CA2058670A1 - Constituent layer harvesting from a centrifuged sample in a tube - Google Patents
Constituent layer harvesting from a centrifuged sample in a tubeInfo
- Publication number
- CA2058670A1 CA2058670A1 CA002058670A CA2058670A CA2058670A1 CA 2058670 A1 CA2058670 A1 CA 2058670A1 CA 002058670 A CA002058670 A CA 002058670A CA 2058670 A CA2058670 A CA 2058670A CA 2058670 A1 CA2058670 A1 CA 2058670A1
- Authority
- CA
- Canada
- Prior art keywords
- tube
- float
- blood
- sample
- centrifuged
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/491—Blood by separating the blood components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5021—Test tubes specially adapted for centrifugation purposes
- B01L3/50215—Test tubes specially adapted for centrifugation purposes using a float to separate phases
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/2575—Volumetric liquid transfer
Abstract
INVENTORS: Robert A. Levine Stephen C. Wardlaw TITLE: "CONSTITUENT LAYER HARVESTING
FROM A CENTRIFUGED SAMPLE IN
A TUBE"
DOCKET NO.: H-1170 ABSTRACT OF THE DISCLOSURE
Constituent layers are harvested from a centrifuged multi-constituent material in an evacuated glass or clear plastic tube which contains a float. When possibly contaminated materials, such as blood, are being tested, the use of an evacuated tube allows the measurements to be made without the technician being exposed to the blood. The tubes are large enough to hold approximately one ml of blood, and arefilled with an inert gas at low pressure. The floats are formed with a through bore into which cell bands to be harvested will settle during centrifugation. The cell bands are stabilized by a layer of a flowable material which settles onto the plasma layer during centrifugation and forms a pellicle thereon. The cell layers to be harvested areaspirated from the float bore by means of a hypodermic needle or cannula inserted into the tube and float bore.
FROM A CENTRIFUGED SAMPLE IN
A TUBE"
DOCKET NO.: H-1170 ABSTRACT OF THE DISCLOSURE
Constituent layers are harvested from a centrifuged multi-constituent material in an evacuated glass or clear plastic tube which contains a float. When possibly contaminated materials, such as blood, are being tested, the use of an evacuated tube allows the measurements to be made without the technician being exposed to the blood. The tubes are large enough to hold approximately one ml of blood, and arefilled with an inert gas at low pressure. The floats are formed with a through bore into which cell bands to be harvested will settle during centrifugation. The cell bands are stabilized by a layer of a flowable material which settles onto the plasma layer during centrifugation and forms a pellicle thereon. The cell layers to be harvested areaspirated from the float bore by means of a hypodermic needle or cannula inserted into the tube and float bore.
Description
2 ~ 7 ~
This invention rela~es to paraphenalia and a method for determining information about a constituent layer in a centrifuged sample of a material such as blood. The constitu~nt layer is harvested from an evacuated ~ube containing a float which expands the consti~uent layers being harvested, and which contains a through bore or passage into which the particuiar constituent layers settle during centrifugation.
A technique has been developed to measure constituent layers in a complex materia!
mixture by centrifuging a sarnpl~ of the material mixture in a capillaiy or other tube which oontains a float. The ~ioat is pr~erably cylindrical and of a specifio gravi~ which eauses it to settle into the centrifuged mixture to a degree which creates a free volume annulus in the tube into which the layer, or layers to be measured will settle. The layers to be measured arc thus physically elongated, and can be more easily and accurately rneasured. This technique is described in U. S. Patents Nos. 4,027,660 issued ~une 7, 1977; 4,082,085 issued April 4, 1978; 4,156,570 issued May 29,1979;
and others.
When the material being ~ested is a possibly contaminated material such as biood, it is desirable to make provisions for protecting the technician against exposure to ~he blood. When the aforesaid prior art techniques are pei~ormed with capillai~ tubes, the person pei~orming the test is exposed to the blood since the capillaiy tubes areopen-ended. Thus, despite taking normal precautions in handling ot the samples, the chance of being contaminated by a blood sample exists. The aforesaid prior art also does not readily lend i~elf to harvesting of any of the centrifuged blood cell bands ~rom the tub~. .
This invention is directed to a me~hod and paraphenalia ~or use in the collecting ~nstituent cells or other particles from a possib!y contamin~ted material swh asblood, wherein the person doing the colleoting is never exposed to the blood. Thus, the possibility of becoming infeoted by a contaminated blood sample is elimin~ted.
When the tube and float of this invention ara used, the blood sample is collected in a sealed tube; then concentration o~ cells to be harves~ed is made in the tube; and the 2 ~ 7 0 cells can be aspirated from the tube without ever exposing the ~echnician to the blood sample. An addi~ional advantage of th~ invention resides in ~he fact that it entails the use of a unitary sealed tube which contains all of the required components for use in performing the cell concentration and harvesting, and those components are disposed in a stable, inert environment. The tub~ used in this invention is preferably a giass tube with an integral closed end. It will be the same length as a capillary tube but will hav0 a ~arg~r diameter so as to be able to con~ain about 0.9 ml of blood. A cylindrical float is disposed inside of the tube, which float has an accurately controlled outside diameter so as ~o fit snugly in the tube bore under static conditions. When used in harvesting blood cells the float is forrned with an axial through bore which rec~ives and expands the white cell and pla~elet layers in the blood sample af~r centrifugation thereof. The float is made from a plastic material having a specific gravity that causes it to float in the packed red cells after centrifugatian of the blood sample in the tube.
Required reagents, such as a stain and a red cell densifier, preferably potassium oxalate, may be disposed in the tube, preferably in liquid form. An elastomeric stopper closes the open end of the tube, and the interior of the tube is filled with an inert gas at low pressure. The low pressure in the tube is used to draw the blood sample into the tube, preferably from a primary blood collection device, such as ~hat sold by Becton Dickinson and Company under ghe tra~emark "Vacutainer".
The float may preferably be a compound structure made from plastics which have aspecific gravity which causes the float to be buoyed up in ~he centrifuged red cell layer.
The float is formed with a core portion which has the through bore, and an annular sleeve portion which will expand and contract responsive to the magnitude of dynamic forces imposed on ~he float during performance of the sample centrifugation. The float ~r~ must be formed from a plastic material, such as ~ transparent styrene, which is dimensionally stable during centrifugation. The peripheral sleeve portion o~ ~he float can be formed from a ~ransparent pliable vinyl plas~ic. The ~No components of the float can be joinecl together by c~extrudin~ or hy co-molding the float cvmponents. The tu~e can be provided with a lubricant coa~ing, such as a silicone coating to enhance movement of the float in ~he tube during centrifugation. Specific plastics which can be 2~5$~70 used for the core and sleeve of the float are polystyrene and polyvinylchloride (PVC) respectively. The float may also be formed from a single plastic material iF so desired.
The primary blood ~ollection tube will be provided with a needle which is used to pierce the elastomeric stopper in the tube of this invention, whereupon ~he blood will flow from the collection tube, through the needie, into the testing tubA. In order to preserve cell band formation in the tube when the tube and ~lood are cen~rifuged, a thixotropic gel would be disposed in the top of the ~ube. During centrifugation, the gel will flow down the wall of the tube and settle on top of the plasrna layer to form a viscous peliicle on the plasma. Obviously, the gel must have a specific gravity which is less than that of the plasma. A thin plastic cup may be used in lieu of the gel.
When the larger bore diameter tube and the larger float with an axi~l bore are used per this invention, there occurs a relaxation in the diameter dimensional tolerances in the tube bore ID. It is d~sirable to achieve a ten fold expansion of the whito cell and platelet layers when perForming the cell harvesting with the tube-float combination of the aforesaid prior art. When using the enlarged tubes and floats of this invention, the ten fold expansion can be obtained frorn a through bore diameter of 1.265 mm wherl a 4.0 mm diameter tube bore is used. This compares with a free space uf about 43 microns with the prior art capillary tubes and floats. The ~I- variation in the bore diameter is 20 rnicrons when using the paraphPnalia of this invention.
A benefit deriving from the use of the larger tube and float paraphenalia is an improvement in the hydrodynamics of the centrifuga~ion. A~ter blood is added ~o the tube, the tube is centrifuged at 10,000 G, as is the usual practice. With a float of this type, several forces are brought in~o play. First, the centripedal accelleration forces the float to the end of the tube at the same time as th~ blood cells are separating.Secondly, a tidal force is exerted on the float be~use the accelleration is unequal at the e~dg of the float. This tidal force is about 2,0V0 G at near the csnter of th~ tube.
This exerts a stretching or con~racting force on ~he float of about 500 G, which is enough to sufficiently elongate the pliable elastomeric portion of the float and sliyhtly ', 2 ~ 7 ~1 decrease its diameter, aliowing it to easily slip down ~he ~ube. AMer the float settles according to its density into the R~C layer, and ~he centrifuge slows to a s~op, the tidal forces cease, and the float relaxes to its normal diameter thereby reassuming its close approximation to the walls of the tube.
The cells and components of the bu~y coat ~ayer are expantied linearly in the narrow bore channel in the float and thus can be easily harvested ~herefrom.
It is therefore an object of this invention lo provide an improved blood sampling paraphenalia which allows for the blood cell harvesting to be made without exposing the technician to contamination from the blood sample.
It is a further object of this invention to provide blood sampling paraphenalia of the character described wherein dimensional tolerances are relaxed while providing the necessary cell layer expansion.
It is an additional object of this invention to provide blood sampling paraphenalia of the character described wherein larger biood samples are used.
It is still another object of this invention to provide blood sampling paraphenaiia o~ the character described wherein the formation of cell bands a~ter centrifugation, isstabilized and preserved.
It is yet an additional object of this invention to provide blood sampling paraphenalia of the character describe~ wherein improved hydrodynamics during centrifugation is achievecl.
These and other obje~e. and adYantages of ~he invention will become more readilyapparent from the following deseription of a preferred embodiment ther~of when eonsidered in oonjunction wim ~he accompanying drawings, in which:
2 ~ 7 ~
FIGURE 1 is an axial sectional view of a preferred embodiment of a tube and float assembiy formed in accordance with this in~/ention.
FIGURE 2 is an axial sectional view of the float;
FIGURE 3 is an axial sectional view showing how the assembly can b used to draw a bloo~l sample from a primary blood collecting tube;
Fl(3URE 4 is a view similiar to FIGURES 1 ~nd 3 but showing the assembly of FIGURE
1 after the blood sample has been drawn and centrlfuged, and FIGURE 5 is a fragmented sectional view showing how a cell layer can be harvested from the centrifuged sarnple.
Referring now to the drawings, there is shown in FIGURE 1 a preferred embodiment of the blood sampling paraphenalia formed in accordanc~ with this invention. The blood sampling paraphenalia includes a transparent ~ube 2 formed preferably ot glass, and having an in~egrally closed end 4. A plastic float member 6 is disposed in the tube 2, as are the stain and red cell densifier reagents 8. An elastomeric plug 10 closes the open end of the tube 2, and a supply of a thixotropic gel 12 is disposed inside of the tube 2 around the plug 10 In placc of thP gel 12, a thin plastic disc or cup 17 can be used. The tube is preferably about 75 mm long, the same length as a capillary tube, and has a bore diameter of abou~ 40 mm. Its capacity for blood is about a.s ml. The float wili be about B rnm in length and about 4 mm in diameter when static in the tube.
.
The float 6 is a compund structure which has a central through bore 7 into which the white cells and platele~s layer out during centrifugation. The bore 7 is preJerably about 1.265 mm in diameter so as to achiev~ the necessary c~ll band elonga~ion to ailow haNesting of the target cell band. The float 6 is formed with a core part 9 made from a dimensionally stable transparent plastic, such as a rigid styrene plastic. A sleeve part 11 surrounds the core 9 and is bond~d thereto. The sleeve 11 is formed from a pliable ~ .i.~
2~$~7~
transparent plastic such as PVC. The ends o~ the sleeve 11 are flared, as at 13, ~nd the ends of the bore 7 are also fiared as ag 15 to allow movernent of the ~lood in the tube 2 during filling and centrifugation.
FIGURE 3 shows how the tube 2 can be filled with blood from a primary blood collecting tube 14 by means of a transferring device 16 having a double piercingneedle or cannula 18. The transfer device 16 inciudes an outer shroud 20 with a needle-carrying plug 22 telescoped thereinto. The needle 18 extends into a first weli 24 in the plug 22 sized to receive the stoppered end of the blood sarnpling tube 2. The shroud 20 forms a second well 26 which is sized to receive the stoppered end of the primary blood collecting tube 14. The transfer needle 18 pierces the plug 28 in the tube 14 and also pierces the plug 10 in the sampling tube 2. The low pressure in the tube 2 causes blood to be drawn from the tube 14 through the needle 18 into the tube 2, the flow of blood continuing until the tube 2 is substantially filled. Once filled, the tube 2 is withdrawn from the well 24 and centri~uged. While trans~erring blood to the testing tube 2 frorn a collection tube 14 is one way to fill the tube 2, it is readily apparent that the sample could be taken directly from a patient using a needle and the evacuated tube 2.
When the blood enters the tu~e 2, the reagents 8 wiil mix with ~he blood, and the tube 2 will be ready to centrifu~e. The tubes 2 are oriented in the centrffuge with the closed end 4 out, so tha~ thP red cells will settle in the closed encl of the tube 2 and the plasma wilt be adjacent to the stoppered end of the tube 2 after centrifugation. FIGURE 4 shows the condition of the tube 2 and blood after the cen~rifu~ation has been completed. The red cells 30 collect in the closed end of the tube 2 and the float 6 becomes embedded in, and projects above the top of the red cell layer. The whitecells and plate~et layers which make up ~he bu~fy coat 32 set~le into the axial through bore 7 in the float 10 and the plasma 34 is disposecl above the buffy coa~ and floa~ 10.
The thixotropic gel 12 (or plastic disc 17) covers and floats on the plasma layer 34 thereby holding the centrifuged blbod constituent layers in place in th~ bore 7 when the tube 2 is handl~d af~er the centrifugatiorl step during harvesting ~f th~ target cell band from the floa~ bore 7.
FIGURE 5 shows the manner in which the target cells can be harvested from the float bore 7 with an aspirating needle 31. The needle 31 is inserted into the tube 2 ~hrough the plug 10 so that its tip 23 may be positioned in ~he target cell ~and B, ~he other ~ell bands being desiynated A, (:~, D and E. Suction is applied to the band B via theneedle 31 causing the cells to move in the directivn of the arrow 33 into the needle 31.
When the filled tube 2 is subjected to centrifugation forces of 10,000 G, which is the force at which the prior art capillary tubes are centri~uged, the pliable sleeve part 11 of the float 6 radially contracts whereby the effective diameter of ~he float 6 decreases.
Thus the float 6 is forced through the blood sample until the float encounters the centrifuged red cell layer which, because of its specific gravity, resists fu~ther movement of the float 6. Once this occurs, the float 6 will be stabilized and the sleeve part 11 will expand back outwardly into snug engagement with the tube bore. The tube bore wall may be coated with a siiicone lubricant to enhance the slidability of the float 6 in the tube 2.
It will be readily appreciat~d tha~ the tubes of this invention can be used to draw blo~
samples frorn pa~ients or from blcod collecting tubes, and the blood cell measurements c~n then be made directly in ~he stoppered, closed tubes without exposing anyone to the possibitity of contact with cont~minated blood. Thus the blood testing procedure can even be used with patients who are known to have contaminated blood with no danger to the person doing the testing. The dimensional tolerances observed in producing the tubes and floa~s are relaxed, and the test assemblies have a longer shelf l~e since the interior of the evaouated tubes is filled with an inert gas. Cell layer band ~rmation is preserved during handling of the tube ~ter centrifugation due to thepellicle formed on top of ~he plasma by the thixotropic material or by a piastic disc in the tube during centrifugation. Target cells can be easily harvested from the readily visible, elon~ated bands of cells in the float bore.
2 ~ 7 ~
Since many changes and variations of the disclosed embodinnen~ of the invention may be rnade without ~eparting from the inven~ive concept, it is not intended to limit the invention otherwise than as requirec by the appended claims.
This invention rela~es to paraphenalia and a method for determining information about a constituent layer in a centrifuged sample of a material such as blood. The constitu~nt layer is harvested from an evacuated ~ube containing a float which expands the consti~uent layers being harvested, and which contains a through bore or passage into which the particuiar constituent layers settle during centrifugation.
A technique has been developed to measure constituent layers in a complex materia!
mixture by centrifuging a sarnpl~ of the material mixture in a capillaiy or other tube which oontains a float. The ~ioat is pr~erably cylindrical and of a specifio gravi~ which eauses it to settle into the centrifuged mixture to a degree which creates a free volume annulus in the tube into which the layer, or layers to be measured will settle. The layers to be measured arc thus physically elongated, and can be more easily and accurately rneasured. This technique is described in U. S. Patents Nos. 4,027,660 issued ~une 7, 1977; 4,082,085 issued April 4, 1978; 4,156,570 issued May 29,1979;
and others.
When the material being ~ested is a possibly contaminated material such as biood, it is desirable to make provisions for protecting the technician against exposure to ~he blood. When the aforesaid prior art techniques are pei~ormed with capillai~ tubes, the person pei~orming the test is exposed to the blood since the capillaiy tubes areopen-ended. Thus, despite taking normal precautions in handling ot the samples, the chance of being contaminated by a blood sample exists. The aforesaid prior art also does not readily lend i~elf to harvesting of any of the centrifuged blood cell bands ~rom the tub~. .
This invention is directed to a me~hod and paraphenalia ~or use in the collecting ~nstituent cells or other particles from a possib!y contamin~ted material swh asblood, wherein the person doing the colleoting is never exposed to the blood. Thus, the possibility of becoming infeoted by a contaminated blood sample is elimin~ted.
When the tube and float of this invention ara used, the blood sample is collected in a sealed tube; then concentration o~ cells to be harves~ed is made in the tube; and the 2 ~ 7 0 cells can be aspirated from the tube without ever exposing the ~echnician to the blood sample. An addi~ional advantage of th~ invention resides in ~he fact that it entails the use of a unitary sealed tube which contains all of the required components for use in performing the cell concentration and harvesting, and those components are disposed in a stable, inert environment. The tub~ used in this invention is preferably a giass tube with an integral closed end. It will be the same length as a capillary tube but will hav0 a ~arg~r diameter so as to be able to con~ain about 0.9 ml of blood. A cylindrical float is disposed inside of the tube, which float has an accurately controlled outside diameter so as ~o fit snugly in the tube bore under static conditions. When used in harvesting blood cells the float is forrned with an axial through bore which rec~ives and expands the white cell and pla~elet layers in the blood sample af~r centrifugation thereof. The float is made from a plastic material having a specific gravity that causes it to float in the packed red cells after centrifugatian of the blood sample in the tube.
Required reagents, such as a stain and a red cell densifier, preferably potassium oxalate, may be disposed in the tube, preferably in liquid form. An elastomeric stopper closes the open end of the tube, and the interior of the tube is filled with an inert gas at low pressure. The low pressure in the tube is used to draw the blood sample into the tube, preferably from a primary blood collection device, such as ~hat sold by Becton Dickinson and Company under ghe tra~emark "Vacutainer".
The float may preferably be a compound structure made from plastics which have aspecific gravity which causes the float to be buoyed up in ~he centrifuged red cell layer.
The float is formed with a core portion which has the through bore, and an annular sleeve portion which will expand and contract responsive to the magnitude of dynamic forces imposed on ~he float during performance of the sample centrifugation. The float ~r~ must be formed from a plastic material, such as ~ transparent styrene, which is dimensionally stable during centrifugation. The peripheral sleeve portion o~ ~he float can be formed from a ~ransparent pliable vinyl plas~ic. The ~No components of the float can be joinecl together by c~extrudin~ or hy co-molding the float cvmponents. The tu~e can be provided with a lubricant coa~ing, such as a silicone coating to enhance movement of the float in ~he tube during centrifugation. Specific plastics which can be 2~5$~70 used for the core and sleeve of the float are polystyrene and polyvinylchloride (PVC) respectively. The float may also be formed from a single plastic material iF so desired.
The primary blood ~ollection tube will be provided with a needle which is used to pierce the elastomeric stopper in the tube of this invention, whereupon ~he blood will flow from the collection tube, through the needie, into the testing tubA. In order to preserve cell band formation in the tube when the tube and ~lood are cen~rifuged, a thixotropic gel would be disposed in the top of the ~ube. During centrifugation, the gel will flow down the wall of the tube and settle on top of the plasrna layer to form a viscous peliicle on the plasma. Obviously, the gel must have a specific gravity which is less than that of the plasma. A thin plastic cup may be used in lieu of the gel.
When the larger bore diameter tube and the larger float with an axi~l bore are used per this invention, there occurs a relaxation in the diameter dimensional tolerances in the tube bore ID. It is d~sirable to achieve a ten fold expansion of the whito cell and platelet layers when perForming the cell harvesting with the tube-float combination of the aforesaid prior art. When using the enlarged tubes and floats of this invention, the ten fold expansion can be obtained frorn a through bore diameter of 1.265 mm wherl a 4.0 mm diameter tube bore is used. This compares with a free space uf about 43 microns with the prior art capillary tubes and floats. The ~I- variation in the bore diameter is 20 rnicrons when using the paraphPnalia of this invention.
A benefit deriving from the use of the larger tube and float paraphenalia is an improvement in the hydrodynamics of the centrifuga~ion. A~ter blood is added ~o the tube, the tube is centrifuged at 10,000 G, as is the usual practice. With a float of this type, several forces are brought in~o play. First, the centripedal accelleration forces the float to the end of the tube at the same time as th~ blood cells are separating.Secondly, a tidal force is exerted on the float be~use the accelleration is unequal at the e~dg of the float. This tidal force is about 2,0V0 G at near the csnter of th~ tube.
This exerts a stretching or con~racting force on ~he float of about 500 G, which is enough to sufficiently elongate the pliable elastomeric portion of the float and sliyhtly ', 2 ~ 7 ~1 decrease its diameter, aliowing it to easily slip down ~he ~ube. AMer the float settles according to its density into the R~C layer, and ~he centrifuge slows to a s~op, the tidal forces cease, and the float relaxes to its normal diameter thereby reassuming its close approximation to the walls of the tube.
The cells and components of the bu~y coat ~ayer are expantied linearly in the narrow bore channel in the float and thus can be easily harvested ~herefrom.
It is therefore an object of this invention lo provide an improved blood sampling paraphenalia which allows for the blood cell harvesting to be made without exposing the technician to contamination from the blood sample.
It is a further object of this invention to provide blood sampling paraphenalia of the character described wherein dimensional tolerances are relaxed while providing the necessary cell layer expansion.
It is an additional object of this invention to provide blood sampling paraphenalia of the character described wherein larger biood samples are used.
It is still another object of this invention to provide blood sampling paraphenaiia o~ the character described wherein the formation of cell bands a~ter centrifugation, isstabilized and preserved.
It is yet an additional object of this invention to provide blood sampling paraphenalia of the character describe~ wherein improved hydrodynamics during centrifugation is achievecl.
These and other obje~e. and adYantages of ~he invention will become more readilyapparent from the following deseription of a preferred embodiment ther~of when eonsidered in oonjunction wim ~he accompanying drawings, in which:
2 ~ 7 ~
FIGURE 1 is an axial sectional view of a preferred embodiment of a tube and float assembiy formed in accordance with this in~/ention.
FIGURE 2 is an axial sectional view of the float;
FIGURE 3 is an axial sectional view showing how the assembly can b used to draw a bloo~l sample from a primary blood collecting tube;
Fl(3URE 4 is a view similiar to FIGURES 1 ~nd 3 but showing the assembly of FIGURE
1 after the blood sample has been drawn and centrlfuged, and FIGURE 5 is a fragmented sectional view showing how a cell layer can be harvested from the centrifuged sarnple.
Referring now to the drawings, there is shown in FIGURE 1 a preferred embodiment of the blood sampling paraphenalia formed in accordanc~ with this invention. The blood sampling paraphenalia includes a transparent ~ube 2 formed preferably ot glass, and having an in~egrally closed end 4. A plastic float member 6 is disposed in the tube 2, as are the stain and red cell densifier reagents 8. An elastomeric plug 10 closes the open end of the tube 2, and a supply of a thixotropic gel 12 is disposed inside of the tube 2 around the plug 10 In placc of thP gel 12, a thin plastic disc or cup 17 can be used. The tube is preferably about 75 mm long, the same length as a capillary tube, and has a bore diameter of abou~ 40 mm. Its capacity for blood is about a.s ml. The float wili be about B rnm in length and about 4 mm in diameter when static in the tube.
.
The float 6 is a compund structure which has a central through bore 7 into which the white cells and platele~s layer out during centrifugation. The bore 7 is preJerably about 1.265 mm in diameter so as to achiev~ the necessary c~ll band elonga~ion to ailow haNesting of the target cell band. The float 6 is formed with a core part 9 made from a dimensionally stable transparent plastic, such as a rigid styrene plastic. A sleeve part 11 surrounds the core 9 and is bond~d thereto. The sleeve 11 is formed from a pliable ~ .i.~
2~$~7~
transparent plastic such as PVC. The ends o~ the sleeve 11 are flared, as at 13, ~nd the ends of the bore 7 are also fiared as ag 15 to allow movernent of the ~lood in the tube 2 during filling and centrifugation.
FIGURE 3 shows how the tube 2 can be filled with blood from a primary blood collecting tube 14 by means of a transferring device 16 having a double piercingneedle or cannula 18. The transfer device 16 inciudes an outer shroud 20 with a needle-carrying plug 22 telescoped thereinto. The needle 18 extends into a first weli 24 in the plug 22 sized to receive the stoppered end of the blood sarnpling tube 2. The shroud 20 forms a second well 26 which is sized to receive the stoppered end of the primary blood collecting tube 14. The transfer needle 18 pierces the plug 28 in the tube 14 and also pierces the plug 10 in the sampling tube 2. The low pressure in the tube 2 causes blood to be drawn from the tube 14 through the needle 18 into the tube 2, the flow of blood continuing until the tube 2 is substantially filled. Once filled, the tube 2 is withdrawn from the well 24 and centri~uged. While trans~erring blood to the testing tube 2 frorn a collection tube 14 is one way to fill the tube 2, it is readily apparent that the sample could be taken directly from a patient using a needle and the evacuated tube 2.
When the blood enters the tu~e 2, the reagents 8 wiil mix with ~he blood, and the tube 2 will be ready to centrifu~e. The tubes 2 are oriented in the centrffuge with the closed end 4 out, so tha~ thP red cells will settle in the closed encl of the tube 2 and the plasma wilt be adjacent to the stoppered end of the tube 2 after centrifugation. FIGURE 4 shows the condition of the tube 2 and blood after the cen~rifu~ation has been completed. The red cells 30 collect in the closed end of the tube 2 and the float 6 becomes embedded in, and projects above the top of the red cell layer. The whitecells and plate~et layers which make up ~he bu~fy coat 32 set~le into the axial through bore 7 in the float 10 and the plasma 34 is disposecl above the buffy coa~ and floa~ 10.
The thixotropic gel 12 (or plastic disc 17) covers and floats on the plasma layer 34 thereby holding the centrifuged blbod constituent layers in place in th~ bore 7 when the tube 2 is handl~d af~er the centrifugatiorl step during harvesting ~f th~ target cell band from the floa~ bore 7.
FIGURE 5 shows the manner in which the target cells can be harvested from the float bore 7 with an aspirating needle 31. The needle 31 is inserted into the tube 2 ~hrough the plug 10 so that its tip 23 may be positioned in ~he target cell ~and B, ~he other ~ell bands being desiynated A, (:~, D and E. Suction is applied to the band B via theneedle 31 causing the cells to move in the directivn of the arrow 33 into the needle 31.
When the filled tube 2 is subjected to centrifugation forces of 10,000 G, which is the force at which the prior art capillary tubes are centri~uged, the pliable sleeve part 11 of the float 6 radially contracts whereby the effective diameter of ~he float 6 decreases.
Thus the float 6 is forced through the blood sample until the float encounters the centrifuged red cell layer which, because of its specific gravity, resists fu~ther movement of the float 6. Once this occurs, the float 6 will be stabilized and the sleeve part 11 will expand back outwardly into snug engagement with the tube bore. The tube bore wall may be coated with a siiicone lubricant to enhance the slidability of the float 6 in the tube 2.
It will be readily appreciat~d tha~ the tubes of this invention can be used to draw blo~
samples frorn pa~ients or from blcod collecting tubes, and the blood cell measurements c~n then be made directly in ~he stoppered, closed tubes without exposing anyone to the possibitity of contact with cont~minated blood. Thus the blood testing procedure can even be used with patients who are known to have contaminated blood with no danger to the person doing the testing. The dimensional tolerances observed in producing the tubes and floa~s are relaxed, and the test assemblies have a longer shelf l~e since the interior of the evaouated tubes is filled with an inert gas. Cell layer band ~rmation is preserved during handling of the tube ~ter centrifugation due to thepellicle formed on top of ~he plasma by the thixotropic material or by a piastic disc in the tube during centrifugation. Target cells can be easily harvested from the readily visible, elon~ated bands of cells in the float bore.
2 ~ 7 ~
Since many changes and variations of the disclosed embodinnen~ of the invention may be rnade without ~eparting from the inven~ive concept, it is not intended to limit the invention otherwise than as requirec by the appended claims.
Claims (9)
1. A blood sampling assembly for use in harvesting target blood cells from a centrifuged sample of blood contained in the assembly without exposing one performing the test to the blood being sampled, said assembly comprising: a transparent tube for holding the sample of blood; an elongated float member disposed in said tube, said float member having an outer surface which, during static conditions, closely conforms to the bore of said tube, and said float member being operable to settle into the red blood cell layer of the centrifuged blood sample and said float member having a restricted through passage into which the blood sample buffy coat layers out thereby to physically expand the white cell and platelet layers in the buffy coat; an elastomeric stopper sealing one end of said tube; and the interior of said tube having a subatmospheric pressure whereby the blood will be automatically drawn into said tube when said stopper is pierced by a blood sampling needle.
2. The assembly of Claim 1 further comprising means for forming a pellicle on top of the centrifuged blood sample in the tube whereby centrifuged cell layers are rendered stable for handling of the tube during harvesting of the target cells.
3. The assembly of Claim 1 wherein said tube is filled with an inert gas prior to use of the assembly to draw a blood sample.
4. The assembly of Claim 1 wherein said float member is a compound structure which includes a dimensionally stable transparent plastic core containing said throughpassage, and a pliable transparent plastic sleeve bonded to and surrounding saidcore, said sleeve providing radially contractible means for allowing said float member to freely move through the tube bore when said float member is subjected to centripetal forces during centrifugation of the blood sample.
5. The assmebly of Claim 4 wherein said through passage has a diameter of approximately 1.265 mm.
6. A method for harvesting target cells from a centrifuged sample of blood contained in a tube which also contains a float having a through passage for receiving and elongating layers of blood cell components, the float having an outer diameter which ensures that the float fits snugly in the tube when under static conditions, said method comprising the steps of:
a) drawing a blood sample into the tube; and b) centrifuging the blood, tube and float at sufficient G forces to move the float toward one end of the tube, and concurrently exerting a tidal force on the float duringcentrifugation to contract the outer diameter of the float sufficiently to allow the float to slide through the tube during the centrifugation step.
a) drawing a blood sample into the tube; and b) centrifuging the blood, tube and float at sufficient G forces to move the float toward one end of the tube, and concurrently exerting a tidal force on the float duringcentrifugation to contract the outer diameter of the float sufficiently to allow the float to slide through the tube during the centrifugation step.
7. The method of Claim 6 further comprising the step of lubricating the interface between the tube and float to enhance movement of the float through the tube.
8. The method of Claim 7 further comprising the step of evacuating said tube to an extent needed to automatically draw blood into said tube.
9 . A method for harvesting a target constituent from a centrifuged sample of a multi-constituent material sample contained in a tube which tube also contains a float having a through passage for receiving and elongating a layer of the target constituent, the float having an outer diameter which ensures that the float fits snugly in the tube when under static conditions, said method comprising the steps of:
a) drawing the material sample into the tube;
b) centrifuging the material sample, the tube and the float at adequate G forces to move the float toward one end of the tube sufficiently that the target constituent layers out in the through passage in the float and is thereby physically elongated; and c) aspirating the target constituent out of the float through passage without removing any of the rest of the material sample from the tube.
a) drawing the material sample into the tube;
b) centrifuging the material sample, the tube and the float at adequate G forces to move the float toward one end of the tube sufficiently that the target constituent layers out in the through passage in the float and is thereby physically elongated; and c) aspirating the target constituent out of the float through passage without removing any of the rest of the material sample from the tube.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63626090A | 1990-12-31 | 1990-12-31 | |
| US07/636,260 | 1990-12-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2058670A1 true CA2058670A1 (en) | 1992-07-01 |
Family
ID=24551137
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002058670A Abandoned CA2058670A1 (en) | 1990-12-31 | 1991-12-31 | Constituent layer harvesting from a centrifuged sample in a tube |
Country Status (13)
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| US (1) | US5393674A (en) |
| EP (1) | EP0493838B1 (en) |
| JP (1) | JPH0774772B2 (en) |
| CN (1) | CN1033056C (en) |
| AT (1) | ATE153761T1 (en) |
| CA (1) | CA2058670A1 (en) |
| DE (1) | DE69126293T2 (en) |
| DK (1) | DK0493838T3 (en) |
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| MX (1) | MX9102860A (en) |
| NO (1) | NO303800B1 (en) |
| RU (1) | RU2088921C1 (en) |
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- 1991-12-30 DE DE69126293T patent/DE69126293T2/en not_active Expired - Fee Related
- 1991-12-30 NO NO915134A patent/NO303800B1/en unknown
- 1991-12-31 CA CA002058670A patent/CA2058670A1/en not_active Abandoned
-
1993
- 1993-03-08 US US08/025,343 patent/US5393674A/en not_active Expired - Fee Related
-
1997
- 1997-06-26 GR GR970401562T patent/GR3023914T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN1063163A (en) | 1992-07-29 |
| NO915134L (en) | 1992-07-01 |
| JPH04303730A (en) | 1992-10-27 |
| MX9102860A (en) | 1992-07-01 |
| ATE153761T1 (en) | 1997-06-15 |
| JPH0774772B2 (en) | 1995-08-09 |
| DE69126293D1 (en) | 1997-07-03 |
| US5393674A (en) | 1995-02-28 |
| GR3023914T3 (en) | 1997-09-30 |
| RU2088921C1 (en) | 1997-08-27 |
| NO303800B1 (en) | 1998-08-31 |
| ES2104654T3 (en) | 1997-10-16 |
| DK0493838T3 (en) | 1997-07-28 |
| EP0493838A1 (en) | 1992-07-08 |
| DE69126293T2 (en) | 1998-01-15 |
| NO915134D0 (en) | 1991-12-30 |
| CN1033056C (en) | 1996-10-16 |
| EP0493838B1 (en) | 1997-05-28 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |