Immunoassay dr. jayesh

Interferences in Immunoassay

1. Interferences and Interpretations in Immunoassay Dr. Jayesh Warade

2. Few things in life are perfect, and unfortunately this also applies to medical diagnostic procedures such as clinical laboratory testing .

3. A 32 years female with repeated high human chorionic gonadotropin ( hCG ) result, prompted to undergo a hysterectomy, chemotherapy, radiotherapy, and a partial pneumonectomy . Only on subsequent testing with another method the beta HCG was found to be falsely high because of interference A patient with an Escherichia coli septicemia was found to have increased immunoassay results for cardiac troponin I, thyroid stimulating hormone, hCG , a-fetoprotein, and CA-125, but none of these results was consistent with the clinical findings. Serum protein electrophoresis revealed that the patient also had a restricted IgM l peak. This was identified as the cause of the falsely increased values in the immunometric assays .

4. Immunoassay An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule in a solution through the use of an antibody or immunoglobulin Immunoassays may be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Multi-step assays are often called separation immunoassays or heterogeneous immunoassays. Some immunoassays can be carried out simply by mixing the reagents and sample and making a physical measurement. Such assays are called homogenous immunoassays or less frequently non-separation immunoassays . 12/4/2015

5. Immunoassay 12/4/2015 Direct Indirect Sandwitch Competitive

6. Techniques CLIA CMIA ELISA ECLIA RIA Lateral Flow Test 12/4/2015

7. Immunoassay Immunoassays are analytically sensitive and measurements can frequently performed without prior extraction. However, immunoassays may lack adequate specificity and accuracy. Specificity of an immunoassay depend on the binding property of the antibody composition of the antigen and its matrix is important. reagent composition and immunoassay format

8. Substances that alter the measurable concentration of the analyte in the sample or alter antibody binding can potentially result in assay interference

9. Immunoassays Commonly Affected Endocrine Cortisol Estradiol Free thyroxine FSH LH Progesterone Prolactin Testosterone Thyroglobulin Thyroxine Triiodothyronine TSH Tumour Marker AFP CA 125 CA 15-3 CA 19-9 CEA hCG PSA Others CK-MB Ferritin Hepatitis B surface antigen Troponin I 12/4/2015

10. Incidence Heterophile antibodies may be present in all patients The potential for immunoassay interference much lower. The frequency of immunoassay interferences resulting from these antibodies depends on what magnitude of bias in the analytical method constitutes a significant interference. The prevalence of potentially interfering antibodies has been reported to be as high as 40%, the incidence of immunoassay interference is estimated to be less than 2%. 12/4/2015

11. Types of Interferences 12/4/2015 analyte-dependent analyte–independent positive interference negative interference

12. Common Interferences – Analyte Independent hemolysis anticoagulants sample storage icterus lipemia

13. Common Interferences – Analyte Dependent 12/4/2015 heterophilic antibodies auto-analyte antibodies rheumatoid factor other proteins human anti-animal antibodies

14. Interference in Immunoassays 12/4/2015 Cross reactivity Hook Effect Antibody Interference Signal Interference Matrix Effect

15. Properties of Interfering Substances Unique to an individual Concentration can change over time within the same individual Low affinity polyspecific antibodies can be present in high concentrations or high affinity in low concentrations Can produce falsely high (false-positive) or falsely low ( false-negative) results may interfere within one or more manufacturers' immunoassay systems but not necessarily in all assays May interfere in a number of immunoassays for different analytes The inclusion of one or more interference blocking agents in manufacturers immunoassay reagents may be insufficient to overcome the interference

16. Cross Reactivity M ost common interference - mostly in competitive assay Compete for binding site on antibody, resulting in over- or underestimation of analyte concentration Cross-reaction is a problem in diagnostic immunoassays where endogenous molecules with a similar structure to the measured analyte exist or where metabolites of the analyte have common cross-reacting epitopes, or where there is administration of structurally similar medications . 12/4/2015

17. Cross Reactivity The most common examples can be seen during determinations of hormone concentration, Hormones TSH (thyroid-stimulating hormone), LH ( luteinising hormone) and hCG (human chorionic gonadotrophin ) carry an analogue alpha-chain, and the beta-chain determines the specificity of the respective hormone. drugs and allergen-specific IgE . 12/4/2015

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19. High-Dose Hook Effect Based on the saturation curve of antibody with antigen. Excessively high concentrations of analyte simultaneously saturating both capture and detector antibodies. Occure mostly (but not exclusively) in one-step immunometric (sandwich) assays, giving a decrease in signal at very high concentration of analyte . Immunoassays with very large analyte concentration ranges (ferritin, growth hormone, hCG , PRL, Tg , tumor markers PSA, CA19.9, CA125); antigen-antibody reactions can go into antigen excess and result in falsely decreased results and potential misdiagnosis. In one step two-site immunoassays, capture and detection antibody are added simultaneously, free analyte and analyte bound to the labeled antibody compete for the limited number of antibody-binding sites of the detector and in the presence of very high analyte concentration will decrease in stead of increase label bound to the solid phase. 12/4/2015

20. High-dose Hook Effect High-dose hook effect can be avoided by increasing the quantity of the reagent antibodies and by reducing the amount sample required for analysis or by sample dilution. Careful assay design is necessary to ensure that the concentrations of both capture and detector antibodies are sufficiently high to cope with levels of analytes over the entire pathological range. It is common practice to re-assay samples at several dilutions as a check on the validity of the result 12/4/2015

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22. Practical Encounters C ortisol assays can show significant cross-reactivity with fludrocortisone derivates and result in falsely elevated cortisol levels in patients using these drugs. The problem of cross-reactivity in active vitamin D (1,25(OH)2D) determination due to possible positive interference of 25(OH) D is well known. In competitive immunoassays for drugs of abuse screening, positive interference may result from medications or their metabolites that have similar chemical structures Immunoassays for cyclosporine A show cross-reactivity for cyclosporine metabolites with levels up to 174% higher In digoxin immunoassays, the presence of digoxin-like immunoreactive factors that are commonly found in renal failure, liver disease and hypertension, cause interference by cross reaction 12/4/2015

23. Antibody Interference Heterophile Antibodies Human Anti-animal Antibodies 12/4/2015

24. Heterophile Antibodies E ndogenous antibodies produced against poorly defined antigens. no history or medical treatment with animal immunoglobulins or other well-defined immunogens , multi-specific (reacts with immunoglobulin from two or more species) or exhibit rheumatoid activity. These antibodies react with many antigens and the variable region of other antibodies (anti- idiotypic antibodies) In two-site IMA’s, heterophilic antibodies can bridge two assay antibodies together and falsely elevates the patient value by producing an assay signal. 12/4/2015

25. Heterophilic Antibodies In case of rheumatoid factor (RF), false elevated results arise from the binding of RF to the Fc constant domain of antigen-antibody complexes. The presences of RF in serum can cause falsely elevated analyte levels in troponin assays, thyroid function tests, tumour marker assays and falsely detected HCV-specific IgM . IgM antibodies play a key role in interfering sera from rheumatic patients as they can bind Fc fragments of human antibodies . 12/4/2015

26. Heterophilic Antibodies Assays using either polyclonal or monoclonal antibodies can be affected. The same heterophilic may react differently for different antibody combinations hence causing rise one assay but a lower result in another assay. The presence of excess non-human immunoglobulin in the assay buffers reduces the possibility of the interfering substances binding to the capture and detection antibody by binding instead to the interfering immunoglobulin Both IgG and IgM heterophilic antibodies are reported to occur 12/4/2015

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28. 12/4/2015 False Negative From Heterophilic Antibody

29. Human Anti-animal Antibodies endogenous antibodies history of medical treatment with animal immunoglobulin and immunoglobulin from the same species used in the immunoassay 12/4/2015

30. Human Anti-animal Antibodies high-affinity, specific polyclonal antibodies generated after contact with animal immunoglobulin. They show strong binding and are produced in a high titer. Can be of the IgG , IgA, IgM , or rarely, the IgE class. They compete with the test antigen by cross-reacting with reagent antibody of the same species to produce a false signal. Most common are human antimouse antibodies (HAMA), but also antibodies to rat, rabbit, goat, sheep, cow, pig, horse may occur. Especially prevalent in serum of animal workers and in patients on mouse monoclonal antibody for therapy or imaging . 12/4/2015

31. Human Anti-animal Antibodies interference has been reported for cardiac markers assays, thyroid function tests, drugs and tumour markers. Two-site (sandwich) immunoassays more prone to interference From antibodies to animal IgG in serum and may cross-react with reagent antibodies especially from the same species. HAMAs is responsible for both false positive as well as false negative results . Methods that use only one mouse monoclonal in IMA assays are less prone to interference from HAMA. 12/4/2015

32. Autoanalyte Antibodies Autoantibodies have been described that can cause interference for a number of analytes including thyroid hormones in both free and total forms, thyroglobulin, insulin, prolactin and testosterone. Positive or negative influence 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. Autoantibodies against thyroid hormones, especially anti-T4 and anti-T3 antibodies, have been reported in patients with Hashimoto’s thyroiditis, Graves’ disease, hyperthyroidism after treatment, carcinoma, goitre and non-thyroid autoimmune conditions. 12/4/2015

33. Autoanalyte Antibodies These endogenous factors particularly interfere in total T4, free T4, total T3 and free T3 methods. Thyroid hormone antibody interferences are difficult to predict and can occur even with frequently used and well- characterised methods. Antibody prevalence depends on the detection method used: it is low in healthy subjects but may be as high as 10% in patients with autoimmune disease although only a minority of such samples demonstrate substantial thyroid assay interference. Their presence should be suspected when FT4 and TSH results appear to be discordant to the clinical findings. 12/4/2015

34. Interference is also a serious problem in Tg assays largely due to endogenous Tg antibodies (TgAb). Serum TgAbs are present in up to 25% of differentiated thyroid cancer (DTC) patients and in 10% of the general population. It is important to use a Tg method that provides measurements that are concordant with the tumour status in DTC patients. IMA methods are prone to underestimate serum Tg when TgAb is present, increasing the risk that persistent or metastatic DTC will be missed. Because falsely low Tg results can occur by IMA and falsely elevated results by RIA, anti-Tg antibodies should be measured in all samples analysed for Tg and a possible interference should be retained in all TgAb positive samples. 12/4/2015

35. A nti-prolactin autoantibodies can be present in serum in the form of macroprolactin (macroPRL). The presence of macro-PRL can cause macroprolactinemia with normal prolactin (PRL) concentrations and may lead to unnecessary medical or surgical procedures . Macro-PRL is a macro-molecular complex of prolactin (PRL) with an IgG antibody directed against specific epitope(s) on the PRL molecule. Macro-PRL is considered biologically inactive in vivo because of its decreased bioavailability. Macro-PRL is cleared more slowly than monomeric PRL and hence accumulates in the sera of affected subjects. The incidence of macro-PRL is up to 26% of all reported cases of hyperprolactinemia depending on the immunoassay system. Macro-PRL is detected in various degrees by different immunoassays. Laboratories should know the reactivity of the PRL assay with macroPRL and ideally test for the presence of macro-PRL in all patients with hyperprolactinemia by gel filtration chromatography or pre-treatment with polyethylene glycol. It is important to both recognize the presence of macro-PRL and provide an estimate of the monomeric PRL concentration because some patients with macroprolactinemia may have clinically significant, elevated monomeric PRL levels also. 12/4/2015

36. Other Proteins Interfering proteins of general relevance include albumin, complement, lysozyme, fibrinogen and paraprotein . They can affect antibody binding and can cause interference in immunoassays. Albumin may interfere as a result of its high concentration and its ability to bind or release large proportions of ligand. Complement binds to the Fc fragment of immunoglobulins and can block the analyte -specific binding sites of antibodies. Lysozyme can form a bridge between the solid-phase IgG and the detector antibody. IgG kappa paraprotein can bind to a TSH assay antibody and sterically block the binding of TSH and lead to falsely lowered TSH values. 12/4/2015

37. Endogenous Signal-Generating Substances The presence of endogenous signal-generating substances can interfere in the signal detection of an immunoassay. Diagnostic or therapeutic administration of radioisotopes can be carried over to the final counting tube, altering radioimmunoassay results. Endogenous europium can interfere in time-resolved fluorescence. With fluorescent immunoassays, interference can result from endogenous fluorescent substances, fluorescent drugs or fluorescein administration for the performance of retinal angiography 12/4/2015

38. Testing for Suspected Samples Use of an alternate immunoassay that preferably uses antibody raised to a different species Measurement before and after addition of a blocking reagent, especially bovine, or a series of concentrations of the blocker, or a combination of blockers from different species Measurement of dilutions of the sample using the manufacturer's diluent containing non-immune globulin Sample pre-treatment Radioimmunoprecipitation of labelled thyroid hormones to detect anti-T3 and anti-T4 autoantibodies 12/4/2015

39. Detecting Interference retroactive and proactive approaches exist for detecting interfering antibodies. Retroactive refers to instances when the laboratory result is questioned because it is inconsistent with the clinical findings or exceeds extreme limits for the analyte . A proactive approach would implement a mechanism or procedure that would detect the presence of interfering antibodies prior to obtaining or reporting the result. 12/4/2015

40. Retroactive Approach 12/4/2015

41. Proactive Approach Looking for discrepancies among alternative methods of measurement, serial dilutions to reveal nonlinearity, screening for antimouse antibodies with the Tandem ICON ImmunoConcentration human chorionic gonadotropin assay or pretreating specimens with blocking reagents. A study investigating the general usefulness of some of these approaches concluded that introducing a protocol to prescreen all samples for the presence of endogenous interfering antibodies is not warranted because the approaches were associated with too low an event rate to justify routine implementation or with too high a prevalence and were too nonspecific to be useful. Another proactive approach would be to request pertinent patient history, such as previous treatment with monoclonal antibody preparations or a history of erroneous laboratory results. This approach would be difficult to implement and is likely to be an ineffective strategy because it is unknown how many cases of interference could be predicted with this type of information. 12/4/2015

42. Clinical Laboratory Responsibility (CLSI) Ensure the personnel performing the assay have the required knowledge of endogenous antibody interference. Contact clinicians when there is suspicion of interference in a patient specimen. Follow up on complaints about clinically inconsistent results. Notify the manufacturer/vendor of assay interference problems. Investigate mismatches between assay results and clinical information in conjunction with the manufacturer/vendor. Inform clinicians regarding the nature of the interfering antibody, including how it affects the immunoassay in question and how it may affect other immunoassay tests ordered on the patient. 12/4/2015

43. Troubleshooting Obtaining patient history regarding therapy with a monoclonal antibody preparation, animal exposure, or transfusions may be helpful. The linearity of the in-house assay for the specimen in question can be measured, taking care to use appropriate diluents. However, linearity might still be observed even in the presence of interfering antibodies. The specimen may be sent to another laboratory for retesting using an alternate method, such as an immunoassay that uses a different technology (homogeneous vs. heterogeneous; competitive vs. non-competitive) and a different reagent antibody source than the original immunoassay suspected to display the interference, or a nonimmunometric method . 12/4/2015

44. Troubleshooting Certain analytes also appear in urine. Because endogenous antibodies are not usually present in urine, a discrepancy between urine and serum concentrations may suggest interference. A specimen may be preincubated with commercially available blocking reagents (some involve blocking antibodies immobilized on the inside surface of a test tube ). Compare values of related analytes if applicable: for example, creatine kinase–muscle and brain (CK-MB) and troponin. 12/4/2015

45. 12/4/2015 Interpretation in Immunoassay PSA I ncreased by prostatitis, Irritation , B enign prostatic hyperplasia (BPH), and Recent ejaculation, Digital rectal examination (DRE) Trans-rectal ultrasound Acute Renal Failure Bypass Surgery Prostate biopsy TURP Radiation treatment (transient) Antiandrogen drug therapy Prolonged exercise

46. 12/4/2015 Interpretation in Immunoassay HCG Certain drugs such as diuretics and promethazine (an antihistamine) may cause false-negative urine results. Other drugs such as anti- convulsants , anti- parkinson drugs, hypnotics, and tranquilizers may cause false-positive results. The presence of protein in the urine (proteinuria), blood in the urine (hematuria), or excess pituitary gonadotropin may also cause a false positive

47. 12/4/2015 Interpretation in Immunoassay Prolactine diurnal variations Prolactin levels peak during REM sleep, and in the early morning. Levels can rise after exercise, high-protein meals, sexual intercourse, breast examination, minor surgical procedures, following epileptic seizures due to physical or emotional stress

48. 12/4/2015 Interpretation in Immunoassay TSH Increases , decreases, and changes (inherited or acquired) in the proteins that bind T4 and T3 Pregnancy Estrogen and other drugs Liver disease Systemic illness Resistance to thyroid hormones Nonthyroidal illnesses Extreme stress and acute illness

49. 12/4/2015 Interpretation in Immunoassay Ferritin A cute-phase reactant O xidative damage - isoferritins - contribute to an overall increase in ferritin concentration

50. 12/4/2015 Interpretation in Immunoassay FSH results can be increased with use of certain drugs, including cimetidine, clomiphene, digitalis, and levodopa. FSH results can be decreased with oral contraceptives, phenothiazines , and hormone treatments

51. 12/4/2015 Interpretation in Immunoassay LH increases, anticonvulsants, clomiphene, and naloxone, while others cause LH to decrease, digoxin, oral contraceptives, and hormone treatments