URINE ERYTHROPOIETIN (EPO) TEST

Part A: INTRODUCTION AND MANDATE

Erythropoietin (EPO) n Stimulates erythropoiesis and the increase in red blood cells improves the amount of oxygen the blood can carry to the muscles. n 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.

n 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. n All EPO products comprise multiple isoforms which differ in charge and isoelectric point. n Isoforms of rhEPO were more alkaline than those of hEPO.

n 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. n 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.

n 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 n 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. n It has already shown the benefits, since athletes have reduced the use of EPO.

2-Quality of EPO test is high n 6 laboratories are experts in performing EPO urinary test. n Very few modifications of the original method, which are improvements. n To standardize and maintain the same quality of the test, worldwide, inter- laboratory tests performed regularly and following a well defined protocol.

3-Improvements at several steps of the method (1) n 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

3-Improvements at several steps of the method (2) n Improvements in reducing cost and labor, so test is economic and can be widely used. n Improvements with the participation of all accredited laboratories and by sharing the research. n A substantial international financial effort should support this development.

4-Improving sensitivity of the urine EPO test n The test never gives false positive results. n Improvements in order to gain in sensitivity (fewer false negatives). n 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 n 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. n 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 n 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 n Urine EPO detection by electrophoretic assay with immunochemical detection. n Discrimination between physiological EPO (endogenous EPO) and catabolized recombinant human EPO and hyperglycosylated darbepoetin alfa (exogenous EPO) used as doping agents. n Endogenous and exogenous EPO-based glycoprotein hormones differ in electric charge and isoelectric points of the different isoforms.

n Isoform separation from concentrated urine sample using a special gel electrophoretic method referred to as isoelectric focusing (IEF). n Protein separation is based upon differences in isoelectric points (pI). n Protein identification is based upon immunodetection employed specific mouse monoclonal antibodies to human EPO.

n 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. n Time-consuming (36 hours), requires well trained technicians, strict multi-step protocol, expensive ( Euro per sample).

2. High quality of urine EPO test n 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: n Urine samples should be characterized by its total protein content and their state of dilution. n Assay sensitivity can be related to the concentration of urine. n Urine dilution can be assessed by monitoring its conductivity, osmolality and/or creatinine concentration.

2) More selective urine preconcentration step: n Preconcentration of urine sample to achieve sensitivity. 700 to 1000-fold concentration may not show measurable EPO concentration. n Samples unusually rich in proteins, after competition induce some artifacts (poor electrophoretic migration inducing smears, background staining). n 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: n 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: n 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. n 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: n 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. n 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: n 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: n 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.

n 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.

n 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. n Thus, all 3 methods should be compared carefully on the same urine samples and the best procedure should then be adopted for routine use.

4. Improving sensitivity of urine EPO test n Sensitivity refers to the ability of the assay to correctly identify EPO abusers. n Ultimate goal is to have no false negatives. n Test should not lead to false positives. n 100% sensitivity cannot be reached, 95% are usually statistically acceptable.

5. EPO profile from each athlete and research of EPO isoforms n To avoid variation from individual to individual in the endogenous EPO isoform pattern being of genetic origin. n 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 n Development of totally new urinary EPO tests should be encouraged and funded eg based upon immunoassays and capillary electrophoresis. n Carbohydrate chain analysis (sugar profiling) could be an elegant way to monitor the presence of exogenous EPO in serum and/or urine:

1) Immunoassays: n The membrane assisted isoform immuno assay technology, based on charge separation and immunoassay detection, can determine the presence of recombinant EPO in biological fluids. n Technique is rapid (10 min) and is developed into a simple-to-use field test procedure. n It has the required sensitivity to detect urinary EPO (0.2 to 1.2 pM) in urine that was not concentrated. n Applied on transferrin isoforms and can analyze an isoform pattern ranging from pI 5.2 to 5.7.

2) Capillary electrophoresis: n Can characterize the glycosylation heterogeneity of rhEPO and thus the characterization of different EPO products. n Appropriate concentration of the sample prior to electrophoresis. n Applied on transferrin isoforms in human serum. n 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): n 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.