Sample prep method considerations for sample-to-answer systems

So, what is ‘sample prep’? ‘Sample prep’ is the catchall name we give to methods that improve analytic performance by extraction, purification, and concentration. Concentrating the analyte increases signal, and purifying it reduces noise. Some samples are collected in an encased form—for example, nucleic acids inside a cell—and in these cases extracting the analyte makes it available for analysis. A given sample prep method may include any combination of these three elements, in any order.

Porting a benchtop method to a cartridge can be a challenge, but changing a sample prep method mid-stream is often worse. For this reason, it’s important to pick sample prep methods that can go the distance. Thinking through the choices for the long term will pay dividends and help to avoid such risks. If a kit is being developed now, is an instrument development envisioned for the future? If an assay is being deployed in a laboratory today, will there be a need to port it onto a cartridge later?

With these answers in hand, it’s possible to qualify how well different sample prep strategies will serve the development team’s needs.

Know the metrics of sample prep

Recovery. Every analytic system has a minimum amount of analyte the system can measure. To characterize the quality of our sample prep, one needs to know whether the amount of sample that comes out of the prep—the recovery—is sufficient. Typically, recovery is stated as a percentage of the sample that went into the prep.

Concentration. The detector utilized will also have some physical limitations. If it’s an imaging system, maybe the limit is the field of view. If it’s a fluorescence system, maybe it’s the volume of the chamber. Whatever the specific limit is, it will place an upper bound on the total amount of sample that can be measured at once. Dividing the smallest number of analytes the detector can see by the largest sample volume it can measure, the minimum concentration can be determined.

When thinking about sample prep, it’s again useful to compare the concentration before and after the prep, which results in unitless concentration factor. If the prep method concentrates the sample, the concentration factor is greater than one. If it dilutes the sample, it’s less than one.

Purity. Even with 100% recovery and a highly concentrated sample, an analyte might be so awash in background that the detector cannot see it. So, it is important to know the purity a particular sample prep method can achieve. To gauge the purity a prep method can obtain, it is good to prepare and measure a real sample first. Then prepare and measure a sample with no analyte. Divide the first answer by the second, and a signal-to-noise ratio is determined.

Speed. Finally, there’s how long a test user is willing to wait. This is a function of the method’s speed—the volume of sample we process in a fixed period of time.

Metric interdependency

In any given sample prep system, these metrics are linked in subtle, and technology-dependent ways. Much of the skill of optimizing an assay comes down to understanding these subtle dependencies. A couple of examples:

• Magnetic beads: Higher speed (shorter incubation) reduces recovery (less binding). Increasing cost (adding beads) can reduce this tradeoff.

• Filtration: Higher speed (faster perfusion, larger filter area) can reduce purity (unwanted lysis) and can either improve or reduce recovery (higher pressures may force more fluid through, but larger areas may have a higher fluid loss).

• Lysis: Higher recovery usually reduces purity, because the more cells you burst, the more cell debris you release into the sample.

• Centrifugation: Concentrating the sample reduces speed, because you have to spin for longer. Alternatively, you can spin faster, but this may reduce your purity as you can degrade the sample or get your analyte mixed with unwanted material.

Once these interdependencies are understood, it’s easy to see why changing a sample prep method mid-stream in development can throw an assay project into real chaos. This is why thoughtful sample prep design is so important: it is just too costly (time, money and effort) to make changes later.

Confidently call the shot

Thinking ahead will help rule out sample prep methods that will only cause pain down the road, and help keep focus on getting quality results from methods that are worth investing in. For example, if there is a plan to transition an assay from a benchtop format to a sample-to-answer cartridge, then there is likely value in looking into cartridge-ready tools.

In addition, one should consider whether a particular sample prep method needs to be able to handle multiple sample types. Are swabs being used today, but there are plans to add blood tomorrow? Many sample prep methods are sample-type specific, especially in the microfluidic or cartridge context. A few insights about biospecimens to ponder:

• Blood has a staggering background of cells, metabolites and (when lysed) genomic and proteomic material

• Urine comes in large volumes, with many analytes (especially genomic markers) in very low concentration

• Saliva is heterogeneous and analyte concentration is unpredictable

• CSF is collected in small volumes

So, if handling multiple sample types is a must, it is good to make sure a sample prep method can accommodate them.

If sample prep methods that are working now but there are concerns these will not work in  future products, look for tools that can help you make the transition. Now, back to magnetic beads as the example. Widely used in lab assays, they , but STR™BeadPak chips enable the transition, enabling comparable or better performance in the cartridge as on the bench (see STR chip results in a recent study).

The bottom line is that the risk is too great not to take a long, hard look at the sample prep method being considered to ensure it’s the right one for a given application. And, to avoid project chaos down the line, both the current system development needs as well as needs for future ones should be kept in mind.