M Protein Interference in Renal Function Assays: A Quest for True Kidney Function

Which renal function assay(s) should be considered when interference by a monoclonal protein is potentially present? Which clinical consequences could be avoided when detection of M protein is timely? How can M protein interference in routine clinical chemistry assays be better dealt with in the future? An 82-year-old woman goes for biannual cardiovascular risk management check-ups at the general practitioner (GP), where her kidney function is evaluated by measuring creatinine (enzymatic assay), from which an estimated glomerular filtration rate (eGFR) is calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) 2009 formula. She has a history of hypertension, reduced left ventricular ejection fraction at 45%, osteoporosis, choledocholithiasis, subclinical hypothyroidism, chronic renal insufficiency, and a monoclonal gammopathy of undetermined significance, type IgM kappa (original M protein concentration in 2012: 10 g/L). In 2020, the GP notices a slight deterioration in kidney function as measured by laboratory A (serum creatinine from 1.52 to 1.73 mg/dL [134 to 153 µmol/L], eGFR 32 to 27 mL/min/1.73 m2). This prompts the GP to send the patient to the internist–nephrologist at hospital B. A serum creatinine level of 1.30 mg/dL (115 µmol/L, eGFR 38 mL/min/1.73 m2) is measured. In Fig. 1A, the creatinine values from both laboratories over time are shown. Even though the creatinine assays from laboratories A and B are from different analyzer platforms (Fig. 1B for assay details), it is assumed that a possible coexisting heart failure could be causing kidney function fluctuations. However, in March 2022, an even larger and clinically significant discrepancy is found between the laboratories, with a creatinine of 2.14 mg/dL (189 µmol/L, eGFR 21 mL/min/1.73 m2) in laboratory A vs 1.15 mg/dL (102 μmol/L, eGFR 44 mL/min/1.73 m2) in laboratory B (Fig. 1A). Besides this creatinine discrepancy, a small increasing M protein fraction (IgM kappa) of approximately 15 g/L (Fig. 1C), a hemoglobin of 13.05 g/dL (8.1 mmol/L), no abnormal blood counts or urine screening, and a normal calcium value are found. (A), The patient's creatinine over the years measured at laboratories A and B. The discrepancy between laboratories A and B is seen between January 2020 and March 2023 (between the dashed lines); (B), Overview of creatinine and cystatin method principles from the different laboratories (1–4), specifying the type of standardization used in each assay and the March 2022 value; (C), M protein fraction over the years measured at laboratory B. The area between the dashed lines shows the M protein fraction at the time of the creatinine discrepancy between laboratory A and laboratories B. (D), (E), and (F): Creatinine and cystatin C were measured upon dilution, and the results presented were obtained through multiplication by the dilution factor. The creatinine or cystatin C value after recalculation (y-axis) is displayed vs the x-fold dilution (x-axis). (D), Creatinine dilution series with NaCl and PEG at laboratory A; (E), Creatinine dilution series with NaCl and PEG at laboratory C; (F) Cystatin C dilution series with NaCl and PEG at laboratory C. In search of the true kidney function and an explanation for the measurement discrepancy between laboratories A and B, the GP, in consultation with the clinical chemist, requests a cystatin C measurement (turbidimetric assay) from hospital laboratory C. Cystatin C is above the upper limit of detection (>10.2 mg/L) and a creatinine of 1.13 mg/dL (100 μmol/L, eGFR 45 mL/min/1.73 m2, Fig. 1E) is measured, meaning no definitive answer can be given regarding renal function. M protein interference is thought to play a role, and additional experiments are performed to demonstrate this. For the most commonly used creatinine enzymatic assays several interferences are known, including hemolysis, icterus, lipemia, and also monoclonal immunoglobulins, especially IgM pentamers (approximately 900 kDa) (1, 5–8). For example, McGill and co-workers have elegantly demonstrated monoclonal IgM kappa interference, by spiking normal patient plasma with extracted M protein, resulting in increased creatinine values (7). Although it has been suggested to measure cystatin C in case of M protein interference (8), such interference could likewise also apply to cystatin C assays—to date, however, no case reports are available (2, 5, 6). Several mechanisms for M protein interference have been described in the literature, most commonly clouding (5, 9). Each M protein is a unique monoclonal molecule with a distinctive variable region and characteristic intrinsic properties, like immunoglobulin type (mostly IgG, IgM, IgA, or IgD), molecular weight, solubility, hydrophobicity, and isoelectric point. Due to the M proteins’ distinctiveness, the possibility of interference with clinical chemistry assays must be evaluated on an individual patient basis. First, the reaction conditions (e.g., low ionic strength, high and low pH) have been suggested as possible causes of interference (6). Second, various chromogens used in spectrophotometric assays might bind to specific variable regions of IgM proteins (7). To prove M protein interference for the current case, dilution series are made (Fig. 1D–F). Both creatinine tests in laboratories A and C (Fig. 1D and E) and the turbidimetric cystatin C assay (Fig. 1F) show a decrease upon NaCl dilution, consistent with possible interference. The slight decrease for creatinine upon dilution may be attributed to increased measurement inaccuracy at lower concentrations, given a limit of detection of 0.06 mg/dL (5 µmol/L) (1). For cystatin C, a striking nonlinear behavior is found (Fig. 1F). However, if dilution is performed with addition of the highly hydrophilic polymer polyethylene glycol (PEG 6000 g/mol), it is possible to produce a reliable cystatin C result and remove the M protein interference. It is noteworthy that dilution and/or PEG precipitation studies should be validated by each laboratory for clinical use. The cystatin C result upon 2× PEG dilution is 1.48 mg/L, consistent with an eGFR [Caucasian, Asian, pediatric, and adult (CAPA) formula (10)] of 44 mL/min/1.73 m2 and corresponding to the previous creatinine of 1.15 mg/dL (102 μmol/L, eGFR 44 mL/ min/1.73 m2) of hospital B. Hence, 44 to 45 mL/min/1.73 m2 seems the “true” eGFR, which is further confirmed using a specific liquid chromatography–mass spectrometry (LC-MS) method at hospital laboratory D resulting in a creatinine of 1.18 mg/dL (104 μmol/L, eGFR 45 mL/min/1.73 m2). PEG therefore seems able to precipitate the M protein from the serum. The mechanism by which the polymer induces protein precipitation is based on excluded volume creating an attractive force (entropic in nature) between the protein molecules (11). For comparison, creatinine and cystatin C dilution series from two positive controls are also performed. Here, we observe less effect with PEG dilution (data not shown); thus, the patient's M protein in particular is causing the interference. In addition to PEG precipitation, removal of the M protein ( approximately 900 kDa) is attempted by filtration (100 kDa filter). Removal of proteins >30 kDa by ultrafiltration was previously shown for monoclonal IgM kappa in creatinine measurements. (7) However, protein gel electrophoresis followed by immunofixation, quantification by total IgM assay, and cystatin C measurements (Fig. 2A and B) confirm not all M protein is removed using our filter, possibly due to filter cutoff, overly forceful centrifugal conditions (11 423 × g) and/or membrane type (regenerated cellulose, low binding): monoclonal IgM kappa bands are still visually present, and 8.5 g/L (from 19.5 g/L) total IgM remains, in contrast to the PEG method with no monoclonal IgM kappa bands remaining, and 0 g/L total IgM in the quantitative assay. Additionally, the reaction monitor graphs from the analyzer software display no upward absorption trend after adding PEG in comparison to the untreated sample and the sample after filtration (Fig. 2D–F). The reaction mixture also appears less cloudy after adding PEG (Fig. 2C—right). Hence, the abovementioned clouding mechanism seems the most plausible reason for M protein interference, especially given the reaction kinetics with increasing absorption for the untreated sample (Fig. 2D) and the clouding in the vial (Fig. 2C—left). For further comparison, cystatin C measurement is also carried out using a nephelometric method at hospital E, yielding 1.89 mg/L (eGFR 32 mL/min/1.73 m2). Although the eGFR does not entirely correspond to the “true value” of 44–45 mL/min/1.73 m2, there appears to be less interference than with the turbidimetric method. (A), Results from protein gel electrophoresis and immunofixation of immunoglobulins in serum (left lane: protein mixture in serum, right lanes: immunofixation of immunoglobulin type G, A, or M and light chains: kappa [K] and lambda [L]), showing the IgM kappa bands for the untreated sample (lower left) and after filtration (upper left). After PEG precipitation, no bands are visible anymore (right); (B), IgM (quantified by total IgM turbidimetric assay) and cystatin C quantifications before and after filtration and after PEG addition. Experiments performed at laboratory C; (C), Image of the cystatin C reaction mixtures before (left) and after PEG treatment (right). (D to F), Reaction monitor graphs for the cystatin C assay from the analyzer software: untreated, after filtration, and after PEG precipitation. The turbidity signal on the y-axis represents the value of the absorbance at primary wavelength 546 nm minus the absorbance at secondary wavelength 700 nm. On the x-axis, the reaction time is represented by the increasing number of assay points. In this case, both creatinine and cystatin C assays show M protein interference, with some assays being more affected than others (Fig. 1B, assay overview). For creatinine, possible interference by M proteins is generally known, yet this case affirms that cystatin C can also be significantly impacted by M proteins. The testing principles of the creatinine and cystatin C assays among suppliers differ significantly (Fig. 1B), possibly contributing to different assay performance in the presence of M proteins. The creatinine assays of laboratory A vs laboratories B and C (same enzymatic Roche assay) all measure light intensity, yet at different wavelengths and using different chromogens. The method at laboratory A (measuring at 600 nm) may be more severely affected by turbidity from M protein at higher wavelengths; likewise, the different dye could be more susceptible to M protein binding. Our finding, that positive control samples with IgM-type M proteins are not subject to critical interference, suggests that the binding phenomenon is governed by the M protein variable region. Hence, M protein interference can emerge on an individual patient basis. The LC-MS creatinine method of laboratory D is unaffected by M protein interference and results in the true creatinine value, corresponding to the enzymatic assay results from laboratories B and C. The turbidimetric cystatin C assay from laboratory C does show M protein interference, whereas the nephelometric test in laboratory E suffers less from interference. This might be explained by the difference between polystyrene and latex particles, which could have different interactions with the M protein based on their different hydrophobic/hydrophilic polymer nature. Because of her falsely increased creatinine value, the patient in this case was mistakenly referred to the internist-nephrologist, potentially leading to unnecessary additional diagnostics and needless worry. Hence, it is paramount that clinicians apply caution when evaluating the kidney function of patients with monoclonal gammopathy. At the laboratory, when a measurement discrepancy is suspected, analytical interference has to be ruled out. (Non)linear behavior upon serial dilutions and creatinine and cystatin C assays on different analyzer platforms can provide insight into possible discrepancies. A confirmatory test using an LC-MS creatinine method (no interference) is also recommended. Ultrafiltration can also avoid M protein interference with creatinine measurements (7). Since this method may not always be successful or available, a PEG dilution series is a suitable alternative. Although it was recently shown that cystatin C can be more reliable than creatinine for kidney function evaluation of older patients (12), our case demonstrates that cystatin C can also be subject to M protein interference. When unexpected results are found, it is recommended to consult the clinical chemist about possible M protein interference and determine a clinical management strategy on an individual patient basis. Ethics statement: Informed consent has been received for writing this case. M proteins can distort routine laboratory tests for kidney function determination, including both creatinine and cystatin C assays. Performing kidney function assays on different analyzer platforms can reveal monoclonal protein interference, most notably type IgM. Both LC-MS creatinine measurement and PEG dilution series can confirm the actual patient kidney function. Each M protein is unique and can behave in different ways in routine laboratory assays. Individual clinical management is thus mandatory and clinicians should apply caution when evaluating kidney function in patients with a monoclonal gammopathy. Timely consultation with the laboratory (i.e., clinical chemist) about possible M protein interference is paramount to avoid adverse clinical consequences for the patient. With confirmed M protein interference, future clinical management strategies should be worked out between physician and laboratory to prevent discrepancies until the interference is resolved. More generally, stakeholders including clinical chemists and in vitro diagnostic companies need to focus on precision diagnostics to avoid test suboptimalities by design such as interference by M proteins. Nonstandard Abbreviations: CAPA, Caucasian, Asian, pediatric, and adult; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; GP, general practitioner; eGFR, estimated glomerular filtration rate; PEG, polyethylene glycol. Author Contributions: The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Michelle van der Helm (Conceptualization-Lead, Data curation-Lead, Investigation-Equal, Project administration-Lead, Visualization-Lead, Writing—original draft-Lead, Writing—review & editing-Lead), Brigitte Wevers (Conceptualization-Equal, Data curation-Equal, Methodology-Equal, Supervision-Equal, Writing—review & editing-Equal), Cees van Beek (Resources-Supporting, Writing—review & editing-Supporting), Paul Schenk (Conceptualization-Lead, Data curation-Equal, Methodology-Equal, Project administration-Equal, Supervision-Lead, Writing—review & editing-Lead), and Angela Bikker-Koornneef (Conceptualization-Equal, Data curation-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Resources-Lead, Writing—review & editing-Equal) Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest. The authors would like to acknowledge Jan H. Didden, Sonja Didden-Buitendijk, Zeliha Mengi (laboratory technicians, LUMC), Hans de Graaf, and Marquerite de Bie (laboratory technicians, St. Antonius Hospital) for performing additional confirmation experiments. Dr. Rebecca Heiner-Fokkema (clinical chemist, University Medical Center Groningen—UMCG) is acknowledged for creatinine LC-MS determination, and Dr. Hans Kemperman (clinical chemist, University Medical Center Utrecht—UMCU) for confirmatory nephelometric cystatin C measurement.

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