Evaluation of residual humoral immune response against SARS-CoV-2 by a surrogate virus neutralization test (sVNT) 9 months after BNT162b2 primary vaccination.

The humoral response to SARS-CoV-2 vaccination has shown to be temporary, although may be more prolonged in vaccinated individuals with a history of natural infection. We aimed to study the residual humoral response and the correlation between anti-Receptor Binding Domain (RBD) IgG levels and antibody neutralizing capacity in a population of health care workers (HCWs) after 9 months from COVID-19 vaccination. In this cross-sectional study, plasma samples were screened for anti-RBD IgG using a quantitative method. The neutralizing capacity for each sample was estimated by means of a surrogate virus neutralizing test (sVNT) and results expressed as the percentage of inhibition (%IH) of the interaction between RBD and the angiotensin-converting enzyme. Samples of 274 HCWs (227 SARS-CoV-2 naïve and 47 SARS-CoV-2 experienced) were tested. The median level of anti-RBD IgG was significantly higher in SARS-CoV-2 experienced than in naïve HCWs: 2673.2 AU/mL versus 610.9 AU/mL, respectively (p <0.001). Samples of SARS-CoV-2 experienced subjects also showed higher neutralizing capacity as compared to naïve subjects: median %IH = 81.20% versus 38.55%, respectively; p <0.001. A quantitative correlation between anti-RBD Ab and inhibition activity levels was observed (Spearman's rho = 0.89, p <0.001): the optimal cut-off correlating with high neutralization was estimated to be 1236.1 AU/mL (sensitivity 96.8%, specificity 91.9%; AUC 0.979). Anti-SARS-CoV-2 hybrid immunity elicited by a combination of vaccination and infection confers higher anti-RBD IgG levels and higher neutralizing capacity than vaccination alone, likely providing better protection against COVID-19.

Short- and medium-term biological variation estimates of red blood cell and reticulocyte parameters in healthy subjects.

BACKGROUND: The integrated evaluation of traditional and innovative red blood cell (RBC) and reticulocyte parameters is a rapid, inexpensive and non-invasive diagnostic tools for differential diagnosis and follow-up of anemia and other pathological conditions needing bone marrow erythropoiesis assessment. Therefore, estimating the biological variation (BV) of these parameters is essential for evaluating the analytical performance of hematological analyzers, and for enabling accurate data interpretation and appropriate clinical management. This study aims to define short- and medium-term BV estimates and reference change value (RCV) of RBC and reticulocyte parameters. METHODS: Twenty-one healthy volunteers participated in the assessment of medium-term BV (blood sampling once/week, five consecutive weeks) and 22 volunteers in the assessment of short-term BV (blood sampling once/day, five consecutive days) using Sysmex XN. Outlier analysis was performed before CV-ANOVA, to determine BV estimates with confidence intervals (CI). RESULTS: Medium- and short-term within-subject BV were between 0.3% and 16.4% and 0.2%-10.4% (MCH and IRF), respectively, whereas medium and short-term between-subjects BV ranged between 0.9% and 66.6% (MCHC and Micro-R) and 1.4%-43.6% (MCHC and IRF), respectively. The RCVs were similar for all parameters in both arms of the study, except for hemoglobin, RDW-CV and MCV. CONCLUSIONS: This study allowed for estimating the BV of many RBC and reticulocyte parameters, some of which have not been currently explored. For RBC, hemoglobin, RDW-CV and MCV it seems advisable to use RCV calculated according to monitoring time and/or differentiated by sex. As regards analytical goals, we suggest using the most stringent targets found in the short-term arm of this study.

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

BACKGROUND: 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). METHODS: This study was based on 1013 peripheral blood samples (PB) referred for routine hematological testing. Results of testing on CAL-8000 were analyzed using both locally derived and International Consensus Group for Hematology (ICGH) validation rules, and then compared with data obtained by optical microscopy (OM). A workflow analysis was also completed. RESULTS: The overall agreement with locally derived and ICGH criteria was 91% and 85%, but a higher sensitivity was observed for locally derived criteria (0.97 vs 0.95). The percentage of false negative and false positive samples was 2.1% and 7.1% using ICGH criteria, and was 1.4% and 14% using locally defined rules. The throughput of CAL-8000 system was 208 samples/h, with a percentage of OM analysis comprised between 14% and 17%, and sensitivity of 0.97. As regards personnel activity, we estimated 0.8 full-time equivalent (FTE) of technical staff and 0.7 FTE of personnel for clinical validation of data and blood smear review. CONCLUSION: These results show that customization of validation rules is necessary for enhancing the quality of hematological testing and optimizing workflow.

Automated Cerebrospinal Fluid Cell Counts Using the New Body Fluid Mode of Sysmex UF-1000i.

BACKGROUND: We evaluated the new body fluid module on Sysmex UF1000-i (UF1000i-BF) for analysis of white blood cell (WBC) and red blood cell (RBC) in cerebrospinal fluid (CSF). METHODS: WBC and RBC counting were compared between UF1000i-BF and Fuchs-Rosenthal counting chamber in 67 CSF samples. This study also included the evaluation of between-day precision, limit of blank (LoB), limit of detection (LoD), functional sensitivity (limit of quantitation, LoQ), carryover and linearity. Diagnostic agreement for differentiation between normal and increased WBC counts (≥5.0 × 10(6) /L) was also assessed. RESULTS: The agreement between UF1000i-BF and manual WBC counts was otpiaml in all CSF samples (r = 0.99; y = 1.05x + 0.09). A modest overestimation was noticed in samples with WBC < 30 × 10(6) /L (r = 0.95; y = 1.21x - 0.15). A good agreement was observed for RBC counts (r = 0.98; y = 1.15x + 0.55), particularly in samples with RBC ≥ 18 × 10(6) /L (r = 0.98; y = 1.01x + 8.90). Between-day precision was good, with coefficient of variations (CVs) lower than 7.2% for both WBC and RBC. The LoBs were 0.1 × 10(6) WBC/L and 1.2 × 10(6) RBC/L, the LoDs were 0.7 × 10(6) WBC/L and 5.5 × 10(6) RBC/L, the LoQs were 2.4 × 10(6) WBC/L and 18.0 × 10(6) RBC/L, respectively. Linearity was excellent (r = 1.00 for both WBC and RBC). Carryover was negligible. Excellent diagnostic agreement was obtained at 4.5 × 10(6) WBC/L cut-off (sensitivity, 100%; specificity, 97.4%). CONCLUSION: The UF1000i-BF provides rapid and accurate WBC and RBC counts in clinically relevant values of CSF cells. The use of UF1000i-BF may hence allow to replace routine optical counting, except for samples displaying abnormal WBC counts or abnormal scattergram distribution, for which differential cell counts may still be required.

JAK2V617F mutation and hydroxyurea treatment as determinants of immature platelet parameters in essential thrombocythemia and polycythemia vera patients.

Immature platelets (IPFs), which are hemostatically more active than mature platelets, have been found elevated in essential thrombocythemia and polycythemia vera, 2 myeloproliferative neoplasms (MPN) characterized by an increased risk of thrombosis. It is not known whether the IPF levels are influenced by pathogenetic factors, including JAK2V617F mutational status, or by treatment regimen. To address this point, in 46 essential thrombocythemia and 38 polycythemia vera consecutive patients, we measured IPF and correlated the results to JAK2V617F mutation and myelosuppressive treatment with hydroxyurea. This analysis provides 2 new elements regarding IPF and MPN. The first finding is that the JAK2V617F mutation is linked to the quantity of IPF in patients with MPN, which might contribute to the prothrombotic phenotype in these patients. The second finding is that IPF is susceptible to myelosuppressive treatment, which may additionally explain the favorable effect of hydroxyurea therapy on MPN outcome as well as the associated thrombotic risk.

SARS-CoV-2 antibody responses before and after a third dose of the BNT162b2 vaccine in Italian healthcare workers aged ≤60 years: One year of surveillance.

This study monitored the anti-spike-receptor-binding domain (RBD) and neutralizing antibodies induced by the Pfizer/BioNTech mRNA BNT162b2 vaccine in a cohort of 163 healthcare workers aged ≤60 years. We have taken advantage of two study groups, both of whom received the first two doses in the same time window, but Group 1 (54 HCWs) received the third dose 2 months before Group 2 (68 HCWs) did. The cohorts were monitored from the 12th day after the first vaccine dose up to 1 month after the third vaccine dose for a total of eight time points and about 1 year of surveillance (T1 = 12 days after the first dose; T2 = 10 days after the second dose; T3 = 1 month after the second dose; T4 = 3 months after the second dose; T5 = 4 months after the second dose; T6 = 5 months after the second dose; T7 = 7 months after the second dose; T8 = 1 month after the third dose for Group 1; T8* = 9 months after the second dose for Group 2; T9 = 1 month after the third dose for Group 2). The mean value of anti-spike antibodies decreased faster over time, but at T7, its decline was significantly slowed (T7 vs. T8*). After the third dose, the anti-spike titer rose about 34-fold (T7 vs. T8 and T8* vs. T9) and the booster improved the anti-spike titer by about three times compared with that of the second dose (T3 vs. T8 and T3 vs. T9), and no difference was noted between the two groups. The neutralizing titer was evaluated at T3, T7, T8, and T9. Anti-spike and neutralizing antibodies were found to be strongly correlated (r2 = 0.980; p < 0.001). At T3, 70% of the participants had a neutralizing antibody titer >91% of total anti-spike antibodies that increased to 90% after the third dose (T8 and T9). However, when the anti-spike titer reached its lowest value (T7), the neutralizing antibody levels decreased even further, representing only 44% of total anti-spike antibodies (p < 0.0001). Our findings show that the third vaccine dose improves the humoral response, but the wane of the anti-spike and neutralizing antibody titers over time is more marked in the neutralizing antibodies.

Inflammation and thrombosis in essential thrombocythemia and polycythemia vera: different role of C-reactive protein and pentraxin 3.

We tested the hypothesis that levels of pentraxin high sensitivity C-reactive protein and pentraxin 3 might be correlated with cardiovascular complications in patients with essential thrombocythemia and polycythemia vera. High sensitivity C-reactive protein and pentraxin 3 were measured in 244 consecutive essential thrombocythemia and polycythemia vera patients in whom, after a median follow up of 5.3 years (range 0-24), 68 cardiovascular events were diagnosed. The highest C-reactive protein tertile was compared with the lowest (>3 vs. <1 mg/L) and correlated with age (P=0.001), phenotype (polycythemia vera vs. essential thrombocythemia, P=0.006), cardiovascular risk factors (P=0.012) and JAK2V617F allele burden greater than 50% (P=0.003). Major thrombosis rate was higher in the highest C-reactive protein tertile (P=0.01) and lower at the highest pentraxin 3 levels (P=0.045). These associations remained significant in multivariate analyses and indicate that blood levels of high sensitivity C-reactive protein and petraxin 3 independently and in opposite ways modulate the intrinsic risk of cardiovascular events in patients with myeloproliferative disorders.

A risk-analysis approach to the evaluation of analytical quality.

BACKGROUND: Setting specifications for analytical quality is always difficult. The risk-management approach might be a way to do so. In this approach, the definition of the required analytical quality is based on the evaluation of patient risk. Risk derives from the probability of error and from the damage that such an error might cause. METHODS: Eight Italian laboratories took part in this experiment. Measurements of glucose and total calcium were taken as examples. Analytical quality was evaluated using a specific ring trial with a frozen serum pool and by means of internal quality-control data. The total allowable error was defined according to biological variation specifications. The probability of error was extracted from the imprecision and comparative bias data of each laboratory. The damage caused by a wrong result was evaluated using the absolute probability judgment approach. RESULTS: According to the iso-risk plots (standardized hyperboles on a graph where the x-axis represents damage and the y-axis represents probability) for glucose, all the laboratories were working with an analytical quality that guaranteed low risk for patients. On the contrary, for total calcium none of the laboratories exhibited sufficient quality to guarantee low risk for patients, the presence of bias being the most relevant problem. CONCLUSIONS: The results seem to demonstrate the applicability of the risk approach to the analytical phase, indicating a new possible way to define analytical quality targets.

Mid-stream vs. first-voided urine collection by using automated analyzers for particle examination in healthy subjects: an Italian multicenter study.

BACKGROUND: In analogy with other areas of laboratory diagnostics, the pre-analytical phase is the leading source of variability also in urinalysis. We carried out a multicentric study for comparing results obtained from first-voided and mid-stream urine samples. METHODS: Each of the six hospital-based clinical laboratories participating to this study recruited 50 healthy subjects among laboratory staff and/or their relatives. Two consecutive samples of the first morning micturition were collected by vacuum system, the first from the first-void and the second from the mid-stream. Routine urinalysis was performed using dip-stick automated analyzers for chemical examination and automated analyzers for formed particle examination (Sysmex UF-100, Sysmex UF-1000i and Iris iQ-200). RESULTS: Counts of epithelial cells (EC), erythrocytes (ERY) and leukocytes (LEU) but not for cylinders (CAS) were significantly higher in the first-voided samples. A significantly higher count of EC, ERY and LEU was also observed between females and males in first-voided samples, whereas no significant difference could be found in mid-stream samples. Health related analyzer specific upper reference limits (URL) were CAS≤1, EC≤5, ERY≤19, Leu≤13 for UF-100; CAS≤1, EC≤4, ERY≤15, Leu≤11 for UF-1000i; CAS≤1, EC≤4, ERY≤18, Leu≤10 for iQ200. The overall prevalence of subjects with cellular elements count exceeding URL was also higher in first-voided than in mid-stream samples. CONCLUSIONS: Mid-stream urine was confirmed as the most appropriate sample, since the presence of contaminating elements, such as bacteria, analytes and formed particles are minimized.