CN102036753B - 免疫磁性富集稀少细胞的改进的成像 - Google Patents
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Abstract
本发明提供一种去除过量未结合的铁磁流体以及使免疫磁性富集循环肿瘤细胞成像的方法。在观察表面中具有预制凹槽的器皿优选地被设计用于细胞排列和成像。通过离心法分离未结合的粒子之后,施加外部施加的力,从而将磁性反应性粒子-CTC络合物朝着透明收集壁传送。所述室的观察面的具有凹槽的内表面提供粒子的均匀分布,以使成像容易。本发明还用于在疾病中的CTC定量分析中结合自动细胞计数技术进行定量分析和样品制备。
Description
相关专利申请的交叉引用
本专利申请要求于2008年5月19日提交的共同待审的美国临时专利申请序列No.61/054,219的优先权和利益,该专利申请以引用的方式全文并入本文。
背景技术
本发明涉及用于对微观生物样本进行定性和定量分析的改进的设备和方法。具体地讲,本发明涉及用于分离、收集、固定和/或分析微观生物样本或物质的设备和方法,所述微观生物样本或物质容易与具有结合剂的磁响应粒子免疫特异性或非特异性地结合,所述结合剂用于在流体介质内产生磁性标记的物质。如本文所用,例如“磁性标记的标本”应当指容易受这种磁标记影响的具有临床研究兴趣的这种生物标本或物质。
美国专利No.5,985,153描述了一种设备和方法,其中采用外部磁梯度来将存在于收集室中的磁性标记的目标样本吸引至其表面之一,并且采用内部磁梯度来获得在所述表面上的那些样本的精确排列(alignment)。通过施加竖直磁梯度以使磁性标记的生物样本运动至收集表面来获得磁性标记的生物样本至收集表面的运动。收集表面设有铁磁体捕获结构,例如在样本室的光学透明(观察)面上支撑的多条铁磁体线。
一旦磁性标记的生物样本通过外部施加的梯度被拉得足够靠近所述表面,它们就受到通过铁磁体收集表面产生的局部强梯度的影响并且横向地固定在在它们附近的位置。所述局部梯度优选地超过在生物样本碰撞透明表面之后可将生物样本保持到所述透明表面上的粘附力。或者,所述表面的粘附性可足够弱以允许水平磁力将磁性标记的生物样本朝着铁磁体结构移动。所述表面的平滑度和疏水性或亲水性特性是可影响为收集表面选择的材料或影响对所述表面的处理以获得平滑表面的因素。
U.S.10/733829和U.S.6,790,366描述了用于分离、固定和量化流体样品中的生物物质的方法和设备,包括上述外部施加的梯度的原理,还包括在所述透明收集壁上的高内部梯度磁捕获结构。所述捕获结构激励捕获的生物物质的均匀排列,以便于利用自动计数技术的定量分析。
US11/447562描述了所述室的光学透明(观察)面的流体侧上的小V形凹槽,以排列用于自动光学分析的目标样本。所述室的光学透明(观察)面的流体侧上的小V形凹槽与磁性标记的样本的最佳稀释液一起提供用于自动光学分析的排列表面。在外部施加的磁梯度的影响下,磁性标记的样本和未结合的磁粒子朝着所述室的观察面的内表面运动。当它们接近所述表面时,它们与所述V形凹槽的斜坡接触,迫使磁性标记的样本和未结合的磁粒子运动到所述凹槽的顶部。
US11/344757描述了用于可检测的标记的稀少目标细胞的自动集合和图像分析的装置和方法。这些磁性标记的稀少细胞在CellTracks(细胞轨迹)平台中受到时间延长积分成像(TimeDelayIntegrationImaging,TDI)。但是,当估计癌症患者的血液中的循环肿瘤细胞(CTC)以与疾病活性关联起来时,由免疫磁性分离留下的未结合的磁粒子将使图像变形并妨碍确定捕获的目标为CTC。
本发明提供一种小的凹槽设计,其允许在利用CellTracks平台中的TDI分析标记的稀少细胞的过程中完全去除未结合的磁粒子。
附图说明
图1是细胞排列结构的示意图。
图2在平滑排列和成像表面上利用扫描电镜显示了PDMS印记图像。
图3显示了1.3mm厚度的PDMS块和0.5mm厚度的PDMS块与1.0mm厚的典型的玻璃显微镜载片比较的透射光谱。
图4显示了过量未结合的磁粒子对图像强度的影响。EpCAM铁磁流体的含量增加导致图像强度降低以及估计强度的变化增加。
图5显示了在存在40ug/ml铁磁流体和不存在40ug/ml铁磁流体的情况下的总强度差别。存在铁磁流体时,分布更差。
图6显示了显示排列的SKBR3细胞的明视野、DAPI和PE结合的图像。
具体实施方式
已知癌症患者血液中的循环肿瘤细胞(CTC)与疾病活性相关。CellTracks平台提供了对可疑目标细胞的磁富集和图像分析,以进行计数和与疾病状态关联。例如细胞、细胞碎片和细胞组分的目标集合在器皿的收集表面上,用不着与铁磁体收集表面相邻的后续排列。这些细胞包括白血细胞、上皮组织细胞、内皮细胞、真菌细胞和细菌细胞。所述收集表面被取向为垂直于外部磁体产生的磁场梯度。所得图像包括磁纳米粒子和磁性标记的生物样本,按照基本均匀的分布方式集合在所述室的光学透明面上,而未选择的实体在流体介质中保持低水平。
在稀少细胞分析的过程中将时间延长积分(TimeDelayIntegration)(US11/344757)结合到CellTracks平台中可用于快速检测和描绘CTC。目标稀少细胞(CTC)的免疫磁性分离通过EpCAM阳性选择来实现。表达上皮细胞粘附分子(EpCAM)的抗原的循环细胞通过单克隆抗体特异性或EpCAM选择,并且共价地连接到磁纳米粒子。当暴露到磁场时,所述络合物从剩余血液组分中分离。
捕获的目标的后续分析通过细胞中组分的一系列荧光标记来确定。使用荧光染料DAPI来标记CTC的核以及非特定选择的白细胞,CTC的细胞骨架用Cytokeratine-PE标记,而白细胞用CD45-APC标记。标记的细胞被磁操纵以沿着分析表面排列。然而,在该系统中,由免疫磁性分离留下的未结合的磁粒子将导致图像变形并且降低确认CTC存在的能力。
为了提供目标样本的空间图案化集合以便定性和定量分析微观生物样品,本发明涉及在成像室的内表面上制造和使用预制结构。一般来讲,预制凹槽和长v形凹槽被预制到成像室的观察表面的内部中。这些结构提供与先前报道的Ni线一样好甚至更好的细胞排列。此外,它们由高度透明材料制成,光学上适于整个细胞的成像。
图1显示了利用本发明的凹槽的细胞排列原理。在磁梯度存在的情况下发生在室中的磁诱导细胞运动。这里,磁性标记的细胞将与所述结构的斜面(排列表面)碰撞并滑入凹槽的顶部(表示成像表面),或者它们将直接撞击成像表面的顶部。在任一种情形下,细胞将在凹槽中排列,从而允许后续成像。
为了沿着凹槽的斜面充分运动,所述表面应当平坦并且细胞被防止粘到壁上。为了实现平滑精确的设计,使用公知的晶圆蚀刻技术。但是,因为花费和光学要求,硅晶圆不合适,相反,聚二甲基硅氧烷(PDMS)复制品模制提供将满足这些需要的组合物。将满足所述标准的组合物也在本发明的考虑中。蚀刻到硅晶圆上的结构是最终设计的反相,并当浇铸到硅模具上时为PDMS模具提供正确的形状。在固化之后,所述形状被切割成能更换成像室的玻璃表面的尺寸。
可在可用于制造室的任何光学透明的材料上实现蚀刻。例如,硅晶圆可用于蚀刻,这是因为它易精密、细节精细并且容易再生。本领域公知的具有类似特性的任何材料在本发明的考虑之中。这种结构的蚀刻使用了两种通用蚀刻技术。首先,创造蚀刻凹槽需要的蚀刻掩模。利用BHF(缓冲的氢氟酸)蚀刻技术创造所述掩模。一旦完成BHF蚀刻,在不应当蚀刻凹槽的地方,SiO2薄层就留在硅晶圆上。各向异性蚀刻也用于蚀刻凹槽。这里,KOH用作蚀刻剂。当该方法被应用到合适取向的晶圆上时,在通过硅晶圆的结晶平面限制的情况下,蚀刻V形凹槽。因此,产生高度可再生和恒定的蚀刻角度。在图1的实施例中显示取决于晶圆取向的角度处于70.5度的角度。另一技术是深反应离子蚀刻(DRIE)。通过利用这种技术,可利用大的长宽比(结构的长度和宽度之间的比率)蚀刻结构。DRIE在蚀刻和沉积步骤之间循环交替,以形成有圆齿的(scalloped)侧壁。
使用PDMS模制以获得加工的晶圆上的正印记。PDMS或聚二甲基硅氧烷(道康宁公司(Sylgard184,DowCorning,Midland,MI,USA))是一种在Si(硅)和O(氧)之间含有硅氧烷键的聚合物。聚合物分子链接在一起以形成平均数在50至100左右的更长的聚合物。
通过添加交联剂获得最终的PDMS。交联剂与聚合物连接以形成聚合物的长网络,得到透光、弹性、化学惰性、热稳定的材料。在聚合反应之后,PDMS形成附着到非常稀少的材料并且不吸湿的透光柔性物质,因此防止细胞由于PDMS附着到非常稀少的材料的事实而粘合到侧部。此外,其从大约300至900nm是热稳定且透明的。这些特性对于其使用在荧光成像系统中和透射可见光来说都是非常重要的。在形成之后,凹槽被切割成所述室的观察面的尺寸。
为了在最小细胞损耗的情况下去除大多数过量的粒子,针对观察表面进行研发图1所示的细胞排列结构。图1提供了细胞排列结构的概况。图1中所示的PDMS微观结构被发展为将预定区域中的细胞排列,并减少成像时间。所述设计包括含有80um宽30mm长的六个沟道的30×2.7mm2的PDMS薄片。这些PDMS薄片从晶圆模具印制。我们选择PDMS是因为它的优异的透射性质、复制能力和易用性。这种构造将样品的成像时间从32分钟缩短为12分钟。
图2显示了具有本发明使用的结构的PDMS印记的扫描电子显微图。该图片显示了观察表面上的平滑排列和成像表面。
图3将本发明使用的PDMS的厚度与典型的玻璃显微镜载片的透射光谱进行比较。当厚度为0.5mm时,PMMA追踪400nm和800nm波长之间的玻璃显微镜载片的透射。
过量结合的磁粒子(EpCAM-铁磁流体)被用于确保最大CTC恢复。但是,在成像过程中,过量的铁磁流体形成成像表面上的细长的聚集体,其影响细胞的定量和定性成像。图4显示了过量的未结合的磁粒子对成像的影响。对于浓度高于2ug/ml(圆形)的铁磁流体来说,总核强度剧烈下降,而对于通常存在于患者样品中的浓度(例如,40ug/ml(三角形))来说,CV升高至40%。
在图5中显示了过量的铁磁流体对成像的影响以及所述去除方法的结果。SKBR-3细胞利用40ug/ml浓度的铁磁流体和0ug/ml铁磁流体成像。对于具有过量的铁磁流体的样品来说,总强度的分布明显很差。
本发明避免了这个问题,并且提供了一种方法,即,将样品在锥形管内以900rpm进行旋转。这迫使所述管中的所有细胞向外。铁磁流体粒子非常小(直径为~175nm)并在样品中保持无规则分布。在几分钟之后,60%的样品从旋转管的底部自动抽出。这个过程重复5次,且SKBR-3细胞利用本发明的PDMS结构沿着观察表面排列。图6显示了沿着观察表面排列的捕获的SKBR-3细胞的明视野、DAPI和PE组合图像。
利用本发明的方法,可去除95%至97%的过量的铁磁流体,使患者样品的最终浓度降低至小于2ug/ml。这样,去除了足够多的过量的铁磁流体,以获得最佳定量测量和图像质量的明显提高。另外,CTC的回复在95%和99%之间。因此利用本发明的去除铁磁流体的方法充分地去除了过量的铁磁流体,从而获得最佳定量测量。图像的质量提高并且可能进行明视野成像,从而获得个体CTC的额外形态学数据。
尽管上文已经描述和具体例证了本发明的某些优选实施例,但并不意味着本发明将局限于这些实施例。在不脱离本发明精神的情况下可以对这些实施例进行各种修改,后附的权利要求书涵盖了全部改进范围。
Claims (8)
1.一种用于光学分析悬浮于流体介质中的稀少细胞的改进的方法,所述方法包括:
a.从疑似患有癌症的患者身上获得血样;
b.将所述血样与铁磁流体粒子混合,所述铁磁流体粒子连接到针对上皮细胞表面上的EpCAM抗原具有特异性的抗体;
c.从结合的铁磁流体中去除未结合的铁磁流体;以及
d.在观察室中利用时间延长积分观察所述血样中的磁响应组分,其中在所述观察室的光学透明的面的内表面上,所述组分沿着观察表面均匀地分布在预制排列结构中。
2.根据权利要求1所述的方法,其中所述排列结构为散兵坑形状的凹槽。
3.根据权利要求1所述的方法,其中所述排列结构包括具有图像观察表面和排列表面的凹槽。
4.根据权利要求1所述的方法,其中所述观察表面为80um。
5.根据权利要求1所述的方法,其中所述去除未结合的铁磁流体的步骤包括:
a.在锥形管中以900rpm旋转具有铁磁流体粒子的所述血样;
b.从所述锥形管的底部抽吸所述血样;以及
c.重复步骤(a)和(b)5次。
6.根据权利要求1所述的方法,其中所述观察的步骤还包括确定所述结合的上皮细胞为CTC。
7.根据权利要求6所述的方法,其中所述确定的步骤包括:
a.利用DAPI阳性第一标记核;
b.利用Cytokeratine-PE阳性第二标记细胞骨架;以及
c.利用CD45-APC阳性第三标记白细胞。
8.根据权利要求7所述的方法,还包括明视野检查捕获的CTC。
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