Guidance

Referral of antenatal samples for molecular analysis

Updated 10 October 2024

Applies to England

Most pregnancies at risk of clinically significant β thalassaemia or sickle cell disease are identified initially through the antenatal screening programme. The diagnosis of α thalassaemia is more complicated because DNA analysis is the only way to distinguish between α⁺ thalassaemia, non deletional α thalassaemia and α⁰ thalassaemia.

Due to the high frequency of α⁺ thalassaemia in some populations and the low cost benefit ratio in a screening programme, DNA confirmation is not required for all possible cases of α thalassaemia. The screening programme relies on a strategy for diagnosis of α⁰ thalassaemia by combining haematological tests and details of family origin^{[footnote 1]}.

Although the antenatal screening programme guidelines are designed to identify most carriers for serious haemoglobinopathies, including thalassaemias, the screening protocols will not identify every pregnancy at risk for every haemoglobinopathy. For example, they are not designed to pick up pregnancies at risk for Haemoglobin H (HbH) disease or the extremely rare cases of pregnancies where the family origins on the family origin questionnaire (FOQ) are low risk for Hb Bart’s hydrops fetalis syndrome.

1. Testing of the baby’s biological father

This must be performed according to screening protocols as for maternal phenotype testing. If the baby’s biological father has a phenotype that can interact with the maternal phenotype – see Referral guidelines for antenatal screening specimens – then they should be counselled. If prenatal diagnosis (PND) is being considered, refer to the following guidelines for sample requirements and recommendations. Seek advice if there is concern around timeliness of PND testing.

If the baby’s biological father is unavailable for testing or his haemoglobinopathy status is unknown the programme supports the women being offered PND. PND can be undertaken without the DNA of the baby’s biological father, although in some circumstances the diagnosis will not be able to be given with such a high degree of certainty as when the baby’s biological father’s mutation is known.

1.1 Referral of blood samples for DNA analysis

The blood samples must be accompanied by an appropriately completed laboratory referral form (obtained from the referral laboratory) and the laboratory notified before sending samples to the genetic hubs, and ultimately to the commissioned hubs for specialist haematology. Screening results must be included in the referral information. This includes full blood count (FBC), HPLC or CE results with the percentage of all haemoglobins detected and any relevant clinical information.

Prior notification for PND samples is essential, as is inclusion of screening results (which must include FBC, HPLC or CE results providing the percentage of all haemoglobins detected) in the referral information.

DNA laboratories must take the phenotypic information into account when compiling the final interpretive report.

2. Haemoglobin variants

If both biological parents carry HbS, or one carries S and one carries C then blood samples are not required immediately to confirm the mutation by DNA analysis.

If PND is accepted, fresh blood samples from both biological parents should be sent at the same time or before the fetal sample.

If one biological parent carries HbS and the other is a suspected carrier of HbO^Arab, HbD^Punjab, HbE, Hb Lepore or a type of β thalassaemia (including δβ thalassaemia) then:

• confirmation of the biological parents’ mutations before PND testing is advisable, provided it does not limit options available by affecting timelines

• if PND is accepted, a fresh maternal sample (and paternal sample), if not previously tested as above, should be sent at the same time, or before the fetal sample

If one biological parent carries HbS and the other is thought to carry deletional hereditary persistance of fetal haemoglobin (HPFH) then:

• PND is not usually indicated
• it is important to make sure that deletional HPFH is differentiated from δβ thalassaemia (these cases may require mutational analysis)

Carriers of deletional HPFH have a raised HbF level of approximately 20 to 30% and are usually associated with normal red cell indices. It is important to differentiate deletional HPFH from δβ thalassaemia. The carrier state for δβ thalassaemia is associated with a reduced mean cell haemoglobin (MCH) and an HbF level usually in the range of 5 to 15%. Non-deletional HPFH usually results in a more modest increase of HbF (less than 10%) in adults and is found in many populations. It is usually associated with normal red cell indices.

If one biological parent carries HbE and the other carries β thalassaemia, Hb Lepore, δβ thalassaemia, or is thought to carry α⁰ thalassaemia, then:

• confirmation of the biological parents’ mutations before fetal sampling is advisable, provided it does not limit options available by affecting timelines

• if PND is accepted, a fresh maternal sample (and paternal sample), if not previously tested as above, should be sent at the same time or before the fetal sample

Note that in people of high-risk family origins for α⁰ thalassaemia, there is a possibility of a hidden risk to the fetus of homozygous α⁰ thalassaemia, as the carrier state for α⁰ thalassaemia can be masked. It is important to determine the α genotype by DNA analysis.

3. Beta thalassaemias

If one biological parent carries β thalassaemia, Hb Lepore, δβ thalassaemia and the other carries β thalassaemia, Hb Lepore, δβ thalassaemia, HbO^Arab or HbS, then:

• confirmation of the biological parents’ mutations before fetal sampling is advisable, provided it does not limit options available by affecting timelines

• if PND is accepted, a fresh maternal sample (and paternal sample), if not previously tested as above, should be sent at the same time or before the fetal sample

Note that in people of high-risk family origins for α⁰ thalassaemia, the carrier state for α⁰ thalassaemia may also be present. It is important to determine the α genotype by DNA analysis.

4. Alpha thalassaemias

4.1 Coinheritance of α thalassaemia with other haemoglobin variants

Blood samples should not be sent to confirm and identify α thalassaemia, unless both biological parents are of high-risk family origins for α⁰ thalassaemia. There is rarely clinical interaction between α thalassaemia and other haemoglobin variants. Unless there are high-risk family origins, the type of α thalassaemia is almost always the α⁺ thalassaemia type, which usually poses no serious genetic risk to the fetus.

4.2 Alpha plus thalassaemia (α⁺ thalassaemia or -α/αα and -α/-α)

This is found in all ethnic groups, with a high (10 to 30%) carrier frequency in some parts of Africa, in African-Caribbeans and in South and Southeast Asia. Even if both biological parents are carriers, there is no risk to the fetus. Homozygous α⁺ thalassaemia (-α/-α) is not a clinically significant disorder with respect to genetic or obstetric complications, but can cause diagnostic confusion with α⁰ thalassaemia or iron deficiency.

Heterozygotes (-α/αα) generally have an MCH of 25 to 28pg and a normal HbA₂ level. Approximately one third of cases will be silent.

Homozygotes (-α/-α) generally have an MCH below 25pg and some have HbH inclusions, as do carriers for α⁰ thalassaemia (- -/αα).

Individuals may be heterozygous for an α globin gene deletion (carrier genotype: -α/αα) or a rarer point mutation in one alpha globin gene affecting gene expression (carrier genotype denoted: α^Tα/αα if the mutation is on alpha 1, or αα^T/αα if mutation is on alpha 2), commonly called a non-deletional mutation. There are 2 common deletions (-α^3.7 and –α^4.2) and a number of less common non-deletional mutations, some of which also produce unstable haemoglobin variants which may be observed as minor peaks in relatively fresh blood samples.

5. Alpha zero thalassaemia

This carries the potential for a clinically significant condition.

If both biological parents are carriers of α⁰ thalassaemia (- -/αα), the pregnancy has a 1 in 4 risk of producing a fetus with Hb Bart’s hydrops fetalis syndrome (- -/- -) and the mother is at risk of obstetric complications, particularly in the third trimester of pregnancy. The mutations leading to Hb Bart’s hydrops fetalis syndrome are almost always alpha globin gene deletions.

If one biological parent carries α⁰ thalassaemia (- -/αα) and the other α⁺ thalassaemia (either -α/αα or -α/-α), there is a risk of having a child with HbH disease (- -/-α). This disorder is usually associated with a moderate anaemia but typically requires little clinical intervention. Therefore PND is not usually indicated for HbH disease unless there are known higher risk mutations that can result in HbH hydrops such as Hb Constant Spring or Hb Adana.

1. Sprour Y, Heppinstall S, Porter N, Wilson G, Goodeve A, Rees D, Wright J. Is routine molecular screening for common a thalassaemia deletions necessary as part of an antenatal screening programme? Journal of Medical Screening 2007: volume 14, pages 60 to 61 ↩