Evaluation of survival and functionality in frozen platelets

ABSTRACT Objective: Prolong platelet survival and functionality up to 28 days. Methods: A sample of apheresis platelets was evaluated, distributed in 3 groups according to the cryopreservative solution used: DMS05%+2%albumin; DMSO5%+NaC10,9% and DMS05%+Dextrose2%. They were then frozen at -80 °C and thawed at 7, 14 and 28 days. The in vitro survival and viability were assessed by the post-thaw platelet count and the CD41, CD61 and CD42a staining percentages by flow cytometry. The functionality was determined with the percentage of post-stimulation aggregation with INm-thrombin using the Chromo-Log490 aggregometer. The control group (CG) consisted of fresh platelets under constant agitation at 22 °C. Results: A total of 72 platelet aliquots was analyzed. The CG presented a platelet-count of 1934 ± 0.5 × 109/L and a 100% viability. The percentages of CD41, CD61 and CD42a labeling were 99, 98.5 and 96.5%, respectively. The percentage of aggregation was 99%. On day 7 of the post-freezing, the platelet count for groups 1, 2 and 3 was 1,844 ± 102, 1,856 ± 76 and 1,752 ± 226, with the viability of 98, 96 and 95%, respectively. On day 14, the counts were 1,722 ± 238, 1,649 ± 215 and 1,578 ± 223 with the viability of 96, 95 and 94% and, on day 28, they were 1,602 ± 374, 1,438.6 ± 429 and 1,406.6 ± 436, with the viability of 96, 94 and 93%, respectively. Groupl presented a higher expression of membrane antigens. Aggregation percentages were 90, 98 and 89% at day 7, 88%, 98 and 87% at day 14 and 84%, 95 and 82% at day of the 28 post-freezing, respectively, with group2 presenting the best results. Conclusion: The results support cryopreservation as a reasonable method to prolong platelet survival up to 28 days, maintaining its functionality and viability greater than 50%.

Evaluation of survival and functionality in frozen platelets.

OBJECTIVE: Prolong platelet survival and functionality up to 28 days. METHODS: A sample of apheresis platelets was evaluated, distributed in 3 groups according to the cryopreservative solution used: DMSO5%+2%albumin; DMSO5%+NaCl0,9% and DMSO5%+Dextrose2%. They were then frozen at -80 °C and thawed at 7, 14 and 28 days. The in vitro survival and viability were assessed by the post-thaw platelet count and the CD41, CD61 and CD42a staining percentages by flow cytometry. The functionality was determined with the percentage of post-stimulation aggregation with 1Nm-thrombin using the Chromo-Log490 aggregometer. The control group (CG) consisted of fresh platelets under constant agitation at 22 °C. RESULTS: A total of 72 platelet aliquots was analyzed. The CG presented a platelet-count of 1934 ± 0.5 × 109/L and a 100% viability. The percentages of CD41, CD61 and CD42a labeling were 99, 98.5 and 96.5%, respectively. The percentage of aggregation was 99%. On day 7 of the post-freezing, the platelet count for groups 1, 2 and 3 was 1,844 ± 102, 1,856 ± 76 and 1,752 ± 226, with the viability of 98, 96 and 95%, respectively. On day 14, the counts were 1,722 ± 238, 1,649 ± 215 and 1,578 ± 223 with the viability of 96, 95 and 94% and, on day 28, they were 1,602 ± 374, 1,438.6 ± 429 and 1,406.6 ± 436, with the viability of 96, 94 and 93%, respectively. Group1 presented a higher expression of membrane antigens. Aggregation percentages were 90, 98 and 89% at day 7, 88%, 98 and 87% at day 14 and 84%, 95 and 82% at day of the 28 post-freezing, respectively, with group2 presenting the best results. CONCLUSION: The results support cryopreservation as a reasonable method to prolong platelet survival up to 28 days, maintaining its functionality and viability greater than 50%.

Evaluación in vitro de la eficacia de plaquetas convencionales, atemperadas y congeladas. Posible empleo en el medio militar / In vitro evaluation of the efficacy of fresh, temperatured and frozen platelets. Possible employment in the military environment

Introducción: La hemorragia exanguinante es la primera causa de muerte prevenible del combatiente en los conflictos armados, convirtiendo así la posibilidad de transfundir hemocomponentes de manera precoz en una prestación absolutamente fundamental durante la asistencia sanitaria a las bajas generadas en las operaciones militares. A lo largo de los numerosos conflictos acontecidos durante el pasado siglo, se han producido importantes cambios en el tratamiento hemoterápico a las bajas junto con una evolución de la doctrina sanitaria respecto a este tema. En algunos protocolos de transfusión masiva se ha empleado la técnica diagnóstica de la tromboelastometría (TE). La TE es una prueba que muestra las propiedades viscoelásticas de la sangre desde la formación del coágulo hasta su fibrinólisis, evalúa la función plaquetaria y se correlaciona con el proceso fisiológico de la hemostasia de una forma rápida. El objetivo principal de este estudio es evaluar in vitro la capacidad hemostática de los diversos concentrados de plaquetas frías, congeladas y frescas mediante pruebas de coagulación estandarizadas y tromboelastometría, esclareciendo si se mejora significativamente la contribución al coágulo con los pool de plaquetas frías (conservadas a 4ºC), en comparación con las plaquetas frescas y congeladas. También se pretende determinar qué ventajas supondría la incorporación de las plaquetas frías en la medicina transfusional realizada en el medio militar. Material y método: Se diseñó un estudio experimental para comparar in vitro plaquetas frías (refrigeradas), congeladas y frescas (convencionales), analizando su rendimiento y efectividad mediante análisis sistemático de sangre, mecanismos de laboratorio de coagulación rutinarios (Tiempo de Protrombina, Actividad de Protrombina, Tiempo de Cefalina y Fibrinógeno) y Tromboelastometría rotacional (ROTEM)...(AU)

Introduction: Exsanguinating hemorrhage is the first preventable cause of death for combatants in armed conflicts, thus making the possibility of transfusing blood components early an absolutely essential benefit during health care for casualties generated in military operations. Throughout the numerous conflicts that have occurred during the past century, there have been important changes in the hemotherapy treatment of casualties along with an evolution of the health doctrine regarding this issue. In some massive transfusion protocols, the diagnostic technique of thromboelastometry (TE) has been used. TE is a test that shows the viscoelastic properties of blood from clot formation to fibrinolysis, evaluates platelet function and correlates quickly with the physiological process of hemostasis. The main objective of this study is the evaluation in vitro of the hemostatic capacity of the various cold, frozen and fresh platelet concentrates through standardized coagulation tests and thromboelastometry, clarifying whether the contribution to the clot is significantly improved with cold platelet pools (preserved at 4ºC), compared to fresh and frozen platelets. It is also intended to determine what advantages would be the incorporation of cold platelets in transfusion medicine performed in the military environment. Material and methods: An experimental study was designed to compare cold (refrigerated), frozen and fresh (conventional) platelets in vitro, analyzing their performance and effectiveness through systematic blood analysis, routine laboratory coagulation mechanisms (Prothrombin Time, Prothrombin Activity, Cephalin and Fibrinogen) and Rotational Thromboelastometry (ROTEM).A sample of 20 healthy patients was recruited, after informing them in writing and obtaining the mandatory informed consent, they donated 6 tubes with 10 ml citrate. of blood per patient...(AU)

Frozen Platelets-Development and Future Directions.

Storage requirements and outdating of platelets represent a continued challenge for blood banks. These hurdles are confounded for rural area hospitals or in military deployments. Over 60 years of research and development into frozen platelets have generated a stable and reproducible product. Valeri's method to freeze platelets in 6% dimethyl sulfoxide (DMSO) and storage at -80°C allows for long-term storage alleviating burdens placed on blood banks. Clinical studies show that frozen platelet transfusions are safe with no related thrombotic or other serious adverse events. There are ongoing efforts to demonstrate cryopreserved platelet (CPP) superiority in efficacy studies designed in trauma or cardiac surgery patients. Technical advances in CPP manufacturing including closed system manufacturing, applications of pathogen reduction technology and potency standard characterization add to the appeal of CPP as an alternative to traditional liquid-stored platelets (LP) in settings of supply shortages, mass casualty, active bleeding, rapid provision of HLA-compatible platelets, and remote care.

Emergency Supply Policy of Cryopreserved RBC and PLT: The Czech Republic Concept.

Supply of blood for urgent substitution is a strategic logistical problem for the military medical services across the world. The limited shelf life of blood- derived bioproductsin the liquid state and the need for special transport and use conditions, apart from donor and donations availability are among the causes for concern. To solve these problems many national health-care authorities implemented the national emergency blood crisis policy, to get a large amount of blood at any time at any place in the case of disaster, terrorist attack or war. The civil therapeutic problems in immunohematolgy cases can also be solved by stocks of fresh and cryopreserved homologous or autologous blood for patients with rare RBCs antigens or HLA / HPA platelet refractoriness with no chance to use common blood. The short shelf life of fresh platelets limits their efficient inventory management and availability during a massive transfusion protocol. Building an inventory of frozen blood components can mitigate the risk of insufficient availability. Since the beginning of the century in the Czech Republic, used, like other countries, the use of of cryopreserved blood-derived bioproducts has become the current method used to overcome the shortages of a timely supply. The Military University Hospital, Prague, and its bank of cryopreserved blood have been operating under this policy since 2006. There is currently a stock of frozen RBCs for military reserve, for a national blood crisis and, also, a stock of rare RBC units. For crisis management there are also stored, frozen PLTs, which are used in the treatment of heavily bleeding polytrauma patients. Both the containment and research development mitigation policy programs are in place for civil / military emergency situations. Even pathogen reduced frozen PLTs and frozen RBCs were successfully investigated for clinical use if demands arose. Currently, it is possible to meet operational demand while reducing the number of resupply transports and loss of products due to expiration. A lesson has been learned from the current containment, reseach and mitigation programs of efficient blood supply management with cryopreserved blood and blood derived bioproducts.

Optimizing the supply of whole blood-derived bioproducts through the combined implementation of cryopreservation and pathogen reduction technologies and practices: An overview.

The essential historical knowledge and expertise developed over the past 5-6 decades on the safety / efficacy of conventional blood components therapy by blood transfusion establishments have guided the development of validated methods which have ensure optimal safety margins for frozen blood and its bioproducts with or even without pathogen reduction. Newer generations of pathogen reduced frozen red blood cell, plasma and platelet products and the standardised and safer pooling of human platelet lysate are now become available for potential clinical use. These types of whole blood-derived bioproducts not only reduce the risk of transmission of range of pathogenic blood-borne pathogen. As cryopreservation can be combined with PRT without significantly compromising in vitro quality characteristics or physiological capabilities, it allows us to maximize the available inventory of these blood products in both civil and military trauma settings. The main objective of this overview is to update readers and scientific / medical communities of the various building blocks needed to optimally grantee the pathogen safety of whole blood-derived bioproducts, with minimal untoward events to the recipients. While this is an emerging area, we are seeing the numerous potential opportunities that cryopreservation and pathogen inactivation can have on the transfused patient outcomes. This manuscript is informed by recent publications on this topic.

Novel platelet products under development for the treatment of thrombocytopenia or acute hemorrhage.

Controlling hemorrhage has been a focus of survival since man recognized that the loss of blood led to death. Papyri from 1600 BCE describe methods for hemorrhage control including; direct pressure, ligature and the use of sutures. Multiple studies have demonstrated the survival advantage of early transfusion of whole blood or red cells and plasma. The added survival impact of early transfusion of platelets was recently reported in a substudy of the prospective Pragmatic, Randomized Optimal Platelet and Plasma Ratios (PROPPR) trial. Early transfusion of platelets demonstrated a statistically significant survival benefit at 24 h and 30 days post-injury. [1] Platelet availability is limited due to the short shelf life (5-7 days) and storage requirements (room temperature with constant agitation). Providing platelets or platelet derived products for prehospital treatment and to rural and some urban hospitals is an unmet medical need. The interest in novel and alternative platelet products has grown over the past decade and the status of novel platelet products is presented herein. Development, approval, and distribution of hemostatically effective approved platelet products for prehospital use and routine stockage in rural and urban centers could significantly increase survival rates in bleeding patients.

Frozen platelets.

The technical limitations on platelet shelf life and storage have driven research for alternatives, including cryopreservation of platelets. Over the past 60 years, product development has adopted freezing platelets in 6% dimethyl sulfoxide (DMSO) and storage in mechanical freezers at -80 °C for up to 2 years. Frozen platelets show a primed, hypercoagulable in vitro phenotype post-thaw when assayed using morphology, flow cytometry for marker expression, and thrombin capacity. In vivo studies show a role for frozen platelets in the maintenance of hemostasis and data from limited clinical trials show frozen platelets are safe and appear beneficial. As research continues to address the functional role of in vitro assays for clinical outcomes, frozen platelet product development represents a good alternative to room temperature platelets for many applications.

Inhibitory role of S-nitrosoglutathione in the aggregation of frozen platelets, and its effect on the expression of membrane glycoproteins.

The aim of this study was to explore the effects of S-nitrosoglutathione (GSNO) on the aggregation of frozen platelets, the platelet nitric oxide (NO) content and the expression of membrane glycoproteins. The level of frozen platelet aggregation was measured using a platelet aggregation analyzer, while the content of NO was measured by the nitrate reductase method and the expression of frozen platelet membrane glycoproteins was determined by flow cytometry. The level of frozen platelet aggregation was reduced from 35.47±2.93 to 24.43±3.07% following treatment with GSNO. The mean NO concentration in the 32 samples of frozen platelets treated with GSNO was 45.64±6.31 µmol/l, which was significantly higher compared with the concentration in the fresh liquid platelet group. There were no significant differences in the levels of PAC-1 in the fresh liquid platelet, frozen platelet and GSNO-treated frozen platelet groups; by contrast, significant differences were observed in the CD42b and CD62P levels. The platelet membrane glycoprotein expression levels in the frozen platelet and the GSNO-treated frozen platelet groups were not significantly different. The results of the study indicate that GSNO has potential as a cryoprotectant, due to its ability to increase the NO concentration in frozen platelets, inhibit platelet aggregation and maintain platelet function. It is likely that the molecular arrangement and structure of the frozen platelets were altered following GSNO treatment, or that the frozen platelets were affected by alternative mechanisms.

Flow cytometric human leukocyte antigen-B27 typing with stored samples for batch testing.

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: WE COMPARED FOUR STORAGE METHODS: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.

Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.

Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.