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Pre-staining standardization is critical.

Any pathologist, lab manager or histotechnologist will readily acknowledge that preparation for IHC staining begins the moment tissue is acquired. The literature documents optimal conditions for tissue fixation, processing and sectioning to ensure morphology and antigenicity are maintained. Further improvements in maintaining consistency to correctly control for these factors could be pursued. This may involve a laboratory establishing itself at the site of collection, recognizing that accessioning and sample preparation begins here. Automated accession, LIS and sample tracking infrastructure should extend out of the laboratory and into surgery/point of biopsy, enabling standardized prep-equipment (”controlled” pots, fixatives) to be monitored for optimal processing downstream. A parallel industry that recognized this value is the blood analysis laboratory, which utilizes standardized coated collection vials with bar coded patient information for tracking and accessioning.

Linking fixation, tissue processing and IHC staining will add value to control quality. Laboratories that can monitor and record fixation and tissue processing conditions and link these with IHC staining protocols will be able to report a diagnosis under a tighter quality controlled environment. The frequent use of controls for pre processing variability (eg. control every 100 slides) may also provide a good measure for staining instrument tolerances and performance. These workflows and technologies will evolve as the IHC industry matures. However there are a number of good practices that are known to establish and maintain high quality and consistent results from IHC staining (Table 1).

Process Location Common Issues that Impact IHC
Excision/Biopsy      Surgery/Physician Office     »» Mechanical tissue damaged by excision equipments
»» Margins not clean or area of interest missed
»» Ischemia and cell death due to delay between excision and fixation, as well as surface area to fixative ratios
»» Mislabeling of samples
»» Over-fixation
Accessioning      Laboratory     »» Sample mislabeling
»» Overlooked samples
Grossing      Laboratory     »» Slices too thick
»» Specimen trauma caused by incorrect handling
»» Cross-contamination with other cases
»» Inappropriate cassette type
»» Overloaded cassettes
Tissue processing      Laboratory     »» Poor decalcification (where required)
»» Incompete fixation or over-fixation (see Figure 2)
»» Inappropriate schedule chosen
»» Poor quality reagents
»» Dirty reagents
Embedding      Laboratory     »» Incorrect orientation
»» Inappropriate or overfilled mold
»» Mechanical trauma due to rough handling or excessive heat
»» Inappropriate cassette type
»» Overloaded cassettes
Microtomy      Laboratory     »» Poor quality knife or incorrect knife profile
»» Incorrect knife tilt angle
»» Damage due to freezing
»» Rushed sectioning causing "chatter"
Flotation      Laboratory     »» Dirty or contaminated water
»» Cross-contamination caused by incorrect bath cleaning or floating-out sections for multiple blocks
»» Incorrect water temperature
»» Over-expanded sections
»» Bubbles forming under the tissue or sections lifting
»» Time before staining commences

Table 1: Pre-staining practice to support quality IHC staining

Figure 2: Uneven fixation (zonal fixation) has resulted in uneven staining in this section (breast tumor, ER)

A vision for high-quality IHC and ISH.

At Leica Biosystems our vison is to advance cancer diagnostics and improve lives. One way we can achieve this vision is by helping improve staining quality. As we recognize that IHC and ISH quality doesn‘t begin at the stainer, this series looks at many different aspects of staining quality, and considers how future tests will influence improved diagnosis.

This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature. Although every effort has been made to report faithfully the information, Leica cannot be held responsible for the correctness. This document is not intended to be, and should not be construed as medical advice. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document.

Copyright © by Leica Biosystems Melbourne Pty Ltd, Melbourne, Australia, 2016. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH. 95.14104 Rev A ∙ 04/2016