Predictors and consequences of non-suicidal self-injury in relation to life, peer, and school factors.

INTRODUCTION: Numerous life, peer, and school-related factors have been found to be associated with non-suicidal self-injury (NSSI) among adolescents; however, most studies have not explored the possible reciprocal nature of these associations. The aim of the current study was to examine bidirectional and longitudinal associations between NSSI and several life, peer, and school-related factors (i.e., stressful life events, peer relationships, academic achievement, and attitudes towards school). METHOD: Community-based adolescents completed questionnaires assessing the variables of interest at three time points; age 12 (T1; 55.09% girls), age 13 (T2; 56.95% girls), and ages 14-15 (T3; 57.41% girls). In total, 529 adolescents provided complete data across all three-time points. RESULTS: Analyses showed a bidirectional association between NSSI and both attitudes towards school and stressful life events. Specifically, stressful life events at T2 predicted engagement in NSSI at T3, and NSSI at T2 predicted increased risk of stressful life events at T3. Similarly, having negative attitudes towards school predicted NSSI at T2, which, in turn, predicted negative attitudes towards school at T3. Further, academic achievement at T1 was negatively associated with NSSI at T2. Peer relationships were neither a predictor nor a consequence of NSSI. CONCLUSIONS: Our results suggest that NSSI can be both a predictor and a consequence of various life, and school factors. Focus on these factors in prevention and intervention efforts for NSSI among adolescents may be warranted.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

A proposed method for assessing plasma hypertonicity in vivo.

Indices of plasma hypertonicity, elevated plasma concentrations of solutes that draw fluid out of cells by osmosis, are needed to pursue hypertonicity as a possible risk factor for obesity and chronic disease. This paper proposes a new index that may be more sensitive to mild hypertonicity in vivo at a point in time than traditional measures. The index compares mean corpuscular volume (MCV) estimates from diluted (in solution by automated cell counter) and nondiluted blood (calculated from manual hematocrit, MCV=Hct/RBC*10(6)). A larger Auto vs Manual MCV (>2 fl) in vitro indicates hypertonicity in vivo if the cell counter diluent is isotonic with the threshold for plasma vasopressin (PVP) release and PVP is detectable in plasma (>0.5 pg/ml). To evaluate this principle of concept, hypertonicity was induced by 24-h fluid restriction after a 20 ml/kg water load in four healthy men (20-46 years). Unlike serum and urine indices, the MCV difference-&-PVP index detected hypertonicity in all participants.

International Council for Standardization in Haematology (ICSH) recommendations for "surrogate reference" method for the packed cell volume.

The spun packed cell volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination, and assignment of values to calibrators/controls. In 2001, the International Council for Standardization in Haematology (ICSH) recommended a Reference PCV method, which is fully traceable to the ICSH reference hemoglobin method. Because of its complexity, however, this method is impractical for occasional use in routine laboratories and is therefore intended primarily for use by manufacturers of capillary microhematocrit tubes, liquid calibrators, and multichannel analyzers. In response to the need for a simpler method--accessible to all routine laboratories--the ICSH offers this "Surrogate Reference" PCV procedure. It is traceable to the original ICSH Reference PCV method and is based on spun PCVs obtained using borosilicate capillary tubes with an already-known relationship to this reference procedure. This ICSH "Surrogate Reference" PCV method is substantially simpler, thus putting it within the reach of most routine hematology laboratories.

The ability of apoplastic ascorbate to protect poplar leaves against ambient ozone concentrations: a quantitative approach.

Shoots of a sensitive (Populus nigra 'Brandaris') and a more tolerant (Populus euramericana 'Robusta') poplar clones were exposed for 30 days to Filtered Air or ambient O3-concentrations in fumigation cabinets. At regular intervals were determined: gas exchange of the leaves, the internal air space (Vair) and apoplastic water volume (Vapo) and the reduced (ASA) and oxidized (DHA) ascorbate concentration in the apoplast and in the mesophyll cells. The apoplastic ASA-concentration was 0.2 mM at the start of the experiment for both cultivars, while the effective cell wall thickness, estimated from Vapo, varied from 0.3 to 0.6 micron. Model calculations revealed that only 30% of the O3 molecules entering the apoplast was intercepted at these values. The O3-treatment induced a decline in stomatal conductance, an increase in Vapo and in the apoplastic ASA-concentration. As a result the estimated O3-flux to the cell membrane strongly declined. However, these responses occurred after the O3-induced reduction in photosynthesis. Moreover, they did not prevent early senescence of the leaves at a prolonged exposure. Therefore, it is concluded that the increase in apoplastic ASA-concentration was rather a general stress reaction of the affected poplar leaf than a (specific) defence reaction induced by O3. Our results suggest that other factors than the scavenging efficiency of apoplastic ASA were responsible for the difference in O3 sensitivity between both poplar cultivars.

An interlaboratory study of a candidate reference method for platelet counting.

A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 x 10(3)/microL (1-400 x 10(9)/L). Pooled analysis of the serial dilutions showed that RBC-platelet and RBC-RBC coincidence events became negligible at sufficiently high dilutions (i.e., > 1:1,000). All laboratories demonstrated excellent intra-assay and acceptable interlaboratory precision. Two antibodies (CD61 and CD41) were used for identifying platelets and individually gave acceptable results, but in a minority of samples, staining differences were observed. The optimum method thus uses a double-labeling procedure with a final dilution factor of 1:1,000. The study demonstrated that this method meets the criteria for a reference platelet count.

Guidelines for organisation and management of external quality assessment using proficiency testing. Expert Panel on Cytometry of the International Council for Standardization in Haematology.

This document is intended to assist towards the WHO objective that external quality assessment (EQA) schemes be established at national and/or regional levels world-wide. Quality assurance is defined as all steps taken by the director of a laboratory to ensure reliability of laboratory results and to increase accuracy, reproducibility and between-laboratory comparability. This includes the use of internal quality control procedures and participation in external quality assessment. Internal quality control provides the means for evaluation of analytic test results at the time of testing in order to decide whether they are reliable enough to be released to the requesting clinicians. EQA, on the other hand, refers to a system of retrospective and objective comparison of results from different laboratories by means of proficiency testing (PT) organised by an external agency. The main purpose is to establish between-laboratory and between-method (including between-instrument) comparability, and agreement with a reference standard where one exists. Internal quality control and EQA complement each other and must never be considered as alternatives.

Improved automated leucocyte counting and differential in newborns achieved by the haematology analyser CELL-DYN 3500.

Automated leucocyte counts in newborns generated by the impedance principle are artificially affected by the high osmotic resistance of some newborn RBC and possibly by the high normoblast numbers present during the neonatal period. Erroneously high WBC counts may result. The haematology analyser CELL-DYN 3500 (Abbott Diagnostika GmbH, Wiesbaden-Delkenheim, Germany) has two different channels for the WBC count, an electrical resistivity (impedance) channel and a laseroptical channel. In combination with facultative extended lysis of resistant RBC before WBC count, this instrument is claimed to be very suitable for newborn blood analysis. We measured the WBC count and differential of 165 blood samples from newborns and cord blood on the CELL-DYN 3500. Reticulocyte count and manual differential including normoblasts were determined. Furthermore, some technical aspects of neonatal blood analysis were evaluated: precision, cell stability, effect of incorrect blood-anticoagulant ratio of small blood collecting tubes. The internal decision making process of the CELL-DYN 3500 selects the result either from the optical channel (identifies and excludes normoblasts) or from the resistivity channel (eliminates resistant RBC). This instrument gives a reliable and accurate WBC count and differential of neonatal samples even in blood samples with normoblasts and lytic resistant RBC. The result given by the CELL-DYN 3500 can be confirmed by a subsequent run in extended lyse mode.

Recommended procedures for the classification of acute leukaemias.

The classification of acute leukaemias is now widely based on a combined morphological, cytochemical and immunophenotyping approach. Difficulties are frequently encountered however in reaching an acceptable degree of diagnostic concordance between different laboratories because of variations in the techniques used (in terms of methodologies, reagents and equipment) and diagnostic interpretation. The International Council for Standardization in Haematology (ICSH) convened an expert panel to consider currently available diagnostic techniques with the aim of defining a minimum cytochemical and immunological diagnostic panel that could be used as core components for the classification of acute leukaemia. The proposed ICSH scheme, which attempts to balance the basic requirement for providing precise and informative diagnostic information without limiting its use to only those laboratories with sophisticated facilities, is based on three sequential levels of investigation; primary cytochemistry, intracellular phenotyping and membrane immunophenotyping. The minimum ICSH recommended cytochemistries comprise myeloperoxidase (MPO), chloroacetate esterase (ChlorE) and alpha-naphthyl acetate esterase (ANAE), and standardised methods for these cytochemistries are detailed in this communication. For cases of acute leukaemia that remain unclassified by primary cytochemistry, subsequent immunological analyses for cytoplasmic CD3, CD22, MPO and nuclear TdT are recommended. The ICSH panel considers that the use of these minimum primary cytochemical and intracellular phenotyping procedures will lead to the consistent classification of most acute leukaemias, and that the third level of investigation (membrane immunophenotyping) should be used for the purposes of confirmation, diagnostic clarification of atypical leukaemias, and the subtyping of acute lymphoblastic leukaemias (ALL). The ICSH panel also recognised that there are a number of additional technologies which can provide definitive diagnostic information, such as cytogenetics and DNA genotyping, but these were excluded from the minimum panel because of their restricted availability. While many specialised laboratories, particularly in the areas of diagnostic research, will continue to use individual investigatory protocols, it is considered that the inclusion of the ICSH scheme as core components would lead to greater consistency when comparing independent studies of acute leukaemia.

Serum erythropoietin levels in elderly inpatients with anemia of chronic disorders and iron deficiency anemia.

OBJECTIVE: To analyze the relationship between serum erythropoietin levels and hemoglobin levels in elderly patients with anemia of chronic disorders related to cancer or acute infection when compared with anemic patients with iron deficiency. DESIGN: Prospective survey with comparison groups. SETTING: Tertiary care center. PATIENTS: An elderly group aged 70 and above (mean 84, range 70-96) was divided into subgroups of 45 with anemia of chronic disorders (23 with cancer and 22 with acute infection), 24 with iron-deficiency anemia, and 27 with no anemia. Thirty non-anemic younger adults were also studied. MEASUREMENTS: Serum erythropoietin (radioimmunoassay), complete blood count, serum iron, B12, folate and ferritin, liver and kidney function tests, blood gas analyses, and bacteriological and radiological tests. RESULTS: The serum erythropoietin levels were significantly lower in the elderly non-anemic hospitalized group than in the healthy younger group. A significant negative relationship between the log serum erythropoietin and hemoglobin levels was found in patients with iron deficiency, but not in the other groups. For any given hemoglobin level, the response of erythropoietin was significantly higher in anemic patients with iron deficiency when compared with the neoplastic and infectious group. CONCLUSION: Erythropoietin response to anemia is blunted in elderly patients with anemia of chronic disorders related to cancer or acute infection. Erythropoietin level is lower in non-anemic elderly inpatients than in healthy younger persons.

Evaluation of flow cytometry for multidrug resistance detection in low resistance K562 cells using daunorubicin and monoclonal antibodies.

We compared the effectiveness of two flow cytometric methods for the detection of the multidrug resistance (MDR) phenotype. The sensitivity of both methods depended on the ability to discriminate low resistance cells from sensitive ones. Therefore, K562 cells with decreasing vinblastine (VLB) resistance levels were examined, the lowest resistance level being nonmeasurable with a colorimetric MTT assay. The fluorescent drug daunorubicin (DNR) was measured in combination with two modulators of MDR, cyclosporin-A (CsA) and verapamil (Vp) in a functional flow cytometric assay. When compared to sensitive cells, DNR uptake levels at steady state were reduced in all resistant cell lines, except for the lowest resistant cell line. The effect of modulator, CsA, on DNR uptake was seen in all resistant sublines, compared to sensitive cells, except for the lowest resistant cells. In another assay, the P-glycoprotein (Pgp) expression was analysed with monoclonal antibodies, MRK16 and C219. MRK16 was found to be the most sensitive antibody to screen for MDR+ cells, since we could show Pgp hyperexpression in all resistant cells. C219 reactivity became evident in cells possessing resistance factors higher than 5. These results indicate that both the functional assay and the Pgp assay are sensitive to be used for screening of MDR+ cells.

Recommended procedures for the classification of acute leukaemias. International Council for Standardization in Haematology (ICSH).

The classification of acute leukaemias is now widely based on a combined morphological, cytochemical and immunophenotyping approach. Difficulties are frequently encountered however in reaching an acceptable degree of diagnostic concordance between different laboratories because of variations in the techniques used (in terms of methodologies, reagents and equipment) and diagnostic interpretation. The International Council for Standardization in Haematology (ICSH) convened an expert panel to consider currently available diagnostic techniques with the aim of defining a minimum cytochemical and immunological diagnostic panel that could be used as core components for the classification of acute leukemia. The proposed ICSH scheme, which attempts to balance the basic requirement for providing precise and informative diagnostic information without limiting its use to only those laboratories with sophisticated facilities, is based on three sequential levels of investigation; primary cytochemistry, intracellular phenotyping and membrane immunophenotyping. The minimum ICSH recommended cytochemistries comprise myeloperoxidase (MPO), chloroacetate esterase (ChlorE) and alpha-naphthyl acetate esterase (ANAE), and standardised methods for these cytochemistries are detailed in this communication. For cases of acute leukaemia that remain unclassified by primary cytochemistry, subsequent immunological analyses for cytoplasmic CD3, CD22, MPO and nuclear TdT are recommended. The ICSH panel considers that the use of these minimum primary cytochemical and intracellular phenotyping procedures will lead to the consistent classification of most acute leukaemias, and that the third level of investigation (membrane immunophenotyping) should be used for the purposes of confirmation, diagnostic clarification of atypical leukaemias, and the subtyping of acute lymphoblastic leukaemias (ALL). The ICSH panel also recognised that there are a number of additional technologies which can provide definitive diagnostic information, such as cytogenetics and DNA genotyping, but these were excluded from the minimum panel because of their restricted availability. While many specialised laboratories, particularly in the areas of diagnostic research, will continue to use individual investigatory protocols, it is considered that the inclusion of the ICSH scheme as core components would lead to greater consistency when comparing independent studies of acute leukemia.

A new CD43 monoclonal antibody induces homotypic aggregation of human leucocytes through a CD11a/CD18-dependent and -independent mechanism.

We describe a monoclonal antibody (mAb), designated 1.C1, that causes rapid and vigorous aggregation among normal leucocytes and among T and myeloid/monocytic cell lines. As shown by competitive binding and sequential immunoprecipitation experiments, the antigen recognized by mAb 1.C1 is a 115,000 MW sialoglycoprotein, that corresponds to the human CD43 antigen, also known as leukosialin or sialophorin. The aggregation process starts within minutes and reaches maximum level 6-18 hr after addition of the antibody. It is dependent on active cell metabolism (inhibited at low temperatures and by a mixture of the metabolic poisons azide and 2-deoxy-D-glucose), a fluid plasma membrane (inhibited by pretreatment of the cells with paraformaldehyde) and an intact cytoskeleton (inhibited by cytochalasin B). Two reference CD43 antibodies (MEM-59 and DF-T1), both binding the same or closely related sialic acid-dependent epitope as mAb 1.C1, are also capable of inducing cell clump formation. CD11a/CD18 mAb block the 1.C1-induced adhesion of resting peripheral blood leucocytes, but not of haematopoietic cell line cells. In addition, mAb 1.C1 induces homotypic aggregation of K-562 cells, which do not express members of the beta 2 integrin subfamily on their surface. These data suggest that triggering of the CD43 antigen promotes homotypic cell adhesion that is mediated by both CD11a/CD18-dependent and -independent pathways.

Comparative evaluation of three flow cytometers for reticulocyte enumeration.

Flow cytometric determination of reticulocytosis is progressively replacing the manual microscopic method. We evaluated three flow cytometers (Becton Dickinson FACScan, Coulter EPICS Profile II, Ortho Cytoron Absolute) for reticulocyte enumeration, using thiazole orange. For each sample, 30,000 cells were analysed. In order to comparatively evaluate the three instruments, reticulocytes were counted by manually gating the erythroid population and evaluating the gated population for fluorescence characteristics. The different instruments showed good linearity and precision. No carry-over was observed. Orthogonal regression analysis of reticulocyte counts of 100 healthy blood donor samples and 108 haematological patient samples showed a good mutual comparability between all three instruments tested, although the paired t-test showed a significant difference between the Cytoron and both the FACScan and the Profile. Despite minor statistical differences, the three instruments can be considered equivalent for daily routine reticulocyte enumeration.

Correlation of cytogenetic patterns and clinicobiological features in adult acute myeloid leukemia expressing lymphoid markers.

Cytogenetic, biomolecular, and clinicopathologic features were retrospectively studied in 34 adult patients with acute myelogenous leukemia expressing one or more of the following lymphoid-associated markers (LMs): CD7, CD2, CD10, CD19, CD22, TdT. Six patients showed 11q23 rearrangements (group I); three patients had the classic Ph chromosome (group II); 15 patients had aberrations of the myeloid type (group III), including four patients with structural aberrations of 13q or trisomy 13, three patients with 7q and 1q anomalies, and two patients with trisomy 11q. Ten patients had a normal karyotype (group IV). Anomalies exclusively associated with lymphoid malignancies were not seen. Ig H and/or T-cell receptor genes were found to be rearranged in 50% and 66% of patients in cytogenetic groups I and II, respectively, versus 8% in group III and 12% in group IV. Likewise, more than one LM was more frequently detected in groups I and II. In group III, two of four patients with aberrations of chromosome 13 expressed two or more lymphoid features. Clinically, patients belonging to cytogenetic groups I and II were generally young, presented with a high white blood cell (WBC) count, and had a low complete remission rate. Survival in Ph chromosome-positive cases was uniformly short. We conclude that although there is no cytogenetic anomaly specifically associated with acute myelogenous leukemia expressing LM, a Morphologic, Immunologic, and Cytogenetic classification may constitute a working basis for further studies aimed at a better definition of clinicopathologic features and optimal treatment strategies for these leukemias.

Recombinant human erythropoietin for the treatment of anemia in the myelodysplastic syndromes: a clinical and erythrokinetic assessment.

The clinical and ferrokinetic effects of escalating doses of subcutaneously administered recombinant human erythropoietin (rh-EPO) were studied in ten patients with myelodysplastic syndromes and severe transfusion-dependent anemia. Red blood cell transfusion requirements diminished in four patients, and one of the patients eventually became transfusion independent with an EPO-induced rise of Hb from 7.7 g/dl to 12.3 g/dl. Endogenous serum levels of EPO were significantly increased in all patients (100-5700 mU/ml), but three of four responders had a relatively low baseline level. The effective red cell iron turnover (RCIT) improved in two responding patients and even normalized in one patient. This increase in RCIT was accompanied with a decline in the ineffective red cell iron turnover (IIT). The other responding patients had a relatively preserved RCIT before EPO treatment. EPO therapy further increased the fraction of IIT in the latter patients. Red cell survival time did not increase during EPO therapy, even in the responding patients. One transient and one maintained increase in platelet count were observed. Disease progression with a sustained increase in blast cells in one patient and a transient elevation of blasts in another patient was seen. No other side effects of EPO therapy were observed. These results suggest that anemic MDS patients with low serum EPO levels and relatively spared effective erythropoiesis as measured by ferrokinetic studies may be the best candidates for treatment with recombinant human EPO.

Lymphocyte subset reference ranges in adult Caucasians.

We report here the distributions of lymphocyte populations bearing the following antigens: CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic and some NK cells), and CD3-, CD16+, and/or CD56+ (NK cells). At four sites, analyses were performed on healthy, normal subjects between the ages of 18 and 70, using identical flow cytometry systems and techniques. Reference ranges (unadjusted for sex differences and age variation) are CD3 (61 to 85%), CD19 (7 to 23%), NK (6 to 29%), CD4 (28 to 58%), and CD8 (19 to 48%). The lymphocyte subpopulation distributions for all antigens were found to be similar at all sites. By combining data from all sites, it has been possible to estimate age variation and sex differences for each of these subpopulations. Age and sex associated differences are substantial for some lymphocyte subsets (CD3, CD4, NK cells), and proper accounting of these effects is essential in evaluating the individual patient, if further disease-related variation is to be accurately and consistently assessed. It appears possible to recommend reference ranges for lymphocyte population parameters applicable across national and laboratory boundaries. These ranges provide a basis for comparing results from different institutions and for combining such results on subjects and patients from several institutions, provided the methodology and equipment are identical at all sites.

Monocyte counting: discrepancies in results obtained with different automated instruments.

To determine the accuracy of several methods for measuring the monocyte count, the results obtained by a number of different automated cell counters were analysed. Considerable discrepancies occurred for monocyte counts obtained in normal blood among the counters. The results of a visual monocyte count on a total of 800 leucocytes were used as the reference method. The technique of measuring the monocyte count by using dual staining with monoclonal antibodies CD45 and CD14 provided the closest agreement with the reference method. Six other automated counting systems were assessed. Two of these systems (Coulter VCS and Technicon H1) gave results, which, although under-estimating monocytosis, correlated well with the results obtained by the reference technique. A third system (Toa Sysmex NE-8000) gave unreliable results. Three of the automated systems evaluated measured a "third population"--that is, monocytes together with other leucocytes. One of these systems (Ortho ELT 1500), overestimated the count, as expected, but correlated well with the reference method. The second of these "third population counters" (Coulter S Plus IV) correlated moderately well with the reference monocytosis, while the Toa Sysmex E-5000 correlated poorly. It is clear that problems exist in the evaluation of different instruments for counting monocytes. An accurate and reliable reference method is a pre-requisite to evaluate this aspect of cell counters. As the visual method is too cumbersome a different reference method would be useful. Based on the results of this study, it is suggested that the technique using fluorescence labelled monoclonal antibodies should be regarded as an acceptable alternative.

In vitro modulation of normal and diseased human neutrophil function by pentoxifylline.

The influence of pentoxifylline on normal and diseased neutrophil function has been studied in vitro. In high concentrations pentoxifylline stimulated human neutrophil chemotaxis toward both bacterial oligopeptides and complement components. Pentoxifylline was also shown in vitro to restore the normal chemotactic capacity of neutrophils from patients with known functional defects, i.e. myelodysplastic syndromes, lazy leucocyte syndrome, juvenile parodontitis, hyper-IgE-syndrome and liver cirrhosis. Pentoxifylline was also shown to strongly inhibit the release of primary and secondary granule release of granulocytes. Moreover, pentoxifylline inhibits both basal and stimulated neutrophil adhesion to both aortic and pulmonary artery calf endothelium. The mechanism whereby pentoxifylline exerts this action is not adequately understood. While our results partially imply interference of pentoxifylline with neutrophil cyclic AMP and/or prostaglandin metabolism, down-regulation of neutrophil functional antigen (e.g. CD11, CD18) expression seems to play a key role in the observed drug effects. Finally, these results indicate that pentoxifylline may be useful in the treatment of granulocyte mediated diseases and symptoms.