Abstract
Background
Sickle cell disease is a heterogenous disorder characterized by an abnormal haemoglobin and sickling phenomena of red cells. It is prevalent among certain nomadic tribes in Sudan. Painful, aplastic and haemolytic crises mark sickle cell anaemia. Haemoglobin S (HbS) is detected using haemoglobin electrophoresis, iso-electric focusing and/or high-performance liquid chromatography techniques with high sensitivity, but entails cost and expertise. This study aimed to determine the sensitivity, specificity and positive predictive values (PPV) of the ID-particle gel diffusion technique for screening, diagnosis and phenotyping of HbS in patients with a provisional diagnosis of abnormal haemoglobin.
Methods
Following informed consent, 100 sequential individuals who reported to a central referral haemoglobinopathy clinic were enrolled. ID-particle gel diffusion technique was compared with cellulose acetate electrophoresis to determine haemoglobin phenotypes.
Results
The ID-particle gel test detected HbAA with 100% sensitivity and 100% specificity. Sensitivity for HbS was 100%, whether as HbSS or as a mixed pattern. HbSS was identified in all cases where this is the only haemoglobin present. Other patterns were detected with <100% specificity and these would require further testing by other means to definitively identify abnormal haemoglobins.
Conclusions
Although the ID-particle technique is a simple and cheap technique with high sensitivity, specificity and PPV compared with cellulose acetate electrophoresis in detecting HbSS, it could not differentiate HbAS from HbSS with high levels of HbF. High environmental temperatures could melt the test microtubes. Cellulose acetate electrophoresis remains the technique of choice for the screening of abnormal haemoglobins in the Sudan.
Introduction
Sickle cell disease is a collective name for a group of conditions due to abnormal haemoglobins that share the sickling phenomena of red cells when exposed to low oxygen tension. 1 The conditions include sickle cell anaemia, sickle cell/thalassaemia, sickle cell/haemoglobin C disease, sickle cell haemoglobin/O Arab disease and sickle cell haemoglobin/D(Punjab) disease. The clinical features of sickle cell disease are often variable in patients from the same geographical location with an identical molecular defect. 2 Severe sickle cell disease is seen in Africans, while a mild form compatible with normal life is seen among the Arabs of eastern Saudi Arabia. 3,4 Patients with homozygous or double heterozygous HbS are constantly plagued by painful episodes of pallor, dactylitis hand-foot syndrome and jaundice. Dactylitis is the most common initial symptom in some African infants, while pallor, jaundice and painful crises are the most persistent features in all ages. 5–7 In Sudan, patients usually present with severe anaemia, hand-foot syndrome, fever, jaundice and vaso-occlusive crises. 8,9 The symptoms usually start after the age of 6 months, but some cases have been reported as early as 3 months of age. In sickle cell trait, patients are asymptomatic, but when exposed to severe and prolonged low oxygen tension, the clinical features are similar to that of sickle cell disease. 10 Abnormal haemoglobins are diagnosed using different techniques of variable costs and sensitivities. Haemoglobin electrophoresis 11 is a simple cheap test, which is routinely carried out on cellulose acetate paper with Tris–EDTA–borate buffer at pH 8.5 or on agar gel with a citrate buffer with a pH of 6.0. Iso-electric focusing 12 and high-performance liquid chromatography (HPLC) are other valuable diagnostic tools with very high sensitivities and specificities, but are not always available and are costly. Molecular diagnosis of sickle cell haemoglobin is a recent approach in the diagnosis that is carried out by specialized centres presently. 13–15 ID-particle gel technique is a relatively recent technique that is used extensively in blood banks and haematology laboratories for ABO, Rh blood grouping, Rh phenotyping and haemoglobin S (HbS) detection. 16,17 ID-particle gel technique provides a simple and cheap alternative for screening, diagnosis and phenotyping of HbS. Its principle relies on the fact that HbS has a reduced solubility in its deoxygenated form, which will keep the haemoglobin molecules on the top of the gel, while other soluble haemoglobins sink to the bottom of the tube following centrifugation. 16 In this communication, we tested the sensitivity, specificity and the positive predictive value (PPV) of ID-particle gel technique as a simple and cheap technique for screening, diagnosis and phenotyping of HbS in patients with a provisional diagnosis of an abnormal haemoglobin.
Materials and methods
Following informed consent, patients with symptoms and signs suggestive of HbS disease who reported to Khartoum's Paediatrics Emergency Hospital and the Haemoglobinopathy Clinic at the Institute of Endemic Diseases, University of Khartoum were enrolled. Five millilitres of blood were collected from each patient into K2–EDTA containers using disposable syringes. Each sample was divided into two portions: one portion was used as whole blood for particle gel diffusion technique, and the other was processed into haemolysate for Hb electrophoresis.
Cellulose acetate electrophoresis at pH 8.5
Haemolysates were prepared by lysing two volumes of washed packed cells in one volume of distilled water with the addition of one volume of carbon tetrachloride. The mixture was shaken vigorously and centrifuged at 3000 rpm (1200
Tris–EDTA borate (TEB) buffer was used as the electrophoresis buffer and cellulose acetate membranes were soaked in TEB buffer for at least 5 min and blotted with absorbent paper.
Ten microlitres of diluted sample was placed into the sample wells and samples were applied to the cellulose acetate approximately 3 cm from one end of the membrane. The membrane was placed across the bridge of the tank to establish contact with the buffer, with the line of application at the cathode end. The electrophoresis was run at 250–300 V for 20 min. The membrane was stained with Ponceau S for 3–5 min. The membrane was eluted with a de-staining solution (5% v/v glacial acetic acid in distilled water), dehydrated in absolute methanol for 2–3 min, immersed in cleaning solution (25% glacial acetic acid in methanol) for 6–8 min and dried at 65°C for 4–6 min.
ID-sickle cell test
ID-particle gel diffusion technique was carried out according to the manufacturer's instructions (DiaMed, Morat, Switzerland).
Principle of the test
Cells containing HbS that have been induced to sickle by exposure to a reducing agent in a phosphate buffer, do not pass through a gel contained in microtubules. Twenty microlitres of blood were gently re-suspended in 200 μL of working solution prepared by adding 1 mL of distilled water into a vial of lyophilized reducing agent. Twenty microlitres of the mixture were dispensed into the appropriate microtube of ID-card. The card was centrifuged at 1000 rpm for 10 min. Known haemoglobin controls (HbAA, HbSS and HbF) were included. The patient was considered positive for HbS when the red precipitated is formed on the surface of the gel. When the red colour is dispersed in the gel with a small button forming at the bottom of the microtube, then the possibility of HbS with another haemoglobin is considered (HbAA, HbC, HbF). HbAA, HbA2, HbF samples formed compacted buttons on the bottom of the microtubes.
Results
One hundred (54 females and 46 males) displaced patients from Western Sudan with provisional diagnoses of haemoglobinopathies, were referred to the haemoglobinopathy clinic at the Institute of Endemic Diseases and Khartoum Paediatric Hospital. Forty-two percent were typed as HbAS, 26% as HbSS and 32% as HbAA with cellulose acetate technique. The prevalence of HbS was high among the Misseria tribe, where 13/32 (40.6%) of the patients were typed as HbAS, while 13/32 (40.6%) were typed as HbSS and the rest were reported as HbAA. Seven patients were diagnosed as HbSS + HbF by Hb electrophoresis and HbAS by the ID-gel technique. ID-particle gel technique could not differentiate HbF from HbAA (Table 1).
Comparison between methyl cellulose electrophoresis as a standard technique and ID-particle gel in detection of haemoglobins A, S and F
*HbS + HbF was reported as HbAS in ID-particle gel technique
The ID-particle gel test detected HbAA with 100% sensitivity and 100% specificity. Sensitivity for HbS was 100%, whether as HbSS or as a mixed pattern. HbSS was identified in all cases where this is the only haemoglobin present. Other patterns were detected with <100% specificity and these would require further testing by other means to definitively identify abnormal haemoglobins. PPV and negative predictive values (NPV) of the test for the presence of HbSS were 100% and 80.5%, respectively.
Discussion
Sickle cell disease is an important cause of morbidity and mortality among indigenous and immigrant ethnic groups in Sudan. Most of these groups live in remote areas with meagre health-care facilities. Diagnostic tests for such a serious disease need to be user-friendly, cheap, sensitive and specific. ID-particle gel diffusion technique was introduced as a simple diagnostic test for HbS disease. Although it is easy and takes less time to perform compared with cellulose acetate electrophoresis, it is more expensive for each sample. The micro-gel tubes can be stored at room temperatures (22–24°C) in temperate regions, but high room temperatures in some parts of Sudan can reach 35°C during the summer months and can liquefy the gel and render the tubes unusable. This may necessitate the kits storages in cold rooms that may not be available in remotes areas where electricity is in short supply. On the other-hand, cellulose acetate plates are easy to handle and can be stored for long times at temperatures up to 35°C.
In this study, HbA and HbS detection results for the two techniques were comparable. The ID-particle gel technique could not detect HbF in seven patients with HbSS and a high percentage of HbF and were reported as HbAS (Table 1). Some sicklers have high percentage of HbF (Shitti Arabs, Saudi Arabia) who are usually asymptomatic and could easily be diagnosed as HbS trait rather than HbSS disease by the ID-particle gel technique.
ID-particle gel technique is not a suitable diagnostic test for patients with hereditary persistent HbF, because these individuals will be typed as HbAA. Our results are concordant with a previous study, which reported that the ID-particle technique is not suitable for differentiating HbF from HbA and HbAS from HbSS + HbF. 17
This study confirmed the presumed high prevalence of HbS in the immigrant Misseiria tribe, but with higher figures than previous reports. 18 The reason could be that, our samples were biased towards patients with symptoms and signs of HbSS disease.
In conclusion, ID-particle gel technique is easy to perform and has a high sensitivity, specificity and PPV in screening for HbSS/HbAS in remote areas. The technique is not useful in differentiating HbSS with high HbF levels, a minority among patients with sickle cell haemoglobin in Sudan, from HbAS. The technique is unlikely to differentiate sickle cell/HbC disease and sickle cell/HbD(Punjab) from HbAS. In addition, the possibility of meltdown of the micro-tubes at relatively high room temperatures in remote areas makes it an unsuitable screening test for abnormal haemoglobins in Sudan and cellulose acetate electrophoresis remains the technique of choice.