Hemoglobinopathies are the most common monogenic disorders in the world with an ever increasing global disease burden each year. As most hemoglobinopathies show recessive inheritance carriers are... Show moreHemoglobinopathies are the most common monogenic disorders in the world with an ever increasing global disease burden each year. As most hemoglobinopathies show recessive inheritance carriers are usually clinically silent. Programmes for preconception and antenatal carrier screening, with the option of prenatal diagnosis are considered beneficial in many endemic countries. With the development of genetic tools such as Array analysis and Next Generation Sequencing in addition to state of the art screening at the hematologic, biochemic and genetic level, have contributed to the discovery of an increasing number of rare rearrangements and novel factors influencing the disease severity over the recent years. This review summarizes the basic requirements for adequate carrier screening analysis, the importance of genotype-phenotype correlation and how this may lead to the unrevealing exceptional interactions causing a clinically more severe phenotype in otherwise asymptomatic carriers. A special group of patients are beta-thalassemia carriers presenting with features of beta-thalassemia intermedia of various clinical severity. The disease mechanisms may involve duplicated alpha-globin genes, mosaic partial Uniparental Isodisomy of chromosome 11p15.4 where the HBB gene is located or haplo-insufficiency of a non-linked gene SUPT5H on chromosome 19q, first described in two Dutch families with beta-thalassemia trait without variants in the HBB gene. Show less
Introduction The definition of the International Normalized Ratio (INR) depends on a reference measurement procedure for the prothrombin time (PT) determined with international standards for... Show moreIntroduction The definition of the International Normalized Ratio (INR) depends on a reference measurement procedure for the prothrombin time (PT) determined with international standards for thromboplastins. The agreed water bath temperature for PT determination in the reference measurement procedure is 37 degrees C. The aim of the study was to assess the influence of small deviations of the agreed reaction temperature on PT and INR determined with World Health Organization international standards for thromboplastins rTF/16 (recombinant human) and RBT/16 (rabbit brain). Methods Prothrombin time was determined, with a manual hook technique, in glass test tubes in a water bath at a controlled temperature. The PT reaction temperatures were varied between 28 and 40 degrees C. Pooled normal plasma and pooled coumarin plasma (INR approximate to 2.8) were used as test plasmas. The data were fitted to a quadratic relationship between PT and temperature. Results Prothrombin times with rTF/16 were shortened by increasing the reaction temperature up to approximately 39-40 degrees C. PTs with RBT/16 were shortened by increasing the reaction temperature up to approximately 34-37 degrees C, but were prolonged at higher temperatures. The apparent INR change of the coumarin plasma at 37.0 degrees C was 0.06/degrees C and 0.11/degrees C for rTF/16 and RBT/16, respectively. Conclusions Reaction temperature had a significant effect on PT and the apparent INR with the International Standards. At 37.0 degrees C, the apparent INR of coumarin plasma determined with RBT/16 was more responsive to temperature change than the apparent INR determined with rTF/16. The required accuracy of the water bath temperature should be 37.0 +/- 0.1 degrees C. Show less
Introduction The high-sequence homology of the alpha-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion... Show moreIntroduction The high-sequence homology of the alpha-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. Methods Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing. Results Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. Conclusions The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the --(gb), but as it was not registered correctly in the available databases, it was not initially recognized as such. Show less
Wild, B.J.; Chohan, D.K.; Harteveld, C.L.; Salle, B. de la 2020
Introduction The accurate measurement of HbA(2) is essential for the detection of beta-thalassaemia carriers and as no single calibrant is used by the various manufacturers of analysers,... Show moreIntroduction The accurate measurement of HbA(2) is essential for the detection of beta-thalassaemia carriers and as no single calibrant is used by the various manufacturers of analysers, differences are seen in results obtained. The World Health Organization International Reference Reagent for HbA(2) (WHO IRR 89/666) was made available to diagnostic laboratories in the 1980s and remains the only international reference material available. A previous study (2015) demonstrated that the WHO IRR remained suitable for use as an HbA(2) standard as tested by 52 participants in the UK NEQAS Haematology Abnormal Haemoglobins Programme. This study was undertaken to include simultaneous analysis of three whole blood specimens over a range of HbA(2) values with the WHO IRR and to include participants from laboratories outside of the UK.Method Three whole blood specimens with HbA(2) levels ranging from 2.4% to 5.7% and the WHO IRR were distributed to 56 laboratories located in 14 different countries. Participants were requested to test the specimens at defined intervals and return results accompanied by chromatograms or electropherograms produced.Results Differences found in results from different analyser groups reflect the bias found in the 2015 study in that bias is seen according to the methodology used and also varies in relation to the level of analyte being measured.Conclusion Results of measurements from whole blood specimens and the lyophilized WHO IRR standard did not show any deterioration of the IRR, and it remains suitable for use. Linearity and calibration of analysers remain a problem. Show less
Salle, B. de la; Stephens, A.D.; Wild, B.J.; Harteveld, C.L.; Hyde, K. 2019
IntroductionThe accurate determination of Hb A(2) is a key marker when screening for a -thalassaemia carrier. Data from external quality assessment (EQA) exercises have shown a lack of alignment of... Show moreIntroductionThe accurate determination of Hb A(2) is a key marker when screening for a -thalassaemia carrier. Data from external quality assessment (EQA) exercises have shown a lack of alignment of Hb A(2) quantitation both within and between methods. The only reference material available for Hb A(2) quantitative assay at the time of writing is the World Health Organization International Reference Reagent (89/666; WHO IRR) prepared in the 1980s and not validated for all current methodologies.MethodThe WHO IRR was analysed for Hb A(2) concentration by 52 laboratories using a representative range of high-performance liquid chromatography and capillary electrophoresis analysers. The results of the analysis were compared to those of a whole blood EQA specimen of similar Hb A(2) concentration, distributed in the same week.ResultsThe mean Hb A(2) value obtained for the WHO IRR was 5.17%, compared to the assigned value of 5.3%. The range of results returned was wide (4.0%-6.2%), with differences in the results observed by between and within analyser groups. A similar range of results was seen with the whole blood sample, although the bias observed between analyser types was different from that seen with the WHO IRR.ConclusionThe results may indicate a lack of commutability of the WHO IRR material, resulting from deterioration, matrix effects or changes in reagent formulation or calibration parameters. Further examination of the suitability of the WHO IRR (89/666) for continued use is required. Show less
INTRODUCTION The aim of this review is to study the frequency of common and the occurrence of rare and novel mutations of the delta-globin gene and of Hb Lepore defects that might interfere with... Show moreINTRODUCTION The aim of this review is to study the frequency of common and the occurrence of rare and novel mutations of the delta-globin gene and of Hb Lepore defects that might interfere with thalassemia diagnostics and to report the rationale of HbA2 estimation in the presence of delta- or alpha-gene mutations. METHODS A total of 135 cases suspected to have a delta-globin gene defect collected in a diagnostic center in the USA and in a reference laboratory in the Netherlands were characterized by molecular analysis. RESULTS Hb B2 was found at a frequency of at least 0.5% in the USA and 0.87% in the Netherlands. Known variants such as Hb A2-Babinga, Hb A2-Sphakia, Hb A2-Fitzroy, Hb A2-Flatbush, Hb A2-NYU, Hb A2-Grovetown, HbA2-Yialousa, Hb A2-Indonesia and several delta-thalassemia mutations were found together with 13 new mutations and two new polymorphisms, while Hb Lepores were regularly observed. CONCLUSION HbA2 mutations either structurally stable and visible or undetectable because of a thalassemia effect or instability are clinically asymptomatic but may compromise the diagnosis of beta-thalassemia minor. Stable mutations result in two HbA2 fractions of about half of the expected value. Expression defects are undetectable as a protein fraction but reduce the amount of HbA2 by half. Show less
