Search

Background:Newborn screening (NBS) is an established screening procedure in many countries worldwide, aiming at the early detection of inborn errors of metabolism. For decades, dried blood spots have been the standard specimen for NBS. The procedure of blood collection is well described and standardized and includes many critical pre-analytical steps. We examined the impact of contamination of some anticipated common substances on NBS results obtained from dry spot samples. This possible pre-analytical source of uncertainty has been poorly examined in the past.Methods:Capillary blood was obtained from 15 adult volunteers and applied to 10 screening filter papers per volunteer. Nine filter papers were contaminated without visible trace. The contaminants were baby diaper rash cream, baby wet wipes, disinfectant, liquid infant formula, liquid infant formula hypoallergenic (HA), ultrasonic gel, breast milk, feces, and urine. The differences between control and contaminated samples were evaluated for 45 NBS quantities. We estimated if the contaminations might lead to false-positive NBS results.Results:Eight of nine investigated contaminants significantly altered NBS analyte concentrations and potentially caused false-positive screening outcomes. A contamination with feces was most influential, affecting 24 of 45 tested analytes followed by liquid infant formula (HA) and urine, affecting 19 and 13 of 45 analytes, respectively.Conclusions:A contamination of filter paper samples can have a substantial effect on the NBS results. Our results underline the importance of good pre-analytical training to make the staff aware of the threat and ensure reliable screening results.

Background: Quantitative assays using dried filter paper bloodspots (DBS) may be adversely affected by hematocrit (HCT) as an unknown variable. Studies have demonstrated the utility of the measurement of potassium (K) from DBS punches to estimate HCT. Because there is significant accrual of RBCs at the DBS perimeter, we investigated whether K measurement from ring-shaped specimens inclusive of the perimeter might provide an advantage over conventional interior circular sub-punch samples to estimate HCT. Methods: Primary samples were Li-heparin whole blood, with HCT as measured on concurrently-drawn K-EDTA specimens. DBS were formed by bolus addition of 40 μL whole blood to filter paper cards. Total bloodspot area was determined by image analysis. Removal of center sub-punch (P) samples of fixed area produced remainder ring (R) samples inclusive of the perimeter. Samples were extracted in K-EDTA (2.5 mmol/L) and measured for diluent-corrected K per area (α, μmol K/cm2). Results: Forty-three patient samples were utilized. α was normally distributed: α(P)=1.23±0.26 μmol K/cm2; α(R)=1.86±0.41 μmol K/cm2; α(R)/α(P)=1.51±0.15. α was correlated with HCT: α(P)=0.030 HCT(%)+0.015 μmol K/cm2 (r2=0.795); α(R)=0.052 HCT(%)+0.010 μmol K/cm2 (r2 = 0.912), but with higher resolution and lesser error for α(R). Conclusions: K per area (α) was significantly higher in R samples vs. P samples, with higher resolution for α(R) vs. HCT. Use of ring samples inclusive of the perimeter to estimate HCT for DBS via K measurement can provide an advantage over use of center sub-punch samples.

Background: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. Methods: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. Results: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. Conclusions: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.