Michela Pelloso

Performance criteria and quality indicators for the pre-analytical phase

Abstract The definition, implementation and monitoring of valuable analytical quality specifications have played a fundamental role in improving the quality of laboratory services and reducing the rates of analytical errors. However, a body of evidence has been accumulated on the relevance of the extra-analytical phases, namely the pre-analytical steps, their vulnerability and impact on the overall quality of the laboratory information. The identification and establishment of valueable quality indicators (QIs) represents a promising strategy for collecting data on quality in the total testing process (TTP) and, particularly, for detecting any mistakes made in the individual steps of the pre-analytical phase, thus providing useful information for quality improvement projects. The consensus achieved on the developed list of harmonized QIs is a premise for the further step: the identification of achievable and realistic performance targets based on the knowledge of the state-of-the-art. Data collected by several clinical laboratories worldwide allow the classification of performances for available QIs into three levels: optimum, desirable and minimum, in agreement with the widely accepted proposal for analytical quality specifications.

VKORC1, CYP2C9 and CYP4F2 genetic-based algorithm for warfarin dosing: an Italian retrospective study.

AIM A total of 371 patients under stable warfarin therapy were retrospectively selected to develop a pharmacogenetic algorithm to identify the individual maintenance dose. MATERIALS & METHODS The variables that were entered into the algorithm were: VKORC1, CYP2C9 and CYP4F2 polymorphisms, body surface area and age. RESULTS The percentage of cases whose predicted mean weekly warfarin dose was within 20% of the actual maintenance dose was 51.8% considering patients overall, and were 36.2, 66.2 and 55.4%, respectively, taking into account patients requiring low (≤25 mg/week), intermediate (25-45 mg/week) and high (≥45 mg/week) doses. The algorithm could correctly assign 73.8 and 63.2% of patients to the low- and high-dose regimens, respectively. We developed and validated a pharmacogenetic algorithm in a series of Italian patients, we then tested, in the same series of italian patients, the formulas of three published algorithms. These three algorithms were developed and validated by their authors in a series of patients different from our own. The performance of our algorithm in our patients series was slightly higher than that achieved when using the three other algorithms in our patients series. CONCLUSION The high predictive accuracy of low and high warfarin requirements of our algorithm warrants its application in prospective studies for clinical validation.

Pancreatic Tumors and Immature Immunosuppressive Myeloid Cells in Blood and Spleen: Role of Inhibitory Co-Stimulatory Molecules PDL1 and CTLA4. An In Vivo and In Vitro Study

Background Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4. Methodology and Principal Findings 103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8+ lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33+CD14−HLA-DR− were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33+CD14+HLA-DR− IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive. Conclusion In PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

Laboratory critical values: automated notification supports effective clinical decision making.

OBJECTIVE Failure to adequately communicate a laboratory critical value (CV) is a potential cause of adverse events. The harmonization of CV reporting is increasingly recognized as a key issue in ensuring patient care and minimizing harm. With ongoing improvements in CV reporting, the patients outcome should be audited to assess the effectiveness of CV notification. DESIGN AND METHODS We report the data audited throughout a six month-period during which an analysis was made of CVs, and we describe the approach of clinicians and general practitioners (GPs), and their decision making following CV reporting. RESULTS CV notification led to a change of treatment in 98.0% of patients admitted to surgical and in 90.6% of those admitted to medical wards. Clinicians made a further evaluation of new complications in patients in 70.0% and 60.4% of cases, in surgical and medical wards respectively. In more than 40.0% of cases, CVs were unexpected findings. In the primary care setting, critical hyperkalemia was managed by GPs in 55% of patients, thus sparing patients hospitalization. For all outpatients with critical INR (international normalized ratio), the GPs changed or stopped warfarin dosage. Twenty-four percent of patients were checked for an additional INR, whereas a further medical examination by a consultant in the hospital setting was requested for 5% of patients. CONCLUSIONS The laboratory plays a key role in ensuring patient safety, especially in CV reporting. An evaluation should be made of the patients outcome and clinical decision making in order to assess the effectiveness of the CV process.

New screening tests enrich anti-transglutaminase results and support a highly sensitive two-test based strategy for celiac disease diagnosis.

BACKGROUND The identification of specific serological algorithms allowing the diagnosis of celiac disease (CD) is a new challenge for both the clinic and the laboratory. We compared the diagnostic accuracy of three new tests proposed for CD screening with that of the well established IgA tTG, and ascertained whether any combination of these tools might enhance accuracy in diagnosing CD. METHODS In sera from 329 CD and 374 control children, the following were assayed: IgA tTG; IgA/IgG, which identify tTG-gliadin complexes (Aeskulisa Celi Check and CeliCheck IgGA); IgA/IgG, which identify deamidated gliadin peptides and tTG (QUANTA Lite(TM) h-tTG/DGP Screen). RESULTS When specificity was set at 100%, the most sensitive index of CD was IgA tTG (75.7%, cut-off=100U), followed by QUANTA Lite(TM) h-tTG/DGP Screen (65.3%, cut-off 145U), Aeskulisa Celi Check (62.6%, cut-off 909U/mL) and CeliCheck IgGA (59.6%, cut-off 977U/mL). Three algorithms were obtained by combining IgA tTG with each of the new tests. The algorithm obtained by measuring IgA tTG and QUANTA Lite(TM) h-tTG/DGP Screen allowed the correct identification of CD in 78.7% of cases (negative predictive value=97.3%). CONCLUSIONS The two-test based strategy could be used for the cost effective diagnosis of CD.

A Randomized Trial of Pharmacogenetic Warfarin Dosing in Naïve Patients with Non-Valvular Atrial Fibrillation

Genotype-guided warfarin dosing have been proposed to improve patient’s management. This study is aimed to determine whether a CYP2C9- VKORC1- CYP4F2-based pharmacogenetic algorithm is superior to a standard, clinically adopted, pharmacodynamic method. Two-hundred naïve patients with non-valvular atrial fibrillation were randomized to trial arms and 180 completed the study. No significant differences were found in the number of out-of-range INRs (INR<2.0 or >3.0) (p = 0.79) and in the mean percentage of time spent in the therapeutic range (TTR) after 19 days in the pharmacogenetic (51.9%) and in the control arm (53.2%, p = 0.71). The percentage of time spent at INR>4.0 was significantly lower in the pharmacogenetic (0.7%) than in the control arm (1.8%) (p = 0.02). Genotype-guided warfarin dosing is not superior in overall anticoagulation control when compared to accurate clinical standard of care. Trial Registration ClinicalTrials.gov NCT01178034

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1

BackgroundIn order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines.ResultsNT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFβ1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFβ1 canonical signaling pathway. TGFβ1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFβ1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFβ1 (MALDI-TOF/MS and co-immuno-precipitation).ConclusionsThe effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFβ1 on cell signaling, and the formation of complexes between TGFβ1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

Computer-based-limited and personalised education management maximise appropriateness of vitamin D, vitamin B12 and folate retesting

Aim To identify the best management strategy for improving the appropriateness of vitamin D, vitamin B12 and folate retesting. Methods The study was conducted between 3 November 2012 and 8 June 2015, with inpatients and outpatients being considered separately. After an observational reference period (3 November 2012 to 14 September 2013), an information technology (IT)-based permissive strategy (16 September 2013 to 27 July 2014) followed by a limiting strategy was used to manage the demand for inpatient retesting. For outpatients, an educational strategy period (28 July 2014 to 16 December 2014) with direct contact between medical personnel and general practitioners (GPs) was followed by a post-educational period without any restriction. Data from a total of 66 496 patients for vitamin D, 14 618 for vitamin B12 and 14 445 for folate were retrieved from the laboratory IT system. The main outcomes measures were inappropriate vitamin D, vitamin B12 and folate retesting. The minimal retesting intervals were 90 (vitamin D) or 180 days (vitamin B12 and folate). Results In the absence of a laboratory demand strategy, the frequency of inappropriate retesting for vitamin D, vitamin B12 and folate was 60%, 94% and 93%, respectively, for inpatients, and 27%, 87% and 87%, respectively, for outpatients. A limiting IT-based demand management strategy reduced inappropriate retesting for vitamin D (36%), but not for vitamin B12 and folate. The educational strategy was followed by a reduction in inappropriate retesting among outpatients (16% for vitamin D, 72% for vitamin B12 and folate). Conclusions Laboratory demand management based on an IT-limiting management strategy or on education of the referring physicians appears helpful in maximising appropriate retesting.

Effectiveness of the Combined Evaluation of KLK3 Genetics and Free-to-Total Prostate Specific Antigen Ratio for Prostate Cancer Diagnosis

PURPOSE Of serum prostate specific antigen variability 40% depends on inherited factors. We ascertained whether the knowledge of KLK3 genetics would enhance prostate specific antigen diagnostic performance in patients with clinical suspicion of prostate cancer. MATERIALS AND METHODS We studied 1,058 men who consecutively underwent prostate biopsy for clinical suspicion of prostate cancer. At histology prostate cancer was present in 401 cases and absent in 657. Serum total prostate specific antigen and the free-to-total prostate specific antigen ratio were determined. Four polymorphisms of the KLK3 gene (rs2569733, rs2739448, rs925013 and rs2735839) and 1 polymorphism of the SRD5A2 gene (rs523349) were studied. The influence of genetics on prostate specific antigen variability was evaluated by multivariate linear regression analysis. The performance of total prostate specific antigen and the free-to-total prostate specific antigen ratio alone or combined with a genetically based patient classification were defined by ROC curve analyses. RESULTS For prostate cancer diagnosis the free-to-total prostate specific antigen ratio index alone (cutoff 11%) was superior to total prostate specific antigen (cutoff 4 ng/ml) and to free-to-total prostate specific antigen ratio reflex testing (positive predictive value 61%, 43% and 54%, respectively). Prostate specific antigen correlated with KLK3 genetics (rs2735839 polymorphism p = 0.001, and rs2569733, rs2739448 and rs925013 haplotype combination p = 0.003). In patients with different KLK3 genetics 2 optimal free-to-total prostate specific antigen ratio cutoffs (11% and 14.5%) were found. For free-to-total prostate specific antigen ratio values between 11% and 14.5% the prostate cancer probability ranged from 30.0% to 47.4% according to patient genetics. CONCLUSIONS The free-to-total prostate specific antigen ratio is superior to total prostate specific antigen for prostate cancer diagnosis, independent of total prostate specific antigen results. Free-to-total prostate specific antigen ratio findings below 11% are positively associated with prostate cancer and those above 14.5% are negatively associated with prostate cancer, while the interpretation of those between 11% and 14.5% is improved by patient KLK3 genetic analysis.

Blood expression of matrix metalloproteinases 8 and 9 and of their inducers S100A8 and S100A9 supports diagnosis and prognosis of PDAC-associated diabetes mellitus.

BACKGROUND Based on the knowledge that matrix metalloproteinases (MMPs) and S100A8/A9 synergistically work in causing PDAC-associated type 2 diabetes mellitus (T2DM), we verified whether tissue and blood MMP8, MMP9, S100A8 and S100A9 expression might help in distinguishing PDAC among diabetics. METHODS Relative quantification of MMP8, MMP9, S100A8 and S100A9 mRNA was performed in tissues obtained from 8 PDAC, 4 chronic pancreatitis (ChrPa), 4 non-PDAC tumors and in PBMCs obtained from 30 controls, 43 T2DM, 41 ChrPa, 91 PDAC and 33 pancreatic-biliary tract tumors. RESULTS T2DM was observed in PDAC (66%), in pancreatic-biliary tract tumors (64%) and in ChrPa (70%). In diabetics, with or without PDAC, MMP9 tissue expression was increased (p<0.05). Both MMPs increased in PDAC and MMP9 increased also in pancreatic-biliary tract tumors PBMCs. In diabetics, MMP9 was independently associated with PDAC (p=0.025), but failed to enhance CA 19-9 discriminant efficacy. A highly reduced S100A9 expression, found in 7 PDAC, was significantly correlated with a reduced overall survival (p=0.015). CONCLUSIONS An increased expression of tissue and blood MMP9 reflects the presence of PDAC-associated diabetes mellitus. This finding fits with the hypothesized role of MMPs as part of the complex network linking cancer to diabetes.