Separation of lymphocytes and monocytes from blood samples

Claim 3

A method according to claim 1 wherein said incubating said sample of blood with said incubation fluid is maintained at least about 10 minutes.

Claim 4

A method according to claim 1 wherein said hypertonic fluid containing a low molecular weight ionic substance is an aqueous saline solution.

Claim 5

A method according to claim 1 wherein said ionic substance has a molecular weight of less than about 1500.

Claim 6

A method according to claim 1 wherein said high molecular weight organic substance contains molecules having a lipophilic substituent in their structure.

Claim 7

A method according to claim 5 wherein said high molecular weight organic substance is selected from the group consisting of metrizoic acid, derivatives thereof and combinations thereof.

Claim 8

A method according to claim 6 wherein said derivative of metrizoic acid is selected from the group of sodium metrizoate and metrizamide.

Claim 9

A method according to 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 10

A method according to claim 1 wherein said culture medium for blood cells is selected from the group of Roswell Park Memorial Institute medium and McCoy's medium.

Claim 11

An assembly for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood and for at least one of inhibiting any apparent shift in the buoyant density of the granulocytes 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 first layer of a gradient separation medium, the gradient separation medium being in the form of a water insoluble, thixotropic gel-like substance, which is chemically inert to blood constituents, the thixotropic gel-like substance being positioned in a first position adjacent said closed end; (c) a second layer of an incubation fluid for incubating the sample of unseparated whole blood, the second layer being separate from and unmixed with the first layer and being positioned above the surface of said water-insoluble, thixotropic gel-like substance of the first layer so as to contact the sample of whole blood, said second layer of incubation fluid 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, the second layer of incubation fluid being maintained in contact with the sample of whole blood for a sufficient length of time to inhibit the apparent shift in the buoyant density of the granulocytes and restore any loss in the buoyant density of the granulocytes; and (d) a free space initially adjacent and above said second layer of incubation fluid, said free space being of sufficient volume to contain said sample of unseparated whole blood, the first layer of thixotropic gel-like substance being a means for moving to a second position away from the closed end of the container upon centrifugation of the assembly to form a barrier at the second position between the lymphocytes and monocytes, which reside on top of the barrier, and the granulocytes thereby allowing the lymphocytes and monocytes to be removed from atop the barrier.

Claim 12

An assembly for separating lymphocytes and monocytes from granulocytes in a sample of unseparated whole blood and for at least one of inhibiting any apparent shift in the buoyant density of the granulocytes 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 first layer of a gradient separation medium, the gradient separation medium being in the form of a water insoluble, thixotropic gel-like substance, which is chemically inert to blood constituents, the thixotropic gel-like substance being positioned in a first position adjacent said closed end; (c) a second layer of an incubation fluid for incubating the sample of unseparated whole blood, the second layer being separate from and unmixed with the first layer and being positioned above the surface of said water-insoluble, thixotropic gel-like substance of the first layer so as to contact the sample of whole blood, said second layer of incubation fluid 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, the second layer of incubation fluid being maintained in contact with the sample of whole blood for a sufficient length of time to inhibit the apparent shift in the buoyant density of the granulocytes and restore any loss in the buoyant density of the granulocytes; and (d) a free space initially adjacent and above said second layer of incubation fluid, said free space being of sufficient volume to contain said sample of unseparated whole blood, the first layer of thixotropic gel-like substance being a means for moving to a second position away from the closed end of the container upon centrifugation of the assembly to form a barrier at the second position between the lymphocytes and monocytes, which reside on top of the barrier, and the granulocytes thereby allowing the lymphocytes and monocytes to be removed from atop the barrier.

Claim 13

The assembly of claim 10 which further comprises a closure means for sealing said open end of said container.

Claim 14

The assembly of claim 11 wherein said closure means is suitable for vacuum sealing said open end of said container.

Claim 15

The assembly of claim 11 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 16

The assembly of claim 10 wherein said sample of unseparated whole blood is anticoagulated.

Claim 17

The assembly of claim 10 wherein a reagent is added to or placed on top of said incubation fluid.

Claim 18

The assembly of claim 15 wherein said reagent is an anticoagulent.

Claim 19

The assembly of claim 10 wherein said incubation fluid is a hypertonic fluid containing a low molecular weight inorganic ionic substance which is essentially chemically compatible with the blood cells.

Claim 20

The assembly of claim 17 wherein said hypertonic fluid is an aqueous saline solution.

Claim 21

The assembly of claim 17 wherein said inorganic ionic substance has a molecular weight of less than about 1500.

Claim 22

The assembly of claim 10 wherein said incubation fluid is an isotonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells.

Claim 23

The assembly of claim 20 wherein said high molecular weight organic substance is selected from the group consisting of metrizoic acid, derivatives thereof and combinations thereof.

Claim 24

The assembly of claim 21 wherein said high molecular weight organic substance is selected from the group consisting of sodium metrizoate and metrizamide and combinations thereof.

Claim 25

The assembly of claim 20 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 26

The assembly of claim 10 wherein said incubation fluid is a culture medium for blood cells.

Claim 27

The assembly of claim 24 wherein said culture medium is selected from the group consisting of Roswell Park Memorial Institute medium and McCoy's medium.

Claim 28

The assembly of claim 10 wherein said thixotropic gel-like substance has a specific gravity between about 1.055 to about 1.075 g/cm 3 .

Claim 29

The assembly of claim 26 wherein said thixotropic gel-like substance has a specific gravity between about 1.06 to about 1.065 g/cm 3 .