Chemistry, toxicology & urinalysis


Assay interferences

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Felix Leung, Ph.D.

Last author update: 11 May 2021
Last staff update: 11 May 2021

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PubMed Search: Assay interferences[TIAB]

Felix Leung, Ph.D.
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Cite this page: Leung F. Assay interferences. website. Accessed June 1st, 2023.
Definition / general
  • Interference is defined as a cause of clinically significant difference in the assay result, due to another component or property of the sample
  • Most interferences are missed by quality control processes and can lead to undetected discrepant test results, which can lead to patient harm
Essential features
  • Analyte: component represented in the name of a measurable quantity
  • Matrix: all components of a material system, except the analyte
  • Measurand: particular quantity subject to measurement
Types of interferences
  • Interferents can originate from both exogenous and endogenous sources (Clin Chem Lab Med 2020;58:350)
  • Endogenous:
    • Metabolites that arise from pathological conditions (free hemoglobin, bilirubin, lipidemia)
    • Macrocomplexes (macroprolactin, macroenzymes)
    • Antianalyte antibodies (antithyroglobulin)
    • Paraproteins
    • Specimen matrix itself
  • Exogenous:
    • Compounds given to patient for treatment (e.g. drugs, anticoagulants, intravenous saline or dextrose solutions)
    • Ingested substances (biotin, a type of vitamin B)
    • Environmental contaminants (powder from gloves, atmospheric air)
    • Sample additives from phlebotomy processes (anticoagulants, preservatives)
  • Identifying the presence of most interferents depends on clinicians recognizing discordant test results and communicating with laboratorians
Mechanisms of interference
  • Broad categories of interference include:
    • Chemical interference: interferent disrupts assay reaction
    • Spectral interference: interferent has similar spectral properties to the measurand
    • Physical interference: interferent alters physical properties of the sample or measurand
    • Enzymatic interference: interferent alters activity of enzyme(s) used in the assay reaction
    • Nonselectivity: interferent mimics measurand in the assay reaction
    • Additive interference: interferent or additional measurand introduced into sample
  • The same interferent can interfere differently, depending on the assay
    • In vitro hemolysis can cause chemical interference (hemoglobin inhibits certain reactions), spectral interference (hemoglobin has characteristic red color) and additive interference (red blood cells have high intracellular concentrations of certain analytes, e.g. potassium, lactate dehydrogenase, magnesium, phosphorus and AST)
  • Critical to assess interferent in context of assay in question
  • Reference: Rifai: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 6th Edition, 2017
Serum indices

Common tests affected by HIL interference:
Falsely increased values
Falsely decreased values

  • Potassium
  • Aspartate aminotransferase
  • Lactate dehydrogenase
  • Phosphate
  • Magnesium

  • Insulin

  • Creatinine (JaffĂ©)
  • Hydrogen peroxide based assays
    (cholesterol, triglycerides, glucose)

  • Plasma / serum electrolytes
    (sodium, chloride, potassium)
  • Turbidimetric / nephelometric assays (immunoglobulins,
    C reactive protein, serum free light chains, immunosuppressants)
Other common interferents
  • Biotin (vitamin B7) will interfere with immunoassays that use the streptavidin - biotin system
    • Interference can be negative or positive, depending on assay format (i.e. competitive or noncompetitive)
  • Heterophilic antibodies can interfere with any type of immunoassay
    • Defined as antibodies that can react nonspecifically with different molecules
    • Typically causes false positive sandwich immunoassay result but may also cause false negative results
    • Often increased in patients with autoimmune or inflammatory conditions
  • Human antianimal antibodies can interfere with immunoassay, if it uses antibodies produced by the animal in question
    • Most frequent cause is human antimouse antibodies
  • Macrocomplexes (endogenous analytes that self polymerize or complex with immunoglobulins) will cause discrepantly high results that are not indicative of patient status
    • Most commonly affected tests are aspartate aminotransferase, creatine kinase, amylase and prolactin
  • Reference: Rifai: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 6th Edition, 2017
Strategies to handle interferents
Board review style question #1
Which of the following statements about assay interferences is true?

  1. An interferent typically exhibits the same interference properties across different assays
  2. Interferents exhibit similar interference properties across different assays
  3. The recognition of assay interference by biotin and heterophilic antibodies often depends on awareness by clinicians
  4. Most automated analyzers are unable to detect HIL (hemolysis, icterus, lipemia) interferences
Board review style answer #1
C. The recognition of assay interference by biotin and heterophilic antibodies often depends on awareness by clinicians

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