Clinical variables associated with late-onset thrombotic and cardiovascular events, after SARS-CoV-2 infection, in a cohort of patients from the first epidemic wave: an 18-month analysis on the "Surviving-COVID" cohort from Bergamo, Italy.

Importance: Population studies have recorded an increased, unexplained risk of post-acute cardiovascular and thrombotic events, up to 1 year after acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To search for clinical variables and biomarkers associated with late post-acute thrombotic and cardiovascular events after SARS-CoV-2 infection. Design: Retrospective cohort study. Setting: Third-level referral hospital in Bergamo (Italy). Participants: Analysis of an existing database of adult patients, who received care for SARS-CoV-2 infection at our institution between 20 February and 30 September 2020, followed up on a single date ("entry date") at 3-6 months. Exposure: Initial infection by SARS-CoV-2. Main outcomes and measures: Primary outcome: occurrence, in the 18 months after entry date, of a composite endpoint, defined by the International Classification of Diseases-9th edition (ICD-9) codes for at least one of: cerebral/cardiac ischemia, venous/arterial thrombosis (any site), pulmonary embolism, cardiac arrhythmia, heart failure. Measures (as recorded on entry date): history of initial infection, symptoms, current medications, pulmonary function test, blood tests results, and semi-quantitative radiographic lung damage (BRIXIA score). Individual clinical data were matched to hospitalizations, voluntary vaccination against SARS-CoV-2 (according to regulations and product availability), and documented reinfections in the following 18 months, as recorded in the provincial Health Authority database. A multivariable Cox proportional hazard model (including vaccine doses as a time-dependent variable) was fitted, adjusting for potential confounders. We report associations as hazard ratios (HR) and 95% confidence intervals (CI). Results: Among 1,515 patients (948 men, 62.6%, median age 59; interquartile range: 50-69), we identified 84 endpoint events, occurring to 75 patients (5%): 30 arterial thromboses, 11 venous thromboses, 28 arrhythmic and 24 heart failure events. From a multivariable Cox model, we found the following significant associations with the outcome: previous occurrence of any outcome event, in the 18 months before infection (HR: 2.38; 95% CI: 1.23-4.62); BRIXIA score ≥ 3 (HR: 2.43; 95% CI: 1.30-4.55); neutrophils-to-lymphocytes ratio ≥ 3.3 (HR: 2.60; 95% CI: 1.43-4.72), and estimated glomerular filtration rate < 45 ml/min/1.73 m2 (HR: 3.84; 95% CI: 1.49-9.91). Conclusions and relevance: We identified four clinical variables, associated with the occurrence of post-acute thrombotic and cardiovascular events, after SARS-CoV-2 infection. Further research is needed, to confirm these results.

Evaluation and comparison of automated hematology analyzer, flow cytometry, and digital morphology analyzer for monocyte counting.

INTRODUCTION: This study was aimed to evaluate monocyte counts on Sysmex XN-9000, Sysmex CyFlow Space System, and Sysmex DI60 and compare the performance of these systems with the reference optical microscopy (OM) assessment. METHODS: In all, 55 peripheral blood samples, collected in K3 EDTA tubes, were analyzed with XN-9000, CyFlow System (FlowDiff1 and 2), DI60, and OM. Within-run imprecision was carried out using normal samples. Data comparison was performed with Passing-Bablok regression and Bland-Altman plots. RESULTS: The within-run imprecision of monocyte count on XN, FlowDiff, OM, and DI60 ranged between 1.9% for FlowDiff 2 and 22.1% for DI60. The Passing-Bablok regression analysis of absolute count yielded slopes comprised between 0.93 (FlowDiff2 vs DI60) and 1.21 (DI60 vs OM), whereas the intercepts ranged between -0.002 (FlowDiff 1 vs FlowDiff 2) and 0.13 (FlowDiff1 and 2 vs DI60). Bland-Altman plots in absolute values yielded absolute bias comprised between -0.01 × 109 /L (FlowDiff 1 vs FlowDiff 2; DI60 vs OM) and 0.15 × 109 (XN-module vs DI60). CONCLUSION: The results of this analytical evaluation suggest that flow cytometry generates monocyte counts suitable for routine clinical use. OM or DI60 analysis may be useful for identifying morphologic abnormalities, but does not achieve a satisfactory level of accuracy for enumerating blood cells types such as monocytes, which are usually very low in peripheral blood.

Abnormal scattergrams and cell population data generated by fully automated hematological analyzers: New tools for screening malaria infection?

INTRODUCTION: Malaria is a life-threatening infectious disease, which has been for long confined to specific endemic areas. Nevertheless, the recent increase in immigration flows from endemic regions and imported cases has reemphasized many diagnostic challenges in Western countries, thus paving the way to introduce rapid and accurate strategies for screening subjects with suspected Malaria infection. Therefore, the aim of this article was to describe our recent experience with Sysmex XN-module for rapid screening of subjects with suspected Malaria. METHODS: Fourteen patients admitted to the Emergency Department (Papa Giovanni XXIII Hospital Bergamo, Italy) with a clinical suspicion of Malaria infection were evaluated, along with 1047 control samples. The analysis of peripheral blood was performed with XN-module, and results were then compared to optical microscopy. RESULTS: Nine patients were positive to Plasmodim falciparum, 3 to Plasmodim vivax, one to Plasmodim ovale, and one to Plasmodim malarie. Characteristic abnormalities could be observed in both white blood cell differential (WDF) and white cell nucleated (WNR) scattergrams (sensitivity 0.64 and specificity 1.0) in 9 samples with parasites at gametocyte or schizos stage irrespective of Plasmodium species and parasitic index, while characteristic scattergram abnormalities could not be seen in the 5 samples containing only parasites at the trophozoites stage. In these cases, specific variations of some cell population data (CPD) could be recorded (sensitivity 1.00 and specificity 0.91). CONCLUSION: The peculiar abnormalities observed in CPDs, WDF, and WNR-scattergrams may raise a definite suspicion of Malaria infection. Further studies should then be planned for validating these preliminary findings and assessing whether these specific abnormalities may be incorporated in rapid and inexpensive Malaria diagnostic algorithms.

Two-site evaluation of the diagnostic performance of the Sysmex XN Body Fluid (BF) module for cell count and differential in Cerebrospinal Fluid.

INTRODUCTION: Cellular analysis in cerebrospinal fluid (CSF) provides important diagnostic information in many pathological settings. The aim of this two-site study was to evaluate the Sysmex XN Body Fluid mode (XN-BF) for cell analysis of CSF compared to light microscopy (LM). METHODS: Two hundred and seven consecutive CSF samples were analyzed in parallel with XN-BF and LM. The study also included the estimation of the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), carry-over and linearity of XN-BF module. RESULTS: LoQ of white blood cells (WBC) was 3×106  cells/L; linearity was good and carry-over negligible. XN-BF parameters were compared to LM for the following cell classes: total cells, WBC, polymorphonuclear (PMN), and mononuclear (MN) cells. The bias ranged from 1.3 to 15.2×106  cells/L. The receiver operating characteristics curve analysis for WBC showed an area under the curve of 0.98, and the global diagnostic agreement was 95% at a cutoff of 5×106  cells/L. CONCLUSIONS: XN-BF provides rapid and accurate counts in clinically relevant ranges of CSF values, thus providing a valuable alternative to conventional LM analysis. However, microscopic review remains advisable in samples with abnormal cell counts or high fluorescent (HF-BF) cell parameter exceeding 5×106  cells/L.

Performance evaluation of the automated nucleated red blood cell count of five commercial hematological analyzers.

INTRODUCTION: Recent automated hematology analyzers (HAs) can identify and report nucleated red blood cells (NRBC) count as a separate population out of white blood cells (WBC). The aim of this study was to investigate the analytical performances of NRBC enumeration on five top of the range HAs. METHODS: We evaluated the within-run and between-day precision, limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) of XE-2100 and XN-module (Sysmex), ADVIA 2120i (Siemens), BC-6800 (Mindray), and UniCel DxH 800 (Beckman Coulter). Automated NRBC counts were also compared with optical microscopy (OM). RESULTS: The limits of detection for NRBC of the BC-6800, XN-module, XE-2100, UniCel DxH 800, and ADVIA 2120i are 0.035×109 /L, 0.019×109 /L, 0.067×109 /L, 0.038×109 /L, and 0.167×109 /L, respectively. Our data indicated excellent performance in terms of precision. The agreement with OM was excellent for BC-6800, XN-module, and XE-2100 (Bias 0.023, 0.019, and 0.033×109 /L, respectively). ADVIA 2120i displayed a significant constant error and UniCel DxH 800 both proportional and small constant error. CONCLUSION: Regards to NRBC counting, the performances shown by BC-6800, XN-module, and XE-2100 are excellent also a low count, ADVIA 2120i and UniCel DxH 800 need to be improved.

Comparing the performance of three panels rules of blood smear review criteria on an Italian multicenter evaluation.

BACKGROUND: The aims of this study were to compare the diagnostic accuracy of blood smear review criteria, by means of three different panel rules, those proposed by: the International Consensus Group for Hematology [41-ICGH rules], the Italian Survey [IS rules] and the Working Group on Hematology-SIBioC (WGH) consensus rules (WGH rules). METHODS: This study is based on 2707 peripheral blood (PB) samples referred for routine hematological testing to the WGH-associated laboratories displaced all over the Italian territory. The PB samples were processed on seven different hematology analyzers (HAs): Advia 2120i, XE-2100, BC-6800, ABX Pentra, XN-1000, Cell-DYN Sapphire, and DxH800, respectively. All the results provided by the HAs were analyzed through the application of three different blood smear review criteria: that is, the 41-ICGH, IS, and WGH rules. Finally, data were compared with those obtained by optical microscopy (OM), as the current gold standard. RESULTS: The overall the agreement OM classification with ICGH, IS, and WGH panel rules is 0.83, 0.83, and 0.85, respectively. The false negatives are 2.1%, 3.0%, and 2.9%, while false positives are 15.1%, 13.7%, and 11.7%, respectively. All the seven HAs showed variable interinstrument performance, as three different criteria for OM review were adopted on each of them from time to time. CONCLUSION: These results presented show that the customization of validation rules is necessary for enhancing the quality of hematological testing and optimizing workflow.

Reliability of automated synovial fluid cell counting with Mindray BC-6800 body fluid mode.

INTRODUCTION: The enumeration and differentiation of nuclear elements in synovial fluid is a cornerstone for diagnosis and follow-up of many orthopedic and rheumatologic diseases. In this study, we evaluated the analytical performance of Mindray BC-6800 BF mode (BC-6800-BF) for synovial fluid analysis. METHODS: Overall, 78 synovial fluids were collected and analyzed with both BC-6800-BF and light microscopy. The study also entailed the assessment of limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), carryover and linearity. RESULTS: The LoB for the parameters total cells and white blood cells was 6 × 106 cells/L, and the LoD and LoQ were instead 15 and 16 × 106 cells/L, respectively. Linearity was excellent and carryover was negligible. The agreement between BC-6800-BF and light microscopy was satisfactory for all samples pretreated with hyaluronidase, displaying a bias between -5.9% and 8.2%. CONCLUSIONS: The use of BC-6800-BF for synovial fluid analysis enables rapid and accurate assessment, especially for total cell and polymorphonuclear counts. The use of BC-6800-BF may therefore allow the replacement of optical analysis, especially in samples pretreated with hyaluronidase, thus allowing its routine use for the screening of synovial specimens.

Analytical comparison between two hematological analyzer systems: CAL-8000 vs. XN-9000.

INTRODUCTION: This study was aimed to compare the analytical performance of traditional and new parameters and morphological flags of CAL-8000 and XN-9000. The automated differential leukocyte count (DIFF profile) and morphological flags were compared with optical microscopy (OM). METHODS: A total of 1025 peripheral blood samples, collected in K3 EDTA tubes, were analyzed by CAL-8000, by XN-9000, and by OM. Within-run imprecision was performed in low cellularity samples. The comparison was made using Spearman's correlation, Passing-Bablok regression, Bland-Altman bias, and Cohen's K test. RESULTS: Within-run imprecision in low cellularity samples yielded reproducible data between the instruments (imprecision was higher than 10% on samples with platelet count <21 × 109 /L using impedance technology). Passing-Bablok regression (CAL-8000 vs. XN-9000) yielded slopes ranging between 0.2 to 1.16 and intercepts from -6.54 to 21.63. The bias for leukocytes parameters ranged from -1.8% to -82.2%, the red blood cell parameters from -2.9% to 3.1%, platelets parameters from -27.8% to 26%, and reticulocyte parameters from -115.3% to 4.5%. The comparison of morphological flags yielded a K value always <0.55. The DIFF profile vs. OM had a Passing-Bablok regression with slopes ranging between 0.34 to 1.00 and intercepts from -0.01% to 0.11 and bias ranging from -42.9% to 2.6% for XN-9000 parameters and from -2.7% to 35.0% for CAL-8000 parameters. The comparison of morphological flags showed a K value ranging from 0.35 to 0.77 for XN-9000 and from 0.17 to 0.54 for CAL-8000. CONCLUSION: Differences exist between the two analyzers, especially in the generation of morphology flags, thus emphasizing the need of pursuing a major degree of harmonization and/or adopting instrument-specific reference ranges.

Mindray BC-6800 body fluid mode, performance of nucleated cells, and differential count in ascitic and pleural fluids.

INTRODUCTION: An accurate and rapid analysis of cells in body fluids (BFs) is important for diagnosis and follow-up in many pathological conditions. We evaluated the analytical performance of the module BF Mindray BC-6800 (BC-6800-BF) for cytometric analysis of ascitic and pleural fluids. METHODS: A total of 99 ascitic and 45 pleural samples were collected and assessed with BC-6800-BF and optical microscopy. This study also includes the evaluation of limit blank (LoB), limit detection (LoD), limit quantitation, (LoQ), carryover, linearity, and diagnostic concordance between the two methods. RESULTS: For TC-BF, LoB was 1 × 10(6) cells/L, LoD was 3 × 10(6) cells/L, and LoQ was 4 × 10(6) cells/L. Linearity was excellent (r(2) = 0.99) and carryover was negligible. TC-BF performed with the two methods showed Pearson's correlation of 0.99 (P < 0.0001), Passing-Bablok regression y = 1.04x - 1.17, and bias 33.7 cells. In ascitic fluids, polymorphonuclear cells (PMN) showed an area under curve (AUC) of 0.98 (P < 0.0001). In pleural fluids, mononuclear cells (MN) and PMN % displayed an AUC of 0.79 (P < 0.0001) and 0.93 (P < 0.0001), respectively. CONCLUSIONS: BC-6800-BF in ascitic and pleural fluids offers rapid and accurate cell and differential counts in clinically relevant concentration ranges. The use of BC-6800-BF may allow to replace routine optical counting, except for samples displaying abnormal cell counts or abnormal DIFF scattergram.