Mabruk M J, Flint S R, Coleman D C, Shiels O, Toner M, Atkins G J
University of Dublin, Moyne Institute of Preventive Medicine, Department of Microbiology, Republic of Ireland.
J Oral Pathol Med. 1996 Apr;25(4):170-6. doi: 10.1111/j.1600-0714.1996.tb00215.x.
As a diagnostic technique, in situ hybridization requires a long processing time, a degree of expertise and may be difficult to handle routinely in some laboratories. To simplify the in situ hybridization method, we have modified a microwave in situ hybridization technique and applied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV-seropositive patients (definitively diagnosed by a conventional in situ hybridization technique) with appropriate controls. It was necessary to design a novel chamber to avoid drying of sections during the hybridization step. This modified microwave in situ hybridization technique was equispecific and equisensitive to the conventional technique and it shortens the hybridization time from overnight incubation to 14 minutes. To determine the sensitivity of our microwave in situ hybridization method we applied it to previously documented tongue tissue obtained from an AIDS autopsy without clinical evidence of OHL, but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridization. This tissue specimen acted as a low EBV copy number, positive control. The sensitivity of immunohistochemistry using three different commercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (antibody double cycling). Clearly positive signals for EBV were detected in all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was evaluated at immunohistochemistry level by their application to the low-EBV copy number positive control specimen. Signals for EBV antigen in the low copy number positive control specimen were obtained only with the Vectastain Elite ABC kit. This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for immunohistochemistry to that found by in situ hybridiation.
作为一种诊断技术，原位杂交需要较长的处理时间，需要一定的专业知识，并且在一些实验室中可能难以常规操作。为了简化原位杂交方法，我们改进了一种微波原位杂交技术，并将其应用于从10例HIV血清阳性患者（通过传统原位杂交技术明确诊断）获取的口腔毛状白斑（OHL）活检组织，并设置了适当的对照。有必要设计一种新颖的腔室，以避免在杂交步骤中切片干燥。这种改进的微波原位杂交技术与传统技术具有同等特异性和同等敏感性，并且将杂交时间从过夜孵育缩短至14分钟。为了确定我们的微波原位杂交方法的敏感性，我们将其应用于从艾滋病尸检中获得的先前记录的舌组织，该组织无OHL的临床证据，但通过传统原位杂交发现含有EB病毒（EBV）。该组织标本用作低EBV拷贝数的阳性对照。在进行优化步骤后，将使用三种不同商业检测试剂盒的免疫组织化学敏感性与同一组织上的原位杂交敏感性进行比较。这包括使用两轮一抗和生物素化二抗（抗体双循环）。使用Vectastain Elite ABC试剂盒和Histostain-SP试剂盒在所有OHL活检组织中均检测到明显的EBV阳性信号。通过将三种商业检测试剂盒应用于低EBV拷贝数阳性对照标本，在免疫组织化学水平上评估了它们的敏感性。仅使用Vectastain Elite ABC试剂盒在低拷贝数阳性对照标本中获得了EBV抗原信号。这表明，在该应用中，使用Vectastain Elite ABC试剂盒进行免疫组织化学的敏感性与原位杂交相当。
