Comparison Between a Manual Squamous Cell Carcinoma Antigen Assay and an Automated Assay in a Clinical Setting

Comparison Between a Manual Squamous Cell Carcinoma Antigen Assay and an Automated Assay in a... Abstract Objective To compare a manual squamous cell carcinoma antigen (SCCA) assay and an automated SCCA assay in a clinical setting. Methods We included, in this study, a total of 158 specimens that had been tested using the manual SCCA assay. The CanAg SCC EIA assay and the Elecsys SCC were compared for their clinical settings. Results Within-run and between-day coefficients of variation (CVs, %) were lower than 3%. Comparison of the manual and automated SCCA assays yielded good correlation. The correlation coefficient (R2) between the CanAg SCC EIA and Elecsys SCC assay results was 0.989 (P <.001) and the overall concordance rate was 94.3%. Conclusions We report that the Elecsys SCC automated SCCA assay yielded performance comparable to that of the manual SCCA assay: the automated assay reduced the number of manual steps and test turnaround time. squamous cell carcinoma antigen (SCCA), comparison, performance, Elecsys SCC, CanAg SCC, clinical setting Squamous cell carcinoma antigen (SCCA) is a glycoprotein with a molecular weight ranging from 45 kDa to 55 kDa that is produced in normal squamous epithelial skin cells1 and acts as a serine protease inhibitor.2 SCCA was first isolated from squamous carcinoma tissue of the uterine cervix1 and has more than 10 isoforms that are classified as acidic (pI <6.25) or neutral (pI≥ 6.25).3 Neutral SCCA proteins normally reside in cytosol, but acidic SCCA proteins are easily released from cytosol and are the main cause of elevated levels of SCCA in serum,2,3 which are observed in epithelial cancer of the neck, lungs, and cervix.4 Further, SCCA seems to be related to the invasion and metastasis of squamous cell cancers5 and thus, serum SCCA is used to monitor patients with squamous-cell carcinoma.6,7 However, some controversy exists regarding associations between serum SCCA level and tumor stage, size, and progression in squamous cell carcinoma of the cervix or esophagus.8–10 Therefore, accurate measurement of serum SCCA concentration is an important management issue for patients with cancer. In this study, we compared the diagnostic performance of the manual CanAg SCC enzyme immunoassay (EIA) (Fujirebio Diagnostics AB) and automated Elecsys SCC assay performed with a Modular Analytics E170 analyzer (Roche Diagnostics) in a clinical setting. Materials and Methods Patients and Specimens A total of 158 specimens from 140 individuals who had been tested using the manual SCCA assay were included; for validation purposes, their test results were compared with those obtained using the automated SCCA test (126 women, 32 men). The 140 individuals were classified into 2 groups by clinical diagnosis obtained by medical record review. Group 1 consisted of 104 patients with cancer and group 2 was comprised of 36 healthy control individuals. In group 1, patients had the following diagnoses: malignant neoplasm of the cervix (n = 84), endometrium (n = 3), esophagus (n = 7), lung (n = 3), anal canal (n = 3), stomach (n = 1), glottis (n = 1), neck (n = 1), and skin (n = 1). Manual SCCA Assay: CanAg SCC EIA CanAg SCC EIA is a test that uses antibodies and a color change to identify SCCA in serum. Specimen antigen material is attached to the primary antibody. Additional specific antibodies are then applied to the surface to bind the antigen. A substance containing enzyme substrate is added, and subsequent reactions cause a color change in the substrate. Reagents and specimens were allowed to reach an ambient temperature 20° to 25° before use, and the assay was performed at 20° to 25° according to the manufacturer-provided instructions. First, microplate strips were placed in a strip frame. Second, each strip was washed once with wash solution. After washing, strips were marked for identification purposes, and SCC calibrators (CAL A, B, C, D, or E; 25 µL) and patient specimens were pipetted into the strip wells. Antibody solution (100 µL) was then added to each well using a 100-µL precision pipette, and the microplate was incubated for 1 hour at room temperature (20° to 25°) with constant shaking using a microplate shaker. Strips were then washed 6 times as instructed. Tetramethylbenzidine-horseradish peroxidase (TMB HRP) substrate (100 µL) was then added as quickly as possible (the time between adding substrate to the first and last wells was <5 minutes). The plate was then incubated for 30 minutes at room temperature with constant shaking, while avoiding direct sunlight. Finally, absorbance was read at 620 nm using a microplate spectrophotometer. The measurement range of this assay is 0.3 to 50 ng per mL, and results of 1.5 ng per mL or greater are deemed to be positive. Elecsys SCC Automated SCCA Assay The Elecsys SCC is an assay intended for the in vitro determination of SCCA levels in serum or plasma by electrochemiluminescence immunoassay (ECLIA) using the Modular Analytics E170. During the first incubation, 15 µL of serum is incubated with biotinylated monoclonal SCC-specific antibodies. Streptavidin-coated microparticles and SCC-specific antibodies marked with ruthenium complex are then added to form a complex by solid-phase interaction between biotin and streptavidin. Reaction mixtures are placed in measuring cells and microparticles are held on the surface of an electrode by a magnet. Unbound reagent components are removed by washing with Elecsys ProCell M. Current flow through the surface of the electrode generates an electrochemiluminescence signal, which is quantified using a photomultiplier. The measurement range of this assay is 0.1 to 70 ng per mL, and results of 2.6 ng per mL or greater are deemed to be positive. The basic characteristics of the 2 assays are described in Table 1. Table 1. Characteristics of Elecsys SCC Assay and the CanAg SCC EIA Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer SCC, squamous cell carcinoma; EIA, enzyme immunoassay. View Large Table 1. Characteristics of Elecsys SCC Assay and the CanAg SCC EIA Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer SCC, squamous cell carcinoma; EIA, enzyme immunoassay. View Large Precision The precision of the Elecsys SCC was determined according to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 using 2 quality control materials: PreciControl Lung Cancer, version 1 (PC LC1) and version 2 (PC LC2), provided by Roche Diagnostics. Replicate testing was performed 5 times per day for 5 days. Linearity Linearity of the Elecsys SCC was determined according to the CLSI EP6-A guideline. For the linearity study, low and high serum concentrations (0.535 and 42.075 ng/mL, respectively) were serially diluted at 1:0, 3:1, 1:1, 1:3, and 0:1 and analyzed twice. Method Comparison We compared the performance of the Elecsys SCC assay with that of the CanAg SCC EIA. The results of the 158 specimens were analyzed and compared. The true-positive SCCA results were determined by the diagnosis, based on considerations of clinical, radiological, and pathology evaluations by physicians. Statistical Analysis We performed statistical analyses using EP Evaluator software, version 11.2.0.23 (Data Innovations LLC) and MedCalc for Windows, version 17.9.2 (MedCalc Software). Passing-Bablok and Bland Altman analysis were used to compare the automated assay and the manual method. Agreement between the 2 methods was evaluated using κ coefficients, which were categorized as follows: very good (0.81–1.0), good (0.61–0.8), moderate (0.41–0.6), fair (0.21–0.4), or poor (0–0.2).11 Results Serum SCCA Concentrations Results obtained using the CanAg SCC EIA and the Elecsys SCC assay were 0.2 to 50.47 ng per mL and 0.468 to 70 ng per mL for group 1 and 0.2 to 3 ng per mL and 0.525 to 7.2 ng per mL for group 2, respectively. Precision In this study, within-run coefficient of variation (CV, %) variabilities of Elecsys SCC were 2.3% at 1.725 ng per mL and 2.0% at 19.904 ng per mL (Table 2). Between-day CVs (%) were 1.9% and 2.5% for PC LC1 and PC LC2, respectively. Table 2. CVs of Elecsys SCC Assaya Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 CVs, coefficients of variance; PC LC1, PC LC1, PreciControl Lung Cancer control material, version 1; PC LC2, PreciControl Lung Cancer control material, version 2. aManufactured by Roche Diagnostics. View Large Table 2. CVs of Elecsys SCC Assaya Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 CVs, coefficients of variance; PC LC1, PC LC1, PreciControl Lung Cancer control material, version 1; PC LC2, PreciControl Lung Cancer control material, version 2. aManufactured by Roche Diagnostics. View Large Linearity The linearity of Elecsys SCC was analyzed on a Modular E170 over the range 0.5350 to 42.075 ng per mL. In this range, Elecsys SCC produced linear results. Regression analysis gave a slope of 1.021 and an intercept of 0.0011 (Figure 1) with a R2 value of 0.999 (P <.001). Figure 1 View largeDownload slide Results of linearity and regression analysis for the Elecsys SCC instrument (Roche Diagnostics). Figure 1 View largeDownload slide Results of linearity and regression analysis for the Elecsys SCC instrument (Roche Diagnostics). Method Comparison The positive predictive value of Elecsys SCC for the diagnosis of malignant neoplasms was 81.82% (95% confidence interval [CI], 66.81% to 90.96%), compared with 86.11% (72.21% to 93.67%) for the CanAg SCC EIA. Passing-Bablok regression analysis showed no significant deviation from linearity between the manual and the automated SCC assay (P = .07) and a slope of 1.89 (95% CI, 1.75 to 2.05) and intercept of 0.366 (95% CI, 0.272 to 0.454) (Figure 2A). The Bland-Altman plot of the 158 values obtained using the 2 assays revealed a mean ratio of 2.81 (Figure 2B). The R2 value between the CanAg SCC EIA and the Elecsys SCC assay was 0.989 (P<.001). The percentage agreement between the 2 SCCA assays was 94.3%, and the κ value was 0.833 (95% CI, 0.728 to 0.938). Accordingly, agreement between the automated and manual assay was considered to be very good. Figure 2 View largeDownload slide Analysis results for Elecsys SCC assay (Roche Diagnostics) vs CanAg SCC enzyme immunoassay (EIA; Fujirebio Diagnostics AB). A, Results of Passing-Bablock regression analysis. B, Bland-Altman plot. Figure 2 View largeDownload slide Analysis results for Elecsys SCC assay (Roche Diagnostics) vs CanAg SCC enzyme immunoassay (EIA; Fujirebio Diagnostics AB). A, Results of Passing-Bablock regression analysis. B, Bland-Altman plot. Discussion The first SCCA assay was conducted by Kato and Torigoe in 1977.1 Today, SCCA expression increases in many squamous cell carcinomas, including uterine, cervix, lung, head, neck, esophagus, and anal canal, as well as in several nonmalignant skin disorders and renal failure.12–17 In our hospital, the manual CanAg SCC EIA was used for determining serum SCCA levels before the automated Elecsys SCCA assay became available. However, this manual assay has some weak points. First, it requires more time than the automated assay; for instance, it requires a 1-hour incubation step and another 30 minutes for washing. Second, assay results are dependent on the profiency of each tester. Therefore, we evaluated the automated Elecsys SCC assay using a Modular Analytics E170. The precision CV (%) of the Elecsys SCC was determined to be lower than 3%, and the R2 value and overall concordance between the 2 assays was rated as being good. The κ agreement between the 2 assays was considered to be very good. Some limitations of this study warrant consideration. First, a relatively small number (n = 39) of SCCA specimens yielded positive results. Serum SCCA concentration diminishes after disease remission, and this tendency contributes to the difficulties in obtaining SCCA specimens with positive results. Second, test specimens were not age- and sex-matched, and as such, the results of pathologic biopsies of specimens could not be thoroughly matched in this study. Of note, the 9 cases from 8 individuals showed discordant results between the manual and automated assays (Table 3). Of the 9 cases, 3 specimens tested negative via manual assay but positive via automated assay, and the other 6 tested positive via manual assay and negative via automated assay. These discordant cases originated from 6 individuals with a diagnosis of cervical cancer, 1 individual with a diagnosis of endometrial and esophageal cancer, and 1 individual with normal results on annual general health checkup. Of the 9 cases, we were able to test the 6 discrepant specimens using a chemiluminesecent microparticle immunoassay method using ARCHITECT i2000 (Abbott Laboratories, Inc.) for confirmation. Table 3. Agreement Between Elecsys SCC Assay and CanAg EIA SCC Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 SCC, squamous cell carcinoma; EIA, enzyme immunoassay; SCCA, squamous cell carcinoma antigen; EIA, enzyme immunoassay. aCanAg SCC EIA is manufactured by Fujirebio Diagnostics AB. bManufactured by Roche Diagnostics. View Large Table 3. Agreement Between Elecsys SCC Assay and CanAg EIA SCC Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 SCC, squamous cell carcinoma; EIA, enzyme immunoassay; SCCA, squamous cell carcinoma antigen; EIA, enzyme immunoassay. aCanAg SCC EIA is manufactured by Fujirebio Diagnostics AB. bManufactured by Roche Diagnostics. View Large Those 6 discrepant specimens showed all positive SCC results via the manual assay and negative SCC results via the automated assay but all positive SCC results via CMIA testing with the ARCHITECT i2000. Of these 6 discrepant results, we could only obtain 2 biopsy results within 2 days from SCCA tests: 1 was a squamous cell carcinoma and the other was a reactive cellular change. When we thoroughly reviewed the discrepant cases of manual and automated SCC, the levels of each test were almost near the cutoff range. In fact, the 6 manual tests yielded slightly higher levels (mean [SD], 1.8 [0.4]) than the CanAg SCC cutoff level (1.5 ng/mL), and the automated tests showed slightly lower levels (mean [SD], 1.7 [0.3]) than the Elecsys SCC cut-off level (2.6 ng/mL). The false negativity and positivity of SCC test results should be warranted, although those results may have been caused by specimen contamination. In fact, Chauvel et al18 reported the possibility of false positives due to specimen contamination by saliva, skin particles, sweat, or sneeze-generated aerosol particles during SCC testing. Also, the variances in enrolled patients might impact these discrepancies. These discrepancies need to be explored in further large-scale prospective studies. In a previous study, the performance of the Elecsys SCC assay was compared with that of another automated assay, the BRAHMS SCC assay, which was conducted on a BRAHMS Kryptor (Thermo Fisher Scientific Inc.) based on time-resolved amplified cryptate emission.18 It was reported that the Elecsys SCC assay had within-run CVs of 3% for PreciControl Lung Cancer, version 1 (PC LC1) and 2.4% for PreciControl Lung Cancer, version 2 (PC LC2), between-day CVs of 7% for PC LC1 and 3.4% for PC LC2, exhibited linearity (y = 1.193x + 0.14), and had an analytical performance similar with those of our present study. To our knowledge, the automated Roche Elecsys SCC was not widely compared with various SCC assays. This report constitutes a performance comparison of SCC tests in clinical settings. In conclusion, we compared the manual CanAg SCC and automated Elecsys SCC assay and discovered that they have comparable performances. The automated assay reduces turnaround time and workload to a greater degree than the manual assay. Also, the automated SCCA assay probably can reduce the variability of each tester. However, the possibility of false-negative and false-positive results warranted further well-designed prospective large-scale studies. Abbreviations: SCCA squamous cell carcinoma antigen EIA enzyme immunoassay TMB HRP tetramethylbenzidine-horseradish peroxidase ECLIA electrochemiluminescence immunoassay CLSI Clinical and Laboratory Standards Institute PC LC1 PreciControl Lung Cancer, version 1 PC LC2 PreciControl Lung Cancer, version 2 CV coefficient of variation CI confidence interval References 1. Kato H , Torigoe T . Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma . Cancer . 1977 ; 40 ( 4 ): 1621 – 1628 . 2. Suminami Y , Nawata S , Kato H . Biological role of SCC antigen . Tumour Biol . 1998 ; 19 ( 6 ): 488 – 493 . 3. Kato H , Nagaya T , Torigoe T . Heterogeneity of a tumor antigen TA-4 of squamous cell carcinoma in relation to its appearance in the circulation . Gan . 1984 ; 75 ( 5 ): 433 – 435 . 4. Soyemi OM , Otegbayo JA , Ola SO , Akere A , Soyemi T . Comparative diagnostic efficacy of serum squamous cell carcinoma antigen in hepatocellular carcinoma . BMC Res Notes . 2012 ; 5 : 403 . 5. Erickson JA , Lu J , Smith JJ , Bornhorst JA , Grenache DG , Ashwood ER . Immunoassay for quantifying squamous cell carcinoma antigen in serum . Clin Chem . 2010 ; 56 ( 9 ): 1496 – 1499 . 6. Torre GC . SCC antigen in malignant and nonmalignant squamous lesions . Tumour Biol . 1998 ; 19 ( 6 ): 517 – 526 . 7. Micke O , Bruns F , Schäfer U , Prott FJ , Willich N . The impact of squamous cell carcinoma (SCC) antigen in patients with advanced cancer of uterine cervix treated with (chemo-)radiotherapy . Anticancer Res . 2005 ; 25 ( 3A ): 1663 – 1666 . 8. Gadducci A , Cosio S , Carpi A , Nicolini A , Genazzani AR . Serum tumor markers in the management of ovarian, endometrial and cervical cancer . Biomed Pharmacother . 2004 ; 58 ( 1 ): 24 – 38 . 9. Shimada H , Nabeya Y , Okazumi S , et al. Prediction of survival with squamous cell carcinoma antigen in patients with resectable esophageal squamous cell carcinoma . Surgery . 2003 ; 133 ( 5 ): 486 – 494 . 10. Bolger BS , Dabbas M , Lopes A , Monaghan JM . Prognostic value of preoperative squamous cell carcinoma antigen level in patients surgically treated for cervical carcinoma . Gynecol Oncol . 1997 ; 65 ( 2 ): 309 – 313 . 11. Landis JR , Koch GG . The measurement of observer agreement for categorical data . Biometrics . 1977 ; 33 ( 1 ): 159 – 174 . 12. Ayude D , Gacio G , Páez de la Cadena M , et al. Combined use of established and novel tumour markers in the diagnosis of head and neck squamous cell carcinoma . Oncol Rep . 2003 ; 10 ( 5 ): 1345 – 1350 . 13. Crombach G , Scharl A , Vierbuchen M , Würz H , Bolte A . Detection of squamous cell carcinoma antigen in normal squamous epithelia and in squamous cell carcinomas of the uterine cervix . Cancer . 1989 ; 63 ( 7 ): 1337 – 1342 . 14. Kosugi S , Nishimaki T , Kanda T , Nakagawa S , Ohashi M , Hatakeyama K . Clinical significance of serum carcinoembryonic antigen, carbohydrate antigen 19-9, and squamous cell carcinoma antigen levels in esophageal cancer patients . World J Surg . 2004 ; 28 ( 7 ): 680 – 685 . 15. Molina R , Filella X , Auge J , et al. Tumor markers (CEA, CA 125, CYFRA 21-1, SCC and NSE) in patients with non-small cell lung cancer as an aid in histological diagnosis and prognosis . Tumour Biol . 2003 ; 24 ( 4 ): 209 – 218 . 16. Petrelli NJ , Palmer M , Herrera L , Bhargava A . The utility of squamous cell carcinoma antigen for the follow-up of patients with squamous cell carcinoma of the anal canal . Cancer . 1992 ; 70 ( 1 ): 35 – 39 . 17. Trapé J , Filella X , Alsina-Donadeu M , Juan-Pereira L , Bosch-Ferrer À , Rigo-Bonnin R . Increased plasma concentrations of tumour markers in the absence of neoplasia . Clin Chem Lab Med . 2011 ; 49 ( 10 ): 1605 – 1620 . 18. Chauvel C , Jordan P , Popovici T , Bories P-N . Evaluation of the new Elecsys SCC assay: comparison with the Kryptor SCC assay . Clin Chem Lab Med . 2018 ; 56 ( 2 ): e56 – e58 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Laboratory Medicine Oxford University Press

Comparison Between a Manual Squamous Cell Carcinoma Antigen Assay and an Automated Assay in a Clinical Setting

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Abstract

Abstract Objective To compare a manual squamous cell carcinoma antigen (SCCA) assay and an automated SCCA assay in a clinical setting. Methods We included, in this study, a total of 158 specimens that had been tested using the manual SCCA assay. The CanAg SCC EIA assay and the Elecsys SCC were compared for their clinical settings. Results Within-run and between-day coefficients of variation (CVs, %) were lower than 3%. Comparison of the manual and automated SCCA assays yielded good correlation. The correlation coefficient (R2) between the CanAg SCC EIA and Elecsys SCC assay results was 0.989 (P <.001) and the overall concordance rate was 94.3%. Conclusions We report that the Elecsys SCC automated SCCA assay yielded performance comparable to that of the manual SCCA assay: the automated assay reduced the number of manual steps and test turnaround time. squamous cell carcinoma antigen (SCCA), comparison, performance, Elecsys SCC, CanAg SCC, clinical setting Squamous cell carcinoma antigen (SCCA) is a glycoprotein with a molecular weight ranging from 45 kDa to 55 kDa that is produced in normal squamous epithelial skin cells1 and acts as a serine protease inhibitor.2 SCCA was first isolated from squamous carcinoma tissue of the uterine cervix1 and has more than 10 isoforms that are classified as acidic (pI <6.25) or neutral (pI≥ 6.25).3 Neutral SCCA proteins normally reside in cytosol, but acidic SCCA proteins are easily released from cytosol and are the main cause of elevated levels of SCCA in serum,2,3 which are observed in epithelial cancer of the neck, lungs, and cervix.4 Further, SCCA seems to be related to the invasion and metastasis of squamous cell cancers5 and thus, serum SCCA is used to monitor patients with squamous-cell carcinoma.6,7 However, some controversy exists regarding associations between serum SCCA level and tumor stage, size, and progression in squamous cell carcinoma of the cervix or esophagus.8–10 Therefore, accurate measurement of serum SCCA concentration is an important management issue for patients with cancer. In this study, we compared the diagnostic performance of the manual CanAg SCC enzyme immunoassay (EIA) (Fujirebio Diagnostics AB) and automated Elecsys SCC assay performed with a Modular Analytics E170 analyzer (Roche Diagnostics) in a clinical setting. Materials and Methods Patients and Specimens A total of 158 specimens from 140 individuals who had been tested using the manual SCCA assay were included; for validation purposes, their test results were compared with those obtained using the automated SCCA test (126 women, 32 men). The 140 individuals were classified into 2 groups by clinical diagnosis obtained by medical record review. Group 1 consisted of 104 patients with cancer and group 2 was comprised of 36 healthy control individuals. In group 1, patients had the following diagnoses: malignant neoplasm of the cervix (n = 84), endometrium (n = 3), esophagus (n = 7), lung (n = 3), anal canal (n = 3), stomach (n = 1), glottis (n = 1), neck (n = 1), and skin (n = 1). Manual SCCA Assay: CanAg SCC EIA CanAg SCC EIA is a test that uses antibodies and a color change to identify SCCA in serum. Specimen antigen material is attached to the primary antibody. Additional specific antibodies are then applied to the surface to bind the antigen. A substance containing enzyme substrate is added, and subsequent reactions cause a color change in the substrate. Reagents and specimens were allowed to reach an ambient temperature 20° to 25° before use, and the assay was performed at 20° to 25° according to the manufacturer-provided instructions. First, microplate strips were placed in a strip frame. Second, each strip was washed once with wash solution. After washing, strips were marked for identification purposes, and SCC calibrators (CAL A, B, C, D, or E; 25 µL) and patient specimens were pipetted into the strip wells. Antibody solution (100 µL) was then added to each well using a 100-µL precision pipette, and the microplate was incubated for 1 hour at room temperature (20° to 25°) with constant shaking using a microplate shaker. Strips were then washed 6 times as instructed. Tetramethylbenzidine-horseradish peroxidase (TMB HRP) substrate (100 µL) was then added as quickly as possible (the time between adding substrate to the first and last wells was <5 minutes). The plate was then incubated for 30 minutes at room temperature with constant shaking, while avoiding direct sunlight. Finally, absorbance was read at 620 nm using a microplate spectrophotometer. The measurement range of this assay is 0.3 to 50 ng per mL, and results of 1.5 ng per mL or greater are deemed to be positive. Elecsys SCC Automated SCCA Assay The Elecsys SCC is an assay intended for the in vitro determination of SCCA levels in serum or plasma by electrochemiluminescence immunoassay (ECLIA) using the Modular Analytics E170. During the first incubation, 15 µL of serum is incubated with biotinylated monoclonal SCC-specific antibodies. Streptavidin-coated microparticles and SCC-specific antibodies marked with ruthenium complex are then added to form a complex by solid-phase interaction between biotin and streptavidin. Reaction mixtures are placed in measuring cells and microparticles are held on the surface of an electrode by a magnet. Unbound reagent components are removed by washing with Elecsys ProCell M. Current flow through the surface of the electrode generates an electrochemiluminescence signal, which is quantified using a photomultiplier. The measurement range of this assay is 0.1 to 70 ng per mL, and results of 2.6 ng per mL or greater are deemed to be positive. The basic characteristics of the 2 assays are described in Table 1. Table 1. Characteristics of Elecsys SCC Assay and the CanAg SCC EIA Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer SCC, squamous cell carcinoma; EIA, enzyme immunoassay. View Large Table 1. Characteristics of Elecsys SCC Assay and the CanAg SCC EIA Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer Parameters Elecsys SCC CanAg SCC EIA Manufacturer Roche Diagnostics Fujirebio Diagnostics AB Specimen type Serum, plasma Serum Specimen volume 15 µL 25 µL Measuring range (ng/mL) 0.1–70 0.3–50 Cutoff value (ng/mL) 2.6 1.5 Results within 18 min 2 h Analyzing device Modular Analytics E170 PR 3100 microplate spectrophotometer SCC, squamous cell carcinoma; EIA, enzyme immunoassay. View Large Precision The precision of the Elecsys SCC was determined according to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 using 2 quality control materials: PreciControl Lung Cancer, version 1 (PC LC1) and version 2 (PC LC2), provided by Roche Diagnostics. Replicate testing was performed 5 times per day for 5 days. Linearity Linearity of the Elecsys SCC was determined according to the CLSI EP6-A guideline. For the linearity study, low and high serum concentrations (0.535 and 42.075 ng/mL, respectively) were serially diluted at 1:0, 3:1, 1:1, 1:3, and 0:1 and analyzed twice. Method Comparison We compared the performance of the Elecsys SCC assay with that of the CanAg SCC EIA. The results of the 158 specimens were analyzed and compared. The true-positive SCCA results were determined by the diagnosis, based on considerations of clinical, radiological, and pathology evaluations by physicians. Statistical Analysis We performed statistical analyses using EP Evaluator software, version 11.2.0.23 (Data Innovations LLC) and MedCalc for Windows, version 17.9.2 (MedCalc Software). Passing-Bablok and Bland Altman analysis were used to compare the automated assay and the manual method. Agreement between the 2 methods was evaluated using κ coefficients, which were categorized as follows: very good (0.81–1.0), good (0.61–0.8), moderate (0.41–0.6), fair (0.21–0.4), or poor (0–0.2).11 Results Serum SCCA Concentrations Results obtained using the CanAg SCC EIA and the Elecsys SCC assay were 0.2 to 50.47 ng per mL and 0.468 to 70 ng per mL for group 1 and 0.2 to 3 ng per mL and 0.525 to 7.2 ng per mL for group 2, respectively. Precision In this study, within-run coefficient of variation (CV, %) variabilities of Elecsys SCC were 2.3% at 1.725 ng per mL and 2.0% at 19.904 ng per mL (Table 2). Between-day CVs (%) were 1.9% and 2.5% for PC LC1 and PC LC2, respectively. Table 2. CVs of Elecsys SCC Assaya Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 CVs, coefficients of variance; PC LC1, PC LC1, PreciControl Lung Cancer control material, version 1; PC LC2, PreciControl Lung Cancer control material, version 2. aManufactured by Roche Diagnostics. View Large Table 2. CVs of Elecsys SCC Assaya Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 Control Materials Within-Run CV (%) Between-Day CV (%) Total CV (%) PC LC1 2.3 1.9 2.9 PC LC2 2.0 1.4 2.5 CVs, coefficients of variance; PC LC1, PC LC1, PreciControl Lung Cancer control material, version 1; PC LC2, PreciControl Lung Cancer control material, version 2. aManufactured by Roche Diagnostics. View Large Linearity The linearity of Elecsys SCC was analyzed on a Modular E170 over the range 0.5350 to 42.075 ng per mL. In this range, Elecsys SCC produced linear results. Regression analysis gave a slope of 1.021 and an intercept of 0.0011 (Figure 1) with a R2 value of 0.999 (P <.001). Figure 1 View largeDownload slide Results of linearity and regression analysis for the Elecsys SCC instrument (Roche Diagnostics). Figure 1 View largeDownload slide Results of linearity and regression analysis for the Elecsys SCC instrument (Roche Diagnostics). Method Comparison The positive predictive value of Elecsys SCC for the diagnosis of malignant neoplasms was 81.82% (95% confidence interval [CI], 66.81% to 90.96%), compared with 86.11% (72.21% to 93.67%) for the CanAg SCC EIA. Passing-Bablok regression analysis showed no significant deviation from linearity between the manual and the automated SCC assay (P = .07) and a slope of 1.89 (95% CI, 1.75 to 2.05) and intercept of 0.366 (95% CI, 0.272 to 0.454) (Figure 2A). The Bland-Altman plot of the 158 values obtained using the 2 assays revealed a mean ratio of 2.81 (Figure 2B). The R2 value between the CanAg SCC EIA and the Elecsys SCC assay was 0.989 (P<.001). The percentage agreement between the 2 SCCA assays was 94.3%, and the κ value was 0.833 (95% CI, 0.728 to 0.938). Accordingly, agreement between the automated and manual assay was considered to be very good. Figure 2 View largeDownload slide Analysis results for Elecsys SCC assay (Roche Diagnostics) vs CanAg SCC enzyme immunoassay (EIA; Fujirebio Diagnostics AB). A, Results of Passing-Bablock regression analysis. B, Bland-Altman plot. Figure 2 View largeDownload slide Analysis results for Elecsys SCC assay (Roche Diagnostics) vs CanAg SCC enzyme immunoassay (EIA; Fujirebio Diagnostics AB). A, Results of Passing-Bablock regression analysis. B, Bland-Altman plot. Discussion The first SCCA assay was conducted by Kato and Torigoe in 1977.1 Today, SCCA expression increases in many squamous cell carcinomas, including uterine, cervix, lung, head, neck, esophagus, and anal canal, as well as in several nonmalignant skin disorders and renal failure.12–17 In our hospital, the manual CanAg SCC EIA was used for determining serum SCCA levels before the automated Elecsys SCCA assay became available. However, this manual assay has some weak points. First, it requires more time than the automated assay; for instance, it requires a 1-hour incubation step and another 30 minutes for washing. Second, assay results are dependent on the profiency of each tester. Therefore, we evaluated the automated Elecsys SCC assay using a Modular Analytics E170. The precision CV (%) of the Elecsys SCC was determined to be lower than 3%, and the R2 value and overall concordance between the 2 assays was rated as being good. The κ agreement between the 2 assays was considered to be very good. Some limitations of this study warrant consideration. First, a relatively small number (n = 39) of SCCA specimens yielded positive results. Serum SCCA concentration diminishes after disease remission, and this tendency contributes to the difficulties in obtaining SCCA specimens with positive results. Second, test specimens were not age- and sex-matched, and as such, the results of pathologic biopsies of specimens could not be thoroughly matched in this study. Of note, the 9 cases from 8 individuals showed discordant results between the manual and automated assays (Table 3). Of the 9 cases, 3 specimens tested negative via manual assay but positive via automated assay, and the other 6 tested positive via manual assay and negative via automated assay. These discordant cases originated from 6 individuals with a diagnosis of cervical cancer, 1 individual with a diagnosis of endometrial and esophageal cancer, and 1 individual with normal results on annual general health checkup. Of the 9 cases, we were able to test the 6 discrepant specimens using a chemiluminesecent microparticle immunoassay method using ARCHITECT i2000 (Abbott Laboratories, Inc.) for confirmation. Table 3. Agreement Between Elecsys SCC Assay and CanAg EIA SCC Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 SCC, squamous cell carcinoma; EIA, enzyme immunoassay; SCCA, squamous cell carcinoma antigen; EIA, enzyme immunoassay. aCanAg SCC EIA is manufactured by Fujirebio Diagnostics AB. bManufactured by Roche Diagnostics. View Large Table 3. Agreement Between Elecsys SCC Assay and CanAg EIA SCC Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 Manual vs Automated SCCA CanAg SCC EIA Resulta Positive Negative Total Elecsys SCCb Positive 30 3 33 Negative 6 119 125 Total 36 122 158 SCC, squamous cell carcinoma; EIA, enzyme immunoassay; SCCA, squamous cell carcinoma antigen; EIA, enzyme immunoassay. aCanAg SCC EIA is manufactured by Fujirebio Diagnostics AB. bManufactured by Roche Diagnostics. View Large Those 6 discrepant specimens showed all positive SCC results via the manual assay and negative SCC results via the automated assay but all positive SCC results via CMIA testing with the ARCHITECT i2000. Of these 6 discrepant results, we could only obtain 2 biopsy results within 2 days from SCCA tests: 1 was a squamous cell carcinoma and the other was a reactive cellular change. When we thoroughly reviewed the discrepant cases of manual and automated SCC, the levels of each test were almost near the cutoff range. In fact, the 6 manual tests yielded slightly higher levels (mean [SD], 1.8 [0.4]) than the CanAg SCC cutoff level (1.5 ng/mL), and the automated tests showed slightly lower levels (mean [SD], 1.7 [0.3]) than the Elecsys SCC cut-off level (2.6 ng/mL). The false negativity and positivity of SCC test results should be warranted, although those results may have been caused by specimen contamination. In fact, Chauvel et al18 reported the possibility of false positives due to specimen contamination by saliva, skin particles, sweat, or sneeze-generated aerosol particles during SCC testing. Also, the variances in enrolled patients might impact these discrepancies. These discrepancies need to be explored in further large-scale prospective studies. In a previous study, the performance of the Elecsys SCC assay was compared with that of another automated assay, the BRAHMS SCC assay, which was conducted on a BRAHMS Kryptor (Thermo Fisher Scientific Inc.) based on time-resolved amplified cryptate emission.18 It was reported that the Elecsys SCC assay had within-run CVs of 3% for PreciControl Lung Cancer, version 1 (PC LC1) and 2.4% for PreciControl Lung Cancer, version 2 (PC LC2), between-day CVs of 7% for PC LC1 and 3.4% for PC LC2, exhibited linearity (y = 1.193x + 0.14), and had an analytical performance similar with those of our present study. To our knowledge, the automated Roche Elecsys SCC was not widely compared with various SCC assays. This report constitutes a performance comparison of SCC tests in clinical settings. In conclusion, we compared the manual CanAg SCC and automated Elecsys SCC assay and discovered that they have comparable performances. The automated assay reduces turnaround time and workload to a greater degree than the manual assay. Also, the automated SCCA assay probably can reduce the variability of each tester. However, the possibility of false-negative and false-positive results warranted further well-designed prospective large-scale studies. Abbreviations: SCCA squamous cell carcinoma antigen EIA enzyme immunoassay TMB HRP tetramethylbenzidine-horseradish peroxidase ECLIA electrochemiluminescence immunoassay CLSI Clinical and Laboratory Standards Institute PC LC1 PreciControl Lung Cancer, version 1 PC LC2 PreciControl Lung Cancer, version 2 CV coefficient of variation CI confidence interval References 1. Kato H , Torigoe T . Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma . Cancer . 1977 ; 40 ( 4 ): 1621 – 1628 . 2. Suminami Y , Nawata S , Kato H . Biological role of SCC antigen . Tumour Biol . 1998 ; 19 ( 6 ): 488 – 493 . 3. Kato H , Nagaya T , Torigoe T . Heterogeneity of a tumor antigen TA-4 of squamous cell carcinoma in relation to its appearance in the circulation . Gan . 1984 ; 75 ( 5 ): 433 – 435 . 4. Soyemi OM , Otegbayo JA , Ola SO , Akere A , Soyemi T . Comparative diagnostic efficacy of serum squamous cell carcinoma antigen in hepatocellular carcinoma . BMC Res Notes . 2012 ; 5 : 403 . 5. Erickson JA , Lu J , Smith JJ , Bornhorst JA , Grenache DG , Ashwood ER . Immunoassay for quantifying squamous cell carcinoma antigen in serum . Clin Chem . 2010 ; 56 ( 9 ): 1496 – 1499 . 6. Torre GC . SCC antigen in malignant and nonmalignant squamous lesions . Tumour Biol . 1998 ; 19 ( 6 ): 517 – 526 . 7. Micke O , Bruns F , Schäfer U , Prott FJ , Willich N . The impact of squamous cell carcinoma (SCC) antigen in patients with advanced cancer of uterine cervix treated with (chemo-)radiotherapy . Anticancer Res . 2005 ; 25 ( 3A ): 1663 – 1666 . 8. Gadducci A , Cosio S , Carpi A , Nicolini A , Genazzani AR . Serum tumor markers in the management of ovarian, endometrial and cervical cancer . Biomed Pharmacother . 2004 ; 58 ( 1 ): 24 – 38 . 9. Shimada H , Nabeya Y , Okazumi S , et al. Prediction of survival with squamous cell carcinoma antigen in patients with resectable esophageal squamous cell carcinoma . Surgery . 2003 ; 133 ( 5 ): 486 – 494 . 10. Bolger BS , Dabbas M , Lopes A , Monaghan JM . Prognostic value of preoperative squamous cell carcinoma antigen level in patients surgically treated for cervical carcinoma . Gynecol Oncol . 1997 ; 65 ( 2 ): 309 – 313 . 11. Landis JR , Koch GG . The measurement of observer agreement for categorical data . Biometrics . 1977 ; 33 ( 1 ): 159 – 174 . 12. Ayude D , Gacio G , Páez de la Cadena M , et al. Combined use of established and novel tumour markers in the diagnosis of head and neck squamous cell carcinoma . Oncol Rep . 2003 ; 10 ( 5 ): 1345 – 1350 . 13. Crombach G , Scharl A , Vierbuchen M , Würz H , Bolte A . Detection of squamous cell carcinoma antigen in normal squamous epithelia and in squamous cell carcinomas of the uterine cervix . Cancer . 1989 ; 63 ( 7 ): 1337 – 1342 . 14. Kosugi S , Nishimaki T , Kanda T , Nakagawa S , Ohashi M , Hatakeyama K . Clinical significance of serum carcinoembryonic antigen, carbohydrate antigen 19-9, and squamous cell carcinoma antigen levels in esophageal cancer patients . World J Surg . 2004 ; 28 ( 7 ): 680 – 685 . 15. Molina R , Filella X , Auge J , et al. Tumor markers (CEA, CA 125, CYFRA 21-1, SCC and NSE) in patients with non-small cell lung cancer as an aid in histological diagnosis and prognosis . Tumour Biol . 2003 ; 24 ( 4 ): 209 – 218 . 16. Petrelli NJ , Palmer M , Herrera L , Bhargava A . The utility of squamous cell carcinoma antigen for the follow-up of patients with squamous cell carcinoma of the anal canal . Cancer . 1992 ; 70 ( 1 ): 35 – 39 . 17. Trapé J , Filella X , Alsina-Donadeu M , Juan-Pereira L , Bosch-Ferrer À , Rigo-Bonnin R . Increased plasma concentrations of tumour markers in the absence of neoplasia . Clin Chem Lab Med . 2011 ; 49 ( 10 ): 1605 – 1620 . 18. Chauvel C , Jordan P , Popovici T , Bories P-N . Evaluation of the new Elecsys SCC assay: comparison with the Kryptor SCC assay . Clin Chem Lab Med . 2018 ; 56 ( 2 ): e56 – e58 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Laboratory MedicineOxford University Press

Published: Mar 16, 2018

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