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Ask the Expert: Overcoming the Challenges of Sample Preparation

Byron Brehm-Stecher, Ph.D., Associate Professor in the Department of Food Science and Human Nutrition at Iowa State University, heads the Rapid Microbial Detection & Control Laboratory that works to ensure the safety of the food supply through improved detection and inactivation of food-borne pathogens. While the laboratory uses a combination of several different analytical tools for rapid detection, a key emphasis lies on pre-analytical sample preparation (sample prep), which is the separation and concentration of target cells from complex samples and removal of interfering matrix components prior to detection. He talks about the common challenges associated with sample prep and what an ideal sample prep process should involve.

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Q: What does sample prep typically involve, and what are the common challenges encountered?

A: Sample prep is problematic on many levels. Typically there is always the need to manipulate the sample in some way prior to the analysis. Usually there are three critical things that need to be done. First, there is the sampling step. If you are working with, say, a water sample from a lake or a big container of ground beef or some bodily fluid for clinical analysis, you need to first obtain a statistically valid sample or you are going to limit your chances of finding what you are looking for right off the bat, with the result that your downstream efforts may be futile or just “hit and miss.” If you have an adequate sampling procedure in place, then the next aspect is sample prep. These two steps are often confused with each other, but they are distinct. The sample prep step is when you take your sample and do what is needed to concentrate it—to make it homogenous or to remove any substances that may interfere with the assay. The accompanying figure gives an overview of the six critical properties characteristic of an ideal sample prep method. These concepts were first articulated by Dr. Mary Lou Tortorello, Chief, Food Technology Branch, Division of Food Processing Science & Technology, U.S. Food and Drug Administration, and later published in the Journal of Food Protection review on sample prep that we co-authored with Lee-Ann Jaykus of North Carolina State University and Chuck Young of the Johns Hopkins Applied Physics Laboratory. After sample prep is done, it’s time to move to the detection step. In the past, the majority of the focus has been on the detection step alone, with published assays often performed only under perfectly controlled laboratory conditions. However, in the past few years, there has been greater recognition that all three steps— sampling, sample prep and detection—are critical and each must be operating at their best to ensure an optimal result. Limitations at any step will propagate through the system, impacting the final result.

Click here to see the six properties of an ideal sample prep method.

Q: Why is there now a greater need for sample prep?

A: I think that recognition of the importance of sample prep has reached a critical point. For a while, especially in the early days of molecular detection techniques, it was enough to focus solely on the detection step. Sampling methods have been around for a very long time—people have been working on these since the 1930s or earlier and how to get your best samples has been well published, at least in the food space. But now people are realizing that if they want to translate their laboratory-tested method into reality and have it used practically in the field, then they have to focus on sample prep and try to integrate all three aspects that we talked about. People now recognize that it’s an important area that they need to focus on. There is no one sample prep method that applies to everything, and that’s one of the challenges, especially for a food microbiologist. If you are a clinical microbiologist, there are a few different types of samples you have to look at, such as body fluids, blood, vomit, feces and tissues—but the list is still fairly short. However, foods are incredibly complex and diverse. Therefore, sample prep for foods is liable to keep us occupied for a while.

Q: Is there also a push from the technical side with more sensitive and accurate detection tools allowing more quantitative versus qualitative detection?

A: If you are going to do anything with risk assessment, then you need to know how much of your target is present in the sample, and therefore, strictly qualitative analyses won’t help. But if you have a system that is sensitive enough to detect a single cell, you are definitely limited by the fact that you cannot apply that platform to its fullest ability, especially if you have an unsolved upstream sample prep issue. Surely that is a motivation to dovetail an excellent sample prep method together with the newest generation of detection instrumentation.

Q: Are there any innovative approaches being applied to sample prep in recent years, and have you had experience working with any of them?

A: There are a few things that have proved very helpful for sample prep. Some of them are not off-the-shelf technologies but just procedures that you can integrate into your own process flow. For example, there are different types of centrifugation methods and exposure to different chemical reagents to break up food emulsions. There are other techniques, such as the recirculating immunomagnetic separation approach from Matrix MicroScience, that are very interesting. We are working with InnovaPrep, LLC, a company that is developing some novel methods for foam and vacuum-based extraction of microbes from very large surfaces. These are methods that were initially developed for the biodefense area, but they can easily be translated to the food science arena. Microbiologists in both areas seek to detect low levels of target organisms distributed, perhaps heterogeneously, over large surface areas, so we’re finding this approach to be very useful when coupled with real-time PCR for detection of salmonella. There are also technologies that concentrate samples, which could be anything from simple hollow fiber filtration to techniques like SCODA (synchronous coefficient of drag alteration), a method developed by Dr. Andre Marziali at Boreal Genomics for selective concentration of nucleic acids in complex samples. That’s an entirely different technology for concentrating a sample based on electrophoresis rather than physical enrichment.

Q: Do you have any advice for lab managers who are struggling with sample prep issues?

A: Lab managers working in industry who have pressing problems and not enough time and resources to do the basic research themselves need answers and need to better their processes now. For them, reaching out to companies is good. Of course, you have to look carefully and decide if any given technology is right for your application, because everyone is going to claim that they have the answers that you are looking for. But I believe that there are a number of companies that are doing some very interesting basic research and are developing some methods that can be readily picked up and applied. Many of these companies are small, so you can often simply pick up the phone and start a discussion with the right person. Reaching out to people who have the answers—whether it’s something packaged commercially or something being done at a university such as Iowa State—and communicating with people facing similar problems or who are in the middle of solving problems similar to yours is the best thing to do.

Q: What, in your mind, is the Holy Grail when it comes to sample prep? What is the one thing that people are striving to achieve?

A: It comes back to the accompanying figure about the six elements that are characteristic of an ideal sample prep method. You don’t necessarily need all those properties for every single assay. At the end of the day, it depends on what you are looking at. There are some combinations of platforms and reagents that allow you to cut through complex samples with minimal sample prep. For instance, we routinely use fluorescence in situ hybridization (FISH) and flow cytometry to analyze very complex samples like alfalfa sprouts contaminated with low levels of salmonella. Here, you are working with high levels of particulate matter and background microflora that are closely related to salmonella. When we combine FISH and flow cytometry, we can cut through all that mess. This is a unique situation, but it highlights the fact that every sample and every need is different. Some systems may need multiple sample prep steps and some may need very few.

Q: What role are the regulatory agencies playing in this field?

A: There is definitely a clear interest in sample prep on the regulatory side. In fact, the USDA, in its latest request for proposals, has indicated that sample prep is extremely important to them. They recognize that if you are going to have some way of intervening with pathogens in food then you have to be able to detect them, and that involves some amount of sample prep. The fact that they have put this on their docket is very heartening.