ORIGINAL ARTICLE Factor VIII and fibrinogen recovery in plasma after Theraflex methylene blue-treatment: effect of plasma source and treatment time André Rapaille1, Stefan Reichenberg2, Tome Najdovski1, Nicolas Cellier1, Nicolas de Valensart1, Véronique Deneys1 1
Belgian Red Cross, Blood Service, Suarlée, Belgium; 2Macopharma International GmbH, Langen, Germany
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Background. The quality of fresh-frozen plasma is affected by different factors. Factor VIII is sensitive to blood component storage processes and storage as well as pathogen-reduction technologies. The level of fibrinogen in plasma is not affected by the collection processes but it is affected by preparation and pathogen-reduction technologies. Materials and methods. The quality of plasma from whole blood and apheresis donations harvested at different times and treated with a pathogen-reduction technique, methylene blue/light, was investigated, considering, in particular, fibrinogen and factor VIII levels and recovery. Results. The mean factor VIII level after methylene blue treatment exceeded 0.5 IU/mL in all series. Factor VIII recovery varied between 78% and 89% in different series. The recovery of factor VIII was dependent on plasma source as opposed to treatment time. The interaction between the two factors was statistically significant. Mean levels of fibrinogen after methylene blue/light treatment exceeded 200 mg/dL in all arms. The level of fibrinogen after treatment correlated strongly with the level before treatment. There was a negative correlation between fibrinogen level before treatment and recovery. Pearson's correlation coefficient between factor VIII recovery and fibrinogen recovery was 0.58. Discussion. These results show a difference in recovery of factor VIII and fibrinogen correlated with plasma source. The recovery of both factor VIII and fibrinogen was higher in whole blood plasma than in apheresis plasma. Factor VIII and fibrinogen recovery did not appear to be correlated.
Introduction
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Keywords: factor VIII, fibrinogen, methylene blue, MB-FFP.
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The quality of fresh-frozen plasma is affected by numerous factors such as the source of the plasma (whole blood/apheresis), preparation time and treatment with a pathogen-reduction technique1-8. Pathogen-reduction techniques are used to reduce the residual risk of transmission of infections after serological and nucleic acid testing9. Treatment with methylene blue and light (MB) is a well-known procedure for inactivating bloodborne viruses in fresh-frozen plasma. The aim of this study was to investigate the effect of plasma source and preparation time on the quality of MB-treated fresh-frozen plasma in terms of factor VIII (FVIII) and fibrinogen levels and recovery. Moreover, we aimed to study a possible correlation between FVIII and fibrinogen recovery.
Material and methods Plasma collection and processing Whole blood units were collected into "top and bottom" configuration quadruple bags sets (Composelect®, Fresenius Kabi, Bad Homburg,
Germany) with 63 mL of CPD anticoagulant (sodium citrate 26.3 g/L, citric acid 3.3 g/L, dextrose 25.5 g/L, sodium biphosphate 2.5 g/L, pH 5.3-5.9). The centrifuged units were processed in accordance with standard procedures to obtain red blood cell concentrate, buffy-coat and plasma (T-ACE II, Terumo BCT Europe, Zaventem, Belgium). Apheresis plasma collections were harvested in accordance with standard procedures using Autopheresis-CTM (Fenwal, Mont Saint Guibert, Belgium) equipment. A container for plasma collection was docked to the disposable set. The blood was anticoagulated with a 4% citrate solution (sodium citrate dehydrate 40g/L, pH 6.4-7.5) at a ratio of 1:16 (6%). Plasma selection is based on gender (mainly male donors), no history of past transfusions and/or pregnancy and volume. By visual in-process control, lipaemic or haemolytic specimens were excluded from the MB treatment process. All plasma collections were treated using the MacoPharma Theraflex MB-Plasma Bag system with PLAS4 filter and Bluexflex filter (MacoPharma, Tourcoing, France). Plasma units were connected to the Theraflex kit and filtered by gravity. Blood Transfus 2014; 12: 226-31 DOI 10.2450/2014.0176-13 © SIMTI Servizi Srl
226 All rights reserved - For personal use only No other uses without permission
FVIII and fibrinogen recovery in MB-treated FFP
Fibrinogen and factor VIII measurement The concentration of fibrinogen (factor I) was measured following Clauss's method with thrombin reagent (Dade Behring) on a CA-7000 automated coagulation analyser (Sysmex Ltd., Milton Keynes, UK). FVIII was quantified using one-stage clotting times with Actin-FS (Dade-Behring) on a CA-1500 automated instrument (Sysmex Ltd.). Clotting factor-deficient plasmas consisted of human immune-adsorbed plasmas and were obtained from Dade-Behring.
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Statistical analysis The values were analysed using the Anderson-Darlin test for normal distribution. All results are expressed as the mean±standard deviation (SD) and range for each study arm. Comparisons of FVIII and fibrinogen recovery between the various groups were performed using analysis of variance (ANOVA) for independent groups and two factors: source (WB or apheresis) and treatment time (D0, D1, F-T). Subsequent statistical analyses were performed with Student's t-test to uncover which groups were different. Correlations were examined using Pearson's correlation coefficient (r). The rates of FVIII exceeding 0.5 UI/mL and fibrinogen exceeding 200 mg/dL between different groups were evaluated with Pearson's chi-square association test. Differences and correlations were considered to be statistically significant when the P value was lower than 0.05. All analyses were performed using Minitab® Statistical Software (State College, Pennsylvania, USA).
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Anhydrous MB chloride (85 μg) in the form of a dry pill was sufficient to obtain a concentration of 1 μM based on a plasma unit having a volume of 266 mL (range, 230318 mL). The pill, in the transfer line, dissolved in the plasma during transfer. The packs were illuminated using the MacoTronic (Macopharma) plasma illumination system. The peak emission wavelength for the lamps was 590 nm. This procedure delivers the required dose of 180 J/cm2. Illuminated plasma was filtered through a Blueflex filter to eliminate at least 90% of MB and its photoresidues. The whole procedure was performed in accordance with the manufacturer's instructions. Plasma collected by apheresis was split into two or three units and leucodepleted with the Plasmaflex PLAS4 filter before MB-treatment. All plasma units (MB-treated or not) were frozen to −30 °C with the same procedure within 60 minutes using a shock freezer (MBP42, Dometic, Hosingen, Luxembourg) in respect of the Council of Europe recommendations10. WB plasma collections were frozen within 18 hours of collection; apheresis plasmas within 6 hours of collection. They were stored for 2 to 20 days (mean 7 days for WB plasmas and 12 days for apheresis plasmas) at −30 °C. They were thawed in a water bath at 30 °C for 60 minutes and immediately treated by the MB procedure. The study comprised six study arms each involving 30 plasmas (Table I). In one branch, whole blood-derived (WB) plasmas were separated and treated within 6 hours of collection (D0), separated after whole blood storage on eutectic plates at 20 °C and processed within 18 hours of collection (D1) or treated after freezing and thawing (F-T). In the other branch, apheresis plasmas (Aph) were treated within 6 hours (D0), stored on eutectic plates at 20 °C and treated within 18 hours (D1) or treated after freezing and thawing (F-T).
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Plasma sampling For each plasma unit, samples were taken just before MB-treatment and immediately after treatment. All samples were snap-frozen and stored at a temperature below −70 °C before FVIII and fibrinogen were assessed.
Results Plasma factor VIII Detailed results including mean SD, minimum and maximum, are presented in Table II for both FVIII and fibrinogen. The means of FVIII after MB treatment from all arms of the study exceeded 0.50 IU/mL. The percentage of plasmas in which the FVIII level exceeded 0.5 IU/mL varied between 83% for the WB and frozenthawed plasma and 100% for the WB plasma on day 0. The Pearson's chi-square result (0.258, p=0.879) showed no difference in the proportion of plasma with FVIII exceeding 0.50 UI/mL between the different groups.
Table I - Sampling design. WB plasma Study arm Treatment time
Apheresis plasma
A
B
C
D
E
F
Day 0
Day 1
Frozen-thawed
Day 0
Day 1
Frozen-thawed
Number units
30
30
30
30
30
30
Group O
10
12
0
13
13
10
Group A, B, AB Volume before treatment (mL)
20
18
30
17
17
20
272±16
279±13
273±13
267±35
244±6
276±40
Sampling taken before and after treatment.
Blood Transfus 2014; 12: 226-31 DOI 10.2450/2014.0176-13 227 All rights reserved - For personal use only No other uses without permission
Rapaille A et al Table II - Plasma factor recovery in MB-treated apheresis and WB plasma. Whole blood Study arm
Apheresis
WB D0
WB D1
WB F-T
Before treatment
1.20±0.30 (0.67-1.79)
0.79±0.19 (0.44-1.10)
0.81±0.16 (0.42-1.24)
After treatment
1.08±0.27 (0.57-1.67)
0.70±0.16 (0.37-0.96)
Recovery (%)
89±6 (76-100) 100
Aph D0
Aph D1
Aph F-T
1.04±0.27 (0.57-1.71)
0.98±0.23 (0.54-1.57)
1.04±0.33 (0.33-2.03)
89±7 (76-99)
0.67±0.18 (0.30-1.22) 82±10*† (53-98)
0.83±0.24 (0.49-1.37) 80±10*† (54-97)
0.76±0.21 (0.44-1.30) 78±8*† (59-91)
0.85±0.27 (0.29-1.66) 83±5*†‡ (73-97)
87
83
93
93
93
FVIII (IU/mL)
% units >0.5 IU/mL
Fibrinogen (mg/dL) 258±49 (143-400)
260±53 (172-386)
269±49 (171-372)
257±38 (184-353)
254±43 (198-411)
284±66 (171-414)
After treatment
235±40 (137-347)
231±47 (146-319)
Recovery (%)
92±4 (83-100)
89±6 (77-99)
229±48 (141-342) 85±7*§ (67-97)
219±35 (178-353) 85±7*§ (70-100)
219±35 (170-324) 87±5* (79-99)
244±53 (149-361) 86±4*§ (76-93)
90
70
70
70
63
80
% units >200 mg/dL
Sr l
Before treatment
result was obtained in apheresis plasma treated on day 1 (78±8%). All treated plasmas of the six arms had a FVIII recovery above 50%. ANOVA results showed a significant effect of plasma source (p
Belgian Red Cross, Blood Service, Suarlée, Belgium; 2Macopharma International GmbH, Langen, Germany
TI
Se
rv
iz i
Sr l
Background. The quality of fresh-frozen plasma is affected by different factors. Factor VIII is sensitive to blood component storage processes and storage as well as pathogen-reduction technologies. The level of fibrinogen in plasma is not affected by the collection processes but it is affected by preparation and pathogen-reduction technologies. Materials and methods. The quality of plasma from whole blood and apheresis donations harvested at different times and treated with a pathogen-reduction technique, methylene blue/light, was investigated, considering, in particular, fibrinogen and factor VIII levels and recovery. Results. The mean factor VIII level after methylene blue treatment exceeded 0.5 IU/mL in all series. Factor VIII recovery varied between 78% and 89% in different series. The recovery of factor VIII was dependent on plasma source as opposed to treatment time. The interaction between the two factors was statistically significant. Mean levels of fibrinogen after methylene blue/light treatment exceeded 200 mg/dL in all arms. The level of fibrinogen after treatment correlated strongly with the level before treatment. There was a negative correlation between fibrinogen level before treatment and recovery. Pearson's correlation coefficient between factor VIII recovery and fibrinogen recovery was 0.58. Discussion. These results show a difference in recovery of factor VIII and fibrinogen correlated with plasma source. The recovery of both factor VIII and fibrinogen was higher in whole blood plasma than in apheresis plasma. Factor VIII and fibrinogen recovery did not appear to be correlated.
Introduction
SI M
Keywords: factor VIII, fibrinogen, methylene blue, MB-FFP.
©
The quality of fresh-frozen plasma is affected by numerous factors such as the source of the plasma (whole blood/apheresis), preparation time and treatment with a pathogen-reduction technique1-8. Pathogen-reduction techniques are used to reduce the residual risk of transmission of infections after serological and nucleic acid testing9. Treatment with methylene blue and light (MB) is a well-known procedure for inactivating bloodborne viruses in fresh-frozen plasma. The aim of this study was to investigate the effect of plasma source and preparation time on the quality of MB-treated fresh-frozen plasma in terms of factor VIII (FVIII) and fibrinogen levels and recovery. Moreover, we aimed to study a possible correlation between FVIII and fibrinogen recovery.
Material and methods Plasma collection and processing Whole blood units were collected into "top and bottom" configuration quadruple bags sets (Composelect®, Fresenius Kabi, Bad Homburg,
Germany) with 63 mL of CPD anticoagulant (sodium citrate 26.3 g/L, citric acid 3.3 g/L, dextrose 25.5 g/L, sodium biphosphate 2.5 g/L, pH 5.3-5.9). The centrifuged units were processed in accordance with standard procedures to obtain red blood cell concentrate, buffy-coat and plasma (T-ACE II, Terumo BCT Europe, Zaventem, Belgium). Apheresis plasma collections were harvested in accordance with standard procedures using Autopheresis-CTM (Fenwal, Mont Saint Guibert, Belgium) equipment. A container for plasma collection was docked to the disposable set. The blood was anticoagulated with a 4% citrate solution (sodium citrate dehydrate 40g/L, pH 6.4-7.5) at a ratio of 1:16 (6%). Plasma selection is based on gender (mainly male donors), no history of past transfusions and/or pregnancy and volume. By visual in-process control, lipaemic or haemolytic specimens were excluded from the MB treatment process. All plasma collections were treated using the MacoPharma Theraflex MB-Plasma Bag system with PLAS4 filter and Bluexflex filter (MacoPharma, Tourcoing, France). Plasma units were connected to the Theraflex kit and filtered by gravity. Blood Transfus 2014; 12: 226-31 DOI 10.2450/2014.0176-13 © SIMTI Servizi Srl
226 All rights reserved - For personal use only No other uses without permission
FVIII and fibrinogen recovery in MB-treated FFP
Fibrinogen and factor VIII measurement The concentration of fibrinogen (factor I) was measured following Clauss's method with thrombin reagent (Dade Behring) on a CA-7000 automated coagulation analyser (Sysmex Ltd., Milton Keynes, UK). FVIII was quantified using one-stage clotting times with Actin-FS (Dade-Behring) on a CA-1500 automated instrument (Sysmex Ltd.). Clotting factor-deficient plasmas consisted of human immune-adsorbed plasmas and were obtained from Dade-Behring.
rv
iz i
Sr l
Statistical analysis The values were analysed using the Anderson-Darlin test for normal distribution. All results are expressed as the mean±standard deviation (SD) and range for each study arm. Comparisons of FVIII and fibrinogen recovery between the various groups were performed using analysis of variance (ANOVA) for independent groups and two factors: source (WB or apheresis) and treatment time (D0, D1, F-T). Subsequent statistical analyses were performed with Student's t-test to uncover which groups were different. Correlations were examined using Pearson's correlation coefficient (r). The rates of FVIII exceeding 0.5 UI/mL and fibrinogen exceeding 200 mg/dL between different groups were evaluated with Pearson's chi-square association test. Differences and correlations were considered to be statistically significant when the P value was lower than 0.05. All analyses were performed using Minitab® Statistical Software (State College, Pennsylvania, USA).
SI M
TI
Se
Anhydrous MB chloride (85 μg) in the form of a dry pill was sufficient to obtain a concentration of 1 μM based on a plasma unit having a volume of 266 mL (range, 230318 mL). The pill, in the transfer line, dissolved in the plasma during transfer. The packs were illuminated using the MacoTronic (Macopharma) plasma illumination system. The peak emission wavelength for the lamps was 590 nm. This procedure delivers the required dose of 180 J/cm2. Illuminated plasma was filtered through a Blueflex filter to eliminate at least 90% of MB and its photoresidues. The whole procedure was performed in accordance with the manufacturer's instructions. Plasma collected by apheresis was split into two or three units and leucodepleted with the Plasmaflex PLAS4 filter before MB-treatment. All plasma units (MB-treated or not) were frozen to −30 °C with the same procedure within 60 minutes using a shock freezer (MBP42, Dometic, Hosingen, Luxembourg) in respect of the Council of Europe recommendations10. WB plasma collections were frozen within 18 hours of collection; apheresis plasmas within 6 hours of collection. They were stored for 2 to 20 days (mean 7 days for WB plasmas and 12 days for apheresis plasmas) at −30 °C. They were thawed in a water bath at 30 °C for 60 minutes and immediately treated by the MB procedure. The study comprised six study arms each involving 30 plasmas (Table I). In one branch, whole blood-derived (WB) plasmas were separated and treated within 6 hours of collection (D0), separated after whole blood storage on eutectic plates at 20 °C and processed within 18 hours of collection (D1) or treated after freezing and thawing (F-T). In the other branch, apheresis plasmas (Aph) were treated within 6 hours (D0), stored on eutectic plates at 20 °C and treated within 18 hours (D1) or treated after freezing and thawing (F-T).
©
Plasma sampling For each plasma unit, samples were taken just before MB-treatment and immediately after treatment. All samples were snap-frozen and stored at a temperature below −70 °C before FVIII and fibrinogen were assessed.
Results Plasma factor VIII Detailed results including mean SD, minimum and maximum, are presented in Table II for both FVIII and fibrinogen. The means of FVIII after MB treatment from all arms of the study exceeded 0.50 IU/mL. The percentage of plasmas in which the FVIII level exceeded 0.5 IU/mL varied between 83% for the WB and frozenthawed plasma and 100% for the WB plasma on day 0. The Pearson's chi-square result (0.258, p=0.879) showed no difference in the proportion of plasma with FVIII exceeding 0.50 UI/mL between the different groups.
Table I - Sampling design. WB plasma Study arm Treatment time
Apheresis plasma
A
B
C
D
E
F
Day 0
Day 1
Frozen-thawed
Day 0
Day 1
Frozen-thawed
Number units
30
30
30
30
30
30
Group O
10
12
0
13
13
10
Group A, B, AB Volume before treatment (mL)
20
18
30
17
17
20
272±16
279±13
273±13
267±35
244±6
276±40
Sampling taken before and after treatment.
Blood Transfus 2014; 12: 226-31 DOI 10.2450/2014.0176-13 227 All rights reserved - For personal use only No other uses without permission
Rapaille A et al Table II - Plasma factor recovery in MB-treated apheresis and WB plasma. Whole blood Study arm
Apheresis
WB D0
WB D1
WB F-T
Before treatment
1.20±0.30 (0.67-1.79)
0.79±0.19 (0.44-1.10)
0.81±0.16 (0.42-1.24)
After treatment
1.08±0.27 (0.57-1.67)
0.70±0.16 (0.37-0.96)
Recovery (%)
89±6 (76-100) 100
Aph D0
Aph D1
Aph F-T
1.04±0.27 (0.57-1.71)
0.98±0.23 (0.54-1.57)
1.04±0.33 (0.33-2.03)
89±7 (76-99)
0.67±0.18 (0.30-1.22) 82±10*† (53-98)
0.83±0.24 (0.49-1.37) 80±10*† (54-97)
0.76±0.21 (0.44-1.30) 78±8*† (59-91)
0.85±0.27 (0.29-1.66) 83±5*†‡ (73-97)
87
83
93
93
93
FVIII (IU/mL)
% units >0.5 IU/mL
Fibrinogen (mg/dL) 258±49 (143-400)
260±53 (172-386)
269±49 (171-372)
257±38 (184-353)
254±43 (198-411)
284±66 (171-414)
After treatment
235±40 (137-347)
231±47 (146-319)
Recovery (%)
92±4 (83-100)
89±6 (77-99)
229±48 (141-342) 85±7*§ (67-97)
219±35 (178-353) 85±7*§ (70-100)
219±35 (170-324) 87±5* (79-99)
244±53 (149-361) 86±4*§ (76-93)
90
70
70
70
63
80
% units >200 mg/dL
Sr l
Before treatment
result was obtained in apheresis plasma treated on day 1 (78±8%). All treated plasmas of the six arms had a FVIII recovery above 50%. ANOVA results showed a significant effect of plasma source (p
