Grants

Over $4.5 million in federal funding secured and executed

Since its 2017 inception, In Vitro Diagnostic Solutions has successfully secured and executed over $4.5 million in federal funding from the National Institutes of Health and the National Science Foundation. Through these programs, we have successfully demonstrated strong scientific feasibility and launched commercialization of several innovative chemistries in a universal platform that will transform the market and increase accessibility for point of care diagnostics across a wide spectrum of challenge diseases.

A User-Friendly Point of Care Device, Glucose-6-Phosphate Dehydrogenase 

Grant Number: 1R43AI129057-01A1
Grant Agency and Type: NIH NIAID, Phase I SBIR
Amount: $191,220
Principal Investigator: Robert Harper
Disease Indication: Malaria

Overview: Malaria caused by Plasmodium vivax threatens over 2 billion people globally and sickens tens of millions annually. Radical cure for P. vivax malaria includes therapy aimed both at the acute attack (blood schizontocidal) and against future attacks (hypnozoitocidal). The only hypnozoitocide available is 8- aminoquinolines such as Primaquine or tafenoquine. However, clinicians often do not prescribe 8- aminoquinolines due to the high prevalence (8%) of individuals with various levels of inherited Glucose-6- phosphate dehydrogenase (G6PD) deficiencies, because these drugs can cause life-threatening acute hemolytic anemia in patients with moderate to severe G6PD deficits. There is an urgent need to quantify both Hgb and G6PD for patients stricken with malaria or prior to the administration of 8-aminoquinilones. All current quantitative methods for G6PD determination are laboratory-based spectrophotometric methods, requiring diluents, reagents, pipettes, and trained personnel. There are currently no commercially available point-of-care (POC) tests that can quantify both Hgb and G6PD directly from a fingerstick sample. Developing a robust, quantitative assay for field use in low resource areas is a high priority for overall malaria control and elimination.

Technology Solution: In Vitro Diagnostic Systems (IVDS) will establish the feasibility of developing a POC test, the PreQuine Test, in which Hgb and G6PD levels can be quantitated simultaneously from a 30 mL blood fingerstick sample. In pilot studies, we have successfully assessed Hgb and G6PD levels using independent test strips. To develop a dual Hgb and G6PD test strip, we propose developing a lysing protocol to maximize liberation of Hgb and G6PD from blood samples (Aim 1), optimize Hgb and G6PD assay conditions (Aim 2) and then combine the Hgb and G6PD assays into a single test strip (Aim 3). The PreQuine Test results will be generated with a calibrated hand-held meter; demonstration of concordance of the results from the PreQuine Test strip and calibrated meter to a suitable reference method will indicate success.

Results and Commercialization Status: We achieved all of our Phase I Aims and filed for a Phase II application, 2R44AI129057-02A1

A User-Friendly Point of Care Device, Glucose-6-Phosphate Dehydrogenase 

Grant Number: 2R44AI129057-02A1
Grant Agency and Type: NIH NIAID, Phase II SBIR
Amount: $1,447,330
Principal Investigator: Robert Harper
Disease Indication: Malaria

Overview: Malaria caused by Plasmodium vivax threatens over 2 billion people globally and sickens tens of millions annually. Radical cure for P. vivax malaria includes therapy aimed both at the acute attack (blood schizontocidal) and against future attacks (hypnozoitocidal). The only hypnozoitocide available is 8- aminoquinolines such as Primaquine or tafenoquine. However, clinicians often do not prescribe 8- aminoquinolines due to the high prevalence (8%) of individuals with various levels of inherited Glucose-6- phosphate dehydrogenase (G6PD) deficiencies, because these drugs can cause life-threatening acute hemolytic anemia in patients with moderate to severe G6PD deficits. There is an urgent need to quantify both Hgb and G6PD for patients stricken with malaria or prior to the administration of 8-aminoquinilones. All current quantitative methods for G6PD determination are laboratory-based spectrophotometric methods, requiring diluents, reagents, pipettes, and trained personnel. There are currently no commercially available point-of-care (POC) tests that can quantify both Hgb and G6PD directly from a fingerstick sample. Developing a robust, quantitative assay for field use in low resource areas is a high priority for overall malaria control and elimination.

Technology Solution: To complete the development of the PreQuine System, we will: (1) Finalize Assay Development by Assessing Bioactive Components. Three commercially available diaphorase will be tested to ensure (a) a high degree of activity at the optimal pH, (b) long-term stability; and (c) no inhibition in the presence of maleimide; (2) Transition from Hand Assembly to Semi-Automated Assembly of Test Strips. This will be achieved by optimizing process capabilities of coating, slitting, and laminating the strips into 5-up card stock; (3) Incorporate a Temperature Correction Factor (TFC). The PreQuine system must be able to function in a working temperature of 18°C to 40°C. The TFC will be incorporated into the software and correct for temperature differences. Finally, (4) Validation of the PreQuine System. Validation for G6PD and Hgb levels will demonstrate concordance between the manufactured test strips and reference methods. Once validated and commercialized, the PreQuine System will provide a point-of-care diagnostic tool to identify G6PD-deficient individuals who cannot tolerate 8-aminoquinoline treatment, which will transform malaria treatment strategies and aid in the eradication of P.vivax and P. oval malaria.

Results and Commercialization Status:
Graph 1. is a plot of Percent Reflectance (%R) versus Glucose-6-Phosphate Dehydrogenase (G6PD) concentrations from patient samples; samples spiked with G6PD  and samples that went through a freeze-thaw process to ensure the samples encompassed the analytical range of  0.8-12.5  Units  G6PD /g Hgb. Reflectance values were obtained by the hand-held PreQuine Meter, and G6PD concentrations were obtained via the Pointe Scientific G6PD Spectrophotometric assay.  

Graph 1. Third-degree Polynomial of Spectrophotometric Concentration of G6PD vs % Reflectance

Graph 2. is a linear regression of retrospectively calculated results from the PreQuine Meter versus results obtained by the Pointe Scientific G6PD Spectrophotometric assay.  The graph reveals excellent agreement. Once we have completed the calibration software, the output will be displayed as  Hgb g/dL and G6PD U/g Hgb.

Graph 2. First-order Linear Regression Spectrophotometric Concentration of G6PD vs PreQuine Meter Output Calculation

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

Overview: 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 3. 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 3. First-order Linear Regression of Tandem Mass Spectral Analysis (U of Wisconsin) vs PKU Now results

Graph 4. 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 4. Comparison of L-Phenylalanine Detection with and without L-Tyrosine Suppression 

Graph 5. 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 5. Bland-Altman Plot of Amino Acid Analysis versus PKU Now results 60 determinations spike PHE Samples

A Point-Of-Care Device for Phenylalanine Determination

Grant Number: 2 R44 GM126593-02A1
Grant Agency and Type: NIH NIGMS, Phase II SBIR
Amount: $1,946,957
Principal Investigator: Robert Harper
Disease Indication: Phenylketonuria

Overview: Phenylketonuria (PKU) is an inherited disorder that increases the levels of L-phenylalanine (Phe) in the blood. Without proper monitoring and treatment, patients can develop permanent intellectual disabilities, seizures, behavioral problems, and psychiatric disorders. In Vitro Diagnostic Solutions (IVDS) proposes to develop the first point-of-care (POC) test, the PKU Now, for the determination of Phe from a finger or heel-stick sample. Like the glucose model, the PKU NowTM System is comprised of a hand-held meter and test strip. The PKU Now will be used as both a home monitoring device and a diagnostic device in countries that do not currently offer newborn screening. In Phase II, we propose to test a range of commercially available diaphorase and phenylalanine dehydrogenases (PheDHs) and select the pair that provides the greatest delta (optimally 45-47 units %R) and the shortest read time (ideally under 3 minutes). The diaphorase is critical for the stability and rate of color development of the reagent. It must (1) have a high degree of activity in conjunction with PheDH; (2) have specificity for B-NAD+; and (3) provide long-term reagent stability (20-25°C for 18 months).

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 Solutions (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.

A Point-Of-Care Device for the Determination of the Bilirubin, the BiliNow 

Grant Number: 1R43HD095727-01
Grant Agency and Type: NIH NICHD, Phase I SBIR
Amount: $224,700
Principal Investigator: Robert Harper
Disease Indication: Kernicterus

Overview: Neonates with high bilirubin levels can develop a fatal disorder known as Kernicterus. Kernicterus has a death rate of 2-3% and causes brain damage and serious long-term complications. High bilirubin levels are the leading cause of readmission of newborns to the hospital in the first two weeks of life. Current laboratory analyzers require a venipuncture for neonates, which is difficult for the care-provider, patient, and parent. “With the onset of illness, it is not uncommon for neonates to lose 15-30% of circulating blood volume to laboratory testing.” A NICHD workshop on NICUs concluded that there is an urgent need for metabolic analysis methods that require minimal volumes of blood, can measure analytes in parallel, and are accurate, reliable, and cost-effective. A recent report by Market Research Future, states that by 2024 the Bilirubin Testing market will reach 2.5 billion dollars. 

Technology Solution: In Vitro Diagnostic Solutions (IVDS) proposes the development of the first and only point-of-care (POC) system, the “BiliNow”, for the determination of bilirubin from a finger or heel-stick. The “BiliNow” will be used in the Neonatal Intensive Care Unit (NICU), emergency room (ER), intensive care units (ICU), and clinician’s office, providing immediate feedback to expedite treatment. The BiliNow utilizes a microfluidic chip format that is minimally invasive, requiring only 30 microliters of whole blood, which is collected from a heel-stick or fingerstick. In Phase I studies, we will: 1) Test solubilizing agents to ensure complete solubility of unconjugated bilirubin; 2) Test and eliminate hematocrit bias in the neonatal range of 32 to 60%; and 3) Evaluate a dose-response of bilirubin with whole blood samples. Microchips will be dosed and dried with chromophore-sensors specific for conjugated and unconjugated bilirubin. Whole blood will be spiked with conjugated and unconjugated bilirubin levels over the analytical ranges. Plasma will be separated, in-situ, and the end color of each sensing-well will be quantitated using percent reflectance and converted to a concentration in mg/dL, via a hand-held, portable analyzer, similar to the glucose model. The “BiliNow” provides results for both conjugated and unconjugated bilirubin in a single step within 3 minutes. The “BiliNow” technology has a clear advantage in the neonatal blood chemistry market, as no other device can measure bilirubin from a single drop of blood.

Results and Commercialization Status: Results showed excellent agreement with spiked blood samples for both conjugated and unconjugated bilirubin. IVDS will be applying for a Phase II application in Q1 of 2021. We are finalizing our Phase I Close-Out Report.


A Point-of-Care Device for Simultaneous G6PD and Hemoglobin

Grant Number: 1746309
Grant Agency and Type: National Science Foundation, Phase I SBIR
Amount: $225,000
Principal Investigator: Robert Harper
Disease Indication: Malaria

Overview: This SBIR Phase I project sought to develop an optical detection system to quantitate Hgb and G6PD from a test strip. This would transform treatment regimens for individuals suffering from malaria infections. Malaria, particularly caused by Plasmodium vivax (P. vivax) and Plasmodium oval  (P. oval) remains a potential cause of morbidity and mortality amongst the 2.85 billion people living at risk of infection. The only drug currently available for treatment therapy for P. vivax and P. oval is Primaquine, which can cause life-threatening anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Clinicians often do not prescribe Primaquine due to the high prevalence (8%) of individuals who are born with G6PD deficiency. The World Health Organization (WHO) and the Program for Appropriate Technology in Health (PATH) are urgently searching for a reliable assay for the diagnosis of G6PD deficiency to effectively treat patients and aid in the eradication of P. vivax and P. oval malaria. This novel assay will quantify G6PD and hemoglobin (Hgb) concentrations simultaneously from a fingerstick sample. This system comprises a single test strip coupled with a new prototype reflectance-based meter and cell phone application with Bluetooth connectivity to incorporate a patient’s I.D., test results, global tracking, and history of treatment. It is projected that this point-of-care assay will be used to screen >23 million people for G6PD within 5 years.

Technology Solution: The proposed novel platform will quantify both Glucose-6-Phosphate Dehydrogenase (G6PD) and hemoglobin (Hgb) concentrations simultaneously from a single finger stick sample using a point-of-care (POC) prototype reflectance-based meter. There is currently no such device on the market, which is urgently needed to screen patients being treated for P. vivax and P. oval malaria. A significant portion (8%) of the world population is G6PD deficient, which places these individuals at risk for life-threatening anemia after treatment with current therapeutics such as Primaquine against malaria. The POC assay utilizes a novel lysis and reagent layer membrane platform to enable a reflectance-based meter to measure non-overlapping wavelengths to quantify G6PD and Hgb concentrations. In this proposal, a functional prototype reflectance-based meter and data collection software will be constructed and compared to readings obtained using the current gold standard method (Konica Minolta spectrophotometer).  The POC assay will then be validated through performance testing using a G6PD-deficient whole blood specimen bank provided by the Program for Appropriate Technology in Health (PATH). Data obtained from 20 samples using the proposed assay will be compared to the World Health Organization’s (WHO) approved spectrophotometric method for measuring G6PD, and an FDA approved method for measuring Hgb. Success will be indicated by an R2 > 0.95, which demonstrates linear correlation, as well as a demonstration that 90% of the differences in individual measurements fall within 2 SD of the mean difference. A POC assay that can simultaneously screen patients for both G6PD deficiency and Hgb levels will allow clinicians to treat patients with P. vivax and P. oval malaria infections effectively and aid in the eradication of malaria.

Results and Commercialization Status: IVDS is continuing to develop a reflectance-based optical system with 3 LEDs to read three analytes. The meter is approximately 2” x 3” in width and length. The meter will have Bluetooth connectivity, QAR code, RFID, and a cell phone application. We are expecting to have the working prototype in September of  2020.

In Vitro Diagnostic Solutions is extremely grateful for the strong commercialization support provided by both the National Institute of Health and National Science Foundation Small Business Innovative Research Programs.

ABOUT IVDS
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.
CONTACT INFO
  • In Vitro Diagnostic Solutions
    900 N. Lenola Rd., Bldg. 7, Unit L
    Moorestown, NJ 08057
  • (856)-343-5098
  • Robert@ivd.solutions