Resumen Objetivos La calprotectina (CP) es una proteína de unión a calcio y zinc que se suele determinar en muestras fecales, aunque su cuantificación en otros fluidos biológicos podría ser de interés. El objetivo del presente estudio es validar la determinación de CP en líquido pleural mediante quimioluminiscencia. Métodos Para la cuantificación de CP en líquido pleural, se utilizó LIAISON ® XL, un autoanalizador de quimioluminiscencia. Se diseñó un protocolo de validación empleando tanto materiales de control de calidad suministrados por el fabricante, como soluciones de muestras de líquido pleural. Se evaluaron la estabilidad, imprecisión, sesgo, linealidad, capacidad de detección y efecto de arrastre. Resultados La CP permaneció estable en líquido pleural refrigerado durante al menos una semana, y durante cuatro semanas a −80 °C. La imprecisión intradía e interdía observada fue del 2,2 % y del 6,49 %, respectivamente, con un sesgo negativo del 5,51 %. La linealidad del método se verificó hasta los 2000 ng/mL. El límite de cuantificación (LoQ) de la prueba fue de 48,52 ng/mL. Se observó un efecto de arrastre estadísticamente significativo tras medir concentraciones de CP superiores al límite máximo de linealidad. Sin embargo, dada la magnitud observada, no se debe esperar un impacto clínicamente relevante. Conclusiones La prueba Liaison ® Calprotectin de DiaSorin es fiable para la determinación de CP en líquido pleural.

Objectives: Calprotectin (CP) is a calcium and zinc binding protein that is widely measured on faecal samples but its determination in other biological fluids might be of interest. The aim of this work was to validate the measurement of CP in pleural fluid by chemiluminescence.Methods: LIAISON (R) XL, a fully automated chemiluminescence analyzer, was used for CP quantification on pleural fluid. A validation protocol was designed using both quality control materials provided by the manufacturer and pools of pleural fluid samples. Stability, imprecision, bias, linearity, detection capability and carry over effect were evaluated.Results: CP was stable on pleural fluid at least one week, under refrigerated conditions, and four weeks at -80 80 degrees C. The observed intra- and inter-day imprecision was between 2.2 and 6.49 %, with a negative bias under 5.51 %. The linearity of the method was verified up to 2,000 ng/mL. The LoQ for the assay was 48.52 ng/mL. A statistically significant carry-over effect was observed after measuring CP concentrations above the upper limit of linearity, but given the observed magnitude, a clinically relevant impact should not be expected.Conclusions: Diasorin Liaison (R) calprotectin assay allows reliable measurement of CP in pleural fluid.

Allogeneic hematopoietic stem cell transplantation (HSCT) represents the best therapeutic option for many patients with acute myeloid leukemia (AML). However, relapse remains the main cause of mortality after transplantation. The detection of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in AML, before and after HSCT, has been described as a powerful predictor of outcome. Nevertheless, multicenter and standardized studies are lacking. A retrospective analysis was performed, including 295 AML patients undergoing HSCT in 4 centers that worked according to recommendations from the Euroflow consortium. Among patients in complete remission (CR), MRD levels prior to transplantation significantly influenced outcomes, with overall (OS) and leukemia free survival (LFS) at 2 years of 76.7% and 67.6% for MRD-negative patients, 68.5% and 49.7% for MRD-low patients (MRD < 0.1), and 50.5% and 36.6% for MRD-high patients (MRD ≥ 0.1) (p < 0.001), respectively. MRD level did influence the outcome, irrespective of the conditioning regimen. In our patient cohort, positive MRD on day +100 after transplantation was associated with an extremely poor prognosis, with a cumulative incidence of relapse of 93.3%. In conclusion, our multicenter study confirms the prognostic value of MRD performed in accordance with standardized recommendations.

BACKGROUND:Multidimensional flow cytometry (MFC) is routinely used for the diagnosis and follow-up of hematolymphoid neoplasms but its contribution to the identification of non-hematolymphoid malignant tumors is limited. METHODS:The presence of non-hematolymphoid cells in clinical samples obtained via minimally invasive methods was ascertained by using a panel of monoclonal antibodies previously developed in our laboratory comprising a mixture of antibodies: CD9-PacB/CD45-OC515/CD57-FITC/CD56-PE/CD3-PerCP-Cy5.5/CD117-PE-Cy7/CD326-APC/CD81-APC-C750. Histopathological studies were performed using standard techniques. RESULTS:164 specimens of different origins were included. Malignancy was finally confirmed in 142 (86.5%), while 22 non neoplastic samples were identified. The most frequent diagnosis was small cell lung carcinoma (SCLC) (50%). High sensitivity (S = 98.6%) was reached combining MFC and conventional pathology. Individual markers differed according to the cellular origin of the neoplasm, with neuroendocrine tumors showing a unique immunophenotypic profile (CD56+ CD326+ CD117-/+ and variable tetraspanins expression). Principal component analysis efficiently distinguished SCLC from other tumor samples. In immune cell populations, differences between reactive and malignant biopsies were found in different cell compartments, especially in B cells and Plasma cells. Differences also emerged in the percentage of CD4+ CD8- T cells, CD4-CD8+ T cells and NK cells and these were dependent on the origin of the tumor cells. CONCLUSIONS:These results support the use of MFC as a rapid and valuable technique to detect non-hematolymphoid tumoral cells in clinical specimens, providing an initial orientation to complement hystopathological studies and allow a more precise diagnosis, especially in neuroendocrine neoplasms. The impact of different immune cell patterns warrants further research.

High levels of inflammation play an important role in chronic heart failure (CHF). Patients with CHF have elevated levels of pro-inflammatory cytokines circulating systemically, mainly TNF and IL-6. However, there are almost no studies that relate these levels to the functional status of patients in CHF, much less to their CMV serostatus. In this study, patients with CHF (n=40; age=54.9 ± 6.3; New York Heart Association functional classification (NYHA, I-III) and healthy controls (n=40; age=53.5 ± 7.1) were analyzed. The serum concentrations of nine pro- and anti-inflammatory cytokines were measured by Luminex® xMap Technology and the basal level of mRNA expression of some immune molecules was quantified by TaqMan™ Array in CD4+ T-lymphocytes. The concentration of these cytokines in culture supernatants in response to anti-CD3 and LPS was also measured. The percentage of CD28null T-cells was determined, as well as the antibody titer against CMV. We found a higher concentration of all cytokines studied in CHF serum compared to healthy controls, as well as a direct correlation between functional status in CHF patients and levels of inflammatory cytokines. Moreover, the highest cytokine concentrations were found in patients with higher concentrations of lymphocytes lacking CD28 molecule. The cytokine production was much higher in CMV+ patients, and the production of these cytokines was found mainly in the T-lymphocytes of CMV+ patients in response to anti-CD3. Anti-CMV antibody levels were positively correlated with cytokine levels. The baseline expression of specific mRNA of the main molecules involved in the Th1 response, as well as molecules related to the CD4+CD28 null subset was higher in CMV+ patients. The cytokine concentrations are higher in CHF CMV+ patients and these concentrations are related to the production of antibodies against CMV. These high levels of cytokines are also associated with the more differentiated CD28null lymphocyte populations. All this, together with the dynamics of the pathology itself, makes CMV+ patients present a worse functional status and possibly a worse evolution of the pathology.

Introduction: Acute myeloblastic leukaemia (AML) is an heterogeneous disease with different molecular and prognostic characteristics. According to the comorbidities and the revised 2017 European Leukaemia genetic risk stratification (ELN17), allogeneic hematopoietic cell transplantation (HCT) is the best therapeutic option for many patients with AML (Grimm, Blood Adv 2020). However, relapse remains the main cause of mortality after transplantation. Impact of MRD on the outcome of patients is well recognized and ELN2017 introduced the new response category complete remission (CR) without MRD (Döhner H, Blood 2017). Detection of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in AML before allogeneic HCT could be a powerful predictor of outcome and decisive when establishing strategies that modify the prognosis of these patients. Methods: Retrospective multicentre analysis of MRD by MFC of patients undergoing transplantation allogeneic in 4 centres during the period from 2012 to 2020. Both Leukaemia Associated Aberrant Immunophenotype (LAIP) and different from normal (DFN) approach were used to analyse the MRD. The MRD was carried out with 8-color panels based on Euroflow protocols. The samples were acquired in 8-color digital cytometers (FACSCanto II) calibrated and compensated according to Euroflow protocols. Results: 295 of 318 patients were evaluated. Table 1 shows the characteristics of the patients. 285 (96.7%) were in complete remission (CR), 207 had negative MRD, in 21 MRD was less than 0.1% (MRD-low) and in 57 greater than or equal to 0.1% (MRD-high). At 2 years, the overall survival (OS) and leukaemia-free survival (LFS) in the whole group were 69% (95% CI 63.18-74.18) and 58.4% (95% CI 52.4-63.9) respectively. In CR patients, MRD levels significantly influenced on outcomes, with OS and LFS of 76.7% and 67.6% for negative MRD, 68.5% and 49.7% MRD-low and 50 % and 36.6% in MRD-high, p <0.001) (Figure 1). Considering only MRD-high as positive, according to ELN17, cumulative incidence of relapse (CIR) at 2 and 5 years were significantly lower among those with positive MRD: 22% (95% CI 17-28.1%) and 27% (95% CI 21%-33.5%) for negative MRD vs 46,5% (95% CI 32.4%-59.5%) and 50% (95% CI 34.8%-63.2%) in positive MRD, p 0.0005. No differences were observed in terms of non relapse mortality (p 0.2).. Likewise, positive MRD also identified different prognostic subgroups within the ELN2017 subgroups: OS and LFS among high-risk ELN2017 patients of 63.6% and 52.3% in negative MRD vs 35.7% and 18.2% in positive MRD patients, p = 0.0085 and p = 0.0094, respectively; for intermediate risk: 77% and 67.6% in negative MRD vs 67% and 50.5% in positive MRD patients, p = 0.23 and p = 0.056; and for favourable: 84% and 77.7% in negative MRD vs 48% and 39.2% in patients with positive MRD, p = 0.0051 and p = 0.0341. Considering the conditioning regimen, patients with MRD negative before transplant had better OS and LFS at 2 years (82% and 71.4% among those received myeloablative conditioning and 65% and 57.6% among those who received reduced intensity, respectively) than those who had positive MRD prior to transplant (56% and 44.4% in myeloablative and 43% and 25.5% in reduced intensity) (p <0.001) (Figure 2). In multivariate time-dependent analysis, age (HR 1.019 p = 0.024-95% CI 1.001-1,038), adverse risk group according to ELN17 (HR 2.13 p = 0.033 CI95 1.54-3.93 ) and MRD before transplant (HR 3.8 p <0.001 95 CI 1.55-3.93) significantly influenced survival. Conclusions: Detection of MRD prior to transplant by MFC identifies a group of patients with a worse prognosis and could be key when selecting the most appropriate therapeutic strategy.

BACKGROUND:Diagnosis of hematolymphoid neoplasm (HLN) requires different technologies which are performed on a patient basis instead of per protocol. We hypothesize that integration of hematimetric and cytological analysis along with multiparametric flow cytometry (MFC) provides a framework to evaluate peripheral blood (PB) samples from Primary Care. METHODS:Samples from patients with persistent (>3 months) lymphocytosis (>5 × 109/L) and/or monocytosis (>109/L) or the presence of atypical and/or blast cells upon the smear review were analyzed by MFC concurrent to cytological analysis. MFC studies were carried out following standardized procedures. RESULTS:In a 3-year period, smear review and MFC were performed simultaneously in 350 samples, demonstrating HLN in 194 cases (55.4%). In 156 cases, reactive cell populations were found. The combination of age, absolute lymphocyte count (ALC), hemoglobin and platelets provided the best correlation with MFC for the presence of a chronic lymphoproliferative disorder (CLPD) in lymphocytosis [area under the curve (AUC) 0.891, p < 0.05]. A model evaluating the probability of CLPD has been proposed and validated in an independent cohort. CONCLUSIONS:A strategy to perform MFC studies following standardized procedures has proven to be useful to evaluate samples from patients in Primary Care centers for HLN diagnosis or reactive conditions, providing a sensitive and rapid clinical orientation and avoiding unnecessary consultations in routine clinical practice. The probability for the presence of CLPD in PB can be calculated and help guide decision-making regarding further testing.

In chronic kidney disease (CKD), hyperphosphatemia-induced inflammation aggravates vascular calcification (VC) by increasing vascular smooth muscle cell (VSMC) osteogenic differentiation, ADAM17-induced renal and vascular injury, and TNFα-induction of neutral-sphingomyelinase2 (nSMase2) to release pro-calcifying exosomes. This study examined anti-inflammatory β-glucans efficacy at attenuating systemic inflammation in health, and renal and vascular injury favoring VC in hyperphosphatemic CKD. In healthy adults, dietary barley β-glucans (Bβglucans) reduced leukocyte superoxide production, inflammatory ADAM17, TNFα, nSMase2, and pro-aging/pro-inflammatory STING (Stimulator of interferon genes) gene expression without decreasing circulating inflammatory cytokines, except for γ-interferon. In hyperphosphatemic rat CKD, dietary Bβglucans reduced renal and aortic ADAM17-driven inflammation attenuating CKD-progression (higher GFR and lower serum creatinine, proteinuria, kidney inflammatory infiltration and nSMase2), and TNFα-driven increases in aortic nSMase2 and calcium deposition without improving mineral homeostasis. In VSMC, Bβglucans prevented LPS- or uremic serum-induced rapid increases in ADAM17, TNFα and nSMase2, and reduced the 13-fold higher calcium deposition induced by prolonged calcifying conditions by inhibiting osteogenic differentiation and increases in nSMase2 through Dectin1-independent actions involving Bβglucans internalization. Thus, dietary Bβglucans inhibit leukocyte superoxide production and leukocyte, renal and aortic ADAM17- and nSMase2 gene expression attenuating systemic inflammation in health, and renal injury and aortic calcification despite hyperphosphatemia in CKD.

Introduction CD47 over expression has been reported in several tumor subtypes. CD47 interacts with SIRPalpha on macrophages inhibiting phagocytic signal, providing a survival advantage to tumor. CD47, therefore, represents a valuable target for immunotherapy and is currently under clinical investigation. We aimed to study CD47 expression in Hodgkin Reed Sternberg cells (HRS). Methods We tested a polyclonal CD47 antibody (LifeSpan Biosciences, Seattle, WA) expression along with classical HRS cell markers on a tissue array of 16 classical Hodgkin Lymphoma (CHL) tumor biopsies obtained from newly diagnosed, non-selected patients (8 Female, 8 Male patients) in our institution from October 2016 to January 2018. Histologic subtypes were nodular sclerosis in 11 cases, mixed Cellularity in 3 cases and lymphocyte rich in 2 additional cases. Median age was 53 years (Range: 8, 74). Early stage disease was found in three patients without unfavorable prognostic factors according to EORTC and GHSG criteria, one patient with unfavorable prognostic factors and nine patients had advanced disease. Bulk disease was present in one patient. Normal lymphoid tissue and normal prostate epithelium were used as normal controls as recommended by manufacturer. Approval from the Local Ethical committee was obtained before any analysis. Results CD47 was overexpressed on all HRS cells with a characteristic dot-like pattern in 13/13 cases of CHL. HRS clearly expressed CD47 more intensely than infiltrating T and stromal cells. Discussion We propose that HRS cells, by up-regulating CD47, might avoid innate immunity check on tumor growth, which could be circumvented using blocking monoclonal antibodies.

Leishmaniasis is rising in many countries, including Iran, due to climate change, refugee crises, urbanization and etc. The aim of this study was to explore the epidemiology, extent and identity of Leishmania species in a newly emerged focus in Abdanan County, Ilam Province, South-western Iran. This study was performed as a descriptive cross-sectional study by a systematic house-to-house approach. The Leishmania species was identified by RFLP-PCR and sequencing. Altogether, 46799 individuals consisting of 22907 (48.9) female and 23892 (51.1%) male were interviewed and physically examined for the presence of skin lesions. Overall, the incidence rate was 0.34% (n = 160). All age groups were affected and the incidence rate was the highest in <10 years of age group (0.49%) and the lowest in >50 years old individuals (0.15%), although there was no significant difference regarding the sex and age. The majority of patients had one lesion (47.5%) on hands (56%) and most of the cases occurred in Abdanan city (%54) in summer. Based on the RFLP-PCR analysis, all the Leishmania isolates were L. major of single genotype. A newly emerged focus of zoonotic CL caused by L. major occurred in South-western of Iran. Multiple risk factors created this epidemic area. Further studies on the vector and reservoir are crucial needed to provide evidences to select the prophylactic and therapeutic measures for future control strategies.

Introduction. Nucleophosmin gene mutation (NPM1mut) occurs in around 30% of acute myeloid leukemia (AML) patients, frequently linked with favourable prognosis in the absence of FLT3-ITDmut, which occurs in around 40% of NPM1mutAML. Therefore, more expeditious diagnostic approaches may contribute to early diagnosis and prognostic stratification of these patients. Herein, we investigated the association of immunophenotypic features of leukemic and monocytic cells with the presence of NPM1mut in AML. Methods. A total of 404 bone marrow (BM) samples from newly-diagnosed AML patients according to WHO 2017 classification were retrospectively studied by 8-color flow cytometry, including 225 AML with NPM1mut and 179 cases wild type gene (NPM1wt). Information on FLT3-ITD could be obtained from 397/404 cases. Thus, FLT3-ITDmut was present in 85/397 (21%), being concomitant with NPM1mutin 62/85 AML cases (73%). Logistic regression analysis was used to identify predictive phenotypes for the presence of NPM1mut. Results. Overall, blast cell with immunophenotypic features of monocytic differentiation (corresponding to FAB M4 and M5 AML subtypes) were observed among 135/225 (60%) and 69/179 (38.5%) AML patients with NPM1mutand NPM1wt, respectively (p<0.001). In the remaining AML cases without monocytic differentiation (FAB M0, M1 and M2; n=200), both immature/myeloid blasts and remaining monocytic cells were immunophenotypically characterized. Among AML with monocytic blast cell differentiation, altered monocytic phenotypes were more frequent among NPM1mut vs. NPM1wtcases (98% vs. 36%) (p<0.001). In detail, these phenotypic alterations consisted of asynchronous expression of CD300e prior CD14 (79% vs. 5% NPM1wtcases, respectively; p<0.001) and/or CD35 prior CD14 (84% vs. 30%), which were observed independently of FLT3-ITD. Of note, coexistence of the latter two asynchronous monocytic patterns (CD300e prior CD14 and CD35 prior CD14) on monocytic blast cells was specific for NPM1mut (64% vs. 0% NPM1wtpatients; p<0.001). Noteworthy, in AML cases without blast cell monocytic differentiation, remaining monocytic cells showed similar asynchronous phenotypic patterns, which were also more frequent among NPM1mut cases (78% vs. 23% of NPM1wtcases, respectively; p <0.001). Thus, remaining monocytic cells from NPM1mut AML cases also showed a higher frequency of asynchronous CD300e prior CD14 (64% vs. 8% of NPM1wt cases; p<0.001) and CD35 prior CD14 expression (58% vs. 20% of NPM1wt cases, respectively; p<0.001). In addition, aberrant CD9 blast cell expression was found in a significant proportion of all AML cases studied (124/222, 56%). However, altered CD9 was more frequent on (either monocytic or immature/myeloid) blast cells from AML cases with NPM1mut (76% vs. 46% NPM1wtcases; p<0.001), but not in AML with only-FLT3-ITDmut(39% vs. 46% of NPM1wtFLT3-ITDwt; p>0.05). In turn, aberrant CD25 expression on blast cells was otherwise linked to FLT3-ITDmut (61% vs. 20% of FLT3-ITDwtcases; p<0.001), being more frequent in AML with FLT3-ITDmutNPM1wt and double mutated AML cases vs. AML with FLT3-ITDwtNPM1mut and FLT3-ITDwtNPM1wt (79% and 47% vs. 20% and 20% of cases, respectively; p<0.001). In addition, overall, FLT3-ITDmut was associated with a significantly higher proportion of immature (i.e. CD34+) blasts, as compared to FLT3-ITDwt cases (median of 10% vs. 0.3% CD34+ blasts; p<0.001). In multivariate analysis, baseline detection of monocytic-lineage blast cells with asynchronous expression of CD300 prior CD14 -C-index= 0.954, odds ratio (OR), 78.8; 95% confidence interval (CI), 13.1-471; p<0.001- and CD35 prior CD14 (OR, 24.5; 95% CI, 4.8-123; p<0.001) and detection of these asynchronous patterns on remaining monocytic cells from AML without monocytic differentiation (C-index= 0.816, OR, 14.7; 95% CI, 6.4-34; p<0.001 and, OR, 2.5; 95% CI, 1.2-5.3; p=0.01, respectively) showed the highest predictive value for NPM1mut in AML. In turn, CD25 aberrant blast cell expression was the only immunophenotypic parameter with predictive value for FLT3-ITDmut (OR, 6.4; 95% CI, 2.9-14.5; p<0.001). Conclusions. Detection of specific aberrant immunophenotypic patterns among blast cells and/or remaining monocytic cells from AML patients is highly predictive for NPM1mut, which may contribute to early diagnosis and follow-up of these patients. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.