Scott Murphy

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.

A Simple Nonradioisotope Technic for the Determination of Platelet Life-Span

Acetylsalicylic acid was shown both in vivo and in vitro to prevent the platelet lipid peroxidation normally induced by the aggregating agents thrombin and epinephrine, and the sulfhydryl inhibitor N-ethylmaleimide. After aspirin ingestion, there was a significant reduction (p smaller than 0.005) in platelet lipid peroxidation, with a gradual return to base-line values over a 10-day period. With these values, a normal platelet survival curve was constructed with a mean half-life of 4.4 days (range of 2.9 to 5.9 days). These values agree with the standard 51-Cr survivals in three patients with chronic idiopathic thrombocytopenic purpura. Half-lives of 1.0, 2.5, and 4.1 days by lipid peroxide technic compared with 1.9, 2.5, and 3.9 days by the concurrent use of 51-Cr. Thus, the technic may be used to measure platelet survival.

Platelet dysfunction in uremia. Multifaceted defect partially corrected by dialysis

In an attempt to elucidate the nature of the bleeding tendency in uremia, some in vitro functions of platelets from eight patients undergoing long-term hemodialysis were studied. None of the patients had diabetes. All had bleeding times longer than eight minutes. Threshold aggregating concentrations for collagen, adenosine diphosphate, and epinephrine, when used singly or in pairs, were two to three times higher than normal in platelet-rich plasmas from these patients. In contrast, those for arachidonic acid and U-46619, a cyclic endoperoxide/thromboxane A2 analogue, were within the normal ranges. Thromboxane B2 formation was normal in response to arachidonic acid (0.2 to 1 mM), whereas it was decreased by 30 to 50 percent in response to thrombin (0.5 to 10 units/ml), collagen (0.5 to 10 micrograms/ml), and the combination of collagen with adenosine diphosphate or epinephrine. There was a partial (about 35 percent) reduction of the platelet granular content of adenosine diphosphate. Secretion of adenosine triphosphate by 5 units/ml of thrombin was 25 to 50 percent less than in normal subjects. Thus, there was a storage pool defect as well. Similar but less severe defects were found in platelets from uremic patients who had never undergone hemodialysis. Partial correction of aggregation and thromboxane B2 formation was seen after dialysis, although platelet adenosine diphosphate content did not increase. It is concluded that the platelet dysfunction in uremia is multifaceted. There appears to be an aggregation and secretion defect related to impaired arachidonic acid release from platelet phospholipids as well as a storage pool defect. The first is improved with dialysis; the second is not.

Platelet Size and Kinetics in Hereditary and Acquired Thrombocytopenia

Abstract Measurements of platelet size and life-0 span were obtained to classify a variety of thrombocytopenic states. The mean size index in idiopathic thrombocytopenic purpura was significantly increased, whereas platelets in the Wiskott–Aldrich syndrome were small. The platelet defect in a family characterized by thrombocytopenia, sex-linked recessive inheritance, small platelet size and shortened platelet life-span closely resembled that of the Wiskott–Aldrich syndrome, but only mild eczema was present and immunologic abnormalities were absent. In a second family, thrombocytopenia was associated with increased platelet size suggesting a young cell population similar to idiopathic thrombocytopenic purpura. However, autologous platelet life-span was normal. Size measurements are thus useful in the classification of thrombocytopenias. Although in acquired conditions increased platelet size suggests a young platelet population, in hereditary disease platelet size is determined more by the nature of the in...

A critical comparison of platelet preparation methods.

Purpose of reviewPlatelet concentrates may be prepared from whole blood or by plateletpheresis. Currently, the non-evidence-based preponderance of apheresis units in the United States and the 50: 50 ratio in Europe may not optimize the gifts of whole-blood donors or minimize healthcare costs. Post-storage pooled, whole-blood-derived platelets, on the other hand, do not provide the convenience of or an equivalent level of safety as apheresis platelets. Recent findingsSome data suggest that different methods of manufacture of whole-blood-derived platelets (platelet-rich plasma or buffy coat intermediate steps) result in differing degrees of platelet activation, which may impact on the quality of stored concentrates. Recent studies have observed superior radiolabel recovery and post-transfusion increments for platelets derived from apheresis compared with platelet-rich plasma whole-blood-derived platelets. A pre-storage pooling system for whole-blood-derived platelets has just been licensed in the USA, and may eventually combine the benefits of apheresis-derived and whole-blood-derived platelets. The advantages of the European method of manufacture of buffy coat whole-blood-derived platelet concentrate have convinced the Canadian Blood Services to abandon platelet-rich-plasma-derived concentrates. SummaryWe present a literature-based review of the relative merits of apheresis-derived and whole-blood-derived platelets. Additional studies are needed in order to define the optimal proportion of the platelet supply from apheresis collections and the choice of whole-blood-derived production method for US blood providers.

Storage of platelets in additive solutions: a multicentre study of the in vitro effects of potassium and magnesium

Background and Objectives  In a preliminary study, the presence of potassium and magnesium in a modified synthetic medium (PAS‐III) was found to have a significant influence on platelet metabolism (using apheresis‐derived, as well as buffy‐coat‐derived platelets) when compared with standard PAS‐III. The differences included reduced glycolysis, as evidenced by lower consumption of glucose and lower production of lactate, but also better preservation of pH and hypotonic shock response reactivity. The results suggested that storage in modified PAS‐III containing 20% plasma was comparable to storage in standard PAS‐III containing 30% plasma. To confirm the preliminary results and to evaluate the effects of different preparation protocols, an international multicentre study, which included 11 different sites, was conducted.

Platelet storage solution effects on the accuracy of laboratory tests for platelet function: a multi‐laboratory study

Background and Objectives  Extent of shape change (ESC) and hypotonic shock response (HSR) have been widely used to characterize the in vitro function of platelets and have been shown to correlate with in vivo viability. These assays have been routinely performed using platelet‐poor plasma (PPP) as the test sample diluent. Because of the increasing popularity of storing platelets in synthetic media, it is important to understand the effects of using these synthetic media as test diluents for ESC and HSR measurements. The objective of this study was to determine the effect of using platelet storage solutions vs. plasma for the in vitro testing of ESC and HSR.

Role of acetate during platelet storage in a synthetic medium

It has previously been shown that buffy coat platelet concentrates (BC‐PCs) stored in a medium made up of approximately 70 percent platelet storage medium (Plasma‐Lyte A, PL) and 30 percent plasma (BC‐PC‐P) are effective in vivo after 9 to 12 days of storage. In addition to sodium, potassium, magnesium, and chloride, PL contains 27 m/W (27 mmol/L) sodium acetate and 23 m/W (23 mmol/L) sodium gluconate. This study investigated the effect of acetate and gluconate on platelet metabolism. Identical BC‐PCs were stored at 22 ± 2°C in PL (BC‐PC‐P); PL with gluconate but without acetate, termed PL‐A (BC‐PC‐A); or PL with acetate but without gluconate, termed PL‐G (BC‐PC‐G). On Day 1 of storage, no significant differences were seen between the three groups of BC‐PCs. In both BC‐PC‐P and BC‐PC‐G, pH and bicarbonate were stable at 7.0 ± 0.03 and 8.4 ± 0.9 mEq per L throughout 10 days of storage, whereas in BC‐PC‐A, they fell to 6.7 ± 0.05 and 5.5 ± 0.8 mEq per L on Day 5 (p<0.01 vs. Day 1) and to 6.1 ± 0.1 and 1.2 ± 0.4 mEq per L, respectively, on Day 10. The buffering capacities of 70 percent PL, PL‐A, or PL‐G and 30 percent plasma were similar in a platelet‐free setting when incremental additions of lactic acid were made. The role of acetate was further studied by adding 14C‐ or 3H‐labeled acetate to BC‐PC‐P. On Day 7 of storage, radioactivity decreased to 79 ± 1 percent of baseline in 14C‐labeled BC‐PC‐P (p = 0.02), while it was unchanged in 3H‐labeled BC‐PC‐P and in control samples in which 14C‐ and 3H‐acetate were added to platelet‐free plasma or PL. These studies indicate that platelets metabolize acetate to CO2 during storage and that acetate metabolism plays a major role in pH control during storage of BC‐PCs.

Extension of platelet concentrate storage.

Extension of the storage time of platelet concentrates in a satellite bag which is part of a new blood bag system was studied by reinfusing autologous 51Cr‐labeled platelets into normal volunteers, and measuring postinfusion platelet counts and bleeding times in patients requiring platelet transfusions. This satellite bag, made of polyvinylchloride plasticized with a new agent, was found to protect platelet concentrates against fall of pH better than other containers studied. This protection was felt to be due to the greater gas permeability of the new plastic. Mean in vivo recovery and half‐life (greater than 31% and 3.3 days, respectively) of autologous reinf used platelets were satisfactory following 5 days of storage. Following 7 days of storage, mean recovery was 41 percent and half‐life was 2.8 days. Peripheral platelet count increments in patients following platelet transfusions with concentrates stored 4 to 7 days in the new plastic were comparable to increments following transfusion of platelets stored 2 to 3 days in the other plastics studied. Bleeding times shortened in three of four patients receiving platelet concentrates stored from 4 to 6 days in the new plastic. Platelet concentrates stored in the new bag at 20 to 24 ° C with flat‐bed or elliptical agitation could be transfused for up to 5 days following phlebotomy with acceptable clinical results. The new plastic container is promising for storage of platelet concentrates for up to 7 days. Due to the higher pH of 50‐ml platelet concentrates stored in bags made with the new plastic, the concentrates were superior at any storage interval to those stored in bags made of the other plastics studied.

The efficacy of synthetic media in the storage of human platelets for transfusion

] ~ O R THREE DECADES, specialists in transfu. ] ~ sion medicine have become accustomed to preparing, storing, and transfusing red blood cells suspended in synthetic storage solutions. The idea of a simple nutrient solution containing adenine and glucose for this purpose was apparently first described in 1965.1 It is now widely accepted practice. During storage at 4~ red cells maintain their viability longer in these solutions than they do in plasma. Since the first report of Rock et al 2 in 1985, many investigators have directed their efforts toward developing :an analogous solution for platelets. Potential advantages include superior platelet quality after storage; availability of more plasma for fractionation; and the reduction in frequency and severity of transfusion complications related to components of the suspending plasma. Although 9 progress has been slower than was originally hoped, some succ;essful solutions have been developed and are in use in some transfusion services in Europe. The purpose of this review is to describe the events of the last 15 years that have led to our current circumstances. Inevitably, it will reflect the experience (and the biases) of the author. Where strong disagreements have been expressed, these are noted and addressed. Attention is confined to the storage ofplatelets with appropriate agitation at 20~ to 24~ 3 within plastic containers with gas permeability adequate to maintain the oxidative metabolism of the platelets being stored. 4 The optimal method for assessing the quality of stored platelets is the measurement of their capacity to circulate in vivo after reinfusion. Because of the complexity and expense of such in vivo studies, some of the reports described have used in vitro correlates for in vivo viability. 5 Most of the studies reviewed have used platelets prepared by the platelet-rich plasma (PRP) method from units of whole blood. In this method, whole blood is centrifuged at low speed to prepare PRP, which is then centrifuged at high speed to prepare a platelet button, which is resuspended in approximately 50 mL of autologous plasma, yielding a platelet concentrate (PC). A PC made by this method is referred to as PRP-PC. Typically, when PRP-PC have been used with synthetic media, the button is resuspended in medium, leaving a 10% to 20% plasma carryover. In later studies, the buffy coat (BC) method was used, in which the whole blood is centrifuged at high speed to prepare a BC from which a BC-PC is prepared. Finally, two studies used platelet concentrates prepared by apheresis (AP-PC).