Monitoring of Donor/recipient T-cell Engraftment Kinetics in Myeloablative Allogeneic Stem Cell Transplantation Using Short Tandem Repeat Amplification from Cell Lysates

Abstract Abstract 4004 Background: Mitochondrial DNA (mtDNA) is widely used in forensic identification and anthropologic studies on account of its abundance resulting in preferential amplification, sequencing and inherent variability. We developed mtDNA markers to monitor donor cell engraftment after allogeneic stem cell transplantation(SCT), then compared with nuclear short tandem repeat (STR) markers. Patients and methods: The mtDNA control regions and six mtDNA minisatellites (mtMS) (303 poly C, 16184 poly C, 514 (CA) repeat, 3566 poly C, 12385 poly C and 12418 poly A) from the total DNA samples of 215 cases (donor, recipient and after allogeneic SCT) were amplified using the designated specific primers and PCR. The results were compared with those from the six short tandem repeat (STR) markers (D12S391, D18S51, F13A1, HUM RENA-4, HUM FABP2 and Amelogenin). Results: Polymorphisms in the mtDNA control region identify an informative marker in 88% (189 cases) of all cases. Among the six mtMS markers, the informativeness of 303 poly C and 16184 poly C mtMS was 63% and 67% respectively. A combination of direct sequencing through the mtDNA control region, 303poly C and 16184 poly C mtMS could completely distinguish the donor cells from the recipient cells. The results from a typical mixing experiment to determine the sensitivity revealed a detection limit (DL) of the gene scan analysis in a mtDNA mixture to be visible at 1% heteroplasmy in 303 poly C mtMS marker. However, the DL from D12S391 in the same mixing experiment was 5–10% heteroplasmy. Conclusions: mtMS markers, especially 303 poly C and 16184 poly C markers, can provide a sensitive, accurate and quantitative determination of mixed chimerism after a SCT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Quantitative analysis of chimerism after allogeneic stem cell transplantation using multiplex PCR amplification of short tandem repeat markers and fluorescence detection

Leukemia ◽

10.1038/sj.leu.2402008 ◽

2001 ◽

Vol 15 (2) ◽

pp. 303-306 ◽

Cited By ~ 19

Keyword(s):

Stem Cell ◽

Quantitative Analysis ◽

Stem Cell Transplantation ◽

Fluorescence Detection ◽

Allogeneic Stem Cell Transplantation ◽

Tandem Repeat ◽

Short Tandem Repeat ◽

Pcr Amplification ◽

Short Tandem Repeat Markers ◽

Short Tandem

Download Full-text

The Campath-Level at the Day of Transplant Predicts T-Cell Engraftment after Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v108.11.2951.2951 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2951-2951

Author(s):

Johannes Schetelig ◽

Christian Thiede ◽

Martin Bornhaeuser ◽

Brigitte Mohr ◽

Uta Oelschlägel ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Cd8 T Cell ◽

Cell Engraftment ◽

Gvhd Prophylaxis ◽

Donor Type

Abstract Purpose: To analyse hematopoietic engraftment after allogeneic stem cell transplantation (SCT) in patients with chronic lymphocytic leukemia (CLL) who had received Campath prior to SCT. Patients and Methods: 40 patients with relapsed CLL were included into the analysis. All patients received Busulphan 8 mg/kg and Fludarabine 150 mg/m2 as conditioning regimen. The first 20 patients (Bu/Flu/Cam) received a cumulative dose of 75 mg Campath on days -9 to -5 and Cyclosporine (CSA) as GVHD-prophylaxis. The second 20 patients (preCam/Bu/Flu) received a conventional treatment (3 × 30 mg weekly) with Campath prior to SCT. Transplantation was scheduled 14 days from the last dose of Campath and GVHD-prophylaxis consisted of CSA and Methotrexate. The two groups did not differ in age, median number of prior chemotherapy regimens, prior fludarabine treatment or donor type. The observation of late secondary graft failure during the first phase of the study led to the analysis of lineage-specific engraftment (CD4+T-cells, CD8+T-cells, NK-cells and Neutrophils) and of Campath levels with an ELISA at the day of transplant. Complete donor chimerism was defined as more than 95% donor-DNA in a specimen. Results: Campath levels are available for 22 patients. The median antibody level was 307 ng/mL (range, 41 to 1820 ng/mL) after Bu/Flu/Cam and 62 ng/mL (detection limit to 490 ng/mL) after preCam/Bu/Flu. All patients reached neutrophil engraftment (>0.5/nl) after a median of 18 days (range, 14 to 27). The cumulative incidence of complete leukocyte chimerism at day +100 was 75% after Bu/Flu/Cam and 78% after preCam/Bu/Flu (p=0.858). The cumulative incidences of CD4+ and CD8+ T-cell donor chimerism at day +100 were 45% after Bu/Flu/Cam and 70% after preCam/Bu/Flu for both T-cell subsets (CD4+subset, p=0.481; CD8+subset, p=0.398). Of note, the Campath-level at the day of transplantation discriminated better between patients with prompt versus impaired T-cell engraftment. The incidences of complete CD4+ and CD8+ T-cell chimerism at day +100 were 14% and 29% in patients with Campath levels >250 ng/mL compared to 82% and 82% in patients with antibody levels <250 ng/mL (CD4-subset, p=0.006 (Figure 1); CD8-subset, p=0.017). Other potential factors like prior transplant, CD34-count and CD3-count of the graft or donor type did not have a significant impact on the kinetics T-cell engraftment. Of note, 3 out of 7 patients with Campath levels > 250 ng/mL compared to 0 out of 17 patients with Campath levels <250 ng/mL needed donor lymphocyte infusions to convert incomplete T-cell chimerism (p=0.017). Conclusions: T-cell chimerism should be analysed in patients who have received Campath recent to SCT. The level of Campath at the day of transplant may be an important factor for delayed T-cell engraftment. Figure Figure

Download Full-text