Analytical platforms and immunoassay interference

Dr Les PerryConsultant Clinical ScientistCroydon University Hospital

ACB course: Warwick 2013

Introduction

Immunoassay & Historical introduction

Consequences of interference

Pre-analytical factors

Antibody interference

Idenitification of analytical interference and corrective actions

Introduction

Introduction immunoassays revolutionised endocrine hormone analysisBased on Ab recognition of moleculeManual IA use polyclonal (many animal species) Ab v automated platforms use mAb (murine)IA label: radioactive (3H/125I); later fluorescent, chemiluminescent or enzyme reactionIA methods gave sense of security in value of lab result Sometimes the result not fit the clinical / endocrine picture. Clinicians need to liaise with lab !!

Basic principle of immunoassay

Ab + Ag* Ab –Ag *Fixed 10,000 cpm 10,000 100% B

Ab + Ag* Ab -Ag* + Ag*

+ Ag Ag 9000 90%B

Ab + Ag* Ab -Ag* + Ag*+ Ag Ag 5,000

50%B

Ab + Ag* Ab -Ag* + Ag*+ Ag Ag 2,000

20%B

Calibration curve

0

100

10 100 1000 10000Concentration of Ag

% A

b bi

ndin

g

Immunoassays characteristicsSensitivity ◦ measure minute concentrations of an analyteSpecificity◦ Discriminate between closely-related cpdsAccuracy ◦ Using reference stds can relate results between

different labs to derive reference rangesPrecision◦ minimal variation between measurements; singlet

or duplicate measurement trusted & reproducable

Characteristics inter-related eg sensitivity+precision◦ Sensitivity = unacceptable imprecision

Sensitivity

0.1 0.2 0.3 0.5 1.0

Classically do paired t-test with zero calibrator and low concn analyte to find conc at which analyte statistically diff from zero

x

Problem: Sensitivity will decrease with increase in the number of replicates (standard error is inversely proportional to the sq root of n)

Sensitivity: Analytical v Functional

Analytical: measure zero std n=20 and take >3 SD above the meanHence probability that result not part of distribution of zero std = Analytical sensitivity

Prob: precision maybe v poor/ unacceptable

Functional: lowest concentration in the assay which has a <20%CV◦Arbitrary value

Sensitivity example

Bayer Immuno-1 analyser◦ ?use 25nmol/L as cut off for diagnosis of Cushing’s

syndrome following LDDST◦ Cortisol assay kit insert quoted sensitivity as 10nmol/L◦ Took patient sample with F=53 nmol/L◦ made approx 15 aliquots and froze then analysed daily◦ Mean = 52.6 nmol/L CV=3.8%◦ Therefore happy to use <50 as limit of reporting

◦ Took a patient sample measured as ~25nmol/L◦ Did same as above◦ Mean was ~25 nmol/L but CV = 28% !◦ Conclusion – NOT use 25 nmol/L

Leads to concept of precision profile

Precision

Derivation of functional sensitivity more complex than analytical sensitivityManufacturer / kit leaflets not stated more likely that analytical sensitivity quoted

Precision describes reproducability. Imprecision is opposite and its use is widely recommended.

Imprecision = estimate of error expressed as %CV (SD)

Assay imprecision (1)

Caused by the combined effects of several sources of variationAb◦ reach equilibrium (most platforms not)◦ High affinity reach eq’brium quicker◦ Factors that affect Ab-Ag reaction which if

incorrectly optimized have impact on rate of reaction and therefore precision

Ab concAssay temppH of the final reaction mixturevariation in density of Ab coating on solid-phase

Assay imprecision (2)

Incubation / instrumentation variations◦ Temp variation across analyser incubator◦ Sample / reagent temp variation prior to assay◦ Edge effects of µ-well assys (heat unevenly)◦ Incomplete suspension of solid-phase particles prior

to or during assay incubationSeparation◦ Requires effective separation of unbound material

from particles or wells; usually washing. Physical separation. Suffers from problem of mechanical, fluidic or pressure variations or blockages

Assay imprecision (3)

Detection errors◦ Radioactive tracer counting errors. ◦ Error =√ of total counts. ◦ eg 1000c=10%; 10000c=1%; 40000c= 0.1%

3H / 14C require scintillation countingGlass tubes with 125I add 1-2%imprecionMultiwell gamma counters: calibration & contamination

Opitical readers : Turbidity, fluorophoretic and reagents involved in signal generation may lose activity over time so S/N ratio

Interferences in Immunoassay

Anti-analyte AbAutoantibodiesBlood substitutesCarryoverCollection tubeContaminationContrast agentsComplementDrugsDrug metabolitesFibrinHaemolysisHerbal remedies

Heterophilic AbHigh-dose hook effectHuman anti-animal AbImaging agentsImmune complexesLipaemiaMicroclotsParaproteinsPartially filled collection tubeRFSample storageSample matrix

Testosterone assays

RIA was introduced 1970Large volume serum3H radioligandOrganic solvent extractionChromatographic separation stepDextran-coated charcoal separation stepCalculation using graph paper, sharp pencil & flexicurve Result in 2-3 daysRun in small batch (n=20-30)Tended to be analysed in specialist labs where HoD had an interest /expertise

Hexane

Hexane:MeOH

Diethyl ether

Why chromatographic separation?

Testosterone

Many steroids circulate in blood with the C=C

and the C=O

5α-dihydrotestosterone

Testosterone

Testosterone assays progression1980’sImprovements in antibody specificity Introduction of 125I-tracersPreparation of in-house standards in serumObviate chromatographic step (direct assays) ◦ eg ANS in TFT / Danazol

in ‘F’ assays◦ Use of buffer pHImprovements in separation step (sac-cel; magnetised charcoal)Data reduction packagesAssay in large batch modeResults same day

1990’s – to dateIntroduction of non-isotopic assaysIntroduction of solid-phase Ab assisting in separation stepAutomation of analytical processReagent packs for platformContinuous workflowResults in hoursSmall sample volumeNow performed in almost every lab whether it be DGH or Teaching Hospital

Automation of endocrine assays

AdvantagesImproved precision Faster turn-around timeMultiple analytesAutodilutionObviate isotopes & extractionFewer lab staff

DisadvantagesHook & heterophilic Ab interferencePoor specificitySome assays not very good (poor bias/var)Prone to lot–to-lot variationWhen they go wrong serious impact on service

Calibration

• Standards in ethanol, buffer, serum

• ‘Steroid free’ serum?

• For steroids weigh out powder and make up in steroid free serum

• Problem of no IRP or now very old

• Proteins may have different molecular forms eg HCG

• Tumours can produce different molecular forms

CalibrationWhat is ‘zero’ standard for endogenous proteins?

◦ TSH assay• 1st generation measured down to 0.1 mU/L• 2nd generation measured down to 0.02 mU/L• 3rd generation measure down to 0.002 mU/L

Traceability

Lab results will be influenced by systematic and random errors

Agreement of measurements between labs or agreement over time in one lab can be a problem

To ensure agreement of results between methods traceability becomes a key issue

Traceability is a link of measurement comparisons back to a known reference value

Calibration: from primary reference method to routine method

eg Calcium

‘Gold standard’ or Primary reference procedure –isotope dilution mass spectrometry (ID-MS)

Atomic absorption spectrophotometry

Manufacturer ‘reference sample’

Lot 1 Lot 2 Lot 3

Labs 1 - 50

Lab 1 IQC Lab 2 IQC etc

EQA scheme organiser

Calibration (PTH calibration from UKNEQAS)

Calibration (PTH calibration from UKNEQAS)

Recommendations from the Working Group of Senior Scottish Clinical Biochemists on Parathyroid Hormone

(PTH) Targets in the Management of Renal Failure (2009)

5 different PTH assays which serve the renal units in Scotland. In the absence of an agreed international PTH standard assay comparability is a problem. In particular, UK NEQAS data shows that there is a wide method-dependent bias around the ALTM Problem is compounded by over-recovery of synthetic PTH1-84 and method-dependent differences in sensitivity towards PTH7-84. Despite the assay bias differences, which are not reflected in the upper limit of the reference range, Scottish biochemists andrenal physicians adopted target PTH conc of x2-4 ULNConsequence: patient management, using the agreed PTH target, ≠ across the different renal units. Some units likely to be under- and others over-treating. Recommendation : set new targets accomodating method differences to standardise RX. Targets are as follows: ◦ Abbott Architect 16 - 31 pmol/L ◦ Beckman Access DxI 13 - 25 pmol/L ◦ DiaSorin Liaison 12 - 24 pmol/L ◦ Roche Elecsys 14 – 28 pmol/L ◦ Siemens ADVIA Centaur 15 - 31 pmol/L ◦ Siemens Immulite 2000 22 - 45 pmol/L

Consequences of interferences

Incorrect result produced then used for clinical decisionMedico-legal action

eg elevated result

*HCG -?gonadal tumour SurgeryPRL ?prolactinoma SxF or UFC Ix for CS when on medsfemale testosterone inappropriate Ix

eg elevated & low resultsFalse TSH or thyroid hormone Inappropriate Rx

Clinical consequence – Rufer casePatient (Ms Rufer) presented with abdo pain & vaginal bleeding.

serum HCG by commercial lab. ectopic by O&G Cons

Rx with low dose chemo- HCG not fall.

Referred to Gynae Onc at Univ Hosp

Continued to have serum HCG in Univ lab so with trophoblstic disease so Rx with high dose chemo, hysterectomy and medastinal split (lung) surgery for suspected metstatic disease

Commercial lab and Uni lab both used HCG assay from same manufacturer

Ix later identified patients blood was only one of 50 tested that failed the dilution analysis test. Manufacturer informed but didnot respond to request for help from Univ lab

Univ Hosp Dr concluded HCG result due to interference and Rx unnecessary

Sued and eventually won $16 million 50% hospital lab 50% manufacturer (other labs had reported problem to manufacturer)

Pre-analytical ‘interference’: Patient

?Age (newborn), sex ; day of cycle

Pregnant : E2 → binding hormones ; F + TT4: structural similarity → TSH in 1st trimester

Time: eg ACTH + cortisol; testosterone, random hGH

Patient : NTI ‘sick euthyroid’; stress ;ITU TFT request – if definite hypothyroid will still

have raised TSH

Food : insulin and Gastrin

Medications : in-vivo interaction with analyte or in-vitro interference in IA; heparin ( FFA compete with T4 for binding sites), OCP, amiodarone

Pre-analytical interference : Sample collection

Blood collection tubes + additives

◦ Serum v plasma: EDTA chelatesSiemens Immulite if use ACTH EDTA sample for A4: >35nmol/L

◦ Serum unstable due to proteolytic enzymesACTH, GI hormones use EDTA or Li/Hep with

additional antiptoteolytic agent (Trasylol) still need to cold spin and snap freeze

◦ Serum matrix of choice for most IA (? PTH)◦ Haemolysis: invalidates PTH, ACTH, insulin, Gastrin

assays (release of proteolytic enzymes)◦ ‘Gel separator’ tubes problem with some drug assays

(?other IA when stored over cells over several days eg 50% P4). Stopper plasticizer did interfere but since removed.

Antibody interference

◦ Heterophilic

◦ Anti-animal (HAMA)

◦ Autoantibodies

Heterophilic Ab

Characterised by substantial non-specificity, produced in response to no clear immunogen

Associated with autoimmune or inflammatory disease BUT also present in healthy

Interference in both competative and non-competative IA (>in latter)

Can falsely or result

Heterophilic antibodies

+ +

RF binds to Fc portion of Ig → steric hindrance → or binding to Ag

TFT interference – What does lab do?Repeat

Do dilution of TSH to see if it dilutes linearly

Send sample to Cambridge?Do additional analyses on x2 2-step immunoassay platforms plus also measure Total T4, TT3, FT3, FT4 and if required will perform Equilibrium dialysis analysis on sample. Without the latter = ~£50; £90 with latter. This is expensive

Plan is to test on another 2 step assay platform (Abbott) before send to Cambridge

If abnormal in Cambridge then another sample sent to investigate for possible TSH’oma (measure α-subunit Birmingham) or thyroid hormone resistance syndrome / TSH-receptor abnormality (Cambridge)

TFT assay platforms

One-step◦ Roche Modular E170 & Cobas◦ Seimens Immulite◦ Seimens Centaur◦ Tosoh

Two-step◦ Abbott Architect◦ Wallac Delphia◦ Beckman

Immunoassay – one step

+

Labelled 2nd

Ab (signal Ab)

+

Immunoassay – 2-step

W

A

S

H

+

W

A

S

H

+

+ +

Equilibrium dialysis problems

Semi-permeable membrane

Sensitive assay to measure [FT4]

Problem to find a radioactive molecule that will behave the same as natural FT4

Process is very temp dependent

Anti-animal antibodies

Antibodies produced in response to immunization

More commonly interfere in sandwich assays by linking the capture to the detection Ag →falsely high result

Incidence of HAMA as use mouse mAb as vehicle to delivery agents for immunoscintigraphy and chemotherapy

HAMA a big problem for IA as the major platforms nearly all use mouse monoclonal Ab

Antibody specificity

Lack of is major source of inaccuracy in IA

Specificity problem > if recognition requires one epitope

Polyclonal >monoclonal

Manufacturers obliged to provide x-reaction data for an assay. Usually a single concn, at extreme not physiological.

Assumes a parallel displacement at all levels of interference. Rarely case.

Cortisol assay & x-reactivity with commonly prescribed steroids

Dexamethasone

11-Deoxycortisol

Prednisolone

CortisolHO

Cortisol assay & x-reactivity with commonly prescribed steroids

X reactivity= amount of cortisol required to lower binding by 50%amount of cross-reactant to lower binding by 50%

Sign

al (

1/c

once

ntr

atio

n)

100 250 1000 10,0000

Steroid concentration (nmol/l)

11-deoxycortisol

cortisol

For example

% X-reactivity of 11-deoxycortisol in cortisol assay

= 100 x 100 = 10%1000

100 250 1000

0

0

cortisol

11-deoxycortisol

Steroid concentration (nmol/l)

Sign

al (

1/c

once

ntr

atio

n)

In Cushings syndrome treated with metyrapone:[11-deoxycortisol] can reach ~400 nmol/L so apparent contribution to cortisol ~ 40nmol/l

In health: Normal range 11-deoxycortisol ~10nmol/L @0900h so contribution to cortisol ~1 nmol/L

X-reactivity in Roche cortisol assay:•Corticosterone = 5.8%•11-Deoxycortisol = 4.1%•Dexamethasone = 0.01%•Prednisolone = 170%•Methylprednisolone = 390%•Betamethasone = 0.08%•Beclamethasone = not tested •Triamcinolone = 0.32%

Cortisol assay & x-reactivity with commonly prescribed steroids

Cortisol and prednisolone

Prednisolone X-reactivity = 170%Our assay is unsuitable for assessing adrenal function in patients on prednisolone→ Royal Brompton

High performance liquid chromatographySeparates cortisol and prednisolone

SC, 26yo female, AsthmaCortisol (Roche)= 301 nmol/LCortisol (HPLC) = <20 nmol/LPrednisolone (HPLC)

= 415 nmol/L

Antibody specificity (2)

Lack of specificity sometimes advantageous◦ eg HCG has many different forms and an assay that is

specific for one form (intact HCG as in preg test) maybe disadvantageous if used as a tumour marker test as the tumour may produce other molecular forms not detected

Examples of components affecting assay specificity

Cross-reactant AnalytePrecursors Pro-insulinFragments hCG, PTHMetabolites SteroidsDimers hGHProtein complex MacroprolactinRelated molecules Steroids

High – dose ‘hook effect’

• This issue is not seen in two-step sandwich immunoassays ie. those with a wash step between the addition of sample and labelled antibody

• To detect the hook effect samples are analysed neat and diluted. If the result on dilution is higher than that for the neat sample, the neat sample is most likely affected by high dose hook effect.

The hook effect – clinicians dilemma

• 26 year old male

• Bitemporal hemianopia

• Imaging shows large

pituitary lesion

• Prolactin measured

= 900 miu/L

• Dilution?

• Analytical range of assay • 10 – 10,000 mU/L• Automatic 10x dilution if result is >10,000 mU/L

• Manufacturer kit insert states that no hook effect is seen up to 200,000mU/L

Prolactin high-dose hook effect

* *

*

*

Sign

al

* ***2.5K 5K 10K 40K 160K 1280K

Prolactin concentration (mu/L)

*

**

*

Point of ‘hook’~500,000 mu/L

5,600 mu/L

Manual dilution of all results >5,000 and <10,000mU/L

Identifying interference

Clinician is crucial. Identify if result at odds with clinical picture. Interferences in general are rare

Important that clinician liaises with lab

Clinical evidence must NOT be over ruled by numerical number

Patient with identified interference must have this recorded in notes to prevent future misinterpretation of result

Troubleshooting suspected interference

Excellent chapter in ‘The Immunoassay Handbook’. Ed by David Wild. Published by Elseiver Feb 2013.

Chapter 5.3 by Jason Park & Larry Kricka◦ Covers interferes, strategies to prove presence,

measures to prevent interference & clin consequences

Chapter 6.5 by David Wild & Jianwen He◦ Covers: -impact of reagent lot change

-impact of commercial QC material biasedfrom target mean

-EQAS Bias of method with ALTM

Corrective actions

Check reagents, calibration IQC and all pre-analytical factors (where possible) are correctEnsure platform can ‘do what it says on the tin’ eg insulin assaysRerun the sample to confirm result(s)Perform dilution (with appropriate diluent). Non-linearity strongly indicative of interferenceRun the sample on another platform that uses different technology ◦ eg TFT interference one step v 2-step assay v

Equilibrium dialysis◦ eg IA v LCMSRe-analyse the sample after using Ab-blocking tube. Perform PEG-precipitation of sample

Siemens Immulite 2500 Insulin assayX-reaction with pro-insulin = 8%

X-reactivity with different synthetic insulin analogs is variable

?insulin overdoseLocal assay NOT recommendedSample should be sent to Guildford SAS lab.

Mercodia ELISA kitshown to detect every synthetic insulin currently in BNF (pers comm. Dr Gwen Wark – Consultant Clinical Scientist)

NB : Patients on synthetic insulin analogs may also develop anti-insulin Ab’s - these may also interfere with insulin assay

Testosterone clinical case 1

Infant born 37/40 in Sheffield1st child of unrelated parentsUneventful pregnancy, no maternal virilizationBW 5lb 4ozCliteromegaly and somewhat rugose labial skin noticed at birth No family history of note

What tests would you advise?

Case 1

Steroid Age : 24h

Cortisol nmol/L 517

Testosterone nmol/L 10.3

Androstenedione nmol/L

18

DHEAS umol/L 9.5

Oestradiol pmol/L 304

Any other tests?

17-hydroxyprogesterone = 22.6 nmol/L Karyotype 46XX

Case 1 : clinical dilemmaSteroid Age :

1/7Age : 1/12

Age : 3/12

Cortisol nmol/L 517 600

Testosterone nmol/L 10.3 2.2 0.4

Androstenedione nmol/L

18 5.9 <0.3

DHEAS umol/L 9.5 3.1 <0.8

Oestradiol pmol/L 304

•At 24h problems of steroids from maternal circulation present plus poor analytical specificity

•Degree of x-reactivity will depend on analytical platform

•Outcome: Infant at 10months age was normal 46 XX, with normal external genitalia (clitoris & vagina present) and no palpable gonads