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Are laboratories reporting serum quantitative hCG results correctly?

Human chorionic gonadotropin human chorionic gonadotropin (HCG): see gonadotropic hormone.  (hCG) is a heterodimeric glycoprotein glycoprotein (glī'kōprō`tēn), organic compound composed of both a protein and a carbohydrate joined together in covalent chemical linkage.  hormone composed of an [alpha]-subunit (hCG[alpha]) and a [beta]-subunit (hCG[beta]). HCG[alpha] is essentially identical to that of lutropin (LH), follitropin (FSH FSH follicle-stimulating hormone.

FSH
abbr.
follicle-stimulating hormone


Facioscapulohumeral muscular dystrophy (FSH) 
), and thyrotropin thyrotropin (thī'rätrō`pĭn) or thyroid-stimulating hormone (TSH), hormone released by the anterior pituitary gland that stimulates the thyroid gland to release thyroxine.  (TSH TSH thyroid-stimulating hormone; see thyrotropin.

TSH
abbr.
thyroid-stimulating hormone


Thyroid-stimulating hormone (TSH) 
), whereas hCG[beta] is unique to hCG and confers its biological and immunological specificity (1). hCG is synthesized and secreted by trophoblastic tro·pho·blast  
n.
The outermost layer of cells of the blastocyst that attaches the fertilized ovum to the uterine wall and serves as a nutritive pathway for the embryo. Also called trophoderm.
 tissue of the placenta in pregnancy, gestational trophoblastic diseases, and certain other pathological conditions (2-5). At least 6 different forms of hCG have been identified in serum: non-nicked and nicked intact hCG (hCG and hCGn), non-nicked and nicked free [beta]-subunit (hCG[beta] and hCGn[beta]), and regular and hyperglycosylated (or large) free hCG[alpha] (4). In pregnancy, intact hCG predominates, whereas free hCG[beta] accounts for <1% of total [beta] hCG (hCG + hCG[beta]) in serum (4, 6). However, the free hCG[beta] concentration in patients with trophoblastic diseases or testicular testicular /tes·tic·u·lar/ (tes-tik´u-lar) pertaining to a testis.

tes·tic·u·lar
adj.
Of or relating to a testicle or testis.



testicular

pertaining to the testis.
 tumors is disproportionately increased, and the ratio of free hCG[beta] to intact hCG or to hCG + hCG[beta] is markedly increased (7, 8). Therefore, measurements of hCG + hCG[beta] and free hCG[beta] are important in diagnosis and management of trophoblastic diseases and testicular tumors (8-12). Free hCG[beta] and hCG + hCG[beta] have also been suggested to be better biomarkers than intact hCG in prenatal screening for Down syndrome Down syndrome, congenital disorder characterized by mild to severe mental retardation, slow physical development, and characteristic physical features. Down syndrome affects about 1 in every 730 live births and occurs in all populations equally.  (13).

There are > 100 different commercial immunoassay Immunoassay

An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus.
 test kits for measuring hCG. Most procedures detect intact hCG (nicked + nonnicked hCG) or hCG + hCG[beta], and a few immunoassays detect free hCG[beta] alone or with other forms (4). In clinical laboratories in the US, fewer than 20 immunoassay methods are commonly used. These assays vary in immunospecificity toward different forms of hCG, and results of measurements may have distinct clinical applications. It is important that laboratorians, who perform the test and consult with physicians about test procedures and results, be aware of the heterogeneity of hCG and the specificity of the method in service.

The Wadsworth Center of the New York New York, state, United States
New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of
 State Department of Health conducts CLIA'88-approved proficiency testing programs, and CLIA'88 regulations define acceptable performance criteria for qualitative and quantitative test results for hCG (14). Based on information from manufacturers' product inserts for the packages of test kits commonly used by clinical laboratories in the US, the method principles, and the occurrence of test kit names in peer-reviewed articles, we request participant laboratories to report results as "Intact hCG" and/or "Total [beta] hCG" for quantitative results of hCG as appropriate.

In the proficiency test event of January 2006, we prepared 1 of 5 specimens by addition of intact hCG and free hCG[beta] to final concentrations of 50 IU/L and 20 IU/L, respectively, as well as other non-hCG-related hormones, into human serum matrix that had essentially no endogenous hCG. Intact hCG, WHO standard (code 75/589), was purchased from the National Institute for Biological Standards and Control, and free hCG[beta] was from AspenBio Pharma. The addition of both intact hCG and free hCG[beta] was intended to mimic pathophysiological conditions and to challenge the specificity of various immunoassays. Along with 4 other proficiency test specimens prepared by the sole addition of intact hCG, the samples were delivered overnight on ice to participant laboratories. Results of total [beta] hCG were reported by 266 laboratories. (Complete details on results from the proficiency test events discussed in this paper are available at http://www.wadsworth.org/chemheme/chem/endo/ptframes.htm.)

[FIGURE 1 OMITTED]

Total [beta] hCG results for specimens with both intact hCG and hCG[beta] showed higher overall variability (16% CV) than the samples spiked with only intact hCG (9.6% CV). We also noticed a bimodal distribution bimodal distribution

a distribution with two peaks separated by a region of low frequency of observations.
 of results reported by users of the Roche Elecsys system for total [beta] hCG, with the specimen containing both forms of hCG: 2 medians were centered at approximately 47 IU/L and 67 IU/L (Fig. 1). In contrast, we found normal distributions of results for the other 4 specimens with this method. Further investigation revealed that 2 different reagent systems are provided for the Roche Elecsys instrument, reagent "HCG STAT" for measuring intact hCG and reagent "HCG + [beta]" for measuring total [beta] hCG (hCG + hCG[beta]). Those laboratories that reported results of approximately 47 IU/L used the reagent HCG STAT, and those that reported values of approximately 67 IU/L used the reagent HCG + [beta]. Unfortunately, many laboratories that used the HCG STAT procedure reported their results as total [beta] hCG.

We carried out further studies in the proficiency test event of May 2007. We prepared sample E03 by the sole addition of intact hCG at approximately 45 IU/L; sample E04 by the sole addition of free hCG[beta] at approximately 30 IU/L; and sample E05 by spiking with both intact hCG (approximately 40 IU/L) and free hCG[beta] (approximately 20 IU/L). These preparations, plus other non-hCG related analytes, were added to an essentially hCG-free human serum matrix. In sample E04, the intact hCG values should be undetectable, since no intact hCG was added, and total [beta] hCG values should be close to the calculated concentration of 30 IU/L. Results for total [beta] hCG were reported by 235 participant laboratories using 12 FDA-approved immunoassay methods and ranged from < 1 to 49.6 IU/L for sample E04 (Table 1). In contrast, results ranged from 33.3 to 53.9 IU/L for sample E03 and 40 to 83 IU/L for sample E05. Results for intact hCG were reported by 61 laboratories using 7 immunoassay instruments and ranged from 1 to 50 IU/L for sample E04, in comparison to results ranging from 34 to 53.3 IU/L for sample E03 and 35 to 77.2 IU/L for sample E05 (Table 1). Investigation of the sources of the discrepancy revealed that the Dade Behring Dimension Flex reagent cartridge hCG detects only the intact hCG molecule, and therefore the hCG values measured on this instrument were close to zero in sample E04, close to 45 IU/L in sample E03, and about 40 IU/L in sample E05. However, 20 of 65 users of this method reported results as total [beta] hCG, rather than the correct intact hCG. Errors were observed with the following 3 automated immunoassay instruments, which detect both intact hCG and free hCG[beta] (total [beta] hCG or hCG + hCG[beta]): Bayer Advia Centaur centaur (sĕn`tôr), in Greek mythology, creature, half man and half horse. The centaurs were fathered by Ixion or by Centaurus, who was Ixion's son.  (Total hCG), Vitros ECi/ECiQ (Total [beta]-hCG), and Siemens Immulite series (Immulite 1000 HCG and Immulite 2000 HCG). For these instruments, however, a total of 8 laboratories reported results as intact hCG. Two Elecsys HCG STAT users also reported intact hCG results as total [beta] hCG (Table 1).

In addition, the recovery of total [beta] hCG differed among these immunoassay methods (Table 1). The variation is likely due to the difference in immunoreactivity of these immunoassays toward hCG[beta] and/or differences in calibration.

CLIA CLIA Clinical Laboratory Improvement Amendments of 1988 Congressional legislation that promulgated quality assurance practices in clinical labs, and required them to measure performance at each step of the testing process from the beginning to the end-point of a  requires that proficiency test specimens be treated in the same manner as patient specimens (14). Erroneous reporting of proficiency test results will thus reflect errors in reporting of patient results that could cause misdiagnosis mis·di·ag·no·sis
n. pl. mis·di·ag·no·ses
An incorrect diagnosis.



mis·diag·nose
 and mismanagement mis·man·age  
tr.v. mis·man·aged, mis·man·ag·ing, mis·man·ag·es
To manage badly or carelessly.



mis·manage·ment n.
 of trophoblastic diseases, although such errors may not affect the diagnosis of normal pregnancy, in which both intact hCG and total [beta] hCG increase dramatically (4, 5). Given the overall reporting error rate of 13.1% (8 of 61) for intact hCG and 9.3% (22 of 235) for total [beta] hCG, the occurrence of such errors is likely to be a significant problem worldwide. Such errors also occur in the literature; for instance, publications have reported "total hCG" determined on the Dade Dimension instrument and "intact hCG" measured on the Immulite system (15, 16).

The utility of monitoring hCG and its various forms reaches beyond the detection of pregnancy to various disease conditions, e.g., ectopic pregnancy ectopic pregnancy
 or extrauterine pregnancy

Condition in which a fertilized egg is imbedded outside the uterus (see fertilization). Early on, it may resemble a normal pregnancy, with hormonal changes, amenorrhea, and development of a placenta.
, choriocarcinoma choriocarcinoma: see neoplasm. , testicular cancer, trophoblastic diseases, and nontrophoblastic tumors (2, 3, 5, 7, 9-12, 17). National Academy of Clinical Biochemistry (NACB NACB National Academy of Clinical Biochemistry
NACB National Association of College Broadcasters (trade association for student radio and TV stations)
NACB National Accreditation of Certification Bodies
NACB Non-Aligned Coordinating Bureau
) and European Group on Tumor Markers (EGTM) guidelines emphasize that both intact hCG and free hCG[beta] be detected by assay kits for tumor markers (18, 19).

To provide reliable services, laboratorians and clinicians must know which forms of hCG the method in service detects. We conclude that at least 4 factors have contributed to the incorrect reporting of hCG results by laboratories: (a) the complexity of the hCG molecule and confusion of nomenclature on the various forms of hCG; (b) laboratory personnel's lack of awareness of the distinctions of the forms of hCG and failure to recognize the specificity of assays for their measurement; (c) manufacturers' lack of clarity and uniformity in reagent labeling; and (d) most product inserts' lack of information on the specificity of each method to the various forms of hCG.

Solving this problem will require involvement of multiple parties in reaching a consensus on nomenclature and clarity of information provided in package inserts. The availability of new WHO reference materials for hCG and its 5 other forms makes this task feasible (20). An urgent need is for manufacturers to clearly state what forms of hCG are detected by each assay. Appropriate labeling and information on assay specificity should then be provided to the user, since current labeling is both inadequate and confusing (4). Furthermore, it has been noted that when hCG[beta] is measured by assays detecting hCG and hCG[beta] together, the results are erroneous as they are based on the IU for hCG; it has been recommended that assay procedures report results in molar concentrations (5).

Grant/Funding Support: None declared.

Financial Disclosures: None declared.

References

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(2.) Young RC. Gynecologic gynecologic /gy·ne·co·log·ic/ (gi?ne-) (jin?e-kah-loj´ik) pertaining to the female reproductive tract or to gynecology.  malignancies. In: Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL, Isselbacher KJ, eds. Harrison's Principles of Internal Medicine. 16th ed. New York: McGraw-Hill, 2007;553-8.

(3.) Motzer RJ, Bosl GJ. Testicular cancer. In: Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL, Isselbacher KJ, eds. Harrison's Principles of Internal Medicine. 16th ed. New York: McGraw-Hill, 2007;550-3.

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(6.) Thomas CMG CMG Coastal & Marine Geology (USGS)
CMG Chipotle Mexican Grill, Inc. (stock symbol)
CMG Companion (of the Order Of) St Michael and St George
CMG Computer Measurement Group
, Reijnders FJL FJL Franz Josef Land (Russian Arctic Archipelago) , Segers MFG MFG Manufacturing
MFG Manufacturer
MFG Mit Freundlichen Grüßen (German: With Best Regards)
MfG Mitfahrgelegenheit (German)
MFG Marithe Francois Girbaud (French clothing company) 
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(7.) Fan C, Goto S, Furuhashi Y, Tomoda Y. Radioimmunoassay of the serum free beta-subunit of human chorionic gonadotropin in trophoblastic disease. J Clin Endocrinol Metab 1987;64:313- 8.

(8.) Rinker AD, Tietz NW. [beta]-hCG vs intact hCG assays in the detection of trophoblastic disease. Clin Chem 1989;35:1799-800.

(9.) Cole LA, Shahabi S, Butler SA, Mitchell H, Newlands ES, Behrman HR, Verrill HL. Utility of commonly used commercial human chorionic gonadotropin immunoassays in the diagnosis and management of trophoblastic diseases. Clin Chem 2001;47:308-15.

(10.) Cole LA, Khanlian SA, Muller CY, Giddings A, Kohorn E, Berkowitz R. Gestational trophoblastic diseases: 3. Human chorionic chorionic

pertaining to the chorion.


chorionic girdle
a circular band of cells of placental origin that invade the endometrium and form the endometrial cups in the mare.
 gonadotropin-free [beta]-subunit, a reliable marker of placental site trophoblastic tumors. Gynecol Oncol 2006;102: 160-4.

(11.) Sailer B, Clara R, Spottl G, Siddle K, Mann K. Testicular cancer secretes intact human choriogonadotropin (hCG) and its free [beta]-subunit: evidence that hCG (+ hCG- [beta]) assays are the most reliable in diagnosis and follow-up. Clin Chem 1990;36: 234-9.

(12.) Cole LA, Sutton JM. Selecting an appropriate hCG test for managing gestational trophoblastic disease Gestational trophoblastic disease
A rare, aggressive, malignant, often metastatic (spreading to other organs) cancer in women of childbearing age in which cancer cells grow in the tissues that are formed in the uterus after conception
 and cancer. J Reprod Med 2004;49:545-53.

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(14.) US Department of Health and Human Services Noun 1. Department of Health and Human Services - the United States federal department that administers all federal programs dealing with health and welfare; created in 1979
Health and Human Services, HHS
. Medicare, Medicaid and CLIA programs: regulations implementing the Clinical Laboratory Improvement Amendments Clinical Laboratory Improvement Amendments (CLIA) of 1988 are United States federal regulatory standards that apply to all clinical laboratory testing performed on humans in the United States, except clinical trials and basic research.  of 1988 (CLIA), final rule. Fed Regist 1992;57:7002-186.

(15.) Massart C, Lucas C, Rioux-Leclercq N, Fergelot P, Pouvreau-Quillien V, Volatron AC. Importance of the detection method for intact dimeric human chorionic gonadotropin without interference with the free human chorionic gonadotropin [beta] subunit for pregnancy exclusion before liver transplantation in a woman with cholangiocarcinoma. Clin Chem Lab Med 2005;43:886-9.

(16.) Hallahan T, Krantz Krantz is the name of two persons:
  • Kermit E Krantz Physician and inventor
  • Grover Krantz Bigfoot researcher
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(17.) van Trommel trom·mel  
n.
A revolving cylindrical sieve used for screening or sizing rock and ore.



[German, from Middle High German trummel, diminutive of trumme, drum,
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(20.) Birken S, Berger P, Bidart JM, Weber M, Bristow A, Norman R, Sturgeon C, Stenman UH. Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites. Clin Chem 2003;49:144 -54.

DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.1373/clinchem.2007.098822

Zhimin (Tim) Cao, [1] * Robert Rej [1,2]

[1] Division of Molecular Medicine, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY; 2 Department of Biomedical bi·o·med·i·cal
adj.
1. Of or relating to biomedicine.

2. Of, relating to, or involving biological, medical, and physical sciences.
 Sciences, School of Public Health, University at Albany, Albany, NY; * address correspondence to this author at: Division of Molecular Medicine, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509. Fax 518-474-9185; e-mail tim@wadsworth.org.
Table 1. Summary of laboratory responses for quantitative hCG
measurements.

                             Sample E03
                             (only intact hCG added)

Instrument, label            Intact HCG,      Total [beta] HCG,
of reagent name              IU/L             IU/L                   %

Abbott Architect, total                       35.9 (2.4)            80
  [beta]-hCG                                  (n = 7)
Abbott AxSym, total                           46.8 (2.7)           104
  [beta]-hCG                                  (n = 4)
Bayer ADVIA:Centaur,         42, 44 (n = 2)   42.0 (2.5)            93
  Total hCG                                   (n = 60)
Bayer ACS:180, total hCG                      45.0 (1.0)           100
  (ThCG)                                      (n = 8)
Beckman Coulter UniCel                        34.6 (1.6)            77
  DxI 800, total [beta]hCG                    (n = 17)
Beckman Coulter Access,                       33.3 (1.8)            74
  total [beta]hCG                             (n = 47)
Diagnostic Systems           43 (n = 1)
  Laboratories, intact
  hCG ELISA
Dade Behring Dimension,      46.5 (1.7)       46.3 (1.9)
  Flex reagent cartridge     (n = 45)         (n = 20)
  [beta]hCG
Dade Behring Stratus CS,                      42.3 (1.4)            94
  [beta]HCG TestPak                           (n = 3)
Vitros ECi/ECiQ, total       43 (n = 1)       43.6 (2.5)            97
  [beta]-hCG                                  (n = 12)
Roche Elecsys, Cobas and     42.6 (3.1)       41, 43
  Modular, HCG STAT          (n = 5)          (n = 2)
Roche Elecsys, Cobas and                      41.3 (2.7)            92
  Modular, HCG + [beta]                       (n = 24)
Siemens IMMULITE series,     53.3 (3.7)       53.9 (4.3)           120
  HCG                        (n = 5)          (n = 31)
BioMerieux Vidas, VIDAS      34, 41
  HCG                        (n = 2)
Target, based on             45               45                   100
  additions

                             Sample E04
                             (only intact hCG[beta] added)

Instrument, label            Intact HCG,      Total [beta] HCG,
of reagent name              IU/L             IU/L                   %

Abbott Architect, total                       18.6 (2.1)            62
  [beta]-hCG                                  (n = 7)
Abbott AxSym, total                           36.5 (1.2)           122
  [beta]-hCG                                  (n = 4)
Bayer ADVIA:Centaur,         23, 24 (n = 2)   21.5 (1.6)            72
  Total hCG                                   (n = 60)
Bayer ACS:180, total hCG                      22.3 (0.9)            74
  (ThCG)                                      (n = 8)
Beckman Coulter UniCel                        36.8 (1.5)           123
  DxI 800, total [beta]hCG                    (n = 17)
Beckman Coulter Access,                       35.7 (2.2)           119
  total [beta]hCG                             (n = 47)
Diagnostic Systems           1 (n = 1)
  Laboratories, intact
  hCG ELISA
Dade Behring Dimension,      1 (0.0)          1.5 (0.7)
  Flex reagent cartridge     (n = 45)         (n = 20)
  [beta]hCG
Dade Behring Stratus CS,                      29.0 (0.9)            97
  [beta]HCG TestPak                           (n = 3)
Vitros ECi/ECiQ, total       50 (n = 1)       48.8 (2.9)           160
  [beta]-hCG                                  (n = 12)
Roche Elecsys, Cobas and     <3.3 (n = 5)     <1, 1.0
  Modular, HCG STAT                           (n = 2)
Roche Elecsys, Cobas and                      30.7 (1.9)           102
  Modular, HCG + [beta]                       (n = 24)
Siemens IMMULITE series,     48.9 (3.0)       49.6 (6.4)           165
  HCG                        (n = 5)          (n = 31)
BioMerieux Vidas, VIDAS       2,  2 (n = 2)
  HCG
Target, based on             1                30                   100
  additions

                             Sample E05
                             (only intact hCG and hCG[beta] added)

Instrument, label            Intact HCG,      Total [beta] HCG,
of reagent name              IU/L             IU/L                   %

Abbott Architect, total                       48.0 (2.2)            80
  [beta]-hCG                                  (n = 7)
Abbott AxSym, total                           67.2 (4.9)           112
  [beta]-hCG                                  (n = 4)
Bayer ADVIA:Centaur,         55, 56 (n = 2)   53.8 (2.8)            90
  Total hCG                                   (n = 60)
Bayer ACS:180, total hCG                      56.7 (2.8)            95
  (ThCG)                                      (n = 8)
Beckman Coulter UniCel                        59.2 (3.7)            99
  DxI 800, total [beta]hCG                    (n = 17)
Beckman Coulter Access,                       58.6 (2.9)            98
  total [beta]hCG                             (n = 47)
Diagnostic Systems           35 (n = 1)
  Laboratories, intact
  hCG ELISA
Dade Behring Dimension,      40.8 (1.6)       40.8 (1.7)
  Flex reagent cartridge     (n = 45)         (n = 20)
  [beta]hCG
Dade Behring Stratus CS,                      59.7 (1.4)           100
  [beta]HCG TestPak                           (n = 3)
Vitros ECi/ECiQ, total       71 (n = 1)       68.7 (4.9)           115
  [beta]-hCG                                  (n = 12)
Roche Elecsys, Cobas and     42.8 (2.3)       40, 43 (n = 2)
  Modular, HCG STAT          (n = 5)
Roche Elecsys, Cobas and                      59.6 (3.6)            99
  Modular, HCG + [beta]                       (n = 24)
Siemens IMMULITE series,     77.2 (4.9)       83.0 (9.5)           138
  HCG                        (n = 5)          (n = 31)
BioMerieux Vidas, VIDAS      40, 44 (n = 2)
  HCG
Target, based on             40               60                   100
  additions

Results are expressed as mean (SD) or each result when n < 3.
Percentages of calculated target values are based on addition of known
quantities of hCG. Results of intact hCG and that of erroneously
reported as total [beta] hCG are not calculated for % recovery.
Complete details on results from the proficiency test events
discussed in this paper are available at http://www.wadsworth.org/
chemheme/chem/endo/ptframes.htm.
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Title Annotation:Brief Communications
Author:Cao, Zhimin "Tim"; Rej, Robert
Publication:Clinical Chemistry
Date:Apr 1, 2008
Words:3059
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