Vox Sang. 35: 207-214 (1978)
Collection of Granulocytes for Transfusion The Effect of Collection Methods on Cell Enzyme Release Neil Blumberg, Petrina Genco, Alfred Katz and Joseph Bove Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Conn., and Connecticut Red Cross Blood Center and University of Connecticut School of Medicine, Farmington, Conn.
Abstract. When granulocytes are collected by either discontinuous-flow centrifugation or filtration leukapheresis, lysozyme is released. More lysozyme is released during the filtration procedure than during the centrifugation procedure. A small amount of red cell lysis occurs during the centrifugation but not the filtration procedure. A rise in lactate dehydrogenase levels consonant with the amount of hemolysis is observed. These findings suggest that granulocytes collected by either filtration or discontinuous-flow centrifugation undergo degranulation but not lysis sufficient to cause cytoplasmic enzyme release during the donation.
Introduction The collection of polymorphonuclear leukocytes from normal donors has become a widespread, albeit investigational practice. While there is both human and animal evidence that granulocyte transfusion is beneficial [l, 8, 13, 15, 25, 261 controversy exists concerning optimal collection techniques. Yields are greater with filtration leukapheresis (FL), while cells collected by continuous-flow centrifugation leukapheresis (CFCL) may provide greater posttransfusion increments [12], cause fewer recipient reactions and show fewer in vitro functional abnormalities [21, 291. Discontinuous-flow centrifugation leukapheresis (DFCL), a relatively newer technique, yields neutrophils
with similar properties to the CFCL cells, as far as has been determined [4]. Sciziffer et al. [24] have reported that lysozyme is released when granulocytes are exposed to nylon fibers. Some investigators claim that lysozyme release is a measure of cellular disintegration in vivo [lo]. However, since lysozyme can be released by stimuli as disparate as calcium ion [ 5 ] , complement [6], phorbol myristate acetate [7] and conconavalin A [16], its release does not necessarily indicate cell lysis or membrane damage. To evaluate the role of cell damage as a cause of lysozyme release during FL or DFCL, we compared levels of lysozyme with those of the cytoplasmic enzyme lactate dehydrogenase (LDH) during the collection of leukocytes. We also
208
Blumberg/GencolKaWBove
measured hemoglobin to determine whether LDH release might be due to red cell lysis. LDIl release without increased plasma hemoglobin would suggest platelet or white cell lysis.
Metbods Collection of Neutrophils by DFCL and by FL Collection was performed by previously dexribed methods [14, 181. DFCL was performed cmploying the Haemonetics 30 blood processor. ACD was employed as the anticoagulant and hydroxyethyl starch (6%) as a rouleaux-inducing agent [14]. 6-8 cycles of centrifugation using the 225-m1 disposable bowl were completed. Residual red cells were returned to the donor at the completion of the collection so that the net loss of packed red cells was less than 20ml. FL was performed using a dual nylon wool filter harness and peristaltic roller pump, with anticoagulation by heparin [18]. Saline was used to wash residual red cells from the filters at the collection’s completion and return them to the donor. Granulocyte elution was performed with a solution of ACD, CPD plasma and saline, with gentle tapping of the filters [18]. All donors received either oral prednisone (40 mg) the night immediately previous to donation or intravenous dexamethasone (6 mg) at the beginning of leukapheresis. None of the donors were studied more than once during the experiments to be described. Collection of Samples Donors undergoing leukapheresis for patient care needs gave informed consent to the collection of blood samples. All samples were collected in plastic syringes through three-way stopcocks and then emptied down the sides of tubes containing 3.8% sodium citrate. Specimens were centrifuged at 1,250 g for 10 min, the supernatant plasma removed and stored frozen at -20 OC. Samples were transported on dry ice from the collection site in Farmington, CT, to New Haven and immediately stored at -20 OC until assayed. In 10 donors undergoing FL and in 10 undergoing DFCL, pre- and postdonation samples were
collected from the withdrawal arm. In a second experiment involving an additional 10 F L and 13 DFCL donors, samples were obtained simultaneously from the withdrawal and reinfusion lines. In the DFCL donors, reinfusion line samples were collected simultaneously with withdrawal line samples after the plasma had been returned to the donor red cells, while reinfusion was in progress. Blood samples were obtained at least 1 h after the start of the donation (FL) or after at least two centrifugation cycles (DFCL) to ensure some degree of donor equilibration. This latter experiment (withdrawal and reinfusion line sampling) was repeated on an additional 10 FL and 14 DFCL donors with identical procedures except that blood was collected in tubes without anticoagulant, allowed to clot at room temperature, centrifuged and the ‘serum’ separated. Since these samples contained either heparin (FL) or ACD (DFCL) they cannot correctly be called serum, but since clotting did occur within 0-2 h, we will use the term ‘serum’ to differentiate these samples from the plasma samples previously described. Lysozyme, Lactate Dchydrogenase and Hemoglobin The lysoplate method [22], which combines gel diffusion and bacterial lysis was used to measure plasma lysozyme. Precautions were taken to obviate difficulties that others have experienced with this method [9]. These included assaying samples on plates made from one lot of agar at the same time, and running controls to assure day-to-day comparability. Human lysozyme standard was the gift of Dr. Eliott F. Osserman. The plasma lysozyme concentrations of 30 healthy persons were all less than 14 pglml. Plasma LDH was measured by a kinetic ’ method utilizing the oxidation of NADH [ l l ] adapted to the Perkin-Elmer KA-150. The LDH levels of 30 healthy persons were all less than 600 spectrophotometric Ulml. Plasma hemoglobin was measured using the oxidation of o-tolidine [2]. Employing a two-syringe technique healthy people have levels less than 5 mg/dl. Neither sodium citrate (3.8%), ACD nor hydroxyethyl starch interfere with the above assays. ‘Serum’ LDH and hemoglobin were measured by the above methods and have identical ranges to plasma levels in healthy individuals. To mea-
Cell Enzyme Release during Granulocyte Collection
sure ‘serum’ lysozyme we used the turbidometric method of Litwack [20]. Utilizing a standard of egg-white lysozyme, healthy persons have levels less than 14 pg/ml. In the DFCL procedure one part of ACDhydroxyethyl starch is mixed with eight parts of donor blood during each centrifugation cycle. A correction for this dilution was made for all data from the DFCL reinfusion line samples. The average donor hematocrit during DFCL was 40%. The plasmacrit was therefore 60%. Thus, the fraction of diluted blood that is plasma is approximately 0.6 (819) = 0.533. The fraction that is ACD-hydroxyethyl starch is 119 = 0.111. The plasma space for lysozyme, LDH and hemoglobin has been expanded by approximately
0.533 + 0.111
= 1.2
0.533 in the reinfusion line samples. Analysis of variance (utilizing unweighted means when there were unequal numbers of donors in the groups compared) and the NewmanKeuls test (when individual comparisons of means was appropriate and desired) were used to evaluate data [28]..
Results Pre- and Postpheresis Data from Donors Data and results of the statistical analysis for the first study are shown in table I. Neutrophil yields averaged 1.35 ? 0.32 x 1O1O (SEM) by FL and 1.23 k 0.18 x 10’0 (SEM) by DFCL. Hemodilution, as documented by fall in hematocrit, occurred with both collection methods and was significantly greater with DFCL. The mean fall in hematocrit was 3.4% with FL and 6.7% with DFCL. Plasma lysozyme fell in the DFCL but not the FL donors. Plasma LDH and white count did not change in either group of donors. Plasma hemoglobin was increased postdonation in the DFCL donors.
209
Withdrawal and Reinfusion Line Data from Donors Results of the study of simultaneous samples from the withdrawal and reinfusion lines are shown in table 11. Plasma lysozyme was significantly higher in the reinfusion line than in the withdrawal line for both leukapheresis techniques. Despite the greater change in plasma lysozyme with FL than with DFCL, there was no significant difference between the two methods in the degree of withdrawal to reinfusion line change they produce, at least in this group of donors. Plasma LDH was decreased in the reinfusion line from the level in the withdrawal line by the DFCL but not the FL method, but this difference was not significant. Plasma hemoglobin was increased in the reinfusion line from the level in the withdrawal line by the DFCL but not the FL method. Individual plasma hemoglobin values in the withdrawal and reinfusion line samples are given in table 111. Levels are low in all cases but one when filtration was used but were much higher during centrifugation leukapheresis. In four donors extremely high values in both the reinfusion and withdrawal lines were observed. It was determined retrospectively that these samples had been drawn by needle puncture of the withdrawal and reinfusion lines and then injected into evacuated tubes through the needle. In order to confirm our findings of lysozyme, LDH and hemoglobin levels in the withdrawal and reinfusion lines of FL and DFCL donors we measured ‘serum’ levels in an additional 10 FL and 14 DFCL donors. These results are shown in table IV. Individually, all 10 FL and 12 of 14 DFCL donors showed increases in lysozyme level in the reinfusion line compared with that in the withdrawal line. In this group of donors,
Blumberg/Genco/Katz/Bove
210
Table I. Pre- and postpheresis data from donors undergoing filtration (FL) or centrifugation leukapheresis (CL); all data are f 1 SEM Filtration leukapheresis (n= 10) predonation
postdonation
Centrifugation leukapheresis (n = 10) significance
predonation
postdonation
significance
Significance of differences between FL and CL'
Hematocri t
42.6f1.0
39.2f0.7
White count
7,100f900
6,500f700
NS
Plasma lysozyme, pg/ml
8.44Z0.6
8.7f0.5
NS
9.9f0.7
7.7f0.8
p
Collection of Granulocytes for Transfusion The Effect of Collection Methods on Cell Enzyme Release Neil Blumberg, Petrina Genco, Alfred Katz and Joseph Bove Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Conn., and Connecticut Red Cross Blood Center and University of Connecticut School of Medicine, Farmington, Conn.
Abstract. When granulocytes are collected by either discontinuous-flow centrifugation or filtration leukapheresis, lysozyme is released. More lysozyme is released during the filtration procedure than during the centrifugation procedure. A small amount of red cell lysis occurs during the centrifugation but not the filtration procedure. A rise in lactate dehydrogenase levels consonant with the amount of hemolysis is observed. These findings suggest that granulocytes collected by either filtration or discontinuous-flow centrifugation undergo degranulation but not lysis sufficient to cause cytoplasmic enzyme release during the donation.
Introduction The collection of polymorphonuclear leukocytes from normal donors has become a widespread, albeit investigational practice. While there is both human and animal evidence that granulocyte transfusion is beneficial [l, 8, 13, 15, 25, 261 controversy exists concerning optimal collection techniques. Yields are greater with filtration leukapheresis (FL), while cells collected by continuous-flow centrifugation leukapheresis (CFCL) may provide greater posttransfusion increments [12], cause fewer recipient reactions and show fewer in vitro functional abnormalities [21, 291. Discontinuous-flow centrifugation leukapheresis (DFCL), a relatively newer technique, yields neutrophils
with similar properties to the CFCL cells, as far as has been determined [4]. Sciziffer et al. [24] have reported that lysozyme is released when granulocytes are exposed to nylon fibers. Some investigators claim that lysozyme release is a measure of cellular disintegration in vivo [lo]. However, since lysozyme can be released by stimuli as disparate as calcium ion [ 5 ] , complement [6], phorbol myristate acetate [7] and conconavalin A [16], its release does not necessarily indicate cell lysis or membrane damage. To evaluate the role of cell damage as a cause of lysozyme release during FL or DFCL, we compared levels of lysozyme with those of the cytoplasmic enzyme lactate dehydrogenase (LDH) during the collection of leukocytes. We also
208
Blumberg/GencolKaWBove
measured hemoglobin to determine whether LDH release might be due to red cell lysis. LDIl release without increased plasma hemoglobin would suggest platelet or white cell lysis.
Metbods Collection of Neutrophils by DFCL and by FL Collection was performed by previously dexribed methods [14, 181. DFCL was performed cmploying the Haemonetics 30 blood processor. ACD was employed as the anticoagulant and hydroxyethyl starch (6%) as a rouleaux-inducing agent [14]. 6-8 cycles of centrifugation using the 225-m1 disposable bowl were completed. Residual red cells were returned to the donor at the completion of the collection so that the net loss of packed red cells was less than 20ml. FL was performed using a dual nylon wool filter harness and peristaltic roller pump, with anticoagulation by heparin [18]. Saline was used to wash residual red cells from the filters at the collection’s completion and return them to the donor. Granulocyte elution was performed with a solution of ACD, CPD plasma and saline, with gentle tapping of the filters [18]. All donors received either oral prednisone (40 mg) the night immediately previous to donation or intravenous dexamethasone (6 mg) at the beginning of leukapheresis. None of the donors were studied more than once during the experiments to be described. Collection of Samples Donors undergoing leukapheresis for patient care needs gave informed consent to the collection of blood samples. All samples were collected in plastic syringes through three-way stopcocks and then emptied down the sides of tubes containing 3.8% sodium citrate. Specimens were centrifuged at 1,250 g for 10 min, the supernatant plasma removed and stored frozen at -20 OC. Samples were transported on dry ice from the collection site in Farmington, CT, to New Haven and immediately stored at -20 OC until assayed. In 10 donors undergoing FL and in 10 undergoing DFCL, pre- and postdonation samples were
collected from the withdrawal arm. In a second experiment involving an additional 10 F L and 13 DFCL donors, samples were obtained simultaneously from the withdrawal and reinfusion lines. In the DFCL donors, reinfusion line samples were collected simultaneously with withdrawal line samples after the plasma had been returned to the donor red cells, while reinfusion was in progress. Blood samples were obtained at least 1 h after the start of the donation (FL) or after at least two centrifugation cycles (DFCL) to ensure some degree of donor equilibration. This latter experiment (withdrawal and reinfusion line sampling) was repeated on an additional 10 FL and 14 DFCL donors with identical procedures except that blood was collected in tubes without anticoagulant, allowed to clot at room temperature, centrifuged and the ‘serum’ separated. Since these samples contained either heparin (FL) or ACD (DFCL) they cannot correctly be called serum, but since clotting did occur within 0-2 h, we will use the term ‘serum’ to differentiate these samples from the plasma samples previously described. Lysozyme, Lactate Dchydrogenase and Hemoglobin The lysoplate method [22], which combines gel diffusion and bacterial lysis was used to measure plasma lysozyme. Precautions were taken to obviate difficulties that others have experienced with this method [9]. These included assaying samples on plates made from one lot of agar at the same time, and running controls to assure day-to-day comparability. Human lysozyme standard was the gift of Dr. Eliott F. Osserman. The plasma lysozyme concentrations of 30 healthy persons were all less than 14 pglml. Plasma LDH was measured by a kinetic ’ method utilizing the oxidation of NADH [ l l ] adapted to the Perkin-Elmer KA-150. The LDH levels of 30 healthy persons were all less than 600 spectrophotometric Ulml. Plasma hemoglobin was measured using the oxidation of o-tolidine [2]. Employing a two-syringe technique healthy people have levels less than 5 mg/dl. Neither sodium citrate (3.8%), ACD nor hydroxyethyl starch interfere with the above assays. ‘Serum’ LDH and hemoglobin were measured by the above methods and have identical ranges to plasma levels in healthy individuals. To mea-
Cell Enzyme Release during Granulocyte Collection
sure ‘serum’ lysozyme we used the turbidometric method of Litwack [20]. Utilizing a standard of egg-white lysozyme, healthy persons have levels less than 14 pg/ml. In the DFCL procedure one part of ACDhydroxyethyl starch is mixed with eight parts of donor blood during each centrifugation cycle. A correction for this dilution was made for all data from the DFCL reinfusion line samples. The average donor hematocrit during DFCL was 40%. The plasmacrit was therefore 60%. Thus, the fraction of diluted blood that is plasma is approximately 0.6 (819) = 0.533. The fraction that is ACD-hydroxyethyl starch is 119 = 0.111. The plasma space for lysozyme, LDH and hemoglobin has been expanded by approximately
0.533 + 0.111
= 1.2
0.533 in the reinfusion line samples. Analysis of variance (utilizing unweighted means when there were unequal numbers of donors in the groups compared) and the NewmanKeuls test (when individual comparisons of means was appropriate and desired) were used to evaluate data [28]..
Results Pre- and Postpheresis Data from Donors Data and results of the statistical analysis for the first study are shown in table I. Neutrophil yields averaged 1.35 ? 0.32 x 1O1O (SEM) by FL and 1.23 k 0.18 x 10’0 (SEM) by DFCL. Hemodilution, as documented by fall in hematocrit, occurred with both collection methods and was significantly greater with DFCL. The mean fall in hematocrit was 3.4% with FL and 6.7% with DFCL. Plasma lysozyme fell in the DFCL but not the FL donors. Plasma LDH and white count did not change in either group of donors. Plasma hemoglobin was increased postdonation in the DFCL donors.
209
Withdrawal and Reinfusion Line Data from Donors Results of the study of simultaneous samples from the withdrawal and reinfusion lines are shown in table 11. Plasma lysozyme was significantly higher in the reinfusion line than in the withdrawal line for both leukapheresis techniques. Despite the greater change in plasma lysozyme with FL than with DFCL, there was no significant difference between the two methods in the degree of withdrawal to reinfusion line change they produce, at least in this group of donors. Plasma LDH was decreased in the reinfusion line from the level in the withdrawal line by the DFCL but not the FL method, but this difference was not significant. Plasma hemoglobin was increased in the reinfusion line from the level in the withdrawal line by the DFCL but not the FL method. Individual plasma hemoglobin values in the withdrawal and reinfusion line samples are given in table 111. Levels are low in all cases but one when filtration was used but were much higher during centrifugation leukapheresis. In four donors extremely high values in both the reinfusion and withdrawal lines were observed. It was determined retrospectively that these samples had been drawn by needle puncture of the withdrawal and reinfusion lines and then injected into evacuated tubes through the needle. In order to confirm our findings of lysozyme, LDH and hemoglobin levels in the withdrawal and reinfusion lines of FL and DFCL donors we measured ‘serum’ levels in an additional 10 FL and 14 DFCL donors. These results are shown in table IV. Individually, all 10 FL and 12 of 14 DFCL donors showed increases in lysozyme level in the reinfusion line compared with that in the withdrawal line. In this group of donors,
Blumberg/Genco/Katz/Bove
210
Table I. Pre- and postpheresis data from donors undergoing filtration (FL) or centrifugation leukapheresis (CL); all data are f 1 SEM Filtration leukapheresis (n= 10) predonation
postdonation
Centrifugation leukapheresis (n = 10) significance
predonation
postdonation
significance
Significance of differences between FL and CL'
Hematocri t
42.6f1.0
39.2f0.7
White count
7,100f900
6,500f700
NS
Plasma lysozyme, pg/ml
8.44Z0.6
8.7f0.5
NS
9.9f0.7
7.7f0.8
p
