title: High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA.
abstract: BACKGROUND
  Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome.
  
  
  METHODS
  High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene.
  
  
  RESULTS
  High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection.
  
  
  CONCLUSIONS
  High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.
authors.affiliation: Infectious Diseases Unit, Karolinska Institutet, Center for Molecular Medicine, L8:01, Karolinska University Hospital, Solna, SE-171 76 Stockholm, Sweden. klara.lundblom@ki.se
authors.forename: Klara
authors.initials: K
authors.lastname: Lundblom
authors.forename: Alex
authors.initials: A
authors.lastname: Macharia
authors.forename: Marianne
authors.initials: M
authors.lastname: Lebbad
authors.forename: Adan
authors.initials: A
authors.lastname: Mohammed
authors.forename: Anna
authors.initials: A
authors.lastname: Färnert
mesh_terms: D001769:Blood / Q000469:parasitology*; D002675:Child, Preschool; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005615:Freezing; D006447:Hemoglobin H / Q000235:genetics*; D006801:Humans; D007223:Infant; D007630:Kenya; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology; D010961:Plasmodium / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D017085:alpha-Thalassemia / Q000175:diagnosis*
chemical_list: D004247:DNA; D006447:Hemoglobin H