Hb Mutation Research Articles

Differential Effects on HbA1c Detection by HPLC and Capillary Electrophoresis in Five Types of Hb Variants in China.

Hemoglobin A1c (HbA1c) can be used to evaluate blood glucose control. Its measurement will be affected by many factors, but Hb variation is the most critical factor. This study aimed to explore the types of variants found in routine work and their impact on test results. Samples with abnormal HbA1c chromatograms found in routine testing were tested with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) and then further tested with polymerase chain reaction (PCR) and sequencing. Five recessive heterozygous mutations were identified after PCR and sequencing. Hb Riccarton-II (a mutation in the HBA2 gene), Hb E, Hb G-Coushatta, Hb G-Taipei, and Hb North Manchester (a mutation in the HbB gene) were identified. All HbA1c values of these variants detected by HLC-723 G8 (HPLC method) were lower than those of Sebia Capillarys 2 FP (C2FP, CE method) with P < .0001. Five Hb mutations were identified in our routine HbA1c test, and their HPLC detection values were significantly lower than those obtained with the CE method.

Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano-LC Coupled MALDI MS/MS.

Background: Hemoglobin (Hb) variants arise due to point mutations in globin chains and their pathological treatments rely heavily on the identification of the nature and location of the mutation in the globin chains. Traditional methods for diagnosis such as HPLC and electrophoresis have their own limitations. Therefore, the present study aims to develop and optimize a specific method of sample processing that could lead to improved sequence coverage and analysis of Hb variants by nano LC−MALDI MS/MS. Methods: In our study, we primarily standardized various sample processing methods such as conventional digestion with trypsin followed by 10% acetonitrile treatment, digestion with multiple proteases like trypsin, Glu−C, Lys−C, and trypsin digestion subsequent to 2,2,2 trifluoroethanol (TFE) treatment. Finally, the peptides were identified by LC−MALDI MS/MS. All of these sample processing steps were primarily tested with recombinant Hb samples. After initial optimization, we found that the TFE method was the most suitable one and the efficiency of this method was applied in Hb variant identification based on high sequence coverage. Results: We developed and optimized a method using an organic solvent TFE and heat denaturation prior to digestion, resulting in 100% sequence coverage in the β−chains and 95% sequence coverage in the α−chains, which further helped in the identification of Hb mutations. A Hb variant protein sequence database was created to specify the search and reduce the search time. Conclusion: All of the mutations were identified using a bottom−up non−target approach. Therefore, a sensitive, robust and reproducible method was developed to identify single substitution mutations in the Hb variants from the sequence of the entire globin chains. Biological Significance: Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. Hb variants generally arise due to point mutation in the globin chains. There is high sequence homology between normal Hb and Hb variant chains. Due to this high homology between the two forms, identification of variants by mass spectrometry is very difficult and requires the full sequence coverage of α− and β−chains. As such, there is a need for a suitable method that provides 100% sequence coverage of globin chains for variant analysis by mass spectrometry. Our study provides a simple, robust, and reproducible method that is suitable for LC−MALDI and provides nearly complete sequence coverage in the globin chains. This method may be used in the near future in routine diagnosis for Hb variant analysis.

Investigating the suitability of high-resolution mass spectrometry for newborn screening: identification of hemoglobinopathies and β-thalassemias in dried blood spots.

A fast and reliable method for the determination of hemoglobinopathies and thalassemias by high-resolution accurate mass spectrometry (HRAM/MS) is presented. The established method was verified in a prospective clinical study (HRAM/MS vs. high-pressure liquid chromatography [HPLC]) of 5335 de-identified newborn samples from the Hamburg area. The analytical method is based on a dual strategy using intact protein ratios for thalassemias and tryptic digest fragments for the diagnosis of hemoglobinopathies. Due to the minimal sample preparation and the use of flow injection, the assay can be considered as a high-throughput screening approach for newborn screening programs (2 min/sample). Using a simple dried blood spot (DBS) extraction (tryptic digest buffer), the following results were obtained: (1) a carrier incidence of 1:100 newborns (35 FAS, nine FAC, eight FAD and two FAE), and (2) no homozygous affected patient was detected. Using the HRAM/MS protocol, an unknown Hb mutation was identified and confirmed by genetic testing. In addition to greater specificity toward rare mutations and β-thalassemia, the low price/sample (1-2€) as well as an automated data processing represent the major benefits of the described HRAM/MS method.

Involvement of hemoglobins in the pathophysiology of Alzheimer's disease.

Hemoglobins (Hbs) are heme-containing proteins binding oxygen, carbon monoxide, and nitric oxide. While erythrocytes are the most well-known location of Hbs, Hbs also exist in neurons, glia and oligodendroglia and they are primarily localized in the inner mitochondrial membrane of neurons with likely roles in cellular respiration and buffering protons. Recently, studies have suggested links between hypoxia and neurodegenerative disorders such as Alzheimer Disease (AD) and furthermore suggested involvement of Hbs in the pathogenesis of AD. While cellular immunohistochemical studies on AD brains have observed reduced levels of Hb in the cytoplasm of pre-tangle and tangle-bearing neurons, other studies on homogenates of AD brain samples observed increased Hb levels. This potential discrepancy may result from differential presence and function of intracellular versus extracellular Hbs. Intracellular Hbs may protect neurons against hypoxia and hyperoxia. On the other hand, extracellular free Hb and its degradation products may trigger inflammatory immune and oxidative reactions against neural macromolecules and/or damage the blood-brain barrier. Therefore, biological processes leading to reduction of Hb transcription (including clinically silent Hb mutations) may influence intra-erythrocytic and neural Hbs, and reduce the transport of oxygen, carbon monoxide and nitric oxide which may be involved in the (patho)physiology of neurodegenerative disorders such as AD. Agents such as erythropoietin, which stimulate both erythropoiesis, reduce eryptosis and induce intracellular neural Hbs may exert multiple beneficial effects on the onset and course of AD. Thus, evidence accumulates for a role of Hbs in the central nervous system while Hbs deserve more attention as possible candidate molecules involved in AD.

Double Heterozygosity for Hb Durham-N.C. (HBB: c.344T>C) [β114(G16)Leu→Pro] and the IVS-I-110 (HBB: c.93-21G>A) Causing a Severe β-Thalassemia Phenotype

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two β-globin gene defects, a β-thalassemia (β-thal), mutation at IVS-I-110 (HBB: c.93-21G>A) and a variant hemoglobin (Hb) mutation at codon 114 (HBB: c.344T>C) on the other allele, reported as Hb Durham-N.C. (also known as Hb Brescia) [β114(G16)Leu→Pro] in the HbVar database. A very low Hb level (Hb 3.5 g/dL), microcytosis [mean corpuscular volume (MCV) 63.2 fL] and hypocromia [mean corpuscular Hb (MCH) 19.6 pg], increased red blood cell (RBC) distribution width (RDW) (36.0%), higher reticulocytes (6.2%), anisocytosis, poikilocytosis, hypocromia, basophilic stippling and inclusion body formation, were present in the affected subject. Analysis of other family components showed the presence of HBB: c.93-21G>A defect in the mother and in her brother, while Hb Durham-N.C. was absent in all other relatives, thus, this mutation has arisen as a de novo defect. This is the first case described as a severe thalassemic phenotype in a compound heterozygote carrier of this unstable Hb and a common β-thalassemic allele. The important information gained from this case is that a rare dominant or recessive mutation may arise in every individual, even if this is a very rare event.

Hemoglobin A1c Levels Are Slightly but Significantly Lower in Normoglycemic Subjects With the Hemoglobin E Phenotype.

Hb mutations can alter the structure, behavior, stability, or quantity of the globin chain produced. Some Hb variants shorten the erythrocyte life span, resulting in physiologically lower hemoglobin A1c (HbA1c) levels. The hemoglobin E (HbE) phenotype involves a single-nucleotide polymorphism that reduces β-globin chain synthesis. We compared the HbA1c levels of subjects with normal Hb (HbAA; N=131) and HbE (N=148) phenotypes, examining potential hematological and biochemical factors contributing to differences in HbA1c levels. All had normal fasting plasma glucose (<5.6 mmol/L), AST, ALT, and creatinine levels. Mean±SD HbA1c levels differed between HbAA and HbE subjects: 5.5±0.3% and 5.3±0.3% (P<0.001) according to an immunoassay, and 5.5±0.3% and 5.3±0.3% (P<0.001) according to cation-exchange HPLC, respectively. In multiple logistic regression, only mean corpuscular volume (P<0.001) contributed to the difference in HbA1c levels between groups. Although a 0.2% difference in HbA1c is relatively small and unlikely to alter clinical decisions, epidemiologically, this can lead to misclassification of a significant proportion of the population, especially since the threshold of non-diabetes HbA1c (≤5.6%) falls very close to the HbA1c median of the general population.

A Rapid, Affordable and Feasible Method for Detection of the HBG1: g.-225_-222delAGCA Polymorphism

Single point mutations or small deletions in the Aγ - and Gγ-globin gene promoter region are associated to the nondeletional hereditary persistence of fetal hemoglobin (HPFH). Currently, DNA sequencing is most common technique adopted for detection of hemoglobin (Hb) mutations. However, some can be rapidly detected because they either destroy or create a recognition site for a restriction enzyme. Here we show that the 4 bp deletion, HBG1: g.-225_-222delAGCA in the Aγ-globin gene promoter can be easily detected using the Tru1I (MseI) restriction enzyme that cuts only in the absence of this deletion. This approach utilizes ordinary instrumentations (thermocycler and agarose gel electrophoresis) available in any basic molecular genetics laboratory, providing a reliable and inexpensive method of genetic screening.

The Use of Dried Blood Spots: A Potential Tool for the Introduction of a Neonatal Screening Program for Sickle Cell Anemia in Zambia.

Context:Sickle cell disease is a group of hemoglobin (Hb) disorders resulting from the inheritance of the sickle β-globin gene. It is the most common pathological Hb mutation worldwide with 75% being born in Sub-Saharan Africa.Aims:This study aims to determine if dried blood spots (DBSs) can be used for diagnosis of sickle cell in newborns. In Zambia, there is no neonatal screening program for sickle cell anemia (SCA), yet it has been proved that early diagnosis by newborn screening (NBS) using DBSs and access to comprehensive care results in survival to adulthood of over 96% of sickle cell patients.Settings and Design:A cross-sectional study was carried out at the University Teaching Hospital to determine whether DBSs can be used to diagnose sickle cell using Hb electrophoresis.Subjects and Methods:Results from DBSs stored for 2 weeks were then compared to those obtained using freshly collected whole blood.Statistical Analysis Used:To evaluate performance characteristics, the following values were used: true positive, false positive, true negative, and false negative.Results:Ninety-seven participants were included in this study. DBSs had a sensitivity of 100%, a specificity of 94.7%, positive predictive value of 96.7%, negative predictive value of 100%, overall efficiency of 97.9%, and a Kappa r2, P < 0.0001 in comparison to fresh whole blood which we used as the gold standard.Conclusions:The use of DBSs can be recommended for NBS of SCA in Zambia due to its high sensitivity, specificity, and stability of hemoglobin.

The first validated criteria for effective screening and a new simplified method for α-globin gene sequencing for diagnosis of uncommon α-globin mutations.

No well-defined phenotypes that distinguish between unknown α- and β-globin mutations have been reported to date. Direct DNA sequencing of α-globin genes can be technically challenging, as α1- and α2-globin genes are nearly indistinguishable. To detect hemoglobin variants (HbXs) on Hb analysis, the entire β- and α-globin genes were directly sequenced using a newly developed sequencing protocol for α-globin genes. An algorithm to distinguish between α- and β-HbXs was constructed and subsequently validated in the independent validation group. Distinctive characteristics that can distinguish 39 α-HbXs from 24 β-HbXs were the presence of unidentifiable variants of HbA2 and/or HbX of <37% on isoelectric focusing and <31% on high-performance liquid chromatography. Another set of 67 HbXs was employed to validate our algorithm. This accurately predicted 33 α-HbXs with 100% sensitivity and 97.1% specificity. Our sequencing protocol for α-globin genes was able to identify 11 rare mutations among all exons of both α-globin genes from 72 subjects. Six of these variants were first discovered in Thais. This is the first well-characterized algorithm for distinguishing unknown Hb variants in a large cohort. Our validated criteria and DNA sequencing procedure are highly efficient for molecular characterization of rare Hb mutations.

Hemoglobinopathies in the Çukurova Region and Neighboring Provinces

To contribute to the creation of a mutation map of the region, we aimed to determine the mutation spectrum of thalassemias and abnormal hemoglobins (Hbs) in the Çukurova region and surrounding provinces. In this study, a total of 8135 samples from Adana, Hatay, Mersin, Konya and Kayseri provinces between 1993 and 2014 were analyzed. Complete blood cell (CBC) counts and Hb typing were carried out using automatic cell counters, cellulose acetate membrane electrophoresis and high performance liquid chromatography (HPLC), respectively. For the molecular analyses, genomic DNA was extracted using both manual and automated DNA extraction devices. Determination of Hb mutations were done by microarray, restriction fragment length polymorphism (RFLP), amplification refractory mutation system (ARMS) and gap-polymerase chain reaction (gap-PCR) methodologies. Samples were analyzed for abnormal Hb and thalassemia mutations. Out of 8135 samples, 1382 were observed to be carrying Hb mutations. It was identified that 826 mutation carriers included abnormal Hbs with a frequency of 59.7%, 416 carriers included β-thalassemia (β-thal) mutations with a frequency of 30.7% and 136 carriers included α-thalassemia (α-thal) mutations with a frequency of 9.9%. In this study, the most frequently observed abnormal Hb in the region was Hb S [β6(A3)Glu→Val (GTG > GAG), HBB: c.20T > A], whereas the most commonly observed mutations were the IVS-I-110 (G > A) (HBB: c.93–21G > A) point mutation in β-thal and the 3.7 kb deletion in α-thal.

135 Cardiovascular Complications in Sickle Cell Disease

<h3>Introduction</h3> Sickle cell disease (SCD) is a genetic disorder affecting the production of haemoglobin (Hb). It has life-long consequences with multi-organ involvement including the cardiopulmonary circulation and an increased mortality. Although all SCD patients share the same genetic defect, the phenotype varies. This suggests other factors, unrelated to the Hb mutation, play an important role particularly in later decades. This retrospective study examined patient survival attending a specialist tertiary centre in the United Kingdom based on Tricuspid Regurgitant Jet Velocity (TRJ) measured by echocardiography and associated laboratory studies. <h3>Method</h3> A retrospective analysis of TRJ and diastolic function (lateral E/E’ ratio) assessed by echocardiography in 127 intensively treated steady-state SCD patients (mean age 40 +/- 12 years) screened for cardiovascular complications. Laboratory studies were also analysed. <h3>Results</h3> The clinical characteristics of patients in this study is summarised in Table 1. 15% of patients had a TRJ between 2.6–2.99 m/s and 9% with TRJ &gt;3.0 m/s. 11% of patients had diastolic dysfunction using lateral E/E’ ratio. When compared to patients with a TRJ &lt;2.5 m/s, the risk of death is 4 times higher in the 2.6–2.99 TRJ group and 24 times higher in the &gt; 3.0 TRJ group. See Figure 1. Lateral E/E’ ratio (beta = 0.88, p = 0.015), left atrial area (beta = 0.38, p &lt; 0.001) and microalbuminuria (beta = 0.10, p = 0.05) were independently associated with TRJ. <h3>Conclusion</h3> Mortality in SCD patients with raised TRJ remains high despite intensive therapy, such as exchange transfusion, directed at correcting Hb. Screening with echocardiography remains an important tool in identifying high-risk patients. Additional therapies directed at the premature ageing of the cardiopulmonary circulation are urgently needed.

Intraspecific Polymorphism, Interspecific Divergence, and the Origins of Function-Altering Mutations in Deer Mouse Hemoglobin

Major challenges for illuminating the genetic basis of phenotypic evolution are to identify causative mutations, to quantify their functional effects, to trace their origins as new or preexisting variants, and to assess the manner in which segregating variation is transduced into species differences. Here, we report an experimental analysis of genetic variation in hemoglobin (Hb) function within and among species of Peromyscus mice that are native to different elevations. A multilocus survey of sequence variation in the duplicated HBA and HBB genes in Peromyscus maniculatus revealed that function-altering amino acid variants are widely shared among geographically disparate populations from different elevations, and numerous amino acid polymorphisms are also shared with closely related species. Variation in Hb-O2 affinity within and among populations of P. maniculatus is attributable to numerous amino acid mutations that have individually small effects. One especially surprising feature of the Hb polymorphism in P. maniculatus is that an appreciable fraction of functional standing variation in the two transcriptionally active HBA paralogs is attributable to recurrent gene conversion from a tandemly linked HBA pseudogene. Moreover, transpecific polymorphism in the duplicated HBA genes is not solely attributable to incomplete lineage sorting or introgressive hybridization; instead, it is mainly attributable to recurrent interparalog gene conversion that has occurred independently in different species. Partly as a result of concerted evolution between tandemly duplicated globin genes, the same amino acid changes that contribute to variation in Hb function within P. maniculatus also contribute to divergence in Hb function among different species of Peromyscus. In the case of function-altering Hb mutations in Peromyscus, there is no qualitative or quantitative distinction between segregating variants within species and fixed differences between species.

Hb Papanui [α99(G8)Lys→Arg; HBA2: c.299A>G]: A Novel Silent Substitution Interfering in Hb A1c Determination

We report a novel hemoglobin (Hb) mutation [Hb Papanui [α99(G8)Lys→Arg; HBA2: c.299A>G] that was identified in an individual with an inappropriately low Hb A1c value of 2.8% when using an ion exchange method. Although occurring at a well conserved site, the conservative substitution was not associated with any adverse clinical features.

Treatment with hydroxyurea in a patient compound heterozygote for a high oxygen affinity hemoglobin and β‐thalassemia minor

Compound heterozygotes for β-thalassemia and high oxygen affinity hemoglobin (Hb) have been documented, but experience in the management of such rare cases is minimal. Although hydroxyurea (HU) has never been used in a heterozygote with high oxygen affinity Hb and β-thalassemia, we hypothesized that it would decrease erythrocytosis through a lowered production of abnormal cells and increase of P 50 by induction of fetal hemoglobin (HbF). We present the case of a patient with compound high oxygen affinity Hb mutation with β-thalassemia. PCR analysis revealed combined Hb Regina and IVS1-110 G/A mutations. Treatment with HU caused a decrease in Ht (61.1% to 38.6%) and erythrocyte volume (74.87 mUkg to 40.65 mUkg), as well as an increase in P 50 (6 mmHg to 10 mmHg ) and HbF level (3.6% to 29.8%) at 12-month follow-up. Effects of HU on 2,3 DPG and HbNO levels did not appear to be predictable. Corroboration with other cases is needed to establish solid evidence on the clinical efficacy of HU in this population.

Electrospray Ionization Mass Spectrometric Analysis of the Globin Chains in Hemoglobin Heterozygotes Can Detect the Variants HbC, D, and E

We would like to point out that 2 recent articles in this journal about human hemoglobin (Hb) analysis (1)(2) give the false impression that variant globin chains with <6 Da mass difference from normal cannot be detected in heterozygotes by electrospray ionization mass spectrometry (ESI-MS). Kleinert et al. (1) state: “Two important drawbacks of the MS methods should be mentioned. First, its insufficient resolution prevents the detection of Hb mutations with small mass differences of the globin chains. The precision of normal low-resolution mass measurements was insufficient to distinguish the wild-type β-chain from several β-chain variants such as HbC, D, or E”. Brennan (2) comments similarly by stating that whereas traditional methods readily detect the majority of common variants, such as HbC, HbD, or HbE, “the substitutions involved in these, and similar charge variants (Glu↔Lys, Glu↔Gln, Asp↔Asn, and Lys↔Gln) involve mass changes of 1 Da or less, and are not detectable by …

Rapid restructuring of bicoid-dependent hunchback promoters within and between Dipteran species: implications for molecular coevolution.

Interacting genetic elements need to coevolve if their joint function is to be maintained; for example, the correct binding of transcriptional regulators to defined binding sites in gene promoters needs to be maintained during evolution to ensure proper function. As part of a wider investigation into the molecular coevolution of the Dipteran homeodomain-bearing regulator bicoid (bcd) and Bcd-dependent promoters, we present data on the functional, structural, and sequence differences between the promoters of the segmentation gene hunchback (hb), in several species of Cyclorrhaphan (higher) Diptera. The result of phenocopying hb mutations using RNA interference (RNAi) in Musca domestica shows broadly similar functions to the hb gene in Drosophila melanogaster. However, the Bcd-binding sites in the hb promoters of Drosophila, Musca, and the two blowfly species Lucilia sericata and Calliphora vicina differ in copy number, sequence, orientation, and spacing. Furthermore, all promoters are subject to rapid turnover by slippage-like processes leading to high densities of short repetitive motifs. A study of polymorphism among six strains of M. domestica reveals that turnover by slippage also occurs in the promoter, untranslated leader, and exonic coding sequences of hb, but to different extents. We discuss these results in terms of the known interspecific differences in bcdand the potential coevolution of selected compensatory mutations in trans and cis in response to continuous promoter restructuring.

β‐thalassemia intermedia caused by compound heterozygosity for Hb Malay (β codon 19 AAC→AGC; Asn→Ser) and codons 41/42 (‐CTTT) β0‐thalassemia mutation

We report a case of β-thalassemia intermedia caused by compound heterozygosity for hemoglobin (Hb) Malay and codon 41/42 (-CTTT) β0-thalassemia mutation in a 38-year-old Chinese woman. This patient has long-standing anemia with a baseline Hb level of around 70 g/L. She worked as a full-time cashier and had not required regular blood transfusions. Nevertheless, she had splenomegaly necessitating splenectomy, cholelithiasis, and iron overload. This case illustrates the varied phenotypic expression associated with compound heterozygosity for Hb Malay and other β-thalassemia mutations. Since Hb Malay migrates as Hb A on electrophoresis and chromatography, this variant Hb mutation ought to be included in the differential diagnosis for β-thalassemia major or intermedia patients of Southeast Asian descent who are reported to have Hb A on the basis of Hb analysis. The possible presence of this mutation should also be considered in appropriate cases for genetic counseling in couples at risk of conceiving fetuses with β-thalassemia major or intermedia. Am. J. Hematol. 64:206–209, 2000. © 2000 Wiley-Liss, Inc.

β-thalassemia intermedia caused by compound heterozygosity for Hb Malay (β codon 19 AAC→AGC; Asn→Ser) and codons 41/42 (-CTTT) β0-thalassemia mutation

We report a case of beta-thalassemia intermedia caused by compound heterozygosity for hemoglobin (Hb) Malay and codon 41/42 (-CTTT) beta(0)-thalassemia mutation in a 38-year-old Chinese woman. This patient has long-standing anemia with a baseline Hb level of around 70 g/L. She worked as a full-time cashier and had not required regular blood transfusions. Nevertheless, she had splenomegaly necessitating splenectomy, cholelithiasis, and iron overload. This case illustrates the varied phenotypic expression associated with compound heterozygosity for Hb Malay and other beta-thalassemia mutations. Since Hb Malay migrates as Hb A on electrophoresis and chromatography, this variant Hb mutation ought to be included in the differential diagnosis for beta-thalassemia major or intermedia patients of Southeast Asian descent who are reported to have Hb A on the basis of Hb analysis. The possible presence of this mutation should also be considered in appropriate cases for genetic counseling in couples at risk of conceiving fetuses with beta-thalassemia major or intermedia.

Transcervical cells and the prenatal diagnosis of haemoglobin (Hb) mutations.

Prenatal diagnoses of haemoglobin (Hb) mutations were performed using transcervical cells, retrieved by aspiration from the endocervical canal of ten selected pregnant women at about 10 weeks of gestation, prior to chorionic villus sampling (CVS). Both parents were carriers of haemoglobinopathies (thalassaemia or HbS). Clumps of fetal cells were isolated by micromanipulation under an inverted microscope and aliquots of the extracted DNA tested separately for the presence of paternally derived chromosome markers and Hb mutations by quantitative fluorescent polymerase chain reaction (PCR). The correct prenatal diagnosis of Hb diseases, using selected single clumps of trophoblastic cellular elements free of maternal contaminating cells, was achieved in six out of ten cases.

Hemoglobin-Specific Antibody in a Multiply Transfused Patient With Sickle Cell Disease

AbstractHuman hemoglobins (Hbs) are known to be immunogenic, and both normal and variant forms of Hb have been shown to stimulate antibody formation in a variety of animal species. In patients who are homozygous for the sickle Hb (HbS) mutation, transfusion of normal, HbA-containing erythrocytes provides a potential stimulus for HbA alloimmunization. We tested serum samples for the presence of anti-Hb antibody by a solid-phase enzyme-linked immunosorbent assay (ELISA) using Hb-coated polystyrene microtiter plates. Hb-bound antibody was identified using an antihuman IgG antibody. Serum samples from 89 patients with sickle cell disease were initially tested for evidence of Hb antibody. The serum from three individuals exhibited antibody activity against HbA with little or no activity against HbS. Only one of them, a multiply transfused adult with HbSS, was available for further study. When this patient's antibody was tested against a variety of normal and mutant Hbs using antibody either to human IgG or to κ chains, the anti-Hb antibody demonstrated specificity for the region of the Hb β chain corresponding to the site of the amino acid substitution of HbS. The level of activity of the patient's anti-HbA showed no significant change over 1.5 years of observation. The transfusion of erythrocytes containing Hb structurally different from that of the recipient appeared to be capable of stimulating the production of Hb-specific alloimmune antibody.