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A national neonatal screening programme for hemoglobinopathies in the Netherlands

Figure 1. HPLC result of a healthy newborn with ≈80% HbF and ≈20% HbA.
Figure 2. HPLC result indicating sickle cell disease either by homozygote HbS or by compound heterozygote HbS/β-thalassemia.
Figure 3. HPLC result indicating an HbS carrier.
Figure 4. HPLC result indicating HbH disease.
Table 1. Results of pilot study and nationwide screening in 2007 and 2008.

On 1 January 2007 neonatal screening for sickle cell disease commenced in the Netherlands. This implementation was preceded by a pilot study in two of the five Dutch screening laboratories. During this pilot study 21,969 dried blood spots from the regular neonatal screening programme were analysed for hemoglobinopathies. In the first two years (2007-2008) of nationwide screening, 83 children with presumed sickle cell disease and four children with b-thalassemia major were referred to a pediatrician. During these two years 82 children were also referred because of screening results indicating hemoglobin H (HbH) disease. The referred children benefited from screening because it allowed early treatment, which reduced complications.

by Dr M. J. Bouva and Dr B. Elvers

Sickle cell disease (SCD), β-thalassemia major (βTM) and hemoglobin H (HbH) disease are hemoglobinopathies with intermediate or severe pathology. Some of the patients with the most severe disease can be cured by bone marrow transplantation, but for most patients treatment consists of transfusion/chelation therapy, pain relief and management of the complications, such as organ damage in the case of sickle cell disease.

The World Health Organisation has estimated that each year over 330,000 babies are born worldwide with a severe form of hemoglobinopathy [1]. Early detection of sickle cell patients for instance, allows treatment with antibiotic prophylaxis to prevent life-threatening complications from Streptococcal pneumonia.

As well as preconceptional and prenatal screening for hemoglobinopathies, neonatal screening using iso-electrofocusing (IEF), electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS) is being performed in an increasing number of countries, such as the United States [2], United Kingdom [3, 4] and France [5]. In 2005 the Dutch Minister of Health decided to expand the national neonatal screening programme to cover seventeen diseases (previously three) including screening for sickle cell disease. After a pilot screening study [6], nationwide screening started in January 2007 using the Bio-Rad Variant Newborn Screening (Vnbs). This article summarises the results of the pilot study and reports on the results of the screening programme in the first two years.

Method
The Variant Newborn Screening (Vnbs) is a cation exchange HPLC-method. A three mm punch from a dried blood spot is extracted in 230 µL deionised water in a well of a microtitration plate. Normal blood (pattern FA) in a newborn consists of about 80% fetal hemoglobin (F) and 20% adult hemoglobin (A) [Figure 1]. A newborn with sickle cell disease produces hemoglobin S instead of A (pattern FS) while an HbS carrier produces both F, A and S (pattern FAS). [Figures 2 and 3]. All HPLC patterns from neonates also have a small first peak, called the FAST-peak, which mainly consists of degradation material. Children with alpha-thalassemia also produce HbBart’s and this HbBart’s appears in the FAST-peak [Figure 4]. Children with a FAST-peak ≥ 20% are also referred to the pediatrician for suspected HbH disease.

Results
Pilot study
Between March and June 2006 a pilot study was performed in the laboratories of the IJsselland Hospital in Capelle aan den IJssel and the National Institute for Public Health and the Environment in Bilthoven (RIVM), The Netherlands. A total of 21,969 random dried blood samples (DBS) from the regular neonatal screening programme were analysed on the Vnbs. Parents had given their consent to use the spots for anonymous scientific research purposes. The HPLC results indicated three sickle cell disease (SCD) patients, two β-thalassemia major (βTM) patients and 83 carriers of sickle cell disease [6].

From this pilot study, it became clear that the method of integration of the HPLC pattern was not always consistent. ‘Valley to valley’ integration and ‘drop–line’ integration were used alternately. This prevented the assessment of normal values in relation to gestational age (see discussion).

Nationwide screening in 2007 and 2008
Neonatal screening in the Netherlands is a nationwide programme carried out by five screening laboratories, with the RIVM in Bilthoven being the reference laboratory. This screening programme has a compliance of 99.5%, and the birth rate is around 185.000 children per year. Between 72 and 168 hours after birth a heel prick is performed and blood is collected on filter paper and sent to the regional laboratory. Commencing on 1 January 2007, all dried blood spots (DBS) were analysed on the Vnbs HPLC system. Children with screening results indicating SCD, βTM and HbH disease are referred to a pediatrician for confirmation and treatment. Parents of the affected children are guided by a genetic counsellor. These parents are informed about their own carrier status and the consequences, and educated to deal with the complications of their child’s disease.

Carriers of HbS are reported to the general practitioner. Parents of these carriers are advised to have their own carrier status determined. If both parents are carriers, the neonatal screening programme can thus also help prevent subsequent children being born with a hemoglobinopathy.

In the first year of screening for hemoglobinopathies (2007; 182,302 newborns) 41 children were referred with suspected SCD. In two cases the HPLC results indicated a βTM, but at least two children with βTM were not referred because the HPLC result only detected a small amount of HbA. Nineteen children were presumed to have HbH disease because of a high HbBart’s concentration (≥20%). Of all screened children, 0.441% (805) were informed about their HbS carrier status.

In 2008 187,075 children were screened, 32 children were referred to the pediatrician for confirmation of SCD, two children for βTM and 62 for HbH disease.

Conclusions
Prior to the implementation of neonatal screening for sickle cell disease a pilot screening study was performed by the RIVM and IJsselland Hospital. Based on the results of this pilot study, Bio-Rad was convinced to adjust its integration software. This resulted in a more reliable determination of the peak percentages. Because of this the Bio-Rad Vnbs HPLC system, which was in principle developed to screen for SCD only, also became a potential candidate method for screening for thalassemia [6].

Based on the results of the pilot screening study, it was expected that each year 40 to 50 newborns with SCD or βTM would be born in the Netherlands. Furthermore, around 1200 carriers of one of the most prevalent hemoglobin variants (HbS, HbC, HbD-Los Angeles and HbE) were expected [6]. These figures were confirmed by the results of screening in 2007 and 2008 [Table 1].

The primary goal of the Dutch screening programme was to detect children with sickle cell disease and provide them with early treatment. Because the HPLC technique also detects other variants besides HbS, the screening programme was able to report all clinically relevant diseases (βTM and HbH disease) and carriers of HbS. For the screening of βTM and HbH disease cut off values were necessary. Children with no HbA and only HbF were putatively reported as βTM patients. When the HPLC showed an HbBart’s peak ≥ 20%, the newborn was referred for HbH disease. Within the first year of screening at least two children with βTM were not referred to the pediatrician because they showed traces of HbA. To improve screening of βTM, a cut-off value was set for HbA after some scrutiny of results. Starting on April 2009, all children with less than 1.0% HbA were referred. Furthermore, all children of at least 32 weeks gestational age and with 2.5% HbA or less were also referred.

In 2008 a significant increase in number of referrals for HbH disease was noticed. In part this was caused by a new resin used in the Vnbs. Because the system was developed to detect hemoglobin variants such as HbS, the resin was not tested for accuracy of detection of HbBart’s. This problem was quickly solved by Bio-Rad. However, most of the children referred for HbH disease were diagnosed with a mild alpha-thalassemia. Starting on January 2010, the cut off value for HbBart’s to detect HbH disease was raised to 22.5%, hopefully resulting in less referrals and higher true positive rate.

In the near future the target of the screening programme will be changed, from screening for SCD only to screening for SCD and βTM. HbH disease and carriers of HbS will still be reported, and probably carriers of the other hemoglobinopathies will also be reported to the general practitioner.

Acknowledgment
We thank the screening laboratories (Dr A. Boelen, Academical Medical Center, Amsterdam; Dr E.A. Kemper-Proper, IJsselland Hospital, Capelle a/d IJssel; Dr R. Triepels; Elisabeth Hospital, Tilburg; Dr J.M.M. Rondeel. Isala clinics, Zwolle) and Dr M. Peters (Advisory Committee on Neonatal Screening for Sickle Cell Disease) for their contribution in the screening and Dr JG Loeber RIVM for helpful discussions.

References
1. Modell B and Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organisation 2008;86.
2. Benson JM and Therrell BL, Jr. History and current status of newborn screening for hemoglobinopathies. Semin Perinatol 2010;34:134-44.
3. Streetly A, Clarke M, Downing M et al. Implementation of the newborn screening programme for sickle cell disease in England: results for 2003-2005. J Med Screen 2008;15:9-13.
4. Streetly A, Latinovic R, Hall K and Henthorn J. Implementation of universal newborn bloodspot screening for sickle cell disease and other clinically significant haemoglobinopathies in England: screening results for 2005-7. J Clin Pathol 2009;62:26-30.
5. Bardakdjian-Michau J, Bahuau M, Hurtrel D et al. Neonatal screening for sickle cell disease in France. J Clin Pathol 2009;62:31-3.
6. Bouva MJ, Mohrmann K, Brinkman HB et al. Implementing neonatal screening for haemoglobinopathies in the Netherlands. J Med Screen 2010;17:58-65.

The authors
Dr MJ Bouva and Dr B Elvers
National Institute for Public Healh and the Environment,
Laboratory for Infectious Diseases and Perinatal Screening,
Bilthoven,
The Netherlands
e-mail: marelle.bouva@rivm.nl


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