Newborn screening for abnormal haemoglobins in Jamaica: Practical issues in an island programme.

OBJECTIVE: To report the diagnostic challenges of newborn screening for abnormal haemoglobins. SETTING: Cord blood samples from 13 hospitals in southwest Jamaica taken in 2008-2019. METHODS: Blood spots, collected from the umbilical cord, were analysed by high pressure liquid chromatography (HPLC) to reveal phenotypes for HbSS and HbCC, but genotype confirmation may require parental studies or gene sequencing. Such cases that were successfully traced were analysed in this follow-up study. RESULTS: HPLC screening of 121,306 samples detected HbAS in 11,846 (9.8%), HbAC in 4508 (3.7%) and other electrophoretic abnormalities in 1090 babies. Among 101 previously unconfirmed cases, 34/90 (38%) with HPLC evidence of a HbSS phenotype had other genotypes, and 7/11 (64%) with a HbCC phenotype had other genotypes. Syndromes from the interaction of ß thalassaemia occurred in 112 babies (85 with HbS, 27 with HbC) and of genes for hereditary persistence of fetal haemoglobin (HPFH) in 18 (12 with HbS, 6 with HbC). Variants other than HbS and HbC occurred in 270 babies, 16 in combination with either HbS or HbC, and 254 as traits. Most variants are benign even when inherited with HbS, although HbO Arab, HbD Punjab, or Hb Lepore Washington, which occurred in 6 cases, may cause sickle cell disease. CONCLUSIONS: Genes for ß thalassaemia and HPFH are common in western Jamaica and when associated with HbS may present diagnostic challenges in newborns, as HbF and HbA2 have not reached diagnostic levels. Family and DNA studies may be necessary for genotype confirmation.

Recent Advances in the Treatment of Sickle Cell Disease.

Sickle cell anemia (SCA) was first described in the Western literature more than 100 years ago. Elucidation of its molecular basis prompted numerous biochemical and genetic studies that have contributed to a better understanding of its pathophysiology. Unfortunately, the translation of such knowledge into developing treatments has been disproportionately slow and elusive. In the last 10 years, discovery of BCL11A, a major γ-globin gene repressor, has led to a better understanding of the switch from fetal to adult hemoglobin and a resurgence of efforts on exploring pharmacological and genetic/genomic approaches for reactivating fetal hemoglobin as possible therapeutic options. Alongside therapeutic reactivation of fetal hemoglobin, further understanding of stem cell transplantation and mixed chimerism as well as gene editing, and genomics have yielded very encouraging outcomes. Other advances have contributed to the FDA approval of three new medications in 2017 and 2019 for management of sickle cell disease, with several other drugs currently under development. In this review, we will focus on the most important advances in the last decade.

Interactions of an Anti-Sickling Drug with Hemoglobin in Red Blood Cells from a Patient with Sickle Cell Anemia.

The pathophysiology associated with sickle cell disease (SCD) includes hemolytic anemia, vaso-occlusive events, and ultimately end organ damage set off by the polymerization of deoxygenated hemoglobin S (HbS) into long fibers and sickling of red blood cells (RBCs). One approach toward mitigating HbS polymerization is to pharmacologically stabilize the oxygenated (R) conformation of HbS and thereby reduce sickling frequency and SCD pathology. GBT440 is an α-subunit-specific modifying agent that has recently been reported to increase HbS oxygen binding affinity and consequently delay in vitro polymerization. In addition, animal model studies have demonstrated the potential for GBT440 to be a suitable therapeutic for daily oral dosing in humans. Here, we report an optimized method for detecting GBT440 intermediates in human patient hemolysate using a combination of HPLC and mass spectrometry analysis. First, oxygen dissociation curves (ODCs) analyzed from patient blood showed that oxygen affinity increased in a dose dependent manner. Second, HPLC and integrated mass spectrometric analysis collectively confirmed that GBT440 labeling was specific to the α N-terminus thereby ruling out other potential ligand binding sites. Finally, the results from this optimized analytical approach allowed us to detect a stable α-specific GBT440 adduct in the patient's hemolysate in a dose dependent manner. The results and methods presented in this report could therefore potentially help therapeutic monitoring of GBT440 induced oxygen affinity and reveal critical insight into the biophysical properties of GBT440 Hb complexes.

Interim assessment of liver damage in patients with sickle cell disease using new non-invasive techniques.

We explored transient elastography (TE) and enhanced liver fibrosis (ELF™ ) score with standard markers of liver function to assess liver damage in 193 well patients with sickle cell disease (SCD). Patients with HbSS or HbSß0 thalassaemia (sickle cell anaemia, SCA; N = 134), had significantly higher TE results and ELF scores than those with HbSC (N = 49) disease (TE, 6·8 vs. 5·3, P < 0·0001 and ELF, 9·2 vs. 8·6 P < 0·0001). In SCA patients, TE and ELF correlated significantly with age and all serum liver function tests (LFTs). Additionally, (weak) positive correlation was found with lactate dehydrogenase (TE: r = 0·24, P = 0·004; ELF: r = 0·26 P = 0·002), and (weak) negative correlation with haemoglobin (TE: r = -0·25, P = 0·002; ELF: r = -0·25 P = 0·004). In HbSC patients, correlations were weaker or not significant between TE or ELF, and serum LFTs. All markers of iron loading correlated with TE values when corrected for sickle genotype (serum ferritin, ß = 0·25, P < 0·0001, total blood transfusion units, ß = 0·25, P < 0·0001 and LIC ß = 0·32, P = 0·046). The exploratory study suggests that, while TE could have a role, the utility of ELF score in monitoring liver damage in SCD, needs further longitudinal studies.

Tissue Iron Distribution Assessed by MRI in Patients with Iron Loading Anemias.

Bone marrow, spleen, liver and kidney proton transverse relaxation rates (R2), together with cardiac R2* from patients with sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNH) and non-transfusion dependent thalassemia (NTDT) have been compared with a control group. Increased liver and bone marrow R2 values for the three groups of patients in comparison with the controls have been found. SCD and PNH patients also present an increased spleen R2 in comparison with the controls. The simultaneous measurement of R2 values for several tissue types by magnetic resonance imaging (MRI) has allowed the identification of iron distribution patterns in diseases associated with iron imbalance. Preferential liver iron loading is found in the highly transfused SCD patients, while the low transfused ones present a preferential iron loading of the spleen. Similar to the highly transfused SCD group, PNH patients preferentially accumulate iron in the liver. A reduced spleen iron accumulation in comparison with the liver and bone marrow loading has been found in NTDT patients, presumably related to the differential increased intestinal iron absorption. The correlation between serum ferritin and tissue R2 is moderate to good for the liver, spleen and bone marrow in SCD and PNH patients. However, serum ferritin does not correlate with NTDT liver R2, spleen R2 or heart R2*. As opposed to serum ferritin measurements, tissue R2 values are a more direct measurement of each tissue's iron loading. This kind of determination will allow a better understanding of the different patterns of tissue iron biodistribution in diseases predisposed to tissue iron accumulation.

How we treat delayed haemolytic transfusion reactions in patients with sickle cell disease.

Transfusion therapy is effective in the prevention and treatment of many complications of sickle cell disease (SCD). However, its benefits must be balanced against its risks, including delayed haemolytic transfusion reactions (DHTR). Not only is the relative rate of alloimmunization higher in patients with SCD than in other patient populations, but attendant risks associated with DHTR are even greater in SCD. Clinicians' awareness of DHTR events is poor because symptoms of DHTR mimic acute vaso-occlusive pain and immunohaematology findings are often negative. Transfusions delivered in the acute rather than elective setting appear to confer a higher risk of DHTR. Management of DHTR in SCD depends on the clinical severity, ranging from supportive care to immunosuppression, and optimization of erythropoiesis. DHTR must be considered in any recently transfused patient presenting with acute sickle cell pain. Meticulous documentation of transfusion and immunohaematology history is key. We anticipate an increase in DHTR events in SCD patients with the increasing use of red blood cell transfusion therapy.

Delayed haemolytic transfusion reaction in adults with sickle cell disease: a 5-year experience.

Delayed haemolytic transfusion reactions (DHTR) are potentially life-threatening complications in patients with sickle cell disease (SCD). Between 1 August 2008 and 31 December 2013, 220 of 637 adult patients in our centre had at least one red blood cell (RBC) transfusion in 2158 separate transfusion episodes. Twenty-three DHTR events occurred in 17 patients (13 female) including 15 HbSS, one HbSC and one HbSß(0) thalassaemia, equating to a DHTR rate of 7·7% of patients transfused. Mean interval from RBC transfusion to DHTR event was 10·1 ± 5·4 d, and typical presenting features were fever, pain and haemoglobinuria. Twenty of the 23 (87·0%) DHTR episodes occurred following transfusion in the acute setting. Notably, 11/23 (47·8%) of DHTRs were not diagnosed at the time of the event, most were misdiagnosed as a vaso-occlusive crisis. 16/23 DHTRs had 'relative reticulocytopenia', which was more common in older patients. Seven of 23 episodes resulted in alloantibody formation, and three caused autoantibody formation. DHTRs are a severe but uncommon complication of RBC transfusion in SCD and remain poorly recognized, possibly because they mimic an acute painful crisis. Most of the DHTRs are triggered by RBC transfusion in the acute setting when patients are in an inflammatory state.

A randomized phase I/II trial of HQK-1001, an oral fetal globin gene inducer, in ß-thalassaemia intermedia and HbE/ß-thalassaemia.

ß-thalassaemia intermedia (BTI) syndromes cause haemolytic anaemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anaemia. Sodium 2,2 dimethylbutyrate (HQK-1001), an orally bioavailable short-chain fatty acid derivative, induces γ-globin expression experimentally and is well-tolerated in normal subjects. Accordingly, a randomized, blinded, placebo-controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/ß(0) thalassaemia and seven with ß(+)/ß(0) thalassaemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK-1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg per day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t(1/2) of 10-12 h. Adverse events with HQK-1001 treatment were not significantly different from placebo treatment. The 20 mg/kg treatment doses increased median HbF above baseline levels by 6·6% and 4·4 g/l (P < 0·01) in 8/9 subjects; total haemoglobin (Hb) increased by a mean of 11 g/l in 4/9 subjects. These findings identified a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK-1001 with longer dosing to definitively evaluate its haematological potential appears warranted.

Renal iron load in sickle cell disease is influenced by severity of haemolysis.

Spin density projection-assisted R2-magnetic resonance imaging (R2-MRI; FerriScan(®)) scans from 40 chelation-naïve sickle cell patients were used to assess renal iron load by measuring renal R2 (R-R2). Clinical data were collected retrospectively for the 2-year period preceding the scan. R-R2 showed no significant correlation with transfusional iron load (assessed by liver iron concentration), but correlated significantly with serum bilirubin (R = 0·61, P < 0·0001) and lactate dehydrogenase (R = 0·58, P < 0·0001). Mean (±standard deviation) R-R2 was higher (P = 0·02) in patients with renal hyperfiltration (29·8 ± 10·3/s) than those without (23·11 ± 6·6/s). Five patients had significantly lower signal intensity in the renal cortex, as compared to the medulla. These patients had a significantly higher (P < 0·0001) mean R-R2 than those showing no cortico-medullary difference. We postulate that the increased R-R2 is associated with haemolysis rather than transfusional iron load in sickle cell disease.

Sickle cell nephropathy - a practical approach.

Despite its apparently simple molecular aetiology, sickle cell disease (SCD) has long been known to have a remarkably variable clinical course, with complications involving many organs including the kidneys. Whilst many affected individuals show no evidence of renal involvement into late adulthood, others develop renal dysfunction in childhood or early adult life with a significant proportion eventually requiring renal replacement therapy. This review explores the pathophysiology and clinical manifestations of sickle cell nephropathy (SCN) and discusses how each complication can be investigated, monitored and managed in the outpatient setting. We summarize current knowledge of genetic modulation of sickle-related renal dysfunction. We outline the evidence for various treatment options and discuss others for which little evidence currently exists.

A twins heritability study on alpha hemoglobin stabilizing protein (AHSP) expression variability.

Cytotoxic precipitation of free α-globin monomers and its production of reactive oxygen species cause red cell membrane damage that leads to anemia and eventually ineffective erythropoiesis in ß-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) was found to bind only to free α-globin monomers creating a stable and inert complex which remains soluble in the cytoplasm thus preventing harmful precipitations. Alpha hemoglobin stabilizing protein was shown to bind nascent α-globin monomers with transient strength before transferring α-globin to ß-globin to form hemoglobin tetramer. A classical twin study would be beneficial to investigate the role of genetics and environment in the variation of alpha hemoglobin stabilizing protein expression as this knowledge will enable us to determine further investigations with regards to therapeutic interventions if alpha hemoglobin stabilizing protein is to be a therapeutic agent for ß-thalassemia. This study investigates the heritability influence of alpha hemoglobin stabilizing protein expression and factors that may contribute to this. Results indicated that a major proportion of alpha hemoglobin stabilizing protein expression was influenced by genetic heritability (46%) with cis-acting factors accounting for 19% and trans-acting factors at 27%.

Outcome of children with sickle cell disease admitted to intensive care - a single institution experience.

We retrospectively audited children with sickle cell disease (SCD) admitted to paediatric intensive care (PICU) at King's College Hospital between January 2000 and December 2008. Forty-six children with SCD were admitted, on 49 separate occasions. Ages ranged from 4 months to 15 years (median 7.6 years). Three children died in PICU, however two presented to hospital in cardiorespiratory arrest; overall mortality was 6%. The most common reason for admission was acute chest syndrome (43%). 88% of admissions required blood transfusion, of which 74% had exchange blood transfusions. The mortality among children with SCD admitted to PICU is low.

Outcome of adults with sickle cell disease admitted to critical care - experience of a single institution in the UK.

Sickle cell disease (SCD) patients are perceived to have a high mortality when admitted to the Critical Care Unit (CCU). We performed a retrospective analysis of all adult sickle admissions to CCU at a single centre over an 8-year period (1 January 2000 to 31 December 2007). Thirty-eight patients (14 male) were admitted 46 times to CCU; the commonest reasons for admission were acute chest syndrome (14, 30%), multi-organ failure (8, 17%) and planned post-elective surgery (7, 15%). CCU mortality for SCD patients was 19.6%, comparable to a CCU-wide mortality of 17.6% during the study period in the same institution. Re-admission to CCU was high (16% over the 8-year period) but did not increase mortality risk.

The associations between air quality and the number of hospital admissions for acute pain and sickle-cell disease in an urban environment.

The clinical severity of sickle-cell disease (SCD) is dependent on genetic and environmental variables. Environmental factors have been poorly studied. We have investigated possible links between air pollution and acute pain in SCD. We retrospectively studied the numbers of daily admissions with acute sickle-cell pain to King's College Hospital, London, in relation to local daily air quality measurements. We analysed 1047 admissions over 1400 d (1st January 1998-31st October 2001). Time series analysis was performed using the cross-correlation function (CCF). CCF showed a significant association between increased numbers of admissions and low levels of nitric oxide (NO), low levels of carbon monoxide (CO) and high levels of ozone (O(3)). There was no association with sulphur dioxide (SO(2)), nitrogen dioxide or PM(10) (dust). The significant results were further examined using quartile analysis. This confirmed that high levels of O(3) and low levels of CO were associated with increased numbers of hospital admissions. Low NO levels were also associated with increased admissions but did not reach statistical significance on quartile analysis. Our study suggests air quality has a significant effect on acute pain in SCD and that patients should be counselled accordingly. The potential beneficial effect of CO and NO is intriguing and requires further investigation.

Alpha-haemoglobin stabilising protein is a quantitative trait gene that modifies the phenotype of beta-thalassaemia.

It has been suggested that altered levels or function of alpha-haemoglobin stabilising protein (AHSP), an erythroid-specific protein that binds specifically to free alpha-(haemo)globin, might account for some of the clinical variability in beta-thalassaemia. To assess the variation of AHSP expression, mRNA levels in circulating reticulocytes of 103 healthy individuals were measured by quantitative reverse transcription-polymerase chain reaction. AHSP expression varied up to threefold, and did not correlate with age or sex. A systematic survey of the AHSP locus identified eight sequence variants, of which six were common. Four common variants, including the longer homopolymer (T18) in the putative promoter, are strongly associated with AHSP expression. Reporter assays in K562 cells showed that the activity of the shorter (T15) reporter was relatively lower than that of the T18 reporter. In a study of nine anaemic patients who were heterozygous for beta-thalassaemia and also heterozygous for the triplicated alpha-globin gene (alpha alpha alpha/alpha alpha), frequency of the shorter homopolymer was higher than expected. AHSP expression is variable, with cis control accounting for some of its variance. In some families, the subtle altered levels in AHSP related to the AHSP genotype appears to be a relevant contributory factor in the haematological phenotype.