Sickle Cell Testing: Electrophoresis and Stroke Screening

Sickle cell testing starts with haemoglobin electrophoresis, a blood test that separates the different haemoglobins and shows whether a person is unaffected, a carrier or affected. It can be done at birth, and in pregnancy the diagnosis can be made from amniotic fluid or placental tissue. From two years of age, transcranial Doppler ultrasound screens affected children for stroke risk.

Each of these tests answers a different question, and each changes what happens next. This article sets out how Dr. Sukriti Gupta, Sr. Consultant, BMT, Haematopoietic Stem Cell Transplant, Hematology, Oncology, Paediatric Haemato-Oncology & BMT, Artemis Hospitals, Gurugram, India, approaches the diagnostic work-up: the first-line test, how to read the haemoglobin S percentage, when the older sickling tests mislead, family and prenatal screening, and the stroke surveillance that she regards as essential for every affected child. For the biology behind the results, see the complete guide to sickle cell disease.

Why early diagnosis decides the outcome

Most of the organ damage in sickle cell disease, to the kidneys, heart, lungs and eyes, becomes apparent only in adulthood, which, Dr. Gupta noted, is why early screening of children matters. A child identified in infancy can start penicillin prophylaxis, vaccination, hydration and hydroxyurea early, can be screened for stroke risk, and can be assessed for a curative transplant while the organs are still well preserved, one of the prerequisites she lists for a successful transplant.

Haemoglobin electrophoresis: the first-line sickle cell test

Haemoglobin electrophoresis is the first-line sickle cell test in Dr. Gupta's practice, because it is definitive and widely available. A blood sample is run through a gel, classically cellulose acetate with an alkaline buffer, under an electric current. Each type of haemoglobin carries a different charge and so migrates a different distance, producing a pattern of bands that identifies haemoglobins A, A2, F, C, D, E and S.

From that pattern the laboratory can distinguish:

• a normal individual (predominantly HbA)
• a sickle cell carrier, with sickle cell trait (HbA plus HbS)
• sickle cell disease (HbSS)
• compound conditions in which HbS pairs with another variant, such as HbSC or HbSE, and other haemoglobinopathies

Three features make it the right first-line test. It is definitive, it is now easily available even in resource-limited settings, and results come back within a day. Many countries already run it as part of routine newborn screening. Once the pattern is known, the genetic basis of the condition can be explained to the family.

Reading the haemoglobin S percentage

A doctor from Cameroon asked whether the haemoglobin S threshold for diagnosis is the same in all countries. The diagnosis rests on the whole pattern, especially whether normal HbA is present, rather than on one HbS cut-off. The usual patterns in an untransfused patient are:

She added an important caveat. Severity tends to rise with the proportion of HbS, but there is no exact mathematical correlation between the percentage and the clinical picture. The number guides the diagnosis; it does not predict how a given patient will fare. In sickle cell trait, normal haemoglobin A remains the dominant haemoglobin, which is a further point of distinction on the electrophoresis pattern.

Sickling tests: false positives and false negatives

Dr. Gupta no longer prefers sickling tests, because they can give false results in both directions. Before electrophoresis became widely available, screening relied on these tests, which force red cells to sickle in the laboratory, most commonly the sodium metabisulphite test.

• False positives occur, for example with technical error or very high plasma protein levels. A positive sickling test also cannot distinguish sickle cell trait from sickle cell disease, so it never establishes the diagnosis on its own.
• False negatives also occur, particularly in infants under about six months, whose high fetal haemoglobin prevents sickling, in severe anaemia and after a recent transfusion. A child can have a negative sickling test and a positive electrophoresis.

Her conclusion was firm. Where electrophoresis is available it should always be preferred, and it is essential whenever there is a family history of the disease.

Family screening and genetic counselling

A diagnosis in one child is a diagnosis for the family. Once a child with sickle cell disease is identified, Dr. Gupta screens all other family members, particularly the siblings and the parents. Siblings matter twice: they may themselves be affected or be carriers, and they are the most likely source of a fully matched bone marrow donor if a transplant is needed later.

Prevention, she argued, is the key to eradicating the disease, and prevention begins with genetic counselling for the next generation. Carrier couples need to understand the 25% chance, in every pregnancy, of a child with sickle cell disease, and the options for testing during pregnancy.

Prenatal diagnosis of sickle cell disease

Where both parents have been tested and found to carry HbS, or where a previous child is affected or there is a strong family history, the diagnosis can be made before birth. Two procedures are used. In both, the sample is examined by genetic testing for the change in the HBB gene; electrophoresis cannot be run on these samples.

The advantage of CVS is time. A result in the first trimester gives a couple more room to consider their options.

Stroke screening with transcranial Doppler

Transcranial Doppler (TCD) ultrasound, started at two years of age, identifies children with sickle cell disease who are at high risk of stroke before a stroke occurs. Stroke is one of the most feared complications of sickle cell disease in children, and its risk can be predicted. Asked during the session to explain how strokes develop in these children, Dr. Gupta pointed to TCD as the key tool.

How transcranial Doppler works

TCD uses ultrasound waves, sent through the thin bone at the side of the skull, to measure how fast blood flows in the major arteries of the brain. In sickle cell disease, the large arteries at the base of the brain gradually narrow. Where an artery narrows, blood speeds up to pass through it, so an abnormally high flow velocity marks the child whose arteries are at risk, before any stroke has occurred.

Dr. Gupta noted that TCD is not yet carried out frequently in India, but that it is recommended for every patient with sickle cell disease.

The screening schedule

Her schedule for TCD surveillance is:

1. First scan at two years of age.
2. If the scan shows any abnormality, repeat it sooner, at an interval of two to three months, and act on the result.
3. If cerebral blood flow is normal, repeat the scan every six months to one year.

The standard against which these results are judged comes from the STOP trial and the guidance that followed it. Children with HbSS are screened from 2 to 16 years of age. A time-averaged mean velocity of 200 cm/s or more is abnormal and, once confirmed, is treated with regular blood transfusion, which in the STOP trial reduced the risk of a first stroke by about 90%. Hydroxyurea can later replace transfusion in selected children.

Which strokes children with sickle cell disease suffer

Another doctor asked what types of stroke these patients are most likely to suffer and how to prevent them. The narrowing typically affects the end of the internal carotid artery and the start of the middle and anterior cerebral arteries, so the middle cerebral artery territory is the most commonly involved, as it is in adults.

On prevention, Dr. Gupta's message was realistic. Regular TCD from two years of age picks out the children at risk early, so that preventive treatment can be started. It may not prevent every stroke, but regular follow-up and monitoring are what make prevention possible. A child who does have a stroke has, in her framework, an absolute indication for a bone marrow transplant, covered in bone marrow transplant versus gene therapy.

Routine surveillance after diagnosis

Diagnosis is the start of a monitoring relationship, not a single event. Dr. Gupta recommends that every child with sickle cell disease see a general practitioner or paediatrician every two to three months, even when well. These visits are where TCD is scheduled and where early signs of organ damage, such as kidney or heart changes, are picked up. Regular screening for heart muscle disease (cardiomyopathy) and tricuspid valve regurgitation, which can signal raised pressure in the lungs, is part of that surveillance, because either finding strengthens the case for a curative transplant.

What follows diagnosis, from penicillin and hydration to transfusion and hydroxyurea, is covered in sickle cell treatment: supportive care, hydroxyurea and pregnancy. The doctors' questions on haemoglobin S thresholds, sickling tests and stroke are collected in the Live Discussion.