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Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry
Published on: July 26, 2019
A reference image-based method for optimization of clinical immunohistochemistry
Marcel Kap1, King H Lam1, Patricia Ewing-Graham1
1Department of Pathology, Erasmus MC, Rotterdam, The Netherlands.
Insights
Optimizing immunohistochemistry (IHC) protocols by adjusting analytical factors, not pre-analytical ones like cold ischaemic and formalin fixation time (CIT and FFT), improved staining consistency. This method aids in reducing interlaboratory variation for reliable IHC results.
Area of Science:
- Anatomical Pathology
- Biochemistry
- Immunology
Background:
- Intralaboratory variation in immunohistochemistry (IHC) is often attributed to pre-analytical factors such as cold ischaemic time (CIT) and formalin fixation time (FFT).
- Standardized control tissues with known pre-analytical histories are essential for method development and validation.
Purpose of the Study:
- To develop and evaluate a novel method for optimizing IHC protocols.
- To assess the impact of CIT and FFT on IHC staining using control tissue blocks.
- To compare IHC outcomes with digitized reference slides for quality assessment.
Main Methods:
- Control tissue specimens were divided and subjected to varying CIT and FFT.
- Immunohistochemistry was performed using 34 antibodies and standard operating procedures.
- Staining intensity was scored, and IHC protocols were adapted by modifying antibody dilution and antigen retrieval methods.
Main Results:
- Seven antibodies were affected by CIT, 13 by FFT, and five by both.
- Protocol optimization, primarily through analytical adjustments, improved staining consistency for most antibodies.
- Nine protocols remained un-optimizable, but optimized results met adequate quality standards when compared to reference slides.
Conclusions:
- Optimizing the analytical phase of IHC protocols is more effective than altering pre-analytical conditions for improving consistency.
- This method, if supported by global references, has the potential to significantly reduce interlaboratory variation in IHC.
- Further standardization of pre-analytical workflows is recommended to complement analytical optimization efforts.
Aims:
Cold ischaemic and formalin fixation time (CIT and FFT) are considered to be crucial parameters for intralaboratory variation in immunohistochemistry (IHC). Here we describe a new method to optimize IHC, by using control tissue blocks with known pre-analytical history and comparing the IHC outcome with digitized reference slides.
Methods And Results:
Tissue specimens (two per tissue type) were divided into eight samples, which were subjected to different CIT and FFT. Immunohistochemistry was performed with 34 routinely used antibodies, following standard operating procedures. Relative staining intensity of four sections per slide was scored. Of the antibodies studied, seven were influenced by CIT, 13 by FFT and five by both parameters. IHC protocols were adapted until most sections on the slide showed the same intensity. Changing the antibody dilution for 10 protocols and the antigen retrieval method for six protocols improved the consistency of the IHC staining. Nine protocols could not be optimized. The optimized staining results were compared to reference slides and were found to be of adequate quality.
Conclusions:
It was possible to optimize most IHC protocols by adapting the analytical, rather than the pre-analytical, phase. If global references can be established, this method could decrease interlaboratory variation, preceding standardization of the pre-analytical workflow.
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