bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kolmogorov–Smirnov statistical test for analysis of ZAP‐70 expression in B‐CLL, compared with quantitative PCR and IgVH mutation status
Authors: Van Bockstaele Femke , Janssens Ann , Piette Anne , Callewaert Filip , Pede Valerie , Offner Fritz , Verhasselt Bruno , Philippé Jan 
Published: August 2006

AbstractBackground:ZAP‐70 has been proposed as a surrogate marker for immunoglobulin heavy‐chain variable region (IgVH) mutation status, which is known as a prognostic marker in B‐cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP‐70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov–Smirnov (KS) statistical test to make analysis more straightforward.Methods:We examined ZAP‐70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764‐1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver‐operating‐characteristics (ROC)‐curve analyses were performed to determine the optimal cut‐off values for ZAP‐70 measured by the two approaches. ZAP‐70 protein expression was compared with ZAP‐70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgVH mutation status.Results:Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgVH mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo‐method.Conclusions:The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP‐70 expression in CLL. © 2006 International Society for Analytical Cytology
Discordant results of flow cytometric ZAP‐70 expression status in B‐CLL samples if different gating strategies are applied
Authors: Wilhelm Christian , Neubauer Andreas , Brendel Cornelia 
Published: August 2006

AbstractBackground:Recent studies have identified ZAP‐70 expression status as an excellent prognostic parameter in chronic lymphocytic leukemia (CLL). ZAP‐70 expression can be determined by direct antibody staining followed by flow cytometric analysis. However, there are currently several different gating strategies for analysis. We compared those strategies for ZAP‐70 expression analysis.Methods:One hundred and one patients with B‐CLL were analyzed employing a directly labeled alexa‐fluor‐488‐ZAP‐70‐antibody. In 27 cases, we additionally measured and analyzed ZAP‐70 expression, together with healthy controls as described previously.Results:Applying either T‐/NK‐cell isotype or healthy control analysis strategies on patient samples that were processed in parallel revealed discrepant results in 48% (12/25) of all cases. Taken together with the 74 B‐CLL patients, who were analyzed with regard to average reference values, disconcordant results were obtained in 58% of the samples. We demonstrate that high variances in ZAP‐70 T‐/NK‐cell staining within B‐CLL patients, paired with a close proximity of ZAP‐70 B‐cell values to the suggested cut‐off levels, may lead to interpretation difficulties of ZAP‐70 status.Conclusions:We conclude that different gating strategies for determining flow cytometric ZAP‐70 expression status produce highly discordant results. Further standardization is required before ZAP‐70 can be used as a reliable prognostic parameter in immunophenotyping of B‐CLL. © 2006 International Society for Analytical Cytology
What is ZAP‐70?
Authors: Béné Marie Christine 
Published: August 2006

AbstractZap‐70, a crucial molecule for the selective activation of T cells, through its interaction with the ζ chain of the TCR/CD3 complex, is a tyrosine kinase. This well studied molecule has gained a renewed interest upon the demonstration of its transduction and expression in B cells from patients with chronic lymphocytic leukemia. Here the major characteristics of this cytosolic protein are reviewed, as well as its expression in various cell types and some indications about its detection. © 2006 International Society for Analytical Cytology
Use of a blocking antibody method for the flow cytometric measurement of ZAP‐70 in B‐CLL
Authors: Shenkin Mark , Maiese Russell 
Published: August 2006

AbstractBackground:In this study we developed a method to measure the amount of ZAP‐70 [zeta accessory protein] in B‐CLL cells without relying on the ZAP‐70 expression of patient B or T cells to normalize fluorescence intensity.Methods:B‐CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP‐70 antigen was blocked in one tube with unlabeled antibody to ZAP‐70 [clone 1E7.2]. Zap‐70‐PE was then added to this tube. ZAP‐70‐PE was added to a second tube without unlabeled antibody to ZAP‐70. The mean fluorescence intensity of the ZAP‐70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP‐70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non‐specific ZAP‐70 fluorescence in the B‐CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP‐70 fluorescence intensity in the tube with unlabeled antibody to ZAP‐70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP‐70 cells in the tube without unlabeled antibody to ZAP‐70. The brighter the ZAP‐70 fluorescence above the non‐specific background, the higher the %POS.Results:Due to the varying amount of non‐specific staining between patient B‐CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP‐70 in B‐CLL cells than other methods, which use the ratio of B‐CLL fluorescence to normal B or T‐cell fluorescence. Using this improved method, ZAP‐70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP‐70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut‐off value lower than previous values published, due to exclusion of non‐specific staining. Both cut‐offs were based upon patient specimen distribution profiling.Conclusions:Use of a blocking antibody resulted in a robust, reproducible clinical B‐CLL assay that is not influenced by the need to measure the amount of ZAP‐70 in other cells. ZAP‐70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. © 2006 International Society for Analytical Cytology
Flow cytometry for ZAP‐70: New colors for chronic lymphocytic leukemia
Authors: Wiestner Adrian 
Published: August 2006

AbstractZAP‐70 has become one of the most studied prognostic markers in Chronic Lymphocytic Leukemia (CLL). ZAP‐70 is remarkable in many ways: ZAP‐70 has been identified as the best discriminating gene between prognostically distinct CLL subtypes using large scale gene expression profiling; ZAP‐70 has been shown to enhance signal transduction in CLL B‐cells and therefore could contribute to disease progression; and ZAP‐70 is one of the rare examples of an intracellular target considered for clinical flow cytometry. This issue attests to the enormous effort and the steady progress made in overcoming technical challenges of testing for ZAP‐70 expression and sets the foundation for a successful translation of this important marker into clinical practice. Despite the best effort, one will likely have to accept that not all cases can be clearly assigned to one or the other group, given that ZAP‐70 expression between CLL patients falls along a continuum from absent to high. Nevertheless, ZAP‐70 expression could become a key parameter to guide patients towards risk adapted treatment strategies in prospective clinical trials. © 2006 International Society for Analytical Cytology
ZAP‐70 expression in B‐cell chronic lymphocytic leukemia: Evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgVH mutations
Authors: Zucchetto Antonella , Bomben Riccardo , Bo Michele Dal , Nanni Paola , Bulian Pietro , Rossi Francesca Maria , Del Principe Maria Ilaria , Santini Simone , Del Poeta Giovanni , Degan Massimo , Gattei Valter 
Published: August 2006

AbstractBackground:Expression of T cell specific zeta‐associated protein 70 (ZAP‐70) by B‐cell chronic lymphocytic leukemia (B‐CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgVH) mutations, although a standardization of the cytometric protocol is still lacking.Methods:Flow cytometric analyses for ZAP‐70 were performed in peripheral blood samples from 145 B‐CLL (124 with IgVH mutations) by a standard three‐color protocol. Identification of ZAP‐70+ cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed.Results:While 86/145 cases were concordant as for ZAP‐70 expression according to the two methods (ISO+TNK+ or ISO−TNK−), 59/145 cases had discordant ZAP‐70 expression, mainly (56/59) showing a ISO+TNK− profile. These latter cases express higher levels of ZAP‐70 in their normal T cell component. Moreover, discordant ISO+TNK− cases had a IgVH gene mutation profile similar to that of concordantly positive cases and different from ZAP‐70 concordantly negative B‐CLL.Conclusion:Analysis of ZAP‐70 expression by B‐CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP‐70 by residual T cells and yields a better correlation with IgVH gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut‐off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP‐70 expression, as determined according to the ISO and TNK methods, is still needed. © 2006 International Society for Analytical Cytology
Comparison of bone marrow and peripheral blood ZAP‐70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia
Authors: Sheridan Rachel , Mounajjed Taofic , Ehrmann Diane E. , Hurtubise Paul E. , Schrager Jeffrey A. 
Published: August 2006

AbstractBackground:The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP‐70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP‐70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP‐70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP‐70 expression in CLL patients.Methods:We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP‐70 positivity as greater than 30% of the CD19+ B‐cells above the isotype control value that coexpress ZAP‐70. Statistical analyses were performed using the Fisher exact test and student t‐test.Results:A significantly greater number of bone marrow specimens were positive for ZAP‐70 when compared with the number of peripheral blood specimens. Of all the ZAP‐70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP‐70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP‐70 positive in the bone marrow but ZAP‐70 negative in the peripheral blood.Conclusions:These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP‐70 results from either specimen type, it is prudent to further examine this observation. © 2006 International Society for Analytical Cytology
ZAP‐70 expression is low in normal precursor B cells or hematogones
Authors: Davis Bruce H. , Schwartz Michael 
Published: August 2006

AbstractBackground:Zeta‐associated protein (ZAP‐70) expression has been associated with a less favorable prognosis in chronic lymphocytic leukemia (CLL). The role of ZAP‐70 in immature B‐cells is not well understood. Immature or precursor B lymphocytes (hematogones) often occur in clinical samples and coexpress CD10, CD38, and CD19 lacking surface immunoglobulin expression. The present study was carried out in an attempt to further elucidate the role of ZAP‐70 in the spectrum of B‐cells seen in clinical specimens.Methods:ZAP‐70 expression was evaluated on 25 patient samples that expressed greater than 2% of CD10+/CD38bright/CD19+ coexpression during routine evaluation. In our sample set, the hematogone expression ranged from 2 to 18% of total leukocytes and occurred in a variety of conditions, including CLL, NHL, AML, MDS, Hodgkins Disease, and Multiple Myeloma. The method of ZAP‐70 detection was that routinely applied to our clinical testing of CLL samples supplemented with the determination of ZAP‐70 levels in the CD38bright/CD19+ coexpressing cells.Results:In all cases ZAP‐70 was not expressed on the CD5 negative, CD38/CD19 coexpressing precusor B cells. In all cases hematogones were found to not express significant levels of ZAP‐70.Conclusion:The presence of hematogones in clinical samples should be recognized so as to not adversely influence prognostic studies of ZAP‐70 or CD38 in CLL. © 2006 International Society for Analytical Cytology
Variations in the detection of ZAP‐70 in chronic lymphocytic leukemia: Comparison with IgVH mutation analysis
Authors: Sheikholeslami M. R. , Jilani I. , Keating M. , Uyeji J. , Chen K. , Kantarjian H. , O'Brien S. , Giles F. , Albitar M. 
Published: August 2006

AbstractLack of immunoglobulin heavy chain genes (IgVH) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta‐chain (T‐cell receptor) associated protein kinase 70 kDa (ZAP‐70) has been reported to be a surrogate marker for IgVH mutation status, and its expression in leukemic cells correlates with unmutated IgVH. However, ZAP‐70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP‐70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP‐70 by comparing results with clinical outcome and the mutation status of the IgVH. Using stored samples, we show significant correlation between ZAP‐70 expression and clinical outcome as well as IgVH mutation at a cut‐off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgVH mutation status when 15% cut‐off was used. In these samples, cut‐point of 10% correlated with the IgVH mutation (P = 0.0001). This data suggests that although ZAP‐70 positivity correlates with IgVH mutation status and survival, variations in sample handling and preparation may influence results. We show that IgVH mutation results, unlike ZAP‐70 remain correlated with CD38 expression and β‐2 microglobulin in shipped samples, and ZAP‐70 testing should not be used as the sole criterion for stratifying patients for therapy. © 2006 International Society for Analytical Cytology
A standardized ZAP‐70 assay—Lessons learned in the trenches
Authors: Shults Keith E. , Miller David T. , Davis Bruce H. , Flye Leanne , Hobbs Leigh Ann , Stelzer Gregory T. 
Published: August 2006

AbstractBackground:The disease of chronic lymphocytic leukemia (CLL) has been shown to exhibit varying clinical outcomes based on reported laboratory parameters. One of these parameters involves the measurement of the protein levels of zeta‐associated protein (ZAP‐70) in CLL cells. A standardized assay has not yet reached consensus in the clinical cytometry community.Methods:We developed a system using the 8‐peak Rainbow beads as our fluorescence calibrator along with a fixed cell control. Using a panel of CD19‐PE, CD5‐FITC, and ZAP‐70‐Alexa 647, we stained normal whole blood, and blood and bone marrow from patients with CLL to determine the level of ZAP‐70 expression in T‐cell, B‐cell, and CLL‐cell populations. ZAP‐70 expression was reported in molecules equivalent fluorescence (MEFL) based on the calibration of the flow cytometer with the 8‐peak Rainbow beads.Results:Daily assay performance as well as operating MEFL defined ranges for ZAP‐70 detection in CLL were developed. A rank‐order, nonparametric approach to reference ranges was used to assign a cutoff for “negative” as well as ranges for “intermediate” and “positive” staining using T and B cells from a pool of 50 normal subjects, and CLL cells from 395 patients. The assay was validated in a multi‐institutional study and has demonstrated correlation with published techniques. Since its initial development, the assay has been implemented at two additional laboratory sites and has been shown to produce reproducible, correlated data at all sites.Conclusions:Strict adherence to standardization can yield an assay that is predictable, reliable, and reproducible as well as capable of multisite implementation. The Rainbow beads provide a common platform for system calibration. The fixed cell culture controls provide a common target to test antibody. The final level of control tests the sensitivity of ZAP‐70 detection in a normal peripheral blood sample stained along with the submitted CLL patients. The acceptance/rejection of test results must meet all three levels of control before patient results are reported. © 2006 International Society for Analytical Cytology
Induction of IgE and allergic‐type responses in fur mite‐infested mice
Authors: Pochanke Veronika , Hatak Sarah , Hengartner Hans , Zinkernagel Rolf M. , McCoy Kathy D. 
Published: August 2006

AbstractHigh serum IgE levels are characteristic of allergic diseases and immune responses to nematode parasites. A murine allergy model based on infestation with the fur mites Myocoptes musculinus and Myobia musculi was investigated. Analysis of mite infestation in various knockout mice revealed that IgE production in response to these ectoparasites was dependent on T cells, IL‐4, and CD40L. Secretion of IL‐4 by CD4+ T cells obtained from peripheral LN draining mite‐infested skin sites was increased with progressing mite infestation and correlated with the serum IgE induction. A time course analysis of the mRNA expression of switched IgE, activation‐induced cytidine deaminase (AID), and ϵ germ‐line transcripts (ϵGLT) suggested that switching to IgE in response to fur mites occurred initially in skin‐draining LN. In addition, mite infestation induced mast cell degranulation in the skin as well as mast cell infiltration into skin‐draining LN. Analysis of the immune response generated in mite‐infested mice is a valuable model for the investigation of allergic disorders and provides information for better understanding of mechanisms involved in IgE induction and regulation in a physiological way of allergen‐exposure resembling atopic sensitization in humans.
Binding Constant of VIVO to Transferrin
Authors: Kiss Tamás , Jakusch Tamás , Bouhsina Saâd , Sakurai Hiromu , Enyedy Éva Anna 
Published: August 2006

AbstractThe interactions of bis‐VIVO‐1,2‐dimethyl‐3‐hydroxy‐4(1H)‐pyridinone (DHP) with apotransferrin (apoTF) were followed by means of electron paramagnetic resonance spectroscopy (EPR) at room temperature and at liquid nitrogen temperature. Partial ligand displacement between DHP and apoTF was observed, and the reaction was used to determine the binding constant of VIVO to apoTF. A value of log K = 14.3 ± 0.6 was obtained for the binding of the first VIVO to apoTF. In order to confirm the validity of the binding constant, UV spectroscopy was used to monitor the displacement reaction between VIVO–apoTF and nitrilotriacetic acid (NTA). The difference between the binding constants obtained by the two different methods (ca. one log unit) was explained in terms of the possible ternary complex formation between VIVO–apoTF and the displacing ligand. The binding constants obtained exhibit a reasonably good agreement with the linear free energy relationship (LFER) estimation. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
Twisted Intercalating Nucleic Acids – Intercalator Influence on Parallel Triplex Stabilities
Authors: Filichev Vyacheslav V. , Gaber Hatem , Olsen Thomas R. , Jørgensen Per T. , Jessen Carsten H. , Pedersen Erik B. 
Published: August 2006

AbstractPhosphoramidites of several new twisted intercalating nucleic acid (TINA) monomers and the previously discovered (R)‐1‐O‐[4‐(1‐pyrenylethynyl)phenylmethyl]glycerol (1) were synthesized and used in DNA synthesis. Stabilization of Hoogsteen‐type triplexes was observed in cases of insertion of the novel (R)‐1‐O‐[3‐(naphthalen‐1‐ylethynyl)phenylmethyl]glycerol (2) as a bulge into homopyrimidine oligodeoxynucleotides (ONs), whereas phenylethynyl and 4‐(biphenylylethynyl) derivatives of TINAs resulted in destabilization of parallel triplexes relative to the wild‐type triplex. It was concluded that TINA monomers should possess at least two fused phenyl rings attached through the triple bond at the 4‐position of bulged (R)‐1‐O‐(phenylmethyl)glycerol in homopyrimidine ONs in order to stabilize parallel triplexes. Slight destabilization of DNA/DNA Watson–Crick type duplexes (ΔTm = 1.0–4.5 °C) was detected for 2 inserted as a bulge, while RNA/DNA duplexes and duplexes with other TINA analogues were considerably destabilized (ΔTm > 6.0 °C). In cases of double insertion of 1 opposite to base inversions in dsDNA, the thermal stabilities of the triplexes were higher than that of the wild‐type triplex, which is a new solution to overcome the problem of targeting homopurine stretches with single base pair inversions. A DNA three‐way junction was considerably stabilized (ΔTm in a range of 10.0–15.5 °C) upon insertion of TINA monomers in the junction point as a bulge. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
Formation of 4H‐Azepine by the Electrophilic Reaction of a 2‐Methoxyazepinium Ion and Analysis of the Sigmatropic Isomerization
Authors: Cordonier Christopher E. J. , Satake Kyosuke , Okamoto Hideki , Kimura Masaru 
Published: August 2006

Abstract2‐Aryl‐2H‐, 3‐aryl‐3H‐, and 4‐aryl‐4H‐azepine were formed by the novel, electrophilic, πLUMO‐controlled reaction of the 2‐methoxyazepinium ion, generated in situ by the reaction of TiCl4 with 2,7‐dialkoxy‐2H‐azepine and an aryl compound, for which the kinetic parameters of the sigmatropic hydrogen rearrangement of the 4H‐azepine was measured. The substitution and hydrogen shift of the azepinium ion were analyzed with DFT studies. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)