User`s Manual and Instructions
Contents
1. OF RESULTS 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 Calibrators A D E and F Assign the concentration for each calibrator stated on the vial in mU 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 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 the penultimate 2 to the highest calibrator i e Calibrator E EPO concentrations above the concentration of the penultimate calibrator in the example shown below as 120 mU mL should be interpolated using the 405 nm reading Automated Method 4 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 Ca2. a C gt Biocnaine wwwbiochaineom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com User s Manual and Instructions Human EPO Erythropoietin ELISA Kit Specific quantitative assay for the determination of erythropoietin in serum Catalog Number Z6040001 INTENDED USAGE The BioChain 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 chains The recombinant EPO is a good substitute for the native protein for use in an immunoassay 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 oxygenation There is evidence indicating that the protein on these cells which detects oxygen saturation of t
3. 3 2 04 523 Control 1 0 046 0 044 0 045 lt 156 Control 2 0 649 0 626 0 638 184 Patient Sample 1 0 000 __ 0 000 lt 156 Patient Sample 2 0 007 0 007 lt 156 Patient Sample 3 0 023 0 023 lt 156 Patient Sample 4 0 14 0 14 lt 156 Patient Sample 5 1 161 1 161 302 For samples with concentrations lt the concentration of Calibrator E e g 156 mU 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 QUALITY CONTROL Control samples 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 m
4. 7 352 359 Jacobson L O Goldwasser E Fried W Pizak L F The Role of the Kidney in Erythropoiesis Nature 1957 179 633 634 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 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 Erslev A J Caro J Birgegard G Silver R Miller O The Biogenesis of Erythropoietin Experimental Hematology 1980 Suppl 8 1 13 Spivak J L The Mechanism of Action of Erythropoietin Int J Cell Cloning 1986 4 139 166 Erslev A J Erythropoietin New Eng J Med 1991 324 1339 1344 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 99 624 635 Wild D editor The Immunoassay Handbook Stockton Press 1994 p 428 Wide L Bengtsson C Birgegard G Circadian Rhythm of Erythropoietin in Human Serum Br J Haematol 1989 72 85 90 F 753 3UMRevC Z6040001UC Active Date 06252013 a C gt Biocnaine wwwpbiochaineom 12 13 14 15 16 17 18 19 20 21 Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Cahan C Decker M J Arnold J L Washington L H Veldhuis J D Goldwasser E Stro
5. 88 762 2568 510 783 5386 cs bioChain com www bioChain com F 753 3UMRevCG Z6040001UC Active Date 06252013
6. It has been reported that serum samples clotted at room temperature 22 C to 28 C caused a decrease in EPO value as assessed by radioimmunoassay of about 30 over clotting on ice Then the serum should be promptly separated preferably in a refrigerated centrifuge and stored at 15 C or lower Serum samples may be stored up to 24 hours at 2 8 C Serum samples frozen at 15 C are stable for up to 12 months Do not store samples in self defrosting freezers Avoid repeated freezing and thawing of samples For long term storage of samples it is recommended that samples should be aliquoted into sample tubes or vials prior to freezing Prior to use allow all specimens to come to room temperature 22 C to 28 C and mix by gentle inversion or swirling Avoid grossly hemolyzed or grossly lipemic samples REAGENT PREPARATION AND STORAGE Store all kit components at 2 8 C except the Wash Concentrate 1 All reagents except the calibrators kit controls and the Wash Concentrate are ready to use Store all reagents at 2 8 C except the Wash Concentrate which should be kept at room temperature 22 C to 28 C until dilution to avoid precipitation 2 For Zero Calibrator Calibrator A reconstitute vial with 4 mL of distilled or deionized water and mix For each of the non zero calibrators Calibrator B through F and kit controls 1 and 2 reconstitute each vial with 2 mL of distilled or deionized water and mix Allow the vials to stand for 10 minutes
7. ade 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 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 F 753 3UMRevC Z6040001UC Active Date 06252013 a C S Biocnaine wwwpiochaineom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com antibodies Nonetheless there can be no assurance that the heterophile interference has been completely eliminated Therefore it is recommended that at least three dilutions ot any elevated and or suspect positive results be assayed to detect nonparallelism compared to reference standards Because results obtained with one commercial EPO assay may differ significantly from those obtained with any other it is recommended th
8. and then mix thoroughly by gentle inversion to insure complete reconstitution Use the calibrators and controls as soon as possible upon reconstitution Freeze 15 C the remaining calibrators and controls as soon as possible after use Standards and controls are stable at 15 C for 6 weeks after reconstitution with up to 3 freeze thaw cycles when handled as recommended in Procedural Notes section 3 ELISA Reagent A Wash Concentrate Mix contents of wash concentrate thoroughly If precipitate is present in the Wash Concentrate due to storage at lower temperature such as 4 C dissolve by placing the vial in a 37 C water bath or oven with swirling or stirring Add wash concentrate 30 mL to 570 mL of distilled or deionized water and mix The diluted working wash solution is stable for 90 days when stored at room temperature ASSAY PROCEDURE 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 2 Pipet 200 uL 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 3 Add or dispense 25 uL of Reagent 1 Biotinylated Antibody into each of the wells which already contain the calibrators controls and samples 4 Add or dispense 25 uL of Reagent 2 Enzyme Labeled Antibody into each of the same wel
9. at any serial testing performed on the same patient over time should be performed with the same commercial EPO test This test may not be sufficiently sensitive to consistently discriminate abnormally low EPO values from normal levels of EPO Lower EPO levels than expected have been seen with anemias associated with the following conditions rheumatoid arthritis acquired immunodeficiency syndrome cancer and ulcerative colitis sickle cell disease and in premature neonates After allogeneic bone marrow transplant impaired erythropoietin response may delay erythropoietin recovery 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 altitudes EXPECTED VALUES EPO levels were measured in 120 apparently normal individuals in the U S with the BioChain 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 Further the EPO values do not appea
10. h gloves and eye protection and appropriate protective clothing Any spill should be wiped immediately with copious quantities of water Do not breath vapor and avoid inhalation 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 ELISA 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 SAMPLE COLLECTION AND STORAGE The determination of EPO should be performed on human serum To assay the specimen in duplicate 400 uL of human serum is required It is highly recommended that the specimen be collected between 7 30 a m to 12 00 noon because F 753 3UMRevC Z6040001UC Active Date 06252013 94 gt sai A BioChain Ss wwthiochaincom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com diurnal variation of erythropoietin has been reported in literature Collect whole blood without anticoagulant and allow blood to clot between 2 8 C if possible
11. he blood is a heme containing moiety As the pO2 of the plasma a function of the hematocrit decreases EPO concentration will increase There are also observations suggesting that normally there is an inverse correlation between serum EPO levels and red blood cell mass 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 EPO Low concentrations of EPO may give an early warning of kidney transplant rejection 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 apliasia 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 EPO Secondary polycythemias which are also characterized by an increase in t
12. he 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 BioChain EPO ELISA has a calculated sensitivity of 1 1 mU mL Hence patient sample results below 1 1 mU mL should be reported as Less than 1 1 mU mL Precision and Reproducibility The Intra assay precision of the BioChain EPO ELISA Test was calculated from 22 replicate determinations on each of the two samples F 753 3UMRevG Z6040001UC Active Date 06252013 C3 Biocnaine wwwbiochaincom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Intra Assay Variation Coefficient of Variation The inter assay precision of the BioChain EPO ELISA Test was calculated from data on two samples obtained in 22 different assays N Coefficient mU E of Variation Specificity and Cross Reactivity Cross reactivity in the EPO was studied by the addition of various substances to the Zero Calibrator Calibrator A c t Amount of Crossreactant rossreactan Added Fume Teter Human iin enconjugne Inter Assay Variation Human emozione Has Alp Global Human a 1 Acid Glycoprotein Human Gamma Globulin ACTE intact molecule ammo acid sequencel 39 Human Alpha Macroglobulin 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 t
13. he statistical limits of intraassay variation Recovery Various amounts of EPO were added to four different patient sera to determine the recovery The results are described in the following table F 753 3UMRevC Z6040001UC Active Date 06252013 d C gt Biocnaine wwwbiochaincom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com s Endogenous EPO Expected Measured R ears EPO added Value ery ample mU mL mU mL mU mL i Linearity of Patient Sample Dilutions Parallelism Three patient serum samples were diluted with Calibrator A Zero Calibrator Results in mU mL are shown below High Dose Hook Effect The BioChain EPO ELISA kit has exhibited no high dose hook effect in standard diluent spiked with 200 000 mU mL of EPO Additionally three samples with known high EPO values 1 920 mU mL 1 520 mU mL and 966 mU 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 REFERENCES 1 2 ooN 10 11 Sawyer S T Krantz S B Sawada K Receptors for Erythropoietin in Mouse and Human Erythroid Cells and Placenta Blood 1989 74 103 109 Imai N Kawamura A Higuchi M et al Physicochemical and Biological Comparison of Recombinant Human Erythropoietin with Human Urinary Erythropoietin J Biochem 1990 10
14. he 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 treatment Some tumors produce EPO and in these cases EPO may be used as a tumor marker to monitor the effectiveness of treatment PRINCIPLE OF THE TEST The BioChain 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 Streptavidin Well Biotinylated Anti EPO mouse monoclonal EPO HRP conjugated Anti EPO mouse monoclonal In this assay calibrators controls or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidincoated 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 co
15. hl K P Diurnal Variations in Serum Erythropoietin Levels in Healthy Subjects and Sleep Apnea Patients J Appl Physiol 1992 72 2112 7 Goldwasser E and Sherwood J B Annotation Radioimmunoassay of Erythropoietin Br J Haematol 1981 48 359 63 Kricka L J Human Anti Animal Antibody Interferences in Immunological Assays ClinChem 1999 45 942 956 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 Jelkmann W Renal Erythropoietin Properties and Production Rev Physiol Biochem Pharmacol 1986 104 139 215 Cotes M P Anomalies in Circulating Erythropoietin Levels Annals of NY Acad Sci 1994 718 103 9 Wintrobe s Clinical Hematology ninth edition edited by Lee G R Bithell T C Foerster J Athens J W Lukens J N Lea amp Febiger Philadelphia 1993 Fairbanks V Q amp A CAP Today Nov 1996 pg 88 Spivak JL Erythrocytosis Hematology Basic Principles and Practice editors Hoffman R Benz EJ Jr Shattil SJ Furie B Cohen Hu Silberstein LE 1995 Chapter 37 484 491 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 ORDERING INFORMATION BioChain Institute Inc 39600 Eureka Dr Newark Phone Fax E mail Website CA 94560 USA 8
16. ilized synthetic h EPO Lyophilized Zero calibrator is a buffered 1x 4mL for A 0 mU mL protein solution and all other calibrators consist of synthetic h EPO 1 165 the zero B in buffered protein solution These standards have been calibrated against calibrator C Refer to vial labels the World Health Organization erythropoietin 1 international standard D for exact recombinant DNA derived EPO 87 684 Each calibrator contains the 1x2 mL for E concentration preservative ciprofloxacin hydrochloride all other F calibrators CTRL Controls 1 amp 2 Lyophilized 2 Levels Synthetic h EPO 1 165 in 1x2 mL per Refer to vial labels for a buffered protein solution Each control contains level exact ranges the preservative ciprofloxacin hydrochloride MATERIAL AND EQUIPMENT REQUIRED BUT NOT PROVIDED e Microplate reader capable of reading at 450nm and 405nm Microplate washer if washer is unavailable manual washing is acceptable Precision Pipettors to deliver 25 200 100 and 150 pL Optional A multi channel dispenser or a repeating dispenser for 25 100 and150 uL Timer capable of 2 minute accuracy Distilled or Deionized water Orbital rotator or shaker WARNINGS AND PRECAUTIONS FOR USERS For in vitro diagnostic use Potential Biohazardous Material Caution Stopping Solution consists of 1 N Sulfuric Acid This is a strong acid Although diluted it still must be handled with care It can cause burns and should be handled wit
17. l the analyst or technician has gained sufficient experience as evidenced by the coefficient of variation duplicate being less than 10 except for the values below the 2 non zero lowest standard and the ability to obtain results for the kit controls within the suggested acceptable ranges The samples should be pipetted into the well with minimum amount of airbubble 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 gt 494 mU mL Reagents from different lot numbers must not be interchanged If preferred mix in equal volumes in sufficient quantities for the assay Reagent 1 Biotinylated Antibody and Reagent 2 Enzyme Labeled Antibody in a clean amber bottle The combined reagent is stable for seven 7 days when stored at 4 C Then use 50 uL of the mixed antibody into each well This alternative method should replace Step 3 and 4 to be followed with the incubation When mixing avoid splashing of reagents from wells This will affect assay precision and accuracy CALCULATION
18. librators A D E and F Sample Data at 450 nm raw A U readout against distilled or deionized water F 753 3UMRevC Z6040001UC Active Date 06252013 49 gt sai P BioChain wwwiochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Microplate Well 1 Reading 2 Reading Average EPO Absorbance Absorbance Absorbance mIU mL Unit Unit Unit Calibrator A 0 006 0 006 0 006 0 Calibrator B 0 094 0 092 0 093 10 3 Calibrator C 0 232 0 219 0 226 24 8 Calibrator D 0 509 0 474 0 492 48 Calibrator E 1 918 1 799 1 859 156 Control 1 0 171 0 170 0 171 18 2 Control 2 2 27 2 20 2 24 184 Patient Sample 1 0 012 0 012 1 1 Patient Sample 2 0 031 0 031 3 2 Patient Sample 3 0 089 0 089 9 6 Patient Sample 4 0 508 0 508 50 1 Patient Sample 5 3 283 3 283 gt 156 Because the Concentration of these samples are gt the Concentration of Calibrator E e g 156 mU mL it is recommended to use the data obtained at 405 nm as shown in Sample Data at 405 nm in the table below Sample Data at 405 nm raw A U readout against distilled or deionized water Microplate Well 1 Reading 2 Reading Average EPO Absorbance Absorbance Absorbance mIU mL Unit Unit Unit Calibrator A 0 0 0 0 Calibrator D 0 14 0 13 0 135 48 Calibrator E 0 538 0 508 0 523 156 Calibrator F 2 06 2 0
19. ls 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 22 28 C 5 First aspirate the fluid completely and then wash aspirate each well five 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 uL of the ELISA 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 s on an orbital shaker or rotator set at 170 10 rpm for 30 5 minutes at room temperature 22 28 C 8 Add or dispense 100 uL 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 uL of distilled or deionized water Read the plate again with the reader set to 405 nm against distilled or deionized water No
20. nverts the color to yellow F 753 3UMRevC Z6040001UC Active Date 06252013 49 gt sai A BioChain Ss wwthiochaincom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com 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 BioChain Inc 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 1 international standard 87 684 KIT COMPONENTS Kit Components Description Quantity RGT 1 Reagent 1 Biotinylated EPO Antibody mouse monoclonal anti human EPO containing 1 x 3 5 mL Proclin 300 as preservative RGT 2 Reagent 2 Peroxidase Enzyme labeled EPO Antibody mouse monoclonal anti 1x 3 5 mL human EPO RGT A Reagent A ELISA Wash Concentrate Saline with surfactant with the preservative 1x 30 mL ciprofloxacin hydrochloride RGT B Reagent B TMB Substrate tetramethylbenzidine 1 x 20 mL SOLN Stopping ELISA Stop Solution 1 N sulfuric acid 1x20 mL Solution PLA Microplate One holder with Streptavidin Coated Strips 12 x 8 well strips CAL Calibrators Lyoph
21. r 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 mU 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 PERFORMANCE CHARACTERISTICS Accuracy Eighty five 85 patient samples with EPO values ranging from 3 8 to 304 mU mL were assayed by the BioChain ELISA procedure and an ELISA EPO kit Linear regression analysis gives the following statistics BioChain ELISA 0 94 ELISA Kit 0 41 mU mL r 0 989 N 85 Sensitivity T
22. te 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 mU mL the exact concentration is printed on the vial label and will change slightly from one lot to another Hence patient samples with EPO gt 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 Patient and control samples should be read using the 450 nm for EPO concentrations up to the concentration of Calibrator E EPO concentrations reading above that of Calibrator E should be interpolated using the 405 nm reading F 753 3UMRevCG Z6040001UC Active Date 06252013 a C gt Biocnaine _ wwwbiochaineom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com 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 PROCEDURAL NOTES Samples that have values below the limit of detection 1 2 mU mL should be reported as lt 1 2 mU mL It is recommended that all calibrators controls and patient samples are assayed in duplicate unti
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