Quantification of circulating activated protein C in human plasma by immunoassays--enzyme levels are proportional to total protein C levels.

We have developed a simple assay that measures the circulating activated protein C (APC) in plasma. The assay requires collection of duplicate blood samples, one in citrate plus heparin and the other in citrate plus inhibitors of the enzyme. In the heparin tube, APC reacts completely and irreversibly with its major plasma inhibitors, protein C inhibitor (PCI) and alpha 1-antitrypsin (alpha 1AT), and the complexes formed are measured by ELISAs. The amount of circulating APC is calculated from the difference between the total amount of complexed APC (sample in citrate plus heparin) and the amount of APC complexed in vivo (sample in citrate plus inhibitor). Over 95% of the APC added to blood collected with heparin was recovered in the assay. The assay can easily be performed in four hours, and had a detection limit of 0.1 ng/ml APC. The mean APC level in 18 protein C heterozygous members from seven kindreds was significantly lower (0.6 +/- 0.3 ng/ml) than in 20 healthy controls (1.1 +/- 0.3 ng/ml) (p < 0.001), whereas the mean level in 10 non-affected members from the kindreds studied was 1.5 +/- 0.3 ng/ml. In the group of 12 nonanticoagulated heterozygous protein C-deficient individuals, the three patients with a history of venous thrombosis had a mean APC level significantly lower than the nine asymptomatic members (p < 0.01), both subgroups showing similar protein C levels. There was a significant correlation in all groups between the levels of APC and the levels of protein C antigen (r = 0.758, p < 0.0001) and activity (r = 0.745, p < 0.0001), which means that APC circulating levels are proportional to protein C levels and suggests that the protein C level is the limiting factor in the rate of protein C activation in vivo.

Activation of the protein C pathway in acute sepsis.

To obtain quantitative information on the in vivo activation of the protein C system during the acute phase of sepsis, several components of the protein C pathway were studied in 18 patients. Blood samples were obtained one day after diagnosis (day 1) and, in 11 patients, also on the fourth and tenth days after diagnosis (days 4 and 10). On day 1, patients showed laboratory signs of haemostatic alterations such as positive fibrinogen/fibrin degradation products, and increased thrombin:antithrombin-III (TAT) complex levels. Compared with the control group, patients on day 1 had significantly decreased (p < 0.001) antigenic protein C (69 +/- 28%) and protein C inhibitor (PCI) (33 +/- 22%) whereas a significant increase in the levels of activated protein C (APC) complexed with alpha 1-antitrypsin (alpha 1AT) (APC:alpha 1AT, 26 +/- 15 ng/mL) and APC:PCI complex (3.0 +/- 2.0 ng/mL), and in the level of plasma kallikrein (KK) complexes with PCI (KK:PCI) (31 +/- 22 ng/mL) was observed. There was a positive correlation between APC:alpha 1AT and TAT complex levels (r = 0.597, p = 0.009). In the follow-up a trend toward normal values in antigenic protein C and PCI, and in APC:PCI and KK:PCI complex levels was found. However, PCI remained significantly decreased compared to normal values. C4b-binding protein, alpha 1AT, and TAT and APC:alpha 1AT complexes did not show any significant variations during the course of the disease, suggesting the contribution of the inflammatory and haemostatic responses, in spite of the good recovery of the patients. This study shows that in the course of sepsis, patients experience a generalized activation of the protein C pathway which was more prominent on day 1, resulting in the consumption of protein C and PCI and in the increase of APC:inhibitor complexes. Moreover, these data provide further evidence that KK:PCI circulating complexes occur in vivo.

[Subcutaneous administration of desmopressin to plasma donors: a controlled study]. / Administración subcutánea de desmopresina a donantes de plasma: estudio controlado.

BACKGROUND: The aim of the present controlled study was to assess the effect of subcutaneous administration of desmopressin acetate (DDAVP) on the yield of Factor VIII (FVIII) and von Willebrand Factor (vWF) obtained in cryoprecipitates after plasmapheresis collection. SUBJECTS AND METHODS: Fifteen plasma donors underwent twice plasmapheresis collections using an "Autophereses C. model A-201" and cryoprecipitates were prepared using standard methods. In every subject of study one of the procedure was performed 35 min after subcutaneous administration of DDAVP (0.3 micrograms/kg), obtained from a highly concentrated preparation of the drug (40 micrograms/ml). Prior to the other apheresis procedure no chemicals were administered. Evaluation of FVIII and vWF levels was performed in peripheral blood samples obtained at different time points and cryoprecipitates product samples. RESULTS: Administration of DDAVP led to two-fold increase the yield of FVIII and vWF immediately before the performance of plasmapheresis. DDAVP stimulation resulted in an increase in the yield of FVIII and vWF in cryoprecipitates, compared with levels in the absence of the drug (703 +/- 53 and 768 +/- 51 U versus 227 +/- 12 and 362 +/- 20 U, respectively). No adverse effects but flushing were observed after DDAVP administration. CONCLUSIONS: Subcutaneous administration of DDAVP prior to plasma collection is a safe method to improve drastically the content of FVIII and vWF in cryoprecipitates. This procedure should be considered as an useful tool to help in providing self-sufficiency of plasma.

[Association of acute leukemia with disseminated intravascular coagulation in adults. Analysis of 14 cases]. / Asociación de leucemias agudas y coagulación intravascular diseminada en adultos. Análisis de 14 casos.

The association of acute leukemia (AL) and disseminated intravascular coagulation (DIC) in 89 patients with a de novo diagnosis of AL made in our center during the last 8 years was retrospectively evaluated. DIC was demonstrated in 14 patients (15.7%) (7 AML-M3, 1 AML-M3, 1 AML-M2, 1 AML-M4, 2 AML-M5, and 2 ALL-L1). In 5 of them ICD was diagnosed after the beginning of chemotherapy. The factors predisposing to the development of DIC were: 1) the type of AL (p less than 0.01), as 70% of AML-M3 had DIC; 2) the intensity of granulation in leukemia cells (p less than 0.004); 3) the presence of Auer's rods and/or splinters in these cells, and 4) the presence of hemorrhagic diathesis (p less than 0.007). Eight of the 14 patients with DIC received heparin at a prophylactic dosage. No significant differences in the clinical course were in the group of patients with DIC who received heparin and in those who did not, excepting that in the former the platelet requirements were higher (p less than 0.005). Mortality rate during the first month was higher in the group of AL with DIC than in AL without DIC (p less than 0.025). Long term mortality was similar in both groups. The control of hemostasis is fundamental in AL, even in those patients without DIC at the time of diagnosis. The administration of blood derivatives has a high priority in AL with DIC. The role of heparin is still controversial.

[The value of the initial bone biopsy in chronic myeloproliferative syndromes. Review of 80 cases]. / Valor de la biopsia ósea inicial en los síndromes mieloproliferativos crónicos. Revisión de 80 casos.

In order to review the histological characteristics and to evaluate the presence of valuable findings for diagnostic or prognostic purposes, 80 patients diagnosed of chronic myeloproliferative syndromes (CMPS) who had undergone initial bone marrow biopsy were studied in retrospect. The patients were distributed into 26 cases of chronic myeloid leukaemia (CML), 22 of myelofibrosis (MF), 16 of polycythaemia vera (PV) and 16 of essential thrombocythaemia (ET). The histological findings in the 26 cases of CML consisted of hypercellular bone marrow with fat depletion in all cases; marked thickening of trabeculae was seen in 4 cases and neoformation-like osseous tissue in 7 others. Erythroblastic nests were found in 11 patients, eosinophilia in 23 instances, and abnormal location of immature precursor cells (ALIP) was present in 16 cases. Reticulinic fibrosis was found in 22 patients, collagen fibrosis in 6 and sinusoidal dilatation in 5 cases. Of the 22 MF patients, 8 had normal bone trabeculae, while 14 had trabecular thickening, of moderate degree in 6 cases and marked in 8. Osteoid tissue formation was observed in 21 instances; diminished or absent fat was appreciated in 19 cases. Erythroblastic nests were present in 12 instances, ALIP in 5 and eosinophilia in 14. Reticulin fibres were increased in 22 instances, collagen fibrosis was present in 18 cases, sinusoid dilatation in 12, and lymphoid follicles were seen in 4 patients. Trabecular thickening was found in 15 cases of the PV group (16 patients) and osteoid tissue formation in 2. Fatty tissue was decreased or absent in 11 instances Erythroblastic nests plus eosinophilia were seen in 15 cases, and ALIP in 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Conservative treatment of large rectus sheath hematoma in patients undergoing anticoagulant therapy.

BACKGROUND: The purpose of this study was to describe our experience in the conservative management of large rectus sheath hematoma (RSH) in patients undergoing anticoagulation therapy. METHODS: This is a retrospective study of the medical histories of 12 cases of RSH (11 female, one male; mean age = 67.6 years). Seven patients were taking oral anticoagulants, three were taking intravenous unfractionated heparin, and two were taking subcutaneous low-molecular-weight heparin. Six patients had a history of coughing fits. Ultrasound examination and computed tomography (CT) was performed in all cases. RESULTS: Clinically, the majority of patients presented acute abdominal pain, infraumbilical masses, and anemic syndrome. Ultrasonography demonstrated nine of the 12 cases of RSH, and CT showed the hematoma in all 12 cases. Type II (five cases) and type III (seven cases) indicate moderate and severe hematomas, respectively. Excessive anticoagulation was observed in eight cases, and coagulation within correct ranges was seen in the remaining four cases. In five patients the normalization of coagulation was achieved by administering vitamin K1 and fresh frozen plasma. All cases of type III hematoma required blood transfusion. Conservative treatment was effective in all cases. CONCLUSIONS: RSH must be suspected in women of advanced age undergoing treatment with anticoagulants who present the clinical triad of acute abdominal pain, infraumbilical mass, and anemic syndrome. CT is the examination of choice for the diagnosis of RSH. Early diagnosis of RSH permits conservative management, even in the case of large hematomas with hemodynamic repercussions and avoids unnecessary surgical intervention.

[Comparative multicenter study of a rabbit high-sensitivity thromboplastin and a recombinant thromboplastin with synthetic phospholipids]. / Estudio multicéntrico comparativo entre una tromboplastina de conejo de alta sensibilidad y una tromboplastina recombinante con fosfolípidos sintéticos.

PURPOSE: The purpose of the present study was to compare the results obtained with a human recombinant thromboplastin (Innovin, Baxter) (IN) and a high-sensitivity rabbit brain reagent (Thromboplastin IS, Baxter) (IS), on the performance of prothrombin time (PT) test and the functional assay of factors included in the extrinsic coagulation system, in order to establish possible differences on imprecision, diagnostic accuracy and sensitivity to the oral anticoagulant defect, between the two products. MATERIAL AND METHODS: Six Spanish hospital took part in the study. Plasma samples from 221 healthy subjects, 100 patients with severe liver disease, 27 with dysfibrinogenaemia, 10 with lupus anticoagulant and from 13 individuals propositus and their relatives with congenital deficiencies of the extrinsic coagulation pathway, and their relatives were studied; 188 patients stabilized on oral anticoagulant therapy and 82 on heparin therapy were also included. The in vitro effect of heparin was tested by addition of increasing amounts of heparin (0.3 to 10.0 IU/mL) to aliquots of normal plasma. RESULTS: Both in the intra-assay and in the inter-assay imprecision study, a better coefficient of variation was obtained with IN when the PT was performed on abnormal samples. Prothrombin time ratio from patients with liver disease had significantly higher values with IS. On the contrary, IN had a higher sensitivity in samples from patients with dysfibrinogenaemia or from those stabilized on oral anticoagulant therapy. In showed a very low sensitivity to heparin at concentrations corresponding to the therapeutic range. CONCLUSIONS: The results of this field study indicate that IN, compared with a high-sensitivity rabbit brain thromboplastin, is a suitable reagent for PT determination in normal subjects, patients with liver disease or with congenital deficiencies of clotting factors. It shows a higher sensitivity in cases of dysfibrinogenaemia and in patients on oral anticoagulant therapy. In addition, the recombinant reagent had better reproducibility when the PT was performed on abnormal samples, and it was hardly affected by heparin within the therapeutic range.