Measurement of factor VIII activity using one-stage clotting assay: a calibration curve has not to be systematically included in each run.

Coagulation factor VIII (FVIII) is usually evaluated using activated partial thromboplastin time-based one-stage clotting assays. Guidelines for clotting factor assays indicate that a calibration curve should be included each time the assay is performed. Therefore, FVIII measurement is expensive, reagent- and time-consuming. The aim of this study was to compare FVIII activities obtained using the same fully automated assay that was calibrated once (stored calibration curve) or each time the assay was performed. Unique lots of reagents were used throughout the study. We analysed 255 frozen plasma samples from patients who were prescribed FVIII measurement including treated and untreated haemophilia A patients. Twenty-six runs were performed on a 28-week period, each including four lyophilized control and at most 10 patient plasma samples. In control samples, FVIII activities were not significantly different when the assay was performed using the stored calibration curve or was daily calibrated. The same applied to FVIII activities in patient plasma samples that were not significantly different throughout the measuring range of activities [68.3% (<1-179) vs. 67.6% (<1-177), P=0.48] and no relevant bias could be demonstrated when data were compared according to Bland and Altman. These results suggest that in the studied technical conditions, performing the FVIII assay using a stored calibration curve is reliable, for at least 6 months. Therefore, as far as the same lots of reagents are used, it is not mandatory to include a calibration curve each time the FVIII assay was performed. However, this strategy has to be validated if the assay is performed in different technical conditions.

Acquired factor V inhibitor: Success of steroids in a patient with primary sclerosing cholangitis, ulcero haemorragic rectocolitis, biological Biermer's disease.

We report the case of a 43-years-old Turkish man with acquired deficiency of factor V (FV) diagnosed in a usual screening before a (recto) colonoscopy. In the biologic explorations, activated partial prothrombin time (APTT) was abnormally high and prothrombin time (PT) was low 18IU/dL with no anticoagulant drugs (the PT was normal 6 months ago). The controlled level of factor V was 3IU/dL with FV antibodies (9 Bethesda Units/mL). This patient had a previous history of primary sclerosing cholangitis (2000) and ulcero haemorrhagic rectocolitis (2002) and a fortuitous biological Biermer's disease was revealed. Corticosteroids were prescribed at 1mg/kg/day with decreasing during 6 months, patient had gradual regression of the caused bleeding and FV became greater than 90%, F V antibodies decreased to less than 0.7 Bethesda Units/mL. This case illustrates the presence of FV inhibitor in an autoimmune gastrointestinal context with regression of clinical (caused) signs and antibodies with corticosteroids.

Use of D-dimer in oral anticoagulation therapy.

Individualized anticoagulation management and improvement of the safety and effectiveness of oral anticoagulant have always been the focus of clinicians' attention. D-dimer, a sensitive marker of thrombosis and coagulation activation, is not only traditionally used in the diagnosis of venous thromboembolism, acute aortic dissection, and disseminated intravascular coagulation but can also be used as a helpful marker in the management of oral anticoagulant, including evaluating the anticoagulation quality, predicting clinical outcomes, and determining the optimal duration and intensity of anticoagulation.

Impact of different storage times at room temperature of unspun citrated blood samples on routine coagulation tests results. Results of a bicenter study and review of the literature.

INTRODUCTION: A maximum delay between blood collection and coagulation testing of 4 hours is recommended by most guidelines. As information on optimal storage times is limited, we investigated the potential effect of different storage times of unspun tubes, that is, ≤2, 4, 6, and 8 hours, on routine coagulation test results. METHODS: Four evacuated polymer tubes containing 0.109 mol/L tri-Na citrate were drawn from 144 patients, including 39 patients on vitamin K-antagonists. Except for storage time, all tubes underwent the same preanalytical process. Prothrombin time (PT)/international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, factor V (FV), FVIII, and D-dimer were evaluated in two centers using the same technical conditions. RESULTS: Analytical comparison of aPTT, fibrinogen, FV, and FVIII results evaluated after prolonged storage times vs a <2-hours storage demonstrated significant difference, whereas PT/INR and D-dimer remained unchanged up to 8 hours. Mean bias between test results obtained after prolonged storage times remained below the desirable values for all studied parameters except for FVIII evaluated after 6- and 8-hours storages, but only in patients with FVIII above 100 IU/dL. Even though the corresponding bias of -5.2% and -8.5%, respectively, remained within the GEHT recommended limits of variation, its evaluation after an 8-hours storage could lead to significant underestimation of FVIII. CONCLUSION: These results suggest that, in the studied technical conditions, PT/INR, aPTT, fibrinogen, FV, and D-dimer can be reliably evaluated in tubes stored unspun at room temperature for up to 8 hours after blood collection. That optimal delay should be of 6 hours for FVIII.

Developmental hemostasis: laboratory and clinical implications.

The pediatric hemostatic balance, which is different from that in adults, is an evolving process as the hemostatic system changes and matures throughout the time from fetal to adult life, particularly in the first months of life. The concept of developmental hemostasis was confirmed by several studies evaluating different patients' population in various technical conditions. All these studies demonstrated that, at birth, the plasma levels of most coagulation factors were around half that found in adults, the preterm newborns having lower levels than full-term newborns. Adult values were usually reached between a few months of age and up to above 16 years for specific parameters. If the global trends are consistent across the studies, differences in absolute values could be demonstrated that are likely due to differences in the reagents and/or the instruments used. Accordingly, it is recommended by the Perinatal and Pediatric Haemostasis Subcommittee of the Scientific and Standardization Committee of the ISTH for each laboratory to define the age-dependent reference ranges using its own technical condition. The understanding of that concept of developmental hemostasis, which is now universally accepted, is critical to ensure optimal prevention, diagnosis, and treatment of thrombotic and hemorrhagic diseases in children. Actually, developmental hemostasis could affect the interaction between anticoagulant drugs and the coagulation system and so explain in part the discrepancy between anticoagulation in adults and in children. Finally, developmental hemostasis could probably provide a protective mechanism for neonates and children, contributing to the decreased risk of thrombosis and/or bleeding in these age-groups.

In vitro sensitivity of different activated partial thromboplastin time reagents to mild clotting factor deficiencies.

INTRODUCTION: Activated partial thromboplastin time (aPTT) is a routine clotting assay that is widely used to globally screen for coagulation abnormalities. It is commonly admitted that a prolonged test result, may trigger the need for specific assays to be performed, particularly factor measurement. However, the sensitivity of aPTT reagents to deficiencies of clotting factors varies. METHODS: We evaluated, according to the recommendation of the CLSI H47-A2 guideline, the responsiveness to single factor levels of five aPTT reagents by using factor-deficient plasmas spiked with a calibration plasma to produce individual factor activities ranging from <1 to ~100 Unit (U)/dL. Test results were expressed as the sample-to-control ratio, the latter was defined as the clotting time obtained in the calibration plasma containing ~100 U/dL factor activity. The factor activity producing a prolongation of aPTT above the upper limit of its specific normal range (in ratio) was assigned as the factor responsiveness in U/dL to that reagent. RESULTS: Responsiveness ranged from 34 to 47 U/dL to FVIII: C, from 18 to 57 U/dL to FIX, from 38 to 52 U/dL to FXI, from 29 to 50 U/dL to FXII, from 40 and 59 U/dL to FV, from 7.5 to 49 U/dL to FX, and from 9.1 to 10.5 U/dL to FII. CONCLUSIONS: These results suggest that the sensitivity of the tested aPTT reagents to single factor deficiency is highly variable. Moreover, for one given aPTT reagent, its sensitivity was very different depending on the deficient factor. This must be considered when analyzing clinical materials.

Prevalence of antiphospholipid antibodies in systemic sclerosis and association with primitive pulmonary arterial hypertension and endothelial injury.

OBJECTIVE: To investigate the prevalence and clinical significance of antiphospholipid antibodies in patients with systemic sclerosis (SSc). METHODS: Autoantibodies against cardiolipin (aCL) and beta2-glycoprotein 1 (beta2-GPI) were detected by enzyme-linked immunoabsorbent assays (ELISAs) in successively hospitalised SSc patients admitted during a 24-month period. These patients were compared to patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA). RESULTS: 108 SSc patients were included: 61 had limited cutaneous SSc, 47 had the diffuse sub-type, 16 had primitive pulmonary arterial hypertension (PAH) and 34 had digital ulcerations. The control groups consisted of 37 RA and 38 SLE patients. The prevalence of aCL positivity was lower in SSc patients vs SLE patients (14 vs 47%; p < 0.001), lower in RA patients vs SLE patients (19 vs 47%; p < 0.001), and not different in SSc vs RA patients (14 vs 19%; NS). The mean aCL titer was also lower in SSc vs SLE patients (8+/-10 vs 15+/-20; p < 0.001). In SSc patients, positivity for aCL was associated with PAH (p = 0.009) and the aCL titer correlated with that of the von Willebrand antigen factor (r= 0.23; p = 0.045). The prevalence of anti beta2-GPI positive patients (IgG and/or IgM) was 5% in the SSc group, 18% in the SLE group and 5% in the RA group (SLE vs SSc and SLE vs RA; p = 0.005). CONCLUSION: We found that the prevalence of antiphospholipid antibodies in SSc patients was low. However, aCL antibodies were associated with PAH and endothelial injury.

Increased heparin cofactor II levels in women taking oral contraceptives.

Heparin cofactor II (HCII) is a thrombin inhibitor present in human plasma whose activity is enhanced by heparin. HCII exhibits important homologies with antithrombin III, the main heparin-enhanced thrombin inhibitor. Cases of recurrent thromboembolism have been recently reported in patients with HCII deficiency. Since the use of oral contraceptives (OC) is associated with an increased risk of thrombosis, the study of the plasma levels of HCII was undertaken in women taking contraceptive pills. Plasma HCII levels were found significantly higher in 62 women taking low-estrogen content OC (1.20 +/- 0.28 U/ml) than in 62 age matched women not taking OC (0.94 +/- 0.16 U/ml) or in 62 men (0.96 +/- 0.19 U/ml). Significant correlations between HCII and fibrinogen levels were reported in the three groups. From the pooled data of the two control groups (men and women not taking OC), the normal range for plasma HCII levels was defined to be between 0.60 and 1.30 U/ml (mean +/- 2 SD). Two cases of low HCII levels (less than 0.60 U/ml) were found in the control groups, but none in the group of women taking OC. It is concluded that the use of oral contraceptives is associated with a rise in HCII levels and that the screening for HCII deficiency has to be performed at distance of any OC therapy.

PIP: Heparin cofactor II (HCII) is a thrombin inhibitor present in human plasma whose activity is enhanced by heparin. HCII exhibits important homologies with antithrombin III, the main heparin-enhanced thrombin inhibitor. Cases of recurrent thromboembolism have been reported recently in patients with HCII deficiency. Since the use of oral contraceptives (OCs) is associated with an increased risk of thrombosis, the study of the plasma levels of HCII was undertaken in women taking OCs. Plasma HCII levels were found to be significantly higher in 62 women taking low-estrogen OCs (1.20 +or- 0.28 U/ml) than in 62 age-matched women not taking OCs (0.94 +or- 0.16 U/ml) or in 62 men (0.96 +or- 0.19 U/ml). Significant correlations between HCII and fibrinogen levels were reported in 3 groups. From the pooled data of the 2 control groups (men and women not taking OCs), the normal range for plasma HCII levels was defined to be between 0.60-1.30 U/ml (mean +or- 2 SD). 2 cases of low HCII levels (0.60 U/ml) were found in the control groups, but none in the group of women taking OCs. It is concluded that the use of OCs is associated with a rise in HCII levels and that the screening for HCII deficiency has to be performed at some point during the OC therapy.

Heparin cofactor II deficiency in renal allograft recipients: no correlation with the development of thrombosis.

Heparin cofactor II (HC II) is a thrombin inhibitor in human plasma which displays great similarities with antithrombin III (AT III). Hereditary HC II deficiency was recently reported to be associated with thrombophilia. Since thromboembolism constitutes an important post-operative complication after renal transplantation, we measured HC II and AT III in the plasma of 118 healthy renal allograft recipients (RAR) and found stable low HC II and high AT III levels. Administration of azathioprine (AZA), cyclosporine A (CSA) or both as immunosuppressive therapy did not affect HC II levels, but CSA seems to have raised plasma AT III. The proportion of patients with HC II deficiency was significantly higher in RAR than the proportion we previously found (11) in healthy individuals (16.9% vs 1.5%). However, the proportions with low plasma HC II were not different in healthy RAR and in ten RAR with thrombotic events, suggesting that in transplanted patients, HC II deficiency is not in itself a risk factor for the development of thrombosis.

Heparin cofactor II deficiency in patients infected with the human immunodeficiency virus.

In human plasma, heparin cofactor II (HCII) is a thrombin inhibitor, whose deficiency has been reported to be associated with recurrent thrombosis. The finding of two cases of low plasma HCII activity in two patients infected with the human immunodeficiency virus (HIV) led us to investigate this coagulation inhibitor in the plasma of a larger population of HIV-infected patients. The mean plasma HCII activity was significantly lower in 96 HIV-infected patients than in 96 age- and sex-matched healthy individuals (0.75 +/- 0.24 vs 0.99 +/- 0.17 U/ml, p < 0.0001). HCII antigen concentration was decreased to the same extent as the activity. The proportion of subjects with HCII deficiency was significantly higher in the HIV-infected group than in healthy individuals (38.5% vs 2.1%). In addition, HCII was significantly lower in AIDS patients than in other HIV-infected patients, classified according to the Centers for Disease Control (CDC) on the basis of an absolute number of circulating CD4+ lymphocytes below 200 x 10(6)/l. The link between HCII and immunodeficiency is further suggested by significant correlations between HCII activity and both the absolute number of CD4+ lymphocytes and the CD4+ to CD8+ lymphocyte ratio. Nevertheless, the mean HCII level was not different in the various groups of patients classified according to clinical criteria, except in CDC IVD patients in whom HCII levels were significantly lower. In addition, no correlation could be demonstrated between HCII and protein S activities, another coagulation inhibitor whose plasma level was also found to be decreased in HIV-infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin polymerization defect in HIV-infected patients--evidence for a critical role of albumin in the prolongation of thrombin and reptilase clotting times.

Thrombin clotting time (TCT) and reptilase clotting time (RCT) were found significantly prolonged in a series of 72 HIV-infected patients drawn for routine coagulation testing. Both TCT and RCT were highly significantly correlated with albumin (r = -0.64, and r = -0.73 respectively, p < 0.0001). TCT and RCT were significantly higher (p < 0.0001) in a series of 30 other HIV-infected patients selected on their albumin level below 30.0 g/l (group 1) than in 30 HIV-infected patients with albumin level above 40.0 g/l or in 30 HIV-negative controls; the two latter groups were not different. In vitro supplementation of plasma from group 1 patients with purified human albumin up to 45.0 g/l (final concentration) lead to a dramatic shortening effect on both TCT and RCT, which reached normal values. The TCT and RCT of the purified fibrinogen solutions (2.0 g/l final concentration) were not different in the three groups, and normal polymerization curves were obtained in all cases. This further ruled out the presence of any dysfibrinogenemia in the plasma from group 1 patients. Using purified proteins, highly significant correlations were demonstrated between the albumin concentration and the prolongations of both TCT and RCT, which were of the same magnitude order than those found in the patients plasma. These results suggest that hypoalbuminemia is responsible for the acquired fibrin polymerization defect reported in HIV-infected patients. The pathophysiological defect reported in HIV-infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring heparin therapy using activated partial thromboplastin time--results of a multicenter trial establishing the therapeutic range for SILIMAT, a reagent with high sensitivity to heparin.

APTT is widely used for laboratory monitoring of treatment with unfractionated heparin (UFH). However, since its sensitivity to heparin varies significantly from one reagent to another, the therapeutic range had to be defined for each brand of APTT reagent. As an example, SILIMAT (bio-Mérieux) is a new APTT reagent containing rabbit brain phospholipids and micronized silica as an activator. Since its high sensitivity to heparin has been previously reported, a multicenter trial was carried out in an attempt to define the therapeutic range of APTT performed using this new reagent. For that purpose, 170 blood samples drawn for routine coagulation testing from 170 different patients treated with UFH were analyzed. A single batch of two different APTT reagents were used on KC10 coagulometers: SILIMAT and Automated APTT (Organon-Teknika) whereas the anti-Xa activity was evaluated by a chromogenic substrate-based assay. The same methodology was used in all the centers. In order to obtain a plasma anti-Xa activity within the therapeutic range i.e. between 0.30 and 0.70 IU/ml, the APTT ratios were found between 1.90 and 5.40 for SILIMAT, which corresponded to clotting times of the patients plasma between 63 and 178 s. The APTT ratios were significantly lower when evaluated using Automated APTT (between 1.70 and 4.10), with clotting times between 53 and 127 s. In addition, a good correlation was found between the Anti-Xa activity and APTT for both reagents (r > 0.65). However, it is not possible to make recommendations regarding the therapeutic ranges without restrictions. Although about 70% of the patients with an anti-Xa activity between 0.30 and 0.70 IU/ml had an APTT in the above defined ranges, the degree of concordance between the two assays is not absolute. Actually more than 30% of the patients had discordant anti-Xa activity and APTT and more than a quarter of the patients included in the above defined therapeutic range for APTT had an anti-Xa activity outside the 0.30-0.70 IU/ml range, whatever the reagent used. In conclusion, to define the therapeutic ranges of APTT using the recommended method is practicable but some critical points could be raised, suggesting that a better method is awaited in order to improve the standardization.

The relationship between plasma microparticles, protein S and anticardiolipin antibodies in patients with human immunodeficiency virus infection.

The high prevalence of free protein S deficiency in human immunodeficiency virus (HIV)-infected patients is poorly understood. We studied 38 HIV seropositive patients. Free protein S antigen values assayed using the polyethylene-glycol precipitation technique (PEG-fS) were statistically lower in patients than in controls. These values using a specific monoclonal antibody-based ELISA (MoAb-fS) and the values of protein S activity (S-act) were not statistically different between patients and controls. C4b-binding protein values were not different from control values. In patients, PEG-fS values were lower than MoAb-fS values. Ten patients had a PEG-fS deficiency, 4 patients had a MoAb-fS deficiency and 8 had a S-act deficiency. Protein S activity and MoAb-fS were lower in clinical groups with poor prognosis and in patients with AIDS but PEG-fS was not. A trend for reduced S-act/MoAb-fS ratios was observed in patients. PEG-fS was negatively correlated with anticardiolipin antibody titers whereas MoAb-fS was not. The plasma of PEG-fS deficient HIV-patients contained high amounts of flow cytometry detectable microparticles which were depleted from plasma by PEG precipitation. The microparticles were partly CD42b and CD4 positive but CD8 negative. These micro-particles were labelled by an anti free protein S monoclonal antibody. The observed differences between MoAb-fS and PEG-fS values were correlated with the amount of detectable plasma microparticles, just like the differences between MoAb-fS and S-act. Plasma microparticles correlated with anticardiolipin antibody titers. In summary, free protein S antigen in HIV infected patients is underestimated when the PEG precipitation technique is used due to the presence of elevated levels of microparticles that bind protein S. The activity of free protein S is also impaired by high levels of microparticles. The prevalence of free protein S deficiency in HIV positive patients is lower than previously published (4/38, approximately 10%) and is correlated with poor prognosis. By implication, use of a PEG precipitation technique might give artefactually low free protein S antigen values in other patient groups if high numbers of microparticles are present. In HIV patients, high titers of anticardiolipin antibodies are associated with high concentrations of cell-derived plasma microparticles.

Antithrombin III and heparin cofactor II in patients with chronic renal failure undergoing regular hemodialysis.

Heparin enhances the inhibition rate of thrombin by both antithrombin III (AT III) and heparin cofactor II (HC II). We studied the activity of these two plasma proteins in patients with chronic renal failure (CRF) undergoing regular hemodialysis as their heparin requirements varied widely. In 77 normal blood donors, normal ranges (mean +/- 2 SD) were 82-122% for AT III and 65-145% for HC II. When compared with these controls 82 dialyzed CRF patients had a subnormal AT III activity and a significantly (p less than 0.001) lower HC II activity. To evaluate the effect of hemodialysis we compared AT III, HC II and total proteins in plasma before and after dialysis in 24 patients (12 with normal and 12 with low basal HC II activity). AT III and HC II activities significantly (p less than 0.001) increased in absolute value. When related to total plasma proteins, in order to suppress the influence of hemoconcentration induced by dialysis, AT III decreased significantly (p less than 0.01) whereas HC II increased slightly but significantly (p less than 0.01) in the 12 patients with low initial HC II activity. The decrease of AT III induced by heparin administrated during dialysis is likely to account for this relative decrease of AT III activity. A modification of the distribution of both HC II and heparin between the vascular wall and the circulating blood is evoked to explain the relative increase in HC II activity and the need for higher heparin dosage in patients with low HC II levels.

Molecular basis of antithrombin type I deficiency: the first large in-frame deletion and two novel mutations in exon 6.

We report three novel mutations accounting for cases of inherited type I antithrombin (AT) deficiency. Using the polymerase chain reaction (PCR) and direct sequencing of the coding sequences of the AT gene, we found one mutation in exon 4 and two in exon 6. A deletion of 105 bp causing an in-frame deletion of 35 amino acids between Tyr 240 and Gly 276 was found in exon 4. In a second kindred, deletion of two adenines in codon 412-413 introduced a frameshift and a stop codon at position 431. The last mutation was an insertion of ACCG in codon 387, generating a frameshift with a stop codon located at the normal position. The finding of a sequence repeat of nine residues located at the 5' and 3' ends of the deleted fragment might explain the 105 bp deletion by slippage and mispairing at the replication fork during DNA synthesis. The second mutation is the fourth described within a region of six amino acids (between Phe 408 and Arg 413), which seems to be a cluster of mutations. In this case, the presence of a double repeat sequence--TTCCT and AACA--flanking this region could be particularly favorable for slipped mispairing. These results confirm that human gene mutations are not random events but are strongly influenced by DNA flanking sequences.

Circulating activities during constant infusion of heparin or a low molecular weight derivative (enoxaparine): failure to demonstrate any circadian variations.

We compared in six patients successively treated with an unfractionated heparin (UFH) and a low molecular weight heparin (LMWH) the variations in plasma anti-Xa activity, measured in a chromogenic assay, during a 36 h constant infusion. The values varied in a wider range during UHF infusion, but remained in the therapeutic range except once in one patient. No circadian rhythm could be demonstrated in our six patients. LMWH infusion yielded very constant anti-Xa circulating activities. In both cases, there were no significant modifications of three proteins with high heparin affinity (antithrombin III, heparin cofactor II, histidine-rich glycoprotein). Our results suggest that the circadian rhythm of the biological activities previously observed in patients treated with constant heparin infusion using clotting method is due to other factors than heparin itself.

A study of fibrinogen and fibrinolysis in 10 adults with nephrotic syndrome.

In 10 patients with nephrotic syndrome (NS), the coagulation inhibitors, the fibrinolytic system and several functions of the fibrinogen-fibrin molecule were studied. Among the coagulation inhibitors, only antithrombin III (AT III) was found decreased and correlated with serum-albumin levels. Venous occlusion test provoked a normal tissue plasminogen activator (tPA) release in all patients. The plasminogen activator inhibitor (PAI) had an increased activity in 5 out of the 10 patients. Thrombin and reptilase times were found abnormal in most patients. The thrombin time (TT) prolongation correlated with serum albumin levels and was corrected by adding purified albumin. The fibrinogen was purified from each of the 10 patients' plasma. Only 2 of them showed abnormal polymerization in purified system, suggesting dysfibrinogenaemia. Other functions (thrombin binding, tPA stimulating activity, lysis by purified plasmin) were found normal except in one of the 2 patients with dysfibrinogenaemia whose fibrinogen lysis by plasmin was delayed. It is concluded that an abnormal fibrinogen molecule is not the most frequent explanation for thrombin time prolongation in NS.

Effects of aprotinin on hemorrhagic complications in ARDS patients during prolonged extracorporeal CO2 removal.

The effects of aprotinin, a broad-based proteinase inhibitor, in the management of hemorrhagic complications during prolonged venovenous extracorporeal CO2 removal in patients with adult respiratory distress syndrome are not evaluated. In two patients, aprotinin infusion was added to heparin to treat bleeding, occurring after few days of bypass and responsible for respiratory and hemodynamic deterioration. After aprotinin infusion (loading dose of 2 x 10(6) kIU followed by a continuous infusion of 5 x 10(5) kIU/h) combined with heparin, bleeding vanished until the end of bypass.

Screening for risk factors for thrombosis using a new generation of assays developed to evaluate the functionality of the protein C anticoagulant pathway.

These results, obtained in a small series of patients, suggest that both the ProC Global assay and the PCP Test would be suitable, using well-defined cut-off levels, to identify all the carriers of the Factor V Leiden mutation and all the patients with a protein C deficiency or with combined defects of the protein C pathway. For both assays, however, the sensitivity for protein S deficiency was below 60%. These results in selected patients are congruent with those previously reported in the literature about the ProC Global assay, the PCP Test, and other assays evaluating the functionality of the protein C anticoagulant pathway. All demonstrated a weak sensitivity to protein S deficiency, suggesting that protein S plays only a minor role as an APC cofactor in such global assays. A major discrepancy between the two evaluated assays was obtained in the group of patients without abnormality of the protein C pathway. Actually, using the ProC Global assay, more than 40% of the patients had a decreased PCAT-NR while presenting with none of the three tested abnormalities, whereas none of the studied patients had a ratio below 1.80, and only 5 of 143 (3.5%) had a ratio below 2.00 when using the PCP Test. The observation that around 40% of the control patients had a decreased PCAT-NR could suggest the influence of currently unknown defects of the protein C/protein S pathway on the ProC Global assay. It could also be hypothesized that the higher factor VIII levels already reported in patients with a history of thrombosis than in controls had a significant role in the low responsiveness, but this parameter was not tested in the authors' series. In that connection, it is also well established that elevated factor VIII levels both shorten the APTT and reduce the anticoagulant effect of heparin when evaluated using APTT. Actually, some of the samples investigated in this study were obtained during the acute phase of thrombosis. It is not possible to draw out the hypothesis of an association of biologically undetectable minor changes in various factors involved in the protein C anticoagulant pathway; all the individual factors would remain within their normal ranges. Finally, because 40% of the patients without abnormalities of the protein C pathway had a decreased PCAT-NR, the question arises whether the ProC Global assay might in itself be a biologic marker of thrombophilia, independent of its sensitivity for abnormalities of the protein C anticoagulant pathway. In that connection, the correlation between the result of the ProC Global assay and the risk for thromboembolism was recently evaluated by two different groups. In both cases, the preliminary results suggested that a decreased response to the ProC Global assay might be an independent risk factor for venous thrombosis. The two global assays could therefore have distinctly different applications. If the global assays are used in the hemostasis laboratory to screen for abnormalities of the protein C pathway, and thus to rationalize the use of specific assays, the PCP Test should be chosen, because of its high specificity. Because only 3.5% of the control patients had a ratio below 2.00 (and none had a ratio below 1.80), the PCP Test could be accurately used as a first-step assay in the laboratory screening for these abnormalities of the protein C anticoagulant pathway. Using such a flow chart, the specific assays for APC resistance or the identification of the factor V Leiden mutation and protein C would be performed only in case of a ratio below a cut-off defined using receiver operating characteristic (ROC)-analysis in unselected patients. Because of the weak sensitivity of this assay to both constitutional and acquired protein S deficiencies (below 15% using 1.80 as the cut-off level, or 60% using 2.00), the measurement of this parameter had to be performed in all cases. If, on the other hand, the assay is used to screen for risk factors for thrombosis, the ProC Global assay could b