A two-step approach (Enzyme-linked immunosorbent assay and confirmation assay) to detect antibodies against von Willebrand factor in patients with Acquired von Willebrand Syndrome.

BACKGROUND: Acquired von Willebrand Syndrome is a rare bleeding disorder, which arises in individuals with no personal or family history of bleeding, associated with lymphoproliferative and myeloproliferative disorders or other diseases. AIM: To develop a two-step approach assay to detect autoantibodies against VWF and to verify their prevalence in AVWS. METHODS: AVWS definition: negative personal or family history of bleeding diathesis, VWF below normal range and recent history of bleeding manifestations. Twenty-three consecutive patients affected by AVWS were enrolled. An ELISA assay (first step) using recombinant VWF protein immobilized on plates and sheep/goat polyclonal anti-human IgG or IgM labelled with peroxidase was developed. A group of 40 healthy subjects was tested to calculate the floating cut point value. A confirmation assay (with addition of purified VWF vs buffer) was performed (second step). RESULTS: Twenty-one patients (93%) had an associated disease, two patients had idiopathic AVWS. Anti-VWF autoantibodies were detected in 9 patients (39%). Of these, eight (89%) had VWF:RCo levels <10%, but none of them resulted positive using Bethesda assay (neutralizing antibodies). The confirmation test confirmed the positive results obtained with ELISA and resulted negative in those patients with negative results and in the controls. CONCLUSION: With the present two-step approach assay nine out of 23 (39%) patients affected with AVWS resulted positive for anti-VWF autoantibodies. This ELISA assay might be used as an additional confirmation tool in the diagnostic procedure in patients affected by AVWS or in the follow-up of congenital and acquired patients exposed to replacement therapy.

Salvage therapy with high dose Intravenous Immunoglobulins in acquired Von Willebrand Syndrome and unresponsive severe intestinal bleeding.

A 91-year-old woman affected with acquired Von Willebrand (VW) syndrome and intestinal angiodysplasias presented with severe gastrointestinal bleeding (hemoglobin 5 g/dl). Despite replacement therapy with VW factor/factor VIII concentrate qid, bleeding did not stop (eleven packed red blood cell units were transfused over three days). High circulating levels of anti-VW factor immunoglobulin M were documented immunoenzimatically. Heart ultrasound showed abnormalities of the mitral and aortic valves with severe flow alterations. When intravenous immunoglobulins were added to therapy, prompt clinical and laboratory responses occurred: complete cessation of bleeding, raise in hemoglobin, VW factor antigen, VW ristocetin cofactor and factor VIII levels as well as progressive reduction of the anti-VWF autoantibody levels.

Normal reference ranges of antithrombin, protein C and protein S: effect of sex, age and hormonal status.

INTRODUCTION: Antithrombin (AT), protein C (PC) and protein S (PS) deficiencies are risk factors for venous thromboembolism. Overlapping values between heterozygous carriers and normal individuals often make a correct classification of a deficiency difficult. The aim of this study was to investigate the effect of sex, age, menopause and hormone therapy on natural anticoagulant plasma levels in a large group of healthy individuals, and to evaluate the need of separate reference ranges. MATERIALS AND METHODS: AT and PC were measured with a chromogenic assay, antigenic free PS with an ELISA test. To evaluate the effect of sex, age, oral contraception, hormonal status (and their interaction) on AT, PC and PS levels, linear regression models were used. Biological relevance and the value of the normal deviate z were chosen as rules to decide for separate reference ranges. RESULTS: The study population consisted of 1837 healthy adult individuals (741 men, 1096 women), aged 18-85 years (median age: 44 years). In men AT levels decreased after the age of 50 years. Men had higher levels of PS than women, particularly at young ages. In women, after correction for menopause, only PC levels increased with age. Menopause affected AT and PS, but not PC levels. Oral contraceptive intake was associated with a decrease of AT and PS, and an increase of PC levels. CONCLUSIONS: For AT, PC and PS, sex- and age-specific normal reference ranges can be useful, in order to better discriminate true carriers of a natural anticoagulant deficiency.

Risk factors for postpartum hemorrhage in a cohort of 6011 Italian women.

INTRODUCTION: Postpartum hemorrhage is responsible for 25% of maternal pregnancy-related deaths and it is the first cause of maternal morbidity and mortality worldwide. OBJECTIVE: To define the prevalence of postpartum hemorrhage and associated risk factors after vaginal birth and to develop a risk model that improves postpartum hemorrhage prediction. PATIENTS AND METHODS: All women who underwent a vaginal delivery at the Obstetric Unit of a large University hospital in Milan (Italy) between July 2007 and September 2009 were enrolled. Postpartum hemorrhage was defined as ≥ 500 mL blood loss. A nomogram tailored to predict postpartum hemorrhage was developed, summarizing the impact of each covariate on the probability of postpartum hemorrhage. RESULTS: 6011 women were studied (24% had blood loss ≥ 500 mL and 4.8% ≥ 1000 mL). Nulliparity, episiotomy, retained placenta and high neonatal body weight were confirmed as risk factors for postpartum hemorrhage. The odds ratio of postpartum hemorrhage was 0.86 (95%CI 0.78-0.90) for each 1 gr/dL increase in ante-partum hemoglobin. An extensive internal validation of the developed nomogram demonstrated a good stability of the risk model. CONCLUSIONS: Low ante-partum hemoglobin is a new potentially modifiable risk factor for postpartum hemorrhage. A nomogram to predict the probability of postpartum hemorrhage is now available for external validation.

Coagulation testing before epidural analgesia at delivery: cost analysis.

BACKGROUND: The usefulness of coagulation tests performed before epidural analgesia for surgery or to alleviate labour pain is controversial. The aims of this study were: (1) to evaluate the prevalence of abnormal tests in a large cohort of healthy pregnant women and their association with epidural hematoma; (2) to assess the approach of the anesthesiologists to women with abnormal tests; (3) to evaluate the cost of performing coagulation tests before epidural analgesia in all healthy pregnant women. METHODS: Data regarding epidural analgesia, epidural hematoma, PT, APTT, fibrinogen and platelet count were extracted from medical charts. RESULTS: There was no case of epidural hematoma in 2546 pregnant women undergoing epidural analgesia. PT and APTT results were obtained in 2871 women; fibrinogen in 4063 women; platelet count in 5090 women. Three of them (0.1%) had a prolonged PT, 4 (0.14%) had a prolonged APTT, 27 (0.53%) had platelets ≤ 100 × 10(9)/L and 37 (0.91%) had plasma fibrinogen levels <3 g/L. No further tests were requested by the anesthesiologists in these women. Only women with platelets <80 × 10(9)/L were denied epidural analgesia. Based on the data from the literature on the frequencies of epidural hematoma after epidural analgesia, a total cost ranging from 4.5 to 40 million Euros to perform coagulation tests would be necessary to avoid one case of epidural hematoma. DISCUSSION: Unselected coagulation tests before epidural analgesia are not recommended, because epidural hematoma is extremely rare in healthy pregnant women and the cost of screening is not justified.

Maternal and fetal thrombophilia in intrauterine growth restriction in the presence or absence of maternal hypertensive disease.

Intrauterine growth restriction (IUGR) depends on the placental capacity to transfer oxygen and nutrients from the maternal to the fetal circulation. Placental insufficiency may be caused by impairment of the maternal or fetal circulation by a thrombotic event, possibly associated with thrombophilic disorders. The goals of our study were to define the role of maternal/fetal gain-of-function factor V Leiden and prothrombin G20210A mutations in the development of IUGR and to evaluate whether maternal pregnancy-induced hypertensive diseases would modify any such association. This is a case-control study: controls were 259 normal pregnancies, cases were 77 IUGR, 28 with and 49 without preeclampsia (PE) or pregnancy-induced hypertension (PIH). An association was found between IUGR and fetal thrombophilia (OR 2.09 CI 95% 1-4.5). The association was stronger in IUGR without PE and PIH (OR 2.9 CI 95% 1.3-6.6). This suggests a role for the fetal genotype in the development of IUGR.

Endothelial protein C receptor plasma levels increase in chronic liver disease, while thrombomodulin plasma levels increase only in hepatocellular carcinoma.

INTRODUCTION: Thrombomodulin (TM) and endothelial protein C receptor (EPCR) are two transmembrane endothelial receptors involved in the protein C pathway, that regulates coagulation and inflammation processes. We postulated that soluble thrombomodulin and EPCR are plasmatic markers of progression to hepatocellular carcinoma (HCC) and prognostic indicators in cirrhotic patients. MATERIALS AND METHODS: Plasma levels of TM and EPCR were measured in 104 patients affected by different stages of liver diseases (66 patients with HCC, and 38 without HCC), and in 52 healthy controls. RESULTS: EPCR levels were higher in patients than in controls (239+/-1.8 ng/mL vs. 127+/-1.5 ng/mL, p<0.0001). TM levels were higher in patients with HCC than in those without (42.1+/-2.0 ng/mL vs. 28.3+/-2.1 ng/mL; p=0.039), while EPCR levels were similar in the two groups. No association between TM and clinical outcome was found, while high levels of EPCR were associated with death and thrombosis of the portal vein. CONCLUSIONS: We surmise a possible role for high levels of TM as a marker of HCC development in patients with cirrhosis, whereas high levels of EPCR are a possible marker of worse HCC prognosis, being a sign of endothelial damage of large vessels.

Annexin V C/T-1 polymorphism and pregnancy complications.

The -1T variant of the annexin V gene, which has been suggested to have a protective role against thrombotic disease, was evaluated in 140 women with pregnancy complications and 317 control women with uncomplicated pregnancies. The presence of the -1 CT or TT genotype did not show a protective effect in normal pregnancies.

Expression of endothelial protein C receptor and thrombomodulin in the intestinal tissue of patients with inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel diseases are characterized by disorders of immunity, thrombosis of large vessels, and microthrombosis of mucosal vessels. The expression of endothelial protein C receptor (EPCR) and thrombomodulin-two receptors of the protein C pathway involved in thrombin scavenging and inflammation-was studied in intestinal resection specimens or mucosal biopsies from patients with inflammatory bowel disease and from controls. The soluble forms of the receptors in plasma were measured. DATA SOURCE: This study involved patients from two large university hospitals. After surgery or biopsy, tissue samples were either frozen or fixed in formalin and embedded in paraffin. Sections for immunohistochemistry examination were cut and tested with the specific antibodies to EPCR and thrombomodulin. RNA was extracted from frozen tissue for amplification via reverse-transcriptase polymerase chain reaction. Normal intestinal and diverticulitis tissue was used as a control. Resection samples from 36 patients with ulcerative colitis, 38 with Crohn's disease, 38 with colonic cancer, and 32 with diverticulitis were studied by immunohistochemistry, and frozen sections from the same patients were studied by immunofluorescence. Twelve biopsy specimens of adjacent intestinal areas from six patients with inflammatory bowel disease were included in the study for reverse-transcriptase polymerase chain reaction. Soluble receptors were measured in the plasma of 52 inflammatory bowel disease patients and 52 controls. DATA SUMMARY: EPCR and thrombomodulin were expressed on the mucosal endothelium of controls, and the intensity of the signal decreased in inflammatory bowel disease patients. EPCR was expressed by dendritic-like cells in controls, which also stained positive for CD21. The EPCR/CD21 dendritic-like cells were not as commonly observed in sections from ulcerative colitis patients as they were in sections from control patients (12.0 +/- 3.6 cells per high-power field vs. 23.8 +/- 10.4 cells per high-power field, p =.03), and this decrease was less evident in sections from Crohn's disease patients. Levels of messenger RNA for EPCR paralleled protein expression. Soluble thrombomodulin and EPCR levels were both higher in patients than in controls: 41.5 vs. 26.0 ng/mL (p <.0001) and 141 vs. 130 ng/mL (p <.05), respectively. CONCLUSIONS: EPCR expression on dendritic-like cells that bear the key complement receptor CD21 suggests a role for EPCR in innate immunity. The reduced expression of thrombomodulin and EPCR in the mucosal vessels in inflammatory bowel disease impairs protein C activation, favoring microthrombosis.

Intrauterine growth restriction and genetic predisposition to thrombophilia.

BACKGROUND AND OBJECTIVES: Intrauterine growth restriction is an important cause of morbidity and mortality. Its pathogenesis is still a matter of debate. The aim of this study was to evaluate the association between intrauterine growth restriction (diagnosed in utero by serial ultrasound examinations and characterized by abnormal umbilical arterial Doppler velocimetry) and thrombophilic polymorphisms (factor V Leiden, prothrombin G20210A) or methylenetetrahydrofolate reductase C677T carried by mothers and/or neonates. DESIGN AND METHODS: This was a case-control study with prospective enrollment. Fetuses with intrauterine growth restriction were included if they had three characteristics: 1) reduced intrauterine growth (measured in utero by ultrasound); 2) birth weight below the 10th percentile; 3) abnormal Doppler velocimetry of the umbilical artery. The three polymorphisms were evaluated in 48 cases and in 98 controls by polymerase chain reaction (PCR) and restriction analysis. RESULTS: Factor V Leiden was present in 2/48 (4%) mothers or neonates among cases and 7/98 (7%) among controls. Prothrombin G20210A was present in 0/48 (0%) mothers or neonates among cases and 4/98 (4%) among controls. Methylenetetrahydrofolate reductase C677T was present in 16/48 (33%) mothers or neonates among cases and 22/98 (22%) controls. Overall the prevalence of the polymorphisms in mothers and/or neonates was 18/48 (37%) in cases and 33/98 (34%) in controls. INTERPRETATION AND CONCLUSIONS: No association was found in this study between intrauterine growth restriction with abnormal umbilical blood flow and thrombophilic polymorphisms or methylenetetrahydrofolate reductase C677T.

Expression of endothelial protein C receptor and thrombomodulin in human coronary atherosclerotic plaques.

BACKGROUND: The expression of selected genes in human coronary atherosclerotic plaques may help to clarify the evolution of atherogenesis and the causes of thrombogenesis on some fissured plaques. The aim of this study was to analyze the expression of the genes known to participate in inflammation and hemostasis: thrombomodulin and endothelial protein C receptor, E- and P-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor and plasminogen activator inhibitor-1 (PAI-1). METHODS: RNA was extracted and reverse-transcribed from 27 atherectomized human coronary atherosclerotic plaques. The genes were specifically amplified together with a housekeeping gene. RESULTS: Thrombomodulin was not expressed in the 8/27 plaques from which RNA could be obtained. The levels of expression of tissue factor, ICAM-1, P- and E-selectin, and PAI-1 were low, whereas those of endothelial protein C receptor and VCAM-1 were high. CONCLUSIONS: RNA may be extracted from ex vivo atherosclerotic plaques. In addition to anticoagulation, endothelial protein C receptor may play an important inflammation-related role in plaque development.

Coagulation activation and long-term outcome in acute coronary syndromes.

After an episode of unstable angina or myocardial infarction, a high proportion of patients show biochemical signs of coagulation activation, expressed as persistently elevated thrombin generation, in their blood. It is not known whether this has any influence on long-term outcome. In this prospective multicenter cohort study, we assessed the relation of persistently elevated thrombin generation to outcome in 319 consecutive patients with acute coronary syndromes enrolled in the Global Use of Strategies To Open occluded coronary arteries (GUSTO) IIb trial. Plasma prothrombin fragment 1 + 2 levels, an index of "in vivo" thrombin generation, was measured during the acute phase and after 1, 6, and 12 months, and its relation to outcome was assessed during a median 29-month follow-up period. The primary end point of cardiac death or myocardial (re)infarction occurred in 61 patients (19%). There was a U-shaped relationship between plasma prothrombin fragment 1 + 2 levels and the risk of developing the primary end point; intermediate levels (1.5-1.9 nM) were associated with the lowest risk, whereas both higher (> 1.9 nM) and lower (< 1.5 nM) values were associated with an increased risk (relative risk [RR] 1.56 and 95% confidence interval [CI], 1.25-2.28; RR, 1.35 and 95% CI, 1.11-1.86, respectively). After an episode of acute coronary syndrome, both high and low levels of thrombin generation are predictors of an increased risk of an unfavorable outcome.

Mutations in the thrombomodulin gene are rare in patients with severe thrombophilia.

Because thrombomodulin plays a key role in the protein C pathway, we evaluated the contribution of thrombomodulin gene mutations to venous thrombosis. We examined 38 patients with recurrent, documented thrombotic events at a young age and a positive family history. Twelve individuals with low levels of soluble thrombomodulin in plasma were also studied. Finally, the allelic frequency of the Ala455Val polymorphism was estimated in 192 patients with at least one thrombotic event and in 369 age- and sex-matched asymptomatic controls. Two mutations were identified; G/A-201, in a severely thrombophilic patient and G/T 1456, in a patient with low soluble thrombomodulin levels. The first mutation has been reported by some, but not others, to be associated with moderately reduced levels of thrombomodulin. The second was identified previously in a patient with low soluble thrombomodulin, but expression studies failed to show functional changes in the mutant. Thrombomodulin gene mutations thus appear to be rare even in highly selected thrombophilic patients, and possibly functionally irrelevant. The allelic frequency of the Ala455Val polymorphism was identical in patients and controls. Considering the lack of a phenotype and the costly screening procedure, we recommend that thrombomodulin defects be sought only for research purposes.