CA2346975C

CA2346975C - Analytical rotors and methods for analysis of biological fluids

Info

Publication number: CA2346975C
Application number: CA002346975A
Authority: CA
Inventors: Tammy Leigh Burd; Carol T. Schembri; Boris Braynin; Vladimir Ostoich
Original assignee: Abaxis Inc
Current assignee: Abaxis Inc
Priority date: 1990-06-04
Filing date: 1991-05-31
Publication date: 2004-11-16
Anticipated expiration: 2011-05-31
Also published as: CA2346974C; CA2346975A1; CA2346974A1; CA2347669A1; CA2347669C

Abstract

An analytical rotor (10) for separating cellular components from a biological sample includes a plurality of internal chambers and passages for combining the cell-free sample with one or more reagents and distributing the sample to a plurality of individual test wells (92). The chambers and passages necessary for separating cellular components from a sample such as whole blood include a measuring chamber (40), an overflow chamber (44), a separation chamber (60), and a reagent chamber (80). The measuring chamber (40) and overflow chamber (44) have capillary dimensions so that an initial volume of whole blood partitions therebetween, with the measuring chamber filling first to provide a preselected volume. The separation chamber (60) is located radially outward from both the measuring chamber (40) and the reagent chamber (80) so that spinning of the rotor causes both the reagent and the measured blue volume to flow outward into the separation chamber (60). A collection chamber (90) is formed to receive the plasma from the separation chamber (60). Further spinning of the rotor causes the plasma to flow radially outward into a plurality of test well (92) formed around the periphery of the collection chamber (90). Testing of the separated plasma volumes may be performed without removing the plasma from the rotor.

Description

ANALYTICAL ROTORS AND METHODS
FOR ANALYSIS OF BIOLOGICAL FLUIDS
BACKGaROUND OF THE INVENTION
The present invention relates generally to devices and methods for optically analyzing biological fluids. In particular, it relates to analytical rotors Which allow for sample metering, separation of cellular material from fluid in the sample, and distribution of the separated fluid to a plurality of test wells for various biochemical analyses.
Blood tests frequently require that potentially-interfering cellular components.of the blood be separated from the blood plasma prior to testing of the plasma. It is also frequently desirable to divide the separated blood plasma into a plurality of discrete: aliquots so that a variety of tests or assays may be performed on the blood. Such separation and division steps have heretofore been typically performed by centrifugation to separate the blood plasma from the cellular components, followed b~~ manual or automated pipetting of the blood plasma into separate test wells. Such procedures are labor intensive and time-consuming, and various automated systems and methods have been proposed for providing multiple aliquots of plasma suitable for testing in a more efficient manner.
Of particular- interest to the present invention are centrifugal rotors which have been modified both to separate plasma from whole blood and to distribute the separated plasma into separate test wel7.s. The use of such rotors can provide a plurality of discrete plasma volumes which may be tested or evaluated, all present within the centrifugal rotor, greatly enhancing the efficiency of automated testing procedures.
Although a significant improvement over prior manual or partly manual procedures, previous modified centrifugal rotors have suffered foom a number of deficiencies. Such rotors have frequently required tha application of relatively VVO 91/18656 PGT/USyl/03840 large volumes of whole :blood in order to achieve the desired separation and distribution. The efficiency of separation has frequently been low, typically on the order of 5% based on the initial amount of plasma available. Moreover, such rotors have frequently utilized complex designs which are difficult and costly to manufacture. Often, the rotors require various separable parts or components which are brought together or separated at different ;points in the centrifugation procedure.
Previous centrifugal rotors have often been limited in the number of discrete samples and test wells which they can provide, and in some cases require the use of a separate displacement fluid to effect flow of blood and plasma through the system.
For these reasons, it would be desirable to provide improved centrifugal rotors and methods suitable for separating blood into plasma and cellular components and for further distributing the separated plasma into a plurality of discrete test wells within~the rotors. The rotors should be capable of separating relatively small volumes of blood and should not require the use of a displacement fluid for effecting such separation. In particular, it would be desirable to have a separation efficiency greater than 10%, preferably greater than 20%, and more preferably greater than 30%. The rotors should be able to accommodate relatively large numbers of test wells, and the rotor design should be simple and amenable to low-cost manufacturing procedures. In particular, it would be desirable if the rotors were of unitary construction with no separable or movable parts. Plasma separation methods should be simple and be capable of being performed in relatively short times. In particular, the methods should require relatively few steps and should be capable of being performed with little or no intervention or manipulations by the operator. It would be particularly desirable if the methods required only rotation of the rotor in order to effect both the separation and distribution of the plasma.

SUMMARY OF TFiE INVENTION
According to the present invention, an improved cell separator comprises a c:entrifugal rotor having top and bottom surfaces and a central axis therethrough. A separation chamber is disposed at a first level within tie rotor whereby a biological fluid (e. g., whole blood) can be separated into cell-free fluid (e.g., plasma) and cellular components in response to spinning o!: the rotor. The separation chamber usually includes a receptacle region, a cell trap spaced radially outward from t:he receptacle region, and a capillary region between the receptacle region and the cell trap. In this way, spinning of t:he rotor causes cells in the biological fluid to pass from the receptacle region through the capillary region into the cell trap. After spinning is stopped, the capillary region inhibits backflow of the cellular material from the cell trap into the receptacle region where the clear biological fluid, e.g., plasma, remains.
Alternativel~~, the separation chamber may be located radially outward from a~ sample chamber and connected thereto by a flow restrictive charnel. Thus, biological fluid which is introduced to the samp~.e chamber will flaw radially outward into the separation chamber as the rotor is spun. The dimensions of the flow restrictive channel are selected to provide a desired flow rate into the separation chamber when the rotor is spun at a predetermined speed. .
The sample chamber may be a sample receptacle having an inlet opening or poz-t which allows the user to introduce a volume of sample to be tested. Alternatively, the sample chamber may be a mixing chamber which receives sample from a 3o sample chamber and diluent from a diluent chamber. Optionally, such a mixing chamber may include a cell retention region at or near its radially outward periphery.
The separation chamber will have dimensions selected to allow cellular components to separate from the biological fluid as the fluid continuously flows toward a means for collecting cell-free fluid within the chamber. Typically, the separation chamber will. be annularly elongated with the flow restrictive channel located at or near one annular-extremity and the means for collecting cell free fluid located at or near the opposite annular extremity. In this way, the separation chamber provides a sufficiently long flow path so that the residence time of the biological fluid (i.e., the time prior to the collection of cell-free fluid) is adequate to allow the cellular components to migrate radially outward into a retention region which is located at the radially outward periphery of the separation chamber.
The analytical rotors of the present invention preferably contain means for measurement and delivery of a predetermined volume of fluid to a receiving chamber, such as the separation chamber. Thus, the rotor typically comprises a bulk fluid chamber containing a bulk amount of fluid to~be partitioned and a metering chamber connected to the bulk fluid chamber positioned radially outward from the bulk fluid chamber such that fluid enters the metering chamber as the rotor is~ spun. An overflow chamber is connected to the metering chamber for receiving excess fluid after the metering chamber is filled. The volume of the fluid remaining in the metering chamber corresponds to the predetermined volume. Either a biological fluid, such as whole blood, or a reagent, such as a diluent, may be placed in the bulk fluid chamber.
A receiving chamber is positioned radially outward from the metering chamt~er and is connected to the metering chamber through a connecting means which prevents flow of fluid into the receiving cha~sber until the metering chamber contains the predetermined voluxae. The fluid can be held in the metering chamber for as long as desired before delivery to the receiving chamber. Ty~~ically, the connecting means is a capillary exit duct in which capillary forces prevent flow at a first rotational speed. At a second, higher rotational speed, centrifugal force exceeds the capillary force and the metering chamber is emptied. The connecting means can also be a siphon having an elbow that i:~ substantially the same distance from the center of the rotor as the radially most inward point of the metering chamber. As the rotor is spinning the fluid does not flow past the elbow. After the rotor stops, capillary forces "prime" the siphon by pulling fluid just around the elbow. When the rotor is restarted, the combination of centrifugal and capillary forces draw the remaining fluid out of the metering chamber into the receiving chamber.

5 After a predetermined volume of fluid is separated from cellular components, fluid is typically delivered to a plurality of peripheral test wells or cuvettes for optical analysis. In one embodiment, each of the cuvettes is connected to a central collection chamber by means of a generally radial inlet channel which has a discrete flow path for flow of liquid into the cuvette and a second discrete flow path for flow of gas out of the cuvette as the rotor is spun. Preferably, the liquid flow path is on the side of the inlet channel in the direction of the rotation of the rotor and the gas evacuation flow path is on the side away from the direction of the rotation. The liquid is channeled into its flow path by differences in depth or surface texture between the two flow paths.
The rotor may also comprise a plurality of reflective surfaces positioned radially inward from the cuvettes, the reflective surfaces being capable of deflecting a light beam by about 90°. Typically, a light beam oriented generally parallel to the axis of rotation of the rotor is deflected so that it passes horizontally through a fluid in the cuvette. The reflective surface is preferably oriented at about a 45° angle to the vertical axis of the rotor and is produced by a total internal reflection ccndition at the rotor material/air interface.
The rotor of the present invention is preferably made of clear plastic, more: preferably acrylic. Each cuvette typically contains reagents necessary for a biochemical analysis of the fluid in the cuvette. The biochemical analysis preferably produces an optical effect when exposed to the light beam which is then dete<:ted and analyzed.
In accordance with the present invention, there is provided an analytical rotor for continuously separating fluid from a cell-containing 7_iquid sample, said rotor comprising a rotor body having an ax~~.s of rotation, a sample chamber, a separation chamber, a cell retention region located generally at the radially outward periphery of the separation chamber, a flow restrictive channel. connecting the sample chamber to the separation chamber, and means annularly offset from the flow restrictive channel and radially inward from the cell retention region for collecting cell-free fluid from the separation chamber, whereby spinning the rotor causes liquid sample in the sample chamber to flow into the separation chamber where cells in the sample move-radially outward into the retention region and cell-free fluid flows annularly into the collecting means, wherein said flow restrictive channel and said collecting means are annularly separated by a distance sufficient to permit cells to separate from t:he fluid before the fluid reaches the collecting means.
In accordance with the present invention, there is also provided a method i=or separating cells from a biological fluid, said method comprising: introducing the fluid into a receptacle region within a rotor; spinning the rotor to effect radially outward flow oi= the fluid from the receptacle region through a flow restrictive passage into a separation chamber, whereby cells collect iii a retention region at the radially outward periphery of the' separation chamber; and continuously removing the resulting separated fluid through a port which is disposed at a position .Located radially inward from the 6a retention region and annularly displaced from the flow restrictive passage.
Other advantages of the subject invention will be apparent to those skillE:d in the art from consideration of the detailed description of embodiments of the subject invention set forth and of the attached drawings BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a perspective view of a centrifugal rotor constructed in accordance with the principles of the present invention, with portions broken away.
Figs. lA and .LB illustrate alternate geometries for a separation chamber of the type employed in a centrifugal rotor constructed in accordance with the principles of the present invention.
Fig. 2 is a top plan view of the centrifugal rotor of Fig. 1.
Fig. 3 is a vertical cross-sectional view of the rotor of Figs. 1 and 2, taken along line 3-3 in Fig. 2.
Fig. 4 is a vE~rtical cross-sectional view of the rotor of Figs. 1 and 2, taken along line 4-4 in Fig. 2.
Fig. 4A is a cross-sectional view through a portion of a centrifugal rotor constructed in accordance with an alternative embodiment of the present invention.
Fig. 5 is a horizontal cross-sectional view of the rotor of Figs. 1-3, takfen along line 5-5 in Fig. 3.
Fig. 6 is a horizontal cross-sectional view of the rotor of Figs. 1-3, taken along line 6-6 in Figs. 3 and 4.

6b Figs. 7-11 il7.ustrate the method of the present invention utilizing the centrifugal rotor of Fig. 1.
Fig. 12 illustrates an alternate embodiment of the centrifugal rotor of the: present invention.
Fig. 13 is a perspective view of an analytical rotor constructed in accordant;e with the principles of the present invention, with portion:c broken away.
Fig. 14 is a plan view of the centrifugal rotor of Fig. 13.
Fig. 15 is a cross-sectional view taken along line 15-15 of Fig. 14.
Fig. 16 is a cross-sectional view taken along line 16-16 of Fig. 14.
Fig. 17 is a plan view of an alternate rotor design.
Fig. 18 is a cross-sectional view taken along line 18-18 of Fig. 17.
Fig. 19 is a cross-sectional view taken along line 19-19 of Fig. 17.
Fig. 20 is a. plan view of a second alternate rotor design.
Fig. 21 is a cross-sectional view taken along line 21-21 in Fig. 20.
Fig. 22 is a plan view of a rotor designed in accordance with one embodiment of the present invention in which the metering chamber is used to measure a precise volume of blood.
l0 Fig. 23 is a plan view of a rotor in which the metering chamber is used for diluent.
Fig. 24 is a plan view of a rotor in which.a siphon is used to control flow between the metering chamber and the separation chamber.
Fig. 25 is a plan view of the bottom layer of a rotor made in accordance witlh the present invention.
Fig. 26 is a plan view of the bottom layer of the rotor of Fig. 25 showing a cuvette, a curved inlet channel and a reflective surface.
Fig. 27 show, two cross-sectional views along line 27-27 in Fig. 26.
Fig. 28 is a cross-sectional view along line 28-28 of the bottom layer of thin rotor in Fig. 25.
Fig. 29 is perspective view of an irriet channel showing the direction of flow in the discrete flow paths.
Fig. 30 is a cross-sectional view of the bottom layer of the rotor of the pre=sent invention showing the light path through the fluid in tie cuvette.
Fig. 31 is a cross-sectional view alonq_ l~,,xi~,.31-31 in Fig. 32.
Fig. 32 is a plan view of the rotor showing a cuvette, a straight in7Let channel and a reflective surface.

DESCRIPTION OF TFIE PREFERRED EMBODIMENT
The present invention provides apparatus and methods for separating cellular components from biological fluids, and in particular for separating whole blood into plasma which may then be subjected to a wide variety of analytic procedures.
Conveniently, the apparatus and methods will also provide for distribution of the separated plasma into a plurality of test wells within the rotor ao that different analytic procedures may be performed without having to transfer aliquots of the plasma from the apparatus. The apparatus and method are able to separate very low volumes of blood, usually as low as about 0.03 cc, frequently as low as about 0.015 cc, and sometimes as low as about 0.005 cc, although the present invention is suitable for separating much larger volumes as well. The present invention does not require the use of a displacement medium for effecting the desired separation and distribution, and the apparatus design is very simple with no separate or moving parts required. Of course, it may be desirable in certain circumstances to provide such separate or moving parts, but they are not required in order to achieve the blood separation according to the method of the present invention.
As a result, the apparatus is very easy to manufacture and can be produced at a very low cost, making the apparatus suitable for use as a disposable. in testing whole-blood samples. The apparatus and method are able to separate precise volumes of blood without the need to premeasure the amount applied to the apparatus. The apparatus can further provide for automatic combination of the separated plasma with a reagent or diluent and can apportion substantially equal volumes of plasma among the plurality of test wells. Various diluents known to those skilled in the art are suitable for use in the present invention. For instance, standard diluents such as normal saline solution (0.5% rraCl in water), phosphate buffered solution, and Ringer's lactate solution and the like may be used.
In addition, the apparatus is suitable for use with a variety of conventional analytic measurement devices, such as spectrophotometers and fluorometers, which allow tire plasma in the test wells to be individually examined without the need to remove the plasma from the wells.
Although the present invention is particularly suitable for separating cells from blood to produce plasma, it will be useful with a wide variety of other biological fluids, such as urine, sputum, semen, saliva, ocular lens fluid, cerebral fluid, spinal fluid, amniotic fluid, and tissue culture media, as well as food and industrial chemicals, and the like, where it may be desirable to separate cells and other interfering substances prior to analysis or assay.
The apparatus, of the present invention includes a centrifugal rotor which is capable of being mounted on a conventional laboratory centrifuge of the type which is commercially available from suppliers, such as Beckman Instruments, Inc., Spinco Division, Fullerton, California;
Fisher Scientific, Pittsburgh, Pennsylvanian VWR Scientific, San Francisco, California, and the like. Generally, the centrifugal rotors will. include a receptacle or other coupling device suitable for mounting on a vertical drive shaft within the centrifuge. The particular design of the receptacle or coupling device will depend on the nature of the centrifuge, and it will be appreciated that the centrifugal rotor of the present invention may he adapted to be used with most types of centrifuges which are now available or which may become available in the futurE:.
The centrifugal rotor comprises a body structure which maintains a desired geometric pattern or relationship between a plurality of chambers and interconnecting passages, as described in more detail hereinbelow. Usually, the body will be a substantiall;t solid plate with the chambers and passages fonaed as spaces or voids in an otherwise solid matrix. Conveniently, such solid plate structures may be formed by laminating a plurality of separately formed layers together into a composite structure where the chambers and passages are generally formed between adjacent layers. The individual layers may he formed by injection molding, machining, and combinations thereof, and will usually be joined together, typically using a suitable adhesive or by ultrasonic welding. The final enclosed volumes are formed when the layers are brought together. Of course, the centrifugal rotor could also be formed as a plurality of discrete components, such as tubes, vessels, chambers, etc., arranged in a suitable 5 structural framework. Such assemblies, however, are generally more difficult to manufacture and are therefore less desirable than those formed in a substantially solid plate.
The centrifugal rotor may be formed from a wide variety of materials and may optionally include two or more l0 materials. Usually, the materials will be transparent so that the presence and distribution of blood, plasma, and other reagents, may be observed within the various internal chambers and passages. Also, it is generally required that the test wells formed within the rotor have suitable optical paths formed therethrough so that the contents of the test well may be observed spectrophot.ometrically, fluorometrically, or by other visual assessment. instruments. In the exemplary embodiment described below, the rotor is formed from acrylic resins having the required optical properties, at least in those areas which define the optical paths.
The apparatus. and method of the present invention are suitable for performing a wide variety of analytic procedures which are beneficially or necessarily performed on blood plasma. The analytic procedures will generally require that the blood plasma be combined with one or more reagents so that some visibly detectable: change occurs in the plasma which may be related to measurement of a particular component or characteristic of the F~lasma. Preferably, the plasma will undergo a reaction or other change which results in a change in color, fluorescence, luminescence, or the like, which may be measured by conventional spectrophotometers, fluorometers, light detectors, etc. In some cases, immunoassays and other specific binding assayer may be performed in the test wells.
Generally, however, such assay procedures must be homogeneous and not require a separation step. In other cases, it will be possible to accommodatEa heterogeneous assay systems by providing a means to separate blood plasma from the test wells after an immunological reaction step has occurred.--Conventional blood assays which may be performed include glucose, lactai~e dehydrogenase, serum glutamic-oxaloacetic transamina:~e (SGOT), serum glutamic-pyruvic transaminase (SGPT), blood urea (nitrogen) (BUN), total protein, alkalinity, phosphatase, bilirubin, calcium, chloride, sodium, potassium, mag~zesium, and the like. This list is not exhaustive and is intended merely as being exemplary of the assays which may be performed using the apparatus and method of the present invention. Usually, these tests will require that l0 the blood plasma be combined with one or more reagents which result in a visually deatectable, usually photometrically detectable, change in 1=he plasma. The reagents which are required are well know~z and amply described in the patent and scientific literature.
In one embod:invent, the separation chamber will include a capillary barrier which divides the cell retention region from the fluid ~Elow path between the flow restrictive channel and the cell-free fluid collection means. In this way, the cells which pass into the retention region as the rotor is spun may be retained within the retention region even during subsequent handling of the rotor.
In another embodiment of the present invention, the rotor will contain a p:Lurality of sample receptacles, separation chambers, and optionally mixing chambers and collection chambers. :Cn this way, multiple blood samples may be simultaneously separated and optionally analyzed with all cellular separation stE~ps being performed simultaneously.
In yet another preferred embodiment, the flow rate restrictive channel wi:Ll include a siphon structure having a path which extends radially inward and which (prior to "priming") prevents flow from the sample chamber into the separation chamber whi:Le the rotor is spinning. The use of such a siphon barrier is particularly advantageous in embodiments which employ a mixing chamber to allow initial introduction of sample and diluent without carryover into the separation chamber.
In an altern<ite embodiment, the mixing chamber may be sufficient to provide a desired level of separation without a separation chamber. Ths~ provision of a flow restrictive outlet on the mixing chamber allows a sufficient fluid residence time within the mixing chamber so that the cellular components of the fluid can be separated radially outward into a retention region as the rotor is ::pun.
According to a method of the present invention, the cell-containing biological fluid is introduced to the receptacle region within the analytical rotor. Spinning of the rotor causes a radially outward flow of the biological fluid through the flow restri<aive channel into the separation chamber. Within the separation chamber, continued spinning of the rotor causes cellular components of the fluid to migrate radially outward into the retention region at the periphery of the chamber where they sire entrapped. The resulting cell-free fluid, in contrast, flows along a flow path which is radially inward from the retention region and is continuously removed from the separation chaiaber through the collection means which is located at a position annularly spaced-apart from the flow channel entry point. In this way, sufficient residence time is provided so that the ce:Llular components may be substantially completely separated prior to removal of the fluid fraction.
By properly selecting the volume of the biological fluid, the flow rate of the restri~~tive flow channel, and the volume of the cell retention reginn, the rotor can be designed to allow separation of a predetermined fluid volume without substantial carryover of the cellular components.
In an alternate method, the cell-containing biological fluid is transferred to a mixing chamber where it is combined with a diluent. The biological fluid and diluent are mixed in the mixing chamber, typically by reversing the rotational direction of the rotor or by alternately accelerating and decelerating the velocity of rotation in a single rotational direction. The cells can then be separated outward into a peripheral retention region by spinning the rotor. A flow restrictive outlet on the mixing chamber assures that there is sufficient residence time to effect a desired degree of separation. optionally, the fluid from the mixing chamber flows through the restrictive channel and into a separation chamber as described above. In this way, two stages of separation are achieved, assuring substantially complete separation of cellular components.
The present invention also provides devices and methods for measuring and delivering predetermined volumes of fluid, such as a diluent or a biological fluid, to a receiving chamber in a centrifugal rotor. The measurement of the fluid is provided by a metering chamber of predetermined volume which is connected to a bulk fluid chamber. The bulk fluid chamber may contain a diluent, or other reagent, which is preloaded in the rotor for storage c~ntil the rotor is used. Alternatively, the bulk fluid chamber may be a capillary chamber which receives a biological t:luid to be tested, for example blood.
The fluid flowing into the metering chamber from the bulk fluid chamber fills the metering chamber, while excess fluid flows out of the metering chamber into an overflow chamber.
The predetermined volume in the metering chamber is delivered to the receiving chamber through at least one connecting means which controls flow out of the metering chamber so that fluid is delivered to the receiving chamber only after some predetermined time. Typically, delivery is delayed until after the: metering chamber is filled and it contains the predeterm~~.ned volume of fluid. The connecting means is designed such that essentially no detectable fluid escapes from the metering chamber until it is emptied. No .
detectable fluid is considered to have escaped from the metering chamber if thE~ total volume of fluid ultimately delivered to the receiving chamber is sufficiently accurate such that subsequent analyses are not adversely affected.
The prevention of flow between the metering chamber and the receiving chamt~er can be accomplished in a number of ways. For instance, a capillary exit duct in which capillary forces prevent flow at a first rotational speed can be used.
When the speed is incrcaased to a second, higher rotational speed, centrifugal force exceeds the capillary force and the metering chamber is emptied. Other means can also be used to block flow at the first. speed, for example, a membrane which ruptures only at the higher rotational speed can be inserted in the duct.
Other embodim~ants utilize a siphon having an elbow which is at substantially the same distance from the center of the rotor as the minimwa radial point (i.e., the radially most inward point) of the me~tering chamber. After the metering chamber is filled and the rotor is stopped, capillary action pulls the fluid just beyond the elbow. A siphon in which the fluid has moved to this point as a result of capillary action is considered to be "pr.imed". The rotor is restarted and the l0 combination of centrifugal and capillary forces pulls the fluid out of the metering chamber and into the receiving chamber.
It is also possible to serialize several metering chambers in a single rotor. Fluid overflowing from a first metering chamber enters a second metering chamber of a precise, predetermined size. The excess from the second metering chamber then overflows into a third metering chamber, etc.
Each metering chamber in the rotor typically feeds a separate receiving chamber.
The present invention also provides devices and methods for optically analyzing biological fluids, and in particular for analyzing blood plasma after first separating it.
from cellular material in the separation chamber. The apparatus and methods provide for distribution of the separated plasma or diluted plasma into a plurality of cuvettes within the rotor so that different optical analytic procedures may be performed without having to transfer aliquots of the fluid from the apparatus. Conveniently, the present invention decreases variability in the analyses by providing uniform optical paths in each cuvette and avoiding the creation of air bubbles when filling the cuvette. Uniform optical paths are provided through the use of reflective surfaces in the rotor which deflect a light beam oriented parallel with the axis of rotation so that the beam passes horizontally through the cuvette. Alternatively, a horizontal (ela., radial) light beam may be deflected vertically after passing through the fluid.
Using either embodiment:, variations in the amount of fluid in the cuvette, distortions in the light path due to welding seams, or matter floating on the top of the fluid will not affect results. The creation of air bubbles is also avoided by the use of novel inlet channels which allow gas to exit through one flow path in the channel, while fluid enters through another flow path in the same channel.
5 Referring now to Figs. 1-6, a centrifugal rotor 10 constructed in accordance with the principles of the present invention will be described in detail. The rotor 10 is in the form of a substantiall;~ solid disk including a top layer 12, middle layer 14, and bottom layer 16 laminated together to form 10 a composite structure. Typically, each of the layers 12, 14, and 16 will be composed of the same material, usually a transparent plastic such as an acrylate, but it is possible that the layers will b.e composed of different materials and that each layer may include two or more different materials 15 forming different portions of the layer. The exposed surface of top layer 12 will be referred to as the top surface while the exposed surface of the bottom layer 16 will be referred to as the bottom surface. A receptacle 18 is formed in the bottom surface of layer 16 and is generally aligned with the vertical axis 20 of the rotor, as best observed in Figs. 3 and 4. The receptacle 18 is formed to mate with the drive shaft of a.
conventional centrifuge system, as described previously.
The top surface 12 includes a blood application port 22 and four vent ports 24, 26, 28, and 30. The blood application port 22 and vent ports 24, 26, 28, and 30, penetrate the entire thickness of the top layer 12 and, as described in more detail hereinbelow, are aligned with various chambers fonaed in the middle layer 14 of the rotor 10. These penetrations may conveniently be formed in the top layer 12 by machining, e.g., drilling. The upper surface of middle layer 14 includes a plurality of chambers and passages formed therein. The chambers. and passages may be formed by machining a disk having generally flat surfaces or may be formed by injection molding of a~ suitable plastic resin in order to initially form the disk.
The middle layer 14 includes a metering chamber 40 having an inlet segment 42 which is generally aligned with the blood application port: 22 in top layer 12. The metering chamber 40 is connected to an overflow chamber 44 by a connecting passage 46, with the overflow chamber being located radially outward from, the metering chamber. A vent connector passage 48 extends from the radially-outward end of overflow chamber 44, first in a generally annular direction and thereafter in a generally radially-inward direction. The distal terminus 50 of passage 48 is aligned with vent port 28 in top layer 12 so that the outward radial extremity of overflow chamber 44 will be vented to the atmosphere during use of the rotor 10.
The depth of metering chamber 40 and overflow chamber 44 will be selected to provide for capillary dimensions when the chambers are completed by lamination of the top layer 12. Typically, the depth will be in the range from about 0.1 to 1.0 mm, more typically being in the range from about 0.25 to 0.75 mm. Usually, the depth will be uniform for both chambers 40 and 46 as well as the connecting passage 46, although it will be possible to vary the depth so long as capillarity is maintained. Alternate embodiments for the metering chamber 40 and overflow chamber 44 are described in Figs. 22-24 and the accompanying text, below.
A separation chamber 60 is forZaed in the upper surface of middle. layer 14 and is disposed radially outward from the metering chamber 40. The separation chamber 60 includes a cell trap 62 formed at its radially-outward periphery and a receptacle region 65 formed along its radially-inward perimeter. A capillary region 66 is formed between the receptacle region 65 and the cell trap 62 in order to inhibit the backflow of cells after they have entered the cell trap 62 as a result of centrifugal separation. The receptacle.~region 65 provides a volume which is capable of receiving whole blood or other biological fluid (optionally combined with a diluent or reagent) and which retains the blood plasma or other separated fluid after' centrifugation has been completed. An axial port 64 is conveniently formed as an annular passage which penetrates the entire thickness of middle layer 14 so that separated plasma may flow downward from receptacle region 65 of chamber 60 into a collection chamber 90 formed in bottom layer 16, as described in more detail hereinafter. The geometry of the separation chamber 60 may be varied considerably, as discussed in more detail in connection with Figs. lA and 1B, below.
The metering chamber 40 is connected to the separation chamber 60 by a short capillary passage 70 which terminates in a vertical wall 72 which forms the inner surface of axial port 64. Such termination of passage 70 will, of course, terminate the capillarity which would otherwise draw fluid through the passage.
The volume of metering chamber 40 will vary depending on the desired application, but will usually be selected to be as low as possible to provide a desired amount of plasma to each of the test wells formed in bottom layer 16, as described in more detail hereinafter. Typically, the volume of metering chamber 40 will be in the range.from about 0.005 to 0.05 cc, more typically being in the range from about 0.030 to 0.040 cc.
The volume of overflow chamber 44 will generally be larger than that of the: metering chamber 40 in order to accommodate excess blood which may be applied through blood application port 42. Generally, the volume of the overflow chamber 44 will be at least twice that of the metering chamber 40, typically being three or more times larger.
The volume o!: separation chamber 60 will be selected to accommodate the expected volume of plasma and optionally reagent or diluent which can flow from the metering chamber 40 and reagent chamber 80 (as described below). Typically, the volume of the receptacJ.e region 65 will be in the range from about 0.1 cc to 1.0 cc, more typically being in the range from about 0.25 cc to 0.50 c:c. The volume of the cell trap 62 will depend at least in part. on the volume of the receptacle region 65. In order to maxim~lze the efficiency of separation, i.e., increase the amount of plasma obtained from a fixed amount of whole blood, it is des:~rable that the volume of the cell trap 62 be just large enough to accommodate the largest expected volume of cellular material. For whole blood this can be calculated based on th~a highest expected hematocrit, where the volume of cell trap 62 will then be the expected percentage of the volume of metering chamber 40.
A reagent chamber 80 is also formed in the upper surface of middle layer 14 and connected to the separation chamber 60 through a capillary passage 82. The reagent chamber 80 will be disposed radially inward from the separation chamber 60 so that flow of reagent or diluent from the reagent chamber to the separation chamber 60 may be effected by spinning the rotor 10, as will be described in more detail hereinafter. As illustrated, the capillary passage 82 terminates with an open channel in wall 72. In this way, flow of reagent from chamber 80 will not occur in the absence of outward centrifugal force resulting from spinning of the rotor 10. In many cases, however, it may be desirable to provide a removable seal or barrier in chamber 82, or contain the reagent within a pouch or other package, to preserve the reagent and further assure that the reagent will not leak from chamber 80. Such a barrier, seal or package will be particularly desirable. when the reagent is "prepackaged" into the centrifugal rotor 10 at a central preparation facility and later subjected to shipping, storing, and other handling procedures which might. otherwise cause the reagent to degrade or leak.
As best observed in Fig. 3, the reagent chamber 80 may have substantially greater depth than the metering chamber 40 since the ability to provide capillary flow is not necessary. Thus, it i.s easy to store volumes of reagent which are substantially~grea~ter than the volume of blood or plasma which is provided to separation chamber 60 from metering chamber 40.
~ A collection chamber 90 is formed in the~upper surface of bottom layer 16 and is disposed.to receive plasma from the axial port 64~. A plurality of test wells 92 is formed about the periphery o!: the collection chamber 90 and connected by short radial passages 94. The test wells 92 and radial passages 94 are described in more detail in Figs. 25-32 and the accompanying text, below. Generally, the test wells 92 will be spaced equally about t:he periphery of layer 16 in order to enhance the equal distribution of plasma to each of the test rV0 91/18656 PCT/U5y1/03840 wells. The material above and below each test well 92 will usually be optically transparent in order to provide a clear optical path for visual assessment of the plasma in each well.
Alternate optical paths through the rotor 10 may also be provided.
The volume of the test wells 92 will usually be relatively low, typically being in the range from about 0.005 cc to 0.015 cc, more usually being in the range from about 0.008 cc to 0.010 cc. It is possible that liquid, dried, l0 or lyophilized reagents may be provided within the individual test wells so that combination occurs with the plasma when it is introduced. Alternatively, the walls or bottom of the test well 92 may be derivatized with various active components, such as antibodies, antigens,, receptors, or the like, which are intended to take part in the analytic procedure.
Referring now to Figs. lA and 18, the geometry of the separation chamber 60 may be varied considerably within the scope of the present invention. The central feature of the separation chamber 60 i:a the capillary region 66, which is preferably an annular space having an inner arcuate boundary 200 and an outer arcuate boundary 202. The capillarity of region 66 is broken at peach boundary 200 and 202 as the size of the adjoining regions, i.e., receptacle region 65 and cell trap 62, are increased to break the capillarity. Thus, fluid will be unable to flow through the capillary region 66 except when sufficient centrifugal force is applied by centrifugation.
The shapes of the receptacle region 65 and cell trap 62 may vary substantially. The receptacle region 65 will generally be tapered so that the distance between opposed 3o horizontal surfaces increases in the radially inward direction.
Such increasing distance provides the desired capillarity break, as discussed above. The taper may be provided by inclining the lower surface relative to the horizontal plane (Fig. 1), inclining the upper surface relative to the horizontal plane (Fig. lA), or inclining both surfaces (Fig.
18). The angle between the opposed surfaces of receptacle region 65 is not critical, typically being between 0' and 40', and usually being between 18' and 22'. The inner arcuate boundary 200 of the capillary region is usually formed contiguously with the narrow end of the tapered receptacle region which defines an arcuate aperture.
The cell trap 62 is typically formed as an annular 5 well which penetrates axially downward in the rotor and which is disposed contiguously with the outer arcuate boundary 202 of the annular space of the capillary region 66. The cell trap 62, however, may also extend upwardly, as illustrated in Fig. 1B, need not have a true annular shape.
10 Referring now to Fig. 4A, the geometry of collection chamber 90 may be modified to promote mixing of the separated biological fluid, e.g., plasma, with a diluent or reagent combined in separation chamber 60. In particular, the volume of the collection chamber 90 may be 15 increased and a peripheral vertical wall 91 may be provided inside of radial passages 94. Conveniently, the radial passage 94 will be capillaries which serve to prevent loss of fluid from the test wells 92 after the separation and distribution steps are completed. The increased volume of 20 collection chamber 90 and peripheral wall 91 both act to increase the retention time of liquid in chamber 90 as the rotor 10 is spun. Such increased retention time allows more thorough mixing prior to distribution.
In some cases, downward flow of plasma or other separated fluid through axial port 64 may be restricted by surface tension. In such case, it may be desirable to provide means, such as wicking fibers 93, which can disrupt the surface tension and allow the desired flow from receptacle region 65 into the collection chamber 90.
Alternatively, the surface tension can be disrupted by abruptly stopping the spinning of the rotor 10 after 20a separation has been achieved. Such cessation of spinning will cause the fluid to wet the wall of the region 65, allowing downflow.
Referring now to Figs. 7-11, the method of the present invention using the centrifugal rotor 10 as just described will be described in detail. Initially, reagent chamber 80 will be filled with reagent to a desired volume.
As illustrated, the chamber 80 is entirely filled, but it is also possible that the chamber will be partially filled.
The reagent may be loaded into rotor 10 either at a central preparation facility or immediately prior to use by the user.
In the later case, the: reagent may be filled using a pipette through vent port 24.
Whole blood may be loaded onto the rotor 10 through application port 22 in a volume greater than that which can be accommodated by measuring chamber 40. As soon as the blood is applied through port 2.2, it will begin to flow laterally both into the main portion of chamber 40 and through passage 46 into overflow chamber 44 b~~ capillary action. .Since the flow area into measuring chamber 40 is substantially larger than that through passage 46, the measuring chamber will quickly fill with blood, with the overflow passing into overflow chamber 44.
In this way, the blood applied through poz-t 22 need not be carefully measured prior to application. After a time sufficient for the blood to partition between measuring chamber 40 and overflow chamber 44, the distribution of blood will be as illustrated in Fig. 7 with the capillary portion of chamber 40 being completely filled and overflow chamber 44 being partially filled.
Referring now to Fig. 8, after the reagent has been added to chamber 80 and the whole blood has partitioned between chamber 40 and 44, the: rotor 10 will be centrifuged or spun at a rate sufficient to cause the blood from chamber 40 and reagent from chamber f~0 to flow into separation chamber 60.
Additionally, the blood in overflow chamber 44 will flow radially outward, as illustrated. Conveniently, the rotor 10 will be spun at a rate: in the range from about 1500 rpm to 5000 rpm, more usuall~~ from about 2500 rpm to 4000 rpm, for a time.in the range from about 20 seconds to 5 minutes,-'more typically being about 1 minute to 3 minutes, so that the cellular components o!: the blood will flow into trap 66 while the plasma will remain generally in the open portion of separation chamber 60.
After the sE:paration of plasmas from the cellular components of the whole blood has been completed, spinning of the rotor 10 will be :copped and the separated plasma will flow downward through axial. passage 64, as illustrated--in Figs. 9 and 10. The cellular components remain in cell trap 66, and the overflow blood remains in overflow chamber 44 while the plasma has flowed downw~~rd into a pool P in collection chamber 90. The plasma may then be distributed substantially equally into the individual test wells 92 by further rotation of the rotor 10, typica:Lly at a rate in the range from about 900 rpm to 5000 rpm for a time in the range from about seconds to 1 minute. After the desired distribution has been achieved, the rotor 10 may be removed from the centrifuge 10 and the rotor transferred to an appropriate instrument, such as a spectrophotometer or :Eluorometer, for testing.
Referring now to Fig. 12, an alternate rotor construction 100 will be described. The rotor 100 will generally be a laminate structure similar to rotor 10, with only a middle layer 102 being illustrated in Fig. 12. The upper layer will include an application port (not illustrated) which is aligned with a:n entry chamber 104 formed in the upper surface of layer 102. 'The entry chamber 104 is generally aligned with the vertical (spinning) axis of the rotor 100, and a pair of passages 106 .and 108 extend radially outward from said entry port. Chamber 106 serves as the measuring chamber and has a larger cross-sectional area than passage 108 so that it will fill more rapidly. Chamber 108 serves as the overflow chamber so that it can take up any excess blood which is applied through entry chamber 104. A reagent chamber 110 is located radially outward from the entry chamber 104 and connects with a non-capillary passage 112, which is connected with the distal end of chamber 106 and extends generally radially outward.
After blood is applied through entry chamber 104 so that measuring passage 106 is filled and reagent is loaded into chamber 110, the rotor 100 may be spun to cause both the blood from passage 106 and reagent from chamber 110 to flow outward through passage 112 into a separation chamber 114. Continued spinning of the rotor 100 causes the cells generally to collect along the radially-outward wall 116 of chamber 114, and further to flow down a spirally-outward path 118 to collect in cell trap 120. The separation chamber 114 and cell trap--120 are vented through the terminal end 122 of vent path 124. Once the desired separation. of plasma has been achieved, spinning of the rotor 100 will be stopped, and the plasma allowed to flow downward by gravity through a drainage port 126 formed at the radially-inward periphery of separation chamber 114. Usually, the bottom floor of chamber 114 will be sloped downward in the inward radial direction to promote the drainage of plasma through port 126. A callection chamber will be formed beneath the drainage port 126 in a manner similar to that illustrated in Figs. 1-6.
Referring now to Figs. 13-16,.an alternate embodiment of the analytical rotor 10' is described in detail. The rotor 10' includes a mounting receptacle 225 formed in the bottom surface of lower layer 206. The mounting receptacle 225 is suitable for mounting the rotor 10' on the spindle of a conventional centrifuge (not shown), as described hereinabove.
A first separation assembly within the rotor 10' includes a sample receptacle 224 formed through the top layer 204 and into the bottovm layer 206.which is joined to a separation chamber 210 by a flow restrictive channel 212. The separation chamber 210 includes a radially inward region 214 which generally defines a fluid flow path from the entry point of the flow channel 212 to a fluid outlet port 216. The separation chamber further includes a radially outward region 218 which forms a cell retention region which receives cells which are separated from the biological fluid as the rotor 10' is spun.
As illustrated, rotor 10' includes a second separation assembly including sample receptacle 224.;, __ separation chamber 210', flow restrictive channel 212', and collection chamber 220'. The chambers and passageways in the second separation assembly are arranged in a pattern identical to that of the first separation assembly so that two equivalent separation and analytic procedures may be performed simultaneously. It will be appreciated that the rotor could be adapted to include three or more similar or identical separation assemblies -to allow additional separation procedures to be performed simult;3neously. w It is necessary that the point at which flow channel 212 enters the separation chamber 210 be annularly-spaced apart from the collection port 216. In this way, the biological fluid will have a sufficient residence time within the separation chamber as the fluid flows from the entry point to the collection port. In the preferred embodiment, the flow passage 212 will be connected at one annular extremt~y of the collection chamber 210 while the collection port 216 will be located at or near the other annular extremity.
Usually, the receptacle 224 will be sized to receive the entire volume of biological fluid which is~to be separated.
The cell retention region 218 within the separation chamber 210 will be sized to accommodate the maximum possible volume of cellular material which may be present in the sample. For blood samples, this will depend on the blood volume as well as the maximum expected hemocrit to be processed. Usually, the cell retention region will have a volume which is equal to about 4k to 10%, more usually being about 7~, of the volume of the sample region 214.
The dimensions of the flow channel 212 will be selected to provide a flow rate of the biological sample into the separation chamber 210 which is sufficiently low to allow time for the cellular components to be separated from the fluid prior to the fluid reaching the outlet port 216. The particular dimensions will depend, of course, on the precise characteristics of the fluid as well as the speed at which the rotor is to be spun. For most purposes, flow channel having a width in the range from about 0.1 to 0.4 mm, more usually from about 0.15 to 0.25 mm, and a depth in the range from about 0.01 to 0.2 mm, more usually in the range from about 0.03 tor0.06 mm, will be suitable.
The outlet port 216 may be connected to an annular overflow passage 222 which in turn is connected to an annularly spaced-apart collection chamber 220. Thus, after sufficient sample has entered the separation chamber 210 to fill the chamber 210 back to the inner peripheral wall thereof, cell-free fluid will begin flowing laterally through the connecting channel 222 into the collection chamber-220. It will be appreciated that initially, the separation chamber 210 will be filled primarily with cell-free fluid, having a thin layer of separated cells formed adjacent the outer peripheral wall of the chamber. Over time, however, the thickness of the 5 layer of cells will increase, with the interface between cells and the cell free fluid moving radially inward over time. The volume of the separation chamber 210, however, will be sufficient so that the entire sample will be separated before the cellular interface can move sufficiently close to the l0 outlet port 216 to cause overflow of the cellular material.
The cell-free fluid entering chamber 220 will be ~-available immediately to react with any reagents which may be present within the chamber. In this way, an analytical reaction may be initiated prior to complete separation of the 15 biological fluid. By the time the separation is complete, the desired analytical reaction may be substantially completed, requiring only a small additional reaction time. Optionally, the cell-free fluid within collection chamber 220 may be observed directly through the rotor 10' without removal from 20 the chamber 220.
A vent port 226 will normally be provided near the inner peripheral wall of the collection chamber 220 in order to allow gases to vent as the chamber is filled with fluid.
The rotor 10' is used by applying a biological sample 25 to be separated into sample receptacle 224, usually while the rotor is at rest. The rotor 10' is then spun on a conventional centrifuge, typically a.t a speed in the range from about 1,500 rpm to 5,000 rpm, more usually being in the range from about 2,500 to 4,000 rF~m, for a time in the range from about 20 seconds to five minutes., depending on the volume of fluid being separated. The direction of rotation is not critical but will usually be counterclockwise (i.e., the direction of arrow 211 in Fig. 1) so that cellular build-up will be more likely to move away from the outlet port 216. Cell-free fluid will begin entering the collection chamber 220 as soon as the level of fluid in the separation chamber 210 has moved radially inward to reach the collection port 216. The collection chamber 220 may include reagents) which are selected to effect desired detection reactions, and reaction with the reagents may begin as soon as the cell-freEa fluid enters the collection chamber while the rotor continueas to spin and additional fluid continues to be separatEad.
Referring now to Figs. 17-19, another embodiment 227 of the analytical rotor of the present invention is illustrated. A sample receptacle 228 is connected to a separation chamber 230 by means of a flow restrictive channel 232. Separation chamber 230 includes a cell retention region 234 at its outer periphery and a collection port 236 located near its inner peripher~r. The collection port 236 is annularly spaced apart from the inlet of the flow restrictive channel 232 in order to allow a suf:Eiciently long flow path to provide sufficient residence time for the desired cellular separation.
The collection port 236 is vertically disposed and connected to an underlying collection chamber 238 which in turn is connected to a plurality of analytical cuvette 240 located about the periphery of 'the rotor 227. A vent path 242 will be provided in order to allow gases to escape from the separation 2o chamber 230 as the fluid enters.
In use, a biological sample is introduced to the sample receptacle 228, and the rotor is spun to cause the sample to enter the separation chamber 230 where the cellular components collect in the retention region 234. After a short time, cell-free fluid will reach the collection port 236 and will begin to flow downward into the collection chamber 238.
From the collection chamber, the cell-free fluid will move radially outward into the individual cuvettes where it may undergo reaction and subsequent analysis.
Referring now to Figs. 20 and 21, another embodimEnt 244 of the analytical rotor of the present invention will be described. The analytical rotor 244 comprises a disk-shaped rotor body 246 generally similar to the rotor 10' and rotor 227 described previously. The rotor 244 comprises a sample receptacle 248 having a.n inlet port 250. A diluent chamber 252 is formed adjacent the sample chamber 248 and will typically hold a container of "pre-packaged" diluent. Conveniently, the diluent container will be introduced at the time o~ rotor fabrication, but means may be provided for inserting such containers immediately prior to use.
A mixing cha~aber 254 is located radially outward from both the sample chamber 248 and diluent chamber 252. The sample chamber 248 is c=onnected to the mixing chamber 254 through a port 256, and the diluent chamber 252 is connected to the mixing chamber 254 through a port 258. Both the ports 256 and 258 are sufficiently large so that flow will~not be substantially restrictcad between the chambers 248 and 252 and the mixing chamber 254.. Thus, after introduction of the sample to be tested and the d:~luent, both the sample and the diluent may be substantially iimaediately transferred to the mixing chamber 254 by spinning the rotor 244.
The mixing chamber 254 is connected to separation chamber 260 by a flow restrictive channel 262. The flow restrictive channel 262, typically a capillary channel, will prevent immediate transfer of the contents of mixing chamber 254 to the separation c:hamber 260. Thus, the sample and the diluent may be thoroughly mixed while present in chamber 254, typically by reversibl~r rotating the rotor 244 or by alternately accelerating and decelerating the velocity of rotation in a single direction. An exemplary pattern of reversible rotation is to accelerate from 0 to 1200 rpm in a first rotational direct=ion over a period of three seconds, followed by stopping ttie rotation and accelerating from 0 to 1200 rpm in the opposit=a rotational direction over a period of three seconds. Such a pattern can be repeated until the desired mixing is achie=ved, with five repetitions usually being sufficient. An exemplary pattern of acceleration and _ deceleration is to spin the rotor 244 at 500 rpm for a short period, e.g., about 1.E~ seconds, followed by rapid acceleration to 4000 rpm for about 7..6 seconds. This pattern of acceleration and decelsaration will also be repeated a sufficient number of t~~mes to effect a desired degree of mixing.
Conveniently,. the mixing chamber 254 may itself be formed as a separation chamber including a cell retention region 263 (best illust=rated in Fig. 21) at its radially outward periphery. The cell retention region 263 is isolated by a capillary restriction 269, similar to capillary region 66 described in connection with Fig. 1. Thus, after mixing of the sample and diluent is completed, a first stage of cellular separation may be effected by spinning the rotor to cause the more dense cells to flow through capillary restriction 269 into the cell trap 263.
While a substantial proportion of the cellular components may be removed as just described in the mixing chamber 254, the fact that the mixing chamber is filled and agitated prior to separation increases the likelihood that.
there will be carryover of cellular material from the mixing chamber. Thus, a separation chamber similar to chambers 210 and 40 described previously is still necessary. The flow restrictive channel 262 is connected to a first annular extremity 264 of the separation chamber 260, while an outlet channel 266 is connected to the opposite annular extremity. A
cell retention region 268 is formed at the radially outward periphery of chamber 260, so that cell-containing fluid entering the chamber through flow restrictive channel 262 will have sufficient residence time for the cellular material to separate out into the cell retention region 268 as the rotor is spun.
The flow restrictive channel 262 will have an inlet port 280 (opening into separation chamber 260) which is spaced radially-outward from the outlet port 282 (which is connected to the mixing chamber 254). Thus, rotation of the rotor 244 will cause a fluid to flow at a controlled rate from the mixing chamber 254 to the separation chamber 260.
~ In a preferred aspect of the present invention, the flow restrictive channel 262 may include a siphon structure defined by a path segment 284 which extends radially inward.
Such a siphon structure will initially prevent flow from the mixing chamber 254 to the separation chamber 260. That is, so long as the path 284 i.s not filled with liquid, spinning of rotor 244 will not cause fluid from mixing chamber 254 to flow radially inward around the path 284 which defines the size and structure. Once spinning of the rotor 244 is stopped, however, capillary forces will cause fluid from the mixing chamber 254 to fill the flow restrictive channel 262, as described in connection with the previous embodiments. Once the channel 262 is filled, spinning of the rotor 244 will then cause fluid to flow without interruption from the mixing chamber 254 to the separation chamber 260. Use of the siphon structure is advantageous since it inhibits flow of the sample and diluent during the initial transfer from the sample chamber 248 and diluent chamber 252 which is effected by high speed spinning.
to Some carryover may occur, however, during the subsequent agitation step.
In use, a biological sample is introduced to sample receptacle 248 through port 250. Diluent will either be introduced or will be present in a package within the diluent chamber 252. In some eases, it may be necessary to open or pierce a diluent container in order to allow transfer of the diluent to the mixing chamber 254. In any event, the rotor 244 is then spun in order t:o effect transfer of the sample and diluent into the mixings chamber 254. At this point, the siphon' structure in the flow restrictive channel 262 will substantially prevent any flow or carryover of fluids into the separation chamber 260.
After the transfer of sample and diluent to the mixing chamber 254 has been completed, the contents of the chamber will be thoroughly mixed by subjecting the rotor 244 to a constantly reversing rotation or by alternately increasing and decreasing the velocity of rotation in the same direction, as described above. Such agitation will be continued for a time sufficient to assure complete mixing of the diluent sample. During such agitation, there may be some inadvertent loss or transfer of fluid from the mixing chamber 254 into the separation chamber 260,, although the flow restrictive nature of channel 262 as well as the presence of the siphon structure will largely inhibit such transfer.
After the mi:King is complete, the rotor 244 is held stationary for a time :sufficient to allow fluid from the mixing chamber 254 to fill the flow restrictive channel 262 by capillary action. Once the channel 254 is filled, the rotor can be spun in order to effect the fluid flow from the mixing chamber to the separation chamber 260 by centrifugal force. The separation chamber 260 will fill with the fluid from the mixing chamber 254, and any cells which may be 5 present will be trapped within the cell retention region 268, in a manner similar to that described for previous embodiments. Cell-free fluid from the separation chamber 260 will then flow into the distribution channel 266 and into a plurality of analytical chambers 286, e.g., optical 10 cuvettes as described previously.
Referring now to Figs. 22-24, an analytical rotor having flow partition in accordance with the principles of the present invention is shown in detail. Fig. 22 presents the middle layer 288 of a rotor such as is illustrated in I5 Fig. 1. The middle layer 288 comprises a blood capillary 290 and a metering chamber 292 connected to the blood capillary 290 by a connecting channel 294. An overflow chamber 296 is connected to the metering chamber 292 through the overflow channel 298. The blood capillary 290, the 20 metering chamber 292 and overflow chamber 296 preferably have capillary dimensions. An initial volume of fluid, such as whole blood, is introduced into the blood capillary 290 through blood application port 22 (shown in Fig. 1). As the rotor spins the initial volume partitions between the 25 metering chamber 292 and the overflow chamber 296. The metering chamber 292 is sized to accept the predetermined amount of fluid desired to be split from the blood capillary 290. The first fluid entering the metering chamber 292 will fill the chamber, while excess fluid will overflow the 30 metering chamber 292 and flow through connecting channel 294 and overflow channel 298 into overflow channel 296. The 79.437-11F
30a overflow feature causes the original bulk amount of fluid to be split into two amounts, the first precisely measured amount and the excess fluid.
The fluid entering the metering chamber 292 is delivered to a separation chamber 300 through an exit duct 302. The exit duct is typically of capillary dimensions which prevent flow of fluid at rotational speeds which cause filling of the metering chamber 292. For whole blood or diluent, the diameter of the capillary exit duct 302 is typically between about 0.05 mm and about 0.25 mm, preferably between about 0.075 mm and about 0.125 mm. The rotational speeds to fill the metering chamber typically generate a centrifugal force of about 5xg to about 42xg, preferably about 20xg to about 27xg.
To deliver fluid to the separation chamber 300 after the metering chamber is filled, the rotor's speed is increased sufficiently to cause the centrifugal force to exceed the capillary force and thus drain the metering chamber 292 into the separation chamber 300. The higher rotational speeds typically generate a centrifugal force exceeding about 45xg.
Fig. 23 shows another embodiment of the middle layer 288 the present invention in which a diluent chamber 304 containing a bulk~amount of diluent is connected to a diluent metering chamber 306, 'which is connected by a plurality of exit ducts 308 to the separation chamber 300. Typically, the diluent is preloaded i:n the rotor and stored in the rotor until use. A biological fluid, such as blood, is also delivered to the separation chamber 300 through a blood metering chamber 316.
The diluent :metering chamber operates on the same principles as the metering chamber described in Fig. 22. As the rotor spins the initial-.bulk volume of diluent partitions between the diluent metering chamber 306 and the overflow chamber 310. The dilu~ent metering~chamber 306 is sized to accept the predetermined amount of fluid desired to be split from the diluent chamber 304. The diluent entering the diluent metering chamber 306 will fill the chamber, while excess diluent will overflow 'the diluent metering chamber 306 and flow through connecting channel 312 and overflow channel~31~.4_ into overflow chamber 310. The overflow feature causes the original bulk diluent to be split into two amounts, the precisely measured amount and the excess fluid.
To deliver the diluent to the separation chamber 300 after the diluent metering chamber-306 is filled, the rotor's speed is increased sufficiently to cause the centrifugal force to exceed the capillary force and thus drain the diluent metering chamber 306 into the separation chamber 300. The rotational speeds to fill and empty the diluent metering 79437-lIF

chamber 306 are preferably the same as those to fill and empty the metering chamber 292 and to measure the blood.
Turning now to Fig. 24, a rotor using a siphon 318 as the connecting means to control flow between the metering chamber 292 and separation chamber 300 is shown. The elbow 320 of the siphon 318 is positioned so that it is substantially the same distance from the center of the rotor as the radially most inward point of the metering chamber 292.
As the rotor spins and the metering chamber 292 is filled, fluid in the siphon 318 does not move past the elbow 320. The rotor is then stopped and capillary action pulls fluid just beyond the elbow and the siphon is "primed".
When the rotor is spun again, centrifugal and capillary forces pull the fluid out of the metering chamber 292 into the separation chamber 300.
Referring now to Figs. 25-32 an analytical rotor comprising inlet channels, cuvettes, and reflective surfaces of the present invention are described in detail. The bottom layer 322 of a rotor as described in Fig. 1 is shown in Fig. 25. In some embodiments, however, the structures described below may be positioned radially outward from the separation chamber. Typically, the bottom layer 322 is composed of a transparent plastic, such as acrylic. The bottom layer 322 comprises a sample collection chamber 324 spaced radially inward from a plurality of peripheral cuvettes 326. Each cuvette 326 is connected to the collection chamber 324 by an inlet channel 328. The collection chamber may be formed in any shape, for instance, as a circle, a ring, or the like.

32a Each inlet channel 328 comprises two discrete flow paths, a first flow path 340 for the flow of liquid into the cuvette 326 and a second flow path 342 for the flow of gas out of the cuvette. The term "discrete" as used herein refers to the fact that the two flow paths 340 and 342 are separately defined and distinct from each other. The inlet channels 328 are preferably curved so as to prevent backwash or carryover when the contents of the cuvettes are agitated to effect mixing of the contents. Thus, cross contamination between cuvettes is avoided. The use of the flow paths 340 and 342 allow gas to escape easily from the c:uvette 326 as it is filled and thus prevent the formation of: bubbles in the cuvette 326, Which can deleteriously affect the: results of optical analyses.
There are a number of ways to create two discrete flow paths in the inlet channel 328. For instance, Figs. 27A, 27B and 31 show three possible configurations in which liquid flow path 340 has a greater depth than the gas flow path 342.
Because of its greater depth, the fluid will preferentially flow down path 340, leaving path 342 available for the evacuation of gas from t:he cuvette 328. The liquid flow path 340 may be on the side of the inlet channel 328 toward the direction of rotation oi: the rotor, as shown in Figs. 27A
and 278. In this configuration, centrifugal force will urge the liquid along the "leading" wall. Alternatively, if the inlet channel is not cw:ved, as shown in Fig. 32, the fluid flow path 340 may be in the center of the inlet channel 328, as shown in Fig. 31.
The inlet channel 328 is conveniently formed such that it passes around a reflective surface 330 (described more fully, below). If a re:Elective surface 330 is present, the inlet channel 328 will i~ypically pass around the reflective surface 330 on the side in the direction of rotation of the rotor. In the absence of a reflective surface 330, the inlet channel may be formed i~a any other generally radial configuration.
Other embodiments of the present invention utilize.
inlet channels 328 having regions with different surface textures. For instance, the gas flow path 342 may be left unpolished, leaving a rough surface texture in that region, while the fluid flow path 340 is polished. Alternatively, the liquid flow path 340 mayy be treated~so as to be hydrophilic whereas the gas flow path is treated so as to be hydrophobic.
The manner of treatment to make the surfaces hydrophilic or hydrophobic is well known in the art and need not be recited here. Any known surface treatment may be used as desired so long as it is chemically inert to the fluids passing through the inlet channel 328.
The rotor of this invention thus permits rapid filling of the cuvettes., Each cuvette is filled completely leaving little or no gas to interfere with subsequent optical analysis of the cuvettes contents.
Turning now to Fig. 30, it can be seen that optical analysis of the cuvette contents is facilitated by reflective surfaces 330 positioned radially inward from each cuvette 326 such that they are capa~~le of deflecting a light beam between a generally vertical and ~i generally horizontal direction and which are oriented at about 45' from the vertical axis of the rotor. As used herein, the "horizontal" and "vertical"
directions are determined in relation to the axis of rotation of the rotor. The horiaontal direction (typically radial) is perpendicular to the axis and the vertical direction is parallel to the axis.
The reflectiv~_ surface._330 need not be oriented directly radially inward from the cuvette. The reflective surface 330, however, most be parallel to the side of the cuvette in the optical pathway. For instance, a horizontal light beam which does not pass radially through the rotor may be used. Thus, the reflective surface 330 will be placed on a radial plane different from that of the cuvette 326, as shown in Fig. 32.
In an exemplified embodiment, the reflective surface deflects a vertical light beam 332 from a light source 334 so that it passes radially through a fluid 336 in the cuvette 326.
A light is then detected by the detector 338. The orientation of the reflective surfaces 330 is such that the positions of the detector 338 and light source 334 can be reversed. In the reversed configuration a horizontal light beam passes through the cuvette contents and is then deflected so that it passes vertically through the rotor where it is detected below the rotor. The reflective surfaces can be composed of any reflective surface known in the art which provides total internal reflection, an,d are typically air mirrors in which light is reflected at the acrylic-air interface.
Alternatively, the surface can be coated or backed with.a light reflective material.
Although the foregoing invention has beep described in detail for purposes of clarity of understanding, it will be obvious that certain modifications may be practiced within the scope of the appended claims. In particular, it will be appreciated that two or more metering chambers, separation 5 chambers, and collection chambers may be provided in order to run simultaneous tests and assays which require different test conditions. For example, multiple metering chambers may be provided to allow combination with different reagents or diluents in isolated separation chambers. Alternatively, a 10 single metering chamber may be connected by separate capillary passages to control flow into separate separation chambers. In either case, assays and tests requiring different protocols can be carried out in a single rotor system.

Claims

CLAIMS:

1. An analytical rotor for continuously separating fluid from a cell-containing liquid sample, said rotor comprising a rotor body having an axis of rotation, a sample chamber, a separation chamber, a cell retention region located generally at the radially outward periphery of the separation chamber, a flow restrictive channel connecting the sample chamber to the separation chamber, and means annularly offset from the flow restrictive channel and radially inward from the cell retention region for collecting cell-free fluid from the separation chamber, whereby spinning the rotor causes liquid sample in the sample chamber to flow into the separation chamber where cells in the sample move-radially outward into the retention region and cell-free fluid flows annularly into the collecting means, wherein said flow restrictive channel and said collecting means are annularly separated by a distance sufficient to permit cells to separate from the fluid before the fluid reaches the collecting means.

2. An analytical rotor of claim 1, wherein the sample chamber is a mixing chamber having a volume greater than that of the separation chamber.

3. An analytical rotor of claim 1, further comprising a sample receptacle and a diluent chamber spaced radially-inward from the mixing chamber and connected thereto with non-restrictive connecting means, whereby sample in the sample receptacle and diluent in the diluent chamber may be rapidly transferred to the mixing chamber but will be retained in the mixing chamber by the flow restrictive channel.

4. An analytical rotor of claim 1, wherein the flow restrictive channel includes a siphon structure having a path segment which extends radially inward.

5. A method for separating cells from a biological fluid, said method comprising:
introducing the fluid into a receptacle region within a rotor;
spinning the rotor to effect radially outward flow of the fluid from the receptacle region through a flow restrictive passage into a separation chamber, whereby cells collect in a retention region at the radially outward periphery of the separation chamber; and continuously removing the resulting separated fluid through a port which is disposed at a position located radially inward from the retention region and annularly displaced from the flow restrictive passage.

6. A method of claim 5, wherein the biological fluid continues to be introduced to the receptacle region while the separated fluid is removed through the port.

7. A method of claim 5, wherein the port is connected to an annular channel which allows the separated fluid to pass to an annularly-displaced collection chamber.

8. A method of claim 5, further comprising exposing the separated fluid to a reagent after the fluid is removed through the port.

9. A method of claim 8, further comprising examining the separated fluid after exposure to the reagent.

10. A method of claim 5, wherein the biological fluid is blood and the separated fluid is plasma.