Paired Marrow Aspirations to Assess Assays in Sensitized Renal Allograft Recipients

The aim of this risk protocol is to determine the variability of the AlloElispot and
Allospecificities assay.

Our group has developed two novel assays to determine: 1) the number of donor-specific
alloantibody (DSA) secreting bone marrow derived plasma cells (AlloELISPOT assay); and 2) the
function of DSA-secreting Plasma cells (Allospecificities assay).

These assays were developed over the past 3 years and already have provided an important
means of testing new therapeutic protocols aimed at controlling DSA production. It is
important to note that repeated attempts to isolate PCs from peripheral blood have been
unsuccessful (PCs are extremely rare in peripheral blood) and the bone marrow is the only
accessible source of PCs.

It is now clear to that we have reached a point that we must validate these assays
(coefficient of variation, etc), in order to appropriately evaluate data derived from these
assays. Inter-assay variability can be assessed by performing two paired assays in the same
patient. This could be done in two ways—paired bone marrow aspirations separated by time or
two bone marrow aspirations performed at the same time. We have decided to pursue the latter
approach. We will do both marrows either at the time of transplantation when the subjects are
under general anesthesia or in the Clinical Research Unit (CRU) using conscious sedation.. We
believe that this is safe and will be well-tolerated and will provide the data that we need
to validate the assays.

Inclusion criteria.

- Pre or post renal transplant recipients who are "sensitized", having allo antibodies
as evidenced by single antigen bead analysis).

- Renal transplant donors.

- Those who give voluntary written informed consent before performance of any
study-related procedures, which are not part of normal medical care, with the
understanding that consent may be withdrawn by the subject at any time without
prejudice to future medical care.

Exclusion criteria.

- Any patient currently receiving systemic anticoagulation therapy with heparin or
coumadin.

- Patient has a platelet count of <30 x 10(9)/L within 14 days before enrollment.

- Patient has an absolute neutrophil count of ANC<1.0 x 10(9)/L within 14 days before
enrollment.

- Patient has received other investigational drugs within14 days before enrollment.

- Serious medical or psychiatric illness likely to interfere with participation in this
clinical study.

- Diagnosed or treated for malignancy within 5 years of enrollment, with the exception
of complete resection of basal cell carcinoma or squamous cell carcinoma of the skin,
an in situ malignancy, or low-risk prostate cancer after curative therapy.

- Contraindication to kidney transplantation or donation—active infection, comorbid
medical conditions, etc