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Comparison of manual and machine plasmapheresis techniques
Plasma Ther Transfis Technol 1988; 9:337-342 Printed in Great Britain. AI1 rights reserved
027%6222188 $3.00+0.00 Copyright @ 1988 Pergamon Press plc
Comparison of Manual and Machine Plasmapheresis Techniques G. Rock N. McCombie V. FuIler P. Tittley
n While the benefits of automated plasmapheresis have been described, little data is available to compare it to manual plasmapheresis. We therefore compared standard manual techniques (MP) with three devices (API, automated Haemoneticb Models V50 and PCS, and the HemaScience Autopheresis-CTM (HA). Plasma protein concentrations were 56 + 4 g/L for MP, 63 + 2 g/L for the VSO,59 + 5 g/L for the PCS and 63 + 5 g/L for the HA. Factor WI recoveries averaged 105 + 23 U/dL for the MP, 91 + 18 U/dL and 125 f 25 U/dL for the V50 (PPP and PRP modes) 95 * 27 U/dL for the PCS and 121+ 22 U/dL for the HA, showing a statistically significant difference (P < 0.05) between manual and automated procedures. Percentage recovery of activity in the cryoprecipitate was similar, giving 337 f 80 U or 63% for MP, 262 f 70 U or 57% for VSO,252 + 60 U or 57% for PCS and 367 + 110 U or 59% for HA. The plasma from MP and HA contained relatively few cells with 12 f 5 x 109/L and 6 rt 1 x 109/Lplatelets; the V50 plasma (PPP mode j contained an average of 103 + 4 x 109/L platelets whereas the PCS plasma had 51 f 22 x 109/L. (3Thromboglobulin levels were significantly elevated in the plasma from the V50. Automated procedures took only
3MO min whereas the manual procedures took a minimum of 2 hr. While different economic factors will affect the choice of methodology, there is considerable benefit to the use of certain automated devices rather than the widely used manual method for the collection of plasma. l
INTRODUCTION In many countries plasmapheresis is carried out to provide plasma for fractionation with the major purpose being to generate adequate quantities of Factor VIII to meet national requirements for self-sufficiency. There are a number of methods available for plasmapheresis and these differ both in the technique and the type and amount of anticoagulant which are used. In the standard manual technique, a double bag is used to remove whole blood then, following centrifugation, the red cells are returned to the donor, the plasma is retained and the procedure is repeated to obtain a total of 500-600 mL of plasma. Recently, several automated devices which operate by both centrifugal and membrane techniques have become available for plasmapheresis. These methods have certain advantages over manual procedures in that they are faster and avoid the risk of returning the wrong red cells to a donor.14 Nonetheless, the manual method is used in the
From the Ottawa Centre, Canadian Red Cross, Blood Transfusion Service, 85 PI 011th Street, Ottawa and Departments of Medicine, r lochemIstry and Pathology, Umversity of Ottawa, Canada. Received 6/88, Accepted S/SE. 337
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majority of centers, particularly in the United States. We have reported that different levels of Factor VIII:C are obtained depending on the amount and type of citrate anticoagulant which is used, with a direct relationship between the level of divalent calcium and the Factor VIII:C recovery? as the level of ionized calcium decreases, so does the Factor VIII:C recovery.5 Another important factor in the choice of anticoagulant is the known relationship between pH of the solution and Factor VIII:C recovery.6 The loss of as much as 15% of the Factor VIII:C has been reported when blood is collected into ACD-A which has a pH of 4.9. This loss has been attributed to the “anticoagulant shock” which occurs when the first few millilitres of blood are introduced into the large volume of anticoagulant. The method of adding anticoagulant to blood is also important: it has been shown that constant mixing of the blood during collection, with little generation of fibrinopeptide A (FPA), contributes to better Factor VIII:C recovery.’ Since these various parameters are different in manual and automated plasmapheresis procedures, we have examined the plasma product and the Factor VIII:C recovery using five available methods of plasmapheresis. We have also assessed, during manual plasmapheresis, the effect of the return of the anticoagulated red cell concentrate on the circulating levels of Factor VIII:C in the donor, since it is commonly believed that during manual plasmapheresis the Factor VIII:C level is lower in the second bag. Sequential samples were also taken from donors during some of the automated procedures to determine any changes. METHODS AND MATERIALS The plasmapheresis procedures were carried out on normal, healthy donors who gave informed consent and met the usual criteria for donation of the Canadian Red Cross. According to manufacturers’ recommended standard operating procedures, a total of 500 mL of plasma product
(i.e. plasma + anticoagulant) were collected by manual plasmapheresis (MP) (n = 10) using a 1 in 10 ratio of 4% sodium citrate (Cutter Laboratories, Berkeley, automated plasCalifornia); by mapheresis using the Haemonetick VSO machine (V.50) (Haemonetics Corporation, Braintree, Massachusetts), both in the platelet-rich plasma (PRP) mode using ACD-A as the anticoagulant in a ratio of 1 in 12 (n = 5) and in the platelet-poor plasma (PPP) mode using a 4% sodium citrate anticoagulant in a 1 in 15 ratio (n = 5); by the more recently developed Haemonetics PCS device using the Latham bowl and a 1 in 16.6 ratio of sodium citrate (n = 10) and by the machine (HA) Autopheresis-CTM (HemaScience Laboratories, Santa Ana, California) using ACD-A as the anticoagulant in a 1 in 12.5 ratio (n = 10). Samples were taken from the donor before the procedure and from the plasma product. To permit direct comparison of these values between procedures, the donor samples were drawn into the standard laboratory anticoagulants for each test. Various measurements were made as follows: protein and albumin by a multi-channel autoanalyzer (model SMA18 Technicon Corporation, Tarrytown, New York); platelet count using an electronic particle counter (model ZB, Coulter Electronics, Hialeah, Florida); total microaggregate volume in the size range of l&100 ~1diameter (model TA2, Coulter Electronics); Factor VIII:C content using a one-stage assays against a Factor VIII:C reference plasma calibrated in WHO units (Thromboscreen, Pacific Hemostasis, Bakersfield, California); (3thromboglobulin (BTG), platelet factor 4 (PF4) and fibrinopeptide A (FPA) using commercial kits (Amersham Intemational, U.K., Abbott Laboratories, Montreal, Quebec and Mallinckrodt, St Louis, Missouri). The volume of the final product included the volume of anticoagulant as well as the collected plasma. A 20 mL aliquot was removed from the apheresis plasma product, placed in small amounts and frozen at -60°C until assayed for the above parameters. The
Comparisonof Plasmapheresis
plasma product was frozen at - 80°Cin a blast freezer (model PCF, Harris Manufacturing, N. Bellerica, Massachusetts) and cryoprecipitates were subsequently made using a rapid-thaw method.9 The Factor VIII:C level of the cryoprecipitate was determined and the percentage recovery was calculated based on the volume of plasma made into cryo (i.e. post sampling for baseline values]. The effect of returning the anticoagulant on the circulating level of Factor VIII:C in the donor was assessed during both MP and HA. Serial samples were taken from the donor’s free arm during the course of the procedure into the standard anticoagulant in use in clinical laboratories (3.8% sodium citrate in a 1 in 10 ratio) and the Factor VIII:C levels were measured. Statistical analysis was carried out using the Student t-test; P-values of less than 0.05 were considered significant. All values are given as mean + standard deviation. RESULTS Manual plasmapheresis lo), which use a double contain 50 mL of 4% anticoagulant in each
procedures (n = bag system and sodium citrate of the primary
Techniques
339
blood bags, produced a final product with a volume of 583 + 15 mL including both plasma and anticoagulant. The average total protein was 56 g/L; albumin was 35 g/L (Table 1). The Factor VIII:C levels averaged 105 U/dL with 337 + 80 units in the cryoprecipitate, representing a recovery of 63 f 3%. The FPA levels varied considerably, whereas BTG values were relatively low. Some microaggregate formation was noted. Collection and processing of the 2 bags took an averge of 120 + 30 min. Samples taken from the donor into the standard anticoagulant, following removal of the first bag of blood, and prior to the re-infusion of the first bag of packed red cells, showed a slight decrease in circulating Factor VI&C levels, from the pre-plasmapheresis values of 117 +_ 20 U/dL to 110 f 22 U/dL (P = 0.45). Following completion of the procedure, i.e. after return of the second bag of packed red cells, the circulating Factor VIII:C level was 104 + 20 U/dL. This was not significantly reduced (P > 0.05). Correction for dilution by anticoagulant of these and other values was not necessary since the same type and volume of anticoagulant was used for donor sampling. Comparison of the contents of the
Table 1. Comparison of Apheresis Products
Donor pre-procedure3 Factor VIILC (U/dL) Total protein (g/L] Product Volume (mL) Factor VIII:C (U/dL) Total protein (g/L] Albumin (g/L) Citrate (mM) BTG (ng/mL) PF4 (ng/mL FPA (ng/mL) Platelet count (X 109/L) Microaggregates ( x lOQ/mL) No. Tested
Manual’
V-50 PPP Mode
117k20 70*5
95f19 73+3
5835 15 105+23 56+4 35f2 20f2 56+38 19t-25 1225 12+6 10
514-t4
V-50 PRP Mode PRP PPP2
PCS
AutoDheresis C
123+24 73*3
123f24 73+3
95+27 69?6
121+22 75+3
598+20
467+47 125?25 63+2 43+2 1751 194*40 83+47 3+1 20+5 8+2 5
493+5 95+_27 59+5 39+4 23+3 440+200 87+59 3k2 51k22 18+3 10
504f26 121f22 63+5 4122 17-1-2 66f18 29+9 4+3 6fl 3+1 10
91+18 125f25 63f2 63f2 39+3 43+2 12+1 17+1 443+252 115*28 144599 72f20 2+1 3&l 103+4 393+32 8Ot-3 5 s
’ Mean of both plasma bags. 2 PPP was prepared by centrifugation of the PRP product.’ 3 Blood was taken from the donor into the standard anticoagulant.
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first and second plasma bags showed only slight differences for protein, albumin and Factor VIII:C levels. The differences for Factor VIII:C (106 f 24 and 101 + 22 U/dL) were not statistically significant (P > 0.05); however, the total protein {SS + 3 and 54 + 4 g/L) and albumin (36 + 2 and 34 f 2 g/L) levels were significantly different (P< 0.05). When the VSOmachine was used in the PPP mode (n = 5) with an average of three passes to collect 500 mL of plasma with 4% sodium citrate at a 1 in 15 ratio, the protein level was 63 Ifr2 g/L and the albumin was 39 f 3 g/L (Table 1). The Factor VIII:C level was 91 t- 18 U/dL with a recovery of 262 _+70 units in the cryoprecipitate, representing 57 + 6%. The BTG and PF4 level were high at 443 + 252 ng/mL, and 144 + 99 ng/mL. The FPA was 2 f 1 ng/mL and a small amount of microaggregate formation was noted. Analyses of samples taken from the plasma collection bag showed slight decreases (P > 0.05) in the level of Factor VIII:C in the collected plasma over time as earlier reported (data not shown).1 An average total of 75 mL of anticoagulant was used to collect the 500 mL of plasma which required 30 min. Using the V50 in the PRP mode (n = 5) and after removal of platelets from the plasma by subsequent centrifugation in the laboratory,2 467 + 47 mL of plasma were collected with an average total protein of 63 f 2 g/L and an albumin content of 43 + 2 g/L (Table 1J. The total volume of anticoagulant used was 137 + 33 mL with the amount required depending on donor hematocrit, as is the case for all automated apheresis procedures. Total collection time was 45 min. The Factor VIII:C level was 125 +_25 U/dL and the cryoprecipitate contained 333 f 156 units, representing 56 f 17% recovery. Prior to centrifugation to obtain the platelet concentrate (PC), the BTG was 115f28ng/mLandthePF4was72+20 ng/mL. After centrifugation, the BTG level in the PPP product was 194 f 40 ng/mL and the PF4 was 83 +- 47 ng/mL. The PCS machine, used with the Latham bowl, produced a plasma with 5 1
+ 22 x 109/Lplatelets, and averge protein level of 59 g/L and 95 + 27 U/dL of VIILC. In the cryoprecipitate, 57 f 11% FVIII (252 f 60U) was recovered. The BTG level in the plasma was 440 ng/mL (Table 1). An average of 100 mL of anticoagulant was used to collect 493 f. 5 mL of plasma. When the Autopheresis machine was used (n = lo), the plasma protein and albumin levels were 63 + 5 g/L and 41+ 2 g/L (Table 1). The Factor VIII:C levels averaged 121 U/dL. The cryoprecipitate contained 367 + 110 units with 59 + 10% recovery. BTG and PF4 levels were 66 f 18 ng/mL and 29 + 9 ng/mL respectively with little microaggregate or FPA formation. The plasma collection was completed in 30 min. Sequential samples taken into the standard anticoagulant from the opposite arm of five donors during the process showed a progressive decrease in the circulating levels of Factor VIII:C from 137 + 38 U/dL before plasmapheresis to 117 + 34 U/dL at 15 min after completion of the procedure (P > 0.05). An average of 150 + I3 mL of anticoagulant was used during collection of the 500 mL of plasma volume. DISCUSSION Better Factor VIII:C recoveries are seen in plasma obtained by AP rather than MP. One advantage of all of the machines is the automated metering of anticoagulant to blood near the site of venepuncture. This permits delivery of a constant volume of anticoagulant and mixing with the blood to avoid denaturation of Factor VIII:C (FVIILC) caused by exposure to low pH6 or by activation of coagulation. This could explain, in part, the improved recovery of FVIII:C. Another major determinant of the plasma FVIII:C level seems to be the absolute amount of anticoagulant used and the resultant amount of citrates per mL of plasma. Both the albumin and FVIII:C concentrations were higher with the automated devices. This is due simply to dilutional factors since all the machines use less anticoagulant per mL
Comparison of Plasmapheresis Techniques
of blood. Another recent study of automated plasmapheresis devices has also reported that the level of VIII:C was comparable in the Haemonetics and HemaScience machines,10 although, in that study, no comparison was made with the manual method. Another potential explanation for the higher FVIII values seen, is the use of different anticoagulants since ACD-A contains considerably less citrate per mL (21 mg) then does 4% sodium citrate (26 mg). Studies by Mustard’1 and our laboratory4 have shown an inverse relationship between the level of citrate and FVIII:C with an increase in the FVIILC as the citrate level decreases. However, in this case there is very little difference in the final citrate concentrations in the plasma (20 + 2 mM for MP and 17 _+ 2 mM for HA). Therefore, it is unlikely that the absolute amount of citrate accounted for the differences in FVIII:C noted. The increase in the BTG values in the plasma obtained with the VSO machine indicates release from the platelet a-granules during processing. This increase in BTG using the V50 has been previously noted by others,12 however, the significance is not known. These platelets are reported to have normal survival and recovery and be acceptable for in vitro function.2J2 Although not significantly different (P = 0.45), there was a slight decrease in the circulating levels of Factor VIII:C in the donors during plasmapheresis. This is likely due to the combined effects of the anticoagulant and dilution. Thus, both logistically and in terms of Factor VIII:C recovery, there are benefits to the use of automated devices rather than manual procedures to collect plasma. In some cases, the ultimate determining factor in conversion to automated methods will be the cost. Software costs are higher for automated devices (US $10-15) than for manual procedures (US $6-8). However, the labour-intensive monitoring required with MP, plus the requirement for additional centrifugation, offset the higher software costs of the automated procedures. Reduction of
341
costs through bulk purchasing will also affect the final cost of the plasma. In countries such as Canada, where a principle of national self-sufficiency in blood products and plasma has been adopted, the automated devices have considerable appeal, particularly in ensuring protection of the volunteer donor and reducing the required time commitment such that plasmapheresis becomes an acceptable form of routine volunteer donation. Acknowledgements The authors wish to acknowledge the cooperation and assistance of the Nursing Department and the Apheresis Section of the Ottawa Centre of the Canadian Red Cross. Thanks are also due to Carol McCoshen and Rena Tennant for their assistance in typing the manuscript.
REFERENCES I. Rock G, McCombie N, Tittley P: A new
2.
3.
4.
5.
6.
7.
8.
technique for the collection of plasma: machine plasmapheresis. Transfusion 1981; 21:241-246. Rock G, Herzig R, McCombie N, Lazarus H, Tittley P: Automated platelet production during plasmapheresis. Transfusion 1983; 23:29&293. Rock G, Tittley P, McCombie N: Plasma collection using an automated membrane device. Transfusion 1986; 26:269271. Rock G, Cruickshank W, Tackaberry E, Ganz P, Palmer D: Stability of VIII:C in plasma: the dependence on protease activity and calcium. Thomb Res 1983; 29:521-535. Rock G, Tackaberry E, Palmer D: Factors affecting the relative distribution of high and low molecular weight forms of Factor VIILC. Thromb Res 1979; 14:573-587. Vermeer C, Soute B, Ates G, Hellings J, Brummelhuis H: Contributions to the optimal use of human blood. VIII. Stability of blood coagulation Factor VIILC during collection and storage of whole bloodandplasma. VOXSang 1976; 31:5567. Pflugshaupt R, Kurt G: FPA content-a criterion of quality for plasma as Factor VIII:C source. VOX Sang 1983; 45:224-232. Hardistry R, Macpherson J: A one-stage Factor VIII:C (antihemophilic globulin) assay and its use on venous and capillary plasma. Thromb Diath Haemorrh 1962; 7:215-228.
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9. Rock G, Tittley P: The effect of temperature variations on cryoprecipitate. Transfusion 19793 19:86-89. 10. Vezon G. Piauet Y. Manier C. Schooneman F, iesnier F, Ivloulinier J:‘Technical aspects of different donor plasmapheresis systems and biological results obtained in collected plasma. VOXSang 1986; 51 (Suppl. 1):40-44. 11 Mustard J: Somein vitro effects of various concentrations of disodium ethylene-
diamine tetracetate, potassium oxalate, and sodium citrate on coagulation of blood. Am I Clin Path01 19583 30:498506. 12. Penny A, Townley A, Angela E, Robinson A, Warrington R, Learoyd P, Gilkes L, Tovey L: Pilot study of an automated dual component plasmapheresis collection system. Apheresis Bull 1984; 2:312.
027%6222188 $3.00+0.00 Copyright @ 1988 Pergamon Press plc
Comparison of Manual and Machine Plasmapheresis Techniques G. Rock N. McCombie V. FuIler P. Tittley
n While the benefits of automated plasmapheresis have been described, little data is available to compare it to manual plasmapheresis. We therefore compared standard manual techniques (MP) with three devices (API, automated Haemoneticb Models V50 and PCS, and the HemaScience Autopheresis-CTM (HA). Plasma protein concentrations were 56 + 4 g/L for MP, 63 + 2 g/L for the VSO,59 + 5 g/L for the PCS and 63 + 5 g/L for the HA. Factor WI recoveries averaged 105 + 23 U/dL for the MP, 91 + 18 U/dL and 125 f 25 U/dL for the V50 (PPP and PRP modes) 95 * 27 U/dL for the PCS and 121+ 22 U/dL for the HA, showing a statistically significant difference (P < 0.05) between manual and automated procedures. Percentage recovery of activity in the cryoprecipitate was similar, giving 337 f 80 U or 63% for MP, 262 f 70 U or 57% for VSO,252 + 60 U or 57% for PCS and 367 + 110 U or 59% for HA. The plasma from MP and HA contained relatively few cells with 12 f 5 x 109/L and 6 rt 1 x 109/Lplatelets; the V50 plasma (PPP mode j contained an average of 103 + 4 x 109/L platelets whereas the PCS plasma had 51 f 22 x 109/L. (3Thromboglobulin levels were significantly elevated in the plasma from the V50. Automated procedures took only
3MO min whereas the manual procedures took a minimum of 2 hr. While different economic factors will affect the choice of methodology, there is considerable benefit to the use of certain automated devices rather than the widely used manual method for the collection of plasma. l
INTRODUCTION In many countries plasmapheresis is carried out to provide plasma for fractionation with the major purpose being to generate adequate quantities of Factor VIII to meet national requirements for self-sufficiency. There are a number of methods available for plasmapheresis and these differ both in the technique and the type and amount of anticoagulant which are used. In the standard manual technique, a double bag is used to remove whole blood then, following centrifugation, the red cells are returned to the donor, the plasma is retained and the procedure is repeated to obtain a total of 500-600 mL of plasma. Recently, several automated devices which operate by both centrifugal and membrane techniques have become available for plasmapheresis. These methods have certain advantages over manual procedures in that they are faster and avoid the risk of returning the wrong red cells to a donor.14 Nonetheless, the manual method is used in the
From the Ottawa Centre, Canadian Red Cross, Blood Transfusion Service, 85 PI 011th Street, Ottawa and Departments of Medicine, r lochemIstry and Pathology, Umversity of Ottawa, Canada. Received 6/88, Accepted S/SE. 337
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Plasma Ther Transfus Technol
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majority of centers, particularly in the United States. We have reported that different levels of Factor VIII:C are obtained depending on the amount and type of citrate anticoagulant which is used, with a direct relationship between the level of divalent calcium and the Factor VIII:C recovery? as the level of ionized calcium decreases, so does the Factor VIII:C recovery.5 Another important factor in the choice of anticoagulant is the known relationship between pH of the solution and Factor VIII:C recovery.6 The loss of as much as 15% of the Factor VIII:C has been reported when blood is collected into ACD-A which has a pH of 4.9. This loss has been attributed to the “anticoagulant shock” which occurs when the first few millilitres of blood are introduced into the large volume of anticoagulant. The method of adding anticoagulant to blood is also important: it has been shown that constant mixing of the blood during collection, with little generation of fibrinopeptide A (FPA), contributes to better Factor VIII:C recovery.’ Since these various parameters are different in manual and automated plasmapheresis procedures, we have examined the plasma product and the Factor VIII:C recovery using five available methods of plasmapheresis. We have also assessed, during manual plasmapheresis, the effect of the return of the anticoagulated red cell concentrate on the circulating levels of Factor VIII:C in the donor, since it is commonly believed that during manual plasmapheresis the Factor VIII:C level is lower in the second bag. Sequential samples were also taken from donors during some of the automated procedures to determine any changes. METHODS AND MATERIALS The plasmapheresis procedures were carried out on normal, healthy donors who gave informed consent and met the usual criteria for donation of the Canadian Red Cross. According to manufacturers’ recommended standard operating procedures, a total of 500 mL of plasma product
(i.e. plasma + anticoagulant) were collected by manual plasmapheresis (MP) (n = 10) using a 1 in 10 ratio of 4% sodium citrate (Cutter Laboratories, Berkeley, automated plasCalifornia); by mapheresis using the Haemonetick VSO machine (V.50) (Haemonetics Corporation, Braintree, Massachusetts), both in the platelet-rich plasma (PRP) mode using ACD-A as the anticoagulant in a ratio of 1 in 12 (n = 5) and in the platelet-poor plasma (PPP) mode using a 4% sodium citrate anticoagulant in a 1 in 15 ratio (n = 5); by the more recently developed Haemonetics PCS device using the Latham bowl and a 1 in 16.6 ratio of sodium citrate (n = 10) and by the machine (HA) Autopheresis-CTM (HemaScience Laboratories, Santa Ana, California) using ACD-A as the anticoagulant in a 1 in 12.5 ratio (n = 10). Samples were taken from the donor before the procedure and from the plasma product. To permit direct comparison of these values between procedures, the donor samples were drawn into the standard laboratory anticoagulants for each test. Various measurements were made as follows: protein and albumin by a multi-channel autoanalyzer (model SMA18 Technicon Corporation, Tarrytown, New York); platelet count using an electronic particle counter (model ZB, Coulter Electronics, Hialeah, Florida); total microaggregate volume in the size range of l&100 ~1diameter (model TA2, Coulter Electronics); Factor VIII:C content using a one-stage assays against a Factor VIII:C reference plasma calibrated in WHO units (Thromboscreen, Pacific Hemostasis, Bakersfield, California); (3thromboglobulin (BTG), platelet factor 4 (PF4) and fibrinopeptide A (FPA) using commercial kits (Amersham Intemational, U.K., Abbott Laboratories, Montreal, Quebec and Mallinckrodt, St Louis, Missouri). The volume of the final product included the volume of anticoagulant as well as the collected plasma. A 20 mL aliquot was removed from the apheresis plasma product, placed in small amounts and frozen at -60°C until assayed for the above parameters. The
Comparisonof Plasmapheresis
plasma product was frozen at - 80°Cin a blast freezer (model PCF, Harris Manufacturing, N. Bellerica, Massachusetts) and cryoprecipitates were subsequently made using a rapid-thaw method.9 The Factor VIII:C level of the cryoprecipitate was determined and the percentage recovery was calculated based on the volume of plasma made into cryo (i.e. post sampling for baseline values]. The effect of returning the anticoagulant on the circulating level of Factor VIII:C in the donor was assessed during both MP and HA. Serial samples were taken from the donor’s free arm during the course of the procedure into the standard anticoagulant in use in clinical laboratories (3.8% sodium citrate in a 1 in 10 ratio) and the Factor VIII:C levels were measured. Statistical analysis was carried out using the Student t-test; P-values of less than 0.05 were considered significant. All values are given as mean + standard deviation. RESULTS Manual plasmapheresis lo), which use a double contain 50 mL of 4% anticoagulant in each
procedures (n = bag system and sodium citrate of the primary
Techniques
339
blood bags, produced a final product with a volume of 583 + 15 mL including both plasma and anticoagulant. The average total protein was 56 g/L; albumin was 35 g/L (Table 1). The Factor VIII:C levels averaged 105 U/dL with 337 + 80 units in the cryoprecipitate, representing a recovery of 63 f 3%. The FPA levels varied considerably, whereas BTG values were relatively low. Some microaggregate formation was noted. Collection and processing of the 2 bags took an averge of 120 + 30 min. Samples taken from the donor into the standard anticoagulant, following removal of the first bag of blood, and prior to the re-infusion of the first bag of packed red cells, showed a slight decrease in circulating Factor VI&C levels, from the pre-plasmapheresis values of 117 +_ 20 U/dL to 110 f 22 U/dL (P = 0.45). Following completion of the procedure, i.e. after return of the second bag of packed red cells, the circulating Factor VIII:C level was 104 + 20 U/dL. This was not significantly reduced (P > 0.05). Correction for dilution by anticoagulant of these and other values was not necessary since the same type and volume of anticoagulant was used for donor sampling. Comparison of the contents of the
Table 1. Comparison of Apheresis Products
Donor pre-procedure3 Factor VIILC (U/dL) Total protein (g/L] Product Volume (mL) Factor VIII:C (U/dL) Total protein (g/L] Albumin (g/L) Citrate (mM) BTG (ng/mL) PF4 (ng/mL FPA (ng/mL) Platelet count (X 109/L) Microaggregates ( x lOQ/mL) No. Tested
Manual’
V-50 PPP Mode
117k20 70*5
95f19 73+3
5835 15 105+23 56+4 35f2 20f2 56+38 19t-25 1225 12+6 10
514-t4
V-50 PRP Mode PRP PPP2
PCS
AutoDheresis C
123+24 73*3
123f24 73+3
95+27 69?6
121+22 75+3
598+20
467+47 125?25 63+2 43+2 1751 194*40 83+47 3+1 20+5 8+2 5
493+5 95+_27 59+5 39+4 23+3 440+200 87+59 3k2 51k22 18+3 10
504f26 121f22 63+5 4122 17-1-2 66f18 29+9 4+3 6fl 3+1 10
91+18 125f25 63f2 63f2 39+3 43+2 12+1 17+1 443+252 115*28 144599 72f20 2+1 3&l 103+4 393+32 8Ot-3 5 s
’ Mean of both plasma bags. 2 PPP was prepared by centrifugation of the PRP product.’ 3 Blood was taken from the donor into the standard anticoagulant.
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first and second plasma bags showed only slight differences for protein, albumin and Factor VIII:C levels. The differences for Factor VIII:C (106 f 24 and 101 + 22 U/dL) were not statistically significant (P > 0.05); however, the total protein {SS + 3 and 54 + 4 g/L) and albumin (36 + 2 and 34 f 2 g/L) levels were significantly different (P< 0.05). When the VSOmachine was used in the PPP mode (n = 5) with an average of three passes to collect 500 mL of plasma with 4% sodium citrate at a 1 in 15 ratio, the protein level was 63 Ifr2 g/L and the albumin was 39 f 3 g/L (Table 1). The Factor VIII:C level was 91 t- 18 U/dL with a recovery of 262 _+70 units in the cryoprecipitate, representing 57 + 6%. The BTG and PF4 level were high at 443 + 252 ng/mL, and 144 + 99 ng/mL. The FPA was 2 f 1 ng/mL and a small amount of microaggregate formation was noted. Analyses of samples taken from the plasma collection bag showed slight decreases (P > 0.05) in the level of Factor VIII:C in the collected plasma over time as earlier reported (data not shown).1 An average total of 75 mL of anticoagulant was used to collect the 500 mL of plasma which required 30 min. Using the V50 in the PRP mode (n = 5) and after removal of platelets from the plasma by subsequent centrifugation in the laboratory,2 467 + 47 mL of plasma were collected with an average total protein of 63 f 2 g/L and an albumin content of 43 + 2 g/L (Table 1J. The total volume of anticoagulant used was 137 + 33 mL with the amount required depending on donor hematocrit, as is the case for all automated apheresis procedures. Total collection time was 45 min. The Factor VIII:C level was 125 +_25 U/dL and the cryoprecipitate contained 333 f 156 units, representing 56 f 17% recovery. Prior to centrifugation to obtain the platelet concentrate (PC), the BTG was 115f28ng/mLandthePF4was72+20 ng/mL. After centrifugation, the BTG level in the PPP product was 194 f 40 ng/mL and the PF4 was 83 +- 47 ng/mL. The PCS machine, used with the Latham bowl, produced a plasma with 5 1
+ 22 x 109/Lplatelets, and averge protein level of 59 g/L and 95 + 27 U/dL of VIILC. In the cryoprecipitate, 57 f 11% FVIII (252 f 60U) was recovered. The BTG level in the plasma was 440 ng/mL (Table 1). An average of 100 mL of anticoagulant was used to collect 493 f. 5 mL of plasma. When the Autopheresis machine was used (n = lo), the plasma protein and albumin levels were 63 + 5 g/L and 41+ 2 g/L (Table 1). The Factor VIII:C levels averaged 121 U/dL. The cryoprecipitate contained 367 + 110 units with 59 + 10% recovery. BTG and PF4 levels were 66 f 18 ng/mL and 29 + 9 ng/mL respectively with little microaggregate or FPA formation. The plasma collection was completed in 30 min. Sequential samples taken into the standard anticoagulant from the opposite arm of five donors during the process showed a progressive decrease in the circulating levels of Factor VIII:C from 137 + 38 U/dL before plasmapheresis to 117 + 34 U/dL at 15 min after completion of the procedure (P > 0.05). An average of 150 + I3 mL of anticoagulant was used during collection of the 500 mL of plasma volume. DISCUSSION Better Factor VIII:C recoveries are seen in plasma obtained by AP rather than MP. One advantage of all of the machines is the automated metering of anticoagulant to blood near the site of venepuncture. This permits delivery of a constant volume of anticoagulant and mixing with the blood to avoid denaturation of Factor VIII:C (FVIILC) caused by exposure to low pH6 or by activation of coagulation. This could explain, in part, the improved recovery of FVIII:C. Another major determinant of the plasma FVIII:C level seems to be the absolute amount of anticoagulant used and the resultant amount of citrates per mL of plasma. Both the albumin and FVIII:C concentrations were higher with the automated devices. This is due simply to dilutional factors since all the machines use less anticoagulant per mL
Comparison of Plasmapheresis Techniques
of blood. Another recent study of automated plasmapheresis devices has also reported that the level of VIII:C was comparable in the Haemonetics and HemaScience machines,10 although, in that study, no comparison was made with the manual method. Another potential explanation for the higher FVIII values seen, is the use of different anticoagulants since ACD-A contains considerably less citrate per mL (21 mg) then does 4% sodium citrate (26 mg). Studies by Mustard’1 and our laboratory4 have shown an inverse relationship between the level of citrate and FVIII:C with an increase in the FVIILC as the citrate level decreases. However, in this case there is very little difference in the final citrate concentrations in the plasma (20 + 2 mM for MP and 17 _+ 2 mM for HA). Therefore, it is unlikely that the absolute amount of citrate accounted for the differences in FVIII:C noted. The increase in the BTG values in the plasma obtained with the VSO machine indicates release from the platelet a-granules during processing. This increase in BTG using the V50 has been previously noted by others,12 however, the significance is not known. These platelets are reported to have normal survival and recovery and be acceptable for in vitro function.2J2 Although not significantly different (P = 0.45), there was a slight decrease in the circulating levels of Factor VIII:C in the donors during plasmapheresis. This is likely due to the combined effects of the anticoagulant and dilution. Thus, both logistically and in terms of Factor VIII:C recovery, there are benefits to the use of automated devices rather than manual procedures to collect plasma. In some cases, the ultimate determining factor in conversion to automated methods will be the cost. Software costs are higher for automated devices (US $10-15) than for manual procedures (US $6-8). However, the labour-intensive monitoring required with MP, plus the requirement for additional centrifugation, offset the higher software costs of the automated procedures. Reduction of
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costs through bulk purchasing will also affect the final cost of the plasma. In countries such as Canada, where a principle of national self-sufficiency in blood products and plasma has been adopted, the automated devices have considerable appeal, particularly in ensuring protection of the volunteer donor and reducing the required time commitment such that plasmapheresis becomes an acceptable form of routine volunteer donation. Acknowledgements The authors wish to acknowledge the cooperation and assistance of the Nursing Department and the Apheresis Section of the Ottawa Centre of the Canadian Red Cross. Thanks are also due to Carol McCoshen and Rena Tennant for their assistance in typing the manuscript.
REFERENCES I. Rock G, McCombie N, Tittley P: A new
2.
3.
4.
5.
6.
7.
8.
technique for the collection of plasma: machine plasmapheresis. Transfusion 1981; 21:241-246. Rock G, Herzig R, McCombie N, Lazarus H, Tittley P: Automated platelet production during plasmapheresis. Transfusion 1983; 23:29&293. Rock G, Tittley P, McCombie N: Plasma collection using an automated membrane device. Transfusion 1986; 26:269271. Rock G, Cruickshank W, Tackaberry E, Ganz P, Palmer D: Stability of VIII:C in plasma: the dependence on protease activity and calcium. Thomb Res 1983; 29:521-535. Rock G, Tackaberry E, Palmer D: Factors affecting the relative distribution of high and low molecular weight forms of Factor VIILC. Thromb Res 1979; 14:573-587. Vermeer C, Soute B, Ates G, Hellings J, Brummelhuis H: Contributions to the optimal use of human blood. VIII. Stability of blood coagulation Factor VIILC during collection and storage of whole bloodandplasma. VOXSang 1976; 31:5567. Pflugshaupt R, Kurt G: FPA content-a criterion of quality for plasma as Factor VIII:C source. VOX Sang 1983; 45:224-232. Hardistry R, Macpherson J: A one-stage Factor VIII:C (antihemophilic globulin) assay and its use on venous and capillary plasma. Thromb Diath Haemorrh 1962; 7:215-228.
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9. Rock G, Tittley P: The effect of temperature variations on cryoprecipitate. Transfusion 19793 19:86-89. 10. Vezon G. Piauet Y. Manier C. Schooneman F, iesnier F, Ivloulinier J:‘Technical aspects of different donor plasmapheresis systems and biological results obtained in collected plasma. VOXSang 1986; 51 (Suppl. 1):40-44. 11 Mustard J: Somein vitro effects of various concentrations of disodium ethylene-
diamine tetracetate, potassium oxalate, and sodium citrate on coagulation of blood. Am I Clin Path01 19583 30:498506. 12. Penny A, Townley A, Angela E, Robinson A, Warrington R, Learoyd P, Gilkes L, Tovey L: Pilot study of an automated dual component plasmapheresis collection system. Apheresis Bull 1984; 2:312.