scholarly journals Thermolability of factor VIII in donor fresh frozen blood plasma

2021 ◽  
Vol 66 (4) ◽  
pp. 593-609
Author(s):  
V. N. Lemondzhava ◽  
A. V. Chechetkin ◽  
A. G. Gudkov ◽  
V. Yu. Leushin ◽  
A. D. Kasianov ◽  
...  

A criterion of the quality of fresh frozen blood plasma (FFP) is the activity of clotting factor VIII (FVIII).Aim — to identify technological barriers in the study of FVIII thermolability and to describe the requirements for experiments, providing new knowledge about the thermolability of this factor.Basic information. An analysis of domestic and foreign publications devoted to the study of the mechanisms responsible for reducing the value of FVIII activity in donor blood plasma from the moment of donation to the moment of transfusion was carried out. Data on the decrease in FVIII activity at various stages of work with blood plasma are presented. An analysis of methods for preparing samples for studying changes in the values of FVIII in donor blood plasma was performed. The existence of contradictory conclusions about the infl uence on the change in FVIII at the thawing stage of various values of the effects on FFP and poor knowledge of the change in the indicator at the stage of heating to the transfusion temperature after the end of the phase transition in the samples was established. The fundamental differences in the methods of preparing and conducting experiments in previous works are determined. Methods for increasing the reliability of experimental results for studying the thermal lability of FVIII are proposed.

Author(s):  
Kourosh Kabir ◽  
Hassan Hosseini ◽  
Mehdy Jahed Zargar ◽  
Zeynab Mandeh ◽  
Fatemeh Amrollahi ◽  
...  

Background and Aims: Hemophilia is a rare autoimmune disorder caused by autoantibodies directed in the majority of the cases against clotting factor VIII (FVIII). FVIII is extracted from human plasma or engineered from mammalian cell cultures using recombinant DNA technology. In Iran, most of the used FVIII is prepared from human plasma in Iranian Blood Transfusion Organization. It was seen important to estimate its stability and activity from bleeding time until product preparation.Material and Methods: In the analytical study, 60 healthy male donors (20-50 years old), 15 donors from each blood group, were selected after obtaining informed consent. Donors' blood was collected in QUADRI-PACKs and centrifuged after keeping at 24°C for 2 hours. The separated plasma was divided into three groups and incubated in the lab (22°C) for 0, 90, and 180 minutes, respectively. Then, samples were stored at -20°C for one month. Afterward, the plasma was thawed, and FVIII activity was assayed.Results: The activity of FVIII significantly (p<0.05) reduced by delay in freezing; after the time of 0 min: 134.84%±42, after 90 min: 126.88%±38, after 180 min: 120.22%±34. At all incubation times, the highest and the lowest FVIII activity were observed in A and O blood groups, respectively (p<0.05). FVIII activity was increased along with increasing age up to 35-40, but it decreased in subjects of 40-50 years old. These experiments confirmed that the longer the delay in freezing fresh frozen plasma, the greater the decrease in FVIII stability.Conclusions:. According to the results of this study, the best blood donors for FVIII product are those with blood group A in the age range of 40-35 years.


2020 ◽  
pp. 56-57
Author(s):  
V.L. Novak ◽  
B.O. Kondratsky ◽  
S.V. Primak ◽  
O.O. Tarasyuk ◽  
O.M. Tushnitsky ◽  
...  

Objective. Analysis of issues related to the safety and quality of donated blood and its components. Materials and methods. Many years of experience of hematologists, immunologists, isoserologists, morphologists, biochemists in studying the composition, morpho-functional properties of cells and blood plasma, the use of donor blood and its components in clinical practice have made it possible to reconsider method of chemotherapy “multilateral action”. Results and discussion. One of the main axioms of modern transfusion medicine: chemotherapy should be performed strictly according to the indications and those blood components that are needed to ensure the viability of the body. The development of transfusiology has proved, with few exceptions, the inexpediency and even harmfulness of the use of whole donor blood. When using blood and its components, it is necessary to clearly consider extremely important point: the use will be in a planned manner, or in special circumstances. Blood components and blood plasma preparations have unique medicinal properties and there is currently no alternative to them. Each country is recommended to switch to self-sufficiency of blood components and their derivatives, to organize their own production structures that would meet the country’s domestic needs in blood components and preparations. The main components of the ideology of component chemotherapy are: recovery of blood component deficiency is not achieved on a “drop by drop” basis; no need to completely replace the existing deficiency of a cellular or protein component. Transfusions of blood components should be treated as a responsible invasive medical procedure – an operation that can have both immediate and long-term complications and consequences. Unreasonable transfusions of whole canned blood, especially after long periods of storage, are not only ineffective, but often pose a danger. In canned blood, during storage, complex biochemical metabolic processes take place both in cells and in plasma, which ultimately reduce the quality of both the blood itself and the morpho-functional properties of its individual components. Thus, 8-day storage of erythrocytes is the threshold after which erythrocytes begin to adversely affect the patient. Morpho-functional properties of blood components are directly dependent on the shelf life and distance of transportation. During the storage of blood and erythrocyte mass, the level of ATP decreases, on which the elasticity of the erythrocyte membrane depends. The magnitude of the negative electrical charge of the surface membrane of blood cells decreases. The number of prehemolytic forms of erythrocytes and cells that are not capable of reverse transformation increases. Within 1-4 days, leukocytes die, bacteria are released, so after this period, the greatest number of complications and reactions. Leukocytes and platelets form microaggregates at an early stage of storage, which can cause microembolism and distress syndrome. Microclots are formed, which include lysed blood cells and fibrin. The number of microclots increases every day, reaching on day 21 to 100 thousand/ml, so when transfusing it is advisable to use antiplatelet filters, rather than leukocyte. The use of bed leukofilters after a long period of storage of erythromass is not advisable, because there are no leukocytes. In addition, up to 2 % of erythrocytes are lost. The pH decreases, the content of 2,3-DFG, which is responsible for oxygen transport function (decreases by 50 % on the third day), hemolysis increases (up to 200 mg% of free hemoglobin). The concentration of potassium and ammonium ions increases. The recommended threshold for erythrocyte concentrate transfusions is a hemoglobin level of less than 70 g/l in adults and most children. It is important to use fresh-frozen plasma, erythrocyte concentrate and platelets obtained from one donor. Modern blood separators technically provide such an opportunity. Conditionally acceptable number of platelets in patients with injuries is more than 50×109/l cells, and in patients with combined brain injury is 100×109/l. Platelet concentrate obtained by the manual method from 4-5 donors leads to the development of refractoriness. With increasing shelf life of platelets, the functional properties of cells deteriorate significantly. All attempts to create the so-called artificial blood in the 19th century ended at the level of scientific developments. Synthetic and bioengineered cellular components of blood, hematopoietic factors, as well as hematopoietic stem cells are considered promising in the future. One of the possible ways to solve the problem of long-term storage of blood components, especially liquid groups, is cryopreservation of individual cells (erythrocytes) at moderately low (-20; -40; -80 ºС) and ultra-low (-165-196 ºС) temperatures, followed by deglycerization (washing), the use of special solutions for resuspension. With the development of low-temperature electric refrigeration equipment in Ukraine, this has become a reality. Conclusions. Practice has shown that both the blood itself and its components can neither be produced nor extracted as minerals, it can only be shared. All of the above indicates that blood donation is and will remain the main source of cellular components of the blood for at least the next decades.


2019 ◽  
Vol 12 (4) ◽  
pp. e227974
Author(s):  
Antonio Liras ◽  
Luis Romeu

Haemophilia is a hereditary X-linked recessive disorder caused by a deficiency of either clotting factor VIII (haemophilia A) or IX (haemophilia B). Conventional treatment is currently based on the use of either plasma derived or recombinant coagulation factors. This paper reports on the case of a patient with severe haemophilia who presented with mesial decay and interproximal tartar build-up, for which extraction and scaling to remove tartar deposits were indicated. Following extraction, the usual haemostasis techniques were applied, and postoperative prophylactic antihaemophilic treatment was indicated for 2 or 3 days. The patient presented with moderate bleeding for a few minutes immediately after the procedure. Administration of factor VIII before surgery as well as the patient’s favourable pharmacokinetic response allowed for an optimal result. This treatment has afforded patients with haemophilia a better quality of life, and safe and efficient access to invasive surgical procedures.


2016 ◽  
Vol 62 (06/2016) ◽  
Author(s):  
Piotr Kuta ◽  
Barbara Hauck-Dlimi ◽  
Julian Strobel ◽  
Robert Zimmermann ◽  
Reinhold Eckstein

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2039-2039
Author(s):  
Shuxian Song ◽  
James Harrang ◽  
Bryn Smith ◽  
Carol H. Miao

Abstract Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 285-289 ◽  
Author(s):  
VS Blanchette ◽  
E Vorstman ◽  
A Shore ◽  
E Wang ◽  
M Petric ◽  
...  

Abstract Antibodies to hepatitis C virus (anti-HCV) were quantitated in stored sera from selected groups of hemophilic children (less than or equal to 18 years of age). During the period 1987 to 1989, seropositivity rates were as follows: untransfused hemophiliacs 0% (0 of 11 cases), hemophiliacs treated exclusively with vapor-heated factor VIII or IX concentrates 0% (0 of 9 cases), hemophiliacs treated only with cryoprecipitate or single donor blood products 0% (0 of 9 cases), and hemophiliacs regularly treated with unheated or dry heat-treated factor VIII or IX concentrates 95% (21 of 22 cases). Corresponding alanine aminotransferase (ALT) results were similar: values were always below the upper limit of laboratory normal (40 U/L) in untransfused hemophiliacs, hemophiliacs treated with vapor-heated factor concentrates, or those who received only cryoprecipitate or single donor blood products. By contrast ALT values were greater than 40 U/L in 82% (18 of 22 cases) of hemophilic children regularly infused with unheated or dry heat-treated factor concentrates. Three conclusions are drawn from this data: (1) HCV is a major cause of chronic hepatitis in multitransfused hemophilic children, (2) unheated and dry heat-treated clotting factor concentrates carry a very high risk of transmitting HCV infection, and (3) clotting factor concentrates inactivated by vapor heating carry a very low and perhaps zero risk of transmitting HCV infection. These findings are of therapeutic significance for previously untransfused hemophiliacs susceptible to HCV infection.


2021 ◽  
Vol 273 ◽  
pp. 02031
Author(s):  
Natalia Kovalyuk ◽  
Natalia Basova ◽  
Mikhail Staroselov ◽  
Ludmila Yakusheva ◽  
Julia Shakhnazarova ◽  
...  

As a result of the study, new knowledge was obtained for the first time about the effect of the FXI locus polymorphism on the quality of reproduction in dairy cattle, the degree of influence of the FXI polymorphism on the frequency of development of pathologies of the reproductive organs, their clinical manifestation in individuals - carriers of the anomaly and healthy animals was established. The influence of the FXI locus polymorphism on embryonic and postembryonic mortality, clinical condition and safety of newborn calves in comparison with individuals without a genetic defect was determined.


2021 ◽  
Author(s):  
Michela Milani ◽  
Cesare Canepari ◽  
Tongyao Liu ◽  
Mauro Biffi ◽  
Fabio Russo ◽  
...  

Abstract Liver gene therapy with adeno-associated viral (AAV) vectors delivering a clotting factor transgene into hepatocytes has shown multi-year therapeutic benefit in adults with hemophilia. However, anti-AAV pre-existing immunity and the mostly episomal nature of AAV vectors, currently challenges application of AAV-vector mediated liver gene therapy to people with anti-AAV neutralizing antibodies and young pediatric patients. We have developed lentiviral vectors (LV), which integrate in the host cell genome, which achieve stable and efficient liver gene transfer in mice, dogs and non-human primates (NHP), by intravenous (i.v.) delivery. Here we show long-term coagulation factor VIII (FVIII) activity and restoration of hemostasis, by LV i.v. gene therapy to newborn and adult hemophilia A mice and normal-range FVIII activity in NHP, paving the way for potential clinical application.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4234-4234
Author(s):  
Hande Kizilocak ◽  
Elizabeth Marquez-Casas ◽  
Joshua Brown ◽  
Jemily Malvar ◽  
Guy Young

Abstract Introduction Emicizumab is a recombinant, humanized bispecific monoclonal antibody that mimics the function of factor VIII (FVIII) which results in a significant reduction in the annualized bleeding rate in patients with hemophilia A (HA), however, the degree with which emicizumab corrects the coagulation defect remains unclear. The objective of this study was to compare the current available laboratory methods in clinical practice; one-stage clotting factor assays (OSCA), bovine and human chromogenic FVIII activity (bovCHR and humCHR, respectively) and FVIII Equivalency of Emicizumab by Thrombin Generation (F8EmT). Aims The aim of this study is to address the differences of FVIII activity with different techniques in patients with severe HA with inhibitors on emicizumab. Materials and Methods Factor VIII levels are determined with an activated partial thromboplastin time (aPTT), OSCA using SynthASil on the ACL TOP 500 (Instrumentation Laboratory, Bedford, MA). Factor VIII activity is also determined photometrically via the Chromogenix Coatest® SP4 FVIII chromogenic assay kit (bovCHR, Diapharma Group, West Chester, OH) and the Biophen FVIII:C chromogenic assay kit (humCHR, Aniara Diagnostica, West Chester, OH). For F8EmT, linear regression was utilized to model the FVIII levels as a function of the endogenous thrombin potential (ETP) and peak thrombin values for patients with mild/moderate hemophilia. Then, we used the ETP and peak thrombin results of the severe HA patients on emicizumab with the calibration curve to calculate their F8EmT. Association between patient weight and their F8EmT were also examined and evaluated by linear regression. Results Data is presented for eight patients with severe HA with inhibitors on emicizumab in the non-bleeding state (Table-1). All patients' FVIII levels measured with OSCA are in or above the normal range (94.0-289.1). Bovine chromogenic FVIII activity is in the severe hemophilia range for five out of eight patients, for the rest it is in the moderate hemophilia range. Human chromogenic FVIII activity ranged between 12.5-49.8%. Factor VIII Equivalency of Emicizumab by Thrombin Generation is either in the mild hemophilia or normal range in all participants of the study. Conclusion One-stage clotting factor assays demonstrated falsely high results as expected since it is activated partial thromboplastin time based. Bovine chromogenic FVIII activity results were consistent with the severe HA range of the patients though a few had results slightly above that level. Previous literature has stated that the humCHR in patients on emicizumab results in FVIII levels of ∼30% when emicizumab is at its therapeutic concentration (∼50 mcg/ml). This study also demonstrated similar results with 5/8 patients having levels 30-50%. F8EMT levels were mostly consistent with the humCHR. In conclusion, understanding the degree to which emicizumab corrects the coagulation defect of is an important goal as it has clinical implications.Certainly, additional studies with higher participant numbers are needed to confirm these findings. Figure 1 Figure 1. Disclosures Young: Apcintex, BioMarin, Genentech/Roche, Grifols, Novo Nordisk, Pfizer, Rani, Sanofi Genzyme, Spark, Takeda, and UniQure: Consultancy; Genentech/Roche, Grifols, and Takeda: Research Funding.


Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 285-289
Author(s):  
VS Blanchette ◽  
E Vorstman ◽  
A Shore ◽  
E Wang ◽  
M Petric ◽  
...  

Antibodies to hepatitis C virus (anti-HCV) were quantitated in stored sera from selected groups of hemophilic children (less than or equal to 18 years of age). During the period 1987 to 1989, seropositivity rates were as follows: untransfused hemophiliacs 0% (0 of 11 cases), hemophiliacs treated exclusively with vapor-heated factor VIII or IX concentrates 0% (0 of 9 cases), hemophiliacs treated only with cryoprecipitate or single donor blood products 0% (0 of 9 cases), and hemophiliacs regularly treated with unheated or dry heat-treated factor VIII or IX concentrates 95% (21 of 22 cases). Corresponding alanine aminotransferase (ALT) results were similar: values were always below the upper limit of laboratory normal (40 U/L) in untransfused hemophiliacs, hemophiliacs treated with vapor-heated factor concentrates, or those who received only cryoprecipitate or single donor blood products. By contrast ALT values were greater than 40 U/L in 82% (18 of 22 cases) of hemophilic children regularly infused with unheated or dry heat-treated factor concentrates. Three conclusions are drawn from this data: (1) HCV is a major cause of chronic hepatitis in multitransfused hemophilic children, (2) unheated and dry heat-treated clotting factor concentrates carry a very high risk of transmitting HCV infection, and (3) clotting factor concentrates inactivated by vapor heating carry a very low and perhaps zero risk of transmitting HCV infection. These findings are of therapeutic significance for previously untransfused hemophiliacs susceptible to HCV infection.


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