TABLE 2.

Issues to be considered and sources of possible error in connection with sample preparation and DNA extraction for NAATs

Step	Issue(s) to consider and indicate	Pitfalls and comments
A	Method	State all modifications and the version of a kit protocol; most commercially available kits provide numerous diverging protocols
B	Amt of specimen	Use concn and/or wt; unusual metrics do not allow for evaluating sensitivity and data comparison
C	Elution vol	Missing information does not allow for back calculations and makes interstudy comparison impossible
D	No. and quality of ENCsa	“Water only” ENCs without carrier DNA might miss low amplicon carryover; the same is true for too-small no. of ENCs
E	No., quality, and concn of EPCsb	Too many and/or too highly concentrated EPCs can be a contamination source, as can certain formulations (e.g., amplicon, irrespective of whether cloned or not)
F	Integrity and amt of DNA	DNA might get lost during repeated thawing-freezing cycles or extraction; amplification of genes other than the target does not prove integrity of an intact target region and rather provides an indication of specimen quality and quantity
G	Quality and concn of ICOc if used	To check for a successful DNA extraction and/or inhibition, ICO might be included at the extraction level; primers and probes used to amplify ICO should have attributes similar to those of targets; if the ICO concn is too high, complete or partial inhibition is not ruled out; if the concn is too low, ICO might get lost during extraction and inhibition is falsely assumed
H	Anticontamination strategy	Basic recommendations include separate rooms with laboratory coats, safety cabinet, pipettes plus aerosol-resistant pipette tips, gloves, and racks; wrong airway pressure conditions favor contamination; all reagents used should be subdivided into small aliquotsd

^aENC, extraction-negative control.

^bEPC, extraction-positive control.

^cICO, internal control.

^dA good and still up-to-date review on NAAT laboratory design and workflow can be found in reference 24.