Guidance

Interpretation and reporting of antenatal screening results

Updated 5 September 2022

Applies to England

These guidelines should provide enough information to allow all normal, and over 95% of abnormal, results to be reported in a standardised manner. There will always be some situations that need further tests, or family studies, before a useful clinical result can be achieved because of the diversity of haemoglobin variants and thalassaemia syndromes. Appropriate clinical referral is needed in these cases to make sure the risk to the pregnancy is fully and correctly assessed.

If an individual had a blood transfusion and any of the transfused red cells are still present, misleading data and conclusions may result. This is also true for undeclared bone marrow transplants and gene therapy. It is essential that clinicians realise this fact. Laboratories must have a universal footnote on all haemoglobinopathy results, for example, ‘unless otherwise stated, result assumes no bone marrow transplant, gene therapy or recent transfusion’ or ‘result valid if not transfused and no history of bone marrow transplant or gene therapy’.

Laboratories should treat haemoglobin values of less than 80g/L with caution. There are multiple conditions that can cause severe anaemia in pregnancy, including iron deficiency, haemolytic anaemia, HbH disease, or β thalassaemia intermedia. Laboratories should review these on a case-by-case basis. If necessary, they should discuss such cases with a senior scientist or clinician to consider appropriate further action, including molecular testing and/or testing of the baby’s biological father.

It is not possible to separate all haemoglobin variants using screening methods and a second haemoglobin variant may be migrating/eluting with any other haemoglobin, including HbA. This is a particular issue in HPLC instruments which use a stepwise buffer gradient, notably the co-elution of Hbs S and C. Conversely, such gradients may also split other haemoglobin variants into 2 peaks, for example HbD^Punjab. In other cases, such as when thalassaemia and haemoglobin variants are co-inherited, one condition may be masking another.

HPLC instruments separate adducted fractions of haemoglobins. These fractions must not be added to the assumed parent peak. Addition of adducted peaks can lead to incorrect results. No additional testing is necessary if the HbA₂ is raised (but not greater than 8%) and the red cell indices are typical of a carrier of β thalassaemia. It is important to be aware that HbF levels are highly variable in β thalassaemia carriers.

Antenatal screening results must be interpreted and 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.

1. Screening algorithms

1.1 Scenario 0

Low prevalence areas only, initial screening tests are the MCH and FOQ information.

Note if MCH <27 pg, either biological parent is of high risk or unknown family origins or there are other special circumstances the mother’s sample must be tested by HPLC or CE. See section 4 of Antenatal screening.

If initial screening tests show:

• MCH is ≥ 27pg

• And both the baby’s mother and biological father are of low risk family origins as determined by the FOQ.

No further action. Use report format 0. See Recommended report formats.

1.2 Screening shows the presence of HbA only

The following list of countries considered high risk for alpha zero thalassaemia is included for reference:

• Southeast Asian origin (China (including Hong Kong)
• Thailand
• Taiwan
• Cambodia
• Laos
• Vietnam
• Indonesia
• Myanmar (formerly Burma)
• Malaysia, Singapore or Philippines
• East Mediterranean (Cyprus, Greece, Sardinia or Turkey)

1.3 Scenario 1

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is ≥ 27pg

• HbA₂ is <4.0%

• HbF is <10%

• Hb is ≥80g/L

Then no further testing is required. There is negligible risk in the pregnancy associated with an abnormal haemoglobin or thalassaemia. Use report format 1. See Recommended report formats.

1.4 Scenario 2

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is ≥ 27pg

• HbA₂ is ≥ 4.0% but < 8.0%

Then there is a risk in the pregnancy associated with the presence of β thalassaemia. Use report format 4b. See Recommended report formats

If the HbA₂ ≥ 8.0%, check for other variants using an alternate technique.

1.5 Scenario 3

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is ≥ 27pg

• HbA₂ is < 3.5%

• HbF is ≥ 10%

• Hb is ≥ 80g/L

There is a risk to the pregnancy associated with the presence of HPFH. Use report format 5a. See Recommended report formats.

If Hb is < 80g/L ensure that all other potential risks have been considered.

1.6 Scenario 4

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 27pg

• HbA₂ is ≥ 4.0% but < 8.0%

Then there is a risk in the pregnancy associated with the presence of β thalassaemia. Use report format 4a. See Recommended report formats.

If the HbA₂ is ≥ 8.0% check for other variants, particularly Hb Lepore. Consider risk of α⁰ thalassaemia.

1.7 Scenario 5

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 27pg

• HbA₂ is ≥ 3.5% but < 4.0%

Then there is a risk in the pregnancy associated with the presence of β thalassaemia. Use report format 4b. See Recommended report formats.

Consider risk of α⁰ thalassaemia.

1.8 Scenario 6

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 27pg

• Hb is < 80g/L

• HbA₂ is ≥ 3.0% but < 4.0%

Then there is a risk in the pregnancy associated with the presence of β thalassaemia. Use report format 4b. See Recommended report formats

Consider risk of α⁰ thalassaemia.

1.9 Scenario 7

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 27pg

• HbA₂ is < 3.5%

• HbF is ≥ 5.0%

There is a risk to the pregnancy associated with the presence of δβ thalassaemia. Use report format 5b. See Recommended report formats.

Consider risk of α⁰ thalassaemia.

1.10 Scenario 8

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 27pg

• HbA₂ is < 3.5%

Consider whether this is the measurement of the total HbA₂.

If the patient has a δ chain variant or an α chain variant, a second HbA₂ peak/band may be present and must be included in the total HbA₂ where appropriate.

If total HbA₂ (HbA₂ plus HbA₂ variant) ≥ 3.5% then there may be a risk in the pregnancy associated with the presence of β thalassaemia.

If β thalassaemia is suspected use report format 4a or 4b as described in scenarios 4 to 6. See Recommended report formats

Consider risk of α⁰ thalassaemia.

If the HbA₂ measurement is correct and < 3.5% please see scenarios 9 to 11. In these scenarios the FOQ forms part of the screening tests.

1.11 Scenario 9

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 27pg but ≥ 25pg

• HbA₂ is < 3.5%

• HbF is < 5.0%

• Hb is ≥ 80g/L

This indicates possible iron deficiency or α⁺ (-α or α^T) thalassaemia. There is negligible risk to the pregnancy. Use report format 7b. See Recommended report formats

1.12 Scenario 10

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 25pg

• HbA₂ is < 3.5%

• HbF is < 5.0%

• Hb is ≥80g/L

• biological parents are both of high risk family origins for α⁰ thalassaemia or unknown or one has family origins of high risk and the other is unknown

This indicates possible α⁰ thalassaemia or iron deficiency. Use report format 6a. See Recommended report formats

If the woman has HbH disease, the Hb may be <80g/L. Use report format 6b. See Recommended report formats

1.13 Scenario 11

If screening tests show only a single major Hb peak (band) in the HbA position and the:

• MCH is < 25pg

• HbA₂ is < 3.5%

• HbF is < 5.0%

• Hb is ≥80g/L

• neither, or only one, biological parent has family origins of high risk for α⁰ thalassaemia

This indicates possible iron deficiency, homozygous α⁺ (-α or α^T) thalassaemia, or heterozygous α⁰ thalassaemia (use report format 7a) or HbH disease, note Hb may be < 80g/L (use report format 6b). See Recommended report formats.

1.14 First test suggests HbA and either HbS, HbC, HbD, HbE or HbO^Arab

Further tests required: specific HbS test.

It is recommended to perform a validated specific HbS test on all haemoglobin variants detected. If the variant has features suggestive of HbS and the test is positive, then no further testing is usually required. See section 5.1 of Antenatal screening for more information on the need for caution when using the sickle solubility test for confirmatory purposes with certain instruments. If the variant has features suggestive of HbS and the test is negative further investigations must be undertaken.

2. Interpretation of relative proportions

2.1 Hb Variant (V) between 30 and 40% (25 and 35% if HbV is HbE).

These values include both typical ratios and possible α⁺ thalassaemia carriers. The screening algorithm does not aim to detect the latter.

2.2 Hb Variant (V) between 40 and 50% (35 and 50% if HbV is HbE).

Is the band/peak in the HbA position actually HbA? If the HbV > HbA the possibility exists of Hb V/β⁺ thalassaemia with an unusually high expression of HbA. Test biological father, and consider referral for molecular testing, depending on his results.

2.3 HbV ≥ 50%

Consider Hb V/β⁺ thalassaemia. Cases of Hb V/β⁺ thalassaemia will usually have a reduced MCH. Consider the HbA₂, although the presence of a haemoglobin variant may affect the values obtained on many systems. Ensure that red cell indices are reviewed and where V/β⁺ thalassaemia is suspected further investigation is required. In the case of V/β⁺ thalassaemia the antenatal risk is for both HbV and β thalassaemia.

2.4 HbV ≤ 30% (≤ 25% if HbV is HbE) and MCH < 25pg

HbV carrier with possible co-existing α thalassaemia or iron deficiency. The α thalassaemia risk needs to be considered in the light of the family origins of both biological parents – see algorithm for α thalassaemia. The major risk is for HbV, but the risk of Hb Bart’s hydrops fetalis syndrome must not be overlooked especially if HbV is HbE.

In all the above cases use report format 2 or 8 as appropriate. See Recommended report formats.

3. Variant specific considerations

Refer to section 8 of Antenatal screening.

3.1 HbS

Be aware that with some HPLC systems adducts of HbS may elute in the HbA window and may be mistaken for a small amount of HbA, so a person with sickle cell disease may appear to have Hb S/β⁺ thalassaemia. If there is doubt as to the identity of this small peak an alternate method to confirm or refute the presence of HbA must be used.

3.2 HbC

The position of HbC relative to other haemoglobins can vary considerably depending upon the system used. On some HPLC systems, HbC gives a small additional peak of adducts of HbC in the HbS window. Other systems underestimate the HbA₂ level in the presence of HbC. This should not cause a diagnostic problem except when differentiating between Hb C/β⁰ thalassaemia and Hb CC when it is essential to review the red cell indices.

3.3 HbD

The position of HbD will depend upon the system in use. Some systems underestimate the HbA₂ level in the presence of HbD^Punjab. This should not cause a diagnostic problem except when differentiating between Hb D/β⁰ thalassaemia and Hb DD when it is essential to review the red cell indices.

3.4 HbE

With some HPLC methods, HbE appears as a peak in the HbA₂ window, so the levels of both HbE and HbA₂ cannot be estimated accurately. The percentages given above can vary depending on the analyser in use. The HbE mutation creates an alternate splicing site in the messenger RNA resulting in reduced expression. This, together with the instability of HbE, means there is less of the variant when compared with other β chain mutations phenotypically. There is often associated microcytosis and slightly raised HbA₂. Where they are measured separately the reference range for HbA₂ is unlikely to apply.

3.5 HbO^Arab

HbO^Arab appears in different positions on different analysers but frequently appears in close proximity to HbC.

3.6 Hb Lepore

Hb Lepore appears in different positions depending on the analyser used. It frequently appears as an oddly shaped elevated HbA₂ peak with values up to 15% and therefore the levels of neither Hb Lepore nor HbA₂ can be estimated accurately. By IEF there is a band in HbG ^Philadelphia position and by alkaline electrophoresis, a band just faster than HbS, but this may be difficult to separate from HbS. The MCH is usually < 27pg.

The α thalassaemia risk needs to be considered in the light of the family origin of both biological parents – see algorithm for α thalassaemia. The major risk is for Hb Lepore, but the risk of Hb Bart’s hydrops fetalis syndrome must not be overlooked.

There is a risk in the pregnancy associated with the presence of Hb Lepore. Use report format 2 or 8 as appropriate. See Recommended report formats.

4. Homozygotes and compound heterozygotes

Where a single major peak (band) is detected, co-existing β thalassaemia must always be considered. The red cell indices and HbA₂ level need to be reviewed. Remember to allow for the variable nature of HbA₂ levels in the presence of Hb variants. Refer to Problems with the measurement and interpretation of HbA₂. Once coexisting β thalassaemia has been excluded, the presence of single peak/band is most likely due to homozygosity for a Hb variant. Compound heterozygotes for haemoglobin variants usually have 2 peaks/bands equating to the 2 haemoglobin variants present, but it is possible for these to co-migrate.

The α thalassaemia risk needs to be considered in the light of the family origins of both biological parents. See section 10 of Antenatal screening.

The risk in these pregnancies is associated with the haemoglobin variant detected and possible co-existing thalassaemia. Use report format 8. See Recommended report formats