Macroprolactinaemia – some progress but still an ongoing problem

The study by Professor Hattori and colleagues reported in this volume of the Journal, demonstrates the presence of IgA-prolactin complexes in cases of macroprolactinaemia [1]. It is another contribution from a group which has been active in this field of research for more than 30 years and has made an outstanding contribution to our understanding of the nature of macroprolactin and the clinical significance of macroprolactinaemia. All immunoassays for prolactin (PRL) aim to detect the 23 kDa monomeric bioactive form of the hormone secreted by the pituitary, which, when present in excess, may be responsible for the symptoms of the hyperprolactinaemic syndrome and may indicate uncontrolled secretion by a prolactinoma. It has been known for over 50 years that two additional fractions of immunoreactive prolactin can be demonstrated in serum by size exclusion chromatography; big-PRL (50 kDa) and big-big PRL (>150 kDa), the latter now most commonly referred to as macroprolactin [2]. The macroprolactin fraction has received much attention since it was identified as an apparent cause of hyperprolactinaemia (macroprolactinaemia) and a potential source of clinical confusion with ‘true hyperprolactinaemia’ due to monomeric PRL [3]. An IgG component of macroprolactin was detected by Leite et al. [4] and Hattori et al. showed that in many cases the PRL component could be exchanged for [¹²⁵I]-labelled PRL [5], 6] and displaced by excess unlabelled PRL, indicating a PRL-autoantibody complex. The major epitopes recognised by the anti-PRL antibodies were located in both N- and C-terminal residues of the PRL molecule [7]. In a series of 44 cases of macroprolactinaemia, binding to protein G indicated the presence of PRL-IgG complexes in 87 % with anti-PRL autoantibodies detected in 67 % and the remainder classified as non-IgG macroprolactin [8], 9] including extensively glycosylated forms [10]. The identification of IgA-PRL forms of macroprolactin [1] further clarifies the heterogeneous nature of macroprolactin, especially the non-IgG component, and Hattori et al. suggest that the IgA and IgG forms of macroprolactin may in fact be related as a result of an immunoglobulin class switching mechanism. In many studies, Hattori et al. have emphasised the PRL-autoantibody nature of macroprolactin. They have presented further evidence of an autoimmune mechanism for macroprolactinaemia, demonstrating the predominance of the IgG4 subclass in cases of macroprolactinaemia indicative of chronic antigen stimulation [11] and developed a model of macroprolactinaemia in rats following immunisation with homologous pituitary PRL [12]. However, some uncertainty exists concerning the significance of the results of [¹²⁵I]-PRL binding studies. Binding was not detected in all cases of the IgG form of macroprolactin [7] and while binding may indicate the presence of anti-PRL autoantibodies, the results do not provide a quantitative estimate of the proportion of IgG-PRL behaving as a PRL-autoantibody complex. The fraction of autoantibody-bound PRL may be relatively small and the major form of macroprolactin may be the result of non-specific binding of IgG to PRL. Hattori et al. have pointed out that the antigenicity of PRL may be altered during iodination with consequences for the validity of [¹²⁵I]-PRL binding studies [7]. Scatchard analysis in cases of macroprolactinaemia [5], 6] indicated a low affinity antibody but there is no evidence of dissociation of macroprolactin in the presence of the assay anti-PRL antibodies exhibiting high avidity. While the nature of macroprolactin requires further study the clinical significance of macroprolactinaemia, defined as hyperprolactinaemia due to macroprolactin with normal levels of monomeric PRL, is firmly established and has been the subject of a number of comprehensive reviews over a period of 20 years [9], [13], [14], [15], [16]. All concur on three points. First, macroprolactin is not bioactive in vivo, probably because the bound IgG prevents the prolactin component binding to cellular prolactin receptors and, because of its size, the complex is confined to the intravascular space and unable to reach tissue receptors. Second, macroprolactinaemia is common, contributing to 5–25 % of results indicating hyperprolactinaemia in routine clinical laboratory practice and frequently observed in patients with symptoms of the hyperprolactinaemic syndrome. This is because PRL is more likely to be measured in patients with such symptoms and the finding of hyperprolactinaemia and symptoms in cases of macroprolactinaemia is coincidence rather than cause and effect. Third, all reviews conclude that the clinical significance of macroprolactinaemia is that, if it is not recognised initially as the cause of hyperprolactinaemia it leads to clinical confusion. The review by Gibney et al. [14] examined eight case series of female patients with macroprolactinaemia reported between 1992 and 2003. Symptoms and signs of hyperprolactinaemia were common amongst cases in all series and 22–87 % received treatment with dopamine agonists. Gibney et al. [14] concluded that the misdiagnosis of hyperprolactinaemia in these cases led to patient mismanagement involving inappropriate imaging investigations and treatment, and delay in making a correct diagnosis and instituting appropriate treatment. One of the series of patients considered by Gibney et al. [14] was followed up over a 10 year period with the clinical conclusion that macroprolactinaemia is a persistent but benign condition with no pathological significance [17]. There has been progress, in our understanding of the nature of macroprolactin, in establishing the clinical significance of macroprolactinaemia and also in the laboratory detection of macroprolactinaemia. A relatively simple test, precipitation of macroprolactin by polyethylene glycol (PEG) has been validated against the gold standard chromatographic procedure for the detection of macroprolactinaemia and quantitation of monomeric PRL when applied to the most widely used automated immunoassays [18]. The problem is that, despite the evidence reviewed above, there has been little change in practice and reports of misdiagnosis and inappropriate treatment of patients with macroprolactinaemia continue to appear [19], [20], [21]. The cross-reactivity with macroprolactin of assays purported to measure the 23 kDa bioactive form of PRL is a form of assay interference which should primarily be a concern for both assay manufacturers and clinical laboratories. Ideally, assays for PRL should be specific for the biologically active 23 kDa monomeric form. At present all assays cross-react with macroprolactin to a variable degree depending on the nature of the macroprolactin [22], 23] and the specificity of the assay antibodies. While there has been progress and some manufacturers (Mindray, Roche, Tosoh) have modified their PRL assays to reduce cross-reactivity, some residual cross-reactivity persists. For example, by using different antibodies and reaction conditions in the Roche PRL II assay the prevalence of macroprolactinamia in cases of hyperprolactinaemia was reduced from 15 to 6 % [24]. It appears that while cross-reactivity can be reduced it cannot be eliminated. This fact, and much of the information concerning interference by macroprolactin discussed above is not included by the majority of manufacturers in Information for Users (IFU) documents accompanying assays for PRL [25]. In the European Union the In Vitro Diagnostic Medical Devices Regulation (IVDR) specifies that devices should control known relevant endogenous and exogenous interference, but this stipulation appears to have had little effect [25]. The information provided by Roche in their IFU is more comprehensive than any other manufacturer and includes a protocol for PEG precipitation to detect macroprolactinaemia. In the United Kingdom External Quality Assessment Scheme for Peptide Hormones the proportion of Roche users reporting the results of PEG precipitation (66 %) is greater than for other manufacturers (19–41 %) indicating that the provision of information to users may be an encouragement to do so. In the United States, relatively few laboratories screen for macroprolactinaemia because of regulatory concerns that PEG precipitation is classified as a Laboratory Developed Test and its application to a commercial, CE marked assay would be considered off-label in the absence of manufacturer guidelines [23]. There is a paucity of information on the proportion of clinical laboratories which operate a screening policy to detect macroprolactinaemia in cases of hyperprolactinaemia. External quality assessment schemes for PRL, which include the results of PEG precipitation give some indication and, in the UK, and Australian schemes a minority of laboratories report the results of monomeric PRL. The failure of the majority of manufacturers to provide users with adequate information to understand the nature of the problem of interference by macroprolactin in assays for PRL, and a protocol for detecting macroprolactinaemia is one reason why laboratory screening for macroprolactinaemia is not more widely practiced and this also contributes to the considerable variation in practice amongst laboratories which do screen [26]. It is also likely that many highly automated laboratories find it difficult to accommodate the additional workload and expense of the manual PEG precipitation procedure. Finally, there are conflicting recommendations from reviews. It has been argued that the clinical confusion caused by macroprolactinaemia indicates that it cannot be distinguished from true hyperprolactinaemia on clinical grounds and therefore screening of all cases of hyperprolactinaemia to exclude macroprolactinaemia has been advocated as best laboratory practice [9], 13], 16], 27], 28]. In contrast clinical guidelines continue to advise a case-finding approach with testing for macroprolactinaemia in cases with atypical clinical features [29], [30], [31]. There has been little debate on the different recommendations [32], 33]. 40 years after the first report of macroprolactinaemia causing clinical confusion [3] interference by macroprolactin in assays for PRL continues to lead to misdiagnosis and mismanagement of patients. Clinical Biochemists, Endocrinologists and manufacturers should collaborate to formulate consensus, evidence-based, best practice guidelines for all involved.