US4957638A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for separating the cellular components of blood samples
Assignee: Becton Dickinson and Co
•
Filing: 1989-05-09
•
Granted: 1990-09-18
•
Expires: 2009-05-09
Abstract
Abstract An assembly for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood. A water insoluble, thixotropic gel-like substance, which is chemically inert to blood constituents is provided in a container. A fluid capable of altering blood cell densities and diameters is positioned above the surface of the water-insoluble thixotropic gel-like substance. Means for preventing the absorption of water by the thixotropic gel-like substance from the fluid and/or the sample of unseparated whole blood prior to separation are provided to substantially eliminate the influence of water absorption on the cell separation performance characteristics of said thixotropic gel-like substance. A method for separating lymphocytes and monocytes from granulocytes which inhibits the apparent shift of the buoyant density of the granulocytes and which substantially eliminates the influence of water absorption on the cell separation performance characteristics of the thixotropic gel-like substance is also disclosed.
Inventors
- 👤 Ward C. Smith
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 07:56
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/483; G01N33/487; G01N33/49; G01N33/491; Y; Y10; Y10T; Y10T436/00; Y10T436/25; Y10T436/25375
Claims Analysis
Total Claims: 38
Claim 1
Independent Claim
184 words
An assembly for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood and inhibiting any apparent shift in the buoyant density and restoring any loss in buoyant density of the granulocytes which comprises: (a) a container having an open end and a closed end; (b) a water insoluble, thixotropic gel-like substance, which is chemically inert to blood constituents, positioned adjacent said closed end; (c) a chemical reagent positioned adjacent said thixotropic gel-like substance, said chemical reagent being provided to alter the osmolarity of said blood, thereby changing cell diameters and cell densities of said granulocytes; (d) a free space initially adjacent and above said chemical reagent, said free space of sufficient volume to contain said sample of unseparated whole blood; and (e) means for preventing the absorption of water by said thixotropic gel-like substance from at least one of said chemical reagent and said sample of unseparated whole blood prior to separating said lymphocytes and monocytes from said granulocytes so as to substantially eliminate the influence of water absorption on the cell separation performance characteristics of said thixotropic gel-like substance.
Claim 2
Dependent Claim
184 words
An assembly for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood and inhibiting any apparent shift in the buoyant density and restoring any loss in buoyant density of the granulocytes which comprises: (a) a container having an open end and a closed end; (b) a water insoluble, thixotropic gel-like substance, which is chemically inert to blood constituents, positioned adjacent said closed end; (c) a chemical reagent positioned adjacent said thixotropic gel-like substance, said chemical reagent being provided to alter the osmolarity of said blood, thereby changing cell diameters and cell densities of said granulocytes; (d) a free space initially adjacent and above said chemical reagent, said free space of sufficient volume to contain said sample of unseparated whole blood; and (e) means for preventing the absorption of water by said thixotropic gel-like substance from at least one of said chemical reagent and said sample of unseparated whole blood prior to separating said lymphocytes and monocytes from said granulocytes so as to substantially eliminate the influence of water absorption on the cell separation performance characteristics of said thixotropic gel-like substance.
Claim 3
Dependent Claim
52 words
The assembly of claim 1 wherein said means for preventing the absorption of water is provided by fabricating said thixotropic gel-like substance from an organic resin which allows high density and high viscosity polymers to form, so that said thixotropic gel-like substance is substantially devoid of inorganic fillers which can absorb water.
Claim 4
Dependent Claim
31 words
The assembly of claim 1 wherein the thixotropic gel is formed at least in part from one of a silicone oil, a butadiene resin, a polyester resin, and a butylene resin.
Claim 5
Dependent Claim
37 words
The assembly of claim 1 wherein said means for preventing the absorption of water is provided by at least one of presaturating said thixotropic gel-like substance with water during manufacture and curing of said thixotropic gel-like substance.
Claim 6
Dependent Claim
43 words
The assembly of claim 1 wherein said means for preventing the absorption of water by said thixotropic gel-like substance includes a barrier interposed between said thixotropic gel-like substance and at least one of said chemical reagent and said sample of unseparated whole blood.
Claim 7
Dependent Claim
14 words
The assembly of claim 5 wherein said barrier includes a plastic or elastomeric partition.
Claim 8
Dependent Claim
35 words
The assembly of claim 6 wherein said plastic or elastomeric partition further comprises channels extending therethrough, said channels being adapted to become opened to allow passage of a predetermined substance or substances through said barrier.
Claim 9
Dependent Claim
84 words
The assembly of claim 1 wherein said chemical reagent is selected from the group consisting of a hypertonic fluid containing a low molecular weight organic ionic substance which is essentially chemically compatible with the blood cells, a hypertonic fluid containing a low molecular weight inorganic ionic substance which is essentially chemically compatible with the blood cells, an isotonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells, a culture medium for blood cells and combinations thereof.
Claim 10
Dependent Claim
17 words
The assembly of claim 1 having a closure means for sealing said open end of said container.
Claim 11
Dependent Claim
20 words
The assembly of claim 9 wherein said closure means is suitable for vacuum sealing said open end of said container.
Claim 12
Dependent Claim
29 words
The assembly of claim 9 wherein said closure means is pierceable by a needle for supplying a blood sample to said container which is adapted to draw said sample.
Claim 13
Dependent Claim
14 words
The assembly of claim 1 wherein said sample of unseparated whole blood is anticoagulated.
Claim 14
Dependent Claim
14 words
The assembly of claim 1 wherein said hypertonic fluid is an aqueous saline solution.
Claim 15
Dependent Claim
19 words
The assembly of claim 1 wherein said inorganic ionic substance has a molecular weight of less than about 1500.
Claim 16
Dependent Claim
26 words
The assembly of claim 1 wherein said high molecular weight organic substance is selected from the group consisting of metrizoic acid, derivatives thereof and combinations thereof.
Claim 17
Dependent Claim
26 words
The assembly of claim 1 wherein said high molecular weight organic substance is selected from the group consisting of sodium metrizoate and metrizamide and combinations thereof.
Claim 18
Dependent Claim
15 words
The assembly of claim 1 wherein said high molecular weight organic substance is N,N'-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl) acetamido]-2,4,6-triiodo-isophthalamide.
Claim 19
Dependent Claim
24 words
The assembly of claim 1 wherein said culture medium is selected from the group consisting of Roswell Park Memorial Institute medium and McCoy's medium.
Claim 20
Dependent Claim
23 words
The assembly of claim 1 wherein said thixotropic gel-like substance has a specific gravity between about 1.055 to about 1.080 g/cm 3 .
Claim 21
Dependent Claim
20 words
The assembly of claim 1 wherein said thixotropic gel-like substance has a specific gravity of about 1.077 g/cm 3 .
Claim 22
Independent Claim
208 words
A method for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood wherein an apparent shift in the buoyant density of the granulocytes is inhibited and any loss in the buoyant density of the granulocytes is restored, said method comprising the steps of: (a) introducing a water insoluble, thixotropic gel-like substance into a vessel having an open end and a closed end, said thixotropic gel-like substance being positioned adjacent said closed end; (b) introducing a chemical reagent that is adapted to alter the osmolarity of said blood resulting in a change in cell diameters and cell densities of said granulocytes into said vessel; (c) providing within said vessel, means for preventing the absorption of water by said thixotropic gel-like substance from at least one of said chemical reagent and said sample of unseparated whole blood so as to substantially eliminate the influence of water absorption on the cell separation performance characteristics of said thixotropic gel-like substance; (d) introducing said sample of unseparated whole blood into said vessel; and (e) centrifuging said vessel at a force and for a sufficient length of time to cause said thixotropic gel-like substance to flow in order to form a barrier between said lymphocytes and monocytes, and the granulocytes.
Claim 23
Independent Claim
208 words
A method for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood wherein an apparent shift in the buoyant density of the granulocytes is inhibited and any loss in the buoyant density of the granulocytes is restored, said method comprising the steps of: (a) introducing a water insoluble, thixotropic gel-like substance into a vessel having an open end and a closed end, said thixotropic gel-like substance being positioned adjacent said closed end; (b) introducing a chemical reagent that is adapted to alter the osmolarity of said blood resulting in a change in cell diameters and cell densities of said granulocytes into said vessel; (c) providing within said vessel, means for preventing the absorption of water by said thixotropic gel-like substance from at least one of said chemical reagent and said sample of unseparated whole blood so as to substantially eliminate the influence of water absorption on the cell separation performance characteristics of said thixotropic gel-like substance; (d) introducing said sample of unseparated whole blood into said vessel; and (e) centrifuging said vessel at a force and for a sufficient length of time to cause said thixotropic gel-like substance to flow in order to form a barrier between said lymphocytes and monocytes, and the granulocytes.
Claim 24
Dependent Claim
20 words
The method of claim 21, which further comprises the step of recovering said lymphocytes and monocytes from atop said barrier.
Claim 25
Dependent Claim
52 words
The method of claim 21 wherein said means for preventing the absorption of water is provided by fabricating said thixotropic gel-like substance from an organic resin which allows high density and high viscosity polymers to form, so that said thixotropic gel-like substance is substantially devoid of inorganic fillers which can absorb water.
Claim 26
Dependent Claim
31 words
The method of claim 21 wherein the thixotropic gel is formed at least in part from one of a silicone oil, a butadiene resin, a polyester resin, and a butylene resin.
Claim 27
Dependent Claim
37 words
The method of claim 21 wherein said means for preventing the absorption of water is provided by at least one of presaturating said thixotropic gel-like substance with water during manufacture and curing of said thixotropic gel-like substance.
Claim 28
Dependent Claim
41 words
The method of claim 21 wherein said means for preventing the absorption of water is provided by interposing a second barrier between said thixotropic gel-like substance and at least one of said chemical reagent and said sample of unseparated whole blood.
Claim 29
Dependent Claim
15 words
The method of claim 26 wherein said second barrier includes a plastic or elastomeric partition.
Claim 30
Dependent Claim
84 words
The method of claim 21 wherein said chemical reagent is selected from the group consisting of a hypertonic fluid containing a low molecular weight organic ionic substance which is essentially chemically compatible with the blood cells, a hypertonic fluid containing a low molecular weight inorganic ionic substance which is essentially chemically compatible with the blood cells, an isotonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells, a culture medium for blood cells and combinations thereof.
Claim 31
Dependent Claim
14 words
The method of claim 28 wherein said hypertonic fluid is an aqueous saline solution.
Claim 32
Dependent Claim
19 words
The method of claim 28 wherein said inorganic ionic substance has a molecular weight of less than about 1500.
Claim 33
Dependent Claim
26 words
The method of claim 28 wherein said high molecular weight organic substance is selected from the group consisting of metrizoic acid, derivatives thereof and combinations thereof.
Claim 34
Dependent Claim
26 words
The method of claim 28 wherein said high molecular weight organic substance is selected from the group consisting of sodium metrizoate and metrizamide and combinations thereof.
Claim 35
Dependent Claim
15 words
The method of claim 28 wherein said high molecular weight organic substance is N,N'-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl) acetamido]-2,4,6-triiodo-isophthalamide.
Claim 36
Dependent Claim
24 words
The method of claim 28 wherein said culture medium is selected from the group consisting of Roswell Park Memorial Institute medium and McCoy's medium.
Claim 37
Dependent Claim
23 words
The method of claim 21 wherein said thixotropic gel-like substance has a specific gravity between about 1.055 to about 1.080 g/cm 3 .
Claim 38
Dependent Claim
20 words
The method of claim 21 wherein said thixotropic gel-like substance has a specific gravity of about 1.077 g/cm 3 .
References Cited (Backward Citations)
US3852194A
Cited
US3945928A
Cited
US3920549A
Cited
US3960727A
Cited
US4101422A
Cited
US4153739A
Cited
US4350593A
Cited
US4147628A
Cited
US4190535A
Cited
US4255256A
Cited
US4310430A
Cited
EP0036168A2
Cited
US4295974A
Cited
US4457782A
Cited
US4534798A
Cited
CA1127537A
Cited
US4417981A
Cited
US4436631A
Cited
US4435293A
Cited
US4487700A
Cited
US4640785A
Cited
US4816168A
Cited
US4824560A
Cited
US4844818A
Cited
US4867887A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-09-17 | 1994-03-19 | 1994-09-18 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-09-17 | 1998-03-19 | 1998-09-18 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-09-17 | 2002-03-19 | 2002-09-18 | $7700.00 | Window Closed / Lapsed |