Optimising blood sample storage and instrument alignment

Code: T08M05MA

The activities presented in this report have been directed toward optimising the alignment of Sysmex blood analysers housed in WADA-accredited laboratories throughout the world. As is standard practice in laboratory haematology, between-laboratory comparability of those instruments currently relies upon participation in an external quality assurance program. Each month all laboratories are sent test samples whose results must be in close agreement with the remainder of the laboratories. However those samples must comprise stabilised blood cells in order to yield a useful shelf life, and those stabilised cells respond differently to the reagents and stains used in the analyser. Subsequently, Sysmex analysers use different modes of analysis depending whether samples are comprised of fresh or stabilised blood cells. As an alternate approach, we investigated whether alignment procedures could be modified so as to utilise fresh blood samples instead of stabilised materials.

Experimental work first focused on the stability of fresh blood samples during storage, with the intention of establishing the maximum delay that could be tolerated between sample collection and analysis. This had the dual benefit of not only informing our subsequent experiments, but also providing empirical support for WADA’s interest in extending the Athlete Biological Passport’s

(ABP) current sample collection window (i.e., 36 hrs maximum between collection and analysis). We found that the two key blood parameters utilised in the ABP, namely haemoglobin concentration and reticulocyte percentage, remained stable for up to five days post-collection provided that the sample is stored at approximately 6-8 oC. Moreover the red blood cells were found to swell in a predictable manner, enabling us to develop a nomogram that permits users to reverse extrapolate back to a stored sample’s initial characteristics provided that interim temperature and duration of storage are known.

The second phase of the study was instrument-based. We sought to align the reticulocyte counts of three analysers as closely as possible to the readings provided by a ‘reference’ instrument. We collaborated closely with a Sysmex technical representative, which permitted us to derive a deep appreciation of instrument nuances and subsequently to develop a robust alignment protocol. We found that our prototype re-calibration protocol, which was based upon tight calibration to target QC values, yielded excellent comparability between instruments. As a result, incorporation of our alignment protocol offers the possibility to optimise the comparability of Sysmex instruments located in WADA-accredited laboratories without the need to share fresh blood samples

Main findings

Our study has shown that a 0.31% bias in reticulocyte counts can exist between two XT-2000i instruments which are both operating within manufacturer’s specifications. We also demonstrated that recalibrating instruments toward the assigned value of control material, rather than lying within a tolerance range, brought reticulocyte counts into close alignment.

When contemplating possible explanations as to how two of our Test instruments demonstrated absolute biases of 0.24% and 0.31% when counting reticulocytes in fresh blood compared to our Comparative instrument, we conclude that the most likely origin stems from how the separate instruments were calibrated during installation. Standard installation procedures do not require the technician to recover QC reticulocyte values from the Sysmex XT-2000i (other than to verify that in terms of precision the reticulocyte percentage and absolute numbers have a CV < 15%). Instead, calibration materials are used to establish the other channels (e.g., the 16-parameter haemogram plus 5-part white blood cell differential), then the technician merely confirms using ten normal range fresh blood samples that the average reticulocyte value for those ten samples lie within the instrument’s reference interval (i.e., an XT’s typical reference range is approximately 0.64% – 1.65%). This would seem to provide relatively generous tolerances. For example, in the case of our first Test instrument reporting 0.31% low based on the average of ten fresh blood samples, that instrument would still yield a result that would fall within an acceptable range provided that the true average value of those ten samples lay between 0.95% and 1.96% (i.e., 0.64% + 0.31% and 1.65% + 0.31%, respectively). Under that hypothetical circumstance there would have been no basis for the installing technician to have refined the instrument’s set up during installation. Likewise, our data show that the manufacturer’s tolerance for RBC-X sensitivity adjustments mean that an instrument’s fresh blood reticulocyte counts can span a range of 0.47% without failing the manufacturer’s performance specifications. In other words, it seems tenable that tolerances specified by the manufacturer could enable a bias in the order of 0.3%-0.5% to exist in the reticulocyte percentage reported by two properly calibrated XT-2000i instruments.

We have shown that it is possible to remove bias between instruments down to at least one decimal place, in fact in our hands a Test instrument replicated the Comparative instrument’s reticulocyte counts down to two decimal places. We consider either to be zero bias in the context of the Athlete Biological Passport. Our original hypothesis was that because the XT-2000i uses different approaches depending whether stabilised or fresh samples are tested, alignment of reticulocyte counts would necessarily require the comparison of fresh blood results between instruments. However we found that excellent alignment could also be achieved merely by calibrating each instrument to the assigned value of control materials. This possibility has important implications for those WADA-accredited labs testing athlete samples, because an alignment protocol which utilised surrogate samples would avoid having to transport fresh blood to remote laboratories within the imited shelf life associated with this live tissue specimen. However, as proposed by the CLSI’s standard on validation, verification and quality assurance of haematology analysers, when possible fresh blood should be part of an overall QC program (9). A sensible compromise to enhance linkage between QC-derived data and reportable patient results might be to fortify a QC-based approach with localised fresh blood ring studies. For example, regional laboratories within close proximity may elect to optimise alignment by sharing fresh blood samples with their immediate neighbours. Without too much coordination one member could compare with a different regional cohort of laboratories and therefore propagate the confirmation beyond their localised region.

The benefit that improved between-laboratory comparability of reticulocyte counts brings to antidoping efforts is important but deceptively subtle. Currently, there is a two-step process followed before an athlete can be sanctioned via data derived from CBC results. The first step entails a statistical program which flags abnormal blood values that lie beyond the athlete’s individual reference range. These reference ranges are generated with a tolerance for both within- and between-subject components of variation, which far exceed the magnitude of between-laboratory variation. Decreasing these variance components by modest amounts has surprisingly little impact on the tolerance thresholds. Subsequently, because the between-laboratory error component is dwarfed by the within-subject component, reducing the variance has little impact on the statistical process.

However regardless of the statistics, an athlete is not considered to have committed an antidoping rule violation until and unless during the second step an expert review of the haematological data concludes that the most likely cause of the abnormal blood result was doping (as opposed to, for example, a pathology or analytical issue). This expert review shares a common lineage with how clinical haematologists evaluate serial change of reported results in a given patient, inasmuch as both groups are obliged to factor into their considerations an allowance for between-laboratory differences. A subjective allowance of 0.2 – 0.3% is typical of the buffer afforded in the athlete’s favour when blood is tested in different laboratories. Reducing the between-laboratory bias to within 0.1% or lower, as we have shown is possible to accomplish, would effectively mean that experts could interpret all results as if they had been collected on the same instrument. This would reduce the subjective tolerances made for potential between-laboratory bias, and thereby provide additional certainty to their opinions.