Microaggregate Formation in Stored Human Packed Cells: Comparison with Formation in Stored Whole Blood and a Method for their Removal JOHN BARRETT, M.B.,* DAVID S. DE JONGH, M.D.,t EDITH MILLER, M.S., MARTIN S. LITWIN, M.D.t
Experiments were performed to compare the formation of microaggregates in stored human whole blood (WB) with that in stored packed cells (PC) and also to compare the effectiveness of standard blood transfusion filters with dacron wool (Swank) micropore transfusion filters in removing such microaggregates. After 5, 10, 15 and 20 days of storage SFP and debris weights of PC's were considerably greater than those of matched WB samples. Passage of either WB or PC's through standard blood transfusion filters resulted in small decreases in SFP and debris weights. Passage of either WB or PC's through dacron wool (Swank) transfusion filters led to striking and highly significant decreases in both SFP and debris weights. When stored PC's were diluted to the same hematocrits as their corresponding WB samples, SFP remained considerably elevated above those of the WB samples. On the basis of this research, it is concluded that centrifugation of blood during component separation leads to a significant increase in microaggregate formation over and above that which progressively occurs during storage and that the risk of pulmonary microembolization during transfusion with stored PC's is greater than that during WB transfusion. For this reason, dacron wool (Swank) filters should always be used when PC's are being transfused. `
MsICROAGGREGATES consisting mainly of platelets and white blood cells in a fibrin matrix form in progressively increasing numbers as the age of stored human whole blood increases.1l5-9"12'14'15'19,21 Transfusion of microaggregate-containing blood through standard blood transfusion filters may be a cause for the development of pulmonary insufficiency.3'5'9'11'14"6"7 Submitted for publication August 27, 1975. Supported by contract no. DADA 17-67-C-7049 with Surgical Directorate, U.S. Army Medical Research and Development Command. * Surgical Research Fellow & Ainsworth Scholar from National University of Ireland. t Director, Department of Hemotherapy. t Professor of Surgery.
From Department of Surgery, Tulane University Medical School and Department of Hemotherapy, Charity Hospital of Louisiana, New Orleans, Louisiana 70112
Even though specific blood components are used extensively in modern surgical practice, the occurrence of microaggregates in stored packed cells has not previously been reported. It was the purpose of this research to compare the formation of microaggregates in human whole blood (WB) with that in human packed cells (PC) prepared and stored under standard blood bank conditions. The effectiveness of standard blood transfusion filters and dacron wool (Swank) micropore blood transfusion filters in removal of microaggregates from stored PC was also compared. Materials and Methods Blood from 48 donors which was unacceptable for human use was included in this study (Table 1). None of the donors was receiving medications which are known to cause alterations in platelet adhesiveness. Donors included 42 males and 6 females, and their ages ranged from 19 to 64 years (average age 32.1 years). Under sterile conditions, one unit (approximately 500 cc) of blood was withdrawn from an antecubital vein of each donor into the collection bag of a plastic triple pack blood storage bag (Fenwal triple pack #HF82P4) containing CPD anticoagulant. Duration of the phlebotomies ranged from 5 to 22 minutes (average 10.1 minutes). Sterile collection methods standard to this center were used. Each unit of blood was then separated into two equal portions using the two other plastic bags of the triple
109
Ann. Surg.
BARRETT AND OTHERS TABLE 1. Reasons for Rejection of Blood from 48 Human Donors
Reasons
No. of Patients
History of hepatitis or possible hepatitis exposure (all radioimmunoassay negative) Venereal disease Medical drug treatment History of malaria Miscellaneous
33 5 4 3 3
pack. One of these aliquots was stored as WB. The other was centrifuged at 5,400 x G for 5 minutes in a RC-3 Sorvall refrigerated centrifuge, the technique standard to this center for separating PC and plasma. The plasma was removed aseptically into a plastic plasma pack. The various aliquots of WB, PC and plasma were stored at 4 C. In this way each separate PC and plasma sample could be compared to its corresponding WB sample throughout the study. The total of 48 aliquots of whole blood together with its corresponding PC and plasma samples was divided equally into four groups of 12 units each. At five day intervals after initial storage (days 5, 10, 15 and 20) one entire group (including WB, PC and plasma) was removed and studied. The filter screen to be used for determining screen filtration pressure (SFP) of each aliquot was weighed. SFP was then determined on WB, PC and plasma using a separate preweighed screen for each.7821 The differential between screen weight before SFP measurement and that after SFP measurement was the debris weight of microaggregates removed from equal aliquot portions of either WB or the corresponding specific component of that WB sample.13 Hematocrit and white blood cell and platelet counts were also determined on each WB and PC sample. After completion of these measurements, one-half of each aliquot was filtered through a standard blood transfusion filter* and the remaining one-half was passed through a dacron wool (Swank) transfusion filter.t All tests were then repeated after filtration of these samples.
February 1976
SFP ac hieved by stored human WB which was acceptable for tranisfusion. Neither was there a statistically significant co]rrelation between the SFP achieved in the stored blood a nd the time length of the donor's phlebotomy. Aver;age SFP after each storage time interval and in each grroup was higher for the PC sample than for the corresponding WB sample (Fig. 1). After 5 days of storage , average SFP of the WB was 67 mm Hg and for the conresponding PC, 320 mm Hg (P < 0.01). After 10 days of storage, SFP of the WB was 84 mm Hg and for the corresponding PC, 401 mm Hg (P < 0.005). When stored for 15 d,ays, SFP ofthe WB was 106 mm Hg and that of the PC wars 348.mm Hg (P < 0.0125). After storage for 20 days, S ,FP of the WB was 284 mm Hg and that of the PC was 5733 mm Hg (P < 0.01). Debriis weights from the PC samples were also significantly ggreater than debris weights from the corresponding WB samples (P < 0.01) (Fig. 1). No siignificant SFP was measured for any of the plasma
sampless. Filtrattion of WB samples through standard blood transfusion fiilters resulted in slight decreases in SFP after all storage time spans studied; none of these decreases achieved statistical significance (Fig. 2). Debris weights also de creased, and these changes were statistically significaant (Table 2). When WB samples were passed through dacron wool (Swank) filters SFP of all stored sampless returned to normal (average 4 mm Hg) (Fig. 2). A strikiing decrease in debris weights of material passing the filteirs was of very high statistical significance (Table AGGREGATE FORMATION IN PACKED CELLS PKiOd clls
6w
(omW SW
40 i_ 3W
DWhoblood
7-
200
Might *w
(
~
cpw..
100
Results Statistical analysis was performed on the SFP achieved by each of the various donor groups to determine if the causes for rejection of their blood might have led to changes in SFP (Table 1). No such correlations were found, and the range of SFP achieved by the stored blood from these 48 donors was compatible with the range of
9
_#
Wa
6W
-
20
L
11
0~
5
10
15
20
Days of Storoe *
Cutter "Saftifilter" blood administration set 814-28, Cutter Labora-
tories, Inc., Berkeley, California 94710. FIG. 1. Screen filtration pressures and debris weights for stored human t Swank transfusion filter, IL-200, Extracorporeal Medical Special- packed cells were considerably greater than those for matched stored ties, Inc., King of Prussia, Pennsylvania 19406. whole blood samples.
Vol. 183 * No. 2
MICROAGGREGA,TE FORMATION AGGREGATE
Sfp
1mmftg) 30 20
FORMATION IN PACKED CELLS
WHOIE BOOD 0 --
Pre-filtration
10 _
/
.,OAfter O
filtrotion
X/,,,,,
t0
o 0
Debri5 3 We,ght (m9j 20
1
__ After
filtration ~~~~~~~~~~~~~~~~~Swank Pre- filtration After ston filt After Swank filt.
0
111
One-half were diluted with normal saline and the other half with their own stored plasma. SFP of these dilutions were then determined. SFP of samples diluted with saline and those diluted with plasma did not differ. SFP of the diluted PC samples were higher than their corresponding WB samples with the same hematocfits (Fig. 4). These were not of statistical significance after 15 days of storage but after 5, 10 and 20 days, P values were
Experiments were performed to compare the formation of microaggregates in stored human whole blood (WB) with that in stored packed cells (PC) and also to compare the effectiveness of standard blood transfusion filters with dacron wool (Swank) micropore transfusion filters in removing such microaggregates. After 5, 10, 15 and 20 days of storage SFP and debris weights of PC's were considerably greater than those of matched WB samples. Passage of either WB or PC's through standard blood transfusion filters resulted in small decreases in SFP and debris weights. Passage of either WB or PC's through dacron wool (Swank) transfusion filters led to striking and highly significant decreases in both SFP and debris weights. When stored PC's were diluted to the same hematocrits as their corresponding WB samples, SFP remained considerably elevated above those of the WB samples. On the basis of this research, it is concluded that centrifugation of blood during component separation leads to a significant increase in microaggregate formation over and above that which progressively occurs during storage and that the risk of pulmonary microembolization during transfusion with stored PC's is greater than that during WB transfusion. For this reason, dacron wool (Swank) filters should always be used when PC's are being transfused. `
MsICROAGGREGATES consisting mainly of platelets and white blood cells in a fibrin matrix form in progressively increasing numbers as the age of stored human whole blood increases.1l5-9"12'14'15'19,21 Transfusion of microaggregate-containing blood through standard blood transfusion filters may be a cause for the development of pulmonary insufficiency.3'5'9'11'14"6"7 Submitted for publication August 27, 1975. Supported by contract no. DADA 17-67-C-7049 with Surgical Directorate, U.S. Army Medical Research and Development Command. * Surgical Research Fellow & Ainsworth Scholar from National University of Ireland. t Director, Department of Hemotherapy. t Professor of Surgery.
From Department of Surgery, Tulane University Medical School and Department of Hemotherapy, Charity Hospital of Louisiana, New Orleans, Louisiana 70112
Even though specific blood components are used extensively in modern surgical practice, the occurrence of microaggregates in stored packed cells has not previously been reported. It was the purpose of this research to compare the formation of microaggregates in human whole blood (WB) with that in human packed cells (PC) prepared and stored under standard blood bank conditions. The effectiveness of standard blood transfusion filters and dacron wool (Swank) micropore blood transfusion filters in removal of microaggregates from stored PC was also compared. Materials and Methods Blood from 48 donors which was unacceptable for human use was included in this study (Table 1). None of the donors was receiving medications which are known to cause alterations in platelet adhesiveness. Donors included 42 males and 6 females, and their ages ranged from 19 to 64 years (average age 32.1 years). Under sterile conditions, one unit (approximately 500 cc) of blood was withdrawn from an antecubital vein of each donor into the collection bag of a plastic triple pack blood storage bag (Fenwal triple pack #HF82P4) containing CPD anticoagulant. Duration of the phlebotomies ranged from 5 to 22 minutes (average 10.1 minutes). Sterile collection methods standard to this center were used. Each unit of blood was then separated into two equal portions using the two other plastic bags of the triple
109
Ann. Surg.
BARRETT AND OTHERS TABLE 1. Reasons for Rejection of Blood from 48 Human Donors
Reasons
No. of Patients
History of hepatitis or possible hepatitis exposure (all radioimmunoassay negative) Venereal disease Medical drug treatment History of malaria Miscellaneous
33 5 4 3 3
pack. One of these aliquots was stored as WB. The other was centrifuged at 5,400 x G for 5 minutes in a RC-3 Sorvall refrigerated centrifuge, the technique standard to this center for separating PC and plasma. The plasma was removed aseptically into a plastic plasma pack. The various aliquots of WB, PC and plasma were stored at 4 C. In this way each separate PC and plasma sample could be compared to its corresponding WB sample throughout the study. The total of 48 aliquots of whole blood together with its corresponding PC and plasma samples was divided equally into four groups of 12 units each. At five day intervals after initial storage (days 5, 10, 15 and 20) one entire group (including WB, PC and plasma) was removed and studied. The filter screen to be used for determining screen filtration pressure (SFP) of each aliquot was weighed. SFP was then determined on WB, PC and plasma using a separate preweighed screen for each.7821 The differential between screen weight before SFP measurement and that after SFP measurement was the debris weight of microaggregates removed from equal aliquot portions of either WB or the corresponding specific component of that WB sample.13 Hematocrit and white blood cell and platelet counts were also determined on each WB and PC sample. After completion of these measurements, one-half of each aliquot was filtered through a standard blood transfusion filter* and the remaining one-half was passed through a dacron wool (Swank) transfusion filter.t All tests were then repeated after filtration of these samples.
February 1976
SFP ac hieved by stored human WB which was acceptable for tranisfusion. Neither was there a statistically significant co]rrelation between the SFP achieved in the stored blood a nd the time length of the donor's phlebotomy. Aver;age SFP after each storage time interval and in each grroup was higher for the PC sample than for the corresponding WB sample (Fig. 1). After 5 days of storage , average SFP of the WB was 67 mm Hg and for the conresponding PC, 320 mm Hg (P < 0.01). After 10 days of storage, SFP of the WB was 84 mm Hg and for the corresponding PC, 401 mm Hg (P < 0.005). When stored for 15 d,ays, SFP ofthe WB was 106 mm Hg and that of the PC wars 348.mm Hg (P < 0.0125). After storage for 20 days, S ,FP of the WB was 284 mm Hg and that of the PC was 5733 mm Hg (P < 0.01). Debriis weights from the PC samples were also significantly ggreater than debris weights from the corresponding WB samples (P < 0.01) (Fig. 1). No siignificant SFP was measured for any of the plasma
sampless. Filtrattion of WB samples through standard blood transfusion fiilters resulted in slight decreases in SFP after all storage time spans studied; none of these decreases achieved statistical significance (Fig. 2). Debris weights also de creased, and these changes were statistically significaant (Table 2). When WB samples were passed through dacron wool (Swank) filters SFP of all stored sampless returned to normal (average 4 mm Hg) (Fig. 2). A strikiing decrease in debris weights of material passing the filteirs was of very high statistical significance (Table AGGREGATE FORMATION IN PACKED CELLS PKiOd clls
6w
(omW SW
40 i_ 3W
DWhoblood
7-
200
Might *w
(
~
cpw..
100
Results Statistical analysis was performed on the SFP achieved by each of the various donor groups to determine if the causes for rejection of their blood might have led to changes in SFP (Table 1). No such correlations were found, and the range of SFP achieved by the stored blood from these 48 donors was compatible with the range of
9
_#
Wa
6W
-
20
L
11
0~
5
10
15
20
Days of Storoe *
Cutter "Saftifilter" blood administration set 814-28, Cutter Labora-
tories, Inc., Berkeley, California 94710. FIG. 1. Screen filtration pressures and debris weights for stored human t Swank transfusion filter, IL-200, Extracorporeal Medical Special- packed cells were considerably greater than those for matched stored ties, Inc., King of Prussia, Pennsylvania 19406. whole blood samples.
Vol. 183 * No. 2
MICROAGGREGA,TE FORMATION AGGREGATE
Sfp
1mmftg) 30 20
FORMATION IN PACKED CELLS
WHOIE BOOD 0 --
Pre-filtration
10 _
/
.,OAfter O
filtrotion
X/,,,,,
t0
o 0
Debri5 3 We,ght (m9j 20
1
__ After
filtration ~~~~~~~~~~~~~~~~~Swank Pre- filtration After ston filt After Swank filt.
0
111
One-half were diluted with normal saline and the other half with their own stored plasma. SFP of these dilutions were then determined. SFP of samples diluted with saline and those diluted with plasma did not differ. SFP of the diluted PC samples were higher than their corresponding WB samples with the same hematocfits (Fig. 4). These were not of statistical significance after 15 days of storage but after 5, 10 and 20 days, P values were
