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Diagnostic accuracy of HemoTypeSC for detecting sickle cell disease in newborns: A multicentric study
For correspondence: Dr Anita Nadkarni, Department of Haematogenetics, ICMR-National Institute of Immunohaematology, Mumbai 400 012, Maharashtra, India. e-mail: anitanadkarni@gmail.com
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Received: ,
Accepted: ,
Abstract
Background & objectives
Sickle cell disease (SCD) is a common, life-limiting genetic disorder in India. It is best managed when diagnosed early through newborn screening, which requires a reliable, rapid and an affordable point-of-care test for diagnosis. The study aimed to evaluate the diagnostic accuracy of HemoTypeSC as point-of care for detecting SCD in newborn screening. Additionally, the study assessed the feasibility of implementing HemoTypeSC in poor resource settings.
Methods
We conducted a prospective multicentre diagnostic accuracy study using an investigational assay HemoTypeSC, and compared it with the reference (‘Gold-standard’) High performance liquid chromatography (HPLC) during the year 2019-20. Six different study centers were selected to evaluate the performance of the test across different climatic and geographical locations.
Results
A total of 1725 newborn babies were screened from six participating centers. The samples were analyzed by both HemoTypeSC and HPLC. HemoTypeSC correctly identified the 14/15 SCD cases [sensitivity 93.3% (95% confidence interval (CI): 68-99.8%) and specificity 100% (95% CI: 99.7-100%)] and 192/200 sickle cell trait cases [sensitivity 96% (92.2-98.2%, CI 95%) and specificity 99.8% (95% CI: 99.5-99.9%)]. The accuracy of HemoTypeSC to detect SCD and sickle cell trait was found to be 99.9 per cent (95% CI: 99.6-100%) and 99.5 per cent (95% CI: 99-99.7%), respectively.
Interpretation & conclusions
HemoTypeSC can be a viable alternative to HPLC in resource-limited regions with a high prevalence of SCD to provide timely diagnosis and support newborn screening programmes.
Keywords
HemoTypeSC
India
newborn
point of care (POC)
sickle cell disease
Sickle cell disease is globally distributed and highly prevalent in sub-Saharan Africa, the Middle East, Mediterranean regions and India1. India contributes to 14.5 per cent of the global burden of sickle cell disease2. The carrier frequency of the HbS varies up to 40 per cent and is more prevalent among the tribal communities. But due to the large population size, lack of awareness and limited screening program the affected babies born remain undiagnosed or are diagnosed late, which ultimately increases the mortality rate3. Hemoglobin electrophoresis using high performance liquid chromatography (HPLC) is a preferred method to detect SCD. However, it requires well equipped laboratory, skilled professional and is cost intensive. HPLC facilities are often unavailable in remote rural areas and transportation of the samples from remote areas poses logistic challenges. These challenges have contributed to the lack of established newborn screening programme in India4-7. Hence there is a need for the rapid, inexpensive and easy-to-use point-of-care (POC) testing devices to diagnose SCD in resource-limited and remote areas.
Various POC testing devices have been developed, which overcome concerns of cost, need for skilled operators and well-established laboratories. Based on their operating principles and detection system, they are classified into four groups, paper-based hemoglobin solubility assay, lateral flow immunoassay (LFA), density-based separation, and micro-engineered electrophoresis. Each of these methods has its own strength and limitations and thus the choice of technique depends on the specific needs of the diagnostic context such as cost, speed, accuracy, and available resources. For large scale screening and low resource settings, simpler methods like paper-based solubility assay or LFA may be appropriate, while for detailed analysis and confirmation more sophisticated methods like micro-engineered electrophoresis and density-based separation are preferred8-12.
Considering the burden of SCD in India there is a need for large scale newborn screening in low resource settings. This study aimed to evaluate the accuracy, specificity and sensitivity of the point of care device HemoTypeSC assay (Silver Lake Research Corporation USA) for newborn screening across six different centers located in the States of Maharashtra, Gujarat, Madhya Pradesh, Rajasthan and Odisha, where the prevalence of sickle cell is more, have high number of tribal population and low resource settings. HemoTypeSC assay works on the principle of LFA, utilizing specific antibodies against HbA, HbS and HbC. This enables the detection of various phenotypes, including normal (HbAA), sickle cell trait (HbAS), sickle cell anaemia (HbSS), hemoglobin C trait (HbAC) and compound heterozygous condition of HbS and HbC (HbSC disease). The assay is blind to HbF, allowing accurate detection in newborns with high HbF levels and very low HbA or HbS levels. Additionally, this test does not require specialized instruments, electricity or cold storage, making it ideal for use in resource limited and remote settings. The interpretation of the result is qualitative and visual, and the result is obtained within 20 min with minimal quantity (2 µL) of blood. The cost of the test is also projected to be low, making it a cost-effective solution for large scale screening programmes11,13.
Materials & Methods
This study was undertaken by the department of Haematogenetics, Indian Council of Medical Research – National Institute of Immunohaematology (ICMR-NIIH), Mumbai, Maharashtra, India after obtaining the ethical clearance from the Institutional Ethical Committee. The study was conducted for a period of one year (May 2019- May 2020) to evaluate prospective diagnostic accuracy of HemoTypeSC against the Bio-Rad variant II (β-thalassemia short programme) as the gold standard for HbS screening in newborns.
Study sites
Six different centers namely ICMR-Regional Medical Research Centre (ICMR-RMRC), Bhubaneswar, Odisha; ICMR-Centre for Research, Management and Control of Hemoglobinopathies (ICMR-CRMCH), Chandrapur, Maharashtra; ICMR-National Institute for Research in Reproductive and Child Health (ICMR-NIRRCH) - Model Rural Health Research Unit (MRHRU), Dahanu, Maharashtra; SEWA Rural Kendra, Jhagadia, Gujarat; ICMR- National Institute of Research in Tribal Health (ICMR-NIRTH), Jabalpur, Madhya Pradesh and ICMR- National Institute for Implementation Research on Non-Communicable Disease (ICMR-NIIRNCD), Jodhpur, Rajasthan were included in the study based on the high prevalence of HbS in these regions. The study sites were selected in semi urban and rural health centers based on the prevalence of SCD, tribal dominated population and poor resource settings. The study was conducted as a part of newborn screening programme and approved by the institutional ethics committee at each centre. HemoTypeSC test kit was provided to each centre free of cost.
Sample size
The total number of participants (sample size) to be enrolled in this study was calculated based on the estimated prevalence of sickle cell (10%) across Maharashtra, Gujarat, Madhya Pradesh, Rajasthan and Odisha along with the expected test sensitivity (95%) and desired absolute precision (0.05)14. This yielded a minimum required sample size of 700 to validate this study and we have tested a total of 1725 newborn babies.
Sample collection
After written informed consent from the parents, 1-2 mL of cord blood sample of the newborn baby was collected at birth in EDTA vacutainer. Heel prick samples were collected when cord blood collection was not possible within 48 h of birth.
Test methods
The samples were run on the Bio-Rad variant machine using β-thalassemia short programme at all centers. Simultaneously all the samples were tested using HemoTypeSC kit and then the results were compared.
For HPLC on Bio-Rad variant machine, 5 μl of EDTA blood sample was added to 1 mL of the lysing buffer provided with the kit, mixed and run on the machine (Bio-Rad Laboratories, Hercules, California, USA). For HemoTypeSC 1.5 μL of anticoagulated blood was absorbed on absorbent pad of sampling device and inserted into test tube containing six drops of water, swirled to ensure transfer of blood to the water and lysis of blood which turn the water reddish pink. The HemoTypeSC test strip was then inserted in the test tube containing reddish pink water, incubated for 10 min at room temperature and finally the results were interpreted based on the presence and absence of the bands.
Assessment
Diagnostic accuracy, sensitivity and specificity were calculated for HemoTypeSC verses the gold standard (HPLC) in detecting normal (AA), sickle cell trait (AS) and sickle cell disease (SS/Sβ) phenotype as per the National Committee for Clinical Laboratory Standards (NCCLS) guidelines15. MadCalc Easy to Use online statistical software was used to calculate sensitivity and specificity. Agreement analysis between POC and HPLC was carried out using Kappa. The Kappa was calculated using online GraphPad Prism software version 9 and value of Kappa results was interpreted as follows: values ≤ 0 as indicating no agreement and 0.01–0.20 as slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61 –0.80 as substantial and 0.81–1 as almost perfect agreement16. Molecular confirmation and parental screening was done for all affected babies identified by POC.
Results
A total of 1725 newborn babies were screened across six cities: Bhubaneswar (301), Chandrapur (318), Dahanu (345), Jhagadia (251), Jabalpur (300) and Jodhpur (210). The site wise comparison of HemoTypeSC test results with HPLC test is mentioned in supplementary table I.
Accuracy of HemoTypeSC to detect variants of hemoglobin (HbA and HbS)
Hemoglobin variant A (HbA) was identified in 1,710 newborn babies and hemoglobin S variant (HbS) in 15 by HPLC. All 1,710 babies with Hb A variant were reported concordantly by HemoTypeSC. Of the 15 newborn babies with Hb S variant 14 were found to be concordant by HemoTypeSC (centre-wise data provided in supplementary table II). Overall sensitivity and specificity of HemoTypeSC to detect Hb variant HbA and HbS was found to be 100 per cent [95% confidence interval (CI): 99.7-100%] and 93.3 per cent (95% CI: 68-99.8%), respectively, with accuracy of 94 per cent (95% CI: 92.7-95%).
Accuracy of HemoTypeSC to detect sickle cell disease and sickle cell trait
Total 15 newborn babies with SCD were identified by HPLC and 14 of them were found to concordant by HemoTypeSC whereas of the 200 newborns with HbAS genotype identified by HPLC, 192 were reported concordantly by HemoTypeSC. Molecular testing confirmed that all 15 babies affected by sickle cell disease had sickle cell anemia. Screening of their parents revealed that all were carriers of the sickle cell trait.
The sensitivity and specificity of HemoTypeSC to detect SCD was found to be 93.3 per cent (95% CI: 68-99.8%) and 100 per cent (95% CI: 99.7-100%), respectively. The sensitivity and specificity of HemoTypeSC for detection of sickle cell trait was found to be 96 per cent (95% CI: 92.2-98.2%) and 99.8 per cent (95% CI: 99.5-99.9%) center wise data are provided in supplementary table III. The accuracy of HemoTypeSC to detect SCD and sickle cell trait was found to be 99.9 per cent (95% CI: 99.6-100%) and 99.5 per cent (95% CI: 99-99.7%), respectively.
Discordant results
Of the 1725 newborn samples screened, 11 (0.6%) showed discordant results between HemoTypeSC and HPLC. Of the 11 samples, two samples with normal (AA) phenotype by HPLC were found to be AS in HemoTypeSC. Of these two samples, we have retested one sample in HemoTypeSC and on retesting the result was found to be concordant with HPLC, i.e., AA. Eight samples with AS phenotype by HPLC were found to be AA in HemoTypeSC and one sample with SS phenotype in HPLC was found to be AA in HemoTypeSC. To confirm the results, retesting was not possible in seven samples.
The POC and HPLC were both tested positive in 1508 (99.9%) for HbAA, 192 (96%) for HbAS and 14 (93.3%) for HbSS. Whereas POC and HPLC were both tested negative for 206 (95.8%) for HbAA, 1522 (99.9%) for HbAS and 1710 (100%) for HbSS. The POC negative and HPLC positive results were obtained in 2 (0.1%) for HbAA and 8 (4%) for HbAS whereas POC positive and HPLC negative results were obtained in 9 (4.2%) for HbAA and 2 (0.1%) for HbAS.POC and HPLC showed a concordance with a perfect agreement for POC positive, HPLC positive with κ = 0.965 and 0.97 for detection of Hb variant HbA and HbS, respectively.
Discussion
The present study assessed the efficacy of POC device HemoTypeSC in detecting different phenotype of HbS (AA, AS and SS) in newborn babies from five different States with high HbS prevalence viz Maharashtra, Gujarat, Madhya Pradesh, Rajasthan and Odisha. The sensitivity of the kit to detect SCD and sickle cell trait was found to be 93.3 per cent and 96 per cent, respectively while the specificity was found to be 100 per cent to detect SCD and 99.8 per cent to detect sickle cell trait. The accuracy of HemoTypeSC to detect SCD and sickle cell trait was found to be 99.9 per cent. Discordant results between HPLC and HemoTypeSC were observed in less than one per cent samples, which could be due to low Hb percentage or high HbF percentage falling outside the reference range. Retesting of all the discordant samples was not possible due to logistic constraints.
The HemoTypeSC, which can differentiate normal (AA), sickle cell trait (AS) sickle cell anemia (SS), HbC trait and HbSC disease has proven to be a highly effective POC for newborn screening. In a study by Nondu et al16, HemoTypeSC was validated against HPLC as the gold standard in newborns, yielding 93.4 per cent sensitivity and 99.9 per cent specificity, with an overall accuracy of 99.1 per cent. A study from Uganda tested 1000 known SCD cases using HemoTypeSC, with results compared to capillary zone electrophoresis (CZE), reported ≥99.5 per cent sensitivity and ≥99.9 per cent specificity. Two HbSS cases were misidentified as HbAS due to recent blood transfusions17. Similarly, in a study from Côte D’Ivoire, HemoTypeSC demonstrated 98.2 per cent sensitivity and 99.7 per cent specificity18. In research by Mukerjee et al19, the test showed 97.1 per cent sensitivity and 98.6 per cent specificity for detecting HbAA, HbAS and HbSS phenotypes in newborns. Overall, the sensitivity and specificity for HbSS detection across both newborns and adults exceeded 99 per cent. In a study conducted in Mali, the diagnostic performance of two point-of-care (POC) devices, Sickle Scan and HemoTypeSC, was evaluated for newborn screening to detect sickle cell disease (SCD). Both devices demonstrated reliable diagnostic accuracy when compared to high-performance liquid chromatography (HPLC). The HemoTypeSC showed a sensitivity of 95 per cent and specificity of 98 per cent, while the Sickle Scan exhibited a sensitivity of 96.1 per cent and specificity of 97.6 per cent20. Our study which focused exclusively on screening newborns, also found the sensitivity and specificity for HemoTypeSC for detecting HbAA, HbAS, and HbSS phenotypes to be more than 99 per cent, consistent with these previous findings. However, HemoTypeSC has certain limitations, due to its inability to differentiate between HbSS and HbS- β-thalassemia, it may yield false negative for HbSS cases, following recent blood transfusions16-19. A recent systematic review by Bagnall et al21 reported pooled sensitivity and specificity of 92.5 per cent and 99.9 per cent, respectively, for HbSS detection in newborns and infants. Further, rapid health technology assessment on cost-effectiveness of rapid diagnostic test for SCD emphasizes that HemoTypeSC offers a more convenient alternative, as it only requires distilled or tap water for test execution, eliminating the need for extra consumables22.
This study has certain limitations, including its one-year duration, during which only a limited number of sickle cell anemia cases were identified using HemoTypeSC. Additionally, during this period, no cases of other sickle cell disorders such as HbS-β-thalassemia, HbS-D disease, or HbS-E disease were encountered. As a result, we were unable to evaluate the effectiveness of HemoTypeSC for detecting these specific conditions.
To conclude, HemoTypeSC has demonstrated effectiveness in accurately identifying SCD in newborns, offering a valuable tool for early diagnosis. The rapid nature of the test makes it especially suitable for large-scale newborn screening programmes, potentially leading to timely interventions and improved long-term outcomes for affected infants. It is important to emphasize that this device functions as a screening tool. For definitive diagnosis and classification of hemoglobin variants, gold standard (HPLC or CZE), should be used. Unlike other complex methods such as HPLC and CZE, HemoTypeSC offers a simple and efficient alternative, particularly in low-resource environments. In light of execution of National Sickle Cell Anemia Elimination Mission to eliminate sickle cell disease by 2047 by National Health Mission (NHM) India, HemoTypeSC may serve as one of the effective POCs for newborn screening in program to achieve preventive management and reduction in SCD.
Financial support & sponsorship
The study received funding support from Indian Council of Medical Research, New Delhi (grant no. Tribal/CFP/1/2018-ECD-II).
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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