Diagnostic comparison between cord blood and filter paper for the screening of congenital hypothyroidism

Abstract Background Cord‐blood and heel‐prick TSH levels are essential in diagnosing and preventing the serious complications of congenital hypothyroidism, which mainly include intellectual disability. The study aimed to compare between cord‐blood and heel‐prick TSH sensitivity and specificity in detecting congenital hypothyroidism (CH) among newborn screened babies. Method The study included 21,012 newborn screened babies for congenital hypothyroidism starting from September 2013 until March 2019. Both cord‐blood and heel‐prick TSH were collected from each newborn. Heel prick and cord‐blood TSH cutoff values of >21 μU/ml and >30 mIU/L respectively were considered positive. Results Out of the total screened newborns, 12 were confirmed for having primary congenital hypothyroidism. Nine cases were positive for cord‐blood TSH (Sensitivity 75%, specificity 99.9%, and a recall rate of 0.004%), while 139 cases were positive for heel‐prick blood TSH (Sensitivity of 100%, specificity of 99.3%, and a recall rate of 0.60%). Conclusion For the screening of CH, heel prick is considered a superior method, but cord blood remains a practical option due to its cost‐effectiveness, immediate action, and lower recall rate. Therefore, whenever recall is difficult and/or early discharge is the practice, cord blood is an alternative method to heel prick but not with cases of prematurity.

congenital hypothyroidism. On the contrary, a temporary deficiency of thyroid hormone which usually improves in a few months is known as transient congenital hypothyroidism. 2 Prevalence of congenital hypothyroidism in infants with low and very low birth weight is significantly high and is said to be around 1 in 400 cases, but in the case of full-term infants, it is 1 in 4000 cases. 3 Almost all infants with congenital hypothyroidism are asymptomatic at birth and show no signs, and thus, the diagnosis is delayed due to a lack of clinical findings in most cases in the newborn period. Eventually, this will prompt the most serious result of congenital hypothyroidism, intellectual disability. 4 Therefore, screening programs and better management plans have been established to prevent and overcome this disease. 3 In the 1970s, hypothyroidism neonatal screening programs were developed worldwide. 1 Cord-blood specimens and spotted heelprick blood on filter papers have been utilized to measure thyroidstimulating hormone (TSH) and free thyroxine (FT4) for the screening of congenital hypothyroidism. 1 The initiation of general screening in the 1970s has effectively reinforced the capacity of North America, Europe, partly Asia, Latin America, and a couple of African nations to surpass congenital hypothyroidism consequences and rise the number of survivors, which helped roughly in diagnosis and treatment of congenital hypothyroidism at an early manner. 1 In 1972 Dussault, Quebec-Canada, the first congenital hypothyroidism screening was performed. Seven hypothyroid infants were detected among 47,000 newborns within 3 years. In the initial report, the method missed 10% of the cases with hypothalamic-pituitary hypothyroidism. It is attributable to under-developed hypothalamicpituitary axis in that group of newborns results in the delayed rise of TSH. 3,4 Neonatal TSH physiological surge increases TSH levels and prompts dynamic thyroxine (T4) and triiodothyronine (T3) changes within 24 to 48 h from birth. Thus, most centers collect heel-prick blood samples following 24 h of age to limit the rate of false-positive high TSH. 4 In congenital hypothyroidism diagnosis, TSH was more specific. On the contrary, in the detection of hypothalamic-pituitary hypothyroidism, T4 was more sensitive. Nevertheless, T4 along with TSH is not cost-effective methods of screening; therefore, generally, TSH and infrequently T4 screening is utilized worldwide. 4 Hence, optimum sensitivity and specificity of the screening method, cord blood, or heel-prick blood, is needed especially for high-risk newborn. 3 In short, advancements in laboratory research have empowered clinicians to enhance the lives of newborns with congenital hypothyroidism. The development of sensitive and specific assays to measure TSH using cord blood and heel-prick blood made it possible to initiate highly cost-effective newborn thyroid screening programs.
Thus, early diagnosis and treatment will save detected children from intellectual disability. Therefore, this study aims to compare and determine the sensitivity and specificity of cord blood and heel-prick blood thyroid-stimulating hormone (TSH) in detecting congenital hypothyroidism among newborn screened babies at King Abdulaziz Medical City, Jeddah, Kingdom of Saudi Arabia.

| Data collection
We conducted a comparative cross-sectional study at King Abdulaziz Medical City (KAMC), Jeddah, Saudi Arabia. All the data included in the study were obtained from samples, which were delivered from the delivery unit in the hospital. All newborn screened babies from September 2013 until the end of March 2019 were included in this study. We excluded transferred patients from other hospitals, neonatal death prior to specimen collection for newborn screening, babies with incomplete screening for congenital hypothyroidism, and all lost to follow-up patients.
As shown in Figure 1 The data of patients were collected using a data collection sheet containing the basic demographic variables (gender and date of birth), gestational age, birth weight, initial cord-blood TSH result, initial heel-prick TSH results, repeated cord-blood TSH result, repeated heel-prick TSH result, confirmed diagnosis, and patient remarks. Heel-prick TSH blood sample levels of more than 21.0 μU/ml were considered positive and required confirmation. Cord TSH levels above 30.0 mIU/L were considered positive.
The ethical committee at King Abdullah Medical City had granted ethics approval for the study (IRB# SP19-457-J). As data would be collected from health records, informed consent was not applicable, and all patients' medical record numbers were anonymized during data analysis.

| Laboratory methods
For cord blood, TSH was performed using i2000 Architect chemiluminescent immunoassay, (Abbott Diagnostics) since 2013 until now.
TSH measurement in heel-prick sample using dry blood spot filter paper was performed using Genetic Screen Processor by Perkin Elmer method.

| Statistical analysis
We used proportions for categorical data, means ± SD or median (range) for quantitative data, chi-square or Fisher exact test for correlating categorical variables, Student's t test for correlating qualitative and quantitative variables, and Spearman's correlation for correlating quantitative variables.

| Demographics and characteristics
Our study had a total of 149 patients who had positive heel prick or positive cord blood or both. Furthermore, it stresses that CH is a rare diagnosis with only 12 true-positive cases. While heel-prick positive cases were comparable in terms of birth weight (53% normal; 42% low birth weight; 4% very low birth weight; and 1% extremely low birth weight), gestational age (50% term; 44% moderate late/ preterm; 5% very preterm; and 1% extremely preterm), and gender distribution with 57% females, cord-blood-positive cases were only term (100%) with normal birth weight (100%) and were predominantly females (70%). Table 1 depicts TSH levels based on gender, birth weight, and gestational age in true-positive cases. Results show that male, term babies with normal birth weight had higher TSH levels in both heel prick and cord blood in comparison with others. On the contrary, Table 2 shows that in false-positive cases, the TSH levels were lower regardless of gender, birth weight, and gestational age. Nevertheless, prematurity and low birth weight increase false-positive rate in heelprick TSH. Table 3 shows three positive CH cases that were detected within cord-blood sampling as false negative. All three cases were below TA B L E 1 True-positive results for thyroid-stimulating hormone levels using heel prick and cord blood  the average birth weight with preterm in the first and second cases and late preterm in the third case.

| Sensitivity and specificity
As shown in Table 4, heel-prick samples have 100% sensitivity with higher recall rate and much lower positive predictive value, while cord-blood samples which have higher specificity (99.9%) but lower sensitivity (75%). Moreover, TSH levels in those true-positive samples were notably higher measuring around 140 uU/ml (Figure 2A). Samples that were collected on the same day had the highest false-positive rates (94%) with only 7 true-positive samples (6%) ( Figure 2B). TSH level in those true-positive samples was also less than the TSH level in samples collected on the same day measuring around 90 uU/ml ( Figure 2C).

| Time of heel-prick sample collection
In comparison, samples that were collected after 24 h or more of birth had lower false-positive rates (72%) with only 5 true-positive samples (28%) ( Figure 2D).
Overall, TSH levels in both heel-prick samples collected at birthday or 24 h after birth were much higher in true-positive cases ( Figure 3A) compared with false-positive cases ( Figure 3B). Truepositive cases in both heel-prick samples collected at birthday or 24 h after were comparable ( Figure 3C); however, false-positive cases were much higher when collected on the same day of birth ( Figure 3D).

| DISCUSS ION
Up to date, this study is one of a few that have compared the sensitivity and specificity between cord and heel-prick TSH neonatal screening for CH. TSH, which is the main screening test, had been measured through both cord and heel-prick blood in order to early detect and diagnose CH.
Our study had a total of 149 cases: 10 were detected by cord blood and 139 were detected by heel brick, who had positive heel prick or positive cord blood or both. Furthermore, it stresses that CH is a rare diagnosis with only 12 true-positive cases. All the cases were true-positive heel-brick samples, but only 9 cases were truepositive cord-blood samples.

| CON CLUS ION AND RECOMMENDATIONS
Cord-blood TSH appears to be more practical option as a screening method for CH disorders due to low cost, low recall rate, and

ACK N OWLED G EM ENTS
The authors acknowledge the support received from King Abdullah Arabia. The authors are also grateful to the hospital information system and medical records departments for their help and cooperation in providing data.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data can be available from the corresponding author upon request.