Analytical platforms and

immunoassay interference
Dr Les Perry
Consultant Clinical Scientist
Croydon 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 analysis
Based on Ab recognition of molecule
Manual IA use polyclonal (many animal species)
Ab v automated platforms use mAb (murine)
IA label: radioactive (
3
H/
125
I); later fluorescent,
chemiluminescent or enzyme reaction
IA 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 10000
Concentration of Ag
%

A
b

b
i
n
d
i
n
g
Immunoassays characteristics
Sensitivity
measure minute concentrations of an analyte
Specificity
Discriminate between closely-related cpds
Accuracy
Using reference stds can relate results between
different labs to derive reference ranges
Precision
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 mean
Hence 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 Cushings
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 sensitivity
Manufacturer / 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 variation
Ab
reach equilibrium (most platforms not)
High affinity reach eqbrium quicker
Factors that affect Ab-Ag reaction which if
incorrectly optimized have impact on rate
of reaction and therefore precision
Ab conc
Assay temp
pH of the final reaction mixture
variation 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 -well assys (heat unevenly)
Incomplete suspension of solid-phase particles prior
to or during assay incubation
Separation
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%

3
H /
14
C require scintillation counting
Glass tubes with
125
I add 1-2%imprecion
Multiwell 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 Ab
Autoantibodies
Blood substitutes
Carryover
Collection tube
Contamination
Contrast agents
Complement
Drugs
Drug metabolites
Fibrin
Haemolysis
Herbal remedies
Heterophilic Ab
High-dose hook effect
Human anti-animal Ab
Imaging agents
Immune complexes
Lipaemia
Microclots
Paraproteins
Partially filled collection
tube
RF
Sample storage
Sample matrix
Testosterone assays
RIA was introduced 1970
Large volume serum

3
H radioligand
Organic solvent extraction
Chromatographic separation step
Dextran-coated charcoal separation
step
Calculation using graph paper, sharp
pencil & flexicurve
Result in 2-3 days
Run 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
5-dihydrotestosterone
Testosterone
Testosterone assays progression
1980s
Improvements in antibody
specificity
Introduction of
125
I-tracers
Preparation of in-house
standards in serum
Obviate chromatographic
step (direct assays)
eg ANS in TFT / Danazol
in F assays
Use of buffer pH
Improvements in
separation step (sac-cel;
magnetised charcoal)
Data reduction packages
Assay in large batch mode
Results same day
1990s to date
Introduction of non-
isotopic assays
Introduction of solid-
phase Ab assisting in
separation step
Automation of analytical
process
Reagent packs for
platform
Continuous workflow
Results in hours
Small sample volume
Now performed in almost
every lab whether it be
DGH or Teaching Hospital
Automation of endocrine assays
Advantages
Improved precision
Faster turn-around
time
Multiple analytes
Autodilution
Obviate isotopes &
extraction
Fewer lab staff
Disadvantages
Hook & heterophilic
Ab interference
Poor specificity
Some assays not
very good (poor
bias/var)
Prone to lotto-lot
variation
When 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
Calibration
What is zero standard for endogenous
proteins?
TSH assay
1
st
generation measured down to 0.1 mU/L
2
nd
generation measured down to 0.02 mU/L
3
rd
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 and
renal physicians adopted target PTH conc of x2-4 ULN
Consequence: 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
decision
Medico-legal action
eg elevated result
*HCG -?gonadal tumour Surgery
PRL ?prolactinoma Sx
F or UFC Ix for CS when on meds
female testosterone inappropriate Ix
eg elevated & low results
False TSH or thyroid hormone Inappropriate Rx
Clinical consequence Rufer case
Patient (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 did
not 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 1
st
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 chelates
Siemens Immulite if use ACTH EDTA sample for
A4: >35nmol/L
Serum unstable due to proteolytic enzymes
ACTH, 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 TSHoma (measure -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 2
nd
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 conc
n
, 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
Cortisol
HO
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%
S
i
g
n
a
l

(
1
/
c
o
n
c
e
n
t
r
a
t
i
o
n
)
100 250 1000 10,000 0
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)
S
i
g
n
a
l

(
1
/
c
o
n
c
e
n
t
r
a
t
i
o
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
chromatography
Separates cortisol and
prednisolone
SC, 26yo female, Asthma
Cortisol (Roche)= 301 nmol/L
Cortisol (HPLC) = <20 nmol/L
Prednisolone (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 Analyte
Precursors Pro-insulin
Fragments hCG, PTH
Metabolites Steroids
Dimers hGH
Protein complex Macroprolactin
Related 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
*
*
*
*
S
i
g
n
a
l
*
*
*
*
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 biased
from target mean
-EQAS Bias of method with ALTM
Corrective actions
Check reagents, calibration IQC and all pre-
analytical factors (where possible) are correct
Ensure platform can do what it says on the tin eg
insulin assays
Rerun the sample to confirm result(s)
Perform dilution (with appropriate diluent). Non-
linearity strongly indicative of interference
Run the sample on another platform that uses
different technology
eg TFT interference one step v 2-step assay v
Equilibrium dialysis
eg IA v LCMS
Re-analyse the sample after using Ab-blocking tube.
Perform PEG-precipitation of sample
Siemens Immulite 2500 Insulin assay
X-reaction with pro-insulin = 8%
X-reactivity with different synthetic
insulin analogs is variable
?insulin overdose
Local assay NOT recommended
Sample should be sent to
Guildford SAS lab.
Mercodia ELISA kit
shown 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 Abs - these may also interfere with
insulin assay
Testosterone clinical case 1
Infant born 37/40 in Sheffield
1
st
child of unrelated parents
Uneventful pregnancy, no maternal
virilization
BW 5lb 4oz
Cliteromegaly 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 dilemma
Steroid Age :
1/7
Age :
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