Effect of Recombinant Granulocyte Colony-Stimulating Factor on Blood

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Original ...... Article Effect of Recombinant Granulocyte Colony-Stimulating Factor on Blood Neutrophil Concentrations among Patients with ‘‘Idiopathic Neonatal Neutropenia’’: A Randomized, Placebo-controlled Trial

Sandra E. Juul, MD, PhD INTRODUCTION Robert D. Christensen, MD A severe but self-limited idiopathic variety of neutropenia, termed ‘‘idiopathic neonatal neutropenia’’, has been described among preterm infants.1,2 Neonates with this condition have several features in common, including the fact that no specific variety of OBJECTIVES: neutropenia is diagnosed, it often appears late in the clinical We previously described a severe, prolonged, idiopathic, but self-resolving, course, can be quite prolonged, and generally occurs in otherwise variety of neutropenia among preterm neonates. In the present study, we well infants.1,2 It is not clear whether this condition is a single sought to assess the marrow neutrophil reserves of these patients by entity or represents multiple disorders with the common feature of serially measuring blood neutrophils following the administration of severe but self-limited neutropenia. As neutropenia in preterm recombinant granulocyte colony-stimulating factor (rG-CSF) or placebo. infants can be a sign of incipient severe illness, particularly sepsis, STUDY DESIGN: this variety of neutropenia in the neonatal intensive care unit (NICU) often causes concern, and sometimes prompts evaluations Prospective, randomized trial of rG-CSF vs placebo for infants with for infection and repeated or prolonged courses of antibiotics.1,2 ‘‘idiopathic neonatal neutropenia’’. We previously reported four patients with this variety of À1 RESULTS: neutropenia, all with blood neutrophil counts less than 500 ml . All responded to a 3-day course of recombinant granulocyte colony- During 36 consecutive months, 2407 neonates were admitted to the stimulating factor (rG-CSF) (10 mg/kg/day) with an increase in neonatal intensive care unit; 429 weighed less than 1500 g at birth, 14 of blood neutrophil counts.1 Our investigations suggested that this these were later diagnosed with ‘‘idiopathic neonatal neutropenia’’; 10 variety was the result of reduced neutrophil production, which was were enrolled in this trial. The five rG-CSF recipients had an immediate, not cyclic, not alloimmune, and not associated with recognized marked increase in blood neutrophil concentration, indicating adequate inborn errors, bacterial or viral infections, or medications. rG-CSF-mobilizable marrow neutrophil reserves. This effect persisted to The marrow neutrophil storage pool is a ready reserve of day 5, but counts were not different from those of the five placebo phagocytes that can be released into the circulation upon demand. recipients on days 12 and 15. Patients who have severe and prolonged neutropenia accompanied CONCLUSIONS: by few neutrophils in the marrow reserve have a significant Patients with ‘‘idiopathic neonatal neutropenia’’ have a substantial rG- deficiency in antibacterial defense. Such patients do not display a CSF-mobilizable marrow neutrophil reserve. On that basis, we speculate rapid increase in blood neutrophils following rG-CSF that this variety of neonatal neutropenia does not constitute a significant administration. It is not clear whether patients with ‘‘idiopathic deficiency in antibacterial defense. neonatal neutropenia’’ have a rapid increase in blood neutrophils Journal of Perinatology (2003) 23, 493–497. doi:10.1038/sj.jp.7210961 following rG-CSF administration, signifying an adequate neutrophil reserve, or rather whether such patients lack this response signifying the likelihood of a significant antibacterial defense deficiency. Our four previous cases appeared to have an intact response to rG-CSF, suggesting adequate neutrophil reserves. However, it can be

Department of Pediatrics (S.E.J.), University of Washington, WA, USA; and Department of problematical to interpret serial blood neutrophil counts in Pediatrics (R.D.C.), University of South Florida, FL, USA. neonates without a comparison group of placebo recipients. This study was Supported by NIH RR00082. Therefore, we conducted a randomized, placebo-controlled trial of

Address correspondence and reprint requests to Sandra Juul, MD, PhD, Department of Pediatrics, rG-CSF administration to patients with ‘‘idiopathic neonatal Division of Neonatology, University of Washington, Box 356320, Seattle, WA 98195, USA. neutropenia’’ to assess the blood neutrophil response and thereby

gain insight into the adequacy or inadequacy of their rG-CSF- Table 1 Clinical Characteristics of Patients <1500 g mobilizable marrow neutrophil reserves. Infants <1500 g

Sex (% male) 240/429 (56%) MATERIALS AND METHODS Ventilator support 317/429 (73.9%) From June 1, 1998 through July 1, 2001, patients in the NICU of Bronchopulmonary dysplasia 115/429 (26.8%) the Shands Children’s Hospital at the University of Florida Intraventricular hemorrhage underwent evaluation for neutropenia in accordance with our All grades 65/429 (15.2%) consensus document, ‘‘A consistent approach to evaluating Grade III 29/429 (6.7%) 3 neutropenia in the NICU’’. Patients with a blood neutrophil count Grade IV 8/429 (1.9%) <500 mlÀ1 for more than two days, or <1000 mlÀ1 for more than 4,5 5 days, had maternal blood testing for antineutrophil antibodies. Retinopathy of prematurity If those tests were negative, yet the neutropenia persisted with All stages 48/429 (11.2%) no diagnosis for the neutropenia, a marrow biopsy was performed Stage III 9/429 (2.1%) using a micro-method we reported.6 If no specific diagnosis for Stage IV 4/429 (0.9%) the neutropenia was still evident, the condition was termed ‘‘idiopathic neonatal neutropenia’’1 and the patient was eligible for performed. After this evaluation, 14 infants were identified with this study. ‘‘idiopathic neonatal neutropenia’’. In total, 10 were enrolled in After parental informed consent was obtained, eligible patients this study, six of whom were male. None of the 10 were clinically were randomized by the Shand’s Hospital Research Pharmacist, ill at the time of study entry. Blood neutrophil counts on the day of using a random-number table, to receive either rG-CSF 10 mg/kg/ study entry were 691±202 mlÀ1. The gestational age of these day i.v. over 30 minutes or placebo for 3 consecutive days. Study infants (mean±SE) was 28.7±0.7 weeks, median 29, range 25 to subjects had blood obtained for neutrophil concentrations at preset 32 weeks, with birth weights of 1067±80 g, median 1136, range intervals: 24 h, 48 h, 72 hours, 5, 12, and 15 days after the first 741 to 1474 g. Apgar scores averaged 6 at 1 minute, and 7 at 5 injection. Nosocomial infections between study entry and hospital minutes. Postnatal age of presentation with neutropenia ranged discharge were diagnosed by the clinicians caring for the patients. from 5 to 13 weeks (7.1±0.8 weeks, median 6.3 weeks). All had the No special infectious surveillance or cultures were part of this diagnosis of ‘‘idiopathic neonatal neutropenia’’, in that none had evaluation. Rather the hospital records were reviewed at discharge positive neutrophil antibody tests, none were delivered to women searching specifically for the diagnosis of nosocomial infection. with pregnancy-induced hypertension, and none had a recognized The study was approved by the Institutional Review Board of the infectious disease at the time of study. University of Florida and by the University of Florida Clinical As part of our ‘‘Consistent approach to neonatal neutropenia’’,3 Research Center Advisory Committee. Informed consent included all of these subjects with severe or prolonged neutropenia had a permission to use relevant clinical data recorded in the chart. marrow biopsy.3 In order to differentiate individuals with Data were analyzed by ANOVA, w2 test, or Pearson’s r correlation pseudoneutropenia due to excessive marginization, lidocaine with as appropriate. Results are reported as mean±SE, range and epinephrine was used as the topical anesthetic for these biopsies. median. Significance was considered at p<0.05. Only one infant had a significant increase in circulating neutrophils after the procedure; the prebiopsy neutrophil count was 913 mlÀ1, rising to 1596 mlÀ1 after the epinephrine exposure. RESULTS Excessive neutrophil marginization (pseudo-neutropenia) was not During the 3-year study period, 2407 infants were admitted to the thought to be a significant issue in these patients. NICU; 429 of these were less than 1500 g birth weight. Marrow findings and the specifics of the neutrophil counts from Demographics of the infants less than 1500 g are shown in Table 1. all 10 subjects are shown in Table 2. No specific diagnoses were Screening CBCs were not routinely performed; however, if an infant evident from any marrow study. Eight had varying degrees of left- was noted to be neutropenic based on a clinically indicated CBC, shifted myeloid maturation consistent with a consumptive process, they were evaluated according to our algorithm, ‘‘A consistent and two had decreased neutrophil precursors consistent with approach to evaluating neutropenia in the NICU’’.3 This decreased neutrophil production. Four of the marrow aspirates had assessment included an evaluation for sepsis if clinically increased B lymphocytes with decreased T lymphocytes. appropriate, and an appraisal for the presence of pregnancy- rG-CSF was administered for 3 consecutive days to five infants, induced hypertension, intrauterine growth retardation, and and placebo to five. Within 2 to 4 hours of rG-CSF administration, maternal antibodies. If no etiology for the neutropenia was the absolute neutrophil count increased, and counts remained apparent and neutropenia persisted, a marrow biopsy was elevated, compared with placebo recipients, for at least 5 days

494 Journal of Perinatology 2003; 23:493–497 Idiopathic Neonatal Neutropenia Juul and Christensen

Table 2 Neutrophil and Bone Marrow Data

Gest. age G-CSF Lowest ANC Peak ANC Day 12 ANC Comments* BM results

26 Y 918 4003 1008 Six sepsis evals, Staph Epi sepsis, Mix of maturing myeloid and erythroid cells F 10% neutrophil Gram neg sepsis precursorsFsomewhat decreased but otherwise normal 32 N 144 7134 7134 Two sepsis evals Myeloid hyperplasia and left shift. Megakaryocytes and erythroid precursors are present 28 N 276 1632 1054 Staph Epi sepsis, Candida sepsis Normocellular marrow for age (greater than 90% cellularity) with multilinear hematopoiesis and adequate maturation. Granulopoiesis is active and shows a moderate left shift 31 Y 754 10,080 10,080 Staph epi sepsis Myeloid precursors present. In some places these cells are scattered, however, focally they form sheets. Left-shifted myeloid maturation, suggestive of a consumptive process rather than a proliferative defect 28 Y 330 9790 2176 Four sepsis evals Immature myeloid cells without a maturing component; that is, Left shifted. Erythroid and megakaryocytes normal 30 N 539 2,070 1,352 Necrotizing enterocolitis, Staph Trilineage hematopoiesis present. Myeloid maturation Epi sepsis, one sepsis eval appears to be left shifted 29 N 782 1184 1184 Five sepsis evals Normocellular marrow with multilineage hematopoiesis. Neutrophils/mature granulocytes are markedly decreased but present. Predominance of B cells over T lymphocytes 25 Y 774 5040 2993 Four sepsis evals Immature granulocytes are present but are markedly decreased. Erythroid and megakaryocytic precursors are normal. B lymphocytes are increased and predominate over T lymphocytes 29 Y 896 9234 7987 Staph Epi sepsis, Four Left-shifted myeloid maturation with increased numbers of sepsis eval small lymphocytes. Megakaryocytes and erythroid precursors are quantitatively normal Predominance of B cells over T lymphocytes 29 N 638 1826 1650 Four sepsis evals Normocellular for age with left-shifted granulocytic hyperplasia. Mature granulocytes are present, but markedly reduced. A predominance of B lymphocytes over T cells

*‘‘Sepsis eval’’ indicates episodes in which the infant was cultured for possible sepsis, treated with 48 to 72 hours of antibiotics, but cultures were negative and antibiotics stopped. Episodes of documented sepsis are listed.

(Figure 1). On days 12 and 15, the counts of the two groups were similar. Prior to study, the neutropenia had persisted for between 18 and 63 days (Table 2). These infants underwent as many as six evaluations for sepsis during their period of neutropenia (range 2 to 6). No nosocomial infections were diagnosed in any of the subjects at the time of the neutropenia; however, sepsis was common during the entire hospitalization (Table 2). After the initial randomization, one infant received multiple courses of rG-CSF for persistent neutropenia with ANC <500 mlÀ1, and was discharged to home on rG-CSF twice a week.

DISCUSSION Severe and prolonged neutropenia can constitute a significant host 7 Figure 1. Effect of rG-CSF on neutrophil count. The Y-axis shows the defense deficiency. Indeed, children who have any of the varieties absolute neutrophil count, and the X-axis shows time in days. The 8–10 of severe chronic neutropenia, such as Kostmann syndrome, horizontal bar shows the duration of rG-CSF treatment. Mean values for cyclic neutropenia,8,11 Barth syndrome,12 or Schwachman each group are shown, flanked by standard error bars. *Indicates Diamond syndrome13 are likely to have repeated bacterial p<0.05.

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infections. Patients with profound but relatively transient information suggests to us that this variety of neonatal neutropenia neutropenia, such as following chemotherapy, can also have does not convey a high risk for infection. If we had observed a problems with bacterial infections during the neutropenic period.14 failure to respond to rG-CSF, we would have predicted an impaired rG-CSF provides benefit to patients with severe chronic neutropenia antibacterial defense. Epidemiological studies will be needed to by reducing their probability of acquiring an infection.8,9,15 The compare the rate of nosocomial infection among neonates who role of rG-CSF is less clear among neonates who have severe but have this condition vs matched controls, but we predict that such self-limited varieties of neutropenia, such as those with rates will be similar and that ‘‘idiopathic neonatal neutropenia’’ alloimmune neutropenia,16–18 autoimmune neutropenia,19,20 the can be considered a benign condition. neutropenia of neonates born to women with severe pregnancy- induced hypertension,21,22 and ‘‘idiopathic neonatal References neutropenia’’.1,2 1. Juul SE, Calhoun DA, Christensen RD. "Idiopathic neutropenia" in very low Many different definitions for neutropenia in neonates have birthweight infants. Acta Paediatr 1998;87:963–8. 23 À1 23 been proposed, including those by Xanthou (1500 ml ), 2. Omar SA, Salhadar A, Wooliever DE, Alsgaard PK. Late-onset neutropenia in 24 À1 25 À1 Manroe et al. (1800 ml ), and Mozinho et al. (1100 ml ). very low birth weight infants. Pediatrics 2000;106E:55. When defined as a circulating neutrophil concentration of less 3. Calhoun DA, Christensen RD, Edstrom CS, et al. Consistent approaches to than 1500 mlÀ1, neutropenia is a relatively common condition in procedures and practices in neonatal hematology. Clin Perinatol the NICU, affecting between 6 and 58% of preterm infants.2,7,26 2000;27:733–53. One of the relevant issues in determining whether a particular 4. Bux J, Hofmann C, Welte K. Serum G-CSF levels are not increased in variety of severe neutropenia conveys a risk for infectious patients with antibody-induced neutropenia unless they are suffering from acquisition is whether or not the marrow neutrophil reserve is infectious diseases. Br J Haematol 1999;105:616–7. adequate. For instance, individuals with ‘‘pseudoneutropenia’’, a 5. Bux J, Stroncek D. Human neutrophil antigens. Transfusion 2002;42:1523. condition where circulating neutrophils are chronically very low, 6. Sola MC, Rimsza LM, Christensen RD. A bone marrow biopsy technique suitable for use in neonates. Br J Haematol 1999;107:458–60. but the marrow reserve is normal, do not have a host defense 7. al-Mulla ZS, Christensen RD. Neutropenia in the neonate. Clin Perinatol deficiency. In vivo studies, measuring neutrophil accumulation in 1995;22:711–39. skin or mucous membranes, indicate that such individuals are 8. Welte K, Dale D. Pathophysiology and treatment of severe chronic capable of mobilizing adequate quantities of neutrophils from the neutropenia. Ann Hematol 1996;72:158–65. 27 marrow reserve into sites of infection. 9. Welte K, Boxer LA. Severe chronic neutropenia: pathophysiology and It is not clear whether the marrow neutrophil reserve is therapy. Semin Hematol 1997;34:267–78. adequate among patients with ‘‘idiopathic neonatal neutropenia’’. 10. Zeidler C, Boxer L, Dale DC, Freedman MH, Kinsey S, Welte K. Management The present study was designed to obtain this information, with of Kostmann syndrome in the G-CSF era. Br J Haematol 2000;109:490–5. respect to the rG-CSF-mobilizable marrow neutrophil reserves. 11. Bonilla MA, Dale D, Zeidler C, et al. Long-term safety of treatment with To accomplish this study, we used a period of 3 years at one center. recombinant human granulocyte colony- stimulating factor (r-metHuG- This was done with the cognizance that the number of study CSF) in patients with severe congenital neutropenias. Br J Haematol subjects would be very small, yet the consistent approach we had 1994;88:723–30. 12. Ades LC, Gedeon AK, Wilson MJ, et al. Barth syndrome: clinical features and adopted at the University of Florida for evaluating neutropenia was 3 confirmation of gene localization to distal Xq28. Am J Med Genet helpful in insuring a uniform evaluation. 1993;45:327–34. We observed that rG-CSF produced an immediate increase in 13. Calhoun DA, Christensen RD. Recent advances in the pathogenesis and blood neutrophil concentration. From this we conclude that the treatment of nonimmune neutropenias in the neonate. Curr Opin Hematol marrow reserves of these patients are not completely depleted. This 1998;5:37–41. interpretation is consistent with the reduced, but not depleted, 14. Murray NA, Acolet D, Deane M, Price J, Roberts IA. Fetal marrow suppression marrow reserve we previously reported among four patients with after maternal chemotherapy for leukaemia. Arch Dis Child Fetal Neonatal this condition.1 We observed that rG-CSF administration for 3 Ed 1994;71:F209–10. consecutive days effectuated a rapid and substantial increase in 15. Morstyn G, Foote M, Nelson S. Clinical benefits of improving host defenses blood neutrophil counts that persisted for at least 5 days, but did with rHuG-CSF. Ciba Found Symp 1997;204:78–85. not persist to 12 or to 15 days. We observed no clear benefit to 16. Gilmore MM, Stroncek DF, Korones DN. Treatment of alloimmune neonatal rG-CSF administration among the five recipients, compared with neutropenia with granulocyte colony-stimulating factor. J Pediatr 1994;125:948–51. the five placebo recipients, other than on blood neutrophil counts. 17. Rodwell RL, Gray PH, Taylor KM, Minchinton R. Granulocyte colony However, the study was not powered to assess possible clinical stimulating factor treatment for alloimmune neonatal neutropenia. Arch benefits, but was focused on whether blood neutrophil counts Dis Child Fetal Neonatal Ed 1996;75:F57–8. increased, which clearly they did. 18. Latini G, Del Vecchio A, Rosati E, Borzini P, Chirico G, Rondini G. Different We conclude that the rG-CSF-mobilizable neutrophil reserves of responses to granulocyte colony-stimulating factor treatment in siblings patients with ‘‘idiopathic neonatal neutropenia’’ are adequate. This with alloimmune neonatal neutropenia. 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