How clinical laboratories select and use Analytical Performance Specifications (APS) in Italy.

OBJECTIVES: Even if the topic of the analytical quality required to provide laboratory results "fit for purpose" exists since the beginning of the modern medical laboratory, there is the suspect that the expression "Analytical Performance Specifications" (APS) is not well-known. To investigate this aspect a survey was conducted. METHODS: A questionnaire with seven questions related to the knowledge about the topic, the sources of information and the criteria used by the laboratories to set the APS and their applications was prepared. It was distributed to all the clinical pathology laboratories of Lombardy Region (143) and to the members of SIBioC Laboratory Medicine (excluding Lombardy). RESULTS: We received 201 replies: 127 from Lombardy and 74 from the rest of Italy. Fifteen percent declared to ignore the meaning of APS and only 64 % of those knowing the meaning of the term declared to use them in the daily practice. The state-of-the-art was the principle used more frequently to set APS (about 48 %) followed by biological variation (41 %), and APS were typically applied to set goals for Internal Quality Control for selected measurands. Usually imprecision or total error APS were used, much less frequently uncertainty APS. In fact only 27 % of the laboratories declared to have calculated the measurement uncertainty for part or the majority of their measurands. CONCLUSIONS: Even considering the limits of a survey that relies upon self-declarations, it appears clearly that, at list in Italy, there is some work to be done to promote the concept and the use of APS.

A territory-wide opportunistic, hospital-based HCV screening in the general population from northern Italy: The 1969-1989 birth-cohort.

BACKGROUND AND AIM: The World Health Organization (WHO) goal of Hepatitis C Virus (HCV) elimination by 2030 rose awareness about the need of screening plans, worldwide. In Italy, graduated screening starting from people born in 1969-1989 might be the most-effective strategy. We performed an opportunistic HCV screening study in the general population attending health facilities in Lombardy region, Northern Italy. METHODS: This is a prospective, multicenter, territory-wide, opportunistic study supported by the Regional Government of Lombardy, Italy. Between June 2022 and December 2022, all subjects born in 1969-1989, hospitalized or accessing blood collection centres were offered anti-HCV and HCV-RNA tests. Patients with known anti-HCV positivity and/or previous anti-HCV treatment were excluded. Demographic features were uploaded into a regional web-based platform. RESULTS: In total, 120 193 individuals were screened in 75 centres. Mean age was 44 (±6) years, 65.2% were females, 83.7% were tested at blood collection centres. Anti-HCV tested positive in 604 (0.50%) subjects: mean age 47 (±5), 51.1% females. HCV seroprevalence was higher in males (p < 0.00001), elderly (p < 0.00001) and in- vs. outpatients (p = 0.0009). HCV-RNA was detectable in 125 out of 441 (28.3%) anti-HCV positive subjects. Actively infected patients were 46 (±6) years old, mainly males (56.8%). The overall prevalence of active HCV infection was 0.10%, higher in elderly (p = 0.0003) and in in-patients (p = 0.0007). Among 93 HCV-RNA positive patients, the median age was 48 years, 58% males, 62% Italian born, median HCV-RNA levels were 6,1 log IU/mL, liver stiffness measurement (LSM) values 5.5 (3.1-29.9) kPa and ALT levels 48 U/L. CONCLUSIONS: The prevalence of active HCV infection in the 1969-1989 population attending health facilities in Lombardy was low. Most viremic patients were Italian-born, with mild liver disease but high-HCV-RNA levels. Due to the higher prevalence in the elderly, the extension of such opportunistic screening programs to lower birth cohorts would be warranted.

Reconstructing the impact of COVID-19 on the immunity gap and transmission of respiratory syncytial virus in Lombardy, Italy.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of hospitalisation and mortality in young children globally. The social distancing measures implemented against COVID-19 in Lombardy (Italy) disrupted the typically seasonal RSV circulation during 2019-2021 and caused substantially more hospitalisations during 2021-2022. The primary aim of this study is to quantify the immunity gap-defined as the increased proportion of the population naïve to RSV infection following the relaxation of COVID-19 restrictions in Lombardy, which has been hypothesised to be a potential cause of the increased RSV burden in 2021-2022. METHODS: We developed a catalytic model to reconstruct changes in the age-dependent susceptibility profile of the Lombardy population throughout the COVID-19 pandemic. The model is calibrated to routinely collected hospitalisation, syndromic, and virological surveillance data and tested for alternative assumptions on age-dependencies in the risk of RSV infection throughout the pandemic. FINDINGS: We estimate that the proportion of the Lombardy population naïve to RSV infection increased by 60.8% (95% CrI: 55.2-65.4%) during the COVID-19 pandemic: from 1.4% (95% CrI: 1.3-1.6%) in 2018-2019 to 2.3% (95% CrI: 2.2-2.5%) before the 2021-2022 season, corresponding to an immunity gap of 0.87% (95% CrI: 0.87-0.88%). We found evidence of heterogeneity in RSV transmission by age, suggesting that the COVID-19 restrictions had variable impact on the contact patterns and risk of RSV infection across ages. INTERPRETATION: We estimate a substantial increase in the population-level susceptibility to RSV in Lombardy during 2019-2021, which contributed to an increase in primary RSV infections in 2021-2022. FUNDING: UK Medical Research Council (MRC), UK Foreign, Commonwealth & Development Office (FCDO), EDCTP2 programme, European Union, Wellcome Trust, Royal Society, EU-MUR PNRR INF-ACT.

Multicentric evaluation of the variability of digital morphology performances also respect to the reference methods by optical microscopy.

INTRODUCTION: Despite the important diagnostic role of peripheral blood morphology, cell classification is subjective. Automated image-processing systems (AIS) provide more accurate and objective morphological evaluation. The aims of this multicenter study were the evaluation of the intra and inter-laboratory variation between different AIS in cell pre-classification and after reclassification, compared with manual optical microscopy, the reference method. METHODS: Six peripheral blood samples were included in this study, for each sample, 70 May-Grunwald and Giemsa stained PB smears were prepared from each specimen and 10 slides were delivered to the seven laboratories involved. Smears were processed by both optical microscopy (OM) and AIS. In addition, the assessment times of both methods were recorded. RESULTS: Within-laboratory Reproducibility ranged between 4.76% and 153.78%; between-laboratory Precision ranged between 2.10% and 82.2%, while Total Imprecision ranged between 5.21% and 20.60%. The relative Bland Altman bias ranged between -0.01% and 20.60%. The mean of assessment times were 326 ± 110 s and 191 ± 68 s for AIS post reclassification and OM, respectively. CONCLUSIONS: AIS can be helpful when the number of cell counted are low and can give advantages in terms of efficiency, objectivity and time saving in the morphological analysis of blood cells. They can also help in the interpretation of some morphological features and can serve as learning and investigation tools.

First Results of an External Quality Assessment (EQA) Scheme for Molecular, Serological and Antigenic Diagnostic Test for SARS-CoV-2 Detection in Lombardy Region (Northern Italy), 2020-2022.

For diagnosing SARS-CoV-2 infection and for monitoring its spread, the implementation of external quality assessment (EQA) schemes is mandatory to assess and ensure a standard quality according to national and international guidelines. Here, we present the results of the 2020, 2021, 2022 EQA schemes in Lombardy region for assessing the quality of the diagnostic laboratories involved in SARS-CoV-2 diagnosis. In the framework of the Quality Assurance Programs (QAPs), the routinely EQA schemes are managed by the regional reference centre for diagnostic laboratories quality (RRC-EQA) of the Lombardy region and are carried out by all the diagnostic laboratories. Three EQA programs were organized: (1) EQA of SARS-CoV-2 nucleic acid detection; (2) EQA of anti-SARS-CoV-2-antibody testing; (3) EQA of SARS-CoV-2 direct antigens detection. The percentage of concordance of 1938 molecular tests carried out within the SARS-CoV-2 nucleic acid detection EQA was 97.7%. The overall concordance of 1875 tests carried out within the anti-SARS-CoV-2 antibody EQA was 93.9% (79.6% for IgM). The overall concordance of 1495 tests carried out within the SARS-CoV-2 direct antigens detection EQA was 85% and it was negatively impacted by the results obtained by the analysis of weak positive samples. In conclusion, the EQA schemes for assessing the accuracy of SARS-CoV-2 diagnosis in the Lombardy region highlighted a suitable reproducibility and reliability of diagnostic assays, despite the heterogeneous landscape of SARS-CoV-2 tests and methods. Laboratory testing based on the detection of viral RNA in respiratory samples can be considered the gold standard for SARS-CoV-2 diagnosis.

Performance evaluation of automated cell counts compared with reference methods for body fluid analysis.

OBJECTIVES: Cellular analysis of body fluids (BFs) can assist clinicians for the diagnosis of many medical conditions. The aim of this work is the evaluation of the analytical performance of the UF-5000 body fluid mode (UF-BF) analyzer compared to the gold standard method (optical microscopy, OM) and to XN-1000 (XN-BF), another analyzer produced by the same manufacturer (Sysmex) and with a similar technology for BF analysis. METHODS: One hundred BF samples collected in K3EDTA tubes were analyzed by UF-BF, XN-BF and OM. The agreement was evaluated using Passing and Bablok regression and Bland-Altman plot analysis. The receiver operating characteristic (ROC) curves were selected for evaluating the diagnostic agreement between OM classification and UF-BF parameters. RESULTS: Comparison between UF-BF and OM, in all BF types, showed Passing and Bablok's slope comprised between 0.99 (polymorphonuclear cells count, PMN-BF) and 1.39 (mononuclear cells count, MN-BF), the intercepts ranged between 26.47 (PMN-BF parameter) and 226.80 (white blood cell count). Bland-Altman bias was comprised between 7.3% (total cell count, TC-BF) and 52.9% (MN-BF). Comparison between UF-BF and XN-BF in all BF showed slopes ranged between 1.07 (TC-BF and PMN-BF) and 1.16 (MN-BF), intercepts ranged between 8.30 (PMN) and 64.78 (WBC-BF). Bland-Altman bias ranged between 5.8 (TC-BF) and 21.1% (MN-BF). The ROC curve analysis showed an area under the curve ranged between 0.9664 and 1.000. CONCLUSIONS: UF-BF shows very good performance for the differential counts of cells in ascitic, pleural and synovial fluids and therefore it is useful to screen and count cells in this type of BF.

Preparing for future waves and pandemics: a global hospital survey on infection control measures and infection rates in COVID-19.

A survey of hospitals on three continents was performed to assess their infection control preparedness and measures, and their infection rate in hospital health care workers during the COVID-19 pandemic. All surveyed hospitals used similar PPE but differences in preparedness, PPE shortages, and infection rates were reported.

Complete Blood Count as point of care testing QBC STAR™: Preliminary evaluation.

INTRODUCTION: Point of care testing (POCT) represents a valuable option when laboratory data shall be urgently available for timely clinical management, with a turnaround time (TAT) that is unfeasible using conventional laboratory instrumentation. This study was aimed to compare the performance of QBC STAR™ compared to Sysmex XN-module and the reference optical microscopy (OM) assessment. MATERIAL AND METHODS: One hundred peripheral blood samples, collected in K3 EDTA tubes, and 50 capillary blood samples obtained by finger stick were analyzed with QBC STAR™, Sysmex XN-module, and OM. Data were compared with Passing-Bablok regression and Bland-Altman plots. RESULTS: The Passing-Bablok regression analysis (QBC STAR™ capillary sample vs XN-module) yielded slopes comprised between 0.30 and 1.37, while the intercepts ranged between -17.57 and 232.6. Bland-Altman plots yielded relative bias comprised between -4.87% (for MN QBC STAR™ capillary sample vs XN-module) and 27% (PLT QBC STAR™ capillary sample vs XN-module). A significant bias was found for all parameters except MN and WBC, RBC in all and pediatric samples, and HB in adults samples. CONCLUSION: The results of this analytical evaluation suggest that QBC STAR™ may not be the ideal tool for performing complete blood count analysis for diagnostic purposes, while it could be more useful in urgent/emergent conditions, such as for rapid monitoring of some hematological parameters (eg, WBC and HB).

Covid-19 and gender: lower rate but same mortality of severe disease in women-an observational study.

BACKGROUND: Gender-related factors might affect vulnerability to Covid-19. The aim of this study was to describe the role of gender on clinical features and 28-day mortality in Covid-19 patients. METHODS: Observational study of Covid-19 patients hospitalized in Bergamo, Italy, during the first three weeks of the outbreak. Medical records, clinical, radiological and laboratory findings upon admission and treatment have been collected. Primary outcome was 28-day mortality since hospitalization. RESULTS: 431 consecutive adult patients were admitted. Female patients were 119 (27.6%) with a mean age of 67.0 ± 14.5 years (vs 67.8 ± 12.5 for males, p = 0.54). Previous history of myocardial infarction, vasculopathy and former smoking habits were more common for males. At the time of admission PaO2/FiO2 was similar between men and women (228 [IQR, 134-273] vs 238 mmHg [150-281], p = 0.28). Continuous Positive Airway Pressure (CPAP) assistance was needed in the first 24 h more frequently in male patients (25.7% vs 13.0%; p = 0.006). Overall 28-day mortality was 26.1% in women and 38.1% in men (p = 0.018). Gender did not result an independent predictor of death once the parameters related to disease severity at presentation were included in the multivariable analysis (p = 0.898). Accordingly, the Kaplan-Meier survival analysis in female and male patients requiring CPAP or non-invasive ventilation in the first 24 h did not find a significant difference (p = 0.687). CONCLUSION: Hospitalized women are less likely to die from Covid-19; however, once severe disease occurs, the risk of dying is similar to men. Further studies are needed to better investigate the role of gender in clinical course and outcome of Covid-19.

At the peak of COVID-19 age and disease severity but not comorbidities are predictors of mortality: COVID-19 burden in Bergamo, Italy.

BACKGROUND: Findings from February 2020, indicate that the clinical spectrum of COVID-19 can be heterogeneous, probably due to the infectious dose and viral load of SARS-CoV-2 within the first weeks of the outbreak. The aim of this study was to investigate predictors of overall 28-day mortality at the peak of the Italian outbreak. METHODS: Retrospective observational study of all COVID-19 patients admitted to the main hospital of Bergamo, from February 23 to March 14, 2020. RESULTS: Five hundred and eight patients were hospitalized, predominantly male (72.4%), mean age of 66±15 years; 49.2% were older than 70 years. Most of patients presented with severe respiratory failure (median value [IQR] of PaO2/FiO2: 233 [149-281]). Mortality rate at 28 days resulted of 33.7% (N.=171). Thirty-nine percent of patients were treated with continuous positive airway pressure (CPAP), 9.5% with noninvasive ventilation (NIV) and 13.6% with endotracheal intubation. 9.5% were admitted to Semi-Intensive Respiratory Care Unit, and 18.9% to Intensive Care Unit. Risk factors independently associated with 28-day mortality were advanced age (≥78 years: odds ratio [OR], 95% confidence interval [CI]: 38.91 [10.67-141.93], P<0.001; 70-77 years: 17.30 [5.40-55.38], P<0.001; 60-69 years: 3.20 [1.00-10.20], P=0.049), PaO2/FiO2<200 at presentation (3.50 [1.70-7.20], P=0.001), need for CPAP/NIV in the first 24 hours (8.38 [3.63-19.35], P<0.001), and blood urea value at admission (1.01 [1.00-1.02], P=0.015). CONCLUSIONS: At the peak of the outbreak, with a probable high infectious dose and viral load, older age, the severity of respiratory failure and renal impairment at presentation, but not comorbidities, are predictors of 28-day mortality in COVID-19.

Standardization and harmonization in hematology: Instrument alignment, quality control materials, and commutability issue.

INTRODUCTION: In the hub and spoke laboratory network, the number of hematology analyzers (HAs) within each core center has increased, and the control of HAs alignment is becoming necessary requirement to ensure analytical quality. In this scenario, HA alignment can be assessed by analyzing the same control material used for internal quality control on multiple HAs, assuming its commutability. The aim of the study was to verify the applicability of a protocol for the alignment of HAs based on control material rather than on fresh whole-blood samples. METHODS: The alignment of five HAs was evaluated for red (RBC, Hb, MCV, RET), white (WBC, NE, LY, MO, EO, BA, IG), and platelet (PLT) series parameters, following a protocol by SIBioC, using human sample (HS) and quality control material (QC), after the verification of commutability, according to the IFCC protocol. Maximum bias was derived from biological variation data. RESULTS: A complete alignment between instruments was confirmed for the majority of the parameters investigated both for HS and QC material. Partial misalignments or inconcludent results were instead evident for MCV, MO, EO, BA, and IG. Interestingly, QC material was found to be not commutable for LY, MO, and BA. CONCLUSION: The alignment of hematologic analyzers for main cell population parameters may be verified with both QC and HS, displaying consistent results and interpretation. The evaluation for some white series parameters (EO, BA, and IG) is critical, and particular attention must be paid to the values of the material used for the alignment.

What is the normal composition of pericardial fluid?

OBJECTIVE: Biochemical and cytological pericardial fluid (PF) analysis is essentially based on the knowledge of pleural fluid composition. The aim of the present study is to identify reference intervals (RIs) for PF according to state-of-art methodological standards. METHODS: We prospectively collected and analysed the PF and venous blood of consecutive subjects undergoing elective open-heart surgery from July 2017 to October 2018. Exclusion criteria for study enrolment were evidence of pericardial diseases at preoperatory workup or at intraoperatory assessment, or any other condition that could affect PF analysis. RESULTS: The final study sample included 120 patients (median age 69 years, 83 men, 69.1%). The main findings were (1) High levels of proteins, albumin and lactate dehydrogenase (LDH), but not of glucose and cholesterol (2) High cellularity, mainly represented by mesothelial cells. RIs for pericardial biochemistry were: protein content 1.7-4.6 g/dL PF/serum protein ratio 0.29-0.83, albumin 1.19-3.06 g/dL, pericardium-to-serum albumin gradient 0.18-2.37 g/dL, LDH 141-2613 U/L, PF/serum LDH ratio 0.40-2.99, glucose 80-134 mg/dL, total cholesterol 12-69 mg/dL, PF/serum cholesterol ratio 0.07-0.51. RIs for pericardial cells by optic microscopy were: 278-5608 × 106 nucleated cells/L, 40-3790 × 106 mesothelial cells/L, 35-2210 × 106 leucocytes/L, 19-1634 × 106 lymphocytes/L. CONCLUSIONS: PF is rich in nucleated cells, protein, albumin, LDH, at levels consistent with inflammatory exudates in other biological fluids. Physicians should stop to interpret PF as exudate or transudate according to tools not validated for this setting.

Multicentric evaluation of analytical performances digital morphology with respect to the reference methods by manual optical microscopy.

AIMS: Optical microscopic (OM) evaluation of peripheral blood (PB) cells is still a crucial step of the laboratory haematological workflow. The morphological cell analysis is time-consuming and expensive and it requires skilled operator. To address these challenges, automated image-processing systems, as digital morphology (DM), were developed in the last few years. The aim of this multicentre study, performed according to international guidelines, is to verify the analytical performance of DM compared with manual OM, the reference method. METHODS: Four hundred and ninety PB samples were evaluated. For each sample, two May Grunwald-stained and Giemsa-stained smears were performed and the morphological evaluation of cells was analysed with both DM and OM. In addition, the assessment times of both methods were recorded. RESULTS: Comparison of DM versus OM methods was assessed with Passing-Bablok and Deming fit regression analysis: slopes ranged between 0.17 for atypical, reactive lymphocytes and plasma cells (LY(AT)) and 1.24 for basophils, and the intercepts ranged between -0.09 for blasts and 0.40 for LY(AT). The Bland-Altman bias ranged between -6.5% for eosinophils and 21.8% for meta-myemielocytes. The diagnostic agreement between the two methods was 0.98. The mean of assessment times were 150 s and 250 s for DM and OM, respectively. CONCLUSION: DM shows excellent performance. Approximately only 1.6% of PB smears need the OM revision, giving advantages in terms of efficiency, standardisation and assessment time of morphological analysis of the cells. The findings of this study may provide useful information regarding the use of DM to improve the haematological workflow.

Age-specific SARS-CoV-2 infection fatality ratio and associated risk factors, Italy, February to April 2020.

We analysed 5,484 close contacts of coronavirus disease (COVID-19) cases in Italy, all tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection fatality ratio was 0.43% (95% confidence interval (CI): 0.21-0.79) for individuals younger than 70 years and 10.5% (95% CI: 8.0-13.6) for older individuals. Risk of death after infection was 62% lower (95% CI: 31-80) in clusters identified after 16 March 2020 and 1.8-fold higher for males (95% CI: 1.03-3.16).

Multicenter evaluation of analytical performances of platelet counts and platelet parameters: Carryover, precision, and stability.

INTRODUCTION: The correctness of the results of automated platelet analysis is still highly debated. The aim of this multicenter study, conducted according to international guidelines, was to verify the analytical performance of nine different types of hematology analyzers (HAs) in the automated platelet analysis. METHODS: Four hundred eighty-six peripheral blood samples (PB), collected in K3 EDTA tubes, were analyzed by ABX Pentra, ADVIA2120i, BC-6800, BC-6800 Plus, Cell-DYN Sapphire, DxH800, XE-2100, XE-5000, XN-20 with PLT-F App. Within-run imprecision and between-run imprecision were carried out using PB and material control, respectively. The carryover, low limit of quantification (LoQ), and the PB stability were evaluated. RESULTS: The carryover was absent for all HAs. The LoQ of PLT ranged between 2.0 (Cell-Dyn Sapphire) and 25.0 × 109 /L (ADVIA 2120i), while immature platelet fraction (IPF) ranged between 1.0 (XN-20) and 12.0 × 109 /L (XE-5000). The imprecision (%CV) increases as the platelet count decreases. No HAs showed desirable CVAPS for PLT counts less than 50.0 × 109 /L, with the exception of Cell-DYN Sapphire (CV 3.0% with PLT-O mean value of 26.7 × 109 /L), XN-20 (CV 2.4% with PLT-F mean value of 21.5 × 109 /L), and BC-6800 Plus (CV 1.9% with PLT-O mean value of 26.5 × 109 /L). The sample stability ranged between under two hours for MPV by ADVIA2120i and 8 hours for other PLT parameters and HAs. CONCLUSION: The findings of this study may provide useful information regarding carryover, precision, and stability of platelet counts and parameters, especially in thrombocytopenic samples. Moreover, the stability of sample for platelet analysis is conditioned by the HA and by temperature and storage time.

Platelet Transfusion Thresholds: How Low Can We Go in Respect to Platelet Counting?

Platelet transfusion is conventionally used to prevent or treat bleeding in patients with low platelet counts or impaired platelet function. The identification of accurate thresholds of platelet count for guiding platelet transfusion practices is a crucial aspect in health care to prevent adverse events, side effects, unwarranted costs for the health care service, and deprivation of supplies. This article is therefore aimed at providing a narrative overview on current guidelines and recommendations for platelet transfusion across many clinical settings, including platelet function disorders, and critically analyzing the available platelet transfusion thresholds according to the current analytical performance of platelet counting with automated hematological analyzers. Overall, universal agreement on the definition of platelet transfusion thresholds has not been reached. The degree of accuracy and imprecision of many fully automated hematological analyzers appears also unsatisfactory, especially at the lower thrombocytopenic range, and this may thus jeopardize the managed care of patients who are candidates for platelet transfusions. Potential solutions to overcome the current shortcomings of automated platelet counting are also discussed, encompassing the use of alternative tests for guiding platelet transfusion (e.g., thrombin generation assays or thromboelastography) along with innovative approaches for platelet enumeration (e.g., fluorescent labeling and flow cytometry).

Preliminary evaluation of a new flow cytometry method for the routine hematology workflow.

Background In a generalist laboratory, the integration of the data obtained from hematology analyzers (HAs) with those from multiparametric flow cytometry (FMC) could increase the specificity and sensitivity of first level screening to identify the pathological samples. The aim of this study was to perform a preliminary evaluation of a new simple hybrid method (HM). The method was obtained by integration between HAs reagents into FCM, with a basic monoclonal antibodies panel for the leukocytes differential count. Methods Eighty-one peripheral blood samples, collected in K3EDTA tubes, were analyzed by XN-module, and CyFlow Space System, using both standard MoAbs and HM method analysis, and with the optical microscopy (OM). Within-run imprecision was carried out using normal samples, the carryover was evaluated, data comparison was performed with Passing-Bablok regression and Bland-Altman plots. Results The within-run imprecision of HM methods ranged between 1.4% for neutrophils (NE) and 10.1% for monocytes (MO) always equal or lower to the OM. The comparison between HM methods vs. OM shows Passing-Bablok regression slopes comprised between 0.83 for lymphocyte (LY) and 1.14 for MO, whilst the intercepts ranged between -0.18 for NE and 0.25 for LY. Bland-Altman relative bias was comprised between -12.43% for NE, and 19.77% for eosinophils. In all 11 pathological samples the agreement between the methods was 100%. Conclusions The new hybrid method generates a leukocytes differential count suitable for routine clinical use and it is also useful for identifying morphological abnormalities with a reduction in cost and improvement of screening for first level hematology workflow.