Magnetic bead technology in viral RNA and DNA extraction from plasma minipools.
BACKGROUND
Nucleic acid testing (NAT) of pooled plasma samples from individual blood donations for viral nucleic acids has become widely established. Full automation of such sample processing can overcome many of the problems associated with methods used so far.


METHODS
In this study an automated extraction method for viral nucleic acids (parvovirus [PAV] B19 DNA, hepatitis B virus [HBV] DNA, and hepatitis A virus [HAV] RNA), starting directly from the minipool sample (n = 96, 9.6 mL), was evaluated. A magnetic separation module I (chemagic, Polymer Laboratories) in combination with the chemagic viral DNA and RNA kit special based on the use of magnetic beads was used for this purpose. More than 144 pools spiked with defined concentrations of reference material and an additional 102 pools negative for the analyte were extracted and amplified. The isolated viral nucleic acids were detected by polymerase chain reaction (PCR).


RESULTS
The assays were highly specific and obtained a 95 percent detection limit of 875 IU per mL of pooled single donation for PAV B19, 260 IU per mL for HAV, and 1274 IU per mL for HBV, respectively. The crossing points showed variation coefficients from 1.49 to 2.76 percent. The turnaround time for the whole process was 3 hours. Testing of subpools to determine an infected single donation would be possible with the same general extraction method. A total of 102 unspiked minipools (96 x 100 microL per donation) were analyzed and none tested positive.


CONCLUSIONS
The automated magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donations for viremic donors to further increase the safety of blood products. Minipools as well as subpools can be directly processed.
Pichl
Lutz
L
German Red Cross Blood Transfusion Service West, Central Laboratory, Hagen, Germany. l.pichl@bsdwest.de
Heitmann
Alke
A
Herzog
Petra
P
Oster
Juergen
J
Smets
Helga
H
Schottstedt
Volkmar
V
D001769:Blood / Q000821:virology; D001782:Blood Donors; D004279:DNA, Viral / Q000097:blood*; D005069:Evaluation Studies as Topic; D016174:Hepacivirus / Q000302:isolation & purification; D017957:Hepatitis A Virus, Human / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000302:isolation & purification; D006801:Humans; D008280:Magnetics; D021141:Nucleic Acid Amplification Techniques; D016732:Parvovirus B19, Human / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood*; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013672:Technology; D013997:Time Factors; D019562:Viral Load
D004279:DNA, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic