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.