Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Analysis of the shelf life and transfusion life of whole blood and erythrocyte concentrates

The shelf life of 60,000 units of whole blood and red cell concentrates in the blood center were analysed before delivering to hospitals as well as the transfusion age of 18,000 units in relation to the stock-size and the outdated units. The mean transfusion age of whole blood units and red blood cell concentrates amounted to 11 to 12 days and 16 to 17 days, respectively. The shelf life of blood transfused within 24 hours after collection was 1.6% and 3.8% transfused within 48 hours according to the total number of whole blood and red blood cell concentrates. Washed red cell concentrates were prepared from buffy coat-free resuspended red cell concentrates on an average at the 7th day of storage. A high stock-size of about 1,000 units rather than of 600 units in the blood center increased the shelf life and also the number of outdated units from less than 0.5% up to more than 3%.     

Comparison of posttransfusion recoveries achieved with either fresh or stored platelet concentrates

The effectiveness of platelet concentrate transfusion depends on such variables as blood bag material, donor--recipient compatibility, and time elapsed between donation and transfusion. To study the latter a corrected thrombocyte increment for recovery in the recipients was evaluated with 108 platelet transfusions in 31 patients. In 83 treatment programs, the mean recovery at the one-hour post-transfusion time point was 8.6 X 10(9) platelets/l with fresh platelets and 5.9 X 10(9) platelets/l with stored platelets. Significantly better recovery was achieved with freshly prepared platelet over the total of platelet concentrates stored for up to 96 hours; however, if the recoveries in different patient groups given stored platelets were considered separately in terms of storage times of up to 48 h or 48-96 h, the good recovery with fresh platelets was significantly better only when compared to the older (p = 0.034) but not to the younger group of stored platelets. In patients with signs indicating enhanced platelet destruction (fever, splenomegaly, disseminated intravascular coagulation) the transfusion with fresh platelet concentrates gave a significantly better recovery compared to stored platelet concentrates (p = 0.028), whereas in the absence of such signs the recovery produced by fresh concentrates was not significantly higher than with stored concentrates. These findings may be relevant for the logistics in blood banking.     

Flow cytometric analysis of CD41-labeled platelets isolated by the rapid, one-step OptiPrep method from human blood.

BACKGROUND: Although platelet-rich plasma is relatively easy to produce by centrifugation of whole blood, yields of platelets may be variable because many of them are trapped within the erythrocyte layer. Although they can be recovered by washing these cells, it is a general rule that the number of centrifugations should be kept to a minimum to avoid activation of platelets. This work describes the rapid, one-step OptiPrep method for the isolation of highly purified platelets from human blood (buffy coat). METHODS: To provide a functionally intact and uncontaminated platelet fraction, a density gradient centrifugation was performed by using a density barrier prepared from OptiPrep. CD41 antibody staining was performed to assess the purity of the obtained platelet population by means of a FACScan flow cytometer. Platelets were identified by a morphologic gate in which events were further studied for CD41 expression. Data were analyzed by CellQuest (Becton Dickinson). RESULTS: Platelet-specific CD41 antibody staining showed that the purity of the platelet population recovered from this density barrier method was greater than 90%. The platelets showed an excellent morphologic state. CONCLUSION: The rapid, one-step OptiPrep density gradient centrifugation is a reliable method for obtaining highly purified platelets from human blood that are ready for further pharmacologic investigations.     

Value of examining buffy coats for intragranulocytic micro-organisms in patients with fever.

To determine the significance of the presence of intragranulocytic micro-organisms in the blood buffy coat in patients with suspected infection, buffy coat examination and blood cultures were simultaneously performed in 455 consecutive patients with fever. There was no general correlation between the finding of intragranulocytic micro-organisms in the buffy coat and positive blood cultures. Patients with persistent bacteraemia and sterile blood cultures were, however, shown to have persistently positive buffy coat findings on repeated examination. These patients, who had culture-negative endocarditis or catheter-associated infections, had sterile blood cultures because of antibiotic treatment. Repeated positive findings in the buffy coat may therefore be valuable in detecting patients with persistent bacteraemia, but sporadic findings of micro-organisms in the buffy coats of acutely ill patients seem to have little diagnostic value.     

Ubiquinol and a coenzyme Q reducing system protect platelet mitochondrial function of transfusional buffy coats from oxidative stress

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ 10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.     

Ubiquinol and a Coenzyme Q Reducing System Protect Platelet Mitochondrial Function of Transfusional Buffy Coats from Oxidative Stress

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ 10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.     

Preparation of leukocyte-poor platelet concentrates from buffy coats by a new type of quadruple bag

A new quadruple blood bag system for collection of platelet concentrates is described. The principle modification consists of a smaller (150 ml) buffy coat bag in conjunction with SAG-mannitol for red cell preservation. Platelet yield and function were comparable to conventional techniques, leukocyte contamination was lower (1.7 +/- 1.3 x 10(7) per unit). The system is recommended for specialized institutions with a high demand of blood components.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

Background

Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells.

Methodology/Principal Findings

In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed.

Conclusion/Significance

Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.     

Improving the quality of washed and buffy coat-free erythrocyte concentrates]

Washing buffy-coat free erythrocyte concentrates three times in bottles used for blood storage will diminish their leukocyte content to 0.22 +/- 0.11 x 10(9) per TE (= 9% of the initial value in whole blood, and the thrombocyte content to 0.3 +/- 0.5 x 10(9) per day (= 2% of the initial value in whole blood). Even 50% of leukocytes (mainly lymphocytes) and 80% of thrombocytes are eliminated simply by buffy coat separation. 30% of erythrocytes are lost by the washing process. Due to increasing haemolysis (0.22%) a subsequent storage of 24 hours should not be exceeded for washed erythrocyte concentrates. Further quality parameters, such as morphological index, pH, ATP, 2,3-P2G and K+ and Na+, were investigated. As far as selected quality parameters are concerned, washing erythrocyte concentrates three times in bottles for blood storage may be compared with washing them once in blood bags. The present findings confirm the conclusion that the washing of erythrocyte concentrates with a solution of sodium chloride in order to eliminate leukocytes may for the most part exclude non-haemolytic febrile transfusion reactions, but not immunization. More effective procedures of eliminating leukocytes, such as filtration, TTK or even glycerin, treatment of erythrocyte concentrates without cryoconservation, are indispensable.     

Metabolomic profiling highlights oxidative damages in platelet concentrates treated for pathogen inactivation and shows protective role of urate

Introduction

Research in transfusion medicine is motivated by the desire to deliver the most-compatible and most-efficient blood product to the patient. It is now well accepted that ex vivo platelet concentrates (PCs) experience biochemical alterations and a functional decline known as storage lesions. Photochemical treatments have been introduced to secure PCs against pathogens but are reported to accelerate these lesions.

Objectives

The objective of this study was to investigate metabolic changes in stored PCs treated for pathogen inactivation with the INTERCEPT Blood system (Cerus, Concord, USA).

Methods

PCs either untreated (uPCs) or INTERCEPT-treated (iPCs) were sampled along the 7-day storage period. First, metabolites were extracted and analyzed using ultra-high pressure liquid chromatography—high resolution mass spectrometry followed by statistical analysis. Secondly, we investigated the role of urate, a major plasma antioxidant, in the platelet function using flow cytometry-based assays.

Results

We observed oxidative damages in stored iPCs compared to uPCs, in particular alteration of the purine and the glutathione metabolism. We showed diminution of antioxidant defenses following INTERCEPT treatment such as the conversion of urate to allantoin, only possible in humans under the action of reactive oxygen species (ROS). Functional assays on platelets in absence or in an excess of urate suggest a protective role of urate in PCs.

Conclusion

Our results indicate oxidative damages occurring at the metabolic level in stored iPCs. Understanding better the role of antioxidants such as urate in ex vivo PCs would definitively provide new insights to ameliorate the storage conditions and preserve the functionality of platelets.

     

Transfusion strategy for patients in therapeutic aplasia. Experience of the Henri Mondor Hospital apropos of 2 successive protocols

Retrospective analysis of two transfusion protocols applied in our institution to the bone marrow transplanted patients was conducted. Granulocyte transfusions should be only proposed as a therapeutic treatment to patients with severe well documented bacterial infection resistant to an adapted antibiotherapy. Leukocyte-depleted blood products reduce the incidence of HLA-immunization but do not influence the frequency of CMV infections. Random single donor platelet concentrates (obtained by cytapheresis) could decrease the incidence of polyspecific HLA-antibodies in comparison with the use of random standard platelet concentrates. The best transfusion protocol should associate leukocyte-depleted blood products with transfusion of prophylactic single donor platelet concentrates. In our institution, this protocol is less expensive than the protocol with prophylactic white blood cell transfusions and has the same cost than other protocols using standard blood products.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

Determining Toxoplasma high-risk autologous and allogeneic hematopoietic stem cell transplantation patients by systematic pre-transplant PCR screening of stem cell originated buffy coat

The diagnosis of Toxoplasma infection or disease in hematopoietic stem cell transplantation (HSCT) patients is achieved mainly by PCR screening; however screening did not find wide field of use in practice due to costly expenditures of PCR. This study aimed to determine patients at high risk of Toxoplasma infection or disease before transplantation by stem cell originated buffy coat PCR and subsequently to screen them. Buffy coats collected from 12 autologous and 18 allogeneic HSCT patients' donors were investigated by PCR before transplantation. After transplantation, blood and sera collected at fixed time intervals were screened by two PCR methods and serological assays. Screening results first time assessed a toxoplasmosis incidence level as 25% in autologous HSCT patients and increased incidence level in allogeneic HSCT patients to 22%. Importantly, buffy coat PCR was first time performed before transplantation, to determine the risk of toxoplasmosis. Buffy coat PCR results showed that four patients were at high risk of toxoplasmosis before transplantation. After transplantation, these patients experienced toxoplasmosis. In conclusion, for the determination of patients at risk of toxoplasmosis, clinicians should consider buffy coat PCR in combination with serology before transplantation. After transplantation, PCR screening can be initiated in high risk patients upon clinical suspicion.     

The use of inhibitors of platelet activation or protease activity in platelet concentrates stored for transfusion.

During storage of platelet concentrates the platelets show signs of activation, and extracellular protease activity becomes evident in the plasma. The consequences of platelet activation and plasma protease activity are potentially detrimental to the preservation of platelet function in vitro. The earlier use of prostaglandins during preparation of platelet concentrates to increase the harvest of platelets from whole blood did little to improve their shelf-life. Other compounds that sustain elevated cyclic AMP levels or that directly inhibit platelet agonists provide more effective inhibition of platelet activation during storage. Also, the inclusion of general or specific protease inhibitors appears to improve platelet preservation over extended storage periods. These studies demonstrate the possibility of prolonging the shelf-life of platelet concentrates stored at 22 degrees C through the addition of non-toxic formulations of inhibitors of platelet activation and protease activity.     

Experience with a blood component program in surgical hemotherapy

Within the component concept applied during the past 6 years at the University Hospital in Berne (Switzerland), 85% of all red cell units are transfused as concentrates with a hematocrit of 70%, and the remaining 15% as fresh whole blood. The rationale in surgical patients is to exploit the different "critical levels" of the blood volume, hematocrit, total serum protein, plasmatic coagulation factors, and platelets. A colloid plasma substitute compensating for the plasma deficit of the red cell concentrates is an integral part of the system. A carefully checked, retrospective study in 372 patients revealed no disadvantages for the postoperative course. During the first 4 1/2 years after its introduction it did not increase the demand for human plasma protein solutions. With the red cells as a pacemaker for the number of blood donations, this system can simultaneously cover a reasonable national demand for albumin and factor VIII.     

Comparison of the effect of calcium gluconate and batroxobin on the release of transforming growth factor beta 1 in canine platelet concentrates

ABSTRACT: BACKGROUND: The clinical use of autologous platelet concentrates (also known as platelet-rich plasma) on the field of regenerative therapy, in the last decade has been the subject of several studies especially in equine medicine and surgery. The objectives of this study was: 1) to describe and compare the cellular population in whole blood, lower fraction (A) and upper fraction (B) of platelet concentrates, 2) to measure and compare the transforming growth factor beta 1 (TGF-beta1) concentration in plasma and both platelet concentrates after be activated with calcium gluconate or batroxobin plus calcium gluconate and, 3) to determine correlations between cell counts in platelet concentrates and concentrations of TGF-beta1. Blood samples were taken from 16 dogs for complete blood count, plasma collection and platelet concentrates preparation. The platelet concentrates (PC) were arbitrarily divided into two fractions, specifically, PC-A (lower fraction) and PC-B (upper fraction). The Platelet concentrates were analyzed by hemogram. After activated with calcium gluconate or batroxobin plus calcium gluconate, TGF-beta1 concentration was determined in supernatants of platelet concentrates and plasma. RESULTS: There were differences statistically significant (P < 0.05) for the platelet count and leukocyte count and TGF-beta1 concentration between whole blood, plasma and both platelet concentrates. A significant correlation was found between the number of platelets in both platelet concentrates and TGF-beta1 concentration. Platelet collection efficiency was 46.34% and 28.16% for PC-A and PC-B, respectively. TGF-beta1 concentration efficiency for PC activated with calcium gluconate was 47.75% and 31.77%, for PC-A and PC-B, respectively. PC activated with batroxobin plus CG showed 46.87% and 32.24% for PC-A and PC-B, respectively. CONCLUSIONS: The methodology used in this study allows the concentration of a number of platelets and TGF-beta1 that might be acceptable for a biological effect for clinical or experimental use as a regenerative therapy in dogs.     

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<10⁶ WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrP^Sc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.