Andreas Weimann

Immature platelet count: a simple parameter for distinguishing thrombocytopenia in pediatric acute lymphocytic leukemia from immune thrombocytopenia.

Platelet counts below normal values define thrombocytopenia. However, platelet counts alone do not reveal the underlying pathomechanism. New blood cell counters provide additional information on platelet size and volume, and enable the distinction of sub‐populations. In this preliminary study, we evaluate whether one of these markers can be used for diagnosis of isolated thrombocytopenia in children.

Immature platelet fraction as novel laboratory parameter predicting the course of neonatal thrombocytopenia

Thrombocytopenia occurs in up to 30% of neonates admitted to neonatal intensive care units (NICUs). In preterm neonates, severe thrombocytopenia is associated with increased risk of intracerebral bleeding. Thus, diagnosis of thrombocytopenia often leads to repeated subsequent analysis of blood cell counts and transfusion of platelets. However, no evidence has been provided that current practice for platelet transfusion prevents bleeding complications and improves clinical outcome. A rapid automated method to assess reticulated platelets, the immature platelet fraction (IPF), is available (Briggs et al, 2000). The aim of this study was to investigate the diagnostic value of IPF as a marker for megakaryopoietic activity in neonates. All 612 neonates admitted to our NICUs over a 6-month period were included in the study. Written parental consent and organisational approval were obtained. During the first postnatal week, blood samples were obtained as clinically indicated. To generate reference values for the IPF, patients were divided in two groups: The control group (n = 456) always displayed normal platelet counts (150–450 · 10/l). In the thrombocytopenic group (n = 156), platelet counts dropped below 150 · 10/l at least once. Blood samples obtained after platelet transfusion were excluded from analysis. Platelet counts were optically measured with the automated analyser XE-2100 (Sysmex, Kobe, Japan) equipped with the software xe-ipf-master. Determination of IPF is based on fluorescence flow cytometry using a nucleic acid specific dye that stains RNA in erythrocytic and platelet reticulocytes. The computed algorithm discriminates mature and immature platelet populations applying a preset gate (fluorescent intensity = RNA-content; forward scatter = cell volume). The IPF is normally expressed as a percentage of the platelet count to indicate the rate of platelet production; additionally, absolute values may be used to differentiate between insufficient platelet production and increased consumption as cause of thrombocytopenia. Regression analysis was used to calculate the correlation between platelet counts and IPF. Student’s t-test was applied to compare group means. Statistical analysis was performed using the software statgraphics (Manugistics Inc., Rockville, MD, USA). A P-value < 0Æ05 was considered to indicate statistical significance. In 1045 out of 1339 blood specimens IPF was routinely determined in addition to platelet counts. Four hundred and fifty-six patients (838 specimens) were assigned to the control group, 25Æ5% of patients (n = 156; 501 specimens) to the thrombocytopenic group. The mean [standard deviation (SD)] birth weight in the control group was significantly higher than in the thrombocytopenic group [2717 g, (SD ± 849) vs. 2188 g, (SD ± 105), P < 0Æ001]; this also refers to mean gestational age [36Æ3 weeks, (SD ± 3Æ7) vs. 34Æ2 weeks, (SD ± 5Æ0), P < 0Æ001]. In controls, the mean IPF value was 4Æ3% [95% confidence interval (CI) 0Æ7–7Æ9%] during the first postnatal week. In the thrombocytopenic group, the mean IPF (SD) during the first postnatal week was always significantly higher than in the control group [e.g. day 1: IPF 8Æ7% (9Æ3) vs. 4Æ53% (1Æ8); P < 0Æ001; Fig 1). 36% of thrombocytopenic neonates displayed an IPF above 7Æ9%. We found no significant correlation between gestational age and IPF. Using simultaneous IPF and platelet measurements of both groups, a significant negative correlation between IPF and platelet counts was found with an exponential decay (r = )0Æ62, P < 0Æ001). Increasing platelet counts were anticipated by an increased IPF, reflecting enhanced platelet production (Fig 1). According to the study aim, we analysed whether the platelet counts on the following day were somehow predicted by means of previous IPF values. In patients whose blood samples were analysed on two subsequent days, the difference in platelet counts was plotted against the corresponding IPF value (Fig 2). In this scatter plot, specimens (n = 398) were divided in to four quadrants according to IPF (>8%, which is outside CI > 95% of controls) and difference in platelet counts (severe

Polymorphisms of the toll-like receptor 2 and 4 genes are associated with faster progression and a more severe course of sepsis in critically ill patients

Objective To determine whether the Arg753Gln polymorphism of the toll-like receptor 2 (TLR2) gene and the Asp299Gly polymorphism of the TLR4 gene in critically ill patients affect their clinical outcomes. Methods Medical and surgical patients in three intensive care units (ICU) were enrolled in this prospective study. TLR2 and TLR4 gene polymorphisms were determined using restriction fragment length polymorphism analysis. Results A total of 145 patients were included in this study: 28 patients carried heterozygous mutations (10 in the TLR2 gene, 19 in the TLR4 gene, and one combined) and 117 patients were wild type. Severe sepsis was observed in 33% of wild types (n = 38), 60% of the TLR2 group (n = 6), and 63% of the TLR4 group (n = 12); the difference was significant between the TLR4 and wild type groups. Both TLR groups demonstrated a shorter time-to-onset of severe sepsis or septic shock. Only the TLR4 group demonstrated significant progression towards septic shock compared with the wild type group. Length of ICU stay was significantly prolonged in the TLR4 group compared with the wild type group, but not in the TLR2 group. Conclusions Two common SNPs of the TLR2 and TLR4 genes – Arg753Gln and Asp299Gly – were associated with a shorter time-to-onset of severe sepsis or septic shock in patients admitted to the ICU.

Profiling of Endo H-released serum N-glycans using CE-LIF and MALDI-TOF-MS – Application to rheumatoid arthritis

High‐mannose and hybrid‐type N‐glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high‐mannose and hybrid‐type N‐glycans in total human serum. To this end, N‐glycans were released using Endo‐β‐N‐acetylglucosaminidase H (Endo H) and analyzed by CE‐LIF and MALDI‐TOF‐MS. We found that the high‐mannose structures Man5–9GlcNAc1 represented the majority of the pool. The monoglucosylated structure Glc1Man9GlcNAc1 as well as four hybrid structures could be identified. Then, we compared the Endo H‐released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose‐binding lectin deficiency (MBL) and modulation of α‐mannosidase activity were previously associated with this disease. Interestingly, we observed that both high‐mannose and hybrid structures were fairly constant, suggesting that circulating MBL and α‐mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans.

Soluble CEACAM8 Interacts with CEACAM1 Inhibiting TLR2-Triggered Immune Responses

Lower respiratory tract bacterial infections are characterized by neutrophilic inflammation in the airways. The carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 8 is expressed in and released by human granulocytes. Our study demonstrates that human granulocytes release CEACAM8 in response to bacterial DNA in a TLR9-dependent manner. Individuals with a high percentage of bronchial lavage fluid (BALF) granulocytes were more likely to have detectable levels of released CEACAM8 in the BALF than those with a normal granulocyte count. Soluble, recombinant CEACAM8-Fc binds to CEACAM1 expressed on human airway epithelium. Application of CEACAM8-Fc to CEACAM1-positive human pulmonary epithelial cells resulted in reduced TLR2-dependent inflammatory responses. These inhibitory effects were accompanied by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and by recruitment of the phosphatase SHP-1, which could negatively regulate Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results suggest a new mechanism by which granulocytes reduce pro-inflammatory immune responses in human airways via secretion of CEACAM8 in neutrophil-driven bacterial infections.

Comparison of six immunohistochemical markers for the histologic diagnosis of neoplasia in Barrett’s esophagus

In esophageal neoplasms, the histopathologic differentiation between Barrett’s esophagus with or without intraepithelial neoplasia and adenocarcinoma is often challenging. Immunohistochemistry might help to differentiate between these lesions. The expression of CDX2, LI-cadherin, mucin 2 (MUC2), blood group 8 (BG8, Lewisy), claudin-2, and villin was investigated in normal gastroesophageal (n = 23) and in Barrett’s (n = 17) mucosa, in low-grade (n = 12) and high-grade (n = 9) intraepithelial neoplasia (IEN) as well as in esophageal adenocarcinoma (n = 16), using immunohistochemistry. For CDX2 and LI-cadherin, the immunoreactivity score was highest in IEN while for MUC2, BG8, and villin, it dropped gradually from Barrett’s via IEN to adenocarcinoma, and expression of Claudin-2 was only weak and focal in all lesions. The expression of MUC2 and LI-cadherin differed significantly between all examined lesions except between low-grade and high-grade IEN. MUC2 and LI-cadherin are useful immunohistochemical markers for the differentiation between normal glandular mucosa, Barrett’s mucosa, IEN, and invasive carcinoma of the esophagus; however, none of the examined markers was helpful for the differentiation between low-grade and high-grade IEN.

CDX2 and LI-Cadherin Expression in Esophageal Mucosa: Use of Both Markers Can Facilitate the Histologic Diagnosis of Barrett’s Esophagus and Carcinoma

Background: Barrett’s mucosa is a risk factor for esophageal adenocarcinoma and should be detected at an early stage. CDX2 and liver—intestine (LI)-cadherin are intestine-specific markers.Aberrant CDX2 expression has been demonstrated in Barrett’s metaplasia, esophagitis, and intestinal metaplasia of the stomach. Methods: The relationship between CDX2 and LI-cadherin expression was investigated in normal gastroesophageal (n = 24) and in Barrett’s (n = 20) mucosa, in low-grade (n = 15) and high-grade (n = 13) intraepithelial neoplasia (IEN) as well as in esophageal adenocarcinoma (n = 16), using immunohistochemistry. Results: Nuclear positivity for CDX2 coupled with membranous expression of LI-cadherin was observed in about 70% of the epithelial cells of Barrett’s mucosa. The intensity of staining and the percentage of positive cells increased within the sequential steps of low-grade to high-grade IEN, whereas the normal cylindric epithelium lacked the expression of both. In adenocarcinoma, the expression of LI-cadherin and CDX2 was significantly weaker or absent. Conclusions: CDX2 and LI-cadherin are sensitive markers of intestinal metaplasia with or without dysplasia in the upper gastrointestinal tract. Both can be helpful for the early histologic diagnosis of Barrett’s esophagus and its subsequent lesions; however, they do not significantly discern between different grades of dysplasia.

Granularity Index of the SYSMEX XE-5000 hematology analyzer as a replacement for manual microscopy of toxic granulation neutrophils in patients with inflammatory diseases.

Abstract Background: When certain inflammatory processes occur, toxic granulation neutrophils (TGNs) appear in the blood showing prominent cytoplasmic granules. Currently, the granularity of TGNs is analyzed by manual microscopy of blood smears. The SYSMEX XE-5000 is an automated hematology analyzer, which can measure toxic granulation of TGNs by calculating the Granularity (GI) Index. In this study we investigated if the GI-Index is suitable as a parameter for the TGN granularity in inflammatory diseases. Methods: An evaluation of the toxic granulation neutrophil (TGN) granularity by manual microscopy, the GI-Index and the C-reactive protein (CRP) concentrations of 158 patients were determined. Blood samples from 40 healthy individuals were incubated with lipopolysaccharide (LPS) for in vitro kinetic measurements of the GI-Index. Furthermore, time course measurements of the GI-Index and CRP concentrations of 100 intensive care unit patients were performed. Results: The GI-Index correlated with the microscopic rating of TGNs (n=158; rs=0.839; p<0.0001). When incubating the blood samples with LPS, the neutrophils displayed hypogranulation 30 min after incubation and a hypergranulation after 90 min. In vivo, the GI-Index indicated changes of the bacterial infection status 1 day earlier than the CRP concentration. The correlation of CRP and GI-Index varied between the patient cohorts (n=158; rs=0.836) (n=100; r=0.177), depending on the cause and extent of inflammation. Conclusions: The GI-Index is suited to quantify the granularity of TGNs. The GI-Index is an automated, standardized parameter available on a 24 h basis. We suggest that it replace the time-consuming, subjective and semiquantitative microscopic procedure.

Is there a role for mannan-binding lectin in the diagnosis of inflammatory bowel disease?

Mannan-binding lectin (MBL) activates the lectin-complement pathway as part of the innate immune defence by binding to the surface of microorganisms. Therefore, MBL2 presents an interesting candidate gene for the inflammatory bowel diseases, ulcerative colitis (UC) and Crohns disease (CD). In our study, we evaluated the MBL serum concentrations and genotypes for diagnostic and classification purposes of patients with CD and UC. The MBL serum concentration was analysed in 98 CD patients and in 83 UC patients. In total, 82 patients with inflammatory rheumatic disorders and 189 healthy individuals served as controls. All study subjects were genotyped for the MBL2 polymorphisms G54D, G57E and R52C and the NOD2 (CARD15) mutations R702W, G908R and L1007fsinsC. Neither the median MBL serum concentration nor the MBL2 genotype distribution differed significantly between cohorts. Measurement of MBL serum concentrations offers no benefit for the diagnosis of CD or UC.

In vitro cytotoxicity of the novel antimyeloma agents perifosine, bortezomib and lenalidomide against different cell lines

SummaryThe novel AKT inhibitor perifosine, a synthetic alkylphospholipid, is currently being investigated in clinical trials for the treatment of different hematological and oncological malignancies. The in vitro cytotoxicity of perifosine, bortezomib and lenalidomide against 6 cell lines derived from hematological malignancies was investigated using trypan blue staining, flow cytometry-based detection of activated caspases, Annexin V assays, immunohistochemistry studies (KI-67 and caspase-3 staining) and the immature-myeloid-information (IMI) technique. Perifosine and bortezomib induced concentration- and time-dependent cytotoxicity in all cell lines tested. Perifosine together with bortezomib largely exerted additive or synergistic effects with combination indices ranging from 1.13 to 0.22 for combined efficacies of 25% to 75% after 24-hour incubation. Lenalidomide-triggered cytotoxicity was low in all cell lines tested with any assay (less than 10% compared to the negative control). Finally, perifosine, but not bortezomib or lenalidomide, significantly increased the number of cells detected in the IMI channel. Perifosine and bortezomib- but not lenalidomide- trigger substantial cytotoxicity by caspase activation and mainly act additively or synergistically. The IMI technique might be a useful tool for studying cytotoxicity of agents like perifosine that interact mainly with the cellular membrane.