Quantification of neutrophil and monocyte CD64 expression: a predictive biomarker for active tuberculosis.

SETTING: QuantiFERON TB assay (QFT) is used to screen tuberculosis (TB) infection, but it cannot distinguish active TB from latent TB infection (LTBI).OBJECTIVE: To evaluate the quantitative expression of the high-affinity FCgamma receptor I (CD64) on neutrophils (NE) and monocytes (MO) in peripheral blood using flow cytometry, measured in antibody binding capacity (ABC) units as a predictive biomarker of TB.DESIGN: Fifty-two patients were enrolled (45 QFT-positive and 7 QFT-indeterminate). Cultures and molecular analyses were performed.RESULTS: Of the 45 QFT-positive patients, 29 were culture-positive (active TB) and 16 were negative (LTBI). The median NE CD64 ABC and MO CD64 ABC expression was significantly higher (P < 0.001) in culture-positive patients. The NE CD64 and MO CD64 area under the receiver operating characteristic curve values were respectively 0.948 (95%CI 0.838-0.992) and 0.989 (0.901-1.000). By setting the cut-off NE CD64 value at >2400 ABC or MO CD64 value >25 800 the assay sensitivity increased to 95.5% with 100% specificity and 100% positive predictive value. In the QFT-indeterminate group, five culture-positive cases had NE CD64 >2400 ABC or MO CD64 value >25 800; two culture-negative cases had lower values.CONCLUSION: The CD64 quantitative expression on peripheral blood cells may be used as a predictive biomarker for active TB.

Epidemiology of hepatitis D virus (HDV) infection in an urban area of northern Italy.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Hexamethylene bisacetamide inhibits malignant phenotype in T-ALL cell lines.

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.

Panleucogating as an accurate and affordable flow cytometric protocol to analyse lymphocyte subsets among HIV-positive patients on HAART treatment in Mozambique.

In Africa tens of millions of people are HIV+. Prevention alone is not effective, and needs to be coupled with anti-retroviral treatment (HAART). Laboratory tests as CD4+ T cell count are fundamental tools in HIV disease monitoring, but they require costly equipment, reagents and specialised manpower. The goal of this study was to minimise and optimise the reagents needed for a reliable routine CD4+ cell count in a resource-poor setting (Mozambique). Panleucogating protocol (PLG), requires two antibodies only, CD45 and CD4, or three if CD8 is requested for special clinical reasons. PLG was compared with the current protocol used in two Mozambique hospitals, based on FSC/SSC gating and CD3/CD4/CD8 staining. 189 samples from HIV+ patients, included in the Community of Sant'Egidio's DREAM program and on HAART were processed with both protocols. The overall correlation of the lymphocyte subsets measurements was satisfactory, with r2 always >0.96. The Bland-Altman analysis of CD4+ cell count showed a negative bias when CD4+ cells were <15%, due to the imprecise FSC/SSC gating used previously. When CD4+ cells were >15% the negative bias tended to zero, further confirming the better quality of the PLG gating strategy. Two- or three color PLG protocol, in double platform, currently seems the most accurate and affordable method to monitor CD4+ lymphocytes and CD4/CD8 ratio by flow cytometry in resource-poor medical settings.

Determination of telomere length by flow-fluorescence in situ hybridization in Down's syndrome patients.

A new method for measuring telomere length in a population of Down's syndrome patients aged 18-60 years old is presented. The method is based on flow cytometry and quantitative fluorescence in situ hybridization (flow-FISH) on whole cells. At least three methods for measuring the length of telomere repeats have been described: (i) Southern blot analysis, and quantitative FISH using either (ii) digital fluorescence microscopy (Q-FISH) or (iii) flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas flow-FISH needed relatively few cells (1.5-2.5 x 106) and could be completed in 24-48 h. The method can be used to rapidly determine telomere length in subsets of nucleated blood cells from patients with age-related diseases such as Down's syndrome, Alzheimer's disease and Werner syndrome.

Successful CD34+ cell mobilization by intermediate-dose Ara-C in chronic lymphocytic leukemia patients treated with sequential fludarabine and Campath-1H.

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.

Diagnostic and prognostic value of flow cytometric immunophenotyping in malignant hematological diseases.

Flow cytometry is a diagnostic cell analysis technique with ever increasing applications in modern hematological practice. To date immunophenotyping of clonal hematological diseases represents one of the primary clinical applications of flow cytometry. Immunophenotyping of abnormal cells is now considered a fundamental tool to establish the cell lineage assignment and to obtain a more precise identification of the various cell subtypes. A number of observations have emerged showing strong association between specific immunophenotypes and genetic recurrent abnormalities underlying the malignant transformation, with prognostic value.

Effect of familial hypertension on glomerular hemodynamics and tubulo-glomerular feedback after uninephrectomy.

Familial hypertension, glomerular hemodynamic alterations, and dysregulation of tubulo-glomerular feedback (TGFB) have all been associated with the development of chronic renal failure. In the present study we evaluated renal and glomerular hemodynamics and TGFB responses in healthy kidney donors either with or without familial hypertension, before and after nephrectomy. Para-amino-hippurate plasma clearance (CPAH) and inulin plasma clearance (CInu) were measured in 15 kidney donors before and 1 year after nephrectomy. All subjects were normotensive and were kept in a sodium-replete state. Both clearances were measured after 40 min of constant infusion of PAH and Inu, as well as 20, 30, 50, and 60 min after the intravenous administration of acetazolamide (5 mg/kg). Glomerular hemodynamics were calculated by means of the Gomez formulae. Nephrectomy caused the expected decreases in CPAH and CInu and increase in the filtration fraction (all P < .0001). The decrease in renal resistances of the remaining kidney was greater at the afferent (-24%, P = .0075) than at the efferent arteriolar level (-17%, P < .0001). The TGFB activation was not altered by nephrectomy or by familial hypertension. Effective renal plasma flow (ERPF) decrease after TGFB activation appeared earlier than glomerular filtration rate (GFR) decrease before (P = .01), but not after, nephrectomy (P = .48). The presence of familial hypertension was associated with increased glomerular pressure (P = .0004). This study suggests that uninephrectomy in healthy human subjects causes a greater decrease in afferent arteriolar resistances, but that TGFB responses are not quantitatively altered. Familial hypertension is associated with a tendency toward higher glomerular pressures.

The "vanishing counting bead" phenomenon: effect on absolute CD34+ cell counting in phosphate-buffered saline-diluted leukapheresis samples.

BACKGROUND: Using a single-platform protocol to count absolute CD34+ hematopoietic precursor cell (HPC) levels with different reference microbeads, we recorded occasionally artifactually high CD34+ HPC counts in some leukapheresis bags, whereas dual-platform calculations were always consistent. Abnormal countings were observed only when phosphate-buffered saline (PBS)-diluted leukapheresis samples were vortexed before analysis. A large series of blood samples analyzed similarly for CD34+ and CD4+ absolute counts did not show any sample or vortexing effect. With the volumetric absolute counting cytometer Partec-PAS, lower counts were also observed when different reference beads were vortexed before the instrument checking procedures. The counting abnormality was caused by a drop in microbead concentration (the "vanishing bead phenomenon"). This phenomenon reduced the total and relative bead event number in experimental and routine samples and in calibration procedures. This altered the bead denominator used to calculate absolute CD34+ HPC levels and it also reduced the concentration of standard calibration beads. METHODS: Using the Partec-PAS to measure volumetrically the actual bead concentration, we studied the vanishing bead phenomenon. Different types of counting and reference microbeads were resuspended in media with or without proteins or cells. Replicates were submitted either to gentle manual mixing or to vortexing before counting. RESULTS: Vortex agitation almost invariably induced the vanishing bead phenomenon when beads were resuspended in saline media or when an insufficient protein concentration was present, such as in diluted leukapheresis samples. Different bead types showed various degrees of sensitivity to vortexing. The bead disappearance was not caused by bubble formation or disruption. The addition of small amounts of protein completely prevented the vanishing bead phenomenon. The causative effect of the electrostatic charging of tube induced by vortexing is hypothesized. CONCLUSIONS: Sample suspensions containing counting beads for single-platform analysis must be resuspended in media with protein supplements to prevent the vanishing bead phenomenon and to ensure accurate counting.

Enumeration of absolute cell counts using immunophenotypic techniques.

Absolute counting of cells or cell subsets has a number of significant clinical applications: monitoring the disease status of HIV-infected patients, enumerating residual white blood cells in leukoreduced blood products, and assessing immunodeficiency in a variety of situations. The single-platform method (flow cytometry alone) has emerged as the method of choice for absolute cell enumeration. This technology counts only the cells of interest in a precisely determined blood volume. Exact cell identification is accomplished by a logical electronic gating algorithm capable of identifying lineage-specific immunofluorescent markers. Exclusion of unwanted cells is automatic. This extensive and detailed unit presents protocols for both volumetric and flow-rate determination of residual white blood cells and of leukocyte subsets.

Incidence and clinical characteristics of posttransplant lymphoproliferative disorders: report from a single center.

In the period 1973-1998, among 2139 allograft recipients treated with standard immunosuppression, posttransplant lymphoproliferative disorders (PTLD) developed in 19 patients (0.9%): one plasmacytic hyperplasia, two polymorphic PTLD, one myeloma, and 15 lymphomas. PTLD developed 1 year after transplantation (tx) in 14 patients. Five patients were diagnosed at autopsy, 2 were lost to follow up, 3 died before therapy could be instituted, and 1 patient has just started chemotherapy. Of the 8 evaluable patients, 2 received acyclovir and are alive in complete remission (CR) and 6 received chemotherapy +/- surgery. Of these 6, 4 died of lymphoma and/or infection, 1 died of unrelated causes in CR, and 1 is alive in CR. PTLD is a severe complication of tx, usually running an aggressive course which may preclude prompt diagnosis and treatment. Nevertheless, therapy is feasible and must be tailored on the histologic subtype. Seventy-four percent of patients were diagnosed with late-onset PTLD stressing the need for long-term follow up.

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions.

BACKGROUND: At present, immunophenotyping of hematological malignancies represents one of the most relevant clinical applications of flow cytometry. In recent years, its use has extended from clinical research to diagnostic laboratories. The aim of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact as well as to highlight its potential future applications. METHODS: The currently available information, including that provided by different international consensus groups on the phenotypic characterization of hematologic malignancies, was reviewed. Additionally, recent reports on the immunophenotypic analysis of hematological malignancies published in hematology, oncology, pathology, immunology, and cell biology journals were also analyzed. RESULTS: A careful review of the literature showed that in spite of the well-established utility of immunophenotyping for the diagnosis, classification, prognostic stratification, and monitoring of hematological malignancies, only a small part of the information on the immunophenotypic characteristics of pathological hemopoietic cells has been used routinely. Specific and sensitive identification of neoplastic cells and their accurate enumeration and phenotypic characterization represent the major aims of these procedures. Similarities between leukemic and healthy cells allow the establishment of the lineage and maturation stage of the pathologic cells, this information being of great utility for the diagnosis, classification, and prognostic evaluation of different subtypes of hematological malignancies. On the other hand, the phenotypic aberrations displayed by leukemic cells could allow the selection of cases carrying specific genetic abnormalities in which further confirmatory molecular studies will be performed. CONCLUSIONS: The information provided by the flow cytometric immunophenotyping of hematological malignancies is of great clinical utility, with a major challenge for the near future being the standardization of technical procedures, data interpretation, and reporting.

Increment of nonreceptor tyrosine kinase Arg RNA as evaluated by semiquantitative RT-PCR in granulocyte and macrophage-like differentiation of HL-60 cells.

The products of the human Arg gene and human, mouse, Drosophila, and nematode Abl genes characterize the Abelson family of nonreceptor tyrosine protein kinase. The Arg gene, expressed as a 12-kb transcript, codes a protein highly related to c-abl in the tyrosine kinase, SH2, and SH3 domains, and both proteins have a myristoylated isoform. The C-terminal domains of Arg and c-abl, poorly similar to each other, may account for their different functions. Arg is cytoplasmic, c-abl also has nuclear localization, and their products have different transforming activity. To gain insight about the role of Arg in myeloid differentiation we investigated Arg gene expression in HL-60 cells differentiated with all-trans retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. With a semiquantitative reverse transcriptase-polymerase chain reaction assay it was evident that the Arg transcript level in HL-60 cells differentiated toward granulocyte and macrophage-like lineage was, respectively, 3.5- and 2.8-fold the Arg level evidenced in undifferentiated HL-60 cells. In the HL-60 cells, under the same differentiating conditions, the c-abl RNA level did not change significantly, showing that Arg and c-abl responded in a different way to the inducers of differentiation used.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis.

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.

Effect of n-3 polyunsaturated fatty acids on cyclosporine pharmacokinetics in kidney graft recipients: a randomized placebo-controlled study.

Highly concentrated marine polyunsaturated fatty acids (n-3 PUFA), affecting the lipids and lipophilic drugs metabolism, can interfere with cyclosporine (CyA) pharmacokinetics. This prospective, randomized and placebo-controlled, double-blind study involved 42 kidney graft recipients. From day +1, 21 pts (E) received 6 g n-3 PUFA (85% EPA + DHA, Esapent, Pharmacia) and 21 pts (P) received placebo (olive oil), both reduced to 3 g from day +30 on. A quadruple immunosuppressive regimen was employed. Plasma creatinine, lipids and CyA pharmacokinetics were investigated 1, 3, 6, 9 and 12 months after graft. The two groups were comparable for age, weight, M/F ratio, hypertension prevalence and baseline lipids. Active treatment did not affect total and HDL-cholesterol, but significantly lowered triglycerides (E:120 +/- 12 vs P:166 +/- 21 mg/dl, p < 0.0001). At one year, E pts had lower creatinine than P (1.26 +/- 0.06 vs. 1.88 +/- 0.2 mg/dl, p < 0.05), comparable CyA dosage, and a larger CyA area under the curve (AUC) (n.s.), with a higher blood peak level (Cmax) (p < 0.04) and less variance in time to peak (n.s.). The larger AUC in the E group at all intervals and the better pattern of plasma creatinine, with no rise in blood pressure, provided evidence of better CyA absorption and metabolism in n-3 PUFA supplemented kidney graft recipients.