Processing method for the long-term stabilization of biological red blood cell volume

• US 8,029,978 B2
• Filed: 12/30/2005
• Issued: 10/04/2011
• Est. Priority Date: 02/25/2005
• 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: Zhang, Hui, Xu, Zuyue, Liu, Mulong
• Primary Examiner(s): Menon; Krishnan S
• Assistant Examiner(s): Fritchman; Rebecca M
• Application Number:
  US11/323,702
• Publication Number:
  US 20060194191A1
• Time in Patent Office: 2,104 Days
• Field of Search: 436/176, 436/10, 436/17, 435/372, 435/2, 435/40.51
• US Class Current: 435/2
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/2525 Stabilizing or preserving

Abstract

The present invention relates to a processing method for long-term stabilization of biological red blood cell volume. The red blood cells can be further used in reference materials and calibrators of a hematology analyzer. The method of the present invention comprises contacting the sample with a solution comprising a membrane-fixation reagent and an aldehyde for a processing period to terminate or slow down the red blood cell metabolism. It is provided by experiments that such process enables the volume of the red blood cells to be stable in a period over 120 days.

Claims

1. A processing method for the long-term stabilization of biological red blood cell volume, comprising:

contacting red blood cells in a blood sample with a processing solution comprising a membrane-fixation reagent and an aldehyde,

wherein the membrane-fixation reagent is selected from a group consisting of dichromate salts, picric acid and salts of cadmium, wherein the membrane fixation reagent is added such that the concentration in the processing solution is 0.02%-0.5% (w/v), and wherein the aldehyde is selected from a group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, olefine aldehyde and combinations thereof; and

stabilizing the red blood cell volume in the blood sample with a coefficient of variation of 1.32% or less for over 30 days, excluding a changing period of the first 5 days.

2. The method according to claim 1, wherein the membrane-fixation reagent is potassium dichromate or cadmium chloride.

3. The method according to claim 1, wherein the aldehyde is formaldehyde, paraformaldehyde, or a mixture of formaldehyde and glutaraldehyde.

4. The method according to claim 1, wherein the contacting is carried out at room temperature.

6. The method according to claim 1, wherein the concentration of the aldehyde is 0.02%-0.3% (v/v).

7. The method according to claim 6, wherein the processing solution comprises 0.05% (w/v) potassium dichromate and 0.08% (v/v) formaldehyde.

8. The method according to claim 6, wherein the processing solution comprises 0.05% (w/v) cadmium chloride, 0.05% (v/v) formaldehyde and 0.02% (v/v) glutaraldehyde.

9. The method according to claim 1, wherein the processing solution is obtained by adding the membrane-fixation reagent and the aldehyde to a common nutrient-free buffer system such as citrate buffer, phosphate-buffered saline (PBS) and Tris buffer, and the final pH is between 7.1 and 8.5.

10. The method according to claim 9, wherein a nutrient-free buffer system comprising sodium citrate (1-10 g/L), sodium azide (1-5 g/L), potassium chloride (0.1-5 g/L), sodium sulfate (0.5-2 g/L), penicillin (0.1-2 g/L), streptomycin (0.1-2 g/L), neomycin (0.1-2 g/L), sodium chloride (1-8 g/L) and agarose or cellulose (0.2-10 mg/L) is used.

11. The method according to claim 1, wherein the biological RBCS are human RBCS, animal RBCS, or a mixed sample comprising RBCS from multiple species.

15. The method of claim 1, wherein the membrane-fixation reagent is selected from the group consisting of dichromate salts and picric acid.

16. The method of claim 1, wherein red blood cell volume is stabilized with a coefficient of variation of 1.32% or less for at least 120 days.

18. The method of claim 1, wherein red blood cell volume is stabilized with a coefficient of variation of 1.32% or less for at least 150 days.

20. The method of claim 1, wherein red blood cell volume is stabilized with a coefficient of variation of 1.32% or less for at least 60 days.

5. A processing method for the long-term stabilization of biological red blood cell volume, comprising:

contacting red blood cells in a blood sample with a processing solution comprising a membrane-fixation reagent and an aldehyde,

wherein the membrane-fixation reagent is selected from a group consisting of dichromate salts, picric acid and salts of cadmium, and wherein the aldehyde is selected from a group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, olefine aldehyde and combinations thereof, and

wherein the contacting of the blood sample with the processing solution lasts for a period of between 30 minutes and 3 hours, and

stabilizing the red blood cell volume in the blood sample with a coefficient of variation of 1.32% or less for over 30 days, excluding a changing period of the first 5 days.

12. A processing method for the long-term stabilization of biological red blood cell volume, comprising:

removing white blood cells from a blood sample to create a red blood cell sample;

stabilizing phospholipids in membranes of red blood cells with a membrane-fixation reagent, wherein the membrane fixation reagent is selected from a group consisting of dichromate salts, picric acid and salts of cadmium; and

stabilizing membrane proteins in membranes of red blood cells with a protein-fixation reagent comprising an aldehyde selected from a group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, olefine aldehyde and combinations thereof;

wherein the combination of the membrane-fixation reagent and the protein-fixation reagent is capable of stabilizing the red blood cell volume in the red blood cell sample with a coefficient of variation of 1.32% or less for over 30 days, excluding a changing period of the first 5 days.

13. The method of claim 12, further comprising buffering an environment of each step of the method at a pH of between 7.1 and 8.5.

14. The method of claim 12, wherein inhibiting the metabolism of red blood cells comprises using a red blood cell nutrient-free system.

17. The method of claim 12, wherein red blood cell volume is stabilized with a coefficient of variation of 1.32% or less for at least 120 days.

19. The method of claim 12, wherein red blood cell volume is stabilized with a coefficient of variation of 1.32% or less for at least 150 days.

21. The method of claim 12, wherein red blood cell volume is stabilized with a coefficient of variation of 1.32% or less for at least 60 days.