Michael N. Fahie-Wilson

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.

Polyethylene glycol precipitation: proceed with care

Two articles in this volume of the Annals demonstrate the utility of polyethylene glycol (PEG) precipitation for the detection of serum macro-analytes. In these cases, elevated aspartate aminotransferase (AST) activity was due to the enzyme being sequestered in serum as a high molecular mass (macro) enzyme complex rather than as a result of increased AST release by damaged tissue. PEG precipitation is now widely used in UK laboratories to detect elevated serum prolactin concentration due to macroprolactin and familiarity with the technique and ready availability of the reagent may encourage laboratories to use this approach for the investigation of other macro-analytes which can cause similar diagnostic confusion and patient mismanagement. PEG precipitation undoubtedly has the potential for wider routine application and has been used to study interference from macro-analytes or immunoglobulins in assays for various cytokines, C-terminal telopeptide of type I collagen, cardiac troponin I, a-fetoprotein, C-reactive protein, IgA, IgM, insulin, thyroid-stimulating hormone (TSH), parathyroid hormone, prolactin, luteinizing hormone, follicle-stimulating hormone (FSH) growth hormone, total triiodothyronine, creatine kinase (CK), amylase, alkaline phosphatase (ALP), AST, alanine aminotransferase (ALT) lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT). However, the PEG precipitation test has limitations and the characteristics of the technique, particularly the specificity and the capacity of PEG to interfere in some assays, must be appreciated if the test is to be applied appropriately and the results interpreted correctly. PEG precipitation is a crude and non-specific technique, which separates proteins by virtue of their solubility. PEG acts as an inert solvent sponge, reducing solvent availability. With increasing concentration of PEG the effective protein concentration is increased until solubility is exceeded and precipitation occurs. When applied to serum PEG precipitation is relatively specific for Igs and Ig complexes. However, it should be noted that precipitation of IgA is only partial and a preliminary report indicates that the rare cases of macroprolactinaemia with an IgA macroprolactin may be missed. Macroamylase is predominantly an IgA complex but similar problems of incomplete precipitation by PEG have not been reported. Unfortunately, PEG precipitation is not entirely specific for Igs and Ig complexes in serum. When used to detect macro-analytes a proportion of the free analyte is invariably precipitated by PEG and this varies considerably between analytes and also for any given analyte. For example, the upper limit of the reference range for PEG precipitated activity (PPA) of seven commonly measured serum enzymes varies from 36% (ALP) to 76% (ALT) and the reference range for PPA for LDH is 12–70%. While the precipitation of free, monomeric prolactin has been shown to be influenced by the concentration of serum gamma globulins this does not account for all of the variability for this analyte and other, as yet unknown factors must be present. Attempts to optimize the concentration of PEG used to selectively precipitate macro-analytes are often thwarted by proportionate effects on the solubility of the uncomplexed analyte as demonstrated for CK in serum. While PEG is relatively inert and the precipitated protein can be re-dissolved for further study, a further limitation of the method is that PEG can interfere with some immunoassays. All of the effects discussed above necessitate the determination of appropriate analyte and method specific reference ranges and the examination of a wide range of serum samples to exclude potential matrix effects. Furthermore, as the elevation in activity or concentration caused by macro-analytes is most frequently modest the cut-offs used with PEG precipitation to detect such cases inevitably sacrifice specificity for sensitivity and confirmatory tests are required. These are usually more complex and expensive and may not be readily available in the routine laboratory. When PEG precipitation has been applied to the detection of macroenzymes, results have usually been reported as the percentage of enzyme activity precipitated, which is directly related to the proportion of macroenzyme present. When applied to detection of macro-hormones, particularly macroprolactin, the results have largely been reported as percentage recovery of hormone, which is inversely related to the proportion of macro-hormone present. Recently, it has been argued that the priority for the laboratory should be to determine whether the bioactive monomeric prolactin is elevated rather than simply detect the presence of macroprolactin and it has been demonstrated that interpretation of results in terms of percentage recovery can be misleading when macroprolactin is present and the monomeric prolactin concentration is elevated. It has been proposed that the prolactin concentration after PEG precipitation be taken as a measure of monomeric prolactin

Circulating Immunoreactive Cardiac Troponin Forms Determined by Gel Filtration Chromatography after Acute Myocardial Infarction

BACKGROUND Cardiac troponin I (cTnI) and cTnT measurements are used in the diagnosis of acute myocardial infarction (AMI). Together with troponin C (TnC), the cTnI and cTnT forms make up the ternary cTnT-cTnI-TnC (TIC) complex found within myocardium. Whether cTn occurs in the circulation after AMI as ternary TIC, binary cTnI-TnC (IC) complexes, or free troponin forms has not been thoroughly investigated. METHODS Blood samples from 10 AMI patients were collected at hospital admission and then at 12, 24, and 48 h after onset of chest pain. Serum was subjected to gel filtration chromatography and cTnT (Roche cTnT) and cTnI (Siemens Centaur UltraTnI and Beckman Access AccuTnI) concentrations were measured in the gel filtration chromatography fractions. RESULTS cTnT was present predominantly as free cTnT and cTnI as binary IC complex. These 2 forms were present at every time point. Lesser quantities of TIC complex (6%-32% of total cTnT and <50% of total cTnI) were detected in 4 patients at varying times. Minor quantities of a high molecular mass form of cTnI were detected occasionally. No free cTnI was found. Both cTnI assays identified a similar pattern of cTnI forms. CONCLUSIONS After AMI, cTnI is present in serum as TIC and IC complexes. cTnT may be present as a combination of TIC and free cTnT or exclusively as free cTnT.

HDL cholesterol and the acute phase reaction following myocardial infarction and acute pancreatitis

The response of HDL in the acute phase reaction following myocardial infarction (MI) (82 subjects) and acute pancreatitis (AP) (30 subjects) has been examined and compared with that in a control group (76 subjects) admitted to hospital with suspected MI but in whom the diagnosis was not subsequently confirmed. The temporal and quantitative characteristics of the changes in concentration of the positive acute phase reactants fibrinogen and alpha 1-antitrypsin and the negative acute phase reactants albumin and LDL were similar in the myocardial infarction and acute pancreatitis subjects. In contrast, the response of HDL was different to that of the other transport proteins both within each experimental group and between the two groups. This indicated that the response of HDL cannot be regarded as simply part of a secondary negative acute phase reaction. After adjustment for changes in plasma volume, the data indicated that hepatobiliary dysfunction was probably a major factor in the negative response of HDL following acute pancreatitis and may have contributed to its response following myocardial infarction.

False-positive polyethylene glycol precipitation tests for macroprolactin due to increased serum globulins

Abstract Background The polyethylene glycol (PEG) precipitation test is widely used to detect hyperprolactinaemia caused by macroprolactin. We report two cases of hyperprolactinaemia in which a low recovery of serum prolactin (PRL) after PEG precipitation indicated the presence of macroprolactin, but no macroprolactin was detected by gel filtration chromatography (GFC). Both cases had elevated concentrations of serum globulin (IgG myeloma and polyclonal hypergammaglobulinaemia due to human immunodeficiency virus [HIV] infection), which prompted us to investigate further the effect of serum globulin on the specificity of the PEG precipitation procedure. Methods The effect of increasing concentrations of gamma globulin on the precipitation of PRL by PEG was studied by adding purified human gamma globulin to serum. Ten samples from HIV-infected patients, which showed a low recovery of PRL after PEG precipitation (<60%) were studied with GFC. Results Addition of gamma globulin decreased the recovery of PRL following precipitation with PEG and gamma globulin concentrations correlated inversely with PRL concentrations (r = 0.9429, P < 0.0167) and percentage recovery of PRL (r = −1.000, P < 0.005). Only one out of 10 samples from HIV-infected patients with PRL recoveries of <60% following PEG precipitation showed a substantial macroprolactin component on GFC. Conclusions Monomeric PRL is co-precipitated with serum globulins by PEG. Increased serum globulin concentrations can increase the amount of monomeric PRL precipitated by PEG giving a false estimate of the monomeric PRL and the erroneous impression that macroprolactin is present. The results of the PEG precipitation test should be interpreted with caution in patients with elevated serum globulin concentrations.

Immunoglobulin interference in serum follicle-stimulating hormone assays: autoimmune and heterophilic antibody interference

Interference in immunoassay caused by endogenous immunoglobulin is a cause of incorrect laboratory results that can drastically affect patient management. Two cases of immunoglobulin interference in serum follicle-stimulating hormone (FSH) assays are presented. These cases illustrate two common mechanisms for false-positive interference in two-site (sandwich) immunoassays. The first case describes a circulating autoimmune FSH immunoglobulin complex (‘macro’-FSH), which has not been previously described for FSH, and the second a cross-linking antibody directed against the assay reagents. Immunoglobulin interference was detected and characterized using a combination of method comparison, immunosubtraction and size exclusion chromatography.

What is the role of cerebrospinal fluid ferritin in the diagnosis of subarachnoid haemorrhage in computed tomography-negative patients?

Abstract Background Spectrophotometry of cerebrospinal fluid (CSF) for bilirubin is the recommended method for investigation in suspected cases of subarachnoid haemorrhage (SAH), when a computed tomography (CT) of the head is negative for blood. There is a potential need for a simpler alternative. Measurement of CSF ferritin might fulfil this need. Method We have measured ferritin in the CSF from 252 patients with suspected SAH who were negative on a CT of the head for blood, recruited on a consecutive intention to recruit basis from four centres. CSF spectrophotometry was performed on all samples. A positive outcome was taken as an aneurysm found on angiography that was treated or a discharge diagnosis of non-aneurysmal SAH. Results A final diagnosis of aneurysmal SAH was made in six patients, an arteriovenous malformation in one and non-aneurysmal SAH in nine. Receiver operating characteristic (ROC) analysis showed that at 6.4 μg/L, sensitivity, specificity, positive and negative predictive values were 1.0, 0.48, 0.12 and 1.0, respectively. At 12 μg/L, these values were 0.81, 0.91, 0.38 and 0.98, respectively. Conclusions At an appropriate negative predictive value (1.0) for a rule-out test, ferritin has too low a specificity to function as a stand-alone test and we cannot recommend it as an initial screen to be followed by spectrophotometry.

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 …

A paediatric case of macro aspartate aminotransferase

Macroenzymes are enzymes in plasma that have a higher molecular mass than the corresponding enzyme normally present under (patho) physiological conditions. Macro species have been described for most routinely measured enzymes, but with only a few reports of macro species with aspartate aminotransferase (AST), and in particular very few reports in children and adolescents. Routine biochemical analysis in a 15-year-old girl presenting with lower back pain revealed an isolated raised AST as part of a liver function test profile. Polyethylene glycol precipitation and gel filtration chromatography showed this to be a macro species.

Prevalence of increased serum creatine kinase activity due to macro-creatine kinase and experience of screening programmes in district general hospitals

Background: Macro creatine kinase type 1 (MCK1) may be the cause of elevated total serum CK activity, which can lead to diagnostic confusion. There is evidence that this problem is poorly recognized perhaps due to a lack of information on its prevalence. Precipitation with polyethylene glycol (PEG) has been described for the detection of MCK1 but has not been fully evaluated. Methods: We introduced a screening programme to detect elevated serum total CK due to MCK1 and determine the prevalence of this problem using PEG precipitation with confirmation by gel filtration chromatography (GFC). The results were compared with those from a laboratory which selected samples for further investigation during the clinical validation process. We also studied characteristics of the PEG precipitation test including sensitivity and specificity when compared with GFC. Results: Over 2 years we screened 368 patients. In 17 cases the proportion of CK activity precipitated by PEG was relatively high and the presence of MCK1 was confirmed in seven by GFC. In a second laboratory, over a period of 5 years, 11 samples were selected during the clinical validation process for further study and MCK1 was the cause of the elevated CK activity in six cases. PEG precipitates a proportion of normal, uncomplexed CK and this is increased by increasing serum globulin concentration and by higher concentrations of PEG. Conclusions: The prevalence of elevated serum CK activity due to MCK1 was approximately 2%. Laboratories should consider introducing a systematic screening programme based on PEG precipitation.