Reference Intervals for and Validation of Recalibrated ...

Letters to the Editor mean peak BNPsp concentration was lower in this group. Again, the differences did not reach statistical significance by ANOVA (hsTnT, P ⫽ 0.104; BNPsp, P ⫽ 0.290; NTproBNP, P ⫽ 0.324). There was a statistically significant relationship between the absolute change (␦) in the hsTnT concentration and the cumulative dobutamine dose (r 2 ⫽ 0.26; P ⫽ 0.015) in the CAD patients. There was no corresponding correlation with ␦ BNPsp or ␦ hsTnT in the CAD group (r 2 ⫽ ⫺0.028; P ⫽ 0.562), and there was no significant correlation between ␦ NT-proBNP and ␦ BNPsp (r 2 ⫽ 0.083; P ⫽ 0.317). To our knowledge, this study is the first to demonstrate the patterns of release of TnT with a highsensitivity assay during DSE testing. Our data suggest stepwise increments in DSE-induced increases in plasma hsTnT and BNPsp in healthy volunteers and CAD patients. CAD patients with inducible ischemia also received the highest dobutamine doses, a finding that must be considered. In contrast with hsTnT and NTproBNP, the release kinetics for BNPsp indicate that it is a much more dynamic marker. The reasons for the attenuated release of BNPsp in individuals with echocardiographically positive test results are unclear. In view of the small sample size, this observation requires verification. If genuine, it is possible that BNPsp release mechanisms are more susceptible than troponin to ischemic preconditioning, or there may be a depletable pool of this peptide. Given the results of this pilot study, we propose that both exaggerated cardiac troponin release and attenuated BNPsp release in patients with inducible ischemia during DSE warrant further investigation with a larger sample size— and with longer follow-up—to establish whether specific threshold 1494 Clinical Chemistry 58:10 (2012)

biomarker responses correspond to worse ischemia and/or a worse prognosis.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: The Canterbury Medical Research Foundation (grant 10/04). M. Sirwardena, National Heart Foundation of New Zealand (grant 1422); C.J. Pemberton, Health Research Council of New Zealand (grant 07/114). Expert Testimony: None declared. Patents: A.M. Richards, New Zealand patent PCT/NZ2007/000265-BNP-sp; C.J. Pemberton, United States patent US 2012/ 381,100 (granted September 1, 2012). Acknowledgments: A report on this study was previously presented at the Cardiac Society of Australia and New Zealand (CSANZ) 2011, Perth, Australia.

3. Reichlin T, Hochholzer W, Bassetti S, Steuer S, Stelzig C, Hartwiger S, et al. Early diagnosis of myocardial infarction with sensitive cardiac troponin assays. N Engl J Med 2009;361:858 – 67. 4. Keller T, Zeller T, Peetz D, Tzikas S, Roth A, Czyz E, et al. Sensitive troponin I assay in early diagnosis of myocardial infarction. N Engl J Med 2009;361:868 –77. 5. Siriwardena M, Kleffmann T, Ruygrok P, Cameron V, Yandle T, Nicholls G, et al. B-type natriuretic peptide signal peptide circulates in human blood: evaluation as a potential biomarker of cardiac ischemia. Circulation 2010;122:255– 64.

Maithri Siriwardena* Vicki Campbell A. Mark Richards Christopher J. Pemberton Christchurch Cardioendocrine Research Group Department of Medicine University of Otago Christchurch, New Zealand *Address correspondence to this author at: Christchurch Cardioendocrine Research Group Department of Medicine 2 Riccarton Ave. University of Otago P.O. Box 4345 Christchurch 8140, New Zealand Fax ⫹643-364-0818 E-mail [email protected]

Previously published online at DOI: 10.1373/clinchem.2012.187682

References 1. Douglas P, Khandheria B, Stainback R, Weissman N, Peterson E, Hendel R, et al. ACCF/ASE/ ACEP/AHA/ASNC/SCAI/SCCT/SCMR 2008 appropriateness criteria for stress echocardiography: a report of the American College of Cardiology Foundation Appropriateness Criteria Task Force, American Society of Echocardiography, American College of Emergency Physicians, American Heart Association, American Society of Nuclear Cardiology, Society for Cardiovascular Angiography and Interventions, Society of Cardiovascular Computed Tomography, and Society of Cardiovascular Magnetic Resonance endorsed by the Heart Rhythm Society and the Society of Critical Care Medicine. J Am Coll Cardiol 2008;51:1127– 47. 2. Gianrossi R, Detrano R, Mulvihill D, Lehmann K, Dubach P, Colombo A, et al. Exercise-induced ST depression in the diagnosis of coronary artery disease. A meta-analysis. Circulation 1989;80: 87–98.

Reference Intervals for and Validation of Recalibrated Immunoassays for Trypsinogen-1 and Trypsinogen-2 To the Editor: Serum trypsinogen assays are used as diagnostic and prognostic tools for cystic fibrosis and acute pancreatitis (AP) (1, 2 ). Calibrator stability is a challenge in these assays, because trypsinogen readily autoactivates and subsequently autodegrades. Furthermore, the reference

Letters to the Editor

Table 1. Trypsinogen-1 and -2 concentrations in serum from healthy volunteers. Trypsinogen-1

Trypsinogen-2

n

Median, ␮g/L

Range, ␮g/L

n

Median, ␮g/L

Range, ␮g/L

18–30 years

27

19.6

10.0–30.5

27

6.5

2.9–12.8

31–50 years

26

22.9

11.8–38.2

27

7.6

2.6–17.4

51–70 years

26

25.0

11.0–49.1

27

8.2

3.8–16.9

⬎70 years

20

26.4

7.2–43.9

22

8.8

3.3–17.7

18–30 years

27

25.3

17.3–45.8

26

7.2

4.2–13.8

31–50 years

25

24.9

16.8–45.1

24

9.1

3.7–14.8

51–70 years

25

27.9

17.4–42.6

25

8.7

6.4–17.9

⬎70 years

21

26.8

13.2–47.2

19

9.4

5.8–15.6

Men

Women

intervals described thus far have been based on samples from a limited number of individuals (1, 3 ). We recalibrated a new immunoassay for trypsinogen-2 and a previously described assay for trypsinogen-1 (1 ) with stable calibrators (2, 4 ) and report reference intervals for these 2 analytes in serum. We have produced new monoclonal antibodies and developed a time-resolved immunofluorometric assay for trypsinogen-2, as previously described (1 ). Monoclonal antibodies F87–9E6 and F88 – 8F7 were used as capture antibody and tracer antibody, respectively. To prevent activation, we produced stable mutated (Lys23Gln) trypsinogen-1 and trypsinogen-2 by recombinant technology (2, 4 ) and used them to recalibrate both the previously described trypsinogen-1 assay (1 ) and the new trypsinogen-2 assay. In this assay, the calibrators covered the concentration interval of 1.0 to 1000 ␮g/L, the limit of detection was 0.24 ␮g/L, and the limit of quantification was 1.0 ␮g/L. The intraassay CV was 6.0%–9.5%, and the interassay CV was 8.7%– 12.6%. The recovery of recombinant trypsinogen-2 added to serum was 87% to 135%, and the

cross-reactivity with trypsinogen-1 was ⬍0.1%. The cross-reactivity with the trypsin-2/␣1-protease inhibitor complex was 71% (w/w) as calculated on the basis of trypsinogen-2 (␮g/L) and the trypsin-2/␣1-protease inhibitor complex (␮g/L) immunoreactivity in gel filtration fractions of serum from a patient with AP. Trypsinogen-2 was stable in serum for at least 7 days at room temperature (CV, 8%–15%), at least 5 weeks at 4 °C (CV, 7%– 17%), and at least 6 weeks at ⫺20 °C (CV, 8%–13%). The immunoreactivity did not decrease after 6 freeze–thaw cycles repeated at 1-week intervals (CV, 7%–13%). Recombinant trypsinogen-2 calibrators were stable in assay buffer for 28 days at 4 °C and ⫺20 °C (CVs, 8%–17% and 7%–14%, respectively). The effect of a breakfast meal on serum trypsinogen-1 and -2 concentrations was studied in 21 volunteers among the laboratory staff (1 man, 20 women). Blood samples were drawn within 1 week before and after a regular Finnish breakfast that consists of some of these: coffee, tea, milk, juice, bread, cheese, ham, porridge, cereals, or yogurt. The breakfast had no effect on the trypsinogen-2 concentra-

tion, but trypsinogen-1 concentrations were slightly higher (5.5%; P ⫽ 0.0349, paired t-test). Reference intervals were established with serum samples from 197 healthy volunteers. Samples from men and women were separately divided into age groups comprising 19 to 27 participants. In the age group 18 –30 years, the concentrations of trypsinogen-1— but not of trypsinogen-2— were significantly lower in men than in women (P ⫽ 0.0015, Mann–Whitney U-test; Table 1). Trypsinogen-2 concentrations were significantly lower in men and women 18 –30 years of age than in older (31–50 years) volunteers (P ⫽ 0.0354). Despite this finding, we combined sex and age groups for calculating reference intervals. In adults, the central 95% reference interval (5 ) was higher for trypsinogen-1 (13.0 – 46.2 ␮g/L) than for trypsinogen-2 (3.8 – 17.4 ␮g/L). The trypsinogen-1 concentrations are in line with those obtained in previous studies (1, 3 ); however, serum trypsinogen-2 concentrations reported for various assays have shown greater variation (1, 3 ). The form of the trypsinogen-2 calibrator used is the most likely source of these differences. Clinical Chemistry 58:10 (2012) 1495

Letters to the Editor We also analyzed trypsinogen1 and -2 immunoreactivities in serum samples from 40 patients with mild AP and 22 patients with severe AP (2 ). The median trypsinogen-1 concentration for patients with mild AP was 253 ␮g/L (95% CI, 105–361 ␮g/L), whereas that for trypsinogen-2 was 522 ␮g/L (95% CI, 377–1047 ␮g/ L). For patients with severe AP, the corresponding median concentrations were 364 ␮g/L (95% CI, 187– 523 ␮g/L) and 1074 ␮g/L (95% CI, 661–1261 ␮g/L). The area under the ROC curve for differentiating between AP (n ⫽ 62) and healthy volunteers (n ⫽ 197) was 0.93 for trypsinogen-1 and 1.00 for trypsinogen-2. The values for the area under the curve for differentiating between mild and severe disease were 0.65 and 0.68, respectively. In conclusion, we produced stable calibrators and used them to calibrate immunoassays for trypsinogen-1 and trypsinogen-2 and established serum reference intervals for these 2 analytes. The reference intervals for trypsinogen-2 were lower than for our earlier method, which used calibrators prepared from tumor-associated trypsinogen-2 (1 ). Our limited study of patients with AP confirms that trypsinogen-2 is a diagnostically sensitive and specific marker for the diagnosis of AP. It will be

1496 Clinical Chemistry 58:10 (2012)

important to determine the commutability of these calibrators in other assays.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: Grants from the Helsinki University Central Hospital Research Fund. Patents: None declared. Acknowledgments: We thank Maarit Leinimaa, Marianne Niemelä, Annikki Löfhjelm, and Helena Taskinen for expert technical assistance.

References 1. Itkonen O, Koivunen E, Hurme M, Alfthan H, Schroder T, Stenman UH. Time-resolved immunofluorometric assays for trypsinogen-1 and 2 in serum reveal preferential elevation of trypsinogen-2 in pancreatitis. J Lab Clin Med 1990;115:712– 8. 2. Oiva J, Itkonen O, Koistinen R, Hotakainen K, Zhang WM, Kemppainen E, et al. Specific immunoassay reveals increased serum trypsinogen 3

in acute pancreatitis. Clin Chem 2011;57: 1506 –13. 3. Kimland M, Russick C, Marks WH, Borgstrom A. Immunoreactive anionic and cationic trypsin in human serum. Clin Chim Acta 1989;184:31– 46. 4. Koistinen H, Koistinen R, Zhang WM, Valmu L, Stenman UH. Nexin-1 inhibits the activity of human brain trypsin. Neuroscience 2009;160: 97–102. 5. CLSI. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline—third edition. Wayne (PA): CLSI; 2008. CLSI document C28 –A3.

Outi Itkonen1,2* Leena Kylänpää3 Wan-Ming Zhang4 Ulf-Håkan Stenman1,2 1

Department of Clinical Chemistry University of Helsinki Helsinki, Finland 2 Laboratory Division HUSLAB and 3 Department of Surgery Helsinki University Central Hospital Helsinki, Finland 4 Department of Clinical Pathology Cleveland Clinic Cleveland, OH *Address correspondence to this author at: HUSLAB P.O. Box 140 FIN-00029 HUS Helsinki, Finland Fax ⫹358-9-471-74806 E-mail [email protected] Previously published online at DOI: 10.1373/clinchem.2012.188706