Using Ultrasensitive Digital Immunoassays for PSA Analysis

Prostatespecific antigen (PSA) measurement after radical prostatectomy (RP) has become a common practice for tracking the recurrence of prostate cancer. Most assay techniques cannot usually detect the levels of plasma PSA after surgery, and it is largely accepted that over time, undetectable postsurgical PSA shows a good prognosis.1

So far, evaluation of the success of surgical and secondary treatment has depended on tracking the increase in PSA using assays that are incapable of measuring PSA at very low concentrations. As long as PSA stays undetectable, the patient assumes that there is no biochemical indication of cancer recurrence. Based on the assay used, the period of undetectable PSA can be short or on the order of years.

If the assay is less sensitive, the assurance offered is longer, although PSA could be increased but simply undetected. The reaction of physicians to the re-emergence of increasing PSA following RP has relied on several factors, such as clinicopathological factors, the time to biochemical recurrence (BCR), and patient life expectancy.2 BCR is usually defined as an established PSA concentration of 0.2 ng/mL (200 pg/mL) and occurs in nearly 40% of patients following RP. In the end, one-third of these patients develop metastatic disease.3

Significantly, though clinical data progressively signifies that early adjuvant and salvage radiation therapies following surgery have greatly enhanced patient outcomes,4 present-day assays do not have the potential to measure PSA at concentrations that could help stratify patients and thereby allow physicians to recognize patients who would gain from early adjuvant treatment.

Tracking of postsurgical PSA with more sensitive assays was at first investigated in the 1990s with the advent of the first ultrasensitive immunoassays, which could quantify the PSA levels down to 10 pg/mL. Several reports defined the prognostic significance of serial ultrasensitive PSA measurements, revealing that an ultrasensitive technique could detect PSA increases well before a first‐generation assay could, thus potentially offering years of early warning.5

In recent times, more sensitive assays with a detection limit of 1 pg/mL have been used to investigate the prognostic importance of the lowest postsurgical PSA (nadir PSA) for biochemical relapse. While PSA was undetectable in about half the patients, recurrence danger could nevertheless be stratified based on nadir PSA concentration, representing the clinical value of ultra‐low PSA measurement for estimating long‐term BCR‐free survival.6

The capability to precisely measure PSA values for all RP patients could enhance the evaluation of patient prognosis and response to treatment, and better target secondary therapy to patients who may gain most.

Quanterix Solution

Quanterix™ has created an ultra‐sensitive system that can measure individual proteins at concentrations 1000 times lower than present-day commercially available immunoassays. The Single Molecule Array (Simoa™) technology at the center of this system allows the detection and quantification of biomarkers that were challenging or impossible to measure earlier.

The ultra-sensitivity of Simoa™ assays makes it exclusive than all other immunoassays available at present, thus delivering PCR‐like detection limits for both current and novel protein biomarkers.

In short, this innovative technology can detect single protein molecules in body fluids, including blood, by trapping the proteins on microscopic beads coated with certain antibodies and labeling the immunocomplexes using a reporter enzyme that can produce a fluorescent product. After separating the beads in 50‐fL reaction chambers engineered to hold a single bead, fluorescence imaging identifies the single protein molecules.

Quanterix™ has confirmed a PSA assay for the completely automated Simoa™ HD‐1 Analyzer. With a limit of detection (LOD) of 0.028 pg/mL, this assay displays a 3‐log enhancement in sensitivity than the most sensitive PSA assays available in the market today.

The assay is powerful, with a functional sensitivity of <0.05 pg/mL (see Figure 1), total imprecision of <10% from 1 to 50 pg/mL, a linear response over 4 logs of dynamic range down to sub‐pg/mL levels, and exceptional agreement with a comparator technique.7 This assay’s analytical sensitivity facilitated dependable quantification of PSA in all post‐RP samples tested.

LOQ of Simoa™ PSA Assay. LOQ was estimated by sample replicate CVs across six weeks of testing. Female serum samples are highlighted in pink.

Figure 1. LOQ of Simoa™ PSA Assay. LOQ was estimated by sample replicate CVs across six weeks of testing. Female serum samples are highlighted in pink.

To additionally measure the clinical utility of the Simoa™ PSA assay, 31 frozen serum specimens were taken from men who underwent RP.8 Overall, 11 of them (35.5%) developed BCR within a mean of 2.1 years. Men without signs of BCR had a minimum of five years of PSA follow‐up.

Figure 2 illustrates the distribution of nadir PSA levels for the BCR and non‐BCR groups. The mean PSA levels in the non‐BCR and BCR groups were 2.3 and 47 pg/mL, respectively (p < 0.001). A value of 3.0 pg/mL was used as a threshold for determining two risk groups (high versus low) for BCR.

Dot plot of Simoa™ PSA nadir (pg/mL) for non‐BCR and BCR groups.

Figure 2. Dot plot of Simoa™ PSA nadir (pg/mL) for nonBCR and BCR groups.

The Kaplan‐Meier survival curves for the risk groups determined by the bifurcated nadir value is depicted in Figure 3 (p = 0.00024). The specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) for the nadir threshold of 3 pg/mL for forecasting BCR within five years were 75%, 100%, 69%, and 100%, respectively.

Kaplan‐Meier time‐to‐BCR curves. Nadir 3.0 pg/mL threshold.

Figure 3. KaplanMeier timetoBCR curves. Nadir 3.0 pg/mL threshold.

The Simoa™ PSA assay exhibits a more than 1000‐fold progress in analytical sensitivity and everyday assay reproducibility that can dependably quantify PSA in prostatectomy patients. Here, five‐year BCR‐free survival was precisely estimated following surgery. Identification of a dependable predictor of BCR immediately following RP has vital implications for PSA testing frequency, choosing candidates for adjuvant therapy, and reassuring a large subgroup of men that they are not prone to the risk of relapse.

The Simoa™ technology and HD‐1 system signify an opportunity to apply high‐sensitivity assays for applications other than tracking cancer recurrence, such as the assessment of cardiovascular, neurological, inflammatory, and infectious diseases.


The Quanterix™ Simoa™ PSA test provides a 1000‐fold improvement in sensitivity over existing commercially available assays. This sensitivity allows dependable measurement of PSA concentrations in plasma from all human subjects who have gone through radical prostatectomy. It also predicts a five‐year BCR-free survival following surgery.


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  2. Gunnar A. Eur Urol 2007;51:1155–58.
  3. Han M, Partin AW, Pound CR, Epstein JI, Walsh PC. Urol Clin North Am 2001;28:555–65.
  4. Trock BJ, Han M, Freedland SJ, Humphreys EB, DeWeese TL, Partin AW, et al. JAMA 2008;299:2760–69.
  5. Yu H, Diamandis EP, Wong PY, Nam R, Trachtenberg J. J Urol 1997;157:913–18.
  6. Hong SK, Park HZ, Lee WK, Kim DS, Lee JS, Doo SH, et al. Urology 2010;76:723–27.
  7. Wilson DH, Hanlon DW, Provuncher GK, Chang L, Song L, Patel PP, Ferrell EP, Lepor H, Partin AW, Chan DW, Sokoll LJ, Cheli CD, Thiel RP, Fournier DR, Duffy DC. Clin Chem 2011;57:1712–21.
  8. Lepor H, Cheli CD, Thiel RP, Taneja SS, Laze J, Chan DW, Sokoll LJ, Mangold L, Partin AW. BJU Int. 2012;109:1770–75.

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Last updated: Aug 13, 2019 at 4:01 AM


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