CHRONO-MOBILIZE: Chronotherapy of G-CSF for CD34+ Mobilization in Healthy Donors

Background and Rationale

Peripheral blood stem cell collection after G-CSF mobilization is the most widely used donation approach for allo-HSCT. Donor mobilization outcomes exhibit inter-individual variability, and donors with lower circulating CD34+ levels may require longer processing volumes, additional collection days, or rescue mobilization. Circadian biology may influence hematopoietic cell trafficking and cytokine responses, suggesting that the administration timing of G-CSF could affect mobilization intensity and collection efficiency. This trial tests whether a simple, operational change in dosing time can improve pre-apheresis CD34+ levels in healthy donors.

Objectives

Primary Objective: To compare the pre-apheresis peripheral blood CD34+ cell count between donors receiving G-CSF in the morning versus the evening.

Secondary Objectives: To compare collection efficiency and CD34+ yield measures, the need for additional collection days, target attainment on day 1, and donor safety outcomes between study groups.

Study Design

This is a prospective, multicenter, stratified randomized controlled trial with two parallel arms. The study is open-label due to the nature of the intervention (fixed dosing times), while the statistician will remain blinded to group allocation until database lock.

Randomization is conducted via a central interactive web response system (IWRS) using block randomization. Allocation is stratified by donor age (<40 vs ≥40 years) and sex to balance key donor characteristics across arms.

Interventions and Procedures

Eligible healthy donors will receive subcutaneous G-CSF at 10 μg/kg once daily for 5 consecutive days (Day 1 to Day 5), assigned to one of the following schedules:

Morning group: 08:00 (±15 minutes) each day

Evening group: 20:00 (±15 minutes) each day

The first apheresis session is scheduled to begin approximately 12 (±1) hours after the final G-CSF dose, and the primary endpoint blood sample is obtained within 60 minutes prior to the start of apheresis. Collection targets are based on recipient weight, with standard operational procedures for conducting a second collection day if the first-day yield does not meet the target. If peripheral blood CD34+ is low after the final G-CSF dose, centers may apply rescue mobilization per local practice, and such use will be recorded for sensitivity analyses.

Outcome Measures

Primary Outcome: Peripheral blood CD34+ cell count (cells/μL) measured approximately 12 (±1) hours after the last G-CSF dose and within 60 minutes before the first apheresis session, assessed by standardized flow cytometry procedures.

Key Secondary Outcomes:

Collection efficiency (CE2)

CD34+ yield normalized to processed blood volume

Proportion of donors achieving the target CD34+ dose on the first collection day

Need for a second collection day

Donor safety outcomes, including adverse events during mobilization, collection, and follow-up

Sample Size and Analysis Overview

A total of 160 donors (80 per group) will be enrolled to provide adequate power for detecting clinically meaningful differences in the primary endpoint while allowing for non-evaluable cases. The primary analysis will follow the intention-to-treat principle, with additional per-protocol and sensitivity analyses as appropriate, including analyses accounting for rescue mobilization when applicable.

Safety Monitoring and Follow-up

Donors will be monitored with clinical assessment and routine laboratory testing during the mobilization period and on collection days. Adverse events commonly associated with G-CSF (e.g., bone pain) and donation-related events will be recorded and managed per standard-of-care practices. Donors will be followed through approximately Day 30 to capture delayed or persistent adverse events.

• G-CSF (Granulocyte colony-stimulating factor) (Drug): Subcutaneous recombinant human G-CSF (10 μg/kg once daily) administered for 5 consecutive days. Participants are assigned to a fixed dosing time: 08:00 (±15 minutes) or 20:00 (±15 minutes). Peripheral blood CD34+ is measured approximately 12 (±1) hours after the last dose and within 60 minutes before the first apheresis. The first apheresis begins \~12 (±1) hours after the final G-CSF dose. A second collection day may be performed per standard practice if the first-day yield does not meet the target; rescue mobilization (e.g., plerixafor) may be used per center practice when pre-defined low CD34+ criteria are met and will be recorded.

Primary outcomes

• Pre-Apheresis Peripheral Blood CD34+ Cell Count [On the day of first apheresis: approximately 12 (±1) hours after the final G-CSF dose and within 60 minutes prior to apheresis start.]: Peripheral blood CD34+ cell count (cells/µL) measured by flow cytometry (ISHAGE single-platform method). Blood is drawn approximately 12 (±1) hours after the last G-CSF dose and within 60 minutes before the start of the first apheresis session.

Sex: All, ages: 18 Years to 55 Years, accepts healthy volunteers

Inclusion Criteria:

1. Age 18-55 years
2. HLA matching appropriate for intended allo-HSCT recipient and meets donor medical evaluation criteria
3. Body weight ≥35 kg and adequate blood volume/BSA for apheresis
4. Karnofsky score ≥80
5. Screening labs meet thresholds (Hb/platelets/ANC, liver/renal/coagulation), no splenomegaly, pregnancy test negative if applicable
6. Written informed consent
7. Stable circadian rhythm before mobilization (no night shift/no ≥3 time-zone travel; avoid melatonin/sedatives or other agents affecting sleep/circadian rhythm)

Exclusion Criteria:

1. Prior/current hematologic or immune diseases (e.g., aplastic anemia, leukemia, lymphoma, autoimmune disease)
2. Significant cardiovascular/structural heart disease, uncontrolled hypertension, uncontrolled diabetes
3. Neurologic/psychiatric disorders affecting adherence
4. Sleep/circadian disorders; recent night shift or ≥3 time-zone travel
5. Prohibited medications (e.g., beta-blockers, systemic steroids >10 mg prednisone-equivalent ≥7 days, melatonin/psychotropics; recent G-CSF/GM-CSF/CXCR4 inhibitor use)
6. Severe allergy to G-CSF or components