By Kellie Madigan

The quality and consistency of immunostaining contributes highly to productivity for pathologists and cellular pathology laboratory managers. An evaluation study among key opinion-leaders in this area of healthcare has sought to understand how today’s pathologists perceive staining quality, its impact on their working life, and what the term ‘quality’ covers. This article discusses the needs of diagnostics in healthcare in relation to a recently launched advanced staining solution.

One of the current challenges in healthcare following the SARS-CoV-2 pandemic is dealing with the resultant backlog of patients presenting with cancer symptoms, and those being monitored for disease progression or remission [1]. This is where diagnostics plays a vital role, and that of the pathologist is pivotal in this regard. However, 21st century cancer diagnostics requires not only accurate processing and interpretation of a tissue sample but also systems that can maintain high quality while handling the challenge of increasing workloads.

There is urgent need for a high-throughput immunohistochemistry (IHC) system that can handle various slide combinations, a wide variety of labelled antibodies, and the different chromogens needed by modern histopathology without any concern over loss of quality.

Melanoma marker HMB45 stained with red alkaline phosphatase (BOND-PRIME)

Developments in IHC

The past eight decades have witnessed the development of direct detection techniques using fluorescein isothiocyanate (FITC)-labelled antibodies [2,3], and the subsequent introduction of indirect techniques resulted in the use of peroxidase–anti-peroxidase [4,5] and, later, streptavidin–biotin complex [6]. The latter methods were developed to label antibodies applied to tissue sections to detect the presence of an increasing range of cellular elements. This has led to the current application of IHC and in situ hybridization (ISH) methods as vital diagnostic tools to detect the presence of diagnostically important cellular features.

The arrival of automated technologies led to the introduction of the first compact systems (BOND-III and BOND-MAX IHC/ISH systems, Leica Biosystems) able to provide fast, flexible and efficient delivery of quality IHC staining [7,8]. Further innovation this year saw the arrival of a technologically enhanced, next-generation staining solution (BOND-PRIME IHC/ISH System, Leica Biosystems).

What a customer wants

A study among pathologists and cellular pathology laboratory managers was carried out to understand how today’s pathologists perceive staining quality, and what quality means to them, as well as its effect on their working day or even their personal time. Moreover, there is the wider impact of poor quality, for example, in terms of overall laboratory costs of increased pathologist time.

The study noted the impact on service delivery when faced with poor-quality slides. This would increase the time it took to complete a case, whether from spending more time at the microscope
to arrive at a diagnosis or from the need to repeat staining.

While automation solutions replaced early, labour-intensive manual IHC methods that required careful, time-consuming technique, that same attention to detail can now be seen through the lens of delivering consistent and reproducible quality. The study noted that when an automated staining solution delivered consistently high quality, this built confidence in its performance. This was not only in terms of personal success, but it was noticeable how highly pathologists valued their institutions for investing in the means to deliver quality slides as standard. This was echoed by the repeated view that quality on one slide alone was insufficient; it required reliance on reproducibility as standard of that quality. This was judged equally important.

What was also interesting was the degree of personal pride expressed as “emotional and visual pleasure” that pathologists experienced from high-quality slides. This personal aspect was reflected when evaluating new systems, with pathologists seeking out colleagues for word-of-mouth endorsement. This was shown to have far greater influence than simply relying on manufacturer brochures and publications.

Never compromising on quality or productivity

As well as maintaining or enhancing the quality of immunostaining, pathologists and cellular pathology staff need a system with the flexibility to adapt rapidly to changing workflow requirements. This type of technological agility means a system that can handle any slide, in any combination, at any time and using any reagent. While several manufacturers can offer aspects of universal access (UA), only the new platform has the flexibility to deliver all four steps. For the laboratory staff this means time is saved by being able to:

• load any slide: any staining protocol, chromogen or technology;
• in any combination: load single slides, complete cases or full batches;
• with any reagent: add markers or detection at any time with 70 reagent positions; and
• at any time: preload and unload drawers let the user load and unload slides according to their schedule, not the instrument’s schedule.

In terms of productivity, they can expect an instrument delivering UA to provide touch time of less than 6 minutes for an operator when processing 96 slides, while loading and unloading 24 slides from the rack can take a fraction over 80 seconds.

Scheduled maintenance tasks are vital to ensure that quality is maintained and any equipment is running optimally. In line with the demands of the diagnostics healthcare sector, the claimed yearly user resource time of less than 6 hours means that laboratory productivity is increased as a result of the reduced time spent maintaining the system, thereby allowing staff to perform other critical laboratory tasks and activities.

Active Reagent Control with individual staining chambers (BOND-PRIME)

Staining that is clear, crisp and vibrant

Another aspect of quality control lies with the technology behind the staining process. This focuses on highly controlled reagent application, incubation and exchange, carried out within a fully sealed, temperature-controlled chamber.

While all systems are expected to deliver even tissue coverage, the Leica Biosystems approach incorporates active reagent control (ARC), and an air-gapped exchange to minimize the risk of carryover. The new ARC technology with a new design covertile prevents reagent evaporation and reduces waste.

To test out the impact of the new technological developments, pathologists from two different US hospital sites carried out an evaluation of the staining and diagnostic quality of the new platform. They were asked to assess sections stained for a wide range of biomarkers using both 3,3’-diamino­benzidine and alkaline phosphatase Red as the chromogen. With regard to the quality of staining, this was rated as “excellent” with the system producing a “crisp and clear” result that the pathologists judged “acceptable for diagnosis”.

Across the entire data set (100 responses) the average score for staining quality was 4.7/5, meeting the description of “excellent”. Eight out of the 20 slides (staining for CD117, CDX2, CK5, CK20, SMA, Vimentin, WT-1 and BCL-2) all received 5/5 for staining quality from every pathologist in the evaluation sites. Six out of the 20 slides (CD117, CDX2, CK5, CK20, TTF-1 and WT-1) all received 5/5 for their aesthetics quality from all pathologists, with the average aesthetics score being 4.61/5 and again meeting the description of “excellent”. One hundred percent of slides were assessed as acceptable for diagnosis (scores greater than 3/5).

Kidney cancer cells stained for vimentin (BOND-PRIME)

Enhanced functionality

For the laboratory, enhanced functionality gives highly trained staff more time to attend to added-value tasks, without the need to constantly monitor the platform. In practical terms, this could mean:

• single-slide loading undertaken without impacting the staining of other slides, with fast TAT (STAT) staining achieved in an average of 60 minutes;
• continuous loading of cases of slides as they arrive for fast IHC staining while keeping cases together, with staining for case-based workflow achieved on average in 95 minutes;
• batch loading when a large number of slides are ready, or run 48 slides overnight or when walkaway automation is the appropriate option; and
• staining of a 24-slide batch on average takes less than 130 minutes.

IHC laboratories and their scientists look to companies such as Leica Biosystems to provide expert training resources and guidance for pathologists. This proves invaluable when staff face problems related to pre-staining issues and conditions that are outside the control of automation. The resources available aim to assist users with troubleshooting any IHC staining issues that may arise in order to maximize quality. Two examples are documents entitled A Guide to Better IHC [9] and 101 Steps to Better Histology [10].

Innovation supports immunohistochemistry

In today’s fast-paced diagnostics healthcare environment, adaptability is essential to ensure that tissue sections are processed and stained to the highest quality and in a timely manner. The arrival of technology with all aspects of UA enables a workflow with the agility to adapt quickly when change is needed. This is achieved while delivering consistent high-quality staining. Laboratories will then have the confidence that they can maintain high immunostaining quality even when faced with the challenge of ever-increasing workloads.

The author

Kellie Madigan, Leica Biosystems,
Melbourne, Victoria, Australia
Email : kellie.madigan@leicabiosystems.com

For further information see Leica
Biosystems: www.leicabiosystems.com

References
1. NHS backlog data analysis. BMA 2023 (https://bitly.ws/39QL3).
2. Arthur G. Albert Coons: harnessing the power of the antibody. Lancet Respir Med 2016;4(3):181–182. doi: 10.1016/S2213-2600(16)00020-5.
3. Coons AH, Creech HJ, Jones RN, Berliner E. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 1942;45:159–170.
4. Mason DY, Sammons R. Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use. J Immunol Methods 1978;20:317–324. doi: 10.1016/0022-1759(78)90267-3.
5. van der Weij JP, van der Veen CJ, de Vries E, Cats A. The use of a peroxidase-antiperoxidase complex for the visualization of monoclonal antibodies on the ultrastructural level. Clin Exp Immunol 1983;54(3):819–826.
6. Musiani M, Zerbini M, Plazzi M et al. Immunocytochemical detection of antibodies to Epstein–Barr virus nuclear antigen by a streptavidin–biotin-complex assay. J Clin Microbiol 1988;26(5):1005–1008. doi: 10.1128/jcm.26.5.1005-1008.1988.
7. Lu DY, Qian J, Easley KA et al. Automated in situ hybridization and immunohistochemistry for cytomegalovirus detection in paraffin-embedded tissue sections. Appl Immunohistochem Mol Morphol 2009; 17(2):158–164. doi: 10.1097/PAI.0b013e318185d1b5.
8. Hsu JD, Yao CC, Lee MY et al. True cytokeratin 8/18 immunohistochemistry is of no use in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study. Int J Gynecol Pathol 2010;29(3):282–289. doi: 10.1097/PGP.0b013e3181c043bc.
9. A guide to better IHC. Leica Biosystems 2023 (https://bitly.ws/39QL9).
10. Rolls G. 101 steps to better histology – a practical guide to good histology practice. Leica Biosystems 2023 (https://bitly.ws/39Utv).