Lessons learnt in the screening and diagnosis of haemoglobinopathies.

For this paper, cases reported formally and anecdotally to the authors in their screening and diagnostic roles have been selected to demonstrate areas where errors have occurred, and caution should be exercised. The cases demonstrate that it is vital that the performance and limitations of the techniques used, along with the phenotypic presentation of cases where haemoglobin variants and/or thalassaemias are coinherited are understood by those performing result interpretation. Those who deliver the service as well as those who receive reports and give results and counselling should be aware of the complexity of the topic.

Significant haemoglobinopathies: A guideline for screening and diagnosis: A British Society for Haematology Guideline: A British Society for Haematology Guideline.

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the National Health Service (NHS) Sickle Cell and Thalassaemia Screening Programme. Newborn screening and, when necessary, follow-up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening Programme. All babies under 1 year of age arriving in the United Kingdom should be offered screening for sickle cell disease (SCD). Preoperative screening for SCD should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with a sickle solubility test must always be followed by definitive analysis. Laboratories performing antenatal screening should utilise methods that are capable of detecting significant variants and are capable of quantitating haemoglobins A2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available for antenatal patients within three working days from sample receipt.

Basophil activation test has high reproducibility and is feasible in the clinical setting.

BACKGROUND: The basophil activation test (BAT) has high accuracy to diagnose peanut allergy and can reduce the need for oral food challenges (OFC); however, so far it has not been incorporated in clinical practice. METHODS: We assessed the reproducibility of BAT within the same laboratory and between two different laboratories and the feasibility of using BAT in the clinical setting. RESULTS: One hundred and two children being assessed for peanut allergy were tested on BAT (72 allergic, 30 sensitized tolerant). There was little internal variation (coefficient of variation <15%) in the BAT and a very strong correlation (Rs > .95) between BAT performed across laboratories. The 2 BAT methods were strongly correlated but not interchangeable. In the cases of discrepancy, our in house BAT method was 100% accurate. BAT was feasible and well-accepted by clinicians: no patient with positive BAT was referred for OFC, leading to reduction in the number of OFC required. Twenty one percent of patients who underwent OFC reacted to peanut. A negative BAT also encouraged the performance of OFC in sensitized children who would otherwise be considered allergic, 50% of whom did not react and incorporated peanut in the diet. CONCLUSIONS: The BAT is a robust test that can reliably be transferred between laboratories; however, different BAT methods are not interchangeable. BAT was well integrated in the clinical decision-making process in a specialized center.

Performance analysis of the compact haematology analyser Sight OLO.

INTRODUCTION: Sight OLO is a compact full blood count (FBC) analyser that uses digital imaging techniques and artificial intelligence to count and assess cellular components of capillary or venous blood. It provides a FBC with a 5-part white blood cell differential count. Our aim was to evaluate its performance against our standard analyser and optical microscopy. METHODS: Comparative studies for the FBC parameters were done between the Sight OLO and the Unicel DxH800 analyser (Beckman Coulter). Evaluation comprised also repeatability studies and reproducibility studies. The flagging efficiency of the Sight OLO was assessed against the reference method (optical microscopy). RESULTS: The SIGHT OLO showed a good comparability with the Unicel DxH800 analyser for most of the FBC parameters (r > 0.9). The biases recorded between both equipments were within the manufacturer's target specifications for all the FBC parameters. The standard deviation and coefficient of variation calculated per parameter for the precision studies were within the manufacturer's target specifications for all FBC parameters, for all the variation components tested. The five alert flags assessed showed an overall efficiency above 75%, however, high frequency of false negatives was noted for some of the flags assessed. CONCLUSION: The evaluation of the Sight OLO showed that it can produce accurate FBC results, making it a suitable option for integration in several setups. Its innovative methodology gives it further potential to refine its capabilities.

A novel non-invasive prenatal sickle cell disease test for all at-risk pregnancies.

Sickle cell disease (SCD) is the most common genetic haematological disorder. The availability of non-invasive prenatal diagnosis (NIPD) is predicted to increase uptake of prenatal diagnosis for SCD, as it has no perceived procedure-related miscarriage risk. We report the development of a targeted massively parallel sequencing (MPS) assay for the NIPD of fetal SCD using fetal cell-free (cf)DNA from maternal plasma, with no requirement for paternal or proband samples. In all, 64 plasma samples from pregnant women were analysed: 42 from SCD carriers, 15 from women with homozygous (Hb SS) SCD and seven from women with compound heterozygous (Hb SC) SCD. Our assay incorporated a relative mutation dosage assay for maternal carriers and a wild type allele detection assay for affected women (Hb SS/Hb SC). Selective analysis of only smaller cfDNA fragments and modifications to DNA fragment hybridisation capture improved diagnostic accuracy. Clinical sensitivity was 100% and clinical specificity was 100%. One sample with a fetal fraction of <4% was correctly called as 'unaffected', but with a discordant genotype (Hb AA rather than Hb AS). Six samples gave inconclusive results, of which two had a fetal fraction of <4%. This study demonstrates that NIPD for SCD is approaching clinical utility.

Sickle cell disease and thalassaemia antenatal screening programme in England over 10 years: a review from 2007/2008 to 2016/2017.

OBJECTIVES: To evaluate the antenatal sickle cell and thalassaemia screening programme in England over 10 years from 1 April 2007 to 31 March 2017. METHODS: Four routine data sources were used: antenatal screening laboratory data; key performance indicator data from maternity trusts; prenatal diagnosis (PND) laboratory data and data from screening incidents. RESULTS: For the 10 years examined a total of 6608 575 booking samples were reported as screened, and 154 196 pregnant women required further testing. There were 3941 reported PND tests of which there were 964 affected fetal results. Antenatal test coverage and Family Origin Questionnaire completion rates are high and increasing; the proportion of tests declined has decreased. However, there is wide variation in the timing of antenatal tests and completeness of follow-up and testing. Since 2014/2015 a lower proportion of PND tests are performed by the programme standard of 12+6 weeks. Results suggest that PND timing affects reproductive choices as those with an affected fetus identified by PND testing earlier are more likely to terminate the pregnancy. CONCLUSIONS: The screening programme appears to be widely accepted as part of routine antenatal care in England. However, the timeliness of screening and subsequent PND testing has consistently not met programme standards. Improving timeliness would enable individuals to consider their options to make informed choices for their pregnancies at the appropriate time. This paper reports carrier rates for an almost complete cohort of women which provides important epidemiological information on the genetic profile of women in England.

Newborn screening for sickle cell disease: an innovative pilot program to improve child survival in Dar es Salaam, Tanzania.

BACKGROUND: Sickle cell disease (SCD) is a recognized cause of childhood mortality. Tanzania has the fifth highest incidence of SCD (with an estimated 11 000 SCD annual births) worldwide. Although newborn screening (NBS) for SCD and comprehensive healthcare have been shown to reduce under-5 mortality by up to 94% in high-income countries such as the USA, no country in Africa has maintained NBS for SCD as a national health program. The aims of this program were to establish and evaluate NBS-SCD as a health intervention in Tanzania and to determine the birth prevalence of SCD. METHODS: Muhimbili University of Health and Allied Sciences conducted NBS for SCD from January 2015 to November 2016. Dried blood spot samples were collected and tested for SCD using isoelectric focusing. RESULTS: Screening was conducted on 3981 newborns. Thirty-one (0.8%) babies had SCD, 505 (12.6%) had sickle cell trait and 26 (0.7%) had other hemoglobinopathies. Twenty-eight (90.3%) of the 31 newborns with SCD were enrolled for comprehensive healthcare. CONCLUSIONS: This is the first report on NBS as a health program for SCD in Tanzania. The SCD birth prevalence of 8 per 1000 births is of public health significance. It is therefore important to conduct NBS for SCD with enrollment into a comprehensive care program.

Reliability of the current newborn screening action value for beta thalassaemia disease detection in England: A prospective study.

OBJECTIVE: Beta thalassaemias are a group of hereditary red cell disorders resulting in a reduced or absent production of the main adult haemoglobin, adult haemoglobin. In England, the NHS Sickle Cell & Thalassaemia Screening Programme recommends reporting newborn beta thalassaemia disease as an incidental finding when detected whilst screening for sickle cell disease. The current action value to initiate further investigations is 1.5% adult haemoglobin, using high-performance liquid chromatography or capillary electrophoresis. We examined the reliability of this action value. METHODS: A 44-month country-wide prospective study using data from 13 newborn screening laboratories in England. RESULTS: There were 81 cases reported with an adult haemoglobin of 1.5% or less at first-line screen, of which nine were lost to follow-up. The six false-positive results were all of 32 weeks' gestation or less. Of the 66 true-positives, 36 had confirmatory molecular results (11 of these cases also have results from tandem mass spectrometry), 19 had clinical confirmation and 11 had the results of both parents available which were consistent with the screening result. There was one false-negative, a confirmed beta thalassaemia major case with an adult haemoglobin of 1.7%, above the action value at first-line screen but known to be at risk from parental results and therefore referred into clinical care by the laboratory. CONCLUSIONS: This study demonstrates a positive predictive value of 91.7%, with a specificity of 99.9% and a sensitivity of 98.5%. These results confirm the reliability of the current action value.

Newborn Screening for Sickle Cell Disease in Europe.

The history of newborn screening (NBS) for sickle cell disease (SCD) in Europe goes back almost 40 years. However, most European countries have not established it to date. The European screening map is surprisingly heterogenous. The first countries to introduce sickle cell screening on a national scale were France and England. The French West Indies started to screen their newborns for SCD as early as 1983/84. To this day, all countries of the United Kingdom of Great Britain and Northern Ireland have added SCD as a target disease to their NBS programs. The Netherlands, Spain and Malta also have national programs. Belgium screens regionally in the Brussels and Liège regions, Ireland has been running a pilot for many years that has become quasi-official. However, the Belgian and Irish programs are not publicly funded. Italy and Germany have completed several pilot studies but are still in the preparatory phase of national NBS programs for SCD, although both countries have well-established concepts for metabolic and endocrine disorders. This article will give a brief overview of the situation in Europe and put a focus on the programs of the two pioneers of the continent, England and France.

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

Newborn Sickle Cell Disease Screening Using Electrospray Tandem Mass Spectrometry.

There is a growing demand for newborn sickle cell disease screening globally. Historically techniques have relied on the separation of intact haemoglobin tetramers using electrophoretic or liquid chromatography techniques. These techniques also identify haemoglobin variants of no clinical significance. Specific electrospray ionization-mass spectrometry-mass spectrometry techniques to analyse targeted peptides formed after digestion of the haemoglobin with trypsin were reported in 2005. Since this time the method has been further developed and adopted in several European countries. It is estimated that more than one million babies have been screened with no false-negative cases reported. This review reports on the current use of the technique and reviews the related publications.

Newborn screening for sickling and other haemoglobin disorders using tandem mass spectrometry: A pilot study of methodology in laboratories in England.

OBJECTIVE: To determine (i) if electrospray mass spectrometry-mass spectrometry with the SpOtOn Diagnostics Ltd reagent kit for sickle cell screening could be integrated into the English newborn screening programme, under routine screening conditions, and provide mass spectrometry-mass spectrometry results which match existing methods, and (ii) if common action values could be set for all manufacturers in the study, for all assessed haemoglobins, to indicate which samples require further investigation. METHODS: Anonymised residual blood spots were analysed using the SpOtOn reagent kit as per manufacturer's instructions, in parallel with existing techniques at four laboratories. Mass spectrometry-mass spectrometry instrumentation at Laboratories A and B was AB Sciex (Warrington, UK) AP4000, and at Laboratories C and D, Waters Micromass (Manchester, UK), Xevo TQMS and Premier, respectively. RESULTS: There were 23,898 results accepted from the four laboratories. Excellent specificity at 100% sensitivity was observed for haemoglobin S, haemoglobin C, haemoglobin E and haemoglobin OArab. A common action value was not possible for Hb C, but action values were set by manufacturer. The two haemoglobin DPunjab cases at Laboratory D were not detected using the common action value. Conversely, false-positive results with haemoglobin DPunjab were a problem at the remaining three laboratories. CONCLUSIONS: This multicentre study demonstrates that it is possible to implement mass spectrometry-mass spectrometry into an established screening programme while maintaining consistency with existing methods for haemoglobinopathy screening. However, one of the instruments investigated cannot be recommended for use with this application.

Sickle-Cell Disease in Nigerian Children: Parental Knowledge and Laboratory Results.

BACKGROUND: Sickle-cell disease (SCD) is the most common inherited genetic disorder in sub-Saharan Africa, and it is associated with early mortality and lifelong morbidity. Early diagnosis is essential for instituting appropriate care and preventive therapy. OBJECTIVE: To compare parental knowledge or perception of their offspring's hemoglobin phenotype prior to testing and actual validated laboratory test results. METHODS: In a prospective community-based survey, we assessed parental knowledge of their children's hemoglobin phenotype and corroborated this with the results from a laboratory confirmatory test determined by high-performance liquid chromatography. RESULTS: We screened 10,126 children aged less than 5 years. A total of 163 (1.6%) parents indicated that their offspring had been previously tested and had knowledge of the child's hemoglobin genotype. However, 51 (31.2%) of 163 parents of children who had been previously tested did not know the result of their offspring's test, and 18 (35.3%) of these 51 children were found to have SCD. Of those who claimed previous knowledge, 25 (15.3%) of 163 reported incorrect results. Overall, we identified 272 (2.76%) new cases from 9,963 children who had not been previously tested. CONCLUSION: There is the need to promote public awareness about SCD and the benefit of early diagnosis, quality assurance in laboratory diagnosis and institution of sustainable patient care pathways.

Evaluation of the validity of Hb A2 and mean corpuscular haemoglobin action values in antenatal screening for beta thalassaemia carriers in England.

National antenatal screening of all pregnant women in England is carried out using standards and guidelines produced by the National Health Service Sickle Cell and Thalassaemia Screening Programme. The algorithms for detection of beta thalassaemia carrier status rely on action criteria, which are set using the percentage Hb A2 and mean corpuscular haemoglobin (MCH) values. Three groups of samples: MCH <27 pg and Hb A2 3·5-3·9%, MCH ≥27 pg and Hb A2 4-4·3% and MCH ≥27 pg and Hb A2 3·5-3·9% were selected from a sample population of 59 500 to assess the validity and predictive value of the action criteria - 25 false positives (0·042% of total) and nine false negatives (0·015% of total) were detected. These findings support the continuation of the current action values.

Newborn bloodspot results: predictive value of screen positive test for thalassaemia major.

AIM: There are limited published data on the performance of the percentage of haemoglobin A (Hb A) as a screening test for beta thalassaemia major in the newborn period. This paper aims to analyse data derived from a national newborn bloodspot screening programme for sickle cell disease on the performance of haemoglobin A (Hb A) as a screening test for beta thalassaemia major in the newborn period. METHODS: Newborn bloodspot sickle cell screening data from 2,288,008 babies were analysed. Data reported to the NHS Sickle Cell and Thalassaemia Screening Programme in England for the period 2005 to 2012 were also reviewed to identify any missed cases (4,599,849 babies). RESULTS: Within the cohort of 2,288,008 births, 170 babies were identified as screen positive for beta thalassaemia major using a cut-point of 1.5% HbA. There were 51 identified through look-back methods and 119 prospectively identified from 4 screening laboratories. Among 119 babies with prospective data, 7 were lost to follow up and 15 were false positive results. Using a cut-off value of 1.5% Hb A as a percentage of the total haemoglobin as a screening test for beta thalassaemia major in the newborn provides an estimated sensitivity of 99% (from the look back arm of the study) with a positive predictive value of 87% (from the prospective arm of the study). Excluding infants born before 32 weeks gestation, the positive predictive value rose to 95%. CONCLUSION: A haemoglobin A value of less than 1.5% is a reliable screening test for beta thalassaemia major in the newborn period.

Sickle cell/ß0-thalassemia associated with the 1393 bp deletion can be associated with a severe phenotype.

In patients who have inherited both the sickle cell gene and the ß-thalassemia (ß-thal) gene, the nature of the ß-thal mutation will impact on the disease phenotype. The ß-thal mutation caused by the 1393 bp deletion has previously been described as having a mild clinical phenotype when inherited with the sickle gene. We describe three members of a family with this deletion who present with a more severe phenotype. The severity cannot be explained by their Hb F levels, or the XmnI-HBG2 polymorphism. This deletion cannot be presumed to be associated with a mild disease phenotype and we recommend that patients with Hb S/ß(0)-thal are screened for this deletion.