© Borgis - Postępy Nauk Medycznych 7/2012, s. 557-562
*Katarzyna Guz1, Barbara Nasiłowska-Adamska2, Kazimierz Hałaburda2, 3, Agnieszka Orzińska1, Bożena Mariańska2, Ewa Brojer1
Analiza wczesnego chimeryzmu przy użyciu PCR w czasie rzeczywistym jako czynnik rokowniczy choroby przeszczep przeciwko gospodarzowi po allogenicznym przeszczepieniu hematopoetycznych komórek krwiotwórczych
Early chimerism analysis using real-time PCR method as a predictor of graft-versus-host-disease following allogeneic haematopoietic stem cell transplantation
1Department of Immunohaematology and Immunology of Transfusion Medicine, Institute of Haematology and Transfusion Medicine, Warsaw
Head of Department: prof. Ewa Brojer, MD, PhD
2Department of Haemopoietic Stem Cell Transplantation, Institute of Haematology and Transfusion Medicine, Warsaw
Head of Department: prof. Bożena Mariańska, MD, PhD
3Department of Haematology, Oncology and Internal Diseases, Medical University of Warsaw
Head of Department: prof. Wiesław Wiktor Jędrzejczak, MD, PhD
Ocena chimeryzmu dawcy odgrywa znaczącą rolę w rokowaniu pacjentów po allogenicznym przeszczepieniu hematopoetycznych komórek krwiotwórczych (alloHSCT).
Materiał i metody. W tej retrospektywnej pracy, chimeryzm oceniano metodą PCR w czasie rzeczywistym z użyciem polimorfizmów insercja/delecja co 5 dni, aż do +30 dnia po alloHSCT u 33. pacjentów z hematologicznym standardowym mieloablacyjnym kondycjonowaniem (CST) lub kondycjonowaniem o zredukowanej intensywności (RIC). Źródłem komórek macierzystych był szpik kostny bez usuwania limfocytów T (BM) lub komórki macierzyste krwi obwodowej (PBSC).
Wyniki. Statystycznie istotne różnice w tempie nabywania chimeryzmu dawcy obserwowano między +5 a +10 dniem u pacjentów z przeszczepieniem CST i PBSC, co wskazywało na szybsze nabywanie chimeryzmu dawcy w porównaniu z tymi po CST i BM lub po RIC. Analiza wczesnego chimeryzmu dawcy u pacjentów po przeszczepieniu z CST i PBSC, u których wystąpiła ostra lub przewlekła choroba przeszczep przeciwko gospodarzowi, wykazała znacząco szybszy zanik autologicznych komórek i ekspansję przeszczepionych komórek dawcy między +5, a +10 dniem w porównaniu z pacjentami bez tej choroby.
Assessment of donor chimerism has an established role in the follow-up of patients after allogeneic haematopoietic stem cell transplantation (alloHSCT).
Material and methods. In this retrospective study, chimerism was measured by real-time PCR using insertion/deletion polymorphisms every 5 days until day +30 following alloHSCT in 33 patients with haematological malignancies conditioned with standard (CST) or reduced intensity regimens (RIC). No T-depleted bone marrow (BM) or granulocyte-colony stimulating factor mobilized peripheral blood (PBSC) were the sources of stem cells.
Results. The statistically important differences in the assessment of early donor chimerism increase were observed between day +5 and +10 in patients transplanted with CST and PBSC (the faster donor chimerism increase) compared to those with CST and BM or after RIC. The analysis of early donor chimerism in patients transplanted with CST and PBCS, who developed acute and chronic graft-versus-host-disease (GvHD), showed significantly faster disappearance of autologous cells and the expansion of transplanted donor cells between day +5 and +10 as compared to patients with no GvHD.
Although donor chimerism assessment is an established part of the follow-up procedure in patients after allogeneic haematopoietic stem cell transplantation (alloHSCT) (1), no sufficient data on kinetics and clinical importance of early chimerism (assessed within first 30 days following alloHSCT) are available. For this purpose routine methods of chimerism analysis cannot be used because of their insufficient sensitivity. New perspectives have been opened with the implementation of the very sensitive real-time PCR method (RQ-PCR). Several RQ-PCRs protocols using different primers and probes (SNP (2, 3) or insertion/deletion polymorphism (4, 5) as well as different methods of product detection [specific labeled probes (4, 5) or the nonspecific detection of PCR product by SYBR-Green (6)] have been proposed for assessment of early chimerism. The aim of our study was to develop a reliable RQ-PCR-based protocol for early chimerism evaluation as predictor of acute (a) and chronic (ch) graft-versus-host-disease (GvHD).
Material and methods
We applied the RQ-PCR method based on TaqMan technology on ABI PRISM 7700 with specific labeled probes and primers (Applied Biosystems, USA).
PCRs were performed in 25 μl reagents: 12.5 μl TaqMan master mix (Applied Biosystems, USA), 200 nM appropriate probes and 300 nM appropriate primers. As stem cell donor and recipient markers we used 26 specific markers, described in other studies (4, 7, 8, 9, 10) and applied by us routinely for noninvasive prenatal diagnostics in RhD negative women (11): 6 null alleles (SRY, S03, S06, GSTM1, GSTT1, RHD), 10 insertion/deletion polymorphisms (ACE, S01, S04, S05, S07, S08, S09, S10, S11, RHC/c) and the housekeeping CCR5 gene for both estimation of DNA concentration in each sample and the „normalizator” for chimerism analysis. The details of our RQ-PCR protocols for chimerism analysis can be read at previous publication (12).
In this retrospective study, we evaluated early chimerism in 33 consecutive patients transplanted in our center for haematological malignancies from fully HLA-matched related or unrelated donors. The baseline characteristics of patients enrolled to the study are presented in table 1. The study protocols were approved by the Ethical Committee.
Table 1. Baseline characteristics of study patiens.
Acute lymphoblastic leukemia
Acute myelogenous leukemia
Chronic myeloid leukemia
Chronic lymphocytic leukemia
Non – Hodgink’s lymphoma
allogeneic (HLA matched):
|WBC > 1,0 x 109/l (days after HSCT)|
|ANC > 0,5 x 109/l (days after HSCT)|
|PLT > 20 x 109/l (days after HSCT)|
|PLT > 50 x 109/l (days after HSCT)|
Standard conditioning (CST) was administered in 28 patients. In this group, no T-depleted bone marrow (BM) in 8 patients or peripheral blood stem cells (PBSC) in 20 patients were the sources of stem cells. In the remaining 5 patients, reduced intensity conditioning (RIC) with PBSC infusion was applied. All patients undergoing CST were administered GvHD prophylaxis with CsA/MTX (15 mg/m2 i.v. on day +1 and 10 mg/m2 on days +3, +6, +11) or CsA/MTX/ATG from related or unrelated HLA-matched donors, respectively. In patients undergoing RIC transplantations, single agent prophylaxis with CsA was applied.
Blood samples were collected from donors and recipients prior to transplantation and from recipients on days +5, +10, +15, +20, +25 and +30 (± 1) following transplantation. Blood samples were also collected from the panel of 51 healthy volunteers. Mononuclear cells (MC) were isolated on Ficoll density gradient (Lymphoprep, Axis Shield PoC, Norway). DNA was extracted according to standard procedure (Qiagen, Germany) and stored at -80°C until RQ-PCR was performed. The concentration and purity of each DNA sample were measured spectrophotometrically at 260 and 280 nm and by RQ-PCR using primers and probes for CCR5 gene.
Prior to transplantation, donor and recipient DNA were tested to identify the specific markers present in the recipient but absent in the donor and vice versa. The healthy volunteer-panel was also tested for the “calibrator” DNA for each transplanted patient. SRY, RHD, RHC/c alleles were chosen as markers in sex- or RhD/C mismatched recipient/ donor pairs. If more than one marker for one or for both individuals were informative, those with the most similar PCR efficiencies were selected.
To evaluate the sensitivity, accuracy and variability of each RQ-PCRs, standard amplification curves were plotted for all specific markers. PCRs with artificial chimeric DNAs were prepared by serial dilution of appropriate positive DNA (heterozygous for specific allele) in negative DNA (without the specific allele) with a constant final DNA amount of 50 ng. Positive and negative DNA were obtained from healthy volunteers pre-genotyped for all markers. The experimentally established detection limit for RQ-PCR-based chimerism analysis was 4/7575 copies of negative DNA (0.05%); Ct value for 1 copy (0,013% chimerism) was between 37.328 and 41.081. The slope of regression curve varied from -3.191 to -4.198 and the R2 correlation coefficient, from 0.972 to 0.999. The inter-and intra-assay variabilities were examined according to Jiménez-Velasco et al. (5) and the CV range was between 0.24-1.42 and 1.00-3.26, respectively. The standardization results of our RQ-PCR protocol for chimerism analysis and comparison with STR-PCR method was previously published on the larger DNA samples from recipient alloHSCT including material collected after +30 day following transplantation (13).
The marker informativity of our system was 100% in unrelated pairs and 97.1% in related individuals presented in this article. In 85.3% of the last group markers were found for both donor and recipient and in 11.8%, only for recipient. In one case (2.9%), the marker was found only for donor and was not further analyzed.
For chimerism analysis, the simultaneous quantitative analysis of the stem cell donor and recipient polymorphisms and the referent CCR5 gene were performed in duplicates using 50 ng of DNA per reaction. As calibrators we used DNA of 6/51 tested volunteer blood donors. The “calibrator” DNA from healthy volunteers were of the same genotype and zygosity as the stem cell donor and recipient prior to transplantation.
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