CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of priority pursuant to 35 U.S.C. § 119(e) of U.S.

provisional patent application No. 62/347,447 entitled "Adjustable Control Of Cell Compositions During Centrifugation," filed on June 8, 2016, which is hereby incorporated by reference in its entirety.

BACKGROUND

Field

[002] The present disclosure relates generally to methods for processing and preparing specific cell populations from whole blood, bone marrow, cord blood, and apheresis blood products.

Aspects described herein identify the cellular components present in the whole blood, bone marrow, cord blood, and apheresis blood products during centrifugation and redirect desired components into containers in preprogrammed amounts so as to generate a user-defined composition having desired amounts of particular components from the whole blood, bone marrow, cord blood, and apheresis blood products. Some systems comprise a sterile,

functionally closed bag set, and a portable battery powered electromechanical device, which are configured to process fluids under centrifugation.

Description of the Related Art

[003] Devices and methods of separating cell types have been described, for example, in U.S. Patent Nos. 9,050,422, 8,167,139, 8,066,127, and 7,241,281. Such devices utilize centrifugation to stratify the fluid sample into layers and then separate and transfer fluids into separate containers.

SUMMARY

[004] The devices, systems, and methods disclosed herein have several features, no single one of which is solely responsible for its desirable attributes. Without limiting the scope as expressed by the claims that follow, its more prominent features will now be discussed briefly. After considering this discussion, and particularly after reading the section entitled "Detailed Description" one will understand how the features of the devices, systems, and methods provide several advantages over traditional devices, systems, and methods.

[005] Disclosed herein are systems, devices, and methods for separating a heterogeneous sample into separate amounts of component fractions. In some embodiments, the sample may be a biological sample selected from one or more of whole blood, bone marrow, cord blood, and apheresis blood products. In many embodiments, the component fractions may be selected from one or more of a red blood cell (RBC) fraction, stem cell fraction, white blood cell fraction, platelet fraction, plasma fraction, etc. In many cases, the fractions may be differentiated by centrifugation of the sample. This may result in stratification of the sample into layers, for example a layer substantially comprising RBCs, a layer substantially comprising white blood cells and platelets, and a layer substantially comprising plasma. These layers may be referred to as the RBC layer, the Buffy Coat Layer, and the Plasma Layer. In most cases, stratification results in the RBC layer on the bottom, the Plasma Layer on the top, and the Buffy Coat Layer between the other two layers. After separation, white blood cells and platelets may fractionate into a fraction referred to as the buffy coat layer.

[006] The disclosed systems, devices, and methods are configured to differentiate the different layers/fractions after stratification (by, for example, centrifugation), and separate the

fractions/layers from a first container into two or more receiving containers. The first container may be referred to as a Sample container. In many cases, the relative amounts and/or actual amounts (i.e. volumes) of the different fractions/layers are unknown prior to the separation. In most embodiments, the majority of the RBC layer/fraction is separated into a second container (or RBC container), until the device and/or system determines that a known amount or volume of the RBC layer/fraction remains in the sample container. This known volume (Volume 3 or V ₃ ) is defined by the design/configuration of the Sample container and location(s) of an optical detector/emitter designed to interrogate the sample. V ₃ is the sum of two sub-volumes, V ₂ and

Vi, that are defined by the volume of sample interrogated and the volume of sample between the optical detector/emitter and a valve. The valve is configured to direct sample into one of the two or more receiving containers, for example a second container, which may be referred to as a RBC container. A third container, for receiving sample, may be referred to as a Cell container. [007] The systems, devices, and methods are configured to determine a flow rate for sample transferred out of the Sample container. In most embodiments, the system is configured to measure the time required to transfer the RBC layer through the area interrogated by the optical detector/emitter. Because this area defines a known volume of sample, and the system has measured the time required for the layer to traverse this area, a flow rate can be calculated

(known volume ÷ measured time = calculated flowrate) for transfer of sample into the one or more containers. This calculated flow rate can then be used to determine the volume of subsequent sample transfers, for example transfers of the RBC layer, the Buffy Coat layer, and/or the Plasma Layer, from the Sample container to the two or more receiving containers, for example the RBC container and the Cell container.

[008] The disclosed systems, devices, and methods allow a user to pre-select a collected volume of the stratified layers. In some embodiments, the user may collect all of, or a set volume of the Buffy Coat layer and/or a set volume of the RBC layer. In many embodiments, the user may program the disclosed systems and/or devices to collect a specific amount of RBC and/or Buffy Coat layer.

[009] In some implementations, a method of accumulating a desired volume from a sample during centrifugation may include one or more of the following. The method may include centrifuging the sample in a first container. At least a portion of the sample may be directed from the first container to a second container during centrifugation. The method may also include calculating a flow rate of the portion directed from the first container to the second container during centrifugation. The method may also include directing a desired volume from the first container to the second container using the calculated flow rate during centrifugation.

[0010] At least a portion of the sample from the first container may be directed to the second container during centrifugation by opening a valve positioned between the first container and the second container during centrifugation. Calculating the flow rate may include measuring a time interval that it takes the portion to pass through a known volume of the first container during centrifugation. In some aspects, measuring a time interval that it takes the portion to pass through a known volume of the first container during centrifugation includes monitoring an intensity of a light passing through the sample. Light passing through the sample may be generated by one or more light sources, in one example the light source is a Light Emitting Diodes (LEDs).

[0011] The method may include calculating a second flow rate that is different from the first flow rate. The second flow rate may be calculated at least in part from detecting a force imparted on a third container. Directing a desired volume from the first container using the calculating flow rate during centrifugation may include opening a valve positioned between the first container and the second container during centrifugation for a calculated time interval. Directing a desired volume from the first container using the calculating flow rate during centrifugation may include opening a valve positioned between the first container and a third container during centrifugation for a calculated time interval.

[0012] In some implementations, a method of separating cells during centrifugation includes one or more of the following. The method may include stratifying a sample containing cellular material in a first container by centrifugation into at least a first layer and a second layer disposed on top of the first layer. The method may also include directing at least a portion of the first layer from the first container to a second container during centrifugation. The method may also include measuring a time interval that it takes a portion of the first layer to pass through a known volume of the first container during centrifugation. The method may also include calculating a flow rate of the first layer based at least in part on the measured time interval and the known volume of the sample container based on sensor positioning during centrifugation. The method may also include directing a volume of the first layer from the first container based at least in part on the flow rate calculated during centrifugation such that a pre- selected volume of the first layer remains in the first container.

[0013] In some implementations, a device for recovering a target volume containing a preselected volume of an RBC layer from a blood or bone marrow sample during centrifugation includes a housing configured for use in a centrifuge. The device may include at least a sample container, a RBC container, and a cell container disposed within the housing. A first flow path may connect the sample container to the RBC container. A second flow path may connect the sample container to the cell container. A motor-controlled valve may be disposed in the first and second flow path. The motor-controlled valve may be configured to open and close the first flow path and configured to open and close the second flow path. A light source may be configured to emit light through the sample container. An optical detector may be configured to detect at least a portion of the light emitted by the light source. An accelerometer and a load sensor may be configured to determine a force imparted on the cell container. Circuitry may be operably coupled to at least the motor-controlled valve, the optical detector, the accelerometer, and the load sensor. The circuitry may be configured to determine a first rate of flow of a fluid passing through the first flow path at least in part by input received from the optical sensor. The circuitry may be further configured to control the motor-controlled valve such that a first volume of fluid passes through the first flow path based at least in part on the determined first rate of flow. The circuitry may be further configured to determine a second rate of flow of a fluid passing through the second flow path at least based in part by input received from the accelerometer and the load sensor. The circuitry may be further configured to control the motor-controlled valve such that a second volume of fluid passes through the second flow path based at least in part on the determined second rate of flow.

[0014] In some implementations, a method of separating cells during centrifugation includes one or more of the following. The method may include stratifying a sample in a sample container with centrifugation into at least an RBC layer, a buffy coat layer, and a plasma layer. The sample container may be in selective fluid communication with an RBC container and a cell container. The selective fluid communication may be controlled by a valve. The method may also include transferring at least a portion of the RBC layer from the sample container to the RBC container. In some aspects, substantially the entire RBC layer is removed. In some aspects, the entire RBC layer is removed. The method may also include determining a first flow rate of the RBC layer from the sample container to the RBC container. The method may also include determining a first transfer time based at least in part on the first flow rate. The method may also include transferring a first volume of the RBC layer from the sample container to the RBC container based at least in part on the first transfer time. The method may also include transferring the remaining RBC layer and at least a portion of the buffy coat layer from the sample container to the cell container. The method may also include determining a second flow rate of the remaining RBC layer and the least a portion of the buffy coat layer from the sample container to the cell container. The method may also include determining a second transfer time based at least in part on the second flow rate. The method may also include transferring a second volume from the sample container to the cell container based at least in part on the second transfer time.

[0015] In some implementations, a device configured to control the flow of fluid from a first container to a second container during centrifugation includes a flow path. A valve may be positioned within the flow path. The valve may be configured to move from a closed position to at least a partially open position and back to the closed position for a time interval. At least one light source may be configured to emit light through the flow path. At least one optical sensor may be configured to detect at least a portion of the emitted light. Circuitry may be coupled at least to the valve and the optical sensor, the circuitry configured to adjust the time interval and the partially open position of the valve least based at least in part on the portion of the emitted light detected by the optical sensor. The circuitry may be configured to adjust the time interval and the partially open position of the valve least based at least in part on the rate of change of the light detected by the optical sensor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following is a brief description of each of the drawings. From figure to figure, the same reference numerals have been used to designate the same components of an illustrated embodiment. The drawings disclose illustrative embodiments and particularly illustrative implementations in the context of separating the components of whole blood. They do not set forth all embodiments. Other embodiments may be used in addition to or instead. Conversely, some embodiments may be practiced without all of the details that are disclosed.

[0017] It is to be noted that the figures provided herein are not drawn to any particular proportion or scale, and that many variations can be made to the illustrated embodiments. Brief

introductions to some of the features, which are common to the described embodiments, are now described.

[0018] FIG. 1 is a two-dimensional lay-out of an embodiment of the flexible bag set of the invention.

[0019] FIGs. 2A and 2B show a semi-rigid bag set in perspective and as it would be inserted into the processing device, FIG. 2B, and in stretched out form to mimic the view in FIG. 1. [0020] FIG. 3A is a schematic diagram showing one fluid path of the metering valve of the bag set of FIG. lA and IB

[0021] FIG. 3B is a schematic diagram showing a second fluid path of the metering valve shown in FIG. 3A.

[0022] FIG. 3C is a schematic diagram showing a third fluid path of the metering valve shown in FIG. 3A.

[0023] FIG. 4A is a schematic diagram showing one fluid path of an embodiment of the metering valve different from the embodiment of FIGS. 3A-3C.

[0024] FIG. 4B is a schematic diagram showing a second fluid path of the metering valve shown in FIG. 4A.

[0025] FIG 4C is a schematic diagram showing one fluid path of an embodiment of the metering valve different from the embodiment of FIGS. 3A - 3C

[0026] FIG 4D is a schematic diagram showing a second fluid path of the metering valve shown in FIG 4C.

[0027] FIG. 5 is a perspective view of an embodiment of a flexible bagset sample processing device.

[0028] FIG 6 is a perspective view of an embodiment of a semi-rigid bagset sample processing device.

[0029] FIG. 7 is a perspective view of the processing device of FIG. 5.

[0030] FIG 8 is a partially exploded view of the processing device of FIG. 6.

[0031] FIG. 9 is a back view of the processing device of FIG. 5.

[0032] FIG. 10 is a perspective view of an embodiment of the base plate of the processing device of FIG. 5, showing the components.

[0033] FIG. 11 is an exploded view of the processing device of FIG. 5.

[0034] FIG. 12 is a perspective view of the flexible bag set of FIG. 1 placed in the processing device of FIG. 5.

[0035] FIG. 13 is a perspective view of the flexible bag set and processing device shown in FIG. 12 With hinged door closed.

[0036] FIG. 14 is a perspective view of the semi-rigid bagset of FIGs. 2A and 2B placed in the processing device of FIG. 6. [0037] FIG. 15 is a perspective view of the flexible bag set and processing device of FIG. 12 in a centrifuge bucket.

[0038] FIG 16 is a perspective view of the semi-rigid bag set and processing device of FIG 14 in a centrifuge bucket.

[0039] FIG. 17 is a partially exploded view showing the loaded centrifuge bucket of FIG. 16 and a centrifuge.

[0040] FIG. 18 is a block diagram of the inputs and outputs of the microcontroller of the processing device of FIG. 5.

[0041] FIG. 19A is a top down view of a device that is substantially similar to the processing device of FIGS. 5-15 configured to calculate the flow rate of the sample leaving a processing bag or sample container.

[0042] FIG. 19B is a cross-sectional view of the device of FIG. 19A taken about the line A-A.

[0043] FIG. 20 is an enlarged view of FIG. 19B. As shown, the device is loaded with unprocessed blood in the sample container and the multiport valve set to an OFF flow position prior to centrifugation.

[0044] FIG. 21A is a cross sectional view of the multiport valve of FIG. 19B. As shown, the valve is in a "PARTIALLY OPEN" RBC position allowing fluid transfer from the sample container to the RBC container.

[0045] FIG. 21B is a cross sectional view of the multiport valve of FIG 19B. As shown, the valve is in a "PARTIALLY OPEN" RBC position allowing fluid transfer from the sample container to the RBC container.

[0046] FIG. 21C as shown valve is in "PARTIALLY OPEN" cell position allowing fluid transfer from the sample container to the cell container.

[0047] FIG. 21D as shown valve is in the "PARTIALLY OPEN" cell position allowing fluid transfer from the sample container to the cell container.

[0048] FIG. 22 shows the stratified blood in the first container under high speed centrifugation. As shown, the valve is set to the "RBC OFF" - no flow position.

[0049] FIG. 23 shows the RBC layer of the stratified blood sample is being directed to the RBC container during centrifugation. [0050] FIG. 24 shows the buffy layer is just about to cross the optical aperture area. Here, the optical transmittance between the RBC layer and buffy coat layer can just start to be detected.

[0051] FIG. 25 shows the buffy coat layer has crossed into the optical aperture area.

[0052] FIG. 26 shows the RBC layer has completely exited the optical aperture area.

[0053] FIG. 27 shows the buffy coat layer has passed below the optical aperture area and the valve is positioned in the Cell Collection ON position such that fluid may flow from the sample container to the cell container during centrifugation.

[0054] FIG. 28 shows the RBC layer and the buffy coat layer have moved into the cell container during centrifugation.

[0055] FIG. 29 A is a combination of graphical waveforms representing the multiport valve position, the light transmittance, and the volume in the cell container according to one implementation using animal blood. In some implementations, certain time intervals may be designated as Stages A-F.

[0056] FIG. 29B is a combination of graphical waveforms presenting the multiport valve position, the light transmittance, and volume in the cell container according to one

implementation using peripheral blood.

[0057] FIG. 30 is an expanded waveform plot of Stage A when the RBC layer is first transferred from the sample container to the RBC container until an optical trigger is reached.

[0058] FIG. 31 is an expanded waveform plot of Stage B when the buffy coat layer passes through the optical aperture area.

[0059] FIG. 32 is an expanded waveform plot of Stage C when the optical transmittance has saturated and the device transitions from Stage B to Stage D.

[0060] FIG. 33 is an expanded waveform plot of Stage D when a pre-set volume of the RBC layer is transferred from the sample container to the RBC container.

[0061] FIG. 34 is an expanded waveform plot of Stage E when a portion of the remaining volume in the sample container is transferred to the cell container.

[0062] FIG. 35 is an expanded waveform plot of Stage F when a portion of the remaining volume in the sample container is transferred to the cell container until the final volume within the cell container is reached.

[0063] FIG. 36 shows the valve in a "BUFFY OFF" no flow position. [0064] FIGs. 37A, 37B, and 37C show a flow chart of a method for separating cells according to one embodiment. FIG. 37 shows Initial RBC Transfer of Stage A to Control of Transfer of RBC, Stage B. FIG. 37B shows Light Transmittance inquiry to cycle counting of Stage E. FIG. 37C shows Stage C, Calculating viscosity and RBC Depletion, to reaching Target Volume.

DETAILED DESCRIPTION

[0065] The following invention generally relates to methods for processing and recovering specific cell populations from whole blood, bone marrow, cord blood, or apheresis blood products, e.g., from humans or other mammals, such as dogs, cats, horses, or domestic or livestock animals. More specifically, aspects described herein sort specific types of fluid content and the components therein (e.g., cells and/or plasma) during centrifugation and redirect specific volumes and/or components in the whole blood, bone marrow, cord blood, apheresis blood products, as defined by an end-user, e.g., in a pre-programmed fashion. While previous devices were capable of sorting fluids (e.g. RBC-containing fluid) from other fluids (e.g. WBC- containing cellular fluid and/or plasma) in a bone marrow sample, e.g., during centrifugation, embodiments described herein accurately direct specific volumes of fluid and/or components present in the whole blood, bone marrow, cord blood, apheresis blood products (e.g., red blood cells and/or stem cells) to a desired container in user- specified amounts so as to allow for the generation of user-defined compositions containing desired volumes of fluid, at desired flow- rates and/or containing desired cellular components. Moreover, rather than rely on a set preprogramed routine, some alternatives allow for the dynamic or manual adjustment of the time intervals that particular valves open or close or remain opened or closed so as to allow for incremental partition of components present in an unrefined sample and the direction of specified flows of these components during centrifugation into one or more collection containers, which allows the user to generate a unique mixture of components from whole blood, bone marrow, cord blood, apheresis blood products at desired volumes and/or viscosities. Alternatives described herein, for example, estimate and/or calculate the flow rate of the RBC-containing fluid and/or stem cell-containing fluid and/or plasma in order to accurately deliver desired volumes of such fluids from the sample to the desired location (e.g., container). Because flow rates vary across samples, the flow rate may be specifically determined for each unique sample such that the desired cells may be removed and/or sorted more accurately. In addition, alternatives described herein provide flexibility to the end user by allowing the user to select the volumes of cells and/or fluid that are sent to a desired container. Thus, the final collected volume of each type of fluid from the sample may be adjusted to result in, for example, a final collected volume having a desired or pre-selected hematocrit and/or viscosity. In some aspects, the overall processing time is also reduced. In some aspects, only a single LED and a single light detector are utilized - thus reducing complexity and expense. In other embodiments, light may be directed to one or more detectors from one or more LEDs. In some embodiments, the LEDs may be able to emit light of one or more wavelengths.

[0066] In some implementations, the system/device includes a sterile, functionally closed bag set, and a portable battery powered electromechanical device both designed to process fluids in a centrifugal field.

[0067] In the context of stem cell partitioning, redirection, and/or removal, it may be desirable to collect a volume that has as many stem cells as possible in the volume while having as few of red blood cells (RBCs) as possible in the same collected volume. However, it may be also desirable to recover a relatively precise volume of stem cells and a relatively precise volume of RBCs and/or RBC containing fluids which make up the total collected volume. For example, it may be desirable to collect a volume of RBCs and/or RBC containing fluid such that the end sample volume has a given or pre-selected hematocrit and/or viscosity. Such a desired hematocrit and/or viscosity may be pre-selected by an end user. Thus, there is a need for new devices and collection methods having enhanced processing capabilities.

[0068] One example of enhanced processing requirements pertains to blood type mismatches in allogeneic transplantation. In brief, improper matching between a bone marrow recipient and a donor may be associated with potentially life-threatening adverse events. Furthermore, clinical outcomes in ABO (i.e. A, B, AB, and O blood types) incompatible transplants are considered to be inferior to better matched procedures. Incompatibility of ABO-blood groups is involved to some degree in nearly one-half of all allogeneic hematopoietic cell transplantations. In particular, pediatric patients are at higher risk of adverse events due to the lower ratio of recipient weight (correlating to lower blood volume) to the quantity of RBC infused as part of the transplant dose. As such, alternatives described herein may be used to reliably achieve a defined reduction of RBCs and neutrophils in whole blood, bone marrow, cord blood, and other apheresis products (e.g., from a human). In some implementations, existing hardware/software may be at least partially utilized to leverage existing hardware.

[0069] Some implementations involve a method to detect, calculate, control, and/or process specific types of cellular content from peripheral blood, bone marrow, cord blood, and other apheresis products (e.g., from a human) during centrifugation with parameters input by an end- user. The method may utilize a system that includes two separate but interacting assemblies; (1) a processing bag set and (2) a microprocessor controlled electromechanical device. The processing bag set may include a multiport valve connected to three separate and sterile disposable flexible or semi-ridged containers: (1) a starting volume sample container, (2) a RBC container, and (3) a cell collection container. A multiport valve may be connected to all three containers which allows for the transfer of fluid through the multiport valve to at least one of the three containers during processing. The multiport valve may be controlled by a processing device which autonomously detects and separates the blood components during centrifugation.

[0070] In some implementations, a device is configured to calculate and record the fluid flow rate of the RBC layer during the transfer of the RBC layer from the sample container to the RBC container. In some implementations, the device is configured to calculate and record the fluid flow rate of the WBC stem/progenitor cell layer during a transfer of the WBC stem/progenitor cell layer to the cell collection container. In some aspects, the device is configured to calculate an optimum valve open time and/or position based at least in part on an optical sensor response during fluid transfer and/or based at least in part on the fluid flow rates calculated during transfer. In some embodiments, the fluid flow rate calculated during transfer may range from about 10 μΏ to about 1000 μΏ$, for example per second transfer volumes greater than about 20 μί, 30 μΐ,, 40 μΐ,, 50 μί, 60 μί, 70 μί, 80 μί, 90 μί, 100 μί, 120 μί, 140 μί, 160 μί, 180 μί, 2000 μί, 250 μί, 300 μί, 350 μί, 400 μί, 450 μί, 500 μί, 550 μί, 600 μί, 650 μί, 700 μί, 750 μί, 800 μί, 850 μί, 900 μί, or 950 μί, 1000 μί, 1100 μί, 1200 μί, 1300 μί, 1400 μί, 1500 μί, 1600 μί, 1700 μί, 1800 μί, or 1900 μί, and less than about 2000 μί, 1900 μί, 1800 μί, 1700 μί, 1600 μί, 1500 μί, 1400 μί, 1300 μί, 1200 μί, 1100 μί, 1000 μί, 950 μί, 900 μί, 850 μί, 800 μί, 750 μί, 700 μί, 650 μί, 600 μί, 550 μί, 500 μί, 450 μί, 400 μί, 450 μί, 300 μΐ,, 350 μΐ,, 250 μί, 200 μί, 190 μί, 180 μί, 170 μί, 160 μί, 150 μί, 140 μί, 130 μί, 120 μί, 110 μί, 100 μί, 90 μί, 80 μί, 70 μί, 60 μί, 50 μί, 30 μί, 40 μί, or 20 μί.

[0071] In some aspects, the device is configured to assess the cellular composition of the sample as the sample is passed or directed into/through one or more light sources that emit and/or detect various wavelengths of light. For example, the wavelength of emitted light may be between about 200nm and 1200nm. In some embodiments the emitted light may be greater than about 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, or 1100 nm, and less than about 1250 nm, 1200 nm, 1150 nm, 1100 nm, 1050 nm, 1000 nm, 950 nm, 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm,

250 nm, 200 nm, and 150 nm. In some embodiments the emitted light may have one or more peak values.

[0072] In some implementations, the device is configured to manage and/or control RBC rich stratum fluid transfers over fixed volumes. In some aspects, the device is configured to control the RBC content (hematocrit) in the final product contained in the cell collection container.

[0073] Some implementations include a method that begins with transferring human whole blood, bone marrow, cord blood, or apheresis product volume into the sample processing container where the multiport valve is set to a closed (no fluid transfer) position. The method may continue by inserting the bag set into a processing device and placed into a centrifuge bucket. The method may continue by spinning the device at high speed to centrifuge the sample and stratify the fluid based on density, size of the cells, and starting volume into, for example, a RBC layer, a stem cellAVBC layer, and plasma layer using sufficient centrifugal force. Once the sample is stratified, the method may include reducing the centrifuge speed. The method may continue by monitoring, calculating, and controlling fluid transfers through the multiport valve in order to separate RBC and stem cellAVBC fluids to each designated container as desired.

[0074] In some implementations, the device is configured to calculate the amount of valve open time or closure time needed to transfer a desired volume of RBC-containing fluids to a RBC container. In some aspects, the device is configured to adjust the valve opening position to actively control RBC fluid transfers in real time. In some aspects, the device is configured to accept and processes to a final target RBC volume that is set by the end-user. Similarly, in some aspects, the device is configured to accept and process to achieve a final target WBC stem cell and/or progenitor cell volume that is set by the end-user. In some aspects, the device is configured to optimally reduce the amount of time required to transfer a target volume of WBCs, stem cells and/or progenitor cells to a cell collection container. In some aspects, the device is configured to calculate and control a plurality of low volume material transfers to reach a final target volume.

[0075] For example, an end user may wish to collect 21 ml from a whole blood sample of 100 ml. The end user may desire that the 21 ml that is collected from the sample includes 0.5 ml of the RBC layer with the remaining 20.5 ml containing the buffy coat layer and potentially a portion of the plasma layer. The user may enter the target sample volume and the volume of the

RBC layer that should be included in the target sample volume. The volume of the RBC layer may be expressed as a volume, percentage, hematocrit, or viscosity. During centrifugation, the device may stratify the sample and direct all but 0.5 ml of the RBC layer to the RBC container. The device may then transfer the remaining 0.5 ml of the RBC layer to the cell container along with 1 ml of buffy coat and any necessary plasma layer until the final volume of 21 ml in the cell container is reached.

[0076] Some implementations include a method for calculating a RBC fluid flow rate based on a predetermined volume and an optical sensor response as the RBC fluid is being transferred from the sample container to the RBC container. The method may begin after the sample fluid has been stratified with a sufficient g force. The method may include separating an RBC layer from a buffy coat layer. In some aspects, the method may be implemented using a microcontroller or other similar circuitry to continually monitor the optical intensity of a beam of light directed through the sample container during the RBC fluid transfer from the sample container to the RBC container. In some aspects, changes in the optical sensor response are detected when the RBC-buffy transition is nearing the upper boundary of the optical aperture area. The method may also include a series of rapid fluid transfers followed by one or more controlled transfers based at least in part on changes in the optical response. In some aspects, the RBC fluid transfer time is accumulated during each transfer and change in optical transmittance. In some aspects, the fluid transfer time elapsed is saved by, for example, the microcontroller until the optical transmittance has reached a steady state. Once the optical transitions reach steady state the microcontroller may be configured to calculate a RBC flow rate based on the fixed volume across the optical sensor aperture and the total transfer time accumulated while the RBC fluid was being transferred. This flow rate may be retained and recorded by, for example, the device microcontroller for additional processing and data analysis.

[0077] Some implementations include a method to selectively reduce the amount of RBC content in a stem cell and/or progenitor cell container based on a calculated RBC flow rate and transfer time of the RBC fluid from the sample container to the RBC container. In some aspects, a target RBC volume in the WBC/stem cell container is pre-programmed or input by an end-user prior to processing the sample. In some aspects, the extraction of the RBC-containing fluid volume begins immediately after the boundary between the RBC layer and buffy coat layer has traversed the optical aperture and the volume from this point to the valve, which is a known volume, is filled with RBC cellular content. In some aspects, the fluid flow rate of the RBC-containing fluid from the sample container to the RBC container is used, at least in part, to determine the length of time the valve between the sample container and the RBC container is opened.

[0078] In some aspects, a run-time calculated RBC flow rate is used to engage a series of valve movements to transfer a desired amount of RBC fluid into the RBC container. In some aspects, the series of valve movements is determined at least in part on calculating and adjusting the amount of valve fluid transfer time that is needed to move the desired amount of RBC fluid into the RBC container. In some aspects, the measuring and/or calculating is performed in real time. In some aspects, one or more steps of the method are performed at least in part by an embedded microcontroller or other circuitry.

[0079] Some implementations include a method to calculate a buffy coat flow rate based at least in part on the amount of transfer time and volume collected by the processing device. Such a method may begin after the RBC transfer is complete and the remaining volume in the processing bag consists of some RBCs, WBC stem cells, and plasma. In some aspects, a microcontroller or other circuitry controls a series of short fluid transfers from the processing container to the cell collection container over a known time period. In some aspects, the microcontroller simultaneously monitors a force sensor and accelerometers to calculate the amount of fluid transferred to the WBC and/or stem cell and/or progenitor cell bag and/or to calculate a flow rate. The flow rate may be used by the microcontroller to dynamically adjust the valve fluid transfer time period such that the WBC and/or stem cell and/or progenitor cell- containing fluid is captured as quickly as possible without under or overshooting a preset target WBC and/or stem cell and/or progenitor cell volume.

[0080] In some implementations, a system for depleting, sorting and/or redirecting a selected volume of red blood cells and/or other cells (e.g., stem cells and/or progenitor cells) from a sample of human peripheral blood, cord blood, bone marrow or apheresis products includes at least three containers. The containers may be bags. The containers may be supplied as a blood bag set. The at least three containers may include a sample container, RBC container, and a cell container. The at least three containers may be configured for use in a centrifuge. The sample container may be in fluid communication with the RBC container and the cell container. The containers may each be configured to fit within a housing that is configured to be inserted into a centrifuge.

[0081] The system may also include at least one valve configured to selectively control the flow of fluid from the sample container to the RBC container and /or the cell container during centrifugation. A motor may be coupled to the valve to control the valve position. In some aspects the motor is operatively coupled to the valve when the containers are placed within the housing.

[0082] The system may also include one or more accelerometers. The one or more

accelerometers may be configured to determine the g force during centrifugation.

[0083] The system may also include one or more electromagnetic wave sources. The electromagnetic wave sources may be light sources. The light sources may be light emitting diodes. The one or more light sources may be configured to emit one or more wavelengths of light through the sample container.

[0084] The system may also include one or more electromagnetic wave detectors. The electromagnetic wave detectors may be optical sensors.

[0085] The system may include circuitry. The circuitry may include one or more of a microprocessor and a storage device. In many embodiments the circuitry may be operatively coupled to one or more input and output devices, for example an actuator, a motor controlled valve, a motor, an accelerometer, a load sensor, a light source, and a light detector. The circuitry may comprise one or more microcontrollers. [0086] In some aspects, the circuitry may be configured to receive input from at least one accelerometer. The circuitry may be configured to monitor the input from the accelerometer and determine when sufficient g forces have been applied to a sample during centrifugation and/or for a sufficient amount of time. That is to say, the circuitry and accelerometer may be capable of determining when the sample is sufficiently stratified in the sample container during

centrifugation of the sample container. In some aspects, the stratified sample includes a RBC layer, a white blood cell layer (e.g. a buffy coat layer containing granulocytes, stem cells, progenitor cells, and platelets), and a plasma layer.

[0087] In some aspects, after the circuitry determines that the sample is sufficiently stratified, the circuitry may instruct the end-user to verify the amount of g force used. Fluid from the sample container may be directed to one or both of the RBC container and/or the cell container during g forces that are less than the g forces required to sufficiently stratify the sample.

[0088] In general, the stratified sample includes a bottom RBC layer, a middle white blood cell layer containing granulocytes, mono-nuclear cells, stem cells, progenitor cells, and platelet (e.g. a buffy coat layer), and an upper plasma layer. It is to be understood that the bottom RBC layer is the densest layer and primarily contains RBCs. The RBC layer may also contain other cell types and/or fluids. The middle layer buffy coat layer is of intermediate density and primarily contains WBCs, stem cells, and progenitor cells and some platelets. The buffy coat layer may also contain other cell types and/or fluids. The upper plasma layer is the least dense layer and is primarily plasma with some additional platelets. The plasma layer may also contain other cell types/and or fluids. The system may be configured to transfer / deliver at least a portion of the bottom RBC layer from the sample container to one of the RBC container or the cell container. The system may also be configured to transfer / deliver at least a portion of the buffy coat layer to the cell container.

[0089] In some aspects, circuitry is configured to cause the motor to adjust the valve such that a fluid path between the sample container and the RBC container is opened. In this way, the lower RBC fluid layer may move from the sample container to the RBC container during

centrifugation. In some aspects, the circuitry/motor causes the valve to open and to close after a short time interval. In some aspects, the circuitry/motor causes the valve to open and to close a plurality of times. In some aspects, the total amount of time that the valve is open between the sample container and the RBC container is tracked and/or stored by the circuitry.

[0090] In some aspects, while the RBC fluid layer moves to the RBC container, the circuitry may be configured to monitor the output of the one or more optical sensors detecting the rate of change in light transmittance of the optical source through a portion of the sample container. In some aspects, the circuitry is configured to adjust the length of time that the valve between the sample container and the RBC container based at least in part on the rate of change of the detected light transmittance. For example, if the detected light transmittance level changes slowly, the circuitry may be configured to leave the fluid path between the sample container and the RBC container open for a relatively longer time interval. If the detected light transmittance level changes slowly, the circuitry may be configured to leave the fluid path between the sample container and the RBC container open for a relatively longer time interval.

[0091] In some aspects, the circuitry is configured to calculate a RBC container fluid flow rate based upon the change in optical light transmittance over a known container volume. For example, in some aspects, the system is configured such that the volume of the RBC layer in the sample is known when circuitry determines that the detected light transmittance is at a stable maximum. The circuitry may be further configured to determine the required valve open time to transfer all, or a portion of, the known remaining RBC layer in the sample container to the RBC container and/or the cell container.

[0092] In some aspects, the circuitry determines the flow rate of the RBC layer from the sample container to the RBC container by dividing a known volume by the total amount of time the valve opens between the sample container and the RBC container in order to transfer the known volume to the RBC container. For example, the circuitry may be configured to detect when the buffy layer first starts to abut an optical aperture volume. The circuitry may also be configured to detect when the RBC layer completely exits the optical aperture volume. Thus, the total amount of time the valve is opened to remove the RBC layer within the known optical aperture volume can be used to determine the flow rate of the RBC layer from the sample container to the RBC container. As the remaining volume below the optical aperture volume is also known, the valve open time to remove all or a portion the remaining RBC layer may be calculated. In some aspects the valve open time includes successive opening and closing valve movements to transfer fluid between the sample container and the RBC container to reach a pre-selected RBC fluid volume in the RBC container, a pre-selected RBC fluid volume in the cell container, and/or to leave behind a pre-selected RBC fluid volume in the sample container.

[0093] In some aspects, the system includes one or more load cells. The one or more load cells may be configured to determine the volume of liquid that is transferred to the RBC container and/or the cell container. The one or more accelerometers may be used in conjunction with the one or more load cells to calculate the mass of the transferred sample. That is to say, circuitry may be able to determine the volume that is transferred to a container at least in part by receiving signals from the one or more load cells indicative of the force applied on the load sensor and at least in part by receiving signals from the one or more accelerometers indicative of the acceleration.

[0094] In some aspects, the circuitry is configured to tare the cell container to zero. The circuitry may be configured to tare the cell container to zero in response to an input received by the microcontroller from the load cell sensor configured to measure a force of the cell container. After the cell container is tared to zero, the circuitry may be configured to cause the motor to move the valve to a position where a fluid flow path between the sample container and the cell container is opened. In some aspects, the circuitry is configured to open the flow path for a set length of time. In some aspects, the circuitry is configured to open and close the flow path a plurality of times and record the total time that the flow path was opened. In some aspects, the circuitry may then be configured to determine the volume of fluid that was transferred to the cell container by receiving signals from the load sensor(s) and accelerometer(s). In this way, the flow rate of the fluid that is transferred from the sample container to the cell container may be determined by dividing the volume recovered in the cell container by the total time that the flow path between the sample container and the cell container was open.

[0095] In some aspects, the circuitry may then be configured to determine the remaining desired open time for the valve between the sample container and the cell container in order to transfer the desired amount of fluid volume to the cell container. In some aspects, the valve between the sample container and the cell container may be opened and closed multiple times to reach the desired total valve open time as calculated by the circuitry. In some aspects, the circuitry may also continue to monitor input from the load sensor(s) and accelerometer(s) to determine when the desired volume in the cell container is reached.

[0096] The following description and examples illustrate preferred embodiments of the device in the context of separating a sample of whole blood. Of course other samples such as, for example, bone marrow or cord blood sample may be similarly processed. It will also be understood that the inventive aspects disclosed herein can be applied to other procedures and/or devices. For example, the inventive aspects can be utilized to sort various cell types and/or direct other non-cellular and/or non-organic fluids during centrifugation. Those of skill in the art will recognize that the disclosed aspects and features are not limited to any particular cell processing system or device, which may include one or more of the inventive aspects and features described herein. Furthermore, the disclosed embodiments can be used in a variety of medical procedures and in connection with a variety of commercially available devices.

[0097] Moreover, the disclosed systems, methods, and devices may be used to sort materials having different densities using centrifugation. Such materials may include beads. The beads may be paramagnetic beads. The beads may be configured to bind to a target molecule, protein, cell, and the like. For example, the beads may be coated with one or more antibodies capable of binding a particular cell type. Multiple beads having various densities may be utilized and sorted by the techniques described herein.

[0098] In addition, while the described implementations include transferring all or all but a pre- selected volume of the RBC layer from the sample container to the RBC container, additional volumes of the sample may also be transferred to the RBC container. For example, the RBC layer and a portion of the buffy coat layer may both be transferred to the RBC container. In some aspects, all of the RBC layer and all of the buffy coat layer are transferred to the RBC container and a pre- selected volume of plasma is transferred to the cell container. In other aspects, all of the RBC layer, all of the buffy coat layer, and at least a portion of the plasma layer are all transferred to the RBC container.

[0099] While the implementation described below generally includes a bag set that is inserted into a device that is then inserted into a centrifuge, other configurations are contemplated. For example, the device may include its own centrifuge. The centrifuge may be a desk-top centrifuge or micro centrifuge. Furthermore, while samples are typically collected in blood bag like containers, any suitable container may be utilized.

[00100] Some aspects include monitoring and/or recording and/or analyzing electronic signals. It is to be understood that such devices may include analog and digital signals. Thus, while a condition and/or sensor may be monitored and/or connected to circuitry, the circuitry may only intermittently sample, record, and or process such data. In some aspects, continuous monitoring may include intermittent monitoring at set intervals. Such intervals may correspond to the clock speed of the particular circuitry that is implemented.

System for Separating Samples

[00101] As shown in FIGS. 1 - 2A/2B, one system that can be modified to implement the inventions described herein may include a flexible bag set 100 or rigid bag set 1100 and a processing device 200 or 1200 (refer to FIG. 6). The flexible bag set 100 and rigid bag set have similar features, which are similarly numbered in the figures, with the exception of the rigid backbone structure 1300 shown in FIG. 2B. The bag set 100 or (1100 of FIGs. 2A and 2B) may be functionally closed and may be disposable. The bag set 100 or 1100 of (FIGs. 2A and 2B) may include three or more bags connected by lines, ports or tubing to a metering valve, with inlet lines, clamps, filters, and sampling sites. The bag set 100 or (1100 of FIGs. 2A and 2B) may include three bags: a flexible processing bag 102 or rigid processing bag 1102 of FIGs. 2A and 2B), a flexible red blood cell (RBC) concentrate bag 104 or rigid RBC concentrate bag (1104 of FIGs. 2A and 2B), and a flexible stem cell bag 106 or (1106 of FIGs. 2A and 2B).

[00102] The processing bag 102, (1102 of FIG 2) may be made of ethylene vinyl acetate

(EVA), but may also be made of poly vinyl chloride (PVC) or other plastics. The RBC concentrate bag may be made of PVC or other plastics. The stem cell bag 106 may be made of EVA, although other plastics may be used. The bags 102, (1102 of FIG 2) and 106, (1106 of FIG 2) may be blow-molded or injection molded. The RBC concentrate bag may be RF-welded

(radio frequency- welded), although it may be blow-molded or injection molded.

[00103] The processing bag 102 may be three-dimensional bag that may have an asymmetric shape, including top edge 108, curved side 110, straight side 112, tapered bottom 113, and bottom outlet 114. Top edge 108 includes inlet 115 and two holes 116. Alternatively, processing bag 102 may be shaped symmetrically such that its sides taper symmetrically towards bottom outlet 114. The total volume of processing bag 102 may be about 240 mL, although in use, it is typically filled with about 50-150 mL of bone marrow or cord blood. Processing bag 102 may be supplied through inlet line 118 which connects to inlet 115. Inlet line 118 includes a female luer 120 which allows bag set 100 to be connected to a syringe containing the bone marrow or cord blood to be transferred into processing bag 102. Female luer 120 is connected to male luer 122 which is connected to spike 124, which is covered by cap 126, which allows bag set 100 to be connected to a collection bag containing the bone marrow or cord blood to be transferred into processing bag 102. Inlet line 118 includes clot and bone chip filter 128 (about 200-300μ mesh). Line or tubing clamp 130 is located between clot and bone chip filter 128 and female luer 120. Inlet line 118 may optionally also include sampling site 132, sampling pillow

134, and sampling site 136, all located below clot and bone chip filter 128. Sampling sites 132 and 136 each include a needleless female luer and a non-breathing luer cap. Bottom outlet 114 directs output from processing bag 102 into metering valve 138.

[00104] The RBC concentrate bag 104 may be a flat bag, having top edge 105, bottom edge 107, and two side edges 109, and includes butterfly spike port 140 which is used to remove an aliquot of the RBCs at the end of the process should that be desired. Bottom edge 107 includes inlet 111 at one corner. The volume of RBC concentrate bag 104 is about 100 mL, although in use, it is typically filled with about 30-80 mL. RBC concentrate bag 104 is connected at inlet 111 to supply line 142 which is connected to metering valve 138 at one of metering valve's 138 connectors 139.

[00105] The stem cell bag 106 may be a three-dimensional bag that is rectangular in shape.

Stem cell bag 106 includes top edge 117, bottom edge 119, large compartment 144, and small compartment 146, with compartments 144 and 146 connected by two channels 148. Top edge 117 includes inlet 121 and two spike ports 150, which are used to remove the stem cells at the end of the process. The volume of stem cell bag 106 is about 30 mL, although in use, it is typically filled with about 25 mL, with about 20 mL in large compartment 144 and about 5 mL in small compartment 146. Stem cell bag 106 is connected at inlet 121 to stem cell bag inlet line 152 which is connected to supply line 156 through F connector 154. Supply line 156 is connected to metering valve 138 at one of metering valve's 138 connectors 139. F Connector 154 connects supply line 156 and stem cell bag inlet line 152 to branch line 158. Branch line 158 is connected to sampling line 162 and cryoprotectant supply line 165 through T connector 160. Sampling line 162 includes line clamp 164 and sampling site 166, and terminates in sampling pillow 168. Cryoprotectant supply line 165 includes sampling site 170 and line clamp 172, and terminates in sterile filter 174 (e.g., about 0.2 μ mesh). Sampling sites 166 and 170 each include a needleless female luer and a non-breathing luer cap.

[00106] Lines 118, 142, 156, and 162 are tubing which may be made of PVC, EVA or other material. Lines 152 and 158 are tubing made of EVA. Cryoprotectant supply line 164 is co-extruded tubing made with PVC on the outside and EVA on the inside. Because plastic materials that come into contact with the cryoprotectant could, if they leach out into the cryoprotectant, enter stem cell bag 106 and contaminate the final product, it is advantageous to use a material, such as EVA, that does not contain plasticizers that could leach into the cryoprotectant for the lines that will be in contact with the cryoprotectant.

[00107] The metering valve 138 may be a stopcock. In one embodiment, metering valve

138 is a three-way stopcock in that it has three connectors 139 such that it can be connected to three bags: processing bag 102, RBC concentrate bag 104, and stem cell bag 106. Other types of metering valves or stopcocks will also work, such as a four-way stopcock having four connectors. Metering valve 138 includes an outer portion 141 having three connectors 139 and an inner portion 143 (See FIG. 12). Outer portion 141 may be made of polycarbonate. Inner portion 143 includes handle 145 and barrel 147, integrally molded, which may be made of polyethylene. Barrel 147 moves between several positions, including a closed position, defined as a position that does not allow any fluid flow through metering valve 138, and two open positions defined as positions that permit fluid flow through metering valve 138. The two open positions include one that permits fluid flow from processing bag 102 through metering valve 138 to RBC concentrate bag 104 and one that permits fluid flow from processing bag 102 through metering valve 138 to stem cell bag 106.

[00108] In one embodiment, shown in FIGS. 3A-3C, barrel 147 of metering valve 138 may be configured to contain three openings, permitting fluid flow along three possible fluid paths: an intended path from processing bag 102 to RBC concentrate bag 104 as shown in FIG. 3 A; an intended path from processing bag 102 to stem cell bag 106 as shown in FIG. 3B; and an unintended transient path between RBC concentrate bag 102 and stem cell bag 106 as shown in FIG. 3C that may occur when metering valve 138 is moving between its two intended positions. Such transient fluid flow is undesirable as it could allow some additional RBCs to flow into stem cell bag 106, thereby reducing the purity of the resulting stem cell composition.

[00109] To address this concern, in another embodiment, shown in FIGS. 4A-4D, barrel 147 of metering valve 138 may alternatively be configured to contain only two openings, permitting fluid flow along only two possible fluid paths: an intended path from processing bag 102 to RBC concentrate bag 104 as shown in FIG. 4A or 4C; and an intended path from processing bag 102 to stem cell bag 106 as shown in FIG. 4B or 4D. This configuration would preclude any possible transient fluid flow between RBC concentrate bag 104 and stem cell bag 106. Metering valve 138, 1138 of Fig 2 is designed to be able to withstand the high pressures that occur during centrifugation; for example, a valve rated to about 300 psi is sufficient. Supply line 142 leads from metering valve 138 to RBC concentrate bag 104. Supply line 156 leads from metering valve 138 to F connector 154, which leads to stem cell bag 106 via stem cell bag inlet line 152. Lines 142, 152, and 156 may each be heat sealed and separated from bag set 100.

[00110] If more bags are needed to separate additional components from the bone marrow or cord blood, bag set 100 may include additional bags. If additional bags are included, metering valve 138 will have additional connectors to accommodate each bag and the bag set will include additional supply lines to connect the metering valve to each bag. For example, if it is desired to separate the last portion of the RBCs in processing bag 102 from those RBCs transferred into RBC concentrate bag 104, a fourth bag is used. The fourth bag is connected to metering valve

138 by a separate supply line. In that case, metering valve 138 would be a four-way stopcock having four connectors and would include a barrel that permits fluid flow from processing bag 102 to the each of the other three bags.

[00111] FIGS. 5-14 shows two embodiments of a processing device 200 and 1200 (of FIG 2). As described above, structures that are similar between the processing device 200 for the flexible bagset 100 are similarly numbered in the processing device 1200 for the rigid bag set 1100. Processing device 200 is somewhat cylindrical, having top 202, bottom 204, front 206, back 208, side 210, and side 212. Front 206 includes front door 214 and front walls 216 and 217. Processing device 200 has body 218, stem cell bag compartment 220, base plate 222, support bracket 224, and processing bag hanger 226. Body 218 and stem cell bag compartment 220 may be made of molded urethane, although other moldable polymeric materials may be used. Base plate 222 and bracket 224 may be made from stainless steel, aluminum, and the like. In some aspects, the base plate 222 and/or bracket 224 include plastics. Processing device 200 is sized to fit inside a centrifuge bucket with a minimum capacity of 1 L, which may be circular or oval in cross-section. FIG. 15 shows processing device 200, containing bag set 100, inside a centrifuge bucket 228. FIG 16 shows processing device 1200, containing bag set 1100, inside a centrifuge bucket 228. FIG. 17 shows how a loaded centrifuge bucket 228 fits into a centrifuge with five other loaded centrifuge buckets already in place.

[00112] Body 218 of processing device 200 includes main compartment 230, which has an elongated oval shape dimensioned to receive processing bag 102. Main compartment 230 is open at the top and is accessed by opening front door 214 which is attached to front 206 of processing device 200 by hinge 232. Main compartment 230 has side walls 234 and 236 and back wall 238 that conform to and support processing bag 102, and tapers down to channel 240, which is dimensioned receive tapered bottom 113 of processing bag 102. Side walls 234 and 236 cradle processing bag 102 loosely around the middle and more tightly at tapered bottom 113. Closer tolerance near tapered bottom 113 of processing bag 102 is advantageous in order to provide support for processing bag 102 and its contents during the high pressures of centrifugation and to minimize disturbance to the contents of the bag. Front door 214 includes a concave inner recess 242 on its inside surface that corresponds to side walls 234 and 236 and channel 240 of main compartment 230 and provides a continuous surface, such that when front door 214 is closed it conforms to and supports processing bag 102.

[00113] Recess 244 is located on front 206 of processing device 200 below front walls 216 and 217, and is configured to support metering valve 138's connectors 139. Valve actuator cuff 246 is located inside recess 244, and is sized and configured to receive metering valve 138's handle 145. Valve actuator cuff 246 is attached to the shaft of servo motor 248 (FIG 10) by screw 250. A corresponding recess 252 is located on the inside of front door 214 and is sized and configured to receive the protruding end and connectors 139 of metering valve 138.

[00114] One or more optical sensors 254 are assembled through front wall 217 of main compartment 230, having their apertures located on side wall 236, with an equal number of LEDs 256 located on opposite side wall 234. LED 256 may be configured to emit a red LED light (e.g. about 620-630 nm light), although other wavelengths emitted from LEDs may be used. One optical sensor 254 and one LED 256 may be located about 2 cm above channel 240 such that the volume in processing bag 102 between the level of optical sensor 254 and LED 256 and the level of metering valve 138 is about 2 mL, although other distances and corresponding volumes may also be used. In some embodiments, the one or more LEDs may be located from about 0.1 to about 5 cm above the channel, for example more than about 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, 2.0 cm, 2.1 cm, 2.2 cm, 2.3 cm, 2.4 cm, 2.5 cm, 2.6 cm, 2.7 cm, 2.8 cm, 2.9 cm, 3.0 cm, 3.5 cm, 4.0 cm, or 4.5 cm, and less than about 5.0 cm, 4.5 cm, 4.0 cm, 3.5 cm, 3.0 cm, 2.9 cm, 2.8 cm, 2.7 cm, 2.6 cm, 2.5 cm, 2.4 cm, 2.2 cm, 2.3 cm, 2.1 cm, 2.0 cm, 1.9 cm, 1.8 cm, 1.7 cm, 1.6 cm, 1.5 cm, 1.4 cm, 1.3 cm, 1.2 cm, 1.1 cm, 1.0 cm, 0.9, 0.8 cm, 0.7 cm, 0.6 cm, 0.5 cm, 0.4 cm, 0.3 cm, or 0.2 cm.

[00115] More than one LED and or optical detector may be utilized. If additional optical sensors and LEDs are included, they may be located above or below optical sensor 254 and LED 256. In some aspects, two or more LEDs and/or optical detectors may be used to obtain two or more calculated flow rates. Multiple flow rate calculations may lead to more reliable and/or accurate results.

[00116] In some aspects, the LED 256 is capable of emitting more than one wavelength of light. For example, the LED 256 may be capable of emitting red (e.g. about 620-630 nm light) and green (e.g. about 495-570 nm light) selectively or at the same time. Similarly, the optical sensor 254 may be configured to detect the transmittance of one or more wavelengths. In some aspects, green light may be utilized for determining when the buffy coat has entered and/or exited the optical aperture area 257 (not shown).

[00117] Side 212 of processing device 200 includes notch 258, storage cavity 260, channel 262, RBC concentrate bag recess 264, support shelf 266, and hook 268. Notch 258 is sized and configured to receive spike port 140 of RBC concentrate bag 104. RBC concentrate bag recess 264 extends from notch 258 down to support shelf 266, and is sized and configured to receive RBC concentrate bag 104. Storage cavity 260 is located below notch 258 and is behind and continuous with RBC concentrate bag recess 264. Channel 262 extends along the inside edges of RBC concentrate bag recess 264, adjacent to front door 214, top 202, and back 208. Support shelf 266 is a horizontal shelf which forms the floor of RBC concentrate bag recess 264.

[00118] As depicted in Fig. 7, the stem cell bag compartment 220 is a horizontal, hollow rectangular compartment having a larger side 270 located under hinge 232 of front door 214. Stem cell bag compartment 268 is located below base plate 232 and is attached to load cell 272.

Stem cell bag compartment 220 is accessed through hinged door 274 which opens downward, and snaps into place via latch 276 located inside stem cell bag compartment 220 at its top edge. The inside of hinged door 274 contains recess 278 that is sized and configured to accommodate stem cell bag 106's spike ports 150 and inlet 121. The outside of hinged door 274 contains slot 280 and channel 282 sized to accommodate stem cell bag inlet line 152.

[00119] Bottom channel 284 extends above and parallel to base plate 222, from front 206 under hinge 232 across side 210 and back 208 to side 212. Bottom channel 284 is sized to accommodate line 142.

[00120] Processing device 200 includes processing bag hanger 226 that extends above top 202 at side 210. Processing bag hanger 226 has tabs 227 that engage holes 116 on processing bag 102, maintaining processing bag 102 in position inside processing device 200. LED windows 229 are located on top 202 at side 210. Support bracket 224 is a U-shaped bracket that is attached to base plate 222 by screws 225 and is oriented parallel to processing device's 200 sides 210 and 212.

[00121] Processing device shown in Fig 6 is somewhat cylindrical, having a top, bottom front, back, left and right side. The upper body of the processing device includes a handle and two compartments dimensioned to receive the rigid processing bag set snapped in from the front. The lower portion of the device consists of a base plate, plastic based stem cell container and standing bracket. The base plate includes slots to mount and house the internal electronics, wiring and sensors. The stem cell container is attached to a sensor and bracket mounted to the base plate. The standing bracket contains a direct or wireless contact to communicate electronically with the device microprocessor. The base plate and bracket may be made from stainless steel, aluminum, and the like. In some aspects the base plate and/or bracket includes plastics. The device body and stem cell bag compartment may be made of molded urethane, although other moldable polymeric materials may be used. [00122] The front of the body includes a center compartment designed to accept a rigid shell processing container. The front includes two slots on either side of the center compartment for snapping in two clips from the rigid bag set assembly. The clips ensure the rigid bag set assembly is secure during handling. The center cavity also includes two or more apertures openings to house LEDs and optical sensors.

[00123] The left side of the body houses a compartment to store the removable

rechargeable battery pack inserted from the top and optical sensors positioned next to the processing container slot. The battery packet assembly includes electrical mating contacts on the bottom of the assembly to interface with internal connectors housed inside the body when inserted completely. A protruding handle on top of the battery pack is provided to allow the user to remove and insert rechargeable portable batteries. The left side of the body also includes a compartment to house two more optical sensors mounted to a printed circuit board assembly which interfaces with the internal electronics of the device. The optical sensor board assembly mounted on the left side of the body is accessible through a cover and two screws.

[00124] The right side of the body is designed to receive the rigid RBC container part of the semi-rigid bag set. The RBC container geometry fills the open cavity on the right side of the main body resting on the base plate. The right side of the body includes a compartment to house two or more LEDs mounted to a printed circuit board assembly which interfaces with the internal electronics of the device. The LED circuit board assembly may be mounted on the right side of the body and may be accessible through a cover and two screws. The LEDs may be configured to emit a red light, although other wavelengths emitted form LEDs may be used. More than one LED or optical detector may be utilized. In some aspects, two or more LEDs and/or optical detectors may be used to obtain two or more calculated flows. Multiple flow rate calculations may lead to more reliability and/or accurate results.

[00125] The device handle contains a slot to house a series of LED indicators to display the battery status, high speed status and low speed status via a multi-color LEDs (Red, Green, Blue & White) mounted to a printed circuit board assembly. The LED circuit board is controlled via interconnect wires routed down the handle to the internal electronics.

[00126] The device design allows the metering valve actuator cuff to meet the bagset valve once inserted completely and snapped in. The valve actuator cuff is attached to the shaft of a servo motor mounted to the base plate. Fig. 15 shows the semi-rigid bagset loaded and snapped into the device. The ability to snap in the semi-rigid bagset into the device reduces the handling time to load a sample into the device for processing. The flexible cell container part of the semi rigid bagset is placed into the stem cell compartment mounted to the base plate. The semi-rigid bagset inserted into the device assembly are sized to fit inside a centrifuge bucket with a minimum capacity of 1L, which may be circular or oval in cross-section as shown in Figure 17.

[00127] The bottom bracket includes a printed board assembly to provide power and means to communicate with the device microprocessor. The board assembly is electrically connected to the main board assembly housed on the base plate. The bottom bracket may include electronics to wirelessly provide power to the device. As shown in FIG. 10, base plate 222 may include the following components mounted to it: printed circuit board 286, servo motor 248, load cell 272, and interface contact printed circuit board 288. Printed circuit board 286 contains programmable read only memory microcontroller 290. Microcontroller 290 may require temperature compensation due to heat generation during centrifugation. Load cell 272 is a temperature compensated strain gauge load cell. Servo motor 248 is a gear reduction motor.

One or more accelerometers 292 are mounted on printed circuit board 286. The accelerometers 292 may include a low g accelerometer 291 which measures about 0-200xg and a high g accelerometer 293 which measures about 1000-2000xg. Printed circuit board 286 may also include status LEDs 294 that are visible through LED windows 229 (see FIG. 19A). One or more status LEDs 294 may indicate the charge status of battery 296 and the current step being performed in the process, as well as other information. Interface contact printed circuit board 288 is provided to connect to an external battery charger and to provide a communication connection with a personal computer. Base plate 222 is attached to body 218 by screws 298 (see FIG. 11).

[00128] FIG. 18 is a block diagram showing the inputs and outputs of microcontroller 290. Optical sensor 254, battery voltage 302, load cell 272, and accelerometers 291 and 293, generate analog outputs which are converted to digital inputs received by microcontroller 290.

Microcontroller 290 records, stores, and analyzes those inputs at predetermined timed intervals. Microcontroller controls servo motor 248, LED 256 for optical sensor 254, and status LEDs 294. When bag set 100 including metering valve 138 is properly placed into processing device 200, servo motor 248 is connected through its drive shaft to valve actuator cuff 246 and, in response to a signal from microcontroller 290, servo motor 248 causes metering valve 138 to move to different positions to open or close fluid flow to the bags of bag set 100.

[00129] Battery 296 is located in battery cavity 300 on top 202 at side 210 of processing device 200. Battery 296 is a rechargeable nickel metal hydride battery including three cells with a total of about 3.6-4 volts, which voltage is regulated by circuitry 286 to provide constant voltages of 5-5.5 volts. Battery 296 powers microcontroller 290, optical sensor 254, accelerometers 291 and 293, load cell 272, optical sensor LED 256, status LEDs 294, servo motor 248, and all electrical circuitry in processing device 200 including serial communication with a personal computer.

[00130] Processing unit 200 may be placed in a separate docking station, not shown, which includes a battery charger and which may be connected to storage and processing device, for example a personal computer. Data from microcontroller 290 may be transferred to the, storage and processing device or personal computer, and commands may be received from storage and processing device or personal computer, via the docking station through interface contact printed circuit board 288.

[00131] As shown in FIGS. 12-15, bag set 100 is inserted into processing device 200 as follows. Stem cell bag 106 is placed into stem cell bag compartment 220 such that bottom edge 119 fits in first, and small compartment 146 is folded over and placed in larger side 270. Inlet 121 and spike ports 150 are placed inside recess 278 of hinged door 274. Stem cell bag inlet line 152 is placed in slot 280 of hinged door 274. Hinged door 274 is then closed and latched with latch 276. Stem cell bag inlet line 152 is placed in channel 282. Metering valve 138's handle 145 is placed into valve actuator cuff 246. Processing bag 102 is oriented in main compartment 230 such that front door 214 closes over processing bag 102's straight side 112. F connector 154 is placed inside RBC concentrate bag recess 264. Line clamp 165 is closed and placed inside channel 262 along with sampling line 162. Cryoprotectant supply line 165, line clamp 172

(closed), and sampling site 170 are placed into storage cavity 260, with sterile filter 174 placed into the right side wall receptacle of storage cavity 260. Sampling site 166 is anchored at hook 268. Sampling pillow 168 fits into recess directly below the filter receptacle in the right side of RBC concentrate bag recess 264. Then, supply line 142 is placed into bottom channel 284 and RBC concentrate bag 104 is placed into RBC concentrate bag recess 264. Inlet line 118 and sampling site 136 are folded downward and toward processing bag hanger 226 at side 210. Holes 116 of processing bag 102 are attached to hanger 226's tabs 227. Front door 214 is closed. Spike port 140 fits into notch 258, with bottom edge 107 on support shelf 266, such that RBC concentrate bag 104 fits over storage cavity 260. Branch line 158 is placed vertically along the left side of storage cavity 260.

Method And Device For Recovering A Desired Volume From A Sample Container

[00132] FIG. 19A is a top downward view of a device that is substantially similar to the processing devices of FIGS. 5-14 configured to calculate the flow rate of the sample leaving a processing bag or sample container. FIG. 19B is a cross-sectional view of the device of FIG. 19A taken about the line A-A.

[00133] As shown in FIG. 19B, a sample container 302 or processing bag 102 may be disposed in the approximate center and upper portion of a centrifuge housing 305. The sample container 302 (or 102) may be fluidly connected to a valve 139 via a bottom outlet 114. The valve 139 is also coupled to a supply line 156 which may be fluidly connected to a cell container 300. In some aspects, the cell container 300 includes a stem cell bag 106 and/or assorted collection bags and tubing as described above. The valve 139 may also be coupled to supply line 142 which may be fluidly connected to a RBC container 304. The RBC container 304 may include a RBC concentrate bag 104 and associated tubing as described above.

[00134] The valve 139 may be configured to selectively open and close a flow path from the sample container 302 to the cell container 300 or to the RBC container 304. In this way, centrifugal force may cause the fluid to flow from the sample container 302 to either the cell container 300 or to the RBC container 304 when the valve 139 is in the desired open position while the housing is housing 305 rotating.

[00135] As shown in FIG. 19B, the valve 139 is in a closed position such that fluid may not flow from the sample container 302 to either the cell container 300 or to the RBC container

304. An un-processed whole blood sample 400 is contained by the sample container 302. As shown, the device is positioned upright where the blood sample 400 fills the lower end of the sample container 302. In addition, the blood sample 400 fills less than the maximum volume of the sample container 302 such that the top portion of the container 300 is empty space representing air.

[00136] Turning to FIG. 20, an enlarged view of FIG. 19B, illustrates that in some implementations an LED 256 and an optical sensor 254 may be positioned within the housing 305 such that the LED 256 is projected through the sample container 302. As will be discussed further below, the LED 256 may project onto an optical aperture area 257. The amount of light that passes through the sample container 302 and/or sample 400 may be detected by the optical sensor 254. The volume Vi within the optical aperture area 257 may be a first known volume. Similarly, the volume V ₂ beneath the optical aperture area 257 may be a second known volume. In many cases, the first known volume, Vi, and the second known volume, V ₂ , may sum to be equal to a third known volume, V ₃ . The LED 256 and/or optical sensor 254 may be electrically connected to circuitry, such as a microcontroller (not shown).

[00137] Valve 139 may include a rotating "T shaped" or other-shaped tubing 199. In various embodiments the tubing may be shaped to connect 0, 1, 2, 3, 4, or more lines simultaneously. In some embodiments the tubing may be substantially straight and may be used to connect two or more lines at least partially, as shown in FIG. 21B. The T- or other-shaped tubing 199 may be rotated by a motor (not shown). The motor may be controlled by circuitry, such as a microcontroller (not shown). The T- or other-shaped tubing 199 may be selectively rotated such that the sample 400 may flow from the sample container 302 to one or more or none of the cell container 300 or the RBC container 304. More or fewer valve openings sub- containers, fluid paths, and/or may be provided as desired.

[00138] FIGS. 21A-21D illustrate that the valve 139 may be configured to be positioned in a partially open position allowing fluid transfer. FIGs. 21A and 21B illustrate the valve 139 in a position such that fluid may flow from the sample container 302 to the RBC container 304. FIGs. 21C and 21D illustrate the valve 139 in a position such that fluid may flow from the sample container 302 to the cell collection container 300. In such partially open positions, the fluid flow rate from the sample container 302 is reduced in comparison to a fully opened position. Thus, the valve 139 may be used to adjust the fluid flow rate to the RBC container or cell collection container or both during processing. Furthermore, the direct fluid path geometry of 2 IB and 2 ID improves the control and accuracy of fluid rate calculation compared to the path configurations shown in Figs. 21A and 21C.

[00139] In some aspects, the valve 139 may be set to anywhere between 100% open and

100% closed. For example, the valve 139 may be set to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% open. Depending on the dimensions of the containers, flow paths, and the like, the valve 139 may be adjusted such that the fluid is not transferred too quickly. That is to say, the valve 139 may be set in a partially open position to ensure that the fluids and or layers are separated in a controlled manner. In some aspects, the valve 139 may be configured to quickly move from a partially open to a partially closed position for discrete time intervals. For example, the valve may 139 be capable of moving from a 10% open position to a fully closed position in about 80 ms. Repeated opening and closing of the valve 139 may allow for fluid to be transferred in a controlled and predictable manner.

[00140] FIG. 22 is similar to FIG. 20, but depicts devices and samples after the device has been sufficiently centrifuged to stratify the sample 400. As shown, the blood sample 400 now includes a bottom RBC layer 401, a middle buffy coat layer 402, and a top plasma layer 403.

[00141] Briefly, centrifugation sufficient to stratify the blood sample 400 may be accomplished by weighing the device, providing a counter balance, and spinning at a sufficient g force or relative centrifugal force (RCF) for a sufficient time interval. In some aspects, the spin time and spin rate is set by a user. As an example the centrifugation speed is typically set to about 1400 RCF for about 20-40 minutes. In some aspects, the valve 139 is set to an all closed position such that the fluid remains in the sample container 302.

[00142] In some implementations, the device includes an accelerometer and/or a RCF pressure sensor. The accelerometer and/or the RCF pressure sensor may be part of the device's circuitry and/or internal electronic board assembly and may be configured to monitor and transmit one or more signals to electronic memory for recording the RCF forces experienced by the device during stratification. An embedded microprocessor or microcontroller may calculate an elapsed time that the device is spun above a sufficient RCF level for stratification. In some aspects, if the speed or time elapsed is less than the input thresholds set by the end-user, the device may notify the operator that the centrifugation was insufficient. The device may notify the user with one or more visual alerts. Centrifugation may be restarted and may continue until the sample 400 has sufficiently stratified into multiple layers based on, for example, density and cell size.

[00143] Returning to FIG. 22 the bottom RBC layer 401 is the densest layer and is composed primarily of RBC and accompanied by neutrophils. The buffy coat layer 402 is composed primarily of WBCs (leukocytes) and platelets. In decreasing order of density, the cellular components of the buffy coat layer are granulocytes, mono-nuclear cells, stem and progenitor cells, and platelets. The plasma layer 403 is the least dense layer and is composed primarily of plasma. At this processing stage the valve 139 may be adjusted to a "RBC off position to prepare for the initial fluid flow into the RBC container 304.

[00144] With reference to FIG. 29A, one implementation of an overall process for removing the layers of the stratified sample 400 is illustrated. The waveform of FIG. 29A graphically illustrates the valve 139 position over time and the detected optical transmittance over time. FIG. 29A also shows a buffy coat volume line that represents the fluid volume present in the cell container 300 from zero until the target desired volume. Thus, the vertical axis represents multiple scale values which increase with distance above the horizontal axis of time. Stages A-F may be summarized as follows:

[00145] Stage A: transfer the RBC layer 401 from the sample container 302 to the RBC container 304 until the bottom edge of the buffy coat layer 402 abuts the upper edge of the optical aperture area 257 (see FIGS. 22-24). FIG. 29 B shows another implementation of an overall process similar to that shown in FIG. 29A.

[00146] Stage B: transfer the RBC layer 401 from the sample container 302 to the RBC container 304 until buffy coat layer 402 has traversed the optical aperture area 257 (see FIGS. 25-27).

[00147] Stage C: determining the flow rate of the RBC layer 401 from the sample container 302 to the RBC container 304.

[00148] Stage D: transfer the RBC layer 401 from the sample container 302 to the RBC container 304 until a known volume of the RBC layer 401 remains in the sample container 302 (see FIG. 26). [00149] Stage E: transfer the remaining RBC layer 401 and at least a portion of the buffy coat layer 402 from the sample container 302 to the cell container 300 (see FIG. 27).

[00150] Stage F: transfer the remaining the buffy coat layer 402 and plasma layer 403 from the sample container 302 to the cell container 300 until a final volume in the cell container is reached (see FIG. 28).

[00151] In some aspects, the circuitry is configured to continuously monitor at least one accelerometer sensor signal throughout the process given the clock speed of the

circuitry/microprocessor. For example, if the centrifuge speed drops above or below a range of 65 to 90 xgs, the device may be configured to terminate the process. Termination may include resetting the valve 139 into an ail-off position and/or notifying an end user of a fault. The notification may be in the form of an illuminated light on the exterior of the device. A user may be notified of a fault in other cases, for example where the battery malfunctions or is drained prior to finishing the collection, or the valve malfunctions or fails to open or close, or the g-force exceeds or drops below a pre-determined value or range.

[00152] Turning now to FIG. 23, after the sample 400 is sufficiently stratified, the valve

139 may be positioned to a "RBC container open" position. In some aspects, the centrifuge is deaccelerated to a lower g force during the transfer time period. The centrifuge may be preprogramed such that the device is spun at a lower rate than the spin rate for stratification. A slower centrifugation speed may induce fluid flow out of the sample container 302. The reduced centrifugation speed may provide a sufficient pressure gradient to transfer fluid from the bottom of the sample container 302, through the valve 139, and into the RBC collection container 304. In most embodiments, centrifugation speed, during transfer of sample, is typically set to between about 65 to 90 RCF.

[00153] An accelerometer and/or the RCF pressure sensor may be used to detect the RCF experienced by the device. In some aspects, circuitry contained within the device may monitor and record the RCF experienced by the device continuously. For example, the circuitry may be configured to determine the RCF using signals received form the accelerometer and/or the RCF pressure sensor every 10 ms.

[00154] In some aspects, after the circuitry (e.g. microprocessor or microcontroller) detects a stable RCF force in a preset range or above a preset limit, the circuitry initiates the removal process as described below and illustrated in FIGS. 29-35. In addition, in some aspects, after the circuitry detects a stable RCF force in a preset range or above a preset limit, the circuitry is configured to turn on the LED 256.

[00155] The LED 256 may be configured to emit one or more wavelengths of light. In some aspects, the LED 256 is configured to emit a combination of different light wavelengths. In some implementations, a plurality of light sources may be provided. For example, two or three light sources may be vertically stacked and may also include parallel light sensors on the opposite side of the processing chamber.

[00156] In some implementations, after the LED 256 is illuminated, the circuitry is configured to monitor the optical transmittance through the sample 400 in the sample container

302. Transmittance from the LED 256 may be detected by optical sensor 254. Optical sensor 254 may be a photonic light sensor aligned opposite to the LED 256 and configured to detect light from the LED. The intensity of the wavelength emitted from the LED 256 may be calibrated such that the maximum intensity does not oversaturate the optical sensor 254. In some aspects, the light intensity emitted by the LED 256 is calibrated about 10-15% below the maximum intensity detection level of the optical sensor 254 to prevent sensor saturation. In some aspects, the optical sensor 254 is positioned such that the volume in said sample container 302 between the level of the optical sensor 254 and the level of the valve 139 is about 2 mL. In some aspects, the optical sensor 254 is positioned such that the volume in said sample container 302 between the level of the optical sensor 254 and the level of the valve 139 is about 8 mL. In most embodiments, the sample volume between the level of the optical sensor 254 and the level of the valve 139 is known and pre-programmed into the circuitry, for example the

microprocessor and/or storage device. In many embodiments, the sample volume (that is Vi and/or V ₂ ) between the level of the optical sensor 254 and the level of the valve 139 is greater than about 0.2 mL, 0.3 mL, 0.4 mL, 0.6 mL, 0.5 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.5 mL,

2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 5.0 mL, 6.0 mL, or 7.0 mL, and less than about 8.0 mL, 7.0 mL, 6.0 mL, 5.0 mL, 4.0 mL, 3.5 mL, 3.0 mL, 2.5 mL, 2.0 mL, 1.5 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, or 0.3 mL.

[00157] For example, to detect a RBC-buffy coat transition at the optical sensor 254, a red dominate wavelength spectrum may be utilized given that packed RBCs absorb this photonic energy range. If the optical transmittance detected by the optical sensor 254 is below a preset programmed intensity threshold, the circuitry can detect the presence of packed RBC content and the RBC-buffy coat transition is above the LED 256 - optical sensor 254 (e.g. the optical aperture area 257).

[00158] To begin reducing the RBC layer 400 in the sample container 302 and thus lower the RBC-buffy coat transition interface level, the circuitry may instruct the motor to move the valve into the "RBC container open" position shown in FIG. 23 (or the partial on position shown in FIG. 21A). A partial valve opening, shown in FIG. 21A, can be used to reduce fluid flow of the initial RBC transfer if the sample 400 is of low viscosity. A full valve opening is illustrated in FIG. 23.

[00159] As the RBC layer 401 is transferred from the sample container 302 to the RBC container 304 the RBC layer 401 buffy coat layer 402 interface drops towards the optical aperture area 257. During this time, the circuitry may continuously read, record, and analyzes the light transmittance level it receives as input from the optical sensor 254 as a function of time. As the RBC layer 401 buffy coat layer 402 interface nears the upper boundary of the optical aperture area 257, the light transmittance begins to rise quickly.

[00160] FIG. 30 is a detailed example waveform of the light transmittance level over time during this stage. The waveform also includes the instantaneous valve position as a function of time.

[00161] FIG. 24 illustrates the moment that the RBC layer 401 buffy coat layer 402 interface upper boundary of the optical aperture area 257. At this stage, the detected light transmittance rises above a threshold as illustrated, for example, by the light transmittance waveform in FIG. 30. At this moment, the circuitry may instruct the motor to change the valve 139 position to "RBC Valve Closed" (marked as time point 500 in waveform of FIG. 30). In other aspects, the circuitry may be configured to end Stage A when the detected light

transmittance rises above a value of about twice the threshold as shown, for example, at time point 501 in waveform of FIG. 30.

[00162] In some aspects, the circuitry is configured to wait for at least 30 seconds to allow the RBC layer 401 buffy coat layer 402 interface to settle and ensure the RBC layer 401 buffy coat layer 402 interface has reached the optical aperture boundary illustrated in FIG. 24. If the detected light transmittance level drops below the threshold, a second RBC fluid transfer may commence until the detected light transmittance level is stable and above the threshold. At this stage the volume V ₃ of the RBC layer 401 may be known due to the known geometry of the sample container 302.

[00163] Once the light transmittance threshold is achieved, the device may take a series of

ON/OFF pulses to determine the best valves for the RBC open time and valve RBC open position for Stage B. In this way, more controlled transfers of the RBC layer 401 to the RBC container 304 can be achieved. The device typically utilizes up to about five pulses to determine the best RBC valve open time interval and/or RBC valve percent open position (e.g., between 100% fully open and 10% open). In some aspects, if the light transmittance level changes relatively slowly, the circuitry is configured to position the valve in the open or partially open position for relatively longer time intervals. In some aspects, the circuitry may also be configured to increase the relative position that the valve moves to when opened (e.g., adjust between a 10% open to a 20% open position). If the light transmittance level changes relatively quickly, the circuitry may be configured to position the valve in the open or partially open position for relatively shorter time intervals. In some aspects, the circuitry may also be configured to decrease the relative position that the valve moves to when opened (e.g., between 100% fully open and 10% open). Such adjustments of how much the valve opens and how long each interval of time that the valve remains in such an open position may lead to a more precise transfer of fluid and may cause a more step like detection of transmittance as shown in FIG. 31.

This completes Stage A.

[00164] Once the light transmittance level is stable and above the threshold or a set value above the threshold, the circuitry may instruct the motor to cause a series of rapid valve 139 movements that cycle back and forth between RBC container open (or RBC container partially open) and RBC container closed (beginning Stage B). This rapid on-off sequence may help accurately lower the RBC layer 401 as shown in FIG. 25 until the upper most level of the RBC layer 401 is a position just under the optical aperture area 257 as shown in FIG. 26. At the stage in FIG. 26, the volume V ₂ of the RBC layer 401 may be known due to the known geometry of the sample container 302. [00165] The circuitry may be configured to continuously read, record, and analyze the light transmittance level it receives from the optical sensor 254 as a function of time as the RBC layer 401 buffy coat layer 402 interface moves through the optical aperture area 257. In some aspects, the detected light transmittance level change is analyzed by the circuitry after each valve 139 open-close cycle. The circuitry may also be configured to adjust the valve 139 open time to properly control RBC layer 401 transfers.

[00166] An implementation using rapid RBC open-closed cycles and light transmittance changes are illustrated in FIG. 31.

[00167] FIG. 25 illustrates a RBC valve open moment mid-way through Stage B where approximately 50% of light transmittance is observed given that the RBC layer 401 at least partially blocks the optical aperture area 257.

[00168] The stair step waveform of light transmittance shown in FIG. 31 may be preferred as it represents a proper and fixed control of RBC layer 401 movements across the optical aperture area 257 into the RBC container 304. Each time the circuitry instructs the valve 139 to open and close the fluid flow path between the sample container 302 and the RBC container 304 the circuitry is configured to compare the change in detected light transmittance with previous pulses and calculate if the change in level remains controlled and stable. If the change (delta) in detected light transmittance appears unstable after the valve 139 has closed or increases by more than 50% from the previous three pulses, the circuitry may be configured to adjust the time the valve is in the open position to stabilize the next transfer of the RBC layer 401. In some aspects, when the detected light transmittance reaches a stable maximum after three consecutive transfers of the RBC layer 401 to the RBC container 304, the circuitry may be configured to cease the periodic valve 139 open-close movement and the device may proceed to Stage C.

[00169] In some aspects, after a number of open-close valve 139 cycles in Stage B, the circuitry is configured to determine the flow rate of the RBC layer 401 during Stage C. The flow rate may be calculated by dividing the known volume change from V ₃ to V ₂ by the total amount of time that the valve was in the 139 RBC container open (or RBC container partially open) position. This flow rate may be used to help optimize the removal of the remaining portion of the RBC layer 401. [00170] In some implementations, Stage C includes a short period where the circuitry calculates the RBC fluid flow rate. During this short period (e.g. about 50 ms) the circuitry stops monitoring other inputs, instructs the motor to position the valve 139 in the off position, and calculate a RBC fluid flow rate. The RBC flow rate is used to accurately deplete a desired RBC layer 401 volume such that a desired RBC layer 401 volume remains in the sample container

302.

[00171] In some aspects, the desired final RBC volume delivered to the cell container 300 is preset by the end user prior to centrifugation. The desired final RBC volume delivered to the cell container 300 may be set, for example, from a max of about 3.0 mL to a minimum of about 0.2 mL.

[00172] In some implementations the circuitry recalls from embedded memory the total number of seconds that lapsed when the RBC valve was set to open in Stage B. The fluid flow rate is calculated and recorded by the microcontroller by dividing the known fixed volume of the processing container subtending the optical aperture area (Vi in FIG. 26) by the RBC valve open time. The RBC fluid flow rate, which varies between blood samples, is then used to accurately configure a custom depletion sequence by adjusting the RBC valve open time for Stage D. This configuration uses the total fluidic volume of the container up to the optical aperture (V ₂ in FIG. 26) in and the maximum RBC valve off-on-off speed of the valve motor. Once the configuration is programmed and recorded, the circuitry may initiate Stage D.

[00173] FIG. 32 illustrates an example waveform of Stage C where the detected red wavelength light transmittance is constant and the RBC valve open/closed timing updates between Stage B and Stage D. The red light transmittance is constant in this example because the RBC layer 401 is below the optical aperture area 257 and buffy coat layer 402 does not absorb this type of wavelength.

[00174] Turning now to FIG. 33, detailing Stage D, the valve 139 opens and closes the flow path between the sample container 302 and the RBC container 304 without changing the detected light transmittance as explained above. FIG. 33 shows an example of the total sequence of RBC valve open transfers over time for Stage D. The number of valve transfers and RBC open valve time is tailored such that the remaining RBC layer 401 volume matches the target specified by the end user. That is to say, using the calculated RBC flow rate, the circuitry instructs the motor to open and close the flow path between the sample container 302 and the RBC container 304 for the precise amount of time to deplete V ₂ (FIG. 26) such that the target RBC layer volume remains in the sample container 302, as shown for example in FIG. 27. The circuitry also instructs the motor to narrow the valve position linearly by a predetermined percentage during valve movements to counteract RBC flows above 250uL/sec. The narrowing method ensures the RBC depletion is adjusted such that stem cellular content is transferred inadvertently to the cellular bag. This completes Stage D.

[00175] In Stage E the circuitry begins a series of buffy coat layer 402 transfers to the cell container 300 to achieve a final target volume. The final target volume contained in the cell container 300 may be preselected or preprogrammed by an end user. In some aspects, the final target volume may be set from between about 5 ml to about 25 ml. To accurately measure the volume being transferred to the cell container 300, a load cell force sensor in conjunction with an accelerometer may be utilized. The circuitry may determine the accumulated volume in the cell container 300 by calculating the mass from the force applied on the load cell sensor divided by the accelerometer response.

[00176] Before Stage E begins, the circuitry may be configured to tare the mass (volume) of the empty cell container 300 to zero and may instruct the motor to position the valve 139 in the buffy off position shown in FIG. 36. Once the calculated mass from the load cell force sensor and accelerometer has registered a value of zero, the circuitry may initiate a series of three rapid fluid transfers into the cell container 300 to determine the buffy coat layer 402 flow rate. The buffy coat layer 402 flow rate may be calculated by the circuitry by dividing the volume reported by the load cell force sensor over the length of time the valve 139 was set to the open buffy position. This flow rate may be is stored and recorded by the circuitry. The flow rate may be used to calculate the viscosity of the material transferred into the cell container 300. This viscosity may be displayed to an end user.

[00177] FIG. 34 is an example waveform of at least a portion of Stage E. Three buffy valve open pulses are shown at the beginning of the stage with small increases in the amount of buffy volume reported by the load cell / accelerometer calculated mass. FIG. 27 illustrates the valve in a fully open position such that a flow path between the sample container 320 and the cell container 300 is opened. This allows the remaining RBC layer 401 and buffy coat layer 402 to move from the sample container 302 to the cell container 300. In some aspects, after the three pulses are completed, the circuitry calculates and schedules a series of three to four fluid transfers to rapidly transfer up to 75% of desired final volume to the cell container 300. The circuitry may be configured to dynamically adjust the valve buffy open time based at least in part on the calculated buffy coat layer 402 fluid flow rate. As shown in FIG. 34, three valve buffy open transfers are executed in the middle of this stage where the load cell/accelerometer calculated mass reports rapid increases in the volume transferred to the cell container 300. The transfer may be accomplished efficiently by utilizing a known buffy flow rate. Once the volume in the cell container 300 has reached about 75% of the target volume final volume desired, the device may initiate Stage F.

[00178] Stage F may include transferring the necessary remaining volume from the sample container 302 to the cell container 300 to reach the desired final volume in the cell container. Stage F may include the transfer of the any remaining buffy layer 402 and/or a portion of the plasma layer 403 from the sample container 302 into the cell container 300.

[00179] FIG. 28 illustrates the fluid transfer path when the valve 139 is set in the cell container open position. Transfers may be executed by using a series of rapid valve openings and closing. In some aspects, the valve 139 is set in the cell container open position for about 80 ms before it is closed. Short intervals for valve 139 open times may enable the fine metering of the remaining necessary fluid to reach the target volume in the cell container. In some aspects, the final accumulated volume is accurate to about 0.3 ml or better with respect to the pre-selected volume.

[00180] FIG. 35 is an example waveform of Stage F where valve 139 open-close cycle provide a discrete controlled increase in volume within the cell container 300. In some aspects, after each valve open-close cycle, the circuitry records the volume in the cell container 300 and calculates a running average of the changes in volume. This average may be monitored by the circuitry after each fluid transfer such that the cell container 300 is not overfilled.

[00181] FIGs. 37A-C depict an example flow chart illustrating circuitry logic 600 for an exemplary implementation. The method may start at block 601 by opening the RBC valve to begin the transfer of the RBC layer from the sample container to the RBC container. As shown in block 603, the circuitry may analyze the input from the optical sensor at a set interval of, for example every 10 ms. If the detected transmittance is below a threshold, the circuitry may continue to monitor the detected transmittance. Once the detected transmittance is above the threshold the method may move to block 604. At block 604, the circuitry may initiate a series of five rapid RBC open - RBC closed valve movements. The change in light transmittance may be analyze after each cycle in order to determine the optimum partial valve open position for the best controlled fine transfer of RBC layer. The valve open-close internal may be set to a maximum of about 500 ms and a minimum of about 80 ms. The valve position may be set from a range of about 30% partially open to 100% open. The open-open close cycles may be counted at block 605 until five cycles have completed and the optimum valve open interval and % opening of the valve may be determined. In some aspects, Stage A is completed after block 605.

[00182] The method may continue at block 606 with the controlled transfer of the buffy coat layer through the optical aperture area may begin. At block 607, the circuitry may open and close the valve for the time intervals and % open position determined at block 604 until the circuitry detects a steady state input from the optical detector.

[00183] The method may continue at block 608 where the circuitry may calculate the RBC layer flow rate and the remaining RBC valve open time in order to transfer a precise volume of the RBC layer to the RBC container - leaving a precise volume of the RBC layer in the sample container. The total RBC valve open time may be divided by, for example, eighteen (or any appropriate number between about 5 and about 25) and the circuitry may instruct the valve to open and close, for example, eighteen times such that the total calculated RBC valve open time has elapsed at block 610.

[00184] The method may continue at block 612 by a first transfer to the cell container.

The first transfer may include transferring the remaining RBC layer and at least a portion of the buffy coat layer that is in the sample container. This may be performed by three cell container valve open-close movements at block 613.

[00185] The method may continue at block 614 where the flow rate of the fluid transfer to the cell container may be calculated by the circuitry. The circuitry may use the flow rate to determine the viscosity of the sample in the cell container at block 614. The transfer of fluid to the cell container may continue at block 615 until the cell volume in the cell container is about 75% of the desired final volume is detected at block 616. [00186] The method may continue at block 618 transferring small volumes of fluid to the cell container using the calculated flow rate and recording of the volume transferred to the cell container. The current volume in the cell container may be compared to the target volume at block 620 and the method may end when the target volume in the cell container is reached.

[00187] Once the target volume is reached the circuitry may cause the motor to rotate the valve 139 into an off position. The circuitry may then be configured to finalize the sequence by readying the recorded data for transfer to a removable memory.

[00188] In some implementations, the volume within the cell container 300 may include a solution of human plasma, MNCs, and platelets that were isolated from whole blood, bone marrow, cord blood, or apheresis product. The solution may include at least 70% of the MNCs in the original whole blood, bone marrow, cord blood, or apheresis product (e.g. if the target RBC volume is set to 0.6 ml in the output). In some aspects, the solution may include at least 95% of the MNCs in the original whole blood, bone marrow, cord blood, or apheresis product. In some aspects, the solution includes 2-30% of the RBC's in the original whole blood, bone marrow, cord blood, or apheresis product. In some aspects, the solution is isolated from the whole blood, bone marrow, cord blood, or apheresis product during a single two-step

centrifugation cycle. In some aspects, the solution is isolated using a single disposable cartridge. In some aspects, at least 95% of the RBC's are depleted from the sample by the methods and devices described above. In some aspects, the methods and devices disclosed herein do not require the use of a density gradient medium or other buffer. The solution may include at least

70% MNCs by weight.

[00189] In some implementations, the methods and devices may be used to deplete at least

90% of the RBCs from a sample of whole blood, bone marrow, or cord blood. The depletion may occur under more than one gravity of acceleration, within a single disposable device, and without the use of a density gradient medium or a buffer.

Example 1

[00190] Horse blood was drawn one day prior to being run in prototype devices disclosed herein. Heparin was added. The valve 139 was set to be 45% open in the valve open positions described above. The detected light threshold was set to 12, and the circuitry was configured to smart-predict the number of extra sips required to reach the target remaining RBC volume of 0.6 mL. In other words, the device was configured such that 0.6 mL of the RBC layer would be present in the cell container 300 and a final volume within the cell container was set to 21 mL. 150 mL of the horse blood was loaded into each of six different disposable sample containers each in the configured devices. The prototype devices were spun at 1400 RCF for 40min and 80 RCF for 20 min on a Sorval 3BP. The results are shown in Table 1 below.

Table 1

[00191] Overall, the devices performed very well and delivered the expected amount of

RBC into the product bag. Device 4 had a battery performance issue and was not able to finish the separation process. Device 2 had lower than expected hematocrit (and therefore lower recovery). Average recovery for the 0.6 ml target was 0.5ml +/-0.12.

[00192] The disclosed devices, methods, and systems may be configured to recover various target volumes from one or more layers. In some embodiments recovery of one or more layers may be between about 0.05 mL and 30 mL, for example greater than about 0.04 mL, 0.05 mL, 0.06 mL, 0.07 mL, 0.08 mL, 0.09 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.4 mL, 1.3 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 5.5 mL, 6.0 mL, 6.5 mL, 7.0 mL, 7.5 mL, 8.0 mL, 8.5 mL, 9.0 mL, 9.5 mL, 10 mL, 11 mL, 15 mL, 20 mL, or 25 mL, and less than about 30 mL, 25 mL, 20 mL, 15 mL, 12 mL, 11 mL, 10 mL, 9.5 mL, 9.0 mL, 8.5 mL, 8.0 mL, 7.5 mL, 7.0 mL, 6.5 mL, 6.0 mL, 5.5 mL, 5.0 mL, 4.5 mL, 4.0 mL, 3.5 mL, 3.0 mL, 2.5 mL, 2.0 mL, 1.9 mL, 1.8 mL, 1.7 mL, 1.6 mL, 1.5 mL, 1.4 mL, 1.3 mL, 1.2 mL, 1.1 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.09 mL, 0.08 mL, 0.07 mL, 0.06 mL, or 0.05 mL.

Example 2

[00193] Using the same set-up of Example 1, the target RBC volume was set to 1.0 mL. The results are shown in Table 2 below.

Table 2

[00194] Overall, the devices performed very well and delivered the expected amount of

RBC into the product bag. Device 2 had lower than expected hematocrit (and therefore lower recovery) which was likely due to poor battery performance. Average recovery for the 1 mL target was 1.0mL +/-0.05

[00195] All of the devices took about the same number of calculated extra-sips (either 16 or 17). All devices were able to time their measuring sips such that at least 15 sips were taken before finding "flat," indicating that the buffy-plasma transition had been reached correctly. Larger numbers of measuring sips improve the circuity's ability to accurately calculate flow rates. All final RBC volumes were either exactly on-target, or slightly below target.

Example 3:

[00196] Three different peripheral blood samples were drawn one day prior on three separate processing days to being run in two prototype devices disclosed herein. The valve 139 was set partially open between 35% and 55% in the valve open position described above. The detected light threshold was set to 12, and the circuitry was configured to marked-predict the number of extra sips required to reach the target remaining RBC volume of 0.6 mL. In other words, the device was configured such that 0.6ml of the RBC layer would be present in the cell container 300 and a final volume within the cell container was set to 6 mL. 60ml up to 85ml of human peripheral blood was loaded in the disposable sample container. The prototype device was spun at 2000 RCF for 10 minutes and 85 RCF for 10 min on a Cesca POC Centrifuge. The results are show in Table 3 below:

Table 3

[00197] At 0.6ml RBC target volume the MNC % was on average 79.8% with a RBC depletion above 96%. If the starting volume remained at 60 ml, the total processing time was less than 15 minutes.

[00198] The foregoing description details certain embodiments of the systems, devices, and methods disclosed herein. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the devices and methods can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re- defined herein to be restricted to including any specific characteristics of the features or aspects of the technology with which that terminology is associated. The scope of the disclosure should therefore be construed in accordance with the appended claims and any equivalents thereof.

[00199] It will be appreciated by those skilled in the art that various modifications and changes may be made without departing from the scope of the described technology. Such modifications and changes are intended to fall within the scope of the embodiments, as defined by the appended claims. It will also be appreciated by those of skill in the art that parts included in one embodiment are interchangeable with other embodiments; one or more parts from a depicted embodiment can be included with other depicted embodiments in any combination. For example, any of the various components described herein and/or depicted in the Figures may be combined, interchanged, or excluded from other embodiments.

[00200] Those of skill would further appreciate that any of the various illustrative schematic drawings described in connection with the aspects disclosed herein may be

implemented as electronic hardware (e.g., a digital implementation, an analog implementation, or a combination of the two, which may be designed using source coding or some other technique), various forms of program or design code incorporating instructions, or combinations of both.

[00201] The various circuitry, controllers, microcontroller, or switches, and the like, that are disclosed herein may be implemented within or performed by an integrated circuit (IC), an access terminal, or an access point. The IC may comprise a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, electrical components, optical components, mechanical components, or any combination thereof designed to perform the functions described herein, and may execute codes or instructions that reside within the IC, outside of the IC, or both.

[00202] The functions described may be implemented in hardware, software, firmware, or any combination thereof. If implemented in software, the functions may be stored on or transmitted over as one or more instructions or code on a tangible, non-transitory computer- readable medium. Computer-readable media includes both computer storage media and communication media including any medium that facilitates transfer of a computer program from one place to another. A storage media may be any available media that can be accessed by a computer. By way of example, and not limitation, such computer-readable media can include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to carry or store desired program code in the form of instructions or data structures and that can be accessed by a computer. A computer-readable medium may be in the form of a non-transitory or transitory computer- readable medium. [00203] The above description is provided to enable any person skilled in the art to make or use embodiments within the scope of the appended claims. Various modifications to these aspects will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other aspects without departing from the scope of the disclosure. Thus, the present disclosure is not intended to be limited to the aspects shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.