Mitochondrial DNA levels in plasma products: A comparative study of apheresis plasma from COVID-19 convalescent donors and apheresis and fresh frozen plasma from non-infected donors.

The purpose of our study was to determine prospectively relationships between plasma mitochondrial deoxyribonucleic acid (mtDNA) concentration and clinical outcome in patients with intra-abdominal infections (IAIs) induced by severe abdominal trauma. The DNA was isolated from serum samples taken from patients with IAIs at hospital days zero, one, and two. Plasma mtDNA concentration was assessed by real-time polymerase chain reaction (PCR). The study population's clinical and laboratory data were analyzed. The mtDNA damage-associated molecular patterns were expressed as a PCR threshold cycle number using four selected sequences. The patients with IAIs had significant higher plasma mtDNA than healthy control subjects. Patients with IAIs with sepsis apparently had elevated mtDNA levels compared with non-septic patients with IAIs (30.9 ± 2.0 vs. 28.7 ± 2.4; 33.3 ± 2.6 vs. 28.9 ± 2.4; 32.9 ± 1.6 vs. 31.2 ± 2.2; 33.1 ± 3.6 vs. 28.1 ± 2.2, respectively). Patients with IAIs in whom multiple organ dysfunction syndrome (MODS) developed also had increased mtDNA concentration compared with those who did not (31.0 ± 1.8 vs. 27.9 ± 1.8; 32.9 ± 2.4 vs. 27.8 ± 1.7; 32.9 ± 1.5 vs. 29.8 ± 1.7; 32.0 ± 3.8 vs. 27.1 ± 2.1, respectively). Baseline mtDNA concentration had high effectiveness in predicting death for patients with IAIs who had severe trauma using receiver operating characteristic analysis. Furthermore, serum mtDNA levels on admission correlated with the lactate concentration, but no significant correlations were found between mtDNA levels and levels of white blood cells, C-reactive protein, and procalcitonin. Plasma mtDNA was associated with the occurrence of sepsis, MODS, and death in patients with IAIs caused by severe abdominal trauma.

Thawing fresh-frozen plasma (FFP) may cause delay in delivery, and one approach to circumvent this is to store plasma at +4 degrees C. Thawed plasma is commonly discarded after a few days of storage, owing to the assumption that coagulation factor activity decreases to clinically unacceptable levels. Eighteen apheresis plasma (AP) units were collected from blood donors. The collected plasma was divided into two equal parts: one part frozen at -74 degrees C as FFP and one part stored at +4 degrees C as fresh liquid plasma (FLP). Thirty-nine units of whole blood (WB) were collected from blood donors and leukodepleted by inline filtration, followed by plasma separation. Twenty plasma units were frozen at -74 degrees C as FFP and 19 plasma units were stored at +4 degrees C as FLP for 28 days. Plasma aliquots were collected before freezing and immediately after thawing FFP and before and during storage of FLP at Days 14 and 28. Factor (F)V, FVIII, D-dimers, and C1-esterase inhibitor levels were assessed. No significant differences in coagulation factor levels were assessed between FLP prepared from AP and FLP prepared from WB. FV and FVIII levels decreased on average 25 and 50 percent, respectively, at Day 14 of storage. C1-esterase inhibitor and D-dimers levels were not affected. Leukodepleted apheresis and WB plasma stored for 14 days retain sufficient levels of FV and FVIII activity for maintenance of normal hemostasis and could therefore be considered useful in selected clinical situations.

Efficacy of Serotherapy on an N501Y Variant of SARS-CoV-2 in a Patient With Chronic Lymphocytic Leukemia.

Study question Do alterations in mitochondrial DNA levels in luteal granulosa cells (LGCs) affect the quality of mature oocytes in infertile women? Summary answer Alterations in the mitochondrial DNA levels in LGCs may modify the characteristics of the first polar body in oocytes and subsequently decrease their fertilization potential. What is known already Assisted reproductive treatment outcomes are closely linked to the oocyte quality as shown in several systematic reviews and meta-analyses. Particularly, the oocyte quality is highly affected by the surrounding luteal granulosa cells (LGCs). This is mainly due to the crucial role of the mitochondria in the LGCs in fueling the metabolic processes required for oocyte maturation. Therefore, modifications in the quality of LGCs may have direct effects on the developmental competence of oocytes. However, the exact mechanisms by which this could happen are not fully understood. Study design, size, duration A retrospective multicenter study was conducted on 303 mature oocytes retrieved from 51 women undergoing intracytoplasmic sperm injection (ICSI). It was conducted in Lebanon at Al Hadi IVF center and Azoury IVF clinic, between January 2019 and January 2020. G-Power 3.1 was used to determine the sample size for Generalized Linear Mixed Models through a one-sample t-test power analysis with alpha 0.05, power 0.8, medium effect size (f2 = 0.15), and 5 predictors. Resulting sample size: 43. Participants/materials, setting, methods This study excluded cases of premature ovarian failure, severe oligozoospermia (<2 x 102/ml), or cases using frozen gametes. Mature oocytes, from young women (< 36 years old), were injected and cultured in the Embryoscope where morphometric measurements were performed. LGCs vitality and mitochondrial DNA (mtDNA) levels were analyzed using trypan blue exclusion dye and next-generation sequencing, respectively. Possible associations between these independent variables and the size and integrity of the first polar body were evaluated. Main results and the role of chance The included women presented with primary infertility. Their mean age was 29.98 ± 5.5 years old and their mean body mass index was 23.33 ± 3.32 Kg/m2. 10% of the included women had polycystic ovary syndrome, and 48% were smokers. Regarding the LGCs parameters, a statistically significant negative correlation was found between the mtDNA levels in LGCs and their vitality percentage (r = −0.313, p = 0.029). Interestingly, the average size of the first polar body (PBI) was 346.67 µm. Here we found that the PBI size decreased by 15.05 units when the mtDNA level in LGCs increased by one unit above its average. In parallel, the median percentage of oocytes having a fragmented PBI was 33.33 (0-100). This percentage increased by 4.26% for every one unit increase in the mtDNA level in LGCs (p < 0.0001). In contrast, this percentage decreased by 0.86% for every unit increase in the percentage of LGCs vitality (p < 0.0001). Moreover, the mean percentage of fertilization rate was found to be 70.19 ± 26.7. A higher percentage of fragmented PBI (38.3%) was found among oocytes that did not fertilize compared to those that did successfully fertilize (24%) (p = 0.019). Limitations, reasons for caution This study only analyzed the effects of alterations in mtDNA levels in LGCs on oocytes. It would be worthwhile to also analyze the effects on foetal development and offspring health. Furthermore, it would be interesting to study the relationship between alterations in LGCs and oocyte quality within individual follicles. Wider implications of the findings Several studies have suggested that abnormal size and fragmentation of the PBI may impair embryo development. Therefore, treating factors that affect LGCs, such as obesity and polycystic ovary syndrome, may benefit infertile women. This highlights the need for new clinical trials in this context. Trial registration number Not applicable

Recruitment Strategy for Potential COVID-19 Convalescent Plasma Donors

Plasma can be manufactured by multiple methods. Few studies have compared quality parameters between plasma products that may affect efficacy and safety. Four different plasma products were analyzed to include fresh frozen plasma (FFP), liquid plasma (LP), solvent detergent plasma (SDP), and a spray-dried, solvent detergent-treated plasma (SD-SDP) at multiple time points of storage. Parameters measured included red blood cell, platelet, and white blood cell counts; microparticle phenotypes; thrombin generation; and thrombelastography. These parameters were compared in 10 samples of each product. SDP and SD-SDP contained the smallest number of residual cells compared with FFP and LP. Platelets were the most common residual cell in all products and were highest in LP. FFP contained the greatest number of residual red blood cells. Total microparticle counts were elevated in LP and FFP compared with SDP and SD-SDP. Cell-derived microparticles in both LP and FFP were mostly platelet in origin. Microparticle counts in SDP and SD-SDP were negligible. Thrombelastography results demonstrated similar thrombin, fibrinogen, and platelet function on Day 28 LP compared with Day 5 thawed FFP. Thrombin generation assays revealed that the total, lag time to, and peak thrombin formation were higher in SDP and SD-SDP compared with FFP and LP. All parameters in FFP and LP products were characterized by a large degree of variability. The differences in cellular, microparticle, and functional hemostatic parameters measured between plasma products have the potential to affect efficacy and safety. Further study is needed to elucidate the potential immune effects of the cellular and microparticle differences noted as well as the clinical implications of altered thrombin generation kinetics in SD products.

Background: Mitochondrial dysfunction is increasingly recognized as an important feature of multiple sclerosis (MS) pathology and may be relevant for clinical disease progression. However, it is unknown whether mitochondrial DNA (mtDNA) levels in the cerebrospinal fluid (CSF) associate with disease progression and therapeutic response. Objectives: To evaluate whether CSF concentrations of mtDNA in MS patients can serve as a marker of ongoing neuropathology and may be helpful to differentiate between MS disease subtypes. To explore the effect of disease-modifying therapies on mtDNA levels in the CSF. Methods: CSF mtDNA was measured using a digital polymerase chain reaction (PCR) CSF mtDNA in two independent MS cohorts. The cohorts included 92 relapsing-remitting multiple sclerosis (RRMS) patients, 40 progressive multiple sclerosis (PMS) patients (27 secondary progressive and 13 primary progressive), 50 various neurologic disease controls, and 5 healthy controls. Results: Patients with PMS showed a significant increase in CSF mtDNA compared to non-inflammatory neurologic disease controls. Patients with higher T2 lesion volumes and lower normalized brain volumes showed increased concentration of mtDNA. Patients treated with fingolimod had significantly lower mtDNA copy levels at follow-up compared to baseline. Conclusion: Our results showed a non-specific elevation of concentration of mtDNA in PMS patients. mtDNA concentrations respond to fingolimod and may be used to monitor biological effect of this treatment.

Transfusion‐related acute lung injury (TRALI) is one of the most serious complications of blood transfusion. We have been gathering information about TRALl in Japan since 1997 through our national haemovigilance system. Recently, we have 20–40 TRALI cases per year (five million bags issued per year). Although TRALI is a rare complication of transfusion, it may become severe and may sometimes be fatal. A substantial number of TRALI cases have been caused by donor HLA or HNA antibodies, which were generated in alloimmunized, i.e. parous females. Considering this mechanism, TRALI mitigation strategies such as the use of male‐only‐plasma have been implemented in many blood centres or countries. Because of the availability of male plasma and the time limit for the production of fresh frozen plasma (FFP), we first tried to prepare FFP‐LR2 derived from 400 ml whole blood only from male donors. FFP‐LR2 accounts for 75% of FFP in Japan. (Six per cent of FFP products are from 200 ml whole blood and 19% from apheresis plasma.) A preliminary project on male‐predominant plasma was started at several core blood centres. To minimize the burden on nurses regarding marking bags collected from males or females, which might lead to serious errors in collection sites, we updated the computer system that allows the staff members in the production department to sort male and female blood automatically. In a month after the system implementation nationwide, we successfully increased the production rate of male plasma for FFP‐LR2 product by up to more than 98%. Regarding FFP‐LR1 derived from 200 ml whole blood, the percentage of female donors is approximately 75%. One of the core blood centres has successfully implemented the same strategy for producing FFP‐LR1, but others have not done so yet. Although male‐only‐plasma strategies achieved good results without any expensive screening tests to reduce TRALI incidence in some countries, the standard measures for other plasma rich blood products such as platelet concentrates or apheresis plasma have not been established yet. There are some measures to consider reducing the risk of TRALI caused by other plasma‐rich products – recruiting only males as apheresis platelet donors, testing a certain number of donors for HLA antibodies, replacing the supernatant of a platelet pool with male plasma and excluding female donors from apheresis donations. Some blood centres adopted some of these measures, but so far there is little evidence for the efficacy of these measures for reducing the risk of TRALI. We have recently conducted research about the relationship between strength of HLA antibodies and TRALI development. On the basis of the results of the research, we can set the cut‐off level in HLA antibody tests, which can reduce the risk of TRALI by eliminating strong HLA antibodies that presumably cause TRALI development if the specificity of an antibody matches the patient’s antigen. Preliminary screening for donor HLA antibodies is ongoing in three core blood centres in Japan, results of which enable us to establish effective and feasible screening strategies for HLA antibodies in the near future.

The von Willebrand factor (VWF)-cleaving protease, ADAMTS13, is often deficient in cases of thrombotic thrombocytopenic purpura (TTP). The primary treatment of TTP is therapeutic plasma exchange (TPE) utilizing a variety of plasma products that help restore ADAMTS13 activity. However, multiple replacement products are available to choose from. Thawed plasma products have a variable refrigerated shelf life depending on the product type; stability of ADAMTS13 in thawed products stored at 1 to 6 degrees C has not been determined. ADAMTS13 activity was measured in three types of plasma products and cryoprecipitate. Fresh-frozen plasma (FFP) aliquots and cryoprecipitate-poor plasma (CPP) products were produced from 10 whole-blood (WB) donations. Twenty-four-hour plasma products were manufactured from 10 additional WB donations. ADAMTS13 activity in these products at time of thaw and after 5 days of storage at 1 to 6 degrees C was measured with a modified version of the FRETS-VWF73 fluorogenic assay. ADAMTS13 activity at time of thaw was measured in 10 units of cryoprecipitate and five related CPP products. ADAMTS13 is present in similar amounts in FFP, CPP, and 24-hour plasma products. Storage at 1 to 6 degrees C for up to 5 days did not significantly diminish ADAMTS13 activity. The concentration of ADAMTS13 in cryoprecipitate was significantly higher than that observed in plasma products. FFP, CPP, and 24-hour plasma products should be equally effective for ADAMTS13 restoration through TPE and should remain so for the duration of the shelf life of the thawed products.

Viral inactivation of fresh frozen plasma.

ObjectivePlasma nuclear and mitochondrial DNA (mtDNA) levels are altered in many diseases. However, it is not known whether they are also altered in acute myocardial infarction (AMI). In the present study, we examined plasma nuclear and mtDNA levels in the patients with AMI before and after a percutaneous coronary intervention (PCI) to explore their potential as biomarkers.Methods and resultsPlasma nuclear and mtDNA levels were measured by quantitative PCR in 25 AMI patients, 25 non-myocardial infarction (MI) control participants (with MI risk), and 20 healthy individuals during the study period. The concentrations of nuclear and mtDNA were significantly higher in the AMI group on hospital day 1 than that in the non-MI controls (nuclear: 0.4948±0.0830 vs. 0.2047±0.0222 ng/μl, P<0.05; mitochondrial: 3.754±0.384 vs. 1.851±0.3483 ng/μl, P<0.05) and healthy individuals (nuclear: 0.4948±0.0830 vs. 0.1683±0.0254 ng/μl, P=0.001; mitochondrial: 3.754±0.384 vs. 0.1517±0.0924 ng/μl, P<0.05) and decreased shortly after PCI.ConclusionBoth plasma nuclear and mtDNA levels are elevated in AMI patients, but return to normal levels immediately after PCI, suggesting that they are potentially novel biomarkers for AMI.

Supplementation of fibrinogen concentrate in children with severe acquired hypofibrinogenaemia during chemotherapy for acute lymphoblastic leukaemia: our experience.

Increased levels of cell-free mitochondrial DNA in the cerebrospinal fluid of patients with multiple sclerosis

Data published during the last decade are suggestive of a role for mitochondrial dysfunction in the pathogenesis of schizophrenia, bipolar disorder and other psychiatric diseases. In order to determine if the mitochondrial deficits reported in the literature are caused by abnormalities in the mitochondrial DNA of psychiatric patients, we quantified mitochondrial DNA (mtDNA) levels and the 5 kb common mitochondrial deletion (CD) in postmortem frontal cortex tissue. The mitochondrial CD and mtDNA levels were measured in tissue obtained from the frontal cortex (Brodmann Area 46) of 144 individuals (45 patients with schizophrenia, 40 patients with bipolar disorder, 44 controls, and 15 patients with major depression). These variables were measured using newly developed SYBR green and TaqMan real time PCR assays. Both the TaqMan and the SYBR green assays gave similar results. There was no statistically significant difference for the quantity of the common mitochondrial deletion between controls and patients. We also did not detect a difference in the mtDNA levels amongst the diagnosis groups. There were statistically significant differences for the evaluated parameters for smokers, schizophrenic patients on antipsychotic drugs at time of death, and bipolar patients with antidepressant use and alcohol abuse. Based on this study and other reports, we conclude that neither the common mitochondrial deletion nor changes in mitochondrial DNA levels are likely to account for the mitochondrial changes associated with bipolar disorder or schizophrenia. The effect of premortem agonal factors and medication on mitochondrial dysfunction still needs further elucidation.

Tissue mitochondrial DNA (mtDNA) levels have been proposed as a marker of nucleoside analouge reverse transcriptase inhibitor (NRTI) toxicity. However, clinical studies have yielded conflicting data regarding possible associations with mtDNA levels. This study examined mtDNA levels in matched samples of peripheral blood mononuclear cells (PBMCs) and subcutaneous fat from a large Australian cohort to examine treatment, clinical, and demographic associations with mtDNA depletion. mtDNA was quantified by real-time polymerase chain reaction. Results were compared across patient treatment and demographic details using linear mixed models. One hundred sixty-three PBMCs and 161 fat samples were available from 61 individuals. Current NRTI exposure was the major determinant of mtDNA levels. Both ddI (didanosine) and d4T (stavudine) exposures were associated with mtDNA depletion in fat (P < or = 0.0001 vs. those not on NRTIs). DdI exposure (P = 0.003), but not d4T exposure (P = 0.5), was associated with mtDNA depletion in PBMCs. No association between patient demographics or time on current therapy and mtDNA was observed. Current NRTI exposure is the major determinant of tissue mtDNA, but the precise determinants are tissue specific. Both ddI and d4T exposure are associated with fat mtDNA depletion, whereas ddI exposure was the only observed association with mtDNA depletion in PBMCs.