Newborn screening blood spot analysis in the UK: influence of spot size, punch location and haematocrit

Abstract

Objective: In dried blood spot analysis, punch location and variations in applied sample volume and haematocrit can produce different measured concentrations of analytes. We investigated the magnitude of these effects in newborn screening in the UK. Methods: Heparinized blood spiked with thyroid stimulating hormone (TSH), phenylalanine, tyrosine, leucine, methionine, octanoyl carnitine (C8), and immunoreactive trypsinogen (IRT) was spotted onto filter paper: (i) at a constant haematocrit of 50% at various volumes, and (ii) at a range of haematocrits using a constant volume. Subpunches (3.2 mm) of the dried blood spots were then analysed. Results: Compared with a central punch from a 50 mL blood spot with 50% haematocrit, 10 mL spots can have significantly lower measured concentrations of all analytes, with decreases of 15% or more observed for leucine, methionine, phenylalanine, and tyrosine. Punching at the edge of a spot can increase measured concentrations up to 35%. Higher haematocrit decreased measured TSH and C8 yet increased amino acids and IRT by 15% compared with 50% haematocrit. Lower haematocrits had the opposite effect, but only with higher concentrations of some analytes. Conclusions: Differences in blood spot size, haematocrit and punch location substantially affect measured concentrations for analytes used in the UK newborn screening programme, and this could affect false positive and negative rates. To minimize analytical bias, these variables should be controlled or adjusted for where possible.

Effect of Dried Blood Spot Quality on Newborn Screening Analyte Concentrations and Recommendations for Minimum Acceptance Criteria for Sample Analysis

“…We confirmed previous findings that a smallervolume DBS will produce lower analyte concentrations (4,10,15,16 ). The smaller the volume applied to the card, the further the blood spreads relative to a sample of higher volume.…”

“…Previous studies have assessed other factors that may affect results for some analytes, including punch location (10,15 ), hematocrit (10,15 ), sample volume (6,10,15,16 ), and filter paper type (17 ).…”

“…In the UK, PerkinElmer 226 filter paper, which contains four 10-mm-diameter circles, is used (10 ). DBS quality is assessed subjectively by visual inspection in the screening laboratory, and repeat samples are requested on those deemed unsuitable for analysis.…”

What Is the Best Blood Sampling Time for Metabolic Control of Phenylalanine and Tyrosine Concentrations in Tyrosinemia Type 1 Patients?

“…Second, amino acid analysis was done in blood spots. Measurements of amino acids in blood spots are influenced by factors such as humidity, hematocrit, and size and location of punches in the blood spot (Holub et al 2006;Lawson et al 2016). Patients' hematocrit was not measured, but can be considered normal as no illness or trauma occurred in our patients during the time of the study.…”

“…This was performed 4 times a day (pre-breakfast: 7-8 a.m., pre-midday meal: 12-1 p.m., before evening meal: 5-6 p.m. and bedtime) for 3 days with 12 samples per participant. Phenylalanine and tyrosine concentrations of all blood specimens were analyzed by LC-MS/MS in the laboratory of Metabolic Diseases of the UMCG in Groningen following a standardized protocol taking into account differences such as the spot size and localization of the punch (Holub et al 2006;Lawson et al 2016). …”