EP0801543A4

EP0801543A4 - Device and method for transferring fluids for analysis

Info

Publication number: EP0801543A4
Application number: EP96904431A
Authority: EP
Inventors: Wallace H Coulter; Charles R Shambaugh; James H Carter; Ravindra Shukla
Original assignee: Coulter International Corp
Current assignee: Beckman Coulter Inc
Priority date: 1995-01-13
Filing date: 1996-01-11
Publication date: 1999-07-28
Also published as: WO1996021399A1; JPH10512368A; EP0801543A1

Abstract

A device and method for transferring fluids between closed vials is provided. In one embodiment, a reagent transfer vial (42) is provided. In a further embodiment, the reagent transfer vial is used in conjunction with a transfer device (10) which includes a cylinder having a pair of opposing open tubes or tubular portions into which a sample vial (12) and the reagent transfer vial (42) can be inserted to transfer fluid therebetween. The tubes are separated by a wall through which extends a fluid transfer needle (40) and a vent needle (24) which first is inserted into a sample vial to vent the sample vial into a vent chamber (36) or reservoir formed in the transfer device before the transfer needle is inserted into the sample vial.

Description

DEVICE AND METHOD FOR TRANSFERRING FLUIDS FOR ANALYSIS

Technical Field

This invention relates generally to transferring a fluid from one vial to another. More particularly, the invention is directed to a method of making a device for fluids and a method of transferring a fluid between two closed vials. The device comprises a reagent transfer vial which preferably contains a test reagent. In a further embodiment, the device further contains a sample vial which contains the fluid which is to be analyzed. The method of transferring a fluid between two closed vials enables the transferring of an aliquot of a fluid, such as blood, from one closed vial (the sample vial) to another closed vial (the reagent transfer vial) without exposing an operator to the fluid. Background Art

Automated blood and blood cell analyzers are well known. These analyzers typically utilize a portion of a whole or pre-prepared blood sample. When the blood sample is taken from a subject, it usually is placed into a vial or test tube. With the potential of exposure to highly infectious diseases by an operator, such as the HIV virus or hepatitis, the vial preferably is closed, typically by a resealable rubber stopper. Many types of blood sample sampling devices have been developed, generally following the procedure of piercing the vial stopper to aspirate a portion or aliquot of the blood sample. The needle probe or cannula then is removed from the vial and the stopper reseals to maintain the remainder of the blood sample sealed in the vial. For some operations, the blood sample is combined or mixed with another fluid or reagent outside of the analyzer prior to being aspirated into the analyzer. If the vial is opened or handled for a transfer operation, the risk of exposure is greatly increased. The vials, typically made of glass can break, removing the stopper can result in aerosols, the contaminated stopper then must separately be disposed of and the risk of spillage also increases. It therefore would be desirable to provide a method of transferring fluids between closed vials without exposing the operator to the fluids. Further, it would be desirable to transfer a precise desired volume of fluid between the vials.

Disclosure of the Invention

The invention provides a reagent transfer vial utilized for transferring fluids. The reagent transfer vial includes a predetermined vacuum and preferably includes a reagent sealed under vacuum in the vial. The reagent preferably is in a lyophilized form and the vacuum in the vial is formed during the lyophilization process. The reagent transfer vial includes a pierceable stopper, which also preferably is inserted in the lyophilization process. The pierceable stopper preferably is secured to the reagent transfer vial to insure that the pierceable stopper is not driven into or pulled out of the reagent transfer vial during the fluid transfer operation.

The reagent transfer vial preferably is utilized with a transfer device. The transfer device includes a cylinder having a pair of opposing open tubes or tubular portions into which a pair of vials can be inserted to transfer fluid therebetween. The tubes are separated by a wall through which extends a vent needle and to a lesser distance, a fluid transfer needle. A sample fluid containing vial, stopper first, is inserted into a first tube until seated onto an exposed end of the vent needle. This allows the sample vial to be vented before it further is inserted onto the transfer needle. The reagent transfer vial, stopper first, then is inserted into the second tube until seated onto the opposite exposed end of the transfer needle. The negative pressure differential between the closed vials is utilized to transfer a desired fluid portion between the vials. The transfer device wall can include a vent chamber or reservoir into which the vent needle is connected to retain any expelled fluid or aerosol.

More specifically, the invention relates to a method of making a device for fluids comprising providing a reagent transfer vial having an open end and a stopper securable in said open end; inserting a reagent comprising an aqueous solution of heat sensitive monoclonal antibodies into said reagent transfer vial; freeze drying said stopper and said reagent transfer vial and said reagent to form a predetermined vacuum in said reagent transfer vial to transfer a predetermined amount of a sample fluid into said reagent transfer vial, said predetermined amount of said sample fluid is between 25 to 2,000 microliters; and securing said stopper into said reagent transfer vial open end to close said reagent transfer vial and maintain said predetermined vacuum for utilization in transferring said predetermined amount of fluid into said reagent transfer vial through said stopper.

In another embodiment, the method further comprises providing a closed sample vial containing a blood sample therein; and providing a housing including a first substantially tubular portion having a vent needle enclosed in said tubular portion for piercing said closed sample vial when inserted into said first tubular portion in a first position to vent said closed sample vial, said first tubular portion further enclosing a transfer needle having a first end for piercing said closed sample vial after said vent needle in a second position; and said housing further including a second tubular portion having a second end for piercing said closed reagent transfer vial when inserted into said second tubular portion to transfer a fluid aliquot from said closed sample vial to said reagent transfer vial utilizing said pressure differential.

In a further embodiment, the invention is further directed to a method of transferring a fluid between two closed vials comprising providing a closed reagent transfer vial having an internal pressure less than atmospheric pressure; providing a closed sample vial with at least a first fluid therein; piercing said closed sample vial with at least a first vent needle to vent said closed sample vial substantially to atmospheric pressure; piercing said closed sample vial with at least a first end of a substantially integral transfer needle spaced from said vent needle; and piercing said closed reagent transfer vial with at least a second end of said transfer needle to transfer a fluid aliquot from said closed sample vial to said reagent transfer vial by a negative pressure differential between said reagent transfer vial and sample vial.

Brief Description of Drawings

FIG. 1 is an exploded perspective of one embodiment of a transfer device;

FIG. 2 is a perspective view of a tube adapter which can be utilized with the transfer device;

FIG. 3 is a side-plan view partially in section of the transfer device with a reagent vial and a blood sample vial inserted therein;

FIG. 4 is a side view partially in section of a transfer wall and needle assembly of the transfer device;

FIG. 5 is a top-plan view of a fixture which can be utilized with the transfer device; FIGS. 6-8 are side views of the transfer operation of the transfer device utilizing the fixture of FIG. 5; and

FIG. 9 is an exploded side view, partially in section, of the reagent vial. Modes for Carrying out the Invention

Referring to FIGS. 1 and 3, a first embodiment of a closed vial transfer device of the present invention is designated generally by the reference numeral 10. As illustrated in FIGS. 1 and 3, the transfer device 10 has inserted therein a sample vial or sample tube 12. The vial 12 is inserted into a first tubular portion or tube 14 of the transfer device 10 through an open end 16. The tubular portion 14 includes a slot 18 formed there through and a tab 20 mounted in the slot 18.

The tab 20 preferably is formed or secured in a first position (FIG. 6) adjacent a first edge 22 of the slot 18. The vial 12 is inserted against the tab 20, which forms a vent position and a vent needle 24 pierces a tube stopper 26 in the vial 18. The stopper 26 is conventionally formed and mounted in the open end of the vial 12.

The vent needle 24 is mounted in a needle assembly 28 (FIGS. 1 , 3, and 4). The needle assembly 28 is formed in a closed end 30 of a second tubular portion or tube 32. The end 30 can snap fit with a mating flange 34 on the tubular portion 14 to form a transfer housing or body. The tubular portions 14 and 32 can also be welded or adhered to one another or molded in one piece as desired.

The vent needle 24 opens into a vent chamber or reservoir 36 formed in the closed end 30 of the tubular portion 32. A second wall of the vent chamber 36 is formed by a hydrophobic member or pad 38. The vial 12 is vented into the vent chamber 36, which will trap any fluid or aerosol which can be expelled from the vial 12. The pad 38 is formed of a conventional high-surface tension material, which is selected to allow the air to pass through the pad 38, but prevents the fluid from passing there through. The fluid then is trapped in the reservoir 36. The tab 20 preferably is formed as an integral snap tab with the edge

22, but could also be adhered to the edge 22. The tab 20 also could be rotatable from the vent position into the slot 18 for movement to the aspirating position. Once the vial 12 is vented, the tab 20 is broken away or released from the edge 22 and the vial 12 then can be moved to the aspirate position, as illustrated in FIGS. 1 and 3.

As the vial 12 is moved into the aspirate position, a second transfer needle 40 pierces the stopper 26. The transfer needle 40 is mounted through the needle assembly 28 and is sealed from the chamber 36. The vial 12 then is ready for a sample aliquot to be transferred to a reagent transfer vial 42. The reagent transfer vial 42 has a stopper 44 and as the reagent transfer vial 42 is inserted into the tubular portion 32, the stopper 44 is pierced by a free end 46 of the transfer needle 40. The reagent transfer vial 42 will be more fully described with respect to FIG. 9.

The vial 42 can include a reagent or can be empty, but in any case is maintained under a predetermined vacuum, which vacuum will draw the desired amount of the fluid in the vial 12 into the vial 42. The sample fluid is thus withdrawn simply from the vial 12 into the vial 42 without exposing the operator to the sample fluid or aerosols from the sample fluid. The tab 20 includes an opening 47 through which the needles 24 and 40 pass.

FIG. 2 illustrates the utilization of a smaller diameter sample vial 48, which can be a pediatric vial, when utilized with blood samples. An adapter sleeve 50 is utilized to properly insert the vial 48 into the center of the tubular portion 14 of the transfer device 10.

Referring now to FIGS. 5-8, a transfer operation is illustrated utilizing a fixture 52. The fixture 52 can include a plurality of sample vial wells 54 and the sample vial 12 is inserted into one of the wells 54. The vial 42 preferably is inserted in another well 56.

To transfer fluid, such as blood, between the vials 12 and 42, the vials 12 and 42 preferably first are inserted into the respective wells 54 and 56 of the fixture 52. The transfer device 10 then is mounted onto the vial 12 with the vial 12 being inserted into the first tubular portion 14. The vial 12 includes the stopper 26 into which the vent needle 24 first is inserted. The vial 12 is allowed to vent and the vent position is defined by a first vent position of the transfer device 10, as illustrated in FIG. 6. The vent position is defined by the tab 20. As stated, the tab 20 can be a snap tab or can be a rotatable tab (not illustrated) mounted into the vent position of the slot 18. Once the vial 12 is vented the tab 20 is snapped off or broken (not illustrated) or the tab 20 is rotated into the slot 18. Snapping the tab 20 or rotating it into the slot 18 allows the transfer device 10 to be inserted onto the vial 12 a further distance which then inserts a first end of the transfer needle 40 through the stopper 26 into the vial 12, as illustrated in FIG. 7.

The fluid in the vial 12 then is ready to be transferred. The transfer device 10 then is inverted and is inserted over the reagent transfer vial 42. The vial 42 also includes the stopper 44. The free end 46 of the transfer needle 40 is inserted through the stopper 44 and the fluid then preferably is transferred from the vial 12 into the vial 42 via a negative pressure differential between the two vials 12 and 42. The pressure is equalized, to allow the fluid transfer, by air passing back through the pad 38 and the transfer needle 40.

The vial 42 then can be removed from the transfer device 10 and the fluid mixture then can be utilized/aspirated through the stopper 44 into an analyzer (not illustrated) without exposing the operator to the sample or reagent fluids. The vial 12 can be removed from the transfer device 10 for further utilization or for disposal, also still in a sealed non-exposing mode, with the stopper 26 still intact.

Once the transfer operation is completed, the tab 20 can be moved back to the lip 22, which frees the stopper 26 from the transfer needle 40. The vial 12 then can be grasped and removed from the transfer device 10 with the stopper 26 secure in the vial 12. The tubular portion 14 preferably is sized such that the vial 12 cannot easily be twisted when it is seated on both the vent needle 24 and the transfer needle 40.

Although the transfer device 10 can be utilized in any transfer operation, it generally can be utilized to add a specific fluid aliquot from the sample vial 12 to a reagent contained in the vial 42. The reagent can be any type of reagent which is to be combined with the sample aliquot. The reagent can be a chemical, a fluorescent labelled antibody or a monoclonal antibody bound to microspheres. The microspheres can be magnetic or non-magnetic and can be utilized for a variety of operations, such as disclosed in U.S. Patent Nos. 5,223,398; 5,231 ,005 and 5,260,192. One specific utilization of the transfer device 10 is to obtain a lymphocyte subset determination, such as the CD4 or CD8 subsets, as described for example in U.S. Application Serial No. 08/303,924, entitled METHOD AND APPARATUS FOR SCREENING MICROSCOPIC CELLS UTILIZING LIGHT SCATTER TECHNIQUES, which is incorporated herein by reference. This application describes a procedure wherein, microspheres having a CD4 or CD8 monoclonal antibody bound thereto are first mixed with a whole blood sample, where the microspheres bind to the CD4 or CD8 positive cells, and then operated on in the instrument, such as a STKS analyzer sold by the assignee of the present invention, Coulter Corporation. In this operation, the transfer device 10 can be utilized to transfer the desired aliquot of the whole blood sample from the vial 12 into the vial 42. The vial 42 has the reagent, CD4 or CD8 antibody bound microspheres, contained therein under vacuum. The vacuum is selected to draw a precise desired volume of the blood sample from the vial 12. The microspheres preferably are in a lyophilized form, but also can be in a liquid, if desired. The amount of vacuum established for a lyophilized reagent is much easier to determine and establish than that for a liquid reagent.

In a vial containing a liquid reagent containing an evaporable solvent, such as water, establishment of a particular partial vacuum as in a lyophilizer chamber, followed by stoppering the vial, results in a vacuum equal to that of the chamber, less the partial pressure of the solvent vapor at the particular temperature prevailing at the moment. An increase in the vial's temperature will result in an increase in the partial pressure due to the solvent vapor, thus reducing the vacuum in the vial, thereby reducing the amount of sample that it will draw. Conversely, a decrease in the vial's temperature will cause an increase in the vacuum in the vial, causing an increase in the amount of sample drawn. This problem, plus the complication of calculating complex partial pressures for the solvent, would result in much more variability in the performance of the vial 42 than using lyophilized reagents. Once the aliquot of blood is transferred into the vial 42, the vial 42 then is moved to a mixer, where the blood and reagent are mixed and then the vial 42 then can be utilized in the desired analyzer, all without exposing an operator to the sample fluid.

The transfer device 10 and one or more vials 42 having a reagent under vacuum can be provided in a kit form for utilization in the above- referenced blood cell analysis operations. In one preferred embodiment, the vial 42 can include about eighty (80) microliters of antibody bound microspheres in a liquid form or an equivalent lyophilized amount. The vacuum can be about 10-11 inches of mercury and the resultant transferred aliquot of whole blood will be on the order of eight hundred (800) microliters of blood, which then can be utilized for further blood cell analysis. The kit (not illustrated) can include a transfer device 10 and one or more vials 42, each containing a different reagent. One transfer device 10 can be utilized more than once, as long as the same sample is being transferred to the different transfer vials. If more than one sample is to be operated on, then a second set of one or more transfer vials and a transfer device will be included for the different samples.

The tubular portions 14 and 32 of the transfer device 10 and the vials 12 and 42 preferably are labeled, color coded, or otherwise identified for proper operation of the transfer device 10. The vials 12 and 42 also can be of different outer diameters, such that the primary or sample tube 12 only can be inserted into the tubular portion 14. The identification preferably is readily apparent for proper operation of the transfer device 10.

Referring to FIG. 9, a reagent transfer vial of the present invention is designated generally by the reference numeral 60. The reagent transfer vial 60 is a preferred embodiment of the reagent transfer vial 42 for utilization with the transfer device 10. The reagent transfer vial 60 preferably includes the reagent in a lyophilized form, generally including monoclonal antibodies. The prior art has utilized vacuum in EDTA tubes or vials, however, not with the precision necessary for utilization in the reagent transfer vial 60. Further, and more critically, the prior art has established the vacuum by heating the vials or tubes during manufacturing. The tubes are heated to a certain temperature to roughly expand the air inside to balance the prevailing atmospheric pressure at the time and place of manufacture. When stoppered and cooled, the EDTA tube will draw about 90% of its total volume with air occupying the remainder of the tube space. The air bubble is required for proper mixing of the blood and the EDTA. Prior art manufacturers are only concerned with drawing a relatively approximate amount of blood, sufficient to result in a blood-to-anticoagulant ratio of 1.0 to 1.5 mg EDTA per microliter of blood. Ratios above or below this range cause distortions of the blood sample and result in mismeasurements of hematological parameters. Utilizing the reagent transfer vial 60 with a monoclonal antibody reagent, it is desired to draw approximately 800 microliters of blood into the reagent transfer vial 60. Utilizing the ideal gas laws, the pressure, volume and temperature of an ideal gas are related according to the formula:

PV = nRT Two gas states require two simultaneous equations:

P₁V₁ = ΠRT_T for a gas in state 1

P V₂ = nRT₂ for a gas in state 2

Changes in any one parameter result in equivalent changes in the other parameters, such that equality of this equation is always maintained. In utilizing the reagent transfer vial 60, atmospheric air can be considered an ideal gas.

Applicants have found, by both calculation and experiment, that a vacuum of -11.5 inches of mercury, as measured by a calibrated vacuum gauge, is required in the reagent transfer vial 60 to draw the required 800 microliters of blood samples into the tube. Thus, for air as the gas in the tube: The volume of the tube is fixed and constant.

Standard sea level atmospheric pressure = 760 mm mercury

11.5 inches mercury x 25.4 mm/inch = 292.1 mm mercury

Pressure in the reagent transfer vial 60 required to draw 800 microliters of blood sample:

760 - 292.1 = 467.9 mm mercury

Room temperature = 20°C = 293°Kelvin

Since n and R are constants and \/^ = V₂ the simultaneous formulas set forth above can be reduced to:

T, T₂

293° T₂

222.680

T₂ = 468 = 475°K - 273°K = 203°C

In order to establish a pressure of 468 mm of mercury inside the vial 60 when the vial is at 20°C, one would have to heat the vial 60 to 203°C prior to stoppering. Upon cooling to room temperature, the above gas law results in the residual pressure in the vial 60 being the required 468 mm mercury.

Thus, to draw 800 microliter of blood into the reagent transfer vial 60, the reagent transfer vial 60 would have to be heated to greater than 200°C. When utilizing a monoclonal antibody containing reagent, the monoclonal antibodies are heat sensitive and would effectively be destroyed at temperatures greater than about 40°C. Clearly heating of the reagent transfer vial 60 to produce the desired vacuum is not an option with monoclonal antibodies or other heat sensitive reagents. Further, the transfer method is utilizable for a range of small sample fluid volumes on the order of 25 to 2000 microliters of sample fluid. Higher transfer volumes would require even higher processing temperatures. Applicants have solved the problem of providing a vacuum without heating the reagent transfer vial 60 and of providing the reagent in a dry, non-liquid form by utilizing a lyophilization or freeze drying process. One specific example utilizing the reagent transfer vial 60 with a monoclonal antibody containing reagent is set forth in Example I.

EXAMPLE I Materials:

1. Latex particle microsphere suspension, 2 micron diameter nominal, 4% solids suspension 2. Phosphate buffered saline, 0.85% (PBS)

3. CD4 and CD8 monoclonal antibody

4. Conjugation reagents (various types)

5. Bovine serum albumin

6. Dextraπ, 100,000 - 200,000 molecular weight 7. 13x65 mm polypropylene (PP) or polyethylene (PET) screw-cap tubes

8. "Split leg" rubber stoppers, ca. 10 mm diameter

9. Cyanoacrylate adhesive ("Super Glue")

10. Screw caps, 13 mm, color coded

Method:

1. Volumetrically measure the quantity of microspheres to be prepared, then add PBS to wash away the microsphere-suspending solution and any broken microspheres (fines). 2. CD4 and CD8 is then chemically bound (conjugated) to the microsphere surface by one of several methods well known to those skilled in the art.

3. After the reaction is complete, the CD4/CD8 conjugated microspheres are washed again to remove all conjugation chemicals, then resuspended to 1% solids content in PBS with 1 % BSA.

4. Dextran is added to the microsphere suspension at 50 mg per microliter and the mixture is gently mixed in a tube roller apparatus for 30 minutes at room temperature. 5. The 13x65mm vials are placed in a rack suitable for use in a lyophilizer and vacuum chamber, then aliquot 80 microliters of the microsphere suspension into each vial with a pipette or dispensing device, mixing the microsphere suspension constantly.

6. Place the rubber stoppers loosely on top of each vial, leaving the split above the rim of the vial, then place the rack into the lyophilizer with the shelf temperature set at -40 to -50°C and allow the microsphere suspension in each tube to freeze solidly. (Note: For technical reasons, freeze drying and stoppering may be carried out as two separate steps; in this case, stoppers would be added after the lyophilization of the microsphere suspension.)

7. Place temperature measurement probes into selected vials to monitor the temperature during the lyophilization process.

8. When the probes indicate a product temperature of -40°C (approximately 3 hours, depending on equipment used and number of vials), evacuate the lyophilization chamber and start the condenser; after 24 to 48 hours, the cycle will be complete.

9. Turn off the shelf refrigeration and allow the product to warm to approximately +20°C while still under vacuum, then slowly release the vacuum by bleeding in dry room air or dry nitrogen to a vacuum reading of - 11.5 inches of mercury; maintain this reading for at least 5 minutes to allow vacuum in the vials to completely equilibrate. 10. Activate the stoppering mechanism to stopper the vials; check visually to see that all vials are well sealed and repeat the stoppering, if necessary.

11. Release vacuum on the chamber completely, open the lyophilizer and remove the vials.

12. Place two drops of cyanoacrylate adhesive on the tops of the stoppers and screw cap tightly.

13. Label vials with appropriate identity labeling.

Referring again to FIG. 9, it was determined in developing the reagent transfer vial 60 that a number of issues had to be addressed. The vial 60 includes a tube portion 62 in to which a stopper 64 is inserted. The stopper 64 is vented so that the liquid can be drawn out around the stopper 64 during the lyophilization process. Once the lyophilization process is completed, the stopper 64 is seated into the tube portion 62.

A conventional stopper could be inserted as a seal into the tube portion 62, but is not, preferred for several reasons. The vials 60 are to be handled and stored and the vacuum must be maintained for proper operation. Thus, an upper portion 66 of the tube portion 62 is threaded to receive an overcap 68. The overcap 68 is screwed onto the tube portion 62, securing the stopper 64 to the vial 60. This insures both that the stopper 64 will be maintained in the vial 60 during handling and storage to maintain the vacuum and that the transfer needle 40 or other access needle will not dislodge the stopper 64. The overcap 68 includes an opening 70 (shown in a top view) in the center of the overcap 68 for access to the stopper 64 by the needles. The overcap 68 also preferably is secured to the stopper 64 by an adhesive to insure that an access needle does not collapse the stopper 64 into the tube portion 62.

The reagent preferably will utilize a dispersing agent, especially when the sample fluid is undiluted blood. The dispersing agent insures that the microsphere, are resuspended into the blood driving the transfer operation without clumping of the microspheres which would cause an inaccurate operation. A preferred dispersing agent is Dextran having 100,000 - 200,000 molecular weight. Alternate dispersing materials could be polyethylene glycol, 6,000 molecular weight, albumin (bovine source) and non-hemolytic detergents, e.g. Pluronic F-68 (BASF Wyandotte) - a polyethoxylate of the condensate of propylene oxide and propylene glycol or Surfynol (Air Products) - a group of methylated alkyne-diols, such as tetramethyl decynediol.

Many modifications and variations of the present invention are possible in light of the above teachings. The invention includes utilization of the reagent transfer vial 60 without a reagent to transfer fluid between closed vials. The vials 12 and 42 can be conventional types of vials, formed of either glass or plastic. The microspheres can be of various types and sizes, generally in the range of 1 to 5 micron diameter, both magnetic and non¬ magnetic. The vacuum is selected depending upon the volume of the sample fluid to be transferred and the size of the vials utilized and effectively can be utilized within about 20% of the selected volume. It is therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims

1. A method of making a device for fluids comprising: a. providing a reagent transfer vial having an open end and a stopper securable in said open end; b. inserting a reagent comprising an aqueous solution of heat sensitive monoclonal antibodies into said reagent transfer vial; c. freeze drying said stopper and said reagent transfer vial and said reagent to form a predetermined vacuum in said reagent transfer vial to transfer a predetermined amount of a sample fluid into said reagent transfer vial, said predetermined amount of said sample fluid is between 25 to 2,000 microliters; and d. securing said stopper into said reagent transfer vial open end to close said vial and maintain said predetermined vacuum for utilization in transferring said predetermined amount of fluid into said vial through said stopper.

2. The method of claim 1 wherein the monoclonal antibodies are CD4 and CD8.

3. The method as defined in claim 2 including providing said monoclonal antibodies in said reagent bound to a plurality of microspheres.

4. The method as defined in claim 3 including providing a dispersing agent in said reagent.

5. The method as defined in claim 2 including providing said reagent transfer vial open end with screw threads, providing a screwable overcap for said vial and screwing said overcap over said stopper to prevent said stopper from being pulled from said closed vial. 6. The method as defined in claim 5 including adhering said stopper to said overcap to prevent said stopper from being pushed into said vial.

7. The method of claim 2 which further comprises: a. providing a closed sample vial containing a blood sample therein; and b. providing a housing including a first substantially tubular portion having a vent needle enclosed in said tubular portion for piercing said closed sample vial when inserted into said first tubular portion in a first position to vent said closed sample vial, said first tubular portion further enclosing a transfer needle having a first end for piercing said closed sample vial after said vent needle in a second position; and said housing further including a second tubular portion having a second end for piercing said closed reagent transfer vial when inserted into said second tubular portion to transfer a fluid aliquot from said closed sample vial to said reagent transfer vial utilizing said pressure differential.

8. The device as defined in claim 7 which further comprises forming a reservoir in said second tubular portion into which said closed sample vial is vented.

9. The device as defined in claim 8 which further comprises providing said reservoir with hydrophobic means for trapping fluid in said reservoir while allowing air to pass through the hydrophobic means and out of said reservoir and out of said second tubular portion.

10. The device as defined in claim 7 which further comprises providing a needle assembly formed in said second tubular portion, said tubular portions formed as a pair of opposed open tubes and said closed sample vial to be inserted into a first one of said tubes having said vent needle and a first end of said transfer needle therein and said closed reagent transfer vial to be inserted into the second one of said tubes having a second end of said transfer needle therein. 11. The device as defined in claim 10 which further comprises providing a reservoir formed in said needle assembly and said closed sample vial is vented into said reservoir by inserting said vent needle into said closed sample vial prior to inserting said transfer needle therein.

12. The device as defined in claim 11 which further comprises providing said reservoir with a hydrophobic means for trapping fluid in said reservoir while allowing air to pass through the hydrophobic means and out of said reservoir and out of said second tube.

13. The device as defined in claim 10 which further comprises including a means for identifying said tubes and said reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

14. The device as defined in claim 10 which further comprises including said needle assembly having a physical stop to form a first vent position for said closed sample vial and said first tube.

15. The device as defined in claim 14 which further comprises including a reservoir in said needle assembly into which said closed sample vial is vented, said reservoir including hydrophobic means for trapping fluid in said reservoir while allowing air to pass through the hydrophobic means and out of said reservoir, and means for identifying said tubes and said closed reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

16. The device as defined in claim 14 which further comprises including said physical stop being movable from said first vent position after venting said closed sample vial to a second transfer position. 17. The device as defined in claim 16 which further comprises including said physical stop being movable to said first vent position to release said closed sample vial after said fluid aliquot is transferred.

18. The device as defined in claim 16 which further comprises including said physical stop is formed as a tab in said first vent position.

19. A method of transferring a fluid between two closed vials comprising: a. providing a closed reagent transfer vial having an internal pressure less than atmospheric pressure; b. providing a closed sample vial with at least a first fluid therein c. piercing said closed sample vial with at least a first vent needle to vent said closed sample vial substantially to atmospheric pressure; d. piercing said closed sample vial with at least a first end of a substantially integral transfer needle spaced from said vent needle; and e. piercing said closed reagent transfer vial with at least a second end of said transfer needle to transfer a fluid aliquot from said closed sample vial to said reagent transfer vial by a negative pressure differential between said reagent transfer vial and sample vial.

20. The method as defined in claim 19 including providing a reservoir and venting said closed sample vial into said reservoir.

21. The method as defined in claim 20 including providing said reservoir with at least a hydrophobic portion to trap fluid in said reservoir while allowing air to pass through the hydrophobic portion and out of said reservoir. 22. The method as defined in claim 19 including providing a needle assembly including a pair of open tubes and inserting said closed sample vial into a first one of said tubes having said vent needle and a first end of said transfer needle therein and inserting said closed reagent transfer vial into the second one of said tubes having a second end of said transfer needle therein.

23. The method as defined in claim 22 including providing a reservoir in said needle assembly and venting said closed sample vial into said reservoir by inserting said vent needle into said closed sample vial prior to inserting said transfer needle therein.

24. The method as defined in claim 23 including providing said reservoir with at least a hydrophobic portion to trap fluid in said reservoir while allowing air to pass through the hydrophobic portion and out of said reservoir.

25. The method as defined in claim 22 including identifying said tubes and said closed reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

26. The method as defined in claim 22 including providing said needle assembly with a physical stop to form a first vent position for said closed sample vial and said first tube. 27. The method as defined in claim 26 including providing a reservoir in said needle assembly and venting said closed sample vial into said reservoir, said reservoir including at least a hydrophobic portion to trap fluid in said reservoir while allowing air to pass through the hydrophobic portion and out of said reservoir, and identifying said tubes and said reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

28. The method as defined in claim 26 include moving said physical stop from said first vent position after venting said closed sample vial to a second transfer position.

29. The method as defined in claim 28 including returning said physical stop to said first vent position to release said closed sample vial after said fluid aliquot is transferred.

30. The method as defined in claim 28 including providing said physical stop as a tab in said first vent position.

31. The method as defined in claim 19 including forming a vacuum in said closed reagent transfer vial to form said negative pressure differential between said reagent transfer vial and sample vial.

32. The method as defined in claim 31 including providing a reagent in said closed reagent transfer vial.

33. The method as defined in claim 32 including providing a plurality of microspheres having a predetermined monoclonal antibody bound thereto as at least a portion of said reagent.

34. The method as defined in claim 33 including providing said reagent in a lyophilized form. AMENDED CLAIMS

[received by the International Bureau on 10 June 1996 (10.06.96); original claims 1 and 7-34 amended; new claim 35 added; remaining claims unchanged (7 pages)]

1. A method of making a reagent vial for transferring fluids comprising: a. providing a tubular vial having an open end and a stopper securable in said open end; b. inserting a reagent containing a monoclonal antibody in the reagent vial prior to freeze drying; c. freeze drying said reagent; d. forming a vacuum for utilization in transferring between 25 and 2,000 microliters of a fluid; and e. securing said stopper into said tubular vial open end to close said tubular vial to maintain said vacuum for utilization in transferring_between 25 and 2,000 microliters of fluid into said tubular vial through said stopper.

2. The method of claim 1 wherein the monoclonal antibodies are CD4 and CD8.

5. The method as defined in claim 2 including providing said reagent transfer vial open end with screw threads, providing a screwable overcap for said vial and screwing said overcap over said stopper to prevent said stopper from being pulled from said closed vial.

6. The method as defined in claim 5 including adhering said stopper to said overcap to prevent said stopper from being pushed into said vial.

AMENDED SHEET (ARTICLE 19) 7. A method of transferring a fluid between two closed vials comprising: a. providing a closed reagent transfer vial containing a reagent comprising a freeze dried antibody and having an internal pressure less than atmospheric pressure; b. providing a closed sample vial with at least a first fluid of blood contained therein; c. piercing said closed sample vial with at least a first vent needle to vent said closed sample vial substantially to atmospheric pressure; d. piercing said closed sample vial with at least a first end of a substantially integral transfer needle spaced from said vent needle; and e. piercing said closed reagent transfer vial with at least a second end of said transfer needle to transfer between 25 to 2,000 microliters of a fluid aliquot from said closed sample vial to said reagent transfer vial by a negative pressure differential between said reagent transfer vial and sample vial.

8. The method as defined in claim 7 including providing a reservoir and venting said closed sample vial into said reservoir.

9. The method as defined in claim 8 including providing said reservoir with at least a hydrophobic portion for trapping aerosol or liquid or both in said reservoir while allowing air to pass through the hydrophobic portion and out of said reservoir.

10. The method as defined in claim 7 including providing a needle assembly including a pair of open tubes and inserting said closed sample vial into a first one of said tubes having said vent needle and a first end of said transfer needle therein and inserting said closed reagent transfer vial

AMENDED SHEET (ARTICLE 19) into the second one of said tubes having a second end of said transfer needle therein.

11. The method as defined in claim 10 including providing a reservoir in said needle assembly and venting said closed sample vial into said reservoir by inserting said vent needle into said closed sample vial prior to inserting said transfer needle therein.

12. The method as defined in claim 11 including providing said reservoir with at least a hydrophobic portion to for trapping aerosol or liquid or both in said reservoir while allowing air to pass through the hydrophobic portion and out of said reservoir.

13. The method as defined in claim 10 including identifying said tubes and said closed reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

14. The method as defined in claim 10 including providing said needle assembly with a physical stop to form a first vent position for said closed sample vial and said first tube and inserting said sample vial to said first vent position.

15. The method as defined in claim 14 including providing a reservoir in said needle assembly and venting said closed sample vial into said reservoir, said reservoir including at least a hydrophobic portion to trap fluid in said reservoir while allowing air to pass through the hydrophobic portion and out of said reservoir, and identifying said tubes and said reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

AMENDED SHEET (ARTICLE 19) 16. The method as defined in claim 14 including moving said physical stop from said first vent position after venting said closed sample vial to a second transfer position.

17. The method as defined in claim 16 including returning said physical stop to said first vent position to release said closed sample vial after said fluid aliquot is transferred.

18. The method as defined in claim 16 including providing said physical stop as a tab in said first vent position.

19. The method as defined in claim 7 including forming a vacuum in said closed reagent transfer vial to form said negative pressure differential between said reagent transfer vial and sample vial.

20. The method as defined in claim 19 including providing a reagent in said closed reagent transfer vial.

21. The method as defined in claim 20 including providing a plurality of microspheres having a predetermined monoclonal antibody bound thereto as at least a portion of said reagent.

22. The method as defined in claim 21 including providing said reagent in a lyophilized form.

23. A reagent vial made according to method claim 1.

24. A transfer device for transferring a fluid between a first closed sample vial containing said fluid and second closed reagent vial having a internal pressure less than atmospheric pressure comprising:

AMENDED SHEET (ARTICLE 19) a. a housing including a first substantially tubular portion having a vent needle enclosed in said tubular portion and extending to a first position for piercing said first closed sample vial when inserted into said first tubular portion to a first position to vent said firs closed vial, said first tubular portion further enclosing a substantially integral transfer needle having a first end extending to a second position removed from said first position for piecing said first closed sample vial after said vent needle in a second position, and b. said housing including a second tubular portion having a second end of said transfer needle enclosed therein and extending to a transfer position for piercing said second closed vial when inserted into said second tubular portion to said transfer position to transfer a fluid aliquot from said first to said second closed vial utilizing said pressure differential.

25. The device as defined in claim 24 which further comprises forming a reservoir in said second tubular portion into which said closed sample vial is vented.

26. The device as defined in claim 25 which further comprises providing said reservoir with hydrophobic means for trapping liquid or aerosol or both in said reservoir while allowing air to pass through the hydrophobic means and out of said reservoir and out of said second tubular portion.

27. The device as defined in claim 26 which further comprises providing a needle assembly formed in said second tubular portion, said tubular portions formed as a pair of opposed open tubes and said closed sample vial to be inserted into a first one of said tubes having said vent needle and a first end of said transfer needle therein and said closed reagent transfer vial to be inserted into the second one of said tubes having a second end of said transfer needle therein.

AMENDED SHEET (ARTICLE 19) 28. The device as defined in claim 27 which further comprises providing a reservoir formed in said needle assembly and said closed sample vial is vented into said reservoir by inserting said vent needle into said closed sample vial prior to inserting said transfer needle therein.

29. The device as defined in claim 28 which further comprises providing said reservoir with a hydrophobic means for trapping liquid or aerosol or both in said reservoir while allowing air to pass through the hydrophobic means and out of said reservoir and out of said second tube.

30. The device as defined in claim 27 which further comprises including a means for identifying said tubes and said reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

31. The device as defined in claim 27 which further comprises including said needle assembly having a physical stop to form a first vent position for said closed sample vial and said first tube.

32. The device as defined in claim 31 which further comprises including a reservoir in said needle assembly into which said closed sample vial is vented, said reservoir including hydrophobic means for trapping liquid or aerosol or both in said reservoir while allowing air to pass through the hydrophobic means and out of said reservoir, and means for identifying said tubes and said closed reagent transfer vial and closed sample vial to match only said closed sample vial with said first tube and said closed reagent transfer vial with said second tube.

33. The device as defined in claim 31 which further comprises including said physical stop being movable from said first vent position after venting said closed sample vial to a second transfer position.

AMENDED SHEET (ARTICLE 19) 34. The device as defined in claim 33 which further comprises including said physical stop being movable to said first vent position to release said closed sample vial after said fluid aliquot is transferred.

35. The device as defined in claim 33 which further comprises including said physical stop is formed as a tab in said first vent position.

AMENDED SHEET (ARTICLE 19)