Fibrin monomer assay

Quantitative assay for fibrin monomer was done by use of a chromogenic substrate (S-2390, Coa set fibrin monomer). Samples from DIC prone patients with the underlying disease were assayed and classified into four groups. The pre DIC group showed higher FM values than the control with no laboratory coagulation abnormality, although the FDP . D-dimer showed no significant rise. FM assay is a useful marker for the detection of early coagulopathy in DIC. Administration of the AT III concentrate in the case of low level of plasma ATIII, thrombin . antithrombin complex I (TAT) caused a significant transient rise. The clinical course of DIC by TAT is often affected by the fluctuation of ATIII level in plasma, the usefulness of FM is that it reflects the real thrombin generation in DIC.     

Plasma factor VII levels in disseminated intravascular coagulation

To evaluate the activation of the extrinsic pathway of coagulation in disseminated intravascular coagulation (DIC), plasma factor VII coagulant activity (FVIIc) and antigen levels (FVIIag) were measured in 81 blood samples obtained from the 56 patients with DIC together with various hemostatic parameters. Plasma FVIIc (77 +/- 40%, range: 11-200%) and FVIIag (76 +/- 43%, range: 16-175%) were significantly lower in DIC subjects than in age-matched controls (FVIIc: 128 +/- 28%, FVII: 128 +/- 31%, p less than 0.01) and correlated significantly with both the antithrombin III and plasminogen activities (p less than 0.001). These results indicated that a decrease in factor VII levels is due to the consumption. However, there were several exceptions which showed elevated factor VII levels. This seems to be due to enhanced liver synthesis of factor VII compensating for the consumption. The level of tPA-PAI-I complex, a marker of pathologic endothelial stimulation, was negatively correlated with FVIIag (r = 0.45, p less than 0.05). Thus, the more the endothelium is pathologically stimulated, the more the extrinsic pathway is activated in DIC. The FVIIc/FVIIag ratio, an index of activation of factor VII zymogen, correlated with FDP and fibrin monomer levels (p less than 0.01). There were no correlations between the thrombin-antithrombin III complex. D-dimer, and alpha 2 antiplasmin-plasmin complex levels and factor VII levels. Considering the underlying diseases. the FVIIc and FVIIag levels were markedly lower in liver cirrhosis, but not significantly different in other diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical usefulness of the measurement of plasma D-dimer levels

To evaluate the clinical usefulness of D-dimer, various effects on the measurement of D-dimer were examined. Although both fibrinolytic and fibrinogenolytic products were detected by the measurement of FDP, only fibrinolytic products were detected by the measurement of D-dimer. In patients with DIC and other thrombo-embolic diseases, plasma D-dimer levels were significantly higher than in normal persons. A significant positive correlation between plasma D-dimer and serum FDP was found in DIC patients. In patients with DIC associated with acute promyelocytic leukemia, which is thought to be an increased fibrinogenolysis state, serum FDP was higher than the plasma D-dimer which suggests that increased fibrinogenolysis affects the result of serum FDP measurement. Plasma D-dimer significantly increased 5 minutes after endoscopic embolization with thrombin in the patients with esophageal varices. However serum FDP increased 30 minutes after the treatment, which suggests that the D-dimer is more useful for rapid detection of coagulo-fibrinolytic change than serum FDP. Plasma D-dimer was significantly higher in patients with cerebral infarction and increased with age. These finding suggest the usefulness of plasma D-dimer measurement for the specific and rapid evaluation of coagulo-fibrinolytic activation and thrombo-embolic state.     

Study on cases of D dimer values were dissociated from FDP-E

Determination of FDP D-dimer (D-dimer) has been recently developed for the diagnosis of thrombotic diseases with secondary fibrinolysis. We have studied the correlation between D-dimer and FDP-E concentrations in plasma from 282 patients with 630 samples. A linear correlation (r = 0.9269) was observed between the values of FDP-E and D-dimer. However, 13 out of 282 cases revealed an apparent dissociation of D-dimer concentrations from FDP-E values. Among them, 4 of these 13 cases (Group A) have shown to possess higher level of D-dimer when compared with the expected values from FDP-E, while 9 of 13 cases (Group B) revealed lower levels of D-dimer than that expected from FDP-E. All of Group A patients have been diagnosed as disseminated intravascular coagulation (DIC). On the other hand, in Group B patients, 6 of 9 were shown to have a widespread metastasis of cancer and 2 of them were under treatment with urokinase. To study whether Group B patients were under hypercoagulable or hyper-fibrinolytic state, we have examined ratios of AT III/alpha 2 PI and PIC/TAT in these cases. It has been shown that 4 of 9 patients in Group B have higher ratios of both AT III/alpha 2 PI and PIC/TAT if compared with other patients than Group B. This suggests that patients in Group B have been under hyper-fibrinolytic states.     

Measurement of plasma fibrin D-dimer levels with the use of a monoclonal antibody coupled to latex beads

Recently, monoclonal antibody (DD-3B6) to fibrin D-dimer was prepared and coupled to latex beads to provide a specific test (Dimertest) for fibrinolysis. The purpose of this study was to evaluate the Dimertest assay as a clinical laboratory test for the measurement of plasma fibrin D-dimer derivatives. The Dimer-test assay specifically detected 2 micrograms/mL of purified fibrin D-dimer or fibrin D-dimer/fragment E complex added to afibrinogenemic plasma but did not detect 500 micrograms/mL of either fibrinogen fragments X, D, E, or 160 micrograms/mL cross-linked fibrinogen. The fibrin(ogen) degradation product (FDP) assays of American Dade or Wellcome Diagnostics detected 5.0 micrograms/mL of fibrin D-dimer and from 1 to 10 micrograms/mL of the other FDPs. Twenty-eight percent of 150 random plasma samples assayed from hospitalized patients were positive for fibrin D-dimer derivatives. Plasma samples from 152 patients suspected of having disseminated intravascular coagulation (DIC) were assayed for serum FDP (Wellcome Diagnostics) and plasma fibrin D-dimer derivatives. Samples from 69% of patients with serum FDP levels less than 10 micrograms/mL, and more than 90% of those with serum FDP levels greater than 10 micrograms/mL, were positive for fibrin D-dimer derivatives. Dimertest results were not modified by heparin, streptokinase, freeze-thawing, or clotting plasma. Serum fibrinogen-related antigens were immunoadsorbed from Dimer-test positive sera by anti-fibrinogen antibody and formalin-fixed Cowan I strain Staphylococcus aureus. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein blotting with the use of monoclonal antibody DD-3B6 demonstrated a protein band with similar mobility to purified D-dimer. The measurement of plasma fibrin D-dimer derivatives by the Dimertest assay is a rapid, sensitive, and specific laboratory test for fibrinolysis. The Dimertest assay has proven to be a useful addition to the clinical laboratory and should be helpful in the diagnosis and management of patients with diseases associated with fibrinolysis.     

Fluorometric assay of antithrombin III. Diagnostic value in 112 patients with abnormal hemostasis

The authors assessed the diagnostic value of the plasma anti-thrombin III (AT III) assay in 112 adult hospitalized patients with abnormal hemostatic parameters, using the Protopath procedure. In 100 patients tested only once, low AT III was observed in 25 of 29 cases of acute disseminated intravascular coagulation (DIC); 7 of 10 with infections; 8 of 11 with acute liver disease; 19 of 20 with chronic liver disease; and 16 of 30 with other illnesses. The authors conclude that the assay cannot distinguish among disease categories, although it is a sensitive index of DIC. AT III was also repeatedly measured at various time intervals in 12 additional patients. The results suggest enhancement of the diagnostic and prognostic value of the assay with serial testing.     

Clinical application of laboratory diagnosis: leukemia and DIC]

Plasma levels of molecular markers of hemostatic activation were investigated in 205 samples from patients with haematopoietic malignancies. These markers included thrombin/antithrombin III complex (TAT), D-dimer, plasmin/alpha 2plasmin inhibitor complex (PIC) and thrombomodulin (TM), and were assayed by EIA methods. Samples were divided into 4 groups according to the level of FDP: group A; FDP 10 greater than, group B; 10 less than or equal to less than 20 group C; 20 less than or equal to less than 40, and group D; less than 40. The mean level of each marker except TM increased in the order of group A, B, C and D. However, in many samples belonging to group A the plasma TAT or PIC levels and both were increased in spite of low FDP level. Furthermore, levels of TAT and PIC in several samples belonging to groups C and D were within the normal range. Also, the mean levels of each marker except TM increased in the order of 2, 3, 4, 5 and over 6 points in DIC score according to the criteria of DIC diagnosis by the research committee on DIC of the Ministry of Health and Welfare in Japan. Eight of the 11 samples (72.7%) obtained from cases with a DIC score of 3 points had high plasma levels of TAT, PIC and D-dimer. Plasma levels of these markers were increased after chemotherapy. These findings lead to the following conclusions: 1) FDP reflexed activation of coagulation and fibrinolysis, but 2) FDP was not more sensitive than TAT and PIC, and 3) the increase of FDP rarely resulted from fibrinogenolysis or non-plasmin mediated fibrinolysis. Furthermore, 4) TAT, D-dimer and PIC may serve as sensitive parameters of hemostatic activation in circulating blood and be valuable markers for early diagnosis of DIC.     

Monitoring of plasma concentration of low molecular weight heparin--comparative evaluation with chromogenic and clotting assays

The efficacy of low molecular weight heparin (LMWH) in the treatment of thrombosis is increasing interest in its clinical potential. However, the measurement of in vitro anticoagulant activities of LMWH has been controversial for its appropriate clinical dose. The study has been carried out to compare two methods for measurement of anti-factor Xa activity of LMWH (fragmin). One is 'HEPTEST' recently developed as a new clotting assay method and the other is an authentic chromogenic assay using S-2222. The coefficients of variation in intra-assay were 1.87-2.75% in clotting assay, and 0.61-0.89% in chromogenic assay. The sensitivity to detect minimum concentration was 0.02 IU/ml in clotting assay, and 0.04 IU/ml in chromogenic assay. The correlation between two methods was good (r = 0.935), whereas clotting assay has been revealed as a very simple rapid method. LMWH (fragmin) with 75 IU/kg/day was administered to three patients with coagulopathy; two disseminated intravascular coagulopathy (DIC) and one veno-occlusive disease (VOD). Hemostatic abnormalities have been improved serially after treatment in all patients. During treatment, the plasma concentration of LMWH was measured, showing 0.05-0.23 IU/ml in DIC and 0.02-0.10 IU/ml in VOD. These results suggest that measurement of plasma concentration of LMWH using the easy and rapid clotting assay method as 'HEPTEST' is clinically useful for monitoring to detect clinical dose of LMWH for DIC and thrombosis.     

Antithrombin III in patients on long-term oral anticoagulants.

The antithrombin III (AT III) concentration in plasma was measured in 63 patients on oral anticoagulant treatment (mean age 57.7 years), 26 healthy laboratory controls (mean age 28 years), and 21 patients attending the hypertensive clinic who had never been on oral anticoagulants (mean age 50 years). Three methods were used to measure AT III: a coagulation assay, a chromogenic substrate assay, and an immunological assay. In patients on oral anticoagulants, the mean values for AT III in the three assays were: 124%, 107%, and 96% respectively. The mean AT III concentration in laboratory staff was 103.4%, 94%, and 104.1% for the three assays; patients attending the hypertensive clinic had AT III concentrations indistinguishable from those in patients on oral anticoagulants: 117.9%, 110.5%, and 93.9%. The difference between both patient groups and laboratory staff was statistically highly significant, but no difference was demonstrated between patients on anticoagulant treatment and those not receiving it. Our results show that the increase in the functional AT III concentration (measured by coagulation and chromogenic assays) observed in patients on oral anticoagulants is probably due to the effects of age and underlying disease rather than to the anticoagulant treatment itself.     

红花注射液对脂多糖诱导的兔弥散性血管内凝血的作用

 目的：研究红花注射液对脂多糖（LPS）诱导的兔弥散性血管内凝血（DIC）的作用。方法：兔耳缘静脉持续滴注LPS建立兔DIC模型。采用全自动凝血分析仪检测活化部分凝血活酶时间（APTT）和凝血酶原时间（PT）;凝固法测定纤维蛋白原含量;全自动血细胞分析仪进行血小板计数;发色底物法测定蛋白C及抗凝血酶Ⅲ的活性;全自动血浆分析仪测定丙氨酸氨基转移酶（ALT）和血尿素氮（BUN）;ELISA法检测血浆纤维蛋白（原）降解产物（FDP）和肿瘤坏死因子 α（TNF-α）含量。结果：DIC模型组APTT和PT进行性延长,ALT活性和BUN含量显著升高,蛋白C和抗凝血酶Ⅲ的活性显著降低,血小板计数进行性减少,血浆FDP和TNF-α含量显著升高。给予红花注射液后,DIC模型兔APTT和PT的延长明显缩短,血小板计数、纤维蛋白原含量、蛋白C及抗凝血酶Ⅲ活性均明显恢复,血浆ALT、BUN、FDP和TNF-α水平均明显降低。结论: 红花注射液对LPS诱导的兔DIC有较好的拮抗作用。     

Heparin reduction with the use of cardiotomy suction is associated with hyperfibrinolysis during distal aortic perfusion with a heparin-coated semi-closed cardiopulmonary bypass system

We sought to elucidate the effects of different anticoagulation levels and the use of cardiotomy suction on the postoperative coagulatory and fibrinolytic systems in patients undergoing distal aortic perfusion using a fully heparin-coated (semi-)closed cardiopulmonary bypass (CPB) system incorporating a soft reservoir bag. Thirty-two patients were divided into two groups: those who underwent cardiotomy suction (S group, 18 patients) and those who did not (N group, 14 patients). We administered 1–2 mg/kg heparin in the S group, which achieved an activated clotting time (ACT) of 345 ± 71 s. In the N group, we administered 0.7–1 mg/kg heparin, which achieved an ACT of 297 ± 52 s. Data on platelet counts and serum levels of fibrinogen, antithrombin III, D-dimer, and fibrin degradation products (FDP) were collected, and factors influencing these variables were analyzed by multiple regression analysis. Both the patient group and the initial ACT level were independent factors influencing postoperative levels of FDP and D-dimer, whereas peak ACT level and the use of selective visceral/renal shunt/perfusion, but not the patient group, were independent factors influencing the postoperative platelet counts. In the S group, a significant inverse correlation was found between the ACT and levels of FDP or D-dimer, whereas no correlation was found in the N group. The use of cardiotomy suction was associated with elevated FDP and D-dimer levels even when a fully heparin-coated semi-closed CPB system was used. Lower ACT levels with the use of cardiotomy suction were associated with higher FDP and D-dimer levels, whereas such a relationship did not exist when cardiotomy suction was not used.     

Diagnosis of disseminated intravascular coagulation. Role of D-dimer

Detection of the cross-linked fibrin degradation fragment, D-dimer, in patients at risk for disseminated intravascular coagulation (DIC) is strong evidence for the diagnosis. D-dimer confirms that both thrombin generation and plasmin generation have occurred. Patients at risk for DIC (58) and normal controls (7) were studied. Thirty-three patients had DIC--with fragment D-dimer identified in their serum by immunoblotting. Latex agglutination measurements of fibrin(ogen) degradation products (FDPs) and D-dimer were compared with immunoblotting in the detection of D-dimer. FDP measurement was extremely sensitive but not specific. D-dimer measurement was less sensitive but highly specific. Used in tandem, screening with FDP and confirming with D-dimer, sensitivity and specificity were maximized, rendering a predictive value of a confirmed FDP of 100% in this cohort. D-dimer is a valuable adjunct for the laboratory diagnosis of DIC but is most appropriately used as a confirmatory test for the very sensitive FDP test.     

Clinical evaluation of a test for plasma fibrin/fibrinogen degradation products (FDP) based on monoclonal anti-FDP antibody technology: an application for the scoring system of the disseminated intravascular coagulation (DIC) diagnostic criteria

Fibrin/fibrinogen degradation products(FDP) have been measured using serum samples which were specially prepared for the FDP test because of the usage of anti-human fibrinogen antibody for the assay. Since diagnostic criteria for DIC were established by the study group on thrombosis and hemostasis which is supported by the Japanese Ministry of Health and Welfare(JMHW), serum FDP assay have been used as standard methods to diagnose DIC in Japan. Recently, a reagent using an anti-human FDP monoclonal antibody was developed and this has enabled the use of plasma samples for FDP measurement. The comparability, especially of the DIC score, of a new assay, Latex test BL-2 P-FDP, using plasma samples with a conventional assay for serum was investigated. Two sets of DIC scores based on data from the two tests were compared and the correlation was high with 97.5% of the patients being diagnosed with the same DIC status. In four disease groups--DIC, thrombosis, leukemia and solid cancer--high comparability between the two tests was also shown and no significant difference was observed in the correlation coefficient and the slope coefficient between serum and plasma samples. To conclude, it is suggested that "Latex test BL-2 P-FDP" is applicable to the diagnostic criteria for DIC from JMHW without any difficulty.     

肺炎衣原体感染与冠心病相互关系的研究

探讨肺炎衣原体(CP)感染与冠心病(CHD)的相互关系。应用ELISA法检测150例CHD患者及50名健康者(对照组)血清CP特异性抗体IgG,同时检测超灵敏C反应蛋白(hsCRP)、D二聚体(DDimer)及纤维蛋白降解物(FDP)。CHD患者血清检测结果:CP阳性率占72%;hsCRP阳性率占73%;DDimer、FDP指标在CP阳性组与CP阴性组比,P<0.05。实验证明,CP感染与CHD发病机理存在着一定的关系。     

30例D-二聚体假性增高的数据分析

目的探讨30例临床检测中血浆D-二聚体水平>FDP水平的原因及相关资料分析。方法收取临床检测过程中首次出现血浆D-二聚体水平>FDP水平的标本30例。分别用血浆D-二聚体检测试剂(STA-LIATEST D-DI和STA-LIATEST D-DI PLUS)检测。并对其进行数据比较和原因分析。结果血浆D-二聚体检测试剂STA-LIATEST D-DI与STA-LIATEST D-DI PLUS数据相比较,[3.63(2.89~10.49)μg/mL vs 0.36(0.26~1.00)μg/mL,P<0.0001],两组数据的差别具有统计学意义。30例患者中21例患者类风湿因子(rheumatoid factor,RF)水平异常,>20 IU/mL(正常参考值上限)。9例患者类风湿因子水平正常,<20 IU/mL。结论血浆D-二聚体检测试剂STA-LIATEST D-DI PLUS对于D-二聚体水平假性增高的标本有很强的的纠正能力。血浆D-二聚体水平>FDP水平时,绝大多数是由于类风湿因子干扰造成的,少部分原因不明确,可能由其他异嗜性抗体导致。     

Incidence and possible reasons for discordant results between positive FDP and negative D-dimer latex assays in clinical specimens.

In general, FDP and D-dimer values have a correlation in clinical conditions associated with disseminated intravascular coagulation(DIC) or coagulation activation. However, there are some patients with discordant results who demonstrate elevated FDP and negative D-dimer results by latex agglutination assays. The incidence and possible reasons for the discordance between FDP and D-dimer results were investigated through simultaneous measurements (n = 763) from clinical patients with suspected DIC or coagulation activation. 24.8% (189/763) of samples with elevated FDP were negative for D-dimer assays by the latex agglutination method. Further detailed analysis on randomly-selected discordant samples (n = 41) revealed that the most common reason for the discordance was the lower sensitivity of the semiquantitative latex agglutination method for D-dimer, compared with quantitative enzyme or other latex immunoassay. The other contributing factors to the discordance were accelerated fibrinogenolysis without secondary fibrinolysis, elevated soluble fibrin monomer and rheumatoid factor.     

Comparison of three methods for the estimation of plasma antithrombin.

Plasma antithrombin levels were measured by clotting, immunological, and amidolytic methods on two groups of subjects: 20 normal individuals and nine patients studied serially post-operatively (hip replacement). The postoperative patients were observed for the emergence of deep-vein thrombosis using 125I-fibrinogen uptake measurements (FUT). The three methods gave similar ranges for the normal subjects, were reproducible (cv less than 5%), and detected early postoperative reduction of antithrombin levels. All three methods failed to show any significant differences in preoperative antithrombin levels between the positive and negative FUT groups. Correlation studies were performed on the pooled data from the normal and postoperative group (range 60-130% of normal; 100 samples). The best correlation (r = 0.75; P less than 0.01) was achieved with the chromogenic kit assay method versus the Mancini immunoassay technique. The thrombin agarose (total antithrombin) gel diffusion technique correlated less well with the chromogenic (r = 0.65; P less than 0.01) and Mancini immunoassay (r = 0.45; P less than 0.01) methods. It is concluded that the chromogenic kit method gives a rapid, reproducible, and specific measurement of antithrombin III. The thrombin agarose diffusion method, though not specific for antithrombin III, is a cheap and simple method to perform. The potential of the three methods for detecting the prethrombotic stage and early thrombosis is discussed.     

Analysis of antibody reactivity for FDP D-dimer fragments by Western blotting

We evaluated three test kits for fibrin degradation products (FDP) D-dimer. We found that six of 217 plasma sample values obtained by Nanopia test were markedly higher than the values obtained using the other two kits. The regression equation for 211 samples (excluding six) was y=0.64x+3.05 (y: Nanopia, x: LIAS AUTO) and the correlation coefficient was 0.915. Therefore, we classified these samples into three categories, namely correlated(y< 1.0x), incompatible (y= 1.0x-2.9x) and markedly incompatible (y> or =3.0x). Selected samples, eight correlated, four incompatible and four markedly incompatible, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting(WB). WB analysis using anti-fibrinogen antibody showed that both high molecular weight fragments of cross-linked fibrin (HMW-XDP) and DD/E fragments were present in the correlated samples, but there was less HMW-XDP than DD/E in the incompatible samples and mostly DD/E (HMW-XDP was significantly less than DD/E) in the markedly incompatible samples. These data suggest that plasma FDP samples that contain mostly DD/E and little HMW-XDP demonstrated markedly incompatible values using the three D-dimer test kits. These data was reflected by markedly elevated plasmin alpha2-plasmin inhibitor complex values in the incompatible and markedly incompatible samples. Unfortunately, we did not directly demonstrate these phenomena by WB analysis with two anti-D-dimer antibodies used Nanopia or LPIA reagent. In the near future, we expect that standardization of FDP D-dimer assay will be accomplished.     

Factor VIII activity as measured by an amidolytic assay compared with a one-stage clotting assay

Factor VIII amidolytic activity was measured by a commercially available method and compared with clotting activity measured in a one-stage assay. Parallel assays with both methods were used for plasma samples from 40 blood donors with normal or high Factor VIII levels, 22 patients with hemophilia before or after treatment with Factor VIII concentrates, 10 patients with chronic liver disease, and 10 normal subjects with high Factor VIII levels after treatment with desmopressin. The results obtained with the amidolytic assay were highly correlated (r = 0.97) with those obtained in the one-stage clotting assay. There were no significant differences in the results with the two methods in any of the patient groups, although in two cases of mild hemophilia the amidolytic assay gave lower values than the clotting assay. The reproducibility of the amidolytic assay (coefficient of variation = 6%) was better than that of the clotting assay (12%) at both normal and low levels of Factor VIII.     

Blood coagulation and fibrinolysis in patients with Beh?et's disease

In patients with Beh?et's disease, venous thrombosis has often been described as a complication. The pathogenesis of this complication, however, has not been fully understood. In this work, various parameters of blood coagulation and fibrinolysis were studied in 20 patients with Beh?et's disease and 13 sex-matched healthy volunteers. Patients were classified into three subgroups according to the number of clinical signs involved; group I (no sign): 4 patients; group II (one or two signs): 11 patients; group III (more than three signs): 5 patients. Patients with Beh?et's disease, showed an activation of blood coagulation, such as the shortening of prothrombin time (p less than 0.001), decreases in concentrations and activities of plasma antithrombin III (AT-III) (p less than 0.01) and elevated levels of plasma thrombin-antithrombin-III complex (TAT) (p less than 0.01), compared to the control group. Plasma levels (p less than 0.01) and activities (p less than 0.01) of protein C (PC) and total protein S (PS) levels (p less than 0.05) were increased in the patients. Decreased levels of alpha 2-plasmin inhibitor (p less than 0.001) also indicated an activation of fibrinolysis in the patients. When analyzed among the subgroups, patients belong to group II and III showed higher levels of plasma FDP D-dimer (p less than 0.05) and lower levels of plasminogen (p less than 0.05), as compared with patients in group I or control group.(ABSTRACT TRUNCATED AT 250 WORDS)