Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples

Abstract

The field of molecular pathology has advanced over the past 15 years with the integration of laser-based microdissection systems and a variety of associated biological assays and analysis methods (Bonner et al. 1997; EmmertBuck et al. 1996;Burgess and Hazelton 2000;Iyer and Cox 2010;Craven and Banks 2001;Mustafa et al. 2008;Lawrie and Curran 2005;Charboneau et al. 2002;Gillespie et al. 2004;Bichsel et al. 2001;Callagy et al. 2005;Kurihara et al. 2002) SummaryLaser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno-laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods. (J Histochem Cytochem 59:591-600, 2011)

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…Microdissected tissue pieces can also be investigated after their status linked to markers of interest. This approach was named immune laser capture microdissection (iLCM), where IHC is performed (Fend et al, 2000;Tangrea et al, 2011) prior to LCM. It was used for multiple clinical applications such as proteomic studies of prolactin cells from pituitary adenoma (Liu et al, 2010) or CD24 + / -cells from pancreatic adenocarcinoma, this marker being directly correlated to cancerous tumorigenesis, progression, and metastasis of pancreatic cancer (Zhu et al, 2013).…”

Semiautomated Laser Capture Microdissection of Lung Adenocarcinoma Cytology Samples

“…Thus, computer-aided IHC (TTF-1) LCM ensured we identified the majority of tumor cells (single cells as well as clusters of tumor cells) and increased our specificity. While this provided a high-throughput semiautomated method to isolate tumor cells for molecular assays, the downside is that IHC can negatively affect the DNA recovered from tissue sections [30]. …”

“…Studies from our group and others have shown that, although the quantity of DNA retrieved is reduced, the quality and integrity is not affected and DNA can be successfully amplified by PCR [30] with no obvious difference in performance between IHC-stained and unstained samples. However, in the context of clinical molecular assays one does have to be cautious of introducing false-positive results when amplifying very small amounts of DNA.…”

“…To date, several studies using immunoguided LCM have been published that use: (1) human operator-based dissection, (2) computer-assisted stain recognition software program targeting, or (3) expression microdissection [27,28,29,30,31,32]. The first method allows for the selection of specific cell types by aiming the laser at the stained target cells under direct microscopic visualization, whereas the second approach uses a computer algorithm to target cell selection for LCM.…”

“…It is important to note, however, that there are two main drawbacks to these procedures. The first is the time, expense, and technical troubleshooting required for the immunostaining step, and the second is the detrimental effect of IHC on molecular recovery, including a reduced DNA yield from immunostained samples, posing a potential limitation of studying cytology cell blocks where the tumor cells are often scant and limited [30]. …”