Normal Red Cell Indices Research Articles

Autosomal Dominant Hereditary Spherocytosis In An Elderly Patient

Hereditary spherocytosis (HS) is an autosomal dominant inherited hemolytic anemia that usually manifests in early adolescence. A very small proportion (5%) may undergo de novo mutations and present at an elderly age without any positive family history. The disease is characterized by anemia, jaundice, splenomegaly, and the presence of spherocytes in peripheral blood, which are osmotically fragile. A 55-year-old elderly male presented with generalized weakness and a history of repeated blood transfusions for 5 months. He had recurrent jaundice for 8 years. There was no relevant family or drug history. He was anemic, icteric, and had mild splenomegaly. Investigation revealed persistently low Hb with normal red cell indices. There were a lot of spherocytes with polychromasia on the peripheral blood film. There was also evidence of hemolysis with a high reticulocyte count and higher levels of total and indirect bilirubin. Autoimmune hemolysis was excluded by negative direct and indirect Coomb’s tests, and Hb defect was excluded by normal Hb electrophoresis. We also ruled out other infectious causes. The ultrasound confirmed splenomegaly. The osmotic fragility test showed increased osmotic fragility of the patient’s red cells. History, symptoms, and test results strongly point to autosomal dominant hereditary spherocytosis with new mutations. An eosin-5-Maleimide binding test was advised, along with family screening of the patient. Bangladesh J Medicine 2024; 35: 110-113

A mysterious appearance at zone 1

We present the case of a 76-year-old male with history of Type II diabetes presenting for routine HbA1c testing. The HbA1c profile via capillary electrophoresis (CE) revealed the presence of an unknown haemoglobin peak of 19.3% in Zone 1. Blood film showed microcytosis, hypochromia, poikilocytosis and red cell dimorphism. The unknown haemoglobin migrated in the HbH position on the Sebia Capillarys3 Hemoglobin(E) program and on High Pressure Liquid Chromatography (HPLC). Many HbH bodies were detected on Brilliant Creysl Blue (BCB) staining and biochemistry was consistent with haemolysis. A presumptive diagnosis of acquired HbH disease, or alpha thalassaemia-MDS (AT-MDS), was determined. The acquired nature was determined by previously normal red cell indices and HbA1c chromatogram. AT-MDS is an acquired disorder that most commonly occurs due to rare somatic mutations in the ATRX gene, or less commonly chromosome 16p. There is a male predominance (6:1) and the median diagnosis age is 68. These mutations occur in 0.2–‘.8% of MDS cases and produce a more severe alpha thalassaemia phenotype when compared to germ line mutations of ATRX. This case highlights a novel diagnostic pathway for a rare disease and emphasises the importance of investigating ‘incidental’ haemoglobin variants detected during HbA1c testing.

Trends in ferritin measurements in children and adolescents: AFinnish 9-year observational study.

AimA lack of stored iron, indicated by low serum ferritin, has been associated with various clinical symptoms. There are no longitudinal data on the frequency of ferritin measurements in children and adolescents.MethodsA total of 2834 children aged <18 years with serum ferritin and other anaemia‐related blood parameters taken during an outpatient visit between 2012 and 2019 were investigated. Patients with acute infections were excluded. Nationwide temporal and regional variations and correlations with public information searches through Google were analysed.ResultsA significant increase in the frequency of ferritin measurements was seen starting in 2018, with a 47‐fold rise in 2019 compared to 2012. A simultaneous escalation in Google Search activity was seen. Deficiency of stored iron was relatively common: 21.6% of children with normal haemoglobin and 14.9% of non‐anaemic children with normal red cell indices exhibited ferritin levels below 15 μg/L.ConclusionFerritin measurement has increased greatly among children and adolescents. Our results suggest that public interest and popular trends can significantly influence health care practices. This calls for further investigation into the causes and consequences of such a phenomenon. Prospective randomised intervention studies are needed to evaluate the utility of iron supplementation in patients with low iron storage levels.

P104: BASE EDITING REPAIRS THE HBE MUTATION RESTORING THE PRODUCTION OF NORMAL GLOBIN CHAINS IN SEVERE HBE/β-THALASSEMIA PATIENT HEMATOPOIETIC STEM AND ERYTHROID CELLS

Aims: HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide1. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any β0-thalassemia mutation on the other. There is a reduction in β-globin production, resulting in a relative excess in α-globin chains that leads to ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype shared by 1.5% of the world’s population2. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies rely on non-homologous end joining targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Methods: Adenine base editors (ABEs) circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C transitions. Conversion of the HbE codon to WT through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions. ABEs are able to convert the HbE codon (AAG) to wild-type (GAG), but also to GGG or AGG (Fig A). GGG at codon 26 is found in a naturally occurring haemoglobin, Hb Aubenas3. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors4 as mRNA into 3 different severe HbE/β-thalassemia donor HSPCs with sgRNAs targeting the HbE codon. Results: The mean conversion from the HbE codon to a normal or normal variant in unselected cells was 86.2 (SD±8.1%, Fig B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure C), indicating a reduction in the excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Fig D & E). In serial NSG-murine xenotransplantation experiments, base edited cells were found to persist in secondary transplants, showing editing is possible in long-term HSCs (mean editing efficiency 34.5%, Fig F). Potential off-target effects were assessed in-vitro by CIRCLE-seq5; most candidate sites were in intergenic and intronic regions (Fig G & H). The top 250 sites were sequenced using deep targeted NGS. Only 5 sites showed OT deaminations at low levels in patient cells (mean 1.5%). We developed a machine learning model to assess potential OT-effects on chromatin accessibility, at all candidate sites in 49 different blood cell types6. Only 17 potential edits were predicted to result in a significant change in chromatin state (Fig I). 3 of these were in the top 250 sites sequenced previously, and none showed deamination in-vivo. Conclusion: Together these data provide robust evidence for base editing being used as an effective and safe therapeutic strategy for HbE/β-thalassemia.

Base Editing Repairs the HbE Mutation Restoring the Production of Normal Globin Chains in Severe HbE/β-Thalassemia Patient Hematopoietic Stem Cells and Erythroid Cells

HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal.We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D).To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing.Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Large β Globin Gene Cluster Deletions and Implications on Transcription Factor Regulation in Hemoglobin Switching - Α Novel ε Gγ Αγ Deletion with Intact LCR

The εγδβ thalassemias (EGDBT) are infrequently occurring deletions of the β globin gene cluster on chromosome 11. EGDBTs can be an unsuspected cause of severe neonatal anemia that resolves during the first year of life into a normal Hb A2 thalassemic phenotype, presumed to be due to an imbalance of β-like chains during development. First described in 1972, they are categorized into two groups, both with loss of the five DNase I hypersensitivity sites upstream of the β gene (HBB) called the locus control region (LCR). In group 1 deletions, all or most of the β globin gene cluster including HBB is deleted; in group 2 deletions, removal of the LCR silences an intact HBB.Large deletions that do not involve LCR but remove the embryonic (HBE) and fetal genes (HBG2/HBG1) [LCR(ε Gγ Aγ) 0δβ] have not been reported. It is not known whether a deletion with intact LCR as well as δ and β genes would similarly manifest as neonatal anemia that resolves into a normocytic phenotype with normal hemoglobin fraction percentages after infancy.Four individuals with an LCR(ε Gγ Aγ) 0δβ deletion have been evaluated. These include monochorionic, diamniotic twin girls whose newborn screen (NBS) returned significant for Hb A percentage greater than Hb F for both twins. NBS was repeated two weeks later with the same result. The newborns were induced late pre-term (36 weeks and 6 days) after an uncomplicated pregnancy, including no features of twin to twin transfusion. Both infants had no anemia and bilirubin levels remained in the low to low-intermediate risk zones. Neither twin received transfusions or phototherapy during admission.At a follow up visit at 20 months of age a hemoglobin electrophoresis was performed which showed borderline Hb A2 and no Hb F in both twins (Table 1). Ferritin levels were low-normal in Twin A (14 µg/L) and low in Twin B (9 µg/L). Both infants were found to have a heterozygous large deletion affecting HBE, HBG2, HBG1, and HBBP1 loci by Multiplex Ligation-dependent Probe Amplification (MLPA), a method that uses multiple DNA probe pairs to determine the size of the DNA segment deleted. MLPA deletion/duplication testing on the α genes was negative. No β globin variants were identified by DNA sequencing. An evaluation was performed on the 25 year-old healthy mother and showed similar findings including the heterozygous large deletion. Mother and infants continue to be asymptomatic with normal red cell indices for age (Table 1).The fourth was an unrelated 40-year-old female with mild anemia screened by hemoglobin electrophoresis for obstetric evaluation who was found to have borderline Hb A2 (see table 1). MLPA showed a heterozygous deletion involving the same loci. Again, no β globin variants were identified by DNA sequencing.All four cases showed the same MLPA pattern except one non-specific probe commonly altered in common gamma gene conversion events. Long range sequencing performed on one case confirmed a single contiguous 32,599 bp deletion that matched the MLPA data (g.5262276-5294875). The detected deletion has not been reported in literature and differs from EGDBT mutations in that the LCR is intact. As the LCR controls expression of the entire gene cluster, this mutation is expected to display different phenotypic features than classic EGDBT. HBD and HBB are not deleted and therefore adequate transcription of Hb A2 and Hb A are expected with no imbalance of α/β chains after the neonatal period. It is postulated this finding explains the decreased Hb F levels in the NBS test result. The Hb A2 increase is likely a product of this deletion and does not indicate β-thalassemia.The heterozygous absence of HBE and HBG2/HBG1 may result in upregulation of HBD and HBB expression through loss of FKLF and FKLF-2 binding. In addition, HBB-EKLF binding would have less competition as the gamma promoter elements have been heterozygously deleted.In summary, in contrast to classic EGDBTs which cause transient severe neonatal anemia and resolve to normal Hb A2 thalassemic indices, this novel LCR(ε Gγ Aγ) 0δβ deletion was associated with normal CBC values with an absence of severe neonatal anemia, inverted Hb F/Hb A percentages at birth and borderline Hb A2 levels. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PB2413 HAEMOGLOBIN AGHIA SOPHIA (HBA1:C.187_189DELGTG) IN NORTHERN GREECE. IMPLICATIONS IN CLINICAL PHENOTYPE OF HBH

Background: Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and the clinical phenotype varies from almost asymptomatic to a lethal haemolytic anaemia. It is probably the most common monogenic gene disorder in the world. Several mutations have been described that can affect globin gene expression in any step and many of those mutations are rare causes of alpha thalassaemia. Aims: 28 patients have been diagnosed with Hb H disease (14 males and 14 females) from 1986 to 2019 at the regions of Central and Western Macedonia in Northern Greece with a population of around 2.5 million. The most prevalent genotype was the -α 3.7/—med, (5 cases), 2 cases of –α 3,7/-α5,2, 1 case of –med/αPoly A, 1 case of –α 3,7/Hb Adana and 1 case of Hb Aghia Sophia/--med. The phenotype suggests that HbH disease in Greece has in general a moderate presentation. The molecular characterization of the patients with Hb H disease is useful for the prediction of the clinical outcome and the genetic counselling of the couples at risk. Methods: Hb Aghia Sophia carriers are asymptomatic with normal or nearly normal Hb levels and red cell indices similar to α+ thalassaemia carrier as it was shown in 2 members of a family that this mutation was also detected in our region. Haemoglobin (Hb) Aghia Sophia in interaction with deletional α-thalassaemia mutations leads to HbH. We report a case of Hb Aghia Sophia with co-inheritance with the a-thalassaemia – med deletion, a rare double heterozygosity case. Results: The case presented, concerns a 40 year old woman followed in our Thalassemia Clinic who was diagnosed at the age of 8 with Hb H. At diagnosis, totally incidental findings were compatible with a typical phenotype of Hb H, anaemia being mild and growth not having been impaired. As a girl, she retained a mild hypochromic microcytic anaemia until the age of 10 that a splenectomy was decided due to marked splenomegaly and anemia. During the following years her hemoglobin was maintained around 8gr/dl and she had normal growth. At the age of 35 she had an abortion. She was transfused each month during the next two pregnancies. Lately she was complaining for fatigue and her hemoglobin value was 7gr/dl. The investigation of hemoglobin deterioration did not reveal any significant cause. Bone marrow aspiration showed increased erythroid hyperplasia. Molecular examination revealed Aghia Sophia with co-inheritance with the a-thalassaemia – med deletion. As she denied transfusion, erythropoietin therapy was started with good results maintaining Hb level to 9gr/dl. Additionally, she receives deferasirox due to iron overload. Summary/Conclusion: The co-inheritance of Hb Aghia Sophia with –med, is extremely rare and the severity is due to the interaction of a hyper unstable mutant of the a1 gene with an α0-thal determinant. Hb Aghia Sophia causes instability to the polypeptide chain and is rapidly catabolized. Long follow-up of such rare cases is necessary in order to gain as much information as possible, so as to offer the best management to the patients and the most accurate counseling to their families.

IRON DEFICIENCY AND THALASSAEMIA

Objectives: To study the importance of normal or low mean corpuscular volume in vitamin B12 deficiency due to co-existence of iron deficiency or beta thalassaemia trait masking a rise in mean corpuscular volume. Study Design: Observational non-probability cross sectional study. Setting: DDRRL. Period: January 2014 to September 2014. Methods: 105 vitamin B12 deficient cases (vitamin B12 less than 200ng/l) who presented with normal or low mean corpuscular volume (MCV less than 95 fl) on complete blood count (CBC) were determined from Dow diagnostic research and reference laboratory (DDRRL). Serum ferritin, red blood cell folate (RBC Folate) level and Hemoglobin electrophoresis for beta thalassaemiatrait were analysed in these patients. Results: Total of 105 vitamin B12 deficient patients who fulfilled the inclusion criteria were enrolled in this study from which 39 (37.14%) were male and 66 (62.85%) were females. Amongst them 36.19% had microcytosis with the mean age of 37±16.2 years while 63.8% were normocytic with mean age of 41.58±15.65 years. In microcytic group, iron deficiency, beta thalassaemia trait, combined deficiency of B12, iron and beta thalassaemia trait and RBC folate deficient were 52.6%,34.21%, 7.8% and 2.63% respectively. In normocytic group, iron deficiency, beta thalassaemia trait and RBC Folate deficient were 13.4%, 00% and 11.9% respectively. Conclusion: There is a significant coexistent frequency of iron deficiency and beta thalassaemia trait in vitamin B12 deficiency with normal or low MCV especially in females of 20-40 years of age. There should be a high index of suspicion for B12 deficiency when investigating anaemia with normal or altered red cell indices.

A Unique Interaction of IVS-I-1 (G>A) (HBA2: c.95+1G>A) with Hb Adana (HBA2: c.179G>A) Presenting as Transfusion-Dependent α-Thalassemia

Nondeletional α-globin mutations are known to cause more serious clinical effects than deletional ones. A rare IVS-I-1 (G>A) (HBA2: c.95+1G>A) donor splice site mutation interferes with normal splicing of pre mRNA and results in activation of a cryptic splice site as well as a frameshift mutation. Hb Adana [HBA2: c.179G>A (or HBA1)] is a highly unstable variant hemoglobin (Hb) resulting from a mutation at codon 59 on the HBA2 or HBA1 gene, recognized to cause severe α-thalassemia (α-thal) syndromes. We report a unique case of compound heterozygosity for these two mutations in a 9-year-old boy who presented with a Hb level of 5.3 g/dL and hepatomegaly at the age of 15 months. He required regular blood transfusions in view of a Hb level of <7.0 g/dL and failure to thrive. He had thalassemic red cell indices and peripheral blood film. The Hb electrophoresis only showed a raised Hb F level (3.3%) and a pre run peak but the Hb H inclusion test was negative. His father had thalassemic red cell indices but a normal Hb level. His mother had almost normal Hb levels and red cell indices. Hb Adana involving the HBA2 gene was detected by mutiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in the proband and his father. DNA sequencing of the HBA2 gene confirmed the IVS-I-1 mutation in the proband and his mother. This case highlighted the unique interaction of the IVS-I-1 mutation with Hb Adana in a young Malay boy presenting with transfusion-dependent α-thal.

Iron status and anaemia in Sri Lankan secondary school children: A cross-sectional survey

BackgroundIron deficiency, the most common micronutrient disorder and cause of anaemia globally, impairs growth, cognition, behaviour and resistance to infection.Methods/ResultsAs part of a national survey of inherited haemoglobin variants in 7526 students from 72 secondary schools purposefully selected from the 25 districts of Sri Lanka, we studied 5912 students with a normal haemoglobin genotype. Median age was 16.0 (IQR 15.0–17.0) years and 3189 (53.9%) students were males. Most students were Sinhalese (65.7%), with fewer Tamils (23.1%) and Muslims (11.2%). Anaemia occurred in 470 students and was more common in females (11.1%) than males (5.6%). Haemoglobin, serum ferritin, transferrin receptor and iron were determined in 1196 students with low red cell indices and a structured sample of those with normal red cell indices (n = 513). The findings were weighted to estimate the frequencies of iron deficiency and iron deficiency anaemia classified according to WHO criteria. Iron depletion (serum ferritin <15ug/ml) occurred in 19.2% and cellular iron deficiency (low serum ferritin and transferrin receptor >28.1 nmol/l) in 11.6% students. Iron deficiency anaemia (cellular iron deficiency with low haemoglobin) occurred in only 130/2794 (4.6%) females and 28/2789 (1.0%) males. Iron biomarkers were normal in 83/470 (14.6%) students with anaemia. In multiple regression analysis, the odds for iron depletion and cellular iron deficiency were about one-third in males compared with females, and the odds for iron deficiency anaemia were about one fifth in males compared to females. Tamil ethnicity and age <16 years increased the risk of all three stages of iron deficiency and living at high altitude significantly reduced the risk of iron depletion.ConclusionsLow iron status and anaemia remain common problems in Sri Lankan secondary school students especially females, younger students and the socioeconomically disadvantaged Tamil population. More research is needed to identify factors other than low iron status that contribute to anaemia in adolescents.

Iron deficiency and red cell indices in patients with heart failure.

To investigate the prevalence of iron deficiency (ID) in heart failure (HF) patients with normal vs. abnormal red cell indices (RCI), the associations between iron parameters and RCI, and prognostic consequences of ID independently of RCI. We analysed clinical data of 1821 patients with HF [mean age 66 ± 13 years; 71% men; New York Heart Association class I/II/III/IV (11%/39%/44%/6%); left ventricular ejection fraction >45%: 19%]. Iron deficiency (ferritin <100 µg/L or ferritin 100-299 µg/L with transferrin saturation <20%) was common irrespective of the presence of anaemia (haemoglobin <12 g/dL in women and <13 g/dL in men) or low RCI, from 75% in anaemic subjects with low mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and MCH concentration (MCHC), to 36% in non-anaemic subjects with MCV, MCH, and MCHC above the lower limit of normal. After adjustment for clinical variables, iron parameters remained independently associated with haemoglobin, MCV, MCH, MCHC, mean reticulocyte haemoglobin content (CHR), and red cell distribution width (RDW). In multivariable Cox proportional hazard regression models there was a trend towards higher mortality in patients with vs. without ID when adjusted for relevant HF prognosticators and MCH or MCHC (but not haemoglobin, CHR or RDW). Patients with HF should be routinely screened for ID irrespective of the presence of anaemia or abnormal RCI. The detrimental impact of ID on long-term survival in HF is partially independent of RCI.

The Use of NESTROFT for Screening Pregnant Women for Detection of β-Thalassemia Carriers

The thalassemias and sickle cell disorders are widely distributed in India and cause a significant health burden [1, 2]. Community control programmes are the only way to reduce this burden. Screening for identification of carriers forms an integral component of a prevention programme [1, 2]. The often-debated questions are whom to screen and which would be the most appropriate technology to be used. The individuals targeted for screening in most studies include school or university students, newly-married couples, pregnant women, extended family members of a thalassemia major child, or members of communities where the prevalence of b-thalassemia carriers is higher than the average of 3 %–4 % in the Indian population. Pregnant women would be the most appropriate group as they are the ones at immediate risk but they often come late in the second trimester of pregnancy to the antenatal clinic for their first visit, especially in public hospitals, and it is often difficult to get their husbands for testing [3]. All these target groups have been screened in India by different centers [4–7]. Premarital screening is largely not acceptable due to fear of stigmatization. The technologies used for screening and diagnosis, include the following: Naked eye single tube red cell osmotic fragility test (NESTROFT); red cell indices; discriminant functions; flow cytometric osmotic fragility; and hemoglobin analysis by high-performance liquid chromatography (HPLC), capillary electrophoresis, or cellulose acetate electrophoresis for estimation of Hb A2, Hb F or other hemoglobin variants. A combination of these methods has been used by different investigators and each one has its advantages and limitations [8–13]. NESTROFT has been used as a primary screening test for population screening in many studies after Kattamis et al. first showed that a 0.36 % saline solution was sensitive and effectively detected more than 96 % of b-thalassemia carriers on screening [14]. The ideal approach would be to measure the red cell indices and quantitate Hb A2, Hb F, and any other hemoglobin variant by HPLC or capillary electrophoresis in every individual to ensure that all b-thalassemia heterozygotes as well as carriers of abnormal hemoglobins are identified. However, this is not a cost-effective strategy for massscreening programmes in a large country. Moreover, even with this ideal strategy, around 2 % of b-thalassemia heterozygotes who have normal red cell indices, and/or normal or borderline Hb A2 levels will be missed. NESTROFT has a high sensitivity but lower specificity as shown in several studies [8–13]. Using NESTROFT as a single first-line screening test has some limitations. Although it has a high negative predictive value, few bthalassemia carriers have been missed in different studies. In a quality control evaluation done during the large multicenter study of Indian Council of Medical Research (ICMR) under the Jai Vigyan programme, the main reasons for higher number of false negatives at some centers were the quality of water used, inaccurate dilution of the buffer, and frequent change of technicians. It was also shown that if NESTROFT was combined with measuring red cell indices in the first step itself using mean corpuscular volume (MCV) \80 fL and mean corpuscular hemoglobin (MHC)\27 pg as cut-off values, the b-thalassemia carriers & Roshan B. Colah colahrb@gmail.com

HbA1C An Inaccurate Measure of Glycemic Control in a Female with Hereditary Spherocytosis Despite Normal Hemoglobin Levels

ABSTRACT Objective: Earlier cases have documented a discrepancy between glycated hemoglobin A1c (HbA1C) levels and glycemic control in patients with diabetes and previously diagnosed hereditary spherocytosis (HS). However, this has not been reported in patients with normal hemoglobin (Hb) and red blood cell (RBC) indices. Methods: A 22-year-old Caucasian woman with type 1 diabetes since age 11 was referred to Endocrinology for persistent hyperglycemia. Results: The initial HbA1C was 5.1% with normal Hb, hematocrit, mean cell volume, mean corpuscular Hb concentration, and RBC distribution. Given self-reported hyperglycemia, fructosamine was checked and was elevated (358 μmol/L, normal [nl] 200–285 μmol/L). Peripheral smear was normal. Professional retrospective continuous glucose monitoring (CGM) demonstrated marked hyperglycemic excursions, particularly in the evenings, with mean sensor glucose of 195 mg/dL ± 48, corresponding HbA1C of 5.5%, and fructosamine of 370 μmol/L. She later reported new diagnosis...

Congenital Dyserythropoietic Anaemia: Case Report of a Rare Blood Disorder in a Nigerian Child

IntroductionThe congenital dyserythropoietic anaemias (CDA) are a rare group of inherited haemolytic anaemias characterized by ineffective erythropoiesis and dyserythropoiesis. Due to the rarity of this disorder (and in Africa where haemoglobinopathies are the commonest cause of congenital haemolytic anaemias), the diagnosis can be missed. We report a six year old girl with recurrent anaemia, jaundice, hepatosplenomegaly and history of multiple transfusions who was diagnosed with CDA.Case ReportA six year old girl presented to the paediatric clinic with a two year history of passing coke-coloured urine, recurrent anaemia requiring regular three-monthly transfusions and persistent jaundice. She was not a known sickle cell anaemia or thalassaemia patient. History of prenatal period, birth and delivery were normal (birth weight 3.5kg/ 7.7lbs). There was no history of neonatal jaundice or family history of similar illness.At presentation, she was acutely ill-looking, conscious but weak. She was afebrile, severely pale, icteric, tachypneic and in respiratory distress (respiratory rate of 52 cycles/minute). She was small for age; weight 15kg / 33.1lbs (below the 3rd percentile), height 104cm (below the 3rd percentile) with frontal bossing and gnathopathy. The liver was 6cm below the right costal margin and tender, with splenomegaly- 5cm below the costal margin. There was no lymphadenopathy. Her chest was clear, heart rate 140 beats/minute, and blood pressure of 100/50mmHg with presence of a haemic murmur. A preliminary clinical diagnosis of recurrent severe haemolytic anaemia in heart failure secondary to haemoglobinopathy was made.Full blood count showed severe anaemia with haemoglobin (Hb) concentration of 5.1g/dL, normal red cell indices, white cell count- 13.6 X 109/L, platelets- 191 X 109/L and reticulocyte count- 1.3%. The Hb genotype was AA, direct and indirect antiglobulin test were negative. Liver functions tests showed normal enzyme and protein values; but high total bilirubin (104mmol/L) and unconjugated bilirubin- 17.4umol/L. Serum uric acid was normal. HIV, hepatitis B and C were negative. Urinalysis was positive for blood, urobilinogen, bilirubin, with a pH of 8.0. Urine microscopy showed granular, epithelial and red blood cell casts. Glucose-6-phosphate dehydrogenase assay- 12.5 μ/Hb.She was admitted, placed on oxygen, transfused and discharged on the third day in stable condition. However she was brought back to the hospital about 3 months later for similar symptoms. Urgent Hb concentration was 6.0g/dL. Peripheral blood film revealed anisopoikilocytosis, some macrocytes, tear drop cells, polychromasia, basophilic stippling, fragmented red cells and presence of nucleated red cells (12/100 white cells)- a few of which were multinucleated (Fig 1). HPLC showed low HbA (74.1%), severely increased HbF (23.8%) and HbA2 (2.1%). Bone marrow (BM) aspiration showed a hypercellular marrow, erythroid hyperplasia (myeloid/erythroid ratio 1:2), dyserythropoiesis with erythroid multinuclearity in >10% of late erythroid precursors and significant karyorrhexis. Myelopoiesis and megakaryopoiesis were essentially normal (Fig 2). Serum ferritin was elevated (2,658ng/ml). In the absence of availability of electron microscopy or molecular studies, a diagnosis of CDA type II was made based on clinical, laboratory and characteristic bone marrow findings. She was transfused, placed on iron chelation therapy, her parents counseled on treatment options and she is being followed up.DiscussionThe CDAs are classically grouped into 3 types based on bone marrow morphology. Type I has erythroblasts joined by an internuclear bridge. Type II erythroblasts have multinuclearity of late erythroblasts while type III has gigantoblasts (erythroblasts with ≥8 nuclei). Inheritance is autosomal recessive and diagnosis is usually in childhood or early adult life. Common clinical findings are anaemia, jaundice and splenomegaly; however these are seen in other more common inherited haemolytic anaemias. BM examination is the gold standard for diagnosis. Patients with CDA usually have high serum ferritin that may require iron chelation therapy.CDA although rare must be considered in a child who has recurrent anaemia in whom other causes have been excluded. BM examination remains a key diagnostic tool in identification of the CDAs. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Screening of Thalassaemia Carriers and Its Limitations

The α and β-thalassaemias (thal) are common genetic disorders of globin chain synthesis where the carriers have deficiency of α or β globin chain respectively. In resource limited settings, the naked eye single tube red cell osmotic fragility (NESTROFT) has been used for screening of thalassaemia. It has low specificity compared with its sensitivity. Screening of carriers encompasses automated/semi-automated techniques and manual methods for presumptive diagnosis of thalassaemia. The current strategy in screening involves a step by step process simplified into the following acronym, BHES. B, refers to blood counts and a film; H, haemoglobin (Hb) subtyping; E, Hb electrophoresis; and S, stability (H-inclusion and Sickle cell test). Blood counts and red blood cell indices are now available by automated instrumentation where a cut-off value of mean corpuscular Hb (MCH)<27 pg necessitates further investigation for thalassaemia or iron deficiency. The distribution of normal Hb subtypes (HbA, HbA2 and HbF) is done commonly by high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). The thalassaemia trait phenotype is indicated by a near normal Hb, microcytic and hypochromic red cell indices. In classical β-thalassaemia trait, the cut-off value of HbA2 value is 4% and above. However, milder mutations can result in HbA2 values between 3.2 to 3.9%. In alpha thalassaemia trait the HbA2 falls within the normal range. In a carrier of α0-thalassaemia, occasional or few H-inclusions suggests its presence. However, absence of H-inclusions does not rule out its presence. Recently, it has been shown that the current screening method of thalassaemia carriers has its limitations. Mild mutations may show normal or near normal red cell indices and be missed in the screening process. Carriers with these mutations can cause thalassaemia intermedia. In addition there are mutations that have more profound effects resulting in more severe outcome but have carriers with normal red cell indices. In α-thalassaemia intermedia, HbH disease, those with nondeletional mutations are clinically more severe. These non-deletion mutations include Hb Constant Spring, Hb Pakse and Hb Adana. Carriers with these non-deletional α-thal mutations have normal red cell indices. Cases of hydrops foetalis have been reported with a homozygous state of Hb Adana and also in α0 with Hb Adana. Coinheritance of α or β-thal also effects the red cell indices making them near normal. Thalassaemia intermedia and major have serious implications and prevention requires strategies to detect couples at risk. Mutational spectrum of the globin genes is updated continuously with improved diagnostic technology. Clinical programs preventing thalassaemia are presently based upon prospective screening and prenatal diagnosis. In order for prevention to be effective, there is a need to be aware that mutations can exist in thalassaemia carriers where the red cell indices are normal.

English

INTRODUCTION: Hemoglobin D Punjab was first discovered in the early fifties in a mixed British and American family of Indian origin from the Los Angeles area; hence it is also sometimes called as “D Los Angeles”. Hemoglobin D is the 4th common hemoglobin variance. It developed as a response to the selective pressures of malaria in these regions. The substitution in Hb D is a glutamic acid to glutamine at the 121st amino acid of the beta -globin chain (121Glu Gln). Hb D has an S-like mobility on alkaline electrophoresis but co-migrates with HbA on acid pH. Osmotic fragility may be decreased. Blood films may show target cells. [1] Two to 3 percent of the population of Punjab carries the Hb D gene. Subsequently, it has also been found in a number of other populations including Europeans, Mediterranean region, and Americans of African descent.[2] Hb D heterozygotes (Hb D trait) are usually asymptomatic, not anemic, and have normal red cell Indices.[3]Hb D homozygotes (Hb D disease) manifests as mild hemolytic anemia and splenomegaly.[4]But here we present a case of symptomatic Hb D trait, who presented with hemolytic anemia and splenomegaly from Hubli, Karnataka which is a rare phenomenon.

The Prevalence of Anaemia, Hypochromia and Microcytosis in Preoperative Cardiac Surgical Patients

This retrospective study aimed to determine the prevalence of preoperative anaemia, hypochromia and microcytosis in cardiac surgery patients. Data was analysed for 943 patients (over a two-year period) undergoing coronary artery bypass graft, valve or combined coronary artery bypass graft and valve surgery at a tertiary hospital in South Australia. Overall prevalence of preoperative anaemia was 25.2%, greater in males than females (27.6 vs 19.9%, P <0.01). Of patients with preoperative anaemia, 19.3% had reduced red cell indices (mean corpuscular haemoglobin and/or mean corpuscular volume) compared to 4% of patients without anaemia. The proportion of anaemic patients with low red cell indices was significantly higher in women <50 years and 50-65 years, compared to those >65 years of age (P=0.003). Anaemic patients with low red cell indices had lower preoperative haemoglobin than anaemic patients without low red cell indices (median haemoglobin 112 vs 120 g/l, P=0.008). Compared to non-anaemic patients, anaemic patients had higher transfusion rates (79.8 vs 46.4%, P <0.0001), which were greater in those with reduced red cell indices compared to those with normal red cell indices (93.5 vs 76.6%, P=0.01). This study demonstrated a high prevalence of preoperative anaemia, microcytosis and hypochromia in cardiac surgical patients.

A novel GATA1 mutation (Stop414Arg) in a family with the rare X‐linked blood group Lu(a‐b‐) phenotype and mild macrothrombocytic thrombocytopenia

A novel <i><scp>GATA</scp>1</i> mutation (<scp>S</scp>top414<scp>A</scp>rg) in a family with the rare <scp>X</scp>‐linked blood group <scp>L</scp>u(a‐b‐) phenotype and mild macrothrombocytic thrombocytopenia

Manifestaciones neurológicas por déficit aislado de vitamina B12

Isolated vitamin B12 deficiency is a common condition in elderly patients but uncommon in patients younger than 30 years, with an average age of onset between 60 and 70 years. This is because the dietary cobalamin, which is normally split by enzymes in meat in the presence of hydrochloric acid and pepsin in the stomach, is not released in the stomachs of elderly patients, usually due to achlorhydria. Although the body may be unable to release cobalamin it does retain the ability to absorb vitamin B12 in its crystalline form, which is present in multivitamin preparations. Other causes are due to drugs that suppress gastric acid production.Neurological signs of vitamin B12 deficiency can occur in patients with a normal haematocrit and red cell indices. They include paresthesia, loss of sensation and strength in the limbs, and ataxia. Reflexes may be slowed down or increased. Romberg and Babinsky signs may be positive, and vibration and position sensitivity often decreases. Behavoural disorders range from irritability and memory loss to severe dementia. The symptoms often do not fully respond to treatment.A case is presented of an isolated vitamin B12 deficiency in 27 year-old female patient who was seen in primary health care. During anamnesis she mentioned low back pain, to which she attributed the loss of strength and tenderness in the right side of the body, as well as the slow and progressive onset of accompanied headache for the previous 4 days.

α0-Thalassemia Trait with Normal Red Cell Indices: A Report of Two Cases

Thalassemia screening usually involves cut-off values based on a mean corpuscular volume (MCV) of less than 80 fL or a mean corpuscular hemoglobin (MCH) of less than 27 pg. These strategies are able to detect almost all heterozygous carriers of the α-thalassemia (α-thal) (– –SEA) deletion in the Chinese population. However, an exception can occur. We describe an α0-thal trait with normal red cell indices in two Chinese individuals.