Platelet Biochemistry and Morphology after Cryopreservation

Platelet cryopreservation has been investigated for several decades as an alternative to room temperature storage of platelet concentrates. The use of dimethylsulfoxide as a cryoprotectant has improved platelet storage and cryopreserved concentrates can be kept at −80 °C for two years. Cryopreserved platelets can serve as emergency backup to support stock crises or to disburden difficult logistic areas like rural or military regions. Cryopreservation significantly influences platelet morphology, decreases platelet activation and severely abrogates platelet aggregation. Recent data indicate that cryopreserved platelets have a procoagulant phenotype because thrombin and fibrin formation kicks in earlier compared to room temperature stored platelets. This happens both in static and hydrodynamic conditions. In a clinical setting, low 1-h post transfusion recoveries of cryopreserved platelets represent fast clearance from circulation which may be explained by changes to the platelet GPIbα receptor. Cryopreservation splits the concentrate in two platelet subpopulations depending on GPIbα expression levels. Further research is needed to unravel its physiological importance. Proving clinical efficacy of cryopreserved platelets is difficult because of the heterogeneity of indications and the ambiguity of outcome measures. The procoagulant character of cryopreserved platelets has increased interest for use in trauma stressing the need for double-blinded randomized clinical trials in actively bleeding patients

A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

Background and objectives: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0 center dot 0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 +/- 9% versus 76 +/- 11%, respectively (P < 0 center dot 0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7 center dot 4 platelet-like particles per input cell from PB compared to 4 center dot 2 from UCB (P = 0 center dot 02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank

A microfluidic flow chamber model for platelet transfusion and hemostasis measures platelet deposition and fibrin formation in real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay

Impact of cold storage on platelets treated with Intercept pathogen inactivation

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogen-inactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22 degrees C), cold (4 degrees C, n = 6), or cryopreservation (-80 degrees C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS Platelet aggregation of cold-stored AS-PCT PLTs was 44% +/- 11% compared to 57% +/- 14% for cold-stored standard PLTs and 58% +/- 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% +/- 9% compared to 77% +/- 6% for cold-stored standard PLTs and 69% +/- 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 +/- 140 sec) compared to cold-stored standard PLTs (960 +/- 192 sec) and RT-stored AS-PCT PLTs (1134 +/- 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 +/- 47 nmol/L thrombin compared to 159 +/- 33 nmol/L for cold-stored standard PLTs and 83 +/- 25 nmol/L for RT-stored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% +/- 10%) was not significantly different from cryopreserved standard PLTs (25% +/- 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation

A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

Background and objectives: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0 center dot 0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 +/- 9% versus 76 +/- 11%, respectively (P < 0 center dot 0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7 center dot 4 platelet-like particles per input cell from PB compared to 4 center dot 2 from UCB (P = 0 center dot 02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank

Impact of cold storage on platelets treated with Intercept pathogen inactivation

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogen-inactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22 degrees C), cold (4 degrees C, n = 6), or cryopreservation (-80 degrees C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS Platelet aggregation of cold-stored AS-PCT PLTs was 44% +/- 11% compared to 57% +/- 14% for cold-stored standard PLTs and 58% +/- 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% +/- 9% compared to 77% +/- 6% for cold-stored standard PLTs and 69% +/- 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 +/- 140 sec) compared to cold-stored standard PLTs (960 +/- 192 sec) and RT-stored AS-PCT PLTs (1134 +/- 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 +/- 47 nmol/L thrombin compared to 159 +/- 33 nmol/L for cold-stored standard PLTs and 83 +/- 25 nmol/L for RT-stored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% +/- 10%) was not significantly different from cryopreserved standard PLTs (25% +/- 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation