Prospective cohort study of the circadian rhythm pattern in allogeneic sibling donors undergoing standard granulocyte colony-stimulating factor mobilization
© Shi et al.; licensee BioMed Central Ltd. 2013
Received: 20 November 2012
Accepted: 12 March 2013
Published: 20 March 2013
Prior in vivo murine studies suggest circadian oscillations for hematopoietic stem cell release, which are maintained following administration of granulocyte colony-stimulating factor (G-CSF) or plerixafor. Furthermore, retrospective data analysis of healthy donors who underwent G-CSF-induced mobilization demonstrated significantly increased CD34+ cell yields when collected in the afternoon compared with the morning.
A prospective study was conducted to directly examine the number of peripheral blood CD34+ and CD34+CD38– progenitor/stem cells at baseline and then every 6 hours for 24 hours on days 4 to 5 of G-CSF (10 μg/kg/day in the morning) mobilization in 11 allogeneic donors. Data were analyzed using mixed-model analysis of repeated measures.
Whereas we observed a significant increase in CD34+ cell counts toward the evening, counts were then sustained on the morning of day 5. The correlation between CD34+CD38– cell counts and the less defined CD34+ populations was weak.
Our results suggest that the pharmacodynamic activity and timing of G-CSF may alter endogenous progenitor rhythms. Donor age, medical history, and medications may also impact circadian rhythm. Further studies should examine the circadian rhythm at the peak of G-CSF mobilization and should consider potential confounders such as the time of G-CSF administration and the age of the subjects.
KeywordsAntigens CD34 Antigens CD38 Circadian rhythm Granulocyte-colony stimulating factor Hematopoietic progenitor cells Hematopoietic stem cell mobilization
Hematopoietic progenitor cell (HPC) mobilization using granulocyte colony-stimulating factor (G-CSF) is currently the most frequent method to obtain HPC for allogeneic transplantation. The optimal dose and schedule remains uncertain , but the National Marrow Donor Program uses a schedule of 10 μg/kg for 5 days, with the fifth dose ~1 hour prior to the initiation of the first leukapheresis . CD34+ cell doses >4.5 to 5×106/kg are associated with improved overall survival and reduced transplant-related mortality in the allogeneic setting [3, 4]. Although obtaining this goal in a single apheresis is preferable for donor convenience and safety, only 68% of G-CSF mobilized allogeneic donors reach a CD34+ cell dose ≥4×106/kg in a single apheresis, with a similar percentage for pegfilgrastim [5, 6], and about 70% of allogeneic donors actually undergo two leukaphereses . In addition, the timing of leukapheresis collection on days 5 and 6 is standardized based on staffing convenience rather than on kinetic data regarding the CD34+ cell number.
Based on preclinical and clinical data showing circadian oscillations of hematopoietic progenitor and stem cell release that are maintained with G-CSF or plerixafor mobilization [7–10], we conducted a single-center, prospective cohort study in allogeneic donors undergoing standard National Marrow Donor Program G-CSF mobilization (10 μg/kg/day for 5 days, administered between 06:00 and 07:00 hours), with the hypothesis that evening CD34+ concentrations are significantly higher than morning CD34+ concentrations. This study is important because hematopoietic progenitor collections typically occur in the morning due to convenience. The National Marrow Donor Program regimen was chosen because it is the most frequently used G-CSF regimen for unrelated allogeneic donors in the United States.
This trial was a single-center, prospective cohort IRB-approved (Mount Sinai School of Medicine IRB, #08-0446) study in allogeneic sibling donors. Written and signed consent was obtained from both donors and recipients. All sibling donors for allogeneic transplants at Mount Sinai Medical Center from January 2009 to December 2011 were approached to participate. Baseline CD34+ concentrations prior to starting G-CSF were assessed with two separately collected samples. Donors were admitted the evening of day 3 of G-CSF to the Clinical Research Center at Mount Sinai and stayed through two nights until the morning of day 5 of G-CSF mobilization, when they were discharged to the apheresis center to start leukapheresis. All donors reported normal sleep habits on specific review of symptoms and, except for one from Wisconsin, lived in the New York City area and had no history of travel within the past month outside their normal time zone.
Statistical analysis was performed using the following software: Excel® 2007 (Microsoft Corporation, Redmond, WA. USA), SPSS Statistics® 18 (IBM Corporation, Armonk, NY, USA), and SAS version 9.2 (SAS Institute Corp., Cary, NY, USA).
Summary of allogeneic sibling donor characteristics
Medical diseases (number of patients)
Coronary artery disease: 1
Range: 20 to 63
Diabetes mellitus type II: 2
Summary of recipient characteristics
Hematologic malignancy (number of patients)
CD34 doseaper recipient kg (×106)
Acute lymphoblastic leukemia: 1
Grade I: 1
Acute myelogenous leukemia: 3
Grade II: 4
Range: 23 to 60
Hodgkin disease: 1
Reduced intensity: 8
Grade III: 2
Range: 3.6 to 10.0
Myelodyplastic syndrome: 2
Non-Hodgkin lymphoma: 3
Plasma cell leukemia: 1
Post-hoc Kaplan–Meier analysis (although hypothesis-forming only) showed a significant correlation (P = 0.02) between PB [CD34+CD38–] and relapse (Figure 3B), of interest since donor CD34+CD38– stem cells may compete against leukemic CD34+CD38– stem cells that influence relapse [15, 16]. Although a product CD34+CD38– count was not performed, the correlation coefficient between product CD34+ count/donor weight and 06:00 hours PB [CD34+] was 0.86.
In previous human studies, there was an average >2-fold difference in baseline (nonmobilized) circadian variation in HPCs [7, 9, 10]. In this prospective cohort study of 11 G-CSF mobilized allogeneic sibling donors, there was a highly significant rise in PB [CD34+] and [CD34+CD38–] throughout day 4 (P <0.0001), but no reductions in the morning of day 5, as might be expected with a circadian rhythm pattern.
The expected trough on the morning of day 5 may be mitigated by the effect of G-CSF. Previous pharmacodynamic studies show that the peak of G-CSF-induced mobilization occurs on days 5 and 6 [17, 18]. The significant difference in absolute mean 06:00 hours HPC concentrations (P = 0.004 for [CD34+], P = 0.037 for [CD34+CD38–], paired t tests) between day 4 and day 5 is consistent with a previous report .
On the contrary, with a G-CSF effect alone one might expect the day 5 06:00 hours [CD34+] / [CD34+CD38–] to be significantly higher than the day 4 evening values; thus the absence of this finding is consistent with a circadian rhythm. Ethical considerations precluded postponing leukapheresis to evaluate the day 5 to day 6 PB HPC concentrations, which might effectively address the conundrum of dissecting the circadian rhythm from the G-CSF pharmacodynamic effect, which plateaus during this time period .
The timing of G-CSF administration in the morning may have overridden endogenous evening peak HPC counts except in the youngest donors. In a study of serial administration of a 4-day G-CSF course at different hours of the day , the peak of the CD34+ count on the fifth day occurred within several hours of the G-CSF administration time on the previous 4 days (Georg Bjarnason, personal communication). On day 5 of daily morning G-CSF administration, there was also a nonsignificant trend in 10 healthy volunteers towards a peak CD34+ count 2 hours after the G-CSF dose . In the mouse model, where circadian rhythms were maintained with G-CSF mobilization , human G-CSF was injected subcutaneously at 125 μg/kg every 12 hours for 4 days, and blood collection was always timed at 3 hours after the last dose of G-CSF. Perhaps the steadier serum levels of G-CSF obtained with every 12-hour dosing  is important for maintenance of the circadian rhythm, given G-CSF pharmacokinetics of a maximum serum concentration after subcutaneous administration for 2 to 8 hours and a half-life of ~3.5 hours.
The circadian pattern of stem cell egress may be altered in older people, as it is notable that the three youngest donors had the highest [CD34+] mobilization, and reached or maintained their peak [CD34+CD38–] at day 4 midnight. Older patients do not mobilize hematopoietic progenitors as well as young subjects  and previous studies finding an endogenous circadian rhythm used as their subjects young mice aged 7 to 8 weeks old  or healthy volunteers with mostly an age range in their 20s and 30s [7–10].
Donor ailments or medications may have affected their circadian rhythm, as stem cell mobilization may be affected by diabetes  or by drugs affecting sympathetic tone [24, 25]. The two diabetic patients had relatively low CD34+ mobilization, while the asthmatic donor on the β2-adrenergic agonist albuterol (and α-adrenergic agonist pseudoephedrine) had the highest mobilization.
Finally, the circadian peak may have occurred between 18:00 and 00:00 hours and was thus missed. Previously reported ~2-fold differences in PB [CD34+] / [CD34+CD38–] were seen at 20:00 hours compared with 08:00 hours collection times . Previous mouse data suggest that the circadian HPC concentrations can fluctuate significantly over 4-hour intervals .
The relatively poor correlation of [CD34+CD38–] with [CD34+] and its possible prognostic importance suggest that [CD34+CD38–] might be further explored as a determinant of the optimal time for collection.
This is the first prospective cohort study based on preclinical mouse as well as retrospective clinical data to examine whether circadian oscillations to hematopoietic stem cell release are maintained with G-CSF (or plerixafor) mobilization. Such data are important because a simple change of collection time to coincide with the peak of circadian egress might theoretically optimize the number of HPC collected. Our data suggest that endogenous progenitor rhythms may be altered by pharmacodynamic effect and the timing of G-CSF, and also donor age, medical history, and medications. These findings have important implications for future study design using G-CSF or possibly other mobilizing agents to study HPC biology.
granulocyte colony-stimulating factor
hematopoietic progenitor cell
This study was funded by Grant Number #UL1RR029887 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). The study’s contents are solely the responsibility of the authors and NCRR/NIH had no role in study design or collection, analysis, and interpretation of data. The authors would like to thank the study subjects; Daniel Lucas for providing helpful manuscript review; and Erlinda Sacris, our research nurse for the study.
- Kroger N, Zander AR: Dose and schedule effect of G-GSF for stem cell mobilization in healthy donors for allogeneic transplantation. Leuk Lymphoma. 2002, 43: 1391-1394. 10.1080/10428190290033323.View ArticlePubMedGoogle Scholar
- Pulsipher MA, Chitphakdithai P, Miller JP, Logan BR, King RJ, Rizzo JD, Leitman SF, Anderlini P, Haagenson MD, Kurian S, Klein JP, Horowitz MM, Confer DL: Adverse events among 2408 unrelated donors of peripheral blood stem cells: results of a prospective trial from the National Marrow Donor Program. Blood. 2009, 113: 3604-3611. 10.1182/blood-2008-08-175323.PubMed CentralView ArticlePubMedGoogle Scholar
- Collins NH, Gee AP, Durett AG, Kan F, Zhang MJ, Champlin RE, Confer D, Eapen M, Howard A, King R, Laughlin MJ, Plante RJ, Setterholm M, Spellman S, Keever-Taylor C, Wagner JE, Weisdorf DJ: The effect of the composition of unrelated donor bone marrow and peripheral blood progenitor cell grafts on transplantation outcomes. Biol Blood Marrow Transplant. 2010, 16: 253-262. 10.1016/j.bbmt.2009.10.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Pulsipher MA, Chitphakdithai P, Logan BR, Leitman SF, Anderlini P, Klein JP, Horowitz MM, Miller JP, King RJ, Confer DL: Donor, recipient, and transplant characteristics as risk factors after unrelated donor PBSC transplantation: beneficial effects of higher CD34+ cell dose. Blood. 2009, 114: 2606-2616. 10.1182/blood-2009-03-208355.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe MJ, Champlin RE, Korbling M: Allogeneic blood progenitor cell collection in normal donors after mobilization with filgrastim: the M.D. Anderson Cancer Center experience. Transfusion. 1999, 39: 555-560. 10.1046/j.1537-2995.1999.39060555.x.View ArticlePubMedGoogle Scholar
- Kroschinsky F, Holig K, Poppe-Thiede K, Zimmer K, Ordemann R, Blechschmidt M, Oelschlaegel U, Bornhauser M, Rall G, Rutt C, Ehninger G: Single-dose pegfilgrastim for the mobilization of allogeneic CD34+ peripheral blood progenitor cells in healthy family and unrelated donors. Haematologica. 2005, 90: 1665-1671.PubMedGoogle Scholar
- Abrahamsen JF, Smaaland R, Sothern RB, Laerum OD: Variation in cell yield and proliferative activity of positive selected human CD34+ bone marrow cells along the circadian time scale. Eur J Haematol. 1998, 60: 7-15.View ArticlePubMedGoogle Scholar
- Mendez-Ferrer S, Lucas D, Battista M, Frenette PS: Haematopoietic stem cell release is regulated by circadian oscillations. Nature. 2008, 452: 442-447. 10.1038/nature06685.View ArticlePubMedGoogle Scholar
- Lucas D, Battista M, Shi PA, Isola L, Frenette PS: Mobilized hematopoietic stem cell yield depends on species-specific circadian timing. Cell Stem Cell. 2008, 3: 364-366. 10.1016/j.stem.2008.09.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Verma DS, Fisher R, Spitzer G, Zander AR, McCredie KB, Dicke KA: Diurnal changes in circulating myeloid progenitor cells in man. Am J Hematol. 1980, 9: 185-192. 10.1002/ajh.2830090206.View ArticlePubMedGoogle Scholar
- Kamel AM, El-Sharkawy N, Mahmoud HK, Khalaf MR, El Haddad A, Fahmy O, El Fattah RA, Sayed D: Impact of CD34 subsets on engraftment kinetics in allogeneic peripheral blood stem cell transplantation. Bone Marrow Transplant. 2005, 35: 129-136. 10.1038/sj.bmt.1704755.View ArticlePubMedGoogle Scholar
- Pastore D, Mestice A, Perrone T, Gaudio F, Delia M, Albano F, Russo Rossi A, Carluiccio P, Leo M, Liso V, Specchia G: Subsets of CD34+ and early engraftment kinetics in allogeneic peripheral SCT for AML. Bone Marrow Transplant. 2008, 41: 977-981. 10.1038/bmt.2008.87.View ArticlePubMedGoogle Scholar
- Specchia G, Pastore D, Mestice A, Liso A, Carluccio P, Leo M, Casanova M, Sibilla S, Giannoccaro M, Liso V: Early and long-term engraftment after autologous peripheral stem cell transplantation in acute myeloid leukemia patients. Acta Haematol. 2006, 116: 229-237. 10.1159/000095872.View ArticlePubMedGoogle Scholar
- Henon P, Sovalat H, Becker M, Arkam Y, Ojeda-Uribe M, Raidot JP, Husseini F, Wunder E, Bourderont D, Audhuy B: Primordial role of CD34+ 38– cells in early and late trilineage haemopoietic engraftment after autologous blood cell transplantation. Br J Haematol. 1998, 103: 568-581. 10.1046/j.1365-2141.1998.01066.x.View ArticlePubMedGoogle Scholar
- Cobaleda C, Gutierrez-Cianca N, Perez-Losada J, Flores T, Garcia-Sanz R, Gonzalez M, Sanchez-Garcia I: A primitive hematopoietic cell is the target for the leukemic transformation in human philadelphia-positive acute lymphoblastic leukemia. Blood. 2000, 95: 1007-1013.PubMedGoogle Scholar
- Gerber JM, Smith BD, Ngwang B, Zhang H, Vala MS, Morsberger L, Galkin S, Collector MI, Perkins B, Levis MJ, Griffin CA, Sharkis SJ, Borowitz MJ, Karp JE, Jones RJ: A clinically relevant population of leukemic CD34(+)CD38(−) cells in acute myeloid leukemia. Blood. 2012, 119: 3571-3577. 10.1182/blood-2011-06-364182.PubMed CentralView ArticlePubMedGoogle Scholar
- Stroncek DF, Clay ME, Petzoldt ML, Smith J, Jaszcz W, Oldham FB, McCullough J: Treatment of normal individuals with granulocyte-colony-stimulating factor: donor experiences and the effects on peripheral blood CD34+ cell counts and on the collection of peripheral blood stem cells. Transfusion. 1996, 36: 601-610. 10.1046/j.1537-2995.1996.36796323059.x.View ArticlePubMedGoogle Scholar
- Anderlini P, Przepiorka D, Huh Y, Lauppe J, Miller P, Sundberg J, Seong D, Champlin R, Korbling M: Duration of filgrastim mobilization and apheresis yield of CD34+ progenitor cells and lymphoid subsets in normal donors for allogeneic transplantation. Br J Haematol. 1996, 93: 940-942. 10.1046/j.1365-2141.1996.d01-1747.x.View ArticlePubMedGoogle Scholar
- Stroncek DF, Clay ME, Herr G, Smith J, Jaszcz WB, Ilstrup S, McCullough J: The kinetics of G-CSF mobilization of CD34+ cells in healthy people. Transfus Med. 1997, 7: 19-24. 10.1046/j.1365-3148.1997.d01-75.x.View ArticlePubMedGoogle Scholar
- Bjarnason G, Reis M, Robinson JB: Circadian variation in CD34+ count and response to G-CSF therapy in healthy volunteers: implications for harvesting efficiacy. Blood. 1996, 10: 395a-Google Scholar
- Kroger N, Sonnenberg S, Cortes-Dericks L, Freiberger P, Mollnau H, Zander AR: Kinetics of G-CSF and CD34+ cell mobilization after once or twice daily stimulation with rHu granulocyte-stimulating factor (lenograstim) in healthy volunteers: an intraindividual crossover study. Transfusion. 2004, 44: 104-110. 10.1111/j.0041-1132.2004.00618.x.View ArticlePubMedGoogle Scholar
- Richa E, Papari M, Allen J, Martinez G, Wickrema A, Anastasi J, Van Besien K, Artz A: Older age but not donor health impairs allogeneic granulocyte colony-stimulating factor (G-CSF) peripheral blood stem cell mobilization. Biol Blood Marrow Transplant. 2009, 15: 1394-1399. 10.1016/j.bbmt.2009.07.005.View ArticlePubMedGoogle Scholar
- Ferraro F, Lymperi S, Mendez-Ferrer S, Saez B, Spencer JA, Yeap BY, Masselli E, Graiani G, Prezioso L, Rizzini EL, Mangoni M, Rizzoli V, Sykes SM, Lin CP, Frenette PS, Quaini F, Scadden DT: Diabetes impairs hematopoietic stem cell mobilization by altering niche function. Sci Transl Med. 2011, 3: 104ra101-10.1126/scitranslmed.3002191.PubMed CentralView ArticlePubMedGoogle Scholar
- Mendez-Ferrer S, Battista M, Frenette PS: Cooperation of beta(2)- and beta(3)-adrenergic receptors in hematopoietic progenitor cell mobilization. Ann N Y Acad Sci. 2010, 1192: 139-144. 10.1111/j.1749-6632.2010.05390.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Lucas D, Bruns I, Battista M, Mendez-Ferrer S, Magnon C, Kunisaki Y, Frenette PS: Norepinephrine reuptake inhibition promotes mobilization in mice: potential impact to rescue low stem cell yields. Blood. 2012, 119: 3962-3965. 10.1182/blood-2011-07-367102.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.