The VES-Matic 5 system: performance of a novel instrument for measuring erythrocyte sedimentation rate.

OBJECTIVES: The VES-Matic 5 is an automated analyzer that assesses erythrocyte sedimentation rate based on a modified Westergren sedimentation technique. Instrument performance was established by addressing the recommendations of the International Council for Standardization in Haematology. METHODS: Comparison against the reference Westergren method was performed for all samples, and further for the low, middle, and upper third of the analytical range. Intra-run precision, inter-run precision, and interference studies were further assessed. This study included the evaluation of reference ranges. RESULTS: The comparison of methods by Passing-Bablok analysis has shown a good agreement without systematic or proportional differences. The regression equation was y=-0.646 + 0.979x. The mean bias of -0.542 was obtained by Bland-Altman analysis and the upper limit of 8.03 with the lower limit of -9.11 can be considered clinically acceptable. Intra-run and inter-run precision were good for each parameter and interference studies did not show any significant bias with exception of anemia samples, which showed a proportional difference when comparing high erythrocyte sedimentation rate values. Using the local adult reference population, we verified the reference ranges in comparison to those available in the literature, and according to the Clinical Laboratory Standards Institute (CLSI) EP28-A3C document. We determined the upper limit partitioned by gender and the following age groups: from 18 to 50, from 50 to 70, and over 70. CONCLUSIONS: The VES-Matic 5 analyzer presented good comparability with the reference method. As there are commercial quality control and suitable external quality assessment (EQA) material and programs, the VES-Matic 5 can be employed appropriately for routine purposes.

Monocyte distribution width (MDW) parameter as a sepsis indicator in intensive care units.

OBJECTIVES: Patients in Intensive Care Units (ICU) are a high-risk population for sepsis, recognized as a major cause of admission and death. The aim of the current study was to evaluate the diagnostic accuracy and prognostication of monocyte distribution width (MDW) in sepsis for patients admitted to ICU. METHODS: Between January and June 2020, we conducted a prospective observational study during the hospitalization of 506 adult patients admitted to the ICU. MDW was evaluated in 2,367 consecutive samples received for routine complete blood counts (CBC) performed once a day and every day during the study. Sepsis was diagnosed according to Sepsis-3 criteria and patients enrolled were classified in the following groups: no sepsis, sepsis and septic shock. RESULTS: MDW values were significantly higher in patients with sepsis or septic shock in comparison to those within the no sepsis group [median 26.23 (IQR: 23.48-29.83); 28.97 (IQR: 21.27-37.21); 21.99 (IQR: 19.86-24.36) respectively]. ROC analysis demonstrated that AUC is 0.785 with a sensitivity of 66.88% and specificity of 77.79% at a cut-off point of 24.63. In patients that developed an ICU-acquired sepsis MDW showed an increase from 21.33 [median (IQR: 19.47-21.72)] to 29.19 [median (IQR: 27.46-31.47)]. MDW increase is not affected by the aetiology of sepsis, even in patients with COVID-19. In sepsis survivors a decrease of MDW values were found from the first time to the end of their stay [median from 29.14 (IQR: 26.22-32.52) to 25.67 (IQR: 22.93-30.28)]. CONCLUSIONS: In ICU, MDW enhances the sepsis detection and is related to disease severity.

Automated measurement of the erythrocyte sedimentation rate: method validation and comparison.

Background Development of automated analyzers for erythrocyte sedimentation rate (ESR) has imposed the need for extensive validation prior to their implementation in routine practice, to ensure comparability with the reference Westergren method. The aim of our study was to perform the analytical validation of two automated ESR analyzers, the Ves-Matic Cube 200 and the TEST1. Methods Validation was performed according to the recent International Council for Standardization in Hematology recommendations and included determination of intrarun and inter-run precision, assessment of sample carryover, hemolysis interference, sensitivity to fibrinogen, method comparison with the gold standard Westergren method and stability test. Results The highest intrarun imprecision was obtained for the low ESR range (33.5% for Ves-Matic Cube; 37.3% for TEST1) while inter-run coefficients of variation on three levels were much better for the TEST1 (0%, 2% and 1.2%) compared to the Ves-Matic Cube 200 on two levels (24.9% and 5.8%). Both Ves-Matic Cube 200 and TEST1 showed no statistically significant difference when compared with Westergren. Bland-Altman analysis yielded overall insignificant mean biases for all comparisons, but a wider dispersion of results and 95% limits of agreement for comparisons including the Ves-Matic Cube 200. Carryover was considered insignificant, while hemolysis had a negative effect on all assessed ESR methods. The highest sensitivity to fibrinogen was observed for the Ves-Matic Cube 200, followed by Westergren and the least sensitive was the TEST1. Conclusions The obtained results proved the analytical validity of the TEST1 and the Ves-Matic Cube 200, and high comparability with the gold standard Westergren method, showing obvious improvements in standardization of ESR methods.

Performance criteria and quality indicators for the post-analytical phase.

BACKGROUND: Quality indicators (QIs) used as performance measurements are an effective tool in accurately estimating quality, identifying problems that may need to be addressed, and monitoring the processes over time. In Laboratory Medicine, QIs should cover all steps of the testing process, as error studies have confirmed that most errors occur in the pre- and post-analytical phase of testing. Aim of the present study is to provide preliminary results on QIs and related performance criteria in the post-analytical phase. METHODS: This work was conducted according to a previously described study design based on the voluntary participation of clinical laboratories in the project on QIs of the Working Group "Laboratory Errors and Patient Safety" (WG-LEPS) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). RESULTS: Overall, data collected highlighted an improvement or stability in performances over time for all reported indicators thus demonstrating that the use of QIs is effective in the quality improvement strategy. Moreover, QIs data are an important source for defining the state-of-the-art concerning the error rate in the total testing process. The definition of performance specifications based on the state-of-the-art, as suggested by consensus documents, is a valuable benchmark point in evaluating the performance of each laboratory. CONCLUSIONS: Laboratory tests play a relevant role in the monitoring and evaluation of the efficacy of patient outcome thus assisting clinicians in decision-making. Laboratory performance evaluation is therefore crucial to providing patients with safe, effective and efficient care.

Performance criteria and quality indicators for the pre-analytical phase.

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.

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFß1.

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFß1 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. RESULTS: NT-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 TGFß1. 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 TGFß1 canonical signaling pathway. TGFß1 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 TGFß1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFß1 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFß1 on cell signaling, and the formation of complexes between TGFß1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

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.

T Cell Senescence by Extensive Phenotyping: An Emerging Feature of COVID-19 Severity.

OBJECTIVE: To identify the potential prognostic value of lymphocyte subsets in COVID-19 patients, where lymphopenia is a common finding. METHODS: In 353 COVID-19 inpatients and 40 controls T cell subsets with markers of senescence and exhaustion were studied by flow cytometry. RESULTS: In severe illness, total lymphocytes B, NK, and all T subsets were dampened. Senescent CD4+, but mainly CD8+ T cells, increased in patients with respect to controls. The most significant index predicting fatal outcome was neutrophils/CD3+ T ratio. CONCLUSION: In conclusion, an altered T cell pattern underlies COVID-19 severity and is involved in predicting the outcome.

Reduced muscle mass as predictor of intensive care unit hospitalization in COVID-19 patients.

PURPOSE: To evaluate if reduced muscle mass, assessed with Computed Tomography (CT), is a predictor of intensive care unit (ICU) hospitalization in COVID-19 patients. METHODS: In this Institution Review Board approved study, we retrospectively evaluated COVID-19 patients treated in our tertiary center from March to November 2020 who underwent an unenhanced chest CT scan within three weeks from hospitalization.We recorded the mean Hounsfield Unit (Hu) value of the right paravertebral muscle at the level of the 12th thoracic vertebra, the hospitalization unit (ICU and COVID-19 wards), clinical symptoms, Barthel Index, and laboratory findings.Logistic regression analysis was applied to assess if muscle loss (Hu<30) is a predictor of ICU admission and outcome.Fisher's exact and Student's tests were applied to evaluate if differences between patients with and without muscle loss occurred (p<0.05). RESULTS: One-hundred-fifty patients matched the inclusion criteria (46 females; mean age±SD 61.3±15 years-old), 36 treated in ICU. Patients in ICU showed significantly lower Hu values (29±24 vs 39.4±12, p = 0.001). Muscle loss was a predictor of ICU admission (p = 0.004).Patients with muscle loss were significantly older (73.4±10 vs 56.4±14 years), had lower Barthel Index scores (54.4±33 vs 85.1±26), red blood-cell count (3.9±1 vs 4.6±1×1012L-1), and Hb levels (11.5±2 vs 13.2±2g/l) as well as higher white blood-cell count (9.4±7 vs 7.2±4×109L-1), C-reactive protein (71.5±71 vs 44±48U/L), and lactate dehydrogenase levels (335±163 vs 265.8±116U/L) (p<0.05, each). CONCLUSIONS: Muscle loss seems to be a predictor of ICU hospitalization in COVID-19 patients and radiologists reporting chest CT at admission should note this finding in their reports.

Assessment and monitoring of agreement among professionals for morphological evaluation in compliance with International accreditation standard requirements.

OBJECTIVES: Standardized criteria guaranteeing harmonized interpretation among morphologists in the provision of morphology results represent an important tool to be adopted for risk management and patient safety. Aim of this work is to assess agreement among morphologists in the microscopic evaluation of the peripheral blood smear. METHODS: 17 morphologists participating in the external quality assessment (EQA) program individually evaluated the blood smear and recorded the results using a personal username and password. Agreement among operators was evaluated. RESULTS: The overall agreement rate in microscopic differential was 95% in 2016 and 97% in 2017 (acceptance limit 90%), with 6/120 and 4/120 incongruent results, respectively. The agreement for the diagnostic hypothesis was satisfactory with a full agreement being reached in 5 out of 16 cases. CONCLUSIONS: The creation of a tool to assess the agreement of readers providing morphological evaluations is a valuable step forward in ensuring patient safety and quality laboratory medicine.

Validation of a composed COVID-19 chest radiography score: the CARE project.

OBJECTIVES: The aim of this study was to validate a composed coronavirus disease 2019 (COVID-19) chest radiography score (CARE) based on the extension of ground-glass opacity (GG) and consolidations (Co), separately assessed, and to investigate its prognostic performance. METHODS: COVID-19-positive patients referring to our tertiary centre during the first month of the outbreak in our area and with a known outcome were retrospectively evaluated. Each lung was subdivided into three areas and a three-grade score assessing the extension of GG and Co was used. The CARE was derived from the sum of the subscores. A mixed-model ANOVA with post hoc Bonferroni correction was used to evaluate whether differences related to the referring unit (emergency room, COVID-19 wards and intensive care unit (ICU)) occurred. Logistic regression analyses were used to investigate the impact of CARE, patients' age and sex on the outcome. To evaluate the prognostic performance of CARE, receiver operating characteristic curves were computed for the entire stay and at admission only. RESULTS: A total of 1203 chest radiographs of 175 patients (120 males; mean age 67.81±15.5 years old) were examined. On average, each patient underwent 6.8±10.3 radiographs. Patients in ICU as well as deceased patients showed higher CARE scores (p<0.05, each). Age, Co and CARE significantly influenced the outcome (p<0.05 each). The CARE demonstrated good accuracy (area under the curve (AUC)=0.736) using longitudinal data as well as at admission only (AUC=0.740). A CARE score of 17.5 during hospitalisation showed 75% sensitivity and 69.9% specificity. CONCLUSIONS: The CARE was demonstrated to be a reliable tool to assess the severity of pulmonary involvement at chest radiography with a good prognostic performance.

Ves-Matic CUBE 200: is modified Westergren method for erythrocyte sedimentation rate a valid alternative to the gold standard?

Ves-Matic CUBE 200 is an automated erythrocyte sedimentation rate (ESR) analyser based on the modified Westergren principle of measurement. In this study, we aimed to assess its analytical performance following the key points addressed by the International Council for Standardization in Haematology and the comparability with the gold standard Westergren method. Comparison of the two methods yielded a correlation coefficient of 0.852, no significant bias and a small constant difference between compared results. Intrarun coefficients of variation (CV) ranged from 2.2% to 22.2%, the higher being for lower ESR values, while inter-run CVs were 19.7% for the normal range and 3.0% for the abnormal range. This study proved the analytical validity of the Ves-Matic CUBE 200 and its high comparability with the Westergren method, showing obvious improvements in the technology applied for automated determination of ESR and a valuable step forward in standardisation of ESR methods.

Performance specifications of critical results management.

Formerly defined "critical values", the importance of critical results (CRs) management in patient care has grown in recent years. According to the George Lundberg definition the result becomes "critical" when, exceeding actionable thresholds, it suggests imminent danger for the patient, unless appropriate therapy is initiated promptly. As required in most important accreditation standards, such as the ISO:15,189 or the Joint Commission standards, a quality reporting system should deliver the correct result to the appropriate clinician in a time-frame that ensures patient safety. From this point of view, medical laboratories should implement a process that assures the most effective communication in a timely manner, to the referring physician or care team member. Failure in communication, particularly in this type of situation, continues to be one of the most common factors contributing to the occurrence of adverse events. In the last few decades, Information Technology (IT) in Health Care has become increasingly important. The ability to interface radiology, anatomic pathology or laboratory information systems with electronic medical records is now a real opportunity, offering much safer communication than in the past. Future achievements on performance criteria and quality indicators for the notification of CRs, should ensure a comparable examination across different institutions, adding value to clinical laboratories in controlling post-analytical processes that concern patient safety. Therefore, the novel approach to CRs should combine quality initiatives, IT solutions and a culture to strengthen professional interaction.

Relevance of pre-analytical blood management on the emerging cardiovascular protein biomarkers TWEAK and HMGB1 and on miRNA serum and plasma profiling.

BACKGROUND: Disease-independent sources of biomarker variability include pre-analytical, analytical and biological variance. The aim of the present study was to evaluate whether the pre-analytical phase has any impact on the emerging heart disease TWEAK and HMGB1 protein markers and miRNA biomarkers, and whether peptidome profiling allows the identification of pre-analytical quality markers. METHODS: An assessment was made of sample type (serum, EDTA-Plasma, Citrate-Plasma, ACD-plasma, Heparin-plasma), temperature of sample storage (room temperature or refrigerated), time of sample storage (0.5, 3, 6 and 9h) and centrifugation (one or two-step). Aliquots of all processed samples were immediately frozen (-80°C) before analysis. Proteins were assayed by ELISAs, miRNA expression profile by microarray and peptidome profiling by MALDI-TOF/MS. RESULTS: Temperature, time and centrifugation had no impact on TWEAK and HMGB1 results, which were significantly influenced by matrix type, TWEAK levels being significantly higher (F=194.7, p<0.0001), and HMGB1 levels significantly lower (F=36.32, p<0.0001) in serum than in any other plasma type. Unsuitable miRNA results were obtained using Heparin-plasma. Serum miRNA expression profiles depended mainly on temperature, while EDTA-plasma miRNA expression profiles were strongly affected by the centrifugation method used. MALDI-TOF/MS allowed the identification of seven features as indices of pre-analytical serum (m/z at 1206, 1350, 1865 and 2021) or EDTA-plasma (m/z 1897, 2740 and 2917) degradation. CONCLUSIONS: Serum and EDTA-plasma allow the analysis of both proteins and miRNA emerging biomarkers of heart diseases. Refrigerated storage prevents an altered miRNA expression profile also in cases of a prolonged time-interval between blood drawing and processing.

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.