EP0112868A1

EP0112868A1 - Ultracentrifuge tube with multiple chambers

Info

Publication number: EP0112868A1
Application number: EP19830902064
Authority: EP
Inventors: Steven T. Nielsen; Carleton C. Lee
Original assignee: Beckman Instruments Inc
Current assignee: Beckman Coulter Inc
Priority date: 1982-07-06
Filing date: 1983-05-16
Publication date: 1984-07-11
Also published as: WO1984000313A1; JPS59500008U

Abstract

Tube centrifugeur (10) à paroi mince possèdant des chambres multiples (24, 26) pour une utilisation dans l'examen d'un échantillon liquide avec ultracentrifugeuse. Plus particulièrement, le tube centrifugeur (10) à chambres mltiples peut être utilisé par exemple pour une séparation lipoprotéinique. Le tube ultracentrifugeur (10) peut possèder deux chambres (24, 26) séparées, ou plus, quii, après la centrigation, peuvent être séparées hermétiquement l'une de l'autre afin de retenir les composants séparés (50, 54) de l'échantillon examiné. Les chambres (24, 26) du tube (10) sont unie par une zone étranglée (20) permettant une communication de liquide entre les chambres (24, 26). Le tube (10) est destiné à être utilisé conjointement avec un organe d'espacement de soutien (32) attenant à l'étranglement (20) du tube (10) si bien que le tube (10) est soutenu correctement lors d'une ultracentrifugation.Thin wall centrifuge tube (10) having multiple chambers (24, 26) for use in examining a liquid sample with an ultracentrifuge. More particularly, the centrifuge tube (10) with multiple chambers can be used for example for lipoprotein separation. The ultracentrifuge tube (10) may have two or more separate chambers (24, 26) which, after centralization, can be hermetically separated from each other to retain the separate components (50, 54) of the 'sample examined. The chambers (24, 26) of the tube (10) are joined by a constricted area (20) allowing liquid communication between the chambers (24, 26). The tube (10) is intended to be used in conjunction with a support spacer (32) adjoining the neck (20) of the tube (10) so that the tube (10) is properly supported during a ultracentrifugation.

Description

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ULTRACENTRIFUGE TUBE WITH MULTIPLE CHAMBERS

Background of the Invention The present invention is directed to ultracen- 5 trifuge tubes and, more particularly, is directed to a multi-chamber ultracentrifuge tube.

One of the more important areas in medical research is directed to an understanding of the causes of ° heart attacks and strokes as the result of arterioscler¬ osis. Incalculable studies over many years have revealed that one of the primary causes of death or incapacitation in adults over their mid-thirties is attributable to heart attacks as well as strokes. Heart disease has reached epidemic proportion in modern civilization.

One of the important research studies being conducted in the medical field is to learn not only how to prevent the occurrence of heart attacks, but also how to predict the likelihood of a heart attack occurring for a particular individual. Such predictions will allow the patient to undergo a program to avoid the occurrence of heart attack. Large research studies have found that the quantitation of cholesterol in each of the lipoprotein density classes provides patient information that is extremely helpful in predicting the risk of coronary heart disease. The lipoproteins in the blood are classi¬ fied by their buoyant density. It is generally well known that the relative magnitudes of density for the various lipoproteins can be classified as follows: very low density lipoproteins (VLDL) , 0.95 to 1.006 grams per ml; low density lipoproteins (LDL) , 1.006 to 1.063 grams per ml; and high density lipoproteins (HDL) , 1.063 to 1.21 grams per ml. In view of the relative gradations of density between the various lipoproteins, centrifugation provides an obvious choice to accomplish the separation of the various lipoproteins from each other under a high cen- trifugal force field. Therefore, lipoprotein investi¬ gation involves the utilization of a preparative ultra¬ centrifuge after which the separated lipoproteins are subjected to analytical measurements to determine the cholesterol concentration. Since the sample is normally placed in a typical single chamber centrifuge tube for insertion in a fixed angle or vertical tube ultracentri¬ fuge rotor, the separate bands formed during centrifuga¬ tion will reorient after the centrifugation is com¬ pleted. Some residue from one band may be left on the tube wall and contact other bands as they reorient to a new position affecting the purity of the separation. One type of single chamber tube used is that which is dis¬ closed in U.S. Patent 4,301,963 issued to Steven T. Nielsen on November 24, 1981 and entitled "Integral One piece Centrifuge Tube."

After centrifugation each separated lipoprotein fraction must be physically isolated so that it is not contaminated and will not result in an incorrect reading of the cholesterol concentration for that particular density lipoprotein. Typical techniques utilized to accomplish this separation are centrifuge tube slicing or aspiration. In the tube slicing technique the cutting blade must be carefully positioned and be maintained in an extremely clean condition. Furthermore, depending upon the particular type of tube utilized, a significant amount of force may have to be applied by the user to pierce the tube. Once the tube is sliced, the upper portion of the tube is isolated and the separated lipo- protein can be aspirated out of the sliced-off portion of the tube without disturbing the other separated lipopro¬ tein bands in the remainder of the tube.

In the straight aspiration technique, bands are pipetted or suctioned off layer by layer. This operation may be simple and relatively convenient, but it poses a strong possibility of contamination. This is because lipoproteins from the removed fraction may adhere to the side of the tube and contaminate the remaining fraction.

The need exists in the process of measuring the cholesterol concentration in lipoprotein separations for a means to conveniently isolate the separated lipopro¬ teins so that the subsequent investigation of cholesterol concentration in the separated lipoproteins can be ac¬ complished with a minimum of effort and a minimum of contamination.

Summary of the Invention The present invention is directed to a multi- chamber ultracentrifuge tube. The separate chambers in the centrifuge tube are joined by a constricted area which forms, a conduit to provide fluid communication between the chambers during centrifugation of the tube. A fluid sample placed into the centrifuge tube will, during centrifugation, be separated into lighter material in the chamber closest to the rotational axis of the rotor, while the heavier material will be separated into the chamber farthest from the spin axis of the rotor.

Since the chambers are divided by a constricted area having an outer dimension significantly smaller than the outer dimension of the main portion of the tube with its chambers, a support spacer is placed adjacent the constriction to provide support to the tube at the con¬ stricted area during centrifugation.

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Various configurations of the constricted area are applicable to the present invention. Further, the number of separate chambers that can be formed is limited only by the length of the tube.

Use of the present invention allows an inves¬ tigator of blood serum lipoproteins to accomplish an efficient separation of the various density lipoproteins without contamination. Subsequent to the centrifugation run, the constricted area of the tube can be severed and sealed to ensure the isolation of the different density lipoproteins.

Brief Description of the Drawings Figure 1 is an elevational view of the ultra¬ centrifuge tube of the present invention;

Figure 2 is an elevation sectional view of the ultracentrifuge with an annular support spacer and a top support spacer;

Figure 3 is a top view of the split annular spacer of Figure 2;

Figure 4 is a sectional view of the annular spacer taken along the lines 4-4 in Figure 3;

Figure 5 is a partial sectional view of a cen¬ trifuge rotor with the present invention in place, show¬ ing the orientation of the various separated fluid sample bands during centrifugation;

Figure 6 shows the reorientation of the fluid sample bands in Figure 5 when the tube is at rest in a vertical position; Figure 7 shows an alternate embodiment of the present invention utilizing a wedge spacer in a centri¬ fuge rotor with the wedge facing the spin axis of the rotor;

Figure 8 is similar to the centrifuge tube shown in Figure 7 with the wedge-shaped spacer facing away from the spin axis of the rotor;

Figure 9 is a second alternate embodiment of the present invention showing several chambers in the centrifuge tube with several annular support spacers; and

Figure 10 is a third alternate embodiment of the present invention showing several chambers within the centrifuge tube separated by multiple wedge spacers.

Detailed Description of the Invention A multi-chamber ultracentrifuge tube 10 of the present invention is shown in Figure 1 having a generally uniform cylindrical wall 12 enclosed with a hemispherical bottom 14 and a bell-shaped upper portion 16. Located between the upper portion 16 and the hemispherical bottom end 14 is a constricted area 20 forming an interior con- duit 22 that provides fluid communication between an upper chamber 24 and a lower chamber 26. Positioned on the upper portion 16 is a fill port 28 which is open (shown in phantom) for receipt of a fluid sample prior to centrifugation. After the tube is filled with the sample to be centrifugated, the fill port 28 is hermetically sealed to form a solid seal 30. The tube 10 is prefera¬ bly an integral one-piece tube of a thin-wall polyallomer of the type explained more fully in the above-referenced U.S. Patent 4,301,963. As shown in Figure 2, the centrifuge tube 10 is designed so that its constricted area 20, having the shape generally of an hour glass, compatibly receives an annular support spacer 32. The diameter of the outside surface 34 of the annular spacer is approximately the same as the diameter of the cylindrical wall 12 of the tube. The spacer 32 provides support to the thin-wall polyallomer tube 10 during centrifugation, so that there is no distortion of the tube or constriction of the con- duit 22 which would affect fluid communication between the upper chamber 24 and the lower chamber 26. Posi¬ tioned on the upper portion 16 of the tube is a floating spacer 36 that is designed to provide exterior support to the upper portion 16 of the tube when in a rotor during centrifugation. For further discussion concerning the floating spacer 36, reference is made to U.S. Patent 4,304,356 issued to Steven J. Chulay and Steven T. Nielsen on December 8, 1981 entitled "Supporting Cap for Sealed Centrifuge Tube".

The configuration of the spacer 32 is shown in Figure 3 with two half sections 38 and 40 that form the entire circular arrangement of the split annular spa¬ cer. The central opening 41 formed by the spacer sec- tions 38 and 40 is approximately the same size as the exterior diameter of the constricted area 20 on the tube 10 in Figure 1. The sectional configuration of one por¬ tion 38 of the split annular spacer 32 is shown in Figure 4. The sectional cross area 42 is designed to occupy and conform to the constricted area 20 in the tube 10.

As explained previously, one type of fluid sample which is investigated to help predict the risk of coronary heart disease is serum lipoproteins. It is desirable to determine the amount of cholesterol found in each of the lipoprotein classes in a patient's blood serum. Since the different lipoproteins have differing density characteristics, the process of centrifugation provides the necessary separation for individual analysis and measurement of the cholesterol in each of the types of lipoproteins. In this type of an investigation, it is preferable that the centrifuge tube 10 in Figure 1 have two chambers 24 and 26 with a constriction 20 between the chambers that forms a conduit 22 which will be large enough to allow fluid communication between the cham- bers. It has been found through experimentation that in a two-chamber tube the upper chamber volume should be approximately 31% of the total tube volume and the lower portion be 69% of the total tube volume. This chosen relative volume for each of the upper and lower chambers is for analysis of a serum sample to ensure that during centrifugation the very low density lipoprotein fraction will remain in the upper chamber after centrifugation, while the combination low density and high density lipo¬ protein fraction will remain in the lower chamber.

Once the centrifuge tube 10 is filled with the blood sample, the fill port 28 in Figure 1 is sealed. The tube 10 is then partially lowered into a tube cavity 44 in the rotor 46 in Figure 5. The split annular spacer 32 is placed around the constricted area 20 of the tube 10 and the tube is lowered completely into the cavity 44 as shown in Figure 5. The top floating spacer 36 is positioned above the tube in contact with its upper por¬ tion 16. The rotor lid 48 is placed on the rotor and the centrifugation process is initiated. During centrifuga¬ tion the very low density lipoproteins 50 will band in an area of the tube closest to the spin axis 52 of the rotor 46. On the other hand, the low density and high density lipoprotein portion 54 will form a band in the portion of the tube farthest from the spin axis 52 of the rotor. As discussed above, the respective upper and lower chambers are sized so that the entire very low density lipopro¬ teins 50 will remain in the upper chamber 24, while the low density and high density lipoproteins will remain in the lower chamber 26. The lipoprotein fractions 50 and 54 will be separated by an inner band of the blood sample 56 that does not contain any lipoproteins.

Once the centrifugation process has been com¬ pleted and the lipoproteins have been separated, tube 10 is removed from the centrifuge rotor and the constricted area 20 of the tube is pinched with a clamp, hemastats or possibly heated hemastats to seal off the conduit 22 and maintain the isolation of the separated lipoprotein con¬ tents in the respective chambers. The constricted area 20 may also be pinched off by twisting one chamber 360^β along its longitudinal axis with respect to the other chamber. To obtain a cholesterol value for the very low density lipoprotein fraction in the upper chamber 24, the contents from the chamber are transferred to another tube for the cholesterol analysis. There is no requirement of tube slicing or aspiration to separate the lipoprotein bands.

The content of the lower chamber 26 which con- tains both the low density lipoproteins and high density lipoproteins is transferred to another two-chamber tube 10 and the centrifugation process is repeated so that the lighter low density lipoprotein fraction will flow to the top of the upper chamber 24 and the heavy density lipo- protein will be in the lower chamber 26. After centrifu¬ gation, the constricted area 20 is pinched off to seal the conduit 22 and maintain the separation of the respec¬ tive lipoprotein constituents.

Attention is directed to Figure 7 showing an alternate embodiment of the present invention wherein the — —

ultracentrifuge tube 60 has an upper chamber 62 and a lower chamber 64 which are joined by a restricted area 66 forming a conduit 68. While the embodiment of the pres¬ ent invention shown in Figure 1 has the conduit formed symmetrically around the longitudinal center of the tube 10, the embodiment shown in Figure 7 has the conduit portion 68 closely adjacent the cylindrical wall 70 of the tube 60. The bottom hemispherical portion 72 of the tube 60 and its upper bell-shaped upper portion 74 estab- lish an enclosed centrifuge tube. A wedge-shaped spacer 76 is placed in the constricted area 66 to form exterior support to the upper chamber 62 and the lower chamber 64 during centrifugation. The orientation of the tube 60 in Figure 7 positions the conduit 68 so that it is as far away from the spin axis 52 of the rotor as possible. In this orientation the heavier components will move outward and collect on the straight interior wall 73 of the cen¬ trifuge tube and move smoothly into the lower chamber 64. However, the lighter material must follow a more oblique path around the wedge-shaped surface 78 of the constricted portion 66.

In Figure 8 the same centrifuge tube 60 is shown rotated 180° in the tube cavity 44 of the rotor 46 as compared to the tube position in Figure 7. In this orientation the constricted portion 66 is oriented in a position as close as possible to the spin axis 52 of the rotor. This orientation of the centrifuge tube 60 has the advantage that the light fractions will collect on the straight surface 73 of the tube closest to the spin axis of the rotor and move smoothly up into the upper chamber 62. However, the constricted wedge-shaped por¬ tion 78 on the interior of the tube will result in a more oblique or circuitous path for the heavier fractions to move down into the lower chamber 64 which is farther away from the spin axis of the rotor. It should be noted with respect to not only the split annular spacer 32 of the embodiment shown in Figure 2, but also the wedge-shaped spacer 76 shown in Figure 7 that each are preferably made of a polyphenylene oxide material known as Noryl developed by General Electric. This material is advantageous, because its density is approximately 1.06 grams per ml which is closely approxi-

•» mate bulk serum density. During investigation of lipo¬ proteins this material will not cause undue pressure to be exerted on the top of the lower chamber because of the centrifugal loading of the spacer.

Reference is made to Figure 9 showing a second alternate embodiment of the present invention wherein the centrifuge tube 80 has four chambers 82, 84, 86, and

88. These chambers are separated by respective constric¬ ted areas 90, 92, and 94. The chambers are in fluid communication with each other by the respective conduits 96, 98, and 100. At each of the respective restricted areas 90, 92, and 94 is located a split ring spacer 34 as is shown used with the centrifuge tube 10 in Figure 2. In particular applications depending upon the size of the centrifuge tube and the size of the respective chambers 82, 84, 86, and 88, it is possible to facilitate separa- tion of the bands of a fluid sample into three or four zones.

Similarly, with respect to Figure 10 the centri¬ fuge tube 110 is shown having a plurality of chambers 112, 114, 116, and 118 which are separated respectively by constricted areas 120, 122, and 124. Each of the respective chambers 112, 114, 116, and 118 are in fluid communication with each other through the respective conduits 126, 128, and 130. The respective restricted areas 120, 122, and 124 receive wedge spacers 76 similar to that arrangement as shown in conjunction with the

-£1 REΛ O PI centrifuge tube 60 in Figure 7. Although the alternate embodiments shown in Figures 9 and 10 show four chambers, it is envisioned that any number of multiple chambers could be utilized depending upon the size of the centri- fuge tube and the application to which it is directed.

It should be noted that although the tube of the present invention is preferably made of a polyallomer material because of its density proximity to blood serum, it is possible to consider other thermoplastic polymers such as thermoplastic polyester, polypropylene and poly¬ ethylene.

It is envisioned that embodiments of centrifuge tubes other than those specifically shown in this appli¬ cation could be designed within the scope and spirit of the present invention.

Claims

1. A multi-chamber centrifuge tube (10) for placement in a centrifuge rotor C46) , characterized by an upper cylindrical chamber (24) ; a lower cylindrical cham¬ ber (26); a constricted area (20) joining the chambers and forming a channel (22) between the chambers, the channel having a sectional perimeter smaller than the sectional perimeter of each of the chambers; and a spacer (32) mounted adjacent the constricted area to support the chambers during centrifugation.

2. A multi-chamber tube according to claim 1, characterized in that the constricted area comprises a cylindrical channel having its longitudinal center aligned with the longitudinal center of the upper and lower chambers.

3. A multi-chamber tube according to claim 2, characterized in that the spacer comprises an annular ring.

4. A multi-chamber tube according to claim 1, characterized in that the constricted area comprises the channel positioned adjacent and in alignment with a por¬ tion of the wall (70) of the upper and lower chambers.

5. A multi-chamber tube according to claim 4, characterized in that the spacer comprises a wedge-shaped spacer (76) .

6. A multi-chamber tube according to claim 1, characterized in that the upper cylindrical chamber, the lower cylindrical chamber and the constricted area are integrally formed from one piece of material.

7. A multi-chamber centrifuge tube according to claim 1, characterized in that the upper and lower

OMPI ° cylindrical chambers have a generally uniform exterior and interior cross-sectional area.

8. A centrifuge rotor assembly (46) character¬ ized by a rotor having a plurality of cavities (44); at least one sample carrying generally cylindrical centri¬ fuge tube (10) placed in one of the cavities, the tube having at least two chambers divided by a reduced diam¬ eter area located at a specified position between the top and bottom of the tube, the restricted area forming an upper and a lower chamber, the reduced diameter area establishing an orifice to provide fluid communication between the chambers; and a support spacer (32) mounted adjacent said reduced diameter area.

9. A method for separating lipoproteins using a one-piece multi-chamber centrifuge tube with a con¬ stricted area between the chambers characterized by the steps of placing a lipoprotein sample into the tube; sealing the tube; positioning the tube in a centrifuge rotor; centrifuging the tube to accumulate very low den¬ sity lipoproteins (50) in one of the chambers and to accumulate low and high density lipoproteins (54) in another of the chambers; removing thesaid tube from the rotor after centrifugation; and sealing the constricted area to seal the chambers from each other to isolate the very low density lipoproteins from the low and high den¬ sity lipoproteins.

10. A method according to claim 10, character¬ ized by the additional steps of transferring the low and high density lipoproteins to a second multi-chamber cen¬ trifuge tube with a restricted area between the chambers; sealing the second tube; centrifuging the tube to accumu¬ late the low density lipoproteins in a first chamber of the second tube and to accumulate the high density lipo- proteins in a second chamber of the second tube; and sealing the restricted area of the second tube to isolate- the low density lipoproteins from the high density lipo¬ proteins.

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