Effects of Physical Exercise on Markers of Cellular Immunosenescence: A Systematic Review.

Aging affects negatively the immune system, defined as immunosenescence, which increases the susceptibility of elderly persons to infection, autoimmune disease, and cancer. There are strong indications that physical exercise in elderly persons may prevent the age-related decline in immune response without significant side effects. Consequently, exercise is being considered as a safe mode of intervention to reduce immunosenescence. The aim of this review was to appraise the existing evidence regarding the impact of exercise on surface markers of cellular immunosenescence in either young and old humans or animals. PubMed and Web of Science were systematically screened, and 28 relevant articles in humans or animals were retrieved. Most of the intervention studies demonstrated that an acute bout of exercise induced increases in senescent, naïve, memory CD4+ and CD8+ T-lymphocytes and significantly elevated apoptotic lymphocytes in peripheral blood. As regards long-term effects, exercise induced increased levels of T-lymphocytes expressing CD28+ in both young and elderly subjects. Few studies found an increase in natural killer cell activity following a period of training. We can conclude that exercise has considerable effects on markers of cellular aspects of the immune system. However, very few studies have been conducted so far to investigate the effects of exercise on markers of cellular immunosenescence in elderly persons. Implications for immunosenescence need further investigation.

[Rivaroxaban: Xarelto--recommendations for pharmacists]. / Le Rivaroxaban (Xarelto): guide de la pratique officinale.

Rivaroxaban is one of the new oral anticoagulants (NOACs). It has many potential advantages in comparison with Vitamin K Antagonists (VKA). It has a predictable anticoagulant effect and does not theoretically require biological monitoring. It is also characterized by less food and drug interactions. However, due to major risks associated with over- and under-dosage, its optimal use in patients should be carefully followed by health care professionals. The aim of this article is to provide recommendations for pharmacists on the practical use of Xarelto in its different approved indications. This document is adapted from the practical user guide of rivaroxaban which was developed by an independent group of Belgian experts in the field of thrombosis and haemostasis.

Influence of apixaban on commonly used coagulation assays: results from the Belgian national External Quality Assessment Scheme.

INTRODUCTION: The Belgian national External Quality Assessment Scheme performed a survey to assess the effect of the direct oral anticoagulant apixaban on the coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin as performed with a large number of reagent/instrument combinations. METHODS: Four lyophilized plasma samples spiked with apixaban (0, 41, 94 and 225 ng/mL) were sent to the 195 Belgian and Luxembourg clinical laboratories performing coagulation testing. RESULTS: PT and aPTT were barely influenced at the concentrations tested. At 225 ng/mL apixaban, PT and aPTT clotting times were only 1.15 times longer than at 0 ng/mL. Among PT reagents, RecombiPlasTin 2G® showed a slightly higher sensitivity with 225 ng/mL apixaban prolonging the PT clotting time 1.3-fold. Among aPTT reagents, there was no appreciable difference in sensitivity. Fibrinogen results were unaffected by the presence of apixaban, but antithrombin activity was considerably overestimated when measured with a FXa-based assay. At 225 ng/mL apixaban, the median percentage increase in antithrombin level was 31% when measured with the Liquid Antithrombin® reagent and 44% with the Innovance Antithrombin® reagent. CONCLUSION: Our data provide clinical laboratories with useful information on the impact of apixaban on their routine coagulation assays.

Flow cytometric immunophenotyping of leukemia cells in clotted blood and bone marrow.

Immunophenotyping of leukemia cells is generally performed on cells in suspension. These suspensions are usually prepared from anticoagulated peripheral blood or bone marrow samples but when anticoagulation is suboptimal clotted samples may reach the laboratory. In this study cell suspensions were prepared from clotted blood and bone marrow samples of leukemia patients by lysis of the clots with streptokinase. The cell suspensions were then labeled with monoclonal or polyclonal antibodies and examined by flow cytometry or fluorescence microscopy. Enough cells could be isolated from small volumes of clotted blood or bone marrow aspirate to determine the immunophenotype of the cells. The morphology of the cells was well preserved and accurate identification of the cells was possible. The immunophenotypes determined on clotted samples from four patients with acute myeloblastic leukemia, five with acute lymphoblastic leukemia and two with chronic lymphocytic leukemia were identical to those established using EDTA anticoagulated samples of the same patients. When no unclotted samples are available this approach may avoid the necessity for a new sample and the concomitant delay in treatment of the patient.

An immunogold-silver staining method for detection of cell surface antigens in cell smears.

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy.

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies.

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.

The molecular basis of antithrombin deficiency in Belgian and Dutch families.

The molecular basis of hereditary antithrombin (AT) deficiency has been investigated in ten Belgian and three Dutch unrelated kindreds. Eleven of these families had a quantitative or type I AT deficiency, with a history of major venous thromboembolic events in different affected members. In the other two families a qualitative or type II AT deficiency was occasionally diagnosed. DNA studies of the AT gene were performed, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and intron-exon junction regions. Six novel point mutations were identified: four missense, one nonsense mutation and a single nucleotide deletion near the reactive site, causing a frameshift with premature translation termination. In two kindreds the underlying genetic defect was caused by a whole gene deletion, known as a rare cause of AT deficiency. In these cases, Southern blot and polymorphism analysis of different parts of the AT gene proved useful for diagnosis. In another kindred a partial gene deletion spanning 698 basepairs could precisely be determined to a part of intron 3B and exon 4. In two type I and in both type II AT deficient families a previously reported mutation was identified. In all cases, the affected individuals were heterozygous for the genetic defect.

Hematologic values and lymphocyte subsets in fetal blood.

Hematologic values and lymphocyte subpopulations were determined in normal fetal blood during the second trimester of gestation. In these samples the platelet, erythrocyte, and leukocyte counts were significantly lower than in adults. Large red blood cells with a high hemoglobin content were present. Before the twentieth week of gestation, erythroblasts made up about half of the nucleated elements. Lymphocytes formed most of the leukocytes, and their absolute numbers were comparable to those in adults. Most of the fetal blood lymphocytes expressed T- or B-cell surface differentiation antigens. The percentage of T-cells was lower and that of B-cells was higher than in the adult. A high OKT4/OKT8 ratio was present. It was due to a low percentage of OKT8-positive cells. Lymphocytes with a natural killer cell phenotype were rare. Most lymphocytes were OKT10 positive, but almost none reacted with the antithymocyte antibody OKT6. These results give additional information about the development of blood cells in early human life. They can be used as reference values for the prenatal diagnosis of hereditary or acquired anomalies of the hematologic and immunologic systems.

Southern blot analysis in a case of Richter's syndrome. Evidence for a postrearrangement heavy chain gene deletion associated with the altered phenotype.

Richter's syndrome (RS) can be defined as the emergence of an aggressive lymphoma in patients suffering from chronic lymphocytic leukemia (CLL). The authors performed immunophenotypic and Southern blot analysis of the peripheral blood and tissue specimen of a patient with RS. Using immunoperoxidase and immunogold-silver staining techniques and a panel of monoclonal antibodies, the authors found that the large cells characteristic of RS showed an altered immunophenotype as compared with the CLL cells and did not express mu heavy chain. Southern blot analysis revealed identical kappa light chain rearrangements in both tumoral cell populations consistent with a common clonal origin. Using the JH probe and several restriction enzymes, the authors also found evidence for a postrearrangement deletion of the heavy chain mu gene. These findings suggest that in this case of RS, a deletion of the heavy chain mu gene resulted in loss of mu expression by the larger cells that were characteristic of RS and was associated with their altered phenotype.

Leukemia and lymphoma immunophenotyping in cell smears with immunogold-silver staining.

The potential of the immunogold-silver staining (IGSS) technique for immunophenotyping leukemia and lymphoma cells in cell smears was examined. Peripheral blood, bone marrow aspirates, lymph node biopsy specimens, fine-needle aspirates, and biologic fluids of 83 patients with acute or chronic leukemias, non-Hodgkin's lymphomas, or Hodgkin's disease were labeled. Cell smears, cytocentrifuge preparations, or imprints were fixed, incubated with the reagents, and counterstained with May-Grünwald-Giemsa. Stable immunostaining and good morphologic characteristics allowed accurate cell identification and rapid enumeration of the positive cells. The immunophenotypes obtained with the use of 35 monoclonal antibodies with different specificities were similar to those determined by flow cytometry or immunohistochemical studies on the same samples. This IGSS method was especially useful for the examination of poor samples or complex cell suspensions with rare malignant cells. It could be an alternative to the immunoenzyme methods that generally are used for this purpose.

Anaesthetic management of a parturient with severe congenital factor XI deficiency undergoing caesarean section for triplet pregnancy.

The report describes the anaesthetic management of a Jewish patient of Ashkenazi descent with severe factor XI deficiency complicated by thrombocytopenia for caesarean section for triplets at the 35th week of gestation. Perioperative management consisted of sustained replacement therapy with fresh frozen plasma and platelets until the sixth postoperative day. General anaesthesia was used for the procedure. No other maternal or neonatal complications occurred.

[Antithrombin deficiency and thrombosis in a young child]. / Déficit en antithrombine et thromboses chez le jeune enfant.

BACKGROUND: Thromboses represent a rare event in children and may be due to a deficiency of antithrombin. CASE REPORT: A 10-year-old boy developed thrombosis due to a congenital quantitative deficiency in antithrombin, confirmed by molecular biology. His father was diagnosed with the same deficiency. The child was first treated with heparin and is now on antivitamin K. He is well 26 months after diagnosis. CONCLUSION: When a young patient presents with a thrombotic event, a congenital deficiency in one of the inhibitors of coagulation, one of which is antithrombin, should be looked for and the condition treated as soon as possible.

Guidelines for the use of fresh frozen plasma.

Recommendations, which aim at standardising and rationalising clinical indications for the transfusion of fresh frozen plasma (FFP) in Belgium, were drawn up by a working group of the Superior Health Council. For this purpose the Superior Health Council organised an expert meeting devoted to "Transfusion Guidelines: Pathogen reduction, products and indications for the transfusion of plasma" in collaboration with the Belgian Haematological Society.The experts discussed the indications for the transfusion of FFP, pathogen reduction for FFP and the practical issues of administering FFP and plasma-derived concentrates. The recommendations formulated by the experts were validated by the working group with the purpose of harmonising FFP transfusion in Belgian hospitals.

External quality assessment in the measurement of haemoglobin by blood gas analysers in Belgium.

OBJECTIVE: The Belgian national External Quality Assessment Scheme (EQAS) for haematology organized a survey to assess the reliability of haemoglobin (Hb) measurements with the blood gas analysers (BGAs) currently available in Belgian hospitals. MATERIAL AND METHODS: All hospital laboratories received two specimens of fresh EDTA anticoagulated whole blood and were asked to determine the Hb concentration using both the conventional haematology analyser (HA) and all BGAs in the hospital. Ninety-seven hospital laboratories participated in the study and a total of 166 results were reported. The BGAs used (grouped according to technology) were Rapidlab 845, 855, 865 (Bayer 1, n = 41), Rapidlab 1245, 1265, Rapidpoint 405 (Bayer 2, n = 19), GEM Premier 3000 (Instrumentation Laboratory, IL, n = 13), ABL 500 and 600 series (Radiometer 1, n = 13), ABL 700 and 800 series (Radiometer 2, n = 35), Omni C, S5 (Roche 1, n = 7), Omni 3, 6, 9, S2, S4, S6 (Roche 2, n = 21). RESULTS: For the BGAs from Bayer, Radiometer and Roche, interlaboratory variation ranged from 0.6 % to 4.1 %, indicating good precision and close agreement between centres. A significant negative bias observed on the GEM Premier 3000 using the EDTA anticoagulated blood samples did not appear to be present in fresh heparinized whole blood samples. There was no significant difference in imprecision and bias between Hb measurements on BGA situated in and outside the central laboratory.

Antithrombin-Gly 424 Arg: a novel point mutation responsible for type 1 antithrombin deficiency and neonatal thrombosis.

Inherited type 1 antithrombin (AT) III deficiency is characterized by a decrease of immunoreactive and functional protein levels to about 50%. The disorder is associated with a significantly increased risk of thromboembolism. We have investigated the molecular basis of type 1 AT deficiency in a Belgian family. The diagnosis of the disease was primarily made in a newborn girl with unusually severe thrombotic complications. Using the polymerase chain reaction and single-strand conformation polymorphism analysis, followed by direct sequencing of AT gene fragments, we identified a novel point mutation in exon 6. We detected a G to C substitution in the first position of codon 424 leading to a glycine to arginine substitution. The modification at this highly conserved position in the serine protease inhibitor gene family probably leads to an unstable mutant-gene product. The mutation creates a unique restriction site for the enzyme Hha I in exon 6. This change permitted a rapid and accurate screening of the kindred with identification of the molecular defect in five other family members.

Plasma D-dimer concentrations in different clinical conditions.

D-dimers (DD), specific degradation products of crosslinked fibrin, are markers for activation of plasma coagulation and/or fibrinolysis. DD results below the cut-off level are proven to be useful to rule out the probable diagnosis of deep venous thrombosis (DVT) and/or pulmonary embolism (PE). Our objective was to demonstrate that positive DD occur in many diseases and certain physiological conditions as high age and pregnancy and to look for gradations in positivity between different pathological conditions. We wanted to investigate the request attitude of our clinicians concerning DD. Positive DD results still confuse some physicians. Retrospectively, we examined medical records of 574 consecutive patients, in whom plasma DD were measured in daily routine. Both outpatients (n = 423) and inpatients (n = 151) were included. We noted their clinically predominant disease. Measurement of DD concentration is too often requested by clinicians, in any medical condition, and is not always clinically relevant. The relation of a positive result and the clinical problem is sometimes not understood. Overall, we found 64% DD positivity with a median concentration of 775 micrograms/L. We found elevated DD concentrations in various clinical conditions.