Systematic Screening of Neonatal Sickle Cell Disease with HemoTypeSCTM Kit-Test: Case Study and Literature Review

HemoTypeSCTM test is a new cheap, faster, and appropriate screening method for neonatal diagnosis of sickle cell disease. The literature reports a few cases of its applicability. This study extends the cases study and reviews the available literature. The sample consisted of 99 subjects, including 87 newborns (36 girls and 51 boys; 1.9 4.9 kg BW) sampled among 566 babies bone at six hospitals in Kisangani city (Democratic Republic of Congo) during March-April 2019; height infant-adolescents (<18 years); and four adults. Duplicate blood samples of 75 newborns, spotted on filter paper, were transferred to Liège in Belgium for LC-MS test confirmation. Of 99 subjects, 74.74% tested HbAA, 24.26% HbAS and 1% HbSS. The prevalence of HbAS compared to the HbAA phenotype was 15/60 (20%) by HemoTypeSCTM and 14/61 (18.7%) by LC-MS. The concordance between the two methods was 98.3% or a discordance of 1.7%. The findings support the validity of the HemoTypeSCTM test as a sensitive, specific point of care test, cheap and reliable for poor African populations.


Introduction
Sickle cell disease (SCD) is an autosomal recessive inherited hemoglobinopathy due to the substitution of glutamic acid by valine at the sixth amino acid in the β-globin chain [1]- [6]. The resulting abnormal hemoglobin (HbS) is poorly soluble and rapidly polymerizes when deoxygenated [2]. SCD is probably the first genetic disease in the world [3] [7] and one of the most common high-prevalence hemoglobinopathies in sub-Saharan Africa [5] [8].
Although data on child mortality are not widely available today, several authors report that 50% to 90% of sickle cell children die before the age of 5, undiagnosed [5] [9] [10]. There is a consensus that early diagnosis of SCD in neonates is necessary for initial management [4] [5] [8] [11]. In many resource-rich areas, universal neonatal screening programs associated with prophylactic interventions have significantly reduced SCD morbidity and mortality in the first 20 years of patients' life [4] [5] [12] [13] [14]. However, in sub-Saharan Africa and central India, where more than 90% of sickle-cell births occur, neonatal screening programs are not systematically applied, if at all, mainly because of the cost and logistical burden of laboratory diagnostic tests [11] [13].
There are many rapid tests also called Point of care (POC) rapid tests on the market with bedside results (SickleScan, HemoTypeSC TM , and Sickledex) [9] [13] [15] and others are quite robust but do not give immediate results (capillary electrophoresis, high-performance liquid chromatography (HPLC), mass spectrometry (MS) and PCR polymerase chain reaction) [9] [15]. There are intermediate tests such as the Lamp Human Hemoglobin S & C mutation Kit (LaCAR), which have demonstrated their effectiveness in the diagnosis of hemoglobinopathies. However, these tests are not as fast as the first ones [6]. Rapid tests exploit either solubility properties of HbS and the cell density, or immunological approaches [15] [16]. All POC rapid tests have the advantage of saving time in terms of results and facilitating application in rural areas without electricity or qualified personnel [9] [11]. Several studies report that robust tests are available in high-income countries where they are considered worldwide as a "gold standard" for the diagnosis of sickle cell disease [18]. Their sensitivities and specificities have been studied and evaluated at 100% [18]. Nevertheless, all these studies recognize some of the weaknesses of these robust technologies for implementing them in Africa [6] [11] [12] [13] [17] [18].
HemoTypeSC TM is a competitive side-flow immunoassay that uses monoclonal antibodies to detect hemoglobin A, S, and C in a 1.5 μL whole blood sample [11] [17]. HemoTypeSC TM provides point-of-care determination of hemoglobin phenotypes HbAA (normal), HbSS and HbSC (sickle cell disease), HbCC (hemoglobin C disease), and HbAS and HbAC (carrier or trait). It does not detect other hemoglobin variants (such as Hb D or E), these variants will give the same result as Hb A. Its diagnostic accuracy oscillates around 99 or even 100% when performed on a sample containing more than 80% of HbF [11] [17]. In January 2018, HemoTypeSC TM was utilized at Holy Family Hospital in Techiman, Ghana, to screen over 400 newborns, infants, children, and adults for hemoglobins A, S, and C. All 14 carriers of hemoglobin C were successfully identified. The multicentric study by Steele et al. [11] and that of Charles Quinn [17] indicate that only the HemoTypeSC TM remains the rapid test mentioned for neonatal screening [11].
While all these considerations point to the use of simple, rapid, inexpensive, and easy-to-use diagnostic tools [13], the clinical evaluation of these tests remains necessary in many African countries. In the Democratic Republic of Congo (DRC), neonatal screening for sickle cell disease is not a common practice due to a lack of adequate infrastructure [19]. Only a few centers in Kinshasa or Lubumbashi practice it [4] [15] [20]. In Kisangani, updated data on neonatal screening for sickle cell disease are not available due to a lack of appropriate diagnostic tools [13] [15]. The available SickleSCAN assay (Biomedomics, Inc., Research Triangle Park, North Carolina) is better for screening beyond the age of 6 months and not before this period because of a high concentration of fetal hemoglobin (HbF) in the newborn's blood that would interfere with the outcome of this test [13] [21].
To implement a systematic neonatal diagnosis, we designed this preliminary study to explore the possibility of using HemoTypeSC TM as a systematic neonatal screening method in the Kisangani environment.

Study Design and Sample
To test the applicability of the test HemoTypeSC Adjustment for 566 births in two weeks gave around 100 subjects.
Neonates transfused during the study period, and those whose mothers did not consent to participate did not enter the study. We collected blood samples in

Ethical Consideration
The ethics committee of the University of Kisangani approved the study, Ref.
UNIKIS/CER/005/2018. Parents of the children included in the study gave informed consent.

Procedures for HemoTypeSC TM
Following the manufacturer's instructions (Figure 1), a sample of 1.5 μL of blood was collected by a puncture in the heel from the neonates or in the finger from adults using a thin needle (lancet) regularly used locally for the blood sample.

Procedures for LC-MS Method
In Belgium, they used the TQ5500 triple quadrupole mass spectrometer source (Sciex) system (AB Sciex, Njeuwerkerkaan den Ijssel, Netherlands) for LC-MS analysis following the manufacturer's technical specifications as described elsewhere [22]. Briefly, the blood samples are desorbed from the disc by gentle rotation with 200 µL deionized water for one hour in 90 microplates and then 100 µL of each plate transferred to fresh microplates. Following a 10 minutes' denaturation step with ACN 17 µL) and 1% aqueous formic acid (17 µL), proteins are treated overnight with 10 µL of a TCPK-treated trypsin solution. After centrifugation and incubation at 37˚C, 20 µL of the digested solution is diluted with 180 µl of ACN/deionized water (1:1) with formic acid 0.1% and centrifuged again. This working solution is ready for injection into ACN: H 2 O (50:50) with 0.1% formic acid mobile phase in Waters Acquity-UPLC system and directly introduced into the source without prior chromatographic separation.
The MS analysis operated in the multiple reaction monitoring (MRM) mode, with total acquisition time 60 seconds. The first tryptic peptide of the β globin chain (T1β) was analyzed to identify HbS and HbC variants while the twelfth tryptic peptide of the δ-globin chain (T12g) was selected to check the efficiency of digestion and to calculate HbA/HbF ratio. For each peptide, the system acquires four transitions and calculates cumulated MRM ratios variant/HbA, which allows Phenotype classification.

Statistical Analysis
We compared results from HemoTypeSC TM and LC-MS methods to measure the concordance between the two, using a paired t-test with 0.05 precision. The demographic characteristics concerning gender, time of birth, and weight of neonates were determined to anticipate the potential influence on the prevalence of HbAS and the discordance between the two methods with ANOVA.    could predict the positivity of HbAS, such as sex, the timing of delivery, and newborn weight. It is evident that those factors do not predict the status but can perhaps affect the specificity of the test. The discordant subject was a male, born at term, weighing less than 3 kg. No statistical differences were found (p > 0.05).

Discussion
According to the literature point of view, the value of neonatal screening is the early detection of newborns with HbSS to receive penicillin prophylaxis between 2 -3 months, preferably before the onset of SCD symptoms [ The prevalence of HbSS in this study was 1% for the small sample examined. One percent for a population of 100,000 people will be at least 1000 children suffering SCD. The best-recommended strategy to curve the tendencies of SCD in a population is counseling HbAS carriers not getting married between them. Thus, testing should also concern all adolescents before marriage. There is, therefore, a need for availing simple, specific, and sensitive testing methods.
The specificity of a clinical test refers to the ability of the test to exclude the event from unaffected subjects [24]. The multicenter study conducted by Cindy Steele et al. [11] showed the sensitivity and specificity of HemoTypeSC TM greater than 99%, which led the authors to qualify this test for neonatal screening of SCD. In Uganda, Nankanja et al. [28] emphasized that the value of sensitivity, specificity, positive, and negative value of HemoTypeSC TM is around 100%. The concordance in 98.3% of cases or 1.7% of discordance between HemoTypeSC TM and LC-MS method in our study supports the literature reports even though accuracy, positive and negative values of HemoTypeSC TM could not be provided here since the objective was not to validate a method (we did not use control samples). We added specificity and sensitivity in comparison with LC-MS result. This test falsely diagnosed a patient as sick when he was not, but a double check by HemoTypeSC TM would have been necessary to re-check the accuracy.
We anticipated factors that could predict the positivity of HbAS, such as sex, the timing of delivery, and newborn weight. It is evident that those factors do not predict the status but can perhaps affect the specificity of the test.
Other rapid tests exist, like Sickle SCAN and Sickledex, but they are not effective in neonates [17] [21]. The study by Luke et al. [19] reports that SickleS-CAN TM was able to simultaneously detect SCD and anemia in children less than 21 years of age, while it was less valid in the newborns. The simplicity of Hemo-TypeSC TM lies in the fact that the blood sample is taken from the soles of the feet of newborns. Venous blood is not ideal in these cases. In this regard, Nanjela [24] emphasizes that venous blood collection for neonatal screening is not perfect, especially for the testing of patients with SCD. The concept that capillary blood, obtained by stinging on the heel or finger and transferred to a filter paper, is the best method to use for detecting metabolic diseases in large populations of newborns, was introduced in Scotland by Guthrie and Susie since 1963 [12]. As a result, newborn blood samples have been regularly collected in many countries to detect many disorders, including SCD, which makes HemoTypeSC TM the best "point of care" test for neonatal screening in poor populations.
Currently, HemoTypeSC TM is an inexpensive test than other known POC tests, making it the cheapest known test nowadays. Both the study conducted by Cindy Steele et al. [11] and by Nankanja R et al. [28] confirmed the POC He-moTypeSC TM as inexpensive, competitive lateral-flow immunoassay test because the straightforward design of this test leads to an end-user cost less than $2 per test [11].
The reading of the test by the newborn's mother was consistent with that of the test performer. This conformity of the result as well by the mother as by the investigator denotes the ease of realization of the test, even by an inexperienced person. Given the diagnostic accuracy of HemoTypeSC TM , this test also remains the cheapest.
The limitations of the study are the sample size and the fact that there was only one case of HbSS. That could not allow performing rigorous statistics.

Conclusion
HemoTypeSC TM is a sensitive and specific point of care test which would open the neonatal screening program of sickle cell disease for resource-poor countries. This test is adapted to the tropical climatic conditions and doesn't need the use of electricity; that could be a solution for Kisangani because it is rapid, cheap, and reliable. For these reasons, we aim to widespread this pilot study to all of the