|Publication number||US3513976 A|
|Publication date||May 26, 1970|
|Filing date||Mar 19, 1968|
|Priority date||Mar 19, 1968|
|Publication number||US 3513976 A, US 3513976A, US-A-3513976, US3513976 A, US3513976A|
|Inventors||William C James|
|Original Assignee||William C James|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (3), Referenced by (42), Classifications (12)|
|External Links: USPTO, USPTO Assignment, Espacenet|
y 6, 1970 w. c. JAMES 3,513,976
LEUKOCYTE FLASH AND METHOD OF OBTAINING WHITE CELLS FROM WHOLE BLOOD Filed March 19, 1968 INVENTOR WILLIAM C. JAMES ATTORNEY US. Cl. 21078 2 Claims ABSTRACT OF THE DISCLOSURE This specification discloses a flask forpse in obtaining leukocyte from whole blood containing an ant1coagulant with the flask including a lower portion of substantially horizontal extent that receives the red blood cells, a narrow vertical column of small horizontal crosssectional area upstanding from the lower portion and which receives the leukocyte or white cells and an upper portion of large horizontal cross section that receives blood plasma. A tube is provided at one side of the lower portion for introducing mercury into the bottom thereof to raise the blood components to a desired level. A pipette is inserted through the plasma into the narrow column to withdraw the white cells aspiration.
The present invention relates to the harvesting of white blood cells from heparinized whole blood and is concerned primarily with a novel method and apparatus for achieving the harvest.
Present day practice in this field may be characterized as requiring two centrifugations of the blood with a substance of high molecular weight being added before the first centrifugation.
The present invention has as a highly important object the provision of a method and apparatus whereby the white cells of heparinized whole blood may be separated and harvested in a pure state with but a single centrifugation and without the addition of any susbtances to the blood.
Red blood cells constitute about forty percent of whole blood. Leukocyte or white blood cells are present in a proporotion of about one-half of one percent. Plasma makes up the remaining fifty-nine to sixty percent. When heparinized whole blood is centrifuged in accordance with present day practice, the small percentage of white cells forms a thin layer commonly known as a buify coat immediately over the top of the red blood cells. An important object of the present invention is to provide in a method of the type noted the step of transforming this thin layer into a narrow vertical column of leukocytes which may be readily harvested by aspiration.
In more detail, the invention has as an object the provision of a method of the character indicated which includes the step of centrifuging heparinized whole blood to form a mass of red blood cells at the bottom, a narrow column of white blood cells upstanding from the red blood cells, and a mass of plasma above the column and then harvesting the white blood cells by aspiration through the plasma.
Another object is to provide in a method of the type noted the step of accurately controlling the level constituting the line of demarcation between the red and white cells so that the latter may be harvested with the definite assurance that they will be in a pure state. This step may be carried out by introducing a required amount of a liquid of high density and chemically inert with respect to the blood, such as mercury, beneath the red blood cells.
As mentioned above, it is of course important to maintain a clear line of demarcation between the red blood United States Patent 3,513,976 Patented May 26, 1970 cells and the leukocytes. To this end, the method of this invention provides for the gradual diminution of the horizontal cross-sectional area of the red blood cells to their uppermost level at which point the narrow column of white blood cells begins.
Another important object in view is to provide apparatus for carrying out the above method with the apapparatus taking the form of a flask of what might be called a vertical dumbbell like formation. This flask comprises a bottom portion having an upper conical wall, the narrow end of which is joined to a tubular neck having a small bore with the upper end of the neck being joined into the small end of the lower conical section of an upper portion. The upper and lower portions and the intermediate neck are preferably integrally formed from any suitable transparent material such as glass.
Another object is to provide in a flask of the character aforesaid a lower portion or member the conical wall of which is formed with a passage through which extends a vertically disposed tube the lower free end of which is spaced slightly from the bottom of the flask. This tube is adapted to receive the mercury aforesaid.
In accordance with the present invention, the upper flask member terminates in an open top. A pipette of an external diameter less than the bore of the neck is passed through this open top and plasma in the upper portion into the neck with the lower end of the pipette being disposed slightly above the joinder of the neck to the conical end of the bottom section. Leukocytes are aspirated through this pipette.
Various other more detailed objects and advantages of the invention such as arise in connection with carrying out the above noted ideas in a practical embodiment will in part become apparent and in part be hereinafter stated as the description of the invention proceeds.
For a more complete understanding of the invention, reference may be had to the following description and accompanying drawing, wherein:
FIG. 1 is a side elevation of a leukocyte flask designed in accordance with the principles of this invention.
FIG. 2 is a vertical section through the flask of FIG.
1 with the components of blood illustrated as being received therein and the pipette in position for harvesting the leukocytes.
FIG. 3 is a horizontal section taken about on the plane represented by the line 33 of FIG. 1.
Referring now to the drawing, the leukocyte flask of this invention is referred to in its entirety by the reference character F. It comprises three main portions or elements which are integrally connected. It may be made of glass or any other material having the required property of transparency so that a user may readily ascertain the condition of the blood components which obtain therewithin.
The flask F comprises a lower section 10, an intermediate neck 11, and an upper section 12.
The lower section 10 includes a flat bottom wall 13, the peripheral edge of which is integrally joined to a cylindrical wall portion 14. Integrally joined to the upper edge of the latter along the line represented at 15 is a conical section 16 having a small end at 17.
The neck 11 is joined to the conical section 16 at 17 and has a small bore 18. By way of example, it is noted that this bore 18 may have a diameter of 2.5 to 3 mm.
The neck 11 will have a vertical extent that is related to the volume of blood that is handled in any single operation. Thus, with a cylindrical portion 14 having a vertical extent of 27 mm. and the conical portion 16 a vertical extent of 18 mm., the neck 11 should have an extent of about 45 mm.
The upper end of the neck 11 is indicated at 19 and integrally joined thereto is the smaller end of the conical section 20 constituting a part of the upper member 12."'
The wider end of the conical section 20 is represented by the line 21 and integrally joined thereto is a cylindrical portion 22. The upper end of the latter is rounded as indicated in 23 and joined to a cylindrical portion 24 of reduced diameter. The latter terminates in a bead 25 which defines an open top.
To complete the specifications for the flask, it is noted that with the dimensions for the lower member 10 and neck 11 as given, the conical section 20 should have a vertical extent of 18 mm., the cylindrical portion 22 a vertical extent of 31 mm., and the reduced cylindrical portion 24 a vertical extent of 11 mm. Thus, with the flask F having an overall vertical extent of about 160 mm., the neck 11 has an extent of about 45 mm. It is also notable that the cylindrical sections 14 and 22 have outside diameters of about 35 mm. and a wall thickness of about 2 mm.
The conical section 16 of the bottom member 10 is formed with a vertical passage 26 through which passes a tube 27, the lower free end 28 of which is spaced slightly above the bottom wall 13.
In use, whole blood containing an anticoagulant, such as heparin or sodium citrate, is introduced into the flask F and centrifuged. This results in red blood cells such as represented at 29, in FIG. 2, filling the lower member 10 substantially up to the line 17. Luekocytes or white blood cells indicated at 30 fill the bore 18 of the neck 11 substantially up to the point 19, and plasma represented at 31 fills the upper section 12 up to the level represented at 32.
Before this separation is achieved, mercury is introduced through the tube 27 to build up a layer 33 of mercury of about one centimeter in thickness. After the blood is centrifuged, mercury may be added or withdrawn so that the lower level of the white cells 30 in the neck 11 will be positioned substantially at the line 17. A pipette 34 is then passed through the open top defined by the bead 25 and plasma 37 is passed into the bore 18 so that the lower end of the pipette 34 is represented at 35 and is disposed just above the line 17. The white blood cells 30 may then be withdrawn by aspiration and harvested in a substantially pure state.
While a preferred specific embodiment of the invention is hereinbefore set forth, it is clearly understood that the invention is not to be limited to the exact constructions, inventions and steps illustrated and described, because various modifications of these details may be provided the lower mass, the white blood cells assume the position of the narrow column, and plasma occupies the space of the upper mass, adjusting the line of demarcation between the 'red blood cells and the white blood cells by varying 'the quantity of mercury, and then withdrawing the white blood cells from the narrow column by aspiration.
2. In a leukocyte flask for separating white blood cells from whole blood containing an anticoagulant, a lower section having an upper conical portion, a vertical neck having a bore with its lower end integrally joined to the upper end of said conical portion, and an upper section having a lower conical portion, the smaller end of which is integrally joined to the upper end of said neck of said upper and lower portions and said neck being proportioned so that after blood received therein is centrifuged, red blood cells will substantially fill the lower portion, white blood cells will fill the bore of the neck, and plasma will be contained in the upper portion, the said lower section having a flat bottom wall together with a tube passing through the conical portion of said lower section and terminating substantially near above said bottom wall.
References Cited UNITED STATES PATENTS 2,854,143 9/1958 Novak 2l0 36l X 2,928,591 3/1960 Deaver 233-26 X 3,291,693 12/1966 Brown 23326 X OTHER REFERENCES German printed application No. 1,014,348, Aug. 22, 1957, Beyerle.
JAMES L. DECESARE, Primary Examiner US. Cl. X.R.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US2854143 *||Sep 16, 1957||Sep 30, 1958||Ohio Commw Eng Co||Centrifugal stratifier with plural filter means|
|US2928591 *||Dec 27, 1956||Mar 15, 1960||Lee Deaver George||Method and apparatus for separating particles in a fluid dispersion|
|US3291693 *||Jul 14, 1959||Dec 13, 1966||American Optical Corp||Method for determining values of components of whole blood|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US3750645 *||Oct 20, 1970||Aug 7, 1973||Becton Dickinson Co||Method of collecting blood and separating cellular components thereof|
|US3914985 *||Mar 29, 1974||Oct 28, 1975||American Hospital Supply Corp||Centrifuging device and method|
|US3977598 *||Apr 17, 1975||Aug 31, 1976||Mcdonald Bernard||Centrifuge tube|
|US4027660 *||Apr 2, 1976||Jun 7, 1977||Wardlaw Stephen C||Material layer volume determination|
|US4040959 *||Jun 22, 1976||Aug 9, 1977||Berman Irwin R||Multi-purpose blood bag|
|US4077396 *||Dec 2, 1976||Mar 7, 1978||Wardlaw Stephen C||Material layer volume determination|
|US4082085 *||Sep 20, 1976||Apr 4, 1978||Wardlaw Stephen C||Blood constituent testing methods|
|US4255256 *||Nov 23, 1979||Mar 10, 1981||Antonio Ferrante||Medium for the separation of human blood leucocytes|
|US4511349 *||Feb 27, 1984||Apr 16, 1985||Beckman Instruments, Inc.||Ultracentrifuge tube with multiple chambers|
|US4639242 *||Feb 4, 1985||Jan 27, 1987||Babson Arthur L||Vessel and procedure for automated assay|
|US4663032 *||May 14, 1985||May 5, 1987||Npbi Nederlands Produktielaboratorium Voor Bloedtransfusieapparatuur En Infusievloeistoffen B.V.||Apparatus for decanting of a blood-plasma layer and a buffy-coat layer from a centrifuge-blood bag|
|US4824560 *||Apr 4, 1986||Apr 25, 1989||Assaf Pharmaceutical Industries Ltd.||Separation of materials from a liquid dispersion by sedimentation|
|US4861477 *||Apr 21, 1988||Aug 29, 1989||Shiro Kimura||Tubular container for centrifugal separation|
|US5152905 *||Nov 6, 1990||Oct 6, 1992||Pall Corporation||Method for processing blood for human transfusion|
|US5360545 *||Aug 3, 1993||Nov 1, 1994||Pall Corporation||Filter for obtaining platelets|
|US5399268 *||Jul 6, 1994||Mar 21, 1995||Pall Corporation||Method for processing blood for human transfusion|
|US5445736 *||Apr 25, 1994||Aug 29, 1995||Pall Corporation||Device and filter element for processing blood for human transfusion|
|US5474687 *||Aug 31, 1994||Dec 12, 1995||Activated Cell Therapy, Inc.||Methods for enriching CD34+ human hematopoietic progenitor cells|
|US5543060 *||Jun 6, 1995||Aug 6, 1996||Pall Corporation||Method for processing blood for human transfusion|
|US5577513 *||Aug 31, 1994||Nov 26, 1996||Activated Cell Therapy, Inc.||Centrifugation syringe, system and method|
|US5580465 *||Dec 3, 1993||Dec 3, 1996||Pall Corporation||Method for preparing platelets|
|US5646004 *||Aug 31, 1994||Jul 8, 1997||Activated Cell Therapy, Inc.||Methods for enriching fetal cells from maternal body fluids|
|US5648223 *||Aug 31, 1994||Jul 15, 1997||Activated Cell Therapy, Inc.||Methods for enriching breast tumor cells|
|US5656154 *||Jun 7, 1995||Aug 12, 1997||Organ, Inc.||Method and apparatus for separating a fluid into components and for washing a material|
|US5663051 *||Dec 11, 1995||Sep 2, 1997||Activated Cell Therapy, Inc.||Separation apparatus and method|
|US5770069 *||Feb 21, 1997||Jun 23, 1998||Organ, Inc.||Collapsible container for holding a fluid during a centrifugation operation|
|US5840502 *||Aug 31, 1994||Nov 24, 1998||Activated Cell Therapy, Inc.||Methods for enriching specific cell-types by density gradient centrifugation|
|US6309606 *||Oct 29, 1998||Oct 30, 2001||Giammaria Sitar||Device and method for the separation of human or animal cells of different densities from cellular dispersions which contain them|
|US8048678 *||Jun 29, 2010||Nov 1, 2011||Ecw Therapeutics, Inc.||Cell separation method and apparatus|
|US8241592||Aug 14, 2012||Endocellutions, Inc.||Cell separation method and apparatus|
|US9168528 *||Apr 1, 2014||Oct 27, 2015||Alliance Partners, Llc||Fluids concentration cup assembly with hourglass shape|
|US20100120596 *||May 8, 2008||May 13, 2010||Ge Healthcare Bio-Sciences Ab||Separation device|
|US20110033925 *||Jun 29, 2010||Feb 10, 2011||Duffy Jr Neil F||Cell Separation Method and Apparatus|
|US20150004080 *||Apr 1, 2014||Jan 1, 2015||Alliance Partners Llc||Fluids concentration cup assembly with hourglass shape|
|CN102844121A *||Dec 15, 2010||Dec 26, 2012||全玟墉||Centrifuge tube|
|EP0069053A1 *||May 26, 1982||Jan 5, 1983||Battelle Memorial Institute||Test tube for separating, by centrifugation, a physiological liquid, and for collecting one of the constituents with a given density|
|EP0742735A1 *||Jan 23, 1995||Nov 20, 1996||Applied Imaging Corp.||Centrifuge tube and adaptor|
|WO1982004201A1 *||May 25, 1982||Dec 9, 1982||Ringrose Anthony||Test-tube for the centrifuging and collecting of a liquid|
|WO1984000313A1 *||May 16, 1983||Feb 2, 1984||Beckman Instruments Inc||Ultracentrifuge tube with multiple chambers|
|WO1991004088A1 *||Sep 11, 1990||Apr 4, 1991||Pall Corporation||Device and method for processing blood for human transfusion|
|WO2008143570A1 *||May 8, 2008||Nov 27, 2008||Ge Healthcare Bio-Sciences Ab||Separation device|
|WO2011071353A2||Dec 15, 2010||Jun 16, 2011||Min-Yong Jeon||Centrifuge tube|
|U.S. Classification||210/782, 210/361, 494/16, 422/918, 494/902|
|International Classification||G01N33/49, B01L3/14|
|Cooperative Classification||G01N33/491, Y10S494/902, B01L3/5021|
|European Classification||B01L3/5021, G01N33/49C|