Monoclonal—and sometimes high concentrations of polyclonal—immunoglobulins known as paraproteins can interfere with many laboratory tests on different automated chemistry, nephelometry/turbidimetry, immunochemistry, and hematology platforms, causing erroneous results. By understanding the mechanisms behind and patterns associated with this phenomenon, clinical labs will be better able to recognize when it occurs.

Examples of paraprotein interference include when direct bilirubin results are higher than total bilirubin, or when high creatinine results do not correspond with a patient’s clinical picture. However, published reports describe paraprotein interference in tests for many other analytes such as inorganic phosphate, calcium, C-reactive protein, total bilirubin, albumin, glucose, iron, uric acid, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, insulin, and total protein. Often, these spurious results are irreproducible upon repeat testing, and repeat testing on the same sample may show fluctuating results.

Assay- and Patient-Specific

Most reported cases have involved individual paraprotein (IgG, IgM, or IgA) interference in assays on particular platforms. Testing other samples with the same type of paraprotein via the same assay on the same platform may or may not produce the interference. Similarly, testing the original problematic specimen for the same analyte on a different platform may not exhibit paraprotein interference. This suggests that the mechanisms for paraprotein interference depend on a paraprotein’s unique properties that determine its conformational changes under particular assay conditions. Paraproteins may have antibody-like activities that bind to analytes/reagents. They might also behave like a heterophilic antibody that causes erroneous immunoassay results, or like a cryoglobulin that induces red cells to agglutinate, thereby producing incorrect hematology results.

In the case of chemistry tests that measure via absorbance or turbidity, the most important mechanism for paraprotein interference is precipitation of paraprotein under certain assay conditions. Our own research strongly suggests that the assay’s pH and a low ionic strength are key factors in this circumstance.

The Precipitation Challenge

Paraprotein concentrations in patients’ samples can be high (7%–65%), so precipitating them can change the specimen’s turbidity and cause significant alterations in measurements of transmitted or scattered light. If the precipitates are small and evenly suspended in the solution, background subtraction may correct the errant light to a certain degree. This does not work in the case of large precipitates. The protein aggregates can move in and out of the light path due to random Brownian motions. This generates a fluctuation pattern in the spectrometer-measured absorbance, causing erroneous and sometimes irreproducible results. We have described our experience with this using the kinetic Jaffé method for creatinine that uses the difference between two measurement points (Practical Laboratory Medicine 2015;3:8–16).

Going to Extremes

Extreme pH conditions—either alkaline or acidic—also sometimes induce protein conformational changes that promote protein aggregations. Most reported cases of paraprotein interference have been in assays with extreme acidic conditions like inorganic phosphorous, iron, and direct bilirubin, or extreme alkaline conditions such as creatinine (Jaffé method), total protein, and lithium.

Our lab encountered paraprotein interference in measuring one patient’s sample for creatinine and total protein. This patient had a unique IgM paraprotein, and when we processed the patient’s samples under very acidic pH (1-2), very alkaline pH (12-13), or low ionic strength conditions, we saw large aggregates, but not in the samples of eight other patients with elevated IgM or IgG. This is because proteins tend to precipitate at their isoelectric points—determined by their amino acid sequences—and thus have different set points for conformational changes in response to pH and ionic strength.

Since paraproteins precipitate under particular assay conditions, whether basic or acidic, or even neutral pH, it would be very difficult to fashion an ideal assay that prevents all such instances. Still, most manufacturers have optimized their assays to minimize this problem. That is why only a small percentage of paraprotein immunoglobulins interfere with any particular assay. Because assays on different platforms are not formulated exactly the same, interference exhibited by one paraprotein with one assay on one platform might not be duplicated on another.

Even with the work they’ve already done, we strongly encourage manufacturers to continue taking steps to improve their assay conditions by increasing ionic strength and by adding protein stabilizers/surfactants. Recently one laboratory reported success using a new function to detect abnormal reaction data on an automated chemistry analyzer. If this new function does not significantly hinder the throughput of the instrument, it certainly would be a step forward toward solving the problem.

In the meantime, technologists working with automated analyzers in busy clinical laboratories remain challenged to know which samples have paraprotein interferences. In our lab, we programmed two flags in our laboratory information system to alert us when conjugated bilirubin results exceed those of total bilirubin or when there is a negative result on any test. When labs suspect a paraprotein interference, they should measure the questionable sample with a different method or platform to see if this yields valid results. For analytes that do not bind to proteins, removal of protein (including immunoglobulins) using ultrafiltration or precipitation prior to testing may be helpful. However, the effect of protein removal by a particular method needs to be validated beforehand or concurrently by testing the analyte in a sample that does not contain paraproteins before and after the procedure.

Lu Song, PhD, DABCC, FACB, is associate director of clinical chemistry at Ronald Reagan Medical Center and associate professor of pathology and laboratory medicine at University of California Los Angeles. +Email: