Pancreas collagen digestion during islet of Langerhans isolation-a prospective study.

The success of pancreas islet isolation largely depends on donor characteristics, including extracellular matrix composition of which collagen is the main element. We hypothesized that isolation yields are proportional to collagen digestion percentage, and aimed to determine a threshold that predicts isolation success. The amount of pancreas collagen (I-V) was determined using colorimetry prior to and after the digestion process in 52 human islet isolations. Collagen I-V and VI were also assessed histologically. We identified a collagen digestion threshold of ≥ 60% as an independent factor beyond which an islet preparation has a ninefold increased odds of yielding ≥ 250 000 islet equivalents (IEQ) (P = 0.009) and a sixfold increased odds of being transplanted (P = 0.015). Preparations with ≥ 60% collagen digestion (n = 35) yielded 283 017 ± 164 214 IEQ versus 180 142 ± 85 397 in the < 60% collagen digestion group (n = 17) (P = 0.016); respectively 62.9% versus 29.4% of those were transplanted (P = 0.024). Common donor characteristics, initial collagen content, enzyme blend, and digestion times were not associated with collagen digestion percentage variations. Donor age positively correlated with the amount of collagen VI (P = 0.013). There was no difference in islet graft survival between high and low digestion groups. We determined that a 60% pancreas collagen digestion is the threshold beyond which an islet isolation is likely to be successful and transplanted.

HIV drug resistance surveillance using pooled pyrosequencing.

BACKGROUND: Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals. METHODOLOGY/PRINCIPAL FINDINGS: The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies >/=5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods. CONCLUSIONS/SIGNIFICANCE: We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required.