Introduction

Have you ever wondered how the Theriome’s Aristotle test can just use just a few drops or 20 µL of blood to accurately quantify 126 metabolites, unlike traditional blood tests?

Dried blood spot tests are much easier to collect and more stable, eliminating the need for traditional blood draws or specialized overnight shipping. Furthermore, the dried blood spots(DBS) work best for Theriome Aristotle test. 

Beyond the obvious logistical advantages, this article delves into the technical details of our clinically validated GC/MS technology with dried blood spot samples. We also highlight the key differences from common blood tests, explaining why this innovation is not another Theranos.

Why Traditional Blood Tests Require a Lot of Blood

Generally, how much blood you need for each test depends on the analyte being measured and the type of assay you need to quantify the analyte. For example, albumin and other large proteins often require whole blood to achieve accurate measurements. Here's why traditional blood tests typically need milliliters of blood:

Lower Sensitivity and Specificity

Traditional techniques, such as enzymatic assays and immunoassays, often exhibit limited sensitivity because they are indirect detection methods, where analyte concentration is inferred rather than directly measured.

These methods also suffer from low specificity, because they can be prone to cross-reactivity and interference from similar compounds. Therefore, for analytes present in naturally low concentrations, you will need higher initial sample volume to concentrate the analyte and produce a detectable signal. Additionally, these techniques typically operate within a narrow linear range. This Means the analyte must be present at or above a specific threshold concentration for accurate quantification, necessitating larger sample volumes.

Analyte Dilution and Stability

Many biological analytes are unstable, requiring larger starting amounts to ensure accurate detection. Many substances, peptides, and hormones are susceptible to rapid degradation, oxidation, or binding interactions in whole blood. 

In traditional blood tests, you need more blood to account for these potential losses during sample handling and processing. 

Furthermore, analyte stability is often compromised in small sample volumes due to surface adsorption effects and enzymatic activity. These issues make larger sample volumes necessary to preserve the integrity of low-abundance compounds.

Limited Analytical Range

Conventional methods, such as spectrophotometry and fluorescence-based assays, operate within narrow analytic ranges—they are only reliable for analyte concentrations that fall within such ranges. Outside these ranges, whether due to high or low concentrations, these methods can become unreliable or unresponsive. 

As a result, larger samples ensure that low abundance analytes fall within the detectable range of the chosen method, particularly for low-abundance compounds.

Sample Processing Needs

Certain types of liquid chromatography, which is often used in traditional lab settings, involve multiple sample preparation steps, including extraction, purification, and concentration. Each of these steps introduces sample loss due to adsorption, degradation, or phase separation. 

To mitigate these losses, you need to start with large sample volumes that are typically required to ensure that sufficient analytes remain available for accurate detection and quantification.

Why Theriome’s Aristotle Test Can Use Small Sample Volumes

The Aristotle Test capitalizes on the inherent strengths of GC-MS, which has been optimized to handle complex biological samples with extremely high sensitivity and specificity [1]. Here’s how we achieve this with just a 6mm DBS sample.

 

High Efficiency of GC Columns

Modern capillary GC columns, with a high theoretical plate number, enables the separation of complex mixtures of metabolites with an unprecedented resolution. Even from a tiny sample, we can achieve baseline resolution for structurally similar compounds. This reduces the likelihood of co-elution and enhances analytical sensitivity. The efficiency of the GC columns ensures each analyte is distinctly separated, leading to improved signal intensity, even from minimal sample amounts.

Electron Impact (EI) Ionization

As a hard ionization technique, electron impact (EI) ionization produces highly reproducible fragmentation patterns, making it ideal for the identification and quantification of metabolites in complex biological matrices. Despite the intense fragmentation, resulting in multiple ion fragments, these fragments can be leveraged to identify specific metabolites with high certainty. Our methodology is fine-tuned to enhance molecular ion detection at low sample volumes, ensuring high signal intensity for trace metabolites.

Mass Selective Detection

The mass spectrometer operates as a highly selective detector, focusing on ions of a specific mass-to-charge ratio (m/z). This selectivity reduces background noise and ensures that only the ions of interest are detected, which significantly increases the signal-to-noise ratio (SNR) and enhances the limit of detection (LOD). By selectively monitoring specific m/z ratios of known metabolites, our system achieves exceptional sensitivity and specificity, even from minute sample volumes.

Pre-Concentration and Derivatization

Prior to GC-MS analysis, samples undergo derivatization to increase volatility and detection efficiency. This step improves the detectability of metabolites with low volatility or thermal stability, enabling the  analysis of a broader range of compounds from small samples. Solid phase microextraction (SPME) further enhances sensitivity by pre-concentrating analytes and minimizing sample loss during preparation. This process is particularly critical for low-volume samples, as it maximizes the recovery and detection of trace metabolites.

Data Acquisition and Processing

Our GC-MS instruments are equipped with sophisticated software that processes large datasets, identifies peaks of interest, and filters out noise. This enables us to detect low-abundance compounds with high accuracy. Advanced peak integration algorithms and machine learning techniques distinguish true signals from baseline noise, enabling robust detection of metabolites in small-volume samples.

Elimination of Interferences

GC-MS combines the separation power of gas chromatography with the detection power of mass spectrometry, effectively eliminating interferences that may mask low-abundance metabolites. This reduces matrix effects and allows precise quantification of target metabolites without requiring large sample volumes.

High Vacuum System

The mass analyzer operates under a high vacuum, minimizing interference from air and other gases. This reduces background noise and improves the sensitivity of the analysis particularly for small sample volumes, where noise can significantly impact the detection limits.

Regular Tuning and Calibration

To ensure the highest sensitivity and accuracy, our GC-MS instruments undergo regular tuning and calibration using reference standards. This ensures that even trace amounts of compounds produce  significant and recognizable signals, enabling accurate quantification from small sample volumes.

Addressing Concerns in the Post-Theranos Era

In the wake of the Theranos scandal, skepticism has grown regarding claims of accurate results from small sample volumes. Theriome’s approach is grounded in validated science, leveraging GC-MS technology that is extensively peer-reviewed and well documented [1, 2]. 

Our co-founder and Chief Science Officer, Dr. Paniz Jasbi, has published numerous peer-reviewed publications, such as those on metabolomic profiling and the application of mass spectrometry in clinical and environmental studies. These studies confirm the reliability of our methods, including comparisons of results from our DBS-based Aristotle Test with traditional venipuncture-based tests. Results demonstrate  strong correlation and high concordance for a wide range of metabolites (R2 95-99%, with coefficient of variation <5% for most metabolites and <10% for all metabolites). Detection limits for most metabolites are within micromolar detections, while others are within picomolar or femtomolar ranges. 

Unlike unproven technologies, GC-MS has been a gold standard for decades in fields where precision is critical, such as forensic science, drug testing, and environmental monitoring.

What Does a Dried Blood Spot Test for? The Basics of DBS Testing

The Accuracy and Advantages of Dried Blood Spot Testing

One common question among healthcare professionals is, “How accurate is dried blood spot testing?” Currently, dried blood spot tests are commercially available and reliable for:

• Monitoring viral types and load in infections such as HIV and Hepatitis [3]
• Genomic sequencing [4]
• Chronic disease monitoring [5]
• Vitamin D Levels [6]
• Omega-3 and other fatty acids [7]
• HbA1C measurements

Due to the logistical ease and increasing consumer demands for more accessible health monitoring from home, lab companies are also developing dried blood spot tests for other kinds of tests. Most of these tests, however, require several blood spots due to the constraints mentioned above.

The primary advantages of DBS testing include:

• Convenience and Accessibility: Patients can collect samples at home, enabling better compliance and regular monitoring.
• Sample Stability: DBS samples remain stable longer than wet blood over time and can be transported without special handling.
• Reduced Sample Volume: A single drop of blood suffices, which is particularly useful for pediatric or geriatric patients and those with limited venous access.

Summary

The reason a small amount of blood is sufficient for the Aristotle test lies in the power and sensitivity of gas chromatography-mass spectrometry (GC-MS) [8].

Unlike traditional blood tests that require larger samples, GC-MS can detect and quantify trace amounts of metabolites with detection limits in the micrograms per liter range, which is a typical method for detecting drugs in blood and urine samples.

GC-MS separates and identifies compounds at the molecular level, allowing it to detect a broad range of metabolites in a single, small sample [4]. 

This is particularly advantageous for dried blood spot testing, where a single drop can yield enough data to identify metabolic markers with high accuracy [4]. By focusing on metabolomic profiling through GC-MS, the Aristotle test achieves a comprehensive analysis without the need for larger, more invasive blood samples typically required in traditional tests.

By integrating advanced GC-MS technology with a commitment to integrity, Theriome’s Aristotle test represents the next generation in comprehensive metabolic analysis, offering unprecedented accuracy and convenience.

[1]  Fiehn, O. (2016) Metabolomics by gas chromatography-mass spectrometry: Combined targeted and untargeted profiling. Curr. Protoc. Mol. Biol., Wiley 114,

[2]  Beale, D. J., Pinu, F. R., Kouremenos, K. A., Poojary, M. M., Narayana, V. K., Boughton, B. A., et al. (2018) Review of recent developments in GC-MS approaches to metabolomics-based research. Metabolomics, Springer Science and Business Media LLC 14, 152 

[3]  Tuaillon, E., Kania, D., Pisoni, A., Bollore, K., Taieb, F., Ontsira Ngoyi, E. N., et al. (2020) Dried blood spot tests for the diagnosis and therapeutic monitoring of HIV and viral hepatitis B and C. Front. Microbiol., Frontiers Media SA 11, 373 

[4]  McBride, D. J., Fielding, C., Newington, T., Vatsiou, A., Fischl, H., Bajracharya, M., et al. (2023) Whole-genome sequencing can identify clinically relevant variants from a single sub-punch of a dried blood spot specimen. Int. J. Neonatal Screen., Int J Neonatal Screen 9 

[5]  Lim, M. D. (2018) Dried blood spots for global health diagnostics and surveillance: Opportunities and challenges. Am. J. Trop. Med. Hyg. 99, 256–265 

[6]  Heath, A. K., Williamson, E. J., Ebeling, P. R., Kvaskoff, D., Eyles, D. W. and English, D. R. (2014) Measurements of 25-hydroxyvitamin D concentrations in archived dried blood spots are reliable and accurately reflect those in plasma. J. Clin. Endocrinol. Metab., J Clin Endocrinol Metab 99, 3319–3324 

[7]  Wood, J., Minter, L. J., Stoskopf, M. K., Bibus, D., Ange, D., Tollefson, T. N., et al. (2021) Investigation of dried blood spot cards for fatty acid analysis using porcine blood. Vet. Med. Int., Hindawi Limited 2021, 6624751 

[8]  Ingels, A.-S., De Paepe, P., Anseeuw, K., Van Sassenbroeck, D., Neels, H., Lambert, W., et al. (2011) Dried blood spot punches for confirmation of suspected γ-hydroxybutyric acid intoxications: validation of an optimized GC-MS procedure. Bioanalysis, Informa UK Limited 3, 2271–2281