Rapid Assay for Measurement of Serum Ferritin GHALEB A. SAAB, M.D., RALPH GREEN, M.D., AND WILLIAM H. CROSBY, M.D.
From the Division of Hematology-Oncology, Scripps Clinic and Research Foundation, La Jolla, California
Saab, Ghaleb A., Green, Ralph, and Crosby, William H.: Rapid assay for measurement of serum ferritin. Am J Clin Pathol 70: 275-279, 1978. The authors present a rapid modification of the tube immunoradiometric assay for serum ferritin quantitation. The method involves gentle rotatory mixing of the tube contents at ambient temperature and can be performed in a few hours. Variables affecting different stages of the assay were systematically investigated. The performance characteristics of the shortened assay in terms of reproducibility, sensitivity and recovery were then validated. Results with this method correlate well with those obtained by the conventional assay. (Key words: Ferritin; Immunoassay; Immunoradiometric assay.) SERUM FERRITIN DETERMINATION is carried out by a two-site immunoradiometric assay. In the first reaction, ferritin binds to rabbit antihuman ferritin coating the wall of a polystyrene tube; in the second reaction, purified l25 I-labeled antihuman ferritin completes the "sandwich." After each phase unbound reactants remaining in solution are washed away. The amount of radioactivity in the solid phase ("tube counts") is proportional to the amount of ferritin in the sandwich. The original method described by Addison and coworkers' and later modified by Miles and associates" is a three-day assay, in which each reaction, including the preparatory coating, requires 24 hours of incubation at 4 C. By enhancing the kinetics of the antigen-antibody reaction, the method described here shortens the time needed for each step, so that the entire assay takes only a few hours. The results show excellent correlation with the conventional method.
Tube Coating Polystyrene tubest 12 x 75 mm, are coated with rabbit antihuman hepatic ferritin antibodies by delivering 1 ml of antihuman ferritin serum (diluted 1:10,000 in 0.2 M N a H C 0 3 , p H 9.2) to the bottom of each tube. The styrofoam tube holder rotates for 30 minutes at room temperature and then is carefully removed from the rotator and placed flat on the bench. The antibody solution is aspirated and each tube is washed twice by delivering wash solution with an Oxford pipettor§ and simultaneously aspirating with vacuum suction. The first wash is with 2 ml BSA buffer (0.05 M Veronal buffer,pH 8.0, containing 4.5 g/1 NaCl, 100 mg/1 sodium azide, 1 g/1 bovine serum albumin11), and the second wash with 2 ml distilled water.
Materials and Methods For preparation of the human ferritin, the antihuman ferritin antibody, and the purified 125I-labeled anti-ferritin, we follow the methods described by Miles and associates." The source of ferritin used to establish the reference standard and to raise the antibody is human liver. Following ferritin extraction 2 and purification by passage through a 100 x 2.5-cm Sephadex G200 column, ferritin protein concentration is determined using the method of Lowry 8 with a human albumin protein standard.*
Reaction I
Received March 7, 1977; received revised manuscript May 6. 1977; accepted for publication May 6, 1977. Supported by Grant #AM 16452 from the National Institutes of Health, Bethesda, Maryland. Address reprint requests to Dr Green: Division of Hematology Oncology, Scripps Clinic and Research Foundation, 10666 N. Torrey Pines Road, La Jolla, California 92037. * Hycel Inc., Houston, Texas.
Standards: Hepatic ferritin standard is prepared from purified human hepatic ferritin by diluting it in 1:20 normal non-immune rabbit serum-BSA buffer." The highest concentration on the reference curve is 100 ng/0.2 ml. The other points on the curve are established by serial doubling dilutions using the same diluent so that the lowest concentration on the curve is 0.05 ng/0.2 ml. Volumes of 200 p\ of each standard solution are pipetted into the coated polystyrene tubes. Standard solutions are stored at - 2 0 C. t BBL 60448, Division of BioQuest, Cockeysville, Maryland. t Falcon Products, Los Angeles, California. § Scientific Products, McGaw Park, Illinois. 11 Sigma Chemical Co., St. Louis, Missouri.
0002-9173/78/0800/0275 $00.75 © American Society of Clinical Pathologists
275
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Each ferritin determination is run in duplicate. In the rapid method we rotate the tubes during each stage of the assay on a turntable rotator (21 cycles/min) in such a way that the long axis of the tubes remains perpendicular to the face of the turntable, which is positioned at approximately 120 degrees to the horizontal plane. The tubes fit in a square styrofoam holder (23.5 x 23.5 cm) adapted to fit on the tube rotator (Fig. l).t
276
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cal decline (the "hook" effect9) in the higher range (e.g., Fig. 2). For this reason, unknowns are treated in the manner described above. Results Factors Affecting the Assay We examined a number of variables for their effects on different phases of the assay. Unless otherwise stated, complete reference standard curves were run in these experiments (100 to 0.05 ng in serial doubling dilutions).
Unknowns Into duplicate tubes are pipetted 190-/xl volumes of non-immune rabbit serum-BSA buffer, followed by 10 fj.\ volumes of each undiluted unknown serum. Into a further set of duplicates, 190 /A1 non-immune rabbit serum-BSA buffer and 10 fj\ serum diluted 1:10 in nonimmune rabbit serum-BSA buffer are pipetted. Additional dilutions are used when there is a paradoxical increase of counts in the tubes containing diluted serum 4 or when tube counts of diluted and undiluted unknowns fall above the working range of the assay. A blank containing 200 /A1 of non-immune rabbit serum BSA buffer is prepared in place of the ferritin solution to serve as a control for nonspecific binding of radiolabeled antibody during the next stage of the assay. Then the tubes are rotated again in the styrofoam rack for 90 minutes, and washed once with 2 ml BSA buffer as before. Reaction II A 200-/U.1 volume of purified labeled ,25 I antihuman hepatic ferritin antibody containing 60,000-80,000 counts ml/min is delivered to every tube. The tubes are rotated for 90 minutes and then washed with 2 ml BSA buffer, aspirated, and radioactivity counted in a gamma scintillation spectrometer for 5 minutes. The dose-response curve usually begins to plateau in tubes containing 6.25 ng/0.2 ml and more, and then often shows a paradoxi-
(2) Effect of rotation. We examined the effect of rotation with tubes standing 90 minutes at room temperature, and with tubes rotating in the manner described. An appreciable decrease in dose response was demonstrated when tubes were not rotated (50% of control). In order to amplify the effect of rotation, an experiment was carried out in which a 15-minute incubation time was used for each reaction. Tubes that were not rotated showed a minimal dose-response curve, whereas tubes rotated showed a distinct dose response, although far less than the optimal response obtained at 90 minutes (Fig. 3). Finally, an experiment in which each reaction was carried out for 24 hours at 4 C was done. Rotated tubes showed a slight increase in dose response over the tubes that stood, but this difference was insignificant (3%). On the other hand, a striking increase in nonspecific binding in the blank tubes occurred when tubes rotated for that length of time. An increase in nonspecific binding also occurred when tubes rotated for longer than 90 minutes in each reaction, regardless of the temperature. (3) Effect of area of contact. The method of rotation results in a greater total area of contact between the liquid and the tube wall. Con-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
FIG. 1. Tube rotator, shown with the styrofoam tube holder in place.
(/) Effect of time of incubation. (a) Both reactions I and II were rotated at room temperature for 60 minutes, 90 minutes, 120 minutes, and 180 minutes each. Comparing the dose-response curves with the conventional 24-hour method (4 C) done at the same time, optimal binding occurs at 90 minutes' rotation for each reaction (Fig. 2). (b) Reaction I was rotated for 90 minutes at room temperature followed by reaction II for 24 hours (standing) at 4 C as in the conventional assay. In a simultaneous experiment, the reverse conditions applied, i.e., reaction I stood 24 hours at 4 C and reaction II rotated 90 minutes at room temperature. The two curves were similar in every respect, indicating that each reaction had proceeded optimally under the altered conditions of incubation.
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277
RAPID ASSAY FOR SERUM FERRITIN 7-j 6.»5-
FIG. 2. Effects of varying time of rotation on the hepatic ferritin dose-response curve. A = conventional method (standing, 24 hr, 4C); A = 180 minutes: • = 120 minutes; • = 90 minutes; O = 60 minutes (rotating, room temperature).
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sequently, the effect of a simple increase in the area of tube wall contact was examined. A threefold increase in the volume of reagents in each reaction, without altering the absolute amount of either ferritin or labeled antibody, did not increase the dose response. Next, we increased the area of contact without altering the volumes or concentration of any of the reagents. We compared the 12 x 75-mm tubes routinely used with (a) 10 x 75-mm polystyrene tubes, (b) 17 x 10mm polystyrene tubes, (c) inserting 10 x 75-mm tubes into 12 x 75-mm tubes after addition of the reagents in the conventional way. Again, there was no significant increase in dose response.
run in the cold. However, quality-control sera run at the same time gave comparable results.
(4) Effect of temperature. The effect of temperature was examined by carrying out the rapid assay at room temperature and at 4 C. Figure 4 shows that there was a considerable reduction in the efficiency of binding when the assay was
Validation of the Assay
(5) Tube coating. Tubes coated by standing for 12 hours at 4 C gave dose-response curves identical to those obtained using tubes that were coated by standing for 24 hours at 4 C. Moreover, 60-minute or 30-minute coating with rotation at room temperature was as efficient and again gave identical dose-response curves. However, 5-minute rotation of the polystyrene tubes with antiserum gave inadequate coating, as reflected by a very low efficiency of binding (less than 30%).
(I) Correlation with the conventional method. We measured the ferritin concentrations of 28 human sera and compared results with values obtained using the conventional 72-hour method. The results in Figure
Fie. 3. Effects of rotation on the hepatic ferritin dose-response curve. • = 15 min,utes, standing; O = 15 minutes, rotation; • = 90 minutes, rotation, room temperature.
0.05
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1.0 Ferritin (ng)
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1.0 Ferritin (ng)
SAAB, GREEN AND CROSBY
278
A.J.C.P. • August 1978
FIG. 4. Effects of temperature on the hepatic ferritin dose-response curve in the rapid assay (with rotation). • = room temperature; O = 4 C.
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(3) Sensitivity. The smallest amount of ferritin that could be detected in the assay was 0.05 ng, corresponding to a serum ferritin concentration of 5 ng/ml. This is comparable to the sensitivity of the original assay described by Miles and associates (0.03 ng).
• / / / / / / / /
140
(4) Recovery. To assess recovery, the following experiments were carried out: (a) A 190-/xl volume of each standard ferritin solution was added to (i) 10 /xl ferritin-deficient human serum or to (ii) 10 /u,l human serum immunochemically depleted of ferritin by repeated incubation of the serum in antihuman ferritin antibody-coated tubes till a zero dose response was obtained or to (iii) 10 /xl of normal serum (ferritin 58 ng/ml). Mean recoveries ranged from 95 to 105%.
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1 i 1 i r r i 20 40 60 80 100 120 140 Serum Ferritin (ng/ml) by Conventional Method
(2) Reproducibility. (a) Within-batch variation: We measured the ferritin contents of six different sera ten times in the same assay. The results are shown in Table 1. Coefficients of variation ranged from 1.8 to 7.8% and, in general, related inversely to the ferritin concentration. Tube position in the styrofoam holder had no effect on reproducibility. (b) Between-batch variation: We also performed duplicate determinations on ten different days on each of the same six sera assayed above, and results again showed good reproducibility (Table 2), with coefficients of variation ranging from 2.2 to 8.8%.
i
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Discussion 9
FIG. 5. Correlation between the results of rapid and conventional assays for serum ferritin in 28 sera. The broken line is the theoretical line of perfect correlation. The solid line is the regression line calculated by the method of least squares (y = 1.081x - 3.8; r = 0.979).
In existing serum ferritin assays, the reference standard consists of hepatic or splenic ferritin. Because of immunologic non-identity between serum ferritin and ferritin from these organs, the assay may not provide
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
5 demonstrate excellent correlation between methods (r = 0.979;/' < 0.001). Using the conventional method of Miles and associates,9 the 95% confidence ranges for serum ferritin concentration in our laboratory are 20250 ng/ml (geometric mean 86 ng/ml) for normal males and 10-200 ng/ml (geometric mean 40 ng/ml) for normal females.10 To test correlation in the low range of the assay, we also assayed six sera with ferritin concentrations less than 20 ng/ml by both methods. Agreement between the two methods was again excellent (r = 0.934; P < 0.001).
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Table I. Within-batch Reproducibility of the Rapid Serum Ferritin Assay
Table 2. Between-batch Reproducibility of the Rapid Serum Ferritin Assay
Ferritin (ng/ml)
Ferritin (ng/ml)
Coefficient
Coefficient of Variation
Serum No.
Mean*
Range
(%)
Serum No.
Mean*
Range
(%)
1 2 3 4 5 6
118.9 163.8 17.7 19.8 73.7 73.2
105-125 158-170 16-20 18-21 70-78 71-76
4.2 1.8 7.8 6.8 4.1 2.5
1 2 3 4 5 6
113.4 165.0 17.2 19.5 74.0 72.5
100-118 160-170 15-19 18-21 70-78 70-75
5.0 2.2 8.8 6.9 4.3 2.3
' Mean for ten determinations.
a somewhat lower efficiency of binding, results obtained on quality-control sera still correlated well with results of the conventional assay. The method of rotation we describe is inexpensive and saves considerable time. Shortening of the assay time brings serum ferritin determinations more within the scope of routine clinical testing for patients with suspected disorders of iron metabolism. The technic of rotation would be of value in similar assays where reduction in assay time is desirable. References 1. Addison GM. Beamish MR, Hales CN, et al: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol 25:326-329. 1972 2. Granick S: Ferritin. I. Physical and chemical properties of horse spleen ferritin. J Biol Chem 146:451-461. 1942 3. Green R, Saab GA, Watson LR. et al: Differences between liver and serum ferritin dose response curves suggest immunologic non-identity (abstr). Blood 48:990. 1976 4. Green R. Watson LR. Saab GA, et al: " N o r m a l " serum ferritin values in hemochromatosis — A caution (letter). Blood 50: 545-547. 1977 5. Hazard JT. Yokota M. Arosio P, et al: Immunologic differences in human isoferritins: Implications for immunologic quantitation of serum ferritin. Blood 49:139-145. 1977 6. Jacobs A, Worwood J: Ferritin in serum. N Engl J Med 292: 951-956. 1975 7. Lipschitz DA. Cook JD. Finch CA: A clinical evaluation of serum ferritin as an index of iron stores. N Engl J Med 290: 1213-1217. 1974 8. Lowry OH, Rosebrough NJ. Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265. 1951 9. Miles LEM. Lipschitz DA. Bieber CP, et al: Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 61:209-224, 1974 10. Ryan S. Watson LR. Tavassoli M. et al: Methods for establishing a working immunoradiometric assay for serum ferritin. Am J Hematol (in press)
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precise quantification of the serum ferritin concentration.:,,s Nonetheless, measurement of serum ferritin provides a useful laboratory index in the diagnosis of disorders of iron metabolism.6-7 However, a major drawback of the assay from the clinical standpoint is the length of time taken to complete it. The method described here shortens the time of the assay from three days to less than one day. We have shown that by the expedient of rotating tubes at ambient temperature the incubation time of each phase of the assay may be appreciably shortened without sacrificing binding efficiency or assay performance characteristics. In the modified assay, tube coating may be accomplished in 30 min, and antigen-antibody binding reactions in 90 min each. Further time may be saved when tubes are coated ahead of time and stored at 4 C. Tubes coated in this way may be used for as long as two weeks. The fundamental modification to the conventional technic described by Miles and associates is that of rotating the assay tubes in a novel fashion at room temperature rather than leaving them to stand at 4 C. This method of rotation has several advantages. It does not involve tube inversion, and this obviates the risks of radioactive spillage or contamination. Also, reproducible symmetry of contact between the liquid and tube wall is maintained. Our data indicate that this maneuver enhances the kinetics of the antigen-antibody reaction sufficiently for near-equilibrium binding to be attained within 90 min rather than 24 hours as in the conventional method. From our experiments, it appears that agitation rather than an increase in the area of contact is responsible for the enhancement of assay kinetics. We have also found that the assay may be carried out at 4 C rotating for the same length of time. Although this results in
Mean for duplicate determinations carried out on ten different days.
From the Division of Hematology-Oncology, Scripps Clinic and Research Foundation, La Jolla, California
Saab, Ghaleb A., Green, Ralph, and Crosby, William H.: Rapid assay for measurement of serum ferritin. Am J Clin Pathol 70: 275-279, 1978. The authors present a rapid modification of the tube immunoradiometric assay for serum ferritin quantitation. The method involves gentle rotatory mixing of the tube contents at ambient temperature and can be performed in a few hours. Variables affecting different stages of the assay were systematically investigated. The performance characteristics of the shortened assay in terms of reproducibility, sensitivity and recovery were then validated. Results with this method correlate well with those obtained by the conventional assay. (Key words: Ferritin; Immunoassay; Immunoradiometric assay.) SERUM FERRITIN DETERMINATION is carried out by a two-site immunoradiometric assay. In the first reaction, ferritin binds to rabbit antihuman ferritin coating the wall of a polystyrene tube; in the second reaction, purified l25 I-labeled antihuman ferritin completes the "sandwich." After each phase unbound reactants remaining in solution are washed away. The amount of radioactivity in the solid phase ("tube counts") is proportional to the amount of ferritin in the sandwich. The original method described by Addison and coworkers' and later modified by Miles and associates" is a three-day assay, in which each reaction, including the preparatory coating, requires 24 hours of incubation at 4 C. By enhancing the kinetics of the antigen-antibody reaction, the method described here shortens the time needed for each step, so that the entire assay takes only a few hours. The results show excellent correlation with the conventional method.
Tube Coating Polystyrene tubest 12 x 75 mm, are coated with rabbit antihuman hepatic ferritin antibodies by delivering 1 ml of antihuman ferritin serum (diluted 1:10,000 in 0.2 M N a H C 0 3 , p H 9.2) to the bottom of each tube. The styrofoam tube holder rotates for 30 minutes at room temperature and then is carefully removed from the rotator and placed flat on the bench. The antibody solution is aspirated and each tube is washed twice by delivering wash solution with an Oxford pipettor§ and simultaneously aspirating with vacuum suction. The first wash is with 2 ml BSA buffer (0.05 M Veronal buffer,pH 8.0, containing 4.5 g/1 NaCl, 100 mg/1 sodium azide, 1 g/1 bovine serum albumin11), and the second wash with 2 ml distilled water.
Materials and Methods For preparation of the human ferritin, the antihuman ferritin antibody, and the purified 125I-labeled anti-ferritin, we follow the methods described by Miles and associates." The source of ferritin used to establish the reference standard and to raise the antibody is human liver. Following ferritin extraction 2 and purification by passage through a 100 x 2.5-cm Sephadex G200 column, ferritin protein concentration is determined using the method of Lowry 8 with a human albumin protein standard.*
Reaction I
Received March 7, 1977; received revised manuscript May 6. 1977; accepted for publication May 6, 1977. Supported by Grant #AM 16452 from the National Institutes of Health, Bethesda, Maryland. Address reprint requests to Dr Green: Division of Hematology Oncology, Scripps Clinic and Research Foundation, 10666 N. Torrey Pines Road, La Jolla, California 92037. * Hycel Inc., Houston, Texas.
Standards: Hepatic ferritin standard is prepared from purified human hepatic ferritin by diluting it in 1:20 normal non-immune rabbit serum-BSA buffer." The highest concentration on the reference curve is 100 ng/0.2 ml. The other points on the curve are established by serial doubling dilutions using the same diluent so that the lowest concentration on the curve is 0.05 ng/0.2 ml. Volumes of 200 p\ of each standard solution are pipetted into the coated polystyrene tubes. Standard solutions are stored at - 2 0 C. t BBL 60448, Division of BioQuest, Cockeysville, Maryland. t Falcon Products, Los Angeles, California. § Scientific Products, McGaw Park, Illinois. 11 Sigma Chemical Co., St. Louis, Missouri.
0002-9173/78/0800/0275 $00.75 © American Society of Clinical Pathologists
275
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Each ferritin determination is run in duplicate. In the rapid method we rotate the tubes during each stage of the assay on a turntable rotator (21 cycles/min) in such a way that the long axis of the tubes remains perpendicular to the face of the turntable, which is positioned at approximately 120 degrees to the horizontal plane. The tubes fit in a square styrofoam holder (23.5 x 23.5 cm) adapted to fit on the tube rotator (Fig. l).t
276
SAAB, GREEN AND CROSBY
A.J.C.P. • August 1978
cal decline (the "hook" effect9) in the higher range (e.g., Fig. 2). For this reason, unknowns are treated in the manner described above. Results Factors Affecting the Assay We examined a number of variables for their effects on different phases of the assay. Unless otherwise stated, complete reference standard curves were run in these experiments (100 to 0.05 ng in serial doubling dilutions).
Unknowns Into duplicate tubes are pipetted 190-/xl volumes of non-immune rabbit serum-BSA buffer, followed by 10 fj.\ volumes of each undiluted unknown serum. Into a further set of duplicates, 190 /A1 non-immune rabbit serum-BSA buffer and 10 fj\ serum diluted 1:10 in nonimmune rabbit serum-BSA buffer are pipetted. Additional dilutions are used when there is a paradoxical increase of counts in the tubes containing diluted serum 4 or when tube counts of diluted and undiluted unknowns fall above the working range of the assay. A blank containing 200 /A1 of non-immune rabbit serum BSA buffer is prepared in place of the ferritin solution to serve as a control for nonspecific binding of radiolabeled antibody during the next stage of the assay. Then the tubes are rotated again in the styrofoam rack for 90 minutes, and washed once with 2 ml BSA buffer as before. Reaction II A 200-/U.1 volume of purified labeled ,25 I antihuman hepatic ferritin antibody containing 60,000-80,000 counts ml/min is delivered to every tube. The tubes are rotated for 90 minutes and then washed with 2 ml BSA buffer, aspirated, and radioactivity counted in a gamma scintillation spectrometer for 5 minutes. The dose-response curve usually begins to plateau in tubes containing 6.25 ng/0.2 ml and more, and then often shows a paradoxi-
(2) Effect of rotation. We examined the effect of rotation with tubes standing 90 minutes at room temperature, and with tubes rotating in the manner described. An appreciable decrease in dose response was demonstrated when tubes were not rotated (50% of control). In order to amplify the effect of rotation, an experiment was carried out in which a 15-minute incubation time was used for each reaction. Tubes that were not rotated showed a minimal dose-response curve, whereas tubes rotated showed a distinct dose response, although far less than the optimal response obtained at 90 minutes (Fig. 3). Finally, an experiment in which each reaction was carried out for 24 hours at 4 C was done. Rotated tubes showed a slight increase in dose response over the tubes that stood, but this difference was insignificant (3%). On the other hand, a striking increase in nonspecific binding in the blank tubes occurred when tubes rotated for that length of time. An increase in nonspecific binding also occurred when tubes rotated for longer than 90 minutes in each reaction, regardless of the temperature. (3) Effect of area of contact. The method of rotation results in a greater total area of contact between the liquid and the tube wall. Con-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
FIG. 1. Tube rotator, shown with the styrofoam tube holder in place.
(/) Effect of time of incubation. (a) Both reactions I and II were rotated at room temperature for 60 minutes, 90 minutes, 120 minutes, and 180 minutes each. Comparing the dose-response curves with the conventional 24-hour method (4 C) done at the same time, optimal binding occurs at 90 minutes' rotation for each reaction (Fig. 2). (b) Reaction I was rotated for 90 minutes at room temperature followed by reaction II for 24 hours (standing) at 4 C as in the conventional assay. In a simultaneous experiment, the reverse conditions applied, i.e., reaction I stood 24 hours at 4 C and reaction II rotated 90 minutes at room temperature. The two curves were similar in every respect, indicating that each reaction had proceeded optimally under the altered conditions of incubation.
Vol. 70 • No. 2
277
RAPID ASSAY FOR SERUM FERRITIN 7-j 6.»5-
FIG. 2. Effects of varying time of rotation on the hepatic ferritin dose-response curve. A = conventional method (standing, 24 hr, 4C); A = 180 minutes: • = 120 minutes; • = 90 minutes; O = 60 minutes (rotating, room temperature).
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sequently, the effect of a simple increase in the area of tube wall contact was examined. A threefold increase in the volume of reagents in each reaction, without altering the absolute amount of either ferritin or labeled antibody, did not increase the dose response. Next, we increased the area of contact without altering the volumes or concentration of any of the reagents. We compared the 12 x 75-mm tubes routinely used with (a) 10 x 75-mm polystyrene tubes, (b) 17 x 10mm polystyrene tubes, (c) inserting 10 x 75-mm tubes into 12 x 75-mm tubes after addition of the reagents in the conventional way. Again, there was no significant increase in dose response.
run in the cold. However, quality-control sera run at the same time gave comparable results.
(4) Effect of temperature. The effect of temperature was examined by carrying out the rapid assay at room temperature and at 4 C. Figure 4 shows that there was a considerable reduction in the efficiency of binding when the assay was
Validation of the Assay
(5) Tube coating. Tubes coated by standing for 12 hours at 4 C gave dose-response curves identical to those obtained using tubes that were coated by standing for 24 hours at 4 C. Moreover, 60-minute or 30-minute coating with rotation at room temperature was as efficient and again gave identical dose-response curves. However, 5-minute rotation of the polystyrene tubes with antiserum gave inadequate coating, as reflected by a very low efficiency of binding (less than 30%).
(I) Correlation with the conventional method. We measured the ferritin concentrations of 28 human sera and compared results with values obtained using the conventional 72-hour method. The results in Figure
Fie. 3. Effects of rotation on the hepatic ferritin dose-response curve. • = 15 min,utes, standing; O = 15 minutes, rotation; • = 90 minutes, rotation, room temperature.
0.05
0.1
1.0 Ferritin (ng)
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1.0 Ferritin (ng)
SAAB, GREEN AND CROSBY
278
A.J.C.P. • August 1978
FIG. 4. Effects of temperature on the hepatic ferritin dose-response curve in the rapid assay (with rotation). • = room temperature; O = 4 C.
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(3) Sensitivity. The smallest amount of ferritin that could be detected in the assay was 0.05 ng, corresponding to a serum ferritin concentration of 5 ng/ml. This is comparable to the sensitivity of the original assay described by Miles and associates (0.03 ng).
• / / / / / / / /
140
(4) Recovery. To assess recovery, the following experiments were carried out: (a) A 190-/xl volume of each standard ferritin solution was added to (i) 10 /xl ferritin-deficient human serum or to (ii) 10 /u,l human serum immunochemically depleted of ferritin by repeated incubation of the serum in antihuman ferritin antibody-coated tubes till a zero dose response was obtained or to (iii) 10 /xl of normal serum (ferritin 58 ng/ml). Mean recoveries ranged from 95 to 105%.
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(2) Reproducibility. (a) Within-batch variation: We measured the ferritin contents of six different sera ten times in the same assay. The results are shown in Table 1. Coefficients of variation ranged from 1.8 to 7.8% and, in general, related inversely to the ferritin concentration. Tube position in the styrofoam holder had no effect on reproducibility. (b) Between-batch variation: We also performed duplicate determinations on ten different days on each of the same six sera assayed above, and results again showed good reproducibility (Table 2), with coefficients of variation ranging from 2.2 to 8.8%.
i
160
Discussion 9
FIG. 5. Correlation between the results of rapid and conventional assays for serum ferritin in 28 sera. The broken line is the theoretical line of perfect correlation. The solid line is the regression line calculated by the method of least squares (y = 1.081x - 3.8; r = 0.979).
In existing serum ferritin assays, the reference standard consists of hepatic or splenic ferritin. Because of immunologic non-identity between serum ferritin and ferritin from these organs, the assay may not provide
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
5 demonstrate excellent correlation between methods (r = 0.979;/' < 0.001). Using the conventional method of Miles and associates,9 the 95% confidence ranges for serum ferritin concentration in our laboratory are 20250 ng/ml (geometric mean 86 ng/ml) for normal males and 10-200 ng/ml (geometric mean 40 ng/ml) for normal females.10 To test correlation in the low range of the assay, we also assayed six sera with ferritin concentrations less than 20 ng/ml by both methods. Agreement between the two methods was again excellent (r = 0.934; P < 0.001).
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Vol. 70 . No. 2
Table I. Within-batch Reproducibility of the Rapid Serum Ferritin Assay
Table 2. Between-batch Reproducibility of the Rapid Serum Ferritin Assay
Ferritin (ng/ml)
Ferritin (ng/ml)
Coefficient
Coefficient of Variation
Serum No.
Mean*
Range
(%)
Serum No.
Mean*
Range
(%)
1 2 3 4 5 6
118.9 163.8 17.7 19.8 73.7 73.2
105-125 158-170 16-20 18-21 70-78 71-76
4.2 1.8 7.8 6.8 4.1 2.5
1 2 3 4 5 6
113.4 165.0 17.2 19.5 74.0 72.5
100-118 160-170 15-19 18-21 70-78 70-75
5.0 2.2 8.8 6.9 4.3 2.3
' Mean for ten determinations.
a somewhat lower efficiency of binding, results obtained on quality-control sera still correlated well with results of the conventional assay. The method of rotation we describe is inexpensive and saves considerable time. Shortening of the assay time brings serum ferritin determinations more within the scope of routine clinical testing for patients with suspected disorders of iron metabolism. The technic of rotation would be of value in similar assays where reduction in assay time is desirable. References 1. Addison GM. Beamish MR, Hales CN, et al: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol 25:326-329. 1972 2. Granick S: Ferritin. I. Physical and chemical properties of horse spleen ferritin. J Biol Chem 146:451-461. 1942 3. Green R, Saab GA, Watson LR. et al: Differences between liver and serum ferritin dose response curves suggest immunologic non-identity (abstr). Blood 48:990. 1976 4. Green R. Watson LR. Saab GA, et al: " N o r m a l " serum ferritin values in hemochromatosis — A caution (letter). Blood 50: 545-547. 1977 5. Hazard JT. Yokota M. Arosio P, et al: Immunologic differences in human isoferritins: Implications for immunologic quantitation of serum ferritin. Blood 49:139-145. 1977 6. Jacobs A, Worwood J: Ferritin in serum. N Engl J Med 292: 951-956. 1975 7. Lipschitz DA. Cook JD. Finch CA: A clinical evaluation of serum ferritin as an index of iron stores. N Engl J Med 290: 1213-1217. 1974 8. Lowry OH, Rosebrough NJ. Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265. 1951 9. Miles LEM. Lipschitz DA. Bieber CP, et al: Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 61:209-224, 1974 10. Ryan S. Watson LR. Tavassoli M. et al: Methods for establishing a working immunoradiometric assay for serum ferritin. Am J Hematol (in press)
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precise quantification of the serum ferritin concentration.:,,s Nonetheless, measurement of serum ferritin provides a useful laboratory index in the diagnosis of disorders of iron metabolism.6-7 However, a major drawback of the assay from the clinical standpoint is the length of time taken to complete it. The method described here shortens the time of the assay from three days to less than one day. We have shown that by the expedient of rotating tubes at ambient temperature the incubation time of each phase of the assay may be appreciably shortened without sacrificing binding efficiency or assay performance characteristics. In the modified assay, tube coating may be accomplished in 30 min, and antigen-antibody binding reactions in 90 min each. Further time may be saved when tubes are coated ahead of time and stored at 4 C. Tubes coated in this way may be used for as long as two weeks. The fundamental modification to the conventional technic described by Miles and associates is that of rotating the assay tubes in a novel fashion at room temperature rather than leaving them to stand at 4 C. This method of rotation has several advantages. It does not involve tube inversion, and this obviates the risks of radioactive spillage or contamination. Also, reproducible symmetry of contact between the liquid and tube wall is maintained. Our data indicate that this maneuver enhances the kinetics of the antigen-antibody reaction sufficiently for near-equilibrium binding to be attained within 90 min rather than 24 hours as in the conventional method. From our experiments, it appears that agitation rather than an increase in the area of contact is responsible for the enhancement of assay kinetics. We have also found that the assay may be carried out at 4 C rotating for the same length of time. Although this results in
Mean for duplicate determinations carried out on ten different days.
