Cultured Red Blood Cells : Life Span in Vivo Study
Condition: Healthy Volunteers
Study type: Interventional

Summary: 
      To study the in vivo life span in healthy volunteers of red blood cells generated in vitro
      from autologous peripheral stem cells. The study will be carried out in 4 phases :

        -  production in vitro of the equivalent of 1 ml of blood, i.e. 5x10e9 enucleated RBC from
           mononuclear cells isolated from an HSC graft obtained from a healthy volunteers donor.

        -  labeling of these cRBC with 51 Cr

        -  reinjection of the autologous cRBC

        -  follow up of the receivers for a maximum 33 days to measure the half-life span of the
           injected cells.
    

Detailed description: 
      1. GENERALITIES

           1.1 Clinical grade production of cRBC In this study the cRBC is both the active
           principle and the final product subjected to radioactive chromium labeling.

           The quantity of 5x109 cRBC to be reinjected is determined by the necessity to have a
           quantity of radioactivity lying between 2500 and 5000 kBq in order to have sufficient
           sensitivity to measure the life span.

           1.2 Hypothesis tested Since the cRBC have in vitro all the functional characteristics of
           native RBC, it is hypothesized that they will be viable in vivo.

           This viability will be evaluated by measuring their life span. It will be compared to
           the reported life span of native RBC, i.e. 30+3 days.

        2. OBJECTIVES OF THE STUDY

           2.1 Principal objective Study of the life span in vivo of cultured Red Blood Cells.

           2.2 Evaluation criteria

           2.2.1 Principal: The principal criterion is the measured life span of the cRBC. It will
           be compared to the reported life span of native RBC, i.e. 30+3 days.

           The study involves labeling a cohort of cRBC: after administration, this cohort of cRBC
           will age in the circulating blood and then disappear at the end of its "life span". The
           results will be interpreted in the following manner:

           - if the radioactivity of the first 3 blood samples remains constant (after correction
           for decay), this will be taken as evidence in favor of an absence of premature cell
           destruction.

           The following 3 samples will be drawn later [within the limit of 30 days, after which
           measurement of the radioactivity of the samples will no longer be possible due to the
           decay of chromium 51, elimination of chromium from the cells at the rate of 1.2% per day
           and lack of precision of the radioactive count for very low activities]. One will
           conclude that the life span of the cRBC is superior to 33 days.

           - if, in the first 3 samples, the radioactivity diminishes in a significant manner and
           earlier than on the reference curves, three other samples taken close together will
           enable estimation of the mean life span of the cRBC.

        3. EXPERIMENTAL PLAN

           3.1 Type of study Clinical feasibility assay.

           3.2 Experimental plan

           The study will be carried out in 4 phases:

             1. production in vitro of the equivalent of 1mL of blood, i.e. 5x109 enucleated RBC,
                from mononuclear cells isolated from an HSC graft obtained from a healthy
                benevolent donor,

             2. labeling of these cRBC with 51Cr, according to the classical protocol used to study
                the life span of red blood cells,

             3. reinjection of the autologous cRBC into the same donor,

             4. follow-up of the receiver for a maximal duration of 33 days to measure the
                half-life of the injected cells.

        4. CHARACTERISTICS OF THE RECEIVERS 4.1 Description of the study population The choice of
           conducting the study in healthy volunteers and not in patients is based on the necessity
           to have a healthy physiological context avoiding any situation which could lead to
           hemolysis. As the protocol requires mobilization with a growth factor, the donors of
           peripheral stem cells (PSC) receive G-CSF.

      The voluntary receivers will be recruited among intra-familial donors of PSC, in order to
      avoid the unnecessary exposure of a healthy donor to G-CSF.

      In the context of the mobilization of HSC, they will receive for 5 days subcutaneous
      injections of G-CSF at a dose of 10 µg/kg body weight.

      4.1.1 Collection of mononuclear cells by cytapheresis

      Characteristics of the starting material:

      Peripheral blood hematopoietic stem cells will not be taken for the production of cultured
      RBC unless the graft destined for the patient contains > 7x106 CD34+ cells/kg body weight of
      the patient receiver, the cell dose established in the literature as being sufficient to
      reconstitute the hematopoiesis of a patient receiving an allogenic graft. Thus the research
      protocol will incur no loss of chance for the patient.

      4.1.2 Isolation of CD34+ cells for the protocol The CD34+ cells to be used for research will
      be selected on a positive immunomagnetic selection column using an Isolex® system. A minimum
      of 45x106 CD34+ cells is necessary for good functioning of the system. These limiting factors
      oblige us to use as starting material stem cells mobilized with G-CSF. After removal of this
      sample for the study, the graft will have to contain a minimum of 7x106 CD34+ cells/kg body
      weight of the patient.

      The post-selection starting material will consist of clinical grade CD34+ cells purified by
      immunomagnetic selection. A purity of more than 50% CD34+ cells should be obtained after
      immunomagnetic selection. The percentage of viable cells will be determined by staining with
      trypan blue. The purified CD34+ cells will be frozen in a 4% albumin solution containing 10%
      DMSO and conditioned in 2.5 mL cryotubes containing 1x106 CD34+ cells. After thawing, the
      cell viability should be more than 50%.

      A minimum of 1 million viable CD34+ cells is necessary for seeding in culture on day 0.

      4.1.3 Biological characterization of the cRBC

      The active principle is filtered cRBC and the final product cRBC labeled with 51Cr. As the
      product should be reinjected immediately after labeling, characterization will be performed
      on the active principle and will involve the following elements:

      The active principle should consist of:

        1. RBC characterized by:

             -  their morphology (on slides stained with May-Grunwald-Giemsa),

             -  their hemoglobin content (MHC > 25 pg measured in a Sysmex XE-2100 automate) (see
                3.2.S.4.3);

        2. acidophilic erythroblasts < 1.2% (in > 10,000 cells counted on a slide);

        3. to the exclusion of all other cell types in > 10,000 cells counted on a slide.

      4.1.4 Sufficient criteria of the active principle (cRBC)

      The product will be labeled with chromium if it presents the following characteristics:

        -  5 to 10x109 RBC with MHC > 25 pg,

        -  < 1.2% acidophilic erythroblasts,

        -  an absence of leukocytes. The remaining microbiological controls will be known a
           posteriori.

      4.1.5 Labeling with chromium 51

      This step will be performed in the nuclear medicine clinic. Labeling is carried out:

        -  using a suspension of cRBC,

        -  and a sterile solution of [51Cr] sodium chromate having an activity of 37 MBq/mL on
           calibration.

      The labeling procedure itself is performed under clinically sterile conditions. The
      suspension is injected in 0.3 mL fractions (constraint linked to measurement of the 51Cr
      activity of the syringes before injection) using 2 mL syringes closed with a stopper. The
      maximal volume reinjected will be 5 mL by intravenous route.

      4.1.6 Sufficient criteria of the final product labeled with 51Cr

      The labeled product will be injected into the healthy volunteer if it presents the following
      characteristics:

        -  51Cr activity between 2,500 KBq (minimum) and 5,000 KBq (maximum),

        -  an absence of 51Cr not bound to cells.

      4.1.7 Injection into receivers This step will take place within 2 months after stem cell
      collection. Direct intravenous injection of labeled cRBC in a volume of 1 mL, in a room
      reserved for the administration of radioactive drugs, situated in the nuclear medicine clinic
      and adjacent to the preparation laboratory, for regulatory reasons.

      Maintenance of a venous route until the end of the period of surveillance. Collection of 2
      samples of 10 mL of blood into EDTA by venous puncture at the bend of the elbow, at T+1h and
      T+3h after injection.

      Criteria for discharge from the clinic: absence of fever or shivering, normal blood pressure
      and normal clinical examination.

      Total duration of the post-injection surveillance: 4 hours.

      4.2 Planning of the appointments for measurement of the life span of cRBC Collection of 10 mL
      samples by venous puncture at the bend of the elbow and follow-up of the receivers will be
      carried out at the CIC St Antoine.

      Transfer of the tubes to the nuclear medicine clinic.

        -  The third sample will be taken T+24h after injection.

        -  The following 3 will be taken on days chosen according to the decay estimated from the
           first samples (between T+48h and T+30 days after injection).

      The lack of establishment of fixed dates of sampling is justified by the necessity to adapt
      to the kinetics of this homogenous population of cRBC, at present unknown.

      A clinical examination will be carried out at each appointment. After performing the
      measurements, the samples will be destroyed.

      In conclusion, this approach has several advantages:

        1. It avoids injecting a growth factor, G-CSF, into a healthy volunteer for the sole
           objectives of the study.

        2. The autologous situation avoids any immunological risk and any risk of transmission of
           infectious agents.

        3. 51Cr is an emitter of gamma radiation of energy 320 keV and has a radioactive period of
           27.7 days. The irradiation resulting from this study involves no measurable risk. No
           particular radioprotective precautions need to envisaged, either for the receiver or for
           those in contact with him.
    

Intervention name(s): intravenous injection of labeled cRBC
Intervention type(s): Biological
Intervention description(s): This step will take place within 2 months after stem cell collection. Direct intravenous injection of labeled cRBC in a volume of 1 mL, in a room reserved for the administration of radioactive drugs, situated in the nuclear medicine clinic and adjacent to the preparation laboratory, for regulatory reasons.
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Health Services Research
Keywords: peripheral stem cells (PSC) donors; cultured red blood cells; In vivo half life