1. A Simple Protocol for Identification of Heterophile Antibody Interferences in the Abbott AxSYM hCG Assay. P Giannopoulos, G Jones, E-M Lim Department of Chemical Pathology St Vincent’s Hospital, Sydney

2. Heterophile Antibodies? Immunoglobulins found in some patient’s serum that bind antibodies from other species in 2 site immunoassays Heterophilic Antibodies can mimic the presence of analyte by binding the two antibodies in the assay produce false results

3. Why? Interference with Abbott AxSYM hCG assay Dr Laurence Cole, USA hCG Reference Laboratory –over 40 cases false positives –Average “hCG” 81 U/L –Laparoscopy, surgery, chemotherapy Consistent with heterophile antibodies

4. Abbott? Letter from Worldwide Marketing Manager January 2001 –Urine hCG –Re-measure serum on alternate analyser –Identify clinical inconsistency

5. Why are we telling you? RCPA AACB Quality Assurance Program -37% use AxSYM hCG assay

6. How did we do it? AxSYM:

7. How do we do it now? Sydpath protocol hCG levels: 10 and 100U/L manual 1 in 2 hCG levels: 100 and 1000U/L auto dilution 1 in 10 Dilution performed in hCG specific diluent

8. What is the Rationale for the Sydpath Protocol? Increased amounts of animal proteins may block heterophile antibody interference

9. Diluent 11 uL Sample 52 uL

10. What we found? 2400 hCG measurements 133 dilution protocol (5.6%) 2 patients with discrepant results on dilution (0.08%)

11. Discrepant results? Patient A ?Pregnant hCG = 87 U/L (46-122) 1 in 2 = 0 U/L Patient B ? Pregnant hCG = 83 U/L(52-121) 1 in 2 = 0 U/L

12. What we did next? Obtained urine samples –Negative on Hybritech Icon and AxSYM Measured serum on another analyser –hCG not detected Issued results: <2 U/L hCG

13. Further Investigation? Other analysers Protein G agarose precipitation Serial dilution

14. Summary of Measurements

15. Protein G Agarose Protein G: An Immunoglobulin G binding protein

16. Dilutions in buffer Protein-free buffer

17. Abbott Letter Discrepant results with clinical findings Urine HCG measurement Measure on another analyser

18. What we learnt? The Abbott recommendation is limited for identifying false positive results. SydPath Protocol: a fast, simple, cost- effective solution. We prevented two incorrect results from being issued Acknowledge Dr Laurence Cole. These powerpoint slides will be available at www.sydpath.stvincents.com.au after the conference.