Stein Holme

A multi-laboratory evaluation of in vitro platelet assays: the tests for extent of shape change and response to hypotonic shock. Biomedical Excellence for Safer Transfusion Working Party of the International Society of Blood Transfusion

BACKGROUND: There is no consensus regarding the use of specific in vitro tests for the assessment of the quality of platelet components. A literature review found that the platelet discoid shape as measured photometrically by the extent of shape change (ESC) and hypotonic shock response (HSR) correlated well with in vivo viability. The purpose of this study was to determine whether multiple research laboratories can perform the ESC and HSR assays in an accurate, reproducible manner, with acceptable sensitivity and comparable results.

Synthetic, plasma-free, transfusible storage medium for red blood cells and platelets

The invention is a sterile, plasma-free storage medium for blood components including red blood cells and for platelets processed separately or together. The red cell storage medium includes a physiologically compatible, aqueous electrolyte solution. In one liter of this electrolyte solution there is between about 3.0 grams and about 25.0 grams of dextrose, between about 3.0 grams and about 6.0 grams of sodium citrate, and between about 2.0 grams and about 4.2 grams of sodium bicarbonate. The red cell storage medium is isotonic and has a pH in a range of between about 6.8 and about 7.4. The red cell storage medium is capable of storing and preserving red cells for at least 49 days. The medium can also contain adenine.

Concepts About Current Conditions for the Preparation and Storage of Platelets

Substantial experimentation over the last 20 years has led to the conditions that are currently used to prepare and store platelets. Although platelet rich plasma is used in most instances to prepare platelet concentrates, there may be some benefit associated with the use of buffy coats as the source component. Extension of the maximum allowable storage time for platelets to 5 days has been possible as a result of defining the conditions which allow for the better retention of platelet properties. Storage temperature, permeability of the storage container, volume of platelet suspension, and the need to agitate platelets have been identified as key parameters that maintain platelet viability and functional properties. Storage in the 20 to 24 degrees C range prevents a reduction in posttransfusion viability that occurs when platelets are maintained at lower temperatures. Adequate influx of oxygen through container walls to support platelet metabolism and, to a lesser degree, adequate efflux of produced carbon dioxide are essential for maintaining pH levels, a key parameter that also influences posttransfusion viability. Permeability is influenced by container size and material, by use of a satisfactory volume of plasma and by agitating the container. Although platelet concentrates prepared from whole blood have been primarily used to delineate appropriate storage conditions, they also apply to platelets harvested by aphersis technology. Storage under currently used conditions, although providing products with acceptable clinical efficacy, is associated with a reduction in viability and functional characteristics. The development of storage media, specific for platelets, may minimize the occurrence of deleterious changes.

Detection of bacteria in WBC-reduced PLT concentrates using percent oxygen as a marker for bacteria growth

BACKGROUND:  The risk of receiving a PLT concentrate (PC) contaminated with bacteria may be 1000‐fold greater than that of pathogenic viral transmission, yet surveillance for this risk is not generally practiced. A novel bacteria detection system (BDS) that overcomes the limitations of current systems is described. The BDS monitors percent oxygen (%O2) in air above aliquots of PCs that have been filtered to remove the confounding effect of respiring PLTs and residual WBCs.

Morphological changes associated with pH changes during storage of platelet concentrates in first-generation 3-day container.

Abstract. The platelet injury and loss of viability that has been shown to occur with storage of platelet concentrates (PC) under conditions with increasing or falling pH were examined using scanning and transmission electron microscopy. After storage, samples were taken for measurement of pH value, platelet count and size distribution, release of lactate dehydrogenase (LDH) into plasma, and for SEM and TEM. Increased levels of LDH were observed in PC with pH above 7.3 and below 6.1. In PC with pH above 7.3 this was related to an increased number (23%) of platelets that were lysed or had a swollen disintegrated internal structure (balloons) as seen with TEM. SEM and Coulter counter studies also showed that platelet fragmentation and formation of microvesicles were prominent in PC with pH above 7.3. The electron microscopic pictures confirmed previous suggestions that platelet disc‐to‐sphere transformation and cytoplasmic swelling occur when pH falls below 6.7‐6.8 during storage. SEM studies showed that concomitant with this change, folds and bulky projections appeared on the platelet surface. In PC with pH below 6.1 the morphological change was irreversible with the appearance of more than 90% lysed and balloon platelets. In conclusion, these studies suggest that the loss of viability observed with PC with pH above 7.3 or below 6.1 after storage is related to an increased percentage of lysed and balloon platelets.

Platelet Storage Lesions in Second-Generation Containers: Correlation with in vivo Behavior with Storage up to 14 Days

Abstract. The relationship between in vivo behavior and in vitro characteristics of 59 platelet concentrates (PC) stored for up to 14 days in a synthetic medium or in CPDA‐1 plasma was systematically investigated. 25 paired studies (1 study was incomplete) were performed comparing platelets suspended either in the synthetic medium or CPDA‐1 plasma with 5 days (n = 5); 7 days (n = 10); 10 days (n = 5); and 14 days (n = 5) of storage. In addition, 10 control studies were performed with freshly prepared PC (6–24 h) in CPDA‐1 plasma. Both percent recovery and survival estimations showed decreases with increasing storage duration, irrespective of storage medium used. In both media, with prolonged storage, the platelet survival curves not only became shorter, but also increasingly exponential, suggesting that in vitro storage caused progressive damage to the platelets present in circulation. Survival curves of platelets suspended in synthetic medium remained more linear, indicative of less random damage during storage. Mean population lifespan (MPL) of the stored PC was determined by the area below the survival curve divided by the mean percent recovery for the fresh PC, which was 55%. MPL decreased from 4.5 days (fresh PC) to 0.4 days after 14 days of storage in plasma, with a 50% reduction (t½) estimated at 7.2 days of storage. MPL t½ for PC stored in the synthetic medium was estimated to be 8.8 days. The decrease in in vivo viability with prolonged storage was paralleled with loss of energy‐dependent in vitro parameters such as hypotonic shock response, shape change with ADP, and ATP levels, and with increased lactate levels. Although the length of the storage period was shown to be the major factor responsible for the variability in MPL observed in this study, multiple linear‐regression analysis showed also that platelet discoid shape, as measured by the shape change, and lactate production were independent in vitro parameters with significant predictability of the in vivo viability. Combined with days of storage in the regression equation, these parameters were found to ‘explain’ 78% of the variability of the MPL found in this study.

In vitro pH effects on in vivo recovery and survival of platelets: an analysis by the BEST Collaborative.

BACKGROUND: The pH environment of stored platelet (PLT) products is recognized as an important factor and is generally used as a key surrogate measure of PLT viability. It is the only in vitro measurement that has been translated into industry standards and regulatory rules or specifications for storage of PLT products. The objective of this study was to evaluate the effect of in vitro pH on the in vivo recovery and survival of autologous PLT products.

Platelet microvesicles adhere to subendothelium and promote adhesion of platelets.

A role in hemostasis has been suggested for platelet membrane microvesicles (mv). The objectives of the studies reported here include functional analysis of platelet mv in models developed for study of platelet adhesion, as well as investigation of possible interactions between mv and intact platelets in these same adhesion models. Microvesicles were prepared from washed platelet concentrates by repeated freezing and thawing. Adhesion to subendothelium was measured quantitatively by radiolabelling mv with 111-In, and morphologically by scanning electron microscopy. Platelet mv adhered to subendothelium quantitatively over time. Using a modified Baumgartner chamber, we found adhesion of mv to subendothelium significantly increased with increasing shear rates. With this same model we found that prior exposure of subendothelium to mv greatly increased subsequent adhesion of platelets to the same everted vessel, compared to platelet adhesion in the absence of mv. All of these experiments were conducted with mv suspended in ACD/saline, indicating that plasma components are not essential for adhesion of mv. Our studies show that platelet mv adhere to subendothelium in much the same way as do platelets, and support the concept of a hemostatic role for mv in that they appear to increase platelet adhesion.

Enhancement of a culture-based bacterial detection system (eBDS) for platelet products based on measurement of oxygen consumption

BACKGROUND: An enhanced bacterial detection system (Pall eBDS) was developed that distinguishes itself from its predecessor (Pall BDS) by removal of the platelet (PLT)‐retaining filter allowing for optimal bacterial transfer, modification of the culture tablet to reduce the confounding effects of respiring PLTs while enhancing bacterial growth, and facilitation of nutrients and gas exchange by agitating the sample pouch during incubation at 35°C. The objective was to evaluate the performance of the new eBDS.

Extended storage of platelets in an artificial medium with the platelet activation inhibitors prostaglandin E1 and theophylline.

Abstract. The addition of platelet activation inhibitors to the anticoagulant and the replacement of plasma with a fortified electrolyte medium have been shown separately in previous work to improve the storage of platelets during a 2‐week period. In the present study, we have combined these strategies to investigate whether a synergistic improvement could be obtained. A total of 85 concentrates was studied with 300nM prostaglandin E1 (PGE1) and 1.9mM theophylline added to the whole blood, platelet‐rich plasma (PRP), and/or the storage medium during the preparation of platelet concentrates. In vitro markers of platelet aggregation, respiration, and cell integrity were measured over a 20‐day storage period and evaluated in an analysis of variance. We found that a single‐step addition of PGE1 and theophylline to the PRP prior to centrifugation was not sufficient in terms of preventing a rapid fall in pH, rise in pO2, fall in pCO2, loss of hypotonic shock response, and loss of aggregation response, compared to the addition of the inhibitors to the storage medium used to resuspend the platelet pellet. Factorial analysis showed that a reduction in the surface‐to‐volume ratio of the storage container further improved the maintenance of platelet respiration and, for three in vitro markers (hypotonic shock response, released lactic dehydrogenase, and surface glycoprotein Ib levels) displayed an interactive effect with the inhibitors. The addition of protease inhibitors to the formulation of PGE1 and theophylline showed further improvement in several markers. These findings demonstrate the possibility of preserving platelets for 15–20 days with the synergistic effects of activation inhibitors and an electrolyte storage medium fortified with citrate, buffers, and dextrose. In addition, these data suggest that platelet agonists generated in plasma accelerate the in vitro aging process of stored platelets.