534 Technical report

Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity Elena Campelloa, Luca Spieziaa, Claudia M. Radua, Sabrina Gavassoa, Barry Woodhamsb and Paolo Simionia Microparticles are now considered as critical effectors involved in numerous biological processes (coagulation, inflammation, and vascular biology). Microparticle can be measured using a quantitative and descriptive approach by flow cytometry (FCT) or by a functional approach assessing microparticle biological activities by a factor Xa-based clotting assay. FCT can be used to determine the cellular origin of the different microparticles, although there are concerns about the detection limit of this approach. Functional assays measure only the procoagulant activity of isolated microparticles and give no information on the cellular source or the physical properties of the microparticles. The advantage of the functional assays is that the assays use well defined reagents, and they are readily automated with high sensitivity and simplicity. In this study, we analyzed samples from 60 patients with active cancer of different type, 60 patients with a BMI more than 25 kg/m2, and 49 carriers of Factor V Leiden with and without a prior venous thromboembolic episode. The study showed a significant correlation (P < 0.05) between

Introduction Growing evidence suggests that microparticles present in peripheral blood contribute to the development and progression of cancer and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases [1,2]. Microparticles have a large diagnostic potential as biomarkers, and their measurement in the laboratory is increasing. However, because of the current technological limitations in the purification of microparticles and an absence of standardized methods of microparticles detection, challenges remain in validating the potential of microparticles as a noninvasive and early diagnostic platform. Ideally, for a full characterization of the microparticles a combination of methods is needed, but this at present is not a practical proposition. Published methods for microparticles measurement differ widely with regard to analytical and preanalytical variables such as plasma preparation, blood drawing, storage, freezing, thawing, labeling of microparticles, and instrument adjustments [3]. This remains the major problem inhibiting the wider use of these assays. Microparticles can be measured by flow cytometry (FCT) [4] or by assessing functional procoagulant 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins

microparticles determined by annexin V-microparticlebased FCT and a procoagulant activity-based clotting assay. This indicates that the procoagulant assay could be used as a routine screening test to screen out the normal samples so that only the abnormal samples need further testing by FCT. Blood Coagul Fibrinolysis 25:534–537 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Blood Coagulation and Fibrinolysis 2014, 25:534–537 Keywords: flow cytometry, microparticles, procoagulant assays a

Department of Cardiologic, Thoracic and Vascular Sciences, University of Padua Medical School, Padua, Italy and bHaemaCon Ltd, Bromley, GB, UK Correspondence to Professor Paolo Simioni, MD, PhD, Department of Cardiologic, Thoracic, and Vascular Sciences, University of Padua, Via Ospedale Civile 105, 35100 Padova, Italy Tel: +39 049 8212667; fax: +39 049 8212661; e-mail: [email protected] Received 17 September 2013 Revised 1 November 2013 Accepted 04 December 2013

activity [5]. A number of other methods are available using different methodology such as capture assays (Zymuphen, Hyphen Biomed) or under evaluation (atomic force microscopy [6], zeta potential analysis [7], and nanotracking [8]). At present, FCT is the most widely used method. FCT determines both the number and the cellular origin of the different microparticles, although there are major concerns about the detection limit of FCT [9] as it misses all the smaller microparticles. Functional assays, such as the Procoagulant Phospholipid assay (STA-procoag-PPL, Stago, Asnieres, France) or XaCT (Haemtex, Sydney, Australia) measure only the procoagulant activity and give no information on the source or the physical properties of the microparticles. The advantage of the procoagulant activity assays is that they are rapid and easily performed using well defined reagents that are readily automated with high sensitivity. For this study, we selected an assay that could be automated on a routine coagulation analyzer – STA-procoagProcoagulant Phospholipid (PPL) to compare with FCT. The basis of both measurements is the same as the annexin V used in FCT binds to the same phospholipid surface as used in the PPL assay, so some correlation DOI:10.1097/MBC.0000000000000068

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Microparticle assay comparison Campello et al. 535

between FCT and procoagulant activity would be expected. Here, we correlated annexin V–microparticles (AMP) measurement by FCT with procoagulant activity evaluated by means of a commercially available factor Xa (FXa)-based clotting assay (PPL) in different clinical settings referred to our Medical and Thrombotic Units, at Padua University.

Materials We used a cross-section of samples collected for other FCT studies by our group [10,11]. Samples from 60 patients with different types of cancer (M/F, 30/30; age 40–92), 60 overweight/obese patients with a BMI more than 25 kg/m2 (M/F 30/30, age 18–66), and 142 heterozygous and homozygous carriers of Factor V Leiden (FVL) with or without a previous venous thromboembolism episode (VTE) (M/F 19/30, age 26–64) were analyzed. These patients had been previously enrolled in the studies by our group and had given prior informed consent [10,11]. Citrated blood samples (collected in 3.2%/109 mmol/l citrate) were processed within 1 h of collection by double centrifugation (2  15 min at 2500g) at room temperature. Platelet free plasma (PFP) was stored in 1.5 ml aliquots at –808C until use. Frozen plasma was thawed in a waterbath at 378C for 5 min immediately before use. As it is well documented [3,4] that preanalytical variables can influence results, for these experiments both the FCT and PPL assays were performed simultaneously on the same sample. This ensures that the same population of microparticles is being measured in both assays. All samples were tested blind. Microparticle levels were assessed by FCT (FC500; Beckman Coulter, Miami, Florida, USA), as previously described [10,11]. Thirty microliters of PFP were incubated for 15 min with 10 ml of annexin V- fluorescein isothiocyanate (FITC) (Bender MedSystems GmbH, Vienna, Austria) to measure AMP. Platelet-derived microparticles (PMP) were measured using 10 ml of CD61 – phycoerythrin (Beckman Coulter) in addition of 10 ml of annexin V – FITC. The samples were diluted in 500 ml of annexin-V kit binding buffer before analysis. PMP were identified 30 ml of counting beads with an established concentration (Flow Count TM Fluorospheres; Beckman Coulter) were added to each sample in order to calculate microparticles as absolute numbers per microliter of PFP (AMP). The microparticle gate was established using a blend of mono-dispersed fluorescent beads of three different diameters (0.5, 0.9, and 3 mm) (Megamix, BioCytex; Stago, France). The PPL assay measures the clotting time in a system dependent on the procoagulant phospholipid content of

the sample giving the procoagulant activity of the sample. The assay is performed using a phospholipid-depleted substrate plasma to eliminate the influence of any coagulation factors upstream. FXa, in the presence of calcium, triggers the coagulation cascade, and a shortening clotting time of the sample indicates an increased concentration of procoagulant phospholipids and procoagulant activity [12,13]. The PPL clotting time correlates linearly with the functional activity of microparticle present in the sample. The manufacturers claim that the assay has an inter-assay and intra-assay coefficient of variation of less than 2.5%. The assay has two reagents – a lyophilized citrated human plasma depleted of all procoagulant phospholids (reconstituted with 1 ml of distilled water), and a lyophilized mix of bovine factor Xa in a calcium medium (reconstituted with 4 ml of distilled water). Both reagents are stable after reconstitution for 8 h. The reagents are insensitive to unfractionated heparin levels up to 1.5 IU/ml and low molecular weight heparin levels up to 2.0 anti-Xa IU/ml. The assay can be automated on any coagulation analyzer. For this study, the BCT instrument (Siemens AG, Erlangen, Germany) was used. To assay on the BCT system, 0.05 ml of sample were mixed with 0.05 ml of phospholipid free plasma and incubate for 2 min at 378C. 0.2 ml of the FXa/Ca2þ reagent was added, and the clotting time was measured. Results were expressed as medians with interquartile range (IQR). Comparisons between cases were performed using Spearman’s rank correlation test.

Results Totally 262 individuals were enrolled: 60 patients with different types of solid active cancer; 60 patients suffering from overweight/obesity; 142 carriers of FVL (49 with previous VTE). AMP median (IQR) level was 3540 (2976–4501) microparticle/ml in cancer patients, 2679 (2080–3618) microparticle/ml in overweight/obese patients, 3284 (2725–3742) microparticle/ml in FVL carriers with previous VTE, and 2668 (1974–3080) microparticle/ml in FVL carriers without previous VTE. The median (IQR) PPL clotting time was 55 (48–69) s in cancer patients, 63 (50–75) s in overweight/obese individuals, 37 (34–51) s in FVL carriers with previous VTE, and 43 (32–47) s in FVL individuals without previous VTE. In all three groups of patients tested, we found the same inverse correlation between the FCT analysis of AMP counts and procoagulant activity obtained by PPL: in cancer patients’ r 0.685, P < 0.001, in overweight/ obese patients’ r 0.478, P < 0.05, and in the FVL patients group r 0.521, P ¼ 0.0043 (Table 1). A significant inverse correlation was found between AMP counts and procoagulant activity in plasma samples, suggesting that the microparticle count is strongly associated with

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536 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 5

Table 1 Levels of circulating microparticle, procoagulant phospholipid-clotting time, and correlations in patients with cancer, overweight/ obesity, and factor V Leiden Study population Cancer Overweight/obesity FVL carriers With previous DVT Without previous DVT

AMP (MPs/ml)

PPL (s)

Correlation

3540 (2976–4501) 2679 (2080–3618)

55 (48–69) 63 (50–75)

r 0.685, P < 0.001 r –0.478, P < 0.05

3284 (2725–3742) 2668 (1974–3080)

37 (34–51) 43 (32–47)

r 0.521 P ¼ 0.0043

Values are expressed as median and IQR. AMP, annexin Vþ MP; DVT, deep vein thrombosis; FVL, factor V Leiden; PPL, phospholipid-clotting time; s, seconds.

sample procoagulant activity. This inverse correlation is expected as a shortening clotting time indicates increased procoagulant activity. In addition, PMP median level was 1968 (968–2976) microparticle/ml in cancer patients, 1120 (867–2403) microparticle/ml in overweight/obese patients, 1061 (573–1268) microparticle/ml in FVL carriers with previous VTE, and 855 (310–1021) microparticle/ml in FVL carriers without previous VTE. A significant inverse correlation was found between PMP counts and procoagulant activity obtained by PPL: in cancer patients’ r 0.60, P ¼ 0.003; in overweight/obese patients’ r 0.57, P < 0.01; and in FVL patients group r 0.491, P < 0.05.

Discussion Previously, investigations on PPL have reported good correlation between microparticle and procoagulant activity [5,12–15]. Connor et al. [5] performed the functional test in a single whole blood sample in which known concentrations of microparticles were added and showed that the addition of serial dilutions of microparticles decreased procoagulant activity, and this was dependent on the number of microparticles added. The PPL assay is not affected by the addition of antitissue factor antibodies, suggesting that it is insensitive to tissue factor [12]. Van Dreden et al. [13] showed a large and highly significant shortening in PPL clotting times corresponding to an increase in circulating platelet microparticles in different clinical setting (diabetes mellitus, sickle cell disease, thyroid cancer, and multiple trauma). In sickle cell disease, Noubouossie et al. [14] demonstrated that procoagulant activity may contribute to high kinetic and high peak profiles of thrombin generation. Moreover, according to the recent results reported by Stagnara et al. [15], we confirmed also that platelet microparticles enumeration and procoagulant activity correlated and that the majority of annexin-Vþ microparticles were platelet derived. However, it was observed by the authors that with a more intense centrifugation protocol, the number and procoagulant activity of the platelet microparticles were reduced, and correlations between techniques were not as good as those obtained with less intense centrifugation protocol (1500g  15 min and 13 000g  2 min). Ultracentrifugation removes microparticles causing an increase in the clotting time of the PPL

assay. This should be the same for both assays, but as the FCT procedure is responsive only to the larger subpopulation of microparticles, it should rapidly lose sensitivity with increasing centrifugation. For this reason, we followed the Scientific and Standardization Committee guidelines, which were designed basically for FCT [16]. We confirmed that the two different methods used for microparticles evaluation had good correlation even if the principles for the microparticles analysis were completely different (number vs. activity), probably because they both rely on the presence of charged phospholipids. However, we cannot exclude that in some situations major differences between the two methods may be found, for example, there are several conditions in which excess phospholipid from damaged cells other than platelets may be released (sickle cell crisis, thalassaemia after splenectomy, or disseminated intravascular thrombosis). Our analysis was limited to the study of annexin V positive microparticles. Therefore, we cannot exclude the presence of circulating annexin V negative microparticles, but their role and their possible activity are still unclear [17]. FCT is considered the ‘gold standard’ of microparticles detection, but there are many technical limitations reducing its wide spread use that is complexity of the assay by itself and concerns about detection of the small and very small microparticles. The simplicity, reliability, reproducibility of the PPL assay makes it a useful initial automated screening test to detect patients with abnormal microparticles activity. These abnormal samples can then be further characterized using FCT.

Acknowledgements Conflicts of interest

Conflict of interest statement: None except B.W. who also acts as a consultant via HaemaCon for Diagnostica Stago.

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Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity.

Microparticles are now considered as critical effectors involved in numerous biological processes (coagulation, inflammation, and vascular biology). M...
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