In the present study, we analysed seven surveys of a French EQA program for seminal biomarkers that have been conducted over the last 6 years. We provided new data on the biomarkers panel offered by laboratories, the kind of assays used and their performance.
Nowadays, EQA programs represent a fundamental pillar of a laboratory’s quality management system. The first programs were introduced in laboratory medicine more than 70 years ago as educational tools to reveal that results for a same aliquot were different between laboratories . Progressively, their scope was extended and some of them have reached a high level of design sophistication as those proposed by EQA providers that meet ISO 17043 requirements. Miller et al. proposed a classification of EQA programs into 6 categories according to how well they are able to evaluate performance . Three characteristics underpin this evaluation capability: sample commutability, process for target assignment and inclusion or non-inclusion of replicate samples. Commutability of an EQA sample is a complex and much discussed property [9, 17]. A commutable sample has the same numeric relationship between measurement procedures than that of a representative set clinical samples. There are consensus procedures to validate this commutability but they are heavy and expensive to perform in current practice . Since our EQA samples were obtained by pooling native and unmodified SP according to a well-described preparation, we reasonably assumed their commutability. For target values assignment (accuracy), we used the all-participants median value because there was no reference measurement procedures to trace results to. Finally, we did not include replicate samples that would permit to calculate individual intra-laboratory reproducibility. According to these characteristics, our EQA program belongs to the 4th category of Miller’s classification. For performance assessment of each biomarker assay, we set the limits around the target (ALP) values according to literature data and our own knowledge of seminal biochemistry. We will see below that those chosen ALP (S1) were fairly accurate. This state-of-art based approach fits to the model 3 of the Consensus Statement from the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine (or Milan Consensus) . The 10 primary purposes of an EQA scheme have been recently recalled by Badrick et al.  amongst which the present EQA program fulfilled six (Supplementary Table 3). This overall examination highlights some strengths of the scheme (sample commutability, satisfying regulatory requirements) as well as some opportunities of improvement (moving to clinical and statistical-based performance assessment instead of state-of-art , assessing intra-lab reproducibility). However, since seminal biochemistry is an “analytical niche” involving a limited number of laboratories, it is unlikely that reference measurement procedures will be developed. This issue prevents our EQA to evaluate efficiently both accuracy and assays standardization.
A small number of laboratories (median of 6 by survey) have been regularly involved in the EQA programme, indicating that they routinely execute seminal plasma explorations and their willingness to accredit them. By way of comparison, around 90 laboratories routinely perform spermograms in France . Thus, we could estimate two ratios: for 11.2 million French inhabitants, there is one laboratory performing biochemical assays versus 15 performing spermograms. Further studies are needed to determine whether this analytical offer matches the demand and to compare these ratios to those of other countries. Regarding the panel of biomarkers, we identified that a total of seven are routinely carried out: three for the prostate (citrate, zinc, prostatic acid phosphatase), one for the seminal vesicles (fructose) and three for epididymis (free L-carnitine, α-1,4 glucosidase and GPC). Interestingly, these biomarkers are those reported by the last WHO manual . However only four assays are shared by all laboratories and could be evaluated in this EQA program: citrate, zinc, fructose, and α-1,4 glucosidase. Thus, each annex gland is explored by at least one biomarker. The EQA program allowed us to have also an overview on the used methods and their performances. There is a mix of in-house assays and industrial kits and their proportions are likely to vary from a biomarker to another. Citrate, PAP, Fructose and free-L Carnitine are mainly assayed with CE-IVD methods. For α-1,4 glucosidase, most of reported assays are in-house but a CE-IVD kit is available. For GPC, there is only an in-house method. The distinction between the two categories is of importance because the use of CE-IVD methods facilitates the accreditation process, standardization and methods traceability that are the gold standards of laboratory medicine. The case of zinc is particular since the most widely used methods are spectroscopy-based. Although they are not CE-IVD marked, they are recognized as efficient and valid than colorimetric methods for assessing zinc levels in human fluids . Thus, citrate, PAP, fructose, α-1,4 glucosidase and free L-carnitine, for which CE-IVD assays are available can theoretically be carried out under ISO 15189 accreditation. In fact, according to the gathered data on the field, this may concern only citrate, fructose and zinc by extension. This unexpectedly provides a first clue on the fragility of the biochemical exploration of epididymis in France.
Regarding overall analytical performances, the median CD were below 10% for citrate, zinc and fructose and slightly above for α-1,4 glucosidase. Of interest, the reported values were very close to those of ALP initially expected to set laboratories performance (Supplementary data S1). Our EQA allows evaluating methods performance around the normal ranges as confirmed by Table 2 that reports seminal biomarkers concentrations for control patients i.e. fertile and/or normozoospermic men with normal ejaculate volume. It confirms that mixing a high number of seminal plasma samples provides physiological concentrations of biomarkers. Of interest, we noticed that reported interquartile ranges for total and neutral α-1,4 glucosidase were highly overerlapping. This point calls into question the value of distinguishing between total and neutral form of the enzyme in the clinical setting. In order to improve the EQA, samples with pathological levels, i.e. below thresholds proposed by the WHO manual , should be assayed in future surveys.
Finally, we undertook to synthesize all gathered data and to build a scoring system (Supplementary Table 2). We aimed to understand which biomarkers support laboratories efforts to meet ISO 15189 requirements and which do not. We reasoned that a “good” seminal biomarker: (i) is assayed by a high number of laboratories allowing a robust inter-laboratory comparisons; (ii) there are several CE-IVD methods with a good inter-method agreement; (ii) most laboratories provide consistent results indicating good skills and a mastery of methods. This reasoning is the rational of our scoring system. It appears that methods for citrate, zinc and fructose pave the way for meeting ISO 15189 requirements. It is less certain for α-1,4 glucosidase because it is more frequently analyzed with in-house methods that may extend its validation process. It seems completely out of the question for PAP, free L-carnitine and GPC that could not be evaluated in our EQA survey. Thus, one can consider the impact of these findings on andrological decision-making in France. Prostate and seminal vesicles are biochemically explored with a good level of confidence. Conversely, the situation is still far from satisfactory for epidydimis as we noted it above. To overcome this issue, it would be pertinent that each laboratory performs simultaneous assays of α-1,4 glucosidase and free-L carnitine with available CE-IVD methods.