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33 Apheresis quality and donor safety and tolerance in triple platelet apheresis. Final results from the DGTI hemapheresis multicentre trial
Abstracts of Swisstransfusion 2010, Joint Congress of WAA, ESFH, BTS/SRC, SATM / Transfusion and Apheresis Science 43 (2010) S1–S27
S11
22 The predictive markers of response for cytapheresis in active ulcerative colitis Y. Yokoyama, K. Fukunaga, K. Kamikozuru, K. Nagase, K. Katou, R. Kikuyama, N. Takeda, T. Matsumoto. Nishinomiya, JP
33 Apheresis quality and donor safety and tolerance in triple platelet apheresis. Final results from the DGTI hemapheresis multicentre trial
Objectives: The predictive parameter of response for Cytapheresis (CAP) in active ulcerative colitis (UC) patient is unclear although this unique therapeutic strategy has been accepted as an effective adjunct therapy for steroid-refractory UC patients with active flare in Japan. This retrospective study has been hence designed to detect the predictive marker for response to CAP by evaluating the correlation between patients’ demographic background and clinical efficiency in patients who received CAP, including either filtration Leukocytapheresis (LCA) or Granulocyte/ Monocyte Adsorption (GMA) from 2007 to 2009. Method: Seventy-one of patients with mild to severe active UC, which is defined as ≥5 points in the Lichtiger’s clinical activity index (CAI), were enrolled in this study. And their CAI ≥ 12, 7–11 and ≥6 were defined as severe, moderate and mild, respectively. All patients have received weekly CAP for 5 consecutive weeks. The mean CAI (points), age (years old), and UC duration (years) prior to start the 1st CAP were 9.0±2.1 (5–16), 40.3±13.2 (19–78), and 8.1±6.6 (1–33), respectively. The primary end point was CAI after 5 CAP sessions. Clinical efficiency of CAP was determined by categorizing the patient into 2 groups of; Remission (R-) group (CAI ≤ 4) and Non-remission (N-) group (CAI ≥ 5), at the primary end point. Degeneration was defined if patient has been required to commence or increase corticosteroid during the observation period. A comparison analysis has been conducted between the efficiency of CAP and the patients’ demographic background, including CAI, C-reactive protein (CRP), UC duration, daily corticosteroid dose, and age. Result: The ratio (number) of R-group treated with LCA and GMA were 50.0% (17/34) and 35.1% (13/37), respectively. Degeneration was obtained in 3 patients (LCA; 1/34, GMA; 2/37). After 5 weekly sessions, the average CAI score significantly decreased in the R-group (LCA: 8.0±1.6 to 3.7±0.6; P < 0.005, GMA: 8.6±2.1 to 3.7±0.5; P < 0.005). The UC activity prior to start LCA was significantly severe in the N-group comparing with that of R-group (CAI score; 10.2±2.3 vs 8.0±1.6; P < 0.005). However, other parameter, such as UC duration, age, daily corticosteroid dose, and CRP value, were not significantly different between the 2 groups receiving LCA. In the patients treated with GMA, the UC duration of R-group was significantly shorter than that of N-group (3.8±3.3 vs 10.0±8.3; P < 0.005). However, other parameters such as CAI, CRP value, daily corticosteroid dose, and age were not significantly differenced in the 2 groups receiving GMA. Conclusion: In this study, UC patient with moderate activity has been determined as a good candidate for the remission induction therapy with weekly LCA; whereas, GMA has been proven to have a significant correlation between UC duration and its response. We have expected that the potential efficacy of this unique therapy could be improved by applying optimum case according to the obtained results.
Objective: Increasing demand for blood products, decreasing donor availability, and economic aspects cause blood centres for collecting more than one product during one apheresis procedure. While the collection of two platelet (PLT) units is a well known standard procedure, poor knowledge exists on potential donor risks when collecting 3 PLT units. For determining potential donor risks as well as the impact on the product quality, a multicentre trial was organized by the DGTI Haemapheresis working party. Design and Methods: From January, 5th to December 31th, 2007, 2249 PLT aphereses (PA) from 411 donors in 11 (9 German, 2 Austrian) haemapheresis centres were included (median 6 PA/donor, range 1 to 12 PA/donor). Inclusion criteria were at least 280x109 PLTs/L prior to apheresis and a body weight of at least 80 kg. Donor randomization was done at a 1:1.2 ratio to donate either two (double unit = DU) or three (triple unit = TU) PLT units with PLT yields of at least 2.5×1011 PLTs per unit (DU: 5.0×1011 PLT, TU: 7.5×1011 ). The predominant evaluation parameters were the donors’ postapheresis PLT counts, the products’ PLT yields, and the apheresis related adverse events. Results: Aphereses were completed using the following devices: Amicus (n = 348), ComTec (n = 395), Trima Accel (n = 1327), Spectra (n = 61), Haemonetics MCSplus (n = 22). There was no difference for flow rate (DU: 63.7±11.8 mL/min, TU: 63.9±12.0 mL/min; n.s.), PLT yield (DU: 2.9±0.4×1011 PLTs, TU: 2.8±0.4×1011 PLTs; n.s.), collection efficiency (DU: 69.2±9.1%, TU: 70.9±9.0%; n.s.), collection rate (DU: 10.4±2.3×109 PLTs/min, TU: 10.8±2.3×109 PLTs/min; n.s.), or WBC contamination (DU: 38±144×103 WBCs, TU: 22±67×103 WBCs; n.s.). The donors presented moderate PLT declines of 86×109 /L for DU (n = 1133) and 114×109 /L for TU (n = 1020); thus, the decline was 25.00±7.90% for DU and 33.99±9.09% for TU. This difference was statistically significant (p < 0.001). Twenty-four out of 1032 completed triple donations (2.3%) from 18 out of 227 triple apheresis donors (7.9%) achieved postapheresis PLT counts of less than 150×109 /L. The lowest PLT value was 106×109 /L. A total of 265 adverse events have been observed. There was no significant difference between DU and TU aphereses regarding venous access problems (VAPs) without abortion (3.8%), but DU aphereses showed less VAPs with abortion than TU aphereses (1.1% vs. 3.0%, resp.; p < 0.01). Furthermore, other reactions occurred more often in TU procedures: citrate toxicity (1.7% vs. 3.9% of DU vs. TU, resp.; p < 0.01), circulatory reactions (0.4% vs. 2.2% of DU vs. TU, resp.; p < 0.01), other problems (1.5% vs. 2.8% of DU vs. TU, resp.; p < 0.04). However, these differences are based on the occurrences of mild reactions, whereas reactions leading to abortion were equally distributed. Conclusions: The implementation of a triple PLT apheresis program does not impair the product quality. Furthermore, triple PLT apheresis is apparently rarely associated with postapheresis PLT counts below 150×109 /L (probability 2%).
J. Zingsem, R. Moog, E.G. Fischer, H.G. Heuft. Erlangen, Bernau, Aachen, Hanover, DE
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Abstracts of Swisstransfusion 2010, Joint Congress of WAA, ESFH, BTS/SRC, SATM / Transfusion and Apheresis Science 43 (2010) S1–S27
In no case TU apheresis resulted in postapheresis PLT counts below 100×109 /L. Adverse events such as mild citrate toxicity, mild circulatory reactions and abortion due to VAP may increase in triple PLT aphereses.
Orals (national) 50 Ageing and storage of erythrocyte concentrates J. Delobel, O. Rubin, D. Crettaz, JD. Tissot, N. Lion. Epalinges, CH Introduction: Red blood cell (RBC) concentrate is the main labile blood product used in transfusion medicine. Numerous efforts are made to ensure quality and security of blood products from collection to transfusion. The standard preparation of RBC concentrates is as follows: whole blood is collected into anticoagulant solution. Plasma, platelets and leukocyte are then removed. And finally, RBCs are stored at 4°C in a conservative solution from 35 up to 49 days depending on the solution and protocols. Non-physiological cold-storage may provoke RBC alterations which are well described in the literature and known as “RBC storage lesions”. Those lesions include a decrease of ATP, 2,3-DPG and due to transient loss of function of cation pumps, there is a consequent accumulation of sodium and loss of potassium. Moreover, there are oxidative damages changing band 3 structure, lipids peroxidation or accumulation of met-hemoglobin. RBC membranes become more rigid, there are externalization of negative phospholipids, lipid raft formation, vesiculation and microparticles release. It takes few hours to some weeks to observe physico-chemical changes such as pH decrease, or increase in potassium or lactate in the concentrate medium. Those changes affecting transfusion efficiency are not yet clearly understood. Studying storage lesions, we already demonstrate an accumulation of microparticles during storage of RBC concentrates. As mentioned above, among the different storage lesions, RBCs are subjected to oxidative stress, which leads to the oxidation of part of their protein content. Oxidative lesions can be investigated by different ways. Results presented here are based on spectrophotometric detection and quantitation of protein carbonylation in cytoplasmic protein extracts from RBCs of different time of storage at 4°C in the SAGM additive solution. Results: Protein carbonylation is an irreversible oxidative post-translational modification consisting in the addition of a carbonyl group (C = 0) on lateral side chain of amino acids, particularly on Lysine, Arginine, Proline and Threonine. Carbonyl group could either be an aldehyde or a ketone one. The assay used here is based on derivatization of carbonylated proteins with 2,4-dinitrophenylhydrazine (2,4-DNPH). The reaction leads to formation of a proteinDNP hydrazone complex. Hydrazone optic density at 375 nm permits to quantify carbonylation. Preliminary results show a storage time-related increase of the carbonylated protein content (see figure1) which seems to present an important increase at day 14 (from 1.7 nmol per mg of protein to 3.2 nmol/mg), with a maximum at day 22
(3.7 nmol/mg), and seems to stay relatively constant with a little decrease until day 42 (2.7 nmol/mg). Since RBCs from a same donation are not all at the same lifetime, it’s interesting to investigate the protein carbonylation content of population of different ages, and from RBC concentrates of different storage periods. Such fractionation is performed by density centrifugation through a Percoll gradient. Low density RBCs have a higher MCV compare to high density RBCs. Thus, upper fraction is thought to be composed of younger RBCs and lower fraction of older ones. Perspective: Contrary to expected, results show that it’s the population of lower density (supposed to be composed of younger RBCs) which have higher carbonylation content. In our hands, it appears that number of microparticles released by RBCs increase during storage. A possible explanation could be that the microparticulation process allows RBC to eliminate harmful components, of which carbonylated proteins possibly belongs. These interesting finding have to be confirmed and new experiments on microparticles and oxidation have to be conduct to support these results.
Figure 1. Cytoplasmic carbonylation content in comparison with the time of storage. Carbonylation assay of cytoplasmic extracts of red blood cells of different storage time at 4°C in SAGM additive solution. For each extract, assay is performed in triplicate. Data are presented as the mean value of the triplicates ± standard deviation. 28 Serology helps moleculardiagnostics to solve a rare case of homozygous FY*X/FY*X – consequences for transfusion recommendations C. Engstrom, ¨ B. Grossrieder, C. Gassner, B.M. Frey. Schlieren, CH Background: The DUFFY (FY) blood group system is expressed on the DARC protein (Duffy antigen/receptor for chemokines, CD234), which is a 336 AA multipass glycoprotein type I of the red blood cell (RBC) membrane and contains 6,000–13,000 copies of DARC. DARC is encoded by two exons on chromosome 1q22–23 and expresses 7 antigens, namely Fya (G125), Fyb (A125), Fyx (A125, T265, A298), Fy3, Fy4, Fy5, Fy6. FY*X is a variant FY*B allele with dramatically weakened antigen expression. The Fy(a-b-) phenotype is also prevalent among Africans and as such mainly due to an upstream promoter mutation T-67C (GATA-1, assigned as FY*Fy(Bnull-67C)) which prevents expression of DARC on erythropoietic tissue. In our case, however, we observed a patient with Fy(a-b-) phenotype
S11
22 The predictive markers of response for cytapheresis in active ulcerative colitis Y. Yokoyama, K. Fukunaga, K. Kamikozuru, K. Nagase, K. Katou, R. Kikuyama, N. Takeda, T. Matsumoto. Nishinomiya, JP
33 Apheresis quality and donor safety and tolerance in triple platelet apheresis. Final results from the DGTI hemapheresis multicentre trial
Objectives: The predictive parameter of response for Cytapheresis (CAP) in active ulcerative colitis (UC) patient is unclear although this unique therapeutic strategy has been accepted as an effective adjunct therapy for steroid-refractory UC patients with active flare in Japan. This retrospective study has been hence designed to detect the predictive marker for response to CAP by evaluating the correlation between patients’ demographic background and clinical efficiency in patients who received CAP, including either filtration Leukocytapheresis (LCA) or Granulocyte/ Monocyte Adsorption (GMA) from 2007 to 2009. Method: Seventy-one of patients with mild to severe active UC, which is defined as ≥5 points in the Lichtiger’s clinical activity index (CAI), were enrolled in this study. And their CAI ≥ 12, 7–11 and ≥6 were defined as severe, moderate and mild, respectively. All patients have received weekly CAP for 5 consecutive weeks. The mean CAI (points), age (years old), and UC duration (years) prior to start the 1st CAP were 9.0±2.1 (5–16), 40.3±13.2 (19–78), and 8.1±6.6 (1–33), respectively. The primary end point was CAI after 5 CAP sessions. Clinical efficiency of CAP was determined by categorizing the patient into 2 groups of; Remission (R-) group (CAI ≤ 4) and Non-remission (N-) group (CAI ≥ 5), at the primary end point. Degeneration was defined if patient has been required to commence or increase corticosteroid during the observation period. A comparison analysis has been conducted between the efficiency of CAP and the patients’ demographic background, including CAI, C-reactive protein (CRP), UC duration, daily corticosteroid dose, and age. Result: The ratio (number) of R-group treated with LCA and GMA were 50.0% (17/34) and 35.1% (13/37), respectively. Degeneration was obtained in 3 patients (LCA; 1/34, GMA; 2/37). After 5 weekly sessions, the average CAI score significantly decreased in the R-group (LCA: 8.0±1.6 to 3.7±0.6; P < 0.005, GMA: 8.6±2.1 to 3.7±0.5; P < 0.005). The UC activity prior to start LCA was significantly severe in the N-group comparing with that of R-group (CAI score; 10.2±2.3 vs 8.0±1.6; P < 0.005). However, other parameter, such as UC duration, age, daily corticosteroid dose, and CRP value, were not significantly different between the 2 groups receiving LCA. In the patients treated with GMA, the UC duration of R-group was significantly shorter than that of N-group (3.8±3.3 vs 10.0±8.3; P < 0.005). However, other parameters such as CAI, CRP value, daily corticosteroid dose, and age were not significantly differenced in the 2 groups receiving GMA. Conclusion: In this study, UC patient with moderate activity has been determined as a good candidate for the remission induction therapy with weekly LCA; whereas, GMA has been proven to have a significant correlation between UC duration and its response. We have expected that the potential efficacy of this unique therapy could be improved by applying optimum case according to the obtained results.
Objective: Increasing demand for blood products, decreasing donor availability, and economic aspects cause blood centres for collecting more than one product during one apheresis procedure. While the collection of two platelet (PLT) units is a well known standard procedure, poor knowledge exists on potential donor risks when collecting 3 PLT units. For determining potential donor risks as well as the impact on the product quality, a multicentre trial was organized by the DGTI Haemapheresis working party. Design and Methods: From January, 5th to December 31th, 2007, 2249 PLT aphereses (PA) from 411 donors in 11 (9 German, 2 Austrian) haemapheresis centres were included (median 6 PA/donor, range 1 to 12 PA/donor). Inclusion criteria were at least 280x109 PLTs/L prior to apheresis and a body weight of at least 80 kg. Donor randomization was done at a 1:1.2 ratio to donate either two (double unit = DU) or three (triple unit = TU) PLT units with PLT yields of at least 2.5×1011 PLTs per unit (DU: 5.0×1011 PLT, TU: 7.5×1011 ). The predominant evaluation parameters were the donors’ postapheresis PLT counts, the products’ PLT yields, and the apheresis related adverse events. Results: Aphereses were completed using the following devices: Amicus (n = 348), ComTec (n = 395), Trima Accel (n = 1327), Spectra (n = 61), Haemonetics MCSplus (n = 22). There was no difference for flow rate (DU: 63.7±11.8 mL/min, TU: 63.9±12.0 mL/min; n.s.), PLT yield (DU: 2.9±0.4×1011 PLTs, TU: 2.8±0.4×1011 PLTs; n.s.), collection efficiency (DU: 69.2±9.1%, TU: 70.9±9.0%; n.s.), collection rate (DU: 10.4±2.3×109 PLTs/min, TU: 10.8±2.3×109 PLTs/min; n.s.), or WBC contamination (DU: 38±144×103 WBCs, TU: 22±67×103 WBCs; n.s.). The donors presented moderate PLT declines of 86×109 /L for DU (n = 1133) and 114×109 /L for TU (n = 1020); thus, the decline was 25.00±7.90% for DU and 33.99±9.09% for TU. This difference was statistically significant (p < 0.001). Twenty-four out of 1032 completed triple donations (2.3%) from 18 out of 227 triple apheresis donors (7.9%) achieved postapheresis PLT counts of less than 150×109 /L. The lowest PLT value was 106×109 /L. A total of 265 adverse events have been observed. There was no significant difference between DU and TU aphereses regarding venous access problems (VAPs) without abortion (3.8%), but DU aphereses showed less VAPs with abortion than TU aphereses (1.1% vs. 3.0%, resp.; p < 0.01). Furthermore, other reactions occurred more often in TU procedures: citrate toxicity (1.7% vs. 3.9% of DU vs. TU, resp.; p < 0.01), circulatory reactions (0.4% vs. 2.2% of DU vs. TU, resp.; p < 0.01), other problems (1.5% vs. 2.8% of DU vs. TU, resp.; p < 0.04). However, these differences are based on the occurrences of mild reactions, whereas reactions leading to abortion were equally distributed. Conclusions: The implementation of a triple PLT apheresis program does not impair the product quality. Furthermore, triple PLT apheresis is apparently rarely associated with postapheresis PLT counts below 150×109 /L (probability 2%).
J. Zingsem, R. Moog, E.G. Fischer, H.G. Heuft. Erlangen, Bernau, Aachen, Hanover, DE
S12
Abstracts of Swisstransfusion 2010, Joint Congress of WAA, ESFH, BTS/SRC, SATM / Transfusion and Apheresis Science 43 (2010) S1–S27
In no case TU apheresis resulted in postapheresis PLT counts below 100×109 /L. Adverse events such as mild citrate toxicity, mild circulatory reactions and abortion due to VAP may increase in triple PLT aphereses.
Orals (national) 50 Ageing and storage of erythrocyte concentrates J. Delobel, O. Rubin, D. Crettaz, JD. Tissot, N. Lion. Epalinges, CH Introduction: Red blood cell (RBC) concentrate is the main labile blood product used in transfusion medicine. Numerous efforts are made to ensure quality and security of blood products from collection to transfusion. The standard preparation of RBC concentrates is as follows: whole blood is collected into anticoagulant solution. Plasma, platelets and leukocyte are then removed. And finally, RBCs are stored at 4°C in a conservative solution from 35 up to 49 days depending on the solution and protocols. Non-physiological cold-storage may provoke RBC alterations which are well described in the literature and known as “RBC storage lesions”. Those lesions include a decrease of ATP, 2,3-DPG and due to transient loss of function of cation pumps, there is a consequent accumulation of sodium and loss of potassium. Moreover, there are oxidative damages changing band 3 structure, lipids peroxidation or accumulation of met-hemoglobin. RBC membranes become more rigid, there are externalization of negative phospholipids, lipid raft formation, vesiculation and microparticles release. It takes few hours to some weeks to observe physico-chemical changes such as pH decrease, or increase in potassium or lactate in the concentrate medium. Those changes affecting transfusion efficiency are not yet clearly understood. Studying storage lesions, we already demonstrate an accumulation of microparticles during storage of RBC concentrates. As mentioned above, among the different storage lesions, RBCs are subjected to oxidative stress, which leads to the oxidation of part of their protein content. Oxidative lesions can be investigated by different ways. Results presented here are based on spectrophotometric detection and quantitation of protein carbonylation in cytoplasmic protein extracts from RBCs of different time of storage at 4°C in the SAGM additive solution. Results: Protein carbonylation is an irreversible oxidative post-translational modification consisting in the addition of a carbonyl group (C = 0) on lateral side chain of amino acids, particularly on Lysine, Arginine, Proline and Threonine. Carbonyl group could either be an aldehyde or a ketone one. The assay used here is based on derivatization of carbonylated proteins with 2,4-dinitrophenylhydrazine (2,4-DNPH). The reaction leads to formation of a proteinDNP hydrazone complex. Hydrazone optic density at 375 nm permits to quantify carbonylation. Preliminary results show a storage time-related increase of the carbonylated protein content (see figure1) which seems to present an important increase at day 14 (from 1.7 nmol per mg of protein to 3.2 nmol/mg), with a maximum at day 22
(3.7 nmol/mg), and seems to stay relatively constant with a little decrease until day 42 (2.7 nmol/mg). Since RBCs from a same donation are not all at the same lifetime, it’s interesting to investigate the protein carbonylation content of population of different ages, and from RBC concentrates of different storage periods. Such fractionation is performed by density centrifugation through a Percoll gradient. Low density RBCs have a higher MCV compare to high density RBCs. Thus, upper fraction is thought to be composed of younger RBCs and lower fraction of older ones. Perspective: Contrary to expected, results show that it’s the population of lower density (supposed to be composed of younger RBCs) which have higher carbonylation content. In our hands, it appears that number of microparticles released by RBCs increase during storage. A possible explanation could be that the microparticulation process allows RBC to eliminate harmful components, of which carbonylated proteins possibly belongs. These interesting finding have to be confirmed and new experiments on microparticles and oxidation have to be conduct to support these results.
Figure 1. Cytoplasmic carbonylation content in comparison with the time of storage. Carbonylation assay of cytoplasmic extracts of red blood cells of different storage time at 4°C in SAGM additive solution. For each extract, assay is performed in triplicate. Data are presented as the mean value of the triplicates ± standard deviation. 28 Serology helps moleculardiagnostics to solve a rare case of homozygous FY*X/FY*X – consequences for transfusion recommendations C. Engstrom, ¨ B. Grossrieder, C. Gassner, B.M. Frey. Schlieren, CH Background: The DUFFY (FY) blood group system is expressed on the DARC protein (Duffy antigen/receptor for chemokines, CD234), which is a 336 AA multipass glycoprotein type I of the red blood cell (RBC) membrane and contains 6,000–13,000 copies of DARC. DARC is encoded by two exons on chromosome 1q22–23 and expresses 7 antigens, namely Fya (G125), Fyb (A125), Fyx (A125, T265, A298), Fy3, Fy4, Fy5, Fy6. FY*X is a variant FY*B allele with dramatically weakened antigen expression. The Fy(a-b-) phenotype is also prevalent among Africans and as such mainly due to an upstream promoter mutation T-67C (GATA-1, assigned as FY*Fy(Bnull-67C)) which prevents expression of DARC on erythropoietic tissue. In our case, however, we observed a patient with Fy(a-b-) phenotype