Guidance

Antenatal screening

Updated 5 September 2022

Applies to England

The aim of the antenatal screening programme is to offer timely antenatal sickle cell and thalassaemia (SCT) screening to all women (and couples), to enable personal informed choice. We aim to:

• screen women by 10 weeks + 0 days of pregnancy
• offer prenatal diagnosis (PND) to at risk women and couples by 12 weeks + 0 days of pregnancy
• perform PND before 12 weeks + 6 days of pregnancy

The antenatal and newborn SCT screening programme aims to:

• promote an appropriate level of understanding about screening for these genetically inherited conditions among professionals involved with the programme
• review the results from antenatal testing before, during and after the newborn test is offered, and to check that the results are congruent
• prepare parents for their baby’s screening result

1. Provision of screening

There are 2 approaches to the provision of the screening programme. The approach depends on whether a provider is defined as high prevalence or low prevalence. Each laboratory must use only one screening algorithm. Where high and low prevalence trusts merge, the high prevalence algorithm should be used. In all cases, a failsafe system must be in place to make sure all eligible women have been offered screening, and those who accept are tested.

1.1 High prevalence

Providers are considered high prevalence if 2% or more of antenatal screening test results for SCT received by the laboratory are screen positive. The high prevalence algorithm is viewed as the gold standard. In this case, laboratory testing for haemoglobin variants and thalassaemia must be carried out on all women who have accepted screening.

1.2 Low prevalence

Providers are considered low prevalence where less than 1% of the antenatal screening test results for SCT received by the laboratory are screen positive. Areas defined as low prevalence must perform screening for thalassaemia on all women who have accepted screening using the routine Hb and red cell indices. Further laboratory testing must be carried out on all women with defined abnormalities of the full blood count, those with high risk family origins in either biological parent as determined by the family origin questionnaire (FOQ) and those women who request testing.

We advise providers that have a prevalence between these 2 values to continue to use their current high or low prevalence algorithm.

1.3 Testing in subsequent pregnancies

If a woman is booked for antenatal care for a subsequent pregnancy, the healthcare professional must complete the FOQ, irrespective of previous screening. If the woman accepts screening, the healthcare professional must take the blood sample. They must make sure the sample is clearly labelled and sent to the laboratory accompanied by the completed FOQ. In both high and low prevalence settings this blood sample must be tested according to the screening algorithm. In low prevalence settings HPLC or CE must be performed if there is indication from the FOQ or FBC that it is necessary even if this has been performed previously. If, on repeat testing by HPLC or CE, a variant is found with identical characteristics to one previously confirmed, second line testing does not need to be performed again.

If a woman with the condition or who is a carrier is identified, the baby’s biological father must be offered a screening test, irrespective of previous screening history (see section 4.5 below).

Due to the complexities of the testing algorithm, screening for SCT must be repeated in every pregnancy. Laboratories must be aware that screening safety incidents have resulted when this policy has not been followed.

2. Organisation of screening services

Providers must have a risk assessment of the entire pathway and protocols defining where responsibility falls for all aspects of the process. Laboratories must have protocols detailing receipt of samples, analytical procedures and reporting of results in a timely manner. If some or all samples are referred to, or received from, other laboratories, agreements must be in place as per ISO 15189:2012 requirements. Laboratory staff must follow the analytical guidelines, algorithms and service specifications produced by the NHS SCT Screening Programme. Additionally, there must be an agreement outlining who is responsible for the provision of key performance indicators (KPIs) and annual return data.

Where opinions on results obtained are sought from third parties, laboratories must make sure they meet and comply with screening programme requirements. This is particularly important when using any service provided by a commercial organisation. When reports received from third parties are transcribed into internal laboratory information systems, a full and exact copy of the report must be made. The transcribed report must be checked by a second person who is a registered scientist to ensure accuracy. Laboratories should scan these reports if possible or retain the original in line with local laboratory record keeping policies.

3. Family origin questionnaire (FOQ)

The FOQ is used in all areas. In low prevalence areas it is the basis for determining which women must be tested for haemoglobin variants.

The woman’s sample must be tested if she or the baby’s biological father is in a high risk group.

In high prevalence areas, all women choosing to be screened must be tested. If a woman has declined screening then testing should not be carried out and this must be documented.

Local policy must be in place for situations where an FOQ has not been received or is incomplete to make sure test reporting is not delayed beyond the 3-day turnaround time. If necessary, the sample must be treated as high risk family origins and tested to achieve this. Where multiple family origins are selected the presence of a high risk family origin has priority and must result in a test. The FOQ must not be rejected if the only missing information is the gestational age.

All women must be screened for thalassaemia using the mean cell haemoglobin (MCH) measurement, which has been shown to be stable for up to 4 to 5 days at room temperature ^{[footnote 1] [footnote 2]}. The family origin also assists laboratories with the interpretation of laboratory screening results, particularly the interpretation of results indicating possible alpha (α) thalassaemia. People originating from certain areas of the Far East and Eastern Mediterranean are at higher risk of α⁰ thalassaemia (see section 10 below), which in the homozygous state results in Hb Bart’s hydrops fetalis syndrome.

The FOQ was developed after a literature review and research^{[footnote 3]}. The questions aim to determine family origins and are different from census-based self-assigned ethnic group questions.

Providers are responsible for producing an FOQ which is consistent with the version that has been tested rigorously in clinical practice. See guidance on developing a digital, electronic FOQ (e-FOQ), a paper FOQ form or a combined FOQ booking blood request form for all antenatal screening.

4. Situations requiring particular care

4.1 Fertility treatment – donor gametes

If the pregnancy was achieved using a donor egg then the screening results on the woman will not be informative. The baby’s biological father must be offered testing and, if screen positive, the report must recommend that the fertility clinic is contacted to obtain the biological mother’s haemoglobinopathy results.

If donor sperm was used and the woman has a positive screening result then the report must recommend that the fertility clinic is contacted to obtain the biological father’s haemoglobinopathy results.

In the case of surrogacy, the report must recommend that the fertility clinic is contacted to obtain haemoglobinopathy results of both biological parents.

Test the pregnant woman according to your current high or low prevalence status to ensure optimal maternal care. The report must provide the appropriate advice for the circumstances of the pregnancy.

4.2 Adoption

If either biological parent was adopted, the FOQ information may not accurately reflect the true family origins. Such cases must be treated as high risk and have full laboratory screening.

4.3 Bone marrow transplant (BMT)

Where either biological parent has had a BMT it is likely the results obtained will reflect the BMT donor rather than the biological parent and so will not accurately represent the genetic status of the fetus. If the biological mother has had a BMT, the baby’s biological father must be offered testing to make sure this is not a high risk pregnancy. If DNA confirmation of the biological mother’s status is required, or if the baby’s biological father is post BMT and requires testing, then options that can be considered include:

• use of pre-transplant DNA

• obtaining DNA from samples other than blood if there is sufficient time and choice is not compromised (extraction of DNA from skin biopsy is currently the only accredited procedure).

• referral for specialist counselling and offer of prenatal diagnosis (PND)

4.4 Gene therapy

Where either biological parent has had gene therapy for a haemoglobinopathy it is likely the results obtained will be misleading. Results will vary depending on the strategy used. If the biological mother has had gene therapy, the baby’s biological father must be offered testing to make sure this is not a high risk pregnancy.

If DNA confirmation of the biological mother’s status is required or if the baby’s biological father is post gene therapy and requires testing, then options that can be considered are:

• use of pre-transplant DNA

• DNA obtained from samples other than blood, if there is sufficient time and choice is not compromised (note extraction of DNA from skin biopsy is currently the only accredited procedure)

• referral for specialist counselling and offer of PND

4.5 Maternal conditions requiring testing of the baby’s biological father

Antenatal screening should detect the following significant maternal haemoglobinopathies that are important for maternal care:

• Hb SS
• Hb SC
• Hb SD^Punjab
• Hb SE
• Hb SO^Arab
• Hb S/Lepore and Hb Lepore/β thalassaemia
• Hb S/β thalassaemia
• Hb S/δβ thalassaemia
• HbH disease (- -/-α)
• β thalassaemia major/intermedia
• Hb E/β thalassaemia

Antenatal screening should also detect the following carrier states in the biological mother:

• HbS
• HbC
• HbD^Punjab
• HbE
• HbO^Arab
• Hb Lepore
• β thalassaemia
• δβ thalassaemia
• α⁰ thalassaemia (- -/αα)
• Hereditary Persistance of Fetal Haemoglobin (HPFH)

In addition, antenatal screening should detect:

• any compound heterozygote state including one or more of the above conditions

• any homozygous state of the above conditions

5. Laboratory screening techniques

In low prevalence areas the first-line screen for thalassaemia is the full blood count.

The following techniques are suitable for use in first-line screening for haemoglobin variants and for HbA₂ quantitation:

• high performance liquid chromatography (HPLC), preferably with continuous gradient elution
• capillary electrophoresis (CE)

Both these techniques provide provisional results. Abnormal results must have further testing with a minimum of one alternate technique with a different analytical principle to the original and which is appropriate for the suspected variant (section 5.1 below). See minimum requirements for quality control material for these techniques. In some cases more than one technique will be required. If results from all techniques are interpreted together laboratories can achieve a result which is sufficiently reliable for screening purposes. Only mass spectrometry and DNA analysis provide a definitive result.

Devices which primarily detect HbS or have a limited repertoire are not suitable for use as a first line screen.

5.1 Acceptable laboratory techniques

Initial method is high performance liquid chromatography (HPLC)

Alternate method:

• Alkaline electrophoresis
• Acid electrophoresis
• Isoelectric focusing (IEF)
• CE
• Mass spectrometry
• DNA

Initial method is capillary electrophoresis (CE)

Alternate method:

• Acid electrophoresis
• Alkaline electrophoresis
• HPLC
• IEF
• Mass spectrometry
• DNA

Please note, results from alkaline electrophoresis are very similar to CE, so this should not be used as the only alternate test. In the absence of IEF it may be useful in combination with acid electrophoresis.

In most circumstances, a validated specific sickle test can be used as confirmation of an initial screen that suggests the presence of HbS. There is an inherent unreliability in the sickle solubility test with the risk of false positive and false negative results. Special care must be taken when using an instrument where HbS co-elutes with another haemoglobin variant, such as HbC. For example, in cases of co-eluting haemoglobin variants S and C, a negative solubility test cannot be presumed to indicate HbC and a positive solubility alone cannot be presumed to indicate HbS. In all cases where haemoglobins co-elute, it is necessary to use an additional technique, for example alkaline electrophoresis or IEF.

5.2 HbA₂ measurement

HPLC and CE methods are acceptable. Isoelectric focusing (IEF) and scanning densitometry are not acceptable.

5.3 HbF measurement

HPLC, CE or 2-minute alkali denaturation are acceptable. The Kleihauer test is not appropriate for measurement but is useful to support the identification of HbF.

6. General laboratory considerations

This handbook highlights common analytical and diagnostic issues but every laboratory must follow the principles of good laboratory practice. Laboratories must make sure they understand the capabilities and limitations of their chosen technique. The equipment and protocol chosen must fulfil the requirements of the screening programme and demonstrate suitable performance on external quality assurance (EQA). The manufacturer’s published recommendations should be followed. Where an instrument operates with more than one analytical programme designed for haemoglobin variant analysis then both programmes must be validated to make sure there are no significant differences where a numerical value is to be reported. If significant differences are found a single programme should be chosen based on the validation data, from which all results are reported.

It may be necessary to return to the original specimen tube to check the identification details. For this reason, it should be standard practice to make sure that any labels affixed when the specimen is received in the laboratory do not obscure the written identity or identity label already attached.

For all analytical techniques, appropriate controls must be included wherever possible. If IEF or electrophoresis is used, then control haemoglobins must be run with each plate.

See Recommended quality control for antenatal screening for sickle cell disease.

If a column is used this must be replaced once it has performed the recommended number of analyses as stated in writing by the manufacturer or if the performance is observed to deteriorate.

If software rules are used to screen samples for further action and reporting, it is essential that the process is risk assessed and there are failsafe mechanisms in place.

Raw data, including analytical traces, must always be reviewed and any post analytical procedures, including algorithms, must be fully documented and traceable to make sure there is consistency of quality. All raw data, including analytical traces and interpretative comments, must be reviewed by 2 people, one of whom must be a registered scientist.

Final validation must be by a person with suitable expertise, as demonstrated by current competency testing.

6.1 Selecting an analytical system

Co-ordination of pathology services across chemistry and haematology can allow the sharing of equipment, for example with that used for measurement of HbA1c. In all situations, it is essential that HbA₂ is analysed by a protocol that calibrates and clearly separates HbA₂ from HbA. The required antenatal turnaround time of 3 working days for the issue of a report must be considered when this approach is used.

6.2 High performance liquid chromatography (HPLC)

HPLC uses an ion exchange resin, held in a column, in conjunction with a buffer gradient. As the ionic strength and/or pH of the buffer changes, certain haemoglobins are eluted from the column and the presence of haemoglobin is detected using a spectrophotometric technique. The time from injection to the point at which the haemoglobin fraction elutes is known as the retention time of the haemoglobin and is a reproducible measurement for a particular column, buffer, exchange resin and temperature.

It is common for different haemoglobins to elute at the same retention time. Thus the retention time is not a unique identifier. HbF and HbA₂ elute separately and the relative proportion is calculated with the use of calibrators. Ideally haemoglobins S, C, D, E and O^Arab should have separate retention times and characteristic chromatographic profiles. In addition, the relative proportions of the different haemoglobins are recorded. HPLC analysers that use step-wise buffer gradients are not recommended if they produce co-elution of common haemoglobin variants which can lead to misinterpretation.

When selecting an analyser for quantitation of HbA₂ the first consideration is good separation. It is important to make sure the analyser clearly separates peaks to ensure accurate quantitation as shown in Figure 1 below. Laboratories should understand how the integration takes place and be aware that peaks measured on sloping baselines or on shoulders of adjacent peaks are likely to be less reliable. Sophisticated integration and the use of calibration factors cannot make up for poor chromatography.

Figure 1: examples of integration

6.3 Capillary electrophoresis (CE)

CE uses a combination of ion migration and electro-osmotic flow to separate protein molecules. When a voltage is applied across the capillary tube filled with an electrolyte solution, the solution begins to move towards one of the electrodes due to electro-osmotic flow. This drives the bulk flow of materials past the detector in the same way that a pump pushes the liquid in HPLC. The haemoglobin molecules move towards the detector at different speeds depending on their ionic charge and electrophoretic mobility. Both electro-osmotic flow and electrophoretic mobility are occurring at the same time, working in opposite directions to provide greater resolution.

This method of separation should not be confused with simple electrophoretic mobility as seen in cellulose acetate electrophoresis. Combining electro-osmotic flow and electrophoretic mobility is a separate phenomenon and is exploited in CE for maximum separation power. Even so, it is quite common for different haemoglobins to migrate at the same rate and appear at the same position, so position is not a unique identifier. HbF and HbA₂ elute separately and the relative proportion of each is calculated. Haemoglobins S, C, D, E and O^Arab also have separate retention times and characteristic profiles. In addition, the relative proportions of the different haemoglobins are recorded.

Optical density (OD) levels greater than 0.07 and the presence of either HbA and HbA₂ or HbF and HbA₂ are required to determine the migration position and thus permit ‘zoning’ and a provisional identification of haemoglobins present in the sample. If failure to zone is due to low OD, this is usually related to the amount of haemoglobin in the sample. This should be corrected by increasing the haemoglobin to diluent ratio. Extreme care is needed if extraneous haemoglobin is added to a clinical sample to allow zoning. The addition of haemoglobins which were not present in the initial sample may lead to misinterpretation of the results.

6.4 Action values

A HbA₂ action value of greater than or equal to 3.5% is set for carriers of β thalassaemia ^{[footnote 4]}.

There are 2 action values for HbF:

• if the MCH is greater than or equal to 27pg, the action value is greater than or equal to 10%

• if the MCH is less than 27pg, the action value is greater than or equal to 5.0%

The chosen system must be able to measure HbA₂ and HbF with accuracy and precision at these action values and detect the haemoglobin variants as specified by the antenatal screening programme. Instrument validation protocols must assess these requirements. Quantitation at different levels may be needed for other clinical purposes.

UK National External Quality Assurance Scheme (UK NEQAS) data and published literature have demonstrated biases between different analysers for HbA₂. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the International Council for Standardization in Haematology (ICSH) have recognised the need to develop a new certified reference material for HbA₂. The screening programme supports the objectives of this work and continues to work with UK NEQAS to monitor the impact of inter-method variability.

Laboratories in the UK should be aware of these problems and make sure they have optimised their methods. When considering replacement purchases, laboratories should review all available evidence, including UK NEQAS data.

Analytical systems should be able to detect and measure at least the most common HbA₂ variant (HbA₂^Prime (A₂’)) to enable an approximate calculation of total HbA₂ (for example HbA₂ plus A₂’).

If using equipment or an elution programme for more than one analyte, for example HbA₂ and HbA1c, laboratories should make sure that the quantitation of HbA₂ and HbF is not compromised.

7. Good practice and troubleshooting

When operating analytical systems the following points of good practice should be followed and may be used as a troubleshooting guide if problems are identified by internal and/or external quality assurance.

1. Make sure the HPLC column count is within manufacturer’s written guaranteed limit.

2. Make sure the calibration factor does not drift significantly or fall outside recommended limits.

3. Monitor internal quality control for drift.

4. Follow the manufacturer’s recommended maintenance and cleaning protocols.

5. Make sure that reagents, controls and calibrators are stored in the correct conditions, are within date, have appropriate lot numbers and are in the correct positions.

6. Avoid pooling and mixing reagents, do not pool reagents with different lot numbers.

7. When reviewing analytical traces, make sure the appearance is as it should be with the correct peak shape, baseline and separation from adjacent haemoglobin peaks.

8. Make sure maintenance visits are carried out at the recommended intervals.

9. If the machine is moved or laboratory conditions change, appropriate revalidation procedures must be performed.

10. If problems occur that cannot be resolved contact the instrument manufacturer.

If samples require modification by the addition of extraneous haemoglobin an aliquot must be used. This must be analysed with a unique identifier distinguishable from both the original specimen and from any other clinical sample.

In the event of an out of consensus EQA result a look back exercise must be considered. Look back may also be required when internal quality assurance falls outside acceptable limits or when indicated for any other reason. See Governance, quality assurance and accreditation.

8. Problems with the measurement and interpretation of HbA₂

In the presence of an α chain variant it can be difficult to obtain a reliable HbA₂ value. Where the biological mother has an MCH of less than 27pg and is found to have a suspected α chain variant or a haemoglobin variant that coelutes with, or compromises the HbA₂, the baby’s biological father must be offered screening. If the biological father has an MCH of less than 27pg with a suspected α chain variant or a variant which co-elutes with, or compromises the HbA₂ and the biological mother is known to have β thalassaemia, or has Hb S, E, O^Arab or Lepore and/or is at risk of alpha zero (α⁰) thalassaemia, DNA studies should be performed.

If any small peaks which could be an HbA₂ variant elute separately from the main HbA₂ peak in a patient with an MCH below the action value (< 27pg), further investigation will be required if the addition of these peaks brings the total HbA₂ greater than or equal to 3.5%.

HbA₂ values of 4.0% and above with normal indices may indicate a carrier of β thalassaemia. In this case the findings including the FBC should be confirmed by re-running the existing samples. If the results remain the same then the baby’s biological father must be tested. Other factors such as HIV infection, B12/folate deficiency or liver disease/alcohol can increase the HbA₂.

HbA₂ values less than 4.0% with normal red cell indices and an HbF level of less than 10% are regarded as not significant for screening purposes.

With many HPLC systems, HbA₂ is overestimated in the presence of HbS. This is not a problem where the percentage of HbA is greater than HbS. See section 2 of Interpretation and reporting of antenatal screening results.

With many HPLC systems, HbA₂ is underestimated in the presence of HbD. This is not a problem where the percentage of HbA is greater than HbD but may make discrimination between Hb DD and D/β⁰ thalassaemia more difficult. Make sure red cell indices are reviewed.

When using CE, HbA₂ is underestimated in the presence of HbC. This is not a problem where the percentage of HbA is greater than HbC but may make discrimination between Hb CC and C/β⁰ thalassaemia more difficult. Make sure red cell indices are reviewed.

For analysers that separate and measure HbA₂ in the presence of HbE, caution should be used when interpreting this value.

9. Iron deficiency

There are varying reports of the impact of iron deficiency on the HbA₂ level, some report a mild reduction in cases of severe iron deficiency anaemia ^{[footnote 5]} but screening for haemoglobin variants and thalassaemia should proceed without regard to iron deficiency, suspected or proven.

Any decrease in MCH should be regarded as potentially due to a haemoglobinopathy and the HbA₂ should be measured. It may be appropriate to simultaneously investigate pregnant women for iron deficiency, using ferritin or zinc protoporphyrin but this is not specifically part of the screening protocols.

In pregnant women there is no justification for delaying the investigation of haemoglobinopathies while treating iron deficiency, as this will delay the process of identifying at risk pregnancies where PND should be offered.

10. Screening for carriers of alpha zero thalassaemia

10.1 Methods of screening

The lack of a specific biomarker for the detection of α thalassaemia carriers creates problems, particularly in the context of a screening programme. In this context only alpha zero (α⁰) thalassaemia is regarded as significant and policies are designed to detect pregnancies at risk of Hb Bart’s hydrops fetalis syndrome. Policy is designed to increase the positive predictive value of the screening algorithm and reduce false positives.

10.2 Diagnosis

Molecular techniques must be used for the confirmation and diagnosis of α⁰ thalassaemia when suspected in both biological parents. Alpha thalassaemia mutations may be deletional (α⁺, α⁰) or non-deletional (α^T), requiring the use of different diagnostic techniques.

10.3 Population estimates and ethnic distribution of α⁰ thalassaemia

It is estimated that only a small number of cases of Hb Bart’s hydrops fetalis syndrome can be expected each year in England, with approximately half of these of Chinese family origin.

Alpha zero thalassaemia is most commonly found in those of Southeast Asian origin (China (including Hong Kong), Thailand, Taiwan, Cambodia, Laos, Vietnam, Indonesia, Myanmar (formerly Burma), Malaysia, Singapore or Philippines) and East Mediterranean (Cyprus, Greece, Sardinia or Turkey). There are 2 common Mediterranean (- -MED, - -α^(20.5)) and 3 common Southeast Asian deletional mutations (- -SEA, - -THAI, - -FIL).

Alpha zero thalassaemia is also reported to occur at low frequencies in some Middle Eastern countries: the - -MED allele in the UAE, Iran, Yemen, Kuwait, and Jordan; the - -α^(20.5)) allele in Iran; the - -YEM allele in the Yemen.

Alpha zero thalassaemia is rarely reported in patients of African, Pakistani and Indian origin. Alpha zero thalassaemia is rarely observed in patients of British origin and no pregnancy at risk of Hb Bart’s hydrops fetalis syndrome has been reported in this population. In these family origin groups the risk is small and in the context of screening, the cost benefit ratio is poor. Therefore the FOQ does not identify these groups as at high risk for α⁰ thalassaemia ^{[footnote 6]}.

11. Further investigation of alpha zero thalassaemia

Hb Bart’s hydrops fetalis syndrome (α thalassaemia major) is invariably fatal without treatment due to severe fetal anaemia. If not detected, it can result in a stillbirth. A mother carrying a fetus with Hb Bart’s hydrops fetalis syndrome is at risk of obstetric complications, such as pre-eclampsia and hypertension, particularly in the third trimester of pregnancy.

If a fetus with Hb Bart’s hydrops fetalis syndrome is transfused in utero, survival is possible but the baby will be transfusion-dependent. BMT and gene therapy options are also being investigated for these cases. Usually a baby only has Hb Bart’s hydrops fetalis syndrome if both biological parents are carriers of α⁰ thalassaemia.

11.1 Deletional α thalassaemia genotypes

Gentotype	Phenotype
αα/αα	Normal
-α/αα	Alpha plus (α+) thalassaemia (heterozygote)
-α/-α	Alpha plus (α+) thalassaemia (homozygote)
- -/αα	Alpha zero (α0) thalassaemia (heterozygote)
- - /-α	Haemoglobin H disease
- -/- -	Hb Bart’s hydrops fetalis syndrome

11.2 The approach to screening and α⁰ thalassaemia

Two sets of information from the pregnant woman are combined as the screen:

1. Is the MCH <25pg?

2. Is the woman’s family origin identified as high risk from the FOQ: China (including Hong Kong), Southeast Asia (especially Thailand, Taiwan, Cambodia, Laos, Vietnam, Myanmar (formerly Burma), Malaysia, Singapore, Indonesia or Philippines, Cyprus, Greece, Sardinia, Turkey, or unknown?

If the answer to both questions is yes, testing of the baby’s biological father must be offered if he is also from a high risk area or his family origins are unknown.

If both biological parents are identified to be at risk for α⁰ thalassaemia DNA analysis is required for confirmation.

Please note coinheritance of another thalassaemia mutation such as beta or delta beta thalassaemia can complicate screening for α⁰ thalassaemia carriers. Where there are high risk family origins, in such cases, the possibility of α⁰ thalassaemia should be considered, even if the MCH is >25pg.

11.3 DNA analysis of samples and α⁰ thalassaemia

The policy guidance developed by the screening programme reduces the number of cases with α+ (-α or α^T) referred for DNA analysis.

11.4 Evidence for above approach

Published studies have shown that 99% of α⁰ thalassaemia cases have an MCH <25pg. In a series of 270 carriers of α⁰ thalassaemia diagnosed by DNA analysis in the UK, only 2 patients had an MCH between 25 and 26pg – one with liver disease ^{[footnote 7]}. Findings in a study from Sheffield ^{[footnote 8]} which undertook DNA analysis in 425 pregnant women with an MCH <27pg showed that all cases of α⁰ thalassaemia had an MCH <25pg and would have been detected using an FOQ alone, which supports the screening programme’s approach.

12. Risk assessment for antenatal screening in high and low prevalence areas

It is inevitable there will be some false positives and false negatives as a result of screening because of the way screening programmes are designed, particularly with the use of action values. False positives have a positive result from the screening test but, when further tests are performed, do not have one of the designated haemoglobins or thalassaemia. Potential causes of false negatives are shown below.

Conditions that may be missed using the screening scenarios (assuming that the FOQ is completed accurately) in high and low prevalence areas include:

• ‘silent’ or ‘near silent’ β thalassaemia carriers. Some β thalassaemia carrier genotypes are associated with borderline HbA₂ levels and an action value of 3.5% with an MCH of < 27pg will miss some cases. Examples of such mutations include the c.-50 A>C [CAP+1 (A>C)], c.92+6 T>C [IVSI-6 (T>C)], c.-151C>T [-101 (C>T)] and c.111 A>G or c.110 T>C [Poly A (A>G) or (T>C)]
• some β thalassaemia carriers obscured by severe iron deficiency anaemia
• thalassaemia carriers where the MCH is raised, for example B12/folate deficiency, liver disease, HIV therapy
• β thalassaemia carriers with a co-existing δ chain mutation which is silent or masked by co-eluting/migrating peaks in the first line screening technique, or who have co-existing delta thalassaemia
• β thalassaemia carriers with co-existing HbH Disease as some cases have normal HbA₂ values
• α⁰ thalassaemia occurring outside the defined at risk family origins masked by another condition or outside the defined MCH ranges (see section 10 above)
• δβ thalassaemia carriers with HbF ≤5%
• γδβ thalassaemia carriers
• dominant haemoglobinopathies in the baby’s biological father when the woman is Hb AA, these are very rare and should be suggested by the family history
• any significant condition silent with the first line screening
• any significant condition masked by a blood transfusion
• any significant condition masked by an unreported bone marrow transplant or gene therapy
• any significant condition present in donor egg or sperm where the donor is undeclared or untested
• incorrect family origins assigned due to undeclared adoption
• incorrect family origins declared or recorded

Conditions that may be missed using the screening scenarios (assuming that the FOQ has been completed accurately) in low prevalence areas only:

• Hb S, C, D^Punjab, E, O^Arab outside the defined at-risk family origins

1. de Baca ME, Gulati G, Kocher W, Schwarting R. Effects of Storage of Blood at Room Temperature on Hematologic Parameters Measured on Sysmex XE-2100. Laboratory Medicine 2006: volume 37, issue 1, pages 28 to 35 ↩

2. Cohle S, Saleem A, Maddaoui D. Effects of storage of blood on stability of hematologic parame-ters. American Journal of Clinical Pathology 1981: volume 76, issue 1, pages 67 to 69 ↩

3. ETHNOS. Evaluation of the Family Origin Questionnaire. March 2006 ↩

4. Daniel Y, Cartwright R, Rennie K, Streetly A, Howard J. Evaluation of the validity of HbA2 and mean corpuscular haemoglobin action values in antenatal screening for beta thalassaemia carriers in England. British Journal of Haematology 2014: volume 166, issue 4, pages 607 - 611 ↩

5. Mosca A , Paleari R, Ivaldi G, Galanello R, Giordano P. The role of haemoglobin A(2) in the diagnosis of thalassaemias and related haemoglobinopathies. Journal of Clinical Pathology 2009: vol-ume 62, pages 13 to 17 ↩

6. Piel F, Weatherall D. The α-Thalassaemias, New England Journal of Medicine 2014: volume 371, issue 20, pages 1908 to 1916 ↩

7. England J, Rowan R, Dawson D, Lewis S, Shinton N, Stephens A, et al. Guidelines for haemoglobinopathy screening. Clinical Laboratory Haematology 1988: volume 10, pages 87 to 94 ↩

8. 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 pro-gramme? Journal of Medical Screening 2007: volume 14, pages 60 to 61 ↩