Newborn Thyroid Screening: Influence of Pre-Analytic Variables on Dried Blood Spot Thyrotropin Measurement.

BACKGROUND: Measuring thyrotropin (TSH) eluted from a dried blood spot (DBS) is used to screen an estimated 30 million newborns annually for congenital hypothyroidism (CH). Newborn thyroid screening has eliminated cretinism from the industrialized world and decreased the adverse effects of unrecognized CH on neurocognitive development. Hematocrit, a pre-analytic variable that affects the measurement of TSH from a DBS, contributes to the imprecision of DBS TSH measurement and could account for false-negative and false-positive DBS newborn screening test results. To assess whether variations in hematocrit found in newborns have a clinical effect in DBS-based newborn thyroid screening, the effects of hematocrit variability on the measurement of DBS TSH were studied. METHODS: U.S. Centers for Disease Control and Prevention procedures for manufacturing DBS performance testing standards were used to generate DBSs from blood samples, with hematocrits of 35%, 40%, 45%, 50%, 55%, 60%, and 65% and serum TSH concentrations of 6.3 ± 0.4 and 26.6 ± 8.0 mIU/L. TSH was measured in the eluates of four replicate DBS 3 mm punches at each hematocrit using the Thailand Ministry of Public Health Newborn Screening Operation Center enzyme-linked immunosorbent assay. Data were analyzed using a linear mixed-effects model. RESULTS: Based on the mixed-effects model, hematocrit significantly affected DBS TSH measurement (p < 0.001). A 1% increase in hematocrit resulted in a 0.06 mIU/L decrease in eluate TSH when TSH was 6.3 + 0.4 mIU/L, and a 0.21 mIU/L decrease in eluate TSH when TSH was 26.6 + 8.0 mIU/L. CONCLUSIONS: DBS TSH is significantly affected by the blood sample hematocrit. The pre-analytic variability due to hematocrit is independent of TSH assay sensitivity, specificity, precision, repeatability, and reference intervals. The effect of hematocrit on DBS TSH measurement is clinically relevant, could account for geographic and ethnic variation in the incidence of CH, and may result in both false-positive and false-negative CH screening results. Individual newborn and population-specific hematocrit correction factors may improve the precision of DBS TSH measurement.

Neonatal screening program in Thailand.

The Neonatal Screening Program for congenital hypothyroidism (CHT) and phenylketonuria (PKU) commenced in 1996 with the objective of bringing better quality of life to people throughout the country, especially in the remote areas. This involved the implementation of routine services to the public health infrastructure all over the country. The plan of action has been designed so that by the year 2000 all public health service units throughout the country may provide screening services which can cover 1.2 million babies/ annum. Implementation of the screening program has been performed through public health sectors all over the country. These involved: education of the health personnel and communities, implementation of routine specimens collection and delivery systems to the central laboratories, establishment of central laboratory screening services, routine follow up and case management. Local in-house reagents using ELISA and IRMA techniques have been developed and utilized as screening and confirmation tests for CHT. In addition, Guthrie's test has been used for PKU screening and the automated Fluorometry has been selected for PKU confirmation. All 724 community hospitals have provided newborn screening services as one of the basic requirements for newborns according to public health policy. Of 1,425,025 babies screened, 3,450 (0.24%) were above the first screening cut off for CHT (TSH > 25 mU/l) and 321 (0.02%) for PKU (PKU > 4mg/dl). With a 63.10% follow up rate, the incidences were 1:3,314 for CHT and 1:237,504 for PKU. Newborn screening has been implemented as routine practice for all public health sectors of the country for CHT and PKU. It is expected that by the year 2003, all Thai newborns will be provided with screening services resulting in a better quality of life for the next generation.

Congenital adrenal hyperplasia: should nationwide screening be implemented in Thailand?

A project to establish the Thailand National Neonatal Screening Program was started in 1996 with the objective of screening every newborn for congenital hypothyroidism and phenylketonuria. Over a million newborns were screened and over 430 abnormal cases were detected. A study was also conducted to determine the feasibility of including CAH screening in the program. The incidence of this disease has not yet been clearly determined. Since 1999, 58,563 newborns have been screened for CAH and 144 newborns with serum 17-OHP higher than 40 ng/mL were recalled for confirmatory tests. Of those, 68 were retested and 6 were found to have elevated 17-OHP levels. Two were confirmed with salt wasting CAH one month after birth, two others were diagnosed with another disease that caused electrolyte imbalance, one patient died, and the sixth required further clinical diagnosis. Five other babies were reported dead before the second specimens could be collected for confirmation. It appears that CAH may be one of the underlying causes of death among Thai newborns and the incidence may be higher than thus far shown due to incomplete confirmation of positive screens and deaths to some infants.

A simple method to detect and differentiate Burkholderia pseudomallei and Burkholderia thailandensis using specific flagellin gene primers.

We have previously shown that Burkholderia pseudomallei, the causative pathogen of melioidosis, may be discriminated from the closely related non-pathogenic species Burkholderia thailandensis by the presence of a 15 base pair deletion in the flagellin gene of B. thailandensis. Using specific flagellin gene primers flanking the distinctive region, PCR products of 191 and 176 bp in size were detected for B. pseudomallei and B. thailandensis, respectively. The sensitivity of detection is 20-80 colony forming units/reaction of B. pseudomallei and B. thailandensis cell suspension. To mimic the expected environmental situation, mixed populations of the two species were analyzed. The results showed that the PCR-based method could be use to distinguish the two species in a duplex reaction. In addition, we have developed a simplified method for direct PCR-based detection from soil samples. The result indicated that about 200 colonies of bacteria per reaction could be detected. This method can be applied to epidemiological studies, especially for investigating the ecological relationship between these two species in the environment.