RESUMEN
BACKGROUND: The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach. STUDY DESIGN AND METHODS: Small volumes (50 mL) of single apheresis PLT components (APCs), collected on either CaridianBCT Trima or Haemonetics MCS+ instruments, were aliquoted on Days 2, 3, 4, and 5 postcollection into Fenwal PL1240 or 4R2014 bags or 60-mL polypropylene syringes. Samples were tested for in vitro quality at their recommended expiry times (4 hr for 4R2014 bags and syringes or Day 5 for PL1240 bags). Assays included pH, CD62P expression, and metabolic measures. RESULTS: CD62P expression increased throughout storage in all containers. Among the small-volume containers, pH, pCO(2) , lactate, and bicarbonate varied considerably. Regardless of the day of aliquoting, pCO(2) was significantly higher and pO(2) was significantly lower in gas-impermeable syringes than other containers. No bacterial growth was detected in any sample. CONCLUSION: The quality of APCs aliquoted into small-volume containers meets regulatory requirements and is generally equivalent to that of full-volume APCs at expiry.
Asunto(s)
Bancos de Sangre/normas , Plaquetas/citología , Plaquetas/metabolismo , Conservación de la Sangre/normas , Transfusión de Plaquetas/normas , Antígenos de Plaqueta Humana/metabolismo , Bicarbonatos/metabolismo , Conservación de la Sangre/instrumentación , Conservación de la Sangre/métodos , Dióxido de Carbono/metabolismo , Niño , Citometría de Flujo , Glucosa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ácido Láctico/metabolismo , Oxígeno/metabolismo , Selectina-P/metabolismo , Recuento de Plaquetas , Transfusión de Plaquetas/métodos , Guías de Práctica Clínica como Asunto , Almacenamiento de Sangre/métodosRESUMEN
BACKGROUND: A quality monitoring program (QMP) for platelet concentrates (PCs) was implemented at Canadian Blood Services (CBS) to improve standards and to better understand platelet (PLT) products by supplementing routine quality control (QC). STUDY DESIGN AND METHODS: Annual surveys of PCs from CBS production sites were conducted, with four completed to date (QMP Cycles 1-4) spanning two different PC production methods: PLT-rich plasma (PRP) and buffy coat (BC). Randomly selected PCs were sent to a central laboratory and tested 1 day after expiry. An expanded panel of tests including CD62P expression by flow cytometry, mean PLT volume, PLT count and morphology, extent of shape change, and PLT metabolic parameters, were applied. RESULTS: QMP data on the implementation of the BC production method across CBS indicated that BC PCs have less variable in vitro quality measures than PRP PCs. For the QC parameters pH and PLT count per unit, the range of mean values from each site for QMP 3 and 4 fell well within the range defined by regulatory standards, a first step in defining quality benchmarks for PCs. Of the extended panel of quality parameters, CD62P expression was the most sensitive indicator of change and identified an issue with the implementation of the BC PC production method at one site, which was subsequently remedied. CONCLUSION: A QMP was found to be useful to monitor production processes across sites and highlights best practice approaches while deepening understanding of the quality of PLT products at CBS.
Asunto(s)
Plaquetas/fisiología , Eliminación de Componentes Sanguíneos/normas , Plaquetas/química , Canadá , Concentración de Iones de Hidrógeno , Selectina-P/análisis , Recuento de Plaquetas , Control de CalidadRESUMEN
The metabolic conversion of glucose to energy and reducing power by platelets is examined. Although platelets concurrently metabolize glucose aerobically and anaerobically, the balance between the cytosolic and mitochondrial pathways is affected not only by physiological activation but also by conditions prevailing during in vitro storage. The development of platelet additive solutions and pathogen reduction technologies point to increased glucose metabolism and consequent high levels of lactate production as the effect of platelet damage, rather than the cause. Consequently a different perspective of the data suggests that reduction rather than support of platelet metabolism in vitro would result in a better quality of stored platelets.
Asunto(s)
Plaquetas/citología , Plaquetas/metabolismo , Conservación de la Sangre/métodos , Glucosa/farmacología , Conservadores Farmacéuticos/farmacología , Edulcorantes/farmacología , Humanos , Soluciones Farmacéuticas/farmacologíaRESUMEN
BACKGROUND: Platelet additive solutions (PASs) are an alternative to plasma for the storage of platelet concentrates (PCs). However, little is known about the effect of PAS on the growth dynamics of contaminant bacteria. Conversely, there have been no studies on the influence of bacteria on platelet (PLT) quality indicators when suspended in PAS. STUDY DESIGN AND METHODS: Eight buffy coats were pooled, split, and processed into PCs suspended in either plasma or PAS (SSP+, MacoPharma). PCs were inoculated with 10 and 100 colony-forming units (CFUs)/bag of either Serratia liquefaciens or Staphylococcus epidermidis. Bacterial growth was measured over 5 days by colony counts and bacterial biofilm formation was assayed by scanning electron microscopy and crystal violet staining. Concurrently, PLT markers were measured by an assay panel and flow cytometry. RESULTS: S. liquefaciens exhibited an apparent slower doubling time in plasma-suspended PCs (plasma-PCs). Biofilm formation by S. liquefaciens and S. epidermidis was significantly greater in PCs stored in plasma than in PAS. Although S. liquefaciens altered several PLT quality markers by Days 3 to 4 postinoculation in both PAS- and plasma-PCs, S. epidermidis contamination did not produce measurable PLT changes. CONCLUSIONS: S. liquefaciens can be detected more quickly in PAS-suspended PCs (PAS-PCs) than in plasma-PCs by colony counting. Furthermore, reduced biofilm formation by S. liquefaciens and S. epidermidis during storage in PAS-PCs increases bacteria availability for sampling detection. Culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs, while changes of PLT quality can herald S. liquefaciens contamination when in excess of 10(8) CFUs/mL.
Asunto(s)
Biopelículas/efectos de los fármacos , Conservación de la Sangre/métodos , Transfusión de Plaquetas , Soluciones/farmacología , Infecciones Estafilocócicas/prevención & control , Staphylococcus epidermidis/crecimiento & desarrollo , Acetatos/farmacología , Capa Leucocitaria de la Sangre/citología , Plaquetas/citología , Cloruros/farmacología , Citratos/farmacología , Humanos , Técnicas Microbiológicas , Plasma Rico en Plaquetas , Infecciones por Serratia/prevención & control , Serratia liquefaciens/crecimiento & desarrollo , Citrato de SodioRESUMEN
Preserving cell viability and function is an essential component in the translation and delivery of existing and emerging cell-based therapeutics from the research lab to the patient bedside. This workshop provided a summary of the advances and challenges that currently face the preservation sciences, together with a glimpse at the future applications and instrumentation that will enhance our ability to process, preserve, and store red blood cells (RBCs), platelets, and stem cells. It is clear from the presentations made during the workshop and the discussions that ensued after that, for us to overcome the challenges that face blood biopreservation, it will require a concerted effort from clinicians, scientists, and engineers from a variety of disciplines. Through this interdisciplinary research effort, significant progress will be made to improve the safety, quality, and potency of the blood products that are used in reparative medicine. As the need for effective preservation technologies will be the motivation for more concerted efforts in the biopreservation sciences, there are encouraging prospects for the future applications of biopreserved blood cells.
Asunto(s)
Plaquetas , Conservación de la Sangre/métodos , Conservación de la Sangre/tendencias , Eritrocitos , Células Madre , Educación , HumanosRESUMEN
We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95+/-0.02 degrees C/min), medium (73+/-3 degrees C/min) and fast (265+/-12 degrees C/min) cooling rates and storage in liquid nitrogen, followed by a 37 degrees C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%+/-5% vs 29%+/-4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.
Asunto(s)
Conservación de la Sangre/métodos , Membrana Celular/química , Criopreservación/métodos , Crioprotectores , Eritrocitos/efectos de los fármacos , Trehalosa , Supervivencia Celular , Crioprotectores/química , Portadores de Fármacos/química , Eritrocitos/citología , Hemólisis , Humanos , Liposomas/química , Fosfolípidos , Trehalosa/químicaRESUMEN
Conventional antithrombotic drug discovery requires testing of large numbers of drug candidates. We used computer-aided macromolecular interaction assessment (MIAX) to select antithrombotic molecules that mimic and therefore block platelet GPIb's binding to von Willebrand factor (vWf), an early step in thrombus formation. We screened a random array of 15-mer D-amino acid peptides for binding vWf. Structures of 4 candidate peptides were inferred by comparison to sequences in protein databases, conversion from the L to D conformations and molecular dynamics (MD) determinations of those most energetically stable. By MIAX, we deduced the amino acids and intermolecular hydrogen bonds contributing to the GPIb-vWf interaction interface. We docked the peptides onto vWf in silico to localize their binding sites and consequent potential for preventing GPIb-vWf binding. In vitro inhibition of ristocetin-initiated platelet agglutination confirmed peptide function and suitability for antithrombotic development, thereby validating this novel approach to drug discovery.
Asunto(s)
Fibrinolíticos/química , Péptidos/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Factor de von Willebrand/química , Sitios de Unión , Diseño de Fármacos , Descubrimiento de Drogas , Integrinas/antagonistas & inhibidores , Integrinas/química , Integrinas/metabolismo , Modelos Moleculares , Agregación Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Conformación Proteica , Factor de von Willebrand/antagonistas & inhibidores , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Buffy coat (BC) production of platelets (PLTs) has been successfully used in Europe for more than two decades. Currently, Canadian Blood Services is implementing the BC method. This article summarizes results of the validation testing performed to qualify the process of PLT production from whole blood and compares the quality of PLTs produced in routine production by either the PLT-rich plasma method (PRP-PCs) or the BC method (BC-PCs). STUDY DESIGN AND METHODS: Validation data included variables used for routine quality control (QC; pH, PLT count, volume, sterility, residual white blood cell count) as well as nonroutine testing of PLTs for PLT activation, metabolic changes during storage, and PLT responsiveness to hypotonic shock and the extent of shape change induced by adenosine 5'-diphosphate. BC-PCs were tested on Days 1 and 6. QC of production runs included the same routine tests performed on Day 6. RESULTS: PLTs produced by the BC method during validation and pilot implementation met all Canadian Standards Association standards with respect to yield, volume, pH, and leukoreduction. Additional validation testing indicated a moderate level of PLT storage lesion development. In comparison to PRP-PCs, in vitro variables of BC-PCs, either pH in this study, or other markers compared to the literature were better, suggesting that BC-PCs have less evidence of production-related damage and improved PLT quality during storage. CONCLUSIONS: PLT concentrates produced from whole blood by the BC method after an overnight hold have laboratory variables suggestive of a higher quality than those concentrates produced by the PRP method.
Asunto(s)
Plaquetas , Conservación de la Sangre/métodos , Recolección de Muestras de Sangre/métodos , Separación Celular/métodos , Centrifugación/métodos , Plasma Rico en Plaquetas/citología , Plaquetas/metabolismo , Separación Celular/instrumentación , Separación Celular/normas , Metabolismo Energético , Humanos , Concentración de Iones de Hidrógeno , Recuento de Leucocitos , Procedimientos de Reducción del Leucocitos , Recuento de PlaquetasRESUMEN
BACKGROUND: Buffy-coat processing allows for the use of platelet additive solutions (PASs). PASs reduce plasma-associated transfusion reactions and conserve plasma for transfusion or fractionation. Platelet (PLT) storage in plasma was compared to storage in three commercially available PASs compared to assess their influence on in vitro laboratory variables. STUDY DESIGN AND METHODS: Platelet concentrates (PCs) were prepared from leukoreduced pools of four buffy coats (BCPs) suspended in autologous plasma or one of PASs (Composol, Fresenius-Kabi; T-Sol, Baxter Corp.; or SSP+, MacoPharma). On Days 1, 2, 3, 5, and 7 of storage, samples were tested for PLT concentration, mean PLT volume (MPV), CD62P, morphology, pO2, pCO2, glucose, lactate and total protein concentration, pH, extent of shape change (ESC), and hypotonic shock response (HSR). Data were analyzed by analysis of variance (ANOVA) with repeated measures and t tests. RESULTS: PLT recoveries from BCPs were higher (p < 0.05) with plasma than any PAS. Storage medium and duration did not affect PLT concentration or MPV over time. CD62P expression and morphology were significantly different among PCs pooled with different media. ANOVA showed (p < 0.05) differences among the rates of change of pCO2, pH, glucose consumption, lactate production, and ESC; PASs such as Composol and SSP+ offered excellent maintenance of pH and low rates of glucose consumption. PAS performed poorly in ESC and HSR compared to plasma. Correlation studies reveal far more significant correlations between variables of PLTs in PAS than in plasma. CONCLUSION: Newer PASs, for example, SSP+ and Composol, can maintain PLT integrity and moderate metabolism similarly to plasma but offer consistently lower PLT recoveries and limited osmotic balance.
Asunto(s)
Almacenamiento de Sangre/métodos , Plaquetas/metabolismo , Conservación de la Sangre/métodos , Plasma Rico en Plaquetas/metabolismo , Plaquetas/citología , Tampones (Química) , Dióxido de Carbono/metabolismo , Gluconatos/farmacología , Glucosa/farmacología , Humanos , Soluciones Hipotónicas/farmacología , Ácido Láctico/farmacología , Magnesio/farmacología , Presión Osmótica , Oxígeno/metabolismo , Fosfatos/farmacología , Transfusión de Plaquetas , Potasio/farmacologíaRESUMEN
Immune thrombocytopenic purpura's diagnosis (ITP) is based on low platelet count and exclusion of clinical conditions rather than a specific diagnostic test. We used the reticulated platelet (RP) assay to study ITP and thrombocytopenia associated with HIV infection (HIV-ITP). Data from 96 ITP and 23 HIV-ITP patients showed low platelet counts (PC) with both high or low %RP suggesting that individuals have different degrees of thrombopoiesis. About 20% of ITP and 46% of HIV-ITP patients had %RP in the 'low' or 'normal' ranges. Grouped by platelet count <30x10(9)/L, 24% ITP and 36% HIV-ITP patients had 'low' to 'normal' %RP. The patient population did not show correlation between PC and %RP, but individuals showed an inverse relationship. Within a week of receiving IVIG, 18 ITP and 9 HIV-ITP patients' PC increased, %RP decreased. Patients with %RP measured within 24 h of IVIG treatment had lower %RP than expected, suggesting dilution by an older platelet population. ITP and HIV-ITP patients' responses to i.v. gammaglobulins were similar. Thrombopoietin levels of ITP patients did not correlate with PC, %RP, or RP count. Estimation of thrombopoiesis by RP assay provides useful information for differentiation among thrombocytopenias.