High Oxygen Affinity Hemoglobin Research Articles

Influence of Hemoglobin Strasbourg, a Rare High Oxygen Affinity Hemoglobin Variant, on Different Methods of HbA1c Measurement

Hemoglobin Strasbourg is a rare high oxygen affinity hemoglobin variant which leads to secondary erythrocytosis. This variant is caused by a HBB gene mutation c.71T > A resulting in an amino acid exchange on position 23 of the β globin chain (p.Val23Asp.). The influence of Hb Strasbourg on HbA1c measurement has not been studied to date. For patients with hemoglobin variants it is important to know whether possible interferences exist with the measurement of HbA1c. We therefore investigated the influence of Hb Strasbourg on HbA1c measurement with two different HPLC (high-performance liquid chromatography) systems and one turbidimetric immunoassay in two non-diabetic brothers who are heterozygous carriers of Hb Strasbourg. The examined tests are all used in routine diagnostics. In the case of Hb Strasbourg, the HbA1c measured by HPLC showed lower results than those obtained by the immunoassay. We conclude that HbA1c is underestimated when measured with these methods as glycated Hb Strasbourg is most likely not co-eluting with HbA1c in HPLC.

First Observation of Hemoglobin Ypsilanti in Saudi Arabia: A High Oxygen Affinity Hemoglobin Presenting with Extreme Erythrocytosis

Abstract High oxygen affinity hemoglobin (HOAH) variants are rare hemoglobinopathies sometimes associated with congenital erythrocytosis. Patients with HOAH are usually asymptomatic and are incidentally discovered when unexplained erythrocytosis is observed in the complete blood count. Here, we report the first patient with hemoglobin (Hb) Ypsilanti from high-altitude Southern Saudi Arabia, who presented with chronic extreme erythrocytosis (polycythemia) with an Hb concentration of 23 g/dL and hematocrit of 70%. The patient lacked any finding suggestive of secondary erythrocytosis, and the evaluation for acquired polycythemia vera was negative. Hb protein analysis and genetic testing confirmed the presence of a high-affinity Hb variant. Living at high altitude contributed to the presentation of extreme erythrocytosis. Besides highlighting important clinical and diagnostic characteristics, this case illustrates two important diagnostics caveats: high-affinity Hb can be misdiagnosed as another more common qualitative hemoglobinopathy such as Hb S or Hb D carrier state, and it can interfere with HbA1C analysis.

Syracuse Hemoglobinopathy Presenting With Tophaceous Gout: A Case Report.

Hemoglobinopathies are inherited disorders of hemoglobin that alter oxygen binding capacity by affecting the production of a specific subset of globin chains or their entire structure. A lesser-known subtype, Syracuse hemoglobinopathy, was first identified in 4 generations of a family in the 1970s. Gout has been reported in several forms of hemoglobinopathy, such as thalassemia and hemoglobin C disorder. A 44-year-old man with a known history of Syracuse hemoglobinopathy, tobacco use disorder, and shoulder osteoarthritis presented with diffuse nodular masses on his joints along with joint pain. His laboratory tests and imaging showed elevated uric acid, urate crystals in his synovial fluid, and bony erosions. These findings were concerning for gout, which was treated with allopurinol, prednisone, and colchicine, resulting in improvement in his symptoms. Syracuse hemoglobinopathy, a rare disorder of high oxygen affinity hemoglobin, can present itself with findings of elevated serum uric acid and tophaceous gout. Most patients with hyperuricemia never go on to develop gout. However, having a condition that increases serum levels of uric acid should raise an astute clinician's suspicion when a patient presents with a history of hemoglobinopathy and joint pain.

JAK2 unmutated erythrocytosis: 2023 Update on diagnosis and management.

JAK2 unmutated or non-polycythemia vera (PV) erythrocytosis encompasses a heterogenous spectrum of hereditary and acquired entities. Foremost in the evaluation of erythrocytosis is the exclusion of PV through JAK2 (inclusive of exons 12-15) mutation screening. Initial assessment should also include gathering of previous records on hematocrit (Hct) and hemoglobin (Hgb) levels, in order to streamline the diagnostic process by first distinguishing longstanding from acquired erythrocytosis; subsequent subcategorization is facilitated by serum erythropoietin (Epo) measurement, germline mutation screening, and review of historical data, including comorbid conditions and medication list. Hereditary erythrocytosis constitutes the main culprit in the context of longstanding erythrocytosis, especially when associated with a positive family history. In this regard, a subnormal serum Epo level suggests EPO receptor mutation. Otherwise, considerations include those associated with decreased (high oxygen affinity Hgb variants, 2,3-bisphosphoglycerate deficiency, PIEZO1 mutations, methemoglobinemia) or normal oxygen tension at 50% Hgb saturation (P50). The latter include germline oxygen sensing pathway (HIF2A-PHD2-VHL) and other rare mutations. Acquired erythrocytosis commonly results from central (e.g., cardiopulmonary disease, high-altitude habitat) or peripheral (e.g., renal artery stenosis) hypoxia. Other noteworthy conditions associated with acquired erythrocytosis include Epo-producing tumors (e.g., renal cell carcinoma, cerebral hemangioblastoma) and drugs (e.g., testosterone, erythropoiesis stimulating agents, sodium-glucose cotransporter-2 inhibitors). Idiopathic erythrocytosis is an ill-defined terminology that presumes the existence of an increased Hgb/Hct level without an identifiable etiology. Such classification often lacks accounting for normal outliers and is marred by truncated diagnostic evaluation. Current consensus treatment guidelines are not supported by hard evidence and their value is further undermined by limited phenotypic characterization and unfounded concerns for thrombosis. We are of the opinion that cytoreductive therapy and indiscriminate use of phlebotomy should be avoided in the treatment of non-clonal erythrocytosis. However, it is reasonable to consider therapeutic phlebotomy if one were to demonstrate value in symptom control, with frequency determined by symptoms rather than Hct level. In addition, cardiovascular risk optimization and low dose aspirin is often advised. Advances in molecular hematology might result in better characterization of "idiopathic erythrocytosis" and expansion of the repertoire for germline mutations in hereditary erythrocytosis. Prospective controlled studies are needed to clarify potential pathology from JAK2 unmutated erythrocytosis, as well as to document the therapeutic value of phlebotomy.

The Clinical Profile of JAK2 Negative,Normal Erythropoeitin,Normal Arterial Oxygen Saturation Erythrocytosis in an Indian Tertiary Centre

Introduction: Polycythemia Vera (PV) is a myeloproliferative syndrome characterized by JAK2 mutation with evidence of panmyelosis in the bone marrow [WHO 2016]. PV has risk of thrombosis and transformation to myelofibrosis /leukemia. Etiologies enlisted for JAK2 negative erythrocytosis include high-oxygen affinity hemoglobin variants, 2,3 biphosphoglycerate deficiency, and germline (e.g., PHD2, HIF2A, VHL) or EPO signaling (e.g., EPOR) mutations. Investigating for such causes is not feasible in rural India. JAK2 negative erythrocytosis is the category comprising these mutations and idiopathic causes. The treatment recommendations for JAK2 negative erythrocytosis is limited. The risk of thrombosis in this group is unknown. We present the clinical profile of subgroup of JAK2 Negative erythrocytosis with normal Erythropoietin [EPO] and normal arterial oxygen saturation [PaO2] from a tertiary care center in India. Method of study: Retrospective analysis of all the patients presented to the hematology outpatient clinic with high Hb /Hematocrit >16.5/49 for males and >16/48 for females from August 2019 to November 2021 were evaluated. Secondary causes evaluated. They underwent clinical examination, Complete blood count, ABG analysis for arterial oxygen saturation, Serum EPO estimation by chemiluminescence immunoassay [Normal: 2.6 - 18.5 mIU/mL], blood JAK2 V617F and JAK2 exon 12 mutation analysis by NGS at Medgenome Labs Pvt Limited, Bengaluru, Unilateral bone marrow aspiration and biopsy if willing. Inclusion criteria: 1.Hb /HCT >16.5gram%/ 49 for men and >16gram% /48 for women. Exclusion Criteria: 1.History of smoking, cyanosis or obstructive sleep apnea, COPD or cyanotic heart disease or high altitude, anabolic steroids. 2.Family history. 3.PaO2 below normal. 4.WBC > 11000/microliter, Platelets > 450000/microliter. 5.Palpable splenomegaly. 6.Erythropoietin levels <2.6mIU/ml or>8.5mIU/ml. 7.Jak2 V617F or Exon 12 mutation in NGS. 8.Panmyelosis in bone marrow [WHO 2016 criteria] Results: Total 256 patients were with elevated Hemoglobin.122 patients were excluded in view of meeting one of the exclusion criteria. Additional 58 patients were excluded because of lack of JAK2 mutational analysis. 76 patients who met the inclusion criteria of JAK2 negative erythrocytosis with normal EPO and normal PaO2.75 patients were males, mean age was 40 years [Range:15 -75]. Mean Hemoglobin at presentation was 18gram% [Range:16.8 to 22]. Mean erythropoietin was 6.69mIU/ml [Range 3-16.3]. The median follow up was for 8 months [Range:1 month to 24 months]. 37 patients were asymptomatic at presentation, for whom high Hb was detected while screening for blood donation or as a part of routine checkup. 16 patients developed thrombosis at the presentation. Median age of the patient's developing thrombosis was 42 [range 24 to 68 years].8 patients developed stroke,3 patients developed coronary artery disease,4 patients developed venous thrombosis.15 patients had hypertension at presentation. Other presenting symptoms were headache, redness of eyes, giddiness. Only 13 patients underwent bone marrow examination, in which 9 had normal bone marrow, 4 had isolated erythrocytosis. 47 patients took aspirin regularly. 32 patients underwent phlebotomy once a month if Hb/HCT >15gram/45. 13 of the 16 patients with thrombosis at presentation were continuing aspirin and were adhering to phlebotomy during follow up.3 patients received hydroxyurea as they were not tolerating phlebotomy. No patients developed new thrombotic events during the follow up. There was no correlation with level of Hb or EPO with thrombosis. Discussion: Our retrospective of JAK2 negative erythrocytosis data showed that the majority of patients were male,48 % patients were asymptomatic and had no thrombotic complications during the follow up. 21% developed thrombotic events at presentation. The duration of follow up is short in our study. Ideal treatment for thrombosis in this group needs further clinical trials. Treatment protocol for asymptomatic patients also need further clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Influence of High Hemoglobin-Oxygen Affinity on Humans During Hypoxia.

Humans elicit a robust series of physiological responses to maintain adequate oxygen delivery during hypoxia, including a transient reduction in hemoglobin-oxygen (Hb-O2) affinity. However, high Hb-O2 affinity has been identified as a beneficial adaptation in several species that have been exposed to high altitude for generations. The observed differences in Hb-O2 affinity between humans and species adapted to high altitude pose a central question: is higher or lower Hb-O2 affinity in humans more advantageous when O2 availability is limited? Humans with genetic mutations in hemoglobin structure resulting in high Hb-O2 affinity have shown attenuated cardiorespiratory adjustments during hypoxia both at rest and during exercise, providing unique insight into this central question. Therefore, the purpose of this review is to examine the influence of high Hb-O2 affinity during hypoxia through comparison of cardiovascular and respiratory adjustments elicited by humans with high Hb-O2 affinity compared to those with normal Hb-O2 affinity.

Importance of Sequencing HBA1, HBA2 and HBB Genes to Confirm the Diagnosis of High Oxygen Affinity Hemoglobin.

High oxygen affinity hemoglobin (HOAH) is the main cause of constitutional erythrocytosis. Mutations in the genes coding the alpha and beta globin chains (HBA1, HBA2 and HBB) strengthen the binding of oxygen to hemoglobin (Hb), bringing about tissue hypoxia and a secondary erythrocytosis. The diagnosis of HOAH is based upon the identification of a mutation in HBA1, HBA2 or HBB in specialized laboratories. Phenotypic studies of Hb are also useful, but electrophoretic analysis can be normal in 1/3 of cases. The establishment of the dissociation curve of Hb can be used as another screening test, a shift to the left indicating an increased affinity for Hb. The direct measurement of venous P50 using a Hemox Analyzer is of great importance, but due to specific analytic conditions, it is only available in a few specialized laboratories. Alternatively, an estimated measurement of the P50 can be obtained in most of the blood gas analyzers on venous blood. The aim of our study was therefore to determine whether a normal venous P50 value could rule out HOAH. We sequenced the HBB, HBA1 and HBA2 genes of 75 patients with idiopathic erythrocytosis. Patients had previously undergone an exhaustive medical check-up after which the venous P50 value was defined as normal. Surprisingly, sequencing detected HOAH in three patients (Hb Olympia in two patients, and Hb St Nazaire in another). A careful retrospective examination of their medical files revealed that (i) one of the P50 samples was arterial; (ii) there was some air in another sample; and (iii) the P50 measurement was not actually done in one of the patients. Our study shows that in real life conditions, due to pre-analytical contingencies, a venous P50 value that is classified as being normal may not be sufficient to rule out a diagnosis of HOAH. Therefore, we recommend the systematic sequencing of the HBB, HBA1 and HBA2 genes in the exploration of idiopathic erythrocytosis.

High-Oxygen-Affinity Hemoglobinopathy-Associated Erythrocytosis: Clinical Outcomes and Impact of Therapy in 41 Cases

▪BackgroundApproximately 100 high oxygen affinity (HOA) hemoglobin (Hgb) variants have been reported to date; review of the Mayo Clinic laboratory database (1974-2018) identified 80 distinct variants including 12 novel variants (60 β, 20 α). One-third of HOA Hgb variants result in secondary erythrocytosis, provoking concern regarding increased risk for thrombosis. Current management guidelines lack supporting evidence regarding the utilization of phlebotomy in such cases. We describe presenting features, treatment strategies and follow-up events involving 41 consecutive cases seen at our institution.MethodsStudy patients were recruited from our institutional laboratory and clinical Hgb variant database. Initial evaluation for Hgb variants was conducted by capillary electrophoresis and high-performance liquid chromatography. Additional testing which included mass spectrometry and isoelectric focusing was pursued as necessary. Since the majority of variants were difficult to identify by protein studies, DNA sequencing of HBB, or HBA1, HBA2 genes was performed for confirmation. Symptoms and thrombosis, both at presentation and during follow up, were recorded. Therapeutic interventions were based on physician discretion and mostly included phlebotomy and/or aspirin therapy; a careful response assessment was performed to determine the impact of each therapy on symptoms and/or thrombosis.ResultsA total of 41 patients with HOA Hgb variant-associated erythrocytosis (median age 39 years, range 1-81; 54% males) were seen at our institution between January 1973 and February 2020. The majority of the patients carried β-chain variants (n=34; 83%), common variants being Hgb Malmo (n=13), Olympia (n=4), San Diego (n=3), and Wood (n=2). Among the 7 patients with α-chain variants, Hgb Dallas was the most frequent (n=4).Presenting median values (range) for Hgb/Hct, serum erythropoietin and p50 were 18 g/dl/52.9%(16-21.9g/dl/48-66%) 10.4 mIU (4-36.3 mIU), and 20 mmHg (12-25 mmHg), respectively. Family history was documented in 34 patients, of which 24 (71%) reported one or more affected family members. Family history of thrombosis was documented in 7 patients (21%). CV risk factors were present in half of the patients; by contrast, history of thrombosis prior to or at diagnosis was documented in only two patients (5%). Of 23 pregnancies reported in 12 women, live birth rate was 78% (n=18); none of the fetal losses were attributed to erythrocytosis. Active therapies at the time of initial referral consisted of phlebotomy (n=12), aspirin (n=11) and systemic anticoagulation (n=1).At a median follow-up of 10 years (range; 0.04-44), 23 patients had reported one or more symptoms, attributed to hyper-viscosity, such as headaches, fatigue, and lightheadedness. Neither Hct level at diagnosis (p=0.32) nor phlebotomy (p=0.16; 75% patients on vs 52% not on phlebotomy) or aspirin therapy (p=0.75; 55% patients on vs 60% not on aspirin) appeared to influence the occurrence of symptoms. Phlebotomy relieved symptoms in 7 (42%) symptomatic patients; however, 7 (30%) of 23 patients on phlebotomy reported one or more adverse symptoms that were attributed to phlebotomy-induced iron deficiency.Ten patients (24%) experienced thrombosis prior to or following diagnosis: 6 arterial and 4 venous. Median age at thrombotic event was 51 years and median hematocrit 52%; active therapies at the time of event included phlebotomy in 5 patients, aspirin in 4, and systemic anticoagulation in 2. Hct level at diagnosis (p=0.10) or the time of event (p=0.67) did not correlate with occurrence of thrombosis. Additionally, the incidence of thrombosis was no different among patients receiving or not receiving phlebotomy (5/23(22%) vs 5/18(28%), respectively; p=0.66). The presence of CV risk factors was predictive of arterial events (p=0.002). Two of the 4 venous events developed in the context of concomitant thrombophilia. Eight patients (20%), median age 28 years, without CV risk factors, were observed without therapy for a median of 9.5 years (range; 0.4-21) and have not experienced any thrombosis to date.ConclusionsWe found no association between Hct level and either thrombotic or non-thrombotic symptoms in HOA hemoglobinopathy-associated erythrocytosis; furthermore, implementation of aggressive phlebotomy did not provide a clear benefit with respect to thrombosis risk reduction. [Display omitted] DisclosuresPatnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Genetic Background of Congenital Erythrocytosis.

True erythrocytosis is present when the red cell mass is greater than 125% of predicted sex and body mass, which is reflected by elevated hemoglobin and hematocrit. Erythrocytosis can be primary or secondary and congenital or acquired. Congenital defects are often found in those diagnosed at a young age and with a family history of erythrocytosis. Primary congenital defects mainly include mutations in the Erythropoietin receptor gene but SH2B3 has also been implicated. Secondary congenital erythrocytosis can arise through a variety of genetic mechanisms, including mutations in the genes in the oxygen sensing pathway, with high oxygen affinity hemoglobin variants and mutations in other genes such as BPMG, where ultimately the production of erythropoietin is increased, resulting in erythrocytosis. Recently, mutations in PIEZ01 have been associated with erythrocytosis. In many cases, a genetic variant cannot be identified, leaving a group of patients with the label idiopathic erythrocytosis who should be the subject of future investigations. The clinical course in congenital erythrocytosis is hard to evaluate as these are rare cases. However, some of these patients may well present at a young age and with sometimes catastrophic thromboembolic events. There is little evidence to guide the management of congenital erythrocytosis but the use of venesection and low dose aspirin should be considered.

Thromboembolic and Vascular Complications in Patients with Secondary Erythrocytosis Caused By High Oxygen Affinity Variant Hb Coimbra

There are 99 hemoglobin (Hb) variants with increased oxygen affinity inducing compensatory erythrocytosis (Globin Gene Server Web Site: http://globin.cse.psu.edu). One of these mutations, named Hb Coimbra, was described in 1991 in Portugal by Tamagnini et al. (HBB c.300T>A, p.Asp100Glu). Limited amount of published clinical and laboratory data of this condition and the absence of specific guidelines for its management suggest it would be a very benign condition. Here we report clinical and laboratorial findings of carriers of Hb Coimbra with some unexpectedly more severe clinical presentations.We collected peripheral blood venous samples from 10 patients (6 female), aged 31-72 years, upon signed informed consent. All of them were diagnosed with erythrocytosis, with hematocrits in the 46.9-78.1% range at the time of diagnosis, and were heterozygous for the Hb Coimbra mutation as confirmed by direct sequencing of the β-globin gene. The β-globin cluster haplotypes were determined by TaqMan SNP Genotyping Assay (rs7482144, rs968857, rs16911905) and other polymorphisms in the β-globin gene by direct sequencing (rs713040, rs10768683, rs7480526, rs7946748). We were able to identify a common haplotype for the Hb Coimbra allele, which strongly suggests a common origin despite the patients belonging to 5 different unrelated families.Three patients had complications possibly associated with the disease. Patient 1, male, 64 years old, presented with pulmonary embolism in July 2012. Patient 2, male, 60 years old, suffered a left lower limb amputation due to complicated venous thromboembolism in 2010 before he was diagnosed with Hb Coimbra and, in January of 2015, he also developed right pulmonary artery thromboembolism. Patient 3, male, 72 years old, presented with left hemiparesis in 2016 due to intraparenchymatous hemorrhage in the right basal ganglia.Differently from the other patients, elevated levels of serum erythropoietin were observed only in patients 1, 2, and 3 (81.1 mUI/mL, 423.5 mUI/mL, and 35.4 mUI/mL, respectively, reference range 2.6-18.5 mUI/mL). We speculate this abnormality could be associated with the therapeutic phlebotomy treatment performed in these cases.Oxygen affinity was evaluated by determining the p50 in whole blood samples without Hb isolation. O2 dissociation and saturation curves demonstrated that samples containing Hb Coimbra had considerably high affinity for oxygen in comparison to wild type (p50deoxi and p50oxi for Hb Coimbra were, 8.22 [SD 0.64] and 7.94 [SD 0.98], respectively, vs. wild type p50deoxi = 15.56 [SD 0.50] and p50oxi = 15.56 [SD 0.77]). Heme-heme cooperativity was also significantly reduced, tending to a non-cooperative event. Hill coefficients were ndeoxi - Hb Coimbra= 1.51 (SD 0.19) and noxi - Hb Coimbra = 1.48 (SD 0.32), ndeoxi - wild type = 2.52 (SD 0.22) and noxi - wild type = 2.41 (SD 0.32). In spite of the very high O2 affinity of Hb Coimbra, no significant difference was observed between samples from patients with clinical complications and from the remaining Hb Coimbra carriers.Alterations in the α1β2 interface, such as the one in Hb Coimbra, promote conformational changes in the structure of the hemoglobin, and tend to promote O2 binding, reducing oxygen supply to peripheral tissues and resulting in compensatory erythrocytosis (Wajcman et al, 2005). Mutations that generate hemoglobin variants are very rare, but should be considered in the context of familial erythrocytosis or in cases in which more frequent hematological diseases such as polycythemia vera have been excluded.In general, hemoglobin variants with high oxygen affinity manifest exclusively with erythrocytosis and a benign clinical course; however, our data suggest that this might not be the case in some patients, particularly in carriers of Hb Coimbra. Our experience supports that some patients may benefit from the prophylactic use of aspirin or therapeutic phlebotomy as attempts to reduce the incidence of thromboembolic or other vascular complications.Taken together, our data underscore that larger and multicentric studies of the clinical complications secondary to high oxygen affinity hemoglobins are necessary to define guidelines for the optimal management of this type of erythrocytosis. DisclosuresFertrin:Alexion Pharmaceuticals: Consultancy.

Congenital erythrocytosis

Erythrocytosis, or increased red cell mass, may be labeled as primary or secondary, depending on whether the molecular defect is intrinsic to the red blood cells/their precursors or extrinsic to them, the latter being typically associated with elevated erythropoietin (EPO) levels. Inherited/congenital erythrocytosis (CE) of both primary and secondary types is increasingly recognized as the cause in many patients in whom acquired, especially neoplastic causes have been excluded. During the past two decades, the underlying molecular mechanisms of CE are increasingly getting unraveled. Gain-in-function mutations in the erythropoietin receptor gene were among the first to be characterized in a disorder termed primary familial and congenital polycythemia. Another set of mutations affect the components of the oxygen-sensing pathway. Under normoxic conditions, the hypoxia-inducible factor (HIF), upon hydroxylation by the prolyl-4-hydroxylase domain protein 2 (PHD2) enzyme, is degraded by the von Hippel-Lindau protein. In hypoxic conditions, failure of prolyl hydroxylation leads to stabilization of HIF and activation of the EPO gene. CE has been found to be caused by loss-of-function mutations in VHL and PHD2/EGLN1 as well as gain-of-function mutations in HIF-2α (EPAS1), all resulting in constitutive activation of EPO signaling. Apart from these, globin gene mutations leading to formation of high oxygen affinity hemoglobins also cause CE. Rarely, bisphosphoglycerate mutate mutations, affecting the 2,3-bisphosphoglycerate levels, can increase the oxygen affinity of hemoglobin and cause CE. This narrative review examines the current mutational spectrum of CE and the distinctive pathogenetic mechanisms that give rise to this increasingly recognized condition in various parts of the world.

Haemoglobin Olympia {β Codon 20 (B2) G→A, Val→Met}: A Silent Haemoglobin Variant

High oxygen affinity haemoglobin variants are rare and often underdiagnosed in persistent erythrocytosis with no apparent aetiology. Here the author present a 29-year-old Indian male patient with a long-standing history of erythrocytosis which was incidentally detected. The proband had a prothrombotic family history of cerebral vessel stroke in his paternal grandfather at a young age and unexplained erythrocytosis in his father and brother. A review of his haemograms showed persistent high haemoglobin values. Routine tests did not reveal any specific aetiology and haemoglobin electrophoresis by High-Performance Liquid Chromatography (HPLC) showed absence of any abnormal peak or unstable haemoglobin. DNA sequencing of the β globin gene revealed heterozygosity for codon 20 {GTG→ATG, Valine (Val)→ Methionine (Met)} mutation confirming the presence of an electrophoretically silent Hb variant - Haemoglobin Olympia in him and his extended family members. This case study emphasises importance of this rare entity of high oxygen affinity haemoglobin variant as a differential diagnosis while screening for erythrocytosis. This is the first case report of Haemoglobin Olympia from India reported in the literature.

Novel High Oxygen Affinity Hemoglobin Variant in a Patient with Polycythemia: Hb Kennisis [β85(F1)Phe→Leu (TTT>TTG); HBB: c.258T>G

We report the case of a 61-year-old Canadian male of Maltese descent investigated for unexplained polycythemia. Decreased p50 suggested the presence of a high oxygen affinity hemoglobin (Hb) variant. Molecular genetic testing demonstrated that he carries a novel missense mutation (HBB: c.258T>G), resulting in a Phe→Leu substitution at position 85 of the β chain. The novel Hb variant has been designated Hb Kennisis in recognition of where the proband resides. Two other missense mutations have been reported at this position [Hb Bryn Mawr or Hb Buenos Aires, β85(F1)Phe→Ser (HBB: c.257T>C); Hb Grantham, β85(F1)Phe→Cys; (HBB: c.257T>G)], both of which have increased oxygen affinity.

A Kindred with a β-Globin Base Substitution [β89(F5)Ser→Arg (AGT>AGG); HBB: c.270T>G] Resulting in Hemoglobin Vanderbilt

High oxygen affinity hemoglobins (Hbs), characterized by a decreased ability to release oxygen to the tissues and a left-shifted oxygen dissociation curve, are a rare cause of secondary erythrocytosis. Here, we report a base substitution in the β-globin gene at codon 89 (AGT>AGG) in a kindred with familial erythrocytosis resulting in Hb Vanderbilt, a high oxygen affinity variant.

First Description of Hb San Diego (HBB: c.328G>A) in a Chinese Family with Congenital Erythrocytosis

Congenital erythrocytosis is a rare and hereditary disorder of red blood cell (RBC) production that can be caused by high oxygen affinity hemoglobin (Hb) variants. We applied a genetic approach including whole exome sequencing and Sanger sequencing. We identified a heterozygous β-globin gene (Hb San Diego or HBB: c.328G>A) in exon 3 as a causative germline mutation in a Chinese family with congenital erythrocytosis. We concluded that in erythrocytosis with a dominant inheritance and normal or inappropriately high erythropoietin (EPO) levels, the high oxygen affinity Hb variants should be considered. In addition, as a tool for identification of mutations in congenital erythrocytosis, whole exome sequencing improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

A guideline for the management of specific situations in polycythaemia vera and secondary erythrocytosis: A British Society for Haematology Guideline.

The previous BSH guideline for the management of erythrocytosis was published in 2005 (McMullin et al, 2005) and amended in 2007 (McMullin et al, 2007). Here, we re‐evaluate the literature formulate guidance on the management of specific situations encountered in polycythaemia vera (PV) and the management of the other types of secondary erythrocytosis. Recommendations for the diagnostic pathway of investigation of an erythrocytosis, risk stratification and management of PV are in the accompanying guideline (McMullin et al, 2018). We review evidence and outline guidance on management of acute thrombotic events and secondary prevention of thrombosis in PV. The unusual thrombotic events, splanchnic vein and cerebral vein thromboses are discussed and haemorrhage. The specific situations of surgery and pregnancy and guidance on management of pruritus are included. The evidence for the management of other causes of erythrocytosis, including idiopathic erythrocytosis, congenital erythrocytosis, hypoxic pulmonary disease and post‐transplant erythrocytosis, is reviewed and recommendations made.

Crystal Structure Analysis of Great Cormorant (Phalacrocorax carbo) Hemoglobin to Understand its High Oxygen Affinity Characteristics by Special Structural Features.

Hemoglobin (Hb) subunits are composed of the specific functional prosthetic group "heme'' and a protein moiety "globin". Bird Hbs are functionally similar to mammalian Hbs but they are structurally dissimilar with mammalian. The insufficient structural studies on avian Hbs limit us to understand their degree of adaptation to such critical environments. The Great Cormorant (GCT) can fly and swim, the dual characteristic of GCT leads to study the sturcture of hemoglobin. To determine the crystal structure of Great Cormorant Hemoglobin and to compare its three dimensional structure with other high and low oxygen affinity hemoglobin species to understand its characteristic features of high oxygen affinity. The GCT hemoglobin has been purified, crystallized and data sets were processed using iMosflm. The integrated data has been solved using Molecular replacement method using Graylag hemoglobin (1FAW) as the template. The structure has been deposited in Protein Data Bank with PDB code: 3WR1. In order to characterize the tertiary and quaternary structural differences, the structure of cormorant hemoglobin is compared with GLG, BHG and human Hb. The larger variation observed between GCT and human Hb indicates that GCT Hb differs remarkably from human. The α1β1 interface of Great cormorant Hb is similar to bar-headed goose Hb with few amino acid substitutions. It has been found that the interaction which is common among avian hemoglobins (α119 Pro- β55Leu) is altered by Ala 119 in GCT. This intra-dimer contact (α119 Pro - β 55 Leu) disruption leads to high oxygen affinity in BGH Hb. In cormorant, GLG and human the proline is unchanged but interestingly, in cormorant Hb, the β55 position was found to be Thr instead of Leu. Similar kind of substitutions (β 55 Leu - Ser) observed in Andean goose Hb structure leads to elevated oxygen affinity between Hb-O2. To our surprise, such type of substitution at β 55 (Thr) in cormorant Hb confirms that it is comparable with Andean goose Hb structure. Thus the sequence, structural differences at alpha, beta heme pocket and interface contacts confirms that GCT adopts high oxygen affinity conformation. The three dimensional structure of Great cormorant hemoglobin has been investigated to understand its unique structural features to adopt during hypoxia condition. By comparing the sequence and overall structural similarities with high and low oxygen affinity species, it appears that GCT has more possibilities to subsist with low oxygen demand.

Genotype-Phenotype Correlation of Hereditary Erythrocytosis Mutations, a single center experience.

Hereditary erythrocytosis is associated with high oxygen affinity hemoglobin variants (HOAs), 2,3-bisphosphoglycerate deficiency and abnormalities in EPOR and the oxygen-sensing pathway proteins PHD, HIF2α, and VHL. Our laboratory has 40 years of experience with hemoglobin disorder testing and we have characterized HOAs using varied protein and molecular techniques including functional assessment by p50 analysis. In addition, we have more recently commenced adding the assessment of clinically relevant regions of the VHL, BPGM, EPOR, EGLN1 (PHD2), and EPAS1 (HIF2A) genes in a more comprehensive hereditary erythrocytosis panel of tests. Review of our experience confirms a wide spectrum of alterations associated with erythrocytosis which we have correlated with phenotypic and clinical features. Through generic hemoglobinopathy testing we have identified 762 patients with 81 distinct HOA Hb variants (61 β, 20 α), including 12 that were first identified by our laboratory. Of the 1192 cases received for an evaluation specific for hereditary erythrocytosis, approximately 12% had reportable alterations: 85 pathogenic/likely pathogenic mutations and 58 variants of unknown significance. Many have not been previously reported. Correlation with clinical and phenotypic data supports an algorithmic approach to guide economical evaluation; although, testing is expanded if the suspected causes are negative or of uncertain significance. Clinical features are similar and range from asymptomatic to recurrent headaches, fatigue, restless legs, chest pain, exertional dyspnea and thrombotic episodes. Many patients were chronically phlebotomized with reported relief of symptoms. This article is protected by copyright. All rights reserved.

Coinheritance of High Oxygen Affinity Hb Helsinki [HBB: c.248A>T; β82(EF6)Lys→Met] with Hb H Disease

Hb Helsinki [HBB: c.248A>T; β82(EF6)Lys→Met] is a high oxygen affinity hemoglobin (Hb) causing polycythemia, whereas Hb H (β4) disease causes mild to severe chronic hemolytic anemia. The clinical characteristics, gel electrophoresis, capillary electrophoresis (CE) and molecular genotyping of a case of Hb Helsinki coinherited with Hb H disease in an ethnic Malay is described, illustrating the interaction between the β-globin variant and coinheritance of three α gene deletions. The proband was asymptomatic, exhibited microcytosis and a normal with Hb value.

High oxygen affinity hemoglobins

High oxygen affinity hemoglobins are responsible for rare and heterogeneous autosomic dominant genetic diseases. They cause pure erythrocytosis, sometimes accountable for hyperviscosity and thrombosis, or hemolysis. Differential diagnoses must be first ruled out. The diagnosis is based on the identification of a decreased P50, and their possible characterization by cation exchange-high performance liquid chromatography and capillary electrophoresis. Finally, genetic studies of the responsible globin chain gene will confirm the mutation. The prognosis mainly relies on the P50 decrease rate and on the hemoglobin cooperativity impairment. Disease management should be personalized, and it should primarily depend on smoking cessation and physical activity. Phlebotomy and platelet aggregation inhibitors’ prescriptions can be discussed. There is no contraindication to flights, high-altitude conditions, or pregnancy. Nevertheless, blood donation must be prohibited.