Endogenous antibody interference in immunoassays€¦ · Endogenous antibody interference in immunoassays Ellen Anckaert, ... electrochemiluminescence FT3 assay ... l Prevalence of

Endogenous antibody interference inimmunoassays

Ellen Anckaert, M.D., Ph.D.Dienst Klinische Chemie en Radio-immunologieUZ Brussel

To help protect your privacy, PowerPoint has blocked automatic download of this picture.

False positive serum hCG values have lead to cancermisdiagnosis

Jury awards $15.5 million to woman misdiagnosed with cancerUniversity of Washington and diagnostic company share blame

Photo: Grant M. Haller/Seattle Post-IntelligencerPublished June 29, 2001

Interference by endogenous antibodies inimmunoassays: an ongoing story

J Clin Endocrinol Metab 2016


RARE CAUSES


FREQUENT CAUSES

Immunoassay interference by endogenousantibodies

Antibodies againstassay antibodies

Antibodiesagainstanalyte

Antibodies againstsignal molecules

Endogenous antibodies against assay antibodies

Possible clinical consequences:• Misclassification of monitoring results• Unnecessary follow-up examinations• False therapy decisions• Unfavorable patient prognosis

Mechanisms of interference by heterophilicantibodies

Bridging of capture anddetector antibodies=> Falsely elevated result

Exclusive binding of capture or detector antibodyonly=> Falsely lowered result

Endogenous antibodies against assay antibodies

Heterophilic

antibodies

Human anti-mouseantibodies (HAMA)

Rheumatoid factor

Etiology Poorly defined,

no clear immunogen

Known antigenicstimulus

Auto-antibody

Specificity Low:

bind different species Ig

High Low: bind Fcregion of differentspecies Ig

Affinity Low High Low

Titer Low High High in activerheumatic disease

Ig class IgG, IgM IgG, IgA, IgM Usually IgM

Prevalence 40% In 40-70% ofpatients treatedwith mouse Mabs

Up to 10% gen. pop

5-10% gen. pop

70% autoimmunerheumatic disease

1. Addition of a combination of blocking agents

Addition of a “blocking agent” of the same species as the assayantibodies:- animal normal serum- animal nonimmune immunoglobulin- aggregated mouse monoclonal IgG1 (MAK33) to eliminate strong HAMAinterferences, usually therapy induced

2. Fragmentation of antibodies

Use of Fab orF(ab’)2 fragments

Single chainfragments scFv

Variable region from mouse

IgG

C1 constant region from

human IgG

Fc-fragment cleaved off

3. Chimeric antibody fragments

Constructed from 2 different species (mouse / human )

Prevalence of interference

l Prevalence of interference depends on the immunoassay (IA) method

l Bjerner, Clin Chem 2002 (CEA, 11.261 patient samples)g unblocked IA 4%g Fc removal 0.1%g Heat-treated MAK33 0.06%

Assay design: measures against heterophilicantibody interference

No protection

Use of blocking proteins

Fragmentation of Antibodies

Use of chimeric MABs

Interference level:High: <5-15% Low: <= 0,05%

Interference is not completely eleminated

What can the lab do to detect immunoassayinterference?

1. Repeat the analysis with an alternative immunoassay,preferably using assay antibodies from a different species

2. Treat the sample with an additional blocking agent(Heterophilic Blocking Tubes, Scantibodies)

3. Serial dilution: non linearity indicates assay interference

Use several measures: a single negative interference test doesnot exclude interferenceMarks (Clin Chem 2002): blood from 10 donors with interference in one immunoassay

• 8.7% of 3445 immunoassay results were ‘falsely increased’

• half of these were not corrected by HBT





Interference in immunoassays by autoantibodiesagainst analytes

l Antibodies leading to macrohormones:

l Anti-prolactin (macro-prolactin)

l Anti-TSH (macro-TSH)

l Anti-calcitonin (macro-calcitonine)

l Anti-thyroglobulin antibodies

l Anti-insulin antibodies

l ...

Circulating forms of prolactin

MACROPROLACTINEMIA: hyperprolactinemia with an elevated % ofcirculating PRL consisting of BIOLOGICALLY INACTIVE macroprolactin

• 1-4% of general population• 4-40% in patients with hyperPRL• > 90% of cases: macroPRL = PRL-IgG complex• macroPRL accumulates in circulation due to decreased renal clearance

titel17 27-10-2016

Causes of hyperprolactinaemia

Immunoreactivity for macroPRL is assay-dependent

Fahie-Wilson M, Best Practice & Research Clinical Endocrinology & Metabolism 2013

Reactivity is depending on:- Epitopes targeted by the assay antibodies- Affinity of capture antibodies- Incubation time- Sample dilution

PEG-precipitation for detection ofmacroprolactin

• 200 µL serum + 200 µL PEG 6000 25% (g/v) in PBS buffer in conical tube(room temperature)

• vortex , centrifuge and measure PRL in supernatant• % recovery = (PRL supernatant * 2)/PRL serum *100

³ 60% = absence of macroPRL (method-dependent!)• Monomeric PRL after PEG

Macro-TSH

l Macro-molecule composed of TSH and anti-TSHimmunoglobulin

l Reduced renal clearance leads to accumulation ofmacro-TSH

l Macro-TSH is biologically INACTIVEg Patients are typically clinically EUTHYROID

g Typically: (grossly) elevated TSH with normal FT4

Variable detection of macro TSH bycommercial immunoassays

Hattori, 2016

What can the lab do to detect macro-TSH?

1. REPEAT ANALYSIS WITH ALTERNATIVE IMMUNOASSAY:results VARY depending on immunoreactivity with macro-TSH

2. TREAT SAMPLE WITH BLOCKING AGENT: NO EFFECT

3. SERIAL DILUTION:

gMacro-TSH: dissociation ↑ or nl TSH recovery

g Heterophilic antibody interference: ↓ TSH recovery

4. PEG-PRECIPITATION of high molecular weight proteins:

gMacro-TSH: ↓ TSH recovery

g Heterophilic antibody interference ↓ TSH recovery

5. Mix with hypothyroid serum (= high TSH) sample:

gMacro-TSH: free anti-TSH binds TSH ↓ TSH recovery

Confirmation of macro-TSH by gel filtrationchromatography

Patient serum: TSH peak fraction thatapproximates the molecular size of IgG (dots).

Patient serum incubated with hypothyroidserum: HMW fraction, confirming excessTSH binding capacity and macro-TSH(trangles).

Loh T P, JCEM 2012

Clinical characteristics of macro-TSH patients

Case Sex Age Thyroidantibodypositive

Clinicalsigns/symptoms

TSH (mIU/l) Immunoassay

1 F 56 Anti-Tg No 274 Elecsys

2

3

F

-

mother

newborn

-

-

No

No

308

828

Elecsys

Elecsys

4

5

6

F

-

F

28

45

23

Neg

Neg

TRAb

No

No

Graves HT

5.1

22

9.7

Elecsys

Elecsys

Elecsys

7

8

F

F

mother

newborn

-

Neg

No

No

55

103

Elecsys

Delfia

9 F 46 Neg No 24.5 Elecsys

10 M 60 Anti-TPO No 232 Vitros

11 M 29 -

-

No 40-115 RIA

12

13

F

F

53

6

Neg

Neg

No

No

1.4 ->100

2.7 ->100

Immunoassay

Immunoassay

Reviewed by Loh, JCEM 2013

Vertical transmission of anti-TSH antibody

Rix, Acta Paediatr 2011

Prevalence of macro-TSH

Method: systematic screening of samples with elevated TSH by PEGprecipitation and confirmation by GFC

• Mills 2013: TSH > 10 mIU/l (Roche Elecsys)• 3/495 (0,6%)

• Hattori 2015: elevated TSH, normal FT4 (Vitros)• 10/681 (1,5%)

• Hattori 2016: TSH > 4 mIU/L, normal FT4 (EIA)• 15/1901 (0,8%)

Other macro-hormones


Other case reports: macro-LH, macro-FSH, ...

Tg antibody interference in Tg immunoassays

l Sensitive Thyroglobulin (Tg) measurement is important forfollow-up of differentiated thyroid carcinoma (DTC)

l No Tg method is completely free from anti-Tg interference

g Frequent underestimation in non-competitive assay

g Rare false elevation is possible in competitive assay

l Anti-Tg antibody prevalence

g 10% general population

g 25% in DTC

g 60% in autoimmune thyroid diseaseDifferent epitope recognition patterns


Anti-Tg interference in Tg IMA is a common problem


l What can the lab do:g Comment on Tg value ‘not reliable in case of anti-Tg’g Confirm by an alternative method (RIA, LC-MSMS)g Exogenous Tg recovery test

l low recovery indicates interferencel normal recovery does not exclude interference

l TgAbs can be used as a surrogate tumour marker

l Guidelines: measurement of Tg in follow-up of DTC shouldalways be accompanied by anti-Tg measurement using asensitive anti-Tg immunoassay

TgAb immunoassay


Even when cut-off is based on analyticalsensitivity: failure to detect interferencein 20-30% of cases

Analytical sensitivityCut-off provided by manufacturer





Interference by anti-ruthenium antibodies inFT4 – FT3 immunoassays

Anti-Ru antibodies

l Mainly in areas with textile industryg Use of Ru in dying process of clothing

g Ru in environment, clothing or food chain

l Estimated frequency of interference in first generationelectrochemiluminescence FT3 assay: 0.2% (Sapin, Clin ChemLab Med 2007)

Anti-RU interference in FT4 – FT3immunoassay

Protection against anti-Ru antibodies in nextgeneration immunoassay

The sulfo-RU label is less recognized by anti-RU

Current generationOlder generation

Case report

Visit Normal values4 3 2 1

TSH (mIU/l) 0.552 0.344 0.569 0.515 0.27-4.2

FT3 (ng/l) 3.2 5.9 7.0 6.2 2.6-4.4

FT4 (ng/l) 12.6 20.8 21.2 19.5 9.3-17.0

Inappropriate TSH secretion:- Thyrotropinoma (1/1.000.000)- Thyroid hormone resistance (1/50.000)

Switch to current generation FT3 and FT4assays

Biotin

Streptavidin

Antibodies against other assay components

Biotin IgM antibodies present in 3%of Finish population interfere in EIA(Chen, PlosOne 2012)

Case report: interference by anti-streptavidin in ECLIA (Johnson Rulander, ArchPatol Lab Med 2013)

Immunoassay interference

l Frequent causes of interference (macro-PRL, anti-Tg)require a systematic approach

l No method is completely free from interference

g be aware of the susceptibility of a particular commercialimmunoassay to interference

g clinician should be actively encouraged to contact the laboratoryin case of any doubt

l In case of confirmed interference:

g Patient medical record should contain information about thepresence of interfering substances in serum

g Manufacturer should be noticed

Interference by endogenous antibodies inFT4 – FT3 assays

Anti-T4 and anti-T3 antibodies

l Prevalence depends on the selected population and themethod of detection

l £20% in autoimmune thyroid disease

l 6% in non-thyroidal autoimmune disease

l 0-2% in healthy individuals

l women > men

l Mostly IgG subclass, mostly polyclonal

l Most patients also have anti-Tg and/or anti-microsomalantibodies

l Impact on immunoassay (interference) depends onl the assay format

l titer, affinity and specificity of the antibody

One step method - Labeled Analog

SerumBindingProtein

T4

FT4+ + *

Anti - T4 AntibodyBound to Particle

+

*SeparateandCount

X*

ConjugatedAnalog

Clinically inconsistent TSH result

Perform serial dilution of the sample and measure TSH

Repeat the measurement of TSH on an alternative platform

Screen for macro-TSH if unexplainedassay interference

Likely heterophile antibodies interference

Heterophile blocking tube studies

Likely interference by rheumatoid factors

Measure rheumatoid factors

Linear recovery

Non-discrepantresult

Normal recoveryNegative

Assay interference unlikely

Low recovery

Positive

Non-linearrecovery

Discrepantresult

Mix with hypothyroid patient serum

GFC

Heterophilic antibody / HAMA interference

l Prevalence of interference depends on the immunoassay (IA)method

l Bjerner 2002 (CEA, 11.261 patient samples)g unblocked IA 4%g blocked IA (Fc removal) 0.1%g blocked IA (Fc removal – MAK33) 0.06%

l Boscato 1986 (hCG IRMA, 668 healthy subject samples)g unblocked IA 15%g blocked IA 0.6%

l Ward 1997 (TSH, 21.000 patient samples)g blocked IA 0.03%

Þ addition of “blocking reagent” reduces interference, but isno garantee for complete elimination of interference

Þ estimated prevalence: 0.03 – 3%

titel45 27-10-2016

Major forms of PRL in serum

Variant MW %

Monomeric hPRL 23 kDa 85

BigPRL 60 kDa 10

BigBig PRL= MacroPRL

>150 kDa 1 - 5

Macroprolactinemia:• Hyperprolactinemia where an elevated fraction of circulating PRL

consists of biologically inactive macroprolactin• Prevalence: 1-4% of general population, 4-40% in patients with

hyperPRL• > 90% of cases: macroPRL = PRL-IgG complex

Case report macro-TSH (1)

l 60 year old man, clinically euthyroidg TSH1 232 mIU/l (0.45-5 mIU/l)

g FT4 10 pmol/l (10-23 pmol/l)

g TPO Ab 496 IU/ml (0-50 IU/ml)

g Tg Ab Neg

g anti-TSH receptor Abs Neg

l Test with an alternative immunoassay methodg TSH2 122mIU/l

1 Vitros 5600, Ortho Clinical Diagnostics; 2 Advia Centaur, Siemens Healthcare Diagnostics

l Test dilution linearity3:l TSH 1:1 122mIU/l

l TSH 1:10 165 mIU/l (135% recovery)

3 TSH assay diluent and immunoassay: Advia Centaur

l Test for antibodies against assay antibodiesl RF Negative

l Heterophilic blocking tubes No interference detected

Loh T P, JCEM 2012

Endogenous antibody interference in immunoassays€¦ · Endogenous antibody interference in immunoassays Ellen Anckaert, ... electrochemiluminescence FT3 assay ... l Prevalence of

Documents