International Journal of Cell Cloning 83303-313 Suppl 1 (1990)
Use of Granulocyte-Macrophage Colony-Stimulating Factor in Patients with Malignancy and Bone Marrow Failure Richard K. Shadduck, Cmig S. Rosenfeld, Matthew Sulecki, Nabil Phillips, Donna Pnepiorka. Martin Earle, Ronald Stoller, Samuel Jacobs Pittsburgh Cancer Institute and Montefiore Hospital, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
Key Words. Granulocyte-macrophage colony-stimulating factor Hemopoiesis Myelopoiesis Granulopoiesis Myelodysplastic syndromes Bone marrow failure Abstract. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered to 10 patients with refractory malignancies, 2 patients who had myelodysplastic syndromes with severe neutropenia and to a patient who had delayed marrow recovery after 3 cycles of therapy for acute leukemia. A marked neutropenia and monocytopenia was observed within 5 min after an i.v. injection of GM-CSF. This persisted for 1-2h and seemed related to activation of an adhesive glycoprotein (M01)on the surface of these cells. With continued daily i.v. administration of GM-CSF, all patients with refractory malignancies developed a striking leukocytosis. Total leukocyte counts reached 75,0001plwithin 2 weeks of treatment. This was due to an increase in band and segmented neutrophils, eosinophils and monocytes. Accelerated myelopoiesis required the continuous presence of GM-CSF; with pump failure for 24 h or discontinuation after 14 days, leukocyte counts returned to normal levels in 24-48h. GM-CSF also increased myelopoiesis in the patients with myelodysplastic syndromes or following anti-leukemic treatment. These observations suggest that this growth factor should prove a useful adjunct in the treatment of patients with malignancies and bone marrow failure.
Introduction Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of a series of hemopoietic growth factors that appear to be involved in the regulation of granulopoiesis. When added to bone marrow cells in vitro, this factor induces colonies of granulocytes (neutrophils and eosinophils) and macrophages [l]. In Correspondence: Richard K.Shadduck, M.D., Department of Medicine, Montefiore Hospital, 3459 Fifth Avenue, Pittsburgh, PA 15213,USA. Received October 5, 1989;accepted for publication October 5, 1989. 0737-1454/90/$2.0010oAlphaMed Press
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addition, GM-CSF appears to prime other hemopoietic lineages to respond to factors such as erythropoietin . GM-CSF has also been shown to stimulate leukocyte production in vivo. Initial studies with appropriatetypes of GM-CSF (murine and human) showed stimulation of myelopoiesis in both mice  and in non-human primates . These observations were extended to patients with a variety of hematologic disorders, including the acquired immunodeficiencysyndrome . This initial report showed a dose-dependent increase in most circulating leukocytes in response to i.v. therapy. The studies have now been extended to patients with myelodysplastic syndromes [6,7], aplastic anemia [8, 91 and various types of malignancy [10-l2].In addition, GM-CSF has been used as a means of accelerating hemopoietic recovery, either in conjunction with high-dose chemotherapy  or following bone marrow transplantation [B]. We report our experiences with patients who had a variety of refractory malignancies, with far advanced myelodysplastic syndromesand one patient with delayed hematologic recovery after treatment for leukemia.
Patients and Materials GM-CSF was kindly supplied by Immunex Corporation (Seattle, WA) and by HoechstRoussel Pharmaceuticals, Inc. (Somerville, NJ). Ten patients with refractory metastatic carcinoma received this drug as a potential biologic modifier [D]. Leukocyte kinetics were observed in order to define the mechanism of action of this growth factor. These patients were 39 to 70 years old, and all were ambulatory outpatients. They received an initial i.v. bolus of either 5 pg or 25 pg/m* of GM-CSF. Blood counts were obtained at intervals of 5 min to 4 h thereafter. Additional blood was drawn on days 2 and 6 after the injection for leukocyte counts. GM-CSF was then administered as a continuous i.v. infusion at 100 or 500 pg/m*/day for 14 days. An early neutropenia was observed shortly after the i.v. injection. This was characterized by flow cytometry using an antibody directed against the M01 receptor. Two patients with severe advanced myelodysplasia were treated. The first patient was a 56-year-old white male with pancytopenia. The marrow was hypercellular with a striking increase in megakaryocytes, 70%erythroid cells, 10% myeloblasts and prominent ring sideroblasts. He received numerous blood and platelet transfusions over 4 months. He was hospitalized continuously from February through July 1988,requiring daily platelet transfusions and frequent red cell transfusions. The leukocyte count fell progressively to 1,500/& with 20% neutrophils by late May 1988. High-dose i.v. IgG failed to reverse the neutropenia or platelet refractory state. During this hospitalization, the patient developed a severe perirectal abscess that was unresponsive to antibiotics. With progression of his pancytopenia and worsening infection, the patient was treated with 250 pg/m*/day of GM-CSF by continuous i.v. infusion. This was discontinued after 14 days according to protocol. After a 10-day rest period, treatment was reinstituted for a second 14-day cycle of therapy. The second patient with myelodysplasia was a 65-year-old white female who had pancytopenia for 2 months. Her marrow was 40% cellular, but markedly dysplastic. She received transfusions, corticosteroids and androgens. She was readmitted to the hospital in August 1988 with a leukocyte count of 400/pl, a hemoglobin count of 7.8 and a platelet count of 7,000. During this hospitalization, the patient received multiple antibiotics, corticosteroids and i.v. IgG with no improvement. She was transferred to Montefiore Hospital in September 1988 with fever and acute gastrointestinal bleeding. She developed generalized seizures
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and became obtunded; CT scan was negative for central nervous system hemorrhage. Her leukocyte count ranged from 300-800/pl with few neutrophils seen. Treatment was continued with multiple antibiotics. After worsening of pulmonary function, bronchoscopy revealed Cundidu and Aspergillus organisms. She was treated for 1 week with daily 2 h infusions of 500 pg/m2 of GM-CSF. A 37-year-old white male with T cell acute lymphocytic leukemia (ALL) received GM-CSF for severe pancytopenia, following 3 courses of induction therapy. In May 1989, the leukocyte count was 75,00O/pl with 87% immature lymphocytes. Therapy with vincristine, daunorubicin and prednisone failed to yield a remission. He received mitoxantrone, cytosine arabinoside and VP16 starting on June 9, 1989. The marrow remained infiltrated with blast cells through June 23rd, at which time the patient was given 1.2 glmz of cyclophosphamide, followed by 14 days of L-asparaginase and dexamethasone. Over the next 34 days, the leukocyte count ranged from 200 to 400/pl, with rare neutrophils seen. He developed Entembucter sepsis and, subsequently, Staphylococcus epidermidis sepsis. A bone marrow aspirateand a biopsy obtained on July 24,1989, were essentiallydevoid of hemopoietic cells. The patient was treated with 250 pglm’ of GM-CSF S.C. every 12 h for 5 days.
In those patients with malignancies, leukocyte counts fell from 8,900to 3,200 within 30 min of 5 pg/m2 of i.v. GM-CSF and returned to baseline in 2 h. The leukocyte responses to 25 pg/m2of i.v. GM-CSF are shown in Table I. There was a dramatic early decrease in circulating neutrophils, monocytes and eosinophils; lymphocytes were largely unaffected. Leukocyte counts returned to pretreatment values within 2 h after injection. This appeared to result from the release of band and segmented cells from the marrow, rather than a return of the previous cells to the circulation. Bandkegmented rations were increased; several marrow samples obtained 30 min after injection revealed a marked decrease in band and segmented cells. Flow cytometry analysis revealed an increase in the reactivity of the residual neutrophils and monocytes with the M01 monoclonal antibody. The percentage of neutrophils positive for this receptor increased from 75% prior to injection to 95% 2 h after administration of the GM-CSF. There was also a 2-fold increase in the intensity of staining as measured by arbitrary fluorescence units. With continued i.v. infusion of GM-CSF, the leukocyte counts rose within 24 h (Fig. 1). The initial rise was due primarily to band and segmented neutrophils. This was followed by a second increment in cells after 5 days of GM-CSF therapy. At this time, increased eosinophils, monocytes, metamyelocytes and myelocytes were observed. In this patient, there was a 24 h pump failure that led to a marked decline in leukocyte count. With reinstitution of the infusion, a persistent leukocytosis was observed. In this patient and most others, leukocyte counts fell within 24 to 48 h of discontinuation of the infusion. There was no clinically significantchange in hematocrit or reticulocyte values at either dose level of GM-CSF (Table n).Neutrophil, monocyte and particularly eosinophil counts were markedly elevated after 14 days of therapy. Platelet values were unaffected at 100 pg/m*, but a moderate decline was seen in patients receiving the higher dose level. There was a mild increase in bone marrow cellularity
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Table I. Acute leukocyte responses to i.v. bolus GM-CSF
Minutes 0 5
15 30 60 120 240
8,250 1,150 1,425 2,725 4,167 7,500 9,575
6,885 230 505 1,650 3,378 6,392 8,229
542 40 114 154 161 295 528
695 857 772 844 620 733 744
3 3 17 15 64 78
Values are counts/pl after i.v. bolus injection of 25 pg/mzof GM-CSF. Neutrophils include band and segmented cells. ~
Table II. Hematologic values before and after GM-CSF treatment 100 pg/mz
Baseline Hematocrit (%) 29.6 f 1.1 Reticulocytes (%) 0.9 f 0.2 Leukocytes (pl) 7,480 f 1,180 Neutrophils 6,030 f 1,OOO Monocytes 550 f 130 Eosinophils 210 f 80 Platelets (x10-3) 323 f 71
30.5 f 1.0 31.2 f 1.8 28.6 f 2.3 1.3 f 0.3 1.2 f 0.2 1.4 f 0.2 20,100 f 3,740 7,900 f 1,950 44,930 f 8,500 13,530 f 2,790 6,530 f 1,660 25,810 f 6,240 1,020 f 460 500 f 150 3,300 f 1,240 3,620 f 1,150 60 f 30 11,500 f 5,310 316 f 81 420 f 67 245 f 55
Values were obtained on the day following a 14-day infusion of GM-CSF.
at the low-dose level (31% to 40%); cellularity increased from 34% to 76% in patients receiving 500 pg/m2. The 56-year-old patient with severe myelodysplasia was hospitalized continuously from February through July 1988. He remained febrile with a draining perirectal abscess despite administration of antibiotics and required almost daily platelet transfusions. At the time of GM-CSF therapy, the leukocyte count was 1,700 with 320 neutrophils and 240 monocytes/pl. The bone marrow was markedly hypercellular with increased megakaryocytes (Fig. 2) despite essentially no circulating platelets. GM-CSF was administered over 14 days. As shown in Figure 3, there was a striking improvement in the patient’s leukocyte and neutrophil values. The temperature declined with apparent healing of the perirectal abscess. During the 2-week treatment interval, he required only 2 U of red cell transfusions, as compared to 7 U in the 2 weeks preceding therapy and 10 U in the following 2 weeks. According to protocol, therapy was discontinued for 10 days at which time the
Effects of GM-CSF on Myelopoiesis
Fig. 1. Leukocyte response to continuous i.v. infusion of 100 pg/m*of GM-CSF. The infusion pump malfunctioned between days 6 and 7 of the continuous infusion.
Fig. 2. Bone marrow aspirate from a patient with a myelodysplastic syndrome. The marrow is hypercellular and erythroid predominant with increased megakaryqtes.
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Fig. 3. Responses to GM-CSFin a patient with a myelodysplasticsyndrome. The patient received two 14day courses of therapy in a dose of 250 Fg/m*/dayby continuous infusion.
leukocyte count and neutrophil values fell. Treatment with GM-CSF was reinstituted on June 23rd with a second, but less impressive, neutrophil response and a greater increase in eosinophils. Despite this improvement in white count, there was no improvement in red cell or platelet production. The patient experienced increasing peripheral edema and hypotension. After discontinuation of the GMCSF, he expired due to progressive pneumonia and congestive heart failure. A 65-year-old white female who had severe myelodysplasia with marked neutropenia, gastrointestinal bleeding and fungal pneumonia was treated with 7 daily infusions of GM-CSF. As shown in Figure 4, there was a prompt increase in circulating neutrophils and a decline in fever. Despite this, the patient developed progressive hypotension and multiple organ system failure with renal and hepatic insufficiency and expired. A 37-year-old white male with T cell ALL required 3 courses of induction days after treatment with cyclotherapy to induce bone marrow aplasia. *-one phosphamide, the leukocyte count was 200/pl. A bone marrow biopsy revealed 5% cellularity with predominant lymphocytes and plasma cells. Only a rare myeloid or erythroid precursor could be identified. On July 27th. the patient started therapy with 250 pg/m2 of GM-CSF S.C. every 12 h. By the following day, the leukocyte count had risen to 1,600 and achieved normal levels several days later (Table m).GM-CSF was discontinued 5 days later, when the leukocyte count was 5,600 with 81% neutrophils.
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Fig. 4. Responses to GM-CSF in a second patient with a myelodysplastic syndrome. The patient received daily 2 h infusions of 500 pg/m2 of GM-CSF.
Discussion These studies show that GM-CSF is an effective agent for increasing leukocyte counts in patients with malignancy, with myelodysplasia and following intensive chemotherapy. The initial leukopenia seen after i.v. injection or in more recent patients during i.v. infusions of GM-CSF appears to result from a rapid upregulation of the M01 receptor [13, 16, 171. This receptor is one of a series of adhesion-promoting glycoproteins that may be responsible for localization of neutrophils at a site of inflammation . In this regard, Peters et. al. have shown decreased migration of neutrophils into skin windows when patients were receiving GM-CSF after bone marrow transplantation . In future studies it will be important to determine whether this localization of cells can be overcome by appropriate chemotactic stimuli. Leukocyte counts returned to baseline values within several hours of i.v. bolus GM-CSF therapy. This appeared to result from release of new cells from the marrow as judged by an increased bandkegmented ratio and a decrease in mature cells in the marrow 30 min after injection. This raises the question of whether stimulation of myelopoiesis is due to the removal of a negative feedback by repeated release of mature cells from the marrow or to a direct effect on myeloid progenitor cells. Since both effects may occur simultaneously, it may be difficult, even with kinetic studies, to establish all the mechanisms of action of GM-CSF.
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Table III. Peripheral blood response to GM-CSF after prolonged aplasia Date
7/26 7/27 7/28 7/29 7/30 713 1 8/ 1
400 600 1,600 3,100 4,300 5,100
ND 200 1,248 2,325 3,655 4,284 4,536
Recovery from aplasia after anti-leukemic therapy followed by late rescue with GM-CSF. The patient received 250 pg/mZS.C. twice a day starting on 7/27. ND = not done
In several previous studies, GM-CSF appeared to improve red cell and platelet production in some patients. Approximately one-third of patients with myelodysplastic syndromes had an increase in hematocrit values or platelet counts with therapy .Furthermore, patients undergoing autologous bone marrow transplant for lymphoma had more rapid neutrophil and platelet recovery with GM-CSF as compared to historical controls . In the present patients, there was no evidence of stimulation of red cell or platelet production. Hematocrit and reticulocyte values were essentially unchanged in the patients with malignancy. Platelet counts were unchanged after treatment with 100 pg/m2,but declined in those patients receiving 500 pg/m2. Both of the patients with myelodysplastic syndromes had increments in their leukocyte counts, but neither had an increase in platelet counts with therapy. The first patient had evidence for marked ineffective megakaryocytopoiesis, as judged by a striking increase in megakaryocytes in the marrow. There was no reduction in the platelet transfusion requirements throughout the two courses of GM-CSF therapy. This suggests that this factor may have no direct effect on the differentiation of abnormal megakaryocytes. It is possible that longer treatment intervals may lead to stimulation of normal progenitor cells in some patients and suppression of the underlying malignant clone. It is of interest that this one patient had a reduced red cell transfusion requirement during the first course of GM-CSF therapy. Reticulocyte counts were not obtained, so it is difficult to judge whether this may have resulted from an increase in red blood cell production. Unfortunately, both of the patients with myelodysplastic syndromes had far advanced failure of the bone marrow and other organ systems at the time of therapy. Although GM-CSF increased the leukocyte counts and decreased fever, both patients expired from progressive multiple organ failure. A new prospective study is now underway in patients in earlier stages of myelodysplastic syndromes. This study is comparing the relative efficacy of i.v. versus S.C. treatment in these pa-
Effects of GM-CSF on Myelopoiesis
tients. The one patient with ALL appeared to benefit substantially from GM-CSF therapy. He was pancytopenic for eight weeks after three courses of induction therapy. He had just experienced two recent episodes of septicemia and had virtual aplasia of the marrow. There was a prompt rise in neutrophil counts within days of initiating therapy with GM-CSF. Although this may have occurred spontaneously, the tempo of hemopoietic recovery suggested a beneficial effect of the GM-CSF. The results outlined herein suggest that patients with a variety of hematologic disorders may respond to GM-CSF therapy. This may be used as an adjunct to chemotherapy to hasten marrow recovery, to stimulate neoplastic cells to proliferate and difkrentiate, or to rescue patients from severe aplasia after intensive antileukemic therapy.
Acknowledgments We gratefdly acknowledge the supply of recombinant human GM-CSF from Immunex and from Hoechst-Roussel Pharmaceuticals, Inc. We thank Dr. Alan Widelstein for help in performing the flow cytometry studies. We also thank Ann Mkh, Margaret Rosemweig, Mary Jo Santicky and Florence Boegel for providing excellent nursing care and aid in data collection. This research was supported in part by grants ROlCAl5237 and (2,424429 from the National Institutes of Health, and by the Immunex Corporation.
References Metcalf D, Begley CG, Johnson GR, et al. Biologic properties in vitro of a recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1986;6737-45. Sieff CA,Emerson SG, Donahue RE, et al. Human recombinant granulocyte-macrophage colony-stimulating factor: a multilineage hematopoietin. Science 1985;230: 1171-1173. Metcalf D, Begley CG. Williamson DJ, et al. Hemopoietic responses in mice injected with purified recombinant murine GM-CSF. Exp Hematol 1987;l5:1-9. Donahue RE, Wang EA, Stone DK, et al. Stimulationof haematopoiesis in primates by continuous infusion of recombinant human GM-CSF. Nature 1986;321:872-875. Groopman JE,Mitsuyasu RT,Dele0 MJ, Oette DH, Golde DW. Effect of recornbinant human granulocyte-macrophage colony-stimulatingfactor on myelopoiesis in the acquired immunodeficiency syndrome. N Engl J Med 1987317593-598, Vadhan-Raj S,bating M, LeMaistre A, et al. Effects of recombinant human granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 1987;3171545-l552. Thompson JA, Lee DJ, Kidd P, et al. Subcutaneousgranulocyte-macrophagecolonystimulating factor in patients with myelodysplastic syndrome: toxicity, phannacokinetics, and hematological effects. J Clin Oncol 1989;7629-637. Vadhan-Raj S,Buescher S,Broxmeyer HE, et al. Stimulationof myelopoiesis in patients with aplastic anemia by recombinant human granulocyte-macrophagecolonystimulating factor. N Engl J Med 1988;319:1628-1634.
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9 Champlin RE, Nimer SD, Ireland P, Oette DH, Golde DW. Treatment of refractory aplastic anemia with recombinant human granulocyte-macrophagecolony-stimulating factor. Blood 1989;73:694-699. 10 Vadhan-Raj S,Buescher S, LeMaistre A, et al. Stimulation of hematopoiesis in patients with bone marrow failure and in patients with malignancy by recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1988;72:134-141. 11 Henmann F, Schulz G, Lindemann A, et al. Hematopoietic responses in patients with advanced malignancy treated with recombinant human granulocyte-macrophage colony-stimulating factor. J Clin Oncol 1989;7159-167. 12 Lieschke GJ, Maher D, Cebon J, et al. Effects of bacterially synthesized recombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy. AM Int Med 1989;110:357-364. 13 Phillips N, Jacobs S,Stoller R, Earle M, Przepiorka D, Shadduck RK. Effect of recombinant human granulocyte-macrophage colony-stimulating factor on myelopoiesis in patients with refractory metastatic carcinoma. Blood 1989;74:26-34. 14 Antman KS, Griftin JD, Elias A, et al. Effect of recombinant human granulocytemacrophage colony-stimulating factor on chemotherapy-inhuced myelosuppression. N Engl J Med 1988;319:593-598. 15 Brandt SJ, Peters WP, Atwater SK, et al. Effect of recombinant human granulocytemacrophage colony-stimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation. N Engl J Med 1988; 318:869-876. 16 Socinski MA, Cannistra SA, Sullivan R, et al. Granulocyte-macrophage colonystimulating factor induces the expression of the CDllb surface adhesion molecule on human granulocytes in vivo. Blood 1988;72:691-697. 17 Devereux S, Bull HA, Campos-Costa D, Saib R, Linch DC. Granulocyte macrophage colony stimulating factor induced changes in cellular adhesion molecule expression and adhesion to endothelium: in vitro and in vivo studies in man. Br J Haematol 1989;71:323-330. 18 Freyer DR, Morganroth ML, Rogers CE, Arnaout MA, Todd RF III. Modulation of surface CDll/CD18 glycoproteins (Mol, LFA, ~ 1 5 4 9 5by ) human mononuclear phagocytes. Clin Immunol Immunopath 1988;46:272-283. 19 Peters WP, Stuart A, Affronti ML, Kim CS, Coleman RE. Neutrophil migration is defective during recombinant human granulocytemacrophage colony-stimulating factor infusion after autologous bone marrow transplantation in humans. Blood 1988;72: 1310-1315. 20 Nemunaitis J, Singer JW, Buckner CD, et al. Use of recombinant human granulocytemacrophage colony-stimulating factor in autologous marrow transplantation for lymphoid malignancies. Blood 1988;172:834-836.
Discussion Aglietta: I was interested in seeing your data with graft failure. We have treated some patients with GM-CSF after graft failure and in only one patient did we see an increase in neutrophil count that was maintained after GM-CSF discontinuation. In the other patients, the increase was minimal and was only present during GM-CSF therapy. So, we don’t have much evidence that GM-CSF is useful in graft failure. I wonder how many patients you had.
Shadduck: We have now treated 7 patients with poor engraftment at delayed intervals after the marrow transplant. I think four of the seven had marked increments in their neutro-
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phi1 counts towards normal levels. When you stop the GM-CSF, they often fall to about 50% of the latest count, but have not usually fallen back to severely neutropenic levels.
Aglietta: Any effect on platelets? Shadduck: Platelet recovery has been delayed. In the one patient I mentioned with Hodgkin’s disease, he developed an ITP-like picture and underwent splenectomy. In the patients with solid tumors that we treated at the 500 pglm2 dose level, there was a statistically significant decline in platelet count, but not a biologically significant decline. Kaushansky: When you were talking about weakness in the patients receiving the GM-CSF and that TNF was detectable by biological assays but not ELISA was that just a difference in sensitivity of the two assays or did you mean to imply that another molecule, like lymphotoxin was responsible for the weakness? Shadduck: We had only three serum samples before and three after GM-CSF that were measured. Although the values fell within the normal range, they were still threefold elevated as compared to the patient’s baseline by the bioassay. I don’t know the reason for the differences between the bioassay and ELISA. Murphy: In the one patient with a severe thrombocytopenia due to myelodysplasia, did you see any increase in platelets after two courses of GM-CSF? Shadduck: No. We did see a decrease in the red cell transfusion requirement, from about seven or eight units per week in the previous weeks to two units per week while on the GM-CSF. But, that may have been due to control of infection and other factors. However, there was no meaningful increase in baseline platelet counts prior to each daily transfusion. Marmont: Do you see this early sequestration phenomenon in all types of blood-I mean capillary blood, venous blood, and arterial blood? The reason for asking this question is that I once studied the same effect on thrombocytes after heparin, wherein there was a striking difference between capillary blood as opposed to big vessel blood. Shadduck: I can’t answer that. We were only obtaining blood from the central lines in these patients. Marmont: Haw long does the leukopeniapersist and have others observed this phenomenon? Shadduck Many investigatorshave observed the early neutropenia. The phenomenon clears within two hours when the counts return to normal. It seems to occur every day in patients receiving infusions of GM-CSF. I think it’s a repetitive phenomenon of activating the M01 receptors and the cells adhering presumably to the endothelium and then being replaced by a new wave of cells that come out. Griffin: Just a comment. I don’t think they stick to the endothelium. GM-CSF does not make neutrophils adhere very well to the endothelium in vitro. It could happen in vivo, but they adhere very well to each other because of the induction of M01 or CDllb, and I think their aggregates get trapped in small vessels like the lung.