Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update
01 Aug 2000-Clinical Chemistry (American Association for Clinical Chemistry)-Vol. 46, Iss: 8, pp 1284-1290
TL;DR: The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders, and several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation.
Abstract: 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.
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TL;DR: 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.
Abstract: 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.
207 citations
TL;DR: An introduction to the application of mass spectrometry for DNA analysis is given, and an overview of most studies using SNPs as genetic markers and MALDImass spectrometric detection that are related to clinical applications and molecular diagnostics are provided.
157 citations
TL;DR: 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.
Abstract: 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.
147 citations
TL;DR: The epidemiology of thalassemia in North America reflects a heterogeneous group of diseases with new ethnicities, genotypes, and phenotypes and physicians will need to provide education, prenatal diagnosis, counseling, and management of this newly diverse group of patients.
Abstract: 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.
130 citations
Additional excerpts
...White 312 (43) 240 (62) 65 (62) 5 (4) 2 (2) Greek/Italian 216 (30) 182 (47) 32 (30) 2 (2) 0 (0) Other white* 96 (13) 58 (15) 33 (31) 3 (2) 2 (2) Asian 368 (51) 136 (35) 28 (27) 112 (85) 92 (97) Indian/Pakistani 82 (11) 59 (15) 11 (10) 4 (3) 8 (8) Chinese 95 (13) 50 (13) 9 (9) 26 (20) 10 (11) Southeast Asian 147 (20) 10 (3) 5 (5) 70 (53) 62 (65) Other Asian† 44 (6) 17 (4) 3 (3) 12 (9) 12 (13) Other ethnicities‡ 41 (6) 13 (3) 12 (11) 15 (11) 1 (1) Total 721 389 105 132 95...
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TL;DR: A review on the aetiopathogenesis, clinical manifestations, and management of SCD in Nigeria, with a focus on its local patterns and peculiarities is provided, as well as recommendations for improving care of affected persons.
Abstract: 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.
120 citations
Cites methods from "Laboratory Investigation of Hemoglo..."
...Genetic studies such as PCR are used for prenatal and preimplantation diagnosis [170]....
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TL;DR: The ARMS (Amplification Refractory Mutation System) as discussed by the authors is a system that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis.
Abstract: We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.
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TL;DR: Whether patient bleeding history and unselected coagulation testing predict abnormal perioperative bleeding is determined and the positive predictive value and likelihood ratio for coagulating tests indicate that they are poor predictors of bleeding.
Abstract: Summary Unselected coagulation testing is widely practiced in the process of assessing bleeding risk prior to surgery. This may delay surgery inappropriately and cause unnecessary concern in patients who are found to have ‘abnormal’ tests. In addition it is associated with a significant cost. This systematic review was performed to determine whether patient bleeding history and unselected coagulation testing predict abnormal perioperative bleeding. A literature search of Medline between 1966 and 2005 was performed to identify appropriate studies. Studies that contained enough data to allow the calculation of the predictive value and likelihood ratios of tests for perioperative bleeding were included. Nine observational studies (three prospective) were identified. The positive predictive value (0AE03–0AE22) and likelihood ratio (0AE94–5AE1) for coagulation tests indicate that they are poor predictors of bleeding. Patients undergoing surgery should have a bleeding history taken. This should include detail of previous surgery and trauma, a family history, and detail of anti-thrombotic medication. Patients with a negative bleeding history do not require routine coagulation screening prior to surgery.
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654 citations
TL;DR: A rapid approach to detect the two most common α‐thalassemia‐2 determinants by the polymerase chain reaction (PCR) technique, which takes a few hours to complete, is believed to provide a cost‐effective way to screen large numbers of blood samples in a relatively short time and can be used to identifyα‐thal‐2 heterozygote and homozygotes and compound heterozygotes in populations where such α‐gene defects are shown to exist at high frequencies.
Abstract: We have developed a rapid approach to detect the two most common alpha-thalassemia-2 (alpha-thal-2) determinants by the polymerase chain reaction (PCR) technique, which takes a few hours to complete. Specific oligonucleotides selectively amplify appropriate segments of the chromosome with the deletion and the normal chromosome under identical experimental conditions, and the products are identified by electrophoresis on 1.5% agarose. Characterization of the two most prevalent types of the -alpha 3.7 determinant [-alpha 3.7(I) and -alpha 3.7(II)] can be made by Apa I digestion of the PCR product. Two types of alpha-thal-2 determinants, -alpha 3.7 and -alpha 4.2, were tested in numerous samples from various parts of the world. This approach is believed to provide a cost-effective way to screen large numbers of blood samples in a relatively short time and can be used to identify alpha-thal-2 heterozygotes and homozygotes and compound heterozygotes (-alpha 3.7/-alpha 4.2) in populations where such alpha-gene defects are shown to exist at high frequencies.
221 citations
TL;DR: This strategy should be useful in the development of screening programmes to identify carriers of α thalassaemia and prenatal diagnosis of the Hb Bart's hydrops fetalis syndrome for those populations in which this represents a major cause of perinatal death.
Abstract: A rapid and inexpensive polymerase chain reaction (PCR) based strategy is described which detects the three common, severe α thalassaemia determinants observed in southeast Asia (——SEA) and the Mediterranean (——MED and —(α)20.5). Oligonucleotide primers have been chosen which allow specific identification of both normal (αα) and abnormal (——) chromosomes using identical conditions in either the same or parallel PCR reactions. This strategy should be useful in the development of screening programmes to identify carriers of α thalassaemia (——/αα) and prenatal diagnosis of the Hb Bart's hydrops fetalis syndrome (——/——) for those populations in which this represents a major cause of perinatal death.
156 citations