selected from at least one of the following:

H, halogen and C_1-6alkylsulfonate, provided that R₁ and R₂ are not all simultaneously H.

3. The reagent for blood analysis according to claim 1, wherein R₃ is C_1-6alkyl or C_1-6alkylOR₅.

4. The reagent for blood analysis according to claim 1, wherein R₄ is C_1-6alkyl or C_1-6alkylOR₅.

5. The reagent for blood analysis according to claim 1, wherein R₅ is H, C_1-3alkylCO or C_1-6alkyl.

6. The reagent for blood analysis according to claim 1, wherein X is C(CH₃)₂, O or S.

7. The reagent for blood analysis according to claim 1, wherein n is 1 or 2.

8. The reagent for blood analysis according to claim 1, wherein Y⁻

is selected from halogen ions, ClO₄⁻

, PF₆⁻

, CF₃SO₃⁻

, BF₄⁻

, acetate and p-toluenesulfonate anions.

9. The reagent for blood analysis according to claim 1, wherein the compound having the general formula I is:

10. The reagent for blood analysis according to claim 1, wherein the compound having the general formula I has a concentration in the range of 1 to 100 mg/L.

11. The reagent for blood analysis according to claim 1, wherein the compound having the general formula I has a concentration in the range of 5 to 50 mg/L.

12. The reagent for blood analysis according to claim 1, wherein said surfactant is selected from anionic surfactants.

13. The reagent for blood analysis according to claim 1, wherein said surfactant is selected from zwitterionic surfactants.

14. The reagent for blood analysis according to claim 1, wherein said surfactant is selected from cationic surfactants.

15. The reagent for blood analysis according to claim 1, wherein said surfactant is selected from at least one of the following:

sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, cocoamidopropyl betaine, dodecyldimethyl betaine, dodecyltrimethylammonium chloride and decyltrimethylammonium chloride.

16. The reagent for blood analysis according to claim 1, said reagent further comprising a pH buffering agent selected from at least one of the following:

phosphate, Tris, HEPES, borate and combinations thereof.

17. The reagent for blood analysis according to claim 16, wherein said pH buffering agent has a pH value in the range of 6.0-10.0.

18. The reagent for blood analysis according to claim 16, wherein said pH buffering agent has a pH value in the range of 7.5-9.5.

19. The reagent for blood analysis according to claim 1, said reagent further comprising an osmotic regulating agent selected from alkali metal salts, glucose or mannitol.

20. The reagent for blood analysis according to claim 19, wherein said osmotic regulating agent maintains the osmotic pressure in the range of 170-350 mOsm/kg.

21. The reagent for blood analysis according to claim 19, wherein said osmotic regulating agent maintains the osmotic pressure in the range of 200-350 mOsm/kg.

22. The reagent for blood analysis according to claim 1, said reagent further comprising a preserving agent selected from parabens and isothiazolinones.

23. A method for blood analysis, said method comprising the following steps of:

(a) mixing a blood sample with the reagent for blood analysis according to claim 1 to form a cell suspension;

(b) detecting scattered light signals and fluorescence signals from cells in the cell suspension; and

(c) differentiating and counting the cells in the blood in terms of the scattered light signals and fluorescence signals.

24. The method according to claim 23, wherein the cells in said step (b) comprise reticulocytes, mature erythrocytes and leukocytes.

25. The method according to claim 24, wherein the cells comprise reticulocytes.

26. The method according to claim 23, wherein the blood sample and the reagent for blood analysis in said step (a) are mixed in a ratio in the range of 1:

100 to 1;

500.

27. The method according to claim 23, wherein the cell suspension in said step (a) is incubated at a reaction temperature of between 35-45°

C. for between 20-40 seconds.