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Can dried blood spots be used to accurately measure vitamin D metabolites?

Comparison of HIV-1 RNA level estimated with plasma and DBS samples: A pilot study from India (South)

This study aimed at exploring the feasibility of using dried blood spots (DBS) to detect HIV drug resistance genotyping in China by comparing the results of drug resistance from DBS, plasma and whole blood samples. Blood samples were collected from 39 AIDS patients from Anhui (10), Yunnan (13), Hunan (6) and Xinjiang (10) provinces and autonomous regions. The HIV strains that infected these patients covered all the major HIV-1 subtypes prevailing in China (B, CRF01_AE, CRF07_BC). HIV drug resistance genotyping assay was performed on DBS as well as on the whole blood and plasma samples from the same patients simultaneously by using an in-house nest RT-PCR method. Drug resistance levels were determined based on Stanford University HIV drug resistance database, and the results from these three types of samples were compared. The percentages of successful amplification of protease and reverse transcriptase regions in the pol gene were 95% (37/39) from DBS, 92% (36/39) from whole blood and 100% (39/39) from plasma samples. The sequences from the three types of samples showed more than 99% identity.86% (31/36) of the DBS samples had the same set of drug resistance mutations as those which were detected from plasma samples. The differences probably resulted from mixed bases. There was no major difference in detecting HIV drug resistance genotyping among DBS, plasma and whole blood samples. Therefore, DBS is useful for detection of HIV drug resistance genotyping and is particularly valuable in developing countries like China, especially in remote rural regions.

To the Editor: Invasive pneumococcal disease (IPD) causes many cases of severe disease and death among children <5 years of age, mostly in developing countries (1,2). Before conjugate vaccines can be introduced in developing countries, information about disease epidemiology is urgently needed. The lack of laboratories equipped to perform pneumococcal serotyping leads to the need to send isolates to reference laboratories. Good sample preservation is necessary to prevent samples from arriving at the laboratory in poor condition. We evaluated the usefulness of multiplex real-time PCR from strains and blood samples kept at room temperature on dried blood spot (DBS) filter paper for detecting and serotyping Streptococcus pneumoniae. DBS screening is a reliable method that requires only a small amount of blood; it is used for the diagnosis of several human diseases (3,4). To validate the technique, we selected 15 pneumococcus clinical isolates representing 15 serotypes (1, 5, 19A, 19F, 14, 3, 7F, 4, 6A, 6B, 8, 9N, 18C, 23A, 23F) obtained during 2009 from patients at Hospital Sant Joan de Deu, in Barcelona. These isolates, used as controls, had been serotyped by quellung reaction at the Instituto de Salud Carlos III, Majadahonda-Madrid, Spain. These strains were cultured overnight at 35°C in 5% carbon dioxide on Columbia agar plates with 5% sheep blood (bioMerieux SA, Marcy l’Etoile, France). A suspension of each strain was adjusted to match a 0.5 McFarland standard (equivalent to 108 colony-forming units (CFU)/mL). Stock solutions of pneumococcus culture for each previously identified serotype were injected into blood previously extracted from 2 healthy volunteers. Serial dilutions of 100,000 CFU/mL to 1,000 CFU/mL (1,000 to 10 CFU equivalents/PCR) were performed. A total of 100 µL of blood was applied to DBS filter paper, and another 100 µL was used for DNA extraction from fresh blood. All DBS samples were air dried for 1 week. The procedure was also performed on negative control blood samples. DNA was extracted from DBS and fresh blood samples by using the NucliSense easyMAG automated extraction platform (bioMerieux, Boxtel, the Netherlands) according to the manufacturer’s instructions. DNA detection of the pneumolysin (ply) gene by real-time PCR was performed according to a published assay (5). In addition, we performed a multiplex real-time PCR for molecular serotype detection of serotypes 1, 3, 5, 4, 6A, 6B, 7FA, 8, 9VANL, 14, 15BC, 18CB, 19A, 19FBC, 23F, 23A and the conserved capsular gene wzg as described by Tarrago et al. (6). DNA extracts were amplified with the Applied Biosystems 7300 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Negative results were defined as those with cycle threshold >40. To evaluate the reliability obtained with this in vivo approach, we performed identification and serotyping of S. pneumoniae in 25 DBS samples from 25 children at Saint John of God Hospital in Mabesseneh-Lunsar, Sierra Leone. This hospital does not perform blood cultures. IPD was confirmed when DNA of a pneumolysin (ply) gene and an additional capsular gene of S. pneumoniae were detected by multiplex real-time PCR of DBS samples. Detection of ply, wzg, and the specific gene for molecular serotype showed that both fresh blood and DBS samples yielded correctly positive results from the 10-fold serial dilutions analyzed (Table). With respect to the 25 (11 female and 14 male) patients from Sierra Leone who had suspected IPD, the median age was 25.71 months (range 15 days to 96 months); all had a diagnosis of fever without apparent source, and 16 also had malaria. Of these 25 children, DBS samples from 15 (60%) yielded a positive result for the ply and wzg genes, so they were considered confirmed episodes of IPD. A serotype included in 13-valent conjugate vaccine was detected in 6 (40%) of 15 positive samples: serotypes 3, 7FA, 19A, 6A, 6B, and 9VNL (1 sample each). In the remaining 9 samples, the results for ply gene and wzg gene were positive, but none of the 24 tested serotypes was detected. Table Sensitivity of real-time PCR for detecting Streptococcus pneumoniae ply or wzg genes or a specific gene for molecular serotype from fresh or dried blood spot samples* This preliminary study enabled us to demonstrate that DBS screening is a reliable and easy method for diagnosing IPD and also for epidemiologic surveillance of the more frequent serotypes. The main limitation of our study is the small number of DBS samples sent from Saint John of God Hospital in Sierra Leone. In conclusion, the DBS technique enables reproducible transport of samples for identification and serotyping of S. pneumoniae by multiplex PCR. The use of DBS on filter paper is an attractive alternative method for storing samples at room temperature and easily transporting them. Additional studies, including evaluation of the relative sensitivity of this method compared to direct culture, are necessary.

Dried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)–confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance.

Aim: Dried blood spot (DBS) is a sampling approach that offers several advantages over plasma and whole blood (WB) sampling, but several factors, such as hematocrit and temperature, can adversely affect quantitation. Methodology & results: In an open-label, three-way crossover study in healthy subjects, we explored the correlation between DBS, WB and plasma samples, and between DBS samples from finger-prick and venipuncture blood for measuring edoxaban and its metabolite M-4 using LC-MS/MS. The methods were validated comprehensively. The incurred sample reanalysis experiments demonstrated quantitation reproducibility in all three matrices. Overall, there was a good correlation (near perfect concordance for edoxaban) among plasma, WB and DBS measurements. M-4 concentrations in DBS and WB were lower than in plasma. Conclusion: These results indicate using DBS may be used as an alternative methodology to measure edoxaban pharmacokinetics.

HIV-1 viral Load (VL) testing is recommended for the monitoring of antiretroviral treatment. Dried Blood Spots (DBS) are an effective sample type in resource limited settings, where safe phlebotomy and reliable shipping are hard to guarantee. In HIV high burden countries, high throughput assays can improve access to testing services. The Hologic Aptima HIV-1 Quant Dx Assay (Aptima Assay) is a high throughput assay that runs on the CE-IVD approved Panther platform. The objectives of this study were to assess the performance characteristics of Aptima for VL monitoring using plasma and venous DBS specimens and to determine the stability of HIV-1 RNA in DBS. This was a cross-sectional study of 2227 HIV infected adults visiting health facilities in Nairobi and Busia, Kenya. Each provided a venous blood sample; plasma was prepared from 1312 samples while paired DBS samples and plasma were prepared from the remaining 915 samples. The agreement between the Aptima assay and the Abbott RealTime HIV-1 Assay (Abbott RT) was analysed by comparing the HIV-1 VL in both assays at the medical decision point of 1000 copies/mL. To assess stability of HIV-1 RNA in DBS, VL in DBS spotted on day 0 were compared with that from the same DBS card after 21 days of storage at room temperature. Overall, 436 plasma samples had quantifiable results in both Aptima and Abbott RT. The agreement between the two assays at 1000 copies/mL was 97.48% with a Pearson's correlation coefficient (r) of 0.9589 and gave a mean bias of 0.33 log copies/mL on Bland-Altman analysis. For fresh DBS, the agreement in both assays was 94.64% at 1000 copies/mL, with an r of 0.8692 and a mean bias of 0.35 log copies/mL. The overall agreement between DBS tested in Aptima on day 0 versus day 21 was 95.71%, with a mean bias of -0.154. The Aptima HIV-1 Quant Dx assay is an accurate test for VL monitoring of HIV-1 using DBS and plasma sample types in Kenya.

Development of a dried blood spot (DBS) method for golimumab will facilitate sample collection in a study setting and will give a more complete insight in the total drug exposure (area under the curve, AUC). We established a DBS method and assessed its robustness, user-friendliness and clinical usefulness in 10 patients with ulcerative colitis during golimumab induction and maintenance regimens. DBS was obtained through spotting of golimumab spiked in whole citrated blood to a filter paper. Several extraction conditions were evaluated and the selected extraction condition analytically validated. In a clinical setting, DBS and serum samples were taken simultaneously through intensive sampling regimens and a conversion factor was determined. Golimumab concentrations were measured using an in-house-developed ELISA and a CE-marked ELISA kit. User-friendliness was evaluated using a questionnaire. Mucosal healing was evaluated at week 14. A total of 79 matched pairs of serum and DBS sample golimumab concentrations revealed an overall conversion factor of 3.9. DBS golimumab concentrations after conversion correlated strongly with serum golimumab concentrations (ICC = 0.984). During induction, no linear correlation was found between golimumab trough concentration (TC) and AUC (R2= 0.29). Multiple peaks emerged during drug absorption. Patients who achieved mucosal healing appeared to have less fluctuating TC and a constant AUC over time. Nine out of 10 patients reported DBS sampling as user-friendly. The GOUDA study showed that DBS sampling is a robust and patient-friendly alternative to venous blood collection. DBS sampling may provide better insights into golimumab absorption and exposure. ( ClinicalTrials.gov NCT02910375).

IntroductionHIV-1 viral Load (VL) testing is recommended for the monitoring of antiretroviral treatment. Dried Blood Spots (DBS) are an effective sample type in resource limited settings, where safe phlebotomy and reliable shipping are hard to guarantee. In HIV high burden countries, high throughput assays can improve access to testing services. The Hologic Aptima HIV-1 Quant Dx Assay (Aptima Assay) is a high throughput assay that runs on the CE-IVD approved Panther platform. The objectives of this study were to assess the performance characteristics of Aptima for VL monitoring using plasma and venous DBS specimens and to determine the stability of HIV-1 RNA in DBS.Materials and methodsThis was a cross-sectional study of 2227 HIV infected adults visiting health facilities in Nairobi and Busia, Kenya. Each provided a venous blood sample; plasma was prepared from 1312 samples while paired DBS samples and plasma were prepared from the remaining 915 samples. The agreement between the Aptima assay and the Abbott RealTime HIV-1 Assay (Abbott RT) was analysed by comparing the HIV-1 VL in both assays at the medical decision point of 1000 copies/mL. To assess stability of HIV-1 RNA in DBS, VL in DBS spotted on day 0 were compared with that from the same DBS card after 21 days of storage at room temperature.ResultsOverall, 436 plasma samples had quantifiable results in both Aptima and Abbott RT. The agreement between the two assays at 1000 copies/mL was 97.48% with a Pearson’s correlation coefficient (r) of 0.9589 and gave a mean bias of 0.33 log copies/mL on Bland-Altman analysis. For fresh DBS, the agreement in both assays was 94.64% at 1000 copies/mL, with an r of 0.8692 and a mean bias of 0.35 log copies/mL. The overall agreement between DBS tested in Aptima on day 0 versus day 21 was 95.71%, with a mean bias of -0.154.ConclusionThe Aptima HIV-1 Quant Dx assay is an accurate test for VL monitoring of HIV-1 using DBS and plasma sample types in Kenya.

Antibody tests have been extensively used to monitor the prevalence and changes in the HIV-1 epidemic in the general population. In the UK, unlinked anonymous testing of neonatal dried blood spot (DBS) samples has provided unbiased estimates of the prevalence of maternal infection in women with live births [1]. Quality assurance and quality control procedures are necessary to ensure consistent tests resulting in unbiased estimates of prevalence in population-based surveys. As part of the quality assurance programme for neonatal screening laboratories, Centers for Disease Control and Prevention (CDC, Atlanta, USA) has produced blinded (negative and positive) samples which have been sent to laboratories worldwide for 25 years [2]. In addition, CDC has supplied HIV-1-positive quality control DBS specimens produced to a set standard to measure assay performance. As part of the national unlinked anonymous surveillance programme covering the former Thames regions of England [3], we have received HIV-1 quality assurance samples every quarter and quality control samples every 6 months for over 20 years, and have a 100% record of correctly identifying HIV-1 antibody-positive DBS CDC quality assurance samples. In total, we have tested over 3.5 million neonatal DBS samples for HIV-1 maternal antibody. DBS samples are eluted overnight at 4°C in phosphate-buffered saline-Tween 80 and sodium azide (0.0005%). These eluted samples are then tested by making a 1 : 5 dilution in Treponema pallidum particle agglutination (TPPA) buffer into a V-well test plate and adding a 1 : 10 dilution of Serodia HIV-1 gelatin particles (Fujirebio Inc., Tokyo, Japan). After settlement for 4 h at room temperature, the plates are read over a light box, a positive result being shown by a characteristic agglutination pattern, and a negative result by a button of settled particles. Positive samples are titrated to an end point. Although DBS samples containing HIV-1 antibodies have been reported to be stable over time, even in unsuitable climatic and storage conditions [4,5], to our knowledge, no study has re-analysed them after a period of more than 5 years [6]. The stability of DBS samples over longer periods could prove useful for retrospective testing of antibodies for other conditions using long-term stored DBS samples [7]. To analyse antibody stability, we selected the oldest available DBS samples and eluates stored in our laboratory and re-tested them for HIV-1 antibodies. Firstly, we examined four DBS CDC quality control high positive control samples produced in 1991 and stored continuously in a freezer at −20°C in gas-impermeable bags with a dessicant. When tested in February 2014, all of them had the same titres as those measured in 1991. Secondly, we retested 12 DBS samples produced between October 1997 and October 1999 by the CDC quality assurance programme. The samples had been stored at 4°C in a laboratory fridge for at least 14 years and were originally tested when they reached our laboratory. They were re-tested in November 2013 and all 12 samples were HIV-positive; nine had the same titre, two increased by two dilutions, and one decreased by two dilutions. Finally, we re-tested 98 HIV-1-positive neonatal DBS eluates available from the unlinked anonymous anti-HIV-1 survey in pregnant women in North Thames which were originally tested in the first three quarters of 2003 and stored for over 9 years at −20°C. As we were interested in antibody degradation, we selected the DBS eluates with the lowest titres among the oldest specimens stored in our laboratory. When re-tested in March 2014, all remained positive for HIV-1 antibody. In 67 samples, titres remained unchanged, there was one dilution decrease in 1 sample, and an increase between 1 and 5 dilutions in 30 samples; the latter changes may be due to variations in testing conditions. In conclusion, these findings suggest that, over long periods, HIV-1 antibody concentration does not deteriorate in DBS samples stored at 4 and −20°C, or in eluates stored at −20°C. This implies that archived DBS samples could be reliably used in retrospective studies on HIV and potentially other infections. Acknowledgements We are grateful to the Laboratory Quality Assurance and Standardization Programs, Division of Laboratory Sciences, CDC for providing the quality assurance and quality control specimens used in this letter. D.W. curated the samples and carried out the testing. All authors contributed to the final draft and approved it. We are grateful to the Health Protection Agency (now Public Health England), and to the Welton Foundation for additional funding. The UCL Institute of Child Health receives a proportion of funding from the Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme. Conflicts of interest There are no conflicts of interest.

Evaluation of: Limaye AP, Santo Hayes TK, Huang ML, et al. Quantitation of cytomegalovirus DNA load in dried blood spots correlates well with plasma viral load. J. Clin. Microbiol. 51, 2360–2364 (2013).Cytomegalovirus (CMV) represents the major infectious cause of birth defects, as well as an important pathogen for immune-compromised individuals. Several studies described the use of dried blood spots (DBS) for the detection of CMV DNA for late diagnosis of congenital CMV infection in cases of strong clinical suspicion. In the article under evaluation, Limaye et al. perform for the first time the quantification of CMV in pairs of finger-stick DBS and plasma samples collected from transplant patients. The work concluded that finger-stick DBS could be an alternative sample type for quantification of CMV load that correlates well with plasma levels. Prospective trials to evaluate the use of DBS for monitoring CMV load in transplant recipients will be required.

Hepatitis B Virus (HBV) infections represent a major public health problem because of the ability of HBV to cause a chronic carrier state. Even though chronic carriers remain largely asymptomatic, a large number of these individuals subsequently develop cirrhosis and primary hepatocellular carcinomas. Dried Blood Spot (DBS) samples are a simple and inexpensive sampling method, especially useful for blood collection in resource poor settings with limited access to diagnostic facilities. The main advantage of DBS samples over routine blood samples is that only a small quantity of blood, is required. They are easy to obtain, stable and can be transported to a reference laboratory at minimal cost. This study was to evaluate the feasibility of DBS samples for direct amplification of HBV DNA bypassing nucleic acid extraction. Results obtained from DBS samples were compared from those from plasma by routine molecular technique and also with those from whole blood. On the whole results for DBS, whole blood and plasma samples for HBV-DNA semi quantitative PCR monitoring, demonstrated very good agreement. This study also represents the first report in Egypt to evaluate the use of DBS to direct amplification of HBV-DNA and concluded that the use of DBS for direct amplification of HBV DNA without nucleic acid extraction was reliable, specific, sensitive, cheap and appropriate method to monitor the HBV infected patients.

ObjectiveUse of dried blood spots (DBS) for detection of hepatitis B virus (HBV) markers of infection has the potential to facilitate diagnosis of HBV infection especially in resource-limited countries. The aim of this study was to evaluate the feasibility of DBS for detection of various markers of HBV infections.ResultsFifty-four DBS samples were engineered from well-characterized plasma samples. All DBS samples were tested for HBsAg, total anti-HBc and HBV DNA, 20 of 54 samples were also tested for HBeAg using commercially available assays. HBsAg was detected in 24 of 25 (96%), HBV DNA in 22 of 25 (88%), total anti-HBc in all 9 (100%), and HBeAg in all 7 (100%) DBS samples. The average difference in HBV DNA levels between DBS eluates and corresponding plasma samples was 2.7 log10 IU/mL. Fifteen DBS eluates positive for HBV DNA were sequenced and all of them belonged to HBV genotype A. Thirteen samples which were negative for all HBV markers showed HBeAg false positivity. Therefore, DBS is a reliable sample matrix for detection of HBsAg, total anti-HBc and HBV DNA, but not HBeAg. Further feasibility studies of DBS for diagnostic purposes and epidemiologic studies are warranted.

Anlotinib is a multi-target tyrosine kinase inhibitor, approved in China for treating several cancer types. Dose individualization based on therapeutic drug monitoring (TDM) is a useful tool to reduce toxicity. However, it is not convenient for patients to go to hospital for routine TDM via venous blood sampling at a certain time. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determination of anlotinib in human plasma and dried blood spot (DBS), characterized by simple sample preparation, high sensitivity, and short analysis time. The assay was validated in the concentration range of 0.2-200 ng/mL in plasma and 5-1000 ng/mL in DBS. This method was applied to monitor anlotinib exposure levels in patients with advanced biliary tract cancer (BTC) and non-small cell lung cancer (NSCLC). The trough plasma concentration (Ctrough ) of anlotinib was highly variable among BTC patients with coefficients of variation (CV) of 47.5%. DBS and venous blood samples were also collected from NSCLC patients to determine whether DBS sampling is a viable alternative sampling approach. Pearson correlation coefficient (R) between DBS and plasma concentration was 0.985. Bland-Altman plot demonstrated that the difference between estimated and measured plasma concentration was -2.9%. And 87% of sample pairs had a maximal deviation of ±20%. Anlotinib exhibits a high inter-individual variability in plasma exposure, and DBS sampling could be a promising tool for TDM of anlotinib.

Utilisation des prélèvements de sang séché sur papier buvard pour le diagnostic et le suivi thérapeutique des maladies infectieuses