CA2013021C

CA2013021C - Blood collection device

Info

Publication number: CA2013021C
Application number: CA002013021A
Authority: CA
Inventors: Richard Lewis Columbus; Harvey John Palmer
Original assignee: Eastman Kodak Co
Current assignee: Eastman Kodak Co
Priority date: 1989-11-29
Filing date: 1990-03-26
Publication date: 1995-05-09
Anticipated expiration: 2010-03-26
Also published as: JP2989256B2; CA2024891A1; JPH03185356A; CA2024891C; CA2029459A1; EP0430356A2; JPH03270748A; CA2024892C; CA2013021A1; EP0430356A3; US5032288A; EP0430355A3; IE904304A1; IE904303A1; CA2024892A1; EP0430355A2

Abstract

A device is disclosed that causes phase separation of whole blood, using much lower centrifugal forces. As a result, lymphocytes are separated from blood cells having specific gravities of 1.08 g/ml or higher. The device features a separation chamber arranged so that its long dimension or axis is parallel, not perpendicular, to the spin axis, and a valve that allows automatic removal of the lighter phase(s). The valve is constructed to respond only to the head of liquid pressure generated by an increased centrifugal force, and not to that increased force alone.

Description

-1- 2Q~3(~2~
BLOOD COLLECTION DEVI~
FIELD OF THE IN~;r.110N
The invention relates to devices for separating a light phase from a heavier phase in a multi-phase liquid, particularly whole blood, and a method for achieving this separation.
BACKGROUND OF T~F I h~NllON
Blood collection and separating devices have from time immemorial, spun down the whole blood in a container having its long axis oriented parallel, or mostly parallel, to the direction of the centrifugal force. Examples can be seen in, e.g., U.S. Patent No. 4,012,325. There are several reasons for this orientation. One reason is that when centrifugal forces cease, there is a substantial di6tance of ~eparation between the heavier red cells and the lighter serum, and at the same time, an interface between the two phases of reduced surface area. As a result, when serum is drawn off, there is less likelihood that the blood cells will redisperse into the lighter serum phase. To further prevent this undesired event, a gel of intermediate specific gravity is often used, to occupy the surface area between the two phases. The spinning of the container about the long axis insures that the depth of the gel that resists remixing after centrifuging, will be substantial.
Stated from an opposite point of view, it has not been considered feasible to spin such containers about one of the shorter axes. The reason is that the distance between the free surface of the separated ~erum, and its interface with the separated blood cells, becomes very short, with a concommittant large surface area at said interface. This in turn makes blood cell contamination of the serum as it is "poured off" or removed, more likely. Any attempt to ~, -2- 2a ~ ~O 2 uæe a gel to shore up such a large surface area interface is less likely to succeed, since the gel will have only a short depth to it to resist remixing. (The volume of the gel will be distributed primarily over that large surface area of the interface.) However, the conventional approach has paid a price for these conclusions. The price is, that phase separation takes a long time since it has to occur over the longest dimension of the liquid volume. For example, in a blood volume of about 2 mL, using a device similar to that described in the aforesaid '325 patent, the time of separation of the serum from the blood cells is on the order of 5.3 min when spinning at, e.g., 100 g's. It is true, of course, that such separation times are also a function of the centrifugal force applied - the greater the force (e.g., created by higher rpm values), the faster the separation. Thus, typically the forces that are used are well in excess of 1000 g's, as lower forces will cause unacceptable delay in the phase separation. But even at such higher forces, such as 1600 g's, the separation in a 2 mL
volume container has not been generally possible in less than 30 sec. Most importantly, however, is a disadvantage that has now been discovered about such centrifugal forces: at the interface between the blood cells and the serum is a layer called the "buffy coat". Among other things, when formed at centrifugal forces in excess of 100 g's, the buffy coat has as an inseparable part thereof, leukocyte cells such aæ the lymphocyte cells, which contain useful DNA. If those cells could be drawn off, the DNA could be extracted. The problem now i8, the phase separation that occurs using conventional containers and centrifuges therefor, insures that 2(~3021 those lymphocyte cells are irretrievably mixed with the rest of the buffy coat. It will be readily apparent, therefore, that any attempt to speed up phase separation to less than one minute by drastically boosting the force of spinning, will completely interfere with the retrieval of the lymphocyte cells.
Therefore, prior to this invention there has been a substantial need for a blood phase separation device that can be spun about one of its short axes, to allow faster phase separation and/or lower spinning forces, while at the same time somehow æolving the high risk of remixing of the phases, noted above.
One approach to dealing with this need would be, of course, the provision of some mechanism that allows for ready withdrawal of the light serum phase from the container, before the centrifugal force is removed. This in turn will aid in retaining the unwanted blood cells in a capture zone of the container, during serum removal, since the centrifugal force will still be applied. In fact, a blood separator device has been proposed that allows serum removal from the container while spinning still occurs - it even occurs by increasing the spinning speed. The device in question is shown, for example, in Japanese Kokai 63/237368. A valve is provided closing off exit passageway from the container, it being spring biased so that it will open onlv when the centrifugal force is increased beyond the speed used during phase separation, e.g., from 3000 to 5000 rpm. Clearly, in such a device serum can be drawn off with a minimum of risk of red cells remixing with the serum being drawn. However, even in such a device, it was not considered that the "while -centrifuging" serum withdrawal would permit 2~?13(`~21 reorienting the device to spin about its short axis.
Instead, the device once again insists on the conventional spin orientation wherein the phase 8eparation must occur over the long axi8 of the container.
Another diRadvantage of the device shown in the Japanese publication is that the valve will stay open as long as a high centrifugal force is applied, even in the absence of liquid flow. Clearly, a better construction is one in which the valve automatically closes after all serum is removed. The reasons are that a) failure to do so makes it possible that non-serum components, if somehow loosened in the container, can also get out the valve, and b) the still-open valve prevents other processing from being accomplished while spinning, on the blood cells remaining in the container. This disadvantage stems from the fact that the valve of this prior device operates only in response to centrifugal force, and NOT in response to the presence of liquid, e.g., serum, which is to be drawn off.
There has been a need, therefore, prior to this invention, for a two-phase liquid separation device that will more promptly, and at slower speeds, achieve phase separation and automatic removal of the lighter phase, particularly when processing whole blood.
SUMMARY OF THE INVENTION
I have developed a multi-phase liquid separation device and method that meet the aforesaid needs. This is achieved by centrifuging the device about one of the short dimensions of the liguid compartment rather than the long one and by a more judicious use of value means allowing removal of the lighter phase durin centrifugation. In its ~~ -5- 2~13~21 preferred form, the valve means are respongive only to pressure from the lighter pha~e, and not to the centrifugal force. The result is a dramatic reduction in forces used for phase separation, to levels that allow recovery of cellular fractions heretofore lost, without extending the total time of centrifugation unreasonably.
More specifically, in accord with one aspect of the invention there is provided a liquid phase separation device for phase separation by centrifuging, comprising a chamber with a predetermined volume V, a longest dimension 1, and at least one shorter dimension d; the chamber having at least a heavier phase-collecting portion and a lighter phaæe-collecting portion; means permitting liquid introduction into the chamber; and removing means for removing separated lighter phase out of the chamber after separation without decreasing the centrifugal force used to separate the two-phases.
The device is improved in that the heavier phase-collecting portion and the lighter phase-collecting portion are disposed so that the longest dimension of the chamber is generally equal to the length of at least one of the collecting portions, and the dimension ~d~ extends from the lighter phase-collecting portion into said heavier phase-collecting portion whereby phase separation for a liquid volume of 500 ~L can occur for a spin radius of about 2.5 cm, in less than 2 minutes using a centrifuging force no greater than about 30 g~s.
In accord with another aspect of the invention, there is provided a two-phase liquid separation device suitable for phase separation by centrifuging, comprising a chamber with a predetermined volume V, the chamber having a heavier phase-collecting portion, and a lighter 2~13~Zl phase-collecting portion; means permitting liquid introduction into the chamberi and means for removing separated lighter phase out of the chamber including a valve constructed to open at centrifugal forces in excess of tho~e used to separate the lighter phase from the heavier phase. The device is improved in that the device further includes means for opening and maintaining the valve open only in response to a liquid head of pressure.
In accord with yet another aspect of the invention there is provided a method of separating a lighter phase from a heavier phase in a two-phase liquid, comprising a) placing the two-phase liquid of a predetermined volume into a first chamber having one void dimension longer than the other orthogonal void dimensions, and b) spinning the chamber about an axis that is generally parallel to the one dimension, at a rate of no greater than 100 g~s, and c) drawing off the separated lighter phase into a second chamber adjacent to the first chamber with a valve interposed between the chambers.
In accord with still another aspect of the invention, there is provided a method of collecting lymphocytes from whole blood, comprising the steps of a) spinning the whole blood in a container at a centrifugal force less than about 100 g's until serum and lymphocytes have separated from blood cells having a specific gravity of at least 1.08 g/ml, and b) drawing off serum and lymphocytes into a compartment separate from the blood cells.
Accordingly it is an ad~-antageous feature of the invention that a phase separation device and 3S method are provided that give separations of phases such as in whole blood, at drastically reduced 2l)13( ~2~

centrifugal forces that still require about the same centrifuging times as conventional devices using forces that are hundreds of g~s greater.
It is a related advantageous feature of this invention that phase separation by centrifugation can be done under low force conditions that allow the recovery of lymphocytes from the cell fraction that is normally lost. -It i8 another advantageous feature of the invention that such a device and phase separation are provided with valving means that draw off the desired phase while centrifuging is still occurring, only in response to the pressure generated by the liquid to be drawn off.
Other advantageous features will become apparent upon reference to the Description of the Preferred Embodiments, when read in light of the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is an elevational view in section of a serum separation device constructed in accord with the prior art;
Fig. 2A is an elevational view in section of a serum separation device constructed in accord with this invention;
Fig. 2B is a section view taken generallyalong the line IIB-IIB of Fig. 2A;
Fig. 3 is a plot of serum separation time vs. centrifugal force, as practiced by the device of this invention;
Fig. 4 is a graph of recovered lymphocytes versus the centrifugal force used for phase separation;
~ ig. 5 is an elevational view similar to that of Fig. 2, but of an alternate embodiment; and Figs. 6A and 6B are fragmentary sectional views similar to portions of Fig. 5, but illustrating an alternate embodiment in two positions of use.

Zt~3~)21 DESCRIPTION OF THE PREFERRED EMBODI~F.~TS
The invention iæ hereinafter described in light of its use in preferred embodiments, wherein blood serum or plasma is the lighter phase of a two-phase liquid, and particularly preferred chambers are described for collecting the serum and/or lymphocytes valved off from the two separated phases, using a ball check valve. In addition, the invention is useful regardless of the multiple-phase liquid it is used with, regardless of the type or even presence of a subsequent chamber downstream of the valve, and regardless of the valve construction; 80 long as the valve that is used meets the requirements of the lnvent ion .
Many serum separators of the prior art have conventionally used a container 10, Fig. 1, in which the longitudinal axis 12 of the container is parallel to the direction of centrifugal force CF, arrow 14.
As a result, substantial time and force is required to separate the heavier blood cells 16 from the lighter serum 18, into the two fractions shown. In some designs, such as in the Japanese application noted above, a pour-off aperture 20 is provided along with a valve 22, to allow just the serum to flow into a separate-like chamber 24 where it can contact a slide-like test element E, shown in phantom. Valve 22 is constructed to open, arrow 26, only when a centrifugal force greater than the CF used to separate the two phases, is achieved, the valve moving in that event against a return spring, not shown. This construction has all the attendant disadvantages noted above. In addition, whole blood is added through aperture 28 in a pouring step, that requires operator attention or an intermediate machine step after whole blood is collected in a separate operation via a needle.

_g_ In accord with the invention, a phase separation tevice 30, ~ig. 2A, for phase separation of at least 2 phases is constructed with a chamber 32 for phase separation that has its long dimension Q
oriented perpendicular, not parallel, to the direction of centrifugal force CF, arrow 34, and with a ~pecially constructed ~alve SO. Chamber 32 i~
defined by a body member 33 having a blood intake end 36 and an opposite, serum-removal end 38. Chamber 32 extends from end 36 to delivery passageway 56. End 36 has an inta~e aperture 40 filled with a conventional septum 41, chamber~32 being either vented at 43 or evacuated due to attachment at 43 to an external vacuum source, to assist in biood intake. Aperture 40 allows entrance of whole blood via passageway 42 to chamber 32. The width "d" of chamber 32 i8 one of the shorter dimensions, enough blood being drawn in to fill to about the depth d'.
Sidewall 44 of chamber 32 is the sidewall against which the heavier blood cells collect, whereas opposite sidewall 46 is adjacent the lighter serum fraction, during centrifugation. Thus, dimensions d and d' extend from the lighter phase into the heavier phase.
Optionally, fixed porou~ ~echanical means, such as baffles 48, can be positioned along wall 44 80 as to be disposed in the blood cells. As described in Canadian Patent A~plication No. 610,860 filed September 11, 1989, entitled, "Inteqrate~ Blood Coll~ction Syst ~", such means act to retain the hea~,-ier Phase from remixing when the lighter, serum phase i~ drawn off. The plates of the baffles are inclined at an angle alpha that re~ists remixing forces when flow occurs out of chamber 32 in the direction of arrow 49. Preferably, this angle is a value that is -lo- 2013~2~
between about 30C and about 120, most preferably about 60. Preferably, the distance between the individual plates of baffles 48 i8 between about 0.018 cm and about 0.10 cm, most preferably about 0.025 cm. The thickness of each plate i8 not critical, 80 long as a significant number of such plates are present as will create the needed volume between them to collect the blood cells.
In accord with one aspect of the invention, valve 50 is disposed at an end 52 of chamber 32 intermediate ends 36 and 38, positioned to draw off separated, or plasma serum and lymphocytes (discussed hereinafter). Most importantly, valve 50 is constructed to open only in response to a hydraulic head of force, and not to the effects of force CF, regardless of the magnitude of the latter. To this end, valve 50 is preferably a ball check valve with a ball 54 positioned downstream of passageway 56 at chamber end 52. Ball 54 seats against a hemispherical seat 58, and is biased by a spring 60 aligned to act in a direction that is generally perpendicular to the direction of force CF. This alignment tends to ensure that ball 54 will act against spring 60 only in response to forces other than force CF.
A serum or plasma exit passageway 62 is constructed adjacent seat 58, to carry off the liquid when valve 50 opens. Passageway 62 joins a chamber or compartment 64 sized to receive substantially all the liquid that exits chamber 32 via valve 50.
Chamber 64 has a deep well portion 66 designed to collect lymphocytes, and a large opening 68 adapted to allow a pipette access to chamber 64 generally and to well portion 66 in particular. A cover 70 is removably sealed over opening 68 except when access of the pipette or other removal means i~ desired.

;~013~Zl Passageway 62 preferably extends beyond chamber 64 to a trap 74. The function of the trap i8 to collect the few red blood cells that will gather prior to and during centrifuging, in passageway 56, allowing only desired serum, or plasma and lymphocytes, to pass into chamber 64.
Device 30 can be assembled as two plates, ~ig. 2B, using a foil layer 75 to achieve a seal that will allow a vacuum to be drawn using vent 43, as described above.
Such a device 30 can be spun in any convenient centrifuge, not shown, where the long dimension Q is generally parallel to the spin axis 76. Preferred spin radii are about 2.5 cm, although a wide variety can be used.
The method of phase separating, using device 30, will be readily apparent from the preceding discussion. Whole blood is placed into chamber 32 by, e.g., a needle that penetrates septum 41. Device 30 is then spun about axis 76. ~owever, in accord with another aspect of the invention, the speed of rotation that is selected is slow - a speed producing no greater than 400 g's centrifugal force, and most preferably no greater than 30 g's. The reason is that device 30 is capable of achieving phase separation at such forces, using 2 mL of liquid, in less than 2 minuteæ, and in some cases less than 1 minute, due to the (relatively) short distance (about d'/2) that the blood cells have to traverse to be separated. Fig. 3 illustrates the separation times achievable with the invention, using a 2.5 cm spin radius and a total whole blood volume of 500 ~L.
As indicated, the serum, or plasma and lymphocytes, is separated in less than 1 minute if the centrifugal force i~ about 150 g 18 or greater, there being little separation time enhancement occurring at forces above _ -12- 2~1~021 400 g's. At the other end, a separation force of only 30 g's will produce complete phase separation in less than 8 minutes, for example, 5.5 minuteæ. As a comparative example, as described in ~.S. Patent No.
4,818,418 the conditions achieved using a conventional Ficol-Pague/Percoll as an additive are also indicated - a force of 400 g's is effective to achieve separation only after 30 minutes; point FP on Fig. 3.
Whatever centrifugal force that is selected, after serum or plasma separation occurs the lighter phase is then drawn off the stacked liquid in chamber 32, by opening valve 50. This occurs as follows:
spring 60 has a spring constant Kl that is pre-selected to resist movement of ball 54 until a certain head of pressure builds up against ball 54.
The increased head of pressure occurs by increasing the centrifugal force a factor, for example 50%, above the force used to achieve phase separation.
Preferably, the speed of rotation is increased a corresponding amount. Since the serum and blood cells are relatively incompressible against wall 44, the increase in centrifugal force CF translates into an increased force in the direction of arrow 49, which overcomes spring constant Kl of spring 60, and the valve opens. ~owever, this is true only as long as enough serum or plasma remains in chamber 32 to push out aperture 56. When most of the serum or plasma has passed through the val~e, the head of pressure occurring even at the increased speed of rotation, drops. As a result, valve 50 closes automatically even at the higher speeds of rotation, unlike the operation of valve 22 in Fig. 1.
Fig. 4 illustrates that in fact this process does produce the separation of lymphocytes, without the necessity of using a chemical phase separation 20~3021 agent common in conventional lymphocyte separation by centrifuging. (If lymphocytes are the desired end-product, then plasma is the lighter phase, rather than serum. Serum is the same as plasma, except that in serum the fibrinogen has been removed, a step considered detrimental to obtaining lymphocytes.) That is, because the centrifugal forces are at a level below about 100 g's, the lymphocytes do not get irretrievably compacted into the buffy coat, as is 10 the case in prior centrifuges that operate at forces above 100 gls.
More specifically, the graph of Fig. 4 was prepared using a device of the type shown in Fig. 2A, in a centrifuge rotor where ~'r" has the value 2.54 cm (1 inch). Since lymphocytes can all be lost in the red cells if the spin time is allowed to proceed too long before opening valve 50, it is necessary that the process be sampled at varying times for any given centrifugal force Gi. Thus, for, e.g., CF = 50 g~s, many gpin times (between 1 and 10 minutes) were examined to determine what optimal time for that CF
produces the maximum amount of lymphocytes remaining in the plasma. This is the same as the amount transferred by opening valve 50 by increasing the force CF. The amount of the lymphocytes so remaining in the lighter phase at those optimized times, for each different g force, as a fraction of the total original amount of lymphocytes, was then plotted versus the g forces, expressed as a log to the base 10. Fig. 4 is the result, where a band 88 surrounding the curve has been drawn to "fit" the data. This band symbolizes the uncertainty in the data, where each data point is the mean for the tests. No standard deviation has been determined, however. As noted, the important feature i8 the recovery of significant fractions of the lymphocytes available. This occurred where the centrifugal force was less than 100 g's.

2û1~0~1 _ -14-It is not essential that the valve operate on an axis that is neutral to the centrifugal force, as is shown in the alternate embodiment of Fig. 5.
Parts similar to those previously described bear the 5 ~ame reference numeral, to which the distinguishing suffix A has been appended.
Thus, device 30A comprises a body 34A having a chamber 32A, passageway 42A supplying blood thereto as before. Baffles 48A can be included to retain the 10 heavier blood cells, and passageway 56A allows removal of the lighter phases such as lymphocytes and plasma, into covered chamber 64A, from chamber 32A, using valve 60A. The long dimension Q of chamber 23Ais parallel to spin axis 76A. However, in this 15 embodiment ~pring 60Ais oriented to be parallel to the direction of centrifugal force CF. Nevertheless, the spring constant K2 f spring 60Ais selected so that ball 54A ~till opens only in response to a liquid head of pressure, and ~Q~ in response to the 20 centrifugal force. When ball 54A lifts off seat 58A, the lighter phases pour into chamber 64A. In this embodiment, the volume of passageway 74A that is not filled by spring 60Ais just enough to trap any blood cells caught in passageway 56A prior to phase 25 separation.
The careful selection of spring constant K2 of spring 60Ais as follows: It is selected so that valve 50Awill not open at the first centrifugal speed CFl used to achieve phase separation.
30 Moreover, it is strong enough to prevent valve opening even in the presence of the higher centrifugal speed CF2 used to create a head of pressure on the valve, in the absence of any liquid pressing on ball 54A. However, because ball 54A has 35 a surface that is included at a non--90 angle to the force of arrow 49A, ball 54A will incur a force -15- ~13~2~
parallel to CF2 due to a liquid head of pressure ~P generated in the direction of arrow 49A, caused by centrifugal force CF2. (The component of Ap that i8 parallel to CF2 i8 hereinafter designated ~PCF.) That is, spring constant K2 is greater than the force generated by CF2 alone, but less an (CF2 ~ ~PCF). When all of the lighter phase liquid has transferred to chamber 64A, there no longer i8 a liquid head of pressure creating a force 10 ~PcF~ and valve 50A closes automatically, even in the face of a centrifugal force CF2.
The contents of chamber 64A, such as lymphocytes and plasma, are then aspirated out, by removing cover 70A.
The valve for automatic removal of the lighter phase need not be a ball valve, to respond only to the liquid head of pressure. Any valve can be used, if it is constructed to resist forces other than this head of pressure. Another type is shown in 20 Fig. 6, in which parts similar to thoæe previously described bear the same reference numeral, to which the distinguishing suffix "8" is appended.
Thus, device 30B includes blood collection and separation chambers such as chamber 32B, and a 25 valve 50B that operates only in response to a head of liquid pressure to pass the lighter phase into separate chamber 64B, as in the previous embodiments. However, whereas the previous valves used balls, valve 50B comprises a solid rectangular 30 block 90 backed by a spring 91 of a suitable spring constant selected to deform enough to open the valve, only when centrifugal force is increased from CFl, Fig. 6A, to CF2, Fig. 6B. Flow then proceeds via arrows 100, 102.

~01;~21 The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims

1. In a liquid phase separation device suitable for phase separation by centrifuging, comprising a chamber with a predetermined volume V, a longest dimension 1, and at least one shorter dimension d; said chamber having at least a heavier phase-collecting portion and a lighter phase-collecting portion; means permitting liquid introduction into said chamber; and removing means for removing separated lighter phase out of said chamber after separation without decreasing the centrifugal force used to separate the two-phases;
the improvement wherein said heavier phase-collecting portion and said lighter phase-collecting portion are disposed so that said longest dimension of said chamber is generally equal to the length of at least one of said collecting portions, and said dimension "d" extends from the lighter phase-collecting portion into said heavier phase-collecting portion, whereby phase separation for a liquid volume of 500 µL can occur for a spin radius of about 2.5 cm, in less than 2 minutes using a centrifuging force no greater than about 30 g's.

2. A device as defined in claim 1, wherein said removing means includes a valve that opens only in response to a liquid head of pressure.

3. In a two-phase liquid separation device suitable for phase separation by centrifuging, comprising a chamber with a predetermined volume V, said chamber having a heavier phase-collecting portion, and a lighter phase-collecting portion;
means permitting liquid introduction into said chamber; and means for removing separated lighter phase out of said chamber including a valve constructed to open at centrifugal forces in excess of those used to separate the lighter phase from the heavier phase;
the improvement wherein said device further includes means for opening and maintaining said valve open only in response to a liquid head of pressure.

4. A device as defined in claim 3, wherein said opening and maintaining means is constructed to close said valve in the absence of liquid pressure, regardless of the magnitude of the centrifugal force during centrifuging.

5. A device as defined in claim 4, wherein said valve includes biasing means to bias the valve closed, said biasing means being operative in a direction that is substantially perpendicular to the direction of force of said centrifuging, with a biasing constant adjusted to open said valve in response only to a predetermined liquid head of pressure, whereby said valve opens and stays open only as long as a liquid head of pressure is present because liquid is pressing against said valve, even when high centrifugal forces are applied in said perpendicular direction.

6. A device as defined in claim 5, wherein said valve is a ball valve.

7. A device as defined in claim 1, 2, 3 or 4, wherein said liquid is whole blood and said lighter phase is serum or plasma, and further including means for withdrawing and trapping residual blood cells left in said lighter-phase-collecting portion when centrifuging begins.

8. A device as defined in claim 1, 2, 3, or 4, and further including in said chamber means for restricting separated heavier phase from remixing with said separated lighter phase after phase separation.

9. A method of separating a lighter phase from a heavier phase in a two-phase liquid, comprising a) placing the two-phase liquid of a predetermined volume into a first chamber having one void dimension longer than the other orthogonal void dimensions, b) spinning said chamber about an axis that is generally parallel to said one dimension and offset from said chamber, at a rate of no greater than 400 g's, and c) drawing off while spinning, the separated lighter phase into a second chamber adjacent to said first chamber with a valve interposed between said chambers.

10. A method as defined in claim 9, wherein said step c) comprises the step of opening said valve only in response to a predetermined head of liquid pressure generated by the lighter phase.

11. A method as defined in claim 10, wherein said head of pressure is created by increasing the centrifugal force within said chamber above the force used to achieve phase separation.