WO1999006149A1

WO1999006149A1 - Pipetting devices preloaded with standardized control sample materials

Info

Publication number: WO1999006149A1
Application number: PCT/US1998/016137
Authority: WO
Inventors: Stephen F. Mauro; Robert A. Reynolds
Original assignee: Aalto Scientific Ltd
Current assignee: Aalto Scientific Ltd
Priority date: 1997-08-01
Filing date: 1998-07-31
Publication date: 1999-02-11
Anticipated expiration: 2000-02-01
Also published as: AU2034999A

Abstract

Devices and apparatus are disclosed for rapid, consistent, precise and accurate formation of standard solutions for use as reference samples without error, excessive costs, waste and degradations problems. The reference sample is formed by drawing in carrier (26) through tubing (32). Tubing (32) terminates in a tip (34) which is inserted into a body of carrier (26) which is drawn upward through the tubing (32) by a pump within the instrument. As the carrier is drawn up the tubing (32), it encounters a body of analyte (10) retained in place by barriers (12 and 14). The carrier (26) passes through the barriers (12 and 14), simultaneously dissolving the analyte (10), such that when the resulting solution reaches the instrument through the remaining portion of tubing (32), it has formed a measured predetermined reference solution sample, as with the pipette systems. Since the tubing (32) is flexible, it will be preferred that the analyte (10) be in the form of lyophilized or solid particles, granules, solid balls or coated balls rather than being in the form of a rigid solid body.

Description

PIPETTING DEVICES PRELOADED WITH STANDARDIZED CONTROL SAMPLE MATERIALS

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of application Serial No.

08/600,758, filed February 13, 1996, of like title.

BACKGROUND OF THE INVENTION

Field of the Invention: The invention herein relates to pipettes and pipetting equipment and instruments. More particularly, it relates to such devices which are used to collect, store and dispense reference materials for use as primary/secondary standards, calibrators or quality controls in medical, biological and chemical tests. Description of the Prior Art:

In many medical, chemical and biological laboratory and research procedures, it is necessary to calibrate or standardize analytical instruments, analytes and test procedures and to utilize quality control materials of known properties in order to obtain valid results from analytical or comparative testing of patient samples or the like. For instance, in emergency treatment, the properties of fluid (e.g., blood) samples drawn from the patient are compared against standard reference samples to identify the extent to which the patient's fluids exhibit properties differing from normal fluid properties, so that the physician can diagnose the disease or dysfunction from which the patient is suffering.

It will of course be evident that accurate standard reference samples are absolutely necessary if valid comparisons are to be made with patient samples and accurate diagnoses of patient conditions are to be obtained from the resulting data. In conventional practice, reference materials are made up in quantity by dissolving a sample of the reference analyte in a volume of the desired solvent liquid. Analytes are usually provided in solid dehydrated or preferably lyophilized form, since in such forms the analytes, most of which are organic and biological materials, have stable extended shelf lives. The laboratory conventionally mixes a sufficient quantity of solution to enable drawings of reference samples for as long a period as the solution remains effective (which may be from several hours to several weeks). This conventional practice, however, has several drawbacks and allows for the introduction of several different types of error.

For instance, errors can arise during measuring of the quantity of solid analyte and/or the liquid solvent, during withdrawal of individual sample quantities from the formulated solution, and by partial loss of sample during transport of the reference sample from the solution container to the place at which the analysis is to be run. Such errors may be random (such as when a laboratory technician misreads the measured weight of the solid analyte or a portion of sample leaks from the test instrument) or systematic (such as when the scale used to weigh the solid analyte is improperly calibrated). Thus, unless the technician works with extreme skill and carefully checks all measurements and equipment frequently, significant errors in standard reference samples can be anticipated. In actual daily practice, the time demands on laboratory technicians are usually such that there is little time for such thoroughness. Errors therefore consistently creep into reference samples and thus are incorporated into reference and test data.

In addition, conventional practice for preparation of reference materials is wasteful and expensive. Since it is has not been practical to make up an individual solution for each sample, it is common (as noted) for a large quantity of reference solution to be produced on some daily, weekly or monthly basis, from which the individual reference samples are subsequently drawn. Since one can rarely anticipate accurately how many reference samples will be needed in the course of a day, week, etc., an excess quantity of solution must be initially prepared. This results at the end of the service period in a significant quantity of unused reference solution being discarded, since it is no longer effective for tests. Since many of the analytes are quite expensive, this results in a large economic loss. In addition, discard and safe disposal of reference solutions often require special handling, also resulting in significant costs.

Further, while analytes may have extended stability in solid or lyophilized form, stability begins to decrease once the analyte is dissolved in the solvent liquid. Thus, over time the master solution properties change, thus often also changing the reference data of the samples. Therefore the laboratory technicians must apply correction factors to the standard samples, depending on when the samples have been drawn from the master solution. Such corrections can introduce additional errors, since the technician may incorrectly calculate or apply the correction, or the correction value itself may be uncertain, since the exact degree of degradation of the master solution normally can only be roughly estimated.

SUMMARY OF THE INVENTION The devices of this invention avoid the potentials for error, excessive costs, waste and degradation problems of the prior art, and permit the rapid, consistent, precise and accurate preparation of standard solutions for use as reference samples. An analyte (preferably the solute, but also alternatively the solvent) which will form the desired test solution is incorporated ("preloaded") in precisely measured amounts into a pipette tip in a form in which it has long term stability. Preferably the analyte will initially be in lyophilized form or will be lyophilized in situ within the tip, in which form it can be subsequently readily and quickly dispersed or dissolved in the carrier liquid to form the desired standard or reference solution. Alternatively other analyte forms may be used, such as hydrated analytes, if such are also readily and quickly dispersed or dissolved in the carrier liquid to form the desired standard or reference solution. Use of the preloaded pipette tips and calibrated pipetting devices of this invention permits drawing of accurate predetermined quantities of the carrier liquids into contact with the analyte solids, with the rapid dissolution or dispersion of the analyte in the carrier liquid, thus forming a consistent and accurate reference sample for each individual test. The system is useable for formation of many different standardized reference sample materials and with a variety of different analytical instruments. It substantially eliminates the opportunity for operator error. It also operates such that only the amount of analyte and carrier liquid which are needed for each individual test are used, so that there is no preparation of excess material, eliminating sample waste and disposal problems. Since the invention involves pipette tips preloaded with stable analytes, it can be used in many different settings, including emergency rooms and vehicles.

Further, in the present invention it is contemplated that chemical or biological reaction will not occur within the pipette tip between the analyte and the carrier liquid or components thereof. Of course, the reference sample formed by the dissolution or dispersion of the analyte in the carrier liquid is in many cases intended to react with a subsequent experimental, test or calibration material after it is dispensed from the pipette/pipette tip into contact with such material.

It is be understood that while the terms "pipette" and "pipette tip" are used herein for convenience and brevity, the devices of this invention will include not only those devices which are conventionally regarded as pipettes or pipette tips, but also all devices which operate in an equivalent manner and have a generally equivalent structure. This may include, but will not be limited to, liquid communication tubing used with an automated sampling instrument.

Therefore, in one broad embodiment, the invention is a pipette tip for creating and dispensing a liquid sample of known properties from a pipetting device having a liquid reservoir of known volume and liquid transfer means cooperating with the reservoir for alternately drawing a defined volume of a carrier liquid into the reservoir from the exterior of the device and dispensing a corresponding volume of the liquid sample to the exterior from the reservoir, which tip comprises a tubular liquid conduit attachable to the reservoir and providing liquid communication for the carrier liquid and the liquid sample between the reservoir and the exterior of the device; analyte containment means within the conduit for containing a defined quantity of analyte therein and causing effective contact between the carrier liquid and the analyte during passage of the carrier liquid from the exterior to the reservoir, such that if the analyte is a solute, the solute is rapidly and fully dissolved, dispersed or hydrolyzed in the carrier liquid, or if the analyte is a solvent, the solvent rapidly and fully dissolves or disperses a respective solute contained in the carrier liquid, either thereby forming the liquid sample by dissolution or dispersion without significant reaction, the liquid sample having known properties derived from the analyte, with the liquid transfer means causing the liquid sample to be drawn into the reservoir and subsequently dispensed as a reference material of the known properties

The invention is capable of operating effectively with the preloaded analyte being either a solute (analyte) or, alternatively, a solvent with the solute being present in the carrier liquid drawn into the tip during use For the most part, however, it is anticipated that the analyte preloaded into the tip will be the solute or analyte, with the carrier liquid serving as a solvent or suspensant for the analyte

The reservoir may be part of a separate pipetting device or similar instrument or it may be a pipette itself and is formed to contain the desired predetermined volume of carrier liquid, with operation of the device precisely and accurately drawing in that specific quantity of carrier liquid desired for dissolution or dispersion of the analyte to form the sample

The invention also includes means for retaining a readily available supply of carrier liquid adjacent to detached tips containing preloaded solid analyte, such that when the technician connects the pipette tip to the pipette or instrument, the required quantity of carrier liquid can also obtained and the desired solution sample produced in a single operation

Therefore, in another broad embodiment, the invention is of a pipetting device for collecting and dispensing a standard liquid sample as of a control material, which comprises a liquid reservoir of known volume, liquid transfer means cooperating with the reservoir for alternately drawing a defined volume of a carrier liquid into the reservoir from the exterior of the device and dispensing a corresponding volume of the liquid sample to the exterior from the reservoir, through a pipette tip which comprises a tubular liquid conduit attachable to the reservoir and providing liquid communication for the carrier liquid and the liquid sample between the reservoir and the exterior of the device; analyte containment means within the conduit for containing a defined quantity of analyte therein and causing effective contact between the carrier liquid and the analyte during passage of the carrier liquid from the exterior to the reservoir, such that if the analyte is a solute, the solute is rapidly and fully dissolved, dispersed or hydrolyzed in the carrier liquid, or if the analyte is a solvent, the solvent rapidly and fully dissolves or disperses a respective solute contained in the carrier liquid, either thereby forming the liquid sample by dissolution or dispersion without significant reaction, the liquid sample having known properties derived from the analyte, with the liquid transfer means causing the liquid sample to be drawn into the reservoir and subsequently dispensed as a reference material of the known properties. -

Also included within the invention is a container for retaining the detached pipette tips prior to being attached to a pipette or instrument reservoir. The container includes a basin for containing a body of the carrier- liquid and a retainer, such as a rack, for retaining the detached pipette tips disposed above surface level of the body of carrier liquid when the liquid is contained in the basin, the pipette tips also being retained in alignment for attachment to the reservoir, so that the motion of attaching a pipette tip to a pipette or instrument can also be used to immerse the distal end of the tip in the body in the liquid and draw the liquid into the pipette or instrument. Preferably the analyte is loaded and disposed within the tip in lyophilized form, but may be initially loaded in non-lyophilized form and thereafter lyophilized in situ. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1-3 are elevation views, partially cut away, of pipette tips illustrating the incorporation of an analyte in a non-lyophilized liquid form, followed by lyophilizing in situ. Figure 4 is a similar elevation view, partially cut away, of a pipette tip illustrating the containment of an analyte loaded in lyophilized or other solid form. Figures 5-8 are similar elevation views, also partially cut away, illustrating various other embodiments of incorporation of preloaded analytes into pipette tips according to this invention. Figure 9 is a pictorial view of a pipetting machine with a flexible tube connecting the machine to the pipette tip, with an enlarged view of a portion of the tube illustrating the incorporation of a preloaded analyte within the tube.

Figure 10 is a diagrammatic perspective view illustrating apparatus of the invention in which representative pipette tips containing analyte are retained in a package which also contains a quantity of carrier liquid, such that upon attachment of a preloaded pipette tip to a pipette or instrument, the required quantity of carrier liquid can also be drawn into the tip by a single operation.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS The^' devices and apparatus of the present invention will be best understood by reference to the drawings. Considering first Figures 1-8, in a typical operation, the technician manipulates the pipette itself (not shown) which has a hollow tubular appendage 2 (illustrated only in Figure 4) which is inserted into the top end 4 of pipette tip 6 and secured therein, usually by a friction fit. Pipette tips have commonly been packaged in open racks so that the technician can easily secure the pipette to a pipette tip and withdraw the tip from the rack for use. Following use in prior art systems, the pipette tip may then be discarded or cleaned and reused, depending on the protocols of the system. It will of course be understood that in the prior art, pipette tips have either been empty hollow tips or have been intended as reaction vessels. ln the present invention the same techniques for racking pipette tips and joining them to pipettes will be used However, a critical feature of this invention is that, rather than the tips being empty or designed to serve as reaction vessels, within the barrel 8 of the tip 6 there is disposed a preloaded quantity of analyte material 10 which is secured in place for subsequent contact with the carrier liquid 26

Figures 1-3 illustrate a pipette tip embodiment in which the desired analyte is initially loaded in a hydrated or liquid form, and subsequently lyophilized in situ within the tip The analyte material 10 is, in this embodiment, either hydrated or in a fluidized form as indicated at 3 Positioned across the interior of the barrel 8, near the distal end 24 of the tip 6 is a barrier 5 which will be permeable to the carrier liquid 26 but which offers sufficient flow resistance to the hydrated or fluidized form 3 of the analyte 10, so that upon loading the material 3 does not merely run out of the bottom of the tip The tip 6 is then capped by a cap 7, which fits snugly within the top end 9 of the tip 6 The cap 7 has a slot or groove 11 formed in its bottom At the beginning of the lyophihzing process begins after loading the material 3 containing the analyte 10, the cap 7 is inserted into the top 9 and pushed down only far enough to leave a small segment of the slot 11 above the top rim, as illustrated in Figure 2 This positioning of the cap 7 allows for water vapor to escape from the interior of the tip 6 during lyophi zation of the material 3 Optionally, an additional barrier 13, which is permeable to water vapor but not to solid materials, may be positioned between the body of material 3 and the top 9 of the tip Once the lyophihzation of the material 3 is complete, so that only the lyophilized analyte 10 remains, the cap 7 is pushed fully into the top of the tip so that the top end 9 is sealed against moisture intrusion, as illustrated in Figure 3 Preferably the lower end 24 of the tip 6 is also sealed against moisture incursion, as by a second cap 15 Since many pipette tips have quite small interior diameters at the lower end 24, cap 15 may contain an internal guide 17 to aid the technician in placing it on the lower end of the tip In Figure 4, a quantity of analyte 10 in solid form is illustrated as secured in place by a porous distal barrier 12 and a porous proximal barrier 14 ("distal" and "proximal" being with relation to the pipette/reservoir) The particular solid form of the analyte 10 is not specified in Figure 1 , rather, the drawing is intended to illustrate generally a solid analyte This particular barrier configuration, however, is preferably used when the analyte is loaded initially as a lyophilized material or otherwise in the form of particulated or granular material of relatively small particle size The diameters of the pores of the porous barriers 12 and 14 will be smaller than the particle size of the particulated or granular analyte material, such that the analyte particles cannot escape from the containment chamber formed by cooperation of the barriers 12 and 14 and the inner surface of the barrel 8

Figures 5-7 illustrate other convenient embodiments by which the analyte may be contained within the barrel 8 of the pipette tip 6 In Figure 5, there are a plurality of small inert balls 16 If the balls are solid, they may have a coating of the analyte 10 on them Alternatively, if they are porous, analyte 10 may be coated across the outer surface and incorporated within the pores of the balls 16 The "balls" need not actually be spherical, they may be of any convenient physical form For instance, they may be of standard column packing shapes or other shapes which are convenient for receiving the coating of the analyte 10 and being loaded into the pipette tip 6 The balls 16 are retained in place by proximal barrier 14 and distal barrier 12 In an alternative embodiment also illustrated by Figure 2, the analyte 10 need not be coated on each ball 16 Rather some of the balls 16 may be made of an inert nonsoluble material and other balls (designated 16') may be made in whole or in part from the analyte itself, such that the analyte 10 in ball 16' form is mixed in with equivalent but inert balls 16

In Figure 6, the embodiment shown has the analyte in the form of a solid annular plug 18 fitted tightly within the barrel 8 of the pipette tip 6 A hole 20 is left through the annular plug 18 to facilitate drawing in the carrier 26 to the pipette. In this embodiment, only the distal barrier 12 is required, although a proximal barrier 14 may also be used if desired.

In the embodiment shown in Figure 7, the analyte 10 is in the form of a porous solid body 22 (not particulated) and only distal barrier 12 is usually needed, although again both barriers may be present if desired.

Figure 8 illustrates another embodiment of the invention, similar to that previously illustrated in Figure 7. However, Figure 8 illustrates that the analyte 10, whether granulated or as a massive solid, can be secured at any position along the length of barrel 8 of pipette tip 6. It is preferred, however, that the analyte 10 be positioned somewhere centrally of the barrel 8, as generally illustrated in Figures 1-4, rather than being near the distal end 24 or the upper end 4 of the pipette tip 6. If the disposition of the analyte 10 is too close to the distal end 24, there is some potential for analyte material to be lost during the filling of the pipette with the carrier, while if it is near or within the upper or proximal end 4 there is a potential for interference with the attachment or detachment of the pipette tube 2.

Proximal barrier 14 is normally hydrophilic, but in some cases (discussed below) it may initially be at least slightly hydrophobic. Similarly, distal barrier 12 will be either initially hydrophobic (designated as 12 in the Figures) or initially hydrophilic (designated as 12' in the Figures). Whether a barrier 12 or 14 is initially hydrophobic or hydrophilic will normally depend on the manner in which the analyte 10 is initially incorporated into the barrel 8 of the pipette tip 6 and the shelf life conditions of the preloaded pipette tips. For instance, if the solid form of the analyte 10 is created outside the pipette tip, as would commonly be the case in the embodiment of Figure 4 (where a solid body of analyte would be formed and then pulverized to form solid particles or granules), the embodiment of Figure 5 (where the analyte-coated or -impregnated balls 16 or the analyte balls 16' are formed outside the pipette tip) or the embodiment of Figure 7 (where the annular plug 18 is formed outside the pipette tip), both barriers may be hydrophillic. However, if it is possible that the preloaded pipette tips may be stored in a humid environment, it may be desirable to have the barriers be at least slightly hydrophobic, to prevent absorption of moisture through the barriers and into contact with the preloaded solid analyte. This may be accomplished, if the barrier material itself is hydrophilic, by placing a thin hydrophobic coating over the outer surface (i.e., the surface which faces away from the analyte body 10) of each barrier. Conversely, the barriers may be made of material which is initially hydrophobic.

Figure 7 also illustrates an embodiment in which the solid analyte body 10 is to be formed in situ by deposition from or drying of an aqueous solution or slurry, other than as by lyophilization as illustrated in Figures 1-3. Figure 7 illustrates a distal barrier 12' which is initially hydrophobic, so that the liquid can be poured into the barrel 8 and will be blocked from draining out by the hydrophobic barrier 12'. Initial hydrophobicity may be imparted to the barrier 12' either by coating the inner surface (i.e., the surface which will face the analyte body 10) with a thin hydrophobic material, or the barrier 12' itself may be made of initially hydrophobic material. The barrier 12' thus retains the solution or slurry in place while the water carrier evaporates, leaving behind the deposited analyte 10 as porous body 22. Following the drying and solidification of the porous body 10, a proximal barrier 14 may optionally be emplaced. That proximal barrier may be either hydrophilic or hydrophobic, as discussed above. Since the carrier 26 will normally be water or an aqueous body fluid, it will be necessary to incorporate into the carrier an additive which will convert an initially hydrophobic barrier to a hydrophilic barrier. (The additive itself of course must not be detrimental to the analyte or affect the test results.) If the hydrophobicity was provided by a coating on a barrier, the hydrophobic coating must be rapidly dissolved by the additive. If the barrier itself was initially hydrophobic, the material from which it is made must be rapidly made hydrophilic by the additive.

For the purposes of this invention, "hydrophilic" means that the barriers (filters) will wet simultaneously with water, which has a surface tension of approximately 72 dynes/cm² at normal ambient room conditions. "Hydrophobic" means that the barriers have sufficient surface tension of their own that they will not spontaneously wet with water, unless that surface tension is reduced. For instance, a PTFE hydrophobic barrier would require reduction of surface tension to about 50 dynes/cm² to be wetted by pure water. One may use a wetting agent such as detergent or organic solvent with a surface tension of less than 32 dynes/cm² in the carrier to wet the barrier and make it hydrophilic. Various materials which can be converted from hydrophobic to hydrophilic in this or a like manner are well known and can be obtained commercially. The particular materials chosen for the additive and barrier will be determined by the nature of the carrier 26 and the analyte 10, since the additive and the barrier material must both be inert to the analyte and carrier, so that the nature of the resulting reference solution is not adversely affected. For instance, if the barrier material is too strongly hydrophobic, while it may be converted to pass some carrier water it may still bind too much analyte protein and therefore adversely affect test results. Useful barrier materials include, but are not limited to, cellulose acetate, modified polyvinylidene fluoride, and nylon (all hydrophilic) and polytetrafluoro- ethylene (PTFE), polyvinylidene fluoride, and Immobilon XXX (all hydrophobic).

Figures 1-8 illustrate the incorporation of the solid analyte into pipette tips.

Such preloaded tips represent an important end use of the present invention, and will be purchased by many laboratory, hospital, emergency care and other facilities for use in a wide variety of commonly conducted tests in which a pipetted reference standard is required. It will, however, also be recognized that the invention may be used in other test instruments and automated pipetting systems for reference samples where those instruments or systems operate in a manner akin to a typical pipette, by drawing in a predetermined quantity of carrier and forming a standard solution from the carrier and the specific quantity of preloaded analyte. Such a system is illustrated in Figure 9. An instrument 28 is shown generically. This instrument may be any one of many different types which form a reference solution sample and a test solution sample and separate, compare specific predetermined properties of the two samples and then display a differential value reading for a desired property or for some other indication to a technician, such as on a display screen 30. In the instrument shown schematically in Figure 9, the reference sample is formed by drawing in carrier 26 through tubing 32 Tubing 32 terminates in a tip 34 which is inserted into a body of carrier 26 which is drawn upward through the tubing by a pump (not shown) within the instrument As the carrier is drawn up the tubing 32, it encounters a body of analyte 10 retained in place by barriers 12 and 14, in a manner similar to that shown in Figures 1-8 The carrier 26 passes through the barriers 12 and 14, simultaneously dissolving the analyte 10, such that when the resulting solution reaches the instrument through the remaining portion of tubing 32, it has formed a measured predetermined reference solution sample, as with the pipette systems Since the tubing 32 is flexible, it will be preferred that the analyte 10 be in the form of lyophilized or solid particles, granules, solid balls or coated balls rather than being in the form of a rigid solid body

In operation of the embodiments shown in Figures 1-8, the operator will normally place the pipette appendage 2 at the proximal end 4 of the preloaded pipette tip and couple the two together, normally simply by pushing the appendage 2 into the top 4 of the tip 6, as in the prior art procedures It is convenient for the pipette tips for this type of operation to simply be packaged in conventional racks as are the prior art non-preloaded tips The technician thus can use the same procedure that he or she is familiar with to secure the preloaded tip of this invention to the pipette The pipette is then moved to a separate container of carrier 26 and the carrier is drawn in to the pipette by a conventional pipette mechanism, which is calibrated to draw in a precise predetermined quantity of carrier As it is drawn in through the tip 6, the carrier 26 passes over and through the mass of the analyte 10 and rapidly dissolves, disperses or hydrates it completely To facilitate complete dissolution suspension or hydration, it will usually be desirable to have the carrier 26 completely fill not only the pipette chamber, but also enough of the pipette tip to completely surround the remaining initially undissolved portion of the analyte, so that the dissolution can continue until complete The analyte will be sufficiently rapidly soluble or receptive that complete dissolution, suspension or hydration will occur in no more than about 30 seconds, and preferably will occur within about 5-15 seconds. The technician waits this length of time and then transfers the now formed mixture of analyte and carrier to a receptacle (such as test tube, petri dish, test instrument chamber or other similar conventional receptacle) into which is deposited the entire solution to form the control or reference sample. The emptied pipette tip will then commonly be discarded. Alternatively, however, it may in some cases be desirable for emptied tips to be collected for recycle by sterilization and refiling.

Figure 10 illustrates a further refinement of this system in which the preloaded pipette tip and the carrier can be obtained by the technician in a single operation and virtually a single motion. A container 36 has a rack 38 mounted near its top. In the rack 38 are mounted a plurality of pipette tips 6. (In Figure 7, two pipette tips 6, one of which is shown partially cut away, are illustrated as representative.) It will be understood that initially all openings 40 of the rack 38 may, and normally will, also contain preloaded pipette tips. Preferably these will all have the same analyte loading and be of the same size, in order to further eliminate potential errors, but it is also possible to have a container with a variety of different pipette tip sizes, analytes and analyte loadings, as long as they are individually clearly marked by labeling or color coding such that the technician can rapidly determine the correct preloaded tip to be used in a given test. The container 36 will be significantly deeper than the prior art pipette tip rack containers, such that the distal end 24 of a pipette tip 6 is positioned only part way down into the depth of the container 36. This leaves a volume (depth indicated at 42) which is used to contain a quantity of the carrier 26. The carrier 26 may be loaded into the container 32 prior to closing and shipping, but more preferably the volume to house the carrier will be left empty during shipping. Thereafter, upon opening the package and readying it for use, the technician will pour a desired quantity of carrier 26 into the bottom portion of the container 36, normally to the depth 42, to fill the volume. Conveniently, one of the openings 40 in the grid 38 may be left vacant to facilitate the technician's transfer of the carrier 26 from a supply source to the interior of the container 36. The rack or grid 38 will be made of a flexible material such that after the technician brings the pipette and its protrusion 2 into contact with and joins the upper portion 4 of the pipette tip 6, the technician can continue to push the assembled device downward, flexing the rack 38, until the distal end 24 of the pipette tip 6 is submerged in the body of carrier liquid 26. The predetermined quantity of carrier 26 can then be readily drawn into the pipette via the pipette tip and through the body of analyte 10. Once the quantity of carrier liquid has been drawn into the pipette, the technician can merely lift the entire assembly directly out of the rack 38 and transport it to the target receptacle. The rack 38 will also at that time resile to its previous position to keep the other remaining pipette tips out of the body of carrier 26 until each of them is to be submerged. The grid 38 will be made of a material (such as a plastic material) which has some degree of flexibility but is still sufficiently rigid to support the pipette tip 6 while the pipette appendage 2 is being pushed into the upper portion 4, and also to resile immediately upon withdrawal of the pipette tip 6 to its previous position supporting the remaining pipette tips.

It will be evident from this description that the formation of precise and accurate standard reference samples is assured by the present invention. The tip is preloaded with a predetermined quantity of analyte, the pipette is chosen to draw in exactly the quantity of carrier desired, and the analyte dissolves rapidly and completely when contacted with the carrier. Consequently, the resulting solution is of an exactly known combination of carrier and analyte such that the properties of that solution are a precise and consistently repeatable standard. The potential for technician error is essentially eliminated, since the technician does not measure either the amount of analyte or carrier used. While conceivably a technician might not allow sufficient time for complete dissolution of the analyte to take place prior, to dispensing the solution from the pipette into the target receptacle, the dissolution is normally sufficiently rapid that it will be completed during the time it takes the technician to move the pipette from the carrier container to the target receptacle. Indeed, this is desirable wherever possible, since it minimizes the time required for producing each sample, and therefore maximizes the number of samples that a technician can accurately produce within a given time period Of course, if an automated system such as one using instrument 28 is being run, the chance for operator error by premature dispensing of the solution before complete dissolution of the carrier is eliminated Common analyte materials which will be used with the preloaded pipette tips or tubing of the present invention include, but are not limited to, (1) materials obtained from human or animal body fluids such as blood, serum, plasma, urine, cerebrospinal fluid, pleural fluid, ascitic fluid, tears, sweat, saliva and amniotic fluid, (2) processed or purified human or animal proteins, peptides, lipids or carbohydrates in native, processed or synthetic human or animal body fluids, or (3) dried synthetic or semisynthetic human or animal body fluids, buffers or other stabilizing media containing various amounts of actual body fluids as in (1) above or purified or processed materials as in (2) above, or combinations thereof The aqueous carrier may be water, an aqueous buffer, native or processed human or animal body fluid or other solutions containing, where applicable, appropriate concentrations of wetting agents The wetting agents may be nonionic, anionic or cationic, as appropriate to the particular solutions Such wetting agents are well known and readily available commercially Typical nonionic detergents include polyoxyethylene ethers (such as Triton™ X-100, X- 114 or X-405) and polyoxyethylene sorbitans (such as Tween™ 20 or 80), typical cationic detergents include benzethonium chloride, cetylpyπndium XXX chloride and benzalkonium chloride, and typical anionic detergents include alginic acid and "Aerosol™ 22" The aqueous carrier may also include dilute organic carriers in combination with the water, buffer or native or processed body fluid, with or without the presence of a wetting agent Such organic carriers may include, but are not limited to, methanol, ethanol, gylcerol, ethylene glycol and dimethyl sulfoxide (DMSO)

As will be described in greater detail below, the devices and apparatus of the present invention may be used in a wide variety of end uses, including but not limited to analyses of bodily fluids such as blood, semen, urine, spinal fluid, saliva and sweat. It may also be used in any type of chemical or biological analysis where a test solution must be compared with a standard solution. The system will thus find use in chemical and biological laboratories, medical facilities, including emergency rooms and similar locations. It is particularly useful in emergency medical situations and locations, such as being in the inventory of materials available on emergency response vehicles for people such as paramedics. Since both the amount of carrier and of analyte are predetermined, an emergency medical technician can simply use the current invention to withdraw a sample of bodily fluid (such as blood) from the patient into the pipette (usually by having a hypodermic needle tip at the end of the pipette tip) and then dispense the resulting solution into a test device within the emergency vehicle, such that test results can be available for the emergency room physician by the time the patient is delivered to the hospital. This ability has not been available before now, since emergency medical technicians have not had time to manufacture accurate reference solutions while attending to the patient or transporting the patient to the hospital, nor have emergency vehicles such as ambulances been equipped for such manufacture. However, with the present invention, by having all of the components immediately available and simply drawing the fluid through the preloaded sample device, whether a hypodermic or a pipette tip, the technician can quickly obtain valuable data about the patient's condition for the subsequent attending physician.

Because of the rapid dissolution of the analyte, the system is ideal for other applications where rapid results are needed, such as in running tests in an emergency room to make a rapid diagnosis of a received patient's condition, or where a large number of identical samples need to be run in a fixed period of time. Because this system can be repeated quickly and accurately, a laboratory technician can increase his or her productivity significantly by being able to make numerous consistent repeat samples in the time previously required for production of only a single sample. Further, because none of the samples formed by this invention have time before use for the carrier/analyte solution to deteriorate significantly, all of the samples will be of equivalent accuracy and no time consuming and potentially inaccurate time calibrations or corrections need to be made

It will also be seen that the analyte 10 need not be a single material, but in fact can be a plurality of different materials, all of which are rapidly soluble in the same carrier 26, but which can be used for reading of different properties or conditions of a patient Of course, the individual analytes 10 must not interact with each other in a manner which would adversely affect the test readings It is also possible to have different materials within the analyte 10 which are themselves interactive but which do not react until they are both immersed in the carrier 26 This also allows for great stability and long shelf life, since the analyte will have the shelf life of lyophilized materials, and solution or dispersion resulting from the combination of the preloaded analyte and the drawn carrier will not deteriorate significantly in the short time from its formation to its use

As indicated above, the preloaded pipette tips, instrument tubing, and the like products will find use in many test and analysis procedures These include, but are not limited to, blood chemistries tests such as for sodium, potassium, glucose and urea creatinine XXX, enzyme chemistries such as creatine kinase, lactate dehydrogenase, serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphate, cardiac emergency room chemistries such as creatine kinase MS, troponin T&l and myoglobin, serum protein electrophoresis and other electrophoretic tests to serve as position markers for the various protein or other analyte bands for proper identification, standards and control materials for all types of immunological tests, including for arthritis, lupus erythematosis, scleroderma, hepatitis and HIV, diabetes testing for glycated hemoglobin and other abnormal hemoglobins such as fetal and hemoglobin S, amniotic fluid testing for fetal lung maturity and other abnormalities of the fetus urine testing for various pathologies as well as drugs of abuse and toxic chemicals and tests for multiple sclerosis and other neurological pathologies using spinal fluid in conjunction with serum It will thus be evident, as noted, that the invention will find use in a wide variety of medical, research and other facilities, such as hospital labs, emergency rooms, surgery facilities, autopsy labs, medical examiners' labs, DNA/RNA analysis labs, military field hospitals, emergency vehicles, point-of- care treatment facilities, chemistry labs, water testing labs, veterinary offices and labs, physicians' offices, intensive care units, urgent care centers, organ, tissue, fluid and molecular pathology labs, polymerase chain reaction procedure labs, shipboard hospitals and labs, toxicology labs, patient beside testing, environmental labs and agricultural labs. It will also be evident that there are numerous embodiments of this invention which, while not expressly set forth above, are clearly within the scope and spirit of the invention. The above disclosure is therefore intended to be exemplary only, and the actual scope of the invention is to be limited solely by the appended claims.

WE CLAIM:

Claims

1. A pipette tip for creating and dispensing a liquid sample of known properties from a pipetting device having a liquid reservoir of known volume and liquid transfer means cooperating with said reservoir for alternately drawing a defined volume of a carrier liquid into said reservoir from the exterior of said device and dispensing a corresponding volume of said liquid sample to said exterior from said reservoir, which tip comprises: a tubular liquid conduit attachable to said reservoir and providing liquid communication for said carrier liquid and said liquid sample between said reservoir and said exterior of said device; analyte containment means within said conduit for containing a defined quantity of analyte therein and causing effective contact between said carrier liquid and said analyte during passage of said carrier liquid from said exterior to said reservoir, such that if said analyte is a solute, said solute is rapidly and fully dissolved, dispersed or hydrolyzed in said carrier liquid, or if said analyte is a solvent, said solvent rapidly and fully dissolves or disperses a respective solute contained in said carrier liquid, either thereby forming said liquid sample by dissolution or dispersion without significant reaction, said liquid sample having known properties derived from said analyte, with said liquid transfer means causing said liquid sample to be drawn into said reservoir and subsequently dispensed as a reference material of said known properties.

2. A pipette tip as in Claim 1 wherein said conduit comprises a substantially rigid tube having an attachment end for attachment to said reservoir and an opposite exterior end, with an openings at each end.

3. A pipette tip as in Claim 1 wherein said conduit comprises a flexible tube having an attachment end for attachment to said reservoir and an opposite exterior end, with an openings at each end.

4. A pipette tip as in Claim 1 wherein said analyte is in lyophilized form.

5. A pipette tip as in Claim 4 wherein said analyte is initially disposed within said conduit in a non-lyophilized form and is lyophilized in situ.

6. A pipette tip as in Claim 5 wherein said analyte containment means comprises a liquid permeable barrier disposed across the interior of said tubular conduit adjacent said exterior end and forming an analyte containment chamber within said conduit between said barrier and said attachment end.

7. A pipette tip as in Claim 6 further comprising a removable closure cooperating with said attachment end of said conduit, said closure being positionabie in a first position to allow escape of water vapor during in situ lyophilization of said analyte and in a second position to seal said pipette tip against water entry when said in situ lyophilization is complete, said closure further being removable from said tip when said tip to be utilized.

8. A pipette tip as in Claim 1 wherein said reactant containment means comprises a pair of barriers disposed across and secured within said conduit and spaced apart along the axis of said conduit, said barriers being permeable to said carrier^'liquid and defining a chamber therebetween wherein said analyte is contained.

9. A pipette tip as in Claim 8 wherein one of said barriers is initially hydrophobic and the other is hydrophilic.

10. A pipette tip as in Claim 1 wherein said analyte is maintained within said conduit in comminuted solid form.

11. A pipette tip as in Claim 1 wherein said analyte is maintained within said conduit as a coating on a comminuted solid inert material.

12. A pipette tip as in Claim 1 wherein said analyte is maintained within said conduit as a coating on a porous body.

13. A pipetting device for collecting and dispensing a standard liquid sample as of a control material, which comprises: a liquid reservoir of known volume; liquid transfer means cooperating with said reservoir for alternately drawing a defined volume of a carrier liquid into said reservoir from the exterior of said device and dispensing a corresponding volume of said liquid sample to said exterior from said reservoir, through a pipette tip which comprises: a tubular liquid conduit attachable to said reservoir and providing liquid communication for said carrier liquid and said liquid sample between said reservoir and said exterior of said device; analyte containment means within said conduit for containing a defined quantity of analyte therein and causing effective contact between said carrier liquid and said analyte during passage of said carrier liquid from said exterior to said reservoir, such that if said analyte is a solute, said solute is rapidly and fully dissolved, dispersed or hydrolyzed in said carrier liquid, or if said analyte is a solvent, said solvent rapidly and fully dissolves or disperses a respective solute contained in said carrier liquid, either thereby forming said liquid sample by dissolution or dispersion without significant reaction, said liquid sample having known properties derived from said analyte, with said liquid transfer means causing said liquid sample to be drawn into said reservoir and subsequently dispensed as a reference material of said known properties

14 A pipetting device as in Claim 13 wherein said tip is separable from said reservoir

15 A pipetting device as in Claim 13 wherein said tip comprises a substantially rigid tube having an attachment end for attachment to said reservoir and an opposite exterior end, with an openings at each end

16 A pipetting device as in Claim 13 wherein said tip comprises a flexible tube having an attachment end for attachment to said reservoir and an opposite exterior end, with an openings at each end

17 A pipetting device as in Claim 13 wherein said reservoir is formed to contain a predetermined volume of liquid, and said defined quantity of carrier liquid comprises said volume

18 A pipetting device as in Claim 13 wherein said analyte is in lyophilized form

19 A pipetting device as in Claim 18 wherein said analyte is initially disposed within said tip in a non-lyophi zed form and is lyophilized in situ

20 A pipetting device as in Claim 19 wherein said analyte containment means comprises a liquid permeable barrier disposed across the interior of said tip adjacent said exterior end and forming an analyte containment chamber within said tip between said barrier and said attachment end

21 A pipetting device as in Claim 13 wherein said reactant containment means comprises a pair of barriers disposed across and secured within said tip and spaced apart along the axis of said tip, said barriers being permeable to said carrier liquid and defining a chamber therebetween wherein said analyte is contained

22 A pipetting device as in Claim 21 wherein one of said barriers is initially hydrophobic and the other is hydrophilic

23 A pipetting device as in Claim 13 wherein said analyte is maintained within said tip in comminuted solid form

24 A pipetting device as in Claim 13 wherein said analyte is maintained within said conduit as a coating on a comminuted solid inert material

25 A pipetting device as in Claim 13 wherein said analyte is maintained within said conduit as a coating on a porous body

26 A pipetting device as in Claim 14 further comprising a container for retaining a detached tip prior to its being attached to said reservoir

27 A pipetting device as in Claim 26 wherein said container comprises a basin for containing a body of said carrier liquid and retainer means for retaining said detached tip disposed above surface level of said body of carrier liquid when said body is contained in said basin, said tip also being retained in alignment for attachment to said reservoir

28 A pipetting device as in Claim 27 wherein said retainer means is flexible, such that following attachment of said tip is attached to said reservoir flexing of said retainer means with carrier liquid in said basin permits said exterior end of said tip to contact said carrier liquid and said carrier liquid to be drawn through said tip into said reservoir AMENDED CLAIMS

[received by the International Bureau on 22 December 1998 (22.12.98); original claims 4 and 18 cancelled; original claims 1,5,8,13,19 and 21 amended; remaining claims unchanged (5 pages)]

1 . A pipette tip for creating and dispensing a liquid sample of known properties from a pipetting device having a liquid reservoir of known volume and liquid transfer means cooperating with said reservoir for alternately drawing a defined volume of a carrier liquid into said reservoir from the exterior of said device and dispensing a corresponding volume of said liquid sample to said exterior from said reservoir, which tip comprises: a tubular liquid conduit attachable to said reservoir and providing liquid communication for said carrier liquid and said liquid sample between said reservoir and said exterior of said device; and analyte containment means within said conduit for containing a defined quantity of lyophilized solute analyte therein and causing effective contact between said carrier liquid and said lyophilized solute analyte during passage of said carrier liquid from said exterior to said reservoir, such that said lyophilized solute analyte is rapidly and fully dissolved, dispersed or hydrolyzed in said carrier liquid, thereby forming said liquid sample by dissolution or dispersion without significant reaction, said liquid sample having known properties derived from said analyte, with said liquid transfer means causing said liquid sample to be drawn into said reservoir and subsequently dispensed as a reference material of said known properties.

5. A pipette tip as in Claim 1 wherein said analyte is initially disposed within said conduit in a non-lyophilized form and is lyophilized in situ.

7. A pipette tip as in Claim 6 further comprising a removable closure cooperating with said attachment end of said conduit, said closure being positionable in a first position to allow escape of water vapor during in situ lyophilization of said analyte and in a second position to seal said pipette tip against water entry when said in situ lyophilization is complete, said closure further being removable from said tip when said tip to be utilized.

8. A pipette tip as in Claim 1 wherein said analyte containment means comprises a pair of barriers disposed across and secured within said conduit and spaced apart along the axis of said conduit, said barriers being permeable to said carrier liquid and defining a chamber therebetween wherein said analyte is contained.

1 0. A pipette tip as in Claim 1 wherein said analyte is maintained within said conduit in comminuted solid form.

1 1 . A pipette tip as in Claim 1 wherein said analyte is maintained within said conduit as a coating on a comminuted solid inert material.

1 2. A pipette tip as in Claim 1 wherein said analyte is maintained within said conduit as a coating on a porous body.

1 3. A pipetting device for collecting and dispensing a standard liquid sample as of a control material, which comprises: a liquid reservoir of known volume; liquid transfer means cooperating with said reservoir for alternately drawing a defined volume of a carrier liquid into said reservoir from the exterior of said device and dispensing a corresponding volume of said liquid sample to said exterior from said reservoir, through a pipette tip which comprises: a tubular liquid conduit attachable to said reservoir and providing liquid communication for said carrier liquid and said liquid sample between said reservoir and said exterior of said device; and analyte containment means within said conduit for containing a defined quantity of lyophilized solute analyte therein and causing effective contact between said carrier liquid and said analyte during passage of said carrier liquid from said exterior to said reservoir, such that said lyophilized solute analyte is rapidly and fully dissolved, dispersed or hydrolyzed in said carrier liquid, thereby forming said liquid sample by dissolution or dispersion without significant reaction, said liquid sample having known properties derived from said analyte, with said liquid transfer means causing said liquid sample to be drawn into said reservoir and subsequently dispensed as a reference material of said known properties.

1 4. A pipetting device as in Claim 1 3 wherein said tip is separable from said reservoir.

1 5. A pipetting device as in Claim 1 3 wherein said tip comprises a substantially rigid tube having an attachment end for attachment to said reservoir and an opposite exterior end, with an openings at each end.

1 6. A pipetting device as in Claim 1 3 wherein said tip comprises a flexible tube having an attachment end for attachment to said reservoir and an opposite exterior end, with an openings at each end.

1 7. A pipetting device as in Claim 1 3 wherein said reservoir is formed to contain a predetermined volume of liquid, and said defined quantity of carrier liquid comprises said volume.

1 9. A pipetting device as in Claim 1 3 wherein said analyte is initially disposed within said tip in a non-lyophihzed form and is lyophilized in situ.

20. A pipetting device as in Claim 1 9 wherein said analyte containment means comprises a liquid permeable barrier disposed across the interior of said tip adjacent said exterior end and forming an analyte containment chamber within said tip between said barrier and said attachment end.

21 . A pipetting device as in Claim 1 3 wherein said analyte containment means comprises a pair of barriers disposed across and secured within said tip and spaced apart along the axis of said tip, said barriers being permeable to said carrier liquid and defining a chamber therebetween wherein said analyte is contained.

22. A pipetting device as in Claim 21 wherein one of said barriers is initially hydrophobic and the other is hydrophilic.

23. A pipetting device as in Claim 1 3 wherein said analyte is maintained within said tip in comminuted solid form.

24. A pipetting device as in Claim 1 3 wherein said analyte is maintained within said conduit as a coating on a comminuted solid inert material.

25. A pipetting device as in Claim 1 3 wherein said analyte is maintained within said conduit as a coating on a porous body.

26. A pipetting device as in Claim 1 4 further comprising a container for retaining a detached tip prior to its being attached to said reservoir.

27. A pipetting device as in Claim 26 wherein said container comprises a basin for containing a body of said carrier liquid and retainer means for retaining said detached tip disposed above surface level of said body of carrier liquid when said body is contained in said basin, said tip also being retained in alignment for attachment to said reservoir.

28. A pipetting device as in Claim 27 wherein said retainer means is flexible, such that following attachment of said tip is attached to said reservoir flexing of said retainer means with carrier liquid in said basin permits said exterior end of said tip to contact said carrier liquid and said carrier liquid to be drawn through said tip into said reservoir. STATEMENT UNDER ARTICLE 19

This is in response to the PCT Notification of Transmittal of the International Search Report or the Declaration dated 28 October 1 998 in reference to the above- identified international application.

The International Search Report identifies the following documents, all of which are U.S. Patents, as relevant prior art.

Inventor(s) Patent No. Categorv(ies)

Rampal et al. U.S. 5,437,979 X, Y

Filbert, Jr. U.S. 5,249,71 1 Y

Diekmann U.S. 4,956,298 Y

White U.S. 5, 1 56,81 1 Y

Puchinger et al. U.S. 4,999, 1 64 Y

Gazit et al. U.S. 5,496,523 A

Smith U.S.5, 364,595 A

The same references, with equivalent indication of degree of relevance, were cited in the priority U.S. application. The claims as revised herein parallel the amendments made in the priority application, and distinguish the cited references as follows.

The claimed invention is a pipette tip device into which a known lyophyllized solute analyte is preloaded in known quantity to make up a standard control sample. A solvent drawn up into the pipette rehydrates and dissolves the analyte, to form the control solution, which is then ejected. The lyophyllized solute analyte does not undergo any chemical or biological reaction in the tip or pipette.

Rampal et al. disclosed a pipette tip reaction chamber which contains a solid phase reaction support which is active and specific for a subsequent chemical reaction. There is no preloaded analyte. A first reactant which is support-specific is drawn in and immobilized, followed by a second reactant which reacts in situ on the support with the immobilized first reactant to form a reaction product, which is subsequently dissociated from the support and removed from the tip. Rampal et al's tip is entirely different from Applicant's being structured not for dissolution but rather for reaction. Rampal et al. neither disclose nor suggest Applicant's invention.

Filbert's teaching of a flexible tube to which the tip is connected does not alter the fundamental differences between the Rampal et al. tip structure and Applicants' tip structure. Consequently, Filbert combined with Rampal et al. does not suggest or disclose Applicant's invention.

Diekmann's device is essentially either a chromatographic separation column or a reaction column, but is not a hydration tip. Diekmann teaches no structure by which a solvent liquid can be drawn up into a tip, there dissolve a lyophyllized analyte, and then dispense the resultant solution. Rather Diekmann discloses a packed column with a separable or reactable "sample material" which upon being centrifuged is driven through the packing where it is separated or reacted with the products then being collected. Diekmann does not disclose nor suggest Applicant's invention.

Neither White nor Puchinger et al., alone or in combination, does not disclose nor suggest Applicants' invention. White's unique device allows gas to flow through trapped liquid within a tip without escape of liquid. Hydrophyllic barriers and a scavenging liquid block any movement of the trapped liquid. Using Puchinger et al's barriers merely enhance the White tip's liquid trapping function.

Gazit et al. and Smith are background and require no comment.