WO2010091304A1 - Procédé sur filtre pour séparer des ferrofluides dans un échantillon biologique - Google Patents

Procédé sur filtre pour séparer des ferrofluides dans un échantillon biologique Download PDF

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Publication number
WO2010091304A1
WO2010091304A1 PCT/US2010/023388 US2010023388W WO2010091304A1 WO 2010091304 A1 WO2010091304 A1 WO 2010091304A1 US 2010023388 W US2010023388 W US 2010023388W WO 2010091304 A1 WO2010091304 A1 WO 2010091304A1
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WO
WIPO (PCT)
Prior art keywords
cells
unbound
filter
target
ferrofluid
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PCT/US2010/023388
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English (en)
Inventor
Arjan G.J. Tibbe
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Veridex, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Veridex, Llc filed Critical Veridex, Llc
Priority to US13/147,908 priority Critical patent/US20120055854A1/en
Publication of WO2010091304A1 publication Critical patent/WO2010091304A1/fr

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    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Definitions

  • the invention relates generally to imaging target components in a fluidic (biological) sample. More specifically, methods and apparatus are described that provide for the separation of bound and unbound ferrofluid particles during a positive selection of target cells from a blood sample.
  • immunomagnetic separation technology provides greater sensitivity and specificity in the detection of target entities in blood for example, but not limited to, intact circulating cancer cells and endothelial cells.
  • This simple and sensitive diagnostic tool as described (US6, 365,362; US6,551,843; US6,623,982; US6,620,627; US6,645,731 ; WO 02/077604; WO03/065042; and WO 03/019141) can be used in the present invention to correlate the statistical survivability of an individual patient based on a threshold level.
  • a prior diagnostic tool incorporates a blood sample from a cancer patient (WO 03/018757) incubated with magnetic beads, coated with antibodies directed against an epithelial cell surface antigen as for example EpCAM. After labeling with anti-EpCAM-coated magnetic nanoparticles, the magnetically labeled cells are then isolated using a magnetic separator. The immunomagnetically enriched fraction is further processed for downstream immunocytochemical analysis or image cytometry, for example, in the CellSpotter or CellTracks ® System (Immunicon Corp., USA). The magnetic fraction can also be used for downstream immunocytochemical analysis, RT- PCR, PCR, FISH, fl ⁇ wcytometry, or other types of image cytometry.
  • the CellSpotter or CellTracks® System utilizes immunomagnetic selection and separation to highly enrich and concentrate any epithelial cells present in whole blood samples.
  • the captured cells are detectably labeled with a leukocyte specific marker and with one or more tumor cell specific fluorescent monoclonal antibodies to allow identification and enumeration of the captured CTCs as well as instrumental or visual differentiation from contaminating non-target cells.
  • this assay allows tumor cell detection even in the early stages of low tumor mass.
  • EasyCount® system (PCT/US03/04468) is a fluorescent imaging system, designed to make a distinction between lymphocytes, granulocytes and monocytes.
  • the system includes a compact electronic optical instruments, analytical methods, image acquisition, and data reduction algorithms for the detection and enumeration of magnetically labeled target cells or particles.
  • whole blood as an example, blood cells are fluorescently labeled using one or more target specific fluorescent dyes, such as a DNA staining dye.
  • the cells of interest or target cells in the blood sample are labeled by incubation with monoclonal antibodies conjugated to ferromagnetic particles.
  • the sample is then placed into an appropriate optical detection chamber or covet, which in turn is placed into a magnetic field gradient that selectively causes the magnetically labeled cells to move towards the planar viewing surface of the chamber.
  • the target ceils are collected and immobilized substantially uniformly on the optically transparent surface of the chamber. A segment of this surface and the labeled target cells thereon are illuminated by means of one or more LED (light emitting diodes). Subsequently, the light emitted by individual target cells is captured by a CCD (charge coupled device).
  • Image acquisition methods, processing methods, and algorithms, disclosed herein, are used to count the number of captured light-emitting cells and to relate the data output to the target cells per microliter of the analysis sample in the chamber and ultimately to the original specimen.
  • a coated permanent magnetic device is placed within the sample for magnetic manipulation.
  • the system immunomagnetically concentrates the target entity, fluorescently labels, identifies and quantifies target cells by positive enumeration. Subsequent statistical analysis enables the clinician to obtain potential diagnostic information.
  • immuno-magnetic particles conjugated with an antibody against the cells of interest are added.
  • a small permanent magnet is added to the whole blood sample.
  • a small NdFeB magnet is directly added to a sample container. After 10 minutes the small permanent magnet is pulled out of the sample using an iron rod or another magnet. The magnet is positioned within the container to allow for image analysis.
  • a further embodiment of the present invention has the magnet fixed to a floatation device (floater) within the reaction chamber.
  • a blood sample is incubated with immunomagentic particle and other reagents. After incubation the floater is added.
  • the the immunomagnetically labeled target cells are collected on the face of the floater positioned in a single imaging plane for analysis, all within the reaction chamber.
  • One draw-back with this process is the presence of unbound immuno-magnetic particles (ferrofluids), positioned in the same imaging plane.
  • the present invention provides a filter device to achieve such a purpose.
  • the present invention is a method and means for separating unbound ferrofluid from target bound ferrofluid in a biological sample when positive selecting and imaging target entities using permanent magnets.
  • the process involves the addition of a coated permanent magnetic device for magnetic manipulation.
  • the system immunomagnetically concentrates the target entity onto a filter device having porosity such that passage of the target is restricted while the small unbound ferrofluid is allowed to pass toward the collection surface of the permanent magnet.
  • the filtering device allows for the target to be fluorescently labeled, identified and/or quantified by positive enumeration of the target after these have been collected on the filter and the free unbound ferrofluid is separated from the target. Subsequent statistical analysis enables the clinician to obtain potential diagnostic information.
  • a filter is positioned on the collection surface to restrict passage of the target yet allow smaller, unbound ferrofluid to collect on the collection surface of the floater.
  • FIG. 1 Panel A shows a diagram of the floater having a sieve structure positioned on the cover of the collection surface of the permanent magnet (M). Inset shows a magnified view of the side of the sieve and floater device, depicting the orientation of the spacer, filter, and collection surface. Panel B shows a top view of the filter device with an air outlet means.
  • Figure 2 Image displaying an overlay color image of cells that are collected on a nylon woven filter with 3 micron pores (panel A) or 5 micron pores (panel B).
  • Figure 3 Image showing unbound ferrofluid separation is less efficient at the edges of the filter.
  • Panel A is a 5x image of control cells on the filter.
  • Panel B is the circled area of Panel A at a 4Ox image.
  • Figure 4 Image displaying microsieve with collected control cells.
  • Panel A shows control cells on a microsieve with 5 micron pores. Light grey areas are the support structures and the interspaced darker gray area contains the 5 micron pores.
  • Panel B is a 4Ox image of the control cells plus white blood cells on a Whatman filter with 5 micron holes. Control cell are identified by their green cytokeratin staining plus blue colored nucleus. White blood cells are identified by the blue nucleus only. The unbound ferrofluid passed through the filter holes to the magnet
  • Figure 5 Representation of the pillar structure and the separation of unbound ferrofluid from cells.
  • the pillar structure restricts further movement of the cells between the pillars, yet allows unbound ferrofluid to collect between the pillars.
  • the enlarged view shows unbound ferrofluid collecting between the pillars.
  • Figure 6 Fluorescent images acquired using three different objectives.
  • A image acquired using 5x NA 0.12 objective.
  • B Image acquired using 10x, NA 0.25 and a
  • C Image using 40x, NA 0.6 objective. Blue color represent DAPI, green is CD8-PE and red is CD4-APC.
  • Figure 7 Images obtained with a 5x and 4Ox objective with the addition of 20, 40, 60, and 80 microliters of EpCam ferrofluid (20mg/ml).
  • CTC circulating tumor cells
  • Image cytometric analysis such that the immunomagnetically enriched sample is analyzed by the CellSpotter and CellTracks® System utilizes a fluorescence-based microscope image analysis system, which in contrast with flowcytometric analysis permits the visualization of events and the assessment of morphologic features to further identify objects (US 6,365,362).
  • the CellSpotter and CellTracks® System refers to an automated fluorescence microscopic system for automated enumeration of isolated cells from blood.
  • the system contains an integrated computer controlled fluorescence microscope and automated stage with a magnetic yoke assembly that will hold a disposable sample cartridge.
  • the magnetic yoke is designed to enable ferrofluid-labeled candidate tumor cells within the sample chamber to be magnetically localized to the upper viewing surface of the sample cartridge for microscopic viewing.
  • Software presents target cells, labeled with antibodies to cytokeratin and having epithelial origin, to the operator for final selection. Isolation of target cells can be accomplished by any means known in the art. After magnetic separation, the cells bound to the immunomagnetic- linked antibodies are magnetically held at the wall of the tube.
  • Unbound sample is then aspirated and an isotonic solution is added to resuspend the sample.
  • a nucleic acid dye, monoclonal antibodies to cytokeratin (a marker of epithelial cells) and CD 45 (a broad-spectrum leukocyte marker) are incubated with the sample.
  • the unbound fraction is again aspirated and the bound and labeled cells are resuspended in 0.2 ml of an isotonic solution.
  • the sample is suspended in a cell presentation chamber and placed in a magnetic device whose field orients the magnetically labeled cells for fluorescence microscopic examination. Cells are identified automatically and candidate target entities presented to the operator for checklist enumeration.
  • An enumeration checklist consists of predetermined mo ⁇ hologic criteria constituting a complete cell.
  • Another reported means to isolate target cells utilizes a permanent magnet (WO2006/102233) which is added directly to immu ⁇ omagnetically separate the labeled target entity for other components in a blood sample.
  • the target is further labeled with imaging nucleic acid dyes, cell membrane, and/or cytoskeletal immunofluorescent labels.
  • a small neodymium (NdFeB) permanent magnet is added to a whole blood sample after immunomagnetically labeled and fluorescently labeled for CO4. After 10 minutes, the small permanent magnet is separated from the fluid sample and within the sample container to be viewed through a viewing surface.
  • Example 1 demonstrates the ability to collect and image target entities, for example subpopulations of cells, on the collection surface of the floater.
  • Example 2 shows the decline in image quality with an increase in unbound ferrofluid. Cells become buried under a layer of ferrofluid, resulting, in part, in low recoveries.
  • One embodiment provides a means to prevent the interference of unbound ferrofluid by filtrating during the collection on the imaging surface.
  • the unbound ferrofluid is allowed to pass through holes a filtering device, collect while the target entity remains on the collection surface of a filter device.
  • this collection means unbound ferrofluid passes through a filter while the target entity remains on the filter surface.
  • the collection surface of the floater (WO2006/102233) is supplied with a sieve (filter).
  • the filter is separated from the magnet collection surface by a spacer means.
  • One example of a spacer means is shown in Panel A.
  • a filter is positioned with a pore size that will allow unbound ferrofluid to traverse, yet restrict the movement of the target.
  • the unbound ferrofluid will collect on the surface of the magnet while the target entity, bound to ferrofluid, will collect on the filter surface.
  • the present invention further considers the problem of air entrapment beneath the filter. When this occurs, unbound ferrofluid will not be able to travel through the filter and collect on the collection surface of the magnet. Consequently, the filter device must include an air escape means.
  • Panel B depicts one embodiment for removing air by having an outlet means between flanking the spacers.
  • any filter known in the art is considered in the present invention. However, some filters will work better than others, depending upon target and optimization conditions. These include, but not limited to, nylon woven filters (Sefar Filtration, Goor, The Netherlands) and Microsieves (Aquamarijn, Zutphen, The Netherlands).
  • Figure 2 shows an image of the filter surface using nylon woven fibers.
  • CellSearch control cells were removed from the cartridge and transferred to the glass tube. An additional 1.5 ml of system buffer is added.
  • the floater and filter depicted as in Figure 1 the floater and filter device were added to the sample and the sample rotated for 15 minutes on a clinical rotating device. The floater was imaged using a fluorescence microscope with the images shown in Figure 2.
  • Figure 2 shows the results using woven filters. Each panel depicts overlay color images of cells that are collected on an nylon woven (Sefar Filtration, Nitex 03-1/1) filter. Panel A has a porosity of 3 microns and Panel B has a porosity of 5 microns.
  • Panel A restricts the passage of unbound fenrofluid and target cells.
  • One potential explanation for this restriction is that under a magnetic field unbound fenrofluid attach to each other and thereby increases in size. Additionally, they orient themselves along the magnetic field lines forming long needle like structures. This orientation further restricts unbound ferrofiuid movement through the membrane.
  • Panel B has a porosity that will allow passage of unbound ferrofiuid and restriction of the target entity.
  • Figure 3 shows the efficiency of ferrofiuid removal across the filter collection surface using overlay images.
  • Pane A is a 5x image of the collection surface depicting the relative collection of unbound ferrofiuid and target cells.
  • Panel B provides a 4Ox image of the encircled area of Panel A.
  • the needle like dark structures are unbound ferrofiuid oriented in long needle structures. The orientation of these needle structures inhibits passage of unbound ferrofiuid.
  • FIG. 4 Panel A displays a microsieve using an image overlay of collected control cells.
  • the control cells are shown on a microsieve with pores with a diameter of 5 microns.
  • the light grey areas are supportive structures with the height of these bars approximately 600 microns.
  • the dark gray areas are the regions with 5 micron pores. All unbound ferrofiuid moved through the holes towards the magnet and the unbound ferrofiuid is not present in the image anymore.
  • Panel B is a 40 x fluorescence image of control cells collected in whole blood using a floater that was provided with a Whatman membrane filter with 5 micron holes.
  • the bright green events with a blue nucleus are the control cells
  • the white blood cells are only recognized by their blue nucleus.
  • a small pillar structure is used where the pillars are smaller than the size of individual cells.
  • unbound ferrofluid lodges between the pillars while the cells remain along the top.
  • unbound ferrofluid and ferrofluid, bound to target entities are magnetically attracted to the cover of the permanent magnet.
  • the bound ferrofluid (and consequently the target entity) remain on top of the pillar structure while the smaller, unbound ferrofuid particles travel between the pillars and collect within the pillars structure.
  • CD-Chex with known absolute numbers of leukocytes and their phenotypes is used.
  • CD-Chex (lot # 60650071):
  • Figure 7A displays the image acquired using a 5x NA 0.12 objective.
  • Figures 7B and 7C are acquired using a 10X 1 NA 0.25 and a 40X 1 NA 0.6 objective, respectively.
  • the blue color represents the DAPI, green is CD8-PE and red is CD4-APC.
  • the capture efficiency will be 16%.
  • COMPEL Magnetic Microspheres, Dragon green, 2.914 10 7 /ml, diameter 8.44 microns, lot#6548 (Bangs Laboratories Inc, Catalog code UMC4F) were diluted 1:100.
  • System buffer 1.5 ml was added to the glass vial and 50 microliters containing 14570 beads were added together with 20, 40, 60 and 80 microliters of EpCam ferrofluid (20 mg/ml). Fluorescence images were acquired after 15 and 30 minutes of rotation. Test tube rotator was set at 10 rpm, resulting in 150 and 300 rotations. Floater is Corning 1/16" diameter magnet. Results:
  • the use of the floater was evaluated by spiking immuno-magnetc pre labeled and pre-stained control cells in whole blood.
  • the vial with floater are placed under the objective of a fluorescence microscope. Next fluorescent images of the cells collected on the filter are acquired.
  • Panel B of figure 5 shows the result.
  • the control cells are identified by the greencolore of the cytokeratine and the blue nucleus.
  • the white blood cells are only stained by their blue nucleus only.

Abstract

L'invention concerne un système de filtration pour séparer un ferrofluide non lié d'un ferrofluide lié pour l'enrichissement d'un échantillon biologique en une entité cible. Le dispositif de filtration de la présente invention a des applications dans le domaine de l'isolement de cellules cibles d'un ferrofluide non lié lors d'une séparation avec un mécanisme à aimant permanent (flotteur). Ce procédé réduit l'interférence du ferrofluide non lié pendant l'analyse d'image ou le dénombrement des cellules consécutif par cytométrie en image. Le système a des applications dans l'évaluation de populations cibles comme des sous-ensembles de leucocytes dans des fluides corporels différents ou une contamination bactérienne dans des échantillons environnementaux, des produits alimentaires et des fluides corporels. En bref, des cellules cibles marquées par fluorescence sont liées à des particules ou des billes magnétiques. Le procédé de liaison donne un mélange comprenant une population de particules magnétiques non liées contaminantes. Dans un mode de réalisation de la séparation, on insère un petit aimant permanent directement dans le compartiment contenant les cellules marquées. Les aimants sont recouverts de caoutchouc siliconé PDMS pour obtenir une surface lisse et uniforme qui permet l'imagerie dans un seul plan focal. Un filtre est placé sur un couvercle du dispositif de flotteur pour permettre le passage du ferrofluide non lié à travers les pores, mais empêche le passage de l'entité cible. Le flotteur et le filtre sont retirés de l'échantillon et la surface du filtre est éclairée, la lumière fluorescente émise par les cellules cibles étant capturée par une caméra CCD. L'analyse d'image peut être effectuée avec un nouvel algorithme afin d'obtenir un comptage des cellules sur la surface, qui correspond à la concentration de cellules cibles dans l'échantillon originel.
PCT/US2010/023388 2009-02-05 2010-02-05 Procédé sur filtre pour séparer des ferrofluides dans un échantillon biologique WO2010091304A1 (fr)

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Application Number Priority Date Filing Date Title
US13/147,908 US20120055854A1 (en) 2009-02-05 2010-02-05 Filter Method for Separating Unbound Ferrofluid from Target-bound Ferrofluid in a Biological Sample

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US50007809P 2009-02-05 2009-02-05
US611500078 2009-02-05

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Cited By (1)

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EP2694965A2 (fr) * 2011-04-05 2014-02-12 Purdue Research Foundation Système micro-fluidique utilisant des micro-ouvertures pour une détection de haut débit de cellules

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WO2006102233A2 (fr) * 2005-03-18 2006-09-28 Immunivest Corporation Procede et appareil d'imagerie de composants cibles dans un echantillon biologique au moyen d'aimants permanents
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2694965A2 (fr) * 2011-04-05 2014-02-12 Purdue Research Foundation Système micro-fluidique utilisant des micro-ouvertures pour une détection de haut débit de cellules
EP2694965A4 (fr) * 2011-04-05 2014-08-27 Purdue Research Foundation Système micro-fluidique utilisant des micro-ouvertures pour une détection de haut débit de cellules
US9494557B2 (en) 2011-04-05 2016-11-15 Purdue Research Foundation Micro-fluidic system using micro-apertures for high throughput detection of cells
US9500625B2 (en) 2011-04-05 2016-11-22 Purdue Research Foundation Micro-fluidic system using micro-apertures for high throughput detection of cells
EP3193170A1 (fr) * 2011-04-05 2017-07-19 Purdue Research Foundation Système micro-fluidique utilisant des micro-ouvertures pour une détection de haut débit de unitees
US10207267B2 (en) 2011-04-05 2019-02-19 Purdue Research Foundation Micro-fluidic system using micro-apertures for high throughput detection of cells
US10335790B2 (en) 2011-04-05 2019-07-02 Purdue Research Foundation Micro-fluidic system using micro-apertures for high throughput detection of cells
US11077439B2 (en) 2011-04-05 2021-08-03 Purdue Research Foundation Micro-fluidic system using micro-apertures for high throughput detection of cells
US11478797B2 (en) 2011-04-05 2022-10-25 Purdue Research Foundation Micro-fluidic system using micro-apertures for high throughput detection of cells

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