In an earlier blog post we discussed the importance of reliable samples for human genomics, microbiome and infectious disease nucleic acid based applications, and how a lack of trustworthy samples can not only lead to incorrect outcomes, but also keep you from having full confidence in your results. To obtain a truly accurate outcome, a sample must be stabilized and protected from the point of collection until it is analysed downstream.

But just what does it mean for a sample to be stabilized and protected? How can a biospecimen be fully resistant to external factors that may lead to change or degradation? How immediately should a sample be stabilized after collection? Let’s explore these and other questions regarding the concept of immediate sample stabilization.

What does it mean for a sample to be stabilized?

Stabilization encompasses a series of characteristics, including neutrality (the ability to capture unbiased samples/profiles wholly representative of the donor), reproducibility (homogenous sample material from which highly concordant aliquots can be taken), and integrity (molecular weight), as measured over time. Essentially, this means that the sample has not changed since the point of collection.

Why is it necessary to stabilize samples between collection and analysis?

Sample stabilization is essential because if a biospecimen experiences any changes between collection and analysis, the sample may not yield enough quality DNA to conduct analysis. Data that is generated and analysed from a changed biospecimen will not be a true representation of the in vivo state of the donor, leading to flawed and irreproducible results.

What sort of changes can occur in a sample?

A sample may undergo changes in reaction to outside influences. An obvious example of such an influence is temperature—the human body temperature is 37°C, while room temperature is considerably cooler. As soon as the sample leaves its host, it is placed in an environment with a drastically colder temperature than what it is used to. Alternatively samples collected in warmer climates may be exposed to extremely elevated temperatures which can quickly damage a sample and degrade the DNA. The longer a sample remains unstabilized, the greater the likelihood that it will change as a result of environmental impacts and natural biological degradation.

What are some other factors that can contribute to changes in a sample?

Other factors that can cause post-collection changes in an unprotected sample include the rate of freezing, the size of aliquots, storage duration, the type of and time in stabilizing reagent and user/handler error.

Doesn’t freezing the sample protect and stabilize it?

Freezing your samples can potentially protect and stabilize them, but not without accompanying costs, complexity and risks. Shipping and storing your samples at ambient temperature is proven to lead to significant savings, and the increased complexity both inside and outside of the lab associated with frozen samples can affect the efficiency of your study considerably.

Samples may undergo changes if there is any delay between collection and freezing, although even an immediate freezing process at the point of collection is no guarantee of the sample’s safety; the greatest risk involved with frozen samples is that samples can be lost during unexpected power failures or shipping delays.

Doesn’t using a stabilizing reagent protect a sample prior to analysis?

The use of a stabilizing reagent can be a potentially error-prone process; even a mistake-free preservation procedure may alter a sample or leave artifacts that can be misinterpreted as naturally occurring. These artifacts must be carefully accounted for, adding needless complications to your research and potentially impacting results downstream due to carry over or sample damage.

Just what is meant by “immediate” stabilization?

When we say that a sample should be stabilized immediately, we mean within seconds of collection. Collection kits and stabilizing reagents should enable you to stabilize your samples as quickly as possible before any contamination or putrefaction can occur.

What impact does immediate stabilization have in the areas of human genetics, microbiome and infectious disease?

In the field of human genetics, not immediately stabilizing your samples can lead to wasted or lost samples, DNA degradation, and flawed or irreproducible results—an assay’s performance can be significantly affected by the introduction of variables to a biospecimen.

In order for a complete and true microbiome to be observed, the provided sample must be stabilized as soon as possible. The microbiome profile can be quite volatile, and if left unprotected over time, a true representation of the microbiome (“snapshot”) will not be achieved. A lack of reproducibility is a major issue in studying microbiome, predominantly due to changes occurring in a sample between collection and analysis. If the gut microbiota sample is stabilized immediately until it is processed, you can have full confidence that the sample analysed has not experienced any changes since collection.

Infectious disease samples must be stabilized straight away to eliminate the risk of false positives and negatives, thereby creating an incorrect and incomplete understanding of the infection rate among study participants. It is also essential that the sample remain unaltered because any unnatural environmental changes would not reflect the disease progression or associated pharmaceutical impact that you may be intending to study.  

Let’s use tuberculosis (Mtb) as an example. Mtb is reactive to environmental conditions, but at a considerably slower rate than other organisms in a sputum sample. During delays between collection and stabilization, putrefaction and bacterial growth on the sample can obscure the presence of Mtb to the point that it may be completely unobserved and the sample rendered unusable. The sample should be decontaminated while allowing the Mtb to remain viable, providing optimized discovery capability.

From a safety standpoint, unprotected or destabilized biospecimens may carry contagions that can endanger those handling the samples—the samples should be decontaminated without cold chain requirements to reduce the risk for all involved.

What should I look for in a sample stabilization process?

It is necessary that sample stabilizing products be tested extensively to ensure that all samples will maintain their utility over prolonged periods of time and through freeze-thaw cycles. The products’ shelf lives should also be lengthy, meaning that you can have confidence that your samples will be stabilized even if collections or shipments experience delays. Ideally the process would be as simple as possible to reduce the opportunity for user error.

DNA Genotek products remove the need for a separate stabilizing reagent and its accompanying risks to your research. By incorporating the stabilization process into the collection procedure, stabilization and protection are achieved in one easy step.

Our sample-stabilizing products have been designed to be as easy-to-use as possible to lessen the likelihood of user error. In support of this simplicity we provide online instructional videos, as well as instructions printed in multiple languages. This reduces user error in sample collection and stabilization, thereby increasing your trust in both your sample and its performance downstream.

How do DNA Genotek products stabilize a sample more optimally than traditional methods?

Traditionally those who care about sample reliability have coddled the biospecimen with a preservative and attempted to account for any environmental impact. DNA Genotek products are designed to create the optimum chemical environment to control all preanalytical conditions.

All DNA Genotek products are built on a basic principle of cellular disruption and liquid-based stabilization, allowing the sampling process to be standardized and optimized. This is due to our unique stabilizing chemistry, which is the foundation of our company. Each of our collection kits and reagents contain a chemistry that allows you to immediately stabilize a biological sample so that it cannot be affected by external factors, and all desired biological material is protected over time and temperature.

Let’s use our Oragene/saliva collection kit as an example. When a donor has spit the requisite amount of saliva into the Oragene tube and clicked the funnel lid closed, our patented chemistry is discharged into the sample, disrupting the individual cells and releasing their DNA into the solution. This creates a controlled liquid environment, impenetrable by outside influences. The sample can be stored at room temperature for years without worry of DNA degradation or bacterial buildup and will remain unchanged until it is analysed.

Immediate sample stabilization is at the heart of the ‘Right from the start’ concept. By easily stabilizing your samples within seconds of collection, you can ensure that no changes will occur to the biospecimen prior to analysis. A stabilized sample is truly representative of the in vivo state of the donor, allowing you to have complete confidence in the accuracy of your discoveries.