Platelet dysfunction reversal with cold-stored vs. room temperature-stored platelet transfusions.

Platelets are stored at room temperature for 5-7 days (RSP). Due to frequent and severe shortages, the FDA recently approved up to 14-day cold-stored platelets in plasma (CSP). However, the post-transfusion function of CSP is unknown and it is unclear which donors are best suited to provide either RSP or CSP. In this study, we sought to evaluate the post-transfusion platelet function and its predictors for platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). We conducted a randomized cross-over study in ten healthy humans. Subjects donated one platelet unit stored at either 22 °C or 4 °C based on randomization. Before transfusion, subjects ingested ASA to inhibit endogenous platelets. Transfusion recipients were tested for platelet function and lipid mediators. Platelet units were tested for lipid mediators only. A second round of transfusion with the alternative product was followed by an identical testing sequence. RSP reversed platelet inhibition significantly better in αIIbß3 integrin activation-dependent assays. In contrast, CSP in recipients led to significantly more thrombin generation, which was independent of platelet microparticles. Lysophosphatidylcholine-O species levels predicted the procoagulant capacity of CSP. In contrast, polyunsaturated fatty acid concentrations predicted the aggregation response of RSP. In summary, we provide the first efficacy data of extended-stored CSP in plasma. Our results suggest that identifying ideal RSP and CSP donors is possible and pave the way for larger studies in the future. Registration: NCT03787927.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbß3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Evaluating stored platelet shape change using imaging flow cytometry.

Platelets are routinely stored at room temperature for 5-7 days before transfusion. Stored platelet quality is traditionally assessed by Kunicki's morphology score. This method requires extensive training, experience, and is highly subjective. Moreover, the number of laboratories familiar with this technique is decreasing. Cold storage of platelets has recently regained interest because of potential advantages such as reduced bacterial growth and preserved function. However, platelets exposed to cold temperatures change uniformly from a discoid to a spherical shape, reducing the morphology score outcomes to spheroid versus discoid during cooling. We developed a simpler, unbiased screening tool to measure temperature-induced platelet shape change using imaging flow cytometry. When reduced to two dimensions, spheres appear circular, while discs are detected on a spectrum from fusiform to circular. We defined circular events as having a transverse axis of >0.8 of the longitudinal axis and fusiform events ≤0.8 of the longitudinal axis. Using this assay, mouse and human platelets show a temperature and time-dependent, two-dimensional shape change from fusiform to circular, consistent with their three-dimensional change from discs to spheres. The method we describe here is a valuable tool for detecting shape change differences in response to agonists or temperature and will help screening for therapeutic measures to mitigate the cold-induced storage lesion.

What is the context? Platelets for transfusion are currently stored for 5­7 days at room temperature, increasing the risk for bacterial growthCold storage reduces the risk for bacterial growth but reduces circulation timeStored platelet quality can be assessed by the light microscopy-based Morphology Score, first described in the 1970sDownsides of the Morphology Score include subjectivity, extensive training, and reduced availability in platelet laboratories.What is new? In this study, we provide data showing that the Morphology score is reduced to a binary spheres versus discs response in cold-exposed plateletsWe developed an imaging flow cytometry-based approach to quantify platelets' response to cold based on the two-dimensional projection of the three-dimensional shapes, i.e., fusiform (discoid) versus circular (discoid and spherical)We provide validation of this approach in mouse and human plateletsWhat is the impact?This study provides an easy and unbiased tool for laboratories working on circumventing the cold-induced storage lesion or documenting spherical shape change in general.

Repeatability of gaseous measurements across consecutive days in sheep using portable accumulation chambers.

Portable accumulation chambers (PACs) enable gaseous emissions from small ruminants to be measured over a 50-min period; to date, however, the repeatability of consecutive days of measurement in the PAC has not been investigated. The objectives of this study were 1) to investigate the repeatability of consecutive days of gaseous measurements in the PAC, 2) to determine the number of days required to achieve precise gaseous measurements, and 3) to develop a prediction equation for gaseous emissions in sheep. A total of 48 ewe lambs (c. 10 to 11 mo of age) were randomly divided into four measurement groups each day, for 17 consecutive days. Gaseous measurements were conducted between 0800 and 1200 hours daily. Animals were removed from perennial ryegrass silage for at least 1 h before measurements in the PAC, and animals were assigned randomly to each of the 12 chambers. Methane (CH4; ppm) concentration, oxygen (O2; %), and carbon dioxide (CO2; %) were measured at three time points (0, 25, and 50 min after entry of the first animal into the first chamber). To quantify the effect of animal and day variation on gaseous emissions, between-animal, between-day, and error variances were calculated for each gaseous measurement using a linear mixed model. The number of days required to gain a certain precision (defined as the 95% confidence interval range) for each gaseous measurement was also calculated. For all three gases, the between-day variance (39% to 40%) accounted for a larger proportion of total variance compared with between-animal variance, while the repeatability of 17 consecutive days of measurement was 0.36, 0.31, and 0.23 for CH4, CO2, and O2, respectively. Correlations between consecutive days of measurement were strong for all three gases; the strongest correlation between day 1 and the remaining days for CH4, CO2, and O2 was 0.71 (days 1 and 6), 0.77 (days 1 and 2), and 0.83 (days 1 and 5), respectively. A high level of precision was achieved when gaseous measurements from PAC were taken over three consecutive days. The prediction equation overestimated gaseous production for all three gases: the correlations between actual and predicted gaseous output ranged from 0.67 to 0.71, with the r2 ranging from 0.45 to 0.71. The results from this study will aid the refinement of the protocol for the measurement of gaseous emissions in sheep using the PAC.

Storage temperature determines platelet GPVI levels and function in mice and humans.

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature-stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP; however, their rate of aggregation in response to the GPVI agonist collagen was significantly lower. In a mouse model of platelet transfusion, we found a significantly lower response rate to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.

Grazing Cow Behavior's Association with Mild and Moderate Lameness.

Accelerometer-based mobility scoring has focused on cow behaviors such as lying and walking. Accuracy levels as high as 91% have been previously reported. However, there has been limited replication of results. Here, measures previously identified as indicative of mobility, such as lying bouts and walking time, were examined. On a research farm and a commercial farm, 63 grazing cows' behavior was monitored in four trials (16, 16, 16, and 15 cows) using leg-worn accelerometers. Seventeen good mobility (score 0), 23 imperfect mobility (score 1), and 22 mildly impaired mobility (score 2) cows were monitored. Only modest associations with activity, standing, and lying events were found. Thus, behavior monitoring appears to be insufficient to discern mildly and moderately impaired mobility of grazing cows.

Infrared thermography as a tool to detect hoof lesions in sheep.

Lameness has a major negative impact on sheep production. The objective of this study was to 1) quantify the repeatability of sheep hoof temperatures estimated using infrared thermography (IRT); 2) determine the relationship between ambient temperature, sheep hoof temperature, and sheep hoof health status; and 3) validate the use of IRT to detect infection in sheep hooves. Three experiments (a repeatability, exploratory, and validation experiment) were conducted over 10 distinct nonconsecutive days. In the repeatability experiment, 30 replicate thermal images were captured from each of the front and back hooves of nine ewes on a single day. In the exploratory experiment, hoof lesion scores, locomotion scores, and hoof thermal images were recorded every day from the same cohort of 18 healthy ewes in addition to a group of lame ewes, which ranged from one to nine ewes on each day. Hoof lesion and locomotion scores were blindly recorded by three independent operators. In the validation experiment, all of the same procedures from the exploratory experiment were applied to a new cohort of 40 ewes across 2 d. The maximum and average temperature of each hoof was extracted from the thermal images. Repeatability of IRT measurements was assessed by partitioning the variance because of ewe and error using mixed models. The relationship between ambient temperature, hoof temperature, and hoof health status was quantified using mixed models. The percentage of hooves correctly classified as healthy (i.e., specificity) and infected (i.e., sensitivity) was calculated for a range of temperature thresholds. Results showed that a small-to-moderate proportion of the IRT-estimated temperature variability in a given hoof was due to error (1.6% to 20.7%). A large temperature difference (8.5 °C) between healthy and infected hooves was also detected. The maximum temperature of infected hooves was unaffected by ambient temperature (P > 0.05), whereas the temperature of healthy hooves was associated with ambient temperature. The best sensitivity (92%) and specificity (91%) results in the exploratory experiment were observed when infected hooves were defined as having a maximum hoof temperature ≥9 °C above the average of the five coldest hooves in the flock on that day. When the same threshold was applied to the validation dataset, a sensitivity of 77% and specificity of 78% was achieved, indicating that IRT could have the potential to detect infection in sheep hooves.

Investigation of the relationship between udder quarter somatic cell count and udder skin surface temperature of dairy cows measured by infrared thermography.

The objective of the present study was to quantify the relationship between udder skin surface temperature (USST) and somatic cell count (SCC) in lactating dairy cows. Data were recorded on the same 14 Holstein-Friesian cows, at evening (15:00 to 16:00) milking every day over a 2-mo period. Surface temperature measurements of all udders were extracted from thermal images. After imaging, milk was extracted from each quarter and analyzed for SCC. Environmental and cow-related factors (i.e., ambient temperature, humidity, rainfall, wind speed, distance walked to the parlor, number of days since the udder was shaved, parity, and stage of lactation) were recorded on each day of the experiment. A large array of descriptive temperature parameters (DTP) were extracted from every udder image including temperature-based (e.g., maximum, average and minimum USST), pixel count-based, and textural-based DTPs. Several different analytical methods were tested in an attempt to relate any given DTP to SCC; this included investigating the relationship between USST and the log transform of SCC (i.e., somatic cell score; SCS). The temperature range within each udder was also compared with the natural log of the range in SCC of the respective quarters. In a separate analysis, the temperature difference between each DTP and its respective daily baseline (i.e., average of the 5 lowest values of that DTP across the herd) was compared with SCS. Finally, the association between environmental and cow-related factors with each DTP was investigated to create prediction models for each DTP, the residuals of which were compared with SCC. Results from the present study indicate that the correlation between any DTP and SCS was weak (range of -0.16 to 0.19) and so could not be used to identify quarters with high SCC. Although some alternative measures had a significant relationship with SCS, again, the correlation was too weak for practical use on its own. Maximum and average USST could be predicted with a root mean square error of 0.23 and 0.35 °C, respectively, although the residuals from the prediction model could not be used to identify animals with high SCC. This suggests that infrared thermography alone could not be used as a real-time automated tool to detect high SCC for dairy cows in a pasture-based system.