Stability of radioiodinated ferritin

261

Cfinicu Chimicu Acru, 168 (1987) 261-272 Eisevier CCA 03936

Stability of radioiodinated

ferritin

Ann M. Cove11 and James D. Cook Division of Hematoho,

Department of Medicine, University of Kansas Medicai Center, Kansas City, KS fUS.4)

(Received 20 April 1987; accepted 8 May 1987) Key words: Stability; Radioi~ination;

Fe&in

Summary Ferritin which had been radioiodinated using chloramine T exhibited marked instability on storage at 4’ C. Both [‘251]human liver and heart ferritins showed a similar rate of decline in immunoreactivity (tl,Z = 20-23 days) indicating that deterioration with storage was not a function of isoferritin composition. The decrease in radioactivity associated with ferritin was due not only to loss of “‘I from the molecule but also to protein degradation as shown by enzyme-linked i~unoassay and gel filtration. The degradation products had an M, of at least 69000 although low M, material could be identified by gel filtration when a marked reduction in immunoreactivity had occurred. Ferritin instability was much more pronounced than when other proteins such as immunoglobulin and albumin were radioiodinated with chloramine T. These observations indicate that when performing in vivo and in vitro studies with labeled ferritin, degradation of the protein during storage should be carefully monitored and the protein repurified before use.

Introduction The structure and bi~he~cal properties of the iron-storage protein ferritin have been studied extensively [1,2]. Apoferritin is a spherical protein with an M, of 480000, and is comprised of 24 subunits which form a hollow shell. Iron passes through specific channels in this shell and is stored internally. Human tissue ferritin is composed of two types of subunits designated as heavy (H) and light (L) with M,s of 21000 and 19000 respectively. Varying proportions of these two subunits give rise to a family of isoferritin molecules with different isoelectric points. Ferritin

Correspondence and reprint requests to: Ann M. Cove% Ph.D, Division of Hematology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, KS 66103, USA. ~9-8981/87/~03.50

0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

262

isolated from heart or placenta is more acidic on isoelectric focusing and contains a preponderance of H subunits whereas ferritin isolated from liver and spleen focuses at a more basic pH and contains mainly L subunits. Ferritin concentrations in serum correlate well with storage iron levels both in normal subjects and in patients with iron deficiency and iron overload [1,2]. Ferritin radiolabeled with 1251has been used in a variety of in vivo and in vitro studies of ferritin uptake, transport and metabolism in recent years. For example, kinetic studies have been performed in man to measure the in vivo turnover of circulating and tissue ferritins in plasma [3,4] and similar studies have been performed in animals [5-91. Radioiodinated ferritin has also been used to study binding of the protein to the surface of circulating lymphocytes and other cellular membranes (10-171. Indeed, radioferritin has been used to identify a putative ferritin receptor on rat hepatocytes, guinea pig reticulocytes and human placenta [11,13,14,16,17]. All of these investigations require a constant supply of highly immunoreactive labeled proteins. Radiolabeled ferritin has also been used extensively in radioimmunometric assays of the circulating protein [18-221. Many of the studies employing radiolabeled ferritin have used the chloramine T method for iodination [23]. Other methods that have been used include the conjugation method with N-succinimidyl-3-(4-hydroxy, 5-[‘251]iodophenyl)propionate [24], and occasionally the enzymatic lactoperoxidase technique [25,26], the iodine monochloride method [27] and the Iodogen technique [28]. The chloramine T method has a higher potential for damaging proteins because it employs strong oxidizing and reducing agents, although it remains a popular technique. In the present study we have examined the stability of tissue ferritins and other proteins radioiodinated with chloramine T using a variety of immunologic, chromatographic and electrophoretic techniques. Materials and methods

Human liver and heart ferritins were purified by the methods of Ryan et al. [29] and Jones and Worwood [30] respectively. Polyacrylamide gel electrophoresis (PAGE) on 5% (w/v) acrylamide gels [31] stained with coomassie blue showed that the purified proteins contained dimers. Isoelectric focusing (IEF) on 4% (w/v) acrylamide gels [32] confirmed that the liver ferritin preparation consisted only of relatively basic isoferritins and that heart ferritin was comprised mainly of acidic isoferritins. Ferritins (3-5 pg) were labeled with 1 mCi 1251(Amersham Intemational, Arlington Heights, IL, USA) by incubation for 45 s at room temperature with 20 pg chloramine T. Forty pg sodium metabisulphite was added followed by 80-160 pg potassium iodide, diluted in 0.5-l% (w/v) bovine serum albumin (BSA: fraction V powder, Sigma Chemical Co., St. Louis, MO, USA) in 0.05 mol/l phosphate buffer, pH 7.4. The immunoglobulin (IgG) fraction of rabbit anti-human liver ferritin was prepared in our laboratory and radioiodinated with chloramine T [23]. Human serum albumin (HSA) labeled with 1251 using the chloramine T technique (specific activity 1 pCi/mg) was obtained from Mallingkrodt, Inc. (St. Louis, MO, USA).

263

Salt Peak

Void Vdume

t 6

6 F

x

4

i 4

2

Fraction

Fig. 1. Typical elution profile of (‘2SI]liver ferritin on a Sepharose 6B column immediately after chloramine T iodination. Shaded area represents the proportion of radioactivity which could be precipitated with polyclonal antiferritin (W immunoreactivity). Fractions 22-27 were pooled and stored at 4OC for up to 3 mth.

Following iodination, [ 12’Ilferritin was separated from nonincorporated iodine and aggregated or damaged ferritin by gel filtration [3] on a column (25 cm x 1.3 cm) of Sepharose 6B (Pharmacia, Inc., Piscataway NJ, USA) by elution in 0.05 mol/l phosphate, pH 7.4, containing 0.5-l% (w/v) BSA. Fractions (0.5-l ml) were collected at 20-25 ml/h at room temperature. Radioactivity in the eluate was measured by counting fractions in a gamma counter. Normally two radioactivity peaks were obtained by Sepharose 6B gel filtration. A typical elution profile immediately after iodination is shown in Fig. 1. The first broad peak represented protein and the second unincorporated iodine (salt peak). Occasionally, a small initial peak was observed at the void volume. Following gel filtration, the most immunoreactive [1251]ferritin fractions were selected by immunoprecipitation with polyclonal anti-human liver ferritin [3] as follows. Ten-p1 samples of each fraction were mixed with 75-250 ~1 of carrier unlabeled human liver ferritin (100 pg/ml in 0.15 mol/l NaCl) and incubated with 150-500 ~1 of anti-ferritin (diluted optimally in 0.15 mol/l NaCl). The volume was made up to 0.4-l ml with 0.15 mol/l NaCl. Following an overnight incubation at 4” C, the samples were centrifuged at 1500 x g for 20 min and precipitates washed with 0.15 mol/l NaCl. The radioactivity recovered in the precipitates was expressed as a percentage of the total radioactivity added (% immunoreactivity). The maxi-

264

mum immunoreactivity occurred in the major protein peak containing ferritin, although values were always lower on the descending part of the peak (Fig. 1). Those labeled fractions showing the highest immunoreactivity (75-90s) were pooled and stored in glass vials or polypropylene tubes for up to three months. Similar recoveries of radioactivity were usually obtained with heart and liver ferritin when precipitated with polyclonal anti-human liver ferritin and carrier human liver ferritin. ‘*‘I incorporation into immunoreactive ferritin was approximately 9%. The specific activity was about 60 pCi/pg protein. When IgG was radioiodinated, unbound “‘1 was removed by chromatography on a column (26 cm x 0.8 cm) of Sephadex G50 (Pharmacia, Inc.). 125Iincorporation into IgG exceeded 45% and the specific activity was 5-10 pCC;/pg protein. All labeled proteins were stored at 4O C. The mobility of [ “‘1 ] ferritin was compared with that of the unlabeled protein on PAGE and IEF. Protein was detected by coomassie blue stain and autoradiography was carried out with X-Omat AR film (Eastman Kodak Company, Rochester, NY, USA). Purified [‘251]ferritin comigrated with the unlabeled protein. In addition there was little difference in mobility between high and low immunoreactive ferritin obtained immediately after iodination and gel filtration. PAGE showed that there was insignificant 1251 incorporation into BSA which had been present in gel filtration buffer after ferritin iodination. The proportion of radioactivity which remained associated with purified, stored, labeled ferritin was evaluated at weekly intervals by precipitation with anti-ferritin. Stored [‘251]ferritin samples were assayed for ferritin concentration each week with specific enzyme-linked immunoabsorbent assays (ELISA) [33] using monoclonal antibodies to human liver or heart ferritin. The stability of purified [‘251]ferritin, IgG and HSA was also examined by rechromatography of the proteins at intervals throughout the storage period. Gel filtration was performed on a column of Sepharose 6B (25 cm X 1.3 cm) or Sephadex G-100 (Pharmacia, Inc., 77 cm x 1.4 cm) at 20-25 ml/h to determine the relative distribution of radioactivity between protein and free iodine with time. M, marker proteins were used to standardize the columns. Immunoreactivity measurements were performed on fractions of labeled ferritin as described above. In addition, fractions were incubated with goat anti-BSA antibody (Sigma Chemical Co., St. Louis, MO) to determine the degree of incorporation of radioiodine into BSA which was present in the phosphate buffer used immediately after ferritin iodination. [‘251]ferritin instability was also studied by PAGE on 5% gels with autoradiography at approximately weekly intervals after iodination. Results

There was a striking reduction in immunoprecipitable radioactivity of stored liver and heart ferritin with time. The results of a typical study for both proteins are shown in Fig. 2. The t,,, for liver ferritin was 23 days and for heart ferritin 22 days. There was therefore no appreciable difference in instability between labeled liver and heart ferritin. In two additional studies with labeled heart ferritin, t,,, values of 20 and 21 days were obtained.

265

l 0

0 l 0

l

Fig. 2. Typical decline in immunoreactivity of [1251]liver(0) and [ “‘IJheart (0) ferritins with time as measured by precipitation with polyclonal anti-human ferritin. Each point is the mean of duplicate observations.

200

r --

l

a

Weeks

Fig. 3. Comparison between loss of immunoreactivity and immunoassayable [‘251]human heart ferritin with time. Immunoreactivity (0) was measured by precipitation with polyclonal anti-human ferritin and ferritin concentration (0) by a specific ELISA with monoclonal antibodies. A single preparation only is shown and each point is the mean .of duplicate or triplicate observations.

266

Day 1 Storew

32

40

4%

53

84

Fraction

Fig. 4. Change in f “2JT]human liver ferritin radioactivity eluticm profile with time. Ferritin was iodinated and separated from damaged protein and unincorporated “‘1 at day 0, then stored at 4’C. On days 1 and 26 an aliquot was rechromatographed on a Sepharose 6EI column. Shaded area represents the proportion of radioactivity which Could be precipitated with polyclona1 anti-f&tin (percent immunoreactivity). The elution positions of M, marker proteins are also shown from a separate rur~: blue dextran 2 x 106, ferritin 4NWOO,myoglobin 17000.

Parallel s&d&s 0x1 t&x &x& ferritin samples demons~at~ that the loss of i~unopr~ipitable radioa~ti~ty was accompanied by de~adation of the protein. The decline in heart and liver ferritin as measured by an ELISA was at least as rapid as the decline in immunoreactivity. A typical study with labeled heart ferritin is shown in Fig. 3, The t,,, for immunoreactivity was 21 days as compared with 13 days when the protein was measured by ELISA. In a second study with labeled . . heart ferntm (not shown) the tl,l for immunoreactivity was 20 days as compared with 15 days as measured by ELISA. There was a pronounced change in the distribution of radioactivity on Sepharase 6B gel filtration after storage of labeled liver ferritin for 26 days (Fig. 4). Most of the loss in i~~or~Gti~ty could be attributed to the release of free iodine from

Day 1 Storage

Day 26 Stwaqe

Fracthwr Fig . 5. Change in [‘251]humau liver ferritin radioactivity elutiou profile with time. Ferritin was iodinated and stored as described in legend to Fig. 4, then rechromatographed on a Sephadex G-100 column. Shaded area represents percent immunoreactivity. M, marker proteins are shown.

the protein (fractions 54-63). There was an increase in low immunoreactive material on the descending part of the protein peak (fractions 42-50) indicating that some damage to the molecule had occurred. Similar findings were observed when the labeled ferritin was examined with higher resolution on Sephadex G-100 (Fig. 5). The most immunoreactive ferritin fractions eluted at the void volume. After 26 days of storage, an additional peak of nonimmunoreactive protein eluted in fractions 32-39 where albumin with an M, of 69000 is typically seen. When [‘251fferritin was examined by PAGE and autoradio~aphy, no obvious difference was observed in the mobility between the stored, labeled protein and unlabeled ferritin. A much greater degree of protein degradation was observed in a stored preparation of heart ferritin in which immunoreactivity had fallen to less than 10% (Fig. 6). A large quantity of nonimmunoreactive protein was observed in a Sephadex G-100 elution volume corresponding to an M, of 17 000-69000. None of the material eluting between fractions 30-40 (Fig. 6) in the study could be precipitated with an antiserum against BSA. PAGE demonstrated that there was no in~~oration of “‘1 into BSA after 4 wk of storage of labeled ferritin in albumin buffer. There was a striking difference in the stability of labeled ferritin, IgG and HSA (Fig, 7). The most dramatic decline was observed with ferritin, with only 46% of the

269

radioactivity remaining associated with the protein five weeks following iodination. An intermediate loss was observed with labeled IgG which amounted to approximately 20% during the first 5 wk, although no further decline was observed in the subsequent 2 mth. Labeled HSA was highly stable, with more than 95% of the radioactivity remaining associated with the protein after 1 yr of storage at 4” C. Discussion

Several workers have commented on difficulties encountered when preparing radiolabeled ferritin due either to damage of the protein during iodination, or instability of the labeled product on storage [18-221. These prior studies have been concerned with the preparation of reagent for the radioimmunoassay of serum ferritin. Barnett and coworkers [19] found it necessary to purify the iodinated material carefully by chromatography to remove aggregated material and to isolate the monomer peak. They reported that the ‘251-labeled ferritin obtained in this manner was stable for at least 1 mth as judged by immunoreactivity. There was no improvement in stability when [ ‘251]ferritin was stored in the presence of disaggregating agents or at different temperatures. Luxton et al [22] reported a loss of immunoreactivity with a half-time of approximately 3 wk and Deppe and coworkers [21] observed variation in the rate of degradation among different batches of labeled ferritin. Degradation of ‘251-labeled ferritin does not necessarily affect the radioimmunoassay of the circulating protein. The main effect is to increase the nonspecific binding of [‘251]ferritin in the assay, with the result that the zero standard binding decreases with time. If the loss of immunoreactivity is due to a release of “‘1 from an intact protein, the performance of the assay would not be affected. We have added large amounts of free 1251 to labeled ferritin provided in commercially available kits and observed no significant change in the standard curves or quality control serum values. However, if the labeled protein is degraded with storage, some deterioration in assay performance is likely to occur. Several workers have emphasized the importance of repeating the iodination at frequent intervals. The stated shelf life of immunoassay kits for serum ferritin employing radiolabeled protein is usually 6 to 8 wk. Our focus in the present study was not on the initial damage to ferritin during iodination, but on the long term stability of highly immunoreactive material following its purification on Sepharose 6B. There was a pronounced loss of immunoreactivity with time (Fig. 2). Serial chromatographic studies demonstrated that this instability was due at least in part to release of 1251as measured by an increase in radioactivity eluting in the salt peak. Using a membrane with an M, cutoff of about 12000, approximately 15% of the total radioactivity was dialyzable within 1 wk of storage of labeled ferritin. To determine whether [‘251]ferritin protein was functionally intact, we performed serial immunoassays using monoclonal reagents and found an even greater rate of protein degradation than would have been predicted from the loss of immunoreactivity (Fig. 3). This type of instability will have a major effect on metabolic studies designed to examine the uptake and

270

transport of the labeled ferritin. It should be noted that unlabeled ferritin does not deteriorate with storage at 4” C for up to 9 mth [34]. We found no effect of isoferritin composition on the rate of loss of immunoreactivity. Interestingly, Hazard and coworkers encountered considerable difficulty in iodinating HeLa ferritin to the same specific radioactivity as liver ferritin. Although both HeLa ferritin and heart ferritin contain more acidic isoferritins, other differences may exist that explain our observation that the rate of deterioration of heart and liver ferritin on storage is similar. Ferritin damage from iodination and loss of immunoreactivity with time has been observed with ferritins from different tissues and species such as human spleen and plasma ferritin, and rabbit liver and kidney ferritin (Covell AM and Worwood M, unpublished findings). The mechanism for the progressive loss of immunoreactivity of labeled ferritin is not clear. It is possible that during iodination, a proportion of lz51 enters the protein shell and is subsequently released. Alternatively, 1251may be noncovalently bound to the surface of the protein and is then eluted. These explanations alone are unlikely because immunoassay measurements and gel filtration studies demonstrated a progressive structural alteration of the protein with time. The greatest proportion of damaged ferritin had an M, of about 69000, but in a preparation in which immunoreactivity was markedly reduced, low M, peptides appeared which could not be precipitated with polyclonal antiferritin. Electrophoretic studies demonstrated that the greatest proportion of radioactivity comigrated with unlabeled ferritin despite the shift in the elution profile on gel filtration with time. This indicates that damaged and non-damaged proteins had a similar net charge. Despite the appearance of a non-immunoreactive protein with a peak at about M, 69000, both electrophoretic analysis and immunologic measurements failed to demonstrate significant uptake of radioactivity by BSA in storage buffer. An important factor contributing to the instability of labeled ferritin is the iodination technique. Bolton and coworkers [18] compared 3 different methods for iodinating human spleen ferritin and observed that the stability of the labeled material prepared with oxidative methods such as chloramine T was substantially less than when a conjugation technique such as the Bolton-Hunter method was used. In the latter method, radioiodine is incorporated into proteins by conjugation of a radioiodinated moiety to the epsilon amino side chains of lysine residues. Labeled material prepared by the conjugation technique showed greater stability on storage, although no direct assays for degradation were performed as in the present study. Exposure of ferritin, for even brief periods, to strong oxidizing and reducing agents such as chloramine T and sodium metabisulphate may degrade the protein to peptides containing [‘251]tyrosine or histidine residues. This would not explain why ferritin degradation continued long after the offending chemicals had been removed. In our studies, an average of 0.5 radioiodine atoms were incorporated into each ferritin subunit, which is within the optimum range for many proteins. Comparisons with immunoglobulin and human serum albumin indicate that ferritin is much more susceptible to damage with chloramine T. The chloramine T technique of iodination has a number of important advantages. It is rapid, simple and relatively inexpensive. The conjugation procedure has the

271

disadvantage of producing at least one bulky 1251phenyl side chain which could interfere with receptor-mediated uptake of ferritin by cells and tissues. In vivo studies have already demonstrated that the clearance of chloramine T iodinated spleen or liver ferritin from plasma is not affected by the labeling procedure as long as highly immunoreactive protein is selected for injection [4,8]. In summary, we have demonstrated that ferritin radioiodinated by the chloramine T method is highly unstable and that this instability is associated not only with the loss of 12’1, but also with degradation of the protein. Ferritin is far less stable than other labeled proteins such as IgG and albumin. We see no reason to avoid using the chloramine T method of ferritin labeling for kinetic studies [3,4,8], but it is necessary to monitor degradation carefully. An ELISA technique may be more sensitive for detecting deterioration than immunoprecipitation. Because marked protein degradation may occur when immunoreactivity has fallen to less than lo-20%, new radiolabeled material should be prepared at 4-6 wk intervals. In addition, the labeled protein should be carefully repurified by gel filtration prior to use. Acknowledgement

We thank Dr. Ronald Weiner, Division of Nuclear Medicine, KUMC for his gift of [ ‘251]HSA. References 1 Theil EC. Ferritin: structure, function, and regulation. Adv Inorg Biochem 1983;5:1-38. 2 Jacobs A. Ferritin: an interim review. Curr Topics Hematol 1985;5:25-62. 3 Worwood M, Cragg SJ, Williams AM, Wagstaff M, Jacobs A. The clearance of 1311-human plasma ferritin in man. Blood 1982;60:827-833. 4 Cragg SJ, Covell AM, Bunch A, Gwen GM, Jacobs A, Worwood M. Turnover of r3’I-human spleen ferritin in plasma. Br J Haematol 1983;55:83-92. 5 Halliday JM, Mack U, Powell LW. The kinetics of serum and tissue fenitins: relation to carbohydrate content. Br J Haematol 1979;42:535-546. 6 Mack U, Cooksley WGE, Ferris RA, Powell LW, Halliday JW. Regulation of plasma ferritin by the isolated perfused rat liver. Br J Haematol 1981;47:403-412. 7 Frenkel EJ, Van Den Beld B, Marx JJM. Influence of heat-treatment on rabbit liver ferritin kinetics. Br J Haematol 1983;55:449-453. 8 Cove11AM, Worwood M. Turnover and tissue uptake of rabbit ferritin from rabbit plasma. Comp Biochem Physiol (B) 1984;77:829-834. 9 Borges HF. Goldstein C, Rim M, Michael AF. The glomerular mesangium: kinetics using radiolabeled ferritin and the effects of aggregated IgG. Clin Immunol Immunopathol 1984;33:80-86. 10 Moroz C, Lahat N, Biniaminov M, Ramot B. Ferritin on the surface of lymphocytes in Hodgkin’s disease patients: a possible blocking substance removed by levamisole. Clin Exp Immunol 1977;29:30-35. 11 Pollack S, Campana T. Immature red cells have ferritin receptors. Biochem Biophys Res Commun 1981;100:1667-1672. 12 Ulvik RJ. Relevance of ferritin-binding sites on isolated mitochondria to the mobilization of iron from ferritin. Biochim Biophys Acta 1982;715:42-51. 13 Mack U, Powell LW, Halliday JW. Detection and isolation of a hepatic membrane receptor for ferritin. J Biol Chem 1983;258:4672-4675.

272 14 Blight GD, Morgan EH. Ferritin and iron uptake by reticulocytes. Br J Haematol 1983;55:59-71. of ferritin-bearing peripheral mononuclear 15 Bluestein BI, Luderer AA, Hess D, et al. Measurement blood cells in cancer patients by radioimmunoassay. Cancer Res 1984;44:4131-4136. of the binding of ferritin to the rat 16 Mack U, Storey EL, Powell LW, Halliday JW. Characterization liver ferritin receptor. Biochim Biophys Acta 1985;843:164-170. 17 Takami, M, Mizumoto K, Kasuya I, Kino K, Sussman HH, Tsunoo H. Human placental ferritin receptor. Biochim Biophys Acta 1986;884:31-38. 18 Bolton AE, Lee-Own V, McLean RK, Challand GS. Three different radioiodination methods for human spleen ferritin compared. Clin Chem 1979;25:1826-1830. 19 Bamett MD, Gordon YB, Amess JAL, Mollin DL. Measurement of ferritin in serum by radioimmunoassay. J Chn Path01 1978;31:742-748. differences in human isoferritins: 20 Hazard JT, Yokota M, Arosio P, Drysdale JW. Immunologic implications for immunologic quantitation of serum ferritin. Blood 1977;139-145. 21 Deppe WM, Joubert SM, Naidoo P. Radioimmunoassay of serum ferritin. J Clin Path01 1978;31:872-877. 22 Luxton AW, Walker WHC, Gauldie J, Ah MAM, Pelletier C. A radioimmunoassay for serum ferritin. Clin Chem 1977;28:683-689. 23 Greenwood FC, Hunter WM, Glover JS. The preparation of 13’1 labelled human growth hormone of high specific radioactivity. Biochem J 1963;89:114-123. by conjugation to a 24 Bolton AE, Hunter WM. The labelling of proteins to high specific radioactivities ‘251-containing acylating agent. Application to the radioimmunoassay. Biochem J 1973;133:529-539. JJ. An enzymic method for the trace iodination of immunoglobulins and other proteins. 25 Marchalonis Biochem J 1969;113:299-305. BG. Enzymatic iodination of polypeptides with ‘25I to high specific activity. 25 Thorell JI, Johansson Biochim Biophys Acta 1971;251:363-369. 27 McFarlane AS. Efficient trace-labelling of proteins with iodine. Nature 1958;182:53. 28 Salacinski P, Hope J, McLean C, et al. A new simple method which allows theoretical incorporation of radio-iodine into proteins and peptides without damage. J Endocrinol 1979;81:131P. for establishing a working 29 Ryan S, Watson LR, Tavassoli M, Green R, Crosby WH. Methods immunoradiometric assay for serum ferritin. Am J Hematol 1978;4:375-386. M. An immunoradiometric assay for the acidic ferritin of human heart: 30 Jones BM, Worwood application to human tissues, cells and serum. Clin Chim Acta 1978;85:81-88. vol 1. London: Churchill 31 Worwood M. Serum ferritin. In: Cook JD, ed. Methods in hematology, Livingstone, 1980;59-89. 32 Wagstaff M, Worwood M, Jacobs A. Iron and isoferritins in iron overload. Clin Sci 1982;62:529-540. 33 Flowers CA, Kuizon M, Beard JL, Skikne BS, Cove11 AM, Cook JD. A serum ferritin assay for prevalence studies of iron deficiency. Am J Hematol 1986;23:141-151. Committee for Standardization in Hematology (Expert Panel on Iron). Preparation, 34 International characterization and storage of human ferritin for use as a standard for the assay of serum feritin. Clin Lab Haematol 1984:6:177-191.