Red Blood Cell (RBC) Survival Following Transfusion in Infants
Condition: Neonatal Anemia
MeSH terms: Anemia, Neonatal
Study type: Interventional
Phase: Phase 2

Summary: 
      OUR OVERALL HYPOTHESIS is that post-transfusion survival of allogeneic and autologous RBCs
      can be accurately quantified in anemic human infants using biotin-labeled RBCs combined with
      mathematical modeling that adjusts for confounding factors commonly encountered in neonates.
      These confounding factors include 1) dilution of labeled RBC as a result of growth stimulated
      erythropoiesis, anemia stimulated erythropoiesis, and blood transfusion; 2) loss of labeled
      RBC due to laboratory phlebotomy; and 3) variable RBC life spans resulting from RBCs having
      been produced at different developmental periods and under varying rates of erythropoiesis.
      In contrast to infants, adjustment for these factors is not necessary in healthy adults under
      conditions of steady state erythropoiesis. Instead in adults, RBC survival is typified by a
      linear decline in concentration of labeled RBCs over time. When this line is extrapolated to
      zero concentration, the intercept with the time axis represents the mean potential lifespan
      (MPL) of RBCs. (<7 d) and stored (>21 d) allogeneic adult RBCs transfused in the same infant.
    

Detailed description: 
      Phase I: Includes only Aim #1 from Thrasher Foundation Grant in which adult subjects will be
      studied.

      SPECIFIC AIM #1 (Thrasher Foundation Grant): To develop in vitro and validate in vivo in
      adult humans and anemic infants the capability of biotinylating RBCs at up to 5 discrete
      densities for simultaneously determining RBC kinetics of multiple, distinct RBC populations.
      This requires expansion of the RBC biotin labeling technology we have previously developed.
      After refinement, the method will be applied in the subsequent aims conducted in anemic
      infants receiving clinically ordered RBC transfusions.

      As suggested by our previous RBC survival studies, we anticipate that the heaviest biotin
      labeling will alter the intrinsic RBC structural properties of RBCs, shortening their long
      term survival. The in vitro and in vivo validation studies we propose in adults are necessary
      because the conditions for reproducibly obtaining discrete RBC biotin densities at
      appropriate, closely spaced intervals have not been conclusively worked out, nor have we
      determined empirically which of the five densities will not artifactually shorten long-term
      RBC survival. We can use the lighter densities for RBC survival measurements and the heavier
      densities for the simultaneous RBC volume measurements needed to account for RBC volume
      increases caused by growth. It is important to perform these feasibility studies in both
      adults and infants because of the vastly different physiologic states, ie, normal, healthy
      adults are in steady state erythropoiesis while critically ill, anemic infants experience
      multiple clinical circumstances which perturb RBC survival, eg, growth, phlebotomy,
      intervening transfusion, etc.

      In addition to the biotin labeling of RBC method, the "differential agglutination, antigenic
      method" using flow cytometry will be applied for infants receiving allogeneic RBC
      transfusions. The results of the two methods will be compared with one another. The
      differential agglutination method utilizes differences in the RBC surface antigens between
      the donor and the recipient for determining short- and long-term RBC survival. Genotyping
      results of adult donor and the infant recipient RBC antigens (performed at the Mississippi
      Valley Regional Blood Center) permits identification of minor blood group differences between
      donor and recipient RBCs so that appropriately labeled minor RBC antibodies (available
      commercially and used by blood banks for minor blood group RBC typing) can be used in flow
      cytometric determination of RBC survival. RBC survival determined for each of the biotin
      labels and for the RBC antigenic differences will all be compared among one other. The
      differential agglutination/antigen method will considered the "gold standard" as the RBCs are
      labeled ex-vivo, AFTER the RBC transfusion, thus without modifying the RBC membrane surface
      proteins as biotinylation does. Our hypothesis is that the addition of too much biotin can
      lead to artifactually shortened in vivo RBC survival, but that lower doses of biotin to not.
      By also including the differential agglutination/antigen method RBC survival results, we will
      be able to better validate our multi-density biotin hypothesis.

      Our overall objective in this research is to improve red blood cell (RBC) transfusion
      practices for anemic, critically ill infants. This project has received support from two
      granting agencies: 1) The Thrasher Foundation, entitled, "Red Blood Cell Recovery and
      Survival Following Transfusion in Infants;" and 2) NIH PPG Grant P01 HL046925, Project 1
      entitled, "Red Blood Cell Survival Following Transfusion In Infants." Abstracts for both are
      included below. This project will be completed in two phases:

      Phase I includes studies of autologous biotinylated RBCs transfused into normal, health adult
      human volunteers (see Specific Aim #1 in Thrasher Foundation Abstract below); and

      Phase II includes studies of infants requiring physician order red blood cell transfusions
      (see Specific Aims #2, #3, and #4 in Thrasher Foundation Abstract below; and see Specific Aim
      #4 in NIH which is included in its entirety in the three Thrasher Grant specific aims).

      The Phase I studies are completed and we are no longer enrolling adults. We are enrolling
      infants for Phase II studies. We are adding these phases sequentially based on the
      recommendation of Martha Jones at the time our proposal was first submitted to the IRB. We
      have applied the knowledge we have gained from the Phase I studies in adult subject
      volunteers to make modifications of our study design for Phase II to be performed in infant
      study subjects receiving clinically ordered RBC transfusions. At the end of Phase II,
      justification for including a new, comparison "gold standard" method for determining RBC
      survival in infants (the "differential agglutination, antigenic method") is included. We have
      completed enrollment of all adult subjects for phase I.

      ABSTRACT I (FOR THRASHER FOUNDATION GRANT)

      Background:

      Anemic, critically ill newborn infants are among the most frequently transfused groups of
      patients in the US. An estimated 130,000 infants annually receive approximately 1,000,000 RBC
      transfusions. Unlike adults, important data are lacking regarding the transfusion product for
      optimal RBC survival in infants. Contributing to this lack are two major recent changes in
      neonatal blood banking and transfusion practices. These are 1) the use of transfused adult
      donor blood stored for up to the 42 day FDA limit instead of only using blood stored for less
      than 7 days as had been done previously; and 2) potential use of the infant's own blood
      harvested from the placenta to avoid the risks of viral infections and immune transfusion
      reactions from donor blood. Unfortunately, there are no definitive infant studies that
      address these changes by directly measuring RBC survival. Such infant studies have been
      hampered by technical problems. Specifically, RBC survival data must be adjusted for growth,
      laboratory phlebotomy loss, and intervening additional RBC transfusions but in practice have
      not. Moreover, safety issues have precluded many infant RBC survival studies (eg, exposure to
      51Cr radioactivity and removal of too much blood from today's tiny, premature infants).
      Indeed, prior to 1970 (the end of using 51Cr as a RBC label in infant RBC survival studies),
      infants weighing less than 1,500 g at birth did not often survive, and RBC survival data do
      not exist for today's smallest, most frequently transfused infants whose birth weights are
      500-1,000 g.

      Specific Aims:

      Specific Aim #1: To develop in vitro and validate in vivo in adults the capability for
      biotinylating RBCs at up to 5 discrete densities that are measurable by flow cytometry. These
      five RBC biotin density labels will be used in Specific Aims #2, 3, and 4 to simultaneously
      determine RBC survival of multiple, distinct populations of transfused RBCs in premature
      infants.

      Specific Aim #2. To determine whether RBC survival of donor and placental RBCs are
      significantly longer when adjusted by mathematical modeling. We anticipate that the unmodeled
      values substantially underestimate RBC survival.

      Specific Aim #3. To compare long-term RBC survival results for transfused adult donor and
      fetal/placental RBCs in anemic newborn infants. We anticipate that more rapid growth in
      fetuses will result in greater stress erythropoiesis leading to intrinsic RBC "defects" and
      shortened RBC survival compared to adult donor RBCs.

      Specific Aim #4. To quantify the effects of storage on model adjusted RBC survival of adult
      donor RBCs transfused into newborn infants. We anticipate that storage of donor RBCs will not
      alter RBC survival.

      Study Design:

      The studies proposed here build on biotinylation and mathematical modeling methods that our
      research group has developed for accurately measuring RBC survival without exposing the
      infant to radioactivity. The biotin RBC labeling method is well suited to newborn infants
      because it permits the simultaneous tracking of multiple RBC populations on <10 µL blood.
      Survival of RBCs labeled at multiple biotin densities will be quantified using the standard
      RBC survival parameters, ie, post-transfusion short-term 24 h RBC recovery and long-term
      modeled RBC survival, ie, until 50 and 100% of biotin-labeled RBCs have disappeared from the
      circulation. The latter two measurements will be calculated using the required mathematical
      adjustments for growth, laboratory phlebotomy loss, and intervening RBC transfusions.

      Only Specific Aim #4 in the Thrasher Foundation Abstract involves human infant study
      subjects. Specific Aim #4) USE THE RBC BIOTINYLATION AND MATHEMATICAL MODELING METHODOLOGIES
      VALIDATED IN ADULT SHEEP AND NEWBORN LAMBS TO ACCURATELY MEASURE POST-TRANSFUSION RBC
      KINETICS IN ANEMIC NEWBORN INFANTS TRANSFUSED WITH FRESH AUTOLOGOUS, FRESH ALLOGENEIC AND
      STORED ALLOGENEIC RBCS. The use of biotin, a non-toxic, non-radioactive B vitamin, to
      distinguish among different RBC populations simultaneously by flow cytometry is critical for
      accomplishing our aims and holds clear advantages over other RBC labeling methods in both
      safety and accuracy. In utilizing the four Specific Aims to achieve our goal of establishing
      more effective transfusion practices by identifying the optimal RBC transfusion product for
      use in anemic INFANTS.
    

Intervention name(s): Transfused Biotin RBCs - Adults Phase I; Transfused Biotin RBCs - Infants Phase II; Transfused Biotin RBCs - Infants Phase III
Intervention type(s): Biological; Biological; Biological
Intervention description(s): A 3 mL venous blood sample is obtained. 250 mL of blood will be drawn to a blood collection bag containing the anticoagulant CPD. Separate equal volumes of RBCs are labeled with up to five different densities of biotin. The biotinylated RBCs are resuspended in autologous plasma to achieve a 60 to 70% hematocrit. An IV is inserted for the reinfusion of the biotinylated RBCs. Three mL aliquots of blood are sampled at 5, 10, 20, and 60 minutes after infusion. The subject returns ~24 hours and 3 days after the RBC infusion to obtain a 3 mL venous blood sample. Subjects return for weekly 3 mL blood sampling.; After the infant's clinical care team decides that a RBC transfusion is needed, a 15mL/kilogram of body weight is ordered. Transfusion will be given in 2 parts: 1) approximately 80% of the total transfusion to be transfused over 3-4 hours and 2) approximately 20% of the total transfusion will be marked with biotin to be transfused upon completion of the first part. The bedside nurse maintains constant observation of the infant as appropriate for the infant's condition, assessing for signs and symptoms of a transfusion reaction.; Phase III (infants) to be determined upon completion of Phase II (infants).
Intervention MeSH terms: Biotin
Allocation: Non-Randomized
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Basic Science