The Oklahoma Thrombotic Thrombocytopenic Purpura-Hemolytic Uremic Syndrome Registry: the Swiss connection

OBJECTIVES: Thrombotic thrombocytopenic purpura (TTP) was initially described as an uncommon and usually fatal disorder. With effective treatment it is more frequently diagnosed, the clinical presentations are more diverse, and long-term sequelae are becoming recognized. METHODS: Patient data are from The Oklahoma TTP-hemolytic uremic syndrome (HUS) Registry, an inception cohort of 348 consecutive patients with their first episode of clinically diagnosed TTP or HUS, 1989-2006. The Registry enrolls all patients in a defined region who are diagnosed with TTP or HUS and for whom plasma exchange treatment is requested. ADAMTS13 activity has been analyzed on 235 (93%) of 254 patients since 1995 at the University of Berne, Switzerland. Patients are described by clinical categories, related to their associated conditions and clinically apparent etiologies, and by the presence of severe ADAMTS13 deficiency. RESULTS AND CONCLUSIONS: The clinical spectrum of syndromes described as TTP is variable with multiple etiologies. Advances in clinical and laboratory investigation have provided better understanding of the pathogenesis of these syndromes, their clinical evaluation and management, and their long-term outcomes. In addition to new information about TTP, these studies provide a model for translational research to define the complete community spectrum of uncommon disorders.

Significantly shorter telomeres in T-cells of patients with ZAP-70+/CD38+ chronic lymphocytic leukaemia

In contrast to other B-cell neoplasias, chronic lymphocytic leukaemia (CLL) is characterized by increased non-leukaemic T-cells. In order to assess their replicative history, telomere length was analyzed in subsets of leucocytes from CLL patients. Naive and memory T-cells from ZAP-70(+)/CD38(+) patients exhibited significantly shorter average telomere lengths than ZAP-70(-)/CD38(-) patients. Compared to the age-related percentiles of telomere length values from healthy individuals practically all values of the naive and memory T-cells from the ZAP-70(+)/CD38(+) CLL patients fell below the 50th percentile. This indicated an extensive expansion and a role for T-cells in ZAP-70(+)/CD38(+) CLL patients.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

Haematologic data, iron parameters and molecular findings in two new cases of iron-refractory iron deficiency anaemia

Matriptase-2 (Tmprss6), a type II transmembrane serine protease, has an essential role in iron homoeostasis as a hepcidin regulator. Recently, patients with TMPRSS6 mutations and suffering from iron-refractory iron deficiency anaemia (IRIDA) have been reported. We describe two new cases of IRIDA, one patient of Swiss origin and the second of Italian origin. The first case results from a large deletion of 1054 nucleotides corresponding to an in frame deletion of 30 amino acid residues in the low-density lipoprotein receptor-1/-2 (LDLR-1/-2) domains and from a missense mutation in CUB1 (S304L). In the second case, a homozygous G-->C mutation in the last nucleotide of exon 15 and which modified the consensus sequence of the 5' splice donor site of intron 15 (AGgt-->ACgt) was identified. Both patients had a high hepcidin level and low serum iron and transferrin saturation compared to age-matched controls. Continuous perfusion of i.v. iron 4 h/d x 5 d in the first case resulted in a significant rise in haemoglobin. These new cases of IRIDA illustrate the importance of LDLR-1/-2 and CUB1 domains in matriptase-2 function as well as the role of matriptase-2 in hepcidin regulation. Furthermore a deletional form of TMPRSS6 (in LDLR-1/-2 domains) resulting in IRIDA is described for the first time. These cases reinforce the belief that patients suffering from IRIDA have no specific geographical or ethnic distribution and are sporadic secondary to different mutations of the matriptase-2 gene.

Infection prevention strategies in a stem cell transplant unit: impact of change of care in isolation practice and routine use of high dose intravenous immunoglobulins on infectious complications and transplant related mortality

OBJECTIVE: Nursing in 'live islands' and routine high dose intravenous immunoglobulins after allogeneic hematopoietic stem cell transplantation were abandoned by many teams in view of limited evidence and high costs. METHODS: This retrospective single-center study examines the impact of change from nursing in 'live islands' to care in single rooms (SR) and from high dose to targeted intravenous immunoglobulins (IVIG) on mortality and infection rate of adult patients receiving an allogeneic stem cell or bone marrow transplantation in two steps and three time cohorts (1993-1997, 1997-2000, 2000-2003). RESULTS: Two hundred forty-eight allogeneic hematopoetic stem cell transplantations were performed in 227 patients. Patient characteristics were comparable in the three cohorts for gender, median age, underlying disease, and disease stage, prophylaxis for graft versus host disease (GvHD) and cytomegalovirus constellation. The incidence of infections (78.4%) and infection rates remained stable (rates/1000 days of neutropenia for sepsis 17.61, for pneumonia 6.76). Cumulative incidence of GvHD and transplant-related mortality did not change over time. CONCLUSIONS: Change from nursing in 'live islands' to SR and reduction of high dose to targeted IVIG did not result in increased infection rates or mortality despite an increase in patient age. These results support the current practice.

Design and establishment of a biobank in a multicenter prospective cohort study of elderly patients with venous thromboembolism (SWITCO65+)

In the field of thrombosis and haemostasis, many preanalytical variables influence the results of coagulation assays and measures to limit potential results variations should be taken. To our knowledge, no paper describing the development and maintenance of a haemostasis biobank has been previously published. Our description of the biobank of the Swiss cohort of elderly patients with venous thromboembolism (SWITCO65+) is intended to facilitate the set-up of other biobanks in the field of thrombosis and haemostasis. SWITCO65+ is a multicentre cohort that prospectively enrolled consecutive patients aged ≥65 years with venous thromboembolism at nine Swiss hospitals from 09/2009 to 03/2012. Patients will be followed up until December 2013. The cohort includes a biobank with biological material from each participant taken at baseline and after 12 months of follow-up. Whole blood from all participants is assayed with a standard haematology panel, for which fresh samples are required. Two buffy coat vials, one PAXgene Blood RNA System tube and one EDTA-whole blood sample are also collected at baseline for RNA/DNA extraction. Blood samples are processed and vialed within 1 h of collection and transported in batches to a central laboratory where they are stored in ultra-low temperature archives. All analyses of the same type are performed in the same laboratory in batches. Using multiple core laboratories increased the speed of sample analyses and reduced storage time. After recruiting, processing and analyzing the blood of more than 1,000 patients, we determined that the adopted methods and technologies were fit-for-purpose and robust.

Congenital thrombotic thrombocytopenic purpura caused by new compound heterozygous mutations of the ADAMTS13 gene

Upshaw-Schulman syndrome (USS) is due to severe congenital deficiency of von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains, nr 13) activity resulting in the presence of unusually large forms of VWF in the circulation, causing intravascular platelet clumping and thrombotic microangiopathy. Our patient, a 26-year-old man, had attacks of thrombotic thrombocytopenic purpura (TTP) with thrombocytopenia and a urine dipstick positive for hemoglobin (4+), often as the only sign of hemolytic activity. He had ADAMTS13 activity of <1% of normal plasma without the presence of inhibitors of ADAMTS13. ADAMTS13 deficiency was caused by two new mutations of the ADAMTS13 gene: a deletion of a single nucleotide in exon17 (c. 2042 delA) leading to a frameshift (K681C fs X16), and a missense mutation in exon 25 (c.3368G>A) leading to p.R1123H. This case report confirms the importance of the analysis of the ADAMTS13 activity and its inhibitor in patients who have episodes of TTP, with a very low platelet count and sometimes without the classic biochemical signs of hemolysis.

Third degree atrioventricular block and accelerated idioventricular rhythm associated with a heart base chemodectoma in a syncopal Rottweiler.

A 7-year-old male intact Rottweiler was presented with a 1-week history of lethargy, anorexia, vomiting and multiple syncopal events. The results of the clinical examination and electrocardiography were consistent with a third degree atrioventricular block and an intermittent accelerated idioventricular rhythm. Haematology, serum biochemistry, serology for Borrelia burgdorferi, blood culture, total T4, thoracic radiography and echocardiography did not reveal the cause of the arrhythmia. Response to medical treatment with isoproterenol was poor. Pacemaker placement was declined by the owners and the dog was euthanized at their request. Histopathological examination of the heart revealed a chemodectoma at the base of the heart. There was no neoplastic infiltration of the conduction tissue. Potential mechanisms explaining the association of the arrhythmias and the tumour, such as vagal stimulation and neuroendocrine factors are discussed.

Delayed Haematological recovery after autologous stem cell transplantation is associated with favourable outcome in acute myeloid leukaemia.

Autologous stem cell transplantation (ASCT) is applied to consolidate first remission in patients with acute myeloid leukaemia (AML). However, outcome after ASCT widely varies among AML patients. We analyzed the prognostic significance of haematological recovery for neutrophils [absolute neutrophil count (ANC) >1·0 × 10(9) /l] and platelets (platelet count >20·0 × 10(9) /l), stratifying at day 20 after ASCT in 88 consecutive and homogeneously treated AML patients in first remission. We observed that patients with delayed recovery had better overall survival (OS; ANC: P < 0·0001 and platelets: P = 0·0062) and time to progression (TTP; ANC: P = 0·0003 and platelets: P = 0·0125). Delayed recovery was an independent marker for better OS and TTP in a multivariate analysis including age, gender, number of transfused CD34+ cells, cytogenetics, FLT3-internal tandem duplication and NPM1 mutation. Our results suggest that delayed neutrophil and platelet recovery is associated with longer OS and TTP in AML patients consolidated with ASCT in first remission.