Development of a novel, hemolysis-resistant reagent for assessment of α-amylase in biological fluids
-
Luca Ronda
Abstract
Background: Although the assessment of α-amylase is an essential part of the diagnostic workout of several pancreatic and extra-pancreatic disorders, its enzymatic activity is significantly reduced in the presence of cell-free hemoglobin such as in samples with spurious hemolysis, due to chemical and spectrophotometric interference. We developed a new reagent that provides reliable results on hemolyzed biological specimens.
Methods: All tests were performed on Beckman Coulter AU5822. Intra-assay imprecision was assessed on three serum samples with low, intermediate and high α-amylase concentration. Linearity was assessed by serially diluting two samples with low and high values of α-amylase. The comparison with commercial reagent was performed on 40 serum samples. Hemolysis studies were carried out by mechanically hemolysis of 20 lithium-heparin samples.
Results: The intra-assay imprecision was comprised between 1.3% and 2.2%. The linearity was excellent (r=0.998), and highly significant agreement was observed with the commercial assay (r=1.00; mean bias −3.8%). Although a significant correlation between non-hemolyzed and hemolyzed specimens was found with both assays (p<0.001), a much greater agreement was observed with the experimental method (r=0.997 vs. 0.818). No measurement exceeded the total allowable error with the experimental assay, whereas the threshold was exceeded in 85% of samples using the commercial method.
Conclusions: The clinical applications of the experimental reagent include α-amylase assessment in hemolyzed samples, in urine and other biological fluids contaminated with lysed erythrocytes, or in patients under frequent transfusions and hemoglobin-based blood substitutes therapy. The formulation of this reagent could be adapted for other clinical chemistry or immunochemistry assays.
The authors acknowledge Francesco Moretti and Michele Boldi for the skill technical assistance in optimizing the method on the Beckman Coulter AU 5822.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
References
1. Cooper G, DeJonge N, Ehrmeyer S, Yundt-Pacheco J, Jansen R, Ricós C, et al. Collective opinion paper on findings of the 2010 convocation of experts on laboratory quality. Clin Chem Lab Med 2011;49:793–802.10.1515/CCLM.2011.149Search in Google Scholar PubMed
2. Plebani M, Lippi G. Closing the brain-to-brain loop in laboratory testing. Clin Chem Lab Med 2011;49:1131–3.10.1515/CCLM.2011.617Search in Google Scholar PubMed
3. Lippi G, Guidi GC, Mattiuzzi C, Plebani M. Preanalytical variability: the dark side of the moon in laboratory testing. Clin Chem Lab Med 2006;44:358–65.10.1515/CCLM.2006.073Search in Google Scholar PubMed
4. Lippi G, Salvagno GL, Montagnana M, Lima-Oliveira G, Guidi GC, Favaloro EJ. Quality standards for sample collection in coagulation testing. Semin Thromb Hemost 2012;38:565–75.10.1055/s-0032-1315961Search in Google Scholar PubMed
5. Lippi G, Chance JJ, Church S, Dazzi P, Fontana R, Giavarina D, et al. Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med 2011;49:1113–26.10.1515/CCLM.2011.600Search in Google Scholar PubMed
6. Lippi G, Ippolito L, Fontana R. Prevalence of hemolytic specimens referred for arterial blood gas analysis. Clin Chem Lab Med 2011;49:931–2.10.1515/CCLM.2011.136Search in Google Scholar PubMed
7. Lippi G, Plebani M, Di Somma S, Cervellin G. Hemolyzed specimens: a major challenge for emergency departments and clinical laboratories. Crit Rev Clin Lab Sci 2011;48:143–53.10.3109/10408363.2011.600228Search in Google Scholar PubMed
8. Lippi G, Blanckaert N, Bonini P, Green S, Kitchen S, Palicka V, et al. Haemolysis: an overview of the leading cause of unsuitable specimens in clinical laboratories. Clin Chem Lab Med 2008;46:764–72.10.1515/CCLM.2008.170Search in Google Scholar PubMed
9. Lippi G, Plebani M. Continuous flow automation and hemolysis index: a crucial combination. J Lab Autom 2012 Jun 19. [Epub ahead of print]. Doi: 10.1177/2211068212450014.10.1177/2211068212450014Search in Google Scholar PubMed
10. Lippi G, Valentino M, Cervellin G. Laboratory diagnosis of acute pancreatitis: in search of the Holy Grail. Crit Rev Clin Lab Sci 2012;49:18–31.10.3109/10408363.2012.658354Search in Google Scholar PubMed
11. Street HV, Closer JR. Determination of amylase activity in biological fluids. Clin Chim Acta 1956;1:256–68.10.1016/0009-8981(56)90072-9Search in Google Scholar
12. Snozek CL, Mascarenhas RC, O’Kane DJ. Use of cyst fluid CEA, CA19-9, and amylase for evaluation of pancreatic lesions. Clin Biochem 2009;42:1585–8.10.1016/j.clinbiochem.2009.06.020Search in Google Scholar PubMed
13. Frossard JL, Robert J, Soravia C, Mensi N, Magnin A, Hadengue A, et al. Early prediction in acute pancreatitis: the contribution of amylase and lipase levels in peritoneal fluid. JOP 2000;1:36–45.Search in Google Scholar
14. Burkart J, Haigler S, Caruana R, Hylander B. Usefulness of peritoneal fluid amylase levels in the differential diagnosis of peritonitis in peritoneal dialysis patients. J Am Soc Nephrol 1991;1:1186–90.10.1681/ASN.V1101186Search in Google Scholar PubMed
15. Simson JN. Amylase levels in pleural and peritoneal fluid in Meigs’ syndrome. J R Soc Med 1987;80:661.10.1177/014107688708001029Search in Google Scholar
16. Guder WG. Haemolysis as an influence and interference factor in clinical chemistry. J Clin Chem Clin Biochem 1986;24:125–6.Search in Google Scholar
17. Sonntag O. Haemolysis as an interference factor in clinical chemistry. J Clin Chem Clin Biochem 1986;24:127–39.Search in Google Scholar
18. Jay DW, Provasek D. Characterization and mathematical correction of hemolysis interference in selected Hitachi 717 assays. Clin Chem 1993;39:1804–10.10.1093/clinchem/39.9.1804Search in Google Scholar
19. Grafmeyer D, Bondon M, Manchon M, Levillain P. The influence of bilirubin, haemolysis and turbidity on 20 analytical tests performed on automatic analysers. Results of an interlaboratory study. Eur J Clin Chem Clin Biochem 1995;33:31–52.Search in Google Scholar
20. Kazmierczak SC, Catrou PG, Best AE, Sullivan SW, Briley KP. Multiple regression analysis of interference effects from a hemoglobin-based oxygen carrier solution. Clin Chem Lab Med 1999;37:453–64.10.1515/CCLM.1999.074Search in Google Scholar PubMed
21. Steen G, Vermeer HJ, Naus AJ, Goevaerts B, Agricola PT, Schoenmakers CH. Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays. Clin Chem Lab Med 2006;44:413–9.10.1515/CCLM.2006.067Search in Google Scholar PubMed
22. Koseoglu M, Hur A, Atay A, Cuhadar S. Effects of hemolysis interference on routine biochemistry parameters. Biochem Med (Zagreb) 2011;21:79–85.10.11613/BM.2011.015Search in Google Scholar
23. Saruç M, Yuceyar H, Turkel N, Ozutemiz O, Tuzcuoglu I, Ayhan S, et al. The role of heme in hemolysis-induced acute pancreatitis. Med Sci Monit 2007;13:BR67–72.Search in Google Scholar
24. Malinoski DJ, Hadjizacharia P, Salim A, Kim H, Dolich MO, Cinat M, et al. Elevated serum pancreatic enzyme levels after hemorrhagic shock predict organ failure and death. J Trauma 2009;67:445–9.10.1097/TA.0b013e3181b5dc11Search in Google Scholar PubMed
25. Frémont S, Combe AM, Mecrin M, Tronel H, Galland AV, Scholl C, et al. Influence of visible interferences on biochemical assays realised on Au 5231, AU 5223 (Olympus) and CL 7200 (Shimadzu). Ann Biol Clin (Paris) 1996;54:309–20.Search in Google Scholar
26. Lippi G, Dipalo M, Musa R, Avanzini P, Ferrarini C, Pattini A, et al. Evaluation of the analytical performances of the novel Beckman Coulter AU5800. Clin Biochem 2012;45:502–4.10.1016/j.clinbiochem.2012.01.015Search in Google Scholar PubMed
27. Lippi G, Mercadanti M, Aloe R, Targher G. Erythrocyte mechanical fragility is increased in patients with type 2 diabetes. Eur J Intern Med 2012;23:150–3.10.1016/j.ejim.2011.11.004Search in Google Scholar PubMed
28. Lippi G, Musa R, Avanzini P, Aloe R, Pipitone S, Sandei F. Influence of in vitro hemolysis on hematological testing on Advia 2120. Int J Lab Hematol 2012;34:179–84.10.1111/j.1751-553X.2011.01378.xSearch in Google Scholar PubMed
29. Lippi G. Interference studies: focus on blood cell lysates preparation and testing. Clin Lab 2012;58:351–5.Search in Google Scholar
30. Lippi G, Luca Salvagno G, Blanckaert N, Giavarina D, Green S, Kitchen S, et al. Multicenter evaluation of the hemolysis index in automated clinical chemistry systems. Clin Chem Lab Med 2009;47:934–9.10.1515/CCLM.2009.218Search in Google Scholar PubMed
31. Ricós C, Alvarez V, Cava F, García-Lario JV, Hernández A, Jiménez CV, et al. Current databases on biological variation: pros, cons and progress. Scand J Clin Lab Invest 1999;59:491–500.10.1080/00365519950185229Search in Google Scholar PubMed
32. Mozzarelli A, Ronda L, Faggiano S, Bettati S, Bruno S. Haemoglobin-based oxygen carriers: research and reality towards an alternative to blood transfusions. Blood Transfus 2010;8(Suppl 3):s59–68.Search in Google Scholar
33. Mozzarelli A, Bettati S. Chemistry and biochemistry of oxygen therapeutics: from transfusion to artificial blood. Chichester, UK: Wiley & Sons Ltd., 2011.10.1002/9781119975427Search in Google Scholar
34. Caccia D, Ronda L, Frassi R, Perrella M, Del Favero E, Bruno S, et al. PEGylation promotes hemoglobin tetramer dissociation. Bioconjug Chem 2009;20:1356–66.10.1021/bc900130fSearch in Google Scholar PubMed
35. Ronda L, Pioselli B, Bruno S, Faggiano S, Mozzarelli A. Electrophoretic analysis of PEGylated hemoglobin-based blood substitutes. Anal Biochem 2011;408:118–23.10.1016/j.ab.2010.08.043Search in Google Scholar PubMed
36. Portoro I, Kocsis L, Herman P, Caccia D, Perrella M, Ronda L, et al. Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration. Biochim Biophys Acta 2008;1784:1402–9.10.1016/j.bbapap.2008.03.005Search in Google Scholar PubMed
37. Ma Z, Monk TG, Goodnough LT, McClellan A, Gawryl M, Clark T, et al. Effect of hemoglobin- and Perflubron-based oxygen carriers on common clinical laboratory tests. Clin Chem 1997;43:1732–7.10.1093/clinchem/43.9.1732Search in Google Scholar
38. Cameron SJ, Gerhardt G, Engelstad M, Young MA, Norris EJ, Sokoll LJ. Interference in clinical chemistry assays by the hemoglobin-based oxygen carrier, Hemospan. Clin Biochem 2009;42:221–4.10.1016/j.clinbiochem.2008.10.023Search in Google Scholar PubMed
39. Lippi G, Avanzini P, Dipalo M, Aloe R, Cervellin G. Influence of hemolysis on troponin testing: studies on Beckman Coulter UniCel DxI 800 Accu-TnI and overview of the literature. Clin Chem Lab Med 2011;49:2097–100.10.1515/CCLM.2011.703Search in Google Scholar PubMed
©2013 by Walter de Gruyter Berlin Boston
Articles in the same Issue
- Masthead
- Masthead
- Editorials
- Biomarkers in the treatment of cancer: opportunities and pitfalls
- Multicenter reference intervals studies: a promising perspective for the future?
- Hemolysis-resistant reagent: another part of the puzzle for preventing errors in laboratory testing
- Review
- Exhaled breath condensate: a comprehensive update
- Mini Review
- Psoriasis, a new challenge for laboratory medicine
- Opinion Paper
- CA 19-9: handle with care
- General Clinical Chemistry and Laboratory Medicine
- Performance evaluation of human cytokines profiles obtained by various multiplexed-based technologies underlines a need for standardization
- The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study
- Red blood cell distribution width is a potential prognostic index for liver disease
- Development of a novel, hemolysis-resistant reagent for assessment of α-amylase in biological fluids
- Performance of cassette-based blood gas analyzers to monitor blood glucose and lactate levels in a surgical intensive care setting
- Reference Values and Biological Variations
- The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes
- The Asian project for collaborative derivation of reference intervals: (2) results of non-standardized analytes and transference of reference intervals to the participating laboratories on the basis of cross-comparison of test results
- Cancer Diagnostics
- Study of an elevated carbohydrate antigen 19-9 concentration in a large health check-up cohort in China
- Glycoproteomic identification of potential glycoprotein biomarkers in ovarian cancer proximal fluids
- Comparison of serum calcitonin and procalcitonin in detecting medullary thyroid carcinoma among patients with thyroid nodules
- Serum HER-2 predicts response and resistance to trastuzumab treatment in breast cancer
- Elevated tumor markers in patients with pulmonary alveolar proteinosis
- Serum carcinoembryonic antigen is associated with non-alcoholic fatty liver disease in healthy Korean non-smokers
- Assessment of SOX17 DNA methylation in cell free DNA from patients with operable gastric cancer. Association with prognostic variables and survival
- Sensitivity of CA 15-3, CEA and serum HER2 in the early detection of recurrence of breast cancer
- Cardiovascular Diseases
- Association of nucleotide-binding oligomerization domain-like receptor 3 inflammasome and adverse clinical outcomes in patients with idiopathic dilated cardiomyopathy
- Diabetes
- α1-Microglobulin/albumin ratio may improve interpretation of albuminuria in statin-treated patients
- Infectious Diseases
- The role of procalcitonin and IL-6 in discriminating between septic and non-septic causes of ALI/ARDS: a prospective observational study
- Letters to the Editor
- Carryover does not affect results of Beckman Coulter highly-sensitive-AccuTnI assay on Access 2
- Adding calcium to EDTA plasma samples prior to analysis could solve the compatibility issue in commercially available ELISAs that are standardized for serum
- Interference of luteinizing hormone in the urinary point-of-care human chorionic gonadotropin test
- Standardize the serum albumin assay now: calibrate it to 60% of the serum total protein assay
- Interference of M-paraprotein in automated urea assays
- A case of mistaken diagnosis of heparin-induced thrombocytopenia due to pseudothrombocytopenia
- Delta check: a must in the management of hyponatremia
- A novel cost effective algorithm for antinuclear antibody (ANA) testing in an outpatient setting
Articles in the same Issue
- Masthead
- Masthead
- Editorials
- Biomarkers in the treatment of cancer: opportunities and pitfalls
- Multicenter reference intervals studies: a promising perspective for the future?
- Hemolysis-resistant reagent: another part of the puzzle for preventing errors in laboratory testing
- Review
- Exhaled breath condensate: a comprehensive update
- Mini Review
- Psoriasis, a new challenge for laboratory medicine
- Opinion Paper
- CA 19-9: handle with care
- General Clinical Chemistry and Laboratory Medicine
- Performance evaluation of human cytokines profiles obtained by various multiplexed-based technologies underlines a need for standardization
- The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study
- Red blood cell distribution width is a potential prognostic index for liver disease
- Development of a novel, hemolysis-resistant reagent for assessment of α-amylase in biological fluids
- Performance of cassette-based blood gas analyzers to monitor blood glucose and lactate levels in a surgical intensive care setting
- Reference Values and Biological Variations
- The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes
- The Asian project for collaborative derivation of reference intervals: (2) results of non-standardized analytes and transference of reference intervals to the participating laboratories on the basis of cross-comparison of test results
- Cancer Diagnostics
- Study of an elevated carbohydrate antigen 19-9 concentration in a large health check-up cohort in China
- Glycoproteomic identification of potential glycoprotein biomarkers in ovarian cancer proximal fluids
- Comparison of serum calcitonin and procalcitonin in detecting medullary thyroid carcinoma among patients with thyroid nodules
- Serum HER-2 predicts response and resistance to trastuzumab treatment in breast cancer
- Elevated tumor markers in patients with pulmonary alveolar proteinosis
- Serum carcinoembryonic antigen is associated with non-alcoholic fatty liver disease in healthy Korean non-smokers
- Assessment of SOX17 DNA methylation in cell free DNA from patients with operable gastric cancer. Association with prognostic variables and survival
- Sensitivity of CA 15-3, CEA and serum HER2 in the early detection of recurrence of breast cancer
- Cardiovascular Diseases
- Association of nucleotide-binding oligomerization domain-like receptor 3 inflammasome and adverse clinical outcomes in patients with idiopathic dilated cardiomyopathy
- Diabetes
- α1-Microglobulin/albumin ratio may improve interpretation of albuminuria in statin-treated patients
- Infectious Diseases
- The role of procalcitonin and IL-6 in discriminating between septic and non-septic causes of ALI/ARDS: a prospective observational study
- Letters to the Editor
- Carryover does not affect results of Beckman Coulter highly-sensitive-AccuTnI assay on Access 2
- Adding calcium to EDTA plasma samples prior to analysis could solve the compatibility issue in commercially available ELISAs that are standardized for serum
- Interference of luteinizing hormone in the urinary point-of-care human chorionic gonadotropin test
- Standardize the serum albumin assay now: calibrate it to 60% of the serum total protein assay
- Interference of M-paraprotein in automated urea assays
- A case of mistaken diagnosis of heparin-induced thrombocytopenia due to pseudothrombocytopenia
- Delta check: a must in the management of hyponatremia
- A novel cost effective algorithm for antinuclear antibody (ANA) testing in an outpatient setting
