Expert Tips On IHC Optimization

Introduction

Acepix: the leader and visionary in IHC controls design

Acepix Biosciences, Inc., based in the San Francisco Bay Area, is an innovator in developing and providing high quality assays and biological materials for research and diagnostics. The company also provides high quality services in the field of histopathology, digital pathology, biomarker discovery, and translational research. The founders of Acepix believe that high quality research does not need to be accompanied by high cost, but rather should from high quality materials and optimized designs and protocols at every step of the studies. Therefore, the scientific team at Acepix strives to develop, improve, and validate robust reagents and protocols to support biopharma and academic research. The A-FLX technologies developed by Acepix is the most robust method for creating highly consistent, cell-based, FFPE or frozen reference materials for research and diagnostic.

According to our mutual connections, Dr. Su, the co-founder of Acepix, is one of the most knowledgeable person on subject of immunohistochemistry. Rumor has it that he makes the smoothest cell smear slides the world has ever seen via the A-FLX techniques. Personally, Dr. Su is one of the most passionate person we know and has an amazing amount of insights on the subject of IHC. We are glad to have him as our special guest for the interview.

Challenges in the Histopathology Industry

and Dr. Su's vision for tackling these challenges

When asked to list some current challenges in the histopathology world, Nan said one of the most prominent issue is with the standardization of controls. "Both positive control and negative control are needed for diagnostic immunohistochemistry (dIHC). In theory, well-designed controls are supposed to demonstrate the sensitivity and specificity of the assay. However, in day-to-day practice, it is unclear which types of positive and negative controls should be used for a given biomarker. This is particularly true for predictive biomarkers, the number of which has been rising rapidly as personalized medicine continues to develop." Nan explained.

Negative controls In IHC are crucial for the validity of the assay

"I am a big believer of implementing controls, especially given the large number of variables in an IHC assay. Comprehensively designed controls are essential to the validity of the assay since there are many things that can go wrong in IHC," said Nan. "We can easily list out more than a dozen commonly encountered variables when running an IHC assay, and sometimes these variabilities are completely out of our control or don’t immediately raise an alarm, such as lot-to-lot variation of an antibody or reagent, instrument having random dispensing variation or misalignment, ambient temperature fluctuation, etc.," Nan continued. How do we know if an assay performed as it’s supposed to or how it has been performing in the past? "It’s only possible if we include control samples," Nan stated. In an ideal world, every testing sample should have a control section on the same slide so the staining result of every sample can be interpreted with confidence at the analytical level. In practice, according to Nan, it is always recommended that a least one control sample be included for each run to assess the suitability of that particular batch of staining.

Our Secret Juice for IHC Controls

Cell Line Based FFPE Controls can serve as the golden control standard

Controls seem to be at the core of Acepix's expertise. When asked what customers could benefit from their special version of better controls, Nan said, "We developed a special process to create highly consistent cell line based FFPE controls. We have validated over 100 cell lines that express many different biomarkers at various levels, and those also include over a dozen clinically relevant point mutations. These control samples are now being used worldwide in both research and diagnostic settings. For example, we’ve created a universal breast cancer FFPE cell pellet array, which consists of 4 cell lines expressing HER2, ER, PR, and EGFR at various levels, and hospitals use that to assay the quality of routine IHC staining on breast cancer samples." Another area where these cell pellet controls are in high demand is for reagent and diagnostic test developers. At every step of assay improvement, scientists and technicians need to have the confidence that the improvement is real and not just sample variation. Consistent cell pellets can serve that exact purpose.

Best Practices For Antibody Optimization In Immunohistochemistry?

"Typically, with a validated platform and detection system, there are only a couple of parameters to optimize for an IHC assay, such as epitope retrieval and antibody concentration," Nan explained. He pointed out that one can go and further optimize conditions such as antibody incubation time and temperature, or switch to a more sensitive detection system, but we would only do that in one of the following cases:

1) We run out of choices and only have a few available antibodies.

2) We want to maximize signal-to-noise ratio for a particular antibody.

Although it may look like a good idea to go with a comprehensive and large optimization matrix that includes all possible conditions (e.g. for epitope retrieval, there are acidic and basic retrieval, proteolytic retrieval, and sometimes no retrieval), but this might not be the best idea according to Nan. He described, "Well, granted that certain antibodies may work better with protease retrieval or no retrieval at all, experience has told us over and over again that a heated retrieval step almost always works better. Furthermore, the basic ER solution in the majority of cases give stronger staining comparing to acidic ER solution. Of course, acidic ER often yields cleaner background, but with a well-validated detection system, using basic ER provides higher signal-to-noise ratio with lower antibody usage and lower optimization cost for over 80% of antibodies. That’s why most of our customers choose to go with a smaller optimization matrix using basic ER as a start."

How To Design Titration Series As An Initial IHC Optimization?

The dilution of the primary antibody is dependent on the detection system one uses. Nan commented that if you're using one of the polymer systems, then you’d probably want to start with a lower concentration. Many recommendations from an antibody vendor’s website for IHC dilution may not be optimal for a polymer detection system. The concentration might be unnecessarily high. "Based on our experience, the starting concentration should not be higher than 5ug/ml for mouse antibodies or 2ug/ml for rabbit antibodies. If you need to go higher than that, then it’s not a good antibody, period. The results can be complicated by background staining." It is true, according to our experience from Boster's histology lab, that if you throw more than 10ug/ml antibody on to a tissue, you will get some staining, just don't trust those stains.

Troubleshooting IHC: What To Investigate When It Just Does Not Work

Also, When to Call It Quits and Move On to the Next Antibody

There are numerous troubleshooting guides online, and there are many factors one can try to optimize in IHC. Nan suggests to focus only on the few that matters. He mentioned, "If after a couple of rounds of optimization based on a well-designed parameter matrix as previously described, the staining is still poor, then the antibody is probably not suitable for IHC and extensive troubleshooting will not likely take you very far. I’d say the affinity and specificity are inherited properties of an antibody, so optimizing conditions will only push the signal-to-noise ratio to close to the best the antibody can do. Occasionally, altering incubation conditions can make a huge difference. For example, 4°C overnight incubation will typically give higher signal-to-noise ratio as it allows longer time to reach binding equilibrium while minimizing non-specific binding, comparing to RT incubation for 30min to 1hr. Unfortunately, it’s not really a practical workflow for diagnostic assays and almost impossible to automate. Therefore, for research purposes, it’s ok to try everything possible to push for the best results. However, for developing a more routine assay for large scale studies, especially clinically related studies, it’s better to try to find a different and more robust antibody."

The experiment itself sometimes is not to be blamed. According to Nan, the pre-analytical factors are just as important. "Samples that were prepared under sub-optimal conditions may generate very poor staining even when using a robust IHC assay," Nan said, "The major categories of pre-analytical factors include collection, fixation, processing, and storage. It is a whole different topic, but one needs to make sure that the samples are properly prepared before diving into tediously IHC optimization. Basically, garbage in, garbage out."

Another possible cause of poor staining is that maybe the assay just didn’t work on that day, and that can come from numerous reasons, such as reagents or instrument. If one suspects that the assay or instrument itself has some issues and decides to find the root cause, that could be another long journey. "But how do we know if a particular run is sub-optimal? The only way is to include control samples, which we can talk more in depth about another time." Nan said, re-emphasizing the importance of IHC controls.

How to select antibodies for Immunohistochemistry?

Choosing A Commercial Primary antibody for My IHC assay

On the question of how to choose a primary antibody for IHC, Nan said, "First of all, we need to realize that the majority of the commercial antibodies will not work well for IHC."

According to Nan, it doesn’t necessarily mean that they will not detect the target in an FFPE tissue, but the signal-to-noise ratio is often too poor to be considered working at all. Many companies focus on developing antibodies that are compatible with IHC, and those are always the first choices. However, even among the “IHC validated” antibodies, the performance may vary dramatically. Therefore, in order to develop the most robust assay, it’s always good to evaluate as many antibodies as possible. On the other hand, there are only a small percentage of antibodies that work exceptionally well for IHC. For all the other antibodies, the main question is “Does it answer my question?”. If an IHC assay sufficiently addresses a particular question, then extensive screening or optimization might not be necessary.

Choosing Secondary antibodies for IHC

"Nowadays, there are many great-performing polymer or multimer based secondary antibodies available from companies such as DAKO, Leica, Boster Bio, Ventana, Vector Labs, ThermoFisher, Biocare, etc. These detection systems significantly increase detection sensitivity compared to the traditional enzyme conjugated secondary antibodies," said Nan, "however, we need to pay close attention to the compatibility of sample species and the primary antibody host species to avoid non-specific background. For example, using a rat anti-mouse antibody on a mouse tissue will require a secondary antibody that’s highly cross-adsorbed against mouse IgG; otherwise, the secondary antibody may detect endogenous IgG in the mouse tissue due to the similarity between rat and mouse IgG. There are different grades of adsorbed secondary antibodies available. Even for the highly adsorbed detection antibody, it’s always a good idea to include proper negative controls, including Isotype control and no antibody control, to assess the background staining. Another powerful amplification system is tyramide signal amplification (TSA) system. It has outstanding amplification power, but might require more optimization."