Significance of chimerism in hematopoietic stem cell transplantation: new variations on an old theme


The main goal of post-transplantation monitoring in hematopoietic stem cell transplantation (HSCT) is to predict negative events, such as disease relapse, graft rejection and graft-versus-host disease, in order to intervene with appropriate therapy. In this context, chimerism analysis is an important method in monitoring post HSCT outcome. Mixed chimerism (MC) is mainly evaluated to define engraftment and relapse. Detection of MC is a prerequisite in both myeloablative and nonmyeloablative HSCT, in order to assess the graft status and decide later therapeutic strategies such as donor lymphocyte infusion. In this review, we discuss various techniques including erythrocyte phenotyping, cytogenetic analysis, fluorescent in situ hybridization, restriction fragment length polymorphism, STR/VNTR analysis and real-time quantitative PCR, along with the various methods used to detect minimal residual disease (MRD) in different diseases such as chronic myeloid leukemia, acute myelomonocytic leukemia or acute lymphoblastic leukemia. The review mainly highlights the optimal methodological approach, which needs to be informative, sensitive and quantitatively accurate for MC detection. Future of post HSCT graft monitoring lies in the selection of the most accurate and sensitive technique to determine both MC and MRD. Such an approach would be helpful in not only determining relapse or rejection, but also in ascertaining various responses to different treatment modalities.

In Greek mythology, the Chimera was a creature with the head of a lion, the body of a goat and tail of a serpent.1 In medicine, the term chimera is used to designate an individual whose body contains cell populations derived from different individuals of the same or a different species occurring spontaneously or produced artificially.2 The phenomenon of co-existence of cells from two different organisms (evolved from two different zygotes) in one body is called chimerism.

In this review, we consider chimerism in allogeneic hematopoietic stem cell transplantation (HSCT), as it is one of the important states that develop after engraftment, and it is an important indication of disease relapse, graft rejection or graft-versus-host disease (GVHD). Over the past two decades, allogeneic HSCT has become the treatment of choice for patients suffering from certain malignant and nonmalignant hematological disorders.3, 4, 5, 6, 7, 8, 9 Allogeneic HSCT has been effective in the reconstitution of normal hematopoiesis in these patients. Furthermore, allogeneic HSCT is the preferred therapeutic option, primarily because of its intrinsic graft-versus-leukemia (GVL) effect. Most of this GVL effect is usually ascribed to donor T-cell immunoreactivity against host minor histocompatibility antigens, developmentally regulated antigens or leukemia-specific epitopes.10, 11

The success of this treatment modality is mainly affected by the recurrence of the underlying disease. Factors responsible for relapse include insufficient conditioning regimens or, eventually, a deficient GVL effect due to decreasing amounts of effector cells or to their functional ineffectiveness.7 In human HSCT, complete donor-derived hematopoiesis has been considered essential for sustained engraftment and for the prevention of relapse. Successful chemotherapy/radiotherapy causes eradication of all hematopoietic progenitors, which results in stable chimerism. Different states of chimerism are summarized in Table 1. The stage when the patient shows no evidence of recipient cells at any time after transplantation is considered to be complete chimerism (CC). The second stage is mixed chimerism (MC), where the patient shows both recipient as well as donor cells in the peripheral blood. However, the recipient cells in MC could be normal hemopoietic cells or leukemic cells. Persistence of residual leukemic cells is a result of the inefficiency of ablative conditioning regimens. With time, following HSCT, it is possible that these cells will re-emerge, resulting in leukemia relapse. The presence of these small numbers of hematopoietic malignant cells of host origin is associated with minimal residual disease (MRD). However, in MC, there is also the possibility that all leukemia cells in the recipient are destroyed, but a small number of apparently normal recipient hematopoietic cells still remain in the circulation. Patients who develop an increasing number of recipient cells are referred to as having increasing or progressive MC and patients with decreasing recipient cells display decreasing MC.

Table 1 Different states of chimerism

Relapse following HSCT presumably results from the expansion of a small number of recipient leukemic cells, which have survived the conditioning therapy. To define and detect the patients who are at high risk of leukemia relapse, it is vital to detect residual leukemia in the form of MRD and host hematopoiesis in the form of MC. The incidence and significance of MC following HSCT for various hematological diseases has been investigated by several groups over the past two decades.3, 4, 5, 6, 7, 8, 12, 13, 14, 15, 16 With the use of different techniques for the detection of residual recipient cells, MC is frequently observed post allogeneic HSCT. More sensitive and specific techniques are emerging to detect even the lowest fraction of residual recipient cells in patients post allogeneic HSCT.

Moreover, with the emergence of more advanced HSCT procedures, which include nonmyeloablative stem cell transplantation, depletion of the donor's T-cells from bone marrow grafts and donor lymphocyte infusion (DLI), it becomes most important to detect the exact status of chimerism in patients after HSCT. This could help in deciding on therapies to salvage the graft as well as to induce sustained remissions after allogeneic HSCT in patients with graft rejection, leukemia relapse or GVHD.

However, the true incidence and significance of the detection of mixed hematopoietic chimerism post HSCT remains unclear.2, 17 MC was initially thought to indicate impending relapse; however, using more sensitive molecular techniques, it has become clearer that MC is not uncommon, with varying percentages of recipient cells being detected in different study groups.12, 13, 14, 15, 16, 18, 19, 20, 21 This variation in the degree of MC is influenced by a number of factors, including the sensitivity and timing of the assay, the disease indication for HSCT, the stage of disease at the time of HSCT and the choice of conditioning regimen. Several studies have indicated that low levels of persisting recipient cells are not associated with an increased risk of leukemia relapse. However, increasing level of recipient cells over time (progressive or increasing mixed chimerism) appears to predict relapse, especially in recipients of a T-cell-depleted (TCD) transplant.6, 22, 23, 24

Moreover, the correlation of MC with the relapse of disease is varying and depends on the kind of disease involved. Various studies have suggested that MC after allogeneic HSCT might reflect the risk of relapse in chronic myeloid leukemia (CML).25, 26 However, some studies have suggested a correlation between leukemia levels of recipient cells and relapse in acute leukemia,7, 8, 25, 27 while others found no such correlation.26, 28, 29 This may be due to the fact that relapse develops within a very short span of time and, if the chimerism is not detected during this period, it may produce conflicting results.7

Detection of chimerism

The basic principle in the detection of chimerism is the utilization of the differences between donor and recipient polymorphic genetic markers or their products. Detection of these differences by employing a variety of molecular techniques has been used in a large number of studies for the establishment of chimerism status in patients post allogeneic HSCT. The choice of marker and the technique involved varies between different studies depending on the sensitivity and specificity of the technique, sex differences between donor and recipient and also the disease from which patient was suffering before HSCT.

Different researchers have used a variety of techniques to detect the host hematopoiesis in terms of MC. PCR-based amplification of a highly polymorphic STR/VNTR system is considered to be the most informative and sensitive technique.25 STRs and VNTRs are the tandem repetitive blocks of DNA. When the repetitive sequence is 15–50 nucleotides long it is termed VNTR, or minisatellite, and when the repetitive sequence is 2–6 nucleotides long, it is called STR, or microsatellite (Figure 1a and b). However, recently Alizadeh et al30 have proposed the use of real-time quantitative PCR (RT-PCR) and have compared the sensitivity of RT-PCR with the STR system and revealed that the RT-quantitative method is the best to assess and quantify MC status. In sex-mismatched HSCTs, Y-chromosome-specific markers,31 X-chromosome-specific markers32 and amelogenin loci25 have been frequently used, and recently dual-color fluorescent in situ hybridization (FISH) has been introduced as the technique of choice in cases of sex-mismatched HSCTs.33, 34, 35 Also used in routine analysis to establish chimerism status are cytogenetic techniques, erythrocyte phenotyping36 and restriction fragment length polymorphisms (RFLPs). Some of the studies have also used PCR-based allele-specific amplification techniques like the amplified refractory mutation system (ARMS)37 or restriction endonuclease in situ digestion (REISDs)38 to detect the mixed chimerism. The different techniques, their specificities, sensitivity and informativity are shown in Table 2.

Figure 1

Chimerism analysis by STR markers. (a) Tandem repeats of STR, indicating different alleles on the basis of difference in the number of repeats on two chromosomes; F – forward primer, R – reverse primer. (b) STR-based chimerism analysis: the donor and recipient have different number of repeats (alleles) at STR locus amplified by PCR. (c) Informativeness of different STR markers: lanes 1 and 2 are noninformative; lanes 3 and 4 are informative markers with both alleles different, lanes 5 and 6 are informative markers with one different allele and lanes 7 and 8 are informative markers with one different allele as the donor is homozygous. (d) Different states of chimerism detected by STR analysis: lane 1 – recipient before HSCT, lane 2 – donor, lane 3 – complete chimerism, lane 4 – mixed chimerism and lane 5 – graft rejection and disease relapse.

Table 2 Different methods used for detection of chimerism

Despite differences in the protocols of different methods, they are all based on the same basic procedure in which the donor and patient are screened before transplant for those genetic markers at which patient and donor differ: such markers are labeled informative markers (Figure 1c). Subsequently, these informative genetic markers are analyzed in patients post allogeneic HSCT to assess and quantify the amount of the recipient's own cells, as well as of the donor's cells in order to assess the chimerism status (Figure 1d). In the following section, we review the techniques used to study chimerism and minimal residual disease.

In addition to MC analysis, MRD detection is also considered to be an important method of prognosing the relapse of graft rejection. Detection of MRD depends on the disease for which HSCT has been performed, for example BCR-ABL-based RT-PCR is used for detection of MRD in patients suffering from CML.39

Erythrocyte phenotyping

Erythrocyte or red blood cell phenotyping (RCP) has been used to determine autologous hematopoiesis mainly in CML because in CML, at the time of relapse, recipient granulocytes, monocytes and erythrocytes appear and progressively replace their counterparts of donor origin. Other lineages, such as B and NK cells, remain of donor origin.36 Due to this, patients transplanted for CML are monitored frequently by RCP. An increasing percentage of autologous red blood cells and/or a decreasing number of erythrocytes of donor origin always correlates with relapse. The advantages of RCP are clear: it can be performed on blood samples; the assays are simple, accurate and very sensitive; and the results can be obtained within 24 h. Recently developed flow cytometric methods have increased the speed and efficacy of RCP.40 The methodology of RCP is very simple: complete red cell phenotyping of patient and donor is performed using different antigens: A, B, C, c, E, D, K, Fya, Jka, Jkb, M, N, S and s. RCP of patients should be performed before transfusion is given. Afterwards, discriminating markers are used to detect MC with the help of the fluoro microsphere method.36

Cytogenetic analysis

Cytogenetic analysis has frequently been used to differentiate between donor and recipient cells. When donor and recipient are sex matched, characteristic polymorphic regions or satellites are used to distinguish the donor or recipient origin of dividing cells. In different leukemias and other hematological disorders, chromosomal rearrangement with loss of genetic information (monosomies or deletions) occurs, hence cytogenetic analysis is an important aspect in chimerism analysis. These chromosomal studies are mainly done on peripheral blood cells and bone marrow cells of the metaphase stage. Standard G-band cytogenetic analysis (CG) is usually carried out in cases of CML to monitor the presence of Philadelphia chromosome-positive (Ph+) cells.33 However, cytogenetic analysis has been used mainly in cases of sex-mismatched HSCTs, where X–Y-specific probes are used to differentiate donor cells from recipient cells.36

Fluorescent in situ hybridization (FISH)

The FISH technique is mainly employed using sex-specific probes. It is a simple and quantitative method to detect MC following sex-mismatched HSCTs, particularly in cases where only small samples for examination are available.34 Classic cytogenetic detection of X and Y chromosomes in bone marrow cells from sex-mismatched transplant recipients is labor intensive, time consuming and limited by the number of cells that can be analyzed. For instance, to exclude mosaicism at the level of 1%, 300 or more metaphase cells must be examined,41 which is nearly impossible in the routine cytogenetic lab. FISH allows the rapid screening of a large number of cells and is a powerful tool for monitoring engraftment with a high sensitivity and low false positivity rates.

FISH using a Y-specific probe has been proved to be a reliable method in sex-mismatched transplant evaluation, enabling the study of a great number of dividing cells. More recently, dual-color FISH using X- and Y-specific probes seems to be a more efficient method, providing an internal quality control parameter for successful hybridization.42 On the other hand, correlative interphase FISH can detect 1000 interphase cells in just 4 h and is mainly used to detect relapse in allogeneic HSCTs,35 and to assess different lineages in malignant diseases.43, 44

FISH is mainly carried out using peripheral blood cells or bone marrow cells; however, a few studies have also reported the use of cerebrospinal fluid (CSF), as this is another location where leukocytes migrate.34 Various X- and Y-specific probes are used for FISH, including satellite sequences DXZ1, DXZ3 for the X chromosome and DYZ1 and DYZ3 for the Y chromosome. Probes are mainly dual-color X/Y probes and are hybridized to their complementary chromosomal locations at interphase nuclei.

Restriction fragment length polymorphism (RFLP)

Restriction fragment length polymorphism (RFLP) is widely used as a method of choice for detection of autologous hematopoiesis in patients after HSCT. RFLP analysis is based on the differences in the presence or absence of cutting sites for restriction enzymes in patients and donors. The restriction endonucleases mainly used are EcoRI, HindIII, BamHI, XbaI, MspI, etc. Some researchers have carried out southern hybridization to evaluate the RFLP patterns, while others have used electrophoresis to determine the restriction digestion.

These DNA-RFLPs are used more often because they not only allow distinction and quantitation of donor recipient cells in HSCT patients, but also identify the origin of non-dividing cells. There are several reasons why RFLP is a more sensitive method for detection of MC in the early post-transplant period. Firstly, each RFLP analysis includes DNA extracted from at least 106 cells, while cytogenetic analysis only evaluates 20–50 cells in each sample. DNA studies using RFLP can easily detect 5–10% of the chimerism and under optimal conditions can even detect 0.1–1% chimerism. Secondly, RFLP analysis includes DNA from all nucleated cells, whether or not these cells are proliferating, while cytogenetic analysis evaluates only a fraction of cells that are spontaneously dividing.

Short tandem repeats and variable number of tandem repeats

Microsatellite (STR) and minisatellite (VNTR) genotyping has been the best choice to assess chimerism status in post HSCT patients. STRs and VNTRs are the tandem repetitive blocks of DNA. The basic repeat sequence of a STR/VNTR remains the same, but the number of times that a particular sequence is repeated varies between individuals, resulting in different alleles based on different repeat numbers (Figure 1a).

These STRs/VNTRs are highly polymorphic regions. About 106 STRs are known to be present in human genome. STR/VNTR analysis is regarded as the most sensitive and rapid method for determining the chimerism status. Moreover, STR/VNTR genotyping is independent of HLA mismatching or sex mismatching, and can be used immediately after transplant when very few cells are available for analysis. Three methods have been employed for genotyping of STRs and VNTRs: probe-based typing, PCR-based typing and multiplexing. However, probe-based typing is no longer used because PCR amplification is a very rapid, robust and sensitive technique.

PCR-based typing

PCR amplification of individual specific STR/VNTR is a highly sensitive technique which identifies up to 1–0.1% and 0.1–0.01% of an individual's specific DNA, respectively.33 Individual loci are amplified using flanking primers and the amplicon is size fractionated to determine the allele length and eventually to interpret chimerism status. Figure 1b describes the chimerism analysis by amplification of STR/VNTR.

Multiplexing and automated fragment analyzer-based typing

With the introduction of the automated fragment analyzer (ABI377 and ABI310), quantification of chimerism status has become more accurate and rapid, employing the study of a number of STR/VNTR loci together.45 STR/VNTR loci with identical amplification conditions and different allele lengths are selected. Primers of these loci are labeled with different fluorescent dyes. All the loci are amplified together for each sample in a single PCR and the amplified product is size fractionated by capillary electrophoresis. Further analysis is done by Genescanner and Genotyper software. This fluorescent-based PCR technology has become the gold standard for quantitative chimerism analysis.30

Y-chromosome-specific markers

In recent years, the chimeric status after allogeneic HSCT has been studied by the sensitive molecular technique of PCR-based typing of gender-specific gene sequences. Y-chromosome-specific sequences including genes, Y-STRs and Y-biallelic markers are mainly studied by PCR amplification. These polymorphic regions are studied for the establishment of autologous hematopoiesis in sex-mismatched HSCTs and are of maximum utility in the case of male recipient.

Some of the important Y-STRs used are DYS-19, DYS-390, DYS-389I, DYS-389II, DYS-391, DYS-392 and DYS-393. The Y-chromosome-specific gene studied by various researchers is the portion of the testis-determining gene (SRY) located on the short arm of the Y-chromosome, which encodes a testis-specific transcript that influences sex developmental differentiation.31

X-chromosome-specific markers

In sex-mismatched stem cell transplantation with a female donor, Y-chromosome-specific sequences have been proven the most sensitive marker. In the case of a male donor, the X-linked human androgen receptor gene (HUMARA), containing a highly polymorphic CAG tri-nucleotide repeat, is a reliable marker. Near this polymorphic site are methyl-sensitive HpaII restriction enzyme sites. Unmethylated male HUMARA sequences are completely digested, while methylated female sequences remain intact among the cells of male origin. This allows highly efficient detection of even a small number of female cells.32

X–Y chromosome-based amelogenin marker

The amelogenin system is extensively used as a marker of choice in sex-mismatched HSCTs, where the recipient is male and donor is female. Amelogenin displays a 212 bp long X-chromosome-specific band and a 218 bp long Y-chromosome-specific band on PCR amplification of the amelogenin locus.25 This allows discrimination between X and Y chromosomes, and thus detection of even a slight recurrence of autologous cells.

Real-time quantitative PCR

The latest and most sensitive approach for determining mixed chimerism is based on real-time quantitative PCR using the TaqMan technology.30 This method relies on the detection and measurement of the PCR process itself. This is done by means of a sequence-specific probe, inserted between the forward and reverse primers. During the extension phase of the PCR process, the fluorogenic probe is cleaved and therefore emits a fluorescent signal, which is analyzed in a dedicated thermocycler. As amplifiable PCR products, and therefore the amount of cleaved probe, double during each cycle of a regular PCR process, a strict linear relationship is observed between the logarithm of starting amplifiable DNA copy number and the rank of the first PCR cycle where a significant increase in the fluorescent signal is detected. This is termed the cycle threshold (ct).

Real time-PCR is usually used for the biallelic markers and enables rapid, accurate and robust quantification of mixed chimerism with a sensitivity of 0.1% regardless of sex mismatch.

Amplification refractory mutation system

Allele-specific PCR using the ARMS46 is another strategy in which even a minor DNA population belonging to the recipient's cell in patients after allo-HSCT can be selectively amplified. The basis of this system is that a primer with a mismatched 3′ end will not function efficiently as an amplimer under specific conditions. Consequently, an allele with a perfect match with the 3′ terminal of the primer will be preferentially amplified.

In the context of post HSCT chimerism, an ARMS primer is designed such that its 3′ terminal is perfectly matched to an allele at a polymorphic locus possessed by the recipient, but mismatched in relation to the donor's allele(s).37

Image processing and restriction endonuclease in situ digestion (IPA-REISD2)

This is a quantitative sex-independent method, able to discriminate between donor and recipient cells.38 This approach is based on the detection of cryptic polymorphisms of highly repeated DNA sequences which are uncovered by REISD of metaphase chromosomes or interphase nuclei from peripheral blood lymphocytes or bone marrow cells.47 If cells from recipients and donors show different digestion patterns when treated with a particular RE, the origin of each cell in a post HSCT study may be determined and the chimerism in the transplanted patient may thus be monitored. One of the assayed REs, Sau3A, has shown itself to be particularly useful for this purpose because it reveals a polymorphism for the constitutive heterochromatin of chromosome 3. Different RE banding patterns are directly monitored under fluorescence microscopy.

Moreover, an image processing and analysis (IPA)-assisted procedure, which resolves residual fluorescent regions in metaphase chromosome and interphase nuclei after REISD, has been developed.38 The IPA-REISD technique includes basic screening assisted by the processing and analysis of digitalized fluorescence microscopy images.

Detection of minimal residual disease

With the development of more advanced techniques, researchers have started using more than one technique for more accurate and quantitative assessment of MC. A combination of techniques has been employed to detect the persistence of host hematopoiesis (MC) as well as MRD to detect the disease relapse. These techniques involve study of a novel leukemia-specific mRNA transcript (BCR-ABL) in CML patients,39 study of CBFβ/MYH11 fusion transcripts in acute myelomonocytic leukemia (AML) patients positive for inversion 16,48 β-globin synthesis by erythroid colonies in β-thalassemia patients and analysis of immunoglobin heavy chain (IgH) and T-cell receptor (TCR) genes in B-cell AML patients.49 Table 3 summarizes different methods used to detect MRD for different diseases.

Table 3 Different techniques used to detect MRD

BCR-ABL analysis

The chromosome rearrangement derived from the translocation t(9;22) (q34; q11) is an important mutation found in more than 95% of CML cases and 10–15% acute lymphoblastic leukemia (ALL) patients. This nonrandom 9;22 chromosomal translocation is known as Philadelphia chromosome (Ph). At the molecular level, this translocation gives rise to a novel leukemia-specific m-RNA transcript known as BCR-ABL. This reciprocal translocation involves the Abelson oncogene (ABL) on chromosome 9 and the breakpoint cluster gene (BCR) on chromosome 22; hence it is called BCR–ABL transcript.39

Molecular detection of MRD can be performed in CML patients due to the presence of this leukemia-specific BCR–ABL m-RNA transcript.49, 50 BCR–ABL can be detected using reverse transcriptase PCR (RT-PCR).51 On an individual patient basis, a single positive RT-PCR result is not indicative of relapse; however, rising levels of BCR-ABL transcripts or continued PCR positivity are associated with higher relapse risk.

CBFβ/MYH11 fusion transcript analysis

AML with bone marrow eosinophilia (AML-M4Eo), according to the French–American–British (FAB) classification, is a distinct subtype of AML. AML-M4Eo is often associated with rearrangements of chromosome 16, mostly involving 16p13 and 16q22, leading to a pericentric inversion, inv(16) (p13q22), or less commonly, to a translocation between the homologous chromosome, t(16;16) (p13; q22). The pericentric inversion inv(16) (p13q22) is one of the most frequently occurring chromosomal rearrangements detected in the neoplasm, which has been reported to account for approximately 16% of all AMLs.48, 52, 53, 54

The breakpoint involved in the inv(16) has recently been cloned and shown to involve the core binding factor β- gene (CBFβ) on chromosome 16q22 and the smooth muscle heavy chain (MYH11) on 16p13. The formation of the chimeric CBFβ/MYH11 fusion gene results in the disruption of the normal interaction of the transcription factor complex α, β and ETS.48, 52 These CBFβ/MYH11 fusion m-RNAs are used to detect MRD in post HSCT inv(16)-positive AML patients using RT-PCR assay.

β-Globin synthesis analysis

This technique is used for detection of MRD and even MC in post HSCT β-thalassemia patients. The primary objective of HSCT for β-thalassemia is to replace defective erythropoiesis and β-globin synthesis. To evaluate the risk of erythroid graft rejection effectively, it is necessary to examine chimerism in the erythroid cell lineages in these patients. Prior to HSCT, the recipient having β-thalassemia is characterized by the absence of β-globin synthesis.55 Therefore, after HSCT, any colonies which synthesized β-globin must have been derived from the donor's erythroid progenitor cells. By examining β-globin synthesis in individual erythroid colonies, it is possible to determine the percentage of donor-derived stem/progenitor cells that contributed to the erythroid reconstitution in these patients. Analysis of β-globin synthesis by erythroid colonies provides direct analysis of erythroid chimerism.56

The technique involves growing erythroid colonies in methylcellulose cultures and then estimating globin synthesis of an individual colony. Various investigators have also used β-globin gene point mutation analysis57 for detection of MRD in post HSCT β-thalassemia patients.

IgH and TcR analysis

The use of PCR to amplify clonal IgH and TcR gene rearrangements has so far provided the most sensitive means for the analysis of MRD levels in patients with B-cell ALL.58 The use of clonally rearranged VDJ genes is accepted as a reliable disease relapse predictor in ALL.49, 59 This is probably due to greater sensitivity (10−5 level), and to the fact that the DNA sequence achieved is a more specific leukemia marker. The disadvantage of VDJ analysis is the high frequency of changes in rearrangement patterns.49, 60

PCR amplification of IgH genes involves a degenerate primer complementary to framework three (FR3) of the IgH variable (VH) gene segments, together with the consensus joining (JH)1–6 gene segment primer.49 PCR amplification of TcRδ genes involves a specific Vδ2 gene segment primer together with a specific Dδ3 gene segment primer.

Detection of the split chimerism

‘Split’ chimerism refers to individual cell phenotypes of single but differing genotype (for example, T lymphocytes of donor origin and recipient macrophages), in contrast to MC where there is donor–recipient chimerism within one cell phenotype.61 Detection of split chimerism involves separation of different mononuclear cells from peripheral blood such as T-cells by CD3 antibody, myeloid cells by DC15 antibody, B-cells by CD19 antibody and natural killer cells by CD56 antibody. This is followed by chimerism analysis in each subcell population using the STR/VNTR system. Gracia-Morales et al62 have described a powerful method, PCR-Flow, combining the sensitivity of PCR technology and the resolving power of flow cytometry. Antibody bead separation of cells followed by flow cytometry cell sorting before subjecting the cells for PCR analysis has also been used.

Mixed chimerism detection and relapse of disease

The occurrence of MC and its relationship to the chance of relapse is the most debatable issue in HSCT. Even though hundreds of reports are available on the status of chimerism in post HSCT patients suffering from both malignant and nonmalignant hematological diseases, the picture is still unclear. On one hand there is the belief that there is a strong correlation between relapse and hematopoietic MC, while on the other hand there are reports which show no such correlation.

The presence of MC has been shown to be dependent on several factors including the intensity of the conditioning regimen,63 the use of T-cell depletion22, 23, 24 and the number of stem cells infused.64 In addition, the detection of MC also depends on the sensitivity of the technique utilized30, 65 and the timing at which the assay is performed.66 Moreover, correlation of MC and relapse varies with the disease involved.

The exact dating of relapse or graft rejection mainly depends upon the time of detection. In the immediate period post transplant, persistent MC or disappearance of donor alleles have been shown to be associated with graft rejection or early relapse in both unmanipulated or TCD allografts.66 Similarly increasing levels of recipient cells later post transplant have also been shown to be associated with disease relapse or late rejection following transplantation.29

CML and MC status

In case of CML, the detection of host-type hematopoiesis after HSCT is generally considered to reflect increased probability of persistence of malignant cells, and, in consequence, is associated with a higher risk of post-transplant recurrence. However, some investigators have reported that MC is not necessarily associated with an increased risk of leukemic relapse.24, 31 Following TCD HSCT for CML patients, progressive MC appears to predict relapse.39 The use of BCR–ABL analysis for detection of MRD in conjunction with assessment of chimerism is very useful in defining the relapse status. Despite conflicting results, the majority of researchers are of the opinion that increasing MC or even stable MC is predictive of relapse in CML.

AML, ALL and MC status

Some investigators found no correlation between MC and relapse in patients with acute leukemia (AML and ALL),7, 63 while other described patients with high levels of residual and/or a rapidly increasing amount of recipient cells who carried high risk of developing relapse.7, 29 The conflicting results in literature might be explained on the basis of the biology of acute leukemia whereby relapse develops within a short time and absence of this transient stage results in confusing results.7

It has been shown that relapse is preceded by a critical and a transient stage of MC characterized by increasing amounts of autologous cells. This requires close monitoring of graft status, so that this vital transient stage can be detected.

MC detection and nonmalignant disorders

A wide range of nonmalignant disorders is curable by HSCT including immunodeficiencies, acquired and congenital marrow failure syndromes and hemoglobonopathies. The treatment involves stable engraftment of hematopoietic cells with normal enzyme activities. Therefore, CC is not required to predict reversal or cure of underlying disease. This is the reason why conditioning protocols are less myeloablative. Graft rejection is the major cause of HSCT failure in the patients suffering from aplastic anemia (AA) and other nonmalignant disorders.

Long-term MC is a frequent consequence of HSCT in nonmalignant disorders including AA, severe combined immunodeficiencies (SCIDs), β-thalassaemia and Wiskott–Aldrich syndrome. Conventional ablative regimens also lead to 50% MC; however, use of T-cell depletion results in CC establishment. MC prior to 100 days does not predict rejection; however, chimerism can fluctuate over time without specific interventions. In general, more than 10% donor cells may be adequate for functional correction of the underlying disease.2, 67

The correlation of MC and AA is still unclear. Factors influencing graft rejection remain unclear, but the number of cells infused, prior to transfusion and use of cyclosporine and radiation, appears important.2 Most of the studies suggest the presence of CC from the first month onwards in cases of AA. However, incidences of graft rejection have been often found to be associated with MC. But it is important to establish if graft failure in AA is associated with re-establishment of recipient hemopoiesis or purely a failure to sustain donor hemopoiesis. The former would indicate a requirement for a second HSCT, whereas the latter may benefit from infusion of additional bone marrow.2, 67

Chimerism detection and different therapeutic strategy

The traditional myeloablative (immunosuppressive) bone marrow transplantation approach includes toxic chemo-radiotherapy, followed by a syngeneic or allogeneic HSCT. To avoid myeloablation and obtain optimal results with HSCT, a new, nonmyeloablative, strategy has been suggested, involving suppression of the host-versus-graft reaction and enhancement of the GVL effect at the expense of reducing GVHD.49, 68, 69 However, despite the fact that whether transplantation is myeloablative, with or without T-cell depletion or peripheral blood stem cell transplantation (PBSCT) or nonmyeloablative, measurement of chimerism after transplantation is a prerequisite for manipulating engraftment by altering patient immunosuppression and DLI.3, 30, 70, 71 Table 4 shows the comparison of chimerism analysis for myeloablative and nonmyeloablative transplantation with regard to detection time and therapeutic strategies as recommended by Antin et al.72

Table 4 Comparison of different aspects of chimerism analysis in myeloablative and nonmyeloablative HSCT

Assessment of chimerism in unmanipulated myeloablative HSCT

Myeloablative HSCT without T-cell depletion results in more cases of GVHD, but less frequent relapse. Chimerism analysis mainly reveals full donor chimerism, although researchers have also reported MC. Presumably, T cells in the graft contribute to the establishment of full donor chimerism by a GVL effect; however early CC indicates GVHD, and should be followed by GVHD prophylaxis.72

Assessment of chimerism in manipulated myeloablative HSCT

Patients receiving marrow that has been T cell depleted are commonly found to have MC. Studies have revealed that MC was found to occur in 11–57% of patients who underwent allogeneic HSCT without T-cell depletion and in as many as 50–100% of patients who received bone marrow grafts after the in vitro T-cell depletion.36 A TCD graft is used in HSCTs to avoid the chances of GVHD. Until recently, therapeutic options for CML were limited; however, a new form of adoptive immunotherapy, DLI, appears to be extremely useful especially in cases of TCD graft transplantation.36, 73, 74, 75, 76

Assessment of chimerism in nonmyeloablative HSCT

Nonmyeloablative HSCT is based on two approaches: (1) a low-intensity chemo-radiotherapy regimen before HSCT, in order to reduce the host immune reactivity and (2) DLI after HSCT in order to achieve disease eradication. This approach relies on the GVL effects of the graft rather that the myeloablative effects of irradiation to kill cancer cells. After the transplant, the recipient typically experiences MC.3 If the patient does not experience GVHD, the donor's lymphocytes are infused, leading to a conversion of complete donor chimerism due to GVL effect of the donor's lymphocytes. Therefore, in nonmyeloablative HSCT, the induction of MC, which has to be precisely evaluated, provides a platform for the delivery of adoptive cellular immunotherapy with DLI, whose effects also require monitoring by quantitative analysis of chimerism. Figure 2 describes the importance of chimerism analysis in nonmyeloablative HSCT.70, 71, 72 Lineage-specific chimerism is the best method for post HSCT graft monitoring in nonmyeloablative HSCT. Various interpretations made on the basis of lineage-specific chimerism analysis in nonmyeloablative chimerism72 have been summarized in Table 5.

Figure 2

Chimerism analysis in myeloablative and nonmyeloablative HSCT. D – donor; R – recipient. (a) Myeloablative HSCT without T-cell depletion. GVHD is the main cause of transplant failure. (b) T-cell-depleted myeloablative HSCT, chimerism analysis and MRD detection. (c) Nonmyeloablative HSCT, chimerism analysis and MRD detection results for administering DLI.

Table 5 Lineage-specific chimerism analysis in nonmyeloablative HSCT and its prediction for graft status

DLI and chimerism

DLI is the latest therapeutic strategy used in nonmyeloablative HSCT and TCD myeloablative HSCT.36, 73, 74, 75, 76 Infused donor lymphocytes become sensitized to surface antigens that are expressed on the leukemic cells. This expression is either on polymorphic minor histocompatibility antigens or leukemia-associated antigens. This transforms into cytotoxic lymphocytes that kill the leukemic cells. DLI has been most successful in cases of CML (about 75% cases), which could be due to host antigen-presenting cells that are part of the malignant clones. However, results are less impressive in patients with AML and multiple myelomas. The outcome of DLI is more favorable if the number of leukemia cells is low at the time of infusion. This implies that it requires both chimerism studies after HSCT to provide information about residual autologous hematopoiesis, as well as discrimination between malignant and nonmalignant autologous hematopoiesis in the form of MRD.3, 30, 36, 73, 74, 75, 76 Subsequently, chimerism detection is again required to monitor the effect of DLI in terms of establishment of complete donor chimerism.


The main goal of post-transplantation monitoring is to predict unwanted events such as disease relapse, graft rejection and GVHD, in order to initiate the relevant treatment. In this context, chimerism analysis is, beyond doubt, an important method in monitoring post HSCT outcome. In fact, several previous works suggest that an accurate quantitative analysis of chimerism kinetics would permit early differentiation between the absence of engraftment and a delay in engraftment, as well as early detection of patients with a high risk of GVHD or those liable to relapse.

The presence of MC is to a major extent considered as being a phenomenon parallel to disease evolution post HSCT. The occurrence of MC is dependent on several factors such as the disease for which HSCT has been performed, the intensity of the conditioning regimen, the use of T-cell depletion, the number of stem cells infused, the sensitivity of the technique utilized and the timing at which the assay is performed.

The chimerism detection technique, therefore, should be selected with extreme care, so that an accurate status can be detected and exact information obtained regarding relapse, GVHD or GVL effect. For all these clinical applications, the optimal methodological approach needs to be informative, sensitive and quantitatively accurate. Quantitative real-time PCR using biallelic markers seems to be the best method for detection of autologous hematopoiesis. However, a combination of techniques such as STR/VNTR analysis and BCR–ABL detection is useful in CML patients with sex-matched HSCTs. For sex-mismatched HSCTs, the most suitable techniques are Y-chromosome-specific markers in case of female donor, X-chromosome-specific markers in case of male donor and AMG markers with BCR–ABL detection. This makes it possible to detect not only MC but also MRD, which eventually helps in monitoring graft status as well as disease relapse. Detection of MC is therefore a very important exercise in the light of the latest therapeutic strategies of nonmyeloablative stem cell transplantation and DLI, when early diagnosis is an important aspect of prognosis.


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Khan, F., Agarwal, A. & Agrawal, S. Significance of chimerism in hematopoietic stem cell transplantation: new variations on an old theme. Bone Marrow Transplant 34, 1–12 (2004).

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  • chimerism
  • hematopoeitic stem cell transplantation
  • short tandem repeats
  • donors' lymphocyte infusion
  • nonmyeloablative transplant

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