An On-card Approach for Assessment of Hematocrit on Dried Blood Spots which Allows for Correction of Sample Volume

Miller, John H.; Poston, Philip A.; Rutan, Sarah C.; H, Thomas Karnes

doi:10.4172/2155-9872.1000162

Cited by 29 publications

(24 citation statements)

References 20 publications

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“…The average haematocrit for a healthy 5 day old baby may ordinarily be less than 55%, and a lower haematocrit for calibration and EQA materials for this age could be more appropriate. Our observed increasing trend in leucine, methionine, phenylalanine, and tyrosine concentrations with increasing haematocrit concord with Holub's work, 15 and may be due to changes in plasma volume 20 or reduced sample spreading on the filter paper. 2 The decreasing concentration with increasing haematocrit observed for C8 differs from Holub's 15 findings, and merits substantiation.…”

Section: Discussionsupporting

confidence: 87%

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

2015

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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.

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Section: Discussionsupporting

confidence: 87%

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

2015

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“…During routine collection of DBS for screening, blood from a heel prick is applied to filter paper attached to a screening card (2,4,5 ). In the UK, PerkinElmer 226 filter paper, which contains four 10-mm-diameter circles, is used (10 ).…”

confidence: 99%

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

George¹,

Moat

2016

Clinical Chemistry

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BACKGROUND The analysis of dried blood spots has been used routinely for newborn screening since the early 1970s, and the number of disorders screened has expanded substantially in recent years. However, there is a lack of evidence regarding minimum blood spot quality acceptance criteria for sample analysis. METHODS Blood pools were spiked with phenylalanine, tyrosine, leucine, methionine, octanoylcarnitine, decanoylcarnitine, isovalerylcarnitine, glutarylcarnitine, thyroid-stimulating hormone, and immunoreactive trypsinogen to concentrations at the analytical cutoffs used in UK screening protocols. We evaluated the effect of sample volume applied to the card (10, 20, 50, 75, and 100 μL), punch location (central vs peripheral), and sample quality (double layering, applying blood to both sides of the filter paper, multispotting, applying insufficient sample, and compressing the sample after application). RESULTS Compression of blood spots produced significantly lower results (14%–44%) for all analytes measured (P < 0.001). Smaller blood spots produced significantly lower results (15%–24% for 10-μL vs 50-μL sample size) for all analytes at all concentrations measured (P < 0.001). Results obtained from peripheral punches were higher than those from a central punch, although this did not reach statistical significance for all analytes. Insufficient and multispotted samples demonstrated heterogeneous results. CONCLUSIONS All blood spots containing ≤20 μL (blood spot diameter <8 mm), those in which blood has not fully penetrated the filter paper, and all samples with evidence of compression should be rejected, since there is a risk of producing false-negative results.

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“…Different hematocrit levels often lead to a diverse spread of blood onto the spot of the collection card, consequently the use of a center punch may result in the transfer of different blood volumes to sample preparation tubes. The current assay procedures involved the use of a whole-spot punch to avoid this effect ( 26 ); indeed, no influence of hematocrit levels was observed for the samples analyzed by this method, even with different levels of blood spread between the samples ( 27 ). The whole-spot approach with a fixed volume transferred by pipette can be considered a useful alternative that avoids hematocrit issues ( 15 ).…”

Section: Discussionmentioning

confidence: 99%

“…Human blood with EDTA as an anticoagulant was used to prepare samples at 20%, 30%, 40%, 50%, 60%, and 70% hematocrit. After centrifugation at 1,300 relative centrifugal force for 20 min, an appropriate volume of serum was added or removed to adjust the hematocrit ( 27 ).…”

Section: Methodsmentioning

confidence: 99%

Dried Blood Spot Technique-Based Liquid Chromatography-Tandem Mass Spectrometry Method as a Simple Alternative for Benznidazole Pharmacokinetic Assessment

Bedor

Bedor²,

Silva

et al. 2018

Antimicrob Agents Chemother

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An On-card Approach for Assessment of Hematocrit on Dried Blood Spots which Allows for Correction of Sample Volume

Cited by 29 publications

References 20 publications

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

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

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

Dried Blood Spot Technique-Based Liquid Chromatography-Tandem Mass Spectrometry Method as a Simple Alternative for Benznidazole Pharmacokinetic Assessment

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