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.

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.

Individualización del tratamiento ortodontico. Importancia de los tipos rotacionales y las categorias auxologicas

El objetivo del presente trabajo consiste en proporcionar herramientas diagnósticas útiles para la individualización terapéutica de cada paciente según sus características individuales de crecimiento y desarrollo cráneo facial. Se describen diversos tipos rotacionales mandibulares así como las categorías auxológicas propuestas por Petrovic, Lavergne y Stutzmann como indicadores del potencial de crecimiento y la modalidad que permite su identificación a través del análisis cefalométrico. En el presente trabajo se evaluaron 800 pacientes que acudieron al servicio de Ortodoncia de la Universidad Católica Sacro Cuore (Roma) a quienes se les determinó el tipo rotacional y la categoría auxológica de crecimiento según Petrovic.

The aim of the authors is to furnish of the indication useful to guideline for orthodontics treatment. We describe rotational type and auxology categories Petrovic's, Lavergne and Stutzmann as indication of growth potential and the modality to identify with cephalometric studies.

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.