Vox Sanguinis (2015) 108, 89–95 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12202
ORIGINAL PAPER
Buffy coat volume reduction for optimization of leucapheresis harvests produced by the AUTOMNC program S. Burk,1 M. Erdmann,2 D. Weiss,1 R. Eckstein1 & E. F. Strasser1 1
Department of Transfusion Medicine and Hemostaseology, University Hospital Erlangen, FAU Erlangen-N€ urnberg, Erlangen, Germany Dermatology Clinic, University Hospital Erlangen, FAU Erlangen-N€ urnberg, Erlangen, Germany
2
Background and Objectives Buffy coat (BC) volume reduction was evaluated in leucapheresis (LA) harvests due to the target monocyte yield and the red blood cell (RBC) content. A packed erythrocyte volume (PEV) of 75 ml should not be exceeded to avoid RBC debulking with loss of leucocytes (WBCs) and the monocyte fraction during monocyte counterflow elutriation, a next step of monocyte enrichment prior to cell culture. Materials and Methods Two hundred and fifty-three 5-l leucaphereses (autoMNC program) performed in 102 healthy blood donors (24 female and 78 male donors) were retrospectively analysed. Different categories of BC volumes were compared due to the quality of the LA products measured by blood counts and flow cytometry. Results Collection of maximum BC volume of 10 ml and more each collection cycle (product volume: 169 – 21 ml) resulted in 158 – 041 9 10e9 CD14+ monocytes and high volume of packed erythrocyte (184 – 88 ml). Low BC volume collection below 6 ml each collection cycle produced only 107 – 040 9 10e9 CD14+ monocytes but reduced PEV significantly by 64% (67 – 41 ml).
Received: 18 January 2014, revised 11 July 2014, accepted 22 July 2014, published online 21 October 2014
Conclusion By reduction of the BC volume, the PEV in LA products could be reduced, which is a precondition for counterflow elutriation of monocytes. A BC volume between 7 and 8 ml per collection cycle should be adjusted to reduce PEV to 75 ml without relevant monocyte loss. Key words: apheresis, cellular therapy, component production.
Introduction Optimization of MNC collection programs towards tailored leucapheresis (LA) products for subsequent cell processing reduces workload for the staff. Optimum target cell yields are of prime importance due to cost-effectiveness in cellular therapy [1, 2]. As previously reported by Erdmann et al., the elutriation apheresis device allows to enrich a monocyte fraction with a purity of more than 80% prior to DC vaccination. The program of the elutriation device, a well-known standard procedure for monoCorrespondence: Erwin F. Strasser, Department of Transfusion Medicine and Hemostaseology, University Hospital of Erlangen-N€urnberg, Krankenhausstr. 12, D-91012 Erlangen, Germany E-mail: [email protected]
cyte enrichment in a closed system, however, performs debulking, if the volume of packed erythrocytes (PEV) in the elutriation chamber derived from the original LA harvest exceeds a volume of 75 ml. As a consequence of this, abundant harvest volume containing monocytes is removed before counterflow elutriation is starting, which results in an unnecessary loss of monocytes. Packed erythrocyte volume (PEV) is calculated from LA volume and RBC concentration [3]. The AUTOMNC program enables the collection of high monocyte yields required for DC vaccination with the disadvantage of high yields of residual platelets (PLTs) and red blood cells (RBC) in the product [4–6]. While the AUTOMNC program software automatically adjusts the spillover volume as previously reported, the operator has to optimize the setting of the buffy coat (BC) volume to
89
90 S. Burk et al.
reduce the part of unwanted cells in the LA product [6]. This manually performed adjustment should assure high monocyte yields during LA as starting population to finally obtain high yields of monocyte-derived DCs (MoDCs) for vaccination. For DC vaccines, leucocyte products should yield more than 10 9 109 CD14+ cells [5–7]. If the target monocyte yield is not achieved, an additional LA is required. The elutriation procedure is an important subsequent preparation step to reduce unwanted PLTs in the original leucocyte product efficiently but is limited due its red cell content. Thus, LA products may require an additional reduction of RBCs prior to elutriation, because low RBC load is prerequisite for optimum MNC yields recovered from elutriation [7, 8]. The AUTOMNC program with the PL-1 single chamber for LA is actually one of the most effective devices for WBC harvest [6]. This retrospective analysis compares yields of WBC, CD14+ monocytes and unwanted RBCs and PLTs in LA products depending on different categories of BC volumes manually adjusted by the operator of the LA.
Materials and methods Two hundred and fifty-three 5-l LA procedures performed in 102 healthy blood donors (78 male donors and 24 female donors) between January 2008 and December 2009 in the Transfusion Medicine and Haemostaseology Department of the University Hospital of Erlangen (Germany) were retrospectively analysed. For leucocyte collection, the AUTOMNC program (software version 4.02.00)
of the COM.TEC cell separator (Fresenius HemoCare GmbH, Bad Homburg, Germany) was used [6]. WBCs, CD14+ monocytes, residual PLTs and red cells were measured pre- and postdonation, as well as in the LA product (Fig. 1).
Donors and apheresis procedures The healthy blood donors gave a written informed consent for LA and met the German guidelines for blood donation [9]. Repeatedly LA procedures were performed in an interval between 2 weeks and 15 months. The AUTOMNC program uses the PL1 single stage chamber and collects the BC after the adjustable ‘spillover phase’ (removal of BC via plasma port) during the ‘BC phase’ (collection of BC in a harvest bag) [6]. An inlet blood flow rate of 50 ml/min was adjusted that complies with a [coupling parameter (CP) of 212]. Centrifugal force of 297 g, coupling parameter (CP) of 212]. The ratio of ACD-A to blood was initially set to 1:10 as basic adjustment. On average, 15 cycles were collected in each donation with a constant cycle blood volume of 350 ml, which showed consistent collection results and thus met the customers’ needs. The Spillover volume is initially set to 150 ml and automatically adjusted by sensor and AUTOMNC program. The BC detection by an interface monitor is controlled by eight optical ports on the template of the first stage of the chamber. The cell to plasma interface was adjusted to a constant ratio of 7:1. The BC volume was 100 ml at the beginning of the LA. For optimum BC collection, the operator adjusted the setting of the BC
Fig. 1 Yields of CD14-positive monocytes related to the reduction of the BC volume. BC, buffy coat.
© 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 89–95
Optimizing buffy coat volume in LA 91
volume manually during LA. All procedures were conducted following the manufacturer’s recommendations.
Table 1 Short overview about the processing data of 253 leucapheresis procedures Measure
Mean – SD
Donors Male/Female Apheresis products (n) Blood volume (ml) Blood processed (ml) ACD-A volume (ml) Collection cycles Buffy coat volume (ml) Collection time (min) Collection volume (ml)
102 78/24 253 5190 – 5093 – 511 – 1501 – 810 – 120 – 128 –
Blood sampling Peripheral blood samples (2 ml, EDTA) were drawn from each donor by the inlet line to the cell separator before and after apheresis as well as from the product. The samples were assayed for WBCs, RBCs, haematocrit and PLT counts on an automated blood cell counter (Sysmex K 1000; TAO Medical, Kobe, Japan). CD14+ monocytes were analysed with a flow cytometer (FACSCalibur; Becton Dickinson, BD, San Jose, CA, USA). For monocyte counts, the samples were prepared following the Lyse/No-Wash method for direct immunofluorescence. Fifty microlitre of each EDTA blood sample was stained with fluorochrome-labelled monoclonal antibodies, CD 45-FITC and CD 14-PE (Becton Dickinson, BD). After incubation for 15 min in the dark at room temperature and addition of 1 ml FACS-Lysing Solution, the samples were analysed by cytometry. The monocytes expressing CD 14 were defined as the percentage of CD45-positive WBCs.
828 309 3146 094 207 639 28
(4) Calculation of cell loss: Cell loss (%) = (1-(postcellcount/precellcount)) 9 (-100) (5) Calculation of Coupling Parameter (CP): Centrifuge velocity (rpm) CP ¼ pffi Inlet blood flow (ml/min) (6) Calculation of packed erythrocytes volume (ml): RBC concentration of LA 9 LA volume 9 0095021
Statistical methods Statistical calculations were performed using a statistical program (SPSS for Windows, version 19; SPSS Inc., Chicago, IL, USA). The results were tested for normal distribution by means of the Shapiro–Wilks tests. In the case of normally distributed data, we performed the Student’s t-test for paired or unpaired samples. If the variables were not distributed normally, statistical analysis was performed using the U-test. Linear associations between two variables were ascertained with the Pearson’s correlation test. The Pearson correlation coefficient was employed to investigate the correlation of variables. Formula: (1) Yield (Y) of a cell population:
Results The mean age of donors was 33 – 10 years (18–58 year) with a mean body weight of 79 – 13 kg and a total blood volume of 512 – 083 l, which also was affected by the gender. The number of collection cycles ranged from 11 to 17, the processed blood volume from 363 to 563 l, respectively. The procedure time is dependent on the number of the collection cycles (mean: 15 cycles) and amounted on average 120 min with a mean BC volume of 8 ml. The processing data of all LA procedures are presented in Table 1. The maximum cell loss affected the donor lymphocytes by 2339% and the PLTs by 3026%. The donor WBC concentration decreased by 93%. The concentration of the donor CD14-positive monocytes diminished from 658% to 572% (Table 2).
Yield [cells] ¼ Cell concentration in the product (per ml) harvest volume (ml)
Collection results of LA procedures
(2) Collection rate (CR): CR = Yield/separation time (min) (3) Collection efficiency (CE):
The WBC concentration of all LA harvests produced with the AUTOMNC program ranged from 137 9 109 WBCs per
CEð%Þ ¼
Yield of cells in the product 100 Processed blood volume*[ml] 0 5 (predonation cell count + postdonation cell count [per ml])
*Processed blood volume = Total volume processed [ACD-A] volume (ml) © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 89–95
l to 823 9 109 WBCs per l (WBC yields: 173 9 109 to 1141 9 109 WBCs per unit) and contained an average
92 S. Burk et al.
Table 2 Donor blood counts, comparing pre- and postdonation counts together with the calculation of cell loss Measure WBCs (9103/ll) Before After WBC loss (%) CD14+ cells (%) Before After CD14+ cells (/ll) Before After CD14+ loss (%) LYMs (%) Before After LYMs (9103/ll) Before After LYM loss (%) PLTs (9103/ll) Before After PLT loss (%)
Mean – SD
P-value
530 – 115 477 – 104 -930 – 1175
ORIGINAL PAPER
Buffy coat volume reduction for optimization of leucapheresis harvests produced by the AUTOMNC program S. Burk,1 M. Erdmann,2 D. Weiss,1 R. Eckstein1 & E. F. Strasser1 1
Department of Transfusion Medicine and Hemostaseology, University Hospital Erlangen, FAU Erlangen-N€ urnberg, Erlangen, Germany Dermatology Clinic, University Hospital Erlangen, FAU Erlangen-N€ urnberg, Erlangen, Germany
2
Background and Objectives Buffy coat (BC) volume reduction was evaluated in leucapheresis (LA) harvests due to the target monocyte yield and the red blood cell (RBC) content. A packed erythrocyte volume (PEV) of 75 ml should not be exceeded to avoid RBC debulking with loss of leucocytes (WBCs) and the monocyte fraction during monocyte counterflow elutriation, a next step of monocyte enrichment prior to cell culture. Materials and Methods Two hundred and fifty-three 5-l leucaphereses (autoMNC program) performed in 102 healthy blood donors (24 female and 78 male donors) were retrospectively analysed. Different categories of BC volumes were compared due to the quality of the LA products measured by blood counts and flow cytometry. Results Collection of maximum BC volume of 10 ml and more each collection cycle (product volume: 169 – 21 ml) resulted in 158 – 041 9 10e9 CD14+ monocytes and high volume of packed erythrocyte (184 – 88 ml). Low BC volume collection below 6 ml each collection cycle produced only 107 – 040 9 10e9 CD14+ monocytes but reduced PEV significantly by 64% (67 – 41 ml).
Received: 18 January 2014, revised 11 July 2014, accepted 22 July 2014, published online 21 October 2014
Conclusion By reduction of the BC volume, the PEV in LA products could be reduced, which is a precondition for counterflow elutriation of monocytes. A BC volume between 7 and 8 ml per collection cycle should be adjusted to reduce PEV to 75 ml without relevant monocyte loss. Key words: apheresis, cellular therapy, component production.
Introduction Optimization of MNC collection programs towards tailored leucapheresis (LA) products for subsequent cell processing reduces workload for the staff. Optimum target cell yields are of prime importance due to cost-effectiveness in cellular therapy [1, 2]. As previously reported by Erdmann et al., the elutriation apheresis device allows to enrich a monocyte fraction with a purity of more than 80% prior to DC vaccination. The program of the elutriation device, a well-known standard procedure for monoCorrespondence: Erwin F. Strasser, Department of Transfusion Medicine and Hemostaseology, University Hospital of Erlangen-N€urnberg, Krankenhausstr. 12, D-91012 Erlangen, Germany E-mail: [email protected]
cyte enrichment in a closed system, however, performs debulking, if the volume of packed erythrocytes (PEV) in the elutriation chamber derived from the original LA harvest exceeds a volume of 75 ml. As a consequence of this, abundant harvest volume containing monocytes is removed before counterflow elutriation is starting, which results in an unnecessary loss of monocytes. Packed erythrocyte volume (PEV) is calculated from LA volume and RBC concentration [3]. The AUTOMNC program enables the collection of high monocyte yields required for DC vaccination with the disadvantage of high yields of residual platelets (PLTs) and red blood cells (RBC) in the product [4–6]. While the AUTOMNC program software automatically adjusts the spillover volume as previously reported, the operator has to optimize the setting of the buffy coat (BC) volume to
89
90 S. Burk et al.
reduce the part of unwanted cells in the LA product [6]. This manually performed adjustment should assure high monocyte yields during LA as starting population to finally obtain high yields of monocyte-derived DCs (MoDCs) for vaccination. For DC vaccines, leucocyte products should yield more than 10 9 109 CD14+ cells [5–7]. If the target monocyte yield is not achieved, an additional LA is required. The elutriation procedure is an important subsequent preparation step to reduce unwanted PLTs in the original leucocyte product efficiently but is limited due its red cell content. Thus, LA products may require an additional reduction of RBCs prior to elutriation, because low RBC load is prerequisite for optimum MNC yields recovered from elutriation [7, 8]. The AUTOMNC program with the PL-1 single chamber for LA is actually one of the most effective devices for WBC harvest [6]. This retrospective analysis compares yields of WBC, CD14+ monocytes and unwanted RBCs and PLTs in LA products depending on different categories of BC volumes manually adjusted by the operator of the LA.
Materials and methods Two hundred and fifty-three 5-l LA procedures performed in 102 healthy blood donors (78 male donors and 24 female donors) between January 2008 and December 2009 in the Transfusion Medicine and Haemostaseology Department of the University Hospital of Erlangen (Germany) were retrospectively analysed. For leucocyte collection, the AUTOMNC program (software version 4.02.00)
of the COM.TEC cell separator (Fresenius HemoCare GmbH, Bad Homburg, Germany) was used [6]. WBCs, CD14+ monocytes, residual PLTs and red cells were measured pre- and postdonation, as well as in the LA product (Fig. 1).
Donors and apheresis procedures The healthy blood donors gave a written informed consent for LA and met the German guidelines for blood donation [9]. Repeatedly LA procedures were performed in an interval between 2 weeks and 15 months. The AUTOMNC program uses the PL1 single stage chamber and collects the BC after the adjustable ‘spillover phase’ (removal of BC via plasma port) during the ‘BC phase’ (collection of BC in a harvest bag) [6]. An inlet blood flow rate of 50 ml/min was adjusted that complies with a [coupling parameter (CP) of 212]. Centrifugal force of 297 g, coupling parameter (CP) of 212]. The ratio of ACD-A to blood was initially set to 1:10 as basic adjustment. On average, 15 cycles were collected in each donation with a constant cycle blood volume of 350 ml, which showed consistent collection results and thus met the customers’ needs. The Spillover volume is initially set to 150 ml and automatically adjusted by sensor and AUTOMNC program. The BC detection by an interface monitor is controlled by eight optical ports on the template of the first stage of the chamber. The cell to plasma interface was adjusted to a constant ratio of 7:1. The BC volume was 100 ml at the beginning of the LA. For optimum BC collection, the operator adjusted the setting of the BC
Fig. 1 Yields of CD14-positive monocytes related to the reduction of the BC volume. BC, buffy coat.
© 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 89–95
Optimizing buffy coat volume in LA 91
volume manually during LA. All procedures were conducted following the manufacturer’s recommendations.
Table 1 Short overview about the processing data of 253 leucapheresis procedures Measure
Mean – SD
Donors Male/Female Apheresis products (n) Blood volume (ml) Blood processed (ml) ACD-A volume (ml) Collection cycles Buffy coat volume (ml) Collection time (min) Collection volume (ml)
102 78/24 253 5190 – 5093 – 511 – 1501 – 810 – 120 – 128 –
Blood sampling Peripheral blood samples (2 ml, EDTA) were drawn from each donor by the inlet line to the cell separator before and after apheresis as well as from the product. The samples were assayed for WBCs, RBCs, haematocrit and PLT counts on an automated blood cell counter (Sysmex K 1000; TAO Medical, Kobe, Japan). CD14+ monocytes were analysed with a flow cytometer (FACSCalibur; Becton Dickinson, BD, San Jose, CA, USA). For monocyte counts, the samples were prepared following the Lyse/No-Wash method for direct immunofluorescence. Fifty microlitre of each EDTA blood sample was stained with fluorochrome-labelled monoclonal antibodies, CD 45-FITC and CD 14-PE (Becton Dickinson, BD). After incubation for 15 min in the dark at room temperature and addition of 1 ml FACS-Lysing Solution, the samples were analysed by cytometry. The monocytes expressing CD 14 were defined as the percentage of CD45-positive WBCs.
828 309 3146 094 207 639 28
(4) Calculation of cell loss: Cell loss (%) = (1-(postcellcount/precellcount)) 9 (-100) (5) Calculation of Coupling Parameter (CP): Centrifuge velocity (rpm) CP ¼ pffi Inlet blood flow (ml/min) (6) Calculation of packed erythrocytes volume (ml): RBC concentration of LA 9 LA volume 9 0095021
Statistical methods Statistical calculations were performed using a statistical program (SPSS for Windows, version 19; SPSS Inc., Chicago, IL, USA). The results were tested for normal distribution by means of the Shapiro–Wilks tests. In the case of normally distributed data, we performed the Student’s t-test for paired or unpaired samples. If the variables were not distributed normally, statistical analysis was performed using the U-test. Linear associations between two variables were ascertained with the Pearson’s correlation test. The Pearson correlation coefficient was employed to investigate the correlation of variables. Formula: (1) Yield (Y) of a cell population:
Results The mean age of donors was 33 – 10 years (18–58 year) with a mean body weight of 79 – 13 kg and a total blood volume of 512 – 083 l, which also was affected by the gender. The number of collection cycles ranged from 11 to 17, the processed blood volume from 363 to 563 l, respectively. The procedure time is dependent on the number of the collection cycles (mean: 15 cycles) and amounted on average 120 min with a mean BC volume of 8 ml. The processing data of all LA procedures are presented in Table 1. The maximum cell loss affected the donor lymphocytes by 2339% and the PLTs by 3026%. The donor WBC concentration decreased by 93%. The concentration of the donor CD14-positive monocytes diminished from 658% to 572% (Table 2).
Yield [cells] ¼ Cell concentration in the product (per ml) harvest volume (ml)
Collection results of LA procedures
(2) Collection rate (CR): CR = Yield/separation time (min) (3) Collection efficiency (CE):
The WBC concentration of all LA harvests produced with the AUTOMNC program ranged from 137 9 109 WBCs per
CEð%Þ ¼
Yield of cells in the product 100 Processed blood volume*[ml] 0 5 (predonation cell count + postdonation cell count [per ml])
*Processed blood volume = Total volume processed [ACD-A] volume (ml) © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 89–95
l to 823 9 109 WBCs per l (WBC yields: 173 9 109 to 1141 9 109 WBCs per unit) and contained an average
92 S. Burk et al.
Table 2 Donor blood counts, comparing pre- and postdonation counts together with the calculation of cell loss Measure WBCs (9103/ll) Before After WBC loss (%) CD14+ cells (%) Before After CD14+ cells (/ll) Before After CD14+ loss (%) LYMs (%) Before After LYMs (9103/ll) Before After LYM loss (%) PLTs (9103/ll) Before After PLT loss (%)
Mean – SD
P-value
530 – 115 477 – 104 -930 – 1175
