Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

An estimated 5–7% of the world's population harbours a haemolytic anaemia-associated allele of haemoglobin. In as many as 30 million people, autosomal recessive sickle cell disease (SCD) accompanies the homozygous missense mutation E6V in the β-globin polypeptide encoding sickle haemoglobin (HbS). SCD is especially prominent in equatorial Africa where, under strong positive evolutionary selection pressure of the malaria parasite Plasmodium (Bunn, 2013), regional HbS allele frequencies range from 1–2% to as high as 45%. Resulting rates of SCD can exceed 2% of a nation's population. In contrast to the steadily increasing life expectancy of SCD patients in the developed world, the 150,000–400,000 African infants born each year with sickle cell disease have a median survival under 5 years, constituting ∼16% of deaths before age 5, and accounting for 12–20% of hospitalized pediatric patients (WHO, 2010). Decades and more before Herrick's 1904 report of ‘peculiar elongated and sickle-shaped’ cells in patient blood, the disease was described in 19th century West Africa as ogbanjes (Igbo for ‘children who come and go’), malevolent, vengeful reincarnates visiting misfortune upon their families (Nzewi, 2001; Ebrahim et al. 2010). Unlike the α2β2 tetramers of wild-type HbA, the α2βS2 tetramers of HbS respond to deoxygenation in the peripheral vasculature by polymerizing into long strands that associate laterally to form rigid fibres. These fibres impinge upon, distort and damage the red cell membrane lipid bilayer and its constituent proteins. The rate of HbS polymerization is independent of its intracellular concentration ([HbS]), but the lag time between sickle cell deoxygenation and the onset of HbS polymerization is exquisitely and inversely dependent upon [HbS]. After completion of erythropoiesis, [HbS] in circulating red cells is an inverse function of red cell volume, regulated primarily by the Ca2+-activated K+ channel (Gardos channel/IK1/KCa3.1/KCNN4) and by SLC12 K+–Cl− cotransporters. Overactivity of these K+ efflux pathways in SCD generates dense, dehydrated red cells with elevated [HbS]. This dense cell fraction constitutes the population most prone to sickle, adhering to and activating endothelial cells, and leading to the chronic, recurrent ischaemic insults and vaso-occlusive symptoms of SCD (Lew & Bookchin, 2005). Overactivity of the Gardos channel is attributable in part to Psickle, a deoxygenation-activated non-specific cation permeability of still unknown molecular identity, unique to sickle red cells. Psickle was first characterized as a property of sickle red cells by ion flux studies in the early 1980s, and more recently documented as a deoxygenation-activated ion channel activity in whole-cell (Browning et al. 2007b; Ma et al. 2012) and cell-attached patch clamp experiments (Vandorpe et al. 2010). Inhibitors of the Gardos channel, the K+–Cl− cotransporters, and Psickle, by suppressing the overactivity of the K+ leak pathways in SCD red cells, should greatly decrease the fraction of high-density, dehydrated SCD red cells in circulation. Thus, development of such inhibitors remains of considerable promise for adjunct treatment of SCD. Activation of Psickle by deoxygenation is accompanied by slower activation of a poorly understood, non-specific permeability pathway for a broad range of small, non-ionic solutes (Browning et al. 2007a). Polymerization of intracellular HbS appears to be required for activation of both the non-ionic solute pathway and Psickle in sickle red cells. Effective, inexpensive treatment in the form of hydroxyurea has long been available to SCD patients, 50–70% of whom are clinically responsive to the drug. However, despite the proven safety to date of chronic hydroxyurea therapy, the drug is a mutagen, and its responsible use requires accurate diagnosis. In the developed world, SCD diagnosis entails microscopic examination of the blood smear, combined with detection of HbS protein by electrophoresis, isoelectric focusing, or HPLC, and/or polymerase chain reaction detection of the HbS β-globin mutation. But these are not realistic diagnostic options for most African SCD patients residing in regions without access to trained health care providers and clinical laboratories. Induction of sickling typically is accomplished by deoxygenation. However, normoxic sickling of SCD red cells can be triggered by hyperosmotic solution or by acid pH. In this issue of The Journal of Physiology, Milligan and colleagues in the laboratory of John Gibson exploit this property to advantage (Milligan et al. 2013). Building upon their previous demonstration that deoxygenation in isosmotic, low-ionic strength sucrose solution leads to reproducible, incomplete haemolysis of sickle but not normal red cells (Browning et al. 2007a), Milligan et al. now present experimental conditions reproducing this haemolysis in room air. At neutral pH and 37°C, 40–60 min exposure to isosmotic sucrose in deoxygenated conditions lyses ∼15% of HbSS blood cells and ∼10% HbSC cells, allowing discrimination of both pathological genotypes from red cells of asymptomatic genotypes HbAS and HbAA. This haemolysis is blocked by non-specific inhibitors of HbS polymerization such as o-vanillin, hydroxymethylfurfural and urea, without altering Hb oxygen saturation. Deoxygenation-induced haemolysis is also blocked by actin cytoskeleton disruptor, cytochalasin B. Haemolysis is reduced in blood taken from SCD patients after 5 months of treatment with hydroxyurea. Most importantly, from the perspective of broad diagnostic utility, acidification of the isosmotic sucrose solution to pH 6.0 is permissive for normoxic haemolysis with unchanged inhibitor sensitivity. Liberated from the need for a deoxygenation step, a promising version of a point-of-care diagnostic test can now be envisioned. Although Milligan et al. show that sickle red cell haemolysis under these conditions does not occur at room temperature, ambient temperature in the field may in many endemic areas suffice in many areas to allow the requisite diagnostic discrimination. The molecular identity of the sickle red cell non-ionic solute pathway studied by Milligan et al. remains unknown. However, the non-ionic solute pathway shares with Psickle its sensitivity to block by non-specific inhibitors of HbS polymerization, its activation by acidic pH, and its sensitivity in some settings to cytochalasins. Thus, the work of Milligan and colleagues on an unusual property of the sickle red cell membrane may provide novel approaches to identification of Psickle and inhibitors of sickle cell dehydration, while also suggesting a novel path towards a point-of-care test for sickle disease suitable for the currently underserved majority of SCD patients worldwide.