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Technical Briefs |
1
Department of Clinical Chemistry, Southend Hospital, Prittlewell Chase, Westcliff-on-Sea, Essex SSO ORY, United Kingdom
2
Department of Clinical Chemistry, Basildon Hospital, Nether Mayne, Basildon, Essex SS16 5NL, United Kingdom
a author for
correspondence: fax 44-1702-221059, e-mail
mike.fahie-wilson{at}hospital.southend.nhs.uk
Prolactin occasionally is present in serum in a macromolecular complex. The most common form is macroprolactin, an antibody-antigen complex of prolactin (PRL) and immunoglobulin G with a molecular mass of 150–170 kDa (1)(2)(3)(4). The PRL component remains reactive (to various degrees) in immunoassays for PRL (5)(6)(7), and macroprolactin is cleared more slowly than PRL from the circulation, leading to apparent hyperprolactinemia (8).
Macroprolactin is bioactive in vitro but has minimal bioactivity in vivo, probably because of the failure of the high-molecular mass complex to cross the capillary membrane to reach target PRL receptors (9). Hyperprolactinemia attributable to macroprolactin may cause diagnostic confusion and lead to inappropriate treatment, and it is important that laboratories characterize the response of their assay to macroprolactin and identify macroprolactin when this is a cause of hyperprolactinemia (1)(5).
The Wallac Delfia immunofluorometric PRL assay reacts strongly with macroprolactin, and this response is well characterized (5)(6)(10)(11). High concentrations of polyethylene glycol (PEG) precipitate macroprolactin from serum, and this principle has been used in a screening test for the presence of macroprolactin validated with the Delfia assay (5).
We studied the reaction of the PRL assay on the Roche Elecsys 2010 analyzer to macroprolactin and compared it with that of the Delfia assay. We also evaluated the PEG precipitation technique as a means of detecting macroprolactin as the cause of hyperprolactinemia in the Elecsys assay.
Precipitation with PEG was carried out by adding 200 µL of serum to 200 µL of 250 g/L PEG 6000 (product no. 29577; Merck Ltd.). After thorough mixing and centrifugation at 1500g for 30 min, the supernatant was removed for analysis. Recovery of PRL after precipitation with PEG was determined by comparison with a dilution of 200 µL of serum in either 200 µL of 10 mmol/L Tris buffer (pH 7.4) containing 140 mmol/L sodium chloride (Delfia assay) or in Elecsys system diluent.
PRL in the supernatant after precipitation of serum with PEG and in the diluted serum was measured as for untreated serum, using the Elecsys and AutoDelfia analyzers according to the manufacturers’ instructions. Gel-filtration chromatography of serum PRL was carried out on a 1.5 x 50 cm Sephacryl S-300 (Pharmacia) column as described previously (5). Monomeric PRL and macroprolactin were quantified from the relative areas under the peaks and the total serum PRL results obtained by the Wallac AutoDelfia PRL assay.
In a retrospective study with stored samples, the Elecsys assay gave
results similar to Delfia in samples containing only monomeric PRL,
whereas in samples containing macroprolactin demonstrated by
gel-filtration chromatography, the Elecsys assay gave higher results
(Table 1
).
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Mean recovery of PRL from standards (IS 84/500 in phosphate buffer;
Wallac) after precipitation with PEG with the Elecsys assay was 106%
(range, 103–110%). Mean recovery of PRL from serum (n = 35)
containing only monomeric PRL was 86% (range, 71–106%), indicating
variable coprecipitation of PRL with serum proteins. The recovery of
PRL after precipitation with PEG from serum containing macroprolactin
was lower and showed a quantitative relationship proportional to the
percentage of macroprolactin present, as determined by
gel-filtration chromatography (Fig. 1
A). This relationship is emphasized by the line in Fig. 1A
, which shows
the recovery of PRL after precipitation with PEG in one sample,
containing 93% immunoreactive PRL as macroprolactin, that had been
diluted with various amounts of a sample containing only monomeric
PRL. Repeated assays (n = 12) over 3 months of PRL
recovery after PEG precipitation, using a pool of frozen serum
containing only monomeric PRL, gave a mean recovery of 85.1% and a CV
of 4.1%.
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PEG precipitation was applied prospectively to 139 consecutive serum samples with PRL >600 mIU/L identified over a period of 10 months in two laboratories using the Elecsys assay.
Recovery of PRL showed a bimodal frequency distribution (Fig. 1B
). A
cutoff of 50% has been used and validated with the Delfia assay to
identify the presence of macroprolactin (11).
Application of this cutoff to the data in Fig. 1
indicated that
macroprolactin was the cause of hyperprolactinemia in 11 of the 69
samples from Southend (16%) and 12 of the 70 samples from Basildon
(17%). All 11 samples with recoveries <50% from the Southend
population were subjected to gel-filtration chromatography, which
confirmed that macroprolactin was the predominant immunoreactive form
of PRL in all of these samples.
Two samples were distributed to seven laboratories that use the Elecsys PRL assay and the PEG precipitation procedure: sample A, with a total PRL of 2982 mIU/L and containing macroprolactin that contributed 90% of the total PRL immunoreactivity (Delfia); and sample B, with a total PRL of 2004 mIU/L and containing only monomeric PRL. All laboratories found low recovery of PRL after PEG precipitation in sample A (6.5–9.0%) and normal recovery in sample B (81.3–92.0%).
We surmise that the Roche Elecsys PRL assay reacts strongly with macroprolactin and that macroprolactin is a common cause of hyperprolactinemia with this assay. Macroprolactin can be detected by low PRL recovery after precipitation with PEG, and the recovery allows an estimate of the monomeric PRL. We recommend that users of the Elecsys assay make their clinical colleagues aware that macroprolactin may cause apparent hyperprolactinemia and that samples from all patients with increased PRL be examined for the presence of macroprolactin by precipitation with PEG.
It is interesting to note that the PRL assay on the Roche Enzymun system reacts less strongly with macroprolactin (7) but uses the same pair of antibodies as the Elecsys assay. However, the antibodies are coupled to different solid phases and use different signal-generation systems in the two assays.
Acknowledgments
We thank Roche Diagnostics (Lewes, UK) for providing the reagents for this work.
References
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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 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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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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