Evaluation of the Presage™ ST2 ELISA Jun Lu1, David G. Grenache1,2

1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT2Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT

Background: Soluble ST2 (sST2) is a protein in the interleukin-1 receptor family secreted by myocytes in response to mechanical strain. Like NT-proBNP, elevated serum concentrations of sST2 are strongly prognostic in patients with heart failure. Although analogous for prognosis the two markers have been shown to be independent and complementary. Objective: To perform an analytical validation of the Presage™ ST2 enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of sST2 in human serum or plasma.Methods: sST2 was measured using the Presage ST2 (Critical Diagnostics, New York, NY) ELISA. Performance characteristics including imprecision, linearity, recovery, analytical sensitivity, limit of quantification, stability, and sample type comparisons were established using residual specimens sent to ARUP Laboratories. Gender-specific reference intervals were established by non-parametric analysis using serum specimens obtained from 120 healthy males and 120 healthy females. The project was approved by the University of Utah’s Institutional Review Board.Results: Within day and total precision were determined by testing three specimens in triplicate each day for five days. At sST2 concentrations of 11.6, 26.9, and 88.0 U/mL, the within-day CV was 7.6, 2.4, and 3.8%, respectively and the total CV was 11.5, 14.0, and 6.3%, respectively. Linearity of sST2 was determined by serially diluting a specimen with an sST2 concentration of 214.8 U/mL and testing each sample in duplicate. The assay was linear over a concentration range of 2.8-161.1 U/mL (y=0.95x+1.37; r=1.0). Recovery was evaluated by combining two specimens with sST2 concentrations of 107.4 (high) and 29.8 U/mL (low) in ratios of 2:3 and 3:2 (high:low). Measured concentrations were 57.8 and 73.2 U/mL, which were 95.0 and 95.9%, respectively of the expected concentrations. Analytical sensitivity was determined to be 1.6 U/mL by measuring 10 replicates of the zero calibrator, calculating the mean, and adding 3 standard deviations. The limit of quantification, defined as the concentration that produced a CV <20%, was determined to be 3.3 U/mL. sST2 stability was determined using pooled patient specimens with an sST2 concentration of 12.3 U/mL. After storing aliquots for 2 days at room temperature, 10 days at 4 C, and 30 days at -20 C, sST2 concentrations were 94.3, 97.7, and 96.0% of the original concentration, respectively. The mean (SD) concentration of sST2 determined in serum and plasma obtained from each of 13 patients was 141.9 (194.7) and 155.6 (217.2) U/mL, respectively and were not significantly different (p=0.43) and showed excellent agreement (y=1.07x+3.2; r=0.96). Concentrations of sST2 were significantly greater in males compared to females (24.2 vs. 17.5 U/mL, respectively; p<0.0001) but were not correlated by age in either gender (r=-0.09; p=0.31). Reference intervals for sST2 assay were determined to be 10.4-52.1 U/mL for males and 8.4-33.6 U/mL for females.Conclusions: The Presage ST2 ELISA demonstrates excellent performance characteristics for quantifying sST2 in serum or plasma. The assay is linear over a wide sST2 concentration range and can precisely measure low concentrations of sST2. Concentrations of sST2 are unaffected by age but are greater in males compared to females.

• The Presage sST2 ELISA demonstrates excellent performance characteristics for quantifying sST2 . The assay is linear over a wide sST2 concentration range and can precisely measure low concentrations of sST2.

• Concentrations of sST2 are unaffected by age but are greater in males compared to females.

• US healthy population reference values are considerably higher than those in Austria.

Abstract

Figure 1. Linearity of sST2 was determined by serially diluting a specimen with an sST2 concentration of 214.8 U/mL and testing each sample in duplicate. Error bars represent the SD.

1. Dieplinger B, Januzzi Jr JL, Steinmair M, Gabriel C, Poelz W, Haltmayer M, Mueller T. Analytical and clinical evaluation of a novel high-sensitivity assay for measurement of soluble ST2 in human plasma—the Presage ST2 assay. Clin Chim Acta 2009;409:33-40.

Reference Intervals

Conclusions

References

AcknowledgementSupport for this study was provided by the ARUP Institute for Clinical and Experimental Pathology and Critical Diagnostics.

Analytical Validation

N Median sST2, U/mL Reference interval, U/mL Male 120 21.7 10.4-52.1 Female 120 17.3 8.4-33.6 Overall 240 18.9 8.9-41.3

Table 4. Summary of US self-reported healthy cohort reference intervals calculated using a non-parametric percentile method (95% double-sided).

Figure 3. sST2 serum concentrations in US self-reported healthy 120 male and 120 female individuals . Concentrations of sST2 were not correlated by age in either gender (r=-0.09; p=0.31 for all). Mean sST2 values show significant difference between males and females (24.2 U/mL vs. 17.5 U/mL, respectively, p<0.0001), and are significant higher than those reported for an Austrian cohort (13.0 U/mL vs. 8.0 U/mL, respectively) (1).

Mean Activity Within Day Total U/mL CV, % CV, %

High 88.0 3.8 6.3 Medium 26.9 2.4 14.0 Low 11.6 7.6 11.5

Table 1. Within day and total precision were determined by testing three specimens in triplicate each day for five days.

Introduction The Presage ST2 sandwich immunoassay (Critical Diagnostics, New York, NY) was developed to quantify the concentration of ST2 in human serum or plasma, a novel prognostic marker of heart failure. We evaluated the assay’s performance characteristics including precision, linearity, recovery, analytical sensitivity, limit of quantification, stability, and sample type comparisons. Reference intervals were established from serum obtained from 120 male and 120 female self-reported healthy individuals.

Analytical sensitivity:Limit of Absence (LOA) was investigated by measuring 10 replicates of the zero calibrator and calculating as:LOA (Mean + 3SD) = 0.94 + 3x0.22 = 1.6 U/mL

Limit of Detection (LOD) was investigated by measuring 10 replicates of a concentration near the expected LOD and calculating as:LOD (LOA + 3SD) = 1.6 + 3x0.14 = 2.0 U/mL

Limit of Quantification (LOQ) was investigated by measuring 10 replicates of a serial dilution of a low sample and determining the concentration which produces a CV 20%. At concentrations of 2.5, 3.3 and 4.0 U/mL, CVs are 23, 20 and 12%, respectively. LOQ = 3.3 U/mL

Percent of sST2 compared with base line after

RT Refrigerated Frozen

1 day 2 days 10 days 30 days

96.4% 94.3% 97.7% 96.0%

Table 2. sST concentration of a patient sample was tested at time 0, sample pools were stored at room temperature, 4 C and -20 C respectively, sST2 was tested after storage.

Figure 2. Different sample types for sST2 were compared by testing serum and plasma specimens obtained from each of 13 patients.

Table 3. Recovery was evaluated by combining two specimens with sST2 concentrations of 107.4 (high) and 29.8 U/mL (low) in ratios of 2:3 and 3:2 (high:low) in singlet.

ST2, U/mL

Linearity

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

180

Expected concentration, U/mLM

easu

red

co

nce

ntr

atio

n,

U/m

L y = 0.95x + 2.25R2 = 1.00

Paired samples

y = 1.07x + 3.20

R2 = 0.93

0

100

200

300

400

500

600

700

800

0 100 200 300 400 500 600 700 800Serum, U/mL

Pla

sm

a,

U/m

L

Research and Development -- ARUP

Lower UpperValue 95% CI Value 95% CI Ratio

Confidence

Nonparametric (CLSI C28-A) 8.367 <7.087 to 9.808 33.603 25.539 to >39.843 >0.34

Alternatives:Transformed Parametric 8.841 8.009 to 9.758 31.243 28.306 to 34.486 0.18Parametric 6.193 4.426 to 7.961 28.792 27.024 to 30.560 0.16

True Gaussian SDI

3210-1-2-3

0

30

20

10

Nonparametric 95% CI Parametric

True Gaussian SDI

3210-1-2-3

0

33.1

20.1

12.2

7.4

Parametric

Selection Criteria:BoundsFilter

Statistics:MeanSDMedianRangeNDistinct valuesZeroesCentral 95% Index

AnalystExpt. Date

0

50403020100-10

25%

20%

15%

10%

5%

0%

Histogram

Frequency plots of 120 females Frequency plots of 120 males

ST2, U/mL

Research and Development -- ARUP

Lower UpperValue 95% CI Value 95% CI Ratio

Confidence

Nonparametric (CLSI C28-A) 10.363 <8.543 to 13.218 52.139 39.542 to >66.320 >0.38

Alternatives:Transformed Parametric 10.547 9.373 to 11.866 47.621 42.324 to 53.580 0.19Parametric 4.487 1.409 to 7.566 43.842 40.763 to 46.920 0.16

True Gaussian SDI

3210-1-2-3

0

60

50

40

30

20

10

Nonparametric 95% CI Parametric

True Gaussian SDI

3210-1-2-3

0

54.6

33.1

20.1

12.2

Parametric

Selection Criteria:BoundsFilter

Statistics:MeanSDMedianRangeNDistinct valuesZeroesCentral 95% Index

AnalystExpt. Date

0

806040200-20

40%

30%

20%

10%

0%

Histogram

Specimen High Low High Low

sST2, U/mL 107.4 29.8 107.4 29.8

Volume, µL 8 12 12 8

Expected sST2,U/mL 61.6 76.6

Measured sST2, U/mL

57.8 73.2

Recovery, % 93.8 95.6