The present technology relates generally to systems for touchless processing of dried blood spots, and methods of using same.
Dried filter paper dried blood spots (“DBS”) represent attractive sample matrices for many diagnostic tests. The DBS requires only a finger, toe or heel puncture, thereby eliminating the need for venipuncture by skilled phlebotomists. If properly dried and stored, many analytes are stable across a wide range of temperatures. DBS cards generally include multiple DBS configured to store blood samples in dry form. The DBS cards are more easily transported than anti-coagulated liquid blood samples. As such, the use of DBS is increasing worldwide, especially in resource-poor regions and in clinical trial settings.
However, processing of DBS samples using conventional methods introduces risk of cross-contamination. Typically, hole punchers are used to punch a disc (hereafter called a ‘spot’) from the card into a tube. Sometimes manual manipulation with scissors and/or forceps is required. While cross-contamination is generally less problematic in chemistry-based assays (where analyte concentrations usually vary <10-fold between patients), cross-contamination can be especially problematic for molecular assays, where small amount of DNA or RNA carried over from a high positive sample can result in later false-positive results. Furthermore, where conventional automatic punching is not possible, there is significant risk of repetitive stress injuries from manual punching of DBS.
Improved systems and methods for processing DBS samples, especially when molecular assays are involved, are therefore needed.
Many aspects of the present technology can be better understood with reference to the following drawings. The relative dimensions in the drawings may be to scale with respect to some embodiments. With respect to other embodiments, the drawings may not be to scale. For ease of reference, throughout this disclosure identical reference numbers may be used to identify identical or at least generally similar or analogous components or features.
The present technology is generally directed to systems for touchless processing of DBS and methods of using such systems. DBS processing systems configured in accordance with embodiments of the present technology are expected to reduce the risk of cross-contamination, enhance the efficacy, and/or reduce the costs associated with processing DBS.
Touchless DBS processing systems consistent with the present technology may be configured to cut (e.g., laser cut) at least a portion of a DBS from a DBS card without contacting the DBS. In some embodiments, the cut portion of the DBS is deposited in a receptacle, such as a sample tube or a well of a multi-well plate, without contacting the cut portion of the DBS. Specific details of several embodiments of the present technology are described herein with reference to
For ease of reference, throughout this disclosure identical reference numbers are used to identify similar or analogous components or features, but the use of the same reference number does not imply that the parts should be construed to be identical. Indeed, in many examples described herein, the identically-numbered parts are distinct in structure and/or function.
Generally, unless the context indicates otherwise, the terms “distal” and “proximal” within this disclosure reference a position or direction with respect to the treating clinician or clinician's surgical tool (e.g., a surgical navigation registration tool). “Distal” or “distally” are a position distant from or in a direction away from the clinician or clinician's surgical tool. “Proximal” and “proximally” are a position near or in a direction toward the clinician or clinician's surgical tool.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.
Specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
The present technology includes systems configured to process a DBS card by cutting at least a portion of a DBS and depositing the cutting into a receptacle, wherein the system does not physically contact the DBS (e.g., “touchless” systems), and methods of using such systems to process DBS cards with minimal or no risk of cross-contaminating samples. In some embodiments, the touchless system includes a laser configured to cut at least a portion of the DBS from the DBS card. In one embodiment, the system includes a DBS card support, a laser, a receptacle support and a controller operably connected to the laser and configured to cause the laser to cut at least a portion of a DBS from a DBS card placed in the DBS card support using a beam of light.
Referring now to
The laser 115 can be any laser capable of cutting through the DBS card 140. In general, higher powered lasers are capable of cutting the DBS card 140 while producing a minimal amount of charring and/or smoke byproduct. However, lower powered lasers are also suitable and particularly advantageous for portable embodiments of the systems described herein. In some embodiments, the laser 115 is stabilized by a laser housing 110. The laser housing 110 may include mechanisms for enabling the laser 115 to move in any direction (e.g., x, y, and/or z) relative to the DBS card support 130, and may also enable the laser 115 to rotate in one or more axes relative to the DBS card support 130. The laser 115 is operably connected to the controller 120, for example via a wired or wireless connection 125, which may control power and/or movement of the laser 115, for example according to a set of instructions stored on a memory device incorporated into the controller 120. In some embodiments, the laser housing 110 includes a cabinet, a box, a rack, a bracket, an enclosure, or any other suitable support framework for stabilizing the position of the laser 115 relative to the DBS card support 130.
The DBS card support 130 is positioned proximately to the laser 115 such that a beam emitted by the laser 115 can accurately and effectively cut a shape out of one or more of the DBSs 142 on the DBS card 140. In some embodiments, the DBS card support 130 includes one or more pairs of DBS card mounts 132 configured to enable accurate placement of DBS cards 140 in a consistent position relative to the laser 115. In the embodiment shown in
The receptacle support 134-136 can be in any configuration suitable for holding one or more receptacles 150 in alignment with the DBSs 142. In the embodiment shown in
The controller 120 may be configured to cause the laser 115 to emit a beam that contacts the surface of the DBS card 140 at a particular location. For example, the controller 120 in some embodiments may cause the laser 115 to emit a beam that contacts the surface of the DBS card 140 at one or more of DBSs 142a-e. The controller 120 may also be configured to cause the laser 115 to emit a beam in a pattern that, upon completion, causes at least a portion of the DBS card 140 to automatically separate from the DBS card 140. In some embodiments, the pattern is a regular or common shape such as a circle, oval, ellipse, polygon, triangle, quadrilateral, square, rectangle, rhombus, parallelogram, trapezoid, pentagon, hexagon, heptagon, octagon, nonagon, decagon, etc. In some embodiments, the pattern is an irregular curved shape or an irregular polygon. In some embodiments, the pattern is a combination of curves and straight lines.
The system 100 is configured to enable the excised portions of the DBS 140a-e to be deposited into a receptacle 150a-e. In some embodiments, the excised portion of the DBS 140a-e fall into the receptacle 150a-e via gravity. In some embodiments, the excised portion of the DBS 140a-e is directed into the receptacle 150a-e by another touchless force, such as forced gas (e.g., a jet or puff of air or an inert gas such as nitrogen, argon, etc.).
The controller 120 may be configured to select a beam pattern based on one or more parameters of the DBS card 140 and/or of the assay to be performed on the DBS. For example, the controller 120 may be configured to select a beam pattern that cuts a portion of the DBS card 140 that has a predetermined area. In some embodiments the controller 120 is configured to select a beam pattern that cuts a portion of the DBS card 140 that has an area of about 10 mm2 to about 200 mm2, about 10 mm2 to about 100 mm2, about 20 mm2 to about 80 mm2, or about 50 mm2 to about 75 mm2, for example 10 mm2, about 11 mm2, about 12 mm2, about 13 mm2, about 14 mm2, about 15 mm2, about 16 mm2, about 17 mm2, about 18 mm2, about 19 mm2, about 20 mm2, about 21 mm2, about 22 mm2, about 23 mm2, about 24 mm2, about 25 mm2, about 26 mm2, about 27 mm2, about 28 mm2, about 29 mm2, about 30 mm2, about 31 mm2, about 32 mm2, about 33 mm2, about 34 mm2, about 35 mm2, about 36 mm2, about 37 mm2, about 38 mm2, about 39 mm2, about 40 mm2, about 41 mm2, about 42 mm2, about 43 mm2, about 44 mm2, about 45 mm2, about 46 mm2, about 47 mm2, about 48 mm2, about 49 mm2, about 50 mm2, about 51 mm2, about 52 mm2, about 53 mm2, about 54 mm2, about 55 mm2, about 56 mm2, about 57 mm2, about 58 mm2, about 59 mm2, about 60 mm2, about 61 mm2, about 62 mm2, about 63 mm2, about 64 mm2, about 65 mm2, about 66 mm2, about 67 mm2, about 68 mm2, about 69 mm2, about 70 mm2, about 71 mm2, about 72 mm2, about 73 mm2, about 74 mm2, about 75 mm2, about 76 mm2, about 77 mm2, about 78 mm2, about 79 mm2, about 80 mm2, about 81 mm2, about 82 mm2, about 83 mm2, about 84 mm2, about 85 mm2, about 86 mm2, about 87 mm2, about 88 mm2, about 89 mm2, about 90 mm2, about 91 mm2, about 92 mm2, about 93 mm2, about 94 mm2, about 95 mm2, about 96 mm2, about 97 mm2, about 98 mm2, about 99 mm2, about 100 mm2, about 101 mm2, about 102 mm2, about 103 mm2, about 104 mm2, about 105 mm2, about 106 mm2, about 107 mm2, about 108 mm2, about 109 mm2, about 110 mm2, about 111 mm2, about 112 mm2, about 113 mm2, about 114 mm2, about 115 mm2, about 116 mm2, about 117 mm2, about 118 mm2, about 119 mm2, about 120 mm2, about 121 mm2, about 122 mm2, about 123 mm2, about 124 mm2, about 125 mm2, about 126 mm2, about 127 mm2, about 128 mm2, about 129 mm2, about 130 mm2, about 131 mm2, about 132 mm2, about 133 mm2, about 134 mm2, about 135 mm2, about 136 mm2, about 137 mm2, about 138 mm2, about 139 mm2, about 140 mm2, about 141 mm2, about 142 mm2, about 143 mm2, about 144 mm2, about 145 mm2, about 146 mm2, about 147 mm2, about 148 mm2, about 149 mm2, about 150 mm2, about 151 mm2, about 152 mm2, about 153 mm2, about 154 mm2, about 155 mm2, about 156 mm2, about 157 mm2, about 158 mm2, about 159 mm2, about 160 mm2, about 161 mm2, about 162 mm2, about 163 mm2, about 164 mm2, about 165 mm2, about 166 mm2, about 167 mm2, about 168 mm2, about 169 mm2, about 170 mm2, about 171 mm2, about 172 mm2, about 173 mm2, about 174 mm2, about 175 mm2, about 176 mm2, about 177 mm2, about 178 mm2, about 179 mm2, about 180 mm2, about 181 mm2, about 182 mm2, about 183 mm2, about 184 mm2, about 185 mm2, about 186 mm2, about 187 mm2, about 188 mm2, about 189 mm2, about 190 mm2, about 191 mm2, about 192 mm2, about 193 mm2, about 194 mm2, about 195 mm2, about 196 mm2, about 197 mm2, about 198 mm2, about 199 mm2, or about 200 mm2. In some embodiments, the cut area of the portion of the DBS card is divided between two or more cuttings, for example in two cuttings, three cuttings, four cuttings, five cuttings, six cuttings, seven cuttings, eight cuttings, nine cuttings, ten cuttings, or more than ten cuttings.
Referring now to
Referring now to
As shown in
Referring now to
As shown in
Alternatively or in addition to the configurations described above, the controller 120 may be configured to store information about the DBS card 140 (e.g., identifying information about the subject and/or the blood sample), the location of the portion of the DBS 142 to be excised, the pattern of light beam to be executed by the laser 115, the power voltage and/or amperage supplied to the laser 115, the image obtained by the camera 116, notes from an operator of the system 100, and/or any error messages (e.g., power faults, laser faults, and/or possible defects in the DBS card 140a determined from the image obtained by the camera 116) that are generated by the system 100 during processing of the DBS card 140. In some embodiments, the controller 120 is configured to store the information in association with a unique identifier assigned to the DBS card 140, for example a machine-readable feature such as a 1-dimensional bar code, a 2-dimensional bar code, an RFID code, or any other suitable identifier.
The present technology also includes systems configured to process multiple DBS cards by cutting at least a portion of a DBS from each DBS card without physically contacting the DBS, and depositing each cutting into a receptacle, wherein the system does not physically contact the DBS, and methods of using such systems to process DBS cards with minimal or no risk of cross-contaminating samples. In some embodiments, the touchless system includes a laser configured to cut at least a portion of the DBS from the DBS card using a beam of light.
Referring now to
Systems 200 for automated touchless processing of DBS cards 140 may include components similar or identical to those described above with respect to touchless processing systems 100. For example, the laser 115 may be configured similarly or identically in system 200 as described above with respect to system 100. Similarly, the system 200 may include a laser housing 110 and/or a camera 116 configured similarly or identically to laser housing 110 and camera 116 as described above with respect to system 100.
The system 200 may include a DBS card hopper 260 configured to hold multiple DBS cards 140, for example, in a stack. The DBS card hopper 260 may include walls, edges, guides, rails, rollers, trays, or other components for storing the DBS cards 140 without physically contacting any DBSs 142. For example, the DBS card hopper 260 does not include any rollers, guides, treads, or other components in locations that are likely to come into contact with a DBS 142 on a DBS card 140 during the storage process. Typically, DBS cards 140 are stored in a folded configuration to prevent cross-contamination from contact with other DBS cards 140. In the folded configuration, at least one additional layer of DBS card material separate each layer of DBSs 142. The DBS cards 140 must be unfolded to expose the DBSs 142 before processing. Accordingly, in some embodiments, the DBS cards 140 are placed in the DBS card hopper 260 in an unfolded configuration. In other embodiments, the DBS cards 140 are placed in the DBS card hopper 260 in a folded configuration, for example to prevent cross-contamination caused by physical contact of a DBS 142 from one DBS card 140 with a DBS 142 from an adjacent DBS card 140. In such embodiments, the DBS card hopper 260 may include a DBS card unfolder 270 configured to accept a folded DBS card 140 from the DBS card hopper portion 260 and unfold the DBS card 140.
The DBS card feeder 280 is configured to accept a DBS card 140 (e.g., in an unfolded configuration) from the DBS card hopper 260 or the DBS card unfolder 270. The DBS card feeder 280 may include rollers, treads, gears, or other mechanisms for transporting the DBS card 140 without physically contacting any DBSs 142. For example, the DBS card feeder 280 does not include any rollers, guides, treads, or other components in locations that are likely to come into contact with a DBS 142 on a DBS card 140 during the transport process. To affect transport of the DBS card 140 from the DBS card hopper 260 and/or the DBS card unfolder 270, the DBS card feeder 280 may include opposing sets of rollers (e.g., motorized rollers) configured to pinch the edges of the DBS card 140 in the margin between the edge of the DBS card 140 and the DBSs 142 nearest the edge. In such embodiments, the DBS card feeder 280 contacts only portions of the DBS card 140 that do not include dried blood, thus reducing or eliminating the risk of cross-contaminating one DBS card 140 with dried blood from another DBS card 140.
The DBS card support 232 is configured to accept a DBS card 140 from the DBS card feeder 280 and position the DBS card 140a to be cut by the laser 115, and to enable the excised portion of the DBS 142 to be placed in a receptacle 150. Similar to the DBS card hopper 260, the DBS card unfolder 270, and the DBS card feeder 280, the DBS card support 232 may include walls, edges, guides, rails, rollers, trays, or other components for accurately and repeatably positioning the DBS card 140a without physically contacting any DBSs 142. For example, the DBS card support 232 may include two opposing rails with rollers (e.g., motorized rollers) and/or guides configured to receive the edges of the DBS card 140a and position the DBS card 140a at a predetermined location relative to the laser 115 and/or the receptacles 150. The DBS card support 232 may also be configured to transport the DBS card 140a to the DBS card depository 290 after the laser completes cutting a portion of the DBS 142. Alternatively, the DBS card depository may include a feeder portion configured to retrieve the DBS card 140a as described more fully below.
In some embodiments, the DBS card support 232 is configured to adjust the distance D1 between the DBS card 140a and the laser 115, and/or the distance D2 between the DBS card 140a and the receptacles 150. In other embodiments, the DBS card support 232 positions the DBS card 140a at a fixed elevation, and the laser 115/laser housing 110 can be adjusted to provide the desired distance D1, as described more fully with respect to
In embodiments wherein the DBS card support 232 is configured to (i) transport the DBS card 140a from the DBS card feeder 280, (ii) transport the DBS card 140a to the DBS card depository 290, and/or (iii) adjust the position of the DBS card 140a relative to the laser 115 and/or to the receptacle support 230, the DBS card support 232 may be operably connected to the controller 120, which may control power and/or movement of the DBS card support 232, for example according to a set of instructions stored on a memory device incorporated into the controller 120. In some embodiments, the position of the DBS card support 232 is predetermined based at least in part on information about the DBSs 142 obtained by the camera 116. In some embodiments, the position of the DBS card support 232 is predetermined based at least in part on information about the assay to be performed on the DBS, and/or the size and/or shape of the light beam pattern to be executed by the laser 115.
Although
In other embodiments, the DBS card hopper 260, the DBS card unfolder 270, the DBS card feeder 280, the DBS card support 232, and the DBS card depository 290 are each configured to transport and support the DBS card 140/140a in a configuration other than that shown in
The receptacle support 230 is configured to hold one or more receptacles 150 similar to receptacle support 130/136 as described above with respect to
The DBS card depository 290 is operably connected to the DBS card support 232, and is configured to store one or more DBS cards 140b after they have been processed. In some embodiments, the DBS card depository receives the DBS card 140a from the DBS card support 232. In other embodiments, the DBS card depository retrieves the DBS card 140a from the DBS card support 232 and stores the retrieved DBS card 140b. In such embodiments, the DBS card depository 290 may include rollers, gears, treads, or any other suitable transport mechanism for transporting the DBS card 140a from the DBS card support 232 to the DBS card depository 290. In such embodiments, the DBS card depository 290 is operably connected to the controller 120, which may control power and/or movement of the components of the DBS card depository 290, for example according to a set of instructions stored on a memory device incorporated into the controller 120.
In some embodiments, the system 200 additionally includes an exhaust system 190 configured to intake fumes generated by the touchless system 200 and vent the fumes to an appropriate exhaust location. In some embodiments, the exhaust system 190 includes a fume collector portion 192 and a vent portion 194 connected to the collector portion 192 and configured to safely release the collected fumes, such as described above with respect to
Optionally, the system 200 may include a component configured to scan a machine-readable feature 144 included on the DBS card 140a. For example, in some embodiments, the camera 116 may be configured to obtain an image of the machine-readable feature 144, which then may be translated (e.g., by the controller 120) into information about the DBS card 140a and/or the subject who provided the blood sample stored on the DBS card 140a, such as the subject's name, the date the sample was obtained, a subject identification number (e.g., for blind trials or other subject-identification protective purposes), and/or the assay(s) to be performed.
Alternatively or in addition to the configurations described above, the controller 120 may be configured to store information about the DBS card 140a (e.g., identifying information about the subject and/or the blood sample), the location of the portion of the DBS 142 to be excised, the pattern of light beam to be executed by the laser 115, the power voltage and/or amperage supplied to the laser 115, the image obtained by the camera 116, notes from an operator of the system 200, and/or any error messages (e.g., power faults, laser faults, faults in transporting (e.g., feeding) the DBS card 140a through the components of the system 200, and/or possible defects in the DBS card 140a determined from the image obtained by the camera 116) that are generated by the system 200 during processing of the DBS card 140a.
The present technology also includes DBS cards including machine-readable identification feature which enables automated processing of multiple DBS cards and interpretation of the resulting assay data. In some embodiments, the DBS cards comprise a machine-readable identification feature such as a linear barcode, a matrix barcode (e.g., a QR code), an alphanumeric code, or other suitable type of machine-readable code.
Referring now to
In embodiments in which the DBS card 340 includes the machine-readable feature 344, the system 100/200 may be configured to obtain an image (e.g., scan) of the machine-readable feature 344 and determine one or more operating parameters, such as the location of the cut to be made, the size of cut and/or light beam pattern to be executed by the laser 115, the type and/or location of the receptacle 150 to be used, and the like. For example, in some embodiments, the camera 116 may be configured to obtain an image of the machine-readable feature 344, which then may be translated (e.g., by the controller 120) into information about the DBS card 140a and/or the subject who provided the blood sample stored on the DBS card 140a, such as the subject's name, the date the sample was obtained, a subject identification number (e.g., for blind trials or other subject-identification protective purposes), and/or the assay(s) to be performed.
In some embodiments, the DBS card 140 is encased or at least partially enclosed in a protective container, such as a plastic case. In such embodiments, the system 100/200 may be configured to remove the DBS card 140 from the container before the system 100/200 excises a portion of a DBS 142 from the DBS card 140. In some embodiments, the system 100/200 is additionally configured to return the DBS 140 to its initial encased or at least partially enclosed configuration after the portion of the DBS 142 is excised. For example, system 200 may be configured to store a plurality of encased or at least partially enclosed DBS cards 140 in the DBS card hopper 260 in the encased or at least partially enclosed configuration, and the DBS card unfolder 270 is configured to expose at least a portion of the DBS cards 140 before the DBS card feeder 280 positions the exposed portion of the DBS card 140a for processing by the laser 115. The DBS card depository 290 may be configured to receive and return the processed DBS cards 140b to their initial encased or at least partially enclosed configuration before storage.
The present technology also includes methods for processing one or more DBS cards by cutting at least a portion of a DBS from each DBS card and depositing each cutting into a receptacle, wherein the method does not include physically contacting the DBS in order to minimize or eliminate a risk of cross-contaminating the dried blood samples (e.g., “touchless” processing).
In some embodiments, the method comprises positioning a DBS card in alignment with a receptacle, wherein the DBS card has at least one DBS comprising dried blood from a subject; contacting the DBS with a beam of light from a laser in a pattern sufficient to excise at least a portion of the DBS from the DBS card; and depositing the excised portion of the DBS into the receptacle.
In some embodiments, the method further comprises obtaining an image of the DBS card before contacting the DBS with the beam of light from the laser. In such embodiments, the image may include information about one or more of the DBSs on the DBS card, and/or a machine-readable code. In some embodiments, the pattern for the beam of light is determined by the system based at least in part on the image. Alternatively or in addition, the location of the DBS to be contacted with the beam of light is determined by the system based at least in part on the image. In embodiments wherein the DBS card comprises a plurality of DBSs, one of the plurality of DBSs may be selected to be contacted with the beam of light based at least in part on the image. In some embodiments, the method further comprises storing information comprising the location and/or the light beam pattern in association with the machine-readable code in a database. For example, the controller 120 of system 100 or system 200 may include a database configured to store information about the location and/or the light beam pattern in association with the machine-readable code corresponding to a DBS card.
The method may further comprise analyzing the excised portion of the DBS for the presence of one or more diseases. In some embodiments, the disease is HIV. In some embodiments, the disease is malaria. Any suitable method of analyzing the excised portion of the DBS may be used. For diseases detectable by analyzing blood for specific genetic material (e.g., viral or bacterial diseases), the analysis may include PCR, RT-PCR, LAMP, NASBA or other similar genetic amplification method known to those of skill in the art. In some embodiments, the result of the disease testing is stored in a database in association with the machine-readable code described above. For example, the controller 120 of system 100 or system 200 may include a database configured to store a test result in association with the machine-readable code corresponding to a DBS card.
In embodiments wherein the receptacle is housed in a receptacle support comprising a plurality of receptacles, the method may further comprise determining an assay to be performed on the DBS; selecting a receptacle from among the plurality of receptacles after positioning the DBS card; and (i) if the selected receptacle is in alignment with a first DBS having a sufficient area comprising dried blood for the determined assay, contacting the first DBS in alignment with the selected receptacle with a beam of light from the laser in a pattern sufficient to excise at least a portion of the first DBS from the DBS card, or (ii) if the selected receptacle is not in alignment with a DBS having a sufficient area comprising dried blood for the determined assay: (a) repositioning the DBS card and/or the receptacle to align a second DBS having a sufficient area comprising dried blood for the determined assay, and (b) contacting the second DBS in alignment with the selected receptacle with a beam of light from the laser in a pattern sufficient to excise at least a portion of the second DBS from the DBS card.
Methods of the present technology may further comprise processing a calibration DBS card. In such embodiments, the method may comprise providing a calibration DBS card including a plurality of calibration DBSs each having a different concentration of one or more analyte; positioning the calibration DBS card such that each calibration DBS is in alignment with a single receptacle; contacting each of the calibration DBSs with a beam of light from a laser in a pattern sufficient to excise at least a portion of each calibration DBS from the calibration DBS card; and depositing each of the excised portions of the calibration DBSs into the aligned receptacles. One example embodiment of a calibration DBS card 440 is shown in
The present technology also provides methods for processing a plurality of DBS cards without touching any of the DBSs of the DBS cards (e.g., “touchless” automatic bulk processing of DBS cards). In some embodiments, the method comprises: (i) providing a plurality of DBS cards; (ii) selecting a first DBS card from the plurality of DBS cards, the first DBS card having at least one DBS comprising dried blood; (iii) positioning the first DBS card in alignment with a first receptacle; (iv) contacting the first DBS with a beam of light from a laser in a pattern sufficient to excise at least a portion of the first DBS from the first DBS card; (v) depositing the excised portion of the first DBS into the first receptacle; (vi) depositing the first DBS card into a DBS card depository; and (vii) depositing an excised portion of a second DBS into a second receptacle by repeating steps (ii) to (vi) for a second DBS card selected from the plurality of DBS cards.
In some embodiments, the method is suitable for efficiently analyzing a large number of subject samples simultaneously (e.g., “pooled” analysis), for example for high throughput screening of low-prevalence diseases. Typically, pooled screening methods include combining a large number of samples into a single pooled sample and then analyzing the combined samples for the presence of the target analyte of interest. If no analyte associated with the disease-causing organism is detected (e.g., no genetic material of a selected pathogen or virus is detected by PCR or RT-PCR), then none of the individual samples is likely to be infected with that pathogen. Accordingly, the method of the present technology may include pooling excised portions of multiple DBSs before analyzing the pooled spots for the presence of a disease. In such embodiments, the second receptacle configured to receive the excised portion of the second (and subsequent) DBS is the same as the first receptacle configured to receive the excised portion of the first DBS. The system 100/200 may be configured to pool a predetermined number of DBSs before providing a new receptacle to receive additional (optionally pooled) DBSs. For example, the controller 120 may be configured to pool 2 to about 5,000 samples (e.g., excised portions of DBSs), about 50 to about 2,500 samples, about 100 to about 2,000 samples, about 250 to about 1,000 samples, or about 500 to about 750 samples in a single receptacle. In such embodiments, the controller 120 may additionally be configured to store information about each DBS card, the location and/or the light beam pattern associated with each DBS card, for example by associating the stored information with a machine-readable code located on each processed DBS card. In some embodiments, the information is stored in a database incorporated in the controller 120.
In other embodiments, each DBS is processed into a separate receptacle. Such methods are useful, for example, in detecting the presence of a disease-associated analyte for diseases with relatively high incidence rates, or for re-analyzing individual DBS cards previously analyzed in a pooled method (e.g., as described above). In such embodiments, the second receptacle (and each subsequent receptacle) is separate from the first receptacle.
In any automated touchless processing method described herein, an image of the each DBS card may be obtained before contacting the DBS with the beam of light from the laser. In such embodiments, the image may include information about one or more of the DBSs on the DBS card, and/or a machine-readable code. In some embodiments, the image is used at least in part to determine a location of the first DBS to be contacted with the beam of light. In embodiments wherein the DBS card comprises a plurality of DBSs, the DBS is selected from the plurality of DBSs, based at least in part on the image, to be contacted with the beam of light. In some embodiments, the method further comprises storing information comprising the location and/or the light beam pattern for each or at least some of the DBS cards in association with the machine-readable codes in a database. For example, the controller 120 of the system 200 may include a database configured to store information about the location and/or the light beam pattern in association with the machine-readable code corresponding to a DBS card.
Alternatively, the system 200 may be configured to periodically obtain an image of only some of the plurality of DBS cards to be analyzed, such as for quality control assessments. In such embodiments, an image of the DBS card may be obtained before, during, and/or after the laser executes the light beam pattern. The image(s) may be stored for later review, such as in a database incorporated in the controller 120. In some embodiments, the system 200 is configured to provide a quality report based on the image(s). The quality report may be generated by the controller 120, and may be provided to a user in any suitable form, such as in a printout, in an electronic format, and/or displayed on a screen.
Malaria infection can be diagnosed by demonstrating the causative Plasmodium parasite in red blood cells by microscopy, by rapid antigen detection or by molecular methods. Microscopy is time consuming and not amenable to high-throughput use, and rapid antigen detection kits are insufficiently sensitive for many settings. While more sensitive, most molecular methods require larger sample volumes than DBS can accommodate to achieve sufficiently high sensitivity. A first-generation highly sensitive quantitative RT-PCR assay targeting the P. falciparum 18S rRNA from total nucleic acids demonstrated sensitive detection from only 50 μL of liquid whole blood (Murphy, S. C., et al., Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. Am. J. Trop. Med. Hyg., vol. 86(3), pages 383-94 (2012)). Since each parasite contains ˜3.4-4.0 log10 RNA copies, the assay can detect as few as 20 parasites per mL of whole blood, similar to other high volume DNA-only assays used for vaccine trial monitoring. The small sample volume of the present assay afforded the possibility of using DBS for detecting patent and pre-patent (sub-microscopic) parasitemia.
The first-generation RT-PCR assay described above was modified to use TaqMan probe chemistry on a high-throughput instrument as a second generation assay. Data generated from testing synthetic RNA standards (5×102 to 1×109 copies per RT-PCR reaction) in an extracted whole blood internal control RNA-containing matrix were used to evaluate the standard curve, reportable range and carryover. Data generated from testing parasite-containing whole blood samples (4×101 to 4×107 parasites per mL of blood) containing internal control RNA were used to evaluate accuracy, precision, analytical sensitivity, analytical specificity, reportable range and carryover. A ‘synthetic standard curve’ diluted in negative whole blood (data not shown) was compared against cultured blood-stage parasites in whole blood (
Parasite-containing specimens (high, medium and low concentration) and negative control specimens were tested in triplicate over a 5-day timespan to calculate diagnostic sensitivity and diagnostic specificity. There were no false positive or false negative results in this dataset. The difference between the nominal (expected) and observed estimates was determined and plotted against the nominal value. Of 54 samples in this data set, the average log10 difference (bias) across all 54 samples was 0.143 log10 RNA copies/mL (95% CI −0.379 to 0.367 log10 RNA copies/mL) and no samples showed a difference in recovery >0.5 log10 RNA copies/mL. The correlation across all samples was linear (r2=0.9911; slope 1.03) with no concentration-dependent differences in recovery based on a Bland-Altman plot (not shown, r2=0.1278). Within-run (repeatability) and between-run (within lab) precision was determined by testing triplicate high, medium, low and negative samples daily for 5 days judged against the in vitro standard curve from the overall validation. Intra- and inter-assay components of variation were calculated as described in S. C. Murphy, Am. J. Trop. Med. Hyg., vol. 86(3), pages 383-94 (2012). The standard deviation (log10 copy number) and the percent coefficient of variation (% CV=Standard deviation/mean) are reported in Table 1. Based on repeated testing of samples containing 500 copies of the synthetic malaria control RNA per reaction (data not shown), the analytical sensitivity was determined to be 20 parasites/mL.
The reportable range (
The assay described in Example 1 was adapted to utilize blood samples stored on DBS cards. However, because the dynamic range of the P. falciparum 18S rRNA assay [˜2×105 copies/mL to 4×1012 copies/mL (2×101 parasites/mL to 4×108 parasites/mL) includes samples with much higher template concentrations than observed for HIV-1 assays, DBS samples for malaria testing could have been more prone to cross-contamination from hole punching than HIV-1 DBS. Indeed, analysis of DBS samples processed by hole punching showed a significant rate of false positives due to cross-contamination for malaria samples. To overcome this problem, a laser cutting approach described substantially as above was used to process DBS without touching the blood-containing sections of the DBS card. Cross-contamination was not observed for samples processed using the laser cutting approach as described below.
When processing DBS samples using conventional punching, numerous false positives were detected in samples originating from malaria-negative whole blood (Table 2). Such samples were processed after a high or medium concentration malaria-positive samples, indicating template cross-contamination. Despite using the conventional HIV-compatible approach of punching the entire circle into a sample tube using a standard office supply-type hole puncher then cleaning the puncher by punching five clean DBS sheets before proceeding to the next sample, carryover occurred in >50% of conventionally processed, sequentially tested malaria-positive (+, ++, +++) and malaria-negative (−) DBS samples 1 to 18 in the order shown in the left-most column (Table 2). All samples were first processed using the conventional punch method. All samples were then processed using a laser configured as described herein.
Compiled data on standardized whole blood samples processed by conventional liquid processing, by punch DBS processing or by laser cut DBS processing showed that only the punch processed DBS were susceptible to false positives. Of the samples that were processed by interspersing negative samples (0 parasites/mL) with high (4×107 parasites/mL), medium (8×103 parasites/mL) and low positive (80 parasites/mL) samples, cross-contamination was not detected in laser-cut DBS or conventional liquid samples, but was detected following punched samples amongst known negative samples in 7 of 8 instances following a high positive sample and in 2 of 6 instances following medium positive samples (Table 2;
Additional samples that are not included in Table 2 are also displayed in
Since recovery for DBS samples was less than for liquid samples, DBS samples require a different calibration standard curve than liquid samples. A DBS-derived standard curve may therefore be obtained (for example using a standard DBS card as shown in
Some laser-cut low positive samples were not detected by RT-PCR. In such instances, two laser-cut discs were processed in a single tube (equivalent to 54.9 microliters of whole blood) in an attempt to overcome this qualitative detection problem. Amongst low positive samples (80 parasites/mL) where this approach was used, 13 of 13 samples were positive (mean parasite density 117 parasites/mL; data not shown), which essentially overcame the false negatives observed when one disc was used. Since detection of low-positives was restored by using two laser cut discs per sample, the false negative findings depicted in
A series of 108 de-identified samples collected from subjects in a clinical trial were tested using both the laser-cut DBS method of the present disclosure and a standard liquid blood (LB) method. The number of positive samples for each assay are shown below in Table 3.
The source samples were collected from subjects who were in the initial stages of malaria infection (e.g., the number of parasites was exceedingly low and near the limit of detection for the assay). The results show general agreement between laser-cut DBS cards and LB with 33/108 positive by both methods and 62/108 negative by both methods. The incongruent values likely have more to do with the very low parasite load (e.g., Poisson statistics affecting sampling proportions) than with actual false-positives and/or false-negatives resulting from defects in the liquid blood assay or the DBS processing assay.
The performance of DBS whole blood collection and testing methods for detecting HIV-1 antibodies and HIV-1 nucleic acid (NA) in a population-based HIV surveillance study were assessed. Plasma and DBS were collected from multiple subject cohorts that included known HIV-negative and known HIV-positive subjects. Plasma is processed by standard methods. DBS samples are processed using a laser configured consistently with the present disclosure. Samples are analyzed by three HIV-1/2 molecular diagnostic tests and a fourth syphilis antibody test. A total of 1200 samples from phases I and II are analyzed.
A preliminary analysis was conducted to determine HIV-1 infection status on 316 finger-prick DBS cards collected in Chicago, Ill. between July 2013 and April 2014. DBS samples (50 μL) venous blood per spot) were obtained from HIV-1 viremic and HIV-1 seronegative patients. Samples were excised with the laser cutter as described herein. From each DBS card, one 50 μL spot was eluted in 0.5-mL phosphate buffered saline for HIV serological tests and another 50 μL spot was eluted in 2.5 mL BioMérieux NucliSENS lysis buffer for HIV NA testing. HIV-1 tests included the Abbott Architect HIV Ag/Ab Combo Assay: HIV-1/-2 Ab and HIV-1 Ag; the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test: differentiation of HIV-1 and -2 Ab and the Abbott RealTime HIV-1 assay: quantification of HIV-1 NA. Analysis revealed that 185/316 subjects were HIV-negative, while 2/316 were acutely infected with HIV. 68/316 had low viremia established infection and 61/316 had high viremia established infection (Table 4). A subset of samples (33) were tested to compare whole blood DBS performance against plasma samples using the Abbott Architect HIV Ag/Ab Combo Assay, and all samples showed good quantitative concordance including an absence of false positives in the HIV-1 negative control subjects. The Abbott RealTime HIV-1 assay was determined to have a DBS sensitivity of 2000 copies HIV-1/mL whole blood using a single DBS sample. To improve the sensitivity of this assay, a two-spot assay was evaluated. Optimal two-spot assay performance was obtained by extracting the samples on the bioMerieux miniMag extraction instrument for subsequent quantification by the enzymatic amplification of Abbott RealTime HIV-1 assay—the sensitivity of this approach was 520 copies HIV-1/mL whole blood, which is sufficiently sensitive to detect WHO-defined virological failures at the defined threshold of 1000 HIV-1 RNA copies/mL plasma. Thus, laser-cut DBS provide an inexpensive and patient-friendly way to collect, store and transport patients' blood samples. Laser cut DBS can be tested for HIV-1/2 using the Abbott Architect HIV Ag/Ab Assay, Bio-Rad Multispot Rapid Test and Abbott RealTime HIV-1 assay.
As part of a large collaborative project, samples were collected from up to 4100 HIV-positive patients receiving antiretroviral therapies (ART) in 15 health facilities across Uganda. HIV-1/-2 viral loads were analyzed and the data used as part of a broad ART cost-effectiveness study in Uganda. Blood samples were transported from the rural health facilities to the nearest urban health facilities for refrigeration and then transported onward to a central laboratory in Kampala for plasma viral load testing. Dried blood spots (DBS) were transported to a central laboratory and then transferred to our facility (University of Washington) for DBS viral load testing. DBS were cut using the laser cutter as described in Murphy et al. 2012, and a two-spot sample was extracted using the bioMérieux miniMag system and quantified using the Abbott RealTime HIV-1 assay; for comparison, the viral load from plasma samples were measured entirely by the FDA-approved Abbott RealTime HIV-1 assay. To date, >1300 samples have been tested. When the study is completed, the laser cut DBS viral load results will be compared to the corresponding plasma viral load values.
As of June 2014, 1349 samples have been processed using the laser cutting system and subsequently tested for whole blood viral load and have a paired plasma sample that was separately tested by the FDA-approved assay. Interim analysis of these samples alone showed that 135/192 plasma-positive samples were also DBS-positive for HIV and that 1122/1157 plasma-negative samples were also DBS-negative for HIV (Table 5). Using the WHO-defined indicator of virological failure (≧1000 copies/mL plasma) as the sensitivity cutoff, the DBS approach had a sensitivity of 70.3% and a specificity of 97.0%.
Performance characteristics of the DBS viral load assay are shown in Table 6.
Examples 4 and 5 demonstrate that the laser cutting system disclosed herein provides rapid excision of samples. A slightly modified racking system currently in use allows the technologist to deposit two laser cut DBS discs into a single tube for onward use in the two-spot assay. This modification is facilitated by moving the destination tube independent of the static DBS card, although either component could be moved relative to the other in future generations of laser cutting DBS devices.
This disclosure is not intended to be exhaustive or to limit the present technology to the precise forms disclosed herein. Although specific embodiments are disclosed herein for illustrative purposes, various equivalent modifications are possible without deviating from the present technology, as those of ordinary skill in the relevant art will recognize. In some cases, well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the present technology. Although steps of methods may be presented herein in a particular order, alternative embodiments may perform the steps in a different order. Similarly, certain aspects of the present technology disclosed in the context of particular embodiments can be combined or eliminated in other embodiments. While advantages associated with certain embodiments of the present technology may have been disclosed in the context of those embodiments, other embodiments can also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages or other advantages disclosed herein to fall within the scope of the present technology. Accordingly, this disclosure and associated technology can encompass other embodiments not expressly shown or described herein.
Throughout this disclosure, the singular terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, unless the word “or” is expressly limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of “or” in such a list is to be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list. Additionally, the terms “comprising” and the like are used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and/or additional types of other features are not precluded. Directional terms, such as “upper,” “lower,” “front,” “back,” “vertical,” and “horizontal,” may be used herein to express and clarify the relationship between various elements. It should be understood that such terms do not denote absolute orientation. Reference herein to “one embodiment,” “an embodiment,” or similar formulations means that a particular feature, structure, operation, or characteristic described in connection with the embodiment can be included in at least one embodiment of the present technology. Thus, the appearances of such phrases or formulations herein are not necessarily all referring to the same embodiment. Furthermore, various particular features, structures, operations, or characteristics may be combined in any suitable manner in one or more embodiments.
This application claims priority to pending U.S. Provisional Application No. 61/846,494, filed Jul. 15, 2013, the entire contents of which are incorporated herein by reference and relied upon.
Number | Date | Country | |
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61864494 | Aug 2013 | US |