Patent Application
20210129135
The present invention relates to disposable lab supplies, and more specifically, to a blood fractionation tube and system having a unique hourglass shape.
Blood fractionation is the process of separating whole blood into its component parts. For example, simple centrifugation is a common fractionation method that results in plasma in the upper phase, the buffy coat in the middle phase, and erythrocytes at the bottom of the centrifuge tube. This is shown in
Density gradient media may be added to whole blood before centrifugation when it is desirable to isolate more specific components than is possible with simple centrifugation alone. For example, the density gradient medium FICOLL facilitates the separation of whole blood into a top layer of plasma, followed by a fraction of peripheral blood mononuclear cells (PBMCs), a fraction of polymorphonuclear cells such as neutrophils and eosinophils, and finally erythrocytes. This is shown in
Harvesting a pure sample of isolated PBMC from a standard blood fractionation tube is difficult because the PBMC layer is relatively thin and the borders are not always clearly distinguishable to the human eye, so pipetting and the like often compromises a sample and results in low PBMC recovery. As can be seen, there is a need for an improved blood fractionation tube design that is easy to use, results in samples with less contamination by surrounding fractions, yields more viable cells, and is more efficient overall.
An hourglass shaped blood fractionation tube is sized and shaped to elongate or widen the PBMC layer of whole blood that has been centrifuged with a density gradient medium. Elongating or widening the PBMC layer allows technicians to harvest higher purity and volume PBMC samples versus samples harvested with a standard cylindrical blood fractionation tube. The hourglass shaped blood fractionation tube according to the present invention preferably includes a cap and can be used in conventional centrifuges and robotic pipetting stations.
The following detailed description is of the best currently contemplated modes of carrying out exemplary embodiments of the invention. The description is not to be taken in a limiting sense but is made merely for the purpose of illustrating the general principles of the invention, since the scope of the invention is best defined by the appended claims.
The following structure numbers shall apply to the following structures among the various FIGS.:
10—tube system;
20—cap;
30—vessel;
31—threads;
32—upper cylindrical portion;
33—upper tapered portion;
35—neck;
37—lower tapered portion;
38—lower cylindrical portion;
39—bottom;
40—whole blood;
42—plasma;
44—PBMC's;
46—granulocytes;
48—erythrocytes; and
50—density gradient media.
Referring to
In a preferred embodiment vessel 30 has a volume of approximately 31 mL to approximately 36 mL, with approximately 11 mL to approximately 12 mL of total vessel volume attributed to upper cylindrical portion 32; approximately 6 mL to approximately 7 mL of total vessel volume attributed to upper tapered portion 33; approximately 4 mL to approximately 5 mL of total vessel volume attributed to neck 35; approximately 3 mL to approximately 4 mL of total vessel volume attributed to lower tapered portion 37; and approximately 7 mL to approximately 8 mL of total vessel volume attributed to lower cylindrical portion 38. The volume attributed to neck 35 is approximately 10% to approximately 17% of the total volume of vessel 30.
In a preferred embodiment the outer surface of upper cylindrical portion 32 defines threads 31 that releasably engage with cap 20.
The approximate dimensions of a preferred vessel are: 114.3 mm-116 mm total height; 30 mm height of upper cylindrical portion 32; 13 mm height of upper tapered portion 33; 50 mm height of neck 35; 10 mm of lower tapered portion 37; and 13 mm of lower cylindrical portion 38. The height of the elongated central neck is preferably approximately 38% to approximately 48% the total length of the vessel.
In a preferred embodiment the diameter of upper cylindrical portion 32 is approximately 28 mm; diameter of lower cylindrical portion 38 is approximately 11 mm; diameter of neck 35 is approximately 3.9 mm; and thickness of material is approximately 0.29 mm. The diameter of neck 35 is preferably approximately 9% to approximately 19% the diameter of said upper cylindrical portion.
In a preferred embodiment the vessel is constructed of known materials such as polypropylene, polystyrene, and/or glass and is manufactured by known methods such as injection molding and/or blow. Examples of automated systems that the tube is compatible with include Hamilton Microlab Star. Hamilton Microlab Vantage, Hamilton Microlab Nimbus, Beckman 15, Beckman 17 Tecan EVO and Tecan Fluent liquid handlers; and Beckman Coulter centrifuges.
In use, a technician dispenses approximately 4 mL to approximately 10 mL of a density gradient media such as FICOLL into vessel 30, adds approximately 10 mL to approximately 20 mL diluted whole uncoagulated blood, and seals the sample within vessel with cap 20. The ratio of the volume of blood to the volume of density gradient media is preferably approximately 1:1 to approximately 5:1.
The sample is centrifuged at 400 to 800 g at room temperature for 25 to 40 minutes with centrifugation brakes turned off. Following centrifugation, the PBMC layer is transferred to a 15 ml or 50 ml centrifugation tube using a transfer pipet or equivalent. The PBMC is diluted with a suitable wash solution to 14 ml or 40 ml for 15 ml or 50 ml centrifugation tubes, respectively, and centrifuged at 200 to 400 g at room temperature for 10 to 15 minutes, with the brakes turned on. After centrifugation, the supernatant is decanted and the PBMC pellet is broken up, then diluted in the media of choice, for example Phosphate-buffered saline.
Specifications of certain structures and components of the present invention have been established in the process of developing and perfecting prototypes and working models. These specifications are set forth for purposes of describing an embodiment, and setting forth the best mode, but should not be construed as teaching the only possible embodiment. Rather, modifications may be made without departing from the spirit and scope of the invention as set forth in the following claims. An example of a modification is a different scale of tube size while keeping similar ratios in the dimensions. It should be understood that all specifications, unless otherwise stated or contrary to common sense, are +/−10%, and that ranges of values set forth inherently include those values, as well as all increments between. Also, “substantially” and similar language means “generally” but allowing for variations due to factors such as materials and manufacturing, and human interference.
This application claims the benefit of U.S. Provisional Patent Application 62/929,165 entitled HOURGLASS SHAPED BLOOD FRACTIONATION TUBE AND SYSTEM, which was filed Nov. 1, 2019. The provisional application is incorporated in its entirety into the present application.
| Number | Date | Country | |
|---|---|---|---|
| 62929165 | Nov 2019 | US |
