Lysing Reagent For Simultaneous Enumeration Of Different Types Of Blood Cells In A Blood Sample

• US 20080085560A1
• Filed: 10/20/2005
• Published: 04/10/2008
• Est. Priority Date: 10/20/2004
• Status: Abandoned Application

• Current Assignee: Chempaq A/S
• Original Assignee: Chempaq A/S
• Inventors: Ekberg, Bjorn
• Application Number:
  US11/665,753
• Publication Number:
  US 20080085560A1
• Time in Patent Office: Days
• US Class Current: 436/17
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

A combined diluent and lysing reagent for simultaneously counting or analysis of various blood cells such as thrombocytes, leukocytes and leukocyte subpopulations, as well as analysis of hemoglobin. The reagent comprises a hemoglobin stabilizing/methemoglobin ligand compound selected from the group consisting of imidazole, imidazole derivatives, and combinations thereof, at least two quaternary ammonium salts, 1,3-dimethylurea and/or salts thereof; and an organic buffer. Accordingly, the reagent can be used in a method for determining the content of blood cells in a blood sample comprising: mixing a blood sample with a reagent according to the invention; and analyzing the content of the blood sample by employment of the Coulter principle and optionally an spectrophotometric analysis of the hemoglobin content.

Claims

1-32. -32. (canceled)

33. A reagent comprising

a hemoglobin-stabilizing compound selected from the group consisting of imidazole, imidazole derivatives, and combinations thereof;

at least two quaternary ammonium salts;

1,3-dimethylurea and/or salts thereof; and

an organic buffer.

34. A reagent according to claim 33, wherein the concentration of the hemoglobin-stabilizing compound selected from the group consisting of imidazole, imidazole derivatives, and combinations thereof in the reagent is in the range from about 10 mmol/L to about 100 mmol/L, such as from about 25 mmol/L to about 75 mmol/L, from about 40 mmol/L to about 60 mmol/L, and preferably about 50 mmol/L.

35. A reagent according to claim 33, where the hemoglobin-stabilizing compound is imidazole in a concentration of about 50 mmol/L.

36. A reagent according to claim 33, wherein the at least two quaternary ammonium salts comprise a combination of dodecyltrimethylammonium chloride and hexadecyltrimethylammonium bromide.

37. A reagent according to claim 33, wherein the total concentration of the at least two quaternary ammonium salts in the reagent is in the range from about 1.0 mmol/L to about 10 mmol/L, such as from about 3.0 mmol/L to about 9.0 mmol/L, from about 4.5 mmol/L to about 8.5 mmol/L, and preferably about 6.5 mmol/L.

38. A reagent according to claim 33, wherein the at least two quaternary ammonium salts is a combination of dodecyltrimethylammonium chloride and hexadecyltrimethylammonium bromide in concentrations of about 5.6 mmol/L and about 0.9 mmol/L, respectively.

39. A reagent according to claim 33, wherein the concentration of 1,3-dimethylurea and/or salts thereof in the reagent is in the range from about 10 mmol/L to about 25 mmol/L, such as from about 12 mmol/L to about 23 mmol/L, from about 14 mmol/L to about 20 mmol/L, from about 16 mmol/L to about 18 mmol/L, and preferably about 17 mmol/L.

40. A reagent according to claim 33, wherein the 1,3-dimethylurea and/or salts thereof is 1,3-dimethylurea in a concentration of about 17 mmol/L.

41. A reagent according to claim 33, wherein the reagent further comprises procaine, procaine hydrochloride or other salts thereof.

42. A reagent according to claim 41, wherein the concentration of procaine hydrochloride in the reagent is in the range from about 0.2 mmol/L to about 1.6 mmol/L, such as from about 0.4 mmol/L to about 1.4 mmol/L, from about 0.6 mmol/L to about 1.2 mmol/L, from about 0.8 mmol/L to about 1.0 mmol/L, and preferably about 0.9 mmol/L.

43. A reagent according to claim 33, wherein the organic buffer is a compound such as ADA (N-2-(acetamido)iminodiacetic acid), HEPES (2-(4(2-hydroxyethyl)-1-piperazine)ethanesulphonic acid), MOPS (3-(N-morpholino)propane sulphonic acid), PIPES (piperazine-N,N′

-bis(2-ethane sulphonic acid) or combinations thereof, with a concentration in the range from about 2 mmol/L to about 8 mmol/L, such as from about 4 mmol/L to about 6 mmol/L, from about 5 mmol/L to about 5.6 mmol/L, and preferably about 5.3 mmol/L.

44. A reagent according to claim 33, wherein the organic buffer is ADA (N-2-(acetamido)iminodiacetic acid) with a concentration in the range from about, 2 mmol/L to about 8 mmol/L, such as from about 4 mmol/L to about 6 mmol/L, from about 5.0 mmol/L to about 5.6 mmol/L, and preferably about 5.3 mmol/L.

45. A reagent according to claim 33, wherein the organic buffer is ADA (N-2-(acetamido)iminodiacetic acid) with a concentration of about 5.3 mmol/L.

46. A reagent according to claim 33, wherein the osmolality of the reagent is adjusted with at least one inorganic salt, such as sodium chloride, and/or sodium sulphate.

47. A reagent according to claim 33, wherein the osmolality of the reagent is adjusted with sodium chloride and sodium sulphate.

48. A reagent according to claim 46, wherein the osmolality of the reagent is in the range from about 200 mosmol/L to about 400 mosmol/L, such as from about 250 mosmol/L to about 380 mosmol/L, from about 300 mosmol/L to about 350 mosmol/L, and preferably about 330 mosmol/L

49. A reagent according to claim 33, wherein the pH of the reagent is adjusted with an acid, and the pH of the reagent is in the range from about 6 to about 8, such as from about 6.5 to about 7.5, and preferably about 7.1.

50. A reagent according to claim 33, comprising imidazole in a concentration of about 50 mmol/L;

dodecyltrimethylammonium chloride in a concentration of about 5.6 mmol/L;

hexadecyltrimethylammonium bromide in a concentration of about 0.9 mmol/L;

1,3-dimethylurea in a concentration of about 17 mmol/L;

ADA (N-2-(acetamido)iminodiacetic acid) in a concentration of 5.3 mmol/L;

procaine hydrochloride in a concentration of 0.9 mmol/L; and

de-ionized water;

wherein pH is adjusted to 7.1 with diluted sulfuric acid and the osmolality is adjusted with sodium chloride and sodium sulphate.

51. A coating comprising a reagent as defined in claim 33.

52. A method of determining the content of blood cells in a blood sample, the method comprising the steps of:

i) Mixing a blood sample with a reagent as defined in claim 33, and

ii) Analyzing the content of the blood sample by employment of the Coulter principle.

53. A method according to claim 52, wherein the thrombocyte content is determined by step ii).

54. A method according to claim 52, wherein the leukocyte content is determined by step ii).

55. A method according to claim 52, wherein one or more leukocyte subpopulations such as, e.g., the lymphocytes, monocytes and/or granulocytes are determined by step ii).

56. A method according to claim 52, wherein the method further comprises

iii) Analyzing the content of the blood sample by spectrophotometric quantification of the hemoglobin content.

57. A method according to claim 52, wherein the blood:

reagent-ratio by volume is in the range from about 1;

10,000 to about 1;

200, such as from about 1;

1,000 to about 1;

300, from about 1;

600 to about 1;

400, from about 1;

550 to about 1;

450, and preferably about 1;

500.

58. A method according to claim 52, wherein

step i) is performed in a cartridge suitable for use in an apparatus for enumeration of cells in a blood sample, in which cartridge the reagent is present in a holding chamber that is connected with a mixing chamber in which the reagent is mixed with the blood sample;

and

step ii) is performed by subsequent passage of the blood sample comprising cells through an orifice positioned between the mixing chamber and a collection chamber, whereby the content of the blood is analyzed by employment of the Coulter principle by cell characterization means.

59. A method according to claim 52, wherein step iii) is performed in a volume metering chamber connected to the collection chamber and adapted to spectrophotometric quantification.

60. Use of a reagent as defined in claim 33, for determination of the content of thrombocytes in a blood sample.

61. Use according to claim 60, for simultaneous determination of the content of leukocytes in the same blood sample.

62. Use according to claim 60, for simultaneous determination of one or more leukocyte subpopulations in the same blood sample.

63. Use according to claim 62, wherein the leukocyte subpopulation includes lymphocytes, monocytes and/or granulocytes.

64. Use according to claim 60, for determination of the hemoglobin content in the same blood sample.