Outcome of SARS CoV-2 inpatients treated with convalescent plasma: One-year of data from the Veneto region (Italy) Registry.

OBJECTIVES AND BACKGROUND: Convalescent plasma (CP) has been used worldwide to contrast SARS-CoV-2 infection. Since April 2020, it has also been used in the treatment of patients with COVID-19 in the Veneto region (Italy), along with all the other available drugs and therapeutic tools. Here we report data analysis and clinical results in 1,517 COVID-19 inpatients treated with CP containing high-titre neutralizing anti-SARS-CoV-2 antibodies (CCP). Mortality after 30 days of hospitalization has been considered primary outcome, by comparing patients treated with CCP vs all COVID-19 patients admitted to hospitals of the Veneto region in a one-year period (from April 2020 to April 2021). PATIENTS AND METHODS: Adult inpatients with a severe form of COVID-19 have been enrolled, with at least one of the following inclusion criteria: 1) tachypnea with respiratory rate (RR) ≥ 30 breaths/min; 2) oxygen saturation (SpO2) ≤ 93% at rest and in room air; 3) partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) ≤ 200 mmHg, 4) radiological picture and/or chest CT scan showing signs of interstitial disease and/or rapid progression of lung involvement. Patients received a maximum of three therapeutic fractions (TFs) of CCP with a neutralizing antibody titre of ≥ 1:160, administered over a period of 3-5 days. If TFs of CCP with titre ≥ 1:160 were unavailable, 2 with antibody titre of ≥ 1:80 have been administered. RESULTS: Of the 1,517 patients treated with CCP, 209 deceased at the 30-day follow-up (14%). Death was significantly associated with an older age (p<0.001), a longer time of hospitalization before CCP infusion (p<0.001), a greater number of inclusion criteria (p<0.001) and associated comorbidities (p<0.001). Conditions significantly associated with an increased frequency of death were PaO2/FiO2 ≤ 200 (p<0.001) and tachypnea with RR>30 (p<0.05) at entry, concurrent arterial hypertension (p<0.001), cardiovascular disease (p<0.001), chronic kidney disease (p<0.001), dyslipidemia (p<0.05) and cancer (p<0.05). Moreover, factors leading to an unfavorable prognosis were a life-threatening disease (p<0.001), admission to Intensive Care Unit (p<0.001), high flow oxygen therapy or mechanical ventilation (p<0.05) and a chest X-ray showing consolidation area (p<0.001). By analyzing the regional report of hospitalized patients, a comparison of mortality by age group, with respect to our series of patients treated with CCP, has been made. Mortality was altogether lower in patients treated with CCP (14% v. 25%), especially in the group of the elderly patients (23% vs 40%,), with a strong significance (p<0.001). As regards the safety of CCP administration, 16 adverse events were recorded out of a total of 3,937 transfused TFs (0,4%). CONCLUSIONS: To overcome the difficulties of setting up a randomized controlled study in an emergency period, a data collection from a large series of patients with severe COVID-19 admitted to CCP therapy with well-defined inclusion criteria has been implemented in the Veneto region. Our results have shown that in patients with severe COVID-19 early treatment with CCP might contribute to a favourable outcome, with a reduced mortality, in absence of relevant adverse events.

Preparedness and activities of the anti-SARS-CoV-2 convalescent plasma bank in the Veneto region (Italy): An organizational model for future emergencies.

BACKGROUND: Convalescent plasma (CP) has been used in the past in various pandemics, in particular in H1N1, SARS and MERS infections. In Spring 2020, when ongoing the SARS-CoV-2 pandemics, the Veneto Region (V-R) has proposed setting-up an anti-SARS-CoV-2 CP (CCP) Bank, with the aim of preparing a supply of CCP immediately available in case of subsequest epidemic waves. MATERIALS AND METHODS: Key-points to be developed for a quick set-up of the V-R CCP Bank have been recruitment of donors recovered from COVID-19 infection, laboratory analysis for the biological qualification of the CCP units, including titre of neutralizing antibodies and reduction of pathogens, according to National Blood Centre (CNS) Directives, adaptation of the V-R Information Technology systems and cost analysis. Some activities, including diagnostic and viral inactivation processes, have been centralized in 2 or 3 sites. Laboratory analysis upon preliminary admission of the donor included all tests required by the Italian laws and the CNS directives. RESULTS: From April to August 2020, 3,298 people have contacted the V-R Blood Transfusion Services: of these, 1,632 have been evaluated and examined as first time donors and those found to be suitable have carried out 955 donations, from which 2,626 therapeutic fractions have been obtained, at a cost around 215,00 Euro. Since October 2020, the number of COVID-19 inpatients has had a surge with a heavy hospital overload. Moreover, the high request of CCP therapy by clinicians has been just as unexpected, showing a wide therapeutic use. CONCLUSIONS: The organizational model here presented, which has allowed the rapid collection of a large amount of CCP, could be useful when facing new pandemic outbreaks, especially in low and middle income countries, with generally acceptable costs.

[PHYSICAL, CHEMICAL AND MORPHOLOGICAL URINE EXAMINATION: RECOMMENDATIONS FOR THE POST ANALYTICAL PHASE FROM THE INTERDISCIPLINARY URINALYSIS GROUP (GIAU)].

With these recommendations the Interdisciplinary Urinalysis Group (GIAU) aims to stimulate the following aspects : improvement and standardization of the post analytical approach to physical, chemical and morphological urine examination (ECMU); emphasize the value added to ECMU by selection of clinically significant parameters, indication of analytical methods, of units of measurement, of reference values; improvement of interpretation of dip stick urinalysis with particular regard to the reconsideration of the diagnostic significance of the evaluated parameters together with an increasing awareness of the limits of sensitivity and specificity of this analytical method. Accompanied by the skills to propose and carry out in-depth investigations with analytical methods that are more sensitive and specific;increase the awareness of the importance of professional skills in the field of urinary morphology and their relationships with the clinicians. through the introduction, in the report, of descriptive and interpretative comments depending on the type of request, the complexity of the laboratory, the competence of the pathologist;implement a policy of evaluation of the analytical quality by using, in addition to traditional internal and external controls, a program for the evaluation of morphological competence. The hope is to revalue the enormous potential diagnostic of ECMU, implementing a urinalysis on personalized diagnostic needs that each patient brings with it.

Harmonization protocols for TSH immunoassays: a multicenter study in Italy.

BACKGROUND: Systematic difference between thyroid-stimulating hormone (TSH) immunoassays may produce misleading interpretation when samples of the same patients are measured with different methods. The study aims were to evaluate whether systematic differences are present among TSH immunoassays, and whether it is possible to obtain a better harmonization among TSH methods using results obtained in external quality assessment (EQA) schemes. METHODS: Seven Italian clinical laboratories measured TSH in 745 serum samples of healthy subjects and patients with thyroid disorders. These samples were also re-measured by two reference laboratories of the study with the six TSH immunoassays most popular in Italy after 2 months of storage at -80 °C. Moreover, these data were compared to 53,823 TSH measurements, obtained by laboratories participant to 2012-2015 EQA annual cycles in 72 quality control samples (TSH concentrations from about 0.1 mIU/L to 18.0 mIU/L). TSH concentrations were recalibrated using a mathematical approach based on the principal component analysis (PCA). RESULTS: Systematic differences were found between the most popular commercially available TSH immunoassays. TSH concentrations measured by the clinical laboratories were very closely correlated to those measured with the same method by reference laboratories after 2 months of storage at -80 °C. After recalibration using the PCA approach the variation of TSH values significantly decreased from a median pre-calibration value of 13.53% (10.79%-16.53%) to 9.63% (6.90%-13.21%) after recalibration. CONCLUSIONS: Our data suggest that EQA schemes are useful to improve harmonization among TSH immunoassays and also to produce some mathematical formulas, which can be used by clinicians to better compare TSH values measured with different methods.

Effect of dabigatran on a prothrombinase-based assay for detecting activated protein C resistance: an ex vivo and in vitro study in normal subjects and factor V Leiden carriers.

BACKGROUND: The aim of this study was to evaluate ex vivo and in vitro interference of a direct factor IIa inhibitor, dabigatran, on a prothrombinase-based assay to detect activated protein C resistance. MATERIALS AND METHODS: An ex vivo study was performed in six heterozygous factor V Leiden carriers and 12 normal subjects without the factor V Leiden mutation who were treated with dabigatran. An in vitro study was also performed considering 12 plasma samples (six from normal subjects and six from heterozygous factor V Leiden carriers) spiked with dabigatran. The dabigatran concentration was evaluated using a diluted thrombin time assay, activated protein C resistance was evaluated using a prothrombinase-based assay. RESULTS: In both the ex vivo and in vitro studies dabigatran interfered significantly with activated protein C resistance ratios observed in normal subjects and in factor V Leiden heterozygous carriers. DISCUSSION: The results reported in this paper seem to confirm that dabigatran is able to interfere with the Penthafarm prothrombinase-based assay used to study activated protein C resistance, significantly increasing observed ratios. This effect appears to be present already at low concentrations of dabigatran (6 ng/mL) and affects both normal subjects and heterozygous carriers of factor V Leiden. In this group of patients, dabigatran, at concentrations in the therapeutic range (100-200 ng/mL), could markedly increase the activated protein C resistance ratio, bringing it up to within the reference range for normal subjects, thus potentially leading to misclassification of patients.

Multicenter evaluation of the new immunoassay method for TSH measurement using the automated DxI platform.

AIM OF THE STUDY: Recently, Beckman Coulter Diagnostics set up a new TSH immunoassay for the automated DxI platform. The aim of this study was to evaluate and compare the analytical performance and clinical results of this method with those of previous method. MATERIAL AND METHODS: A multicenter study (named TSH ELAS Study) was organized using 593 serum samples, collected from healthy subjects and patients with thyroid disorders, and 13 control samples, circulated in an External Quality Assessment (EQA) scheme. RESULTS: The values of LoB and LoD, and LoQ at 20% CV were 0.0004mIU/L, 0.001mIU/L and 0.0023mIU/L, respectively. Moreover, TSH concentrations >0.01mIU/L actually show imprecision values lower than 5% CV. This new TSH assay showed a systematic underestimation (on average of 6.25%) compared to old method, which is mainly due to larger differences between methods for samples with low TSH concentrations, related to the better analytical sensitivity of new compared to old method. In a reference population, including 279 apparently healthy adult subjects, Caucasian volunteers (mean age 43.6years, age 20-63years, 138 women and 141 males) the distribution of TSH concentrations was: mean (CI 95%) 1.694mIU/L (1.588-1.779), median 1.495mIU/L (1.412-1.588mIU/L), 97.5th percentile 3.707mIU/L. CONCLUSIONS: The new TSH immunoassay for DxI platform shows some relevant improvements compared to the previous one: use of the most recent WHO 3rd IRP 81/563 standard and monoclonal antibodies (instead of polyclonal antibodies of the old method), and better analytical sensitivities and reproducibility.

Perioperative ROTEM and ROTEMplatelet monitoring in a case of Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia is a rare, inherited disease, which consists in abnormality of the glycoprotein IIa/IIIb genes that causes impaired platelet function. Glanzmann's thrombasthenia patients show prolongation of bleeding time and may experience life-threatening hemorrhage after surgery. We present a case of a young woman having Glanzmann's thrombasthenia, who underwent gynecological operation for ovarian cyst. Perioperative monitoring included thromboelastometry with ROTEM and impedance aggregometry with ROTEMplatelet. In this case, despite acceptable ROTEM results after platelet transfusion, ROTEMplatelet was more indicative of hemostatic impairment and reflected the bleeding occurred in the postoperative period. When treating patients with congenital or acquired platelet dysfunction, some intrinsic limits of thromboelastometry to assess platelet function may be overcome with the combined use of a point-of-care device that measures platelet aggregation.

Estimated glomerular filtration rate calculated using different creatinine and cystatin based formulas in prediction of trough plasma Dabigtran concentration.

BACKGROUND: In this paper we evaluated the relationship between trough DAB concentration with eGFR calculated using CRE and CYS based formulas. MATERIALS AND METHODS: We considered 100 patients. eGFR was calculated using CKD-EPIcreat, CG, MDRD, CKD-EPIcys and CKD-EPIcombined equations. DAB dosage was selected on the basis of CKD-EPIcreat and relationship between trough DAB concentration and eGFRs was evaluated. RESULTS: Trough DAB concentration roughly correlates with eGFR calculated using various formulas. CKD-EPIcreat eGFR was higher than CKD-EPIcys. In patients receiving a DAB dosage considered adequate using CKD-EPIcreat eGFR but considered excessive using CKD-EPIcys, we observed higher DAB trough concentration and an increased prevalence of subjects with drug concentration >200ng/mL. CONCLUSION: These results suggest that eGFR alone was unable to fully explain trough DAB plasma concentration. Therefore a drug's prescription schedule based on eGFR only should be inadequate. We observed a better correlation between trough DAB concentration and CKD-EPIcys rather than CKD-EPIcreat eGFR. Thus, in patients chronically treated with DAB for thromboprophylaxis in nonvalvular atrial fibrillation evaluation of eGFR using a cystatin base formula should be considered.

[Physical, chemical and morphological urine examination guidelines for the Analytical Phase from the Intersociety Urinalysis Group]. / Esame fisico, chimico e morfologico delle urine proposta di linee guida per la fase analitica del Gruppo Intersocietario Analisi delle Urine (GIAU).

With these guidelines the Intersociety Urinalysis Group (GIAU) aims to stimulate the following aspects: Improvement and standardization of the analytical approach to physical, chemical and morphological urine examination (ECMU). Improvement of the chemical analysis of urine with particular regard to the reconsideration of the diagnostic significance of the parameters that are traditionally evaluated in dipstick analysis together with an increasing awareness of the limits of sensitivity and specificity of this analytical method. Increase the awareness of the importance of professional skills in the field of urinary morphology and the relationship with the clinicians. Implement a policy of evaluation of the analytical quality by using, in addition to traditional internal and external controls, a program for the evaluation of morphological competence. Stimulate the diagnostics industry to focus research efforts and development methodology and instrumental catering on the needs of clinical diagnosis. The hope is to revalue the enormous diagnostic potential of 'ECMU, implementing a urinalysis on personalized diagnostic needs for each patient. Emphasize the value added to ECMU by automated analyzers for the study of the morphology of the corpuscular fraction urine. The hope is to revalue the enormous potential diagnostic of 'ECMU, implementing a urinalysis on personalized diagnostic needs that each patient brings with it.

Postpartum thrombotic complication in a patient with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal nonneoplastic hematopoietic stem cell disease characterized by an acquired mutation of the PIG-A gene with reduction or absence of CD55 and CD59. The absence of these proteins renders PNH erythrocytes susceptible to complement-mediated hemolysis. We report the case of a PNH patient before and during pregnancy until delivery. We observed and treated some postpartum thrombotic complications. Eculizumab should be used with caution in pregnancy. There are several reports supporting its use in these patients. This case should be considered paradigmatic of a series of clinical situations that may occur in the course of a pregnancy in patients with PNH: increased need for transfusion, need to increase the dose of Eculizumab, and insurgence of fetal sufferance. Moreover, after delivery, the patient, despite adequate prophylaxis with low-molecular-weight heparins, presented severe complications: development of pleural and peritoneal effusion, pulmonary embolism, bilateral upper limbs thrombophlebitis, and a possible abdominal angina with a transient paralytic ileus. All these complications were overcome and now the baby is healthy and the mother has returned to the usual therapeutic regimen.

Does flow cytometry have a role in preliminary differentiation between urinary tract infections sustained by gram positive and gram negative bacteria? An Italian polycentric study.

BACKGROUND: Urine culture is the most frequently requested test for a Microbiology Lab. A reliable screening tool would be of paramount importance both to clinicians and laboratorians, provided that it could get fast and accurate negative results in order to rule-out urinary tract infection (UTI). MATERIALS AND METHODS: We evaluated 1907 consecutive urine samples from outpatients. Culture was performed on chromogenic agar with 1µL loop, using 10(5)CFU/mL as a limit of positive growth. Using Sysmex Uf-1000i analyzer we evaluated bacteria forward scatter (B_FSC) and fluorescent light scatter (B_FLH) in a preliminary discrimination step for UTI caused by Gram+ or Gram- bacteria. RESULTS: We got 512 positive samples. A mono-microbial infection was observed in 490 samples; two bacterial strains were isolated in 22 samples, so 534 bacterial strains were found: 392 Gram-, 133 Gram+ and 9 yeasts. Comparing Gram+ and Gram- bacteria we observed a statistically significant difference for B_FSC but not for B_FLH. In this application experimental cut-off value for B_FSC was 25ch. Using this cut-off to perform a presumptive identification of UTI sustained by Gram-+ bacteria, we observed a SE 0.68, SP 0.84. CONCLUSION: Our data although preliminary suggest that B_FSC could be useful in presumptive exclusion of UTI caused by Gram-positive bacteria.

Significance of anti-HBc only in blood donors: a serological and virological study after hepatitis B vaccination.

BACKGROUND: Blood donors positive only for anti-HBc may have a resolved hepatitis B virus (HBV) infection, low grade chronic infection or infection with variant strains of HBV. We aimed to assess the significance of this serological pattern after hepatitis B vaccination in such cases. MATERIALS AND METHODS: Twenty-four anti-HBc only blood donors were vaccinated with the Engerix HBV vaccine and a serological and virological evaluation was performed before HBV vaccination and 7-10 days after each dose. Subjects were classified as non-responders if their anti-HBs levels stayed below 10 IU/L after full vaccination, while the response was considered secondary (anamnestic) if anti-HBs levels rose over 10 IU/L after the first vaccine dose, and primary if anti-HBs levels rose over 10 IU/L only after the second or third vaccine dose. RESULTS: Of the 21 fully evaluable donors, six had no response, eight showed a primary response and seven had an anamnestic response. One non-responder had transient positivity for HBV-DNA at low levels (12 IU/mL) with persistent negativity for HBsAg. DISCUSSION: Anti-HBc-only positive blood donors are a heterogeneous population including HBV naïve subjects with a likely false-positive anti-HBc reactivity, subjects with a resolved HBV infection, and subjects with persistent low-level HBV replication. The analysis of the anti-HBs response after a dose of HBV vaccine may help to distinguish among the different causes of the isolated anti-HBc positivity, thereby enabling proper counselling and potential readmission to blood donation.

Gender's equality in evaluation of urine particles: Results of a multicenter study of the Italian Urinalysis Group.

OBJECTIVE: We performed a multicenter study to calculate the upper reference limits (URL) for urine particle quantification in mid-stream samples by using automated urine analyzers. DESIGN & METHODS: Two laboratories tested 283 subjects using a Sysmex UF-100, two other laboratories tested 313 subjects using Sysmex UF-1000i, whereas two other laboratories tested 267 subjects using Iris IQ®200. RESULTS: The URLs of UF-100 in females and males were 7.8/µL and 6.7/µL for epithelial cells (EC), 11.1/µL and 9.9/µL for red blood cells (RBC), 10.2/µL and 9.7/µL for white blood cells (WBC), and 0.85/µL and 0.87/µL for cylinders (CAST). The URLs of UF-1000i in females and males were 7.6/µL and 7.1/µL for EC, 12.2/µL and 11.1/µL for RBC, 11.9/µL and 11.7/µL for WBC, and 0.88/µL and 0.86/µL for CAST. The URLs of Iris IQ®200 in females and males were 7.8/µL and 6.6/µL for EC, 12.4/µL and 10.1/µL for RBC, 10.9/µL and 9.9/µL for WBC, and 1.1/µL and 1.0/µL for CAST. CONCLUSION: The URLs obtained in this study were comparable to the lowest values previously reported in the literature. Moreover, no gender-related difference was observed, and analyzer-specific upper reference limits were very similar.

Pediatric reference values for urine particle quantification by using automated flow cytometer: results of a multicenter study of Italian urinalysis group.

OBJECTIVES: The purpose of this Italian multicenter study was to define pediatric upper reference values for urine particle quantification by using automated flow cytometry. DESIGN AND METHODS: Four hospital-based clinical laboratories participated in this multicenter investigation, which included a total study population of 161 Italian children aged from 1 to 12years. Two laboratories used Sysmex UF-100 and analyzed 86 children, whereas the other two used Sysmex UF-1000i and analyzed 75 subjects. Particle quantification included the analysis of white blood cells (WBC), red blood cells (RBC), squamous epithelial cells (EC), transitional epithelial cells (TC), casts (CAST) and bacteria (BACT). RESULTS: The upper reference values in subjects tested with the Sysmex UF-100 were 9.7WBC/µL, 10.1RBC/µL, 7.5EC/µL, 2.5TC/µL, 0.7CAST/µL and 3090BACT/µL, whereas the upper reference values in subjects tested with the Sysmex UF-1000i were 10.5WBC/µL, 8.3RBC/µL, 7.2EC/µL, 2.9TC/µL, 0.7CAST/µL and 48BACT/µL. No statistically significant differences between genders were found in the value distribution of any of the parameters tested. Similarly, no statistically significant differences were observed between the two urine analyzers, except for BACT. CONCLUSIONS: Automated analysis of urine particles appears a suitable means to optimize the workflow of routine urinalysis of children specimens. The upper reference limits for pediatric subjects obtained in this study were comparable to those previously reported in the literature, with no significant differences between genders and analyzers.

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.