Annals of Clinical & Laboratory Science 32:287-291 (2002)
© 2002 Association of Clinical Scientists
Filter Paper Collection of Plasma for IGF-I Test in Patients with Acromegaly
Ta-Jen Wu1,
Eugene Hsin Yu1,
Lu Song2,
Robert L. Taylor2 and
Pai C. Kao2
1 Section of Endocrinology and Metabolism, Department of Internal Medicine, National Cheng Kung University Hospital, Taiwan, ROC
2 Division of Clinical Biochemistry and Immunology, Mayo Clinic, Rochester, Minnesota
Address correspondence to Pai C. Kao, Ph.D., Emeritus Office, Plummer N10, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; e-mail: kao.pai{at}mayo.edu.
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Abstract
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The concentration of insulin-like growth factor I (IGF-I) was determined in paired specimens of liquid plasma and of plasma that had been applied to a filter paper disk, dried, and eluted; the measurements were performed by an immunochemiluminometric assay (ICMA). The 129 paired assays showed good correlation: y = 1.04x + 4.99; R2 = 0.934, where y is the result on dried samples and x is that on liquid samples. In 18 patients with acromegaly who lived on an island off the West Pacific Rim, plasma specimens were collected, applied to filter paper, dried, and mailed to a laboratory in the Upper Midwest of the United States. IGF-I was eluted from the filter paper disks and measured by ICMA. IGF-I concentrations ranged from 101 to 254 ng/ml in 11 patients with newly diagnosed but untreated acromegaly, 138 to 503 ng/ml in 4 patients with recurrent acromegaly, and 27 to 88 ng/ml in 3 patients with surgically cured acromegaly. These data demonstrate that plasma specimens of patients with acromegaly can be collected on filter paper disks and mailed to a laboratory on a distant continent for measurement of IGF-I. Such use of laboratory resources in one continent to help another continent, "Laboratories Without Borders," is somewhat analogous to the program "Doctors Without Borders."
(received 11 January 2002; accepted 29 January 2002)
Keywords: acromegaly, plasma transport, filter paper, IGF-I, immunochemiluminometry
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Introduction
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Increased serum concentration of growth hormone is pathophysiologically the cardinal feature of acromegaly. Growth hormone stimulates the secretion of insulin-like growth factor I (IGF-I) by the liver. IGF-I is bound to its major binding protein IGFBP-3 [1,2]. Serum IGF-I has a longer half-life than growth hormone [3,4]. Serum IGF-I is uniformly increased in untreated cases of acromegaly [5,6]. Measurement of IGF-I in serum or plasma is more precise and cost-effective than that of growth hormone because the growth hormone levels tend to fluctuate. For diagnosis of acromegaly, measurement of total IGF-I seems to be the most sensitive parameter [7]. Mean serum growth hormone levels and single serum IGF-I levels are reliable biochemical predictors of the adequacy of surgical treatment [8]. Similar findings were obtained by Kao et al [9,10] using an in-house procedure in which plasma IGF-I was first dissociated by acid, extracted with a C-2 cartridge, and measured by radioimmunoassay.
For countries in which the plasma IGF-I test is unavailable, adoption of a method for collecting plasma and shipping it over long distances without the need for solid CO2 would overcome the logistical problems of specimen shipping. It is relatively impractical to ship frozen plasma specimens from a distant clinical site. The most economical way is to spot plasma onto a paper disk, mail the dry disk to a distant laboratory, and then elute the plasma from the disk for measurement. In a pilot study, plasma specimens collected from subjects in Inner Mongolia were processed in this fashion and IGF-I was satisfactorily measured by radioimmunoassay [11]. In the current study, we used a nonradioisotopic immunochemiluminometric assay, which further reduced the cost of the assay, to test the feasibility of IGF-I assays for the diagnosis of acromegaly by mailing dried samples from a distant site. Our goal is to establish a program in which the resources of one area of the world can help another area, creating so-called "Laboratories Without Borders," similar to the program "Doctors Without Borders."
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Materials and Methods
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Eighteen patients (6 men, 12 women; age 32–68 yr) with acromegaly were registered at the National Cheng Kung University Hospital, Tainan, Taiwan. They included 11 patients with untreated active acromegaly, 4 with recurrent acromegaly, and 3 with surgically cured acromegaly. Fifteen patients (11 with untreated active acromegaly, 4 with recurrent acromegaly) were evaluated and the diagnosis was confirmed by 3 tests, including the insulin hypoglycemia test, L-dopa test, and thyrotropin-releasing hormone test. In 12 of these patients, the results of all tests were positive; in the other 3 patients, at least 2 tests yielded positive results.
Plasma specimens (100 µl) from the 18 patients were applied to Whatman filter paper (GF/DVA), dried, and mailed to the Mayo Clinic laboratory in Rochester, MN, USA. The paper spot with the specimen was cut out, soaked in 2.0 ml of 75% acetonitrile and 1% trifluoroacetic acid mixture, and incubated for 2 hr on a shaker to extract IGF-I. The supernatant was transferred to a plastic tube and dried overnight. To the dried residue, 500 µl of 12.5% hydrochloric acid/87.5% ethanol was added, mixed, incubated for 15 min, and centrifuged to remove any precipitate. The supernatant (250 µl) was neutralized with 100 µl of 0.855 M Tris base, shaken with a vortex mixer, and recentrifuged.
The supernatant (20 µl) was assayed by an IGF-I immunochemiluminometric assay. For preparation of the signal antibody, an affinity-purified IGF-I goat antibody (R&D Systems, Minneapolis, MN) was labeled with acridinium ester and prepared in-house. The signal antibody was diluted with assay buffer (0.04 M sodium phosphate, pH 7.4) to 1.2 x 106 relative light units (RLU)/200 µl and incubated either with 20 µl of IGF-I standards (0.666–21.3 ng/ml, Amgen, Thousand Oaks, CA) or 20 µl of plasma extract, and with monoclonal anti-IGF-I antibody (Biodesign, Saco, ME), 0.5 mg/50 µl. At the same time, a bead (Hoover, 1/4-in. diameter, grade 2, frosted) coated with sheep antimouse antibody (Biodesign, #W992728) was added, and the tubes were incubated for 3 hr at room temperature. The beads were washed with phosphate buffered saline (PBS) containing 0.1% Tween 20. After washing, the chemiluminescence was measured by a luminometer. This assay is similar in principle to our rapid PTH assay [12].
Paired plasma aliquots were assayed for IGF-I by the routine "wet" procedure for liquid plasma and by the dried disk procedure in specimens from 129 patients (69 male, age 0.5–77 yr; 60 female, age 1–88 yr) who were registered at the Mayo Clinic. In the routine procedure, 100 µl of plasma was mixed with 500 µl of 12.5% hydrochloric acid/87.5% ethanol mixture, incubated for 15 min, and centrifuged to dissociate IGF-I from its binding proteins and to precipitate the binding proteins. After centrifugation, 250 µl of the supernatant was neutralized with 100 µl of 0.855 M tris base, vortexed, and centrifuged. The supernatant (20 µl) was assayed for IGF-I by the immunochemiluminometric assay. The correlation between the paired results obtained with liquid plasma and the dried disk specimens was measured by Pearson’s correlation test.
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Results
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Liquid and dried specimens.
For 129 plasma specimens that were assayed by both the liquid and dried disk procedures, the correlation of IGF-I results was good: y = 1.04x + 4.99; R2 = 0.934, where y is the result using a dried disk and x is the result using a liquid sample (Fig. 1
).
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Fig. 1. Correlation of insulin-like growth factor-I (IGF-I) concentrations assayed in 129 plasma specimens that were dried on filter paper disks, versus paired specimens of "wet" (liquid) plasma.
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Taiwanese patients with acromegaly.
The results of IGF-I assays using the dried plasma samples from the 18 Taiwanese patients were all consistent with their clinical diagnoses (Fig. 1
). In 11 patients with newly diagnosed acromegaly, plasma IGF-I levels ranged from 101 to 254 ng/ml (upper limit of the reference range = 100 ng/ml). The 4 patients with recurrent acromegaly had plasma IGF-I levels from 138 to 503 ng/ml. The 3 patients with surgically cured acromegaly had plasma IGF-I levels from 27 to 88 ng/ml, which were within the reference range.
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Discussion
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The accepted laboratory criteria for cure of acromegaly are normalization of IGF-I and growth hormone concentrations in serum or plasma after an oral load of glucose. Plasma IGF-I is a stable marker for acromegaly and its measurement is clinically more useful than measurement of serum growth hormone for the follow-up of acromegaly. Determination of free IGF-I offered no advantage over that of total IGF-I [7]. Plasma IGF-I, but not IGFBP-3, is a suitable biochemical parameter for screening of acromegaly [13].
Measurement of plasma IGF-I is a low-volume test that is available in only a few medical centers; it is not cost-effective to have it widely available in hospitals or clinics. In a pilot study, plasma specimens from young, healthy individuals were collected on filter paper in Inner Mongolia and mailed to Rochester, MN, USA for IGF-I assay [11]. The previous study indicated that specimens from patients with acromegaly could be dried on filter paper and satisfactorily shipped for IGF-I assay. Using dried specimens on filter paper avoided the logistical problem of shipping frozen specimens. In the present study, we demonstrated that plasma IGF-I levels in patients with acromegaly can be assessed correctly by the filter-paper collection and transport method.
Plasma IGF-I level remains increased at 1 day after successful removal of a pituitary tumor and by 4 days postoperatively, the plasma IGF-I level decreases to the normal range [9]. Thus, it is necessary to wait at least 4 days after removal of pituitary tumor to collect samples for determining the success of the operation. If there is no recurrence, the plasma IGF-I value remains normal for up to 23 yr [9]. We speculate that other peptide hormones also can be measured by the filter-paper collection and transport technique; however, further evaluation studies need to be done.
In conclusion, plasma specimens from patients with acromegaly can be collected on filter paper, dried, mailed, and assayed at a distant laboratory for measurement of IGF-I. The method potentially allows access to the plasma IGF-I assay throughout the world. This study shows that "Laboratories Without Borders" is a feasible program.
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Fig. 2. Plasma insulin-like growth factor (IGF-I) levels in 18 Taiwanese patients with acromegaly. The plasma specimens were dried on filter paper disks and transported by mail to the USA, where the IGF-I was eluted from the disks and assayed by chemiluminometry.
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References
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- Wass JA. Growth hormone, insulin-like growth factor-I and its binding proteins in the follow-up of acromegaly. J Endocrinol 1997;155(Suppl 1):S17–S19.[Abstract/Free Full Text]
- Barkan AL. New options for diagnosing and treating acromegaly. Cleve Clin J Med 1998;65:343, 347–349.[Medline]
- Veldhuis JDP, Blizzard RM, Rogol AD, Martha PM, Jr., Kirkland JL, Sherman BM. Properties of spontaneous growth hormone secretory bursts and half-life of endogenous growth hormone in boys with idiopathic short stature. J Clin Endocrinol Metab 1992:74:766–773.[Abstract]
- Miell JP, Taylor AM, Jones J, Buchanan CR, Rennie J, Sherwood R, Leicester R, Ross RJ. Administration of human recombinant insulin-like growth factor-I to patients following major gastrointestinal surgery. Clin Endocrinol (Oxford) 1992;37:542–551.[Medline]
- Bates AS, Evans AJ, Jones P, Clayton RN. Assessment of GH status in acromegaly using serum growth hormone, serum insulin-like growth factor-1 and urinary growth hormone excretion. Clin Endocrinol (Oxford) 1995;42:417–423.[Medline]
- Stoffel-Wagner B, Springer W, Bidlingmaier F, Klingmuller D. A comparison of different methods for diagnosing acromegaly. Clin Endocrinol (Oxford) 1997;46:531–537.[Medline]
- Arosio M, Garrone S, Bruzzi P, Faglia G, Minuto F, Barreca A. Diagnostic value of the acid-labile subunit in acromegaly: evaluation in comparison with insulin-like growth factor (IGF) I, and IGF-binding protein-1, -2, and -3. J Clin Endocrinol Metab 2001;86:1091–1098.[Abstract/Free Full Text]
- Kaltsas GA, Isidori AM, Florakis D, Trainer PJ, Camacho-Hubner C, Afshar F, Sabin I, Jenkins JP, Chew SL, Monson JP, Besser GM, Grossman AB. Predictors of the outcome of surgical treatment in acromegaly and the value of the mean growth hormone day curve in assessing postoperative disease activity. J Clin Endocrinol Metab 2001;86:1645–1652.[Abstract/Free Full Text]
- Kao PC, Tateishi K, Abboud CF, Zimmerman D, Randall RV, Li CH. Assay of somatomedin C by cartridge extraction prior to radioimmunoassay with antiserum developed against synthetic somatomedin C. Ann Clin Lab Sci 1988;18:120–130.[Abstract]
- Kao PC, Laws ER Jr., Zimmerman D. Somatomedin C/insulin-like growth factor I levels after treatment of acromegaly. Ann Clin Lab Sci 1992;22: 95–99.[Abstract]
- Song L, Eastvold M, Kao PC. Comparison of dried and liquid plasma for IGF-I determination: impact on shipping and sampling IGF-I specimen (abstract). Ann Clin Lab Sci 1997;27:463.
- Kao PC, van Heerden JA, Taylor RL. Intraoperative monitoring of parathyroid procedures by a 15-minute parathyroid hormone immunochemiluminometric assay. Mayo Clin Proc 1994;69:532–537.[Medline]
- van der Lely AJ, de Herder WW, Janssen JA, Lamberts SW. Acromegaly: the significance of serum total and free IGF-I and IGF-binding protein-3 in diagnosis. J Endocrinol 1997;155(Suppl 1):S9–S13.[Abstract/Free Full Text]
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Abstract
Introduction
