immunoassay
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immunoassay pdfTRANSCRIPT
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