# Verification of Reference Intervals and Reportable Range ## Quick Answer - Verify a reference interval by collecting 20 samples from healthy individuals in your local population and comparing results against the manufacturer's stated interval. - Verify the reportable range by testing samples with known analyte concentrations across the claimed linear interval and comparing measured values to expected values. - Verification with small sample sizes cannot fully replace a de novo reference interval study, and a failed verification requires a larger confirmatory study. ## At a Glance | Verification Component | Minimum Sample Size | Acceptance Criterion | Action on Failure | | --- | --- | --- | --- | | Reference interval verification | 20 reference individuals | No more than 2 of 20 results fall outside the stated interval | Recruit more samples or perform a full reference interval study | | Reportable range verification | 5 to 7 concentration levels | Measured values within acceptable deviation from expected values | Narrow the reportable range or investigate calibration and linearity | | Analytical measurement range | 5 to 7 levels spanning the claimed range | Linear regression slope near 1.0 and intercept near zero | Recalibrate, adjust the range, or contact the instrument manufacturer | ## Purpose of Verification Reference intervals and reportable ranges are central to diagnostic assay validation. A reference interval defines the range of values expected in a healthy population, and the reportable range defines the span of analyte concentrations that an assay can measure accurately without dilution or concentration steps. Both parameters must be verified before a laboratory introduces a new assay or changes its patient population. Verification is distinct from validation. Validation establishes that an assay works as intended for its intended use. Verification confirms that an already validated assay performs correctly in a specific laboratory setting. The distinction matters because a manufacturer may have validated an assay for a general population, but the laboratory must confirm that the assay works for its own patient population and instruments. The verification process uses small sample sizes and limited measurements to confirm that the assay performs as expected. This approach is practical for routine laboratory operations because a full validation study is expensive and time-consuming. However, the small sample size means that verification has limited statistical power. A failed verification requires a more comprehensive study to determine whether the assay is truly unsuitable or whether the verification sample was not representative. ## Core Principles of Reference Interval Verification ### The 20-Sample Rule The most widely used approach for reference interval verification is the 20-sample rule. This rule requires collecting samples from 20 healthy individuals who represent the laboratory's patient population. The laboratory measures the analyte in each sample and compares the results to the manufacturer's stated reference interval. The acceptance criterion is straightforward. If no more than 2 of the 20 results fall outside the stated reference interval, the interval is considered verified for the local population. If 3 or more results fall outside the interval, the laboratory cannot verify the reference interval and must take corrective action. The statistical basis for the 20-sample rule comes from the binomial distribution. With 20 samples, the probability of observing 3 or more results outside the reference interval is low if the true percentage of healthy individuals outside the interval is 10% or less. This gives the rule reasonable sensitivity for detecting a reference interval that does not fit the local population. ### Sample Selection The 20 samples must come from individuals who represent the laboratory's patient population. This means the samples should reflect the age, sex, and health status of the population that the laboratory serves. The individuals should be healthy, meaning they do not have conditions that would affect the analyte being measured. The selection process should be documented. The laboratory should record the inclusion and exclusion criteria used to select the reference individuals. Common exclusion criteria include recent illness, pregnancy, and use of medications that affect the analyte. The laboratory should also record the demographic characteristics of the reference individuals so that the verification can be repeated if needed. ### Comparison to the Stated Reference Interval The comparison of the 20 results to the stated reference interval is a direct process. Each result is classified as inside or outside the reference interval. The laboratory then counts the number of results outside the interval and applies the 2-out-of-20 rule. This comparison does not require statistical software or complex calculations. The laboratory can perform the comparison manually or with a simple spreadsheet. The simplicity of the process is one of its strengths, because it allows verification to be performed in any laboratory setting. ## Core Principles of Reportable Range Verification The reportable range is the range of analyte values that the assay can measure accurately. It is also called the linear range or the analytical measurement range. The reportable range is established by the manufacturer during validation, and the laboratory must verify that the range is valid for its own instrument and reagents. Reportable range verification uses samples with known analyte concentrations. The laboratory measures these samples and compares the measured values to the expected values. The comparison determines whether the assay is linear across the claimed range. ### Sample Preparation The samples used for reportable range verification should span the entire range of the assay. The laboratory should use samples with concentrations at the low end, the high end, and several points in between. The samples can be prepared by diluting a high-concentration sample or by using commercially available calibration materials. The preparation of samples must be documented. The laboratory should record the source of the samples, the method used to prepare them, and the expected concentrations. This documentation is necessary for evaluating the verification results and for troubleshooting if the verification fails. ### Measurement and Comparison Each sample is measured in duplicate or triplicate, and the mean measured value is compared to the expected value. The comparison is typically expressed as a percentage deviation. The laboratory should have a predetermined criterion for acceptable deviation, which is often based on the manufacturer's recommendations or on the laboratory's quality requirements. The results are also evaluated for linearity. The measured values should be plotted against the expected values, and the relationship should be linear. The slope of the line should be close to 1.0, and the intercept should be close to zero. Deviations from linearity indicate that the assay cannot measure accurately across the entire range. ## Practical Workflow for Reference Interval Verification ### Step 1: Define the Reference Population The first step is to define the reference population. The laboratory must decide which individuals are eligible to serve as reference individuals. The definition should be based on the laboratory's patient population and the intended use of the assay. The laboratory should document the inclusion and exclusion criteria. The criteria should be specific enough to ensure that the reference individuals are healthy and representative. The documentation should also include the demographic characteristics of the reference individuals. ### Step 2: Collect Samples The laboratory collects samples from 20 eligible individuals. The samples should be collected and handled according to the assay manufacturer's instructions. The laboratory should record the collection date, the collection method, and any relevant information about the individuals. The samples should be processed and stored according to the manufacturer's instructions. Improper processing or storage can affect the results and lead to a failed verification. ### Step 3: Measure the Analyte The laboratory measures the analyte in each of the 20 samples. The measurements should be performed using the same instrument and reagents that will be used for patient testing. The laboratory should follow the manufacturer's instructions for the assay. The laboratory should also run quality control samples with the verification samples. The quality control results should be within the acceptable range before the verification results are evaluated. ### Step 4: Compare Results to the Reference Interval The laboratory compares each of the 20 results to the stated reference interval. The laboratory counts the number of results outside the interval. If 2 or fewer results are outside the interval, the reference interval is verified. If 3 or more results are outside the interval, the reference interval is not verified. ### Step 5: Document the Verification The laboratory documents the verification process and the results. The documentation should include the reference population definition, the sample collection details, the measurement results, and the verification decision. The documentation should be retained for the laboratory's records. ## Practical Workflow for Reportable Range Verification ### Step 1: Obtain Samples with Known Concentrations The laboratory obtains samples with known analyte concentrations. The samples should span the entire reportable range of the assay. The laboratory can use commercially available samples, samples prepared by dilution, or samples with values assigned by a reference method. ### Step 2: Measure the Samples The laboratory measures each sample in the assay. The laboratory should measure each sample in duplicate or triplicate to reduce the effect of random error. The laboratory should record the measured values for each sample. ### Step 3: Compare Measured Values to Expected Values The laboratory compares the measured values to the expected values. The comparison should be performed using a defined criterion for acceptable deviation. The laboratory should also evaluate the linearity of the relationship between the measured and expected values. ### Step 4: Evaluate the Results The laboratory evaluates the results to determine whether the reportable range is verified. The evaluation should consider the deviation of each measured value from the expected value and the overall linearity of the relationship. The laboratory should document the evaluation and the conclusion. ### Step 5: Escalate on Failure If the reportable range is not verified, the laboratory should escalate the issue. The escalation should include a review of the calibration, the reagents, and the instrument. The laboratory may need to contact the manufacturer for support or narrow the reportable range. ## Options and Tradeoffs in Verification ### Sample Size The 20-sample rule is the most common approach for reference interval verification. The rule is practical because it requires a relatively small number of samples. However, the rule has limited statistical power. A larger sample size would provide more confidence in the verification, but it would also require more resources. The laboratory should consider the tradeoff between sample size and confidence. For most applications, the 20-sample rule provides a reasonable balance. The laboratory should use a larger sample size when the reference interval is critical for patient care or when the local population is known to differ from the manufacturer's population. ### Sample Source The samples used for verification can come from different sources. The laboratory can use samples from healthy volunteers, samples from patients, or commercially available samples. Each source has advantages and disadvantages. Samples from healthy volunteers are the most appropriate for reference interval verification because they represent the healthy population. However, recruiting healthy volunteers can be time-consuming and expensive. Samples from patients may be easier to obtain, but they may not represent the healthy population. Commercially available samples are convenient, but they may not be representative of the local population. ### Statistical Approach The 20-sample rule is a simple statistical approach that does not require complex calculations. The rule is easy to apply and interpret. However, the rule does not provide a quantitative estimate of the confidence in the verification. A more sophisticated statistical approach would involve calculating the confidence interval for the percentage of results outside the reference interval. This approach would provide more information, but it would also require more complex calculations. The laboratory should choose the statistical approach that is appropriate for its needs and resources. ## Observations and Measurements The verification process generates several types of observations and measurements. The laboratory should record all of these data for the verification report. ### Reference Interval Verification Data The laboratory should record the following data for reference interval verification: - The reference interval stated by the manufacturer - The demographic characteristics of the reference individuals - The measured value for each of the 20 samples - The number of results outside the stated reference interval - The verification conclusion ### Reportable Range Verification Data The laboratory should record the following data for reportable range verification: - The reportable range stated by the manufacturer - The expected concentration for each sample - The measured concentration for each sample - The deviation between the measured and expected concentrations - The linearity of the relationship between the measured and expected concentrations ## Records and Documentation The laboratory should maintain records of all verification activities. The records should be complete enough to allow the verification to be evaluated by an external reviewer. The records should include the following: - The date of the verification - The personnel who performed the verification - The instrument and reagents used - The samples used and their sources - The measurement results - The verification conclusion - Any corrective actions taken The records should be retained according to the laboratory's policies and any applicable requirements. The records should be available for review by the laboratory director and by external inspectors. ## Common Failure Patterns ### Reference Interval Verification Failures A reference interval verification can fail for several reasons. The most common reason is that the reference population is not representative of the laboratory's patient population. This can happen when the reference individuals are not selected using the appropriate criteria or when the sample size is too small. Another common reason is that the assay is not performing as expected. The assay may have a calibration problem, or the reagents may be degraded. The laboratory should review the quality control data to determine whether the assay is performing correctly. A third reason is that the manufacturer's reference interval is not appropriate for the local population. This can happen when the local population has a different demographic composition than the manufacturer's population. In this case, the laboratory may need to establish its own reference interval. ### Reportable Range Verification Failure A reportable range verification can fail for several reasons. The most common reason is that the assay is not linear across the entire range. The assay may be linear at the low end but not at the high end, or the assay may have a nonlinear response in the middle of the range. Another common cause is that the calibration is not correct. The calibration may be off at one end of the range, which would cause the measured values to deviate from the expected values. The laboratory should review the calibration data to determine if the calibration is the cause. A common cause is that the samples used for verification are not prepared correctly. The samples may have been diluted incorrectly, or the expected concentrations may be incorrect. The laboratory should review the sample preparation to determine if the samples are the cause. ## Limitations of Verification Verification has several limitations that the laboratory should understand. The most important limitation is that verification with a small sample size cannot fully validate a reference interval. The 20-sample rule provides a practical check, but it does not provide the same confidence as a full reference interval study. The verification also cannot detect all problems with an assay. The verification is designed to detect problems with the reference interval and the reportable range, but it cannot detect problems with the assay's sensitivity, specificity, or other performance characteristics. The laboratory should perform a full validation study for a new assay. The verification is also limited by the quality of the samples used. If the samples are not representative of the patient population or if the samples are not prepared correctly, the verification may not be valid. The laboratory should use the appropriate samples and follow the appropriate procedures. ## Safety and Regulatory Context The verification process involves handling biological samples. The laboratory should follow the biosafety principles for handling samples. The laboratory should use the appropriate personal protective equipment and should follow the containment procedures for the samples. The laboratory should also follow the institutional oversight requirements for the use of human samples. The laboratory should have the appropriate approvals for the use of human samples in the verification process. The laboratory should follow the requirements for the protection of human subjects. The laboratory should also follow the requirements for the use of recombinant or synthetic nucleic acid molecules if the assay involves these materials. The laboratory should follow the guidelines for the use of these materials and should have the appropriate oversight. The laboratory should follow the measurement science principles for the verification process. The laboratory should use the appropriate standards and should follow the calibration procedures for the instrument. The laboratory should follow the quality principles for the verification process. ## Professional Escalation Criteria The laboratory should escalate the verification issue when the verification fails or when the laboratory cannot resolve the issue. The escalation should be to the laboratory director or to the appropriate supervisor. The escalation should include the following: - A description of the verification failure - The data from the verification - The steps taken to resolve the issue - The recommendation for the next step The laboratory should escalate the issue when the verification fails and the laboratory cannot determine the cause. The laboratory should also escalate the issue when the verification fails and the laboratory cannot resolve the issue with the available resources. The laboratory should escalate the issue when the verification indicates that the reference interval or the reportable range is not valid for the local population. The laboratory should escalate the issue to the appropriate authority to determine the next step. ## Decision Framework for Verification Under Constrained Resources Verification of reference intervals and reportable ranges is often performed under real-world constraints that the basic protocols do not fully address. A laboratory may have limited access to healthy volunteers, a tight budget for commercial samples, or an urgent need to introduce an assay before a full verification can be completed. The standard 20-sample rule and the 5 to 7 level reportable range check assume that the laboratory can obtain ideal materials and dedicate sufficient time. When these assumptions do not hold, the laboratory needs a structured way to decide whether to proceed, defer, or modify the verification plan. This section provides a practical decision framework for allocating verification effort based on risk, sample availability, and the consequences of an incorrect verification decision. ### Risk-Based Verification Tiers The intensity of verification should match the risk associated with the assay and its intended use. A laboratory can classify each new assay into one of three verification tiers. The classification is based on the clinical impact of an incorrect result, the stability of the assay chemistry, and the degree to which the local patient population may differ from the manufacturer's validation population. Tier 1 applies to assays with low clinical risk. These are assays where an incorrect result is unlikely to lead to a serious patient outcome, where the assay chemistry is well established and stable, and where the local population is expected to closely match the manufacturer's population. Examples include routine chemistry analytes that are not used for critical treatment decisions. For Tier 1 assays, the laboratory may proceed with the minimum verification protocol of 20 reference samples and 5 concentration levels. The verification can be completed in a single batch if sample availability permits. Tier 2 applies to assays with moderate clinical risk. These include assays used for diagnosis or monitoring of conditions where an incorrect result could lead to delayed treatment or unnecessary intervention. The local population may have demographic characteristics that differ from the manufacturer's population, such as a different age distribution or a higher prevalence of a condition that affects the analyte. For Tier 2 assays, the laboratory should use the full 20-sample reference interval verification and the full 5 to 7 level reportable range verification. The laboratory should also document the demographic characteristics of the reference individuals and compare them to the manufacturer's population description. Tier 3 applies to assays with high clinical risk. These include assays used for critical care decisions, for monitoring of therapies with narrow therapeutic windows, or for conditions where a false result could lead to serious harm. The assay chemistry may be new or poorly characterized, or the local population may be known to differ substantially from the manufacturer's population. For Tier 3 assays, the laboratory should consider a more extensive verification. This may include collecting more than 20 reference samples, using a larger number of reportable range levels, and performing a more detailed statistical analysis of the results. The tier classification should be documented and reviewed periodically. The laboratory director or a designated supervisor should approve the tier assignment for each assay. The classification should be revisited when the assay is changed, when the patient population changes, or when quality control data indicate a problem with the assay. ### Risk-Based Sample Size Adjustment The 20-sample rule is a minimum standard, not an optimal sample size for all situations. The laboratory can adjust the sample size based on the tier classification and on the expected difference between the local population and the manufacturer's population. The adjustment should be made before the verification begins, and the rationale for the adjustment should be documented. For Tier 1 assays, the laboratory may use fewer than 20 samples if the assay is well established and the local population is expected to match the manufacturer's population. However, the laboratory should not use fewer than 10 samples, because the statistical power of the verification becomes too low to detect a meaningful difference. With 10 samples, the probability of detecting a reference interval that does not fit the local population is substantially lower than with 20 samples. For Tier 2 assays, the laboratory should use at least 20 samples. If the local population is known to differ from the manufacturer's population, the laboratory should consider using 30 to 40 samples. The larger sample size provides more confidence in the verification and reduces the risk of a false verification. For Tier 3 assays, the laboratory should use at least 30 samples. If the assay is new or the local population is known to differ substantially, the laboratory should consider a full reference interval study with 120 or more samples. The decision to perform a full study should be made in consultation with the laboratory director and should be documented. The sample size adjustment should also consider the expected percentage of results outside the reference interval. If the laboratory expects that the local population will have a higher percentage of results outside the manufacturer's interval, the laboratory should use a larger sample size. The expected percentage can be estimated from published data, from the laboratory's own patient data, or from the clinical judgment of the laboratory director. ### Sample Availability and Alternative Sources The availability of reference samples is a common constraint. The laboratory may not be able to collect 20 fresh samples from healthy volunteers within the required time frame. The decision framework should address this constraint by providing options for alternative sample sources. The first option is to use leftover samples from routine patient testing. These samples are not ideal for reference interval verification because they come from patients who may have conditions that affect the analyte. However, the laboratory can use these samples if the patient population is well characterized and if the laboratory can apply the appropriate exclusion criteria. The laboratory should document the exclusion criteria and the demographic characteristics of the patients whose samples are used. The second option is to use commercially available reference samples. These samples are prepared from pooled human serum or plasma and have assigned values for the analyte. The laboratory should verify that the commercial samples are appropriate for the assay and that the assigned values are traceable to a reference method. The laboratory should also verify that the commercial samples are representative of the local population, which is often not the case. The third option is to use samples from a neighboring laboratory or from a reference laboratory. These samples may be available if the laboratory has a relationship with another institution. The laboratory should verify that the samples are appropriate for the local population and that the samples have been handled and stored correctly. The decision to use an alternative sample source should be documented. The documentation should include the source of the samples, the number of samples used, the exclusion criteria applied, and the rationale for using the alternative source. The laboratory should also document the limitations of the alternative source and the impact of these limitations on the verification conclusion. ### Sequential Verification Approach A sequential verification approach can reduce the total sample requirement while maintaining confidence in the verification. The laboratory collects samples in batches and evaluates the results after each batch. The verification is stopped early if the results are clearly acceptable or clearly unacceptable. The sequential approach begins with a small batch of 10 samples. The laboratory measures the analyte in these samples and compares the results to the stated reference interval. If all 10 results fall within the interval, the laboratory may proceed to the second batch of 10 samples. If 3 or more results fall outside the interval, the verification fails and the laboratory does not need to collect additional samples. If the first batch has 1 or 2 results outside the interval, the laboratory should collect the second batch of 10 samples. The laboratory then evaluates the combined results from both batches. If the combined results have 2 or fewer results outside the interval, the verification is successful. If the combined results have 3 or more results outside the interval, the verification fails. The sequential approach has a statistical advantage. The probability of a false verification is lower with the sequential approach than with a single batch of 20 samples, because the sequential approach allows the laboratory to stop early when the results are clearly unacceptable. The sequential approach also allows the laboratory to use fewer samples when the results are clearly acceptable. The sequential approach requires a clear stopping rule. The laboratory should document the stopping rule before the verification begins. The stopping rule should specify the number of samples in each batch, the number of batches, and the criteria for stopping early. ### Reportable Range Verification Under Material Constraints The reportable range verification requires samples with known concentrations that span the entire range. The laboratory may not have access to commercial samples at all required concentration levels. The decision framework should address this constraint by providing a structured approach to sample preparation and verification. The laboratory can prepare samples by diluting a high-concentration sample with a diluent. The dilution should be performed using a calibrated pipette and a documented procedure. The expected concentration of each diluted sample is calculated from the concentration of the stock sample and the dilution factor. The laboratory should verify the dilution procedure by measuring the diluted samples in duplicate and comparing the measured values to the expected values. The laboratory can also prepare samples by spiking a low-concentration sample with a known amount of the analyte. The spiking procedure should be documented and the expected concentration should be calculated from the concentration of the base sample and the amount of analyte added. The laboratory should verify the spiking procedure by measuring the spiked samples in duplicate. The laboratory should use at least 5 concentration levels for the reportable range verification. If the laboratory cannot obtain samples at all 5 levels, the laboratory should use the levels that are available and should document the limitation. The laboratory should also document the range of concentrations that were verified and the range that was not verified. The decision to narrow the reportable range should be based on the verification results. If the measured values deviate from the expected values at the high end of the range, the laboratory should narrow the reportable range to the highest concentration that was verified. The laboratory should document the narrowed range and the rationale for the narrowing. ### Escalation and Consultation Criteria The decision framework should include clear criteria for escalating the verification issue to a higher authority. The laboratory should escalate the issue when the verification fails and the laboratory cannot determine the cause. The laboratory should also escalate the issue when the verification indicates that the reference interval or the reportable range is not valid for the local population. The escalation should be to the laboratory director or to the appropriate supervisor. The escalation should include a description of the verification failure, the data from the verification, the steps taken to resolve the issue, and the recommendation for the next step. The escalation should be documented and retained for the laboratory's records. The laboratory should also escalate the issue when the verification cannot be completed due to a lack of samples or materials. The laboratory should document the reason for the incomplete verification and the steps taken to obtain the necessary materials. The laboratory should not introduce the assay for patient testing until the verification is complete. ### Comparison of Verification Approaches The decision framework provides a structured approach to verification that is distinct from the basic protocol. The basic protocol assumes that the laboratory can collect 20 reference samples and 5 to 7 concentration levels without difficulty. The decision framework recognizes that these assumptions may not hold and provides a structured approach for making decisions under constraints. The decision framework also provides a more nuanced approach to sample size. The basic protocol uses a fixed sample size of 20. The decision framework allows the laboratory to adjust the sample size based on the risk classification and the expected difference between the local population and the manufacturer's population. The decision framework also provides a more structured approach to the use of alternative sample sources. The basic protocol assumes that the laboratory can collect samples from healthy volunteers. The decision framework recognizes that the laboratory may need to use alternative sources and provides a structured approach for evaluating the suitability of these sources. The decision framework is not a replacement for the basic verification protocol. The basic protocol provides the minimum requirements for verification. The decision framework provides a structured approach for applying the basic protocol under real-world constraints. The laboratory should use the decision framework to plan the verification and the basic protocol to execute the verification. ### Implementation Steps for the Decision Framework The laboratory should implement the decision framework in a structured manner. The implementation should include the following steps: 1. Classify each assay into a verification tier based on the clinical risk, the assay chemistry, and the expected difference between the local population and the manufacturer's population. Document the tier assignment and the rationale for the assignment. 2. Determine the sample size for the reference interval verification based on the tier classification and the expected percentage of results outside the reference interval. Document the sample size and the rationale for the sample size. 3. Identify the sample sources for the reference interval verification. Determine whether the laboratory can collect samples from healthy individuals or whether the laboratory needs to use alternative sources. Document the sample sources and the rationale for the sources. 4. Determine the sample preparation method for the reportable range verification. Determine whether the laboratory can obtain samples at all required concentration levels or whether the laboratory needs to prepare samples by dilution or spiking. Document the preparation method and the rationale for the method. 5. Perform the verification according to the documented plan. Record the results and the verification conclusion. 6. Evaluate the verification results and determine whether the verification is successful. If the verification fails, investigate the cause and escalate the issue if necessary. 7. Document the verification process and the results. Retain the documentation for the laboratory's records. The implementation steps should be reviewed by the laboratory director or the responsible supervisor. The review should confirm that the verification plan is appropriate for the assay and the laboratory setting. The review should also confirm that the verification plan is consistent with the laboratory's quality requirements. ### Records for the Decision Framework The laboratory should maintain records of the decision framework and the verification process. The records should include the tier classification for each assay, the sample size used for the verification, the sample sources used, the sample preparation methods used, and the verification results. The records should also include the rationale for each decision and the limitations of the verification. The records should be retained according to the laboratory's policies and any applicable requirements. The records should be available for review by the laboratory director and by external inspectors. The records should be complete enough to allow the verification to be evaluated by an external reviewer. The records should also include the escalation decisions and the outcomes of the escalation. The records should document the issue that was escalated, the person to whom the issue was escalated, and the outcome of the escalation. The records should be retained for the laboratory's records. ### Common Failure Patterns in the Decision Framework The decision framework can fail if the laboratory does not follow the documented plan. The most common failure is the use of an inappropriate sample source. The laboratory may use samples from patients who have conditions that affect the analyte, which can lead to a false verification failure or a false verification success. Another common failure is the use of an inappropriate sample size. The laboratory may use a sample size that is too small for the risk classification, which can lead to a false verification success. The laboratory may also use a sample size that is too large for the risk classification, which can lead to unnecessary resource use. A third common failure is the use of an inappropriate sample preparation method for the reportable range verification. The laboratory may use a dilution method that is not accurate, which can lead to a false verification failure. The laboratory may also use a spiking method that is not accurate, which can lead to a false verification success. The laboratory should review the decision framework process periodically to identify and correct these failure patterns. The review should include a review of the tier classifications, the sample sizes, the sample sources, and the sample preparation methods. The review should also include a review of the verification results and the verification conclusions. ### Comparison of Verification Approaches The decision framework provides a structured approach for verification under resource constraints. The framework is based on the principles of risk assessment and the principles of measurement science. The framework is consistent with the principles of the basic verification protocol and provides a structured way to apply the protocol under constraints. The framework is not a replacement for the basic verification protocol. The basic protocol provides the minimum requirements for verification. The framework provides a structured way to apply the requirements under constraints. The laboratory should use the framework to plan the verification and the basic protocol to apply the verification. The framework is also not a replacement for a full reference interval study. The framework provides a structured way to verify a reference interval with a small sample size. The framework does not provide the same confidence as a full reference interval study. The laboratory should perform a full reference interval study when the verification fails or when the risk classification indicates that a full study is necessary. The framework is consistent with the principles of the measurement science. The framework uses the principles of risk assessment and the principles of measurement uncertainty. The framework also uses the principles of quality control and the principles of documentation. The framework is consistent with the principles of the laboratory quality system. ## Frequently Asked Questions ### What is the difference between reference interval verification and a full reference interval study? Reference interval verification uses a small sample of 20 individuals to confirm that a manufacturer's stated reference interval is valid for the local population. A full reference interval study uses a larger sample of at least 120 individuals to establish a new reference interval for the local population. Verification is a practical check, while a full study is a more comprehensive and statistically robust process. ### How many samples are needed for reference interval verification? The standard approach uses 20 samples from healthy individuals. The laboratory compares the results to the stated reference interval and counts the number of results outside the interval. If 2 or fewer results are outside the interval, the reference interval is verified. If 3 or more results are outside the interval, the reference interval is not verified. ### What should a laboratory do when reference interval verification fails? The laboratory should first review the quality control data to determine if the assay is performing correctly. The laboratory should also review the reference population to determine if the samples were representative. If the assay is performing correctly and the samples were representative, the laboratory may need to perform a full reference interval study to establish a new reference interval. ### What is the reportable range and why does it need verification? The reportable range is the range of analyte values that an assay can measure accurately without dilution or concentration. The range is established by the manufacturer and must be verified by the laboratory to confirm that the assay can measure accurately across the range on the laboratory's instrument. Verification involves measuring samples with known concentrations and comparing the measured values to the expected values. ### How is the reportable range verified? The reportable range is verified by measuring samples with known concentrations that span the entire range. The laboratory measures each sample in duplicate or triplicate and compares the measured values to the expected values. The laboratory evaluates the linearity of the relationship between the measured and expected values to determine if the range is verified. ### What is the difference between the reportable range and the analytical measurement range? The reportable range is the range of analyte values that the laboratory can report for patient samples. The analytical measurement range is the range of values that the assay can measure directly without dilution or concentration. The reportable range may be wider than the analytical measurement range if the laboratory uses dilution or concentration to extend the range. ### What are the limitations of verification with a small sample size? Verification with a small sample size has limited statistical power. The 20-sample rule can detect large differences between the manufacturer's reference interval and the local population, but it cannot detect small differences. A failed verification requires a full reference interval study, but a passed verification does not guarantee that the reference interval is optimal for the local population. ### What records should be kept for the verification process? The laboratory should keep records of the verification date, the personnel who performed the verification, the instrument and reagents used, the sample collection details, the measurement results, the verification conclusion, and any corrective action taken. The records should be retained for the laboratory's records and should be available for review by the laboratory or by external inspectors. ## Using the Evidence | Source | Best use in this topic | Important limitation | |---|---|---| | [Molecular Biology and Laboratory Methods](https://www.ncbi.nlm.nih.gov/books) | official guidance | Check the linked page for current local requirements | | [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html) | official guidance | Check the linked page for current local requirements | | [NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules](https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules) | official guidance | Check the linked page for current local requirements | ## Related Diagnostic Guides - [Reference Intervals in Clinical Chemistry: Establishment and Verification](/knowledge/diagnostics/clinical-pathology/reference-intervals-in-clinical-chemistry-establishment-and-verification) - [Establishing Reference Intervals in Veterinary Clinical Chemistry](/knowledge/diagnostics/clinical-chemistry/establishing-reference-intervals-in-veterinary-clinical-chemistry) - [Method Validation in Clinical Laboratories: A Practical Protocol](/knowledge/diagnostics/emerging-tech/method-validation-in-clinical-laboratories-a-practical-protocol) - [Cell Viability Assays: Choosing the Right Method for Your Experiment](/knowledge/diagnostics/emerging-tech/cell-viability-assays-choosing-the-right-method-for-your-experiment) ## References and Further Reading - [Molecular Biology and Laboratory Methods](https://www.ncbi.nlm.nih.gov/books). National Library of Medicine. - [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html). CDC and NIH. - [NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules](https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules). National Institutes of Health. - [Addgene Protocols](https://www.addgene.org/protocols). Addgene. - [MIQE Guidelines](https://academic.oup.com/clinchem/article/55/4/611/5631762). Clinical Chemistry. - [NIST Laboratory Programs](https://www.nist.gov/laboratories). National Institute of Standards and Technology. - [Laboratory Biosafety Manual](https://www.who.int/publications/i/item/9789240011311). World Health Organization. - [NCBI Literature Resources](https://www.ncbi.nlm.nih.gov/). National Center for Biotechnology Information. - [Verification of reference intervals in routine clinical laboratories: practical challenges and recommendations.](https://pubmed.ncbi.nlm.nih.gov/29729142). Clinical chemistry and laboratory medicine, 2018. - [Verification and quality control of routine hematology analyzers.](https://pubmed.ncbi.nlm.nih.gov/27161194). International journal of laboratory hematology, 2016. - [Pediatric reference interval verification for 17 specialized immunoassays and cancer markers on the Abbott Alinity i system in the CALIPER cohort of healthy children and adolescents.](https://pubmed.ncbi.nlm.nih.gov/36117243). Clinical chemistry and laboratory medicine, 2023. > This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.