Dried Serum Spot Research Articles

Advancing Global Health Surveillance of Mycotoxin Exposures using Minimally Invasive Sampling Techniques: A State-of-the-Science Review.

Mycotoxins are a heterogeneous group of toxins produced by fungi that can grow in staple crops (e.g., maize, cereals), resulting in health risks due to widespread exposure from human consumption and inhalation. Dried blood spot (DBS), dried serum spot (DSS), and volumetric tip microsampling (VTS) assays were developed and validated for several important mycotoxins. This review summarizes studies that have developed these assays to monitor mycotoxin exposures in human biological samples and highlights future directions to facilitate minimally invasive sampling techniques as global public health tools. A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted. Key assay performance metrics were extracted to provide a critical review of the available methods. This search identified 11 published reports related to measuring mycotoxins (ochratoxins, aflatoxins, and fumonisins) using DBS/DSS and VTS assays. Multimycotoxin assays adapted for DBS/DSS and VTS have undergone sufficient laboratory validation for applications in large-scale population health and human biomonitoring studies. Future work should expand the number of mycotoxins that can be measured in multimycotoxin assays, continue to improve multimycotoxin assay sensitivities of several biomarkers with low detection rates, and validate multimycotoxin assays across diverse populations with varying exposure levels. Validated low-cost and ultrasensitive minimally invasive sampling methods should be deployed in human biomonitoring and public health surveillance studies to guide policy interventions to reduce inequities in global mycotoxin exposures.

Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC-MS/MS from Dried Serum Spots.

One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.

Dried serum spots on pre-punched filter paper discs are ready-to-use storage and shipping devices for blood-borne antigens and antibodies

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the “Submerge and Dry” protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.

Mass Spectrometry of Transferrin and Apolipoprotein CIII from Dried Blood Spots for Congenital Disorders of Glycosylation.

Dried blood spot (DBS) is the standard specimen for the newborn screening of inborn errors of metabolism (IEM) by tandem mass spectrometry. Availability of DBS for the mass spectrometric analysis of the diagnostic marker proteins, transferrin (Tf) and apolipoprotein CIII (apoCIII), of congenital disorders of glycosylation (CDG) was examined. Recovery of Tf from DBS was only slightly reduced compared with fresh serum. Although oxidation of the core polypeptides was observed, glycans of Tf and apoCIII were unaffected by storage of DBS in the ambient environment for at least 1 month. The combination of DBS and the triple quadrupole mass spectrometer used for IEM screening was sufficient to characterize the aberrant glycoprofiles of Tf and apoCIII in CDG. DBS or dried serum spot on filter paper can reduce the cost of sample transportation and potentially promote mass spectrometric screening of CDG.

A pilot study for colorectal carcinoma screening by instant metabolomic profiles using conductive polymer spray ionization mass spectrometry

BackgroundThe rapid and accurate discrimination of colorectal carcinoma (CRC) and polyps at the molecular level enables early intervention of CRC, which can greatly improve the 5-year survival rate of patients. Here we reported the potential of conductive polymer spray ionization mass spectrometry (CPSI-MS) in successfully screening CRC according to the serum metabolic profile. MethodsTrace intravenous blood (50 μL) was collected from 60 colorectal carcinoma (CRC) and 60 polyp patients, respectively. After centrifugation, serum (2 μL) was loaded onto the tip of conductive polymer to form a dried serum spot. When the 5 μL methanol-water (1:1, v/v) extraction solvent was spiked onto the dried serum spot followed with +4.5 kV high voltage applied on the polymer tip, the extracted components will be ionized and carried into the MS system for direct metabolic profiling. FindingsThere were 51 metabolites discovered to be significantly changed in CRC serum compared to polyps. Combining these metabolites as the characteristic panel, the ideal diagnostic performance was achieved by Lasso regression model with the accuracy of 88.3%. InterpretationThis pilot study demonstrated the potential of CPSI-MS as a cost-effective tool in large-scale CRC screening in the high-risk population.

Profiling of intact blood proteins by matrix-assisted laser desorption/ionization mass spectrometry without the need for freezing - Dried serum spots as future clinical tools for patient screening.

To open up new ways for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based patient screening, blood serum is the most preferred specimen because of its richness in patho-physiological information and due to ease of collection. To overcome deleterious freeze/thaw cycles and to reduce high costs for shipping and storage, we sought to develop a procedure which enables MALDI-MS protein profiling of blood serum proteins without the need for serum freezing. Blood sera from patients/donors were divided into portions which after pre-incubation were fast frozen. Thawed aliquots were deposited on filter paper discs and air-dried at room temperature. Intact serum proteins were eluted with acid-labile detergent-containing solutions and were desalted by employing a reversed-phase bead system. Purified protein solutions were screened by MALDI-MS using standardized instrument settings. MALDI mass spectra from protein solutions which were eluted from filter paper discs and desalted showed on average 25 strong ion signals (mass range m/z 6000 to 10,000) from intact serum proteins (apolipoproteins, complement proteins, transthyretin and hemoglobin) and from proteolytic processing products. Semi-quantitative analysis of three ion pairs: m/z 6433 and 6631, m/z 8205 and 8916, as well as m/z 9275 and 9422, indicated that the mass spectra from either pre-incubated fast-frozen serum or pre-incubated dried serum spot eluted serum contained the same information on protein composition. A workflow that avoids the conventional cold-chain and yet enables the investigation of intact serum proteins and/or serum proteolysis products by MALDI-MS profiling was developed. The presented protocol tremendously broadens the clinical application of MALDI-MS and simultaneously allows a reduction in the costs for storage and shipping of serum samples. This will pave the way for clinical screening of patients also in areas with limited access to health care systems, and/or specialized laboratories.

A Pilot Study for Colorectal Carcinoma Screening by Instant Metabolomic Profiles Using Conductive Polymer Spray Ionization Mass Spectrometry

Background: The rapid and accurate differentiation of colorectal carcinoma (CRC) and polyps at the molecular level enables early intervention of CRC, which can greatly improve the 5-year survival rate of patients. Here we reported the diagnostic potential of conductive polymer spray ionization mass spectrometry (CPSI-MS) in successfully differentiating CRC by the serum metabolic profile. Methods: Trace intravenous blood (50 μL) was collected from 60 colorectal carcinoma (CRC) and 60 polyp patients, respectively. After centrifugation, serum (2 μL) was loaded onto the tip of conductive polymer to form a dried serum spot. When the 3 μL methanol-water (1:1, v/v) extraction solvent was spiked onto the dried serum spot followed with + 4.5 kV high voltage applied on the self-conductive tip, the extracted components will be ionized and carried into the MS system for direct metabolic profiling. Findings: There were 51 metabolites discovered to be significantly changed in the metabolic profiles between the CRC and polyps. Combining these significantly metabolites as the characteristic panel, the diagnostic accuracy was over 90% with high sensitivity and specificity using Lasso regression. Interpretation: This pilot study demonstrated CPSI-MS can be a cost-effective tool in discriminating CRC from polyp which has the potential in large scale CRC screening at the high-risk population. Funding: This work was supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2017zx09101001002002) and the Natural Science Foundation of China (No. 81303216). Declaration of Interest: None to declare. Ethical Approval: Patients had signed consent form for the blood collection which was approved by the medical ethics committee of the Sixth Medical Center of PLA General Hospital.

Utility of dried serum spots for triglyceride estimation

In a developing country like India, screening the population of remote areas for Non Communicable Diseases is difficult because of less availability of infrastructure and technical staff. It is ideal to carry out the analysis in a good quality central laboratory, but the transportation of samples in cold chain is of concern. Dried serum spot technology has various advantages compared to conventional serum analysis, it helps in convenient shipment of samples at low cost without the requirement of cold chain. Problems with leaking and spilling of liquid serum samples will not happen. Stability of analytes will be improved. Serum spot analysis is therefore a convenient method for screening the population presenting with health risks. This study was undertaken to look for stability of triglycerides in serum dried on filter paper at room temperature for different time intervals.100 Samples of Patients coming to the laboratory of KLE Hospital Belgaum were analysed for Triglyceride levels. The remaining serum was spotted onto 3M Whattman filter paper and left at room temperature for an hour for drying. Subsequently dried serum spots were analysed for Triglyceride levels on 0, 7, 14, 21, 28 and 35 days in a Erba semi autoanalyser using commercially available kit.The comparable values obtained from dried serum spots makes it a reliable and convenient method for screening modifiable risk factors like Triglyceride levels. Keywords: Dried serum spots, 3M Whatmann filter paper, Screening, Triglyceride estimation

Serum lipidomics study of Ding-Zhi-Xiao-Wan effect on Alzheimer's disease using online liquid extraction surface analysis coupled to direct infusion mass spectrometry

Disorder of lipid metabolism is closely related to the incidence of Alzheimer's disease (AD). Regulation of lipid disorder is one of the important mechanisms of drug therapy for AD. In this study, serum lipidomics study based on an online liquid extraction surface analysis coupled to direct infusion mass spectrometry (Online-LESA-direct infusion MS) was established and applied to assess the efficacy of Ding-Zhi-Xiao-Wan (DZXW) for treating AD in rats by regulating lipid metabolism disorders. Dried serum spot (DSS) samples of 10 health control (HG), 10 AD, 10 DZXW, and 10 donepezil rats were directly detected by Online-LESA-direct infusion MS without prior treatments. And the acquired data was analyzed by multivariate statistical analyses such as PCA and PLS-DA to identify potential lipid biomarkers in serum. By this method, 15 compounds were screened and identified, belonging to phosphatidylcholines (PCs), sphingomyelins (SMs) and lysophosphatides (LPCs), which were considered as potential lipid biomarkers with AD mechanisms. Among them, the levels of PCs and SMs were markedly declined, and the levels of LPCs were increased in the AD group. Moreover, compared with administrating donepezil, the observed PCs and SMs were more up-regulation to normal levels with the treatment of DZXW in AD rats, which indicated that DZXW can play a better role in the treatment of AD by regulating lipid metabolism. And the results of the study also showed that the Online-LESA-direct infusion MS on DSS analytical strategy has a potential to become a high-throughput analysis method for large-scale clinical diagnostics analysis.

Development of dried serum spot sampling techniques for the assessment of trace elements in serum samples by LA-ICP-MS

A novel approach for serum analysis by dried matrix spot (DMS) technique is proposed. The methodology consists of sampling filter paper discs (2.7 mm in diameter) containing the large amount of serum retained after a single spotting. Several oxidizers (sodium chlorate, sodium azide, acetic acid, formic acid, 1-butyl-3-methylimidazoliumm chloride/bromide) were tested (oxidizers premixed with the sample before spotting, and papers previously soaked in concentrated additive/oxidizer solutions). Direct multi-element determination (Al, Be, Ca, Cu, Fe, K, Li, Mg, Mn, Mo, Na, P, Rb, Se, V, and Zn) in dried serum spots at very low levels was therefore assessed by laser ablation (LA) coupled with inductively coupled plasma - mass spectrometry (ICP-MS). Laser ablation was performed using a focused Nd: YAG laser beam in lineal scan mode (wavelength 213 nm, laser fluency 2.2 J cm−2, repetition rate 20 Hz, laser spot diameter 90 µm, depth 0 µm, scanning speed 12 µm s−1). Matrix-matched calibration mode and 13C as internal standard (for signal intensities normalization) was used throughout the work. Limits of quantification were found to be from 21 µg L−1 to 221 mg L−1. Repeatability (seven ablations of the same dried serum spot) and reproducibility (two ablations of seven dried serum spot from the same material) offered RSDs below 12% for all analytes, which seems satisfactory for clinical purposes. The method was validated by analyzing several certified reference materials (Seronorm™ level I and II trace elements in serum), and it was applied to several DMS from serum samples from healthy adults.

Evaluation of the Analytical and Clinical Concordance of 25-Hydroxyvitamin D Levels in Dried Blood Spots, Dried Serum Spots, and Serum as Potential Biorepository Specimens

The promising perspective of storing dried blood spot (DBS) for evaluating 25-hydroxyvitamin D (25OHD) levels is increasingly being realized. While strong correlations have been demonstrated between 25OHD levels measured in DBS and in systemic serum samples in earlier works, the clinical concordance of the assay results has not been evaluated. Moreover, the utility of dried serum spot (DSS), a highly suitable matrix for sample archiving, has not been investigated in this respect. 25-hydroxycholecalciferol and 25-hydroxyergocalciferol levels were established selectively in DBS (n = 73) and DSS (n = 67) specimens obtained from deidentified whole blood and serum using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In addition, total 25OHD levels were determined in the serum samples using the LIAISON 25OH Total Vitamin D Assay (LIA, n = 73). The analytical and clinical performance of the three approaches was compared pairwise. Deming regression, Bland-Altman analysis, and concordance correlation coefficients consistently demonstrated the lack of analytical equivalence among the three result sets. The overall percentage agreement of the clinical classifications (hypovitaminosis or euvitaminosis) was moderate (67.1%-83.6%). The delivery of positive cases was decreasing significantly in the order LIA>DSS>DBS (p < 0.05). The approaches tested did not deliver equivalent outputs either in an analytical or a clinical context. Therefore, specific reference ranges must be established for each matrix to avoid false clinical evaluation. 25OHD can be quantified when assay results are scaled by a factor of 1.60-1.67. Considering the convenience and efficiency of the storage and processing of DSS, along with the difficulties of quantifying 25OHD in real-life DBS samples accurately, DSS is proposed as an alternative for the long-term archiving of specimens.

National molecular surveillance of recently acquired HIV infections in Germany, 2013 to 2014.

To enable an up-to-date molecular analysis of human immunodeficiency virus (HIV) genotypes circulating in Germany we have established a surveillance system based on recently acquired HIV infections. New HIV infections are reported to the Robert Koch Institute as a statutory duty for anonymous notification. In 2013 and 2014, a dried serum spot (DSS) sample was received from 6,371 newly diagnosed HIV-cases; their analysis suggested that 1,797 samples originated from a recent infection. Of these, 809 were successfully genotyped in the pol region to identify transmitted drug resistance (TDR) mutations and to determine the HIV-1 subtype. Total TDR was 10.8%, comprising 4.3% with mono-resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 2.6% to non-NRTIs, 3.0% to protease inhibitors and 0.6% and 0.2%, respectively, with dual- and triple-class resistances. HIV-1 subtype B was most prevalent with 77.0%. Non-B infections were identified more often in men and women with heterosexual transmission compared with intravenous drug users or men who have sex with men (79% and 76%, 33%, 12%; all p < 0.05). Non-B subtypes were also more frequently found in patients originating from countries other than Germany (46% vs 14%; p < 0.05) and in patients infected outside of Germany (63% vs 14%; p < 0.05).

Use of dried blood spot as an alternative method to estimate serum triglyceride in field studies

Introduction: Cardiovascular diseases are showing an increasing trend in both developing as well as developed countries. Raised triglycerides is considered as an important risk factor of cardio vascular diseases. Collection, storage and transportation of the blood samples from the field becomes a major limiting factor in areas lacking modern laboratory facilities. In such situations, dry blood or serum spots can become an alternative for the estimation of triglyceride. The objectives of our study was to evaluate the use of dry blood or dry serum spot to estimate triglyceride levels as an alternative method to estimate serum triglyceride and also to study the effect of storage time and storage temperature on the triglyceride content in the dry blood and serum spots. Methodology: Blood samples were collected from 100 study participants. Seven spots each of blood and serum from these samples were made on Whatman paper 3. After drying, one dried spot each from blood and serum was analysed on same day. Three of blood and three of serum dried spots were kept at room temperature and remaining at a temperature of 2-8° in separate re-sealable bags which were analysed on days 7, 15 and 30. Triglyceride was measured by Glycerol-3-phosphate oxidase-Peroxidase method. For estimating triglycerides in the eluate, 100 microlitre of the extract from the test-tube was added to 1 ml of the commercially available enzymatic reagent kit and incubated at 37°C for 15 min, and the absorbance was measured at 500 nm using a spectrophotometer. The data collected was analysed by Pearson’s correlation using SPSSv15. Results: Study showed a good correlation between the serum triglyceride values and the dry serum or blood spot triglyceride values. Dried serum spot triglyceride was found to be stable for about 15 days at room temperature and 7 days at 2-8°C with good correlation with serum value. The triglyceride concentration in the dry blood spots remained stable for 7 days at both room temperature and at 2-8°C. Conclusion: Dry blood/serum spots can be used as an alternative to serum for the estimation of triglyceride level which can be used to estimate various biochemical parameters at community level in remote areas which lack modern laboratory facilities. Key words: Serum triglycerides, Dry blood spot, Dry serum spot, cardiovascular diseases, surveillance

Performances of fourth generation HIV antigen/antibody assays on filter paper for detection of early HIV infections

BackgroundPoint-of-care testing and diagnosis of HIV acute infections play important roles in preventing transmission, but HIV rapid diagnosis tests have poor capacity to detect early infections. Filter paper can be used for capillary blood collection and HIV testing using 4th generation immunoassays. ObjectivesAntigen/antibody combined immunoassays were evaluated for their capacity to identify early HIV infections using filter paper in comparison with rapid test. Study designThirty nine serum samples collected from HIV seroconverters were spotted onto filter paper and tested by the Roche Elecsys® HIV Combi PT test and the DiaSorin Liaison XL Murex HIV Ab/Ag assay. ResultsFourth generation immunoassays identified 34 out of 39 HIV early infections using dried serum spot, whereas the Determine™ HIV-1/2 rapid test detected 24 out of 39 HIV positive serum (87.2% vs 61.5% respectively, p=0.009). p24 antigen was detected by the Liaison XL in 19 dried serum samples (48.7%). In the group characterized by a negative western blot, 7 out of 8 (87.5%) and 6 out of 8 (75.0%) samples were found positive for HIV using the Elecsys and the Liaison XL, respectively. None of these eight samples classified in this group of early acute infections were found positive by the rapid test. ConclusionFourth generation Ag/Ab immunoassays performed on dried serum spot had good performance for HIV testing during the early phases of HIV infection. This method may be useful to detect HIV early infections in hard-to-reach populations and individuals living in remote areas before rapid tests become positive.

Supercritical fluid extraction as a preparation method for mass spectrometry of dried blood spots.

The potential of supercritical fluid extraction (SFE) as a preparation method for mass spectrometry of dried blood spots (DBS) was examined. SFE is generally used for the extraction of hydrophobic compounds, but hydrophilic metabolites such as amino acids, amines, and nucleic-acid-related metabolites could be extracted by adding a low level of methanol as a modifier. Under the optimized conditions, over 200 metabolites were detected from a dried serum spot, of which over 160 metabolites could be analyzed stably (RSD <20%). These results show that SFE is an effective extraction method of metabolites with a wide range of polarity in DBS.

Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77%-92% within replicates and the majority of these repeated proteins (70%) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

RNA amplification of the HIV‐1 Pol and env regions on dried serum and plasma spots

Dried sample spots have molecular applications, but few data are available on conditions of HIV-1 RNA amplification. To determine the impact of (i) the sample type (plasma or serum), (ii) various storage periods, (iii) transfer at ambient temperature of the dried spots via postal mail, and (iv) two different methods of elution-extraction, to amplify partial pol and env genes with a view to both phylogenic and resistance analyses. Fourteen samples (dried plasma spot and dried serum spot) from seven patients were stored at 20-25 degrees C for 2, 5 and 7 days. Two extraction buffers were tested on these samples, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) on pol and env genes. Sixteen spots from eight other patients were sent by postal mail at ambient temperature between two laboratories, and were analysed at both sites. We observed no influence of the 2-7 day storage period, whatever the sample type and viral load, but the choice of the extraction procedure is a critical step. Analysis of the mailed spots resulted in a good agreement between the two laboratories. This study shows that amplification of HIV-1 RNA on spots is possible in various conditions by different operators and using appropriate reagents. This finding could be particularly useful for large-scale molecular and resistance epidemiological studies in resource-rich and resource-limited countries alike.

A dried serum spot assay for vitamin B12

A newly developed dried serum spot (DSS) vitamin B(12) assay compares well with a conventional reference serum vitamin B(12) microbiological assay (r=0.97, n=161) and demonstrates adequate within (CV% <6) and between assay (CV% <10) reproducibility. The consistency of long-term vitamin B(12) assay performance was supported and validated using a reconstituted International Reference Serum (IRR 81/563) stored at -70 degrees C as both whole serum aliquots and as DSS. The inclusion of such reference sera also allows accurate comparisons to be made with data from other laboratories and studies. The DSS matrix displays excellent characteristics of pre-analytical serum vitamin B(12) stability at ambient temperatures with less than 5% loss of activity occurring at 4 degrees C, 20 degrees C and 37 degrees C after 7 days of storage in the dark. These qualities underline the suitability of the DSS matrix for epidemiological screens of serum vitamin B(12) levels by obviating the need for costly refrigeration and specialised handling of serum samples and allowing economic transportation using the basic postal service.

Ferritin concentrations in dried serum spots prepared by standard compared with simplified approaches: a validation study in Guatemala City

Spot ferritin assay on dried serum spot (DSS) samples provides reliable and accurate assessment. Standard DSS preparations, however, involve precise serum aliquots and require some skill and training of field personnel. We evaluated the validity of the spot ferritin assay on DSS samples prepared by simplified approaches and standard technique in Guatemala City. Venous blood (5 mL) was obtained from 104 subjects aged 24 +/- 15 y (x +/- SD) and transferred into nonheparin-containing (2 plain and 2 self-sealing) capillary blood collection tubes. Three DSS samples were prepared: A (standard, 20 microL serum), B (blot, approximately 30-35 mm serum column), and C (dispenser, 20 microL serum pushed directly from self-sealing capillary tubes with a dispenser). Spots were air-dried and placed in hermetic plastic bags with a desiccant. Two weeks later, entire spots for DSS A and C samples and a circle in the center for DSS B samples were analyzed. DSS ferritin A, B, and C correlated strongly with traditional ferritin (r = 0.71-0.88, P < 0.001). The geometric mean (-1 SD and +1 SD) values for the DSS A, B, and C and traditional ferritin methods were 27.5 (12.6, 60.2), 32.4 (13.5, 77.6), 27.5 (11.7, 64.6), and 30.2 (13.8, 66.1) microg/L, respectively, and did not differ significantly. The difference in ferritin values by various DSS approaches compared with the traditional approach was small (<4 microg/L; P > 0.05). Simplified and standard DSS methods provide accurate iron-status assessment in population studies. The simplified DSS approaches for serum ferritin measurement need to be evaluated further in populations in whom iron deficiency is prevalent.

HIV-1 resistance genotyping on dried serum spots

To assess the feasibility of HIV-1 group M resistance genotyping on dried serum spots, by testing samples from previously untreated patients, patients on treatment, and patients having stopped treatment, representing a wide genetic diversity panel. Serum samples from 62 HIV-1-infected Caucasian and African patients, with viral load values from 715 copies/ml to more than 750,000 copies/ml, were deposited on filter paper. After elution and RNA extraction, nested RT-PCR was used to amplify the protease and RT regions of the pol gene. Resistance sequencing was performed on all the protease and RT amplicons. The sequences obtained for resistance genotyping were used for subtyping by phylogenetic analysis. Amplification was successful in the protease region in 53/62 cases (85.5%) and in the RT region in 51/62 cases (82.3%). All samples with viral loads of at least 5 Log (17 of 62) were successfully amplified in both the RT and protease regions. Of the 29 samples with viral loads between 4 Log and 5 Log, 28 (97%) were amplified in the RT region and 25 (86%) in the protease region. The detected mutations were in keeping with the treatment status. Marked natural polymorphism was observed in the protease region, but no major consequences were deduced in terms of resistance. The results showed a broad diversity of the panel, including subtype B (n = 36) and non B or recombinant forms (n = 20). Our results show the feasibility of this dried serum spot method for monitoring resistance to antiretroviral drugs and the molecular epidemiology of HIV diversity. The simplicity of sample preparation, storage and transport potentially makes this an importance tool for individual and epidemiological monitoring throughout the world.