Recurrent finding of the FIP1L1-PDGFRA fusion gene in eosinophilia-associated acute myeloid leukemia and lymphoblastic T-cell lymphoma.

The FIP1L1-PDGFRA fusion gene has been described in patients with eosinophilia-associated myeloproliferative disorders (Eos-MPD). Here, we report on seven FIP1L1-PDGFRA-positive patients who presented with acute myeloid leukemia (AML, n=5) or lymphoblastic T-cell non-Hodgkin-lymphoma (n=2) in conjunction with AML or Eos-MPD. All patients were male, the median age was 58 years (range, 40-66). AML patients were negative for common mutations of FLT3, NRAS, NPM1, KIT, MLL and JAK2; one patient revealed a splice mutation of RUNX1 exon 7. Patients were treated with imatinib (100 mg, n=5; 400 mg, n=2) either as monotherapy (n=2), as maintenance treatment after intensive chemotherapy (n=3) or in overt relapse 43 and 72 months, respectively, after primary diagnosis and treatment of FIP1L1-PDGFRA-positive disease (n=2). All patients are alive, disease-free and in complete hematologic and complete molecular remission after a median time of 20 months (range, 9-36) on imatinib. The median time to achievement of complete molecular remission was 6 months (range, 1-14). We conclude that all eosinophilia-associated hematological malignancies should be screened for the presence of the FIP1L1-PDGFRA fusion gene as they are excellent candidates for treatment with tyrosine kinase inhibitors even if they present with an aggressive phenotype such as AML.

Diagnosis of invasive fungal infections in haematological patients by combined use of galactomannan, 1,3-ß-D-glucan, Aspergillus PCR, multifungal DNA-microarray, and Aspergillus azole resistance PCRs in blood and bronchoalveolar lavage samples: results of a prospective multicentre study.

High mortality rates of invasive fungal disease (IFD), especially invasive aspergillosis (IA), in immunocompromised haematological patients and current diagnostic limitations require improvement of detection of fungal pathogens by defining the optimal use of biomarkers and clinical samples. Concurrent bronchoalveolar lavage (BAL) and peripheral blood samples of 99 haematological patients with suspected IFD were investigated within a multicentre prospective study. Diagnostic performance of a galactomannan (GM) enzyme immune assay (EIA), a 1,3-ß-D-glucan assay (BDG), an Aspergillus PCR, and a multifungal DNA-microarray (Chip) alone or in combination were calculated. IFD were classified as proven (n=3), probable (n=34), possible (n=33), and no IFD (n=29) according to EORTC/MSG criteria. GM, PCR, and Chip showed superior diagnostic performance in BAL than in blood, whereas specificity of BDG in BAL was poor (48% (14/29)). The combination of GM (BAL) with BDG (blood) showed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and DOR (diagnostic odds ratio) of 92% (34/37), 93% (27/29), 94%, 90%, and 153.0, respectively. Combining GM (BAL) with PCR (BAL) showed convincing diagnostic potential for diagnosing IA with sensitivity, specificity, PPV, NPV, and DOR of 85% (17/20), 97% (28/29), 94%, 90%, and 158.7. Addition of the DNA-microarray resulted in further detection of two mucormycetes infections. In 1 out of 15 Aspergillus DNA-positive samples a triazole resistance-mediating Cyp51A mutation was found. Combination of biomarkers is superior to their sole use in diagnosing IFD, particularly IA. Integrating blood and BAL samples into a diagnostic algorithm is an advantageous approach.

Screening Fanconi anemia lymphoid cell lines of non-A, C, D2, E, F, G subtypes for defects in BRCA2/FANCD1.

Biallelic mutations in BRCA2/FANCD1 were recently recognized as a rare cause of Fanconi anemia (FA). Using immunodetection with an antiserum directed against the carboxyterminus of the BRCA2 protein, we screened 38 lymphoid cell lines from FA patients whom we could not previously assign, via retroviral complementation analysis, to any of six known FA complementation groups (FA-A, -C, -D2, -E, -F, or -G). Three of these 38 cell lines lacked the 380-kDa BRCA2 signal on immunoblots. DNA sequencing showed biallelic compound and truncating mutations in two of the immuno-negative cell lines, whereas a monoallelic frameshift mutation and an amino acid substitution were detected in the third cell line. Our data show that less than 10% of unassigned FA cell lines harbor truncating mutations in BRCA2/FANCD1. This finding strongly suggests the existence of (an) additional, as yet unknown FA gene(s).

Autologous red cell recovery and relapse of multiple myeloma after bone marrow transplantation: is this a graft-versus-myeloma effect?

We report a patient who, following an allogeneic bone marrow transplant for multiple myeloma, recovered autologous erythropoiesis which was rapidly followed by relapse of her multiple myeloma. We postulate that the loss of the graft (as demonstrated by loss of donor erythropoiesis) and subsequent relapse of the multiple myeloma may be support for the existence of a graft-versus-myeloma effect.

In vivo comparison of THC:YAG laser welding to sutured closure of biliary tissue.

Percutaneous endoscopic approaches to cholelithiasis are an alternative to extracorporeal shockwave lithotripsy which offer advantages of fewer restrictions on stone size and type as well as avoidance of fragmented stone passage complications. Endoscopic techniques would be facilitated by methods of welding gallbladder tissues. The technical constraints imposed by the endoscopic approach favor nonsuture methods of fusing gallbladder tissues. To evaluate a laser method for fusing biliary tissue, we have compared the healing response of laser-welded versus polyglycolic acid suture-closed incisions in canine gallbladder tissue in vivo. The laser used was a thulium-holmium-chromium:YAG laser producing 200-microsecond, 300-millijoule pulses at 2.15 microns. Serial sacrifice of dogs that underwent laser or suture closure of incisions made in the fundus of the gallbladder revealed that all repairs healed without evidence of leakage or infection. Laser-welded cholecystotomy sites had complete fibrous healing of the wound by two weeks postoperatively and reepithelialization by three weeks after operation. Suture-closed wounds were still without complete epithelization four weeks after the procedure. Laser welding may be a useful technique in endoscopic biliary surgery.

Development of a flow cytometric test for the detection of D-positive fetal cells after fetomaternal hemorrhage and a survey of the prevalence in D-negative women.

A sensitive test for the presence of D-positive fetal red blood cells (RBCs) in the maternal circulation of D-negative women has been developed. It was used to investigate the possibility that the occasional failure in preventing alloimmunization might be due to the administration of inadequate amounts of prophylactic anti-D Rh immune globulin. The standard dose in Australia contains 125 microg of antibody, and can suppress immunization by an estimated 6 mL of packed D-positive RBCs. A fetomaternal hemorrhage (FMH) of this volume is detectable in the maternal circulation as approximately 0.25 percent of the total RBCs. Our test utilizes a commercially available human monoclonal IgG anti-D that has been biotinylated and used with a dye-conjugated streptavidin. Flow cytometry is used to quantitate fluorescing D-positive RBCs. To date, 2,288 tests have been performed on blood samples from D-negative women attending local antenatal clinics or at the time of delivery. Evidence for an FMH has been obtained in six cases (0.26%). In one case, the FMH was only 0.1 percent, and in another (confirmed by the Kleihauer-Betke method), fetal cells constituted only 0.2 percent. Additional Rh immune globulin was not given to these patients. In the other four cases, the D-positive fetal cells were estimated to be 0.7,0.5,0.5, and 0.4 percent, and additional prophylactic Rh immune globulin was administered. Although the prevalence of FMH is low, screening D- negative women at risk of alloimmunization has proved to be simple, fast, and inexpensive.

Quantifying the loss of ABO antigenicity in a patient with acute myeloid leukemia by flow cytometric analysis.

The loss of B antigenicity from the red blood cells of a patient with acute myeloid leukemia is reported. The patient had normal B transferase levels, but had reduced levels of H transferase. Flow cytometry was used to quantify the loss of B antigenicity and monitor the expression of the B antigen throughout the progression of the disease.

A knowledge-based approach to intelligent alarms in anesthesia.

The combination and aggregation of separate information sources to generate more ;intelligent' alarms using a knowledge-based approach is described. The approach gas developed as part of the knowledge-based anesthesia decision support system, AES-2, which monitors the patient's physiological state during anesthesia and eventually suggests therapeutic actions. Integration of the alarm system into a conventional information system is discussed. The state variable model used and the modeling of uncertainty are examined. The tools that support knowledge acquisition, design of knowledge-base prototypes. and test of the knowledge base of the AES-2 are described. A simulation example is given.

Technical development of UV-C- and VUV-photochemically induced oxidative degradation processes.

Technical development work is presented, where the VUV photochemically induced oxidative degradation is used: (i) for analytic purposes, and (ii) for small to medium scale (< 10 m2/d) waste water treatment processes or ultrapure water production. In the first case, small Xe-excimer radiation sources with an integrated reaction space designed for optimal conditions, as far as incident photon flux density, turbulence and concentration of dissolved molecular oxygen are concerned, have been built and tested. Under conditions of exhaustive oxidation and/or mineralization of pollutants in a continuous regime, they may be used for sample pre-treatment modules prior TOC, TOX and electrochemical trace metal analysis. Under conditions of partial oxidation or mineralization, the same lamp/reactor combination may be used for functionalization purposes prior to e.g. GC or HPLC analyses. In the second case, mass transfer limitations between the non-irradiated bulk volume and the irradiated volume are overcome by the electrochemical generation of molecular oxygen within or close to the irradiated volume and by the design of the photochemical part of the reactor.

Identification of recurring tumor-specific somatic mutations in acute myeloid leukemia by transcriptome sequencing.

Genetic lesions are crucial for cancer initiation. Recently, whole genome sequencing, using next generation technology, was used as a systematic approach to identify mutations in genomes of various types of tumors including melanoma, lung and breast cancer, as well as acute myeloid leukemia (AML). Here, we identify tumor-specific somatic mutations by sequencing transcriptionally active genes. Mutations were detected by comparing the transcriptome sequence of an AML sample with the corresponding remission sample. Using this approach, we found five non-synonymous mutations specific to the tumor sample. They include a nonsense mutation affecting the RUNX1 gene, which is a known mutational target in AML, and a missense mutation in the putative tumor suppressor gene TLE4, which encodes a RUNX1 interacting protein. Another missense mutation was identified in SHKBP1, which acts downstream of FLT3, a receptor tyrosine kinase mutated in about 30% of AML cases. The frequency of mutations in TLE4 and SHKBP1 in 95 cytogenetically normal AML patients was 2%. Our study demonstrates that whole transcriptome sequencing leads to the rapid detection of recurring point mutations in the coding regions of genes relevant to malignant transformation.

ELDAR, a knowledge base system on microcomputer for electrolyte solutions. The factual knowledge of ELDAR.

The knowledge base system ELDAR (ELectrolyte DAta Regensburg), consisting of data base, method base, rule base, and communication manager, classifies the knowledge of electrolyte solutions into factual, algorithmic, and rule knowledge. In this paper information is given on the factual knowledge of ELDAR and the mapping of facts in Codd's relational data model with an extension of its "1st Normal Form" to repeating attributes. ELDAR offers equal user interfaces for all factual knowledge services, such as literature, data, thesaurus, module, parameter, basic data, and rule retrieval.

Rapid detection of Rh(D)- or K-positive fetal red cells in chorion villus samples by a flow cytometric technique.

Rh(D)- and K-negative women who have become severely isoimmunized by pregnancy are at risk of fetal loss or damage in subsequent pregnancies. A flow cytometric method is described whereby the presence of Rh(D) or K antigen on fetal erythrocytes may be determined using chorion villus samples taken during the first trimester. This method has the advantage of speed and sensitivity with results being available within 2 h. Decisions as to management of the pregnancy or termination may thus be made with minimal delay.

Rapid quantitation of mixed red cell populations using flow cytometry.

Methods are described for the identification and quantitation of mixed red cell populations using flow cytometry. Antibodies specific for a wide range of blood group antigens have been used and examples are given in which these analyses have proved to be of clinical use. These examples include monitoring of erythropoiesis following engraftment in allogeneic bone marrow transplant recipients and the detection of chimaeric states months or years after transplantation. The techniques involved are fast, simple and inexpensive.

A flow-cytometric equivalent of the Kleihauer test.

BACKGROUND AND OBJECTIVES: The Kleihauer slide test is in general use to screen obstetric patients for possible fetomaternal haemorrhage. Since 1993, Rh(D)-negative patients have been tested in our laboratory by a flow-cytometric method detecting Rh(D)-positive fetal cells, a method which offers improved sensitivity and accuracy. We report another flow-cytometric method of broader application which quantitates cells according to haemoglobin F (HbF) content. MATERIALS AND METHODS: The red cells are fixed with glutaraldehyde and permeabilized by exposure to Triton X-100. A polyclonal sheep antibody to HbF is incubated with the cells followed by a fluorescein-labelled anti-sheep antibody. RESULTS: Quantitation of the percentage of fetal cells following a FMH can be achieved irrespective of the blood groups of either mother or infant, and the presence of maternal F cells need not interfere since the intensity of staining is usually less than that of fetal cells. Two of 19 transfusion-dependent patients with beta-thalassaemia have been found to have red cells indistinguishable from fetal cells on the basis of HbF content, but these patients also have been found to give positive results by the Kleihauer test. CONCLUSIONS: The flow-cytometric method may serve to replace the traditional Kleihauer test since it appears to offer improved accuracy and objectivity.

An interactive computer simulator of the circulation for knowledge acquisition in cardio-anesthesia.

Knowledge-based decision support systems for use in cardio-anesthesia can provide online support to the anesthesiologist by generating intelligent alarms. However, the acquisition and validation of a consistent knowledge base for this application bears problems related to the transfer of clinical experiences into a rule system. An interactive simulator of the human circulation is presented that supports the process of knowledge acquisition and testing. The simulator can be controlled in realtime by an anesthesiologist during the simulation run thus providing a basis for interdisciplinary discussion of routine as well as critical situations. The output data can be transferred to a knowledge-based system for test purposes. The simulator is currently being used for the development of the Anesthesia Expert Assist System AES-2. With regard to the special application a model of the heart-function was integrated which enables the simulation of heart insufficiency. Simulation runs under various conditions are presented and discussed. The simulator was implemented on an ATARI ST personal computer.

Continuous-wave laser pump light sources: new concepts.

The relatively short lifetime of rare-gas discharge pump light sources for high-power cw solid-state lasers is caused mainly by vaporized cathode emitter material reacting with the thermally highly loaded quartz wall. The introduction of new bipolar electrodes without special emitter materials mounted on hot-ended molybdenum-cup seals, together with 60-kHz ac operation, makes possible the application of a tungsten halide cleaning cycle within the lamp bulb. This new type of high-power rare-gas pump light source exhibits 20% better radiation efficiency, 20% lower Nd:YAG laser threshold, and considerably extended lifetime.