In vitro evaluation of pathogen inactivated platelet quality: An 8 year experience of routine use in Galicia, Spain

BACKGROUND: Platelet concentrates (PCs) treated by the pathogen inactivation technology (PI) using amotosalen and UVA illumination (PI-PCs) can be manufactured in additive solutions (PAS-III and PAS-IIIM) or in 100% Plasma. Quality control (QC) is an integral part of the production. We capitalized on our ongoing QC program to capture 8 years-worth of data on parameters related to the quality of 116,214 PI-PCs produced under different manufacturing methods. MATERIALS AND METHODS: Selected in vitro parameters of metabolism, activation, and storage were analyzed for the different manufacturing periods to compare PI-PCs versus conventional PCs (C-PCs) resuspended in different PAS. RESULTS AND DISCUSSION: All BC-PCs met quality standards for pH and dose and residual leucocytes. As expected, storage time correlated with increased lactate, LDH, Annexin V, CD62, sCD40 L levels and decreased glucose and pH. With PAS-IIIM, higher levels of glucose were observed toward the end of shelf life (p < 0.0001) with lower platelet activation markers Annexin V (p = 0.038) and CD62 (p = 0.0006). Following PI implementation, a low expire rate of <0.5% was observed. While a 2.3% mean increase in the production of PCs occurred from 2011 to 2015, the distribution of red blood cell concentrates dropped by 4.4%. A mean incidence of 0.14% for transfusion-related adverse reaction was observed while PI-PCs were distributed, similar to the one observed with C-PCs. Overall, PI-PCs prepared in additive solutions consistently met quality standards. Those prepared in PAS-IIIM appeared to have better retention of in vitro characteristics compared to PAS-III though all demonstrated functionality and clinical effectiveness

Treatment of buffy coat platelets in platelet additive solution with the mirasol(®) pathogen reduction technology system

BACKGROUND: The Mirasol pathogen reduction technology (PRT) system uses riboflavin and ultraviolet light and is currently approved and used in Europe for the treatment of platelets and plasma. Mirasol treatment is intended to reduce the infectious pathogen load and to inactivate leukocytes in blood products. Our objective was to evaluate buffy coat platelet concentrates (BCPCs) prepared with platelet additive solution (PAS) and treated with the Mirasol system and to examine the effects on platelet cell quality during storage. METHODS: 26 BCPCs were prepared and split, creating 13 paired control and test units. The test units were treated with the Mirasol system and the platelet quality was assessed in all units over 7 days of storage. RESULTS: All products met the incoming specifications for Mirasol treatment, and the pH of all Mirasol-treated BCPCs in PAS met the requirements of the Council of Europe guidelines throughout storage. Analysis of lactate production and glucose consumption rates, CD62p expression and cytokines indicates enhanced cellular metabolism in treated platelets, but the levels were within previously published ranges. CONCLUSION: While Mirasol-treated BCPCs in PAS had increased metabolism and activation compared to controls, the results indicate that these units can be stored for 7 days with acceptable cell quality

Irradiación de componentes sanguíneos

La enfermedad del injerto contra el huésped asociada a transfusión (EICH-AT) es una complicación postransfusional infrecuente, a menudo fatal. Sucede cuando linfocitos T viables del donante (D) injertan y proliferan en el receptor (R), y éste es incapaz de rechazarlos. El riesgo de cada transfusión depende del número y viabilidad de los linfocitos (L) transfundidos, la competencia del sistema inmunológico del R y el grado de homología antigénica HLA entre D y R. Todos los componentes sanguíneos (CS) que contengan L viables (generalmente >105) pueden potencialmente provocar EICH-AT, como los hematíes, plaquetas y granulocitos, y la sangre total. No hay descritos casos con componentes congelados. La EICH-AT se manifiesta casi siempre con fiebre, así como exantema maculopapular, complicaciones gastrointestinales y/o aplasia medular. El riesgo de EICH-AT es mayor en pacientes con inmunodeficiencias congénitas o adquiridas, o con enfermedades oncohematológicas, en los que la inmunidad se afecta por la propia enfermedad o por tratamientos como radioterapia, análogos de las purinas (fludarabina, deoxycoformicina y otros) o alemtuzumab (anti-CD52). Los neonatos prematuros y los que reciben transfusión intrauterina o exanguinotransfusión, muestran un mayor riesgo de EICH-AT. También hay pacientes inmunocompetentes de riesgo, por transfusión procedente de D homocigotos para uno de los haplotipos del sistema HLA del R. Esta situación se da en la población con cierta homogeneidad en el sistema HLA y también en las transfusiones de D familiares. La irradiación gamma de los L-T viables de los CS es la clave para la prevención de la EICH-AT. La Guía del Consejo de Europa aconseja la aplicación de una dosis de 25 a 50 Gy, lo cual inhibe la proliferación de L sin afectar la integridad de los CS. Los CS irradiados deben ser monitorizados con una etiqueta radiosensible u otro sistema que indique la exposición a rayos γ. Se recomienda irradiar los hematíes en los primeros 14 días tras la extracción y conservarlos un máximo de 28 días. La irradiación produce aumento del potasio extracelular durante las primeras horas, por lo que, en neonatos, es aconsejable transfundir los hematíes irradiados antes de 24 horas. Las diferentes sociedades de transfusión actualizan periódicamente las indicaciones clínicas de irradiación de CS, intentando establecer criterios uniformes basados en la experiencia y en los trabajos publicados recientemente (ver tabla con un resumen de indicaciones actuales). Los rayos X constituyen una alternativa a la irradiación γ, aceptada desde 2011 en el Reino Unido. Ofrecen las ventajas de usar una fuente no radiactiva, con menos requerimientos legales, coste, entrenamiento y medidas de protección, y menores consecuencias en caso de mal uso. Los pacientes que requieren CS irradiados deben tener una alerta en los registros de su historia clínica, del laboratorio y de enfermería. En los últimos años se han introducido tecnologías de reducción de patógenos (RP) para plaquetas, como Intercept®(amotosaleno+UVA) y Mirasol® (Riboflavina+UV), que inhiben la replicación de ácidos nucleicos. Reducen más de 5 log10 los L-T e inhiben casi completamente la síntesis de citocinas. Son relevantes en la prevención de la EICH-AT, como una alternativa clara a la irradiación γ. Publicaciones recientes sobre el seguimiento de pacientes transfundidos con plaquetas tratadas (RP) y sin irradiar, no registran ningún caso de EICH-AT. Indicaciones de irradiación de CS aceptadas 1) Donaciones dirigidas (familiares de 1er ó 2º grado), incluso si el R es inmunocompetente 2) Transfusión de CS HLA idénticos y/o compatibles 3) Transfusión de granulocitos 4) Transfusión intraútero y exanguinotransfusión 5) Síndromes de inmunodeficiencia celular congénita 6) Trasplante de células progenitoras hematopoyeticas, alogénico y autólogo 7) Linfoma de Hodgkin 8) Pacientes a tratamiento con análogos de las purinas y/o alemtuzumab (anti CD52) Posibles indicaciones 1) Recién nacido prematuro o de bajo peso (≤ 1500g) 2) Todos los recién nacidos 3) Linfoma no Hodgkin 4) Neoplasias de células T 5) Altas dosis de quimioterapia y/o radioterapia, que causen linfopenia <0.5x109 /L 6) Anemia aplásica con tratamiento inmunosupresor 7) Transfusión masiva (microquimerismo) No indicaciones 1) SIDA (si ninguno de los puntos anteriores se puede aplicar) 2) Síndromes de inmunodeficiencia humoral congénita 3) Trasplante de órganos sólidos, a no ser que las circunstancias clínicas lo aconsejen Dada la ausencia de efectos nocivos por la irradiación y la gravedad de la EICH-AT, en caso de duda es prudente irradiar los C

Analysis of Blood Components and Usage in the Region of Galicia

Background/Case Studies: The distribution section of the Transfusion Center of Galicia (CTG) distributed daily blood components (CB) to the dif-ferent transfusion services of the 34 hospitals in the Galician health care network. The actual donation rate in our region is 41.5 units per thousand inhabitants per year. We have analyzed the evolution of the donation, dis-tribution and usage of CB in the region of Galicia during the period 2001-2010. Study Design/Methods: Population data were obtained from the National Institute of Statistics (www.ine.es), the number of donations and the number of CB distributed were obtained from computer systems e-Del-phyn and NetBank. The units transfused are reported by different hospitals in the annual statistics. Transfusion rates of each CB were defi ned as number units per thousand inhabitants per year. The units of FFP (fresh frozen plasma) are treated with methylene blue. The platelet concentrates (PCs) from apheresis and from standard donation have been treated by Intercept since March 2008. Results/Findings: From 2001 to 2010 the population increased by only 1 percent and the number of donations has increased by 5.3%. During the study period, the supply of RBC (red blood cells) increased by 9.7% and the supply of PCs increased by 62.4%. The causes of this increase are largely associated with tumor diseases, cardio-vascular surgery, hematological disorders including allogeneic hematopoi-etic progenitor cell transplant, as well as ageing population. However, the provision of FFP was reduced by 24.7%. Transfusion data are shown with more detail in the attached Table. From 2001 to 2006 we do not have reg-isters of transfused PCs. Conclusion: Data show an increase in demand for RBC and PCs, to remain constant or decrease FFP needs. There is variability in the use of CB among different regions of the country and with other Western countries with health systems similar to ours. Further studies are needed to analyze the variables that infl uence the donation and usage in order to apply corrective measures to ensure its sustainabilit

Can the range of pH be a predictive value in the quality of platelet concentrates?

Background: We can evaluate in vitro quality of platelet concentrates (PC) by mea-suring different parameters to know what in vitro tests, best determine platelet quality. The pH is one of these parameters and is easy to measure. In 2010 an article was published that described a rating system to evaluate the quality of PC (Van der Meer etal. Vox Sang 2010; 98:517–524). Following up on their work, we studied if the pH ofPC on day 5 of storage can give a predictive value for the quality PC on day 7. Material and methods: In this study we included only PC from buffy coat (BC). A PC-pool was produced from 5 BC isogroup and processed by Orbisac*, the platelets weresuspended in additive solution (AS) in a ratio 35:65 (plasma:AS). In group A) 25 PCs were produced, the AS added was SSP plus. In group B) 14 PCs were treated with pathogen reduction technology (PRT), Mirasol* (riboflavina+UV light, AS: SSP plus). In group C) 22 PCs were treated with PRT Intercept* (amotosalen + UVA light,AS: InterSol), this last group represents our daily routine . All PCS were stored on a flatbed shaker at 22°C. Samples were taken at days 2, 5, 7 and 9 for in vitro test: pH (22°C), CD62p expression, Annexin V binding, swirl, LDH, glucose and lactate concentration. Results: We took into account three parameters (CD62p, annexin V, and lactate concentration), the score of these were combined into one rating value (6 = goodquality, 0 = poor quality). In general PCs in SSP plus had a good score on day 7, 68%of PCs had rating value‡5 and 100% of PCs‡3. When PCs are treated with Mirasol* PRT, using the same AS, the rating value 3, was reached in 50% of PCs on day 7. PCs treated with Intercept* PRT and AS: InterSol, had the rating value 3, in 64% of PCs onday 7. We group together pH ranges on day 5, for three group and analized CD62p, Annexin V and lactate on day 7, this gave a numeric score, rating value. The attached table shows the percentages of PCs within the specified pH ranges on day 5 that meetthe requirements on day 7. All PCs showed ‘‘swirling’’ at the end of storage (swirl‡2). Inour current practice swirl are checked after PCs preparation and the time of issue, when we found PCs with poor swirl (score 1), the value of pH is £6.8 Conclusion: In our experience the AS: SSP plus gives a good metabolic and functional parameters during storage. When PRT was applied there are more activation and lactateproduction in PCs. We can say a pH‡7.1 on day 5, generally provides PCs with acceptable quality on day 7. More studies are needed to check other tests, that could beuseful in a rating syste

Annexin V binding during storage of platelet concentrate from buffy coat, after patogen reduction treatments

Background:Pathogen reduction technology (PRT) is important for limiting transfusion transmitted infections. Intercept* treated platelet concentrates (PC) have been implemented in our centre since 2008, and other systems have been evaluated. Aim:To determine if PRT for PC increase cellular injury or apoptosis during storage, Annexin V was measured after three PRTs. Methods: Platelet concentrates from buffy coat (BC-PC) were obtained by pooling 5BC from standard whole blood donations, after PRT was applied. With Intercept* (amotosalen+UVA light) 12 BC-PC were studied, using Intersol as additive solution(AS). Thirteen BC-PC were evaluated using MirasolÒ(Riboflavin + UV light). TwelveBC-PC were evaluated using Teraflex* (UVC light). In both of these the AS used wasSSP plus. In all PRTs the ratio plasma: AS was approximately 35:65. All PCs were storedat 22°C until day 9 after donation. In vitro parameters tested on days 2, 5, 7 and 9 wereswirling, MPV, pH, CD62p, glucose, lactate and LDH. Annexin V binding was measuredby FC (labeled with FITC) to determine the cellular injury during storage. Correlationbetween values of Annexin V (in three PRTs) and other parameters, was performedusing Spearman test correlation. Results: Annexin V as an apoptotic marker was correlationated with other plateletmetabolic and functional parameters tested. The percentage of platelets binding ann-exin V tended to be lower in PCs treated with InterceptÒand Teraflex*. In general thecorrelation was weak with all parameters tested, but some tests showed good corre-lation (r‡0.60) like MVP, CD62p, glucose, LDH in different days of storage. When PCs were evaluated on day 7 of storage: (i) in Intercept* units no correlation was shown, (ii) in Mirasol* units good correlation (r‡0.71) was found with MVP and glucose (iii) inTeraflex* units, correlation (=0.50) was only detected with LDH. During storage, Annexin V binding of all units increased steadily. But Intercept* and Teraflex* PCsremained comparable to control units (previous study). Mirasol* PCs had higher valuesduring terminal storage. Platelet activation and apoptosis are evident from day 5 ofstorage but they show a degree of overlap as we can see. Swirling scores were goodthroughout the 7 days of storage. Residual leucocytes by FC were in all PC <1·106.Conclusion:The correlation between Annexin V binding and other parameters wasbetween weak and intermediate. Mirasol* units exhibited the highest values for Annexin V on day 7 of storage. During storage all units showed a steady increase in Annexin V although there were differences in PRTs and it could be due to specificprocess of PRTs and storage media. The question whether markers of platelets apoptosiswill be relevant for clinical efficacy should be determined in future studies

Two Automatic Methods to Prepare Buffy Coat Platelet Concentrates: Evaluation of Impact on the Organization of Production in a Transfusion Centre

Background/Case Studies: Basically, the automatic techniques OrbiSac® (Caridian BCT) and TACSI® (Terumo) perform in one shift many manual operations: centrifugation, separation and leucoreduction by fi ltration of the buffy coat (BC pool). Last year we produced 50% of the platelet concentrate (PC) production from BC (7,650 BC-PC per year).We tried to evaluate some special conditions of the two systems and the impact on the organization of the laboratory. Study Design/Methods: We routinely use the Orbisac® equipment, but we prepared more than 500 BC-PC by TACSI®. Whole blood (WB) units were collected on day 0 and stored overnight at 22°C. On day 1, WB was processed to obtain RCC, FFP and BC. The BC units had a mean volume of 52.2 ± 3.5 ml; mean hematocrit 35.2 ± 6.1%. We have 4 Orbisac® devices at our disposition and 1 TACSI® device.The main steps in the preparation of the BC-pool are:a) The BCs must remain without agitation at 22 ± 2°C, at least 2 hours.b) Assign a number to a pool made up of 5 BC (entered into a computer).c) Five BC ABO isogroup were connected by sterile sealing (TSC-II, Terumo®) to processing set and 280 ml of InterSol (AS) bag.d) Manual rinsing of BCs with AS.e) BC-Pools were packed and placed in the automatic devices.All steps were executed in “a chain”, one technician did step b), other did steps c) and d), another completed step e). Results/Findings: We register the mean time of each cycle in both machines, from step 1 to step 4, their descript ion and the duration are shown in attached table. The prior work included in steps a), b), c) and d) is the same using either machine. The mean platelet content in fi nal platelet concentrate (PC) in routine conditions for Orbisac® and TACSI® were respectively 3.88 ± 0.4 × 1011 (n = 180) and 3.92 ± 0.5 × 1011(n = 80). Following this procedure, work in “a chain”, with 4 Orbisac® we can obtain 20 PCs per hour, with 5 we could get 25 PCs. Our experience with TACSI® is less than with the other system. We obtained 12 PCs per hour (54 minutes) using 1 TACSI® device. Conclusion: Two automatic methods can be easily implemented in a transfusion center. Using both technologies we are able to prepare high quality products reducing the variation due to manual operation and improving standardization and pro-ductivity of PCs process. We had been able to reach high platelet concentra-tion to applied pathogen reduction treatment, implemented in our center since 200

Production of Standardized Labile Products from Single Whole Blood Donations: Evolution throughout Last Three Years

Background/Case Studies: Last year this transfusion center collected approximately 108,000 whole blood (WB) donations and 7,000 from apheresis. WB is the raw material for production of labile blood products, necessary to cover the regional transfusion requirements. In 2012, 109,584 red blood concentrates (RBCC), 17,932 plasma units (inactivated by methy-lene blue), 6,515 platelet concentrates from apheresis, and 8,053 buffy-coatderived platelet concentrates (BCPC) were issued to hospitals. All platelet products are inactivated by the INTERCEPT® technology. All labile blood products (LBP) are leukodepleted. It was attempted to provide clinicians with standardized LBP, thus WB collection, blood components production, and preparation are checked and quality control were performed in different phases.Study Design/Methods:Two types of WB bag systems (MacoPharma) are used one, with an integrated filter for WB 1) and the other top and bottom configuration 2) with an integrated filter for leukodepletion of RBCC. In general WB from 1) is filtered and separated after 5-8 hours fromdonation. All WB from 2), more than 60% WB collected, was stored overnightin a temperature controlled room (22±2 C). In the morning (15 to 18 hoursfrom donation) WB is centrifuged at 4497 g for 18 minutes. After, WB isseparated on Compomat G-4 (Fresenius). It was changed to Compomat G-5 last year. In this way, it is possible to obtain RBCC, plasma and buffycoat (BC) with a volume 52±3 ml and a haematocrit of 37±3%, these parameters of BC are critical to pooling BC (5BC) preparation with Orbi Sacsystem (Terumo BCT). Results/Findings: From 2010-2012 the production of blood components from WB only was registered, number of units tested for quality control. The automation offered by some systems during component preparation eliminates the deviation caused by manual operations and increases productivity and yield of the platelet preparation/treatment process. Conclusion: Standardization of WB processing procedure allows the ability to obtain final blood components containing a reproducible quantity of activeprinciples that meet the required standards. The results are consistent and continuous, thus facilitating therapeutic use of blood components and also making it easier to introduce new processes and technologies

In Vitro Evaluation of Platelet Concentrates from Buffy Coat Treated with Two Pathogen Reduction Technology (PRT)

Background/Case Studies: In the past decade, technology for inactivation of potential pathogens and leukocytes in labile blood components has been introduced into Blood Centres to reduce the risk of transfusion-associated adverse effects. Intercept® treatment of platelet concentrates (PC) has been used in our Center since 2008. Last year, PRT with Mirasol® was evaluated. We have assessed platelet activation and other parameters for extended periods post-treatment with both PRTs. Study Design/Methods: Intercept® PC was obtained by pooling 5 buffy coat (BC), suspended in InterSol addi-tive solution (AS, 280 ml) with a ratio plasma: AS (37:63) and amotosalen plus illumination UV was applied after. Mirasol® treated PC was obtained the same way, but SSP plus (AS, 250 ml) was used. Then, ribofl avin 500 μM plus light UV were applied to PC. In both strategies, pool-BCs were pro-cessed in an OrbiSac System. All products were stored at 22°C until day 7th.The parameters tested were: pH, glucose, lactate and LDH, as markers of platelet metabolism; CD62p, as an indicator of platelet activation, and annexin V binding, as a measure of phosphatidylserine exposure, were determined by fl ow cytometry. Soluble CD62p, TGF-β1 and RANTES were analyzed by specifi c ELISA assays. Results/Findings: The fi nal cell con-centration in Intercept PC was 850 ± 90 × 106/ml (2nd day); in Mirasol® PC was 1073 ± 70 × 106/ml (2nd day). Swirling scores were good throughout 7 days of storage. Cell loss during processing was 8% and 1% for the Inter-cept® and Mirasol® system, respectively. Other results are shown with more detail in the attached table. On day 7th, 40% of the units in Mirasol contained low glucose levels, but the platelet concentration was higher in Mirasol PCs. Conclusion: In vitro results showed that in both treatments PRTs applied to PCs in the presence of AS generates units that can be stored for seven days with acceptable cell quality. Clinical studies have shown “in vivo” effi -cacy, currently large scale studies are in progress with Mirasol® platelet in AS. The treatment with PRT provided some advantages logistics, elimination gamma irradiation and protection from emerging pathogens