Measurement of prostate specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a 2–logs–lower limit of quantification than current ultrasensitive third–generation PSA assays. We developed reagents for a bead–based ELISA for use with high–density arrays of femtolitervolume wells. Anti–PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. The robust 2–log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.

• Proof of concept study to evaluate a novel digital single molecule immunoassay (AccuPSA) which detects PSA 1000 fold more sensitive than current PSA methods.
• To determine the ability of the AccuPSA assay to predict 5-year Biochemical recurrence (BCR)-free survival following radical prostatectomy (RP).

• 31 frozen serum specimens obtained from specimen logs maintained at New York University Langone Medical Center and the Johns Hopkins University School of Medicine on men who had undergone RP with a minimum of 5 years PSA follow up for those without evidence of BCR.
• For all cases, pre-operative and pathological information were available and a serum specimen between 3 and 6 months following RP with PSA level of <0.1 ng/mL measured by conventional PSA methods at the time of serum collection.
• Specimens were tested with the AccuPSA method.
• A Cox proportional hazard model and Kaplan Meier analysis was performed to determine whether AccuPSA predicted risk of biochemical recurrence.

• Overall, 11 out of 31 (35.5%) of men developed a BCR.
• Mean AccuPSA nadir levels were significantly different (p<0.001) between the non- recurrent group (2.27 pg/ml) versus recurrent group (46.99 pg/mL).
• Using a multivariate Cox proportional hazards model, AccuPSA nadir level was a significant predictor of BCR-free survival (p<0.01).
• Kaplan Meir analysis up to 5 years showed that 100% of men with AccuPSA nadir values <3 pg/mL did not develop a BCR, whereas 62.5% of men with values >3 pg/mL developed a BCR (p=0.00024).
• The sensitivity, specificity, PPV and NPV of the AccuPSA method was 100%, 75%, 69% and 100%, respectively.

• AccuPSA assay predicts 5-year BCR-free survival following RP.
Identifying a reliable predictor of BCR soon after RP has important implications for frequency of PSA testing, selection of candidates for adjuvant therapy, and reassuring a large subset of men they are not at risk of recurrence.
• Larger studies are needed to validate these findings.