Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 477–484
ORIGINAL ARTICLE
Stability of HE4 and CA125 in blood samples from patients diagnosed with ovarian cancer
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NOREEN SANDHU1, MONA A. KARLSEN2, CLAUS HØGDALL3, INGA A. LAURSEN1†, IB J. CHRISTENSEN4 & ESTRID V. S. HØGDALL2 1Department
of Clinical Biochemistry, Immunology & Genetics, Statens Serum Institut, Copenhagen, Denmark, 2Department of Pathology, Molecular Unit, Herlev Hospital, University of Copenhagen, Denmark, 3Gynecologic Clinic, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, and 4The Finsen Laboratory, Rigshospitalet, and Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Denmark Abstract Objective. To investigate the influence of handling and storage on HE4 and CA125 serum and EDTA plasma levels to clarify any important consequences for a clinical setting. Methods. Blood samples from 13 ovarian cancer (OC) patients were collected and allowed to clot or sediment for up to 72 hours at 4°C or 20°C, then processed into serum and EDTA plasma. Furthermore, the effects of up to eight repetitive cycles of freeze/thaw were investigated. HE4 and CA125 were analyzed using a Chemiluminescent Microparticle Immunoassay on the Architect i2000sr System. Results. No significant effect of processing time for HE4 could be shown. HE4 EDTA plasma levels were insignificantly lower (3%) than serum levels (p ⫽ 0.41). Similarly, no significant effect of processing time for CA125 could be demonstrated. CA125 levels at 4°C were significantly reduced compared to levels at 20°C (p ⫽ 0.024). No significant difference between CA125 serum and plasma levels were found (p ⫽ 0.46). Serum and EDTA plasma samples were stable during the eight cycles of freezing and thawing (CA125: all p ⬎ 0.2; HE4: all p ⬎ 0.5). Conclusion. No systematic difference could be demonstrated for HE4. CA125 is not dependent on processing time, EDTA plasma or serum. Levels of CA125 are significantly reduced at 4°C compared to levels at 20°C, but this difference was less than 6% and is not considered clinically relevant. Key Words: Biomarker, cancer antigen 125, chemiluminescent immunoassay, EDTA plasma, Human epididymis protein 4, serum, specimen handling
Introduction Ovarian cancer (OC) accounts for approximately 4% of all cancer cases and is the seventh most common cancer in women in the United States [1]. In Denmark, approximately 440 new cases of OC are found per year [2]. OC causes approximately 215 deaths each year which makes OC the fourth most frequent cancer-causing death among Danish women [3,4]. The high mortality rate of OC is primarily attributable to diagnosis of the disease when the cancer spreads beyond the ovaries. Early diagnosis is an important prognostic factor for substantial improvement in the survival rate of patients with OC.
Today the Cancer Antigen 125 (CA125) is the most common tumor marker used in clinical handling of OC. CA125 is encoded by the MUC16 gene [5,6] and is an antigenic determinant on a large transmembrane glycoprotein expressed by epithelial ovarian tumors [7,8]. Serum CA125 is used for risk estimation of women with a pelvic mass [9–11] as well as for monitoring of OC treatment response and progression [12–14]. However, CA125 has a low sensitivity for early stage disease in OC [13,15]. Human epididymis protein 4 (HE4) is a promising biomarkers for early detection of OC. Recent studies indicate that HE4 is highly expressed in OC tissues, and it has been detected in high levels in
†Deceased 10 December 2011. Correspondence: Estrid Høgdall, Department of Pathology, Molecular Unit, Herlev Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark. Tel: ⫹ 45 3868 9132. E-mail: [email protected]
(Received 6 June 2013 ; accepted 27 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.903430
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Figure 1. Flowchart for the handling procedure for the time study.
serum from OC patients [16–18]. HE4 is a single polypeptide containing two whey acidic fourdisulfide core (WFDC) domains [18]. The HE4 gene (WFDC2) codes for a 13kD protein, although in its mature glycosylated form the relative molecular weight is approximately 20–25 kD. It has been found that HE4 alone or in combination with CA125 has higher sensitivity and specificity for patients with early stage OC compared to CA125 alone [18–21]. The potential role in a differential setting should be examined in future trials. Before implementation of HE4 in clinical practice, alone or in combination with CA125 and/or other relevant markers/clinical information (e.g. menopausal status, age or other) the stability of the biomarkers must be clarified. The stability of HE4 has not been investigated in earlier studies but a study has shown that the stability of CA125 in serum samples was significantly affected by longterm frozen storage at ⫺40°C but least influenced by three short cycles of freezing and thawing [22]. For clinical determination, samples are often stored
at different conditions and transported from different hospitals to a central laboratory and up to a 3-day time period may be required for postal delivery. In view of this it is important to allow the blood samples to clot and sediment for up to 72 hours at different temperature, before being processed into serum or EDTA plasma. Our aim was to investigate the influence of up to eight freeze/thawing cycles of serum and EDTA plasma samples and the storage conditions at different temperatures on the level of the biomarkers CA125 and HE4. Materials and methods Patients All patients were recruited from the Gynecologic Clinic, Rigshospitalet, Denmark. All patients included in the study had pre-operative blood samples taken within 2 weeks prior to their operation for OC. We investigated the stability of HE4 and CA125 measurements in both serum and EDTA
Figure 2. Flowchart for the handling procedure for the freeze and thaw study.
463 55–10000 461 54–10000 433 51–10000 448 55–10000 435 50–10000 425 52–10000 435 54–10000 435 54–10000 429 46–10000 426 49–10000 433 50–10000 423 47–10000 435 49–10000 416 48–10000 435 48–10000 465 52–10000 456 51–10000 433 51–10000
440 52–10000 441 51–10000
332 97–4245 321 92–4004 319 83–4158 316 89–4279 310 88–3629 322 86–3603 305 88–4105 339 83–3592 314 83–3792
324 86–4641
328 94–3768 335 93–3873 328 92–3593 327 92–4103 327 91–3590 334 91–3926
24 Geometric mean (range) 8 Geometric mean (range) 72 Geometric mean (range)
1 Geometric mean (range)
3 Geometric mean (range)
20°C
24 Geometric mean (range) 8 Geometric mean (range)
331 89–3796 333 89–4008 337 93–4287
For each patient, a total of 40 measurements of HE4 and CA125 in serum and EDTA plasma were performed in duplicate using a Chemiluminescent Microparticle Immunoassay (CMIA) on ARCHITECT i2000sr System (Abbott Diagnostics, Wiesbaden, Germany). The assays and handling of reagents were carried out as recommended by the manufacturer. Analytical precision given by Abbott Diagnostics are for the Architect CA125 assay ⱕ 10% total CV with a measurement range of 1.0–1000 kU/L. The
3 Geometric mean (range)
Biochemical analysis
1 Geometric mean (range)
This study is approved by Copenhagen and Frederiksberg Scientific Ethical Committee by the journal number 01-101/02.
Temperature Hours
Ethics
4°C
The study material was comprised of a series of paired serum and EDTA plasma samples from 13 OC patients. About 30 ml of crude blood and 15 ml of blood added to 10 mM EDTA were collected from a cubical vein of each patient. The blood samples were collected in paired EDTA- tubes (K3E EDTA tubes from Vacuette, Greiner bio-one, Kremsmünster, Österreich) and serum tubes coated with micronized silica particles, which activate clotting (Vacuette, Greiner bio-one, Kremsmünster, Österreich). After separation, the sera and EDTA plasma were stored for 5 years in tightly capped Eppendorf tubes. The paired sets of these samples were allowed to clot and sediment in 1, 3, 8, 24 and 72 hours at 4°C or 20°C, before being processed into serum or EDTA plasma samples. Serum and EDTA plasma were separated by centrifugation at 2000 g for 10 mins, and stored at ⫺80°C in aliquots until further analysis. The aliquots were made after each time at the two different temperatures, a total of 10 aliquots (Figure 1). To investigate the effect of repetitive freezing (⫺80°C) and thawing, the serum and EDTA plasma samples were divided into eight paired 1 ml aliquots and stored overnight at ⫺80°C. The primary samples were analyzed and the following day seven of the sample sets were thawed at room temperature and re-frozen 1 hour after thawing. The following day, six sample sets were thawed and re-frozen. This was repeated for 5 additional days, each day taking one less sample set out for thawing and freezing until the last sample set had been frozen and thawed 8 times, a total of 16 aliquots (Figure 2).
Table I. HE4 and CA125 concentrations (mean and range) in EDTA plasma and serum blood samples handled and stored after 1, 3, 8, 24 and 72 hours at 4°C and 20°C.
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Sample handling
HE4 serum [pmol/L] HE4 EDTA plasma [pmol/L] CA125 serum [kU/L] CA125 EDTA plasma [kU/L]
72 Geometric mean (range)
plasma samples from the ‘Danish Heterogeneity study’ [23].
332 107–3821
Stability of HE4 and CA125 in blood samples
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Figure 3. Stability of HE4 in blood samples. This plot shows the ratio of HE4 concentration for each test condition compared to HE4 concentration in samples processed at 1 hour at 20°C with 95% confidence intervals.
dilution linearity was done by Abbott and the recoveries were from 96–110% (Abbott ref. 2K45). CA125 control ranges by Abbott (Ref. 2K45-10) are 28.0–52.0 kU/L (Low control), 210.0–390.0 kU/L (Medium control) and 455.0–845.0 kU/L (High control). Analytical precision for the Architect HE4 assay ⱕ 10% total CV is with a measurement range of
20.0–1500.0 pmol/L. The dilution linearity of HE4 was done by Abbott and the recoveries were from 91–105% (Abbott ref. 2P54). HE4 control ranges by Abbott (Ref. 2P54-10) are 35.0–65.0 pmol/L (Low control), 122.5–227.5 pmol/L (Medium control) and 490.0–910.0 pmol/L (High control). The study was conducted using one reagent lot. A calibration curve was performed for the kit before
Figure 4. Stability of CA125 in blood samples. This plot shows the ratio of CA125 concentration for each test condition compared to CA125 concentration in samples processed at 1 hour at 20°C with 95% confidence intervals.
Stability of HE4 and CA125 in blood samples
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Table II. HE4 and CA125 concentrations during eight repetitive freezing (⫺ 80°C) and thawing cycles measured in serum and EDTA plasma samples. Number of freeze cycles
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HE4 serum [pmol/L] HE4 EDTA plasma [pmol/L] CA125 serum [kU/L] CA125 EDTA plasma [kU/L]
1 2 3 4 5 6 7 8 Geometric Geometric Geometric Geometric Geometric Geometric Geometric Geometric mean (range) mean (range) mean (range) mean (range) mean (range) mean (range) mean (range) mean (range) 236 80–1530
239 81–1551
217 86–1556
244 81–1527
231 85–1558
237 81–1457
229 83–1555
228 81–1570
257 79–1409
239 86–1356
238 89–1386
232 87–1448
233 84–1454
236 93–1405
238 87–1441
235 88–1485
423 67–5584 382 67–6190
413 66–6439 427 66–5737
377 64–5159 427 61–5872
417 65–6342 420 63–5788
407 60–6842 413 65–5379
419 78–6191 419 63–6195
396 65–5543 421 64–6136
400 57–6098 411 65–6199
the start of the experiment. Subsequently, the three-level quality controls from Abbott for both CA125 and HE4 were tested each day to validate the calibration before analyzing the study samples. The measured concentrations of all the quality control samples were within the variation of maximum 10% during the study, which is our laboratory accept limit. Abbott recommends much larger ranges for their controls, as described. As a control we also compared CA125 values of six primary samples with results already measured in our Routine laboratory at time of diagnosis. Comparisons of the CA125 showed that the CA125 result were concordant (data not presented). Statistical analysis The statistical analysis was carried out using a linear model with repeated measures. Fixed effects are serum/EDTA plasma, temperature, and time, as well as the number of thaws. The estimated effects are
shown with 95% confidence limits and p-values. CA125 and HE4 concentrations were log transformed for analysis in order to obtain normally distributed data and back transformed for presentation. Any p values less than 5% were considered significant. Calculations were done using SAS (v9.2, SAS Institute, Cary, NC, USA). Results The mean values (geometric means) and ranges of HE4 and CA125 concentrations in EDTA plasma and serum samples from the 13 OC patients are shown in Table I. Analyzing HE4 on the log scale as a function of processing time, temperature, and sample type (EDTA plasma or serum), using mixed modelling with an interaction term between time and sample type could not demonstrate a significant interaction (Figure 3). In a model without interaction, no significant effect of processing time could be shown for times up to 72 hours compared
Figure 5. Stability of HE4 to freeze/thaw cycles. This plot shows the ratio of HE4 concentration at thaw eight times compared to HE4 concentration in samples stored at ⫺80°C and thawed one time, with 95% confidence intervals.
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Figure 6. Stability of CA125 to freeze/thaw cycles. This plot shows the ratio of CA125 concentration at thaw 8 times compared to CA125 concentration in samples stored at ⫺80°C and thawed one time, with 95% confidence intervals.
to HE4 level at 1 hour (Table I). The concentrations of HE4 EDTA plasma and HE4 serum did not differ significantly (Figure 3). No significant differences for processing time was detected (p ⫽ 0.17). Analyzing CA125 on the log scale as a function of processing time, temperature, and sample type (EDTA plasma or serum) using mixed modelling with an interaction term between processing time and sample type could not demonstrate a significant interaction (Figure 4). In a model without interaction, no significant effect of processing time could be demonstrated although a difference at 72 hours was detected (p ⫽ 0.049) (Figure 4). CA125 levels at 4°C were significantly reduced compared to levels at 20°C (p ⫽ 0.003), but this difference was less than 6%. No significant difference between CA125 serum and plasma levels were found (p ⫽ 0.46) (Figure 4). The estimated differences in EDTA plasma and serum levels for HE4 as well as for CA125 are relatively small, with most deviations less than 5% (Figures 3 and 4). Results of HE4 and CA125 concentrations (geometric mean and ranges) after eight repetitive freezing (⫺80°C) and thawing cycles of serum and EDTA plasma samples are shown in Table II. Serum and EDTA plasma samples were stable during the eight cycles of freezing and thawing as shown in Figure 5 and 6. The coefficient of variation for the concentration of HE4 after up to eight cycles of freezing and thawing in both serum and EDTA plasma samples were found to be 19.9% and 5.7%, respectively. In the case of CA125, after up to eight cycles of freezing and thawing the coefficient of variation were found to be 20.0% and 8.6% in serum and EDTA plasma samples, respectively. Results for the thaw/freeze for HE4 showed that there was no difference for the number of thaws (p ⫽ 0.66) or between sample type (p ⫽ 0.90). The
same analyses for CA125 gave non-significant results (p ⫽ 0.55 and p ⫽ 0.85 respectively). Again the differences for HE4 and CA125 are less than 5% for almost all cases (Figures 5 and 6). Discussion Before a biomarker is adopted by a clinical laboratory, it is mandatory to consider a possible impact of handling and storage conditions on the biological material, which may influence the level of the biomarker analyzed. Therefore, we initiated this study to examine stability of both HE4 and CA125 before initiating a larger research study. In this stability study of the biomarkers HE4 and CA125, we found both HE4 and CA125 to be very stable. No significant difference of HE4 was found dependent of handling temperature in contrast to CA125 where a significantly lower concentration was found in samples handled at 4°C compared to samples handled at room temperature. Therefore, it is important that the blood samples are strictly handled in order to compare analytical results. Both HE4 and CA125 concentrations were not impacted by up to eight freeze and thaw cycles. Our findings of a very high degree of stability for the markers CA125 and HE4 is very important and makes HE4 a relevant marker to study in collected and stored blood samples, as well as in future collections as no special demands for handling and storage are needed. The stability of HE4 has not been investigated in earlier studies, but the stability of CA125 has been investigated in freshly separated serum samples. These samples were frozen at ⫺40°C for three short repetitive freeze-thaw cycles and a long frozen storage of 10 months at ⫺40°C. The study showed that CA125 was significantly affected by the long-term frozen storage but least influenced by the three short
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Stability of HE4 and CA125 in blood samples cycles of freezing and thawing [22]. The significant decrease of CA125 concentration may indicate a degradation of the antigen. The limitations of our study could be the limited number of patients or the fact that samples were stored at ⫺80°C for 5 years before analysis. But since the samples were kept at ⫺80°C in tight capped Eppendorf tubes, the sample degradation and sample evaporation would probably be limited. Sample evaporation would result in an increase of the biomarker level as from six samples of serum CA125 which were measured at the time of the diagnoses. We also performed CA125 analyses together with the HE4 analyses. Comparisons of the CA125 showed that the CA125 results were concordant (data not presented). Studies concerning other cancer-related biomarkers have shown an increase of CA19-9 level after one freezing cycle at ⫺40°C [22]. In the study, the choice of tubes was also shown to have an important role in sample storage. It showed that when sera were stored in plain tubes the tumor markers CA125 and CA19-9 were stable for up to 72 hours (from blood collection) at 4°C but the levels were significantly increased when stored in thixotropic gel-tubes for more than 24 hours at 4°C [24]. Studies of YKL-40, Tetranectin (TN) and macrophage colony-stimulating factor (OVX1) have shown the importance of understanding the sample handling and storage conditions for clinical determination [25–28]. For YKL-40 analyses the blood samples must be processed into serum within 3 hours or into EDTA plasma within 8 hours otherwise the blood samples need to be stored and shipped at 4°C [25]. Another study demonstrated that the OVX1 radioimmunoassay was highly dependent on sample handling, as the concentrations of OVX1 rose significantly when blood samples were allowed to clot at room temperature, but this effect was not seen if the samples were kept at 4°C [26]. Similar to our findings, both markers investigated in the above studies showed that serum samples once separated were stable for up to eight repetitive thawing and freezing cycles, and it is also important to store the samples at a very low temperature, such as ⫺80°C, to avoid protein degradation and sample evaporation. The previous studies, together with our study, show that it may be of importance to clarify if any pre-analytical factors influence the level of a biomarker before being examined in research studies. Also there must be a demand before implementing it in daily clinical use. In conclusion, no systematic difference could be demonstrated for HE4. CA125 is not dependent on processing time, EDTA plasma or serum. Levels of CA125 are significantly reduced at 4°C compared to levels at 20°C, but this difference was less than 6% and is not considered clinically relevant.
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Acknowledgements Abbott GmbH & Co. KG, Germany, provided study reagents for this study. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29. [2] Høgdall CK, Nielsen MLS. Taaning L. Year rapport 2011. Danish Gynecologic Cancer Database. A nationwide clinical database for cancer in ovaries, uterus and cervix. Lægeforeningens forlag, Copenhagen. ISNN: 1903-0223 (online) 2012; Table 14.1.1. [3] Engholm G, Storm HH, Ferlay J, Christensen N, Gjerstorff ML, Johannesen TB, Klint Å, Milter MC, Køtlum JE, Pukkala E, Ólafsdóttir E, NORDCAN: Cancer incidence, mortality, prevalence and survival in the Nordic countries, Version 4.0. Association of the Nordic Cancer Registries. Danish Cancer Society 2011. http://www-dep. iarc.fr/nordcan.htm. [4] Kræftprofil æggestokkræft 2000–2006. 2009. Sundhedsstyrelsen. http://www.sst.dk/publ/Publ2009/EPT/ Kraeftprofiler/Kraeftprofil_Aeggestokkraeft2000-2006.pdf. [5] O’Brien TJ, Beard JB, Underwood LJ, Dennis RA, Santin AD, York L. The CA 125 gene: an extracellular superstructure dominated by repeat sequences. Tumour Biol 2001;22:348–66. [6] Yin BW, Lloyd KO. Molecular cloning of the CA 125 ovarian cancer antigen: identification as a new mucin, MUC16. J Biol Chem 2001;276:27371–5. [7] Bast RC Jr, Feeney M, Lazarus H, Nadler LM, Colvin RB, Knapp RC. Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981;68:1331–7. [8] Kabawat SE, Bast RC Jr, Bhan AK, Welch WR, Knapp RC, Colvin RB. Tissue distribution of a coelomic-epitheliumrelated antigen recognized by the monoclonal antibody OC125. Int J Gynecol Pathol 1983;2:275–85. [9] Bast RC Jr, Knapp RC. Use of the CA 125 antigen in diagnosis and monitoring of ovarian carcinoma. Eur J Obstet Gynecol Reprod Biol 1985;19:354–6. [10] Einhorn N, Bast RC Jr, Knapp RC, Tjernberg B, Zurawski VR Jr. Preoperative evaluation of serum CA 125 levels in patients with primary epithelial ovarian cancer. Obstet Gynecol 1986;67:414–6. [11] Høgdall CK, Mogensen O, Tabor A, Mogensen B, Jakobsen AK, Nørgaard-Pedersen B, Larsen SO, Clemmensen I. The role of serum tetranectin, CA 125, and a combined index as tumor markers in women with pelvic tumors. Gynecol Oncol 1995;56:22–8. [12] Santillan A, Garg R, Zahurak ML, Gardner GJ, Giuntoli RL 2nd, Armstrong DK, Bristow RE. Risk of epithelial ovarian cancer recurrence in patients with rising serum CA-125 levels within the normal range. J Clin Oncol 2005;23:9338–43. [13] Duffy MJ, Bonfrer JM, Kulpa J, Rustin GJ, Soletormos G, Torre GC, Tuxen MK, Zwirner M. CA125 in ovarian cancer: European Group on Tumor Markers guidelines for clinical use. Int J Gynecol Cancer 2005;15:679–91. [14] Riedinger JM, Bonnetain F, Basuyau JP, Eche N, Larbre H, Dalifard I, Wafflart J, Ricolleau G, Pichon MF. Change in CA 125 levels after the first cycle of induction chemotherapy is an independent predictor of epithelial ovarian tumour outcome. Ann Oncol 2007;18:881–5.
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[15] Goonewardene YI, Hall MR, Rustin GJ. Management of asymptomatic patients on follow-up for ovarian cancer with rising CA 125 concentrations. Lancet Oncol 2007; 8:813–21. [16] Bouchard D, Morisset D, Bourbonnais Y, Tremblay GM. Proteins with whey-acidic-protein motifs and cancer. Lancet Oncol 2006;7:167–74. [17] Drapkin R, von Horsten HH, Lin Y, Mok SC, Crum CP, Welch WR, Hecht JL. Human epididymis protein 4 (HE4) is a secreted glycoprotein that is overexpressed by serous and endometrioid ovarian carcinomas. Cancer Res 2005;65: 2162–9. [18] Hellström I, Raycraft J, Hayden-Ledbetter M, Ledbetter JA, Schummer M, McIntosh M, Drescher C, Urban N, Hellström KE. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Res 2003;63:3695–700. [19] Moore RG, Brown AK, Miller MC, Skates S, Allard WJ, Verch T, Steinhoff M, Messerlian G, DiSilvestro P, Granai CO, Bast RC Jr. The use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol Oncol 2008;108:402–8. [20] Moore RG, McMeekin DS, Brown AK, DiSilvestro P, Miller MC, Allard WJ, Gajewski W, Kurman R, Bast RC Jr, Skates SJ. A novel multiple marker bioassay utilizing HE4 and CA125 for the prediction of ovarian cancer in patients with a pelvic mass. Gynecol Oncol 2009; 112:40–6. [21] Karlsen MA, Sandhu N, Høgdall C, Christensen IJ, Nedergaard L, Lundvall L, Engelholm SA, Pedersen AT, Hartwell D, Lydolph M, Laursen IA, Høgdall EV.
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Evaluation of HE4, CA125, risk of ovarian malignancy algorithm (ROMA) and risk of malignancy index (RMI) as diagnostic tools of epithelial ovarian cancer in patients with a pelvic mass. Gynecol Oncol 2012;127:379–83. Gao YC, Yuan ZB, Yang YD, Lu HK. Effect of freezethaw cycles on serum measurements of AFP, CEA, CA125 and CA19-9. Scand J Clin Lab Invest 2007;67:741–7. Sparholt MH, Høgdall CK, Nedergaard L, Heeran MC, Christensen IJ, Høgdall EV. Is tissue CA125 expression in epithelial ovarian adenocarcinoma heterogenic? APMIS 2013;121:38–44. Banfi G, Parma P, Pontillo M. Stability of tumor markers CA 19.9, CA 125, and CA 15.3 in serum obtained from plain tubes and tubes containing thixotropic gel separator. Clin Chem 1997;43:2430–1. Høgdall EV, Johansen JS, Kjaer SK, Price PA, Blaakjaer J, Høgdall CK. Stability of YKL-40 concentration in blood samples. Scand J Clin Lab Invest 2000;60:247–51. Hogdall EV, Hogdall CK, Kjaer SK, Xu F, Yu Y, Bast RC, Blaakaer J, Jacobs IJ. OVX1 radioimmunoassay results are dependent on the method of sample collection and storage. Clin Chem 1999;45:692–4. Begum FD, Høgdall E, Christensen IJ, Kjaer SK, Blaakaer J, Christensen L, Høgdall C. Serum tetranectin is a significant prognostic marker in ovarian cancer patients. Acta Obstet Gynecol Scand 2010;89:190–8. Høgdall CK, Høgdall EV, Hørding U, Clemmensen I, Nørgaard-Pedersen B, Toftager-Larsen K. Pre-operative plasma tetranectin as a prognostic marker in ovarian cancer patients. Scand J Clin Lab Invest 1993;53:741–6.
ORIGINAL ARTICLE
Stability of HE4 and CA125 in blood samples from patients diagnosed with ovarian cancer
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NOREEN SANDHU1, MONA A. KARLSEN2, CLAUS HØGDALL3, INGA A. LAURSEN1†, IB J. CHRISTENSEN4 & ESTRID V. S. HØGDALL2 1Department
of Clinical Biochemistry, Immunology & Genetics, Statens Serum Institut, Copenhagen, Denmark, 2Department of Pathology, Molecular Unit, Herlev Hospital, University of Copenhagen, Denmark, 3Gynecologic Clinic, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, and 4The Finsen Laboratory, Rigshospitalet, and Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Denmark Abstract Objective. To investigate the influence of handling and storage on HE4 and CA125 serum and EDTA plasma levels to clarify any important consequences for a clinical setting. Methods. Blood samples from 13 ovarian cancer (OC) patients were collected and allowed to clot or sediment for up to 72 hours at 4°C or 20°C, then processed into serum and EDTA plasma. Furthermore, the effects of up to eight repetitive cycles of freeze/thaw were investigated. HE4 and CA125 were analyzed using a Chemiluminescent Microparticle Immunoassay on the Architect i2000sr System. Results. No significant effect of processing time for HE4 could be shown. HE4 EDTA plasma levels were insignificantly lower (3%) than serum levels (p ⫽ 0.41). Similarly, no significant effect of processing time for CA125 could be demonstrated. CA125 levels at 4°C were significantly reduced compared to levels at 20°C (p ⫽ 0.024). No significant difference between CA125 serum and plasma levels were found (p ⫽ 0.46). Serum and EDTA plasma samples were stable during the eight cycles of freezing and thawing (CA125: all p ⬎ 0.2; HE4: all p ⬎ 0.5). Conclusion. No systematic difference could be demonstrated for HE4. CA125 is not dependent on processing time, EDTA plasma or serum. Levels of CA125 are significantly reduced at 4°C compared to levels at 20°C, but this difference was less than 6% and is not considered clinically relevant. Key Words: Biomarker, cancer antigen 125, chemiluminescent immunoassay, EDTA plasma, Human epididymis protein 4, serum, specimen handling
Introduction Ovarian cancer (OC) accounts for approximately 4% of all cancer cases and is the seventh most common cancer in women in the United States [1]. In Denmark, approximately 440 new cases of OC are found per year [2]. OC causes approximately 215 deaths each year which makes OC the fourth most frequent cancer-causing death among Danish women [3,4]. The high mortality rate of OC is primarily attributable to diagnosis of the disease when the cancer spreads beyond the ovaries. Early diagnosis is an important prognostic factor for substantial improvement in the survival rate of patients with OC.
Today the Cancer Antigen 125 (CA125) is the most common tumor marker used in clinical handling of OC. CA125 is encoded by the MUC16 gene [5,6] and is an antigenic determinant on a large transmembrane glycoprotein expressed by epithelial ovarian tumors [7,8]. Serum CA125 is used for risk estimation of women with a pelvic mass [9–11] as well as for monitoring of OC treatment response and progression [12–14]. However, CA125 has a low sensitivity for early stage disease in OC [13,15]. Human epididymis protein 4 (HE4) is a promising biomarkers for early detection of OC. Recent studies indicate that HE4 is highly expressed in OC tissues, and it has been detected in high levels in
†Deceased 10 December 2011. Correspondence: Estrid Høgdall, Department of Pathology, Molecular Unit, Herlev Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark. Tel: ⫹ 45 3868 9132. E-mail: [email protected]
(Received 6 June 2013 ; accepted 27 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.903430
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Figure 1. Flowchart for the handling procedure for the time study.
serum from OC patients [16–18]. HE4 is a single polypeptide containing two whey acidic fourdisulfide core (WFDC) domains [18]. The HE4 gene (WFDC2) codes for a 13kD protein, although in its mature glycosylated form the relative molecular weight is approximately 20–25 kD. It has been found that HE4 alone or in combination with CA125 has higher sensitivity and specificity for patients with early stage OC compared to CA125 alone [18–21]. The potential role in a differential setting should be examined in future trials. Before implementation of HE4 in clinical practice, alone or in combination with CA125 and/or other relevant markers/clinical information (e.g. menopausal status, age or other) the stability of the biomarkers must be clarified. The stability of HE4 has not been investigated in earlier studies but a study has shown that the stability of CA125 in serum samples was significantly affected by longterm frozen storage at ⫺40°C but least influenced by three short cycles of freezing and thawing [22]. For clinical determination, samples are often stored
at different conditions and transported from different hospitals to a central laboratory and up to a 3-day time period may be required for postal delivery. In view of this it is important to allow the blood samples to clot and sediment for up to 72 hours at different temperature, before being processed into serum or EDTA plasma. Our aim was to investigate the influence of up to eight freeze/thawing cycles of serum and EDTA plasma samples and the storage conditions at different temperatures on the level of the biomarkers CA125 and HE4. Materials and methods Patients All patients were recruited from the Gynecologic Clinic, Rigshospitalet, Denmark. All patients included in the study had pre-operative blood samples taken within 2 weeks prior to their operation for OC. We investigated the stability of HE4 and CA125 measurements in both serum and EDTA
Figure 2. Flowchart for the handling procedure for the freeze and thaw study.
463 55–10000 461 54–10000 433 51–10000 448 55–10000 435 50–10000 425 52–10000 435 54–10000 435 54–10000 429 46–10000 426 49–10000 433 50–10000 423 47–10000 435 49–10000 416 48–10000 435 48–10000 465 52–10000 456 51–10000 433 51–10000
440 52–10000 441 51–10000
332 97–4245 321 92–4004 319 83–4158 316 89–4279 310 88–3629 322 86–3603 305 88–4105 339 83–3592 314 83–3792
324 86–4641
328 94–3768 335 93–3873 328 92–3593 327 92–4103 327 91–3590 334 91–3926
24 Geometric mean (range) 8 Geometric mean (range) 72 Geometric mean (range)
1 Geometric mean (range)
3 Geometric mean (range)
20°C
24 Geometric mean (range) 8 Geometric mean (range)
331 89–3796 333 89–4008 337 93–4287
For each patient, a total of 40 measurements of HE4 and CA125 in serum and EDTA plasma were performed in duplicate using a Chemiluminescent Microparticle Immunoassay (CMIA) on ARCHITECT i2000sr System (Abbott Diagnostics, Wiesbaden, Germany). The assays and handling of reagents were carried out as recommended by the manufacturer. Analytical precision given by Abbott Diagnostics are for the Architect CA125 assay ⱕ 10% total CV with a measurement range of 1.0–1000 kU/L. The
3 Geometric mean (range)
Biochemical analysis
1 Geometric mean (range)
This study is approved by Copenhagen and Frederiksberg Scientific Ethical Committee by the journal number 01-101/02.
Temperature Hours
Ethics
4°C
The study material was comprised of a series of paired serum and EDTA plasma samples from 13 OC patients. About 30 ml of crude blood and 15 ml of blood added to 10 mM EDTA were collected from a cubical vein of each patient. The blood samples were collected in paired EDTA- tubes (K3E EDTA tubes from Vacuette, Greiner bio-one, Kremsmünster, Österreich) and serum tubes coated with micronized silica particles, which activate clotting (Vacuette, Greiner bio-one, Kremsmünster, Österreich). After separation, the sera and EDTA plasma were stored for 5 years in tightly capped Eppendorf tubes. The paired sets of these samples were allowed to clot and sediment in 1, 3, 8, 24 and 72 hours at 4°C or 20°C, before being processed into serum or EDTA plasma samples. Serum and EDTA plasma were separated by centrifugation at 2000 g for 10 mins, and stored at ⫺80°C in aliquots until further analysis. The aliquots were made after each time at the two different temperatures, a total of 10 aliquots (Figure 1). To investigate the effect of repetitive freezing (⫺80°C) and thawing, the serum and EDTA plasma samples were divided into eight paired 1 ml aliquots and stored overnight at ⫺80°C. The primary samples were analyzed and the following day seven of the sample sets were thawed at room temperature and re-frozen 1 hour after thawing. The following day, six sample sets were thawed and re-frozen. This was repeated for 5 additional days, each day taking one less sample set out for thawing and freezing until the last sample set had been frozen and thawed 8 times, a total of 16 aliquots (Figure 2).
Table I. HE4 and CA125 concentrations (mean and range) in EDTA plasma and serum blood samples handled and stored after 1, 3, 8, 24 and 72 hours at 4°C and 20°C.
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Sample handling
HE4 serum [pmol/L] HE4 EDTA plasma [pmol/L] CA125 serum [kU/L] CA125 EDTA plasma [kU/L]
72 Geometric mean (range)
plasma samples from the ‘Danish Heterogeneity study’ [23].
332 107–3821
Stability of HE4 and CA125 in blood samples
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Figure 3. Stability of HE4 in blood samples. This plot shows the ratio of HE4 concentration for each test condition compared to HE4 concentration in samples processed at 1 hour at 20°C with 95% confidence intervals.
dilution linearity was done by Abbott and the recoveries were from 96–110% (Abbott ref. 2K45). CA125 control ranges by Abbott (Ref. 2K45-10) are 28.0–52.0 kU/L (Low control), 210.0–390.0 kU/L (Medium control) and 455.0–845.0 kU/L (High control). Analytical precision for the Architect HE4 assay ⱕ 10% total CV is with a measurement range of
20.0–1500.0 pmol/L. The dilution linearity of HE4 was done by Abbott and the recoveries were from 91–105% (Abbott ref. 2P54). HE4 control ranges by Abbott (Ref. 2P54-10) are 35.0–65.0 pmol/L (Low control), 122.5–227.5 pmol/L (Medium control) and 490.0–910.0 pmol/L (High control). The study was conducted using one reagent lot. A calibration curve was performed for the kit before
Figure 4. Stability of CA125 in blood samples. This plot shows the ratio of CA125 concentration for each test condition compared to CA125 concentration in samples processed at 1 hour at 20°C with 95% confidence intervals.
Stability of HE4 and CA125 in blood samples
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Table II. HE4 and CA125 concentrations during eight repetitive freezing (⫺ 80°C) and thawing cycles measured in serum and EDTA plasma samples. Number of freeze cycles
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HE4 serum [pmol/L] HE4 EDTA plasma [pmol/L] CA125 serum [kU/L] CA125 EDTA plasma [kU/L]
1 2 3 4 5 6 7 8 Geometric Geometric Geometric Geometric Geometric Geometric Geometric Geometric mean (range) mean (range) mean (range) mean (range) mean (range) mean (range) mean (range) mean (range) 236 80–1530
239 81–1551
217 86–1556
244 81–1527
231 85–1558
237 81–1457
229 83–1555
228 81–1570
257 79–1409
239 86–1356
238 89–1386
232 87–1448
233 84–1454
236 93–1405
238 87–1441
235 88–1485
423 67–5584 382 67–6190
413 66–6439 427 66–5737
377 64–5159 427 61–5872
417 65–6342 420 63–5788
407 60–6842 413 65–5379
419 78–6191 419 63–6195
396 65–5543 421 64–6136
400 57–6098 411 65–6199
the start of the experiment. Subsequently, the three-level quality controls from Abbott for both CA125 and HE4 were tested each day to validate the calibration before analyzing the study samples. The measured concentrations of all the quality control samples were within the variation of maximum 10% during the study, which is our laboratory accept limit. Abbott recommends much larger ranges for their controls, as described. As a control we also compared CA125 values of six primary samples with results already measured in our Routine laboratory at time of diagnosis. Comparisons of the CA125 showed that the CA125 result were concordant (data not presented). Statistical analysis The statistical analysis was carried out using a linear model with repeated measures. Fixed effects are serum/EDTA plasma, temperature, and time, as well as the number of thaws. The estimated effects are
shown with 95% confidence limits and p-values. CA125 and HE4 concentrations were log transformed for analysis in order to obtain normally distributed data and back transformed for presentation. Any p values less than 5% were considered significant. Calculations were done using SAS (v9.2, SAS Institute, Cary, NC, USA). Results The mean values (geometric means) and ranges of HE4 and CA125 concentrations in EDTA plasma and serum samples from the 13 OC patients are shown in Table I. Analyzing HE4 on the log scale as a function of processing time, temperature, and sample type (EDTA plasma or serum), using mixed modelling with an interaction term between time and sample type could not demonstrate a significant interaction (Figure 3). In a model without interaction, no significant effect of processing time could be shown for times up to 72 hours compared
Figure 5. Stability of HE4 to freeze/thaw cycles. This plot shows the ratio of HE4 concentration at thaw eight times compared to HE4 concentration in samples stored at ⫺80°C and thawed one time, with 95% confidence intervals.
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Figure 6. Stability of CA125 to freeze/thaw cycles. This plot shows the ratio of CA125 concentration at thaw 8 times compared to CA125 concentration in samples stored at ⫺80°C and thawed one time, with 95% confidence intervals.
to HE4 level at 1 hour (Table I). The concentrations of HE4 EDTA plasma and HE4 serum did not differ significantly (Figure 3). No significant differences for processing time was detected (p ⫽ 0.17). Analyzing CA125 on the log scale as a function of processing time, temperature, and sample type (EDTA plasma or serum) using mixed modelling with an interaction term between processing time and sample type could not demonstrate a significant interaction (Figure 4). In a model without interaction, no significant effect of processing time could be demonstrated although a difference at 72 hours was detected (p ⫽ 0.049) (Figure 4). CA125 levels at 4°C were significantly reduced compared to levels at 20°C (p ⫽ 0.003), but this difference was less than 6%. No significant difference between CA125 serum and plasma levels were found (p ⫽ 0.46) (Figure 4). The estimated differences in EDTA plasma and serum levels for HE4 as well as for CA125 are relatively small, with most deviations less than 5% (Figures 3 and 4). Results of HE4 and CA125 concentrations (geometric mean and ranges) after eight repetitive freezing (⫺80°C) and thawing cycles of serum and EDTA plasma samples are shown in Table II. Serum and EDTA plasma samples were stable during the eight cycles of freezing and thawing as shown in Figure 5 and 6. The coefficient of variation for the concentration of HE4 after up to eight cycles of freezing and thawing in both serum and EDTA plasma samples were found to be 19.9% and 5.7%, respectively. In the case of CA125, after up to eight cycles of freezing and thawing the coefficient of variation were found to be 20.0% and 8.6% in serum and EDTA plasma samples, respectively. Results for the thaw/freeze for HE4 showed that there was no difference for the number of thaws (p ⫽ 0.66) or between sample type (p ⫽ 0.90). The
same analyses for CA125 gave non-significant results (p ⫽ 0.55 and p ⫽ 0.85 respectively). Again the differences for HE4 and CA125 are less than 5% for almost all cases (Figures 5 and 6). Discussion Before a biomarker is adopted by a clinical laboratory, it is mandatory to consider a possible impact of handling and storage conditions on the biological material, which may influence the level of the biomarker analyzed. Therefore, we initiated this study to examine stability of both HE4 and CA125 before initiating a larger research study. In this stability study of the biomarkers HE4 and CA125, we found both HE4 and CA125 to be very stable. No significant difference of HE4 was found dependent of handling temperature in contrast to CA125 where a significantly lower concentration was found in samples handled at 4°C compared to samples handled at room temperature. Therefore, it is important that the blood samples are strictly handled in order to compare analytical results. Both HE4 and CA125 concentrations were not impacted by up to eight freeze and thaw cycles. Our findings of a very high degree of stability for the markers CA125 and HE4 is very important and makes HE4 a relevant marker to study in collected and stored blood samples, as well as in future collections as no special demands for handling and storage are needed. The stability of HE4 has not been investigated in earlier studies, but the stability of CA125 has been investigated in freshly separated serum samples. These samples were frozen at ⫺40°C for three short repetitive freeze-thaw cycles and a long frozen storage of 10 months at ⫺40°C. The study showed that CA125 was significantly affected by the long-term frozen storage but least influenced by the three short
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Stability of HE4 and CA125 in blood samples cycles of freezing and thawing [22]. The significant decrease of CA125 concentration may indicate a degradation of the antigen. The limitations of our study could be the limited number of patients or the fact that samples were stored at ⫺80°C for 5 years before analysis. But since the samples were kept at ⫺80°C in tight capped Eppendorf tubes, the sample degradation and sample evaporation would probably be limited. Sample evaporation would result in an increase of the biomarker level as from six samples of serum CA125 which were measured at the time of the diagnoses. We also performed CA125 analyses together with the HE4 analyses. Comparisons of the CA125 showed that the CA125 results were concordant (data not presented). Studies concerning other cancer-related biomarkers have shown an increase of CA19-9 level after one freezing cycle at ⫺40°C [22]. In the study, the choice of tubes was also shown to have an important role in sample storage. It showed that when sera were stored in plain tubes the tumor markers CA125 and CA19-9 were stable for up to 72 hours (from blood collection) at 4°C but the levels were significantly increased when stored in thixotropic gel-tubes for more than 24 hours at 4°C [24]. Studies of YKL-40, Tetranectin (TN) and macrophage colony-stimulating factor (OVX1) have shown the importance of understanding the sample handling and storage conditions for clinical determination [25–28]. For YKL-40 analyses the blood samples must be processed into serum within 3 hours or into EDTA plasma within 8 hours otherwise the blood samples need to be stored and shipped at 4°C [25]. Another study demonstrated that the OVX1 radioimmunoassay was highly dependent on sample handling, as the concentrations of OVX1 rose significantly when blood samples were allowed to clot at room temperature, but this effect was not seen if the samples were kept at 4°C [26]. Similar to our findings, both markers investigated in the above studies showed that serum samples once separated were stable for up to eight repetitive thawing and freezing cycles, and it is also important to store the samples at a very low temperature, such as ⫺80°C, to avoid protein degradation and sample evaporation. The previous studies, together with our study, show that it may be of importance to clarify if any pre-analytical factors influence the level of a biomarker before being examined in research studies. Also there must be a demand before implementing it in daily clinical use. In conclusion, no systematic difference could be demonstrated for HE4. CA125 is not dependent on processing time, EDTA plasma or serum. Levels of CA125 are significantly reduced at 4°C compared to levels at 20°C, but this difference was less than 6% and is not considered clinically relevant.
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Acknowledgements Abbott GmbH & Co. KG, Germany, provided study reagents for this study. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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