[Hemoglobin C Variant Affecting Glycated Hemoglobin Test Results: A Rare Case Report].

Hemoglobin (Hb) variants are common factors that affect the results of glycosylated hemoglobin (A1C) tests. Hemoglobin variants react differently to different testing methods. Herein, we presented the first ever report of the effect of hemoglobin C (Hb C) on the test results of A1C in the Chinese population. High performance liquid chromatography (HPLC) and capillary electrophoresis were performed to measure A1C. Hemoglobin electrophoresis was conducted to identify the hemoglobin variants. Hb sequencing was performed to determine the mutation sites on the ß chain. HPLC showed decreased A1C results, which could be corrected by electrophoresis, but the electrophoresis graph still showed abnormal peaks. The hemoglobin electrophoresis results suggested that there were hemoglobin variants, which hemoglobin sequencing results revealed to be Hb C. Uncommon variations in a specific population tend to be overlooked. To avoid clinical decision-making being affected by the results of a single test, we recommend that an explanatory reporting model be routinely adopted for A1C tests so that all reports always contain explanatory notes for the testing methodology and analysis of the graphs.

Southeast Asian Ovalocytosis and Hemoglobinopathies in Newborns: Prevalence, Molecular, and Hematologic Analyses.

OBJECTIVES: Southeast Asian ovalocytosis (SAO) is an inherited red blood cell (RBC) membrane disorder, whereas hemoglobinopathies are inherited globin gene disorders. In an area where both diseases are prevalent, the interaction between them resulting in variable hematologic parameters can be encountered. However, little is known about the genetic interaction of SAO and thalassemia. We investigated the prevalence of SAO and hemoglobinopathy genotypes among newborns in southern Thailand. PATIENTS AND METHODS: This study was carried out on 297 newborns recruited consecutively at Naradhiwas Rajanagarindra Hospital in the south of Thailand. The SAO was identified on blood smear examination and polymerase chain reaction analysis. Thalassemia genotypes were defined. Hematologic parameters and hemoglobin (Hb) profiles were recorded and analyzed. RESULTS: Among 297 newborns, 15 (5.1%) carried SAO, whereas 70 (23.6%) had thalassemia with 15 different thalassemia genotypes. Abnormal Hb including Hb C, Hb Q-Thailand, and Hb D-Punjab were observed in 5 newborns. It was found in the nonthalassemic newborns that RBC count, Hb, and hematocrit of the nonthalassemic newborns with SAO were significantly lower than those without SAO. The same finding was also observed in the thalassemic newborns; RBC count, Hb, and hematocrit of the thalassemic newborns with SAO were significantly lower than those without SAO. However, the mean corpuscular volume, mean corpuscular Hb, and RBC distribution width of the SAO-newborns were significantly higher. CONCLUSIONS: Both SAO and hemoglobinopathy genotypes are common in southern Thailand. One should take this into consideration when evaluating neonatal anemia and other hematologic abnormalities. Identification of both genetic defects and long-term monitoring on the clinical outcome of this genetic interaction should be essential to understand the pathogenesis of these common genetic disorders in the region.

Nanofocused Scanning X-ray Fluorescence Microscopy Revealing an Effect of Heterozygous Hemoglobin S and C on Biochemical Activities in Plasmodium falciparum-Infected Erythrocytes.

While there is ample evidence suggesting that carriers of heterozygous hemoglobin S and C are protected from life-threatening malaria, little is known about the underlying biochemical mechanisms at the single cell level. Using nanofocused scanning X-ray fluorescence microscopy, we quantify the spatial distribution of individual elements in subcellular compartments, including Fe, S, P, Zn, and Cu, in Plasmodium falciparum-infected (P. falciparum-infected) erythrocytes carrying the wild type or variant hemoglobins. Our data indicate that heterozygous hemoglobin S and C significantly modulate biochemical reactions in parasitized erythrocytes, such as aberrant hemozoin mineralization and a delay in hemoglobin degradation. The label-free scanning X-ray fluorescence imaging has great potential to quantify the spatial distribution of elements in subcellular compartments of P. falciparum-infected erythrocytes and unravel the biochemical mechanisms underpinning disease and protective traits.

Point-of-care screening for sickle cell disease in low-resource settings: A multi-center evaluation of HemoTypeSC, a novel rapid test.

Sickle cell disease (SCD) is a common, life-threatening genetic disorder that is best managed when diagnosed early by newborn screening. However, SCD is most prevalent in low-resource regions of the world where newborn screening is rare and diagnosis at the point-of-care is challenging. In many such regions, the majority of affected children die, undiagnosed, before the age of 5 years. A rapid and affordable point-of-care test for SCD is needed. The diagnostic accuracy of HemoTypeSC, a point-of-care immunoassay, for SCD was evaluated in individuals who had SCD, hemoglobin C disease, the related carrier (trait) states, or a normal hemoglobin phenotype. Children and adults participated in low-, medium- and high-resource environments (Ghana [n = 383], Martinique [n = 46], and USA [n = 158]). Paired blood specimens were obtained for HemoTypeSC and a reference diagnostic assay. HemoTypeSC testing was performed at the site of blood collection, and the reference test was performed in a laboratory at each site. In 587 participants, across all study sites, HemoTypeSC had an overall sensitivity of 99.5% and specificity of 99.9% across all hemoglobin phenotypes. The test had 100% sensitivity and specificity for sickle cell anemia. Sensitivity and specificity for detection of normal and trait states were >99%. HemoTypeSC is an inexpensive (<$2 per test), accurate, and rapid point-of-care test that can be used in resource-limited regions with a high prevalence of SCD to provide timely diagnosis and support newborn screening programs.

New approach to accurate interpretation of sickle cell disease newborn screening by applying multiple of median cutoffs and ratios.

BACKGROUND: The main goal of newborn screening (NBS) for sickle cell disease (SCD) is to detect affected neonates so that specific preventive care can be implemented. High-performance liquid chromatography (HPLC) used for NBS has high sensitivity and specificity, but we lack guidelines for quantitative hemoglobin (Hb) fraction interpretation. The purpose of this study was to determine cutoff values to standardize quantitative interpretation in SCD NBS for different clinical situation such as, red blood cell transfusion or beta-thalassemia, which can be real screening pitfalls. METHODS: Retrospective study of 75,026 samples from the neonatal screening program analyzed in our laboratory. Precise HbA and HbS percentages at birth were recorded and median values established for each gestational age, allowing percentage results to be expressed in normal gestation-specific multiples of the median (MoM). Three threshold values of clinical interest were determined. RESULTS: High levels of HbA (>2.5 MoM) allowed identification of newborns who received transfusions. Low levels of HbS (≤0.7 MoM) allowed detection of the association between HbS and other mutations of the beta-globin gene (i.e., HbHope, ß0-thalassemia, etc.). An HbA/HbS ratio <0.5 to distinguish healthy carriers from SCD with S/ß+-thalassemia. The screening accuracy for each threshold was established. The screening accuracy of low-level HbA, which is determinant in identifying the subgroup of patients at risk of ß-thalassemia, will be determined prospectively. CONCLUSIONS: This new approach introduces tools for a quantitative interpretation in SCD NBS by HPLC methods and could allow standardization of interpretation between centers.

Hemoglobinosis C in Morocco : A report of 111 cas.

BACKGROUND: - Hemoglobin C is a hemoglobin variant encountered worldwide. The regionswith high prevalence are West Africa and South-East Asia.The objective of this study is to report cases of hemoglobin C disease brought together during these last twelve years in the Laboratory of Biochemistry and Toxicology of RabatMilitary Hospital Mohammed V (MHIMV). METHODS: - This was a retrospective study including111 cases of hemoglobin C disease collected in the Laboratory of Biochemistry of the MHIMVover the past 12 years. A questionnairewasfulfilledwith the epidemiological data,clinical data and the results of the biological explorations. The screening of the hemoglobin variant in this study included several biochemical (hemoglobin electrophoresis at acid and alkalinepH) and hematological tests. RESULTS: - Sex-ratio was equal to 1,22. The age at the time of diagnosis ranges between 4 and 80years old, with the mean of 38. North-West regions of Morocco seem most affected. The most frequent reasons for prescription of the hemoglobin's studywere: biological abnormalities, splenomegaly and anemic syndrome. Blood smear reveals frequently anisopoikilocytosis and red blood target. The biochemical tests contribute to the diagnosis and reveal various and varied etiological groups: heterozygous A/C (75%),homozygous C/C (8%), double heterozygous S/C (9%),C/ß+-thal (6%) andC/O-Arab (2%). Conclusion - The results of the present descriptive study are in line with the literature data. The importance of genetic counseling and the installation of a national card of systematic neonatal tracking seemto be unavoidable.

Characteristics of a rapid, point-of-care lateral flow immunoassay for the diagnosis of sickle cell disease.

Sickle cell disease (SCD) is a common and life-threatening hematological disorder, affecting approximately 400,000 newborns annually worldwide. Most SCD births occur in low-resource countries, particularly in sub-Saharan Africa, where limited access to accurate diagnostics results in early mortality. We evaluated a prototype immunoassay as a novel, rapid, and low-cost point-of-care (POC) diagnostic device (Sickle SCAN) designed to identify HbA, HbS, and HbC. A total of 139 blood samples were scored by three masked observers and compared to results using capillary zone electrophoresis. The sensitivity (98.3-100%) and specificity (92.5-100%) to detect the presence of HbA, HbS, and HbS were excellent. The test demonstrated 98.4% sensitivity and 98.6% specificity for the diagnosis of HbSS disease and 100% sensitivity and specificity for the diagnosis of HbSC disease. Most variant hemoglobins, including samples with high concentrations of HbF, did not interfere with the ability to detect HbS or HbC. Additionally, HbS and HbC were accurately detected at concentrations as low as 1-2%. Dried blood spot samples yielded clear positive bands, without loss of sensitivity or specificity, and devices stored at 37°C gave reliable results. These analyses indicate that the Sickle SCAN POC device is simple, rapid, and robust with high sensitivity and specificity for the detection of HbA, HbS, and HbC. The ability to obtain rapid and accurate results with both liquid blood and dried blood spots, including those with newborn high-HbF phenotypes, suggests that this POC device is suitable for large-scale screening and potentially for accurate diagnosis of SCD in limited resource settings.

Blood typing profile of a school-aged population of a North Togo township.

The aim of this study was the determination of hemoglobin (Hb) variants and ABO blood groups in a school population aged 6 to 9 years in the township of Agbandé-Yaka in North Togo. A cross-sectional study was carried out on 570 children of four primary schools at Agbande-Yaka, between March and July 2010. Hemoglobin characterization was done by alkaline buffer electrophoresis and the blood types ABO-Rhesus (Rh) D by immuno-hematological methods. A Hb variant was detected in 37.0% of the schoolchildren. Among them, the AS trait accounted for 11.9% and the AC trait for 20.2%. Homozygous Hb S (HBB: c.20A>T) was not found but Hb C (HBB: c.19G>A) appeared at a frequency of 3.3%, while compound heterozygotes carrying Hb SC were seen at a frequency of 1.6%. The O, B and A blood groups accounted for 49.0, 26.8 and 21.9%, respectively. The Hb anomalies reached a high prevalence in this school population. These results are remarkable by the absence of homozygous Hb S individuals compared to homozygous Hb C individuals, which were as numerous as expected. The frequencies of the ABO blood groups are similar to what has been found in other West African populations.

Identification and quantification of hemoglobins in whole blood: the analytical and organizational aspects of Capillarys 2 Flex Piercing compared with agarose electrophoresis and HPLC methods.

BACKGROUND: The present study was conducted to evaluate the analytical performance and the organizational aspects of Capillarys 2 Flex Piercing system (CFP) respect to agarose electrophoresis and HPLC methods in hemoglobinopathies screening. METHODS: The measurement of imprecision in HbA 2 and HbF quantification was verified on HbA 2 CFP control and on three samples; 74 whole blood samples were used to evaluate migration time imprecision of hemoglobin variants S, C and E (HbS, HbC, and HbE); to compare methods, 451 samples were tested on CFP and HPLC; reference values were verified as value distribution in 160 blood donors and at ROC curve analysis on 449 samples from routine analysis. RESULTS: Imprecision: the analytical CV % s ranged from 1.25 to 3.9 at HbA 2 quantification, the CV % was 3.78 at HbF quantification; the running time imprecision for HbS and HbC and HbE ranged from 0.20 to 0.69 % . Method comparison: at regression analysis findings were HbA 2: CFP=1.21×HPLC­0.64, HbF: CFP=1.31×HPLC−0.75, HbS: CFP=1.10×HPLC−3.24. Reference values: the HbA 2 95th percentile range was 2.5­2.8; HbF was undetectable in 154 out 160 samples tested; at ROC curve analysis the best combination of sensitivity and diagnostic efficiency was obtained using 2.2 and 3.0, as reference values, for HbA 2 and 1.1 as the upper reference limit for HbF. Organizational aspects: with respect to the procedures currently implemented in our laboratory CFP requires 2 h less time and obviates the need for some manual steps. CONCLUSIONS: The quantification, reproducibility and diagnostic efficiency provided by CFP in identification and quantification of hemoglobins appear accurate. In addition, the use of primary tubes allows improved safety, and the avoidance of some manual steps, that prolong working time and are a source of possible errors.

Molecular mechanism of hemolytic anemia in homozygous hemoglobin C disease. Electron microscopic study by the freeze-etching technique.

Erythrocytes from a patient with homozygous hemoglobin C disease were subjected to gradual osmotic dehydration by incubation in hypertonic saline. Serial observations of these cells before and after 4 and 12 hr incubation were carried out by means of interference, Soret absorption, polarization microscopy, and the electron microscope employing the freeze-etching technique. Light microscopic studies showed a progressive contraction of cellular contents into central masses which, after 12 hr dehydration, formed birefringent intracellular hemoglobin crystals in 50-75% of the cells. Electron microscopic study of freeze-etched replicas of these cells at 0, 4, and 12 hr of dehydration reveals progressive aggregation, alignment, and crystallization of hemoglobin molecules. Molecular aggregation found in C-C cells prior to osmotic dehydration was not seen in normal erythrocytes. Aggregation and packing varied from cell to cell. Reticulocytes showed a loosely packed aggregate mesh-work; older cells showed variation of molecular packing, which appeared tightest in cells corresponding to microspherocytes. With further loss of intracellular water, aggregates coalesced into patterns of tighter molecular packing with small regions of alignment, and, finally, crystallization occurred. Hemoglobin molecules measuring 70 A in diameter were readily identified within the period patterns of intracellular crystals. These findings suggest that the hemoglobin C molecules within C-C erythrocytes exist in an aggregated state. As the cell ages, intracellular water is lost and intermolecular distance decreases, hemoglobin C molecules polymerize into intracellular crystals. This pathological behavior of hemoglobin C is associated with a charge alteration conferred by the substitution of beta-6-lysine for glutamic acid on the external surface in the A-helix region of the beta-chain of the molecule, possibly increasing intermolecular attraction. Molecular aggregation accounts for the increased rigidity of C-C cells which leads to accelerated membrane and water loss with resultant microspherocyte formation. The microspherocyte, with highest intracellular hemoglobin concentration, rapidly undergoes intracellular crystallization, and is sequestered and destroyed by reticuloendothelial elements.

Radioimmunoassay for abnormal hemoglobins.

A sensitive and specific radioimmunoassay has been developed for the identification or quantification of the human hemoglobin variants S, C, D-Los Angeles, E, G Philadelphia, Russ, O Arab, Beograd, J Paris I, G San Jose, Q Iran, Korle Bu, and F Malta I. In the immunoassay, monospecific antibody preparations are used which recognize the single amino acid substitution in the variant polypeptide chail and do not cross-react with normal hemoglobins or hemoglobin variants containing a different amino acid exchange at the same position.

Hemoglobin interaction: modification of solid phase composition in the sickling phenomenon.

Direct analyses of solid phase formed by deoxygenating solutions of sickle-cell hemoglobin (Hb S) in the presence of certain other hemoglobin species show that hemoglobins A and C can participate in the filamentous fine structure characteristic of the sickling phenomenon. In contrast, fetal hemoglobin (Hb F) is nearly completely excluded.

Newborn screening for sickle cell disease using tandem mass spectrometry.

BACKGROUND: Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening. METHODS: We minimized sample preparation and analysis times by avoiding prior purification, derivatization, or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C, and Hb E) and beta-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, we selected 4 transitions with good signal intensities for each specific peptide and calculated variant/Hb A ratios for each. Method validation included intra- and interseries variability, carryover, and limit of detection. We also performed a comparative study with isoelectric focusing results on 2082 specimens. RESULTS: Intraassay imprecision values (CVs) varied between 2.5% and 30.7%. Interassay CVs were between 6.3% and 23.6%. Carryover was <0.03%, and the limit of detection was fixed at 1% of Hb S. According to the MS/MS settings (detection of Hb S, Hb C, Hb E, and beta-globin production defects), the comparative study did not yield any discrepant results between the 2 techniques. CONCLUSIONS: MS/MS is a reliable method for hemoglobinopathy neonatal screening.

Pathogenesis of hemolytic anemia in homozygous hemoglobin C disease.

Hemoglobin C is less soluble than hemoglobin A in red cells, in hemolysates, and in dilute phosphate buffer. Its relative insolubility may be explained by electrostatic interactions between positively charged beta6-lysyl groups and negatively charged groups on adjacent molecules. Red cells from patients with homozygous hemoglobin C (CC) disease exhibit aberrant physical properties which suggest that the cells are more rigid than normal erythrocytes. They pass through membrane filters less readily than normal red cells do, and their viscosity is higher than that of normal cells. Differences from normal cells are exaggerated if mean corpuscular hemoglobin concentration (MCHC) is increased, by suspension in hypertonic salt solution. Increased rigidity of CC cells, by accelerating their fragmentation, may be responsible for formation of microspherocytes. These small dense cells are exceptionally rigid, and probably are even more susceptible to fragmentation and sequestration. Rigidity of CC cells can be attributed to a "precrystalline" state of intracellular hemoglobin, in which crystallization does not occur, although the MCHC exceeds the solubility of hemoglobin in hemolysates.

Subunit dissociation of certain abnormal human hemoglobins.

The extent of dissociation of various hemoglobins into subunits was estimated from their elution volumes (V(e)) on G-100 Sephadex. Under the same controlled conditions carboxyhemoglobins A, A3 (A(1)), F, S, and C all had the same elution volumes. The carboxy and cyanmet derivatives of hemoglobin Kansas (a variant with very low oxygen affinity) had a relatively high V(e), indicating a decreased mean molecular weight and therefore an increased tendency to form dimers and even monomers. Conversely, the liganded derivatives of hemoglobin Chesapeake (a variant with high oxygen affinity) had a relatively low V(e), suggestive of an impaired degree of subunit dissociation. Deoxyhemoglobin Chesapeake had a V(e) identical with that of deoxyhemoglobin A. Cat hemoglobin, known to have an unusually low oxygen affinity, was found to have a higher V(e) than human, dog, rabbit, rat, or guinea pig hemoglobins. Haptoglobin is thought to bind alphabeta dimers in preference to the alpha(2)beta(2)-tetramer. The comparative haptoglobin affinities of the human hemoglobins were measured by competition between the test hemoglobin and radioactive reference hemoglobin for haptoglobin binding sites. Hemoglobins A, F, S, and C all seemed to bind equally readily, but hemoglobin Kansas and cat hemoglobin showed a higher affinity, and hemoglobin Chesapeake a lower affinity. These results are in accord with recently proposed models which predict that hemoglobins which have an increased degree of subunit dissociation will have a low oxygen affinity, and vice versa.

Hemoglobin CC disease: rheological properties or erythrocytes and abnormalities in cell water.

Suspensions of erythrocytes from patients with hemoglobin (Hb) CC disease showed an increased viscosity and decreased filterability suggesting a less deformable cell. Hemolysates prepared from Hb CC erythrocytes had an increased viscosity compared with hemolysates of normal cells, suggesting that the increased viscosity of Hb CC cells in serum was the result of an increased internal viscosity of the cell. These abnormal rheological properties of Hb CC erythrocytes were associated with a decreased content of cations and an abnormality of cell water. The fraction of the cell volume, which is water in Hb CC cells, was 95.5% of normal. The amount of cell water in Hb CC cells available for osmotic equilibrium, termed solvent water, was only 67% of that in normal cells. The smaller amount of solvent water in Hb CC cells indicates a greater amount of water bound to protein. Altered rheological properties of erythrocytes with Hb AA, CC, and SC also were observed during pregnancy. Suspensions of erythrocytes in serum or plasma from pregnant patients resulted in an increased viscosity compared with suspension in serum or plasma from nonpregnant individuals. An increased viscosity during pregnancy is consistent with the increased severity of the hemolytic anemia in patients with these hemoglobinopathies during pregnancy. The studies reported here suggest that in Hb CC disease the mechanism of erythrocyte destruction, splenic sequestration, results from the increased viscosity, and less deformability of the erythrocyte with on increased internal viscosity.

Hemoglobin variants and disease manifestations in severe falciparum malaria.

CONTEXT: The geographical distributions of hemoglobin S (HbS), hemoglobin C (HbC), and alpha+-thalassemia (-alpha) strongly suggest balancing selection with malaria. However, whereas several studies indicate that the HbS carrier state protects against all major forms of clinical malaria, malaria protection on clinical grounds has been more difficult to confirm for HbC and -alpha, and questions remain as to whether it applies to all forms of the disease. OBJECTIVE: To assess the association between major clinical forms of severe falciparum malaria and HbS, HbC, and -alpha. DESIGN, SETTING, AND PARTICIPANTS: Case-control study of 2591 children with severe falciparum malaria enrolled at a tertiary referral center in Ghana, West Africa, and 2048 age-, sex-, and ethnicity-matched control participants recruited by community surveys. MAIN OUTCOME MEASURES: Frequencies of HbS, HbC, and -alpha in patients and controls, including stratifications of patients for signs of disease. RESULTS: Patients presented with partly overlapping signs of disease, including severe anemia (64%), cerebral malaria (22%), respiratory distress (30%), hyperparasitemia (32%), prostration (52%), acidosis (59%), and hyperlactatemia (56%). Carrier states of HbS, HbC, and -alpha were found in 1.4%, 9.4%, and 25.2% of the patients, respectively, and 14.8%, 8.7%, and 27.3% of controls. The HbS carrier state was negatively associated with all forms of the disease studied (overall odds ratio [OR], 0.08; 95% confidence interval [CI], 0.06-0.12). The HbC carrier state showed a negative association selectively with cerebral malaria (OR, 0.64; 95% CI, 0.45-0.91), and the -alpha carrier state showed a negative association selectively with severe anemia (OR, 0.82; 95% CI, 0.69-0.96). CONCLUSION: Whereas the HbS carrier state was found to be negatively associated with all major forms of severe falciparum malaria, the negative associations of the carrier states of HbC and -alpha appeared to be limited to cerebral malaria and severe anemia, respectively.