Idea Transcript
White Paper VIP 6 AI® validated protocols Ultimate quality and the benefits to the laboratory
By: Simon Cutts Product Marketing Manager Sakura Finetek Europe B.V.
VIP 6 AI Ultimate Quality – Validated protocols
SUMMARY The processing quality of a tissue block will affect all downstream activities within the laboratory as well as the ease of which a patient sample can be diagnosed. High quality processing will reduce the time and costs associated with activities such as the original microtomy and the elimination of rework due to poor processing. This time and cost saving can have a positive impact on the laboratories budgets and ability to cope with workloads. This paper examines the development of the validated protocols for the Tissue-Tek VIP 6 AI processor. It brings together the theory behind processing protocols and the testing that took place to achieve the ultimate quality of tissue processing.
Method This study was conducted in collaboration with Radboud University Medical Center Nijmegen based in the Netherlands, where an efficient, set of standardized processing protocols was developed and validated. These Protocols are designed to be used with and were validated upon the Tissue-Tek VIP 6 AI processor.
The VIP 6 AI has a set of four validated protocols including xylene based and xylene free based methodology
Wide range of routine tissue tested and high quality blocks produced
The first protocol development and testing was performed at Sakura Finetek Japan and their customers. The literature review and further development of the protocols was conducted at Sakura Finetek Europe testing laboratory. A second significant period of testing was performed at Radboud University Medical Center Nijmegen in the Netherlands to identify and validate the protocol to be used for the next phase. This next phase of testing verified the quality, repeatability and solution replacement rates for multiple tissue types whilst pushing the limits in a laboratory situation. The new validated protocols are designed to yield continuous high quality processing results whether fast biopsy or overnight processing with high fatty tissue content, with or without Xylene A wide range of different tissue samples, from biopsies to breast tissue, were grossed according specifications, including prostate, uterus, skin, colon, breast and lipoma. The tissue samples were all properly fixed in neutral-buffered formalin, as this is the current standard for fixation in routine histology. Consequently the samples where processed on the VIP 6 AI using the developed protocols. The protocols were run over several weeks to determine the refreshment rate for the reagents. The slides were then evaluated by a senior lab technician and a Sakura applications specialist. This paper shows the results of those tests and provides insight on how to achieve high quality fatty tissue processing on a shorter protocol.
Results A series of four validated protocols have been developed. These protocols include a short biopsy protocol of 3 hrs. and a routine overnight protocol of 10hrs. using Xylene based methodology. Sakura Finetek Europe B.V. VIP6AI.sakura.eu
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VIP 6 AI Ultimate Quality – Validated protocols
Excellent quality found in difficult fatty tissue
Risk of misdiagnosis reduced due to microsatellite tumours being more easily visible
Sakura Finetek Europe B.V. VIP6AI.sakura.eu
A short biopsy protocol (4hrs.) and an overnight protocol (12 hrs.) using Tissue-Clear (xylene free) based methodology. The overnight protocols also allow the notoriously difficult fatty tissue to be processed within these times alongside normal routine overnight tissue whilst producing excellent quality processing. The benefit of this quality of processing especially on fatty tissue is that the microtomists will find it easier and quicker to section, producing less rework and risk of misdiagnosis through missing microsatellite tumours is reduced. The additional benefits of allowing the Fatty tissue to be processed at the same time as the overnight tissue a reduction in sorting time and turnaround time related to these samples.
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VIP 6 AI Ultimate Quality – Validated protocols
To ensure the continued quality of the processing it is imperative to know when to change the reagents. These refreshment rates were established during the testing to produce validated refreshment rates. These refreshment rates differ between xylene and TissueClear due to the effectiveness of the clearing agent. Refreshment rate xylene protocol
Validated reagent refreshment rates provide confidence in quality on first and last day Refreshment rate Tissue-Clear protocol
The results showed no deterioration in the high quality achieved on the first or last days between the change cycles. The additional benefit to these refreshment rates and the low volume of reagent used in each bottle on the VIP 6 AI may further reduce the cost of reagent usage within the laboratory compared to competitor instruments and current regimes.
Discussion How can such high quality be achieved, including the fatty tissue in a reduced timeframe? A combination of the VIP 6 AI instrument itself and the reactions within the protocol allow the change in quality to be achieved. Most common protocols use a standard formalin in the first station of the protocol to act as additional time in fixative and a holding reagent. Standard formalin solution is the norm for fixing samples and even with these validated protocols the normal fixation protocol should apply, as the start of good processing is fixation.
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VIP 6 AI Ultimate Quality – Validated protocols
It is well documented that formalin although the routine fixative in use across the world is not ideal due to the fact that its penetration into the tissue is slow, Its reactivity with the tissue in forming crosslinks is slow and ineffective on lipids.
More effective dehydration and defatting leads to excellent quality results in quicker turn-around times
The literature (Bancroft and Wick) shows that an alcohol/formalin mix has advantages in processing when compared to the standard formalin. Firstly as a combined solution the alcohol elements speeds the penetration of formalin into the tissue above formalin alone. This means that the fixative powers of formalin can start sooner and be more effective. Secondly the use of alcohol in this step means that dehydration can start sooner and therefore is more effective. Thirdly the alcohol in the mixture allows extensive defatting assisting in the production of high quality fatty tissue blocks. The Sakura validated protocols also use a two protocol steps where a mixture of alcohol and xylene are used during the transition from dehydration to clearing steps of the protocol. The literature shows that this methodology achieves a higher success rate of defatting of the tissue as well as more effective dehydration. As the defatting and dehydration effect is greater, there also follows that there is an enhancement in the infiltration of the paraffin wax into the tissue. The studies performed by Sakura Japan confirmed and supported the literature findings. The effects seen above in the Xylene are also replicated when using the Tissue-Clear Xylene substitute protocol. The VIP 6 AI can formulate this mix of reagents automatically thus making it easy for the users to produce and use these mix reagents without being exposed to the risks of performing this manually. The VIP 6 AI also aids the chemistry behind the reaction between the solvents and tissue samples in the system via its regulation of the temperature, vacuum/pressure and agitation of the solutions throughout the processing protocol. When added together this creates the ultimate quality.
Reduction of rework saves time and costs within the laboratory
The VIP 6 AI tissue processor, is a cost efficient method for the production of superior quality tissue blocks, enabling easy and accurate diagnosis. Enabling the laboratory to get the right result, the first time, allows the laboratory to reduce costs by eliminating rework. Removing requirement for rework improves the turnaround time of the result and eliminates the additional stress to the patient due to re-biopsy. Reduced processing time for fatty tissue specimens ensures that also these specimens fit within the smooth workflow of the laboratory. Pathologists can benefit from high quality processing which means that diagnosis is easier as any microsatellite tumours are visible and easy to identify.
Conclusion: Sakura’s Tissue-Tek VIP 6 AI used in conjunction with the Sakura validated protocols and reagent refresh schedule produces ultimate processing quality. This ultimate processing quality reduces rework time and costs for the laboratory by getting it right first time and enables easier diagnosis for pathologists on the notoriously difficult cases involving fatty tissue.
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VIP 6 AI Ultimate Quality – Validated protocols
References Pien van der Heijden, VIP 6 AI validated protocols, webinar and Application Bulletin Validated protocols Tissue-Tek VIP 6 AI. Theory and Practice of Histological Techniques by John D Bancroft Diagnostic histochemistry by Mark R. Wick Understanding Fixation, Ada Feldman http://philschatz.com/anatomy-book/contents/m46006.html Iwadare, T.,Basic theories in dehydrating, defatting and paraffininfiltrating fixed tissues, Pathology Technical Manual No.3, Pathology Specimen Preparation Technologies, Vol. #1, IshiyakuShuppan, Tokyo, 1981; 63-76. Takeishi, M., Basic course for pathological techniques (3) Dehydration and defatting, Pathology and Clinical Medicine, 1988; 6(6):697-701. Kawashima, T., Evaluation of Immunoenzyme Method for Mammary Tumor No.1 Tissue Processing (centered on defatting methods), Pathology Technologies, 1991; Vol.44 (8):8-10. Nishikawa, T., et al, Paraffin infiltration effect of xylene/ethanol pre-dehydration on automated tissue processor, Japanese Journal of Medical Technology, 2011; Vol.60 No.6: 857-863. Ross MH, Pawlina W., Histology: A text and atlas. 5th ed., (Aiiso S., Uchiyama Y., A Japanese version of Histology, 2. Nonmembranous organelles, Tokyo: Nanko-Do, 2010; 23-70) Uedaira, H., Chap.5 Water in Living Organisms, What is Water – Its Microscopic Actions, Kodansha, Tokyo, 2009: 142-177 Tamura, T., Part I Basis for Cell Biology, Basic Cell Biology, Tokyo Kagaku Doujin, Tokyo, 2010; 2-42 Fuji, H. editor, Chap. 2 Physical Chemistry of Protein Structured, A Guide of Protein Conformation, Kodansha, Tokyo, 2010: 18-52
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