Interferences are defined and categories listed. There is an extensive section on anti-animal and other types of antibodies that can cause immunoassay interference. Interferences by paraproteins, immune complexes, drugs, herbal remedies, blood substitutes and imaging agents are explained. Types of interference arising from the sample are reviewed (icterus, hemolysis, lipemia, sampling problems and compounds leaching from blood collection tubes). Assay-related interferences (cross-reactivity and high-dose hook) are described. Strategies to identify potential cases of assay interference, prove that interference is affecting the results and treat the sample to remove the interference are provided. A case study of the clinical and legal consequences of immunoassay interference (the Rufer case) is presented. Other examples of interference are also presented.

¹Department of Pathology, Children’s Medical Center, and University of Texas Southwestern Medical School, 1935 Medical District Drive, Dallas, Texas 75235, USA
²Department of Pathology & Laboratory Medicine, 7.103 Founders Pavilion, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA

In the year 2015, immunoassay interference (not surprisingly) remains a significant issue and a number of editorials and case reports continue to draw attention to this persistent problem (1-3) . Recently reported interferents include substances endogenous to the patient (heterophilic antibodies, macrohormones, immunoglobulins), iatrogenic (imaging contrast reagents, drugs) and genetic changes in encoded proteins. Genetic changes were not explored in our originally published chapter, but a recent report on false positive malaria diagnostic assays is a reminder of the significance of changes in protein coding genes (4) . Nucleotide variation or whole gene deletion may both result in falsely negative constitutional genetic testing. But these types of genetic variations may also lead to interference in biomarker assays as well as infectious disease assays.

The causative organism for malaria, Plasmodium falciparum, can be detected by rapid immunoassays targeting P. falciparum specific histidine rich protein 2 (5) . Deletion of the Pfhrp2 gene with associated false negative immunoassay testing has now been reported in multiple studies (4-6) . Similarly, false negative immunoassays have been identified for various single nucleotide variants of the gene encoding Hepatitis B surface antigen (HBsAg) (7).

In addition to nucleic acid changes leading to interference in infectious disease immunoassays, there has been a report of a false negative PSA immunoassay secondary to deletion of the PSA encoding gene KLK3 (8). Indeed, genetic variations that encode amino acid changes and pathogenic enzymatic activity (e.g., BRAF V600E) are targeted by clinical immunohistochemistry assays (9).

Potentially, asymptomatic genetic variation can lead to changes in protein expression and also immunogenicity of the protein, and this may be a source of false negative assay results, as suggested for cardiac biomarkers such as BNP (10) as well as troponins (11).

Immunoassay interferents are substances that adversely affect the performance of an immunoassay and often cannot be accounted for in the development of an immunoassay. Traditionally, the focus has been on heterophilic antibodies, but we should also consider other sources of assay interference such as genetic variation of the DNA encoding the proteins targeted by the immunoassays.

1. Halsall, D.J. Antibody interference in immunoassay: know your enemy. Annals of Clinical Biochemistry. 50 (Pt 5), 397-9 (2013). PubMed PMID: 23888058.
2. Lippi, G., Daves, M., Mattiuzzi, C. Interference of medical contrast media on laboratory testing. Biochemia Medica. 24 (1), 80-8 (2014). PubMed PMID: 24627717. Pubmed Central PMCID: 3936969.
3. Rulander, N.J., Cardamone, D., Senior, M., Snyder, P.J., Master, S.R. Interference from anti-streptavidin antibody. Archives of Pathology & Laboratory Medicine. 137(8), 1141-6 (2013). PubMed PMID: 23899071.
4. Kumar, N., Pande, V., Bhatt, R.M., Shah, N.K., Mishra, N., Srivastava, B., et al. Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test. Acta Tropica 125 (1),119-21 (2013). PubMed PMID: 23041541.
5. Lee, N., Baker, J., Andrews, K.T., Gatton, M.L., Bell, D., Cheng, Q., et al. Effect of sequence variation in Plasmodium falciparum histidine-rich protein 2 on binding of specific monoclonal antibodies: Implications for rapid diagnostic tests for malaria. Journal of Clinical Microbiology. 44 (8), 2773-8 (2006). PubMed PMID: 16891491. Pubmed Central PMCID: 1594627.
6. Gamboa, D., Ho, M.F., Bendezu, J., Torres, K., Chiodini, P.L., Barnwell, J.W., et al. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PloS one. 5 (1): e8091 (2010). PubMed PMID: 20111602. Pubmed Central PMCID: 2810332.
7. Coleman, P.F. Detecting hepatitis B surface antigen mutants. Emerging Infectious Diseases, 12 (2),198-203 (2006). PubMed PMID: 16494742. Pubmed Central PMCID: 3293431.
8. Rodriguez, S., Al-Ghamdi, O.A., Burrows, K., Guthrie, P.A., Lane, J.A., Davis, M., et al. Very low PSA concentrations and deletions of the KLK3 gene. Clinical Chemistry, 59 (1), 234-44 (2013). PubMed PMID: 23169475.
9. Kuan, S.F., Navina, S., Cressman, K.L., Pai, R.K. Immunohistochemical detection of BRAF V600E mutant protein using the VE1 antibody in colorectal carcinoma is highly concordant with molecular testing but requires rigorous antibody optimization. Human Pathology, 45 (3):464-72 (2014). PubMed PMID: 24529329.
10. Lanfear, D.E., Stolker, J.M., Marsh, S., Rich, M.W., McLeod, H.L. Genetic variation in the B-type natiuretic peptide pathway affects BNP levels. Cardiovascular Drugs and Therapy / sponsored by the International Society of Cardiovascular Pharmacotherapy, 21 (1), 55-62 (2007). PubMed PMID: 17340039.
11. Lippi, G., Targher, G., Franchini, M., Plebani, M. Genetic and biochemical heterogeneity of cardiac troponins: clinical and laboratory implications. Clinical Chemistry and Laboratory Medicine: CCLM / FESCC, 47 (10),1183-94 (2009). PubMed PMID: 19754353.

Jason Y. Park, MD, PhD, FCAP is an Assistant Professor in the Department of Pathology at UT Southwestern Medical School. He is also the Medical Director of the Advanced Diagnostics Laboratory at the Children’s Medical Center (Dallas, Texas). He trained in a combined MD and PhD program at Thomas Jefferson University (Philadelphia, PA) and was a pathology resident and chief resident in the Department of Pathology and Laboratory Medicine at the Hospital of the University of Pennsylvania. After residency, he was a clinical fellow in gastrointestinal and liver pathology at Johns Hopkins Hospital. He has published over fifty journal articles and book chapters. He has 11 issued U.S. patents and 2 pending U.S. patent applications. His clinical and research interests are in the development of clinical laboratory technologies with a focus on pediatric and gastrointestinal diseases. For more details visit http://profiles.utsouthwestern.edu/profile/115607/jason-park.html/

Larry J. Kricka, D. Phil., F.A.C.B., F.R.S.C. C.Chem., F.R.C.Path, is Professor of Pathology & Laboratory Medicine at the University of Pennsylvania and Director of the General Chemistry Laboratory at the Hospital of the University of Pennsylvania. Awards include the Ullman Award, the American Association for Clinical Chemistry (AACC) Award for Outstanding Contributions to Clinical Chemistry (Selected Area of Research), Rank Prize for Opto-Electronics, Queen’s Award for Technological Achievement, and the Society of Analytical Chemistry Silver Medal (Royal Society of Chemistry). He was President of the AACC and a Distinguished Visiting Scholar at Christ's College, Cambridge, England. He is currently a member of the Executive Board of the International Federation of Clinical Chemistry and Laboratory Medicine. His research interests include bioluminescence/chemiluminescence, analytical microchips, heterophile antibodies and direct to consumer testing. Dr. Kricka holds over 30 U.S. patents and is the author/co-author of over 450 articles, abstracts, book chapters, papers and 22 books, and is Editor-in-chief of Luminescence. For more details visit http://pathology.uphs.upenn.edu/Faculty/FacultyInfo.aspx?site=FacultyList&dept=Faculty&id=15422/

Interference, false positive, false negative, anti-animal antibody, human anti-mouse antibody, heterophile antibody, autoantibodies, rheumatoid factor, paraproteins, drugs, blood substitutes, contrast agents, icterus, hemolysis, lipemia, carryover, contamination, blood collection tubes, cross-reactivity, high-dose hook, dilution, blocker.