Christoph Robier

Nitric Oxide-Related Biological Pathways in Patients with Major Depression.

Background Major depression is a well-known risk factor for cardiovascular diseases and increased mortality following myocardial infarction. However, biomarkers of depression and increased cardiovascular risk are still missing. The aim of this prospective study was to evaluate, whether nitric-oxide (NO) related factors for endothelial dysfunction, such as global arginine bioavailability, arginase activity, L-arginine/ADMA ratio and the arginine metabolites asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) might be biomarkers for depression-induced cardiovascular risk. Methods In 71 in-patients with major depression and 48 healthy controls the Global Arginine Bioavailability Ratio (GABR), arginase activity (arginine/ornithine ratio), the L-arginine/ADMA ratio, ADMA, and SDMA were determined by high-pressure liquid chromatography. Psychiatric and laboratory assessments were obtained at baseline at the time of in-patient admittance and at the time of hospital discharge. Results The ADMA concentrations in patients with major depression were significantly elevated and the SDMA concentrations were significantly decreased in comparison with the healthy controls. Even after a first improvement of depression, ADMA and SDMA levels remained nearly unchanged. In addition, after a first improvement of depression at the time of hospital discharge, a significant decrease in arginase activity, an increased L-arginine/ADMA ratio and a trend for increased global arginine bioavailability were observed. Conclusions Our study results are evidence that in patients with major depression ADMA and SDMA might be biomarkers to indicate an increased cardiovascular threat due to depression-triggered NO reduction. GABR, the L-arginine/ADMA ratio and arginase activity might be indicators of therapy success and increased NO production after remission.

Branched-Chain Amino Acids as New Biomarkers of Major Depression - A Novel Neurobiology of Mood Disorder.

Background The proteinogenic branched-chain amino acids (BCAAs) valine, leucine and isoleucine might play an unrecognised crucial role in the development of depression through their activation of the mammalian target of rapamycin (mTor) pathway. The aim of this research project is to evaluate whether BCAAs are altered in patients with major depression and might thus be appropriate biomarkers for major depression. Methods The concentrations of valine, leucine and isoleucine were determined in 71 in-patients with major depression and 48 healthy controls by high-pressure liquid chromatography. Psychiatric and laboratory assessments were obtained at the time of in-patient admittance. Results The BCAAs are significantly decreased in patients with major depression in comparison with healthy subjects (valine: Mann-Whitney-U: 968.0; p <0.0001, leucine: Mann-Whitney-U: 1246.5; p = 0.013, isoleucine: Mann-Whitney-U: 1252.5; p = 0.014). Furthermore, as shown by Spearmans rank correlation coefficients, there is a significant negative correlation between valine, leucine and isoleucine concentrations and the Hamilton Depression Rating Scale (HAMD-17) as well as Beck Depression Inventory (BDI-II) scores. Conclusions Our study results are strong evidence that in patients with major depression, BCAAs might be appropriate biomarkers for depression. Reduced activation of the mammalian target of rapamycin (mTor) due to a reduction of BCAAs might play a crucial unrecognised factor in the etiology of depression and may evoke depressive symptomatology and lower energy metabolism in patients with major depression. In the future, mTor and its up- and downstream signalling partners might be important targets for the development of novel antidepressants.

Thrombotic Microangiopathy and Disseminated Intravascular Coagulation Associated With Carcinocythemia in a Patient With Breast Cancer

Case Report A 66-year-old white female was admitted to the emergency department because of increasing dyspnea in September 2008. Medical history included lobular and ductal invasive carcinoma of the right breast with hepatic, skeletal, and pulmonary metastases already present at the time of diagnosis in August 2007. Six cycles of adjuvant chemotherapy with epirubicin and cyclophosphamide were administered between August 2007 and December 2007 but failed to induce remission. Pulmonary computed tomography and computed tomography angiography revealed carcinomatous lymphangiosis and intrapulmonal metastases but no evidence of pulmonary embolism, lung edema, or pneumonia. Laboratory evaluation showed hemolytic anemia with hemoglobin concentrations of 8.8 g/dL, RBC count of 2.41 10/L, mean corpuscular volume of 104 fl, mean corpuscular hemoglobin of 36.3 pg, reticulocyte count of 0.283 10/L (normal range: 0.032 to 0.082 10/L), lactate dehydrogenase concentration of 1,661 U/L (normal range: 120 to 240 U/L), total bilirubin concentration of 3.25 mg/dL (normal range: 0.3 to 1.2 mg/dL) and haptoglobin concentration less than 7 mg/dL (normal range: 30 to 200 mg/dL). Furthermore, thrombocytopenia of 109 10/L and leukocytosis of 18.5 10/L were found. The activated partial thromboplastin time was 44 seconds (normal range: 26 to 37 seconds), quick value of 39% (normal range: 70% to 130%), fibrinogen concentration of 116 mg/dL (normal range: 210 to 400 mg/dL), antithrombin 55% (normal range: 75% to 125%) and D-dimer concentration of 28,640 g/L (normal range: 0 to 550 g/L), which indicated disseminated intravascular coagulation (DIC). Examination of a peripheral blood smear stained with May-Grünwald-Giemsa revealed leukoerythroblastosis, severe microangiopathic changes with 16% schistocytes, and atypical blastlike cells less than 1%. These cells showed large nuclei with one or more prominent nucleoli and slightly gray to basophilic cytoplasm and tended to occur in clusters of up to four cells (Fig 1A). Immunocytochemistry by using the cytokeratin antibody CK AE1/3 (Dako, Copenhagen, Denmark) disclosed the epithelial origin or the cells (Fig 1B). The clinical course was characterized by progressive dyspnea and extensive subcutaneous hemorrhage. Laboratory follow-up showed a marked decrease of the platelet count (20 10/L) and fibrinogen concentration (94 mg/dL) as well as an increase in the D-dimer (31,320 g/L), lactate dehydrogenase (2,388 U/L) and bilirubin (4.64 mg/dL) concentrations. The patient was treated with fresh frozen plasma, platelet transfusions, and packed red cells. Nevertheless, she died on the third day after admission. On autopsy, widespread occlusions of lung capillaries with fibrin thrombi and cytokeratin-19–positive, epithelial tumor cells were demonstrated.

Evaluation of platelet function and pharmacological platelet inhibition in patients with myeloproliferative disorders using multiple electrode aggregometry

BACKGROUND The aim of this study was to describe platelet aggregation characteristics by multiple electrode aggregometry (MEA) and to evaluate MEA for its potential to detect platelet dysfunction and response to anti-aggregatory drugs in patients with myeloproliferative disorders (MPD). METHODS We compared the platelet response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) in hirudin-anticoagulated blood of 55 patients with polycythaemia vera and essential thrombocythaemia and 75 controls. RESULTS Comparing MPD patients and controls no statistically significant difference indicative of platelet dysfunction was found in MPD patients. Analysis of covariance revealed platelet- and leukocyte count as a significant influencing factor on MEA function. Furthermore we could demonstrate that ASA and clopidogrel treatment results in a statistically significant lower ASPI (Controls: p<0.0001, MPD: p<0.0001) and ADPtest value (MPD: p=0.00125) compared to untreated patients thereby validating the method for monitoring of anti-aggregatory therapy. CONCLUSION In this study MEA was confirmed as a valid method for monitoring of ASA and clopidogrel treatment in patients with MPD and normal control subjects. The platelet and leukocyte count were identified as major influencing factors on MEA aggregation tests both in MPD patients and controls. No functional platelet abnormalities were detected in MPD patients.

Comparison of the clinical utility of the Elia CTD Screen to indirect immunofluorescence on Hep-2 cells.

Abstract Background: We compared the Elia CTD Screen (ECS), a fluoroenzymeimmunoassay incorporating 17 human antinuclear antigens (ANA), with indirect immunofluorescence (IIF) on Hep-2 cells in order to determine the clinical utility of the ECS in additon to or without IIF. Methods: We examined 1708 consecutive serum samples submitted for ANA testing using the ECS and IIF in parallel. Positive screen results were further examined by quantitative fluoroenzymeimmunoassays and/or immunoblots for antibody identification. The medical records were evaluated for systemic rheumatic disorders. Results: Concordance between ECS and IIF was observed in 1344 (78.8%) samples. ECS had a better detection rate for anti-dsDNA, -SSA/Ro, -SSB/La, -U1RNP and -Jo-1 antibodies, whereas IIF was superior in the detection of anti-CENP-B antibodies as well as anti-histone, -nucleosome and -Pl-12 antibodies, which are not included in the ECS antigen panel. ECS had a 100% sensitivity for Sjögren’s syndrome, systemic sclerosis and Sharp syndrome. The sensitivity for Sjögren’s syndrome was slightly higher for ESC than for IIF (94%). IIF had a higher diagnostic sensitivity for systemic lupus erythematosus, indeterminated connective tissue disease, Raynaud’s syndrome and limited scleroderma, compared to ESC (100% vs. 80%, 100 vs. 75%, 89 vs. 57%, 100 vs. 88.9%). Conclusions: Our results suggest that the ECS represents an appropriate diagnostic tool for ANA screening. However, since some antigens are not incorporated in the ECS panel, and some ANA can also be missed by IIF, sequential or parallel screening with ECS and IIF may be reasonable when the clinical suspicion for connective tissue disease is high.

The anti-VLA-4 antibody natalizumab induces erythroblastaemia in the majority of the treated patients with multiple sclerosis.

The presence of erythroblasts in the peripheral blood is generally associated with severe underlying disorders. The anti-very late antigen-4 (anti-VLA-4) antibody natalizumab, which is approved for treatment of multiple sclerosis, mediates an increase in circulating haematopoietic stem cells and may also trigger erythroblastaemia. We investigated the prevalence of erythroblastaemia in sequential blood smears of 14 natalizumab-treated and 14 interferon-treated patients with multiple sclerosis. Erythroblastaemia was found in 13 natalizumab-treated subjects (93%), whereas all controls were negative (p<0.0001). Knowledge of this frequent side effect is crucial for the correct interpretation of blood smears in natalizumab-treated patients and to avoid unnecessary diagnostic procedures.

Hirudin-induced pseudothrombocytopenia in a patient with EDTA-dependent platelet aggregation: report of a new laboratory artefact

Sir, anticoagulant-induced pseudothrombocytopenia (PTP) is an important cause of spuriously low platelet counts described for all haematology analysers used in clinical routine diagnostics. If misinterpreted, it may lead to serious diagnostical and therapeutical consequences like bone marrow biopsy, initiation of corticosteroid therapy, platelet transfusion or even splenectomy (Zandecki et al., 2007). PTP has been reported for different anticoagulants such as ethylenediamine tetra-acetic acid (EDTA), citrate, heparin and oxalate, occurring with an incidence of 0.1–0.2% for EDTA, which is the most commonly used agent for automated blood counts (Schrezenmeier et al., 1995). We describe the case of an 82-year-old male patient admitted to hospital for trauma surgery. Initial laboratory exploration performed with EDTA (Sarstedt, Nuembrecht, Germany) as anticoagulant on a Cell-Dyn-Sapphire (Abbott, Abbott Park, IL, USA) showed a platelet count of 98 000/ll. A platelet flag and a note reporting platelet aggregates was given, the white blood cell (WBC) scattergrams yielded small dots above and within the lymphocyte area. Blood smear investigation confirmed the suspected occurrence of platelet aggregates. In suspicion of EDTA-induced PTP, further work-up is including the collection of a capillary blood specimen and a venous sample anticoagulated by sodium citrate for performing an automated platelet count and examination of blood smears for revelation of platelet aggregates (Payne & Pierre, 1984). In addition to these routine procedures, we collected a hirudin-anticoagulated venous blood specimen (Sarstedt, 25 lg/ml hirudin) searching for alternative anticoagulants in diagnosis of PTP. In the hirudinsample, the automated platelet count was 41 000/ll, a platelet aggregation flag was given and the WBC histograms corresponded to the results of the EDTA-examination. Exploration of a blood smear revealed large platelet aggregates. The citrate-anticoagulated specimen (Sarstedt) showed the platelet count within the reference range (206 000/ll) without reporting of any flags, the blood smear did not show platelet aggregates. In accordance to these findings, the diagnosis of EDTAand hirudininduced PTP was made. Anticoagulants and associated platelet counts are summarized in Table 1. To our knowledge this is the first description of hirudin-induced PTP. Anticoagulant-induced pseudothrombocytopenia because of chelating anticoagulants like EDTA, citrate and oxalate has been extensively studied. Chelation-dependent depletion of membrane calcium leading to antibody reactions with newly formed membrane antigens has been postulated as potential mechanism (Onder, Weinstein & Hoyer, 1980). Such immunoglobulines (Ig) are mostly of IgG or IgM type and represent so called ‘natural autoantibodies’ with antiplatelet activity, but without any pathological property in vivo (Bizzaro, 1995). The background of PTP development because of direct thrombin inhibitors like hirudin is not clear. Potential mechanisms are antibody reactions, including either unspecific or specific antibodies after contact with recombinant hirudin or the medicinal leech (hirudo medicinalis), as well as nonimmunologically mediated platelet agglutination caused by complex building of hirudin with other non-Ig proteins like thrombin. Thrombin is known to cause platelet agglutination in vitro (Watkins & Shulman, 1970). Lepirudin, a recombinant hirudin analogue, is used as alternative anticoagulant in patients with heparin-induced thrombocytopenia (HIT) II (Lubenow et al., 2004). In a study population of 196 patients with HIT II under prolonged lepirudin therapy, 44.4% developed antihirudin antibodies causing enhanced anticoagulatory drug activity (Eichler et al., 2000). Thrombocytopenia because of hirudin is a rarely reported, severe LETTER TO THE EDITOR INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

Revisiting the tryptophan-serotonin deficiency and the inflammatory hypotheses of major depression in a biopsychosocial approach

Background The aim of this cross-sectional study was to identify important biopsychosocial correlates of major depression. Biological mechanisms, including the inflammatory and the tryptophan-serotonin deficiency hypotheses of major depression, were investigated alongside health-related quality of life, life satisfaction, and social support. Methods The concentrations of plasma tryptophan, plasma kynurenine, plasma kynurenic acid, serum quinolinic acid, and the tryptophan breakdown to kynurenine were determined alongside health-related quality of life (Medical Outcome Study Form, SF-36), life satisfaction (Life Satisfaction Questionnaire, FLZ), and social support (Social Support Survey, SSS) in 71 depressive patients at the time of their in-patient admittance and 48 healthy controls. Results Corresponding with the inflammatory hypothesis of major depression, our study results suggest a tryptophan breakdown to kynurenine in patients with major depression, and depressive patients had a lower concentration of neuroprotective kynurenic acid in comparison to the healthy controls (Mann–Whitney-U: 1315.0; p = 0.046). Contradicting the inflammatory theory, the concentrations of kynurenine (t: −0.945; df = 116; p = 0.347) and quinolinic acid (Mann-Whitney-U: 1376.5; p = 0.076) in depressive patients were not significantly different between depressed and healthy controls. Our findings tend to support the tryptophan-serotonin deficiency hypothesis of major depression, as the deficiency of the serotonin precursor tryptophan in depressive patients (t: −3.931; df = 116; p < 0.001) suggests dysfunction of serotonin neurotransmission. A two-step hierarchical linear regression model showed that low tryptophan concentrations, low social support (SSS), occupational requirements (FLZ), personality traits (FLZ), impaired physical role (SF-36), and impaired vitality (SF-36) predict higher Beck Depression Inventory (BDI-II) scores. Discussion Our study results argue for the validity of a biopsychosocial model of major depression with multiple pathophysiological mechanisms involved.

Marked elevation of procalcitonin in a patient with a drug related infusion reaction to rituximab.

*Corresponding author: Christoph Robier, MD, Central Laboratory, Hospital of the Brothers of St. John of God, Graz, Bergstr. 27, 8020 Graz, Austria, Phone: +43 316 5989 6671, Fax +43 316 5989 1505, E-mail: christoph.robier@bbgraz.at; and Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria Manfred Neubauer and Gerhard Reicht: Department of Internal Medicine, Hospital of the Brothers of St. John of God, Graz, Austria Letter to the Editor

Crystal-associated pseudoeosinophilia of synovial fluid

Determination of the number of white blood cells (WBCs) in the synovial fluid (SF) is the most important laboratory method for the discrimination between inflammatory and non-inflammatory joint diseases (1). The SF leukocytes can be counted microscopically in a Neubauer chamber or with automated hematology analysers (2). Knowledge of laboratory artefacts, such as pseudoleukocytosis is crucial for the correct interpretation of automated leukocyte counts. Misinterpretation may lead to associated clinical errors. SF pseudoleukocytosis, an artificially increased SF leukocyte count, may result from synovial cells, crystals, fat globules, cartilage fragments or chondrocytes (3, 4). To our knowledge, pseudoeosinophilia of SF has not been described previously. We report five cases of patients with crystal-related arthropathies who showed marked pseudoeosinophilia using automated examination of SF. Using polarized microscopy, three where diagnosed with acute gouty arthritis while the two other patients had arthropathies associated with calcium pyrophosphate dihydrate (CPPD) crystals, each with a high concentration of crystals. Articular calcium deposits found on X-rays, being indicative of chondrocalcinosis, were present in one case. The other patient with CPPD crystals had been diagnosed with osteoarthritis of the shoulder. Two of the patients with gout, each presenting with clinical signs of acute gonarthritis, had increased serum urate concentrations (11.0 and 9.1 mg/dL). For the third patient, no laboratory data apart from SF analysis were available. Automated SF differential counts performed with a CellDyn 3200 (Abbott, Abbott Park, IL, USA) showed eosinophilia of 41.5%–54.8% of leukocytes. Microscopic examination of May-Grünwald-Giemsa stained slides showed an acute inflammatory state in four patients and non-inflammatory SF in one case; no eosinophils were found. The clin-