In vitro evaluation of platelets stored in CDP-adenine formulations

Little information is available about the effect of adenine and added glucose on stored platelets. Two new formulations, CPDA-2 and CPDA-3, contain 34 mg adenine per 63 ml preservative and extra glucose (1.75 and 2.0 times the glucose in standard CPD). We have studied the in vitro integrity of platelet concentrates stored in CPD, CPDA-1, CPDA-2, and CPDA-3 at 22 C for 72 hours. Morphology score, pH, platelet size, population distribution parameters, and electron microscopic ultrastructure did not show any adverse effects which could be ascribed to the presence of adenine or extra glucose or both. No differences in platelet adenosine triphosphate (ATP) concentration or plasma glucose utilization during storage were found between CPD and CPDA-1 platelets. The results suggest that adenine and added glucose in these preservatives are not detrimental to platelets in vitro by the measures employed.     

Circulation and function of human platelets isolated from units of CPDA- 1, CPDA-2, and CPDA-3 anticoagulated blood and frozen with DMSO

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate-phosphate-dextrose-adenine solutions (CPDA-1, CPDA-2, CPDA-2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2–3 degrees C per minute and stored in a -80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at -80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid-citrate-dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 ± 17 percent (mean ± one SD). In vitro freeze-thaw-wash recovery determined by phase microscopy was 78.5 ± 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 ± 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin- prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.     

Effects of storage on in vitro platelet responses: Comparison of ACD and Na citrate anticoagulated samples

The present study was designed to evaluate whether the use of acid citrate dextrose (ACD) Formula A may enhance the survival of platelets during storage, thus allowing the continuance of platelet studies over the period of 2–3 hours usually recommended. For this purpose the effects of time on in vitro platelet response to several agonists have been investigated in platelet-rich plasma (PRP) obtained from blood samples anticoagulated with either Na citrate or ACD Formula A. The analysis of the data obtained in in vitro platelet aggregation studies using various parameters and at different time points demonstrated that storage of PRP obtained from citrated samples caused a marked reduction of platelet responses. This reduction was already evident after 6 hours, and a strong decrease was observed after 8 hours with all the agonists used. On the other hand, storage of ACD anticoagulated blood did not cause any significant decrease of platelet responsiveness up to 6 hours. A reduction of platelet aggregation became evident only after 8 hours, but not to the same extent as the one observed in citrated samples. Therefore, it may be concluded that the use of ACD Formula A as anticoagulant is capable of maintaining a normal platelet responsiveness up to 6–8 hours, thus permitting the investigation of platelet function for periods of time over those commonly recommended. © 1996 Wiley-Liss, Inc.     

The in vitro evaluation of modifications in CPD-adenine anticoagulated- preserved blood at various hematocrits

Erythrocytes stored in the new CPD-adenine anticoagulant (CPDA-1) barely met the 70 per cent 24-hour postinfusion 51Cr recoveries on day 35 when stored at hematocrit greater than or equal to 75 per cent. CPDA- 1 differs from CPD in that it has 1.25 times the glucose concentration plus 17.3 mg adenine/63 ml. In an effort to improve the survivability (or viability) of red blood cells following extended storage (35+ days), two new CPD-adenine anticoagulants have been tested in vitro. CPDA-2 and CPDA-3 (both of which contain 34.6 mg/63 ml of anticoagulant or 0.50 mM adenine [final blood concentration], and either 1.75 times or 2.0 times respectively the amount of glucose used in CPD) have been tested for whole blood or red blood cell storage to 42 days. Red blood cell ATP concentrations were better maintained throughout 42 days of storage in both of these formulations than in CPDA-1 at hematocrits that ranged from 40 to 85. Other biochemical parameters (2,3-DPG, pH, plasma hemoglobin) were similar to those of blood stored in CPD or CPDA- 1.     

Factors in the liquid portion of stored blood inhibit the proliferative response in mixed lymphocyte cultures

The transfusion of blood may suppress the immune responses of patients with renal transplants and with malignant disorders. To study the in vitro suppressive effects of banked blood, 4 units of blood were stored in CPDA-1 and ADSOL at 4 degrees C for 14 days. Lymphocytes and plasma or ADSOL supernatants were harvested on Days 0, 4, 7, 10, and 14. Subpopulations of lymphocytes were enumerated by flow cytometry. Recalcified and heat-treated plasma and supernatants from the units of blood were added to mixed lymphocyte cultures (MLC) composed of cells from normal individuals. No significant changes were noted in the proportions of T or B cells from blood stored under these conditions. A 60 +/− 3 percent inhibition in the proliferative response was observed when plasma from CPDA-1 units was added to MLCs (p less than 0.02). Supernatants from ADSOL units demonstrated a 29 +/− 4 percent inhibition (p less than 0.10) of the proliferative response, and this inhibition of response was observed on all 14 days of the study. When appropriate concentrations of dextrose or adenine were added to other MLCs, adenine (at the concentration found in ADSOL) caused a significant inhibition of the proliferative response. This inhibition was not, however, as marked as that observed with recalcified, heat- treated plasma from CPDA-1 units. We conclude that adenine plus some additional factor(s) found in the liquid portion of stored blood inhibits the proliferative response of normal lymphocytes. It is possible that these factors contribute to the immune suppression observed in vivo in some patients who receive blood transfusions.     

Inhibition of activated protein C by platelets.

Activated protein C (APC), an anticoagulant that acts by inactivating Factors Va and VIIIa, is dependent on a suitable surface for its action. In this study we examined the ability of human platelets to provide this surface and support APC-mediated anticoagulant effects. The activity of APC was examined in three systems: the Factor Xa recalcification time of Al(OH)3 adsorbed plasma, studies of thrombin generation in recalcified plasma, and assessment of the rate of inactivation of purified Factor Va. In comparison with phospholipid, intact platelets required significantly greater concentrations of APC to achieve a similar degree of anticoagulation. When washed platelet membranes were substituted for intact platelets, adequate support of APC was observed and the anticoagulant effect was similar to that obtained with phospholipid. Platelet releasate obtained by stimulation of platelets with thrombin and epinephrine contained an inhibitor that interfered with the ability of phospholipid and washed platelet membranes to catalyze the anticoagulant effects of APC. A noncompetitive inhibition was suggested by Dixon plot analysis of the interaction between platelet releasate and APC. The activity of the platelet APC inhibitor was immediate and was not enhanced by heparin, distinguishing it from the circulating protein C inhibitor. The presence of this inhibitor in the platelet and its release with platelet stimulation emphasizes the procoagulant role of this cell.     

An in vivo comparison of CPD and CPDA-2 preserved platelet concentrates after an 8-hour preprocess hold of whole blood

To see if citrate-phosphate-dextrose-adenine-two (CPDA-2) anticoagulant- preservative had an effect on the viability of platelets, we studied autologous in vivo recovery and survival in humans for platelet concentrates prepared from six units of blood drawn into CPDA-2 and compared them to six units drawn into citrate-phosphate-dextrose (CPD). These units were prepared from whole blood held at room temperature for 8 hours after collection and were then stored for 3 days at 22 ± 2 degrees C. The recovery for platelets preserved in CPD was 39.0 +/0 4.8 percent and for platelets preserved in CPDA-2, 32.5 ± 4.4 percent. The difference was not significant (p greater than 0.10). In order to estimate population differences, in vitro effects on in vivo viability were also evaluated. Six in vitro variables were studied but only pH at 72 hours (r = 0.77), platelet count (r = 0.64), and morphology score (r = 0.66) correlated to recovery. Only pH at 72 hours significantly influenced recovery (p = 0.007). By adjusting for individual pH differences, mean recovery for platelets stored in CPD was 37.5 percent, and for platelets stored in CPDA-2, 34.0 percent. The mean lifespan was 6.7 ± 0.7 days for platelets preserved in CPD and 6.1 ± 1.0 days for those preserved in CPDA-2. Although hemostatic function was not studied, these data support in vitro observations that platelets preserved with CPDA-2 are not different from platelets preserved with CPD, even after 8-hours of storage of whole blood at room temperature prior to platelet concentrate preparation.     

Exposure to ultrasound decreases the recalcification time of platelet rich plasma

Human blood was withdrawn, anticoagulated with citrate, and centrifuged, yielding platelet rich plasma (PRP). Recalcification times (i.e. the time taken to form a clot following the addition of sufficient calcium ions) were measured with a semiautomatic device. There were no changes in the recalcification time of PRP sample immediately following exposure to continuous wave 1 MHz ultrasound at intensities in the range 0·065–2W/cm^?2. However, subsequent measurements showed an irreversible time dependent decrease of the recalcification time of an asymptotic value which was invariably less than that of the controls. This behavior can be interpreted as a time dependent alteration to the platelet population.     

Citrate anticoagulation and the dynamics of thrombin generation

BACKGROUND: Sodium citrate has been used as an anticoagulant to stabilize blood and blood products for over 100 years, presumably by sequestering Ca(++) ions in vitro. Anticoagulation of blood without chelation can be achieved by inhibition of the contact pathway by corn trypsin inhibitor (CTI). OBJECTIVE: To evaluate the influence of citrate anticoagulation on the performance of blood, platelet-rich and platelet-poor plasma assays. METHODS: Blood was anticoagulated in three ways: by collection into citrate, CTI and citrate with CTI. Plasma was prepared using each anticoagulation regimen. Functional analyses included calibrated automated thrombography, thromboelastography, plasma clotting, the synthetic coagulation proteome and platelet aggregation. Coagulation reactions were initiated with tissue factor-phospholipid and Ca(++) (when indicated). RESULTS: In all cases, citrate anticoagulation resulted in reaction dynamics significantly altered relative to blood or plasma stabilized with CTI alone. Subsequent experiments showed that calcium citrate itself impairs coagulation dynamics. CONCLUSION: Coagulation analyses using blood that has been exposed to citrate and recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes.     

乙二胺四乙酸盐对肝素钠抗凝后血小板聚集的影响

目的 探讨乙二胺四乙酸盐（EDTA-K2）对肝素钠抗凝后血小板聚集的影响.方法 对38名血小板数量正常的体检者抽取静脉血标本,分别使用EDTA-K2、肝素钠抗凝20min、肝素钠抗凝20 min加入EDTA-K2、肝素钠抗凝60 min加入EDTA-K2的抗凝标本,进行计数及血涂片瑞氏染色观察.结果 肝素钠抗凝静脉血血小板计数结果为（93.7±32.18）× 109/L;EDTA-K2抗凝血血小板计数结果为（210＋58.39）×109/L.组间差异有统计学意义（t=16.74,P＜0.001）.肝素钠抗凝静脉血静置20min后加入EDTA-K2抗凝剂结果为（212.05 ＋60.20）× 109/L;与EDTA-K2抗凝血结果（210＋58.39）× 109/L比较,组间差异无统计学意义（t=1.844,B0.05）.肝素钠抗凝静脉血静置60 min后加入EDTA-K2抗凝剂结果为（56.74＋ 15.33）× 109/L,与EDTA-K2抗凝血结果（210t58.39）× 109/L比较,组间差异有统计学意义（t=22.88,P＜0.001）.结论 EDTA-K2钙离子螯合剂可可逆性地使初发聚集的血小板解聚.     

Effects of some pre-analytical conditions on the measurement of homocysteine and cysteine in plasma.

The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2 h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72 h at room temperature induced a small (approximately equal to 6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72 h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.     

Platelet aggregation and clot formation in comatose survivors of cardiac arrest treated with induced hypothermia and dual platelet inhibition with aspirin and ticagrelor; a prospective observational study

Introduction

We conducted a prospective observational study in cardiac arrest survivors treated with mild induced hypothermia, evaluating different platelet function tests at hypo- and normothermia. We also investigated the relation between gastric emptying and vasodilator stimulated phosphoprotein (VASP).

Methods

Comatose survivors of out of hospital cardiac arrest were included and divided into two groups, depending on whether dual platelet inhibition with peroral ticagrelor and aspirin was given or not. The first blood samples (T1) were collected 12–24 hours after reaching target temperature (33°C) and were compared to blood samples collected 12–28 hours after reaching normothermia (37°C) (T2) within each group. All samples were analysed by Sonoclot viscoelasticity, flow cytometry based VASP and with multiple electrode aggregometry, Multiplate®; adenosine diphosphate (ADP), collagen (COL), thrombin receptor agonist peptide (TRAP) and arachidonic acid (ASPI). Sonoclot and Multiplate® instruments were set on in vivo temperatures. Gastric secretion from the nasogastric tube was measured to assess absorption of per orally administered antiplatelet drugs. Differences between T1 and T2 within each group were calculated using Wilcoxon matched pairs signed test. Significance levels were set at P <0.01.

Results

In total, 23 patients were included. In patients with dual platelet inhibition (n =14) Multiplate®-analyses showed no changes in ADP stimulated platelets. COL, TRAP and ASPI aggregations were higher at T2 compared to T1. Sonoclot-analyses showed that activated clotting time (ACT) was unchanged but both clot rate (CR) and platelet function (PF) were higher at T2 compared to T1. VASP decreased from 53 ± 28(T1) to 24 ± 22(T2), (P <0.001). The average volume of gastric secretion aspirated before T1 correlated well with VASP (T1), r =0.81 (P <0.001). In patients with no platelet inhibition, (n =9) similar changes between T1 and T2 were seen as in patients with dual platelet inhibition while VASP was unchanged.

Conclusions

We have demonstrated increased platelet aggregation and strengthened clot formation over time in out of hospital cardiac arrest patients treated with hypothermia. In patients on oral dual platelet inhibition, the effect of ticagrelor was delayed, probably due to slow gastric emptying.     

血细胞分析仪测定血小板时抗凝剂等影响因素分析

目的为了解血细胞分析仪测定全血中血小板的影响因素,特别是不同抗凝剂的影响程度,以便达到选择合适的抗凝剂,采用正确的操作方法。方法对采集的100份全血样本分别使用3种抗凝剂[肝素、乙二胺四乙酸二钾(EDTA-K2)、枸橼酸钠]抗凝,分别于0、15min时测定血小板计数。经统计学处理,2次结果作配对t检验。结果取标本15min后测定的血小板计数结果高于0min测定的结果 ,差异有统计学意义(P0.001),且采用EDTA-K2作为抗凝剂,对全血中血小板计数的影响最小。结论 EDTA-K2应用于血小板计数,且在室温静置15min后测定结果最适合于机采献血者采前筛查实验用。     

Investigations of Platelet Function in Whole Blood with BAPA as Anticoagulant

BACKGROUND: Citrate anticoagulation of blood results in non-physiologically low calcium concentration and rapid deterioration of platelet viability. Benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide (BAPA) as anticoagulant maintains the physiological calcium level and seems to retain platelet function (PF) over a time period of at least 24 h. We evaluated PF in BAPA-anticoagulated peripheral whole blood (WB) between 0.5 and 30 h after blood collection. METHODS: In WB from 21 healthy volunteers (15 women, 6 men, age range 19-57 years) platelet aggregation (PA) was determined by impedance method and ATP release by luminometry. Platelet response was tested by ADP (10 and 20 μmol/l) and collagen (1 and 2 μg/ml) between 0.5 and 30 h after blood collection. RESULTS: Parameters of ADP-induced PA showed stable values until 6.5 h after blood collection followed by a significant decline. PA in response to collagen was stable up to 25 h of storage. ATP release induced by collagen displayed a continuous, significant decrease over time. CONCLUSION: Preservation of platelet response in BAPA-anticoagulated blood depends on the applied agonist showing that collagen-induced PA is more stable compared to ADP known as a weak agonist in WB.     

The effect of citrate anticoagulants on apheresed plasma

Platelet-rich plasma (PRP) was apheresed from the same donors into four anticoagulant formulations favoured for preparing platelet concentrates (PC), and platelet-poor plasma (PPP) harvested by a second centrifugation. A fifth control collection was made by direct apheresis of PPP. Matched 3–4 kg pools of PPP underwent partial fractionation to determine the effects of plasma anticoagulant on factor VIII recovery and the potential thrombogenicity of prothrombin complex Although an excellent factor VIII recovery was achieved in each case, the cryoprecipitate from plasma apheresed into acid citrate dextrose formula B (ACD-B) showed evidence of incipient clotting, and the factor IX concentrate from the same plasma contained a high concentration of activated factors. It was concluded that ACD-B, in the ratio used, provides insufficient citrate for secure anticoagulation and should not be used when PPP is destined for fractionation of coagulation factor concentrates.     

透析膜的抗凝药物修饰及体外抗凝性能评价

【目的】对透析膜进行抗凝药物修饰，并初步评价其抗凝性能。【方法】以聚丙烯腈透析膜为基材，采用分子自组装技术进行膜修饰，通过亲水角测量法以及抗凝药物吸光度测定跟踪组装过程；复钙化时间检测、血小板粘附试验评价修饰膜的抗凝性能。【结果】亲水角随分子自组装的交替进行而呈锯齿状规律性的变化，抗凝药物阿加曲班的吸光度随组装的进行基本呈线性增加；修饰膜的复钙化时间较未修饰膜明显延长，血小板粘附数目也明显减少，且活力降低。【结论】抗凝药物膜修饰技术基本可行，所得修饰膜具有较好的抗凝性能。     

Perfusion method for thoracoabdominal aneurysm repair using the open distal technique

Challenges related to perfusion support of thoracoabdominal aneurysm repair include maintenance of distal aortic perfusion, rapidity of fluid resuscitation, and avoidance of both hypothermia and excessive hemodilution. Using available technology, we have devised a circuit and protocol that addresses these issues. To accomplish such support a bypass circuit consisting of 3/8 inch tubing connected to a centrifugal pump and low-prime heat exchanger was constructed. The circuit was primed via 1/4 inch spiked connectors attached to a 3-liter bag of normal saline. After initial de-airing, the solution was recirculated through this bag. Patients were anticoagulated with 1 mg/kg of heparin prior to initiation of support. Left atrial-descending aorta bypass was used primarily. A cell salvage device was used for autotransfusion. All blood products were delivered via a rapid infusion device. During partial exsanguination, shed blood was not processed, but directed to the rapid infusor for immediate retransfusion. Any packed cells given were washed prior to transfusion. Citrate dextrose solution was used as an anticoagulant for the cell scavenger. This configuration was used successfully in 50 procedures during an 18-month period. Use of this low-prime, custom circuit reduced both hemodilution and cost. A connection off the cell salvage pump offers fast retransfusion of shed blood during partial exsanguination. Minimal heparinization and citrate anticoagulation appears to reduce coagulopathy.     

Storage of human platelet concentrates in an artificial medium without dextrose

It was shown previously that human blood platelets stored in an artificial medium (PCD) for up to 5 days remain functional in vitro and have normal survival and recovery in vivo. This report demonstrates that the medium can be simplified further by the removal of dextrose, leaving for study a medium consisting simply of balanced salts and citrate anticoagulant (PC). Some dextrose, 3.2 mM, was present in the fresh PC platelet concentrates due to plasma carryover in the production of platelet concentrates, but this dextrose concentration was considerably less than the 22.6 to 25.5 mM present in platelet concentrates in PCD or plasma. Platelet count, pH, PCO2, and PO2, as well as platelet aggregation and release responses to stimulation, in vitro, were as well preserved in the PCD or PC media as in the plasma controls. In the PC medium, platelets consumed 2.5 mM dextrose over 5 days and left 0.7 mM dextrose. The same consumption of dextrose was noted in PCD platelet concentrates, while platelets in plasma metabolized twice as much dextrose and formed twice as much lactate. Thus, the rate of glycolysis in platelet concentrates was independent of the dextrose concentration in the medium, and the platelet functions were well preserved.     

In vivo viability of red blood cells stored in CPDA-2

The marginal viability of erythrocytes stored for 35 days as red blood cell concentrates in citrate-phosphate-dextrose-adenine-one (CPDA-1) was attributed to inadequate nutrient support with adenine and glucose. In an effort to improve the viability of red blood cells following extended storage, a new CPD-adenine and 1.4 times more glucose than CPDA-1. The efficacy of CPDA-2 was evaluated in vivo by measurement of 24-hour postinfusion recovery of 51Cr-labeled erythrocytes which had been stored as whole blood or red blood cell concentrates for 5 to 8 weeks. All red blood cell concentrates, and the whole blood units stored for 35 and 42 days, were held at room temperature for 8 hours prior to processing and/or refrigeration. CPDA-2 yielded significantly higher 51Cr survivals than CPDA-1 and exceeded the accepted criterion for anticoagulant preservative efficacy of 70 percent postinfusion survival of red blood cells after storage for a period of 42 days. Preliminary data supports possible usage to 49 days. Plasma glucose and red blood cell ATP concentration were maintained better in CPDA-2 than in CPDA-1. When compared to historical controls for CPD and CPDA-1 the data suggest that red blood cells stored in CPDA-02 will have superior viability throughout the entire storage period. CPDA-2 is a candidate to replace CPDA-1 as the anticoagulant-preservative solution of choice for red blood cell concentrates.     

BAPA, a synthetic dual inhibitor of Factor Xa and Thrombin, extends the storage-time to a maximum of 12 hours in ADP- and 24 hours in arachidonic acid-induced impedance aggregometry

Impedance aggregometry is mainly limited by the time-dependent instability of platelets in whole blood samples, caused by the influence of ex-vivo anticoagulants. The synthesis of a new anticoagulant, Benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide (BAPA), has recently been introduced. Data from recent studies suggest stable results for up to 32 h. In view of this data we were interested in the results of ADP- and arachidonic acid (AA)-induced measurements after a prolonged storage time depending on the use of citrate or BAPA. Blood samples from 24 healthy individuals were anticoagulated with either citrate or BAPA. ADP- and AA-induced measurements were carried out between 2 h and up to 48 h after blood samples were taken. Results of ADP-induced measurements remained stable for 8 h in citrated and for a maximum of 12 h in BAPA-anticoagulated samples. AA-induced measurements yielded stable results up to 12 h in citrated and up to 24 h in BAPA anticoagulated blood. These data demonstrate that the storage time for whole blood impedance aggregometry can be prolonged when BAPA is used as an anticoagulant. However, in contrast to previous studies implicating considerably longer potential storage times we could not see a clear overall purpose in using BAPA. Further studies including blood samples of patients and the comparison of BAPA to other anticoagulants are necessary to define the potential role of BAPA anticoagulated samples.