Transcript urine epo test - e

URINE ERYTHROPOIETIN
(EPO) TEST
Part A: INTRODUCTION AND
MANDATE
Erythropoietin (EPO)
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Stimulates erythropoiesis and the
increase in red blood cells improves the
amount of oxygen the blood can carry to
the muscles.
Clinically used in treatment of anemia
related to chronic renal disease, and
patients who undergo dialysis to elevate
red blood cell production. Human EPO
(hEPO) is an acidic, 165 amino acid
glycoprotein with 4 sites of carbohydrate
attachment and is synthesized in kidney.
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Different rhEPO are currently available
with the same amino acid sequence but
differ in the procedure they were
produced and thus in their sugar
content and structure.
All EPO products comprise multiple
isoforms which differ in charge and
isoelectric point.
Isoforms of rhEPO were more alkaline
than those of hEPO.
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Darbepoetin alfa (Aranesp) is
hyperglycosylated rhEPO with two extra
N-linked carbohydrate sites in primary
sequence that stimulates erythropoiesis
by the same mechanism as endogenous
hormone, but has longer half-life. It is
more acidic than hEPO and rhEPO.
Urinary test is based upon isoelectric
focusing of a protein concentrate in a gel
featuring a pH 2-6 gradient and
immunochemical EPO detection of the
resolved isoforms with a double blotting
technique.
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The test is adopted for urine analysis of
athletes suspected to have taken
rhEPO and Aranesp for performance
enhancement. It is expensive, time
intensive and requires highly trained
technicians and a well equipped
laboratory and can be performed in a
few specialized laboratories.
Part B: GENERAL CONCLUSIONS
1-The urine EPO test: its use and
limitations
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Method to detect rhEPO and
darbepoetin alfa (Aranesp) uptake.
Highly innovative and specific, but very
sophisticated and time consuming,
expensive, requires well trained
technicians, performed in specialized
laboratories.
It has already shown the benefits, since
athletes have reduced the use of EPO.
2-Quality of EPO test is high
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6 laboratories are experts in performing
EPO urinary test.
Very few modifications of the original
method, which are improvements.
To standardize and maintain the same
quality of the test, worldwide, interlaboratory tests performed regularly and
following a well defined protocol.
3-Improvements at several steps
of the method (1)
Improvements must be listed, tested, validated.
1-assessment of urine samples prior to analysis
2-more selective urine preconcentration step
3-controlled solubilization of the urine concentrate
4-improved electrophoretic separation
5-use of more appropriate membranes and
antibodies
6-use of more recent luminescence kit
7-new approach for the interpretation of the
scanned EPO profiles
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3-Improvements at several steps
of the method (2)
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Improvements in reducing cost and
labor, so test is economic and can be
widely used.
Improvements with the participation of
all accredited laboratories and by
sharing the research.
A substantial international financial
effort should support this development.
4-Improving sensitivity of the
urine EPO test
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The test never gives false positive
results.
Improvements in order to gain in
sensitivity (fewer false negatives).
Aim is to reduce rhEPO abuse in sport. A
higher sensitivity will force athletes to
lower the amount of rhEPO uptake per
injection or/and increase the time interval
between the last injection and urine
collection.
5-EPO profile from each athlete and
basic research of EPO isoforms should
be performed
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Urinary EPO profile upon time is obtained
for each athlete in different physical
activities including effort and rest. Any
abnormal deviation from this profile must
be investigated and explained.
More research in EPO isoforms must be
performed: function, catabolism, half-life,
exact pI and age, gender and genetic
influences, etc.
6-Discussion among accredited
laboratories
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An international data bank of the
existing EPO profiles should document
the effects of race, physio-pathology,
physical effort etc and monitor the
evolution of the different misuses of
rhEPO.
Part C: TECHNICAL POINTS
1. Urine EPO test: its use and
limitations
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Urine EPO detection by electrophoretic
assay with immunochemical detection.
Discrimination between physiological EPO
(endogenous EPO) and catabolized
recombinant human EPO and
hyperglycosylated darbepoetin alfa
(exogenous EPO) used as doping agents.
Endogenous and exogenous EPO-based
glycoprotein hormones differ in electric
charge and isoelectric points of the different
isoforms.
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Isoform separation from concentrated
urine sample using a special gel
electrophoretic method referred to as
isoelectric focusing (IEF).
Protein separation is based upon
differences in isoelectric points (pI).
Protein identification is based upon
immunodetection employed specific
mouse monoclonal antibodies to human
EPO.
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High concentration of urine samples
(700 to 1000-fold) by 2 ultrafiltration
steps, the well adapted acidic narrow
pH range IEF process which gives a
high pI resolution of urinary EPO
isoforms, and 2-step immunodetection
of the EPO bands using a clever and
patented double blotting method are the
major achievements of this method.
Time-consuming (36 hours), requires
well trained technicians, strict multi-step
protocol, expensive (400-600 Euro per
sample).
2. High quality of urine EPO test
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Few modifications of the original
method, including the total duration of
the test varying from 24 to 36 hours, the
anti-protease treatment modified or
omitted, reagents from different sources
being used, etc.
3. Improvements at several steps
1) Assessment of urine samples prior to
analysis:
 Urine samples should be characterized
by its total protein content and their
state of dilution.
 Assay sensitivity can be related to the
concentration of urine.
 Urine dilution can be assessed by
monitoring its conductivity, osmolality
and/or creatinine concentration.
2) More selective urine preconcentration
step:
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Preconcentration of urine sample to achieve
sensitivity. 700 to 1000-fold concentration may
not show measurable EPO concentration.
Samples unusually rich in proteins, after
competition induce some artifacts (poor
electrophoretic migration inducing smears,
background staining).
Further removal of EPO related proteins by a
rapid ion exchange filtration step, eliminating
all proteins more basic than the EPO isoforms,
reducing sample viscosity and unwanted
potential protein-protein interactions.
3) Controlled solubilization of urine
concentrate:
 By using mixtures of chaotropic agents
(eg urea 7M, thiourea 2M) and
detergents (eg CHAPS 2-5%) that are
compatible with IEF and avoid partial
insolubilization of the sample on
electrophoretic gel.
4) Improved electrophoretic separation:
 IEF in narrow pH ranges, using
immobilized pH gradients in gel, to try to
distinguish discrete modifications of
isoelectric points (pI) between
exogenous and endogenous EPO
isoforms in their overlapping pH range.
 Method can accept higher protein load
than the classical carrier ampholyte gels
and thus has the potential of increasing
the sensitivity.
5) Use of appropriate blotting membranes
and antibodies:
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EPO is acidic and hydrophilic protein. Its transfer and
binding is better onto hydrophilic nylon membranes
instead of hydrophobic PVDF membranes that are
used now. Nylon membranes do not stick to
polyacrylamide gels and so avoiding the hydrophilic
Durapore membranes between the gel and PVDF
blotting membrane. Omitting the Durapore membranes
simplifies the method, improves the sharpness of the
EPO band patterns and allows the detection of small pI
differences between endogenous and exogenous EPO
isoforms and thus improves discrimination.
Monoclonal and polyclonal antibodies to EPO should
be tested for specificity and potential capacity in
distinguishing between endogenous and exogenous
EPO.
6) Use of more recent luminescence kit:
 Kits for luminescence detection should
be carefully evaluated and the approach
providing the highest sensitivity should
be adopted for EPO band detection.
7) New approach for interpretation of the
scanned EPO profiles:
 Scanned EPO profiles are evaluated by
integrated optical densities (OD) of
exogenous and endogenous isoform
band patterns. The presence of Aranesp
on acidic side can be recognized,
because no significant overlap exists
between the endogenous and
exogenous EPO isoforms.
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Data evaluation is based upon:
% basic isoforms=(sum of areas of basic
isoforms / sum of areas of all
isoforms)x100
where:
sum of areas of basic isoforms is the sum
of the areas of the bands with pI above
that of the less basic isoform of the
reference rhEPO standard.
Value > 80% is positive for rhEPO.
Other approaches for data evaluation:
1) Based on the comparison of the OD of
only 2 representative bands of the
exogenous and endogenous EPO
isoform patterns.
2) Based on the alteration of the normal
EPO electrophoretic profile by
exogenous EPO bands.
 Thus, all 3 methods should be
compared carefully on the same urine
samples and the best procedure should
then be adopted for routine use.
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4. Improving sensitivity of urine
EPO test
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Sensitivity refers to the ability of the
assay to correctly identify EPO abusers.
Ultimate goal is to have no false
negatives.
Test should not lead to false positives.
100% sensitivity cannot be reached,
95% are usually statistically acceptable.
5. EPO profile from each athlete
and research of EPO isoforms
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To avoid variation from individual to
individual in the endogenous EPO
isoform pattern being of genetic origin.
To register a normal EPO pattern as a
reference for any further EPO testing, it
is advised to conduct a longitudinal
study on the evolution, as a function of
time and physical activity, of urinary
EPO profiles on individual athletes.
6. Development of new urine
EPO tests
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Development of totally new urinary EPO
tests should be encouraged and funded
eg based upon immunoassays and
capillary electrophoresis.
Carbohydrate chain analysis (sugar
profiling) could be an elegant way to
monitor the presence of exogenous
EPO in serum and/or urine:
1) Immunoassays:
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The membrane assisted isoform immuno
assay technology, based on charge
separation and immunoassay detection, can
determine the presence of recombinant EPO
in biological fluids.
Technique is rapid (10 min) and is developed
into a simple-to-use field test procedure.
It has the required sensitivity to detect urinary
EPO (0.2 to 1.2 pM) in urine that was not
concentrated.
Applied on transferrin isoforms and can
analyze an isoform pattern ranging from pI
5.2 to 5.7.
2) Capillary electrophoresis:
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Can characterize the glycosylation
heterogeneity of rhEPO and thus the
characterization of different EPO products.
Appropriate concentration of the sample
prior to electrophoresis.
Applied on transferrin isoforms in human
serum.
Capillary electrophoresis similar to
determination of carbohydrate-deficient
transferrin (CDT), the most specific marker
available for detection of chronic,
excessive alcohol intake.
3) Carbohydrate chain analysis (sugar
profiling):
 Sugar profiling of human serum EPO
differs from rhEPO and this can
establish a direct method to detect the
misuse of EPO in sports. Eg, following
immunological extraction of EPO from
serum or urine, assays based upon
HPLC with fluorescence or mass
spectrometric detection could be
developed.