SIVQ-LCM Protocol for the ArcturusXT Instrument

Summary

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

Abstract

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process. It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

Introduction

Originally developed in the mid-1990s, laser capture microdissection (LCM) enables the user to precisely capture specific cells or cellular regions from a histological tissue section via microscopic visualization^1,2. Many studies comparing molecular analysis of LCM versus tissue scrapes illustrate the value of the method^3-12. In addition, there are three video protocol publications on the technology that are available for viewing^13,14. However, despite its proven value, LCM can be tedious and laborious when the target of interest is a dispersed cell population in a heterogeneous tissue sectio....

Protocol

The described protocol was employed in accordance with NIH rules on the use of human tissue samples.

1. Tissue Preparation

1. Prior to beginning, obtain human tissue specimens according to Institutional Review Board (IRB) protocols.
2. Choose the type of tissue/cell block and corresponding processing method [formalin-fixed paraffin-embedded (FFPE), frozen, or ethanol-fixed paraffin-embedded (EFPE)]. Formalin fixation provides optimal histology, followed by ethanol fixation and flash frozen. However, fixation and tissue processing methods can affect DNA, RNA, and protein quantity and quality for downstream molecular analysis ....

Results

A FFPE human breast tissue section was immunostained for cytokeratin AE1/AE3 using a standard IHC protocol²³. After staining, the tissue slide was placed on the ArcturusXT stage and the SIVQ-LCM protocol was initiated as described above. Since the tissue cannot be coverslipped for microdissection, the IHC+ stained cells can be difficult to discern visually (Figure 1A). Thus, to provide better index matching and an improved image, xylenes were added to the tissue section to create a pseudo-cove.......

Discussion

We present a protocol for the application of SIVQ-LCM to microdissect immunostained epithelial cells from FFPE human breast tissue. The use of an image analysis algorithm, such as SIVQ, reduces the amount of hands-on time required for the microdissection process. This is a potentially important advance for the field since operator time and effort is typically the rate-limiting step for the precise dissection of cells of interest. In the present protocol, we specifically adapted our procedure to the ArcturusXT instrument,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Positive Charged Glass Slides	Thermo Scientific	4951Plus-001
Xylenes, ACS reagent, ≥98.5% xylenes + ethylbenzene basis	Sigma Aldrich	247642	CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.
Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous	The Warner-Graham Company	6.505E+12	CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.
Arcturus CapSure Macro LCM Caps	Life Technologies	LCM0211
ArcturusXT Laser Microdissection Instrument	Life Technologies	ARCTURUSXT
AutoScanXT Software	Life Technologies		An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.
Spatially Invariant Vector Quantization (SIVQ)	University of Michigan		This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]