Detectable Microchimerism Research Articles

Does Transfusion of Red Blood Cells Impact Germline Genetic Test Results?

Background: Molecular genetic testing is increasingly utilized in clinical care, typically with peripheral blood as the preferred specimen. Despite the prevalence of therapeutic blood transfusions, whether donor DNA is present in a sufficient quantity to interfere with recipient genetic testing has not been systematically studied. Microchimerism secondary to transfusion of blood products with donor leukocytes has been well-documented in trauma patients receiving whole blood; however, most medical centers currently transfuse leukoreduced (LR) red blood cell products for non-trauma patients. The degree of leukoreduction varies among centers, but to meet AABB standards must be <5x106 white blood cells (WBC) in 95% of units tested. We sought to determine what quantity, if any, of infused LR or non-LR (NLR) products leads to detectable microchimerism that may impact genetic test results. Methods: We performed an in vitro spiking study utilizing four whole blood units collected from different anonymous donors. Three were leukoreduced at varying levels in order to establish a LR (per our institutional guidelines and standards), a partially leukoreduced (PLR) and a NLR unit, which were then considered "donors". The 4th unit was divided to generate two separate "recipients". The first half was left untreated simulating an immunocompetent recipient while the other was leukoreduced to mimic a leukopenic recipient. Based on a 70 kg patient, we calculated the volume of blood from each "donor" to mix with aliquots of each "recipient" to represent a transfusion of 2, 5, or 16 units, with 2 units corresponding to a double apheresis red blood cell (RBC) transfusion and 16 units corresponding to a near total volume transfusion. DNA was extracted from each individual unit as well as all "transfused" combinations within 24 hours of mixing. Chimerism analysis was then performed by STR analysis using the GlobalFiler PCR amplification kit followed by ChimerMarker analysis software. Results: None of the LR units, despite volume transfused, revealed any level of microchimerism in either the immunocompetent or leukopenic "recipients". The PLR transfused combinations displayed levels ranging from 0.14% to 1.52% of donor chimerism for the immunocompetent "recipient", which is below the limit of detection for most clinical assays evaluating germline genetic variation, and 6.3% to 27.78% of donor chimerism for the leukopenic "recipient", which would be expected to impact a subset of clinical genetic assays. The NLR transfused combinations displayed levels ranging from 13.28% to 95.66% of donor chimerism. Discussion:In vitro "transfusion" of LR RBCs into simulated immunocompetent or leukopenic samples does not lead to detectable donor DNA. However, "transfusion" of PLR and NLR units in an in vitro model reveals significant levels of microchimerism dependent on volume transfused and immune status of the "recipient" which implies possible risk for impact on clinical genetic tests. The minimum time required for clearance of donor leukocytes in the recipient is unknown; we were unable to fully evaluate this and other variables in our in vitro system, but follow-up in vivo studies addressing this question are planned. Disclosures No relevant conflicts of interest to declare.

Infusion of Unrelated-Donor Partially HLA-Matched Cells Results in Detectable Microchimerism in Patients with Acute Myeloid Leukemia: Early Post-Infusion Reactions Are Common but Self-Limiting

AimRelapse remains the leading cause of death for patients with acute myeloid leukemia (AML). Some patients at high risk of relapse are ineligible for allogeneic stem cell transplantation due to age, comorbidities or the absence of a suitable donor. Infusion of fresh haploidentical cells from family donors (microtransplantation) has resulted in improvement in disease-free survival without the development of graft-versus-host disease (GVHD). However, obtaining family donor haploidentical G-CSF primed cells at short notice places a strain on transplant centres because of the requirement for rapid donor evaluation, testing and apheresis. In a Phase I study, we investigated the use of cryopreserved partially HLA-matched cells from unrelated donors as an immediately available alternative to family donors in AML patients ineligible for transplant.MethodA bank of G-CSF primed and steady state apheresis products from 31 normal stem cell transplant donors that were no longer required for their originally intended recipients was established at two transplant centres. Patients in first complete remission (CR) of AML following induction therapy who were not candidates for allogeneic transplant were infused with partially HLA-matched (2/8 HLA-A, -B, -C or DR antigens) unrelated donor apheresis products at a dose of 0.5 x108 CD3 cells/kg following 3 days of cytarabine 2g/m2 BD. No GVHD prophylaxis was given. Up to three cycles were given at 2-month intervals. The presence of microchimerism was assessed via droplet digital PCR using primers/probes targeting 29 bi-allelic insertion/deletion loci.ResultsA total of eight cell infusions have been administered to three patients (Table 1). Non-neutropenic fever occurred within 24 hours of 5 of 8 infusions. Two febrile reactions were associated with transient rashes that were not consistent with GVHD on biopsy. These resolved spontaneously. There were no cases of GVHD following infusion. Median time to neutrophil recovery was 21.5 days (range 13-34) and platelets 17 days (9-39) post commencement of chemotherapy. One patient relapsed 256 days after his 3rd infusion and subsequently died, the other two remain in CR on trial. Of six infusions analysed, microchimerism has been detected in all cases up to 57 days post infusion (Figure 1).ConclusionInfusion of partially HLA-matched cells from unrelated donors results in minimal toxicity and engraftment of donor cells lasting up to 2 months post-infusion. Early non-neutropenic fevers and rashes are common. The effect of donor cells on disease recurrence will be determined through recruitment of additional patients and longer follow up. [Display omitted] [Display omitted] DisclosuresGottlieb:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Indee: Membership on an entity's Board of Directors or advisory committees.

Haploidentical cellular therapy in elderly patients with acute myeloid leukemia: Description of its use in high risk patients

Guo et al. reported an innovative approach for the treatment of acute myeloid leukemia (AML) in a 2011 issue of Blood 1. The report showed for the first time that the infusion after chemotherapy of peripheral blood stem cells (PBSC) from haploidentical donors significantly improved the outcome in elderly AML patients, without major toxicities. After an initial engraftment, haploidentical donor cells are rejected by the host immune response. Indeed, the precise mechanism of action of this cell-therapy procedure is not fully understood and several mechanisms have been hypothesized to explain its beneficial effects: (i) donor allogeneic Natural killer (NK)- or T-cells might exert a direct cytotoxicity on leukemic cells; (ii) a proinflammatory effect of the allogeneic host–donor interaction might trigger an autologous antitumor immunity; and (iii) cell-therapy might induce modifications in leukemic cells or in stromal/vascular cells that ultimately elicit an autologous immune responses. So far, the data obtained, Guo et al. have not been confirmed by other groups worldwide. We recently treated with this protocol three elderly patients with high-risk AML. Compared to the Chinese experience, the patients were all affected by high-risk AML. As summarized in Table 1, Patient #1 had treatment-related AML following prolonged chemotherapy (six total lines) for ovarian cancer and her blasts had a mixed phenotype with complex karyotype. Patients #2 and #3 had AML with myelodysplasia-related changes, one of them (Patient #2) had received four azacitidine courses, with poor response and worsening of both hematological and clinical conditions. Institutional ethics approval was obtained for the use of this treatment protocol. All three patients received an induction regimen (a “3+7” regimen with mitoxantrone and ara-C) followed by the infusion of Granulocyte colony-stimulating Factor (G-CSF)-mobilized PBSC from haploidentical familial donors, according to the original protocol 1. The potential reactivity of patient T-cells against their haploidentical counterparts was proved in vitro by mixed lymphocyte reaction. This in vitro reactivity resulted in vivo in graft-rejection, with detectable microchimerism in only one patients and absence of acute or chronic Graft versus Host Disease (GVHD) in all patients. As detailed in Table 1, Patient #1 had a partial response, with Bone Marrow (BM) blast reduction from 90 to 45%. She then received an intensified salvage regimen (high-dose ara-C and idarubicin plus cyclosporine to overcome Minimal Residual Disease (MRD) resistance) 2, followed by a second infusion of haploidentical PBSC. After this cycle, she had a complete hematological recovery, and the BM aspirate showed complete remission of disease at flow cytometry. Six weeks later, AML relapsed; the patient was not further treated and died shortly thereafter. Patient #2 and #3 received 1 infusion of haploidentical PBSC after induction chemotherapy. Patient #2 was refractory, he refused further treatment and died few weeks later. Patient #3 achieved Complete Remission (CR); however, she was unable to receive consolidation therapy due to the development of fungal endophthalmitis and chronic sinusitis by Fusarium. She completely recovered within 3 months, with a mild residual chronic renal insufficiency. Despite lack of consolidation therapy, she is still in CR at 11 months of follow up. Our observation in high-risk elderly AML patients confirms that infusion of haploidentical-mobilized PBSC following induction chemotherapy is feasible and safe. Compared to Guo et al. 1, 3, we applied the strategy in AML patients with very unfavorable prognosis, two of them previously treated either with multiple chemotherapy lines or with repeated azacitidine courses. In spite of previous treatments, the haploidentical infusion did not induce acute GVHD. Considering the presenting features, the achievement of CR in two of three patients was encouraging and somehow unexpected, especially for the long-surviving patient who achieved CR after a single course of chemotherapy followed by haploidentical PBSC infusion. Finally, the two patients achieving CR had a leukothrombocytopenia duration shorter than one might have expected, suggesting a possible role of the infused cells in favoring hematopoietic recovery. We conclude that the “nonengraftment haploidentical cell-therapy” is feasible also in high-risk and previously treated AML patients, although the real efficacy in this setting needs to be further investigated 4. Indeed, based on our preliminary experience, a prospective trial will be launched soon to evaluate the procedure in elderly AML patients, including AML secondary to myelodysplasia, either untreated or after azacytidine, therapy-related AML as well as de novo AML A. Cignetti,1,2* M. Ruella,1,2 A. R. Elia,2 V. Tassi,3 V. Redoglia,4 D. Gottardi,1C. Tarella1,2 1University Division of Hematology and Cell Therapy, Mauriziano Hospital and University of Torino, Torino, Italy 2Molecular Biotechnology Center (M.B.C.), University of Torino, Torino, Italy 3Blood Bank, Molinette Hospital and University of Torino, Torino, Italy 4Division of Hematology, Molinette Hospital and University of Torino Torino, Italy

Increased fetal cell trafficking in murine lung following complete pregnancy loss from exposure to lipopolysaccharide

To determine whether chemically induced miscarriage affects fetomaternal trafficking in a mouse model, we measured the amount of fetal DNA present in various maternal organs by polymerase chain reaction amplification following exposure to lipopolysaccharide (LPS). As the frequency of fetal cells and the number of animals with detectable microchimerism following LPS injection were significantly increased, particularly in lung tissue compared to controls, with no signs of an inflammatory response, we conclude that LPS-induced miscarriage results in increased murine fetomaternal cell trafficking, supporting a relationship between fetal loss and the establishment of fetal cell microchimerism.

PCR-Based Methodology for Molecular Microchimerism Detection and Quantification

Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied, but a consensus on its detection has not yet been adopted. The objective of this study was to establish a panel of reproducible molecular polymerase chain reaction (PCR)-based methods for detection and quantification of foreign cells in an individual. We analyzed length polymorphisms generated by short tandem repeat (STR) and variable number tandem repeat (VNTR) markers. Human leukocyte antigen (HLA)-A and -B polymorphisms were detected by reference strand conformation analysis (RSCA). Class II polymorphisms on HLA-DRB1 locus were analyzed both by classical PCR-sequence-specific primers (SSP) and by quantitative PCR (Q-PCR). Also, sex-determining region-y gene (SRY) gene allowed specific male donor discrimination and quantification by Q-PCR in female recipients. Binomial statistical distribution analysis was used for each molecular technique to determine the number of PCR replicates of each sample. This analysis allowed the detection of the lowest detectable microchimerism level, when present. We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:10(5) and 1:10(6) donor per recipient cells (DPRC), respectively, using Q-PCR for SRY or for nonshared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified because of technique limitations. However, classical PCR methods allowed detection down to 1:10(4) DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10(4) to 1:10(6) DPRC after kidney or heart transplantation, and 1 log higher (1:10(3) to 1:10(6) DPRC) after liver transplantation. In conclusion, the standardization of molecular microchimerism detection techniques will allow for comparable interpretation of results in microchimerism detection for diagnostic or research studies.

Microchimerism in immune competent patients related to the leukocyte content of transfused red blood cell concentrates

Background. Microchimerism may play a part in transfusion complications. The aim of this study was to examine whether establishment of post-transfusion microchimerism was related to leukocyte content. Methods. Twenty non-pregnant female patients, without known malignant or immunological diseases, mean age 68 years, receiving 2–4 units of red blood cell concentrates during elective surgery, were included. One or two of the units were from male donors. Ten patients received buffy-coat depleted red blood cell concentrates, leukocyte count 108–109 per unit, and 10 patients received red blood cells leukoreduced by prestorage leukocyte filtration, with a leukocyte count of <106 per unit. EDTA samples were collected in vacuum tubes before and after 1 week and 6 months after transfusion. The tubes were frozen and stored at −400 °C. Genomic DNA was isolated and PCR performed using four primer sets amplifying markers on the Y-chromosome. Results. Microchimerism was detected in a total of eight out of the 20 patients. In three patients microchimerism was detected only before transfusion. These patients had given birth to one or two boys each, and had no history of previous transfusion. Two patients receiving buffy-coat depleted red blood cell concentrates and two patients receiving leukoreduced red blood cell concentrates had detectable microchimerism 1 week after transfusion. The age of the transfused red blood cell concentrates was 6, 24, 8 and 7 days, respectively. One patient receiving leukoreduced red blood cell concentrates had detectable microchimerism after 6 months. The age of this concentrate was 22 days. Discussion. This study demonstrates that microchimerism after transfusion does not seem to be dose dependent, and can be induced even by a >3 week old leukoreduced red blood cell concentrate with a very low leukocyte content.

Clinical Tolerance: the end of the beginning.

Clinical Tolerance: the end of the beginning.

Adenoviral transfer of a single donor-specific MHC class I gene to recipient bone marrow cells can induce specific immunological unresponsiveness in vivo

We investigated the delivery of a donor-specific MHC class I gene, H-2K(b), using a newly constructed replication-defective recombinant adenovirus (AdSV40K(b)) to recipient tissue before transplantation as a means of inducing donor-specific immunological unresponsiveness. AdSV40K(b) was able to transduce both a fibroblast cell line and freshly isolated bone marrow cells (BMCs) resulting in cell surface expression of H2-K(b) protein. Intravenous infusion of AdSV40K(b)-transduced syngeneic CBA/Ca (H-2(k)) BMCs into CBA recipient mice treated with an anti-CD4 monoclonal antibody 27 days before transplantation of a fully MHC-mismatched, C57BL/10 (H-2K(b+)), cardiac allograft resulted in significant long-term graft survival when compared with mice receiving the same dose of syngeneic BMCs transduced with a control adenovirus, AdRSVbetagal. Despite the induction of H-2K(b)-specific hyporesponsiveness following pretreatment with AdSV40K(b)-transduced CBA BMCs, persistence of H-2K(b) mRNA in central or peripheral tissues could not be demonstrated by RT-PCR. This result was in contrast to the observed persistence of K(b) mRNA both in the periphery and thymus following the infusion of transgenic CBK (H-2(k) + K(b)) BMCs. We conclude that ex vivo adenoviral gene transfer of a single donor MHC class I gene to recipient BMCs in combination with transient depletion of CD4(+) cells is sufficient to induce long-term graft survival of a fully allogeneic cardiac graft. In addition, detectable microchimerism is not a prerequisite for graft survival.

Microchimerism after heart transplantation: prevalence, predisposing factors, natural history, and prognosis

THE SUCCESS of heart transplantation (HT) is still marred by the need for chronic immunosuppression using agents that display not only limited efficacy and specificity but also considerable toxicity. Overall 1-year survival is only 81%, and patient half-life (the time at which the survival rate has dropped to 50%) is 9.8 years. The induction of tolerance to transplanted organs, might make transplantation safer as well as more successful. Certain studies of recipients of solid organs, including liver, kidney, lung, and heart seem to be associated with apparent induction of tolerance and with the persistence of low levels of donor cells of bone marrow origin (microchimerism). Other studies, however, have not found an association between microchimerism and tolerance, and there have also been cases of apparently tolerant recipients who do not show detectable microchimerism. These discrepancies may be explained by differences in the prevalence of microchimerism depending on the organ type and the time after transplantation. Despite these controversies, some workers have attempt to increase the possibility of tolerance by induction enhancing microchimerism via donor bone marrow infusion (DBMI) at the time of transplantation. The work described herein includes studies of the prevalence and natural history of microchimerism after HT, as well as predisposing factors to establish the state, and its relevance to the outcomes of survival, rejection and steroid requirements.

Persisting alloantigen prevents primed CD45RC CD4 T cells from inducing allograft rejection: implications for immunological memory

Antigen stimulation induces specific CD4 T cells to change from a resting phenotype (CD45RC + ) to a “memory” phenotype (CD45RC − ), an isoform switch that is reversible and regulated by persisting antigen. We show here that CD4 T cells responsible for mediating allograft rejection undergo a similar CD45RC + to CD45RC − switch irrespective of whether antigen priming results in sensitization or tolerance in vivo. Thus, skin allograft priming, designed to induce second set rejection, and a donor-specific blood transfusion (DST), designed to prolong cardiac allograft survival, will generate CD45RC − CD4 T cells that induce acute rejection when adoptively transferred to T cell-deficient athymic nude recipients. The ability of CD45RC − T cells, obtained from DST donors, to induce graft rejection was prevented by giving nude recipients a DST 14, 28 or even 56 days before grafting and T cell transfer. Thus, prolonged allograft survival in rats after DST was found to be strongly linked with persisting alloantigen from the blood transfusion but was not associated with detectable microchimerism. Importantly, CD45RC − T cells from skin graft-primed animals were similarly prevented from inducing rejection by residual DST-derived alloantigen. The investigation shows (1) that an allogeneic blood transfusion primes (not tolerizes) alloreactive CD4 T cells and (2) that residual DST-derived alloantigen can block the action of specifically primed “memory” CD4 T cells. These findings have implications for understanding immunological memory.

Persisting alloantigen prevents primed CD45RC- CD4 T cells from inducing allograft rejection: implications for immunological memory.

Antigen stimulation induces specific CD4 T cells to change from a resting phenotype (CD45RC+) to a "memory" phenotype (CD45RC-), an isoform switch that is reversible and regulated by persisting antigen. We show here that CD4 T cells responsible for mediating allograft rejection undergo a similar CD45RC+ to CD45RC- switch irrespective of whether antigen priming results in sensitization or tolerance in vivo. Thus, skin allograft priming, designed to induce second set rejection, and a donor-specific blood transfusion (DST), designed to prolong cardiac allograft survival, will generate CD45RC- CD4 T cells that induce acute rejection when adoptively transferred to T cell-deficient athymic nude recipients. The ability of CD45RC- T cells, obtained from DST donors, to induce graft rejection was prevented by giving nude recipients a DST 14, 28 or even 56 days before grafting and T cell transfer. Thus, prolonged allograft survival in rats after DST was found to be strongly linked with persisting alloantigen from the blood transfusion but was not associated with detectable microchimerism. Importantly, CD45RC- T cells from skin graft-primed animals were similarly prevented from inducing rejection by residual DST-derived alloantigen. The investigation shows (1) that an allogeneic blood transfusion primes (not tolerizes) alloreactive CD4 T cells and (2) that residual DST-derived alloantigen can block the action of specifically primed "memory" CD4 T cells. These findings have implications for understanding immunological memory.

No detectable microchimerism in long-term tolerant recipients of living-donor kidneys

No detectable microchimerism in long-term tolerant recipients of living-donor kidneys

Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism.

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.