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J.P. Van Geffen et al.
and an Olympus UPLSAPO 60x oil-immersion objective. Images were analyzed for the parameters described in Table 1, using semi- automated scripts written in Fiji. Test variability was 5-8%, depending on the type of surface and parameter.
Platelet immunophenotyping and platelet activation by flow cytometry
Platelet immunophenotyping and platelet activation were per- formed basically as described previously.21 The platelet activation parameters that were analyzed are presented in Table 1.
Results
Multiparameter assessment of whole-blood thrombus formation on microspots under flow
High-throughput microfluidics has been used for in- depth characterization of platelet dysfunction in patients with bleeding disorders. The technique uses microspot- coated flow chambers for multiparameter measurement of thrombus formation during whole-blood perfusion at defined wall-shear rates.7,9 In this study, we further stan- dardized this method for inter-subject analysis of platelet function, using blood samples from healthy subjects, with results expected to represent the normal range observed in a population. Procedures included (see also the Online Supplement and de Witt et al.22): (i) microspot coating using a high-precision mold; (ii) strictly controlled conditions of blood drawing, anticoagulation and storage; (iii) pulse-free blood perfusion through a Maastricht flow chamber at a defined shear rate; (iv) defined time proto- cols for rinsing, staining and capturing of brightfield and fluorescence microscopic images; (v) pre-defined scripts for consistent analysis of all image sets; (vi) a gallery of exemplary images for the scoring of thrombus parame- ters; and (vii) comparative analysis by trained personnel performing flow runs and blind image analysis. Out of a list of 52 different surfaces,7 we selected six microspots (M1-6) to provide the most discriminative information on small changes in thrombus formation. For each surface, eight outcome parameters (P1-8) were defined, as indicat- ed in Table 1.
Employing this standardized procedure, after whole- blood perfusion over each of the microspots, representa- tive images were taken to visualize and assess: platelet adhesion (P1), platelet aggregation and thrombus mor- phology (P2-5); platelet phosphatidylserine exposure, as a marker of procoagulant activity (P6); P-selectin expres- sion, to measure secretion (P7); and fibrinogen binding, to report on integrin αIIbb3 activation (P8). Image sets for a typical healthy control subject are given in Figure 1A.
Blood perfusion over microspot M1 (collagen type I) resulted in the formation of large thrombi with contract- ed aggregates of platelets, high in activation markers (phosphatidylserine exposure, P-selectin expression, αIIbb3 activation), linked to a relatively high GPVI signal.7 Microspot M2 (collagen type III) produced smaller platelet aggregates with less pronounced activation mark- ers, corresponding to more limited GPVI signaling. Microspot M3 [von Willebrand factor (VWF) + laminin] gave a monolayer of platelets with P-selectin expression and αIIbb3 activation, but essentially no phosphatidylser- ine exposure, indicative of the primary adhesive role of the laminin receptor, integrin α6b1. Microspot M4 con- tained a combination of collagen-derived peptides [VWF-
BP + GFOGER-(GPO)n] giving similar thrombi as on colla- gen type I. Microspot M5 (VWF-BP + rhodocytin) also produced full aggregates with contraction of platelets expressing activation markers. Microspot M6 (VWF-BP + fibrinogen) triggered mainly platelet adhesion with the scattered presence of small platelet aggregates, showing limited P-selectin expression and αIIbb3 activation. The observations for M1, M4 and M5 are in agreement with the capability of GPVI and CLEC-2 adhesive surfaces to support full thrombus formation in flow assays.7
To evaluate the performance of the standardized method, we collected three different blood samples from ten healthy donors (cohort 1), and determined the coeffi- cients of variation for each of the parameters per microspot (coated M1, M2 and M6). For the majority of the parameters, intra-assay variability of duplicate meas- urements was 5-8%. For microspots M1 and M2, the median intra-individual coefficients of variation over the three bleeds were 15% and 18%, respectively, which is considered acceptable for whole-blood assays (Figure 1B). For microspot M6, a higher median intra-individual coef- ficient of variation of 37% was obtained, likely due to the fact that parameter values on this ‘weak’ surface are low. This initial analysis indicated that inter-individual coeffi- cients of variation for the microspots were about twice as high as the intra-individual coefficients of variation (Online Supplementary Table S3).
High-throughput platelet phenotyping by multiparameter assessment of thrombus formation in combination with platelet count and platelet activation markers
Subsequently, thrombus formation was assessed on microspots M1-6 with blood samples from 94 genotyped healthy subjects from the NIHR BioResource (cohort 2, all blood type O). Donors of either sex had a median age of 64 years (Online Supplementary Table S1). Using the same microfluidics device, brightfield and fluorescence images were recorded and analyzed for all eight parameters (P1- 8) per microspot. Comparison of the inter-individual coefficients of variation for the 94 samples to the intra- individual ones for a subset of ten of these samples indi- cated, for the parameters of microspots M1-3, approxi- mately 2-4 times higher inter-individual coefficients of variation. This ratio was 2.5-1.5 times higher for the M4- 6 microspots (Figure 2A). Heat mapping of the normal- ized values per parameter and microspot showed major differences between the 94 blood samples (Figure 2B). Taken together, these data indicated that a substantial part of the measured values contained a subject-depen- dent component.
For all 94 subjects, parallel measurements were per- formed to assess: (i) hematologic parameters using a Sysmex XN-1000 analyzer; (ii) expression levels of platelet membrane proteins; and (iii) platelet activation tendencies by flow cytometry. Hematologic parameters included white and red blood cell counts, hematocrit, platelet count, platelet crit, and mean platelet volume. Surface membrane proteins assessed in unstimulated platelets included major adhesive glycoprotein complex- es: integrin αIIbb3 (CD41a, CD41b, CD61), GPIb-V-IX (CD42a, CD42b), integrin α2b1 (CD29, CD49b), GPIV (CD36), GPVI and the surface-expressed protein tyrosine phosphatase (CD148). Platelet activation tendency (inte- grin activation and α-granule secretion) was measured
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haematologica | 2019; 104(6)