Screening for hemoglobinopathies during routine hemoglobin A1c testing using the Tosoh G7 Glycohemoglobin Analyzer

Approximately 5.1% of the US population has diabetes mellitus, and hemoglobin (Hb) A1c levels are routinely measured to monitor long-term glycemic control in these patients. Many laboratories use ion exchange chromatography for such measurements, and the presence of hemoglobin variants and hemoglobinopathies often results in abnormal peaks on the chromatogram. The goal of this study was to evaluate the potential that detection of these abnormal peaks provides as a screening tool for Hb variants and hemoglobinopathies. We examined 366 specimens with abnormal peaks observed during routine Hb A1c measurements using the G7 Glycohemoglobin Analyzer (Tosoh Bioscience, Inc.). Hb variants and hemoglobinopathies were characterized by alkaline and acid electrophoresis, solubility testing for Hb S, and clinical parameters. In 252 cases, sickle cell trait was identified with a mean retention time (RT) of 1.44 (SD +/-0.02) min. In 82 cases, Hb C trait was identified with a mean RT of 1.66 +/-0.03 min. RTs for other Hb abnormalities, including sickle cell disease, homozygous Hb C disease, C Harlem trait, alpha-chain Hb variants, Hb D trait, Hb G trait, Hb J trait, Hb Raleigh, and Hb Lepore were also determined. Our results demonstrate that routine Hb A1c testing provides a potential screening tool for the detection of common hemoglobin variants and hemoglobinopathies.The previously unreported RTs for the G7 Glycohemoglobin Analyzer are provided, which can facilitate further testing in previously undiagnosed patients and confirm the cause of abnormal peaks in patients with known hemoglobin abnormalities.     

Prediction of plasma hemoglobin concentration by near-infrared spectroscopy

The estimation of plasma hemoglobin concentration (Hb) is among one of the daily activities in the practice of clinical anesthesiology. The near-infrared spectroscopy of the brain (rSO(2)) represents a balance between cerebral oxygen delivery and consumption. This study was designed to assess the value of rSO(2) in the prediction of the Hb level while other variables were mathematically controlled. Thirty healthy adult patients undergoing spine surgery, expected to have a moderate degree of intraoperative bleeding, were enrolled in this study. General anesthesia was given and ventilation was mechanically controlled. Measurement of Hb and PaCO(2) were performed at random periods of time. We obtained a total of 97 data combinations for the 30 study patients. The Hb was regressed by independent variables including rSO(2) and PaCO(2). A multilinear regression analysis was performed and the final regression equation was expressed only with statistically significant variables. The measured Hb was tightly regressed with three variables. The final regression equation was Hb=+8.580+0.238.rSO(2)-0.338.PaCO(2)-0.004.anesthetic exposure duration (Tmin) (p=0.000, r(2)=0.809). Near-infrared spectroscopy was shown to be a valuable predictor of plasma Hb in the clinical anesthesiology setting.     

Analysis of polyethylene-glycol-polylactide nano-dimension artificial red blood cells in maintaining systemic hemoglobin levels and prevention of methemoglobin formation

We have recently reported our study on novel nano-dimension red blood cell (rbc) substitute based on ultrathin PEG-PLA membrane nanocapsules (80-150 nanometer diameter) containing hemoglobin (Hb) and enzymes. These have a markedly increased the circulation half-times as compared to our earlier PLA membrane nanocapsules. In the present study to be reported here, instead of looking at this from a pharmacodynamic point of view, we design the Hb nanocapsules from the point of view of transfusion medicine. For instance, the maximal levels of systemic non-red blood cell (rbc) Hb that can be attained after one infusion of 30% blood volume of 10 gm/dl Hb in the form of different types of PEG-PLA Hb nanocapsules or polyHb. Also the length of time one infusion can maintain a given systemic non-rbc hemoglobin Hb level. Of the two types of polyhemoglobins similar to those in clinical trials but prepared in this laboratory, the maximal levels of Hb reached were 3.35 gm/dl and 3.10 gm/dl respectively. The times for the hemoglobin level to fall to 1.67 gm/dl were 14 hours and 10. hours respectively, corresponding to 24 hours and 17 hours in human. The best PEG-PLA Hb nanocapsules are prepared using a combination of the following 4 factors: use of polymerized Hb, the use of higher M.W. PLA, the use of higher concentrations of PEG-PLA and the crosslinking of the newly formed PEG-PLA Hb nanocapsules. With this, the maximal non-rbc systemic Hb reached was 3.66 gm/dl and the time to reach 1.67 gm/dl was 24.2 hours, or 41.5 hours in human if extrapolated using the results obtained with polyHb in rats.     

Analytical interference of lipid parenteral feeding with the determination of methemoglobin levels by an autoanalyzer

We report here an unusual cause of analytical interference observed in methemoglobin values of patients parenterally given nutritive lipid emulsions. In these patients, harboured in intensive care department, T Hb, % O2 Hb, % COHb, Met Hb values are systematically measured using IL 282 CO-Oximeter autoanalyser in addition to daily blood gas determination (pH, PaCO2, PaO2). An increase in % Met Hb rate up to 10-20% was observed in lipid emulsions receiving patients. We first verified that washing red blood cells with saline instantaneously lowers Met Hb values to less than 1%. The mechanism of this interference was addressed by performing three Met Hb determination methods in normal blood in vitro added with variable amounts of lipid emulsions: CO-Oximeter determination, classical Evelyn Malloy method, analysis of continuous absorption spectra between 480 and 640 nm. Results corroborate the spectral origin of the analytical interference observed with CO-Oximeter and leading to false positive values. Blood lipids increase unspecifically the wave length proportional absorption between 480 and 640 nm. Evelyn Malloy's technique suppresses this interference since it uses the ratio of differences in optical densities. Our results emphasize the necessity of knowing patients therapeutics when performing laboratory investigations.     

Evaluation of a dual hemoglobin A(2)/A(1c) quantitation kit on the bio-rad variant II automated hemoglobin analyzer

CONTEXT: Quantitation of hemoglobin (Hb) A(1c) and investigation of hemoglobinopathy on the Bio-Rad Variant analyzers require a switch between 2 separate kits that is time consuming and causes errors. OBJECTIVE: Evaluation of a new Variant II HbA(2)/HbA(1c) Dual kit capable of both Hb A(1c) quantitation and hemoglobinopathy investigation on a single kit. DESIGN: We evaluated Hb A(1c), Hb A(2), and Hb F quantitation for precision, linearity, and correlation with current methodology. We also evaluated detection of Hb variants and correlation of Hb Barts quantitation. SETTING: Hamilton Regional Laboratory Medicine Program, Provincial Hemoglobinopathy Laboratory, St Joseph's Healthcare Site, Hamilton, Ontario. PATIENTS: Patient blood samples submitted for Hb A(1c) quantitation or hemoglobinopathy investigation. MAIN OUTCOME MEASURES: Precision, linearity, linear regression, and reference interval validation. RESULTS: We provide tables and figures illustrating precision, linearity, linear regression, and quantitation of Hb variants. We validated reference intervals for Hb A(1c), Hb A(2), and Hb F. CONCLUSIONS: The dual kit provides precise Hb A(1c), Hb A(2), and Hb F quantitation. The results show good linearity and correlate well with the results of current methods. We detected all clinically important Hb variants and a wide variety of rare variants. The dual kit has several advantages: it eliminates the need for extensive kit switch over; improves utility for newborn screening because of its quantification of Hb Barts; permits quantification of Hb A(1c) using the beta-Thal method; and eliminates the need for separate Hb A(2) reference intervals for patients with Hb S because of its accurate quantitation of Hb A(2) in the presence of Hb S.     

Hemoglobin S/hemoglobin City of Hope compound heterozygote with a SubSaharan genetic background and severe bone marrow hypoplasia

Hemoglobin City of Hope (Hb CH) (HBB: c.208G>A, beta 69 (E13)Gly>Ser) is a rare, anomalous change. Seven independent carriers reported so far, had not displayed any hematological manifestations. The ethnic origin of the known instances is presumably heterogeneous, although they are mainly Mediterraneans or equatorial West Africans. We describe the case of a compound heterozygote in trans for Hb S (Glu6Val) and Hb City of Hope (Gly69Ser) in an anemic two year-old boy with a severe immune-deficient phenotype and fatal chronic parvovirus B19 infection. Haplotype with the Hb S was Bantu; while it was a mixed atypical Benin/Cameroon for Hb CH. Remote ancestral origin of the City of Hope mutation in this family seems to be SubSaharan African. The compound heterozygosis in trans for hemoglobins S and City of Hope, jointly with an unfavorable HBB control region background and a viral chronic infection, seemed the cause of the fatal outcome in the patient. When accompanied by other Hb deleterious mutations in trans, Hb CH should not be considered any longer as an innocuous or functionally silent variant.     

Haemoglobin typing and its variations in Iranian domestic dogs

The study of canine haemoglobin (Hb) components can help to forecast Hb changes during many pathological and physiological processes such as responsive anaemia. The aims of our study were to show canine Hb electrophoretic pattern on cellulose acetate and identify Hb types similar to the human Hb pattern. Blood samples from 78 different canine breeds were randomly collected in tubes containing anticoagulant EDTA. Animals were brought to the Small Animal Teaching Centre for a check-up and vaccination. All blood samples underwent electrophoresis on cellulose acetate paper strips to determine Hb types. Haematocrit and Hb were measured simultaneously. The Hb electrophoresis results showed that Hb A₁ was assigned to most of Hb components on cellulose acetate paper. Also, in some blood samples, Hb A₂ was detected at the cathode end of cellulose acetate paper similar to human Hb A₂, by densitometer. Small amounts of Hb F were detected in some dogs which was not significant. According to our study, Hb A₁ composes most of the total Hb percent in canine blood. Two types of Hb, A₂ and F, were detected in a few animals, but the majority did not possess Hb F. It seems that Hb F is not significant in these animals. We concluded that one or two Hb types could be seen in dogs. There is no difference in electrophoretic pattern between male and female dogs.     

Extraerythrocytic hemoglobin--a possible oxygen transporter in human malignant tumors

We hypothesize that extraerythrocytic hemoglobin (Hb) serves as an oxygen transporter for human malignant tumors. According to our hypothesis, oxygen transport via intraerythrocytic hemoglobin (Hb), meaning Hb found within erythrocytes, is complemented by oxygen transport via extraerythrocytic Hb, meaning Hb found outside erythrocytes, which circulates in intercellular channels of the tumor. The channels may be derived from processes, including vasculogenic mimicry or endothelial cell retraction. We propose the following scenario: Firstly, hemolysis is caused by the irregular and disruptive endothelial cell-lined tumor vasculature, thus generating extraerythrocytic Hb-O₂. Secondly, this Hb-O₂ is transported together with plasma through the intercellular channels of the tumor. Thirdly, extra-erythrocytic Hb-O₂ delivers oxygen to the hypoxic tumor cells. Finally, oxygen passes from the intraerythrocytic Hb-O₂ in endothelial cell-lined tumor vessels to extraerythrocytic Hb due to the higher affinity of extraerythrocytic Hb for oxygen, thus starting a new cycle of oxygen delivery to the tumor tissue. Based on this hypothesis, we predict that inhibiting oxygen binding to extraerythrocytic Hb inhibits malignant tumor growth.     

Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography-mass spectrometry.

We first detected glutathionyl hemoglobin (Hb) beta-chain in hemodialysis patients and healthy subjects using electrospray ionization liquid chromatography-mass spectrometry. The ratio of glutathionyl Hb beta-chain to total beta-chain was markedly increased in the hemodialysis patients as compared with healthy subjects. Glutathionyl Hb will be used as a new clinical marker of oxidative stress.     

Absolute quantification of deoxyhaemoglobin concentration in tissue near infrared spectroscopy

We describe a simple technique for non-invasively determining the absolute concentration of deoxyhaemoglobin (Hb) in living tissue using near infrared spectroscopy (NIRS). The technique uses second-differential spectroscopy to determine the relative concentration of Hb to tissue water by fitting of the spectral features of these two chromophores in the 710 nm to 840 nm region of the near infrared (NIR) spectrum. Since the concentration of tissue water is generally known with accuracies of a few per cent, one can then obtain the absolute concentration of Hb ([Hb]). The validity and likely accuracy of the technique is assessed by applying it to artificially generated NIRS data. Some clinical validation is presented by comparing, during an in vivo study, changes in [Hb] obtained by this method and those calculated using more conventional techniques of relative quantification. Finally, we discuss the likely clinical significance of the measurement of absolute Hb concentration.     

Variation of haemoglobin extinction coefficients can cause errors in the determination of haemoglobin concentration measured by near-infrared spectroscopy

Near-infrared spectroscopy or imaging has been extensively applied to various biomedical applications since it can detect the concentrations of oxyhaemoglobin (HbO(2)), deoxyhaemoglobin (Hb) and total haemoglobin (Hb(total)) from deep tissues. To quantify concentrations of these haemoglobin derivatives, the extinction coefficient values of HbO(2) and Hb have to be employed. However, it was not well recognized among researchers that small differences in extinction coefficients could cause significant errors in quantifying the concentrations of haemoglobin derivatives. In this study, we derived equations to estimate errors of haemoglobin derivatives caused by the variation of haemoglobin extinction coefficients. To prove our error analysis, we performed experiments using liquid-tissue phantoms containing 1% Intralipid in a phosphate-buffered saline solution. The gas intervention of pure oxygen was given in the solution to examine the oxygenation changes in the phantom, and 3 mL of human blood was added twice to show the changes in [Hb(total)]. The error calculation has shown that even a small variation (0.01 cm(-1) mM(-1)) in extinction coefficients can produce appreciable relative errors in quantification of Delta[HbO(2)], Delta[Hb] and Delta[Hb(total)]. We have also observed that the error of Delta[Hb(total)] is not always larger than those of Delta[HbO(2)] and Delta[Hb]. This study concludes that we need to be aware of any variation in haemoglobin extinction coefficients, which could result from changes in temperature, and to utilize corresponding animal's haemoglobin extinction coefficients for the animal experiments, in order to obtain more accurate values of Delta[HbO(2)], Delta[Hb] and Delta[Hb(total)] from in vivo tissue measurements.     

Physiologic responses of cross-linked hemoglobin in endotoxin-treated rats

Purified human cross-linked hemoglobin (alpha alpha Hb) as well as recombinant human hemoglobin is undergoing clinical trials in the setting of acute blood loss and perioperative hemodilution. We have previously demonstrated that in rabbits with circulating plasma Hb, such as alpha alpha Hb, infusion of endotoxin (LPS) impairs myocardial contractility which results in hypotension, tissue hypoperfusion and increased mortality. The untoward cardiovascular effects occurring after the combined infusion of LPS and alpha alpha Hb in this model are similar to those reported for other agents that inhibit nitric oxide (NO) availability. To determine if the deleterious effects of alpha alpha Hb and LPS were species specific, we performed similar studies in rats. Anesthetized Sprague-Dawley rats received LPS (4 mg/kg or 40 mg/kg) alone or in combination with alpha alpha Hb (0.7 g/kg). Mean arterial blood pressures (MAP) increased in the group that received alpha alpha Hb alone (105 +/- 8 to 120 +/- 7 mm Hg, p = 0.2) and a decrease was noted in the groups that received low dose LPS (4 mg/kg, p = 0.5) and high dose LPS (40 mg/kg, p = 0.016). MAP in rats treated with the LPS at either dose combined with alpha alpha Hb remained unchanged. Levels of urine nitrite, which was measured as a surrogate marker for plasma NO, were significantly decreased at 2 hr in groups that received the combination of alpha alpha Hb and LPS at 4 mg/kg (p = 0.022) and 40 mg/kg (p = 0.003). No significant decrease was observed in animals treated only with alpha alpha Hb (p = 0.21) or LPS (4 mg/kg; p = 0.78 and 40 mg/kg; p = 0.65). Survival was evaluated during 72 hr in animals that were infused with high dose LPS (40 mg/kg) alone or in combination with alpha alpha Hb and then allowed to recover. The survival of rats treated with LPS alone or the combination was 29% at the end of 24 hr and was 100% for rats receiving only alpha alpha Hb. The data suggest that the toxicity of alpha alpha Hb appears to be a species specific phenomenon.     

Cord blood study on beta-thalassemia and hemoglobin E

We describe hematologic data from 18 newborn infants including follow-up data. Of these, ten were the offspring of patients with beta-thal/Hb E disease and the remainder were infants who were found to have a decrease in red cell osmotic fragility during a random cord blood examination. The results of the cord blood study showed that two infants having normal red cell osmotic fragility with about 2% Hb E + Hb A + Hb F at birth represented Hb E heterozygosity. Eleven babies had slightly decreased red cell osmotic fragility, a mild degree of microcytosis and poikilocytosis, and hemoglobin types of Hb A + Hb F with no elevation of Hb A2 at birth. They subsequently had hematologic findings consistent with the beta-thal heterozygosity. The means of hematological values of cord blood in the beta-thal trait infants appeared to be statistically different from those in the normal infants only with respect to increased red cell count and reduced MCH. One infant was thought to have the beta-thal trait but had a greater degree of thalassemic changes in red cells; subsequently he turned out to have homozygous beta-thalassemia. Four newborn infants with hypochromia and numerous target cells had 4-7% Hb E + Hb F without Hb A. Follow-up examination showed two cases of Hb E homozygosity; however, the others, who had obvious microcytosis and poikilocytosis in cord blood, finally developed beta-thal/Hb E disease. Thus, a careful study on red cell osmotic fragility, morphology and starch gel electrophoresis at birth allows detection and diagnosis of beta-thal heterozygosity, beta-thal homozygosity, Hb E heterozygosity, Hb E homozygosity and double heterozygosity for beta-thal and Hb E.     

Tyrosyl radical in haemoglobin and haptoglobin-haemoglobin complex: how does haptoglobin make haemoglobin less toxic?

One of the difficulties in creating a blood substitute on the basis of human haemoglobin (Hb) is the toxic nature of Hb when it is outside the safe environment of the red blood cells. The plasma protein haptoglobin (Hp) takes care of the Hb physiologically leaked into the plasma – it binds Hb and makes it much less toxic while retaining the Hb's high oxygen transporting capacity. We used Electron Paramagnetic Resonance (EPR) spectroscopy to show that the protein bound radical induced by H₂O₂ in Hb and Hp-Hb complex is formed on the same tyrosine residue(s), but, in the complex, the radical is found in a more hydrophobic environment and decays slower than in unbound Hb, thus mitigating its oxidative capacity. The data obtained in this study might set new directions in engineering blood substitutes for transfusion that would have the oxygen transporting efficiency typical of Hb, but which would be non-toxic.     

Combined α-thalassemia and Hemoglobin J-Iran (β77 His → Asp). A Family Study in southern Iran

We report a 23-year-old man and three members of his family with Hb J-Iran confirmed by electrophoresis, chain separation by high performance liquid chromatography and sequencing. Alpha thalassemia was also confirmed in two family members. The substitution at β77 led to a higher negative charge of the βJ-Iran subunit, which enhanced its electrostatic attraction for the normal positively-charged α subunit. Therefore, more Hb J-Iran than Hb A forms in the red blood cells of heterozygotes. In α-thalassemia, the more attractive βJ-Iran subunit outcompetes βA subunits in forming assemblies with deficient α subunits, so even more Hb J-Iran was formed.     

Differential diagnosis of adult hemoglobin A, F, and S conditions. A case of G gamma-beta(+)-hereditary persistence of fetal hemoglobin.

Hemoglobin (Hb) S/beta(+)-thalassemia is a hemoglobinopathy of variable but potentially severe clinical course. The condition is usually confirmed by the presence of a microcytic anemia and elevated levels of Hbs S, F, and A2 by electrophoresis. However, other less common disorders of Hb structure and synthesis may exhibit laboratory findings that mimic Hb S/beta(+)-thalassemia but have a more favorable prognosis. We present a case occurring in a man with clinical and laboratory features that were suggestive of Hb S/beta(+)-thalassemia but with normocythemia. Although nonmicrocytic variants of beta(+)-thalassemia, including concomitant nutritional deficiencies, were considered, high-pressure liquid chromatography revealed nearly all of the patient's fetal Hb to contain only G gamma chains. This pattern is most consistent with the rate but clinically benign condition of Hb S/G gamma-beta(+)-hereditary persistence of fetal Hb, a nondeletional type of hereditary persistence of fetal Hb. We discuss a diagnostic approach to adult Hb A, F, and S conditions, including thalassemias and thalassemia-like syndromes.     

The epidemiology of abnormal hemoglobins in Mediterranean high-level athletes

The aim of this study was to determine the prevalence and nature of hemoglobin (Hb) defects in a Mediterranean high-level (HL) athlete population. Five hundred and ninety-four HL male and female athletes were recruited during the annual follow-up of the members of Tunisian national teams. Hematological data, Hb electrophoresis, and DNA analysis were assessed using conventional techniques. Sporting discipline, type of sport, and performance levels were assessed using a questionnaire. The results showed that 32 HL athletes had abnormal Hb (5.4%): β-thalassemia (2.2%), α-thalassemia (0.5%), HbAS (1.5%), HbAC (0.5%), and rare Hb variants (0.7%). Of the 32 defect carriers, all but one (a α-thalassemia) were heterozygous. All the detected hemoglobinopathies but one (an Hb Hope) had already been reported in the country. The prevalence of Hb defect in the HL athletes was similar to that described in the general Tunisian population (P > 0.05). The percentage of Hb defect in the athletes was not dependent on gender, or performance level (P > 0.05). Within each type of sport the percentages of athletes with normal and abnormal Hb were similar (P > 0.05). The hematological data revealed the diversity of anemia, microcytosis, and hypochromia in thalassemic HL athletes. We concluded that HL athletes in Tunisia were a representative sample of the general Tunisian population regarding the prevalence and nature of benign abnormal Hb. The hematological data of the thalassemia carriers exhibited high variability and raised the question of genetic and sporting counseling, as well as biological follow-up for these carriers.     

Secondary and tertiary structure aberration of alpha globin chain in haemoglobin Q-India disorder

Hemoglobinopathies are important inherited disorders with considerable high prevalence in Asia. Hemoglobin Q-India is a hemoglobinopathy that was first identified in India. Hb Q-India is caused by the mutation GAC --> CAC at codon 64 of the alpha-1 globin gene. The correlation between this hemoglobinopathy and thalassemia was reported. Although primary structure of disorder Hb Q-India is well documented, the secondary and tertiary structures, which can help explain the pathogenesis of the Hb Q-India disorder is not known. In this study, amino acid sequence of human alpha globin was searched using ExPASY and used for further mutation to Hb Q-India disorder. The derived sequences, alpha globin chains in both normal and Hb Q-India disorder, were used for further investigation for secondary and tertiary structures. Modeling of these proteins for secondary and tertiary structures was done using the NNPREDICT server and CPHmodels 2.0 Server, respectively. In this study, the secondary and tertiary structures of human alpha globin chains of normal and hemoglobin Q-India disorder are calculated and presented. Based on this information, the main difference between the predicted alpha globin secondary structures of normal and Hb Q-India is an extra helix in the Hb Q-India. The predicted tertiary structure also supports this finding. The results from this study can be good data for further study on Hb Q-India disorder, which can bring to the further understanding on this hemoglobinopathy.