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WO2025038491A1 - Systèmes et procédés d'analyse de fluides biologiques - Google Patents

Systèmes et procédés d'analyse de fluides biologiques Download PDF

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Publication number
WO2025038491A1
WO2025038491A1 PCT/US2024/041820 US2024041820W WO2025038491A1 WO 2025038491 A1 WO2025038491 A1 WO 2025038491A1 US 2024041820 W US2024041820 W US 2024041820W WO 2025038491 A1 WO2025038491 A1 WO 2025038491A1
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WIPO (PCT)
Prior art keywords
sample reception
reception region
equal
channel
sample
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WO2025038491A9 (fr
Inventor
Charles R. Mace
Allison J. TIERNEY
Keith BAILLARGEON
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Tufts University
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Tufts University
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Publication of WO2025038491A9 publication Critical patent/WO2025038491A9/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation

Definitions

  • Fluidic devices comprising porous materials are generally described.
  • the subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles. Some aspects of the present disclosure relate to fluidic devices.
  • the fluidic device comprises a first layer comprising a porous material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, and wherein the channel places the first sample reception region in fluidic communication with the second sample reception region; and wherein, upon application of a fluid comprising white blood cells and red blood cells to the first sample reception region, the porous material is configured to retain at least 75% of white blood cells upstream from the second sample reception region and allow transport of the red blood cells to the second sample reception region via the channel.
  • the fluidic device comprises a first layer comprising a porous material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, and wherein the channel places the first sample reception region in fluidic communication with the second sample reception region; and wherein, upon application of a fluid comprising white blood cells and pathogens to the first sample reception region, the porous material is configured to retain at least 75% of white blood cells upstream from the second sample reception region and allow transport of the pathogens to the second sample reception region via the channel.
  • the fluidic device comprises a first layer comprising a porous material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, and wherein the channel places the first sample reception region in fluidic communication with the second sample reception region, wherein: the second sample reception region is positioned along the channel greater than or equal to 10 mm away and less than or equal to 30 mm from the first sample reception region, the channel has a length greater than or equal to 10 mm and less than or equal to 42 mm, the channel has a thickness greater than or equal to 0.4 mm and less than or equal to 0.8 mm, the channel has a width greater than or equal to 2 mm and less than or equal to 10 mm, and the channel has a porosity greater than or equal to 50 and less than or equal to 90%.
  • the fluidic device comprises a first layer comprising a porous, absorbent material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, and wherein the channel places the first sample reception region in fluidic communication with the second sample reception region; and a second layer, wherein the second layer comprises a vertical transport region in fluidic communication with the first sample reception region and the second sample reception region, and wherein the first layer and/or the second layer comprises a nucleic acid stabilizer.
  • the fluidic device comprises a first layer comprising a porous, absorbent material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, wherein the channel places the first sample reception region in fluidic communication with the second sample reception region, and wherein the first layer comprises a nucleic acid stabilizer.
  • the fluidic device comprises a first layer comprising a porous, absorbent material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, wherein the channel places the first sample reception region in fluidic communication with the second sample reception region, and wherein the porous, absorbent material is configured to transport red blood cells therethrough to a higher degree than white blood cells.
  • the fluidic device comprises a first layer comprising a porous, absorbent material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, and wherein the channel places the first sample reception region in fluidic communication with the second sample reception region; and a second layer, wherein the second layer comprises a vertical transport region in fluidic communication with the first sample reception region and the second sample reception region, and wherein the first layer and/or the second layer comprises an RNA stabilizer.
  • the fluidic device comprises a first layer comprising a porous, absorbent material, wherein the first layer comprises a channel, a first sample reception region, and a second sample reception region, wherein the channel places the first sample reception region in fluidic communication with the second sample reception region, and wherein the first layer comprises an RNA stabilizer.
  • the method comprises transporting a fluid comprising white blood cells and red blood cells through a channel from a first sample reception region to a second sample reception region, wherein at least 75% of the white blood cells are retained upstream from the second sample reception region and a portion of the red blood cells is retained in or on the second sample reception region.
  • the method comprises transporting a fluid comprising white blood cells and pathogens through a channel from a first sample reception region to a second sample reception region, wherein at least 75% of the white blood cells are retained upstream from the second sample reception region and a portion of the pathogens is retained in or on the second sample reception region.
  • the method comprises transporting a fluid comprising white blood cells and red blood cells through a channel from a first sample reception region to a second sample reception region, wherein: the second sample reception region is positioned along the channel greater than or equal to 10 mm away and less than or equal to 30 mm from the first sample reception region, the channel has a length greater than or equal to 0.1 mm and less than or equal to 42 mm, the channel has a thickness greater than or equal to 0.4 mm and less than or equal to 0.8 mm, the channel has a width greater than or equal to 2 mm and less than or equal to 10 mm, and the channel has a porosity greater than or equal to 50 and less than or equal to 90%.
  • the method comprises transporting a blood sample comprising a plurality of white blood cells and a plurality of red blood cells through a fluidic device to a first region and a second region, wherein the second region is a sample reception region, wherein at least a first portion of the fluid is retained in or on the first region and a second portion of the fluid is retained in or on the second region, wherein the first portion of the fluid is enriched in white blood cells, and wherein the second portion of the fluid is enriched in red blood cells; and assaying the first region or the second region.
  • the method comprises transporting a fluid comprising a plurality of white blood cells and a plurality of red blood cells through a fluidic device to a first region and a second region, wherein the second region is a sample reception region, wherein at least a first portion of the fluid is retained in or on the first region and a second portion of the fluid is retained in or on the second region, wherein the first portion of the fluid is enriched in white blood cells, and wherein the second portion of the fluid is enriched in red blood cells; and assaying the first region or the second region for the bloodborne pathogen.
  • the method comprises laterally transporting a fluid comprising a plurality of cells through a channel, wherein: the channel is positioned in a first layer comprising a porous, absorbent material, the first layer further comprises a first sample reception region and a second sample reception region, the channel places the first sample reception region in fluidic communication with the second sample reception region, and the first layer comprises an RNA stabilizer.
  • the method comprises laterally transporting a fluid comprising a plurality of cells through a channel, wherein: the channel is positioned in a first layer comprising a porous, absorbent material, the first layer further comprises a first sample reception region and a second sample reception region, the channel places the first sample reception region in fluidic communication with the second sample reception region, and the first layer comprises a nucleic acid stabilizer.
  • FIG. 1A is a schematic showing a cross-section of a fluidic device, according to some embodiments.
  • FIG. 1B is a schematic showing a plan view of a fluidic device, according to some embodiments.
  • FIG. 1C is a schematic showing a plan view of a fluidic device comprising a channel extending beyond a second sample reception region, according to some embodiments.
  • FIG. 2A is a schematic showing a cross-section of a fluidic device comprising two layers, according to some embodiments.
  • FIG. 2B is a schematic showing a cross-section of a fluidic device comprising two layers, according to some embodiments.
  • FIG. 3 is a schematic showing a cross-section of fluidic device comprising three layers, according to some embodiments.
  • FIG. 4 is a diagram showing DBS cards for collection and enrichment of Plasmodium nucleic acids in infected RBCs (iRBC) by depletion of host WBCs, wherein Whatman 903 cards (left) offer negligible cell separation, while pDBS cards with either 1 (middle) or 2 layers (right) of Leukosorb substantially purify iRBCs, according to some embodiments.
  • FIG. 5 is a plot showing relatively successful amplification of P. falciparum RNA isolated from punches of pDBS cards, according to some embodiments.
  • FIG. 6A is a diagram depicting renderings of device pre- and post-sample addition showing the multi-layer, multi-material design containing white blood cell-depleting inlet zone (arrow) and channel, as well as an outlet zone that can be excised for downstream analysis, according to some embodiments.
  • FIG. 6B is an image of a fluidic device, according to some embodiments.
  • FIG. 7 is a qualitative analysis of an exemplary fluidic device to assess white blood cell depletion wherein white blood cells were isolated, stained with DiI, returned to (a) buffer or (b) whole blood, and analyzed using a fluorescence microscope and wherein white blood cells in buffer and in whole blood were depleted from the sample as the sample wicked along the device, according to some embodiments.
  • FIG. 8 depicts data related to qPCR quantification (CT value) of human 18s rRNA (leukocyte) target, according to some embodiments.
  • FIG. 9 depicts data related to ⁇ C T Values (C T device – C T whole blood ) human 18s rRNA (leukocyte) target, according to some embodiments.
  • FIG. 10 depicts data related to the preparation of various parasitemia ranges using synchronized culture and whole blood, according to some embodiments.
  • FIG. 11A depicts a rendering of card prototype 1 with a two-layer design: a bottom layer of wax-patterned TFN channel and a top layer with a 7 mm TFN circle, according to some embodiments.
  • FIG. 11B depicts a scanned image of prototype 1, according to some embodiments.
  • FIG. 11C depicts filling across 25–55% hematocrit of prototype 1 having a 6 mm punch zone denoted by the arrow, according to some embodiments.
  • FIG. 12A depicts a rendering of prototype 2 with a two-layer design: a bottom layer of wax-patterned TFN channel and a top layer with a 7 mm Leukosorb circle, according to some embodiments.
  • FIG. 12B is an image of prototype 2, according to some embodiments.
  • FIG. 12C depicts filling across 25–55% hematocrit of prototype 2 having a 6 mm punch zone is denoted by the arrow, according to some embodiments.
  • FIG. 13A depicts a rendering of prototype 3 with a three-layer design: a bottom layer of wax-patterned TFN channel and two top layers with 7 mm Leukosorb circles, according to some embodiments.
  • FIG. 13B is an image of prototype 3, according to some embodiments.
  • FIG. 13C depicts filling across 25–55% hematocrit of prototype 3 having a 6 mm punch zone is denoted by the arrow, according to some embodiments.
  • FIG. 14A depicts a rendering of prototype 4 with a laser cut, single layer Leukosorb channel, according to some embodiments.
  • FIG. 14B is an image of prototype 4, according to some embodiments.
  • FIG. 14C depicts filling across 25–55% hematocrit of prototype 3 having a 6 mm punch zone is denoted by the arrow, according to some embodiments.
  • FIG. 15 depicts data related to the analysis of Punch Volume using Drabkin’s Assay, according to some embodiments.
  • FIG. 16A depicts a plot representing amplification curves for human ⁇ -actin and Plasmodium sbp1 genes, according to some embodiments.
  • FIG. 16B depicts a plot representing melt curves for human ⁇ -actin and Plasmodium sbp1 genes, according to some embodiments.
  • FIG. 17 depicts data related to an analysis of WBC Depletion from 903 Card, Prototype 3, and Prototype 4 (LDC), using B-actin, according to some embodiments.
  • FIG. 18 depicts data related to raw Ct and calculated ⁇ Cts, ⁇ Cts, and fold change for in vitro contrived samples, according to some embodiments.
  • FIG. 20 depict data related to patient demographics of clinical study population in order of ascending parasite count, according to some embodiments.
  • FIG. 21 depict scan images of Whatman 903 Protein Saver Cards from clinical patients 01–16 wherein 50 ⁇ L venous blood was added to each spot by pipette, according to some embodiments.
  • FIG. 22 depicts scan images LDCs from clinical patients 01–16. 50 ⁇ L venous blood was added to each inlet zone by pipette, according to some embodiments.
  • FIG. 23 depicts data related to uncorrected Ct values for clinical samples in order of ascending parasite count. according to some embodiments.
  • FIG. 24 depicts data related to corrected Cts for clinical samples in order of ascending parasite count, according to some embodiments.
  • FIG. 25 depicts data related to ⁇ Cts and ⁇ Cts using corrected Ct values, according to some embodiments.
  • FIG. 26C depicts data related to an analysis of covariance (ANCOVA) of linear regressions, according to some embodiments.
  • FIG. 28B is a plot describing liquid calibration curves for parasite counts obtained using a serially diluted liquid clinical sample (
  • FIG. 30 depicts data related to clinical WBC recovery from 903 Card and LDC, according to some embodiments.
  • FIG. 31 depicts data related to clinical parasite recovery from 903 Card and LDC, according to some embodiments.
  • FIG. 32 depicts data related to clinical WBC and parasite recovery from 903 Card and LDC, according to some embodiments.
  • FIG. 33A is a plot depicting sample read count from Illumina sequencing as a function of parasites ⁇ L-1 wherein patients 09, 02, and 11 were analyzed to cover a wide range of parasite counts (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001), according to some embodiments.
  • FIG. 33B is a plot depicting sample read coverage from Illumina sequencing as a function of parasites ⁇ L-1 wherein patients 09, 02, and 11 were analyzed to cover a wide range of parasite counts (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001) and read coverage was calculated by dividing the read count by the number of probes (185) in each sample, according to some embodiments.
  • FIG. 34 is a diagram depicting malaria pDBS card design iterations, according to some embodiments.
  • FIG. 35A is a scanned image of card B, according to some embodiments.
  • FIG. 35B is a rendering of card B, according to some embodiments.
  • FIG. 35A is a scanned image of card B, according to some embodiments.
  • FIG. 35B is a rendering of card B, according to some embodiments.
  • FIG. 35C is a rendering of a single channel of laser cut TFN, according to some embodiments.
  • FIG. 35D is a Drabkin’s assay analysis to determine extraction zone volume and shown generally no significant difference between hematocrits, according to some embodiments.
  • FIG. 36A is a rendering of card A, according to some embodiments.
  • FIG. 36B is a diagram of card A comprising two layers of Leukosorb and a single layer of TFN, according to some embodiments.
  • FIG. 36C is a plot depicting the results of a Drabkin’s assay analysis to determine extraction zone volume, according to some embodiments.
  • FIG. 37 shows WBC depletion analyzed by qPCR for card A, card B, and 903 card, according to some embodiments.
  • FIG. 38 depicts plots related to the matched volume of liquid reference samples, according to some embodiments.
  • FIG. 39 depicts data related to WBC depletion and iRBC movement, according to some embodiments.
  • FIG. 40 depicts data related to WBC depletion and iRBC movement specifically to difference in Ct value between genes and between sample types as well as iRBC enrichment in Card B compared to reference samples and a 903 card, according to some embodiments.
  • FIG. 41 depicts instrument cycling conditions when analyzing 903 cards and LDC with a QuantStudio3 Real Time PCR system, according to some embodiments.
  • DETAILED DESCRIPTION Malaria remains one of the greatest threats to health stability throughout the tropical regions in Africa, South America, and Asia.
  • DBS dried blood spot
  • DBS cards Separate zones on some DBS cards are loosely defined by a dashed circle to guide manual sample application by a user, which often results in heterogeneous sample spots that vary in quality—the distribution of cells is not controllable.
  • biopsy punches may be used to isolate sample discs for processing (e.g., sample elution). Contamination of samples with host DNA can be a challenging obstacle, especially in limited resource settings.
  • host genetic material e.g., white blood cells (WBCs)
  • WBCs white blood cells
  • blood with a low hematocrit i.e., high plasma volume
  • a higher hematocrit i.e., low plasma volume
  • Inconsistencies in sample processing using DBS cards may limit the kinds of samples and analyses that are possible once cards are delivered to a lab.
  • preservation methods such as treatment with protein denaturation reagents can limit later analysis (e.g., by preventing serological analysis).
  • Improved drying times may also be desirable, since without wishing to be bound by any particular theory, long drying times can lead to degradation of nucleic acids and make measurements no longer quantitative or commensurate with the standard liquid blood sample.
  • the devices provided herein may use porous materials to transport fluids by wicking. In some embodiments, this form of fluid transport helps make the devices suitable for operation in field settings where RDT or DBS cards may be used.
  • the devices may be assembled from multiple layers of patterned paper and other porous materials and may be configured to allow segregation of blood components, including red cells, white blood cells and plasma.
  • the devices provided herein may improve sensitivity and specificity of detection for a wide range of blood- borne pathogens, including but not limited to malaria, including Leishmania, Babesia, trypanosomes, filarial diseases, and innumerable viral blood-borne pathogens.
  • Each layer in the device can be modified to either allow or impede the transport of specific types of cells in whole blood in a single step without the use of external equipment.
  • a drop of blood may be applied to the device to initiate a separation without further input from a user.
  • a device provided herein may allow the white cells in a blood sample to quickly and efficiently be separated from the red cells while remaining available for subsequent analysis.
  • the separation of red and white blood cells may have advantages for next- generation sequencing approaches, since without wishing to be bound by any particular theory, contamination with human genomic DNA or ribonucleic acid (RNA) may hamper the detection of less-abundant parasite genomes in blood samples.
  • fluidic devices provided herein comprise one or more layers (e.g., a first layer) comprising a porous material.
  • the device comprises a first layer comprising two or more sample receptions regions (e.g., a first sample reception region and a second sample reception region) wherein the sample reception regions are in fluidic communication with each other through a channel.
  • the first sample reception region is configured to receive a fluid (e.g., a sample) comprising white blood cells and red blood cells.
  • the porous material configured to retain at least a portion of the white blood cells and allow the transport of at least a portion of the red blood cells, may allow for the fluid to flow through the fluidic device such that the second sample reception region is enriched with red blood cells after application of the fluid to the first sample reception region.
  • Any of variety of properties of the flood may be determined by evaluating (e.g., via an assay) the red blood cells in the second sample reception region, as discussed elsewhere in this disclosure.
  • FIG. 1A shows a cross-section of fluidic device 100.
  • Fluidic device 100 comprises first layer 105.
  • first layer 105 comprises a porous material which will be discussed in detail below.
  • First layer 105 comprises first sample reception region 110 in fluidic communication with second sample reception region 120 via channel 115. That is, first sample reception region 110 is in fluidic communication with channel 115, and channel 115 is in fluidic communication second sample reception region 120 thereby placing first sample reception region 110 in fluidic communication with second sample reception region 120. Accordingly, upon application of a fluid to first sample reception region 110, the fluid may flow from first sample reception region 110 through the channel 115 into second sample reception region 120.
  • a first sample reception region and a second sample reception region are positioned apart such that a channel is positioned therebetween. For example, as shown in FIG.
  • first sample reception region 110 is positioned apart from second sample reception region 120 such that channel 115 is positioned therebetween and in fluidic communication with the first and second sample reception region.
  • FIG. 1B shows a plan view of fluidic device 100, according to some embodiments.
  • cells e.g., white blood cells
  • a portion of a channel is positioned between a first and second sample reception region while another portion of the channel extends beyond the second sample reception region.
  • first portion 115A of channel 115 is positioned between first sample reception region 110 and second sample reception region 120 while second portion 115B of channel 115 extends beyond second sample reception region 120.
  • the second portion of the channel extending beyond the second sample reception region may extend in a direction that is substantially parallel to the direction of flow within the fluidic device (e.g., direction 125).
  • the channel may extend beyond the second sample reception region to limit concentration of one or more types of cells (e.g., white blood cells and/or red blood cells) in an irregular manner across the second sample reception region relative to the concentration of cells across the channel and/or the first sample reception region.
  • a channel extends through at least a portion of the thickness of one or more layers of a fluidic device.
  • the channel extends through at least a portion of the thickness of the first layer. For example, as shown in FIG. 1A, channel 115 extends through the entire thickness T1 of first layer 105. When first sample reception region 110 receives the fluid, the fluid may flow from first sample reception region 110 to channel 115 throughout thickness T1 of first layer 105.
  • a fluidic device comprises exactly one layer, such as exactly one layer having one or more of the features shown in one or more of FIGS. 1A-1C (e.g., first and second sample reception regions and a channel).
  • fluidic devices described herein comprise more than one layer. For example, as shown in FIG.
  • fluidic device 200 comprises two layers. Fluidic device 200 comprises second layer 205 disposed on first layer 105. As used herein, when a layer is referred to as being “on” or “disposed on” another layer, it can be directly disposed on the layer, or an intervening layer also may be present. A layer that is “directly on” or “directly disposed on” another layer is positioned with respect to the layer such that no intervening layer is present.
  • Second layer 205 as shown in FIG. 2A, comprises vertical transport region 210 in fluidic communication with first sample reception region 110 and second sample reception region 120 (the latter via first sample reception region 110 and channel 115). As also shown in FIG.
  • the vertical transport region 210 is positioned between an environment external to the fluidic device and the first sample reception region.
  • a fluid may be applied to vertical transport region 210, rather than directly to first sample reception region 110. The fluid may then flow from vertical transport region 210 to first sample reception region 110 through channel 115 to second sample reception region 120.
  • the vertical transport region may serve as a filter for cells (e.g., white blood cells) as described elsewhere in this disclosure.
  • a second layer, and/or any additional layers may be disposed on a first layer such that only a portion of the first layer overlaps the second layer (i.e., is positioned between the first layer and an environment external to the fluidic device) and/or any additional layers.
  • first layer 105 overlaps second layer 205.
  • all of the first layer may overlap the second layer and/or any additional layers.
  • a surface of the second layer and/or any additional layers may have the same or a different overall geometric size compared to the first layer.
  • a second layer partially or fully overlaps a first layer, it may overlap one or more sample reception regions therein (e.g., a first sample reception region, a second sample reception region) and/or a channel therein.
  • fluidic devices herein comprise three or more layers. For example, as shown in FIG.
  • fluidic device 300 comprises second layer 205 disposed on first layer 105.
  • Third layer 305 is disposed on second layer 205. Similar to second layer 205, third layer 305 comprises second vertical transport region 310 in fluidic communication with first vertical transport region 210, first sample reception region 110, channel 115, and second sample reception region 120. Similar to the first vertical transport region, in some embodiments, the fluid may be applied to the second vertical transport region such that a portion of the fluid flows through the fluidic device (e.g., through the first vertical transportation region, first sample reception region, and the channel) to the second sample reception region.
  • layers of a fluidic device comprise the porous material.
  • the porous material may, upon exposure to a fluid sample, wick the fluid sample into the layer and/or wick the fluid sample through the layer.
  • layers comprising channels comprise a porous, absorbent material
  • the porous, absorbent material may wick the fluid sample into the channels therein and/or through the channels therein.
  • a fluid may flow into and/or through a porous material due to capillarity (capillary action) or by wicking.
  • a fluid sample may flow into and/or through a porous material due to capillarity.
  • a porous material will, upon exposure to a fluid sample (e.g., a fluid sample of interest, a fluid sample for which it is absorbent), transport the fluid sample into the interior of the porous material (i.e., the fluid sample may penetrate into the interior of the material in which the pores are positioned, such as into the interior of fibers making up a porous material that comprises fibers).
  • a porous material will, upon exposure to a fluid sample, experience an increase in mass due to the fluid sample absorbed therein. It should be understood that some layers comprising porous absorbent materials may have one or more of the properties described above with respect to porous materials.
  • a porous material is configured to retain a portion of white blood cells.
  • the white blood cells when a fluid comprising white blood cells is provided to the fluidic device (e.g., via the first sample reception region), a portion of the white blood cells are retained within the porous material.
  • the white blood cells may be filtered and/or separated from the rest of the fluid such that, as the fluid flows through the fluidic device, white bloods cells immobilize within the porous material prior to the fluid reaching the second sample reception region. That is, white blood cells may not flow through the porous material.
  • the fluid in the second sample reception region may comprise a lower amount of white blood cells than the amount of white blood cells in the fluid when initially provided to the fluidic device.
  • the first sample reception region, the channel, and/or any vertical transport regions, each comprising the porous material may be enriched with white bloods cells while the second sample reception region may be relatively depleted of white blood cells.
  • the separation of white blood cells from the fluid may advantageously facilitate further processing (e.g., assaying) of the second sample reception region.
  • a porous material is configured to retain a relatively large amount of white blood cells upstream from the second sample reception region upon application of a sample comprising red and white blood cells thereto.
  • some methods comprise retaining a relatively large amount of white blood cells upstream from the second sample reception region upon application of a sample comprising red and white blood cells thereto.
  • the porous material is configured to retain greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 99%, or greater than or equal to 99.9% of white blood cells upstream from the second sample reception region. In some embodiments, the porous material is configured to retain less than or equal to 99.9%, less than or equal to 99%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, or less than or equal to 75% of white blood cells upstream from the second sample reception region.
  • a porous material is configured to allow transport of red blood cells to a second sample reception region via a channel. That is, the porous material may allow for red blood cells in the fluid to flow through the fluidic device, without being immobilized, such that at least a portion of the red blood cells in the fluid are capable of moving through the porous material to enter the second sample reception region.
  • the porous material may promote the immobilization of white blood cells while allowing for the flow and/or transport (e.g., movement) of red blood cells.
  • the porous material may be configured to transport red blood cells therethrough to a higher degree than white blood cells.
  • the separation of white blood cells and red blood cells in a fluidic device may facilitate relatively greater efficiency for further processing (e.g., genetic sequencing) as host contamination of the sample (e.g., a punch of the second sample reception region) may be generally limited.
  • the porous material may allow for the transport of, in addition to red blood cells, pathogens (and/or nucleic acids and/or other genetic materials derived from pathogens) that may be present in the fluid to the second sample reception region.
  • a porous material is configured to allow red blood cells in a fluid to flow (e.g., move) through the device away from the first sample reception region.
  • the porous material is configured to allow transport of greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 99%, or greater than or equal to 99.9% of red blood cells in the fluid to flow through the device away from the first sample reception region.
  • the porous material is configured to allow transport of less than or equal to 99.9%, less than or equal to 99%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80% of red blood cells in the fluid to flow through the device away from the first sample reception region. Combinations of these ranges are possible (e.g., greater than or equal to 80% and less than or equal to 99.9%). Other ranges are also possible.
  • a porous material has a relatively high porosity.
  • the median pore size of the porous material is greater than or equal to 5 micrometers, greater than or equal to 10 micrometers, greater than or equal to 15 micrometers, greater than or equal to 20 micrometers, greater than or equal to 25 micrometers, greater than or equal to 30 micrometers, greater than or equal to 35 micrometers, or greater than or equal to 40 micrometers.
  • the median pore size of the porous material is less than or equal to 40 micrometers, less than or equal to 35 micrometers, less than or equal to 30 micrometers, less than or equal to 25 micrometers, less than or equal to 20 micrometers, less than or equal to 15 micrometers, less than or equal to 10 micrometers, less than or equal to 5 micrometers. Combinations of these ranges are possible (e.g., greater than or equal to 5 micrometers and less than or equal to 40 micrometers). Other ranges are also possible.
  • a porous material has any of a variety of suitable thicknesses. In some embodiments, the porous material has a relatively large thickness.
  • the porous material has a thickness of greater than or equal to 300 micrometers, greater than or equal to 400 micrometers, greater than or equal to 500 micrometers, greater than or equal to 600 micrometers, or greater than or equal to 700 micrometers. In some embodiments, the porous material has a thickness of less than or equal to 700 micrometers, less than or equal to 600 micrometers, less than or equal to 500 micrometers, less than or equal to 400 micrometers, or less than or equal to 300 micrometers. Combinations of these ranges are possible (e.g., greater than or equal to 300 micrometers and less than or equal to 700 micrometers). Other ranges are also possible. In some embodiments, a porous material comprises any of a variety of suitable compositions.
  • the porous material comprises a material configured to retain a portion of white blood cells (e.g., while allowing red blood cells to flow and/or move therethrough).
  • the porous material comprises a polyester having this property.
  • the porous material comprises a partially hydrolyzed polyester.
  • the porous material comprises a white blood cell isolation medium and/or a white blood cell filter such as Leukosorb.
  • the porous material comprises cellulose acetate and/or a cellular acetate filter.
  • a wide variety of porous materials may be used for this purpose such as those manufactured by Pall Corporation, those manufactured by Sartorius, and/or those manufactured by Cytiva.
  • the porous material comprises a plurality of fibers that may allow for advantageous connectivity of pores within the porous material.
  • the porous material may be functionalized and/or subjected to any of a variety of chemical treatments that may alter the surface properties of the porous material.
  • the porous material when exposed to the fluid, functions in a manner similar to a size exclusion filter. That is, a portion of the fluid (e.g., white blood cells) having a size greater than a threshold may be retained by the porous material while a portion of the fluid have a size less than the threshold (e.g., red blood cells) may flow through the porous material.
  • the porous material is a membrane and/or a polymer mesh.
  • a porous material comprises a material that allows white blood cells to flow therethrough.
  • the porous material may comprise a synthetic material and/or a glass.
  • suitable synthetic materials include poly(ether sulfone), polyesters, and nylons.
  • a porous material that allows white blood cells to flow therethrough is a cellulose-based material.
  • the cellulose-based material may comprise cellulose derived from wood (e.g., it may be a wood-based material), cellulose derived from cotton (e.g., it may be a cotton-based material), and/or nitrocellulose.
  • Porous materials described herein may have a variety of designs.
  • a fluidic device comprises a porous material that is a fibrous material (e.g., a fibrous material comprising fibers formed from a cellulose-based material).
  • the fibrous material may be a non- woven material, or may be a woven material.
  • the fibers may have a variety of suitable diameters and distributions of diameters, and, if woven, may be woven in a variety of suitable weaves.
  • the non-woven material is a paper. For instance, in designs that are configured to allow white blood cells to flow through the porous material, cellulose-based papers may be used.
  • a porous material may allow for relatively quick drying times.
  • the sample when a 50 microliter sample of the fluid is provided to the first sample reception region, the sample is capable of drying less than or equal to 60 minutes, less than or equal to 55 minutes, less than or equal to 50 minutes, less than or equal to 45 minutes, less than or equal to 40 minutes, less than or equal to 35 minutes, or less than or equal to 30 minutes while in an environment having a relative humidity of 40%.
  • the above- described ranges may characterize the porous material upon application of a sample having a hematocrit of greater than or equal to 25% and less than or equal to 55% thereto.
  • the sample when a 50 microliter sample of the fluid is provided to the first sample reception region, the sample is capable of drying greater than or equal to 30 minutes, greater than or equal to 35 minutes, greater than or equal to 40 minutes, greater than or equal to 45 minutes, greater than or equal to 50 minutes, greater than or equal to 55 minutes, or greater than or equal to 60 minutes while in an environment having a relative humidity of 40%.
  • the above-described ranges may characterize the porous material upon application of a sample having a hematocrit of greater than or equal to 25% and less than or equal to 55% thereto. Combinations of these ranges are possible (e.g., greater than or equal to 30 minutes and less than or equal to 60 minutes). Other ranges are also possible.
  • the aforementioned drying times may be achieved in an environment having any of a variety of suitable relative humidities. In some embodiments, the environment may have a relative humidity greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 35%, and greater than or equal to 40%.
  • the environment may have a relative humidity less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, or less than or equal to 20%. Combinations of these ranges are possible (e.g., greater than or equal to 20% and less than or equal to 40%). Other ranges are also possible.
  • a porous material may retain one or more types of cells electrostatically.
  • the porous material may separate portions of the fluid based on electrostatic charge differentials between portions of the fluid (e.g., red and/or white blood cells, pathogens) and the porous material.
  • the porous material may retain white blood cells via electrostatic attraction between the porous material and the white blood cells.
  • the porous material may allow for red blood cells to flow as there may be little to no electrostatic attraction between the red blood cells and the porous material.
  • the retention and/or flow of cells through the fluidic device may, at least in part, be electrostatically mediated.
  • a porous material described herein may have any of a variety of suitable porosities.
  • the porous material has an advantageously high porosity.
  • the porous material has a porosity greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%.
  • the porous material has a porosity less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, or less than or equal to 50%. Combinations of these ranges are possible (e.g., greater than or equal to 50% and less than or equal to 90%). Other ranges are also possible.
  • a fluidic device comprises a porous material that is hydrophilic and/or may comprise a layer that is hydrophilic (e.g., a layer comprising a hydrophilic porous material).
  • the hydrophilic material or layer may have a water contact angle of less than or equal to 90°, less than or equal to 85°, less than or equal to 80°, less than or equal to 75°, less than or equal to 70°, less than or equal to 65°, less than or equal to 60°, less than or equal to 55°, less than or equal to 50°, less than or equal to 45°, less than or equal to 40°, less than or equal to 35°, less than or equal to 30°, less than or equal to 25°, less than or equal to 20°, less than or equal to 15°, less than or equal to 10°, or less than or equal to 5°.
  • the hydrophilic material or layer may have a water contact angle of greater than or equal to 0°, greater than or equal to 5°, greater than or equal to 10°, greater than or equal to 15°, greater than or equal to 20°, greater than or equal to 25°, greater than or equal to 30°, greater than or equal to 35°, greater than or equal to 40°, greater than or equal to 45°, greater than or equal to 50°, greater than or equal to 55°, greater than or equal to 60°, greater than or equal to 65°, greater than or equal to 70°, greater than or equal to 75°, greater than or equal to 80°, or greater than or equal to 85°.
  • the water contact angle of a hydrophilic material or layer may be measured using ASTM D5946-04, which comprises positioning a water droplet on a planar solid surface of the hydrophilic material or layer.
  • the water contact angle is the angle between the planar solid surface of the hydrophilic material or layer and the tangent line drawn to the water droplet surface at the three-phase point.
  • a contact angle meter or goniometer can be used for this determination.
  • the hydrophilicity of the hydrophilic material or layer may be such that a water droplet placed on the surface completely wets the surface (e.g., the water droplet is completely absorbed into the material, making the water contact angle 0°).
  • a device may comprise a porous material that is hydrophobic and/or may comprise a layer that is hydrophobic. The hydrophobic material or layer may have a water contact angle outside the ranges described above.
  • a porous material described herein is a porous, absorbent material.
  • the porous, absorbent material may be absorbent and also have any of the properties of the porous material described herein.
  • the porous, absorbent, material upon exposure to a fluid sample, wick the fluid sample into the material itself (e.g., in addition to wicking the fluid into any pores therein). For instance, if a porous, absorbent material is fibrous, it may wick a fluid sample into the fibers therein.
  • a porous material described herein is non-absorbent. Such porous, non-absorbent materials, in some embodiments, do not, upon exposure to a fluid sample, wick the fluid sample into the material itself. Porous, non-absorbent materials may, however, wick the fluid sample into pores therein and/or therethrough (e.g., via pores therein).
  • fluidic devices described herein comprise a first sample reception region.
  • the first sample reception region is configured to receive the fluid either directly or via one or more vertical transport regions.
  • the first sample reception region comprises the porous material, and accordingly, the first sample reception region may transport a portion of the fluid through the fluidic device while immobilizing white blood cells.
  • the first sample reception region may be enriched with white bloods cells. That is, the first sample reception region may have a higher concentration of white bloods cells than in one or more other portions of the fluidic device, such as the second sample reception region.
  • the first sample reception may comprise a material other than the porous material forming the channel.
  • the first sample reception region in accordance with certain embodiments, may comprise cellulosic paper and the porous material may be a material other than cellulosic paper or may be a different type of cellulosic paper.
  • fluidic devices described herein comprise a second sample reception region.
  • the second sample reception region is enriched with one or more components of the fluid.
  • red blood cells and/or when the fluid initially comprises pathogens, pathogens (e.g., bloodborne pathogens).
  • pathogens e.g., bloodborne pathogens.
  • white blood cells may be immobilized by the porous material of the fluidic device, as described above, thereby enriching the fluid that enters the second sample reception region with red blood cells and/or pathogens.
  • a portion of the red blood cells and/or pathogens may be retained in or on the second sample region.
  • the biological material When biological material (e.g., red blood cells, white blood cells, and/or pathogens) are retained in or on a region of the fluidic device, the biological material may be positioned in an interior of the region such that the biological material is within the geometric confines of the corresponding layer of the fluidic device where the region resides and/or the geometric confines of the region itself.
  • the biological material retained in or on the region may be disposed on the region, positioned within pores of the region, and/or wicked into the interior of any absorptive component (e.g., the porous material) of the region such that the biological material is immobilized in the interior of the region.
  • the second sample region may, in some such embodiments, be enriched with red blood cells and/or pathogens (if initially present in the fluid) retained in or on the second sample region (e.g., in comparison to another region of the fluidic device).
  • the red blood cells and/or pathogens may be retained on the second sample reception region after drying of the second sample region.
  • the second sample reception region provides a region of the fluidic device that may be punched such that the second sample reception region may be removed from the fluidic device for further analysis.
  • the second sample reception region may be considered a punch region where a portion of the fluidic device is removed from further processing (e.g., assaying).
  • fluidic devices described herein having a second sample reception region enriched with red blood cells and/or pathogens may be advantageous.
  • the enrichment of the second sample reception region with red blood cells and/or pathogens may facilitate assaying of the second sample reception region with assays involving red blood cells and/or pathogens (e.g., Drabkin’s assay) with limited contamination from white blood cells.
  • the enrichment of the first sample reception region with white blood cells may facilitate assaying of the first sample reception region with assay involving white blood cells with limited contamination from red blood cells and/or pathogens.
  • a fluidic device may comprise a first sample reception region that is enriched with pathogens.
  • a sample applied to a fluidic device described herein may comprise a pathogen that is retained by a porous material present in a first sample reception region and/or a channel.
  • the fluidic device may serve to separate the pathogen from red blood cells.
  • the first sample reception region may be enriched in the pathogen.
  • a second sample reception region is positioned along a channel at any variety of suitable distances away from the first sample region.
  • first sample reception region 110 is positioned at distance D1 away from second sample reception region 120.
  • the second sample reception region is positioned along the channel greater than or equal to 10 mm, greater than or equal to 15 mm, greater than or equal to 20 mm, greater than or equal to 25 mm, or greater than or equal to 30 mm from the first sample reception region.
  • the second sample reception region is positioned along the channel less than or equal to 30 mm, less than or equal to 25 mm, less than or equal to 20 mm, less than or equal to 15 mm, or less than or equal to 10 mm from the first sample reception region. Combinations of these ranges are possible (e.g., greater than or equal to 10 mm and less than or equal to 30 mm). Other ranges are also possible.
  • a first and/or second sample reception region is configured to contain any of a variety of suitable volumes.
  • the first and/or second sample reception region is configured to contain a volume of greater than or equal to 40 microliters, greater than or equal to 45 microliters, greater than or equal to 50 microliters, greater than or equal to 55 microliters, greater than or equal to 60 microliters, greater than or equal to 65 microliters, or greater than or equal to 70 microliters of a fluid sample (e.g., a liquid sample).
  • a fluid sample e.g., a liquid sample.
  • the first and/or second sample reception region is configured to contain a volume of less than or equal to 70 microliters, less than or equal to 65 microliters, less than or equal to 60 microliters, less than or equal to 55 microliters, less than or equal to 50 microliters, less than or equal to 45 microliters, or less than or equal to 40 microliters of a fluid sample (e.g., a liquid sample). Combinations of these ranges are possible (e.g., greater than or equal to 40 microliters and less than or equal to 70 microliters). Other ranges are also possible.
  • a second sample reception region is positioned along a channel such that a portion of cells (e.g., red and/or white blood cells) in the fluid is distributed on the second sample reception region such that a concentration of cells in the second sample region is relatively similar to the concentration of the cells at another position along the channel outside the second sample region.
  • the concentration of red blood cells in the second sample region is greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 100%, greater than or equal to 110%, or greater than or equal to 120% of the concentration of the red blood cells at another position along the channel outside the second sample reception region.
  • the concentration of red blood cells in the second sample region is less than or equal to 120%, less than or equal to 110%, less than or equal to 100%, less than or equal to 90%, or less than or equal to 80% of the concentration of the red blood cells at another position along the channel outside the second sample reception region. Combinations of these ranges are possible (e.g., greater than or equal to 80% and less than or equal to 120%). Other ranges are also possible.
  • fluidic devices described herein comprise a channel. In some embodiments, the channel places the first sample reception region in fluidic communication with the second sample reception region such that a portion of the fluid, when provided to the fluidic device, may flow through the fluidic device to the second sample reception region.
  • the channel comprises the porous material thereby immobilizing at least some of the white blood cells in the fluid. Accordingly, in some embodiments, some or all of the channel may be enriched with white blood cells.
  • a channel of fluidic devices described herein may have any of a variety of suitable lengths. For example, as shown in FIG. 1C, channel 115 has length L1 extending from first sample reception region 110 to the end of channel 115. In some embodiments, where the channel does not extend beyond the second sample reception region, the length of the channel may correspond to the length between the first and second sample reception regions. For example, as shown in FIG. 1B, channel 115 has length L2 extending from first sample reception region 110 to second sample reception 120.
  • the channel has a length greater than or equal to 10 mm, greater than or equal to 15 mm, greater than or equal to 20 mm, greater than or equal to 25 mm, greater than or equal to 30 mm, greater than or equal to 35 mm, or greater than or equal to 42 mm. In some embodiments, the channel has a length less than or equal to 42 mm, less than or equal to 35 mm, less than or equal to 30 mm, less than or equal to 25 mm, less than or equal to 20 mm, less than or equal to 15 mm, or less than or equal to 10 mm. Combinations of these ranges are also possible (e.g., greater than or equal to 10 mm and less than or equal to 42 mm). Other ranges are also possible.
  • a channel is positioned between a first sample reception region and a second sample region, some embodiments of the fluidic devices described herein do not comprise the channel. That is, the first and second sample reception regions may not be positioned apart with a channel therebetween. In such embodiments, the length of the channel may correspond to the summation of the lengths of the first and second sample reception regions.
  • a channel of fluidic devices described herein may have any of a variety of suitable thicknesses. In some embodiments, the channel may have a thickness greater than or equal to 400 micrometers, greater than or equal to 500 micrometers, greater than or equal to 600 micrometers, greater than or equal to 700 micrometers, or greater than or equal to 800 micrometers.
  • the channel may have a thickness less than or equal to 800 micrometers, less than or equal to 700 micrometers, less than or equal to 600 micrometers, less than or equal to 500 micrometers, or less than or equal to 400 micrometers. Combinations of these ranges are possible (e.g., greater than or equal to 400 micrometers and less than or equal to 800 micrometers). Other ranges are also possible.
  • a channel of fluidic devices described herein may have any of a variety of suitable porosities. In some embodiments, the channel has a porosity greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%.
  • the channel has a porosity less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, or less than or equal to 50%. Combinations of these ranges are possible (e.g., greater than or equal to 50% and less than or equal to 90%). Other ranges are also possible.
  • a channel of fluidic devices described herein may have any of a variety of suitable widths. For example, as shown in FIG. 1B, channel 115 has width W1.
  • the width of the channel may influence flow (e.g., flow may be arrested if the width of the channel is unsuitably narrow or flow may be slow if the width of the channel is unsuitably large).
  • the channel has a width greater than or equal to 2 mm, greater than or equal to 3 mm, greater than or equal to 4 mm, greater than or equal to 5 mm, greater than or equal to 6 mm, greater than or equal to 7 mm, or greater than or equal to 8 mm. In some embodiments, the channel has a width less than or equal to 8 mm, less than or equal to 7 mm, less than or equal to 6 mm, less than or equal to 5 mm, less than or equal to 4 mm, less than or equal to 3 mm, or less than or equal to 2 mm. Combinations of these ranges are also possible (e.g., greater than or equal to 2 mm and less than or equal to 8 mm). Other ranges are also possible.
  • the fluid when exposed to a volume of a fluid comprising whole blood that is greater than or equal to a volume of a channel, the fluid fills both first and second sample reception regions. In some embodiments, when exposed to a volume of whole blood that is greater than or equal to a volume of the channel and that has a hematocrit value of greater than or equal to 25% and less than or equal to 55%, the whole blood fills both the first and second sample reception regions. In some embodiments, when exposed to a volume of whole blood that has a hematocrit value of greater than or equal to 25% and less than or equal to 55%, the amount of the second sample reception region filled by the whole blood is substantially independent of the hematocrit value.
  • fluids may be provided to fluidic devices described herein.
  • the fluid comprises a biological fluid (e.g., blood).
  • the fluid comprises whole blood.
  • the fluid comprises white blood cells and red blood cells.
  • the fluid comprises pathogens.
  • the fluid comprises white blood cells and pathogens.
  • the fluid comprises white blood cells, red blood cells, and pathogens.
  • the fluid comprises a bloodborne pathogen and/or biological material-derived from a bloodborne pathogens.
  • the fluidic device may advantageously facilitate the detection of bloodborne pathogens in a blood sample by separating white blood cells, which may interrupt and/or interfere with the detection of the bloodborne pathogens, from the rest of the fluid.
  • the fluid further comprises nucleic acids from one or more pathogens (e.g., parasites, bacteria, viruses).
  • the bodily fluid comprises bloods, tears, saliva, wound exudate, urine, cerebrospinal fluid, and/or sweat.
  • the liquid comprises a fluid derived from the bodily fluid (e.g., plasma derived from blood).
  • the liquid comprises an aqueous solution.
  • fluids may be provided to fluidic devices described herein.
  • the fluid may be provided to the first sample reception region and/or to any vertical transport regions of the fluidic device.
  • a volume of fluid may be provided to the fluidic device sufficient for the fluid to through the channel to the second sample reception region such that the concentration of cells (e.g., red blood cells) is not significantly different than the concentration of cells at a location in the channel proximate to the second sample reception region.
  • fluidic devices described herein comprise a second layer.
  • the second layer comprises a porous material.
  • the second layer comprises the same material (e.g., the porous absorbent material) as the first layer. In some embodiments, the second layer comprises a porous material that is different than the first layer. In some embodiments, the second layer may guide and/or direct flow of the fluid through the fluidic device as the second layer may comprise wax portions that may not wet in the presence of the fluid. The wax portions may then direct the fluid to wet other portions of the first and/or second layer comprising a porous material. In some embodiments, the second layer comprises a vertical transport region in fluidic communication with the first sample reception region and the second sample reception region.
  • a fluid may be provided to the vertical transport region of the fluidic device such the vertical transport region transports a portion of the fluid vertically toward another layer of the fluidic device.
  • the vertical transport region is a white blood cell filter in fluidic communication with the first sample reception region, the second sample reception region, and/or the channel. The vertical transport region may then facilitate the separation and/or the immobilization of white blood cells when provided the fluid, thereby allow red blood cells to flow throughout the fluidic device.
  • the vertical fluid transport region is positioned external to the fluidic device and the channel such that the vertical fluid transport region may separate and/or immobilize at least some white blood cells prior to entry of the fluid into the fluidic device and/or the channel.
  • both a vertical transport region in a second layer of a fluidic device described herein and a porous material present in one or more portions of a first layer of a fluidic device described herein are both white blood cell filters, they may be different white blood cell filters or may be the same type of white blood cell filter.
  • fluidic devices described herein comprise a nucleic acid stabilizer.
  • the nucleic acid stabilizer may be present in any suitable portion of the fluidic device, such as in a layer thereof, a first sample reception region, a second sample reception region, a channel (e.g., a channel fluidically connecting two sample reception regions), and/or a vertical transport region.
  • the nucleic acid stabilizer may advantageously stabilize nucleic acids (e.g., DNA, RNA) such that nucleic acids may be recovered after a relatively long duration of time after the fluid has dried on and/or within the fluidic device.
  • the nucleic acid stabilizer allows for nucleic acids to be recovered after a period of greater than or equal to 1 month, greater than or equal to 3 months, greater than or equal to 6 months, or greater than or equal to 12 months of drying the device. In some embodiments, the nucleic acid stabilizer allows for nucleic acids to be recovered after a period of less than or equal to 12 months, less than or equal to 6 months, less than or equal to 3 months, or less than or equal to 1 month. Combinations of these ranges are possible (e.g., greater than or equal to 1 month and less than or equal to 12 months). Other ranges are also possible.
  • a method comprises recovering a nucleic acid (e.g., DNA, RNA) from a fluidic device and/or a component thereof (e.g., a sample reception region, a channel, a vertical transport region, a layer). This recovery may be performed in one or more of the time periods provided in the preceding paragraph.
  • the nucleic acid stabilizer comprises an RNA stabilizer. Exemplary RNA stabilizers include but are not limited to FTA reagent and RNAlater.
  • the nucleic acid stabilizer comprises a DNA stabilizer.
  • the nucleic acid stabilizer comprises DNA Shield, RNA Shield, RNA Later, DNase and RNase inhibitors, denaturants such as guanidinium salts or surfactants (SDS), quaternary ammonium salts (such as the active ingredient in Cavicide), Qiagen RNAprotect, and/or silk. Other nucleic acid stabilizers may also be used.
  • fluidic devices described herein may facilitate the determination of a property of a fluid. In some embodiments, the fluidic device determines and/or facilitates the determination of a property of a fluid comprising a blood sample.
  • determining a property of the fluid may involve transporting (e.g., laterally transporting) the fluid through the fluidic device such that cells (e.g., a plurality of cells) are transported through the channel of the fluidic device to the second sample reception region.
  • the transporting the fluid involves transporting cells through the channel from the first sample reception region to the second sample reception region (e.g., via a channel fluidically connecting the first and second sample reception regions).
  • the transporting comprises transporting a plurality of white blood cells and a plurality of red blood cells through a fluidic device to a first region and a second region, wherein the second region is a sample reception region.
  • a first portion of the fluid e.g., a portion comprising a portion of the plurality of white blood cells
  • a second portion of the fluid is retained in or on the second region (e.g., the second sample reception region).
  • the first portion of the fluid may be enriched in white blood cells (e.g., in comparison to the amount of white blood cells present in the second sample reception region, as a fraction of the fluid in comparison to the fraction of the fluid occupied by white blood cells in the second sample reception region) while the second portion of the fluid may be enriched in red blood cells (e.g., in comparison to the amount of red blood cells present in the first sample reception region, as a fraction of the fluid in comparison to the fraction of the fluid occupied by red blood cells in the first sample reception region).
  • the second portion of the fluid is enriched in a bloodborne pathogen (e.g., in comparison to the amount of the pathogen present in the first sample reception region, as a fraction of the fluid in comparison to the fraction of the fluid occupied by the pathogen in the first sample reception region).
  • a bloodborne pathogen e.g., in comparison to the amount of the pathogen present in the first sample reception region, as a fraction of the fluid in comparison to the fraction of the fluid occupied by the pathogen in the first sample reception region.
  • the first portion of the fluid to be enriched in a bloodborne pathogen (e.g., in comparison to the amount of the pathogen present in the second sample reception region, as a fraction of the fluid in comparison to the fraction of the fluid occupied by the pathogen in the second sample reception region).
  • fluidic devices described herein may determine and/or facilitate the determination of the presence of a bloodborne pathogen.
  • some methods may comprise determining the presence of a bloodborne pathogen (e.g., using a fluidic device described herein and/or on a sample present in a device described herein).
  • the bloodborne pathogen comprises parasites such as malaria, bacteria, and/or viral agents.
  • the property of a fluid involves biological material derived from a bloodborne pathogen such as malarial nucleic acids such as malarial RNA.
  • the fluidic device provides one or more samples of blood and/or samples of blood components, via a punch of the first sample reception region and/or the second sample reception region, such that the punch may be evaluated.
  • Any of variety of suitable assays may be employed, such as RNA assays and/or Drabkin’s assay.
  • assays and/or methods that may be used to evaluated the second sample region and/or another portion of the fluidic device comprise molecular amplification assays (DNA or RNA) such as those for blood borne pathogens, selective or whole genome sequencing, serological tests, metabolite profiling, and/or hematological indices.
  • an assay may be performed that is suitable for detecting the presence and/or remnants of a bloodborne pathogens.
  • a bloodborne pathogens e.g., biological fluids such as saliva.
  • the fluidic device may be dried for a period of time.
  • the fluidic device may be dried in the first and/or second sample reception region such that either or both reception regions may punched and evaluated for various properties of the fluid (e.g., the presence of bloodborne pathogens).
  • nucleic acids may be recovered from either sample reception region, and in some embodiments, such recovery may occur at least a month after the initial lateral transport of the fluid.
  • LDC Leukocyte Depletion Card
  • WBC depletion and subsequent parasite enrichment for LDC compared to 903 card as well as improved Plasmodium sequencing was demonstrated.
  • An advantage of at least some devices provided in this example is that they may help provide sample cleanup or enrichment of Plasmodium DNA. Such devices may leave samples with high ratios of host DNA (e.g., which may relate to the number of white blood cells in the device) compared to parasite DNA, affecting downstream sequencing.
  • devices provided in this example are capable of depleting host white blood cells (WBCs) from whole blood and/or (optionally simultaneously) storing the sample in a dried format such that the Plasmodium DNA is preserved for downstream analysis.
  • WBCs host white blood cells
  • the hematocrit-independence of at least some devices provided in this example across 25–55% hematocrit may allow for a reproducible punch volume across different patients.
  • Parasite enrichment may be detected, in some embodiments, in blood samples, with 0.001% or more parasitemia (e.g., up to 5%, up to 10%, up to 20%, or more parasitemia) using a device provided in this example as quantified by qPCR, according to some embodiments.
  • the devices provided in this example produce substantial enrichment of parasitic DNA relative to venous blood, depending on the embodiment.
  • the average enrichment of parasitic DNA is greater than or equal to 100-fold, greater than or equal to 150-fold, greater than or equal to 200-fold in a device described herein relative to venous blood.
  • the average enrichment of parasitic DNA in a device described in this example relative to venous blood is as high as 250-fold. Similar enrichments in parasitic DNA may be observed, e.g., relative to a conventional DBC card such as the Whatman 903 card or a generic equivalent thereof.
  • Quantitative cellular analysis of patient-collected, dried specimens may be improved, according to some embodiments, by: (i) control of cell distribution within the collection media, (ii) precision metering of collected volumes, and/or (iii) autonomous sample processing.
  • the devices provided in this example may allow control of cell distribution within the device and/or the precision metering of collected volumes, in some embodiments.
  • the devices provided in this example may be configured for precision metering of collected volumes.
  • a device provided in this example may be suitable for characterizing the nucleic acids of intraerythrocytic parasites, e.g., at least in part as a result of one of the above-mentioned advantages.
  • a device described in this example may be useful for the preservation of RNA in the field (e.g., without use of RNA preservation solutions). Improved and field-friendly technologies would assist efforts in host nation health security, protection and treatment of deployed troops, and global efforts to study, treat, and eradicate malaria.
  • a device provided in this example may allow both transport of RBCs (red blood cells) and also removal of WBCs. In some embodiments, transport of RBCs and removal of WBCs can help to remove host nucleic acid contaminants.
  • a device may exhibit reduced loss of parasitized RBCs relative to a traditional DBC, thereby improving sensitivity. In some embodiments, a device increases the depletion of host WBCs, providing greater measurement specificity than a traditional DBC (see FIG. 7).
  • a device may comprise a single or double layer of a white blood cell isolation medium (e.g., Leukosorb) before filling a patterned channel of DBS cardstock (FIG. 4 and FIG. 6A-6B).
  • a white blood cell isolation medium e.g., Leukosorb
  • a sample of whole blood having an appropriate volume e.g., 40 microliters
  • Donor WBCs may be quantified using the 18S rRNA gene.
  • P. falciparum may be quantified using sbp1, a conserved single copy gene.
  • a ⁇ Ct may be calculated for each gene against an equal volume of whole blood. The ⁇ Ct may allow measurements of parasite loss, enrichment, and purity.
  • P. falciparum DNA purity increased to 23% while offering a 7-fold enrichment over Whatman 903.
  • P. falciparum DNA purity increased to 84% in the double Leukosorb layer device while offering a 50-fold enrichment over Whatman 903.
  • the improved purity observed may facilitate efforts to identify drug resistant strains by next generation sequencing.
  • RNA stabilization may improve RNA stabilization.
  • a device comprises one or more zones (e.g., portions of a layer of a device) comprising an RNA stabilizer.
  • Exemplary RNA stabilizers include but are not limited to FTA reagent and RNAlater.
  • a device comprises one or more zones comprising silk fibroin. Silk fibroin can, in some embodiments, improve RNA stabilization.
  • the devices provided in this example may provide improved RNA stabilization in some cases by providing rapid drying.
  • a device provided in this example may be dry within 60 minutes of use, in some embodiments.
  • a device provided in this example may stabilize RNA (e.g., from intraerthyrocytic parasites) such that it can be recovered after at least one month of storage without any spurious amplification.
  • RNA e.g., from intraerthyrocytic parasites
  • qPCR was used to successfully amplify P. falciparum RNA isolated from punches of pDBS cards without evidence of spurious amplification from contaminating human nucleic acids (FIG. 5).
  • a fluidic device comprises two layers of porous materials.
  • a device further comprises a third layer.
  • the use of a third layer may enhance white blood cell separation relative to the white blood cell separation that could be achieved in a device with only two layers.
  • Layers of a device are, in some embodiments, adhered together (e.g., using double-sided adhesive).
  • One or more layers of the device may be filters.
  • filters may be used to deplete the whole blood sample of white blood cells while permitting other fluid (e.g., comprising red blood cells) to be transported to a bottom layer (e.g., a layer comprising a porous material) of the fluidic device.
  • the filter(s) may reduce the concentration of white blood cells in the fluid transported to the bottom layer by any of a variety of appropriate amounts (e.g., at least 500-fold, relative to the concentration of white blood cells in fluid initially provided to the fluidic device.
  • the separation of the white blood cells may be achieved using any of a variety of appropriate mechanisms, such as size exclusion and/or electrostatic attraction.
  • the bottom layer of the device may receive fluid flow from the top layer(s) in a first sample reception region to and may channel the fluid from the first sample reception region to a second sample reception region. Fluid in the second sample collection region can be excised and analyzed during downstream analysis, according to some embodiments.
  • the device may be operated by applying a fluid (e.g., an undiluted whole blood sample) to a top layer of the device. According to some embodiments, the device is used by waiting ⁇ 5 minutes from the fluid application for at least a portion of the fluid to travel through the device and fill the second sample collection region. The device is, according to some embodiments, then dried and stored.
  • a fluid e.g., an undiluted whole blood sample
  • the device may be stored for long-term downstream applications as discussed above, or for immediate application (e.g., immediate analyte detection via analytical methods).
  • the ability to remove white blood cells specifically from fluids may provide a number of advantages for analyzing patient samples for a wide variety of red blood cell-focused diseases (e.g., several parasitic diseases including malaria) without contamination from unwanted white blood cells.
  • red blood cell-focused diseases e.g., several parasitic diseases including malaria
  • the devices provided in this example advantageously requires a minimal amount of whole blood (e.g., 50–100 ⁇ L) relative to other analytic techniques.
  • a device described herein may be used to obtain a variety of patient health information via downstream methods like qPCR. Moreover, a device described herein may have a relatively small footprint and low cost, making it advantageous for use in the field. A device according to the embodiments described herein may advantageously allow for whole blood collection and separation of white blood cells at point-of-care settings. A device as described herein may, in some embodiments, offer advantages for long-term storage for downstream applications (e.g., biobanking of emerging parasites). The size of and stacking of devices described in this example may provide a method of white blood cell separation from whole blood that is faster and easier to use then other separation methods, according to some embodiments.
  • the separation may occur passively, e.g., (providing user-free separation of white blood cells).
  • devices and methods provided in this example can be used for microsampling of blood via lancet in remote or field settings or the home, without requiring a trained phlebotomist to collect large volumes of blood for transport via cold chain.
  • a device may be configured to fill any of a wide variety of hematocrit ranges (e.g., 25– 55%) to accommodate different patients.
  • a device is configured to dry quickly (e.g., within 30 minutes). Quick drying may promote DNA stability.
  • Drying may also provide the advantage of hematocrit-independent, single extraction punch volume across the tested hematocrit range to allow for matched liquid reference comparisons.
  • Materials and Methods Chemical reagents and materials Munktell TFN paper was purchased from Laboratory Sales and Services (Somerville, NJ). Leukosorb sheets were purchased from Pall Corporation (Port Washington, NY). Fellowes and Avery laminates, and 6 mm hole punch were purchased from Amazon. 1 ⁇ 4” clear acrylic sheets were purchased from McMaster-Carr. Sterile pipette tips were purchased from Mettler Toledo (Columbus, OH). 40-mm microhematocrit capillary tubes were purchased from LW Scientific.
  • Samples of whole blood collected in potassium EDTA vacutainers were obtained from Research Blood Components (Watertown, MA). Drabkin’s reagent, Brij 35 (30% w/w), and ASTM Type I water were purchased from Ricca Chemical (Arlington, TX). Critoseal vinyl plastic putty and 2 mL microcentrifuge tubes were purchased from VWR. QiAamp DNA Mini kits were purchased from Qiagen (Germantown, MD). Whatman 903 Protein Saver cards were purchased from Fisher Scientific (Hampton, NH). 100% ethanol, 96-well qPCR plates, MicroAmp optical adhesive film, and Fast SYBR Green Master Mix were purchased from Thermo Fisher (Waltham, MA).
  • ⁇ -actin forward (5’- CAC CAT TGG CAA TGA GCG GTT C- 3’) and reverse (5’- AGG TCT TTG CGG ATG TCC ACG T-3’) primers
  • sbp1 forward (5’- GGC ATC TGC AAC TAC CGA AT-3’) and reverse (5’- GCT TGA AAA ACC GTC ATC GT- 3’) primers
  • sbp1 forward (5’- GGC ATC TGC AAC TAC CGA AT-3’
  • reverse 5’- GCT TGA AAA ACC GTC ATC GT- 3’
  • top and bottom designs were printed onto Avery laminate sheets using a Xerox ColorQube 8580 wax printer.
  • a sheet of TFN was aligned with the top and bottom designs using a custom acrylic alignment jig.
  • a VEVOR P8200 T-shirt press 50 s at 142 °C was employed to transfer the wax from the laminate sheets to the paper to form hydrophobic barriers through the full thickness of the paper.
  • Leukosorb circles or channels were cut using a VEVOR SH-G35050W laser engraving machine (Rancho Cucamonga, CA). Each card was fabricated by attaching each layer with laser-cut adhesive sheets and sealed each card using Fellowes laminates.
  • parasites were maintained in an atmosphere of N2/CO2/O2: 90/5/5 and complete RPMI medium (RPMI 1640, 25 mM HEPES, 100 ⁇ g mL -1 hypoxanthine, 0.3 mg mL -1 glutamine (KD Biomedical, Columbia, MD)) supplemented with 25 mM NaHCO3 (pH 7.3), 5 ⁇ g/ml of gentamicin, and 10% human serum (Interstate Blood Bank, Memphis, TN) or 0.5% Albumax II (Gibco, Waltham, MA). Parasitemia (Infected RBCs/Total RBCs*100) was evaluated through microscopy after Giemsa staining of smears (10%, 15 minutes).
  • Sorbitol treatment (5%, 10–30 min at 37 °C) was used to synchronize parasites in ring-stage.
  • Sorbitol-synchronized ring stage parasites were harvested and the hematocrit was adjusted to 40% using culture medium.
  • a range of 0%, 0.001%, 0.01%, 0.1%, 1% and 5% parasitemia was prepared. Calculations are shown below. 50 ⁇ L of each parasitemia dilution was applied to LDC 4 and 903 cards, and stored an additional 50 ⁇ L as a whole blood pellet.
  • gDNA was extracted from each punch and the liquid controls using a Qiagen QIAamp DNA Mini kit and according to Qiagen’s dried blood spot and liquid whole blood extraction protocols.
  • 100 ⁇ L of Qiagen QIAamp DNA Mini Kit Buffer AE water-based elution buffer
  • the ⁇ -actin and sbp1 genes were amplified from each purified DNA sample using a QuantStudio3 Real Time PCR system.
  • each 20 ⁇ L qPCR reaction mix contained 10 ⁇ L Fast SYBR Green Master Mix, 1.6 ⁇ L of mixed forward and reverse ⁇ -actin or sbp1 primers (5 ⁇ M total per reaction), 6.4 ⁇ L Type I water, and 2 ⁇ L of purified DNA.
  • RNase P was used as the non-template control. Instrument cycling conditions are shown in FIG. 41.
  • Clinical sample collection Approximately 1 mL of blood from each participant was collected. For each sample, five 50 ⁇ L drops of blood were spotted onto individually labeled Whatman 903 Protein Saver Cards and LDCs. Both card types were dried at room temperature. Upon drying, both card types were placed in a foil bag containing a desiccant and stored at room temperature pending analysis.
  • the remaining blood samples were stored at -20 o C. Determining parasite density of clinical samples using microscopy Blood films were processed and stained according to WHO guidelines. Two independent malaria microscopists read each smear, with any discordant calling of positive or negative smears broken by a third malaria microscopist. Parasite density was estimated as the number of parasites counted per 200 WBCs, multiplied by 40 based on the assumption that 1 ⁇ L of blood contains 8000 WBCs.
  • sWGA is an enrichment method to selectively amplify target genome (here Plasmodium falciparum DNA) over background DNA (human genome) using a pool of primers designed to amplify frequently occurring motifs of short nucleotides in P. falciparum reference genome.
  • the sWGA experiment was performed in two steps. First, 8 ⁇ L of each purified gDNA sample, 0.25 ⁇ L (final solution concentration of 20 ⁇ M of each primer in the pool), 0.5 ⁇ L of 10X ThermoFisher EquiPhi29 reaction buffer, and 1.25 ⁇ L of nuclease-free water was combined. The resulting 10 ⁇ L for 3 minutes at 95o was then denatured.
  • the denatured product was then mixed with 1 ⁇ L (10 units) of EquiPhi29 DNA polymerase, 2 ⁇ L reaction buffer 0.2 ⁇ L of 100 ⁇ M MDT, 2 ⁇ L of 10mM dNTPs, and nuclease-free water to make a total pool volume of 20 ⁇ L and isothermally amplified 45oC for 3 hours, then 65oC for 10 minutes to suspend further enzyme activity.
  • Amplification success was validated with measuring DNA quantity before and after enrichment using Qubit.
  • MIP sequencing and data analysis sWGA amplified DNA was targeted, captured, and sequenced using a molecular inversion probe (MIP) targeting key P.
  • MIP molecular inversion probe
  • falciparum resistance genes associated with artemisinin and partner drug resistance including pfkelch13, pfmdr1, pfcrt, pfdhfr and and pfdhps genes.
  • MIP capture and library preparation was performed.
  • sequencing using an Illumina NextSeq 550 instrument 150 bp paired-end reads was conducted at Brown University (RI, USA).
  • the raw data was generated using MIPs was demultiplexed using MIPtools software (https://github.com/bailey- lab/MIPTools), which is a computationally suitable tool for MIP data processing and analysis.
  • FIGS. 11A-11B, 12A-12B, 13A-13B, and 14A-14B provide a few schematic illustrations and photographs of non-limiting devices, according to some embodiments.
  • Some such devices may be referred to as leukocyte depletion cards (“LDCs”), cards, and/or prototypes.
  • LDCs leukocyte depletion cards
  • Four card prototypes were designed and tested: prototype 1 (Figure 11A-11B), prototype 2 (Figure 12A-12B), prototype 3 ( Figure 13A-13B), and prototype 4 ( Figure 14A-14B), with various differences in material for different purposes (e.g., to promote RBC movement while restricting WBC movement to the extraction punch zone).
  • the material of a device described herein may comprise a single layer or one or more layers.
  • Prototype 4 Single layer of Leukosorb in the shape of the channel inlaid within a TFN support
  • FIGS. 8-10 include data regarding qPCR quantification of human 18s rRNA (leukocyte) target, associated ⁇ C T values, and various parasitemia ranges using synchronized culture and whole blood.
  • a single layer of Leukosorb may be used as is or with additional treatments (e.g., to allow for faster drying and/or improve DNA/RNA stability).
  • Each prototype was challenged with 50 ⁇ L of blood with contrived hematocrits of 25– 55%. All four prototypes filled the entirety of the extraction zone over the hematocrit range tested ( Figures 11C, 12C, 13C, 14C). In some embodiments, the hematocrit ranges do not have a significant effect on the punch volume.
  • Table 4 A non-limiting example is shown in Table 4. This is believed to be advantageous because it may allow for the use of a single volume as a liquid reference for comparison to a sample from any subject by comparing the extracted liquid punches.
  • Prototypes 1, 2, and 3 provided average punch volumes of 13.0, 18.4, and 9.7 ⁇ L, respectively ( Figures 11D, 12D, 13D), across the hematocrit range (25–55%, FIG. 15); however, these numbers varied significantly between hematocrits (ANOVA, p ⁇ 0.001 for all three prototypes).
  • the variation between average punch volumes and hematocrit range may be characterized through statistical parameters such as p values. For example, low p values may represent a high variation.
  • the average punch volume does not vary between hematocrit ranges (See FIGS. 11D, 12D, 13D, and 14D).
  • Prototype 3 did not provide hematocrit independence across the entire hematocrit range tested.
  • Such embodiments may be suitable when the subject does not present a wide range of hematocrit ranges, such as when the subject is a malaria patient.
  • Venous blood was obtained from 16 malaria-infected patients in Cape Coast, Ghana with cell and parasite counts ranging from 3,800–20,100 WBC ⁇ L-1 and 33–251,100 parasites ⁇ L-1, respectively (FIG. 20).
  • DNA was extracted from both cards and qPCR was performed thereon to obtain ⁇ -actin and sbp1 Ct values for each sample (FIGS. 23-24). Unlike in vitro samples, extracted DNA from clinical samples was run across multiple PCR plates.
  • the 903 card had a 1.2-fold change enrichment over the liquid reference while LDC had a 32.5-fold and 36.6-fold change enrichment (FIG. 27) over the liquid reference and 903 card, respectively.
  • raw Ct values were converted into WBC and parasite counts using calibration curves (FIG. 27). WBC and parasite counts are reported as total counts per extracted sample (FIG. 29). The recovery of WBC cells and parasites from each card punch was then calculated using the liquid reference sample (FIGS. 30-31).
  • FIG. 32 illustrates average recovery of WBCs and parasites from each card.
  • such fluidic devices can be uses for applications related to the diagnosis and/or detection of malaria.
  • Arrows in FIG. 34 indicate the position of the inlet allowing for a sample (e.g., a blood sample) to enter the fluidic device.
  • the sample may travel and/or flow along channels in the fluidic device.
  • FIGS. 35A-35C shows a fluidic device having a first layer comprising laser cut TFN and a second layer comprising leukosorb.
  • the fluidic device is configured to receive a sample (e.g., a blood sample)
  • FIGS. 35D shows the results of Drabkin’s assay depicting the extraction zone volume and indicating that an analysis of variance shows no significant difference in extraction zone volume with samples having different hematocrits.
  • FIGS. 36A-36B shows a fluidic device having a first layer comprising TFN and at least one layer comprising Leukosorb.
  • FIG. 36C shows the results of a Drabkin’s assay indicating extraction zone volume as a function of the hematocrit of each sample tested.
  • FIGS. 37 depicts the results fluidic devices in FIGS. 35A-35C and FIGS. 36A-36B as compared to a typical 903 card.
  • the fluidic devices described herein generally have a lower ⁇ Ct than the 903 Card, and moreover, the data in FIG. 37 shown that the fluidic devices (e.g., “Card A” and “Card B” in FIG. 37) deplete WBCs better than 903 cards.
  • FIG. 38 show raw Ct values for liquid references including for the fluidic device of FIGS. 35A-35C and a 903 Card. Such data was derived from control experiments from samples of whole blood supplemented with RBCs infected with P. falciparum at different %parasitemia.
  • the data shows the ability of the fluidic device to remove WBC from the extraction zone. Such an ability is desirable especially if a ⁇ Ct, 18S value greater than 0.
  • the data also shows the ability of the fluidic device to allow RBCs to flow to the extraction zone. Such an ability is desirable especially if a ⁇ Ct, sbp1 value less than or equal to 0.
  • FIG. 37 the fluidic device depicted in FIGS.
  • FIG. 40 compare the WBC depletion and iRBC movement abilities between the fluidic device of FIGS. 35A-35C (“Card B”) and the Whatman 903 card. Briefly, Card B enriches iRBCs, on average, 58-fold better than liquid samples and 78-fold better than 903 cards.
  • a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • wt% is an abbreviation of weight percentage.
  • at% is an abbreviation of atomic percentage.

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Abstract

Dans certains modes de réalisation, des dispositifs fluidiques selon la présente invention comprennent une ou plusieurs couches (par exemple, une première couche) comprenant un matériau poreux. Dans certains modes de réalisation, le dispositif comprend une première couche comprenant au moins deux régions de réception d'échantillon (par exemple, une première région de réception d'échantillon et une seconde région de réception d'échantillon), les régions de réception d'échantillon étant en communication fluidique l'une avec l'autre par l'intermédiaire d'un canal. Dans certains modes de réalisation, la première région de réception d'échantillon est conçue pour recevoir un fluide (par exemple, un échantillon) comprenant des globules blancs et des globules rouges. Le matériau poreux, conçu pour retenir au moins une partie des globules blancs et permettre le transport d'au moins une partie des globules rouges, peut permettre au fluide de s'écouler à travers le dispositif fluidique de telle sorte que la seconde région de réception d'échantillon est enrichie en globules rouges.
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Citations (5)

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Publication number Priority date Publication date Assignee Title
US5164087A (en) * 1988-03-03 1992-11-17 Terumo Kabushiki Kaisha Leukocyte separator
US20060261005A1 (en) * 2002-12-30 2006-11-23 Dao Kinh-Luan Lenny D Porous spun polymeric structures and method of making same
US20200264133A1 (en) * 2017-04-03 2020-08-20 Groupe E.N. Biomédical Inc. Methods and devices for the separation, detection and measurement of molecules in liquid samples
US20210263027A1 (en) * 2018-06-22 2021-08-26 Trustees Of Tufts College Patterned dried blood spot cards and related articles and methods
WO2023167995A2 (fr) * 2022-03-03 2023-09-07 Trustees Of Tufts College Articles et procédés de transport de cellules

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5164087A (en) * 1988-03-03 1992-11-17 Terumo Kabushiki Kaisha Leukocyte separator
US20060261005A1 (en) * 2002-12-30 2006-11-23 Dao Kinh-Luan Lenny D Porous spun polymeric structures and method of making same
US20200264133A1 (en) * 2017-04-03 2020-08-20 Groupe E.N. Biomédical Inc. Methods and devices for the separation, detection and measurement of molecules in liquid samples
US20210263027A1 (en) * 2018-06-22 2021-08-26 Trustees Of Tufts College Patterned dried blood spot cards and related articles and methods
WO2023167995A2 (fr) * 2022-03-03 2023-09-07 Trustees Of Tufts College Articles et procédés de transport de cellules

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