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IMMUNORADIOMETRIC ASSAY FOR QUANTITATIVE DETERMINATION OF FERRITIN IN HUMAN SERUM. FOR IN VITRO DIAGNOSTIC USE ONLY CLINICAL APPLICATIONS The Ferritin molecule consists of 24 protein subunits, each of them with molecular weight of 20 kDa. Ferritin functions mainly as the intracellular site of iron storage (from which iron may be rapidly mobilized), thereby protecting cells from the toxic effects of unbound iron. Most Ferritin in the body is found in the liver cells as well as in the cells of the reticuloendothelial system of liver, spleen and bonemarrow. Small amounts are also found in the heart, pancreas and kidneys. Small but significant amounts of Ferritin are found in the human serum. The exact function of serum Ferritin is unknown but there is a well established correlation between serum Ferritin and the body's iron stores. Serum Ferritin testing thus represents the simplest and less invasive method in monitoring any iron store change in the body. Ferritin serum levels are also affected by body changes due to age as well as sex. Average Ferritin levels in normal subjects (slightly higher at birth) tend to decrease during childhood, until the age of puberty is reached. From then on, a progressive increase of the body iron stores is observed in males, with a proportional rise in serum Ferritin. Females in the reproductive age instead tend to have lower amounts with more stable values. A Ferritin rise is only observed after menopause. Ferritin levels below normal ranges clearly indicate a lack of iron and allow to differentiate iron deficiency anemia from other forms of anemia. An iron overload, either due to an increase in iron uptake (as in idiopathic hemochromatosis) or else due to multiple transfusions, causes serum Ferritin to rise, often beyond normal ranges. High Ferritin levels are also observed for other clinical conditions such as liver disease, infectious as well as inflammatory processes, leukemia, Hodgkin's disease and other forms of malignancy. PRINCIPLE OF THE ASSAY The present method employs two anti-Ferritin monoclonal antibodies which recognize two different epitopes of the molecule. One antibody is adsorbed in solid phase (coated tube), the other (labeled with iodine-125) is used as conjugate. The sample to be tested and the labeled antibody are incubated simultaneously in the coated tube. The amount of bound conjugate will thus be directly proportional to the antigen concentration in calibrators and samples. At the end of the incubation, the unbound material is removed by aspiration and washing. The radioactivity in the tubes is measured in a gamma counter. REAGENTS PROVIDED WITH THE KIT: PREPARATION AND STABILITY − The reagents are sufficient for 100 tubes. − Store the kit at 2-8°C. − The expiry date of each reagent is shown on the vial label. 1 - CT Coated Tubes: 100 tubes coated with mouse monoclonal anti-Ferritin antibody. Unused tubes should be stored at 2-8°C in the appropriate bag and accurately sealed. 2 - CAL Calibrators: 7 vials (1 mL) of Ferritin (human liver) in phosphate buffer at the following concentrations: 0, 5, 20, 50, 250, 1000 and 2000 ng/mL. Preservative: NaN3 (
− Orbital shaker, adjustable at 150 rpm. − Aspiration pump or automated tube washing device. − Scintillation gamma counter. − Millimetric graph paper. − Distilled H2O. RADIOACTIVE MATERIAL Radionuclide: 125 I ITALIAN DECREES (D.Lgs. 230/95 – D.Lgs. 241/00 art. 19) AND RULES OF PREVENTION AND SECURITY In order to obtain correct and reproducible results, the following rules must be observed: − Do not mix reagents of different lots. − Do not use reagents beyond their expiry date. − Use thoroughly clean glassware, free from metal ion contamination or oxidizing substances. − Use distilled water, stored in perfectly clean containers. − Carefully avoid any contamination among samples; for this purpose, disposable tips should be used for each sample and reagent. − Follow exact incubation times, as described in the "Assay Procedure". In order to reduce physical, biological and chemical risks, the following precautions must be observed: − Use protective individual articles (ex.: disposable gloves, lab coats, etc.) while handling radioactive materials and/or potentially infectious material as well as during the assay. − Do not pipette reagents by mouth. − Do not smoke, eat, drink or apply cosmetics during the assay. − All radiological work should be done in a designated area. − Radioactive materials should be stored in their original container in a designated area. − A record book for logging receipt and disposal of all radioactive materials should be kept. − Any radioactive spills should be taken care of immediately in accordance with established procedures. − All material of human origin used for the preparation of this kit was tested negative for HBsAg, anti- HIV and anti-HCV. Since no test at present can guarantee complete absence of these viruses, all samples and reagents containing biological material used for the assay must be considered potentially infectious. − Avoid splashing and aerosol formation; in such cases, carefully wash with a 3% sodium hypochlorite solution. Any cleaning material used for that purpose must be treated as potentially infectious and disposed accordingly. − The sodium azide used as preservative in some reagents may react with lead and copper plumbing; to prevent build-up of explosive metal azides, the reagents should be discarded by flushing the drain with large amounts of water. − The reagents for which a Safety Data Sheet is not supplied do not contain hazardous chemical substances or, if they do, these are below the concentration limits established by the Italian decree D.Lgs.285/98 in compliance with EEC directive 91/155. − According to Italian decree D.L. no. 22 dated 05.02.97, in compliance with EEC directives (91/156/EEC, 91/689/EEC, 94/62/EEC), all waste products originating from either manual and/or automated processing are classified as hazardous special waste material (European classification code 180103, infectious waste or potentially infectious waste). As such, they must be eliminated (by) delegating them to special enterprises (companies), qualified for waste collection and disposal. − All radioactive waste originating from either manual and/or automated processing, must be eliminated delegating them to special enterprises, qualified for waste collection and disposal, according to D.Lgs.241/2000. − Acquisition, storage, use and disposal of radioactive material (liquid and solid) are subjected to regulation and ordination of local authorities. Radioactivity contents to the reference date is shown on the kit external label. KP33CT – FERRITINA IRMA M99 – Rev.09 – 12/2004 – Pag. 9/32
Page 1 and 2: FERRITINA IRMA CT REF KP33CT 100 It
Page 3 and 4: DOSAGGIO IMMUNORADIOMETRICO PER LA
Page 5 and 6: − Ai sensi del D.L. italiano n. 2
Page 7: ACCURATEZZA L'accuratezza del metod
Page 11 and 12: PERFORMANCES OF THE ASSAY SPECIFICI
Page 13 and 14: IMMUN-RADIOMETRISCHER ASSAY ZUR QUA
Page 15 and 16: − Liegt kein Sicherheitsdatenblat
Page 17 and 18: 160.5 ± 8.8 5.4 10 c 1120 ± 91.0
Page 19 and 20: 6 - WASH Solution de Lavage (concen
Page 21 and 22: La lecture de la concentration des
Page 23 and 24: ENSAYO INMUNORADIOMÉTRICO PARA LA
Page 25 and 26: − La azida sódica contenida como
Page 27 and 28: a 27.6 ± 2.05 7.4 10 b 160.5 ± 8.
Page 29 and 30: RDATE Data di Riferimento / Referen
Page 31 and 32: Schema del Dosaggio, Assay Scheme,

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