Sickling Test Research Articles

Potential pitfalls in the diagnosis of Hb Handsworth in areas with high prevalence of HbS

Hb Handsworth is a rare α-globin structural variant caused by a missense mutation either on the α2 or α1-globin gene (HBA2 or HBA1: c.55G>C, p.Gly18Arg). This variant might be erroneously diagnosed as HbS unless secondary confirmative tests are carried out. We encountered a child with a prominent peak eluting in the 'S' window on high-performance liquid chromatography (HPLC). Sickle solubility test, gel electrophoresis, and selective direct nucleotide sequencing of α1, α2, and β globin genes were performed on the patient's sample. In addition, previous HPLC results on a cord blood sample were retrieved. Sickle solubility test was negative. Gel electrophoresis revealed a band migrating at the S region with an extra faint band seen on acid gel electrophoresis. Molecular analysis of α2 globin gene revealed heterozygous state of Hb Handsworth. Hb Handsworth is a rare variant that can mimic HbS on HPLC. Failure to recognize this rare variant in regions where HbS is highly prevalent may result in serious misdiagnosis and subsequent incorrect genetic counseling.

Clinical and molecular characterization of Hb Hofu in eastern India

Hb Hofu (HBB:c. 380T>A) is a rare inherited hemoglobin abnormality with few case reports in the world literature. Screening for the sickle cell gene mutation and other hemoglobinopathies was carried out using the sickle slide test, Hb electrophoresis, and HPLC under an ongoing central government project. We detected twelve Hb Hofu heterozygotes and three sickle Hb Hofu compound heterozygotes. The heterozygotes were asymptomatic except for one individual who had chronic kidney disease and moderate anemia. Only one HbS-Hofu case was symptomatic and presented with intermittent attacks of painful crisis. In the carrier state, the Hb Hofu eluted as a hump at the beginning of the HbA(0) window. But in HbS-Hofu cases, Hb Hofu eluted as a single peak in the HbA(0) window, with the HbA(2) levels being >4% consistently. HbS-Hofu has a variable clinical presentation. The retention time of Hb Hofu on HPLC is very close to that of HbA(0) and often elutes in the A0 window. Thus, there is every possibility of the HbS-Hofu chromatogram to be misinterpreted as that of a sickle cell trait/transfused sickle cell-beta-thalassemia case. This is the first time where Hb Hofu has been detected by HPLC, which is the widely accepted screening technique for hemoglobinopathies around the world.

α-chain hemoglobin variants with electrophoretic mobility similar to that of hemoglobin S in newborn screening programs

Hemoglobin (Hb) S is a β-chain structural variant of human hemoglobin that undergoes polymerization in its deoxygenated form, making red blood cells stiff and undeformable and causing hemolysis and vaso-occlusion, with tissue damage and pain. Heterozygotes (Hb AS) are generally asymptomatic, while homozygotes (Hb SS) have sickle cell anemia (SCA). Combinations of Hb S and other hemoglobin variants, such as Hb C and D, or β-thalassemia can produce clinical pictures similar to that of SCA and are known as sickle cell diseases (SCD)(1). Around 1 to 2% of the world's population are Hb S heterozygotes, but in some regions of Africa that are endemic for malaria (against which Hb AS individuals are protected) the frequency of the βS gene can be greater than 40%(2). In Brazil, the Southeast and Northeast regions have the greatest prevalence (around 8% of individuals of African descent are heterozygotes), although in some populations, such as that in Salvador, Bahia, the prevalence can be higher than 10%(3,4). Correct diagnosis of this hemoglobinopathy is extremely important, particularly during the neonatal period, as early detection of SCD is fundamental if morbidity and mortality of these diseases are to be reduced. Laboratory diagnosis of SCD generally involves techniques based on the electric charge of the variants, such as cellulose acetate or gel electrophoresis, isoelectric focusing, high-performance liquid chromatography (HPLC) and capillary electrophoresis(5). However, as more than 1,000 variants have been described to date(6), the probability of finding another variant with electrophoretic behavior similar to that of Hb S (Hb S-like hemoglobins) is quite high, particularly in a population with a high degree of miscegenation, like that of Brazil. For this reason it is important to use confirmatory tests (which are also frequently used in screening programs), such as the sickling test and the hemoglobin solubility test (based on the insolubility of deoxy-HbS in high molarity phosphate buffer)(5). In the case of Hb S in association with other variants, whether Hb S-like or not, correct identification of the second variant is fundamental to distinguish between those variants that will lead to SCD, those that evolve without any symptoms and those that produce specific symptoms(7). Molecular techniques, such as restriction enzyme analysis and globin gene sequencing, are the most commonly used techniques for known mutations and new or rare mutations, respectively(8,9). It should also be mentioned that some Hb S-like variants can cause red blood cell sickling, hemolysis and vaso-occlusion even in a heterozygous state. Examples include Hb Jamaica Plain(10) and Hb S Sao Paulo(11), the latter recently described in the Brazilian population. It should also be stressed that Hb S in association with an Hb S-like hemoglobin can lead to an incorrect diagnosis of SCA if the appropriate confirmatory tests are not carried out(12).

Plasma Membrane-Derived Vesicles in Sickle Cell Disease: A Possible Indicator of the Continuous Endothelial Stimulation and/or Injury to Blood Cells

Plasma membrane-derived vesicles (PMVs) are released into circulation in response to normal and stress/pathogenic conditions. They are of tremendous significance for the prediction, diagnosis, and observation of the therapeutic success of many diseases. Knowledge of their functional properties would contribute to a better understanding of the pathological mechanisms leading to various diseases in which their levels are raised. The objective of this study was to quantify and compare the levels of PMVs in sickle cell disease patients (Hb SS and Hb SC) with non-sickle cell (Hb AA) subjects. The comparison will help us research and understand the processes that lead to their constitutive release in sickle cell disease patients than in their normal counterparts. A total of one hundred and fifty (150) sickle cell disease patients (study group) and blood donors (control group) that consented to partake in the study were recruited. There were 82 males and 68 females. Fifty (50) each of Hb SS, Hb SC and Hb AA samples were obtained. Sodium metabisulphite (sickling) test, Haemoglobin electrophoresis and quantification of PMVs were carried out on all the samples. The sickle cell disease patients had elevated levels; SS (38.89 ± 0.73 × 104/ml; p= 0.01) and SC (32.62 ± 1.18 × 104/ml; p = 0.01) as against the control subjects (Hb AA) who had average PMVs of 11.28 ± 0.29 × 104/ml PFP (mean ± SEM). It was concluded that both SS and SC (study) samples showed an increased count of PMVs as compared to the AA (control) samples, suggesting persistent endothelial stimulation and/or injury of blood cells leading to continuous shedding of PMVs in sickle cell disease patients.

Hb Haaglanden: a new nonsickling β7Glu>Val variant. Consequences for basic diagnostics, screening, and risk assessment when dealing with HbS‐like variants

To report a new hemoglobin variant undistinguishable from the common HbS on HPLC. To show the efficiency of the simplest confirmation method for HbS and to discuss the implications that may occur if HbS-like variants are wrongly reported as HbS. Basic hematology, separation and measurement of the Hb fractions, 'sickle test,' and molecular analysis. The abnormal Hb fractions were eluting in the HbS window on HPLC, sickle test was however negative, and DNA sequencing of the beta globin gene revealed an unclassified variant HBBc.23A>T, p.Glu8Val in heterozygous form. Although the amino acid substitution of this new variant is identical to that of HbS and shifted of a single amino acid position, no polymerization occurs in vitro. The sickle test is a valid method to confirm or exclude HbS trait in individual cases. Whenever the case is part of a possible couple at risk, then one has to use full DNA analysis in both partners not to miss hidden concomitant defects important for genetic risk predictions.

Continuing Diagnostic Relevance of the Sickling Test in the Era of CE-HPLC

Dear Editor, Cation-exchange high performance liquid chromatography (CE-HPLC) has become the most common method used to screen for hemoglobin variants. However, identification is only provisional and current guidelines emphasize that all chromatographically detected abnormal hemoglobins must subsequently be verified by an independent second technique [1]. Sickle cell hemoglobin (HbS)-related disorders are common in India [2]. CE-HPLC is highly specific for HbS in the Indian setting since the S-window co-eluting variants (retention time 4.3–4.7 min) like HbQ-Thailand, HbG-San Jose, and Hb-Manitoba are few and moreover, are extremely rare in Indians [2–4]. We recently encountered two related patients with a non-sickling hemoglobin eluting in the S-window where a routine time-tested sickling test with sodium metabisulfite–sodium dithionite prevented a misdiagnosis. A Sudanese mother and daughter, who were in India on a business visit, underwent a routine health check-up that revealed very mild hypochromic anemia (Table 1). Their serum iron parameters were within normal limits. CE-HPLC (BioRad variant II system, β-thal short programme) revealed peaks in the HbS-window in both mother (32.9%) and daughter (32.7%) with an identical retention time of 4.61 min (Fig. 1). Although, a diagnosis of sickle cell trait was entirely consistent with their African ancestry, sickling tests with sodium metabisulfite–sodium dithionite were done as is routine in our laboratory for all HbS-window peaks. To our surprise, no sickling occurred at 24 h in either patient. Repeat testing using freshly constituted reagents was also consistently negative. Table 1 Blood count and CE-HPLC parameters in the two patients Fig. 1 CE-HPLC of the mother showing an abnormal hemoglobin peak with a retention time of 4.61 min in the S-window. HbA and HbA2 were also present. The chromatogram of the daughter was very similar Since the second-line modality had failed to confirm HbS, Hb electrophoreses at pH 8.4 was done (SAS-II, Helena BioSciences, Europe). This showed a prominent band for HbA along with a slow-moving band corresponding to the region of Hb S/D/G/Q (Fig. 2). The differential diagnoses at this stage therefore were HbG-San Jose (RT 4.61) and HbQ-Thailand (RT 4.67). Hb-Manitoba was excluded as it migrates just anodal to HbA at alkaline pH [5–7]. Fig. 2 High resolution image of the Hb electrophoresis at alkaline pH. The patients’ samples are in lanes 1 and 2. The other lanes (diagnoses indicated) as well as the adult and fetal controls (lanes 11 and 12) serve as references. The abnormal Hb of ... To differentiate Q-Thailand from G-San Jose, Hb electrophoresis at pH 6.4 was performed. This revealed a band for HbA and another band that merged with it but was faster than HbC and HbS (Fig. 3). This definitively excluded HbS and HbG-San Jose (as G-San Jose migrates between HbC and HbS at acid pH [8]), thereby leading to a presumptive identification of the variant as HbQ-Thailand [9]. Genetic testing was offered, however, the patients declined on being counseled that the variant was unlikely to be of clinical significance to the probands. Fig. 3 High resolution image of the Hb electrophoresis at acid pH. The patients’ samples are in lanes 1 and 2. The other lanes (diagnoses indicated) as well as the adult and fetal controls (lanes 11 and 12) serve as references. The index cases show bands ... HbQ-Thailand occurs due to the mutation α74 (EF3) Asp > His in the α1-globin gene. Its co-inheritance with α2-globin gene deletion is well recognized to yield a high abnormal variant of 30–35% with thalassemic red cell indices as in our cases [9]. In conclusion, our finding of this unusual variant hemoglobin makes a strong argument in favor of the routine confirmation of all S-window CE-HPLC peaks by the simple, cost-effective and rapid test for sickling to avoid misdiagnoses. In addition, it is a reminder to hematopathologists that in this era of medical tourism, one needs to consider rare Hb variants in their differential diagnoses irrespective of ethnic boundaries.

Mouse models for studying pain in sickle disease: effects of strain, age, and acuteness

The clinical management of severe pain associated with sickle cell disease (SCD) remains challenging. Development of an optimal therapy would be facilitated by use of murine model(s) with varying degrees of sickling and pain tests that are most sensitive to vaso-occlusion. We found that young (≤3 months old) NY1DD and S+S(Antilles) mice (having modest and moderate sickle phenotype, respectively) exhibited evidence of deep tissue/musculoskeletal pain. Deep tissue pain and cold sensitivity in S+S(Antilles) mice increased significantly with both age and incitement of hypoxia/reoxygenation (H/R). C57/BL6 mice (genetic background strain of NY1DD and S+S(Antilles) ) were hypersensitive to mechanical and heat stimuli, even without the sickle transgene. H/R treatment of HbSS-BERK mice with severe sickle phenotype resulted in significantly decreased withdrawal thresholds and enhanced mechanical, thermal and deep tissue hyperalgesia. Deep hyperalgesia incited by H/R in HbSS-BERK was ameliorated by CP 55940, a cannabinoid receptor agonist. Thus, assessment of deep tissue pain appears to be the most sensitive measure for studying pain mechanisms across mouse models of SCD, and HbSS-BERK mice may be the best model for vaso-occlusive and chronic pain of SCD.

Impact of sickle cell trait on physical growth in tribal children of Mandla district in Madhya Pradesh, India

Background: Reliable reports on growth impairment in sickle cell trait (SCT) children in India are lacking despite contradictory findings reported earlier.Aim: The present study assessed the impact of SCT on physical growth of tribal children of Mandla district.Subjects and methods: Weight, height, circumferences, breadths, lengths and skinfolds were recorded on 6190 children, inclusive of 732 SCT children, from birth to 12 years of age using a cross-sectional design. The sickle test was conducted in the field using 2% sodium metabisulphite followed by electrophoresis.Results: No significant difference in mean values was observed in the majority of the age groups between SCT and normal children for all 11 body measurements. However, inconsistent growth patterns in these measurements among SCT children were evident. Body weight was more deficient than height or other body measurements in the children when compared to Indian and National Centre for Health Statistics (NCHS) standards, while bicristal breadth was comparable with Indian standards.Conclusions: There was no significant impact of SCT observed on growth of children irrespective of sex. Notably, growth of SCT girls was comparable to their normal counterparts. The actual growth difference between normal and SCT children may have been masked on account of poor attainment of annual gain in each successive age group.

Idiopathic pulmonary hemosiderosis

Idiopathic pulmonary hemosiderosis (IPH) is a rare disorder (triad of iron-deficiency anemia, hemoptysis, and alveolar infiltrates). A 3-year-old male presented with mild fever, breathlessness, dry cough, and bluish nail discoloration for 8 days. He had required five blood transfusions in the past 1 year (last transfusion was given 4 months ago). He had a respiratory rate of 58/min with respiratory distress, cyanosis, and grade III clubbing. Respiratory system examination was normal. Several previous reports of hemoglobin were as low as 3.6 g/dl with hypochromic and microcytic anemia. There were transient increases in the hemoglobin and normalization of red cell morphology with blood transfusions. Serum iron, G6PD enzyme assay, hemoglobin electrophoresis, the sickling test, Coomb's test, stool and urine analysis, and a Meckel's scan were normal. HIV antibody and dsDNA were negative. The chest radiograph revealed symmetrical patchy infiltrates sparing lung apices (confirmed on high-resolution computed tomography). Lung biopsy diagnosed pulmonary hemosiderosis (interstitial lung disease with hemosiderin-laden macrophages scattered in the alveoli and areas of fibrosis in the alveolar septa). The patient showed marked clinical improvement in 10 days of therapy with prednisolone. IPH should be listed in the differential diagnosis of a child presenting with unexplained hypochromic, microcytic anemia and respiratory symptoms.

Significant haemoglobinopathies: guidelines for screening and diagnosis

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Newborn screening and, when necessary, follow up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. All babies under 1 year of age arriving in the UK should be offered screening for sickle cell disease. Preoperative screening for sickle cell disease should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with sickle solubility tests must always be followed by definitive analysis. Laboratories performing antenatal screening should utilize methods capable of detecting significant variants and be capable of quantitating haemoglobins A 2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available within three working days from sample receipt.

The Reliability of Sickling and Solubility Tests and Peripheral Blood Film Method for Sickle Cell Disease Screening at District Health Centers in Uganda

Although sickling and solubility tests and peripheral blood film methods are today available for sickle cell disease screening in Uganda, their reliability and ease of applicability have not been determined. This study was therefore carried out to determine the reliability of sickling and solubility tests and peripheral blood film method for screening for SCD in Uganda. This was descriptive laboratory based study which was carried out at Makerere University College of Health Sciences. The 200 samples from children aged between 6 months and five years were independently analyzed using sickling and solubility tests and peripheral blood film method. Hemoglobin electrophoresis cellulose acetate was used as the gold standard. Sickling and solubility tests had sensitivities of 65.0% and 45.0%, respectively and peripheral film had 35.0%. Sickling, solubility and peripheral film had specificities of 95.6%, 90.0% and 96.7%, respectively. Sickling had diagnostic accuracy of 92.5%, solubility (85.5%) and peripheral film (90.5%). Sickling had a Cohen’s Kappa of 0.6, solubility 0.3 and peripheral film 0.4. Sickling test had turn around time (TAT) of 38 minutes, solubility 70 minutes and peripheral 44 minutes. In conclusion, the sickling test was more reliable and easier to perform than solubility test and peripheral blood film method. It would therefore be a recommended test for preliminary screening of children for SCD at district health centers IV and confirming only positives using hemoglobin electrophoresis.

Diamond Blackfan Syndrome

We report a case of Diamond Blackfan syndrome in 6yr old girl who was detected to have severe anaemia on D4 of life. The baby was detected to have polydactyly right hand (preaxial) and weak radial pulse on right side. On examination there was severe pallor without hepatosplenomegaly. The investigations revealed haemoglobin of 1.9 gm% with reticulocyte count of 0.3%. Other investigations were done to establish the cause of anaemia. The sickling test was negative, Peripheral blood smearrevealed macrocytic anaemia, Hb electrophoresis revealed fetal haemoglobin of 2.7 %. Bone marrow examination revealed markedly reduced erythroid series, stress cytogenetics study done later was negative for any chromosomal breakage. Based on the clinical profile and investigation reports the diagnosis of Diamond Blackfan Syndrome was made. The child was put on corticosteroids which were gradually tapered. Subsequently any attempt at withdrawl of steroids resulted in fall inhaemoglobin levels. Hence the child has been maintained on low dose steroids and has remained symptom free.

Solubility tests and the peripheral blood film method for screening for sickle-cell disease: a cost benefit analysis

Objective . To determine the cost benefit of screening for sicklecell disease among infants at district health centres in Uganda using sickling, solubility tests and the peripheral blood film method. Methods . Pilot screening services were established at district health centres. Cost benefit analysis (CBA) was performed in four scenarios: A1 – where there are no sickle-cell screening services at district health centres and all children are referred either to Mulago national referral hospital or A2 – a regional hospital for haemoglobin (Hb) electrophoresis; B1 – when there are screening services at district health centres, only positive samples are taken either to Mulago Hospital or B2 – the regional hospital for confirmation using haemoglobin electrophoresis. Calculations were done in Uganda shillings (USh). Results . Initial operational costs were high for all scenarios but variably reduced in the subsequent years. Scenarios A1 and A2 were very sensitive compared with B1 and B2. Scenario A1 had the highest screening costs in the subsequent years, costing over 62 000 USh per test in both eastern and western Uganda. Scenario B2 was sensitive and cheaper when using the sickling test, but was expensive and insensitive when using the solubility test and more insensitive though cheaper when using the peripheral blood film method. Conclusions and recommendatio n. Screening children in Mulago hospital using haemoglobin electrophoresis (A1) was very expensive although it was sensitive. Screening the children at four health centres using the sickling method and confirming positive samples at a regional hospital (B2) was both cheap and sensitive and is therefore recommended

Distribution of haemoglobin genotypes and anaemic status of HIV positive pregnant women in Enugu: a hospital based study

This present 4 – year comparative study investigated the distribution pattern of Haemoglobin genotypes and anaemic status of HIV positive pregnant women in Enugu. Haemoglobin variants(HbAA, HbAS, HbSS) in pregnant women attending antenatal clinics at the study site were determined by electrophoreisis. HIV screening tests were done using the Quick test kits. Packed Cell Volume (PCV) was estimated by using standard procedures. Results showed a prevalence rate of 83.3% was obtained for HbAA HIV+ pregnant women as against a 70.1% for HIV- pregnant women; HbAS rates were 16.7% for HIV+ pregnant women and 29.9% for HIV- pregnant women. For the non- pregnant women (control), the rates were 78.63% for HbAA and 21.4% for HbAS. No HbSS genotype was observed in the HIV+ pregnant women and HIV- pregnant women and the control respectively. The HIV seroprevalence rate of 3.67 +- 0.40%(SEM) was obtained for this study. The PCVs of the HIV+ pregnant women with HbAA and HbAS (1999 – 2002) fell into levels indicative of anaemia when compared with those of HIV- pregnant women and the control. Increased awareness, compulsory pre-marital sickling and HIV tests could be the possible reason for the low HIV seroprevalence and the absence of HbSS observed in this population. Nigerian Journal of Health and Biomedical Sciences Vol. 7 (1) 2008 pp. 27-30

Predictors of serum ferritin and haemoglobin during pregnancy, in a malaria-endemic area of western Kenya

Between 2000 and 2004, a cross-sectional survey was conducted, as part of a prospective cohort study, among the women attending antenatal-care clinics in Bondo district, a malaria-endemic area of western Kenya. The aim was to assess the prevalence of iron deficiency and determine the predictors of haemoglobin and serum ferritin concentrations in the women who had a gestational age between 14 and 24 weeks. A standardized questionnaire was used to collect and store the relevant bio-data for the study. Haemoglobin and ferritin concentrations were evaluated, sickle-cell status was determined, and malarial parasitaemias were detected and evaluated, using blood samples collected at enrollment. Multiple regression analysis was then used to test for significant predictors of the haemoglobin and serum ferritin concentrations. Although 842 women were enrolled in the prospective cohort study, haemoglobin concentrations were evaluated for only 828 of them, serum ferritin levels for 621, and levels of parasitaemia for 812. The mean haemoglobin concentration recorded was 10.9 g/dl. Although 37.9% of the subjects had mild-moderate anaemia (7.0-10.5 g haemoglobin/dl), only 0.5% were severely anaemic (<7.0 g haemoglobin/dl). The geometric mean serum ferritin concentration recorded was 18.9 microg/litre, and 32.3% of the subjects evaluated had low serum concentrations of ferritin (<12 microg/litre). Among the parasitaemic primigravidae (but not the parasitaemic multigravidae), those found positive for sickle-cell trait had significantly lower haemoglobin concentrations than those found negative in a sickling test (P=0.01). Among the pregnant women of Bondo district, gravidity, malarial infection and sickle cell appear to be key predictors of haemoglobin concentration.

Double heterozygous for hemoglobin S and hemoglobin E — a case report from central India

Double heterozygosity for HbS and HbE is rare. HbS and HbE are seen in SC, ST and OBC communities from this part of country. Inter caste marriages amongst these communities have resulted into this compound heterozygous condition. Double heterozygous state for HbS and HbE is clinically silent as compared to HbS-β Thalassaemia and HbSS cases. At Regional Hemoglobinopathy Detection and Management Center, we report a case of 15-year-old male, Teli (OBC) by caste who came for screening for sickle cell disorder. Sickling, solubility test and Hb electrophoresis on agar gel at alkaline pH was carried out. His sickling and solubility tests were positive and on hemoglobin electrophoresis it showed two bands one at Hb A(2) position and another at HbS position. For further confirmation sample was subjected for quantitation of haemoglobin on high performance liquid chromatography (HPLC), Bio-Rad. On quantitation he was having HbS 59.8%, HbE 33.5% and HbF 3.2% confirming his double heterozygous state for HbS and HbE. On family screening his father turned out to be sickle cell trait and mother as hemoglobin E trait.

Hemoglobin San Martin: A New Unstable Variant Associated with Hemoglobin S in an Argentinean Boy.

A new unstable hemoglobin (Hb) detected in a 10 year-old boy from San Martin, Buenos Aires, Argentina, is hereby described. The patient (pt) was born at term, after a normal pregnancy, by cesarean section. He had a history of multiple episodes of pallor, jaundice and dark urine related to infections, since 6 months of age. The physical examination only revealed pallor and icterus, with no hepatosplenomegaly. The pt and his mother showed the presence of an anomalous band on both alkaline and acid pH electrophoresis. Isopropanol and sickling tests were positive. In order to identify the abnormal Hb variant the β globin gene was analysed. Three exons were amplified and automatically sequenced with direct and reverse probes.Table 1Hematological dataPatientMotherHb g/dl/PCV %10.9/3610.5/34MCV fl/MCH pg79.4/24.180.7/24.6RDW %15.718.2Reticulocyte count %5.28.0Electrophoresis (pH 9)A/X/A2A/X/A2Electrophoresis (pH 5)X/A-A2X/A-A2Sickling/Isopropanol testsPositive/PositivePositive/PositiveInclusion bodiesPositivePositiveTwo mutations were identified in the same β globin gene. One in Exon 1, corresponding to Hb S [Ex1 β6 Glu→Val (GAG→GTG)]; and the second one in Exon 2 [Ex2 β85 (F1) Phe→Leu (TTT→CTT)]. This second mutation is responsible for a new unstable Hb that has not been described so far, in association with Hb S. The substitution occurs in an external position between two hydrophobic residues of different size.

Sickle liver disease—An unusual presentation in a compound heterozygote for HbS and a novel β‐thalassemia mutation

A 38-year-old Ghanaian man presented with a 6-month history of worsening pruritus, jaundice, and ascites. He was previously fit and well and rarely drank alcohol. Screening tests for chronic liver disease including viral, autoimmune, and other metabolic causes including iron overload were unremarkable. A liver biopsy performed at the referring hospital demonstrated intralobular cholestasis and cirrhosis. He was listed for liver transplantation but subsequently developed sepsis with multiple organ failure and died. The sickle solubility test was positive. Blood smear showed cells consistent with liver failure and no sickle cells. Hemoglobin electrophoresis revealed HbA2 2.8%, HbF 0.5%, and HbS greater than HbA (49.6% vs. 41.3%) in the absence of blood transfusion. Sequence analysis of the beta-globin genes showed he was a compound heterozygote for the Hbs mutation at codon 6 (CAG --> GTG) and a novel mutation at position 844 of intron 2 (betaIVS2-844 C --> A). A diagnosis of sickle hepatopathy causing decompensated cirrhosis was made. This case is unusual insomuch as this patient was asymptomatic for over 35 years and represents a novel presentation of sickle cell disease. Sickle cell disease should be considered in appropriate patients when unusual presentations of liver disease arise.

Antisickling agent in an extract of unripe pawpaw (Carica papaya): Is it real?

Investigations into antisickling and reversal of sickling activities of an aqueous extract of unripe pawpaw (Carica papaya) were carried out on blood from sickle cell patients (Haemoglobin SS, HbSS) using 2% sodium metabisulphite in a sickling test. The minimum concentration of the extract that achieved maximum antisickling in vitro and the fraction of the extract where the antisickling agent resides were determined. Our findings confirmed both antisickling and reversal of sickling activities of the extract. It was established that 1.0 g of unripe pawpaw in 1.0 ml of physiological saline was the minimum concentration that achieved maximum antisickling. Solvent partitioning of the extract with ethyl acetate and butanol revealed that the antisickling agent in the extract of unripe pawpaw resides in the ethyl acetate fraction as this fraction prevented sickling of Hb SS red cells and reversed sickled Hb SS red cells in 2% sodium metabisulphite whereas the butanol and aqueous fractions had none of these properties. We concluded that extract of unripe pawpaw really has antisickling agent and that this antisickling agent lies in the ethyl acetate fraction of the extract.

Haemoglobin‐S in sickle cell trait with papillary necrosis

Sickle cell trait (SCT) is the carrier state of sickle cell anaemia (Flint et al, 1993; Davies & Oni, 1997). However, in SCT, only 20–40% of the total haemoglobin is Hb S, and the abundance of normal Hb A (60–80%) prevents sickling under most physiological circumstances (Barbedo & McCurdy, 1974). Furthermore, the level of Hb S is lower when SCT co-exists with α-thalassaemia (Barbedo & McCurdy, 1974). Nonetheless, SCT red cells do occasionally sickle under the hyper-osmolar conditions encountered in the renal papillae, resulting in renal papillae necrosis (RPN) and haematuria (Barbedo & McCurdy, 1974). This study aimed to identify any association between Hb S levels and the development of RPN by determining the Hb- S levels of SCT individuals in Maiduguri, Nigeria. A total of 52 subjects with SCT who had haematuria because of RPN diagnosed by renal ultrasonography between 1996 and 2004 at the University of Maiduguri Teaching Hospital, Nigeria, were evaluated with respect to leucocyte count, platelet count, total haemoglobin concentration, Hb S levels, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). All parameters were determined by Beckman Coulter AcT-8 except for Hb S, which was determined by a manual elution technique (Dacie et al, 1991) and expressed as the percentage of total haemoglobin. Equal numbers of age- and sex-matched individuals with SCT who had no history of haematuria and had no ultrasonographic features of RPN were also recruited as controls and evaluated with respect to similar parameters in a case–control study. All the subjects included in this study were confirmed cases of SCT, established by a positive sickling test and Hb AS electrophoretic pattern (Dacie et al, 1991). Student t-test was used to compare the mean values of the studied parameters between the two groups; a probability level of P < 0·05 was taken as significant. Conditional logistic regression using the Statistical Package for the Social Sciences (spss) software, version 11.0 (SPSS Inc., Chicago, IL, USA), was performed to assess the risk of RPN because of Hb S levels among the subjects studied. The results of this study are shown in Table I. All subjects had normal haematological parameters. The SCT subjects with a history of RPN did not significantly differ from the control group with respect to mean age, sex ratio, leucocyte and platelet count. The red cell indices, MCV, MCH and MCHC, were normal and did not significantly differ between the two groups of SCT subjects studied (P > 0·05). However, SCT subjects with a history of RPN had a significantly higher mean Hb S level (34%) than that seen in control subjects (25%) (P < 0·05). Conditional logistic regression for the two age- and sex-matched groups with respect to the risk of RPN because of Hb S levels gave an odds ratio (OR) of 1·58 (95% confidence interval: 0·33–6·61) with a significant P-value of 0·03. The finding of normal total haemoglobin concentrations among the SCT subjects studied in this report confirmed the absence of haemolysis in persons with SCT, which was consistent with earlier reports (Beutler, 1991). Furthermore, the finding of normal values of red cell indices among our SCT subjects would strongly suggest the absence of α-thalassaemia (Dacie et al, 1991). This study further revealed that SCT subjects with a history of RPN had a higher mean Hb S level, indicating that RPN was associated with higher levels of Hb S in SCT. This result would suggest that high Hb S level is a risk factor for the development of RPN in SCT subjects. The significant OR (1·58) would suggest a higher relative risk as the level of Hb S increases. However, it should be noted that the actual magnitude of the relative risk would be best determined by a cohort study. The present study found that SCT subjects with a history of RPN were identifiable as a subgroup of SCT subjects that had higher Hb S levels and hence would be at greater risk of sickling under low oxygen tension. Therefore, SCT subjects with a history of RPN would need closer monitoring during surgical anaesthesia or any procedure that carries a risk of deoxygenation.