Abstract
BackgroundDried blood spots are a common medium for collecting patient blood prior to testing for malaria by molecular methods. A new shaped filter device for the quick and simple collection of a designated volume of patient blood has been designed and tested against conventional blood spots for accuracy and precision.MethodsShaped filter devices were laser cut from Whatman GB003 paper to absorb a 20 μl blood volume. These devices were used to sample Plasmodium falciparum infected blood and the volume absorbed was measured volumetrically. Conventional blood spots were made by pipetting 20 μl of the same blood onto Whatman 3MM paper. DNA was extracted from both types of dried blood spot using Qiagen DNA blood mini or Chelex extraction for real-time PCR analysis, and PURE extraction for malaria LAMP testing.ResultsThe shaped filter devices collected a mean volume of 21.1 μl of blood, with a coefficient of variance of 8.1%. When used for DNA extraction by Chelex and Qiagen methodologies the mean number of international standard units of P. falciparum DNA recovered per μl of the eluate was 53.1 (95% CI: 49.4 to 56.7) and 32.7 (95% CI: 28.8 to 36.6), respectively for the shaped filter device, and 54.6 (95% CI: 52.1 to 57.1) and 12.0 (95% CI: 9.9 to 14.1), respectively for the 3MM blood spots. Qiagen extraction of 200 μl of whole infected blood yielded 853.6 international standard units of P. falciparum DNA per μl of eluate.ConclusionsA shaped filter device provides a simple way to quickly sample and store a defined volume of blood without the need for any additional measuring devices. Resultant dried blood spots may be employed for DNA extraction using a variety of technologies for nucleic acid amplification without the need for repeated cleaning of scissors or punches to prevent cross contamination of samples and results are comparable to traditional DBS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0558-x) contains supplementary material, which is available to authorized users.
Highlights
Dried blood spots are a common medium for collecting patient blood prior to testing for malaria by molecular methods
The flexibility of laser cutting means that the device could be and cheaply adapted to allow different blood volumes or additional replicate samples to be collected according to a user’s particular requirements. Under these set of controlled conditions, it can be seen that the shaped filter paper provides a quick and simple way to sample and store blood in the form of dried blood spots
The device performed well with Chelex, Qiagen and PURE DNA extraction protocols compared to traditional dried blood spots (DBS) and provides a level of precision and accuracy comparable to a set volume of blood applied to 3MM paper using small volume pipettes
Summary
Dried blood spots are a common medium for collecting patient blood prior to testing for malaria by molecular methods. A number of different matrices have been available for creation of malaria DBS, such as the cotton cellulose 3MM, 903, GB002, GB003, GB004 papers (Whatman, GB) and 226 sample collection devices (Perkin Elmer, USA), the glass fibre Wallac filter mat A (Perkin Elmer, USA) and GF/C (Whatman, GB) papers and the non cellulose BondElut Dried Matrix Spotting paper (Agilent Technologies, USA). Newer matrices such as FTA cards (Whatman, GB), HemaForm (available as a stand alone paper or within the HemaSpot device, Spot On Sciences, USA) and Mitra (Phenomenex, USA) have been developed to facilitate sample stabilization or recovery of biologically active molecules, a great many of the dried blood spots used in malaria programmes continue to employ cotton cellulose papers
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