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Falsely increased hCG in patients with high leukocyte counts.

To the Editor:

Falsely increased plasma troponin (Tn) I and T results have been reported for several Tn methods, including the Beckman Coulter UniCel DxI AccuTnI assay (Beckman Coulter, Chaska, MN) (1-3). We and others have also observed falsely increased human chorionic gonadotropin (hCG) using the Beckman Coulter Access Total [beta]hCG on the UniCel DxI (1). These false increases were identified by the finding of substantially lower results following high-speed recentrifugation and reanalysis, suggesting a particulate interference. However, the sources of these false increases were not previously determined. In reviewing clinical data for patients from whom plasma samples were found to generate falsely high hCG results on the UniCel DxI in our laboratories, we found these patients had high blood leukocyte counts. Based on this finding, we investigated the interference of leukocytes in the Beckman Coulter Access Total [beta]hCG assay. This study was approved by the Emory Institutional Review Board.

First, we measured hCG in leftover plasma samples from patients with known high blood leukocyte content of >25 X [10.sup.3]/[micro]L. The thirteen randomly selected samples (10 male, 3 female) were inverted several times in the original lithium heparin plasma separator tubes (PSTs) (Becton Dickinson) to disturb any particulates settled during storage (up to 24 h refrigerated) and then measured for hCG on the Unicel DxI. Surprisingly, eleven (11/13) of these samples had markedly increased hCG concentrations exceeding the institutional reference interval of 5 mIU/mL (range: 10 - 256 mIU/ mL, mean: 106 mIU/mL) (Table 1). However, after transferring the plasma to aliquot tubes and recentrifugation, hCG decreased by an average of 93% in the 11 previously increased samples, all to [less than or equal to] 10 mIU/mL (Table 1). Recentrifugation and testing of the original PSTs resulted in less notable reductions in the interference (average decrease of 64%). False increases were not observed in eight PSTs collected from patients with low blood leukocyte content of [less than or equal to] 1 X [10.sup.3]/[micro]L (Table 1).

After finding a high incidence of falsely increased plasma hCG in the samples tested with high blood leukocyte content, we hypothesized that addition of leukocytes in known hCG-negative plasma would lead to false hCG increases. At three separate institutions, an hCG-negative plasma sample with undetectable hCG on the Unicel DxI was identified, transferred to an aliquot tube, spiked with a leukocyte-enriched buffy coat (125 [micro]L added to 700 [micro]L plasma), and returned to the original PST. The same buffy coat (leukocyte concentration of 40 X 103/[micro]L), prepared from potassium EDTA preserved-blood, was used across institutions. The final spiked plasma leukocyte concentration of 6 X [10.sup.3]/uL approximated the cellular content found in PST plasma (after 10 inversions) from patients with high blood leukocyte counts. Untreated hCG-negative plasma served as controls. In one lab, an additional hCG-negative sample was spiked with the same buffy coat in the same ratio but after incubating the buffy coat at 58[degrees]C for 60 min. Both the spiked and control samples, in the original PSTs, were gently inverted 10 times, recentrifuged and measured for hCG on the Unicel DxI at the respective institution. All samples were also tested for hCG on either an Abbott Architect i2000 (Architect Total [beta]hCG, Abbott Diagnostics) or a Siemens Advia Centaur[R] Total hCG (Siemens Medical Solutions Diagnostics) for comparison.

The control plasma samples were consistently negative throughout the experiment, as was the heat-treated buffy coat-spiked sample. The three other spiked samples produced markedly increased hCG results on the Unicel DxI (range of positive results: 16 - 144 mIU/mL), but remained hCG negative (<5 mIU/mL) by the Architect or Centaur. After transferring plasmas to aliquot tubes and recentrifuging, the Unicel DxI results for the spiked samples all decreased to <5 mIU/mL.

The Beckman Coulter immunoassays are two-site immunoenzymatic assays, utilizing antiantigen antibody bound paramagnetic particles and antiantigen antibody: alkaline phosphatase (ALP) conjugate. The immobilized antibody-antigen-antibody:ALP complex is washed, ALP substrate is added, and the resulting amount of light production is measured. Increased plasma concentrations of ALP have previously been demonstrated as a source of positive interference in immunoassays which used an ALP conjugate (4). In our experiments, leukocyte ALP maybe the source of the interference. This is supported by our data showing that the hCG interference was not observed in the sample spiked with heat-treated buffy coat, suggesting a heat labile interference. ALP is not used in the Architect and Centaur assays, both of which were free from interference in our studies.

Herein we identified clinically significant false increases of hCG in plasma of patients with high leukocyte counts and in leukocyte-enriched plasma when tested on the Unicel DxI platform. Aliquoting and recentrifugation of the errant plasmas resulted in the most marked reduction in erroneous results. It is reasonable to assume that the more modest reduction observed after recentrifuging the primary PST tube may be attributed to leakage of cellular material from underneath the gel barrier during recentrifugation. Strathmann, et al., who also saw false increases using the Beckman Coulter hCG, TnI, CK-MB and TSH assays on the Unicel DxI, found reductions in erroneous increases using rapid serum tubes (RSTs) compared to PSTs (1). Serum separator tubes have been shown to contain fewer leukocytes in the supernatant after centrifugation than corresponding PSTs (5). We conclude that leukocytes or leukocyte membrane fragments present in patient plasma samples may not be completely washed away during the assay, and that a heat labile interference contained within the leukocytes, presumably ALP (6), is contributing to the falsely increased Unicel DxI results. Laboratories should consider the possibility of leukocyte-related interference in these immunoassays.

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 author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: C. Wiley, Beckman Coulter, Inc.; J.C. Ritchie, Beckman Coulter, Inc.; C.R. Fantz Beckman Coulter, Inc.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Patents: None declared.

Acknowledgments: We would like to thank James Ford in the flow laboratory for preparing the buffy coats used in this study.


(1.) Strathman FG, Ka MM, Rainey PM, Baird GS. Use of BD Vacutainer Rapid Serum Tube Reduces False-Positive Results for Selected Beckman Coulter Unicel DxI Immunoassays. Am J Clin Pathol 2011;136:325-9.

(2.) Lum G, Solarz DE, Farney L. False Positive Cardiac Troponin Results in Patients Without Myocardial Infarction. LabMedicine 2006;37:546-50.

(3.) Koch CD, Wockenfus AM, Saenger AK, Jaffe AS, Karon BS. BD Rapid Serum Tubes Reduce False Positive Plasma Troponin T Results on the Roche Cobas e411 Analyzer. Clin Biochem 2012;45:842-4.

(4.) Dasgupta A, Chow L, Wells A, et al. Effect of Elevated Concentration of Alkaline Phosphatase on Cardiac Troponin I Assays. J Clin Lab Anal 2001;15:175-7.

(5.) Lin F, Cohen R, Losada R, Bush V. Cellular Sedimentation and Barrier Formation under Centrifugal Force in Blood Collection. LabMedicine 2001;32:588-94.

(6.) Rosenblum D, Petzold SJ. Granulocyte Alkaline Phosphatase: Studies of Purified Enzymes from Normal Subjects and Patients with Polycythemia Vera. J Clin Invest 1973;52:1665-72.

Marion L. Snyder [1] *

Carmen Wiley [2]

Ross J. Molinaro [3]

James C. Ritchie [3]

Corinne R. Fantz [3]

[1] Department of Chemistry Greenville Health System Greenville, SC

[2] Providence Sacred Heart Medical Center Spokane, WA

[3] Department of Pathology and Laboratory Medicine Emory University Atlanta, GA

* Address correspondence to this author at: Greenville Memorial Hospital 701 Grove Rd. Greenville, SC 29605 E-mail:

Previously published online at DOI: 10.1373/clinchem.2013.202911
Table 1. Plasma samples from 13 patients with high
(>25 x [10.sup.3]/[micro]mL) and 8 patients with low
([less than or equal to] 1 x [10.sup.3]/[micro]mL) blood
leukocyte counts were measured for hCG (Beckman Coulter
Access Total [beta]hCG) on the DxI in the original lithium
heparin PST and after transferring plasma to an aliquot tube
and recentrifuging. (a)

                                     hCG, mIU/mL

Sample       Leukocytes,         Original     Aliquot
         x [10.sup.3]/[micro]L     tube     tube-respun

1                68.7            224 (b)         1
2                68.2            256 (b)         3
3                67.9                            3
4                66.7             10 (b)         2
5                52.0              5             4
6                39.3            101 (b)         1
7                39.1            148 (b)         2
8                34.5             91 (b)         1
9                34.3                            2
10               33.8             89 (b)         1
11               32.6            154 (b)         1
12               31.0              1             0
13               27.7             27 (b)         9 (b)
14                1.0              3             0
15                0.9              0             0
16                0.9              2             1
17                0.7              3             2
18                0.7              1             0
19                0.6              0             0
20                0.6              2             2
21                0.5              2             0

(a) Samples are listed in order of decreasing blood leukocyte

(b) hCG results >5 mIU/mL
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Article Details
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Title Annotation:human chorionic gonadotropin
Author:Snyder, Marion L.; Wiley, Carmen; Molinaro, Ross J.; Ritchie, James C.; Fantz, Corinne R.
Publication:Clinical Chemistry
Article Type:Report
Date:Jul 1, 2013
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