Autologous haematopoietic stem cell transplantation as a first-line disease-modifying therapy in patients with 'aggressive' multiple sclerosis.

BACKGROUND: Autologous haematopoietic stem cell transplantation (AHSCT) is an effective treatment for patients with multiple sclerosis (MS) who have highly active disease, despite the use of standard disease-modifying therapies (DMTs). However, the optimal time for offering AHSCT to patients with 'aggressive' MS is yet to be established. OBJECTIVES: The objective was to explore the safety and efficacy of AHSCT as a first-line DMT in patients with 'aggressive' MS. METHODS: All patients with 'aggressive' MS who received AHSCT as a first-line DMT in five European and North American centres were retrospectively evaluated. RESULTS: Twenty patients were identified. The median interval between diagnosis and AHSCT was 5 (1-20) months. All had multiple poor prognostic markers with a median pre-transplant Expanded Disability Status Scale (EDSS) score of 5.0 (1.5-9.5). After a median follow-up of 30 (12-118) months, the median EDSS score improved to 2.0 (0-6.5, p < 0.0001). No patient had further relapses. Three had residual magnetic resonance imaging (MRI) disease activities in the first 6 months post-transplant, but no further new or enhancing lesions were observed in subsequent scans. CONCLUSION: AHSCT is safe and effective as a first-line DMT in inducing rapid and sustained remission in patients with 'aggressive' MS.

Autologous hematopoietic stem cell transplantation for pediatric multiple sclerosis: a registry-based study of the Autoimmune Diseases Working Party (ADWP) and Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT).

Autologous hematopoietic stem cell transplantation (aHSCT) is a promising therapy for multiple sclerosis (MS), which has mainly been used in adults. The purpose of this study was to investigate efficacy and adverse events of aHSCT in the treatment of children with MS using data from the European Society for Blood and Marrow Transplantation registry. Twenty-one patients with a median follow-up time of 2.8 years could be identified. PFS at 3 years was 100%, 16 patients improved in expanded disability status scale score and only 2 patients experienced a clinical relapse. The procedure was generally well tolerated and only two instances of severe transplant-related toxicity were recorded. There was no treatment-related mortality, although one patient needed intensive care. aHSCT may be a therapeutic option for children with disease that does not respond to standard care.

Onset and outcome of pregnancy after autologous haematopoietic SCT (AHSCT) for autoimmune diseases: a retrospective study of the EBMT autoimmune diseases working party (ADWP).

Autologous haematopoietic SCT (AHSCT) is increasingly used to control severe and refractory autoimmune diseases (AD). Many patients are women of reproductive age with a potential desire for children. We present a multicentre retrospective analysis of pregnancy and childbirth in patients who underwent AHSCT for AD. The databases of the European Blood and Marrow Transplantation and University of Sao Paulo, Ribeirão Preto, Brazil were searched for female patients aged 18-50 years who had received AHSCT for AD between 1994-2011. In 324 adult female patients, 22 pregnancies were reported in 15 patients between 1997-2011. Indications for AHSCT included multiple sclerosis (n=7), systemic sclerosis (n=5), rheumatoid arthritis (n=1), juvenile idiopathic arthritis (n=1) and Takayasu disease (n=1). Of the 22 reported pregnancies, 20 followed natural conception. 15 pregnancies (68%) resulted in healthy life births, whereas 7 (32%) failed. Exacerbations of AD occurred in two patients during second pregnancies. No maternal mortality was associated with pregnancy or postpartum. There were no reports of congenital, developmental or any other disease in the children. This retrospective analysis confirms the possibility of pregnancy and childbirth following AHSCT for severe AD. The outcome of pregnancy is generally good and most led to the birth of a healthy child.

Thrombophilia in children with cystic fibrosis.

In some children with cystic fibrosis (CF), percutaneous long lines occlude sooner than expected (due to thrombophlebitis or thrombosis), and many have a totally implantable venous access device (TIVAD), a recognized complication of which is thrombosis. This complication is more likely if the child has an underlying thrombotic tendency, which may be enhanced in the presence of inflammatory lung disease. There are no reports of an identified association of heritable thrombophilia with CF, although individual cases have been recognized. Our aim was to determine the incidence of thrombophilia in children with CF. In a tertiary pediatric CF center, blood was screened for thrombophilia at annual review, and retested if abnormal. A thrombotic abnormality was found in 41/204 (20%) patients. These included activated protein C resistance (10/204, 5%) with a prevalence similar to that expected, but the following abnormalities had an increased prevalence: antithrombin deficiency (2/204, 1%), protein S deficiency (11/204, 5%), protein C deficiency (8/204, 4%), and lupus anticoagulant (18/204, 9%). There were no differences found in those with thrombophilia for the following parameters: age, gender, genotype, lung function, presence of Pseudomonas aeruginosa, prothrombin time, serum IgE, aspergillus-specific IgE, liver function, and blood inflammatory markers. Fifteen children had TIVADs, 4 of whom had evidence of thrombophilia. In conclusion, a significant proportion of patients had a thrombophilic abnormality. We recommend that thrombophilia screening be performed prior to insertion of a TIVAD, and also in those with a history of venous thrombosis, blocked TIVADs, or recurring problems with long lines.

Prospective double-blind randomized study of the effects of four intravenous fluids on platelet function and hemostasis in elective hip surgery.

A prospective randomized double-blind study was performed to determine the effects of three colloids, Haemaccel, Gelofusine and albumin, and also saline on platelet activation, platelet aggregation (induced by adenosine diphosphate (ADP), epinephrine, collagen) platelet agglutination by ristocetin and other hemostatic variables in 55 patients undergoing primary unilateral total hip replacement. The fluids were administered according to normal clinical practice and assessments were made immediately before, at the end, and 2 h after the end of surgery. Surgery was accompanied by thrombin generation (increases in thrombin/antithrombin III complex, prothrombin F1 +2 fragment) platelet activation (betaTG) and compromised coagulation. Generally, the platelet activation appeared to result in platelet desensitization and brought about a persistent reduction in platelet aggregation to ADP and epinephrine, irrespective of the fluid used. Additionally, Haemaccel and Gelofusine inhibited ristocetin-induced platelet agglutination and albumin inhibited collagen-induced platelet aggregation. Gross inhibitory effects of Haemaccel that had been predicted from an earlier in vitro study did not occur. Particular fluids had selective additional effects on the hemostatic system. Albumin infusion served to maintain plasma albumin at normal concentrations postsurgery. The two gelatin preparations, Haemaccel and Gelofusine, maintained plasma viscosity. All three colloids led to a transient increase in activated partial thromboplastin time postsurgery and also a transient fall in the concentration of factor VIII, which were accompanied by a transient increase in bleeding time, but there was no measurable increase in blood loss. Inhibition of platelet aggregation by certain colloids may provide additional protection against the increased thrombotic risk in patients following major surgery.

Study of five cell salvage machines in coronary artery surgery.

We evaluated the effectiveness, ease of use and safety of five machines for blood salvage during coronary artery surgery. All were equally effective in concentrating red cells. We measured haemoglobin, packed cell volume, free haemoglobin, white cells, neutrophil elastase, platelets, thrombin-antithrombin complex (TAT), prothrombin activation peptide F1.2, fibrin degradation product (d-dimers), tissue plasminogen activator (tPA) and heparin in wound blood, in washed cell suspensions and in a unit of bank blood prepared for each patient. All machines were equally safe and easy to use and were equally effective in removing heparin and the physiological components measured. There were no adverse effects on patients. Clotting factors are severely depleted both in salvaged blood, even before washing, and in bank blood. Cell savers are a valuable adjunct to coronary artery surgery, but careful monitoring of coagulation is required when the volumes of either bank blood or salvaged blood are large.

Systemic platelet effects of contrast media: implications for cardiologic research and clinical practice.

BACKGROUND: Angiographic contrast media cause platelet activation and decrease aggregability in vitro. We have previously shown in vitro a significant antiplatelet effect of contrast media at the concentrations obtained locally in the coronary artery during angioplasty. It is not known, however, whether a systemic effect is present. METHOD: Thirty patients undergoing diagnostic coronary angiography were prospectively randomized to receive the nonionic medium iohexol, ionic low-molecular-weight medium ioxaglate, or ionic high-molecular-weight medium diatrizoate. Platelet aggregability was measured before and after the investigation with whole blood electrical impedance aggregometry (WBEA) with collagen agonist and the PFA-100 (Dade, Miami, Fla) platelet function analyzer with combined shear, collagen, and adenosine diphosphate as agonists. RESULTS: With WBEA, with iohexol no difference in impedance change was seen: (medians and ranges) before, 9.8 Omega (4.8-19.2 Omega) versus after, 9.6 Omega (2-19.2 Omega) (P not significant [NS]). With ioxaglate a significant fall was seen: before, 8.6 Omega (6.4-15.2 Omega) versus after, 6.6 Omega (0-12.4 Omega) (P =.004). With diatrizoate a significant and greater fall was seen: before, 10.8 Omega (6.4-17.6 Omega) versus after, 6.6 Omega (0-10.8 Omega) (P =.002). With PFA, no difference in closure time was seen with any medium: iohexol before, 99 seconds (79-142 seconds) versus after, 142 seconds (63-128 seconds) (P NS); ioxaglate before, 120 seconds (75-258 seconds) versus after, 95 seconds (74-258 seconds) (P NS); and diatrizoate before, 114.5 seconds (65-250 seconds) versus after, 100.5 seconds (72-300 seconds) (P NS). CONCLUSIONS: Ionic but not nonionic contrast media have a systemic antiplatelet effect at diagnostic angiographic doses when measured with WBEA. Such an effect has not been shown before. This may explain the observed improved clinical outcome with ionic contrast media but also might confound platelet studies in coronary angioplasty.

High molecular weight kininogen deficiency: a patient who underwent cardiac surgery.

A 66 year old male, referred for cardiac surgery, was found to have high molecular weight kininogen deficiency (activity <1%). Apart from activated partial thromboplastin time (APTT) >300 s, tests of haemostasis were otherwise normal (factors VIII, IX, XI, XII and prekallikrein). No inhibitor of coagulation was found. The activated coagulation time (ACT) was 800 s pre-operatively and >1000 s after heparin. Heparin levels were measured directly by an anti-Xa chromogenic assay, with values of between 2.9 and 3.2 u/ml during cardiopulmonary bypass. Thrombin-antithrombin levels rose from 2.3*g/l before surgery to a peak of 83.5*g/l at the end of cardiopulmonary bypass. Cross linked fibrin d-dimers (XDP) levels rose from 100 ng/ml before operation to 600 ng/ml after protamine administration. The patient had no excess bleeding and no thrombotic complications from surgery. This patient shows that high molecular weight kininogen is not required for thrombin formation or fibrinolysis during cardiac surgery and illustrates the need to measure heparin directly in patients with such contact factor deficiencies.

Tissue factor is rapidly elevated in plasma collected from the pericardial cavity during cardiopulmonary bypass.

There is growing evidence that the tissue factor/factor VIIa pathway of coagulation is enhanced during cardiopulmonary bypass. Hitherto, available evidence has suggested that upregulated monocyte bound tissue factor is made available, either in the blood collected from the site of surgery or on circulating cells. However, cellular upregulation is slow, while generation of factor VIIa in blood collected from the pericardial cavity is rapid. We have therefore investigated the possibility of an alternative source of tissue factor, plasma (as opposed to cellular) tissue factor in blood samples taken from the central vein catheter (systemic circulation) and collected from the pericardial cavity during cardiopulmonary bypass. Six patients undergoing first time cardiopulmonary bypass grafting were studied. Tissue factor antigen was found to be rapidly elevated (by 15 min) in the pericardial plasma, approximately 5-fold above systemic levels (p <0.004). Similar elevations were found in markers of coagulation activation, factor VIIa antigen (p = 0.066), prothrombin fragment F(1+2) (p <0.003) and thrombin-antithrombin complex (p <0.03). To explore whether plasma tissue factor was (or had been) functionally active, factor VIIa was measured also with the soluble tissue factor functional assay after removal of heparin. Functional factor VIIa activity fell significantly in the systemic circulation, probably due to the heparin-induced increase (approximately 15-fold) in tissue factor pathway inhibitor (TFPI), but was elevated in pericardial blood compared with that taken from the central line catheter (p <0.006). These results demonstrate that both components of the activation complex for the extrinsic pathway of coagulation are rapidly generated in pericardial blood during bypass.

Two-chain factor VIIa generated in the pericardium during surgery with cardiopulmonary bypass : relationship to increased thrombin generation and heparin concentration.

Several recent studies have proposed that coagulation is triggered during cardiopulmonary bypass surgery by extrinsic pathway activation involving factor VIIa generation, but the methodology was indirect. Therefore, 12 patients were studied during routine cardiac and cardiopulmonary bypass surgery. Samples were taken before, during, and after bypass from the perfusate, from the aorta (retrograde cardiac drainage), pericardium, and collected suction fluid originating from the whole operative field. These samples were analyzed by enzyme-linked immunosorbent assay for 2-chain factor VIIa, by prothrombin F1+2 assay, by thrombin-antithrombin (TAT) assay, and for heparin concentration. Factor VIIa, F1+2, and TAT levels in samples from the pericardium were greatly elevated (mean, 0.92 to 1.01, 227 to 334, and 399 to 526 microg/L, respectively; preoperative mean, 0.33, 32.3, and 1.90 microg/L, respectively; P<0. 05 for all), whereas levels in suction fluid were less consistently high. Factor VIIa and both F1+2 and thrombin-antithrombin levels in samples from the aorta, pericardium, and suction fluid were significantly correlated (r=0.57, P<0.001, n=111; and r=0.51, P<0. 001, n=105, respectively), and all were inversely correlated with heparin levels (r>-0.35, P<0.001, n>92). There was no evidence of factor VIIa generation in the circuit during bypass surgery, and both F1+2 and thrombin-antithrombin levels rose only approximately 2-fold, probably because heparin levels were higher than they were in the pericardium (P<0.05). We concluded that appreciable activation of factor VII occurs on the pericardium and that this is associated with increased thrombin generation. Ineffective local heparinization may be partly responsible. These results suggest that pericardium-induced activation of factor VII should be the target of anticoagulant strategies during cardiopulmonary bypass surgery.

Increased mature and immature CCR3 messenger RNA+ eosinophils in bone marrow from patients with atopic asthma compared with atopic and nonatopic control subjects.

BACKGROUND: Eosinophil infiltration of the bronchial mucosa is characteristic of asthma. Eosinophils differentiate from CD34(+) progenitors. Animal models suggest cooperation between IL-5 and eotaxin to allow rapid mobilization of a pool of bone marrow eosinophils followed by recruitment to the airway mucosa. OBJECTIVE: The purpose of this study was to enumerate CD34(+) cell numbers in blood and bone marrow from atopic asthmatics and control subjects and to test the hypothesis that there is an increased bone marrow pool of CCR3(+) eosinophils in patients with atopic asthma, as compared with control subjects. METHODS: Bone marrow aspirates and peripheral blood were obtained from volunteers with asthma and control volunteers. CD34(+) cell numbers were evaluated by flow cytometry, and eosinophil colony-forming activity was evaluated by methylcellulose cultures. Mature eosinophils, eosinophil myelocytes, metamyelocytes, and band forms (immature eosinophils) were enumerated by morphologic findings and immunocytochemistry for eosinophil cationic protein. CCR3 and eotaxin mRNA expression was examined by in situ hybridization, and protein expression was examined by immunocytochemistry. CCR3(+) cells were further identified with Chromotrope 2R staining. RESULTS: CD34(+) cell numbers in bone marrow were increased in atopic subjects. Numbers of eosinophil colony-forming units in blood and bone marrow did not differ between groups. Percentages of both mature and immature eosinophils were increased in bone marrow from patients with atopic asthma, but not atopic patients with no asthma or normal control subjects. CCR3 was expressed by immature and mature bone marrow eosinophils. Eotaxin was expressed by bone marrow cells from all 3 groups, but there was no increase in subjects with asthma. CONCLUSION: These findings suggest that in humans there is an increased bone marrow pool of CCR3(+) mature and immature eosinophils available for rapid mobilization in subjects with asthma but not in atopic subjects with no asthma.

Role of factor XII in thrombin generation and fibrinolysis during cardiopulmonary bypass.

During cardiopulmonary bypass, thrombin is generated, which is thought to be initiated by activation of factor XII on the surface of the bypass equipment. We present a patient with severe factor XII deficiency who underwent cardiac surgery. As much thrombin was formed during cardiopulmonary bypass (measured by the prothrombin activation fragment F1 + 2 and thrombin-antithrombin complexes) as in normal patients, showing that factor XII was not necessary for thrombin generation. Factor X, but not factor IX, was activated (as measured by their activation peptides), and this activation correlated with F1 + 2 and thrombin-antithrombin complexes, suggesting that the tissue-factor/factor-VIIa pathway is the trigger for thrombin formation.

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

Review of blood transfusion practices in thoracic surgery.

Between September 1, 1989, and August 31, 1990, 516 patients were admitted to the Royal Brompton National Heart and Lung Hospital for thoracic operations. A prospective audit recorded the nature and extent of operation, the histologic diagnosis, and the number of units of blood prepared and transfused during hospitalization. Cross-matched blood was requested in 243 patients but only 16.1% of these received transfusion. In total, 1,295 units of whole blood or red cell concentrate were cross-matched and made immediately available in the operating suite at the time of operation. Only 322 units were administered (cross-match to transfusion ratio of 4.02:1). Almost half of the patients who received transfusions received 2 units or less, a third received 3 or 4 units, 10% between 5 and 10 units, and 8.4% required more than 10 units during their hospital stay. The nature and extent of resection was an indicator of the need for transfusion. Other important predisposing factors included a previous thoracic operation, resection for inflammatory disease, decortication of empyema thoracis, chest wall resection, or thoracoplasty. Other thoracic procedures such as pleurodesis, pleurectomy, open lung biopsy, pectus correction, operation for bullous lung disease, and mediastinoscopy had a negligible transfusion requirement. The data suggest that understanding risk factors for transfusion requirements of patients undergoing thoracic surgical procedures should optimize present resources. This is critical when exploiting the limited availability of donated blood and blood products. Similarly, anticipation of transfusion requirements takes best advantage of manpower within the blood bank and minimizes unnecessary and avoidable blood wastage and expenditure.

Haemostatic changes and thromboembolic risk during tamoxifen therapy in normal women.

Tamoxifen has been implicated as a risk factor for venous thrombosis in advanced breast cancer although the evidence for increased arterial or venous thrombosis with tamoxifen in early breast cancer is less clear. The effect of tamoxifen on haemostasis, and thereby possible thromboembolic risk, was investigated in normal women enrolled in a placebo controlled trial of tamoxifen as a chemopreventative agent for breast cancer. There was an initial reduction in fibrinogen levels in all women on tamoxifen over the first year of follow-up and a marginal reduction in antithrombin III and Protein S in postmenopausal women at 6 months. There were no changes in cross linked fibrinogen degradation products or Protein C for pre or post-menopausal women. There was no increase in the incidence of thromboembolic events on tamoxifen. This study demonstrates that tamoxifen has only marginal effects on factors involved in haemostasis reported to affect the incidence of arterial or venous thromboembolic disease. The follow-up time is relatively short (maximum 36 months) and careful long term follow-up is necessary to detect clinically significant morbidity.

Protein C deficiency associated with massive cerebral thrombosis following open heart surgery.

We describe a case of massive cerebral venous thrombosis following open heart surgery in a patient with a reduced level of Protein C (40% of mean level). Protein C deficiency is an inherited disorder which in the homozygous form may result in massive fatal venous thrombosis in the newborn. A Protein C level below 55% is highly suggestive of heterozygous deficiency and has been associated with a tendency to venous thrombosis although its clinical penetrance is variable. This is the first reported case of massive venous thrombosis in a patient following open heart surgery associated with Protein C deficiency.

Immunohistochemical localization of basement membrane type IV collagen in invasive and metastatic squamous carcinomas of the head and neck.

The immunohistochemical localization of basement membrane type IV collagen was investigated with a mouse monoclonal antibody in major surgical resections from 25 patients with invasive squamous carcinomas of the head and neck. Irrespective of site, size or stage of the disease, the 16 primary invasive tumours were almost completely surrounded by a layer of type IV collagen. Focal abnormalities were regularly present, consisting of thickening and aggregation of type IV collagen together with attenuation and segmental loss. Similar changes were seen in metastatic squamous carcinomas in 36 cervical lymph nodes. It is suggested that the probable formation of a normal basement membrane protein by these squamous carcinomas indicates the preservation of a normal function of differentiating squamous epithelia. The results indicate that a major basement membrane component, type IV collagen, continues to co-exist with invasive and metastatic squamous carcinomas.

Lysis of type-I collagen by squamous carcinomas of the head and neck.

Lysis of type-I collagen by squamous carcinomas of the head and neck has been studied in freshly excised tissues, xenografts and established cell lines. Investigations with 35 freshly excised tumours showed only low levels of active and total collagenase in both carcinomas and controls. A difference became apparent when the tissues were set up in explant organ culture where a significant (p less than 0.05) increase in total collagenase was found in 13/19 tumours compared with paired control tissues over a 4-week culture period. Two xenografts showed little capacity to lyse collagen in vitro and there was only limited evidence of an increase in total collagenase after explantation and growth in organ culture. Twenty tumour cell lines showed low levels of active collagenase. Total collagenase levels were significantly increased (p less than 0.05) in 4 of the cell lines derived from cancers of the tongue; this activity was sustained in subsequent passages. Six control fibroblastoid cell lines also showed low levels of active collagenase. Levels of total collagenase were consistently high, but this activity was transient and declined in subsequent passages. Co-cultivation experiments with II tumour-cell lines and 5 fibroblastoid cell lines showed some enhanced, synergistic destruction of collagen. Parallel experiments with supernatant media from the carcinoma and fibroblastoid lines showed no enhancement, indicating that intact carcinoma cells and fibroblastoid cells are required for synergistic collagenolysis to take place.