Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization

The activated partial thromboplastin time (aPTT) is widely used as a screening coagulation test and for monitoring unfractionated heparin therapy. Various commercial reagents are available, with different performance characteristics, particularly responsiveness to the lupus anticoagulant (LA). Because aPTT reagent selection significantly affects the interpretation of results, we reviewed College of American Pathologists proficiency testing data involving approximately 4,000 coagulation laboratories, and conducted a survey of coagulation laboratories (n = 93) using The Fritsma Factor hemostasis Web site to determine the basis for aPTT reagent selection. The data demonstrate that for routine aPTT testing, most laboratories use reagents with high/moderate responsiveness to LA. Significant misunderstanding was apparent regarding the use of appropriate aPTT reagent for routine testing and LA identification. We recommend aPTT reagents with low LA responsiveness to screen for coagulation factor deficiencies and heparin monitoring, and suggest continued education of laboratory professionals and reagent manufacturers about appropriate aPTT reagent use.     

Activated partial thromboplastin time reagent sensitivity to the presence of the lupus anticoagulant.

The lupus anticoagulant (LA) is an acquired abnormality that is associated with a prolonged activated partial thromboplastin time (aPTT). It is one of the most frequent coagulation abnormalities seen in the routine clinical laboratory. The sensitivity of various commercial aPTT reagents varies in their ability to detect the LA. We undertook this evaluation by using a single coagulation instrument to determine the sensitivity of five different commercial aPTT reagents to the presence of the LA. We evaluated 23 patients with known LA using five different reagents, two of which were marketed as having enhanced LA sensitivity. All samples and testing were performed under the same conditions in a timely manner. Based on these data, essentially all of the commercial reagents that were tested could detect patients with known LA by at least minimally prolonging the aPTT. Some reagents were slightly better than others in their ability to detect the LA. For most hospitals, the detection of the LA is not the highest priority for the use of the aPTT assay. In most cases, heparin anticoagulation monitoring is the most common use of the aPTT assay. Since all five reagents are sensitive to the LA, then the best overall reagent will be the one with the best sensitivity to the most important need for the laboratory (usually heparin monitoring). Therefore, a reagent should be chosen based on the primary monitoring requirements of the aPTT assay, and greater than 90% of the patients with the LA will be detected.     

Ion interaction reagent reversed-phase high-performance liquid chromatography determination of anti-tuberculosis drugs and metabolites in biological fluids

New methods of ion interaction reagent (IIR) RP-HPLC are presented for the determination of anti-tuberculosis drugs and their metabolites, singly or in multi-component mixtures, in biological fluids. The following analytes are considered: isoniazid, ethionamide, pyrazinamide, morphazinamide, p-aminosalicylic acid, nicotinic and isonicotinic acids. Aqueous solutions of three different ion interaction reagents are alternatively or comparatively used as the mobile phases, namely: (A) 5.00 mM octylamine at pH 3.00 for o-phosphoric acid, (B) 5.00 mM octylamine at pH 8.00 for o-phosphoric acid, and (C) 5.00 mM 1,6 diaminohexane at pH 6.00 for o-phosphoric acid. The response linearity between peak area and analyte concentration is verified for all the analytes in the concentration range within the determination limits and 2.00 mg/l. Detection limits are always lower than 82 microg/l for standard solutions; in the analysis of samples of rat serum, rat plasma and human serum, the matrix effect is negligible, the detection limits are always lower than 94 microg/l and the average recovery yield is always greater than 96%.     

A rapid method for the determination of cyclosporin A in blood by high performance liquid chromatography

The purpose of this paper is to propose a method for the determination of cyclosporin in whole blood by means of HPLC with UV detection. After a one step extraction into ether, the organic layer is washed NaOH and evaporated to dryness. The residue is reconstituted in a mixture of acetonitrile and water (40/60) (V/V) and injected into the chromatograph. The C8 reversed phase column is warming at 75 degrees C temperature. The mobile phase consists of acetonitrile and water (60/40) (V/V) and the wavelength is set at 210 nm. The technique is rapid, sensitive, reproducible and well suited for therapeutic monitoring and for pharmacokinetic studies.     

Use of an ion-pairing reagent for high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry determination of anionic anticoagulant rodenticides in body fluids.

The on-line combination of high-performance liquid chromatography with mass spectrometry (HPLC-MS) has become a powerful tool for trace analysis thanks to the developments in interface techniques. However, non-volatile salts such as ion-pairing reagents are considered to be incompatible with HPLC-MS systems; they cause drops in analyte signals because of contamination of mass analyzers and also because of blocking of the capillary transferring ions from atmospheric pressure to the vacuum manifold. In this work, a new type of ion-pairing reagent, di-n-butylammonium acetate (DBA), was evaluated for use in HPLC-MS. DBA did not cause these problems to HPLC-MS systems; a possible explanation might be that DBA decomposed to volatile compounds under APCI conditions. In addition, DBA was very useful for obtaining sharp peaks, which resulted in high sensitivity. With this ion-pairing reagent, we developed a procedure for the measurement of five (including internal standard) anticoagulant rodenticides in whole blood and urine samples by SIM detection of [M-H]- ions. Calibration range, recoveries and precision of the method were examined; detection limits as low as 1-5 ng/ml blood sample or 0.5-2.5 ng/ml urine sample were achieved.     

A general reagent for amplifying ELISAs

This paper describes a method to enhance the sensitivity of enzyme immunoassays by the use of solid tagged latex beads. Various bead sizes (0.21 and 0.81 micron) were tested using particles which had been covalently modified (or coated) with biotinylated rabbit anti-mouse immunoglobulin antibodies. Detection limits of mouse monoclonal antibodies or antigens (which have been reacted with specific mouse antibodies) were compared to those obtained using the general homogeneous 'sandwich' ELISA assay. Compared to the homogeneous biotin-avidin ELISA an increase in sensitivity level of about 6-10 times was observed in antigen detection assays. The stability of the reagent was excellent. The method gave low backgrounds and was as simple to use as the standard procedure.     

An improved method for the simple and accurate colorimetric determination of urea with Ehrlich's reagent

An improved method for the colorimetric determination of urea is described. The method is simple, requiring a deproteinized filtrate obtained after treatment of a small quantity of whole oxalated blood, serum, plasma or urine with 12·5% trichloracetic acid containing 50 g. activated carbon. The addition of Ehrlich's reagent to this filtrate results in the immediate production of a stable yellow colour. The determination is accurate, very sensitive and rapid, taking place in only 10 minutes.     

Quantitative estimation of preanalytical variables which may influence the determinations of prothrombin time (PT) and activated partial thromboplastin time (APTT)

Prothrombin time (PT) and activated partial thromboplastin time (APTT) tests principally measure the time for a fibrin clot developed in citrated plasma after activation. For the complexity of chemical reactions, a number of preanalytical variables potentially influence the outcome of results. In the present study, we evaluated some preanalytical variables frequently encountered in clinical settings. The volumes of citrated whole-blood specimens collected from inpatients widely varied from 0.99 ml to 2.90 ml indicating 1.6% of unacceptable rate, whereas none of the specimens from outpatients was out of acceptable range. The citrated whole-blood volume significantly affected the determinations of both PT and APTT; the results indicating the more volume the longer clotting time. Also, whole-blood specimens collected in EDTA2K revealed significantly prolonged PT and APTT values in healthy subjects and the patients with anticoagulant therapy of heparin and of warfarin. Storage conditions, time and temperature might influence the PT and APTT values. In particular, citrated whole-blood specimens stood at room temperature revealed the prolonged clotting time in APTT assay by hours. The effects of other variables evaluated such as a half-volume adjustment, needle gauge or syringe type were negligible. With these results, it was concluded that; first, an accurate venipuncture is critical, particularly venipuncture from patients in wards where many different physicians and nurses are in charge and in changing by days. Secondly, the citrated whole-blood specimens should be assayed quickly without any unnecessary storage at room temperature beyond four hours.     

A comparison between two commercially available activators for determining the partial thromboplastin time

Activators that are used to determine the activated partial thromboplastin time should be sensitive enough to detect mild deficiencies of coagulation factors in a reproducible fashion. We compared two commercially available activators--a newly modified preparation of ellagic acid activator and a silica activator. Both reagents produced results that were reproducible over a broad range of concentrations of factors involved in the intrinsic coagulation pathway. The ellagic acid activator was more sensitive in detecting a partial deficiency of nearly all of these factors.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid-liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative-reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS-MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.