Red Blood Cell (RBC) Survival Following Transfusion in Infants

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.

Adult Study:

Inclusion Criteria:

- Males or post-menopausal females

- 18-65 years of age.

- Weight >110 lbs.

- Healthy- the subject feels well and can perform normal activities.

- Hemoglobin at or above 12.5 g/dL or hematocrit at or above 38%.

- Note: Members of the research team that are not supervised or under the employee
of the PI may participate in the study.

Exclusion Criteria:

- Presence of chronic illness unless the subject is being treated and the condition is
under control.

- Consumption of biotin supplements or raw eggs.

- Premenopausal women.

- Blood donation in the previous 8 weeks (single donation) or 16 weeks (double red cell
donation).

- Blood loss in the previous 8 weeks due to epistaxis, gastrointestinal blood loss,
trauma, significant diagnostic phlebotomy loss (i.e., > 30 mL total), or other
significant bleeding

- Treatment with antibiotics within the last 7 days. Antibiotics for prevention of an
infection or treatment of acne are not exclusion criteria.

- Note: If study subjects experience any of these conditions associated with blood
loss or donate any blood products, they will not be included in the primary
analysis but will be replaced.

Infant Study:

MOTHERS FOR PLACENTAL BLOOD COLLECTION AND MOTHERS OF INFANT STUDY SUBJECTS

Inclusion Criteria:

1. >/= 24 weeks gestation

2. mothers who deliver through the birthcanal or by c-section can be included in the
study.

Exclusion Criteria:

1. Pregnant with fetus with major congenital anomaly.

2. Clinically suspected or documented maternal chorioamnionitis (This only applies to
infant study subjects receiving autologous RBCs from the placenta).

3. Viral or bacterial infection (e.g. HIV, Hepatitis B, Hepatitis C, Primary Herpes,
Tuberculosis) based on clinically available prenatal or postnatal test results in the
mother's medical record. (This only applies to infant study subjects receiving
autologous RBCs from the placenta.)

4. minor mothers (<18 years old) are excluded from the study.

INFANT STUDY SUBJECTS

Inclusion Criteria:

Newborns >/=24 weeks gestation who are patients in the Neonatal Intensive Care Unit (NICU)
at UIHC that:

1) Are being treated with the expectation of survival.

Exclusion Criteria:

1. Difference of more than 5% in the percentage of HbF cells (measured by flow cytometry
in the Widness lab) between blood harvested from the placenta and that from discarded
neonatal blood in the first day of life and before the first neonatal blood
transfusion. This is done to exclude the rare possibility of transfusing newborns with
blood that is contaminated with a significant proportion of their mother's blood if a
maternal-to-placenta bleed occurs after umbilical cord clamping is done. (This only
applies to infant study subjects receiving autologous RBCs from the placenta.)

2. Need of emergent blood transfusion as determined by the subject's medical care team.

3. Hematological diseases (except for anemia associated with phlebotomy loss and
prematurity)

4. Alloimmune hemolytic anemia, diffuse intravascular coagulation, and thrombosis.

5. Major congenital anomaly.

6. Sepsis with positive blood or spinal fluid culture.

7. Receiving treatment with erythropoietin (r-HuEPO) or cardiorespiratory bypass support
(ECMO).

8. Overt clinical bleeding.