[show abstract][hide abstract] ABSTRACT: The implementation of mass vaccinations against hepatitis B virus (HBV) has significantly reduced the prevalence of HBsAg-positive subjects. At the same time, the prevalence of the other markers of infection has decreased, but there has been an increase in the percentage of subjects with markers of a successful vaccination. It has been suggested that increasing immigration from countries in which this virus is highly endemic is changing the epidemiology of HBV infection. The aim of this study was to assess the prevalence of the serological markers of HBV in Italian and non-Italian HBsAg-negative subjects.
In the years 2007-2008, 8,018 samples from HBsAg-negative subjects (7,521 Italians and 497 non-Italians) were received for detection of anti-HBs and/or anti-HBc. The findings in the 1,358 samples from candidate blood donors were compared with those obtained in 1991 and 1999.
The rate of anti-HBc positivity was 18.3% in the Italian samples and 32.8% in the non-Italian samples; the corresponding percentages of anti-HBs/anti-HBc positive samples (indicating past infection), anti-HBs positive only samples (vaccination) and anti-HBc positive only were, 11.3% vs. 22.5%, 25.8% vs. 17.2%, and 6.9% vs. 9.9% in Italians and non-Italians, respectively. The differences were more marked when stratified by age. In relation to candidate blood donors, simultaneous positivity for anti-HBs and anti-HBc decreased from 11.0% in 1991 to 8.1% in 1999 and 3.9% in 2007-2008, whereas isolated anti-HBs positivity increased from 2.2% in 1991 to 21.4% in 1999 and 42.9% in 2007-2008.
The frequency of markers of past infection among Italians has decreased over time as a result of mass vaccination and is significantly lower than that observed in non-Italians. The increasing number of immigrants from countries in which HBV is highly endemic is changing the epidemiology of HBV infection in Italy.
[show abstract][hide abstract] ABSTRACT: The introduction of vaccination against hepatitis B initially reduced the number of HBV (hepatitis B virus) and HDV (hepatitis delta virus) infections, but the decreasing trend of HDV infection seems to have stopped. The aim of this study was to assess the prevalence of HDV infection in the general population living in the catchment area of Legnano Hospital in northern Italy.
Of the 22,758 subjects tested in 2007-2008, the 488 who were HBsAg (hepatitis B surface antigen)-positive [including 107 (21.9%) of non-Italian origin] were subsequently tested for anti-HDV antibodies.
Of the 488 subjects who tested positive for HBsAg, 24 (4.9%) were anti-HDV positive, all aged between 30 and 60 years. The difference in prevalence between males (7.1%) and females (1.9%) was statistically significant (p < 0.05), but not that between Italian (5.0%) and non-Italian patients (4.7%). The differences in anti-HDV seropositivity between the patients with acute (0%) and chronic infections (6.3%), and between the incident (2.5%) and prevalent cases (7.4%), were not statistically significant, but there was a significant difference (p < 0.01) between those with asymptomatic (2.1%) and clinically symptomatic infections (10.3%). Intravenous drug abuse was the main source of infection.
In the catchment area of our hospital, the prevalence of HDV infection does not seem to be due to patients of non-Italian origin, but to Italian patients who are not vaccinated against HBV and who survived the HDV epidemic of the 1970s and 1980s. Nevertheless, the increase in the number of immigrants from non-EU countries in recent years is soon likely to lead to a change in the epidemiology of HDV.
[show abstract][hide abstract] ABSTRACT: An independent clinical assessment was compared with flow cytometry (FCM) and cytomorphology results obtained on 227 cerebrospinal fluids investigated for hematologic malignancy, in a retrospective longitudinal study with a median observation time of 11 months. A combined method assessment (CMA), defining "positive" a sample if at least one method gave "positive" results, was also tested. Eleven out of 55 screening samples and 53 out of 166 follow-up samples resulted positive at clinical evaluation. FCM and CM were concordant with positive clinical assessment in 68.5% and 51.5% of cases, respectively. According to CMA, 10.5% of samples (resulting false negative by either FCM or cytomorphology) were rescued as true positive. FCM retained significantly higher accuracy than cytomorphology (p=0.0065) and 100% sensitivity when at least 220 leukocytes were acquired. CMA accuracy was higher than FCM accuracy and significantly higher than cytomorphology accuracy in the analysis of all samples (p<0.0001), samples from mature B/T cell neoplasms (p=0.0021), and samples drawn after intrathecal treatment (p=0.0001). When acquiring ≤220 leukocytes, FCM accuracy was poor, and combining cytomorphology added statistically significant diagnostic advantage (p=0.0043). Although FCM is the best diagnostic tool for evaluating CSF, morphology seems helpful especially when clinically positive follow-up samples are nearly acellular.
Annals of Hematology 01/2011; 90(7):827-35. · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flow cytometry and cytomorphology results on 92 body cavity fluids [61 effusions and 31 bronchoalveolar lavage fluids (BALF)] from hematologic malignancy were compared with retrospective clinical outcome. We observed double true positive/negative results in 67 cases (73%), and double false negative results in 2 cases (2%). Immunophenotyping accounted for true positive/negative results in 22 out of 23 mismatched cases (25%), and retained significantly higher accuracy than that of cytomorphology especially in effusions and differentiated lymphoma. In BALF analysis, immunophenotyping and cytomorphology sensitivity was 75% and 0%, respectively. Flow cytometry retains the highest accuracy in detecting neoplastic cells in body cavity fluids.
Leukemia research 03/2010; 34(8):1027-34. · 2.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: In patients with ST-segment elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary interventions (PCIs), we sought to correlate circulating CD34+ and CD34+ CD133+ cell levels with clinical and laboratory findings that are known to affect prognosis in such patients.
Although recent studies have focused on circulating adult peripheral blood stem cells in those patients, the possible relations between their circulating number and the various factors that may influence STEMI outcome have never been reported.
In 74 patients with STEMI presenting within 12 h from symptoms onset and treated with successful primary PCI, blood samples were collected before PCI (baseline) and 5-8 days thereafter (post-PCI). Myocardial blush was used as an index of effective myocardial reperfusion. Left ventricular functional recovery was assessed with echocardiography at 4-6 months.
In STEMI patients, baseline CD34+ cell as well as CD34+ CD133+ cell numbers were lower than that of age-matched participants without history of ischemic heart disease. Both cell populations however increased post-PCI (P < 0.0001). A significant inverse relation was found between both CD34+, CD34+ CD133+ cell numbers and age, whereas both cell populations were directly related to myocardial blush grade (CD34+ r = 0.39, P = 0.002; CD34+ CD133+ r = 0.37, P = 0.003). By multiple regression analysis, a significant myocardial blush (grade 2-3) was the only predictor of left ventricular functional recovery (OR 10.77, 95% CI 3.1-22.8).
CD34+ and CD34+ CD133+ cell number rises 5-8 days after STEMI, such increase being hampered by old age and favoured by effective myocardial reperfusion after primary PCI.
Journal of Cardiovascular Medicine 07/2008; 9(7):677-82. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: T acute lymphoblastic leukemia cell lines treated with hexamethylene bisacetamide (HMBA) undergo a delay in cell cycle progression and increase susceptibility to apoptosis, although they never overcome the differentiation block. In accordance with changes in cell cycle and apoptosis, transitory p53 pathway activation commonly occurs. Bcl-2 inhibition further favours the pro-apoptotic effect of HMBA. Notch1 expression is down regulated by reduction of its transcription level. Accordingly, Notch1 protein and transcriptional activity were affected. Even if HMBA generally reduces Notch1 level in T acute lymphoblastic leukemia (T-ALL) cell lines, this does not commonly influence the biological response; in fact all the analysed cell lines, except CEM cells, display no biological effect following DAPT-induced Notch inhibition.
Leukemia Research 06/2008; 32(5):791-7. · 2.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: We studied circulating (C)CD34(+) cells by flow cytometry in 96 patients with myelodysplastic syndromes (MDS) at diagnosis, and in a subset of 35 cases during follow-up. CCD34(+) counts were stratified within both International Prognostic Scoring System (IPSS) and World Health Organization (WHO) categories. Counts >10/microl were associated with poorer leukemia-free survival, a prognostic value for evolution independent from that of WHO, and a higher progression probability within intermediate-risk IPSS and WHO classes. When serial measurements were performed, counts >10/microl more frequently correlated to evolution. Separating newly diagnosed patients on the basis of 10/microl cut-off of circulating CD34(+) cells retains prognostic utility, especially in intermediate-risk MDS.
Leukemia Research 05/2008; 32(11):1715-23. · 2.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate zeta chain and Zap 70 expression in T lymphocytes of patients with laryngeal cancer in relation to surgical treatment.
This study investigated, by dual-color flow cytometry, zeta chain and Zap 70 expression in the circulating T lymphocytes of 13 patients with laryngeal cancer patients before and after surgical treatment.
Patients exhibited a significant lower expression of both zeta chain and Zap 70 compared to healthy normal controls; no statistical differences were observed after surgical treatment.
The results of this investigation seem to indicate that both the zeta chain and the Zap 70 expression in circulating T lymphocytes are down-regulated in patients with laryngeal cancer and that these changes do not immediately return to normal after surgery. Flow cytometry analysis may represent an easy-to-use procedure for monitoring the immune status of patients with laryngeal cancer.
Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 03/2007; 29(1):57-62. · 0.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: External quality assurance (EQA) programs in clinical cell analysis are now a consolidated item of laboratory practice. All the flow cytometric testings with an impact on clinical decision making have been submitted to regular EQA programs during the last 20 years, and this has produced internationally homogeneous guidelines, with a remarkable improvement in result reproducibility.Jean-Luc D'Hautcourt was a pioneer in this field, and his valuable contributions to flow cytometric method standardization and to the dissemination of the educational aspects of EQA programs are recognized. The different methodological approaches undertaken in the United States and Europe are discussed. The educational role of SIHON in the Benelux Countries and of UKNEQAS for Leucocyte Immunophenotyping worldwide is emphasized. Accredited and accreditating EQA programs require an impressive degree of organization and technical knowledge, so that only major international providers can afford such a task nowadays. However, small local studies still provide the necessary stimulus to the continuous improvement of the scientifical aspects of EQA schemes.
Cytometry Part B Clinical Cytometry 02/2007; 72(1):2-7. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.
Cytometry Part B Clinical Cytometry 07/2005; · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model.
Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method.
Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets.
The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.
Cytometry Part B Clinical Cytometry 12/2004; 62(1):1-13. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although flow cytometry is increasingly used as a tool to diagnose hematologic malignancies, the reporting format of acute leukemia immunodiagnosis is still imprecise and sometimes vague, often reflecting old guidelines. Thus, the purpose of the present work was to make the reporting format for the immunological diagnosis of acute leukaemia easy and clear to understand. This work represents part of a more articulated series of technical guidelines that the Italian Society for Cytometry (GIC) is currently processing. Thirteen separate recommendations, covering all aspects of an acute leukemia cytometry report, are listed. According to our suggestions, the report must contain clear statements about: 1. demographic identification of patient; 2. identification of the hospital or division sending the sample; 3. type of specimen (bone marrow aspirate, peripheral blood, other biological fluids); 4. timing of observation (first diagnosis or follow-up); 5. diagnostic hypothesis made by the sender; 6. list of antigens and type of immunofluorescence analysis carried out; 7. absolute number of cells in the sample; 8. quality of the sample, in terms of viability; 9. general description of the gating procedure; 10. immunophenotype of blast cells; 11. description of cells surrounding blasts; 12. diagnostic conclusions; 13. definition of an antigen panel (when applicable) for the detection of minimal residual disease. As an example of a final report we present a case of acute myeloid leukaemia with t(8;21) translocation; in filling this report, we followed all the 13 points of the checklist described in the paper.
[show abstract][hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) cells could be undetectable by flow cytometry or polymerase chain reaction after sequential treatment with fludarabine and Campath-1H. Concern has been raised regarding the ability to mobilize sufficient peripheral blood progenitor cells (PBPCs) for autografting after purine analogues, and there are few data about PBPC collection after Campath-1H. In all, 16 CLL patients responding to sequential chemo-immunotherapy entered the study. In 10, mobilization regimen consisted of granulocyte colony-stimulating factor (G-CSF) 5-10 microg/kg/die. Patients failing mobilization or not achieving the target of 2.5 x 10(6) CD34+ cells/kg underwent a second attempt using intermediate-dose (ID) Ara-C, 800 mg/m(2) every 12 h for six doses+G-CSF. PBPC collection after G-CSF alone was successful in two out of 10 patients. An adequate number of CD34+ cells were collected after ID Ara-C+G-CSF in eight patients failing the mobilization with G-CSF alone and in five out of six who did not receive G-CSF before. Greater yields of PBPCs were collected with Ara-C+G-CSF compared with G-CSF alone (13.8 vs 3.3). The extrahematologic toxicity was manageable. In conclusion, PBPC collection is feasible in CLL patients treated with sequential therapy including fludarabine and Campath-1H. Excellent yields were obtained in 92.8% of patients primed with ID Ara-C+G-CSF.
[show abstract][hide abstract] ABSTRACT: Inexpensive antiretroviral therapy (ART) might soon be available to treat human immunodeficiency virus (HIV) infections in resource-restricted areas of the globe. The number of CD4 T-cells in the blood is the single most important laboratory parameter to select patients for therapy at the right time and to monitor the effect of ART. The question is asked whether flow cytometry is adaptable to change from an expensive and complicated scientific analytical instrumentation to become a practical diagnostic tool that can be widely operated. Recent studies indicate that a branch of clinical/practical flow cytometry is gaining a new identity by a confident simplification and improvement of clinical protocols. These new observations can be marshaled into three new areas of development. First, the changes in the choices of reagents and staining protocols have been initiated by the preferred use of primary immunological gating during the flow-cytometric analysis, exploiting the exquisite discriminating power of antibodies. The recent National Institutes of Health (NIH) and Centers for Disease Control and Prevention (CDC) guidelines introduced CD45-assisted protocols that are also at the heart of the PanLeucogating protocol that revitalized the testing of absolute CD4 counts on double-platform. With CD45 staining the leucocyte populations can be reliably studied in aging samples. It is now also documented that minimal technology, that is primary gating with CD4 (or CD8) antibodies, provides excellent CD4 (or CD8) T-cell counts. Consequently the optimal affordable protocol uses CD45 and CD4 antibodies in combination. The second area of development is the absolute counting facility on flow cytometers. Most frequently, microbeads are added to blood to define the sample volume in instruments that are not equipped with a volumetric microsyringe. The beads are, however, expensive, and the utilization of the stable flow rate of the excellent instruments might soon provide (G. Janossy).
Clinical and Applied Immunology Reviews 01/2003; 4:91-107.
[show abstract][hide abstract] ABSTRACT: Recent observations suggested that targeted monoclonal antibodies might be best employed in lymphoid malignancies under conditions of minimal residual disease. This prompted us to investigate the role of Campath-1H as treatment for patients with chronic lymphocytic leukemia (CLL) in whom fludarabine (FAMP) had produced a marked disease debulking with persistence of bone marrow (BM) infiltration or a complete remission (CR) without the disappearance of the molecular aberration (IgH monoclonal expression). As intravenous Campath-1H is almost invariably associated with reactions, sometimes of WHO grade 3-4, we adopted the subcutaneous route of administration, which proved to induce rare and mild adverse reactions but had comparable efficacy. DESIGN AND METHODS. Nine patients (7 males, 2 females) with a median age of 55 years (range 41-61) who responded to FAMP (1 had a CR, 5 a nodular partial remission [PRN], and 3 a partial remission [PR]), according to NCI Working Group Criteria, received subcutaneous Campath-1H, three times a week for 6 weeks in escalating doses up to 10 mg. Monoclonal rearrangement of IgH was present in all patients before immunotherapy. Patients received acyclovir and cotrimoxazole as infection prophylaxis. Granulocyte colony-stimulating factor (G-CSF), at the dosage of 5-10 microg/kg/die, or intermediate-dose Ara-C (800 mg/m(2)/q 12h x 6 doses), was administered to obtain peripheral blood stem cell (PBSC) mobilization.
All patients were evaluable for response. Five patients, 2 in PR and 3 in PRN after FAMP treatment, reached a CR. Three patients, one in PR, one in PRN and one in CR, converted to a molecular remission. In four out of seven patients PBSC harvesting was successful; more than 2.5 x 10(6) cells/kg were collected from all these patients. Collection was polyclonal for IgH gene rearrangement in three cases. One patient has been transplanted after cyclophosphamide and total body irradiation as conditioning regimen, without complications and with rapid hemopoietic engraftment. All patients were evaluable for toxicity. A WHO grade 1-2 skin reaction was observed in 5 patients at the site of injection. No infectious episodes were recorded. Two out of three patients presenting cytomegalovirus reactivation, without pneumonia, were successfully treated with oral gancyclovir.
Subcutaneous Campath-1H administered to CLL patients with residual BM disease after FAMP proved to be safe and effective. Of nine patients, three obtained a molecular CR and five converted into a morphologic and immunophenotypic CR. In four of seven patients submitted to PBSC mobilization, this treatment also allowed a harvest uncontaminated by CD5/CD19 double-positive CLL cells, which was polyclonal for IgH gene rearrangement in three cases.
[show abstract][hide abstract] ABSTRACT: We addressed the definition of limits of error of %CD4+ and CD4+ counts (AbsCD4+) typical of laboratories of excellence, as well as the grading of laboratories based on the decision to take these limits as boundaries of unacceptable data.
We studied the 99.9% confidence intervals of the means of 24 human immunodeficiency virus (HIV)+ and HIV- blood samples analyzed by 18 laboratories of the Liguria Region Quality Assessment Program (Liguria Region QALI). Regression equations of lower (L1) and upper (L2) confidence limits over the means of data cleared of unusual results were used to interpolate limits of error for mean values in the tested range.
L1 and L2 were symmetric around the mean and a single absolute difference (Abs Res) between the limits and the mean was found. Abs Res significantly increased over mean values (P = 0.0005 for %CD4+, P < 0.0001 for AbsCD4+). Limits were compatible with errors shown with blind replicates. Unacceptable results, outside the limits, accounted for 25% and 30% of %CD4+ and for 18% and 35% AbsCD4+ in the Liguria Region QALI and in the Piemonte Region QA Program, respectively. Limits interpolated over the median showed a similar grading. A comparable fraction of unacceptable data was also found with the method used in the U.K. National External Quality Assessment Scheme (NEQAS) immune monitoring scheme.
We propose the general use of these regression equations to determine bounds for unacceptable data in proficiency testing and to identify laboratories of excellence.