Evaluating Factor VIII Concentrates Using Clot Waveform Analysis.

Whenever unfractionated heparin (UFH) is administered i.v. in therapeutic doses, therapeutic drug monitoring of the anticoagulant response should be man datory and the dose should be adjusted accordingly. UFH therapy is usually monitored by the activated partial thromboplastin time (APTT). A 1.5- to 2.5-fold prolonga tion of APTT has become generally accepted as an indi cator of effective i.v. anticoagulation, but it has become common to recommend this ratio without testing the APTT reagents used for their heparin sensitivity and for their actual therapeutic APTT ratio (actual APTT: normal control APTT). Internal heparin sensitivity testing of APTT reagents is managed using heparin sensitivity curves obtained from heparin-spiked normal plasma pool. The most common APTT reagents in Austria and a newly developed double activated APTT reagent were tested for their heparin sensitivity and therapeutic ratios on differ ent automated analyzers, depending on their optical and chemical conditions. Also, newly developed, commer cially prepared heparin standards (0.19, 0.52, 0.86 IU heparin/ml plasma) were tested. APTT reagents differ in their heparin sensitivity and therapeutic ratio; variations in heparin sensitivity are also seen between different an alyzers. Therefore, therapeutic ratio should regularly be checked and the literature should always state which APTT reagent was used on which instrument.

Accurate measurement of coagulation factors is essential, especially for diagnosis of deficiency. Clinical laboratories use commercially available plasma calibrators, which should be traceable to the relevant plasma International Standard (IS). This study assessed the relationship between the plasma IS for factors IX (FIX) and VIII (FVIII) and some commonly used commercial calibrators. Calibrators from seven manufacturers were assayed for FIX and FVIII activity by one‐stage clotting assay (OSCA) using different activated partial thromboplastin time (APTT) reagents and deficient plasmas, or chromogenic assay (CA). Results were calculated relative to the 4th IS Factors II,VII,IX,X, Plasma or the 6th IS Factor VIII/VWF, Plasma. Results for each calibrator were similar across the APTT reagents and deficient plasmas used. All calibrators showed a recovery of 90%‐111% of the manufacturers’ values, except calibrator C, which had recovery of around 85%. CA gave similar results, with good recovery for all but calibrator C. Similar low recoveries for OSCA and CA were found for a different lot of calibrator C and for a different calibrator product from manufacturer C. When all calibrators from manufacturer C were assayed by OSCA using the manufacturer's own deficient plasmas and APTT reagents, the mean recovery was still below 90%. Overall, there was good traceability of the international unit between the IS and commercial calibrator plasmas. Calibrators from one manufacturer consistently yielded lower than expected values for FIX and FVIII. This could lead to an over‐estimation of the coagulation factor content in patient samples and demonstrates the importance of careful choice of calibrator.

Teaching an old dog new tricks?

Guidelines on the investigation and management of the antiphospholipid syndrome.

The phospholipid content of different activated partial thromboplastin time (APTT) reagents was determined and compared to heparin sensitivity. The seven reagents included were those most widely used amongst participants of the U.K. National External Quality Assessment Scheme (NEQAS) at the time of study. Heparin sensitivity was assessed using the APTT ratios obtained by more than 300 NEQAS participants on five plasmas prepared from patients receiving unfractionated heparin. The concentrations of three neutral lipids and six phospholipids present in the seven APTT reagents were determined by high-performance thin-layer chromatography (HPTLC) and densitometry. Both the concentrations and the relative percentages of individual phospholipid components varied markedly between reagents. The total phospholipid concentration included a 12-fold range from 16 to 205 microgram/ml. Phosphatidylserine (PS) was completely lacking from one reagent prepared from vegetable material and ranged from 3 to 22 microgram/ml in the other six reagents containing extracts from animal tissue. The concentration of phosphatidylcholine ranged from 3 to 109 microgram/ml. There was no demonstrable relationship between the concentration of any individual lipid components and heparin sensitivity. However, the relative percentage phospholipid composition was important since a lower % of PS or phosphatidylinositol (PI) correlated with increasing heparin sensitivity.

To build and evaluate intrinsic coagulation factor assays which can eliminate the effect of lupus anticoagulants (LAC). Commercial silica clotting time confirmatory (SCT-C) reagent containing sufficient synthetic phospholipid and routine activated partial thromboplastin time (APTT) reagent were each used for one-stage detection of FVIII, FIX, and FXI activities, in samples with or without LAC, and the results were compared. For samples without LAC, consistent results of FVIII, FIX, and FXI using both SCT-C reagent and APTT reagent were obtained. For samples with LAC, the assays with SCT-C reagent not only could eliminate the effect of strong lupus anticoagulants but also needed fewer dilutions than that with routine APTT reagent. The intrinsic factor detections by SCT-C reagent are credible and convenient to be used for samples with LAC.

Lepirudin (r-hirudin) is one of the two alternative anticoagulants licensed to treat patients with heparin-induced thrombocytopenia (HIT). Manufacturer's guidelines state that lepirudin should be monitored using the activated partial thromboplastin time (APTT) ratio. However, several studies have demonstrated a plateau effect of higher concentrations of lepirudin on APTT ratios and variable results when comparing different APTT reagents. This study compares APTT ratios (using two different APTT reagents) with two other commercially available methods for directly quantifying plasma lepirudin levels: ecarin chromogenic assay and prothrombinase-induced clotting time in 95 samples from five patients receiving lepirudin anticoagulation for HIT.

Aprotinin, a broad spectrum serine proteinase inhibitor, is becoming increasingly used to control bleeding during surgery. Heparinized patients treated with aprotinin for cardiac surgery have a much longer activated clotting time (ACT) than expected for the dose of heparin used and a similar effect has been observed with the activated partial thromboplastin time (APTT). Since APTT reagents vary in their sensitivity to heparin we compared the effect of aprotinin on 25 commercially available products with respect to the APTT response to heparin. Aprotinin (Bayer) was added to normal, pooled, citrated plasma at concentrations of 0, 100, 200, 300 and 400 kallikrein inhibition units (KIU)/ml and mucosal heparin was then added to give concentrations of 0, 0.25, 0.5, 0.75 and 1.0 IU/ml. Duplicate APTTs were performed on each of these 25 plasma samples according to the manufacturers' instructions. As expected, different commercial reagents exhibited different sensitivities to heparin. There was also variation in the sensitivity to aprotinin but this did not correlate with the heparin sensitivity. The prolongation of the APTT by heparin was markedly increased by aprotinin. For example, APTT ratios at 0.5 IU/ml heparin increased up to eight-fold in the presence of 'therapeutic' levels of aprotinin (200 KIU/ml) and this effect was even more pronounced at higher heparin levels. The activator used did not significantly influence the effect of aprotinin on the APTT although there was a trend for kaolin-activated reagents to be less affected by the addition of aprotinin to heparinised plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

There is considerable variation in available methods for the activated partial thromboplastin time (APTT), giving widely differing results with patients on heparin treatment. The study is primarily concerned with the assessment of five of the widest used APTT reagents. The heparin response of these reagents has been related to their lipid composition and physical properties. Of the various correlations between lipid composition of the reagents and clotting performance only electrophoretic mobility was associated with the APTT response to heparin. There was a highly significant negative correlation between the APTT prolongation with heparin and electrophoretic mobility. When plasma is heparinized in vitro a differing order of ranking for APTT reagents is obtained than when heparinized patients are tested. The APTT response in patients with recent thrombosis must therefore be the best guide to the clinical dose of heparin. The therapeutic range of conventional heparin therapy is generally regarded as 1.5-2.5 times the control. External quality assessment programmes in the UK and USA have shown considerable differences between heparin dosage according to the APTT test systems. The definition of the therapeutic range must be derived from randomized clinical studies. The need for progress in standardization of the APTT monitoring of heparin is demonstrated.

We report a case of pseudo-prolongation of activated partial thromboplastin time (APTT), which was suspected to be caused by an animal-derived phospholipid. A 78-year-old woman was referred to our hospital because of an unexplained APTT prolongation. She had compensated alcoholic liver cirrhosis, with modestly decreased platelet count and normal prothrombin time, and no bleeding tendency. The APTT was 66 seconds in a test using phospholipid extracted from rabbit brain but was 34.9 seconds with synthetic phospholipids. The artifactual pseudo-prolongation of the APTT was seemingly attributable to the susceptibility of the test reagents to low factor XII levels. Thus, tests with different APTT reagents would be useful to physicians in the diagnosis of similar cases.

Overestimation of N‐glycoPEGylated factor IX activity in a one‐stage factor IX clotting assay owing to silica‐mediated premature conversion to activated factor IX

Verification of the guidelines for lupus anticoagulant detection: Usefulness of index for circulating anticoagulant in APTT mixing test

The Activated Partial Thromboplastin Time (APTT) test is performed on a large scale, in laboratories to monitor the functioning of intrinsic and common pathways of blood coagulation cascade and the concentration of anticoagulants such as Heparin among patients. A PDMS based microfluidic device has been fabricated and tested to perform APTT as a point-of-care device using whole blood samples by detecting the change in electrical impedance of blood during coagulation. The two chambered PDMS device integrated with microelectrodes is pre-coated with APTT reagent and heparin respectively for better repeatability. The results indicate a repeatable pattern of change in whole blood impedance during coagulation and confirm the theoretical explanation by Hartert [1]. The devices were also tested for their sensitivity to different concentrations of heparin and found to exhibit a favorable increase in coagulation time for increasing heparin concentration.

Human hepatocyte engraftment in F8KO TK-NOG mice: Creating a novel Hemophilia A mouse model with functional human coagulation cascade

The activated partial thromboplastin time (APTT)–clot waveform analysis (CWA) was previously reported to be associated with the early detection of disseminated intravascular coagulation and was also reported to be able to measure very low levels of coagulation factor VIII activity. The software program for the analysis for the APTT-CWA allows the associated first and second derivative curves (first and second DCs) to be displayed. The first and second DC reflect the velocity and acceleration, respectively. The height of the first DC reflects the “thrombin burst” and bleeding risk, while that of the second DC is useful for detecting any coagulation factor deficiency and abnormal enhancement of coagulation by phospholipids. Activated partial thromboplastin time-CWA aids in making a differential diagnosis which is difficult to do using only the routine APTT. The CWA is currently used for many applications in the clinical setting, including the monitoring of hemophilia patients and patients receiving anticoagulant therapy and the differential diagnosis of diseases.