The Measurement and Interpretation of Serum Ferritin*

DONALD T. FORMAN, P h .D. and SHARON L. PARKER, P h .D.!

Departments of Pathology b- Biochemistry, and \Medical Allied Health Professions, University of North Carolina, School of Medicine Chapel Hill, NC 27514

ABSTRACT

Determination of serum ferritin is an important means of assessing body iron stores. Trace amounts of ferritin normally present in serum are detecta ble by sensitive radioimmunoassay techniques or an enzyme immunoassay procedure. Ferritin normally accounts for no more than a very small fraction of the total iron in serum, but generally maintains a stable concentration that is proportional to the much larger pool of storage iron in tissues. The serum ferritin assay, in contrast to other measurements of iron status such as hemo globin, serum iron and iron-binding capacity, can distinguish differences in iron stores within the physiological range. In iron deficiency anemia, the concentration is below 10 ngper ml. Increased concentrations (above 200 ng per ml) are found in conditions with increased iron stores. The information it provides is similar to that obtained from bone-marrow aspirates stained for iron. In contrast to the percent transferrin-saturation and concentration of erythrocyte protoporphyrin, ferritin concentrations become abnormal be fore exhaustion of mobilizable iron stores and before the onset of anemia. Serum ferritin also provides a practical means of assessing new programs of iron supplementation, since it reflects various degrees of iron deficiency and overload.

Introduction and the remainder as hemosiderin and iron containing enzymes.12 Ferritin is F erritin is the major iron storage protein found predominantly in the liver, spleen, of mammalian tissues.4 Of the four to five and bone marrow where it functions in grams of total body iron in the normal recycling iron for hematopoiesis.7 It is adult, approximately 65 percent is bound ubiquitously distributed in many other as hemoglobin, 15 to 20 percent as ferritin, tissues where its highly specialized pro three to five percent in bound myoglobin tein structure enables it to sequester and store iron intracellularly. Increases in Please send reprint requests to Dr. Forman at the Clinical Chemistry Laboratory, North Carolina cellular iron result in the rapid synthesis Memorial Hospital, Chapel Hill, NC 27514. of apoferritin, whereas iron depletion 345 0091-7370/80/0700-0345 $00.90 © Institute for Clinical Science, Inc. 346 FORMAN AND PARKER syndromes result in the mobilization of anemia from the various anemias of iron from its storage proteins with a sub chronic diseases. sequent decrease in tissue ferritin. Ferritin consists of a large spherical shell Methodological Aspects of of 24 single protein subunits surrounding Serum Ferritin Assays an inner core of insoluble ferric phos Several procedures are available for the phate. The iron-free protein, apoferritin, immunological quantitation of serum has a reported molecular mass of about ferritin. These include a two-site im- 450,000, which increases about twofold munoradiometric assay,* radioimmuno when the protein is fully saturated with assay—competitive binding assay,f and iron. The iron appears to pass to and from an enzyme immunoassay procedure in the inner core through small channels in developm ent.2,19 the protein shell.11 Recent data indicate The principle of the modified two-site that the ferritin can exist in multiple immunoradiometric assay originally in molecular forms (isoferritins) in different troduced by Addison1 is a two-stage reac tissues.5 The various isoferritin molecules tion. Stage 1 involves the binding of may represent varying percentage com human serum ferritin to a solid phase positions of two types of subunits in the 24 (plastic beads) and antihuman ferritin. subunit structure of the protein. The more Stage 2 involves the binding of radio basic isoferritins (L subunit) predominate labelled (125I) antihuman ferritin with the in liver and spleen while the acidic forms insoluble antihuman ferritin complex (H subunit) are present in highest concen generated in Stage 1. The solid phase is tration in heart, kidney and certain malig then washed, counted in a gamma nant tissues. The physiological signifi counter, and the ferritin concentration is cance of these tissue isoferritins is not calculated by comparison to a standard clear; however, the analytical significance curve. may be very important because the isofer The radioimmunoassay technique is a ritins appear immunologically distinct,8 competitive binding assay which utilizes and these immunological differences in a precipitating antiserum reagent to sepa isoferritin populations may have impor rate antibody-bound tracer from unbound tant consequences. tracer. The procedure is based on the Small, but clinically important concen competitive binding principles of radio trations of ferritin are detectable in serum immunoassay as described by Yalow and in both normal and pathological condi Berson.20 Non-radioactive ferritin from tions,13 with the concentration of serum patient samples, ferritin standards, and ferritin being directly proportional to the controls compete with a constant amount level of body iron stores and the stainable of (125I) ferritin tracer for binding sites on iron in bone marrow.6 Consequently, the the ferritin antibody, which is held at a principal use of serum ferritin is in the limiting concentration. The amount of diagnosis and management of iron defi labelled ferritin tracer which will bind to ciency and iron overload. This non- the antibody is inversely proportional to invasive technique, which is relatively the amount of non-radioactive ferritin in simple and sensitive, can also provide the the assay tube. A precipitating reagent so clinician and patient more information lution containing a second antibody in a than serum iron levels, transferrin satura tion, hemoglobin, hematocrit, red-cell in * Fer-Iron, Ramco Laboratories, Inc., Houston, TX dicies, and erythrocyte protoporphyrin 77098. levels. The assay is particularly useful in t GammaDab-Ferritin RIA, Clinical Assays, differentiating true iron deficiency Cambridge, MA 02139. MEASUREMENT AND INTERPRETATION OF SERUM FERRITIN 3 4 7 polymer solution is used to separate the antibody with radioactive iodine. Intra antibody bound (125I) ferritin from un assay variation showed a reproducibility bound labelled ferritin by immuno of less than 11 percent.19 The sensitivity of précipitation. The antibody-bound and this technique appears sufficient for clini labelled ferritin is counted. A standard cal purposes. curve is prepared in a like manner and the Each type of assay has its own attributes concentrations of the patients samples are and problems with as large and complex determined from the standard curve. an antigen as ferritin.14 In addition, there The enzyme immunoassay tech is a specificity problem owing to the im nique2,19 for human serum ferritin munological differences in the tissue employs an antibody adsorbed on a solid ferritins used as immunizing agents or phase. Adsorbed antibody against human standards in the serum ferritin procedure. ferritin is first allowed to react with Since ferritin occurs in multiple molecu ferritin. A second antiferritin antibody, lar forms (isoferritins), and these can which is labelled with an enzyme, is change markedly during development, added. At least three enzymes, alkaline iron status, and in malignancy, these dif phosphatase, B-galactosidase, and horse ferences can have important conse radish peroxidase, have been used. The quences for the standardization and in binding of the enzyme labelled antibody terpretation of serum ferritin assays and to the ferritin alters the activity of the en for attempts to quantitate selectively dis zyme. The resultant enzyme activity is tinct isoferritin populations (figure 1). correlated to ferritin concentration by means of a standard curve. D iscussion This technique has the advantage of using stable reagents that can be stored for Ferritin in serum was first reported17 in many months at 4°C, and it eliminates the patients with liver damage in 1956. Its need for constant labelling of antigen or presence as a component of normal serum

100

K. \ \ 75 \ l\ \ \ \ \ \ \ F i g u r e 1. C r o s s « \ reactivity of ferritin an- S \ \ tiserum with human

has been established after the develop In normal individuals, the levels of ment of sensitive radioimmunological serum ferritin depend on age and sex. At techniques. birth, serum ferritin levels are approxi The positive correlation of serum mately 100 ng per ml.18 This level falls ferritin levels with body iron stores, as during the first few months of life, but assessed by marrow or liver biopsy Prus then returns to the 100 ng per ml range in sian Blue staining,6 has resulted in its in young adults.3 Serum ferritin levels can creased use as a diagnostic aid in asses also vary in females during the menstrual sing the physiological status of iron stores. cycle and pregnancy.1 The reference The mechanism of serum ferritin release range in norm al adult m ales is 15 to 200 ng is unknown. It may result from an active per ml with a mean of 90 ng per ml. secretory process or from normal cell Females have a lower range of 10 to 200 ng turnover or lysis. Several factors suggest per ml and a mean of 50 ng per ml.3 Values that serum ferritin differs from tissue in post-menopausal women and men tend ferritin8: (1) serum ferritin binds to Con- to converge and continue to increase conavalin A, whereas tissue ferritins have slightly with age. Serum ferritin levels little binding affinity; (2) most of the under 10 ng per ml are found in iron defi serum ferritin contains little ferric iron ciency and values in excess of3,000 ng per even though tissue ferritins may be satu ml are frequently found in iron overload. rated with iron; (3) serum ferritin consists Clinically, the principal use of serum largely, if not entirely, of the relatively ferritin is in the diagnosis and manage basic L subunit, but tissue ferritin con ment of patients with iron deficiency and tains both basic and acidic (H) subunits; iron overload. Iron deficiency is present and (4) approximately 25 percent of the in more than 10 percent of women in the liver ferritin is synthesized within childbearing age. It is convenient to clas membrane-bound polysomes. These dif sify iron deficiency into three stages of ferences in serum and tissue ferritins increasing severity. The first is iron deple might suggest an active secretory process. tion, which is defined as the absence of However, it cannot be ruled out that these free iron in the marrow. This early stage is differences in physiocochemical proper reflected by the absence of Prussian Blue ties are simply a matrix effect. stainable iron in the bone marrow or a fall in serum ferritin below 10 ng per ml. With TABLE I continuing iron loss, the transferrin sat

Laboratory Diagnosis of Iron Deficiency uration (serum iron/total iron-binding And Anemia of Chronic Disease capacity) falls and the second stage of iron deficient erythropoiesis develops. While Serum Iron there is some impairment of hemoglobin and Total Serum Marrow Iron-binding Heno- synthesis at this stage, resulting in hypo Ferritin Iron Capacity globin chromic microcytic red cells, the hemo globin concentration is maintained in the Iron deficiency 4- 4- and iron normal range. As red cell production is depletion further decreased, the final stage of iron Iron deficient + 4- erythropoiesis deficiency anemia occurs as reflected in a Iron deficiency 4- 4- + 44 significant fall in circulating hemoglobin. anemia Anemia of + + j . j. The laboratory features of the three stages chronic of iron deficiency are summarized in disease table I.

+ - Increased levels --- = No change There are several aspects regarding 4- = Decreased levels serum ferritin levels in iron deficiency MEASUREMENT AND INTERPRETATION OF SERUM FERRITIN 349 that should be emphasized. The first is trols. However, even under these condi that the level falls below 10 ng per ml in tions the correlation between marrow iron the earliest stage of iron depletion; how and serum ferritin is still valid. When iron ever, as the free iron stores become deficiency occurs in patients with infec exhausted, the level no longer reflects the tion or liver disease, a normal serum severity of the iron deficiency state. The ferritin can be found despite absent iron second point is that if iron deficiency stores. Serum ferritin assays have found anemia is not complicated by other clini an important role in monitoring iron bal cal disorders, serum ferritin is highly ance in patients with renal failure treated specific for iron depletion. In one study of by hemodialysis9 where iron deficiency 32 patients with iron deficiency anemia, commonly occurs as a result of blood loss. none had a value which exceeded 14 ng The inherited form of iron overload, per m l.13 idiopathic hemochromatosis, is quite rare Because serum ferritin is essentially as compared to iron deficiency.11 Clinical equivalent to a marrow examination for detection of this condition is important stainable iron, its diagnostic value is in because it can be effectively treated, and clinical situations where the inconven tissue damage from iron excess can often ience, expense, and discomfort of a mar be reversed. Preclinical iron overload has row examination is not desired. Another been difficult to detect in the past because application for the measurement of serum the available tests were unreliable or dif ferritin is in the anemic patient with labo ficult to perform. Tests which have been ratory evidence of iron deficient eryth- utilized include transferrin saturation ropoiesis (microcytic hypochromic ane (serum iron/total iron-binding capacity), mia and low transferrin saturation or w hich is the least invasive, b u t it is a poor serum iron/total iron-binding capacity). measure of body iron stores. Other tests This hematologic picture occurs not only that have been employed include urinary in true iron deficiency but also in clinical iron excretion following desferroxamine, disorders such as chronic infection, in which is a complex technique, and liver flammation, or malignancy where there is biopsy for stainable iron. decreased iron release from the re Serum ferritin measurements can play a ticuloendothelial (RE) cell.11 This anemia major role in the early detection of idio of chronic disease is distinguished from pathic hemochromatosis.15 In patients true iron deficiency by an increase rather with proven disease, the level is invari than a decrease in marrow iron, and the ably greater than 2,000 ng per ml, and thus serum ferritin rises in parallel.11 Bone provides a distinct separation from normal marrow sampling to exclude a defect in individuals. RE iron release in patients w ith suspected Once iron storage disease is diagnosed, iron deficiency anemia may not be neces serum ferritin has been found to be useful sary when serum ferritin is less than 10 ng in monitoring iron stores during therapeu p er ml. tic phlebotomy. There is an orderly de There are certain disorders in which the cline in the level during treatment which serum ferritin concentration is dispropor reaches the low range of normal when ex tionately increased in relation to marrow cess iron has been removed. Transferrin iron stores (e.g., patients with infection or saturation is not usually helpful for this inflammation13 and patients with acute purpose because the levels often remain or chronic liver disease16). In these in elevated in these patients despite normal stances, serum ferritin at each level of body iron stores. Serum ferritin, on the marrow iron was increased about three other hand, can detect precisely the fold, as compared with hospitalized con therapeutic end-point. 3 5 0 FORMAN AND PARKER

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