Structural hemoglobin (Hb) variants typically are based on a point mutation in a globin gene that produce a single amino acid substitution in a globin chain. Although most are of limited clinical significance, a few important subtypes have been identified with some frequency. Homozygous Hb C and Hb S (sickle cell disease) produce significant clinical manifestations, whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although heterozygotes for these variants are typically asymptomatic, diagnosis may be important for genetic counseling. Thalassemia, in contrast, results from quantitative reductions in globin chain synthesis. Those with diminished β-globin chains are termed β-thalassemias, whereas those with decreased α-chain production are called α-thalassemias. Severity of clinical manifestations in these disorders relates to the amount of globin chain produced and the stability of residual chains present in excess. The thalassemia minor syndromes are characterized clinically by mild anemia with persistent microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate, variably compensated hemolytic anemia that may present with clinical symptoms during a period of physiologic stress such as infection, pregnancy, or surgery. The thalassemia major syndromes produce severe, life-threatening anemia. α-Thalassemia major usually is incompatible with extrauterine life; β-thalassemia major presents in infancy and requires life-long transfusion therapy and/or bone marrow transplantation for successful control of the disease. Double heterozygosity for certain structural variants and/or thalassemia syndromes may also lead to severe clinical disease. Several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation. The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders.

Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update

Congenital erythropoietic porphyria due to a mutation in GATA1: the first trans-acting mutation causative for a human porphyria

A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in approximately 90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "beta Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.

/pdf/cation-exchange-hplc-evaluated-for-presumptive-w52ja2siac.pdf

Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants

Hemoglobin A2 (Hb A2) and hemoglobin F (Hb F) are important analytes in the diagnosis and follow up of Hb diseases. We evaluated a new capillary zone electrophoresis (CZE) kit for Hb A2 and Hb F measurements. The imprecision ranged from 3% to 6% for Hb A2 and Hb F at physiological and pathological concentrations. The method compared well with cation-exchange HPLC for Hb A2 and Hb F and with anion-exchange chromatography in microcolumns (MAEC), for Hb A2. Nevertheless, higher results were obtained [Hb A2 CZE (%) = 1.233 Hb A2 HPLC - 0.2; Hb A2 CZE (%) = 1.190 Hb A2 MAEC + 0.1; Hb FCZE (%) = 1.118 Hb FHPLC + 0.4], and new reference values had to be determined (Hb A2 2.7-3.8%; Hb F <1.2%). Quantification of Hb A2 was not influenced by Hb S. Measurement of Hb F was accurate and precise except at low concentrations in Hb AS patients. This new CZE kit is rapid, precise, and reliable, and seems appropriate for use in clinical laboratories.

Evaluation of a Capillary Electrophoresis Method for Routine Determination of Hemoglobins A2 and F

Cerebrovascular accidents (CVA) as a complication of sickle cell disease occur most frequently in childhood. Life-long transfusion prevents recurrent stroke, but inevitably leads to iron overload. Although effective chelation exists, many patients are not compliant. Erythrocytapheresis, an automated method of red blood cell exchange, was evaluated as an alternative to control transfusion-related iron load. Eleven patients with sickle cell anemia and a history of stroke were converted from simple transfusion to pheresis. Total time on pheresis for the group averaged 19 months (range 4-36 months). No significant complications occurred with a mean pre-pheresis hemoglobin S (Hb S) level of 44%. Blood utilization increased by an average of 50%. The effect of pheresis on serum ferritin depended on the patient's pre-pheresis ferritin level and chelation regimen. Ferritin levels remained stable for chelated patients with ferritin levels > or = 5,000 ng/ml, but decreased in a chelated patient with a pre-pheresis ferritin level of 4,000 ng/ml. For non-chelated patients with significant pre-pheresis iron load, ferritin levels remained stable. No patient on chelation prior to pheresis was able to discontinue deferoxamine. However, one patient with pre-pheresis ferritin of 500 ng/ml maintained serum ferritin levels < 200 ng/ml for 36 months of pheresis without chelation. Pheresis is more expensive than simple transfusion unless the cost of chelation and organ damage from iron overload are considered. Erythrocytapheresis is a safe method of controlling Hb S levels and limiting or preventing iron load in chronically transfused sickle cell patients.

Erythrocytapheresis limits iron accumulation in chronically transfused sickle cell patients

The Bio-Rad Variant Hemoglobin Testing System is an automated HPLC analyzer marketed with a Beta-thalassemia Short Program to quantify hemoglobins (Hbs) F and A2 and assist in detecting Hbs A, S, C, D, and E. We evaluated this system to replace several traditional methods for Hb in our hospital laboratory. Analytical performance relevant to quantifying Hbs A, S, C, and F was assessed with blood samples obtained from our local patient population. Studies of precision (CVs 0.93) with results by traditional methods. The satisfactory performance and efficiency led us to implement this system for routine quantification of clinically significant Hbs.

J C Cate

Papers

Identification and quantification of hemoglobins A, F, S, and C by automated chromatography.