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Instructions for Use
Erythropoietin ELISA
Enzyme immunoassay for the quantitative determination of
Erythropoietin (EPO) in human serum.
Flughafenstrasse 52a
Phone: +49 (0)40-53 28 91-0
D-22335 Hamburg, Germany Fax: +49 (0)40-53 28 91-11
Erythropoietin ELISA (NM56011) INTENDED USE
EPO ELISA is intended for the quantitative determination of Erythropoietin (EPO) in human serum. This assay is intended for in vitro diagnostic use, as an aid in the diagnosis of anemias and polycythemias. With the advent of the administration of recombinant erythropoietin as a biologic therapy to increase red blood cell mass, an erythropoietin assay may be used also to aid in the prediction and monitoring of response to recombinant erythropoietin treatment in persons with anemias. SUMMARY AND EXPLANATION
Erythropoietin (EPO) is a heavily glycosylated protein with a molecular weight of about 30.000–34.000 Daltons. Human EPO is a polypeptide consisting of 165 amino acids, containing one O-linked and three N-linked carbohydrate chains1. The recombinant EPO is a good substitute for the native protein for use in an immunoassay2. Serum EPO levels are dependent on the rate of production and the rate of clearance of the protein. Ninety percent of EPO is produced in the peritubular cells of the adult kidney in response to a decrease in tissue oxygenation3,4. There is evidence indicating that the protein on these cells which detects oxygen saturation of the blood is a heme-containing moiety5. As the pO2 of the plasma, a function of the hematocrit decreases, EPO concentration will increase6. There are also observations suggesting that normally there is an inverse correlation between serum EPO levels and red blood cell mass7. Quantitation of serum erythropoietin concentration serves as a diagnostic adjunct in determining the cause of anemia or erythrocytosis. Aplastic anemia, hemolytic anemia and anemia due to iron deficiency all result in serum EPO elevation. Whereas, EPO levels in patients with secondary anemia due to renal failure and other disorders such as acquired immune deficiency syndrome (AIDS) are generally inappropriately low for the degree of anemia. This is mostly likely caused by an impaired ability of the diseased kidney to produce adequate quantities of EPO8. Low concentrations of EPO may give an early warning of kidney transplant rejection10. EPO also can be used to monitor AIDS patients undergoing Zidovudine (AZT) therapy. An increased concentration of EPO verifies that anemia associated with AZT therapy is due to red cell hypoplasia or apliasia10. Polycythemia rubra vera, or primary erythrocytosis (an increase of red blood cell mass) results from unstimulated over production of erythrocytes. Hence, the increase in the hemoglobin causes decreased production of EPO, which results in subnormal levels of serum EPO9. Secondary polycythemias, which are also characterized by an increase in the total red blood cell mass, occur as a physiological response to elevated levels of circulatory EPO caused by tissue hypoxia. The hypoxia may be due to such factors as pulmonary fibrosis, cardiovascular disease, prolonged exposure to high altitude, abnormal forms of hemoglobin or drug treatment10. Some tumors produce EPO and, in these cases, EPO may be used as a tumor marker to monitor the effectiveness of treatment. TEST PRINCIPLE
The EPO Immunoassay is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of the biologically active 165 amino acid chain of EPO. It utilizes two different mouse monoclonal antibodies to human EPO specific for well-defined regions on the EPO molecule. One mouse monoclonal antibody to human EPO, is biotinylated and the other mouse monoclonal antibody to human EPO is labeled with horseradish peroxidase [HRP] for detection. In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidin-coated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of EPO in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of EPO present in the controls and patient samples are determined directly from this curve. The standards have been calibrated against the World Health Organization (WHO) erythropoietin international standard that consists of recombinant DNA derived EPO. The WHO reference standard used was erythropoietin 1st international standard (87/684). Erythropoietin ELISA (NM56011) WARNINGS AND PRECAUTIONS
1. For in-vitro diagnostic use only. For professional use only. 2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 3. In case of severe damage of the kit package please contact IBL or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 4. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 5. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 6. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. Material Safety Data Sheets for this product are available on the IBL-Homepage or upon request directly from IBL. 7. Chemicals and prepared or used reagents have to be treated as hazardous waste according to national biohazard and safety guidelines or regulations. 8. Avoid contact with Stop solution. It may cause skin irritations and burns. 9. Reagent A, Wash Concentrate, and EPO Calibrators and Controls all contain ciprofloxacin hydrochloride as a preservative. Keep from personnel who have demonstrated a sensitivity to Quinoline based drug products. Females who are, or may be pregnant should avoid any contact with Ciprofloxacin. 10. ELISA Reagent 1, Biotinylated EPO Antibody contains ProClin 300 as a preservative. Avoid contact and wear gloves while handling with this reagent. Promptly wash skin with mild soap and water if accidental skin contact should occur. Flush eyes with water for 15 minutes, if reagent should be in contact with eye(s). If ingested, avoid vomiting and give large amount of water. Contact a physician immediately. 11. All reagents of this kit containing human serum or plasma have been tested and were found negative for anti-HIV I/II, HBsAg and anti-HCV. However, a presence of these or other infectious agents cannot be excluded absolutely and therefore reagents should be treated as potential biohazards in use and for disposal. STORAGE AND STABILITY
The kit is shipped at ambient temperature and should be stored at 2-8°C. Keep away from heat or direct sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2–8°C. SPECIMEN COLLECTION AND STORAGE
The determination of EPO should be performed on human serum. To assay the specimen in duplicate,
400 µL of human serum is required. It is highly recommended that the specimen be collected between 7:30
a.m. to 12:00 noon, because diurnal variation of erythropoietin has been reported in literature11,12. Collect
whole blood without anticoagulant and allow blood to clot between 2-8°C, if possible. It has been reported
that serum samples clotted at room temperature (18°C to 25°C) caused a decrease in EPO value as
assessed by radioimmunoassay of about 30% over clotting on ice13. Do not store samples in self-defrosting
freezers. Prior to use, allow all specimens to come to room temperature (18°C to 25°C) and mix by gentle
inversion or swirling. Avoid grossly hemolyzed or grossly lipemic samples.
2-8°C (Aliquots) -15°C (Aliquots) Keep away from heat or direct sun light. Avoid repeated freeze-thaw cycles. Erythropoietin ELISA (NM56011) MATERIALS SUPPLIED
Ready to use. Break apart strips. Coated with Streptavidin.
Reagent 1
Ready to use. Contains: Biotinylated EPO Antibody [mouse monoclonal anti human
EPO] containing ProClin 300 as preservative
Reagent 2

Ready to use. Contains: Peroxidase (Enzym) labeled EPO Antibody [mouse
monoclonal anti human EPO]
Reagent A

Ready to use. Contains: Wash Concentrate (Saline with surfactant with the preservative ciprofloxacin hydrochloride). Reagent B
Ready to use. Contains: TMB Substrate.
Control 1+2
Contains: 2 Levels. Synthetic h-EPO (1-165) in a buffered protein solution. Each
control contains the preservative ciprofloxacin hydrochloride.
Refer to vial labels for exact ranges.
Standard A-F (lyophilized)
Standard A: 0 mIU/mL
Lyophilized Zero calibrator is a buffered protein solution and all other calibrators
consist of synthetic synthetic h-EPO (1-165) in buffered protein solution. These
standards have been calibrated against the World Health Organization
erythropoietin 1st international standard [recombinant DNA derived EPO] (87/684).
Each calibrator contains the preservative ciprofloxacin hydrochloride.
Refer to vial labels for exact concentrations.
G-H (lyophilized)
Extra low Calibrators.
Contains: synthetic h-EPO (1-165) in buffered protein solution. These standards
have been calibrated against the World Health Organization erythropoietin 1st international standard [recombinant DNA derived EPO] (87/684). Each calibrator contains the preservative ciprofloxacin hydrochloride. Refer to vial labels for exact concentrations. Stopping Solution
Ready to use. Contains: 1 N sulphuric acid.
1. Micropipettes (Multipette Eppendorf or similar devices, < 3% CV). Volume: 50; 100 and 150 µL 2. Orbital shaker 3. 8-Channel Micropipettor with reagent reservoirs 4. Wash bottle, automated or semi-automated microtiter plate washing system 5. Microtiter plate reader capable of reading absorbance at 450 nm and 405 nm (reference wavelength 600-650 nm) 6. Bidistilled or deionised water 7. Paper towels, pipette tips and timer PROCEDURE NOTES
1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to reach room temperature (18-25°C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each component and specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. 4. It is advised to determine samples in duplicate to be able to identify potential pipetting errors. Erythropoietin ELISA (NM56011) 5. Use a pipetting scheme to verify an appropriate plate layout. 6. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipettor for pipetting of solutions in all wells. 7. Microplate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microplate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 8. Humidity affects the coated wells/tubes. Do not open the pouch until it reaches room temperature. Unused wells/tubes should be returned immediately to the resealed pouch including the desiccant. 9. Each test run needs a standard curve. 10. Samples that have values below the limit of detection (1.2 mU/mL) should be reported as " <1.2 mU/mL" 11. Patient samples with values greater than the highest calibrator (Calibrator F), which is approximately 450 mU/mL (see exact concentration on vial label, because it can vary from one lot to another), must be diluted with Calibrator A (Zero Calibrator) and re-assayed. Multiply the result by the dilution factor. Alternatively, the result may be reported as greater than the highest calibrator concentration (Calibrator F). For example, if the Calibrator F has an assigned EPO value of 494 mU/mL, the report should be " > 494 mU/mL". PRE-TEST SETUP INSTRUCTIONS
10.1. Preparation of lyophilized or concentrated components
Allow the vials to stand for 10 minutes and then CAL B-H LYO
mix thoroughly by gentle inversion to insure CTRL 1+2 LYO
complete reconstitution. Warm up at 37°C to dissolve crystals, if RGT A CONC
Erythropoietin ELISA (NM56011) TEST PROCEDURE
For enhanced low end sensitivity include Calibrator G and Calibrator H in assay.
1. Place sufficient Streptavidin Coated Strips in a holder to run all six (6) calibrators, A – F of the EPO
CALIBRATORS [Exact concentration is stated on the vial label], Controls and patient samples.
NOTE: for enhanced low end sensitivity include sufficient Streptavidin Coated Strips to include extra
low calibrators G and H.
2. Pipet 200 µL of calibrators, controls and samples into the designated or mapped well. Freeze (-15°C)
the remaining calibrators and controls as soon as possible after use.
NOTE: For enhanced low end sensitivity pipet in the following sequence: Calibrator A,
Calibrator G, Calibrator H, Calibrator B, Calibrator C, etc. until Calibrator F.

3. Add or dispense 25 µL of Reagent 1 (Biotinylated Antibody) into each of the wells, which already
contain the calibrators, controls and samples. 4. Add or dispense 25 µL of Reagent 2 (Enzyme Labeled Antibody) into each of the same wells. Tap
the microplate firmly against a rigid object, such as a pen, to achieve thorough mixing of the sample
with Reagents. For complete assurance of mixing, repeat the tapping for a minimum of 5 times for
each of the remaining three of the four sides of the plate. Be careful to avoid spillage. Cover the
microplate(s) with aluminum foil or a tray to avoid exposure to light, and place it on an orbital shaker
or rotator
set at 170 + 10 rpm for 2 hours + 15 minutes at room temperature (18°-25°C).
5. First aspirate the fluid completely and then wash/aspirate each well 5 times with the Working Wash
Solution (prepared from Reagent A), using an automatic microplate washer. The wash solution
volume should be set to dispense 0.35 mL into each well.
6. Add or dispense 150 µL of the Reagent B (TMB Substrate) into each of the wells. Tap the microplate
as described in Step 4. 7. With appropriate cover to avoid light exposure, place the microplate on an orbital shaker or rotator
set at 170 + 10 rpm for 30 +5 minutes at room temperature (18-25°C).
8. Add or dispense 100 µL of the Stopping Solution into each of the wells. Tap the microplate as
described in Step 4. Be careful to avoid spillage. 9. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to
450 nm against 250 µL of distilled or deionized water. Read the plate again with the reader set to
405 nm against distilled or deionized water.*
10. By using the final absorbance values obtained in the previous step, construct two calibration curves using 405 nm reading and 450 nm reading via cubic spline, 4 parameter logistics, or point-to-point interpolation to quantify the concentration of EPO * Note: The second reading is designed to extend the analytical validity of the calibration curve to the value
represented by the highest calibrator, which is approximately 450 mIU/mL (the exact concentration is printed on the vial label and will change slightly from one lot to another). Hence, patient samples with EPO > the penultimate (2nd to the highest) calibrator, i.e. Calibrator E can be quantified against a calibration curve consisting of the readings all the way up to the concentration equivalent to the highest calibrator using the 405 nm reading, away from the wavelength of maximum absorbance. In general, patient and control samples should be read using the 450 nm for EPO concentrations up to the concentration of Calibrator E. EPO concentrations above that of Calibrator E should be interpolated using the 405 nm reading. QUALITY CONTROL
Control serum or serum pools should be analyzed with each run of calibrators and patient samples. Results generated from the analysis of the control samples should be evaluated for acceptability using appropriate statistical methods. When the laboratory first introduces this EPO assay, the release of patient sample results should be based on whether the kit Control results fall within the suggested acceptable ranges. If one or more of the quality control sample values lie outside the acceptable limits, the assay should be repeated. Once the laboratory has generated data of its own, the quality control parameters should be based on the statistical data by the laboratory, using either kit Control and/or serum pools made by the laboratory. Levy-Jenning plots on control results should be used. If the results for all the control samples are within mean + 2 standard deviations, with no definitive trend or bias of the quality control data, the assay should be deemed acceptable. The Westgard rule should be followed to be compliant with CLIA 88 regulations. If the control results do not fall within the stated parameters as described, assay results are invalid. Erythropoietin ELISA (NM56011) CALCULATION OF RESULTS
13.1. Manual Method
1. For the 450 nm readings, construct a dose response curve (calibration curve) using the first five
calibrators provided, i.e. Calibrators A, B, C, D and E. For the 405 nm readings, construct a second dose response curve using the three calibrators with the highest concentrations, i.e. Calibrators D, E and F. For enhanced low end sensitivity include CALIBRATOR G and CALIBRATOR H for calculation of
Construct a dose response curve (calibration curve) using Calibrators A, G, H, B, C, D and E.
2. Assign the concentration for each calibrator stated on the vial in mIU/mL. Plot the data from the calibration curve on linear graph paper with the concentration on the X-axis and the corresponding A.U. on the Y-axis. 3. Draw a straight line between 2 adjacent points. This mathematical algorithm is commonly known as the "point-to-point" calculation. Obtain the concentration of the sample by locating the absorbance unit on the Y-axis and finding the corresponding concentration value on the X-axis. Patient and control samples should be read using the 450 nm for EPO concentrations up to up to the penultimate [2nd to the highest] calibrator, i.e. Calibrator E. EPO concentrations above the concentration of the penultimate calibrator (in the example shown below as 156 mIU/mL) should be interpolated using the 405 nm reading. 13.2. Automated Method:
Computer programs using cubic spline or 4 PL [4 Parameter Logistics] or Point-to-Point can generally give a good fit. For the 450 nm readings, construct a dose response curve (calibration curve) using the first five calibrators provided, i.e. Calibrators A, B, C, D and E. For the 405 nm readings, construct a second dose response curve using Calibrators A, D, E and F. For enhanced low end sensitivity include CALIBRATOR G and CALIBRATOR H for calculation of

Construct a dose response curve (calibration curve) using Calibrators A, G, H, B, C, D and E.
Sample Data at 450 nm [raw A.U. readout against distilled or deionized water]
1st Reading
2nd Reading
Microplate Well
Patient Sample 1 Patient Sample 2 Patient Sample 3 Patient Sample 4 Patient Sample 5 * Because the concentration readout is > the concentration of Calibrator E, e.g. 156 mIU/mL, it is
recommended to use the data obtained at 405 nm as shown in Sample Data at 405 nm in the table below.
Erythropoietin ELISA (NM56011) Sample Data at 405 nm [raw A.U. readout against distilled or deionized water]
1st Reading
2nd Reading
Microplate Well
Patient Sample 1 Patient Sample 2 Patient Sample 3 Patient Sample 4 Patient Sample 5 ** For samples with concentrations < than the concentration of Calibrator E, e.g. 156 mIU/mL, it is
recommended to use the data obtained at 450 nm as shown in Sample Data at 450 nm in the table
above. This practice should give the results with optimum sensitivity of the assay.
NOTE: The data presented are for illustration purposes only and must not be used in place of data
generated at the time of the assay.
EPO levels were measured in 120 apparently normal individuals in the U.S. with the EPO ELISA. The
samples consist of 61 males and 59 females, ranging from 18 to 96 years of age. There is no significant
statistical difference on the reference ranges obtained from the female and male population of data. This
finding, that there is no gender difference, is consistent with the literature 21. Further, the EPO values do not
appear to have significant age dependence, except higher values were obtained in samples from early
phases of adulthood, i.e. approximately 22 to 42 years of age). Using the nonparametric method for the
analysis of reference values outlined in the NCCLS publication "How to Define, Determine, and Utilize
Reference Intervals in the Clinical Laboratory" (NCCLS Document C28-A, Vol. 15 No. 4) the reference
ranges (2.5–97.5 percentile) were
3.22-31.9 mIU/mL for EPO in serum. Each laboratory should establish
their own range of expected normal values.
"In patients with erythrocytosis due to uncompensated hypoxia, serum immunoreactive EPO is elevated; in those with compensated hypoxia, the serum immunoreactive EPO level is usually within the range of normal, and in patients with polycythemia vera, serum immunoreactive EPO is either normal or low. Thus, while an elevated serum EPO level suggests that erythrocytosis is a secondary phenomenon and a low EPO level supports the possibility of autonomous erythropoiesis, a normal serum EPO level excludes neither hypoxia nor autonomous EPO production as the cause of erythrocytosis." 20 LIMITATIONS OF THE PROCEDURE
Like any analyte used as a diagnostic adjunct, EPO results must be interpreted carefully with the overall
clinical presentations and other supportive diagnostic tests.
Purified IgG proteins of the same species as the ones for which the capture and the label antibodies, were
derived, in addition to one commercial heterophile antibody blocker, have been incorporated in the reagents
to minimize the heterophile antibodies.14 Nonetheless, there can be no assurance that the heterophile
interference has been completely eliminated. Therefore, it is recommended that at least three dilutions of
any elevated and/or suspect positive results be assayed to detect non-parallelism compared to reference
Because results obtained with one commercial EPO assay may differ significantly from those obtained with
any other, it is recommended that any serial testing performed on the same patient over time should be
performed with the same commercial EPO test.16 This test may not be sufficiently sensitive to consistently
discriminate abnormally low EPO values from normal levels of EPO.
Erythropoietin ELISA (NM56011) Lower EPO levels than expected have been seen with anemias associated with the following conditions: rheumatoid arthritis, acquired immunodeficiency syndrome, cancer, and ulcerative colitis17, sickle cell disease, and in premature neonates18. After allogeneic bone marrow transplant, impaired erythropoietin response may delay erythropoietin recovery17. Patients with hypergammaglobulinemia associated with multiple myeloma or Waldenstrom's disease have impaired production of erythropoietin in relation to hemoglobin concentration. This has been linked to increased plasma viscosity. No drugs have been investigated for assay interference. EPO levels of persons living at high altitudes with erythrocytosis may rapidly fall to normal after returning to low altitudes19. PERFORMANCE
Eighty five patient samples, with EPO values ranging from 3.8 to 304 mIU/mL, were assayed by the ELISA
procedure and an ELISA EPO kit. Linear regression analysis gives the following statistics:
ELISA = 0.94 ELISA Kit - 0.41 mIU/mL Sensitivity
The sensitivity, or minimum detection limit, of this assay is defined as the smallest single value, which can
be distinguished from zero at the 95% confidence limit. The EPO ELISA has a calculated sensitivity of 1.2
mIU/mL when using Calibrator B as the lowest calibrator. Hence, patient sample results below 1.2 mIU/mL
should be reported as "Less than 1.2 mIU/mL".
The EPO ELISA with Calibrator G and Calibrator H has a calculated sensitivity of 0.6 mIU/mL Hence, patient
sample results below 0.6 mIU/mL should be reported as "Less than 0.6 mIU/mL".
Precision and Reproducibility
The Intra-assay precision of the EPO ELISA Test was calculated from 22 replicate determinations on each
of the two samples.
Intra-Assay Variation
Mean Value (pg/mL) The inter-assay precision of the EPO ELISA Test was calculated from data on two samples obtained in 22 different assays. Inter-Assay Variation
Mean Value (pg/mL) Erythropoietin ELISA (NM56011) Recovery
Various amounts of EPO were added to four different patient sera to determine the recovery. The results are
described in the following table:
Specificity and Cross-Reactivity
Cross-reactivity in the EPO was studied by the addition of various substances to the Zero Calibrator
(Calibrator A).
Amount of Crossreactant Added
Human Transferrin Human Bilirubin (unconjugated) Human Hemoglobin Human α –Globulin Human α 2-Macroglobulin Human α 1-Acid Glycoprotein, Human α 1-Antitrypsin Human Gamma Globulin ACTH (intact molecule: amino acid sequence1-39) None of the cross reactants interferes with this EPO ELISA in the concentrations studied. The very small changes in EPO seen for some cross reactants were well within the statistical limits of intraassay variation. Linearity of Patient Sample Dilutions: Parallelism
Three patient serum samples were diluted with Calibrator A (Zero Calibrator). Results in mIU/mL are shown
% Observed ÷

Erythropoietin ELISA (NM56011) High Dose Hook Effect
The EPO ELISA kit has exhibited no "high dose hook effect" in standard diluent spiked with 200.000 mIU/mL
1.520 mIU/mL, and 966 mIU/mL) were tested without dilution and their results read much greater than the highest standard. Samples with EPO levels greater than the highest calibrator, however, should be diluted and re-assayed for correct values. PRODUCT LITERATURE REFERENCES
1. Sawyer, S.T., Krantz, S.B., Sawada, K. Receptors for Erythropoietin in Mouse and Human Erythroid Cells and Placenta. Blood 1989; 74: 103-109.
2. Imai, N., Kawamura, A., Higuchi, M., et al. Physicochemical and Biological Comparison of Recombinant Human Erythropoietin with Human Urinary Erythropoietin. J Biochem 1990; 107: 352-359.
3. Jacobson, L.O., Goldwasser, E., Fried, W., Pizak, L.F. The Role of the Kidney in Erythropoiesis. Nature
1957; 179: 633-634. 4. Koury, S.T., Bondurant, M.C., Koury, M.J. Localization of Erythropoietin Synthesizing Cells in Murine Kidney by in-situ Hybridization. Blood 1988; 71: 524-527
5. Goldberg, M.A., Dunning, S.P., Bunn, H. F. Regulation of the Erythropoietin Gene: Evidence that the Oxygen Sensor is a Heme Protein. Science 1988; 242: 1412-1415
6. Erslev, A.J., Caro, J., Birgegard, G., Silver, R., Miller, O. The Biogenesis of Erythropoietin. Experimental Hematology 1980; Suppl 8: 1-13.
7. Spivak, J.L. The Mechanism of Action of Erythropoietin. Int J Cell Cloning 1986; 4: 139-166.
8. Erslev, A.J. Erythropoietin. New Eng J Med 1991; 324:1339-1344.
9. Garcia, J.F., Ebbbe, S.N., Hollander, L., Cutting, H.O., Miller, M.E., Cronkits, E.P. Radioimmunoassay of
Erythropoietin: Circulating Levels in Normal and Polycythemic Human Beings. J Lab Clin Med 1982; 99:
10. Wild, D., editor. The Immunoassay Handbook, Stockton Press, 1994, p. 428.
11. Wide L., Bengtsson C., Birgegard G. Circadian Rhythm of Erythropoietin in Human Serum. Br J
Haematol 1989; 72: 85-90.
12. Cahan C., Decker M.J., Arnold J.L., Washington L.H., Veldhuis J.D., Goldwasser E., Strohl K.P. Diurnal Variations in Serum Erythropoietin Levels in Healthy Subjects and Sleep Apnea Patients. J Appl
1992; 72: 2112-7.
13. Goldwasser E. and Sherwood J.B. Annotation, Radioimmunoassay of Erythropoietin. Br J Haematol
1981; 48: 359-63. 14. Kricka L.J. Human Anti-Animal Antibody Interferences in Immunological Assays. Clin Chem 1999; 45:
15. Cotes P.M. and Spivak J.L. Erythropoietin in Health and Disease. Erythropoietin Molecular, Cellular
and Clinical Biology, Editors: Erslev A.J., Adamson J.W., Wschbach J.W., Winearls C.G. 1991;
Chapter 11:184-207.
16. Jelkmann W. Renal Erythropoietin: Properties and Production. Rev Physiol Biochem Pharmacol 1986;
17. Cotes M.P. Anomalies in Circulating Erythropoietin Levels. Annals of NY Acad, Sci 1994; 718:103-9.
18. Wintrobe's Clinical Hematology, ninth edition, edited by Lee G.R., Bithell T.C., Foerster J., Athens
J.W., Lukens J.N. Lea & Febiger, Philadelphia 1993. 19. Fairbanks V. Q & A. CAP Today Nov 1996, pg. 88.
20. Spivak, JL. "Erythrocytosis", Hematology: Basic Principles and Practice; editors: Hoffman R, Benz EJ
Jr., Shattil, SJ; Furie B., Cohen HJ; Silberstein LE; 1995; Chapter 37:484-491 21. Miller, ME, Chandra M, Garcia JF: "Clinical applications of measurement of serum immunreactive levels of erythropoietin", Ann. N.Y.Acad. Sci. 459: 375-381, 1985. Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύµβολα
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