[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.

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.

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.

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.

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.

Preparation of autologous bone marrow grafts for cryopreservation using the AS104 cell separator.

We evaluated the AS104 cell separator (Fresenius AG, Bad Homburg, Germany) for ex vivo processing of bone marrow (BM) grafts of 43 patients suffering from germ cell cancer (GCC, n = 22), acute lymphocytic leukemia (ALL, n = 13) and malignant lymphoma (ML, n = 8). Recoveries of total nucleated cells (TNC), mononuclear cells (MNC) and colony-forming units granulocyte-macrophage (CFU-GM) were determined in the BM concentrates prepared for cryopreservation. Hematopoietic reconstitution was analyzed in patients who underwent autologous transplantation following high-dose radio-/chemotherapy (HDRCT). Processing of the BM suspension with a median volume of 1,013 ml (range: 422-1,574) resulted in 156 ml (80-186) within 50-120 min (median: 90). In the BM concentrates, medians of 28.6% TNC (10.6-69.6), 37.9% MNC (22.3-86.4), and 52.4% CFU-GM (20.8-96.4) were recovered. Twenty-six patients underwent HDRCT with reinfusion of autologous BM and were evaluable for engraftment. They received a median of 0.8 x 10(8) MNC/kg (0.3-1.6 x 10(8)) and 2.2 x 10(4) CFU-GM/kg (0.6-12.8 x 10(4) for hematopoietic rescue. Engraftment with neutrophils > 500/microliter occurred in a median time of 12 days (8-33) in all patients. We conclude that ex vivo processing of autologous BM with median recovery rates of 37.9% for MNC, and 52.4% for CFU-GM, results in a cell population that can rescue patients from HDRCT. The described technique is convenient, time-efficient, and provides reliable results in preparing BM autografts for cryopreservation.

Living epithelial-mesenchymal compounds formed in vitro suitable for autografting.

Three different, human epithelial-mesenchymal compounds (EMC) were generated in vitro for prospective grafting in epithelial defects. All compounds consisted of a fibroblast-populated collagen lattice as a mesenchymal component seeded with different types of cultured epithelial cells isolated from biopsies of healthy skin, oral mucosa and respiratory mucosa. Maturation of the epithelial cells was enabled by the presence of a high calcium concentration (1.8 mM) when cultures were lifted to the air-liquid interface. Light and electron microscopy revealed moderate differentiation of the multilayered epithelium in all compounds as well as basement membrane development at the epithelial-mesenchymal junction after 2-3 weeks. A coherent, tissue-like consistency of the collagen lattice and the presence of a basement membrane preventing detachment of the epithelium permitted easy handling and even loose suturing of the compounds produced.

[Prognostic significance of plasminogen activator inhibitor-1 (PAI-1) in primary breast carcinoma]. / Prognostische Bedeutung des Plasminogenaktivator Inhibitor-1 (PAI-1) beim primären Mammakarzinom.

The plasminogen activator system plays a key role in the proteolysis of malignant tumours. In 155 patients with primary breast cancer levels from PAI-1 and uPA were measured in cytosol with monoclonal antibodies and by an enzyme-linked immunosorbent assay. 35 tumour tissue samples form benign breast were also examined. Malignant tumours contain higher levels of PAI-1 (8.6 ng/mg) than benign tumours (1.28 ng/mg) (p < 0.01). Also the median level of uPA was significantly higher (p < 0.01) in malignant tissue (2.38 ng/mg) in comparison to benign disease (0.54 ng/mg). No correlation was found between the proteases and the classical prognostic parameters like axillary lymph node involvement, tumour size and menopausal status. However, a significant correlation (p < 0.01) was found in tumours with lymphangiosis carcinomatosa, negative hormone receptors, grade III tumour cells and high S-phase fractions (> 5%). After a median follow-up of 46 months we found that high levels of PAI-1 correlated with short DFS (p = 0.0005) and OAS (p = 0.003). However, in the Cox multivariate regression analysis only PAI-1 was significantly independent for OAS and could therefore give additional information. We conclude that levels from PAI-1 antigen measured in cytosol of primary breast cancer is an independent prognostic parameter to identify patients with high and low risk for relapse and for individualised treatment.

Analysis for recovery and loss of mononuclear cells and colony-forming units granulocyte-macrophage during ex vivo processing of autologous bone marrow.

During ex vivo processing of autologous bone marrow (BM) substantial loss of stem and progenitor cells should be avoided to achieve rapid and sustained hematopoietic reconstitution after high-dose radio-/chemotherapy. We processed 25 autologous BM grafts with the Fresenius AS104 cell separator for cryopreservation and we determined recoveries for mononuclear cells (MNC) and colonyforming units granulocyte-macrophage (CFU-GM) in the BM concentrates. To identify cell loss in BM fractions not cryopreserved, we investigated the MNC and CFU-GM content of BM fat and BM blood. MNC and CFU-GM recovery yielded a mean ( +/- SEM) of 42 +/- 12 and 54 +/- 20% in the BM concentrate. BM fat showed a mean loss of 7 +/- 5% for MNC and 4 +/- 3% for CFU-GM, BM blood 30 +/- 12% for MNC and 13 +/- 13% for CFU-GM, respectively. CFU-GM recovery was significantly higher in the BM concentrate of patients with hematologic malignancy (HM) compared with patients suffering from germ cell cancer (GCC): 66 +/- 21 vs. 43 +/- 12% (p < 0.02). Seventeen patients (7 GCC, 10 HM) underwent high-dose chemotherapy or radio-/chemotherapy and were autografted with 0.8 +/- 0.2 x 10(8) MNC/kg and 3.7 +/- 2.0 x 10(4) CFU-GM/kg. All patients achieved engraftment with neutrophils > 0.5 x 10(9)/l at a mean of 14 +/- 6 days. We conclude that: (1) ex vivo processing of autologous BM with a mean of recovery of 42% for MNC and 54% for CFU-GM in the BM concentrate can result in a cell population capable of sustained hematopoietic reconstitution, (2) CFU-GM recovery is significantly higher in patients with HM than in patients with GCC and (3) 37% MNC and 17% CFU-GM represent in fact cell losses recovered from BM fractions not cryopreserved (BM fat, BM blood). Furthermore, it is likely that MNC and CFU-GM not recovered from BM concentrate, BM fat and BM blood are cell losses related to the cell separator.

Identification of antibodies toward private and public class I HLA epitopes in sensitized patients.

BACKGROUND: Antibodies to HLA determinants may decrease the increment after platelet transfusion and are correlated with increased rates of rejection in renal transplantation. Cross-match tests and HLA antibody specification are used to identify compatible donors for sensitized patients. However, search for cross-match negative donors may be ineffective, and many sera containing antibodies toward public HLA epitopes give no clear results in conventional specificity analysis. MATERIALS AND METHODS: We tested a series of 4,625 sera from 1,073 patients with hematological, malignant or other diseases using a fluorescence lymphocytotoxicity test (LCT). We applied an evaluation program incorporating a list of public antigens belonging to known cross-reacting groups (CREGs) to detect antibodies toward private and just as well public epitopes. RESULTS: In 694 sera (15.0%) from 240 patients the panel reactivity (PRA) in LCT assay was higher than 5%. PRA was > or = 50% in 258 (37.2%) and < 50% in 436 sera (62.8%). In both groups we identified specific antibodies toward public HLA class I epitopes. Overall antibody specification was successful in 429 of 694 sera (61.8%) and in 175 of 240 patients (72.9%), respectively. The rate of antibodies against public epitopes shared by more than one HLA class I gene product was 203/694 (29.3%) with respect to tested sera and 83/240 (34.5%) with respect to patients. The rate of public epitope antibodies was highest in sera with PRA values from 30 to 90% showing public epitope specificity in 159 of 353 sera (45.0%). CONCLUSIONS: We conclude that antibodies toward public HLA class I determinants are detectable not only in highly sensitized patients. The described program may increase the rate of antibody specification and facilitate the platelet supply in platelet refractory patients.

[18 months experience with a new single needle cytapheresis system (Fresenius AS 104 SN)]. / 18 Monate Erfahrung mit einem neuen Single-needle-Zytapheresesystem (Fresenius AS 104 SN).

BACKGROUND: Since October 1991 the third-generation cell separator Fresenius AS 104 offers a single-needle (SN) option for thrombocytapheresis. Here we present our first 18-month experience with this new single-needle system. MATERIALS AND METHODS: We performed 395 thrombocytaphereses in 225 donors. The influence of blood flow, cycle volume, and interface position on efficacy and leukocyte contamination was evaluated. RESULTS: 395 thrombocytaphereses were performed with an average thrombocyte yield of 3.00 +/- 0.69 x 10(11) platelets and a leukocyte contamination of 1.66 +/- 3.1 x 10(8) per concentrate. Blood flow rates of 50 ml/min (vs. 60 ml/min) and an interface position of 6:2 (vs. 7:1) resulted in a lower leukocyte contamination, but without statistical significance. CONCLUSION: The SN program of the AS 104 provides high thrombocyte yields and less leukocyte contamination than other SN systems, but 77% of the concentrates still contain more than 0.5 x 10(8) leukocytes.

[MLC reactivity demonstrates transfusion-induced immunosuppression]. / Die MLC-Reaktivität zeigt die transfusionsinduzierte Immunsuppression an.

The improved graft survival in preoperatively transfused renal transplant recipients led to the hypothesis that blood transfusions have an immunosuppressive effect. We examined 12 patients undergoing cardiac surgery, who received autologous red cells and one homologous HLA class-I-matched platelet concentrate. None of these patients produced red cell or lymphocytotoxic antibodies. Using the mixed lymphocyte culture (MLC) we observed a reduced lymphocyte responce to cells of the platelet donor during the first 4 days after transfusion. The MLC reactivity recovered on days 4-5 to the initial strength and reached over 200% of the initial strength from day 6 on. It must be assumed that these changes in MLC reactivity represent the early signs of the transfusion-induced immunosuppression. As intraoperative transfusions might correlate with cancer relapse, further studies must show whether this immunosuppressive effect can be avoided by the application of filtered leucocyte-depleted red cell transfusions.

[Effect of various rabbit complement batches of HLA class I antigen determination in lymphocytotoxicity testing]. / Der Einfluss verschiedener Kaninchenkomplement-Chargen auf die HLA-Klasse-I-Antigenbestimmung im Lymphozytotoxizitätstest.

HLA class I lymphocytotoxicity testing is strongly complement-dependent. We tested 13 commercially available rabbit complement batches (7 distributors) using a panel of 14 donors with decisive patterns of HLA antigens. We defined a scoring system that weights the different possible faulty reactions. The rating score ranged from 12.45 to 46.55 (median 16.09). Comparing the reactions in batches with low and high rating scores gave statistically significant differences (chi 2 test, p < 0.05). Our complement reaction score may be helpful to find the optimal complement batch for HLA class I lymphocytotoxicity testing.

Effect of muscarinic receptor stimulation on release of cysteinyl-leukotrienes and thromboxane B2 from anaphylactic guinea-pig hearts.

The effects of muscarinic receptor stimulation by infusions of methacholine (6.25 x 10(-8) mol/min or 1.9 x 10(-7) mol/min) into isolated perfused, spontaneously beating sensitized guinea-pig hearts on the anaphylactic release of cysteinyl-leukotrienes (LT) and thromboxane (TX) B2 were investigated. Methacholine increased coronary flow and decreased heart rate under basal conditions. Furthermore, infusions of methacholine (1.9 x 10(-7) mol/min) significantly increased the anaphylactic release of TXB2 as well as of immunoreactive cysteinyl-LT, which were demonstrated by reversed phase high pressure liquid chromatography to consist of a mixture of LTC4, LTD4 and LTE4. Infusions of atropine (1.3 x 10(-7) mol/min) alone did not significantly affect coronary flow and heart rate prior to ovalbumin injection nor anaphylactic release of cysteinyl-LT. The anaphylactic release of TXB2 was, however, significantly decreased in the presence of atropine. Atropine (1.3 x 10(-7) mol/min) infused in addition to methacholine (1.9 x 10(-7) mol/min) abolished the effects of the muscarinic receptor agonist on spontaneous heart rate and significantly antagonized the increase in coronary flow prior to ovalbumin injection. Similarly, the simultaneous infusion of atropine abolished the effects of methacholine on the anaphylactic release of TXB2 and cysteinyl-LT. After antigen challenge hearts infused with methacholine, atropine or the combination of both drugs did not exhibit any differences with respect to anaphylactic changes of heart rate or the time course of anaphylactic coronary flow reduction. Thus, in the isolated perfused anaphylactic guinea-pig heart, muscarinic receptor stimulation significantly enhanced the release of the arachidonic acid-derived mediators TXB2 and cysteinyl-LT.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of cysteinyl-containing leukotrienes by human arterial tissues.

Human arterial rings incubated in modified Tyrode solution released small amounts of leukotriene (LT) C4-like material spontaneously and larger amounts upon stimulation with the ionophore A23187 as determined by radioimmunoassay. By reversed phase high pressure liquid chromatography (HPLC) LTC4-like material was found to comigrate with authentic LTC4, LTD4 and LTE4. Nordihydroguaiaretic acid (NDGA) significantly inhibited the ionophore A23187-induced release of LTC4-like material, while indomethacin was without effect. Simultaneously the arterial rings released much larger amounts of 6-keto-prostaglandin (PG) F1 alpha, which were significantly decreased by indomethacin. The results demonstrate that human arterial tissue has the capacity to synthesize cysteinyl-containing LT from endogenous arachidonic acid.