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    Methods and Algorithms For Cell Enumeration in a Low-Cost Cytometer
    1.
    发明申请
    Methods and Algorithms For Cell Enumeration in a Low-Cost Cytometer 有权
    低成本细胞计数器细胞计数的方法和算法

    公开(公告)号：US20110052037A1

    公开(公告)日：2011-03-03

    申请号：US12707578

    申请日：2010-02-17

    申请人： Frank A.W. Coumans ,  Jan Greve ,  Frank P. Modica ,  Leon W.M.M. Terstappen ,  Arjan G.J. Tibbe ,  John A. Verrant

    发明人： Frank A.W. Coumans ,  Jan Greve ,  Frank P. Modica ,  Leon W.M.M. Terstappen ,  Arjan G.J. Tibbe ,  John A. Verrant

    IPC分类号： G06K9/00

    CPC分类号： G01N15/1475 ,  G01N33/5094 ,  G01N33/54333

    摘要： The enumeration of cells in fluids by flow cytometry is widely used across many disciplines such as assessment of leukocyte subsets in different bodily fluids or of bacterial contamination in environmental samples, food products and bodily fluids. For many applications the cost, size and complexity of the instruments prevents wider use, for example, CD4 analysis in HIV monitoring in resource-poor countries. The novel device, methods and algorithms disclosed herein largely overcome these limitations. Briefly, all cells in a biological sample are fluorescently labeled, but only the target cells are also magnetically labeled. In addition, non-magnetically labeled cells are imaged for viability in a modified slide configuration. The labeled sample, in a chamber or cuvet, is placed between two wedge-shaped magnets to selectively move the magnetically labeled cells to the observation surface of the cuvet. An LED illuminates the cells and a CCD camera captures the images of the fluorescent light emitted by the target cells. Image analysis performed with a novel algorithm provides a count of the cells on the surface that can be related to the target cell concentration of the original sample. The compact cytometer system provides a rugged, affordable and easy-to-use technique, which can be used in remote locations.

    摘要翻译： 通过流式细胞术对流体细胞的计数被广泛应用于许多学科，例如评估不同体液中的白细胞亚群或环境样品，食品和体液中的细菌污染。 对于许多应用，仪器的成本，尺寸和复杂性阻止了更广泛的使用，例如资源贫乏国家的艾滋病毒监测中的CD4分析。 本文公开的新颖的装置，方法和算法在很大程度上克服了这些限制。 简言之，生物样品中的所有细胞都被荧光标记，但只有靶细胞也被磁性标记。 另外，非磁性标记的细胞在修改的载玻片配置中的存活力被成像。 将标记的样品置于室或铜管中，放置在两个楔形磁体之间，以选择性地将磁性标记的细胞移动到铜管的观察表面。 LED照亮细胞，CCD相机捕获由靶细胞发射的荧光的图像。 用新算法进行的图像分析提供了与原始样品的靶细胞浓度相关的表面上的细胞计数。 紧凑型细胞仪系统提供坚固，经济实惠且易于使用的技术，可用于偏远地区。

    Method and apparatus for imaging target components in a biological sample using permanent magnets
    2.
    发明申请
    Method and apparatus for imaging target components in a biological sample using permanent magnets 审中-公开
    用于使用永磁体对生物样品中的目标成分进行成像的方法和装置

    公开(公告)号：US20110014686A1

    公开(公告)日：2011-01-20

    申请号：US12888562

    申请日：2010-09-23

    申请人： Arjan G.J. Tibbe ,  Leon W.M.M. Terstappen

    发明人： Arjan G.J. Tibbe ,  Leon W.M.M. Terstappen

    IPC分类号： C12M1/42

    CPC分类号： G01N33/582 ,  G01N33/54326 ,  G01N33/56972 ,  G01N2333/70514 ,  G01N2446/00

    摘要： A system for enumeration of cells in fluids by image cytometry is described for assessment of target populations such as leukocyte subsets in different bodily fluids or bacterial contamination in environmental samples, food products and bodily fluids. Briefly, fluorescently labeled target cells are linked to magnetic particles or beads. In one embodiment, a small, permanent magnet is inserted directly into the chamber containing the labeled cells. The magnets are coated with PDMS silicone rubber to provide a smooth and even surface which allows imaging on a single focal plane. The magnet is removed from the sample and illuminated with fluorescent light emitted by the target cells captured by a CCD camera. In another embodiment, a floater having a permanent magnet allows the target cells to line up along a single imaging plane within the sample solution. Image analysis can be performed with a novel algorithm to provide a count of the cells on the surface, reflecting the target cell concentration of the original sample.

    摘要翻译： 描述了通过图像细胞计数法检测流体细胞的系统，用于评估目标群体，例如不同体液中的白细胞亚群或环境样品，食品和体液中的细菌污染物。 简言之，荧光标记的靶细胞与磁性颗粒或珠粒连接。 在一个实施例中，将小的永磁体直接插入到包含标记电池的腔室中。 这些磁体用PDMS硅橡胶涂覆，以提供平滑和均匀的表面，允许在单个焦平面上进行成像。 从样品中除去磁体，并用由CCD照相机俘获的靶细胞发射的荧光照射。 在另一个实施例中，具有永磁体的浮子允许靶细胞沿着样品溶液内的单个成像平面排列。 可以用新颖的算法进行图像分析，以提供表面上的细胞计数，反映原始样品的靶细胞浓度。

    Method and apparatus for imaging target components in a biological sample using permanent magnets
    3.
    发明申请
    Method and apparatus for imaging target components in a biological sample using permanent magnets 有权
    用于使用永磁体对生物样品中的目标成分进行成像的方法和装置

    公开(公告)号：US20090061477A1

    公开(公告)日：2009-03-05

    申请号：US12031807

    申请日：2008-02-15

    申请人： Arjan G.J. Tibbe ,  Leon W.M.M. Terstappen

    发明人： Arjan G.J. Tibbe ,  Leon W.M.M. Terstappen

    IPC分类号： C12Q1/06 ,  C12M1/00

    CPC分类号： G01N33/582 ,  G01N33/54326 ,  G01N33/56972 ,  G01N2333/70514 ,  G01N2446/00

    摘要： A system for enumeration of cells in fluids by image cytometry is described for assessment of target populations such as leukocyte subsets in different bodily fluids or bacterial contamination in environmental samples, food products and bodily fluids. Briefly, fluorescently labeled target cells are linked to magnetic particles or beads. In one embodiment, a small, permanent magnet is inserted directly into the chamber containing the labeled cells. The magnets are coated with PDMS silicone rubber to provide a smooth and even surface which allows imaging on a single focal plane. The magnet is removed from the sample and illuminated with fluorescent light emitted by the target cells captured by a CCD camera. In another embodiment, a floater having a permanent magnet allows the target cells to line up along a single imaging plane within the sample solution. Image analysis can be performed with a novel algorithm to provide a count of the cells on the surface, reflecting the target cell concentration of the original sample.

    摘要翻译： 描述了通过图像细胞计数法检测流体细胞的系统，用于评估目标群体，例如不同体液中的白细胞亚群或环境样品，食品和体液中的细菌污染物。 简言之，荧光标记的靶细胞与磁性颗粒或珠粒连接。 在一个实施例中，将小的永磁体直接插入到包含标记电池的腔室中。 这些磁体用PDMS硅橡胶涂覆，以提供平滑和均匀的表面，允许在单个焦平面上进行成像。 从样品中除去磁体，并用由CCD照相机俘获的靶细胞发射的荧光照射。 在另一个实施例中，具有永磁体的浮子允许靶细胞沿着样品溶液内的单个成像平面排列。 可以用新颖的算法进行图像分析，以提供表面上的细胞计数，反映原始样品的靶细胞浓度。

    IMAGING OF IMMUNOMAGNETICALLY ENRICHED RARE CELLS
    4.
    发明申请
    IMAGING OF IMMUNOMAGNETICALLY ENRICHED RARE CELLS 有权
    免疫组织化学显微成像

    公开(公告)号：US20090286264A1

    公开(公告)日：2009-11-19

    申请号：US12467932

    申请日：2009-05-18

    申请人： Tyco M. Scholtens ,  Frederik Schreuder ,  Jan Greve ,  Arhjan G.J. Tibbe ,  Leon W.M.M. Terstappen

    发明人： Tyco M. Scholtens ,  Frederik Schreuder ,  Jan Greve ,  Arhjan G.J. Tibbe ,  Leon W.M.M. Terstappen

    IPC分类号： G01N33/53

    CPC分类号： G01N33/54326

    摘要： A method for removing excess unbound ferrofluid and imaging immunomagnetically enriched circulating tumor cells is provided. A vessels having a preformed grooves in the viewing surface is optimally designed for cell alignment and imaging. After separating the unbound particles by centrifugation, an externally-applied force is applied to transport magnetically responsive particle-CTC complex toward the transparent collection wall. The grooved inner surface of the viewing face of the chamber provide uniform distribution of the particles for easy imaging. The invention is also useful in conducting quantitative analysis and sample preparation in conjunction with automated cell enumeration techniques as in quantitative analysis of CTC in disease.

    摘要翻译： 提供了一种去除过量未结合的铁磁流体和成像免疫磁性富集的循环肿瘤细胞的方法。 在观察表面中具有预成形凹槽的容器被最佳设计用于细胞对准和成像。 通过离心分离未结合的颗粒后，施加外部施加的力以将磁响应颗粒-CTC复合物输送到透明收集壁。 室的观察面的带槽的内表面提供颗粒的均匀分布以便于成像。 本发明还可用于与自动细胞计数技术结合进行定量分析和样品制备，如在疾病中CTC的定量分析中。

    Methods and Algorithms for Cell Enumeration in a Low-Cost Cytometer
    5.
    发明申请
    Methods and Algorithms for Cell Enumeration in a Low-Cost Cytometer 有权
    低成本细胞计数器细胞计数的方法和算法

    公开(公告)号：US20110044527A1

    公开(公告)日：2011-02-24

    申请号：US12917000

    申请日：2010-11-01

    申请人： Arjan Tibbe ,  Jan Greve ,  Dhanesh Gohel ,  Erik Droog ,  Leon W.M.M. Terstappen

    发明人： Arjan Tibbe ,  Jan Greve ,  Dhanesh Gohel ,  Erik Droog ,  Leon W.M.M. Terstappen

    IPC分类号： G06K9/00

    CPC分类号： G01N15/1475 ,  G01N15/1463 ,  G01N33/5094 ,  G01N33/54333 ,  G01N2015/008 ,  G01N2015/1486 ,  Y10T436/101666 ,  Y10T436/25375

    摘要： The enumeration of cells in fluids by flow cytometry is widely used across many disciplines such as assessment of leukocyte subsets in different bodily fluids or of bacterial contamination in environmental samples, food products and bodily fluids. For many applications the cost, size and complexity of the instruments prevents wider use, for example, CD4 analysis in HIV monitoring in resource-poor countries. The novel device, methods and algorithms disclosed herein largely overcome these limitations. Briefly, all cells in a biological sample are fluorescently labeled, but only the target cells are also magnetically labeled. The labeled sample, in a chamber or cuvet, is placed between two wedge-shaped magnets to selectively move the magnetically labeled cells to the observation surface of the cuvet. An LED illuminates the cells and a CCD camera captures the images of the fluorescent light emitted by the target cells. Image analysis performed with a novel algorithm provides a count of the cells on the surface that can be related to the target cell concentration of the original sample. The compact cytometer system provides a rugged, affordable and easy-to-use technique, which can be used in remote locations.

    摘要翻译： 通过流式细胞术对流体细胞的计数被广泛应用于许多学科，例如评估不同体液中的白细胞亚群或环境样品，食品和体液中的细菌污染。 对于许多应用，仪器的成本，尺寸和复杂性阻止了更广泛的使用，例如资源贫乏国家的艾滋病毒监测中的CD4分析。 本文公开的新颖的装置，方法和算法在很大程度上克服了这些限制。 简言之，生物样品中的所有细胞都被荧光标记，但只有靶细胞也被磁性标记。 将标记的样品置于室或铜管中，放置在两个楔形磁体之间，以选择性地将磁性标记的细胞移动到铜管的观察表面。 LED照亮细胞，CCD相机捕获由靶细胞发射的荧光的图像。 用新算法进行的图像分析提供了与原始样品的靶细胞浓度相关的表面上的细胞计数。 紧凑型细胞仪系统提供坚固，经济实惠且易于使用的技术，可用于偏远地区。

    Analysis of Circulating Tumor Cells, Fragments, and Debris
    6.
    发明申请
    Analysis of Circulating Tumor Cells, Fragments, and Debris 有权
    循环肿瘤细胞，片段和碎片的分析

    公开(公告)号：US20110104718A1

    公开(公告)日：2011-05-05

    申请号：US12917630

    申请日：2010-11-02

    申请人： Galla Chandra Rao ,  Christopher Larson ,  Madeline Repollet ,  Herman Rutner ,  Leon W.M.M. Terstappen ,  Shawn Mark O'Hara ,  Steven Gross

    发明人： Galla Chandra Rao ,  Christopher Larson ,  Madeline Repollet ,  Herman Rutner ,  Leon W.M.M. Terstappen ,  Shawn Mark O'Hara ,  Steven Gross

    IPC分类号： G01N33/574 ,  G01N33/566

    CPC分类号： G01N33/574 ,  G01N33/54326 ,  Y10S436/813 ,  Y10T436/101666 ,  Y10T436/25125 ,  Y10T436/25375

    摘要： The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and the CellSpotter® fluorescent microscopy imaging system. Analyzing damaged cells has shown to be important. However, there are two sources of damage: in vivo and in vitro. Damage in vivo occurs by apoptosis, necrosis, or immune response. Damage in vitro occurs during sample acquisition, handling, transport, processing, or analysis. It is therefore desirable to confine, reduce, eliminate, or at least qualify in vitro damage to prevent it from interfering in analysis. Described herein are methods to diagnose, monitor, and screen disease based on circulating rare cells, including malignancy as determined by CTC, clusters, fragments, and debris. Also provided are kits for assaying biological specimens using these methods.

    摘要翻译： 本发明描述的方法和试剂用于分析循环肿瘤细胞，簇，碎片和碎片。 使用许多平台进行分析，包括流式细胞术和CellSpotter®荧光显微镜成像系统。 分析损伤的细胞已被证明是重要的。 然而，有两个损害来源：体内和体外。 体内损伤是通过细胞凋亡，坏死或免疫应答发生的。 在样品采集，处理，运输，加工或分析过程中发生体外损伤。 因此，期望限制，减少，消除或至少限定体外损伤，以防止其干扰分析。 本文描述了基于循环稀有细胞（包括由CTC，簇，碎片和碎片确定的恶性肿瘤）来诊断，监测和筛选疾病的方法。 还提供了使用这些方法测定生物样品的试剂盒。

    Automated Enumeration and Characterization of Circulating Melanoma Cells in Blood
    8.
    发明申请
    Automated Enumeration and Characterization of Circulating Melanoma Cells in Blood 审中-公开
    血液中循环黑色素瘤细胞的自动计数和表征

    公开(公告)号：US20090136946A1

    公开(公告)日：2009-05-28

    申请号：US12254188

    申请日：2008-10-20

    申请人： Mark Carle CONNELLY ,  Galla Chandra Rao ,  Leon W.M.M. Terstappen

    发明人： Mark Carle CONNELLY ,  Galla Chandra Rao ,  Leon W.M.M. Terstappen

    IPC分类号： C12Q1/68 ,  C12Q1/02 ,  G01N33/574

    CPC分类号： G01N33/5743 ,  G01N33/57488 ,  G01N2333/70589 ,  G01N2333/70596

    摘要： The CellTracks® System provides a system to enumerate circulating melanoma cells in blood. The system immunomagnetically concentrates epithelial cells, fluorescently labels the cells and identifies and quantifies circulating melanoma cells. The absolute number of circulating melanoma cells detected in the peripheral blood tumor load is, in part, a factor in prediction of survival, time to progression, and response to therapy. Diagnosis and monitoring of melanoma has been limited by the inability to monitor circulating melanoma cells. The present invention provides a method to enumerate circulating melanoma cells in blood samples. Accordingly, this technology provides a means and device for monitoring disease progression in patients with melanoma.

    摘要翻译： CellTracks（R）系统提供了一种系统来枚举血液中的循环黑素瘤细胞。 该系统免疫磁性浓缩上皮细胞，荧光标记细胞，并鉴定和量化循环黑素瘤细胞。 在外周血肿瘤负荷中检测到的循环黑素瘤细胞的绝对数量部分地是预测存活时间，进展时间和对治疗反应的因素。 黑色素瘤的诊断和监测受到无法监测循环黑素瘤细胞的限制。 本发明提供了一种列举血液样品中循环黑素瘤细胞的方法。 因此，该技术提供了用于监测黑素瘤患者疾病进展的手段和装置。

    Method and apparatus for imaging target components in a biological sample using permanent magnets
    9.
    发明申请
    Method and apparatus for imaging target components in a biological sample using permanent magnets 有权
    用于使用永磁体对生物样品中的目标成分进行成像的方法和装置

    公开(公告)号：US20090061476A1

    公开(公告)日：2009-03-05

    申请号：US11897471

    申请日：2007-08-30

    申请人： Arian G.J. Tibbe ,  Leon W.M.M. Terstappen

    发明人： Arian G.J. Tibbe ,  Leon W.M.M. Terstappen

    IPC分类号： C12Q1/06

    CPC分类号： G01N33/54326 ,  G01N33/56972 ,  G01N2333/70514

    摘要： A system for enumeration of cells in fluids by image cytometry is described for assessment of target populations such as leukocyte subsets in different bodily fluids or bacterial contamination in environmental samples, food products and bodily fluids. Briefly, all cells in a biological sample are fluorescently labeled, but only the target cells are also magnetically labeled. A small, permanent magnet is inserted directly into the chamber containing the labeled sample. The magnets are coated with PDMS silicone rubber to provide a smooth and even surface which allows imaging on a single focal plane. The cells are illuminated and the images of the fluorescent light emitted by the target cells are captured by a CCD camera. Image analysis performed with a novel algorithm provides a count of the cells on the surface that can be related to the target cell concentration of the original sample.

    摘要翻译： 描述了通过图像细胞计数法检测流体细胞的系统，用于评估目标群体，例如不同体液中的白细胞亚群或环境样品，食品和体液中的细菌污染物。 简言之，生物样品中的所有细胞都被荧光标记，但只有靶细胞也被磁性标记。 将一个小的永久磁铁直接插入含有标记样品的室中。 这些磁体用PDMS硅橡胶涂覆，以提供平滑和均匀的表面，允许在单个焦平面上进行成像。 细胞被照射，并且由CCD照相机捕获由靶细胞发射的荧光的图像。 用新算法进行的图像分析提供了与原始样品的靶细胞浓度相关的表面上的细胞计数。