Table 1. Simulations to assess carryover contamination of HIV-1 at multiple steps of testing dried whole blood spots (DBS) by nested PCR for infant diagnosis.

Rates of false positive results when processing blank filter paper following DBS spiked with an average or a high concentration of HIV-1 DNA are shown by potential risk factors for contamination. The sampling procedure was to first punch a disc from a DBS containing HIV-1 followed by six punches of clean filter paper (blank discs 1-6) using the same instrument without any cleaning between punches.

Variations in Processing DBS	# of series tested	Sequence of Blank Disc punched following HIV-1(+) Disc
		#1	#2	#6
DBS containing “high” (500 copies/punch) concentration of HIV-1 DNA
Use of non-corroded hole punchers, and discs transferred on clean surface	40	0 (0%)	0 (0%)	0 (0%)
DBS containing “very high” (5000 copies/punch) concentration of HIV-1 DNA
Optimized protocolb	40	1c (2.5%)	0 (0%)	1c (2.5%)
Discs punched onto shared surface	4	2 (50%)	1 (25%)	0 (0%)
Use of corroded hole punchers	5	1 (20%)	0 (0%)	1c(20%)
No “precautions”a when transferring 1st to 2nd round PCR product	33	1c (3%)	0 (0%)	0 (0%)

^a“Precautions” refers to centrifugation of tubes post PCR prior to opening, and using a tissue to open the tubes, especially important to minimize 1^st round PCR product at top of tubes that can contaminate gloves or splash into adjacent tubes upon opening lids to transfer amplicon to 2^nd round PCR

^bOptimized protocol refers to using sharp punch instrument, individual clean disposable surface to collect disc punched from DBS, and precautions described above

^cRepeat gels of second-round PCR revealed persistent false (+), however when the original first-round PCR product was re-amplified in new 2^nd round PCR, these yielded negative results, suggesting the original false positive result was due to inadvertent transfer (cross contamination) of PCR product between tubes between 1^st- and 2^nd-round PCR