Assessment of Erythroid Chimerism in Sickle Cell Patients Undergoing HSCT By Digital Droplet PCR

Sickle cell disease (SCD) is an inherited autosomal recessive blood disorder associated with significant morbidity. Nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is increasingly used in severely affected patients with SCD. These transplants result in mixed hematopoietic chimerism so accurate chimerism testing is important for monitoring the status of the transplant. Reconstitution of the erythroid compartment is essential. Since red cells lack a nucleus, DNA-based chimerism assays do not directly assess the chimerism in the erythroid compartment. Several studies have shown that the chimerism in the white cells and erythroid cells can be very different (W, C., etal. Exp. Hematol. 2003, 31:924; Andreanni, M., etal. Haematologica, 2011, 96:128). We developed a procedure for quantification of chimerism in the erythroid compartment of blood using RNA-based digital droplet PCR (ddPCR). The sensitivity and specificity of the assay was evaluated then tested post-transplant samples from sickle cell patients.

Total RNA is reverse transcribed and amplified in a two-step RT-PCR approach. The PCR reaction containing allele-specific hydrolysis probes is partitioned into ~15,000 droplets then amplified. The copy number of HbA and HbS transcripts from cells of the erythroid lineage is determined with ddPCR (BioRad, QX-200). The %HbA and the %Donor can be calculated using the donor genotype (A/A or A/S). The assay is designed to have a sensitivity of 1% with donor genotype of A/A and 5% with donor genotype of A/S. Each post-transplant sample is tested in duplicate along with an AA control, SS control, AS control and 1% sensitivity controls (5 controls total).

Thirty AA or SS samples were tested to assess assay specificity. The average %HbA detected in a SS sample was 0.03%; the average %HbS in a AA sample was as 0.02%. The background signal is significantly below the cutoff for 1% sensitivity. Using contrived samples with low, medium, and high %HbA, we demonstrate the assay is accurate and linear to 0-2%HbA across the reportable range of 0%HbA to 100%HbA. The % CV of the minor allele for samples in the range of 10%-90%HbA, were equal or below 15%.

A total of 11 post-transplant samples from 7 transplanted sickle cell patients were tested, and the results were compared to DNA-based/FISH chimerism, if available. Our results were comparable with DNA-based chimerism (Table 1). Five of the samples had slightly higher %donor in the erythroid compartment compared to the white cell compartment. The erythroid chimerism reflected changes in chimerism status: the decrease then increase in chimerism (P2), stable chimerism (P5) and graft failure (P7).

Assessment of the chimerism in the erythroid lineage may be a better indicator of donor erythropoiesis. We describe an accurate and sensitive assay for monitoring erythroid chimerism and the effects of post-transplant therapies in sickle cell patients undergoing HSCT. This assay also demonstrates the feasibility measuring erythroid chimerism detection in other hematologic disorders, such as thalassemia.