Hyperprolactinaemia (hyperPRL) is the pres-ence of too much prolactin (PRL) in the bloodand is the most common endocrine disorder ofthe hypothalamic-pituitary axis. The present-ing symptoms depend on the sex of the patient;in women there is typically oligomenorrhoea-/amenorrhoea and galactorrhoea, sexual disor-ders and infertility, and in men decreased libidoand gynaecomastia. Fast and accurate diagnosisof hyperPRL is essential to enable the disorderto be treated promptly and to restore thepatient's quality of life.

Pregnancy and certain neuroleptic drugs thatreduce dopaminergic effects on the pituitary canbe responsible for hyperPRL and these causes canbe ruled out through comprehensive history tak-ing and simple tests. Where neither is implicated,a prolactin-secreting pituitary adenoma (pro-lactinoma) is usually suspected. Diagnosticimaging techniques can be used to confirm someadenomas, but small adenomas will not bedetected. However, imaging is not specific, socannot be relied upon alone for diagnosis; lesionsindicative of adenoma can be detected in approx-imately 10% of the normal population.

Detection of hyperprolactinaemiaDopamine agonists (bromocriptine, pergolide,cabergoline) are very effective in restoringmenses/gonadal function in patients withhyperPRL and, in the case of prolactinoma,reducing tumour size. With prolactinoma,pituitary surgery is also an option. Macro-prolactinaemia (hyperprolactinaemia due toexcess of macroprolactin) is responsible forhyperPRL in up to 25% of the cases, with noneed for further investigation or treatment.

HyperPRL is typically identified through acombination of clinical symptoms and a quan-titative serum PRL immunoassay. The symp-toms of hyperPRL are less common in macro-prolactinaemia than in true hyperPRL, but theycan occur. Their presence cannot therefore berelied upon as indicating true hyperPRL, ratherthan macroprolactinaemia. In addition 15-26%of patients who appear hyperprolactinaemic(PRL >700mIU/L) actually have raised levels ofmacroprolactin rather than the bioactive PRLmonomer [1]. If routine screening for macro-prolactin and estimation of monomeric PRL levelsbecame the norm in European laboratories,

this would help ensure that further investiga-tions and treatments for hyperPRL were onlygiven to the appropriate patients [1].

Several forms of prolactinIn serum, PRL exists in several forms, as a bioac-tive monomer with a molecular mass of 23kDa, and in two higher molecular mass forms -big prolactin (40-60 kDa) and big-big prolactinor macroprolactin (>100 kDa). In healthy indi-viduals, approximately 85% of circulating PRLis in the bioactive monomer form. However inhyperPRL, the level of any or all of these formscan be raised and their relative proportions canvary considerably.

Macroprolactin has a variable composition andstructure, but it is most frequently a complex ofPRL monomer and IgG with a molecular massof around 150-170 kDa range. The PRLmonomer in the macroprolactin complexremains reactive to a variable extent inimmunoassays, although it has minimal bioac-tivity in vivo and therefore is not of pathologi-cal significance. Owing to its high molecularmass, macroprolactin appears to be confined tothe vascular space and thus the PRL monomerin this complex does not come into contactwith PRL receptors. In comparison to macro-prolactin, little is known about big prolactin.Big prolactin is present to a larger extent thanmacroprolactin in the blood of normal patientsand is rarely, if ever, the cause of hyperPRL.Normal blood contains 60-90% monomericPRL, 15-30% big prolactin and 0-10% macro-prolactin [1].

Patients risk misdiagnosisIn the majority of patients, little or no macro-

prolactin can be detected in the serum.However, in others macroprolactin may be thepredominant form of immunoreactive PRL. Insome cases it is present at such high levels thatit results in apparent hyperprolactinaemia.

The patient faces being misdiagnosed andreceiving unnecessary additional investiga-tions and treatment if the possibility of reac-tivity to macroPRL is not taken into account.A further complication is that certain assays

Macroprolactin - a possible cause of hyperprolactinaemia

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Up to a quarter of patients who are hyperprolactinaemic according to quantitative prolactinimmunoassays (prolactin >700mIU/L) actually have raised macroprolactin rather than thebioactive prolactin monomer [1]. Macroprolactin reacts to a varying extent with in vitroimmunoassays but is minimally bioactive in vivo and so not of pathological significance.Laboratories need to be aware of immunoassay reactivity with macroprolactin, because ofthe potential for misdiagnosis. Routine screening of hyperprolactinaemia samples for macro-prolactin and an estimation of monomeric prolactin levels is also recommended.

by Dr V. Jarrige

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are more likely to react to macroprolactinthan others. As macroprolactin has a longerhalf life and is therefore cleared from theblood circulation more slowly than mono-meric PRL, those immunoassays that react

strongly with the macroprolactin complexmay report high levels of PRL. It is advisablethat laboratories test samples with elevatedimmunoreactive PRL for macroprolactin andto determine the monomeric PRL componentthat is active in vivo.

PRL immunoassays and macro-prolactinSome manufacturers have developed PRL assaysthat react less strongly with macroprolactin. Thisis confirmed by several studies, which have meas-ured the variance in this reactivity to macropro-lactin. In one study by Smith et al, 10 macropro-lactinaemic sera were circulated to 18 laborato-ries and the samples tested for PRL using thenine most widely available immunoassay systems[1]. The serum PRL levels reported for theimmunoassays varied by 2.3 to 7.8-fold betweenthe highest and lowest estimations [Figure 2].The highest serum PRL levels were reported withthe Elecsys assay (Roche) (mean range, 828-4604mIU/L) Lower values were reported by DELFIA

(Wallac, Inc) (range, 743-4133 mIU/L), followedby Immuno-1 (Bayer Corp, 640-3690 mIU/L),AxSYM (Abbott, 448-3021 mIU/L) and Architect(Abbott, 452-2982 mIU/L). The Immulite 2000assay (DPC) generated PRL levels equivalent toapproximately 50% of those reported by thehigh-reading methods, and the lowest PRL levelswere reported by the ACS:180 (Bayer Corp, 290-1189 mIU/L), Centaur (Bayer Corp, 243-947mU/L) and Access (Beckman Coulter, 228-940mIU/L) systems [1].

Reactivity of assaysImmunoassays that react only with the bioac-tive monomeric PRL component are mostdesirable because they will reduce misdiagnosisof apparent hyperPRL related to macropro-lactin, which is not bioactive in vivo. The reac-tivity of immunoassays to macroprolactinvaries markedly.

Although the Access (Beckman Coulter) andCentaur (Bayer Corp) immunoassays did notgive normal PRL levels for all of the 10 macro-prolactinaemic samples tested in the Smith etal study, they gave a reading above 700 mIU/Lfor only one sample [1]. In contrast, theACS:180 system (Bayer Corp) gave values inexcess of 700 mIU/L for two of the macropro-lactinaemic samples, the Immulite 2000(DPC) for four, the Architect (Abbott) forseven, and the AxSYM (Abbott) and theImmuno-1 (Bayer Corp) for eight. TheDELFIA (Wallac, Inc) and Elecsys (Roche)assays gave readings in excess of 700 mIU/Lfor all ten samples. It is therefore importantthat laboratories are aware of the reactivity oftheir assays with macroprolactin and take pre-cautionary steps to identify macroprolactin insamples with elevated PRL levels.

The variable immunoreactivity of assays is dueto a number of factors. The assay antibodiesclearly have a major influence on immunoreac-tivity, probably related to the different epitopeson PRL with which they react and the availabil-ity of these epitopes on the macroprolactincomplex [1]. However, other factors are alsoinvolved because the same pair of antibodiescoupled to different solid phases and signalgenerating systems show different reactivitywith macroprolactin [2], and incubation timehas also been shown to be directly related toreactivity with macroprolactin [3]. Althoughsome assays react with macroprolactin to a lesserextent, all assays react with it to some degree,so it is sound practice to screen all hyperPRLsamples for macroprolactin using other tech-niques. If the site required for recognition isoccupied by the endogenous IgG in themacroprolactin complex the assay will notrecognise macroPRL even when it is present inlarge amounts [1].

Additional tests to detect and quantify macroprolactin There are several methods for detecting andquantifying macroprolactin in hyperPRL sam-ples. The most widely applied techniques are gelfiltration chromatography and polyethylene gly-col (PEG) precipitation.

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Figure 1. Schematic of a typical prolactin immunoassay.

1 step sandwich technique

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Gel filtration chromatography (GFC)GFC is the reference method for the detectionof high molecular mass forms of PRL and thequantification of monomeric PRL, but it is atime-consuming and expensive technique thatrequires a considerable amount of technicalskill.

Precipitation with PEGEven if it is neither perfectly specific nor sensi-tive, this is a simple and reliable screening testin which macroprolactin present in the sampleis precipitated by high concentrations of PEG.The recovery of PRL from serum after PEGprecipitation is low when macroprolactin ispresent, and the percentage recovery is propor-tional to the relative amount of macroprolactinpresent. It has been recommended that the PRLconcentration after PEG precipitation bereported as a measure of the bioactive serumPRL which can be compared with a referencerange determined by PEG precipitation in anormal population [4]. The PEG precipitationtechnique has been successfully applied to anumber of commercial immunoassays for PRLand many manufacturers have issued guidancenotes on the use of PEG precipitation withtheir assay. In some systems PEG precipitationenhances or contributes to the PRL signal,resulting in PRL recoveries >100%. In most ofthese instances the problem can be overcomeby simply adjusting the decision limits for thepercentage recovery of PRL, or by diluting thesupernatant after PEG precipitation to reducethe interference [5].

It is critical that laboratories are aware of thereactivity of their individual assay with macro-prolactin because some react more stronglythan others and a 2.3 to 7.8-fold variance hasbeen reported, between the highest and lowesttotal PRL estimations of different assays [1].

While there are no commercial quality assur-ance materials containing known proportionsof macroprolactin and monomeric PRL [5],

hyperPRL samples should be screened formacroprolactin [5, 6]. A technique that detectsthe presence of macroprolactin and also esti-mates the concentration of monomeric PRLshould be used, because some macroprolacti-naemic patients may also have elevatedmonomeric PRL of pathological origin. PEGprecipitation or GFC fulfil these criteria.

New screening protocolsThe routine use of PEG precipitation to differen-tiate macroprolactinaemia in hyperprolacti-naemic samples is cost-effective and can altermanagement in up to 20% of hyperprolacti-naemic patients. This is shown by an audit ofhyperPRL samples routinely screened for macro-prolactin using PEG precipitation over a period offive years [6]. The audit found that macropro-lactin was entirely responsible for hyperPRL in22% (453) of the 2,089 samples screened by onecentre. Further analysis revealed that the percent-age of hyperPRL explained by macroprolactin wassimilar across all levels of total PRL >700mIU/L.

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Figure 2. Mean serum prolactin levels reported by nine different immunoanalyser user groups in specimenscollected from ten macroprolactinaemic subjects. For comparative purposes the PRL level in each specimenfollowing removal of macroprolactin by gel filtration is shown [1]. Copyright 2002, The Endocrine Society.

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Before routine screening for macroprolactin, most macroprolactinaemicpatients had undergone pituitary imaging and were treated withdopamine agonists. Even when the costs of routine screening for macro-prolactin were taken into account, screening resulted in substantial sav-ings through a 15-17% reduction in imaging requests and prescription ofdopamine agonists [6].

Improving healthcare resourcesManufacturers of PRL immunoassays are beginning to formulate assayswhich react less strongly with macroprolactin, but because the structureof macroprolactin is so variable it is unlikely that there will beimmunoassays that do not react with any form of macroprolactin. Anumber of simple screening tests are available for the detection ofmacroprolactin and estimation of monomeric PRL, and although fur-ther work is required to improve their sensitivity and specificity evenmore, it is advisable to use them to routinely screen for macroprolactinin hyperPRL samples.

It is now recommended that clinical biochemistry laboratories providinga routine service for the measurement of serum PRL follow the proce-dure in Table 1 [5]. There is no doubt that by introducing such screeningprotocols to detect hyperPRL due to macroprolactin, clinical laboratoriescould contribute to improving patient care and the best use of healthservice resources.

References1. Smith TP, Suliman AM, Fahie-Wilson MN and McKenna TJ. Gross variability

in the detection of prolactin in sera containing big big prolactin (macropro-lactin) by commercial immunoassays. J Clin Endocrinol Metab 2002; 87(12):5410-5415.

2. Fahie-Wilson MN, Brunsden P, Surrey J, Everitt A. Macroprolactin and theRoche Elecsys Prolactin Assay: characteristics of the reaction and detection byprecipitation with polyethylene glycol. Clin Chem 2000; 46: 1993-5.

3. Hekim C, Alfthan H, Leinonen JT, Stenman U-H. Effect of incubation time onrecognition of various forms of prolactin in serum by the DELFIA assay. ClinChem 2002; 48: 2253-6.

4. Suliman AM, Smith TP, Gibney J, McKenna TJ. Frequent misdiagnosis & mis-management of hyperprolactinaemic patients before the introduction ofmacroprolactin screening: application of a new strict laboratory definition ofmacroprolactinaemia. Clin Chem 2003; 49: 1504-9.

5. Fahie-Wilson MN, John R and Ellis AR. Macroprolactin; high molecularmass forms of circulating prolactin. Ann Clin Biochem 2005; 42(Pt 3):175-192.

6. Gibney J, Smith TP, McKenna TJ. The impact on clinical practice ofroutine screening for macroprolactin. J Clin Endocrinol Metab 2005;90(7): 3927-32.

The authorDr Véronique JarrigeEuropean Scientific Manager, Immunodiagnostics,Beckman Coulter Europe,Nyon,Switzerland.

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Table 1. Guidelines for laboratories performing routine serum prolactin assays [5].

• Determine the reactivity of the prolactin assay with macroprolactin.

• Determine the prevalence of hyperprolactinaemia due to macroprolactin

and discuss this with clinical colleagues.

• If the prevalence is relatively high, consider the introduction of a screening

programme to detect hyperprolactinaemia due to macroprolactin.

• If macroprolactin is detected, report this with a comment on its significance

and an estimate of the monomeric prolactin concentration.

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