The analysis of body fluids is crucial for diagnosing and managing diverse pathological processes. These fluids are categorized as off-label specimens because they fall outside the manufacturer’s intended use claims. Consequently, they undergo validation procedures to meet regulatory standards, but interference studies in body fluids are typically are carried out separately during this validation process. The rationale behind this is that if accuracy studies via mixing serum and body fluid samples find no significant difference, both are considered to be similar matrices. As a result, the manufacturer’s interference cutoffs for serum and plasma are transferable for body fluid testing.
A review of hemolysis, icterus, and lipemia (HIL) interference in body fluids suggests variations in the frequency and magnitude of these interferences across different fluid types (1). For instance, drains and pericardial fluids often yield higher H- and I-indexes compared with serum and other body fluid types. Of note, these fluids are less frequently included in validation studies compared with the more commonly used serous fluids, such as ascitic or pleural fluids. This raises questions about whether the current approach of extending HIL rules oversimplifies the complexity of body fluids, prompting considerations for the development of HIL-based body fluid specific flagging rules.
MEASURING INTERFERENCES
Studies on interference are commonly conducted to mimic endogenous interference caused by hemoglobin, bilirubin, and lipids. For hemolysis interference, hemolysate typically is prepared from red blood cells and spiked into the body fluid specimen at known concentrations. To simulate icterus interference, a solution of bilirubin conjugate is added to the fluid specimens. Reproducing interference by lipemia or turbidity is challenging due to the absence of standards that can account for the variety of lipoproteins. One approach often employed is to spike the body fluid with a lipid emulsion, such as intralipid. In all cases, the spiking solution represents 5−10% of the total volume (2,3). Because there are no defined total allowable error (TAE) thresholds for analytes in body fluids, conservative thresholds of 10−15% typically are adopted for the comparison with the control body fluid. Another approach for assessing interference involves dilutions using body fluids presenting naturally high HIL indexes and comparing results between the diluted and the undiluted body fluids.
REPORTING RESULTS AND CLINICAL UTILITY
Different approaches can be taken when it comes to reporting results in the presence of interference. Some labs may opt to use the same flagging rules used for serum and release the results regardless of the magnitude of the interference with a comment indicating the type of interference and suggesting that the result be interpreted with caution. Other labs may embark on establishing their own flagging rules to set interference thresholds that, if exceeded, will trigger review to determine if the result should be released based on clinical utility or canceled if interference is abnormally high. Given the irreplaceable and irretrievable nature of body fluids, it is important that any result reported be as informative as possible despite the interference.
Published studies on interferences in body fluids suggest that cutoff values are slightly different for certain analytes compared with serum (2,3). For example, an H-index limit causing a 10% increase in a pleural fluid protein can occur at less than 50% of the H-index listed for serum. Special attention should be directed towards lactate dehydrogenase (LDH) as this is the most affected analyte in the presence of hemolysis (similar to serum) and results spike rapidly with increasing amounts of hemolysis. On the other hand, analysis of amylase, creatinine, and lipase in serous fluids are reported to have markedly lower thresholds for icterus interference than in serum samples. In contrast, most analytes present similar tolerance as in serum for lipemic interference. Integrating these results with clinical utility is paramount to ensuring appropriate patient management. For example, the presence of hemolysis in a pleural fluid would falsely elevate LDH results and increase the risk of misclassifying a transudate (<60% URL serum) as an exudate (≥60% URL serum) using Light’s criteria. Likewise, the presence of icteric interference in pleural fluid may falsely decrease cholesterol values, increasing the risk of misclassifying an exudate (>45 mg/dL) as a transudate (≤45 mg/dL).
Creating flagging rules for all analytes and fluid types is a tough job that demands many resources from the lab, which many may not be able to afford. A possible solution to this issue could be taking an analyte- and fluid-type-specific approach to determine the scenarios where further interference studies are warranted. This would require identifying the most common interferences for each fluid type, determining the analytes with the lowest thresholds that could be affected by these specific interferences, and integrating all of this with clinical significance based on published studies. For example, a Jackson Pratt (JP) drain fluid submitted to assess for a pancreatic leakage will be prone to icterus interference. In this context, amylase has been extensively documented as an indicator of a pancreatic leak if its value is three times higher than serum. Therefore, it is more relevant to pursue icteric interference studies in amylase when validating JP drains than in pericardial fluid where clinical utility has not been reported.
Regardless of the chosen approach to reporting results in the presence of interference in body fluids, close collaboration between the lab and clinicians is essential for interpreting results, especially for analytes with significant known clinical utility.
REFERENCES
- Eigsti RL, Krasowski MD, Vidholia A, et al. Review of interference indices in body fluid specimens submitted for clinical chemistry analyses. Pract Lab Med 2020; doi: 10.1016/j.plabm.2020.e00155.
- Block DR, Ouverson LJ, Wittwer CA, et al. An approach to analytical validation and testing of body fluid assays for the automated clinical laboratory. Clinical Biochemistry 2018; doi: 10.1016/j.clinbiochem.2018.05.002.
- Lo SY, Saifee NH, Mason BO, et al. Filling in the gaps with non-standard body fluids. Pract Lab Med 2016; doi: 10.1016/j.plabm.2016.03.003.
Daisy Unsihuay, PhD, is a clinical chemistry fellow at a joint program of the department of pathology and laboratory medicine at the Hospital of the University of Pennsylvania and the Children’s Hospital of Philadelphia in Philadelphia. +Email: [email protected]
Ping Wang, PhD, D(ABCC), FADLM, is chief of clinical chemistry and director of the core laboratory at the Hospital of the University of Pennsylvania, and associate professor of pathology and laboratory medicine at the University of Pennsylvania in Philadelphia. +Email: [email protected]