A Point-Of-Care Device for Phenylalanine Determination 

Grant Number: 1R43GM126593-01A1
Grant Agency and Type: NIH NIGMS, Phase I SBIR
Amount: $224,700
Principal Investigator: Robert Harper
Disease Indication: Phenylketonuria

Phenylketonuria (commonly known as PKU) is an inherited disorder that increases the levels of a substance called phenylalanine in the blood. PKU is the most common disorder of amino acid metabolism, affecting 1 in 8,000 people globally. Most cases of PKU are detected shortly after birth by newborn screening. Individuals affected by PKU must monitor and control Phe levels throughout their lives to avoid neurological complications, including permanent intellectual disabilities, seizures, delayed development, behavioral problems, and psychiatric disorders. Whole blood is collected in EDTA tubes or spotted onto Dried Blood Spot (DBS) Cards by parents, patients, or caregivers. These samples are sent to laboratories for measurement by tandem mass spectrometry, and results can take days to weeks. This complicated process for monitoring and controlling Phe levels results in non-compliance, a decrease in quality of life, as well as increased healthcare costs for treating complications. 

Technology Solution
The PKU Now will provide real-time Phe levels, allowing parents and caregivers to promptly adjust their diets to help maintain optimal phenylalanine levels. In Vitro Diagnostic Systems (IVDS) has developed a prototype POC test, in which Phe can be quantified from a 20-microliter sample. “PKU Now” will be the only point-of-care testing device (POCT) that will allow the accurate measurement of L-phenylalanine (Phe) levels from a finger or heel-stick sample. The point-of-care test, the “PKU Now,” will be used as a home monitoring device for PKU patients and for diagnostic testing. The PKU Now will provide immediate feedback of whole blood Phe concentration, allowing for stricter dietary compliance, improve the quality of life, and reduce healthcare costs. In Phase I studies, we will resolve three outstanding issues before commercializing the system, namely eliminating interferences (Aim-1), eliminating hematocrit bias in the range of 32% to 60% (Aim-2), and establishing an agreement between the “PKU Now” and a laboratory reference method (Aim-3).

Results and Commercialization Status
We achieved all of our Phase I Aimed and filed for a Phase II application.

Figure 1. Shows the PKU Now test kit containing; 1 white vial (20 test strips); 1 white vial (20 capillary tubes); a capillary stand, a PKU Now Meter and 2 levels of controls

Figure 1. PKU Now test kit (Unpacked)



Graph 1. is a linear regression of PKU Now results versus results from tandem mass spec analysis of Phe-spiked blood samples. The plot reveals excellent agreement.

Graph 1. First-order Linear Regression of Tandem Mass Spectral Analysis (U of Wisconsin) vs PKU Now results

Graph 2. is a plot of 3 samples, i.e., one sample at 1 mg/dL Phe (native sample not spiked) and two samples spiked to 3 and 6 mg/dL Phe. All samples were also spiked with 3 mg/dL tyrosine. Tyrosine has a very similar structure to Phe and is recognized by (the enzyme) PHEdH as a substrate. The elimination of tyrosine has been the pitfall of previous development efforts. As you can see at low concentrations of Phe, the presence of tyrosine almost doubles the Phe reading (false positive or over-recovering for Phe). This is concerning when trying to keep Phe levels well maintained. The patent-pending suppression technology eliminates tyrosine interference.

Graph 2. Comparison of L-Phenylalanine Detection with and without L-Tyrosine Suppression 

Graph 3. is a Bland-Altman plot of the difference between two paired measurements (PKU Now and amino acid analysis method) versus the mean of the two measurements.  The plot reveals only 2 points outside of 2 standard deviations (SD) from the mean for 60 determinations.  We met the acceptance criterion. (i.e., ≥ 95% of the data points should lie within ± 2 SD of the mean difference). 

Graph 3. Bland-Altman Plot of Amino Acid Analysis versus PKU Now results 60 determinations spike PHE Samples

In Vitro Diagnostic Solutions (IVDS) develops novel testing technologies for the healthcare, veterinary, and consumer markets. IVDS scientists have helped bring 10 FDA-approved products to market over their careers in diagnostics.
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