Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing.@*METHODS@#The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method.@*RESULTS@#The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance.@*CONCLUSION@#MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Severe aplastic anemia treated with unrelated cord blood combined with matched sibling allogeneic hematopoietic stem cell transplantation / 中国组织工程研究

BACKGROUND: Sibling matched allogeneic hematopoietic stem cell transplantation is still the first-line treatment for severe aplastic anemia. However, with the increase of patients’ age, the effect of transplantation decreases significantly. OBJECTIVE: To explore the efficacy and safety of unrelated cord blood combined with matched sibling hematopoietic stem cell transplantation in the treatment of severe aplastic anemia. METHODS: A total of four severe aplastic anemia patients who underwent unrelated cord blood combined with matched sibling hematopoietic stem cell transplantation in First Affiliated Hospital (Yijishan Hospital) of Wannan Medical College from July 2017 to February 2018 were enrolled and their clinical data were used for retrospective analysis. RESULTS AND CONCLUSION: (1) All the four patients were male. The median age was 40 years old and the median time from diagnosis to transplantation was 2.5 months. Bone marrow and peripheral blood stem cells from a sibling donor as well as a unit of unrelated cord blood with HLA matching ≥ 4/6 were applied. (2) The median stem cells of total nucleated cells and CD34+ were 13.67×108/kg and 2.7×106/kg of the sibling donor, 2.1×107/kg and 1.21×105/kg of the unrelated cord blood, respectively. (3) The median implantation time of neutrophils and platelets was +10 days and +20 days, respectively. (4) After transplantation, one patient was sibling donor chimerism while that of another three patients was double donors. At the follow-up date, among the three cases of mixed chimerism from two donors, one was completely implanted with unrelated cord blood; one was completely implanted with sibling donor; the third case had mixed chimerism with sibling donor and recipient giving up treatment because of infection. (5) Only one patient developed grade II acute graft versus host disease. The incidence of III-IV acute graft versus host disease and chronic graft versus host disease was 0%. (6) Two-year post-transplant disease free survival and overall survival rates were both 75% and graft versus host disease-free and relapse-free survival was 100%. (7) The results show that sibling allogeneic hematopoietic stem cell transplantation combined with unrelated cord blood transfusion for the treatment of older patients with severe aplastic anemia has a low incidence of graft versus host disease and a positive effect, but the dynamics for the implantation of two kinds of donor stem cells is worthy of further study.

Impact of Day 14 Peripheral Blood Chimerism after Allogeneic Hematopoietic Stem Cell Bone Transplantation on the Treatment Outcome of Non-Malignant Disease

BACKGROUND: The impact of early peripheral blood chimerism on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear. We aimed to determine whether day 14 peripheral blood chimerism after allo-HSCT predicts outcomes in patients with non-malignant diseases. METHODS: Data from 56 patients who received allo-HSCT between April 2007 and March 2016 were retrospectively analyzed. Chimerism was evaluated using short-tandem repeat polymerase chain reaction, with mixed chimerism (MC) defined as greater than 1% recipient cells which was further categorized into low-level MC (> 1% and < 15% of recipient-derived cells) and high-level MC (≥ 15% of the recipient-derived cells). RESULTS: Thirty-six patients showed complete donor chimerism (CC), 14 low-level MC, and 6 high-level MC at day 14 post-transplant. The estimated 5-year event-free survival (EFS) was higher in the CC or low-level MC groups than in the high-level MC group (86.1% vs. 71.4% vs. 33.3%; P = 0.001). In BM or peripheral blood stem cell (BM/PBSC) transplants, the 5-year EFS was higher in the CC or low-level MC group than in the high-level MC group (93.1% vs. 66.7% vs. 0%; P < 0.001). However, in cord blood transplants, the 5-year OS and EFS according to the day 14 peripheral blood chimerism did not reach statistical significance. CONCLUSION: Although CC is not always necessary after allo-HSCT for non-malignant diseases, our data suggest that day 14 peripheral blood chimerism may predict outcomes in patients with non-malignant diseases who underwent BM/PBSC transplants.

Cellular and molecular genetic analysis of sex chromosome chimerism and dicentric isochromosome structural abnormalities: a report of two cases / 中华围产医学杂志

Objective@#To investigate the value of karyotype analysis, bacterial artificial chromosomes-on-beads (BoBs), chromosome microarray analysis (CMA) and fluorescence in situ hybridization (FISH) in the diagnosis of sex chromosome numerical and structural abnormalities.@*Methods@#Conventional G-banding staining technique was used to analyze the karyotypes of amniotic fluid cells and parental peripheral blood cells in two pregnancies with prenatal diagnosis indications. Sex chromosome numerical and structural abnormalities were analyzed based on the results of G-banding, BoBs, CMA and FISH.@*Results@#The results of G-banding karyotype analysis showed that there were mosaics in amniotic fluid cells collected from both cases. Karyotype of Case A was 45,X[25]/46,X,idic(Y)(q11.2?)[6], and Case B was 45,X[39]/46,X,psu idic(X)(q21.32?)[44]. Parental peripheral blood karyotypes of both families were normal. Prenatal BoBs indicated copy number abnormalities in sex chromosomes (Y chromosome in Case A and X chromosome in Case B). CMA results suggested a 20.1 Mb duplication in Yp11.32q11.222, and a 7.7 Mb deletion in Yq11.222q11.23 in fetus A with possible karyotype of 46,X,idic(Y)(q11.222); for fetus B, a 92.0 Mb duplication in Xp22.33q21.32, and a 63.0 Mb deletion in Xq21.32q28 were detected, and the karyotype might be 46,X,psu idic(X)(q21.32). The mid-term FISH test of amniotic fluid cells showed that 90% of the amniotic cells from Case A were 45,X, and 10% were 46,X,idic(Y)(q11.2); about 38% were 45,X, and 62% were 46,X,psu dic(X)(q21.3) from Case B.@*Conclusions@#Numerical and structural abnormalities of sex chromosomes could be accurately diagnosed by combination of several methods including G-banding karyotype analysis, prenatal BoBs, CMA and FISH, which would help to effectively reduce birth defects.

Prognostic value of donor chimerism at +90 days after allogeneic hematopoietic stem cell transplantation in young patients with intermediate-risk acute myeloid leukemia / 中华血液学杂志

Objective@#To investigate the relationship between donor chimerism and relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) .@*Methods@#The clinical data of 105 patients with acute myeloid leukemia (AML) who underwent allo-HSCT and recurrence-free survival>90 days from January 2010 to January 2019 were retrospectively analyzed. The bone marrow samples were collected at 15, 30, 60, 90, 180, 270, 360 days after transplantation. Donor chimerism was detected by single nucleotide polymorphism (SNP) -PCR.@*Results@#Of the 105 patients, 43 cases were male and 62 cases were female, with a median age of 38 (16-60) years. Till April 2019, the median follow-up was 843 (94-3 261) days. Ninety days after transplantation, 18 cases relapsed, 33 cases died, and 72 cases survived. The 3-year overall survival (OS) rate was (66.8±5.1) %, and the recurrence-free survival (RFS) rate was (65.1±5.0) %. Pre-transplant disease status, pre-transplant minimal residual disease (MRD) , and 90 day post-transplantation chimerism were independent risk factors related to RFS. The risk of recurrence was significantly increased in patients with a donor chimerism rate ≤97.24% at 90 days after transplantation[HR=6.921 (95%CI 2.669-17.950) , P<0.001], which was considered as a sign of early relapse.@*Conclusion@#SNP-PCR is an applicable method for detecting donor chimerism in patients after allo-HSCT. Chimerism rate equal or less than 97.24% at 90 days after transplantation predicts a higher risk of relapse.

Application of multiple nucleotide polymorphism analysis in chimerism detection after allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

Objective@#To establish a new method for chimerism analysis after allogeneic hematopoietic stem cell transplantation by using multiple nucleotide polymorphism sequencing (MNPseq) , and to explore its feasibility and superiority.@*Methods@#One hundred MNP fragments were screened and chimeric analysis was performed by high-throughput sequencing technology. The accuracy and sensitivity of the method were verified by simulating chimeric samples and post-transplant samples and comparing them with short tandem repeat (STR) analysis, fusion gene quantitative detection and flow cytometry for minimal residual disease.@*Results@#The accuracy and sensitivity of MNPseq were better than those of STR, in which the sensitivity could reach 0.01%, about 100 times more sensitive than STR. MNPseq could further distinguish 42 STR fully chimeric samples, and after corrected by cutoff value, it was correlated with the quantitative detection of fusion gene. MNPseq could correct false positive of STR caused by the shadow peak, and could be used to detect chimeric samples lacking pre-transplant information from donors and recipients.@*Conclusion@#MNPseq analysis based on high-throughput sequencing is a more accurate and sensitive chimerism detection method, and it solves the problem that chimerism cannot be detected due to the lack of pre-transplant information, which has extremely high clinical application value.

Renal transplantationplus hematopoietic stem cell transplantation as Induction therapy :a single-center 10-year experience / 中华器官移植杂志

Objective To explore the efficacy of renal transplantation plus hematopoietic stem cell transplantation on inducing immune tolerance and summarize its long-term follow-up outcomes . Methods From 2009 to 2018 ,a total of 11 cases of living related donor kidney transplantation plus hematopoietic stem cell transplantation were performed .Two of them were HLA-matched and the remainder were mismatched for one HLA haplotype . The donor hematopoietic stem cells were mobilized using granulocyte colony-stimulating factor at 5 days pre-transplantation and collected at 1 day pre-operation .The recipients received total lymphoid irradiation for 3 days pre-transplantation and received anti-thymocyte globulin induction during transplantation .The donor hematopoietic stem cells were infused at 2 ,4 and 6 postoperative day .Postoperative regulatory T cells ,chimerism ,B cell activating factor and mixed lymphocyte culture and other parameters were detected and long-term follow-up outcomes tracked .Results The immune tolerance-inducible recipients had a significant increase in activated Treg .One HLA-matched recipient achieved 30％-50％ of chimerism and lost after 6 months .However ,other recipients did not achieve mixed chimerism .The BAFF of recipient spiked sharply after transplantation .Mixed lymphocyte culture indicated that a donor-specific low response was induced .The recipients were followed up for 717 to 3612 days .The first recipient lost renal function and another ten recipients had stable renal function . None of the recipients had myelosuppression or graft-versus-host disease .Allograft biopsy confirmed only one case of mild acute rejection . The dose of immunosuppressive agents was lowered in 5 patients .Conclusions Hematopoietic stem cell transplantation for inducing tolerance is safe during renal transplantation . And chimerism is essential for inducing immune tolerance .

Late chimerical status after bone marrow transplantation in severe aplastic anemia according to two different preparatory regimens

ABSTRACT Background: This study investigated the influence of two conditioning regimens on the chimerical status of 104 patients with acquired severe aplastic anemia. Methods: Patients were monitored for at least 18 months after related bone marrow transplantation and reaching partial or complete hematologic recovery. Group I patients (n = 55) received 200 mg/kg cyclophosphamide alone and Group II (n = 49) received 120 mg/kg cyclophosphamide associated with 12 mg/kg busulfan. Patients were classified in three chimerism levels according to the percentage of donor cells in the peripheral blood. Results: Chimerism ≤50% occurred in 36.4% of Group I and none of Group II; chimerism 51-90% was found in 20.0% of Group I and 10.2% of Group II; and chimerism >90% was found in 43.6% of Group I versus 89.8% of Group II. A significant association (p-value < 0.001) was found between conditioning type and chimerism levels. A higher number of infused cells was associated with higher levels of chimerism only in Group I (p-value = 0.013). Multivariate analysis showed that chimerism >90% is associated with the cyclophosphamide plus busulfan conditioning (p-value < 0.001) and higher number of infused cells (p-value = 0.009), suggesting that these factors are predictive of graft outcome. Regarding hematological recovery, higher chimerism levels were associated with higher neutrophil (p-value = 0.003) and platelet counts (p-value < 0.001) in Group I only. These results show that myeloablative conditioning favors full donor chimerism and non-myeloablative conditioning predisposes to mixed chimerism or autologous recovery of hematopoiesis. Conclusion: These data show that autologous recovery depends on the intensity of immunosuppression and that the immunosuppressive function of cyclophosphamide alone can induce this type of hematopoietic recovery.

Prognostic significance of early phase donor chimerism after allogeneic peripheral blood stem cell transplantation / 中华血液学杂志

Objective@#To evaluate the prognostic significance of early phase full donor chimerism (FDC) after myeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT).@*Methods@#The clinical data of 72 hematological patients received myeloablative allo-PBSCT from Feb. 2016 to Jul. 2017 were analyzed retrospectively. The median age was 36.5 years (range 4-59), 44 were males and 28 females. Of the donors, there were 35 HLA matched sibling donors, 27 haploidentical donors and 10 unrelated donors. Polymerase chain reaction amplification of short tandem repeat sequence (PCR-STR) was used to detect donor cell chimerism (DC) rate of recipient bone marrow at one, two and three months after transplantation.@*Results@#The median follow-up was 462 d (range: 47-805 d), 55 cases were still alive, and 45 cases were disease-free survival (DFS) at the end of follow-up. The 2-year overall survival (OS) and DFS were (68.9±7.7)% and (59.5±6.3)%, respectively. A number of 16 cases underwent relapses, with 2-year cumulative incidence of (24.1±5.3)%. The median time of recurrence was 157(32-374) d. Forty cases (55.6%) developed acute graft-versus-host diseases (aGVHD), with median time of 35.5 (13-90) d. Chronic GVHD (cGVHD) occurred in 23 patients (31.9%), with median time of 169 (94-475) d. Univariate analysis found the following factors were not related to OS, DFS or relapse rate (RR), including age, sex, blood type and sex of donor-recipient, occurrence of aGVHD and cGVHD. The OS and DFS in cases reached FDC and no FDC at two months after transplantation were (85.2±6.9)% vs (66.1±7.7)% (P=0.051) and (76.7±7.7)% vs (48.9±8.1)% (P=0.021), respectively. The RR rate in FDC group was lower than that in no FDC group [(16.6±6.8)% vs (30.4±7.8)%, P=0.187, respectively].@*Conclusion@#The present study confirmed the important value for predicting the prognosis with whether or not the patients reached FDC at the early phase after allo-PBSCT. The OS and DFS in cases with FDC at two months after transplantation were significantly higher than those of no FDC patients.

The study of establishing the chimerism through vascularized ilium transplantation animal model / 中华泌尿外科杂志

Objective To construct an animal model of vascularized iliac bone transplantation which could establish the immune chimerism.Methods The experiment was divided into three groups.In experiment group we slected the male SD rats as donors and the female SD rats as recipients.Then the experiment group was performed the improved vascularized ilium transplantation operation.The positive group was male SD rats without handling.The negative group was female SD rats without handling.On the twenty-eighth day after surgery, the rat SRY gene was detected by PCR.Dynamically monitoring the changes of the ratio of CD4+/CD8+ T cells by flow cytometry after operation.Dynamically monitoring the changes of the levels of serum IL-2 by enzyme linked immunosorbent assay after operation.Imaging and pathology were used to detect the transplanted iliac bone.Results Ten transplantations were performed in the experiment group and 7 flaps survived with a successful rate of 73 ％.SRY gene was detected in female rats that receive the male rats'iliac bone transplantation by PCR in the experiment group.After Micro-CT scanning and three-dimensional reconstruction, it showed that the transplanted iliac bone mineral density was decreased.Comparing rats that establish the chimerism with normal rats, the ratio of CD4 +/CD8 + T cells and the levels of serum IL-2 had no difference.Pathological results showed that the transplanted iliac bone of rats with chimerism were normal and the rats without chimerism were necrotic.Conclusions From the improved vascularized ilium transplantation operation, we successfully detected the SRY gene in female rats which received the male rats' iliac bone transplantation.It proved that the improved vascularized ilium transplantation operation could establish the chimerism.Through the immunosuppressant, the immune status of the rat after operation was well.

The clinical characteristics of adult hemophagocytic lymphohistiocytosis treated with haploidentical donor hematopoietic stem cell transplantation / 中华内科杂志

Objective To analyze the clinical characteristics of adult patients with hemophagocytic lymphohistiocytosis (HLH) receiving haploidentical donor hematopoietic stem cell transplantation (HID HSCT).Method We retrospectively reviewed 20 adult patients with HLH from August 2009 to August 2014.The clinical features and outcome were analyzed.Results Conditioning regimens consisted of total body irradiation/etoposide/cyclophosphamide (TBI/VP-16/CTX) and busulfan (Bu)/VP-16/CTX in HLH with anti-thymocyte globulin (ATG) 8 mg/kg.The stem cells were mobilized from donors' peripheral blood.Median time to white blood cell engraftment was 13 (9-27) days.Median time to platelet engraftment was 14 (10-28) days.Mixed chimerism after transplantation developed in 4 patients and no patient presented graft failure.Eight patients developed grade Ⅱ to Ⅲ acute graft-versus-host disease (GVHD),while as chronic GVHD occurred in 9 patients.Among 12 patients with EB virus (EBV) reactivation,2 patients developed post-transplant lymphoproliferative disorder (PTLD),7 were suspected as PTLD and 3 were considered as relapse of primary disease.With a median follow-up of 20 months (range:0.5-108 months) after transplantation,the estimated 2-year overall survival (OS) rate was (60.0 ± 11.0) ％ in all patients.During the follow-up,12 patients survived,8 died including 5 within 100 days after HSCT.Among 5 non-remission patients before HSCT,4 patients died within 100 days after HCT.Conclusions HID HSCT is an effective treatment for adult patients with HLH to achieve remission and long-term survival.High proportion of mixed chimerism has been seen at early stage after transplantation.EBV reactivation and early transplant-related mortality are common.

Diagnóstico molecular, citogenético y anatomohistopatológico del Síndrome Freemartin en hembras bovinas en Colombia / Molecular, cytogenetic and anatomohistopatologic diagnostic of freemartin syndrome on females cattle in

El síndrome Freemartin es un estado de intersexualidad de muchas de las hembras bovinas provenientes de parto múltiple heterosexual (macho - hembra). Éste se origina en la vida fetal entre los 30 y 40 días de gestación producto del intercambio transplacentario de células mediante anastomosis vasculares, presentándose fenómenos de quimerismo 60XX/XY en varios tejidos, y esterilidad consecuente. En el presente trabajo se tomaron 106 muestras de sangre de terneras provenientes de parto múltiple heterosexual, se realizó extracción de ADN de leucocitos y se buscó la amplificación del gen SRY asociado al cromosoma "Y" mediante PCR y lectura en gel de agarosa. 90 terneras (84.9%) de las 106 amplificaron SRY, verificando el quimerismo 60XX/XY, y 16 terneras (15.1%) que no amplificaron el gen, libres del síndrome quimérico y por lo tanto, aptas reproductivamente. El análisis citogenético realizado mediante cultivo de linfocitos demostró la presencia del cromosoma "Y" en linfocitos de hembras positivas a SRY y la ausencia del quimerismo en hembras SRY negativas. El análisis anatómico post mortem de tractos reproductivos de hembras positivas a SRY detectó anormalidades características del síndrome tales como, clítoris hipertrofiados y atresias ductales cervicales. El análisis histopatológico de placas de gónadas de estos animales evidenció la presencia de ovotestículos. El presente estudio confirma la utilidad de las técnicas de biología molecular como herramientas diagnósticas del síndrome, para el aprovechamiento de hembras de reemplazo al servicio del hato bovino.

Freemartin syndrome is a intersexuality condition developed in many of the female calfs born from heterosexual multiple calvings (male female). This originates in the fetal development between 30 and 40 days of gestation with transplacental exchange of cells through vascular anastomosis, occurring 60XX/XY chimerism in various tissues, and consequent sterility. In this paper was took blood samples from 106 female calves born from a multiple heterosexual calving. DNA was extracted from leucocytes and searched the SRY gene amplification associated with the Y chromosome by PCR and reading in agarose gel. 90 of 106 samples (84.9%) was amplified the SRY gene, verifying the 60XX/XY chimerism, and 16 samples (15.1%) that did not amplify the gene. Cytogenetic analysis using lymphocytes cell cultures showed the presence of Y chromosome in samples positive for SRY and absence of chimerism in SRY negative samples. The postmortem analysis of female reproductive tracts of SRY positive calves, shows anatomical abnormalities such as clitoris hypertrophia and cervical atresia. Histological examination of gonads of these animals confirmed the presence of ovotestis. This study confirms the usefulness of molecular biology techniques as diagnostic tools of the syndrome, for the use of replacement females in cattle.

Implicaciones médico legales del quimerismo / Medicolegal implications of chimerstry

Los recientes avances en tecnologías biomédicas proporcionan herramientas a la Medicina Legal para el esclarecimiento de casos complejos y ejercer justicia basada en evidencia científica. ¿Pero qué ocurre cuando se imponen obstáculos como el quimerismo que podrían llevar a decisiones equivocadas? A pesar de que este fenómeno se creía casi inexistente, se han reportado interesantes casos, que han puesto a prueba la utilidad de las pruebas de ADN. El quimerismo se define como la presencia de líneas celulares con distinto material genético proveniente de diferentes orígenes en un único cuerpo. Esto tiene grandes implicaciones medico legales, principalmente en la investigación criminal. Por ejemplo en una escena de crimen, se pueden encontrar muestras de tejidos de un mismo individuo pero estas podrían tener ADN distinto si se trata de un individuo quimérico llevando a una mala interpretación de la información. También cabe resaltar las implicaciones del quimerismo en las pruebas de paternidad, ya que este fenómeno puede ocasionar falsos negativos en estas pruebas y por lo tanto un diagnóstico incierto de paternidad. El objetivo de esta revisión es describir las diferentes implicaciones médico legales del quimerismo y proponer posibles soluciones a los conflictos que podrían presentarse ante tales casos.

Recent advances in biomedical technologies are able to clarify today’s complex cases that are faced by Legal Medicine; allowing this branch of medicine to exercise justice based in scientific evidence. But what happens when an obstacle such as chimerism is present and may lead to false decisions? Although this phenomenon was thought to be inexistent, interesting cases have been reported. Chimerism is defined as the presence of different cell linings in a unique organism. This phenomenon has important implication in Legal Medicine, especially in criminal investigation. For example, in a crime scene the samples gathered may present different DNA and lead to misinterpretation of the information. On the other hand, paternity tests are also implicated in the presence of a chimera since it may originate a false negative result. The purpose of this review is to describe different Legal Medicine’s implications linked to chimerism and propose possible solutions to the conflicts that may arouse from a case of a chimera.

Limited immune tolerance induced by transient mixed chimerism

No abstract available.

Engraftment of bone marrow-derived cells after nonlethal radiation in syngeneic C57BL/6 mice / 中华放射医学与防护杂志

Objective To study the characteristics of cell engraftment in mice at a lower dose under nonlethal radiated condition.Methods A syngeneic C57BL/6 mouse model,transplanted with 1 × 107 bone marrow cells and exposed to 2.5 Gy whole body irradiation (WBI),was selected to study the chimerism of cells from green fluorescent protein positive (GFP +) transgenic mice.The control group was injected with GFP + cells without receiving irradiation.In addition,an allogenic transplantation model of BALB/c mice was also investigated which was infused by GFP + cells from C57BL/6 mice.The engraftment of bone marrow-derived cells (BMDCs) was detected by immunohistochemistry in bone marrow,liver,lung,small intestine and spleen.Results The transplanted bone marrow cells successfully grafted in the haematopoietic tissues from syngeneic GFP transgenic mice.The transplanted GFP+ cells were also detected in the non-haematopoietic tissues,such as the small intestine,liver,spleen and lung,after irradiation.However,a lethal dose irradiation of 8 Gy was required to establish successful chimerism in allogeneic transplantation model by infusing the bone marrow cells from C57BL/6 mice to BALB/c mice.Conclusions Bone marrow-derived cells can be successfully grafted into various recipient tissues receiving a 2.5 Gy dose of radiation in syngeneic mice,but not in allogeneic mice.This nonlethal model may help to further study the plasticity and mechanism of bone marrow-derived cells in tissue repair and regeneration after radiation injury.

Quantification of mixed chimerism allows early therapeutic interventions

Hematopoietic stem cell transplantation is the curative option for patients with myelodysplastic syndrome; however, it requires a long post-transplantation follow-up. A 53-year-old woman with a diagnosis of myelodysplastic syndrome underwent related donor allogeneic hematopoietic stem cell transplantation in July 2006. Three months after transplantation, a comparative short tandem repeat analysis between donor and recipient revealed full chimerism, indicating complete, healthy bone marrow reconstitution. Three years and ten months after hematopoietic stem cell transplantation, the patient developed leukopenia and thrombocytopenia. Another short tandem repeat analysis was carried out which showed mixed chimerism (52.62%), indicating relapsed disease. A donor lymphocyte infusion was administered. The purpose of donor lymphocyte infusion is to induce a graft-versus-leukemia effect; in fact, this donor's lymphocyte infusion induced full chimerism. Successive short tandem repeat analyses were performed as part of post-transplantation follow-up, and in July 2010, one such analysis again showed mixed chimerism (64.25%). Based on this finding, a second donor lymphocyte infusion was administered, but failed to eradicate the disease. In September 2011, the patient presented with relapsed disease, and a second related donor allogeneic hematopoietic stem cell transplantation was performed. Subsequent short tandem repeat analyses revealed full chimerism, indicating complete bone marrow reconstitution. We conclude that quantitative detection of mixed chimerism is an important diagnostic tool that can guide early therapeutic intervention...

Research on molecular chimeric mice pre-T cells infusion to reduce response to allogeneic T cells stimulation / 国际生物医学工程杂志

Objective To research the effect of molecular chimeric mice pre-T cells on proliferation ability of allogeneic mouse T cells.Methods The MHC-Ⅰ gene (H-2Kband H-2Db gene) were extracted and amplified by RT-PCR,the identified pre-T cells were transfected by the constructed eukaryotic expression vector of C57BL/6mouse MHC-I (pIRES-H-2Db and pIRES-H-2Kb),non-transfected group and sham pIRES-transfected control group were set.The molecular chimeric cells were transfused back to BALB/c mouse.After 7 days,T lymphocyte cells of each group were extracted,the ability of molecular chimeric cells inducing spleen T lymphocyte response to allogeneic T cells was observed through mixed lymphocyte culture (MLC).Results Sequencing of the plasmid we have constructed showed that insertion sequence contained C57BL/6 mice H-2Kb and H-2Db series which could be retrieved from GenBank.The result of flow cytometry analysis indicated that H-2Kb and H-2Db protein had an increased expression in pre-T cells,the difference with other two groups was statistically significant (P＜0.05).The result of MLC demonstrated that the stimulation index (SI) of T lymphocyte in co-injection transfected pIRES-H-2Kb and pIRES-H-2Db pre-T cells group (0.764±0.074) were significantly decreased compared with non-transfected group(0.983±0.081)and sham pIRES-transfected group(0.994±0.142) (P＜0.05).Conclusions The molecular chimeric pre-T cells infusion can reduce spleen T lymphocyte response to allogeneic T cells and it may induce immune tolerance in vivo.

Analysis of ABO Blood Discrepancies and Transfusion Experiences in Chonnam National University Hospital / 대한수혈학회지

BACKGROUND: ABO blood group discrepancy occurs when the results of red cell tests do not agree with those of the serum test. In order to select the proper blood units for transfusion, clarification of the cause of ABO discrepancies is essential. We analyzed the cases and recent actual transfusion experiences at Chonnam National University Hospital (CNUH). METHODS: In total, among pre-transfusion blood samples at CNUH between January 2012 and July 2013, 55 cases of ABO discrepancies were analyzed retrospectively. RESULTS: The discrepancy incidence was 0.14%. Problems with serum were the most common cause of ABO discrepancies, with 31 cases (56.4%), and extra serum reactivity due to cold allo-antibodies accounted for the highest frequency (n=7). There were three cases of non-specific aggregations caused by commercial RBC constituents and aggregation was not observed when a re-test was performed with other commercial RBCs or self-prepared human RBCs. Two of three cases with mix-field aggregations involved a pair of twins after in vitro fertilization - embryo transfer (IVF-ET). Among 55 patients, 20 were actually transfused, and all but four cases had weaker or identical RBC units and stronger or identical plasma units. CONCLUSION: There were newly revealed ABO discrepancies caused by non-specific aggregations of commercial RBCs and in twins after IVF-ET. In addition, investigation of actual transfusion experiences in patients with ABO blood group discrepancies would be helpful.

Rapid Determination of Chimerism Status Using Dihydrorhodamine Assay in a Patient with X-linked Chronic Granulomatous Disease Following Hematopoietic Stem Cell Transplantation

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.

The impact of chimerism in DNA-based forensic sex determination analysis

Sex determination is the most important step in personal identification in forensic investigations. DNA-based sex determination analysis is comparatively more reliable than the other conventional methods of sex determination analysis. Advanced technology like real-time polymerase chain reaction (PCR) offers accurate and reproducible results and is at the level of legal acceptance. But still there are situations like chimerism where an individual possess both male and female specific factors together in their body. Sex determination analysis in such cases can give erroneous results. This paper discusses the phenomenon of chimerism and its impact on sex determination analysis in forensic investigations.