Table 1 DBS method development studies.

From: A state-of-the-science review and guide for measuring environmental exposure biomarkers in dried blood spots

Study (sample type)	Instrument	Precision^a	Reliability^b	Accuracy^c	Sensitivity^d	Stability^e	Sample^f	Notes
ETS (cotinine)
Spector 2007 (NDBS, 1999) [30]	GC-MS				NR	Samples stored for 7 y at 4 °C prior to analyses	¼ of DBS (~12.5 µLⁱ)
Sosnoff and Bernett 2008 (DBS) [33]	LC-MS			X	NR	X (4 y at 4 °C)	6.35-mm discs (~12.5 µLⁱ)
Murphy 2013 (DBS)^g [28]	LC-MS	X		R = 0.99, p < 0.001 (plasma)	LOQ: <0.2 ng/mL^j	X (10 m at room T)	3.2-mm discs (~3.2 µLⁱ) for high exposures; 4.8-mm discs for low exposures (~7.1 µLⁱ)	Study 1: 100% DF for plasma cotinine >0.08 ng/mL Study 2: 58.7% DF (comparable to DF % in NHANES) Measured Trans-3’-HCOT
Yang 2013 (NDBS, 1999–2003) [31]	LC-MS	X	X	R = 0.99, p < 0.001 (umbilical cord blood)	LOQ: 3.13 ng/mL	X (7 m at room T)	1 disc × 6.35-mm (~12.5 µLⁱ)	23% (100/428) DF amongst all samples 97.8% sensitivity and 98.2% specificity
Searles Nielsen 2014 (NDBS, 2007) [32]	LC-MS	X			Estimated reporting limit: 0.17 ng/mL		2 discs × 6.35-mm (~12.5 µLⁱ)
Tretzel 2016 (DBS) [29]	LC-MS	X	X	X	LOD: 5 ng/mL LOQ: 15 ng/mL	X (30 d at room T)	6-mm diameters (~11.2 µLⁱ)	Hematocrit Measured Trans-3’-HCOT
Ladror 2018 (DBS)^g [34]	LC-MS	X		R = 0.94 (plasma)	LOQ: <0.25 ng/mL^j		3.2-mm discs (~3.2 µLⁱ)	100% sensitivity and 94% specificity in predicting smoking status Hematocrit effects negligible
Lead (Pb)
Chaudhuri 2009 (NDBS, NR) [62]	ICP-MS		X	X	0.36 µg/dL^h	X (8.5 m at room T)	6.35-mm discs (~12.5 µLⁱ)	Filter blank contamination: 0.3–0.8 µg/dL
Langer 2011 (NDBS, >7 y) [65]	ICP-MS	X (CV >30%)	X	X	NR^l		½ DBS (~50 µLⁱ)	10% DF
Archer 2012 (NDBS, 2002–2006) [50]	ICP-MS			R = 0.48, p < 0.0001 (infant BLLs)	NR		3/16-inch discs (~7.0 µLⁱ)
Funk 2013 (NDBS, 2003–2009) [53]	ICP-MS	X	X	X	NR^l	X (no overall trends across collection years)	½ DBS (~ 30 µL)	100% DF Filter paper contamination: median = 5.7 ppb DBS card acid-cleaned
Funk 2015 (DBS) [52]	ICP-MS	X	X	R = 0.99 (venous)	NR^l		Whole DBS (~50 µLⁱ)	DBS card acid-cleaned
Nyanza 2019 (DBS) [48]	ICP-MS	X	X	R > 0.9, p < 0.0001 (venous)	0.08 µg/L		8-mm diameter (~19.8 µLⁱ)	100% DF Field filter blanks: 0.02 ± 0.02 µg/L Lab filter blanks: 0.009 ± 0.017 µg/L
Rodríguez-Saldaña 2021 (DBS)^g [46]	TXRF	X	X	R = 0.814 in university members and 0.911 in e-waste workers (venous)	LOD: 0.28 µg/dL LOQ: 0.69 µg/dL		DBS whole (~50 µLⁱ) or 3-mm discs (~2.8 µLⁱ)	Filter paper contamination: ~0.03 ± 0.016 µg/dL
Specht 2021 (DBS) [63]	EDXRF	X		R = 0.98, p < 0.001 (same sample of whole blood measured by AAS)	1.7 µg/dL		Non-destructive (i.e., whole blood spot card) (~50.0 µLⁱ)	Potential hematocrit bias not applicable
Mercury (Hg)
Chaudhuri 2009 (NDBS, NR) [62]	ICP-MS		X	X	LOD: 0.65 µg/L^h	X (8.5 m at room T)	6.35-mm discs (~12.5 µLⁱ)	Undetectable filter paper contamination
Funk 2013 (NDBS, 2003–2009) [53]	ICP-MS	X	X	X	NR^l	X (no overall trends across collection years)	½ DBS (~30 µL)	33% DF at 1.9 ppb^k DBS card acid-cleaned
Funk 2015 (DBS) [52]	ICP-MS	X	X	R = 0.98 (venous)	NR^l		Whole DBS (~50 µLⁱ)	DBS card acid-cleaned
Nelson 2016 (NDBS, 4–7 m) [51]	ICP-MS	X		R = 0.82 (umbilical cord blood)	T-Hg: 0.7 µg/L		2 discs × 3-mm (~2.8 µLⁱ)	Hematocrit effects negligible 38% DF for DBS vs 62% for cord blood
Basu 2017 (1st: DBS, 2nd: NDBS, <14 y)^g [64]	GC-CVAFS	X	X	X	Me-Hg: 0.313 µg/L		Batches #1–6: 3-mm discs (~2.8 µLⁱ); Batches #7–20: 2-mm × 6-mm rectangular punches (~4.7 µLⁱ)	Study 2: 98% DF, comparable to NHANES 2011–2012 Me-Hg assay performance
Nyanza 2019 (DBS) [48]	ICP-MS	X	X	R > 0.9 (venous)	T-Hg: 0.012 µg/L		8-mm diameter (~19.8 µLⁱ)	100% DF Field filter blanks: 0.006 ± 0.002 µg/L Lab filter blanks: 0.003 ± 0.003 µg/L Used ultrapure HNO₃ digestion for Hg extraction
Santa-Rios 2020 (DBS)^g [47]	GC-CVAFS	X	X	R > 0.85 (venous)	Me-Hg: 0.3 µg/L; I-Hg: 1.9 µg/L	X (Me-Hg: 1 y at room T)	Whole DBS (12.7-mm) (~40 µL blood, volume controlled)	Study 2: 94% DF
Schweizer 2021 (DBS) [49]	Direct Hg analysis		X	R = 0.95, p < 0.001 (venous)	T-Hg: 0.14 µg/L (LOD); 0.28 µg/L (LOQ)	X (4 w at room T and 40 °C)	3 discs × 0.5-inches (~50.0 µLⁱ)
Cadmium (Cd)
Chaudhuri 2009 (NDBS, NR) [62]	ICP-MS		X	X	NR		6.35-mm discs (~12.5 µLⁱ)	Low recovery rates (e.g., 53% recovery at lower spiked concentrations)
Langer 2011 (NDBS, >7 y) [65]	ICP-MS	X	X (CV ~50%)	X	NR^l		½ DBS (~25 µLⁱ)	100% or 0% DF depending on statistical method used
Funk 2013 (NDBS, 2003–2009) [53]	ICP-MS	X	X	X	NR^l	X (no overall trends across collection years)	½ DBS (~30 µL)	67% DF^k DBS card acid-cleaned
Funk 2015 (DBS) [52]	ICP-MS	X	X	R = 0.94 (venous)	NR^l	X	Whole DBS (~50.0 µLⁱ)	DBS card acid-cleaned
Nyanza 2019 (DBS) [48]	ICP-MS	X	X	R > 0.9 (venous)	0.004 µg/L		8-mm diameter (~19.8 µLⁱ)	100% DF Field filter blanks: 0.0011 ± 0.001 µg/L Lab filter blanks: 0.001 ± 0.001 µg/L
Arsenic (As)
Funk 2013 (NDBS, 2003–2009) [53]	ICP-MS	X	X	X	NR^l	X (no overall trends across collection years)	½ DBS (~30 µL blood)	18% DF^k DBS card acid-cleaned
Funk 2015 (DBS) [52]	ICP-MS	X	X	R = 0.66 (venous)	NR^l	X	Whole DBS (~50.0 µLⁱ)	DBS card acid-cleaned
EDCs and POPs
Burse 1997 (NDBS, 1997) [89]	GC-MS			X	NR		Whole DBS (~50.0 µLⁱ)	DDE (p,p’-) only analyte detected
Kato 2009^g (NDBS, 2007) [88]	LC-MS	X	X	X	LODs: PFHxS: 0.1 ng/mL; PFOS: 0.4 ng/mL; PFOA: 0.2 ng/mL; PFNA: 0.1 ng/mL	X (up to 61 days at 37 °C)	Whole DBS (~50.0 µLⁱ)	100% DF for PFOS and PFOA at concentrations >0.4 ng/mL; 98% DF for PFNA; 70% DF for PFHxS
Ma 2013^g (NDBS, 2008–2011) [86]	LC-MS	X (27.0% RSD for PFOS)	X (28.2% RSD for PFOS)	X	LODs: PFOS: 0.03 ng/mL; PFOA: 0.05 ng/mL; BPA: 0.3 ng/mL		16-mm discs (~79.4 µLⁱ)	100% DF for PFOS and PFOA; 86% DF for BPA Analyte recoveries low for BPA (~39%) Background contamination: 0.01, 0.1, and 0.6 ng/mL for PFOS, PFOA, and BPA, respectively
Batterman and Chernyak 2014 (DBS) [87]	GC-MS	X		R = 0.80 (venous)	LODs: PCB-138, -153, -180: 10, 10, 17 ng/L, respectively; BDE-47, -99: 30 and 30 ng/L, respectively; p,p’-DDE (pesticide): 90 ng/L	X (1 m at room T with exception of PBDE; 1 y for refrigeration)	15-mm discs (~69.8 µLⁱ)	Background contamination: PCB-180: 35 ng/L; PCB-105: 17 ng/L; PCB-194: 24 ng/L; BDE-47: 35 ng/L; not detectable for other POPs
Poothong 2019 (DBS)^g [91]	LC-MS	X	X	R values: PFHXs: 0.90 PFOS: 0.97 PFOA: 0.95 PFNA: 0.90 PFDA: 0.72 PFUnDA: 0.94 PFOSA: 0.84 (p < 0.0001)	LODs: 0.0075–0.3 ng/mL		10 discs × 3-mm (~2.8 µLⁱ)	85% DF for PFHxS, PFOS, PFNA, PFDA, PFUnDA, PFOSA
Fipronil and metabolites
Raju 2016 (DBS) [101]	LC-MS	X	X	X	LODs: Fipronil, Fipronil Sulfone: 0.01 ng/mL; Fipronil desulfinyl: 0.03 ng/mL	X (30 days at room T)	Disc size covering ~10 µL blood
Benzene
Funk 2008 (NDBS, DBS) [100]	GS-MS	X		R = 0.732	NR		NR	Benzene-oxide adducts
Parabens
Mulla 2015 (NDBS) [102]	LC-MS	X	X	X	LOD: 10 ng/mL		Whole DBS (8-mm disc) (~19.8 µLⁱ)	55% and 25% DF for MPB and PPB
Acrylamide
Starlin 2020 (DBS) [103]	LC-MS	X	X	X	LOQ: 2.5 µg/mL	X (1 day at −4 °C and room T)	Whole DBS (~50.0 µLⁱ)	Internal standard: propranolol

These studies were primarily related to the development and validation of DBS methods for measuring environmental exposure biomarkers.
AAS atomic absorption spectroscopy, BDE brominated diphenyl ethers, CV coefficient of variation, DF (%) detection frequency, EDCs endocrine-disrupting chemicals, EDXRF energy-dispersive X-ray fluorescence, GC-CVAFS gas chromatography-cold vapor atomic fluorescence spectrometry, m months, MPB methyl-parabens, NR not reported, PPB propyl-parabens, T temperature, TXRF total reflection X-ray fluorescence, POPs persistent organic pollutants, y years.
^aPrecision: coefficient of variation, %CV, of a single sample with multiple determinations measured in a single assay.
^bReliability: %CV for a single sample with multiple determinations measured on different days.
^cAccuracy: analyte recovery rates, comparison to matched gold standard (venous blood) or plasma, umbilical cord blood, or infant blood lead levels. Correlation coefficients were reported only when studies regressed matched DBS values to one of the above comparator values.
^dSensitivity: limit of detection (LOD), limit of quantification (LOQ), method detection limits (MDLs).
^eStability: across different storage conditions, such as temperature, humidity, and time.
^fSample requirements: reported in diameter punches and estimated blood volumes (see SI for calculations).
^gIncluded an application of the DBS method to measure concentrations of analyte(s) in a population-based study, usually with a relatively small sample size.
^hBased on Supplementary Table 1 for Rodríguez-Saldaña et al. [46] and Santa-Rios et al. [47].
ⁱBlood volume estimates for each disc size were calculated using whole blood applied to a blank filter paper spot. Our careful estimation demonstrates 50 µL of whole blood application corresponds to filling a half-inch (12.7-mm) spot. More details on methods for these calculations are provided in the SI.
^jEstimated plasma values can be converted to whole blood equivalents by multiplying by 0.58 (i.e., 1 – the average hematocrit for men and women, 42%) (ref. Mayo Clinic. Hematocrit test. 2021. https://www.mayoclinic.org/tests-procedures/hematocrit/about/pac-20384728), assuming no biomarker partitioning across the red blood cell membrane (see discussion in Ladror et al. [34]).
^kAmong a sample from a population with no known exposure(s).
^lInstrument detection limits, which differ from method or assay detection limits.
X indicated whether the study reported values for this key quality-control assay parameter. For accuracy, the highest-ranking mode of accuracy is listed with correlation coefficients and p values if available (for example, matched venous whole blood is considered superior to using reference materials for analyte recovery rates).

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Table 1 DBS method development studies.

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