Tommaso Mecca

Cell Population Data and reflex testing rules of cell analysis in pleural and ascitic fluids using body fluid mode on Sysmex XN-9000

BACKGROUND Although optical microscopy (OM) remains the reference technique for analysis of ascitic (AF) and pleural (PF) fluids, novel hematological analyzers are equipped with modules for body fluid (BF) analysis. This study was aimed to analyze the performance of XN-BF module in Sysmex XN-9000, and to develop validation rules for automated cell counts in BFs. METHODS The evaluation of XN-BF module included assessment of carryover, Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ), linearity, data comparison with OM, and development of rules for assisting the validation of automated analysis of BFs and activating reflex testing. RESULTS The carryover was negligible. The LoB, LoD, LoQ and linearity were always excellent. The comparison with OM was characterized by Pearsons correlations ranging from r=0.50 to r=0.99 (p<0.001), modest bias and high diagnostic concordance (Area Under the Curve between 0.85 and 0.99). The use of instrument-specific cut-offs further increased diagnostic concordance. The implementation of reflex testing rules based on XN-BF data increased sensitivity and specificity of BFs classification to 0.98 and 0.95. CONCLUSIONS Our results suggest that the XN-BF module on Sysmex-9000 may be a suitable alternative to OM for screening BF samples, especially when specific validation rules are used.

Reflex Testing Rules for Cell Count and Differentiation of Nucleated Elements in Pleural and Ascitic Fluids on Sysmex XE-5000

Flow cytometry is widely used in many laboratories for automated nucleated cell counts and their differentiation in body fluids. The implementation of new reflex testing rules on these automated instruments could open new frontiers in laboratory workflow, improving characterization of body fluids and clinical diagnosis and decreasing costs. Ascitic (150) and pleural (33) fluids were collected and assessed by XE-5000 and optical microscopy. Cell counts performed with the methods showed a Pearson’s correlation of 0.98 (p < 0.0001), Passing–Bablok regression y = 0.99x + 2.44, and bias of 32.3. In ascitic fluids, the best diagnostic performance was found for polymorphonuclear and neutrophil counts on XE-5000, which exhibited areas under the curve (AUCs) 0.98 (p < 0.0001) and 0.99 (p < 0.0001), respectively. In pleural fluids the best diagnostic performance was found for polymorphonuclear percent parameter, which displayed 0.97 (p < 0.0001). Specific reflex test rules based on these parameters were characterized by 92% diagnostic concordance, 1.00 sensitivity, and 0.84 specificity with optical microscopy. The application of a set of reflex testing rules may improve the diagnostic performance of XE-5000, increasing its reliability for routine automated cell count in body fluids. We acknowledge that further studies should be planned to validate our findings according to clinical data.

Assessment of blood sample stability for complete blood count using the Sysmex XN-9000 and Mindray BC-6800 analyzers

Background Different hematological analyzers have different analytical performances that are often reflected in the criteria for sample stability of the complete blood count. This study aimed to assess the stability of several hematological parameters using the XN-9000 Sysmex and BC-6800 Mindray analyzers. Methods The impact of storage at room temperature and 4 °C was evaluated after 2, 4, 6, 8, 24, 36 and 48 h using ten normal and 40 abnormal blood samples. The variation from the baseline measurement was evaluated by the Steel–Dwass–Critchlow–Fligner test and by Bland–Altman plots, using quality specifications and critical difference as the total allowable variation. Results Red blood cells and reticulocyte parameters (i.e. hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, immature reticulocyte fractions, low-fluorescence reticulocytes, middle-fluorescence reticulocytes, high fluorescence mononuclear cells) showed less stability compared to leukocyte and platelet parameters (except for monocyte count and mean platelet volume). The bias for hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration and red blood cell distribution width coefficient of variation was higher than the critical difference after 8 h using both analyzers. Conclusion Blood samples measured with both analyzers do not show analytically significant changes in up to 2 h of storage at room temperature and 4 °C. However, the maximum time for analysis can be extended for up to 8 h when the bias is compared to the critical difference.

Optimization of Cellular analysis of Synovial Fluids by optical microscopy and automated count using the Sysmex XN Body Fluid Mode.

BACKGROUND This study was planned to assess the impact of pre-treating synovial fluid (SF) samples with hyaluronidase (HY), defining the best procedure for optical microscopy (OM) analysis and evaluating the performance of Sysmex XN-9000 Body Fluid module (XN-BF). METHODS The cell count by OM was carried out both with and without HY pre-treatment, and using 3 different types of staining reagents. The evaluation of XN-BF included data comparison with OM (100 SFs), carryover, Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ) and linearity. RESULTS Unlike cell count in Burkers chamber and staining with Stromatol, pre-treatment with HY and staining with Methylene Blue and Turks promoted cell clustering. The SF samples pre-treated with HY displayed excellent morphological quality, contrary to samples without HY pre-treatment. Excellent correlation was found between total cells counting with both OM and XN-BF. Satisfactory agreement was also observed between polymorphonuclear neutrophils compared to XN-BF parameter, whereas mononuclear cell count on XN-BF had suboptimal agreement with OM. The carryover was negligible. The LoB, LoD, LoQ and linearity were excellent. CONCLUSION XN-BF displays excellent performance, which makes it a reliable and practical alternative to OM for SF samples analysis in clinical laboratories.

Erythroblastaemia in natalizumab-treated patients with multiple sclerosis

BACKGROUND Natalizumab is a monoclonal antibody that significantly reduces the occurrence of relapses in relapse-remitting multiple sclerosis (RRMS) patients. Early papers on the clinical use of natalizumab in RRMS patients reported erythroblastemia as occasional and transient. OBJECTIVES to determine the prevalence and absolute count of erythroblasts (nucleated red blood cells, NRBCs) in peripheral blood of RRMS patients in different treatment groups and healthy controls from the same geographic area using the same equipment for laboratory analysis. METHODS We retrospectively evaluated the samples of 203 consecutive RRMS patients including 26 subjects on natalizumab, 17 on fingolimod, 72 on interferon, 41 on glatiramer acetate, 47 treatment-naïve and 240 healthy controls from the same geographic area. Blood samples were processed using an XN-9000-Hematology Analyzer and subsequent microscopic verification. In the natalizumab-treated patients we performed an additional analysis in order to detect the expression of CD34+ cells in peripheral blood, as confirmation of a bone marrow mobilization. RESULTS The prevalence of patients with NRBCs positivity was significantly higher in natalizumab-treated patients (92%) compared with the other treatment groups and healthy controls (0%) (p<0.0005). The median absolute NRBCs count was significantly higher in natalizumab-treated patients (median 0.020, p<0.0005) than in the other treatment groups and healthy controls. Natalizumab-treated patients also had higher levels of white blood cells than all other groups and lower haemoglobin levels than healthy subjects (p<0.01), but no morphologic alterations were evident at a subsequent review of red blood cells, platelets and white blood cells. CD34+ cells levels were consistent with mobilization of haematopoietic stem cells from the bone marrow (median 8 cells/µL, IQR 5-12). CONCLUSIONS We confirm erythroblastaemia as a frequent finding of natalizumab treatment in RRMS patients. More extended knowledge and adequate long-term observation of this phenomenon are essential to better understand any pathological implication.

Extended leukocyte differential count and C-reactive protein in septic patients with liver impairment: diagnostic approach to evaluate sepsis in intensive care unit

BACKGROUND Sepsis is still a major cause of death in intensive care units (ICUs) worldwide. Patients with liver impairment express an imbalanced cytokine response which alters common sepsis biphasic nature. Cytokines measurement is expensive, often unavailable, whereas leukocytes (WBC) evaluation performed through hematology analyzers can provide a practical strategy for monitoring inflammatory response. METHODS A total of 200 healthy subjects (HS) and 84 patients (18 with, 66 without liver impairment) admitted to ICU, were assessed for International Sepsis Definitions, Sequential Organ Failure Assessment (SOFA) and Model for End-Stage Liver Disease (MELD) scores. We tested 1,022 peripheral blood samples using Sysmex XN-9000, estimating diagnostic accuracy of leukocyte differential count and nontraditional parameters through receiver operating characteristics (ROC) curves analysis compared to clinical classification. RESULTS Median value of all-leukocyte parameters was different in ICU patients compared to HS. Leukocytes, neutrophils (NE) and immature granulocytes (IGs) in sepsis and septic shock (SS) were higher than no sepsis (NS), with an area under the curve: 0.81, 0.82 and 0.78 respectively. Lymphocytes (LY) and monocytes (MO) were significantly associated with liver impairment. CONCLUSIONS Diagnostic accuracy of all-leukocyte parameters may provide valuable information for diagnosis and follow-up of sepsis in ICU patients, especially those with liver impairment.

Validation rules for blood smear revision after automated hematological testing using Mindray CAL-8000.

This article was aimed to test the use of validation rules for blood smear review after automated hematological testing using Mindray CAL‐8000 (two hematological analyzers and one autoslider).

Immature granulocyte count on the new Sysmex XN-9000: performance and diagnosis of sepsis in the intensive care unit

quantified as the band or immature granulocyte (IG) count. The aim of this study was to analyze the IG count performance of the Sysmex XN-9000 hematology analyzer in intensive care unit (ICU) patients.Methods. 480 peripheral blood samples from adult patients admitted to the ICU (301 control, 119 sepsis and 60 septic shock) were analyzed with Sysmex XN-9000. Serum C reactive protein (CRP) was measured on Siemens ADVIA 2400. IG count in peripheral blood was determined either by XN-9000 or optical microscopy (OM). Agreement between the two methods was assessed with Pearson’s correlation, Passing-Bablok regression and Bland Altman bias. Diagnostic accuracy was estimated through ROC curves analysis. Sysmex XN-9000 imprecision and within-run precision were also evaluated.Results. Pearson’s correlation (r) relative to IG count, as absolute and percentage values, was 0.89 (p <0.0001) and 0.74 (p <0.0001), respectively, with a Bias of 0.22 and 1.69 respectively. The Area Under the curve (AUC) for the IG count for diagnosing sepsis was greater on XN-9000 than OM and equal to the serum CRP. The diagnostic accuracy of IG counts improves when taking into account the conventional criteria for diagnosing sepsis.Conclusion. IG count appears suitable and reliable when performed using XN-9000. Even if a modest overestimation was found, the diagnostic accuracy showed by IG analysis on XN-9000 may represent a valid alternative to OM count for diagnosing sepsis in ICU patients.