The concept that for desirable performance, analytical variation (CV_{a}) should be less than half the within-subject biological variation (CV_{i}) was developed over 30 years ago, i.e.,

By adopting 0.5 CV_{i}as the goal for expanded uncertainty of measurement [obtained by multiplying the relative combined standard uncertainty by a coverage factor of 2 (95% level of confidence)], it can be calculated that the true result variability due to biological variation would not be increased by more than 12% due to the analytical uncertainty.

As described above, bias in individual measurements can also have a significant effect on a patient result [7]. For desirable assay performance, it has been accepted that the analytical bias (B_{a}) should be less than one-quarter of the total biological variation, where total biological variation is made up of within-subject (CV_{i}) and between-subject biological variation (CV_{g}), i.e.,

These two equations can then be used to define general desirable quality specifications for total allowable error (TE_{a}) as

where 1.65 is the Z-score at 95% probability [21].

Different levels of analytical quality can be defined by modulating the described theory [22] so that, minimum analytical performance goals can be defined as

expanded uncertainty <0.75 CV_{i}

B_{a}<0.375 (CV_{I}^{2}+CV_{g}^{2})^{1/2}

TE_{a}<1.65 (0.75 CV_{i})+0.375 (CV_{I}^{2}+CV_{g}^{2})^{1/2}

and optimum desirable performance as:

expanded uncertainty <0.25 CV_{i}

B_{a}<0.125 (CV_{I}^{2}+CV_{g}^{2})^{1/2}

TE_{a}<1.65 (0.25 CV_{i})+0.125 (CV_{I}^{2}+CV_{g}^{2})^{1/2}

If for being acceptable, the degree of uncertainty (expanded) of a measurand in the clinical laboratory (including the accumulated uncertainty of the corresponding traceability chain) using unbiased assays should stay within ±0.25 CV_{i}, ±0.50 CV_{i} or ±0.75 CV_{i} (optimum, desirable or minimum quality level, respectively), decisions need to be made on what proportion of this budget can be used up in the traceability chain to ensure enough budget is left for use in routine analysis. In the past, only the contribution of the uncertainty related to the reference procedures has been considered. In particular, Stöckl etal. [23, 24] proposed that this uncertainty should be <0.2 times the maximal tolerated limit, i.e., the clinically allowable uncertainty of measurements.

We have selected three measurands to analyze the current situation and evaluate if traceability is compatible with the application of the biological variation approach.

## Example 1

The desirable and minimum expanded uncertainty for serum creatinine are 3.0% and 4.5%, respectively [25]. Currently, NIST provides a reference material, SRM 967a (frozen human serum), in which there are two concentration levels of creatinine [26]. The certified concentration values for each creatinine level assigned by the reference procedure using isotope dilution liquid chromatography/mass spectrometry (LC-IDMS) are as follows: 74.9±1.6 μmol/L for level 1 and 342.7±7.2 μmol/L for level 2. This corresponds to an expanded uncertainty of 2.1% for both levels, which is lower than both desirable (3.0%) and minimum (4.5%) goals of expanded uncertainty derived from biologic variability. If this reference material is used to transfer trueness to manufacturer’s calibrators, there is still 30% (if desirable goal is used) or approximately 53% (if minimum goal is used) of the total uncertainty budget available for the remainder of the chain. Only for laboratories working with an uncertainty for serum creatinine (i.e., the random effects of measurement) <2.0%, would a remaining uncertainty budget for the rest of the traceability chain be available to comply with the desirable expanded uncertainty limit of 3.0%. Otherwise, the minimum goal should be employed.

## Example 2

Another reference material provided by NIST is the SRM 909c, which contains glucose at a concentration of 5.05±0.059 mmol/L as measured by isotopic dilution gas chromatography-mass spectrometry (GC-IDMS), corresponding to an expanded uncertainty of 1.2%, which is well within the desirable expanded uncertaintyfor measurement of serum glucose derived from biologic variability (3.1%) [24]. If this reference material is used in the traceability chain for glucose, it would leave more than 60% of the uncertainty budget. In one of our laboratories, the CV for serum glucose is around 1.8%, still leaving more than 30% uncertainty budget for the remaining parts of the traceability chain.

One manufacturer who provided traceability data for glucose to the WG-AETR, used NIST SRM 917b as the higher-order reference material. This is a powder material of D-glucose of purity 99.7±0.2%, which was used to prepare their reference calibration curve by weighing into a volumetric flask. Three final calibrators were prepared that gave the following concentrations: 1.83±0.055 mmol/L, 16.50±0.267 mmol/L, and 33.13±0.587 mmol/L. These equate to expanded uncertainties of 3.0%, 1.6% and 1.8%, respectively, that, at least for the lower glucose concentration, already used all of the desirable uncertainty budget for all the traceability chain of 3.1%.

## Example 3

The NIST reference material for sodium in human serum is SRM 956c, which contains sodium at a concentration level of 118.8±1.0 mmol/L, i.e., an expanded uncertainty of 0.84%. The concentration of sodium in this material was assigned by high-performance inductively coupled plasma-optical emission spectrometry (ICP-OES) and ISE potentiometry. As the minimum performance goalfor expanded uncertainty of measurement of sodium in serum is 0.50% [24], it is evident that in order to apply the biologic variability approach this reference material is not appropriate because its displays approximately 70% more uncertainty than the allowable total uncertainty.

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