Guidance

Interpretation and reporting of results

Published 24 November 2021

Applies to England

© Crown copyright 2021

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This publication is available at https://www.gov.uk/government/publications/sct-screening-handbook-for-newborn-laboratories/interpretation-of-results

1. Overview

Results of sickle cell screening are interpreted according to the different haemoglobin fractions present. In unaffected infants, these are fetal haemoglobin (HbF) (as the major fraction) and HbA.

If the results of the first-line screen show the presence of any abnormal fraction, second-line testing must be considered in line with the guidelines.

It is the responsibility of the laboratory performing the second test to make sure this test is appropriate for the suspected condition and that both first and second-line screening tests are evaluated and results are consistent. Inconsistent results must be further investigated. Particular care must be taken when the first screen is performed by tandem mass spectrometry (MSMS).

For MSMS, any ratio falling outside the action value limits must have second-line testing. Extreme care must be taken if discrepancies are found when interpreting second-line results following MSMS analysis. Incident monitoring has identified cases where MSMS results indicated the correct diagnosis and a discrepant second test was not further investigated. This resulted in families being given incorrect results, particularly in cases where the MSMS results indicated the possibility of beta thalassaemia, both with and without HbS.

Other examples where results from second testing following MSMS analysis may give inconsistent results include but are not limited to:

  • cases with fetal haemoglobin, HbS and an unknown variant (FSV)

  • variants which result from 2 mutations in cis, one of which is the mutation under investigation

  • beta thalassaemia disease with alpha and/or gamma chain variants – there are 2 gamma experiments to ensure that beta thalassaemia disease with a gamma chain variant is not missed by this technique

FSV cases will almost invariably give a ratio indicative of a sickle carrier, whereas second-line testing should reveal the variant. These individuals need to be referred into clinical follow-up.

Variants which result from 2 mutations in cis, one of which is the mutation under investigation, will give a positive ratio. These variants may not be detected by second-line testing as they may not separate from HbA, so it is essential that clear positive first line MSMS results for HbS are not discounted.

All discrepant results must be further investigated and cases should be referred for follow-up if one result indicates a potentially clinically significant condition. This is the responsibility of the laboratory performing the second test which must review each case individually and arrange molecular testing or clinical referral.

If the concentration of HbA on the blood spot is abnormally high, then the possibility of a transfusion must be considered. Using MSMS, such a sample would show a low gamma/ beta ratio. This presentation may also be observed when there is contamination of the blood spot with adult blood, as a result of poor practice, and in cases of gamma thalassaemia. It is important to exclude both transfusion and contamination by adult blood.

2. Status code 04

Results usually show the presence of HbF and HbA. It is usual in a term baby for the percentage of HbA to be between 5% and 30%. This figure varies according to gestation and the laboratory must consider gestational age when requesting repeat sampling (see figures 1 and 2 in Newborn screening chapter). Additionally, a small number of other variants may be detected when using high performance liquid chromatography (HPLC), capillary electrophoresis (CE) or isoelectric focusing (IEF). These may not be immediately identifiable and most will be benign. The number of neonates with one of these other variants is likely to be small. Variants other than S, C, DPunjab, E and OArab must not be reported unless co-inherited with HbS. For MSMS all ratios would be within action value limits.

The following guidelines require both first and second-line results to be considered.

3. Sickle cell disease

Results from babies with sickle cell disease (SCD) show the presence of HbF and HbS in the absence of HbA (that is FS); or HbF and HbS with another haemoglobin variant (for example, FSC); or HbF, HbS and HbA where the quantity of HbS is greater than HbA (Hb S/β+ thalassaemia).

In cases of HbF and HbS only, the quantity of HbS found is usually between 4% and 10% for a full-term baby and less in a premature baby. With some HPLC systems a very small peak elutes in the HbA0 window. So a baby with Hb SS may appear to have Hb S/β+ thalassaemia. If the HbA is less than 1.5% it should be regarded as artefact. Some cases may require molecular testing to confirm underlying mutational status. Expected results from infants with SCD are shown in the table below.

3.1 Newborn screening results for sickle cell disease

Newborn screening results Disease
FS Hb SS
FS Hb S/β0 thalassaemia
FS Hb S/δβ thalassaemia
FS Hb S/Lepore
FS Hb S/HPFH
FSA or FS Hb S/β+ thalassaemia
FSC Hb SC
FSD Hb S/DPunjab
FSE Hb S/E
FSOArab Hb S/OArab

4. Hb S/β thalassaemia

Caution must be exercised in the interpretation of results where the amount of HbS is greater than HbA, as the cause may be Hb S/β+ thalassaemia. If HbS is greater than HbA, check the biological parents’ results if these are available. If the biological parents’ results are not available, the HbS would normally be expected to be greater than 120% of the HbA result for a diagnosis of Hb S/β thalassaemia to be made. Again, the actual quantities of HbA and HbS found will vary according to gestational age.

5. Sickle cell carrier

Results from infants who are sickle cell carriers will show the presence of HbF, HbA and HbS. The quantity of HbA should exceed the quantity of HbS, but in some cases the amounts of HbS and HbA are almost equal. The HbS would normally be expected to be greater than 120% of the HbA result for a diagnosis of Hb S/β thalassaemia to be made.

6. Heterozygotes for other haemoglobin variants

Results from babies who are carriers for other haemoglobin variants will show the presence of HbF and HbA and the haemoglobin variant. Again, the quantity of the variant is usually less than the quantity of the HbA and the exact quantities will vary with gestational age. As indicated with Hb S, for Hb C, D and O, a ratio of greater than 120% of the HbA (for Hb E the value is greater than 100%) suggests coinheritance of beta thalassaemia. Some cases may require molecular testing to confirm underlying mutational status.

7. Homozygotes or compound heterozygotes for non-sickling conditions

Results from babies who are homozygotes or compound heterozygotes for conditions other than SCD will show the presence of HbF and the haemoglobin variant(s) only. No HbA will be detected. Results from some automated analysers may be misleading in that an apparent small HbA0 peak may be present. If the HbA is less than 1.5% it should be regarded as artefact. Some cases may require molecular testing to confirm underlying mutational status.

8. β thalassaemia major

Results from a baby with severe β thalassaemia will usually have only HbF present and no HbA. In all cases where the apparent HbA concentration is 1.5% or less of the total haemoglobin, the result must be reported as F only, and followed up[footnote 1] [footnote 2].

For MSMS a unique pattern is usually present due to the low wild-type beta expression. This is a high gamma/beta ratio along with results above the action values for some of the other haemoglobins under investigation.

DNA analysis is required to confirm the diagnosis.

9. Risk assessment

The NHS SCT screening programme is not a diagnostic service and will not detect:

  • β thalassaemia major or intermedia with an HbA or gamma/beta ratio value outside designated action limits
  • any target haemoglobin whose result falls outside designated action limits
  • β, δβ and γδβ thalassaemia carriers
  • Hb H disease and α thalassaemia carriers
  • Hb S/β thalassaemia with a high expression of HbA
  • rare clinically significant haemoglobins (for example, high affinity haemoglobins, unstable haemoglobins and methaemoglobin forming variants)
  • some rare haemoglobin variants that are clinically significant in the presence of HbS
  • undeclared transfused babies whose HbA level is below the action values shown in figures 1 and 2
  • any significant variant which is misinterpreted as a result of an undeclared blood transfusion, in particular the risk that an Hb FS may be reported as Hb FAS

10. Reporting results

The following guidelines on reporting analytical data and the genetic implications (conclusions) of that data, plus the reporting newborn sickle cell results table, should provide enough information to allow all unaffected, and over 95% of abnormal cases to be reported in a standardised manner.

All reports must include the sample date. The haemoglobins present must be reported in the order of the greatest to the least percentage/proportion and analytical fact must be separated from interpretative opinion. The factual results must be given together with a clear conclusion, which may include recommendations. The conclusion must always be given in full text, for example, ‘Results consistent with sickle cell carrier’ is the conclusion for Hb FAS or ‘Results consistent with sickle cell disease’ is the conclusion for an analytical result of Hb FS. Reports and templates used must be clearly presented and laid out so that misinterpretation is avoided.

Status codes are required when reporting newborn screening results to child health systems and for administrative use. These codes are used to support the transfer of information between the respective computer systems and are not intended to be used for clinical purposes. The codes are designed to be generic for all the conditions that are screened for in the NHS Newborn Blood Spot (NBS) screening programme. The Reporting newborn sickle cell results table provides examples of the main status codes for the different types of results.

If the newborn baby has had a blood transfusion and any of the transfused red cells are still present, misleading data and conclusions may result. This includes in utero transfusions which a fetus may have received. It is essential clinicians understand this and they should consider including a footnote on all haemoglobinopathy results such as ‘Results valid if not transfused’ or similar.

11. Issuing laboratory reports

The baby’s parents and GP must be informed of all the outcomes of screening. The approach adopted must follow general guidance from the NHS NBS screening programme.

Laboratories are responsible for sending all screening results to child health departments, or their equivalent, with the relevant status codes.

This information will be used to assess coverage of the screening programme and to provide a mechanism for reporting ‘normal’ results to parents and other healthcare professionals. Presumptive positive results must be reported without delay by the laboratory to the designated healthcare professional.

12. Actions required for particular categories of results

12.1 Babies with status code 08

Screen positive results must be reported as per local pathways, for example, to the clinician and/or designated sickle cell and thalassaemia (SCT) centre, and confirmation of receipt documented. The designated SCT centres ensure that affected babies enter the care pathway and return the confirmatory diagnostic results back to the newborn screening laboratories to confirm enrolment into care and for validation of the screening result.

Local protocols must be in place to ensure that all screen positive results are given to parents by a trained healthcare professional face-to-face by 28 days of age. The baby must also enter the care of a specialist haemoglobinopathy centre by 90 days of age.

12.2 Babies found to have a condition other than sickle cell disease which requires follow-up

These results must be reported as per local pathways, for example, to the designated SCT centre and confirmation of receipt documented. Parents should be informed by personal contact. Copies of all reports must be sent to the GP and health visitor. These babies must also be referred to the appropriate specialist network lead.

12.3 Babies homozygous or compound heterozygous for non-sickling haemoglobins which do not require follow-up

These results must be reported as per local pathways, for example to the designated SCT centre and confirmation of receipt documented. Parents and GPs must also be informed by a locally agreed mechanism.

12.4 Babies heterozygous for haemoglobins S, C, D, E or OArab

These results must be reported as per local pathways, for example to the designated SCT centre, and confirmation of receipt documented. Parents and GPs must also be informed by a locally agreed mechanism.

12.5 Babies with no abnormality detected or haemoglobin variants other than S, C, D, E or OArab

The child health department or equivalent must provide the results in written form to the child’s parents and GP. The wording for reporting the results for these babies with no abnormality detected or haemoglobin variants other than S, C, D, E or OArab uses the newborn status code 04 ‘Condition not suspected’. The wording must state that ‘haemoglobins S, C, DPunjab, E and OArab have not been detected. Note that carriers of β thalassaemia and Hb Lepore are not detected by the techniques used for newborn screening’.

12.6 Premature babies

HbA is normally detectable by 30 weeks gestation and is sometimes detected by 24 weeks. Results must be interpreted with caution. Premature babies who show a HbA or gamma/beta ratio value outside designated action limits must have repeat testing with an appropriate technique to check for the presence of sickle cell disease or β thalassaemia major.

13. Annual data returns

Newborn screening laboratories must provide an annual data return, using the template developed in conjunction with the NHS NBS screening programme.

Newborn screening laboratories must also collate and submit the data required for the relevant key performance indicators (KPIs) for the newborn screening programme.

14. Quality assurance and accreditation

All commissioners and service providers must refer to the SCT screening pathway requirements specification and supporting standards and handbook to ensure the service is set up correctly and meets the standards set by the national screening programme.

Laboratories offering screening for the NHS SCT Screening Programme must:

The UK Accreditation Service (UKAS) will assess both the ISO and the screening requirements on behalf of the screening quality assurance service and the NHS SCT Screening Programme.

15. Useful organisations and more information

See UK Newborn Screening Laboratories Network for laboratories involved in newborn screening.

The Oxford Laboratory provides, on behalf of the screening programme, a dedicated SCT laboratory support service for antenatal and newborn laboratories and other healthcare professionals involved in the screening programme. Tel: 01865 572767 or email lab.support@nhs.net.

British committee for standards in haematology

SCT screening e-learning modules

Contact the SCT association of nurses, midwives and allied professionals (STANMAP) for details of local specialist SCT centres.

See NHS.UK information on screening for sickle cell and thalassaemia.

Royal College of Paediatrics and Child Health

15.1 Clinical network arrangements

See NHS standard contract for specialised services for haemoglobinopathy.

15.2 Failsafe arrangements

The NBS failsafe solution helps make sure all babies born in England are offered screening. It identifies babies who have not been screened or who require a repeat sample to be taken early in the postnatal period so that screening can be offered.

  1. Streetly A, Latinovic R, Henthorn J, Daniel Y, Dormandy E, Darbyshire P, Mantio D, Fraser L, Farrar L, Will A, Tetlow L. ‘Newborn blood spot results: predictive value of screen positive test for thalassaemia major’. Journal of Medical Screening 2013: volume 24, issue 4, pages 183 to 187 ↩

  2. Daniel Y, Henthorn J. Reliability of the current newborn screening action value for beta thalassaemia disease detection in England: A prospective study. Journal of Medical Screening 2019: 26(2):67-79 https://doi.org/10.1177/0969141318797373 ↩

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