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MSc Endocrinology > Immunoassay Interference > Flashcards

Immunoassay Interference Flashcards

1
Q

What is an Immunoassay?

A
  • Antibody : antigen reaction
  • Measure complex heterogeneous molecules
  • Inherently vulnerable to interference
  • Frequency of interference is difficult to assess
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2
Q

What are ways to increase detection of interference?

A
  • Awareness of scientist types of interference
  • Excellent proactive communication with clinical staff
  • Together will increase the detection of interference
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3
Q

Which drugs can affect analytes in the body?

A
  • 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
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4
Q

Where are categories of errors in analysis?

A
  • Pre-analytical
  • Exogenous: Random, Systematic
  • Endogenous
  • Post-analytical
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5
Q

What are characterisitics of endogenous interferences?

A
  • Sporadic
  • Specimen dependent
  • Difficult to identify
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6
Q

What are examples of endogenous interferences?

A
  • 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
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7
Q

What are some features to address with cross-reacting substances?

A
  • Important to understand the specificity of your assay
  • Positive or negative interference
  • Specificity of the antibody
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8
Q

What are things to consider with cross-reacting substances?

A
  • Important to understand the specificity of your assay
  • Positive or negative interference
  • Specificity of the antibody
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9
Q

What is the effect of cross reactivity in HCG?

A
  • 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
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10
Q

What is the effects of cross reactivity in GH assays?

A
  • 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
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11
Q

What is the effects of cross reactivity in Insulin assays?

A
  • 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
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12
Q

What is the effects of cross reactivity in LH assays?

A
  • Potential Cross Reactants: hCG
  • Effect of cross reactivity: Apparent measurable LH in pregnancy
  • Clinical Implications: Delay confirmation of pregnancy
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13
Q

What is the effects of cross reactivity in PTH assays?

A
  • 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
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14
Q

What is the effects of cross reactivity in Testosterone assays?

A
  • Potential Cross Reactants: DHEAS, synthetic OCP
  • Effect of cross reactivity: Potentially spuriously raised testosterone
  • Clinical Implications: Establishment of analyte specific reference ranges
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15
Q

How can Antireagent antibodies cause interference?

A
  • 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

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16
Q

What is the aetiology, characterisitics and frequency of HAMA?

A
  • 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
17
Q

What is the aetiology, characterisitics and frequency of Heterophilic Antbodies?

A

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
18
Q

What is the aetiology, characterisitics and frequency of Rheumatoid factor?

A

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
19
Q

What are examples of interference due to Anti-Analyte Abs?

A
  • Antibodies to insulin
  • Antibodies to thyroglobulin
  • Antibodies to thyroxine
20
Q

What are interferences from macrocomplexes?

A
  • Usually IgG complexes: Prolactin, TSH, ACTH, B12, Troponin
  • Falsely raised results
  • Screening recommended
21
Q

How can biotin cause interference?

A

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
22
Q

How can interferences be identified?

A
  • Clinical suspicion
  • Discordant results
  • Understanding limitations of assays
  • Good clinical colloboration/dialogue
  • MDTs
23
Q

What are investigations undertaken for interference?

A

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
24
Q

What are dilution and recovery studies for interferences?

A
  • Non-linearity on dilution using assay diluent
  • Low recovery of added standard
  • Compare to normal sample
25
Q

How is interference with heterophillic antibodies assessed?

A

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
26
Q

How are macro-complexes assessed?

A

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
27
Q

What is the purpose of sample extraction?

A
  • Extraction with diethyl ether
  • Removes water soluble steroid conjugates
  • Steroids: UFC, Testosterone, 17-OHP
28
Q

How is LC-MS used to assess interference?

A
  • Method specific reference ranges
  • Steroids
  • Most common approach for steroids
29
Q

How is High dose effect noticed clinically?

A
  • Good clinical details
  • Good communication with clinicians
  • Dilution of samples
30
Q

How is risk of interferences minimised?

A
  • Communication with Clinicians
  • Communication with diagnostic companies
  • Communication with EQA providers

MSc Endocrinology (16 decks)

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