Using umbilical cord blood to test for total thyroxin

119 ORIGINAL ARTICLE Is umbilical cord blood total thyroxin measurement effective in newborn screening for hypothyroidism? M Abduljabbar, A Al Shahri and A Afifi... J Med Screen 2009;16:119 123 DOI: 10.1258/jms.2009.009035 See end of article for authors affiliations... Correspondence to: Dr M Abduljabbar, Saudi Aramco Medical Services Organization (SAMSO), Dhahran 31311, Saudi Arabia; mohammad.abduljabbar. 1@aramco.com Accepted for publication 15 June 2009... Objective To assess the performance of the use of umbilical cord blood for screening of primary congenital hypothyroidism in the Saudi Aramco Medical Services Organization newborn thyroid screening programme. Methods Umbilical cord blood total thyroxin (CB-TT4) was measured. In samples with low T4 concentrations, an additional measurement of cord blood thyroid-stimulating hormone was made. Results A total of 96,015 newborn infants were screened in the period January 1990 December 2007. Twenty-six cases of primary congenital hypothyroidism, six cases of transient hypothyroidism and 13 cases of central hypothyroidism were detected. This method of screening resulted in 100% sensitivity and 98% specificity (95% CI 84 100, and 95% CI 98 98.2, respectively). However, there was a high mean recall rate of 1.9%. Conclusion The use of CB-TT4 is a valid screening strategy for primary congenital hypothyroidism. It meets the metabolic screening demands of early discharge policy and guarantees screening all newborns delivered in the hospital. INTRODUCTION Using umbilical cord blood to test for total thyroxin has not been a popular newborn thyroid screening methodology. Concerns have been raised regarding false-negative test results and potential effects of maternal conditions and delivery on the interpretations of the results, as these may increase fetal thyroid-stimulating hormone (TSH) levels. 1,2 In Saudi Aramco Medical Services Organization (SAMSO) Hospitals, we use a cord blood total thyroxin (CB-TT4) screening programme with an additional measurement of cord blood thyroid-stimulating hormone (CB-TSH) in those samples with lowest T4 concentrations. This study reviews the results of the newborn thyroid screening programme in SAMSO Hospitals, and whether they are comparable with international standards. In addition, we adopt a higher CB-TT4 cut-off to improve sensitivity. We are aware of the potential increase in recall rate as a result of this practice. CB-TSH. 20 miu/l were recalled. From 2004, the screening policy was modified to decrease the recall rates (Figure 1). Patients with a CB-TT4 level, 6.6 mg/dl, and with CB-TSH level 20 miu/l, were recalled to test venous blood free thyroxin (FT4) and TSH. Furthermore, patients with a CB-TT4 value in the range of 6.6 7.5 mg/dl, and with CB-TSH level 40 miu/l, were called back to test for their venous blood FT4 and TSH, as well (Figure 1). This last approach was adopted to decrease the recall rate. From 2001 onwards, our premature infants routinely have their venous FT4 and TSH tested at four weeks of age (for those preterm infants close to 34 weeks of gestation an early two-week retest is done at discharge time, i.e. 36 weeks corrected age). PATIENTS AND METHODS Screening protocol All infants born at SAMSO Hospitals were screened by measuring their CB-TT4. Where CB-TT4 measurements were below 7.6 mg/dl, CB-TSH was also measured on the same sample (expressed in International Units per litre). Prior to 2004, all cases with CB-TT4, 7.6 mg/dl and Figure 1 Congenital hypothyroid screening pathway

120 Abduljabbar et al. All suspected cases of congenital hypothyroidism were referred to paediatric endocrine services at SAMSO to confirm the diagnosis, start treatment and plan follow-up. to the paediatric endocrine service at SAMSO Hospital. Cases with mild hypothyroidism (normal T4 and high TSH) have not been documented so far. Patients, samples, data collection and analysis The study includes all Saudi newborn infants born in SAMSO facilities in the period between January 1990 and December 2007. A total of 96,015 newborn infants were screened. At delivery, cord blood was collected in a special microcontainer, and was sent to the laboratory where serum was extracted. Samples were run daily for CB-TT4 by Fluorescent Polarization Immunoassay (Abbott Laboratories, IL, USA). If CB-TT4 results were below a set cut-off (,7.6 mg/dl), a CB-TSH from the same cord blood sample (measured by Microparticle Enzyme Immunoassay; Abbott Laboratories, IL, USA) was run, and abnormal results were reported to the programme coordinator. A dedicated database system captures all the results of CB-TT4, CB-TSH and all the repeated venous thyroid tests on a daily basis. After seven days, all cord samples were discarded. Both CB-TT4 and CB-TSH data were analysed for sensitivity and specificity in diagnosing congenital hypothyroidism. Follow-up at screening programme All suspected or diagnosed cases with congenital hypothyroidism were followed up closely within SAMSO hospitals and clinics. The medical care is an exclusively free service for employees and their dependants (including their infants). We expect those infants upon illness to be referred RESULTS During the study period (January 1990 December 2007), 96,015 newborn infants were screened for CB-TT4. Of those, 9490 cases (9.9%) required further testing for CB-TSH, from which 1827 cases were recalled for venous FT4 and TSH retesting (mean recall rate 1.97%). There were 26 cases of primary congenital hypothyroidism (Table 1). The incidence rate was 1:3692 live births with a male-to-female ratio of 1:1.6, and 23% of them (6 cases) were preterm infants (gestation ages less than 38 weeks). Thyroid scans showed ectopic thyroid in 10 cases (38%), thyroid agenesis in 11 cases (42%) and dyshormonogenesis in three cases (12%). No seasonal variation was noted. The mean age of starting treatment was 8.2 days (range 1 35 days). The mean starting dose of thyroxin was 42.3 mg/day (Table 2). The thyroid screening programme showed a sensitivity of 100% (95% CI 84 100%) and a specificity of 98% (95% CI 98 98.2%). None of the 26 identified cases of congenital primary hypothyroidism were from the recalled group. During the period before 2004, the recall rate was 2.18%, all the cord TSH of confirmed cases were above 40 miu/l and none of them was from the recalled group. In the second period from 2004 onwards with screening policy change, the recall rate decreased to 1.36%. This is a 38% reduction of the mean recall rate and we have not missed any case (Table 3). Table1 Cases of primary congenital hypothyroidism detected by CB-TT4 programme Cases Study year Gestational age (weeks) Cord total T4 (mg/dl) Cord TSH (miu/l) Serum FT4 (mg/dl) Serum TSH (miu/l) Thyroid scan 1 1990 Term 3 677 0.29 190.0 ET 2 1991 Term 5.6 345 0.59 382.0 ET 3 1992 Term 3.6 196 0.41 196.0 TA 4 1992 Term 2 209,0.4 209.0 TA 5 1993 Term 2.9 98.9 0.49 117.7 ND 6 1993 36 4.3 1392 0.41 820.6 ND 7 1994 Term 7 58 0.96 28.5 ET 8 1994 34 6.8 288 1.05 159.0 ET 9 1996 Term 6.38 331.8 0.56 258.7 ET 10 1996 Term 3.64 142 0.4 168.0 ET 11 1996 35 2.7 406,0.4 484.0 TA 12 1997 Term 2.3 487 0.4 440.0 ET 13 1998 37 3.63 574,0.4 760.0 DHG 14 1999 37 4.37 195.48 0.46 237.0 TA 15 1999 Term 1.99 643.53,0.4 976.0 TA 16 1999 Term 4.58 662 0.51 233.0 DHG 17 2000 Term 5.8 405 0.81 41.6 ET 18 2000 30 5.46 6.19,0.4 176.0 DHG 19 2001 Term 3.5 310 0.49 375.7 ET 20 2002 Term 2.9 226.0,0.4 383.9 TA 21 2003 Term 5.24 484.9 0.45 389.0 ET 22 2004 Term 4.18 268 0.46 183.0 TA 23 2005 Term 3.96 165 0.48 170.0 TA 24 2006 Term 2.76 953,0.4 806.0 TA 25 2007 Term 4.08 361.2 TA 26 2007 Term 3.64 538 0.74 532.0 TA Thyroid scan: ET ¼ ectopic thyroid; DHG ¼ dyshormonogenesis; TA ¼ thyroid agenesis; ND ¼ not done FT4 ¼ free thyroxin; TSH ¼ thyroid-stimulating hormone Journal of Medical Screening 2009 Volume 16 Number 3 www.jmedscreen.com

Umbilical cord blood total thyroxin measurement and hypothyroidism 121 Table 2 Thyroid function parameters of 26 patients with congenital hypothyroidism Parameters Mean SD Range Gestational age (weeks) 38.7 2.6 30 41 Cord total T4 (mg/dl) 4.1 1.4 1.99 7 Cord TSH (miu/l) 400.8 299.4 6.2 1392 Serum FT4 (mg/dl) 0.6 0.2 0.29 1.05 Serum TSH (miu/l) 348.7 255.7 28.5 976 Age when treated (days) 8.2 7.4 1 35 Start dose (mg) 42.3 8.7 25 50 One preterm infant, 30 weeks gestation with dyshormonogenesis, had an initial CB-TT4 of 5.46 mg/dl, cord TSH of 6.2 miu/l, FT4 of,0.4 mg/dl and a venous serum TSH of 176 miu/l One term infant with ectopic thyroid had an initial CB-TT4 of 7 mg/dl, cord TSH of 58 miu/l, FT4 of 0.96 mg/dl and a venous serum TSH of 28.53 miu/l T4 ¼ thyroxin; TSH ¼ thyroid stimulating hormone Our cord blood screening programme detected 32 cases of primary congenital hypothyroidism. However, with close follow-up, six cases proved to be transient. Thirteen cases of central hypothyroidism were identified. Only three of them proved to be isolated central hypothyroidism, constituting a rate of 1:32,005 live births. The other 10 patients were diagnosed to have hypopituitarism. DISCUSSION Primary congenital hypothyroidism is the most serious cause of an elevated TSH in a newborn. If left untreated, it leads to developmental delay and mental retardation. Traditionally, screening strategies for the detection of congenital hypothyroidism are either a primary TSH/backup T4 method or a primary T4/backup TSH method. A third strategy uses TSH plus T4 as the primary test. 3 The primary TSH and primary T4 strategies both are capable of detecting congenital hypothyroidism of thyroidal origin; however, the primary TSH screening will miss patients with congenital hypothyroidism of central origin. 4 Many hospitals perform newborn screening at 5 6 days of age, and the reference values reflect this postnatal age. In term healthy newborns, there is an initial physiologic surge of TSH (up to 60 miu/l within 30 minutes of delivery), followed by a rapid decline over the first five days of life to,10 miu/l. 5 Currently, a large number of healthy term newborns are discharged early (before 48 hours of age). Thyroid screening during this time is associated with an increasing number of false-positive results, due to this neonatal TSH surge. In addition, it is difficult to call back infants for thyroid testing once discharged. Some programmes have responded by increasing their threshold value for TSH within the first day of life. A potential problem with this practice is the possibility of missing newborns with a slowly rising TSH. 2 All of these factors make use of umbilical cord blood a practical alternative for thyroid screening purposes. 6 8 Interestingly, some countries revert to cord blood screening as the method of choice, when facing difficult patient recall for initial thyroid testing. 9,10 The newborn thyroid screening programme in SAMSO hospitals has utilized umbilical cord blood for more than two decades, mainly due to concerns about calling back infants for thyroid testing. Interestingly, this method is well suited to the early discharge practice adopted by SAMSO hospitals in the last few years. Technically, mixed cord blood samples for TSH values have compared well with filter paper samples taken in the first few days of life. 9 In addition, neonatal thyroid screening using cord blood on filter paper has been described as an easy, practical and effective method. 10 An important concern of any metabolic screening method is the occurrence of false-negative results. This may happen with very mild types of congenital hypothyroidism (having T4 concentrations within the normal range) and premature Table 3 Recall rates (repeat serum TSH percentage) in two periods Study year Number of cord total T4 samples Number of cord TSH samples Percentage of cord TSH samples (%) Number of serum TSH samples Percentage of serum TSH samples (%), recall rate 1990 7605 590 7.76 7 0.09 1991 7210 536 7.43 27 0.37 1992 7613 599 7.87 57 0.75 1993 6977 574 8.23 73 1.05 1994 6311 746 11.82 105 1.66 1995 6601 869 13.16 95 1.44 1996 6277 727 11.58 133 2.12 1997 6140 627 10.21 167 2.72 1998 5968 598 10.02 186 3.12 1999 5475 660 12.05 156 2.85 2000 5158 510 9.89 190 3.68 2001 4818 473 9.82 172 3.57 2002 4492 433 9.64 151 3.36 2003 4249 395 9.3 161 3.78 Total 2.18 2004 3527 320 9.07 41 1.16 2005 2972 269 9.05 35 1.18 2006 2334 281 12.04 34 1.46 2007 2288 283 12.37 37 1.62 Total 1.36 Grand total 96,015 9490 9.88 1827 1.9 TSH ¼ thyroid-stimulating hormone

122 Abduljabbar et al. infants with congenital central hypothyroidism. 11 Our cord blood thyroid programme reported a reassuring 100% sensitivity and 98% specificity. Unavoidably, the recall rate was noted to be higher than the figure reported in the literature (1.97% versus 1.1%). 12 An obvious advantage of screening umbilical cord TT4 with backup cord TSH testing is its application in a closed, well observed and followed newborn population like ours. In the SAMSO laboratory, the cut-off values for CB-TT4 and TSH are reviewed and adjusted every two years to improve the programme s sensitivity and specificity. In the literature, a cut-off of the third percentile blood total thyroxin TT4 (,6 mg/dl or 77 nmol/l) was employed, but many programmes now use the 10th percentile TT4 (9 mg/dl or 116 nmol/l) or even the 20th percentile as a threshold to measure TSH. This has been done in order to detect patients with subclinical hypothyroidism, a frequent finding in infants with ectopic thyroids (the most common cause of permanent congenital hypothyroidism). The higher the cut-off points, the higher false-positive values which lead to higher recall rate. 12 Our umbilical cord blood thyroid screening programme was able to capture all primary congenital hypothyroidism cases born in SAMSO hospitals by using a CB-TT4 cut-off of,7.6 mg/dl and utilizing cord blood TSH cut-off of 40 miu/l (for CB TT4 of 6.6 7.5 mg/dl) and cord blood TSH cut-off of 20 miu/l (for CB TT4 of,6.6 mg/dl). This approach met the burden from the early discharge policy adopted in term newborn nursery in SAMSO. Notably, since this modification was added in 2004, the recall rate has decreased from a mean yearly rate of 1.97% to 1.4% without missing any case with congenital hypothyroidism. Our umbilical cord TT4 screening strategy can pick up cases with central hypothyroidism as well. This is important, especially to perinatal tertiary care centres dealing with high-risk pregnancies. The programme can detect cases with neonatal hyperthyroxinaemia as well as cases with thyroxine-binding globulin deficiency. Most congenital hypothyroid cases seen in our population had ectopic or absent glands upon thyroid scanning. This is similar to the thyroid aplasia/ectopic thyroid predominance reported in the west. 13 Saidan et al. in a large screened population from Riyadh, Saudi Arabia, described more dyshormonogenesis cases than ectopic or agenesis cases. He presumed this was due to high consanguinity or certain genetic background. 7,14 In our study, dyshormonogenesis has ranked third among our congenital hypothyroid cases. The incidence of primary congenital hypothyroidism in our population is 1:3692, which is similar to the published international figures, but lower than those published by other programmes in Saudi Arabia (1:2666 by Ministry of health hospitals and 1:2759 by Military hospitals). 8 This may be explained by the fact that our rate reflects the true incidence of confirmed primary congenital hypothyroidism. Our cord blood screening programme detected 32 cases of primary congenital hypothyroidism. However, with close follow-up, six cases proved to be transient. Indeed, we are suggesting the following two terms to differentiate between two conditions: (a) screening incidence rate of primary congenital hypothyroidism that reflects all the cases captured by the screening programme and (b) true incidence of primary congenital hypothyroidism which reflects only the permanent cases that are declared through follow-up (i.e. excluding transient cases of congenital hypothyroidism). Treatment for primary congenital hypothyroidism has been recommended with a dose of 50 mg daily (or 15 mg per kg body weight daily) to be started as early as possible. This approach aims to normalize serum TSH level in 2 3 weeks and keep the serum FT4 level within its upper range of normal for age. 15 In our study, we followed the same above-mentioned initial oral thyroxin of 50 mg daily dose, especially in the last few years (Table 2). Mean age of thyroxin-treated patients was 8.2 days. Both traditional primary T4 and primary TSH thyroid screening programmes will miss the rare infant whose T4 level on initial screening is normal but who later develops low T4 and elevated TSH concentrations (,0.5% of infants, most commonly premature babies with transient hypothyroidism). This pattern has been also known as atypical congenital hypothyroidism or delayed TSH rise. Some thyroid screening programmes have responded by performing a second screen for all infants at the time of their return visit to their paediatrician at 2 6 weeks of age. 11 AstudybyAreset al. showed the premature infants less than 32 weeks post-menstrual age and sick infants to have negative iodine balance for the first 2 3 weeks. Those infants may fail to adapt to the extrauterine environment, causing iodine deficiency-induced primary hypothyroidism. 16,17 Although programmes using CB-TSH will be able to pick up most cases with congenital primary hypothyroidism, they still may miss cases in very low birthweight preterm infants (,30 weeks gestation). To close this gap, these programmes should retest all preterm infants less than 34 weeks of gestation for congenital primary hypothyroidism at four weeks of postnatal age. CONCLUSION The use of umbilical cord blood CB-TT4 as the neonatal thyroid screening modality at SAMSO hospitals captured all congenital hypothyroidism cases with 100% sensitivity and 98% specificity. Doing CB-TT4 with subsequent CB-TSH testing meets the metabolic screening demands of early discharge policy, guarantees screening all the cases delivered in the hospital and proves to be both sensitive and specific, but with a high recall rate. Needless to mention, any thyroid method needs efficient sampling, diagnosis, analysis, treatment and follow-up. Until T4 and TSH determinations can be performed practically for all newborn infants, one should be aware of the potential limitations of each method of screening for congenital hypothyroidism.... Authors affiliations M Abduljabbar, Paediatric Endocrinology Attending, Department of Pediatrics, Saudi Aramco Medical Services Organization, Eastern A Al Shahri, Supervisor, Clinical Laboratory Medicine, Department of Pathology, Saudi Aramco Medical Services Organization, Eastern A Afifi, Neonatal-Perinatal Medicine Attending, Department of Pediatrics, Saudi Aramco Medical Services Organization, Eastern Journal of Medical Screening 2009 Volume 16 Number 3 www.jmedscreen.com

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