Elementos del SAT en la Fase Informativa:

Because of the extremely high prevalence of hemoglobinopathies and the continuous increase as a result of the population migration these clinical syndromes pose a significant burden on the health system in developed and developing countries today. In many countries including the UK there is a recognised need for a good screening

programme which allows for the quick and accurate detection of

hemoglobinopathies. The timely identification of such genetic disorders make it possible to initiate the necessary and appropriate treatment as early as possible. In the UK the NHS Sickle cell and thalassemia screening programme has been undertaking this role in the last 12 years. The developed program links two screening

47 set-ups, antenatal screening for pregnant women and their partners and recommending neonates to be screened for variant hemoglobins (Ryan et al. 2010). The aim of the newborn screening programme is to identify infants at risk of sickle cell disease within the neonatal period, since early reliable diagnosis is necessary to initiate early treatment and to arrange clinical follow up. The analytical methods will also detect β-thalassemia major and related conditions in the majority of cases. Newborn sickle cell screening is offered to all babies born in England and is also being implemented in the other UK countries (NHS 2012b). The screening is offered at 5-8 days of age as part of the newborn dried blood spot screening programme (Streetly et al. 2009).

The main aim of newborn screening is to identify affected babies and providing treatment to prevent as many deaths as possible from sickle cell disease (NHS 2012a).

The objective of antenatal screening is to offer sickle cell and thalassemia screening to all pregnant women (and their partners) early in the pregnancy – ideally by 8–10 weeks of gestation – so they can request counselling about possible options and can make an informed decision (NHS 2012a). It is desired to initiate all the tests in the first 10 weeks, and finish all necessary additional testing by the 12 weeks of gestation, if termination of pregnancy is required it should happen as early as possible (Bain 2011).

It is extremely important to collect information of family origin and ethnic background, and investigating this is considered a crucial part of the screening process. The screening procedure is dependent on whether the parents are from high or low prevalence area for SCD and thalassemia. High prevalence areas are those which have fetal prevalence of SCD of 1.5 per 10,000 pregnancies or higher, and low prevalence areas are where the incidence is below 1.5. Initial laboratory screening involves the use of the Family Origin Questionnaire (FOQ), and based on ethnicity a full screen may be performed (Ryan et al. 2010, NHS 2012b).

The screening procedure involves measurements with several different techniques. Initial screening techniques are simple and give an indication for the presence of abnormalities but they are not evaluated diagnosis methods. Initial screening involves complete blood count measurements (CBC) and the assessment of important characteristics. The most important parameters are mean corpuscular volume (MCV - measure of the average red blood cell volume) and mean

48 corpuscular hemoglobin (MCH - the average mass of hemoglobin per red blood cell). These can indicate the presence of thalassemia when MCV<80 fL and MCH<27 pg(Hartwell et al. 2005).

Hemoglobin variants are routinely screened using electrophoresis and chromatography techniques.The most commonly used approaches are ion exchange chromatography, and isoelectric focusing (IEF). Electrophoresis has been the method of choice for identification and quantification of variant Hbs, but it is slow, labour- intensive and inaccurate at low concentration range, and it is not suitable for large batches of samples (Clarke and Higgins 2000).

The Hb variants which must be detected under the screening programme are: Sickle cell anemia (HbSS), HbS/β-thalassemia, HbS/HPHF, HbSC disease, HbS/DPunjab, HbS/E, HbS/OArab. It is also considered necessary to detect β-thalassemia major, HbE/β-thalssemia, β-thalassemia intermedia and HbH disease (NHS 2012b). The employed analytical procedures must be capable of detecting all the common clinically significant Hb variants and also to quantify HbA2 and HbF accurately as a

screening tool for thalassemias (Ryan et al. 2010).

High performance liquid chromatography (HPLC) separates different hemoglobins based on their retention times which depend on their interactions with the stationary phase. In cation exchange HPLC (ce-HPLC) the components with a net positive charge are separated from their interaction with a negatively charged stationary phase, while the mobile phase flows through with an increasing concentration of cations (Bain 2006b). Cation-exchange HPLC has been evaluated for presumptive identification of hemoglobin variants, giving unambiguous results in 90% of cases (Chevenne et al. 1999, Riou et al. 1997). HPLC has many advantages, it can be automated and is capable of processing large batches, provides separation of clinically significant variants and allows sufficiently accurate quantitation of HbA2

and HbF (Ryan et al. 2010). It is, however, unable to detect a large number of existing hemoglobin variants and cannot fully characterise any novel ones which may be present. It only provides an indication that a sample is not normal. The obtained retention times are reproducible under specific experimental conditions. Different variants, however, can elute at the same retention time, so retention time does not provide definitive identification.

A HPLC chromatogram from a normal blood sample is shown in Figure 1.17. In this method peaks correspond to hemoglobin heterodimers (αβ dimers), and elution of

49 HbA2 fully separated from HbA allows the quantitation based on integration of the

peak. The different retention windows show the expected ranges for different clinically significant variants. The HPLC measurement only takes 7.5 minutes and sample introduction and report processing is automated.

Figure 1.17. Chromatogram from TOSOH HLC-723 HbG7 analyser (Tosoh Bioscience Ltd., Redditch, UK) from normal adult.

Retention time windows for Hb F, A, A2, D, S and C are shown.

IEF separates variants based on their isoelectric point, different hemoglobins migrate via a pH gradient to the point where the pH corresponds to their pI, and net charges are zero (Hartwell et al. 2005). The bands are sharper than in the case of electrophoretic techniques (cellulose acetate electrophoresis), and hemoglobins which could not be separated by electrophoresis can be distinguished and identified. The precision for quantification at low concentrations is poor and this technique has not been validated for HbA2 quantitation (Bain 2006b).

DNA analysis is required for absolute genetic characterization of mutations resulting in β-thalassemia and quantitation of the HbA2 is crucial for the routine identification

of carriers. Guidelines and recommendations for correct HbA2 measurements can be

found in publications by Stephens et al. Stephens points out a common problem, that many Hb variants partially or totally co-elute with HbA2, making the quantification

in certain circumstances unreliable (Stephens et al. 2011b). The national recommended cut-off for HbA2 (3.5%) is the action point which has been established

for the diagnosis of β-thalassemia carriers (Ryan et al. 2010).

Hb A

αβdimer

Hb A

Hb D

Hb SHb C

Hb F

Glycated Hb

50 Measurement of Hb F is important in the diagnosis of δβ thalassaemia, hereditary persistence of fetal haemoglobin (HPFH) and in the diagnosis and management of sickle cell disease. As HbF is expected to be present up to 1%, elevated levels of HbF are of diagnostic value. In thalassemia trait levels of up to 10% occur, in δβ thalassaemia trait levels between 5% and 20%, and in HPFH heterozygotes from 2.5% up to 30% in the deletional forms are found (Stephens et al. 2011a). Further investigation of elevated HbF is initiated at a measured level of 5% or higher (Ryan

et al. 2010).

Presumptive identification should be based on a minimum of two techniques based on different principles (Ryan et al. 2010). The routine techniques are useful in the screening of the common clinically significant variants but they are unable to identify novel variants and so additional DNA sequencing or tandem mass spectrometry (MS/MS) is required for identification of a variant. DNA analysis for the diagnosis of every patient is not achievable due to high cost and complexity. The use of mass spectrometry has become an alternative for variant identification and has potential to become a future tool in everyday screening(Ryan et al. 2010).

Some lessons and pitfalls about currently used screening methods and algorithms on how to get the most appropriate results can be found in recent review by Barbara J. Bain (Bain 2011).

1.3.4 Mass spectrometry-based identification of hemoglobinopathies and