Background

Untreated and undiagnosed sickle cell disease is the cause of suffering for thousands. Sickle cell disease presents as pain, shortness of breath, and even organ failure for those suffering from vaso-occlusive crisis induced by sickle cell disease. These symptoms are treatable and even preventable if treatment methods such as blood transfusion or administration of drugs are used. Because the symptoms of sickle cell disease are undifferentiable from that of many other diseases, a diagnosis in order to provide proper treatment is imperative. Many patients suffering from sickle cell disease live in low income communities with less access to medical care and regular visits to the doctor. Typical evaluation for sickle cell disease involves taking a sample of blood and sending it to an external laboratory, with reporting of results in several days, or even weeks. This testing setup is not ideal for areas where the majority of sickle cell patients reside, where laboratory access is limited, and having patients return for follow-up appointments for test results is not realistic.

Therefore, there is a need for a sickle cell disease detecting device that does not require use of an external laboratory, that can return disease results within one doctor’s visit, and is accurate. This device must be simple enough for medical professionals to be able to easily operate, and inexpensive enough for communities with limited resources to be able to afford. An advantage to a device design over other similar devices is the addition of quantitative analysis, or a reading of how much of the blood is affected by sickle cell disease. Lastly, an improvement over current laboratory testing would be the use of a smaller sample of blood, rather than the use of a whole tube.

Our aim is to design, develop, and improve on current sickle cell electrophoresis diagnostic tools by effectively quantifying the amount of hemoglobin S in a sample of sickle-cell trait blood. This diagnostics tool will subsequently be equivalent to submitting the samples to lab-based electrophoresis, IEF, and HPLC. Although the sickle solubility test may seem like an ideal methodology to screen for hemoglobin S (simple, inexpensive, and readily available), it lacks additional information provided by other testing modalities to make a specific diagnosis.

Disease Profile

Sickle Cell Disease (SCD) is a condition that is passed down genetically affecting the hemoglobin of red blood cells (RBCs)4. SCD disproportionately affects some populations such as those with Sub-Saharan, Latin American, Mediterranean, Saudi-Arabian, or Indian ancestry. SCD affects about 100,000 Americans. Many more have Sickle Cell Trait (SCT), or have one abnormal hemoglobin allele and one normal one, and remain largely unaffected by the disease6. Worldwide, there are an estimated 300,000 annual births that carry sickle cell disease5. SCD is a lifelong disease, and the only cure is a bone marrow transplant, however there are many treatments in order to lengthen lifetime and lessen pain2. RBC’s that contain sickled hemoglobin (HbS) or sickled hemoglobin in combination with other abnormal β alleles, when exposed to a deoxygenated environment undergo polymerisation and become rigid. The rigid RBC’s are liable to distortion which can result in hemolysis, which can lead to anemia5, organ damage, and decreased ability to oxygenate tissues and organs4. Further symptoms of distorted red blood cell shapes could include microvascular vaso-occlusion, chronic inflammation, and other dysfunctions which could result in death8.

Traditional Techniques

Methods of testing for SCD include isoelectric focusing (IEF), high–performance liquid chromatography (HPLC), cellulose acetate and citrate agar electrophoresis, capillary zone electrophoresis (CZE) and DNA analysis9. In the US, testing of newborns for SCD is now mandatory. However, this screening is not regularly performed in some other countries, some of which have high prevalence of the disease10. A potential solution to this issue is the use of Point-of-care (POC) tests for SCD. POC tests have been used to diagnose infants in remote locations with high-risk populations such as sub-saharan Africa9.

Hemoglobin electrophoresis is the traditional method used for the separation and identification of normal and variant hemoglobins in blood applied to a filter paper matrix. Isoelectric Focusing (IEF) is another electrophoretic technique used to separate hemoglobins based on their net charge within a pH gradient on a gel medium. High-performance liquid chromatography (HPLC) is a method that is also based on the net charge of the hemoglobin molecule at a specific pH, but is fully automated and provides precise quantification of hemoglobins. Another current technology, Capillary Zone Electrophoresis (CZE) is a hybrid technique combining classical electrophoresis with liquid chromatography15.

IEF, CZE and HPLC are currently considered the gold standard methods for diagnosis of SCD. These methods are well suited to screening programs designed around specialized facilities with timely collection and storage of samples, stable electrical power, refrigeration, automated instrumentation and trained personnel to perform testing. IEF is sensitive and specific and low cost, but is labor intensive and expertise is needed. HPLC is also sensitive and specific, but is high cost and requires skilled technicians. CZE is similar to HPLC but also uses expensive equipment. Mass spectrometry is also used sometimes, but the equipment is very expensive. Point of Care, or POC tests, which are not available by any of these methods, are very easy to use, cheap, and do not require electricity, and are good for newborns16.

Contemporary Techniques

HemeChip is a new device available for the early detection of sickle cell disease in children that uses microchip electrophoresis technology in combination with artificial intelligence to replicate a traditional electrophoresis testing machine. A product created by the University of Colorado Boulder can be run in one minute with only a small droplet of blood. The Acousto Thermal Shift Assay utilizes high-amplitude sound waves, or ultrasound, to heat a protein sample while concentrating the proteins that don’t dissolve. This tool is also accessible, not requiring large or expensive equipment21. There is a smartphone-based image acquisition method for capturing RBC images from the SCD patients in normoxia and hypoxia conditions. A computer algorithm was developed to differentiate RBCs from the patient’s blood before and after cell sickling. Hemoglobin electrophoresis and high-performance liquid chromatography (HPLC) are gold standard methods for the detection of SCD, however, they require many expensive resources. Electrical impedance spectroscopy (EIS) based methods have been developed for the detection of SCD in microfluidic chips, resulting in a reliable, accurate, and efficient method that can easily distinguish normal and sickled RBC. The variations in the measured electrical impedance differential of sickle RBCs can work as a new biomarker of SCD22. Another sickle cell diagnostic system uses images in order to diagnose the disease by taking a picture of the patient’s sample and applying a Gaussian filter in order to get a clear image of the edges of the red blood cells. This will then detect the abnormal cell shapes and output a detection message23. Sickle SCAN is a POC test using imaging that is inexpensive and accurate. Sickle SCAN gives results in the form of a band with a label that indicates whether the hemoglobin in the blood is normal, sickle, or other common forms of sickle cell disease (HbSS and HbSC)20.

Another technique implements the tendencies of Sickled RBCs to dehydrate, which increases the density of the cell. This permits the use of centrifugation to precipitate RBC’s to the bottom of the tubes, and then quantitatively analyzed for the fraction of dense cells24. Electrical impedance-based microflow cytometry technique with oxygen control seems potentially useful for SCD diagnosis. Using microfluidics and electrical impedance spectroscopy, it is a label-free flow cytometry for non-invasive measurement of single cells under controlled oxygen level25.

Our Technique

While current diagnostic methods for SCD are effective, they come with challenges such as accessibility, cost, requirement for specialized equipment and expertise, and the potential for false positives or negatives. One of the most common methods currently used in clinical practice is High Performance Liquid Chromatography, or HPLC. This technique involves separating the components dissolved in a liquid and performing quantitative or qualitative analysis on the separated components by insertion through molecular separation columns at stable rates. The samples are then read by the chromatography detector, and electrical signals are then transmitted and analyzed.10 While this process does not take much time to run, about 10-15 minutes, the equipment is expensive and not portable, meaning samples must be sent to an external laboratory for processing using this technique.32 Another common technique for sickle cell detection is agarose gel electrophoresis, where the mobility of charged molecules in an electric field across a supportive medium is analyzed. This test alone takes around 2 hours, also by using expensive equipment that is not portable.15,32

The electrochemical method of cyclic voltammetry has shown promise for being portable, time efficient, and inexpensive for analysis of blood samples for sickle cell disease. This method measures current that is outputted by the electrochemical cell under conditions of voltage that are predicted by the Nernst equation. The hardware of this system consists of a working electrode, a reference electrode, and a counter electrode. Voltage is induced into the tri-electrode system, and the current changes due to the redox reaction occurring on the working electrode. The forward potential induces an oxidation state, and the backward potential induces a reductive state. These current readings can be graphed and analyzed for when significant changes occur, indicative of drops or spikes in redox potential.35

Research on the redox potentials of blood has shown that sickled and wild type blood can be measured and differentiated as a reduction potential, or the tendency to accept or donate electrons. This is because RBCs affected by sickle cell disease have a decrease in NAD redox potential that may be due to their increased oxidant sensitivity. This is resultant of changes from pyridine nucleotides.34 The redox potential in human erythrocytes has been measured as values ranging from −276 mV to −250 mV for wild-type (HbAA) erythrocytes and −220 mV for homozygous sickled erythrocytes (HbSS).31

Page Leader: Lorelai Schoch

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