AbstractEuropean manufacturers of plasma products and German blood transfusion services were the first to introduce nucleic acid amplification testing (NAT) of blood products in the mid-1990s. Their primary goal was to increase the safety of blood by closing as far as possible the diagnostic window, which exists after the onset of viral infection until the appearance of the first detectable antibodies. Sample preparation, transport and storage are crucial steps in a quality-controlled PCR. Sensitivity and contamination rates highly depend on the sample preparation and storage techniques. Anticoagulants must be selected carefully because some may inhibit the PCR. Dilution of samples by pooling needs to be considered and should be compensated for by subsequent virus enrichment procedures, e.g. centrifugation. The whole process of sample preparation, pooling and virus enrichment must be validated and quality control measures must be implemented. Reagents for the extraction of viral nucleic acids should not pose any risk to the laboratory staff. Nevertheless, the reagents should be highly efficient in liberating viral nucleic acids at high yield and purity for the following amplification reactions. At this critical stage, quality control measures should guarantee an efficient extraction process and contain potential sources of contaminations. Several methods are available for the amplification of nucleic acids. PCR is the most common, especially in in-house assays. The amplification of nucleic acids should be performed as far as possible in a closed system, which may be guaranteed best by real-time PCR approaches. Reaction tubes need never be opened during the amplification because detection can be performed through the closed tube. Amplicons that could contaminate the following PCR reactions will not be released. It is of great importance to blood transfusion services to guarantee that negative results un-equivocally indicate virus negative blood donations. Therefore, internal control sequences should be implemented in each individual PCR reaction in order to monitor that the individual PCR has worked correctly. Besides internal control sequences, external negative and positive controls should be implemented in each PCR run to demonstrate false positive reactions as well as to monitor pre-PCR processes like virus enrichment and extraction. The whole process needs to be validated according to the criteria set in national guidelines or by national authorities. External quality assessment programs are highly recommended.
Filter paper-based spin column for low throughput nucleic acid purification
