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Stability of intact parathyroid hormone in samples from hemodialysis patients

2007; Elsevier BV; Volume: 72; Issue: 3 Linguagem: Inglês

10.1038/sj.ki.5002363

1523-1755

Étienne Cavalier, Pierre Delanaye, Agnès Carlisi, Jean-Marie Krzesinski, J.-P. Chapelle,

Pharmacological Effects and Toxicity Studies

The determination of intact parathyroid hormone levels is used for diagnosis and in the management of renal osteodystrophy. Pre-analytical and analytical conditions are important in the overall confidence of the assay. Unfortunately, there are no clear recommendations for the use of serum samples or samples anticoagulated with ethylenediaminotetraacetic acid (EDTA) for the best preservation of intact parathyroid hormone. In our study, the Roche Elecsys assay was used to measure intact hormone in both serum and EDTA plasmas from 16 hemodialysis patients over the span of a month. Parathyroid hormone stability was determined in samples kept frozen for 1–5 days or after 8–24 h at room temperature. There was no difference in hormone stability between serum and EDTA samples after 1 day in frozen storage. After 5 days frozen, hormone degradation was significantly greater after EDTA anticoagulation than in serum aliquots. When samples were stored at room temperature, intact parathyroid hormone was significantly more stable in EDTA-treated samples than in clotted serum samples, especially after 24 h. We conclude that optimum results are achieved in the measurement of intact parathyroid hormone levels depending on the workflow of the lab. If the lab works with intermittent batches of samples, frozen serum is the best. If the lab services general practitioners and/or several hospitals and has a continuous flow of samples, EDTA-treated samples stored at room temperature are the best. The determination of intact parathyroid hormone levels is used for diagnosis and in the management of renal osteodystrophy. Pre-analytical and analytical conditions are important in the overall confidence of the assay. Unfortunately, there are no clear recommendations for the use of serum samples or samples anticoagulated with ethylenediaminotetraacetic acid (EDTA) for the best preservation of intact parathyroid hormone. In our study, the Roche Elecsys assay was used to measure intact hormone in both serum and EDTA plasmas from 16 hemodialysis patients over the span of a month. Parathyroid hormone stability was determined in samples kept frozen for 1–5 days or after 8–24 h at room temperature. There was no difference in hormone stability between serum and EDTA samples after 1 day in frozen storage. After 5 days frozen, hormone degradation was significantly greater after EDTA anticoagulation than in serum aliquots. When samples were stored at room temperature, intact parathyroid hormone was significantly more stable in EDTA-treated samples than in clotted serum samples, especially after 24 h. We conclude that optimum results are achieved in the measurement of intact parathyroid hormone levels depending on the workflow of the lab. If the lab works with intermittent batches of samples, frozen serum is the best. If the lab services general practitioners and/or several hospitals and has a continuous flow of samples, EDTA-treated samples stored at room temperature are the best. In our University hospital, a high percentage of parathormone (PTH) determinations realized in the laboratory are performed in hemodialyzed patients for diagnosis and management of renal osteodystrophy. Thus, analytical and preanalytical conditions are of importance for a good follow-up of the patients. Several authors have studied the stability of PTH in serum and ethylenediaminotetraacetic acid (EDTA) tubes, but mainly on the Immulite 2000 platform.1Glendenning P. Laffer L.L. Weber H.K. et al.Parathyroid hormone is more stable in EDTA plasma than in serum.Clin Chem. 2002; 48: 766-767PubMed Google Scholar, 2Holmes D.T. Levin A. Forer B. Rosenberg F. Preanalytical influences on DPC IMMULITE 2000 intact PTH assays of plasma and serum from dialysis patients.Clin Chem. 2005; 51: 915-917Crossref PubMed Scopus (36) Google Scholar, 3Omar H. Chamberlin A. Walker V. Wood P.J. Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h.Ann Clin Biochem. 2001; 38: 561-563Crossref PubMed Scopus (33) Google Scholar, 4Scharnhorst V. Valkenburg J. Vosters C. Vader H. Influence of preanalytical factors on the immulite intact parathyroid hormone assay.Clin Chem. 2004; 50: 974-975Crossref PubMed Scopus (10) Google Scholar It has been recently shown5Souberbielle J.C. Boutten A. Carlier M.C. et al.Inter-method variability in PTH measurement: implication for the care of CKD patients.Kidney Int. 2006; 70: 345-350Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar that the Roche Elecsys PTH results were the most in accordance with Allegro PTH, even if the antibodies used in these two tests are targeted against different parts of the peptide.6D'Amour P. Brossard J.H. Rakel A. et al.Evidence that the amino-terminal composition of non-(1–84) parathyroid hormone fragments starts before position 19.Clin Chem. 2005; 51: 169-176Crossref PubMed Scopus (46) Google Scholar As the Kidney Disease Outcomes Quality Initiative guidelines7K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease.Am J Kidney Dis. 2003; 42: S1-201PubMed Google Scholar have been established with the no more available Allegro kit, it may be important to evaluate analytical and preanalytical influences on Roche Elecsys PTH determination (Roche Diagnostics, Mannheim, Germany). Our approach has been designed to be as close as possible to the hospital real life. Two series of blood samples were collected in an interval of a month from 16 hemodialyzed patients at 0800 hours immediately before commencing renal dialysis. All the samples were drawn into 5-ml EDTA and gel separator with clot activator tubes purchased from Becton-Dickinson (Erembodegem, Belgium). In all cases, the tubes were filled completely and brought to the laboratory within 30 min. On the first set of samples, drawn in October, we evaluated the stability of PTH when stored frozen as serum or EDTA plasma at -20°C. The stability of the ‘frozen’ PTH was evaluated after 24 h and 5 days. On the second set of samples, drawn in December, we investigated the stability of PTH when conserved in the sample tube as EDTA whole blood or clotted blood at room temperature (21°C). The stability of ‘temperate’ PTH was evaluated at 0, 8, and 24 h. For both series, the reference was the level of the corresponding tube assayed directly after centrifugation at 4°C. In our hands, the coefficients of variation obtained with the Roche Elecsys PTH were <5%. Data were analyzed with the Student test for independent samples, Kruskal–Wallis test and Zerbe method for paired series Considering the two groups of patients as independent samples, the results obtained on the tubes assayed directly after sampling (zero time) did differ neither between October and December nor between EDTA and serum sample tubes (Table 1). The mean percentages of PTH degradation under our experimental conditions are illustrated in Figure 1. Our results have showed that there was no significant difference in PTH degradation between serum and EDTA after 1-day conservation at -20°C (-6.2 and -6.6%, respectively). Nevertheless, when samples were kept frozen for 5 days, PTH degradation was more important in EDTA aliquots than in serum aliquots (-10.8±2.4 vs -7.2±2.2%, P=0.0004). On the other hand, when samples were conserved at room temperature, PTH in EDTA whole blood remained relatively stable (mean loss of activity: -4.1%), whereas its degradation in clotted serum was already important after 8 h (-8.4±4.8%) and even more after 24 h (-20.5±10.1%). This difference in stability between serum and EDTA tubes was very significant (P<0.0001).Table 1Values obtained directly after sampling in the two series of samples, drawn in October and December on EDTA or serum tubesSeriesSample tubesMean (pg/ml)s.d. (pg/ml)Minimum (pg/ml)Maximum (pg/ml)95% CI for the means.e.m. (pg/ml)OctoberEDTA33027119929168–47872Serum31526115895160–45869DecemberEDTA332269121020183–48169Serum3172527934177–45765CI, confidence interval; EDTA, ethylenediaminotetraacetic acid. Open table in a new tab CI, confidence interval; EDTA, ethylenediaminotetraacetic acid. Clinically, by applying the criterion of the Royal Australasian College of Pathologists Quality Assurance Program, 33% of serum samples stored at room temperature failed assurance criteria (<25% difference between the sample and the target) and would have thus lead to a diagnostic misclassification. In frozen series and EDTA kept at room temperature, no transgression of this quality criterion was observed. As previously said, our approach was designed to be as closed as possible as the ‘real life’ of a sample tube in a hospital. The samples drawn in October mimicked the different options usually faced by a laboratory: immediate assay after reception or freezing of the samples for a further determination, usually 24 h to 5 days after reception. The goal of the study on the samples drawn in December was to see what happens if the tubes are left at room temperature from 8 to 24 h before arriving in the laboratory. This kind of ‘pragmatic’ methodology is original. Indeed, reports on PTH stability in serum and plasma are sometimes contradictory. This variability is linked to the great differences in the methodologies used. For instance, the definition of the ‘zero-point’ is, for some authors, a freshly frozen aliquot,3Omar H. Chamberlin A. Walker V. Wood P.J. Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h.Ann Clin Biochem. 2001; 38: 561-563Crossref PubMed Scopus (33) Google Scholar, 8Teal T.K. Wood J.L. Stevens P.E. Lamb E.J. Stability of Bio-Intact (1–84) parathyroid hormone ex vivo in serum and EDTA plasma from hemodialysis patients.Clin Chem. 2004; 50: 1713-1714Crossref PubMed Scopus (15) Google Scholar, 9Walker K.S. Seth J. Stability of parathyroid hormone in blood from renal patients on haemodialysis.Ann Clin Biochem. 2000; 37: 800-801Crossref PubMed Scopus (17) Google Scholar whereas others consider the sample centrifuged and assayed within 3 h.1Glendenning P. Laffer L.L. Weber H.K. et al.Parathyroid hormone is more stable in EDTA plasma than in serum.Clin Chem. 2002; 48: 766-767PubMed Google Scholar, 2Holmes D.T. Levin A. Forer B. Rosenberg F. Preanalytical influences on DPC IMMULITE 2000 intact PTH assays of plasma and serum from dialysis patients.Clin Chem. 2005; 51: 915-917Crossref PubMed Scopus (36) Google Scholar, 4Scharnhorst V. Valkenburg J. Vosters C. Vader H. Influence of preanalytical factors on the immulite intact parathyroid hormone assay.Clin Chem. 2004; 50: 974-975Crossref PubMed Scopus (10) Google Scholar Moreover, conservation time at -20°C before analysis is not mentioned, which could let suppose that this parameter is of minor or no importance. The definition of the term ‘stability’ is not also well defined, as most of the authors let degrade PTH in serum or plasma EDTA and not in whole EDTA blood or clotted serum. The conclusions of these studies are then only applicable to sample management in laboratories and cannot be extrapolated to pre-analytical handling of the tubes in the wards. From a strict point of view, it may be difficult to draw conclusions from these studies. Nevertheless, we agree that EDTA tubes are preferable in situations where rapid delivery of blood to the laboratory cannot be achieved.9Walker K.S. Seth J. Stability of parathyroid hormone in blood from renal patients on haemodialysis.Ann Clin Biochem. 2000; 37: 800-801Crossref PubMed Scopus (17) Google Scholar We have also observed that if PTH was considerably more stable in EDTA than serum when left at room temperature, it was not completely stable.3Omar H. Chamberlin A. Walker V. Wood P.J. Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h.Ann Clin Biochem. 2001; 38: 561-563Crossref PubMed Scopus (33) Google Scholar The in vitro observation that PTH decline is observed in serum and not in EDTA plasma suggests that the increased protease activity may be due to the clotting process. Some authors showed that the addition of aprotinin significantly reduced the decline in PTH at 24 h – even if there was still a difference with EDTA samples.10Anderson N.R. Nicholas J. Holland M.R. Gama R. Effect of a protease inhibitor on in vitro stability of intact parathyroid hormone.Ann Clin Biochem. 2003; 40: 188-190Crossref PubMed Scopus (6) Google Scholar Others found that an addition of two protease inhibitors (aprotinin and leupeptin) eliminated completely the decline of PTH.11Levin G.E. Nisbet J.A. Stability of parathyroid hormone-related protein and parathyroid hormone at room temperature.Ann Clin Biochem. 1994; 31: 497-500Crossref PubMed Scopus (24) Google Scholar We did not find any difference between serum and EDTA plasma at baseline, whereas some authors reported significant higher values for EDTA plasma.2Holmes D.T. Levin A. Forer B. Rosenberg F. Preanalytical influences on DPC IMMULITE 2000 intact PTH assays of plasma and serum from dialysis patients.Clin Chem. 2005; 51: 915-917Crossref PubMed Scopus (36) Google Scholar, 3Omar H. Chamberlin A. Walker V. Wood P.J. Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h.Ann Clin Biochem. 2001; 38: 561-563Crossref PubMed Scopus (33) Google Scholar, 4Scharnhorst V. Valkenburg J. Vosters C. Vader H. Influence of preanalytical factors on the immulite intact parathyroid hormone assay.Clin Chem. 2004; 50: 974-975Crossref PubMed Scopus (10) Google Scholar Teal et al.8Teal T.K. Wood J.L. Stevens P.E. Lamb E.J. Stability of Bio-Intact (1–84) parathyroid hormone ex vivo in serum and EDTA plasma from hemodialysis patients.Clin Chem. 2004; 50: 1713-1714Crossref PubMed Scopus (15) Google Scholar did not either find differences when they compared PTH stability in serum and EDTA plasma with a third-generation PTH kit. They made the hypothesis that amino-PTH, a phosphorylated N-terminal fragment detected by third-generation immunoassays but not second-generation,6D'Amour P. Brossard J.H. Rakel A. et al.Evidence that the amino-terminal composition of non-(1–84) parathyroid hormone fragments starts before position 19.Clin Chem. 2005; 51: 169-176Crossref PubMed Scopus (46) Google Scholar could be more stable than PTH itself. As Roche Elecsys PTH antibodies also recognize amino-PTH, our findings reinforce this hypothesis. When we compared the stability of PTH after 24 h in the ‘frozen’ EDTA tube with the ‘temperate’ EDTA tube, we observed that PTH was more stable in EDTA whole blood than in EDTA plasma conserved at -20°C. This astonishing observation might be attributable to a greater PTH sensitivity to freezing/unfreezing than to PTH degradation by plasmatic metalloproteases. However, a study on PTH stability after four cycles of freezing/unfreezing did not support this hypothesis (data not shown). We have shown here that PTH is not always more stable in EDTA than in serum. Indeed, EDTA is preferable when samples are left in a ward or in a practitioner's office at room temperature, but once the sample is treated in the laboratory and kept frozen, PTH is more stable in serum than EDTA, compared with a ‘fresh’ determination. Our findings are experimental. We do not know why we observed this difference. On the other hand, the greater degradation of PTH observed in serum compared with EDTA plasma still remains unexplained.10Anderson N.R. Nicholas J. Holland M.R. Gama R. Effect of a protease inhibitor on in vitro stability of intact parathyroid hormone.Ann Clin Biochem. 2003; 40: 188-190Crossref PubMed Scopus (6) Google Scholar Our study was designed to be as close as possible to the routine PTH determination in a laboratory. However, in the light of our results, it could be interesting for research purposes to perform such a study to evaluate PTH stability after 1 year of conservation at -20°C or the stability at -80°C. In conclusion, the type of samples used for PTH determination should depend on the way laboratories work. On one hand, if a lab has a practice of general practitioners or works for a multisites hospital, EDTA tubes conserved at room temperature are better. EDTA tubes are also preferable if a lab works in continuous flow. On the other hand, if a lab works in batches with automates, as it used to be with immunoradiometric assay series, and keep samples frozen before determination, serum tubes are the best choice. We thank Drs Nadine Cielniaszek and Jackie Moreaux for providing us with BD tubes as well as Mrs Michèle Focant and Marie-Antoinette Graceffa for their collaboration.