Please note that this is a special order assay.

EPORIA

Radioimmunoassay for quantifying the level of erythropoietin in serum as an aid in the diagnosis of anemias and polycythemias

Catalog no.  E-90

For In-Vitro Diagnostic Use Only

INTENDED USE

The Ramco EPRIA radioimmunoassay is intended for the quantitative determination of erythropoietin in serum as an aid in the diagnosis of anemias and polycythemias.

SUMMARY AND EXPLANATION

Erythropoietin (EPO) is a heavily glycosylated protein with a molecular weight of about 34,000 Daltons. Functionally, it serves as an obligatory growth factor in the process of erythropoiesis.⁶ Ninety percent of erythropoietin is produced in the peritubular cells of the adult kidney in response to a decrease in tissue oxygenation.^4,5 There is evidence which indicates that the protein on these cells which detects oxygen saturation of the blood is a heme-containing moiety.³ As the pO2 of the plasma, a function of the hematocrit decreases, erythropoietin concentrations will rise.¹

Quantitation of serum erythropoietin concentration is an aid in determining the cause of anemia or erythrocytosis. Secondary anemias due to renal disease and other disorders such as acquired immunodeficiency syndrome (AIDS) result from insufficient production of EPO. In primary anemia, due to most other factors, the kidney responds by producing EPO and serum levels are increased from that of normal persons.¹⁵

Polycythemia rubra vera, or primary erythrocytosis results from unstimulated over production of erythrocytes. The resultant increase in the hemoglobin concentration of the blood causes decreased production of EPO and consequently serum levels of the hormone drop below those seen in normal persons.² On the other hand, secondary erythrocytosis can result from increased EPO production caused by hypoxia (pulmonary or cardiac) or tumorous erythropoietin producing cells. Measurement of erythropoietin may aid in determining which mechanism is responsible for erythrocytosis.¹⁶

Previous methods for measuring erythropoietin include bioassays, either in vivo or in vitro. More recently, ligand based assays have been introduced. Ligand assays such as the Ramco assay are much superior in sensitivity and precision to older bioassays.^2,16

PRINCIPLE OF THE TEST

Ramco's EPORIA assay is a two-antibody competitive radioimmunoassay which uses rabbit anti-human erythropoietin as the first antibody and goat anti-rabbit antibody as the second antibody. After adding radiolabeled EPO, sera to be tested and calibrators are allowed to react with a fixed amount of rabbit anti-human erythropoietin (anti-EPO), the second antibody is added to precipitate the anti-EPO; test samples and calibrators are centrifuged and the supernatants discarded. The radioactivity of all precipitates is determined. The EPO content of test sera is determined by comparing their radioactivity with that of the calibrators run concurrently.

KIT CONTENTS

¹²⁵I EPO:    1 vial containing 11 ml of recombinant human erythropoietin radiolabeled with carrier free radioiodine. The isotope in this assay represents not more than 2 microcuries (74 Kilobecquerels) of radioactivity. This tracer is diluted in a borate buffer containing protein as a stabilizer and sodium azide as a preservative. Refer to the Precautionary Statement for radionuclide handling information. Store at 2 - 8C. Due to the nature of radioiodine, optimum results will occur if the ¹²⁵I EPO is used within 60 days of the date of manufacture. In any event, do not use after expiration date on bottle (90 days from the date of manufacture).

Zero Calibrator/Diluent Buffer:    1 vial containing 20 ml of phosphate buffered saline with protein as a stabilizer and 0.1% azide as a preservative. This solution is used to dilute patient samples with greater than 200 mU/ml EPO. Store at 2 - 8C. Do not use after the expiration date on the bottle.

Calibrators:    6 vials, each containing 2 ml of recombinant human EPO in concentrations of 6.25, 12.5, 25, 50, 100, and 200 mU/mL in a phosphate buffered saline solution with protein as a stabilizer and azide as a preservative; calibrated against the World Health Organization (WHO) 2nd IRP Human Erythropoietin Urinary/Bioassay 67/343. Store at 2 - 8C. Do not use after the expiration date on the bottle.

Erythropoietin Antiserum:    1 vial containing 11 ml of rabbit anti-human recombinant EPO diluted in a borate buffer containing protein as a stabilizer and azide as a preservative. Store at 2 - 8C. Do not use after the expiration date on the bottle.

Second Antiserum:    1 vial containing 5.5ml of goat anti-rabbit IgG with sodium azide as a preservative. Store at 2 - 8C. Do not use after the expiration date on the bottle.

Precipitating Solution:    2 vials, each containing 27 ml of a borate buffer with polyethylene glycol, normal rabbit serum, protein as a stablizer and azide as a preservative. Store at 2 - 8C.  Do not use after the expiration date on the bottle.

Control Sera:    3 vials, each containing 2 ml of human sera with known concentrations of EPO and azide as a preservative. Refer to the label on the bottle for level. Refer to the precautionary statements for information on handling human specimens. Store at 2 - 8C. Do not use after the expiration date on the bottle.

• All contents of this kit are ready for use. No reconstitution or dilution is necessary.
• Store all reagents in this kit at 2 - 8C.
• Review the sections in this handout which pertain to the safe handling of radionuclides, human specimens, and azides before opening the reagents.
• This kit provides reagents for 100 assay tubes.

PRECAUTIONARY STATEMENT

This Kit is Intended for In Vitro Diagnostic Use Only

Human Specimens:    Control Sera provided in this kit contain human sera which have been tested by FDA approved methods for the presence of antibody to HIV-1 and hepatitis B surface antigen and found to be negative. However, since no test method can absolutely guarantee the absence of these or other infections agents, all controls and human sera tested with this assay should be treated as potentially bio-hazardous. Wear gloves and avoid contact with skin and mucous membranes.: 2 vials, each containing 27 ml of a borate buffer with polyethylene glycol, normal rabbit serum, protein as a stabilizer and azide as a preservative. Store at 2 - 8C. Do not use after the expiration date on the bottle.

Azides:    Reagents in this kit contain sodium azide. Contact with copper or lead drain pipes may result in the formation of explosive azide deposits. It is important during disposal to flush drains with copious volummes of water to prevent azide accumulation. Plumbing that may be contaminated with azides can be flushed with 10 percent sodium hydroxide solution.

Radionuclides:    This radioactive material may be received, acquired, possessed and used only by physicians, clinical laboratories, or hospitals, and only for in vitro clinical or laboratory tests not involving internal or external administration of the material or the radiation therefrom, to human beings. Its receipt, acquisition, possession, use and transfer are subject to the regulations and a general license of the U.S. Nuclear Regulatory Commission or of a state with which the commission has entered into an agreement for the exercise of regulatory authority.

Storage of radioactive material should be in the original container and as far removed from the work area as practical.

Use radioactive material in designated areas by authorized personnel only. Do not permit persons under 18 years of age to handle these materials or have access to areas containing them.

Never pipet radioactive materials by mouth. Avoid other external or internal contamination by wearing gloves and lab coat. Do not eat, smoke, or drink in designated radioactive work areas.

Avoid contamination of the laboratory and equipment through careful work practices and use of disposable or easily decontaminated laboratory ware and absorbent covers for bench surfaces.

Should external contamination occur, clean the exposed area thoroughly with soap and water or a radiological decontamination solution. Rinse glassware well with water before washing with other laboratory glassware. The receipt, use, transfer, and disposal are subject to the provision of Title 10 of the Code of Federal (USA) Regulations, Part 20 (10 CFR, Part 20), and your specific license.

Other Precautions:

• Avoid splashing or generating aerosols.
• Follow kit recommendations for incubation times and temperatures to avoid possibly erroneous results.
• Microbial contamination of reagents may cause erroneous results.
• Do not use reagents with those from other lots or manufacturers.
• Do not use kit reagents after expiration date.

ADDITIONAL MATERIALS REQUIRED

• Gamma counter.
• Centrifuge, refrigerated and capable of 3000 x g.
• 12 x 75mm polystyrene or glass (borosilicate) tubes.
• Precision pipettes calibrated to deliver 50 ul, 100 ul, 200 ul and 500 ul � 1 percent.
• Aspirator with a catch vessel suitable for radioactive and human biohazard waste. A nine inch pasteur type transfer pipette is recommended as the tip.
• Blood collection supplies for serum samples.

SPECIMEN COLLECTION AND STORAGE

Warning:    Treat all human body fluid specimens as if they are capable of transmitting severe communicable diseases such as AIDS and Hepatitis. Read the Precautionary Statement in this handout concerning the handling of human specimens.

For measurement of erythropoietin, blood should be collected aseptically by venipuncture as described, for example in Brown's Hematology, Principals and Procedures.¹⁷ Collect enough blood to yield at least 800 ul of serum (enough to repeat the analysis if desired). Centrifuge the specimen as soon as the clot is completely formed and remove the serum to avoid hemolysis. The serum should be kept in a sealed tube to avoid contamination or evaporation. The serum specimens are stable at 2 - 8C for at least one week. It is not necessary to have a fasting specimen; however, if the specimen is grossly lipemic, recollect a sample after the subject has fasted. Reject specimens that are markedly hemolyzed or appear to be contaminated. No drugs have been tested for interference with this assay.

PROCEDURE

1)  Bring all reagents to room temperature (20 - 25C) and mix well.

2)  Label plain 12 x 75mm polystyrene or glass tubes in duplicate according to the following list:

• TC (total counts)
• NSB (nonspecific binding)
• � (max binding, or B_�)
• 6.25, 12.5, 25, 50, 100 and 200 (calibrators)
• Controls and patient samples

3)  Pipet 200 ul of the Zero Calibrator into the NSB and � tubes. Pipet 200 ul of the remaining Calibrators, Controls and patient samples into their respective tubes. For this addition we recommend that a disposable tip micropipette be used and that a fresh tip be used for each sample.

4)  Pipet 100 ul of ¹²⁵I EPO into every tube. Do not cross-contaminate tubes by touching the pipet tip to the sample already in the tube.

5)  Set aside the TC tubes as they will not be used again until counting.

6)  Pipet 100 ul of Erythropoietin Antiserum into all tubes EXCEPT the NSB tubes. Gently shake the rack by hand to mix the contents of each tube before incubation.

7)  Cover all tubes and incubate at room temperature (20 - 25C) for 18 to 24 hours. (Place remainder of kit in cold storage.)

8)  Pipet 50 ul of cold Second Antiserum into each tube (except TC). Gently shake the tubes by hand and allow them to incubate at room temperature for 2 to 5 minutes.

9)  Pipet 500 ul of COLD (2 - 8C) Precipitating Solution to every tube (except TC). Gently shake the rack by hand to mix the contents. The assay tubes must be centrifuged within fifteen minutes of this addition.

10) Centrifuge for 30 minutes at 3000 x g in a centrifuge refrigerated to 2 - 5C.

11) Aspirate the supernatant from the tubes. Use care to aspirate all the liquid but not to aspirate any precipitate. Our reference laboratory recommends using a 9 inch pasteur transfer pipette as the aspirator tip. Alternatively, decant the supernatant from the assay tubes into a radioactive waste container. Drain and blot the inverted tubes on absorbent material to remove any remaining drops of supernatant before turning the tubes upright.

12) Count the activity of the precipitate in ALL tubes (including TC) for one minute using a gamma counter.

QUALITY CONTROL

1)  Control Sera:   The laboratory should run the three EPORIA Control Sera in duplicate with every assay. An acceptable range of values is printed on each Control Sera label. If the controls do not perform as expected, assay results are invalid.

2)  Total Count (TC):   Total Counts obtained should approximate the TC shown in the example data after adjustment for counter efficiency and radioactive decay.

3)  Non specific Binding (NSB):   The average counts per minute (CPM) of the NSB tubes divided by the average CPM of the Total Count tubes x 100 percent (NsB = avg CPM_NSB � avg CPM_TC � 100%). NsB should be less than 2% of avg CPM_TC.

4)  Maximum Binding (B_�):   The Net average CPM of the zero calibrators (�) divided by the average CPM of the total count tubes x 100 percent (B_� = Net avg CPM_� � avg CPM_TC � 100%). B_� should be 10% of avg CPM_TC.

DATA REDUCTION

We recommend calculating the results from a logit-log representation of the data.

1)  Calculate the average counts per minute for each Calibrator, Control, and unknown patient sample (avg CPM_a).

2)  Calculate the Net Counts (B_a) for each Calibrator, Control and unknown sample, by subtracting the average counts per minute of the NSB tubes (avg CPM_NSB) from the average counts per minute of the Calibrators,Controls and samples. B_a = avg CPM_a - avg CPM_NSB.

3)  Determine the percent Bound for each pair of tubes (B_a/B_�) by dividing the Net Counts of each set of tubes (B_a) by the Net Counts of the zero calibrator (B_�) and multiplying by 100 percent (%Bound_a = B_a/B_� � 100%). The Zero Calibrator (B_�) will therefore be 100 percent bound and all other Calibrators, Controls, and unknown samples will be less.

4)  On the logit-log graph paper supplied with this kit, plot the percent bound of the Calibrators (B_6.25/B�, B_12.5/B�, B_25/B�...) as a function of erythropoietin concentration. With a straight edge, draw a line most representing the slope and Y-intercept of the points. Erythropoietin concentrations of the unknowns and Control Sera can be interpolated from this line.

5)  Report values as milli Units erythropoietin/milliliter.

6)  Values lower than 3.3 mU/ml (the limit of detection of this assay) should be reported as less than 3.3 mU/ml.

7)  If any unknown sample assay is greater than the highest Calibrator, it should be diluted appropriately with Zero Calibrator/Diluent Buffer and reassayed. After determining the concentration of the diluted specimen, multiply the value by the appropriate dilution factor.

EXAMPLE DATA

LIMITATIONS

1)  No drugs have been tested for interference in this assay.

2)  The results of this assay should be used in conjunction with information available from clinical evaluations and other diagnostic procedures.

3)  Values lower than 3.3 mU/ml (the limit of detection of this assay) should be reported as less than 3.3 mU/ml.

4)  If any unknown sample assayed is greater than the highest Calibrator, the unknown sample should be diluted appropriately with Zero Calibrator/Diluent Buffer and reassayed.

EXPECTED VALUES

The serum erythropoietin levels of 146 normal individuals from the Houston, Texas area were determined using the EPORIA assay. The average EPO level was found to be 16.2 � 7.6 (1SD) mU/ml. The median value was 14.1 mU/ml. Individual values ranged from 6.0 mU/ml to 51.9 mU/ml. Other investigators have found similar mean EPO levels in their studies:

While some investigators report small differences between male and female EPO levels,^2,7 others find no differences related to sex.^10,21 Each testing laboratory should establish its own normal range.

DISEASE STATES

Erythropoietin is produced primarily in the kidney; the rate of production varies inversely with the oxygen content of the blood with which the kidney is perfused. Secondary anemia may result as a consequence of a failure of the kidney to synthesize an adequate amount of EPO.^11,12 In contrast, primary anemia caused by anything other than a problem with erythropoietin production will be associated with an increase in EPO to levels greater than present in normal persons.^10,13,14 This increase occurs because receptors in the kidney are producing extra EPO directed at the stimulation of erythropoiesis. An increase of red blood cell mass (i.e. primary erythrocytosis) may occur because of a fundamental defect of red blood cell precursors in which they proliferate to excess without stimulation from erythropoietin. This is the nature of the defect which occurs with polycythemia rubra vera. The increased red cell mass (and oxygen content of blood) suppresses EPO production and causes the serum levels of erythropoietin to be low.^2,7

In circumstances of secondary polycythemia or erythrocytosis, the red cell mass is increased because of increased levels of EPO in the plasma which stimulate production of red blood cell precursors. Disorders which may cause increased production of erythropoietin include diseases of the heart or lung which result in a decrease in the oxygen content of arterial blood; abnormalities of the hemoglobin molecule that cause oxygen to bind too tightly to hemoglobin, tumors of the kidney or other organ which produce erythropoietin.^2,8,9

PERFORMANCE DATA

1)  Sensitivity (Limit of Detection):    Sensitivity has been defined as the smallest value of erythropoietin that can be distinguished from the zero standard with 95 percent confidence. In a single assay, 8 zero calibrator (maximum binding) tubes were run along with a set of calibrators and controls. The mean and standard deviation of these zeros were calculated. The standard deviation was multiplied by two and subtracted from the mean. This resulting number was read from the standard curve and the apparent concentration of EPO regarded as the smallest amount detectable for this assay. This test was repeated on 3 separate occasions. The table below shows the results of this study.

2)  Specificity:    Potential assay interference by human: serum albumin, hemoglobin, transferrin, alpha-1-antitrypsin, gamma globulin, alpha-2-macro-globulin, alpha-1-acid glycoprotein, and bilirubin was investigated. A solution of each substance containing 100 ug/ml in Zero Calibrator/Diluent Buffer was assayed and no false positive levels of erythropoietin were detected. Erythropoietin was also spiked into these solutions at a concentration of 100 mU/ml, and the resulting assays indicated that these substances do not impair the ability of the assay to quantitate EPO.

3)  Prozone Reaction:    Samples containing up to 200 U/ml of erythropoietin in Zero Calibrator/Diluent Buffer were assayed and no evidence of a prozone reaction was detected.

4)  Correlation:    Seventy-eight serum samples were tested using the EPORIA assay and a commercially available enzyme immunoassay kit. Satisfactory correlation was demonstrated by linear regression analysis of the resulting data.

Y(EPORIA) = 0.92855 (commercial EIA) + 19.763 r² = 0.891

5)  Assay Precision:    Inter-assay and Intra-assay variance have been determined for four serum samples of various concentrations. Results are shown below:

Intra-Assay:

Eight replicates of each serum sample were tested in a single assay.

Inter-Assay:

Four serum samples were tested in duplicate in 8 assay runs over a period of two and one-half weeks.

6)  Spiking Recovery Study:

Three spiking solutions (A,B & C) consisting of Zero Calibrator/Diluent Buffer with erythropoietin added to specified concentrations were added in the Low, Medium and High control sera to make a 5 Percent concentration of the spiking solution. The expected value is determined by adding 5 percent of the value for the spiking solution to 95 percent of the value for the control sera.

7)  Parallelism:    Three serum samples were assayed undiluted as well as diluted with the EPORIA Zero Calibrator/Diluent Buffer. The observed and expected values are presented below in mU/ml. Linear regression analysis on these observed values versus expected concentrations yielded a correlation coefficient of 0.99 for each specimen tested. The results indicate that the standard curve is appropriate for determining patient sample erythropoietin levels.

REFERENCES

1. Erslev, A.J., Caro, J., Birgegard, G., Silver, R., Miller, O. The Biogenesis of Erythropoietin. Experimental Hematology 1980; Suppl 8:1-13.

2. Garcia, J.F., Ebbe, S.N., Hollander, L., Cutting, H.O., Miller, M.E., Cronkits, E.P.. Radioimmunoassay of Erythropoietin: Circulating Levels in Normal and Polycythemic Human Beings. Journal of Laboratory and Clinical Medicine 1982. 99:624-35.

3. 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; 242:1412-15.

4. Jacobson, L.O., Goldwasser, E., Fried, W., Pizak, L.F. The Role of the Kidney in Erythropoiesis. Nature 1957; 179:633-34.

5. Koury, S.T., Bondurant, M.C., Koury, M.J. Localization of Erythropoietin Synthesizing Cells in Murine Kidney by in-situ Hybridization. Blood 1988; 71:524-27.

6. Spivak, J.L. The Mechanism of Action of Erythropoietin. International Journal of Cell Cloning 1986; 4:139-66.

7. Rhyner, K., Egli, F., Niemoller, M., Wieczorek, A., Greminger, P., Vetter, W. Serum Erythropoietin Levels in Various Diseases. Nephron 1989; 51:Suppl 1:39-46.

8. Cotes, P.M., Dore', C.J., Liu Yin, J.A., Lewis, S.M., Messinezy, M., Pearson, T.C., Reid, C. Determination of Serum Immunoreactive Erythropoietin in the Investigation of Erythrocytosis. The New England Journal of Medicine 1986; 315:283-86.

9. Rege, A.V., Brookins, J., Fisher, J.W. A Radioimmunoassay for erythropoietin: serum levels in normal human subjects and patients with hemopoietic disorders. Journal of Laboratory and Clinical Medicine 1982; 100:829-42.

10. Mizoguchi, H., Ohta, K., Suzuki, T., Murakami, A., Ueda, M., Sasaki, R., Chiba, H. Basic Conditions for Radioimmunoassay of Erythropoietin, and Plasma Levels of Erythropoietin in Normal Subjects and Anemic Patients. Acta Haematologica Japonica 1987; 50: 15-24.

11. Caro, J., Brown, S., Miller, O., Murray, T., Erslev, A.J. Erythropoietin Levels in Uremic Nephric and Anephric Patients. Journal of Laboratory and Clinical Medicine 1979; 93:449-58.

12. McGonigle, R.J.S., Wallin, J.D., Shadduck, R.K., Fisher, J.W. Erythropoietin deficiency and inhibition of erythropoiesis in renal insufficiency. Kidney Int. 1984; 25:437-44.

13. Erslev, A.J., Wilson, J, Caro, J. Erythropoietin titers in anemic, non-uremic patients. Journal of Laboratory and Clinical Medicine. 1987; 109:429-33.

14. Erslev, A.J., Caro, J. The clinical usefulness of erythropoietin measurements. Fisher, J.W. editor, Kidney Hormones III. London: Academic Press 535-58 (1986).

15. Braunwald, E, Isselbacher, K.J., Petersdorf, R.G., Wilson, J.D., Martin, J.B., Fanci, A/S. (eds) Harrison's Principles of Internal Medicine, Tenth Edition, McGraw-Hill, pp 282-92 (1983).

16. Zanjani, E.D., Ascensao, J.L. Erythropoietin: A review. Transfusion 1989; 29:46-57.

17. Brown, B.A. Hematology, Principles and Procedures, Fourth Edition, Lea and Febiger, pp 3-9 (1984).

18. Koeffler, H.P., Goldwasser, E. Erythropoietin radioimmunoassay in evaluating patients with polycythemia. Annals of Internal Medicine 1981; 94:44-7.

19. Naets, J.P., Garcia, J.F., Toussaint, C., Buset, M., Waks, D. Radioimmunoassay of erythropoietin in chronic uremia or anephric patients. Scandinavian Journal of Hematology 1986; 37:390-94.

20. Urabe, A., Saito, T., Fukamachi, H., Kubota, M., Takaku, F. Serum erythropoietin titers in the anemia of chronic renal failure and other hematologic states. International Journal of Cell Cloning 1987; 5:202-8.

21. Schlageter, M.H., Toubert, M.E., Podgorniak, M.P., Najean, Y. Radioimmunoassay of Erythropoietin: Analytical Performance and Clinical Use in Hematology. Clinical Chemistry 1990; 36:1731-35.

Issue Date: 12/15/90

Revision date: April 24, 1995