American Association for Clinical Chemistry
Better health through laboratory medicine
May 2007 Clinical Laboratory News: Macroprolactin

May 2007: Volume 33, Number 5

A Common Cause of Interference in Immunoassays for Serum Prolactin
By Michael Fahie-Wilson, MPhil, MRSC

During pregnancy, a woman’s concentration of serum prolactin (PRL) increases approximately tenfold, stimulating milk production in the mammary glands of the breasts. Breast feeding in the postpartum period maintains the high levels of this pituitary hormone and triggers the accompanying period of amenorrhoea and infertility. But sometimes high serum prolactin levels occur in women who are not pregnant or breast feeding and even in men. Symptoms of hyperprolactinaemia include menstrual disturbance, infertility and galactorrhoea in women, and impotence and loss of libido in men. These symptoms can indicate the presence of an autonomous prolactin secreting pituitary adenoma or prolactinoma. In most cases, hyperprolactinaemia patients respond well either to long-term treatment with dopamine agonist drugs that suppress secretion of PRL and reduce the adenoma size, or to pituitary surgery. Therefore, accurate diagnosis of hyperprolactinaemia patients is essential.

However, such symptoms tend to be common and non-specific, so clinicians rely upon confirmatory tests to make treatment decisions. While pituitary imaging techniques exist, they are insensitive and non-specific. In order to make a definitive diagnosis, clinicians also look at the patient’s serum PRL concentration. In fact, the predominant use of laboratory measurement of serum PRL is for detecting and monitoring prolactinomas.

But over the past decade, researchers have recognized that immunoassays for serum prolactin also produce non-specific results. In addition to detecting the free, monomeric, bioactive form of circulating PRL, current immunoassays also react with a high molecular mass complex of PRL called macroprolactin. This large molecule has minimal bioactivity in vivo, but evidence suggests that macroprolactin production is a relatively common cause of apparent hyperprolactinaemia.

Unrecognized macroprolactin as the cause of apparent hyperprolactinaemia and symptoms of hyperprolactinaemic syndrome can lead to misdiagnosis and mistreatment, unnecessary concern for patient and physician, and waste of healthcare resources. Although researchers demonstrated nearly 25 years ago that macroprolactin was a common cause of hyperprolactinaemia, the molecule today remains poorly recognized by both clinicians and laboratorians.

This article describes the current understanding of macroprolactin and macroprolactinaemia and how laboratorians can detect interference from macroprolactin in serum assays for PRL.

Size Heterogeneity of Circulating PRL

Soon after the isolation of human PRL in the early 1970s, investigators sought to determine the size of the hormone circulating in serum. Using gel filtration chromatography (GFC) or size exclusion chromatography, they identified three fractions of immunoreactive PRL with differing molecular masses: a 23-kDa, monomeric protein, free or little PRL; a 40–60 kDa-protein, big PRL; and a >100 kDa-protein, big-big PRL (Figure 1A). The distribution of serum PRL in normal patients consists of 60–90% little PRL, 15–30% big PRL, and 0–10% big-big PRL. This distribution pattern is also observed in subjects with elevated PRL after stimulation with TRH, during sleep, pregnancy, and post partum lactation. Similarly, patients with a variety of physiological and pathological conditions, including subjects with pituitary tumours or symptomatic hyperprolactinaemia, also have this distribution of immunoreactive PRL molecules.

In order to determine its clinical significance, investigators have studied the big-big PRL component extensively. Using immunoassays, researchers observed that this large molecule is the predominant PRL form in some subjects with elevated serum PRL (Figure 1B) who have no symptoms of, or evident cause for, hyperprolactinaemia. Subsequently, Jackson and colleagues introduced the terms macroprolactin and macroprolactinaemia, defining the latter as “the predominance of a circulating prolactin species with a molecular weight greater than 100 kDa.” While laboratorians may still run into the term big-big PRL, for the most part, endocrinologists now commonly refer to this molecule as macroprolactin.

The Nature of Macroprolactin

In order to understand the origin of macroprolactinaemia, researchers have studied the nature of the macroprolactin complex. While it usually consists of PRL and IgG molecules, researchers have also described non-IgG containing and heavily glycosylated forms of macroprolactin. Recent reports have also described forms containing IgA and IgM molecules, suggesting that there is considerable heterogeneity in this immunoreactive form of PRL. Hattori’s group has proposed that the PRL-IgG forms of macroprolactin result from the combination of monomeric PRL secreted by the pituitary with circulating PRL autoantibodies. Currently, scientists do not understand why PRL stimulates what appears to be an autoimmune response, but Hattori and his colleagues have recently reported the presence of acidic isoforms of PRL in subjects with macroprolactinaemia that might give rise to chronic antigen stimulation.

Prevalence of Macroprolactinaemia

Macroprolactinaemia has been most widely reported and studied in women, but this most likely reflects the fact that symptoms of the hyperprolactinaemic syndrome are reported more frequently by females and followed up with measurement of serum PRL. The syndrome has also been described in males, adolescents, and children, however, and has been found in many populations with a prevalence of 1–2%. It is worth noting that macroprolactin occurs coincidentally in patients with prolactinoma and contributes to hyperprolactinaemia, but the monomeric PRL concentration is elevated in such cases and reflects the pathology.

Changes in Serum Concentration

Recent reports suggest that macroprolactinaemia is common among those with hyperprolactinaemia, and these patients typically display changes in the serum concentration of macroprolactin and monomeric PRL. For example, during pregnancy the pituitary secretes monomeric PRL, which leads to an increase in the concentration of monomeric PRL in serum. In patients with macroprolactinaemia, this increase is followed by a slower, proportionate increase in the concentration of macroprolactin. In fact, the total serum immunoreactive PRL can be quite high, and researchers have reported a concentration as high as 2,000 µg/L at 24 weeks’ gestation.

Conversely, suppression of pituitary secretion of PRL by dopamine agonist drugs results in a decrease in the concentration of serum monomeric PRL, which is followed by a fall in macroprolactin concentration. In subjects with macroprolactinaemia such treatment can reduce the total serum PRL to within the reference range, but macroprolactin remains a major serum component.

Is Macroprolactin Bioactive?

Because patients with symptoms of the hyperprolactinaemic syndrome frequently have circulating macroprolactin, researchers have hypothesized that macroprolactin is bioactive and responsible for the symptoms. However, there are several pieces of evidence indicating that this is not the case and that the association of macroprolactin with symptoms of the hyperprolactinaemic syndrome is coincidental rather than causal.

One of the earliest descriptions of an asymptomatic patient who was diagnosed with hyperprolactinaemia and also had circulating macroprolactin was reported in 1981. Since that time, there have been a substantial number of reports of patients who initially presented with some symptoms of the hyperprolactinaemic syndrome and were subsequently found to have macroprolactin in their sera. However, upon further investigation, clinicians discovered that these patients were either ovulating or pregnant.

Another argument that suggests macroprolactin is not a causative agent for hyperprolactinaemia comes from examining the feedback loop of PRL secretion from the pituitary. Although PRL can suppress its secretion in a short-loop feedback mechanism, macroprolactin does not appear to be active in this feedback loop. The PRL feedback loop works by increasing hypothalamic dopamine secretion, which also affects the dynamics of TSH release. But the concentration of serum monomeric PRL in a population with hyperprolactinaemia due to macroprolactin is similar to that of total PRL in the whole population. Furthermore, the pituitaries of individuals with macroprolactin hyperprolactinaemia secrete PRL and TSH normally in response to dopamine antagonists.

Similarly, elevated levels of serum monomeric PRL suppress gonadotrophin secretion, thereby disturbing the menstrual cycle and causing infertility, which are classical symptoms of the hyperprolactinaemic syndrome. But in patients with elevated serum PRL due to macroprolactin, serum luteinizing hormone and estradiol concentrations are significantly higher than in patients with similar levels of elevated, monomeric PRL. In subjects with menstrual irregularity and elevated serum PRL due to macroprolactin, suppression of hyperprolactinaemia with dopamine agonists has little effect on the symptoms. In contrast, substantial improvement follows treatment of subjects with increased serum monomeric PRL.

There is also evidence that macroprolactin is not bioactive in vivo. Early studies of macroprolactin used bioassays based on non-human PRL receptors and indicated that the molecule was bioactive in vitro. However, a recent study using a bioassay based on the human PRL receptor demonstrated reduced bioactivity in vitro for the majority of the forms of macroprolactin that were tested.

Researchers have also suggested that the size of the macroprolactin complex limits its transfer across the capillary membranes. This would limit the molecules access to PRL receptors in tissues. Supporting this argument is evidence that macroprolactin, unlike monomeric PRL, is not found in extra vascular spaces like cerebrospinal fluid.

Interference in PRL Assays

The PRL component in the macroprolactin complex remains reactive, although to a variable extent, in all commercially available immunoassays for serum PRL. Some assays react more strongly than others with macroprolactin, and the pattern of immunoreactivity reflects the prevalence of macroprolactin as a cause of hyperprolactinaemia with different assays (Table 1, below). From this data, it has been estimated that 10% of all elevated results in laboratories in the U.S. and U.K. may be due to macroprolactinaemia. Nevertheless, the pattern of immunoreactivity is quite different with some samples, reflecting the variable nature of the macroprolactin complex. For this reason a discrepancy in the results of different immunoassays may be evidence for the presence of macroprolactin, but the lack of a discrepancy does not exclude it.

Manufacturers of immunoassays have initiated attempts to minimize the reactivity of their PRL assays with macroprolactin. Roche (Indianapolis, Ind.), for example, has recently released a modified PRL assay that uses different antibodies and reaction conditions. The assay appears to have much reduced reactivity with most forms of macroprolactin when compared to the earlier Roche assay and many other widely used PRL assays. This improvement may be as much as manufacturers can be expected to achieve. Given the variable composition of the macroprolactin complexes, it simply may not be possible to formulate an assay for PRL that does not react with the PRL component in any form of macro-prolactin.

Comparison of Commercial Assays for PRL
Elecsys 1
Roche (Indianapolis, Ind.)
Wallac (Waltham, Mass.)
Abbott (Abbott Park, Ill.)
Tosoh (Grove City, Ohio)
Siemens (Tarrytown, N.Y.)
Beckman Coulter (Brea, Calif.)
Elecsys II
Roche (Indianapolis, Ind.)
The numbers represent the approximate prevalence of hyperprolac-tinaemia due to macroprolactin in samples with elevated total serum PRL.

Methods to Detect Macroprolactin

Although GFC is considered the reference procedure for the detection of macroprolactin and determination of monomeric PRL, it is time consuming and expensive and therefore not suited for routine use in clinical labs. Researchers have described a number of simpler approaches to the detection of macroprolactin complexes based on the composition of the complexes.

All the methods have advantages and disadvantages, but precipitation with polyethylene glycol (PEG) is the most widely used and practical technique for removing macroprolactin from serum. In addition to being simple and inexpensive, the PEG method precipitates all forms of macroprolactin. The more specific and expensive immunoprecipitation techniques, such as protein A or G-agarose and anti-human IgG-agarose, do not remove non-IgG containing forms of macroprolactin. Since there is some evidence that big PRL is not bioactive in vivo, a further advantage of the PEG precipitation technique is that it also precipitates big PRL. The two major disadvantages of PEG precipitation are that PEG interferes in some PRL assays and precipitates some monomeric PRL. For these reasons, reference ranges for PEG precipitation are required for each PRL assay.

Labs usually report the results of PEG precipitation method as the percentage of total serum PRL remaining after precipitation. Consequently, a low value indicates macroprolactinaemia. But researchers have demonstrated that this approach is subject to error in cases where both macroprolactin and monomeric PRL both contribute to the elevated total serum PRL concentration. The preferred approach is to precipitate high molecular mass forms of PRL with PEG and report the residual PRL value, which represents a measure of the monomeric PRL, along with an appropriate reference range. Manufacturers are currently working toward providing such reference ranges.

Detection of Macroprolactinaemia: The Lab’s Role

A clear understanding of PRL test results can help clinicians avoid misdiagnosis and mismanagement of hyperprolactinaemic patients, as well as unnecessary further investigation. But clinical awareness alone rarely leads to detection of macroprolactinaemia before misdiagnosis and mistreatment have occurred. Laboratorians could improve the accurate diagnosis of macroprolactinaemia by identifying interference from macroprolactin in PRL assays and helping clinicians understand the results of serum PRL measurements.

When the lab receives a request for measurement of serum PRL, the question that the clinician is attempting to answer is: “Are this patient’s symptoms due to the bioactivity of elevated, circulating PRL?” Accordingly, the objective of the lab should be to answer the clinical question by providing a measurement of monomeric PRL.

Labs can achieve this objective by using PEG precipitation of high molecular mass forms of PRL and reporting the residual PRL value along with an appropriate reference range (See Box, below). As macroprolactinaemia is commonly observed in patients with elevated serum PRL, laboratorians should consider this approach in order to provide clinicians with the most comprehensive laboratory assessment.

Laboratory Protocol for the Further Investigation of Hyperprolactinaemia

  • Apply to all cases with serum PRL greater than the laboratory reference range.
  • Measure serum PRL after precipitation of high molecular mass forms of PRL with PEG and report the result as a measure of monomeric PRL together with an appropriate reference range.
  • If monomeric PRL is within the reference range, the elevated total PRL was due to high molecular mass forms of PRL (macroprolactinaemia) and is not of pathological significance.
  • If the monomeric PRL is elevated, this may be of pathological significance.

Suggested Readings

Casanueva FF, Molitch ME, Schelechte JA et al. Guidelines of the Pituitary Society for the diagnosis and management of prolactinomas. Clin Endocrinol 2006; 65: 265–273.

Ellis AR, Fahie-Wilson MN, Axcell M, Sands K, Hill RP. Macroprolactin(s): composition and reactivity in immunoassays and laboratory interpretation of results of an unusual patient serum. Ann Clin Biochem 2006; 43: 57–62.

Fahie-Wilson M, John R, Ellis AR. Macroprolactin: high molecular mass forms of circulating prolactin. Ann Clin Biochem 2005; 42:175–192.

Gibney J, Smith TP, McKenna TJ. Clinical relevance of macroprolactin. Clinical Endocrinology 2005; 62: 633–643.

Hattori N, Ikekubo K, Nakaya Y, Kitagawa K, Inagaki C. Immunoglobulin G subclasses and prolactin (PRL) isoforms in macroprolactinaemia due to anti-PRL autoantibodies. J Clin Endocrinol Metab 2005; 90: 3036–3044.

Jackson RD, Wortsman J, Malarkey WB. Characterisation of a large molecular weight prolactin in women with idiopathic hyperprolactinaemia and normal menses. J Clin Endocrinol Metab 1985; 61: 258–264.

Jackson RD, Wortsman J, Malarkey WB. Macroprolactinaemia presenting like a pituitary tumour. Am J Med 1985; 78: 346–350.

Smith TP, Suliman AM, Fahie-Wilson MN, McKenna TJ. Gross variability in the detection of prolactin in sera containing big big prolactin (macroprolactin) by commercial immunoassays. J Clin Endocrinol Metab 2002; 87:5410–5415.

Suliman AM, Smith TP, Gibney J, McKenna TJ. Frequent misdiagnosis and mismanagement of hyperprolactinaemic patients before the introduction of macroprolactin screening: application of a new strict laboratory definition of macroprolactinaemia. Clin Chem 2003; 49: 1504–1509.

Whittaker PG, Wilcox T, Lind T. Maintained fertility in a patient with hyperprolactinaemia due to big, big prolactin. J Clin Endocrinol Metab 1981; 53: 863–866.

Michael Fahie-Wilson, MPhil, MRSC, is a Consultant Clinical Scientist in the Clinical Biochemistry Laboratory at Southend Hospital, Westcliff-on-Sea, Essex, U.K. He has published several papers on macroprolactin.