Brigitte Cantinieaux

Mucormycosis during deferoxamine therapy is a siderophore-mediated infection. In vitro and in vivo animal studies.

This study investigates the pathophysiology of mucormycosis caused by Rhizopus, which has been reported in 46 dialysis patients, while treated with deferoxamine (DFO). This drug aggravates mucormycosis, which we experimentally induced in guinea pigs and which lead to a shortened animal survival (P < or = 0.01). The drugs effect on Rhizopus is not mediated through the polymorphonuclear cells. Fe.DFO, the iron chelate of DFO, abolishes the fungistatic effect of serum on Rhizopus and increases the in vitro growth of the fungus (P < or = 0.0001). This effect is present at Fe.DFO concentrations > or = 0.01 microM, at which fungal uptake of radioiron from 55Fe.DFO is observed. A 1,000-fold higher concentration of iron citrate is required to achieve a similar rate of radioiron uptake and of in vitro growth stimulation as observed with Fe.DFO. These in vitro effects of Fe.DFO (1 microM) in serum on radioiron uptake and on growth stimulation are more striking for Rhizopus than for Aspergillus fumigatus and are practically absent for Candida albicans. For these three fungal species, the rates of radioiron uptake from 55Fe.DFO and of growth stimulation in the presence of Fe.DFO in serum are directly related (r = 0.886). These results underscore the major role of Fe.DFO in the pathogenesis of DFO-related mucormycosis. Pharmacokinetic changes in uremia lead to a prolonged accumulation of Fe.DFO after DFO administration, which helps explain the increased sensitivity of dialysis patients to DFO-related mucormycosis.

Staphylococcus aureus phagocytosis: A new cytofluorometric method using FITC and paraformaldehyde

A new method for measuring the uptake of Staphylococcus aureus by human polymorphonuclear neutrophils (PMN) using flow cytometry (FCM) is described. Bacteria were labeled with fluorescein isothiocyanate (FITC) and incubated with PMN in a suspension assay. At the end of the assay, phagocytosis was arrested by the addition of cold paraformaldehyde. Thereafter, phagocytosis was quantitated by FCM, using crystal violet to distinguish adherent versus ingested bacteria. The method was validated in multiple samples by reference to our microscopic technique; correlations of phagocytic indices were in good agreement. The FCM method was also found to be reproducible and provided a mean to detect low phagocyte values. Another feature of the approach is that FCM readings can be delayed without any appreciable alterations in the results.

Performance and Abnormal Cell Flagging Comparisons of Three Automated Blood Cell Counters

OBJECTIVES To compare two hematological analyzers-the DxH-800 (DxH; Beckman-Coulter, Miami, FL) and XN-2000 (XN; Sysmex, Kobe, Japan)-with the Cell-Dyn Sapphire (SAPH; Abbott, Santa Clara, CA). METHODS We analyzed 4,375 samples. Slide reviews were made in the presence of blast, abnormal lymphocyte, and immature granulocyte (IG) flags or nucleated RBC (NRBC) count. RESULTS The analyzers exhibited excellent correlations for CBC and neutrophils but displayed a limit correlation for lymphocytes. The XN did not miss circulating blasts (0.5%-95% in microscopy). For NRBCs, the XN demonstrated a sensitivity of 90%; DxH, 74%; and SAPH, 29%. Only the XN demonstrated a correlation with microscopy, permitting a WBC six-part differential until 15% of NRBCs. The XN and DxH gave useful IG counts with a cutoff less than 5% and a WBC level more than 2,500/mm(3). For abnormal lymphocytes detection, only XN demonstrated sensitivity of more than 95%, but its specificity of 54% requires adaptation. CONCLUSION The XN increases the sensitivity of abnormal cell detection compared with the other counters, permitting a seven-part differential between predefined levels, decreasing the slide review from 20% to 9%.

Granulocyte functions in children with cancer are differentially sensitive to the toxic effect of chemotherapy.

To analyze the toxicity associated to chemotherapy upon granulocytes, different functional assays were performed, within days of drug exposure and at time of bone marrow recovery, on polymorphonuclear neutrophils (PMN) from children with cancer. There were no significant postchemotherapy changes in the expression of the different receptors studied nor in the phagocytosis ofStaphylococcus aureus 42D. By contrast, a significant decrease was observed in H2O2 production in PMN recently exposed to chemotherapy with both cytofluorometric and chemiluminescence assays. There was also a decrease in the production of O[horizontal line over dot]2 and in chemotaxis; finally, the intracellular killing of S. aureus 42D andEscherichia coli was reduced. In patients having recovered from drug-induced bone marrow aplasia, PMN functions were found to be normal except for bactericidal activity which was still defective. These observations indicate that, in patients exposed to chemotherapy, some PMN functions are transiently altered, whereas microorganism cell killing is continuously impaired.

Prolonged but reversible neutrophil dysfunctions differentially sensitive to granulocyte colony-stimulating factor in children with acute lymphoblastic leukaemia.

Treatment of average‐risk acute lymphoblastic leukaemia (ALL) in children consists of 6 months of intensive chemotherapy followed by 18 months of maintenance therapy. Polymorphonuclear leucocyte (PMN) functions from children with ALL were studied in order to evaluate and compare the toxicity of the initial intensive treatment with the toxicity of the subsequent less intensive maintenance treatment. H2O2 and O−2 production, evaluated by chemiluminescence, were significantly decreased during the intensive period but returned to normal values when maintenance therapy began. In contrast, bactericidal activity against Gram‐positive and Gram‐negative micro‐organisms remained at low levels throughout the treatment but returned to normal values in patients off chemotherapy. PMN from patients on maintenance therapy exhibited an excess of morphological changes associated with apoptosis. This was confirmed by standard two‐colour flow cytometry which revealed an increase in the number of hypodiploid cells, and increased expression of membrane phosphatidylserine together with a drastic reduction in the expression of the Fcγ receptor IIIB (CD16). These defective PMN were differentially sensitive to the effects of granulocyte colony stimulating factor (G‐CSF): G‐CSF induced similar increase in chemiluminescence in control and patient PMN; GSF partially corrected the defective bactericidal activity; G‐CSF did not affect the accelerated PMN apoptosis. These observations indicate that ALL children undergoing chemotherapy present PMN defective functions which are partially sensitive or even resistant to G‐CSF.

Ferritin-associated iron induces neutrophil dysfunction in hemosiderosis.

Neutrophils (PMNs) from patients with secondary iron overload have an increased iron and ferritin content as well as a phagocytosis defect. Several serum components might be incriminated in the cellular iron accumulation. We therefore compared the effects on the PMN phagocytosis of total serum as well as the ferritin and transferrin fractions of serum derived from patients with thalassemia major and healthy control subjects. An incubation system of PMNs was developed. PMN phagocytosis was measured before and after incubation. Total serum from patients with thalassemia induced a defect that was prevented by co-incubation with deferoxamine (DFO). Gel-filtration chromatography was performed to separate the serum fraction containing transferrin and albumin from that containing ferritin. The transferrin-albumin fraction had no effect on PMN phagocytosis. On the contrary, the ferritin fraction of normal serum was deleterious to PMN phagocytosis, and the same fraction from thalassemic serum decreased PMN phagocytosis even more. Co-incubation with DFO or catalase improved this defect. Moreover, a cellular increase in the L-type subunit of ferritin was observed after the incubation of PMNs with the ferritin-containing fraction from thalassemic serum. In conclusion, serum from patients with thalassemia is toxic to PMNs, and this toxicity is due to ferritin-associated iron.

Defective functional activity and accelerated apoptosis in neutrophils from children with cancer are differentially corrected by granulocyte and granulocyte-macrophage colony stimulating factors in vitro.

We have previously shown that polymorphonuclear leucocytes (PMN) harvested from children with cancer and exposed to chemotherapy exhibit defective bactericidal activities against both Gram‐positive and Gram‐negative microorganisms as well as accelerated apoptosis. In this study, PMN from children with cancer were evaluated to compare in vitro the corrective effects of the two myeloid colony stimulating factors G‐CSF and GM‐CSF on these defective pathways. Both G‐CSF and GM‐CSF were able to increase the defective bactericidal activities against S. aureus and E. coli. However, GM‐CSF was consistently superior to G‐CSF in correcting PMN microbicidal activity; this correction was incomplete since it did not reach the level observed in normal PMN exposed to GM‐CSF.

Accurate Flow Cytometric Measurement of Bacteria Concentrations

Accurate measurements of bacteria concentrations are required in numerous studies; they raise methodological problems that complicate, for instance, the investigations of polymorphonuclear neutrophil functions. We propose a new flow cytometric method of determining bacteria concentrations by comparison with a standardized fluorescent latex bead solution. Relative counts of beads and bacteria are established in a system using both fluorescence and light scatter for the two types of particles. On the one hand, the latex bead size (0.98 microns in diameter) permits counting on traditional hematological counters and, on the other, a flow cytometric detection with the same conditions for bacteria. The reproducibility of the study of bacteria concentration measurements gave a coefficient of variation of < 5%.

Neutrophils from patients with secondary haemosiderosis contain excessive amounts of autotoxic iron

Abstract: Secondary haemosiderosis may be accompanied by a decrease in the phagocytic function of neutrophils (PMNs). This dysfunction has been attributed to an exaggerated generation of oxidants induced by intracellular iron. However, an accumulation of iron has so far not been reliably demonstrated in neutrophils harvested from iron‐overloaded patients. Six polytransfused haemodialysed patients, with a serum ferritin level higher than 1000 μg/l, and 10 healthy controls were investigated. The iron status of PMNs was evaluated by iron determination using atomic absorption spectrometry and by ferritin measurement using radioimmunoassay. The phagocytic performance was measured by cytofluorometry. The results confirm that PMNs from the haemosiderosis patients have a decreased phagocytosis. Moreover, they demonstrate for the first time that these PMNs have an increased cellular iron and ferritin content. Both latter concentrations were 4 to 5 times more elevated in secondary haemosiderosis than in healthy controls. This iron accumulation may be toxic for the PMNs and may, at least partially, explain the three‐fold higher risk of bacteraemia which has been reported in those patients.

Applying a direct aPTT ratio (PlatelinLS/ActinFS) permits to identify rapidly and reliably a bleeding‐related factor deficiency or a lupus anticoagulant sequential to an isolated prolongation of aPTT in paediatric pre‐operative screening

An isolated prolongation of activated partial thromboplastin time (aPTT) found in paediatric pre‐operative screening could be due to bleeding or non‐bleeding aetiologies. The aim of our study was to evaluate clinical benefits of an additional ActinFS and/or a mixing aPTT study to identify a bleeding‐related factor deficiency (BRFD).