[When A is not A anymore: problems and pitfalls with blood group typing]. / Wenn A nicht mehr A ist: Schwierigkeiten und Fallstricke bei der Blutgruppenbestimmung.

Correct blood group typing is a prerequisite for transfusion. In most cases blood group determination is without problems; however, in individual cases various factors can complicate blood group determination and sometimes lead to confusing findings. For a better understanding the clinician should have basic knowledge of blood typing. Blood group determination usually covers the AB0 blood groups, Rhesus and Kell systems; in addition, a direct Coombs test and an antibody screening test for the detection of irregular antibodies in the recipient are performed. Confusion of patients, blood samples, results or preparations can lead to severe consequences due to incompatible transfusion and must be prevented. In this context, bedside blood type testing before transfusion is of utmost importance. Problems in laboratory analysis as well as patient-related factors, such as the existence of irregular antibodies against red blood cells can complicate the immunohematology diagnostics. Certain medications, such as daratumumab, lead to a significantly increased complexity in laboratory analyses. Massive transfusions can lead to chimerism with more than one population of circulating red blood cells. Hematopoetic stem cell transplantation can also lead to a change in blood groups as well as chimerism. In addition, there are various other rare causes that can result in difficulties in blood group determination, such as rare blood groups or rare disease-associated phenomena. In the case of problems in blood group determination, early and close cooperation with transfusion medicine is essential for the clinician.

[Novel aspects of the guidelines for hemotherapy 2017 : Implications for anesthesiology]. / Novelle der Hämotherapierichtlinien 2017 : Implikationen für die Anästhesiologie.

Based on the German Transfusion Law, the periodically updated guidelines "Richtlinien zur Gewinnung von Blut und Blutbestandteilen und zur Anwendung von Blutprodukten" ("Hämotherapierichtlinien") are intended to provide the current knowledge and state of the art of blood transfusion practice in Germany. The novel update 2017 contains relevant changes for blood donation, especially the extension of the exclusion period of persons at risk for sexually transmitted HBV, HCV and HIV diseases to 12 months. Moreover, the guidelines provide several changes relevant to blood transfusion practice in anesthesiology, such as: all autologous hemotherapy procedures including normovolemic hemodilution, cell saver, and autologous blood donation and transfusion require formal registration at the regulatory authority. A special detailed protocol is required for every cell saver use. A formal quality control procedure for cell saver use is necessary at least every 3 months. Retransfusion of unprocessed shed blood is generally not permitted. Guidance is provided for the clinical situation of lacking consent for blood transfusion in emergency situations (under certain circumstances blood transfusion may still be allowed). For the first time, the concept of "patient blood management" is explicitly mentioned and recommended in the guidelines. Especially the novel regulations regarding autologous blood use impose new challenges in clinical practice in anesthesiology.

Activation of anorexigenic pro-opiomelanocortin neurones during refeeding is independent of vagal and brainstem inputs.

After fasting, satiety is observed within 2 h after reintroducing food, accompanied by activation of anorexigenic, pro-opiomelanocortin (POMC)-synthesising neurones in the arcuate nucleus (ARC), indicative of the critical role that α-melanocyte-stimulating hormone has in the regulation of meal size during refeeding. To determine whether refeeding-induced activation of POMC neurones in the arcuate is dependent upon the vagus nerve and/or ascending brainstem pathways, bilateral subdiaphragmatic vagotomy or transection of the afferent brainstem input to one side of the ARC was performed. One day after vagotomy or 2 weeks after brain surgery, animals were fasted and then refed for 2 h. Sections containing the ARC from vagotomised animals or animals with effective transection were immunostained for c-Fos and POMC to detect refeeding-induced activation of POMC neurones. Quantitative analyses of double-labelled preparations demonstrated that sham-operated and vagotomised animals markedly increased the number of c-Fos-immunoreactive (-IR) POMC neurones with refeeding. Furthermore, transection of the ascending brainstem pathway had no effect on diminishing c-Fos-immunoreactivity in POMC neurones on either side of the ARC, although it did diminish activation in a separate, subpopulation of neurones in the dorsomedial posterior ARC (dmpARC) on the transected side. We conclude that inputs mediated via the vagus nerve and/or arising from the brainstem do not have a primary role in refeeding-induced activation of POMC neurones in the ARC, and propose that these neurones may be activated solely by direct effects of circulating hormones/metabolites during refeeding. Activation of the dmpARC by refeeding indicates a previously unrecognised role for these neurones in appetite regulation in the rat.

Regulation of cocaine- and amphetamine-regulated transcript-synthesising neurons of the hypothalamic paraventricular nucleus by endotoxin; implications for lipopolysaccharide-induced regulation of energy homeostasis.

Infectious diseases and the administration of bacterial lipopolysaccharide (LPS) result in decreased food intake and increased energy expenditure. Because the hypothalamic paraventricular nucleus (PVN) has pivotal roles in the regulation of energy homeostasis and expresses an anorexic peptide, cocaine- and amphetamine-regulated transcript (CART), we hypothesised that increased CART synthesis in this nucleus may contribute to LPS-induced changes in energy homeostasis. Therefore, we studied the effects of intraperitoneal administration of LPS on CART gene expression in the PVN by semiquantitative in situ hybridisation. LPS caused a rapid increase in CART mRNA levels in the PVN. One hour after treatment, the density of silver grains was increased by three-fold in the PVN, and remained elevated 3 h after treatment. Because the dorsal vagal complex, an important vegetative centre in the brainstem, is heavily innervated by CART-containing axons, we determined whether the retrograde tracer, cholera toxin B subunit (CTB), accumulates in CART neurons in the PVN following stereotaxic injection of the tracer into the dorsal vagal complex. One week after injection, CTB accumulated in CART neurons in the ventral, medial, and lateral parvocellular subdivisions of the PVN. In addition, LPS administration induced c-fos expression in a population of CART neurons in the PVN that project to the dorsal vagal complex. These data indicate that increased CART gene expression in neurons of PVN may contribute to LPS-induced anorexia, and suggest that this action may be mediated, at least in part, through a PVN-dorsal vagal complex pathway.

Regulation of hypophysiotrophic corticotrophin-releasing hormone- and thyrotrophin-releasing hormone-synthesising neurones by brainstem catecholaminergic neurones.

Hypophysiotrophic corticotrophin-releasing hormone (CRH)- and thyrotrophin-releasing hormone (TRH)-synthesising neurones are the principal hypothalamic regulators of glucocorticoid and thyroid hormone secretion, respectively. These two neuroendocrine cell populations are closely situated in the hypothalamic paraventricular nucleus and are targets of neuronal afferent pathways that convey important signals for adapting the neurosecretory activity of CRH and TRH neurones to actual demands. The catecholaminergic afferents of CRH and TRH neurones originate from both noradrenaline- and adrenaline-synthesising cell groups located in the brainstem, and collectively represent one of the most well studied neural inputs of these neurones. The present review summarises the data obtained in recent years concerning the functional significance of the catecholaminergic innervation of hypophysiotrophic CRH and TRH neurones in rats.

A novel HLA-DRB1*11 allele, DRB1*1155, was found in a patient through routine DRB1 typing using sequence-specific primers (SSP) and confirmed by allele-specific sequence-based typing.

In comparison with DRB1*1155 allele, DRB1*1103 differs at position 220/221, 'GC' is changed to 'CT', or DRB1*1125 differs at position 210/211, 'AG' is substituted with 'GA'. This results in a single amino acid exchange depending on the closest related allele investigated, whether DRB*1103 codon 74 alanine (GCG) is changed to leucine (CTG) or DRB1*1125 codon 71 arginine (GAG) is replaced with glutamic acid.

Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

Leukapheresis in chronic myelomonocytic leukemia with leukostasis syndrome: elevated serum lactate levels as an early sign of microcirculation failure.

We present the course of three patients suffering from chronic myelomonocytic leukemia (CMML), who presented with a markedly increase of their WBC (>200 G/l). All patients were started on chemotherapy consisting of ARA-C given as continuous infusion. Due to acute respiratory insufficiency, all patients were treated in the ICCU with ventilation support. Respiratory insufficiency was most likely due to pulmonary leukostasis since pulmonary infection or edema were excluded by X-ray in all patients. Therapeutic leukapheresis was therefore initiated and resulted in a dramatic improvement in one patient. Two patients died due to multiorganic failure despite effective leukocyte depletion (>40%) and maximum supportive care. At the onset of symptoms, two patients had markedly elevated serum lactate levels most likely due to microcirculatory failure. Both patients died because of deteriorating sequelae of pulmonary leukostasis, however, the patient with marginally elevated serum lactate levels survived. Leukapheresis is an established therapeutic approach in patients with hyperleukocytosis and leukostasis, which improves the prognosis of high-risk patients. In our opinion, patients presenting with asymptomatic hyperleukocytosis may benefit from early leukapheresis, particularly when increasing serum lactate levels indicate the early onset of microcirculatory failure.

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.

Arthroscopic synovectomy of the knee joint in early cases of rheumatoid arthritis: follow-up results of a multicenter study.

A retrospective multicenter study on a total of 93 knee joints in 81 patients with early forms of rheumatoid arthritis treated by arthroscopic synovectomy was carried out. During the average follow-up period of 33 months, the patients' clinical state showed appreciable improvement. The Lysholm score modified by Klein and Jensen increased from 43.2 points preoperatively to 78.1 points at follow-up. Also, the Insall score (knee score and functional score) showed a highly significant increase of 25.7 and 25.2 points to 71.2 and 80.2 points, respectively. Patients receiving additional radiation synovectomy showed a highly significantly better result than those receiving synovectomy alone. Among the individual variables investigated, pain, swelling, and walking distance in particular were improved. Only radiologically was a mild worsening observed (from Larsen stage 1.57 preoperatively to 1.95 at follow-up). The follow-up examination revealed no synovitis in 80.6% of the patients. Subjectively, 76.4% of the patients assessed the results to be good or very good, with only 7.5% remaining unsatisfied. 90.3% of the patients declared themselves in retrospect willing to undergo the operation again.

[Gel agglutination test--a new test system for semiquantitative detection of feto-maternal transfusion in Rhesus incompatibility]. / Der Gel-Agglutinations-Test--ein neues Testsystem zum semiquantitativen Nachweis einer feto-maternalen Transfusion bei Rhesusinkompatibilität.

BACKGROUND: The postpartum administration of an adequate amount of anti-D immunoglobulin to the mother in cases of Rhesus incompatibility requires the exact quantification of the amount of Rh-positive fetal cells that may be present in the Rh-negative maternal circulation. The classical methods to detect an intrapartum fetomaternal hemorrhage are either time-intensive (such as the Kleihauer-Betke test), of low specificity (such as the indirect Coombs test), or technically cumbersome (such as flow cytometry). The goals of our study were to develop a simple screening test that may be used routinely to quantify fetomaternal hemorrhage in cases of Rhesus incompatability and to evaluate this test in clinical practice. STUDY DESIGN AND METHODS: In cases of Rhesus-negative mothers of Rhesus-positive neonates, 2.5 ml of maternal Rhesus negative blood was sampled in an EDTA tubes immediately postpartum and was incubated with anti-D antibodies. Thereafter, a semiquantitative determination was made of the amount of antibody that remained unbound in the serum via a gel agglutination test (GAT) (DiaMed., Switzerland) after mixing with test red blood cells. The amount of anti-D consumed (bound to fetal cells in the first phase) is the semi-quantitatively indicated by the degree of positivity in the second phase the weaker reaction--the more anti-D absorbed in the first phase--the more Rhesus-positive fetal cells present in the maternal sample--the larger the fetomaternal hemorrhage. Following the development of a discrimination zone using this GAT which could ascertain an Rhesus-positive erythrocyte concentration of over 0.2%, the test was applied in a clinical setting. Between September 1995 and April 1998 in unselected postpartum blood samples from 603 Rhesus negative parturients, the GAT was used to test the same blood samples as those requiring evaluation for HbF concentration using the traditional Kleihauer-Betke test. RESULTS: In 585 of the 603 cases (97%) there was no evidence of a fetomaternal transfusion following testing using both methods. Furthermore, both tests showed significant evidence for a fetomaternal transfusion in five cases. The Kleihauer-Betke test was false-positive in three cases of mothers who had a hereditary elevation of the HbF concentration. The GAT showed three false-positive reaction due to a Dweak maternal varient. In two cases, the disparity between the GAT and the Kleihauer-Betke test could be attributed to an antecedant dose of anti-D antibody. In the two cases, the Kleihauer-Betke test results were 0.3% while the GAT was only 0.2%. CONCLUSION: The GAT may be used as a screening method in routine clinical practice. This is a quick test that allows for the specific determination and semiquantitative evaluation of the Rh-positive erythrocyte concentration in clinically relevant concentrations. Thus, following a positive GAT screening test, a further specific test such as the Kleihauer-Betke test may be utilized to absolutely quantify the amount of blood transfused from fetus to mother. It is also possible to perform such a quantification test with the GAT by eventually using a diluted maternal blood sample.

Antibodies to private and public HLA class I epitopes in platelet recipients.

BACKGROUND: Transfusions or pregnancies can cause immunization against private HLA determinants and public epitopes shared by more than one private HLA antigen. HLA antibodies are correlated with febrile transfusion reactions, lower platelet response following platelet transfusion, and an increased rate of renal transplant rejection. Until now, antibody specificities in alloantisera from platelet recipients have been poorly characterized. STUDY DESIGN AND METHODS: Consecutive serum screens from platelet recipients were analyzed for antibodies against private HLA class I antigens and public HLA epitopes using a serum analysis program based on the 2 x 2 table analysis of correlations. Serum screens of highly immunized patients and of patients with new alloimmunization events were reviewed separately. RESULTS: Of the serum screens from 566 platelet recipients, 1577 indicated alloimmunization (panel-reactive antibodies >5%). The program assigned a specificity in 1024 of these screens (64.9%) and at least once in 522 of 566 patients (92.2%). In 267 patients, antibodies detecting public epitopes in the combined A- or B-locus cross-reacting groups were found; other public markers were detected in 39 patients. Patterns of reactivity were remarkably less stable than in patient groups previously studied. In many patients, antibodies with apparent private epitope specificity preceded the identification of antibodies against a shared marker of the same cross-reactive group. However, the disappearance of antibodies (whether or not this was followed by a new antibody against a private or public marker belonging to another cross-reacting group) was also observed. CONCLUSION: The computerized analysis of microlymphocytotoxicity tests enhances the rate of antibody specification in sera from platelet recipients with lymphocytotoxic antibodies. The identified antibodies should be taken into account in the selection of platelet donors. The data confirm and extend previous observations on HLA class I antibodies and elucidate new alloimmunization events.

Changes in platelet membrane glycoproteins before bone marrow transplantation and after engraftment--a pilot study.

Thrombotic complications are observed in patients undergoing bone marrow transplantation despite thrombocytopenia and impaired coagulation due to liver function disturbances. Endothelial cell damage which is involved in the pathogenesis of major transplant related complications like graft-versus-host disease, veno-occlusive disease, sepsis or microangiopathy may be a contributing factor. Little is known about platelet function in bone marrow transplant recipients. In order to study functional alterations in circulating platelets we investigated unstimulated and ADP-stimulated platelets of 10 bone marrow transplant recipients ex vivo by flow cytometry in a pilot study using a panel of monoclonal antibodies to characterize changes in membrane glycoproteins. Samples were collected before and during conditioning and at three timepoints after engraftment. 10 healthy volunteers served as controls. Platelets of bone marrow transplant recipients showed partly a significant, higher expression of surface bound fibrinogen, activated fibrinogen receptor, and glycoprotein Ib as compared to controls. P-selectin, a marker of platelet degranulation was significantly elevated after ADP-induced stimulation at all timepoints compared to controls. Only marginal differences were found for GP IIb/IIIa surface expression. The data point to an increased platelet activation state in bone marrow transplant recipients which might contribute to the thrombotic phenomena observed in these patients.

Use of the gel agglutination technique for determination of fetomaternal hemorrhage.

BACKGROUND: Adequate administration of Rh immune globulin requires an accurate determination of the number of D-positive cells in the circulation of D-negative women. Although several tests have been described for the detection of fetomaternal hemorrhage, there is still a need for a rapid, simple, and clinically relevant screening test. STUDY DESIGN AND METHODS: Serial dilutions of a monoclonal anti-D were incubated with stock solutions (0.2 mL) of adult D-negative red cells in the absence or presence of various amounts of fetal D-positive cells (0.1, 0.2, 0.3, 0.4, and 0.5%). After incubation, the supernatants were tested against D-positive red cells by using the new, gel agglutination technique (GAT). After the GAT was adapted to detect D-positive cells at concentrations of > or = 0.2 percent, unselected postpartum samples from D-negative women (n = 420) who delivered D-positive infants were analyzed by both the new test and the Kleihauer-Betke test (KBT). RESULTS: Three of a total of 420 postpartum samples were positive (> or = 0.4% fetal cells), and 406 were negative in both tests. One had 0.5-percent fetal cells in the KBT and gave negative results in the GAT. The latter test was, however, performed after administration of Rh immune globulin. The KBT gave false-positive results in two cases, because of hereditary persistence of hemoglobin F, and the GAT gave a false-positive reaction in one case because of a maternal weak D variant. In the remaining seven cases, the KBT results were only weakly positive (0.2%) and could not be attributed solely to D positive red cells. CONCLUSION: The GAT is suited for routine screening. It provides rapid and specific detection of D-positive red cells at clinically relevant concentrations. The test may (rarely) yield false-negative results due to insufficient administration of Rh immune globulin before testing.

Tandem and triple high-dose chemotherapy with autologous stem cell rescue in metastatic breast cancer.

The purpose of this phase II study was to evaluate the therapeutic efficacy and toxicity of a tandem or triple high-dose chemotherapy (HDC) with autologous peripheral blood stem cell transplantation (PBSCT) in patients with metastatic breast cancer (MBC) as first line chemotherapy. Conventional chemotherapy consisted of two cycles of epirubicin 120 mg/m2 and ifosfamide 7500 mg/m2 in the case of tandem HDC and one cycle of paclitaxel 135 mg/m2, epirubicin 90 mg/m2 and ifosfamide 6000 mg/m2 in the case of triple HDC. Tandem HDC was composed of two cycles of epirubicin 180 mg/m2, ifosfamide 12000 mg/m2 and carboplatin 900 mg/m2. In the case of triple HDC, paclitaxel 180 mg/m2, etoposide 1500 mg/m2 and thiotepa 600 mg/m2 was added as the third cycle. Patients with tandem HDC (n = 20) were evaluable for both survival and toxicity, and patients with triple HDC (n = 21) only for toxicity because of short-term follow-up. Both tandem and triple HDC were well tolerated and could be safely administered. Non-hematological WHO grade 3 or 4 toxicities were mucositis (8), temporary renal insufficiency (1), myocardial infarction (1), and neuropathy (1). No toxic death occurred. The Kaplan-Meier estimates for 44-months without progression and the overall survival were 12% and 38% respectively. The median survival was 22 months (95% CI: 7.4-51.7 months) and the median progression-free interval 14 months (95% CI: 5.1-43.7 months). In a population with an unfavorable prognosis, tandem HDC showed similar efficacy as to that described in other phase II studies. Triple HDC seems not to improve patient outcome compared to tandem HDC, but a long-term follow up is required.