Bostick R M, Fosdick L, Lillemoe T J, Overn P, Wood J R, Grambsch P, Elmer P, Potter J D
Department of Public Health Sciences, Epidemiology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Cancer Epidemiol Biomarkers Prev. 1997 Nov;6(11):931-42.
The methodological issues for measuring colorectal epithelial cell proliferation, an intermediate end point for studies of colon neoplasia, in epidemiological studies are deceptively numerous and complex, with few methodological data available. Accordingly, during our experience with measuring colorectal epithelial cell proliferation from nearly 500 participants attending over 1300 study visits over a 6-year period, we recorded data on a variety of measurement variations. Methods investigated included rectal biopsy technique, general histological and labeling procedures [including the tritiated thymidine, 5-bromodeoxyuridine (BrdUrd), and the proliferating cell nuclear antigen (PCNA) immunohistochemical techniques used to label S-phase cells in colonic crypts in rectal biopsy specimens], biopsy scoring procedures, and summary scoring methods. Findings include that the PCNA technique was the simplest, most economical, and least time-consuming. The BrdUrd labeling failure rate was 15% versus < 1% for PCNA. The percentage of labeled cells (labeling index) was highest using PCNA in biopsies processed without prior incubation, intermediate using PCNA in biopsies processed with prior incubation as for BrdUrd, and lowest using BrdUrd. The percentage of labeled cells that were in the upper 40% of the crypt (phi h) was higher using BrdUrd than PCNA; visit-to-visit correlations were higher using PCNA (r = 0.51 versus 0.35), and visit-to-visit variability was lower and between-person variability was higher using PCNA. Intra- and inter-rater reliabilities for the techniques were comparable (PCNA intra-rater r = 0.93, inter-rater r = 0.92). The PCNA technique, compared to the BrdUrd technique, is more feasible and reliable, provides a more accurate estimate of the labeling index, and cell proliferation measures determined with PCNA have statistical properties that are generally more favorable for detecting differences in clinical trials. Thus, the PCNA technique may be preferable to techniques requiring incubation of biopsies. Other methodological findings lead us to recommend that, for larger studies measuring colorectal epithelial cell proliferation on outpatient rectal biopsies, biopsies should be taken 10 cm above the anus using a flexible, preferably jumbo cup, endoscopic forceps through a rigid sigmoidoscope, and histological sections should be 3 microns thick taken 50 microns apart.
在流行病学研究中，测量结直肠上皮细胞增殖这一结肠肿瘤研究的中间终点时，方法学问题多得惊人且十分复杂，可用的方法学数据却很少。因此，在我们对近500名参与者进行了6年多、超过1300次研究访视来测量结直肠上皮细胞增殖的过程中，我们记录了各种测量差异的数据。研究的方法包括直肠活检技术、一般组织学和标记程序[包括用于标记直肠活检标本结肠隐窝中S期细胞的氚标记胸腺嘧啶核苷、5-溴脱氧尿苷（BrdUrd）和增殖细胞核抗原（PCNA）免疫组化技术]、活检评分程序和汇总评分方法。研究结果包括，PCNA技术是最简单、最经济且最省时的。BrdUrd标记失败率为15%，而PCNA的标记失败率小于1%。在未经预先孵育处理的活检标本中，使用PCNA时标记细胞的百分比（标记指数）最高，在像BrdUrd那样经过预先孵育处理的活检标本中使用PCNA时标记细胞的百分比处于中间水平，而使用BrdUrd时标记细胞的百分比最低。使用BrdUrd时位于隐窝上部40%的标记细胞百分比（φh）高于PCNA；使用PCNA时访视间的相关性更高（r = 0.51对0.35），使用PCNA时访视间的变异性更低，个体间变异性更高。这些技术的评分者内和评分者间信度相当（PCNA评分者内r = 0.93，评分者间r = 0.92）。与BrdUrd技术相比，PCNA技术更可行、更可靠，能更准确地估计标记指数，并且用PCNA确定的细胞增殖测量值具有的统计学特性通常更有利于在临床试验中检测差异。因此，PCNA技术可能优于需要对活检标本进行孵育的技术。其他方法学研究结果使我们建议，对于在门诊直肠活检中测量结直肠上皮细胞增殖的大型研究，应使用柔性、最好是大杯状的内镜活检钳，通过硬式乙状结肠镜在肛门上方10 cm处取材，组织切片厚度应为3微米，间隔50微米。
