Measurement of Anti-Mullerian Hormone: Preliminary Evaluation of an ABEI-Based Fully Automated Immunoassay.

Background: The added value of Anti-Müllerian hormone (AMH) measurement is recognized for several clinical applications such as assessment of the ovarian reserve, monitoring of in vitro fertilization protocol or in the field of oncofertility. Our study objective was to determine the performances of a novel fully automated chemiluminescent assay for AMH testing. Methods: We evaluated the performances of the Maglumi® 800 AMH chemiluminescent immunoassay that applies N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) labels. Assay imprecision was assessed with two levels of control materials. Method comparison was performed with an ultrasensitive AMH ELISA assay (Ansh Laboratories, Inc, Webster, TX, USA) with 88 patients' samples. Results: The within-run and between-run coefficients of variation (CVs) were below 3% for both low and high internal quality controls. The automated and ELISA methods were significantly correlated. Bland-Altman plot evidenced a bias between the methods with a mean bias of 0.6 ng/mL. Conclusions: Our preliminary evaluation showed overall good analytical performances for the Maglumi® AMH fully automated immunoassay and good concordance with a routinely used assay.

Neurofilaments: a key new biomarker for clinicians. Part 1: Importance of neurofilaments in the management of neurodegenerative diseases / Les neurofilaments : un nouveau biomarqueur clé pour les cliniciens. Partie 1 : Intérêt des neurofilaments dans la prise en charge des maladies neurodégénératives

Neurological biomarkers are of great use for clinicians, as they can be used for numerous purposes: guiding clinical diagnosis, estimating prognosis, assessing disease stage and monitoring progression or response to treatment. This field of neurology has evolved considerably in recent years due to analytical improvements in assay methods, now allowing the detection of biomarkers not only in cerebrospinal fluid (CSF) but also in blood. This progress greatly facilitates the repeated quantification of biomarkers, the collection of blood being much less invasive than that of CSF. Among the various informative biomarkers of neurological disorders, neurofilaments light chains (NfL) have proven to be particularly attractive in many contexts, in particular for the diagnosis and prognosis of neurodegenerative diseases (which this review will present), but also in other contexts of neurological disorders (which will be detailed in part 2). We further address the added value of NfL compared to other biomarkers commonly used to monitor the diseases described in this review.

Les biomarqueurs neurologiques sont d'une grande utilité, car ils peuvent être utilisés à de nombreuses fins : orienter le diagnostic clinique, estimer le pronostic, évaluer le stade de la maladie et surveiller la progression ou la réponse au traitement. Ce domaine de la neurologie a considérablement évolué ces dernières années grâce à l'amélioration des méthodes de dosage, permettant désormais la détection de biomarqueurs non seulement dans le liquide cérébro-spinal (LCS) mais aussi dans le sang. Ce progrès facilite la quantification répétée des biomarqueurs, le prélèvement de sang étant beaucoup moins invasif que celui du LCS. Parmi les différents biomarqueurs informatifs des troubles neurologiques, la chaîne légère des neurofilaments (NfL) s'est révélée particulièrement intéressante dans de nombreux contextes, notamment pour le diagnostic et le pronostic des maladies neurodégénératives (que cette revue présentera), mais aussi dans d'autres contextes de troubles neurologiques (qui seront détaillés dans la partie 2). La valeur ajoutée du NfL par rapport aux autres biomarqueurs couramment utilisés est analysée.

Neurofilaments: a key new biomarker for clinicians. Part 2: Neurofilaments, an asset beyond neurodegenerative diseases / Les neurofilaments : un nouveau biomarqueur clé pour les cliniciens. Partie 2 : Neurofilaments, un intérêt au-delà des maladies neurodégénératives.

Neurofilaments (Nf) are proteins selectively expressed in the cytoskeleton of neurons, and their increase is a marker of neuronal damage. The potential utility of neurofilament light chain (NfL) has recently increased considerably, well beyond neurodegenerative diseases, due to analytical advances that allow measurement of their concentrations (even low ones) in cerebrospinal fluid and blood. This article completes the first part, in which we presented the interest of NfL in the context of neurodegenerative diseases. Here we focus our review on other clinical contexts of neurological injury (such as traumatic brain injury, multiple sclerosis, stroke, and cancer) and present the potential value of NfL assay in the management of these patients, for both diagnosis and prognosis. We also discuss the added value of the NfL assay compared to other biomarkers commonly used in the described clinical situations.

Les neurofilaments (Nf) sont des protéines sélectivement exprimées dans le cytosquelette des neurones, dont l'augmentation est un marqueur de dommages neuronaux. L'utilité potentielle de la chaîne légère des neurofilaments (NfL) s'est récemment considérablement accrue, bien au-delà des maladies neurodégénératives, grâce aux progrès analytiques permettant de mesurer leurs niveaux (mêmes faibles) dans le liquide cérébro-spinal et le sang. Cet article complète la première partie, dans laquelle nous avions présenté l'intérêt des NfL dans le contexte des maladies neurodégénératives. Nous axons ici notre revue sur d'autres contextes cliniques de lésions neurologiques (tels que les traumatismes crâniens, la sclérose en plaques, les accidents vasculaires cérébraux et le cancer) et présentons l'intérêt potentiel du dosage des NfL pour la prise en charge de ces patients, tant au niveau diagnostique que pronostique. Nous discutons également de la plus-value du dosage des NfL par rapport aux autres biomarqueurs couramment utilisés dans les contextes cliniques décrits.

Neurofilaments contribution in clinic: state of the art.

Neurological biomarkers are particularly valuable to clinicians as they can be used for diagnosis, prognosis, or response to treatment. This field of neurology has evolved considerably in recent years with the improvement of analytical methods, allowing the detection of biomarkers not only in cerebrospinal fluid (CSF) but also in less invasive fluids like blood. These advances greatly facilitate the repeated quantification of biomarkers, including at asymptomatic stages of the disease. Among the various informative biomarkers of neurological disorders, neurofilaments (NfL) have proven to be of particular interest in many contexts, such as neurodegenerative diseases, traumatic brain injury, multiple sclerosis, stroke, and cancer. Here we discuss these different pathologies and the potential value of NfL assay in the management of these patients, both for diagnosis and prognosis. We also describe the added value of NfL compared to other biomarkers currently used to monitor the diseases described in this review.

Impact of rural versus urban setting on kidney markers: a cross-sectional study in South-Kivu, DRCongo.

BACKGROUND: Most studies of chronic kidney disease (CKD) in Sub-Saharan Africa (SSA) have been conducted in urban settings. They relied on GFR estimated from serum creatinine alone and on the inexpensive, convenient urinary dipstick to assess proteinuria. The dipstick for proteinuria has not been directly compared with the gold standard albumin-to-creatinine ratio (ACR) in a large-sized study in SSA. We hereby assessed the influence of rural versus urban location on the level, interpretation, and diagnostic performance of proteinuria dipstick versus ACR. METHODS: In a cross-sectional population-based study of CKD in both urban (n = 587) and rural (n = 730) settings in South-Kivu, Democratic Republic of Congo (DRC), we assessed the prevalence, performance (sensitivity, specificity, positive predictive value and negative predictive value) and determinants of a positive dipstick proteinuria as compared with albuminuria (ACR). Albuminuria was subdivided into: A1 (< 30 mg/g creatinine), A2 (30 to 299 mg/g creatinine) and A3 (≥ 300 mg/g creatinine). RESULTS: The overall prevalence of positive dipstick proteinuria (≥ 1+) was 9.6 % (95 % CI, 7.9-11.3) and was higher in rural than in urban residents (13.1 % vs. 4.8 %, p < 0.001), whereas the prevalence of albuminuria (A2 or A3) was similar in both sites (6 % rural vs. 7.6 % urban, p = 0.31). In both sites, dipstick proteinuria ≥ 1 + had a poor sensitivity (< 50 %) and positive predictive value (< 11 %) for the detection of A2 or A3. The negative predictive value was 95 %. Diabetes [aOR 6.12 (1.52-24.53)] was a significant predictor of A3 whereas alkaline [aOR 7.45 (3.28-16.93)] and diluted urine [aOR 2.19 (1.35-3.57)] were the main predictors of positive dipstick proteinuria. CONCLUSIONS: ACR and dipstick proteinuria have similar positivity rates in the urban site whereas, in the rural site, dipstick was 2-fold more often positive than ACR. The poor sensitivity and positive predictive value of the dipstick as compared with ACR makes it unattractive as a screening tool in community studies of CKD in SSA.

Immune-mediated neurological syndromes in SARS-CoV-2-infected patients.

BACKGROUND: Evidence of immune-mediated neurological syndromes associated with the severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is limited. We therefore investigated clinical, serological and CSF features of coronavirus disease 2019 (COVID-19) patients with neurological manifestations. METHODS: Consecutive COVID-19 patients with neurological manifestations other than isolated anosmia and/or non-severe headache, and with no previous neurological or psychiatric disorders were prospectively included. Neurological examination was performed in all patients and lumbar puncture with CSF examination was performed when not contraindicated. Serum anti-gangliosides antibodies were tested when clinically indicated. RESULTS: Of the 349 COVID-19 admitted to our center between March 23rd and April 24th 2020, 15 patients (4.3%) had neurological manifestations and fulfilled the study inclusion/exclusion criteria. CSF examination was available in 13 patients and showed lymphocytic pleocytosis in 2 patients: 1 with anti-contactin-associated protein 2 (anti-Caspr2) antibody encephalitis and 1 with meningo-polyradiculitis. Increased serum titer of anti-GD1b antibodies was found in three patients and was associated with variable clinical presentations, including cranial neuropathy with meningo-polyradiculitis, brainstem encephalitis and delirium. CSF PCR for SARS-CoV-2 was negative in all patients. CONCLUSIONS: In SARS-Cov-2 infected patients with neurological manifestations, CSF pleocytosis is associated with para- or post-infectious encephalitis and polyradiculitis. Anti-GD1b and anti-Caspr2 autoantibodies can be identified in certain cases, raising the question of SARS-CoV-2-induced secondary autoimmunity.

A biological profile for diagnosis and outcome of COVID-19 patients.

Objectives As severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) pandemic is increasing its victims on a global scale with recurring outbreaks, it remains of outmost importance to rapidly identify people requiring an intensive care unit (ICU) hospitalization. The aim of this study was to identify Coronavirus Disease 2019 (COVID-19) biomarkers, to investigate their correlation with disease severity and to evaluate their usefulness for follow-up. Methods Fifty patients diagnosed with SARS-Cov-2 were included in March 2020. Clinical and biological data were collected at admission, during hospitalization and one month after discharge. Patients were divided into two severity groups: non-ICU (28) and ICU and/or death (22) to stratify the risk. Results Blood parameters in COVID-19 patients at admission showed increased C-reactive protein (CRP) (100%), ferritin (92%), lactate dehydrogenase (LDH) (80%), white blood cell (WBC) count (26%) with lymphopenia (52%) and eosinopenia (98%). There were significant differences in levels of CRP, ferritin, D-dimers, fibrinogen, lymphocyte count, neutrophil count and neutrophil-to-lymphocyte ratio (NLR) among the two severity groups. Mapping of biomarker's kinetics distinguished early and late parameters. CRP, ferritin, LDH, lymphopenia and eosinopenia were present upon admission with a peak at the first week. Late biomarkers such as anemia, neutrophilia and elevated liver biomarkers appeared after one week with a peak at three weeks of hospitalization. Conclusions We confirmed that high-values of CRP, NLR, D-dimers, ferritin as well as lymphopenia and eosinopenia were consistently found and are good markers for risk stratification. Kinetics of these biomarkers correlate well with COVID-19 severity. Close monitoring of early and late biomarkers is crucial in the management of critical patients to avoid preventable deaths.

A rare case of tuberculosis-induced hypercalcemia.

Laboratory investigations of hypercalcemia involve testing of various biochemical parameters such as parathyroid hormone (PTH), 25-(OH) Vitamin D (25-(OH) VitD), 1,25-(OH)2 Vitamin D3 (calcitriol) and PTH related peptide (PTHrp). We herein present an atypical case of severe hypercalcemia in a patient with rheumatoid arthritis who has been treated for years by various biological disease-modifying antirheumatic drugs (DMARDs) and suddenly presented with general state alteration, oedema and ulceration of her right ankle. We illustrate how tuberculosis (TB) can cause high calcitriol concentration and subsequently lead to potentially severe hypercalcemia. Moreover, we highlight the importance of TB testing and follow-up in patients treated with biological DMARDs.

Prevalence and Risk Factors of CKD in South Kivu, Democratic Republic of Congo: A Large-Scale Population Study.

BACKGROUND: The prevalence of chronic kidney disease (CKD) in African American individuals is high but whether this applies to native populations in sub-Saharan Africa is unclear. METHODS: In a cross-sectional study, we assessed the prevalence and risk factors of CKD in rural and urban adults in South Kivu, Democratic Republic of Congo. Glomerular filtration rate (GFR) was estimated using the CKD-Epidemiology Collaboration (CKD-EPI) equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both markers (eGFRcr-cys), without ethnic correction factor. CKD was defined as an eGFR <60 ml/min per 1.73 m2 and/or albuminuria (albumin-to-creatinine ratio ≥30 mg/g). RESULTS: A total of 1317 participants aged 41.1 ± 17.1 years (730 rural, 587 urban) were enrolled. The prevalence of hypertension (20.2%; 95% confidence interval [CI], 18-22.3), diabetes mellitus (4.3%; 95% CI, 3.2-5.4) and obesity (8.9%; 95% CI, 7.4-10.5) was higher in urban than rural participants (all P < 0.05). HIV infection prevalence was 0.41% (95% CI, 0.05-0.78). The prevalence of eGFRcr <60 ml/min per 1.73 m2 was 5.4% (95% CI, 4.2-6.7). The prevalence of albuminuria was 6.6% (95 % CI, 5.1-8.1). The overall prevalence of CKD was 12.2% (95% CI, 10.2-14.2) according to CKD-EPIcr. Factors independently associated with CKD-EPIcr were older age (adjusted odds ratio [aOR], 1.05 [1.04-1.07]), urban residence (aOR 1.86 [1.18-2.95]), female sex (aOR 1.66 [1.04-2.66]), hypertension (aOR 1.90 [1.15-3.12]), diabetes (aOR 2.03 [1.02-4.06]), and HIV infection (10.21 [2.75-37.85]). The results based on eGFRcys or eGFRcr-cys were largely consistent with the preceding. CONCLUSION: Overall, the burden of CKD is substantial (>11%), predominantly in the urban area, and largely driven by classic risk factors (gender, aging, HIV, hypertension, and diabetes).

Biotin interference: evaluation of a new generation of electrochemiluminescent immunoassays for high-sensitive troponin T and thyroid-stimulating hormone testing.

Background Biotin is currently a matter of concern for laboratories using biotin-streptavidin-based immunoassays. Biotin interferences have been reported for high-sensitive troponin T (hsTnT) and thyroid-stimulating hormone (TSH) assays. We aimed to evaluate the new generation of hsTnT and TSH electrochemiluminescent immunoassays announced to be less sensitive to biotin. Methods Firstly, we assessed the analytical performances of new generation assays (imprecision, bias, total error, limit of quantification) and compared previous and new generation assays in the absence of biotin. Secondly, we challenged both generations of assays with samples spiked with seven different biotin levels. The efficiency of new generation assays was also compared to the streptavidin beads treatment. Results New generation assays presented suitable analytical performances. Previous and new generations of hsTnT and TSH assays were commutable in the absence of biotin. In the presence of biotin, we confirmed that previous generation assays were affected by biotin concentration as low as 40.5 ng/mL and that new generation assays were not affected up to the announced tolerance threshold of 1200 ng/mL. After the streptavidin beads treatment, we observed a higher imprecision for both parameters and a constant 10% negative bias for TSH compared to new generation assays. Conclusions New generation of electrochemiluminescent immunoassays appears as a reliable systematic solution to prevent biotin interference for hsTnT and TSH testing.

Biotin interferences: Have we neglected the impact on serological markers?

BACKGROUND: Biotin has been reported to be a leading cause of interference on several immunoassay platforms using the streptavidin-biotin immobilization system. While biotin interferences have now been well characterized for several assays, only few data are available on their impact on serological markers of infectious viral diseases. METHODS: Overall, 10 healthy volunteers (HVs) received a single 100 mg dose of biotin to evaluate its effect on hepatitis B serological markers. Blood samples were taken several times before and after biotin intake. In addition, spiking experiments were applied to investigate biotin's impact on anti-HIV/p24 Ag and anti-HCV antibody levels. Several procedures designed to overcome this interference were evaluated. RESULTS: Biotin intake resulted in a false-negative anti-HBs immunological status (<10 mIU/mL) in 40.0% of cases. According to our anti-HBc and anti-HBe results, biotin intake was associated with 90.0% and 80.0% of false positive results, respectively. At the theoretical biotin peak concentration following a 100 mg intake, 50.0% and 66.6% of anti-HIV and anti-HCV results were false negatives, respectively. All the procedures evaluated to overcome the interference were proven effective. CONCLUSION: HBV, HCV, and HIV serological markers are likely to be highly sensitive to biotin. Our data confirm that the scope of biotin interference is broader than commonly described.

Macro vitamin B12: an underestimated threat.

Background The correct identification of the macro-B12 interference (macroforms) is paramount to avoid potential erroneous clinical decisions. Our objectives were to determine whether immunoassays are affected by the presence of macro-B12 and to validate a polyethylene glycol (PEG) precipitation procedure to detect it. Methods Sixty-two serum samples obtained from healthy volunteers were analyzed to determine recovery and reference intervals (RIs) following PEG precipitation. Thereafter, 50 serum samples with very high levels of B12 (>1476 pmol/L) were randomly selected to search for macro-B12 interferences. Serum samples obtained from healthy volunteers and related PEG aliquots were analyzed on a Cobas® immunoassay. Patients' samples were analyzed on both Cobas® and Architect® immunoassays. Finally, samples suspected to contain macro-B12 were analyzed by size-exclusion chromatography (SEC) to confirm the presence of macro-B12. Results Recovery and post-PEG RIs determined on a Cobas 8000® in healthy volunteers ranged from 68.3% to 108.4% and from 122.1 to 514.4 pmol/L, respectively. Fifteen samples (30%) were found to show macro-B12 while using the recovery criteria, and nine samples (18%) while using the post-PEG RI. The other immunoassay ran on the Architect i2000® was also affected by the presence of macro-B12. Size-exclusion chromatography studies confirmed the presence of macro-B12 (immunoglobulin-B12 complexes). Conclusions The prevalence of macro-B12 in elevated B12 samples is high. We suggest to systematically screen for the presence of macro-B12 with PEG precipitation procedure in samples with elevated B12 levels to avoid potential misdiagnosis or harmful clinical consequences.

Combined endogenous MR biomarkers to assess changes in tumor oxygenation induced by an allosteric effector of hemoglobin.

Hypoxia is a crucial factor in cancer therapy, determining prognosis and the effectiveness of treatment. Although efforts are being made to develop methods for assessing tumor hypoxia, no markers of hypoxia are currently used in routine clinical practice. Recently, we showed that the combined endogenous MR biomarkers, R1 and R2 *, which are sensitive to [dissolved O2 ] and [dHb], respectively, were able to detect changes in tumor oxygenation induced by a hyperoxic breathing challenge. In this study, we further validated the ability of the combined MR biomarkers to assess the change in tumor oxygenation induced by an allosteric effector of hemoglobin, myo-inositol trispyrophosphate (ITPP), on rat tumor models. ITPP induced an increase in tumor pO2 , as observed using L-band electron paramagnetic resonance oximetry, as well as an increase in both R1 and R2 * MR parameters. The increase in R1 indicated an increase in [O2 ], whereas the increase in R2 * resulted from an increase in O2 release from blood, inducing an increase in [dHb]. The impact of ITPP was then evaluated on factors that can influence tumor oxygenation, including tumor perfusion, saturation rate of hemoglobin, blood pH and oxygen consumption rate (OCR). ITPP decreased blood [HbO2 ] and significantly increased blood acidity, which is also a factor that right-shifts the oxygen dissociation curve. No change in tumor perfusion was observed after ITPP treatment. Interestingly, ITPP decreased OCR in both tumor cell lines. In conclusion, ITPP increased tumor pO2 via a combined mechanism involving a decrease in OCR and an allosteric effect on hemoglobin that was further enhanced by a decrease in blood pH. MR biomarkers could assess the change in tumor oxygenation induced by ITPP. At the intra-tumoral level, a majority of tumor voxels were responsive to ITPP treatment in both of the models studied.

Mechanisms of Crystalloid versus Colloid Osmosis across the Peritoneal Membrane.

Background Osmosis drives transcapillary ultrafiltration and water removal in patients treated with peritoneal dialysis. Crystalloid osmosis, typically induced by glucose, relies on dialysate tonicity and occurs through endothelial aquaporin-1 water channels and interendothelial clefts. In contrast, the mechanisms mediating water flow driven by colloidal agents, such as icodextrin, and combinations of osmotic agents have not been evaluated.Methods We used experimental models of peritoneal dialysis in mouse and biophysical studies combined with mathematical modeling to evaluate the mechanisms of colloid versus crystalloid osmosis across the peritoneal membrane and to investigate the pathways mediating water flow generated by the glucose polymer icodextrin.ResultsIn silico modeling and in vivo studies showed that deletion of aquaporin-1 did not influence osmotic water transport induced by icodextrin but did affect that induced by crystalloid agents. Water flow induced by icodextrin was dependent upon the presence of large, colloidal fractions, with a reflection coefficient close to unity, a low diffusion capacity, and a minimal effect on dialysate osmolality. Combining crystalloid and colloid osmotic agents in the same dialysis solution strikingly enhanced water and sodium transport across the peritoneal membrane, improving ultrafiltration efficiency over that obtained with either type of agent alone.Conclusions These data cast light on the molecular mechanisms involved in colloid versus crystalloid osmosis and characterize novel osmotic agents. Dialysis solutions combining crystalloid and colloid particles may help restore fluid balance in patients treated with peritoneal dialysis.

A Large Intraperitoneal Residual Volume Hampers Adequate Volumetric Assessment of Osmotic Conductance to Glucose.

BACKGROUND: In end-stage renal disease patients treated with peritoneal dialysis (PD), the osmotic conductance to glucose (OCG) represents the intrinsic ability of the membrane to transport water in response to a crystalloid osmotic gradient. A progressive loss of OCG in long-term PD patients indicates the development of fibrosis in the peritoneal interstitium, and helps identify patients at risk for encapsulating peritoneal sclerosis. The double mini-peritoneal equilibration test (PET) has been proposed as a simple method to assess OCG using the difference in initial ultrafiltration rates generated by 2 successive dwells using 1.36% and 3.86% glucose-based, 1-h PET. However, the presence of a large peritoneal residual volume (RV) may potentially interfere with the correct evaluation of drained volumes, limiting the reliability of OCG assessed by the double mini-PET. METHODS: We retrospectively reviewed data from 53 peritoneal function tests in 35 consecutive PD patients starting PD at our center between March 2013 and March 2017. The test consisted of a uni-PET (double mini-PET combined with a 3.86%, 4-h PET) performed at PD start, then yearly. In addition to peritoneal solute transport rate and net ultrafiltration, the tests provided information about osmotic water transport (OCG, sodium sieving, and free-water transport) as well as the RV estimated from albumin dilution. RESULTS: Contrary to sodium sieving, net ultrafiltration, and free-water transport, OCG did not correlate with any of the other parameters of osmotic water transport. In multivariate regression analyses, the RV was identified as the only determinant of OCG, while it did not alter the robust association between sodium sieving/free-water transport and their respective determinants. Considering only baseline tests or the whole series of tests, the presence of a large intraperitoneal RV was associated with discrepant values between OCG and sodium sieving, and with an artificial increase in OCG. CONCLUSIONS: A large RV leads to significant overestimation of OCG using the double mini-PET, potentially reducing the ability of OCG to identify patients with progressive fibrosis in the peritoneal interstitium. On the other hand, sieving of the dialysate sodium, a biochemical surrogate for OCG, is independent of the RV and may therefore be more reliable. A call for caution is warranted in patients with a large RV to avoid misinterpretation of OCG values derived from the double mini-PET.

How to optimize the prescription of laboratory tests? Success and failures in an academic hospital.

In the context of the flat-rate reimbursements in healthcare, we reviewed physicians' behavior towards laboratory test ordering. We demonstrated how it could be improved when a specific stage of the patient management is considered. We took a multi-step approach to analyze the laboratory test orders in the context of planned laparoscopic cholecystectomy in a general teaching hospital. A reference order set was defined through a collaborative analysis between clinicians and laboratory physicians. The clinical and financial impacts were then evaluated over a period of 24 months. After the introduction of the reference order set, the number of laboratory tests per order decreased significantly for patients with cholecystitis of low severity. Above the monitoring of repeated orderings during a single stay, the major impacts were achieved by a drastic reduction of inappropriate orders, particularly in the field of bacteriology. The main effects of the order set were maintained throughout a follow-up period of 24 months. Our study demonstrated that, when considering laboratory test ordering optimization, reference order sets could achieve high levels of efficiency. To ensure high compliance to reference order sets, extensive collaboration between clinicians and laboratory physician is mandatory even if very sophisticated information systems are available.