Means for separating lymphocytes and monocytes from anticoagulated blood

• US 4,190,535 A
• Filed: 07/10/1978
• Issued: 02/26/1980
• Est. Priority Date: 02/27/1978
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Corning Glass Works Inc. (Corning Incorporated)
• Inventors: Luderer, Albert A., Odstrchel, Gerald, Zine, Anthony R. Jr.
• Primary Examiner(s): Hart, Charles N.
• Assistant Examiner(s): Cintins, Ivars
• Application Number:
  US05/922,825
• Time in Patent Office: 596 Days
• Field of Search: 23/230 B, 128/2 F, 128/2 G, 128/DIG. 5, 210/83, 210/84, 210/205, 210/207, 210/516, 210/518, 210/DIG. 23, 210/DIG. 24, 233/1 R, 233/1 A, 233/26
• US Class Current: 210/789
• CPC Class Codes: G01N 33/491 by separating the blood components G01N15/05 takes precedence

Abstract

This invention relates to a method for isolating platelets, lymphocytes, and monocytes from anticoagulated blood. The method involves centrifuging at high force a sample of uncoagulated blood with a water-insoluble thixotropic gel-like material having a specific gravity between 1.065-1.077 g/cc for a length of time sufficient to cause the gel-like material to form a barrier between the platelets, lymphocytes, monocytes, and plasma fraction of the blood and the heavier blood cells. Thereafter, the plasma is withdrawn and the platelets, lymphocytes, and monocytes removed from above the barrier in a buffer solution. Where desired, the platelets can be separated from the lymphocytes and monocytes utilizing a similar process but wherein the water-insoluble thixotropic gel-like material has a specific gravity of less than about 1.055 g/cc.

Claims

1. A method for separating platelets, lymphocytes, and monocytes from anticoagulant blood comprising the steps:

(a) a water-insoluble thixotropic gel-like substance which is chemically inert to blood constituents and having a specific gravity between 1.065-1.077 g/cc is placed in a sample of anticoagulated blood;

(b) the gel-blood sample is centrifuged at a force of at least 1200 G'"'"'s for a sufficient length of time to cause the gel-like substance to form a barrier between the plasma, platelets, lymphocytes, and monocytes and the heavier blood cells; and

, thereafter,

(c) the plasma, platelets, lymphocytes, and monocytes are removed from atop said barrier.

2. A method according to claim 1 wherein said gel-like substance consists of a mixture of silicone fluid and a hydrophobic powdered silica.

3. A method according to claim 1 wherein said gel-like substance consists of a mixture of hydrocarbon polymer and a powdered silica.

4. A method according to claim 3 wherein said hydrocarbon polymer is selected from the group of a butylene polymer composed predominantly of high molecular weight mono-olefins with the balance being isoparaffins, and a hydroxyl terminated hompolymer butadiene with the degree of polymerization being about 50.

5. A method according to claim 1 wherein said gel-like substance has a thixotropic index greater than 1 and up to 10.

6. A method according to claim 1 wherein said centrifugation is conducted at a force between 1200-2500 G'"'"'s.

7. A method according to claim 1 wherein said centrifugation is conducted for a period of about 5-10 minutes.

8. A method for separating platelets, lymphocytes, and monocytes from anticoagulant blood comprising the steps:

(a) a water-insoluble thixotropic gel-like substance which is chemically inert to blood constituents and having a specific gravity between 1.065-1.077 g/cc is placed in a sample of anticoagulated blood;

(b) the gel-blood sample is centrifuged at a force of at least 1200 G'"'"'s for a sufficient length of time to cause the gel-like substance to form a barrier between the plasma, platelets, lymphocytes and monocytes and the heavier blood cells;

(c) the plasma is removed from atop said barrier leaving said platelets, lymphocytes and monocytes resting thereupon;

(d) a buffer solution compatible with platelets, lymphocytes, and monocytes, inert to the gel-like substance, and having a specific gravity less than that of the gel-like substance is deposited upon said barrier;

(e) the buffer solution is agitated to cause the said platelets, lymphocytes, and monocytes to become suspended therein; and

then

(f) the suspension of platelets, lymphocytes, and monocytes in buffer is removed from contact with said barrier.

9. A method according to claim 8 wherein said gel-like substance consists of a mixture of silicone fluid and a hydrophobic powdered silica.

10. A method according to claim 8 wherein said gel-like substance consists of a mixture of hydrocarbon polymer and a powdered silica.

11. A method according to claim 10 wherein said hydrocarbon polymer is selected from the group of a butylene polymer composed predominantly of high molecular weight mono-olefins with the balance being isoparaffins, and a hydroxyl terminated homopolymer of butadiene with the degree of polymerization being about 50.

12. A method according to claim 8 wherein said gel-like substance has a thixotropic index greater than 1 and up to 10.

13. A method according to claim 8 wherein said centrifugation is conducted at a force between 1200-2500 G'"'"'s.

14. A method according to claim 8 wherein said centrifugation is conducted for a period of about 5-10 minutes.

15. A method according to claim 8 wherein said buffer solution is an isotonic Ca⁺²,Mg⁺² -free salt solution.

16. A method for separating platelets from lymphocytes and monocytes comprising the steps:

(a) a water-insoluble thixotropic gel-like substance which is chemically inert to blood constituents and having a specific gravity between 1.03-1.055 g/cc is placed in a blood plasma sample of anticoagulated blood containing platelets, lymphocytes, and monocytes;

(b) the gel-plasma sample is centrifuged at a force of at least 1200 G'"'"'s for a sufficient length of time to cause the gel-like substance to form a barrier between the plasma and platelets and the lymphocytes and monocytes; and

, thereafter,

(c) the plasma and platelets are removed from atop said barrier.

17. A method according to claim 16 wherein said gel-like substance consists of a mixture of silicone fluid and a hydrophobic powdered silica.

18. A method according to claim 16 wherein said gel-like substance consists of a mixture of hydrocarbon polymer and a powdered silica.

19. A method according to claim 18 wherein said hydrocarbon polymer is selected from the group of a butylene polymer composed predominantly of high molecular weight mono-olefins with the balance being isoparaffins, and a hydroxyl terminated homopolymer butadiene with the degree of polymerization being about 50.

20. A method according to claim 16 wherein said gel-like substance has a thixotropic index greater than 1 and up to 10.

21. A method according to claim 16 wherein said centrifugation is conducted at a force between 1200-2500 G'"'"'s.

22. A method according to claim 16 wherein said centrifugation is conducted for a period of about 5-10 minutes.

23. A method for separating platelets from anticoagulant blood comprising the steps:

(a) a water-insoluble thixotropic gel-like substance which is chemically inert to blood constituents and having a specific gravity between 1.03-1.055 g/cc is placed in a sample of anticoagulated blood;

(b) the gel-blood sample is centrifuged at a force of at least 1200 G'"'"'s for a sufficient length of time to cause the gel-like substance to form a barrier between the plasma and platelets and the heavier blood cells; and

, thereafter,

(c) the plasma and platelets are removed from atop said barrier.

24. A method according to claim 23 wherein said gel-like substance consists of a mixture of silicone fluid and a hydrophobic powdered silica.

25. A method according to claim 23 wherein said gel-like substance consists of a mixture of hydrocarbon polymer and a powdered silica.

26. A method according to claim 25 wherein said hydrocarbon polymer is selected from the group of a butylene polymer composed predominantly of high molecular weight mono-olefins with the balance being isoparaffins, and a hydroxyl terminated homopolymer butadiene with the degree of polymerization being about 50.

27. A method according to claim 23 wherein said gel-like substance has a thixotropic index greater than 1 and up to 10.

28. A method according to claim 23 wherein said centrifugation is conducted at a force between 1200-2500 G'"'"'s.

29. A method according to claim 23 wherein said centrifugation is conducted for a period of about 5-10 minutes.