Screening and confirmation of hereditary spherocytosis in children using a CELL-DYN Sapphire haematology analyser.

INTRODUCTION: Multidimensional optical scatter of sphered erythrocytes can identify and enumerate hyperchromic erythrocytes, which might be used for hereditary spherocytosis (HS) screening. The flow cytometric eosin-5'-maleimide test (EMA) is highly sensitive and specific for HS as a confirmatory method. The aims of this study were to assess the utility of hyperchromic erythrocytes in HS screening and to evaluate the EMA test performed on CELL-DYN Sapphire analyser compared with the reference method. METHODS: Blood from 740 paediatric patients presenting at our institution was analysed in reticulocyte mode of the CELL-DYN Sapphire haematology analyser (Abbott Diagnostics) to obtain hyperchromic erythrocyte counts. The EMA test was performed using a flow cytometer as a reference, as well as CELL-DYN Sapphire as an investigational method. RESULTS: Hyperchromic erythrocytes were the highest in patients with HS (median 11.5%; range 5.1-29.2%). Patients with autoimmune haemolytic disease had significantly less hyperchromic erythrocytes (median 4.9%; range 0.0-18.3%). Hyperchromic erythrocytes showed a high area under the ROC curve: 0.972. At 4.9% cut-off, hyperchromic erythrocytes detected HS with 96.4% sensitivity and 99.1% specificity. The EMA test on CELL-DYN Sapphire correlated strongly with the reference test and had identical diagnostic power. Stability studies with blood from HS patients showed a significant decrease in hyperchromic erythrocytes after 6 h storage. CONCLUSIONS: Measurement of hyperchromic erythrocytes is highly sensitive and specific for detecting HS and can be used for rapid and inexpensive screening. If required, the EMA test can be performed on CELL-DYN Sapphire or a standard flow cytometer for confirmation of HS.

Differential diagnosis of microcytic anemia: the role of microcytic and hypochromic erythrocytes.

INTRODUCTION: Various indices derived from red blood cell (RBC) parameters have been described for distinguishing thalassemia and iron deficiency. We studied the microcytic to hypochromic RBC ratio as a discriminant index in microcytic anemia and compared it to traditional indices in a learning set and confirmed our findings in a validation set. METHODS: The learning set comprised samples from 371 patients with microcytic anemia mean cell volume (MCV < 80 fL), which were measured on a CELL-DYN Sapphire analyzer and various discriminant functions calculated. Optimal cutoff values were established using ROC analysis. These values were used in the validation set of 338 patients. RESULTS: In the learning set, a microcytic to hypochromic RBC ratio >6.4 was strongly indicative of thalassemia (area under the curve 0.948). Green-King and England-Fraser indices showed comparable area under the ROC curve. However, the microcytic to hypochromic ratio had the highest sensitivity (0.964). In the validation set, 91.1% of microcytic patients were correctly classified using the M/H ratio. CONCLUSIONS: Overall, the microcytic to hypochromic ratio as measured in CELL-DYN Sapphire performed equally well as the Green-King index in identifying thalassemia carriers, but with higher sensitivity, making it a quick and inexpensive screening tool.

New erythrocyte and reticulocyte parameters on CELL-DYN Sapphire: analytical and preanalytical aspects.

INTRODUCTION: Extended RBC and reticulocyte parameters are useful in diagnosing functional iron deficiency and various other clinical conditions. The newest software of the CELL-DYN Sapphire measures extended RBC and reticulocyte parameters. The aims of the present communication were to assess the analytical aspects of these parameters compared with the Siemens Advia 120 analyzer, to study the effect of sample aging and to briefly explore their clinical usefulness in patients with anemia. METHODS: Blood samples were obtained from the routine workload of two hospital laboratories and were run on Siemens Advia and Abbott CELL-DYN Sapphire analyzers in parallel. Data analysis was performed using standard statistics. RESULTS: In total, 1416 patient samples were analyzed. There was close correlation in microcytic and macrocytic RBC (r(2) > 0.97) with small bias. The hypo- and hyperchromic RBC showed reasonable correlations, Advia 120 giving higher values. Reticulocyte MCV showed acceptable agreement, with significant proportional bias (Advia 8-9% higher). CELL-DYN Sapphire MCHr correlated rather well with Advia CHr (r(2) > 0.91) with significant absolute bias. Remarkably, the correlation data differed significantly between the two laboratories. It was found that aging of EDTA samples had significant effect on most of the RBC parameters. CONCLUSIONS: The new RBC parameters of CELL-DYN Sapphire generally correlated well with those of Advia 120, although significant systematic differences were present, particularly in reticulocyte MCH and MCV. These differences necessitate instrument-specific reference ranges and clinical decision values. To minimize preanalytical effects, these parameters should be measured in fresh blood samples.

Performance evaluation of the Abbott CELL-DYN Ruby and the Sysmex XT-2000i haematology analysers.

Two mid-range haematology analysers (Abbott CELL-DYN Ruby and Sysmex XT-2000i) were evaluated to determine their analytical performance and workflow efficiency in the haematology laboratory. In total 418 samples were processed for determining equivalence of complete blood count (CBC) measurements, and 100 for reticulocyte comparison. Blood smears served for assessing the agreement of the differential counts. Inter-instrument agreement for most parameters was good although small numbers of discrepancies were observed. Systematic biases were found for mean cell volume, reticulocytes, platelets and mean platelet volume. CELL-DYN Ruby WBC differentials were obtained with all samples while the XT-2000i suppressed differentials partially or completely in 13 samples (3.1%). WBC subpopulation counts were otherwise in good agreement with no major outliers. Following first-pass CBC/differential analysis, 88 (21%) of XT-2000i samples required further analyser processing compared to 18 (4.3%) for the CELL-DYN Ruby. Smear referrals for suspected WBC/nucleated red blood cells and platelet abnormalities were indicated for 106 (25.4%) and 95 (22.7%) of the XT-2000i and CELL-DYN Ruby samples respectively. Flagging efficiencies for both analysers were found to be similar. The Sysmex XT-2000i and Abbott CELL-DYN Ruby analysers have broadly comparable analytical performance, but the CELL-DYN Ruby showed superior first-pass efficiency.

Diagnostic performance of the variant lymphocyte flag of the Abbott Cell-Dyn 4000 haematology analyser.

BACKGROUND: In addition to differential cell counts, modern haematology analysers generate suspect flags if abnormal cells are detected. Reports on validation of suspect flags are scarce. We have routine experience with the Abbott Cell-Dyn 4000 analyser for over 5 years and have previously demonstrated the utility of the blast flag. Here we report a similar study on the performance of the analyser's Variant Lymphocyte (VL) flag. AIM OF THE STUDY: Evaluation of the diagnostic performance of the Cell-Dyn 4000 VL flag, as compared with lymphocyte morphology in blood smears. In addition, we investigated the usefulness of the numerical VL flag confidence index as provided by the analyser. MATERIALS AND METHODS: All samples generating a VL flag were reviewed over a 5-month period. We also reviewed smears from patients with known lymphoid disorders, even if the analyser did not flag the sample. Two experienced investigators assessed lymphocyte morphology independently. RESULTS: In total, 187 samples were included in the study, of which 183 had a VL flag and four had not. Of the 183 flagged samples, 83 appeared to have abnormal lymphocyte morphology and 100, normal lymphocyte morphology. The sensitivity of the VL flag for detecting abnormal lymphocytes was 0.95 and the positive predictive value was 0.44. Using ROC analysis of the VL flag confidence index, the area under the ROC curve was 0.58 (95% confidence interval 0.50-0.65). CONCLUSIONS: The Cell-Dyn VL flag has reasonable sensitivity but a high false-positive rate. In addition, its performance is insufficient for detecting clinically relevant abnormal lymphocytes. As the VL flag appeared to rely mainly on numerical criteria, it has no added value over numerical criteria defined by the laboratory.

Evaluation of flow cytometric enumeration of foetal erythrocytes in maternal blood.

In pregnant women subject to abdominal trauma or other foetomaternal haemorrhage, foetal red blood cells containing haemoglobin-F (HbF) can be found in the circulation. Recently, a monoclonal antibody to HbF has become commercially available, enabling application of a flow cytometric immunofluorescence method for accurately determining the concentration of HbF+ red blood cells. We demonstrate that white blood cells are included in the cluster selected as red blood cells and that these white blood cells exhibit a level of autofluorescence that coincides with the fluorescence signal from HbF+ red blood cells. However, these white blood cells can be excluded from the analysis, thus preventing spuriously increased HbF+ red blood cell counts. We present the results of patient samples containing HbF+ red cells as illustrations of the technique and as a potential interference by HbF-containing cells of nonfoetal origin. Using samples spiked with cord blood, the method is exactly linear with a high coefficient of correlation (r=0.997). Furthermore, the assay has excellent precision (CV < 2.4%), a low limit of detection (0.12% HbF+ RBC), is independent of Rhesus D and can be completed within 1.5 h. This method is suitable for accurate determination of foetomaternal haemorrhage.

New terpenoids from Haplopappus multifolius.

The chemical investigation of the aerial parts of Haplopappus multifolius afforded the new monoterpene 2,9-epoxy-p-menth-6-en-8-ol (7, haplopappol), the new monoterpenoid ester 9-cis-p-coumaroyloxy-alpha-terpineol (8, haplofolin), the new diterpene 18-hydroxylabda-7,13Z-dien-15-oic acid (6) and its known E-isomer (5). In addition, the known dihydroflavones 3',5-dihydroxy-4',7-dimethoxydihydroflavone and 3',4',5-trihydroxy-7-methoxydihydroflavone and the known dihydroflavonols 3',5-dihydroxy-4',7-dimethoxydihydroflavonol and 3',4',5-trihydroxy-3-acetyl-7-methoxydihydroflavonol were also obtained. The structural assignments of these compounds were made possible by the different spectroscopic measurements.

Implantable defibrillator event rates in patients with idiopathic dilated cardiomyopathy, nonsustained ventricular tachycardia on Holter and a left ventricular ejection fraction below 30%.

OBJECTIVES: This study investigated the incidence of appropriate implantable cardioverter defibrillator (ICD) interventions for ventricular tachycardia (VT) or ventricular fibrillation (VF) in patients with idiopathic dilated cardiomyopathy (IDC) and nonsustained VT in the presence of a left ventricular ejection fraction below 30%, versus in patients with syncope and patients with a history of VT or VF. BACKGROUND: To date, only limited information is available about the prophylactic use of ICDs in patients with IDC. METHODS: From January 1993 to July 2000, 101 patients with IDC underwent implantation of ICDs with electrogram storage capability at our institution. Patients were placed into one of three groups according to their clinical presentation: asymptomatic or mildly symptomatic nonsustained VT in the presence of a left ventricular ejection fraction < or = 30% (49 patients, prophylactic group), unexplained syncope or near syncope (26 patients, syncope group) and a history of sustained VT or VF (26 patients, VT/VF group). RESULTS: During 36 +/- 22 months follow-up, 18 of 49 patients (37%) in the prophylactic group received appropriate shocks for VT or VF, compared with 8 of 26 patients (31%) in the syncope group and with 9 of 26 patients (35%) of the VT/VF group. Multivariate Cox analysis of baseline clinical variables identified left ventricular ejection fraction, atrial fibrillation and a history of sustained VT or VF as predictors for appropriate ICD interventions during follow-up. CONCLUSIONS: Patients with IDC and prophylactic ICD implantation for nonsustained VT in the presence of a left ventricular ejection fraction < or = 30% had an incidence of appropriate ICD interventions similar to that of patients with a history of syncope or sustained VT or VF. These findings indicate that ICDs may have a role in not only secondary but also primary prevention of sudden death in IDC.

Microbiological conversion of a beta- and gamma-eudesmol mixture by Rhizopus.

A mixture of beta- and gamma-eudesmols was microbiologically biotransformed by Rhizopus stolonifer ATCC 6227. Positions-2 and 3, in both substrates, proved to be accessible by hydroxylase enzyme. Four different metabolites were isolated and their structures were elucidated by different spectroscopic methods. The structures of these metabolites were established as eudesma-3-en-2 beta,11-diol; eudesma-4-en-3 beta,11-diol; eudesma-4(15)-en-2 beta,11-diol and eudesma-4(15)-en-3 beta,11-diol.

[BIS monitoring may allow the detection of severe cerebral ischemia]. / Intérêt potential du BIS pour détecter une souffrance cérébrale importante.

PURPOSE: To show that the bispectral index (BIS) is not only a monitor of the depth of anesthesia but that acute decreases of the index may be related to severe cerebral ischemia. CLINICAL FEATURES: Several clinical observations suggest that an unexplained fall of the BIS may be the result of cerebral ischemia. Somatosensory evoked potentials decreased in parallel to the decrease in BIS during carotid clamping in a 58-yr-old patient undergoing carotid endarterectomy. In a 62-yr-old patient undergoing resection of an aortic aneurysm, the BIS decreased from 40-50% to 8% as the cardiac index and central venous O(2) saturation decreased. The BIS returned to normal values when the low cardiac output was corrected pharmacologically. CONCLUSION: While the BIS is a well accepted monitor of the depth of anesthesia, several factors, unrelated to anesthesia, can modify the index. Thus, to adjust the level of anesthesia based solely on the BIS could be inappropriate. While the sensitivity and specificity of the BIS for this indication have not been determined, we suggest that the BIS may be useful to detect severe cerebral ischemia.

Inhibition of Mycobacterium tuberculosis growth by saringosterol from Lessonia nigrescens.

Assay-guided fractionation of an antitubercular extract obtained from Lessonia nigrescens yielded the phytosterol saringosterol as its active component. No appreciable toxicity against Vero cells was observed for this compound. Saringosterol was also synthesized by oxidation of fucosterol. The MIC values for antitubercular activity of saringosterol and its 24S and 24R epimers were determined as 0.25, 1, and 0.125 microg/mL.

Comparison of three methods for measuring PEG-hirudin in blood.

Three methods for measuring pegylated hirudin (PEG-hirudin), a new antithrombotic agent, in blood were compared using clinical samples. The ecarin clotting time (ECT) was performed in whole blood using a point-of-care device (TAS analyzer). The ECT was also performed in plasma, using a clotting assay in a conventional automated coagulation analyzer. Finally, a chromogenic method was used, based on thrombin inhibition and the substrate S-2238. Both clotting assays showed a linear relationship between the ECT and the PEG-hirudin concentration up to 3.0 microg/ml. The chromogenic substrate method was linear only between 0.1 and 1.0 microg/ml PEG-hirudin. The intra-assay coefficient of variation was 3.0% for the automated ECT method, 6.4% for the point-of-care ECT method and 3.4% for the chromogenic method. The inter-assay coefficient of variation was approximately 10% for both clotting methods and 3.2% for the chromogenic method. There was a high correlation (r = 0.954) in PEG-hirudin concentration between both ECT methods over the entire measuring range. The correlation of the chromogenic method with any ECT was significantly less (r < 0.89), even if only PEG-hirudin concentrations < 1.0 microg/ml were taken into account (r < 0.92). Although we clearly prefer the conventional ECT, any of the other methods may be used for monitoring PEG-hirudin in patients treated with this drug, depending on the specific application and local circumstances.

EDTA-induced pseudo-neutropenia resolved with kanamycin.

This report describes a case of spurious neutropenia caused by EDTA-dependent in vitro agglutination of neutrophils. After raising the temperature of the sample to 37 degrees C the agglutination was irreversible, but it resolved completely after addition of kanamycin. Previously this method has been shown to be effective in EDTA-dependent pseudo-thrombocytopenia, but this is the first report demonstrating successful application in EDTA-dependent pseudo-neutropenia.

Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis.

This report describes the isolation and partial purification of novel triterpenoid saponins [Fraction 35 (F035)] and two pure biologically active derivatives (termed avicins D and G) from Acacia victoriae, an Australian desert tree of the Leguminosae family. F035 and the avicins markedly inhibited the growth of several tumor cell lines with minimum growth inhibition in human foreskin fibroblasts, mouse fibroblasts, and immortalized breast epithelial cells at similar concentrations. F035 and the avicins induced cell cycle (G1) arrest of the human MDA-MB-453 breast cancer cell line and apoptosis of the Jurkat (T-cell leukemia) and the MDA-MB-435 breast cancer cell line. The triterpenoid saponins also partially inhibited phosphatidylinositol 3-kinase activity in Jurkat T cells in a time-dependent manner and phosphorylation in the downstream protein Akt, whereas no affect was seen on the Ras/mitogen-activated protein kinase cascade. These observations as well as other work from our laboratory demonstrating mitochondrial perturbation, chemoprevention, and inhibition of nuclear factor kappaB suggest that triterpenoid saponins from A. victoriae have potential as novel anticancer agents. Recent work linking Akt signaling with glucose metabolism, stress resistance, and longevity suggests other potential applications of these compounds.

[Two patients with chronic lymphocytosis of large granular lymphocytes; benign or premalignant?]. / Twee patiënten met chronische lymfocytose van grote granulaire lymfocyten; benigne of premaligne?

In two female patients aged 44 and 86 years, a chronic lymphocytosis was observed caused by a proliferation of large granular lymphocytes (LGL). The first one had been successfully treated for Hodgkin lymphoma long before, the second had diabetes mellitus type 2. Immunophenotyping showed the proliferating lymphocytes to be natural killer (NK) cells. In contrast to the proliferation of B- and T-lymphocytes, data on the prognosis and treatment of NK-lymphocytosis are very scarce. A literature search revealed three major clinical entities in which LGL proliferate: at one end of the spectrum we see the very aggressive NK-LGL leukaemia, at the other, NK-lymphocytosis, a benign chronic disorder, in between is the relatively indolent chronic T-LGL leukaemia. Both patients suffered from chronic NK-lymphocytosis with a favourable course; there were no further symptoms 4 and 2 years, respectively, after the diagnosis. In cases of prolonged lymphocytosis of unknown origin, immunophenotyping of the lymphocytes is essential. Only in this way can one arrive at the proper diagnosis and reach conclusions as to the prognosis and the possible methods of treatment.

Haemostatic effects of low osmolar non-ionic and ionic contrast media: a double-blind comparative study.

In this prospective, double-blind, randomized study the effects of a non-ionic contrast medium (Iopromide) on the haemostatic system were compared with those of a low osmolar ionic medium (Ioxaglate). The aim was to investigate in vivo whether a non-ionic contrast agent is less anticoagulant or more pro-thrombotic than an ionic medium. A large number of haemostatic parameters, including activation markers, were measured. Either Iopromide (n = 16; median volume 102 ml; 95% confidence interval 90-108 ml) or Ioxaglate (n = 15; median 105 ml; 95% confidence interval 95-114 ml) was given to 31 patients scheduled for abdominal and femoral arteriography. Blood for laboratory investigations was collected before, and 5 and 30 min after, administering the contrast medium. Indications for activation of coagulation and platelets were already found in nearly 50% of the patients before any contrast medium was given. Both Iopromide and Ioxaglate caused further increases in thrombin-antithrombin complex, prothrombin fragments 1 + 2 and beta-thromboglobulin. The degree of activation was similar for both agents. Anticoagulant effects were not observed. The haemorheological effects were compatible with haemodilution by 5-8%, again without differences between the contrast agents. Contrary to the findings from in vitro studies, we found no significant differences between the effects of the non-ionic Iopromide and the ionic Ioxaglate on coagulation and platelets. Both agents activated these systems to a limited, but identical, degree. Our results support the notion that the catheterization procedure per se may represent a source of haemostatic activation and that the ionic contrast agent studied has insufficient anticoagulant effect to prevent clotting activation being induced by the contrast medium.