Polyethylene glycol precipitation: proceed with care

Abstract

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