Use of dried blood spots (DBS) – A simple and field-friendly method of collecting blood samples for the measurement of vitamin A status

Abstract In many parts of the world, diagnosis and monitoring of CML patients is limited by the availability and cost of molecular testing. In countries without molecular diagnostic capabilities, blood samples can be shipped to central labs, but this is both hampered by sample degradation, and the high costs of shipping. This study explores the method of directly spotting peripheral blood onto a paper template (dried blood spots), with subsequent shipping, RNA extraction, and BCR-ABL testing. Methods: Blood Spots and Shipment. We received dried blood spots from Australia and African countries by mail or courier, and blood from CML patients from our institution were also used for these experiments. 200μL of blood (PB) was pipetted onto Whatman 503 Protein Saver Cards (PSC; Sigma-Aldrich), where each card contains four 50μL spots. Cards were allowed to dry for at least 24 hours at room temperature. For mailing, PSCs were sealed into glassine envelopes with a packet of desiccant, and then placed inside a mailing envelope following DOT and IATA regulation for shipping non-regulated, exempt human specimens. RNA Extraction from Cards and %BCR-ABL determination. Blood spots were incubated with proteinase K followed by RNA isolation using RNeasy Mini Kits (Qiagen). Extracted RNA was quantified using a NanoDrop spectrometer (Thermo Scientific). %BCR-ABL was determined using the automated Cepheid GeneXpert platform or manual two-step quantitative RT-PCR on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results: Bench top time course: To test for effects of long transit times on RNA quality, we performed a time course study of cards at room temperature (RT) with 5 samples. For each sample, multiple cards were spotted with PB. The cards were then allowed to sit at RT for predetermined amounts of time, up to 42 days, before extracting RNA. We measured RNA integrity for one of the specimens (CML # 5) and found rapid degradation with the RIN number going from 8.7 for the fresh blood to 2.8 after 28 days on the card. However the amplification for both BCR-ABL and ABL differed less than one cycle between the fresh blood and the last time point by manual qRT-PCR (BCR-ABL Ct = 23.63 for fresh blood and 24.06 for day 28 PSC; ABL Ct = 26.69 for fresh blood and 27.64 for day 28 PSC). Figure 1 shows the results of the time course experiment for the 5 samples as a plot of ΔCt versus time in days. BCR-ABL qRT-PCR concordance studies: We compared the %BCR-ABL results obtained in fresh specimen at the institution sending the sample with the %BCR-ABL results we obtained from RNA extracted from PSC using the Cepheid GeneXpert. Paired evaluable results were available for 9 samples with a median WBC = 9.8 x 109/L (range: 3.37x109/L – 85.5x109/L). Samples were 8 to 49 days old at the time of extraction. The amount of RNA input into the GeneXpert reaction ranged from 38.75ng to 1μg. The %BCR-ABL detected ranged from 0.37% to 27% (see Table). The mean absolute difference between fresh blood and PSC BCR-ABL% is 2%; the relative mean percent change for BCR-ABL, using fresh blood as the reference is 13.1% (S.D., 31.2), P = 0.24. Conclusions and future directions: Dried blood spots are relatively inexpensive method to transport blood that preserves enough RNA stability to allow highly accurate BCR-ABL detection, when compared to results performed on an identical platform using fresh peripheral blood samples. Further studies are undergoing to accurately determine the sensitivity of this method and the feasibility of using regular mail for inexpensive transport of specimens. Table 1IDWBC (1000/μL)Sample Age at Spotting (Days)Sample Age at RNA extraction (Days)RNA ng/μlVolume GeneXPert (μL)Paper %BCR-ABL (IS)GeneXpertFresh Blood % BCR-ABL (IS) GeneXpertI1na010426349naI224.101311092745I38009181544naI47.4285102.4*3.1I55.50495241.92I63.61307.4225912I785.5130102102439I812.212912.415128.8I9na1281.5250.37*0.71I103.370273257.85.7I1115.912731102325I126.612714.415na2.3 *%BCR-ABL was manually calculated due to late ABL Cts because of low starting material. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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An Assessment of Dried Blood-Spot Technology for Identifying Iron Deficiency

Blood ◽

10.1182/blood.v92.5.1807 ◽

1998 ◽

Vol 92 (5) ◽

pp. 1807-1813 ◽

Cited By ~ 38

Author(s):

James D. Cook ◽

Carol H. Flowers ◽

Barry S. Skikne

Keyword(s):

Iron Deficiency ◽

Transferrin Receptor ◽

Venous Blood ◽

Dried Blood Spots ◽

Epidemiologic Studies ◽

Capillary Blood ◽

Deficiency Anemia ◽

Blood Samples ◽

Blood Spots ◽

American Society

Abstract The present study was undertaken to assess the feasibility of using ferritin and transferrin receptor measurements on dried capillary blood spots to identify iron deficiency (ID) in public health surveys. Measurements on serum and blood spots prepared from venous blood were performed in 71 healthy subjects, 41 of whom were iron-replete and 30 who had ID, either without (n = 20) or with (n = 10) anemia. Parallel measurements were performed on hemolyzed whole blood and washed hemolyzed red blood cells to assess the erythrocyte contribution of ferritin and transferrin receptor to dried blood samples. The concentration of ferritin in dried blood samples was threefold higher than serum assays due to the release of ferritin from hemolyzed erythrocytes, which diminished the usefulness of ferritin measurements for detecting ID. On the other hand, there was negligible erythrocyte contribution to the measurement of transferrin receptor in dried blood spots. The most sensitive parameter in dried blood spots was the ratio of receptor/ferritin, which was suitable for identifying iron-deficiency anemia (IDA), but less reliable than serum assays for detecting milder ID without anemia. We conclude that tandem measurements of serum ferritin and transferrin receptor in dried blood spots can be used to facilitate the identification of IDA in epidemiologic studies. © 1998 by The American Society of Hematology.

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Differences of Phenylalanine Concentrations in Dried Blood Spots and in Plasma: Erythrocytes as a Neglected Component for This Observation

Metabolites ◽

10.3390/metabo11100680 ◽

2021 ◽

Vol 11 (10) ◽

pp. 680

Author(s):

Dorothea Haas ◽

Jana Hauke ◽

Kathrin V. Schwarz ◽

Lucia Consalvi ◽

Friedrich K. Trefz ◽

...

Keyword(s):

Mass Spectrometry ◽

Venous Blood ◽

Plasma Concentrations ◽

Dried Blood Spots ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Tandem Mass ◽

Blood Samples ◽

Blood Spots ◽

Edta Plasma

Monitoring phenylalanine (Phe) concentrations is critical for the management of phenylketonuria (PKU). This can be done in dried blood spots (DBS) or in EDTA plasma derived from capillary or venous blood. Different techniques are used to measure Phe, the most common being flow-injection analysis tandem mass spectrometry (FIA-MS-MS) and ion exchange chromatography (IEC). Significant differences have been reported between Phe concentrations in various sample types measured by different techniques, the cause of which is not yet understood. We measured Phe concentrations in 240 venous blood samples from 199 patients with hyperphenylalaninemia in dried blood spots, EDTA plasma and erythrocytes by FIA-MS-MS and IEC. Phe concentrations were significantly lower in erythrocytes than in plasma leading to about 19% lower Phe DBS concentrations compared with plasma independent from the method used for quantification. As most therapy recommendations for PKU patients are based on plasma concentrations reliable conversion of DBS into plasma concentrations is necessary. Variances of Phe concentrations in plasma and DBS are not linear but increases with higher concentrations indicating heteroscedasticity. We therefore suggest the slope of the 75th percentile from quantile regression as a correction factor.

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THE STATUS OF BETA-CAROTENE AND VITAMIN A IN QUEBEC DAIRY HERDS: FACTORS AFFECTING THEIR STATUS IN COWS AND THEIR EFFECTS ON REPRODUCTIVE PERFORMANCE

Canadian Journal of Animal Science ◽

10.4141/cjas87-080 ◽

1987 ◽

Vol 67 (3) ◽

pp. 775-788 ◽

Cited By ~ 9

Author(s):

ELLIOT BLOCK ◽

BERTRAND FARMER

Keyword(s):

Vitamin A ◽

Reproductive Performance ◽

Dairy Herd ◽

Beta Carotene ◽

Limiting Factor ◽

Related Factors ◽

Blood Samples ◽

Factors Affecting ◽

Vitamin A Status ◽

The Status

Feed samples and a milk sample from the bulk tank were obtained from 100 randomly selected Holstein herds on the official option of the Dairy Herd Analysis Service, and jugular blood samples were obtained from five cows within each herd. Feed, milk and blood samples were analyzed for beta-carotene and grains, supplements, milk and blood for vitamin A. Haylage appeared to be the most reliable source of beta-carotene in comparison to dry hay and corn silage. The acid detergent fiber content of hay and/or haylage was shown to be negatively correlated to beta-carotene concentration. The concentrations of beta-carotene in the diet, plasma and milk were shown to be positively correlated; the same trends were observed for vitamin A. The concentrations of beta-carotene and vitamin A in plasma were shown to be negatively related. Factors affecting the concentrations of beta-carotene and vitamin A in plasma were examined. The beta-carotene and vitamin A status of a herd were shown to be both negatively correlated to calving interval and number of breedings per conception. Beta-carotene may only be expected to improve the fertility of a dairy herd when it is the most limiting factor. Additionally, the vitamin A status of a herd should always be considered when evaluating its beta-carotene status. Key words: Beta-carotene, vitamin A, reproductive performance, forage quality, cows (dairy)

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Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots Versus Venous Blood Samples

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.14-0614 ◽

2015 ◽

Vol 92 (3) ◽

pp. 573-577 ◽

Cited By ~ 17

Author(s):

Lydie Canier ◽

Nguon Chea ◽

Malen Ken ◽

Philippe Bosman ◽

Nimol Khim ◽

...

Keyword(s):

Venous Blood ◽

Dried Blood Spots ◽

Pcr Detection ◽

Blood Samples ◽

Low Transmission ◽

Blood Spots

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Application of Retinol-Binding Protein Enzyme Immunoassay to Dried Blood Spots to Assess Vitamin A Deficiency in a Population-Based Survey: The Uganda Demographic and Health Survey 2006

Food and Nutrition Bulletin ◽

10.1177/156482650802900406 ◽

2008 ◽

Vol 29 (4) ◽

pp. 297-305 ◽

Cited By ~ 14

Author(s):

Rhona Kezabu Baingana ◽

Denis Kasozi Matovu ◽

Dean Garrett

Keyword(s):

Vitamin A ◽

Enzyme Immunoassay ◽

Health Survey ◽

Vitamin A Deficiency ◽

Population Level ◽

Dried Blood Spots ◽

Retinol Binding Protein ◽

Serum Retinol ◽

Blood Spots ◽

Resource Poor

Background Vitamin A deficiency is a public health problem in most developing countries. The technological challenges associated with the measurement of serum retinol have limited the epidemiologic assessment of vitamin A deficiency. The combination of retinol-binding protein (RBP) enzyme immunoassay and dried blood spots offers a rapid, inexpensive, and reliable tool for the population-level assessment of vitamin A deficiency in resource-poor settings. Objective To report on the application of RBP enzyme immunoassay and dried blood spots to assess serum retinol concentrations as an indicator of vitamin A status in the Uganda Demographic and Health Survey 2006. Methods A total of 5,642 capillary blood spot samples were collected by fingerprick onto filter paper cards from women (15–49 years) and children (6–59 months) in a representative probability sample of 9,864 households between May and October 2006. The cards were dried, packed individually with desiccant, and kept at 4°C in a portable refrigerator in the field and at –20°C in the laboratory. Prior to analysis, the RBP enzyme immunoassay was optimized with the use of matched serum and dried blood spots. Results The correlation between RBP values determined by matching serum and dried blood spots was excellent ( r = 0.79, p < .00001). The prevalence of vitamin A deficiency in women (RBP < 1.24 μmol/L) and children (RBP < 0.825 μmol/L) was 19.4% and 20.4%, respectively. Conclusions The combination of RBP enzyme immunoassay and dried blood spots is a simple, reliable, and cost-effective tool for the estimation of vitamin A deficiency in population-level surveys in resource-poor settings.

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Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood spots in the quantification of miltefosine in dried blood samples

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2016.12.012 ◽

2017 ◽

Vol 135 ◽

pp. 160-166 ◽

Cited By ~ 27

Author(s):

A.E. Kip ◽

K.C. Kiers ◽

H. Rosing ◽

J.H.M. Schellens ◽

J.H. Beijnen ◽

...

Keyword(s):

Dried Blood Spots ◽

Blood Samples ◽

Blood Spots ◽

Volumetric Absorptive Microsampling

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Comparison of Inflammation Adjustment Strategies for Retinol-binding Protein in Acute and Convalescent Serum of Patients with Acute Febrile Illness in Guayaquil, Ecuador (P10-095-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz034.p10-095-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Susannah Colt ◽

Washington Cárdenas ◽

Mildred Zambrano Leal ◽

Joyce Andrade Velasquez ◽

David Erickson ◽

...

Keyword(s):

Vitamin A ◽

Regression Model ◽

Binding Protein ◽

Febrile Illness ◽

Recovery Phase ◽

Retinol Binding Protein ◽

Acute Febrile Illness ◽

Blood Samples ◽

Vitamin A Status ◽

Adjustment Strategies

Abstract Objectives 1. Assess serum retinol-binding protein (RBP) among clinical participants at two time points: during the onset of acute febrile illness and again during the convalescent recovery phase. 2. Apply and compare several inflammation adjustment strategies for determining vitamin A status. Methods Patients presenting with acute febrile illness were enrolled. Participants were asked to return for a follow-up convalescent visit 2–4 weeks later. Comprehensive demographic and clinical information along with blood samples were collected at both visits. RBP, C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP) were measured by ELISA, (R&D Systems, Inc., Minneapolis, MN), and pro-inflammatory cytokines were measured by magnetic bead multiplex assay, (EMD Millipore Corporation, Billerica, MA). Continuous RBP was adjusted using the Thurnham correction factor (TCF), the BRINDA regression correction (BRC), and the CRP-only adjustment factor (CAF). An interleukin 6 (IL-6) regression model was also applied to adjust RBP based on IL-6 concentrations for participants with CRP ≥5 mg/L. Adjusted RBP concentrations were compared between paired visits using non-parametric paired Wilcoxon signed-rank tests, and p values <0.05 were considered significant. Results Blood samples were collected from 18 participants during acute and convalescent phase visits. The unadjusted median (quartile 1-quartile 3) RBP concentration (µmol/L) during the acute visit, 1.02 (0.90–1.21), was lower than the convalescent visit, 1.57 (1.26–1.75), P = 0.0004. After applying the TCF, BRC, and CAF, median RBP between visits differed significantly, similar to the unadjusted RBP. RBP adjusted using the IL-6 regression model was not significantly different between paired visits 1 and 2, 1.40 (1.11–1.76) and 1.62 (1.26–1.97) respectively, P = 0.12. Conclusions Circulating unadjusted RBP concentrations were significantly lower in participants during the acute illness phase compared to the convalescent recovery phase. Under the assumption that underlying vitamin A status remains constant during a 2–4 week period, adjusting RBP for IL-6 in participants with CRP ≥5 mg/L was the only strategy in which adjusted RBP did not differ significantly between paired visits. Funding Sources NIH National Institute of Biomedical Imaging and Bioengineering, NIH Office of Research on Women's Health.

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A Novel LC-MS/MS Method for Improving Long-Term Stability of Retinol in Dried Blood Spots and Quantification of Its Levels

Current Developments in Nutrition ◽

10.1093/cdn/nzaa067_084 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1857-1857

Author(s):

Min Zhang ◽

Fang Wang ◽

Shuyi Zhang ◽

Jian Yang ◽

Xiaodai Cui ◽

...

Keyword(s):

Vitamin A ◽

Research And Development ◽

Development Program ◽

Dried Blood Spots ◽

Calibration Model ◽

Reference Ranges ◽

Blood Spots ◽

Relative Standard ◽

Plasma Retinol ◽

Retinol Concentration

Abstract Objectives To establish an accurate and reliable method for stabilizing vitamin A (retinol) in Dried blood spots (DBS), quantifying and comparing DBS retinol concentrations with their equivalent plasma retinol levels. Methods Antioxidants pretreated on paper combined with vacuum treatment were used to increase retinol stability on DBS. A surrogate matrix of whole blood prepared using mixture of human erythrocytes and 2% BSA in PBS was firstly used in DBS retinol determination based on the fact that retinol is excluded from erythrocytes. Results DBS retinol was stable during 120 min of air drying and 30 days of room-temperature storage. The method was linear in the concentration range of 0.04–300 μg/mL. Accuracy was calibrated using two National Institute of Standards and Technology (NIST) calibrants generated serum at concentrations of 0.1962 and 0.3948 g/mL, relative errors (RE% values) of 0.07% and 4.95% were found, respectively. Both the between-run (n = 5) and within-run (n = 6) precision (relative standard deviations, RSD%) were below 8.42%. The spiked recoveries at 3 concentrations ranged from 86.48% to 98.13%. A reliable calibration model was first developed to convert DBS retinol concentration to the equivalent plasma retinol concentration. Conclusions The validated method can be applied to the nutritional assessment of vitamin A, using the established calibration model, DBS retinol can compare with clinical reference ranges and with studies using serum or plasma samples. Funding Sources The National Key Research and Development Program of China (2018YFC1002503); The National Natural Science Foundation of China (Grant No. 81,400,848, 81,701,441); The CAMS Initiative for Innovative Medicine (2016-I2M-1–008); The Beijing municipal program of medical research (Grant No. 2016–04); The National Key Research and Development Program of China (No. 2016YFC1306204).

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