Preanalytical Factors Affecting the Mean Platelet Volume: a Review

Mean platelet volume (MPV) as an indicator of platelet activation is a subject of many studies. Since the introduction of automated hematology analyzers, many authors described mean platelet volume as a marker for different pathologic conditions. It is also known that numerous preanalytical conditions could affect MPV results. Specimens are usually tested several hours after blood collection. There are some specific hematology analyzers which are in use by the majority of hematology laboratories. This review demonstrates some important aspects related to MPV measurement in routine laboratory

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques

Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process

Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction

RT-qPCR is the standard method for studying changes in relative transcript level in different experimental and clinical conditions and in different tissues. No validated reference genes have been reported for the normalization of transcript level in platelets. The very low level of platelet RNA and the elimination of leukocyte contamination represented special methodological difficulties. Our aims were to apply a simple technique to separate platelets for transcript level studies, and select the most stable reference genes for platelets from healthy individuals and from patients with the history of myocardial infarction. We developed a simple, straightforward method of platelet separation for RNA isolation. Platelet activation was inhibited by using acid-citrate-dextrose for anticoagulation and by prostaglandin E1. Leukocyte contamination was eliminated by three consecutive centrifugations. Samples prepared by this method were free of leukocytes, showed no inhibition in PCR reaction and no RNA degradation. The assay demands low blood volume, which complies with the requirements of everyday laboratory routine. Seventeen potential reference genes were investigated, but eight of them were excluded during optimization. The stability of the remaining genes, EEF2, EAR, ACTB, GAPDH, ANAPC5, OAZ1, HDGF, GNAS, and CFL1, were determined by four different descriptive statistics. GAPDH, GNAS, and ACTB were shown to be the most stable genes in platelets of healthy individuals, while HDGF, GNAS, and ACTB were the most stable in platelets of patients with the history of myocardial infarction. The results confirm that data normalization needs assessment of appropriate reference genes for a particular sample set

Characteristics of platelet count and size and diagnostic accuracy of mean platelet volume in patients with venous thromboembolism. A systematic review and meta-analysis

The purpose of this study was to evaluate the association between platelet (PLT) count and mean platelet volume (MPV) and venous thromboembolism (VTE). Thus, this study reviewed and performed a quantitative synthesis on data from the literature. This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. 18 studies were included in this paper. A random-effect meta-analysis was conducted for the assessment of heterogeneity using thrombosis place, type of analyzer, type of anticoagulant and incubation time of samples as covariates. A mixed-effect meta-regression was performed based on the subgroup for the whole samples using thrombosis place and method of measurement as moderators for MPV and PLT, respectively. The cumulative estimates and 95% confidence interval (95%CI) of specificity, sensitivity, area under the receiver operator characteristic curve (AUC) and diagnostic odds ratio (DOR) for MPV were calculated using a random effect model. The quality assessments were evaluated according to the quality assessment and diagnostic accuracy tool-2 (QUADAS-2). The primary outcome was the occurrence of VTE. Secondary outcomes included PLT and MPV. Patient with deep vein thrombosis is likely to have a higher value of MPV than control group (P < 0.001). The presence of pulmonary embolism (PE) had no significant effect on the standardized mean difference of MPV between patients and controls. Patients are likely to have less PLT than the control group regarding all studies. However, subgroup analysis demonstrated that this effect was significant for patients with PE (P < 0.05). The summary receiver operating characteristic (SROC) curve indicated that AUC was 0.745 (95% CI: 0.672–0.834). The DOR for MPV was 4.76 (95%CI: 2.3–9.85), with diagnostic accuracy of 0.66

High Neutrophil–Lymphocyte Ratio and Low Lymphocyte–Monocyte Ratio Combination after Thrombolysis Is a Potential Predictor of Poor Functional Outcome of Acute Ischemic Stroke

(1) Background: Ischemic stroke is one of the leading causes of death and disability. An inflammatory response is observed in multiple stages of cerebral ischemia, particularly in the acute phase. Recent publications revealed that the neutrophil–lymphocyte ratio (NLR) and lymphocyte–monocyte ratio (LMR) may be used to predict long-term prognosis in acute ischemic stroke (AIS) after thrombolysis. To test whether there is a relationship between the combination of these parameters and long-term prognosis, we analyzed the NLR–LMR combination in AIS patients treated with intravenous recombinant tissue plasminogen activator (rtPA); (2) Methods: The study included 285 adults with a diagnosis of AIS and rtPA treatment within a 4.5 h time window. Blood samples were obtained at admission and 24 h after thrombolysis to calculate pre- and post-thrombolysis NLR and LMR. Clinical data, including NIHSS was registered on admission and day 1. The long-term outcome was defined 90 days post-event by the modified Rankin Scale (mRS). Therapy-associated intracranial hemorrhage (ICH) was classified according to ECASS II. Receiver operating characteristic curve (ROC) analysis was performed to determine optimal cutoffs of NLR and LMR as predictors of therapy outcomes; (3) Results: Patients were stratified by cutoffs of 5.73 for NLR and 2.08 for LMR. The multivariate logistic regression model, including all possible confounders, displayed no significant association between NLR or LMR with 3-months functional prognosis. The combination of high NLR–low LMR vs. low NRL–high LMR as obtained 24 h after thrombolysis was found to be an independent predictor of poor 3-months functional outcome (mRS ≥ 2; OR 3.407, 95% CI 1.449 to 8.011, p = 0.005). The proportion of patients between low NLR–high LMR and high NLR–low LMR groups from admission to day 1 showed no significant change in the good outcome group. On the other hand, in the poor outcome group (mRS ≥ 2), low NLR–high LMR and high NLR–low LMR groups displayed a significant shift in patient proportions from 67% and 21% at admission (p = 0.001) to 36% and 49% at 24 h after thrombolysis (p < 0.001), respectively; (4) Conclusions: Our study demonstrated for the first time that a high NLR–low LMR combination as observed at 24 h after thrombolysis can serve as an independent predictor of 3-months poor outcome in AIS patients. This simple and readily available data may help clinicians to improve the prognostic estimation of patients and may provide guidance in selecting patients for personalized and intensified care post-thrombolysis