Calibration Hierarchies Explained

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-27

Quick Answer

• A calibration hierarchy is a documented chain connecting a laboratory's routine measurements to a national or international measurement standard through successive comparisons.
• Design the hierarchy by selecting a reference material with stated metrological traceability, then calibrating each lower level against the level above it while recording uncertainty at every step.
• The hierarchy is only as valid as its documentation, so a missing uncertainty statement or an unrecorded comparison breaks the traceability chain.

What a Calibration Hierarchy Is and Why It Matters

A calibration hierarchy is the ordered sequence of calibrations that connects a measurement result to a stated reference. In a diagnostic laboratory, this sequence typically runs from a primary reference material through secondary calibrators to the working calibrators used in routine assays. Each step in the sequence involves a comparison between a higher-level standard and a lower-level standard, and each comparison contributes an uncertainty component that must be accounted for.

The purpose of the hierarchy is metrological traceability. A result is traceable when it can be related to a stated reference through an unbroken chain of documented calibrations, each with a stated uncertainty. Without this chain, a result is only a number produced by an instrument. With the chain, the result carries meaning that can be compared across laboratories, instruments, and time.

For diagnostic assays, the hierarchy matters because patient results are interpreted against reference intervals and clinical decision limits. If the calibrator used to set the assay response is not traceable to a recognized reference, the reference intervals themselves become unreliable. The same principle applies to research laboratories that generate quantitative data for publication, where reviewers and readers expect that measurements can be compared with those from other groups.

The National Institute of Standards and Technology operates laboratory programs that support measurement science, standards, and calibration across many fields. These programs provide the infrastructure that makes traceability possible, including reference materials, calibration services, and measurement assurance methods. A laboratory that wants to establish a defensible calibration hierarchy should understand what reference materials and services are available from such institutions before designing its own chain.

Core Principles of a Calibration Hierarchy

Metrological Traceability

Metrological traceability is the property of a measurement result whereby the result can be related to a stated reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty. The chain must be documented, and the documentation must include the reference used, the calibration procedure, the measurement results, and the uncertainty at each step.

A traceability chain has three essential components. First, the reference must be stated. This can be a primary standard, a certified reference material, or a reference method. Second, the chain must be unbroken, meaning that every comparison between the reference and the final result is accounted for. Third, the uncertainty must be evaluated at each step and combined to give the uncertainty of the final result.

Measurement Uncertainty

Measurement uncertainty is a parameter that characterizes the dispersion of values that could reasonably be attributed to a measurand. In a calibration hierarchy, uncertainty accumulates at each step. The uncertainty of the working calibrator is therefore always larger than the uncertainty of the reference material at the top of the chain.

Uncertainty is expressed as a standard uncertainty or an expanded uncertainty. The expanded uncertainty is the standard uncertainty multiplied by a coverage factor, typically 2 for a 95 percent confidence level. When a laboratory reports a result, it should report the uncertainty so that the user of the result knows the range within which the true value is expected to lie.

The Calibration Chain

The calibration chain is the sequence of comparisons that connects the working calibrator to the reference. Each comparison is a calibration. The chain can be represented as a series of levels, with the reference at the top and the working calibrator at the bottom.

A typical chain in a clinical or research laboratory has the following levels:

1. Primary reference material or reference method
2. Secondary calibrator or manufacturer working calibrator
3. Laboratory working calibrator
4. Assay calibration curve
5. Patient or sample result

Each level is calibrated against the level above it. The calibration at each level produces a measurement result and an uncertainty. The uncertainty is propagated down the chain, so the uncertainty at the bottom is the combination of all the uncertainties from the steps above.

At a Glance

Selecting Reference Materials

Types of Reference Materials

Reference materials are materials that have sufficiently homogeneous and well-established properties to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials. They come in several forms.

Certified reference materials have a value that is certified by a procedure that establishes traceability to an accurate realization of the unit in which the property is expressed. The certificate includes the certified value and the uncertainty. These materials are the most reliable basis for a calibration hierarchy.

Reference materials that are not certified may still be useful for internal quality control, but they do not provide traceability. A laboratory that uses a non-certified material as the top of its hierarchy has not established traceability to a recognized reference.

Selection Criteria

When selecting a reference material for the top of a calibration hierarchy, the laboratory should consider the following criteria:

• The material must be relevant to the measurand. A reference material for a different matrix or a different analyte does not establish traceability for the assay.
• The material must have a stated value and uncertainty. Without these, the material cannot serve as a reference.
• The material must be stable under the conditions of use and storage. A material that degrades during the calibration period introduces an unquantified uncertainty.
• The material must be available in sufficient quantity for the intended use. A material that is available only in a small quantity may not be practical for routine calibration.

Sources of Reference Materials

Reference materials are available from national metrology institutes, such as NIST, and from commercial suppliers. The NIST laboratory programs provide reference materials and calibration services that support measurement science. A laboratory should verify that the reference material it selects is appropriate for its intended use and that the certificate of analysis is current.

The certificate of analysis is the document that states the certified value, the uncertainty, and the conditions of use. The laboratory must retain this certificate as part of the calibration documentation. The certificate also states the period of validity, and the laboratory must ensure that the material is used within that period.

Building the Calibration Chain

Step 1: Define the Measurand and the Reference

The first step in building a calibration chain is to define the measurand. The measurand is the quantity intended to be measured, such as the concentration of a specific protein in a serum sample. The definition must be specific enough that the reference material can be selected.

The reference is the material or method that defines the measurand. For a protein, the reference may be a certified reference material with a value assigned by a reference method. For a nucleic acid, the reference may be a reference material with a certified copy number or concentration.

Step 2: Select the Reference Material

The reference material is selected according to the criteria described above. The laboratory must verify that the material is appropriate for the measurand and that the certificate is current.

Step 3: Calibrate the Working Calibrator

The working calibrator is the material that is used in the routine assay. It is calibrated against the reference material. The calibration involves measuring the working calibrator and the reference material in the same assay, or using a reference method to assign a value to the working calibrator.

The calibration must be performed with a sufficient number of replicates to estimate the uncertainty. The result of the calibration is a value for the working calibrator and an uncertainty.

Step 4: Document the Chain

The documentation of the calibration chain is as important as the calibration itself. The documentation must include:

• The identity of the reference material and its certificate
• The identity of the working calibrator and its preparation
• The calibration procedure and the number of replicates
• The calibration results and the uncertainty at each step
• The date of the calibration and the personnel who performed it

The documentation must be retained for the period required by the laboratory quality system.

Step 5: Verify the Chain

The chain is verified by measuring a control material with a known value. The control material is measured in the same assay as the working calibrator, and the result is compared to the expected value. If the result is within the acceptable range, the chain is considered valid.

The verification is repeated at intervals, and the results are recorded. A control result that is outside the acceptable range indicates a problem with the chain, and the laboratory must investigate.

Uncertainty Propagation in the Calibration Chain

Sources of Uncertainty

Uncertainty in a calibration chain arises from several sources. The reference material has an uncertainty stated on its certificate. The calibration procedure introduces uncertainty from the measurement process, including the instrument, the operator, and the environment. The working calibrator has an uncertainty from its preparation and from the calibration.

The uncertainty of the final result is the combination of all these uncertainties. The combination is performed using the rules of uncertainty propagation, which combine the standard uncertainties of the individual components.

Combining Uncertainties

The combined uncertainty is calculated by taking the square root of the sum of the squares of the individual standard uncertainties. This is the root sum of squares method. The combined uncertainty is then multiplied by a coverage factor to obtain the expanded uncertainty.

The uncertainty budget is a table that lists each source of uncertainty, its value, and its contribution to the combined uncertainty. The budget is used to identify the largest sources of uncertainty and to guide improvements.

Reporting Uncertainty

The uncertainty is reported with the result. The report should state the result, the expanded uncertainty, and the coverage factor. The report should also state the reference to which the result is traceable.

The uncertainty is a property of the result, and it is not a property of the instrument or the method. A result without an uncertainty is incomplete.

Practical Workflow for Establishing a Hierarchy

Assessment Steps

1. Define the measurand and the intended use of the result.
2. Identify the reference material that defines the measurand.
3. Verify the reference material certificate and the validity period.
4. Prepare the working calibrator and document the preparation.
5. Calibrate the working calibrator against the reference material.
6. Calculate the uncertainty of the working calibrator.
7. Verify the chain with a control material.
8. Document the entire chain and retain the records.

Records and Measurements

The records of the calibration chain include the certificate of the reference material, the preparation records of the working calibrator, the calibration results, the uncertainty budget, and the verification records. The records must be legible, complete, and retained for the required period.

The measurements are recorded with the units and the uncertainty. The records must be traceable to the instrument and the operator.

Common Failure Patterns

The most common failure in a calibration chain is a break in the documentation. A missing certificate, an incomplete record, or a calibration that was not recorded breaks the chain. The chain is only as valid as its documentation.

Another common failure is the use of a reference material that is not certified or that is not appropriate for the measurand. This failure is often detected when the control material results are outside the acceptable range.

A third failure is the use of a reference material beyond its validity period. The certificate states the validity period, and the laboratory must ensure that the material is used within that period.

Quality Controls and Verification

Internal Quality Control

Internal quality control is the routine monitoring of the assay to ensure that the results are reliable. The control material is measured in each assay, and the results are plotted on a control chart. The control chart is used to detect trends and shifts in the assay.

The control material must be stable and have a value that is within the measuring range of the assay. The control material is not a calibrator, and it does not provide traceability. The control material is used to monitor the assay, not to calibrate it.

External Quality Assessment

External quality assessment is the comparison of the laboratory results with the results of other laboratories. The laboratory receives a sample with a known value, and the laboratory measures the sample and reports the result. The result is compared to the results of other laboratories and to the reference value.

External quality assessment is a check on the calibration chain. If the laboratory result is outside the acceptable range, the laboratory must investigate the cause.

The Role of the MIQE Guidelines

The MIQE guidelines provide a framework for the reporting of qPCR experiments. The guidelines require the reporting of the calibration and the controls used in the experiment. The guidelines are relevant to the calibration hierarchy because they require the transparency that is necessary for the interpretation of the results.

The MIQE guidelines are published in Clinical Chemistry and are available at the provided URL. The guidelines are a reference for the reporting of qPCR data, and they are not a protocol for the calibration of a qPCR assay.

Biosafety and Institutional Oversight

Biosafety Principles

The calibration of a laboratory assay may involve the use of biological materials, including reference materials and control materials. The laboratory must follow the biosafety principles that are appropriate for the materials in use.

The Biosafety in Microbiological and Biomedical Laboratories (BMBL) provides the principles of biosafety, including risk assessment, containment, and institutional oversight. The BMBL is a joint publication of the CDC and the NIH, and it is available at the CDC website.

The laboratory must conduct a risk assessment for the materials in use and select the appropriate containment level. The risk assessment must consider the nature of the material, the procedure, and the potential for exposure.

Institutional Oversight

The laboratory must have institutional oversight for the use of biological materials. The oversight includes the review of the risk assessment and the approval of the procedures.

The NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules provide the oversight for research involving recombinant or synthetic nucleic acids. The guidelines are available at the NIH website.

The laboratory must comply with the institutional requirements for the use of biological materials. The requirements include the training of the personnel and the documentation of the procedures.

Limitations and Professional Escalation

Limitations of the Calibration Hierarchy

The calibration hierarchy is a model of the measurement process. The model is a simplification, and it does not capture all the sources of variation in the laboratory. The uncertainty budget is an estimate, and it may not include all the sources of uncertainty.

The hierarchy is only as valid as the reference material. If the reference material is not appropriate for the measurand, the hierarchy is not valid. The laboratory must verify the appropriateness of the reference material.

The hierarchy is only as valid as the documentation. If the documentation is incomplete, the traceability is broken. The laboratory must ensure that the documentation is complete and retained.

Professional Escalation Criteria

The laboratory should escalate the issue to a supervisor or a quality manager when the following conditions are met:

• The control results are outside the acceptable range and the cause is not identified.
• The reference material is not available or is expired.
• The calibration results are not reproducible.
• The uncertainty of the calibration is larger than the acceptable limit.

The escalation is a professional judgment, and the laboratory should not delay the escalation when the issue is significant.

Decision Framework for Selecting a Calibration Hierarchy Design

The Core Decision Problem

Laboratory personnel often assume that a single calibration hierarchy design can serve all assays in the laboratory. This assumption leads to practical failures when the hierarchy does not match the assay characteristics. The decision framework presented here addresses the specific problem of matching hierarchy design to assay type, available reference materials, and laboratory capability.

The framework uses a structured set of questions that lead to one of four hierarchy designs. Each design has distinct documentation requirements, uncertainty characteristics, and operational constraints. The framework is not a substitute for professional judgment, but it provides a systematic way to evaluate the options before committing resources to a particular design.

Decision Question 1: What Is the Nature of the Measurand?

The first decision point is the nature of the measurand. The measurand is the quantity intended to be measured, and its nature determines which reference materials are available and how the hierarchy can be constructed.

For a small molecule analyte, such as glucose or creatinine, the measurand is a well-defined chemical entity. Reference materials are typically available as certified reference materials with a stated mass concentration or amount of substance concentration. The hierarchy can be constructed with a primary reference material at the top, and the calibration chain is relatively straightforward because the analyte is stable and well characterized.

For a protein analyte, the measurand is more complex. The protein may exist in multiple forms, such as different isoforms or glycosylation states. The reference material must be representative of the clinically relevant form of the protein. The hierarchy may require a reference method in addition to a reference material, because the value of the reference material may depend on the method used to assign it.

For a nucleic acid target, the measurand is a sequence of nucleotides. The reference material may be a plasmid, a synthetic nucleic acid, or a genomic DNA preparation. The hierarchy must account for the fact that the measured quantity may be a copy number, a concentration, or a mass, and the conversion between these units is not always straightforward.

The decision at this step is to identify the measurand class and to verify that the reference material is appropriate for that class. A reference material for a protein cannot establish traceability for a nucleic acid assay, and a reference material for a small molecule cannot establish traceability for a protein assay.

Decision Question 2: What Reference Materials Are Available?

The second decision is the availability of reference materials. The laboratory must determine whether a certified reference material exists for the measurand, whether a reference method is available, or whether the laboratory must rely on a manufacturer calibrator.

The National Institute of Standards and Technology operates laboratory programs that provide reference materials and calibration services across many fields. The laboratory should search the NIST catalog and the catalogs of other national metrology institutes to determine whether a certified reference material is available for the measurand. If a certified reference material is available, the hierarchy can be constructed with that material at the top.

If a certified reference material is not available, the laboratory may use a reference method. A reference method is a method that has been validated to provide accurate results for the measurand. The hierarchy is then constructed with the reference method at the top, and the working calibrator is calibrated against the reference method.

If neither a certified reference material nor a reference method is available, the laboratory must rely on a manufacturer calibrator. The manufacturer calibrator is a material that is provided by the manufacturer of the assay kit, and the value is assigned by the manufacturer. The hierarchy is then constructed with the manufacturer calibrator at the top, and the traceability is to the manufacturer's stated reference.

The decision at this step is the availability of the reference. The laboratory must document the basis for the reference, and the documentation must state whether the reference is a certified reference material, a reference method, or a manufacturer calibrator.

Decision Question 3: What Is the Required Uncertainty?

The third decision is the required uncertainty of the final result. The required uncertainty is determined by the intended use of the result. A result that is used for a clinical decision has a different uncertainty requirement than a result that is used for research.

The laboratory must define the acceptable uncertainty for the intended use. The acceptable uncertainty is typically expressed as a percentage of the result or as an absolute value. The acceptable uncertainty is then used to evaluate the hierarchy design.

The uncertainty of the final result is the combination of the uncertainties at each step of the hierarchy. The uncertainty at the bottom of the hierarchy is always larger than the uncertainty at the top. The laboratory must estimate the uncertainty at each step and determine whether the combined uncertainty meets the requirement.

If the combined uncertainty is larger than the acceptable uncertainty, the laboratory must either reduce the uncertainty at one or more steps or accept a higher uncertainty. The reduction of uncertainty may require a better reference material, a more precise calibration procedure, or a larger number of replicates.

The decision at this step is the acceptable uncertainty. The laboratory must document the acceptable uncertainty and the basis for that value.

Decision Question 4: What Is the Operational Context?

The fourth decision is the operational context of the laboratory. The operational context includes the number of assays performed, the frequency of calibration, the skill of the personnel, and the resources available for documentation.

A laboratory that performs a high volume of assays may require a hierarchy that is efficient and that minimizes the time between calibration and reporting. A laboratory that performs a low volume of assays may be able to use a more complex hierarchy with a longer calibration time.

The operational context also includes the skill of the personnel. A hierarchy that requires a complex calibration procedure may not be appropriate for a laboratory with limited personnel training. The laboratory must ensure that the personnel are trained in the calibration procedure and that the training is documented.

The operational context includes the documentation requirements. A hierarchy with more steps requires more documentation, and the laboratory must have the resources to maintain the documentation. The documentation must be retained for the period required by the laboratory quality system.

The decision at this step is the operational context. The laboratory must choose a hierarchy that is consistent with the operational context.

The Decision Matrix

The four decisions are combined into a decision matrix. The matrix has four columns, one for each decision, and the rows are the possible outcomes. The matrix is used to select the hierarchy design.

The matrix is a tool for the laboratory to evaluate the options. The laboratory selects the option in each row that matches its situation, and the combination of the options determines the hierarchy design.

Hierarchy Design 1: Certified Reference Material with Low Uncertainty

This design is used when a certified reference material is available, the required uncertainty is low, and the laboratory has the resources to maintain a rigorous hierarchy. The hierarchy has the following levels:

1. Certified reference material
2. Secondary calibrator
3. Working calibrator
4. Assay calibration curve
5. Patient or sample result

The certified reference material is the top of the hierarchy. The secondary calibrator is calibrated against the certified reference material, and the working calibrator is calibrated against the secondary calibrator. The calibration at each step is performed with a sufficient number of replicates to estimate the uncertainty.

The documentation for this design includes the certificate of the certified reference material, the calibration records for the secondary calibrator and the working calibrator, and the uncertainty budget. The documentation must be complete and retained.

Case Design 2: Reference Method and Moderate Uncertainty

This design is selected when a certified reference material is not available, but a reference method is available. The required uncertainty is moderate, and the operational context is medium volume.

The hierarchy has the following levels:

1. Reference method
2. Secondary calibrator
3. Working calibrator
4. Assay calibration curve
5. Patient or sample result

The reference method is the top of the hierarchy. The secondary calibrator is calibrated against the reference method, and the working calibrator is calibrated against the secondary calibrator. The uncertainty is estimated from the reference method and the calibration steps.

The documentation for this design includes the validation records for the reference method, the calibration records for the secondary calibrator and the working calibrator, and the uncertainty budget.

Case Design 3: Manufacturer Calibrator and Higher Uncertainty

This design is selected when neither a certified reference material nor a reference method is available. The required uncertainty is higher, and the operational context is high volume with limited staff.

The hierarchy has the following levels:

1. Manufacturer calibrator
2. Working calibrator
3. Assay calibration curve
4. Patient or sample result

The manufacturer calibrator is the top of the hierarchy. The working calibrator is calibrated against the manufacturer calibrator, and the assay calibration curve is constructed from the working calibrator. The traceability is to the manufacturer's stated value.

The documentation for this design includes the manufacturer's certificate or value assignment report, the calibration records for the working calibrator, and the uncertainty budget. The laboratory must verify that the manufacturer's value is appropriate for the intended use.

Implementation Steps for the Decision Framework

The implementation of the decision framework follows a structured sequence. The laboratory must complete each step before proceeding to the next.

Step 1: Define the measurand and the intended use of the result. The definition must be specific enough to identify the measurand class and the required uncertainty.

Step 2: Search for a certified reference material. The search includes the NIST catalog and the catalogs of other reference material producers. The search is documented, and the results are recorded.

Step 3: If a certified reference material is not available, evaluate whether a reference method is available. The reference method must be validated and documented.

Step 4: If neither a certified reference material nor a reference method is available, select the manufacturer calibrator. The manufacturer calibrator is documented, and the traceability is to the manufacturer's stated value.

Step 5: Estimate the uncertainty of the final result. The uncertainty is estimated from the reference, the calibration steps, and the assay. The estimate is compared to the acceptable uncertainty.

Step 6: Select the hierarchy design that matches the decisions. The design is documented, and the documentation is retained.

Step 7: Implement the hierarchy and verify the chain with a control material. The verification is performed at intervals, and the results are recorded.

Records and Measurements for the Decision Framework

The records for the decision framework include the decision matrix, the search results for the reference material, the evaluation of the reference method, the selection of the manufacturer calibrator, and the uncertainty estimate. The records must be legible, complete, and retained for the required period.

The measurements for the decision framework include the calibration results at each step of the hierarchy. The measurements are recorded with the units and the uncertainty. The records must be traceable to the instrument and the operator.

The records must also include the date of the decision and the personnel who made the decision. The decision is a professional judgment, and the basis for the judgment must be documented.

Common Failure Patterns in the Decision Framework

The most common failure in the decision framework is the selection of a hierarchy that does not match the measurand. The laboratory may select a certified reference material for a measurand that does not have a certified reference material, or the laboratory may select a manufacturer calibrator when a certified reference material is available. This failure is detected when the control results are outside the acceptable range or when the uncertainty is larger than the acceptable limit.

Another common failure is the selection of a hierarchy that is too complex for the operational context. The laboratory may select a hierarchy with a reference method when the personnel are not trained to perform the reference method. This failure is detected when the calibration results are not reproducible.

A third failure is the selection of a hierarchy that does not meet the required uncertainty. The laboratory may select a hierarchy with a manufacturer calibrator when the required uncertainty is tight. This failure is detected when the uncertainty of the final result is larger than the acceptable limit.

Troubleshooting the Decision Framework

When a failure is detected, the laboratory must troubleshoot the decision framework. The troubleshooting is a systematic process that identifies the cause of the failure and corrects the cause.

The first step in troubleshooting is to review the decision matrix. The laboratory must verify that the decisions are correct and that the hierarchy design matches the decisions. If the decisions are incorrect, the laboratory must correct the decisions and select a new hierarchy design.

The second step is to review the reference material. The laboratory must verify that the reference material is appropriate for the measurand and that the certificate is current. If the reference material is not appropriate, the laboratory must select a new reference material.

The third step is to review the calibration records. The laboratory must verify that the calibration was performed correctly and that the uncertainty was estimated correctly. If the calibration records are incomplete, the laboratory must repeat the calibration.

The fourth step is to review the control results. The laboratory must verify that the control results are within the acceptable range. If the control results are outside the acceptable range, the laboratory must investigate the cause and correct the cause.

The troubleshooting process is documented, and the documentation is retained. The documentation includes the cause of the failure, the corrective action, and the verification of the correction.

Comparison of the Decision Framework with the Standard Hierarchy

The decision framework is a systematic method for selecting a hierarchy design. The standard hierarchy is a single design that is applied to all assays. The decision framework is distinct because it requires the laboratory to evaluate the measurand, the reference, the uncertainty, and the operational context before selecting a design.

The decision framework is not a replacement for the standard hierarchy. The standard hierarchy is the sequence of calibrations that connects the result to the reference. The decision framework is the method for selecting the design of the hierarchy. The decision framework is used before the hierarchy is constructed, and the standard hierarchy is used after the design is selected.

The decision framework is also distinct because it requires the laboratory to document the basis for the design. The documentation includes the decision matrix, the search results, and the uncertainty estimate. The documentation is the basis for the traceability of the hierarchy.

Professional Escalation Criteria for the Decision Framework

The laboratory should escalate the issue to a supervisor or a quality manager when the following conditions are met:

• The decision framework does not produce a hierarchy that meets the required uncertainty.
• The reference material is not available for the measurand.
• The reference method is not validated for the measurand.
• The manufacturer calibrator is not reliable for the intended use.
• The control results are outside the acceptable range and the cause is not identified.

The escalation is a professional judgment, and the laboratory should not delay the escalation when the issue is significant. The escalation is documented, and the documentation is retained.

Limitations of the Decision Framework

The decision framework is a model of the decision process. The model is a simplification, and it does not capture all the factors that influence the selection of a hierarchy design. The laboratory must use professional judgment in applying the framework.

The framework is only as valid as the information used to make the decisions. If the information is incorrect, the decisions are incorrect, and the hierarchy is not valid. The laboratory must verify the information before making the decisions.

The framework is only as valid as the documentation. If the documentation is incomplete, the basis for the hierarchy is not clear. The laboratory must ensure that the documentation is complete and retained.

The framework is not a substitute for the professional judgment of the laboratory personnel. The laboratory personnel must use the framework as a tool, and they must apply their professional judgment to the specific situation.

Frequently Asked Questions

What is the difference between a calibration hierarchy and a calibration curve?

A calibration hierarchy is the entire chain of comparisons that connects a result to a reference. A calibration curve is the relationship between the signal and the concentration for a specific assay. The calibration curve is one step in the hierarchy, and it is the step that converts the signal to a concentration.

How do I know if my reference material is certified?

A certified reference material has a certificate that states the certified value and the uncertainty. The certificate is issued by the producer, and it is the basis for the traceability. If the material does not have a certificate, it is not a certified reference material.

What is the difference between a reference material and a control material?

A reference material is used to calibrate the assay and to establish traceability. A control material is used to monitor the assay and to detect drift. The control material is not used to calibrate the assay, and it does not provide traceability.

How often should I calibrate my assay?

The calibration frequency is determined by the laboratory quality system and the stability of the assay. The calibration is performed when the assay is set up, and it is repeated when the control results indicate a change. The calibration is also performed when a new lot of reagent is used.

What is the uncertainty of a calibration?

The uncertainty of a calibration is the parameter that characterizes the dispersion of the values that could be attributed to the calibration. The uncertainty is expressed as a standard uncertainty or an expanded uncertainty. The uncertainty is reported with the calibration result.

What is the role of the MIQE guidelines in calibration?

The MIQE guidelines provide a framework for the reporting of qPCR experiments. The guidelines emphasize the reporting of the calibration and the standards used in the experiment. The guidelines are a reference for the transparency of the reporting, and they are not a calibration procedure.

What should I do if my control results are outside the acceptable range?

The control results outside the acceptable range indicate a problem with the assay. The laboratory should investigate the cause, which may be the calibration, the reagent, or the instrument. The laboratory should not report the results until the cause is identified and corrected.

How do I document the calibration hierarchy?

The documentation of the calibration hierarchy includes the certificate of the reference material, the preparation records of the working calibrator, the calibration results, the uncertainty, and the control records. The documentation must be retained for the lifetime of the assay.

Related Diagnostic Guides

• Biosafety Levels 1-4: A Comparative Framework for Diagnostic Laboratory Design
• Laboratory Proficiency Testing: A Guide to Providers and Program Selection
• Measurement Uncertainty in Laboratory Medicine: A Guide to Calculation and Reporting

References and Further Reading

• Molecular Biology and Laboratory Methods. National Library of Medicine.
• Biosafety in Microbiological and Biomedical Laboratories. CDC and NIH.
• NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules. National Institutes of Health.
• Addgene Protocols. Addgene.
• MIQE Guidelines. Clinical Chemistry.
• NIST Laboratory Programs. National Institute of Standards and Technology.
• Laboratory Biosafety Manual. World Health Organization.
• NCBI Literature Resources. National Center for Biotechnology Information.
• Calibration of computational tools for missense variant pathogenicity classification and ClinGen recommendations for PP3/BP4 criteria.. American journal of human genetics, 2022.
• Defining a metrologically traceable and sustainable calibration hierarchy of international normalized ratio for monitoring of vitamin K antagonist treatment in accordance with International Organization for Standardization (ISO) 17511:2020 standard: communication from the International Federation of Clinical Chemistry and Laboratory Medicine-SSC/ISTH working group on prothrombin time/international normalized ratio standardization.. Journal of thrombosis and haemostasis : JTH, 2024.
• A predictive model for progression of chronic kidney disease to kidney failure.. JAMA, 2011.

This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.