Patent Publication Number: US-2020292541-A1

Title: Whole blood staining preparation cartridge and system

Description:
PRIORITY CLAIM 
     This patent application claims priority to U.S. Prov. Pat. Appl. No. 62/308,351 filed Mar. 15, 2016, the contents of which are hereby incorporated by reference as if fully set forth herein. 
    
    
     BACKGROUND 
     In clinical environments, there are often situations in which a patient requires specific white blood cell (“WBC”) counts to be performed as soon as possible, as the results directly impact downstream decisions. For example, a patient may be undergoing a 4- to 6-hour aphaeresis treatment to extract to hematopoietic progenitor cell antigen CD34 (“CD34”) white blood cells for subsequent re-injection. Just prior to starting aphaeresis, two hours into it, and at the end of treatment it is desirable to test the volumetric counts of the CD34 WBCs. There is currently no practical means for performing a rapid CD34 white blood cell count at or near the patient. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
       Preferred and alternative embodiments of the present invention are described in detail below with reference to the following drawing figures. 
         FIG. 1  is a front view of a cartridge according to an embodiment of the invention; 
         FIG. 2  is a side cross-sectional view of the cartridge of  FIG. 1 ; 
         FIG. 3  is a front cross-sectional view of the cartridge of  FIG. 1 : 
         FIG. 4  is  FIG. 2  is a rear view of the cartridge of  FIG. 1 : 
         FIGS. 5-8  illustrate an embodiment of a system for use with the cartridge of  FIG. 1 ; 
         FIGS. 9-10  illustrate a cartridge according to an alternative embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION 
     This patent application is intended to describe one or more embodiments of the present invention. It is to be understood that the use of absolute terms, such as “must,” “will,” and the like, as well as specific quantities, is to be construed as being applicable to one or more of such embodiments, but not necessarily to all such embodiments. As such, embodiments of the invention may omit, or include a modification of, one or more features or functionalities described in the context of such absolute terms. 
     Embodiments of the invention may be described in the general context of computer-executable instructions, such as program modules, being executed by a processing device having specialized functionality and/or by computer-readable media on which such instructions or modules can be stored. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer storage media including memory storage devices. 
     Embodiments of the invention may include or be implemented in a variety of computer readable media. Computer readable media can be any available media that can be accessed by a computer and includes both volatile and nonvolatile media, removable and non-removable media. By way of example, and not limitation, computer readable media may comprise computer storage media and communication media. Computer storage media include volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can accessed by computer. Communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term “modulated data signal” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer readable media. 
     According to one or more embodiments, the combination of software or computer-executable instructions with a computer-readable medium results in the creation of a machine or apparatus. Similarly, the execution of software or computer-executable instructions by a processing device results in the creation of a machine or apparatus, which may be distinguishable from the processing device, itself, according to an embodiment. 
     Correspondingly, it is to be understood that a computer-readable medium is transformed by storing software or computer-executable instructions thereon. Likewise, a processing device is transformed in the course of executing software or computer-executable instructions. Additionally, it is to be understood that a first set of data input to a processing device during, or otherwise in association with, the execution of software or computer-executable instructions by the processing device is transformed into a second set of data as a consequence of such execution. This second data set may subsequently be stored, displayed, or otherwise communicated. Such transformation, alluded to in each of the above examples, may be a consequence of, or otherwise involve, the physical alteration of portions of a computer-readable medium. Such transformation, alluded to in each of the above examples, may also be a consequence of, or otherwise involve, the physical alteration of, for example, the states of registers and/or counters associated with a processing device during execution of software or computer-executable instructions by the processing device. 
     As used herein, a process that is performed “automatically” may mean that the process is performed as a result of machine-executed instructions and does not, other than the establishment of user preferences, require manual effort. 
     An embodiment of the invention includes a whole-blood cell prep cartridge. This cartridge and corresponding system provides an automated means to prepare whole blood for subsequent white blood cell analysis, specifically using flow cytometry. The disclosed blood cartridge also can safely store fluorescent and biological reagents in the cartridge. In order to do this, an embodiment of the invention includes metabolized Mylar fluid pouches. The pouches can hold the required volumes, are liquid- and air-tight, and are opaque (which aids to protect the fluorescent dyes). The disclosed whole-blood cell prep cartridge allows a nurse or lab technician to simply and repeatably prepare a patient&#39;s whole blood to allow for subsequent volumetric counting using a flow cytometer. 
     As illustrated in  FIGS. 1-4 , an embodiment of the invention includes an automated, mesoscale fluidic prep cartridge  100  that includes all of the components necessary to fluorescently label the desired white blood cells for subsequent volumetric counting using a flow cytometer. Specifically, the cartridge  100  can accurately select a volume of blood from a surplus of blood. This ensures accurate downstream volumetric WBC counts. To accomplish this, an embodiment of the invention includes a rotating valve mechanism to select a volume of blood and make it available for subsequent mixing with a fluorescent dye mixture. Specifically, cartridge  100  includes a fluid (e.g., blood) reservoir  10 , which in a preferred embodiment has a volume of 100 μL, configured to receive a blood sample. A rotating first valve  19  contains a duct  22 , which may be a tube, having a first end  101  and a second end  102 . The first end  101  of the duct  22  can be positioned in fluid communication with the blood reservoir  10  by rotating first valve  19 . Cartridge  100  further includes an integrated hydrophobic vent/port  15 . As is discussed in greater detail herein, the hydrophobic vent technology, which may employ hydrophobic membranes, is used in multiple locations within the whole blood prep cartridge  100  to ensure that trapped air does not cause downstream volumetric count inaccuracies. By applying vacuum to port  15 , whole blood can be pulled (or, alternatively, driven via capillary forces) into the cartridge  100  and into duct  22  from reservoir  10  up to port  15  where such blood flow is stopped. The valve  19  may then be rotated 45 degrees from the vertical position (i.e., position in fluid communication with reservoir  10 ), the duct  22  having thereby collected a known volume of blood. This feature enables a user to obtain accurate counts by starting with a precise, and known, volume of whole blood (in an embodiment, 50 μL). 
     Cartridge  100  further includes a fluorescent liquid dye pouch  12  and an RBC lyse pouch  11 . In an embodiment, with the valve  19  still at 45 degrees from vertical, the fluorescent dye pouch  12  can be compressed by a piercing mechanism  17  ( FIG. 2 ). The fluid containing the fluorescent dye is thereby released and flows into a de-gassing chamber port  18  that contains a hydrophobic vent. This intermediate chamber  18  allows as much air as possible to be removed to help minimize counting volumetric counting errors. 
     A rotating second valve  21  includes a duct  103 , which may be a tube, having a first end  104  and a second end  105 . The first end  104  of the duct  103  can be positioned in fluid communication with the second end  102  of duct  22  by rotating first valve  19 . In an embodiment, the valve  19  may be rotated to a horizontal position and a vacuum can be applied to hydrophobic port  16  to draw the blood/dye mixture via duct  103  to a, preferably, 100 μL collection well  20  that is incorporated into the valve  21 . The blood/dye mixture may be allowed to incubate in well  20  for a predetermined amount of time to allow the dye to specifically bind to the WBCs of interest. 
     Once incubation of the blood/dye mixture is complete, pouch  11  filled with, in an embodiment, 900 μL of red blood cell lyse reagent is pierced, and the valve  21  is rotated such that first end  104  is in fluid communication with pouch  11  and second end  105  is in fluid communication with a mixing chamber  23 . By applying a vacuum to a hydrophobic port  13 , the combination of blood/dye mixture and lyse reagent are drawn into chamber  23  where the combination is stirred with, in an embodiment, a miniature magnetic stir bar and an external electric motor to eliminate as many red blood cells as possible. After mixing for a predetermined amount of time, the mixed sample can be extracted from cartridge  100  for volumetric flow cytometry analysis. In an embodiment, a user can insert the entire cartridge  100  into a custom flow cytometer that will automatically extract the prepared blood directly from the cartridge. 
     Cartridge  100  further comprises a housing  110  within which the blood reservoir  10 , rotatable valves  19 ,  21 , pouches  11 ,  12 , ports  13 ,  15 ,  16 ,  18  and mixing chamber  23  are disposed. 
     Referring now to  FIGS. 5-8 , an embodiment of a system  500  for use with the cartridge  100  is completely self-contained and has an integrated, fully programmable digital circuit board  510  with color touch display  2  and an analog circuit board  520 . It may further have a battery and a USB cable for charging and power. 
     An embodiment has five electric gear motors with encoders for position feedback. Two of the motors (one of which is illustrated as element  1 ) are used to pierce the liquid pouches  11 ,  12  in the cartridge  100  via mechanical cam  6  and a spring-loaded follower type mechanism  7 . More specifically, a spring acts to bias follower  7  and associated piston/cylinder  8  upwards against cam  6  that is connected to indexing gear motor  1 . When the gear motors  1  are activated, the cams  6  rotate causing the follower  7  and piston  8  to compress the liquid-containing pouches  11 ,  12 . Two other motors are used to rotate the fluid valves  19 ,  21 . One motor  4  is used (optionally) to provide an automated cartridge tray opening/closing motion. The system  500  may also have a sixth electric gear motor with no encoder for turning the magnetic stir bar inside the mixing chamber  23 . 
     The pneumatic structure  9  of the system  500  has three spring loaded “pistons” with O-rings that apply a controlled force on the cartridge  100  once the tray  5  is loaded. Each piston is connected to the pneumatic system via a flexible tube. An embodiment uses vacuum only, but positive pressure can also be used to move fluids within the cartridge  100 . The pneumatic structure  9  has a pump, a pressure transducer, a vacuum reservoir (optional), and four solenoid vales. Three solenoid values are used to direct the vacuum source to each of the three pistons. The fourth solenoid valve may be used to vent the system. 
       FIG. 8  depicts a schematic for the pneumatic system  9  that can be used in the system that interfaces with the cartridge  100 . System  9  can generate the vacuum pressure necessary to move the fluids around inside the cartridge  100 . 
     Vacuum pressure is directed to the cartridge  100  one port  13 ,  15 ,  16  at a time via activation of one of the three solenoid valves shown. The remainder of the system may comprise one or more combinations of a vacuum pump, a reservoir, a pressure transducer to measure the generated pressure, and a fourth solenoid to vent the cartridge  100  when needed. 
     An alternative embodiment cartridge  900 , illustrated in  FIGS. 9-10 , includes a magnetic microbead-based cartridge. As discussed below, cartridge  900  employs many of the same concepts/structures as that of cartridge  100 , but incorporates magnetic micro particles to isolate the cells using an externally applied permanent magnet and multiple wash steps. 
     In an embodiment, a first valve  901  is placed in a vertical position such that a duct  902  is in fluid communication with a blood reservoir  903 . Vacuum is applied to hydrophobic port  904  until blood from reservoir  903  reaches port  904 . Valve  901  may then be rotated 45 degrees. Reagent pouch  905  containing fluorescent dye and magnetic particles is pierced and compressed, and this reagent flows into the reagent/de-gassing chamber port  906  through which air may be released. Mechanical pressure on pouch  905  is maintained as valve  901  is rotated to a horizontal position such that duct  902  is in fluid communication with port  906 . 
     Valve  907  is placed in a horizontal position such that a duct  908  is in fluid communication with duct  902 . Vacuum is applied to a port  909  of valve  907 , and the blood/reagent mixture are pulled into an incubation well  910 . Once fluid contacts the hydrophobic port  906 , vacuum is discontinued and port  906  is closed. Valve  901  may then be rotated 45 degrees. The blood/reagent mixture is then allowed to incubate for a predetermined amount of time. 
     A permanent magnet (not shown) is moved into position below well  910  for a predetermined amount of time. Consequently, targeted cells are pulled to the bottom of the well  910 . Valve  907  is rotated so as to fluidly connect a wash pouch  911  containing an appropriate wash fluid to a waste well  912  having a port  913 . Wash pouch  911  may be pierced and compressed. Vacuum is applied to port  913  until fluid contact therewith, and then port  913  is closed. 
     The permanent magnet is removed, and valve  907  is rotated to connect wash pouch  914  with a collection well  915  having a port  916 . Wash pouch  914  is pierced, and vacuum is applied to port  916  of collection well  915  until fluid reaches port  916 . Cells of interest are resuspended and moved into collection well  915 . Cells are then ready for counting. 
     From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.