Sickle Cell Disease: A Global Health Problem

Sickle cell trait is a common genetic defect present in an estimated 7% of the world’s population [1]. Sickle cell trait carriers have an increased resistance to malaria, which has enabled the trait to become widespread among the population of malaria-endemic countries [2]. Despite the benefit that having sickle cell trait provides, individuals born with two copies of the gene encoding for sickle cell trait are subject to the lifelong negative effects of sickle cell disease (SCD) [3]. The clinical manifestations of SCD include jaundice, elevated susceptibility to infection, delayed growth and development, acute chest syndrome, splenic sequestration, stroke, avascular necrosis of the femoral or humeral head, pulmonary hypertension, chronic hemolytic anemia and episodic pain in the chest, stomach, arms and legs [4]. Worldwide, SCD affects about 30 million people of African, Mediterranean, and Middle Eastern descent [5].

Existing Methods for Diagnosing SCD

Hemoglobin (Hb) electrophoresis is the primary laboratory test used to detect abnormal forms of Hb and diagnose SCD (and other hemoglobinopathies) definitively [6]. To perform Hb electrophoresis, red blood cells (RBCs) are lysed to release Hb, which is then allowed to migrate through an electrophoresis substrate (gel) under an applied electric field. Different Hb forms migrate at different rates, creating (when appropriately labeled) easily recognizable patterns unique to known hemoglobinopathies. This assay is very accurate and can easily differentiate between the sickle cell trait (heterozygous HbA / HbS) and sickle cell disease (homozygous HbS / HbS), but it requires specialized equipment and reagents to produce and image the electrophoresis gels, and may take as long as 1 hour to complete.

Abnormal forms of Hb can also be distinguished in the laboratory setting with the isoelectric focusing (IEF) technique, in which the conductive gel substrate is treated with an ampholyte solution to create a spatial gradient of pH on the substrate [7]. When the electric field is applied, the Hb molecules migrate through the substrate, but unlike in Hb electrophoresis the different forms of Hb separate based on their isoelectric points, rather than net charge. The advantage of the IEF technique is that it offers a clearer visual readout than Hb electrophoresis, but IEF uses even more specialized reagents, and is a similarly lengthy procedure [8].

Cation-exchange high-performance liquid chromatography (HPLC) is another laboratory technique frequently used to distinguish between the different forms of Hb that may be present in a patient’s blood sample [9]. In cation-exchange HPLC, a sample of Hb from lyzed RBCs is introduced into a chromatography column containing negatively charged resin particles. The positively charged Hb molecules from the sample readily adsorb onto the resin particles, and then are eluted by passing carefully prepared solutions with an increasing concentration of cations through the chromatography column. The rate of Hb elution from the column is continuously monitored using an optical detector by measuring the absorbance of the eluate. The elution rate depends on the electrical affinity of Hb to the resin particles in the column and is sufficiently different for different forms of Hb to enable highly accurate determination of Hb varieties present in the patient’s sample [8, 10]. Cation-exchange HPLC is a very accurate laboratory technique that can easily differentiate between sickle trait and sickle cell disease for a definitive SCD diagnosis. This technique however requires sophisticated laboratory equipment, high-quality reagents, careful sample preparation and handling, and a highly-skilled operator to perform the assay [10, 11].

Although Hb electrophoresis, IEF and cation-exchange HPLC can reliably distinguish between the different forms of Hb and provide the information necessary for objective, definitive SCD diagnosis, currently each of these assays must be performed in a specialized laboratory away from the point of care. In most realistic circumstances, the transfer of samples from the point of care (e.g. ER) to the laboratory for analysis and the delivery of the results back to the point of care can take days, which is prohibitively late for the diagnosis to affect the treatment of patients presenting with suspected SCD painful crisis. Additionally, the specialized equipment, reagents and skills required to perform these assays may be completely unavailable in the low-income regions of the world affected most by the SCD.

The presence of HbS in the blood sample of the patient can be detected at the point of care using the HbS solubility assay (e.g. SICKLEDEX), which is commonly used by the blood centers for routine screening of donated blood for HbS [6, 12]. In this assay, RBCs are exposed to a hemolytic agent (e.g. saponin) to release Hb into the phosphate buffer solution containing a strong reducing agent (e.g. sodium hydrosulfite). Under these conditions, HbS precipitates (unlike HbA which remains dissolved in solution) – the resulting turbidity of the solution is easily detected through simple visual observation [13]. The HbS solubility assay is relatively fast (5-15 minutes), requires minimal reagents and can be easily performed at the point of care. This test has very limited use for definitive diagnosis of SCD, however, because it can only detect the presence of HbS, but can’t differentiate between the sickle cell trait (HbA / HbS) and sickle cell disease (homozygous HbS / HbS) [6, 12]. The relatively short shelf-life of the chemical solutions involved also makes this test a less viable option for low-resource healthcare systems who cannot afford regular restocking and chemical preparation.

Finally, SCD can be diagnosed through direct microscopic observation of the morphology of the patient’s RBCs in a peripheral blood smear preparation [6, 14, 15]. In the absence of hypoxia, the routine peripheral blood smear for SCD patients does not contain sickled RBCs – to enable quantifiable RBC sickling, a deoxygenating agent (e.g. 2% sodium metabisulfite) is added to the preparation. The peripheral blood smear test for SCD is considered positive if >25% of the RBCs have sickle shape [6]. In its current implementation, this testing method is impractical at the point of care for many of the same reasons as the other methodologies given above. Although trained staff, microscopes, and reagents are readily available in industrialized countries, this method may also be practically impossible in low-income areas of the world. In principle, the peripheral blood smear test can produce a definitive SCD diagnosis if performed perfectly. Because performing the peripheral blood smear analysis relies entirely on human interpretation, however, the likelihood of testing inaccuracies through human error is very high. In regular practice, this test (just like the HbS solubility assay) is used primarily for initial screening of blood samples and the definitive diagnosis of SCD or sickle trait is done with Hb electrophoresis [6].

Challenges of Diagnosing SCD in the Developing Countries

Over three quarters of SCD cases occur in low-income regions of sub-Saharan Africa, where the poverty rate remains as high as 50-60% and access to modern diagnostic equipment (and healthcare in general) by a large fraction of the population is either non-existent or severely limited [16]. In these same regions, SCD is particularly harmful to young children – only a quarter of the estimated 150,000 babies born with SCD annually live to their 5th birthday, contributing about 16% of under-five deaths in Africa [17]. Historically, research and treatment in sub-Saharan Africa has focused largely on HIV/AIDS, parasitic infections and malaria, but this narrow view is starting to change: SCD has been recognized as a public health priority as recently as 2005 by UNESCO and the African Union, and 2006 by the World Health Organization [18].

Firsthand accounts by practicing doctors in the Democratic Republic of the Congo and Burkina Faso make a compelling case for the urgent need for an improved method of SCD diagnosis [18]. A practical methodology currently used for SCD diagnosis in Africa is the IEF, which is regarded as more convenient than other definitive tests (such as the HPLC) because IEF instrumentation requires much less maintenance and consumes significantly fewer reagents [18]. Nevertheless, IEF is still overly expensive with the cost of reagents required to perform a single test at roughly US$2.50, and the total costs even higher when expenses associated with drawing blood, maintaining laboratory equipment and compensating a skilled health worker who performs and interprets the test are taken into account. Because a significant fraction of the general population living in sub-Saharan Africa subsist on less than US$2.00 per day,[19] the cost of IEF testing must be greatly reduced before SCD screening using this methodology becomes a viable option for average Africans. Furthermore, as of 2008, only one laboratory in Kinshasa, the capital of the Democratic Republic of the Congo (population of 6 million), and one laboratory in Ouagadougou, the capital of Burkina Faso (population of 1.6 million), were able to carry out screening for SCD using the IEF test [18]. The associated overstraining of resources located in centralized facilities is highly inefficient, making it practically impossible to meet the needs of the entire population of these areas. The development of a low-cost, portable, easy-to-use diagnostic test for SCD could make it possible for more local health clinics to offer SCD screening, decreasing the burden on overcrowded hospitals and centralized laboratories.

A low-cost, rapid SCD diagnostic test could also be useful in order to facilitate the study of SCD and its interactions with other diseases. In sub-Saharan Africa, many infectious diseases are endemic to the population, including malaria, HIV/AIDS, hepatitis B and C [16]. Possible interactions between these diseases and SCD are very difficult to discern without a simpler, portable method of identifying SCD-positive individuals in the larger population of patients. This capability would be especially useful for understanding the effects of co-morbidities on the rates and severity of SCD painful crisis, secondary pulmonary hypertension,[20] and ACS [21]. Such a device would provide more reliable, statistically significant information about the prevalence and clinical manifestations of SCD in the general population with multiple co-morbidities, which could in turn allow doctors to make better decisions when treating these complex patients. Importantly, screening young children for SCD early in life could be a valuable tool for educating the general public about how to better cope with SCD [22]. If parents are informed of their child’s condition and know the risks involved with SCD, they will be able to watch for the warning signs of SCD complications and act quickly to help. Because of the high rate of home births in developing countries, an ideal rapid diagnostic test for SCD would be simple and reliable enough that parents themselves could administer the test and understand its results independently, with little help from a formal healthcare provider.

The Need for Rapid SCD Diagnostics in Developed Countries

A faster diagnostic tool for SCD is also needed in developed countries where financial constraints may pose less of a barrier for the conventional diagnostic methodologies to ensure that a patient presenting with symptoms of SCD actually has SCD before beginning treatment [1]. One of the most prominent clinical presentations of SCD is the vaso-occlusive painful crises, which can cause severe discomfort and organ damage [4, 23]. Erythrocytes of normal healthy individuals are shaped like biconcave disks while at rest, and retain a great degree of flexibility that enables them to deform easily while passing through the smallest blood vessels (capillaries) and deliver oxygen to cells throughout the body [24]. Erythrocytes of SCD patients stiffen, clump, and curve into a crescent (sickle) shape because of HbS polymerization in low-oxygen environments such as in capillaries and post-capillary venules [24]. The plugging of capillaries by sickle erythrocytes reduces microvascular perfusion, depriving tissues of oxygen, preventing prompt removal of metabolic waste products, and prompting intense pain in the chest, abdomen, arms, and legs [2, 23, 25]. Painful crisis episodes vary from person to person in terms of their severity, frequency, and duration [23]. Some painful crises may be managed bedside at home with pain medication, rest, and extra fluids [1]. However, more severe painful crises may cause the patient to seek intravenous fluids and stronger pain medications in the emergency room (ER) of a local hospital. Severe pain associated with vaso-occlusive crisis is the cause of nearly 90% of all hospital admissions among adult SCD patients [4].

Effective management of patients presenting with symptoms of a SCD painful crisis to the ER for emergency treatment can be challenging, particularly in adults for whom medical history records may not be immediately available. The existing testing methodologies capable of diagnosing SCD definitively are complex laboratory-based procedures that rely on specialized equipment and skills, often require blood samples to be sent to an off-site facility for processing, and are generally too slow for their results to affect the decision making process at the point of care in the ER setting. In this paradigm, it may take several days to objectively confirm that SCD is the real cause of a patient’s complaints, at which time the patient has likely been already treated and released from the ER.

Prompt and accurate diagnosis is pivotal to appropriately managing pain caused by sickle cell complications [4]. Because physicians currently have to wait for a laboratory report, diagnosis of SCD painful crisis at the point of care is often done through elimination of other possible illnesses – a lengthy and inefficient process that may result in a significant delay of treatment and undue suffering. Drug regimens and courses of preventative action to manage SCD complications (including the vaso-occlusive crisis) are well known – faster access to diagnostic information would enable doctors to more clearly weigh the risks of different treatment options against the severity of the condition faced by the patient. Availability of a rapid, definitive SCD diagnostic test performed directly at the point of care (e.g. by a triage nurse) would enable the healthcare providers to complete a differential diagnosis of SCD sufficiently fast to affect patient outcomes in the ER and other time-limited settings.

Another significant barrier to effective management of SCD painful crises in developed countries is the nature of pain medications commonly used to treat patients. One of the most effective methods of relieving a patient’s pain is to give them opioid drugs, which raises the question of addiction in patients claiming to have SCD, potentially generating a significant degree of mistrust between the patient and the healthcare provider and creating social stigma towards adult SCD patients seeking pain medication [23]. Unable to quickly confirm SCD diagnosis to objectively substantiate the patient’s claim, physicians may be hesitant to give out these potentially dangerous and highly addictive drugs, increasing the time it takes for the patient to receive proper treatment or even denying the treatment altogether [23]. On the other hand, an opioid addict could potentially take advantage of the significant delays currently associated with obtaining a definitive SCD diagnosis to obtain the drugs, thus feeding their habit. A rapid, point-of-care SCD test would alleviate this situation entirely by revealing the patient’s true condition and relieving the physician’s concern about inadvertently feeding a possible drug addiction.

Finally, sickle cell centers in the United States, NGOs, religious organizations, WHO, UNICEF, and the International Organization for the Fight against Sickle Cell Disease (OILD/SCDIO) continually provide aid to those who cannot purchase basic drugs through drug provisions at subsidized rates [26]. These organizations routinely distribute pain medications such as Paracetamol, Aspirin, Feldene, Morphine, Fortrin, and Buscopan to treat painful crisis episodes [27, 28]. Without a practical ability to validate claims by patients requesting pain medication, this practice enables drug-seekers claiming to experience symptoms of SCD painful crisis to ultimately obtain free pain medication for substance abuse or resale. A rapid, low-cost device for SCD diagnosis would enable hospitals to better control the costs associated with dispensing the medication and treating misdiagnosed patients, in addition to conserving the resources needed to perform the conventional laboratory-based SCD assays.

Overall, the ability to diagnose SCD at the point-of-care within minutes using low-cost, portable, simple-to-use devices will substantially improve patient care by enabling a more accurate differential diagnosis in patients presenting vague common symptoms (such as abdominal pain) and by giving the healthcare providers an opportunity to choose the correct course of treatment for these patients in a timely manner. Importantly, this capability could help the distribution of the costly and highly addictive pain medication only to patients who objectively need it, reducing the possibility of prescription drug abuse and alleviating the social stigma associated with adult SCD patients seeking pain management.

The Future of Rapid SCD Diagnostics

The recent resurgence of the interest in SCD from various healthcare organizations and funding agencies including the World Health Organization, National Heart, Lung, and Blood Institute of the National Institutes of Health and the Bill and Melinda Gates Foundation is poised to spur significant technological innovation towards the development of low-cost, portable devices for rapid SCD diagnosis in resource-limited settings. In the short term, the most significant progress towards this goal is likely to come from the current efforts in miniaturization and simplification of the basic technologies used to perform the currently available SCD assays (e.g. hemoglobin electrophoresis), and from adaptation of these miniaturized assays for use in the harsh reality of low-income parts of the world. Further development of novel highly-selective and efficient approaches for isolation of nucleated fetal cells from mother’s blood could enable prenatal genetic screening for early detection of SCD [29, 30]. Finally, the rapid development of microfluidic devices mimicking the pathophysiology of the vaso-occlusive crisis could allow using the mechanical properties of sickle red blood cells as a novel definitive biomarker, and may even enable predicting the occurrence of the painful crisis episodes in the future [31-33].