A new flow cytometric method for measurement of von Willebrand factor activity.

BACKGROUND: Patients with von Willebrand's disease may have normal levels of von Willebrand factor (VWF) antigen. It is therefore important to measure not only the antigen concentration but also the VWF activity. The most widely used method for measurement of VWF activity is the ristocetin cofactor assay (VWF:RCo), which is still crucial for the laboratory diagnosis of von Willebrand's disease (VWD). However, VWF:RCo has low precision, poor inter-laboratory reproducibility and requires an aggregometer. Many routine laboratories are not equipped with aggregometers but have flow cytometers instead. METHODS: In this study a simple, precise and rapid flow cytometric assay was developed for the determination of von Willebrand factor activity, utilizing formalin-fixed platelets, fluorescein isothiocyanate-conjugated chicken anti-VWF antibodies (Fab-fragments) and phycoerythrine-conjugated anti-GPIIb/IIIa antibodies. RESULTS: In samples from healthy controls and from patients with von Willebrand disease type 1, the flow cytometry assay showed good correlation with the VWF:RCo assay (r2 = 0.69) and the VWF antigen assays (r2 = 0.83), which was better than the correlation between the VWF:RCo assay and VWF antigen assays (r2 = 0.72). The flow cytometry method had good within-assay and total precision, C.V. 4.2%, and C.V. 7.5%, at a mean concentration of 0.40 IU/mL, respectively. Results obtained with the flow cytometric method on samples from two patients with von Willebrand disease 2B were lower than those obtained with the antigen method in accordance with the diagnosis. CONCLUSION: The accuracy and precision of the von Willebrand activity assay may be improved if a flow cytometer is utilized for measurement of the impact of ristocetin on binding of VWF to formalin-fixed platelets instead of measuring agglutination utilizing an aggregometer. In addition, our flow cytometric method assay enables measurement of von Willebrand factor activity at many more hospitals than was previously possible with the traditional ristocetin cofactor platelet aggregometry assay, and this trend is likely to increase in the future when routine hematological instruments are equipped with built-in flow cytometers.

A flow cytometric assay for the study of dense granule storage and release in human platelets.

The clinical manifestations of platelet dense ( delta ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83+/-6 (mean +/- 1 SD, range 69-91). The difference in MFI between resting and stimulated platelets was 28+/-7 (range 17-40). Six members of a family, of whom one had a known delta -storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval.

Impaired platelet function correlates with multi-organ dysfunction. A study of patients with sepsis.

Patients with sepsis often suffer from haemostatic disturbances such as haemorrhage and disseminated intravascular coagulation (DIC). Considering the pivotal role of platelets in haemostasis, we have investigated platelet function by flow cytometry in 16 patients with sepsis for a better understanding of their haemostatic function. We have also investigated whether platelet function correlates with the severity of disease assessed by multiple organ dysfunction (MOD) score and patient outcome. The platelet response ex vivo after stimulation with agonists, measured as platelet fibrinogen, binding was low in comparison with healthy volunteers ( n = 30). This could reflect a previous response to agonists in vivo , which lead to platelet activation and consumption and formation of microthrombi that could then participate in the development of M OD. The platelets that remain in the circulation might be the result of a selection process where the most active platelets have already been consumed, and the remaining population consists of less active platelets. Another explanation might be desensitization of the remaining platelets because of exposure to agonists in vivo . Platelet activation with the agonists ADP and arachidonic acid were predictive of subsequent development of MOD and final patient outcome.

Activated platelets and impaired platelet function in intensive care patients analyzed by flow cytometry.

Intensive care patients often have disturbances in their coagulation and fibrinolysis systems, which may result in haemorrhage or disseminated intravascular coagulation (DIC). DIC is a dreaded complication that may develop rapidly and has a high mortality rate. Platelets play a central role in haemostasis and it is thus important to have assays that rapidly can monitor platelet activation and platelet function. We have used flow cytometry to measure platelet activation and function in intensive care patients. Fluorescein labelled chicken antibodies were used to detect platelet bound fibrinogen as these antibodies have advantages over mammalian antibodies in flow cytometry. We found increased levels of circulating activated platelets and microparticles in vivo and impaired platelet function after stimulation in vitro. The two patients with the highest percentage of microparticles died shortly after blood sampling.