共聚焦激光扫描显微镜术前检测皮肤肿瘤边界的临床意义研究
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摘要
共聚焦激光扫描显微镜(confocal laser scanning microscopy,CLSM)是近年来新兴的具有高分辨率的光学显微镜,其具有原位、实时、动态三维等特点,从而保证可以在细胞生理状态下进行诊断,还可实时动态地对同一组织多次成像,减少了传统切片制作过程中人为因素的影响。由于CLSM具有较好的敏感性和特异性,常被用于皮肤疾病特别是皮肤肿瘤的诊断和鉴别诊断。同时还可以帮助术者快速确定需要切除的皮损边界,方便手术范围的评估。
皮肤肿瘤大多见于老年人,以日光性角化病、基底细胞癌、鳞状细胞癌为多见,肿瘤首先是在细胞水平发生变化,肉眼无法准确地判定恶性病变的切除范围,Mohs显微外科手术是皮肤外科技术与特殊冰冻组织切片相结合的一种手术方法,具有可实时观察手术标本的特点,不仅能保证一次手术完整彻底切净肿瘤组织,使肿瘤复发率相当低和伤口易于愈合,而且能最大限度地减少皮肤缺损面积,为进一步整形修复打下良好的基础。但其手术费用高、手术时间长,限制了在国内的的进一步开展,这就需要我们进一步探索测定皮肤肿瘤边界的新技术。
本研究通过应用CLSM观察日光性角化病、基底细胞上皮瘤患者皮损周边情况,确定肿瘤边界,与组织病理学结果相比较,从而为CLSM测定肿瘤边界提供理论依据。
实验共分三个部分：第一部分应用CLSM观察健康人的皮肤结构,健康志愿者12人,年龄41岁-73岁,平均年龄57岁。检测前臂皮肤,得到正常皮肤CLSM图像,与传统组织病理作对比；第二部分应用CLSM观察日光性角化病患者皮损周边图像,确定肿瘤边界,与组织病理确定的边界进行分析对比,统计学分析应用t检验；第三部分应用CLSM观察基底细胞上皮瘤患者皮损周边图像,确定肿瘤边界,与组织病理确定的边界进行分析对比,统计学分析应用t检验。
第一部分研究获得与病理切片相对应的正常皮肤的CLSM图像特征；第二部分应用CLSM对10例病理已确诊为日光性角化病患者皮损边缘处进行观测、记录,与病理检测边界进行对比,在统计学上无明显差异,将CLSM观测结果与常规手术切除边界进行对比,发现CLSM组明显低于常规值,具有统计学意义；第三部分应用CLSM对11例病理已确诊为基底细胞上皮瘤患者皮损边缘处进行观测、记录,与病理检测边界进行对比,在统计学上无明显差异,将CLSM观测结果与常规手术切除边界进行对比,发现CLSM组明显低于常规值,具有统计学意义。
实验结论：CLSM可有效确定日光性角化病、基底细胞上皮瘤患者皮损边界,指导临床手术切除病灶。CLSM具有实时、无创的特点,在指导皮肤肿瘤切除方面具有广泛的应用前景,值得进一步推广。
The Research on Clinical Significance of Confocal Laser Scanning Microscopy in preoperative detection of skin tumor
Confocal laser scanning microscope (CLSM) is a new high-light optical microscope in recent years, with the in-situ, real-time, dynamic three-dimensional characteristics. It ensures the cell physiological state diagnosis and real-time dynamic imaging of the same organizations repeatedly, and can reduce influence of artificial factors in the traditional production process. For CLSM with good sensitivity and specificity, it is often used in dermatologic diseases, especially skin cancer diagnosis and differential diagnosis. It also helps identify the border of the skin lesions and facilitates the assessment of extent of the operation.
Most of the skin cancer occurred on the elder. The Actinic keratoses, basal cell carcinoma and squamous cell carcinoma is the common. Skin tumors are cells abnormally proliferation firstly and we can not determine the extent of skin lesions accurately with the naked eye. Mohs surgery is a skin micro-surgery Technology cooperated with frozen tissue sections. It can help us observe the characteristics of surgical specimens in time. It can ensure the integrity of a surgical cut and the tumor recurrence rate is very low. Besides it can help us reduce the area of skin damage in order to lay a good foundation for orthopedic rehabilitation. However, the high cost and long operative time limits the further development in our country. This requires us to further explore the new technology in preoperative detection of skin tumors.
Our study utilized CLSM to observe the dermatic structure of basal cell epithelioma and actinic keratoses patients. Be compared with the histopathological results tumor boundaries identified, so as to provide theoretical evidence in preoperative detection of skin tumors with CLSM.
Experiment was divided into three parts:The first part, to research the dermatic structure of healthy people with CLSM. The amount of volunteers was 12, whose age ranges from 41 to 73 years old, average age was 57 years old. We obtained the normal CLSM dermatic imaging from skin of forearms, compared with conventional histopathology; The second part, with CLSM to observe the dermatic structure of actinic keratoses patients and identified tumor boundaries compared with the histopathological results, statistical analysis with T test; The third part, to observe the dermatic structure of basal cell epithelioma patient with CLSM and identify tumor boundaries compared with the histopathological results.
In part one, we got normal CLSM dermatic imaging successfully. In part two, observed the dermatic boundry of skin lesion of the 10 actinic keratoses patients with CLSM and compared with the histopathological results. It showed no significant difference. CLSM result was significantly lower than conventional value; In part two, observed the dermatic boundry of skin lesion of the 11 basal cell epithelioma patients with CLSM and compared with the histopathological results. It showed no significant difference. CLSM result was significantly lower than conventional value.
Results:CLSM can detect tumor boundaries of the basal cell epithelioma and actinic keratoses effectively and can guide surgical resection of clinical lesions. CLSM is a real-time and non-invasive detection method. And it has broad application prospects in guiding surgical resection of skin tumors, which is worth extending forward.
皮肤肿瘤大多见于老年人,以日光性角化病、基底细胞癌、鳞状细胞癌为多见,肿瘤首先是在细胞水平发生变化,肉眼无法准确地判定恶性病变的切除范围,Mohs显微外科手术是皮肤外科技术与特殊冰冻组织切片相结合的一种手术方法,具有可实时观察手术标本的特点,不仅能保证一次手术完整彻底切净肿瘤组织,使肿瘤复发率相当低和伤口易于愈合,而且能最大限度地减少皮肤缺损面积,为进一步整形修复打下良好的基础。但其手术费用高、手术时间长,限制了在国内的的进一步开展,这就需要我们进一步探索测定皮肤肿瘤边界的新技术。
本研究通过应用CLSM观察日光性角化病、基底细胞上皮瘤患者皮损周边情况,确定肿瘤边界,与组织病理学结果相比较,从而为CLSM测定肿瘤边界提供理论依据。
实验共分三个部分：第一部分应用CLSM观察健康人的皮肤结构,健康志愿者12人,年龄41岁-73岁,平均年龄57岁。检测前臂皮肤,得到正常皮肤CLSM图像,与传统组织病理作对比；第二部分应用CLSM观察日光性角化病患者皮损周边图像,确定肿瘤边界,与组织病理确定的边界进行分析对比,统计学分析应用t检验；第三部分应用CLSM观察基底细胞上皮瘤患者皮损周边图像,确定肿瘤边界,与组织病理确定的边界进行分析对比,统计学分析应用t检验。
第一部分研究获得与病理切片相对应的正常皮肤的CLSM图像特征；第二部分应用CLSM对10例病理已确诊为日光性角化病患者皮损边缘处进行观测、记录,与病理检测边界进行对比,在统计学上无明显差异,将CLSM观测结果与常规手术切除边界进行对比,发现CLSM组明显低于常规值,具有统计学意义；第三部分应用CLSM对11例病理已确诊为基底细胞上皮瘤患者皮损边缘处进行观测、记录,与病理检测边界进行对比,在统计学上无明显差异,将CLSM观测结果与常规手术切除边界进行对比,发现CLSM组明显低于常规值,具有统计学意义。
实验结论：CLSM可有效确定日光性角化病、基底细胞上皮瘤患者皮损边界,指导临床手术切除病灶。CLSM具有实时、无创的特点,在指导皮肤肿瘤切除方面具有广泛的应用前景,值得进一步推广。
The Research on Clinical Significance of Confocal Laser Scanning Microscopy in preoperative detection of skin tumor
Confocal laser scanning microscope (CLSM) is a new high-light optical microscope in recent years, with the in-situ, real-time, dynamic three-dimensional characteristics. It ensures the cell physiological state diagnosis and real-time dynamic imaging of the same organizations repeatedly, and can reduce influence of artificial factors in the traditional production process. For CLSM with good sensitivity and specificity, it is often used in dermatologic diseases, especially skin cancer diagnosis and differential diagnosis. It also helps identify the border of the skin lesions and facilitates the assessment of extent of the operation.
Most of the skin cancer occurred on the elder. The Actinic keratoses, basal cell carcinoma and squamous cell carcinoma is the common. Skin tumors are cells abnormally proliferation firstly and we can not determine the extent of skin lesions accurately with the naked eye. Mohs surgery is a skin micro-surgery Technology cooperated with frozen tissue sections. It can help us observe the characteristics of surgical specimens in time. It can ensure the integrity of a surgical cut and the tumor recurrence rate is very low. Besides it can help us reduce the area of skin damage in order to lay a good foundation for orthopedic rehabilitation. However, the high cost and long operative time limits the further development in our country. This requires us to further explore the new technology in preoperative detection of skin tumors.
Our study utilized CLSM to observe the dermatic structure of basal cell epithelioma and actinic keratoses patients. Be compared with the histopathological results tumor boundaries identified, so as to provide theoretical evidence in preoperative detection of skin tumors with CLSM.
Experiment was divided into three parts:The first part, to research the dermatic structure of healthy people with CLSM. The amount of volunteers was 12, whose age ranges from 41 to 73 years old, average age was 57 years old. We obtained the normal CLSM dermatic imaging from skin of forearms, compared with conventional histopathology; The second part, with CLSM to observe the dermatic structure of actinic keratoses patients and identified tumor boundaries compared with the histopathological results, statistical analysis with T test; The third part, to observe the dermatic structure of basal cell epithelioma patient with CLSM and identify tumor boundaries compared with the histopathological results.
In part one, we got normal CLSM dermatic imaging successfully. In part two, observed the dermatic boundry of skin lesion of the 10 actinic keratoses patients with CLSM and compared with the histopathological results. It showed no significant difference. CLSM result was significantly lower than conventional value; In part two, observed the dermatic boundry of skin lesion of the 11 basal cell epithelioma patients with CLSM and compared with the histopathological results. It showed no significant difference. CLSM result was significantly lower than conventional value.
Results:CLSM can detect tumor boundaries of the basal cell epithelioma and actinic keratoses effectively and can guide surgical resection of clinical lesions. CLSM is a real-time and non-invasive detection method. And it has broad application prospects in guiding surgical resection of skin tumors, which is worth extending forward.
引文
[1]Busam KJ, Hester K, et al. Detection of clinically amelanotic malignant melanoma and assessment of its margins by in vivo confocal scanning laser microscopy. Arch Dermatol.2001 Jul;137(7):923-929.
[2]Huzaira M, Rius F, et al.Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol.2001 Jun;116(6):846-852.
[3]李泓馨,张选棉,高天文.“皮肤CT”—皮肤病诊断的新手段.中国皮肤性病学杂志,2007；21(7)：432-434.
[4]Sauermann K, Clemann,S, et al. Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo.Skin Res Technol.2002 Feb;8(1):52-56.
[5]Har-Shai Y,Hai N,Taran A. Sensitivity and positive predictive values of presurgical clinical diagnosis of excised benign and malignant skin tumors:a prospective study of 835 lesions in 778 patients. Plast Reconstr Surg.2001 Dec; 108(7):1982-1989.
[6]Sauermann K, Gambichler T, Wilmert M, et al. Investigation of basal cell carcinoma by confocal laser scanning microscopy in vivo. Skin Res Technol.2002 Aug;8(3):141-147.
[7]马晶波,刘华绪,任秋实.激光扫描共聚焦显微镜原理及其在皮肤科的应用.中南地区皮肤性病学术会议论文集.2006；45-47.
[8]梅立新.张迎春,宫风春.皮肤病理组织制片的体会[J].承德医学院学报.2002,19(2)：136.
[9]Gonzalez S, Tannous Z. Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma. J Am Acad Dermatol.2002 Dec;47(6):869-874.
[10]Gauthie Gr, Ngo H, et al. Mohs surgery-a new approach with a mould and glass discs:review of the literature and comparative study. J Otolaryngol.2006 Oct;35(5):292-304.
[11]Huilgol SC, Selva D, et al.Surgical margins for lentigo maligna and lentigo maligna melanoma:the technique of mapped serial excision. Arch Dermatol,2004 Sep;140(9):1087-1092.
[12]李航.皮肤外科系列讲座(五)—Mohs显微描记手术.中国美容医学,2008；17(12)：1807-1810.
[13]Bricca GM, Brodland DG, et al. Cutaneous head and neck melanoma treated with Mohs micrographic surgery. J Am Acad Dermatol,2005 Jan;52(1):92-100.
[14]Chung VQ, Dwyer PJ, Nehal KS, et al. Use of Ex Vivo Confocal Scanning Laser Microscopy during Mohs Surgery for Nonmelanoma Skin Cancers. Dermatol Surg.2004 Dec; 30(12 Pt 1):1470-1478.
[15]Milind Rajadhyaksha, Gregg Menaker, Thomas Flotte, et al. Confocal Examination of Nonmelanoma Cancers in Thick Skin Excisions to Potentially Guide Mohs Micrographic Surgery Without Frozen Histopathology. J Inyest Dermatol.2001 Nov; 117(5):1137-1143.
[16]Tannous Z, Torres A, Gonzalez S. In vivo real-time confocal reflectance microscopy:a noninvasive guide for Mohs micrographic surgery facilitated by aluminum chloride, an excellent contrast enhancer. Dermatol Surg.2003 Aug; 29(8):839-846.
[17]Florell SR, Boucher KM, Leachman SA, et al. Histopathologic Recognition of Involved Margins of Lentigo Maligna Excised by Staged Excision. Arch Dermatol.2003 May;139(5):595-604.
[18]Tannous ZS, Mihm MC,Flotte TJ, et al. In vivo examination of lentigo maligna and malignant melanoma in situ, lentigo maligna type by near-infrared reflectance confocal microscopy:comparison of in vivo confocal images with histologic sections. J Am Acad Dermatol.2002 Feb;46(2):260-263.
[19]Curiel-Lewandrowski C, Williams CM, et al. Use of in vivo confocal microscopy in malignant melanoma:an aid in diagnosis and assessment of surgical and nonsurgical therapeutic approaches. Arch Dermatol,2004 Sep;140(9):1127-1132.
[20]M. Horn, A. Gerger, S. Koller, et al. The use of confocal laser scanning microscopy in microsurgery for invasive squamous cell carcinoma. Br J Dermatol.2007 Jan; 156(1):81-84.
[21]刘华绪,郑志忠,任秋实.基于光学共聚焦原理的皮肤在体三维成像系统及应用.中华皮肤科杂志,2006,39(10)：616-619.
[22]Marra DE, Torres A,Schanbacher CF, et al. Detection of residual basal cell carcinoma by in vivo confocal microscopy. Dermatol Surg.2005 May;31(5):538-541.
[23]沈柱,陈凌等.寻常性银屑的皮肤CT特征初探.中国麻风皮肤病杂志,2008,24(4)：301-303.
[1]Rigby PJ, Goldie RG. Confocal microscopy in biomedical research. Croat Med J. 1999 Sep; 40(3):346-52.
[2]Huzaira M, Rius F, Rajadhyaksha M, et al. Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol,2001; 116: 846-852.
[3]Rajadhyaksha M, Gonzaiez S, Zavislan JM, et al.In vivo confocal scanning laser microscopy of human skin II:advances in instrumentation and comparison with histology. J Invest Dermatol,1999; 113:293-303.
[4]Sauermann K, Gambichler T, Jaspers S, et al. Histometric data obtained by in vivo confocal laser scanning microscopy in patients with systemic sclerosis. BMC Dermatol.2002 Aug 6; 2:8.
[5]Choi J,Choo J,Chung H, et al. Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy. Biopolymers.2005 Apr 5;77(5):264-72.
[6]Tannous ZS, Mihm MC,Flotte TJ, et al. In vivo examination of lentigo maligna and malignant melanoma in situ, lentigo maligna type by near-infrared reflectance confocal microscopy:comparison of in vivo confocal images with histologic sections. J Am Acad Dermatol.2002 Feb;46(2):260-3.
[7]Chung VQ, Dwyer PJ, Nehal KS, et al. Use of Ex Vivo Confocal Scanning Laser Microscopy during Mohs Surgery for Nonmelanoma Skin Cancers. Dermatol Surg.2004 Dec; 30(12 Pt 1):1470-8.
[8]Milind Rajadhyaksha, Gregg Menaker, Thomas Flotte, et al. Confocal Examination of Nonmelanoma Cancers in Thick Skin Excisions to Potentially Guide Mohs Micrographic Surgery Without Frozen Histopathology. J Inyest Dermatol.2001 Nov; 117(5):1137-43.
[9]Tannous Z, Torres A, Gonzalez S. In vivo real-time confocal reflectance microscopy:a noninvasive guide for Mohs micrographic surgery facilitated by aluminum chloride, an excellent contrast enhancer. Dermatol Surg.2003 Aug; 29(8):839-46.
[10]Sauermann K, Gambichler T, Wilmert M, et al. Investigation of basal cell carcinoma by confocal laser scanning microscopy in vivo. Skin Res Technol.2002 Aug;8(3):141-7.
[11]M. Horn, A. Gerger, S. Koller, et al. The use of confocal laser scanning microscopy in microsurgery for invasive squamous cell carcinoma. Br J Dermatol.2007 Jan; 156(1):81-4.
[12]Har-Shai Y,Hai N,Taran A. Sensitivity and positive predictive values of presurgical clinical diagnosis of excised benign and malignant skin tumors:a prospective study of 835 lesions in 778 patients. Plast Reconstr Surg.2001 Dec;108(7):1982-9.
[13]Marra DE, Torres A,Schanbacher CF, et al. Detection of residual basal cell carcinoma by in vivo confocal microscopy. Dermatol Surg.2005 May;31(5):538-41.
[14]O'goshi K, Suihko C, Serup J.In vivo imaging of intradermal tattoos by confocal scanning laser microscopy. Skin Res Technol.2006 May; 12(2):94-8.
[15]Gerger A, Koller S, Kern T, et al. Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors. J Invest Dermatol.2005 Mar; 124(3):493-8.
[16]Richard G. B. Langley, Elizabeth Burton, BSc, et al. In vivo confocal scanning laser microscopy of benign lentigines:Comparison to conventional histology and in vivo characteristics of lentigo maligna. J Am Acard Dermatol.2006 Jul; 55(1):88-97.
[17]Agero AL, Busam KJ,Benvenuto-Andrade C, et al. Reflectance confocal microscopy of pigmented basal cell carcinoma. J Am Acad Dermatol.2006 Apr;54(4):638-43.
[18]Langley RQ Rajadhyaksha M,Dwyer PJ, et al. Confocal scanning laser microscopy of benign and malignant melanocytic skin lesions in vivo. J Am Acad Dermatol.2001 Sep; 45(3):365-76. echnol,2002,8:52-56.
[2]Huzaira M, Rius F, et al.Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol.2001 Jun;116(6):846-852.
[3]李泓馨,张选棉,高天文.“皮肤CT”—皮肤病诊断的新手段.中国皮肤性病学杂志,2007；21(7)：432-434.
[4]Sauermann K, Clemann,S, et al. Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo.Skin Res Technol.2002 Feb;8(1):52-56.
[5]Har-Shai Y,Hai N,Taran A. Sensitivity and positive predictive values of presurgical clinical diagnosis of excised benign and malignant skin tumors:a prospective study of 835 lesions in 778 patients. Plast Reconstr Surg.2001 Dec; 108(7):1982-1989.
[6]Sauermann K, Gambichler T, Wilmert M, et al. Investigation of basal cell carcinoma by confocal laser scanning microscopy in vivo. Skin Res Technol.2002 Aug;8(3):141-147.
[7]马晶波,刘华绪,任秋实.激光扫描共聚焦显微镜原理及其在皮肤科的应用.中南地区皮肤性病学术会议论文集.2006；45-47.
[8]梅立新.张迎春,宫风春.皮肤病理组织制片的体会[J].承德医学院学报.2002,19(2)：136.
[9]Gonzalez S, Tannous Z. Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma. J Am Acad Dermatol.2002 Dec;47(6):869-874.
[10]Gauthie Gr, Ngo H, et al. Mohs surgery-a new approach with a mould and glass discs:review of the literature and comparative study. J Otolaryngol.2006 Oct;35(5):292-304.
[11]Huilgol SC, Selva D, et al.Surgical margins for lentigo maligna and lentigo maligna melanoma:the technique of mapped serial excision. Arch Dermatol,2004 Sep;140(9):1087-1092.
[12]李航.皮肤外科系列讲座(五)—Mohs显微描记手术.中国美容医学,2008；17(12)：1807-1810.
[13]Bricca GM, Brodland DG, et al. Cutaneous head and neck melanoma treated with Mohs micrographic surgery. J Am Acad Dermatol,2005 Jan;52(1):92-100.
[14]Chung VQ, Dwyer PJ, Nehal KS, et al. Use of Ex Vivo Confocal Scanning Laser Microscopy during Mohs Surgery for Nonmelanoma Skin Cancers. Dermatol Surg.2004 Dec; 30(12 Pt 1):1470-1478.
[15]Milind Rajadhyaksha, Gregg Menaker, Thomas Flotte, et al. Confocal Examination of Nonmelanoma Cancers in Thick Skin Excisions to Potentially Guide Mohs Micrographic Surgery Without Frozen Histopathology. J Inyest Dermatol.2001 Nov; 117(5):1137-1143.
[16]Tannous Z, Torres A, Gonzalez S. In vivo real-time confocal reflectance microscopy:a noninvasive guide for Mohs micrographic surgery facilitated by aluminum chloride, an excellent contrast enhancer. Dermatol Surg.2003 Aug; 29(8):839-846.
[17]Florell SR, Boucher KM, Leachman SA, et al. Histopathologic Recognition of Involved Margins of Lentigo Maligna Excised by Staged Excision. Arch Dermatol.2003 May;139(5):595-604.
[18]Tannous ZS, Mihm MC,Flotte TJ, et al. In vivo examination of lentigo maligna and malignant melanoma in situ, lentigo maligna type by near-infrared reflectance confocal microscopy:comparison of in vivo confocal images with histologic sections. J Am Acad Dermatol.2002 Feb;46(2):260-263.
[19]Curiel-Lewandrowski C, Williams CM, et al. Use of in vivo confocal microscopy in malignant melanoma:an aid in diagnosis and assessment of surgical and nonsurgical therapeutic approaches. Arch Dermatol,2004 Sep;140(9):1127-1132.
[20]M. Horn, A. Gerger, S. Koller, et al. The use of confocal laser scanning microscopy in microsurgery for invasive squamous cell carcinoma. Br J Dermatol.2007 Jan; 156(1):81-84.
[21]刘华绪,郑志忠,任秋实.基于光学共聚焦原理的皮肤在体三维成像系统及应用.中华皮肤科杂志,2006,39(10)：616-619.
[22]Marra DE, Torres A,Schanbacher CF, et al. Detection of residual basal cell carcinoma by in vivo confocal microscopy. Dermatol Surg.2005 May;31(5):538-541.
[23]沈柱,陈凌等.寻常性银屑的皮肤CT特征初探.中国麻风皮肤病杂志,2008,24(4)：301-303.
[1]Rigby PJ, Goldie RG. Confocal microscopy in biomedical research. Croat Med J. 1999 Sep; 40(3):346-52.
[2]Huzaira M, Rius F, Rajadhyaksha M, et al. Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol,2001; 116: 846-852.
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