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Hemoglobin A2 is a batch fractionated on a column containing diethylaminoethyl-cellulose (DE52, Whatman) with a discontinuous buffer system at pH 8.9 and 7.3. A short micro-column method is also presented, which allows separation in less than 30 min with no interference from hemoglobin S. Both methods clearly distinguish normal from thalassemia specimens, and are simpler and more rapid than previously published methods.
We have previously found that the inhibitory effect of hemoglobin F (Hb F) on the polymerization of Hb S proceeds via the formation of asymmetrical hybrid tetramers of the type alpha2betasgamma. Examination of the gelling properties of binary mixtures of Hb S and several Hb variants now shows that, among the gamma chain amino acid residues that differ from those of the beta chain, residues gamma80 (EF4) and gamma87 (F3) are at least partly responsible for this inhibition. Furthermore, we find that mixing Hb A2(alpha2delta2) with Hb S strongly inhibits gelling to an extent similar to that seen with Hb S/Hb F mixtures; this inhibition is attributable to amino acid differences between the delta and beta chain sequences at positions delta22 (B4) and delta87 (F3). Therefore, residues 22, 80, and 87 of the beta chain appear to be involved in intermolecular contact sites that stabilize the deoxy Hb S polymers.
Compared with other methods for determining hemoglobin A2 (Hb A2) levels, the new microchromatography procedures are fast, easy laboratory technics useful for quantitating Hb A2 that can be readily monitored for quality control of results. A reliable method for preparing a stable hemolysate control for monitoring the accuracy and precision of Hb A2 microchromatographic procedures is described. The recombination of purified hemoglobin fractions in known concentrations and the addition of potassium cyanide and carbon monoxide produce a control solution that gives stable hemoglobin A2 values for at least 40 weeks. Variability among six reference laboratories was small.
The absolute hemoglobin A2 concentration in mg. per 100 ml. of blood was calculated from the hemoglobin level in Gm. per 100 ml. and hemoglobin A2 percentage for 38 patients with documented iron deficiency, 37 patients with proven beta-thalassemia minor, 26 patients with simple chronic anemia and 40 normal control laboratory workers. The mean hemoglobin A2 concentration (mg. per 100 ml.) in the control group was 459 plus or minus 60 (2 S.D.) and that in the beta-thalassemia group, 766 plus or minus 99. However, in the iron deficiency group it was 229 plus or minus 58, while in the simple chronic anemia group it was 315 plus or minus 39. The mean corpuscular volume (M.C.V.) in cu. mu was 90 plus or minus 8 (2 S.D.) in the normal controls, 68 plus or minus 10 in beta-thalassemia, 69 plus or minus 9 in iron deficiency, and 90 plus or minus 15 in secondary anemia. It is proposed that the absolute hemoglobin A2 level in mg. per 100 ml. of blood taken in conjunction with the M.C.V. is of value in establishing the diagnosis of iron deficiency.
Hemoglobin A2 is batch fractionated with diethylaminoethyl Bio-Gel A (Bio-Rad Laboratories) equilibrated with a tris(hydroxymethyl)aminomethane HCl buffer (pH 7.68, 8.75 mmol/liter, 6.36 mmol of CL- per liter). Hemoglobin A1 and F become bound to the resin, allowing the separation and quantitation of A1. Hemoglobin S alters the equilibrium condition, but adjustments are easily made so that A2 can be separated in the presence of S. The procedure is simpler than electrophoretic or chromatographic methods, requires 5 min, is accurate (A2 fraction is at least 94% A2, less than 6% A1), precise (SD +/-0.24 for duplicates), and has a normal limit of 2.6 +/-1.02%.
Hemoglobin-A2-Coburg or alpha2delta2-116 Arg leads to His (G18) has been found in members of a family of Sicilian origin. The propositus is heterozygous for hemoglobin-A2-Coburg as well as for beta-thalassemia, and family data indicate that the gene for the delta-Coburg chain is in trans of the beta-thalassemia determinant.
Elevated hemoglobin A2 (Hb A2) levels can be identified conveniently by densitometry after electrophoresis on cellulose acetate strips. Because a recent report questioned the accuracy of this technic, the method was re-evaluated by paired comparison with microcolumn chromatography. Analysis of 100 patient specimens showed high correlation (r = +0.84), but an average Hb A2 concentration 0.7% higher by densitometry than by chromatography (P less than 0.001). With upper limits set at 4.5%, and 3.8%, respectively, results were divided into "normal" and "high" for each method. Concordant results were obtained in 97 of the 100 cases (82 normal, 15 high). Another densitometer of improved design was used for paired analysis of 50 additional specimens, 25 normal and 25 with beta-thalassemia trait. The two groups were well separated by both procedures, and Hb A2 levels were similar (r = +0.92, P greater than 0.6). This study demonstrates that it is possible, with carefully controlled technics and properly calibrated instruments, to use electrophoresis with densitometry as a reliable means of identifying abnormal Hb A2 levels.
Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.
The influence of malaria on the hemoglobin A2 (Hb A2) level in humans was studied in a series of 94 imported cases in Belgium. Sixty-nine of the patients were natives of Western European countries, their results are reported separately since their origin and the results of their hematological examination made it unlikely that they carried the beta-thalassemia trait. The Hb A2 level of the 94 malaria patients (mean 2.76%; S.D. 0.51%) was not statistically different from that found in 60 healthy controls (mean 2.70%; S.D. 0.38%; P greater than 40). Likewise the level of the 65 Western European patients was not statistically different from that of the same controls (mean 2.81%; S.D. 0.42%; P greater than 0.10). There was also no significant difference between the level in patients infected with a particular species of Plasmodium and that of the controls. No correlation was found between the Hb A2 level and the intensity of the parasitemia or the concentration of total hemoglobin in the blood. These results are discussed in comparison with the divergent ones obtained by others and it is suggested that malaria has no significant influence on the results of surveys for the prevalence of beta-thalassemia in regions of malaria endemicity.
Hyperimmune antisera to chromatographically purified hemoglobins F and A2 were produced in rabbits and made specific for the immunogen by adsorption with normal human hemoglobin A conjugated to cyanogen bromide-activated agarose. A radioimmunoassay was established that permitted identification and quantitation of each of these two minor hemoglobins in hemolysates containing other hemoglobin components. The quantities of hemoglobins A2 and/or F present in hemolysates of individuals with beta-thalassemia, sickle cell anemia, Hb-C disease, and other hematological disorders were determined immunochemically, and the results were commpared to values obtained by microcolumn chromatography for the measurement of Hb-A2 or with the alkali denaturation technique in quantitating Hb-F. The immunoassay procedure has a greater sensitivity than other commonly employed techniques and can detect as little as 0.05 mug of these hemoglobins.
An improved method of A2 quantitation by cellulose acetate electrophoresis is describes. Densitometry is performed on uncleared membranes. The height of the peak obtained for the A2 band is compared with a standard curve derived from peaks obtained by serial dilutions of hemoglobin. Noral A2 hemoglobin as measured by this method is 2.55 percent plus or minus 0.60 (2 S.D.). Twenty patients samples were run in parallel with a column chromatographic method. Eight samples were found to contain elevated A2 levels by the column method. The same eight samples were correctly identified by the electrophoretic technic.
Using a method involving elution of hemoglobin bands from cellulose acetate strips following electrophoresis of hemolysates, hemoglobin A2 (Ab A2) was quantitated in bloods from 300 healthy individuals and 904 patients. The percentage of Hb A2 was elevated in beta-thalassemia heterozygotes and some patients who had megaloblastic anemia. In the latter, the highest Hb A2 levels were observed in patients with the most severe anemia. Low Hb A2 percentages were found in iron-deficiency anemia, hereditary persistance of fetal hemoglobin, and Hb H disease. In iron-deficiency anemia, the lowest levels of Hb A2 were observed in association with the most severe anemia. Iron and folate deficiency each suppressed Hb A2 levels in beta-thalassemia heterozygotes; however, vitamin B12 deficiency did not alter the percentage of Hb A2 in thalassemia. Malignant tumors, renal and hepatic insufficiency, chronic infections and inflammation, hemolytic disease, lead poisoning, aplastic anemia, leukemia, myelofibrosis, and hypothyroidism did not change Hb A2 levels. The pathogenesis of altered Hb A2 levels and their clinical significance in various diseases are discussed.
On 20 consecutive work days during four weeks, one technologist performed 24 microchromatographic determinations of hemoglobin A2 (Hb A2) by each of four methods: the Efremov procedure requiring Tris/HCl buffer, the original Huisman technique with use of glycine developer, and two commercial test kits in which a modified glycine developer is used. The bloood samples tested were obtained from 12 adults with no hematological abnormality and from 12 beta-thalassemia carriers previously diagnosed by familial and hematologic studies. Results by the first method and the two commercial kits (one from Helena Laboratories and one from Isolab, Inc.) did not differ significantly in precision for either the normal or beta-thalassemia trait samples. For both sample types, the second method yielded larger coefficients of variation than those obtained with the other methods. Moreover, the second method was the only one with which values overlapped for normal samples and samples with above-normal Hb A2 concentrations.
Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)
In the 1976 hemoglobinopathy proficiency testing survey of the Center for Disease Control (CDC), whole-blood samples from hematologically normal adults and from individuals heterozygous for beta-thalassemia were shipped to survey participants. The object of this survey was to determine the state of the art for technics used to quantitate hemoglobin A2 (Hb A2) and to test the ability of laboratories to differentiate between blood samples having normal Hb A2 levels and those having elevated levels (i.e., those from individuals with beta-thalassemia trait). The results of Hb A2 quantitation obtained from 183 volunteer participant laboratories were compared with those obtained from 24 reference laboratories. Individual values varied greatly among laboratories and among methods for both normal and elevated Hb A2 samples. The results returned by many laboratories were not within 2 SD of the reference laboratory mean and also were not sufficiently accurate to differentiate between the normal blood samples and those with beta-thalassemia trait. The results suggest that methods for quantitating Hb A2 need to be standardized and a suitable method for determining laboratory performance found.
Three patients with Wilson's disease and initial manifestations of intravascular hemolysis also had transient elevations of hemoglobin A2. It is important to recognize such manifestations in establishing an early diagnosis.
Microcolumn chromatography and a new test tube method for quantitation of hemoglobin A2 were compared with column chromatography on DEAE-Sephadex, starch block and cellulose acetate electrophoresis to ascertain their relative accuracy, precision, reproducibility and speed. One hundred seventy-four blood specimens, including 90 samples from genetically proven beta-thalassemia heterozygotes were examined. The mean Hb A2 values in normal and beta-thalassemia heterozygotes were: 2.3% and 4.7%, respectively, determined by microcolumn chromatography; 2.3% and 4.9%, respectively, determined by a new test tube method; 2.5 and 4.6%, respectively, determined by column chromatography on DEAE-Sephadex; 2.6% and 4.8%, respectively, determined by starch block electrophoresis; and 2.4% and 4.8%, respectively, determined by cellulose acetate electrophoresis. Although all five methods were found to be reliable and reproducible, the microcolumn chromatographic method and the newly developed test tube method using DE-52 cellulose are the most rapid, reproducible, economical, and well suited for large scale surveys. By microcolumn chromatography, 7,953 school children and 2,710 other cases were screened for the quantity of Hb A2. In these samples, 578 beta-thalassemia heterozygotes were detected.
Biosynthetic studies were performed in a patient with beta-thalassemia intermedia heterozygous for both beta-thalassemia with normal hemoglobins A2 and F and beta-thalassemia with increased Hb A2, in his both parents, one sister and one brother. In propositus the alpha/beta ratio was 1.68. In his mother with normal Hb A2, this value was 1.21. In contrast, in his father who had increased Hb A2, the alpha/beta ratio was 1.07, possibly due to combination of alpha- and beta-thalassemia. In his sister who had increased Hb A2, alpha/beta ratio was 1.57. In his brother with normal Hb A2 (2.5%) ratio was 0.6 indicating the presence of an alpha-thalassemia gene. Similar beta-thalassemic syndromes found in other countries are discussed.