Immunoassay Interference Flashcards
What is an Immunoassay?
- Antibody : antigen reaction
- Measure complex heterogeneous molecules
- Inherently vulnerable to interference
- Frequency of interference is difficult to assess
What are ways to increase detection of interference?
- Awareness of scientist types of interference
- Excellent proactive communication with clinical staff
- Together will increase the detection of interference
Which drugs can affect analytes in the body?
- Effect of heparin, phenytoin, salicylate on free hormones
- Finasteride and other 5-alpha reductase inhibitors on PSA
- Oral contraceptives, HRT or pregnacy on binding proteins
Where are categories of errors in analysis?
- Pre-analytical
- Exogenous: Random, Systematic
- Endogenous
- Post-analytical
What are characterisitics of endogenous interferences?
- Sporadic
- Specimen dependent
- Difficult to identify
What are examples of endogenous interferences?
- Normal serum components in excess: Haemolysis eg Troponin T, insulin, PTH and Lipaemia
- Cross-reacting substances
- Anti-analytes and anti-reagent antibodies
- High-dose hooking
- Biotin
What are some features to address with cross-reacting substances?
- Important to understand the specificity of your assay
- Positive or negative interference
- Specificity of the antibody
What are things to consider with cross-reacting substances?
- Important to understand the specificity of your assay
- Positive or negative interference
- Specificity of the antibody
What is the effect of cross reactivity in HCG?
- Potential Cross Reactants: hCG beta subunit
- Effect of cross reactivity: Higher hCG results in assays that recognise beta subunit
- Clinical Implications: Cross reactivity essential for assays for oncology purposes
What is the effects of cross reactivity in GH assays?
- Potential Cross Reactants: GH receptor antagonists eg pegvisomant
- Effect of cross reactivity: Falsely elevated or lowered GH results
- Clinical Implications: Only assays that show no cross-reactivity can be used in patients on pegvisomant
What is the effects of cross reactivity in Insulin assays?
- Potential Cross Reactants: Insulin analogues eg lispro
- Effect of cross reactivity: Differences in cross-reactivity depending on specificity of assay
- Clinical Implications: Knowledge of cross-reactivity when investigating exogenous induced hypoglycaemia
What is the effects of cross reactivity in LH assays?
- Potential Cross Reactants: hCG
- Effect of cross reactivity: Apparent measurable LH in pregnancy
- Clinical Implications: Delay confirmation of pregnancy
What is the effects of cross reactivity in PTH assays?
- Potential Cross Reactants: N truncated fragments of PTH
- Effect of cross reactivity: Differences in results in patients in CRF depending on assay used
- Clinical Implications: Establishment of analyte specific reference ranges
What is the effects of cross reactivity in Testosterone assays?
- Potential Cross Reactants: DHEAS, synthetic OCP
- Effect of cross reactivity: Potentially spuriously raised testosterone
- Clinical Implications: Establishment of analyte specific reference ranges
How can Antireagent antibodies cause interference?
- Antireagent Ab binding ab binds to capture Ab and Labelled Ab lead to spurious high result
- Antireagent Ab present blocking both capture and label Ab leading to falsely low result
Addition of blocking antibody ca fix the issue
What is the aetiology, characterisitics and frequency of HAMA?
- Aetiology: Produced in response to a direct antigenic stimulus, Rx with mouse Mabs,
- Characteristics: Maybe IgG, IgA, IgM or IgE which can Persist long-term
- Frequency: Most often found in patients Rx with Mabs or work with animals
What is the aetiology, characterisitics and frequency of Heterophilic Antbodies?
Aetiology
- Poorly defined abs developed in response to no clear immunogen
Characteristics
- One detecting epitope present on rabbit Ig only
- One detecting epitope on goat, cattle, horse and mouse but not rabbit
Frequency
- Variable prevelance with variable analytical relevance
What is the aetiology, characterisitics and frequency of Rheumatoid factor?
Aetiology
- Autoantibodies present in serum from patients with rheumatoid disease
Characteristics
- Bind to multiple antigenic determinants on the Fc region of IgG
Frequency
- Most often in patients with rheumatoid disease but also in patients with infections
What are examples of interference due to Anti-Analyte Abs?
- Antibodies to insulin
- Antibodies to thyroglobulin
- Antibodies to thyroxine
What are interferences from macrocomplexes?
- Usually IgG complexes: Prolactin, TSH, ACTH, B12, Troponin
- Falsely raised results
- Screening recommended
How can biotin cause interference?
Biotin supplementation, usage and impact on testing
- Is the patient taking Biotin
- Vitamins for hair or nails
- How often ? What dose ? When did you last take a dose ?
Despite lack of clinical evidence some patients are takin very high levels (2 mg – 10 mg per day)
Doses of 5 mg per day may cause interference in some assays (biotin-streptavidin as detection)
- Troponin T, TSH, FT4, TPO abs
How can interferences be identified?
- Clinical suspicion
- Discordant results
- Understanding limitations of assays
- Good clinical colloboration/dialogue
- MDTs
What are investigations undertaken for interference?
Initial Investigations
- Check ID on sample
- Repeat from primary sample
- Check previous results
Confirm results in other immunoassay method:
- Send sample to alternative lab
- Look for discrepancy between methods
- Confirm by third method
- Take into account differences in reference ranges/bias of assay
What are dilution and recovery studies for interferences?
- Non-linearity on dilution using assay diluent
- Low recovery of added standard
- Compare to normal sample
How is interference with heterophillic antibodies assessed?
Heterophilic blocking tubes
- Blocking tubes or heterophilic blocking reagent
- Add to blocking tube and reassess
- Differences between both results suggest interference
Using Non Immune Animal Serum
- Addition of non-immune mouse or rabbit serum at suitable dilution
- Reassess
- Difference suggests interference
How are macro-complexes assessed?
Polyethylene Glycol Precipitation:
- Used to detect macro-complexes
- Non-specific and proportion of monomeric analye may also be precipitated
- Monomeric prolactin reference ranges
- Confirmation by gel filtration
What is the purpose of sample extraction?
- Extraction with diethyl ether
- Removes water soluble steroid conjugates
- Steroids: UFC, Testosterone, 17-OHP
How is LC-MS used to assess interference?
- Method specific reference ranges
- Steroids
- Most common approach for steroids
How is High dose effect noticed clinically?
- Good clinical details
- Good communication with clinicians
- Dilution of samples
How is risk of interferences minimised?
- Communication with Clinicians
- Communication with diagnostic companies
- Communication with EQA providers
