RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer.

BACKGROUND: Chronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrPSc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer. RESULTS: A total of 23 platelet samples derived from CWD-positive deer (ELISA + /IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10-2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10-3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels. CONCLUSIONS: Incorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component significantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

Role of Platelet/Lymphocyte, Neutrophil/Lymphocyte, and Interleukin-37/Interleukin-17 Ratios in the Occurrence and Treatment of Rheumatoid Arthritis.

This study was designed to investigate the correlation of neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and interleukin (IL)-37/IL-17 ratio with the incidence/treatment of rheumatoid arthritis (RA). Firstly, fifty-eight patients with RA treated at the first affiliated hospital of Xinjiang Medical University from January 2018 to January 2019 were selected as the RA group; forty-nine healthy volunteers were enrolled in the control group. RA patients were treated with disease-modifying anti-rheumatic drugs (DMARDs). Next, the NLR, PLR, IL-37, IL-17 and 28-joint disease activity score using erythrocyte sedimentation rate (DAS28-ESR) were deleted in two groups. Subsequently, Spearman correlation analysis was adopted for the correlations of various indicators before and after treatment in two groups. According to the analysis results, the levels of NLR, PLR, IL-37, and IL-17 before treatment in the RA group were higher than those in the control group (P < .05), but the difference in the IL-37/IL-17 level between the two groups was not significant (P > .05). After treatment, NLR, PLR, and IL-37/IL-17 levels were significantly reduced in RA patients (P < .05). NLR and PLR were significantly positively correlated with DAS28-ESR, ESR and C-reactive protein (CRP), of which represented the disease activity of RA. NLP was strongly correlated with IL-37/IL-17. Collectively, NLR, PLR, IL-37, and IL-17 are closely related to the occurrence of RA. In addition, NLR and IL-37/IL-17 are more suitable than PLR in reflecting the therapeutic effect. Therefore, IL-37/IL-17 can be considered as a new indicator for reflecting the treatment effectiveness of RA.

Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs.

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

Hematologic and biochemical inflammatory markers increase with body mass and positively correlate in adolescents.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease that has its origins in childhood. The goal of this study was to explore the relationships of hematologic inflammatory markers to body mass, biochemical inflammatory markers and cardiometabolic risk factors. METHODS: Healthy, white, non-Hispanic identifying adolescents (n = 75, age 12 to 18 years) were enrolled. Measures studied included body mass index percentile (BMI%), neutrophil and platelet to lymphocyte ratio (NLR, PLR), pan immune inflammation value (PIV), lipids, augmentation index, reactive hyperemia, inflammatory markers (interleukin 6: IL6, c-reactive protein: CRP), complement (C3, C3a, C4, C4a, C5a) insulin secretion and insulin sensitivity (oral glucose tolerance test: Matusda index, and disposition index (DI)). RESULTS: NLR (rS = 0.31, p < 0.01), PLR (rS = 0.32, p < 0.01), PIV (rS = 0.32, p < 0.01) and CRP (rS = 0.51, p < 0.001) all positively correlated with BMI% but IL-6 did not. NLR, PLR and PIV all positively correlated with each other. NLR correlated with the reactive hyperemia response (rS = 0.29, p < 0.02) but this relationship was lost when BMI% was included. NLR positively correlated with C3a, C4, CRP and IL6 even when BMI% was included. CONCLUSION: In healthy adolescents hematologic markers of inflammation increase with increasing body mass and neutrocyte to lymphocyte ratio is associated with increased complement and inflammatory markers independent of obesity. IMPACT STATEMENT: Hematologic and biochemical markers of inflammation increase with increased body mass in healthy adolescents. Hematologic and biochemical markers of inflammation are positively related independent of body mass in healthy adolescents. Hematologic inflammatory markers are not related to markers of cardiometabolic risk in healthy adolescents.

Exploring systemic inflammation in children with chronic kidney disease: correlates of interleukin 6.

BACKGROUND: Systemic inflammation (SI) is linked to chronic kidney disease (CKD) progression and multiple complications. Data regarding SI biomarkers in pediatric patients are scarce. This case-control and cross-sectional study investigates the correlation of neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), total iron binding capacity (TIBC) and serum albumin to serum interleukin-6 (IL-6). METHODS: NLR and PLR were measured in 53 patients (median age: 12.9 years), including 17 on dialysis and 36 with a median glomerular filtration rate of 39 ml/min/1.73m2, and in 25 age and sex-matched healthy controls. Iron profile, serum albumin and IL-6 were measured in the patient group. IL-6 levels > 3rd quartile were classified as high. RESULTS: Patients presented higher NLR and PLR and particularly those on dialysis (p < 0.001 and p = 0.001). We observed a significant correlation between natural logarithm (ln) of IL-6 (lnIL-6) and NLR (rs = 0.344, p = 0.014), serum albumin (rs = -0.350, p = 0.011) and TIBC (rs = -0.345, p = 0.012) after adjustment for CKD stage, while the correlation between lnIL-6 and PLR was not significant (rs = 0.206, p = 0.151). Combination of NLR, serum albumin and TIBC predicted high IL-6 (13 patients) with an AUC of 0.771 (95% CI 0.608-0.943). Pairing of NLR ≥ 1.7 and TIBC ≤ 300 µg/dL exhibited the highest sensitivity (76.9%), while incorporating serum albumin ≤ 3.8 g/dL along with them achieved the highest specificity (95%) for detecting high IL-6 levels. CONCLUSION: Both NLR and PLR levels increase in CKD, especially in patients on chronic dialysis. NLR, rather than PLR, along with TIBC and serum albumin, are associated with IL-6 in pediatric CKD.

Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.

BACKGROUND: Research on circulating mitochondrial DNA (cir-mtDNA) based diagnostic is insufficient, as to its function, origin, structural features, and particularly its standardization of isolation. To date, plasma preparation performed in previous studies do not take into consideration the potential bias resulting from the release of mitochondria by activated platelets. METHODS: To tackle this, we compared the mtDNA amount determined by a standard plasma preparation method or a method optimally avoiding platelet activation. MtDNA extracted from the plasma of seven healthy individuals was quantified by Q-PCR in the course of the process of both methods submitted to filtration, freezing or differential centrifugation. RESULTS: 98.7 to 99.4% of plasma mtDNA corresponded to extracellular mitochondria, either free or into large extracellular vesicles. Without platelet activation, the proportion of both types of entities remained preponderant (76-80%), but the amount of detected mtDNA decreased 67-fold. CONCLUSION: We show the high capacity of platelets to release free mitochondria in "in vitro" conditions. This represents a potent confounding factor when extracting mtDNA for cir-mtDNA investigation. Platelet activation during pre-analytical conditions should therefore be avoided when studying cir-mtDNA. Our findings lead to a profound revision of the assumptions previously made by most works in this field. Overall, our data suggest the need to characterize or isolate mtDNA associated various structural forms, as well as to standardize plasma preparation, to better circumscribe cir-mtDNA's diagnostic capacity.

Exploring the diagnostic and prognostic value of the C-reactive protein/lymphocyte ratio for dilated cardiomyopathy based on a real-world study.

To determine the risk factors for dilated cardiomyopathy (DCM) and construct a risk model for predicting HF in patients with DCM, We enrolled a total of 2122 patients, excluding those who did not meet the requirements. A total of 913 patients were included in the analysis (611 males and 302 females) from October 2012 to May 2020, and data on demographic characteristics, blood biochemical markers, and cardiac ultrasound results were collected. Patients were strictly screened for DCM based on the diagnostic criteria. First, these patients were evaluated using propensity score matching (PSM). Next, unconditional logistic regression was used to assess HF risk. Furthermore, receiver operating characteristic (ROC) curve analysis was conducted to determine diagnostic efficiency, and a nomogram was developed to predict HF. Finally, the Kaplan‒Meier survival curve was plotted. Of the initial 2122 patients, the ejection fraction (EF) in males was worse. We included 913 patients after the final DCM diagnosis. The results showed that the levels of NT-proBNP, WBC, PLT, neutrophils, lymphocytes, eosinophils, and IL-6, C-reactive protein (CRP) and the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and CRP/lymphocyte ratio (CLR) were higher in males than in females (P < 0.001-0.009). The nomogram showed that factors such as sex, WBC, neutrophils, PLR, and CLR could predict the risk of worsening cardiac function in patients with DCM before and after PSM (P < 0.05). The ROC curve showed that CLR with an 85.6% area demonstrated higher diagnostic efficacy than the NLR (77.0%) and PLR (76.6%, P < 0.05). Survival analysis showed a higher mortality risk in females with higher CLR levels (P < 0.001-0.009). However, high CLR levels indicated a higher mortality risk (P < 0.001) compared to sex. Male EF is lower in DCM patients. CLR could predict the risk of declined cardiac function in patients with DCM. The mortality in females with higher CLR levels was highest; however, the exact mechanism should be investigated.

[Molecular dynamics simulation of force-regulated interaction between glycoprotein Ib α and filamin].

In resting platelets, the 17 th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 rd phenylalanine (PHE 563-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.

Enhanced diabetic wound healing using platelet-derived extracellular vesicles and reduced graphene oxide in polymer-coordinated hydrogels.

Impaired wound healing is a significant complication of diabetes. Platelet-derived extracellular vesicles (pEVs), rich in growth factors and cytokines, show promise as a powerful biotherapy to modulate cellular proliferation, angiogenesis, immunomodulation, and inflammation. For practical home-based wound therapy, however, pEVs should be incorporated into wound bandages with careful attention to delivery strategies. In this work, a gelatin-alginate hydrogel (GelAlg) loaded with reduced graphene oxide (rGO) was fabricated, and its potential as a diabetic wound dressing was investigated. The GelAlg@rGO-pEV gel exhibited excellent mechanical stability and biocompatibility in vitro, with promising macrophage polarization and reactive oxygen species (ROS)-scavenging capability. In vitro cell migration experiments were complemented by in vivo investigations using a streptozotocin-induced diabetic rat wound model. When exposed to near-infrared light at 2 W cm- 2, the GelAlg@rGO-pEV hydrogel effectively decreased the expression of inflammatory biomarkers, regulated immune response, promoted angiogenesis, and enhanced diabetic wound healing. Interestingly, the GelAlg@rGO-pEV hydrogel also increased the expression of heat shock proteins involved in cellular protective pathways. These findings suggest that the engineered GelAlg@rGO-pEV hydrogel has the potential to serve as a wound dressing that can modulate immune responses, inflammation, angiogenesis, and follicle regeneration in diabetic wounds, potentially leading to accelerated healing of chronic wounds.

Correlation between C-reactive protein and the inflammatory ratios in acute schizophrenia inpatients: are they associated?

C-reactive protein (CRP) and inflammatory ratios have been proposed to study immune dysregulation in schizophrenia. Nevertheless, links between CRP and inflammatory ratios in acute SCZ inpatients have been understudied. This study investigated the relationship between CRP and inflammatory ratios (Neutrophil-Lymphocyte Ratio [NRL], Platelet-Lymphocyte Ratio [PLR], Monocyte-Lymphocyte ratio [MLR] and Basophil-Lymphocyte Ratio [BLR]) in a total of 698 acute SCZ inpatients; and analysed how this relationship is affected by sex and type of episode. CRP correlated with NLR (rs = 0.338, p < 0.001), PLR (rs = 0.271, p < 0.001) and MLR (rs = 0.148, p < 0.001) but not with BLR (rs = 0.059, p = 0.121). Multiple lineal regression analysis showed that high levels of NLR, MLR and PLR but not BLR were independently associated with high CRP levels. No sex-related variations were found. Significant associations were maintained for NLR and MLR in first-episode and multiepisode SCZ, although the strength of the association was stronger in multiepisode SCZ. Again, no sex-related differences were found in these associations. In conclusion, inflammatory ratios were low to moderately associated with CRP in acute SCZ inpatients. NLR and multiepisode SCZ showed the highest associations with CRP. Future studies should consider inflammatory ratios not as a substitute for CRP but as a complementary biomarker.

Association of hemoglobin, albumin, lymphocyte, and platelet score with risk of cerebrovascular, cardiovascular, and all-cause mortality in the general population: results from the NHANES 1999-2018.

Background and aims: Cardiovascular and cerebrovascular disease (CCDs) contribute to leading causes of morbidity and mortality in the United States of America (USA). Hemoglobin, albumin, lymphocyte, and platelet (HALP) score, a simple and convenient indicator, could reflect the combination of inflammation and nutritional status. This study was undertaken to evaluate the associations between HALP score and risk of cardiovascular, cerebrovascular, and all-cause mortality in the general population from the National Health and Nutrition Examination Survey (NHANES) 1999-2018. Methods: We identified 21,578 participants during the 1999-2018 cycles of the NHANES in this research. HALP score was calculated as hemoglobin (g/L) × albumin (g/L) × lymphocytes (/L)/platelets (/L). Outcomes were cerebrovascular, cardiovascular, and all-cause mortality determined by the NHANES-linked National Death Index record and followed until 31 December 2019. Survey-weighted Cox regression, restricted cubic spline analysis, and subgroup analysis were applied to investigate relationships between HALP score and risk of mortality. Results: This cohort study comprised 49.2% male and 50.8% female, of which the median age was 47 years old. In multivariate survey-weighted Cox regression adjusting for all confounders, compared with participants with low HALP scores, participants with highest HALP score had a lower risk of all-cause mortality (adjusted HR:0.80, 95% CI: 0.73, 0.89, P < 0.0001) and cardiovascular mortality (adjusted HR:0.61, 95% CI: 0.50, 0.75, P < 0.0001), and mediate HALP score had the lowest risk of all-cause mortality (adjusted HR:0.68, 95% CI: 0.62, 0.75, P < 0.0001) and cardiovascular mortality (adjusted HR:0.60, 95% CI: 0.48, 0.75, P < 0.0001). Restricted cubic spline analysis showed a non-linear relationship between HALP score and cardiovascular and all-cause mortality (all P values <0.001). Conclusion: HALP score was independently associated with risk of cardiovascular and all-cause mortality, but not cerebrovascular mortality.

The hemoglobin, albumin, lymphocyte, and platelet (HALP) is a potent indicator for the prognosis in hemodialysis patients.

The hemoglobin, albumin, lymphocyte, and platelet (HALP) values were marked as a original index of general nutritional and inflammatory conditions. The purpose of this investigation was to evaluate the potential relationship between HALP and prognosis in hemodialysis (HD) patients. Patients with maintenance HD from multiple dialysis centers in China were retrospectively analyzed. The primary poor outcome were cardiovascular disease (CVD) and all-cause death. The computational equation of HALP values as the follows: hemoglobin (g/L) × albumin (g/L) × lymphocytes (/L)/ platelets (/L). All participants were divided into Tertile 1, Tertile 2, and Tertile 3 according to the tertiles of baseline HALP values. The Kaplan-Meier curve and the Cox regression was done to figure out the relationship about HALP and adverse outcomes. The restricted cubic splines further identified the possible associations. The time-dependent receiver operating characteristic curve and C-index were implemented for evaluate the predictive values of the HALP composite model. There were 4796 patients incorporate into ultimate study. Compared with patients in Tertile 1, patients in Tertile 3 had an lower risk of all-cause mortality [hazard ratios = 0.66, 95% confidence intervals: 0.49-0.86, P = .007] and CVD mortality [sub-distribution hazard ratio = 0.51, 95% confidence intervals: 0.34-0.80, P = .005]. The composite model with the supplement of HALP outperformed the traditional factor model in the time-dependent receiver operating characteristic curve. High HALP values at baseline are related to a diminished risk of CVD death and all-cause death in HD patients. HALP is a novel and potent index for the prognosis of HD patients.

Bioengineered tissue and cell therapy products are efficiently cryopreserved with pathogen-inactivated human platelet lysate-based solutions.

BACKGROUND: There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products. METHODS: We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary. RESULTS: Amongst the tissue-specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at - 20 °C for at least 3 months. Regarding cell-specific solutions, CeA maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs and promoted good recovery post-thaw. Besides, both tested solutions were stable at - 20 °C for 18 months. Finally, we established that there is a 3-h window in which thawed NFAHs and FBs maintain optimum viability immersed in the formulated solutions and at least 2 h for BM-MSCs. CONCLUSIONS: Our results show that pathogen-inactivated solutions Ti5 allocated for bioengineered tissues and CeA allocated for cells are efficient and safe candidates to cryopreserve ATMPs and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions.

Fibrinogen to albumin ratio, a novel serum indicator for evaluating the severity of preeclampsia: A single-center retrospective study.

Preeclampsia (PE) is a disorder that affects approximately 5% to 10% of pregnant women. Timely and accurate identification of PE and assessment of its severity are crucial. Therefore, it is necessary to develop predictive indicators which are easily measured in routine antenatal examinations to enable the early detection of PE and assess its severity. We designed a single-center retrospective study in our daily work to assess whether the serum levels of fibrinogen to albumin ratio (FAR), fibrinogen (Fib), albumin (ALB), prothrombin time, calcium (Ca), activated partial thrombin time, creatinine (Cr), D-dimer(D-D), platelet, white blood cell, neutrophil, and lymphocyte counts could help in assessing PE and evaluating its severity. Our findings showed that the serum levels of FAR, Cr, Fib, and D-D were significantly higher in the severe preeclampsia group (sPE) compared with the control and mild preeclampsia groups, whereas the levels of ALB and Ca were significantly lower in sPE patients. In addition, no differences were found between the control and PE groups in terms of prothrombin time, activated partial thrombin time, platelet, white blood cell, neutrophils, and lymphocytes counts. Furthermore, FAR is a novel and better indicator for evaluating the severity of PE, which has not been reported before. And it is an independent risk factor for the development of sPE. In conclusion, the serum levels of FAR, Cr, D-D and Fib were positively correlated with PE, whereas ALB and Ca were negatively correlated with PE severity, which might be valuable in evaluating the severity of PE. FAR proved to be a feasible diagnostic marker for sPE with sensitivity and specificity comparable to those of ALB and Fib.

Characterization of the human platelet N- and O-glycome upon storage using tandem mass spectrometry.

Changes in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and 7-day room temperature-stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry, and liquid chromatography were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire largely comprised sialylated and fucosylated core-1 and core-2 structures. For both N- and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity after platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets after storage.

A mathematical model for assessing shear induced bleeding risk.

PURPOSE: The objective of this study is to develop a bleeding risk model for assessing device-induced bleeding risk in patients supported with blood contact medical devices (BCMDs). METHODS: The mathematical model for evaluating bleeding risk considers the effects of shear stress on von Willebrand factor (vWF) unfolding, high molecular weight multimers-vWF (HMWM-vWF) degradation, platelet activation and receptor shedding and platelet-vWF binding ability. Functions of the effect of shear stress on the above factors are fitted/employed and solved by the Eulerian transport equation. An axial flow-through Couette device and two clinical VADs which are HeartWare Ventricular Assist Device (HVAD) and HeartMate II (HM II) blood pump were employed to perform the simulation to evaluate platelet receptor shedding (GPIbα and GPIIb/IIIa), loss of HWMW-vWF, platelet-vWF binding ability and bleeding risk for validating the accuracy of our model. RESULTS: The platelet-vWF binding ability after being subjected to high shear region in the axial flow-through Couette device predicted by our bleeding model was highly consistent with reported experimental data. As indicated by our CFD simulation results in the axial flow-through Couette device, it can find that an increase in shear stress led to a decrease in the adhesion ability of platelets on vWF, while the binding ability of vWF with platelets first increase and then decrease as shear stress elevates gradually beyond a threshold. The factor of exposure time can enhance the effect of shear stress. Additionally, the shear-induced bleeding risk predicted by our model increases with increasing shear stress and exposure time in an axial flow-through Couette device. As indicated by our numerical model, the bleeding risk in HVAD was higher than HMII, which is highly consistent with the meta-analysis based on clinical statistics. Our simulation investigations in these two clinical VADs also found that HVAD caused a higher rate of platelet receptor shedding and lower damage to HWMW-vWF than HeartMate II. The high shear stress generated in the narrow and turbulent regions of both VADs was the underlying cause of device-induced bleeding. CONCLUSION: In this study, the shear-induced bleeding risk predicted by our bleeding model in axial flow-through Couette device and two clinical VADs is consistent or highly correlated with experimental and clinical findings, which proves the accuracy of our bleeding model. Our bleeding model can be used to aid the development of new BCMDs with improved functional characteristics and biocompatibility, and help to reduce risk of device-induced adverse events in patients.

Complete blood platelet and lymphocyte ratios increase diagnostic accuracy of periprosthetic joint infection following total hip arthroplasty.

INTRODUCTION: Systemically, changes in serum platelet to lymphocyte ratio (PLR), platelet count to mean platelet volume ratio (PVR), neutrophil to lymphocyte ratio (NLR) and monocyte to lymphocyte (MLR) represent primary responses to early inflammation and infection. This study aimed to determine whether PLR, PVR, NLR, and MLR can be useful in diagnosing periprosthetic joint infection (PJI) in total hip arthroplasty (THA) patients. METHODS: A total of 464 patients that underwent revision THA with calculable PLR, PVR, NLR, and MLR in 2 groups was evaluated: 1) 191 patients with a pre-operative diagnosis of PJI, and 2) 273 matched patients treated for revision THA for aseptic complications. RESULTS: The sensitivity and specificity of PLR combined with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), synovial white blood cell count (WBC) and synovial polymorphonuclear leukocytes (PMN) (97.9%; 98.5%) is significantly higher than only ESR combined with CRP, synovial WBC and synovial PMN (94.2%; 94.5%; p < 0.01). The sensitivity and specificity of PVR combined with ESR, CRP and synovial WBC, and synovial PMN (98.4%; 98.2%) is higher than only ESR combined with CRP, synovial WBC and synovial PMN (94.2%; 94.5%; p < 0.01). CONCLUSION: The study results demonstrate that both PLR and PVR calculated from complete blood counts when combined with serum and synovial fluid markers have increased diagnostic sensitivity and specificity in diagnosing periprosthetic joint infection in THA patients. LEVEL OF EVIDENCE: III, case-control retrospective analysis.

Platelet lysate can support the development of a 3D-engineered skin for clinical application.

Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90+FAP+, CD90+FAP- and CD90-FAP+, based on CD90 (reticular marker) and FAP (papillary marker) expression profile. PL drove the expansion of the intermediate CD90+ FAP+ subpopulation in expense of reticular CD90+FAP-, which may be less fibrotic once grafted. The 3D-engineered skin cultured in PL was analysed by immunofluorescence using specific markers. Detection of ColIV and LMN-511 confirmed basement membrane. K10 confirmed near native differentiation pattern of neo-epidermis. CD29- and K5-positive interfollicular stem cells were also sustained. Transmission and scanning electron microscopies detailed the ultrastructure of the neo-dermis and neo-epidermis. To elucidate the underlying mechanism of the effect of PL on skin maturation, growth factor contents in PL were measured, and TGF-ß1 was identified as one of the most abundant. TGF-ß1 neutralising antibody reduced the number of Ki67-positive proliferative cells, suggesting TGF-ß1 plays a role in skin maturation. Moreover, the 3D-engineered skin was exposed to lucifer yellow on days 1, 3 and 5. Penetration of lucifer yellow into the skin was used as a semi-quantitative measure of improved barrier function over time. Our findings support the concept of PL as a safe and effective serum alternative for bioengineering skin for cell therapies.

Effect of mirasol pathogen reduction technology system on immunomodulatory molecules of apheresis platelets.

Pathogen inactivation for platelets by riboflavin system (MIRASOL) efficiently reduces transfusion related pathogen transmission. However little is known about its impact on platelets' immunomodulatory biochemical profile. We aimed was to assess the effects of MIRASOL treatment on platelet quality parameters and immunomodulatory molecules CD62P, RANTES, and CD40L in Single Donor Platelets (SDPs) resuspended in plasma (SDP-P) or T-PAS and additive solution (SDP-A). Twenty nine SDPs (15 SDP-P and 14 SDP-A) were included in the study. Samples were collected before, after MIRASOL treatment and just before transfusion. P-selectin (CD62P), RANTES, and CD40L were tested by ELISA. Platelet products quality assays were also performed. Platelet count/unit decreased after Mirasol treatment by 13 %. The pH of all units decreased over the 5-day storage period but remained above expected limits and the swirling test was positive throughout storage. P-selectin levels were not different between the three different time points in both SDPs-P and SDPs-A while RANTES levels were found to differ statistically significantly at the three different time points in all units and in the SPD-A subgroup. CD40L levels in all SDP products increased slightly during storage but this was not statistically significant. CD62P, RANTES, and CD40L in all time points were elevated in SDPs-A compared to SDPs-P but not at a statistically significant level. In conclusion MIRASOL treatment apart from RANTES increase does not seem to substantially affect platelets associated other cytokines and immunomodulatory molecules namely P-selectin and sCD40L which are implicated in immune transfusion reactions.