Macrocomplexes, typically either self-polymerized analyte or analytes complexed with immunoglobulins, can cause the measured analyte concentration to be discordant with the true functional or biologically active analyte concentration in a patient’s sample (1). To identify macrocomplexes, a laboratory must determine a method- and analyte-specific reference interval or clinical decision limit so that the presence of macrocomplexes can be ruled in or out. This is determined by calculating the difference in analyte concentration in the sample before and after macromolecule removal steps are performed. Two common ways to identify macrocomplexes are polyethylene glycol (PEG)-precipitation or ultrafiltration using a centrifugal filter to remove molecules above a defined molecular weight.
Recently, the Mayo Clinic Central Clinical Laboratory was consulted after several patients with unexpected vancomycin pharmacokinetics, abnormally prolonged elimination half-life, and evidence of treatment failure (2) were identified by the pharmacy service. Vancomycin is an intravenously-delivered glycopeptide antibiotic used in the treatment of systemic infections by gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant, coagulase-negative Staphylococcus species. Vancomycin is typically 50-55 % protein-bound and total vancomycin concentrations in serum are used to guide dosing. Therapeutic drug monitoring (TDM) is recommended due to the nephrotoxic and ototoxic effects of the drug which are both dose- and concentration-dependent. Preliminary laboratory investigations suggested that the vast majority of measurable vancomycin in these patients’ serum was associated with large molecular weight complexes (termed macrovancomycin) and presumably not bioavailable.
To identify macrovancomycin in patient samples we wanted to validate and establish reference intervals for two methods: PEG-precipitation and ultrafiltration using a 30 kilodalton (kDa) centrifugal filter. The principle of both methods is to measure vancomycin in the neat patient sample and then remove the large molecular weight complexes (including macrovancomycin) using either PEG-precipitation or ultrafiltration and measure vancomycin after removal of macromolecular complexes. The calculated percent (%) vancomycin associated with large molecular weight complexes represents macrovancomycin. We collected residual waste serum samples (N=122 from 87 patients) from patients with a clinically-ordered vancomycin over a two week period to reflect the reference population. The % PEG-precipitated vancomycin and % >30kDa-complexed vancomycin were calculated and central 95th percentile reference intervals were established. For PEG precipitation, the central 95th percentile for % PEG-precipitated vancomycin was 0-16% (90% confidence intervals (CI) 0-0%, 12-19%), and for the >30kDa filter the central 95th percentile for % >30kDa-complexed vancomycin was 6-35% (90% CI 3-7%, 32-41%). The three patients with unexpectedly high vancomycin concentrations yielded results >5-fold and >2-fold above the established upper reference limits for PEG-precipitated and >30kDa-complexed, respectively. Additionally, vancomycin recovery studies to rule out matrix effects from PEG and 30kDa filtration showed satisfactory recovery.
These data suggest that these laboratory methods are suitable for identifying macrovancomycin complexes in serum. In addition, residual waste patient samples provided a suitable reference population for establishing method-specific reference intervals for PEG-precipitation and >30kDa centrifugal filtration methods.
References
- Straseski, J. A., Wyness, S. P. When Big Complexes Cause Big Problems. Clinical Laboratory News 2016 https://www.myadlm.org/cln/articles/2016/september/when-big-complexes-cause-big-problems-macromolecule-interference-with-routine-measurement Accessed Dec. 17 2021
- Rice, M. L., Go, J. R., Rivera, C. G., Zeuli, J. D., Saleh, O. A., Baumann, N. A., & Stevens, R. W. (2021). Unexpected Vancomycin Pharmacokinetic Profile Secondary to Macromolecular Complexing: A Case Series. Therapeutic drug monitoring, 43(5), 696–700. https://doi.org/10.1097/FTD.0000000000000888