Thomas P. Smith

Frequent Misdiagnosis and Mismanagement of Hyperprolactinemic Patients before the Introduction of Macroprolactin Screening: Application of a New Strict Laboratory Definition of Macroprolactinemia

BACKGROUND Macroprolactin (big big prolactin) has reduced bioactivity and is measured by immunoassays for prolactin when it accumulates in the plasma of some individuals. We applied normative data for serum prolactin after treatment of sera to remove macroprolactin to elucidate the contribution of macroprolactin to misleading diagnoses, inappropriate investigations, and unnecessary treatment. METHODS We reviewed records of women attending a tertiary referral center who had prolactin >1000 mIU/L. Application of a reference interval to polyethylene glycol (PEG)-treated hyperprolactinemic sera identified 21 patients in whom hyperprolactinemia was accounted for entirely by the presence of macroprolactin. Presenting clinical features, diagnoses, and treatment were compared in these patients and 42 age-matched true hyperprolactinemic patients. RESULTS Prolactin concentrations in sera of 110 healthy individuals ranged from 78 to 564 mIU/L. The range of values for the sera after PEG treatment was 70-403 mIU/L. For macroprolactinemic samples, PEG treatment decreased mean (SD) prolactin from 1524 (202) mIU/L to 202 (27) mIU/L but decreased it only from 2096 (233) mIU/L to 1705 (190) mIU/L in true hyperprolactinemic patients (P <0.01 between groups). Oligomenorrhea or amenorrhea and galactorrhea were the most common clinical features in both groups, although they occurred more frequently in true hyperprolactinemic patients (P <0.05). Serum estradiol and luteinizing hormone concentrations were significantly higher in participants with macroprolactinemia than in those with true hyperprolactinemia (P <0.05). Among participants with retrospectively identified macroprolactinemia, pituitary imaging was performed in 93% and treatment with dopamine agonist was prescribed in 87%. CONCLUSIONS Macroprolactin is a significant cause of misdiagnosis, unnecessary investigation, and inappropriate treatment. The use of an appropriate reference interval for the PEG immunoprecipitation procedure may be of particular importance in those patients who have an excess of both macroprolactin and monomeric prolactin.

Clinical relevance of macroprolactin

The anterior pituitary hormone PRL was identified in animal species as early as 1933 1 but only purified in humans in 1972. 2 Since then, the clinical syndrome of hyperprolactinaemia has been characterized extensively, the predominant symptoms being galactorrhoea, oligomenorrhoea or amenorrhoea and infertility in women and reduced libido, impotence and galactorrhoea in men. 3–8 Hyperprolactinaemia has an estimated prevalence of 15% in women with secondary amenorrhoea, 9,10 a condition that affects at least 3% of women of reproductive age. 11

Serum Total Prolactin and Monomeric Prolactin Reference Intervals Determined by Precipitation with Polyethylene Glycol: Evaluation and Validation on Common ImmunoAssay Platforms

BACKGROUND Macroprolactin is an important source of immunoassay interference that commonly leads to misdiagnosis and mismanagement of hyperprolactinemic patients. We used the predominant immunoassay platforms for prolactin to assay serum samples treated with polyethylene glycol (PEG) and establish and validate reference intervals for total and monomeric prolactin. METHODS We used the Architect (Abbott), ADVIA Centaur and Immulite (Siemens Diagnostics), Access (Beckman Coulter), Elecsys (Roche Diagnostics), and AIA (Tosoh) analyzers with samples from healthy males (n = 53) and females (n = 93) to derive parametric reference intervals for total and post-PEG monomeric prolactin. Concentrations of immunoreactive prolactin isoforms in serum samples from healthy individuals were established by gel filtration chromatography (GFC). We then used samples from 22 individuals whose hyperprolactinemia was entirely attributable to macroprolactin and 32 patients with true hyperprolactinemia to compare patient classifications and prolactin concentrations measured by GFC with the newly derived post-PEG reference intervals. RESULTS Parametric reference intervals for post-PEG prolactin in male and female serum samples, respectively, were (in mIU/L): 61-196, 66-278 (Centaur); 63-245, 75-381 (Elecsys); 70-301, 92-469 (Access); 72-229, 79-347 (Architect); 73-247, 83-383 (AIA); and 78-263, 85-394 (Immulite). Concordance between GFC and immunoassay-specific post-PEG reference intervals was observed in 311 of 324 cases and for 31 of 32 patients with true hyperprolactinemia and 17 of 22 patients with macroprolactinemia. Results leading to misclassification occurred in a few analyzers for 5 macroprolactinemia patient samples with relatively minor increases in post-PEG prolactin (mean 61 mIU/L). CONCLUSIONS Our validated normative reference data for sera pretreated with PEG and analyzed on the most commonly used immunoassay platforms should facilitate the more widespread introduction of macroprolactin screening by clinical laboratories.

Characterization of macroprolactin and assessment of markers of autoimmunity in macroprolactinaemic patients.

Objective  It has been reported that macroprolactin is a complex of PRL and an immunoglobulin G (IgG). This study further characterizes macroprolactin and evaluates for other markers of autoimmunity using a cohort of macroprolactinaemic sera.

The epidermolytic (exfoliative) toxins of Staphylococcus aureus.

Two epidermolytic toxins, produced by different strains of Staphylococcus aureus, split human skin at a site in the upper epidermis. Clinical effects are most common in infants, but adults are susceptible. Epidermolysis may also be observed in the mouse, in vivo and in vitro, and in a few other mammals. Recent in vitro experiments have demonstrated an inhibition by chelators and point to metal-ion, possibly Ca2+, involvement. The epidermolysis effect is insensitive to a wide range of other metabolic inhibitors. The toxin amino acid sequences are similar to that of staphylococcal proteinase, and new experiments by chemical modification and site-directed mutagenesis have shown that toxicity depends on ‘active serine’ residues of a catalytic triad similar to that found in serine proteases. Furthermore the toxins possess esterolytic activity, also dependent on the ‘active serine’ sites. However, the toxins have low or undetectable activity towards a range of peptide or protein substrates. In histological and related studies, the toxins bound selectively to an intracellular skin protein, profilaggrin, but there was no evidence that the toxin can enter intact epidermal cells. Therefore, although the circumstantial evidence that the toxins act by proteolysis is convincing, a specific skin proteolytic substrate for the toxin has not been identified.

Technology Insight: measuring prolactin in clinical samples

Measurement of prolactin is one of the most commonly undertaken hormonal investigations in evaluating patients with reproductive disorders. Hyperprolactinemia is found in up to 17% of such cases. Diagnostic evaluation of hyperprolactinemia is difficult but is facilitated by a logical approach where a thorough patient history is obtained, secondary causes of hyperprolactinemia are excluded, and the limitations of current prolactin assays are appreciated. Once hyperprolactinemia has been confirmed, attempts to establish the underlying cause can start. Given current workloads, laboratories rely on automated platforms to measure prolactin, most of which employ two-site immunoassay sandwich methods. Although generally robust and reliable, such immunoassays are susceptible to interference, and good collaboration between clinicians and the laboratory helps to minimize problems. A major challenge facing laboratories is correct differentiation of patients with true hyperprolactinemia from those with macroprolactinemia. Macroprolactin is a high-molecular-mass, biologically inactive form of prolactin that is detected to varying degrees by all prolactin immunoassays. Conservative estimates suggest that the presence of macroprolactin leads to misdiagnosis in as many as 10% of all reported instances of biochemical hyperprolactinemia. In the absence of specific testing, macroprolactin represents a diagnostic pitfall that results in the misdiagnosis and mismanagement of large numbers of patients.

Epidermolytic toxin from Staphylococcus aureus binds to filaggrins

The affinity of epidermolytic toxin from Staphylococcus aureus for proteins from the target tissue has been tested by a Western blotting procedure. Particular proteins in a l M phosphate extract of epidermis reacted on nitrocellulose blots with a probe prepared by the conjugation of toxin with peroxidase. Protein extracted into 50 mM Tris‐HCl did not react. The probe detected profilaggrin, filaggrin and a smaller unidentified polypeptide. It is suggested that the interaction is relevant to the mode of action of the toxin.

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

SummaryFluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel ‘patched’ appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.

Reporting of Post–PEG Prolactin Concentrations: Time to Change

Prolactin is the hormone most commonly measured in patients evaluated for reproductive disorders. The biologically active form of prolactin is the 23-kDa monomeric polypeptide secreted by the pituitary gland; however, circulating prolactin exists in a number of additional forms. Big prolactin (60 kDa) and macroprolactin (150 kDa), which are present in serum in varying quantities, can cause apparent hyperprolactinemia, but they have no clinical importance because they exhibit little biological activity. Despite efforts to improve assay specificity, all prolactin immunoassays in routine use detect both big prolactin and macroprolactin to varying degrees (1). The polyethylene glycol (PEG) precipitation test is widely used to detect pseudohyperprolactinemia caused by big prolactin and/or macroprolactin. Current best practice recommends that all sera with increased total prolactin concentrations be subfractionated by PEG precipitation to measure the bioactive monomeric prolactin concentration, a more clinically meaningful variable (2)(3). Subfractionation with PEG allows laboratories to distinguish patients with true hyperprolactinemia, in which there are supraphysiological concentrations of bioactive monomeric prolactin, from those with macroprolactinemia, which is characterized by increased concentrations of macroprolactin and/or big prolactin together with normal concentrations of bioactive monomeric prolactin. In the absence of PEG screening, misdiagnosis and consequent …

Activity requirements of epidermolytic toxin from Staphylococcus aureus studied by an in vitro assay

The activity of epidermolytic toxin from Staphylococcus aureus was studied in vitro using discs of neonatal mouse skin. By assessing the loss of skin integrity as a function of toxin dose and time, it was possible to put the assay on a semi-quantitative basis. Epidermolysis occurred without any change in rate from pH 3.8 to 8.7, and at an increasing rate in the temperature range of 0-37 degrees C. Activity was observed even at the lowest temperature. More than 30 inhibitors of energy metabolism, central metabolic pathways, receptor binding or proteolysis, individually failed to prevent epidermolysis and it is suggested that intoxication cannot be dependent on receptor-mediated endocytosis. Five metal-ion chelators inhibited epidermolysis, due to an effect on the tissue rather than on the toxin. Using X-ray fluorescence and atomic absorption spectroscopy, it was shown that epidermolytic toxins do not contain any essential metal ions. Some transition metals, but not Ca2+ or Mg2+, prevented the chelator-dependent inhibition of epidermolysis.