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.

5. Immunoassay
Direct Indirect Sandwitch Competitive

6. Techniques
 CLIA
 CMIA
 ELISA
 ECLIA
 RIA
 Lateral Flow Test

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

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%.

11. Types of Interferences
analyte-dependent analyte–
independent
positive
interference negative interference

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

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

14. Interference in Immunoassays
Cross
reactivity
Hook
Effect
Antibody
Interference
Signal
Interference
Matrix Effect

15. Properties of Interfering Substances
Ø Unique to an individual
Ø Concentration can change over time within the same individual
Ø Low affinity polyspecific antibodies can be present in high
concentrations or high affinity in low concentrations
Ø 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
Ø May interfere in a number of immunoassays for different analytes
Ø The inclusion of one or more interference blocking agents in
manufacturers immunoassay reagents may be insufficient to overcome
the interference

16. Cross Reactivity
• Most 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.

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.

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.

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

22. Practical Encounters
• Cortisol 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

23. Antibody Interference
• Heterophile Antibodies
• Human Anti-animal Antibodies

24. Heterophile Antibodies
• Endogenous 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.

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.

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

28. 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

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.

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.

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.

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.

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.

35.  Anti-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.

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.

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

38. Testing for Suspected Samples
Ø Use of an alternate immunoassay that preferably uses antibody raised
to a different species
Ø 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
Ø Measurement of dilutions of the sample using the manufacturer's
diluent containing non-immune globulin
Ø Sample pre-treatment
Ø Radioimmunoprecipitation of labelled thyroid hormones to detect anti-
T3 and anti-T4 autoantibodies

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.

40. Retroactive Approach

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.

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.

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.

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.

45. Interpretation in Immunoassay
PSA
Increased by prostatitis, Irritation,
Benign 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. 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. 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. 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. Interpretation in Immunoassay
Ferritin
 Acute-phase reactant
 Oxidative damage - isoferritins - contribute to an overall increase in
ferritin concentration

50. 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. Interpretation in Immunoassay
LH
increases,
 anticonvulsants,
 clomiphene, and
 naloxone,
while others cause LH to decrease,
 digoxin,
 oral contraceptives, and
 hormone treatments