Patients

Investigation of hematopoietic chimerism was performed in ten children (eight girls, two boys) aged 6–16 years. They were diagnosed with acute myelogenous leukemia (n = 4), acute lymphoblastic leukemia (n = 2), chronic myelogenous leukemia (n = 1), myelodysplastic syndrome (n = 1), or Fanconi Anemia (n = 2). All recipients received hematopoietic stem cells from HLA-matched sibling donors, and sex-mismatch between donor and recipient was present in all cases. The material (peripheral blood) for hematopoietic chimerism quantification was collected in different periods after transplantation. In all 10 children reconstitution of hematopoiesis was observed. Out of 8 children transplanted for hematologic malignancies, 4 are well and alive, in complete continuous remission (CCR), while in the other 4, leukemia relapse occurred 4–23 months after transplantation.

DNA isolation

High-molecular-weight DNA was extracted from frozen whole blood (approximately 5 ml) or bone marrow (approximately 3–5 ml) by the standard treatment with sodium dodecyl sulfate (SDS) and proteinase K, and the salting-out method. DNA was isolated from the donors' and patients' blood samples collected before and after transplantation at various intervals in order to determine the chimeric status.

Analysis of PCR products by an ABI 310 Genetic Analyzer

The PCR protocol optimized for the Qiagen polymerase and the PE 9700 thermocycler was performed as described previously [5]. For fragment analysis (after capillary electrophoresis), an ABI 310 Genetic Analyzer (PE) was used [5]. All analyses were performed in the University Children's Hospital, Tuebingen.

Analysis of PCR products by an ALF Express DNA Sequencer

The PCR protocol optimized for the Thermal Controller MJ Research (Watertown, MA) model PTC-100™ was applied. PCR was performed in a volume of 10 μl; the PCR reaction mixture contained: 2.5 pM of each forward and reverse primer, 200 μl of each dNTP, 0.4 U Taq polymerase (Qiagen, Chatsworth, CA), 1x PCR buffer (Qiagen, Chatsworth, CA), and 40 ng of genomic DNA. Conditions for PCR were as follows: 5 min at 94°C for the first denaturation; 26 cycles of amplification with a temperature profile of 45 sec at 94°C, 1 min at 55°C, 1 min at 72°C; with additional 5 min at 72°C in the last cycle. STR loci were amplified with fluorescent PCR primers described previously [6]. Primers for microsatellite markers were labeled with Cy5 dye (TIB MOLBIOL). A 1.5-μl aliquot of PCR reaction was resuspended in 7 μl of loading solution (formamide, bromophenol blue) containing 100 bp and 300 bp internal markers. All samples, after denaturation at 95°C for 5 min, were analyzed on 6% denaturing polyacrylamide gel with 7 M urea in the sequencer. A 50–500 sizer labeled with Cy5 dye was used as an external marker (for calculation of allele sizes). Electrophoresis was carried out in 0.6xTBE buffer at 1500 V/min. The helium-neon laser was operated at a wavelength of approximately 700 nm and laser power value of 2.5 mW. Allele sizes and peak areas of fluorescent products were analyzed and calculated with the use of Fragment Manager software (Pharmacia). PCR and analysis of PCR products by the ALF Express DNA Sequencer was performed at the Institute of Human Genetics, Poznan.

FISH

The experiments were performed on interphase nuclei obtained by standard short culturing of fresh whole blood samples, with probes specific for chromosomes X (locus DXZ1) and Y (locus DYZ1). The FISH procedure according to Cytocell Ltd. was used [7]. The number of scored nuclei was 250 to 550, with a median of 300. FISH experiments were performed at the Institute of Human Genetics, Poznan. Since the material for FISH analysis was not collected in all designated periods after transplantation in some patients, FISH experiments were not performed then.

Quantification of chimerism

After electrophoresis in the ABI 310 Genetic Analyzer (PE), all obtained data were analyzed by GeneScan 3.1 software and then transferred to Genotyper 2.5 software [5]. All data obtained after electrophoresis of fluorescent products in the ALFExpress DNA Sequencer were transferred to Fragment Manager™ software (Pharmacia). For both, calculation of the amount of recipient's DNA was performed using the formula:

% of recipient's DNA = (R1 + R2)/(D1+ D2 + R1 + R2) × 100,

where: R1, R2 = peak areas of recipient's alleles; and D1, D2 = peak areas of donor's alleles.

Only informative markers were used for the analysis. If donor and recipient were heterozygous but shared one allele, only the area of the non-shared alleles was considered for the analysis [8].

To make sure that quantification is accurate, we performed serial dilution experiments, where standardized mixed chimeric samples were created by mixing donor's and pretransplant recipient's DNA in a range between 0 and 100 percent. The sensitivity strongly depends on the size of alleles, the detection level was around 3–5% of patient cells.

The results of chimerism detection by different methods were compared by the Spearman correlation test.

Comparison of methods used

One of the advantages of application of automated DNA sizing techniques for detection of posttransplant chimerism is that the use of radioactivity is not necessary. It is arelatively simple and rapid method, consisting of two steps: polymerase chain reaction with fluorescent primers and automated detection of fluorescently labeled PCR products, separated by electrophoresis. The analysis of fluorescently labeled PCR products provides better accuracy and precision of measurement than traditional electrophoretic methods. The most time-consuming step, which might prolong the examination process, is the search for informative markers.

Analysis of the chimerism status by amplification of STR loci can be performed regardless of donor's and recipient's sex. The most advantageous is the high sensitivity of the detection system used, so that only small amounts of DNA are needed.

Fragment analysis in the ABI 310 Genetic Analyzer (PE) is performed after capillary electrophoresis. The examination of one sample requires about 6 hrs; 144 samples can be analyzed per day if three colors are simultaneously analyzed [5]. The advantage of this method is that thanks to the combination of three-color detection, multiple sets of samples or multiple loci for a single sample can be analyzed on one gel. It is possible to analyze more than one marker at a time after multiplex amplification of informative loci.

In the ALFExpress DNA Analyzer, electrophoresis is carried out in an off-vertical gel cassette specially designed for easy and safe gel casting. The number of samples to be loaded on a gel is limited to 40 per run. The ALFwin Fragment Analyzer is used afterwards for fragment analysis. It is provided with versatile application software for the control of DNA fragment separation runs and subsequent analysis of the data. Collected data are used to accurately size PCR product peaks on the basis of external and internal standards.

One analysis of 40 samples takes 7 hrs, including PCR, electrophoresis in the gel cassette, and paperwork. The appropriate assembling and cleaning of the gel cassette is critical and time consuming.

It is well known that FISH is a good quantitative method of fluorescent signal detection, but requires lots of technical experience and expertise. Fluorescent in situ hybridization for one patient sample lasts at least 5 hrs, including preparation of interphase nuclei, hybridization with specific probes (X, Y dual-color FISH), and analysis. The high cost of the procedure is definitely a disadvantage.