BIOCHEMISTRY


assays utilise fixed amounts of two antibodies that bind to distinct epitopes on the prolactin molecule, enabling accurate quantification of circulating hormone levels.


The problem Circulating prolactin exists in several molecular forms that differ in molecular weight and biological activity (Table 1). Elevated serum prolactin


concentrations do not always reflect an increase in biologically active monomeric prolactin. In some cases, elevated prolactin measurements may result from increased circulating concentrations of macroprolactin, a condition known as macroprolactinaemia. Macroprolactin exhibits reduced


biological activity due to its limited ability to interact with prolactin receptors. This reduced bioactivity is atributed to its large molecular size, which restricts tissue penetration and access to target organs. Current immunoassay-based methods are unable to distinguish between monomeric prolactin and macroprolactin, resulting in potential overestimation of biologically active prolactin concentrations and leading to misdiagnosis of macroprolactinaemia, unnecessary pituitary imaging, and inappropriate initiation of dopamine agonist therapy.


What do we do? Gel filtration chromatography (GFC) is considered the reference technique for the investigation of macroprolactin due to its ability to separate circulating prolactin isoforms according to molecular size. However, despite its high analytical value, GFC is time-consuming, labour-intensive, and associated with substantial costs related to specialised instrumentation, reagents, and technical expertise, which limits its availability. Furthermore, the relatively low


prevalence of hyperprolactinaemia in the general population (approximately 4.04% in women and 4.48% in men) and the proportion of affected individuals with macroprolactinaemia (reported in approximately 10-46% of cases) reduce the cost-effectiveness and practicality of implementing GFC as a routine diagnostic approach.


The next best thing is precipitation


of macroprolactin using polyethylene glycol. PEG precipitation is a relatively simple technique that separates proteins according to their solubility. PEG is a flexible, highly water-soluble polymer that acts by reducing the amount of solvent available to solubilise proteins, creating an excluded-volume or macromolecular crowding effect. This promotes the


Form of prolactin


Monomeric prolactin Big (dimeric) prolactin Big–Big prolactin


(IgG – prolactin complex) macroprolactin


Table 1. Molecular forms of circulating prolactin


precipitation of high-molecular-weight protein complexes. When applied to serum,


PEG preferentially precipitates immunoglobulins and immunoglobulin- containing complexes and is therefore particularly effective at precipitating the most common form of macroprolactin, IgG–prolactin complex. In addition, PEG precipitates other high-molecular-weight prolactin isoforms, including big prolactin, allowing the remaining monomeric prolactin to be measured by routine immunoassay. Although PEG precipitation


demonstrates good specificity for the detection of macroprolactin, it is not absolute. The technique is based on physicochemical properties rather than antigen specificity and does not directly quantify macroprolactin; instead, it estimates its presence by precipitating high-molecular-weight prolactin complexes from solution. Consequently, PEG also co-precipitates other serum components, including immunoglobulins, various protein complexes, and a proportion of biologically active monomeric prolactin, which may lead to underestimation of monomeric prolactin concentrations. For these reasons, PEG precipitation is considered a reliable screening method rather than a definitive diagnostic test. Despite these limitations,


PEG precipitation is technically straightforward, rapid, inexpensive, and readily incorporated into routine laboratory practice. While it may not offer the analytical precision of GFC, its practicality and cost-effectiveness have made it the method of choice for screening


Recovery rate <40%


40–60% >60%


Interpretation


Predominant macroprolactin


Equivocal result


Predominantly monomeric prolactin


Table 2. Prolactin recovery interpretation.


in most clinical laboratories—an example of a technique that is not perfect but is often more than sufficient for the task at hand. A commonly employed laboratory


protocol involves preparation of a 25% PEG 6000 solution by dissolving 25g of PEG 6000 granules isn distilled water to a final volume of 100 mL. Equal volumes of serum and PEG solution are mixed, allowed to incubate at room temperature for 10 minutes and centrifuged at 3000 xg for 30 minutes. Prolactin concentrations are measured before (pre-PEG) and after (post-PEG) precipitation and the prolactin recovery rate (RR) is calculated as:


Molecular


weight (kDa) 23


48 – 56 >150


Prevalence in serum (%)


80 – 95 5 – 10 5 – 10


Biological activity


active


inactive inactive


where multiplication by two corrects for serum dilution during PEG treatment. Interpretation is generally as shown in Table 2.


This protocol had been used in the


laboratory for many years, and it had gained a reputation of its own. Being assigned to the macroprolactin bench was enough to make even the most experienced members of staff sigh. At first glance, the process appears to


be straightforward. In practice, however, it is anything but. So, where does it all go wrong? As it turns out, there are several opportunities for this seemingly simple assay to test both the method and the patience of the scientist performing it. Firstly, the assay has no internal


quality control (IQC), which is far from ideal. Secondly, the 25% w/v PEG solution is highly viscous. As a result, whenever PEG-treated samples are loaded onto the analyser, there is always the anticipation of the dreaded ‘sample integrity’ error message. This indicates that the analyser has rejected the sample and will not process it, regardless of any further atempts. The only option is to repeat the sample preparation process. As a result, multiple rounds of preparation are often required before a coherent and reportable result can finally be achieved. Thirdly, the assay requires a relatively large sample volume of 200 µL, with each


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