Introduction 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) - hCGb (free b subunit) -hCGbcf (the core fragment of hCGb Urine & serum Urine only

Introduction POC hCG devices differentially recognize the many hCG variants hCG hCGa hCGbcf hCGbn hCGb hCGn Sigel & Grenache Clin Chem 2007;53:989-90

Question What potential problems could arise given that different qualitative hCG devices detect different hCG variants? Sigel & Grenache Clin Chem 2007;53:989-90

- reported pregnancy ≥10 wks Materials 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

Materials 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) - hCGbcf, IRR 99/708 - hCGbn, IRR 99/692 - hCGn, IRR 99/642 - hCGb, IRR 99/650

Materials and Methods hCG Variant Assays POC Devices - intact hCG+hCGn- time-resolved IFMA (Auto DELFIA, Perkin-Elmer Wallac) (MAb5008 + MAb5501) - hCGb – in house IFMA mAb1B2 + mAb9C11 - hCGbcf- in house IFMA mAb1B2 + mAb3C11 - Total hCG (hCG + hCGb) Siemens Centaur Total hCG POC Devices - OSOM® hCG Combo test (Genzyme Diagnostics) - ICON ® 25 hCG (Beckman Coulter) - hCG Combo SP® Brand (Cardinal Health)

Question 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

Results Patient Samples using OSOM devices (lot#081299) Patient 1: Neat = neg 1:5 = pos Patient 2: Neat = neg 1:3 = pos Neat on OSOM lot # 071717 & 081334 = weak pos Patient 3: Neat = neg 1:5 = weak pos 1:10 = 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

Results Patient 1 using other qualitative hCG devices ICON 25 hCG Device hCG Combo SP Neat Faint pos pos 1:3 According to Sigel & Grenache Clin Chem 2007;53:989-90 hCG Combo recognizes hCGbcf, but ICON 25 and OSOM do not Could excess hCGbcf be causing the false negative results in the ICON 25 & OSOM devices?

Results hCGbcf was present in molar excess of intact hCG in these patients, representing 64-78% of immunoreactive hCG

Results hCGbcf inhibited OSOM and ICON 25 positive signal in a dose-dependent manner

Results hCG, hCGbn and hCGn also inhibited OSOM positive signal in dose-dependent manner to a lesser extent.

Question What do you think is the mechanism for the hCGbcf inhibition of positive hCG results on these qualitative devices? Sigel & Grenache Clin Chem 2007;53:989-90

Discussion Experiments Indicate: 1) OSOM device can detect urine hCG up to 1.6 million IU/L 2) In 3 index patients [hCGbcf] was in molar excess of intact hCG 3) Addition of purified hCGbcf to hCG pos urine can cause false neg in OSOM and ICON hCG devices Hypotheses is: - One of the two Abs in device recognizes hCGbcf - When [hCGbcf] 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 negative interference (Methods 2000;21:41-50)

Discussion Clinical Implications: - hCGbcf is major hCGb 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 hCGbcf 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

Post Script Clin Chem 2009;55:1885-6. 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.

Question 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

Supplemental Data

Results Supplemental Figure 1. Purified hCGcf 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.

Results Supplemental Figure 2. Effect of purified hCG, hCGn and hCGn using the hCG Combo (Cardinal Health) and OSOM (Genzyme Diagnostics) devices.