Effect of haemodilution on LSR and TEG clot viscoelasticity profiles and parameters.

<p>Recalcified and kaolin-activated swine whole blood diluted at 0 (undiluted blood sample) 40, 50, 60 or 70% were measured using LSR (A) and TEG instruments (B) and blood coagulation parameters such as the clotting time (R+K) (C), the clot progression (angle) (D) and the maximum amplitude (MA) (E) were extracted at various haemodilution concentrations. Linear regression analysis comparing blood coagulation parameters R+K (F) angle (G), and MA (H) between LSR and TEG are presented. Each data point represents the mean of three replications ± SD (histograms) or standard error of the mean, SEM (linear regression). Comparisons were done using ANOVA followed by the Tukey’s method for multiple comparisons. (*, 0%-…); ($, 40%-…); (&, 50%-…); (#, 60–70%). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.</p

LSR coagulation parameters: Descriptions and definition.

<p>LSR coagulation parameters: Descriptions and definition.</p

Laser speckle rheology (LSR) instrument and coagulation parameters.

<p>(A) The schematic diagram of the LSR instrument used for blood coagulation assessment. Polarized light (690 nm, 9 mW) from a diode laser (Newport Corp., LPM690-30C) was focused (spot size 100 μm) on the imaging chamber containing 127 μl of kaolin-activated blood. Cross-polarized laser speckle patterns were acquired at 180° back-scattering geometry via a beam-splitter using a high speed CMOS camera (Basler AG, acA2000-340km) equipped with a focusing lens (Edmund Optics, NT59-872)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182491#pone.0182491.ref014" target="_blank">14</a>]. The captured speckle patterns were transferred to a computer for further processing. (B) Representative clot viscoelasticity profile derived using LSR. The relative change in clot viscoelasticity (<i>G</i>) is measured during coagulation and plotted as a function of time to retrieve the LSR coagulation parameters, R, K, α-angle and MA. Reprinted from [Tripathi MM, Hajjarian Z, Van Cott ME, and Nadkarni KS. Assessing blood coagulation status with laser speckle rheology. Biomed. Opt. Express. 2014. 5: 817–831] under a CC BY license, with permission from [The Optical Society], original copyright [2014].</p

Effect of argatroban on LSR and TEG coagulation parameters.

<p>Kaolin-activated swine blood spiked with 0 (control), 3.8, 5.7, 7.6, 15.2 μM argatroban was measured for 20–50 minutes and blood coagulation parameters including the clotting time (R+K), the clot progression (angle) and the maximum amplitude (MA) were extracted for each concentration (A-C). Correlation between LSR and TEG was evaluated using linear regression analysis (D-F). Each data point represents the mean of three replications ± SD (histograms) or standard error of the mean, SEM (linear regression). Values were compared between control samples (without treatment) and argatroban treated samples using ANOVA followed by the Tukey’s method for multiple comparisons post-tests. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.</p

Effect of warfarin treatment on correlations between LSR and aPTT, PT, INR and TEG parameters.

<p>Coagulation profiles of 12 patients on warfarin therapy were evaluated using LSR and TEG, and with aPTT and PT/INR assays. LSR clotting time (R+K) parameter was compared to aPTT (A), PT (B), INR (C), and TEG R+K time (D). Furthermore LSR and TEG parameters angle (E) and MA (F) were compared. In Figs (B-F) data from all 12 patients is reported. For Fig (A), N = 11 patients are reported as aPTT was not obtained for one patient.</p

Effect of heparin on LSR and TEG coagulation parameters.

<p>Blood coagulation parameters including the clotting time (R+K), the clot progression (angle) and the maximum amplitude (MA) were measured using LSR and TEG for 20–60 minutes following kaolin-activation of swine whole blood samples spiked with heparin at concentration 0 (control), 0.1, 0.2, 0.25, 0.3 USP/ml (A-C). Linear regression analysis between TEG and LSR coagulation parameters at each concentration was performed (D-F). Each data point represents the mean of three replications ± standard deviation (SD) (histograms) or standard error of the mean, SEM (linear regression). Values were compared between control samples (without treatment) and heparin treated samples using ANOVA followed by the Tukey’s method for multiple comparisons post-tests. * p<0.05,** p<0.01, *** p<0.001, **** p<0.0001.</p

Diagnostic laboratory standardization and validation of platelet transmission electron microscopy

<p>Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2–4.0. Mean DG/plt was associated with IPF (<i>p </i>= .01, R² = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (<i>p </i>> .17, R² < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.</p