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Research paper thumbnail of Massive clotting in red cell concentrates during transfusion is caused by backflow of patient blood

We report three cases of massive ex vivo coagulation in a leukocyte depleted red cell concentrate... more We report three cases of massive ex vivo coagulation in a leukocyte depleted red cell concentrate during
transfusion. Molecular blood typing indicated that patient blood was present in the filter housing of the infusion
set and/or in the blood bag itself. Together with the history of events, this suggested that the observed clotting
was caused by backflow of patient blood. Therefore, we recommend maintaining the blood bag above heart
level during the entire transfusion procedure, until the infusion set is removed.

Research paper thumbnail of Biochemical and functional impact of pathogen inactivation on plasma and platelets for transfusion

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion

Vox Sanguinis, 2014

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Plate... more Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Research paper thumbnail of UVC PI treatment of PCs preserves integrin activation but affects trhombus formation kinetics on collagen in vitro (supp)

Lowering molecular oxygen partial pressures has no effect on platelets. (A) P-selectin, (B) phosp... more Lowering molecular oxygen partial pressures has no effect on platelets. (A) P-selectin, (B) phosphatidylserine/-ethanolamine by annexin V binding, (C) GPIbα and (D) integrin α IIb β 3 activation was not different in normoxic (open bars) versus hypoxic (shaded bars) conditions. (E) Integrin activation in response to 30µM PAR1AP or (F) 6ng/mL convulxin was also not different. The differences with untreated controls (closed bars) were consequently comparable. The results from statistical analyses are indicated on top of each panel (n=5). ns=not significant; *P<.05; **P<.01; ***P<.001.

Research paper thumbnail of Ultraviolet c light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro

Research paper thumbnail of Persistent aggregates in apheresis platelet concentrates

Vox sanguinis, Jan 6, 2015

Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing pr... more Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /μl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytome...

Research paper thumbnail of Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro

Vox Sanguinis, 2014

An ABO-matched blood sample from healthy non-medicated volunteers was collected in heparin vacuta... more An ABO-matched blood sample from healthy non-medicated volunteers was collected in heparin vacutainers® (REF368480, BD Diagnostics, Franklin Lakes, NJ) with measures for preserving platelet quiescence 1 . The blood was centrifuged at 250g to prepare platelet rich plasma (PRP) and red blood cells (RBC). The PRP and buffy coat were transferred to a new tube and centrifuged at 4500g to yield platelet-poor plasma. The cell pellet was discarded. To obtain reconstituted blood, packed RBC and plasma from this fresh blood were mixed with platelets from the respective research conditions aiming at 40 percent hematocrit and 250,000 platelets/µL. In the reconstituted blood a mean (±SD) platelet concentration of 236±21 x10 3 platelets/µL and a hematocrit of 40.4±1.1% was become for RF-PRT and 248±16 x10 3 platelets/µL and a hematocrit of 40.5±1.3% was become for AS-PCT (both n=6).

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion

Vox Sanguinis, 2014

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol-tris Kinase Signal Transduction by Altering Plasma Membrane Packing

Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a trea... more Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma and graft-versus-host-disease (GVHD). DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery these are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like ALPS and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton’s tyrosine kinase effectors following activation of the collagen glycoprotein VI (GPVI) and thrombin protease activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol (3,4,5)-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (EC50 8.4±1.1 μM vs. 4.3±1.1 μM) and GPVI (EC50 1.61±0.85 μg/mL vs. 0.26±0.21 μg/mL) but not PAR4 (EC50 50±1 μM vs 58±1 μM) signal transduction. Our findings were confirmed in T-cells from GVHD patients treated with extracorporeal photopheresis (ECP), a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids thereby inhibiting membrane recruitment of effector kinases.

Research paper thumbnail of Massive clotting in red cell concentrates during transfusion is caused by backflow of patient blood

We report three cases of massive ex vivo coagulation in a leukocyte depleted red cell concentrate... more We report three cases of massive ex vivo coagulation in a leukocyte depleted red cell concentrate during
transfusion. Molecular blood typing indicated that patient blood was present in the filter housing of the infusion
set and/or in the blood bag itself. Together with the history of events, this suggested that the observed clotting
was caused by backflow of patient blood. Therefore, we recommend maintaining the blood bag above heart
level during the entire transfusion procedure, until the infusion set is removed.

Research paper thumbnail of Biochemical and functional impact of pathogen inactivation on plasma and platelets for transfusion

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion

Vox Sanguinis, 2014

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Plate... more Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Research paper thumbnail of UVC PI treatment of PCs preserves integrin activation but affects trhombus formation kinetics on collagen in vitro (supp)

Lowering molecular oxygen partial pressures has no effect on platelets. (A) P-selectin, (B) phosp... more Lowering molecular oxygen partial pressures has no effect on platelets. (A) P-selectin, (B) phosphatidylserine/-ethanolamine by annexin V binding, (C) GPIbα and (D) integrin α IIb β 3 activation was not different in normoxic (open bars) versus hypoxic (shaded bars) conditions. (E) Integrin activation in response to 30µM PAR1AP or (F) 6ng/mL convulxin was also not different. The differences with untreated controls (closed bars) were consequently comparable. The results from statistical analyses are indicated on top of each panel (n=5). ns=not significant; *P<.05; **P<.01; ***P<.001.

Research paper thumbnail of Ultraviolet c light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro

Research paper thumbnail of Persistent aggregates in apheresis platelet concentrates

Vox sanguinis, Jan 6, 2015

Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing pr... more Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /μl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytome...

Research paper thumbnail of Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro

Vox Sanguinis, 2014

An ABO-matched blood sample from healthy non-medicated volunteers was collected in heparin vacuta... more An ABO-matched blood sample from healthy non-medicated volunteers was collected in heparin vacutainers® (REF368480, BD Diagnostics, Franklin Lakes, NJ) with measures for preserving platelet quiescence 1 . The blood was centrifuged at 250g to prepare platelet rich plasma (PRP) and red blood cells (RBC). The PRP and buffy coat were transferred to a new tube and centrifuged at 4500g to yield platelet-poor plasma. The cell pellet was discarded. To obtain reconstituted blood, packed RBC and plasma from this fresh blood were mixed with platelets from the respective research conditions aiming at 40 percent hematocrit and 250,000 platelets/µL. In the reconstituted blood a mean (±SD) platelet concentration of 236±21 x10 3 platelets/µL and a hematocrit of 40.4±1.1% was become for RF-PRT and 248±16 x10 3 platelets/µL and a hematocrit of 40.5±1.3% was become for AS-PCT (both n=6).

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion

Vox Sanguinis, 2014

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol-tris Kinase Signal Transduction by Altering Plasma Membrane Packing

Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a trea... more Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma and graft-versus-host-disease (GVHD). DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery these are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like ALPS and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton’s tyrosine kinase effectors following activation of the collagen glycoprotein VI (GPVI) and thrombin protease activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol (3,4,5)-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (EC50 8.4±1.1 μM vs. 4.3±1.1 μM) and GPVI (EC50 1.61±0.85 μg/mL vs. 0.26±0.21 μg/mL) but not PAR4 (EC50 50±1 μM vs 58±1 μM) signal transduction. Our findings were confirmed in T-cells from GVHD patients treated with extracorporeal photopheresis (ECP), a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids thereby inhibiting membrane recruitment of effector kinases.