Hemoglobin Chains Research Articles

Plasma proteins and vitamin K (PIVKA‐II) status in school aged children in Nepal (804.22)

Vitamin K deficiency (VKD) is known to disrupt blood coagulation. However, broader public health effects of childhood VKD are unknown. We explored the plasma proteome to identify proteins that co‐vary with VK status and that may be associated with VKD in Nepalese children. We measured abundance of plasma proteins using quantitative mass spectrometry and concentration of protein induced by VK absence (PIVKA‐II), an indicator of VK status, in 500 rural children 6‐8 y of age. An elevated PIVKA‐II concentration (>2ng/mL), reflecting VKD and observed in 25% of children, was associated with higher concentrations of low‐density lipoproteins, total cholesterol, and triglycerides, and greater prevalence of vitamin D, E (cholesterol adjusted), and B12 deficiencies (all p<0.05). Among ~980 proteins observed in >10% of children, prostaglandin E synthase 3, coagulation factor II, vascular endothelial cadherin, voltage‐dependent calcium channel, gelsolin, and hemoglobin‐delta abundance were correlated with PIVKA‐II (all p<0.001, q<0.1). With a relaxed discovery threshold, other hemoglobin chains, glycolysis enzymes of erythrocytes and antioxidants were positively correlated with PIVKA‐II (all p<0.02, q<0.4). Since these proteins are involved in synthesis of vasodilators, regulation of Ca2+ influx in smooth muscle cells, and red blood cell metabolism in response to hypoxia, subclinical VKD may be involved in vascular and erythrocyte adaptation to low circulating oxygen in blood.Grant Funding Source: Supported by the Bill and Melinda Gates Foundation (Grant 5241).

The hypotensive effect of intrathecally injected (m)VD-hemopressin(α) in urethane-anesthetized rats

Previous studies suggest that cannabinoids system plays an important role in cardiovascular regulation. (m)VD-hemopressin(α) (VD-Hpα), an 11-residue peptide originating from the α1 chain of hemoglobin, was recently reported as a selective agonist of cannabinoid CB1 receptor. The present study was undertaken to investigate the intrathecal (i.t.) action of (m)VD-Hpα on blood pressure in urethane-anesthetized rats. Our results demonstrated that injections of (m)VD-Hpα (5–30nmol, i.t.) produced a dose-dependent decrease in mean arterial pressure (MAP), similar to that of the non-peptidic cannabinoid receptor agonist WIN55212-2 (1.25–10nmol, i.t.). The hypotensive effect of (m)VD-Hpα was not influenced by the CB1 receptor antagonist AM251 (20nmol, i.t.) or the CB2 receptor antagonist AM630 (20nmol, i.t.). However, WIN55212-2-induced hypotension was almost completely prevented by i.t. administration of AM251, not by AM630. The spinal hypotension of (m)VD-Hpα and WIN55212-2 was significantly reduced by pretreatment with the α-adrenoceptor antagonist phentolamine (1mg/kg, i.v.), but not by the β-adrenoceptor antagonist propranolol (2mg/kg, i.v.) or the muscarinic receptor antagonist atropine (2mg/kg, i.v.). In addition, l-NAME (50mg/kg, i.v.), the inhibitor of nitric oxide (NO) synthase, significantly reduced WIN55212-2-induced hypotension, but had no effect on the hypotensive response to (m)VD-Hpα. Collectively, the results show that i.t. administration of (m)VD-Hpα induces a decrease in MAP via a non-CB1 and non-CB2 mechanism.

Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis

Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.

Glucose and Hemoglobin A1c

Glucose, a monosaccharide, is the primary source of energy for the human body. It is used by the liver and other cells for energy or stored as glycogen for later use. The level of glucose in the bloodstream is regulated by 2 pancreatic hormones, insulin and glucagon. Insulin is released when glucose levels rise. Insulin acts by increasing glycogenesis, lipogenesis, and glycolysis, causing a decrease of glucose in the bloodstream. Glucagon is released when glucose levels fall, causing the liver to release stored glucose (glycogen) into the bloodstream (glycogenolysis), thereby increasing the level of glucose in the bloodstream. Hemoglobin A1c (HbA1c), also called glycosylated hemoglobin, is a hemoglobin compound produced when glucose reacts with the amino group on a hemoglobin molecule forming a ketoamine. The glucose molecule is attached to one or both N-terminal valines of the β-polypeptide chains of normal adult hemoglobin.1 The HbA1c formation is proportional to the blood glucose concentrations. Because the average red blood cell life is approximately 120 days, the glycosylated hemoglobin level reflects the average blood glucose level during the previous 2 to 3 months. Glucose testing is used to determine if an individual has hyperglycemia or hypoglycemia. A high fasting glucose level (≥126 mg/dL) and/or a high HbA1c level (>6.5%) might indicate that an individual has diabetes mellitus. HbA1c is a reliable method of monitoring long-term diabetes mellitus control; it determines the average blood glucose level of an individual during a period of approximately 3 months. Normal values range from 4.0% to 6.0%. Results of a study2 have shown the strong linear relationship between average blood glucose levels and HbA1c levels. Current American Diabetes mellitus Association guidelines recommend that a HbA1c test be performed at least twice yearly on patients who are meeting treatment … [↵][1]* To whom correspondence should be addressed. E-mail: vfreeman{at}utmb.edu [1]: #xref-corresp-1-1

A New Hemoglobin Variant: Hb Henan [β90(F6)Glu → Gln; HBB: c.271G < C

We have identified a new β chain hemoglobin (Hb) variant in a Chinese individual. Sequencing of the β-globin gene revealed a mutation in exon 2 at nucleotide 271, which results in the replacement of a glutamic acid by glutamine at codon 90 [β90(F6)Glu → Gln; GAG > CAG; HBB: c.271G > C] that we have named Hb Henan.

Hb A2-Konz [δ50(D1)Ser → Thr; HBD: c.151T > A]: a New δ Chain Hemoglobin Variant Characterized by Mass Spectrometry and High Performance Liquid Chromatography

We report a new slow-moving δ chain hemoglobin (Hb) variant, named Hb A2-Konz [δ50(D1)Ser → Thr; HBD: c.151T > A]. It was detected during simultaneous measurement of Hb A1C and Hb A2 by high resolution cation exchange high performance liquid chromatography (HPLC) using a PolyCATA column. Hb A2-Konz comprised 0.8% of total Hb. This new variant was identified by peptide mapping using nanoliquid chromatography electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) as a serine to threonine substitution at δ50(D1), indicating that the variant was due to a single base change at codon 51 (TCT > ACT) of the δ-globin gene. The new mutant is clinically silent but could lead to a misdiagnosis of β-thalassemia (β-thal) based on the level of Hb A2.

Red cell alloimmunization in thalassemia patients in a developing country – What can be done?

β-Thalassemia major is the major hemoglobinopathy in Pakistan. It has been recognized as a significant health problem with an incidence of over 5% in our general population [ [1] Hafeez M. Aslam M. Ali A. Rashid Y. Jafri H. Regional and ethnic distribution of beta thalassemia mutations and effect of consanguinity in patients referred for prenatal diagnosis. J Coll Physicians Surg Pak. 2007; 17: 144-147 PubMed Google Scholar ]. This disorder is characterized by reduced synthesis of β-globin chains of hemoglobin with subsequent anemia of variable severity, considerable morbidity affecting various organs of the body and has a high mortality.

Reasons of Unknown HPLC Peaks during Hemoglobin Analysis for Patients with Beta Thalassemias

Reasons of Unknown HPLC Peaks during Hemoglobin Analysis for Patients with Beta Thalassemias

Treatment Adherence and Quality of Life of Thalassemic Children

Thalassemia is a genetic disease in which a defect occurs in the rate of synthesis of α or β chains of hemoglobin leading to premature death of RBCs. Children suffering from thalassemia major may become pale and listless and have poor appetite, unexplained fever within first months after birth. For survival, these children have to undergo blood transfusions every 2–4 weeks depending on the severity of illness combined with iron chelation therapy to remove excess iron from their body due to frequent blood transfusions. These children do not always adhere to the treatment regimen which not only affects their physical health but the overall quality of life and may lead to early death in children. Thalassemia can be cured completely using bone marrow transplantation and cord blood stem cell transplantation. However, this treatment is possible only for a small number of children because of suitable donor and cost of treatment. Care of thalassaemia has been included in 12th Five year Plan of the Government of India. Nurses play a major role in improving the quality of life of children by providing the awareness to parents and children and motivate them to follow the treatment regimen.

Mitral Valve Repair Surgery in a Patient With Sickle Cell Disease

Sickle cell disease (SCD) affects between 70,000 and 100,000 Americans, making it one of the most prevalent genetic disorders in the United States. It is an autosomal, recessive disorder characterized by a single amino acid change in the betaglobin chain of hemoglobin (Hob) leading to its pathological polymerization, red cell rigidity and poor microvascular blood flow. These changes also produce the characteristic sickle shape of the red cells, which lose the pliability required to successfully traverse small capillaries. The use of cardiopulmonary bypass (CPB) during adult cardiac surgery initiates many of the conditions that contribute to the sickling of the hemoglobin S cells. Traditionally during CPB the patients are hypothermic and can become acidotic during intermediate periods of surgery. The experience of adult cardiac surgery with CPB on patients with SCD is limited. The purpose of our manuscript is to review the first case of adult cardiac surgery in a patient with SCD at our institution and also to review the literature on the management of patients with SCD who undergo adult cardiac surgery. J Hematol. 2014;3(1):10-12 doi: http://dx.doi.org/10.14740/jh102w

CXC Chemokines CXCL1, CXCL9, CXCL10 and CXCL12 are Variably Expressed in Patients with Sickle Cell Disease and Carriers: Are They Predictive Tools for Disease Complications?

Sickle cell hemoglobinopathies are amongst a group of genetic disorders resulting from a single base-pair DNA mutation at the beta chain of hemoglobin. Chemokines and cytokines play a part in the pathogenesis of inflammatory and infectious diseases. They are also involved in balancing angiogenesis/angiostasis processes to form new vascular networks. We aimed the present study to measure the circulating CXC chemokines CXCL1, CXCL9, CXCL10, and CXCL12 in the plasma of sickle cell patients (SCD). This cross-sectional study was conducted at the Kerman Special Disease Center and Rafsanjan Molecular Medicine Research Center during 2010 to 2011. Peripheral blood specimens were collected from 77 children with SCD and 70 controls. Serum samples were isolated and CXCL1, CXCL9, CXCL10, and CXCL12 were measured using ELISA. The findings of this study demonstrated that serum concentrations of CXCL1 and CXCL12 were elevated in SCD patients when compared with controls. Results also showed that the circulating levels of CXCL9 and CXCL10 were decreased in SCD patients in comparison to control subjects. However, we found increased levels of CXC chemokines in SCD patients suffering from pain crisis but the difference was not significant. According to the results of this study it can probably be concluded that the balance between angiogenesis/angiostasis CXC chemokines is an important predictive factor for initiation of complications in SCD patients. The elevated level of pro-inflammatory CXC chemokines may also be related to inflammatory responses associated with SCD complication.

Anesthetic management of a patient with β-thalassemia and superimposed pneumonia

Beta-thalassemia is an autosomal hematological disorder that is the result of genetically deficient synthesis of beta-globin chains of hemoglobin. Beta-thalassemia is one of the most common single-gene disorders with >400,000 new borns affected per year worldwide. Patients present with severe microcytic hypochromic anemia, massive hepatosplenomegaly, and bony deformities. Infection is a major complication and the leading cause of death in thalassemia out of which Pulmonary disease was the most common cause of mortality. In this report we aimed to discuss our experience with a case of beta-thalassemia major with massive splenomegaly & superimposed pneumonia in a child presenting for splenectomy.

Biomolecular Analysis and Biological Tissue Diagnostics by Electrospray Ionization with a Metal Wire Inserted Gel-Loading Tip

A metal wire-inserted disposable gel-loading tip was examined as an electrospray emitter. Its performance was similar to that of conventional electrospray ionization (ESI) with a relatively low flow rate (~100 nL/min) and without the need for solvent pumps. It was also used as an emitter for solid probe-assisted ESI (SPA-ESI) (e.g., biofluid was sampled from the biological tissue by a needle and was inserted into the solvent-preloaded gel-loading tip). Selective detection of lipids and proteins, such α and β chains of hemoglobin could be accomplished by choosing appropriate solvents. A suitable protocol for cancer diagnosis was established by this method. A good figure of merit of this method is its applicability to biological tissue diagnostics with high cost efficiency and on a disposable basis.

Mechanism and kinetics modeling of the enzymatic hydrolysis of α1–32 antibacterial peptide

Several antibacterial peptides can be obtained by enzymatic hydrolysis of the α chain of bovine hemoglobin. The kinetics of α1-32 peptide hydrolysis by pepsin was studied at several temperatures (15, 23 and 37°C). Intermediate and final peptides were identified, and their antibacterial activity was assessed against four bacterial species. Evolution of generated peptides concentration enabled to propose a reaction pathway describing the parallel and consecutive reactions taking place during the hydrolysis. A mathematical model, based on the proposed mechanism, was developed to describe the kinetics of generated peptides during α1-32 hydrolysis. The constants of the main reactions were identified based on the experimental data, and their dependence on temperature was established using Arrhenius-type equations. Validation of the proposed model was performed by predicting kinetics of α1-32 peptide hydrolysis at 30°C (all other experimental conditions being unchanged) with a good accuracy. This mathematical model could allow defining the optimal conditions for the production of various intermediate peptides with antibacterial activity from peptic hydrolysis of α1-32 peptide.

Advances in Technologies for Screening and Diagnosis of Hemoglobinopathies

Hemoglobinopathies constitute the most common monogenic disorders worldwide, caused by mutations in the globin genes that synthesize the globin chains of hemoglobin. Synthesis may be reduced (thalassemia) or underlie abnormal hemoglobins. Mutation interactions produce a wide range of disorders. For neonatal and antenatal screening, identification of affected newborns or carriers is achieved by hematological tests. DNA analysis supports definitive diagnosis, and additionally facilitates prenatal diagnosis procedures. Most methods used today have been developed over several decades, with few recent advances in hematology methods. However, DNA methods evolve continuously. With global migration and multiethnic societies the trend is from targeted, population-specific methods towards generic methods, such as Sanger sequencing (point mutations) and multiplex ligation probe amplification (deletions). DNA microarrays constitute an advanced DNA method for some mutation categories. The newest DNA technology is next-generation sequencing. Although not completely ready for routine use currently, next-generation sequencing may soon become a reality for some hemoglobin diagnostic laboratories.

A Novel β Globin Gene Mutation, Reported As An α Chain Hemoglobin Variant By High Performance Liquid Chromatography (HPLC) & As Sickle Cell Trait On New Born Screening: A case Report

IntroductionThere are 827 variants of β thalassemia reported to the registry of human hemoglobin variants and thalassemias registry 1. Genetic mutations of β thalassemia are very diverse but can be broadly divided in to non deletion forms and deletion forms. The new mutation is a frame shift insertion in exon 2 of the β globin gene. To the best of our knowledge this mutation has never been described before and presents as a mild form β thalassemia intermedia. ObjectiveTo describe the phenotypic presentation of the new β globin variant, due to insertion of 9 nucleotides (AAAGTGCTC) between nucleotides c.207 and c.208. Case reportA 22 months-old Hispanic boy who was referred for evaluation of persistent anemia. The new born screening for the child was positive for sickle cell trait. Initial hemoglobin (Hb) was 8.8, hematocrit was 27 and MCV was 65.5, which were decreased. RDW was 25.2, which was increased. Hemoglobin evaluation by acid and alkaline electrophoresis and HPLC revealed a HbS 0%, HbF 6.5% HbA 78.4%, HbA2 4.9% and a Hb variant 10.2%. The interpretation of the results was an α globin variant suggestive of Hb Montefiore and β thalassemia trait. Alpha thalassemia PCR for the 7 most common deletions in the α globin chain was negative. Subsequent α globin gene sequencing revealed no α globin gene mutations. On physical exam there were no bony changes or hepatosplenomegaly.Family HistoryThe mother is 29-year-old with HbAA. The father is 39-years-old with the same mutation on beta globin gene analysis. Hb electrophoresis also suggested an α and β globin mutation. Alpha thalassemia PCR was negative and he had a normal α globin gene copy number. At age 19 he had a splenectomy secondary to splenomegaly and hypersplenism. He is consistently anemic with Hb< 9 and MCV < 70.The child has a paternal half brother, who is 21 years old and has similar problems as father. He had a splenectomy at the age of 17 after admission for abdominal pain. The patient has a 5-year-old sibling, who is normal with HbAA and another paternal half sibling reported as no anemia. DiscussionThough β thalassemia intermedia is most commonly homozygous or compound heterozygous, less frequently it can be due to single locus mutation. DNA sequencing of the α globin gene of the index case was completely normal and there was normal copy number of the α globin gene in the father. No Hb S was found on Hb electrophoresis.The new mutation adds 9 base pairs to exon 2 and 3 amino acids (Lys-Val-Leu) between amino acid 68 and 69 of the protein. This elongates the beta chain which can lead to instability and precipitation of Hb as well as hemolysis and anemia2. Further studies like short time incubation and pulse chase globin chain synthesis experiments are needed to know the stability of the β globin protein3. In addition, an increase of α globin gene copy number can also be a reason for increasing the severity of β thalassemia trait2. However the α gene copy was normal in the father. ConclusionWe present a case of a child with a false positive abnormal newborn screen suggestive of sickle cell trait, as well as a Hb electrophoresis suggestive of an alpha globin mutation. As the father and paternal brother have had a splenectomy in their teen years with noted hepatosplenomegaly, suggestive of increased hemolysis, and the anemia is more severe than usual for β thalassemia trait, this suggests that phenotypically the c.199_207dup variant presents as a mild β thalassemia intermedia. In addition, there does not seem to be any bony abnormalities associated with marrow hyperplasia. As both our patients are heterozygous for this novel mutation with normal α globin gene copy number and alpha globin sequencing, we suspect that elongation of the β globin produces an unstable hemoglobin with a mild β thalassemia intermedia phenotype2.

Minor hemoglobins HbA2 and HbF associate with disease severity in bipolar disorder with a likely protective role of HbA2 against post-partum episodes

BackgroundThere exist studies indicating that bipolar disorder (BD) associates with changes in brain blood flow. Human brain with its high demand to oxygen constitutes 2% of the total body weight, while it receives 20% of cardiac output. α and β globin chains of hemoglobin were recently found in neural tissues, yet no study has questioned blood hemoglobins in BD. MethodsA total of 120 euthymic BD patients (40 males and 80 females) were analyzed via high performance liquid chromatography (HPLC) to measure minor hemoglobin levels, which were statistically compared with disease characteristics. ResultsMinor hemoglobins HbA2 and HbF associated positively with episode density as a measure of disease severity in BD. An increased level of HbA2 meant significantly less postpartum episodes in child bearing women. HbF levels were higher in patients with a positive family history of any psychotic disorder. Sum of HbA2 and HbF correlated with episode density with a stronger significance (p<0.001) supporting intermittent hypoxia hypothesis in BD. LimitationsThe study was conducted only on euthymic patients to avoid likely bigger exogenous effects such as electro-convulsive therapy and diverse drug regimes, yet larger comparative studies are needed to support our current findings. ConclusionsHigher HbA2 and HbF in more severe bipolar disorder may be compensations against intermittent hypoxias in BD. HbA2 increases following myocardial angina and in mountain dwellers, which may indicate protective roles in extreme conditions. HbF increase may act more as a maladaptation or emerge via haplotypal associations of BD genes and gamma-globin locus at 11p15.5.

MiR-451 Up-regulation, Induce Erythroid Differentiation of CD133+cells Independent of Cytokine Cocktails.

Erythropoiesis is regulated by some extrinsic and intrinsic factors as microRNAs (miRNAs). miRNAs are endogenously small non-coding regulatory RNAs which play vital roles in the variety of cellular fate, critical processes; growth, apoptosis, metabolism, survival of the cells and specially differentiation. Several miRNAs such as miR-16 and miR-451 have been shown to be correlated with erythroid differentiation. Taking into account the importance of miRNAs in cellular differentiation, the goal of the present study was to examine the role of miRNAs in hematopoietic stem cells (HSC) differentiation into the erythroid cells in the absence of growth factors and stimulatory cytokines. CD133+ stem cells were infected with lentiviruses containing miR-451/miR-16 precursor sequence, erythroid differentiation was evaluated using RT-PCR for hemoglobin chains and surface antigens, also by banzidine staining. MiR-451up-regulation, but not miR-16, could induce α, β and γ-globin expression in CD133+ cells and have strong correlation with appearance of CD71 and CD235a markers in these cells. Moreover, miR-451 up-regulation increases the banzidine positive cells to ~ %40. Our results provide strong evidence that miR-451 up-regulation strongly induces erythroid differentiation and maturation of CD133+ stem cells. Hence, this method may provide a useful technique for the production of artificial blood RBC and be used as a new strategy for gene therapy of hemoglobinopathies, such as β-thalassemias and sickle cell anemia.

Hemoglobin is present as a canonical α2β2 tetramer in dopaminergic neurons

Hemoglobin is the oxygen carrier in blood erythrocytes. Oxygen coordination is mediated by α2β2 tetrameric structure via binding of the ligand to the heme iron atom. This structure is essential for hemoglobin function in the blood. In the last few years, expression of hemoglobin has been found in atypical sites, including the brain. Transcripts for α and β chains of hemoglobin as well as hemoglobin immunoreactivity have been shown in mesencephalic A9 dopaminergic neurons, whose selective degeneration leads to Parkinson's disease. To gain further insights into the roles of hemoglobin in the brain, we examined its quaternary structure in dopaminergic neurons in vitro and in vivo. Our results indicate that (i) in mouse dopaminergic cell line stably over-expressing α and β chains, hemoglobin exists as an α2β2 tetramer; (ii) similarly to the over-expressed protein, endogenous hemoglobin forms a tetramer of 64kDa; (iii) hemoglobin also forms high molecular weight insoluble aggregates; and (iv) endogenous hemoglobin retains its tetrameric structure in mouse mesencephalon in vivo. In conclusion, these results suggest that neuronal hemoglobin may be endowed with some of the biochemical activities and biological function associated to its role in erythroid cells. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Hb Grove City [β38(C4)Thr→Ser, ACC>AGC; HBB: c.116C>G]: A New Low Oxygen Affinity β Chain Variant

A previously unreported β chain hemoglobin (Hb) variant, Hb Grove City [β38(C4)Thr→Ser, ACC>AGC; HBB: c.116C>G], was discovered in a woman who presented with hypoxia and mild anemia. Her young daughter also tested positive for the variant and displayed similar symptoms. Hemoglobin-oxygen dissociation testing confirmed right-shifted oxygen dissociation curves. A corresponding Hb variant was detected by high performance liquid chromatography (HPLC) and intact mass spectrometry (MS) but was not detected by capillary electrophoresis (CE), isoelectrofocusing (IEF) or alkaline or acid electrophoresis. DNA sequencing analysis confirmed a β-globin gene mutation. All three previous mutations at this locus affect oxygen affinity, as does this new variant. This newly described variant showed variable stability results and therefore may be mildly unstable but is not associated with microcytosis, significant hemolysis or clinically evident cyanosis. It is important to consider hemoglobinopathies in patients who are anemic and have unexplained hypoxia. Arterial blood gas and p50 evaluations may prevent unnecessary diagnostic interventions. Additionally, Hb variants with altered oxygen affinity can be electrophoretically silent; therefore, multiple methods including MS and/or DNA sequencing are warranted when clinical suspicion is high.