© copyright 2009 by the american association for clinical chemistry false-negative results in...
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© Copyright 2009 by the American Association for Clinical Chemistry
False-Negative Results in Point-of-Care Qualitative Human Chorionic Gonadotropin (hCG) Devices Due to Excess hCGβ Core Fragment
Ann M. Gronowski, Mark Cervinski, Ulf-Håkan Stenman, Alison Woodworth, Lori Ashby, and Mitchell G. Scott
July 2009
www.clinchem.org/cgi/content/abstract/55/7/1389
© Copyright 2009 by the American Association for Clinical Chemistry
Journal ClubJournal Club
© Copyright 2009 by the American Association for Clinical Chemistry
IntroductionIntroduction
Misleading results in POC devices can be due to:
- incorrect reading time- insufficient or dilute urine- mislabeled specimens- early pregnancy loss - high dose hook effect
In pregnancy, hCG immunoreactivity due to many variants:
- hCG (intact hCG)- hCG-h (hyperglycosylated hCG)- hCGn (nicked hCG)- hCG (free subunit)
-hCGcf (the core fragment of hCG
Urine & serum
Urine only
© Copyright 2009 by the American Association for Clinical Chemistry
IntroductionIntroductionPOC hCG devices differentially recognize the many hCG variants
Sigel & Grenache Clin Chem 2007;53:989-90
hCG
hCG
hCGcf
hCGn
hCG
hCGn
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion What potential problems could arise given that different qualitative
hCG devices detect different hCG variants?
Sigel & Grenache Clin Chem 2007;53:989-90
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and MethodsMaterials and Methods
3 Index Patients
- reported pregnancy ≥10 wks- live intrauterine pregnancy by ultrasound- negative urine hCG using OSOM Combo test
(Genzyme Diagnostics)- serum hCG ≥99,000IU/L
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Materials and MethodsMaterials and Methods
Sources of hCG- urine from patient with gestational trophoblastic disease
(serum hCG 3,200,000 IU/L)
- random hCG pos urine hCG 17,899 IU/L
- purified hCG variants International Reference Reagents from National Institute for Biological Standards & Controls (Hertfordshire, UK)
- hCGcf, IRR 99/708- hCGn, IRR 99/692- hCGn, IRR 99/642- hCG, IRR 99/650
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and MethodsMaterials and Methods
hCG Variant Assays- intact hCG+hCGn- time-resolved IFMA (Auto DELFIA, Perkin-Elmer
Wallac) (MAb5008 + MAb5501) - hCG – in house IFMA mAb1B2 + mAb9C11
- hCGcf- in house IFMA mAb1B2 + mAb3C11- Total hCG (hCG + hCG) Siemens Centaur Total hCG
POC Devices- OSOM® hCG Combo test (Genzyme Diagnostics)- ICON ® 25 hCG (Beckman Coulter)- hCG Combo SP® Brand (Cardinal Health)
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion Three pregnant patients have negative qualitative hCG results. What are possible explanations for the false negative results? What
experiments could be done to test these possibilities?
Sigel & Grenache Clin Chem 2007;53:989-90
© Copyright 2009 by the American Association for Clinical Chemistry
ResultsResultsPatient Samples using OSOM devices (lot#081299)
Patient 1: Neat = neg1:5 = pos
Patient 2: Neat = neg1:3 = posNeat on OSOM lot # 071717 & 081334 = weak pos
Patient 3: Neat = neg1:5 = weak pos1:10 = strong pos
Hook Effect on OSOM Device
<1,600,000 IU/L = pos>1,900,000 IU/L = neg Hook effect occurs >1,600,000
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ResultsResultsPatient 1 using other qualitative hCG devices
ICON 25 hCG Device hCG Combo SP
Neat Faint pos pos
1:3 pos pos
According to Sigel & Grenache Clin Chem 2007;53:989-90 hCG Combo recognizes hCGcf, but ICON 25 and OSOM do not
Could excess hCGcf be causing the false negative results in the ICON 25 & OSOM devices?
© Copyright 2009 by the American Association for Clinical Chemistry
ResultsResults
hCGcf was present in molar excess of intact hCG in these patients, representing 64-78% of immunoreactive hCG
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ResultsResults
hCGcf inhibited OSOM and ICON 25 positive signal in a dose-dependent manner
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ResultsResults
hCG, hCGn and hCGn also inhibited OSOM positive signal in dose-dependent manner to a lesser extent.
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion What do you think is the mechanism for the hCGcf inhibition of
positive hCG results on these qualitative devices?
Sigel & Grenache Clin Chem 2007;53:989-90
© Copyright 2009 by the American Association for Clinical Chemistry
DiscussionDiscussion
Experiments Indicate: 1) OSOM device can detect urine hCG up to 1.6 million IU/L
2) In 3 index patients [hCGcf] was in molar excess of intact hCG3) Addition of purified hCGcf to hCG pos urine can cause false
neg in OSOM and ICON hCG devices
Hypotheses is:- One of the two Abs in device recognizes hCGcf- When [hCGcf] is in molar excess of intact hCG, Abs are
saturated thus preventing sandwich formation- This confirms the predictions of Madersbacher & Berger that
hCG variants can bind one antibody and cause negativeinterference (Methods 2000;21:41-50)
© Copyright 2009 by the American Association for Clinical Chemistry
DiscussionDiscussion
Clinical Implications: - hCGcf is major hCG subunit-related molecule in urine
after ~5-8 wks of pregnancy. Accounts for up to 90% of immunoreactive urine hCG from mid pregnancy.
-CAUTION should be used when hCG devices in which hCGcf causes a neg interference are used to test women who are beyond 5-8 wks gestation, as false neg results may occur
Take Home Message:- Positive =good evidence patient is pregnant - Negative does not mean patient is not pregnant
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Post ScriptPost Script
The authors later reported that the hCG Combo SP® Brand (Cardinal Health) also demonstrated the same false negative effect with a different lot number.Clin Chem 2009;55:1885-6.
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion In what clinical situations should these qualitative hCG devices be used? What can manufacturers do to help prevent these types of problems?
Sigel & Grenache Clin Chem 2007;53:989-90
© Copyright 2009 by the American Association for Clinical Chemistry
Supplemental DataSupplemental Data
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ResultsResults
Supplemental Figure 1. Purified hCGcf causes hook effect in OSOM (Genzyme Diagnostics) and ICON 25 (Beckman Coulter) devices, but not Cardinal Health hCG Combo when added to hCG-positive urine.
© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry
ResultsResults
Supplemental Figure 2. Effect of purified hCG, hCGn and hCGn using the hCG Combo (Cardinal Health) and OSOM (Genzyme Diagnostics) devices.