Practical Guide to the Analytical Validation of Body Fluid Chemistry Testing
Clinical laboratory testing of nonstandard body fluids is an important part of diagnosis and management for a variety of diseases. These nonstandard body fluids are not derived from blood or urine but include fluids such as cerebrospinal fluid (CSF), drain fluid, wound fluids, and essentially any other body fluid that is typically not cited by the manufacturer of a Food and Drug Administration (FDA)-cleared method in the “Intended Use” portion of the product’s package insert.1 Most body fluids (pleural, peritoneal, pericardial) are collected by ultrasound-guided aspiration (Figure 1), while others such as synovial fluid or amniotic fluid collection employ visually guided needle aspiration. Formation of excessive fluid occurs pathologically for a variety of reasons. Extravascular fluid contained in serous cavities is continually being produced at low levels (~1% of plasma) as the fluid is filtered by capillaries and either reabsorbed locally or transported back into the circulation by lymphatic drainage. (Figure 2) Pathologic increases in extravascular fluid volume occur due to amplification in either production of the fluid or reduction in the rate of fluid absorption. Increased fluid production may be caused by increases in intravascular hydrostatic pressure (congestive heart failure, kidney disease), decreased oncotic pressure (malnutrition, severe burns, nephrotic syndrome, liver cirrhosis), increased capillary permeability (inflammation, infection, burns, nephritis), or trauma. Decreased fluid absorption may occur due to lymphatic obstruction, often secondary to malignancy or impaired drainage due to elevations in systemic venous pressures (for example, congestive heart failure).2
Body fluid testing spans a variety of specialty areas within the clinical laboratory, including chemistry, hematology, microbiology, and cytology. These fluids are largely irreplaceable and often associated with complicated clinical cases, leading the provider to request a large number of tests to minimize the risk of missing a diagnostic clue. Clinical laboratories have been analyzing body fluids for decades, as documented extensively in the scientific literature,3-10 although full validation and interpretive information about body fluid test results vary in peer-reviewed publications and in practice.
The importance of full validation of body fluid specimens was brought to light in 2009-2010 when the College of American Pathologists (CAP) added clear verbiage to the inspection checklists, which enforced appropriate validation of these “alternate specimens.”11 Laboratories were cited for noncompliance with this regulation, resulting in many questions that are difficult to answer, including: 1) Does every fluid type and source require full validation? 2) Are there matrix differences in the same fluid type among patients? 3) How important are differences in terminology used for similar specimens, such as a fluid labeled “abdominal” and one labeled “peritoneal”, or a fluid labeled “knee fluid” versus one labeled “synovial fluid?” 4) What are the matrix interferences identified to suggest a laboratory is providing inaccurate results for body fluid testing? These questions, as well as a practical approach to body fluid validation, will be addressed.
Validation of Body Fluids: Selection of Fluid Type, Source and Assays
The first challenge is to appreciate the unique nature of a body fluid and understand the clinical scenarios and tests relevant to that fluid and source type. It is also important to evaluate your own clinical practice and the requests received for body fluid testing, which ultimately impact which specific fluids and tests are validated and how the results are reported. The more prevalent fluid types and chemistry test requests received in our laboratories are shown in Table 1. Assumptions should not be made that each body fluid test that has been historically ordered provides clinical utility. A test that has clinical utility provides results that influence the management and care of the patient or adds value to the clinical context in which it was ordered.
The specified source of the body fluid may present particular challenges when attempting to gather data on clinical ordering practices. Body fluid tests may be ordered using a paper requisition system that often condones manual write-ins for specimen sources and can make data collection particularly descriptive and consequently diverse. Laboratorians are often left to wonder if there is a difference among abdominal fluid, peritoneal fluid, paracentesis fluid, ascites fluid, etc. In some cases, the answer is no. Peritoneal fluid is often collected by a procedure known as paracentesis. Therefore, peritoneal and paracentesis fluid can often be considered identical, bearing in mind that peritoneal fluid could also be collected during surgery or from tubes and drains placed during surgery or other procedures. In other situations, the answer may not be as straightforward. Abdominal, peritoneal, and ascites fluid are derived from the same location in the body, yet the pathophysiology for their exact location and formation may differ, requiring distinction among the fluids in order to report clinically meaningful results. Furthermore, there is no certainty that these fluids derived from the same anatomical compartment will not differ in their analytical performance. Therefore, in this example, the body fluids should be treated as unique specimens and validated as such until proof of comparability exists.
Another challenge encountered in validation of body fluids is deriving interpretive comments for the specific assay and fluid type. Ideally, the result is interpretable in the context of the patient’s clinical condition and comparison to known normal reference ranges. In addition, the result may be evaluated by comparing it to a matched serum concentration reported for the same assay. However, interpretation can be difficult given the complex and differing composition of body fluids and the availability (or lack thereof) of pertinent scientific literature. A thorough and critical review of the literature is always appropriate. Studies indicating that analysis of a particular body fluid has not been found to be helpful can be useful if questions arise from the physician. An example of this is measurement of chloride in CSF. In most clinical circumstances, the chloride concentration found in body fluids mimics serum chloride concentrations and provides little added value.12,13 This is true for a majority of body fluid electrolytes with few exceptions.14 An excellent resource that addresses the clinical utility for a large majority of fluid types can be found in the most recent version of the Clinical Laboratory Standards Institute (CLSI) guidelines on body fluid chemistry testing.14
Finally, consider whether your laboratory needs to perform this testing in-house or if sending it to a reference laboratory makes more practical and financial sense. Some body fluid tests will always require a very short turnaround time, such as pleural fluid pH, CSF testing for collections in the emergency department, amniotic fluid glucose, etc. These tests must be performed routinely in the local laboratory serving that facility to meet the turnaround time.
Analytical Validation of Body Fluids: the Details
Once the assays and sources of fluid types requiring validation have been determined, assessment of the analytical validation strategy becomes important. For FDA-approved tests such as pleural fluid pH, CSF total protein, CSF glucose, CSF oligoclonal banding/IgG index, and fetal fibronectin, it is sufficient to verify the performance claims provided by the manufacturer. The remaining body fluid tests that use an off-label specimen type are considered FDA-modified and require full validation.
Minimum regulatory requirements of a modified test system or laboratory-developed assay require validation of the following components: accuracy, precision, reportable range, analytical sensitivity, analytical specificity, reference intervals, specimen acceptability, stability, and other performance characteristics relevant to the assay or specimen (carryover, clinical sensitivity, and clinical specificity). Establishing the clinical sensitivity and specificity is particularly helpful for providing useful interpretive information with the test result. However, this can be challenging since it may involve relating laboratory results to clinical outcomes in patients who often have complex medical histories.
Accuracy: Validation and Overcoming Matrix Effects
Assessment of accuracy involves confirmation that an analyte in a body fluid matrix can be measured accurately with instruments and reagents that are FDA-approved for specimen types such as serum, plasma, or urine. The accuracy experiments are important to perform first, and perform well, because they form the basis for the choices made throughout the rest of the analytical validation. The predominant issue contributing to potential inaccuracy and interference is the actual body fluid matrix, due to its heterogeneous nature and composition. Inaccuracy due to matrix composition may be multifactorial and include such variables as pH, ionic strength, and protein and lipid content; the latter can influence the solubility of the analyte of interest and influence assays that rely on enzymatic rate reactions. An example of the pH effect can be seen if a gastric fluid, that has a pH of 4, is submitted for amylase analysis.
If the amylase result is undetectable, it would be unclear if the undetectable result is from true lack of amylase activity in the fluid or due to inhibition of amylase occurring at low pH. Additionally, body fluid samples often are viscous, which may result in sampling errors and samples with insufficient volume. In this situation, results may not be reproducible upon repeat testing.
The recommended approach to establishing accuracy in body fluids includes performing at least 2 of the following experiments: 1) Recovery of a calibrator-spiked low concentration sample at multiple concentrations, 2) Mixing of a high and low sample (also serves to validate the reportable range), 3) Method comparison with an alternate platform, if possible. Accuracy studies should be conducted utilizing a variety of body fluid types and sources, reflecting the most frequent and clinically relevant source types for the test being validated. The percent recovery is calculated using the ratio of measured to expected values for spiking and dilution studies. Linear regression and Bland-Altman analysis of method comparison data should be performed. Acceptance criteria can be determined by performing control experiments in an approved source (plasma, serum, urine) with the expectation that the body fluid results are comparable. In cases where it does not meet these acceptance criteria, the laboratory director must assess the impact that bias and poor recovery have on the clinical decision limits and interpretation of results. Spiking studies can be conducted using calibrators, controls, or serum, provided the volume change is less than 10%. When choosing a diluent, options include manufacturer-recommended diluent or a matrix-similar choice such as 7% bovine serum albumin solution.
Linearity and Analytical Measurable Range
Reportable range experiments are performed to demonstrate the range of concentrations at which the analyte can be accurately measured on a sample prior to dilution or concentration. Linearity of a method can be established concurrently with accuracy studies, by mixing a low and high concentration body fluid pools to create 5 samples equally spaced across the reportable range. Linear regression analysis is performed and acceptance criteria are applied for the slope, y-intercept, and R2.
Maximum Dilution/Reportable Range
The analytical measurable range (AMR) may be supplemented by evaluating the concentration range where the analyte can be accurately measured after dilution. The need for dilution beyond the upper linear range is determined by evaluating the relevant range needed for appropriate clinical interpretation and may or may not be similar to dilutions needed with serum or plasma. The maximum allowed dilution is determined by selecting a high concentration sample near, but not outside, the linear range and performing serial dilutions so that the undiluted concentration can be measured with certainty (within the AMR). An example experiment is shown in Table 2. In practice, further dilutions may be required and should be performed on a case by case basis as demonstrated in Table 3. The goal in these situations is to serially dilute the sample until 3 results are within the linear range and the results agree within predetermined acceptance criteria before reporting. Alternately, a “greater than” result may be reported if clinically indicated and further dilutions are unnecessary. Finally, if an automated instrument or pipetting system performs the dilutions, the accuracy and recovery of those dilutions should be validated and compared to results obtained following manual dilution.
Conducting precision experiments ensures that the assay demonstrates acceptable reproducibility and should be performed with body fluid types and sources applicable to the clinical need. Acceptance criteria for precision studies are often based upon the performance of the assay in serum or plasma and where clinical interpretation may be affected. Inter- and intraassay precision experiments can be performed with a similar protocol that is used for plasma or serum precision studies. An example of a reproducibility study for a body fluid analyte is shown in Table 4.
Reference intervals must be reported and are critical to guide proper interpretation of the test results. Reference intervals give clinicians important information on the normal values or clinical decision limits. Body fluid testing presents additional challenges, given that normal healthy subjects do not donate body fluids because of the invasive procedure required for collection. There are a small number of FDA-approved body fluid assays, and interpretive information contained in the package insert may be used. Peer-reviewed literature is often helpful to establish the clinical utility, provided the methodology and platforms are identical. The number of FDA-approved assays for body fluids is unlikely to grow in the future because of the challenges facing in vitro diagnostics manufacturers and the amount of time and resources required. Ideally, interpretive information is reported specific to the combination of test and body fluid. Many tests simply require a recommendation that interpretation of the result be done by comparing it with results from a serum or plasma sample collected near the same time points. For example, this recommendation is useful for a body fluid submitted for creatinine or urea testing to rule out the presence of urine. The interpretation would be negative if the fluid concentration was similar to the serum creatinine or urea concentration.14
The lower limit of quantitation (LLOQ) is defined as the lowest concentration of an analyte that can be reliably reported. Results of the LLOQ experiment define the lower end of the reportable range and require acceptable precision at the lowest reportable concentration. The low-end precision will often have no impact on the utility of the test. However, conducting these experiments allows confirmation that the assay using a body fluid matrix has similar precision performance characteristics when compared to serum or plasma at the low end.
Analytical specificity evaluates the impact of endogenous and exogenous interfering agents on the accuracy of the assay. Examples include testing body fluid samples before and after hyaluronidase treatment to decrease sample viscosity, spiking with hemoglobin or bilirubin at varying concentrations, or ultracentrifugation to remove lipemia or turbidity. The threshold for tolerance for any interference can be determined by assessing the impact of clinical interpretation at varying concentrations of the interfering agent.
Stability of body fluids is an important component to the test validation to determine appropriate pre- and postanalytical transport and storage conditions. Some body fluids are known to be unstable for performance of certain assays, including pleural fluid pH and lamellar body counts. Pleural fluids are required to be collected anaerobically and the pH should be tested using a blood gas analyzer within 60 minutes of collection.7,15-20Quantitation of lamellar bodies in amniotic fluid is utilized for assessing fetal lung maturity and must be performed with a specimen that has never been frozen because the phospholipid particles quantitated in the assay may degrade.21 Validating appropriate postanalytical storage conditions is important if additional testing needs to be performed on the body fluids or the fluid needs to be retested because of quality control failures.
Handling Nonstandard Requests
Unusual requests for laboratory tests on body fluids occur frequently, and often these requests come for test and fluid combinations that have not been fully validated.
Unusual body fluid requests are often ordered either accidentally or inappropriately. The easiest solution is to contact the provider for guidance on which test is most appropriate for the fluid collected and the question to be answered. Some common scenarios are shown in Table 5.
Contacting the provider often resolves the issue by establishing that either there is a legitimate indication for exceptional testing or that the test should be cancelled, a decision that often lies with the laboratory director. It should be emphasized to the provider that performance of the assay on the specimen type has not been validated and that any results may be inaccurate or misleading. However, if necessary, verification of accuracy can be accomplished in real time by performing additional dilution and recovery studies. An algorithm that describes how such requests are handled by Mayo Clinic Central Clinical Laboratory is shown in Figure 3.
Body Fluid as a Specimen Type: Is Additional Validation Necessary?
Many laboratories wonder if the extra effort required to validate body fluid as a specimen type for laboratory tests is necessary. Minimal published studies on body fluid validation are available in the literature, so information to address this concern is negligible. However, it is clear that accrediting agencies do require documented method validation for all specimen types, and at Mayo Clinic we validate all specimen types that are acceptable for a specific laboratory test. This holds true for FDA-approved, FDA-modified, and laboratory-developed tests. Validation is necessary because specimen stability, interference limits, and choice of diluent are not always comparable between body fluids and serum. It is also important to assess specimen quality before placing on the testing instrument. This practice is essential because increased viscosity can produce sampling errors that do not reliably trigger instrument alarms.
Analytical validation of body fluids can be challenging but, in general, should follow the same processes required for other clinical specimens. The various clinical scenarios must be considered to provide body fluid tests that have pertinent interpretive information. Requests to perform assays using specimen types that have not been validated should be discouraged. Testing should be done only when there are extenuating circumstances, with disclaimers included in the report regarding the possible inaccuracy of the results.
Authored by Darci R. Block, PhD
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