Recipient-Derived Cells after Cord Blood Transplantation: Dynamics Elucidated by Multicolor FACS, Reflecting Graft Failure and Relapse

Article Outline

• Abstract
• Introduction
• Results
  • Analysis of Recipient-Derived Cells Using the HLA-Flow Method to Monitor Engraftment Early after CBT
  • Analysis of Recipient-Derived Cells for Leukemia Cell Markers to Monitor Minimal Residual Disease (MRD)
• Discussion
• Materials and Methods
  • Patient Characteristics
  • Cell Preparations and Staining
  • FACS Analysis
  • STR-PCR
  • AML1/ETO-FISH
• Acknowledgments
• References
• Copyright

Abstract 

Although umbilical cord blood has been increasingly used as an alternative donor source to treat hematologic malignancies, cord blood transplantation (CBT) is frequently complicated by graft failure and relapse of primary diseases. Because persistence or increase of recipient-derived hematopoietic or malignant cells has pathogenic import under these conditions, analysis of recipient-derived cells should be useful to understand the pathogenesis of graft failure and relapse of primary disease. Because most CBT involves human leukocyte antigen (HLA)-mismatched transplantation, we developed a 9-color fluorescence activated cell sorter (FACS)-based method of mixed chimerism (MC) analysis using anti-HLA antibodies to detect mismatched antigens (HLA-Flow method). Among CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells, monocytes, and granulocytes, donor- and recipient-derived cells alike could be individually analyzed simultaneously in a rapid, quantitative and highly sensitive manner, making the HLA-Flow method very valuable in monitoring the engraftment process. In addition, this method was also useful in monitoring recipient-derived cells with leukemia-specific phenotypes, both as minimal residual disease (MRD) and as early harbingers of relapse. Leukemia relapse can be definitively diagnosed by cytogenetic or PCR studies using recipient-derived cells sorted for leukemia markers. Multicolor HLA-fFlow analysis and cell sorting in early diagnosis of graft failure and relapse was confirmed as valuable in 14 patients who had received HLA-mismatched CBT.

Key Words: Chimerism, HLA, Flow cytometry, Cord blood transportation

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Introduction 

Analysis of donor-recipient mixed chimerism (MC) after allogeneic stem cell transplantation has become routine in confirming the engraftment of donor-derived cells. Polymerase chain reaction-based short tandem repeat analysis (STR-PCR) [1] and X/Y chromosome analysis using fluorescence in situ hybridization (X/Y-FISH) [2] after gender-mismatched transplantation are the methods most commonly used for mixed chimerism (MC) analysis. Persistence of recipient-derived cells early after transplantation or increases in their numbers are thought to be risk factors for relapse of leukemia 3, 4, 5. In fact, if immunosuppression is discontinued or donor lymphocyte infusions (DLI) are given after early identification of increased numbers of recipient-derived cells, the outcome of SCT is significantly improved 6, 7. Because lineage-specific analysis of MC is thought to be important in understanding the pathogenesis of graft failure and subsequent relapse of leukemia, several research groups evaluated MC of individual leukocyte subpopulations using magnetic cell separation or fluorescence-activated cell sorting (FACS) techniques followed by PCR-based analyses 8, 9. PCR- or FISH-based methods, however, are complicated, insensitive, and time consuming, especially in the case of lineage-specific MC analysis.

During the last decade, the technologies supporting multicolor FACS analysis have been dramatically developed 10, 11. This methodology is very useful to investigate the pathogenic conditions of human diseases by the simultaneous analysis of many phenotypes and functions of cells 12, 13. If donor- and recipient-derived cells have specific surface markers that are able to be stained by fluorescence-conjugated antibodies, respectively, chimerism analysis may be possibly done by flow cytometry in a rapid, quantitative, and highly sensitive manner.

The number of cord blood transplantations (CBTs) has increased recently. Most CBTs are carried out with human leukocyte antigen (HLA)-mismatched donor-recipient combinations, especially in adult patients 14, 15, 16. If donor- and recipient-specific anti-HLA antibodies, respectively, can stain donor- and recipient-specific HLAs, lineage-specific MC theoretically can be analyzed by multicolor flow cytometry in the setting of HLA-mismatched transplantation 10, 11.

Serologic analysis of HLA was first established by Paul I. Terasaki as a lymphocyte toxicity test using antiserum from a multiparous woman that contained anti-HLA polyclonal antibodies [17]. These polyclonal antibodies cannot be used for flow cytometric analysis because of their low affinity and complicated crossreactivity for HLAs [18]. Because the availability of these anti-HLA polyclonal antibodies is limited and it is difficult to maintain their quality, Terasaki et al. established hybridomas to produce anti-HLA monoclonal antibodies (mAbs) that can be used for serologic analysis of HLA. These anti-HLA antibodies are also useful for flow cytometric analysis 19, 20.

To overcome the problems of current PCR- or FISH-based methods for MC analysis, we have developed a flow cytometry-based method, using fluorescence-conjugated anti-HLA mAbs, of MC analysis after HLA-mismatched transplantation. Here we show that this HLA-Flow method is very useful for analysis of lineage-specific MC after HLA-mismatched CBT.

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Results 

Analysis of Recipient-Derived Cells Using the HLA-Flow Method to Monitor Engraftment Early after CBT 

We analyzed lineage-specific MC in 9 patients early after CBT (Table 1; unique patient numbers [UPNs] 1 through 9). Figure 1A shows typical results of peripheral blood MC analysis at week 2 after CBT for mononuclear cells (PBMCs) and leukocyte subpopulations (UPN 1). Lineage-specific MC could be separately analyzed for CD4⁺ T cells, CD8⁺ T cells, NK cells, monocytes, and granulocytes. The highest frequency of recipient-derived cells existed in the CD4⁺ T cell subset (4.62% of total CD4⁺ T cells). In 7 patients, recipient-derived cells were consistently observed in the CD3⁺ T cell subset before week 3 after CBT (UPNs 1, 3-5, and 7-9). These patients showed complete chimerism at week 4 after CBT (Figure 1B) and all achieved donor-type engraftment successfully (data not shown). Another patient (UPN 2) maintained MC early after CBT, and yet another (UPN 6) died on day 21 (Figure 1B).

Table 1. Patient Characteristics and Outcomes after Cord Blood Transplantation

Conditioning regimen consists of TBI (12 Gy) + Ara-C + CY in all patients except 2 (TBI + Ara-C + Flu in UPNs 8 and 13). GVHD prophylaxis consists of CsA + MTX in all patients except 1 (CsA + MMF in UPN 8). Subtype of HLA-A and B specifically recognized by anti-HLA antibodies are underlined. Both engraftment and minimal residual disease were monitored in UPN 1-4. Only engraftment was monitored in UPN 5-9. Only minimal residual disease was monitored in UPN 10-14. Outcomes were confirmed on August 10, 2007.

UPN indicates unique patient number; AML, acute myelogenous leukemia; MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess of blasts; RAEB-T, RAEB in transplantation; TBI, total body irradiation; Ara-C, cytosine arabinoside; G, granulocyte colony-stimulation factor; CY, cyclophosphamide; Flu, fludarabin; CsA, cyclosporine A; MTX, methotrexate; MMF, mycophenolate mofetil.

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• Figure 1. 

  MC analysis early after CBT in 9 patients. (A) In UPN 1, peripheral blood mononuclear cells were stained with an antibody combination of FITC-HLA-A24 (donor-specific), biotin-HLA-A11 (recipient-specific), PerCP-Cy5.5-CD8, PE-Cy7-CD3, APC-CD56, APC-Cy7-CD19, Pacific Blue-CD4, and Pacific Orange-CD14 followed by staining with SA-PE. Cells were stained with PI just before FACS analysis. After gating overall PBMCs, the lymphocyte subpopulation and the monocyte subpopulation on the FSC-SSC plot, PI-positive dead cells detected by PE-TR (610/20) detector were gated out from each subpopulation. In the lymphocyte fraction, CD4⁺ T cells (CD3⁺CD4⁺ cells), CD8⁺ T cells (CD3⁺CD8⁺ cells), B cells (CD3⁻CD19⁺ cells) and NK cells (CD3⁻CD19⁻CD56⁺ cells) were gated using lineage markers CD3, CD4, CD8, CD19, and CD56. Monocytes were gated using lineage marker CD14. MC was analyzed using HLA-A24 and HLA-A11 for PBMCs overall (upper left), CD4⁺ T cells (upper middle), CD8⁺ T cells (upper right), NK cells (lower left), and monocytes (lower middle). Granulocyte fractions isolated from peripheral blood were stained with an antibody combination of FITC-HLA-A24, biotin-HLA-A11, and APC-CD11b followed by staining with SA-PE. Cells were stained with PI just before FACS analysis. After the granulocyte fraction was gated on the FSC-SSC plot, PI-marked dead cells were gated out. Granulocytes were gated using lineage marker CD11b. MC was analyzed in this subpopulation using HLA-A24 and HLA-A11 (lower right). (B) In UPN 1 to 9, peripheral blood (10 mL) was drawn at week 1, 2, 3, and 4 after CBT and FACS-based MC analysis was performed, using 5 mL of blood (percentage of recipient-derived PBMCs isolated by centrifugation on Ficoll-Paque). In UPN 2 (•), numbers of recipient-derived cells started to increase after week 3 and persisted at ∼10% of total PBMCs. UPN 6 (×) died on day 21 because of human herpesvirus 6B encephalitis and multiple organ failure.

In both UPN 2 and UPN 1, recipient-derived cells decreased to 1.5% of PBMCs at week 2 after CBT. Recipient-derived cells, however, started to increase at week 3 in UPN 2 and persisted at ∼10% of total PBMCs for the next 3 weeks (Figure 1B and Figure 2A); these increased recipient-derived cells were mainly observed in the monocyte and granulocyte fractions, but not in the T cell fractions (Figure 2B). Significant numbers of recipient-derived cells in the CD34⁺CD45^dim (hematopoietic stem/progenitor) fraction (Figure 3B, left), in addition to those in the CD34⁻CD45⁺ (mature cell) fraction (Figure 3B, right), were detected in bone marrow at week 7 after CBT (Figure 3C, left). Because recipient-derived cells were persistently observed in serial MC analyses, from week 4 after CBT we rapidly reduced CsA doses, resulting in a quick decrease in recipient-derived cells in the peripheral blood around week 6 to week 10 after CBT (Figure 2A). Recipient-derived cells also dramatically decreased in bone marrow at week 20 (data not shown). The percentages of recipient-derived cells among all white blood cells were also determined by STR-PCR in 5 patients. Percentages of recipient-derived cells among all mononuclear cells were similar when determined by the flow cytometry-based method (Table 2).

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• Figure 2. 

  MC analysis of peripheral blood cells early after CBT in UPN 2. (A) In UPN 2, mononuclear cells and granulocytes isolated from peripheral blood were stained with the antibody combinations described in Figure 1 (excepting biotin-HLA-A11, replaced by biotin-HLA-A26). Total mononuclear cell MC results at weeks 1, 2, 3, 6, and 10 are shown. (B) CD4⁺ T cell, CD8⁺ T cell, NK-cell, monocyte, and granulocyte MC results at week 3 after CBT are shown.

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• Figure 3. 

  MC analysis of bone marrow cells at week 7 after CBT in UPN 2. (A) In UPN 2, mononuclear cells isolated from aspirated bone marrow at week 7 were stained with an antibody combination of FITC-HLA-A24 (donor-specific), biotin-HLA-A26 (recipient-specific), PerCP-Cy5.5-HLA-DR, PE-Cy7-CD33, APC-CD34, APC-Cy7-CD56, Alexa Fluor 405-CD45, and Pacific Orange-CD14 followed by staining with SA-PE. Cells were stained with PI just before FACS analysis. After gating for mononuclear cells not stained by PI (left), bone marrow cells were analyzed with a combination of anti-CD34 and anti-CD45 (right). On this CD34-CD45 plot, 8 distinct subpopulations were observed (tentative gate 1 [G1] to G8, reflecting different kinds of cells). Because these 8 subpopulations had different levels of autofluorescence and HLA expression, positions of donor- and recipient-derived cells on the HLA-A24 versus HLA-A26 plot were different in each subpopulation. (B) For example, the distribution of recipient-derived cells in G2 (CD34⁺CD45^dim fraction, left; hematopoietic/progenitor cells) differed from that in G8 (CD34⁻CD45⁺ fraction, right; mature white blood cells. (C) To elucidate overall distributions of donor- and recipient-derived cells, MC was first analyzed separately in G1 to G8. Donor-derived cells (light green dots) and recipient-derived cells (blue dots) were then plotted on the CD34 versus CD45 plot (left) or the HLA-A24 versus HLA-A26 plot (right). On the HLA-A24 versus HLA-A26 plot (right), the distributions of donor- and recipient-derived cells overlapped, precluding direct gating on this plot of donor- and recipient-derived cells among total bone marrow mononuclear cells.

Table 2. Percentage of Recipient-Derived Cells of Total Peripheral Blood Cells Determined by the HLA-Flow Method and STR-PCR Method

At the time point as shown in the above, 10 mL peripheral blood was drawn and MC analysis was done by the flow cytometry-based method (HLA-Flow method) using 5 mL of blood. In these 5 patients, another 5 mL was used for MC analysis by the STR-PCR method. The FACS-based method shows the percentage of recipient-derived cells of total mononuclear cells isolated by centrifugation on Ficoll-Paque. The STR-PCR method shows the percentage of recipient cells of total white blood cells in which peripheral blood was lyzed and genomic DNA was extracted from white blood cells.

NT indicates not tested; Pt, patient; CBT, cord blood transplantation.

Analysis of Recipient-Derived Cells for Leukemia Cell Markers to Monitor Minimal Residual Disease (MRD) 

We analyzed MC in bone marrow samples to monitor minimal residual disease (MRD) in 9 patients (UPNs 1-4 and 10-14). Seven patients maintained complete remission at last follow-up (Table 1). UPNs 1 and 4 showed relapse of leukemia on day 281 and day 203, respectively. In UPN 1, we serially analyzed recipient-derived cells in bone marrow 5 months and more after CBT. This patient underwent CBT for acute myelogenous leukemia (AML)-M2 with a chromosomal translocation between chromosome 8 and chromosome 21: t(8;21). The phenotype of leukemia cells was CD34⁺CD45^dimHLA-DR⁺CD56⁺. AML1/ETO served as a leukemia-specific chimeric gene marker for this patient. After gating of CD34⁺CD45^dim cells, having the phenotype of either normal hematopoietic stem/progenitor cells or leukemia cells 21, 22, 23, on the CD34-CD45 plot (Figure 4A-G2), MC in this subpopulation was analyzed in terms of the expression levels of HLA-A24 as a marker of the donor and of HLA-A11 as a marker of the recipient. On day 281, frequencies of recipient-derived cells in the CD34⁺CD45^dim fraction were clearly increased (5.71% of the CD34⁺CD45^dim fraction; Figure 4B, middle) compared with those on day 159 (0.16%; Figure 4B, left). In contrast, recipient-derived cells were not detected on day 281, particularly among CD34⁻CD45⁺ cells, having the phenotype of mature blood cells (Figure 4C-G7), or in peripheral blood (data not shown). On day 320, recipient-derived cells had risen to more than half of CD34⁺CD45^dim cells (Figure 4B, right). In the CD34⁺CD45^dim fraction, donor-derived cells revealed an HLA-DR⁺CD56⁻ phenotype and recipient-derived cells revealed an HLA-DR⁺CD56⁺ phenotype, the same as that of leukemia cells (Figure 4D). Finally, we sorted the donor- and recipient-derived CD34⁺CD45^dim cells on day 320 to investigate whether recipient-derived CD34⁺CD45^dim cells were leukemia cells or not. After fixation of sorted cells on glass slides, the AML1/ETO chimeric gene was analyzed using FISH probes (cf. Methods). Recipient-derived CD34⁺CD45^dim cells but not donor-derived ones showed AML1/ETO fusion signals (Figure 4E, left, and 4E, right, respectively). In UPN 4, we detected 12.1% of recipient-derived cells among the CD34⁺CD45^dim fraction on day 182. Leukemia relapse was subsequently diagnosed, on day 203, by morphologic examination (data not shown).

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• Figure 4. 

  Correlation of results from MC analysis with leukemia cell-associated aberrant antigen expression and leukemia-specific gene expression in the CD34⁺CD45^dim fraction of bone marrow cells in UNP 1. (A) In UPN 1, mononuclear cells isolated from aspirated bone marrow were stained with the antibody combinations described in Figure 3 (excepting biotin-HLA-A26, replaced by biotin-HLA-A11). Cells were stained with PI just before FACS analysis. After gating for mononuclear cells not stained by PI, bone marrow cells were analyzed with a combination of anti-CD34 and -CD45. On this CD34-CD45 plot, 7 distinct subpopulations (G1 to G7) were observed. (B) MC was analyzed in CD34⁺CD45^dim cells (A-G2) using anti-HLA antibodies on days 159, 281, and 320. (C) On day 281, the distributions of donor- and recipient-derived cells in G1-G7 were plotted on the CD34-CD45 configuration. (D) On day 320, expression of cell surface markers HLA-DR and CD56 on donor-derived and recipient-derived CD34⁺CD45^dim cells was analyzed (HLA-DR versus CD56 plot: donor-derived CD34⁺CD45^dim cells, green dot; recipient-derived ones, red dots). (E) Recipient-derived CD34⁺CD45^dim cells and donor-derived ones were sorted separately and analyzed for AML1/ETO fusion signals using FISH (left, recipient-derived CD34⁺CD45^dim cells; right, donor-derived ones).

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Discussion 

Using this FACS-based method, we could not only monitor the engraftment process, but also investigate the mechanism of engraftment failure. We observed 2 different types of kinetics in engraftment of cord blood-derived cells in this study. Seven patients (UPN 1, 3-5, 7-9) showed successful engraftment resulting in complete chimerism at week 4 after CBT. In contrast, UPN 2 showed persistent MC for more than 6 weeks after CBT. Recipient-derived cells in the peripheral blood were detected in the monocyte and the granulocyte fractions but not in the CD4⁺ and CD8⁺ T-cell fractions in this patient (Figure 2B). Because granulocytes and monocytes are terminally differentiated cells with a short half-life and do not proliferate in the periphery, we speculated that the recipient-derived granulocyte-monocyte progenitors, that is, colony-forming units of granulocytes and monocytes (CFU-GM), existed in bone marrow and supplied mature granulocytes and monocytes to peripheral blood. MC analysis of bone marrow cells clearly showed recipient-derived CD34⁺CD45^dim cells (Figure 3C, left), which should include progenitors containing granulocyte-monocyte lineages [24]. In this patient, we tapered cyclosporine A (CsA) to eliminate residual recipient-derived cells. Because MC analysis results were available within 1.5 hours after blood or tissue sampling, HLA-Flow was very helpful in titrating CsA doses.

In this study, we compared HLA-Flow against STR-PCR in detection of MC. The results were comparable as shown in Table 2, but our FACS-based analysis had several advantages. We could obtain results thereby more quickly (<1.5 hours) than with STR-PCR, and with a logarithmic increase in sensitivity (0.1% versus 5%). We could also obtain lineage-specific MC data via HLA-Flow.

Normal hematopoietic stem/progenitor cells²¹ and myeloid and B-lineage leukemia cells belong to the CD45^dim fraction 22, 23, and most of them also express CD34 antigen. Mesenchymal cells (which are not replaced with donor-derived cells) are CD34⁻CD45⁻CD44⁺CD73⁺CD90⁺CD105⁺ [25]. Therefore, to monitor residual leukemia cells correctly, the chimerism in the CD34⁺CD45^dim fraction had to be analyzed, on the basis of a CD34-CD45 plot, separately from the CD34⁻CD45⁻ fraction that contained recipient-derived mesenchymal cells. In UPN 1, we serially analyzed bone marrow cells to monitor MRD. On day 159, using FISH and RT-PCR, we did not detect expression of the AML1/ETO chimeric gene (a tumor-specific marker for this patient). Nor did we detect a significant number of recipient-derived cells among CD34⁺CD45^dim cells (0.16% of total CD34⁺CD45^dim cells) by HLA-Flow. Although the relapse of leukemia could not be confirmed on day 281 by microscopic examination because leukemia cell frequencies were low (0.37% of total mononuclear cells), HLA-Flow could clearly show a significant number of recipient-derived cells in the CD34⁺CD45^dim fraction (Figure 4B, middle). Although FISH and PCR analysis also showed an increase in AML1/ETO expression, it took 7 days to obtain these results. Sorting of donor- and recipient-derived CD34⁺CD45^dim cells followed by FISH analysis clearly showed that only the recipient-derived expressed the leukemia-specific AML1/ETO fusion signal; the donor-derived did not. This result demonstrated that HLA-Flow, especially the contribution made by sorting, is very useful in early detection and confirmation of relapse of leukemia.

In this case, leukemia cells expressed CD34, CD56, and HLA-DR in the study at the time of diagnosis. We also detected donor-derived normal progenitor cells and recipient-derived leukemia cells in the CD34⁺CD45^dim fraction (Figure 4D) as HLA-DR⁺CD56⁻ phenotypes (green dots) and HLA-DR⁺CD56⁺ phenotypes (red dots), respectively. We believe that this approach, using antibodies against leukemia cells' aberrant antigens, can be used to detect malignant cells even when tumor-specific genetic markers are unavailable.

Schumm et al. [26] have recently reported that a 4-color flow cytometry-based method using anti-HLA antibodies was useful for determination of chimerism and MRD. In analyzing MC they stained only for donor-specific HLAs. In our experience, however, clear separation of donor- and recipient-derived cells was difficult when using only a single anti-HLA antibody with a few lineage markers because of several technical problems; nonspecific staining, variable autofluorescence and HLA expression among different cell populations, downregulation of cell surface HLA molecules, etc. In addition, because peripheral blood contains only a few cells early after SCT, to analyze MC in many lineage-specific subsets was very difficult. We therefore introduced a 9-color FACS-based system to permit simultaneous analysis, using just a 5-mL blood sample, of both donor and recipient HLA, 6 additional lineage markers, and dead cells, even early after CBT when leukocyte counts are low. Although the distributions of donor- and recipient-derived cells often overlapped on the direct donor HLA versus recipient HLA plot because of some technical problems as mentioned above, we could accurately elucidate MC in appropriate subpopulations as shown in Figure 3A and 3B. In addition, we used FACS-based sorting to permit further analysis of sorted cells for definitive diagnosis, as described.

More than 20 fluorescence-labeled anti-HLA-A and B antibodies are commercially available from One Lambda Inc. (Canoga Park, CA) and AbD Serotec Ltd. (Kidlington, UK). We investigated the diversity of donor-recipient HLA mismatches in Japanese CBT cohort and found that >45% of CBT cases were covered by HLA-Flow method with these commercial antibodies. Although 9-color flow cytometry is still complicated technique, commercial availability of multicolor FACS machine with autocompensation software and new fluorescence-labeled antibodies make this technique easier. Therefore, applications of this HLA-Flow method is not restricted to a few cases in sophisticated flow cytometry laboratories. Our data indicate that to monitor lineage-specific MC after HLA-mismatched CBT, using a rapid, quantitative, and highly sensitive FACS-based method, can contribute substantially to clinical management by permitting early diagnosis of graft failure as well as of leukemia relapse.

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Materials and Methods 

Patient Characteristics 

We analyzed 14 patients after CBT. Their clinical characteristics are summarized in Table 1. Patients with AML and myelodysplastic syndrome (MDS) underwent CBT at The Institute of Medical Science, The University of Tokyo (IMSUT), between June 2002 and May 2007. Twelve patients underwent a 12-Gy total body irradiation (TBI)-containing myeloablative pretransplantation conditioning regimen of TBI + recombinant human granulocyte colony-stimulating factor (G-CSF) combined with cytosine arabinoside (Ara-C) + cyclophosphamide [16]. One patient (UPN 13) underwent a 12-Gy TBI-containing myeloablative pretransplantation conditioning regimen of TBI + G-CSF combined with Ara-C + fludarabine (Flu). They received a standard CsA and methotrexate (MTX) combination as GVHD prophylaxis. One patient (UPN 8) underwent a 4-Gy TBI-containing nonmyeloablative pretransplantation conditioning regimen of TBI + G-CSF combined with Ara-C + Flu. He received a CsA and mycophenolate mofetil (MMF) combination as GVHD prophylaxis. The institutional review board of IMSUT approved the clinical protocol, and written informed consent was obtained from all patients in accordance with the Declaration of Helsinki.

Cell Preparations and Staining 

For monitoring of engraftment, 10 mL of peripheral blood was collected at 1, 2, 3, and 4 weeks after CBT in 9 patients (Table 1; UPN 1-9). Additional samples were collected when MC continued. For monitoring MRD, peripheral blood specimens and bone marrow aspirates were serially collected 5 months and more after CBT in 9 patients (UPN 1 to 4 and 10 to 14 in Table 1). Mononuclear cells were isolated by centrifugation of peripheral blood and bone-marrow aspirate specimens on Ficoll-Paque (Sigma, St. Louis, MO). Granulocytes were isolated from buffy coat after centrifugation of peripheral blood on Ficoll-Paque followed by lysis using ammonium chloride lysing buffer. The isolated cells were washed once with ice-cold phosphate-buffered saline (PBS, Sigma), suspended in a small volume of PBS containing 5% mouse serum (Dako, Glostrup, Denmark) to block nonspecific binding of fluorescence-labeled antibodies to immunoglobulin Fc receptors, and kept on ice until staining.

For monitoring of engraftment, anti-HLA mAbs in combination with lineage-specific mAbs were used to analyze the lineage-specific dynamics of recipient-derived cells. For example, in UPN 1, mononuclear cells were stained using the combination of fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A24 antibody (FITC-HLA-A24, donor-specific), biotin-HLA-A11 (recipient-specific), Peridinin chlorophyll protein (PerCP)-Cy5.5-CD8, phycoerythrin (PE)-Cy7-CD3, allophycocyanin (APC)-CD56, APC-Cy7-CD19, Pacific Blue-CD4, and Pacific Orange-CD14 followed by staining with streptavidin (SA)-PE. Granulocytes were stained using the combination of FITC-HLA-A24, biotin-HLA-A11, and APC-CD11b followed by staining with SA-PE. When monitoring MRD, anti-HLA mAbs combined with mAbs against antigens expressed on leukemia cells were used to detect recipient-derived cells expressing leukemia markers. For example, in UPN 1, mononuclear cells were stained using the antibody combination of FITC-HLA-A24, biotin-HLA-A11, PerCP-Cy5.5-HLA-DR, PE-Cy7-CD33, APC-CD34, APC-Cy7-CD56, Alexa Fluor 405-CD45, and Pacific Orange-CD14 followed by staining with SA-PE. Samples were stained with fluorescence-conjugated antibodies for 20 minutes on ice, washed twice with ice-cold PBS, and suspended in a small volume of ice-cold PBS.

Anti-HLA mAbs specific for donor and recipient HLAs in all patients are summarized in Table 1. All anti-HLA antibodies were purchased from One Lambda. Pacific Orange-CD14 and Alexa Fluor 405-CD45 were purchased from CALTAG-Invitrogen (Carlsbad, CA). Other antibodies, SA-PE and SA-APC were purchased from Becton-Dickinson (BD) pharmingen (San Jose, CA).

FACS Analysis 

Propidium iodide (PI; 1 μg/mL; Sigma) was added to samples to stain dead cells just before FACS analysis; dead cells were detected using a photo multiplier tube (PMT) with a 610/20-nm bandpass filter (detector for PE-Texas Red conjugates). BD FACS Aria (BD Immunocytometry Systems, San Jose, CA) was used for all multicolor FACS analysis and sorting. Flow cytometry standard data were analyzed using FlowJo software (Treestar, San Carlos, CA). The gating procedures used to identify subsets in peripheral blood and bone marrow are explained respectively in Figure 1 and Figure 3.

STR-PCR 

MC was also analyzed using STR-PCR at SRL (Tachikawa, Tokyo, Japan), to monitor engraftment in 5 patients after CBT. Peripheral blood from the recipient and cord blood were obtained before CBT, and DNA was extracted from these samples. Using this DNA for templates, PCR was carried out with 10 fluorescence-labeled STR primers (AmpFlSTR SGM plus kit, Applied Biosystems, Foster City, CA) to find markers distinguishing donors from recipients. Peripheral blood was collected at defined times after CBT. Samples underwent FACS-based MC analysis followed by DNA extraction. PCR products using donor DNA and recipient DNA obtained before and after CBT were analyzed using a capillary electrophoresis ABI PRISM310 Genetic Analyzer and GeneScan and GenoTyper software (all Applied Biosystems). The sensitivity of this analysis was 5% [1].

AML1/ETO-FISH 

In UPN 1, who had AML-M2, the AML1/ETO chimeric gene was analyzed by FISH at SRL. After separating donor-derived from recipient-derived CD34⁺CD45^dim fractions by FACS, cytospin samples were fixed on slide glasses and hybridized with fluorescence-labeled AML1 and ETO DNA probes (AML1/ETO Dual Color, Dual Fusion Translocation Probe, Cancer Genetics, Abbott Molecular, Des Plaines, IL). In all cases, 100 nuclei were scored per slide to reach 5 × 10⁻² sensitivity.

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Acknowledgments 

The authors thank Ms. Maki Monna-Oiwa for data collection and her skillful secretarial assistance and Dr. Alex Knisely for critical reading of the manuscript. We thank SRL for chimerism analysis using STR-PCR and AML1/ETO analysis using FISH. This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT; N.W.), by the MEXT Project for Realization of Regenerative Medicine (N.W.), and by the Kobayashi Foundation (S.T.). The authors declare no conflict of interest.

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