Evaluation of DNA extracted from blood filter spots and eluates processed for enzyme linked immunosorbent assay (ELISA)

Dried filter blood spots have become a significant blood collection method for screening individuals for clinical purposes. When used for ELISAs, they are normally discarded after the blood has been eluted. However, they may still be useful for extraction of DNA for molecular-based assays. The aim of this work was to determine the integrity of DNA extracted from filter paper spots from which blood has initially been eluted for ELISA with sample dilution buffer (SDB) and phosphate buffered saline (PBS). DNA was extracted from the eluted filter spots, the eluate, and dried blood filter spots (controls) using spin column extraction. The quality and quantity of the extracted DNA was assessed and used for PCR to further evaluate their usefulness in molecular assays. Concentration of DNA obtained was dependent on the buffer used for processing the filter blood blots. Accounting for the DNA concentration obtained from dried blood spots, which were used as controls, DNA extracted from the already eluted blood spots were 32 times higher in PBS than SDB processed filter paper. The ratio was even higher for the eluates, which were 57 times higher in PBS than SDS eluates. SDB eluates had significantly higher average DNA concentration than their eluted filter paper, but their purity ratios were similar. 85% PCR success rate was achieved with the DNA samples. Useful DNA can be extracted from blood spots after it has been eluted with SDB. Although the DNA concentration and purity may be low, the DNA could be useful for rather simple PCR assays. Author Summary Collection of blood onto filter paper has become an accepted method for screening individuals for clinical and public health purposes since the 1960s. This method of blood collection has become increasingly popular due to its ease and convenience in collection and transportation. The use of dried blood spots for clinical evaluations and research has become very significant. For research purposes, DBS when used for ELISAs are discarded after single use. DNA may however be extracted from the used filter blots and used for molecular assays. The concentration of DNA obtained may be low but simple assays like PCR could be done using the DNA extracted from the eluted filter spot.

2 23 usefulness in molecular assays. Concentration of DNA obtained was dependent on the buffer 24 used for processing the filter blood blots. Accounting for the DNA concentration obtained from 25 dried blood spots, which were used as controls, DNA extracted from the already eluted blood 26 spots were 32 times higher in PBS than SDB processed filter paper. The ratio was even higher 27 for the eluates, which were 57 times higher in PBS than SDS eluates. SDB eluates had 28 significantly higher average DNA concentration than their eluted filter paper, but their purity 29 ratios were similar. 85% PCR success rate was achieved with the DNA samples. Useful DNA 30 can be extracted from blood spots after it has been eluted with SDB. Although the DNA 31 concentration and purity may be low, the DNA could be useful for rather simple PCR assays.

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Author Summary 33 Collection of blood onto filter paper has become an accepted method for screening individuals 34 for clinical and public health purposes since the 1960s. This method of blood collection has 35 become increasingly popular due to its ease and convenience in collection and transportation. 36 The use of dried blood spots for clinical evaluations and research has become very significant. 37 For research purposes, DBS when used for ELISAs are discarded after single use. DNA may 38 however be extracted from the used filter blots and used for molecular assays. The concentration 39 of DNA obtained may be low but simple assays like PCR could be done using the DNA 40 extracted from the eluted filter spot.

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The reliability and performance of molecular assays are strongly influenced by the quality and Collection of blood onto filter paper has become an accepted method for screening individuals 48 for clinical purposes. This type of specimen has been used for public health purposes since the 49 1960s [4] and has become increasingly popular due to its ease and convenience in collection and 50 transportation. For example, to obtain blood samples from a baby, a few drops of blood from the 51 baby's heel are made to flow onto and fill a printed circle on a special filter paper. The blood 52 dries under ambient atmospheric conditions, and the filter paper is mailed to a laboratory where a 53 portion of the blood spot is punched out with a paper punch [4]. Biological markers that can be 54 measured from whole blood, serum or plasma can be determined from dried blood spots [5]. This 55 includes DNA, which is important for research or studies in genetics.  the Og4C3 ELISA is performed using serum or plasma, either immediately after the sample has 66 been obtained, or more often, from frozen samples. However, the Og4C3 ELISA also offers an 4 67 alternative application method through the use of dried blood spots (DBS) collected on filter 68 paper; an inexpensive and convenient method that requires less space and less stringent 69 refrigeration for transport and storage [15]. 70 Filter spots used for Og4C3 ELISA assays are normally discarded after the blood has been 71 eluted. However, in the advent of increased use of molecular assays for in-depth understanding 72 of diseases, these eluted filter blots hold more information than what is only required for 73 ELISAs. In some cases where molecular analyses are also required in a study besides ELISA, 74 there may not be enough blood from an individual to have extra dried filter spots for DNA 75 extraction. This study, therefore, aims to determine the integrity (quality and yield) of DNA 76 obtained from filter paper spots from which blood has been eluted and the eluate intended for 77 ELISA. We compared the quality and yield of DNA extracted with that from dried blood filter Research and randomly divided into two groups of 25. Two (2) ears of dried blood spots (DBS) 85 were torn from each disk and an ear placed separately into 1.5 mL microcentrifuge tubes. The 86 DBS were processed for DNA extraction as shown (Fig 1).  The eluate was pipetted into a new 1.5 ml microcentrifuge tube, (leaving behind the eluted filter 96 paper). DNA was extracted from the eluate, eluted filter paper and the dried unprocessed DBS  Ethical approval was obtained form the Ghana Health Service Ethical Review Committee for the 120 samples collected for the main study. All sampled were anonymized before usage.

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Comparison of DNA concentrations between extraction templates 123 The aim of this work was to investigate the possibility of obtaining quality DNA from filter 124 paper blots that have been previously been processed, and/or the eluate. Two different elution 125 buffers (SDB and PBS) were used to obtain the eluate. Samples that were processed with SDB

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The two elution buffers were compared to determine their influence on the resulting DNA 138 concentration. Samples that had been processed with PBS prior to DNA extraction produced 139 higher yields than SDB-treated samples (Fig 3). All SDB-processed samples were however 140 similar to control dried filter blots from the PBS group (Fig 3). Purity was estimated using spectrophotometry as a measure of DNA usefulness in further 148 molecular assays. An A 260 /A 280 ratio between 1.7 and approximately to 2.0 is considered pure.

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None of the groups of samples extracted had an average purity ratio within the expected range 150 (Fig 4). Altogether, 16% (24/150) of extracted DNA samples were estimated to be pure (Fig 4).

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The highest contribution of samples to this overall percentage was obtained from PBS-eluates 152 (36%) and eluted blood spots (52%), and these measured higher in purity than any SDB-153 processed DNA sample in pairwise comparisons (Fig 4). Extractions from all dried blood spots 154 were of low purity (Fig 4). Comparisons between the SDB-processed samples showed no 155 difference in their average purity ratios (p<0.05) (Fig 4). The human 18SrRNA could successfully be amplified in our samples (Fig 5), though DNA were  Obtaining blood samples from human subjects for research studies is expensive. Therefore, it is 174 necessary to ensure that as much information required from samples can be extracted without 175 having to return to the subjects for another blood collection. In the Lymphatic Filariasis 176 Elimination Programme, blood samples are often collected on filter paper for ELISA-based 12 177 assays such as Og4C3, Bm14 and Wb123. Although, the focus is not usually on DNA-based 178 assays, this could later be an important inclusion in studying the molecular biology of parasites 179 or infected humans. In this study, we considered the possibility of extracting useful DNA from 180 filter blood papers that have already been processed for ELISA, and from the eluate which is 181 commonly used for the ELISA assays. The DNA concentration and purity were compared among 182 the starting materials to ascertain which gave better DNA integrity. Our results demonstrated that 183 DNA concentration is dependent on the buffer used for processing the filter blood blots.

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Accounting for the DNA concentration obtained from dried blood spots, which were used as 185 controls, DNA extracted from the already eluted blood spots were 32 times higher in PBS than 186 SDB processed filter paper. The ratio was even higher for the eluates which were 57 times higher 187 in PBS than SDS eluates.

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The stability of double stranded DNA (dsDNA) can be affected by temperature, pH and ionic This study has established that DNA can be extracted from blood spots after it has been eluted 207 with the sample dilution buffer used for ELISA-based assays. Although the DNA concentration 208 (could be improved by reducing the elution buffer added at the end of the extraction process) and 209 purity may be low, the DNA could be useful for simple PCR assays such as parasite DNA 210 detection rather than sequencing which is highly sensitive to DNA concentration and purity.