Haemostatic function and biomarkers of endothelial damage before and after RBC transfusion in patients with haematologic disease.

BACKGROUND AND OBJECTIVES: Transfusion of red blood cells (RBC) is beneficial for the patient but can also be harmful, as randomized trials have demonstrated increased infection rates, bleeding and mortality. The study aim was to investigate the response of the vascular system (the haemostatic function and the endothelium) to RBC transfusion. MATERIALS AND METHODS: Blood was sampled from patients with various transfusion-dependent haematologic diseases before 1 and 24 h after RBC transfusion. Primary and secondary haemostasis was evaluated by whole-blood impedance aggregometry (Multiplate) and by thromboelastography (TEG). Samples were analysed by ELISA for biomarkers reflecting endothelial activation and damage (sICAM-1, syndecan-1, sThrombomodulin (sTM), sVE-Cadherin), platelet activation (sCD40L) and inflammation (hsCRP). RESULTS: A total of 58 patients were enrolled in the study. Median age was 71 years. Compared to before transfusion, patients had slightly reduced coagulability 1 h after RBC transfusion, assessed by TEG. However, transfusion of older RBC products (>14 days) was associated with increased coagulability (all P < 0·05). The level of syndecan-1 increased slightly 24 h after transfusion (median 12·4 (IQR 9-23) vs. 13·2 (9-25) ng/ml, P < 0·01), indicating increased glycocalyx degradation. CONCLUSION: Overall, RBC transfusion was associated with reduced coagulability and endothelial glycocalyx degradation. Transfusion of older RBCs was however associated with increased coagulability. The changes observed were small to moderate and the clinical relevance of these findings should be investigated in larger studies.

Haemostatic function and biomarkers of endothelial damage before and after platelet transfusion in patients with acute myeloid leukaemia.

OBJECTIVES: The beneficial effect of platelet transfusion on haemostasis is well established, but there is emerging evidence that platelet transfusion induces an inflammatory response in vascular endothelial cells. BACKGROUND: We investigated haemostatic function and endothelial biomarkers before and after platelet transfusion in patients with acute myeloid leukaemia. MATERIALS AND METHODS: Blood was sampled before, 1 and 24 h after platelet transfusion. Primary and secondary haemostasis was evaluated by whole blood aggregometry (Multiplate) and thromboelastography (TEG). Endothelial biomarkers (sICAM-1, syndecan-1, sThrombomodulin, sVE-Cadherin) and platelet activation biomarkers (sCD40L, TGF-beta) were investigated along with haematology/biochemistry analyses. RESULTS: Twenty-two patients were included. Despite continued low platelet counts, platelet transfusion normalised the median values of most TEG parameters and slightly increased platelet aggregation (all P < 0·05). Endothelial biomarkers were not significantly affected by transfusion. The 1 h sCD40L level correlated positively with Syndecan-1 and soluble thrombomodulin delta values, biomarkers of endothelial damage (both P = 0·005). CONCLUSION: Platelet transfusion improved haemostasis, whereas post-transfusion increases in sCD40L were associated with endothelial damage, indicating that transfused platelets and platelet-derived pro-inflammatory mediators may have opposite effects on the endothelium.

Measuring the impact of a restrictive transfusion guideline in patients with acute myeloid leukaemia.

Interventions to change physician transfusion behavior are often evaluated by examining the amount of red blood cell (RBC) units transfused or the proportion of patients transfused before and after the intervention. The pre-transfusion haemoglobin concentration is a sensitive measure of transfusion practice, but has not been used to evaluate behavioral interventions. We examined the effect of a Danish National Board of Health December 2007 transfusion guideline on the behavior of clinicians treating acute myeloid leukaemia (AML). We compared the effect of the guideline on pre-transfusion haemoglobin concentrations with other measures of transfusion behavior, including use of RBC units and proportion of patients transfused. No change in transfusion behavior could be demonstrated by examining amount of RBC units transfused and proportion of patients transfused. Conversely, the pre-transfusion haemoglobin concentration fell significantly. Pre-transfusion haemoglobin determination is a sensitive measure of the effect of an intervention to change physician transfusion behaviour.

Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia.

Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1-45 years with Philadelphia chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1-9 years (A), 266 were 10-17 years (B) and 221 were 18-45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1-3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, P<0.001), KMT2A rearrangements (6%/5%/3%, P<0.001) and higher day 29 residual leukemia for B-lineage (P<0.001), but not T-ALL (P=0.53). Event-free survival rates (pEFS5y) were 89±1% (A), 80±3% (B) and 74±4% (C) with significant differences only for non-high risk groups. Except for thrombosis, pancreatitis and osteonecrosis, the risk of 19 specified toxicities was not enhanced by age above 10 years. In conclusion, a pediatric-based protocol is tolerable and effective for young adults, despite their increased frequency of higher risk features.

Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase.

Tetranectin, a protein recently identified in a wide variety of human secretory cells (Christensen, L., and I. Clemmensen. 1989. Histochemistry. 92:29-35) was found to colocalize with latent alkaline phosphatase activity in fractions well separated from azurophil granules, specific granules, gelatinase-containing granules, and plasma membranes when postnuclear supernatants of nitrogen-cavitated neutrophils were fractionated on discontinuous Percoll density gradients. Stimulation of intact neutrophils with nanomolar concentrations of FMLP, leukotriene B4, 10-100 U/ml of tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor resulted in parallel release of tetranectin and translocation of alkaline phosphatase to the plasma membrane. Furthermore, intracellular pools of tetranectin and latent alkaline phosphatase were completely released from neutrophils under conditions that barely induced release of specific granules containing B12-binding protein. These findings indicate that tetranectin and latent alkaline phosphatase define an easily mobilizable population of cytoplasmic storage organelles in human neutrophils which are functionally distinguishable from azurophil, specific, and gelatinase-containing granules. These organelles may play an important role as stores of membrane proteins that are mobilized to the cell surface during stimulation by inflammatory mediators.

In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients.

Of 560 consecutive, newly diagnosed untreated patients with B CLL submitted for chromosome study, G-banded karyotypes could be obtained in 480 cases (86%). Of these, 345 (72%) had normal karyotypes and 135 (28%) had clonal chromosome abnormalities: trisomy 12 (+12) was found in 40 cases, 20 as +12 alone (+12single), 20 as +12 with additional abnormalities (+12complex). Other frequent findings included abnormalities of 14q, chromosome 17, 13q and 6q. The immunophenotype was typical for CLL in 358 patients (CD5+, Slg(weak), mainly FMC7-) and atypical for CLL in 122 patients (25%) (CD5-, or Slg(strong) or FMC7+). Chromosome abnormalities were found significantly more often in patients with atypical (48%) than in patients with typical CLL phenotype (22%) (P < 0.00005). Also +12complex, 14q+, del6q, and abnormalities of chromosome 17 were significantly more frequent in patients with atypical CLL phenotype, whereas +12single was found equally often in patients with typical and atypical CLL phenotype. The cytomorphology of most of the +12 patients was that of classical CLL irrespective of phenotype. In univariate survival analysis the following cytogenetic findings were significantly correlated to a poor prognosis: chromosome 17 abnormalities, 14q+, an abnormal karyotype, +12complex, more than one cytogenetic event, and the relative number of abnormal mitoses. In multivariate survival analysis chromosome 17 abnormalities were the only cytogenetic findings with independent prognostic value irrespective of immunophenotype. We conclude that in patients with typical CLL immunophenotype, chromosome abnormalities are somewhat less frequent at the time of diagnosis than hitherto believed. +12single is compatible with classical CLL, and has no prognostic influence whereas chromosome 17 abnormalities signify a poor prognosis. In patients with an atypical CLL immunophenotype, chromosome abnormalities including +12complex, 14q+, del 6q and chromosome 17 are found in about 50% of the patients, and in particular chromosome 17 abnormalities suggest a poor prognosis.

Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with recombinant human granulocyte-colony stimulating factor.

Pretreatment with haemopoietic cytokines prior to marrow harvest may result in improved quality of bone marrow harvested for autologous bone marrow transplantation (BMT). Such improvements may reduce the risk for graft failure and decrease time to engraftment. Patients undergoing autologous BMT received recombinant human G-CSF (rhG-CSF) immediately prior to marrow harvest. rhG-CSF was administered as daily subcutaneous injections for 5 days at 5 micrograms/kg body weight. Comparison of bone marrow samples before and after rhG-CSF treatment showed an increased bone marrow cellularity and a ninefold increase in the number of marrow leucocytes per volume aspirated. The mean marrow myeloid:erythroid ratio increased from 2.6 to 4.0. The mean numbers of immature (CD38 positive) and proliferating (CD71 positive) myeloid cells increased significantly from 41.6 to 50.8% and from 17.0 to 34.8%, respectively. Other subsets studied, including CD34 positive stem cells, were unchanged. The relative numbers of day 7 and 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) were unchanged. Long-term marrow cultures revealed that the numbers of 'long-term culture initiating cells' were unchanged after rhG-CSF treatment in spite of the ninefold increase in cellularity. To date, five of the patients have been transplanted with autologous marrow harvested after rhG-CSF treatment. Time to trilineage engraftment was unchanged compared with historical controls. We conclude that pretreatment with rhG-CSF prior to marrow harvest may improve the graft by increasing the total number of myeloid lineage restricted progenitor cells, resulting in stable but not accelerated myeloid engraftment of autologous marrow.

Cryptococcus neoformans var neoformans resistant to fluconazole in an HIV-negative patient with chronic lymphocytic leukemia.

Cases of fluconazole-resistant Cryptococcus neoformans have been reported in AIDS patients previously treated with fluconazole. We report a case of fluconazole-resistant cryptococcal meningitis in an HIV-negative patient not previously exposed to fluconazole. The patient experienced a clinical relapse after discontinuation of therapy with amphotericin B and subsequent initiation of fluconazole therapy. In vitro resistance was initially verified by Etest and tablet diffusion and later confirmed by NCCLS broth microdilution.

CHOP Versus Chlorambucil + Prednisolone in Chronic Lymphocytic Leukemia.

One hundred and fifty-seven previously untreated stage B or C B-CLL patients were randomized to treatment with either chlorambucil + prednisolone (CLBP) 5 days every 4 weeks or CHOP every 4 weeks. Significantly more patients achieved complete remission on CHOP, but duration of response and survival were equal in the two regimens. Non-responders on CLBP were switched to CHOP, so that finally most patients received nearly the same amount and quality of treatment, which possibly explains the lack of difference in survival. However, compared to previous studies, the study-designed intensive chemotherapy seems to prolong survival for patients with advanced disease, especially those in stage C.

Measurement of beta-2-microglobulin in serum and plasma by an enzyme-linked immunosorbent assay (ELISA).

A simple technique for the measurement of beta-2-microglobulin (beta 2M) in serum was developed. The method was designed as a sandwich technique using rabbit anti-human antibodies, employing commercially available reagents in an enzyme linked immunosorbent assay (ELISA). The assay was of high specificity, sensitivity, accuracy and reproducibility. beta 2M in serum was strongly correlated with age (p less than 0.005), but independent of sex. Values in heparin and citrate plasma were significantly lower than in serum (p less than 0.001), whereas values in serum and EDTA plasma were similar. Release of beta 2M from normal blood cells was not observed in vitro before the test procedure. An excellent correlation between the results obtained in the ELISA and a RIA was demonstrated (rS = 0.99, p less than 0.0001).

Release of elastolytic activity from human monocytes and granulocytes in vitro by immune complex stimulation.

Elastase from phagocytes are neutral proteolytic enzymes and potent destructors of elastic fibres, proteoglycan and collagen. Using soluble 3H-elastin as substrate in a cell culture assay we examined the ability of live, adherent human blood neutrophils and monocytes to release elastolytic activity following immune complex (IC) stimulation. While monocytes increased their elastolysis 2 1/2 times in response to IC (p less than 0.01), neutrophils did not but released lactoferrin and produced superoxide. Both cell types could be stimulated by phorbol myristate acetate (PMA) to increase elastolysis (p less than 0.02) and produce superoxide. Thus, when in contact with the elastin substrate, the in vitro response of monocytes and neutrophils to IC differed with respect to elastolytic release. These findings might be of interest in the understanding of cartilage destruction in immunocomplex-mediated diseases such as rheumatoid arthritis.

The monocytic receptor for lactoferrin and its involvement in lactoferrin-mediated iron transport.

Several studies suggest biological functions of the iron-binding neutrophilic glycoprotein lactoferrin that imply an initial interaction with cells from the monocyte/macrophage family. Among these, an important role of lactoferrin as responsible for the inflammatory-induced blood hyposideremia and accumulation of iron in the monocyte/macrophage system has been suggested mainly based on experiments in rodents. In a series of experiments we have examined the binding of human lactoferrin to human monocytes. We have demonstrated the presence of a receptor binding including a high-affinity component and a low-affinity component. The affinity of the binding is compatible with a biological significance of this receptor (KD is about 10(-8) M, and the number of receptors about 10(6) per cell). More than 90% of the lactoferrin will dissociate from the cell. The binding is not truly reversible since lactoferrin will lose its receptor-binding property after dissociation from the cell. The only observed change in the molecule is a small decrease in isoelectric point from 8.9 to 8.8. Lactoferrin is able to translocate at least 50% of its bound iron to intracellular ferritin in monocytes. These findings are compatible with the idea that lactoferrin might be involved in the pathogenesis of the disturbances in iron metabolism observed during inflammation.

[Screening for hemoglobinopathies at a knowledge center in Copenhagen. Resources should be allocated to solve a growing health problem in the Nordic countries]. / Hemoglobinopatiscreening vid kunskapscentrum i Köpenhamn. Resurser bör avsättas för att lösa växande hälsoproblem i Norden.

Increased immigration to the nordic countries of people from areas in which hemoglobinopathies are common diseases has resulted in an increased frequency of individuals heterozygous for serious hemoglobin disorders such as beta-thalassemia and sickle cell disease. Thus, in Copenhagen County, about 4 per cent of the immigrants from these countries are carriers of one of these diseases. A center for hemoglobinopathies has been established in Copenhagen County, dealing with diagnostics, screening procedures, genetic counseling, prenatal diagnosis, education and treatment of various hemoglobin disorders. In collaboration with Rigshospitalet and the laboratory serving general practitioners, a screening program for pregnant women of relevant ethnic origin has been established, capable of servicing the entire Copenhagen area.

[Anemia in chronic disease]. / Anaemi ved kronisk sygdom.

Anaemia of chronic disease is that associated with inflammatory disorders such as prolonged infections, auto-immune diseases and some cancers. The pathogenesis of anaemia of chronic disease is complex and includes a reduced erythropoiesis, slightly shortened red cell survival, and changes in the iron metabolism. New experimental data have shown that cytokines released during the inflammatory process are of crucial importance in this context. In particular interleukin-1 and tumor necrosis factor alpha, released from activated macrophages, have been shown to inhibit erythropoiesis and might initiate changes in iron metabolism. Clinically, anaemia of chronic disease is mild and the underlying disease usually dominates the clinical picture. Most often, the anaemia takes the form of a normocytic, normochromic anaemia with low serum iron although the iron stores are normal or increased. Anaemia of chronic disease should be distinguished from anaemia due to iron deficiency, and at the moment measurement of serum ferritin seems to be the best analysis for this purpose.

[Localized diffuse pulmonary AL-amyloidosis]. / Lokaliseret diffus pulmonal AL-amyloidose.

A 56 year-old woman was admitted due to exertional dyspnoea. Her chest x-ray and CT scanning showed widespread diffuse infiltrative changes in both lungs, associated with a pronounced decrease in diffusion capacity. Transbronchial biopsies showed primary (AL) amyloidosis. Systemic AL-amyloidosis was excluded and diagnosis of localised diffuse parenchymal pulmonary amyloidosis was established. Treatment with prednisolone and melphalan for one year has stabilised the condition. Lung transplantation will be considered in case of deterioration.

[Hemoglobinopathies. Current therapeutic possibilities]. / Haemoglobinopatier. Aktuelle behandlingsmuligheder.

In recent years, the number of immigrants has increased considerably in Denmark. Consequently, a series of new clinical pictures has appeared in the Danish health care system. Typical examples are the genetic diseases, the haemoglobinopathies. Most of the immigrants come from areas, where the gene frequency of these disorders is widely distributed, for instance the Mediterranean countries, the Middle East, Southeast Asia and Africa. Most frequent are the heterozygous thalassaemias, but also the number of patients with severe thalassaemia and other clinically important haemoglobinopathies such as sickle cell anaemia has also increased in recent years. The clinical problems concerning these patients focus on two important topics, namely genetic counselling of heterozygous individuals (in some cases combined with prenatal diagnostics) and the treatment of patients with clinically severe haemoglobinopathy. The only curative treatment of the haemoglobinopathies is allogeneic bone marrow transplantation, but this treatment can only be offered to a few of these patients. However, a variety of therapeutic options exist which can improve their prognosis and quality of life. Since the number of patients with these diseases will probably increase over the next years we find it relevant, based on typical case stories, to give a review of the present therapeutic possibilities for these disorders.

[Poisoning by kidney beans (Phaseolus vulgaris)]. / Forgiftning med kidneybønner (Phaseolus vulgaris).

During recent years, numerous new and exotic fruits have become available in Denmark. However, some of these may be potentially hazardous if incorrectly prepared. Some leguminous plants, in particular, contain considerable amounts of toxic lectins. The authors report two persons who developed severe symptoms of poisoning including diarrhoea, vomiting, muscular pain, rhabdomyolysis and toxic myocarditis after consuming raw and insufficiently cooked kidney beans (Phaseolus vulgaris). Meticulous instructions about handling should accompany the sale of potentially hazardous vegetables such as these.

[The cryopathies]. / Kryopatier.

The term cryopathies includes conditions in which abnormal sensitivity to cold is a prominent feature and includes the cold agglutinin syndrome, the cold hemolysin syndrome, the cold urticarias, the cryoglobulinemias, and cryofibrinogenemia. The cryopathies may be secondary to lymphoproliferative, autoimmune, and infectious diseases, but in many patients no underlying disease can be found (essential cryopathy). Avoidance of cold is of prime importance in all patients. Underlying disease should be treated, if possible. Severe therapeutic problems may arise in patients with essential cryopathies.

[Autologous bone marrow transplantation in malignant hematologic diseases]. / Autolog knoglemarvstransplantation ved maligne haematologiske sygdomme.

Autologous bone marrow transplantation (Auto-KMT) involves harvesting of a portion of a patient's bone marrow for subsequent reinfusion and restoration of marrow function following ablative doses of cytotoxic therapy, used in the treatment of various malignancies. The use of autologous rather than allogeneic marrow stem cells reduces the probability of acute graft-versus-host disease and reduces the need for obtaining HLA-matched marrow from limited donor pools. The greatest problem in Auto-KMT involves efficacy of the cytotoxic therapy and the obvious lack of graft-versus-leukemia effect. In addition, a theoretical limitation is that the marrow may contain clonogenic malignant cells, which may be the source of reestablished disease. In absence of phase III clinical trials directly comparing Auto-KMT with conventional therapies in the treatment of most malignancies, its role continues to be poorly defined. In an attempt to identify subsets of patients with leukemia or lymphoma who might benefit from transplantation, we performed this study of recent reports from the literature. It is concluded that the associated mortality is acceptable. At present the indications for Auto-KMT are lymphoma in relapse after conventional therapy and acute myeloblastic leukemia in second remission. It is probable that Auto-BMT will be used in earlier disease stages in the future (first remission).

[Metabolic disorders in the treatment of malignant hematologic diseases--the acute tumor lysis syndrome]. / Metaboliske forstyrrelser ved behandling af maligne haematologiske sygdomme--det akutte tumorlysesyndrom.

The acute tumor-lysis syndrome is a potentially fatal complication which characteristically arises during initial chemotherapy of malignant hematological diseases with large tumor burdens. The syndrome is characterized by hyperphosphatemia, hypocalcemia, hyperuricemia and often acute renal failure. Prior to chemotherapy the patient should be treated for 12-24 hour with intensified diuresis to ensure optimal renal function. The treatment of the fully developed syndrome is hemodialysis. Three cases of TLS which developed during initial chemotherapy of patients with acute lymnphoblastic leukemia and non-Hodgkin lymphoma are presented.