TRANSFUSION Vol. 19
No. 1
January-February 1979
Special Report
Functional Considerations of Granulocyte Concentrates Used for Clinical Transfusions L. GLASSER From the Depurtment of Puthology, College of Medicine. Uni,wsitg oJ' Arizonu Tircson. Arizoncr I
tion in the noninfected Whether GRANu LOCYE CON CENTRATES are the this dose is optimal for human survival is not newest of the blood components to be used known. in transfusion therapy. Clinical studies sugAny discussion of dose must take into gest that they are useful primarily in cases consideration the functional competence of of acute leukemia where the goal of chemocells. The performance of the ~-~~ therapy is bone marrow a p l a ~ i a . ~ , l ~ t-h~e transfused harvested phagocyte must be evaluated Component therapy with red blood cells and 1) when drugs are used to stimulate the platelets prevents the complications of anemia and bleeding, and white blood cell endogenous production of normal donor cells, 2) in the donor with chronic transfusions would be expected to reduce granulocytic leukemia, 3) after the leukothe mortality from infection. cyte has been in contact with red blood Transfusions of granulocytes should be cell sedimenting agents or anticoagulants, given daily.I8 It is generally thought that 4) following the interaction of the granulothe more the better. Experiments with cyte with machines or other collection neutropenic dogs show that daily doses of devices, 5 ) after radiation of the concen2 x lo8 neutrophils per kilogram will trate, and 6) during storage. prevent mortality from pseudomonas septiThe most important factor regarding ~ e m i a .Surprisingly, ~ this would translate neutrophil function is the method of collecto one granulocyte concentrate daily for a tion. Methods using centrifugation preserve 70-kilogram man at our institution where function better than filtration methods using cells are collected by intermittent flow reversible leukoadhesion. With continuous is only one c e n t r i f u g a t i ~ n . This ~ ~ * ~dose ~ flow centrifugation (CFC), the average time tenth of the daily human neutrophil producof exposure of the leukocyte to foreign surfaces is three minutes at the relatively Presented in part at the Haemonetics Research In vitro low centrifugal force of 42 x g.ll*30 Institute Advanced Component Seminar, Boston, Massachusetts, June 20. 1978. studies have shown that cells harvested by Received for publication June 8, 1978: accepted CFC have normal r n o r p h o l ~ g y , via~~ July 19, 1978. b i l i t ~ , quantitative ~ . ~ ~ nitroblue tetrazolium Supported by U.S.Public Health Service Grant No. r e d u ~ t i o n ? ~respiratory burst ?7*49 phagoCA 17094. 0041-1132/79/0100/0001 $00.80 0 J . B. Lippincott Co. '
Transtiision January-February 1979
1
Volume 19 Number I
2
GLASSER
Tranrfbrion January-Febmary 1979
studies showing i r n ~ a i r m e n t .Most ~~*~~ ~ y t o s i sbactericidal , ~ ~ ~ ~ ~ c a p a ~ i t y , ' ~and J ~ * ~ some ~ studies indicate that bactericidal capacity show normal c h e r n o t a x i ~ . ~ ~vivo . ~ ~studies ln ~ * ~ ~than * ~ ~ nor*~~.~~ posttransfusion recovery and intravascular is a b n 0 r m a 1 ~ ~ * ~rather mal.20*42 In vitro chemotaxis has also been survival.37Leukocytes harvested by interreported as both norma120~25*37 and abmittent flow centrifugation (IFC) are subPosttransfusion studies show little jected to higher centrifugal forces and increment in the absolute neutrophil count exposed for a longer period of time to a of granulocytopenic recipient^,^^.^^.^^ deforeign surface. A cell may be in the bowl for as long as 20 minutes. The centrifugal creased recovery37 and shortened half life.37 Experiments with dogs using 51Cr force is approximately 500 x g at the top of labeled cells indicate that these cells are the bowl and 900 x g at the bottom. I n vitro studies show normal morpholsequestered in the spleen.zzI n vivo studies 0gy,12*43 ~ i a b i l i t y , ' ~superoxide , ~ ~ , ~ ~ formaconsistently show poor function. Labeled t i ~ n ,nitroblue ~~ tetrazolium r e d u ~ t i o n , ~ ~granulocytes can not be detected at sites of and in vivo chemotaxis is chemiluminescence,48phagocytosis,12*q fun~ approximately gicidal activity,12 bactericidal capacity,48 d e f e c t i ~ e . ~It. ~ requires and chemotaxis." I n vivo studies also three times as many neutrophils when cells are collected by CFF to protect septicemic give IFC good marks showing normal postneutropenic dogs3 and in man it takes 13 transfusion recovery, a blood half time of times more neutrophils collected by CFF 4.1 hours40 and good migration into skin than from units of whole blood to accumulate It has become increasingly the same number of polymorphonuclear evident that granulocytes collected by conleukocytes in skin chamber^.^^ These results tinuous flow filtration (CFF) do not behave normally. Attachment of polymorphonu- show such poor neutrophil function that it is clear leukocytes to nylon fibers appears to necessary to seriously question the bias of initiate a sequence of events that impairs clinical trials indicating the value of granulofunction. Morphology is abnormal. The cyte transfusions using cells collected by develop cells are vacuolated,32~25~37~41*42-44*51 CFF.1*24The weight of experimental evifissures," and are partially degran~lated.~~." dence indicates that the neutrophil collected Abnormalities are directly related to the by reversible leukoadhesion is partially degranulated, has a mild defect in bactericontact time with the nylon fibers.51Some of these changes may be r e v e r ~ i b l eThere .~~ cidal capacity, is excessively "sticky" and is also some indication that defective when transfused shows a poor increment in function may be improved by pretreating the blood absolute neutrophil count, has a donors with g l ~ ~ o ~ o r t i ~ oThe i d spartial .~~ shortened half life and migrates poorly to sites of inflammation. The most gentle degranulation may be analogous to the interaction of neutrophils with biopolymer separation procedure used for clinical transmembranes coated with specific antibodyz1 fusions is gravity le~kapheresis.~ It requires a red blood cell sedimenting agent such as or an example of frustrated phagocytosis. Viability is well preserved and greater than hydroxyethyl starch which does not impair neutrophil function.12.43 Anticoagulants used !MI per cent of the cells exclude dye.20~22*31,42.44 A s might be expected from a method in these procedures maintain function. dependent upon leukoadhesion for cell Their effect on granulocytes has been separation, the harvested cell is excessively studied in units of whole blood c011ected.~~-~~ "sticky" and studies show a median ACD, CPD, heparin and ion exchange do not adherence of 170 per cent compared to impair bacterial killing, chemotaxis, quanPhagocytosis is usually reported titative nitroblue tetrazolium reduction or as n 0 r m a 1 , ~ ~ * although ~ ~ * ~ ~ there * ~ ~ *are ~ ~ hexose monophosphate shunt stimulation.
Volume 19 Number I
GRANULOCYTE CONCENTRATES
However, there are differences when cells are stored and chemotaxis is best maintained with ACD and CPD.36 One of the more important questions to be resolved is whether the use of glucocorticoids to increase the yield of neutrophils impairs function and produces a steroid l e s i ~ n . ~These * ~ drugs have significant anti-inflammatory actions that could be a contraindication to their use. We examined the effect of a single injection of dexamethasone given to leukapheresis donors on their cells collected by IFC. There were no adverse effects on dye exclusion, phagocytosis, fungicidal activity, bactericidal capacity or chemotaxi~.'~Adherence has been tested and found normal in one and abnormal in another.33 There are no studies of in vivo chemotaxis. Steroid premedication may also be advantageou~'~ because of the redistribution of lymphocytes resulting in fewer numbers of T lymphocytes in the peripheral circulat i ~ n , ~ Otheoretically *~* decreasing the likelihood of a graft-versus-host reaction. Functional competence is the major consideration in determining how long granulocyte concentrates can be stored. When neutrophils are stored they gradually show morphologic alterations such as cytoplasmic vacuoles and round hyperchromatic nuclei. Viability, phagocytosis, microbial killing and in vitro chemotaxis show mild deterioration after 24 hours of storage. At 48 hours the chemotactic defect becomes severe.13 A study of in vivo chemotaxis shows a marked defect in migration after one day of storage .38 Practically, this means that granulocyte concentrates collected by CFC or IFC can be stored overnight only once. Leukocytes collected by CFF should not be stored.30 We examined the possibility of improved storage following donor pretreatment with dexamethasone since glucocorticoids have been used in preserving organs for transplantation. Beneficial effects are considered secondary to stabilized lysosomal membranes. We found that the
3
functional capabilities of the stored neutrophils were neither diminished or enhanced by the steroid re treatment.'^ There are a few preliminary reports on the optimal temperature for storing granulocyte concent r a t e ~ . Cells ~ ~ ~collected ~ . ~ ~ by IFC can be stored at 4-6 C.13 There is no comparative data for room temperature storage. Granulocytes collected by CFC maintain chemotaxis better at 20-24 C than at 1-6 C.50Caution must be used when interpretingin vitro tests of stored cells when purified neutrophil suspensions are used. Marginal cells may not survive the isolation procedures and investigators could inadvertently select a nonrepresentative population of healthy cells for testing. Another area to be considered is function following radiation of concentrates to prevent a graft-versus-hostreaction. A dose of 1500 rads is used because it has been shown to decrease the response of lymphocytes to phytohemagglutinin and abolish the response of lymphocytes in mixed cultures. Mature granulocytes function normally ~ , ~ up to after in vitro r a d i a t i ~ n . " . ~Doses 5000 rads have no effect on bactericidal capacity or c h e m o t a x i ~ . ~ ~ The advantage of using the donor with chronic granulocytic leukemia (CGL) is quantity and the numbers are so obviously impressive that it almost seems unnecessary to question functional competence. In this disease the mature neutrophil is known to be altered and abnormal alkaline phosphatase activity is used for both diagnosis and prognosis. Body surface scanning and radioautography techniques demonstrate that radiolabeled cells from donors with CGL do migrate to sites of inflammation after being transfu~ed.~ The immature granulocytes may also be capable of evolving into mature cells and provide the advantage of a prolonged constant infusion of granulocytes from the blood to the tissues. However, in vitro studies of isolated mature CGL polymorphonuclear leukocytes, i.e., bands and segmented forms, show defective
4
GLASSER
bacterial killing and defective intracellular iodination. These cells also have a decreased amount of l y s o ~ y m e . ~ ~ Experiments of cell function of granulocyte concentrates have brought into focus which functional considerations are of importance in granulocyte transfusion therapy and this should direct future investigative efforts. The method of collection is of primary importance and recent work using the technique of in vivo chemotaxis indicates that reversible leukoadhesion to nylon fibers seriously impairs migration to sites of inflammation. A great deal of work is needed on the preservation of leukocytes. Storage of granulocyte concentrates is limited by an inability to adequately preserve function even though cell numbers are well maintained. Chemotaxis is the most sensitive indicator of functional deterioration during storage. Regarding the question of adverse effects of corticosteroids, in vitro studies indicate that steroidrecruited neutrophils are normal. It is unlikely that a single dose of glucocorticoids given to normal individuals would produce a steroid lesion. However, additional studies using in vivo chemotaxis should be done. The anticoagulants and red blood cell sedimenting agents that are presently used do not impair cell function. Radiation also does not harm the mature neutrophil. Because of the limited pool of donors with CGL it is unlikely that functional studies of these cells, although of interest, will have much of an impact on granulocyte transfusion therapy. References 1. Alavi, J. B., R. K. Root, I. Djerassi, A. E. Evans,
S. J. Gluckman, R. R. MacGregor, D. Guerry, A. D. Scheiber, J. M. Shaw, P. Koch, and R. A. Cooper: A randomized clinical trial of granulocyte transfusions for infection in acute leukemia. N. Engl. J. Med. 2%706, 1977. 2. Appelbaum. F. R., L. Norton, and R. G. Graw, Jr.: Migration of transfused granulocytes in leukopenic dogs. Blood 49483, 1977.
3.
-, C. A. Bowles, R. W.Makuch, and A. B.
Deisseroth: Granulocyte transfusion therapy of experimental pseudomonas septicemia: study of cell dose and collection technique. Blood 50 Suppl. 1:w.1977. 4. Benbunan. M.. A. Bussel. M. J. Grange, J. Reviron, and J. Bernard: Collection by blood cell separator and in virra function of normal granulocytes. In: Proceedings of the International Symposium on Leukocyte Separation and Transfusion, London, September 9- 11, 1974. R. M. Lowenthaland J. M. Goldman, Eds. New York. Academic Press, 1975, p. 84. 5. Boggs, D. R.: Transfusion of neutrophils as prevention or treatment of infection in patients with neutropenia. N. Engl. J. Med. 290: 1055, 1974. 6. Cline, M. J.: Drugs and phagocytes. N. Engl. J. Med. 291: 1187, 1974. 7. Djerassi. 1.: Gravity leucapheresis-anew method
for collection of transfusable granulocytes. Exp. Hematol. 5 Suppl. 1:139. 1977. 8. Eastlund. T., L. Peddada, A. Heller, A. Britten, and L. Loose: The effect of temperature on storage of neutrophils collected by continuous flow filtration leukapheresis. Blood 50 Suppl. 1:305, 1977. 9. Eyre, H. J.. 1. M. Goldstein. S. Perry, and
R. G. Graw, Jr.: Leukocyte transfusions: Function of transfused granulocytes from donors with chronic myelocytic leukemia. Blood 36:432, 1970. 10. Fauci, A. S., and D. C. Dale: The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. Clin. Invest. 53240. 1974. 1. Freireich, E. J., G. Judson, and R. H. Levin:
2.
3.
14.
15.
Separation and collection of leukocytes. Cancer Res. 251516. 1%5. Glasser, L.: Discontinuous flow centrifugation leukapheresis and neutrophil function. Transfusion 17513, 1977. : Effect of storage on normal neutrophils collected by discontinuous-flow centrifugation leukapheresis. Blood 5 0 1145. 1977. -, D. W. Huestis, and J. F. Jones: Functional capabilities of steroid-recruited neutrophils harvested for clinical transfusion. N. Engl. J. Med. 29R1033. 1977. -, and D. W. Huestis: Characteristics of stored granulocytes collected from donors stimulated with dexamethasone. Transfusion
-
1953, 1979. 16. Goldstein, I. M.: Effect of steroids on lysosomes. Trans. Proc. 7:21, 1975. 17. Graw, R. G., C. D. Buckner. J. Whang-Peng.
B. G. Leventhal. G. Kruger, C. Berard. and E. S. Henderson: Complication of bone-marrow transplantation. Graft-versus-host disease re-
Volume 19 Number I
GRANULOCYTE CONCENTRATES
sulting from chronic-myelogenous-leukemia leucocyte transfusions. Lancet 2:338, 1970. 18. -, G. Herzig, S. Perry, and E. S. Henderson: Normal granulocyte transfusion therapy: treatment of septicemia due to gram-negative bacteria. N. Engl. J. Med. 287:367, 1972. 19. -, and G. P. Herzig: Granulocyte transfusion therapy. I n : Leukapheresis and Granulocyte Transfusions. E. Cohen and R. B. Dawson, Eds. Washington, D. C., American Association of Blood Banks, 1975, p. 11. 20. Hams, M. B., I. Djerassi, E. Schwartz, and R. K. Root: Polymorphonuclear leukocytes prepared by continuous-flow filtration leukapheresis: viability and function. Blood 44:707. 1974. 21. Hawkins, D.: Biopolymer membrane: A model
22.
23.
24.
25.
26.
system for the study of the neutrophilic leukocyte response t o immune complexes. J. Immunol. 107:344, 1971. Herzig. G. P., R. K. Root, and R. G. Graw, Jr.: Granulocyte collection by continuous-flow filtration leukapheresis. Blood 39554, 1972. Herzig, R. H., G. Herzig, and R. Graw, Jr.: Impaired transfusion response to granulocytes (PMN) collected by filtration leukopheresis. Proceedings of the 11th Annual Meeting of the Am. SOC. Clinical Oncology 16:1132, 1975. -, G. P. Herzig, R. G. Graw, Jr., M. 1. Bull, and K. K. Ray: Successful granulocyte transfusion therapy for gram-negative septicemia. N. Engl. J. Med. 296:701, 1977. Higby, D. J., T. Mazzone, I . Walczak, J. Hennas, and E. S. Henderson: III vitro studies of granulocytes obtained by filtration leucapheresis. I n : Proceedings of the International Symposium on Leukocyte Separation and Transfusion. London, September 9- 1 I , 1974. R. M. Lowenthal and J. M. Goldman, Eds. New York, Academic Press, 1975. p. 229. -, J. W. Yates, E. S. Henderson, and J. F. Holland: Filtration leukapheresis for granulocyte transfusion therapy. N. Engl. J. Med.
292:761, 1975. 27. Holley, T. R., D. E. Van Epps. R. L. Harvey,
R. E. Anderson, and R. C. Williams Jr.: Effect of high doses of radiation on human neutrophil chemotaxis, phagocytosis, and morphology. Am. J. Pathol. 7561. 1974. 28. Huestis, D. W., R. F. White, M. J. Price, and M. Inman: Use of hydroxyethyl starch to improve granulocyte collection in the Latham blood processor. Transfusion 15559, 1975. 29. -: Production, storage and histocompatibility of granulocytes. I n : Blood Leukocytes Function and Use in Therapy. Scientific Symposium of the International Society of Blood Transfusion and Uppsala University, Sweden, October 10-13, 1977, C. F. Hogman. K. LindahlKiessling. and H. Wigzell Eds. p. 55.
5
30. Judson, G., A. Jones, R. Kellogg, D. Buckner,
R. Eisel, S. Perry, and W. Greenough: Closed continuous-flow centrifuge. Nature 212816, 1%8. 31. Klock, J. C., and D. F. Bainton: Degranulation
32.
33.
34.
35.
and abnormal bactericidal function of granulocytes procured by reversible adhesion t o nylon wool. Blood 4 8 149, 1976. Lane, T. A.: Granulocyte concentrate preservation: 6 C versus room temperature. 30th Annual Meeting American Association of Blood Banks (Abstract S-75). 1977. MacGregor, R. R., P. J. Spagnuolo, and A. L. Lentnek: Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. N. Engl. J. Med. 291642,1974. MacPherson, J. L., T. B. Wiltbank, R. T. Steigbigel, J. Nusbacher, and B. Grapka: Leukapheresis: The effect of single high dose prednisone and dexamethasone on granulocyte adherence and bactericidal capacity. Transfusion 16533, 1976. McCullough, J., S. J. Carter, and P. G. Quie: Effects of anticoagulants and storage on granulocyte function in bank blood. Blood 43207, 1974.
36.
-, B.
37.
-, B.
J. Weiblen, and P. G. Quie: Chemotactic activity of human granulocytes preserved in various anticoagulants. J. Lab. Clin. Med.
84:902, 1974.
J. Weiblen, A. R. Deinard, J. Boen. I. E. Fortuny, and P. G. Quie: In vitro function and post-transfusion survival of granulocytes collected by continuous-flow centrifugation and by filtration leukapheresis. Blood
48315, 1976. 38. Odeberg, H., T. Olofsson, and I. Olsson: Granulo-
cyte function in chronic granulocytic leukemia. Br. J. Haematol. 29:427, 1975. 39. Price, T. H., and D. C. Dale: Neutrophil transfusion: The effect of collection technique and storage on in vivo chemotaxis. Blood 50 Suppl. 1:309, 1977.
-, and
D. C. Dale: Neutrophil transfusion: Effect of storage and of collection method on neutrophil blood kinetics. Blood 51:789, 1978. 41. Roy, A. J., R. A. Yankee, A. Brivkalns, and M. Fitch: Viability of granulocytes obtained by filtration leukapheresis. Transfusion 15539, 40.
1975. 42. Sane], F. T., J. Aisner, C. J. Tillman, C. A.
Schiffer, and P. H. Wiernik: Evaluation of granulocytes harvested by filtration leucapheresis: functional, histochemical and ultrastructural studies. I n : Proceedings of the International Symposium on Leukocyte Separation and Transfusion. London, September 9-1 1, 1974. R. M. Lowenthal and J. M. Goldman, Eds.. New York, Academic Press, 1975, p. 236.
6
GLASSER
43. Schiffer, C. A., J. Aisner, M. Schmukler, C. L.
Whitaker, and J. H. Wolff: The effect of hydroxyethyl starch on in vitro platelet and granulocyte function. Transfusion 15473, 1975. 44. -, D. H. Buchholz, J. Aisner, S. W. Betts, and P. H. Wiernik: Clinical experience with transfusion of granulocytes obtained by continuous flow filtration leukopheresis. Am. J. Med. 58373, 1975. 45. Selvaraj, R. J., R.J. McRipley, and A. J. Sbarra: The effect of phagocytosis and X-irradiation on human leukocyte metabolism. Cancer Res. 2722280, 1967. 46,
-,and A. J. Sbarra: Role of the phagocyte in
host-parasite interactions. VIII Effect of wholebody X irradiation on nicotinamides, lysosomal enzymes, and bacterial activities of leukocytes during phagocytosis. J. Bacteriol. 94: 149, 1%7. 47. Stossel, T. P.: Phagocytosis. N. Engl. J. Med. 290:717, 1974. 48. Strauss, R. G., J. A. Koepke, and L. C. Maguire:
Properties of neutrophils (PMN) prepared for
Transfusion January-February 1979
transfusion by the Haemonetics cell-separator. Blood 50 Suppl. 1:310, 1977. 49. Wade, P. H., E. H. Skrabut, L. Vinciguerra, and C. R. Valeri: In vitro function of granulocytes isolated from blood of normal volunteers using continuous-flow centrifugation in the IBMAminco Celltrifuge and adhesion-filtration leukapheresis using nylon fiber. Transfusion 17:136, 1977. 50. Weiblen, B. J., P. Peterson, and J. McCullough:
Effects of temperature on preservation of granulocyte function. 30th Annual Meeting American Association of Blood Banks (Abstract S-27), 1977. 51. Wright, D. G.. J. C. Kauffmann, M. J. Chusid,
G. P. Herzig, and J. I. Gallin: Functional abnormalities of human neutrophils collected by continuous flow filtration leukopheresis. Blood 46901, 1975. 52. Yu, D. T. Y.. P. J. Clements. H. E. Paulus,
J. B. Peter, J. Levy, and E. V. Barnett: Human lymphocyte subpopulations. Effect of corticosteroids. J. Clin. Invest. 53:565, 1974.
No. 1
January-February 1979
Special Report
Functional Considerations of Granulocyte Concentrates Used for Clinical Transfusions L. GLASSER From the Depurtment of Puthology, College of Medicine. Uni,wsitg oJ' Arizonu Tircson. Arizoncr I
tion in the noninfected Whether GRANu LOCYE CON CENTRATES are the this dose is optimal for human survival is not newest of the blood components to be used known. in transfusion therapy. Clinical studies sugAny discussion of dose must take into gest that they are useful primarily in cases consideration the functional competence of of acute leukemia where the goal of chemocells. The performance of the ~-~~ therapy is bone marrow a p l a ~ i a . ~ , l ~ t-h~e transfused harvested phagocyte must be evaluated Component therapy with red blood cells and 1) when drugs are used to stimulate the platelets prevents the complications of anemia and bleeding, and white blood cell endogenous production of normal donor cells, 2) in the donor with chronic transfusions would be expected to reduce granulocytic leukemia, 3) after the leukothe mortality from infection. cyte has been in contact with red blood Transfusions of granulocytes should be cell sedimenting agents or anticoagulants, given daily.I8 It is generally thought that 4) following the interaction of the granulothe more the better. Experiments with cyte with machines or other collection neutropenic dogs show that daily doses of devices, 5 ) after radiation of the concen2 x lo8 neutrophils per kilogram will trate, and 6) during storage. prevent mortality from pseudomonas septiThe most important factor regarding ~ e m i a .Surprisingly, ~ this would translate neutrophil function is the method of collecto one granulocyte concentrate daily for a tion. Methods using centrifugation preserve 70-kilogram man at our institution where function better than filtration methods using cells are collected by intermittent flow reversible leukoadhesion. With continuous is only one c e n t r i f u g a t i ~ n . This ~ ~ * ~dose ~ flow centrifugation (CFC), the average time tenth of the daily human neutrophil producof exposure of the leukocyte to foreign surfaces is three minutes at the relatively Presented in part at the Haemonetics Research In vitro low centrifugal force of 42 x g.ll*30 Institute Advanced Component Seminar, Boston, Massachusetts, June 20. 1978. studies have shown that cells harvested by Received for publication June 8, 1978: accepted CFC have normal r n o r p h o l ~ g y , via~~ July 19, 1978. b i l i t ~ , quantitative ~ . ~ ~ nitroblue tetrazolium Supported by U.S.Public Health Service Grant No. r e d u ~ t i o n ? ~respiratory burst ?7*49 phagoCA 17094. 0041-1132/79/0100/0001 $00.80 0 J . B. Lippincott Co. '
Transtiision January-February 1979
1
Volume 19 Number I
2
GLASSER
Tranrfbrion January-Febmary 1979
studies showing i r n ~ a i r m e n t .Most ~~*~~ ~ y t o s i sbactericidal , ~ ~ ~ ~ ~ c a p a ~ i t y , ' ~and J ~ * ~ some ~ studies indicate that bactericidal capacity show normal c h e r n o t a x i ~ . ~ ~vivo . ~ ~studies ln ~ * ~ ~than * ~ ~ nor*~~.~~ posttransfusion recovery and intravascular is a b n 0 r m a 1 ~ ~ * ~rather mal.20*42 In vitro chemotaxis has also been survival.37Leukocytes harvested by interreported as both norma120~25*37 and abmittent flow centrifugation (IFC) are subPosttransfusion studies show little jected to higher centrifugal forces and increment in the absolute neutrophil count exposed for a longer period of time to a of granulocytopenic recipient^,^^.^^.^^ deforeign surface. A cell may be in the bowl for as long as 20 minutes. The centrifugal creased recovery37 and shortened half life.37 Experiments with dogs using 51Cr force is approximately 500 x g at the top of labeled cells indicate that these cells are the bowl and 900 x g at the bottom. I n vitro studies show normal morpholsequestered in the spleen.zzI n vivo studies 0gy,12*43 ~ i a b i l i t y , ' ~superoxide , ~ ~ , ~ ~ formaconsistently show poor function. Labeled t i ~ n ,nitroblue ~~ tetrazolium r e d u ~ t i o n , ~ ~granulocytes can not be detected at sites of and in vivo chemotaxis is chemiluminescence,48phagocytosis,12*q fun~ approximately gicidal activity,12 bactericidal capacity,48 d e f e c t i ~ e . ~It. ~ requires and chemotaxis." I n vivo studies also three times as many neutrophils when cells are collected by CFF to protect septicemic give IFC good marks showing normal postneutropenic dogs3 and in man it takes 13 transfusion recovery, a blood half time of times more neutrophils collected by CFF 4.1 hours40 and good migration into skin than from units of whole blood to accumulate It has become increasingly the same number of polymorphonuclear evident that granulocytes collected by conleukocytes in skin chamber^.^^ These results tinuous flow filtration (CFF) do not behave normally. Attachment of polymorphonu- show such poor neutrophil function that it is clear leukocytes to nylon fibers appears to necessary to seriously question the bias of initiate a sequence of events that impairs clinical trials indicating the value of granulofunction. Morphology is abnormal. The cyte transfusions using cells collected by develop cells are vacuolated,32~25~37~41*42-44*51 CFF.1*24The weight of experimental evifissures," and are partially degran~lated.~~." dence indicates that the neutrophil collected Abnormalities are directly related to the by reversible leukoadhesion is partially degranulated, has a mild defect in bactericontact time with the nylon fibers.51Some of these changes may be r e v e r ~ i b l eThere .~~ cidal capacity, is excessively "sticky" and is also some indication that defective when transfused shows a poor increment in function may be improved by pretreating the blood absolute neutrophil count, has a donors with g l ~ ~ o ~ o r t i ~ oThe i d spartial .~~ shortened half life and migrates poorly to sites of inflammation. The most gentle degranulation may be analogous to the interaction of neutrophils with biopolymer separation procedure used for clinical transmembranes coated with specific antibodyz1 fusions is gravity le~kapheresis.~ It requires a red blood cell sedimenting agent such as or an example of frustrated phagocytosis. Viability is well preserved and greater than hydroxyethyl starch which does not impair neutrophil function.12.43 Anticoagulants used !MI per cent of the cells exclude dye.20~22*31,42.44 A s might be expected from a method in these procedures maintain function. dependent upon leukoadhesion for cell Their effect on granulocytes has been separation, the harvested cell is excessively studied in units of whole blood c011ected.~~-~~ "sticky" and studies show a median ACD, CPD, heparin and ion exchange do not adherence of 170 per cent compared to impair bacterial killing, chemotaxis, quanPhagocytosis is usually reported titative nitroblue tetrazolium reduction or as n 0 r m a 1 , ~ ~ * although ~ ~ * ~ ~ there * ~ ~ *are ~ ~ hexose monophosphate shunt stimulation.
Volume 19 Number I
GRANULOCYTE CONCENTRATES
However, there are differences when cells are stored and chemotaxis is best maintained with ACD and CPD.36 One of the more important questions to be resolved is whether the use of glucocorticoids to increase the yield of neutrophils impairs function and produces a steroid l e s i ~ n . ~These * ~ drugs have significant anti-inflammatory actions that could be a contraindication to their use. We examined the effect of a single injection of dexamethasone given to leukapheresis donors on their cells collected by IFC. There were no adverse effects on dye exclusion, phagocytosis, fungicidal activity, bactericidal capacity or chemotaxi~.'~Adherence has been tested and found normal in one and abnormal in another.33 There are no studies of in vivo chemotaxis. Steroid premedication may also be advantageou~'~ because of the redistribution of lymphocytes resulting in fewer numbers of T lymphocytes in the peripheral circulat i ~ n , ~ Otheoretically *~* decreasing the likelihood of a graft-versus-host reaction. Functional competence is the major consideration in determining how long granulocyte concentrates can be stored. When neutrophils are stored they gradually show morphologic alterations such as cytoplasmic vacuoles and round hyperchromatic nuclei. Viability, phagocytosis, microbial killing and in vitro chemotaxis show mild deterioration after 24 hours of storage. At 48 hours the chemotactic defect becomes severe.13 A study of in vivo chemotaxis shows a marked defect in migration after one day of storage .38 Practically, this means that granulocyte concentrates collected by CFC or IFC can be stored overnight only once. Leukocytes collected by CFF should not be stored.30 We examined the possibility of improved storage following donor pretreatment with dexamethasone since glucocorticoids have been used in preserving organs for transplantation. Beneficial effects are considered secondary to stabilized lysosomal membranes. We found that the
3
functional capabilities of the stored neutrophils were neither diminished or enhanced by the steroid re treatment.'^ There are a few preliminary reports on the optimal temperature for storing granulocyte concent r a t e ~ . Cells ~ ~ ~collected ~ . ~ ~ by IFC can be stored at 4-6 C.13 There is no comparative data for room temperature storage. Granulocytes collected by CFC maintain chemotaxis better at 20-24 C than at 1-6 C.50Caution must be used when interpretingin vitro tests of stored cells when purified neutrophil suspensions are used. Marginal cells may not survive the isolation procedures and investigators could inadvertently select a nonrepresentative population of healthy cells for testing. Another area to be considered is function following radiation of concentrates to prevent a graft-versus-hostreaction. A dose of 1500 rads is used because it has been shown to decrease the response of lymphocytes to phytohemagglutinin and abolish the response of lymphocytes in mixed cultures. Mature granulocytes function normally ~ , ~ up to after in vitro r a d i a t i ~ n . " . ~Doses 5000 rads have no effect on bactericidal capacity or c h e m o t a x i ~ . ~ ~ The advantage of using the donor with chronic granulocytic leukemia (CGL) is quantity and the numbers are so obviously impressive that it almost seems unnecessary to question functional competence. In this disease the mature neutrophil is known to be altered and abnormal alkaline phosphatase activity is used for both diagnosis and prognosis. Body surface scanning and radioautography techniques demonstrate that radiolabeled cells from donors with CGL do migrate to sites of inflammation after being transfu~ed.~ The immature granulocytes may also be capable of evolving into mature cells and provide the advantage of a prolonged constant infusion of granulocytes from the blood to the tissues. However, in vitro studies of isolated mature CGL polymorphonuclear leukocytes, i.e., bands and segmented forms, show defective
4
GLASSER
bacterial killing and defective intracellular iodination. These cells also have a decreased amount of l y s o ~ y m e . ~ ~ Experiments of cell function of granulocyte concentrates have brought into focus which functional considerations are of importance in granulocyte transfusion therapy and this should direct future investigative efforts. The method of collection is of primary importance and recent work using the technique of in vivo chemotaxis indicates that reversible leukoadhesion to nylon fibers seriously impairs migration to sites of inflammation. A great deal of work is needed on the preservation of leukocytes. Storage of granulocyte concentrates is limited by an inability to adequately preserve function even though cell numbers are well maintained. Chemotaxis is the most sensitive indicator of functional deterioration during storage. Regarding the question of adverse effects of corticosteroids, in vitro studies indicate that steroidrecruited neutrophils are normal. It is unlikely that a single dose of glucocorticoids given to normal individuals would produce a steroid lesion. However, additional studies using in vivo chemotaxis should be done. The anticoagulants and red blood cell sedimenting agents that are presently used do not impair cell function. Radiation also does not harm the mature neutrophil. Because of the limited pool of donors with CGL it is unlikely that functional studies of these cells, although of interest, will have much of an impact on granulocyte transfusion therapy. References 1. Alavi, J. B., R. K. Root, I. Djerassi, A. E. Evans,
S. J. Gluckman, R. R. MacGregor, D. Guerry, A. D. Scheiber, J. M. Shaw, P. Koch, and R. A. Cooper: A randomized clinical trial of granulocyte transfusions for infection in acute leukemia. N. Engl. J. Med. 2%706, 1977. 2. Appelbaum. F. R., L. Norton, and R. G. Graw, Jr.: Migration of transfused granulocytes in leukopenic dogs. Blood 49483, 1977.
3.
-, C. A. Bowles, R. W.Makuch, and A. B.
Deisseroth: Granulocyte transfusion therapy of experimental pseudomonas septicemia: study of cell dose and collection technique. Blood 50 Suppl. 1:w.1977. 4. Benbunan. M.. A. Bussel. M. J. Grange, J. Reviron, and J. Bernard: Collection by blood cell separator and in virra function of normal granulocytes. In: Proceedings of the International Symposium on Leukocyte Separation and Transfusion, London, September 9- 11, 1974. R. M. Lowenthaland J. M. Goldman, Eds. New York. Academic Press, 1975, p. 84. 5. Boggs, D. R.: Transfusion of neutrophils as prevention or treatment of infection in patients with neutropenia. N. Engl. J. Med. 290: 1055, 1974. 6. Cline, M. J.: Drugs and phagocytes. N. Engl. J. Med. 291: 1187, 1974. 7. Djerassi. 1.: Gravity leucapheresis-anew method
for collection of transfusable granulocytes. Exp. Hematol. 5 Suppl. 1:139. 1977. 8. Eastlund. T., L. Peddada, A. Heller, A. Britten, and L. Loose: The effect of temperature on storage of neutrophils collected by continuous flow filtration leukapheresis. Blood 50 Suppl. 1:305, 1977. 9. Eyre, H. J.. 1. M. Goldstein. S. Perry, and
R. G. Graw, Jr.: Leukocyte transfusions: Function of transfused granulocytes from donors with chronic myelocytic leukemia. Blood 36:432, 1970. 10. Fauci, A. S., and D. C. Dale: The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. Clin. Invest. 53240. 1974. 1. Freireich, E. J., G. Judson, and R. H. Levin:
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Separation and collection of leukocytes. Cancer Res. 251516. 1%5. Glasser, L.: Discontinuous flow centrifugation leukapheresis and neutrophil function. Transfusion 17513, 1977. : Effect of storage on normal neutrophils collected by discontinuous-flow centrifugation leukapheresis. Blood 5 0 1145. 1977. -, D. W. Huestis, and J. F. Jones: Functional capabilities of steroid-recruited neutrophils harvested for clinical transfusion. N. Engl. J. Med. 29R1033. 1977. -, and D. W. Huestis: Characteristics of stored granulocytes collected from donors stimulated with dexamethasone. Transfusion
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1953, 1979. 16. Goldstein, I. M.: Effect of steroids on lysosomes. Trans. Proc. 7:21, 1975. 17. Graw, R. G., C. D. Buckner. J. Whang-Peng.
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Volume 19 Number I
GRANULOCYTE CONCENTRATES
sulting from chronic-myelogenous-leukemia leucocyte transfusions. Lancet 2:338, 1970. 18. -, G. Herzig, S. Perry, and E. S. Henderson: Normal granulocyte transfusion therapy: treatment of septicemia due to gram-negative bacteria. N. Engl. J. Med. 287:367, 1972. 19. -, and G. P. Herzig: Granulocyte transfusion therapy. I n : Leukapheresis and Granulocyte Transfusions. E. Cohen and R. B. Dawson, Eds. Washington, D. C., American Association of Blood Banks, 1975, p. 11. 20. Hams, M. B., I. Djerassi, E. Schwartz, and R. K. Root: Polymorphonuclear leukocytes prepared by continuous-flow filtration leukapheresis: viability and function. Blood 44:707. 1974. 21. Hawkins, D.: Biopolymer membrane: A model
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system for the study of the neutrophilic leukocyte response t o immune complexes. J. Immunol. 107:344, 1971. Herzig. G. P., R. K. Root, and R. G. Graw, Jr.: Granulocyte collection by continuous-flow filtration leukapheresis. Blood 39554, 1972. Herzig, R. H., G. Herzig, and R. Graw, Jr.: Impaired transfusion response to granulocytes (PMN) collected by filtration leukopheresis. Proceedings of the 11th Annual Meeting of the Am. SOC. Clinical Oncology 16:1132, 1975. -, G. P. Herzig, R. G. Graw, Jr., M. 1. Bull, and K. K. Ray: Successful granulocyte transfusion therapy for gram-negative septicemia. N. Engl. J. Med. 296:701, 1977. Higby, D. J., T. Mazzone, I . Walczak, J. Hennas, and E. S. Henderson: III vitro studies of granulocytes obtained by filtration leucapheresis. I n : Proceedings of the International Symposium on Leukocyte Separation and Transfusion. London, September 9- 1 I , 1974. R. M. Lowenthal and J. M. Goldman, Eds. New York, Academic Press, 1975. p. 229. -, J. W. Yates, E. S. Henderson, and J. F. Holland: Filtration leukapheresis for granulocyte transfusion therapy. N. Engl. J. Med.
292:761, 1975. 27. Holley, T. R., D. E. Van Epps. R. L. Harvey,
R. E. Anderson, and R. C. Williams Jr.: Effect of high doses of radiation on human neutrophil chemotaxis, phagocytosis, and morphology. Am. J. Pathol. 7561. 1974. 28. Huestis, D. W., R. F. White, M. J. Price, and M. Inman: Use of hydroxyethyl starch to improve granulocyte collection in the Latham blood processor. Transfusion 15559, 1975. 29. -: Production, storage and histocompatibility of granulocytes. I n : Blood Leukocytes Function and Use in Therapy. Scientific Symposium of the International Society of Blood Transfusion and Uppsala University, Sweden, October 10-13, 1977, C. F. Hogman. K. LindahlKiessling. and H. Wigzell Eds. p. 55.
5
30. Judson, G., A. Jones, R. Kellogg, D. Buckner,
R. Eisel, S. Perry, and W. Greenough: Closed continuous-flow centrifuge. Nature 212816, 1%8. 31. Klock, J. C., and D. F. Bainton: Degranulation
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and abnormal bactericidal function of granulocytes procured by reversible adhesion t o nylon wool. Blood 4 8 149, 1976. Lane, T. A.: Granulocyte concentrate preservation: 6 C versus room temperature. 30th Annual Meeting American Association of Blood Banks (Abstract S-75). 1977. MacGregor, R. R., P. J. Spagnuolo, and A. L. Lentnek: Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. N. Engl. J. Med. 291642,1974. MacPherson, J. L., T. B. Wiltbank, R. T. Steigbigel, J. Nusbacher, and B. Grapka: Leukapheresis: The effect of single high dose prednisone and dexamethasone on granulocyte adherence and bactericidal capacity. Transfusion 16533, 1976. McCullough, J., S. J. Carter, and P. G. Quie: Effects of anticoagulants and storage on granulocyte function in bank blood. Blood 43207, 1974.
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J. Weiblen, A. R. Deinard, J. Boen. I. E. Fortuny, and P. G. Quie: In vitro function and post-transfusion survival of granulocytes collected by continuous-flow centrifugation and by filtration leukapheresis. Blood
48315, 1976. 38. Odeberg, H., T. Olofsson, and I. Olsson: Granulo-
cyte function in chronic granulocytic leukemia. Br. J. Haematol. 29:427, 1975. 39. Price, T. H., and D. C. Dale: Neutrophil transfusion: The effect of collection technique and storage on in vivo chemotaxis. Blood 50 Suppl. 1:309, 1977.
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D. C. Dale: Neutrophil transfusion: Effect of storage and of collection method on neutrophil blood kinetics. Blood 51:789, 1978. 41. Roy, A. J., R. A. Yankee, A. Brivkalns, and M. Fitch: Viability of granulocytes obtained by filtration leukapheresis. Transfusion 15539, 40.
1975. 42. Sane], F. T., J. Aisner, C. J. Tillman, C. A.
Schiffer, and P. H. Wiernik: Evaluation of granulocytes harvested by filtration leucapheresis: functional, histochemical and ultrastructural studies. I n : Proceedings of the International Symposium on Leukocyte Separation and Transfusion. London, September 9-1 1, 1974. R. M. Lowenthal and J. M. Goldman, Eds.. New York, Academic Press, 1975, p. 236.
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GLASSER
43. Schiffer, C. A., J. Aisner, M. Schmukler, C. L.
Whitaker, and J. H. Wolff: The effect of hydroxyethyl starch on in vitro platelet and granulocyte function. Transfusion 15473, 1975. 44. -, D. H. Buchholz, J. Aisner, S. W. Betts, and P. H. Wiernik: Clinical experience with transfusion of granulocytes obtained by continuous flow filtration leukopheresis. Am. J. Med. 58373, 1975. 45. Selvaraj, R. J., R.J. McRipley, and A. J. Sbarra: The effect of phagocytosis and X-irradiation on human leukocyte metabolism. Cancer Res. 2722280, 1967. 46,
-,and A. J. Sbarra: Role of the phagocyte in
host-parasite interactions. VIII Effect of wholebody X irradiation on nicotinamides, lysosomal enzymes, and bacterial activities of leukocytes during phagocytosis. J. Bacteriol. 94: 149, 1%7. 47. Stossel, T. P.: Phagocytosis. N. Engl. J. Med. 290:717, 1974. 48. Strauss, R. G., J. A. Koepke, and L. C. Maguire:
Properties of neutrophils (PMN) prepared for
Transfusion January-February 1979
transfusion by the Haemonetics cell-separator. Blood 50 Suppl. 1:310, 1977. 49. Wade, P. H., E. H. Skrabut, L. Vinciguerra, and C. R. Valeri: In vitro function of granulocytes isolated from blood of normal volunteers using continuous-flow centrifugation in the IBMAminco Celltrifuge and adhesion-filtration leukapheresis using nylon fiber. Transfusion 17:136, 1977. 50. Weiblen, B. J., P. Peterson, and J. McCullough:
Effects of temperature on preservation of granulocyte function. 30th Annual Meeting American Association of Blood Banks (Abstract S-27), 1977. 51. Wright, D. G.. J. C. Kauffmann, M. J. Chusid,
G. P. Herzig, and J. I. Gallin: Functional abnormalities of human neutrophils collected by continuous flow filtration leukopheresis. Blood 46901, 1975. 52. Yu, D. T. Y.. P. J. Clements. H. E. Paulus,
J. B. Peter, J. Levy, and E. V. Barnett: Human lymphocyte subpopulations. Effect of corticosteroids. J. Clin. Invest. 53:565, 1974.
