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Articles |
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Laboratoire de Biochimie et d’Immunopathologie, Centre Hospitalier de Luxembourg, rue Barblé 4, 1210 Luxembourg, Luxembourg
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Laboratoire Clinique Sainte-Marie, rue Wurth-Paquet 7, 4350 Esch-sur-Alzette, Luxembourg
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Laboratoire National de Santé, Division de Biochimie, rue du Laboratoire 42, 1911 Luxembourg, Luxembourg
a Author for correspondence: fax 352-457794, e-mail
gilson.georges{at}chl.lu)
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Introduction |
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 Top Introduction References |
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We used the polyethylene glycol (PEG) precipitation method (4) to screen for the presence of macroprolactinemia in a population of 319 samples with increased serum PRL (>30 µg/L) measured by our routine method [electrochemiluminescence immunoassay (Elecsys 2010; Roche)], an immunoassay that reacts strongly with macroprolactin (high-reading method). The presence of macroprolactin was confirmed by GFC on a Sephacryl S-300 column (Pharmacia) as described elsewhere (4). All samples containing macroprolactin, as well as 48 hyperprolactinemic samples containing exclusively monomeric PRL, were additionally assayed for PRL by two other commercially available automated immunoassay systems: a medium-reading method for macroprolactin [chemiluminescent immunoassay (Immulite; Diagnostic Products Corporation)] and a low-reading method [chemiluminescent immunoassay (ACS:180; Bayer)].
PEG precipitation has been validated as a screening method for the presence of macroprolactin only when used with the Wallac Delfia PRL assay (4). In our experiments with PEG precipitation (250 g/L PEG 6000 at room temperature, freshly prepared PEG reagent every 3 months) and the Elecsys PRL assay, we obtained the following results: over a 2-week period, 10 repeated determinations of PRL recovery after PEG precipitation of a sample containing exclusively monomeric PRL and a sample containing 82% macroprolactin gave mean recoveries of 84% (SD, 5%) and 14% (SD, 0.8%), respectively. To validate a cutoff for PEG precipitation with the Elecsys PRL assay, we used a subset of our hyperprolactinemic samples for an initial study. In 30 hyperprolactinemic samples with no evidence of macroprolactin by GFC, the recovery of PRL after PEG precipitation was 53–96%, whereas in 30 samples in which macroprolactin had been identified recovery was 6–46%. Consequently, when PRL was measured with the Elecsys assay, recovery of <50% of PRL after PEG precipitation was considered a positive screening result for macroprolactin. The PEG precipitation method, however, could not be used with the two other automated PRL immunoassays because the presence of PEG produced negative interference with the ACS:180 assay (4) and positive interference with the Immulite assay (recovery >100% after PEG precipitation for samples containing monomeric PRL).
In our population of 319 hyperprolactinemic samples, 59 sera (18%)
gave a PRL recovery of <50% after PEG precipitation, and in all of
these samples, the presence of macroprolactin was confirmed by GFC.
This finding is consistent with the data of other authors reporting a
prevalence of 15.4–26% for macroprolactinemia among
hyperprolactinemic samples (4)(7). Fig. 1A
compares the Elecsys and the ACS:180 PRL assays. The results
obtained with the Elecsys assay were higher than those measured with
the ACS:180 assay, and the difference was influenced by the presence of
macroprolactin. The results for sera containing only monomeric PRL
were, on average, 40% higher with the Elecsys assay (range, 18–60%)
than with the ACS:180 assay, whereas the results for samples containing
macroprolactin were on average 78% higher (range, 20–143%). The
samples for which the difference between the Elecsys and the ACS:180
results was >60% were exclusively macroprolactinemic samples. Of the
59 samples, 17 (29%) containing macroprolactin had a difference
between the Elecsys and the ACS:180 results of 20–59%, which was
similar to the difference observed in the monomeric PRL population.
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The difference plot for the Elecsys and the Immulite assays (Fig. 1B
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shows that the Elecsys assay gave higher results than the Immulite
assay and that the bias was influenced by the presence of
macroprolactin. In the Elecsys assay, the sera containing only
monomeric PRL gave results that were, on average, 44% higher (range,
14–79%) than in the Immulite assay, and the macroprolactinemic
samples gave results that were 78% higher (range, 46–130%) on
average. All samples presenting a difference >80% between the Elecsys
and the Immulite results were macroprolactinemic samples, and all
samples presenting a difference <45% were samples containing only
monomeric PRL. The overlapping of the macroprolactinemic and the
monomeric PRL populations was greater than for the Elecsys/ACS:180
comparison because 56% (33 of 59) of the macroprolactinemic samples
present a difference of the Elecsys and the Immulite results between
45% and 80%.
Fig. 1C
compares the medium-reading Immulite method and the low-reading
ACS:180 method. Although in some samples the results by the two methods
were quite different, there was little overall bias attributable to the
presence of macroprolactin. We obtained an average difference of
-5.1% (range, -51% to 30%) for the monomeric PRL samples and an
average difference of 2.4% (range, -50% to 86%) for the
macroprolactin-containing samples.
We confirmed in our study that, in general practice, macroprolactinemia
is a common phenomenon in hyperprolactinemic samples. Of the three
automated immunoassay systems tested, the Elecsys assay gave higher
results than those obtained by the Immulite and the ACS:180 assays, and
the bias was influenced considerably by the presence of macroprolactin.
Our data confirmed that the reactivity with macroprolactin is dependent
on the assay used for the PRL measurement, but we additionally showed
that the PRL results obtained for samples containing macroprolactin are
also sample dependent. Thus, even if the average difference between
results of macroprolactinemic samples measured with the Elecsys and
ACS:180 assays (78%) was much higher than the average difference for
monomeric PRL samples (40%), we could identify a subpopulation of sera
containing macroprolactin that behave like monomeric samples and
present only a difference of 20–59%. Consequently, it is not possible
to exclude the presence of macroprolactin by comparison of the results
obtained by a high-reading method (Elecsys 2010; Roche) and a
low-reading method (ACS:180; Bayer), as has been proposed by some
authors (9). In such a comparison, 29% of the
macroprolactinemic samples of our population would not have been
recognized. The difference plot illustrating the Immulite/ACS:180
comparison (Fig. 1C
) shows little overall bias attributable to the
presence of macroprolactin, but the considerable range in the
differences between the Immulite and the ACS:180 results obtained for
macroprolactinemic samples (range, -50% to 86%) indicates a highly
variable, sample-dependent response of the ACS:180 assay to
macroprolactin. The great disparity of values observed when comparing
results from macroprolactinemic samples measured by the Elecsys or the
Immulite assay with the results obtained by a low-reading method such
as ACS:180 may reflect variation in the structure of macroprolactin.
Macroprolactin is most probably not one unique macromolecule but rather
a heterogeneous family of PRL-IgG complexes that react differently
depending on the type of immunoassay used for PRL determination.
In conclusion, our study reinforces the point that PRL assays from different manufacturers give highly variable prolactin results for samples containing macroprolactin (4)(5)(6)(7)(8). Our data additionally show that the reactivity of macroprolactin in a PRL immunoassay, be it a low-, medium-, or high-reading method, is not identical for all macroprolactinemic samples. This finding underscores the necessity of a systematic screening strategy for macroprolactin in all samples with increased PRL (4)(5)(10)(11). With the Elecsys PRL assay, PEG precipitation, with a cutoff value of 50%, was an efficient and easy-to-use screening tool for the presence of macroprolactin. Because of the interference of PEG in some commercially available PRL assays, the confirmation of macroprolactinemia may require time-consuming methods, such as centrifugal ultrafiltration (9) and GFC.
We thank Michael N. Fahie-Wilson, Department of Clinical Chemistry, Southend Hospital, Westcliff-on-Sea, Essex, UK, for expert advice.
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References |
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TopIntroductionReferences |
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The following articles in journals at HighWire Press have cited this article:
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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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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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C. Hekim, H. Alfthan, J. T. Leinonen, and U.-H. Stenman Effect of Incubation Time on Recognition of Various Forms of Prolactin in Serum by the DELFIA Assay Clin. Chem., December 1, 2002; 48(12): 2253 - 2256. [Full Text] [PDF]
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R. Sapin and C. Simon False Hyperprolactinemia Corrected by the Use of Heterophilic Antibody-blocking Agent Clin. Chem., December 1, 2001; 47(12): 2184 - 2185. [Full Text] [PDF]
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