Introduction
In clinical laboratory testing, achieving consistent and comparable measurement results across different instruments, laboratories, time points, and healthcare systems is a fundamental challenge. The ability to trust that a result from a hospital in New York is equivalent to one from a lab in California – or from a different instrument brand – directly impacts disease diagnosis accuracy, treatment efficacy assessment, and the interoperability of medical data. Reference materials (RMs) and standardization are the core tools that address this challenge.
This article systematically covers key technical aspects of reference materials and standardization in the IVD field, including definitions and classifications of reference materials, metrological traceability systems, standardization vs harmonization strategies, regulatory frameworks (FDA, CLIA), key international organizations (NIST, JCTLM, CAP), case studies, and future trends – all tailored for North American IVD professionals.
I. Overview of Reference Materials
1.1 What Are Reference Materials?
A reference material (RM) is a material that is sufficiently homogeneous and stable with respect to specified properties, and whose property values have been confirmed to be fit for their intended use in measurement or nominal property testing. In the IVD field, RMs are typically used to calibrate testing systems, evaluate the accuracy of measurement procedures, and verify the traceability of measurement results.
Main Types of Reference Materials:
| Type | Definition | Typical Examples |
|---|---|---|
| Certified Reference Material (CRM) | RM accompanied by a certificate, with property values certified by a metrologically valid procedure, including uncertainty | NIST SRMs, JCTLM-listed RMs |
| International Conventional Calibrator | Reference materials established by international organizations (e.g., WHO) | WHO International Standards (e.g., for cytokines, hormones) |
| Manufacturer's Working Calibrator | Internally established calibrators from IVD manufacturers | In-house standards from Roche, Abbott, Siemens, Beckman |
| Product Calibrator | Calibrators supplied with commercial kits for end-user calibration | Kit calibrators |
| Quality Control Material | Materials used to monitor assay performance over time | Third-party QC (e.g., Bio-Rad, Randox) |
1.2 Reference Material Formats and Commutability
Reference materials can be classified as pure substance RMs or matrix-based RMs.
| Type | Advantages | Limitations | Applications |
|---|---|---|---|
| Pure Substance RM | Stable, high purity, traceable to SI units | Poor commutability with clinical samples | Reference method calibration, raw material value assignment |
| Matrix-Based RM | Similar matrix to clinical samples, good commutability | Complex preparation, lower stability | Routine clinical assay calibration, PT/EQA |
Formats include lyophilized, frozen liquid, fresh frozen plasma, etc. Organizations like NIST are transitioning from lyophilized materials toward fresh frozen matrices to improve commutability with routine clinical methods.
1.3 Commutability – A Critical Requirement
Commutability is the ability of a reference material to have the same behavior as a clinical sample across different measurement procedures. This is a fundamental requirement for any reference material intended for calibration or trueness verification.
If a reference material lacks commutability, even accurate value assignment cannot ensure correct calibration transfer, leading to bias.
Strategies to Improve Commutability:
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Use pooled human samples rather than spiked or processed materials
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Adopt fresh frozen matrices instead of lyophilized formats
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Validate commutability across multiple commonly used methods
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Follow CLSI EP14 and EP30 guidelines for commutability assessment
II. Metrological Traceability Systems
2.1 What Is Metrological Traceability?
Metrological traceability is the property of a measurement result whereby it can be related to a reference standard through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty.
The top of the traceability chain is the International System of Units (SI) . Through reference measurement procedures (RMPs) and certified reference materials (CRMs), the value is transferred from the SI unit to working calibrators, and finally to patient sample results.
2.2 ISO 17511:2020 Calibration Hierarchy Categories
ISO 17511:2020 defines six scenarios for establishing metrological traceability of values assigned to calibrators, trueness control materials, and human samples:
| Scenario | Description | Traceable to SI? | Typical Analytes |
|---|---|---|---|
| 1 | RMP and primary RM available | ✅ Yes | Electrolytes (K⁺, Na⁺, Ca²⁺), metabolites (glucose, creatinine) |
| 2 | Primary RMP available, no primary RM | ✅ Yes | Enzyme activities (ALT, AST, CK), some coagulation factors |
| 3 | RMP calibrated by a primary calibrator | ✅ Yes | HbA1c, some protein biomarkers |
| 4 | International conventional calibrator available | ✅ Yes (non-SI) | WHO International Standards (renin, CEA) |
| 5 | International harmonization protocol available | ❌ No (harmonized) | Protein hormones, some tumor markers (e.g., CA19-9, CA125) |
| 6 | Manufacturer's internal standard only | ❌ No | Many tumor markers, most antibody-based assays |
Scenarios 1-3 enable traceability to SI units through RMPs and CRMs. Scenarios 4-6 cannot achieve SI traceability and require harmonization protocols.
2.3 FDA Recognition and CLIA Requirements
FDA Recognition:
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FDA recognizes ISO 17511:2020 as a consensus standard for establishing metrological traceability of IVD reagents
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Referenced in FDA guidance documents (e.g., "Principles for Codevelopment of an In Vitro Companion Diagnostic Device")
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Compliance with ISO 17511 is part of the premarket review (510(k), De Novo, PMA) expectations
CLIA '88 Requirements:
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Clinical Laboratory Improvement Amendments require laboratories to follow manufacturers' instructions for calibration and QC
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Calibration must be verified using at least two levels of control materials each day of use
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CMS regulations (42 CFR §493) specify calibration verification requirements
FDA LDT Final Rule (2024) :
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FDA now regulates laboratory-developed tests (LDTs) as IVDs
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Requires traceability and calibration verification for LDTs
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Increased focus on reference material quality for laboratory-developed assays
III. Harmonization vs Standardization
3.1 Key Differences
Standardization and harmonization are complementary strategies for addressing result variability across different methods.
| Comparison | Standardization | Harmonization |
|---|---|---|
| Definition | Achieving comparable results through metrological traceability to higher-order reference systems (SI units) | Achieving equivalent results through international consensus protocols when SI traceability is not feasible |
| Traceability Target | SI units or international conventional RMs | Harmonization protocol itself (non-SI) |
| Applicability | RMP and/or CRM exists; commutability is validated | No RMP/CRM available; or RMs lack commutability |
| Typical Analytes | Electrolytes, enzymes, metabolites (glucose, creatinine, cholesterol) | Protein hormones, many tumor markers, antibodies |
| Key Standards | ISO 17511 | ISO 21151 / CLSI EP30 |
3.2 Harmonization Protocol Framework (ISO 21151)
For measurands that cannot be standardized through traditional traceability (Scenarios 5/6 in ISO 17511), harmonization protocols provide an alternative approach. ISO 21151 (also recognized by FDA) specifies the framework:
| Requirement | Core Content |
|---|---|
| Measurand Definition | Clear definition per ISO 17511 to ensure alignment |
| IVD MD Inclusion/Exclusion Criteria | Based on precision, recovery, and clinical impact |
| Harmonization RM | Manufactured from human sample pools; validated for homogeneity, stability, and commutability |
| Value Assignment & Calibration Adjustment | Explicit method for assigning values; manufacturers adjust calibration to achieve equivalence |
| Validation & Sustainability | Independent sample panels for validation; long-term maintenance strategy |
| New IVD MD Integration | Clear process for including new or improved assays |
3.3 The Role of JCTLM and NIST
| Organization | Role in Standardization/Harmonization |
|---|---|
| NIST (USA) | Develops Standard Reference Materials (SRMs) for clinical diagnostics; maintains reference methods |
| JCTLM (Joint Committee for Traceability in Laboratory Medicine) | Maintains database of higher-order RMPs and CRMs; lists reference materials and methods meeting ISO 15193/15194 requirements |
| CAP (College of American Pathologists) | Provides PT/EQA programs, including accuracy-based PT; accredits laboratories |
| CLSI (Clinical and Laboratory Standards Institute) | Publishes EP series guidelines (EP14 – Commutability; EP30 – Harmonization) |
| BIPM (International Bureau of Weights and Measures) | Maintains SI units and international metrological infrastructure |
IV. NIST Reference Material (SRM) Portfolio
The National Institute of Standards and Technology (NIST) has developed more than 30 Standard Reference Materials (SRMs) for clinical diagnostics, many of which are listed in the JCTLM database.
| SRM Number | Name | Analytes |
|---|---|---|
| SRM 909c | Human Serum (Routine Clinical Analytes) | Electrolytes, glucose, creatinine, uric acid, total protein, albumin |
| SRM 967b | Creatinine in Frozen Human Serum | Creatinine (2 levels) |
| SRM 2921 | Human Cardiac Troponin Complex | Cardiac troponin I and T |
| SRM 1951c | Lipids in Frozen Human Serum | Cholesterol, triglycerides, HDL, LDL |
| SRM 972a | Vitamin D Metabolites in Frozen Human Serum | 25-OH-Vitamin D2, D3 (4 levels) |
| SRM 3669 | Arsenic Species in Frozen Human Urine | Arsenic speciation |
| SRM 3840 | Thyroglobulin in Frozen Human Serum | Thyroglobulin (harmonization standard) |
NIST continues to expand its clinical SRM portfolio and is transitioning from lyophilized to fresh frozen matrices to improve commutability with routine clinical methods.
NIST Reference Material Development Process:
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Commutability assessment using CLSI EP14 guidelines
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Value assignment using reference methods
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Uncertainty estimation per ISO Guide 35
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Collaboration with CDC, NIH, and professional organizations (e.g., AACC, CAP)
V. Case Studies
5.1 Creatinine Standardization: NIST SRM 967
Background: Creatinine is a critical marker for kidney function. Inaccurate creatinine measurements affect estimated glomerular filtration rate (eGFR) calculations and CKD diagnosis.
Standardization Approach:
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NIST SRM 967 (Creatinine in Frozen Human Serum) available at two levels
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Isotope dilution mass spectrometry (ID-MS) reference method
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CDC's Creatinine Standardization Program (CSP) uses NIST SRMs
Impact:
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Significantly reduced inter-laboratory variability
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Improved CKD classification accuracy
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Supports eGFR calculation using CKD-EPI and MDRD equations
5.2 Cholesterol Standardization: NIST SRM 1951c
Background: Accurate LDL cholesterol measurement is essential for cardiovascular risk assessment.
Standardization Approach:
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CDC's Lipid Standardization Program (LSP) uses NIST SRM 1951c
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Reference method: ID-MS
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Manufacturers adjust calibration to NIST SRM values
Impact:
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Nationwide standardization of lipid measurements
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Reliable risk stratification for statin therapy
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Supports ATP III and ACC/AHA guidelines
5.3 Cardiac Troponin I Harmonization: NIST SRM 2921
Background: cTnI is the gold standard for myocardial infarction diagnosis. However, different manufacturer assays produced results that varied up to 200-fold for the same sample, as reported in FDA premarket submissions.
International Challenge: Among 15 FDA-cleared cTnI assays, results from the same sample could differ by a factor of 200 – a major clinical concern.
Innovative Approach:
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NIST SRM 2921 (Human Cardiac Troponin Complex) as a harmonization anchor
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IFCC Working Group on cTnI standardization (WG-TNI)
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Manufacturers calibrate to SRM 2921 to achieve harmonization
Impact:
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Reduced inter-assay variability from 200-fold to approximately 2-3 fold
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Improved clinical decision-making for MI diagnosis
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Supports universal 99th percentile upper reference limits
5.4 Accuracy-Based Proficiency Testing (CAP PT Programs)
The College of American Pathologists (CAP) offers accuracy-based PT programs that use commutable materials with target values assigned by reference methods.
Examples:
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Accuracy-Based Chemistry (AB-Chem) : Uses NIST-traceable materials; target values from reference laboratories
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Accuracy-Based Lipids (ABL) : For cholesterol, triglycerides, HDL, LDL
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Accuracy-Based Creatinine (CRT-AB) : Uses NIST SRM 967
How It Works:
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Commutable PT samples distributed to participating laboratories
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Target values established by reference methods (ID-MS, etc.)
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Laboratory results compared to target values – not just peer group mean
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Identifies systematic bias and calibration issues
Impact:
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Laboratories can assess trueness, not just precision
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Identifies calibration drift across manufacturers
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Supports CLIA calibration verification requirements
VI.Frequently Asked Questions (FAQ)
Q1: What is the difference between standardization and harmonization?
A: Standardization achieves comparable results through metrological traceability to higher-order reference systems (SI units). Harmonization achieves equivalent results through international consensus protocols when SI traceability is not feasible. Harmonization applies to measurands without RMPs or CRMs, or when available RMs lack commutability with clinical samples.
Q2: What are the 6 scenarios defined in ISO 17511:2020?
A:
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Scenario 1: RMP and primary RM available (traceable to SI)
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Scenario 2: Primary RMP available, no primary RM (traceable to SI)
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Scenario 3: RMP calibrated by a primary calibrator (traceable to SI)
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Scenario 4: International conventional calibrator available (non-SI traceable)
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Scenario 5: International harmonization protocol available (harmonized, not SI-traceable)
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Scenario 6: Manufacturer's internal standard only (no external traceability)
Q3: What is commutability? Why is it important?
A: Commutability is the ability of a reference material to have the same behavior as a clinical sample across different measurement procedures. Without commutability, an RM cannot correctly transfer calibration values, leading to bias. Commutability is essential for any RM used for calibration or trueness verification. CLSI EP14 provides the framework for commutability assessment.
Q4: What NIST SRMs are most commonly used in clinical diagnostics?
A: SRM 909c (routine clinical analytes), SRM 967b (creatinine), SRM 2921 (troponin), SRM 1951c (lipids), and SRM 972a (vitamin D) are among the most widely used. These are listed in the JCTLM database and recognized by FDA as traceable reference materials.
Q5: What are the CLIA requirements for calibration verification?
A: CLIA '88 (42 CFR §493.1255) requires laboratories to perform calibration at least every 6 months or when changes affect test performance. Calibration verification using at least two levels of control materials must be performed each day of testing. If calibration verification fails, remedial action (recalibration, service) must be taken.
Q6: How does the FDA LDT final rule affect reference material requirements?
A: The FDA LDT final rule (2024) now regulates laboratory-developed tests as IVDs. This requires LDTs to meet the same traceability and calibration verification requirements as commercial IVDs, increasing the demand for high-quality reference materials and traceability documentation for laboratory-developed assays.
Q7: Where can I find higher-order reference materials and methods?
A: The JCTLM database (www.jctlm.org) lists reference materials and methods that meet the requirements of ISO 15193 and ISO 15194. NIST SRMs and CDC reference methods are among those listed.
Q8: What is accuracy-based PT? How is it different from traditional PT?
A: Accuracy-based PT uses commutable materials with target values assigned by reference methods (e.g., ID-MS). Results are compared to these reference method target values, not just peer group means. This allows laboratories to assess trueness (systematic bias), not just precision. CAP offers several accuracy-based PT programs (e.g., AB-Chem, ABL, CRT-AB).
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