Why is the standardization of prothrombin time a problem?

The standardization of prothrombin time (PT) has been a problem for more than 50 years; by applying the methods to the same clinical material the analysis may explain why. The initial standardization by 2--3 times normal clotting time by Quick's PT assay did not standardize presently used PT assays. The ratio method accepted by the WHO in 1977 was not satisfactory because the intercept was neglected. The revised ratio method (WHO 1983) recommended calibration between log clotting times of normal and abnormal plasmas and a simplified conversion into ratios. However, by including a high proportion of normal plasmas (up to 1/3 of abnormal) an erroneous calibration material was introduced; the conversion from clotting times into ratios was also more complex than predicted. The international sensitivity index, which is based on the simplified method, produced international normalized ratios (INR) that were not more standardized than not corrected ratios. Not corrected coagulation activities showed poor standardization due to the different sensitivity of assays for the protein induced by vitamin K absence (Pivka) inhibitor. The corresponding therapeutic ranges of INR and coagulation activities covered 70 and 10--20% of their scales, respectively. Standardization by Pivka-corrected coagulation activities against an international reference assay may be the preferable method.

[Homocysteine and methylmalonic acid. New tests--for what benefit?]. / Homocystein og metylmalonsyre. Nye prøver--til hvilken nytte?

A serum cobalamin level lower than 250 pmol/l combined with high values of plasma homocysteine and serum methylmalonic acid confirms the diagnosis of vitamin B12 deficiency. A low serum and erythrocyte folate level and high plasma homocysteine confirm folate deficiency. If the cobalamin level is higher than 250 pmol/l no further tests are needed. In patients with neurologic or psychiatric disorders combined with elevated levels of homocysteine and methylmalonic acid, cobalamin deficiency is likely if these levels decrease during vitamin B12 therapy. The causes of elevated homocysteine levels are often obscure, and homocysteine should therefore not be used as a screening test.

Prothrombin time standardization by correction of the Pivka inhibitor.

The standardization of prothrombin time (PT) assays needs two steps: (1) calibration of PT assays towards a reference assay or reference thromboplastin, (2) correction of PT assays according to the calibration. The present recommended calibration by clotting times is favored for the linearity between assays; the clotting times of abnormal plasma are partly prolonged due to the protein induced by vitamin K absence (Pivka) inhibitor. Calibration by coagulation activities also demonstrated linearity between PT assays; the regression line for abnormal plasma deviated from the line of identity due to differences in sensitivity of assays for the Pivka inhibitor. The corrected PT assays demonstrated similar results using calibration by clotting time or coagulation activities, but the correction was simpler for coagulation activities. Patient plasma was the preferable material in calibration by clotting times as well as by coagulation activities. The corrections between the reference assay and other PT assays were equal to the differences in sensitivities for the Pivka inhibitor. The corrected PT assays did not differ from the reference assay by statistical analysis; any of the six PT assays tested might be used as reference assay.

Warfarin resistance due to malabsorption.

A female patient developed warfarin resistance after 2 years of satisfactory therapy. Only a third of orally administered warfarin was absorbed, the clearance of warfarin was normal. The patient was also resistant for dicoumarol, while phenandione had satisfactory effect on the prothrombin time (PT).

Simplified calibration of prothrombin time assays.

The calibration of prothrombin time (PT) assays was studied. A simple calibration between pooled normal plasma and pooled stable plasma was as satisfactory as more complex calibration methods based on linear or orthogonal regression between log clotting times of abnormal and/or normal plasmas.

Simplified prothrombin time standardization.

An analysis of prothrombin time (PT) standardization methods is presented. The present recommended ratio method demonstrated complex calibration and wide therapeutic ranges. Standardization by coagulation activities resulted in different therapeutic ranges due to the different sensitivity of assays for the protein induced by vitamin K absence (Pivka) inhibitor. A new method--the modified coagulation activity method--applied a reference assay against which any PT assay can be calibrated by clotting times, and the results were expressed in coagulation activities by the reference assay. This method was preferable due to simple calibration, narrow therapeutic ranges and identical results by any PT assay.

Which coagulation factors interfere with the one-stage prothrombin time?

Coagulation factors that interfere with the one-stage prothrombin time (PT) were investigated. PT responded with identical activities (adequately) against coagulation factors VII or X, only half as expected against factor IX, less than expected and nonlinearly against factor II. In multiple coagulation factor deficiencies PT did not differ from factor VII, which was the most reduced coagulation factor in warfarin therapy or liver disease. PT may also be influenced by factor VII at high activities.

Haemorrhagic effect of enoxaparin, a low molecular weight heparin. Comparison with unfractionated heparin in humans.

The haemorrhagic effects of unfractionated heparin (UFH) and the low molecular weight heparin (LMWH) enoxaparin were investigated and compared in the gastric mucosa (haemorrhage induced by biopsy) and skin (haemorrhage induced by Simplate) of 12 healthy volunteers. Administration of UFH and LMWH (given in a dose of 75 anti-Xa U/kg intravenously) increased median gastric bleeding time (3.5 min) and geometric mean blood loss (11.5 microliters) to 19 min (p = 0.00003) and 54.1 microliters (p = 0.0021) after UFH and to 13 min (p = 0.008) and 29.0 microliters (p = 0.275) after LMWH. Median skin bleeding time (4.25 min) increased to 6.0 min after UFH (p = 0.003) and to 6.75 min after LMWH (p = 0.0008). Mean heparin activity in plasma was 20% higher after LMWH than after UFH. The calculated gastric bleeding time to heparin activity ratio was significantly lower for LMWH than for UFH (p < 0.05).

Interaction between heparin and acetylsalicylic acid on gastric mucosal and skin bleeding in humans.

The haemorrhagic effect of unfractionated heparin and of the low molecular weight heparin, enoxaparin, on gastric mucosal bleeding induced by acetylsalicylic acid (ASA) and on skin bleeding induced by the Simplate technique was investigated in healthy human volunteers. Endoscopic estimation of gastric bleeding by visual analogue scores was more sensitive than biochemical quantitation of blood in the gastric washing by a modified HaemoQuant method. Contrary to what was expected, the ASA-induced gastric mucosal bleeding was not increased by heparin pretreatment, whereas heparin in combination with ASA, but not ASA alone, significantly increased the skin bleeding time. In the interaction with ASA, enoxaparin and unfractionated heparin appeared to act similarly.

Bolus therapy with mitoxantrone and vincristine in combination with high-dose prednisone (NOP-bolus) in resistant multiple myeloma. Nordic Myeloma Study Group (NMSG).

In a phase II study, 58 patients with resistant multiple myeloma (MM) were treated with a combination chemotherapy (NOP-bolus regimen) consisting of mitoxantrone (16 mg/m2 for the first 25 patients and 12 mg/m2 for the subsequent 33), vincristine (2 mg), both as bolus injections on day 1 and prednisone (250 mg/d on d 1-4 and 17-20). In patients greater than 70 years of age, the mitoxantrone dose was reduced to 12 mg/m2 or 8 mg/m2, respectively. The treatment was repeated every 4 weeks. A response (greater than 50% reduction in M component) was obtained in 26% of the patients and a minor response (clinical improvement but less than 50% reduction in M component) in another 21%. Median response duration was 27 wk and median survival for all patients was 25 wk. There were no differences in response rate or duration between patients receiving the high or low mitoxantrone dose, but patients in the low-dose group had fewer serious infections.

Incomplete reversal of enoxaparin-induced bleeding by protamine sulfate.

The neutralizing effect of protamine sulfate on enoxaparin-induced bleeding was compared in two rat models: one employing the gastric mucosa, and the other the tail skin, using a 2:1 ratio of protamine sulfate to enoxaparin on a weight basis. Whereas protamine sulfate reduced the median bleeding time (from 20 to 9.5 min) and blood loss (80%) in the gastric mucosa, and apparent 'all-or-none' response was seen in the tail skin, in agreement with a different hemostatic mechanism in the two bleeding models. In both models, protamine sulfate incompletely reversed the bleeding induced by enoxaparin.

Haemorrhagic effects of unfractionated and two low molecular weight heparins, enoxaparin and fragmin, in rats.

The effects on primary haemostasis of unfractionated heparin and of the two low molecular weight heparins, enoxaparin and fragmin, were compared in two rat models, one employing the gastric mucosa and the other the tail skin. All three heparin preparations prolonged the bleeding time and increased the blood loss dose dependently. The prolongation of the bleeding time per unit dose caused by unfractionated heparin was significantly greater than the prolongation caused by either one of the two low molecular weight heparins. In the gastric mucosa, but not in the tail skin, enoxaparin prolonged the bleeding time significantly less than fragmin (p less than 0.05).

Two-layer gradient separation of granulocytes for functional tests.

Two gradient separation techniques were compared with the dextran sedimentation method for the separation of granulocytes from blood. Both techniques gave adequate yield and excellent purity, and flow cytometric analysis of surface membrane markers gave no indication of subset selection during the procedure. Cells separated by the three methods behaved comparably in functional tests including random migration, chemotaxis and chemiluminescence. The gradient separation techniques are rapid and efficient methods for the preparation of near 100% pure granulocyte suspensions for functional studies.

Gastric mucosal bleeding after unfractionated and low molecular weight heparin in rats.

The bleeding from an induced gastric mucosal lesion was monitored after intravenous administration of unfractionated heparin and enoxaparin, a low molecular weight heparin. The gastric mucosa was exposed in a chamber, which was superfused with saline and emptied at 1-min intervals for quantitation of bleeding. Both heparins prolonged the bleeding time and increased the blood loss dose-dependently. Five to ten times higher doses of enoxaparin (in terms of anti-Xa units) were required to achieve similar prolongation of the bleeding times as with unfractionated heparin. The results indicate a more favourable antihaemostatic effect of enoxaparin than of unfractionated heparin.

A new model to assess the haemorrhagic potential of various heparin preparations.

In the gastric mucosa, haemostasis is hampered by the acidity and peptic activity of the gastric juice and by the fibrinolytic activity of plasmin. The hostile intragastric environment may be responsible for the unsecure haemostasis, with episodes of rebleeds often seen in gastroduodenal haemorrhage. Secondly, the haemostatic mechanisms of the gastric mucosa are largely independent of platelet aggregation and thus rely mainly on the coagulation system, making the gastric mucosa a unique model to test haemorrhagic effects of anticoagulants. Using a rat gastric chamber technique, we studied bleeding times from induced gastric mucosal lesions after intravenous administration of unfractionated and low molecular weight heparins. The bleeding times were dose-dependently prolonged by all heparins. With unfractionated heparin, significant prolongation of the bleeding times was seen already at a dose of 75 anti-Factor Xa U/kg, proving that the present model is more sensitive than previous models. The bleeding time per unit dose of both Kabi 2165 (p less than 0.05) and enoxaparin (p less than 0.001) was significantly less than that of unfractionated heparin.