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Technical Briefs |
a author for correspon
Serum human prolactin (PRL) is heterogeneous in molecular size, with the 23-kDa monomer being the predominant form in healthy subjects and patients with prolactinomas. From the point of view of molecular size, other circulating forms include the 50-kDa dimer (big-PRL) and the 150- to 170-kDa form (big-big-PRL, or macroprolactin) (1). Recent publications have associated asymptomatic hyperprolactinemia with a predominance of macroprolactin in the circulation; this occurrence appears to be more common than previously thought (2)(3)(4) and can have obvious practical implications. The finding of a predominance of macroprolactinemia can change the focus of the evaluation of a patient, with the possible avoidance of more sophisticated and expensive imaging studies.
We evaluated the polyethylene glycol (PEG) precipitation method to
screen for the presence of macroprolactinemia in a large series of
clinical samples. Serum PRL was measured by immunofluorometric assay
(IFMA; reference range, 2–15 µg/L; Delfia, Wallac Oy), and samples
with values 
30 µg/L were studied. The value of 30 µg/L or higher,
considered as overtly abnormal, was arbitrarily selected. To 250 µL
of serum, we added 250 µL of a 250 g/L PEG 6000 solution (in water,
kept at 4 °C), mixed them for 1 min with a vortex mixer, and
centrifuged them (9500g for 5 min at room temperature). PRL
was determined in the supernatant, using the same IFMA and the recovery
calculated on the basis of the original serum value. Reproducibility of
the PEG precipitation process was evaluated in four different serum
samples, studied seven times each, on different days and in different
assays. The following values were obtained: for a sample with PRL value
of 32 µg/L and mean recovery of 63%, the CV was 15%; for a sample
with PRL value of 45 µg/L and mean recovery of 83%, the CV was 7%;
for a sample with PRL of 68 µg/L and mean recovery of 47%, the CV
was 28%; and for a sample with PRL of 71 µg/L and mean recovery of
5%, the CV was 20%.
The gel filtration used Sephacryl S-200 (Pharmacia) packed in a
0.9 x 30 cm column previously calibrated with monomeric
I PRL (New England Nuclear); 0.5 mL of serum was
applied and eluted with sodium phosphate buffer (0.05 mol/L, pH 7.4),
and 0.7-mL (0.1 mL/min) aliquots were collected and analyzed for PRL.
The samples were classified as having a predominance of monomeric PRL
forms when the analysis of the area under the curve corresponding to
monomeric PRL represented >50% of the total area of PRL elution.
Among 19 228 consecutive clinical samples studied in 12 months, PRL
was 30 µg/L in 1279 (6.7%). Of these, 1220 were submitted to PEG
precipitation, and 171 samples were submitted to the chromatographic
study. They were selected to cover the whole range of recovery. The
values of the percentage of recovery plotted against the percentage of
the area under the curve that was classified as high molecular weight
PRL (macroprolactin) are depicted in Fig. 1
. On the basis of the data presented in Fig. 1
, recoveries of
65% were classified as predominantly monomeric, and recoveries of
30% as predominantly high molecular weight forms. Values between
30% and 65% were classified as indeterminate and submitted to
chromatography. The data also showed negative correlation
(r = -0.84) between the percentage of recovery and the
percentage of high molecular forms. Of the 1220 samples studied,
recoveries were 30% in 441 (36%), 65% in 607 (50%), and
30–65% in 172 (14%). The chromatographic study of these 172 samples
showed 72 (42%) with a predominance of high molecular weight forms.
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Our data support the findings of Bjoro et al. (5), who also reported a large proportion of macroprolactinemia in their clinical population. One of the reasons for our surprisingly high incidence of macroprolactinemia may be the kind of samples we receive. As a reference laboratory, we receive samples for confirmation of an unexpected high value detected elsewhere. The PEG precipitation technique that we use is a convenient and simple procedure to screen for the presence of macroprolactinemia. Recoveries >65% are indicative of the absence of, and those <30% indicative of the presence of, macroprolactinemia. Samples with recoveries between 30% and 65% need gel filtration chromatography to characterize the predominant molecular form. Our data, in spite of the special characteristics of the samples studied, stress the necessity that laboratories offering PRL assay service should include, in all reported increased PRL results, some form of study for the presence of macroprolactinemia, as already suggested by Lindstedt (6).
Acknowledgments
We thank the reviewer for constructive suggestions.
Footnotes
Endocrinology Section, Laboratório Fleury, Rua Cincinato Braga 282, 01333-910 São Paulo, SP, Brazil
2>dence: fax 55-11-287-2482, e-mail jose.vieira{at}fleury.com.br
References
The following articles in journals at HighWire Press have cited this article:
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  L. Beltran, M. N. Fahie-Wilson, T. J. McKenna, L. Kavanagh, and T. P. Smith Serum Total Prolactin and Monomeric Prolactin Reference Intervals Determined by Precipitation with Polyethylene Glycol: Evaluation and Validation on Common ImmunoAssay Platforms Clin. Chem., October 1, 2008; 54(10): 1673 - 1681. [Abstract] [Full Text] [PDF]
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 N Hattori, Y Nakayama, K Kitagawa, T Ishihara, Y Saiki, and C Inagaki Anti-prolactin (PRL) autoantibody-binding sites (epitopes) on PRL molecule in macroprolactinemia. J. Endocrinol., August 1, 2006; 190(2): 287 - 293. [Abstract] [Full Text] [PDF]
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L. Kavanagh, T. J. McKenna, M. N. Fahie-Wilson, J. Gibney, and T. P. Smith Specificity and Clinical Utility of Methods for the Detection of Macroprolactin Clin. Chem., July 1, 2006; 52(7): 1366 - 1372. [Abstract] [Full Text] [PDF]
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 A. Glezer, C. R. J. Soares, J. G. Vieira, D. Giannella-Neto, M. T. C. P. Ribela, V. Goffin, and M. D. Bronstein Human Macroprolactin Displays Low Biological Activity via Its Homologous Receptor in a New Sensitive Bioassay J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 1048 - 1055. [Abstract] [Full Text] [PDF]
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J. Schiettecatte, A. Van Opdenbosch, E. Anckaert, J. De Schepper, K. Poppe, B. Velkeniers, and J. Smitz Immunoprecipitation for Rapid Detection of Macroprolactin in the Form of Prolactin-Immunoglobulin Complexes Clin. Chem., September 1, 2005; 51(9): 1746 - 1748. [Full Text] [PDF]
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J. Gibney, T. P. Smith, and T. J. McKenna The Impact on Clinical Practice of Routine Screening for Macroprolactin J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 3927 - 3932. [Abstract] [Full Text] [PDF]
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J. G. H. Vieira, S. K. Nishida, M. T. Faria de Camargo, O. M. Hauache, R. Monteiro de Barros Maciel, and V. Guimaraes "MacroLH": Anomalous Molecular Form That Behaves as a Complex of Luteinizing Hormone (LH) and IgG in a Patient with Unexpectedly High LH Values Clin. Chem., December 1, 2003; 49(12): 2104 - 2105. [Full Text] [PDF]
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A. M. Suliman, T. P. Smith, J. Gibney, and T. J. McKenna Frequent Misdiagnosis and Mismanagement of Hyperprolactinemic Patients before the Introduction of Macroprolactin Screening: Application of a New Strict Laboratory Definition of Macroprolactinemia Clin. Chem., September 1, 2003; 49(9): 1504 - 1509. [Abstract] [Full Text] [PDF]
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 C. Mounier, J. Trouillas, B. Claustrat, R. Duthel, and B. Estour Macroprolactinaemia associated with prolactin adenoma Hum. Reprod., April 1, 2003; 18(4): 853 - 857. [Abstract] [Full Text] [PDF]
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R. Sapin and G. Kertesz Macroprolactin Detection by Precipitation with Protein A-Sepharose: A Rapid Screening Method Compared with Polyethylene Glycol Precipitation Clin. Chem., March 1, 2003; 49(3): 502 - 505. [Full Text] [PDF]
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T. P. Smith, A. M. Suliman, M. N. Fahie-Wilson, and T. J. McKenna Gross Variability in the Detection of Prolactin in Sera Containing Big Big Prolactin (Macroprolactin) by Commercial Immunoassays J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5410 - 5415. [Abstract] [Full Text] [PDF]
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S. Vallette-Kasic, I. Morange-Ramos, A. Selim, G. Gunz, S. Morange, A. Enjalbert, P.-M. Martin, P. Jaquet, and T. Brue Macroprolactinemia Revisited: A Study on 106 Patients J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 581 - 588. [Abstract] [Full Text] [PDF]
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H. Leslie, C. H. Courtney, P. M. Bell, D. R. Hadden, D. R. McCance, P. K. Ellis, B. Sheridan, and A. B. Atkinson Laboratory and Clinical Experience in 55 Patients with Macroprolactinemia Identified by a Simple Polyethylene Glycol Precipitation Method J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2743 - 2746. [Abstract] [Full Text] [PDF]
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G. Gilson, P. Schmit, J. Thix, J.-P. Hoffman, and R.-L. Humbel Prolactin Results for Samples Containing Macroprolactin Are Method and Sample Dependent Clin. Chem., February 1, 2001; 47(2): 331 - 333. [Full Text] [PDF]
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M. Fahie-Wilson, P. Brunsden, J. Surrey, and A. Everitt Macroprolactin and the Roche Elecsys Prolactin Assay: Characteristics of the Reaction and Detection by Precipitation with Polyethylene Glycol Clin. Chem., December 1, 2000; 46(12): 1993 - 1995. [Full Text] [PDF]
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M. N. Fahie-Wilson Polyethylene Glycol Precipitation as a Screening Method for Macroprolactinemia Clin. Chem., March 1, 1999; 45(3): 436 - 437. [Full Text] [PDF]
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J. G. H. Vieira and R. M.B. Maciel Two of the authors of the Technical Brief cited above respond: Clin. Chem., March 1, 1999; 45(3): 437 - 437. [Full Text] [PDF]
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