CN1302210A - 荧光分光术中以乙酸用作增强信号对比度试剂 - Google Patents
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Abstract
本发明涉及正常和肿瘤组织、具体如上皮组织的荧光分光术中使用乙酸作为增强信号对比度试剂的方法。更具体来说,一方面,本发明包括检查患者诊断组织样本中组织异常的方法,所述方法包括下述步骤:从诊断组织样本中获得第一个荧光强度光谱;之后,把乙酸以足以改变该诊断组织样本对激发电磁辐射的反应的浓度在该诊断组织样本上施用至少一段有效时间;在有效时间内,从该诊断组织样本中获得第二个荧光强度光谱;测定显示第一个和第二个荧光发射强度光谱间变化的参数;分析所测定的参数以确定该诊断组织样本属正常或异常的可能性。
Description
技术领域
本发明涉及用于光学诊断组织异常的方法和仪器，更具体来说，本发明涉及通过荧光分光法检查正常组织和肿瘤组织、尤其是上皮组织来检测组织异常的方法和仪器。
背景技术
全世界女性中，在多种癌症当中，子宫颈癌是第二常见的恶性肿瘤，只有乳腺癌的发病率超过它。在美国，子宫颈癌是第三常见的女性生殖道肿瘤。1994年，据报道，美国新发生了15000例侵袭性子宫颈癌和55000例原位癌(CIS)。同一年，估计在美国仅由于子宫颈癌就导致4600人死亡。最近，子宫颈侵袭前鳞状癌的发生率有显著增长，尤其在年轻女性中更是如此。在患有侵袭性子宫颈癌的患者中，年龄35岁以下的女性所占比例高达24.5％，并且对于该年龄组女性，发病率正持续增加。预计，除非在诊断技术方面取得进一步进展，否则在未来十年中，子宫颈癌的死亡率可增加20％。
早期检测出子宫颈癌、或称为鳞状上皮内损伤(SIL)的癌前期状态可降低该疾病带来的死亡率。通常情况下，女性群体中使用Pap涂片来检查CIS和子宫颈癌。Pap涂片中，是将大量通过刮擦子宫颈上皮所获得的细胞涂在玻片上，然后将玻片固定并染色以进行细胞学检查。由于取样和读取误差，Pap涂片通常不能获得高灵敏度和高特异性。例如，估计Pap涂片检查法的灵敏度和特异性分别为11-99％和14-97％。
此外，读取Pap涂片是高强度工作，并且需要经过高度训练的专业人员。当患者表明存在SIL的异常Pap涂片时，之后要进行称为阴道窥器检查诊断，阴道窥器检查包括阴道镜检查、活组织检查、和临床诊断的组织学确认。阴道窥器检查需要强化训练，并且其诊断的准确度多变且很有限，甚至对于老练的能手也是如此。此外，诊断不能即时进行。因此，需要找到通过改善早期检查方法来降低子宫颈癌发病率的途径。还需要提供能改善特异性和灵敏度、降低操作者解读结果所需技术水平、并缩短确诊所需时间的诊断方法。
体内荧光分光术是能迅速、非侵害性、并且定量地探测出组织肿瘤化时出现的生物化学和形态学变化的技术。可将检测到的光谱信息与组织的病理组织学相互联系，开发出临床有效的检查和诊断技术。通过使用自动数据分析技术，有可能开发出由非专家使用的自动、迅速、非侵害、并且准确的癌前期检查和诊断系统。
最近，用于人组织、和子宫颈癌前期组织的检查和诊断技术、尤其是基于诱导荧光分光谱的检查和诊断技术已经得到了相当发展；例如参见下述美国专利和专利申请，其中所述文献引入本发明以作参考：Richard-Kortums等人的US 5699795，“用荧光分光术和采用荧光分光术仪器检查子宫颈癌的光学检查”；Richard-Kortums等人的US5697373，“用拉曼光谱和荧光分光术诊断子宫颈癌前期的光学方法和仪器”；Ramanuiam等人US 5623932，“用激光诱导的荧光诊断发育异常”；Richard-Kortums等人的US 5612540，“用荧光分光术检查子宫颈癌的光学方法”；Kittrell等人的US 5562100，“激光诱导的荧光检查组织方法”；Richard-Kortums等人的US 5697373，“用拉曼光谱和荧光分光术诊断子宫颈癌前期的光学方法和仪器”；Richard-Kortums等人的US 5612540，“用拉曼光谱和荧光分光术检查子宫颈癌前期的光学方法和仪器”；Richard-Kortums等人的US 5421339，“用激光诱导的荧光诊断发育异常”；Kittrell等人的US 5419323，“激光诱导的荧光检查组织方法”；Rava等人的US 5345941，光谱诊断轮廓图；Rava等人的US 5201318，光谱诊断轮廓图；SN＿，“用荧光分光术体外和体内检查鳞状上皮内损伤的诊断技术”；SN 467993，“用于体外和体内检查子宫颈癌前期的近红外拉曼光谱测定法”；SN 693471，“确定内脏上皮特征的方法和仪器”；和SN 672623，“体内检测拉曼信号的光谱探针”。
大多数光学和光谱学测定系统和技术所存在的问题是，获得显示要测定特征的适当信号。通常将增强对比度试剂用在体外和体内组织中以增强照射组织的光反馈信号，因此有助于从样本中提取诊断有用信息。例如，当使用光学显微镜检查组织样本时，通常采用加亮细胞结构的技术。总的来说，最近已经使用5-氨基乙酰丙酸诱导的原卟啉IX荧光来灵敏显示不同组织位点中正常组织和肿瘤间区别。
乙酸通常在阴道窥器检查(包括用低倍显微镜原位检查子宫颈的方法)中使用，以增强子宫颈上皮正常区域和病变区域间的差异。可发展成子宫颈癌的区域会出现肉眼可见的短暂变白(乙酰变白)。虽然导致该现象的机制尚未完全弄清楚，但是通常认为，在非正常上皮中存在较高的核密度是主要原因。
本发明者们已经确定，通过增强指征异常组织的光反馈信号，提供具有比现有技术更高灵敏度和选择性、用于自动分光术检查子宫颈前期癌的技术是可取的。本发明提供了这种技术。
发明简述
本发明包括乙酸在正常和肿瘤组织、尤其是上皮的荧光分光术中作为增强信号对比度试剂的应用。更具体来说，一方面，本发明包括检查患者诊断组织样本中组织异常的方法，包括下述步骤：从诊断组织样本中获得第一个荧光强度光谱；之后，把乙酸以足以改变该诊断组织样本对激发电磁辐射的反应的浓度，在该诊断组织样本上施用至少一段有效时间；在有效时间内，从该诊断组织样本中获得第二个荧光强度光谱；测定表示第一个和第二个荧光发射强度光谱间变化的参数；分析所测定的参数以确定该诊断组织样本是正常的或异常的可能性。
附图中提供了一个或多个本发明实施方案的详细内容，在下面加以说明。通过阅读本说明书、附图、和权利要求，本发明的其它特征、目的、和优点将显而易见。
附图说明
附图1(a)是病理正常位点施用乙酸前和施用乙酸后的荧光光谱图。
附图1(b)是病理异常位点施用乙酸前和施用乙酸后的荧光光谱图。
附图2表示由于乙酸的作用，正常位点和异常位点的峰值发射波长移位变换图。
附图3(a)和3(b)分别表示各种类间t-试验显著性水平α的非归一化和归一化数据连线图。
附图4(a)和4(b)分别表示每一类别以波长差异的百分比对患者百分比所作的非归一化(α=0.07)和归一化(α=0.05)数据的柱状图。
附图5(a)和5(b)分别表示每一类别有效发射波长对患者百分比所作图的非归一化(α=0.07)和归一化(α=0.05)数据连线图。
附图6表示所有患者类别间强度(用非归一化光谱)平均差异的连线图。
发明详述效果的证实
本发明包括乙酸在正常和肿瘤组织、尤其是上皮荧光分光术中作为增强信号对比度试剂的应用。为了证实使用乙酸所增强的光反馈信号，我们在100位由于患有子宫颈前期癌而进行阴道窥器检查和治疗的患者中测定了子宫颈体内荧光光谱。用设计来收集分辨率为2mm的空间分解光谱的多象素光纤探针获得337nm激发波长处收集的发射光谱。用影像光谱仪将光谱记录到成象阵列(例如CDD积分回路成象阵列)上。在将乙酸(优选为3％溶液)施用在进行阴道窥器检查的子宫颈上之前或之后，通过将探针放在子宫颈的4扇形的每一个上来获得光谱。然后对患者进行常规环形电手术切除术，以切下组织癌前期区域。我们确定了施用乙酸前和施用乙酸后光谱的差异，并把这些差异与阴道窥器检查印象以及病理诊断相互联系。
适用于实施本发明的分光镜系统描述在US 5697373中。该系统包括激光器、光纤探针、和用于阴道窥器检查时从无损伤子宫颈记录荧光光谱的光学多道分析器。
附图1(a)是病理正常位点施用乙酸前和施用乙酸后的荧光光谱图。附图1(b)是病理异常位点施用乙酸前和施用乙酸后的荧光光谱图。在正常位点，加入乙酸对光谱的峰值发射波长只有微弱影响。然而，加入乙酸后，异常位点的光谱峰值红移了。附图1(a)和1(b)举例说明用于映射子宫颈上皮直径1cm区域荧光的多象素探针的单一象素中得到的光谱。
附图2表示由于乙酸的作用，正常位点和异常位点的峰值发射波长移位变换的图。附图2中，将施用乙酸后荧光峰值发射波长的移位作为探针两个放置部位(一个在正常位点(a)，另一个在含高级别前期癌的位点(b))的象素位置的函数绘图。在异常位点(b)，平均位移比正常位点(a)大，这表明光谱位移始终发生在子宫颈的正常或异常位点。以前的研究(Ramanuiam,N.等人，“用337 nm激发的激光诱导荧光法体内诊断子宫颈上皮内肿瘤”，《美国国家科学院院报》(Proc.Natl.Acad,Sci.USA)1994,91:10913-10917)仅获得了施用乙酸后337nm激发荧光光谱，并且已表明，相对于正常组织光谱，前期癌组织的光谱表现出红移。这些结果与本发明提供的结果是一致的，并表明，乙酸在能用于区别正常和异常子宫颈上皮的337nm激发的荧光法中起重要作用。
乙酸在进行荧光分光术期间作为增强信号对比度试剂的作用，可通过统计分析证实。如上所述获得了多个患者的光谱，其中数据是从子宫颈的4个扇形获得的。分析每一患者的所有数据。因为放置了探针并使用了病理学方法，把施用乙酸之前或之后从每一扇形获得的所有光谱按正常或异常分类。确立了光谱类别：·正常/施用乙酸前(NB)·异常/施用乙酸前(AB)·正常/施用乙酸后(NA)·异常/施用乙酸后(AA)
使用统计法(t-试验)来比较各类别间在每一发射波长处的荧光强度：·NB对AB·NB对NA·NA对AA·AB对AB
对归一化(峰值为1)和非归一化光谱均进行比较。
各类别间t-试验的显著性水平(α)按下述确立：对于每一患者，将来自单一类别的光谱随机分组。测定每一组的平均光谱。然后对于至少80％患者，其中两个平均光谱看来基本相同(发射波长差异＜l0％)时确定最大α值。附图3(a)和3(b)分别表示类别间t-试验的非归一化和归一化显著性水平α数据的连线图。附图4(a)和4(b)分别表示，对于每一类别，以波长差异的百分比对患者百分比所作图的非归一化(α=0.07)和归一化(α=0.05)数据柱状图。
然后确定类别间显著性差异的量和光谱相关性。在每一发射波长进行t-试验，以确定对于两个类别，强度的分布是否有显著差异。附图5(a)和5(b)分别表示，对于每一类别，以显著发射波长对患者百分比所作图的非归一化(α=0.07)和归一化(α=0.05)数据连线图。
附图6表示所有患者中类别间强度上的平均差异(用非归一化光谱)。将线NB-AB和NA-AA进行比较，与不施用乙酸的组织获得的数据相比，施用乙酸使得正常和异常组织间的信号几乎在所有波长都增加了。将线NB-NA和AB-AA进行比较，在几乎所有波长，施用乙酸对异常组织的作用比对正常组织的作用大。
优选方法
由于已经证实了乙酸可在光谱和影像中产生可能对诊断有用的差异，下面提供实施本发明的优选方法：(1)通过用激发电磁辐射线照射诊断组织样本，从该诊断组织样本获得第一个荧光发射强度光谱。对于体内应用，该组织优选为上皮。为了检查子宫颈异常，该组织优选为子宫颈上皮。优选的激发波长约为380nm、337 nm、和460 nm。(2)然后，把乙酸以足以改变该诊断组织样本对激发电磁辐射的反应的浓度，在该诊断组织样本上施用至少一段有效时间。乙酸的浓度一般为3％-6％，但是能使反应产生所需变化的所有医用安全浓度都是合适的。(3)在有效时间内，从该诊断组织样本获得第二个荧光强度光谱。优选的激发波长同样约为380nm、337nm、和460nm。不论怎样，对于第一个和第二个荧光发射强度光谱，都使用基本上相同的激发波长。(4)然后确定指示第一个和第二个荧光强度光谱变化的参数。已经发现，使用乙酸使得测定的第一个和第二个荧光发射强度光谱的强度发生改变。乙酸使得测定的荧光强度都降低了，但是对异常组织的影响比对正常组织大。还发现，施用乙酸使得测定的第一个和第二个荧光发射强度光谱的红移发生了改变，尤其是峰值发射波长的红移发生了改变。因此，乙酸增强了正常和异常组织间荧光强度和谱系形状的差异。对于乙酸对组织的这种影响，有这样的假设，即由于增强了光散射，乙酸使更少的激发光(和更少的发射光)“直接”穿过上皮。还据信是由于上皮下方基质中的至少某些荧光团(例如NADH、FAD)和吸收剂(例如血红蛋白)的发射作用被减弱了。(5)一旦确定了所需指示参数，就可分析该指示参数，计算该诊断组织样本是正常或异常的可能性。其中一种分析方法只是简单地将该指示参数与凭经验确定的阈值比较。如果该指示参数超过了阈值，则可认为在该诊断组织样本中可能有组织异常。但可采用其它能利用施用乙酸使得正常和异常组织间荧光光谱信号增强这一事实的分析方法。
已经描述了本发明的多个实施方案。然而，应当理解，可进行不背离本发明实质和范围的不同改变。例如，在本发明方法中，除了已经描述的装置以外，可使用其它装置。因此，其它实施方案也在本发明权利要求书所限定的保护范围内。
Claims (13)
1．以患者诊断组织样本检测组织异常的方法，所述方法包括下述步骤：
(a)从诊断组织样本获得第一个荧光发射强度光谱；
(b)然后，把乙酸以足以改变该诊断组织样本对激发电磁辐射的反应的浓度在该诊断组织样本上施用至少一段有效时间；
(c)在有效时间内，从该诊断组织样本获得第二个荧光发射强度光谱；
(d)测定显示第一个和第二个荧光发射强度光谱间出现变化的参数；
(e)分析该测定的参数，以确定该诊断组织样本是正常或异常的可能性。
2．以患者的诊断组织样本检测组织异常的方法，所述方法包括下述步骤：
(a)用激发电磁辐射照射诊断组织样本；
(b)从经上述照射的该诊断组织样本检测第一个荧光发射强度光谱；
(c)然后，把该诊断组织样本在其浓度足以使该诊断组织样本对激发电磁辐射的反应出现可检测变化的乙酸中露置至少一段有效时间；
(d)在有效时间内，再用激发电磁辐射照射该诊断组织样本；
(e)从经上述再一次照射的该诊断组织样本检测产生的第二个荧光发射强度光谱；
(f)测定显示第一个和第二个荧光发射强度光谱间变化的参数；
(g)将该测定参数与阈值比较，如果所确定的参数超过了该阈值，则表示该诊断组织样本中有组织异常。
3．权利要求1或2的方法，其中显示第一个和第二个荧光发射强度光谱间变化的参数是显示强度改变的参数。
4．权利要求1或2的方法，其中显示第一个和第二个荧光发射强度光谱间变化的参数是显示血红蛋白吸收的参数。
5．权利要求1或2的方法，其中显示第一个和第二个荧光发射强度光谱间变化的参数是显示红移的参数。
6．权利要求5的方法，其中所述参数是显示峰值发射波长移动的参数。
7．权利要求1或2的方法，其中所述诊断组织样本为子宫颈组织样本。
8．权利要求1或2的方法，其中所述诊断组织样本是体内样本。
9．权利要求1的方法，其中分析所测定的参数以确定该诊断组织样本属正常或异常的可能性的步骤包括下述步骤：将该测定的参数与阈值比较，如果所测定的参数超过了阈值，则表示该诊断组织样本中出现组织异常。
10．权利要求2的方法，其中步骤(a)和(d)中的电磁辐射采用基本上相同的波长。
11．权利要求2的方法，其中步骤(a)和(d)中使用的电磁辐射波长约为380nm。
12．权利要求2的方法，其中步骤(a)和(d)中使用的电磁辐射波长约为337nm。
13．权利要求2的方法，其中步骤(a)和(d)中使用的电磁辐射波长约为460nm。
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- 1998-10-20 JP JP2000516708A patent/JP2001520393A/ja not_active Withdrawn
- 1998-10-20 AU AU11084/99A patent/AU1108499A/en not_active Abandoned
- 1998-10-20 EP EP98953808A patent/EP1071473B1/en not_active Expired - Lifetime
- 1998-10-20 DE DE69838560T patent/DE69838560T2/de not_active Expired - Lifetime
- 1998-10-20 US US09/175,234 patent/US6187289B1/en not_active Expired - Lifetime
- 1998-10-20 WO PCT/US1998/022217 patent/WO1999020313A1/en active Application Filing
- 1998-10-20 AU AU11083/99A patent/AU1108399A/en not_active Abandoned
- 1998-10-20 US US09/175,232 patent/US6241662B1/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010006513A1 (zh) * | 2008-07-15 | 2010-01-21 | Zeng Kun | 便携式子宫颈癌癌前病变诊断装置 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2001520393A (ja) | 2001-10-30 |
| DE69838560T2 (de) | 2008-08-14 |
| WO1999020314A1 (en) | 1999-04-29 |
| EP1071473A1 (en) | 2001-01-31 |
| AU1108399A (en) | 1999-05-10 |
| WO1999020313A1 (en) | 1999-04-29 |
| AU1108499A (en) | 1999-05-10 |
| US6187289B1 (en) | 2001-02-13 |
| EP1071473B1 (en) | 2007-10-10 |
| EP1071473A4 (en) | 2005-10-19 |
| US6241662B1 (en) | 2001-06-05 |
| CA2306795A1 (en) | 1999-04-29 |
| DE69838560D1 (de) | 2007-11-22 |
| CA2306795C (en) | 2009-01-20 |
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