[Candida glabrata chorioamnionitis following in vitro fertilization]. / Chorioamniotite à Candida glabrata après fécondation in vitro.

We report one case of severe Candida glabrata chorioamnionitis and septicemy occurring in a twin pregnancies achieved by in vitro fertilization techniques which resulted in pregnancy loss after preterm rupture of the membrane at 22 weeks of gestation despite a treatment with amphotericin B.

A standardized procedure for quantitation of CD11b on polymorphonuclear neutrophil by flow cytometry: potential application in infectious diseases.

An up-regulation of the surface marker CD11b has been demonstrated during polymorphonuclear (PMN) cell activation. CD11b over-expression is often associated with inflammation and is considered as an early marker of infection. However, the absence of standardized assay and the variability of preanalytical settings leading to PMN artifactual activation have compromised the interest of this marker. In the present study a standardized quantitative flow cytometry assay directly performed in whole blood has been used to determine CD11b expression on PMN cells. The results indicate that quantitative flow cytometry can provide consistent CD11b density values between laboratories provided that a calibration system is used including specific calibrators, reagents and protocols. This method allowed us to evidence an up-regulation of CD11b expression for infected patients. This quantitation is a standardized and potentially useful method in clinical situations implying quantitative CD11b expression variations.

Red cell membrane Na+ transport systems in hereditary spherocytosis: relevance to understanding the increased Na+ permeability.

Red blood cells (RBCs) in hereditary spherocytosis (HS) show high sodium (Na+) and potassium (K+) movement across the membrane, resulting in dehydration. In general, these abnormal cation fluxes have been interpreted as "increased leaks" due to passive or electrodiffusional permeability of the RBC membrane. A study to elucidate the contribution of concomitant ouabain-resistant pathways (Na-K-2Cl cotransport and Na-Li countertransport) to abnormal Na+ permeability present in RBCs of subjects with HS has been undertaken. Accordingly, erythrocyte Na+ and K+ content and transmembrane cation movements via the Na-K pump, Na-K-2Cl cotransport, Na-Li countertransport, and Na+ passive diffusion, were measured in 25 non-splenectomized patients with HS and compared with the results obtained from the study of 11 patients with congenital non-spherocytic haemolytic anaemia (CNSHA) due to hereditary elliptocytosis (7 cases) and RBC enzyme defects (4 cases) and of 30 normal controls. Compared to the controls, patients with HS exhibited a highly significant (P<0.001) increase in all the Na+ transmembrane movements via passive diffusion (411+/-243 vs 105+/-40), Na-K pump (2615+/-970 vs 1874+/-359), Na-K-2Cl cotransport (males: 371+/-138 vs 190+/-42; females: 401+/-134 vs 104+/-44) and Na-Li countertransport (207+/-131 vs 98+/-41). This was associated with increased Na+ and decreased K+ content, resulting in a reduction of total cation (Na+ + K+) RBC concentration. Furthermore, significant correlations were also found between the patients' RBC cationic content and the mean corpuscular haemoglobin concentration (MCHC) (r=0.51, P<0.05) and between the Na+ passive leak and the haematocrit value (r=-0.44, P<0.05). In the patients with CNSHA, a less significant (P<0.01) increase of active (Na-K pump) and passive (leak) transmembrane permeability to Na+ was associated with normal transmembrane movements via Na-K-2Cl cotransport and Na-Li countertransport. The present study demonstrates that in HS, RBCs are characterized by a variable, but always significant increase of all the membrane transport systems leading to the extrusion of Na+, and that these abnormalities, regardless of their relation to membrane structural defects, may probably be valuable for the differential diagnosis between HS and other congenital defects of RBCs.

A standardized flow cytometric method for screening paroxysmal nocturnal haemoglobinuria (PNH) measuring CD55 and CD59 expression on erythrocytes and granulocytes.

PNH is a disorder of the pluripotent stem cells resulting in a deficient expression of membrane-bound GPI-anchored proteins in different cell types. Several flow cytometric approaches are designed to detect this antigen deficiency. But they all require drawing and testing of normal samples as control. Therefore, in the present study two flow cytometric assays for the detection of CD55 and CD59 deficiency in erythrocytes (REDQUANT CD55/CD59) and granulocytes (CELLQUANT CD55/CD59) are proposed. Precalibrated beads are used to define the cut off between normal and deficient cell populations. The specificity of the tests has been evaluated in healthy blood donors (n=52) resulting in a clear and reproducible cut off (3%) for the normal percentage of GPI-deficient cells. This cut off has been confirmed in leukaemia and lymphoma patients not suspected for developing PNH. The sensitivity has been tested in patients suffering from known PNH (n=23). Both tests performed in combination allowed a reliable detection of PNH in all patients showing antigen deficiencies in both cell types in most patients (20/23). In contrast, the PNH clones in the investigated patients with MDS (4/19) or AA (4/22) were present in granulocytes or erythrocytes, only. This underlines the necessity of analysing erythrocytes as well as granulocytes. Preliminary data regarding a possible correlation between disease activity and percentage of antigen-deficient cells lead to the assumption that haemolytic crises can only be determined on granulocytes whereas deficient erythrocytes disappeared due to complement-mediated lysis of the PNH clone. In conclusion, the combination of the test kits enables the differential diagnosis of PNH clones in a standardized, simple and rapid approach which may have therapeutic consequences.

[Treatment of hepatitis C virus infection in the Poitou- Charentes region]. / Prise en charge de l'infection par le virus de l'hépatite C dans la région Poitou-Charentes.

OBJECTIVES: The prognosis of hepatitis C virus infection could be improved by early treatment. However, this is only possible if most patients with hepatitis C consult a specialized institution. The aim of this study was to evaluate the modalities of care of hepatitis C virus infection in one French district. METHODS: Between November and December 1997, 89 biological laboratories from the "Poitou-Charentes" district were asked to provide results of hepatitis C virus serology tests performed during this period. A questionnaire concerning epidemiological and follow-up data was sent to the medical practitioner who prescribed the test, for all positive tests. RESULTS: Seventy eight out of 89 (88%) laboratoires agreed to participate in the study. During the study period, 6,168 subjects were tested and 196 (3.2%) were positive. This test was a diagnostic test in 69 cases (53%) and a confirmation test in 61 cases (47%). The epidemiological questionnaire was filled out in 130 cases. The main putative factors of viral contamination were: intravenous or nasal drug addiction in 69 cases (53%), blood transfusion in 39 cases (30%), and a nosocomial risk factor in 16 cases (12%). Treatment and care of virus infection was evaluated in 113 cases from the follow-up questionnaire: a liver biopsy was performed in 30 cases (27%) and interferon therapy was administered in 13 cases (12%). Liver biopsy was not performed in 83 cases (73%) due to normal transaminase levels or a contraindication to interferon therapy. The main causes of an absence of care or follow-up were: fear of complications of liver biopsy and/or side effects to interferon therapy (19%), chronic alcoholism (18%) and active drug addiction (8%). CONCLUSION: The main causes of failure to administer adequate care in hepatitis C patients were chronic alcoholism, drug addiction and fear of liver biopsy or side effects of interferon therapy. These data should be taken into account for future screening or information compaigns for the general population.

In vivo 13C-NMR studies of polymer synthesis in rhizobium meliloti M5N1 strain

The use of in vivo 13C-NMR approach for the monitoring of the synthesis of various polymers within cells of Rhizobium meliloti (M5N1 strain) is reported. Significant differences in polymer biosynthesis have been shown as a function of the metabolic state of the cells and the labeled carbon source used. Consumption of carbon source and produced glycogen was complete with mid-exponential phase harvested cells. This was not the case with stationary phase harvested cells, for which polyhydroxybutyrate synthesis was higher and gluconate synthesis was lower than the former. [1-13C]fructose-grown cells produced more exopolysaccharide and polyhydroxybutyrate, but less beta-(1,2) glucan and gluconate than [1-13C]glucose-grown cells. This approach offers a suitable tool to examine the kinetics of polymer biosynthesis by Rhizobia. Copyright 1998 John Wiley & Sons, Inc.

Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR.

The dehydrogenation of [1-(13)C]- and [2-(13)C]glucose into gluconate was monitored by NMR spectroscopy in living cell suspensions of two Rhizobium meliloti strains. The synthesis of gluconate was accompanied, in the cellular environment, by the formation of two gluconolactones, a gamma-lactone being detected in addition to the expected delta-lactone. These lactones--as well as the gluconate--could be further metabolized by the cells. The delta-lactone was utilized faster than the gamma-lactone. The presence--in significant amounts--and the relative stability of the lactones raise the question of their possible physiological significance.

[Evaluation of the Coulter MAXM. Differential leukocyte count and left-shift alarm]. / Evaluación del autoanalizador hematológico Coulter MAXM. Recuento diferencial leucocitario y alarma de desviación a la izquierda.

PURPOSE: To assess the reliability of the differential leucocyte count (DLC) and the left shift flagging (LSF) system provided by the Coulter MAXM (MAXM) haematology analyzer. MATERIAL AND METHODS: 380 blood specimens (drawn with tri-K EDTA as anticoagulant) were studied. RESULTS: By using the reference method (NCCLS H20-A), 50 out of the 380 blood specimens presented abnormal DLC (bands > 6%). Of from these, in 39 (80%) the MAXM displayed LSF of "bands 1 or 2". In 118 left shift flagged specimens (MAXM) with normal manual DLC, 87 (74%) had the "bands 1" alarm and 31 (26%) the "bands 2" alarm. Accordingly if the LSF "bands 1" is overlooked, the percentage of FP decreases from 36% to 10% but the percentage of false negatives (FN) increases from 22% to 58%. In order to improve the appreciation of LSF by decreasing the need of manual revisions, the visual examination of the leucocyte distribution scattergram (LDS), also provided by the MAXM, was conveniently evaluated. This study was performed on 190 blood specimens from which the MAXM displayed a normal DLC in 122 (64%), the LSF of "bands" in 44 (23%) and the LSF of "bands 2" in 24 (12.6%). Of from the 122 specimens with normal DLC, four were FN, of from the 44 specimens with "bands 1" LSF, 37 were FP and of from the 24 specimens with "bands 2" LSF, 16 were FP. The visual appreciation of the LDS showed in the majority of samples with "bands 1" and "bands 2" a definitely different shape consisting in a sharper image up to the top of the picture when compared to samples with normal DLC (without flags). According to this criteria, all the 122 specimens with normal DLC displayed a normal LDS and all the 24 specimens with "bands 2" flag displayed abnormal LDS. Of from the 44 specimens with "bands 1" flag, 26 (59%) showed an abnormal LDS and 18 (41%) a normal LDS. It is noteworthy that of from the 26 specimens with abnormal LDS only 7 were true positive (TP), whereas the 18 specimens with normal LDS all showed a normal DLC according to the reference method. These data allow us to conclude that manual revision was required in 26 out of 68 specimens with "bands 1" and abnormal LDS (13% of the total) and in all the 24 specimens with "bands 2" flag. Therefore by using the information provided by the LDS the need of manual revision decreases to 73% of the total sample with LSF. CONCLUSION: Our results give further support to the idea that th VCS method used by the Coulter MAXM provides a high quality DLC with specific left shift detection.

Evaluation of the Abbott Cell-DYN 3500 hematology analyzer in university hospital.

The Abbott (R) Cell-Dyn 3500 (Abbott CD 3500, Abbott Diagnostics Division, Mountain View, CA) is a fully automated hematology analyzer capable of providing a complete blood count (CBC) profile, including a five-part differential leukocyte count (DLC) and flagging system in this study. The CBC profile and DLC flagging system of the Abbott CD 3500 were evaluated according to the HA-20 protocol of the National Committee for Clinical and Laboratory Standards (NCCLS) and compared to the Technicon H*2 blood analyzer currently used in the authors' laboratory. Linearity, carryover, precision, and stability were all within the limits established by the manufacturer. No significant break-downs were found during the evaluation period. Evaluation of DLC indicated an excellent correlation with the manual reference method for neutrophils, lymphocytes, and eosinophils (r = 0.916, 0.936, 0.967, respectively), a good correlation for monocytes (r = 0.811) and a poor correlation for basophils (r = 0.224). Overall flagging for morphologic abnormalities displayed higher sensitivity (85%) than specificity (67%), with a false-positive ratio of 33%. In general, these results are in accordance with those obtained by other authors in the same period of time.

Sclerotherapy with or without octreotide for acute variceal bleeding.

BACKGROUND: Sclerotherapy is considered the most effective way to stop bleeding from esophageal varices, but acute variceal bleeding is still associated with a high risk of rebleeding and death. We compared sclerotherapy alone with sclerotherapy and octreotide to control acute variceal bleeding and prevent early rebleeding in patients with cirrhosis. METHODS: In a double-blind, prospective trial, 199 patients with cirrhosis and acute variceal bleeding who underwent emergency sclerotherapy were randomly assigned to receive a continuous infusion of octreotide (25 micrograms per hour) or placebo for five days. The primary outcome measure was survival without rebleeding five days after sclerotherapy. RESULTS: After five days, the proportion of patients who had survived without rebleeding was higher in the octreotide group (85 of 98 patients, or 87 percent) than in the placebo group (72 of 101, or 71 percent; 95 percent confidence interval for the difference, 4 to 27 percent; P = 0.009). The mean number of units of blood transfused within the first 24 hours after sclerotherapy was lower in the octreotide group (1.2 units; range, 0 to 7) than in the placebo group (2.0 units; range, 0 to 10; P = 0.006). A logistic-regression analysis showed that the treatment assignment (P = 0.003) and the number of blood units transfused before any other treatment was undertaken (P = 0.002) were the only two variables independently associated with survival without rebleeding. After adjustment for base-line differences between the two groups, the odds ratio for treatment failure in the placebo group, as compared with the octreotide group, was 3.3 (95 percent confidence interval, 1.5 to 7.3). The mean (+/- SD) 15-day cumulative survival rate (estimated by the Kaplan-Meier method) was 88 +/- 12 percent in both groups. Side effects were minor, and their incidence was similar in the two groups. CONCLUSIONS: In patients with cirrhosis, the combination of sclerotherapy and octreotide is more effective than sclerotherapy alone in controlling acute variceal bleeding, but there is no difference between the overall mortality rates associated with the two approaches to treatment.

Hereditary xerocytosis: a report of six unrelated Spanish families with leaky red cell syndrome and increased heat stability of the erythrocyte membrane.

Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na+ + K+ fluxes with low total cation (Na+ + K+) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60 min) and two different temperature values (46 degrees C and 49 degrees C). Under these conditions control RBCs (50 normal subjects) exhibited at 49 degrees C and 30 min a maximum of 30% fragmented RBCs. This value increased to 80% after 60 min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60 min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality. In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.

[Abnormal changes in erythrocyte membrane proteins in hereditary spherocytosis and their relation to clinical and biological aspects of the disease]. / Alteraciones proteicas de la membrana eritrocitaria en la esferocitosis hereditaria y su relación con la expresividad clínica y biológica de la enfermedad.

BACKGROUND: In the present paper we report a study of 20 patients with hereditary spherocytosis (HS) performed with the aim of provide further information on the electrophoretic abnormalities of red blood cell (RBC) membrane proteins and their putative relationship with the clinical, biological and genetic aspects of the disease. METHODS: General hematological parameters, reticulocyte count, osmotic fragility test and erythrocyte morphology analysis, were performed by routine procedures. Membrane proteins of erythrocyte were analyzed by SDS-polyacrylamide gradient gel electrophoresis (SDS-PAGE) using the Laemmli and Fairbanks methods. RESULTS: In 8 out of 20 cases (40%) a defect of band 3, alone or associated with a slight deficiency of protein 4.2, was observed. In addition to the presence of spherocytes, in all these 8 patients, a peculiar morphologic RBC alteration called pincered RBCs was also observed. Moreover, 2 cases showed a deficiency of protein 4.2, 2 cases a deficiency of ankyrin and 2 cases a deficiency of spectrin. In 6 cases (30%) the electrophoretical pattern of the erythrocyte membrane proteins was normal. A significant (r = -0.6; p < 0.01) correlation between the protein 4.2 (pallidin) and the mean corpuscular haemoglobin concentration (MCHC) was found. Also, the multiple regression analysis showed a correlation (r2 = 0.6; p < 0.0001) between the amount of protein 2.1 (ankyrin) and two hematological parameters: the mean corpuscular volume (MCV) and the red cell distribution width (RDW). CONCLUSIONS: The defect of band 3 is the most frequent membrane protein abnormality associated with HS.

[Bone marrow transplantation from unrelated donors in chronic myeloid leukemia: the results in 15 patients]. / Transplante de médula ósea a partir de donantes no emparentados en la leucemia mieloide crónica: resultados en 15 pacientes.

BACKGROUND: Bone marrow transplantation (BMT) from a histocompatible donor is the only curative treatment in chronic myeloid leukemia (CML). Only a minority of patients dispose of an adequate donor from among his/her relatives. The remaining transplant receptors must look to unrelated donors (URD). The experience of the Escuela de Hematología Farreras Valentí (Farreras Valentí School of Hematology, Barcelona, Spain) in BMT from URD in CML in the first chronic phase is herein reported. METHODS: Fifteen patients (9 males and 6 females, median age 33 years; range 14-48 years) were transplanted from October 1988 to May 1994. Serologic identity was expressed in the A, B and D loci in 9 cases and minor incompatibility in 6. Conditioning included total body irradiation and cyclophosphamide in 14 patients and busulphan plus cyclophosphamide in 1. Partial and selective T lymphocyte depletion was performed by elutriation in 7 cases. RESULTS: Primary implant failure was detected in 2 out of 14 risk patients (14%) and secondary failure was observed in 3 out of 12 cases (25%). The actuarial probability of acute graft versus host disease (GVHD) was 55 +/- 15% at 7 weeks with a probability of appearance with an intensity of II-IV of 31 +/- 13%. Five out of 7 patients with a survival of greater than 100 days, developed chronic GVHD (71%). Ten presented fatal complications. In 5 cases, death was due to pulmonary problems. Recurrence of CML was not observed in any of the patients in the series. The probability of disease free survival at 2 years was 30 +/- 12%. CONCLUSIONS: Bone marrow transplantation from an unrelated donor is an effective treatment for a proportion of patients with chronic myeloid leukemia although severe complications are frequent and originate a high mortality.

[Evaluation of 3 automatic systems for measurement of the erythrocyte sedimentation rate]. / Valoración de tres sistemas automáticos para la determinación de la velocidad de sedimentación globular.

PURPOSE: To evaluate three automated devices for measuring the erythrocyte sedimentation rate (ESR) (VES-MATIC 60, Menarini(R); SEDISCAN Becton-Dickinson(R) y SEDIMATIC, Ral(R)) by comparison with the Westergren method (WM). MATERIAL AND METHODS: A total of 1576 whole blood samples (VM: 694, SC: 316 and SM: 566) from patients of the Hospital Clínic i Provincial de Barcelona were included in this study. In all the specimens, the ESR was determined following the ICSH recommendations (WM). The Student's t test for paired data and linear regression analysis were used for inaccuracy study. Reproducibility was assessed after five measurements of three different samples and establishing the coefficient of variation (CV). RESULTS: Significant correlation was found between the systems studied and th WM. Moreover, for ESR > 21 mm/h (groups II, III and IV) the results provided by VM system were not significantly different from those of WM. Finally, all the systems presented a good reproducibility, although the lower values of CV were obtained with the VM method. CONCLUSIONS: The automatic systems for measurements of the ESR demonstrate important advantages and, from this analysis, we concluded that the VM could be the alternative method to conventional Westergren for the ESR determination.

Behavior of spores of Penicillium roquefortii during fed-batch bioconversion of octanoic acid into 2-heptanone.

The bioconversion of octanoic acid into 2-heptanone by spores of Penicillium roquefortii is performed using a fed-batch technique with pH control by addition of the liquid substrate itself. The early stage of this process takes place with a high bioconversion rate and high yield. These values then decrease as a result of germination and growth the biocatalyst. An optimization strategy for the process would thus be to improve the characteristics of this first period, i.e., increase its duration and the reaction rate. An increase in duration is evidenced in two cases: (I) under oxygen limitation: and (ii) when the spore content in the medium is less than 10(7) spores/mL. These conditions give insufficient overall bioconversion rates: better optimization should be achieved without oxygen limitation and with high spore content. Characterization of the first period by material and bioenergetic balances suggests that an increase in the ethanol content of the medium, which acts as an energy source and a permeabilizer, and the use of specific inhibitor of the Krebs cycle, may be a way to further improve the biocatalyst performance and stability.

Randomised trial of prevention of biliary stent occlusion by ursodeoxycholic acid plus norfloxacin.

Biliary stents are liable to clog. We investigated whether a choleretic plus an antibiotic could delay clogging. 20 consecutive patients with a malignant biliary stricture were randomised after endoscopic insertion of a polyethylene stent to receive ursodeoxycholic acid plus norfloxacin (13-15 mg/kg and 400 mg, daily) or conservative treatment. The drug combination was associated with: a longer median patency of first (49 vs 6 weeks) and all stents (38 vs 7 weeks); a prolonged median survival (67 vs 18 weeks); and a shorter mean hospital stay (0.2 vs 1.0 days per week of survival). Thus ursodeoxycholic acid plus norfloxacin may prevent stent clogging.

[Evaluation of the reproducibility of automated monocyte counting using flow cytometry and monoclonal antibodies]. / Evaluación de la fiabilidad del recuento automático de monocitos mediante citometría de flujo y anticuerpos monoclonales.

PURPOSE: To compare the results of the monocyte count provided by autoanalysers with those attained with flow cytometry. MATERIAL AND METHODS: Fifty-six blood samples (EDTA-K3) from the emergency laboratory were studied. The automatic percentage of monocytes attained by four autoanalysers, STKS (Coulter-IZASA), Technicon H*2 (Bayer Diagnostics), Cell-Dyn-3000 (Abbott Diagnostics) and NE-8000 (TOA-Sysmex) was confronted with that provided by the flow cytometry analysis performed with a FACScan (Becton-Dickinson) using monoclonal antibodies of myeloid-monocytic lineage (CD14, CD16). The morphologic observation of the blood smear in accordance with the H20-A protocol of the National Committee for Clinical Laboratory Standards (NCCLS) was used as a reference method. Descriptive statistics plus linear regression analysis, along with the Student's t test for paired data were used for the statistical evaluation of the results. RESULTS: Significant correlation was found between the systems under study and the flow cytometry method: Technicon H*2 (y = 1.79 +/- 0.61x; r = 0.91, p < 0.0001), Coulter STKS (y = 0.61 +/- 0.93X; r = 0.89, p < 0.0001), Cell-Dyn 3000 (y = 0.88 + 0.64x; r = 0.78, p < 0.0001) and Sysmex NE-8000 (y = 4.60 +/- 0.33x; r = 0.52, p < 0.0001). When comparing the percentage of monocytes by means of the Student's t test it was found that Coulter STKS provided the closest results with regard to flow cytometry (13.78 +/- 9.27 vs 14.19 +/- 8.88, p = 0.49), while the closest findings with respect to the reference method were given by Technicon H*2 (9.29 +/- 4.78 vs 9.71 +/- 4.77, p = 0.30). CONCLUSIONS: From this analysis, it was concluded that flow cytometry could be an alternative method to conventional optic observation in the evaluation of the differential count of leucocytes, including monocytes.