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Fungemia is a life-threatening condition associated with high mortality; the most frequently isolated genus is Candida. Candida glabrata is of particular concern because of its increasing resistance to azoles. We evaluated common lab tests accessible by almost all healthcare professionals to estimate the post-test probability of recovery of C. glabrata from a blood culture collected by venipuncture, positive for fungi identified by microscopic examination. Patients with blood cultures positive for C. glabrata had significantly higher median values of serum creatinine (P=0.006), and a value of ≥1.45 mg/dL was the best cut-off in discriminating C. glabrata from other Candida spp., with 0.67 [95% Confidence Interval (CI): 0.49-0.85] sensitivity and 0.75 (95% CI: 0.66-0.84) specificity; Youden's J statistic: 0.42. The receiver operator characteristic curve analysis showed an area under the curve of 0.718 (95% CI: 0.603-0.833); P=0.001. Therefore, given a pre-test probability of 24% and applying the Bayes' theorem, the post-test probability of C. glabrata fungemia with creatinine values ≥1.45 mg/dL increased to 45.8%. In conclusion, we showed how the probability of recovery of C. glabrata from blood cultures collected by venipuncture and positive for fungi can be better estimated using concurrent creatinine values.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large ( 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Background: Various factors, such as viral infections, can act as triggers for the development of autoimmune diseases.In our recent study we reported the presence of autoantibodies in patients diagnosed with COVID-19. To verify whether these autoantibodies persisted over time and led to the development of chronic autoimmune diseases, we conducted a follow-up study at 3 and 6 months after admission.Methods:Thirteen of 40 patients enrolled in the previous study gave their consent to the analysis. The same autoimmunity tests performed at the time of diagnosis were carried out in these patients during follow up.Results:We showed, compatibly with an acute inflammatory response, that two patients were negative 6 months after diagnosis. In nine patients, autoantibodies were still present at follow-up. Among them, one patient had only ENA positivity. Another patient developed strong positivity for ANA and M2-β and Ku antigens. Currently, the symptoms do not meet full diagnostic criteria for diagnosis of polymyositis, but the patient is closely monitored to check its possible onset.Three patients developed: transient alopecia, autoimmune thrombocytopenia and hearing loss. Other four patients did not show any clinical symptoms.Conclusions:In conclusion, our data show that after 6 months, the autoantibodies are still present in the majority of patients. Further investigations will be necessary to check whether these patients will become negative over time or may develop clinical symptoms compatible with the onset of longer-term chronic autoimmune diseases.

Objective: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL. Methods: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL. Results: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified. Conclusions: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.

Con il termine trombofilia si identifica una condizione clinica che determina un aumento del rischio di malattia tromboembolica venosa e di alcune patologie della gravidanza. Questa condizione puo essere associata ad alterazioni ematologiche, sia congenite sia acquisite, che possono essere studiate con dosaggi quantitativi o con tecniche genetiche di biologia molecolare. L’utilita di eseguire indagini di laboratorio per identificare una condizione ereditaria di trombofilia e a tutt’oggi argomento di discussione. Da parte di diverse Societa scientifiche e Gruppi di Lavoro sono state prodotte Linee guida nelle quali si sostiene che gli unici esami validati per uno studio di screening di primo livello per la trombofilia ereditaria sono: il dosaggio di antitrombina, proteina C e proteina S e la ricerca delle mutazioni per fattore V di Leiden e protrombina G20210A. Cio nonostante, a livello di indagini di screening, viene spesso richiesto o proposto lo studio di molti altri polimorfismi con incerta utilita clinica, notevole aumento dei costi e produzione di ansie e timori ingiustificati nella popolazione esaminata. Il Gruppo di Studio di Coagulazione della SIPMeL ha voluto eseguire una ricognizione della letteratura medica per aggiornare la precedente edizione di Linee guida SIMeL sullo screening della trombofilia (2004) e in particolare per ampliare le raccomandazioni in merito alla ricerca dei polimorfismi nello studio della diagnostica della trombofilia ereditaria.

Infectious meningitis and encephalitis are potentially life-threatening conditions caused mostly by bacterial and viral agents. Rapid diagnosis and prompt treatment are associated with a more favorable outcome. In recent years nucleic acid amplification tests have been developed to speed detection and identification of pathogens directly from cerebrospinal fluid (CSF). The aim of this study was to compare the diagnostic accuracy of a commercially available multiplex PCR assay for etiological diagnosis of infectious meningitis directly from CSF samples with culture. A secondary endpoint was to look for a possible screening threshold based on main CSF indices and urgent blood test results, to define CSF samples with low pre-test probability of PCR and/or culture-positive result. We performed a secondary analysis of results of CSF samples already processed as part of routine clinical care from February 2016 to December 2018. In all, 109 CSF samples were included in the study and a total of 14 bacteria were identified by either PCR, culture or both methods, along with nine samples positive for viruses. The comparison of PCR results with culture showed no significant difference: 7/109 (6.4%) vs 13/109 (11.9%) respectively, p=0.07. After exclusion of the isolates not detectable by the multiplex PCR panel, the diagnostic accuracy was: 100% (95% confidence interval (CI): 54.1% to 100%) sensitivity; 98.9% (95% CI: 93.5% to 99.9%) specificity; 85.7% (95% CI: 42% to 99.2%) positive predictive value; 100% (95% CI: 95.1% to 100%) negative predictive value; 96 (95% CI: 13.6 to 674.6) LR+; Zero LR-; Cohen's kappa: 0.918, p0.0001. CSF protein value ≤ 28 mg/dl and CSF glucose/blood glucose ratio ≥0.78 were associated with both PCR-negative result for bacteria or viruses and culture-negative result. The multiplex PCR evaluated in this study showed a very good diagnostic performance compared to culture, and the thresholds found can be a useful tool to best choose which samples to test.

In Medicina di Laboratorio il momento del referto, oltre a rappresentare un evento essenziale nella validazione clinica di un risultato, assume anche il preciso significato di “giudizio interpretativo” formulato dallo specialista di laboratorio. Deve quindi essere ritenuto l’atto conclusivo di un processo tecnico, che ha prodotto risultati numerici derivanti da analisi (biochimiche, ematologiche, immunologiche ecc.) eseguite su materiali biologici. In un sistema di gestione della qualita il referto costituisce l’atto scritto conclusivo e ufficiale di un iter diagnostico all’interno di un percorso clinico rivolto al beneficio di un paziente. Le applicazioni diagnostiche citofluorimetriche sono numerose e riguardano un ampio spettro di patologie e di substrati da analizzare: emopatie maligne, malattie linfoproliferative, studio della malattia minima residua, sottopopolazioni linfocitarie sono solo alcuni importanti campi di utilizzo delle metodiche citofluorimetriche; molteplici sono anche i substrati analizzabili, potendosi effettuare studi su numerosi comparti cellulari e su diversi costituenti subcellulari. Il GdS Citofluorimetria intende proporre alcune raccomandazioni per la stesura del referto citometrico, che spesso risente troppo della personalizzazione dell’operatore ed e una fase nevralgica nel percorso diagnostico-terapeutico del paziente.

OBJECTIVES Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. METHODS Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia-free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

Abstract 4028 BACKGROUND. The corner stone of the WHO classification and prognostic scores of myelodysplastic syndromes (MDS) is the blast count in bone marrow. The standard cytology evaluation of at least 500 bone marrow cells is easy to perform, but some concerns arise about reproducibility of this method. Nowadays bone marrow trephine biopsy and flow cytometry are frequently considered for the diagnosis of MDS. However there is so far paucity of data comparing cytology, histology and flow cytometry in quantifying bone marrow blasts in order to differentiate non RAEB from BAEB-I and RAEB-II cases. AIM OF THE WORK. The Aim of the work was to analyse the differences and the prognostic impact of cytology, histology and flow cytometry in differentiating non RAEB from BAEB-I and RAEB-II. PATIENTS AND METHODS. Since 1999, clinical and laboratory data from 1256 new cases of MDS were prospectively recorded into the Piemonte MDS Registry. Blast count could be performed with the three different methods: BMC (bone marrow cytology) has been performed in 844 cases, BMH (bone marrow histology) in 874 cases, and BMF (bone marrow flow cytometry) in 636. In order to quantify blasts, immune-histochemistry evaluation of CD34+ cells was used in BMH, while both CD34+ and CD117+ cells were considered in BMF. Out of the total of the 636 patients analysed by BMF only 420 had an accurate and complete registration of CD34 and CD117 positivity and were considered for the present analysis. In two hundred and thirty six cases all three evaluations were contemporary available. The concordance of each diagnostic method with the others and their prognostic value were evaluated in both univariate and multivariate analyses. A comparison between BMC and BMH was available in 571 cases, between BMC and BMF in 228 cases, and between BMH and BMF in 279 cases. RESULTS. The disagreement in classifying patients as non-RAEB or RAEB-I or RAEB-II between BMC and BMH was 156/571 (27%), with BMH over-evaluating blasts in 114/571 cases (20%) and under-evaluating blasts in 42/571 cases (7%). The disagreement between BMC and BMF was 80/228 (35%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMC in 53/228 (23%) and in 27/228 (12%) cases respectively. The disagreement between BMH and BMF was present in 113/279 (41%), with BMF over-evaluating and under-evaluating blast percentage in comparison to BMH in 44/279 (16%) and in 69/279 (25%) cases respectively. In univariate analysis all three methods of quantifing blasts and differentiating non-RAEB from RAEB-I and RAEB-II retained an important prognostic value for both leukemic evolution and survival. However when the three models were tested in multivariate analysis in order to define the best predictor of leukemic evolution, BMC retained the best predictive value. CONCLUSIONS. When BMH or BMF are used instead of BMC in order differentiate non-RAEB from RAEB-I and RAEB-II, the shift to a different WHO category is evident in at least 30% of patients and BMH and BMF do not play the same role as BMC. BMC still remain the standard method to quantify blasts for classification and prognostic evaluation of MDS. Disclosures: Off Label Use: Lenalidomide in Mantle Cell Lymphoma. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 5094 Background. Leptomeningeal involvement is an adverse complication of haematological malignancies, acute leukemias and lymphomas. The diagnostic gold standard to identify cerebrospinal fluid (CSF) involvement is conventional cytological analysis, technique characterized by low sensitivity and specificity. Recent papers make evidence that the efficiency of CSF involvement detection could improve by use of Flow Cytometry (FCM) immunophenotypic analysis of CSF samples. We describe the FCM method used in our hospital and we compare the results with cytological findings. Methods. CSF samples should be processed immediately, within 1 hour after collection to avoid cell's deterioration; in B Non Hodglin Lymphomas (B-NHL) we use two steps FCM approach, in four color staining (FITC/PE/PerCP/APC). The first step was two tubes: kappa/lambda/CD45/CD19 and CD5/CD20/CD45/CD3 or CD3/CD20/CD45/CD10 on the basis of a previous know immunophenotype or not. If a pathological population is identified, we complete immunophenotype characterization with a second step, using antibody panels suitable for the suspected malignancy. In case of suspected meningeal relapse of Acute Leukemia we use a monoclonal antibody combination select for Minimal Residual Disease. Results. Between 2006 and 2009 we studied by FCM analysis 53 CSF samples of 36 patients. 7 CSF of 29 B-NHL newly diagnosed resulted positive at FCM analysis (24,1%), 1 CSF of 7 Acute Leukemias shows disease relapse. The conventional cytology was positive only in 1 case of B-NHL. Conclusion. Our study confirms that immunophenotypic flow cytometric analysis of CSF cells markedly improves sensitivity to identify CSF involvement in haematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Background. Recent studies suggest that Flow Cytometry (FCM) might provide information useful in managing Myelodysplastic Syndromes (MDS) patients. Aim of the work. The purpose of this work is to describe the prognostic relevance of FCM analysis on a cohort of 303 myelodysplastic patients (64 RA; 15 RARS; 59 RCMD; 12 5q-MDS; 20 U-MDS, 72 RAEB-I, 44 RAEB-II, 17 LMMC), registered in the Multicenter Piedmont MDS Registry. Patients, materials, and methods. Marrow of 303 patients was studied by seven local FCM laboratories, using monoclonal antibodies against: CD3, CD8, CD4, CD16, CD19, CD34, CD117, CD33, CD14, CD11b, CD66b, HLADR, GPA and CD45. Myeloid maturation was evaluated according to the expression of both CD11b and CD66b antigens. Immature compartment of myeloblasts was analysed with CD117 and CD11b−CD66b−, and myeloid maturation with CD11b−CD66b+ (promyelocytes), CD11b+CD66b++ (myelocytes and metamyelocytes), CD11b+CD66b+ (neutrophils and bands). This definition of myeloid subpopulations was confirmed by sorting and by citospin. The overall survival (OS) and Leukemia-Free Survival (LFS) was calculated from the time of diagnosis until death or until leukaemia transformation. Survival curves were obtained by the Kaplan-Meier method and compared by the log-rank test. To evaluate the impact of FCM parameters on OS and LFS, we determined quantitative expression of the following antigen: CD117 and CD11b−CD66b− (≤5% vs >5%); CD11b−CD66b+ (≤12% vs >12%); CD11b+CD66b++ (≤15% vs >15%); CD11b+CD66b+ (≤25% vs >25%). Results. In our patients, quantitative expression of CD117 correlated with a shorter OS and LFS: the 40 months-OS was 66,5% versus 26,4% (p Discussion. Cytometric recognitions of dysplasia is difficult to perform, as it requires subjective interpretation by a skilled pathologist. This study emphasizes that FCM analysis of myeloid maturation, expressed as percentage of positive cells, may provide meaningful prognostic information. Both blast percentage expressed as CD117+ or CD11b−CD66b− and the quantitative assessment of myeloid lineage maturation showed, in our experience, a high prognostic value. These FCM parameters should be proposed as a prognostic tool easy to reproduce and perform. Results of quantitative FCM markers FCM variable Cut off 40 months-OS p 40 months-LFS p CD117+ ≤5% vs >5% 66.5% vs 26.4% 0.00001 91.6% vs 27.6% 0.00001 CD11b−CD66b− ≤5% vs >5% 71.5% vs 45.9% 0.0002 90.8% vs 51.3% 0.00001 CD11b−CD66b+ ≤12% vs >12% 49.8% vs 58.1% NS 80.7% vs 76.6% NS CD11b+CD66++ ≤15% vs >15% 33.0% vs 63.3% 0.001 58.9% vs 87.5% 0.002 CD11b+CD66b+ ≤25% vs >25% 30.3% vs 64.3% 0.00003 53.3% vs 76.6% 0.0008 Leukaemia transformation free Survival (Kaplan-Meier) ○ Complete + Censored Leukaemia transformation free Survival (Kaplan-Meier) ○ Complete + Censored

We used the monoclonal antibodies LFO3 (specific for the L subunit of ferritin) and 2A4 (specific for the H subunit) in an indirect immunofluorescence test for enumerating ferritin-bearing lymphocytes (FBL). In 13 normal subjects, the geometric mean value of FBL was 4 % (range 0–13 %) with the monoclonal antibody LFO3, and 3 % (range 0–8 %) with the monoclonal antibody 2A4. Values in 5 subjects with transfusional iron overload and increased plasma L-type ferritin concentration were 5 % (4–7 %) and 3 % (2–4 %), respectively, which is similar to those in normal subjects. Thirteen patients with malignant disease had normal to increased values for plasma ferritin; the circulating protein was largely of L-type with undetectable or very low concentrations of H-type ferritin. In the same patients, the percentage of FBL was greater with the monoclonal antibody 2A4 (geometric mean value 8 %; range 3–12 %) than with the monoclonal antibody LFO3 (geometric mean value 3 %; range, 1–7 %). It is concluded that acidic and basic isoferritins can be differently expressed on the surface of peripheral blood lymphocytes, and that the monoclonal 2A4 could be particularly useful in the measurement of FBL in patients with malignancy.