Most Common Interferences in Immunoassays

REVIEW Libri Oncol., Vol. 43 (2015), No 1-3, 23 – 27

MOST COMMON INTERFERENCES IN IMMUNOASSAYS

MIHAELA GAĆE, ZVJEZDANA ŠPACIR PRSKALO, SANJA DOBRIJEVIĆ and LJILJANA MAYER

Department of Medical Biochemistry in Oncology, University Hospital for Tumors, University Hospital Center Sestre milosrdnice, Zagreb, Croatia

Summary The development of technology has enabled fast and easy determination of tumor markers and hormones at all levels of medical and biochemical diagnostics. Markers and hormones are most commonly determined by immunoassays and methods of molecular diagnostics. Immunoassays are based on the specifi c antibody-antigen reaction. Numerous factors during analysis may aff ect the measurement of tumor markers and hormones in patient samples (1). Although immunoassays are sensitive and reproducible they can miss the specifi city and accuracy. Immunoassays are infl uenced by various interferences and most common of them will be explained in text below. KEY WORDS: interferences, immunoassay, heterophilic antibodies, human anti-mouse antibodies

NAJČEŠĆE INTERFERENCIJE KARAKTERISTIČNE ZA IMUNOKEMIJSKE METODE Sažetak Razvoj tehnologije je omogućio brzo i jednostavno određivanje hormona i markera. Najčešće se određuju imunokemij- skim i metodama olekularne biologije. Imunokemijski testovi se temelje na specifi čnoj reakciji protutijelo-antigen. Brojni faktori mogu utjecati na određivanje tumorskih markera i hormona u uzorku krvi. Iako su imunokemijske metode osjetljive i reproducibilne može im nedostajati specifi čnosti i točnosti. Najčešće interferencije su opisane u tekstu. KLJUČNE RIJEČI: interferencije, imuno testovi, heterofi lna protutijela, ljudska protumišja protutijela

INTRODUCTION simple storage are independent of the analyte concentration. Interference is caused by interaction with one Endogenous interfering substances can occur or more steps in the immunoassay procedure and in both healthy and pathological patient samples. the analyte concentration or the antibody binding Each patient has his own unique sample proper- is inﬂ uenced (2). Interference may increase (posi- ties and interference results from an interaction tive interference) or decrease (negative interfer- with one or more steps in the immunoassay proce- ence) the measured result and can be analyte-de- dure. The measurable analyte concentration in the pendent or analyte-independent. sample or antibody binding is altered by many Analyte-dependent interferences in immu- factors (Table 1) (3). noassays are caused by interaction between com- Exogenous interferences are any interference ponents in the sample with one or more reagent caused by the introduction of external factors or antibodies. The common interferences of hemoly- conditions. Any external factors, in vivo or in vi- sis, icterus, lipemia, eﬀ ects of anticoagulants and tro, which are not normally present in native,

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Table 1. A choice of diff erent interference eff ects NATURE OF INTERFERENCES. which may appear in immunoassays are schemat- 1. Interferences that alter the measurable analyte concentration ically shown in Figure1. Diff erent interference ef- in the sample fect can yield false positive or in some case false • Hormone binding proteins negative results • Pre-analytical factors, e.g., anticoagulants, sample storage One of the most common interference in im- • Autoanalyte antibodies munoassays is a cross-reaction. Cross-reaction is a 2. Interferences that alter antibody binding problem in diagnostic immunoassays where en- • Heterophile antibodies dogenous molecules with a similar structure to • Human anti-animal antibodies • High-dose hook effect the measured analyte exist of where metabolites of the analyte have common cross-reacting epit- opes, and where there is administration of struc- properly collected and stored samples can intro- turally similar medications (7). duce interference. For example, hemolysis, lipemia, icterus, blood collection, tube additives, ad- Pre-analytical factors ministration of radioactive or fl uorescent com- pounds, drugs, herbal medicines, nutritional Blood collection tubes are not inert contain- supplements, sample storage and transport are all ers. Silicone oils present in some blood collection exogenous interferences that can adversely aff ect devices or tubes can physically interfere with anti- immunoassays (4). gen-antibody binding. For the sample type, according to the NACB (National Academy of Clinical Biochemistry) rec- ommendations of quality requirements in the pre- analytical phase (9), serum or plasma are usually equally appropriate for immunochemistry analysis. Hemolysis and icterus do not aff ect immunoassays as other analytes measured by spectral or chemical means. Samples with any sign of hemolysis are not acceptable for immunoassays of relatively labile analytes due to the release of pro- teolytic enzymes from erythrocytes that degrade these analytes (calcitonin, parathyroid hormone, gastrin). Lipemia can interfere in some immunoassays may produce erroneous results in some assays by interfering with antigen binding, even when antibodies are linked to a solid support (6). Stability of the tumor markers are generally stable, but serum or plasma should be separated F igure 1. Diff erent interfaces in immunometric immunoassays (5): Aa – assay without any interference; from the clot and stored at 4° C (short-term) or Ab – cross-reactivity of an interfering substance with capture -30° C (long-term) as soon as possible. antibody, resulting in false negative results Carryover due to inadequate washing or fail- B – positive interference: ure to detect a sample clot can result in over- or Ba – unspecifi c binding of labelled detector antibody to a not under-estimation of values, but with fully auto- blocked solid phase; mated process carryover factor is reduced to mini- Bb – “bridge” binding by heterophilic antibodies or HAMA, re- mum. spectively; C – negative interference: Hormone binding globulins such as albumin Ca – change or sterical confi rmation after binding or interfering (because of its large concentration), sex hormone protein to Fc fragment of detector antibody binding globulin (SHBG), thyroid binding globu- Cb – masking of the epitope on analyte surface by a protein of lin (TBG) and cortisol binding globulin (CBG) can the sample alter the measurable analyte concentration in the

24 Libri Oncol., Vol. 43 (2015), No 1–3, 23 – 27 sample either by removal or blocking of the analyte (6). Autoantibodies have been described that can cause interference for a number of analytes in- cluding thyroid hormones in both free and total forms (10), thyroglobulin (11). Positive or negative inﬂ uence may occur, depending on whether the autoantibody-analyte complex partitions into the free or the bound analyte fraction. Interference from autoantibodies can occur in both immunoassay formats (6).

Antibody interference Figure 2.The hook eff ect - An excessive amount of analyte over- whelms the binding capacity of the capture antibody. This re- There are two types of endogenous antibod- sults in an inappropriately low signal that causes erroneous low ies in patient´s specimen, heterophilic antibodies or normal result (“hooked” result) for a patient with an exces- and human anti-animal antibodies. sively elevated serum analyte concentration (5). Heterophilic antibodies are antibodies pro- duced against poorly defi ned antigens. They are multi-specifi c antibodies of the early immune reagent antibodies must be increased and/or reduce sponse and generally show low affi nity and weak the amount of sample required for analysis. De- binding (8). Human anti-animal antibodies (HAAA) sign assay should ensure that the concentrations are high-affi nity, specifi c polyclonal antibodies of both capture and detector antibodies are suffi - generated after contact with animal immunoglobu- ciently high to manage with levels of analytes over lin (12). the entire pathological range. The most common anti-animal antibodies are human anti-mouse antibodies (HAMA). Mouse CONCLUSION monoclonal antibodies are applying mostly for a therapy and diagnostic purposes. HAMA can in- Interference in immunoassays is a serious terfere with mouse monoclonal antibodies that are problem which can have important clinical conse- the main component of the reagent. quences and may lead to unnecessary clinical in- Heterophilic antibodies interfere with immu- vestigation as well as inappropriate treatment noassays and can bind to the conjugate, enzyme, with potentially unfavorable outcome for the pa- or other parts of the detection system in reagent- tient. There is no single procedure that can rule limited assays. The same heterophile may react out all interferences. It is important to recognize diff erently for diff erent antibody combinations the potential for interference in immunoassay and hence giving rise to a falsely elevated result in one to put procedures in place to identify them wher- assay but a lower result in another assay. Reagent ever possible. Process need to be in place in order manufactures routinely add blocking agent to to make both laboratories and physicians aware of their assay formulations in order to reduce inter- the potential for immunoassay interference, which ference. can lead to clinical misinterpretation. In table 2. are shown some of the most common interferenc- High-dose hook eff ect es (11–14). The hook eff ect is caused by excessively high Most important is a consideration of the clin- concentrations of analyte simultaneously saturat- ical picture. If there is any suspicion of discor- ing both capture and detector antibodies (Figure 2). dance between the clinical and the laboratory data In immunoassays with very large analyte an att empt should be made to reconcile the diff er- concentration ranges antigen-antibody reactions ence. Constant dialogue is required between phy- can go into antigen excess and result in falsely de- sician and laboratory about unexpected immuno- creased results and potential misdiagnosis (6). To assay results. Physicians should be encouraged avoid high/dose hook eff ect the quantity of the re- to communicate specifi cally with the laboratory

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Table 2. MOST COMMON INTERFERENCES AND OTHER SOURCE OF VARIATION IN IMMUNOASSAYS

Possible interference and other sources of variation Analyte Increased or decreased Increased result value Decreased result value value α-1-fetoprotein Benign disease (alcohol hepatitis, cholestasis), Long-term sample HAMA and heterophile AFP pregnancy). preservation. antibodies. Carcinoembryonic Benign disease (chronic hepatitis, renal disease, Long-term sample HAMA and heterophile antigen gastrointestinal diseases), smoking, preservation. antibodies. CEA Li and Na-heparin plasma. Carbohydrate Benign disease (chronic hepatitis, autoimmune disease, High-dose hook effect. HAMA and heterophile Antigen 15-3 gynecologic diseases), other cancer diseases. antibodies. CA 15-3 Carbohydrate Benign disease (endometriosis, ovarium cysts, liver Long-term sample HAMA and heterophile Antigen 125 diseases, pregnancy, hart failure), other cancer diseases, preservation. antibodies. CA 125 menstruation period. Carbohydrate Menstruation period, pregnancy, contamination of sample Patients with blood group HAMA and heterophile Antigen19-9 with secretion (saliva, etc.), benign disease (chronic Levis(a) and Levis(b). antibodies. CA 19-9 hepatitis, cholestasis, gynecologic diseases). Human epididymis Menopause, Pregnancy, protein 4 renal disease High BMI HE4 Total specifi c Hormonal therapy, prostate manipulation and ultrasound Prolonged time to sample HAMA and heterophile prostate antigen examination, ejaculation, other cancer disease (breast spin, thermal sample antibodies. tPSA cancer, salivary gland neoplasms). manipulation. Human chorionic Heterophile antibodies, non-specifi c protein binders, Renal Hook effect, thermal gonadotropin disease, menopause, cannabis consummation. sample manipulation. total hCG Thyroid AntiTPO, HAMA and auto antibodies Stimulating Pregnancy, Hormone Cardio vascular risk TSH Thyroxine T4 autoantibodies, HAMA Non-esterifi ed fatty acids, T4 and free form Hashimoto’s thyroiditis, Graves’ disease thyroid binding globulin fT4 (TBG) Triiodothyronine T3 autoantibodies. HAMA Non-esterifi ed fatty acids, T3 and free form Hashimoto’s thyroiditis, Graves’ disease thyroid binding globulin fT3 (TBG) about discordance between results and clinical out all interferences. It is important to recognize fi ndings. the potential for interference in immunoassay and The process should also include on-going to put procedures in place to identify them wher- education, review of patient results in the clinical ever possible. Process need to be in place in order sett ing, protocols for the testing of suspected in- to make both laboratories and physicians aware of terference, and notifi cation of interferences both the potential for immunoassay interference, which to the physician and to the diagnostic manufac- can lead to clinical misinterpretation. turer. Interference in immunoassays is a serious problem which can have important clinical conse- REFERENCES quences and may lead to unnecessary clinical in- 1. Štraus B, Lada R. Biokemija i dijagnostika zloćudnih vestigation as well as inappropriate treatment tumora. U: Čepelak D, Čvoriščec I, ur. Štrausova with potentially unfavorable outcome for the pa- medicinska biokemija. Zagreb: Medicinska naklada; tient. There is no single procedure that can rule 2009. str. 522-33.

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