Introduction Misleading results in POC devices can be due to: - incorrect reading time - insufficient or dilute urine - mislabeled specimens - early.

Presentation on theme: "Introduction Misleading results in POC devices can be due to: - incorrect reading time - insufficient or dilute urine - mislabeled specimens - early."— Presentation transcript:

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2 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

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

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

5 - 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

6 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

7 Materials and Methods hCG Variant Assays POC Devices
- intact hCG+hCGn- time-resolved IFMA (Auto DELFIA, Perkin-Elmer Wallac) (MAb 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)

8 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

9 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 # & = 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

10 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?

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

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

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

14 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

15 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)

16 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

17 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:

18 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

19 Supplemental Data

20 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.

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