The Reevaluation of Thrombin Time Using a Clot Waveform Analysis

The purpose of this study is to assess platelet and fibrin targeted ultrasound contrast microbubbles ability to adhere to fresh platelet and platelet poor clots in vitro with the assistance of radiation force. Whole blood from healthy volunteers (25 ml) was collected and centrifuged at 1100 rpm for 15 min to separate platelet-rich plasma (PRP), which was separated and centrifuged (3000 rpm, 5 min) to get platelet poor plasma (PPP). Calcium and Thrombin from human plasma were added to form fresh blood clots with upregulated receptors. Platelet targeted (MRX44; ImaRx Therapeutics, Inc., Tucson, AZ), fibrin targeted (MRX802; ImaRx Therapeutics) or untargeted, control bubbles were added to the clots. Radiation force was employed, using a single-element transducer with continuous wave ultrasound (2.5-5 MHz, 100 kHz PRF, 30 s exposure, 50.20-123.65 kPap-p pressure), to push the bubbles onto the thrombi. The adherence of the microbubbles to the clots was facilitated by the targeting ligands present on the microbubbles that bind to receptors present in blood clots or thrombi. Attachment rates between the groups were determined using a microscope setup and compared using unpaired t-tests. The average number of bubbles attached per 1000 mum2 of clot area using MRX44 was 7.0 plusmn 4.21 bubbles and 2.72 plusmn 2.44 bubbles, respectively, in PRP and PPP clots. With MRX802 the corresponding attachment rates were 11.45 plusmn 8.52 and 9.25 plusmn 3.40 bubbles, while typically less than 2 control bubbles adhered per 1000 mum2 of clot area in either clot type. The differences in adherence rates were statistically significant for MRX802 compared to MRX44 and to controls in both PRP and PPP clots (p < 0.05). Relative to controls more MRX44 bubbles adhered in PRP clots (p = 0.003) but not in PPP clots (p = 0.07). In conclusion, the in vitro attachment rates of bubbles targeted to human blood clots depend on target availability (i.e., platelet targeted bubbles do better in PRP than in PPP clots). Fibrin targeted bubbles showed the best adherence rates in this study.

The Challenge of Myeloma-Related Thromboembolic Disease: Can Thrombin Generation Assay May Detect Disease-Related Hypercoagulability?

Effects of Platelet-Rich Plasma on Intestinal Anastomotic Healing in Rats: PRP Concentration is a Key Factor

Background/Objectives: FVIII reagent activity varies across different assays, as well as activated partial thromboplastin time (APTT) reagents. The hemostatic ability of various FVIII reagents was examined via clot waveform analysis (CWA). Methods: APTT was measured using 12 APTT reagents, a small amount of tissue factor-induced FIX activation (sTF/FIXa) and a small amount of thrombin time (sTT) in order to examine 10 FVIII reagents and reference plasma (RP) using CWA. FVIII activity was measured using CWA-APTT, a chromogenic assay, or CWA-sTT. Results: Although the peak time (PT) and peak height (PH) of the CWA-APTT were markedly different in different FVIII reagents using several APTT reagents, the PTs of CWA-APTT were generally normal or shortened and the PHs of CWA-APTT were generally lower than those of RP. The FVIII activity varied, as evaluated using APTT, and was higher when using the CWA-sTT method than the APTT or chromogenic methods. CWA-sTT showed an elevated second peak of first DPH in all FVIII reagents, and both CWA-sTF/FIXa and CWA-sTT were enhanced using APTT reagents. Conclusions: Our evaluation of the hemostatic ability of FVIII reagents varied among APTT reagents. CWA-sTT can be used to further evaluate the hemostatic ability of an FVIII concentrate based on thrombin burst.

Assessment Of a Novel Synthetic Collagen Reagent By Platelet Aggregometry

Clot waveforms are generated by coagulation analyzers, such as the ACL-TOP, that utilize photo-optical turbidimetric methods to measure prothrombin time (PT) and activated partial thromboplastin time (aPTT), which are standard clotting tests (SCTs). Additional quantitative clot waveform analysis (CWA) parameters include maximum velocity of the PT curve (PT1d), maximum acceleration of the aPTT curve (aPTT2d), and their change in optical density (ODdelta). We explored the potential for CWA to contribute unique information compared to SCTs and quantitative fibrinogen assay (QFA) in dogs with clinical indications for laboratory evaluation of coagulation. We retrospectively evaluated the frequency and agreement between normal, high, and low values of CWA and SCTs for 203 dogs relative to reference intervals (RIs). Most dogs had SCTs within RIs, but most CWA values were high regardless of whether SCT values were within, below, or above the RI. Agreement between SCTs and CWA was slight (kappa <30%). Clinically significant bleeding was noted in 28 of 203 (14%) dogs. SCTs were not different between bleeders and non-bleeders. Median QFA, PT1d, aPTTdelta, and aPTT2d values were statistically significantly higher than RIs in non-bleeders compared to bleeders, whose median values were not below RI. Compared with SCTs, CWA identified differences between the 2 groups. However, wide overlap between the 2 groups limits the use of CWA to clinically discriminate between bleeders and non-bleeders in a heterogeneous population of dogs. Further studies on the diagnostic utility of CWA in coagulopathic dogs are needed.

Background Prolonged immobilization is widely recognized as a risk factor for thromboembolism. In this prospective study, we investigated the changes in clot waveform analysis (CWA) parameters in prolonged immobilized patients following lower limb trauma. CWA is an advanced method for assessing global coagulation that involves continuously monitoring changes in light transmittance, absorbance, or light scattering during routine clotting tests. Additionally, we also aim to determine the CWA parameters between day one and after day three of immobilization. Methods A total of 30 patients with prolonged immobilizationwere enrolledin this study. The plasma of these patients was collected on the first day of their admission and subsequently obtained again after day three of immobilization. Prothrombin time (PT)-based CWA and activated partial thromboplastin time (aPTT)-based CWA were performed using the ACL TOP 300 CTS (Werfen: Bedford, USA) coagulation analyzer, which utilizes the optical method for clot detection. Plasma samples for 20 normal controls were recruited from a healthy blood donor. The CWA parameters generated during clot formation were analyzed. For the comparison of CWA parameters between patients with prolonged immobilization and healthy controls, the Mann-Whitney test was used. A paired t-test was used for the comparison of clot wave parameters between day one and after day three of immobilization. This study was approved by the Universiti Sains Malaysia Research Ethics Committee. Result The mean values of PT and aPTT in healthy controls were 11.66 seconds and 33.98 seconds, respectively. There was no statistically significant difference between the patients and the healthy controls in the median values of aPTT (P=0.935). However, patients with prolonged immobilization exhibited significantly higher median PT CWA parameter values than controls (P=0.007). These parameters included the delta change (P<0.001), peak time velocity (P=0.008), and height velocity (P<0.001).On the other hand, the delta change (P<0.001) and height velocity (P<0.001) of the aPTT CWA parameters were significantly higher in patients with prolonged immobilization than in controls. In patients with prolonged immobilization, there was no significant difference in PT CWA parameters between day one and after day three of immobilization, whilefor aPTT CWA, all parameters were higher on day three, except for the endpoint time. Conclusion Patients with prolonged immobilization exhibit increased PT and aPTT CWA parameters compared to normal controls. CWA parameters could aid in identifying patients at risk of developing thrombosis through changes in the clot waveform. However, further study is needed to fully utilize additional information from routine coagulation testing.

The activated partial thromboplastin time (APTT)–clot waveform analysis (CWA) was previously reported to be associated with the early detection of disseminated intravascular coagulation and was also reported to be able to measure very low levels of coagulation factor VIII activity. The software program for the analysis for the APTT-CWA allows the associated first and second derivative curves (first and second DCs) to be displayed. The first and second DC reflect the velocity and acceleration, respectively. The height of the first DC reflects the “thrombin burst” and bleeding risk, while that of the second DC is useful for detecting any coagulation factor deficiency and abnormal enhancement of coagulation by phospholipids. Activated partial thromboplastin time-CWA aids in making a differential diagnosis which is difficult to do using only the routine APTT. The CWA is currently used for many applications in the clinical setting, including the monitoring of hemophilia patients and patients receiving anticoagulant therapy and the differential diagnosis of diseases.

As the demand for non-invasive esthetic procedures to maintain a youthful appearance increases, there has been growing interest in the use of autologous platelet-rich plasma (PRP) and platelet-poor plasma (PPP) for the treatment of facial aging. However, there are few studies directly comparing the efficacy of PRP and PPP for facial rejuvenation. This study aimed to compare the efficacy of PRP and PPP for facial rejuvenation. This single-center, double-blind, randomized controlled trial was conducted from January 1, 2022, to July 31, 2022, and included ten participants who completed the follow-up. The participants were randomly assigned to receive 2.5-mL injections of PRP and PPP on different sides of the face in three sessions with 1-month intervals. The outcome was primarily determined by blinded photographic assessments and secondly by scores of the VISIA® system during the follow-up. Both PRP and PPP treatments resulted in significant improvement in the Global Aesthetic Improvement Scales and Modified Fitzpatrick Wrinkle Scale for periocular Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation wrinkles, with no significant difference between the two groups. However, no improvement was observed in the Wrinkle Severity Rating Scales for nasolabial folds in either the PRP- or PPP-treated groups. Furthermore, no severe adverse events were reported. Both PRP and PPP are effective in treating facial photoaging. PRP exhibited slightly superior efficacy in enhancing overall skin condition, while PPP was slightly more effective in improving shallow wrinkles. This study provides valuable evidence for the use of PRP and PPP in facial rejuvenation procedures. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Purpose:To evaluate and compare the effects of homologous and heterologous PRP (Platelet-Rich Plasma) on the quality and speed of skin wound healing, compared to Poor Platelet Plasma (PPP).Methods:Twenty-one male adult rabbits were used; two for preparing homologous PRP, with the rest of them separated randomly in three groups, according to the treatment received: PPP - control (n=5), homologous PRP (n=7), heterologous (n=7). Excisional skin wounds were made on the back of the animals, for the application of homologous and heterologous PPP and PRP. At the 14th post-operative day (POD), the animals were subjected to a new wound, and the treatments were inverted. The wounds were evaluated macroscopically and histologically.Results:A larger percentage of scar retraction was observed on the group treated with heterologous PRP, compared to homologous PRP, at the third POD, an increase of 25.03% (p=0.01). No other statistically significant differences among treatments were observed. Among every group, skin healing was efficient, without local adverse effects.Conclusions:Heterologous PRP contributed with more tissue retraction at the beginning of the wound healing process. After this, there were no differences on the wound healing skin process treated with PRP or PPP. However, our findings suggest the presence of others plasmatic factors, besides platelets, which could also contribute to the wound healing process, and thus, should be further investigated.

Optimal monitoring of bypass therapy in hemophilia A patients with inhibitors by the use of clot waveform analysis

Melatonin is not stored in platelets

It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix. Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 +/- 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix. Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-beta, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma. Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.

Objectives Available clinical evidence suggests the potential therapeutic use of intracavernosal injections of platelet-rich plasma (PRP) to treat erectile dysfunction (ED). However, the effects of PRP on human cavernosal function have not been elucidated. Our aim was to evaluate the effects of acute exposure to autologous PRP on relaxant capacity of human corpus cavernosum (HCC) from patients with ED. Methods HCC strips were obtained from four ED patients undergoing penile prosthesis insertion and giving informed consent. Previous to intervention, PRP and platelet-poor plasma (PPP) were obtained from patients by apheresis and stored at -20°C until functional procedures. Endothelial relaxation to acetylcholine (ACh) was evaluated before and after 60 min exposure to PRP or PPP (1% v/v) in organ chamber. Relaxant response to the PDE5 inhibitor, tadalafil, was also evaluated in PRP or PPP (1%) treated HCC from the same patient. Results Average age of patients was 67-years-old (range 61-71) while hypertension and smoking history was present in 3/4, diabetes in 2/4, and obesity in 1/4. Mean platelet content in PRP was 1,096x10 9 /L (range 786 to 1,284 x109/L) while negligible platelet content was obtained in any PPP. Exposure to autologous PRP but not PPP significantly enhanced (p&amp;lt;0.0001 by two-factors ANOVA) ACh-induced endothelial relaxations (pEC50 5.75±0.65 vs. 7.16±0.50; Emax 57.3±14.8% vs. 88.5±2.0%; before and after 1%PRP, respectively). Additionally, HCC treated with autologous PRP displayed augmented relaxant responses to tadalafil (p=0.0018 by two-factors ANOVA) with respect to those treated with PPP (pEC50 6.08±0.5 vs. 6.82±0.51 for PPP and PRP, respectively). Conclusions Acute exposure to autologous PRP improves endothelial function and functional response to PDE5 inhibition. These results support the reported clinical improvement of erectile function in patients with ED and suggest that PRP injection might induce a functional effect on cavernosal tissue in addition to the claimed restorative/regenerative effect. Conflicts of Interest None

Comparison of Ufh and Enoxaparin Originated from Bovine, Ovine and Porcine Mucosa with Functional Coagulation Assays