Background: Previous evaluations of HPLC as a tool for detection of hemoglobin variants have done so within newborn-screening programs and/or by use of stored samples. We describe a 32-month prospective study in a clinical diagnostic laboratory in which we evaluated the imprecision of HPLC retention times and determined the retention times for hemoglobin variants seen in a multiethnic setting.

Methods: We analyzed all samples on the Bio-Rad Variant II HPLC system. For normal hemoglobin fractions and hemoglobin variants, we recorded and analyzed their retention times, their proportion of the total hemoglobin (%), and the peak characteristics. We compared the imprecision of retention time with the imprecision of retention time normalized to the retention time of hemoglobin A (HbA) and to the retention time of HbA2. Alkaline and acid hemoglobin electrophoresis, and in certain cases globin chain electrophoresis, isoelectric focusing, and DNA analysis, were performed to document the identities of the hemoglobin variants.

Results: The mean (SD) imprecision (CV) of the retention time was 1.0 (0.7)% with no statistical difference compared with the imprecision for normalized retention times. Among 60293 samples tested, we encountered 34 unique hemoglobin variants and 2 tetramers. Eighteen variants and 2 tetramers could be identified solely by retention time and 3 variants by retention time and proportion of total hemoglobin. Four variants could be identified by retention time and peak characteristics and eight variants by retention time and electrophoretic mobility. One variant (HbNew York) was missed on HPLC. Retention time on HPLC was superior to electrophoresis for the differentiation and identification of six members of the HbJ family, four members of the HbD family, and three variants with electrophoretic mobilities identical or similar to that of HbC. Six variants with electrophoretic mobilities identical or similar to that of HbS could be differentiated and identified by retention time and proportion of total hemoglobin. HPLC detected two variants (HbTy Gard and HbTwin Peaks) missed on electrophoresis.

Conclusions: The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional hemoglobin electrophoresis for the detection and identification of hemoglobin variants. Confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC.

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HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A Study of 60000 Samples in a Clinical Diagnostic Laboratory

Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications

Advances in molecular genetics have provided behavioral scientists with a means of investigating the influence of genetic factors on human behavior. Unfortunately, recent candidate gene studies have produced inconsistent results, and a frequent scapegoat for the lack of replication across studies is the threat of population stratification. This review of the literature on population stratification suggests that the threat may be a red herring. Reliable findings will require improved specification and measurement of the behavioral phenotypes in question, a renewed focus on internal validity, and the specification and testing of genetic factors in the context of longitudinal multivariate models. In this respect, behavioral scientists are well suited to investigating genetic factors that influence psychological mechanisms.

Population Stratification in the Candidate Gene Study: Fatal Threat or Red Herring?

Objective. Changing patterns of immi- gration to North America, along with improved treat- ment, have altered the clinical spectrum of thalassemia, one of the world's most common genetic diseases. The new demography of the disease, with its widely variable phenotypes, has implications for its diagnosis, counsel- ing, and management. Characterization of the new spec- trum of this ancient disease, now predominated by mi- nority groups, is essential for optimizing survival. Methods. The National Institutes of Health-spon- sored North American Thalassemia Clinical Research Network (TCRN) conducted a cross-sectional study of 721 patients with thalassemia syndromes. A detailed chart review was undertaken to define the relationships between ethnic origins, genotype, and phenotype. These results were compared with 3 previous surveys of similar regions. To determine if the TCRN patient epidemiology is representative of North American patients, 87 addi- tional programs were reviewed, and hemoglobinopathy programs from the 2 largest thalassemia regions, Ontario and California, were analyzed. Results. A total of 721 patients completed analysis in the TCRN study, including 389 (54%) patients with -thalassemia major, 105 (15%) patients with -thalasse- mia intermedia, 95 (13%) patients with hemoglobin E-- thalassemia, and 132 (18%) patients with -thalassemia. -Thalassemia predominated in Eastern North America. Hemoglobin E--thalassemia and -thalassemia were common on the Western continent. Genotype broadly correlated with the clinical phenotype. However, there was marked heterogeneity in clinical phenotype among patients with similar globin mutations. In -thalassemia disorders, coinheritance of the -thalassemia trait, tripli- cation of -thalassemia genes, and heterozygosity for the dominant -thalassemia allele affected the clinical phe- notype. In -thalassemia disorders, structural mutations such as hemoglobin H-Constant Spring resulted in a severe hemoglobin H phenotype. Sixty percent of pa- tients received regular transfusions, and 86% received regular iron-chelation therapy. Increased survival and decreasing birth rates of Mediterranean patients resulted in an aging Greek/Italian population being replaced by a young Asian/Middle Eastern population. Now, Asian pa- tients account for >50% of the thalassemia population. Evidence of increasing survival is reflected in an advanc- ing mean age of white patients with thalassemia major (25 years, up from 11 years in 1974). The results of the non-TCRN thalassemia survey confirm these observa- tions and describe a young multiethnic thalassemia pop- ulation distributed throughout North America. New- born-screening results suggest that thalassemia births in North America are increasing and reflect the change in genotype and phenotype observed in the TCRN popula- tions. Conclusions. The epidemiology of thalassemia in North America reflects a heterogeneous group of dis- eases with new ethnicities, genotypes, and phenotypes. In these communities, physicians will need to provide education, prenatal diagnosis, counseling, and manage- ment of this newly diverse group of patients. Pediatrics 2005;116:e818-e825. URL: www.pediatrics.org/cgi/doi/ 10.1542/peds.2005-0843; epidemiology, thalassemia, sur- vival, demography, minority disease, genotype, phenotype.

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Changes in the Epidemiology of Thalassemia in North America: A New Minority Disease

Sickle cell disease (SCD) predominates in sub-Saharan Africa, East Mediterranean areas, Middle East, and India. Nigeria, being the most populous black nation in the world, bears its greatest burden in sub-Saharan Africa. The last few decades have witnessed remarkable scientific progress in the understanding of the complex pathophysiology of the disease. Improved clinical insights have heralded development and establishment of disease modifying interventions such as chronic blood transfusions, hydroxyurea therapy, and haemopoietic stem cell transplantation. Coupled with parallel improvements in general supportive, symptomatic, and preventive measures, current evidence reveals remarkable appreciation in quality of life among affected individuals in developed nations. Currently, in Nigeria and other West African states, treatment and control of SCD are largely suboptimal. Improved knowledge regarding SCD phenotypes and its comprehensive care among Nigerian physicians will enhance quality of care for affected persons. This paper therefore provides a review on the aetiopathogenesis, clinical manifestations, and management of SCD in Nigeria, with a focus on its local patterns and peculiarities. Established treatment guidelines as appropriate in the Nigerian setting are proffered, as well as recommendations for improving care of affected persons.

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Management of Sickle Cell Disease: A Review for Physician Education in Nigeria (Sub-Saharan Africa)

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.

Gwendolyn M. Clarke

Papers

Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update

A case of beta thalassemia major detected using HPLC in a child of Chinese ancestry.