Diagnosis of Thrombotic Thrombocytopenic Purpura by ADAMTS13 Activity Quantification.

BACKGROUND: Rapid quantification of ADAMTS13 activity in plasma is essential for establishing a diagnosis of thrombotic thrombocytopenic purpura (TTP); a rare, but potentially lethal disorder. The current methods for quantitating ADAMTS13 activity are manual and only available at specialized laboratories, which often results in initiation of specific treatments long before a diagnosis of TTP is established. METHODS: We compared the performance of the HemosIL, a novel and rapid automated method, and the current standard FRET (fluorescence resonance energy transfer) method in quantitating ADAMTS13 activity using 706 consecutive plasma samples collected over a period of 14 years. The clinical accuracy of both methods was further examined using 212 diagnostic samples. RESULTS: The correlation between the FRET and HemosIL methods in all 706 samples and in the 212 diagnostic samples was excellent (Pearson's r of 0.919 and 0.912, respectively). Both methods displayed a high degree of clinical accuracy using the current cutoff of ADAMTS13 activity <0.10 kIU/L (<10%) as diagnostic for TTP: the area under the curve (AUC) was 97.7% for the FRET method and 99.5% for the HemosIL method. When applying a lower cutoff (ADAMTS13 activity <0.05 kIU/L or <5%), the diagnostic accuracy of the HemosIL method increased further (AUC = 99.7%). CONCLUSIONS: A novel, rapid method for ADAMTS13 quantification is comparable to the more laborious FRET method in patients with possible TTP. A rapid analysis available in the acute setting assessing patients with possible TTP allows for improved care and optimized treatment of a life-threatening condition.

Unexpected, isolated activated partial thromboplastin time prolongation: A practical mini-review.

A common inquiry in coagulation laboratories is how to interpret an unexpected, isolated prolonged activated partial thromboplastin time (APTT). In this context, isolated means together with a normal prothrombin time (PT) and/or normal international normalized ratio (INR). This finding may lead to contact with laboratory doctors for further advice on a diagnostic strategy. Occasionally, the need for a diagnostic algorithm can be subacute, where surgery has to be postponed until an explanation for the isolated, prolonged APTT has been established. Activated partial thromboplastin time as a coagulation test was developed to monitor patients with hemophilia. Different APTT reagents display considerable differences in their sensitivity to deficiencies of coagulation factors. An isolated, prolonged APTT is seen in (a) individuals/patients with lupus anticoagulants, (b) patients in treatment with anticoagulants, mainly heparin, and (c) patients with deficiencies of specific coagulation factors. In this tutorial review, we summarize what may cause an isolated prolonged APTT and we present a simple diagnostic algorithm to differentiate between lupus anticoagulants (common) and factor deficiencies (rare). The identification of an isolated prolonged APTT as well as the underlying cause can be of the utmost importance in ensuring the correct therapeutic follow-up.

[Diagnosing von Willebrand disease].

Von Willebrand disease (VWD) is an inherited bleeding disorder with abnormal primary haemostasis due to defects in, or decreased concentration of the glycoprotein von Willebrand factor. In Denmark, the estimated prevalence of VWD is 1% corresponding to approximately 50,000 patients, but only a few hundred have been diagnosed, mostly due to prolonged bleeding after a trauma or during surgery. Thus, VWD is underdiagnosed in the general population. Improved anamnestic screening for bleeding disorders such as VWD in certain high-risk groups can facilitate institution of prophylactic treatment.

Comparison of automated von Willebrand factor activity assays.

INTRODUCTION: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder. Measurement of von Willebrand factor (VWF) activity in plasma is often based on platelet agglutination stimulated by the ristocetin cofactor activity. Novel assays, based on latex beads with recombinant glycoprotein Ib instead of platelets, have recently been developed but it is unclear whether these can improve the diagnostic capability for VWD. AIM: To compare four automated VWF activity methods in a mixed population of patients referred for evaluation of bleeding tendency. METHODS: The analytical performances of three ristocetin and one non-ristocetin cofactor activity assays were compared in 170 consecutive plasma samples from patients referred for VWD evaluation. RESULTS: All methods correlated well with concordance correlation coefficients ranging from 0.90-0.95. However, when comparing the VWF activity/antigen ratios in samples classified as having VWD (activity <0.4 IU/mL) the number of samples below a ratio of 0.7 differed between 16 and 8%. CONCLUSION: Despite overall correlation between assays we found that differences in classification power might interfere with the interpretation of individual samples.

Effect of N-acetylcysteine on the accuracy of the prothrombin time assay of plasma coagulation factor II+VII+X activity in subjects infused with the drug. Influence of time and temperature.

OBJECTIVES: The prothrombin time (PT) assay of factor II+VII+X activity is an important predictor of liver damage in paracetamol poisoned patients. It complicates interpretation of results that the antidote, acetylcysteine (NAC) depresses this activity. The aim was to investigate if NAC influences the accuracy of the plasma PT assay. MATERIALS AND METHODS: The accuracy of Nycotest PT was studied using plasma added NAC in vitro and plasma from subjects infused with NAC. The latter results were compared with those obtained by analysis of PT by CoaguChek S. RESULTS: Therapeutic NAC concentrations added to plasma in vitro decreased factor II+VII+X activity at 37 degrees C in a time-dependent manner. This effect was quenched at temperatures <24 degrees C. Activity lost at 37 degrees C could partly be recovered by subsequent incubation at 5 or 20 degrees C. Incubation at 37 degrees C prior to assay led to a significant additional depression of factor II+VII+X activity in plasma from subjects infused with NAC during the first 3h of infusion indicating that it contained reactive NAC. The risk that this NAC interfered with the accuracy of the PT assay was considered minimal with samples stored below 24 degrees C. This was supported by similarity of results obtained by analysis of appropriately stored plasma and simultaneously drawn blood by CoaguChek S. CONCLUSIONS: Residual reactive NAC does not interfere with the accuracy of the PT assay of plasma stored below 24 degrees C, but NAC-induced loss in activity at 37 degrees C may be partly recovered during subsequent storage below 24 degrees C.

Characterization of a novel mutation in the von Willebrand factor propeptide in a distinct subtype of recessive von Willebrand disease.

von Willebrand factor (VWF) is a plasma protein that consists of a series of multimers of which the high-molecular-weight VWF multimers are the most potent in platelet adhesion and aggregation. The propeptide of the VWF (VWFpp) is known to be essential in the process of multimer assembly. Genetic studies were performed in a patient with a phenotype of von Willebrand disease (VWD) characterized by very low plasma factor VIII and VWF levels and a VWF consisting of only a dimeric band and total absence of all multimers in plasma. The patient was found to be homozygous for the novel C570S mutation, caused by a 1709G>C transition in exon 14 of the VWF gene coding for the propeptide. Three asymptomatic relatives were found to be heterozygous. In-vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on VWF multimerization. Our findings show that the C570S mutation in the VWFpp abolishes multimerization of VWF. The mutation probably disrupts the normal configuration of the VWFpp, which is essential for correct orientation of the protomers and ultimately multimerization. The mutant amino acid is located in a region that is highly conserved across several species which underlines its critical role. This variant constitutes a distinct subtype of recessive 2A VWD with the exclusive presence of the dimeric form of VWF in plasma.