Clinical Chemistry 56:12 1839–1844 (2010)
Endocrinology and Metabolism
Falsely Decreased Human Chorionic Gonadotropin (hCG) Results Due to Increased Concentrations of the Free  Subunit and the  Core Fragment in Quantitative hCG Assays David G. Grenache,1† Dina N. Greene,1† Anand S. Dighe,2 Corinne R. Fantz,3 Daniel Hoefner,4 Christopher McCudden,5 Lori Sokoll,6 Carmen L. Wiley,7 and Ann M. Gronowski8*
BACKGROUND: Earlier studies have shown that increased concentrations of certain human chorionic gonadotropin (hCG) variants can cause false-negative results in some qualitative hCG devices. The objective of this study was to determine if increased concentrations of hCG and hCG core fragment (hCGcf) cause falsely decreased results on 9 commercially available quantitative hCG assays.
(⬎50%) hCG results in the presence of hCGcf concentrations found during normal pregnancy. © 2010 American Association for Clinical Chemistry
CONCLUSIONS: Extremely high concentrations of hCG variants can cause falsely decreased results in certain quantitative hCG assays. Of the 9 assays examined, none exhibited falsely decreased results in the presence of hCG concentrations typically associated with hCGproducing malignancies. Two assays exhibited decreased
Human chorionic gonadotropin (hCG)9 is a molecularly heterogeneous molecule. The biologically active hormone (intact hCG) is composed of noncovalently linked ␣ and  subunits. However, partially degraded variants of hCG and other variants with modified protein and/or carbohydrate structures are also detectable in serum and urine. These variants include hyperglycosylated hCG, nicked hCG, hCG missing the -subunit C-terminal peptide, hCG free -subunit (hCG), hyperglycosylated free -subunit, nicked hCG, and the hCG core fragment (hCGcf) (1– 4 ). hCGcf is the variant predominantly detected in urine after 5 weeks of pregnancy. The relative abundance of each of these hCG variants in serum or urine depends on the physiological and/or pathological state of the individual. For instance, hyperglycosylated hCG is predominantly increased in urine and serum during early pregnancy but, as pregnancy progresses, hCGcf becomes the predominant variant in urine (3 ). By gestational day 35, mean concentrations of urine hCGcf have been reported to be 65 000 pmol/L and can reach concentrations of 1 000 000 pmol/L (4 – 6 ). Many trophoblastic and nontrophoblastic tumors produce hCG (7 ). Very high concentrations of this variant suggest aggressive disease and are strongly associated with a poor prognosis (7 ). Lempia¨ninen et al.
1
†
Several concentrations of purified hCG and hCGcf were added to 2 sets of 6 serum samples with and without a fixed concentration of intact hCG. We examined 9 widely used immunoassays to measure immunoreactive hCG. Falsely decreased results were defined as those in which the measured hCG concentration was ⱕ50% of expected. METHODS:
RESULTS: High concentrations of hCG (ⱖ240 000 pmol/L) produced falsely decreased hCG measurements in 2 assays known to detect this variant. Similarly, high concentrations of hCGcf (ⱖ63 000 pmol/L) produced falsely decreased hCG measurements in 3 assays that do not detect purified hCGcf. Two assays were identified that detected both hCG and hCGcf, and neither produced falsely decreased results in the presence of high concentrations of these variants.
University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, UT; 2 Harvard Medical School, Department of Pathology, Cambridge, MA; 3 Emory University, Department of Pathology and Laboratory Medicine, Atlanta, GA; 4 Marshfield Clinic, Division of Laboratory Medicine, Marshfield, WI; 5 University of North Carolina School of Medicine, Department of Pathology and Laboratory Medicine, Chapel Hill, NC; 6 Johns Hopkins Medical Institutions, Department of Pathology, Baltimore, MD; 7 Providence Sacred Heart Medical Center, Department of Laboratory Medicine, Spokane, WA; 8 Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO.
David G. Grenache and Dina N. Greene contributed equally to the work, and both should be considered as first authors. * Address correspondence to this author at: Department of Pathology and Immunology, Washington University School of Medicine, Box 8118, 660 S. Euclid, St. Louis, MO 63110. Fax 314-362-1461; email
[email protected]. Received January 13, 2010; accepted August 25, 2010. Previously published online at DOI: 10.1373/clinchem.2010.143479 9 Nonstandard abbreviations: hCG, human chorionic gonadotropin; hCG, hCG free -subunit; hCGcf, hCG core fragment; IRR, international reference reagent; FDA, Food and Drug Administration.
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Table 1. Concentrations of intact hCG, hCG, and hCGcf present in the 14 serum samples measured by each assay. Intact hCG (99/688), pmol/L
Sample ID
Blank
hCG (99/650), pmol/L
hCGcf (99/708), pmol/L
0
0
Intact hCGa,b
1500
0
0
Intact hCG ⫹ hCG(1)a
1500
61 000
0
Intact hCG ⫹ hCG(2)a
1500
240 000
0
Intact hCG ⫹ hCG(3)
a
0
1500
970 000
0
hCG(1)c
0
61 000
0
hCG(2)
0
240 000
0
hCG(3)
0
970 000
0
a
Intact hCG ⫹ hCGcf(1)
1500
0
63 000
Intact hCG ⫹ hCGcf(2)a
1500
0
250 000
Intact hCG ⫹ hCGcf(3)a
1500
0
1 000 000
hCGcf(1)d
0
0
63 000
hCGcf(2)
0
0
250 000
hCGcf(3)
0
0
1 000 000
a
Because of the low upper end of the analytical measuring range (400 IU/L) for the A1A-1800 assay, the concentration of hCG (99/688) was adjusted to 540 pmol/L for these samples. b Mean (SD) agreement between measured and expected hCG concentrations across assays was 99% (10%). c Mean (SD) agreement between measured and expected hCG concentrations across assays was 110% (32%). d Mean (SD) agreement between measured and expected hCG concentrations across assays was 96% (34%).
have shown that patients with nonseminomatous testicular cancers have hCG concentrations as high as 10 000 –100 000 pmol/L, which correlated with disease-related mortality (8 ). Some qualitative point-of-care hCG devices have been reported to demonstrate false-negative results in the presence of increased concentrations of hCGcf, hCG, nicked hCG, and nicked hCG (9, 10 ). In light of these findings, we hypothesized that the same would be true for quantitative hCG assays. The objective of this study was to investigate whether very high concentrations of hCG and hCGcf would produce falsely decreased hCG concentrations in 9 commonly used quantitative hCG assays. The concentrations were chosen so that at least 1 concentration was in a range encountered in pregnancy or malignancy. The other concentrations were intentionally very high to investigate if negative interference can occur. Materials and Methods SAMPLE PREPARATION
The first WHO international reference reagent (IRR) preparations of intact hCG (99/688), hCG (99/650), and hCGcf (99/708) were obtained from the National Institute for Biological Standards and Controls (Hertfordshire, UK). The molar content of each IRR has 1840 Clinical Chemistry 56:12 (2010)
been assigned (11 ). As described below, these IRRs were added to pooled hCG-free serum prepared from leftover human sera and to hCG-free urine obtained from a premenopausal woman. We determined that the serum pool and the urine were hCG free by testing aliquots of each with the Immulite® 2000 hCG (Siemens Medical Solutions Diagnostics) assay as it detects all major hCG variants (12, 13 ) and is Food and Drug Administration (FDA) cleared as a qualitative test using a urine matrix. One ampoule of IRR (99/688) intact hCG (1.88 nmol/ampoule) was reconstituted in 2 mL hCG-free serum (final concentration 940 nmol/L). Each of 16 ampoules of IRR (99/650) hCG (0.88 nmol/ampoule) were reconstituted in 440 L of hCG-free serum (final concentration 1940 000 pmol/L) and pooled. Each of 15 ampoules of IRR (99/708) hCGcf (1.02 nmol/ampoule) were reconstituted in 510 L hCG-free serum (final concentration 2 000 000 pmol/L) and pooled. One ampoule of hCGcf (IRR 99/708) was reconstituted with 1.02 mL of urine containing 1500 pmol/L of intact hCG (IRR 99/688) (final concentration of hCGcf was 1 000 000 pmol/L). Individual serum samples were prepared by combining volumes of hCG-free serum with the reconstituted IRRs to achieve the final concentrations shown in Table 1. Nine sets of each of these 14 samples were placed into
Falsely Decreased hCG
The activity ratios (IU/nmol) for hCG, hCG, and hCGcf have been previously reported for each of these immunoassays except the Cobas e411 hCG Stat assay (12 ). These ratios were used to calculate the assay-specific, expected hCG concentration (in IU/L) for each of the 14 samples based on the molar concentrations shown in Table 1. Activity ratios for hCG, hCG, and hCGcf obtained by using the Cobas e411 hCG Stat assay were determined from the samples containing only those variants and were calculated from the mean measured hCG concentration (IU/L) and the prepared molar concentration (nmol/L). For each sample and each assay, the percent agreement between the actual measured hCG concentration and the expected hCG concentration was determined. Fig. 1. The percent agreement between the measured and the expected hCG concentrations for intact hCG (1500 pmol/L) in the presence of increasing concentrations of hCGcf in urine measured by using the DxI Total hCG assay. The dotted line represents the 50% cutoff used to identify falsely decreased results.
Eppendorf tubes (0.50 – 0.75 mL), frozen at ⫺80 °C, and shipped on dry ice to the participating laboratories. All samples were tested within 3 weeks of preparation. Individual urine samples were prepared by combining volumes of urine containing intact hCG (IRR 99/688) with the hCGcf reconstituted in urine as described above. Final concentrations of hCGcf are shown in Fig. 1. The concentration of intact hCG was kept constant (1500 pmol/L) in each sample. hCG ANALYSIS
All serum samples were analyzed undiluted and after at least 1 dilution (1:100, 1:200, or 1:1000) to obtain 2 results that were within the analytical measuring range of each assay. Results from diluted samples were multiplied by the appropriate dilution factor. The following 9 hCG immunoassays were used in this study: Advia Centaur® Total hCG (Siemens Medical Solutions Diagnostics), AIA-1800 ST Total -hCG (Tosoh Bioscience), Architect® Total -hCG (Abbott Laboratories), Cobas® e411 hCG Stat (Roche Diagnostics), Dimension® RxL® hCG (Siemens Medical Solutions Diagnostics), DxI® Total hCG (Beckman Coulter), Modular Analytics e170 hCG⫹ (Roche Diagnostics), Immulite 2000 hCG, and Vitros® ECi Total -hCG II (Ortho Clinical Diagnostics). These 9 reagent systems account for 94% of hCG assays represented in proficiency testing surveys administered by the CAPs (14 ).
Results The concentrations of hCG variants present in each sample are shown in Table 1. The footnote for Table 1 shows the mean percent agreement between the measured and expected hCG variant concentrations for intact hCG alone, and for the samples containing the lowest concentrations of hCG and hCGcf alone. These data indicate that concentration estimates (in IU/L) based on previously published activity ratios (12 ) were accurate, although the percent agreement for hCG and hCGcf showed more variability than for intact hCG. For each of the 9 assays, the percent agreement between the measured and the expected concentrations for hCG and hCGcf in the presence and absence of intact hCG was determined (See Figs. 1 and 2 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol56/issue12). A percent agreement cutoff of 50% was selected to identify if hCG or hCGcf produced falsely decreased results either alone or when combined with intact hCG. That is, if the measured hCG concentration was ⱕ50% of the expected concentration, we concluded that this constituted a negative influence on the measured hCG concentration by that hCG variant. Previous studies have examined the ability of many quantitative hCG assays to detect hCG variants (12, 13 ). As expected, and in accordance with product information supplied by the manufacturers, we found that the Dimension RxL hCG and the Cobas e411 hCG Stat assays did not recognize hCG (see online Supplemental Fig. 1) (12, 13 ). At the lowest concentration of hCG tested (61 000 pmol/L), the other 7 assays produced results that were ⬎50% of expected when hCG was measured alone. The same was true for all 9 assays when the sample contained 61 000 pmol/L of hCG and 1500 pmol/L intact hCG. Therefore, 61 000 pmol/L of hCG had no influence on the measured hCG result. As the concentration of hCG was inClinical Chemistry 56:12 (2010) 1841
Table 2. Summary of the effects of high concentrations of hCG and hCGcf on the measured hCG concentration as determined by 9 quantitative hCG assays.a
Assay
Assay measures hCG
Effect of hCG on measurement of intact hCGa
Assay measures hCGcf
Effect of hCGcf on measurement of intact hCGa
Advia Centaur Total hCG
Yesb
Falsely decreased at ⱖ970 000 pmol/L
Nob
No effect
AIA-1800 ST Total -hCG
Yesb
No effect
Nob
Falsely decreased at ⱖ250 000 pmol/L
Architect Total -hCG
Yesb
No effect
Nob
Falsely decreased at ⱖ1 000 000 pmol/L
Cobas e411 hCG Stat
Nob
No effect
Nob
No effect
b
No effect
Nob
No effect
DxI Total hCG
b
Yes
No effect
b
No
Modular Analytics e170 hCG⫹
Yesb
No effect
Yes
Dimension RxL hCG
Immulite 2000 hCG Vitros ECi Total -hCG II
No
Yes
b c
Yes
No effect Falsely decreased at ⱖ240 000 pmol/L
b
Falsely decreased at ⱖ3750 pmol/L No effectb
Yes
No effect
c
No effect
No
Falsely decreased results were defined as those in which the measured hCG concentration was ⱕ50% of expected. Whittington et al. (12 ) and Sturgeon et al. (13 ). c Whittington et al. (12 ). a
b
creased, the Advia Centaur and the Vitros ECi assays demonstrated a dose-dependent hook effect when hCG was measured alone and in the presence of intact hCG. When hCG was combined with intact hCG, the Advia Centaur and the Vitros ECi assays produced results that were 47% and 7% of expected, respectively, at the highest concentration (970 000 pmol/L) of hCG (see online Supplemental Fig. 1). At a lower concentration of 240 000 pmol/L hCG, the Vitros ECi assay produced results that were 27% of expected, whereas the Advia Centaur assay was 68% of expected. Although results for the Cobas e411 hCG Stat and Immulite assays did not drop below the 50% cutoff, these assays produced results that were 63% and 85% of expected, respectively, at the highest hCG concentration of 970 000 pmol/L and demonstrated a trend of decreasing hCG with increasing concentrations of hCG. To examine the negative effects of hCG on the Vitros ECi assay, additional testing was performed. Five samples were prepared, each containing 1500 pmol/L of intact hCG in addition to varying concentrations of hCG (range, 60 000 –250 000 pmol/L). Only samples containing ⱖ250 000 pmol/L of hCG produced hCG results that were ⱕ50% of expected (data not shown). Consistent with previous reports (12, 13 ), only the Modular Analytics e170 hCG⫹ and Immulite hCG assays detected the hCGcf variant, and both of these assays produced results that were ⬎50% of expected at all concentrations tested, even when combined with 1842 Clinical Chemistry 56:12 (2010)
intact hCG (see online Supplemental Fig. 2). Similar to what was observed with hCG, the Cobas e411 hCG Stat assay produced measured hCG results that were decreased to 62% at the highest concentration of hCGcf tested (1 000 000 pmol/L), but this assay was within the criterion of acceptability. Importantly, 3 assays produced falsely decreased hCG measurements in the presence of hCGcf, despite the fact that these assays do not recognize purified hCGcf. The AIA-1800 assay showed a clear negative trend, and the measured hCG results were 59%, 26%, and 19% of expected in the presence of 63 000, 250 000, and 1 000 000 pmol/L hCGcf, respectively (see online Supplemental Fig. 2). The Architect assay was affected only at the highest concentration of hCGcf (1 000 000 pmol/L), which produced an hCG result that was 12% of expected. The DxI assay was very sensitive to the presence of hCGcf and produced measured hCG results that were decreased to ⱕ5% of expected at all concentrations of hCGcf tested (see online Supplemental Fig. 2). Owing to the extreme negative effects of hCGcf on the DxI assay, additional testing was performed. Eight samples were prepared in serum, each containing 1500 pmol/L of intact hCG in addition to varying concentrations of hCGcf (range, 0 – 60 000 pmol/L). Samples containing ⱖ3750 pmol/L of hCGcf produced hCG results that were ⱕ50% of expected (see online Supplemental Fig. 3). This effect was also demonstrated in a urine matrix (Fig. 1).
Falsely Decreased hCG
A summary of the effect of these hCG variants and their clinical implications is shown in Table 2. Discussion This study is, in part, a proof-of-principle study. It is the first to demonstrate the effect of increased concentrations of the hCG variants hCG and hCGcf on the measured hCG concentrations in commonly used, quantitative hCG immunoassays, confirming earlier predictions (15 ). hCG and hCGcf were selected for study because these hCG variants can be present in urine or serum in very high concentrations (10 000 – 1 000 000 pmol/L) in pregnant individuals or in those with hCG-secreting neoplasms (7 ). Although no hCG assay has been cleared by the FDA for use in the diagnosis or monitoring of hCGsecreting malignancies, clinicians routinely use hCG tests for this purpose. Thus, it is important that the hCG assay used in a clinical laboratory be capable of detecting hCG. Although most trophoblastic tumors produce intact hCG, unusually increased concentrations of the other hCG variants (particularly hCG) can also be present (16 ). Furthermore, some hCGproducing germ-cell tumors may secrete hCG alone (17 ). Because they do not detect hCG, neither the Cobas e411 hCG Stat nor the Dimension RxL hCG assays are appropriate for oncology applications, a finding that has been shown previously (12, 13 ). In the study we report here, we demonstrated that the Advia Centaur and Vitros ECi hCG assays, both of which recognize hCG, give falsely decreased measurements of hCG in the presence of extremely high hCG concentrations. However, the effect is not significant until the hCG concentrations are greater than those typically observed in malignancies (i.e., ⬎100 000 pmol/L). Of the 9 assays examined in this study, only the Immulite hCG assay has been FDA cleared for use as a qualitative test that can be used with a urine matrix. However, laboratorians sometimes use quantitative serum assays to detect hCG in urine when investigating discrepant hCG results (9, 18, 19 ). As such, knowledge of the potential influence of hCGcf, a predominant hCG variant in urine after 5 weeks of pregnancy, on quantitative hCG assays is essential for accurate result interpretation. As reported previously, only the Modular Analytics e170 hCG⫹ and Immulite hCG assays can detect hCGcf (12, 13 ). As we report here, neither of these assays produced falsely decreased hCG results when this variant was present at a very high (1 000 000 pmol/L) concentration. In contrast, the remaining 7 assays evaluated in this study did not quantify hCGcf, and 3 of them (AIA-1800, Architect, and DxI assays) produced falsely decreased hCG results in its presence. Most notably, the DxI assay was exquisitely sensitive to
the presence of hCGcf, and results were falsely decreased at concentrations as low as 3750 pmol/L. This concentration of hCGcf is present in urine collected from pregnant women at ⬍28 days gestation (5, 6 ). The high-dose hook effect is a well-known cause of falsely decreased results from immunometric assays (20 ). At high antigen concentrations both the capture and label antibodies become saturated with antigen and the signal response is decreased, giving the appearance of an antigen concentration that is lower than it actually is. This phenomenon explains the falsely decreased results we observed with increased concentrations of hCG, because the affected assays were capable of detecting this variant. However, 3 assays did not detect isolated hCGcf yet produced falsely decreased results when it was present at high concentrations, a phenomenon that is not explained by the classic definition of the hook effect. A likely explanation for the falsely decreased results in the presence of increased hCGcf is the recognition of this variant by only 1 of the 2 assay antibodies. When present at a concentration that is in excess of the hCG variants recognized by both antibodies, hCGcf saturates 1 of the antibodies, which then prevents the formation of the double antibody-antigen “sandwich.” This effect was predicted in 2000 by Madersbacher and Berger (15 ) and highlights the need for appropriate selection of antibodies and assay design (17 ). In 2009 Gronowski et al. demonstrated that this phenomenon occurred with some qualitative hCG devices (9, 10 ). One limitation of this study was that measurements were made in singleton and not in duplicate, due to the expense of the purified materials. Therefore the variation of this effect between assays was not quantified. The Modular Analytics e170 hCG⫹ and Immulite hCG assays detected all hCG variants and did not produce falsely decreased results in the presence of high concentrations of hCG and hCGcf. Therefore, these 2 assays could potentially be used to reliably detect hCG in urine. Of these 2 assays, a validation of the Modular Analytics e170 hCG assay in urine has been described (21 ). The other 7 assays failed to detect hCGcf and should not be relied upon to detect all hCG variants in urine specimens. This is particularly true of the AIA-1800 ST Total -hCG, Architect Total -hCG, and DxI Total hCG assays, which will underestimate hCG in the presence of high concentrations of hCGcf. In oncology applications in which the detection of hCG is essential, the Cobas e411 hCG Stat and Dimension RxL hCG assays should not be used because they do not detect hCG. The remaining assays detect hCG, but the Advia Centaur Total hCG and the Vitros ECi Total -hCG II can produce falsely decreased hCG results when hCG is very high. Clinical Chemistry 56:12 (2010) 1843
In summary, we have 3 important conclusions: (a) Extremely high concentrations of hCG variants can cause falsely decreased measurement of intact hCG in some quantitative hCG assays, as has been observed previously for qualitative assays; (b) None of the 9 hCG assays evaluated in this study demonstrated falsely low results until hCG concentrations were greater than those typically found in hCG-producing neoplasms. This result is reassuring for the clinical use of these assays. However, this effect should be investigated in other quantitative hCG assays used to diagnose and follow cancer patients. (c) In 2 hCG assays (DxI Total hCG and AIA-1800 ST Total hCG), increased concentrations of hCGcf in urine caused falsely low measurements of intact hCG at concentrations of hCGcf that normally occur in pregnancy. Therefore, these assays should not be used with urine for clinical or research purposes. These data provide additional evidence that manufacturers of hCG assays should characterize the ability of their hCG reagent systems to detect specific hCG variants and quantify the effect of these variants on hCG measurements to allow for appropriate assay use in a variety of clinical conditions (17, 22, 23 ). These data also illustrate the importance of validating all hCG
assays, in the presence of hCG variants, for any off-label use.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: A.M. Gronowski, AACC. Consultant or Advisory Role: C.R. Fantz, Beckman Coulter; A.M. Gronowski, NIH and FDA. Stock Ownership: None declared. Honoraria: C.R. Fantz, Beckman Coulter. Research Funding: None declared. Expert Testimony: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: The authors acknowledge the expert technical assistance provided by C.A. Diwan and M.G. Rush.
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