GAMMA STERILIZED DEXTRAN SOLUTIONS AND METHODS OF USE
Provided herein are kit for providing a gamma sterilized aqueous dextran solution that increase the efficiency of blood separation by allowing the dextran solution to be sterilized by exposure to gamma radiation while maintaining sufficient molecular weight to act as a red blood cell aggregate. Also provided are methods of use.
1 . A kit for providing a gamma sterilized aqueous dextran solution for red blood cell aggregation comprising:
solutes, the solutes comprising;
dextran, where the dextran has an initial average molecular weight greater than 500 kD; and
2.0 to 20.0 wt % ascorbic acid or its mineral salt to the dextran;
a mixing vessel for containing and enabling a sample comprising red blood cells (RBC) to separate into two or more distinct layers of sub materials, said mixing vessel having two or more valve ports positions for introducing and extracting materials;
a receptacle in fluid communication with the mixing vessel through one of the valve ports configured to contain a 1 to 10 wt/v % of an aqueous solution of the solutes; and
where the kit is capable of undergoing gamma sterilization upon exposure to gamma irradiation at a dose between 20 and 50 kGy such that the aqueous solution, when contained in the receptacle, has an average molecular weight greater than 200 kD after gamma irradiation.
2 . The kit of claim 1 where the dextran initial molecular weight is greater than 750 kD.
3 . The kit of claim 2 where the dextran initial molecular weight is between approximately 1000 to 1500 kD.
4 . The kit of claim 1 where the mineral salt is sodium ascorbate.
5 . The kit of claim 1 where the solutes further comprise a buffer, a non-toxic enhancer or a combination thereof.
6 . The kit of claim 5 where the non-toxic enhancer is sodium citrate, sodium succinate, or a combination thereof.
7 . The kit of claim 5 where the buffer comprises organic or inorganic salts that maintain a pH of 4.0-9.0.
8 . The kit of claim 1 further comprising a flow device configured to add or remove material from the vessel through one or more valved openings.
9 . The kit of claim 1 where the mixing vessel and receptacle are comprised of disposable materials for single use.
10 . The kit of claim 1 where one or more of the solutes are provided in an aqueous solution.
11 . A method to aggregate cells in a sample comprising red blood cells (RBC), comprising the steps of:
a. obtaining an aqueous dextran solution, the aqueous dextran solution comprising;
1 to 10 wt/v % of dextran where the dextran has an initial average molecular weight greater than 500 kD prior to gamma irradiation; and
2.0 to 20.0 wt % ascorbic acid or its mineral salt to the dextran;
b. exposing the solution to gamma radiation at a dose between 20 and 50 kGy resulting in the dextran having an average molecular weight greater than 200 kD after gamma irradiation; and
c. combining the sample comprising red blood cells with the aqueous dextran solution of step b.
12 . The method of claim 11 where the dextran has an initial molecular weight greater than 750 kD.
13 . The method of claim 12 where the dextran has an initial molecular weight between approximately 1000 to 1500 kD.
14 . The method of claim 11 where the mineral salt is sodium ascorbate.
15 . The method of claim 11 where the aqueous solution further comprises a buffer, a non-toxic enhancer or a combination thereof.
16 . The method of claim 15 where the non-toxic enhancer is sodium citrate, sodium succinate, or a combination thereof.
17 . The method of claim 15 where the buffer comprises organic or inorganic salts that maintain a pH of 4.0 to 8.0.
18 . The method of claim 11 where the combining together the sample comprising red blood cells and the aqueous dextran solution comprises;
adding the sample comprising red blood cells to a mixing vessel, the mixing vessel having two or more valve ports positions for introducing and extracting materials; and
adding the aqueous dextran into the mixing vessel from a receptacle, the receptacle being in fluid communication with the mixing vessel through one of the valve ports.
19 . The method of claim 18 further comprising the steps of incubating the sample to aggregate and sediment of the red blood cells and optionally recovering total nucleated cells (TNC) from the sample.
20 . The method of claim 18 where incubating the sample occurs in the mixing vessel.
21 . The method of claim 20 where recovering the TNC comprises concentration of a liquid phase extracted from the mixing vessel, said liquid phase comprising plasma and dextran using centrifugation, membrane filtration, or a combination thereof.